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the applications of proton magnetic resonance spectroscopic imaging ( 1h mrsi ) of the brain can benefit from technical developments in pulse sequences and hardware advances to overcome various limitations of mrsi , including low snr , long acquisition times , and lipid contamination . in pulse sequence development , 2d press - mrsi and 2d steam - mrsi have been developed to address several of these limitations and are now widely used . by exciting only a small region of interest within the brain , both sequences permit reduction in the field of view ( fov ) , which in turn permits a reduction in the number of phase encoding steps and thereby reduces the scan time required to achieve a given spatial resolution . these pulse sequences also reduce contamination of the metabolite signals by lipid signals from the scalp . these and other advantages have motivated the extension of press- and steam - mrsi from conventional uses to 3d or multiple 2d applications , or to their combination with other techniques , such as echo - planar spectroscopic imaging and spiral mrsi . press and steam mrsi , however , also have several disadvantages , such as relatively small brain coverage and scan times that are still long for human applications , especially when used in 3d or multiple 2d modes . ( 1 ) multiple slices are consecutively excited and sampled within one repetition time ( tr ) , whereas lipid signals from subcutaneous fat are suppressed through the application of oblique saturation bands placed by the user on localizer images . ( 2 ) each slice is dynamically shimmed , and therefore better spectral quality can be achieved than with global shimming . ( 1 ) full echoes , instead of free induction decays ( fids ) , are acquired , so that magnitude spectra can be used without employing phase correction . acquiring full echoes offers an additional advantage for the reconstruction of mrsi signals sampled using a multichannel receiver rf coil , in that the residual water signals can be used as sensitivity references when combining signals from the various channels of the coil . the spacing required between slices fails to acquire information throughout the entire imaging volume , not only losing important information but also creating difficulties for the coregistration of high - resolution mri images with the mrsi data , which is important for the segmentation of mrsi slices and the analysis of the mrsi data . although the use of a 288 ms echo time ( te ) is necessary to accommodate the full number of echoes for 512 data points at a sampling rate of 2000 hz , it also lowers the snr and requires a long tr of 2.3 seconds to sample phase - encoded signals from 4 slices . because each tr is full ( figure 1 ) , any further increase in the number of slices is possible only by linearly increasing the tr , which undermines the fast feature of the pulse sequence . the limited number of slices also limits the volume coverage , requiring an increase in slice thickness and a trade - off with compromising spatial resolution to cover a larger volume with the same 4 slices . hardware development , and the use of multichannel coils in particular , has benefitted the applications of 1h mrsi . the initial and conventional application of multichannel coils in mrs was to improve snr [ 912 ] , as the array of surface coils provides superior sensitivity compared with more conventional quadrature volume coils . in recent years the gains in snr or the reduction in scan time , though valuable in their own right , can be further traded for other potential advantages , such as spatial resolution of mrsi . we note that the limitations of multi - section mrsi are not caused by intrinsic disadvantages of the sequence , but rather are the consequence of technical limitations and compromises that can be addressed powerfully using multichannel coil technology . we hypothesize that the use of multichannel coil technology for multi - section spectroscopic imaging can eliminate the need for gaps between slices and permit an increase in the number of slices without resorting to longer acquisition times or lower spatial resolution for improved brain coverage . therefore , our aim in this report is to synergize the multi - section spectroscopic imaging ( si ) pulse sequence with multichannel coil technology to realize whole brain metabolite mapping by removing the gaps between slices and increasing the number of slices without resorting to trading off longer trs and therefore longer acquisition times or poorer spatial resolution for more brain coverage . the timing of the simplified rf pulse sequence for multi - section mrsi is schematically shown in figure 1 . tp is the time for the preparation period , which includes water suppression and outer volume suppression ; 1 and 2 , are the durations of 90 and 180 pulses , respectively ; te is the spin echo time ; td is the time delay before data acquisition , which is used for slice selection in the z - direction and phase encoding in the x - y plane of the slice ( not shown ) ; tacq is the acquisition time ; tsec is the time for one slice ; the total repetition time tr for n slices is tr = tsec nsec . the times of tp and td , as well as 1 and 2 for example , relate to the hardware and are optimized to the shortest possible times during the design of the pulse sequence . tacq equals n / sw , where n is the number of data points in the echo and sw is the spectral width , both of which are determined by practical considerations and compromises . for example , one way to reduce scan time , tsec , is to reduce the te , which is possible by reducing tacq . increasing spectral width can reduce tacq , but does so at the expense of increasing noise . reducing n reduces tacq , but it will reduce the spectral resolution and produce truncation effects , as the acquisition may begin after the echo signal is fully built up and end before the echo signal is fully decayed , as shown in the dashed box in figure 1 . although zero - padding the truncated echo prior to fourier transformation may improve the spectral resolution , it will also produce wiggling in the spectrum . both low digital resolution and all these factors considered , sw and n were conventionally designated to be 1000 hz and 256 , respectively , for a 1.5 t scanner and 2000 hz and 512 for 3 t scanners . with these parameters , and for pe = 32 32 and nsec = 4 , the te is 280 ms , yielding a tsec of 575 ms , tr = 2300 ms , and a scan time of 30 minutes . together with preparation time , including slice and outer volume suppression ( ovs ) band prescription , shimming , and prescanning , the scan time totals 50 min . further increasing we reduced the number of data points in the echoes acquired on a 3 t scanner from 512 to 256 , so that 7 slices will be covered in a tr of 2300 ms . to avoid the disadvantage of low spectral resolution and the truncation effects on spectral fitting in the frequency domain associated with acquisition of a limited number of data points we performed computer simulations to compare the effects of spectral fitting algorithms on signal truncation in the time domain and spectral resolution or sinc wiggles in the frequency domain . the signal simulated the 3 singlets of ch , cr , and naa with amplitudes of 24 , 36 , and 48 ( a.u . ) , respectively . the linewidth was 10 hz for either a lorentzian or gaussian lineshape . for a voigtian lineshape , the lorentzian decay was 2.5 hz , and the gaussian decay was 7.5 hz for all three lines . spectral width was 2000 hz , the number of data points in the echo was 256 , and it was zero - filled to 512 or 1024 . we also conducted a monte carlo simulation for signal fitting in the time domain and compared the standard deviations of the estimated amplitudes with their cramer rao lower bounds ( crlbs ) , a benchmark for assessing the accuracy of spectral fitting algorithms . we carried out all mr measurements on a spectroscopic phantom and on 3 human volunteers , respectively , using a whole body 3 t scanner ( signa hdx 3.0 t , ge healthcare , waukesha , wi ) , equipped with a quadrature transmit / receive head coil , and an 8-channel receive - only head coil . first , we acquired scout images using a commercial gradient recalled echo - based 3-planar mri sequence and then prescribed the localizer images of the mrsi slices , which were in axial plane for phantom scans and in an oblique axial plane parallel to the anterior commissure - posterior commissure line in human subjects . then , we localized the mrsi slices by copying the location of the localizer images and acquired mrsi data using the multiplanar mrsi pulse sequence . the number of slices , the slice thickness , and spacing varied in accord with those of different mrsi sessions ( vide infra ) . however , typical parameters of the mrsi pulse sequence were as follows for both the phantom and human subjects : number of slices = 7 ; slice thickness = 10 mm ; spacing between slices = 4 or 0 mm ; nominal number of phase encodings ( pes ) = 16 16 or 32 32 ; tr / te = 2300/144 ms ; spectral width = 2000 hz ; number of data points in the echo = 256 . the duration of an mrsi scan was 8 or 30 minutes , depending on the number of pes . total scan time including mri localizer , mrsi slice prescription , ovs band placement , and field shimming was about 26 or 50 minutes . when repeating the mrsi scans with different slice spacings , we changed only the spacing but not the position of the central ( 4th ) slice . therefore , we used signals from this slice to assess the effects of differing slice spacings on the crosstalk between slices . for each mrsi scan with a differing spacing between slices , we performed an autoprescan for field shimming and transmitter gain optimization . then receiver gains ( rgs ) were manually adjusted if needed to retain the same rg values for all mrsi scans . the protocol was approved by the institutional review board of the new york state psychiatric institute . the k - space mrsi data from individual coil elements were transformed to the image domain using a 2d spatial fourier transform after spatial filtering that used a hamming window function . ( 1 ) water signal removalwe used a matrix - pencil - method - based procedure to decompose the signal , identify water components by their frequencies , and remove them from the signal . this method was able to remove water signal almost completely ( > 98% ) without interfering with the metabolite signals of interest . we used a matrix - pencil - method - based procedure to decompose the signal , identify water components by their frequencies , and remove them from the signal . this method was able to remove water signal almost completely ( > 98% ) without interfering with the metabolite signals of interest . ( 2 ) removal of corrupted points we replaced the first 6 echo data points , which were corrupted by the activation of the analogue - to - digit converter , with 6 extrapolated points derived from signal parameters that were estimated from the uncorrupted data points using the matrix pencil method . we replaced the first 6 echo data points , which were corrupted by the activation of the analogue - to - digit converter , with 6 extrapolated points derived from signal parameters that were estimated from the uncorrupted data points using the matrix pencil method . ( 3 ) data apodizationwe next multiplied the cleaned echo data by a gaussian function , g(t ) = e , to suppress noise and reduce baseline distortion , albeit at the expense of line broadening . the line broadening was 10 hz for phantom data and 4 hz for in vivo data . we next multiplied the cleaned echo data by a gaussian function , g(t ) = e ( 4 ) phase alignmentwe eliminated voxelwise phase differences in echoes from individual coil elements by subtracting the phases at the top of their echoes . we eliminated voxelwise phase differences in echoes from individual coil elements by subtracting the phases at the top of their echoes . ( 5 ) weighted summationwe summed the phase - aligned echoes using weighting factors that were proportional to the echo amplitudes and inversely proportional to the noise levels of the coil elements . noise levels were determined by measuring the standard deviations of the data points in the signal - free regions of the frequency domain spectra of a phantom . we summed the phase - aligned echoes using weighting factors that were proportional to the echo amplitudes and inversely proportional to the noise levels of the coil elements . noise levels were determined by measuring the standard deviations of the data points in the signal - free regions of the frequency domain spectra of a phantom . we quantified the spectral components using the following general model function to fit the echoes : ( 1)s(t)=m=1mamei(2fmt+m)em|t|mt2 , where am , fm , m , and m represent the amplitude , frequency , phase , and lorentzian decay of peak m , respectively ; m is the gaussian decay . ( n/2 1 ) , where dt is dwell time and n is the number of data points in the echo . note also that when fitting the spectrum with a pure lorentzian model , we set m to be zeros ; when fitting the spectrum with pure gaussian model , we set the m to be zeros ; when fitting the spectrum with a voigtian model , we set the m to be the same for all m peaks . the signal parameters in ( 1 ) were determined using a nonlinear least squares fitting routine in matlab ( 2007a , the mathworks , natick , massachusetts ) . to ensure that these parameters were real numbers , the real parts and imaginary parts of both the model function in ( 1 ) and the measured echo signal were concatenated , respectively , to form real - number series . it is important to determine accurately the initial values for am , fm , and m , as well as the number of peaks , to ensure the robustness of the fitting . they were estimated from the magnitude spectrum obtained by zero - padding the echo to 4096 points and then performing fft , whereas the global initial phase was obtained by the phase of the top point of the echo . the influence of acquiring contiguous slices on signal intensity was determined by comparing signal amplitudes from the two scans with or without spacing between slices . we selected voxels from a region within the brain in the 4th slice whose location was the same for the scans with or without spacing . the amplitudes of the m - th signal components ( peak areas in the frequency domain ) , am , were used to calculate the relative differences of the signals obtained with or without spacing ( 2)dm=(am , wspam , wosp)am , wsp . we used the means and standard deviations ( s.d . ) of the d 's to evaluate the reductions in signal caused by the tables 1 and 2 show the relative differences of the amplitudes , calculated using ( 2 ) , of the phantom and in vivo mrs signals acquired with and without spacing between slices . the phantom data did not show significant differences in these values ( table 1 ) . the mean values of the differences in amplitudes ( or the signal reduction ) caused by the crosstalk in the in vivo data was < 7.5% for the first two tests , when tr = 2.3 s. on the average , the signal reduction was smaller for the third test when tr = 3.0 s ( table 2 ) . errors can be as large as 4% when fitting the noise - free original data in the frequency domain with a lorentzian or gaussian lineshape , or up to 15% for fitting data with a voigtian lineshape ( table 3 ) . these errors are the consequence of low spectral resolution and a ( voigtian ) model mismatch in the frequency domain . when fitting the zero - padded signals ( from the original 256 to 1024 points ) in the frequency domain , errors were of the same order , whereas errors were reduced to zero when fitting the spectrum with the original 1024 points . this finding indicates that errors using padded signals are caused by wiggles ( figure 2 ) . in contrast , when fitting the original 256 points of noise - free signals in the time domain , the signals can be perfectly recovered , regardless of whether the line shapes are lorentzian , gaussian , or voigtian . monte carlo simulations with 400 noise realizations added to the 256 points time domain signals revealed that the estimated amplitudes of naa , cr , and ch approximated the true values for all three lineshapes . the sds of the lorentzian and voigtian lineshapes were less than 2 times the cramr - rao lower bounds ( crlbs ) , whereas the sds of the gaussian line shapes were approximately 1.5 times the crlb ( table 4 ) . the phase differences caused by the differing positions of coil elements were eliminated , yielding perfect alignment of the array signals in the frequency domain and thereby enhancing snr ( figure 3 ) . the performance of spectral fitting of the data of 256 points in the time domain is further demonstrated using in vivo data . an example of the spectral fitting is shown in figure 4 , in the form of an absolute spectrum . the whole brain mrsi of naa is shown in figure 5 , overlaid on their localizer images . we reported herein improvements of the well - known and widely used multi - section mrsi technique . the modified pulse sequence has several important advantages over its standard implementation : ( 1 ) it eliminates spacing between slices ; ( 2 ) it allows increase in the number of slices ( in our study from 4 to 7 ) without the expense of increasing scan time ; ( 3 ) it permits a reduction in slice thickness and therefore improves spatial resolution , which can be achieved without the expense of reducing overall volume coverage because of the increased number of slices that are acquired ; ( 4 ) it employs a multichannel coil for data acquisition to improve snr . compared with the performance of the original mrsi pulse sequence , each modification individually can have its own unique advantages , limitations , and challenges . spacing between slices , for example , was introduced in the original implementation of the sequence to avoid crosstalk between adjacent slices , a phenomenon of signal interference caused by the imperfect slice profiles in which edges are not clearcut but instead interlace with one another . slice spacing , however , comes at the expense of information loss from the volumes between slices that are not imaged , which can be as large as 30% of the total mrs imaging volume . this reduced volume of coverage also reduces overall spatial resolution . the reduction in signal caused by eliminating spacing between slices was less than 10% in vivo using our modified mrsi sequence ( table 2 ) . this loss of signal would be regarded as substantial if the snr of the original signal was low , but it is in fact an inconsequential loss when using an 8-channel multichannel coil array , which typically doubles snr compared with use of a standard quadrature coil [ 8 , 11 , 12 ] . therefore , even with a 10% signal reduction caused by crosstalk from contiguous slices , the snr using a multichannel coil in conjunction with our modified sequence is still much higher than the snr when using a standard quadrature head coil without crosstalk we note , in fact , that increasing the number of slices from 4 to 7 can actually help to reduce the crosstalk across slices , because interleaved slice excitations are separated temporally by at most one interval if the number of slices is 4 ( i.e. , 1 - 3 - 2 - 4 ) , whereas with 7 slices , the temporal interleave can be 2 or 3 intervals for adjacent slices ( e.g. , 1 - 3 - 5 - 7 - 2 - 4 - 6 , where slices 1 and 2 are temporally separated by 3 intervals , slices 4 and 5 are separated by 2 intervals , and so on ) . in addition , the reduction in slice thickness theoretically would lead directly to a proportional reduction in snr when shimming is perfect . in reality , however , and especially in regions near air - tissue interfaces , the effect of reduced volumes in each slice on snr is not linear because smaller volumes come with narrower line widths . consequently , we did not observe significant signal drop - out in regions of air - tissue interface within the lower frontal lobe ( figure 5 ) . thus , the advantages that our modified multi - section mrsi sequence provides are the whole brain coverage with contiguous slices , potentially improved spatial resolution , and improved spectral line width . the reduction in number of data points in the echo , which is the core of the current modification , poses severe challenges for spectral fitting and spectral quantification . computer simulation ( table 3 ) showed that fitting a spectrum with low spectral resolution in the frequency domain can produce sizeable errors . conversely , increasing the spectral resolution can improve the accuracy of spectral fitting in the frequency domain . when the number of data points in the original echo was 1024 , the frequency domain fitting algorithm perfectly recovered the spectrum . however , significant errors remained if the spectrum was obtained by zero - filling the 256 data points to 1024 ( table 3 ) , reflecting the detrimental effects of truncation or wiggle artifacts on spectral fitting . spectral fitting in the time domain , in contrast , accurately fits the echo using 256 data points , suggesting that time domain spectral fitting is preferable for signals with fewer data points , a possibility that monte carlo simulation of the signal ( table 4 ) and spectral fitting of the in vivo data ( figures 4 and 5 ) are verified . our implementation of whole brain metabolite mapping using a multiple 2d mrsi sequence also affords distinct advantages over 3d mrsi , in which the number of phase encoding ( pe ) steps in the 3rd dimension is typically 8 [ 18 , 19 ] . this small number of data points entails pronounced , long - range signal contamination across slices because of the effects of the point spread function when reconstructing the slice data directly using fft . spatial filtering with hamming , hanning , or kaiser window functions must be applied prior to fft to suppress this signal contamination , but at the expense of increasing the amount of signal bleeding between adjacent slices . therefore , the effective slice thickness of 3d mrsi is approximately 1.4 times that of the nominal slice thickness , which significantly degrades spatial resolution . the effective thickness of slices using multiple 2d mrsi , on the other hand , is close to its nominal value , despite the fact that the slice profile is not ideal and slight crosstalk is present between slices . another limitation of 3d mrsi using press localization is that the first and last slices can not be used , reducing the effective number of slices by 2 [ 18 , 19 ] . our pulse sequence , in contrast , provides high - quality mrsi images in all 7 slices ( figure 5 ) . 3d mrsi has a distinct advantage over multi - section mrsi , however , in that the location of slices in 3d mrsi can be shifted to specific anatomical regions of interest by employing a phase shift of the fourier transform . comparing signal losses in phantom and in vivo mrsi data in the absence of spacing between slices showed that signal loss caused by crosstalk is related to the slice profile , the spatial and temporal separations between successive excitations , and the longitudinal relaxation times of the subjects . our experimental procedures ensured that the spatial and temporal separations of excitations were identical for the phantom and in vivo scans . the longitudinal relaxation times of molecules in the phantom were significantly longer than those in the human brain , and therefore comparatively larger signal loss could be expected if all other conditions remained the same . the fact that signal loss was negligible in the phantom ( table 1 ) but was approximately 10% in the human brain ( table 2 ) when spacing was eliminated as compared with 4 mm spacing can be attributed to a better slice profile in the phantom . comparing spectral fitting in the time domain with fitting in the frequency domain has been of long - standing interest in mrs [ 2025 ] . as the time domain and frequency domain signals are related by the fourier transform , they theoretically have the same information content . however , fitting in the time and frequency domains may have their unique advantages and disadvantages depending on the properties of the signal , such as noise level and phase or baseline distortions [ 2025 ] . time domain methods are preferable in the presence of distortions in the measured signals , including truncation [ 20 , 23 ] . our computer simulation provided a numerical example for the detrimental effects of truncation in the frequency domain and showed that fitting in the time domain is immune to those effects . in conclusion , we have presented a realization of extended brain metabolite mapping using a multiple 2d mrsi pulse sequence in conjunction with use of a multichannel rf coil and spectral fitting in the time domain . these combined techniques have permitted an increase in the number of slices from 4 to 7 , without sacrificing scan time or snr . the extended brain coverage , reduced slice thickness , and increased snr can potentially make the sequence more clinically valuable . this study was funded in part by nimh grants k02 74677 , mh089582 , mh068318 , 1p50mh090966 , niehs grant es015579 , and nida grants da027100 , and da017820 .

multisection magnetic resonance spectroscopic imaging is a widely used pulse sequence that has distinct advantages over other spectroscopic imaging sequences , such as dynamic shimming , large region - of - interest coverage within slices , and rapid data acquisition . it has limitations , however , in the number of slices that can be acquired in realistic scan times and information loss from spacing between slices . in this paper , we synergize the multi - section spectroscopic imaging pulse sequence with multichannel coil technology to overcome these limitations . these combined techniques now permit elimination of the gaps between slices and acquisition of a larger number of slices to realize the whole brain metabolite mapping without incurring the penalties of longer repetition times ( and therefore longer acquisition times ) or lower signal - to - noise ratios .

aptamers are short single - stranded oligomers made up of dna , rna , or peptides that are capable of binding a target ligand ( proteins , small molecules , or even living cells ) with high affinity . they are also known as artificial antibodies because in addition to binding with high affinity , they also bind with high specificity . aptamers have several advantages over antibodies , including ease and low cost of production which does not involve animals . aptamers are less immunogenic than antibodies and are already being used as therapeutic agents in humans . nucleic acid aptamers , unlike antibodies , can be selected for and used under nonphysiological conditions , such as high - salt conditions and varying ph . also , nucleic acid aptamers are able to undergo specific conformational changes that antibodies can not . for example , nucleic acid aptamer binding can be turned off by the addition of the complementary strand . additionally , nucleic acid aptamers can undergo a conformational change when binding to their target and can be used as molecular beacons , fluorescently off when unbound and on when bound . the field of aptamers is rapidly growing as is the number of applications for their use . nucleic acid aptamers are evolved from random sequences of dna / rna by a process known as systematic evolution of ligands by exponential enrichment ( selex ) . the selex procedure involves the use of the random library of dna / rna sequences being incubated with the target , followed by a partitioning step to remove unbound sequences , an elution step to recover the binding sequences , and then an amplification step to generate a library of sequences enriched for binding . the selex procedure generally takes months to complete , with a typical selection requiring 10 or more rounds before completion . also , traditional selex requires a support for the target ( magnetic beads , membranes , etc . ) to bind with . the supports themselves can be targets for selection , and often rounds of negative selection must be done to avoid aptamers for the support . use of capillary electrophoresis ( ce ) allows for selex to be performed in a much shorter amount of time due to much more efficient partitioning and without the aptamers binding to the ligand support ( the ligand flows freely in buffer , there is no support ) . in as little as one round of selection , and generally less than five rounds of selection ce - selex is a new technology first developed for use in 2004 and has yet to be commonly used . one of the main advantages to ce - selex over traditional selex is that the aptamer - target complex can be detected in the first round of selection . this early detection contrasts traditional selex , where several rounds must be done before being able to detect any dna . most ce - selex is done with laser - induced fluorescence ( lif ) to increase the detection sensitivity to the analyzed samples . using ce with lif , a laser excites fluorescently labeled samples passing through the glass capillary tube which then emits light that is captured by an on - board detector attached to the ce machine itself . we have developed a technique for selection of dna aptamers using ce but without the need for an on - board laser / detector system . the system takes advantage of real - time polymerase chain reaction ( rt - pcr ) . rt - pcr is able to sensitively detect dna - target complexes early in the selection procedure with an efficacy greater than that of traditional selex and equal to that of ce - selex with lif . we believe that this system could be beneficial for researchers that have access to ce , but do not have access to ce with lif , and are seeking to perform ce - selex . the protein that we chose to use as a target for aptamer selection was bovine serum albumin ( bsa ) . bsa is widely used for stabilization of enzymes , preventing enzymes of interest from adhering to tubes or pipettes , or as a protein comparison standard . there are several advantages to using bsa in that it is relatively stable , abundantly available from cow 's blood , and is of low cost . due to the low cost and high abundance of bsa , we chose it to serve as the protein target for our rt - pcr - coupled selection system . here , we report the isolation of a dna aptamer with specificity for bsa using the rt - pcr coupled capillary electrophoresis selection system . the target protein of interest , bovine serum albumin ( bsa ) , was purchased as a lyophilized powder through sigma - aldrich and was greater than 98% pure . the bsa was dissolved in rb1 buffer ( 50 mm tris - hcl , ph 8.2 ) and stored at 4c at a stock concentration of 1 mm . the dna library was purchased from alpha dna and contained a sequence of 5_cttctgcccgcctccttcc-(n)36-gacgagataggcggacact_3 ( 36 random nucleotides flanked by two - fixed 19 base regions used later as primers for pcr amplification ) . the protocol for selex using capillary electrophoresis ( ce ) was essentially as described earlier but with a few modifications . the initial bulk affinity assay was performed with 50 m bsa and 25 m dna in order to view any dna - protein complexes and determine the collection window . capillary electrophoresis was done using a beckman coulter proteomelab pa 800 with a photon diode array ( pda ) capable of reading wavelengths in the uv range ( 10 nm400 nm ) . separation was done at a voltage of 10 kv and with 0 psi of pressure , except that during timed fraction collection the machine automatically reaches 0.1 psi of pressure . after determination of the collection window based on the bulk affinity analysis , the first round of selection began . the initial in vitro selection procedure involved 500 nm bsa and 3.3 m dna . the dna library ( 0.3 l at 100 m ) was mixed with 0.3 l of sb3 ( 100 mm tris - hcl at ph 8.2 , 200 mm nacl , and 10 mm mgcl2 ) for a final concentration of 50 m dna library , 50 mm tris - hcl at ph 8.2 , 100 mm nacl , and 5 mm mgcl2 . this mixture was heated in the biorad icycler to 94c for 1 minute and then cooled to 20c at a rate of 0.5c per second . after the folding of the dna library , 5 l of 1 m bsa dissolved in rb1 buffer was added , and additional run buffer was added to make the final volume 10 l . this brought the final concentrations to 3.3 m dna library , 500 nm bsa , 6 mm nacl , 300 m mgcl2 , and 50 mm tris - hcl ( ph 8.2 ) . after each round of selection , fractions were analyzed through real - time pcr ( rt - pcr ) using the abi stepone plus . rt - pcr was done with two primers : the forward aptamer - amplifying primer p1 ( 5_cttctgcccgcctccttcc_3 ) and the reverse primer p2 ( 5_agtgtccgcctatctcgtc_3 ) , respectively . the primers were designed using oligoanalyzer ( http://www.idtdna.com/analyzer/applications/oligoanalyzer/ ) to limit complementarity to each other , in order to decrease nonspecific amplification of self - dimerizing primers . for amplification , 20 l of pcr mix was prepared consisting of 10 l of 2x quanta sybr green pcr master mix ( roche ) , 0.6 l of 10 m p1 , 0.6 l of 10 m p2 , 1 l of collected fraction as template , and 7.8 l h2o . following rt - pcr , the fraction containing the complex was amplified using standard pcr . pcr was done in a 100 l volume consisting of 1 l of 5 u/l ex taq polymerase from takara , 3 l of 10 m forward primer p1 , 3 l of 10 m reverse primer p2 , 10 l of 10x mg buffer ( takara ex taq ) , 8 l of 2.5 mm each dntp , and 5 l of the collected fraction from capillary electrophoresis . pcr was done using primer p1 and p2 as noted previously , except that the number of cycles for pcr amplification was based on 50% of the maximum yield as determined by rt - pcr . the biotin - labeled primer was used subsequent to pcr in order to separate the strand of interest and the nonaptamer strand after pcr . magnetic beads with streptavidin coating from bangs laboratories ( biomag nuclease - free streptavidin ) were used to bind the biotin - labeled dna . strands were separated with 10 mm naoh after three washes with wash buffer ( 10 mm tris - hcl at ph 8 with 500 mm nacl and 1 mm edta ) . post - selection dna cloning of the aptamer pool was done with the zero blunt topo pcr cloning kit ( invitrogen ) . standard pcr with unlabeled primers p1 and p2 was used to generate double - stranded dna containing the aptamer sequence , which was then blunt - end ligated into the pcr - blunt ii - topo vector that contains the kanamycin resistance gene . colonies were selected for growth on kanamycin - containing media ( kanamycin final concentration was 40 g / ml ) , and plasmid dna was isolated using the genejet plasmid miniprep kit ( fermentas ) . asymmetric pcr with unlabeled primers p1 and p2 was used on the plasmid dna to predominately generate the strand of interest which was then analyzed using ce and rt - pcr . twelve colonies were pooled into a group and each group was assayed for bsa binding using ce . individual plasmids from each pooled group that showed binding were sequenced by eurofins mwg operon . based on sequencing results , additionally , mfold ( http://mfold.rna.albany.edu/?q=mfold/dna-folding-form ) was used on each candidate aptamer to identify secondary structure . potential aptamer oligonucleotides and a negative control oligonucleotide were 5 labeled with p32 -atp using t4 polynucleotide kinase ( new england biolabs ) . the negative control consisted of an oligonucleotide of the same length as the random dna library oligonucleotides ( 74 bases ) , contained the same flanking primer regions , and had a fixed sequence for its internal region 5-cttctgcccgcctccttccggtcgggcacacctgtcatacccaatctcgaggccagacgagataggcggacact-3. the internal region was chosen using a random dna sequence generator with a specified gc content of 50% ( http://www.faculty.ucr.edu/~mmaduro/random.htm ) . the binding conditions for the initial emsa were done by adding equal amounts of sb3 buffer to the labeled oligonucleotides ( 20,000 cpm equivalent ) and incubating at 94c for 1 minute in a pcr machine and a gradual cooling to 20c at a rate of 0.5c per second ( total time taken is ~4 minutes ) . bsa stock of 1 mm was made in rb1 buffer ( the run buffer used in the ce - selex protocol ) . different dilutions of bsa , as required for assessing aptamer binding , were also made in rb1 buffer . with fixed concentration of the p32-labeled candidate aptamer , the concentration of the ligand ( bsa ) was varied from 50 to 800 m . the buffer used for binding was a 6x buffer consisting of 600 mm ammonium chloride , 300 mm potassium chloride , 30 mm sodium chloride , 120 mm tris - hcl ph 7.5 , 30% glycerol , and bromophenol blue ( bpb ) 0.25% . the reaction was incubated at room temperature for 30 minutes after which 1.5 l of 10x loading buffer ( 200 mm tris - hcl ph 8.2 , 50% glycerol , and bpb 0.25% ) was added and the reaction was left on ice for 515 minutes before loading into the gel . minigels were made with stock solutions of 40% acrylamide / bis - acrylamide ( 29 : 1 ) , 1x tris - borate edta ( tbe ) , 10% ammonium persulfate ( aps ) , and tetramethylethylenediamine ( temed ) . prerun was done in 1x tbe buffer for 1 hour prior to loading of the samples . the samples were run at 150 v until the bromophenol blue dye reached the bottom of the gel . the radioactivity in the gel was analyzed by phosphor imager ( molecular dynamics - typhoon trio imager ) . apart from the above - mentioned 6x buffer , an additional 5x binding buffer containing 100 mm tris - hcl ph 8.5 , 250 mm nacl , 10 mm mgcl2 , 10 mm zncl2 , and 10% glycerol was added in the incubation mixture . furthermore , the radiolabeled oligonucleotides were added directly to the incubation mixture and not heated to 94c as done previously . samples were left on ice for 515 minutes before being loaded into the 4% nondenaturing gels which had been prerun for 1 hour in 1x tbe as described previously . increasing concentrations of the specific unlabeled oligonucleotide were added during incubation of the bsa - aptamer complex ( conditions described previously ) . three different amounts ( 1.25 , 2.5 , and 5 pmol ) were used . complex formation was allowed to go on for 30 minutes at room temperature before the radioactively labeled oligonucleotide was added and the incubation was continued for another hour and 30 minutes . in the control sample , all the conditions were similar except for the absence of the unlabeled competing oligonucleotide . emsas were set up using 4 g of the anti - bsa polyclonal antibody ( obtained from invitrogen ) . essentially , the antibody ( 4 g ) was mixed with bsa ( 500 m ) along with the 5x binding buffer and left on ice for 1 hour . the radioactively labeled i1 - 5 aptamer and the 6x buffer were added next and the incubation continued for 1 hour and 30 minutes at room temperature before stopping the incubation by leaving the samples on ice for 515 minutes . the general scheme of rt - pcr coupled ce - selex is shown in figure 1 . to test the concept that aptamer selection with ce - selex and rt - pcr could be feasible , we first began by calibrating the capillary electrophoresis ( ce ) machine . the ce machine used , the proteomelab pa 800 , is incapable of running solutions that contain high concentrations of salts . due to this limitation , our buffers had to be tested to find the proper salt balance to ensure that the machine did not fail but at the same time had enough salt to stabilize our aptamer structures . therefore , the buffer condition used for the initial step of folding the dna library was done in a high - salt buffer ( 50 mm tris - hcl at ph 8.2 , 100 mm nacl , and 5 mm mgcl2 ) followed by incubation and running the folded dna library in low - salt concentrations ( 50 mm tris - hcl at ph 8.2 , 6 mm nacl , and 300 nm mgcl2 ) to ensure both proper folding and running of the dna library . although the low - salt problem may have excluded many potential binders to bsa , low - salt concentrations actually mirror the physiological conditions more accurately since the cellular level of magnesium is only ~1 - 2 mm . after optimizing for the salt concentrations , we used ce and ran a large amount ( 100 m ) of bsa alone in order to visualize the free protein peak run timing . next , we performed the same analysis using a large ( 3 m ) amount of dna alone . once the individual free protein and free dna run times were established , we proceeded to combine bsa and dna for a bulk affinity analysis ( figure 2 ) . the bulk affinity analysis allowed us to visualize peaks for the free bsa and the free dna in combination , and this information was used to determine the collection window . the ce machine used was the proteomelab pa 800 from beckman coulter , which did not have laser - induced fluorescence ( lif ) . to sensitively detect protein - dna complexes between the free dna and free protein , we relied on rt - pcr . the collection window started from the end of the free - protein peak to the start of the free - dna peak ( figure 3(a ) ) . the seven fractions , instead of just one fraction , allowed us to analyze with greater precision the region of the dna - protein complex . unlike the bulk affinity assay in which 100 m bsa was used , for the selection , the concentration of bsa was reduced to 500 nm in order to increase the stringency of selection . in the first round of selection , the initial collected fraction of dna detected by rt - pcr needed 36 cycles of amplification to reach 50% of the maximum yield , the midpoint cycle , of pcr ( figure 3(a ) ) . the second fraction analyzed had a higher amount of dna compared to the first fraction collected , taking only 34 cycles of amplification to reach the midpoint cycle . the amount of dna collected for the third and fourth fractions was lower than that of the second fraction collected . the fifth fraction collected showed a marked increase in dna indicative of the beginning of the free - dna peak and so this fraction was not used . the pattern detected by rt - pcr implied a dna - protein complex present in the second fraction collected since this fraction was collected by ce after the free protein peak , but well before the free - dna peak . the collected dna containing the supposed dna - protein complex was rt - pcr amplified using primers flanking the internal random sequence . the forward primer , p1 , amplified the aptamer - containing strand of interest , while the reverse primer , p2 , amplified the nonaptamer complementary strand . in a previous protocol to select dna aptamers , it was shown that overamplification of the random library leads to formation of nonspecific products , therefore the random library was only amplified to ~50% of the maximum yield as measured by rt - pcr . after rt - pcr analysis revealed the optimum number of cycles to amplify the collected dna , a regular pcr using more of the collected dna was performed . in this regular pcr , the p2 primer was labeled at its 5 end with biotin while p1 had no modifications . after the pcr , the dna products were attached to streptavidin - coated beads due to the strong interaction between the streptavidin on the beads and the biotin - labeled primer p2 . after immobilization on the beads and several washing steps , the forward aptamer - containing strand was released from the complementary strand by incubation with 10 mm naoh . after this step , the binding of the new aptamer pool was analyzed using ce and rt - pcr . to assess the binding of the newly generated aptamer pool to the target , two separate ce runs were done , one with a 200-fold molar excess of bsa used for the first round of selection ( 100 m ) ( figure 3(b ) ) and another without any bsa ( figure 3(c ) ) . based upon these ce test runs , it was clear that the second fraction collected in round one of ce - selex did show binding to bsa . the aptamer pool ( 5 l of the collected fraction ) was then subjected to another round of selection . selection continued until the fourth round ; at which point rt - pcr did not show any change in the amount of dna at the complex and we did not continue with any further rounds of selection . potential aptamers collected during the third round of selection were again amplified and then used for cloning . instead of amplifying with a biotin - labeled p2 primer , the potential aptamers were amplified with a nonlabeled p2 primer since the biotin label at the 5 end of the dna might interfere with ligation during cloning . topo blunt - end cloning was performed with the aptamer pool and colonies were selected by growth on lb plus kanamycin plates . two hundred colonies were selected and analyzed for bsa binding using ce and rt - pcr . groups of 12 colonies were combined in order to facilitate faster analysis . in order to amplify the aptamer strand from the vector with the aptamer insert , asymmetric pcr was performed using a 10-fold excess of the aptamer strand primer . four groups : i1 , r2 , g2 , and o3 showed stronger binding than the others by rt - pcr and then the individual clones from these groups were sequenced . after sequencing , in order to confirm that the potential aptamer sequences were binding to bsa , salt - free oligonucleotides containing the potential aptamer sequences were used in emsa gels . emsas work on the principle that dna bound in a dna - target complex will run slower than free dna . the free dna will appear at the bottom of the gel , while the dna bound in the dna - target complex will be shifted higher up in the gel . the potential aptamers , as well as a negative control ( described in section 1 ) , were initially screened with a set of binding conditions similar to that of the ce experiments . only those sequences that were 76 or 75 bases in length , and identical or almost identical in length to the original library length of 76 bases , were ordered and subsequently screened by emsa . the candidate aptamer sequences were analyzed in silico by using clustalw2 and mfold , but no consensus sequence or specific secondary structure between the sequences was found . of the initial group of oligonucleotides screened by emsa , the i1 group , one member of this group of potential aptamers , i1 - 5 , did bind to bsa and a distinct bound complex could be visualized at 500 and 800 m bsa in the initial emsa ( figure 4(a ) ) . also in the initial emsa , at higher concentrations of bsa ( > 500 m ) , a faint band could be seen . this band was considered a nonspecific complex because both the negative control and the i1 - 5 aptamer had this band . in this emsa , and in all subsequent emsas , the appearance of radiolabeled dna at the top of each lane , in the well , can be seen . the dna at the top of the lanes is caused due to the single - stranded nature of the dna . the single - stranded dna is capable of forming aggregates with itself and with the loading dye . the size of these aggregates prevents the dna from entering the gel and thus it remains in the well . after the initial emsa modeled after the ce experiments , binding conditions were changed to include higher salt conditions that were not possible to run with the proteomelab pa 800 . in addition to buffer conditions , we also did not subject the aptamers to the heating at 94c prior to incubation . again the potential aptamer i1 - 5 showed a bound complex using these modified binding conditions ( figure 4(b ) ) . after the initial screen for potential aptamers with the i1 group , a subsequent screen using oligos from the g2 group and the modified emsa conditions revealed several additional aptamers for bsa ( figure 5 ) . the list of the potential aptamers ' sequences ordered is shown in table 2 , among these aptamers , i1 - 5 was chosen for further characterization . only the i1 - 5 oligonucleotide was further pursued as it was the first aptamer discovered by the screen that consistently showed binding ability to bsa in the ce - selex as well as under the two different emsa conditions . to further confirm the specificity of this aptamer to bsa , a supershift assay using an antibody to bsa was performed . in the presence of 4 g of the anti - bsa polyclonal antibody , the binding capacity was reduced as seen by an appreciable reduction in the intensity of the radioactive oligo banding pattern ( figure 6 ) . this supports the idea that the anti - bsa polyclonal antibody and the bsa aptamer i1 - 5 recognize the same epitope . a similar pattern of banding was reported for the human neurofilament mrna binding to superoxide dismutase1 ( sod1 ) in a supershift assay . also , competition assays were performed by incubating bsa with an excess of unlabeled i1 - 5 aptamer of three different amounts ( 1.25 , 2.5 , and 5 pmoles ) before the addition of the labeled i1 - 5 oligo . the bsa - aptamer bound complex was abolished in the presence of higher amounts of the unlabeled competitor ( figure 7 ) . although by emsa the i1 - 5 aptamer showed low binding affinity to bsa , the cold - competitor assay , as well as the supershift assay indicated that a specific interaction was occurring between bsa and the i1 - 5 aptamer , thus supporting the effectiveness of rt - pcr coupled ce - selex . the conclusion drawn from our studies is that we have been able to generate an aptamer for bsa with appreciable specificity in a few rounds of selection from a random library of dna oligonucleotides . the protocol described here is very efficient compared to the traditional selex which takes much more time and reagents . as noted previously , traditional selex makes use of a solid support to which the target must be bound . the coupling of the target to the support is not completely efficient and often much of the target remains unbound and washed away . using ce - selex , there is no need for a solid support and because of the small size of the capillary a much smaller amount of target is required . even though ce - selex is much more efficient than traditional selex , ce - selex still requires additional equipment ( lif ) beyond the standard capillary electrophoresis machine . ce is not restricted to the isolation of aptamers , and while many labs may have a ce machine , much fewer will have one with lif . rt - pcr and lif both work under the principle of fluorescence . very sensitively and directly , rt - pcr has the potential to be more sensitive because unlike lif , which directly detects the fluorescent signal , rt - pcr can amplify the signal . by amplifying a signal undetectable by lif , dna - target complexes that would have been missed by lif taken together the efficiency , time , and sensitivity of the protocol for dna aptamer selection by coupling ce - selex with rt - pcr described here can benefit other researchers that are also interested in selecting for aptamers and would like to use ce - selex , but are unable to do so for lack of lif . bsa was chosen to test our rt - pcr - coupled selection system , but the bsa aptamer we selected may be beneficial itself . due to the high amount of bsa used in many biochemical assays , sometimes the amount of bsa present in a mixture of proteins obscures accurate readings of proteins of interest . it would be useful to have a way to deplete this unwanted bsa from a reaction . also , bsa can be allergenic and in certain situations it might be beneficial to detect small amounts of bsa contaminant present in cow 's milk . currently , antibodies to bsa provide an answer to these issues , but as previously mentioned , there are several advantages to using aptamers over antibodies .

aptamers are short nucleic acid or peptide sequences capable of binding to a target molecule with high specificity and affinity . also known as artificial antibodies , aptamers provide many advantages over antibodies . one of the major hurdles to aptamer isolation is the initial time and effort needed for selection . the systematic evolution of ligands by exponential enrichment ( selex ) is the traditional procedure for generating aptamers , but this process is lengthy and requires a large quantity of target and starting aptamer library . a relatively new procedure for generating aptamers using capillary electrophoresis ( ce ) , known as ce - selex , is faster and more efficient than selex but requires laser - induced fluorescence ( lif ) to detect the aptamer - target complexes . here , we implemented an alternative system without lif using real - time- ( rt- ) pcr to indirectly measure aptamer - target complexes . in three rounds of selection , as opposed to ten or more rounds common in selex protocols , a specific aptamer for bovine serum albumin ( bsa ) was obtained . the specificity of the aptamer to bsa was confirmed by electrophoretic mobility shift assays ( emsas ) , an unlabeled competitor assay , and by a supershift assay . the system used here provides a cost effective and a highly efficient means of generating aptamers .

laryngeal sensation is an important factor in swallowing [ 69 ] , but quantitative evaluation of laryngeal sensation has been difficult . aviv et al . described the use of an air - puff stimulator for quantitative evaluation of laryngeal sensation [ 7 , 10 ] , but such testing has been limited because of the special equipment required . in the present study , a novel method to measure laryngeal sensation using a flexible laryngoscope and probes previously developed by yaguchi et al . evaluated and compared laryngeal sensation in young adults and elderly individuals using this method . in the present study , the same method was used to evaluate changes in laryngeal sensation before and after radiotherapy for laryngeal and hypopharyngeal cancer . this study was approved by the ethics committee and institutional review board of our hospital ( no . 534 , 534 - 2 ) . the study involved 12 patients with laryngeal ( n = 8) or hypopharyngeal cancer ( n = 4 ) who did not undergo surgery for the primary lesion , and underwent either radiotherapy alone or chemoradiotherapy . all patients achieved a complete response of the primary lesion . in two patients , neck dissection on the side of the lesion table 1 shows the patients characteristics.table 1patients characteristicsno.agesextumour sitetmnradiotherapy dose ( gy)chemotherapyoperation173mhypopharynxt1n0m070none278mlarynxt3n2cm066none365mhypopharynxt2n0m060concomitant474mlarynxt2n2bm070concomitantnd563mlarynxt1bn0m060concomitant670mlarynxt1an0m070none758mhypopharynxt1n0m066concomitant869mhypopharynxt4bn2cm060concomitant959flarynxt1an0m070none1073mlarynxt2n0m070concomitant1153mlarynxt1n2bm066concomitantnd1259mlarynxt2n0m060concomitantnd neck dissection patients characteristics the test procedure was as follows . a novel flexible laryngoscope ( enf - y0005 ) , with a maximum diameter of 3.3 mm and a 1.2-mm probe port was co - developed with olympus ( tokyo , japan ) in previous research [ 11 , 12 ] . when compared with a conventional flexible laryngoscope with a forceps port , this flexible laryngoscope is narrower , has a curvature angle of 130 , and has excellent manoeuvrability ( fig . 1 ) . the probes , also co - developed with olympus , have a nylon filament attached to a wire tip and are protected by a sheath to prevent entanglement with the flexible laryngoscope . four types of probes with nylon filament diameters of 0.06 , 0.13 , 0.20 , and 0.30 mm were used ( fig . 1 ) . these were prepared based on semmes weinstein monofilaments , which are used to test sensation , for example , in diabetic peripheral neuropathy . at the time of the study , the flexible laryngoscope and probes had not yet become commercially available , and they were provided by olympus.fig . the top is a conventional flexible laryngoscope , and the bottom is the novel , thinner , and flexible laryngoscope . the nylon filament diameters are a 0.06 , b 0.13 , c 0.2 , and d 0.3 mm flexible laryngoscopes and probes . the top is a conventional flexible laryngoscope , and the bottom is the novel , thinner , and flexible laryngoscope . the nylon filament diameters are a 0.06 , b 0.13 , c 0.2 , and d 0.3 mm to insert the flexible laryngoscope without using intranasal anaesthesia , 1:2,000 epinephrine was gently applied to widen the nasal cavity . the flexible laryngoscope was inserted to the oropharynx , and the probes were placed in contact with the tip of the epiglottis and the arytenoid region mucosa ( at a non - tumour site in cases of hypopharyngeal cancer ) . sufficient contact was made so that there was a slight bending of the probe ( fig . the flexible laryngoscope and probes were manipulated , whenever possible , by the same person . patients were asked to ring a buzzer or tap their hand when they felt the probe . for objective evaluation , the laryngeal adductor reflex was observed . testing started with the thinnest probe , and the sensation level was based on the probe giving two of three positive responses.fig . the nylon filament extending out from the sheath tip is in contact with the epiglottis and the arytenoid region laryngoscopic findings during measurement . the nylon filament extending out from the sheath tip is in contact with the epiglottis and the arytenoid region testing was performed four times ; before radiotherapy , 1 , 3 months , and 1 year after completing radiotherapy . the wilcoxon - signed rank test with bonferroni correction was used to confirm significant differences between each testing time . a correlation between the sensation levels of the epiglottis and comparison of sensation levels between mild mucositis and severe mucositis was analysed using the repeated measures analysis of variance test . the grade of radiation mucositis was classified based on the national cancer institute s common terminology criteria for adverse events v.3 ( table 2).table 2radiation mucositis classification based on the national cancer institute s common terminology criteria for adverse events v.3grade 1erythema of the mucosagrade 2patchy ulcerations or pseudomembranesgrade 3confluent ulcerations or pseudomembranes ; bleeding with minor traumagrade 4tissue necrosis ; significant spontaneous bleeding ; life - threatening consequencesgrade 5death radiation mucositis classification based on the national cancer institute s common terminology criteria for adverse events v.3 laryngeal paralysis did not occur before or after treatment in any patient , and arytenoid adduction during phonation was preserved . in patients 8 and 9 , sensation testing 1 year after radiotherapy figure 3 shows the results of laryngeal sensation testing and the grade of radiation mucositis . mucositis was mild in seven patients ( grade 1 in 1 patient , grade 2 in 6 patients ) and severe in five patients ( grade 3 in 5 patients ) . all patients showed sensory deterioration of the epiglottis and/or the arytenoids until 3 months after radiotherapy . one year after radiotherapy , 60% of patients showed the same sensation levels in the epiglottis as before radiotherapy , or better sensation levels than at 1 or 3 months after radiotherapy . with respect to arytenoids sensation , 90% of patients showed the same sensation levels as before radiotherapy or better sensation levels than at 3 months after radiotherapy.fig . 3the sensation levels of the epiglottis and arytenoid ; before , 1 , 3 months , and 1 year after radiotherapy in each patient . closed symbols ( filled circle , filled triangle , filled square , filled diamond , filled inverted triangle ) are the severe mucositis group ; other symbols are the mild mucositis group the sensation levels of the epiglottis and arytenoid ; before , 1 , 3 months , and 1 year after radiotherapy in each patient . closed symbols ( filled circle , filled triangle , filled square , filled diamond , filled inverted triangle ) are the severe mucositis group ; other symbols are the mild mucositis group with respect to sensation of the epiglottis , the wilcoxon - signed rank test with bonferroni correction showed a significant deterioration ( p < 0.017 ) in sensation 1 and 3 months after radiotherapy compared with before radiotherapy . however , there was no significant difference in sensation 1 year after radiotherapy when compared with before radiotherapy ( p = 0.04 ) . with respect to arytenoids sensation , the wilcoxon - signed rank test with bonferroni correction showed a significant deterioration ( p < 0.017 ) in sensation 1 and 3 month after radiotherapy when compared with before radiotherapy , but at 1 year , the difference was not significant when compared with before radiotherapy ( p = 0.12 ) . there was a significant correlation ( p < 0.0001 ) between the sensation levels of the epiglottis and the arytenoid region on the spearman rank correlation . figure 3 also compares the sensation levels between mild mucositis and severe mucositis ; there were no significant differences on the repeated measures analysis of variance test for both the epiglottis ( p = 0.09 ) and the arytenoid region ( p = 0.58 ) . radiotherapy of the laryngopharynx sometimes leads to functional disabilities including swallowing dysfunction [ 25 ] . the reasons for these disabilities are mucosal and muscular inflammation , fibrosis , reduced saliva secretion and deterioration of laryngeal sensation . laryngeal sensation is an important factor in swallowing , and we decided to evaluate laryngeal sensation . procedures for testing laryngeal sensation by laryngoscope include fiberoptic endoscopic evaluation of swallowing with sensory testing ( feesst ) with an air - puff stimulator , as described by aviv et al . , direct mucosal contact with a flexible laryngoscope , and the use of probes , as discussed in the present report . feesst is a minimally invasive procedure to quantify sensation by air pulses stimulating the laryngeal mucosa at a constant pressure and time [ 1316 ] , but the need for a special air - puff stimulator has limited its use . direct contact with a flexible laryngoscope is simple , but it is not very quantitative , with large inter - operator variability . our method using probes , originally described by yaguchi et al . , permits semiquantitative evaluation , is minimally invasive , and is convenient for use in clinical settings ; it allowed us to evaluate changes in laryngeal sensation before and after radiotherapy . first , the test method used in the present study and its development are described . the novel narrow flexible laryngoscope with probe port was considered more appropriate for sensation testing because it was less irritating and unlikely to touch other sites during testing . in selecting an appropriate diameter of nylon filament to use with this method , yaguchi et al . considered semmes weinstein monofilaments , which are used to measure tactile sensation by pressing nylon fibres against the skin . however , the thinnest commercially available nylon filament ( diameter , 0.13 mm ) , in a kit from arkray inc . therefore , yaguchi et al . decided to develop a thinner nylon filament with half the diameter ( 0.06 mm ) , and selected four sizes of nylon filaments for testing : 0.06 , 0.13 , 0.2 , and 0.3 mm . we considered these sizes optimal for sensation testing , and the same size nylon filaments were used in the present study . our selection of measurement sites ( the epiglottic tip and the arytenoid ) was also based on the previous research . these areas are innervated mainly by the superior laryngeal nerve , have a dense distribution of sensory fibres , and are relatively accessible to testing . in determining the sensation level , used subjective and objective findings , and a positive sensory response was based on the agreement between findings . positive response was based on the agreement between subjective and objective findings . when evaluating laryngeal sensation using a flexible laryngoscope , yaguchi et al . limited the number of contacts to avoid patient discomfort , and estimated the sensation level based on two of three positive responses . accordingly , in the present study , sensation level was based on two of three positive responses . although increasing the number of contacts may improve repeatability and precision , we were concerned that increasing the number of contacts might be painful and uncomfortable for patients and may increase the study dropout rate . next , the measurement results are described . in this study , in both the epiglottis and the arytenoid , laryngeal sensation deteriorated significantly 1 and 3 month after radiotherapy compared with before radiotherapy . . evaluated laryngeal sensation in patients with dysphagia by feesst , and they reported a direct association between radiotherapy of the head and neck and severe laryngeal sensation deficits . on the other hand , 1 year after radiotherapy , there was no significant difference compared with before radiotherapy for both the epiglottis and the arytenoid . as mentioned in our results , most patients showed the same sensation levels as before radiotherapy , or better sensation levels than 3 months after radiotherapy . however , we suggest the testing should be repeated 2 or 3 years after radiotherapy . according to dietz et al . , 16% of patients who underwent chemoradiatiotherapy for larynx / hypopharynx carcinoma still showed dysphagia 3 years after chemoradiotherapy . in the present study , a significant correlation was observed between the sensation levels of the epiglottis and the arytenoid region . therefore , in cases where tests are conducted using a conventional laryngoscope with a rather long diameter with a forceps port , or where measurement in the arytenoid region is difficult due to severe pain , it may be possible to determine deterioration of sensation in the arytenoid region to some degree by measuring the sensation in the epiglottis . the reasons for deterioration of laryngeal sensation after radiotherapy need to be considered . bodin et al . reported that a dose of 64 gy in the head and neck field can damage sensory nerves . another study reported peripheral neuropathy in 75% of patients receiving radiotherapy doses of 63 gy . neuropathologic features in peripheral neuropathy include necrosis and hyalinization of the media of small arteries , fibrous replacement of nerve fibres , demyelination , and fibrosis of the nerve sheath . in the present study , 3 of 12 patients received 60 gy , and the other 9 patients had doses of 6670 gy ; thus , peripheral neuropathy was highly likely . the loss of sensory receptors was also considered because , in a study on taste dysfunction in patients receiving radiotherapy to the oropharynx , pathologic analyses showed diminished taste buds . in the same manner , radiotherapy of the laryngopharynx probably causes loss of sensory receptors . in the present study , there was no significant difference in sensory deterioration and sensory recovery between the severe radiation mucositis group and the mild radiation mucositis group . however , based on the underlying mechanism , a correlation between grade of radiation mucositis and deterioration of laryngeal sensation is possible . finally , there have been reports of peripheral paresthesia caused by anticancer agents [ 2123 ] . in the present study , we did not find a significant difference in deterioration and recovery of sensation with the combination of chemotherapy and radiotherapy , but this issue must also be further investigated by accumulating more cases . a novel procedure using a flexible laryngoscope and probes allowed convenient evaluation of changes in laryngeal sensation in patients before and after radiotherapy . in both the epiglottis and the arytenoid , laryngeal sensation deteriorated significantly after radiotherapy .

radiotherapy of the laryngopharynx sometimes leads to functional disabilities including swallowing dysfunction . one of the reasons for these disabilities is a deterioration of laryngeal sensation . laryngeal sensation is an important factor in swallowing , but quantitative evaluation of laryngeal sensation has been difficult . in this study , we evaluated changes in laryngeal sensation before and after radiotherapy for laryngeal and hypopharyngeal cancer , using a flexible laryngoscope and probes . this study was conducted in 12 patients , 8 with laryngeal cancer and 4 with hypopharyngeal cancer , who received radiotherapy alone or chemoradiotherapy at our medical centre . measurements were performed using a 3.3-mm - diameter flexible laryngoscope with a probe port and four types of probes with 0.06- , 0.13- , 0.20- , and 0.30-mm nylon filaments attached to a wire tip . sensation was evaluated at the tip of the epiglottis and the arytenoid region . measurements were performed before radiotherapy , 1 , 3 months , and 1 year after completion of radiotherapy . sensation of the epiglottis and arytenoid deteriorated significantly 1 and 3 months after radiotherapy compared with before radiotherapy . laryngeal sensation recovered in most cases within 1 year after radiotherapy . the present study clearly demonstrates the deterioration of laryngeal sensation with radiotherapy .

in the 1970s , max cooper , dennis osmond , and others discovered that immunoglobulin ( ig)-bearing b cells arose from ig - negative precursor cells in the bone marrow . these precursor cells dubbed pre - b cells by cooper had synthesized part of the receptor ( ig heavy chains ) , which could be detected in the cytoplasm , but had yet to assemble a complete receptor on the cell surface ( 1 ) . osmond later pinpointed a more primitive population of cells ( pro - b cells ) that had not yet synthesized heavy chains ( 2 ) . but the cellular phenotypes that might be lurking between the pro - b and pre - b cell stages remained mysterious , as there was no way to pick apart this mixed bag of cells . as a result , many of the early insights into b cell development including the ordered rearrangement of the v , d , and j gene segments of the ig locus came from studies using transformed b cell lines . the discovery that stromal cells could support the growth and differentiation of primary b cell precursors soon overcame this obstacle ( 3 , 4 ) . then came multicolor ( multilaser ) flow cytometry , a technique that revolutionized the study of b cell development ( and immunology as a whole ) by allowing single cells to be stained simultaneously with multiple fluorescently tagged mabs . hardy a chemist by training used one of the first multilaser facs ( fluorescence activated cell sorter ) machines to investigate mature b cell subpopulations as a post - doc with leonore and leonard herzenberg in the early 1970s ( 5 , 6 ) . with these techniques in hand , plus a battery of lymphocyte - specific mabs ( generated by the herzenbergs , john kemp , and others ) , hardy by then a new faculty member at fox chase cancer center ( philadelphia)set out to explore the diversity of b cell precursors . he started by dividing b220 bone marrow cells based on their expression of cd43 , bp-1 and cd24 . sorting the cd43 populations of cells and culturing them in vitro revealed their order of differentiation : fraction a ( bp-1/cd24 ) begot fraction b ( bp-1/cd24 ) , which in turn begot fraction c ( bp-1/cd24 ) . later ( cd43 ) populations could be distinguished by their expression of surface igm and igd . it was so striking the way you could sort these cells and see the progression , says hardy . hardy confirmed the developmental order of the cells by amplifying the ig genes from each fraction . the ig genes from fraction a cells were in a germline configuration , whereas those from fractions b and c had d - j , but not v - d - j rearrangements , consistent with previous studies . hardy published these data , along with his fractionation scheme , in 1991 in the journal of experimental medicine ( 7 ) . hardy was not alone in his attempt to bring order to early b cell development . fritz melchers and colleagues in basel , switzerland , who discovered the surrogate light chain , added c - kit and cd25 to the growing list of b cell markers ( 8) . that list now goes on and on ( and facs is up to 17 colors ) , with each new marker revealing new layers of diversity . as melchers puts it , it 's a mess , but it 's a beautiful mess . hardy prefers to call it a beautiful complexity , adding , it 's like a fractal the deeper you look , the more you see .

in the early 1990s , richard ( randy ) hardy and colleagues divided b cell precursors into subpopulations the hardy fractions based on the cells ' expression of various cell surface proteins . this classification helped lay the groundwork for our present - day understanding of the molecular events that control early b cell development .

the diagnostic and statistical manual of mental disorder ( dsm-5 ) used by many mental health practitioners does not define social media addiction as a disorder . however , in the fifth edition of dsm-5 , internet gaming disorder is identified in section 3 as a condition warranting more clinical research and experience before it might be considered for inclusion in the main book as a formal disorder . regardless , a vast number of people show what are apparently symptoms of addiction to cyberspace . this phenomenon seems to afflict young individuals , in particular , with evidence revealing students whose academic achievement is undermined as they spend increasing amount of time online [ 1 , 2 ] . some also experience adverse health consequences resulting from lack of sleep due to being online for a long time , particularly late at night . research into internet addiction has grown significantly since the mid-1990s , especially as more and more cases among college students have been detected by university healthcare staff . in addition to internet addiction , concepts such as internet dependency , compulsive internet use , problematic internet use , dysfunctional internet use , and pathological internet use have been used to describe what is fundamentally the same phenomenon . for the current paper , i will use internet addiction chiefly due to its wide adoption in the research in this area . a study in the uk , for example , found internet addiction to be widespread among 18% of young people . a study in italy found the rate to be only 0.8% . furthermore , a large sample survey in china puts the rate at 12% among male and 5% among female college students . internet addiction is not just limited to college students ; it also extends to high school as well as middle school students . a longitudinal survey conducted in hong kong found the incidence rate of internet addiction as high as 26.7% among high school students . as for the amount of time spent online , research indicates that individuals , who consider themselves internet addicts , reported that it varies greatly from 8.5 hours per week to 21.2 hours per week . other studies found a positive correlation between the amount of time spent online and the severity of the symptoms of internet addiction [ 11 , 12 ] . regarding users ' psychological profile , studies have revealed a correlation between depression , locus of control , loneliness , social anxiety , and self - esteem and internet addiction [ 13 , 14 ] . whang et al . , for example , found that internet addicts had a higher degree of loneliness and depression compared to nonaddicts . other findings showed that computer self - efficacy was a significant correlate of problematic internet use . internet addiction was also related to poor mental health and low self - esteem in adolescents . online addiction is virtually indistinguishable from social media addiction with the single difference of the latter being used primarily on mobile devices . while none of the previous researches dealt with the use of mobile social media as such , it is safe to say that the results of computer related internet addiction also apply to mobile internet since they both use the same medium essentially . the introduction of anytime , anywhere wi - fi in mobile phones and the prevalence of free social media apps make it impossible to differentiate them from personal computers when it came to internet addiction . furthermore , as their name signifies , mobile phones are portable providing easy access to the internet regardless of place and time . mobile social media offer a large number of experiences from a psychological perspective , each with potent features that can lead to problem behavior . for example , the extrovert might spend much time on facebook , repeatedly going over their profile to see the number of likes their latest post received . for others , with a narcissistic predisposition , instagram may prove to be an addictive medium for them to display themselves to others with selfies . social anxiety can also lead to social media addiction . the fear of missing out ( fomo ) can be the key reason for repeated social media use irrespective of time of day to the detriment of other activities . mobile phone addiction is sometimes used to distinguish it from the concept of internet addiction [ 18 , 19 ] . most of the traditional studies of online addiction do not address problematic mobile phone use . mobile phones today offer access to almost all internet applications along with voice and video calls , text messaging , and video recording . also , there is a myriad of engaging apps designed especially for small screens , but their results can also be shown on any screen . furthermore , they have the added dimension of being always available , unlike a desktop or even a laptop . the mobile phone can be used while walking and riding on public transportation and even while driving . these micro time slots in which people can engage in a multitude of online activities were not previously available . micro time slots can lead to obsessive mobile phone usage and can interfere with face - to - face interaction and harm academic performance . a study of taiwanese female university students , for example , found that students , who scored high on a test of mobile phone addiction , showed more extraversion and anxiety and somewhat lower self - esteem . another study found that problematic cell phone usage is associated with age , depression , extraversion , and low impulse control . cellphone addiction also varied considerably across male and female users with women spending far more time using their cell phones and showing more signs of addiction than men . another feature of mobile phones that may prove to be of particular significance to addictive behavior is texting either directly or through social media such as twitter and similar applications . recent surveys indicate that young people are starting to discard facebook in favor of twitter , particularly as their parents create accounts and ask to be these types of applications are growing and enabling more and more features such as vine , which allows users to create six - second videos to share with followers . the common feature of these applications is their stickiness , the tendency to have users utilize the app frequently . stickiness is a result of their business models that rely on the growing mass of data on user behavior to share with advertisers for targeted marketing . the scale used the most in internet addiction research is young 's internet addiction test ( iat ) . the researchers extracted six factors from the iat : salience ( five items ) , excessive use ( five items ) , neglect of work ( three items ) , anticipation ( two items ) , self - control ( five items ) , and neglect of social life ( two items ) . these factors accounted for 35.80 percent , 9.02 percent , 6.51 percent , 6.02 percent , 5.55 percent , and 5.21 percent of the variance , respectively . despite the small sample size , widyanto and mcmurran suggested that the iat has the potential to be a good foundation for constructing a valid measurement . chang and law performed factor analysis of iat using a sample of 410 students from eight universities in hong kong . the researchers then identified three factors : withdrawal and social problems ( 9 items ) , time management and performance ( 5 items ) , and reality substitution ( 3 items ) . these factors accounted for 24.29 percent , 20.80 percent , and 10.64 percent of the variance , respectively . conducted a psychometric analysis of the iat with results from a sample of 215 us undergraduates from two universities . consequently , they concluded that the iat is a valid measurement for assessing internet addiction in us college students . khazaal et al . studied the psychometric properties of a french version of the iat with a sample of 246 adults composed of undergraduate medical students and volunteers from the community . the participants ages ranged from 18 to 54 years ( 81 males and 165 females ) . they reported one factor that had good loadings and goodness of fit . finally , hawi attempted to validate an arabic version of the iat from a sample of 817 from intermediate and secondary school students aged 1022 years . his analysis yielded one factor that fits the data well , something similar to the french version . the present study sought to examine the psychometric properties of an arabic version of the iat customized to assess social media , in particular , not the entire internet space . the word internet was replaced by social media to arrive at social media addiction scale ( smas ) . more specifically , the research aims to answer these questions : what is the internal consistency of the smas?how many factors underlie the smas , and what are they?to what degree is the smas concurrently valid ? what is the internal consistency of the smas ? a total of 1327 undergraduate students enrolled in communication courses in a large state university completed a self - administered survey questionnaire over a period of four months during the academic year in 2014 . the sample was selected based on convenience and therefore it is not a probability sample . a research assistant distributed the questionnaires at the beginning of each class and the time it took to fill it out is just under ten minutes . the age of the respondents ranged from 18 to 31 with 96% ranging from 18 to 25 years of age . this gender distribution reflects the enrollment profile of the university student body that is 70% female . the smas consisted of 14 items adapted from the iat to fit the context of social media usage . the items were rated on a five - point likert scale : strongly agree , agree , neutral , disagree , and strongly disagree , scored 5 , 4 , 3 , 2 , and 1 , respectively . the smeq provides a way of quantifying levels of personal use , measuring the extent to which people 's key daily activities tend to involve social media . it consists of five items measuring social media usage patterns throughout different parts of the day from the time one gets up in the morning to the time they go to bed . the items were rated on a seven - point scale ranging from 0 ( never ) to 7 ( seven times ) ( table 2 ) . table 1 shows the wording of all fourteen items in addition to the means and standard deviations . next , the 14 items of the smas were subjected to exploratory factor analysis using principal access factoring ( paf ) extraction method ( table 3 ) . the first included five items explaining 31.06 percent of the variance , an eigenvalue of 4.34 , and cronbach 's alpha of 0.75 . i call this factor social consequences of sm since the items seem to reflect how sm usage affects one 's daily life activities . the second factor contained three items explaining 11.82 percent of the variance , an eigenvalue of 1.65 , and cronbach 's alpha of 0.66 . i call this factor time displacement since the items reflect the time dimension as it relates to sm usage . the third factor had four items explaining 8.84 percent of the variance , eigenvalue of 1.23 , and cronbach 's alpha of 0.61 . all labels are tentative pending further investigation of the subject based on thorough theoretical grounding . the three - factor result is inconsistent with the arabic version of the iat that had a single factor even though it contained far more items . the result is also different from findings in the us ( two factors ) , the uk ( six factors ) , and france ( one factor ) [ 31 , 32 ] discrepancies are most likely due to sampling procedures and differences inherent in the study sample for each location . these factors can be demographic characteristics , cultural norms , or socioeconomic factors . to assess the concurrent validity of the smas a correlation matrix was needed to assess the relationship between it and a criterion variable that has a theoretical link to it . to do this , a correlation was calculated between the three factors of smas and social media engagement questionnaire ( smeq ) . the smeq measures the extent to which people 's key daily activities tend to involve social media . the smeq consists of five items that were all included in the same survey . we would expect the smeq to be positively related to smas since they both purport to measure the extent of usage . the three - factor scores of the smas were correlated with the five items of the smeq . table 4 shows results of this analysis . as can be seen , out of a total of 15 combinations all items were positively correlated save one . that would be a 93 percent success rate which is very good . this leads to the conclusion that the smas is a valid measure of addiction in our sample . smas is a variant of iat customized to measure addiction to social media instead of the internet as a whole . first , the exploratory factor analysis showed that a three - factor model fits the data well . second , concurrent validity analysis showed the smas to be a valid measure of social media addiction . finally , this study showed that the arabic version of the smas is a valid and reliable instrument for use in measuring social media addiction in the arab world .

this study investigated the psychometric properties of the arabic version of the smas . smas is a variant of iat customized to measure addiction to social media instead of the internet as a whole . using a self - report instrument on a cross - sectional sample of undergraduate students , the results revealed the following . first , the exploratory factor analysis showed that a three - factor model fits the data well . second , concurrent validity analysis showed the smas to be a valid measure of social media addiction . however , further studies and data should verify the hypothesized model . finally , this study showed that the arabic version of the smas is a valid and reliable instrument for use in measuring social media addiction in the arab world .

pulmonary thromboembolism , consisting of deep vein thrombosis ( dvt ) and associated complications , is associated with serious and fatal outcomes . for almost one - quarter of such patients , autopsy is the criterion standard method of establishing death due to pte [ 13 ] . there have been a number of studies on the pathophysiology of pte [ 47 ] . pte causes right ventricle ( rv ) dysfunction , which is associated with a dramatically higher incidence of mortality . few reports have addressed the heart findings of patients with pte by autopsy . to identify the overall effects of pte on the heart , we evaluated the heart findings and clinical characteristics of deceased patients that had been diagnosed with pte . forensic files stored at the council of forensic medicine , ministry of justice in istanbul , turkey , from january 2010 to december 2014 were screened for deaths with autopsy - confirmed pte . the following data were collected : demographic information , medical history , clinical diagnosis , signs and symptoms before death , bmi , point of origin of pte , thicknesses of the lv and rv walls , and heart weight . symptoms and clinical courses of the deceased were determined using reports by the bereaved family recorded in the patient s files . pte were classified into 2 categories : fatal ( no other cause of death found at autopsy , the emboli occluding at least 2 lobar arteries ) and contributory ( factors other than pte also implicated , the emboli occluding at least 1 lobar artery or multiple segmental artery ) . cases with a history of cardiopulmonary disease or a finding of significant disease at autopsy , including valvular heart disease and > 50% coronary artery obstruction , were excluded from the cardiac evaluation . right and left ventricular hypertrophy was quantitatively assessed by autopsy using recommendations of the american society of echocardiography . we defined lv wall thickness 1.2 cm and rv wall thickness 0.8 cm as abnormal . all data were managed and analyzed using the statistical package for the social sciences version 13.0 software . quantitative data are presented as means standard deviations , and categorical data are presented as counts and percentages . all data were managed and analyzed using the statistical package for the social sciences version 13.0 software . quantitative data are presented as means standard deviations , and categorical data are presented as counts and percentages . of the 48 cases with pte , 21 ( 43.8% ) were males and 27 ( 56.3% ) were females . there were 26 cases ( 54.16% ) over the age of 40 years , and 22 ( 45.83% ) under the age of 40 years . obesity , defined as a body mass index ( bmi ) 25 , was detected in 30 patients ( 62.5% ) and the mean value was 27.164.67 . the majority of patients exhibited large , coiled thromboemboli in the pulmonary trunk and its major branches . in 7 patients , dissection of lobar and segmental pulmonary arteries demonstrated large numbers of smaller thromboemboli . pte was the main cause of death in 83.3% ( 40/48 ) of the cases , and was a contributing factor in 16.7% ( 8/48 ) . of the 48 autopsy - confirmed pte cases , 19 ( 39.6% ) had shortness of breath , 13 ( 27.08% ) had syncope , and 6 ( 12.5% ) were in shock ( table 1 ) . in all , 76% ( 32/48 ) had 2 or more risk factors , 52% ( 13/48 ) had 3 or more risk factors , 18 ( 37.50% ) suffered multiple traumas , and 21 ( 44% ) underwent surgery ( table 2 ) . deaths occurred after the second week of certain risk factors for pte in the 24/32 ( 75% ) . of the 48 cases , 5 were excluded due to cardiopulmonary diseases for determining heart findings . in addition , the thickness of the lv and rv walls were not measured in some patients . data about diameter of ventricles are not routinely recorded in autopsy studies . in the 43 cases ( mean age 40.1614.8 years ) , cardiac hypertrophy , defined as a heart weighing 300 g for females and 400 g for males , the mean heart weight was 38783.5 g. the mean thickness of the left ventricle ( lv ) wall was 1.40 0.41 cm in 40 cases , and the mean thickness of the rv wall was 0.410.13 cm in 41 cases . we defined an lv wall 1.2 cm thick and an rv wall 0.8 cm thick as pathogenic . the lv walls of 35 ( 87.5% ) cases and the rv walls of 2 cases met these criteria ( table 3 ) . twenty - four cases ( 56% ) had remarkable histopathological findings ( necrosis , hypertrophy , or fibrosis ) while the other 19 ( 44% ) cases had mild congestion or normal tissue findings . necrosis was identified as myocardial necrosis without evident thrombus at the coronaries or atheroma plaques . cardiac hypertrophy ( enlargement of myocardial cells ) was described in 9 cases ( 21% ) . of these , 2 had hypertrophy of the lv ; however , the side of hypertrophy was not indicated for the other cases . acute myocardial infarction ( ami ) with concomitant pulmonary embolism was diagnosed in 1 case . a sudden increase in pulmonary artery pressure reflects an abrupt increase in rv afterload , with consequent elevation of rv wall tension , followed by rv dilation and dysfunction . in addition , elevated wall tension also increases rv myocardial oxygen demand , resulting in ischemia , which may promote further rv dysfunction and cardiogenic shock . perpetuation of this cycle can lead to rv infarction , circulatory collapse , and death [ 1315 ] . rv tissue obtained at autopsy from humans with pte also showed the presence of inflammation in 2 studies . in the present study , the aim of autopsy was to determine the cause of death , so a detailed examination of the heart was not performed histopathologically . however , the thicknesses of the lv and rv walls and heart weight were measured in most cases , and we found pathogenic thickness of lv wall in the majority of cases . orde et al . reported inflammatory changes within the rv myocardium but not the lv myocardium ; in addition , pte cases had thicker rv walls than controls . iwadate et al . reported a mild increase in the number of macrophages in the lv wall in some cases . in a report of 2 cases by iwadate et al . , the lv wall of 1 case was 17 mm thick in patients with massive pte . in the present study , most cases had lv walls of pathogenic thickness ; thus , the lv might also play a role in the pathogenesis of pte . when the rv dilates , the interventricular septum shifts toward the lv , which may lead to underfilling of this chamber . in addition , rv contractile dysfunction and acute tricuspid regurgitation may decrease rv output and further reduce lv preload . with underfilling of the lv , systemic cardiac output and pressure decrease , potentially compromising coronary perfusion . however , the rv is less susceptible to ischemic injury than the lv because of its relatively thin wall and lower systolic pressure . therefore , decreases in coronary pressure due to diminished cardiac output may also induce ischemia in the lv . when ischemia leads to damage to cardiomyocytes or when there is an increased load on the heart leading to inadequate contraction , the heart undergoes hypertrophy , with a compensatory response . cardiomyocytes become thicker and longer , and the thickness of the ventricular wall increases [ 1820 ] . hypertrophy of the left ventricle was supported in at least 2 of 9 cases histopathologically ; the small number was due to failure to perform detailed histopathological examination of the heart , and this is a limitation of the present study . abnormal lv geometry has been identified in patients who are older ( > 70 years ) and more obese ( > 30 kg / m ) [ 2123 ] . in the present study , the mean age and bmi were 40.16 years ( sd 14.863 years ) and 27.16 ( sd 4.678 ) , respectively . deaths occurred after the second week of certain risk factors for pte in the 24/32 ( 75% ) . clinically , patients might delay going to the hospital for complaints due to pte in our community . thus , physicians should provide appropriate information to patients who have certain risk factors ( e.g. , trauma , surgery , and pregnancy ) to predict acute pte at a preventable stage . pte may cause ventricular myocardial inflammation and necrosis , distinct from that seen in typical myocardial infarction due to atherosclerotic diseases . typical pathological findings of myocardial infarction are transmural or subendocardial coagulation necrosis . in the present study , there was only 1 case that had been diagnosed as having ami with concomitant pulmonary embolism , a 64-year - old woman who presented to the emergency department with acute dyspnea and chest pain . therefore , we determined that pte was a contributing factor of the main cause of death in this case . acute myocardial infarction and pulmonary embolism lead to life - threatening conditions such as sudden cardiac death and congestive heart failure . some studies have shown an association between pulmonary embolism and arterial cardiovascular events [ 2527 ] . we hypothesized that patients with venous thrombosis may be at increased risk for myocardial infarction because a large right ventricle and hypertrophy of the left ventricle were identified in this case by autopsy , potentially compromising coronary perfusion . our study illustrates the importance of considering fatal pulmonary thromboembolism , especially in the differential diagnosis of patients presenting with syncope . physicians should provide appropriate information to patients who have certain risk factors ( e.g. , trauma , surgery , and pregnancy ) to predict acute pte at a preventable stage .

backgroundto identify the overall effects of pulmonary thromboembolism ( pte ) on the heart , we evaluated the heart findings and clinical characteristics of deceased patients diagnosed with pte.material/methodsptes were classified into 2 categories : fatal and contributory . cases with a history of cardiopulmonary disease or a finding of significant disease at autopsy , including valvular heart disease and coronary artery obstruction > 50% , were excluded from the cardiac evaluation . we defined an lv wall 1.2 cm thick and an rv wall 0.8 cm thick as abnormal.resultsforty-eight cases were included to the study ( 21 males and 27 females ) . the mean age was 41.4216.5 years . of the 48 cases , 5 were excluded due to cardiopulmonary diseases for determining heart findings . the thicknesses of the lv and rv walls were not measured in some patients . in the 43 cases , cardiac hypertrophy was detected in 28 patients ( 65.1% ) . the mean heart weight was 38783.5 g. the mean thickness of the left ventricle ( lv ) wall was 1.400.41 cm in 40 cases , and the mean thickness of the rv wall was 0.410.135 cm in 41 cases . the lv walls of 35 ( 87.5% ) cases and the rv walls of 2 cases met criteria for abnormal wall thickness . there were histopathological findings of heart in 24/43 cases ( 56% ) ; these findings were necrosis , fibrosis , and hypertrophy.conclusionsthe rv is affected by massive pulmonary embolism ; however , the lv may also play a role in the pathogenesis of pte .

evidence suggests that experiences or anticipation of disrespect and abuse in maternity care , including hiv - related stigma and discrimination , discourage women from seeking health services and can cause them to drop out of care.13 in the case of hiv - related stigma , anticipation of discrimination , internalized stigma , and having stigmatizing attitudes toward people living with hiv are associated with refusing hiv testing during antenatal care , not returning for posttest hiv counseling , not delivering in a facility , reduced adherence to antiretroviral treatment for prevention of vertical ( mother - to - child ) transmission of hiv , and not enrolling in hiv treatment services.3 in tanzania , hiv - related stigma has been associated with nondisclosure of hiv status , women not accessing hiv care for their own health , and poor postnatal adherence to antiretrovirals.4,5 a recent study found that rural women in tanzania , 60% of whom had their last birth at home , identified respectful attitudes from providers as the most important single factor that would reduce their preference for home delivery relative to facility - based delivery.6 in another study , approximately 40% of rural tanzanian women who had their last birth at a facility reported traveling to more distant health care sites because of perceived measures of facility quality such as having the best providers , drug availability , recommendation by a relative / friend , a good previous experience , and high trust in health workers.7 despite increasing research about and recognition of how disrespect and abuse during maternity care and hiv - related stigma and discrimination may negatively affect women 's engagement with hiv and maternal health care services , few studies have explored relationships between hiv status and disrespect and abuse by comparing the experiences of women living with hiv ( wlwh ) and hiv - negative women during childbirth . our analysis compares the reported and observed experiences of disrespect and abuse during labor and delivery of wlwh with hiv - negative women . additionally , we explore relationships with health care providers ' training , work experience , and conceptualizations of disrespect and abuse and equitable labor and delivery care as they pertain to wlwh . the data analyzed for this article are from a baseline study of disrespect and abuse conducted between april and october 2013 at a large urban regional referral hospital in dar es salaam , tanzania . although this hospital addresses clinical quality and interpersonal communication through its continuous quality improvement program , reducing disrespect and abuse were not specific objectives of quality improvement initiatives at the time of data collection for this study.8 the hospital serves urban and periurban women from the district with the lowest average socioeconomic status in dar es salaam . although the hospital is designated within the health system to serve women with complications and those referred from primary healthcare facilities , in practice many women with low - risk pregnancies bypass the lower level facilities to deliver at this hospital . the labor and delivery ward is often overcrowded and understaffed ; it is staffed by 23 health care providers per shift , averages 60 deliveries per day , and reports , on average , 4 maternal deaths , 125 pregnancy - related complications , and 18 neonatal deaths per month ( facility - based indicators 2013 ) . the hiv prevalence among women attending antenatal and delivery care in dar es salaam is 7%.9 the conditions of overcrowding and understaffing of the maternity ward , and the relatively high hiv prevalence among women of reproductive age , are common in urban settings across sub - saharan africa10 meaning that this research may provide useful insights about the dynamics of disrespect and abuse related to hiv status in other similar settings.11 the study used a mixed - method design to assess disrespect and abuse during maternity care with the objective of designing interventions to reduce incidence . adopting a pragmatic approach , we selected the research methods that seemed best suited to assessing prevalence and potential drivers of disrespect and abuse and to identifying the attitudes , training needs , and working conditions of health care providers that would be amenable to interventions.12 women 's experiences of disrespect and abuse were captured through interviews with postpartum women 36 hours postdelivery ( n = 2000 ) , client provider interactions during childbirth were assessed by direct observation ( n = 208 ) , and health care provider attitudes , training , working conditions , and self - report of practice were assessed through structured questionnaires ( n = 50 ) and in - depth interviews ( n = 18 ) . this mix of methods and our analysis aims to describe disrespect and abuse from different standpoints and to reconcile dualistic objectivist and subjectivist epistemologies through the pragmatic position that knowledge is at the very same time constructed and real.13 we implemented a convergent parallel mixed - method design that collected both quantitative and qualitative data before beginning the analysis , although the in - depth interviews were completed after the surveys and direct observation.12 the study protocol was approved by the institutional review board of the tanzanian national institute of medical research . , women were asked about their experiences with the care provided at the hospital , including overall assessments of satisfaction and quality and more specifically about experienced disrespect and abuse , including physical abuse , nonconsented care , nonconfidential care , lack of privacy , nondignified care , abandonment during or after labor and delivery , and detention in facilities . additionally , basic demographic and household information was collected . nurse midwives observed client provider interactions during the labor and delivery process to identify instances of disrespect and abuse . structured questionnaires asked health care providers about their training related to the clinical management of people living with hiv and prevention of vertical hiv transmission , the frequency with which they provide labor and delivery services to wlwh , and their comfort level with providing such services . in - depth interviews explored health care providers ' perceptions and definitions of disrespect and abuse and any patient , provider , or facility - related factors that lead to such treatment occurring , including women 's hiv status . in line with the convergent parallel mixed - method design , the qualitative and quantitative analyses were conducted independently and the results were mixed during the interpretation of the data.12 the analysis triangulates and examines convergence and divergence in results from the different methods to provide insight into how hiv status affects reported and observed disrespect and abuse during childbirth and to identify how provider perspectives and institutional culture regarding hiv status were related to experiences and practices of disrespect and abuse . categorical variables are presented in the tables as number ( % ) and continuous variables are presented as mean and standard deviation ( sd ) . eight primary outcome variables ( any type of disrespect and abuse , physical abuse , nonconsented care , nondignified care , nonconfidential care , lack of privacy , abandonment and detention in facilities ) were dichotomized and modeled using log binomial regression analysis . a separate binomial regression model was run for each of the 8 primary study outcomes to assess any correlation with the independent variables . the multivariate analysis controlled for age , marital status , employment status , parity , and number of antenatal care visits for the index pregnancy . the analysis was performed using sas ( version 9.3 ; sas institute inc , cary , nc ) . in - depth qualitative analysis was conducted simultaneously with data collection to allow for modification of the interview guide to explore emerging issues , and analytic codes were defined through an inductive process based on the narratives of research participants rather than assigned a priori . to promote critical reflexivity and rigor , a team in tanzania and a team in the united states independently developed codebooks , which were then compared to reach consensus on definitions . the data analyzed for this article are from a baseline study of disrespect and abuse conducted between april and october 2013 at a large urban regional referral hospital in dar es salaam , tanzania . although this hospital addresses clinical quality and interpersonal communication through its continuous quality improvement program , reducing disrespect and abuse were not specific objectives of quality improvement initiatives at the time of data collection for this study.8 the hospital serves urban and periurban women from the district with the lowest average socioeconomic status in dar es salaam . although the hospital is designated within the health system to serve women with complications and those referred from primary healthcare facilities , in practice many women with low - risk pregnancies bypass the lower level facilities to deliver at this hospital . the labor and delivery ward is often overcrowded and understaffed ; it is staffed by 23 health care providers per shift , averages 60 deliveries per day , and reports , on average , 4 maternal deaths , 125 pregnancy - related complications , and 18 neonatal deaths per month ( facility - based indicators 2013 ) . the hiv prevalence among women attending antenatal and delivery care in dar es salaam is 7%.9 the conditions of overcrowding and understaffing of the maternity ward , and the relatively high hiv prevalence among women of reproductive age , are common in urban settings across sub - saharan africa10 meaning that this research may provide useful insights about the dynamics of disrespect and abuse related to hiv status in other similar settings.11 the study used a mixed - method design to assess disrespect and abuse during maternity care with the objective of designing interventions to reduce incidence . adopting a pragmatic approach , we selected the research methods that seemed best suited to assessing prevalence and potential drivers of disrespect and abuse and to identifying the attitudes , training needs , and working conditions of health care providers that would be amenable to interventions.12 women 's experiences of disrespect and abuse were captured through interviews with postpartum women 36 hours postdelivery ( n = 2000 ) , client provider interactions during childbirth were assessed by direct observation ( n = 208 ) , and health care provider attitudes , training , working conditions , and self - report of practice were assessed through structured questionnaires ( n = 50 ) and in - depth interviews ( n = 18 ) . this mix of methods and our analysis aims to describe disrespect and abuse from different standpoints and to reconcile dualistic objectivist and subjectivist epistemologies through the pragmatic position that knowledge is at the very same time constructed and real.13 we implemented a convergent parallel mixed - method design that collected both quantitative and qualitative data before beginning the analysis , although the in - depth interviews were completed after the surveys and direct observation.12 the study protocol was approved by the institutional review board of the tanzanian national institute of medical research . during the postpartum interviews , women were asked about their experiences with the care provided at the hospital , including overall assessments of satisfaction and quality and more specifically about experienced disrespect and abuse , including physical abuse , nonconsented care , nonconfidential care , lack of privacy , nondignified care , abandonment during or after labor and delivery , and detention in facilities . nurse midwives observed client provider interactions during the labor and delivery process to identify instances of disrespect and abuse . structured questionnaires asked health care providers about their training related to the clinical management of people living with hiv and prevention of vertical hiv transmission , the frequency with which they provide labor and delivery services to wlwh , and their comfort level with providing such services . in - depth interviews explored health care providers ' perceptions and definitions of disrespect and abuse and any patient , provider , or facility - related factors that lead to such treatment occurring , including women 's hiv status in line with the convergent parallel mixed - method design , the qualitative and quantitative analyses were conducted independently and the results were mixed during the interpretation of the data.12 the analysis triangulates and examines convergence and divergence in results from the different methods to provide insight into how hiv status affects reported and observed disrespect and abuse during childbirth and to identify how provider perspectives and institutional culture regarding hiv status were related to experiences and practices of disrespect and abuse . categorical variables are presented in the tables as number ( % ) and continuous variables are presented as mean and standard deviation ( sd ) . eight primary outcome variables ( any type of disrespect and abuse , physical abuse , nonconsented care , nondignified care , nonconfidential care , lack of privacy , abandonment and detention in facilities ) were dichotomized and modeled using log binomial regression analysis . a separate binomial regression model was run for each of the 8 primary study outcomes to assess any correlation with the independent variables . the multivariate analysis controlled for age , marital status , employment status , parity , and number of antenatal care visits for the index pregnancy . the analysis was performed using sas ( version 9.3 ; sas institute inc , cary , nc ) . in - depth interviews were translated into english . qualitative analysis was conducted simultaneously with data collection to allow for modification of the interview guide to explore emerging issues , and analytic codes were defined through an inductive process based on the narratives of research participants rather than assigned a priori . to promote critical reflexivity and rigor , a team in tanzania and a team in the united states independently developed codebooks , which were then compared to reach consensus on definitions . of the women who participated in the postpartum interview , 147 ( 7.4% ) were known to be living with hiv , 1807 ( 90.4% ) were hiv negative , and 46 ( 2.3% ) were of unknown hiv status . wlwh were slightly older [ 29.1 years ( sd = 8.1 ) vs. 25.3 years ( sd = 6.9 ) ; p < 0.001 ] , higher parity ( p = 0.005 ) , less likely to be married or cohabitating ( 76.9% vs. 83.1% ; p < 0.001 ) , and more likely to be employed ( 53.7% vs. 38.8% ; p < 0.001 ) . sociodemographic and delivery characteristics of study participants ( n = 1954 ) * overall , wlwh were no more or less likely to report any type of disrespect and abuse during labor and delivery ( 12.2% ) than hiv - negative women ( 15.0% ) ( p = 0.37 ) ( table 2 ) . wlwh reported lower prevalence of each category of disrespect and abuse than hiv - negative women with the exception of detention and nonconsented care ( table 2 ) . after adjusting for age , marital status , employment status , parity , and number of antenatal care visits for the index pregnancy , wlwh were more likely to report nonconsented care than hiv - negative women ( adjusted odds ratio 9.16 , 95% ci 1.73 - 115.00 , p = 0.03 ) . no other statistically significant correlations between living with hiv and reporting disrespect and abuse during maternity care were identified in the multivariate model ( table 3 ) . importantly , none of the 147 wlwh who completed the postpartum survey reported that their hiv confidentiality had been violated or attributed the disrespect and abuse they experienced during labor and delivery to their hiv status . comparison of disrespect and abuse reported by wlwh and hiv - negative women during childbirth bivariate and multivariate analysis of the association between living with hiv and reporting disrespect and abuse during childbirth direct observation of client provider interactions during labor and delivery told a similar story to the interviews completed with postpartum women . nurse midwives observed 18 wlwh and 183 hiv - negative women during childbirth . in table 4 , we present all the statistically significant observed differences and the observed measures of physical abuse , nonconsented care , nonconfidential care , lack of privacy , and selected measures of nondignified care which we hypothesized could be overt expressions of hiv - related stigma such as using nondignified language during history taking , not congratulating the woman after birth , or referring women with hiv to unclean beds in the postnatal ward . first , none of the wlwh were asked for consent before the vaginal examination in the antenatal ward , whereas 80% of hiv - negative women were not asked for consent ( p = 0.04 ) . second , it was less common for health care providers not to call wlwh by their names during labor and delivery than other women ( 35.3% vs. 60.7% p = 0.04 ) . finally , although not significant at the 95% confidence interval , there is a trend toward health care workers being less likely to congratulate wlwh after giving birth than hiv - negative women ( 47.1% vs. 25.8% ; p = 0.06 ) . comparison of directly observed disrespect and abuse toward wlwh and hiv - negative women during childbirth * during in - depth interviews , health care providers working in the maternity ward demonstrated awareness of the right of wlwh to maintain the confidentiality of their hiv status during labor and delivery , spontaneously mentioning violations of hiv confidentiality to exemplify what they considered to be disrespectful or abusive care . disrespect is making abusive statements to patients , or for instance , for the hiv - infected patients , mentioning her hiv positive status while she is with the other women , some of such patients feel bad that is unfair some respondents demonstrated sensitivity to the perceived stigma that women with hiv may feel , for instance saying an hiv infected mother is not supposed to be abused ; she will feel twenty times worse than the other mothers ( registered nurse , int12 ) . providers said that hiv has been normalized to such an extent in the institution that wlwh frequently come and tell you directly , they do n't hide : nurse please help me i have this problem , please help me. and then after she tells you , you will take care of her with all the joy in you because she expressed her problem to you and you know how to help her prevent transmitting the infection to the baby and protect yourself ( medical attendant , int18 ) . during in - depth interviews , health care providers uniformly denied that hiv status affected labor and delivery care . for example , a registered nurse said , an hiv infected mother is cared for as usual , there are no stigmatization issues here ; they get good care like any other patient others noted that the only difference is the medication we give the hiv infected [ women ] , apart from that it is the same ( licensed practical nurse , int14 ) . nurses described taking extra care with women with hiv to prevent occupational exposure and hiv transmission to the infant , but insisted that the carefulness we are describing here is [ done ] in such a way that even the mother herself feels like it is being done for her benefit . because nowadays anything you do to a patient you must explain to them first , so they know that the nurse is doing this to save my baby from getting the infection . it is done in a manner that makes the mother satisfied ( registered nurse int15 ) . during interviews , health care providers stated that nondiscriminatory treatment had not always been the institutional status quo . they noted that in the past , health care providers had less information about preventing hiv transmission during labor and delivery and violated confidentiality , for example indicating the patient to be hiv positive openly while others are listening ( registered nurse , int11 ) such things nowadays they have vanished , nowadays they [ wlwh ] receive the usual care like others , they [ providers ] do n't discriminate against them providers attributed that the change in attitude to training and supervision focused on prevention of vertical hiv transmission and normalization of hiv through provision of labor and delivery services to many wlwh . in general , confidential questionnaires completed by 50 health care providers reinforce the finding that training on hiv , including prevention of vertical hiv transmission , and providing services to women with hiv have normalized hiv in this setting . more than 38% of respondents reported receiving training in prevention of vertical hiv transmission during the past year and 36% received training in clinical management of hiv , with 46% of the providers interviewed receiving training in at least one of these issues . additionally , 88% of providers reported having managed at least 1 wlwh during labor and delivery and 78% reported providing antiretroviral therapy to the mother and newborn to prevent vertical hiv transmission during the past 3 months . of those who provided labor and delivery services to wlwh , 77% said they were comfortable doing so and 79% of those who provided antiretroviral therapy to prevent vertical hiv transmission said they were comfortable with this task . in a section of the confidential survey with a low response rate , a minority of responding health care providers ( n = 5 ) said they avoided or had mixed feelings about doing vaginal examinations or repairing episiotomies for wlwh . these same 5 providers felt that wlwh should be isolated during childbirth or had mixed feelings about this practice . that such discriminatory practices were not admitted to or attributed to other health care providers during in - depth interviews further suggests that overt discrimination based on hiv status is perceived as unacceptable in the workplace culture of this facility . stockouts of standard commodities , such as cotton wool and gloves , and overcrowding were consistently described by providers as their greatest workplace challenges . however , during in - depth interviews , providers did not identify these institutional deficiencies which make implementing universal infection prevention precautions difficult as a motive for providing different care to wlwh . in practice , one provider stated that when you have 4 mothers sharing that one bed , you ca n't say to the hiv infected mother sleep on the floor because you have hivthat is not right , they all share the beds ( licensed practical nurse , int14 ) . in the situation of a wlwh sharing a bed with 3 others , the provider describes a general warning given to the occupants that , there might be someone with hiv and you do n't know so do n't contaminate yourself with another persons ' waters , also the blood should not touch you as a deliberate strategy to promote infection control while preserving confidentiality ( int14 ) . the comments made by providers about what constitutes disrespect and abuse , practices of caring for wlwh described during in - depth interviews , and this imperfect solution to overcrowding , all demonstrate provider awareness that differential treatment of wlwh is discrimination and that confidentiality of hiv status must be respected . providers described making efforts to provide equitable care to wlwh in an institutional setting where guaranteeing universal precautions and confidentiality for any woman is a significant challenge . of the women who participated in the postpartum interview , 147 ( 7.4% ) were known to be living with hiv , 1807 ( 90.4% ) were hiv negative , and 46 ( 2.3% ) were of unknown hiv status . wlwh were slightly older [ 29.1 years ( sd = 8.1 ) vs. 25.3 years ( sd = 6.9 ) ; p < 0.001 ] , higher parity ( p = 0.005 ) , less likely to be married or cohabitating ( 76.9% vs. 83.1% ; p < 0.001 ) , and more likely to be employed ( 53.7% vs. 38.8% ; p < 0.001 ) . sociodemographic and delivery characteristics of study participants ( n = 1954 ) * overall , wlwh were no more or less likely to report any type of disrespect and abuse during labor and delivery ( 12.2% ) than hiv - negative women ( 15.0% ) ( p = 0.37 ) ( table 2 ) . wlwh reported lower prevalence of each category of disrespect and abuse than hiv - negative women with the exception of detention and nonconsented care ( table 2 ) . after adjusting for age , marital status , employment status , parity , and number of antenatal care visits for the index pregnancy , wlwh were more likely to report nonconsented care than hiv - negative women ( adjusted odds ratio 9.16 , 95% ci 1.73 - 115.00 , p = 0.03 ) . no other statistically significant correlations between living with hiv and reporting disrespect and abuse during maternity care were identified in the multivariate model ( table 3 ) . importantly , none of the 147 wlwh who completed the postpartum survey reported that their hiv confidentiality had been violated or attributed the disrespect and abuse they experienced during labor and delivery to their hiv status . comparison of disrespect and abuse reported by wlwh and hiv - negative women during childbirth bivariate and multivariate analysis of the association between living with hiv and reporting disrespect and abuse during childbirth direct observation of client provider interactions during labor and delivery told a similar story to the interviews completed with postpartum women . nurse midwives observed 18 wlwh and 183 hiv - negative women during childbirth . in table 4 , we present all the statistically significant observed differences and the observed measures of physical abuse , nonconsented care , nonconfidential care , lack of privacy , and selected measures of nondignified care which we hypothesized could be overt expressions of hiv - related stigma such as using nondignified language during history taking , not congratulating the woman after birth , or referring women with hiv to unclean beds in the postnatal ward . first , none of the wlwh were asked for consent before the vaginal examination in the antenatal ward , whereas 80% of hiv - negative women were not asked for consent ( p = 0.04 ) . second , it was less common for health care providers not to call wlwh by their names during labor and delivery than other women ( 35.3% vs. 60.7% p = 0.04 ) . finally , although not significant at the 95% confidence interval , there is a trend toward health care workers being less likely to congratulate wlwh after giving birth than hiv - negative women ( 47.1% vs. 25.8% ; p = 0.06 ) . comparison of directly observed disrespect and abuse toward wlwh and hiv - negative women during childbirth * during in - depth interviews , health care providers working in the maternity ward demonstrated awareness of the right of wlwh to maintain the confidentiality of their hiv status during labor and delivery , spontaneously mentioning violations of hiv confidentiality to exemplify what they considered to be disrespectful or abusive care . disrespect is making abusive statements to patients , or for instance , for the hiv - infected patients , mentioning her hiv positive status while she is with the other women , some of such patients feel bad that is unfair some respondents demonstrated sensitivity to the perceived stigma that women with hiv may feel , for instance saying an hiv infected mother is not supposed to be abused ; she will feel twenty times worse than the other mothers ( registered nurse , int12 ) . providers said that hiv has been normalized to such an extent in the institution that wlwh frequently come and tell you directly , they do n't hide : nurse please help me i have this problem , please help me. and then after she tells you , you will take care of her with all the joy in you because she expressed her problem to you and you know how to help her prevent transmitting the infection to the baby and protect yourself ( medical attendant , int18 ) . during in - depth interviews , health care providers uniformly denied that hiv status affected labor and delivery care . for example , a registered nurse said , an hiv infected mother is cared for as usual , there are no stigmatization issues here ; they get good care like any other patient ( registered nurse , int6 ) . others noted that the only difference is the medication we give the hiv infected [ women ] , apart from that it is the same ( licensed practical nurse , int14 ) . nurses described taking extra care with women with hiv to prevent occupational exposure and hiv transmission to the infant , but insisted that the carefulness we are describing here is [ done ] in such a way that even the mother herself feels like it is being done for her benefit . because nowadays anything you do to a patient you must explain to them first , so they know that the nurse is doing this to save my baby from getting the infection . it is done in a manner that makes the mother satisfied ( registered nurse int15 ) . during interviews , health care providers stated that nondiscriminatory treatment had not always been the institutional status quo . they noted that in the past , health care providers had less information about preventing hiv transmission during labor and delivery and violated confidentiality , for example indicating the patient to be hiv positive openly while others are listening ( registered nurse , int11 ) such things nowadays they have vanished , nowadays they [ wlwh ] receive the usual care like others , they [ providers ] do n't discriminate against them providers attributed that the change in attitude to training and supervision focused on prevention of vertical hiv transmission and normalization of hiv through provision of labor and delivery services to many wlwh . in general , confidential questionnaires completed by 50 health care providers reinforce the finding that training on hiv , including prevention of vertical hiv transmission , and providing services to women with hiv have normalized hiv in this setting . more than 38% of respondents reported receiving training in prevention of vertical hiv transmission during the past year and 36% received training in clinical management of hiv , with 46% of the providers interviewed receiving training in at least one of these issues . additionally , 88% of providers reported having managed at least 1 wlwh during labor and delivery and 78% reported providing antiretroviral therapy to the mother and newborn to prevent vertical hiv transmission during the past 3 months . of those who provided labor and delivery services to wlwh , 77% said they were comfortable doing so and 79% of those who provided antiretroviral therapy to prevent vertical hiv transmission said they were comfortable with this task . in a section of the confidential survey with a low response rate , a minority of responding health care providers ( n = 5 ) said they avoided or had mixed feelings about doing vaginal examinations or repairing episiotomies for wlwh . these same 5 providers felt that wlwh should be isolated during childbirth or had mixed feelings about this practice . that such discriminatory practices were not admitted to or attributed to other health care providers during in - depth interviews further suggests that overt discrimination based on hiv status is perceived as unacceptable in the workplace culture of this facility . stockouts of standard commodities , such as cotton wool and gloves , and overcrowding were consistently described by providers as their greatest workplace challenges . however , during in - depth interviews , providers did not identify these institutional deficiencies which make implementing universal infection prevention precautions difficult as a motive for providing different care to wlwh . in practice , one provider stated that when you have 4 mothers sharing that one bed , you ca n't say to the hiv infected mother sleep on the floor because you have hivthat is not right , they all share the beds ( licensed practical nurse , int14 ) . in the situation of a wlwh sharing a bed with 3 others , the provider describes a general warning given to the occupants that , there might be someone with hiv and you do n't know so do n't contaminate yourself with another persons ' waters , also the blood should not touch you as a deliberate strategy to promote infection control while preserving confidentiality ( int14 ) . the comments made by providers about what constitutes disrespect and abuse , practices of caring for wlwh described during in - depth interviews , and this imperfect solution to overcrowding , all demonstrate provider awareness that differential treatment of wlwh is discrimination and that confidentiality of hiv status must be respected . providers described making efforts to provide equitable care to wlwh in an institutional setting where guaranteeing universal precautions and confidentiality for any woman is a significant challenge . overall , wlwh who received labor and delivery services at a large urban hospital in tanzania were no more or less likely to report any type of disrespect and abuse during labor and delivery than hiv - negative women ( 12.2% vs. 15.0% ) . however , wlwh were significantly more likely to report nonconsented care even after controlling for age , marital status , employment status , parity , and number of antenatal care visits for the index pregnancy . no other statistically significant differences in women reporting different types of disrespect and abuse were observed in the multivariate logistic regression model . during direct observation of labor and delivery , midwives recorded that wlwh were less likely to be asked for consent for vaginal examinations than hiv - negative women . it is important to note that this exploratory analysis was not specifically powered to detect differences in experiences of disrespect and abuse between wlwh and other women , and thus these results should be interpreted with caution . more research with larger samples is needed for further analysis of the relationship between hiv status and nonconsented care , and to continue to expand knowledge about associations between hiv status and experiences of disrespect and abuse during childbirth . none of the 147 wlwh in the study reported a breach of the confidentiality of her hiv status or attributed the disrespect and abuse that she experienced to living with hiv . the absence of overt discrimination related to hiv status is likely because of the awareness health care providers demonstrated regarding the importance of maintaining confidentiality of hiv status , their affirmations that hiv status did not affect quality of care , and assertions that discriminatory acts , for example , making a woman sleep on the floor in an overcrowded labor ward because of her hiv status , were unacceptable . although a small number of providers expressed lack of willingness or mixed feelings about providing obstetric services to wlwh in the confidential survey , in - depth interviews with providers signaled clearly that overt discrimination based on hiv status was no longer part of the institutional culture at the research site . their descriptions of a change in institutional culture , from a time when violations of hiv confidentiality and lack of willingness to provide delivery services were common to the current situation , offer important lessons about how to discourage other forms of disrespect and abuse during maternity care . first , providers were sensitized about hiv and the rights of wlwh to confidentiality and equitable treatment , causing providers to identify hiv - related discrimination as not right . second , providers received training about hiv and provided services to wlwh frequently almost half had been trained on hiv or prevention of vertical hiv transmission within the past year , 88% had provided labor and delivery services to a wlwh in the past 3 months , and 79% of these individuals felt comfortable doing so . third , and perhaps most importantly , providers received supervision that focused specifically on the quality of services to prevent vertical hiv transmission . finally , addressing institutional deficiencies , like stockouts of basic commodities , similar to our results , a comparative study of perceived acceptability of obstetric services in south africa found no significant differences in feeling respected by health workers between wlwh and other women.14 nevertheless , reports of equitable treatment in obstetric services in tanzania and south africa do not necessarily extend to other domains of reproductive health in which hiv - related stigma has been identified as a driver of discrimination , such as in the case of coercive and forced sterilization.1517 lack of overt hiv - related discrimination during labor and delivery can not be directly equated with acknowledgment of the rights of wlwh to choose the number and spacing of their children . stigmatization of childbearing among wlwh by providers may be indicated by the trend identified in our research toward providers being less likely to congratulate wlwh after delivery than hiv - negative women . our results suggest that research that allows for comparisons between wlwh and other women is valuable for disentangling disrespect and abuse driven by hiv stigma from other causes , and thereby permitting effective targeting of interventions . finally , it is important to highlight that many women reported and were observed to experience disrespect and abuse during childbirth . abusive practices by health care providers and inadequate infrastructure18 that denies women dignity during childbirth , such as commodity stockouts , lack of adequate privacy partitions , and overcrowding that results in bed - sharing , must be transformed to guarantee respectful maternity care for all women . that none of the wlwh reported violations of confidentiality or attributed the disrespect and abuse that they experienced during maternity care to their hiv status is a cause for optimism , as is the finding that providers were aware of the rights to confidentiality and nondiscrimination of wlwh . furthermore , health care providers ' descriptions of changes in institutional norms and practices that resulted in improved respect for the confidentiality of hiv status and nondiscrimination for wlwh indicate that disrespect and abuse are malleable phenomena . our analysis suggests that creating awareness among health care providers that practices they may take for granted violate women 's rights and implementing supervision and institutional quality improvement measures that take seriously the issue of disrespect and abuse during maternity care can contribute to improving women 's childbirth experiences . the normalization of hiv and improvements in respect for the confidentiality of hiv status during the provision of services to prevent vertical hiv transmission documented in this study are an important achievement in the institution where the study was conducted . the political and institutional priority and resources that have been dedicated to ensuring that hiv - related discrimination is not a barrier to prevention of vertical hiv transmission should be expanded to ensure high - quality respectful maternity care for all women . important future directions for research include conducting similar comparative studies with larger samples to explore associations between hiv - status and nonconsented care and to confirm the lack of association between hiv status and other forms of disrespect and abuse during childbirth ; analysis of the effect of disrespect and abuse on willingness to return to the facility , postnatal care seeking , and intention to refer relatives or friends ; and evaluation of interventions designed to reduce disrespect and abuse during childbirth .

introduction : hiv - related stigma and discrimination and disrespect and abuse during childbirth are barriers to use of essential maternal and hiv health services . greater understanding of the relationship between hiv status and disrespect and abuse during childbirth is required to design interventions to promote women 's rights and to increase uptake of and retention in health services ; however , few comparative studies of women living with hiv ( wlwh ) and hiv - negative women exist.methods:mixed methods included interviews with postpartum women ( n = 2000 ) , direct observation during childbirth ( n = 208 ) , structured questionnaires ( n = 50 ) , and in - depth interviews ( n = 18 ) with health care providers . bivariate and multivariate regressions analyzed associations between hiv status and disrespect and abuse , whereas questionnaires and in - depth interviews provided insight into how provider attitudes and workplace culture influence practice.results:of the wlwh and hiv - negative women , 12.2% and 15.0% reported experiencing disrespect and abuse during childbirth ( p = 0.37 ) , respectively . in adjusted analyses , no significant differences between wlwh and hiv - negative women 's experiences of different types of disrespect and abuse were identified , with the exception of wlwh having greater odds of reporting non - consented care ( p = 0.03 ) . none of the wlwh reported violations of hiv confidentiality or attributed disrespect and abuse to their hiv status . provider interviews indicated that training and supervision focused on prevention of vertical hiv transmission had contributed to changing the institutional culture and reducing hiv - related violations.conclusions:in general , wlwh were not more likely to report disrespect and abuse during childbirth than hiv - negative women . however , the high overall prevalence of disrespect and abuse measured indicates a serious problem . similar institutional priority as has been given to training and supervision to reduce hiv - related discrimination during childbirth should be focused on ensuring respectful maternity care for all women .

human immunodeficiency virus ( hiv ) infection is a problem affecting many patients reporting to health care professionals of many specialties , including cardiologists . taking care of these patients can be challenging due to number of hiv - related comorbidities , as well as possible drug - to - drug interactions which can be detected between antiretroviral medications and drugs commonly used in other conditions . a 52-year - old male with hiv infection , asthma and varicose veins presented to the hospital with first - ever chest pain . initial 12-lead electrocardiography ( ecg ) revealed st - segment elevation in v 2 - 5 leads , and st - segment depression in ii , iii and avf . the patient was immediately transferred to a catheterization laboratory , where coronary angiography was performed , which showed a dominant right coronary artery with no significant stenosis , acute occlusion of the left anterior descending artery and chronic occlusion of the intermediate branch . primary percutaneous coronary intervention ( pci ) with implantation of an integrity bare metallic stent ( medtronic cardiovascular , santa rosa , ca , usa ) 3.01.2 mm into the left anterior descending artery was performed , resulting in complete reperfusion { thrombolysis in myocardial infarction ( timi ) 3 flow}. intravascular ultrasound was not performed during the procedure . initial echocardiography revealed a 1.651.55 cm blood clot , fixed in an area near the apical part of the left ventricle ( lv ) . mmol / l , low density lipoproteincholesterol ( ldl - c ) 2.7 mmol / l and triglycerides 1.37 mmol / l and estimated glomerular filtration rate was > 60 ml / min/1.73 . the patient was prescribed aspirin 75 mg , clopidogrel 75 mg , enoxaparin 60 mg , ramipril 2.5 mg , nebivolol 2.5 mg and atorvastatin 40 mg . because of the retroviral infection , he had been receiving combined antiretroviral therapy ( cart ) for 6 years . the cart regiment consistied of 2 nucleoside reverse transcriptase inhibitors ( emtricitabine and tenofovir ) , and protease inhibitor lopinavir boosted with ritonavir ( lopinavir / r ) . his plasma hiv ribonucleic acid ( rna ) levels , measured by real - time polymerase chain reaction were undetectable . the counts of t lymphocytes cd4 and cd8 were 625 cells/l ( norm , 410 - 1590 cells/l ) and 818 cells/l ( norm , 190 - 1140 cells/l ) , respectively . after 5 days of hospitalization , he was discharged and instructed to continue the prescribed medications . two weeks after discharge , the patient was readmitted to the hospital because of general fatigue and malaise . full examination did not reveal any abnormalities other than the previously described blood clot in lv . the patient was discharged and referred for ambulatory surveillance by a cardiologist when his coagulation parameters including the activated partial thromboplastin time ( aptt ) were in the normal range . after 10 days ( 37 days from the first hospitalization ) , the patient again presented to the hospital with acute chest pain . his troponin i level was 4.74 ng / ml , and in the ecg , a qs complex was seen in leads v 1 - 3 , with new st - segment elevation . with an initial diagnosis of type 4b myocardial infarction ( mi ) ( table 1 ) , the patient again underwent coronary angiography , which showed acute occlusion of the left anterior descending artery because of in - stent thrombosis . after a bolus of eptifibatide , the thrombus was aspirated with good hemodynamic results ( timi 3 flow ) . follow up echocardiography showed a change in the diameter of the clot in the lv ( from 1.651.55 cm to 2.21.2 cm ) , and akinesis of the apex , a part of the intraventricular septum , and the anterior wall , with an ejection fraction of 33% . due to the thrombosis , platelet function tests were performed . the aspirin reaction unit was 394 , and the p2y12 reaction unit was 155 . at this time , his total cholesterol was 2.8 mmol / l , ldl - c 1.4 mmol / l and triglycerides 1.7 mmol / l . the patient was prescribed aspirin 75 mg , prasugrel 10 mg , carvedilol 25 mg , torasemide 5 mg , ramipril 2.5 mg , spironolactone 12.5 mg , atorvastatin 40 mg and warfarin ( to keep the inr at 2.5 ) . the patient still received the combination of emtricitabine and tenofovir , but the protease inhibitor was changed from lopinavir / r to darunavir / r . after 6 months of follow - up , the patient is in good general condition , with undetectable hiv rna levels and no drop of t helper lymphocytes cd4 . in taking care of patients after an mi , we have to take into consideration the patient 's co - morbidities and modify the treatment accordingly . in our patient 's case , during the first mi , he received the proper treatment including double antiplatelet therapy that consisted of aspirin and a p2y12 inhibitor , clopidogrel , along with enoxaparin , a low molecular weight heparin . nevertheless , only after 1.5 months , the patient suffered another major adverse cardiac event due to in - stent thrombosis . referring to the newest universal definition of mi developed in 2012 by the european society of cardiology , we diagnosed the patient with mi type 4b , which covers mi related to stent thrombosis , which was detected in our case by coronary angiography.1 ) after the second event , the patient was prescribed prasugrel instead of clopidogrel . comparison of those two drugs in the triton - timi study showed that the newer drug ( prasugrel ) is more effective in patients with st - elevation myocardial infarction who have undergone pci , in the prevention of ischemic events , without being associated with an excessive risk of bleeding.2 ) the alteration in the antiplatelet therapy was important because of the increased risk of cardiovascular disease and hypercoagulation , which our patient was exposed to through his co - morbidities . for not yet fully disclosed reasons , patients with hiv infection suffer from a large number of non - aids conditions . both , hiv and antiretroviral treatment increase the risk of development of conditions such as kidney and liver dysfunction , cancer , osteoporosis , neurological diseases and other end - organ diseases . hiv - positive patients are also known to not only have a shorter life - expectancy , but also to face accelerated aging.3 ) one of the biggest concerns in this group of patients is diseases of the cardiovascular system . cart therapy is known to cause endothelial dysfunction and alterations in the lipid profile ( including elevated triglyceride levels , a high proportion of small and dense ldl particles , and low high density lipoproteincholesterol levels ) , leading to an increased risk of mi.4 ) specific antiretroviral drugs have different influences on cardiovascular disease risks . it was established that exposure to most of the protease inhibitors is associated with an increased risk of mi . this risk is especially strong when patients receive ritonavir - boosted lopinavir ( odds ratio 1.33 ; 95% confidence interval 1.09 - 1.61 per year).5 ) darunavir / r , which the patient received after the treatment modification , is at least as equally effective as lopinavir / r and is related to fewer adverse effects.6 ) up to this day , there is no convincing evidence for ritonavir increasing the cardiovascular disease risk . patients with untreated hiv infection also have increased cardiovascular risks because of hiv - related proatherogenic mechanisms , like ongoing immune activation , inflammation , increased coagulation , and changes in blood lipids.7)8 ) this along with other commonly underdiagnosed conditions can contribute to instent thrombosis.9 ) probably the total harm on the cardiovascular system done by the untreated hiv is greater than the one from cart . with all this in mind , for hiv - infected patients at high cardiovascular risk , we have to remember certain things . the cart scheme should be chosen from the least harmful drugs , and all patients should be treated individually , taking into account modifications in the cardiovascular risk factors and changes in the aggressive pharmacological treatment made as necessary . for example , simvastatin and lovastatin should not be used in patients who are taking protease inhibitors because of potential interactions via cytochrome p-450 enzyme cyp3a4 . on the other hand , atorvastatin and rosuvastatin are safe to use in this case.10 ) today a cardiologist rarely deals with a patient afflicted only with an isolated disease of the heart or vessels . due to prolonged life expectancy , our patients are more and more often burdened with many comorbidities . now we have to deal with not only diseases related to the fields of cardionephrology , cardiodiabetology , cardiosexology , cardiooncology , but also diseases in other fields like the combination of cardiology with infectious diseases . it is important to remember to look at the whole patient instead of only the heart . there is a need for close cooperation between a cardiologist and doctors of other fields , with an expansion of the heart team concept to everyday practice . cardiologists rarely deal with hiv - infected patients and often do not have much knowledge of cart interactions , so that every time a new drug is added to a patient 's regimen , it can be helpful to consult with an infectious diseases specialist .

we report a case of a 52-year - old human immunodeficiency virus ( hiv)-infected male patient receiving combined antiretroviral therapy ( cart ) , who presented with acute st - elevation myocardial infarction ( stemi ) . he was properly treated ( e.g. , prescribed anti - coagulation drugs : aspirin , clopidogrel , enoxaparin ) and discharged . after 1.5 months , another stemi related with in - stent thrombosis took place . the cart scheme was altered , resulting in no further cardiac events in the follow - up period , with undetectable levels of hiv ribonucleic acid . this case highlights the association between hiv infection and the specific drugs of cart , and the risk of cardiovascular disease development .

guillain barr syndrome ( gbs ) is an acute autoimmune inflammatory demyelinating polyneuropathy that mostly affected the peripheral nervous system . gbs is clinically characterized by progressive symmetrical weakness of limbs , with or without autonomic or sensory disturbances . gbs may be induced by various factors , such as immunization , infection , trauma , or surgery . the pathogenesis of gbs was that neural antigens were cross - reactivated with antibodies to infections including cytomegalovirus , hemophilus influenzae , mycoplasma pneumonia , and herpes simplex . in this study , we reported a case of gbs in a 14-year - old patient with scoliosis . written informed consent was obtained from the patient 's parents on behalf of the child for publication of this case report and any accompanying images . a copy of the written consent is available for review by the editor of this journal . we present a 14-year - old patient admitted for a correction of his progressive scoliosis deformity . his spinal plain radiographs showed that the cobb angle of thoracic scoliosis was 114 ( fig . one week later , pain and weakness in the lower limbs , unsteady gait , and the inability to stand up from squatting position unassisted , developed , and progressed . two years later , the patient presented to the spinal service complaining of asymmetry of his shoulders . his spinal plain radiographs showed that the cobb angle of thoracic scoliosis was 114 , suggesting the need for surgical correction . magnetic resonance imaging revealed that cavernous hemangioma in the t4 ( thoracic 4 ) level of the spinal cord ( fig . , cavernous hemangioma resection was performed in the department of neurosurgery of our hospital and histological examination confirmed the diagnosis of cavernous angioma . magnetic resonance imaging revealed that spinal - cord cavernous hemangioma in the thoracic 4 level of the spinal cord . three months after the cavernous hemangioma resection surgery , a posterior correction and fusion at t5 ( thoracic 5)l5 ( lumbar 5 ) levels was performed , using the legacy spinal system ( usa ) . the total operation time was about 4 h. total amount of blood loss was 1200 ml and the amount of autologous blood transfusion was 650 ml . during the operation , the signal of this patient was normal using intraoperative spinal cord monitoring . postoperative plain x - ray film demonstrated a cobb angles correction from 114 to 45 ( correction rate 60.5% ) ( fig . his follow - up was symptomatic , well balanced in the coronal planes , with solid fusion ( fig . standing anteroposterior and lateral radiographs of 4 d after operation . standing anteroposterior and lateral radiographs of 12 mo after operation . gbs is an acute postinfectious autoimmune polyneuropathy , characterized by rapidly progressive , areflexia , and symmetrical limb weakness . the diagnosis of gbs depends on the clinical features including rapid development of areflexia , muscle paralysis , and albuminocytologic dissociation of cerebrospinal fluid . limited reports are available on neuromuscular scoliosis . in this study , we reported this case of a 14-year - old gbs case with scoliosis . in our case , lower limb pain occurred first , followed by weakness in the lower limbs , unsteady gait , and the inability to stand up from squatting position unassisted . the patient showed significant recovery in strength in lower extremities after treatment with intravenous immunoglobulin and steroids . some patients may develop neuromuscular complications such as neuromuscular scoliosis after acute onset of gbs . in this case , the patient presented to the spinal service with asymmetry of shoulders 2 years after the onset of gbs . as far as we know , there is no specific guideline for operations on patients with scoliosis secondary to gbs . the present case described a successful surgical management of a rapidly progressive scoliosis with contemporary posterior surgical instrumentation and fusion . in conclusion , gbs is a relatively rare syndrome described in recent years . doctors must keep in mind that neuromuscular scoliosis could develop secondary to gbs . when evaluating patients with acute inflammatory polyneuropathy , clinical examination of the spine is essential to identify patients with neuromuscular scoliosis .

abstractguillain barr syndrome ( gbs ) is an acute autoimmune inflammatory demyelinating polyneuropathy that mostly affects the peripheral nervous system . little is reported about spinal deformity associated with gbs . this study aims to present a case of scoliosis occurring in the setting of gbs.case report and literature review.the patient was a 14-year - old male with scoliosis . his spinal plain radiographs showed that the cobb angle of thoracic scoliosis was 114. history review revealed that he developed profound lower extremity pain , weakness , and numbness after catching a cold 5 years ago . these symptoms progressed to unsteady gait and inability to stand up from squatting position . the diagnosis of gbs was confirmed based on these symptoms . he underwent a posterior correction at thoracic 5lumbar 5 ( t5l12 ) levels using the ( legacy , usa ) spinal system . the cobb angle was corrected from 114 to 45 ( correction rate 60.5% ) . his follow - up was symptomatic , well balanced in the coronal planes , with solid fusion 12 months after the operation.neuromuscular scoliosis could develop secondary to gbs . when evaluating patients with acute inflammatory polyneuropathy , clinical examination of the spine is essential to identify patients with rare neuromuscular scoliosis .

postoperative af ( poaf ) carries severe clinical and economic implications , increasing early and late morbidity and mortality , and having a great impact on resource utilization . in addition , the incidence of this complication has been reported to steadily increase , owing mostly to an increasing number of elderly patients with severe comorbidities . in light of the importance of poaf for patient outcomes , several studies have sought to identify patients at highest risk for the development of af , but their number is testament to the failure to effectively prevent af through prophylactic measures in unselected patients . routine pharmacological af prophylaxes could expose as many as 60% to 80% of patients to the side effects of antiarrhythmic drugs for which there is no indication , with unnecessary drugrelated costs . as a result , it is of utmost importance to identify patients whose risk of developing af is very low and who should not receive routine antiarrhythmic therapy . accordingly , efforts have been made to develop risk prediction models to identify patients most likely to develop poaf . however , these models often are statistically underpowered and , because of this , inconclusive . in addition , they are limited by the exclusion of highrisk patients with renal and cardiac failure , as well as those undergoing complex procedures , despite the fact that these patients are those most affected by poaf . these risk models also imply complex calculations without rapid estimation for targeting patients , hampering a prompt prophylactic approach , and confounding the clinical decision making and patient counseling . the present study aimed to derive an efficient and reliable bedside tool to predict poaf and its related complications . between july 1999 and december 2010 , all consecutive patients undergoing isolated coronary artery bypass graft surgery ( cabg ) and valve surgery ( with or without concomitant cabg ) at 3 european cardiac centers ( bristol heart institute , uk ; varese university hospital , and centro cardiologico monzino irccs , italy ) were screened for this observational study . patients with a preoperative history of supraventricular arrhythmias requiring treatment were excluded to avoid overestimation of the poaf incidence . the final sample consisted of a total of 17 622 patients . data used in this analysis were retrieved from institutional databases , which remained consistent over the study period . all data were prospectively collected , and information about demographics , comorbidities , medical and surgical history , operative details , and postoperative events during the hospital stay were registered . the study protocol was in compliance with the local institutional review boards and received full approval ; patient consent was waived . preoperative management , anesthetic , and surgical techniques were standardized for all patients and have been previously reported . preoperative antiarrhythmic therapies were not routinely adopted ; in general , blockers were administered on the day of surgery and restarted in the immediate postoperative period unless contraindicated for clinical reasons . at the end of surgery , patients were transferred to a dedicated intensive care unit and managed according to the unit protocol . amiodarone , either orally or intravenously administered , constituted the standard pharmacological treatment of poaf . in patients without successful rhythm cardioversion and with persistent af , warfarin was administered before discharge , with the aim of achieving an international normalized ratio between 2.0 and 3.0 , planning an electric cardioversion within 30 days . symptomatic patients or with ineffective antiarrhythmic therapy to control the ventricular rate underwent cardioversion at any time during the postoperative period . the primary end point of this study was to identify independent predictors of poaf , deriving an effective bedside tool to predict the arrhythmia and its related complications . briefly , poaf was documented on the basis of a rhythm strip or 12lead ecg as previously described . postoperative acute kidney injury ( aki ) was defined according to the rifle ( risk , injury , failure , loss of function , and endstage renal disease ) criteria ; estimated glomerular filtration rate ( egfr ) was calculated with use of the chronic kidney disease epidemiology collaboration equation . cerebrovascular accidents were diagnosed on the basis of a new neurological deficit with morphological substrate confirmed by using computed tomography or nuclear magnetic resonance imaging . the decision to perform offpump or onpump cabg was based on individual surgeon preference . statistical analysis was computed using spss version 20 statistical software ( ibm spss inc ) . the dataset was randomly divided into a derivation dataset ( 75% of patients ) and a validation dataset ( 25% of patients ) . multivariable analysis of data from the derivation dataset was performed using stepwise logistic regression with backward selection . the significance within the models was evaluated with the wald test , whereas the strength of the association of variables with poaf was estimated by calculating the or and 95% cis . model discrimination was evaluated by using the area under the receiver operating characteristic ( roc ) curve . only variables with a value of p<0.05 in univariable analysis furthermore , dichotomous variables with an or < 1.2 were excluded from the final regression analysis . additive risk score for the prediction of poaf ( poaf score ) was calculated by adding weighting points for each independent risk factor . once the predictive ability of the poaf score was tested in the validation dataset , further analyses were performed only in the overall dataset . in particular , patients in the last stratum of this additive score ( poaf score 3 ) were considered for further adjusted analyses . linear regression was used for adjusted analysis of the length of inhospital stay ( los ) . because the los variable was not normally distributed , the analysis was performed after it was logarithmically transformed . between july 1999 and december 2010 , all consecutive patients undergoing isolated coronary artery bypass graft surgery ( cabg ) and valve surgery ( with or without concomitant cabg ) at 3 european cardiac centers ( bristol heart institute , uk ; varese university hospital , and centro cardiologico monzino irccs , italy ) were screened for this observational study . patients with a preoperative history of supraventricular arrhythmias requiring treatment were excluded to avoid overestimation of the poaf incidence . the final sample consisted of a total of 17 622 patients . data used in this analysis were retrieved from institutional databases , which remained consistent over the study period . all data were prospectively collected , and information about demographics , comorbidities , medical and surgical history , operative details , and postoperative events during the hospital stay were registered . the study protocol was in compliance with the local institutional review boards and received full approval ; patient consent was waived . preoperative management , anesthetic , and surgical techniques were standardized for all patients and have been previously reported . preoperative antiarrhythmic therapies were not routinely adopted ; in general , blockers were administered on the day of surgery and restarted in the immediate postoperative period unless contraindicated for clinical reasons . at the end of surgery , patients were transferred to a dedicated intensive care unit and managed according to the unit protocol . amiodarone , either orally or intravenously administered , constituted the standard pharmacological treatment of poaf . in patients without successful rhythm cardioversion and with persistent af , warfarin was administered before discharge , with the aim of achieving an international normalized ratio between 2.0 and 3.0 , planning an electric cardioversion within 30 days . symptomatic patients or with ineffective antiarrhythmic therapy to control the ventricular rate underwent cardioversion at any time during the postoperative period . the primary end point of this study was to identify independent predictors of poaf , deriving an effective bedside tool to predict the arrhythmia and its related complications . briefly , poaf was documented on the basis of a rhythm strip or 12lead ecg as previously described . postoperative acute kidney injury ( aki ) was defined according to the rifle ( risk , injury , failure , loss of function , and endstage renal disease ) criteria ; estimated glomerular filtration rate ( egfr ) was calculated with use of the chronic kidney disease epidemiology collaboration equation . cerebrovascular accidents were diagnosed on the basis of a new neurological deficit with morphological substrate confirmed by using computed tomography or nuclear magnetic resonance imaging . the decision to perform offpump or onpump cabg was based on individual surgeon preference . statistical analysis was computed using spss version 20 statistical software ( ibm spss inc ) . the dataset was randomly divided into a derivation dataset ( 75% of patients ) and a validation dataset ( 25% of patients ) . multivariable analysis of data from the derivation dataset was performed using stepwise logistic regression with backward selection . the significance within the models was evaluated with the wald test , whereas the strength of the association of variables with poaf was estimated by calculating the or and 95% cis . model discrimination was evaluated by using the area under the receiver operating characteristic ( roc ) curve . only variables with a value of p<0.05 in univariable analysis furthermore , dichotomous variables with an or < 1.2 were excluded from the final regression analysis . additive risk score for the prediction of poaf ( poaf score ) was calculated by adding weighting points for each independent risk factor . once the predictive ability of the poaf score was tested in the validation dataset , further analyses were performed only in the overall dataset . in particular , patients in the last stratum of this additive score ( poaf score 3 ) were considered for further adjusted analyses . linear regression was used for adjusted analysis of the length of inhospital stay ( los ) . because the los variable was not normally distributed , the analysis was performed after it was logarithmically transformed . the final population presented a mean age of 66.310.6 years ( range , 18 to 92 years ) , and 4301 ( 25% ) were women . patients undergoing isolated cabg accounted for 11 685 ( 68% ) patients ; isolated valve surgery , 3672 ( 21% ) ; and valve surgery with concomitant cabg , 1905 ( 11% ) . presenting characteristics according to the occurrence of postoperative af af indicates atrial fibrillation ; bmi , body mass index ; iabp , intraaortic balloon pump ; ami , acute myocardial infarction ; lvef , left ventricular ejection fraction ; copd , chronic obstructive pulmonary disease ; pvd , peripheral vascular disease ; cvd , cardiovascular disease ; egfr , estimated glomerular filtration rate ; ace , angiotensinconverting enzyme ; cpb , cardiopulmonary bypass ; acc , aortic crossclamp ; icu , intensive care unit ; cva , cerebrovascular accident ; aki , acute kidney injury ; rrt , renal replacement therapy . for continuous variables , meansd or median ( 25th to 75th percentile ) ; for categorical variables , % . other variables considered for the final model and not reported : body surface area , prior percutaneous coronary intervention , left mainstem stenosis , right coronary artery disease , left atrial diameter , dyslipidemia , basal hemoglobin , basal creatinine , creatinine values > 2.0 mg / dl , angiotensin receptor blockers . test comparing the prevalence of risk factors between the entire derivation and entire validation cohorts . the incidence of poaf was 26.4% ( 4561 of 17262 patients ) in the overall series , subdivided into 23% ( n=2667 ) , 31% ( n=1133 ) , and 40% ( n=761 ) for isolated cabg , isolated valve surgery , and combined procedures , respectively . the profile for patients affected by poaf was considerably different with regard to demographics , comorbid conditions , and operative data . patients affected by poaf in the overall series were older than those who were not ffected ( 69.78.9 versus 65.110.8 years , p<0.0001 ) and were predominantly men ( 72% versus 76% , p<0.0001 ) with a lower left ventricular ejection fraction ( lvef , 53.412.2% versus 55.511.3% , p<0.0001 ) and egfr ( 63.823.9 versus 66.722.1 ml / min per 1.73 m , p<0.001 ) , having a more severe profile of comorbidities ( additive euroscore : 5.43.1 versus 4.02.8 , p<0.001 ) . remarkably , preoperative drug regimen did not influence the development of af . the use of blockers ( p=0.174 ) , calcium antagonists ( p=0.114 ) , angiotensinconverting enzyme inhibitors ( p=0.321 ) , and statins ( p=0.453 ) was not associated with a decreased af risk . ninetyseven patients ( 0.6% ) in the overall series were not included in the final regression models because of missing values of any of the covariates of interest . results of multivariable analysis for prediction of postoperative af in the derivation cohort af indicates atrial fibrillation ; copd , chronic obstructive pulmonary disease ; egfr , estimated glomerular filtration rate ; iabp , intraaortic balloon pump ; lvef , left ventricular ejection fraction . , logistic regression revealed that age , emergency operation , preoperative intraaortic balloon pump ( iabp ) , lvef < 30% , egfr < 15 ml / min per m or dialysis , heart valve surgery , and chronic obstructive pulmonary disease were independent predictors of poaf ( hosmer lemeshow goodnessoffit test : [ 7 df]=4.73 , p=0.662 ; area under the roc curve : 0.65 , 95% ci 0.64 to 0.66 ) . poaf score for poaf was calculated ( weighting factors are summarized in table 2 ) , and its area under the roc curve was similar to that of the regression model ( 0.64 , 95% ci 0.63 to 0.65 ) . according to this risk score , the incidence of poaf in the derivation cohort was 0 , 11.1% ( 215/1933 patients ) ; 1 , 20.1% ( 799/3970 patients ) ; 2 , 28.7% ( 1141/3982 patients ) ; and 3 , 40.9% ( 1223/2992 patients ) ( p<0.001 , figures 1 and 2 ) . incidence of postoperative atrial fibrillation ( poaf ) according to poaf score in the derivation and validation cohorts ( both cohorts , cochran armitage test : p<0.001 ) . correlation between postoperative atrial fibrillation ( poaf ) score and predicted probability of poafoccurrence . in the validation cohort , af incidence was 0 , 13.2% ( 81/613 patients ) ; 1 , 19.5% ( 266/1363 patients ) ; 2 , 29.9% ( 395/1320 patients ) ; and 3 , 42.5% ( 422/994 patients ) ( p<0.001 , figure 1 ) . the area under the roc curve of this risk score for prediction of poaf in the validation cohort was 0.64 ( 95% ci 0.63 to 0.66 ) . in the overall series , the risk of hospital mortality ( p<0.001 ) , stroke ( p<0.001 ) , aki ( p<0.001 ) , renal replacement therapy ( rrt , p<0.001 ) , and los ( p<0.001 ) increased along with the risk of af and its occurrence . the risk of adverse events was increased in patients with poaf in all strata . however , the absolute difference in the rate of adverse events was most evident in the stratum with 3 risk factors for poaf . the poaf score was predictive also of hospital mortality ( area under the roc curve 0.73 , 95% ci 0.70 to 0.76 ) , cva ( 0.71 , 95% ci 0.68 to 0.73 ) , aki ( 0.67 , 95% ci 0.65 to 0.68 ) , and rrt ( 0.66 , 95% ci 0.61 to 0.72 ) ( figure 3 ) . a significant correlation was observed between poaf score and los ( 0.32 , p<0.001 ) . postoperative complication ( hospital mortality , cerebrovascular accident , acute kidney injury , and renal replacement therapy ) rates according to the postoperative atrial fibrillation ( poaf ) score and the occurrence of poaf . p values are within each risk class . analyzing the prognostic impact of poaf in the last stratum of poaf score ( score 3 ) , which included 3986 patients from the overall series , the arrhythmia was associated in univariable analysis with increased risk of hospital mortality ( 5.5% versus 3.2% , p=0.001 , for mortality after the first postoperative day : 5.1% versus 2.6% , p<0.001 ) , cva ( 7.8% versus 4.2% , p<0.001 ) , aki ( 15.1% versus 7.1% , p<0.001 ) , rrt ( 3.8% versus 1.4% , p<0.001 ) , and los ( mean 13.2 versus 10.2 days , p<0.001 ) . additive euroscore adjusted analysis showed that the last stratum of poaf score ( score for af 3 ) had the highest risk of mortality ( or 3.61 , 95% ci 1.27 to 10.26 ) . the area under the roc curve of additive euroscore for predicting hospital mortality in this stratum was 0.80 ( 95% ci 0.77 to 0.82 ) . when adjusted for additive euroscore , poaf was associated with an increased risk of hospital mortality ( or 1.49 , 95% ci 1.08 to 2.05 ) . similarly , af was an independent predictor of hospital mortality and/or cva ( or 1.59 , 95% ci 1.27 to 1.99 ) . because poaf usually occurs a mean of 2 to 3 days after the procedure , its impact on the hospital mortality occurring > 1 day after surgery was also evaluated . the impact of this arrhythmia was even larger in predicting hospital mortality occurring > 1 day after surgery ( or 1.74 , 95% ci 1.24 to 2.45 ) . when adjusted for additive euroscore , poaf was also associated with an increased risk of cva ( or 1.76 , 95% ci 1.34 to 2.31 ) , aki ( or 2.21 , 95% ci 1.72 to 2.62 ) , rrt ( or 2.68 , 95% ci 1.42 to 5.08 ) , and los ( coefficient 0.09 , 95% ci 0.07 to 0.10 ) in the last stratum of the poaf score . the final population presented a mean age of 66.310.6 years ( range , 18 to 92 years ) , and 4301 ( 25% ) were women . patients undergoing isolated cabg accounted for 11 685 ( 68% ) patients ; isolated valve surgery , 3672 ( 21% ) ; and valve surgery with concomitant cabg , 1905 ( 11% ) . presenting characteristics according to the occurrence of postoperative af af indicates atrial fibrillation ; bmi , body mass index ; iabp , intraaortic balloon pump ; ami , acute myocardial infarction ; lvef , left ventricular ejection fraction ; copd , chronic obstructive pulmonary disease ; pvd , peripheral vascular disease ; cvd , cardiovascular disease ; egfr , estimated glomerular filtration rate ; ace , angiotensinconverting enzyme ; cpb , cardiopulmonary bypass ; acc , aortic crossclamp ; icu , intensive care unit ; cva , cerebrovascular accident ; aki , acute kidney injury ; rrt , renal replacement therapy . for continuous variables , meansd or median ( 25th to 75th percentile ) ; for categorical variables , % . other variables considered for the final model and not reported : body surface area , prior percutaneous coronary intervention , left mainstem stenosis , right coronary artery disease , left atrial diameter , dyslipidemia , basal hemoglobin , basal creatinine , creatinine values > 2.0 mg / dl , angiotensin receptor blockers . test comparing the prevalence of risk factors between the entire derivation and entire validation cohorts . the incidence of poaf was 26.4% ( 4561 of 17262 patients ) in the overall series , subdivided into 23% ( n=2667 ) , 31% ( n=1133 ) , and 40% ( n=761 ) for isolated cabg , isolated valve surgery , and combined procedures , respectively . the profile for patients affected by poaf was considerably different with regard to demographics , comorbid conditions , and operative data . patients affected by poaf in the overall series were older than those who were not ffected ( 69.78.9 versus 65.110.8 years , p<0.0001 ) and were predominantly men ( 72% versus 76% , p<0.0001 ) with a lower left ventricular ejection fraction ( lvef , 53.412.2% versus 55.511.3% , p<0.0001 ) and egfr ( 63.823.9 versus 66.722.1 ml / min per 1.73 m , p<0.001 ) , having a more severe profile of comorbidities ( additive euroscore : 5.43.1 versus 4.02.8 , p<0.001 ) . remarkably , preoperative drug regimen did not influence the development of af . the use of blockers ( p=0.174 ) , calcium antagonists ( p=0.114 ) , angiotensinconverting enzyme inhibitors ( p=0.321 ) , and statins ( p=0.453 ) was not associated with a decreased af risk . ninetyseven patients ( 0.6% ) in the overall series were not included in the final regression models because of missing values of any of the covariates of interest . results of multivariable analysis for prediction of postoperative af in the derivation cohort af indicates atrial fibrillation ; copd , chronic obstructive pulmonary disease ; egfr , estimated glomerular filtration rate ; iabp , intraaortic balloon pump ; lvef , left ventricular ejection fraction . , logistic regression revealed that age , emergency operation , preoperative intraaortic balloon pump ( iabp ) , lvef < 30% , egfr < 15 ml / min per m or dialysis , heart valve surgery , and chronic obstructive pulmonary disease were independent predictors of poaf ( hosmer lemeshow goodnessoffit test : [ 7 df]=4.73 , p=0.662 ; area under the roc curve : 0.65 , 95% ci 0.64 to 0.66 ) . poaf score for poaf was calculated ( weighting factors are summarized in table 2 ) , and its area under the roc curve was similar to that of the regression model ( 0.64 , 95% ci 0.63 to 0.65 ) . according to this risk score , the incidence of poaf in the derivation cohort was 0 , 11.1% ( 215/1933 patients ) ; 1 , 20.1% ( 799/3970 patients ) ; 2 , 28.7% ( 1141/3982 patients ) ; and 3 , 40.9% ( 1223/2992 patients ) ( p<0.001 , figures 1 and 2 ) . incidence of postoperative atrial fibrillation ( poaf ) according to poaf score in the derivation and validation cohorts ( both cohorts , cochran armitage test : p<0.001 ) . correlation between postoperative atrial fibrillation ( poaf ) score and predicted probability of poafoccurrence . in the validation cohort , af incidence was 0 , 13.2% ( 81/613 patients ) ; 1 , 19.5% ( 266/1363 patients ) ; 2 , 29.9% ( 395/1320 patients ) ; and 3 , 42.5% ( 422/994 patients ) ( p<0.001 , figure 1 ) . the area under the roc curve of this risk score for prediction of poaf in the validation cohort was 0.64 ( 95% ci 0.63 to 0.66 ) . in the overall series , the risk of hospital mortality ( p<0.001 ) , stroke ( p<0.001 ) , aki ( p<0.001 ) , renal replacement therapy ( rrt , p<0.001 ) , and los ( p<0.001 ) increased along with the risk of af and its occurrence . the risk of adverse events was increased in patients with poaf in all strata . however , the absolute difference in the rate of adverse events was most evident in the stratum with 3 risk factors for poaf . the poaf score was predictive also of hospital mortality ( area under the roc curve 0.73 , 95% ci 0.70 to 0.76 ) , cva ( 0.71 , 95% ci 0.68 to 0.73 ) , aki ( 0.67 , 95% ci 0.65 to 0.68 ) , and rrt ( 0.66 , 95% ci 0.61 to 0.72 ) ( figure 3 ) . a significant correlation was observed between poaf score and los ( 0.32 , p<0.001 ) . postoperative complication ( hospital mortality , cerebrovascular accident , acute kidney injury , and renal replacement therapy ) rates according to the postoperative atrial fibrillation ( poaf ) score and the occurrence of poaf . analyzing the prognostic impact of poaf in the last stratum of poaf score ( score 3 ) , which included 3986 patients from the overall series , the arrhythmia was associated in univariable analysis with increased risk of hospital mortality ( 5.5% versus 3.2% , p=0.001 , for mortality after the first postoperative day : 5.1% versus 2.6% , p<0.001 ) , cva ( 7.8% versus 4.2% , p<0.001 ) , aki ( 15.1% versus 7.1% , p<0.001 ) , rrt ( 3.8% versus 1.4% , p<0.001 ) , and los ( mean 13.2 versus 10.2 days , p<0.001 ) . additive euroscore adjusted analysis showed that the last stratum of poaf score ( score for af 3 ) had the highest risk of mortality ( or 3.61 , 95% ci 1.27 to 10.26 ) . the area under the roc curve of additive euroscore for predicting hospital mortality in this stratum was 0.80 ( 95% ci 0.77 to 0.82 ) . when adjusted for additive euroscore , poaf was associated with an increased risk of hospital mortality ( or 1.49 , 95% ci 1.08 to 2.05 ) . similarly , af was an independent predictor of hospital mortality and/or cva ( or 1.59 , 95% ci 1.27 to 1.99 ) . because poaf usually occurs a mean of 2 to 3 days after the procedure , its impact on the hospital mortality occurring > 1 day after surgery was also evaluated . the impact of this arrhythmia was even larger in predicting hospital mortality occurring > 1 day after surgery ( or 1.74 , 95% ci 1.24 to 2.45 ) . when adjusted for additive euroscore , poaf was also associated with an increased risk of cva ( or 1.76 , 95% ci 1.34 to 2.31 ) , aki ( or 2.21 , 95% ci 1.72 to 2.62 ) , rrt ( or 2.68 , 95% ci 1.42 to 5.08 ) , and los ( coefficient 0.09 , 95% ci 0.07 to 0.10 ) in the last stratum of the poaf score . af after cardiac surgery remains a vexing complication , resulting in prolonged hospital stay , and causes additional morbidity and mortality in a substantial number of patients . consequently , considerable efforts have been directed toward reduction of the risk and poaf management , mainly focusing on pharmacological agents . however , a caveat with the use of the antiarrhythmic approaches is that most patients undergoing cardiac surgery do not develop poaf , and 60% to 80% of them are exposed to the costs and potential side effects of unnecessary prophylaxes . on the one hand , it is critical that before such pharmacological managements are implemented in all cardiac surgery patients , the safety and effectiveness of these strategies in improving patient outcomes must be proved . on the other hand , a rapid , accurate estimation of individual patients ' risk for poaf may facilitate correct identification of patients who are at the lowest risk of the development of af and should not be treated with preventive strategies . in this setting , our study demonstrated that the poaf score is a simple , accurate bedside risk tool , allowing for the identification of highrisk af patients in whom preventive antiarrhythmic therapies could be justified . the poaf score was also found to be of value in developing an easytouse risk scoring method for afrelated or accompanying complications , suggesting possible simultaneous preventive approaches . other risk prediction models have been previously proposed to identify patients most likely to develop poaf . zaman et al first enrolled 326 elective isolated cabg patients and created a model for preoperative risk stratification in patients affected by af , demonstrating that pwave duration > 155 ms , age , and male sex were able to predict poaf in 59% of their patient population . amar et al reported in 1851 patients undergoing isolated cabg that 4 preoperative and postoperative variables were independently associated with af development . using their prediction model , 3 risk categories for af were identified , suggesting increased af occurrence ( from 14% to 60% ) with category worsening . mathew et al performed a prospective multicenter observational study of 4657 cabg patients , obtaining a final model with 3 af risk classes ( low , medium , and high risk ) based on 17 preoperative , intraoperative , and postoperative variables . more recently , magee et al extracted 19 083 patients undergoing isolated cabg from the society of thoracic surgeons database , using perioperative risk factors to develop a predictive risk algorithm of af development . the final model included 14 preoperative , intraoperative , and postoperative variables , which provided statistically significant coefficients and ors . however , all of these prediction models provided controversial results , revealing important limitations , and limiting their widespread adoption in the clinical practice of af preventive strategies . some of these studies were computationally complex due to the relevant number of variables considered and underpowered due to their small sample size . in addition , considered risk factors of poaf such as pwave duration , male sex , or angiotensinconverting enzyme inhibitor treatment have been demonstrated to be poorly associated with the arrhythmia , while other wellknown af predictors were surprisingly not included . patients with deteriorated left ventricular function , compromised renal function , or complex operations including valve surgery were often excluded from these af prediction models . nevertheless , these subjects are the ones mostly affected by af , as demonstrated in several studies , and would benefit the most from antiarrhythmic measures . on the other hand , the same risk prediction models included postoperative variables such as low cardiac output , postoperative drug treatment , and prolonged ventilation , hampering prompt prophylactic treatment , especially in the case of urgent or emergent operations . in fact , magee et al included prolonged ventilation use as one of the most relevant af predictors , limiting the possibility to adopt preventive measures before such a condition develops and postponing the usefulness of their model to the postoperative period only . however , it is well known that poaf most commonly occurs within the first 48 hours after surgery , and it remains distinctly possible that af occurred as a consequence of the observed prolonged ventilation rather than being a cause of it , confounding the predictive value of this variable and consequently invalidating the final model . furthermore , patients with preexistent af were inopportunely included in the earlier mentioned prediction models , ignoring the fact that these subjects represent a specific surgical subgroup , being at higher risk for mortality and morbidity . preexisting af is often encountered in older patients with significant mitral valve disease and left atrial enlargement , and such patients are certainly more prone to develop af after surgery than are those without this preexisting condition . in fact , both amar et al and mathew et al included the history of af as a relevant risk factor in their prediction models , although patients with preexisting arrhythmia were largely affected by poaf ( 68% and 53% , respectively ) . consonant data are obtained in the prediction model of magee et al , with inclusion of the preoperative arrhythmia variable in the final prediction model , although patients affected by it experienced poaf in 70% of cases . in our study , patients with a preoperative history of supraventricular arrhythmias were excluded to avoid confusing the incidence of poaf with recurrence of a preexisting arrhythmic condition and because these patients have been demonstrated to benefit from extensive antiarrhythmic prophylaxis or a contemporary surgical ablation . the present study has important indirect clinical implications with reference to possible af preventive strategies , although it was not designed to specifically address the complex af preoperative prophylaxis issue . our aim was to identify a patient population more likely to develop af and its related complications who may most benefit from of any preventive strategy . the present prediction model includes variables such as copd , valve surgery , emergency status , preoperative iabp need , advanced renal failure , and reduce lvef , which have normally constituted exclusion criteria in several observational and randomized studies testing different af prophylactic regimens , because the adverse drug effects registered in such patient classes . lowrisk patients ( poaf score <3 ) may benefit from an extensive preoperative af prophylaxis based on blockers and statins . however , highrisk patients ( poaf score 3 ) are likely to be the most appropriate candidates for amiodarone prophylaxis , also for subjects already receiving blockers . amiodarone has demonstrated a large compliance in the routine antiarrhythmic care of cardiac surgery patients , because of its low incidence of acute complications and a large effectiveness independently from dose ( high or low ) or administration time ( preoperative or intraoperative ) . in addition , highrisk patients ( poaf score 3 ) may benefit from a prolonged postoperative surveillance because of the possible occurrence of late af episodes . finally , in comparison with previous prediction models , the poaf score for the first time was also tested in predicting related or accompanying complications of poaf . having the highest af risk as identified by poaf score 3 was strongly associated with higher hospital mortality , postoperative cva , aki , and rrt , suggesting a wider preventive approach based on both antiarrhythmic prophylaxes and other protective measures for afrelated morbidities . in particular , patients with a poaf score 3 and affected by poaf experienced a 2 to 4fold increased risk of postoperative cva compared with those in other poaf score classes . patients with a poaf score 3 may largely benefit from antiarrhythmic prophylaxis accompanied by routine postoperative use of lowmolecularweight heparin , starting in the immediate period after surgery , especially in cabg patients as previously anticipated . the use of oral anticoagulation only at the onset of af has been demonstrated to be inadequate to prevent stroke . interestingly , oral anticoagulation in patients affected by poaf has been demonstrated to reduce early and late mortality associated with thromboembolic af sequelae . therefore , it should be considered in highrisk group patients ( poaf score 3 ) immediately after cardiac surgery . in this patient class , the ligation of the let atrial appendage although the af predictors were highly significant and assessed in a validation cohort , the accuracy of the present model is moderate , a result shared with other similar studies . mahoney et al examined more than 10 000 cardiac surgery patients , investigating predictors of poaf in subjects undergoing isolated cabg and valve surgery with or without concomitant coronary surgery , and obtained 3 different models with an area under the curve of 0.67 , 0.65 , and 0.64 , respectively . similar results were also obtained by thorn et al in a cohort of 7115 isolated cabg patients in an effort to identify patients at high risk of developing af , and the predictive ability of their final prediction model was moderate ( area under the roc curve , 0.62 ) . furthermore , the present data were unable to clearly identify the temporal relationship between the onset of af after surgery and the occurrence of adverse clinical events . it can not be excluded that poaf occurred as a consequence of an observed complication rather than it being a cause of the complication . although the af predictors were highly significant and assessed in a validation cohort , the accuracy of the present model is moderate , a result shared with other similar studies . mahoney et al examined more than 10 000 cardiac surgery patients , investigating predictors of poaf in subjects undergoing isolated cabg and valve surgery with or without concomitant coronary surgery , and obtained 3 different models with an area under the curve of 0.67 , 0.65 , and 0.64 , respectively . similar results were also obtained by thorn et al in a cohort of 7115 isolated cabg patients in an effort to identify patients at high risk of developing af , and the predictive ability of their final prediction model was moderate ( area under the roc curve , 0.62 ) . furthermore , the present data were unable to clearly identify the temporal relationship between the onset of af after surgery and the occurrence of adverse clinical events . it can not be excluded that poaf occurred as a consequence of an observed complication rather than it being a cause of the complication . the poaf score based on routinely available preoperative data is a simple , accurate bedside tool , allowing for identification of patients at high risk for poaf and in whom preventive antiarrhythmic therapies seem justified . permitting identification of patients at high risk for af and related or accompanying complications , the poaf score may also help to develop and update clinical and therapeutic strategies that not only would minimize the occurrence of poaf but also would result in an improvement in the otherwise dismal morbidity and mortality rates observed among patients affected by this arrhythmia .

backgroundatrial fibrillation ( af ) remains the most common complication after cardiac surgery . the present study aim was to derive an effective bedside tool to predict postoperative af and its related complications.methods and resultsdata of 17 262 patients undergoing adult cardiac surgery were retrieved at 3 european university hospitals . a risk score for postoperative af ( poaf score ) was derived and validated . in the overall series , 4561 patients ( 26.4% ) developed postoperative af . in the derivation cohort age , chronic obstructive pulmonary disease , emergency operation , preoperative intraaortic balloon pump , left ventricular ejection fraction < 30% , estimated glomerular filtration rate < 15 ml / min per m2 or dialysis , and any heart valve surgery were independent af predictors . poaf score was calculated by summing weighting points for each independent af predictor . according to the prediction model , the incidences of postoperative af in the derivation cohort were 0 , 11.1% ; 1 , 20.1% ; 2 , 28.7% ; and 3 , 40.9% ( p<0.001 ) , and in the validation cohort they were 0 , 13.2% ; 1 , 19.5% ; 2 , 29.9% ; and 3 , 42.5% ( p<0.001 ) . patients with a poaf score 3 , compared with those without arrhythmia , revealed an increased risk of hospital mortality ( 5.5% versus 3.2% , p=0.001 ) , death after the first postoperative day ( 5.1% versus 2.6% , p<0.001 ) , cerebrovascular accident ( 7.8% versus 4.2% , p<0.001 ) , acute kidney injury ( 15.1% versus 7.1% , p<0.001 ) , renal replacement therapy ( 3.8% versus 1.4% , p<0.001 ) , and length of hospital stay ( mean 13.2 versus 10.2 days , p<0.001).conclusionsthe poaf score is a simple , accurate bedside tool to predict postoperative af and its related or accompanying complications .

avulsion of the aortic valve commissure as a cause of acute aortic valve regurgitation is mostly due to trauma , infective endocarditis , or ascending aortic dissection . an 80-year - old male with hypertension was seen in the emergency department with acute onset dyspnea . echocardiogram revealed left ventricular hypertrophy with adequate systolic function , prolapse of the noncoronary cusp , and incomplete coaptation of the right coronary and noncoronary cusps with severe aortic valve regurgitation . aortic valve replacement was performed , and the patient was alive and well 4 years after surgery . a review of the literature showed that more than three - fourths of the similar cases occurred in males , and about half in patients with hypertension and those 60 years of age or older . in the case of acute aortic regurgitation without a history of trauma , infection , or valvotomy , when 2 prolapsed aortic cusps are observed by echocardiography in the absence of an intimal tear of the ascending aorta , an avulsion of the aortic commissure should be suspected , especially in males with hypertension who are 60 years of age or older . acute aortic valve regurgitation ( ar ) caused by detachment of the aortic valve commissure from the aortic wall , also known as avulsion of the aortic valve commissure , is usually related to blunt chest trauma , infective endocarditis , or ascending aortic dissection . it had not been mentioned in recent guidelines or in cardiology textbooks as a cause of acute ar . we present a case of acute ar resulting from nontraumatic avulsion of the aortic valve commissure , and review the relevant literature with a focus on potential risk factors and echocardiographic findings . an 80-year - old male presented to the emergency department with dizziness and shortness of breath for 1 day . he had hypertension , but denied any history of dyspnea , fever , chills , or chest pain . physical examination revealed tachycardia , a systolic - diastolic murmur grade 2/6 over the aortic area / left lower sternal border , bilateral rales of lungs without lower legs pitting edema , and a distended urinary bladder . echocardiography revealed a hypertrophic interventricular septum , concentric left ventricular hypertrophy ( lvh ) , adequate left ventricular systolic function with an ejection fraction of 68% , prolapse of the noncoronary cusp ( ncc ) ( fig . 1a ) , and incomplete coaptation of right coronary cusp ( rcc ) and ncc ( fig . 1b and c ) with severe ar ( fig . ( a ) echocardiography parasternal long - axis view showed prolapse of the noncoronary cusp in the diastolic phase . ( b ) m - mode at the level of the aortic valve showed incomplete coaptation of right and noncoronary cusps . ( c ) parasternal short - axis view showed incomplete coaptation of right , left , and noncoronary cusps . the patient underwent intubation for impending respiratory failure , and a cardiovascular surgeon was consulted . cardiac catheterization revealed a normal coronary angiogram and left ventricular systolic function , as well as severe ar without aortic dissection . chest computed tomography showed aneurysmal dilatation of the ascending aorta with aortic valve calcifications and moderate bilateral pleural effusions . mild atherosclerotic changes of the aortic cusps and prolapse of the left coronary cusp ( lcc ) and ncc ( fig . 2a ) , as well as a transverse crack measuring approximately 0.8 cm in length over the lcc in addition , concentric lvh and a fusiform aneurysm of the ascending aorta ( with a maximal diameter of 4.8 cm ) were noted . aortic valve replacement was performed with a hancock - ii 25 mm porcine valve and longitudinal aortoplasty . ( a ) intraoperative photograph showing mild atherosclerotic changes of the aortic cusps , and prolapse of left and noncoronary cusps . ( b ) after removal of the aortic cusps , local avulsion of the aortic commissure between left and noncoronary cusps was observed ( arrow ) . histopathology examination of the aortic valve showed myxoid degeneration , fibrosis , and calcification ( fig . examination of the ascending aorta revealed myxoid degeneration and fragmentation of elastic fibers ( fig . postoperatively , the patient was transferred to the intensive care unit with ventilator and inotropic ( dobutamine ) support and continuous intravenous dormicum sedation . he was classified as new york heart association class i when last seen 4 years after the surgery . ( a ) the 3 aortic cusps that were surgically removed exhibited white and firm deformed valvular tissue . ( b ) histopathological examination of the aortic valve showed myxoid degeneration , fibrosis , and calcification ( 40 ) . ( c ) examination of the ascending aorta revealed myxoid degeneration and fragmentation of elastic fibers ( 40 ) . ( d ) examination of the ascending aorta revealed myxoid degeneration and fragmentation of elastic fibers ( 400 ) . acute ar can be secondary to trauma , infective endocarditis , type a aortic dissection , annuloaortic ectasia , rheumatic fever , syphilis , bicuspid aortic valve , iatrogenic secondary to a procedure ( such as aortic balloon valvotomy ) , or a failed surgical valve repair . nontraumatic aortic valve commissure avulsion is rare , but should be considered in a case of ar . the diagnosis of avulsion of the aortic commissure is difficult prior to surgery on the basis of imaging alone . transthoracic or transesophageal echocardiography may demonstrate aortic valve prolapse with severe ar , which is a nonspecific finding . a more specific finding is the presence of 2 prolapsed aortic cusps , and a limited intimal tear on the commissure may be observed just above the valves . in addition to avulsion of a commissure , the possible etiologies of aortic valve prolapse include floppy valve , fraying valve , excessive tissue within a congenital bicuspid valve , ruptured cusp , laceration of the aorta , and ventricular septal defect with displacement of the aortic root . as prolapse of the cusps may be observed on echocardiography , the commissural tear may mimic an intimal tear in a type a aortic dissection , or a mass - like lesion . in such a case , transesophageal echocardiography may demonstrate a normal aortic valve with a dissection flap in the proximal ascending aorta just above the aortic valve , but without the intimal tear extending to the aortic root . this is different from a type a aortic dissection , in which an intimal tear may be noted in the ascending aorta . in a case of ar without a history of trauma , infection , or valvotomy , when 2 prolapsed aortic cusps are observed in the absence of an intimal tear of the ascending aorta , an avulsion of the aortic commissure should be suspected . hypertension plays an important role in the formation of a nontraumatic aortic valve commissure avulsion . using avulsion and dehiscence combining with aortic valve commissure as key words to search literature indexed in pubmed , we found 5 and 10 cases of nontraumatic aortic valve commissure avulsion , respectively , and we identified 9 more cases from the references of the reports retrieved ( table 1 ) . including our case , of the 20 cases in which information regarding hypertension was available , 10 had hypertension . other potential risk factors included male sex ( 17/22 ) and older age ( 13/22 at 60 years or older ) . the possible pathogenesis of avulsion of the aortic commissure includes high blood pressure , especially high diastolic pressure which avulses a weakened commissure that may be normal or have underlying atherosclerosis , fibrosis , hyalinosis , cystic medial necrosis , pseudoxanthoma elasticum , annuloaortic ectasia , granulation , elastic fiber tear or myxomatous changes . the aortic valve cusps may be normal , or have mucoid degeneration , atherosclerosis , or myxomatous changes . medical therapy failed in 3 early cases of avulsion of the aortic commissure , and the patients died . surgery aimed at either repair or replacement of the aortic valve or aortic root is lifesaving , with only 1 death out of 22 cases . in the presence of aortic medial disease , connective tissue disease , atherosclerosis , or myxomatous degeneration of aortic valve , if avulsion of the commissure is accompanied by aortic root dilation or avulsion of the aortic cusp , aortic root replacement is indicated . aortic root replacement was performed in 1 case of 2 dehiscences of the aortic valve commissure and cusp in order to reinforce the aortic wall around the left coronary artery ostium . direct suturing of the degenerated and avulsed commissure to the diseased aortic root usually leads to a poor long - term prognosis . hirata et al sutured the detached commissure to the aortic wall in a patient with pseudoxanthoma elasticum , but the long - term results were unknown . newcomb et al initially used teflon pledgeted polypropylene sutures to suspend the commissure to its previous position in a case with a normal aortic valve , but changed to aortic valve replacement due to persistent mild to moderate aortic regurgitation . charokopos et al suspended the detached commissure to its proper position using 3 teflon pledgeted 20 sutures combined with bioglue to reinforce the site of repair which was sutured in 2 layers . shimamoto et al reported a novel method for repair of commissural detachment in 2 cases with annuloaortic ectasia and 1 case with a dilated sinotubular junction . he reimplanted the root in a valsalva graft , and used 20 polyester sutures for the first row and 40 polypropylene sutures for the second row . therefore , we chose aortic valve replacement using a porcine valve , which achieved a good outcome at 4 years after surgery . in conclusion , transthoracic and transesophageal echocardiography may demonstrate 2 prolapsed aortic cusps with or without a limited intimal tear on the commissure just above the valves . hypertension plays an important role in the formation of an avulsion in patients with atherosclerosis , medial necrosis , myxomatous changes , granulation , fibrosis , hyalinosis , or fibrosis . surgery is the only reliable method available to treat this condition , which includes aortic valve repair , replacement , and aortic root replacement .

abstractbackground : avulsion of the aortic valve commissure as a cause of acute aortic valve regurgitation is mostly due to trauma , infective endocarditis , or ascending aortic dissection . nontraumatic avulsion of the aortic valve commissure is very rare . we reviewed the literature and analyzed potential risk factors of nontraumatic avulsion.case presentation : an 80-year - old male with hypertension was seen in the emergency department with acute onset dyspnea . echocardiogram revealed left ventricular hypertrophy with adequate systolic function , prolapse of the noncoronary cusp , and incomplete coaptation of the right coronary and noncoronary cusps with severe aortic valve regurgitation . surgery revealed an avulsion between the left coronary and noncoronary cusps . histopathology examination of the aortic valve showed myxoid degeneration , fibrosis , and calcification . examination of the ascending aorta revealed myxoid degeneration and fragmentation of elastic fibers . aortic valve replacement was performed , and the patient was alive and well 4 years after surgery . a review of the literature showed that more than three - fourths of the similar cases occurred in males , and about half in patients with hypertension and those 60 years of age or older.conclusions:in the case of acute aortic regurgitation without a history of trauma , infection , or valvotomy , when 2 prolapsed aortic cusps are observed by echocardiography in the absence of an intimal tear of the ascending aorta , an avulsion of the aortic commissure should be suspected , especially in males with hypertension who are 60 years of age or older .

considering sexual disparity in gamete investments ( anisogamy ) males should maximise their reproductive success by mating with many females , while females should choose the highest quality partner and mate only to the extend which will ensure fertilization of all her eggs ( bateman 1948 ; trivers 1972 ) . in monandrous insects and mites , sperm obtained from a single insemination can be sufficient to fertilize eggs produced during the whole female life span ( eberhard 1996 ; simmons 2001 ) . monandry may be beneficial , as multiple matings can incur some serious costs to females : e.g. wasted time and energy used for finding a partner and mating , increased exposure to predation , parasites and diseases , harm from aggressive males , etc . ( simmons 2005 ; arnquist and rowe 2005 ) . nonetheless , polyandry is a taxonomically widespread phenomenon , also common in insects and mites ( e.g. dickinson 1997 ; witaliski 1999 ; simmons 2001 ; hosken and stockley 2003 ) . both genetic and non - genetic benefits can promote the evolution of polyandry , e.g. fertilization by high quality sperm due to sperm competition , increased genetic diversity among offspring , reduced risk of inbreeding and genetic incompatibility ; sperm replenishment , increased female fertility and fecundity due to male - donated nutrients and ejaculate defensive compounds , increased parental care and avoidance of infanticide and predation ( rev . . 0.10.3 mm long ) , four - legged and highly specialized herbivores forming galls or living freely on plants . many species are economically important , causing rusting or various distortions of growth of plant tissue , and transmitting plant viruses ( lindquist et al . eriophyoids transfer sperm indirectly , via spermatophores deposited on a substrate and without pair formations between males and females ( so called sex dissociation or non - pairing ) ( thomas and zeh 1984 ; oldfield and michalska 1996 ; proctor 1998 ) . spermatophores of eriophyoids consist of a head with a central sperm reservoir , a stalk and a foot by which the spermatophore is attached to the substrate . up to date behavioural observations on the visitations of spermatophores by eriophyoid females were conducted in aculus fockeui ( nalepa and trouessart ) , a. schlechtendali ( nalepa ) , epitrimerus pyri ( nalepa ) and phyllocoptruta oleivora ( ashmed ) ( oldfield et al . 1972 ; oldfield and newell 1973 ; oldfield 1988 ) . when mounting a spermatophore , females of various species behave similarly ( oldfield and michalska 1996 ) . first , they sway from side to side a few times on a spermatophore and then they become motionless for several seconds , lying on a spermatophore head at the region of the genital opening ( which is situated ventrally just behind the coxae of the second pair of legs ) while their cauda rested on the leaf surface . finally , they resume gross movements and slightly pull the spermatophore , which remains attached to the leaf surface when the females leaves . ( 1972 ) and oldfield and newell ( 1973 ) revealed that females of a. fockeui pick up sperm from only a single spermatophore in their lifetime and deposit sperm in either the left or right spermatheca only . in virgins of a. fockeui , both spermathecae are similar in size and ellipsoidal . however , after the spermatophore visitation , the sac with sperm becomes larger and spheroid . by contrast , in other eriophyoids , the sperm - filled spermatheca can be turgid but ellipsoidal ( oldfield 1973 ) . the size of the sperm reservoir of the a. fockeui spermatophore corresponds to the size of the sperm - filled spermatheca ( oldfield et al . a similar relationship between sperm reservoir and sperm - filled spermatheca as well as asymmetrical sperm storage , was reported in many other eriophyoid species ( 34 species in total ) , mostly from dicotyledonous host plants . by contrast , symmetrical sperm storage , in which sperm is stored in both spermatheca , prevailed in eriophyoid species that associated with gymnosperm and monocot host plants ( oldfield 1973 , 1999 ) . in three examined species , aceria cynondonis ( wilson ) ( previously eriophyes cynondonis ) , aceria tulipae keifer ( previously eriophyes tulipae ) and trisetacus ehamnni keifer the sperm - filled spermathecae were as large or larger than the sperm reservoir of a spermatophore , which suggested that females with symmetrical sperm storage pick up sperm multiply ( oldfield 1973 ) . however , no behavioural studies confirmed polyandry in these eriophyoids . in eriophyoids , monandry may be advantageous , because soon after insemination from a spermatophore females can migrate to new , more profitable patches and reproduce without wasting time and energy on finding subsequent spermatophores . however , by picking up sperm from only a single spermatophore the eriophyoid females risk becoming inseminated for their whole lifetime with sperm of low quality and quantity . under such conditions , female reproductive success would substantially decrease and repeated visitations of spermatophores , as well as polyandry , might be promoted . the aim of this study is to examine spermatophore visitation behaviour and symmetry of sperm storage in two free - living species : cecidophyopsis hendersoni ( keifer ) from yucca sp . and a. allotrichus ( nalepa ) ( syn . vasates robiniae nalepa ) from the black locust tree robinia pseudoaccacia l. this would confirm monandry or polyandry in those species . in c. hendersoni , the effect of the presence of conspecifics and leaf condition on spermatophore deposition rate has been previously studied ( michalska and shi 2004 ) . on non - infested leaves , males deposit considerably fewer spermatophores than on infested leaves from which conspecifics were washed off . when non - infested leaves are offered , males decrease their spermatophore deposition rate on those leaves which are older or mechanically damaged . virgin females have a stimulating effect on spermatophore deposition rate only on leaves previously infested by conspecifics , in this mite . as c. hendersoni inhabits a monocotyledonous host plant , i suspect that females of this species might pick up sperm from spermatophores multiply in their lifetime . secondly , males deposit spermatophores and guard females on their emergence sites ( michalska 1999 ) . guarding of pre - emergent females strongly suggests that females become inseminated once or only a few times during their lifespan , shortly after emergence ( thornhill and alcock 1983 ) . on the emergence sites , a. allotrichus females visit spermatophores as soon as their genital coverflaps are free and most of their old exuvia are shed after moulting . when they are not surrounded with spermatophores they return to the emergence site several times searching for spermatophores and making greater and greater circles until they finally wandered off ( oldfield and michalska 1996 ) . in this paper , i examined the behaviour of a. allotrichus and c. hendersoni females in relation to spermatophores during the whole female lifespan , from the day of emergence to the death of the female . i also examined spermathecae in squashed females right after the visitation of spermatophores as well in females randomly taken from the population to confirm asymmetrical or symmetrical sperm storage in both species . the original population of c. hendersoni was sampled from infested yucca ( yucca sp . ) the eriophyoids used in the experiments were obtained from potted yucca ( yucca sp . ) plants grown in a climatic room at 26 c and 16/8 l / d photoperiod . aculops allotrichus was collected from black locust trees ( r. pseudoaccacia ) grown on the campus of warsaw university of life science . apart from the leaf - lets populated with a. allotrichus i also collected clean leaf - lets from the black locust trees that were non - infested by a. allotrichus . to keep the sampled leaves of yucca or the leaflets of black locust tree in turgor as soon as leaves were brought to the laboratory , detached leaf cages ( for details see michalska and shi 2004 ; michalska 2000 , 2005 ) were constructed and the eriophyoids chosen for a test were transferred to them . to construct cages for a. allotrichus , clean leaf - lets of black locust were used . in c. hendersoni , males deposited many more spermatophores and females laid many more eggs on the infested yucca leaves from which eriophyoids were washed off than they did on the non - infested leaves ( michalska and shi 2004 ; michalska , pers . obs . ) . therefore , in this study , only the washed off infested leaves of yucca were applied . to estimate whether the eriophyoids pick up sperm from one or more spermatophores in their lifetime , females of each species were exposed to the presence of spermatophores from the day of their emergence until the end of their lives . aculops allotrichus females ( n = 25 ) were exposed to spermatophores on the day of emergence ( 1st day ) and then every other day , on 3rd , 5th , 7th , 9th , 13th and 15th day , until the death of the females . aculops allotrichus was observed at a lower frequency than c. hendersoni , as previous observations on asymmetrical sperm storage and spermatophore visitation by a. fockeui ( oldfield and newell 1973 ) , as well as asymmetrical sperm storage ( oldfield 1999 ) and guarding behaviour in female emergence sites of a. allotrichus ( michalska 1999 ) , all suggested that a. allotrichus females should pick up sperm from single spermatophores in their lives and mostly on the day of emergence . in both species , females were tested in the experimental cages , on the leaf arenas of 5.5 mm in diameter ( michalska 2000 ; michalska and shi 2004 ) , each with an aggregation of 810 fresh spermatophores . the spermatophore aggregations were formed by groups of 68 random males that were collected from the infested leaves of yucca plants or black locust trees . males deposited spermatophores under light conditions for 6 h. after that time , males were removed and the experiments started . to obtain females of c. hendersoni and a. allotrichus the quiescent female nymphs were collected from infested leaves of the appropriate host plant and then placed singly into the rearing cages ( in which females spent their whole life ) , onto leaf arenas 6.5 mm in diameter ( michalska 2000 ; michalska and shi 2004 ) . in both species , female quiescent nymphs are bigger than male quiescent nymphs and therefore they can be easily distinguished from each other . the ethological observations were carried out the next day , in the early afternoon , after all the females had emerged . the females were then transferred into experimental cages , placed in the centre of leaf arenas and observed for the next half hour . at each observation , the number and time of spermatophore visitations were recorded . for 11 spermatophore visitations by c. hendersoni and 7 visitations of spermatophores by a. allotrichus the time of mounting a spermatophore , lying on a spermatophore head and dismounting the observations were made at 50100 magnification under a dissecting microscope fitted with a cold light source olympus highlight 3100 ( equipped with a halogen reflector lamp ; colour temperature : 3,090 k ) . after each observation was completed , the females were transferred back to the rearing cages with new , fresh leaves where the females were maintained until the next observations . every day , mites were manipulated using an eye - lash glued onto the tip of a wooden stick . mites and cages were kept in plastic trays lined with wet cotton , in a sanyo growth chamber at 26 c , 8589 % rh and 16/8 l / d photoperiod . i examined spermathecae for the presence of sperm in ten females of c. hendersoni and a. allotrichus , which had been randomly chosen from a mite population , and in four females of c. hendersoni and five females of a. allotrichus , which had been exposed to spermatophores just after emergence . newly emergent females were obtained from quiescent female nymphs and released to the experimental cages with 810 spermatophores , following the methods i have described above . the females of c. hendersoni were inspected only after a half - hour - long observation , during which the females visited 23 spermatophores . by contrast , a. allotrichus females were examined immediately after the visitation of just one spermatophore . to examine spermathecae for the presence of sperm , the oldfield et al . both , random and newly inseminated females were put singly into a drop of saline solution ( 0.9 % nacl ) , in a dorso - ventral position . their caudal part was then cut off using a needle and the acaudal part was covered by the coverslip . coverslips were gently pressed so most of the body content was extruded through the place of the amputated rear body end . spermathecae were then inspected for the presence of non - motile , ellipsoidal sperm cells ca . similar sperm cells have been observed in a drop of saline solution , into which spermatophores of both species were placed and to which sperm from spermatophores was released ( michalska 2011 ; michalska k. , unpubl . ) . spermathecae were examined under a phase - contrast dissecting microscope ( olympus cx41 ) fitted with a colour view iii ccd camera and the imaging software for life science microscopy , cell ( olympus soft imaging system ) . i compared the time of visitation of the first and the second spermatophore using paired t test , and the time of visitation of the first out of two spermatophores and the single spermatophore using a two - sample t test . the data were examined for normality and homogeneity of variance ( sokal and rohlf 1995 ) . in both species , most females changed their behaviour when they approached a spermatophore . at the distance of approximately one length of a female body from the spermatophore aggregation , females either stopped or markedly slowed down , approaching spermatophores on stiff legs. such behaviour was exhibited by 8 out of 9 females of c. hendersoni prior to the visitation of 18 out of 23 spermatophores and by 15 out of 17 females of a. allotrichus prior to the visitation of single spermatophores . for the rest of the tested females , one female of c. hendersoni , prior to the visitation of three spermatophores and five c. hendersoni females , prior to the visitation of one spermatophore , as well as two females a. allotrichus that visited single spermatophores , slowed down their walking only when they entered the spermatophore aggregation . within the aggregation females began to search for the females of both , c. hendersoni and a. allotrichus examined spermatophores in two ways : ( 1 ) they tapped the spermatophore head with the first pair of legs and/or touched the sperm reservoir using their tarsal claws or ( 2 ) they crouched down , while walking over a spermatophore , and rubbed up against it using their genital cover - flaps . in both species , the visitation of a spermatophore consisted of three phases : ( 1 ) mounting , ( 2 ) lying on the spermatophore head , and ( 3 ) dismounting ( fig . 1 ) . when mounting a spermatophore , females brought a spermatophore closer to them with their front legs , and keeping it in place they moved forward over it . then the females swung their bodies from side to side , as if they tried to fit their genital opening with the sperm reservoir placed within the spermatophore head . in c. hendersoni , the mounting phase lasted on average 11.45 3.19 s ( n = 11 ; min max : 335 ) and in a. allotrichus , 10.5 3.19 s ( n = 7 ; min max : 420 ) . finally , the females of both species became motionless on the spermatophore head with their legs hanging loosely ( fig . 1 ) . in this phase some females waved their legs from time to time . c. hendersoni females spent on average 39.09 7.86 s ( n = 11 ; min max : 396 ) lying on spermatophores while a. allotrichus on average 115.38 35.95 s ( n = 7 ; min max : 12228 ) s. dismounting the spermatophore took a female of c. hendersoni on average 4.55 0.41 s ( n = 11 ; min max : 37 ) and a. allotrichus , 5.26 0.49 s ( n = 7 ; min max : 37 ) . first , the female swung her body vigorously from side to side and then she moved forward slowly until a spermatophore sprang off , returning to a perpendicular position on the leaf ( fig . 1 ) . after dismounting , females of both species usually stopped in the close vicinity of the spermatophore and began feeding . however , some females could also exhibit a sort of excitation with the also , after feeding females of c. hendersoni and a. allotrichus did not abandon the spermatophore aggregation immediately . they returned to the aggregation several times , making greater and greater circles until they finally went away . the females that no longer showed interest in spermatophores walked through the aggregation fast , without stopping and examining the spermatophores.fig . an arrow indicates the direction of female movement phases of spermatophore visitation by an eriophyoid female . an arrow indicates the direction of female movement females of c. hendersoni lived on average 5.67 0.85 days ( n = 12 ; min max : 212 ) . one out of the twelve tested females was not interested in spermatophores and died on the second day of her life . females visited spermatophores multiply , mostly day after day , on average 1.54 0.3 spermatophores per day ( n = 12 ; min max : 06 ) ( fig . the visitation of the spermatophore lasted on average 51.69 2.38 s ( n = 97 ; min max : 10120 ) . four females started to visit spermatophores as soon as the first day , and seven other females on the second day after emergence . the other females usually examined all spermatophores in an aggregation before the final visitation of the preferred spermatophores . in two females , a sort of excitation about three used spermatophores was also observed.fig . 2the average and maximum number of spermatophores visited by cecidophyopsis hendersoni females during the successive days of their lifetimes . in brackets number of females that were observed each day the average and maximum number of spermatophores visited by cecidophyopsis hendersoni females during the successive days of their lifetimes . in brackets number of females that were observed each day five out of twelve females laid eggs , on average 0.12 0.05 eggs per day , 13 eggs during their whole lifetime . the female that lived for the longest period of time ( 12 days ) , visited spermatophores every day except the first , seventh and twelfth days . aculops allotrichus females lived on average 5.84 0.79 days ( n = 25 ; min max : 115 ) . only one female did not show any interest in spermatophores , and she died on the second day after emergence . 15 females visited spermatophores on the first day after emergence , 6 females did so on the third day and 3 females visited spermatophores only on the fifth day after emergence . others , before mounting a spermatophore , examined a few or all the other spermatophores in the aggregation . for 4 of the 5 females that visited the first approached spermatophore , this spermatophore was also the only one they mounted in their lifetime , while for the fifth female it was one of the two spermatophores she visited in her lifetime . in total , 16 out of 24 females visited only one spermatophore in their lifetime ( they were not interested in spermatophores in the days following their exposure to spermatophores ) , while 8 females , which is one - third of all those tested , visited two spermatophores in their lives . the mean time of spermatophore visitations was 115.74 14.51 s ( n = 31 ; min max : 20315 ) . when females visited two spermatophores in their lifetime , either they mounted them both on the same day during the half hour exposure to spermatophores ( six females ) , or the visitation of the second spermatophore occurred at the other exposure day ( two females ) , at an interval of 16 days . visitation of the first and the second spermatophore consisted of similar phases of mounting , lying on a spermatophore and dismounting . also , the time of visitations of the first and the second spermatophore was similar ( paired - sample t test : n = 8 ; t = 1.15371 ; p = 0.29 ) ( table 1 ) . moreover , the time of the visitation of the first out of two spermatophores did not differ significantly from that spent on the visitation of the single spermatophore in a female lifetime ( two - sample t test : n1 = 15 ; n2 = 8 ; t = 1.31292 ; p = 0.20 ) ( table 1 ) . a similar result was obtained when four cases of females , which visited a single spermatophore on the first day of the exposure to spermatophores but died before the second exposure day , at which they theoretically could have visited the second spermatophore , were excluded from the analysis ( two - sample t test : n1 = 11 ; n2 = 8 ; t = 1.32172 ; p = 0.20).table 1the times of single and double visitations of spermatophores by females of aculops allotrichus single visitation of spermatophoresdouble visitation of spermatophores1st spermatophore2nd spermatophoremean se time ( s)129.0 23.2783.75 17.13122.88 31.34minimum and maximum time ( s)203153517025280number of observations15 8 8 the times of single and double visitations of spermatophores by females of aculops allotrichus ten out of 24 females laid eggs during the experiment . one female laid an egg on the first day while the others , at the earliest , on the 4th day after emergence . they lived for six or more days and oviposited at the rate , on average , of 0.25 0.03 eggs per day , from one to six eggs during their whole lifetime . nine females laid eggs only after they visited a spermatophore , while one female already deposited an egg before spermatophore visitation . in all females of c. hendersoni , either randomly chosen from a population ( n = 10 ) or inspected just after the visitation of 23 spermatophores ( n = 4 females ) , sperm were present in both spermathecae ( fig . 3 ) . by contrast , all a. allotrichus females , regardless of whether they were random ( n = 10 ) or just after the visitation of a single spermatophore ( n = 5 ) , had spermatozoa only in one , the left or right , sac while the other sac remained empty ( fig . 4 ) . in both species , both left ( ls ) and right ( rs ) spermatheca filled with spermatozoafig . one spermatheca ( ls ) is filled with sperm and the other spermatheca ( rs ) is empty photomicrograph of the paired spermatecae and symmetrical sperm storage in cecidophyopsis hendersoni . both left ( ls ) and right ( rs ) spermatheca filled with spermatozoa photomicrograph of the paired spermatecae and asymmetrical sperm storage in aculops allotrichus . one spermatheca ( ls ) is filled with sperm and the other spermatheca ( rs ) is empty in both species , most females changed their behaviour when they approached a spermatophore . at the distance of approximately one length of a female body from the spermatophore aggregation , females either stopped or markedly slowed down , approaching spermatophores on stiff legs. such behaviour was exhibited by 8 out of 9 females of c. hendersoni prior to the visitation of 18 out of 23 spermatophores and by 15 out of 17 females of a. allotrichus prior to the visitation of single spermatophores . for the rest of the tested females , one female of c. hendersoni , prior to the visitation of three spermatophores and five c. hendersoni females , prior to the visitation of one spermatophore , as well as two females a. allotrichus that visited single spermatophores , slowed down their walking only when they entered the spermatophore aggregation . within the aggregation females began to search for the females of both , c. hendersoni and a. allotrichus examined spermatophores in two ways : ( 1 ) they tapped the spermatophore head with the first pair of legs and/or touched the sperm reservoir using their tarsal claws or ( 2 ) they crouched down , while walking over a spermatophore , and rubbed up against it using their genital cover - flaps . in both species , the visitation of a spermatophore consisted of three phases : ( 1 ) mounting , ( 2 ) lying on the spermatophore head , and ( 3 ) dismounting ( fig . 1 ) . when mounting a spermatophore , females brought a spermatophore closer to them with their front legs , and keeping it in place they moved forward over it . then the females swung their bodies from side to side , as if they tried to fit their genital opening with the sperm reservoir placed within the spermatophore head . in c. hendersoni , the mounting phase lasted on average 11.45 3.19 s ( n = 11 ; min max : 335 ) and in a. allotrichus , 10.5 3.19 s ( n = 7 ; min max : 420 ) . finally , the females of both species became motionless on the spermatophore head with their legs hanging loosely ( fig . 1 ) . in this phase some females waved their legs from time to time . c. hendersoni females spent on average 39.09 7.86 s ( n = 11 ; min max : 396 ) lying on spermatophores while a. allotrichus on average 115.38 35.95 s ( n = 7 ; min max : 12228 ) s. dismounting the spermatophore took a female of c. hendersoni on average 4.55 0.41 s ( n = 11 ; min max : 37 ) and a. allotrichus , 5.26 0.49 s ( n = 7 ; min max : 37 ) . first , the female swung her body vigorously from side to side and then she moved forward slowly until a spermatophore sprang off , returning to a perpendicular position on the leaf ( fig . 1 ) . after dismounting , females of both species usually stopped in the close vicinity of the spermatophore and began feeding . however , some females could also exhibit a sort of excitation with the also , after feeding females of c. hendersoni and a. allotrichus did not abandon the spermatophore aggregation immediately . they returned to the aggregation several times , making greater and greater circles until they finally went away . the females that no longer showed interest in spermatophores walked through the aggregation fast , without stopping and examining the spermatophores.fig . an arrow indicates the direction of female movement phases of spermatophore visitation by an eriophyoid female . females of c. hendersoni lived on average 5.67 0.85 days ( n = 12 ; min max : 212 ) . one out of the twelve tested females was not interested in spermatophores and died on the second day of her life . females visited spermatophores multiply , mostly day after day , on average 1.54 0.3 spermatophores per day ( n = 12 ; min max : 06 ) ( fig . 2 ) . the visitation of the spermatophore lasted on average 51.69 2.38 s ( n = 97 ; min max : 10120 ) . four females started to visit spermatophores as soon as the first day , and seven other females on the second day after emergence . the other females usually examined all spermatophores in an aggregation before the final visitation of the preferred spermatophores . in two females , a sort of excitation about three used spermatophores was also observed.fig . 2the average and maximum number of spermatophores visited by cecidophyopsis hendersoni females during the successive days of their lifetimes . in brackets number of females that were observed each day the average and maximum number of spermatophores visited by cecidophyopsis hendersoni females during the successive days of their lifetimes . in brackets number of females that were observed each day five out of twelve females laid eggs , on average 0.12 0.05 eggs per day , 13 eggs during their whole lifetime . the female that lived for the longest period of time ( 12 days ) , visited spermatophores every day except the first , seventh and twelfth days . aculops allotrichus females lived on average 5.84 0.79 days ( n = 25 ; min max : 115 ) . only one female did not show any interest in spermatophores , and she died on the second day after emergence . 15 females visited spermatophores on the first day after emergence , 6 females did so on the third day and 3 females visited spermatophores only on the fifth day after emergence . others , before mounting a spermatophore , examined a few or all the other spermatophores in the aggregation . for 4 of the 5 females that visited the first approached spermatophore , this spermatophore was also the only one they mounted in their lifetime , while for the fifth female it was one of the two spermatophores she visited in her lifetime . in total , 16 out of 24 females visited only one spermatophore in their lifetime ( they were not interested in spermatophores in the days following their exposure to spermatophores ) , while 8 females , which is one - third of all those tested , visited two spermatophores in their lives . the mean time of spermatophore visitations was 115.74 14.51 s ( n = 31 ; min max : 20315 ) . when females visited two spermatophores in their lifetime , either they mounted them both on the same day during the half hour exposure to spermatophores ( six females ) , or the visitation of the second spermatophore occurred at the other exposure day ( two females ) , at an interval of 16 days . visitation of the first and the second spermatophore consisted of similar phases of mounting , lying on a spermatophore and dismounting . also , the time of visitations of the first and the second spermatophore was similar ( paired - sample t test : n = 8 ; t = 1.15371 ; p = 0.29 ) ( table 1 ) . moreover , the time of the visitation of the first out of two spermatophores did not differ significantly from that spent on the visitation of the single spermatophore in a female lifetime ( two - sample t test : n1 = 15 ; n2 = 8 ; t = 1.31292 ; p = 0.20 ) ( table 1 ) . a similar result was obtained when four cases of females , which visited a single spermatophore on the first day of the exposure to spermatophores but died before the second exposure day , at which they theoretically could have visited the second spermatophore , were excluded from the analysis ( two - sample t test : n1 = 11 ; n2 = 8 ; t = 1.32172 ; p = 0.20).table 1the times of single and double visitations of spermatophores by females of aculops allotrichus single visitation of spermatophoresdouble visitation of spermatophores1st spermatophore2nd spermatophoremean se time ( s)129.0 23.2783.75 17.13122.88 31.34minimum and maximum time ( s)203153517025280number of observations15 8 8 the times of single and double visitations of spermatophores by females of aculops allotrichus ten out of 24 females laid eggs during the experiment . one female laid an egg on the first day while the others , at the earliest , on the 4th day after emergence . they lived for six or more days and oviposited at the rate , on average , of 0.25 0.03 eggs per day , from one to six eggs during their whole lifetime . nine females laid eggs only after they visited a spermatophore , while one female already deposited an egg before spermatophore visitation . in all females of c. hendersoni , either randomly chosen from a population ( n = 10 ) or inspected just after the visitation of 23 spermatophores ( n = 4 females ) , sperm were present in both spermathecae ( fig . 3 ) . by contrast , all a. allotrichus females , regardless of whether they were random ( n = 10 ) or just after the visitation of a single spermatophore ( n = 5 ) , had spermatozoa only in one , the left or right , sac while the other sac remained empty ( fig . 4 ) . in both species , 3photomicrograph of the paired spermatecae and symmetrical sperm storage in cecidophyopsis hendersoni . both left ( ls ) and right ( rs ) spermatheca filled with spermatozoafig . one spermatheca ( ls ) is filled with sperm and the other spermatheca ( rs ) is empty photomicrograph of the paired spermatecae and symmetrical sperm storage in cecidophyopsis hendersoni . both left ( ls ) and right ( rs ) spermatheca filled with spermatozoa photomicrograph of the paired spermatecae and asymmetrical sperm storage in aculops allotrichus . one spermatheca ( ls ) is filled with sperm and the other spermatheca ( rs ) is empty in this study , visitations of spermatophores by a. allotrichus and c. hendersoni consisted of similar phases of mounting , lying on a spermatophore head and dismounting , which were also observed in other eriophyoid species i.e. a. fockeui , a. schlechtendali , e. pyri or p. oleivora ( oldfield and newell 1973 ; oldfield 1988 ; oldfield and michalska 1996 ) . however , while c. hendersoni females were polyandrous and visited spermatophores almost every day , from the day of emergence until death , in a. allotrichus , the majority of females visited spermatophores once , and the others at most twice in their life . previous investigations ( oldfield 1973 ) suggested that eriophyoid species storing sperm symmetrically should become inseminated from two or more spermatophores in their lives . this relationship has been confirmed for the first time in this study . the females of c. hendersoni visited several spermatophores in their lifetimes and those examined just after the visitation of 23 spermatophores , stored sperm in both spermathecae . by contrast , a. allotrichus females deposit spermatozoa only in one spermathecae ( oldfield 1999 ) , as was also revealed in the microscopic and behavioural observations in this study . however , nearly one - third of females visited two spermatophores , instead of one , in their lives . both the visitation of the first and the second spermatophore consisted of similar phases of mounting , lying on a spermatophore and dismounting ; moreover , the time spent on the visitation of the second spermatophore did not differ significantly from that of the first spermatophore and was also similar to the visitation time of a single spermatophore in a female life . this suggests that a. allotrichus females may pick up sperm from two spermatophores . in this study , i examined the spermathecae of a. allotrichus females that visited just one , not two , spermatophores in their lives , and they all stored sperm asymmetrically . moreover , all females i collected randomly from a population , among which some individuals might have picked up sperm from two spermatophores , also exhibited asymmetrical sperm storage . this is in disagreement with the previous findings on a. fockeui and other eriophyoids ( oldfield et al . 1972 ; oldfield and newell 1973 ; oldfield 1973 ; oldfield and michalska 1996 ) suggesting that species with asymmetrical sperm storage should become inseminated from only one spermatophore in their lifespan . in the study by oldfield and newell ( 1973 ) , all ten virgin a. fockeui females promptly visited and become inseminated by single spermatophores , but they failed to visit any fresh spermatophores in the following trials ( on the 4 , 8 , 12 and 16th days of their adult lives ) , even when they became old and started to produce male offspring only ( which indicates that sperm stores have already run out in these haplodiploids ) . in the other study ( oldfield et al . 1972 ) , however , some newly inseminated females mounted a second spermatophore , but for a few seconds only . oldfield ( personal communication ) argued that such mountings lasted too short a time to facilitate sperm transfer . also in this study , in a. allotrichus and c. hendersoni , short , only 1020 s long visitations of spermatophores , were recorded . it is likely that during mounting the females conclusively examined the quality of a spermatophore and abandoned it quickly ( without insemination or picking up only some portion of sperm ) when it turned out to be of lower quality or inappropriate . one can not exclude , however , the possibility that some females simply failed to transfer sperm properly from the spermatophore . such a case was described in the sex dissociated springtail orchesella cincta ( l. ) , in which females between moultings mostly pick up sperm from a single spermtophore ( gols et al . double mated female revealed that only the visitation of the second spermatophore had been successful . the authors did not exclude , however , the possibility that the female collected sperm from two spermatophores deposited by the same male , in which case the double insemination could not have been detected . it must be stressed that in eriophyoids , quick visitation of a spermatophore may be also a consequence of female excitement after insemination and may lead to the prolonged molesting of some used spermatophores observed in this study . in many species of insects and mites , females that were previously considered to mate only once in their lifetime turned out to re - mate with a certain frequency ( helle 1967 ; rev . eberhard 1985 ; potter and wrench 1978 ; ridley 1988 ; khanh et al . 2005 ; burton - chellew et al . benefits arising from re - mating and polyandry , e.g. sperm replenishment , nutrient donation , genetic diversity of offspring , sperm competition and good genes, as described for copulating females ( rev . hosken and stockley 2003 ) , might be similar for females from sex dissociated taxa . for example , the number of spermatozoa per spermatophore varies greatly among a. allotrichus males ( michalska 2011 ) . thus , a. allotrichus females could have visited the second spermatophore if they did not obtain enough sperm from the first spermatophore to fertilize their eggs . eriophyoid females may also accomplish double or multiple insemination to increase the genetic diversity of their offspring ( and so avoid the risk that all of their offspring will be fathered by a male of bad quality ) . however , repeated visitations of spermatophores can also incur some cost of time and energy that the female has to devote to seeking fresh spermatophores . cecidophyopsis hendersoni form very dense , multi - generational populations on evergreen yucca leaves ( michalska and shi 2004 ; michalska k. , pers . obs . ) , in which the chances that a female will encounter a fresh spermatophore appears to be relatively high . by contrast , a. allotrichus lives in a much lower density ; leaves of the black locust tree quickly become rusted by mite feeding and females have to seek new young leaves for lying eggs . in contrast to females , males usually stay on older leaves until the quiescent female nymphs are present and deposit spermatophores beside them ( michalska k. , pers . thus , females would wait several days on the young leaves for new generation males and their spermatophores . however , when an a. allotrichus female emerges she is usually surrounded with spermatophores of various males ( as a result of joint - guarding ) ( michalska et al . 2010 ) , so the female can accomplish double insemination without the wastage of time and energy involved in searching for spermatophores . indeed , as this study revealed , in a. allotrichus , the majority of re - mating females visited the second spermatophore on the same emergence day , within the short time of a 30 min - long observation . it is likely that the receptivity of a. allotrichus females depends on the amount of secretions contained in spermatophores , and if they do not receive enough of those substances or if their amount in a female spermatheca declines ( e.g. with female aging ) , females may re - mate . such a phenomenon was observed in the mediterranean fruit fly ceratitis capitata ( chapman et al . 1988 ; mossinson and yuval 2003 ) . in this species , females that chose to re - mate possessed fewer sperm in their spermatheca than those that did not choose to re - mate ( mossinson and yuval 2003 ) . interestingly , nearly 50 % of females re - mated soon ( on the 2nd day ) after the first mating while only 010 % on the 12th day ( chapman et al . 1988 ) . this study also showed that females of both species as a rule examined several spermatophores before the mounting of one . it is likely that eriophyoid females need some stimulation from the other spermatophores before mounting the spermatophore and picking up sperm from it . however , one can not also exclude the possibility that the eriophyoid females simply chose among the spermatophores and mounted that preferred one . it must be stressed that receptive females of the other eriophyoid , a. fockeui ( oldfield et al . 1970 ) can distinguish between old and fresh spermatophores and do not pick up sperm from spermatophores that are more than 3 days old . thus , in future work , preference tests should be done to determine whether eriophyoid females are choosy about spermatophores and whether their choosiness is based on the different genetic quality of spermatophores or , perhaps , the various ages of spermatophores they encounter . good genes on the basis of some spermatophore traits , especially their pheromones , as well as other substances within a sperm drop ( proctor 1998 ; zizzari et al . 2009 ) . in the eriophyoid spermatophores , the presence of pheromones has not yet been confirmed , although some behavioural observations , e.g. female response toward spermatophores from a distance , as well as discrimination between old and new spermatophores , strongly suggest it ( michalska et al . 2010 ) . female choice has been demonstrated in the sex dissociated springtail o. cincta ( hedlund et al . females chose spermatophores of a particular male and through this choice they gained more fertile male offspring . in future work on a. allotrichus and c. hendersoni , paternity analysis of female offspring should be carried out to confirm that the females which visited two or more spermatophores did indeed become inseminated by sperm from those males . in this context , the mechanism of sperm competition could also have been determined . in insects and mites , sperm from different males mix randomly in spermatheca , or there is sperm precedence of the first or , most frequently , the last male ( simmons 2001 ) . also , the reproductive success of a non - pairing male may depend on the number of sperm per spermatophore or the sequence of visitations of his spermatophores in relation to the spermatophores of competitor males . moreover , the effectiveness of mating may also rely on the interval between the first and the second insemination . in tetranychus urticae ( helle 1967 ) or t. kanzawai ( oku 2008 ) , for example , the first copulation is effective . first male sperm precedence appears to be adaptive in this case , since , similarly to a. allotrichus , tetranychid males spend time and energy on the prolonged guarding of pre - emergent females . however , when the first copulation by t. urticae males was interrupted or the time between the first and the second copulation was shorter than 24 h , the second insemination was effective in some cases ( potter and wrench 1978 ; satoh et al . 2001 ) . similarly , one can not exclude the possibility of some differences in the effectiveness of the repeated inseminations in a. allotrichus between females that accomplish the second insemination just after the visitation of the first spermatophore and those that visited the second spermatophore much later in their lifetimes . the volumetric comparison of the sperm reservoir and spermatheca in three species of eriophyoids a. cynondonis , a. tulipae and t. ehamnni that store sperm symmetrically revealed that each spermatheca may be filled with sperm from 1 to 2 spermatophores ( oldfield 1973 ) . in this study , c. hendersoni females clearly picked up sperm from many more spermatophores that seem to be required for egg fertilization . they laid a maximum of three eggs and visited up to 33 spermatophores in their lifetime , a maximum of 6 spermatophores at one observation . the females that did not lay eggs also visited up to 7 spermatophores in their lifetime . thus , one can not exclude the possibility that in this species females systematically remove or digest the old sperm and refill their spermatheca with new sperm supplies . numerical and functional elimination of sperm by females at mating is a widespread phenomenon among insect species in which females and males copulate ( eberhard 1996 ; dickinson 1997 ; showalter 2006 ; pai and bernasconi 2007 ) . females can digest or extrude sperm after copulation , block sperm transfer into spermatheca , reduce sperm viability via the hostile environment of their reproductive tract etc . females can benefit from sperm removal by promoting sperm competition , manipulating paternity , obtaining nutrition , eliminating sperm that bear mutations , etc . thus , in future studies , paternity analysis is required to confirm multiple inseminations by c. hendersoni females and the possible mechanism of sperm elimination in this species .

eriophyoid females store sperm either asymmetrically in one spermatheca , or symmetrically in both spermathecae . previous studies have suggested that species in which females store sperm asymmetrically pick up sperm from only one spermatophore , while those with symmetrical sperm storage pick up sperm from two or more spermatophores during their lifetime . the aim of this study was to examine spermatophore visitation behaviour and symmetry of sperm storage in aculops allotrichus from the black locust tree and cecidophyopsis hendersoni from the yucca . this would indicate monandry or polyandry in these species . in both eriophyoids , the spermatophore visitation consisted of three phases : mounting , lying on the spermatophore and dismounting . aculops allotrichus stored sperm asymmetrically . however , nearly one - third of the observed females visited two spermatophores , rather than only one in their lives . when a. allotrichus females visited two spermatophores they spent a similar amount of time at the first and at the second visitation . also , the times of visitation of the first of the two spermatophores and the single spermatophore in a female lifetime did not differ significantly . this would suggest that apart from monandry , double insemination also occurs in this species . by contrast , c. hendersoni females were polyandrous . they stored sperm symmetrically and visited several spermatophores , on average 1.54 ( max 6 ) per day , and up to 33 spermatophores in their lives . the benefits of repeated spermatophore visitation and the possible mechanisms of sperm storage in both species are discussed .

calpain belongs to the group of intracellular cysteine proteases that need calcium ions for their activation ( nakagawa and yuan , 2000 ) . even though their physiological role is still incompletely understood , several lines of evidence supports their implication in a variety of calcium - regulated cellular processes such as signal transduction , cell proliferation , cell cycle progression , differentiation , apoptosis , membrane fusion , and platelet activation ( sorimachi and ishiura , 1997 ; goll et al . , 2003 ; suzuki et al . , 2003 the disturbance in the regulation of calpain activity also contributes to various pathological conditions such as neuronal degeneration and alzheimer 's disease ( huang and wang , 2001 ; suzuki et al . , 2003 ) . calpain 1 ( microcalpain or -calpain ) and calpain 2 ( milli calpain or m - calpain ) are among the best characterized calpains that show widest tissue distribution among the members of this family . these calcium - dependent cysteine proteases play a significant role in a huge set of intracellular processes ( carafoli and molinari , 1998 ; wang , 2000 ; huang and wang , 2001 ) particularly in the selective proteolysis of factors involved in the cell cycle ( raynaud et al . , 2004 ) , during apoptosis in association with caspases ( yamashima , 2004 ) or in the cleavage of membrane - cytoskeleton complexes during cell motility phases ( lebart and benyamin , 2006 ) . there is evidence that the increased production of free radicals and modifications in detoxifying enzymes also lead to apoptosis in cultured rat forebrain neurons exposed to transient hypoxia and can be prevented by antioxidants ( ishige et al . neutraceutical ( polyphenolic phytochemicals i.e. , flavonoids ) such as quercetin has gained much attention for its use as a potent drug targeting hypoxia and ischemic insult . flavonoids have been reported to exhibit antioxidant activity by three mechanisms : ( 1 ) increasing intracellular glutathione ( gsh ) levels , ( 2 ) scavenging reactive oxygen species ( ros ) , ( 3 ) blocking ca influx ( ishige et al . , 2001 ) . the anti - oxidant properties of the flavonoids have shown neuroprotective effects in the brain . in spite of its in vitro anti - oxidant property and cytoprotective activity in cell cultures , it has also been reported that quercetin showed beneficial effects in ischemia mediated cerebral injury ( dajas et al . , 2001 ) . the tremendous potential of quercetin to inhibit cancer is also well documented ( seufi et al . , 2009 ) . therefore , in the present study , the effect of quercetin on -calpain was investigated by molecular docking and molecular dynamics simulation as tools . 2g8j ) was obtained from research collaboratory for structural bioinformatics ( rcsb ) protein data bank ( cuerrie et al . , 2006 ) . the structures and smile notation of ligands quercetin was generated by acdlab free software ( www.acdlabs.com ) and pdb file was built by online smile translator ( https://cactus.nci.nih.gov/translate ) . all hetero atoms , including water molecules , except calcium were removed from pdb file of calpain . addition of hydrogen atoms to the calpain molecule and merging of all non polar hydrogen atoms were performed using autodock program . short range vanderwaal and electrostatic interactions , hydrogen bonding , entropy losses were considered for energy based autodock scoring function ( morris et al . , 1998 ; sudhamalla et al . , 2010 ) . the lamarkian ga parameters used in the study were : number of runs , 30 ; population size , 200 ; maximum no . of evals , 2,500,000 ; number of generations , 27,000 ; rate of gene mutation , 0.02 and crossover rate , 0.8 . blind docking was carried out using grid size 126 , 126 , 126 along the x , y and z axes with 0.0469 nm spacing . molecular dynamics ( md ) simulation of the complex was carried out with the gromacs 4.5.4 package ( http://www.gromacs.org/ ) with gromos96 43a1 force field ( van gunsteren et al . , 1996 ; lindah et al . , 2001 ) . lowest binding energy obtained from autodock i.e. , docking conformation with most negative denomination was taken as initial conformation to carry out the molecular dynamics simulation study . the gromacs program was used to generate topology parameters of proteins while the dundee prodrg server ( http://davapc1.bioch.dundee.ac.uk ) was used to build the topology parameters of quercetin . the complex was immersed in an octahedron box of extended simple point charge ( spc ) water molecules ( grand and owen , 1991 ; schuler et al . , the solvated system included -calpain , quercetin and water was neutralized by adding 8 na ions . energy minimization was performed using the steepest descent method of 10,000 steps followed by the conjugate gradient method for 10,000 steps to release conflicting contacts . as molecular dynamics simulation studies consist of equilibration and production phases therefore in the first stage of equilibration , the solute ( protein , counter ions , and quercetin ) was fixed and the position - restrained dynamics simulation of the system was intervened . lastly , the full system was subjected to md production run at 26.85c temperature and 1 bar pressure with isotropic molecule based scaling for 10,000 ps . 2g8j ) was obtained from research collaboratory for structural bioinformatics ( rcsb ) protein data bank ( cuerrie et al . , 2006 ) . the structures and smile notation of ligands quercetin was generated by acdlab free software ( www.acdlabs.com ) and pdb file was built by online smile translator ( https://cactus.nci.nih.gov/translate ) . all hetero atoms , including water molecules , except calcium were removed from pdb file of calpain . addition of hydrogen atoms to the calpain molecule and merging of all non polar hydrogen atoms were performed using autodock program . short range vanderwaal and electrostatic interactions , hydrogen bonding , entropy losses were considered for energy based autodock scoring function ( morris et al . , 1998 ; sudhamalla et al . , 2010 ) . the lamarkian ga parameters used in the study were : number of runs , 30 ; population size , 200 ; maximum no . of evals , 2,500,000 ; number of generations , 27,000 ; rate of gene mutation , 0.02 and crossover rate , 0.8 . blind docking was carried out using grid size 126 , 126 , 126 along the x , y and z axes with 0.0469 nm spacing . molecular dynamics ( md ) simulation of the complex was carried out with the gromacs 4.5.4 package ( http://www.gromacs.org/ ) with gromos96 43a1 force field ( van gunsteren et al . , 1996 ; lindah et al . , 2001 ) . lowest binding energy obtained from autodock i.e. , docking conformation with most negative denomination was taken as initial conformation to carry out the molecular dynamics simulation study . the gromacs program was used to generate topology parameters of proteins while the dundee prodrg server ( http://davapc1.bioch.dundee.ac.uk ) was used to build the topology parameters of quercetin . the complex was immersed in an octahedron box of extended simple point charge ( spc ) water molecules ( grand and owen , 1991 ; schuler et al . , the solvated system included -calpain , quercetin and water was neutralized by adding 8 na ions . energy minimization was performed using the steepest descent method of 10,000 steps followed by the conjugate gradient method for 10,000 steps to release conflicting contacts . as molecular dynamics simulation studies consist of equilibration and production phases therefore in the first stage of equilibration , the solute ( protein , counter ions , and quercetin ) was fixed and the position - restrained dynamics simulation of the system was intervened . lastly , the full system was subjected to md production run at 26.85c temperature and 1 bar pressure with isotropic molecule based scaling for 10,000 ps . quercetin binds with the active site having higher negative binding energy of 28.74408 kj / mol with a ki value of 35.87 m . binding energy was calculated as the summation of four different energies in terms of intermolecular energy , total internal energy , torsional free energy and unbound system energy . the intermolecular energy includes van der waals , hydrogen bond , desolvation energy , and electrostatic energy . quercetin was found to be engaged in h - bonding with active site residues glu 72 , ser 206 , gln 109 and trp 298 as shown in figure 2 . interaction of quercetin with active site residues of -calpain . ( a ) position of quercetin in binding pocket ( having residues glu 72 , ser 206 , gln 109 and trp 298 ) of -calpain at dii domain for its inhibition . ( b ) interaction of quercetin with catalytic triad of calpain ( green color shows the interacting residue of calpain with quercetin ( rainbow color ) . ( c ) closer view of catalytic site ( having residues glu 72 , ser 206 , gln 109 and trp 298 ) occupied by quercetin in -calpain . ( d ) closer view of quercetin interacting with the side chain of active site residue ( yellow dashes indicate hydrogen bonding with active site residues ) . comparative docking studies resulting from docking with known potent synthetic inhibitor ( alpha - mercaptoacrylic acid derivative , i.e. , pd151746 and pd150606 ) as shown in figure 3 and table 1 , we revealed that the binding energies of pd151746 and pd150606 with -calpain were found to be significantly lower than that of quercetin which clearly indicates the strong inhibitory property of natural polyphenol quercetin . ( 1996 ) previously reported that alpha - mercaptoacrylic acid derivative such as pd151746 and pd150606 were selective nonpeptide cell - permeable calpain inhibitors and had a neuroprotective role . in comparative docking , pd151746 was engaged in h - bonding with active site residue arg 326 of -calpain with 17.82384 kj / mol binding energy and a ki value of 759.23 m whereas pd150606 was engaged in h - bonding with active site residue lys 79 of -calpain with 16.98704 kj / mol binding energy and a ki value of 1.05 mm ( table 1 ) . these findings suggest that the affinity of quercetin with -calpain active site as an inhibitor is higher than that of other synthetic inhibitors which enhances the applicability of quercetin as a potent calpain inhibitor against ischemic injury . on the basis of this comparative study and our previous in vitro study in which we explored the possible neuroprotective role of quercetin under different hypoxic conditions ( pandey et al . , 2013 ) , we further carried out a molecular dynamics simulation study involving -calpain - quercetin complex . interaction of pd150606 with active site residues of -calpain . ( a ) position of pd150606 bound to active site residue lys 79 of -calpain . ( b ) interaction of pd150606 with catalytic domain of calpain ( pink color shows the interacting residue of calpain with pd150606 ( rainbow color ) . ( c ) closer view of catalytic site occupied by pd151746 by forming h - bond with residue arg 326 ( green color shows the interacting residue of calpain with pd151746 ( rainbow color ) . ( d ) closer view of pd151746 interacting with the side chain of active site residue ( yellow dashes indicate hydrogen bonding with active site residue ) . comparative study with known potent synthetic inhibitors of -calpain -calpain - quercetin complex with 26.73576 kj / mol binding energy was obtained by molecular docking using autodock and further processed for a molecular dynamics simulation study . a molecular dynamics simulation study represents a suitable method for examining the transformations in gestures of residues / atoms that are exposed to structural and chemical changes . in the present study , we considered the time - dependent activities of molecular dynamics trajectories for -calpain and ligand including root mean square deviation ( rmsd ) for whole backbone atoms and ligand along with the average residue fluctuations of the residues ( rmsf ) . the rmsd of backbone atoms was calculated with respect to their initial conformation and -calpain - quercetin complex was changed with time to judge the effect of the binding of the ligand with their conformational stability of -calpain - quercetin complex throughout the simulation . the rmsd trajectories were captured at every 0.5 ps until the simulation time reached 10,000 ps . the rmsd trajectories were found to vary from 0.18 nm to 0.39 nm in the unbound state of protein whereas 0.21 nm to 0.29 nm in the bound state of protein i.e. , -calpain - quercetin complex . in case of unbound state of -calpain a sequential increase profile was observed at ~3,770 ps with slight up and down activities . after down movement for very small interval , sequential increase was observed again at 4,300 ps and reached to 0.38 nm at 10,000 ps . the rmsd analysis of -calpain - quercetin complex indicates the stability of complex with minor elevation in activity , i.e. , 0.35 nm on 5,400 ps and 0.29 nm at 10,000 ps while rmsd analysis of quercetin showed notable stability throughout the simulation as shown in figure 4 . plots of root mean square deviation ( rmsd ) of backbone of -calpain unbound ( green ) , -calpain complex ( black ) and quercetin ( red ) . the trajectories were captured at every 0.5 ps until the simulation time reached 10,000 ps . the thermodynamic stability of the complex was determined on the basis of the fluctuation in potential energy . the trajectories of potential energy were found to be higher as compared to unbound protein with persistent values of change up and down for the -calpain - quercetin complex during the entire simulation as shown in figure 5 . the number of h - bonds formed during md simulation between quercetin and -calpain was also calculated and variable profile was observed which fluctuated between 0 and 4 with an average value of 1.12 ( figure 6 ) . potential energy profile of -calpain ( green ) and -calpain - quercetin complex ( red ) during 10,000 ps molecular dynamics simulation . number of h - bonds formed between quercetin and -calpain during 10,000 ps molecular dynamics simulation . significant number of h - bonds is 3 for 10,000 ps . to define the effect of quercetin on the folding of -calpain , radius of gyration ( rg ) of -calpain and quercetin rg values of complexes were observed with approximate similarity with endless ups and downs during simulation run represented in figure 7 . rmsf of backbone residues from its time averaged position was also analyzed to distinguish the flexible zones of the proteins . we observed that -calpain - quercetin complex flourished distinctly higher fluctuations in relation to -calpain itself as shown in figure 8 . radius of gyration ( rg ) of -calpain unbound ( black ) and -calpain - quercetin complex ( red ) . root mean square fluctuations ( rmsf ) of -calpain backbone ( black ) and -calpain - quercetin backbone ( red ) . by reducing the dimensionality of the data obtained from a molecular dynamics simulation study , one can identify the configurational space that contains a few degrees of freedom in which an harmonic motion can occur . principal components analysis ( pca ) helps take the trajectory of a molecular dynamics simulation and extracts the dominant modes in the motion of the molecule ( van aalten et al . , 1995 ; the most important motions of the protein were extracted from the trajectory by principal component analysis of the cartesian coordinate covariance matrix , yielding eigenvectors and corresponding eigenvalues . the eigenvectors with the largest associated eigenvalues describes the indispensable subspace where most of the protein dynamics take place . the instant decay in the plot of sorted eigenvalues indicates that a small number of eigenvectors suffice to describe the motions of the -calpain i.e. , the protein backbone motion can be described by a few essential modes . principal component analysis was applied to the backbone atoms in quercetin bound and unbound -calpain . eigenvalues of the covariance matrix resulting from the simulations of the -calpain - quercetin complex and -calpain are shown in figure 9 . eigen values of the covariance matrix resulting from the simulations of the -calpain - quercetin complex ( black ) and -calpain ( red ) . the correlation matrix describes the linear correlation between any pairs of backbone atoms as they move around their regular location during dynamics . at a qualitative level , a positive correlation between two atoms reflects a concerted motion along the same direction , whereas a negative correlation indicates an opposite direction motion ( van aalten et al . analysis of correlation matrices of backbone revealed that quercetin binding reduces opposing correlated motion in -calpain as compared to unbound form ( figure 10 ) . cross - correlations give us an idea to identify the most correlated , rationally correlated and negatively correlated regions between fluctuated residues . in the backbone of -calpain docked with quercetin ( figure 10 ) , a highly negatively correlated motion was noticed as compared to the backbone of -calpain alone ( figure 10 ) which is due to the folding and conformational changes of hydrophobic groove of domain ii . a large number of negatively charged residues of this loop bound to the calcium ion in distant proximity have been reported ( strobl et al . our findings suggest that quercetin strongly binds to the hydrophobic dii domains and changes the structure with the induction of conformational changes . the color scale runs from blue to red ( 0.443 to 0.125 ) for -calpain whereas scale runs from blue to red ( 0.578 to 0.131 ) for quercetin docked -calpain . quercetin binds with the active site having higher negative binding energy of 28.74408 kj / mol with a ki value of 35.87 m . binding energy was calculated as the summation of four different energies in terms of intermolecular energy , total internal energy , torsional free energy and unbound system energy . the intermolecular energy includes van der waals , hydrogen bond , desolvation energy , and electrostatic energy . quercetin was found to be engaged in h - bonding with active site residues glu 72 , ser 206 , gln 109 and trp 298 as shown in figure 2 . interaction of quercetin with active site residues of -calpain . ( a ) position of quercetin in binding pocket ( having residues glu 72 , ser 206 , gln 109 and trp 298 ) of -calpain at dii domain for its inhibition . ( b ) interaction of quercetin with catalytic triad of calpain ( green color shows the interacting residue of calpain with quercetin ( rainbow color ) . ( c ) closer view of catalytic site ( having residues glu 72 , ser 206 , gln 109 and trp 298 ) occupied by quercetin in -calpain . ( d ) closer view of quercetin interacting with the side chain of active site residue ( yellow dashes indicate hydrogen bonding with active site residues ) . comparative docking studies resulting from docking with known potent synthetic inhibitor ( alpha - mercaptoacrylic acid derivative , i.e. , pd151746 and pd150606 ) as shown in figure 3 and table 1 , we revealed that the binding energies of pd151746 and pd150606 with -calpain were found to be significantly lower than that of quercetin which clearly indicates the strong inhibitory property of natural polyphenol quercetin . ( 1996 ) previously reported that alpha - mercaptoacrylic acid derivative such as pd151746 and pd150606 were selective nonpeptide cell - permeable calpain inhibitors and had a neuroprotective role . in comparative docking , pd151746 was engaged in h - bonding with active site residue arg 326 of -calpain with 17.82384 kj / mol binding energy and a ki value of 759.23 m whereas pd150606 was engaged in h - bonding with active site residue lys 79 of -calpain with 16.98704 kj / mol binding energy and a ki value of 1.05 mm ( table 1 ) . these findings suggest that the affinity of quercetin with -calpain active site as an inhibitor is higher than that of other synthetic inhibitors which enhances the applicability of quercetin as a potent calpain inhibitor against ischemic injury . on the basis of this comparative study and our previous in vitro study in which we explored the possible neuroprotective role of quercetin under different hypoxic conditions ( pandey et al . , 2013 ) , we further carried out a molecular dynamics simulation study involving -calpain - quercetin complex . interaction of pd150606 with active site residues of -calpain . ( a ) position of pd150606 bound to active site residue lys 79 of -calpain . ( b ) interaction of pd150606 with catalytic domain of calpain ( pink color shows the interacting residue of calpain with pd150606 ( rainbow color ) . ( c ) closer view of catalytic site occupied by pd151746 by forming h - bond with residue arg 326 ( green color shows the interacting residue of calpain with pd151746 ( rainbow color ) . ( d ) closer view of pd151746 interacting with the side chain of active site residue ( yellow dashes indicate hydrogen bonding with active site residue ) . -calpain - quercetin complex with 26.73576 kj / mol binding energy was obtained by molecular docking using autodock and further processed for a molecular dynamics simulation study . a molecular dynamics simulation study represents a suitable method for examining the transformations in gestures of residues / atoms that are exposed to structural and chemical changes . in the present study , we considered the time - dependent activities of molecular dynamics trajectories for -calpain and ligand including root mean square deviation ( rmsd ) for whole backbone atoms and ligand along with the average residue fluctuations of the residues ( rmsf ) . the rmsd of backbone atoms was calculated with respect to their initial conformation and -calpain - quercetin complex was changed with time to judge the effect of the binding of the ligand with their conformational stability of -calpain - quercetin complex throughout the simulation . the rmsd trajectories were captured at every 0.5 ps until the simulation time reached 10,000 ps . the rmsd trajectories were found to vary from 0.18 nm to 0.39 nm in the unbound state of protein whereas 0.21 nm to 0.29 nm in the bound state of protein i.e. , -calpain - quercetin complex . in case of unbound state of -calpain a sequential increase profile was observed at ~3,770 ps with slight up and down activities . after down movement for very small interval , sequential increase was observed again at 4,300 ps and reached to 0.38 nm at 10,000 ps . the rmsd analysis of -calpain - quercetin complex indicates the stability of complex with minor elevation in activity , i.e. , 0.35 nm on 5,400 ps and 0.29 nm at 10,000 ps while rmsd analysis of quercetin showed notable stability throughout the simulation as shown in figure 4 . plots of root mean square deviation ( rmsd ) of backbone of -calpain unbound ( green ) , -calpain complex ( black ) and quercetin ( red ) . the trajectories were captured at every 0.5 ps until the simulation time reached 10,000 ps . the thermodynamic stability of the complex was determined on the basis of the fluctuation in potential energy . the trajectories of potential energy were found to be higher as compared to unbound protein with persistent values of change up and down for the -calpain - quercetin complex during the entire simulation as shown in figure 5 . the number of h - bonds formed during md simulation between quercetin and -calpain was also calculated and variable profile was observed which fluctuated between 0 and 4 with an average value of 1.12 ( figure 6 ) . potential energy profile of -calpain ( green ) and -calpain - quercetin complex ( red ) during 10,000 ps molecular dynamics simulation . number of h - bonds formed between quercetin and -calpain during 10,000 ps molecular dynamics simulation . significant number of h - bonds is 3 for 10,000 ps . to define the effect of quercetin on the folding of -calpain , radius of gyration ( rg ) of -calpain and quercetin rg values of complexes were observed with approximate similarity with endless ups and downs during simulation run represented in figure 7 . rmsf of backbone residues from its time averaged position was also analyzed to distinguish the flexible zones of the proteins . we observed that -calpain - quercetin complex flourished distinctly higher fluctuations in relation to -calpain itself as shown in figure 8 . radius of gyration ( rg ) of -calpain unbound ( black ) and -calpain - quercetin complex ( red ) . root mean square fluctuations ( rmsf ) of -calpain backbone ( black ) and -calpain - quercetin backbone ( red ) . by reducing the dimensionality of the data obtained from a molecular dynamics simulation study , one can identify the configurational space that contains a few degrees of freedom in which an harmonic motion can occur . principal components analysis ( pca ) helps take the trajectory of a molecular dynamics simulation and extracts the dominant modes in the motion of the molecule ( van aalten et al . in this methodology , the most important motions of the protein were extracted from the trajectory by principal component analysis of the cartesian coordinate covariance matrix , yielding eigenvectors and corresponding eigenvalues . the eigenvectors with the largest associated eigenvalues describes the indispensable subspace where most of the protein dynamics take place . the instant decay in the plot of sorted eigenvalues indicates that a small number of eigenvectors suffice to describe the motions of the -calpain i.e. , the protein backbone motion can be described by a few essential modes . principal component analysis was applied to the backbone atoms in quercetin bound and unbound -calpain . eigenvalues of the covariance matrix resulting from the simulations of the -calpain - quercetin complex and -calpain are shown in figure 9 . eigen values of the covariance matrix resulting from the simulations of the -calpain - quercetin complex ( black ) and -calpain ( red ) . the correlation matrix describes the linear correlation between any pairs of backbone atoms as they move around their regular location during dynamics . at a qualitative level , a positive correlation between two atoms reflects a concerted motion along the same direction , whereas a negative correlation indicates an opposite direction motion ( van aalten et al . , 1995 ) . analysis of correlation matrices of backbone revealed that quercetin binding reduces opposing correlated motion in -calpain as compared to unbound form ( figure 10 ) . cross - correlations give us an idea to identify the most correlated , rationally correlated and negatively correlated regions between fluctuated residues . in the backbone of -calpain docked with quercetin ( figure 10 ) , a highly negatively correlated motion was noticed as compared to the backbone of -calpain alone ( figure 10 ) which is due to the folding and conformational changes of hydrophobic groove of domain ii . a large number of negatively charged residues of this loop bound to the calcium ion in distant proximity have been reported ( strobl et al . , 2000 ) . our findings suggest that quercetin strongly binds to the hydrophobic dii domains and changes the structure with the induction of conformational changes . the color scale runs from blue to red ( 0.443 to 0.125 ) for -calpain whereas scale runs from blue to red ( 0.578 to 0.131 ) for quercetin docked -calpain . the myriad of calcium - dependent enzymes like neuronal nitric oxide synthase ( nnos ) , calpain , phospholipase , xanthine oxidase , ligases and dnases are activated due to elevation in the level of intraneuronal ca . these enzymes are involved in the synthesis of free radicals and catabolism of proteins , phospholipids and nucleic acids ( mehta et al . , 2007 ) . further , the inhibitory outcome of quercetin on calcium mediated rise in no levels ( pandey et al . , 2012 ) also supports the neuroprotective effect following hypoxic insult . an elevation in intraneuronal ca serves as a signal for the initiation of ca calmodulin and protein kinase c regulated neuronal nitric oxide synthase ( nnos ) . activation of nnos produces nitric oxide ( no ) which is a physiological mediator of vasodilation by elevating the level of 3,5-cyclic guanosine monophosphate ( cgmp ) in vascular smooth muscles ( cornwell et al . , 1994 ) . glutamate neurotoxicity might occur either due to the over activation of glutamate receptor or without activation of glutamate receptor that results in the ca influx through the voltage gated ca channels ( das et al . , these calcium influxes might help to activate calpain , a ca dependent cysteine protease and degrade the key cytoskeleton protein such as spectrin leading to apoptosis ( jatzke et al . cao et al . ( 2007 ) reported that calcium dysregulation is nearly a ubiquitous facet of calpain activation and neuronal cell death . under the hypoxic condition this in turn causes membrane lipid peroxidation and decreased mitochondrial membrane potential which leads to alterations in ca homeostasis , dna damage and apoptosis . lactic acidosis follows as a result of anaerobic metabolism due to which ca gets dissociated from calmodulin , further activating calpain and leading to neuronal damage and apoptosis . concurrently , in the other pathways , changed osmolarity and atp depletion due to hypoxia lead to necrosis . earlier studies also suggest that activation of acid sensing ion channels can lead to an activation of calpain during hypoxic insult ( aki et al . , 2002 ) . calpain activation has a prominent role in neuronal cell death either by the elevation of glutamate level or hypoxia induced oxidative stress mediated calcium influx . in the brain , calpain i ( -calpain ) is mainly neuronal , existing at high levels in the dendrites and cell bodies , whereas relatively higher levels of calpain ii ( m - calpain ) have been observed in axons and glia ( onizuka et al . , 1995 although calpain is also found outside cells yet its function is not known ( adachi et al . , calpain ii is generally activated in response to cellular signals , whereas calpain i is often active constitutively . the larger subunit of calpain i ( catalytic subunit ) consists of 4 domains ( d1d4 ) . domain d2 formed active site which was mainly composed of cys 115 , his 272 , asn 296 and trp residue at position 298 and these residues form a catalytic triad of calpain cysteine proteases ( khorchid and ikura , 2002 ; goll et al . we found that quercetin occupied the active site of calpain by the hydrogen binding with active side residue glu 72 , ser 206 , gln 109 , trp 298 during our docking study . the binding energy and ki value of quercetin - calpain complex advocate their tendency of stable binding . it has been already well documented from our previous study that quercetin has antioxidant and ca adaptable capacity against hypoxic injury ( wang et al . , 1996 ; khorchid and ikura , 2002 ; cuerrier et al . , 2006 ; khatri and man , 2013 ; pandey et al . , 2013 ; machado et al . , 2015 ; sundaramoorthy et al . , 2015 ; lalkovicov and danielisov , 2016 ; tafani et al . , 2016 ; quercetin having antioxidant and anti - inflammatory properties along with asic1a reducing capacity proves to be a future molecule of choice for ischemic stroke therapy ( cuerrier et al . , 2006 ; khatri and man , 2013 ; pandey et al . , 2013 ; machado et al . , 2015 ; sundaramoorthy et al . , 2015 ; lalkovicov and danielisov , 2016 ; tafani et al . , 2016 ; zeitouni et al . , 2016 ) . the comparative binding energies of quercetin with two other known calpain inhibitors ( pd151746 and pd150606 ) as shown in table 1 reveal that quercetin might act as a potential calpain inhibitor . the schematic representation of possible mode of neuroprotection of quercetin in hypoxic injury is shown in figure 11 ( cuerrier et al . , 2006 ; khatri and man , 2013 ; machado et al . , 2015 ; sundaramoorthy et al . , 2015 ; lalkovicov and danielisov , 2016 ; tafani et al . , 2016 ; zeitouni et al . , 2016 ) . lactic acidosis then occurs as a consequence of anaerobic metabolism due to which dissociated ca from the calmodulin activates calpain and leads to neuronal damage and apoptosis . taken together , the mechanism of stable quercetin - calpain complex was proposed in the present study . this stable complex was obtained by molecular dynamic stimulation resulting in 4 h - bonds between -calpain and quercetin at residues glu 72 , ser 206 , gln 109 and trp 298 in binding pocket with the binding energy of 28.74408 kj / mol . such results showed strong binding and inhibition of -calpain which is a potent target in our concern for hypoxia , thus providing full proof evidence to support our efforts . on the basis of these observations , it can be hypothesized that quercetin exhibits neuroprotective property by decreasing calpain activity against hypoxia mediated cellular insult .

the neuroprotective property of quercetin is well reported against hypoxia and ischemia in past studies . this property of quercetin lies in its antioxidant property with blood - brain barrier permeability and anti - inflammatory capabilities . -calpain , a calcium ion activated intracellular cysteine protease causes serious cellular insult , leading to cell death in various pathological conditions including hypoxia and ischemic stroke . hence , it may be considered as a potential drug target for the treatment of hypoxia induced neuronal injury . as the inhibitory property of -calpain is yet to be explored in details , hence , in the present study , we investigated the interaction of quercetin with -calpain through a molecular dynamics simulation study as a tool through clarifying the molecular mechanism of such inhibition and determining the probable sites and modes of quercetin interaction with the -calpain catalytic domain . in addition , we also investigated the structure - activity relationship of quercetin with -calpain . affinity binding of quercetin with -calpain had a value of 28.73 kj / mol and a ki value of 35.87 m that may be a probable reason to lead to altered functioning of -calpain . hence , quercetin was found to be an inhibitor of -calpain which might have a possible therapeutic role in hypoxic injury .

glioblastoma multiforme ( gbm ) is the most common malignant type of primary brain tumor , comprising approximately 52% of all primary brain tumors . despite the use of a multidisciplinary treatment approach , the prognosis of gbm is extremely poor ; most patients die within 2 years after the initial diagnosis [ 2 - 4 ] . currently , the standard treatment for newly diagnosed gbm consists of a surgical resection to the extent feasible , followed by concurrent chemoradiotherapy with temozolomide ( ccrt ) and adjuvant temozolomide ( tmz ) . in 2004 , a randomized phase iii multicenter trial reported that ccrt followed by adjuvant tmz improved the median survival and 2-year survival over postoperative radiotherapy ( rt ) alone . food and drug administration approved the concomitant use of tmz with rt for newly diagnosed gbm patients . through a follow - up study longer than 5 years , stupp et al . reported that ccrt followed by adjuvant tmz improved the long - term survival compared to rt alone , and that methylation of the o - methylguanine - dna methyltransferase ( mgmt ) promoter was correlated with a better overall survival ( os ) recently , yang et al . reported a variance in the correlations between mgmt promoter methylation and both the os and progression - free survival ( pfs ) among an ethnically - diverse sample of gbm patients . in their meta - analysis , they found that subjects of european descent had a better os and pfs in gbm with the mgmt promoter methylation compared to different race according to univariate or multivariate analysis , whereas subjects of east asian descent had a longer os compared to difference race according to multivariate analysis only . only a few studies of os and pfs among korean gbm patients that account for mgmt promoter methylation have been reported . in particular , there are no large case studies of korean gbm patients examining the efficacy and safety of ccrt followed by adjuvant tmz . this study assessed the efficacy and safety of ccrt followed by adjuvant tmz for patients with a newly diagnosed gbm through a multicenter , retrospective study of a large case series for the first time in korea . patients older than 20 years with a newly diagnosed gbm were assessed through retrospective chart reviews . nineteen institutions participated in the study , and chart reviews for the clinical and radiological findings were performed independently and analyzed at each institution . a central pathologic review of histopathological specimens from all patients included in the study was performed by three neuropathologists ( k .- h.l . all patients were treated with ccrt with or without adjuvant tmz according to stupp s regimens . the extent of the resection was classified as a gross total resection ( gtr ) , subtotal resection ( str ) , partial resection ( pr ) , or biopsy alone . the patients were irradiated with a total dose of 60 gy , with daily fractions of 2 gy given 5 days per week for 6 weeks , using conventional three - dimensional conformal rt . alternative rt protocols , such as two - dimensional rt and intensity - modulated rt with or without a simultaneous - integrated boost were included to avoid any selection bias and reflect regular clinical situations . tmz was administered at 75 mg / m every day for 6 weeks during the rt period . after a 4-week break , the patients received adjuvant tmz for the first 5 days of each 28-day cycle provided that they continued to tolerate the treatment . the dose of adjuvant tmz was 150 mg / m / day for the first cycle , which was increased to 200 mg / m / day at the beginning of the second cycle if no hematologic toxicity had occurred . the extent of the resection was evaluated by postoperative brain magnetic resonance imaging ( mri ) with gadolinium enhancement within 48 hours after surgery . the gtr was defined as > 99% removal of the initial tumor , str as 50%-99% , and the pr as < 50% . the mri scans were performed before the first cycle and after every two or three cycles of adjuvant tmz . the primary end - point was the os , and the secondary end - points were pfs and safety . the pfs was defined as the time from the date of the initial operation to documented disease progression or the date of the final follow - up visit . the os time was measured from the date of the initial operation to death or the date of the final follow - up visit . the methylation status of the mgmt promoter was evaluated using a methylation - specific polymerase chain reaction where possible . the toxic effects of the treatment were graded as follows according to the national cancer institute common terminology criteria for adverse events ( nci ctcae ) ver . 4.0.3 : a score of 1 , indicating mild adverse effects ; 2 , indicating moderate adverse effects ; 3 , indicating severe adverse effects ; 4 , indicating life - threatening adverse effects ; and 5 , denoting death related to adverse effects . the prognostic factors associated with survival , including the age at diagnosis , sex , preoperative karnofsky performance status ( kps ) , tumor location , extent of resection , use of adjuvant tmz , and methylation status of the mgmt promoter , were examined by univariate and multivariate analyses . the os and pfs were analyzed using the kaplan - meier method with two - sided log - rank tests . patients older than 20 years with a newly diagnosed gbm were assessed through retrospective chart reviews . nineteen institutions participated in the study , and chart reviews for the clinical and radiological findings were performed independently and analyzed at each institution . a central pathologic review of histopathological specimens from all patients included in the study was performed by three neuropathologists ( k .- h.l . all patients were treated with ccrt with or without adjuvant tmz according to stupp s regimens . the extent of the resection was classified as a gross total resection ( gtr ) , subtotal resection ( str ) , partial resection ( pr ) , or biopsy alone . the patients were irradiated with a total dose of 60 gy , with daily fractions of 2 gy given 5 days per week for 6 weeks , using conventional three - dimensional conformal rt . alternative rt protocols , such as two - dimensional rt and intensity - modulated rt with or without a simultaneous - integrated boost were included to avoid any selection bias and reflect regular clinical situations . tmz was administered at 75 mg / m every day for 6 weeks during the rt period . after a 4-week break , the patients received adjuvant tmz for the first 5 days of each 28-day cycle provided that they continued to tolerate the treatment . the dose of adjuvant tmz was 150 mg / m / day for the first cycle , which was increased to 200 mg / m / day at the beginning of the second cycle if no hematologic toxicity had occurred . the extent of the resection was evaluated by postoperative brain magnetic resonance imaging ( mri ) with gadolinium enhancement within 48 hours after surgery . the gtr was defined as > 99% removal of the initial tumor , str as 50%-99% , and the pr as < 50% . the mri scans were performed before the first cycle and after every two or three cycles of adjuvant tmz . the primary end - point was the os , and the secondary end - points were pfs and safety . the pfs was defined as the time from the date of the initial operation to documented disease progression or the date of the final follow - up visit . the os time was measured from the date of the initial operation to death or the date of the final follow - up visit . the methylation status of the mgmt promoter was evaluated using a methylation - specific polymerase chain reaction where possible . the toxic effects of the treatment were graded as follows according to the national cancer institute common terminology criteria for adverse events ( nci ctcae ) ver . 4.0.3 : a score of 1 , indicating mild adverse effects ; 2 , indicating moderate adverse effects ; 3 , indicating severe adverse effects ; 4 , indicating life - threatening adverse effects ; and 5 , denoting death related to adverse effects . the prognostic factors associated with survival , including the age at diagnosis , sex , preoperative karnofsky performance status ( kps ) , tumor location , extent of resection , use of adjuvant tmz , and methylation status of the mgmt promoter , were examined by univariate and multivariate analyses . the os and pfs were analyzed using the kaplan - meier method with two - sided log - rank tests . a total of 750 patients from 19 institutions were enrolled and treated according to stupp s protocol from january 2006 until june 2011 . of these patients , 415 ( 55.3% ) were male and 335 ( 44.7% ) were female ; the median age at the first operation was 57.5 years ( range , 20 to 86 years ) . three hundred eightyseven patients ( 51.7% ) showed a kps score 90 at diagnosis . supratentorial lesions were observed in 719 patients ( 95.9% ) and infratentorial lesions were noted in 31 patients ( 4.1% ) . among the supratentorial lesions , deep - seated gbms were found in 106 patients ( 4.7% ) and superficial gbms were found in 613 patients ( 85.3% ) . at the first operation , gtr , str , pr , and biopsy alone were achieved in 388 ( 51.7% ) , 159 ( 21.2% ) , 96 ( 12.8% ) , and 107 ( 14.3% ) patients , respectively . a central pathological review confirmed the diagnosis of gbm in all cases reviewed . the median time from diagnosis to the start of rt was 3.6 weeks ( range , 0.7 to 26.6 weeks ) . the median therapeutic radiation dose was 60 gy ( range , 4 to 84 gy ) . among the 750 patients assigned to receive ccrt , 659 ( 87.9% ) completed ccrt as planned . after ccrt , 660 patients ( 88% ) started adjuvant tmz and received a median of six cycles ( range , 0 to 15 cycles ) ; 407 patients ( 61.7% ) completed six cycles . the reasons for not starting or not completing adjuvant tmz therapy were disease progression ( 32.6% ) , toxic effects ( 3.5% ) , and follow - up loss ( 2.3% ) . the median kps in those patients who completed and did not complete adjuvant tmz was 90 ( range , 20 to 100 ) and 80 ( 30 to 100 ) , respectively . the methylation status of the mgmt promoter was evaluated in 217 of the 750 patients ( 28.9% ) . of the 217 patients , 89 ( 41.0% ) had the methylated mgmt promoter , and 128 ( 59.0% ) had the unmethylated mgmt promoter . at the time of the analysis , the median follow - up period was 16.3 months ( range , 0.3 to 105.1 months ) , and the median os was 17.5 months ( 95% confidence interval [ ci ] , 16.5 to 18.5 ) . the actuarial survival rates at 1- , 3- , and 5-year os were 72.1% , 21.0% , and 9.0% , respectively . the median pfs was 10.1 months ( 95% ci , 9.3 to 10.9 ) , and the actuarial pfs at 1- , 3- , and 5-year pfs was 42.2% , 13.0% , and 7.8% , respectively . in univariate analysis for survival in all patients , the age at diagnosis ( p < 0.001 ) , preoperative kps ( p < 0.001 ) , tumor location ( p < 0.001 ) , extent of resection ( p < 0.001 ) , use of adjuvant tmz ( p < 0.001 ) , and methylation status of the mgmt promoter ( p=0.012 ) were correlated with survival . the age at diagnosis ( p=0.003 ) , extent of the resection ( p < 0.001 ) , and use of adjuvant tmz ( p < 0.001 ) were correlated with the survival in both univariate and multivariate analyses . in addition , the methylation status of the mgmt promoter ( p=0.083 ) was not correlated with survival in multivariate analysis . patients who had received gtr showed a significantly longer os ( 21.0 months vs. 15.8 months , p=0.001 ) ( fig . 1a ) and longer pfs ( 10.9 months vs. 9.1 months , p=0.001 ) ( fig . 1b ) than those who had received str , pr , or biopsy alone in both univariate and multivariate analyses . patients with the methylated mgmt promoter had a longer os ( 23.9 months vs. 16.7 months , p=0.012 ) ( fig . 2a ) and longer pfs ( 13.2 months vs. 9.3 months , p=0.006 ) ( fig . when analyzing the extent of the resection according to the methylation status of the mgmt , patients receiving gtr showed a significantly longer os than those receiving str , pr , or biopsy alone in the groups with both the methylated ( 28.6 months vs. 16.7 months , p=0.001 ) ( fig . 3a ) and unmethylated mgmt promoter ( 19.0 months vs. 14.8 months , p=0.001 ) ( fig . was observed in patients receiving gtr compared to those receiving str , pr , or biopsy alone in both groups with the methylated ( 20.7 months vs. 11.1 months , p=0.041 ) ( fig . 3b ) and unmethylated mgmt promoter ( 10.6 months vs. 7.2 months , p=0.030 ) ( fig . the use of adjuvant tmz was associated with survival according to both univariate and multivariate analyses . the os and pfs were compared in patients with the methylated and unmethylated mgmt promoter , considering the degree of completeness of adjuvant tmz ( table 1 ) . patients with the methylated mgmt promoter had a longer os and longer pfs as the number of adjuvant tmz cycles approached six compared to those who had the unmethylated mgmt promoter ( fig . the incidence and degree of hematologic toxicity were analyzed separately according to the period of ccrt and adjuvant tmz . a total of 115 patients ( 15.3% ) showed grade 3 or 4 hematologic toxicity , as evaluated by ctcae ver . , grade 3 or 4 hematologic toxicity was noted in 63 patients ( 8.4% ) , including 41 patients ( 5.5% ) with lymphocytopenia , 10 patients ( 1.3% ) with neutropenia , eight patients ( 1.1% ) with thrombocytopenia , seven patients ( 0.9% ) with leukopenia , and five patients ( 0.7% ) with anemia . during the adjuvant tmz period , grade 3 or 4 hematologic toxicity was reported in 66 patients ( 10.2% ) , including 42 patients ( 6.4% ) with lymphocytopenia , 17 patients ( 2.6% ) with neutropenia , 13 patients ( 2.0% ) with thrombocytopenia , five patients ( 0.8% ) with anemia , and two patients ( 0.3% ) with leukopenia . a total of 750 patients from 19 institutions were enrolled and treated according to stupp s protocol from january 2006 until june 2011 . of these patients , 415 ( 55.3% ) were male and 335 ( 44.7% ) were female ; the median age at the first operation was 57.5 years ( range , 20 to 86 years ) . three hundred eightyseven patients ( 51.7% ) showed a kps score 90 at diagnosis . supratentorial lesions were observed in 719 patients ( 95.9% ) and infratentorial lesions were noted in 31 patients ( 4.1% ) . among the supratentorial lesions , deep - seated gbms were found in 106 patients ( 4.7% ) and superficial gbms were found in 613 patients ( 85.3% ) . at the first operation , gtr , str , pr , and biopsy alone were achieved in 388 ( 51.7% ) , 159 ( 21.2% ) , 96 ( 12.8% ) , and 107 ( 14.3% ) patients , respectively . a central pathological review confirmed the diagnosis of gbm in all cases reviewed . the median time from diagnosis to the start of rt was 3.6 weeks ( range , 0.7 to 26.6 weeks ) . the median therapeutic radiation dose was 60 gy ( range , 4 to 84 gy ) . among the 750 patients assigned to receive ccrt , 659 ( 87.9% ) completed ccrt as planned . after ccrt , 660 patients ( 88% ) started adjuvant tmz and received a median of six cycles ( range , 0 to 15 cycles ) ; 407 patients ( 61.7% ) completed six cycles . the reasons for not starting or not completing adjuvant tmz therapy were disease progression ( 32.6% ) , toxic effects ( 3.5% ) , and follow - up loss ( 2.3% ) . the median kps in those patients who completed and did not complete adjuvant tmz was 90 ( range , 20 to 100 ) and 80 ( 30 to 100 ) , respectively . the methylation status of the mgmt promoter was evaluated in 217 of the 750 patients ( 28.9% ) . of the 217 patients , 89 ( 41.0% ) had the methylated mgmt promoter , and 128 ( 59.0% ) had the unmethylated mgmt promoter . the median follow - up period was 16.3 months ( range , 0.3 to 105.1 months ) , and the median os was 17.5 months ( 95% confidence interval [ ci ] , 16.5 to 18.5 ) . the actuarial survival rates at 1- , 3- , and 5-year os were 72.1% , 21.0% , and 9.0% , respectively . the median pfs was 10.1 months ( 95% ci , 9.3 to 10.9 ) , and the actuarial pfs at 1- , 3- , and 5-year pfs was 42.2% , 13.0% , and 7.8% , respectively . in univariate analysis for survival in all patients , the age at diagnosis ( p < 0.001 ) , preoperative kps ( p < 0.001 ) , tumor location ( p < 0.001 ) , extent of resection ( p < 0.001 ) , use of adjuvant tmz ( p < 0.001 ) , and methylation status of the mgmt promoter ( p=0.012 ) were correlated with survival . the age at diagnosis ( p=0.003 ) , extent of the resection ( p < 0.001 ) , and use of adjuvant tmz ( p < in addition , the methylation status of the mgmt promoter ( p=0.083 ) was not correlated with survival in multivariate analysis . patients who had received gtr showed a significantly longer os ( 21.0 months vs. 15.8 months , p=0.001 ) ( fig . 1a ) and longer pfs ( 10.9 months vs. 9.1 months , p=0.001 ) ( fig . 1b ) than those who had received str , pr , or biopsy alone in both univariate and multivariate analyses . patients with the methylated mgmt promoter had a longer os ( 23.9 months vs. 16.7 months , p=0.012 ) ( fig . when analyzing the extent of the resection according to the methylation status of the mgmt , patients receiving gtr showed a significantly longer os than those receiving str , pr , or biopsy alone in the groups with both the methylated ( 28.6 months vs. 16.7 months , p=0.001 ) ( fig . 3a ) and unmethylated mgmt promoter ( 19.0 months vs. 14.8 months , p=0.001 ) ( fig . was observed in patients receiving gtr compared to those receiving str , pr , or biopsy alone in both groups with the methylated ( 20.7 months vs. 11.1 months , p=0.041 ) ( fig . 3b ) and unmethylated mgmt promoter ( 10.6 months vs. 7.2 months , p=0.030 ) ( fig . the use of adjuvant tmz was associated with survival according to both univariate and multivariate analyses . the os and pfs were compared in patients with the methylated and unmethylated mgmt promoter , considering the degree of completeness of adjuvant tmz ( table 1 ) . patients with the methylated mgmt promoter had a longer os and longer pfs as the number of adjuvant tmz cycles approached six compared to those who had the unmethylated mgmt promoter ( fig . the incidence and degree of hematologic toxicity were analyzed separately according to the period of ccrt and adjuvant tmz . a total of 115 patients ( 15.3% ) showed grade 3 or 4 hematologic toxicity , as evaluated by ctcae ver . , grade 3 or 4 hematologic toxicity was noted in 63 patients ( 8.4% ) , including 41 patients ( 5.5% ) with lymphocytopenia , 10 patients ( 1.3% ) with neutropenia , eight patients ( 1.1% ) with thrombocytopenia , seven patients ( 0.9% ) with leukopenia , and five patients ( 0.7% ) with anemia . during the adjuvant tmz period , grade 3 or 4 hematologic toxicity was reported in 66 patients ( 10.2% ) , including 42 patients ( 6.4% ) with lymphocytopenia , 17 patients ( 2.6% ) with neutropenia , 13 patients ( 2.0% ) with thrombocytopenia , five patients ( 0.8% ) with anemia , and two patients ( 0.3% ) with leukopenia . in previous studies , age , performance status , extent of resection , methylation status of the mgmt promoter , and the use of tmz were identified as prognostic factors for patients with gbm . in the present study , age , extent of the resection , and use of adjuvant tmz were correlated with a better os according to univariate and multivariate analyses , whereas the tumor location and methylation status of the mgmt promoter were prognostic factors of os in univariate analysis alone . table 2 summarizes the results of stupp s trial and other studies in east asian samples . in 2004 , the european organization for research and treatment of cancer ( eortc ) and national cancer institute of canada ( ncic ) trial ( stupp s trial ) demonstrated a median os and pfs of 14.6 and 6.9 months in patients with newly diagnosed and histologically proven gbm , respectively . in the present study , the median os and pfs were 17.5 and 10.1 months , respectively . although the treatment in the present study was based on stupp s protocol , better clinical outcomes the number of tmz - treated patients who received gtr was larger than that in stupp s tria ( 51.7% vs. 39% ) . the literature indicates that gtr is a significant prognostic factor in patients with a primary gbm [ 11 - 15 ] . in the current series , patients who received gtr showed a longer os and longer pfs than those who received str , pr , or biopsy alone , regardless of the methylation status of the mgmt promoter . selection bias may also have occurred due to the differences between a prospective randomized study ( stupp s trial ) and a retrospective multiinstitutional study ( the present study ) . the methylation status of the mgmt promoter is a suggested predictive factor of the survival of patients with newly diagnosed gbm treated with rt and tmz . the epigenetic silencing of the mgmt gene by methylation of the mgmt promoter leads to a loss of mgmt expression and a decrease in dna repair activity , resulting in increased sensitivity to tmz and a longer os . on the other hand , whether methylation of the mgmt promoter is truly a prognostic marker or a predictive marker of the sensitivity to alkylating agents or radiation is unclear [ 18 - 20 ] . in the current study , patients with a methylated mgmt promoter and underwent stupp s protocol showed significantly a longer os and longer pfs than those with the unmethylated mgmt promoter . these results are consistent with those of a previous study . among the 573 patients enrolled in stupp s trial , hegi et al . analyzed the mgmt methylation status in 206 patients for whom the mgmt status could be evaluated . in 106 patients who received ccrt , patients with methylated mgmt promoter ( 43.4% ) showed a significantly longer os and longer pfs than those with the unmethylated mgmt promoter ( 56.6% ) ( os , 21.7 months vs. 12.7 months ; pfs , 10.3 months vs. 5.3 months ) . these results also imply that a methylated mgmt promoter is predictive of a response to rt and is a prognostic factor in gbm . the differences in survival between the present and previous studies could also be explained by the early start of postoperative rt and the number of adjuvant tmz cycles administered . the median cellular doubling time of gbm is 17 days . as the tumor grows and produces satellite growths , the chance of a regional miss increases during radiation treatment . consequently , any delays in time from surgery to the start of rt are correlated with a poor survival . the median time from surgery to the start of rt was 5 weeks in stupp s trial , whereas postoperative rt was started within a median time of 3.5 weeks in the present study . a disparity in the degree of completion of adjuvant tmz was observed between the groups . although adjuvant tmz was administered for a maximum of six cycles in stupp s trial , in the present study , adjuvant tmz was continued provided that it was tolerated ( median cycle , 3 cycles vs. 6 cycles , respectively ) . the number of patients completing six cycles of adjuvant tmz was also greater in the present study than in stupp s trial ( 61.7% vs. 47% ) . compared the os and pfs in patients who received six cycles or fewer and more than six cycles of adjuvant tmz . among a total of 114 patients treated with stupp s protocol , 55 received adjuvant tmz of six cycles or less , and 59 received more than six cycles . correlations were observed between the degree of completion of adjuvant tmz and both os and pfs . in the present study , the administration of adjuvant tmz after ccrt was the strongest prognostic factor of survival , and the degree of completion of adjuvant tmz was correlated with the os and pfs regardless of the methylation status of the mgmt promoter . these results suggest that adjuvant tmz is crucial for prolonging the os and pfs in korean patients with a newly diagnosed gbm . finally , the incidence of hematologic toxicity of grade 3 or 4 during the ccrt period was slightly greater in the present study than in stupp s trial ( 8.4% vs. 6.7% ) . in contrast to the ccrt period , the incidence of hematologic toxicity of grade 3 or 4 during the adjuvant tmz period was lower in the present study than in stupp s trial ( 10.2% vs. 14.3% ) . patients treated with ccrt followed by adjuvant tmz had more favorable survival rates and tolerable toxicity . on the other hand , the present study has limitations because of its retrospective nature , the study range was restricted to adult gbm , and the use of a different treatment protocol from that already recognized as the standard for newly diagnosed gbm . nevertheless , this study is the first large , multicenter , observational study of gbm treatment in a sample of korean patients . a randomized , prospective study of a large case series will be needed explore ways of improving the survival among korean gbm patients .

purposethe purpose of this study was to investigate the feasibility and survival benefits of combined treatment with radiotherapy and adjuvant temozolomide ( tmz ) in a korean sample.materials and methodsa total of 750 korean patients with histologically confirmed glioblastoma multiforme , who received concurrent chemoradiotherapy with tmz ( ccrt ) and adjuvant tmz from january 2006 until june 2011 , were analyzed retrospectively.resultsafter the first operation , a gross total resection ( gtr ) , subtotal resection ( str ) , partial resection ( pr ) , biopsy alone were achieved in 388 ( 51.7% ) , 159 ( 21.2% ) , 96 ( 12.8% ) , and 107 ( 14.3% ) patients , respectively . the methylation status of o6-methylguanine - dna methyltransferase ( mgmt ) was reviewed retrospectively in 217 patients . the median follow - up period was 16.3 months and the median overall survival ( os ) was 17.5 months . the actuarial survival rates at the 1- , 3- , and 5-year os were 72.1% , 21.0% , and 9.0% , respectively . the median progression - free survival ( pfs ) was 10.1 months , and the actuarial pfs at 1- , 3- , and 5-year pfs were 42.2% , 13.0% , and 7.8% , respectively . the patients who received gtr showed a significantly longer os and pfs than those who received str , pr , or biopsy alone , regardless of the methylation status of the mgmt promoter . patients with a methylated mgmt promoter also showed a significantly longer os and pfs than those with an unmethylated mgmt promoter . patients who received more than six cycles of adjuvant tmz had a longer os and pfs than those who received six or fewer cycles . hematologic toxicity of grade 3 or 4 was observed in 8.4% of patients during the ccrt period and in 10.2% during the adjuvant tmz period.conclusionpatients treated with ccrt followed by adjuvant tmz had more favorable survival rates and tolerable toxicity than those who did not undergo this treatment .

in animal cells , the centrosome acts as the main site of microtubule ( mt ) * nucleation ; although , depending on the cell type , mts can grow from other sites ( keating and borisy , 1999 ; schroer , 2001 ) . mts nucleated at the centrosome may either remain anchored or be released , like in neuronal or epithelial cells ( ahmad et al . in these cell types , the function of released mts can be addressed as mt targeting to peripheral sites is correlated with cell differentiation . in migrating cells however , where released mts have also been observed , their function remains unknown . a major riddle is the lack of tools to specifically analyze the role of mts released from the centrosome . dynactin complex , which has been shown to be instrumental for mt anchoring at the centrosome ( quintyne et al . , 1999 ) , participates also in other mechanisms , including the interaction of mt plus ends with the cell cortex ( schroer , 2001 ) . in addition , high concentration of mts in the centrosomal area precludes a direct and detailed analysis of mt behavior in that region . in a previous study , we showed that the daughter centriole , which in steady - state conditions seems devoid of associated mts , could actually nucleate mts that were immediately released ( piel et al . , it was also demonstrated that most mts were nucleated at the centrosome , although no mt could be observed in the centrosomal region , as they were released through the severing activity of the protein katanin ( ahmad et al . , 1999 ) . these two results lead to the prediction that mts should be released while they are still very short , being then transported away from the centrosome before they substantially elongate . thus , to study mt release from the centrosome , we used techniques enabling the observation of short mts . as a tool to specifically perturb the balance between mt capture and release at the centrosome , we also used moderate overexpression of the protein ninein : this protein is specifically associated with mt minus ends in various cell lines and has been proposed to participate in mt anchoring at the mother centriole ( mogensen et al . a direct evualuation of the number and behavior of short mts is actually almost impossible in steady - state conditions , as any tubulin staining gives a signal proportional to the length of individual mts ( in the speckle technique , the number of speckles is proportional to the length of the mt ) . information on short mts would thus be hard to extract with such an approach , which imposes a bias toward long mts . as tubulin background after mt depolymerization is too high to allow a direct observation of mts regrowth with labeled tubulin , we used eb1-gfp as a marker of growing distal tips of mts ( mimori - kiyosue et al . , 2000 ) , followed by extraction / fixation of the recorded cells in order to visualize the mt network by immunostaining . in all cells recorded ( n = 10 ) , the great majority of eb1-gfp dots emanated from the centrosome and spread throughout the cytoplasm in < 2 min ( fig . eb1 dots began to leave the centrosome 20 s post nocodazole washout and were already 10 m away at 1 min . on coverslips fixed after 1 min of recording , all cells contained short mts associated with the centrosome , and numerous mts ( 40 on average ) spread throughout the cytoplasm ( fig . 1 b , left ) , but no mt of more than a few microns could be observed . three minutes after regrowth , both centrosomal and noncentrosomal mts of multiple sizes 10 m were aboundant ( fig . 1 b , right ) , meaning that mt growth was not impaired by time - lapse recording . moreover , similar images could be observed on coverslips fixed in the same conditions that had not been illuminated . we can thereby conclude that during the first minute of regrowth tens of short mts are nucleated at the centrosome and then released . ( a ) mts of l929 cells expressing eb1-gfp were fully depolymerized ( 2 h at 4c , 10 m nocodazole ) . after deconvolution , images have been processed to extract fluorescence structures ( eb1-gfp aggregates ) from the background . note that no eb1-gfp dot is visible in the cytoplasm until 20 s after nocodazole washout , but eb1-gfp accumulates around the two centrioles that are separated , as is almost always the case after microtubule depolymerization . after 2030 s , dots appear in the centrosomal region and spread throughout the cytoplasm during the next 60 s. trajectories are mainly radial and emanate from the centrosome . the boundary of the cell can be estimated from the phase contrast image ( top , left ) . ( b ) coverslips processed as in a were fixed 1 and 3 min immediately after recording and labeled with anti-tubulin antibodies . the fact that these short released mts can be found microns away from the centrosome in < 1 min suggests that they are transported , probably by molecular motors . mt transport has already been demonstrated ( keating et al . , 1997 ; yvon and wadsworth , 2000 ) . it was also proposed that cortical dynein could pull on captured mt plus ends and thus transport them if their minus end is free ( smith et al . , 2000 ) . to ask whether short released mts were transported by dynein mt regrowth experiments showed that very few short mts were found away from the centrosome after 1 min of regrowth in p150 cc1expressing cells ( fig . we therefore conclude that in l929 cells , as reported for other cell types such as ptk1 ( keating et al . , 1997 ) , mts released from the centrosome are transported toward the cell periphery by mt minus end motors rather than by treadmilling only , which on the contrary was observed as the main feature in melanophores ( rodionov and borisy , 1997 ) . furthermore , the fact that this transport could be observed in the absence of a mt network suggests that it is driven by motors anchored in cytoplasmic structures , like actin microfilaments ( garces et al . , 1999 ) , or intermediate filaments ( helfand et al . , 2002 ) . motors would be randomly dispersed ; but if their concentration is high enough , it would be sufficient to explain the rather rectilinear movement of short mts , as observed in in vitro motility assay . released short mts are transported by dynein - dynactin complex . ( a ) inhibition of dynein dynactin complex by p150 cc1-dsred expression in l929 cells . cells were treated as described in the legend to fig . 1 and were fixed 1 , 3 , and 5 min after nocodazole washout . cells were further stained with anti-tubulin antibodies , and p150 cc1-dsred was visualized in the red channel . note that almost no short mts can be observed away from the centrosomal region ( arrowhead ) in the p150 cc1-dsred expressing cell . the number of noncentrosomal mts has been estimated in control cells ( n = 12 ) and in p150 cc1-dsred expressing cells ( n = 10 ) ( histogram ) ; error bars indicate a 95% confidence interval calculated with student 's coefficient . images were obtained by stacking consecutive images acquired every second between times indicated in the upper right corner . examples of eb1-gfp aggregates are shown either leaving the centrosome at a constant speed ( c ) or leaving the centrosome at a higher speed , and then slowing down ( b ) . sequential images on the right are a twofold blow up of the regions depicted on the left image . the speed of the particles is shown in d , as a function of their distance from the centrosome . bars : ( a and b ) 10 m ; ( d and e , left ) 5 m ; ( d and e , right ) 3 m . we then reasoned that mts released from the centrosome in steady - state conditions , when they are transported while elongating , should display an increased plus - end speed compared with those anchored at the centrosome . this technique allowed us to record , with a high temporal and three - dimensional spatial resolution , the speed of a considerable number of mt tips emanating either from the centrosome or from other places in the cell . we were indeed able to identify two subpopulations of mts growing from the centrosome : the first characterized by a constant speed of eb1-gfp dots , compatible with polymerization ( fig . 2 , c and d , blue track ; 1525 m / min , 20% of mt tips emanating from the centrosome ) , and the second characterized by much faster movements away from the centrosome ( fig . 2 , b and d , red track ; 3560 m / min , 40% of mt tips emanating from the centrosome ; the other 40% corresponding to speeds of 2535 m / min , an intermediary speed , slightly faster than the usual reported polymerization speed ) . these mt tips usually slowed down to speeds compatible with polymerization as they approached the cell periphery ( fig . the speeds observed for the two populations suggest that they correspond respectively to polymerizing mts anchored at the centrosome and to released mts transported toward cell periphery by dynein , being finally tethered on other compartments or in the cortical cytoskeleton ( hoffmann et al . , 2001 ) . ( 2002 ) , using various approaches to estimate polymerization speed in the cell interior , reported values compatible with our slow moving population ( 17 13.8 m / min ) . fast - growing mts anchored at the centrosome would have been detected by the techniques they used , ruling out the possibility that some anchored mts could have an increased polymerization speed in the centrosomal area . on the other hand , these techniques could have missed short mts moving at high speeds ( 60 m / min ) , which require at least one image of plus - end labeling every second to be monitored . alternatively , the cells used might have less released mts than l929 cells , as very versatile mt behaviors were reported in various cell types . we thus conclude that , in steady - state conditions as in regrowth experiments , a subset of noncentrosomal mts occurs through release from the centrosome and is subsequently transported toward the cell periphery . we then wanted to specifically perturb that population of mts by overexpressing the protein ninein . in l929 , hela and human diploid primary fibroblasts ( aff11 ) cells , gfp - ninein protein specifically accumulated as a unique mass in the centrosomal area , similar to the endogenous localization at low expression levels ( fig . these features , which were also observed with the untagged protein , rule out the possibility that gfp - ninein accumulation at the centrosome corresponds to aggresomes of misfolded proteins . we also observed that gfp - ninein is functional and that , as expected , its accumulation increases mt anchoring capacity of the centrosome ( fig . s2 , available at http://www.jcb.org/cgi/content/full/jcb.200207076/dc1 ) . to determine whether gfp - ninein overexpression could affect the production of short released mts , we performed regrowth experiments as in fig . 1 . 1 min after nocodazole withdrawal , short mts had been nucleated at the centrosome of both transfected and untransfected cells . but , when in control cells , numerous short mts were observed throughout the cytoplasm , in overexpressing cells they were all confined to the centrosomal ninein accumulation ( fig . subsequently , three minutes after regrowth , both centrosomal and noncentrosomal mts were observed in control cells , whereas in gfp - ninein overexpressing cells , which contained a similar number of mts , all mts were associated with the centrosomal gfp - ninein accumulation ( fig . this shows that accumulation of gfp - ninein at the centrosome does not perturb nucleation or elongation of mts but suppresses released mts . transfected l929 cells were treated as described in the legend to to fig . 1 and were fixed 1 ( a c ) or 3 min ( d f ) after drug removal . mts were decorated with an anti-tubulin antibody ( a and d ) , and gfp - ninein was visualized in the gfp channel ( b and e ) . note that noncentrosomal mts are observed only in control cells and that nascent mts are clustered within the ninein mass in gfp - ninein overexpressing cells ( merge in c and f of the outlined areas shown in a e ) . the number of noncentrosomal mts has been estimated in control cells ( n = 12 ) and gfp - ninein expressing cells ( n = 10 ) ( g ) . ( h m ) eb1-gfp dynamics in control and in gfp - ninein overexpressing cells . ( h and h ) gfp signal in an eb1-gfp expressing l929 cell ; ( i and i ) gfp signal in a l929 cell coexpressing eb1-gfp and gfp - ninein . 2 . images in h and i are a detail of the centrosomal region of the cells shown in h and i at time 0 , to show the actual staining of the centrosome without any stacking . note , in h and i , the increased concentration of eb1-gfp dots in the lamellipodial regions at the front of both the control and the gfp - ninein expressing cell . only moderately overexpressing cells were considered for analysis : the sixfold blow - up of the centrosomal area of the gfp - ninein overexpressing cell at the bottom of i is to compare with endogenous ninein in a control cell using an antibody , shown in j. ( k and l ) speed of the eb1-gfp aggregates , tracked in the cells shown in h and i as a function of their distance from the centrosome . a polynomial fit has been added . note the decrease of the mean speed in the first 10 m around the centrosome in the control cell ; whereas , in the gfp - ninein expressing cell , the curve remains almost flat around 25 m / min . three regions ( 13 ) have been distinguished around the centrosome , to make statistics over several cells . there are fewer points in region 1 or 2 than in region 3 , because region 3 is where mt rescues are mainly observed , and because of a geometric effect ( the surface increases with the square of the radius ) . ( m ) for both control condition and gfp - ninein overexpression , statistics were compiled from 2,000 eb1-gfp dots , corresponding to 200 mts in 5 different cells . note that the repartition of speeds in the gfp - ninein overexpressing cells does not vary with the distance from the centrosome , whereas in control cells , an important population of transported mts exist in the regions close to the centrosome ( 1 and 2 ) . bars : ( h , h ' , i , and i ' ) 5 m ; ( i ' blow - up and j ) 1 m . we then analyzed the effect of ninein overexpression on the mt network in steady - state conditions . in cells exhibiting an accumulation of gfp - ninein at the centrosome , only the population of eb1-gfp dots with a constant lower speed m ) . in ninein overexpressing cells as well as in control cells , trajectories of eb1-gfp dots that did not seem to emanate from the centrosome were observed . this could , in most cases , be interpreted as rescues on mts anchored at the centrosome as their inward prolonged trajectories reached the centrosomal area . accordingly , these dots revealed speeds corresponding to the polymerization rate and were mainly observed in the lamellipodium area where most mt shrinkings and rescues were observed ( see also komarova et al . , 2002 ) , heading toward the cell periphery ( video 2 , available at http://www.jcb.org/cgi/content/full/jcb.200207076/dc1 ) . the proportion of noncentrosomal mts occurring by breaking , spontaneous , or catalyzed nucleation at noncentrosomal sites could not really be estimated by the present technique . nevertheless , we can state that only a minor population of eb1-gfp dots had either a nonradial trajectory or a radial movement toward the centrosome , corresponding to obvious nucleation at noncentrosomal sites or growth of a broken mt ( a few percent of the total amount of eb1-gfp dots trajectories , compared with > 20% for trajectories interpreted as mts released from the centrosome in control cells ) . the cell periphery of ninein - overexpressing cells displayed a similar number of such eb1-gfp dot trajectories . that shows that , in steady - state conditions , ninein overexpression did not affect the dynamics of mts near the cell periphery , or the production of free mts by pathways other than centrosomal release . this suggests that mts released from the centrosome are different from other noncentrosomal mts : they might have a minus - end cap that ensures their stability , or bear particular cargoes because they were nucleated in the centrosome vicinity where many proteins are concentrated or activated . contrast video recording , that a moderate accumulation of gfp - ninein at the centrosome also correlated with a pronounced inhibition of cell migration , with a covered area less than half of that of the nontransfected cells ( fig . b ) . strikingly , inhibition of locomotion was due to a reduction in persistent migration in a given direction , whereas polarized membrane ruffling activity was not perturbed ( fig . 4 , a and c ; and videos 3 and 4 , available at http://www.jcb.org/cgi/content/full/jcb.200207076/dc1 ) . this behavior was significantly different from that observed when cells were video recorded in the presence of low amounts of nocodazole ( 10 m ) , which do not depolymerize mts but do inhibit their dynamic instability , or in the presence of paclitaxel ( 10 m ) . in both of these cases , a diminished ruffling activity was observed associated with a loss of cell polarity and motility ( not shown ) , as also reported by others ( liao et al . , 1995 ) . ( a ) representative examples of migration trajectories of gfp - ninein expressing ( green tracks ) and nonexpressing l929 cells ( black tracks ) . ( b ) histogram showing the distribution of the random migration values for control and gfp - ninein overexpressing cells . cell trajectories were characterized by the area covered by cell locomotion in a given time . note the dramatic inhibition of random migration in gfp - ninein overexpressing cells . ( c ) time - lapse video recording of a gfp - ninein transfected cell . contrast images ; the centrosomal gfp - ninein accumulation is marked by a green cross in all other frames . note that the centrosome remained almost completely static during the whole sequence , whereas the leading edge ( arrow ) rotated continuously . see also video 3 ; other cells could show a back and forth movement ( video 4 ) . these data are thus compatible with a model in which dynamic mts anchored at the centrosome regulate polarized regions of lamellipodial extensions ( wittmann and waterman - storer , 2001 ) . by contrast , mts released from the centrosome would be transported toward regions of lamellipodial extension and captured by the actin cytoskeleton . these noncentrosomal mts are likely to be involved in the stabilization of the lamellipodia , which is necessary for efficient directed cell locomotion . a direct effect of overexpressed gfp - ninein on lamellipodial dynamics is unlikely , as gfp - ninein was never observed at locations other than the centrosome in the cells used . control overexpression of other centrosomal proteins in fusion with the gfp ( centrin or -tubulin ) did not affect cell migration . our model is further supported by our observation that most released mts are transported toward regions of lamellipodial extensions where they finally slow down , probably due to capture ( fig . this fits with the description by others of detyrosinated mts specifically enriched in the leading edge during directed locomotion ( gundersen , 2002 ) . these mts were also described as curly , which could be indicative of a free minus end . according to kaverina et al . ( 1999 ) , mts could be targeted to focal adhesion plaques and modulate their turnover . we wondered whether the inhibition of locomotion observed in the absence of released mts could be correlated to a reduced turnover of adhesion plaques or to an increase in stress fiber formation ( ballestrem et al . , 2000 ) . we found no difference in the size of the adhesion plaques , or in the organization of the actin cytoskeleton , between control and gfp - ninein overexpressing cells . by contrast , cells treated with paclitaxel ( 10 m ) , presented larger adhesion plaques and more numerous stress fibers ( fig . this indicates that the regulation of these structures , which are essential for membrane ruffling activity , depends on the dynamics of mts rather than on their anchoring to the centrosome . it may involve the activity of small gtpases of the rho family ( wittmann and waterman - storer , 2001 ; fukata et al . , 2002 ) . the differential contribution of the daughter and the mother centriole to anchoring and release of mts ( piel et al . , 2000 ) may also , together with the plasticity of the intercentriolar linkage , be exploited in cell motility . in conclusion , this work extends to migrating cells the importance of mts released from the centrosome , already noticed in some differentiated cells . however , although in neurons and some epithelial cells , most of the mts are released from the centrosome ; this is not the case in migrating cells , where at least two distinct subsets of mts coexist , each with specific functions in cell locomotion . whether the balance between capture and release of mts by the centrosome is modified in transformed metastatic cells with enhanced motility will deserve further study . y. barandon , cole normale suprieure , paris , france ) , hela , and l929 cells were grown in dme ( gibco brl ) , supplemented with 10% fetal calf serum ( eurobio ) . exponential growing cells were transfected by electroporation as described ( piel et al . , 2000 ) . cells transfected with gfp - ninein were processed as described ( piel et al . , 2000 ) . images were taken using a leica dmrxa microscope and 63/1.32 or 100/1.40.7 pl - apo objectives , a micromax charge - coupled device camera ( princeton instruments ) , and ip - lab or metaview ( universal imaging corp . ) software , and were processed using adobe photoshop . contrast ( every 5 min ) and fluorescence images ( every hour ) on a leica dmirbe microscope controlled by metamorph software for 36 h. the microscope was equipped with an open chamber equilibrated in 5% co2 and maintained at 37c , and images were taken with a 40/0.70 pl fluotar . to quantify cell motility , the area covered by a cell was determined using the root mean square of the excursion : [ < ( x < x > ) > < ( y < y>)>]/2 t , where t is the total time of tracking , and x and y the position coordinates of the cell . cells cotransfected with gfp - ninein and eb1-gfp to analyze mt dynamics were recorded by collecting fluorescent images every second . at each time point , the whole cell volume was scanned using a piezzo device mounted at the base of a 100/1.4 pl - apo objective . micromax charge - coupled device cameras were used for acquisition , and z stacks were deconvoluted and analyzed with metamorph software . in online supplemental fig . s1 , gfp - ninein specifically accumulates as a unique mass at the centrosome . in online supplemental fig . s2 , overexpressed gfp - ninein is functional and increases mt anchoring capacity in aff11 cells . in online supplemental fig . s3 , inhibition of cell migration is not associated with obvious remodelling of adhesion plaques or actin cytoskeleton . video 2 corresponds to fig . 3 and shows dynamics of growing mt plus ends . videos 3 and 4 correspond to fig . 4 and show inhibition of migration in ninein - gfp y. barandon , cole normale suprieure , paris , france ) , hela , and l929 cells were grown in dme ( gibco brl ) , supplemented with 10% fetal calf serum ( eurobio ) . exponential growing cells were transfected by electroporation as described ( piel et al . , 2000 ) . cells transfected with gfp - ninein were processed as described ( piel et al . , 2000 ) . images were taken using a leica dmrxa microscope and 63/1.32 or 100/1.40.7 pl - apo objectives , a micromax charge - coupled device camera ( princeton instruments ) , and ip - lab or metaview ( universal imaging corp . ) software , and were processed using adobe photoshop . analysis of cell motility was accomplished by collecting phase contrast ( every 5 min ) and fluorescence images ( every hour ) on a leica dmirbe microscope controlled by metamorph software for 36 h. the microscope was equipped with an open chamber equilibrated in 5% co2 and maintained at 37c , and images were taken with a 40/0.70 pl fluotar . to quantify cell motility , the area covered by a cell was determined using the root mean square of the excursion : [ < ( x < x > ) > < ( y < y>)>]/2 t , where t is the total time of tracking , and x and y the position coordinates of the cell . cells cotransfected with gfp - ninein and eb1-gfp to analyze mt dynamics were recorded by collecting fluorescent images every second . at each time point , the whole cell volume was scanned using a piezzo device mounted at the base of a 100/1.4 pl - apo objective . micromax charge - coupled device cameras were used for acquisition , and z stacks were deconvoluted and analyzed with metamorph software . in online supplemental fig . s1 , gfp - ninein specifically accumulates as a unique mass at the centrosome . in online supplemental fig . s2 , overexpressed gfp - ninein is functional and increases mt anchoring capacity in aff11 cells . in online supplemental fig . s3 , inhibition of cell migration is not associated with obvious remodelling of adhesion plaques or actin cytoskeleton . video 2 corresponds to fig . 3 and shows dynamics of growing mt plus ends . videos 3 and 4 correspond to fig . 4 and show inhibition of migration in ninein - gfp transfected l929 cells .

in migrating cells , force production relies essentially on a polarized actomyosin system , whereas the spatial regulation of actomyosin contraction and substrate contact turnover involves a complex cooperation between the microtubule ( mt ) and the actin filament networks ( goode , b.l . , d.g . drubin , and g. barnes . 2000 . curr . opin . cell biol . , 12:6371 ) . targeting and capture of mt plus ends at the cell periphery has been described , but whether or not the minus ends of these mts are anchored at the centrosome is not known . here , we show that release of short mts from the centrosome is frequent in migrating cells and that their transport toward the cell periphery is blocked when dynein activity is impaired . we further show that mt release , but not mt nucleation or polymerization dynamics , is abolished by overexpression of the centrosomal mt - anchoring protein ninein . in addition , a dramatic inhibition of cell migration was observed ; but , contrary to cells treated by drugs inhibiting mt dynamics , polarized membrane ruffling activity was not affected in ninein overexpressing cells . we thus propose that the balance between mt minus - end capture and release from the centrosome is critical for efficient cell migration .

breast cancer is the most common form of cancer among women in north america and the majority of european nations . each year , it is diagnosed in an estimated 1 million women worldwide , and is the cause of death of over 400 000 . the incidence of breast cancer increases with age and doubles every 10 years until the menopause , supporting a link with hormonal status . specific life events associated with an enhanced breast cancer risk include reproductive factors , nulliparity , radiation exposure , hormonal status , obesity , family history , and many others [ 3 , 4 ] . breast cancer is a heterogeneous disease in which genetic and environmental factors interact to initiate carcinogenesis . however , 10% of all breast cancer cases have a strong hereditary component in which half carry a deleterious mutation in the high penetrance genes brca1 or brca2 . these account for over 50% of familial breast cancer cases and confer a lifetime risk of 6080% . in its simplest forms , breast cancer can be subclassified into preinvasive and invasive disease categories . neoplastic conversion to invasive cancer likely occurs sometime during the preinvasive histological phases of usual hyperplasia , atypical hyperplasia , and ductal carcinoma in situ ( dcis ) [ 611 ] . one hypothesis suggests the existence of genetically distinct subgroups of dcis , only some of which subsequently progress to invasive ductal carcinoma ( idc ) [ 1214 ] . an alternate theory proposes that dcis progresses from low to high grades and then to invasive cancer with progressive accumulation of genomic changes . however , the large extent to which the genome is altered in dcis indicates that genomic instability most likely precedes phenotypic evidence of invasion , and highlights the importance of environmental components on the development of invasive cancer . recent data have shown significant reductions in the mortality rates of breast cancer , which have been mainly attributed to improved screening techniques , improved surgical and radiotherapy interventions and also the utilization of traditional chemotherapies in a more efficacious manner . large - scale translational research studies have also identified many important new biomarkers predictive of poor prognosis in breast cancer patients [ 1517 ] . however , much remains to be understood about the development and progression of breast cancer . our review will address the contribution of altered epithelial cell - cell adhesion to the development and progression of breast cancer , with particular emphasis on the role of the tight junction ( tj ) adhesion complex in these processes . cell - cell adhesion is necessary for the assembly of coherent sheets of barrier - forming epithelial cells that line the breast ducts and lobules . however cell - cell contacts are far from being static structures which maintain barriers by simply holding cells together . in fact cell - cell contacts undergo constant remodelling to allow the extrusion of apoptotic cells as well as the incorporation of newly differentiated epithelial cells , derived from progenitor cells , without loss of barrier function . cell - cell contacts must also be remodelled depending on the developmental stage of the breast , whether in response to increased proliferative demands of puberty and pregnancy , increased differentiation during lactation , or increased apoptosis in conjunction with gland remodelling during involution . finally , during wound healing , epithelial cells can undergo coordinated movement and proliferation to bridge the wound , and establish new cell - cell contacts with epithelial cells from the opposing side of the wound . epithelial cell - cell contacts consist of three main adhesive structures : tight junctions ( tjs ) , adherens junctions and desmosomes , as well as gap junctions for cell - cell communication ( figure 1 ) . in polarized epithelial cells the tight junction and adherens junction are asymmetrically distributed at the apical region of the lateral membrane forming the apical junctional complex , which encircles the apex of the cells and marks the border between the apical and basolateral membrane domains . these adhesive structures are composed of integral membrane proteins that link the neighbouring cells through homophilic and heterophilic interactions , and the presence of cytoplasmic scaffolding proteins that organise signalling complexes and anchor cell - cell contacts to the actin cytoskeleton ( or intermediate filaments in the case of desmosomes ) . in this review we will first outline the protein components of the tj and discuss the biological roles of the tj complex , review how alterations in these roles could facilitate breast cancer initiation or progression , and finally mention pharmacological approaches towards targeting tj proteins that could have value in limiting breast cancer progression . proteins within tj can be grouped into integral membrane proteins , scaffolding proteins , or signalling proteins as outlined in what follows . the first protein to be discovered at the tight junction , occludin , is a 68 kda transmembrane protein with two extracellular loops and a long cytoplasmic tail containing several protein - binding domains . it exists in unphosphorylated and serine / threonine and tyrosine phosphorylated forms , with the degree of phosphorylation affecting tight junction assembly , transepithelial resistance , and localisation of occludin to the tight junction [ 24 , 25 ] . several enzymes are involved in regulating these phosphorylation events , including pkc , ck2 , and the nonreceptor tyrosine kinase c - yes . the claudin family of tj transmembrane proteins consists of 24 members between 20 and 27 kda in size , mostly with short cytoplasmic tails which bind to the pdz ( psd-95 , dlg , and zo-1 ) domains of other tj proteins including zo-1 , -2 , and -3 . various claudins are expressed in a tissue - specific manner , with the subtle differences in their extracellular loops determining ion selectivity of the paracellular pathway [ 29 , 30 ] . the junctional adhesion molecule ( jam ) family consists of jam - a , -b , -c , -l and jam-4 , which are found at tjs of epithelial / endothelial cells and on various hematopoietic cells [ 3133 ] . they contain an extracellular region with two ig - like domains , a single transmembrane domain , and a short intracellular tail with a pdz binding motif through which jam - a interacts with the pdz proteins af6 , par-3 , cask , mupp1 , and zo-1 [ 3438 ] . the coxackie and adenovirus receptor ( car ) is a 46 kda integral membrane protein with one transmembrane region , a long cytoplasmic tail , and an extracellular region composed of two ig - like domains . the carboxyl terminal domain of car contains a pdz binding motif which interacts with zo-1 , mupp-1 , magi , and pick1 [ 3941 ] . crumbs3 ( crb3 ) is a single - pass membrane protein located at the apical cell membrane with a small fraction in the upper part of tight junctions of epithelial cells . it is involved in the establishment of cell polarity in mammalian epithelial cells and regulates the morphogenesis of tight junctions . crb3 tricellulin is a 66 kda membrane protein which has multiple phosphorylated states but exists predominantly in its unphosphorylated form . it is enriched only at tricellular tight junctions , where it enforces the barrier function of epithelial cell sheets . zona occludens ( zo ) family members zo-1 , zo-2 , and zo-3 belong to the maguk family of tj - associated scaffolding proteins . zo proteins interact with each other [ 47 , 48 ] as well as with cingulin , claudin-18 , actin , -catenin , and occludin . interactions vary between family members , as illustrated by the fact that zo-3 can interact with patj while only zo-1 can interact with jams a - c [ 34 , 37 ] , egfr , and af-6 . in addition , zo-1 binds the y - box transcription factor zonab and the heat shock protein apg-2 [ 54 , 55 ] . afadin ( af-6 ) is a ras - binding , pdz domain - containing scaffolding protein which interacts with nectin , jam - a , zo-1 , profilin , ponsin , rap1 , and signal - induced proliferation - associated protein 1 ( spa-1 , a rap1 gtpase activating protein ) [ 35 , 5658 ] . the nectin - af-6 complex is involved in the formation of adherens and tight junctions . the membrane associated guanylate kinase inverted ( magi ) family consists of magi-1 , magi-2 , and magi-3 . magi-2 was initially identified in rat as a protein interacting with n - methyl - d - aspartate receptors ( nmda - rs ) and neuronal cell adhesion proteins . magi-3 was identified in a two - hybrid screening as a protein interacting with the tumour suppressor pten , and receptor tyrosine phosphatase beta . domain , a coiled - coil rod domain , and a globular tail domain . in vitro binding studies have revealed that cingulin interacts with various components of tight junctions including jam , zo-1 , zo-2 , zo-3 , myosin and f - actin , suggesting a role for cingulin as a linker between the tj membrane and f - actin [ 49 , 6365 ] . cingulin also functions to sequester and inactivate the rhoa activator gef - h1 at tjs , resulting in inhibited rhoa signalling and g1/s phase transition [ 66 , 67 ] . loss or mutation of cingulin does not perturb the formation of tight junctions , but results in increased claudin-2 expression and cellular proliferation , which are dependent on increased rhoa activity . amot is a scaffolding protein with a coiled - coil region and a pdz binding motif , which forms a complex with the rho gtpase - activating protein rich1 and is targeted to the tight junction interaction with patj or mupp1 [ 70 , 71 ] . two amot - like proteins , jeap and mascot , have also been identified as tj proteins [ 72 , 73 ] which interact with mupp1 . atypical protein kinase c is located at the tj and plays a crucial role in maintaining tight junction structure and cell polarity through phosphorylation and stabilization of junction - associated proteins [ 74 , 75 ] . activation of classical protein kinase c and novel protein kinase c has been shown to disassemble tjs . in polarized epithelial cells the tj forms a belt - like structure at the apical - most region of the lateral membrane , and represents a boundary between apical and basolateral membranes . the main functions attributed to tjs are the regulation of paracellular permeability ( gate or barrier function ) , and the formation of a physical barrier preventing intramembranous movement of lipids and proteins ( fence function ) . gate function regulates the passage of ions and solutes across epithelial sheets in an organ - specific manner , and can be modified depending on the specific requirements of the organ [ 30 , 78 ] . fence function is required to maintain asymmetric distribution of membrane components and to develop membrane polarity . epithelial barrier function relies heavily on the claudin family of tj proteins , which form strands controlling selective permeability by forming size- and charge - selective aqueous pores [ 30 , 78 , 79 ] . epithelial fence function on the other hand is not solely reliant on one subset of integral membrane proteins but instead requires cooperation between integral membrane proteins and several tj scaffolding proteins and signalling molecules . epithelial cells control adhesion to the basement membrane and extracellular matrix to maintain an intact barrier that can reseal quickly in response to injury . breaching of the epithelial barrier stimulates cells to extend protrusions into the wound space , which can result in tj disruption and release of proteins such as patj , par3 , apkc , cdc42 , and par6 from their scaffolds . retargeting these ( and other ) proteins to the migrating edge helps polarize migrating cells in the direction of movement via reorientation of the golgi , centrosome and the microtubule cytoskeleton along the axis of migration [ 82 , 83 ] . tjs regulate epithelial polarity by controlling the assembly of three main polarity complexes ; the crb3 complex , the par complex , and the scrib complex , which will be discussed in the following section . the apical junctional complex serves to restrict the movement of these complexes in order to form distinct apical and basolateral domains . apico - basolateral polarity allows for terminal differentiation of epithelial barriers by apical orientation of the trans - golgi network , which can sort membrane proteins toward either apical or basolateral membranes . specialized membrane trafficking leads to the accumulation of receptors and channels in either apical or basolateral membranes , allowing electrochemical gradients to develop across epithelial sheets [ 84 , 85 ] . from the afore - mentioned information , it can be observed that tj proteins exert fundamental influences over cellular processes that regulate polarity , differentiation and migration ; all of which are processes central to cancer progression . therefore , tj and other cell - cell adhesion proteins are gaining increasing attention in breast cancer research [ 8689 ] . most work to date has focused on adherens junction proteins ( such as cadherins ) in breast cancer progression ; and in fact loss of e - cadherin is a defining feature of lobular breast carcinoma [ 90 , 91 ] . however , tj proteins have also been found to be dysregulated in several human cancers including breast , and have been suggested as promising targets for cancer detection , diagnosis , and therapy . in this review , we will attempt to summarize current knowledge on the impact of tj proteins on breast cancer progression ; based on the ability of tjs to control polarity , differentiation , and migration . our review will focus on three aspects whereby functional alterations in tjs may impact breast cancer progression by altering cell polarity , cell fate , and cell migration . for a broad overview of tj alterations in cancer metastasis of other tumours , formation of the tj adhesion belt allows the targeting of scaffolding proteins which regulate the cellular polarity machinery . this machinery is composed of three polarity complexes which identify separate regions of the cell . these polarity complexes were originally identified in c. elegans and drosophila , but have been found to be highly conserved in mice and higher mammals . the crb complex identifies the apical region due to apical concentration of crb3 , which is targeted to the tight junction by patj and zo-3 [ 51 , 94 ] . the par complex localizes at tjs through interactions between par-3 and jam - a . finally , the scribble ( scrib ) complex identifies the basolateral region of the cell , and is targeted to adherens junctions through interaction of scrib and dlg with e - cadherin [ 9598 ] . the crb complex is the most apically located polarity complex in epithelial cells , and acts as an apical anchor for the targeting of cytoplasmic proteins during polarisation . it is composed of the transmembrane protein crb3 and the scaffolding proteins pals1 and patj . for example , crb3 and patj expressions were shown to be repressed by the transcription factor zeb1 , which is upregulated in invasive ductal and lobular breast cancers . zeb1 has been implicated in epithelial to mesenchymal transition ( emt ) , a dedifferentiation programme associated with cancer metastasis in which epithelial junctions and cell polarity are disrupted , contributing to increased cell motility . patj also binds a negative regulator of mtor called tsc2 , which regulates survival , apoptosis , and cell cycle progression [ 101103 ] . the mtor pathway has been shown to be frequently deregulated in various cancers including breast [ 16 , 18 ] . reported that patj knockdown in intestinal epithelial cells resulted in the upregulation of the mtor pathway ; and it is possible that loss of patj in cancers such as breast could facilitate tumour progression by allowing the prosurvival effects of mtor activation to go unchecked . patj is also important for the proper localisation of claudin-1 , zo-3 , crb3 , occludin , apkc , and zo-1 at tjs [ 51 , 94 , 105 ] . loss of patj could , therefore , also promote leaky junctions , resulting in increased access of luminal growth factors to the basolateral epithelial surface . it is intriguing to speculate that this leakiness could promote tumour progression by feeding the developing tumour , as proposed in the nutritional model of carcinogenesis . this along with the dysregulation of apkc has the potential to induce substantial increases in proliferation as well as a loss of polarity , all of which are hallmarks of cancer progression . other members of the crb polarity complex could also play a role in breast cancer progression . knockdown of the pals1 binding partner lin-7 in renal epithelial cells was shown to reduce expression levels of pals1 , pals2 , dlg2 , dlg3 , and patj . a resulting failure to recruit apkc to tjs resulted in reduced epithelial barrier function , delayed polarization , and impaired lumen clearance in three - dimensional morphogenesis models [ 107 , 108 ] . loss of pals1 also resulted in defects in e - cadherin trafficking , which , taken together , suggests that analagous disruption of the crb3 complex during breast cancer could impair barrier function and polarity , and potentially facilitate occlusion of breast duct lumens with tumour cells . the par complex is made up of par3 , par6 , apkc , and cdc42/rac1 . recently , par6b was reported to be transcriptionally upregulated in breast cancer tissues by quantitative pcr . interestingly , mcf-10a breast epithelial cells overexpressing par6 polarized normally in three - dimensional culture models , but showed higher proliferation rates which were dependent upon par6 interactions with apkc and cdc42 . increased par6 signalling has also been reported in mcf-10a cells overexpressing activated erbb2 [ 111 , 112 ] , the growth factor receptor which is amplified in 2530% of breast cancers and which identifies a subtype of highly aggressive tumour . activation of erbb2 in these cells induces the formation of multiacinar structures with abnormal filled lumens , in a manner dependent on interactions of erbb2 with the par6-apkc complex . mutation of par6 in cells overexpressing activated erbb2 was observed to restore lumen formation , suggesting an inhibitory tone of par6-erbb2 interactions on apoptotic clearance of developing lumens . the role of par6 in apoptosis is suggested to be due to the activation of apkc by par6 . par6-apkc interactions have also been shown to activate rac1 in non - small cell lung cancer cells , resulting in anchorage - independent growth and invasion through activation of matrix metalloproteinase-10 ( mmp-10 ) expression [ 115 , 116 ] . thus dysregulation of par6 in cancer cells has the potential to impact tumour progression via direct effects on polarity , migration , and even apoptosis . in contrast to par6 , par3 expression has been found to be reduced in oesophageal squamous cell carcinomas in association with lymph node metastasis and poor differentiation . certain forms of emt have also been shown to downregulate par3 expression , with par3 overexpression capable of rescuing the loss of e - cadherin during emt in a rat kidney epithelial model . given the putative link between emt induction and breast cancer progression , it will be interesting to uncover whether par3 expression might also be lost in breast cancer . since par3 regulates par6 , loss of par3 , in turn , is likely to exert an influence over the control of proliferation , polarity , and apoptosis resistance by par6 signalling in cancer cells . the scribble complex is an evolutionarily conserved complex consisting of three members , scrib , lethal giant larvae homolog ( lgl ) , and discs large homolog ( dlg ) . loss of function mutations of scrib , dlg , and lgl in drosophila have demonstrated abnormal cell polarity with increased proliferation without tumour cell overgrowth , possibly due to increased apoptosis [ 118120 ] . scrib has been shown to colocalise with dlg and e - cadherin at adherens junctions , and is required for formation of this junction and proper localisation of dlg and lgl as well as apical targeting of crb3 [ 97 , 121 ] . scrib staining was shown by immunohistochemistry to be reduced and mislocalized in human breast cancer tissues [ 98 , 122 ] . zhan et al . have suggested a role for scrib in breast cancer development by reducing apoptosis in c - myc over - expressing breast epithelial cells . activation of c - myc enhanced the formation of a scrib complex which activated the small gtpases rac and cdc42 and increased the expression of a proapoptotic protein bim . conversely , loss of scrib suppressed the ability of c - myc to induce bim expression ; suggesting a mechanism for reduced apoptosis and increased resistance of breast cancer cells to cytotoxic stresses in the event of scrib loss . lgl is a cytoplasmic protein which is targeted to the lateral epithelial membrane during polarisation . like crb3 and patj , lgl1 is repressed by the transcription factor zeb1 , whose expression is upregulated in several forms of breast cancer . therefore , dysregulation of lgl may play a role in emt events associated with breast cancer progression . alterations in the final member of the scrib complex , dlg , may also play a role in cancer progression . dlg3 has been shown to be reduced in gastric carcinoma ; whilst overexpression of dlg1 and dlg3 inhibits cellular proliferation via a block in g1/s phase transition of the cell cycle [ 125 , 126 ] . dlg4 interacts with frizzled proteins to regulate the wnt signalling pathway , inappropriate activation of which has been implicated in oncogenesis due to myriad influences on cell adhesion , migration , proliferation , and cell death . dlg1 and dlg3 also regulate wnt signalling through dlg3-mediated -catenin degradation and the binding of dlg1 to apc and by modulating the antiproliferative effects of apc [ 125 , 126 , 129 ] . in unpolarized cells , dlg1 is ubiquitinated and degraded , and only upon junctional formation is it hyperphosphorylated and stabilised . this indicates that the integrity of cell - cell contacts regulates the scrib polarity complex and that disruption of this complex promotes dysregulated growth and resistance to apoptosis . as illustrated above , alterations in crb3 , scrib , and par polarity complexes can promote proliferation , cell cycle progression and evasion of apoptosis as a result of disrupted apical - basolateral polarity in a variety of models . although several of the seminal observations were originally made in drosophila and c. elegans , many findings have since been confirmed in higher mammals and during in vitro studies on human breast and other carcinoma cell lines . thus insights from simple organismal models of polarity are highly relevant not only to the control of normal human physiology by the polarity machinery but also to the development of many cancers including breast . however , abnormalities in the polarity machinery are only one of several ways in which tj dysfunction can impact upon breast cancer progression . the role of tj - mediated alterations in cell fate will be discussed in the following section . although cancer is frequently considered as a disease of abnormal proliferation , cancer progression is not determined solely by proliferative advantage within tumour cells . other factors such as apoptosis resistance and the ability to bypass senescence pathways contribute to an environment supporting breast cancer progression . the role of individual tj proteins in modulating these aspects of breast cancer progression will be addressed in what follows . occludin expression is known to be downregulated in several cancers including breast ; its loss correlating with glandular dedifferentiation and progression of human endometrial , colorectal , and lung carcinomas [ 132134 ] . in recent studies , occludin overexpression was found to promote detachment - induced apoptosis ( anoikis ) in ac2m2 murine breast carcinoma cells , while endogenous occludin re - expression correlated with downregulation of apoptosis - inhibitory genes ( bcl-2 , survivin ) and upregulation of apoptosis - inducing genes ( apaf-1 , bax ) . tunel assays also revealed that hela cells constitutively overexpressing wild - type occludin exhibited increased sensitivity to oxidant - induced apoptosis . occludin overexpression was also shown to induce premature senescence in ac2m2 cells , as assessed by increased senescence - associated -galactosidase enzymatic activity and the upregulation of negative cell cycle regulators such as p16ink4a , p21waf1/cip1 , and p27kip1 but not p53 . the ability of cells to autoinduce growth arrest based upon the expression of tj proteins such as occludin could has a profound inhibitory effect on tumour growth , and illustrates how significant a loss of such proteins could be for tumour progression . similarly to occludin , claudin-1 , -4 , -6 have also been reported as downregulated in breast cancer [ 131 , 136139 ] . suppression of endogenous claudin-6 expression by sirna in mcf7 breast cancer cells increases resistance to oxidant - induced apoptosis and anoikis , thereby promoting colony formation in two- and three - dimensional cultures . in a complementary approach , forced induction of claudin-1 expression in mda - mb-361 breast cancer cells resulted in elevated apoptosis in three - dimensional cultures . enhanced apoptosis correlated with increased spheroid size , suggesting a positive effect of nutrient and growth factor diffusion into spheroids this supports the hypothesis that cancer formation may be promoted in premalignant epithelial tissues that have become chronically leaky to growth factors . claudin-1 may also play a role in the control of cell fate , with observations of increased expression in senescent breast epithelial cells and reduced expression in invasive breast cancers . in an interesting contrast , expression of claudin-3 , -4 and -7 have actually been observed to increase in both breast and ovarian cancers [ 88 , 142145 ] . overexpression of claudin-3 and -4 in hose - b ovarian cells enhanced cell survival in clonogenic assays , further supporting a role for either upregulation or downregulation of key claudins in the controlling cancer cell fate . occludin - interacting proteins zo-1 and zo-2 have also been shown to be repressed with cancer progression [ 52 , 146 , 147 ] , where decreased zo-1 staining correlates with decreased glandular differentiation of breast tumour specimens . zo-1 and zo-2 regulate cell cycle progression and proliferation in a cell density - dependent manner [ 148151 ] through transcription factors such as zonab . zonab localizes to the nucleus and the tjs in proliferating mdck cells , but is not detectable in the nucleus of nonproliferating high - density cells . evidence indicates that cytoplasmic zonab immunoprecipitates with both cdk4 and cyclin d1 and assists in the nuclear accumulation of cdk4 , promoting g1/s phase transition and cell cycle progression . zonab also upregulates erbb2 expression , which ( as discussed earlier ) could profoundly impact progression of a subset of breast cancers . zo-2 blocks cell cycle progression by downregulating cyclin d transcription and inhibiting cdk2 and cdk4 [ 150 , 152 ] . zo-2 also controls cyclin d expression and interacts with the transcription factors jun , fos , and c / ebp to regulate proliferation . thus , it can be observed that zo proteins control cellular proliferation in a density - dependent manner by sequestering transcription factors at the tight junction . loss of zo proteins during breast cancer may , therefore , promote proliferation via a loss of control over cell cycle progression . finally , in addition to the many tj structural proteins which exert regulatory control over cell fate , signalling proteins affiliated with the tj complex could also play a part in breast cancer progression . for example , many small gtpases have been described to affiliate with the tj complex . as key signalling molecules which regulate actin dynamics , for example , rhoa promotes cell cycle progression through the regulation of p21 and p27 levels . tjs regulate rhoa activity by cingulin - mediated sequestration of the rhoa activator gef - h1 and inhibition of g1/s phase transition . increased gef - h1 levels can arise by mutations in p53 , a frequent genetic alteration observed in breast cancer . these alterations suggest a relationship between tj alterations and the malignant potential of several carcinomas , via deficits in controlled proliferation , regulated cell cycle progression and apoptosis . this suggests that the dysregulation of cell - cell contact machinery may be a prerequisite for cancer progression in order to turn off specific epithelial regulatory pathways . the loss of membrane polarity via tight junction abnormalities may also alter cell - cell and cell - extracellular matrix interactions , and might , therefore , facilitate migration , invasion , and the development of metastasis , which will be reviewed in the following section . as discussed earlier , altered cell - cell adhesion contributes to a loss in polarity and contact inhibition , culminating in uncontrolled proliferation during breast cancer initiation . there is also evidence that altered cell adhesion plays a fundamental role in breast cancer progression by freeing tumour cells from both neighbouring cells and the underlying matrix ; and in parallel by conferring a motile or migratory advantage to cells during invasion and metastasis [ 19 , 157 , 158 ] . in this section , we will attempt to summarize current knowledge on the impact of tj proteins on breast cancer progression . occludin has been linked with cancer progression in endometrial carcinoma ; where decreasing expression was correlated with increasing grade , myometrial invasion , and lymph node metastasis . forced expression of occludin in breast cancer cells has been shown to decrease cancer cell migration and invasion both in vitro and in vivo . interestingly , the occludin gene can be silenced by hypermethylation , and it may be that the methylator phenotype promotes tumourigenic , invasive , and metastatic properties of cancer cells . a wealth of evidence has also implicated the claudin family in breast cancer cell migration . tumours with low expression of claudin-3 , -4 and -7 and high expression of stem cell and epithelial - mesenchymal markers were recently shown to associate with poorer overall survival , as mirrored in other studies . like occludin , claudin-1 protein levels are reportedly reduced in breast tumours and breast cancer cell lines . importantly , claudin-1 has been detected in the membranes of normal breast ductal epithelial cells and in some dcis tumour cells , but is frequently absent from invasive tumours . furthermore , claudin-1 expression has been demonstrated as a good predictor of disease recurrence and malignant potential in breast cancer . demonstrated that recurrent breast tumours displayed significant reductions in claudin-1 expression compared to primary tumours ; while reduced claudin-1 expression has also been associated with lymph node involvement and decreased disease - free survival . taken together furthermore , mda - mb-361 breast cancer cells deficient in claudin-1 grow as multicellular filled spheroids in three - dimensional cultures and re - expression of claudin-1 induces central lumen formation ; perhaps by nutritional deprivation of innermost cells inducing apoptosis as already discussed . other than claudin-1 , the loss of several other family members has been reported in breast cancer [ 142 , 162166 ] . osanai et al . demonstrated that decreased expression of claudin-6 in breast cancer cells ( by sirna or epigenetic silencing ) increases mmp activity , likely facilitating increased cancer cell migration and invasion . expression of claudin-7 has been shown to be reduced in idc cells compared to those of the normal breast , and reduced in fine needle aspirates from idc patients . in both studies , loss of claudin-7 expression also correlated with increasing tumour grade and metastatic disease . in an intriguing study , treatment of mcf7 breast cancer cells expressing high levels of claudin-7 with hgf / scatter factor ( which decreases cell - cell adhesion ) led to a dramatic decrease in claudin-7 expression , further linking the loss of claudin-7 and cell cohesion in breast cancer . most recently , forced expression of claudin-16 in mda - mb-231 breast cancer cells has been reported to induce junctional formation and concurrently reduce aggressive and motile behaviour in vitro and in vivo . in contrast to the reduced expression of several claudins , claudin-3 and -4 have in fact been found to be elevated in breast cancer at both mrna and protein level [ 143145 , 167 ] . interestingly , overexpression of either claudin-3 or claudin-4 in human ovarian epithelial cells has been reported to increase migratory and invasive capabilities . as discussed later in this review , the ability of both claudins-3 and -4 to function as receptors for clostridium perfringens enterotoxin ( cpe ) may provide a unique targeting mechanism to eliminate cancer cells overexpressing these proteins . jam proteins regulate numerous cellular adhesive processes including intercellular junction assembly , cell morphology , and leukocyte migration [ 171 , 172 ] ; while jam - a dysregulation has recently been implicated in breast cancer [ 173 , 174 ] . jam - a has been shown to regulate epithelial cell morphology and enhance 1-integrin expression through modulation of rap1 gtpase activity . since loss of tissue architecture and cell polarity is a prerequisite for breast cancer invasion and metastasis , disruption of jam proteins may , therefore , play key roles in disease progression . indeed , disruption of jam - a in a colonic carcinoma cell line was shown to convert cells from a stationery , polarized state to a migratory phenotype . recently , naik et al . reported that jam - a overexpression decreased migration and invasion in breast cancer cell lines , while knockdown of jam - a expression enhanced invasiveness . it was hypothesized , therefore , that the loss of jam - a may correlate with poor clinical prognosis . however , a subsequent study by mcsherry et al . revealed a significant association between high jam - a gene and protein expression and poor survival in 2 large cohorts of human invasive breast cancer tissue specimens . furthermore , knockdown or antagonism of jam - a significantly decreased migration in mcf7 breast cancer cells expressing high endogenous levels of jam - a . the apparent conflict between these two studies may be resolved by the fact that underexpression of jam - a is likely to impair cellular adhesion and polarity ( favouring tumour initiation ) , whereas overexpression of jam - a could promote integrin - mediated migratory events that favour tumour progression . these data clearly implicate an imbalance of jam - a expression patterns in breast cancer , and , as discussed later , may also form an interesting therapeutic target . few studies have specifically focussed upon the involvement of tj adaptor and signalling proteins in breast cancer progression . however , zo-1 loss has been linked to poor prognosis in breast cancer , with significantly reduced levels of tj - associated zo-1 in patients with metastatic disease compared to those remaining disease - free . polette et al . also showed that expression of the matrix metalloproteinase mt1-mmp in invasive breast tumour cell lines is correlated with cytoplasmic localization of zo-1 and with occludin loss . zo-1 has also been shown to be reduced or lost in ductal breast cancer tissues , in parallel with increased dedifferentiation . reduced zo-1 expression has been significantly associated with reduced e - cadherin expression ; whose loss is inextricably linked with lobular breast cancer . furthermore , zo-1 has been reported to play an important role in controlling expression of the erbb2 gene . downregulation of another family member , zo-2 , has also been reported in breast carcinoma . in addition , zo-2 has been reported as crucial for the tumour - inducing capabilities of the adenovirus type 9 e4 protein . expression of mutant zo-2 protein lacking the e4 binding site inhibits e4-mediated tumour initiation in mammary glands [ 180 , 181 ] . taken together , it can , therefore , be considered that zo proteins play important roles in the migratory events associated with breast cancer progression . the par complex ( par3-par6-apkc ) promotes normal junction assembly by regulation of actin dynamics and is known to be altered in many cancers including breast ( reviewed in ) . indeed , as previously addressed in this review , par6 , through association with apkc and erbb2 , has been shown to disrupt apical - basal polarity and protect cells from apoptosis . other potential links between par proteins and breast cancer involve emt and the regulation of rho family small g proteins including rac , rho , and cdc42 . for example , par6 reportedly interacts with tnf in inducing emt and tj loss via degradation of rhoa . in vitro experiments have revealed that the par complex along with rac signalling stabilizes front - rear polarization of noncontacting keratinocytes , thereby stimulating chemotactic migration . indeed , important biological processes such as migration and invasion are highly regulated by the rho family . the rho guanine nucleotide exchange factor ( gef ) tiam has been shown to increase with increasing breast tumour grade or cell line invasiveness ; and tiam - mediated rac1 activation has been correlated with tumour cell migration and invasion in vitro . integrin - mediated adhesion through rho family gtpase activity has been reported as essential in regulating cell polarity and membrane protrusiveness . specifically , rac1 and cdc42 have been linked with integrin - mediated motility and invasion through pi3k signalling in breast cancer cell lines . furthermore , the rap gtpase rap1 has been identified as a crucial signalling element downstream of 1 integrin , responsible for regulating breast acinar structure and inducing mammary gland lumen formation . yet another signalling protein downstream of tjs , pkc , has also been linked to cancer initiation and progression . pkc overexpression and altered localization has been demonstrated in breast cancer , and pkc signalling is required for egf - induced chemotaxis of human breast cancer cells . this wealth of evidence indicates the potential involvement of several tj adhesion cascades in the migratory events associated with breast cancer progression . further studies on these proteins will allow a more comprehensive understanding of their behaviour and contributions to tumour progression , ultimately defining candidate breast cancer prognostic markers . the study of compounds designed to specifically target and block the action of adhesion proteins involved in cancer invasion could be of substantial therapeutic benefit in preventing breast cancer invasion . targeted therapeutic agents for breast cancer represent a growing proportion of new drugs entering clinical testing . since carcinogenesis is a multistep process characterized by alterations in many key growth and development pathways , selection of appropriate drug targets and the ability to effectively deliver drugs to those targets are pivotal issues in drug development . this section will review current knowledge on tjs as breast cancer drug targets , and as targets for therapeutic modulation of cancer drug delivery . in spite of the regulatory influences exerted by tjs on diverse processes relevant to cancer progression ( as discussed in previous sections ) , there are currently no cancer therapies on the market which specifically target tjs . however , clues to potential tj targets of value have come from many sources , including translational research studies involving patient databases . for example , overexpression of claudin-3 and -4 proteins has been demonstrated in over 90% of primary breast carcinomas in a patient group of 188 , and in 6080% of breast tumours in a tissue microarray of 314 patients . claudin-3 and -4 overexpression has also been noted in other neoplasias including ovarian , prostate , pancreatic , and endometrial [ 193196 ] . these proteins form an intriguing potential target for cancer therapies , since both claudin-3 and -4 have been identified as the receptor for clostridium perfringens enterotoxin ( cpe ) and the only claudin family members capable of mediating cpe binding and cytolysis [ 195198 ] . cpe is a well - known virulence factor responsible for the gastrointestinal symptoms associated with c. perfringens type a food poisoning . by inducing permeability alterations in host intestinal epithelial cells , cpe is thus a multifunctional toxin with cytotoxic , tj - damaging , and proinflammatory activities [ 199 , 200 ] . this ability of cpe to rapidly and specifically lyse cells expressing claudin-3 or -4 could potentially be exploited in the treatment of breast cancers overexpressing these proteins . accordingly , it has been shown that claudin-3 and -4 expressing breast cancer cell lines grown in cell culture and as xenograft tumours underwent rapid and dose - dependent cytolysis in response to cpe treatment . even more promisingly , administration of cpe has been shown to reduce the growth of claudin-4 overexpressing human ovarian and pancreatic tumours . thus , local delivery of native cpe may be useful in the treatment of preneoplastic lesions such as dcis and in neoadjuvant settings such as the locoregional control of locally advanced breast carcinoma , as well as in tumour downstaging to allow breast conservation therapy . in addition to this , the documented ability of cpe to downregulate the epithelial barrier through interference with claudin-3 and -4 may enhance local drug delivery for other treatment modalities . firstly ( as discussed in prior sections ) , loss of tj - based adhesion may imbalance cellular polarity which by itself is likely to be protumourogenic . secondly , claudin-3 and -4 are expressed in several normal human tissues including gut , lung , and kidney ; therefore the potential high toxicity of cpe at doses used for systemic cancer therapy in animal models might limit its use in humans to local treatments [ 194 , 202 ] . claudin-3 and -4 overexpression in breast cancer could alternatively be targeted by toxin- or radionuclide - conjugated antibodies , which would either destroy the cancer cells directly or target them for attack by the host immune system . the high sequence identity between claudin-3 and -4 may allow generation of antibodies recognizing both proteins . potential indications for anticlaudin antibody - based therapeutics include carcinomas of colorectal , breast , ovarian , and prostate origin . in common with the discovery of claudin-3 and -4 as the cpe receptor , the tj protein car acts as the primary site for adenovirus attachment during infection , a feature which has been exploited as a delivery mechanism for gene therapies [ 204 , 205 ] . car expression has been shown to significantly increase in breast tumour tissue along with increasing tumour grade . breast tissue samples showing elevated car expression have been associated with poor patient prognosis . while the biological roles of car are incompletely defined whether car overexpression in breast cancer could be successfully targeted by nonpathogenic components of the virus in order to diminish cancer cell proliferation remains an intriguing question . gumbiner et al . showed that occludin homotypic interactions and turnover , but not synthesis , could be affected by treating cells with peptides to the extracellular loop of occludin [ 208 , 209 ] . also identified occludin peptides capable of binding tj structural and signalling proteins , and demonstrated that the second extracellular loop of occludin regulates cellular transformation by oncogenes such as raf-1 . given that occludin has been reported to be dysregulated in some breast cancers , it is intriguing to speculate that occludin could be a target for peptide - based cancer drugs . another tj protein implicated in breast cancer progression is zo-1 , which as noted , has sequence homology with the drosophila tumour suppressor dlg , implying that zo-1 could possess similar functions as a tumour suppressor gene in mammalian epithelia . observed decreased zo-1 staining in several invasive breast cancer cell lines supporting the tumour - suppressive characteristics of zo-1 . even more recently , it has been observed that increased jam - a expression in human breast cancer tissues correlates with poor patient prognosis . since this mechanism is thought to involve promotion of integrin - mediated migratory events at the cell - matrix interface , it is , therefore , interesting to speculate that targeting jam - a dimerization to reduce signalling could be a promising and novel target to reduce breast cancer cell motility during the early stages of invasion or metastasis . a final point regarding tjs as breast cancer therapeutic targets relates to drug delivery in order for therapeutic agents to reach their target in vivo , they must cross epithelial and/or endothelial barriers . since the tj is the primary regulator of paracellular transport across such cells , successful drug delivery may require modulation of tj proteins to allow drug molecules to pass . however , as before , it must be noted that disruption of tj proteins purely for drug delivery purposes may itself promote cancer progression by upsetting homeostatic mechanisms of polarity , differentiation , cell fate , and migration which are tightly regulated by tjs in normal tissues . to conclude this section , we note that therapeutic modulation of breast cancer via selective targeting of tight junction structural proteins is in its infancy . at present , cpe offers the best - developed strategy via targeting of claudin-3 and -4 . while many signalling proteins and enzymes loosely affiliated with the tj plaque may prove easier pharmacological targets , full discussion of this topic is beyond the scope of this review . it is clear however that further investigations into the cell biology of tight junctions are necessary to provide insights into putative future applications of tj components as candidates for drug discovery to prevent or limit breast cancer progression . finally , we summarize the role of tjs in breast cancer initiation and progression as follows ( see figure 2 ) . during the initiation phase of cancer , fundamental alterations in the tj complex may impair its functional control over important cellular processes such as polarity and cell fate determination , or cell motility characteristics . dysregulation of either of these aspects likely contributes to the pathologies which we recognise as ductal breast carcinoma in situ or invasive ductal carcinoma . further dysregulation of a combination of these ( and other ) events is likely to be required for the most serious step of breast cancer progression , the transition to a metastatic phenotype . it must be noted that there is not a simple relationship between tj protein loss or gain and breast cancer . as we have described in this review , both loss and gain of tj proteins can impart a growth advantage to breast cancer cells , as well as increased resistance to apoptosis , loss of polarity , and increased migratory or invasive characteristics . through these important regulatory influences on polarity , cell fate and cell movement , we suggest that an intact and functional tj complex acts as a barrier to the initiation and progression of breast cancer . however , any imbalance in the protein components of this complex ( whether increased or decreased ) will , in turn , imbalance the strict homeostatic control required to maintain breast tissue in its differentiated state , increasing the risk of inducing a pathologically dedifferentiated state such as breast cancer .

breast cancer is a complex and heterogeneous disease that arises from epithelial cells lining the breast ducts and lobules . correct adhesion between adjacent epithelial cells is important in determining the normal structure and function of epithelial tissues , and there is accumulating evidence that dysregulated cell - cell adhesion is associated with many cancers . this review will focus on one cell - cell adhesion complex , the tight junction ( tj ) , and summarize recent evidence that tjs may participate in breast cancer development or progression . we will first outline the protein composition of tjs and discuss the functions of the tj complex . secondly we will examine how alterations in these functions might facilitate breast cancer initiation or progression ; by focussing on the regulatory influence of tjs on cell polarity , cell fate and cell migration . finally we will outline how pharmacological targeting of tj proteins may be useful in limiting breast cancer progression . overall we hope to illustrate that the relationship between tj alterations and breast cancer is a complex one ; but that this area offers promise in uncovering fundamental mechanisms linked to breast cancer progression .

generally pediatric elbow dislocations are complex traumas that are associated with fractures around elbow , medial epicondyle avulsion fractures and median nerve injuries . the purpose of this study is to present an 8-year - old patient who has open elbow dislocation without fracture accompanying with brachial artery injury . an 8-year - old boy had admitted to our emergency department after falling on his left hand with outstretched upper extremity . the patient had admitted to our clinic 30 min after trauma . in the clinical examination of the patient distal humerus and articular surface could be seen by looking through the wound ( fig . although neurological examination showed us no pathology , distal pulses could not be palpated in vascular examination . in the conventional radiographies , a posterior elbow dislocation without accompanying fracture because of the strong possibility of brachial artery injury , the patient had consulted to the vascular surgeon and an emergent surgical intervention had planned for this patient . the transverse incision had lengthened 1 cm and the buttonhole dislocation had released . following the exploration of the open wound in the elbow , open reduction was performed . brachial artery was identified in the open wound without continuity and distal pulses were still impalpable . the vascular surgeon had attended to the surgery and the brachial artery had repaired primarily . after the treatment of vascular pathology , the stability of the elbow was evaluated and the elbow was stable during 0140 of flexion and extension . due to the stability of the elbow , we decided to follow this patient with splint instead of an external fixator . the wound was closed in a standard fashion with the approval of vascular surgeon ; upper elbow splint was applied at 100. we did not observe any postoperative vascular or wound complications in the follow - up . after 4 weeks follow - up with splint , using angle adjustable hinge elbow orthotics started controlled active assistive exercises . by the end of the 12th week , the orthotics was removed and movement was completely allowed . the patient 's elbow range of motion , in supination pronation arc , has been detected as 8085 and in extension hastings class ii heterotopic ossification was detected between the anterior capsule and the brachialis muscles ( fig . posterior dislocation is the most common direction for these injuries and mostly there are associated fractures around elbow . elbow dislocation without fracture in pediatric patients is a very rare injury . according to our knowledge usually the trauma mechanism of elbow dislocation is falling on outstretched hand with elbow in approximately 30 of flexion . although this type of trauma mechanism is typical for supracondylar humerus fractures in pediatric age group , in our patient an open posterior elbow dislocation without fracture had occurred . concomitant vascular injuries and open dislocations are classified as complex dislocations that are often fracture dislocations . in these complex elbow dislocations associated pathologies like compartment syndrome , neurological injury ( 10% ) ( most often the median nerve ) , vascular injuries ( 68% ) , and complications like residual instability and heterotopic ossifications are observed in the treatment of these rare injuries . in the literature , complex open elbow dislocations without fracture have not been well described in pediatric population . in the pediatric population , sharma et al . presented 12-year - old boy who had a closed posterolateral dislocated elbow with a concurrent milch type ii lateral condyle fracture . medial epicondyle avulsion fractures may be associated with complex pediatric elbow dislocations . in some dislocated elbows the clinician should beware of possible entrapment of medial epicondyle in the joint especially after closed reduction . especially before the ossification of medial epicondyle ( under the age of 7 ) , mistaken of this pathology is more possible . pediatric elbow dislocations are rare injuries and the management of these injuries can be technically demanding due to concurrent neurovascular injuries . an open dislocation without fracture is very rare and it should be treated with immediate intervention , an effective teamwork and good rehabilitation . gkhan polat and gkhan karademir , hasan h ceylan helped in english language editing and writing the manuscript .

introductionelbow dislocations in children are rare injuries . these injuries are often in the form of complex injuries that is accompanied by the median nerve damage and medial epicondyle fracture in the pediatric age group . open elbow dislocation without fracture in the pediatric age group has been reported very rarely in the literature.presentation of casethe purpose of this study is to present an 8-year - old patient who has open elbow dislocation without fracture accompanying with brachial artery injury . in the clinical examination of the patient , there was an open wound in the transverse antecubital region . after repair of brachial artery injury , open reduction was performed under general anesthesia . in the postoperative clinical examination at 6 months , left elbow flexion was 140 , extension was full and there were no deficit in the supination and pronation of the forearm.discussionelbow dislocation without fracture in pediatric patients is a very rare injury . usually the trauma mechanism of elbow dislocation is falling on outstretched hand with elbow in approximately 30 of flexion . however our patient had fallen on outstretched hand with elbow in full extension . although this type of trauma mechanism is typical for supracondylar humerus fractures in pediatric age group , in our patient an open posterior elbow dislocation without fracture had occurred.conclusionpediatric elbow dislocations are rare injuries and the management of these injuries can be technically demanding due to concurrent neurovascular injuries . an open dislocation without fracture is very rare and it should be treated with immediate intervention , an effective teamwork and good rehabilitation .

voltage - dependent potassium channels ( kv channels ) are crucial for the electrical signaling in neurons . kv channels activate upon depolarization of the plasma membrane , constraining the length of action potentials . together with calcium - dependent potassium channels ( kca ) , they are also responsible for the afterhyperpolarization that follows action potentials , thus modulating neuronal firing rates . kv channels are a large family of structurally related proteins with some differences in their biophysical properties , such as voltage range of activation , single channel conductance , kinetics , and behavior of gating [ 1 , 2 ] . in recent years it has been recognized that different types of kv channels are targeted to different regions within the plasmatic membrane [ 35 ] , but the physiological relevance of this differential sorting is poorly understood . since kv channels exhibit different sensitivities to kinases and phosphatases , and their activity can be differentially regulated by extra- and intracellular signaling pathways [ 69 ] , it can be predicted that the specific composition of kv - channel oligomers will influence local excitability . this is especially important at presynaptic sites where the frequency and shape of action potential are fundamental to determining the timing and strength of synaptic transmission . short - term forms of plasticity such as paired - pulse facilitation ( ppf ) are thought to be due to presynaptic modulation , but the mechanisms and molecular targets involved have to be identified precisely [ 1113 ] . among the molecules involved , potassium channels seem to play a major role [ 1416 ] . in corticostriatal synapses , a role for potassium channels from kv family was first suggested by jiang and north , while studying the modulation of neurotransmitter release by opiates in the corticostriatal synapses . later , our group showed that blocking k channels disrupted the opiate - induced downregulation of neurotransmitter release [ 15 , 17 ] . more recently we have also shown that kir3 channels ( also known as girk channels ) are presynaptically located at corticostriatal synapse and that blocking these channel reduces presynaptic paired - pulse facilitation . in this work we further extend the analysis to investigate the presynaptic expression of kv channels in corticostriatal synapses using the ppf protocol . when discussing the functional relevance of kv it is important to keep in mind the extraordinary variety of these channels and the complex stoichiometry of its oligomeric structure . kv channels are tetrameric proteins composed of four alpha subunits with six transmembrane segments each that bind together to form the channel pore . over 40 genes encoding kv alpha subunits have been discovered in mammals , so far . alpha subunits are organized into 12 families ( kv1 to kv12 ) with several members each , according to their similarity in sequence , biophysical properties , and pharmacological profiles [ 68 ] . alpha subunits from families kv14 , kv7 , kv10 , and kv11 can combine within their own family to produce functional homo- or heterotetrameric kv channels , while kv5 - 6 and kv8 - 9 families are unable to form functional homomeric channels but can form heteromeric channels with members from kv1kv4 families [ 3 , 19 ] . the reason for such diversity is yet unknown , but it may be necessary to fine - tune the neuronal excitability , since the expression of kv channels with different properties can influence the initiation , length , and magnitude of action potentials , both reaching and arising at nerve terminals [ 4 , 10 ] . although a high number of combinations are possible , only few combinations have been detected in brain by immunoprecipitation so far ( i.e. , [ 2023 ] ) . here we use a combination of physiological and pharmacological approaches to investigate the composition of kv channels present at the corticostriatal synapse terminals . by using the ppf protocol in combination with selective blockers we set to determine whether a particular isoform of kv is present at the presynaptic site of corticostriatal synapse . male wistar rats ( 100120 g ) provided by the animal facilities at fes iztacala , unam , were maintained in accordance with the guidelines for the use of animals in neuroscience research by the society for neuroscience and the helsinki declaration . sagittal dorsal neostriatal slices ( 400 m ) were obtained on a vibroslicer ( easislicer , pelco ) and incubated at room temperature in saline solution containing ( in mm ) 125 nacl , 3 kcl , 1 mgcl2 , 2 cacl2 , 25 nahco3 , 0.2 ( )-ascorbic acid , 0.2 thiourea , and 11 glucose ( saturated with 95% o2 and 5% co2 , ph : 7.40 ) . field population spikes , a composite of both field excitatory postsynaptic potential and population action potentials , were recorded with micropipettes filled with 0.9% nacl ( 24 m ) and electrical recordings were obtained using an ac amplifier ( p55 , grass instruments co. , w. warwick , ri , usa ) . all recordings were filtered at 13 khz and digitized by pci-6221 national instruments ( austin , tx , usa ) daq ( ni - daq ) board in a pc clone using custom - made programs on labview environment ( national instruments , austin , tx , usa ) . the daq board was used to save the data on ascii or binary files in the computer hard disk for further offline analysis . stimuli consisted of a pair of brief square voltage pulses ( 440 v ; 80200 s ; 0.060.4 hz ) generated with an isolated stimulator and delivered through the bipolar electrode . the time interval between the pair of stimuli was in the range of 15 to 50 ms . bicuculline ( 10 m ) was added to the perfusion in order to eliminate the inhibitory component of the synaptic response due to gabaa receptor activation [ 2426 ] . a paired - pulse protocol was applied and changes in the responses to paired stimuli were evaluated by calculating the ratio of field - spike synaptic potentials ( ppr ) , which is expressed in percentage as shown in the equation , where s1 is the first and s2 is the second orthodromic responses to the stimuli:(1)% ppr=100s2s1 . variations in ppr are related to changes in the neurotransmitter release probability in the synaptic terminals from the recorded area [ 17 , 18 , 2528 ] . this protocol has been widely used to assess changes in neurotransmitter release strength [ 17 , 18 , 25 , 2933 ] . it is generally thought that paired - pulse facilitation ( ppf ) occurs when the first pulse induces a transient increase of the residual ca concentration in the synaptic terminal , so that the terminal is able to release more neurotransmitter when second pulse arrives , increasing the amplitude of the corresponding field - spike potential ( s2 > s1 ) . in contrast , paired - pulse depression ( ppd ) is thought to occur when s1 is strong enough to deplete the pool of ready - to - release synaptic vesicles . here , we set the strength of paired pulses to produce ppf and the reduction in the ppr produced by the action of the k channel blockers was interpreted as a presynaptic effect [ 15 , 17 , 18 , 25 , 3437 ] . ppr was measured throughout the experiment and averaged every 5 minutes before and after the addition of drugs . the specificity of kv blockers has been indicated in different studies and reviews ; we use the following blockers mainly based in the reviews from coetzee and coworkers and iuphar reviews from gutman et al . [ 7 , 8 ] : for kv1 channels , dendrotoxin ( dtx ; cat . number : d-350 ) , agitoxin-1 ( agtx ; cat . number : stt-360 ) , for kv3 channels , blood depressing substance i ( bds-1 ; cat . number : bicuculline , tetraethylammonium ( tea ) , and 4-aminopyridine ( 4-ap ) were purchased from sigma ( st . the chemicals used in saline solutions were purchased from sigma - aldrich or j. t. baker . statistical significance of changes in ppr was assessed with no parametrical tests ( wilcoxon test ) . the results are expressed as mean sem ; other statistical parameters are summarized in table 1 . we investigate if kv channels may be present at cortical synaptic terminals in striatum , whether these channels were presynaptically located , and how their presence influences synaptic strength . we stimulate cortical areas using a paired - pulse protocol and analyzed the orthodromic response elicited in neostriatum recording field potentials . the population spikes thus elicited were then measured before and after addition of kv blockers 4-ap and tea ( figure 1 ) . the corticostriatal synapses were very sensitive to blocking with 4-ap , so that not only was the paired - pulse induced - facilitation abolished , but also paired - pulse depression was induced after the exposure to 4-ap ( figure 1(a ) ) ; ppr decreased from 2.63 0.49 to 0.55 0.12 ( n = 11 ; p < 0.001 ) . figure 1(a ) shows a typical response to 1 mm of 4-ap ; the reduction of ppr was close to 130% . the ppr reduction produced by 4-ap was dependent on drug concentration ( figure 1(b ) ) , with an ec50 = 0.186 mm . this result indicates the presence of kv channels from families kv1 and kv3 , whose sensitivity to 4-ap falls within the submillimolar rank . exposure to tea at several concentrations also induced a reduction in ppr ( figure 1(c ) ) , although the reduction was not as strong as that observed for 4-ap , even after 20 mm of tea was applied ( see dose - effect curves in figure 1(d ) ) . in figure 1(c ) we show a representative recording of population spike from a slice before and after exposure to tea 1 mm . the reduction in % ppr observed with even a little amount of tea evidenced the presence of tea - sensitive channels such as kca1.1 ( also known as slo1 ) , the delayed rectifier kv1.1 , and members of kv3 family ( type - a channels ) , as deducted from ic50 values reported by others researchers [ 6 , 8 ] . taken together , these results show that kv channels involved in the release mechanism at the presynaptic terminals are both tea and 4-ap sensitive , pointing to a kv channel population mainly composed of kv1 and kv3 channels . for these channels , ic50 values for 4-ap blockage or kd values for 4-ap binding below 1 mm have been reported ( reviewed by ) . however , since the tea - induced reduction in ppr is not as strong as seen for 4-ap , we guess that some kv1 isoforms sensitive to tea in the millimolar range ( e.g. , kv1.2 , kv1.3 , and kv1.6 ) may be also present at corticostriatal nerve terminals . on the other hand , the combination of sensitivities to both drugs led us to think that other families are not present . for example , the a - type kv4 channels are insensitive to blockage with tea and 4-ap , with an ic50 within the range of 210 mm [ 6 , 8 ] ; therefore it seems quite improbable that kv4 channels are represented at corticostriatal synapses . consistently with this reasoning , the exposure to kv4.2- and kv4.3-specific blockers heteropodatoxin-2 ( 100 nm ) and phrixotoxin-2 ( 54 nm ) renders no change in ppr ( figure 2 ) . a similar reasoning can be applied to kv2 , kv7 , kv10 , kv11 , and kv12 channels , which are rather tea and 4-ap insensitive . within these families likewise , ic50 for blockage with 4-ap is calculated to be within 1.5100 mm [ 6 , 8 ] . our results , of course , do not rule out the presence of these channels in other membrane subregions of cortical or striatal neurons or that they play important roles in controlling neuron excitability , but they imply that these channels are not related to neurotransmitter control at corticostriatal presynaptic sites . to confirm the presence of kv3 channels we used bds - i , a kv3.4-specific blocker . as shown in figure 3 , exposure to 47 nm of bds - i results in a strong reduction of ppr from 2.71 0.90 to 1.5 0.40 ( n = 3 ; p = 0.25 ) . these results strongly suggest the presence of an a - type current playing a major role in presynaptic modulation of the corticostriatal synapses . all members of kv1 family are very sensitive to 4-ap ; however , sensitivity to blockage by tea is quite variable . while little amounts of tea are needed to block homomeric channels kv1.1 and kv1.6 ( ic50 0.5 to 1.7 mm ) , kv1.2 and kv1.3 are sensitive to tea with ic50 between 10 and 50 mm , and kv1.4 , kv1.5 , and kv1.7 are rather insensitive to tea [ 6 , 38 ] . however , it has been reported that native potassium channels are found as heteromers rather than homomers and that pharmacological properties in heteromeric channels may differ from homomeric channels , including sensitivity to tea [ 3840 ] . therefore , we use several kv1 specific blockers alone ( figure 4 ) or in combination ( figure 5 ) to explore the presence of several kv1 family members . although there are no specific drugs for every type of kv1 channel , the combination of toxins available gave us a good picture of kv1 channels composition . first , we tested agitoxin-1 ( 10 nm ) , a specific blocker for kv1.3 ( figure 4(a ) ) , and recorded field - potential spikes ; once again we observed a reduction on ppr until the point to produce ppd . ppr changed from 1.55 0.05 to 1.01 0.07 ( n = 6 ; p = 0.008 ) . this prompts us to think that kv1.3 channels might not be expressed as homomeric channels but in combination with other kv1 subunits . in support of this idea , when we used margatoxin ( 10 nm ) , a kv1.2- and kv1.3-specific blocker , we observed a reduction in ppr comparable to that observed with agitoxin-1 alone , but the drop on ppr was clearly faster ( figures 4(a ) and 4(b ) ) . margatoxin changed ppr from 1.37 0.20 to 1.10 0.15 ( n = 6 ; p < 0.05 ) . in figures 5 and 6 , we show the effects of hongotoxin ( 10 nm ) and dendrotoxin ( 100 nm ) applied alone or in combination . these toxins block equally kv1.1 and kv1.2 containing channels , but hongotoxin also blocks kv1.3 while dendrotoxin blocks kv1.6 . as expected from the two previous experiments , we observed that hongotoxin renders a significant reduction of ppr , so that ppf becomes ppd ; ppr changed from 1.62 0.28 to 1.34 0.10 ( 17.038% ; n = 6 ; p < 0.05 ) . in turn , dendrotoxin produced a stronger ppd , 1.30 0.10 to 0.83 0.12 ( 79.07% , n = 14 ; p < 0.001 ) . consistently with these two results , when hgtx was added sequentially to dtx , it did not render extra reduction of ppr ( figure 6 ) , pointing out the presence of homomeric kv1.1 channels at presynaptic sites . in contrast , when dendrotoxin was applied sequentially to hongotoxin ( figure 5 ) it produced a further reduction of ppr , strongly suggesting the presence of kv1.6-containing channels . when we blocked kv1.2-containing channels with tityustoxin ( 10 nm ) we observed ppr reduction , but adding hongotoxin ( 100 nm ) to the bath did not produce further reduction of ppr . this result indicates that kv1.2-containing channels are heterotetramers with kv1.1 and/or kv1.3 and that no kv1.1 or kv1.3 homotetramers are present at corticostriatal presynaptic terminals . in contrast , addition of dtx ( 10 nm ) renders a second drop in ppr , which points to a kv1.6-containing channels population that exists independently of the kv1.1/kv1.2/kv1.3-containing channels population . significant ppd was also observed when kv1.2/kv1.3 channels were blocked with mgtx ( 10 nm ) , but little or no further change was observed when applying tytx ( 10 nm ) , as it would be expected if all kv1.2-containing channels were already blocked by mgtx . addition of hgtx ( 10 nm ) did not render further ppr reduction , suggesting again that all kv1.2/kv1.3 channels were already blocked by margatoxin and tityustoxin . once again addition of dendrotoxin ( 100 nm ) produced further reduction of ppr , showing the presence of kv1.6 channels . our results reveal that presynaptic terminals of corticostriatal synapse have several kinds of potassium channel from kv families . previously , we have shown before that kir channels also play an important role in modulating neurotransmitter release . here we showed that kv1 and kv3 channels are present at these synapses and their blockage with either specific or nonspecific drugs modulates synaptic strength . our results also rule out the presence of kv4 channels at these synapses and imply that presynaptic a - type current can only be carried by kv3 channels . on the other hand , our results indicate that delayed rectifier type currents can be carried by heteromeric kv1 channels , resulting from the combination of kv1.2 , kv1.3 , and probably kv1.1 but also from a population of kv1.6-containing channels that are not in combination with kv1.1 , 1.2 , and 1.3 . our observations are in good agreement with previous reports regarding the composition of kv channels . for example , koch et al . reported that all i - margatoxin receptors contain at least one kv1.2 subunit and over 80% also contain kv1.1 ; additionally ~30% of these receptors contain kv1.3 subunits . specific inhibitors for kv1.3 and kv1.2 are able to reduce ppr , but kinetic and strength of blocking are remarkably different . specifically kv1.3 inhibitor agitoxin-1 seems to be less effective than kv1.2 inhibitor tityustoxin , which could be related to the relative abundance of both subunits . we observed that hongotoxin does not increase ppr reduction after kv1.2/kv1.3 channels have been already blocked with tityustoxin and margatoxin ( figure 6 ) ; therefore if kv1.1 is present at presynaptic action site , it should be associated with kv1.2 and/or kv1.3 rather than being homomeric . in contrast , the addition of dendrotoxin after kv1.1/kv1.2/kv1.3-containing channels had been blocked with hongotoxin renders an extra reduction of ppr , pointing toward an independent kv1.6-containing channel population , with a composition that excludes kv1.1 , kv1.2 , and kv1.3 . it has been proposed before [ 6 , 41 ] that the combination of kv1.1/kv1.2/kv1.6 accounts for a slow ia , named d - type current , that is sensitive to low concentrations of dendrotoxin and 4-ap ; the existence and role of such currents in regulating synaptic release surely deserve further investigation . in cortical neurons it has been observed that kv - channel alpha subunits are differentially distributed among dendrites , soma , axon , and axonic terminals . for example , somatic action potentials are sensitive to both tea and 4-ap , but axonic action potential is only sensitive to 4-ap , indicating a different composition of heteromeric kv1 channels . moreover , pharmacological blockage of kv channels with dendrotoxin - i increases the synaptic latency , probably due to a shift of the presynaptic calcium current resulting from the prolonged axonal spike . however , the repertoire of channels at presynaptic boutons should also be taken into account since action potential at presynapsis strongly modifies the action potential width and , consequently , neurotransmitter release . all kv1 channels have been detected by immunocytochemistry in hippocampal stratum pyramidale from mouse , but only kv1.1 , kv1.2 , and kv1.6 are located in the stratum oriens , while the kv1.3 immunoreactivity is absent in this layer . an earlier report in rat hippocampus showed that kv1.1 was prominent in stratum lucidum of ca3 , while immunostaining for kv1.2 , kv1.3 , and kv1.6 was very weak and it was difficult to determine whether the signal belongs to the dendritic area of pyramidal ca3 neurons or the mossy fiber terminals . interestingly , kv1.1 immunoreactivity in stratum lucidum disappears when dentate gyrus neurons are exposed to excitotoxic agents , which means that kv1.1 is located in mossy fibers terminals rather than ca3 dendrites . in contrast , kainic acid lesions of ca3 strongly reduced kv1.1 immunoreactivity in stratum radiatum , indicating that kv1.1 is associated with axons and terminal of schaffer collaterals . in good agreement with these observations , a recent report shows that kv1.1 is present on axon initial segments ( ais ) and axon terminals by freeze - fracture gold labelling and that density is 8 times higher on ais . in contrast with the report from veh et al . , sheng and coworkers have reported that kv1.2 immunoreactivity is concentrated on dendrites in hippocampal and cortical pyramidal cells [ 47 , 48 ] and is conspicuously absent in cell bodies . interestingly , excitotoxic lesion of entorhinal cortex reduced the density of kv1.2 in stratum moleculare of ca1ca3 . our results are in good agreement with these observations , as we have shown that kv1.1 and kv1.2 are present at cortical axon terminals . presynaptic localization of heteromeric kv1.1/kv1.2 channels has been observed in primary cultures ; likewise kv1.3 is thought to be targeted preferentially to the axon ; our results provide functional evidence that kv1.1/kv1.2/kv1.3 complexes are located presynaptically in vivo and that they modulate neurotransmitter release . interestingly , in layers ii / iii from somatosensorial and motor cortex mrna for kv1.1 to 1.6 can be detected , but immunoreactivity for kv1.6 seems to be almost absent within the cortex . a possibility is that kv1.6 is sorted to axon terminals , accounting for a dendrotoxin - sensitive population of kv1.6 channels located at corticostriatal synapses . regarding kv4 we did not find evidence of the presence of these channels at presynaptic terminals ; this is in agreement with previous studies showing that kv4 channels are selectively transported to dendrites in cortical neurons in culture . instead we found evidence for the presence of kv3.4 channels , which are both tea and 4-ap sensitive . in hippocampal pyramidal cells 87% of cells express mrna for kv4 channels , while only 17% express mrna for kv3 ( kv3.1 and kv3.2 ) ; however jinno and coworkers have shown that kv4.2 is located in somatic and dendritic compartment , which is in good agreement with our observations . in support of the interpretation of our experiments regarding the pre- or postsynaptic localization of kvs , it is worth mentioning that in striatal medium spiny neurons mrna for kv1.3 is absent , but kv1.2 is present . in these neurons kv1.2 seems to account for as much as 50% of subthreshold depolarization - activated k current . in contrast , in our experiments margatoxin has a strong effect , supporting the notion that the kv1.2/kv1.3-containing channels are concentrated on the presynaptic side of corticostriatal synapses . kv1 channels have been reported to be presynaptically located in other glutamatergic synapses . for example , mossy fibers from dentate granule cells show prominent immunolabeling for kv1.1 and kv1.4 in preterminal segments [ 21 , 54 ] . accordingly , fast inactivating dendrotoxin - sensitive tea - insensitivity currents are recorded in outside - out patches isolated from mossy fibers . however , while it has been suggested that kv1.6 is not present in mossy fibers boutons , in corticostriatal synaptic terminals we found evidence that there is an important population of kv1.6 channels . on the other hand , kv3 channels have been observed to be located presynaptically in cerebellar stellate interneurons and sympathetic nerves [ 42 , 45 ] . in interneurons from cerebellar molecular layer kv3 channels are segregated from kv1 channels , located in soma and the most proximal axon region , while kv3 channels are located in presynaptically buttons . there is a growing body of evidence highlighting that k conductances located at presynaptic terminals control the release of transmitter [ 3 , 12 , 18 , 5557 ] . for example , it has been reported that dtx - sensitive k currents are modulated by opioid signaling and play an important role regulating the calcium influx into mossy fibers terminals [ 45 , 58 ] . in corticostriatal synapses , glutamatergic release is also regulated by opiates and regulation of k - channels activity is part of the modulation mechanism fibers . - and -opiate receptors facilitate glutamate release from corticostriatal [ 15 , 17 ] . since blocking potassium channels with 4-ap and ba , but not tea ( 1 mm ) , precludes opiate upregulation of glutamate release , it was suggested before that kv4 may be involved in the neuromodulation by opiates . however , our study shows that kv4 specific blockers do not modify ppr , indicating that kv4 channels are not expressed presynaptically . as it has observed in mossy fibers [ 21 , 54 ] , heteromeric kv1.1-containing channels may be the ones involved in opiate - induced ppf . a kv1.1 heterotetramer containing kv1.2 and/or kv1.3 would fit the pharmacological profile of potassium channels involved in opiate - induced modulation , sensitive to 4-ap and ba and relatively insensitive to tea , since the presence of kv1.2 in kv1.1/kv1.2 homotetramers renders an ic50 for tea of 10 mm . neurotransmitter release from vesicles lasts only a few milliseconds , due in part to the balance between the activation of voltage - gated calcium and potassium currents . upregulation of kv - channels activity presumably will lead to a stronger and faster repolarization , reducing the time window for calcium influx trough voltage - dependent ca channels , with the consequent reduction in the neurotransmitter release . the present study shows that kv conductances effectively are present at presynaptic terminals of corticostriatal afferents and modulate glutamate release at these synapses . in a previous report we showed that these terminals are also endowed with kir3 channels , which can be regulated by several g - protein receptors . altogether our results show that corticostriatal synaptic terminals are endowed with multiple types of voltage - gated potassium channels , and this opens a large pool of targets / tools for neuromodulators to regulate the synaptic action potential achieving thereby synaptic plasticity .

in the last years it has been increasingly clear that kv - channel activity modulates neurotransmitter release . the subcellular localization and composition of potassium channels are crucial to understanding its influence on neurotransmitter release . to investigate the role of kv in corticostriatal synapses modulation , we combined extracellular recording of population - spike and pharmacological blockage with specific and nonspecific blockers to identify several families of kv channels . we induced paired - pulse facilitation ( ppf ) and studied the changes in paired - pulse ratio ( ppr ) before and after the addition of specific kv blockers to determine whether particular kv subtypes were located pre- or postsynaptically . initially , the presence of kv channels was tested by exposing brain slices to tetraethylammonium or 4-aminopyridine ; in both cases we observed a decrease in ppr that was dose dependent . further experiments with tityustoxin , margatoxin , hongotoxin , agitoxin , dendrotoxin , and bds - i toxins all rendered a reduction in ppr . in contrast heteropodatoxin and phrixotoxin had no effect . our results reveal that corticostriatal presynaptic kv channels have a complex stoichiometry , including heterologous combinations kv1.1 , kv1.2 , kv1.3 , and kv1.6 isoforms , as well as kv3.4 , but not kv4 channels . the variety of kv channels offers a wide spectrum of possibilities to regulate neurotransmitter release , providing fine - tuning mechanisms to modulate synaptic strength .

premenstrual syndrome ( pms ) is one of the most common problems in women at their reproductive age [ 1 , 2 ] . pms is defined as the recurrent mood and physical symptoms which is usually in the luteal phase , and it remits in the follicular phase of the menstrual cycle [ 36 ] . there is a high prevalence of pms ; about 80% of women reported mild premenstrual symptoms , 20%50% reported moderate symptoms , and about 5% of women had severe symptoms [ 7 , 8 ] . despite the high incidence of premenstrual syndrome , causes of it have not been clear and several etiologies have been suggested ( e.g. , hormonal change , neurotransmitters , prostaglandins , diet , drugs , and lifestyle ) [ 7 , 914 ] . although the exact etiology of pms is not known , a wide range of therapeutic interventions have been tested to treat premenstrual symptoms ( e.g. , lifestyle changes , pharmacological intervention , and nonpharmacological treatments ) [ 1522 ] . due to the side effects of chemical drugs , except severe cases , chemical drugs consumption is not recommended . today , complementary and herbal medicine are commonly used in the treatment of many chronic conditions such as pms , menopausal symptoms , and dysmenorrhea [ 17 , 20 , 2327 ] . ginger is commonly used in the herbal medicine and traditionally it used to treat dysmenorrhea [ 28 , 29 ] . studies showed beneficial effects of ginger on vomiting , nausea , motion sickness , arthritis , migraine , headache , and so forth . in addition , studies have shown that the ginger can modulate prostaglandins system [ 3335 ] . with regard to the role of prostaglandins in pms and effect of ginger on modulation of prostaglandins system , the aim of this study was evaluating the effects of ginger on the severity of the pms symptoms . this clinical trial was a randomized , double - blinded , and placebo - controlled study . the study was done on 70 female students in dormitories of tehran university of medical sciences in the year of 2013 . 97/130/d/92 ) and it was registered at iranian registry of clinical trial ( irct no . inclusion criteria were as follows : being healthy premenopausal women aged between 18 and 35 years , having regular menstrual cycles of 2135 days , being single , lacking sensitivity to ginger , not taking any medication , not drinking alcohol , not smoking , and not having stressful events in the last 3 months . the participants recorded symptoms with daily record questionnaires ( this form contains a table with 19 symptoms of premenstrual syndrome questionnaire based on the dsm - iv ( the fourth edition of the diagnostic and statistical manual of mental disorders of the american psychiatric association ) , self - rating scale ) for two cycles before the intervention . this questionnaire determines the severity of pms using 3 items , including mood symptoms ( restlessness ; irritability ; anxiety , depression , or sadness ; crying ; feeling of isolation ) , physical symptoms ( headache , breast tenderness , backache , abdominal pain , weight gain , swelling of extremities , muscle stiffness , gastrointestinal symptoms , and nausea ) , and behavioral characteristics ( fatigue , lack of energy , insomnia , difficulty in concentrating , increased or decreased appetite ) . then the severity of premenstrual syndrome was evaluated for all participants ( ( 0 ) absence of symptoms , ( 1 ) mild symptoms that may not interfere with everyday activities , ( 2 ) moderate symptoms that interfere with daily activities , and ( 3 ) severe symptoms that impede doing daily activities ) . each participant with at least five symptoms was finally diagnosed as a person with pms . after identifying patients with pms doses of ginger and placebo were 250 mg/12 hours and given from 7 days before and until 3 days after onset of menstrual bleeding . the subjects completed the daily record questionnaire at their first , second , and third menstrual cycles . exclusion criteria were incidence of side effects drugs or drugs allergy , use of other drugs , drug use disorder , identifying any diseases during the study , getting married during the study , menstrual irregularities , and irregular bleeding event during the study . intergroup analyses were performed to compare the total score of pms , mood , and behavioral and physical symptoms before and after the intervention . out of the 70 students who participated in the study 2 persons in the ginger group and 2 persons in the placebo group did not complete the study ( figure 1 ) . in the ginger group two samples were excluded , one because of incidence of side effect and second one due to misuse of ginger . in the placebo group reason for exclusion for each of the 2 people was drug use disorder . no significant differences between two groups were seen with regard to demographic characteristics and menstrual history data ( table 1 ) . there were no significant differences regarding the total score of pms , severity of mood , and physical and behavioral symptoms between the two groups before the intervention ( resp . , p = 0.71 , p = 0.69 , p = 0.56 , and p = 0.76 ) ( table 2 ) . statistical analysis demonstrated that there were significant differences in total score of pms , severity of mood , and physical and behavioral symptoms between the two groups after the intervention , and ginger could reduce severity symptom of pms ( table 2 ) . the findings of this study showed that ginger could significantly reduce the total score of pms , severity of mood , and physical and behavioral symptoms of the first month intervention . so according to the availability and the safety of ginger , the ginger can be an appropriate treatment in reducing symptoms of premenstrual syndrome . one of the proposed mechanisms that lead to premenstrual syndrome is change in prostaglandin system [ 7 , 16 , 36 ] . ginger through the inhibition of the metabolism of cyclooxygenase and lipoxygenase prevents the production of prostaglandins . to our knowledge , this study is the first report about the effect of ginger on premenstrual syndrome . . showed that the use of ginger is effective in reducing symptoms of dysmenorrheal [ 28 , 29 ] . primary dysmenorrhea is caused by excessive production of prostaglandins from the endometrial tissue , and 80% of cases of dysmenorrhea with prostaglandin inhibitors can be improved . because some ginger compounds are prostaglandin inhibitors and are effective on dysmenorrhea , so perhaps through this mechanism they could be effective on other problems of menstruation cycle [ 28 , 29 ] . some of symptoms of pms like pain are common with dysmenorrhea ( such as backache and abdominal pain ) . in our study these symptoms of pms have been reduced . in women with pms , severe headache outbreaks during the luteal phase and an increase in headache resistance to painkillers and anti - inflammatory drugs lead to increase in depression , irritability , anxiety , anger , and food intolerance during the luteal phase [ 3739 ] . cady et al . reported that ginger administration caused a significant deceleration in severity of headache in patients with migraine . one of the symptoms of pms is incidence of headache and exacerbation of migraine . in our study ginger levine et al . reported that the ginger reduced the delayed nausea of chemotherapy and reduced use of antiemetic medications . ginger was effective in treating nausea and vomiting during pregnancy [ 31 , 42 ] . study of k. s. naeine and s. s. naeine showed that the use of 1 gram of powdered ginger , 1 hour before taking contraceptive pills , induced reduction in contraceptive pills nausea . nausea is one of symptoms of pms ; in our study the incidence of gastrointestinal disturbances and nausea after taking ginger reduced . studies showed that ginger has anti - inflammatory properties and is effective in treatment of pain in patients with osteoarthritis , muscle pain [ 30 , 33 , 44 ] . joint and muscle pain are also symptoms of pms ; in the present study , ginger was effective in relieving severity of these symptoms of pms . the only side effect observed in the present study was nausea in the group of ginger . in addition , the use of ginger in traditional medicine to treat colds , fever , sore throat , nausea , stomach upset , muscle aches , and arthritis and for cancer prevention is effective . therefore , the use of ginger in the treatment of pms can also benefit from other advantages of ginger . the findings of the present study demonstrated the likelihood of usefulness of ginger in treating pms while no specific side effects have been seen , and consumption of it is associated with health benefits . ginger is readily available because of its low cost and , so , can be widely used in the treatment of premenstrual syndrome . according to increased tendency in the use of traditional medicine and herbal medicine , especially for people who have no desire to use chemical drugs , with more side effects , use of ginger , with very low side effects , is very useful . furthermore , we can not find study that evaluates effect of ginger on pms ; we suggest doing more studies with larger number of patients concerning the efficacy and safety of different doses of ginger .

premenstrual syndrome ( pms ) is a common disorder . although the etiology of pms is not clear , to relieve from this syndrome different methods are recommended . one of them is use of medicinal herbs . this study was carried out to evaluate effects of ginger on severity of symptoms of pms . this study was a clinical trial , double - blinded work , and participants were randomly allocated to intervention ( n = 35 ) and control ( n = 35 ) groups . to determine persons suffering from pms , participants completed daily record scale questionnaire for two consecutive cycles . after identification , each participant received two ginger capsules daily from seven days before menstruation to three days after menstruation for three cycles and they recorded severity of the symptoms by daily record scale questionnaire . data before intervention were compared with date 1 , 2 , and 3 months after intervention . before intervention , there were no significant differences between the mean scores of pms symptoms in the two groups , but after 1 , 2 , and 3 months of treatment , there was a significant difference between the two groups ( p < 0.0001 ) . based on the results of this study , maybe ginger is effective in the reduction of severity of mood and physical and behavioral symptoms of pms and we suggest ginger as treatment for pms .

low back pain ( lbp ) is a condition experienced at least once by more than 80% of the human race the world over1 . more than 85% of cases of lbp in the usa are classified as nonspecific lbp defined by tension , soreness , and/or stiffness of the lower back with unknown origins of pain2 . back pain causes decreased lumbar extensibility , limited range of motion ( lom ) and gait ability , and change of rhythm in the pelvis , limitations that can restrict social activities3 . one of the most widely studied outcomes in lbp patients is the reduction of hamstring extensibility4 . the hamstring is responsible for hip extension and knee flexion along with providing stability of the hip and knee joints during walking5 . a shortening of the hamstring may result in lom of the knee joint , restricting a person s ability to walk and run . in addition , it increases posterior pelvic tilting and reduces lumbar lordosis , contributing to a flat back that can result in back pain6 . objective evaluation tools to easily assess the extensibility of the hamstring include the sit and reach test ( srt ) , the passive toe touch test , and the straight leg raise ( slr ) test . methods for increasing the length of the shortened hamstring include stretching , eccentric resistance exercise , graston instrument soft tissue mobilization , and self - myofascial release8,9,10 . recently , graston instrument soft tissue mobilization has been reported to be effective in promoting the extensibility of the shortened hamstring11 . graston technique ( gt ) is a soft tissue mobilization treatment method using a tool that generates mechanical micro - traumatic damage to the treated area . it thus creates an inflammatory response to accelerate the healing process and restore flexible , normal tissue . this technique seems to have the therapeutic effects of inhibiting the adhesion of tissue , increasing the number of fibroblasts , and promoting collagen synthesis12 . although there are several studies related to graston instrument soft tissue mobilization and shortened hamstrings , only healthy volunteers have been included in these studies11 . therefore , in this study , nonspecific low back pain ( nslbp ) patients with shortened hamstrings were monitored . the purpose of this study was to examine the effects of the gt on hamstring extensibility and pain intensity in patients with nslbp . this study included 24 patients with lbp selected from the staff and student population of gachon university ( except to the department of physical therapy and occupational therapy ) , located in incheon . for more information about participants , refer to table 1table 1.general characteristics between two groupsgt groupss grouppgender ( male / female)8/48/41.000age ( years)34.17 4.9135.25 5.860.629height ( cm)169.25 9.18168.50 8.680.839weight ( kg)61.92 9.8761.83 11.370.985values are expressed as mean sd .. prior to the experiment , the gender , age , and hamstring length of all subjects were recorded after an interview and screening test . participant selection criteria were as follows : the patient should have ( 1 ) a history of nslbp for at least 2 months ( 2 ) a finger - to - ground distance greater than 0 cm ( 3 ) a passive slr angle of 70 or less ( 4 ) most of the knee touching the chest in the passive hip - flexion test for those with normal movement of the hip joint ( 5 ) no neurological signs , and ( 6 ) a visual analog scale ( vas ) score of more than three . furthermore , all patients who participated in this trial provided a signed written consent form after having the expected outcomes and side effects fully explained . values are expressed as mean sd . the srt was used along with the sitting trunk flexion meter ( takei , japan ) . this test involves sitting on the floor with the legs stretched out straight in front of the body . the soles of the feet are placed flat against a box , and the trunk flexed forward . with both knees locked and the back of the legs pressed flat to the floor , the subject reaches forward along the measuring line as far as possible . after some practice reaches , the subject holds that position for 5 seconds while the distance is recorded . the measurement was taken three times at the same time and place , and the average was recorded . the srt for hamstring flexibility is likely to have moderate criterion - related validity ( 0.390.89)7 . a vas was used to assess the level of lbp in the subjects . a vas is usually a horizontal line , 100 mm in length , anchored by word descriptors at each end . the patient marks on the line the point that subjectively matches his or her pain level and is thus scored from 0 to 10 points13 . all participants were randomly assigned to one of two groups : the graston technique group ( gt group , n=12 ) or the static stretching group ( ss group , n=12 ) . the gt was used on the hamstrings of the gt group , while the ss group performed static stretches on the hamstring muscle . the experiment involved one mediator and one evaluator . during the course of the experiment , the mediator was unaware of the evaluation results . additionally , the evaluator did not know to which group the subjects were assigned . both before and after training , flexibility and pain levels were assessed by the evaluator . the srt was performed three times with the subjects taking 5-minute breaks between each test . then , each group received or performed the intervention for 60 seconds and was retested after a 5-minute break . the gt group received soft tissue mobilization of the hamstring muscle with the graston instrument using gt number 1 ( gt-1 ) . first , the subject lay prone and bent the knee joint to around 30 to 60. after applying massage cream on the hamstring muscle , the graston instrument was rubbed on each subject 30 times for 60 seconds from the gluteal line to the popliteal fossa11 . it was fixed at opposite leg and hip joint bending posture of the subject , the direction of the stretch was a knee joint extension direction . the procedure started with initial stretching , where the posture was maintained for 5 seconds . this was followed by three subsequent increases in knee extension , each with a 5-second hold . the final increase in the knee extension angle was maintained for 45 seconds , and then the antagonist muscle was stimulated . the srt and vas differences within a group before and after the treatment were tested using the mann - whitney u test . for all data , the changes in srt and vas scores before and after application of gt are shown in table 2table 2.difference for srt , vas between groups and within groupsgt groupss groupp - valuepre - testpost - testpre - testpost - testp1p2p3srt5.51 2.545.51 3.186.08 1.792.54 2.780.5060.0020.002vas4.33 1.303.42 0.794.83 1.344.25 1.290.3260.0050.008values are expressed as mean sd . srt : sit and reach test ; vas : visual analog scale ; gt : graston group ; ss : static stretching groupp1=difference at baseline ; p2=differences pre / post tests for experimental group ; p3=differences pre / post tests for control group . after the gt , a significant increase in srt and vas scores was observed ( p<0.05 ) . the gt group had a significant difference in srt measurements before and after the intervention ( p<0.05 ) . there were no statistically significant changes in the vas assessments ( table 3table 3.difference change value for srt , vas between two groupsgt groupss groupp - valuesrt11.03 1.808.62 1.600.002vas0.92 0.670.58 0.510.210values are expressed as mean sd . srt : sit and reach test ; vas : visual analog scale ; gt : graston group ; ss : static stretching group ) . srt : sit and reach test ; vas : visual analog scale ; gt : graston group ; ss : static stretching group p1=difference at baseline ; p2=differences pre / post tests for experimental group ; p3=differences pre / post tests for control group values are expressed as mean sd . srt : sit and reach test ; vas : visual analog scale ; gt : graston group ; ss : static stretching group this study aimed to identify the effect of the gt on hamstring extensibility and pain intensity in patients with nslbp and shortened hamstrings . the gt group had a significant difference in srt measurements before and after the intervention ( p<0.05 ) . there were no statistically significant changes in pain intensity . in a study by kim et al . , using gt on hamstrings in healthy young adults was reported to increase the range of motion ( rom ) of the knee joint11 . since the target population in this study was individuals with nslbp with hamstring shortening , this study will be more clinically applicable . as indicated by the above results , the two interventions were found to have positive effects on extensibility of the hamstring and pain intensity . however , the gt group had more significant changes in the extensibility of the hamstring than the ss group did . in a study by hammer and pfefer , the hamstrings of subjects with lbp were treated with soft tissue mobilization using gt six times , which significantly increased the rom of the knee joint15 . in our research , gt was only applied once for 60 seconds and hamstring extensibility increased in the short term . the static stretching used in the control group is a common physical therapy treatment technique for rom and physical function improvement , and it has a positive effect on pain and extensibility in the hamstring after application16,17,18 . reported an increase in rom when subjects performed static stretches on the hamstring , but there were differences depending on the length of the stretches . since 30 to 60 seconds was found to be most effective , the ss group held static stretches for 60 seconds17 , 18 . clinically , a shortened hamstring limits the flexion of the hip joint or causes lumbar hyperextension , resulting in back pain . therefore , it is essential to improve hamstring extensibility in the recovery of back pain patients6 . thus , if lbp occurs with shortening of the hamstring , knee arthritis , or functional limitation , soft tissue mobilization using the graston instrument is a simple and effective intervention for restoring hamstring extensibility . one limitation of this study is that it is difficult to generalize the results due to the low number of subjects . second , it did not use tests with the highest level of reliability and validity . future studies will need to include various disease groups to address this limitation . in this study , the gt proved to be a more effective intervention to improve hamstring extensibility in nslbp patients than static stretching did . we look forward to its use in clinical practice because of its simplicity and efficacy .

[ purpose ] the purpose of this study was to analyze the effect of graston technique on hamstring extensibility and pain intensity in patients with nonspecific low back pain . [ subjects and methods ] twenty - four patients with nonspecific low back pain ( 2746 years of age ) enrolled in the study . all participants were randomly assigned to one of two groups : graston technique group ( n=12 ) and a static stretching group ( n=12 ) . the graston technique was used on the hamstring muscles of the experimental group , while the static stretching group performed static stretching . hamstring extensibility was recorded using the sit and reach test , and a visual analog scale was used to measure pain intensity . [ results ] both groups showed a significant improvement after intervention . in comparison to the static stretching group , the graston technique group had significantly more improvement in hamstring extensibility . [ conclusion ] the graston technique is a simple and effective intervention in nonspecific low back pain patients to improve hamstring extensibility and lower pain intensity , and it would be beneficial in clinical practice .

dr5v2 was designed by replacing the 9 auxre s in dr5 ( tgtctc ) , with tgtcgg , and synthesized to generate cloning vector puc57/dr5v2 ( genscript ) . dr5 reporter cassette from pgiik / dr5::n3egfp was first excised by bamhi and ecori digestion and cloned into bamhi and ecori digested pgiim / lic_swai - ntdtomato - lic_hpai - n3egfp ( a kind gift from thomas laux , freiburg ) to create pgiim / lic_swai - ntdtomato- dr5::n3egfp . dr5v2 was then cloned into swai - digested pgiim / lic_swai - ntdtomato- dr5::n3egfp via ligation independent cloning . pgiim / lic_swai - ntdtomato- dr5v2::n3egfp and pgiim / lic_swai - ntdtomato- dr5v2::n3venus were created by excising dr5v2 reporter cassette from puc57/dr5v2 via bamhi and ecori digestion followed by cloning into bamhi and ecori digested pgiim / lic_swai - ntdtomato- lic_hpai - n3egfp or pgiim / lic_swai - ntdtomato- lic_hpai - n3venus . the prps5a::dii - venus and prps5a::mdii - venus binary vectors were constructed using the multisite gateway technology ( invitrogen ) and following the provider instructions . to do so , the rps5a promoter was cloned in pdonr p4-p1r using primers listed in supplementary table 1 . this vector was used together with the previously described dii / mdii cloned in pdonr221 and venus fused to the n7 nuclear localization signal cloned in pdonr p2r - p3 for recombination in the binary gateway vector ph7m34gw ( http://gateway.psb.ugent.be/ ) . r2d2 in pgiim/ rps5a : : mdii : ntdtomato- rps5a : : dii : n3venus was created through two subsequent ligation independent cloning events . first , rps5a : : dii reporter cassette amplified from genomic dna of prps5a : : dii : venus using primer set prps5a : : dii was cloned into hpai digested pgiim / lic_swai - ntdtomato- lic_hpai - n3venus ( a kind gift from thomas laux , freiburg ) to create pgiim / lic_swai - ntdtomato- rps5a : : dii : n3venus . rps5a : : mdii control cassette amplified from genomic dna of prps5a : : mdii : venus control line using primer set prps5a : : mdii was then cloned into swai digested pgiim / lic_swai - ntdtomato- rps5a : : dii : n3venus to create r2d2 . arabidopsis plants were grown at 22c in 16 hours light/ 8 hours dark cycle for every experiments . all seeds were surface sterilized , sown on half - strength murashige and skoog medium with 0.8% daichin agar ( duchefa ) ( 1/2 ms plate ) if not mentioned otherwise , and vernalized at 4c for 2 days . for microscopic analysis of root , seedlings were grown vertically for five days after transfer to growth chamber , while this period was decreased to three or four days for microscopic analysis in shoot . methotrexate ( mtx ) selection was conducted by growing sterilized seeds on 1/2 ms plates containing 0.1 mg / l mtx ( sigma ; a6770 ) . for dr5/dr5v2 auxin sensitivity analysis via qrt - pcr , surface sterilized seeds were sown on sterilized nylon mesh placed on 1/2 ms plates after stratification and grown in growth chamber for four days then transferred to 1/2 ms plates with 0.11% dmso and 10 m n-1-naphthylphthalamidic acid ( npa ; chem service ) to inhibit auxin transport . after incubation for 12 hours , seedlings were transferred to plates containing 0.11% dmso and 10 m npa with 0.01 , 0.1 , or 1.0 m indole 3-acetic acid ( iaa ; duchefa ) for treatments , 0.11% dmso and 10 m npa for control for two hours before collection for rna isolation . for dr5/dr5v2 auxin sensitivity analysis via confocal microscopy , surface sterilized seeds were sown on sterilized nylon mesh placed on 1/2 ms plates after stratification and grown in growth chamber for four days then transferred to 1/2 ms plates with 0.11% dmso and 10 m npa with 0.0001 , 0.000316 , 0.001 , 0.00316 , 0.01 , 0.0316 , 0.1 , 0.316 , or 1.0 m iaa for treatments , 0.11% dmso and 10 m npa for control for 12 hours before collection for imaging . for temporal dr5/dr5v2 auxin response analysis , surface sterilized seeds were sown on sterilized nylon mesh placed on 1/2 ms plates after stratification and grown in growth chamber for five days then transferred to 1/2 ms plates with 0.01% dmso and 1.0 m iaa as treatment or 1/2 ms plates with 0.01% dmso as control for given time before collection for rna isolation . images were acquired as 8-bit format using a leica tcs sp5ii confocal laser scanning microscope with 20 na=0.75 and 63 na=1.20 water - immersion objective and pinhole equivalent to 1.0 the airy disk diameter . egfp and venus were excited by argon ion laser while tdtomato and propidium iodide were excited using diode laser , and their emissions were detected sequentially with leica hyd in standard mode to prevent cross - talks between fluorophores . excitation and detection of fluorophores were configured as below , egfp was excited at 488 nm and detected 498 - 530 nm ; venus was excited at 514 nm and detected 524 - 540 nm ; tdtomato was excited at 561 nm and detected 571 - 630 nm ; propidium iodide was excited at excited at 561 nm and detected 571 - 700 nm . in comparisons of egfp and tdtomato fluorescence in the same line , the highest fluorescence signal in reference cells listed below in each channel was used to set the upper limit of pixel intensity . reference cells used to setup the upper limit of pixel intensity are lens shape cell of early heart stage when imagining embryos , quiescent center cells when imaging roots , and distal domain of leaf primordia when imaging shoot meristem and leaf primordial . in comparisons of venus and tdtomato fluorescence in r2d2 line , the highest fluorescence signal in reference cells listed below in each channel was used to set the upper limit of pixel intensity . reference cells used to setup the upper limit of pixel intensity are suspensor cells of early globular stage when imagining embryos , cortex cells when imaging roots , and trichome cells when imaging shoot meristem and leaf primordial . embryos were mounted in 1 phosphate solution saline ( pbs ) containing 4% paraformaldehyde and 5% glycerol as described , and seedlings were mounted in demineralized water unless mentioned otherwise with 10 g / ml propidium iodide for roots and without propidium iodide for shoot meristem , leaf primordial , dr5v2 auxin sensitivity analysis , and r2d2 auxin treatment live imaging . the original plastic mask was replaced by a 15.5 mm 21.0 mm 0.5 mm frame made of bioplastic with 10.0 mm 15.0 mm opening in the center at where was covered with 0.4 m ptfe mesh . only one plastic frame was used , but agarose and culture medium were also omitted . five five - day - after - germination seedlings were placed in a two chamber coverglass containing 100 l demineralized water then covert by ptfe frame with 0.4 m ptfe mesh facing to seedlings followed by adding 900l of demineralized water to cover the roots . time interval and coordinates of regions of interest were first defined , and images referred as 0 sec were taken before adding 111 l of 0.1% dmso or 10 m iaa in 0.1% dmso . calcium imaging calculator built in leica las af lite v2.6.3 or v 3.7.x through calculating ratios between signal intensities of each pixel from two channels after subtracting noise , which was defined as the average signal intensity of six 2.5 - 3.5 m area in the cytoplasm of six epidermal cells from a single image and used for the rest of images taken with the same laser / detection configuration . to monitor auxin gradient in the root apical meristem by r2d2 , images from three z - stacks with 2.0 m interval were acquired . the maximum projection of three images was examined to assure the section of region of interest contains only single cell layer . approximate 10 m area in nucleus of cell of interest was selected via the roi tool , and the ratio of red / yellow signal ratio was calculated by calcium imaging calculator built in leica las af lite v2.6.3 or v 3.7.x after noise subtraction . red / yellow ratio of the first 7 - 10 continuous cells ( depending on number of cells in each frame of images due to the different cell size of each tissue ) from the quiescent center was acquired form 32 roots of r2d2 line . cells from both sides of roots were used if possible to generate 32 to 47 data sets of each tissue . over 100 roots from treatments were collected and rna was extracted with plant rneasy kit ( qiagen ) . poly(dt ) cdna was prepared from 600 ng total rna with an iscript cdna synthesis kit ( biorad ) . primer pairs were designed with beacon designer 8.0 ( premier biosoft international ) . although the fluorescent proteins are tandem repeats ( tandem dimer tomato and 3egfp ) , we designed primers that generate a single amplicon per transcript . primers were tested in qrt - pcr using serial diluted pgiim / dr5v2::ntdtomato - dr5::n3egfp plasmid as template to validate the correspondence between amount of amplicons and actual templates ( see supplementary fig.2 ) . qrt - pcr was conducted with iq sybr green supermix ( biorad ) on cfx384 real - time pcr detection system ( biorad ) according to the manufacturer s instructions . efficiency of primers for subjected cdna , ntdtomato and n3egfp , and concentrations of subjected cdna in all samples have been tested in advance to ensure expressions of n3egfp and ntdtomato are comparable . sequences of primers used for qrt - pcr were listed in supplementary table 1 . to compare qrt - pcr results , normalized data sets acquired form qbase were subjected to two - tailed student s t - test with threshold ( alpha level ) of 0.05 to determine if significance of the difference between each treatment . dr5v2 was designed by replacing the 9 auxre s in dr5 ( tgtctc ) , with tgtcgg , and synthesized to generate cloning vector puc57/dr5v2 ( genscript ) . dr5 reporter cassette from pgiik / dr5::n3egfp was first excised by bamhi and ecori digestion and cloned into bamhi and ecori digested pgiim / lic_swai - ntdtomato - lic_hpai - n3egfp ( a kind gift from thomas laux , freiburg ) to create pgiim / lic_swai - ntdtomato- dr5::n3egfp . dr5v2 was then cloned into swai - digested pgiim / lic_swai - ntdtomato- dr5::n3egfp via ligation independent cloning . pgiim / lic_swai - ntdtomato- dr5v2::n3egfp and pgiim / lic_swai - ntdtomato- dr5v2::n3venus were created by excising dr5v2 reporter cassette from puc57/dr5v2 via bamhi and ecori digestion followed by cloning into bamhi and ecori digested pgiim / lic_swai - ntdtomato- lic_hpai - n3egfp or pgiim / lic_swai - ntdtomato- lic_hpai - n3venus . the prps5a::dii - venus and prps5a::mdii - venus binary vectors were constructed using the multisite gateway technology ( invitrogen ) and following the provider instructions . to do so , the rps5a promoter was cloned in pdonr p4-p1r using primers listed in supplementary table 1 . this vector was used together with the previously described dii / mdii cloned in pdonr221 and venus fused to the n7 nuclear localization signal cloned in pdonr p2r - p3 for recombination in the binary gateway vector ph7m34gw ( http://gateway.psb.ugent.be/ ) . r2d2 in pgiim/ rps5a : : mdii : ntdtomato- rps5a : : dii : n3venus was created through two subsequent ligation independent cloning events . first , rps5a : : dii reporter cassette amplified from genomic dna of prps5a : : dii : venus using primer set prps5a : : dii was cloned into hpai digested pgiim / lic_swai - ntdtomato- lic_hpai - n3venus ( a kind gift from thomas laux , freiburg ) to create pgiim / lic_swai - ntdtomato- rps5a : : dii : n3venus . rps5a : : mdii control cassette amplified from genomic dna of prps5a : : mdii : venus control line using primer set prps5a : : mdii was then cloned into swai digested pgiim / lic_swai - ntdtomato- rps5a : : dii : n3venus to create r2d2 . arabidopsis plants were grown at 22c in 16 hours light/ 8 hours dark cycle for every experiments . all seeds were surface sterilized , sown on half - strength murashige and skoog medium with 0.8% daichin agar ( duchefa ) ( 1/2 ms plate ) if not mentioned otherwise , and vernalized at 4c for 2 days . for microscopic analysis of root , seedlings were grown vertically for five days after transfer to growth chamber , while this period was decreased to three or four days for microscopic analysis in shoot . methotrexate ( mtx ) selection was conducted by growing sterilized seeds on 1/2 ms plates containing 0.1 mg / l mtx ( sigma ; a6770 ) . for dr5/dr5v2 auxin sensitivity analysis via qrt - pcr , surface sterilized seeds were sown on sterilized nylon mesh placed on 1/2 ms plates after stratification and grown in growth chamber for four days then transferred to 1/2 ms plates with 0.11% dmso and 10 m n-1-naphthylphthalamidic acid ( npa ; chem service ) to inhibit auxin transport . after incubation for 12 hours , seedlings were transferred to plates containing 0.11% dmso and 10 m npa with 0.01 , 0.1 , or 1.0 m indole 3-acetic acid ( iaa ; duchefa ) for treatments , 0.11% dmso and 10 m npa for control for two hours before collection for rna isolation . for dr5/dr5v2 auxin sensitivity analysis via confocal microscopy , surface sterilized seeds were sown on sterilized nylon mesh placed on 1/2 ms plates after stratification and grown in growth chamber for four days then transferred to 1/2 ms plates with 0.11% dmso and 10 m npa with 0.0001 , 0.000316 , 0.001 , 0.00316 , 0.01 , 0.0316 , 0.1 , 0.316 , or 1.0 m iaa for treatments , 0.11% dmso and 10 m npa for control for 12 hours before collection for imaging . for temporal dr5/dr5v2 auxin response analysis , surface sterilized seeds were sown on sterilized nylon mesh placed on 1/2 ms plates after stratification and grown in growth chamber for five days then transferred to 1/2 ms plates with 0.01% dmso and 1.0 m iaa as treatment or 1/2 ms plates with 0.01% dmso as control for given time before collection for rna isolation . images were acquired as 8-bit format using a leica tcs sp5ii confocal laser scanning microscope with 20 na=0.75 and 63 na=1.20 water - immersion objective and pinhole equivalent to 1.0 the airy disk diameter . egfp and venus were excited by argon ion laser while tdtomato and propidium iodide were excited using diode laser , and their emissions were detected sequentially with leica hyd in standard mode to prevent cross - talks between fluorophores . excitation and detection of fluorophores were configured as below , egfp was excited at 488 nm and detected 498 - 530 nm ; venus was excited at 514 nm and detected 524 - 540 nm ; tdtomato was excited at 561 nm and detected 571 - 630 nm ; propidium iodide was excited at excited at 561 nm and detected 571 - 700 nm . in comparisons of egfp and tdtomato fluorescence in the same line , the highest fluorescence signal in reference cells listed below in each channel was used to set the upper limit of pixel intensity . reference cells used to setup the upper limit of pixel intensity are lens shape cell of early heart stage when imagining embryos , quiescent center cells when imaging roots , and distal domain of leaf primordia when imaging shoot meristem and leaf primordial . in comparisons of venus and tdtomato fluorescence in r2d2 line , the highest fluorescence signal in reference cells listed below in each channel was used to set the upper limit of pixel intensity . reference cells used to setup the upper limit of pixel intensity are suspensor cells of early globular stage when imagining embryos , cortex cells when imaging roots , and trichome cells when imaging shoot meristem and leaf primordial . embryos were mounted in 1 phosphate solution saline ( pbs ) containing 4% paraformaldehyde and 5% glycerol as described , and seedlings were mounted in demineralized water unless mentioned otherwise with 10 g / ml propidium iodide for roots and without propidium iodide for shoot meristem , leaf primordial , dr5v2 auxin sensitivity analysis , and r2d2 auxin treatment live imaging . the original plastic mask was replaced by a 15.5 mm 21.0 mm 0.5 mm frame made of bioplastic with 10.0 mm 15.0 mm opening in the center at where was covered with 0.4 m ptfe mesh . only one plastic frame was used , but agarose and culture medium were also omitted . five five - day - after - germination seedlings were placed in a two chamber coverglass containing 100 l demineralized water then covert by ptfe frame with 0.4 m ptfe mesh facing to seedlings followed by adding 900l of demineralized water to cover the roots . time interval and coordinates of regions of interest were first defined , and images referred as 0 sec were taken before adding 111 l of 0.1% dmso or 10 m iaa in 0.1% dmso . calcium imaging calculator built in leica las af lite v2.6.3 or v 3.7.x through calculating ratios between signal intensities of each pixel from two channels after subtracting noise , which was defined as the average signal intensity of six 2.5 - 3.5 m area in the cytoplasm of six epidermal cells from a single image and used for the rest of images taken with the same laser / detection configuration . to monitor auxin gradient in the root apical meristem by r2d2 , images from three z - stacks with 2.0 m interval were acquired . the maximum projection of three images was examined to assure the section of region of interest contains only single cell layer . approximate 10 m area in nucleus of cell of interest was selected via the roi tool , and the ratio of red / yellow signal ratio was calculated by calcium imaging calculator built in leica las af lite v2.6.3 or v 3.7.x after noise subtraction . red / yellow ratio of the first 7 - 10 continuous cells ( depending on number of cells in each frame of images due to the different cell size of each tissue ) from the quiescent center was acquired form 32 roots of r2d2 line . cells from both sides of roots were used if possible to generate 32 to 47 data sets of each tissue . over 100 roots from treatments were collected and rna was extracted with plant rneasy kit ( qiagen ) . poly(dt ) cdna was prepared from 600 ng total rna with an iscript cdna synthesis kit ( biorad ) . primer pairs were designed with beacon designer 8.0 ( premier biosoft international ) . although the fluorescent proteins are tandem repeats ( tandem dimer tomato and 3egfp ) , we designed primers that generate a single amplicon per transcript . primers were tested in qrt - pcr using serial diluted pgiim / dr5v2::ntdtomato - dr5::n3egfp plasmid as template to validate the correspondence between amount of amplicons and actual templates ( see supplementary fig.2 ) . qrt - pcr was conducted with iq sybr green supermix ( biorad ) on cfx384 real - time pcr detection system ( biorad ) according to the manufacturer s instructions . efficiency of primers for subjected cdna , ntdtomato and n3egfp , and concentrations of subjected cdna in all samples have been tested in advance to ensure expressions of n3egfp and ntdtomato are comparable . sequences of primers used for qrt - pcr were listed in supplementary table 1 . to compare qrt - pcr results , normalized data sets acquired form qbase were subjected to two - tailed student s t - test with threshold ( alpha level ) of 0.05 to determine if significance of the difference between each treatment .

visualization of hormonal signaling input and output is of key importance for understanding regulation of multicellular development . the plant signaling molecule auxin triggers many growth and developmental responses , but current tools lack sensitivity or precision to visualize these . we developed a set of novel fluorescent reporters that allow sensitive and semi - quantitative readout of auxin responses at cellular resolution in arabidopsis . these generic tools are suitable for any transformable plant species .

cerebrospinal fluid ( csf ) leak is a recognized complication of spinal surgeries . the incidence of csf leak during spine surgery is 2 to 5%.1 in cases of intradural tumor removal or nerve root sacrifice , the dura is opened intentionally , thus exposing patients to the risk of csf leak . although most durotomies are repaired and most patients do not experience symptomatic csf leak , it could nevertheless potentially lead to spinal headache , formation of a pseudomeningocele , wound breakdown , and subsequent leakage of csf through the skin . furthermore , reports exist of intracranial hemorrhage associated with csf leak after spinal surgeries.2 csf leak can occur anywhere from the cervical to lumbar spine . in addition to primary closure of dura , numerous other options have been developed and are used intraoperatively in the management of a csf leak . these include the use of fibrin glue and surgical clips in minimally invasive surgeries.3 4 5 6 7 8 postoperatively , supine bed rest has been instituted to decrease the rate of csf leak . refractory csf leaks have been treated with lumbar drain placement , oversewing the incision and repeat surgery.9 10 here we review our experience at two institutions with utilizing epidural subfascial drains after intentional intraoperative durotomies to prevent csf leak . we take particular interest in cases of concurrent durotomy and arthrodesis , the latter of which is now commonly performed to enhance spinal column stability in patients with iatrogenic or pathologic spinal instability . arthrodesis and instrumentation are associated with a relatively high rate of bleeding , often necessitating drain placement to avoid postoperative hematoma or seroma , the pressure of which could cause neurologic symptoms that may require reoperation.11 12 in patients who undergo durotomies and arthrodesis , the dilemma is whether an epidural subfascial suction drain should be placed , given the potential of exacerbating the csf leak . here we present our experience at two institutions with utilization of epidural subfascial drain placement after intentional intraoperative durotomies . a retrospective review was performed to identify patients undergoing spinal surgery over a 4-year period at university of california , los angeles and university of california , san francisco medical centers . the inclusion criteria include ( 1 ) known intentional durotomy with egress of csf , ( 2 ) intraoperative placement of subfascial epidural drains , ( 3 ) complete documentation of daily drain output available via electronic medical record , and ( 4 ) at least one postoperative follow - up visit . medical records were surveyed for patient age and sex , medical comorbidities , duration of drain placement , daily drain output , perioperative laboratory data , and postoperative complications . the primary outcomes of interest were the development of postoperative csf cutaneous fistula or symptomatic pseudomeningocele formation . following the intradural component of the case , the durotomy was closed with running 40 nurolon or 50 prolene ( ethicon , somerville , new jersey , united states ) to achieve primary watertight closure . one layer of either tissel fibrin glue ( baxter , deerfield , illinois , united states ) or duraseal ( covidien , minneapolis , minnesota , united states ) was applied to the primary closed durotomy site . valsava maneuvers up to 40 mm h2o were performed at the surgeon 's discretion but were not always performed . a medium or 19-french channel davol drain ( bard davol inc . , covington , georgia , united states ) was placed in the epidural space at the site of durotomy and tunneled out through a separate incision distal to the closed wound . the subcutaneous tissue and skin were then reapproximated in standard fashion utilizing interrupted 20 vicryl sutures ( ethicon ) and surgical skin staples ( covidien ) or 40 subcuticular sutures , respectively . the patients were placed in bed rest with the head of bed flat for 48 hours . the patients who underwent simultaneous arthrodesis were placed in external rigid orthotics for a total of 6 weeks . if there was evidence of leakage from the wound , suction was then adjusted to full - bulb suction . the drains were discontinued after daily output became negligible ( < 30 ml/24 h ) or 3 to 5 days following surgery when the incision appeared well healed ( even if output was still high , > 100 ml/24 h ) , prior to discharge . if output remained high prior to drain discontinuation , the drain was clamped first and the wound site evaluated for csf leak . if there was no evidence of csf leak , then the drains were removed . of note , a figure - of - eight stitch was placed at the drain exit site after drain removal if csf leaked out of the drain site . patients were clinically evaluated and the wound sites were examined at follow - up visits . a retrospective review was performed to identify patients undergoing spinal surgery over a 4-year period at university of california , los angeles and university of california , san francisco medical centers . the inclusion criteria include ( 1 ) known intentional durotomy with egress of csf , ( 2 ) intraoperative placement of subfascial epidural drains , ( 3 ) complete documentation of daily drain output available via electronic medical record , and ( 4 ) at least one postoperative follow - up visit . medical records were surveyed for patient age and sex , medical comorbidities , duration of drain placement , daily drain output , perioperative laboratory data , and postoperative complications . the primary outcomes of interest were the development of postoperative csf cutaneous fistula or symptomatic pseudomeningocele formation . following the intradural component of the case , the durotomy was closed with running 40 nurolon or 50 prolene ( ethicon , somerville , new jersey , united states ) to achieve primary watertight closure . one layer of either tissel fibrin glue ( baxter , deerfield , illinois , united states ) or duraseal ( covidien , minneapolis , minnesota , united states ) was applied to the primary closed durotomy site . valsava maneuvers up to 40 mm h2o were performed at the surgeon 's discretion but were not always performed . a medium or 19-french channel davol drain ( bard davol inc . , covington , georgia , united states ) was placed in the epidural space at the site of durotomy and tunneled out through a separate incision distal to the closed wound . the fascia was then closed with either interrupted 0 vicryl or no . 1 polydioxanone sutures ( ethicon ) . the subcutaneous tissue and skin were then reapproximated in standard fashion utilizing interrupted 20 vicryl sutures ( ethicon ) and surgical skin staples ( covidien ) or 40 subcuticular sutures , respectively . the patients were placed in bed rest with the head of bed flat for 48 hours . the patients who underwent simultaneous arthrodesis were placed in external rigid orthotics for a total of 6 weeks . if there was evidence of leakage from the wound , suction was then adjusted to full - bulb suction . the drains were discontinued after daily output became negligible ( < 30 ml/24 h ) or 3 to 5 days following surgery when the incision appeared well healed ( even if output was still high , > 100 ml/24 h ) , prior to discharge . if output remained high prior to drain discontinuation , the drain was clamped first and the wound site evaluated for csf leak . if there was no evidence of csf leak , then the drains were removed . of note , a figure - of - eight stitch was placed at the drain exit site after drain removal if csf leaked out of the drain site . patients were clinically evaluated and the wound sites were examined at follow - up visits . twenty - five spinal surgery cases with known intentional intraoperative durotomies were identified from the review of the authors ' surgical logs ( table 1 ) . thirteen men ( age 52.4 15.7 years ) and 12 women ( age 63.3 12.7 years ) were included in the study . the length of follow - up ranged from 0.5 to 28 months ( 9.5 7.4 months ) . the surgeries range from 1 to 11 levels with an average of 3.3 2.8 levels . of the 25 patients , 13 ( 52% ) the mean duration of the drain was 5.3 3.4 days with a range from 2 to 18 days . abbreviations : fu , follow - up ; lod , length of drain ; mis , minimal invasive surgery ; n , no ; who , world health organization ; y , yes . note : all cases experienced primary closure of dura . we observed a direct correlative trend between the number of spinal surgery levels and the duration of the drain placement . however , this difference was not statistically significant ( r = 0.57 , fig . the average drain duration for fused patients was 6.33 4.0 days , which was significantly longer than that of patients who did not undergo arthrodesis ( 3.7 1.1 days , p = 0.016 ) . similarly , the average daily drain output for patients who underwent arthrodesis ( 153.1 117.4 ml ) was significantly higher than that of patients who did not undergo arthrodesis ( 86.8 63.5 ml , p = 0.04 ) . the relationship between the number of spinal levels involved in surgery and duration of the drain 1 ) experienced a delayed postoperative wound infection requiring reoperation for exploration and wound washout 2 weeks after the surgery . this patient had a complicated history of multiple spinal surgeries at or near the operating site . furthermore , his postoperative care was complicated by his immobility , chemotherapy , radiation , and existing deep venous thrombosis . no seroma or pseudomeningocele were identified on his postoperative magnetic resonance imaging or during the reoperation . csf leak is a known and frustrating complication associated with all spinal surgeries and can cause headache , pseudomeningocele , and wound breakdown.13 14 although primary watertight closure remains the gold standard , numerous other strategies have been developed . dilemmas commonly encountered include whether to leave a closed suction wound drain and whether to encourage early mobilization.15 16 17 many surgeons avoid leaving a closed suction wound drain in patients with known durotomy for fear of worsening the csf leak , which is especially problematic when the surgery involves multilevel instrumentation and a surgical drain is usually required to prevent postoperative hematoma . our mean drain duration was 5.3 3.4 days and mean daily drain output was 126.5 103.2 ml . subgroup analyses revealed that the drain duration was significantly longer in patients who underwent arthrodesis compared with those who did not ( 6.33 4.0 days versus 3.7 1.1 days , p = 0.016 ) . the average daily drain output was also significantly higher in the arthrodesis group ( 153.1 117.4 ml versus 86.8 63.5 ml , p = 0.04 ) . furthermore , we noticed a linear trend in the relationship between drain duration and the number of spinal levels of operation , although the difference was not statistically significant . surgeries involving arthrodesis tend to have more extensive dissection , and the decortication processes tend to induce more seroma or hematoma formation . the seroma or hematoma could be symptomatic because of its compression on the neural structures.11 12 complications associated with closed suction wound drains have been previously described.18 19 these complications include infection , hematoma formation , and additional neurologic deficit . in addition , in cases with known durotomies , there is a concern for worsening of csf leak , possible formation of csf cutaneous fistula , pseudomeningocele , and the potential for intracranial subdural hematoma with excessive drainage of csf . of note , although one patient did have a superficial wound infection that required a washout , he had multiple comorbidities and highly complicated preoperative and postoperative courses , which was an anomaly in our study . our findings expand upon the initial findings of hughes et al for the management of csf leak after lumbar spinal surgery.20 although analogous in principle , our treatment strategy is different in that our intraoperative subfascial epidural drains were placed on an inpatient basis , and patients had their drains removed prior to discharge . despite these differences , we observed similar results in terms of our primary outcomes of interest , namely the formation of postoperative csf fistula and symptomatic pseudomeningocele . of the 25 patients in the present study , none displayed evidence of persistent csf leak due to durotomy at the most recent follow - up . and more importantly , none of the 25 patients suffered negative consequences associated with the drain . specifically , none of the patients had a postoperative csf leak or postoperative symptomatic pseudomeningocele requiring intervention . therefore , our results suggest that the techniques described herein may be employed safely and efficaciously in patients with intentional durotomies . the subfascial epidural closed suction drain likely prevents the formation of the csf fistula / leak in a similar manner to the traditional lumbar drain . the closed suction drain provides a lower resistance pathway for the csf to flow if there is a small leak from the durotomy after the primary watertight closure , which allows time for the dura , soft tissue , and fascia to scar and seal the durotomy , thereby closing the dead space . the small drain tubing tract could eventually be closed with a figure - of - eight stitch if csf continues to leak . the advantage of intraoperative placement of the epidural closed suction drain over a lumbar drain is that the epidural drain is placed under direct visualization at the same time of surgery . furthermore , intentional durotomies are usually associated with a lower rate of csf leak because they usually involve much better - quality dura and the primary closures are of significantly higher quality . therefore , future randomized prospective studies with larger sample sizes are needed to draw further conclusions . we present a review of 25 cases in which epidural subfascial drains were placed intraoperatively in patients undergoing spine surgeries with intentional durotomies . no patients developed a postoperative csf cutaneous leak , symptomatic pseudomeningocele , or complications associated closed suction drains at latest follow - up . we conclude that the placement of an epidural surgical drain after durotomy to divert csf away from the wound prevented postoperative csf leak and pseudomeningocele . however , larger studies would be useful to further evaluate the durability of such a treatment modality after durotomy .

study design retrospective chart review . objective postoperative cerebrospinal fluid ( csf ) leak is a known complication of intraoperative durotomy . intraoperative placement of subfascial epidural drains following primary dural repair has been proposed as a potential management strategy to prevent formation of csf cutaneous fistula and symptomatic pseudomeningocele . here we describe our experience with subfascial drain after intentional durotomy . methods medical records of patients who underwent placement of subfascial epidural drains during spinal procedures with intentional intraoperative durotomies over a 4-year period at two institutions were retrospectively reviewed . primary outcomes of interest were postoperative csf cutaneous fistula or symptomatic pseudomeningocele formation . results twenty - five patients were included . mean length of follow - up was 9.5 months . twelve patients ( 48% ) underwent simultaneous arthrodesis . the average duration of the drain was 5.3 days with average daily output of 126.5 ml . subgroup analyses revealed that average drain duration for the arthrodesis group was 6.33 days , which is significantly greater than that of the nonfused group , which was 3.7 days ( p = 0.016 ) . similarly , the average daily drain output for the arthrodesis subgroup at 153.1 ml was significantly higher than that of the nonfused subgroup ( 86.8 ml , p = 0.04 ) . no patient developed postoperative csf cutaneous fistula or symptomatic pseudomeningocele or had negative sequelae associated with overdrainage of csf . one patient had a delayed wound infection . conclusions the intraoperative placement of subfascial epidural drains was not associated with postoperative development of csf cutaneous fistula , symptomatic pseudomeningocele , overdrainage , or subdural hematoma in the cases reviewed . subfascial closed wound drain placement is a safe and efficacious management method after intentional spinal durotomies . it is particularly helpful in those who undergo simultaneous arthrodesis , as those patients have statistically higher daily drain output and longer drain durations .

glucagon - like peptide-1 ( glp-1 ) is a gut hormone , secreted from the intestine in response to meal ingestion , which stimulates insulin secretion and inhibits glucagon release in a dose - dependent fashion . glp-1 can suppress food intake and appetite and decelerate gastric emptying and induce satiety , so it plays an important role in regulation of blood glucose [ 2 , 3 ] . but the rapid inactivation of glp-1 in vivo and the consequent short half - life prevents its therapeutic use . long acting glp-1 receptor agonists ( glp-1 ras ) that can be administered via subcutaneous injection once or twice a day or even once a week treatment with glp-1 ras improves insulin resistance and glucose homoeostasis for patients with type 2 dm [ 5 , 6 ] . also they have a rather low risk of hypoglycemia because of their mode of action . exenatide and liraglutide are currently successfully being employed in the treatment of type 2 dm . overweight or obesity may increase the risk of cardiovascular complications as well as resulting in serious psychological distress for most patients with type 2 dm . effective interventions designed to achieve weight reduction are a critical part of type 2 dm management to prevent the development of microvascular and macrovascular complications . but many antidiabetic agents ( insulin , thiazolidinediones , and sulfonylureas ) have side - effect of bodyweight increase . glp-1 can stimulate satiety by binding to its receptor on hypothalamic neurons and reduce calorie ingestion by delayed gastric emptying [ 9 , 10 ] . although several meta - analyses have revealed the association between glp-1 ras and weight loss , all of them are head - to - head comparisons of glp-1 ras versus placebo or active comparator drugs [ 7 , 1113 ] , and these analyses did not evaluate the impact of different doses of glp-1 ras on weight loss . therefore , we did network meta - analysis of glp-1 ras in order to provide up - to - date overview of the effects of glp-1 ras on weight control in patients with type 2 dm . in consultation with a medical librarian , we established a search strategy for the medline , embase , and cochrane library ( from 1950 to october 2013 ) . the following search strategy ( ovid ) was adapted for the other databases : ( 1 ) exp glucagon - like peptide-1 agonists/ ; ( 2 ) ( glucagon - like peptide or glp-1).tw . ; ( 3 ) ( exenatide or liraglutide).tw . ; ( 6 ) ( ( 1 ) or ( 2 ) or ( 3 ) ) and ( ( 4 ) or ( 5 ) ) . in addition , we searched the bibliographies of published systematic reviews [ 7 , 1113 ] . all relevant authors and principal investigators were contacted to supplement incomplete reports of the original papers or to provide data for unpublished studies . all the studies included are in english and they are eligible for inclusion only if they were rcts involving glp-1 ras , other hypoglycemic drugs or placebo with complete data on body weight . trials are excluded if only they meet one of the following : ( 1 ) trials are not rct ( e.g. , review , expert comment , editor opinion , new agent introduction , single case report , or case series ) ; ( 2 ) early studies ; ( 3 ) experimentation on animals or in vitro ; ( 4 ) not conducted in type 2 dm ; ( 5 ) pharmacokinetics research ; ( 6 ) trials underway , unfinished , or suspended ; ( 7 ) economical evaluation research ; ( 8) other unrelated researches . these studies were approved by the local ethics committees and written informed consent was obtained from all the patients . we excluded trials or arms using nonstandard doses , which mainly came from dose - ranging studies . so only those dose - arms possibly relevant to clinical application were included in our study . the standard exenatide regimens are 5 g twice daily ( ex5bid ) , 10 g twice daily ( ex10bid ) , and 2 mg once weekly ( ex2qw ) . the standard liraglutide regimens are 0.6 mg once daily ( lir0.6qd ) , 0.9 mg once daily ( lir0.9qd ) , 1.2 mg once daily ( lir1.2qd ) , and 1.8 mg once daily ( lir1.8qd ) , respectively . after obtaining full reports of the candidate trials , two reviewers ( csb and lls ) independently used addis software to extract information concerning population characteristics ( age , t2d course , baseline hba1c ) and weight change of each treatment group . quality of studies was assessed according to jadad scale : adequate method for randomization , appropriate blinding procedures , and detailed report of withdrawals . initially , we performed standard pairwise meta - analyses using the dersimonian - laird random effects model for every treatment comparison . where studies did not report intent - to - treat , we analyzed outcomes as all - patients randomized . the i statistic was calculated as a measure of the proportion of the overall variation that is attributable to between - study heterogeneity . we did a network meta - analysis within a bayesian framework , and we summarized the results using mean difference ( md ) and credible interval ( cri ) . bayesian network meta - analysis was a generalization of traditional meta - analysis that allowed all evidence to be taken into account simultaneously ( both direct and indirect ) . we estimated the posterior densities for all unknown parameters using mcmc ( markov chain monte carlo ) for each model . each chain used 40,000 iterations with a burn - in of 20,000 . to formally check whether a model 's overall fit was satisfactory , we considered an absolute measure of fit : d - res . this is the posterior mean of the deviance under the current model , minus the deviance for the saturated model . we would expect that each data point should contribute about 1 to the posterior mean deviance so that it can be compared with the number of data points for the purpose of checking model fit . we estimated the ranking probabilities for all treatments at each possible rank for every intervention . then , we obtained a treatment hierarchy using the surface under the cumulative ranking curve ( sucra ) and posterior mean ranks . sucra can also be expressed as a percentage interpreted as the percentage of efficacy of a treatment on weight control that would be ranked first without uncertainty . one key assumption of the network meta - analysis models is the consistency between direct and indirect evidence , that is , if the information of both sources of evidence is similar enough in order to be combined . to evaluate the presence of inconsistency this method evaluates the consistency assumption in each closed loop of the network separately as the difference between direct and indirect estimates for a specific comparison in the loop ( inconsistency factor ) . then , the magnitude of the inconsistency factors and their 95% cis can be used to infer the presence of inconsistency in each loop . inconsistency can be evaluated as the disagreement between different sources of evidence within a closed loop . analyses were conducted using stata 11.0 ( pairwise meta - analysis , i calculations , and estimation of inconsistence ) , r 3.0.2 ( sucra graphs ) and winbugs 1.4.3 ( network meta - analysis and model fit ) . 1139 studies were identified at first . finally , 51 studies involving 13 treatments and 17521 participants with a mean duration of 31 weeks were included . publication year varied from 2002 to 2013 and duration ranged from 4 weeks to 234 weeks . the 17521 patients had a mean baseline hba1c of 8.3% , a mean age of 56 y , and a mean diabetes duration of 8.3 y. there were 36 studies about exenatide , 13 studies about liraglutide , and 2 studies for both [ 23 , 24 ] . thirteen treatments were analyzed , including seven glp-1 ras dose - group ( ex10bid , ex2qw , ex5bid , lir0.6qd , lir0.9qd , lir1.2qd , and lir1.8qd ) , five kinds of traditional antidiabetics ( insulin , metformin ( met ) , sulfonylureas ( su ) , sitagliptin , and thiazolidinediones ( tzd ) ) , and placebo . thirty - two trials ( 62.75% ) were two - arm studies and nineteen ( 37.25% ) were multiple - arm studies ( see table 1 and figure 2 ) , totally 127 arms . the proportion of appropriate description of randomization , allocation concealment , blinding , and dropout were 78.43% ( 40/51 ) , 50.98% ( 26/51 ) , 52.94% ( 27/51 ) , and 88.24% ( 45/51 ) , respectively . additionally , 90.20% ( 46/51 ) trials were used intention - to - treat analysis ( see table 1 in supplementary material available online at http://dx.doi.org/10.1155/2015/157201 ) . overall risk of bias was , respectively , low and bias mainly came from allocation concealment and blinding of outcome assessment . compared with placebo , ex10bid and lir1.8qd decreased body weight by 1.38 kg ( 95% ci : 1.74 , 1.03 ) and 1.32 kg ( 95% ci : 2.22 , 0.43 ) , respectively , while there was a statistically significant weight gain observed in the traditional hypoglycemic drugs ( insulin , su , and tzd ) compared with placebo , with mean differences of 2.02 kg ( 95% ci : 1.02 , 3.02 ) , 2.03 kg ( 95% ci : 0.91 , 3.15 ) , and 2.20 kg ( 95% ci : 1.54 , 2.86 ) , respectively . glp-1 ras achieved a greater weight loss than traditional hypoglycemic drugs ; the range of weight reduction was 7.30 kg ( 95% ci : 12.82 , 1.78)~1.38 kg ( 95% ci : 1.74 , 1.03 ) with exenatide and 3.12 kg ( 95% ci : 3.80 , 2.44)~1.40 kg ( 95% ci : 1.95 , 0.85 ) with liraglutide , respectively . table 2 displays the result of network meta - analysis between glp-1 ras and traditional hypoglycemic drugs . as displayed in the preceding pairwise meta - analysis , we found ex10bid and lir1.8qd were associated with a significant reduction in body weight versus placebo , with mean differences of 1.92 kg ( 95% ci : 2.61 , 1.24 ) and 0.98 kg ( 95% ci : 1.94 , 0.02 ) , respectively , while treatment with insulin , su , and tzd resulted in significant weight gain versus placebo ( range from 2.60 kg ( 95% ci : 1.56 , 3.62 ) to 3.37 kg ( 95% ci : 2.29 , 4.48 ) ) . similar to direct comparisons , exenatide and liraglutide showed more advantage on weight control than traditional hypoglycemic drugs . the weight reduction significantly ranged from 5.30 kg ( 95% ci : 6.38 , 4.24 ) to 2.21 kg ( 95% ci : 3.59 , 0.83 ) with exenatide and from 4.35 kg ( 95% ci : 5.45 , 3.24 ) to 2.21 kg ( 95% ci : 3.92 , 0.53 ) with liraglutide . compared with ex5bid , lir0.6qd , lir1.2qd , and placebo treatment , treatment with ex10bid resulted in a significantly greater decrease in body weight , with mean differences of 1.07 kg ( 95% ci : 2.41 , 0.02 ) , 2.38 kg ( 95% ci : 3.71 , 1.06 ) , 1.62 kg ( 95% ci : 2.79 , 0.43 ) , and 1.92 kg ( 95% ci : 2.61 , 1.24 ) , respectively . reductions in body weight treated with lir1.8qd reach statistical significance ( 1.43 kg ( 95% ci : 2.73 , 0.15 ) ) versus lir0.6qd and ( 0.98 kg ( 95% ci : 1.94 , 0.02 ) ) versus placebo . network meta - analyses enable estimation of the probability that each intervention is the best for each outcome . a simple numerical summary supplying the graphical display of cumulative ranking is used to estimate the surface under the cumulative line for each treatment . larger sucra means the drug has lower ranking ( see supplementary figures 1(a ) and 1(b ) ) . table 3 showed the mean sucra values providing the hierarchy of 13 treatments on weight loss : ex10bid ( 98.30% ) , lir1.8qd ( 78.89% ) , ex2qw ( 76.23% ) , ex5bid ( 75.02% ) , met ( 63.32% ) , lir1.2qd ( 58.44% ) , placebo ( 48.50% ) , lir0.9qd ( 42.90% ) , sitagliptin ( 42.47% ) , lir0.6qd ( 38.83% ) , insulin ( 12.86% ) , tzd ( 11.29% ) , and su ( 3.02% ) , respectively . according to sucra values , ex10bid had the highest impact and su had the least impact on weight loss among 13 treatments . the model fit can be evaluated using the posterior mean of the residual deviance d - res . the value of the d - res was 115.41 , close to 127 of the data points for weight control , meaning model 's overall fit was relatively satisfactory . additionally , statistical inconsistency between direct and indirect comparisons was generally low for weight control . most loops ( 90.70% , 39/43 ) of 27 triangular loops and 16 quadratic loops were consistent ( p value > 0.05 ; see supplementary table 2 ) and their 95% cis included 0 according to the forest plots ; that means the direct estimate of the summary effect was not different from the indirect estimate ( see supplementary figure 2 ) . obesity is known to increase the risk of diabetes and complications such as insulin resistance , dyslipidemia , and cardiovascular diseases [ 25 , 26 ] . over 80% of individuals with type 2 dm it is attractive to both doctors and patients to avoid weight gain of type 2 dm during the treatment of glycaemic control . glp-1 ras are a novel class of glucose - lowering drugs which has been shown to improve glycaemic control and promote weight loss in clinical studies of patients with type 2 dm . in 2005 , the us food and drug administration approved the first long acting stable glp-1 ra . exenatide ( byetta ; elililly ) and liraglutide ( victoza ; novonordisk ) are now available on the market . both drugs can be used in combination with oral antidiabetic drugs such as metformin , thiazolidinediones , or sulfonylureas compounds . the treatments are approved for patients with type 2 dm who have not achieved adequate glycemic control after treatment with traditional antidiabetic drugs . the network meta - analysis combines direct and indirect evidences in a single analysis , enabling simultaneously comparison of multiple interventions , which is different from the traditional meta - analysis . this approach makes use of direct comparisons from existing trials comparing 2 treatment strategies and indirect comparisons constructed from 2 trials that have at least 1 treatment in common . this statistical tool preserves the within - trial , randomized comparison of each study . and our study showed that a higher proportion of subjects experienced weight loss with exenatide ( ex10bid , ex2qw , and ex5bid ) and liraglutide ( lir1.2qd , lir0.6qd , and lir1.8qd ) than with insulin , su , and tzd , which were similar to previous meta - analysis [ 7 , 1113 , 28 ] , with weight loss following glp-1 ras treatment ranging from 3.31 to 1.22 kg . our results indicated that subjects experienced greater weight reductions in a higher exenatide dose ( ex10bid ) and liraglutide dose ( lir1.8qd ) compared with insulin , su , tzd , and placebo , suggesting a dose - dependent effect . in accordance with the previous report liraglutide 1.8 mg treated subjects experienced more weight loss than 1.2 mg treated subjects . compared with placebo , treatment with su , tzds , and insulin resulted in a significantly greater increase in body weight , with change from 2.60 kg to 3.37 kg . by contrast , use of ex10bid and lir1.8qd resulted in a significant decrease in bodyweight , with mean changes of 1.92 kg and 0.98 kg , respectively . although the precise mechanisms associated with weight loss have not been elucidated yet , it was believed that gastric emptying was an important factor for weight loss . however , the preclinical study showed that liraglutide 's effect on gastric emptying diminished over time , whereas the effect on body weight was sustained over the treatment period . recently , evidence indicated that glp-1-induced weight reduction required higher glp-1 ras levels . compared with placebo , weight did not decrease substantially either with ex2qw and ex5bid or with lir0.6qd , lir0.9qd , and lir1.2qd . but the results suggested the potential relationship between weight reduction and drug dose . except for ex10bid , body weight loss was not significant for sitagliptin and glp-1 ras in our meta - analysis . like the report that showed subjects taking metformin alone lost more weight than subjects taking an su plus metformin , our study showed that body weight did not change in patients with metformin compared with those with exenatide and liraglutide . there were statistically and clinically significant differences between drug classes in weight changes . in our study , ex10bid , lir1.8qd , and ex2qw the results suggested weight reduction of patients has a close relationship with dose of glp-1 ras . first , our study is the largest evaluation of glp-1 ras on weight control to date . second , the network meta - analysis technique allows dissection of the individual drug to evaluate weight control , especially facing very few rcts which directly compared glp-1 ras in type 2 dm . we applied a bayesian model to explore the effect of indirect comparison between them , and it is thought to be the most appropriate method for multiple - treatments network meta - analysis . additionally , posterior probability from bayesian model can help to apply the rank of glp-1 ras in practice . first , other unpublished literatures on relevant pharmaceutical websites were not searched and only trials in english were included , which may lead to a potential publication bias . second , most trials included in this review were not specially designed to evaluate body weight , with the risk of misdiagnosis and under diagnosis . finally , lack of information about the distribution of clinical and methodological variables may lead to potential sources of either heterogeneity or inconsistency in every comparison - specific group of trials . to sum up , this network meta - analysis provides a useful and complete picture of the associations between glp-1 ras , conventional antidiabetic drugs , and placebo on weight control . glp-1 ras are associated with significant weight loss for the treatment of type 2 dm . however , the application of our results should take into account limitations of the analysis and the specific clinical situation . more long - term large - scale trials will be necessary to confirm potential efficacy on weight loss . meantime , determining the specific subject - related factors associated with greater weight loss with glp-1 receptor agonists may be helpful to clinicians in identifying patients who would likely benefit from their use . glp-1 receptor agonists cause weight reduction , in contrast to insulin , metformin , sulfonylureas , sitagliptin , thiazolidinediones , and placebo . according to the mean sucra values , ex10bid , lir1.8qd , and ex2qw rank the top three drugs .

to evaluate the effectiveness of glucagon - like peptide-1 receptor agonists ( glp-1 ras ) on weight reduction in patients with type 2 diabetes mellitus ( type 2 dm ) , a network meta - analysis was conducted . medline , embase , cochrane library , and clinicaltrials.gov were searched from 1950 to october 2013 . randomized controlled trials ( rcts ) involving glp-1 ras were included if they provided information on body weight . a total of 51 rcts were included and 17521 participants were enrolled . the mean duration of 51 rcts was 31 weeks . exenatide 10 g twice daily ( ex10bid ) reduced weight compared with exenatide 5 g twice daily ( ex5bid ) , liraglutide 0.6 mg once daily ( lir0.6qd ) , liraglutide1.2 mg once daily ( lir1.2qd ) , and placebo treatment , with mean differences of 1.07 kg ( 95% ci : 2.41 , 0.02 ) , 2.38 kg ( 95% ci : 3.71 , 1.06 ) , 1.62 kg ( 95% ci : 2.79 , 0.43 ) , and 1.92 kg ( 95% ci : 2.61 , 1.24 ) , respectively . reductions of weight treated with liraglutide1.8 mg once daily ( lir1.8qd ) reach statistical significance ( 1.43 kg ( 95% ci : 2.73 , 0.15 ) ) versus lir1.2qd and ( 0.98 kg ( 95% ci : 1.94 , 0.02 ) ) versus placebo . network meta - analysis found that ex10bid , lir1.8qd , and ex2qw obtained a higher proportion of patients with weight loss than other traditional hypoglycemic agents . our results suggest glp-1 ras are promising candidates for weight control in comparison with traditional hypoglycemic drugs , and ex10bid , lir1.8qd , and ex2qw rank the top three drugs .

teeth are complex anatomic structures that encounter with developmental anomaly in various aspects like defects in the structure , shape , size and number . fusion is a rare developmental anomaly with a complex morphology that can give rise to reduced esthetics , misalignment , dental caries and periodontal problems . the purpose of this article is to report a rare case of bilateral fusion of mandibular second premolar with supernumerary tooth . a 25-year - old male patient reported to the chettinad dental college hospital with complaint of pain on the left lower posterior tooth ( 35 ) which was decayed and had bizarre morphology similar to its counterpart on the right side ( 45 ) . both the teeth were larger on all aspects than the adjacent normal first premolar and had a pronounced buccal groove [ figures 1 and 2 ] . radiographic examination of 35 revealed pulpal involvement associated with a complex coronal and radicular pulpal anatomy . the orthopantomograph showed no evidence of missing tooth in the mandibular arch but presented bilateral impaction of third molars [ figure 3 ] . tooth 35 showing abnormal crown morphology and pronounced buccal groove partially erupted 45 showing abnormal crown morphology orthopantamograph showing abnormal tooth morphology in 35 and 45 along with impaction of 38 and 48 fusion is a developmental dental anomaly caused by the union of two tooth germs to form a single tooth . fused teeth are usually united by enamel and dentin , whereas the pulp chambers and pulp canals are either unified or separated . the etiology of fusion is still unknown ; however , the crowding of the tooth germs during their development can be an important factor . local metabolic disturbances or developmental aberrations of ectoderm and mesoderm during morphodifferentiation of tooth bud can be considered as etiological factors . they do not show any sex predilection but are more frequent among japanese population and american indians . fusion of teeth is more common in the deciduous dentition than the permanent dentition with a prevalence rate of 0.52.5% and 0.1% , respectively . literatures reveal the prevalence of bilateral fusion in primary teeth to be 0.010.04% and in permanent teeth as 0.05% . invariable of the type of dentition , majority of them are associated with the incisors and canine than the posterior arch . fusion of posteriors is infrequent in secondary teeth with a prevalence range from 0.08% to 0.5% . in general , the present case is a rarity as it occurred in the mandibular arch , in the posterior region with bilateral presentation , caused by the union of second premolar and a supernumerary tooth which was seldom reported . the number of teeth present is usually reduced in fusion , but is normal if the anomaly occur between a regular and supernumerary tooth . in contrast , gemination results in an apparent increase in the number of teeth , as they are caused due to the division of a single tooth germ to form two separate teeth . in these situations , even though there is no variation in the treatment plan , an attempt can be made to differentiate both the anomalies by performing a thorough clinical and radiographic examination . fusion between two teeth usually results in space gain or diastema but may not be the case when the anomaly involves a supernumerary tooth as seen in our case . the portion of the fused tooth that is formed by the supernumerary tooth is conical in morphology and is smaller when compared with other fused component that is separated by a developmental groove , whereas in gemination both the halves are mirror images of each other . the present case also revealed two disproportionate components with dissimilar morphology separated by a groove . according to which if the anomalous tooth is counted as two teeth and teeth count is normal in the arch , then it is considered as fusion . in cases where the anomalous tooth is counted as two teeth and if an extra tooth is present in the region , then it is regarded as gemination or a fusion between a normal and a supernumerary tooth . he also suggested referring teeth joined together by dentin as fused teeth . brook and winter proposed the neutral term intraoral radiographs can be considered but may not be confirmatory in the differentiation of double teeth as geminated teeth have a single large root canal , whereas fused teeth may have separate or fused root canals . in the present case , the anomalous teeth revealed an increased mesiodistal width , a single pulp chamber ; single root canal and normal teeth count in the respective quadrant as evident radiographically . . the buccal and lingual grooves may be deep and extend subgingivally favoring plaque accumulation leading to dental caries and periodontal diseases . the complex tooth morphology and pulpal anatomy , tooth position , and difficulty in rubber dam placement may negate endodontic treatment and necessitate surgical removal of the affected tooth . fusion of primary teeth may lead to hypodontia , malformation , impaction , delayed , or altered path of eruption of permanent successors . in conclusion , fusions are rare developmental anomaly and need to be recorded during routine clinical examination . the abnormal morphology demands prophylactic and early interceptive treatment in order to avoid the complicated pulpal and periodontal treatment related to these teeth . surgical extraction of the affected primary teeth can be an option to avoid its delayed exfoliation and the subsequent delayed or ectopic eruption of the successor .

fusion is the union of two normally separated tooth germs resulting in the formation of a single large tooth . the prevalence of this anomaly is less than 1% and most common in the primary dentition , in the incisor - canine region . fusions are almost always unilateral , but few cases of bilateral fusions have been reported . the purpose of this article is to report a rare case of bilateral fusion of mandibular second premolar with supernumerary tooth .

severe acute respiratory syndrome ( sars ) has infected thousands of people and cost hundreds of deaths globally since it first emerged in guangdong , china , in november 2002 ( ref . 1 ; http://www.who.int/csr/sars/en/ ) . sixteen complete or partial genome sequences of sars - cov isolates have been available since march 14 this year ( ref . 2 . , 3 . , 4 . , 5 . ; http://www.ncbi.nlm.nih.gov/ ; table 1 ) . in the process of surveying all available genomic sequences of the sars - cov , we have obtained the complete genomic sequence of an isolate , named isolate gz01 originally . the sequence contains an extra 29-nucleotide segment in the vicinity of the viral structural proteins , m ( the membrane protein ) and n ( the nucleocapsid protein ) . we have renamed the isolate as gd - ins29 to indicate the origin , length , and the type of mutation . we believe that this novel genotype represents one of the early variants of the sars - cov . the absence of the 29-nt fragment may help the virus to escape the likely interference from locally homologous rna molecules of host origin and to become more prevalent in human hosts . this sequence also holds unusually high number of variations among all the sequenced sars - cov isolates , suggesting a more distant relationship from other sequences sars - cov genomes published thus far . the genome landscape of this isolate is not greatly different from others reported by our group and other laboratories . twelve orfs ( open reading frames ) , including 6 cdss ( coding sequences ) and 6 pups ( postulated uncharacterized proteins ) were predicted in the viral genome ( table 2 ; figure 1 ) . we annotated two new pups , bgi - pup5 ( so named to avoid possible ambiguity in nomenclature ) and bgi - pup6 , located between the previously reported pup4 and the n protein . these pups overlap in a 35-nt region ( position 27,843 and 27,879 , in reference to nucleotide position in the complete sequence of bj01 ) . both pups share the same consensus leader sequence ( 5-agucuaaaagaac-3 ) that is immediately followed by the predicted start codon of bgi - pup5 , and approximately 95-nt away from that of bgi - pup6 . the expression , actual existence of protein products , and possible function of the pups , such as that of bgi - pup6 , remain to be elucidated experimentally . an extra sequence segment of 29-nt ( gd - ins29 ) in length was unambiguously identified within the overlapped region of bgi - pup5 and bgi - pup6 . this sequence was confirmed with 60 high - quality sequencing reads from 35 clones of the site - specific amplicon - library constructed with rt - pcr products from this genomic region . among them , 25 clones were sequenced from both ends . no other sequence variations were found in the sequences from this particular amplicon - library , even though minor variants are occasionally seen in other amplicon - libraries from different sequences due to minor variations from viral populations and , to a much less extent , rt - pcr generated aberrant products . we have also identified another sars - cov isolate that harbors the same sequence segment from a sars patient in guangdong province . these results strongly suggest that we have found a novel yet minor genotype of sars - cov , but not encountered a sequence anomaly . in the gd - ins29 sequence , the 29-nt segment is located in - frame after the second nucleotide of codon 35 in bgi - pup5 ( position 27,863 ) and the first nucleotide of codon 7 of bgi - pup6 . the resulted hypothetical protein is predicted to have 122 amino acids . without affecting the coding frame , the 29-nt sequence theoretically could be spliced out in two different ways since there is uncertainty whether the uridine is at the beginning or at the end of the extra sequence , 5- uccuacugguuaccaaccugaauggaaua-3 or 5-ccuacugguuaccaaccugaauggaauau-3. no obvious sequence signatures were found within and around the sequence except several short stretches of simple repetitive sequences of 6 to 8 nucleotides in unit length . the actual mechanism , as to how such sequence was deleted , is yet to be revealed . since the deletion is very significant in size , we reasoned that it might have been affected by the host factors . upon searching the sequence against public human sequence databases , we have found two seemingly interesting matches of 17-nt segments in the center of the sequence to three human chromosomal locations . one ( 5-gguuaccaaccugaaug-3 ) was aligned to protein coding sequences mapped to two chromosomal locations : 15q23 and 9q22 ; the other ( 5-ugguuaccaaccugaau-3 ) matches to a sequence segment on chromosome 11 , which overlaps with a repetitive sequence of the mammalian interspersed repeat , or mir , and is most likely non - protein coding . the protein - coding sequences are both members of acidic ( leucine - rich ) nuclear phosphoprotein 32 family 6 . , 7 .. exhaustive database searches did not yield any other significant matching sequence in the human genomic sequence databases . since the virus is propagated within the cytosol , only the host sequences , such as processed transcripts , mrnas and other operational rnas , would have possibilities directly interacting with the viral host - dependent cellular processes , such as replication of the viral genome via a negative sense rna intermediates , transcription of genes encoding viral replicase and structural proteins , and , to a limited extent , translation , if the interfering sequences enabling strand - annealing with viral rna products . the unique 17-nt sequence that we have discovered from the human genome as mrna forms is thought to be capable of annealing specifically with the negative sense strand of the viral rna while the virus is replicating its rna genomes from the negative sense rna genome intermediates and transcribing viral transcripts for the replicase and structural proteins , thus interfere with the viral life cycle by reducing the efficiency of both viral replication and transcription . the prerequisite for the interference to happen is that the 17-nt sequence comes from a protein or rna coding sequence that exists in infected human cells with reasonable abundance . it is very suggestive that the interaction of the intermediate viral genome products , namely the negative sense rnas , and the host rna species might have happened in the propagation processes within a host of sars - cov , through intermolecular rna - rna recombination ( 8) . after careful sequence alignment , 137 substitutions were identified in comparison with the other 16 published sars - cov genome sequences , yielded an overall - genome mutation rate of approximately 0.46% ( table 3 ) . special attention was paid to the sequences of the mutation sites and multiple clones from the amplicon - libraries constructed from multiple independent rt - pcr products were sequenced . a substantial portion ( 70.4% , 100/142 ) of the substitutions was predicted to be non - synonymous mutations in the orfs , including 14 in the pups . among the 92 substitutions detected in the r ( replicase ) protein , 70.7% ( 65/92 ) could lead to amino acid changes . although the s ( spike ) protein has a low ratio ( 13/22 ) , the ratio is higher in the other structural proteins , the m ( 4/4 ) and n proteins ( 3/4 ) , as well as in bgi - pup2 ( 4/5 ) , bgi - pup3 ( 2/2 ) , and bgi - pup ( gd - ins29 , 2/2 ) . even though a large fraction of substitutions in the viral genome have been located in the orf for the r protein , its substitution rate is actually the lowest among all the defined cdss , with regard to its large size ( 21,222 nt ) . the mutation rate of sars - cov is quite high if we take into consideration such a short time period since it was identified from human hosts . a high mutation rate is consistent with the high error rate of rna replication ( 9 ) and the high fraction of non - synonymous substitutions also implies the possibility that the selective pressure from the host may have worked on the virus , albeit there is very little statistical power to support the conjecture with current data set . a very important notion from our data ( including tens of thousands of sequencing traces ) is the obvious absence of indels ( insertions and deletions ) , suggesting that the viral polymerase is prone to the replication errors in proofreading but remains relatively accurate in frame - moving conveyed by the rdrp ( rna - dependent rna polymerase ) activity . based on comparative analyses on the complete genome sequences of the 17 sars - cov isolates from patients identified in canada , usa , singapore , and china ( beijing , zhejiang , guangdong , hong kong , and taiwan ) , an unrooted phylogenetic tree of the sars - cov was constructed ( figure 2 ) . the phylogenetic tree positioned isolate gd01 in the group composed of bj01 , 02 , 03 , and 04 , two of the hong kong isolates ( cuhk ) , and the us isolate ( urbani ) . this paradigm suggests a possible transmission path among guangdong , hong kong , beijing and usa . the link between the two isolates ( tor2 and hku ) in another group has been suggested that the toronto patient , from whom the isolate was obtained , was known to have traveled from hong kong ( 3 ) . two relatively distant isolates in taiwan and zhejiang , china , reported more recently , are more distant from the rest . additional molecular epidemiological data , based on genomic sequences of viral isolates identified from different regions , is essential to elucidate the possible routes of global spreading and mutation during its transmission among humans . the sars - cov has nearly identical genome organization , especially its gene order , with other members in coronaviridae found in humans and other animals . the sequences of the sars - cov genome from various isolates are also almost identical with only a few dozen nucleotide substitutions per genome compared with each other . therefore , we are strongly in favor of the hypothesis that the sars - cov may have originated from non - human animals , no matter it is virulent or latent to its host , in unknown reservoirs of the wild and recently moved onto humans through a less frequently established contacting route . it is of great importance that such non - human hosts should be promptly identified in order to prevent further evasion into human and other animal populations . the discovery of the gd - ins29 genotype is very useful in differentiating the origin of sars - cov . the fact that we have only identified a couple of isolates in guangdong province supports the scenario that gd - ins29 is the original form transmitted from non - human host to human ones and later turned into a new genotype after deletion of a dna segment that may hinders its efficiency of propagation . first , the non - human reservoir of sars - cov may harbor genome sequences close to gd - ins29 , not the deleted form or the major genotype found most widespread among its human hosts . second , both variants of the major and the minor genotypes could be found in the same animal reservoir owing to multiple transmissions of the virus back and forth between its human and non - human hosts . alternatively , there might have been at least two genotypes of sars - cov spreading in non - human host populations and it is unlikely but possible that the virus have infected humans multiple times in a not - distant past and the two genotypes co - exist in the non - human hosts but not traceable in current human populations . although we can not rule out another possibility that the viruses of the major genotype had acquired a 29-nt sequence and become gd - ins29 genotype during its propagation in human hosts , such event is deemed extremely rare since insertion or deletion is often lethal to the virus that have a rather compact genome . genomic and genetic knowledge can provide an extraordinary trove of information about the pathogenesis and virulence of sars - cov . more sequences data would significantly expand our knowledge on the etiology and evolution of the virus and are essential for seeking new approaches for diagnostics , vaccine development , and effective therapy of sars . high quality sequence data and comprehensive analysis , as well as more experimental evidences , are still of urgent need to understand such a potent infectious agent . the genome landscape of this isolate is not greatly different from others reported by our group and other laboratories . twelve orfs ( open reading frames ) , including 6 cdss ( coding sequences ) and 6 pups ( postulated uncharacterized proteins ) were predicted in the viral genome ( table 2 ; figure 1 ) . we annotated two new pups , bgi - pup5 ( so named to avoid possible ambiguity in nomenclature ) and bgi - pup6 , located between the previously reported pup4 and the n protein . these pups overlap in a 35-nt region ( position 27,843 and 27,879 , in reference to nucleotide position in the complete sequence of bj01 ) . both pups share the same consensus leader sequence ( 5-agucuaaaagaac-3 ) that is immediately followed by the predicted start codon of bgi - pup5 , and approximately 95-nt away from that of bgi - pup6 . the expression , actual existence of protein products , and possible function of the pups , such as that of bgi - pup6 , remain to be elucidated experimentally . an extra sequence segment of 29-nt ( gd - ins29 ) in length was unambiguously identified within the overlapped region of bgi - pup5 and bgi - pup6 . this sequence was confirmed with 60 high - quality sequencing reads from 35 clones of the site - specific amplicon - library constructed with rt - pcr products from this genomic region . among them , 25 clones were sequenced from both ends . no other sequence variations were found in the sequences from this particular amplicon - library , even though minor variants are occasionally seen in other amplicon - libraries from different sequences due to minor variations from viral populations and , to a much less extent , rt - pcr generated aberrant products . we have also identified another sars - cov isolate that harbors the same sequence segment from a sars patient in guangdong province . these results strongly suggest that we have found a novel yet minor genotype of sars - cov , but not encountered a sequence anomaly . in the gd - ins29 sequence , the 29-nt segment is located in - frame after the second nucleotide of codon 35 in bgi - pup5 ( position 27,863 ) and the first nucleotide of codon 7 of bgi - pup6 . the resulted hypothetical protein is predicted to have 122 amino acids . without affecting the coding frame , the 29-nt sequence theoretically could be spliced out in two different ways since there is uncertainty whether the uridine is at the beginning or at the end of the extra sequence , 5- uccuacugguuaccaaccugaauggaaua-3 or 5-ccuacugguuaccaaccugaauggaauau-3. no obvious sequence signatures were found within and around the sequence except several short stretches of simple repetitive sequences of 6 to 8 nucleotides in unit length . the actual mechanism , as to how such sequence was deleted , is yet to be revealed . since the deletion is very significant in size , we reasoned that it might have been affected by the host factors . upon searching the sequence against public human sequence databases , we have found two seemingly interesting matches of 17-nt segments in the center of the sequence to three human chromosomal locations . one ( 5-gguuaccaaccugaaug-3 ) was aligned to protein coding sequences mapped to two chromosomal locations : 15q23 and 9q22 ; the other ( 5-ugguuaccaaccugaau-3 ) matches to a sequence segment on chromosome 11 , which overlaps with a repetitive sequence of the mammalian interspersed repeat , or mir , and is most likely non - protein coding . the protein - coding sequences are both members of acidic ( leucine - rich ) nuclear phosphoprotein 32 family 6 . , 7 .. exhaustive database searches did not yield any other significant matching sequence in the human genomic sequence databases . since the virus is propagated within the cytosol , only the host sequences , such as processed transcripts , mrnas and other operational rnas , would have possibilities directly interacting with the viral host - dependent cellular processes , such as replication of the viral genome via a negative sense rna intermediates , transcription of genes encoding viral replicase and structural proteins , and , to a limited extent , translation , if the interfering sequences enabling strand - annealing with viral rna products . the unique 17-nt sequence that we have discovered from the human genome as mrna forms is thought to be capable of annealing specifically with the negative sense strand of the viral rna while the virus is replicating its rna genomes from the negative sense rna genome intermediates and transcribing viral transcripts for the replicase and structural proteins , thus interfere with the viral life cycle by reducing the efficiency of both viral replication and transcription . the prerequisite for the interference to happen is that the 17-nt sequence comes from a protein or rna coding sequence that exists in infected human cells with reasonable abundance . it is very suggestive that the interaction of the intermediate viral genome products , namely the negative sense rnas , and the host rna species might have happened in the propagation processes within a host of sars - cov , through intermolecular rna - rna recombination ( 8) . after careful sequence alignment , 137 substitutions were identified in comparison with the other 16 published sars - cov genome sequences , yielded an overall - genome mutation rate of approximately 0.46% ( table 3 ) . special attention was paid to the sequences of the mutation sites and multiple clones from the amplicon - libraries constructed from multiple independent rt - pcr products were sequenced . a substantial portion ( 70.4% , 100/142 ) of the substitutions was predicted to be non - synonymous mutations in the orfs , including 14 in the pups . among the 92 substitutions detected in the r ( replicase ) protein , 70.7% ( 65/92 ) could lead to amino acid changes . although the s ( spike ) protein has a low ratio ( 13/22 ) , the ratio is higher in the other structural proteins , the m ( 4/4 ) and n proteins ( 3/4 ) , as well as in bgi - pup2 ( 4/5 ) , bgi - pup3 ( 2/2 ) , and bgi - pup ( gd - ins29 , 2/2 ) . even though a large fraction of substitutions in the viral genome have been located in the orf for the r protein , its substitution rate is actually the lowest among all the defined cdss , with regard to its large size ( 21,222 nt ) . the mutation rate of sars - cov is quite high if we take into consideration such a short time period since it was identified from human hosts . a high mutation rate is consistent with the high error rate of rna replication ( 9 ) and the high fraction of non - synonymous substitutions also implies the possibility that the selective pressure from the host may have worked on the virus , albeit there is very little statistical power to support the conjecture with current data set . a very important notion from our data ( including tens of thousands of sequencing traces ) is the obvious absence of indels ( insertions and deletions ) , suggesting that the viral polymerase is prone to the replication errors in proofreading but remains relatively accurate in frame - moving conveyed by the rdrp ( rna - dependent rna polymerase ) activity . based on comparative analyses on the complete genome sequences of the 17 sars - cov isolates from patients identified in canada , usa , singapore , and china ( beijing , zhejiang , guangdong , hong kong , and taiwan ) , an unrooted phylogenetic tree of the sars - cov was constructed ( figure 2 ) . the phylogenetic tree positioned isolate gd01 in the group composed of bj01 , 02 , 03 , and 04 , two of the hong kong isolates ( cuhk ) , and the us isolate ( urbani ) . this paradigm suggests a possible transmission path among guangdong , hong kong , beijing and usa . the link between the two isolates ( tor2 and hku ) in another group has been suggested that the toronto patient , from whom the isolate was obtained , was known to have traveled from hong kong ( 3 ) . two relatively distant isolates in taiwan and zhejiang , china , reported more recently , are more distant from the rest . additional molecular epidemiological data , based on genomic sequences of viral isolates identified from different regions , is essential to elucidate the possible routes of global spreading and mutation during its transmission among humans . the sars - cov has nearly identical genome organization , especially its gene order , with other members in coronaviridae found in humans and other animals . the sequences of the sars - cov genome from various isolates are also almost identical with only a few dozen nucleotide substitutions per genome compared with each other . therefore , we are strongly in favor of the hypothesis that the sars - cov may have originated from non - human animals , no matter it is virulent or latent to its host , in unknown reservoirs of the wild and recently moved onto humans through a less frequently established contacting route . it is of great importance that such non - human hosts should be promptly identified in order to prevent further evasion into human and other animal populations . the discovery of the gd - ins29 genotype is very useful in differentiating the origin of sars - cov . the fact that we have only identified a couple of isolates in guangdong province supports the scenario that gd - ins29 is the original form transmitted from non - human host to human ones and later turned into a new genotype after deletion of a dna segment that may hinders its efficiency of propagation . first , the non - human reservoir of sars - cov may harbor genome sequences close to gd - ins29 , not the deleted form or the major genotype found most widespread among its human hosts . second , both variants of the major and the minor genotypes could be found in the same animal reservoir owing to multiple transmissions of the virus back and forth between its human and non - human hosts . alternatively , there might have been at least two genotypes of sars - cov spreading in non - human host populations and they have infected human hosts as separate events . it is unlikely but possible that the virus have infected humans multiple times in a not - distant past and the two genotypes co - exist in the non - human hosts but not traceable in current human populations . although we can not rule out another possibility that the viruses of the major genotype had acquired a 29-nt sequence and become gd - ins29 genotype during its propagation in human hosts , such event is deemed extremely rare since insertion or deletion is often lethal to the virus that have a rather compact genome . genomic and genetic knowledge can provide an extraordinary trove of information about the pathogenesis and virulence of sars - cov . more sequences data would significantly expand our knowledge on the etiology and evolution of the virus and are essential for seeking new approaches for diagnostics , vaccine development , and effective therapy of sars . high quality sequence data and comprehensive analysis , as well as more experimental evidences , are still of urgent need to understand such a potent infectious agent . she was suspected being infected during her hospitalization by indirect contact with one of the superspreaders , who stayed in the same hospital as she did . she was one of the sars cases with known transmission path connecting to the index cases identified in guangdong province . the viral isolate was from the autopsied lung tissue of the patient and propagated in vero - e6 cell culture . the isolates of the same genotype were also discovered from sars patients in guangdong province . aliquots of the rt - pcr products from the viral rna were sequenced directly and the remaining was cloned into a plasmid vector ( amplicon - library ) . multiple clones from each amplicon - library were subsequently sequenced from both directions to confirm the results from those directly acquired from pcr products . high - quality sequences were assembled by using a sequence assembly package , phred - phrap - consed ( 10 ) . the consensus sequences from different amplicon - derived clones are accounted and minor variations among different clones are ignored for the consensus assembly . a total number of 75 amplicons and 2,718 sequencing reads were generated ( 1,538,993 bp of raw data ) , equivalent to approximately 52-fold coverage of the viral genome . a complete sequence of 29.757 kb with an overall error rate of 0.0016% was obtained .

we report a complete genomic sequence of rare isolates ( minor genotype ) of the sars - cov from sars patients in guangdong , china , where the first few cases emerged . the most striking discovery from the isolate is an extra 29-nucleotide sequence located at the nucleotide positions between 27,863 and 27,864 ( referred to the complete sequence of bj01 ) within an overlapped region composed of bgi - pup5 ( bgi - postulated uncharacterized protein 5 ) and bgi - pup6 upstream of the n ( nucleocapsid ) protein . the discovery of this minor genotype , gd - ins29 , suggests a significant genetic event and differentiates it from the previously reported genotype , the dominant form among all sequenced sars - cov isolates . a 17-nt segment of this extra sequence is identical to a segment of the same size in two human mrna sequences that may interfere with viral genome replication and transcription in the cytosol of the infected cells . it provides a new avenue for the exploration of the virus - host interaction in viral evolution , host pathogenesis , and vaccine development .

iga nephropathy ( igan ) is one of the most frequent forms of glomerulonephritis ( gn ) . polycythemia can be associated with igan , focal segmental glomerulosclerosis and membranoproliferative gn . here , we report a case of idiopathic erythrocytosis in a 31-year - old male who was incidentally detected to have hypertension during his pre - employment checkup . biochemical parameters revealed raised serum creatinine , and histological findings of the renal biopsy showed igan . a 31-year - old male was referred to nephrology in view of hypertension , renal dysfunction , proteinuria , and active urinary sediments . his clinical examination revealed evidence of bilateral pitting pedal edema and blood pressure ( bp ) of 160/110 mmhg . there was no history suggestive of polyarthralgia , rash , skin ulcers chronic cough and drug intake . complete blood count revealed hemoglobin of 19.2 g / dl , normal total count , and platelet count . serum creatinine was 2.2 mg / dl and uric acid 10.6 mg / dl . the liver function , uric acid , electrolytes , glucose , complement 3 ( c3 ) , and complement 4 ( c4 ) serum levels were normal . coagulation and the levels of antistreptolysin o and high - sensitivity c - reactive protein were also normal . antinuclear antibody profile was done , and antibodies to anti - myeloperoxidase , proteinase 3 , double - stranded dna , nucleosome and sjgren 's syndrome type a ( ss - a ) and ss - b antibodies tested negative . ultrasound revealed normal - sized kidneys with right kidney of 9.6 cm and left kidney measured 9.4 cm . radioisotope scan revealed a glomerular filtration rate ( gfr ) of 78 ml / min with a right and left differential of 33 ml / min and 35 ml / min , respectively . renal biopsy showed chronic iga nephropathy ( igan ) , with m , mesangial hypercellularity ; e , endocapillary proliferation ; s , segmental glomerulosclerosis / adhesion ; t , tubular atrophy / interstitial fibrosis ( mest ) scoring of m1 , e0 , s1 , t1 . the patient was treated with two sittings of phlebotomy , started on amlodipine 5 mg and ramipril 2.5 mg for control of hypertension along with allopurinol 100 mg and aspirin 150 mg for hyperuricemia and polycythemia , respectively . he was followed up every 2 weeks on outpatient basis . at the end of 8 weeks to the best of our knowledge , there have been 24 cases of polycythemia associated with renal disease described so far and out of them only ten have been associated with igan . the other renal lesions associated with polycythemia were focal segmental glomerulosclerosis , membranoproliferative glomerulonephritis , and rapidly progressive glomerulonephritis . furthermore , of the ten cases of polycythemia associated with igan described so far , all happen to be males . only three cases reported to have normal renal function whereas others had either moderate to severe renal dysfunction . several cytokines and growth factors play a role in the progression of renal disease in polycythemia ; one such report demonstrated abnormally upregulated mrna expression of platelet - derived growth factor and insulin - like growth factor . furthermore , the increased blood viscosity and blood volume leads to vascular microthrombi and glomerular capillary occlusion thus reducing the gfr . capillary occlusion leads to ischemia which eventually leads to chronic renal damage if not reversed on time . this is the 11 case of polycythemia associated with igan being reported and this is the first case of idiopathic erythrocytosis being associated with igan . striking similarities with other similar case reports are the preponderance in males ( all ten case reports being males ) and the presence of hyperuricemia , hypertension , and renal failure . there has been an increased presence of igan worldwide and it has become one of the leading causes of renal failure in young adults . hypertension and active urinary sediment is a common finding in most of these individuals , what makes this case report rare is the presence of idiopathic polycythemia as an additional cofactor . as only nine such reports have been made so far , it is essential to dig further into the pathogenesis of polycythemia in these individuals to know more about this causal relationship

we report a case of idiopathic erythrocytosis in a 31-year - old male who was incidentally detected to have hypertension during his preemployment checkup . urine routine showed proteinuria and hematuria . biochemical parameters revealed raised serum creatinine , and histological findings of the renal biopsy showed igan .

a decrease in the physical activity level has been associated with various chronic diseases ( 1 ) . adolescents and youth are not active on a regular basis and are reported to have low physical activity ( 2 , 3 ) . studies reporting the pattern of pa and inactivity among indian adults and adolescents are limited ( 4 , 5 , and 6 ) and there exists differences in the pa levels among various population ( 7 ) . therefore this study was undertaken to describe the prevalence and patterns of physical activity among young adults studying medical sciences . this study was conducted in a sample of 264 medical students in the age group of 1822 years studying in i mbbs . age ( years ) , height ( meter ) , weight ( kg ) , bp ( mmhg ) were recorded using standardized methods . all participants had given their written consent for participating in the study before they were interviewed . for estimating the level of physical activity , the official long form english version of ipaq was used ( 8) . the questionnaire consists of 27 questions that cover 4 domains of physical activity ( work , transport , domestic and garden , and leisure - time ) and time spent sitting . the items in ipaq are structured to provide separate domain - specific scores for walking , moderate - intensity , and vigorous - intensity activity . the results were presented as the estimation of energy expenditure in metabolic equivalent - minutes per week ( met hours / week ) . according to ipaq scoring protocol total physical activity score was calculated , as well as separate scores for each of the 4 physical activity domains . based on standard scoring criteria the study sample was categorized into three levels of physical activity : high moderate and low [ physically inactive ] , levels of physical activity . the standard scoring criteria is as follows : 1 ) high : meets either of two criteria : ( a ) vigorous - intensity activity on > 3 days / week and accumulating at least 1500 met - minutes / week ; or ( b ) > 5 days of any combination of walking , moderate - intensity , or vigorous - intensity activities achieving at least 3000 met - minutes / week . 2 ) moderate : meets any of the following three criteria : ( a ) 3 days of vigorous activity of at least 20 minutes / day ; ( b ) 5 days of moderate - intensity activity or walking of > 30 minutes / day for > 10 minutes at a time ; or ( c ) 5 days of any combination of walking , moderate - intensity or vigorous - intensity activities achieving at least 600 met - minutes / weeks . 3 ) low : meets neither moderate nor high criteria according to data cleaning rules 8 five participants were excluded from analyses , so the final sample included 259 participants . the prevalence of physical activity was calculated as the percentage of students involved in the activity . median was calculated for each domain of physical activity separately as well as for total physical activity . the analyses were performed on the total sample of participants and separately for men and women . gender differences in physical activity were tested using mann - whitney test . the level of significance for all analyses was set at p < 0.05 . according to categorical scoring 41.3 % ( n=107 ) showed a high levels achieving a minimum of at least 3000 met - min /week.43.2% of students showed moderate level of physical activity achieving a minimum of at least 600met - min /week . 15.4% ( n=40 ) did not meet the above two criteria and considered to have low activity . gender specific prevalence of ipaq categories and for total sample is given in table 1 . it was noted that 84.6% ( n=219 ) were engaged in work related activity , 91.1% of them showed transport related activity . the prevalence of students engaged in vigorous activity was 60.6% ( n=157 ) , in moderate intensity activity and walking was found to be 83.4% ( n=261 ) and 91.9 % ( n=238 ) respectively . table 2 provides data about percentage of students with different level of pa and with no physical activity . about one third of the students ( 32.8% ) were not engaged in leisure time activity and 8.1% did not do walking . based on domain specific continuous scores , the median of the total physical activity for the whole sample was 39.13 met / hour / week . median physical activity for whole sample in met - hour / week was 18.10 for work , 4.40 for transportation , 2.60 for domestic and gardening and 4 for leisure - time activity . the major contribution for total physical activity was from work related activity and the lowest was found in the domestic and gardening activities . table 3 shows the total and gender specific physical activity level in met /hr / week in different domains . men had higher physical activity at work than women [ 25.64 met - hour / week vs12 met - hour / week ( p=0.002 ) ] . in transport men had high activity but with no statistical significance . [ 4.95 met - hour / week for men vs. 3.85 met - hour / week for women ] . in leisure - time domain , men were more active than women [ 6.6 met - hour / week vs. 2.83 met - hour / week ( p= 0.002 ) ] . on the other hand , women were more active than men in the domestic and garden domain [ 3.33 met - hour / week vs. 1.50 met - hour / week , ( p=0.040 ) ] . the time spent in sitting ranged from 00 to 17.71 hrs with average time of 7.06 hrs / day . the average time spent in sitting per day was 7.50 hrs 3.38 in men and 6.52 3.26 in women with a p value of 0.026 . the present study reports the prevalence and patterns of physical activity among young students aged 1822 years . in total 84.5% of young adults were involved in high to moderate levels of pa meeting the ipaq high active and moderate active category . a significant proportion of men showed high pa where as women had moderate levels of activity ( table 1 ) . the prevalence of activity level was higher than the levels reported for indian adults in the ips study and for asian adolescents ( 4 , 7 ) . more than a half ( 61% ) of american medical students reported to have similar activity level ( 9 ) . the median pa score for the total sample was 39.13 met / hour / week . the average score was different from activity scores in adult population of various age groups reported in studies from other european countries ( 10 ) . the main source of physical activity energy expenditure in the present study was from work related activity and least was from domestic and gardening . in other studies the leisure time activity was the major component of total pa ( 1113 ) where as in our study the pa done during domestic and leisure time was low indicating that students did not have enough time for these activities . this difference could be due to less pa programs , recreational facilities and busy curriculum . asian indian guadeloupean adolescents ( 7 ) and adults of taiwan , china ( 14 ) reported lower weekly leisure time activity . in most of the previous studies women found to have lower rates of participation in physical activity than men ( 15 ) . a similar gender difference observed in our study with men spending more time in leisure time activity and women in domestic and gardening . it has been confirmed in the study by dong and colleagues that women are engaged in higher house hold activity than for men ( 16 ) . studies conducted in the united states have consistently shown walking to be the most common leisure - time physical activity among adults of all ages ( 17 ) . in our study walking was the most frequent type of pa . the prevalence of physical inactivity reported was higher ( 15.4% ) compared to indian adults ( 9.4% ) ( 18 ) . but almost similar to inactivity reported in urban indians ( 14% ) ( 6 ) . many studies report in general , physical inactivity highest among youngest age group ( 16 ) . in the hdss survey the gender specific level of physical inactivity in different domains of activities were very high for indian adults compared to young adults in this study because of inclusion of wide age group ( 1564 years ) . in earlier studies total score physical activity and the leisure time activity have shown the different associations with educational level ( 10 , 19 ) . but in the present study among students studying mbbs the leisure time activity is very less in spite of them having high total pa . it is important to note that the above studies relate pa cross sectional with education level but not during the study course . though ipaq collects information on total sitting time , the data and observations are not discussed in other studies . a direct comparison of results could not be made with other indian studies ( 4 , 10 ) because of difference in the methodology , age group studied and the description of results in different domains and patterns of physical activity . the results of this study can not be generalized as the sample is from the medical students and may not represent young adults in the general population . physical activity has been related to socio demographic and environmental variables such as food consumption patterns , location of residence , monthly household income and number of household appliances which was not examined . physical activity being one of the leading health indicators , in the healthy people 201020 one of the main objectives is to increase the proportion of adults who engage regularly preferably in moderate physical activity for at least 30 minutes / day . in the present study therefore efforts should be taken to emphasize the benefits of physical activity to the students so that their activity is maintained in future . the low leisure time activity observed in the present study may demand recommendation and facilities for provision of physical education classes in the curriculum .

background : physical activity is one of the leading health indicators . the objective was to study the prevalence and patterns of physical activity among young adults.methods:259 medical students ( men : women = 116:143 ) in the age group of 1822 yrs were interviewed using the official english long version of the international physical activity questionnaire ( ipaq ) . the total level of physical activity and activity in each of the 4 life domains work , transport , domestic and gardening and leisure - time were estimated and was expressed as metabolic equivalent - hours per week ( met - hour / week).results:41.3 % showed high levels of physical activity , 43.2% and 15.4 % of students showed moderate level and low level of physical activity respectively . 84.6 % ( n=219 ) were engaged in work related activity and 80.7% ( n= 209 ) showed transport related activity . domestic and gardening physical activity represented 63.7 % ( n=165 ) of individuals total activity and 67.2% of students showed leisure time activity . the average time spent in sitting was 7.06 hrs / day . the median of the total physical activity for the whole sample was 39.13 met / hour / week and 18.10 for work , 4.40 for transportation , 2.60 for domestic and gardening and 4 for leisure - time activity . there was significant gender difference observed with women having low physical activity.conclusion:this study provides baseline information about the physical activity levels and patterns including sitting hours among indian young adults using ipaq that can used for comparison of data across different parts of world .

migraine is a primary headache that is characterized by mostly unilateral pulsating head pain , is aggravated by routine physical activity , and is associated with nausea and/or photophobia and phonophobia ( headache classification committee of the international headache , 2013 ) . migraine may be accompanied by a variety of autonomic , cognitive , and emotional disturbances ( grassini and nordin , 2015 ) . epidemiological studies have documented its high prevalence and high socio - economic and personal impact . migraine has a 1-year prevalence of 14% in the general population ( vos et al . , 2012 ) and 9.3% in china ( yu et al . , 2012 ) . it was ranked as the sixth greatest cause of disability worldwide in the global burden of disease survey 2013 ( 2015 ) . it is generally believed that migraine is abnormal brain function that depends on the activation and sensitization of the trigeminovascular pathway ( borsook and burstein , 2012 , noseda and burstein , 2013 ) . cortical spreading depression ( csd ) is the electrophysiological correlate of migraine aura ( ferrari et al . , 2015 , pietrobon and moskowitz , 2013 ) . questions remain , however , concerning the mechanisms of initiation , continuation , and termination of migraine . neuroimaging has led to advances in the understanding of primary headache mechanisms , and to better identification of the causes of headache , as well as the structures that are responsible for initiation of the pain . co - localization of structural changes ( i.e. , an increase in voxel - based morphometry [ vbm ] ) as well as changes in localized brain activity characterized using positron emission tomography ( pet ) have been found in the same area of the brain ( hypothalamus ) in cluster headache patients ( may et al . , 1999 ) . hypothalamus is undoubtedly crucial for the pathogenesis of the trigeminal autonomic syndromes , so anatomical co - localization of functional and structural changes ( especially increase in gm ) raises the possibility that the observed changes may be causal as opposed to a consequence of pain ( may , 2009 ) . combined analysis of structural and functional changes in migraine may be urgent to elaborate the pathophysiological mechanism . because of the episodic nature and unpleasantness of migraine attacks , imaging during spontaneous migraine has proven difficult . therefore , most magnetic resonance imaging ( mri ) studies of migraine have focused on the migraineurs between attacks , during the so - called interictal phase . in recent years , grey matter ( gm ) morphology based on mr , particularly vbm , has been carried out on migraineurs . vbm involving statistical voxel - wise testing of the local concentration of gm is a whole - brain method for analysis of automatically pre - processed structural high - resolution mri data ( draganski and may , 2008 ) . some researchers have meta - analyzed vbm studies in migraine , but they did not combine them with functional changes and had not taken frequency of migraine attacks into account ( dai et al . , 2015 , wenting hu et al . , 2015 ) . variations like nociceptive , olfactory and visual stimuli in the methods used complicate a meta - analysis of all studies showing brain regions with atypical activation in migraine sufferers ( chen et al . , 2015 , schwedt et al . , 2015 ) interictal cutaneous pain thresholds are lower in migraine patients compared to controls , so we chose articles used nociceptive stimuli for a meta - analysis ( schwedt et al . , 2011 , weissman - fogel et al . , 2003 ) . here , we systematically summarize and meta - analyze the voxel - wise changes and functional changes in gm in patients with migraine . combined with previously published studies on pain disorders i ) are there concordant structural changes in the foci ( increase or decrease ) in migraine ? iii ) what can be deduced from gm changes in migraine ? we describe how these studies have helped our understanding of the anatomy and biology of migraine , discuss their limitations , and propose avenues for future research using mri to study migraine . we searched pubmed and embase for manuscripts published between january 1985 and november 2015 using the following search terms : ( mri or magnetic resonance imaging ) and migraine and structur * for vbm analysis . search words : ( fmri or functional magnetic resonance imaging ) and ( migraine disorders or ( migraine and disorders ) or migraine ) were used for functional studies . literatures were screened from the title , abstract , intensive reading full - text . we limited the search to studies published in the english language and where the subjects were human . we also examined the references of relevant primary articles and review articles to identify studies that may have been missed in the search . the meta - analysis included only articles ( i ) that evaluated the association of grey matter changes and migraine based on a case - control or cohort design ; ( ii ) that contained information on the sample sizes , disease conditions ; ( iii ) patients with migraine were diagnosed according to the international classification of headache disorders ( ichd ) ; ( iv ) reported whole - brain results of changes in stereotactic coordinates ; v ) nociceptive stimuli ( either heat or ammonia ) were used for functional studies and ( vi ) used thresholds for significance corrected for multiple comparisons or uncorrected with special extent thresholds . we excluded the following : a ) non - original studies ; b ) studies in which the field of view was confined to a restricted region of the cortex ; c ) studies in which the comparisons between patients with migraine and healthy subjects did not include changes in gm ; d ) comorbidity with other diseases ; e ) migraine - like syndromes that were secondary to other diseases ; f ) articles that concerned other types of headache or a special subtype of migraine ( like vestibular migraine ) ; g ) articles that reported no significant results ; h ) articles in which there was no healthy control ( hc ) group or no comparison with hc ; i ) studies with unspecified vbm analyses ; j ) grey matter functional changes due to other stimuli ( like visual or olfactory ) or combined with other tasks or intervention ; k ) only functional connectivity was conducted ; l ) resting - state functional studies in migraineurs ; m ) duplicate articles ; and n ) case reports . the two authors ( zj and sy ) independently extracted data from each study using a predefined data extraction form , any lack of clarity or disagreement was resolved through discussion . the investigators abstracted data from each study to obtain information on author , publication year , sample size , characteristics of the study population ( age , gender ) , types of migraine , disease information and technical details ( mri scanner , region studied , timing , methods and main findings ) . we used ginger ale 2.3.5 ( http://www.brainmap.org/ ) to evaluate the presence of common patterns of gm alterations . ginger ale is a brainmap application that can be used to perform an ale meta - analysis on coordinates in a talairach or mni space . ale is probably the most widely used algorithm for coordinate - based meta - analyses . the approach treats activation foci reported in neuroimaging studies as spatial probability distributions centered at given coordinates rather than as single points . for each voxel , ginger ale estimates the cumulative probabilities that at least one study reports activation for that locus . ale maps are then obtained by computing the union of activation probabilities for each voxel . differentiation between true convergence of foci and random clustering is tested using a permutation procedure ( eickhoff et al . , 2009 , laird et al . , 2005 , turkeltaub et al . , 2012 ) . with version 2.0 , ginger ale switched the ale methods from fixed effects to random effects , and incorporated variable uncertainty based on the sample size ( eickhoff et al . , 2012 ) . a random effects model assumes a higher than chance likelihood of consensus between different experiments , but not in relation to activation variance within each study . during an ale analysis , each activation focus is modeled as the center of a gaussian probability distribution , and is used to generate a modeled activation ( ma ) map for each study . where we used data that were not expressed in talairach coordinates , these were transformed into talairach space using icbm2tal , as implemented in ginger ale 2.3.5 ( lancaster et al . , 2007 a very conservative threshold of p < 0.001 was chosen , and the minimum cluster size was 100 mm . all of these steps combined help to control for publication bias with regard to reported foci . we searched pubmed and embase for manuscripts published between january 1985 and november 2015 using the following search terms : ( mri or magnetic resonance imaging ) and migraine and structur * for vbm analysis . search words : ( fmri or functional magnetic resonance imaging ) and ( migraine disorders or ( migraine and disorders ) or migraine ) were used for functional studies . literatures were screened from the title , abstract , intensive reading full - text . we limited the search to studies published in the english language and where the subjects were human . we also examined the references of relevant primary articles and review articles to identify studies that may have been missed in the search . the meta - analysis included only articles ( i ) that evaluated the association of grey matter changes and migraine based on a case - control or cohort design ; ( ii ) that contained information on the sample sizes , disease conditions ; ( iii ) patients with migraine were diagnosed according to the international classification of headache disorders ( ichd ) ; ( iv ) reported whole - brain results of changes in stereotactic coordinates ; v ) nociceptive stimuli ( either heat or ammonia ) were used for functional studies and ( vi ) used thresholds for significance corrected for multiple comparisons or uncorrected with special extent thresholds . we excluded the following : a ) non - original studies ; b ) studies in which the field of view was confined to a restricted region of the cortex ; c ) studies in which the comparisons between patients with migraine and healthy subjects did not include changes in gm ; d ) comorbidity with other diseases ; e ) migraine - like syndromes that were secondary to other diseases ; f ) articles that concerned other types of headache or a special subtype of migraine ( like vestibular migraine ) ; g ) articles that reported no significant results ; h ) articles in which there was no healthy control ( hc ) group or no comparison with hc ; i ) studies with unspecified vbm analyses ; j ) grey matter functional changes due to other stimuli ( like visual or olfactory ) or combined with other tasks or intervention ; k ) only functional connectivity was conducted ; l ) resting - state functional studies in migraineurs ; m ) duplicate articles ; and n ) case reports . the two authors ( zj and sy ) independently extracted data from each study using a predefined data extraction form , any lack of clarity or disagreement was resolved through discussion . the investigators abstracted data from each study to obtain information on author , publication year , sample size , characteristics of the study population ( age , gender ) , types of migraine , disease information and technical details ( mri scanner , region studied , timing , methods and main findings ) . we used ginger ale 2.3.5 ( http://www.brainmap.org/ ) to evaluate the presence of common patterns of gm alterations . ginger ale is a brainmap application that can be used to perform an ale meta - analysis on coordinates in a talairach or mni space . ale is probably the most widely used algorithm for coordinate - based meta - analyses . the approach treats activation foci reported in neuroimaging studies as spatial probability distributions centered at given coordinates rather than as single points . for each voxel , ginger ale estimates the cumulative probabilities that at least one study reports activation for that locus . ale maps are then obtained by computing the union of activation probabilities for each voxel . differentiation between true convergence of foci and random clustering is tested using a permutation procedure ( eickhoff et al . , 2009 , laird et al . , 2005 , turkeltaub et al . , 2012 ) . with version 2.0 , ginger ale switched the ale methods from fixed effects to random effects , and incorporated variable uncertainty based on the sample size ( eickhoff et al . , 2012 ) . a random effects model assumes a higher than chance likelihood of consensus between different experiments , but not in relation to activation variance within each study . during an ale analysis , each activation focus is modeled as the center of a gaussian probability distribution , and is used to generate a modeled activation ( ma ) map for each study . where we used data that were not expressed in talairach coordinates , these were transformed into talairach space using icbm2tal , as implemented in ginger ale 2.3.5 ( lancaster et al . , 2007 ) . a very conservative threshold of p < 0.001 was chosen , and the minimum cluster size was 100 mm . all of these steps combined help to control for publication bias with regard to reported foci . lots of studies have been performed on grey matter ( structural or functional ) changes in migraine patients , but the results were not consistent and different areas were found in them . after a careful screen , we identified 8 clinical studies that used vbm to assess changes in gm regions in migraineurs and controls , containing a total of 390 subjects ( 191 patients and 199 controls ) . the patient group was comprised of 24 men and 167 women , where 155 cases of migraine were without aura and 36 were with aura . the clinical manifestations and technical details of the structural changes on migraine are listed in table 1 , table 2 . the studies we analyzed compared the whole brain grey matter volume ( gmv ) of 191 migraine patients ( mean age : 37.94 years ) , and 199 healthy controls ( mean age : 36.71 years ) . there were no significant age or gender differences between the patients with migraine and the hc subjects ( t - test ; p < 0.05 ) . the relationship between changes in gmv and frequency of attack and disease duration were also performed in five out of the eight studies ( kim et al . , 2008 , four of them assessing the correlation of gm changes with clinical data through multiple comparison or pearson correlation test , we chose them for subtraction meta - analysis to explore the correlation of gmv changes with frequency of attack and disease duration ( kim et al . , 2008 , rocca et al . , 2006 , schmitz et al . , 2008a , valfre et al . , 2008 ) . as they are correlation analysis , the concept estimated frequency of migraine attacks ( frequency of migraine attacks or duration of the disease ) was used and a dichotomous grouping based on disease duration -related gmv changes may present the complete picture of development in migraine ( deramus and kana , 2015 , kim et al . , 2008 , nellessen et al . , 2015 ) . using these data , group analyses were performed between patients with migraine and hc subjects , lower estimated frequency of migraine attacks and higher estimated frequency of migraine attacks patients , respectively . only one study included chronic migraine , and the number of migraines with aura was small ; hence , a group analysis among migraine subtypes was not possible . five functional mri studies used nociceptive stimuli were included to assess functional changes in the whole brain gm regions in migraine patients containing a total of 189 subjects ( 93 patients and 96 controls ) . 32 men and 61 women were contained in patient group . the clinical manifestations and technical details of the functional changes on migraine can be found in table 3 , table 4 . there was no significant gender difference between the migraineurs and the hc subjects ( t - test ; p < 0.05 ) . age information are not available in some researches , however , gender and age were exactly matched in each study . although pag increase was found in one study ( rocca et al . , 2006 ) the ale results showed that , compared with hc , migraine was associated with a common core set of decreases in gm volume in the bilateral inferior frontal gyri , the right precentral gyrus , the right cerebellar culmen , the left middle frontal gyrus and the left cingulate gyrus ( see fig . 2 and table 5 ) . changes in the volume of gm in the right claustrum , left cingulated gyrus , right anterior cingulate , amygdala , and left parahippocampal gyrus were negatively related to the estimated frequency of attack ( see fig . 3 and table 6 ) . in individual studies , both activated and deactivated areas were found in migraineurs . several areas containing the bilateral lentiform nucleus , putamen and claustrum , the right precentral gyrus , the right midbrain , the right postcentral gyrus , the left limbic lobe and the left cingulate gyrus were activated in the migraine patients after ale analysis . there were also area deactivations in the left precentral gyrus and the right superior temporal gyrus in the migraineurs ( shown in fig . 4 and table 7 ) . genome - wide association studies of migraine have revealed that genetics are involved in migraine ( anttila et al . , 2010 , chasman et al . , 2011 , freilinger et al . , 2012 ) ; however , only limited variance can be explained by genetics . environmental factors , such as stress , weather , hormonal fluctuations , sleep disturbances , meal skipping , and sensory overload , are clearly involved in migraine ( kelman , 2007 , levy et al . , 2009 ) . whatever the cause , imaging during migraine pain has led to a shift from vascular hypotheses of migraine pathophysiology to neurovascular or central nervous system ( cns ) theories . mri is a non - invasive procedure with the potential to determine a morphological basis of neurological diseases , as well as to investigate functional structural relationships . in idiopathic or primary headaches , including migraine , tension - type headaches , and cluster headaches , the accepted view is that these conditions are due to abnormal brain function that occurs with normal brain structure . patients with chronic tension - type headaches exhibit a decrease in gm volume in the structures that are responsible for transmitting pain ( schmidt - wilcke et al . , 2005 ) . data on cluster headaches describe an increase in the volume of gm in the hypothalamus which is associated with functional areas involved during acute headache attacks ( may et al . , 1999 ) . in recent years , there have been a number of studies on gm morphology using mr ( especially vbm ) to investigate the mechanisms of initiation , continuation and termination of migraine ( hougaard et al . , lots of researches also be conducted on gm function in migraine patients used various methods . nevertheless , single imaging study carries several limitations , including small sample sizes , low reliability and its inherent subtraction logic which is only sensitive to differences between conditions ( feredoes and postle , 2007 , price et al . , 2005 ) ale treats activation foci , assesses the overlap between foci based on modelling them as probability distributions centered at the respective coordinates and finds agreement across subject groups . either way , single study results are driven by dataset 's foci as well as how they are grouped . ale was widely applied to meta - analyze the structural and functional brain changes of various neuropsychiatric disorders , such as autism spectrum disorders , alzheimer 's disease , anhedonia , idiopathic dystonia and physiological conditions , such as aging and touch ( chapleau et al . , 2016 , deramus and kana , 2015 , di et al . , 2014 , lokkegaard et al . , 2016 , morrison , 2016 , nickl - jockschat et al . , after ale meta - analysis of structural and functional changes in the gm in migraine patients , combined with previous studies in pain disorder , our study demonstrates : i ) migraine was associated with a common core set of decreases in the volume of gm in the bilateral inferior frontal gyri , the right precentral gyrus , the left middle frontal gyrus and the left anterior cingulate gyrus ; changes in the volume of gm in the cingulated gyrus were negatively related to the estimated frequency of headache attack ; ii ) the precentral gyrus and the cingulate gyrus were also over - activated in interictal migraineurs during nociceptive stimuli ; and iii)structural changes in the frontal gyrus may predispose a person to pain , and the cingulate gyrus may accumulate damage due to the repeated occurrence of migraine ; functional activation in the precentral and postcentral gyrus , the limbic region , the basal ganglia and the right midbrain may be associated with pain processing and associated symptoms in migraine patients . brain function activation in primary headache syndromes contains two groups : areas generally involved in pain processing , such as the somatosensory , cingulate , thalamus et al . ; and areas specifically activated in primary headaches , such as the hypothalamus , which is activated in cluster headache attacks ( may et al . , 1999 ) . one review of stimulus - induced brain activation in migraine found atypical brain responses to sensory stimuli , an absence of the normal habituating response between attacks , and atypical functional connectivity of sensory processing regions ( schwedt et al . , 2015 ) . migraine sensory hypersensitivities may be induced by a combination of enhanced sensory facilitation and reduced inhibition in response to sensory stimuli ( russo et al . , 2012 , schwedt et al . , 2014 ) , as well as reduced or absent habituation to stimuli interictally ( burstein et al . , 2010 ) . they may also contribute to a transformation from episodic to chronic migraine ( moulton et al . , 2008 ) . a concordant activation was found in the somatosensory , cingulate , limbic lobe , basal ganglia and midbrain in migraine patients in our study . all these areas are related to pain processing , modulation and associated symptoms ( like emotional disturbances ) in migraineurs . the activation areas of somatosensory , basal ganglia and midbrain coincided with previous studies and further confirms that activation and sensitization of the trigeminovascular pathway are involved in migraine pathophysiology ( dasilva et al . , 2007 , noseda and burstein , 2013 , schulte and may , 2016 , stankewitz et al . , 2011 ) . studies of changes in brain function during the ictal phase may improve our understanding of migraine mechanisms . , 2001 , welch et al . , 1998 ) , and others exposed migraine subjects to attack triggers in order to initiate migraine ( afridi et al . , 2005b , cao et al . , 2002 , maniyar et al . , 2014 ) . during the premonitory phase of nitroglycerin - triggered migraine attacks , the following areas of the brain were found to be activated : the posterolateral hypothalamus , the brainstem , and various cortical areas , including the occipital , temporal , and prefrontal cortices ( maniyar et al . , 2014 ) . studies that captured the onset of migraine found an increase in the signal from the brainstem ( bahra et al . , 2001 ) . comparisons of ictal images with interictal scans revealed significant activation in the anterior and posterior cingulate , as well as the cerebellum , thalamus , insula , prefrontal cortex , temporal lobes , and dorsal pons during migraine ( afridi et al . , 2005a ) . several studies found that , following treatment of the migraine with sumatriptan and suboccipital stimulator therapy , the dorsal pons remained active ( bahra et al . , 2001 , matharu et al . , 2004 ) . taken together , these studies show that the brainstem is continuously activated during a migraine , and that this may be specific to migraine and it may act as a generator of migraine . a longitudinal fmri study on a migraine patient found that hypothalamic activity increases towards the next migraine attack and shows altered functional coupling with the spinal trigeminal nuclei and the dorsal rostral pons during the preictal day and the pain phase ( schulte and may , 2016 ) . combined with previous study , this may indicate that the real driver of attacks might be the functional changes in hypothalamo brainstem connectivity ( maniyar et al . , 2014 ) . if there are concordant structural changes , especially increases in the gmv , these areas may be the cause of migraine . mri studies of patients with migraine have consistently shown a distributed pattern of morphological brain abnormalities characterized by regions of decreased and increased gmv ( jin et al . , 2013 , kim et al . , 2008 , , 2012a , rocca et al . , 2006 , schmidt - wilcke et al . , 2008a , schmitz et al . , 2008b , valfre et al . , 2008 ) . the role of such abnormalities in the pathogenesis of this condition remains a matter of debate ; histological measures , such as neuronal density , do not correlate with vbm gm probability maps ( eriksson et al . , 2009 ) , and changes in cerebral blood flow produce this may reflect alterations in the dendritic complexity or changes in the number of synapses , or simply changes in water content . an increase in gmv may reflect structural brain plasticity as a result of exercise and learning ( may , 2009 ) . a decrease in gmv suggest that the central reorganization processes in chronic pain syndromes may involve degeneration of anti - nociceptive brain areas . indeed , relationships between decreases in gmv and disease duration or attack frequency have been reported ( kim et al . , 2008 , liu et al . , 2013 , , 2012a , rocca et al . , 2006 , schmidt - wilcke et al . however , some changes in gmv are not correlated with clinical manifestation , suggesting that they may predispose a person to migraine ( liu et al . , 2013 ) . in our study , one study without significant result was excluded , it was conducted in 2003 and the possibility may be the differences existed in migraine patients included and image pre - processing procedures ( matharu et al . , 2003 ) . we found a set of decreases in the gmv in the bilateral inferior frontal gyri , the right precentral gyrus , the left middle frontal gyrus and the left anterior cingulate gyrus in migraine . our finding of reliable structural alterations in homologous regions associated with pain processing highlights the importance of these areas in the pathogenesis of migraine . we also found changes in gmv in the right claustrum , the left cingulate gyrus , the right anterior cingulate , the amygdala , and the left parahippocampal gyrus , which were negatively related to the frequency of headache attacks . our result further indicate that the gmv changes related to the frequency of headache attacks may occur as a consequence of repeated brain insults during migraine attacks . decreases in gmv in the frontal gyri were not related to the frequency of headache attacks . the frontal cortex is one of the most important areas associated with brain abnormalities in migraine patients . the role of the frontal lobe in pain processing has been established in several recent morphological studies , including subjects with chronic back pain ( apkarian et al . , 2004 ) , fibromyalgia , irritable bowel syndrome , and phantom pain atrophy in the prefrontal cortex ( may , 2008 ) . it has recently been reported that significant gm atrophy of the frontal lobe is associated with migraine in pediatric patients , but is not correlated with the disease duration or frequency of attacks ( rocca et al . , 2014 ) . a recent longitudinal mri study using vbm investigated changes in gm related to medication withdrawal in a group of patients with medication overuse headache ( riederer et al . , 2013 ) . patients with clinical improvement showed a significant reversal of the changes in gm in some regions , and patients without treatment response had less gm in the orbitofrontal cortex . it appears that a decrease in gmv in the frontal gyrus may predispose a person to pain conditions , and may be predictive of poor response to treatment . pet studies have shown that the limbic regions ( the cingulate cortex , amygdala , and parahippocampal gyrus ) are activated during migraine attacks ( afridi et al . , 2005b , matharu et al . , 2004)and cluster headache attacks ( may et al . , 1999 , may et al . these areas of the brain are also believed to be involved in emotional , cognitive , and autonomic functions ( may , 2006 , morgane et al . , 2005 , alterations in cerebral structures that modulate the affective components of pain that are found in migraine suggest a possible neurobiological mechanism , which may explain the link between chronic migraine and psychiatric disturbances ( buse et al . , 2013 , minen et al . , 2016 ) . decreases in gmv in the cingulate cortex were also found in patients with chronic back pain ( schmidt - wilcke et al . , 2006 ) , chronic phantom pain ( henry et al . , 2011 ) , and chronic tension - type headaches ( schmidt - wilcke et al . , 2005 ) . these changes are correlated with disease duration and attack frequency , and may result in brain damage accumulated due to repeated occurrences of pain - related processes , and are not specific to migraine . for better understanding the neuroanatomical and functional brain changes in migraine patients , there are several studies performed on the concurrent functional and structural cortical alterations in migraine ( hubbard et al . higher thickness in the post - central gyrus in the higher frequency of attack ( hf ) correlated with the observed stronger functional activation , suggested adaptation to repeated sensory drive ( maleki et al . , 2012a ) . decreased dorsal anterior cingulate cortex was correlated with increased functional connectivity between several regions indicate that frequent nociceptive input has modified the structural and functional patterns of the frontal cortex in migraine ( jin et al . , 2013 ) . concordant structural and functional changes in hippocampal in lower frequency of attack ( lf ) group relative to the hf and hc groups suggestive of an initial adaptive plasticity that may then become dysfunctional with increased frequency ( maleki et al . , 2013 ) . in our study , no gmv increase was found , so the generator of migraine can not been deduced . increased activation in precentral gyrus and cingulate during nociceptive stimuli opposed to gmv decrease might represent increased effort , possibly due to disorganization of these areas and/or the use of compensatory strategies involving pain processing in migraine . we did not find structural changes in gm in areas that were specifically activated in migraine ( i.e. , the brainstem , hypothalamus ) . however , this negative finding does not imply that the brainstem and hypothalamus are not involved in the pathophysiology of migraine . because most of the studies included in our meta - analysis used whole - brain detection ( only in one study was the brainstem subjected to a small - volume correction , but this employed a 1.5-t mri scanner ) , and the gm volume of the brainstem is relatively small further studies are therefore warranted that focus on the brainstem volume using 3.0-t or 7.0-t mri scanners in order to determine whether there are any significant changes in the volume of the brainstem . at the same time , ale provides a way of applying statistical thresholds to large sets of coordinate data , in order to identify the most consistently - existed and reproducible loci across many studies , the result can provide a priori hypotheses for further experimental testing . however , it filters out less robust or infrequently - reported foci that may have a greater dependence on the details of individual studies . longitudinal and combination of structural and functional imaging studies may be needed to better understand the anatomy of migraine . as animal models are available in migraine and their life spans are short , a longitudinal imaging study can also be completed in rodents and brains can be collected ex - vivo for further analysis . there are no established protocols for imaging studies , and image pre - processing procedures , such as smoothing , registration , the choice of small volume corrections , imaging modalities , and data analysis , differed among studies . the headache characteristics ( i.e. , attack frequency and disease duration ) as well as the hc choices were also inconsistent . our meta - analysis was subject to publication bias because unpublished studies and studies without any significant clusters reported were not included , the studies were limited to those published in english and where the subjects were human . the ale meta - analysis was based on pooling of stereotactic coordinates with significant differences rather than on the raw data from the included studies ; this may have limited the accuracy of the results . nevertheless , previous studies suggested that no individual study could significantly bias the results of ale meta - analyses after including the sample size and number of reported foci into ale algorithm , changing from fixed - effects to random - effects inference , and revising for multiple comparison correction ( eickhoff et al . , 2012 , eickhoff et al . , 2009 , turkeltaub et al . , 2012 ) . the inclusion criteria for fmri were nociceptive stimulation in order to allow for analysis , regardless of stimulation site or stimulus type ( e.g. , hand , forehead , heat , and ammonia ) . the result makes clear that the simultaneous activation and deactivation of the relevant regions are implicated in responses to a range of nociceptive stimuli . the sex ratio was not concordant with the prevalence of migraine , and sex differences in brain structure may be present ( maleki et al . , 2012b ) . there were few chronic migraineurs , and the sample may not be broadly representative of migraineurs overall . migraineurs frequently have several comorbid conditions ( e.g. , anxiety and mood disorders , altered lifestyles , and drug use that might directly contribute to morphological changes ) ; this was not considered in our study . brain function activation in primary headache syndromes contains two groups : areas generally involved in pain processing , such as the somatosensory , cingulate , thalamus et al . ; and areas specifically activated in primary headaches , such as the hypothalamus , which is activated in cluster headache attacks ( may et al . , 1999 ) . one review of stimulus - induced brain activation in migraine found atypical brain responses to sensory stimuli , an absence of the normal habituating response between attacks , and atypical functional connectivity of sensory processing regions ( schwedt et al . , 2015 ) . migraine sensory hypersensitivities may be induced by a combination of enhanced sensory facilitation and reduced inhibition in response to sensory stimuli ( russo et al . , 2012 , schwedt et al . , 2014 ) , as well as reduced or absent habituation to stimuli interictally ( burstein et al . , 2010 ) . they may also contribute to a transformation from episodic to chronic migraine ( moulton et al . , 2008 ) . a concordant activation was found in the somatosensory , cingulate , limbic lobe , basal ganglia and midbrain in migraine patients in our study . all these areas are related to pain processing , modulation and associated symptoms ( like emotional disturbances ) in migraineurs . the activation areas of somatosensory , basal ganglia and midbrain coincided with previous studies and further confirms that activation and sensitization of the trigeminovascular pathway are involved in migraine pathophysiology ( dasilva et al . , 2007 , noseda and burstein , 2013 , schulte and may , 2016 , stankewitz et al . , 2011 ) . studies of changes in brain function during the ictal phase may improve our understanding of migraine mechanisms . , 2005a , bahra et al . , 2001 , welch et al . , 1998 ) , and others exposed migraine subjects to attack triggers in order to initiate migraine ( afridi et al . , 2005b , cao et al . , 2002 , maniyar et al . , 2014 ) . during the premonitory phase of nitroglycerin - triggered migraine attacks , the following areas of the brain were found to be activated : the posterolateral hypothalamus , the brainstem , and various cortical areas , including the occipital , temporal , and prefrontal cortices ( maniyar et al . , 2014 ) . studies that captured the onset of migraine found an increase in the signal from the brainstem ( bahra et al . , 2001 ) . comparisons of ictal images with interictal scans revealed significant activation in the anterior and posterior cingulate , as well as the cerebellum , thalamus , insula , prefrontal cortex , temporal lobes , and dorsal pons during migraine ( afridi et al . , 2005a ) . several studies found that , following treatment of the migraine with sumatriptan and suboccipital stimulator therapy , the dorsal pons remained active ( bahra et al . , 2001 , matharu et al . , 2004 ) . taken together , these studies show that the brainstem is continuously activated during a migraine , and that this may be specific to migraine and it may act as a generator of migraine . a longitudinal fmri study on a migraine patient found that hypothalamic activity increases towards the next migraine attack and shows altered functional coupling with the spinal trigeminal nuclei and the dorsal rostral pons during the preictal day and the pain phase ( schulte and may , 2016 ) . combined with previous study , this may indicate that the real driver of attacks might be the functional changes in hypothalamo brainstem connectivity ( maniyar et al . , 2014 ) . if there are concordant structural changes , especially increases in the gmv , these areas may be the cause of migraine . mri studies of patients with migraine have consistently shown a distributed pattern of morphological brain abnormalities characterized by regions of decreased and increased gmv ( jin et al . , 2013 , kim et al . , 2008 , maleki et al . , 2012a , rocca et al . , 2006 , schmidt - wilcke et al . , 2008 , schmitz et al . , 2008a , schmitz et al . , 2008b , valfre et al . , 2008 ) . the role of such abnormalities in the pathogenesis of this condition remains a matter of debate ; histological measures , such as neuronal density , do not correlate with vbm gm probability maps ( eriksson et al . , 2009 ) , and changes in cerebral blood flow produce this may reflect alterations in the dendritic complexity or changes in the number of synapses , or simply changes in water content . an increase in gmv may reflect structural brain plasticity as a result of exercise and learning ( may , 2009 ) . a decrease in gmv suggest that the central reorganization processes in chronic pain syndromes may involve degeneration of anti - nociceptive brain areas . indeed , relationships between decreases in gmv and disease duration or attack frequency have been reported ( kim et al . , 2008 , , 2012a , rocca et al . , 2006 , schmidt - wilcke et al . however , some changes in gmv are not correlated with clinical manifestation , suggesting that they may predispose a person to migraine ( liu et al . , 2013 ) . in our study , one study without significant result was excluded , it was conducted in 2003 and the possibility may be the differences existed in migraine patients included and image pre - processing procedures ( matharu et al . , 2003 ) . we found a set of decreases in the gmv in the bilateral inferior frontal gyri , the right precentral gyrus , the left middle frontal gyrus and the left anterior cingulate gyrus in migraine . our finding of reliable structural alterations in homologous regions associated with pain processing highlights the importance of these areas in the pathogenesis of migraine . we also found changes in gmv in the right claustrum , the left cingulate gyrus , the right anterior cingulate , the amygdala , and the left parahippocampal gyrus , which were negatively related to the frequency of headache attacks . our result further indicate that the gmv changes related to the frequency of headache attacks may occur as a consequence of repeated brain insults during migraine attacks . decreases in gmv in the frontal gyri were not related to the frequency of headache attacks . the frontal cortex is one of the most important areas associated with brain abnormalities in migraine patients . the role of the frontal lobe in pain processing has been established in several recent morphological studies , including subjects with chronic back pain ( apkarian et al . , 2004 ) , fibromyalgia , irritable bowel syndrome , and phantom pain atrophy in the prefrontal cortex ( may , 2008 ) . it has recently been reported that significant gm atrophy of the frontal lobe is associated with migraine in pediatric patients , but is not correlated with the disease duration or frequency of attacks ( rocca et al . , a recent longitudinal mri study using vbm investigated changes in gm related to medication withdrawal in a group of patients with medication overuse headache ( riederer et al . , 2013 ) . patients with clinical improvement showed a significant reversal of the changes in gm in some regions , and patients without treatment response had less gm in the orbitofrontal cortex . it appears that a decrease in gmv in the frontal gyrus may predispose a person to pain conditions , and may be predictive of poor response to treatment . pet studies have shown that the limbic regions ( the cingulate cortex , amygdala , and parahippocampal gyrus ) are activated during migraine attacks ( afridi et al . , 2005b , matharu et al . , 2004)and cluster headache attacks ( may et al . , 1999 , may et al . these areas of the brain are also believed to be involved in emotional , cognitive , and autonomic functions ( may , 2006 , morgane et al . , 2005 , peyron et al . , 1999 ) . alterations in cerebral structures that modulate the affective components of pain that are found in migraine suggest a possible neurobiological mechanism , which may explain the link between chronic migraine and psychiatric disturbances ( buse et al . decreases in gmv in the cingulate cortex were also found in patients with chronic back pain ( schmidt - wilcke et al . , 2006 ) , chronic phantom pain ( henry et al . , 2011 ) , and chronic tension - type headaches ( schmidt - wilcke et al . , 2005 ) . these changes are correlated with disease duration and attack frequency , and may result in brain damage accumulated due to repeated occurrences of pain - related processes , and are not specific to migraine . for better understanding the neuroanatomical and functional brain changes in migraine patients , there are several studies performed on the concurrent functional and structural cortical alterations in migraine ( hubbard et al . higher thickness in the post - central gyrus in the higher frequency of attack ( hf ) correlated with the observed stronger functional activation , suggested adaptation to repeated sensory drive ( maleki et al . decreased dorsal anterior cingulate cortex was correlated with increased functional connectivity between several regions indicate that frequent nociceptive input has modified the structural and functional patterns of the frontal cortex in migraine ( jin et al . , 2013 ) . concordant structural and functional changes in hippocampal in lower frequency of attack ( lf ) group relative to the hf and hc groups suggestive of an initial adaptive plasticity that may then become dysfunctional with increased frequency ( maleki et al . , no gmv increase was found , so the generator of migraine can not been deduced . increased activation in precentral gyrus and cingulate during nociceptive stimuli opposed to gmv decrease might represent increased effort , possibly due to disorganization of these areas and/or the use of compensatory strategies involving pain processing in migraine . we did not find structural changes in gm in areas that were specifically activated in migraine ( i.e. , the brainstem , hypothalamus ) . however , this negative finding does not imply that the brainstem and hypothalamus are not involved in the pathophysiology of migraine . because most of the studies included in our meta - analysis used whole - brain detection ( only in one study was the brainstem subjected to a small - volume correction , but this employed a 1.5-t mri scanner ) , and the gm volume of the brainstem is relatively small further studies are therefore warranted that focus on the brainstem volume using 3.0-t or 7.0-t mri scanners in order to determine whether there are any significant changes in the volume of the brainstem . at the same time , ale provides a way of applying statistical thresholds to large sets of coordinate data , in order to identify the most consistently - existed and reproducible loci across many studies , the result can provide a priori hypotheses for further experimental testing . however , it filters out less robust or infrequently - reported foci that may have a greater dependence on the details of individual studies . longitudinal and combination of structural and functional imaging studies may be needed to better understand the anatomy of migraine . as animal models are available in migraine and their life spans are short , a longitudinal imaging study can also be completed in rodents and brains can be collected ex - vivo for further analysis . first , we consider limitations that are common to imaging studies . there are no established protocols for imaging studies , and image pre - processing procedures , such as smoothing , registration , the choice of small volume corrections , imaging modalities , and data analysis , differed among studies . the headache characteristics ( i.e. , attack frequency and disease duration ) as well as the hc choices were also inconsistent . our meta - analysis was subject to publication bias because unpublished studies and studies without any significant clusters reported were not included , the studies were limited to those published in english and where the subjects were human . the ale meta - analysis was based on pooling of stereotactic coordinates with significant differences rather than on the raw data from the included studies ; this may have limited the accuracy of the results . nevertheless , previous studies suggested that no individual study could significantly bias the results of ale meta - analyses after including the sample size and number of reported foci into ale algorithm , changing from fixed - effects to random - effects inference , and revising for multiple comparison correction ( eickhoff et al . , 2012 , eickhoff et al . , 2009 , turkeltaub et al . , 2012 ) . the inclusion criteria for fmri were nociceptive stimulation in order to allow for analysis , regardless of stimulation site or stimulus type ( e.g. , hand , forehead , heat , and ammonia ) . the result makes clear that the simultaneous activation and deactivation of the relevant regions are implicated in responses to a range of nociceptive stimuli . the sex ratio was not concordant with the prevalence of migraine , and sex differences in brain structure may be present ( maleki et al . there were few chronic migraineurs , and the sample may not be broadly representative of migraineurs overall . migraineurs frequently have several comorbid conditions ( e.g. , anxiety and mood disorders , altered lifestyles , and drug use that might directly contribute to morphological changes ) ; this was not considered in our study . whole - brain vbm studies identified consistent widespread reduction in the gmv in migraine , specifically in the frontal cortex and the cingulate gyrus . the cingulate gyrus and the parahippocampal gyrus were related to the estimated frequency of headache attacks . frontal cortex and cingulate gyrus were over - activated in interictal phase during nociceptive stimuli . these changes in gm may be related to the mechanisms of pain processing and the associated symptoms of migraine , such as cognitive dysfunction , emotional problems , and autonomic dysfunction . the limbic regions may accumulate damage due to repeated occurrences of pain - related processes . further studies are required to determine how these changes can be used to monitor disease progression or exploited in therapeutic interventions . supplementary material 1structural neuroimaging data in nifti format.image 1supplementary material 2structural neuroimaging data related to headache durations in nifti format.image 2supplementary material 3functional neuroimaging data in nifti format.image 3supplementary material 4structural interactive plots.image 4supplementary material 5structural interactive plots related to headache durations.image 5supplementary material 6functional interactive plots.image 6 structural neuroimaging data in nifti format . structural neuroimaging data related to headache durations in nifti format . functional neuroimaging data in nifti format . structural interactive plots .

objectivesto summarize and meta - analyze studies on changes in grey matter ( gm ) in patients with migraine . we aimed to determine whether there are concordant structural changes in the foci , whether structural changes are concordant with functional changes , and provide further understanding of the anatomy and biology of migraine.methodswe searched pubmed and embase for relevant articles published between january 1985 and november 2015 , and examined the references within relevant primary articles . following exclusion of unsuitable studies , meta - analysis were performed using activation likelihood estimation ( ale).resultseight clinical studies were analyzed for structural changes , containing a total of 390 subjects ( 191 patients and 199 controls ) . five functional studies were enrolled , containing 93 patients and 96 controls . ale showed that the migraineurs had concordant decreases in the gm volume ( gmv ) in the bilateral inferior frontal gyri , the right precentral gyrus , the left middle frontal gyrus and the left cingulate gyrus . gmv decreases in right claustrum , left cingulated gyrus , right anterior cingulate , amygdala and left parahippocampal gyrus are related to estimated frequency of headache attack . activation was found in the somatosensory , cingulate , limbic lobe , basal ganglia and midbrain in migraine patients.conclusiongm changes in migraineurs may indicate the mechanism of pain processing and associated symptoms . changes in the frontal gyrus may predispose a person to pain conditions . the limbic regions may be accumulated damage due to the repetitive occurrence of pain - related processes . increased activation in precentral gyrus and cingulate opposed to gmv decrease might suggest increased effort duo to disorganization of these areas and/or the use of compensatory strategies involving pain processing in migraine . knowledge of these structural and functional changes may be useful for monitoring disease progression as well as for therapeutic interventions .

anatomic and pathologic staging remain the most accurate predictors of clinical outcomes in patients with colorectal cancer , enabling physicians to evaluate the benefit of adjuvant chemotherapy for individual patients . however , supplementing standard clinical and pathologic staging using molecular markers would allow more precise identification of those patients with the highest , or lowest , risk of relapse following colorectal cancer surgery . one of the most promising molecular markers that have been investigated in relation to colorectal cancer is the presence of tumor microsatellite instability . in a meta - analysis , colorectal cancers with microsatellite instability had a significantly better prognosis compared to those with intact mismatch repair , and these tumors may be resistant to treatment with 5-fluorouracil . apoptosis is an essential physiological process in maintaining normal tissue homeostasis , cellular differentiation and development . it is well known that defects in apoptosis facilitate the accumulation of somatic mutations which thereby contribute to tumor initiation , progression , and metastasis [ 4 - 6 ] . caspases ( casps ) , a family of cystein - dependent aspartate - specific proteases , play a crucial role in the regulation and execution of apoptosis [ 7 - 9 ] , and based on their proapoptotic function , they can be separated into initiator and effector casps . initiator casps ( casp8 , casp9 , and casp10 ) transmit apoptotic signals , and activation of effector casps ( casp3 , casp6 , and casp7 ) function to execute the final cell death program [ 7 - 10 ] . many polymorphisms identified in the casp genes are putatively functional , and clinical studies have demonstrated that these polymorphisms are involved in the development of solid tumors such as colorectal , lung , breast , and malignant melanoma . for example , casp9 gene polymorphisms and their haplotypes , -1263a > g ( rs4645978 ) and -712c > t ( rs4645981 ) , have been reported to both affect casp9 expression and modulate lung cancer risk . the v410i ( rs13010627g > a ) and i522l ( rs13006529a > t ) polymorphisms of the casp10 gene have been reported to be associated with the risk of developing cutaneous melanoma and familial breast cancer . furthermore , the casp7 rs2227310 and casp9 rs4645981 polymorphisms have been identified as independent prognostic markers for patients with surgically resected , non - small cell lung cancer ( nsclc ) . given these results , casp gene polymorphisms appear to play a role in the carcinogenesis or prognosis of solid tumors . nonetheless , relatively few studies have investigated the single nucleotide polymorphisms ( snps ) in the casp genes and their relationship to the clinical outcomes of colorectal cancer . accordingly , this study analyzed 10 casp gene polymorphisms and evaluated their impact on the prognosis of colorectal cancer patients . all the tissues investigated in this study were obtained from 397 consecutive , ethnic korean , colorectal cancer patients who had undergone a curative resection between january 2003 and august 2006 , at kyungpook national university hospital ( daegu , korea ) . written informed consent for gene expression analyses was received from all participating patients prior to surgery , and the study was approved by the kyungpook national university hospital institutional research board . the diagnosis and staging of the colorectal cancer data was performed according to world health organization ( who ) classifications and the tumor , node , and metastasis ( tnm ) classifications set by the american joint committee on cancer ( ajcc ) . due to the enormous number of snps in the human genome , an appropriate strategy for efficient selection of those snps most likely to contribute phenotypic effects was our first challenge . thus , a prioritization scheme was created using public databases providing diverse information on potential phenotypic risks associated with specific snps . first , candidate snps from casp genes were collected from web - based databases which included information on the biologic pathways and potential biologic effects of these polymorphisms . next , based on the allele frequencies recorded for east asian populations obtained from fastsnp , those snps with frequencies less than 0.1 were excluded . the remaining casp gene snps were then scored according to particular phenotypic risks , and then , based on the algorithm suggested in a previous report , they were ordered according to the sum of their risk scores . among the 13 polymorphisms in the casp3 , casp6 , casp7 , casp8 , casp9 , and casp10 genes that have been reported to be potentially functional or otherwise associated with cancer risk [ 11 - 16 ] , ten polymorphisms ( casp3rs2705897 ; casp6 rs1042891 , rs2301717 ; casp7 rs2227310 , rs11593766 ; casp8 rs3769818 , rs3834129 ; casp9 rs1052571 , rs4645978 ; casp10 casp8 rs1045485 and casp10 rs13010627 were excluded as they are rare or nonexistent in asian populations . genomic dna was extracted from fresh colorectal mucosal tissue at the time of surgery using a wizard genomic dna purification kit ( promega , madison , wi ) . the 10 selected casp gene polymorphisms were then determined using a reverse transcription polymerase chain reaction ( pcr ) genotyping assay . for quality control , the selected , pcr - amplified dna samples ( n=2 , for each genotype ) were also examined by dna sequencing to confirm the genotyping results . the genotypes for each snp were analyzed as a categorical variable for three - groups ( reference model ) , and also grouped according to a dominant and recessive model . the survival estimates were calculated using the kaplan - meier method . as related to the appearance of snps of the casp genes , the differences in patient overall survival ( os ) or disease - free survival ( dfs ) , cox 's proportional hazard regression model was used for the multivariate survival analyses , whereby the analyses were adjusted for potential prognostic factors including age ( median age , 63 years;63 years vs.>63 years ) , differentiation ( well to poor differentiation ) , preoperative carcinoembryonic antigen ( cea ) level ( normal vs. elevated ) , and pathologic stage ( 0 to iv ) . the hazard ratio and 95% confidence interval 11.5 ( spss inc . , chicago , il ) or sas genetic software ( sas institute , cary , nc ) . all the tissues investigated in this study were obtained from 397 consecutive , ethnic korean , colorectal cancer patients who had undergone a curative resection between january 2003 and august 2006 , at kyungpook national university hospital ( daegu , korea ) . written informed consent for gene expression analyses was received from all participating patients prior to surgery , and the study was approved by the kyungpook national university hospital institutional research board . the diagnosis and staging of the colorectal cancer data was performed according to world health organization ( who ) classifications and the tumor , node , and metastasis ( tnm ) classifications set by the american joint committee on cancer ( ajcc ) . due to the enormous number of snps in the human genome , an appropriate strategy for efficient selection of those snps most likely to contribute phenotypic effects was our first challenge . thus , a prioritization scheme was created using public databases providing diverse information on potential phenotypic risks associated with specific snps . first , candidate snps from casp genes were collected from web - based databases which included information on the biologic pathways and potential biologic effects of these polymorphisms . next , based on the allele frequencies recorded for east asian populations obtained from fastsnp , those snps with frequencies less than 0.1 were excluded . the remaining casp gene snps were then scored according to particular phenotypic risks , and then , based on the algorithm suggested in a previous report , they were ordered according to the sum of their risk scores . among the 13 polymorphisms in the casp3 , casp6 , casp7 , casp8 , casp9 , and casp10 genes that have been reported to be potentially functional or otherwise associated with cancer risk [ 11 - 16 ] , ten polymorphisms ( casp3rs2705897 ; casp6 rs1042891 , rs2301717 ; casp7 rs2227310 , rs11593766 ; casp8 rs3769818 , rs3834129 ; casp9 rs1052571 , rs4645978 ; casp10 casp8 rs1045485 and casp10 rs13010627 were excluded as they are rare or nonexistent in asian populations . genomic dna was extracted from fresh colorectal mucosal tissue at the time of surgery using a wizard genomic dna purification kit ( promega , madison , wi ) . the 10 selected casp gene polymorphisms were then determined using a reverse transcription polymerase chain reaction ( pcr ) genotyping assay . for quality control , the selected , pcr - amplified dna samples ( n=2 , for each genotype ) were also examined by dna sequencing to confirm the genotyping results . the genotypes for each snp were analyzed as a categorical variable for three - groups ( reference model ) , and also grouped according to a dominant and recessive model . the survival estimates were calculated using the kaplan - meier method . as related to the appearance of snps of the casp genes , the differences in patient overall survival ( os ) or disease - free survival ( dfs ) , cox 's proportional hazard regression model was used for the multivariate survival analyses , whereby the analyses were adjusted for potential prognostic factors including age ( median age , 63 years;63 years vs.>63 years ) , differentiation ( well to poor differentiation ) , preoperative carcinoembryonic antigen ( cea ) level ( normal vs. elevated ) , and pathologic stage ( 0 to iv ) . the hazard ratio and 95% confidence interval 11.5 ( spss inc . , chicago , il ) or sas genetic software ( sas institute , cary , nc ) . the median age of the patients was 63 years ( range , 21 to 85 years ) , and 217 ( 54.7% ) of the total patients were male . of the total patients , 218 ( 54.9% ) were diagnosed with colon cancer and the remaining with rectal cancer . laparoscopic surgery was performed on 271 ( 68.3% ) patients , while the others received an open colorectal resection . the pathologic stages after the surgical resection were as follows : stage 0/i ( n=86 , 21.7% ) , stage ii ( n=146 , 36.8% ) , stage iii ( n=145 , 36.5% ) , and stage iv ( n=20 , 5.0% ) . among the 291 patients with stage ii or iii disease , 275 ( 94.5% ) received adjuvant chemotherapy using either 6 cycles of 5-fluorouracil / leucovorin radiotherapy ( n=133 ) , 12 cycles of oxaloplatin , leucovorin , and fluorouracil ( folfox-4 ) ( n=12 ) , 8 cycles of capecitabine ( n=20 ) , or doxifluridine during a treatment period of one year ( n=110 ) ( table 1 ) . at the time of the last analysis , 80 patients had experienced disease relapse and 37 had died as a result of colorectal cancer disease progression . however , the deaths of 2 patients were unrelated to colorectal cancer . at the median follow - up duration of 34.8 months ( range , 1.1 to 65.7 months ) , the estimated 5-year os and dfs for all the patients was 89.31.6% and 72.34.3% , respectively . the frequencies of occurrence for each genotype also conformed to the hardy - weinberg equilibrium ( p>0.05 ) . no correlation was observed between any given frequency of the genotype or allele and clinicopathologic parameters , including for t , n , or m stage patients ( data not shown ) . a multivariate survival analysis including pathologic stage , patient age , differentiation , and cea level demonstrated there was no association between any genotypes with outcomes for dfs or os ( table 2 ) . in haplotype analysis of the casp6 , casp8 and casp9 genes , none were associated with colorectal cancer prognosis . in terms of clinicopathologic parameters in a cox model , patient age , cea level , and tnm stage the median age of the patients was 63 years ( range , 21 to 85 years ) , and 217 ( 54.7% ) of the total patients were male . of the total patients , 218 ( 54.9% ) were diagnosed with colon cancer and the remaining with rectal cancer . laparoscopic surgery was performed on 271 ( 68.3% ) patients , while the others received an open colorectal resection . the pathologic stages after the surgical resection were as follows : stage 0/i ( n=86 , 21.7% ) , stage ii ( n=146 , 36.8% ) , stage iii ( n=145 , 36.5% ) , and stage iv ( n=20 , 5.0% ) . among the 291 patients with stage ii or iii disease , 275 ( 94.5% ) received adjuvant chemotherapy using either 6 cycles of 5-fluorouracil / leucovorin radiotherapy ( n=133 ) , 12 cycles of oxaloplatin , leucovorin , and fluorouracil ( folfox-4 ) ( n=12 ) , 8 cycles of capecitabine ( n=20 ) , or doxifluridine during a treatment period of one year ( n=110 ) ( table 1 ) . at the time of the last analysis , 80 patients had experienced disease relapse and 37 had died as a result of colorectal cancer disease progression . however , the deaths of 2 patients were unrelated to colorectal cancer . at the median follow - up duration of 34.8 months ( range , 1.1 to 65.7 months ) , the estimated 5-year os and dfs for all the patients was 89.31.6% and 72.34.3% , respectively . the frequencies of occurrence for each genotype also conformed to the hardy - weinberg equilibrium ( p>0.05 ) . no correlation was observed between any given frequency of the genotype or allele and clinicopathologic parameters , including for t , n , or m stage patients ( data not shown ) . a multivariate survival analysis including pathologic stage , patient age , differentiation , and cea level demonstrated there was no association between any genotypes with outcomes for dfs or os ( table 2 ) . in haplotype analysis of the casp6 , casp8 and casp9 genes , none were associated with colorectal cancer prognosis . in terms of clinicopathologic parameters in a cox model , patient age , cea level , and tnm stage the prognostic impact of 10 snps of casp genes was investigated in a large population of patients with curatively resected colorectal adenocarcinoma . however , no association was observed between the polymorphisms in the casp genes and survival in these patients . given the homogenous ethnic background of the patients ( korean ) , any potential confounding effect due to ethnicity is likely to be small in the present study . since the casp pathway plays an important role in balancing cell growth and death , it is possible that germ line polymorphisms in these genes may affect an individual 's cancer risk or prognosis . in a previous study by yoo et al . , significant associations between the casp7 rs2227310 and casp9 rs4645981 polymorphisms and prognosis were found in 411 korean patients with early stage nsclc . however , these same polymorphisms were found to have no prognostic significance in the survival rate of colorectal cancer patients in the current study . in the yoo et al . 's study , the casp9 rs4645981 tt genotype was found to be significantly associated with poor survival outcomes , suggesting that a lower production genotype for casp9 may have decreased the capacity for casp9 to mediate the apoptotic activity required to eliminate mutated or transformed cells , which would thus increase the risk of lung cancer development and lead to poor survival outcomes in patients with early - stage nsclc . although it remains to be elucidated whether or not the casp7 rs2227310c > g polymorphism itself affects protein characteristics , interaction sites , or protein solubility or stability , this polymorphism was associated with poor survival outcomes for patients suffering with nsclc . relatively few studies have investigated these polymorphisms and their relationship to the risks or clinical outcomes of other solid tumors , including colorectal cancer , and the results are inconsistent [ 11,14,15,22 - 24 ] . for example liu et al . , reported that 2 caspase-8 snps were not associated with colorectal cancer risk in a study conducted with 373 chinese patients and 838 controls . in our previous study that analyzed 15 snps of apoptosis - related genes and their impact on the response to chemotherapy and survival of colorectal cancer patients , no significant association between patient response or survival and the polymorphisms of the casp3 or casp6 to casp10 genes was observed . given that validation of genotype - phenotype association studies requires replication using an independent data set , additional studies are required to confirm the associations between casp gene polymorphisms and survival outcomes in the cancer patients observed in our previous study . none of the 10 casp gene snps investigated in this study was found to be an independent prognostic marker for korean patients with curatively resected colorectal cancer . however , since genetic polymorphisms often vary between ethnic groups , further studies clarifying the association between these polymorphisms and the prognosis of colorectal cancer in diverse ethnic populations are warranted .

purposethis study analyzed potentially functional polymorphisms in caspase ( casp ) genes and their impact on the prognosis for korean colorectal cancer patients.materials and methodsa total of 397 consecutive patients with curatively resected colorectal adenocarcinoma were enrolled in this study . genomic dna from these patients was extracted from fresh colorectal tissue , and the 10 polymorphisms in the casp3 , casp6 , casp7 , casp8 , casp9 , and casp10 genes were determined using a reverse transcription polymerase chain reaction genotyping assay.resultsthe median patient age was 63 years , and 218 ( 54.9% ) patients had colon cancer , while 179 ( 45.1% ) patients had rectal cancer . univariate and multivariate survival analysis including pathologic stage , patient age , differentiation , and carcinoembryonic antigen level demonstrated that these polymorphisms were not associated with either disease - free or overall survival.conclusionnone of the 10 polymorphisms in the casp genes investigated in this study was found to be an independent prognostic marker for korean patients with curatively resected colorectal cancer .

anchorage has always been a matter of great concern in orthodontics . extraoral and intraoral anchorage methods have stood the test of time in providing the best possible treatment outcomes except for some cardinal limitations like compliance factor , slippage , and so forth . osseointegrated implants have also long been used for a variety of purposes including anchorage . the introduction of skeletal anchorage system ( sas ) by sugawara , microscrews by kanomi , and the micro - implant anchorage ( mia ) by park and park et al . in the orthodontic treatment care has negated the limitation faced by other anchorage devices . clinical applications of microimplants as direct or indirect anchorage methods include correction of deep bite , closure of extraction spaces , correction of canted occlusal plane , alignment of dental midlines , extrusion of impacted canines , extrusion and uprighting of impacted molars , molar intrusion , distalization of either maxillary molars or mandibular teeth , en - masse retraction of anterior teeth , molar mesialization , maxillary third molar alignment , intermaxillary anchorage to correct sagittal discrepancies and correction of vertical skeletal discrepancies . the term temporary anchorage devices ( tads ) has become quite popular today and it connotes the nature of mi as a nonosseointegrated device made of titanium alloy or stainless steel meant only for anchorage purpose during active tooth movement . thus , the common concern revolves around their efficiency , that is , the success of micro - implants is dependent on their holding in the bone as stationary anchors . hence , an evaluation of the success rate in a systematic protocol is absolutely essential . recently , some investigations have evaluated the failure or success rates of micro - implants and risk factors associated with their use as tads for orthodontic purposes [ 613 ] . according to these studies , the success rates have varied long between 75.2% and 90.7% . the aim of this retrospective study was to find factors related to the clinical success of micro - implants in asian patients . case history and treatment detail records for cases treated using micro - implants from 2007 to 2009 at our university orthodontic clinic ; and self - made questionnaire containing the relevant study variables were employed for the study . the questionnaire had been filled by the treating faculty members and post - graduate students who had formal training in microimplant insertion . the database was generated with the details recorded using microsoft office excel 2007 . in total , the ages ranged from 13 to 50 years , with a mean of 22.45 years . before implantation , all these patients were informed of the advantages and disadvantages associated with the use mi as part of orthodontic treatment strategy . the screw implants were placed at 30 to 40 angles to the long axes of the teeth in the maxillary arch and at 10 to 20 angles in the mandibular posterior area according to the recommendations of the clinicians who developed them . the patients were instructed to gently massage the implant area and to keep it clean . a chlorhexidine rinse was prescribed for 7 to 14 days . to check the effect of oral hygiene on success , the amount of food debris and plaque accumulation on the tooth surfaces was assessed ; the sample was divided into 3 groups : good , fair , or poor . inflammation around the screw implant was checked in the following categories : yes or no . redness or swelling around the neck of the screws was a sign of inflammation . orthodontic force was applied either immediately or after initial wound healing depending on the clinical situation and operator 's skill . elastomeric modules ( libral traders , new delhi , india ) , power chain or closed - type niti coil springs ( libral traders , new delhi , india ) , stainless steel closed coil ( h.p . industries , india ) or ligature wire ( libral traders , new delhi , india ) for activation of loop were used as the source of orthodontic traction force . forces were applied either directly to the microimplants ( direct anchorage ) or indirectly ( indirect anchorage ) to achieve better torque and directional control and to prevent gingival impingement . these forces were used for the following orthodontic purposes : ( 1 ) en - masse retraction of the 6 anterior teeth in arches , ( 2 ) distalization of the molar teeth ( 3 ) protraction of the molar teeth ( 4 ) intrusion of the maxillary and mandibular incisors and molars , and ( 5 ) en - masse distalization of the dental arches as per the requirement of the case . the criteria of microimplant success were : ( 1 ) no inflammation of the soft tissues surrounding the microimplant , ( 2 ) no clinically detectable mobility , and ( 3 ) anchorage function sustained until the end of the purpose for which the implant was used . failure was defined as spontaneous loss , severe clinical mobility of the microimplant requiring replacement , or infected , painful , pathologic changes in the surrounding soft tissues . clinical variables influencing the success rates of the microimplants were categorized as patient - related , implant - related , and treatment - related and assessed accordingly . patient - related factors included sex , skeletal and dental relationships ( class i , class ii , or class iii ) , mandibular angle ( high , average or low ) overbite ( increased , decreased , or normal ) , oral hygiene status ( good , fair or poor ) , jaw involved ( maxilla or mandible ) , side involved ( right or left ) , and site involved ( anterior to premolar area , posterior to premolar area , retromolar area or palatal area ) . implant - related factors included implant type ( indian implant or abso anchor ) . treatment - related factors included type of insertion ( self drilling or self tapping ) , vertical position of insertion ( attached or moveable gingiva ) , time of loading , purpose of microimplant insertion , mode of loading , type of anchorage used , direction of forces applied ( horizontal , vertical or both ) , and any inflammation if present . fisher 's exact test was used when there was an expected value < 5 in any cell of the cross - tables while chi - square test was conducted for others . the data were statistically analyzed with spss software ( version 11.5 , spss , chicago , ill ) . the microimplant survival time was ( 8.96 4.8 ) months . the data regarding overall and detailed success rates of microimplants patient- , implant- , and treatment - related factors are shown in tables 1 and 2 . of the patient related factors , there were no significant differences according to sex , jaw , side and site of placement , skeletal or dental relationship , and overbite . however , patients with a poor oral hygiene and high mandibular angle showed significantly less success than those with a good oral hygiene and low mandibular angle . male patients showed a higher success rate than female patients though it was not significant statistically . patients with a skeletal class i relation showed higher success than those with skeletal class ii and class iii relations though there was no statistical significance . for implant - related factors , there were no significant differences in the success rate of the two types of implants studied : type a ( indian implant ) type b ( absoanchor ) . among treatment related factors , there was no significant correlation in success rate according to the type of microimplant insertion ( self drilling or self tapping ) , time of loading , purpose of microimplant placement , mode of loading , direction of forces and type of anchorage used . the implants placed in the attached gingiva had significantly higher success than those placed in the moveable gingiva . since microimplants are a relatively new innovation , little is known about factors that affect the rates of success of screw implants . therefore , in this study , we tried to include as many factors as possible . patient - related , implant - related , and treatment - related factors were evaluated . among them , significant differences were found among patient related and treatment related factors . the success rate of microimplants in the present study was 87.8% . the success rate for microimplants in previous studies varied between 83.9% and 93.3% [ 1214 ] . these rates might be explained by the various types of microimplants , different surgical techniques , and varying management protocols . therefore , a direct comparison of success rates might not be possible . also , the success rate in this study was comparable with the rates in other studies of asian patients ( 75.20%91.60% ) [ 611 ] . although male patients showed higher success than female patients but this was not significant statistically . inflammation can damage the bone surrounding the neck of microimplants . with progressive damage of the cortical bone , screw implants can be endangered . to ensure success , it is important to prevent inflammation around the screw implants . in this study , both oral hygiene and local inflammation significantly affected the success rate . in the study by park et al . local inflammation can be exaggerated not only by oral hygiene but also by weak nonkeratinized soft tissue around the neck of the screw implant . this study showed no difference in success rates of microimplants for maxillary or mandibular placement in contrast to the results of chen et al . and park et al . , who found lower success rates for microimplants in the mandibles of asian patients . cortical bone thickness of the mandible that might cause bone overheating while drilling and the short zone of attached gingiva have been suggested as causes of these significantly lower success rates of microimplants in the mandible . our results were similar to previous studies by wiechmannn et al . and antoszweska et al . , that were not necessarily correlated with anatomic factors , such as cortical bone thickness . placement of microimplants in the left quadrant of the dental arch , although not significant in our evaluation , seemed to have a higher success rate than on the right side as also reported previously . a possible explanation for this might be better oral hygiene on the left side of the oral cavity that is usually observed in right - handed patients , who are the majority . in previous studies , it was found that microimplants on the right side of the mandible between the first and second molars had significantly lower success rates : kuroda et al . and antoszweska et al . . chen et al . found the lowest success rates on either side of the mandible between the first and second premolars . there was no statistical significant difference in success rate according to the skeletal and dental relationships . our study observed lower success rates in patients with increased mandibular angles similar to the previous studies by miyawaki et al . and moon et al . . the lower success rates of microimplants in patients with open bites or increased mandibular angles might be because these patients usually have thin cortical bones , the volume of which significantly contributes to the mechanical retention of nonosseointegrated microimplants . in contrast to the study by antoszweska et al . , where open bite was found to be a negative factor significantly influencing the stability of the microimplants , in our study , no statistically significant difference was found between overbite and success rate . the study showed no significant difference in success rate according to the type of microimplant and type of insertion ( self - drilling or self - tapping ) . the main difference between self - tapping screws and self - drilling , self - tapping screws is that the former requires a predrilled hole of lesser diameter than the thickness of the screw which is placed and tightened either manually or by a power - driven screw driver . in case of self - drilling , self - tapping screws , no predrilled hole is required , because the screw has a built - in drill point and does the drilling in steel and the tapping on its own in a single operation . this shows that by following proper surgical protocol and following the recommended instructions , one can achieve a good success rate . with regard to the vertical position of microimplants , their placement in the attached gingiva of the maxilla was associated with significantly higher success rates , in contrast to the results of park et al . , who found higher success rates when microimplants were placed in the oral mucosa of maxilla . however , this is in accordance with the previous study by antoszweska et al . . there was no significant difference in the success rate with respect to the onset of force application . an animal experiment proved that there was osseointegration after immediate loading of the screw implants and suggested immediate loading to reduce the treatment time . therefore , screw implants can be loaded immediately after placement without a discernible deterioration of stability . no significant difference was found between the success rate and the purpose for which the microimplant was placed . there was no stastically significant difference found in success rate according to the mode of loading ( similar to chen et al . ) , type of anchorage ( similar to antoszweska et al . ) , and direction of forces . however , antoszweska et al . showed that vertical forces applied to microimplants seem to be responsible for more failures than horizontal forces . since most of the microimplants used in the department were 1.3 mm in diameter and 8 mm in length , the effects of the dimensions of microimplant on the clinical success were not considered . majority of the sample comprised of young population ; therefore , age factor and systemic problems were not considered . however , the effect of the above shortcomings should be elucidated in a future study . this shows that microimplants can be used for orthodontic anchorage predictably and consistently in routine orthodontic practice . poor oral hygiene , high mandibular angle , placement in moveable gingival , and inflammation were associated with microimplant failure in this study . thus , proper case selection and following the recommended protocol are extremely essential to minimise failures .

introduction . the purpose of this study was to examine the success rate and find factors affecting the clinical success of microimplants used as orthodontic anchorage . methods . seventy - three consecutive patients ( 25 male , 48 female ; mean age , 22.45 years ) with a total of 139 screw implants of 2 types were examined . success rate was determined according to 18 clinical variables . results . the overall success rate was 87.8% . the clinical variables of microimplant factors ( type ) , patient factors ( sex , skeletal and dental relationships , overbite , jaw involved , side involved and site involved ) , and treatment factors ( type of insertion , time of loading , purpose of microimplant insertion , mode of loading , type of anchorage used , direction of forces applied ) did not show any statistical difference in success rates . mandibular angle , vertical position of implant placement , oral hygiene status , and inflammation showed significant difference in success rates . conclusions . proper case selection and following the recommended protocol are extremely essential to minimise failures .

akathisia is an involuntary movement disorder characterized by a subjective feeling of restlessness and fidgety movements . first - generation antipsychotic agents had drug - induced akathisia due to its high incidence and particularly aversive nature . despite the fact that second - generation antipsychotics have a lower potential to cause extrapyramidal side - effects , including akathisia , their incidence is not negligible . a variety of akathisia subtypes have been described : acute , withdrawal , tardive and chronic . tardive akathisia is characterized by longer - term akathisia manifestations , and is occasionally confused with chronic akathisia . it may persist or may become worse when the antipsychotic treatment is discontinued or reduced . it seems that tardive akathisia may have pharmacological differences from acute akathisia . unlike acute akathisia , neither chronic nor tardive akathisia has been reported with drugs not being neuroleptics , considering that these types of akathisias may be pure neuroleptic - related syndromes , and that this makes them potentially important syndromes to examine with regard to pathophysiology . there appears to be dopamine receptor blockade in the mesocortical dopamine system in acute akathisia , while it has been noted that the delayed akathisia may have resulted from a pharmacodynamic effect through serotonin - dopamine and alfa - receptor interaction . in the present study , we planned to evaluate the nature of delayed - onset akathisia in patients on amisulpride monotherapy . overall , 56 patients on amisulpride treatment for 2 months at a stabilized amisulpride dose for their schizophrenia , delusional or schizoaffective disorder were folowed - up . of them , 18 patients with diagnostic and statistical manual of mental disorders - iv ( dsm - iv ) presented with acute or delayed - onset akathisia , and all of them also met the entry criteria . the inclusion criteria were : taking stabilized amisulpride dose for at least 2 months , having normal physical and neurological examination and normal routine laboratory tests and electrocardiography within the normal range . the exclusion criteria included the presence of a severe physical illness , the history of alcohol and substance abuse or dependence , a previous history of neurologic disease and the presence of any endocrinological state . the patients were evaluated at baseline and at the time when akathisia presented clinically , with respect to the positive and negative syndrome scale ( panss ) and barnes akathisia scale ( bas ) . to confirm the akathisia , a score of 2 on the global scale ( range 05 ) of the bas was required . statistical analysis was performed using the statistical package for social sciences ( spss / pc 10.0 version ) . table 1 shows the demographic and clinical data of the 18 patients who developed akathisia . majority of the patients reported one or more adverse effects during the 2-month period apart from the akathisia , most of which were mild to moderate in intensity . the most frequently reported adverse effects were reduced energy ( n = 6 ) , tremor ( n = 5 ) and nausea ( n = 3 ) . overall , significant changes were noted in the panss and clinical global impression - severity ( cgi - s ) scores after the study period . the mean total panss scores were 69.4 8.1 and 52.1 7.2 at baseline and after 2 months , respectively ( p < 0.01 ) . the mean cgi - s scores were 3.64 1.2 and 2.5 0.9 at baseline and at 2 months , respectively ( p < 0.05 ) . using the primary categorical criterion of akathisia ( 2 points of the bas global scale ) , 12 ( 21.4% ) of the 56 patients experienced delayed - onset akathisia , and six ( 10.7% ) showed acute akathisia . the mean time of onset of acute or delayed - onset akathisia was 5.8 2.1 and 39.4 11.3 days , respectively . the mean bas scores at baseline and after the period of 2 months were 1.3 0.6 and 3.9 2.4 , respectively ( p < 0.01 ) . eight patients ( four with acute and four with tardive akathisia ) responded to dose reduction and four responded to benzodiazepines and propranolol treatment while six did not respond to any treatment . to the best of our knowledge , this is the first study to assess the time course of the occurrence of amisulpride - induced akathisia . the present study revealed that amisulpride could lead to , especially , delayed - onset akathisia rather than acute akathisia . because of its mechanism of action not having any affinity to 5-ht2 receptors , amisulpride , a benzamide derivative , has high and similar affinities for the dopamine d2 and d3 receptor subtypes and is devoid of any significant affinity to other receptor systems . therefore , its high d2 occupancy can account for the high rate of akathisia because of the fact that eight of the patients ( four with acute and four with tardive akathisia ) responded to dose reduction . however , a question arises : why amisulpride induces delayed - onset akathisia ? despite the fact that no known active metabolites in rodents were determined , natesan et al . reported slower brain penetration of the amisulpride , measured by its ability to displace [ h ] raclopride in the striatum when compared with haloperidol , risperidone and clozapine . moreover , natesan et al . found that despite amisulpride 's effect of quick onset ( 1 h ) and decline ( 6 h ) of prolactin elevation , it showed a delayed pattern of d2/3 receptor occupancy : 43 , 60 and 88% after 1 , 2 and 6 h ( 100 mg / kg ) , respectively , unlike the atypicals clozapine and risperidone , supporting its poor blood therefore , we suggest that its delayed penetration into the brain may be a contributing factor for its delayed - onset akathisia effect . on the other hand , the severity and time difference for developing extrapyramidal side - effects , including akathisia , also depend on some pharmacological differences between drugs , such as the rate of dissociation from the d2 receptor , the degree of dopamine-2 ( d2 ) and 5ht2a receptor antagonism and the antimuscarinic potential of the drug . therefore , when thinking on the mechanism of tardive akathisia , we should take into consideration not only the d2 receptor blocking effect but also the other receptor - binding profiles such as minimal muscarinic receptor or the 5-ht2 receptor affinity . second , the small sample may limit the interpretation and generalizability of our findings . thus , although our results revealed that amisulpride could considerably lead to delayed - onset akathisia , studies comprising larger samples receiving different antipsychotics , and more comprehensive assessment , will help in ascertaining the role of amisulpride in delayed - onset akathisia .

despite the fact that second - generation antipsychotics have a lower potential to cause extrapyramidal side - effects , including akathisia , their incidence is not negligible . recent work suggests that tardive akathisia may have pharmacological differences from acute akathisia . in the present study , we have evaluated the nature of delayed - onset akathisia in patients on amislpride monotherapy . overall , we screened 56 patients on amisulpride treatment for 2 months at a stabilized amisulpride dose . however , 18 patients with diagnostic and statistical manual of mental disorders - iv ( dsm - iv ) presented with acute or delayed - onset akathisia , and all of them also met the entry criteria . the patients were evaluated at baseline and at the time when akathisia presented clinically , with respect to the positive and negative syndrome scale and barnes akathisia scale ( bas ) . using the primary categorical criterion of akathisia ( 2 points of the bas global scale ) , 12 ( 21.4% ) of the 56 patients experienced delayed - onset akathisia , and six ( 10.7% ) showed acute akathisia . the mean time for onset of acute or delayed - onset akathisia was 5.8 2.1 and 39.4 11.3 days , respectively . the mean bas scores at baseline and after the period of 2 months were 1.3 0.6 and 3.9 2.4 , respectively ( p < 0.001 ) . our results revealed that amisulpride could considerably lead to delayed - onset akathisia . however , studies comprising larger samples receiving different antipsychotics , and more comprehensive assessment , will help to ascertain the role of amisulpride in delayed - onset akathisia .

angioleiomyoma is a benign soft tissue neoplasm that originates from smooth muscle in the tunica media of arteries and veins . it can occur anywhere in the body , with predilection for the lower extremity , and can be found in the dermis , subcutaneous fat , or fascia . the tumor typically presents as a painful , solitary , small ( < 2 cm ) , slow growing , firm , mobile , subcutaneous nodule . angioleiomyoma of the knee is rare and only few cases have been reported so far . herein , we have described the first case report of giant angioleiomyoma of the knee with atypical presentation as a painless ulcer and have discussed the surgical management of the same . a 22-year - old male presented with a painless ulcer of 2 year 's duration on his left knee [ figure 1 ] . the lesion started as a small ulcer and gradually increased in size over the next 2 years . it was neither preceded by trauma nor associated with systemic symptoms like fever or weight loss . the ulcer was 10 8 cm in size , situated on the left knee , well - demarcated , non - tender , and was not fixed to any underlying structure . pre - operative photograph showing the giant ( 10 8 cm ) ulcerative lesion on the patient 's left knee total excision of the lesion with ligation of the feeder vessel , performed under general anesthesia , resulted in a large defect and exposure of the knee joint capsule [ figure 2 ] . the resulting defect was covered by raising an anteromedial thigh fasciocutaneous flap based on septocutaneous perforator , and the donor site defect was closed primarily [ figure 3 ] . the defect after excision of the tumor immediate post - operative photograph of reconstruction of the defect on microscopic examination , the tumor sections showed interlacing fascicles of smooth muscle cells encircling vascular lumina lined by normal - looking endothelial cells [ figure 4 ] . immunohistochemistry confirmed the diagnosis , the tumor being positive for smooth muscle actin ( sma ) , desmin , and vimentin , and negative for hmb45 . there has been no recurrence at 1 year follow - up , and the aesthetic outcome of surgery is satisfactory [ figure 5 ] . photomicrograph showing the tumor composed of numerous fascicles of smooth muscle cells encircling blood vessels h and e , 40 aesthetic outcome after 1 year of surgery the tumor usually presents in the third to fifth decade of life and has female preponderance . they have a predilection for the lower extremities , and the commonest site is the lower leg . the typical lesion is a solitary , small , slowly growing , firm , mobile , subcutaneous nodule . pain , often paroxysmal , is the most striking clinical feature of angioleiomyoma and is present in about 60% of cases . angioleiomyoma of the knee is rare , with only a few case reports in the literature . a case of intra - articular angioleiomyoma of the knee joint has also been described . our case has the following unique features as compared to the other reported cases of knee angioleiomyoma : ( 1 ) male patient ; ( 2 ) relatively younger age of presentation ; ( 3 ) giant size of angioleiomyoma of knee ; ( 4 ) presentation of angioleiomyoma as ulcerative lesion ; and ( 5 ) absence of pain . to our knowledge , this is the first case report of giant angioleiomyoma of the knee . more importantly , cases of angioleiomyoma presenting with an ulcerative lesion have never been described before . after an extensive literature search , we did not come across a single case report of angioleiomyoma of knee that presented with ulcerative lesion . finally , majority of angioleiomyomas of the lower extremity are painful , whereas , in our case , the tumor grew to a massive size but remained painless throughout . our case highlights for the first time the fact that angioleiomyomas may atypically present as an ulcerative lesion , rather than as a typical subcutaneous nodule . this is the first case report of giant angioleiomyoma of the knee , presenting uniquely as a painless ulcer . this case also highlights that angioleiomyomas may atypically present as ulcerative lesion , rather than as a typical subcutaneous nodule .

angioleiomyomas are benign tumors originating in the vascular smooth muscle . the tumor typically presents as painful , solitary , small ( < 2 cm ) , slow growing , subcutaneous nodule . angioleiomyoma of the knee is rare , and only few cases have been reported so far . we have described herein a giant angioleiomyoma of the knee presenting as a painless ulcer in a 22-year - old man . there was no intra - articular extension of the tumor , and total excision was curative . this is the first case report of giant angioleiomyoma of the knee as well as the first case report of angioleiomyoma presenting as a painless ulcerative lesion .

hormone replacement therapy ( hrt ) in postmenopausal women suppresses the increase in bone resorption expected as circulating levels of endogenous estrogen decline . we tested the hypothesis that bone lead content might remain elevated in women on hrt . fifty six women who at recruitment were on average 35 years postmenopausal were placed on calcium supplementation . six months later 33 of these women were prescribed either low dose or moderate dose hormone replacement in addition to the calcium supplementation . after approximately 4 years of hormone replacement , lead content was measured at the tibia and calcaneus by in vivo fluorescence excitation , and lead concentrations were measured in serum , whole blood , and urine . women not taking hormones had significantly lower lead concentrations in cortical bone compared to all women on hrt ( p = 0.007 ) . tibia lead content ( mean + /- sd ) for women on calcium only was 11.13 + /- 6.22 microgram / g bone mineral . for women on hrt , tibia bone lead was 19.37 + /- 8.62 micrograms / g bone mineral on low - dose hrt and 16.87 + /- 11.68 micrograms / g bone mineral on moderate - dose hrt . there were no differences between groups for lead concentrations measured in trabecular bone , whole blood , serum or urine . hormone replacement maintains cortical bone lead content . in women not on hrt , there will be a perimenopausal release of lead from bone.imagesfigure 1 .

we present a case of a patient with cardiac tamponade , who presented with symptoms six weeks after the initial blunt trauma to his chest . a 63-year - old male presented to the emergency department , short of breath and feeling generally unwell for 2 days prior to admission . six weeks prior to admission , he had fallen off a ladder and fractured 2 ribs in the right hemi - thorax . four weeks later he was seen by his gp feeling chesty and was treated with a short course of antibiotics for a suspected lower respiratory tract infection . two days prior to admission , the patient felt increasingly unwell , with the symptoms of being cold and clammy at times . he had right lumbar and right upper quadrant pain , with the latter being more dominant . on admission to the ed , he was hypotensive ( 70/50 ) , tachycardic ( 112 ) but maintaining his oxygen saturations in air ( 100% ) and was afebrile . chest and abdominal plain film x - rays demonstrated healing rib fractures , with no consolidation , pneumo - thorax or haemo - thorax . he remained slightly tender in his right upper abdomen and lumbar regions but looked generally unwell ( sweaty , pale and restless ) . as a result of his persistent hypotension , tachycardia , partial response to resuscitation and acidosis on the arterial blood gases , a ct scan was arranged . the ct findings were a moderate to severe pericardial effusion , with a fluid rim of approximately 2 cm , around the whole pericardium ( as shown in fig . 1 ) . ct slice showing pericardial effusion the effusion was drained under ultrasound guidance . his blood pressure improved dramatically and stabilised , the patient felt immediately better , his breathing eased , and his shortness of breath settled . this case study appears to be the first report of cardiac tamponade that presented 6 weeks after the initial blunt injury . a search on pubmed ( delayed cardiac tamponade blunt trauma ) gave 28 results , of which only 4 were reports of delayed cardiac tamponade as opposed to other pathologies . of these four a second case , presenting three weeks ( 2 ) after blunt trauma , was believed to have developed secondary the commencement of an anticoagulant treatment . the remaining two cases ( 3,4 ) were at 2 weeks and eight days respectively . it is therefore clear that this presentation at six weeks is rare . in this case , other causes ( cancer , uraemia , spontaneous cardiac rupture and recent heart surgery ) were excluded . this case does demonstrate the need to have an index of suspicion of cardiac tamponade in patients presenting with symptoms and signs of being unwell , even if the insult ( blunt chest trauma ) was sustained a few weeks prior to their presentation .

cardiac tamponade is a recognised complication of blunt trauma to the chest . it usually presents at the time of the acute event but there are rare cases of delayed presentations . we present such a case where the tamponade occurred six weeks following the trauma to the chest wall .

for many decades there has been advocacy for the use of calcium supplements in the prevention and treatment of osteoporosis . in the 1960s and 1970s , this was in the context of having no proven medications for osteoporosis management , a situation which has now changed dramatically . the rationale for advocating calcium is that it is one of the principal constituents of bone , though it should always be remembered that bone is a connective tissue , so its fundamental framework is a protein matrix ( type i collagen ) which is laid down by osteoblasts ( bone forming cells ) and remodelled and removed by osteoclasts ( bone resorbing cells ) . in adult life , this cell - mediated process of bone remodelling takes place at intervals across the skeletal surface , and involves the removal and replacement of small packets of bone . once osteoblasts have laid down new type 1 collagen , calcium and phosphate crystalize between the collagen fibres , providing bone with its compressive strength . thus , bone balance , or the change in bone density over time , is driven by the balance of activities of bone forming cells and bone resorbing cells . providing excess calcium and phosphate will not lead to the creation of more bone , since the amount of bone laid down is determined by the number and activity of osteoblasts . while an adequate supply of calcium and phosphate is important to ensure that bone laid down by osteoblasts is normally mineralised it is often assumed that higher calcium intakes result in greater bone density and fewer fractures . however , there are very few data to support this belief , and it is notable that calcium intake does not figure in any of the fracture risk calculators ( e.g. fracture risk assessment tool [ frax ] , garvan ) currently used . thus , cross - sectional studies that relate customary calcium intake to bone density show no relationship between these variables ( in the national health and nutrition examination survey [ nhanes ] the p value for differences in density across quintiles of calcium intake is 0.84 at the spine and 0.72 at the femoral neck ) and the same is true when fracture risk is related to calcium intake . many clinical trials have now been carried out to determine whether calcium supplements can improve bone density and reduce fractures . there is consistent evidence that the use of calcium supplements reduces bone turnover by about 20% , and this is associated with a reduction in postmenopausal bone loss . a study in which frail women living in institutions for the elderly were randomised to calcium plus vitamin d or to placebo demonstrated reduction in hip fracture risk but such positive results are not found in studies with community dwelling individuals . between 2005 and 2010 , six large randomised controlled trials of calcium ( with or without vitamin d ) in community - dwelling individuals were published - none of these trials showed statistically significant fracture prevention . recent meta - analyses confirm that calcium with or without vitamin d is not effective for the prevention of vertebral fractures ( odds ratio [ or ] for calcium alone 0.74 [ 0.45 , 1.12 ] , calcium+d or 0.99 [ 0.74 , 1.41 ] ) , non - vertebral fractures ( calcium or 1.00 [ 0.82 , 1.22 ] , calcium+d or 0.94 [ 0.84 , 1.02 ] or hip fractures ( fig . , the rationale for continuing to use calcium supplements for osteoporosis management is under question , and this questioning becomes even more acute when the possible side - effects of calcium supplements are considered . a number of observational studies have assessed whether there is a relationship between dietary calcium intake and cardiovascular health , and generally none has been found . however , randomised , controlled trials of calcium supplements have suggested beneficial effects on circulating lipid levels and small decreases in blood pressure . because of these observations , we hypothesised that calcium supplementation might decrease the risk of cardiovascular events , and this was a pre - specified secondary endpoint in the auckland calcium study . to our surprise , we found the opposite , observing a significant increase in the risk of myocardial infarction , and an upwards trend in risk of stroke . in light of this , and the absence of other published data on the subject , we contacted the authors of all of the major randomised , controlled trials of calcium supplements in older adults in order to obtain cardiovascular adverse event data . the protocol for this analysis was finalised before the data were provided to us , and the analysis confirmed the adverse effect of supplements on myocardial infarction , and also demonstrated an adverse but non - significant effect of these supplements on stroke risk . the women 's health initiative ( whi ) investigators also addressed this question in their large trial in which the intervention was calcium plus vitamin d. there were adverse trends in some cardiovascular endpoints , particularly in non - obese women , though the authors concluded that no significant effect was present . the whi differed from the trials included in our 2010 meta - analysis in that the intervention included vitamin d as well as calcium , the trial subjects were a decade younger , and more than half of them were already taking calcium supplements at the time they were randomised . we hypothesised that the high level of self - administration of calcium may have reduced the sensitivity of the study to observe an adverse effect of the supplements on cardiovascular risk , and proposed an analysis which would determine whether there was an interaction between calcium supplement use at the time of randomisation in the study , and the effect of the trial intervention on cardiovascular risk . this protocol was approved by the national institutes of health ( nih ; who held the database ) before the data were provided to us . analysis demonstrated that in calcium - naive women there was a significant adverse effect on myocardial infarction from randomisation to calcium plus vitamin d , whereas in those already taking calcium this was not observed . in fact , the results from the calcium - nave subjects in the whi mirrored very closely those from our previous meta - analysis of calcium supplements alone . in both datasets , the effects on myocardial infarction were seen within the first year of supplementation , whereas effects on stroke had a much slower onset ( fig . 2 ) . also , the size of the adverse effects on both events was very similar in the two datasets . when the data from the calcium - nave subjects in the whi was combined with all other trials of calcium with or without vitamin d , a 24.26% increase in the risk of myocardial infarction and a 15.19% increase in the risk of stroke as a result of these findings , together with the absence of any consistent evidence that high dietary calcium intake is deleterious to cardiovascular health , it is now generally recommended that we should obtain our calcium requirement from diet , rather than from the use of supplements . in spite of the consistency of the data presented in figure 2 and in the bolland meta - analyses , controversy persists regarding the adverse cardiovascular effects of calcium . at the 2013 american society for bone and mineral research ( asbmr ) meeting , a further meta - analysis was presented which confirmed our results for the use of calcium supplements alone . however , for the calcium plus vitamin d analyses , they chose to include the whi data from those women already taking calcium supplements ( even though we have demonstrated that their response to intervention is different from that of calcium - nave women ) and also data from the trial of larsen . this is a cluster randomised study ( i.e. not a true randomised , controlled trial ) in which uptake of the calcium plus vitamin d intervention was very low , and in which baseline cardiovascular risk was significantly different between those agreeing to take the calcium plus vitamin d intervention and those agreeing to act as control subjects . the addition of these two additional cohorts results in a relative risk of myocardial infarction of 1.09 which is no longer statistically significant , but still suggestive of an adverse effect which is greater than any benefit in terms of fracture prevention . thus , this controversial analysis still does not provide evidence of net benefit from taking calcium supplements . the publication of our meta - analyses has led to a large number of observational analyses assessing the relationship between calcium supplement use and cardiovascular risk . a number of positive and a number of negative analyses have resulted , reflecting the much lower power of observational studies to address such questions , and the problem of residual confounding which always makes interpretation of observational data difficult . it is noteworthy that the studies which have suggested a significant adverse effect of calcium supplements have not been balanced by a similar number of studies suggesting a significant benefit . thus , on balance , the observational data are generally consistent with the randomised , controlled trial findings . while there is no randomised , controlled trial of calcium supplements which has cardiovascular events as its endpoint ( and neither is there likely to be ) , there have been several other recent studies which reinforce the suggestion that calcium supplementation may have adverse cardiovascular effects . calcium supplements have long been used in patients with renal failure for their phosphate binding properties , including in patients not yet requiring dialysis . recently published a meta - analysis of trials comparing calcium - containing phosphate binders with other agents not containing calcium . she found that mortality was 22% lower in those using phosphate binders which did not contain calcium , and this meta - analysis was accompanied by an editorial entitled the demise of calcium based phosphate binders . this suggests that in patients with renal impairment , calcium supplements have a significant adverse effect on mortality , most of this mortality being cardiovascular . since many older patients at risk of osteoporosis have glomerular filtration rates < 60 ml / minute ( i.e. stage 3 chronic kidney disease [ ckd ] ) these findings are likely to be directly applicable to the population at risk of osteoporosis . the immediate biochemical consequence of taking a calcium supplement is an increase in circulating calcium levels , which persists for > 8 hours . the mirror image of these changes is produced by the infusion of a calcium chelator , such as the ethylenediaminetetraacetic acid ( edta ) infusions which are used in chelation therapy . this highly controversial intervention was recently the subject of an nih - funded trial which demonstrated an 18% decrease in risk of cardiovascular events . this suggests that lowering serum calcium is associated with decreased cardiovascular risk , and is consistent with a large body of observational data which has shown associations of circulating calcium levels with carotid plaque thickness , calcified plaque in the coronary arteries ( fig . , chelation therapy has the opposite effect to calcium supplementation on serum calcium levels , and this appears to be reflected in its opposite effects on cardiovascular event rates . the third recent piece of data relates to the finding that the use of the calcimimetic ion , strontium , is associated with a 60% increase in the risk of myocardial infarction . strontium is adjacent to calcium in the periodic table , binds to the calcium receptor , displaces calcium in hydroxyapatite crystals in bone , and probably can substitute for calcium in a wide range of other biological functions . thus , the finding that strontium increases myocardial infarction is entirely consistent with the trial data suggesting that calcium also has this effect . as discussed above , circulating calcium levels in the upper part of the normal range are associated with increased cardiovascular risk . the use of calcium supplements increases circulating calcium levels to the upper part of the normal range or above . therefore , these effects on circulating calcium concentrations are likely to mediate the adverse cardiovascular outcomes associated with calcium supplement use . the intermediary mechanisms remain to be determined , but the demonstration of associations between aortic , coronary artery and carotid artery calcification and circulating calcium levels suggest that direct effects on the vessel wall may be involved . studies of vascular cells in culture , have suggested that higher calcium concentrations increase calcification of the cultures , and result in changes in levels of calcification - regulating proteins in matrix vesicles . a further mechanism is suggested by our recent observation that calcium supplement use acutely increases blood coaguability ( bristow & reid , unpublished observation ) . this is consistent with in vitro evidence that blood coagulation is critically dependent on ambient calcium levels . thus , the hypercalcemia associated with calcium supplement use may increase coaguability of the blood , thus increasing the risk of coronary artery thrombosis . the whi demonstrated a 17% increase in the risk of kidney stones associated with randomisation to calcium plus vitamin d. for many years there has been a widespread awareness of the adverse gastrointestinal effects of calcium supplementation , and these agents are commonly associated with constipation and other symptoms of gastrointestinal distress . a recent clinical trial has demonstrated that the likelihood of being admitted to hospital with an acute gastrointestinal condition is doubled as a result of randomisation to calcium , and that this effect is large enough to completely abrogate any anti - fracture efficacy from the use of these agents . it is often assumed that higher calcium intakes result in greater bone density and fewer fractures . however , there are very few data to support this belief , and it is notable that calcium intake does not figure in any of the fracture risk calculators ( e.g. fracture risk assessment tool [ frax ] , garvan ) currently used . thus , cross - sectional studies that relate customary calcium intake to bone density show no relationship between these variables ( in the national health and nutrition examination survey [ nhanes ] the p value for differences in density across quintiles of calcium intake is 0.84 at the spine and 0.72 at the femoral neck ) and the same is true when fracture risk is related to calcium intake . many clinical trials have now been carried out to determine whether calcium supplements can improve bone density and reduce fractures . there is consistent evidence that the use of calcium supplements reduces bone turnover by about 20% , and this is associated with a reduction in postmenopausal bone loss . a study in which frail women living in institutions for the elderly were randomised to calcium plus vitamin d or to placebo demonstrated reduction in hip fracture risk but such positive results are not found in studies with community dwelling individuals . between 2005 and 2010 , six large randomised controlled trials of calcium ( with or without vitamin d ) in community - dwelling individuals were published - none of these trials showed statistically significant fracture prevention . recent meta - analyses confirm that calcium with or without vitamin d is not effective for the prevention of vertebral fractures ( odds ratio [ or ] for calcium alone 0.74 [ 0.45 , 1.12 ] , calcium+d or 0.99 [ 0.74 , 1.41 ] ) , non - vertebral fractures ( calcium or 1.00 [ 0.82 , 1.22 ] , calcium+d or 0.94 [ 0.84 , 1.02 ] or hip fractures ( fig . therefore , the rationale for continuing to use calcium supplements for osteoporosis management is under question , and this questioning becomes even more acute when the possible side - effects of calcium supplements are considered . a number of observational studies have assessed whether there is a relationship between dietary calcium intake and cardiovascular health , and generally none has been found . however , randomised , controlled trials of calcium supplements have suggested beneficial effects on circulating lipid levels and small decreases in blood pressure . because of these observations , we hypothesised that calcium supplementation might decrease the risk of cardiovascular events , and this was a pre - specified secondary endpoint in the auckland calcium study . to our surprise , we found the opposite , observing a significant increase in the risk of myocardial infarction , and an upwards trend in risk of stroke . in light of this , and the absence of other published data on the subject , we contacted the authors of all of the major randomised , controlled trials of calcium supplements in older adults in order to obtain cardiovascular adverse event data . the protocol for this analysis was finalised before the data were provided to us , and the analysis confirmed the adverse effect of supplements on myocardial infarction , and also demonstrated an adverse but non - significant effect of these supplements on stroke risk . the women 's health initiative ( whi ) investigators also addressed this question in their large trial in which the intervention was calcium plus vitamin d. there were adverse trends in some cardiovascular endpoints , particularly in non - obese women , though the authors concluded that no significant effect was present . the whi differed from the trials included in our 2010 meta - analysis in that the intervention included vitamin d as well as calcium , the trial subjects were a decade younger , and more than half of them were already taking calcium supplements at the time they were randomised . we hypothesised that the high level of self - administration of calcium may have reduced the sensitivity of the study to observe an adverse effect of the supplements on cardiovascular risk , and proposed an analysis which would determine whether there was an interaction between calcium supplement use at the time of randomisation in the study , and the effect of the trial intervention on cardiovascular risk . this protocol was approved by the national institutes of health ( nih ; who held the database ) before the data were provided to us . analysis demonstrated that in calcium - naive women there was a significant adverse effect on myocardial infarction from randomisation to calcium plus vitamin d , whereas in those already taking calcium this was not observed . in fact , the results from the calcium - nave subjects in the whi mirrored very closely those from our previous meta - analysis of calcium supplements alone . in both datasets , the effects on myocardial infarction were seen within the first year of supplementation , whereas effects on stroke had a much slower onset ( fig . 2 ) . also , the size of the adverse effects on both events was very similar in the two datasets . when the data from the calcium - nave subjects in the whi was combined with all other trials of calcium with or without vitamin d , a 24.26% increase in the risk of myocardial infarction and a 15.19% increase in the risk of stroke as a result of these findings , together with the absence of any consistent evidence that high dietary calcium intake is deleterious to cardiovascular health , it is now generally recommended that we should obtain our calcium requirement from diet , rather than from the use of supplements . in spite of the consistency of the data presented in figure 2 and in the bolland meta - analyses , controversy persists regarding the adverse cardiovascular effects of calcium . at the 2013 american society for bone and mineral research ( asbmr ) meeting , a further meta - analysis was presented which confirmed our results for the use of calcium supplements alone . however , for the calcium plus vitamin d analyses , they chose to include the whi data from those women already taking calcium supplements ( even though we have demonstrated that their response to intervention is different from that of calcium - nave women ) and also data from the trial of larsen . this is a cluster randomised study ( i.e. not a true randomised , controlled trial ) in which uptake of the calcium plus vitamin d intervention was very low , and in which baseline cardiovascular risk was significantly different between those agreeing to take the calcium plus vitamin d intervention and those agreeing to act as control subjects . the addition of these two additional cohorts results in a relative risk of myocardial infarction of 1.09 which is no longer statistically significant , but still suggestive of an adverse effect which is greater than any benefit in terms of fracture prevention . thus , this controversial analysis still does not provide evidence of net benefit from taking calcium supplements . the publication of our meta - analyses has led to a large number of observational analyses assessing the relationship between calcium supplement use and cardiovascular risk . a number of positive and a number of negative analyses have resulted , reflecting the much lower power of observational studies to address such questions , and the problem of residual confounding which always makes interpretation of observational data difficult . it is noteworthy that the studies which have suggested a significant adverse effect of calcium supplements have not been balanced by a similar number of studies suggesting a significant benefit . thus , on balance , the observational data are generally consistent with the randomised , controlled trial findings . while there is no randomised , controlled trial of calcium supplements which has cardiovascular events as its endpoint ( and neither is there likely to be ) , there have been several other recent studies which reinforce the suggestion that calcium supplementation may have adverse cardiovascular effects . calcium supplements have long been used in patients with renal failure for their phosphate binding properties , including in patients not yet requiring dialysis . recently published a meta - analysis of trials comparing calcium - containing phosphate binders with other agents not containing calcium . she found that mortality was 22% lower in those using phosphate binders which did not contain calcium , and this meta - analysis was accompanied by an editorial entitled the demise of calcium based phosphate binders . this suggests that in patients with renal impairment , calcium supplements have a significant adverse effect on mortality , most of this mortality being cardiovascular . since many older patients at risk of osteoporosis have glomerular filtration rates < 60 ml / minute ( i.e. stage 3 chronic kidney disease [ ckd ] ) these findings are likely to be directly applicable to the population at risk of osteoporosis . the immediate biochemical consequence of taking a calcium supplement is an increase in circulating calcium levels , which persists for > 8 hours . the mirror image of these changes is produced by the infusion of a calcium chelator , such as the ethylenediaminetetraacetic acid ( edta ) infusions which are used in chelation therapy . this highly controversial intervention was recently the subject of an nih - funded trial which demonstrated an 18% decrease in risk of cardiovascular events . this suggests that lowering serum calcium is associated with decreased cardiovascular risk , and is consistent with a large body of observational data which has shown associations of circulating calcium levels with carotid plaque thickness , calcified plaque in the coronary arteries ( fig . 3 ) , cardiovascular event rate , and mortality . thus , chelation therapy has the opposite effect to calcium supplementation on serum calcium levels , and this appears to be reflected in its opposite effects on cardiovascular event rates . the third recent piece of data relates to the finding that the use of the calcimimetic ion , strontium , is associated with a 60% increase in the risk of myocardial infarction . strontium is adjacent to calcium in the periodic table , binds to the calcium receptor , displaces calcium in hydroxyapatite crystals in bone , and probably can substitute for calcium in a wide range of other biological functions . thus , the finding that strontium increases myocardial infarction is entirely consistent with the trial data suggesting that calcium also has this effect . as discussed above , circulating calcium levels in the upper part of the normal range are associated with increased cardiovascular risk . the use of calcium supplements increases circulating calcium levels to the upper part of the normal range or above . therefore , these effects on circulating calcium concentrations are likely to mediate the adverse cardiovascular outcomes associated with calcium supplement use . the intermediary mechanisms remain to be determined , but the demonstration of associations between aortic , coronary artery and carotid artery calcification and circulating calcium levels suggest that direct effects on the vessel wall may be involved . studies of vascular cells in culture , have suggested that higher calcium concentrations increase calcification of the cultures , and result in changes in levels of calcification - regulating proteins in matrix vesicles . a further mechanism is suggested by our recent observation that calcium supplement use acutely increases blood coaguability ( bristow & reid , unpublished observation ) . this is consistent with in vitro evidence that blood coagulation is critically dependent on ambient calcium levels . thus , the hypercalcemia associated with calcium supplement use may increase coaguability of the blood , thus increasing the risk of coronary artery thrombosis . the whi demonstrated a 17% increase in the risk of kidney stones associated with randomisation to calcium plus vitamin d. for many years there has been a widespread awareness of the adverse gastrointestinal effects of calcium supplementation , and these agents are commonly associated with constipation and other symptoms of gastrointestinal distress . a recent clinical trial has demonstrated that the likelihood of being admitted to hospital with an acute gastrointestinal condition is doubled as a result of randomisation to calcium , and that this effect is large enough to completely abrogate any anti - fracture efficacy from the use of these agents . advocacy for the use of calcium supplements arose at a time when there were no other effective interventions for the prevention of osteoporosis . their promotion was based on the belief that increasing calcium intake would increase bone formation . our current understandings of the biology of bone suggest that this is not likely to occur . there is evidence that calcium acts as a weak antiresorptive , through its suppression of parathyroid hormone secretion . this is likely to be the mechanism that contributes to the slowing of postmenopausal bone loss with the use of calcium supplements . despite this , recent meta - analyses suggest no benefit from the use of calcium supplements in fracture prevention , and in fact there is evidence of adverse effects of calcium supplementation on hip fracture risk . in sum , there is little substantive evidence of benefit to bone health from the use of calcium supplements . against this needs to be balanced the likelihood that calcium supplement use increases the risk of cardiovascular events , formation of kidney stones , and gastrointestinal symptoms , including the risk of admission to hospital with acute gastrointestinal problems . thus , the balance of risk and benefit seems to be consistently negative . as a result , most organisations providing advice regarding optimisation of bone health , recommend that individuals should obtain their calcium requirement from diet in preference to supplements . dietary calcium intake has not been associated with the adverse effects associated with supplements , probably because calcium is provided in much less concentrated boluses , and these boluses are absorbed more slowly from the gastrointestinal tract since they come together with quantities of protein and fat , resulting in a slower gastric transit time . it is also important to note that we now have much more effective antiresorptive agents than calcium supplements , which prevent fractures and are safe in long - term use . therefore , osteoporosis prevention should centre on the quantitative assessment of fracture risk , and the targeting of appropriate fracture prevention therapies to those found to be at increased risk . as a result , calcium supplements have little role to play in the context of the modern therapeutics of osteoporosis .

advocacy for the use of calcium supplements arose at a time when there were no other effective interventions for the prevention of osteoporosis . their promotion was based on the belief that increasing calcium intake would increase bone formation . our current understandings of the biology of bone suggest that this does not occur , though calcium does act as a weak antiresorptive . thus , it slows postmenopausal bone loss but , despite this , recent meta - analyses suggest no significant prevention of fractures . in sum , there is little substantive evidence of benefit to bone health from the use of calcium supplements . against this needs to be balanced the likelihood that calcium supplement use increases cardiovascular events , kidney stones , gastrointestinal symptoms , and admissions to hospital with acute gastrointestinal problems . thus , the balance of risk and benefit seems to be consistently negative . as a result , current recommendations are to obtain calcium from the diet in preference to supplements . dietary calcium intake has not been associated with the adverse effects associated with supplements , probably because calcium is provided in smaller boluses , which are absorbed more slowly since they come together with quantities of protein and fat , resulting in a slower gastric transit time . these findings suggest that calcium supplements have little role to play in the modern therapeutics of osteoporosis , which is based around the targeting of safe and effective anti - resorptive drugs to individuals demonstrated to be at increased risk of future fractures .

they have a tendency to recur after initial treatment , including at sites outside the bladder such as the urethra . the urethra may be the site of recurrence after previous cystectomy but occur less frequently prior to cystectomy . patients with noninvasive disease in the bladder may prefer to keep their bladders and urethras to preserve normal voiding function . there have been a variety of treatments including urethral chemotherapy , resection , open surgery , as well as fulguration with the nd : yag or ho : yag laser to treat recurrence of uc . we describe a case wherein a distal urethral noninvasive uc was ablated using holmium laser technique ( ho : yag ) . the patient is a 83-year - old man with an history of noninvasive urothelial carcinoma 6 years prior to presentation previously treated with turbt and an induction course of intravesical of bcg therapy . he presented to the spinal cord injury unit after a cauda equina syndrome developed following lumbar spine surgery . due to his previous uc history , the initial office flexible cystoscopy demonstrated a noninvasive appearing uc covering approximately 50% of his pendulous urethra with frondular lesions . the patient was taken to the operating room for cystourethroscopy under anesthesia , biopsies and upper urinary tract evaluation . the upper urinary tracts were negative for lesions on retrograde pyelogram and bladder and urethral biopsies were obtained with similar pathology as seen on office cystoscopy . on pathology , the biopsies showed low - grade superficial urothelial carcinoma ( pta ) with a small focus of high - grade carcinoma [ figure 1 ] . surgical options were then discussed and the patient wanted a bladder sparing minimally invasive surgery . therefore , the patient was taken back to the operating room for a second look turbt and a staged fulguration of urothelial carcinoma with the holmium laser [ figures 2 and 3 ] . using holmium : yag laser vaporization through a 21-fr cystoscope , the lesions in the pendulous urethra were ablated using a 600 m fiber with settings of 8 hz and 1.0 joules ( 8 watts ) . a total time of 20 minutes was taken for the procedure with minimal discomfort postoperatively . mitomycin c was instilled ( 40 mg in 20 cc of sterile water ) for 1 hour immediately after the procedure . this was done due to his history of uc in the bladder as well as cells from the ablation may have been introduced into the bladder with the irrigation fluid . we subsequently performed the second stage of the procedure which included treatment of the other half of the lesions after one month once an office cystoscopy revealed there was no urethral stricture formation . after the second therapy he elected for bcg therapy and he has now been under surveillance for 18 months without cystoscopic evidence of a urethral recurrence and no formation of urethral stricture [ figure 4 ] . urothelial carcinoma of the pendulous urethra is exceedingly rare with minimal literature to provide accurate incidence . although a different scenario , urethral recurrence after cystectomy is approximately 9% which may give some insight into prevalence . while the primary treatment of recurrent uc is resection and or fulguration with or without the use of intravesical therapy , the efficacy in the urethra is unknown . we were concerned that resection would cause significant bleeding and the use of monopolar energy with the bugbee electrode would cause urethral stricture . therefore , we considered treatment with the holmium laser as it provides tissue destruction with a depth of penetration of about 0.5 mm but is very controlled . ho : yag laser therapy has been accepted as a means of fulguration in superficial bladder cancer . the holmium laser has also provided therapeutic use in urethral stricture disease . despite the use of holmium laser to treat lesions of the ureter , there have been no reports of diffuse fulguration of the urethra to determine if this may cause urethral stricture . previous reports suggest that using the ho : yag for ureteral fulguration of uc is effective and the stricture rate is less than 9% . the case was performed in a staged fashion and provided effective treatment without urethral recurrence .

urothelial carcinoma may occur anywhere in the urinary tract including the pendulous urethra . to prevent urethral stricture after resection and monopolor fulguration we describe the use of the holmium laser to fulgurate recurrent pta uc from the urethra . the surgical approach was staged and provided excellent long term results for management of superficial uc .

the who - unfpa has clearly identified maternal mental health as fundamental in attaining the millennium development goals . postnatal depression ( pnd ) is a significant public health issue , occurring during the perinatal period which is a time of intense change and transition for women . distinguishing between a natural response to motherhood and symptoms of pnd can be difficult both for new mothers and their families [ 2 , 3 ] . detection of and intervention in postnatal depression is crucial to the well - being of mothers , their infants , partners , and families . it occurs at a critical time in a mothers ' life and can persist for long periods . it can have adverse effects on partners and on emotional and cognitive development of infants and children [ 46 ] . public health nurses ( phns ) all over the world have a major role in supporting families with new born babies , and a key concern for public health nursing is the framework provided by the millennium development goals which includes improving maternal health . many cases of postnatal depression are not detected as there is no international agreement on screening for postnatal depression . there are opinions that the screening instruments do not meet the who criteria for when screening should be performed . the marc society for perinatal mental health is an international society for the understanding , prevention , and treatment of mental illness related to childbearing . there is a growing view within the society in favour of undertaking universal psychosocial assessment in perinatal women , as long as it takes place within an integrated care model . ireland and norway have many similarities from a geographic , demographic , and public health care model and public health nursing perspectives . the phn is the primary health care professional providing care to women in the postnatal period in both ireland and norway . the european academy of nursing science ( eans ) is a forum for connecting nurse / midwife scientists within europe through scholarship and research . it offers opportunities to test innovative ideas , pool expertise , and strengthen research capacity in line with the objectives of the european research area . researchers may collaborate across participating countries on any subject which demonstrates a need for international cooperation . to draw attention to important common challenges for nurses , the authors met at an eans conference in the summer of 2012 and identified a need to collaborate and strengthen research capacity and discussion on postnatal depression as a public health nursing issue in both countries . the aim of this paper is to present a discussion on the concept of pnd , prevalence , and outcomes ; similarities and differences in public health and public health nursing models in ireland and norway ; research evidence on identification and screening issues for phns ; and the benefits of social support in facilitating recovery for new mothers . whilst it is acknowledged that other health care professionals such as midwifes , social workers , and psychologists also contribute to care of women with pnd , the focus of this paper is on the role of the phn . this paper will contribute to the discourse on pnd and phns contribution in identification and treatment in the context of primary health care internationally . pnd in women usually occurs 46 weeks after birth , and international studies find that between 8% and 15% of mothers are affected by this condition [ 1215 ] . however , in some studies , the prevalence of postnatal depression ranges from zero to almost 60% , and the prevalence rates vary across and within countries , from as low as 4.4% at 12 months to as high as 73.7% . in some countries , there are few reports of pnd , whereas in other countries reported postnatal depressive symptoms are very prevalent . prevalence rates reported from ireland have also varied from 11.4% to 28.6% with the most recent study with first - time mothers reporting prevalence rates of 13% at 6 weeks and 10% at 12 weeks . these figures indicate a serious clinical issue for phns providing postnatal care to new mothers in the community . there may be many reasons for this variation in prevalence which include using different screening assessments , using varying cut - off scores ( 1013 ) on the edinburgh postnatal depression scale ( epds ) , assorted timescales ( 612 weeks postpartum ) , and different samples . for example , one study included a high representation of a sample of mothers with previous history of depression . however , it is well documented that postnatal depression affects at least 10% to 15% of all mothers within the first postpartum year [ 2 , 3 , 16 , 25 ] . thus , several thousand women are affected by this condition each year and this should be an important issue for public health services . this condition has well - documented health consequences for the mother , child , and family . women who have pnd are significantly more likely to experience future episodes of depression , and infants and children are particularly vulnerable because of impaired maternal - infant interactions and significant cognitive and emotional development [ 3 , 5 ] . the nature and symptoms of pnd are characterised by tearfulness , fatigue , anxiety , despondency , and excessive anxiety over the baby . an indication of pnd is a low mood that causes every day to be experienced as heavy and grey . some women experience loss of control over their existence , which can lead to an increasing feeling of unease , irritability and outbreaks of anger , inability to cope , and thoughts of suicide . depressed mothers report higher parenting stress than nondepressed mothers [ 26 , 27 ] , and maternal depressive symptoms might also contribute to unfavourable parenting practices which can adversely affect child growth and development and thus a concern for phns . ireland and norway have many similarities from a geographic and demographic perspective and both have a strong commitment to primary care and public health . both countries have similar sized populations , but economically there are differences in relation to poverty , life expectancy is lower , and inequalities are higher in ireland . the public health system in ireland is a two - tier system where public and private sectors exist and is governed by the health act of 2004 . following this legislation , the health service executive was established and is responsible for providing health and personal social services to the population . the public health system has a number of on - going issues which could have an impact on primary care services . these include long waiting lists ; over capacity on hospital beds ; patients awaiting admission on trolleys in the emergency departments ; moratorium on staff recruitment leading to staff shortages . ireland 's two - tier health care system has failed in many respects to deliver adequate , fair , and equitable services to meet people 's needs . not all citizens in ireland have free health care at the point of delivery as it is based on income . many health care payment schemes operate such as the general medical services ( gms ) card , pay related social insurance ( prsi ) , and drug payment scheme . nearly 40% of the population are covered by a medical card or a gp visit card . mental health services have not been prioritised by government and the quality of services lag behind international best practice . there is an ongoing recognition for the need for a shift from the medical model and in - patient treatment to a holistic model of care with recovery and community services at its core [ 33 , 34 ] . in contrast to ireland , norway has universal health care for its entire population and free health care at the point of delivery . the norwegian government has recognized the need for public health services to address mental health issues for women during pregnancy and after childbirth and acknowledges that well - child clinics are an especially suited arena for preventive mental and social work . in both the women 's health strategy in st . ( 2002 - 2003 ) and the government 's strategic plan for the mental health of children and adolescents is the commitment to expand and strengthen support for women in this period of their lives . there is also a wish to increase research on women 's mental health during pregnancy and birth , which also reflects the ethos of the vision for change strategy document in ireland . in a recent report from australia , perinatal depression is estimated to cost the australian economy $ 433.52 million in 2012 , in financial costs only ( $ 4,509 per person with perinatal depression ) . in addition to the financial costs , perinatal depression equates to a loss of 20,732 disability - adjusted life year dalys in 2012 , which represents a significant disease burden . there are no comparable figures available for ireland and norway , but it is reasonable to assume similar costs to their economies . guidelines for treatment of postpartum mental disorders are lacking in both ireland and norway [ 33 , 40 , 41 ] , and resources have not been increased either in norway [ 36 , 42 ] or in ireland . furthermore , hospital stay for women after delivery has been dramatically shortened in the last decades , from previous 57 days to currently 1 - 2 days . since primary health care has not received the required amount of resources [ 33 , 43 ] , support for new families is significantly impaired . there is need for clinical nursing service improvement both from a resource and evidence based perspectives specifically for the identification and management of pnd . in ireland and norway , public health nurses ( phns ) are geographically based and provide a nursing service to new mothers and their infants in the community . ireland has generalist public health nurses , which means they care for all persons within their defined geographic area from the cradle to the grave . in contrast , phns in norway are specialists and are responsible for preventive services provided to infants , children , adolescents , and their families . maternity services are free which entitles every woman to general practice ( gp ) and hospital obstetric services . in general , midwives are employed to work in the hospital system with some regions having minimal community based service for up to 10 days postpartum . the work of phns consists of health promotion and primary prevention , which means promoting mental and physical health as well as good social and environmental conditions and preventing disease , injury , and disability [ 44 , 46 ] . phns in ireland are mandated to visit all new mothers within 48 hours of discharge from hospital , and similar to phns in ireland are mandated to visit all new mothers within 48 hours of discharge from hospital , and similar to phns in norway who offer home visits within the early weeks after birth and attendance at well baby clinics until the child is four years or school going age . given the short length of stay at the maternity wards , this home visit is especially important to support the new family . support and information from the phn at the home visit can have a preventive effect on depressive symptoms in postpartum women [ 20 , 47 ] . on a very basic level , norway has far more phns devoted specifically to public health issues , with one client group , compared with phns in ireland providing services to all client groups with a preventative and curative remit . in norway , there are 2069 phns employed in municipal family health clinics and school health services , and in ireland there were 1702 phns employed in the irish health service executive . phns in both countries have the most contact with mothers in the postpartum period and therefore are in a prime position to assess for postnatal depression and facilitate and help mothers to mobilise support from their social network and also to provide support when none are available . in norway , recent reports suggest that there is not enough research of satisfactory quality available to give recommendations for how to work with pnd in the municipalities [ 8 , 48 , 49 ] . in february 2013 , the national council for priority setting in health care in norway recommended that screening for postnatal depression should not be introduced on a national basis at the present time . the decision was based on that the epds screening does not meet the who criteria for when screening should be performed . however , the recent position paper by the marc society recommends undertaking universal psychosocial assessment in perinatal women , as long as it takes place within an integrated care model . in ireland , recommendations are made for interventions to address pnd which may have a wide range of socioeconomic benefits , extending well beyond the impact of the intervention on the mother . screening for pnd is currently not a routine component of the phn postnatal visit , and thus , many women may not be assessed . there is growing evidence that pnd can be effectively treated and possibly prevented [ 27 , 5153 ] . however , according to dennis it is still undetected or untreated in many women . although a number of tools ( essentially self - report questionnaires ) have been developed for the detection of depression , only eight studies assess their use in the postnatal period . only one of these , the edinburgh post depression scale ( epds ) , has been used in a sufficient number of studies to make a judgement on its usefulness . recent studies [ 14 , 25 , 27 , 28 , 51 , 53 ] indicate that epds can be a useful tool to detect pnd in women . cox et al . developed this self - rating scale for detecting depressive symptoms among women who have just given birth . the scale considers the intensity of depressive symptoms that are present in the previous seven days . epds has been used both in clinical settings and in epidemiological studies and is generally well accepted by women [ 56 , 57 ] . although the sensitivity and specificity vary across languages and cultures , the sensitivity and specificity of the epds have been satisfactory in several studies [ 2 , 15 , 21 , 58 ] . the form is described as a reliable screening tool and has been recommended for screening of postnatal women [ 15 , 59 ] . there has been much debate in the literature as to the suitability of using the epds in clinical practice for screening for pnd . this reluctance is primarily related to the epds having reasonable sensitivity but lower specificity , and thus , positive predictive value is poor . this means that many women who do not have pnd are being told of the possibility that they have the condition and then could be subject to further investigation , placing an increased and wasteful burden on resources . however , it is important to be aware that the epds is a screening instrument that indicates the possible presence of depression and not a diagnostic tool . to determine a clinical diagnosis of pnd , it is necessary to use the epds , followed by a clinical assessment and an interview . thus , the clinical assessment done by the phn after the epds is decisive of further followup . phns have described epds as a door opener for talking to new mothers about their mental health . according to seeley , question has also demonstrated poor positive predictive value . nonetheless , the current nice guidelines recommend using them . although little specific evidence exists for their use in the perinatal period , their ease of use and reasonable sensitivity and specificity , particularly if combined with the additional help question from arroll et al . the questions are simple screening methods which can detect postnatal depression and lead to a subsequent referral for a full clinical assessment followup . this screening technique is an opportunity to screen without the need for a more formal assessment . however , all postnatal depression screening and assessment must be combined with a treatment chain and systematic referral procedures [ 2 , 10 , 64 ] . public health nurses have the most contact with mothers and new babies in the postpartum period and therefore are in a prime position to assess for pnd and provide support . according to negron et al . , it is important to identify social support resources needs of new mothers to facilitate their transition to motherhood and recovery after childbirth . international and national policy documents suggest that social support is necessary for maternal and infant well - being and facilitates women 's transition to motherhood . in previous research , mothers in the postnatal period have reported help received from their partners and mothers , both with household chores and infant care , to be of great importance to them . providing support for mothers in caring for their infants in the postnatal period is an important concern for nurses in the community , because research has shown that social support can facilitate women 's transition to motherhood , some of whom find the transition psychologically stressful . furthermore , previous research has indicated that social support from partners , maternal mothers and peers , and home visits from nurses [ 22 , 68 ] have reduced postnatal depressive symptoms . within the irish context , given the importance of social support in facilitating transition to motherhood , leahy - warren conducted research with first - time mothers ( n = 99 ) exploring the relationship between social support and confidence in infant care practices at 6 weeks postpartum . findings revealed that support in the guise of mothers ' receiving positive affirmation with caring for their infant had a significant influence on their confidence in caring for their infants . mothers ' revealed that the sources of this type of support were their partners and own mothers . results also showed that public health nurses and maternal mothers were the primary source of informational support . therefore , it is essential that nurses facilitate the identification of individual mothers ' sources of support and continue to provide them with information that is relevant and appropriate . a more recent irish study examined the relationship between postnatal depression , maternal parental self - efficacy ( confidence ) , and postnatal depression during the first 3 months postpartum with a large sample of first - time mothers ( n = 512 ) [ 14 , 50 ] . the results showed that at 6 weeks , significant relationships were found between functional social support and postnatal depression and informal social support and postnatal depression . this means that support received from mothers ' partner , own mother , family , and friends positively influenced postnatal depressive symptoms at 6 weeks . the types of support that were significant were informational , instrumental ( hands - on help ) , emotional ( caring ) and appraisal ( positive affirmation ) . findings also revealed that the higher the level of maternal parental self - efficacy ( confidence ) the lower the level of depressive symptoms . this means that mothers who have confidence in their own ability to care for their infants are less likely to have postnatal depressive symptoms . nurses need to be aware of and acknowledge the significant contribution of social support , particularly from family and friends in positively influencing first - time mothers ' mental health and well - being . the best predictors of postnatal depression at 12 weeks were at - birth professional support and emotional support . what this means is that mothers who received low levels of professional support at birth were 3.24 times more at risk of pnd at 12 weeks than mothers who received high levels of professional support . furthermore , there was an elevated risk ( 2.92 times ) of pnd at 12 weeks in mothers with low emotional support , compared with those who received high emotional support at birth [ 18 , 44 ] . in a study with first - time mothers ( n = 271 ) , when their babies were 3 months old , tarkka et al . showed that social support and support from public health nurses were important factors in first - time mothers coping with child care . similar findings were reported from taiwan , where findings revealed that nursing interventions enhanced women 's ( n = 122 ) social support and decreased their pnd . razurel et al . interviewed 60 women six weeks after the birth of their first child . the new mothers expressed the need to be supported and counselled when problems arose and regretted the lack of long - term postpartum support . compared the prevalence of depression in the postpartum period and its relationship with perceived stress and social support in first - time mothers and fathers . in this cross - sectional study with a sample of 130 pairs of parents , they found that perceived stress , social support , and partner 's depression were significantly associated with depression in new mothers and suggest that counselling , support , and routine screening for depression should be provided to both mothers and fathers . a qualitative study using focus groups of women ( n = 33 ) participating in a postpartum depression randomised controlled trial explored their experiences of social support in the postpartum period . one of the main themes identified were mothers ' major needs and social support expectations including providers of social support . mothers indicated that support from partners and family was expected and should be provided without asking . furthermore , findings indicated that identifying support needs and expectations of new mothers is critical for mothers ' recovery after childbirth . these findings signify the need for public health nurses to be mindful of the importance of support for mothers in the early postnatal period . recovery can be facilitated by helping mothers identify the types of supports they need and who is best from their social network to provide specific supports . phns can contribute by facilitating and helping mothers to mobilise support from within their social network . glavin discusses a model for prevention , identification , and treatment for pnd in a norwegian municipality . the phns in the intervention municipality undertook specific training related to pnd [ 27 , 52 ] . in this study , 2227 women participated , 437 in the control group and 1790 in the intervention group . at the home visit two weeks postpartum , the phns in the intervention group gave information ( both written and oral ) about pnd and encouraged the mother to contact the well - child clinic before the first appointment if she felt depressed . a significant difference in pnd symptoms this indicates that information and support can prevent some cases of pnd with better outcomes for maternal and infant health and well - being . the phns in the intervention municipality used the epds followed by a clinical assessment and an interview to assess all mothers for pnd at six weeks postpartum . the assessment was followed up by supportive counselling sessions with a phn for women in need of that . a total of 228 women , 64 in the control group and 164 in the intervention group , had an epds score 10 at six weeks . the women who received supportive counselling sessions showed a significant decrease in depression score compared to the usual care group up to 12 months postpartum . [ 27 , 47 , 74 ] showed an effect on depressive symptoms among depressed women as well as among the nondepressed up to 12 months after delivery , and the results are supported by other studies [ 51 , 53 ] . in a prospective cluster trial , randomized by gp practice with 1474 interventions and 767 control women , morrell et al . and brugha et al . used epds and trained health visitors to assess for pnd and give supporting counselling sessions to mothers in need of that . they also reported a decrease in the pnd scores among women who received support from health visitors . a review including fifteen trials , involving over 7600 women , dennis and creedy reported that home visits after birth by public health nurses or midwives helped to prevent pnd . thus , several studies indicate that support from the phn may have a preventive effect on pnd in women . the prevalence of pnd at 1015% is a serious public health issue and consequently a public health nursing clinical concern in the community . the adverse consequences of pnd for mothers and their families necessitate the need for phns to identify those at risk . public health care in the guise of primary care in ireland and norway is an ideal integrated model of care in the community in which universal screening could be achieved by phns with appropriate and adequate resources . research evidence has demonstrated the significant beneficial effects of phn support visits and facilitation and mobilisation of social supports from mothers ' social network . priority needs to be given at a strategic level in both countries to resource a perinatal mental health strategy , embedded in public health policy to ensure that universal psychosocial assessment in perinatal women is undertaken within an integrated care model . this needs to be considered given the benefits to the individual , the family , the community , the health care profession , and financial costs to each country .

the framework provided by the millennium development goals includes maternal health as an area of priority . postnatal depression ( pnd ) is a serious public health issue because it occurs at a crucial time in a mothers ' life , can persist for long periods , and can have adverse effects on partners and the emotional , behavioural , and cognitive development of infants and children . internationally , public health nurses ( phns ) are key professionals in the delivery of health care to mothers in the postpartum period , and international research collaborations are encouraged . two researchers from the european academy of nursing science ( eans ) identified a need to collaborate and strengthen research capacity and discussion on postnatal depression , a public health nursing issue in both countries . within the context of public health and public health nursing in ireland and norway , the aim of this paper is to present a discussion on the concept of pnd , prevalence , and outcomes ; screening issues for phns ; and the research evidence of the benefits of social support in facilitating recovery for new mothers .

alopecia areata ( aa ) is considered as a chronic and common inflammatory disease that affects mostly hair follicles along with nails involvement in few cases . this disorder , which is characterized by the sudden hair loss on the scalp or other hair bearing areas , is seen in both sexes , at all ages . various factors , including genetic , endocrine , immunologic , infectious , spiritual , and psychological factors are supposed to have a role in its pathogenesis . the role that psychological factors play in the pathogenesis of aa has been the subject of discussion for decades . numerous studies have been done in relation to this matter that has resulted in controversial outcomes . the objective of our study was to investigate the severity of anxiety and depression in patient with aa . this case - control study , was carried out on 68 confirmed patients ( > 18 years old ) suffering from aa ( on scalp ) referred to the skin clinic , affiliated to hormozgan university of medical sciences ( bandar abbas , iran ) . sixty eight healthy people from general population selected as control , matched with the case group by age , sex and the level of education . informed consent was obtained , and according to severity of disease on scalp , patients were divided into five groups ( s1 : 0 - 25% , s2 : 26 - 49% , s3 : 50 - 74% , s4 : 75 - 99% and s5 : 100% ) based on olsen / canfield criteria . according to the location of the disease onset , the patients were classified into four groups as follow : temporal , occipital , frontal and multifocal . finally , from the view point of education level , the participants were grouped as primary , secondary and higher education . severity of anxiety and depression were assessed respectively by the use of persian versions of hamilton anxiety rating scale ( ham - a ) and hamilton depression rating scale ( ham - d ) . t - student , analysis of variance and chi - square tests were used to compare the groups . logistic regression was applied to estimate odds ratios of the disease for each of the outcome variables . the age range for the cases was from 19 to 64 with average of 35.47.6 years and for the control group was 20 to 63 with average of 33.88.1 years . when compared the two groups , no significant difference regarding age and sex could be observed . the mean of disease duration in men and women was 22.7 and 20.8 months , respectively . based on ham - a , the mean of anxiety scale in case group was significantly more than that of the control group ( 12.767.21 vs 8.546.37 ; p=0.003 ) . moreover , according to ham - d , there were 38 cases ( 56% ) suffering from a degree of depression . the mean of depression scale in case group was significantly more than the control group ( 12.844.03 vs 6.224.95 ; p=0.001 ) . table 1 demonstrates the frequency of anxiety and depression severity in case and control groups based on sex . the severity of anxiety and depression was significant in both the groups ( p<0.05 ) . odds ratios ( or ) were calculated using a bivariate logistic regression model in order to measure the strength of the relationship between the disease and depression or anxiety . results show that , patients with aa are exposed to depression approximately five times more than normal people ( or=4.48 ; 95%ci : 2.12 - 9.44 ) . however , the odds ratio of anxiety originated from aa was 2.72 ( 95%ci : 1.30 - 5.68 ) . table 1 shows the severity of anxiety and depression in the case and control groups based on sex . a degree of anxiety was observed in 44.9% of men and 52.7% of women in case group compared with 16.3% of men and 42.1% of women in control group . moreover , 53.1% of men and 63.2% of women in case group and 18.4% of men and 31.6% of women in control group were suffering from depression . mean scores for anxiety and depression were significantly different in the case and control groups ( p=0.003 ) . in this study , comparison of prevalence , severity of anxiety , and severity of depression between the two groups revealed no significant difference with respect to age , whereas a highly significant difference was observed from the education level s point of view . table 2 shows the prevalence of anxiety of case and control groups based on education level . the prevalence of anxiety and depression was significantly different between the case and control groups considering the education level in two levels of primary and secondary education ( p - values<0.05 ) ( table 2 ) . regarding the means of anxiety and depression when it comes to the level of severity of the disease ( table 3 ) , the results showed that there was significant difference in anxiety and depression mean scores based on the severity of disease ( p - values<0.05 ) . no significant relationship was observed concerning the mean of anxiety and depression and the onset location of the disease . likewise , there was no significant correlation between duration of the disease , and the severity of anxiety or the severity of depression . alopecia areata , which can be considered as a triggering factor in psychological disorders such as anxiety and depression , have been the subject of many previous studies , with contradictory results . the present study revealed that psychological disorders are prevalent in patients with aa ; this is in accordance with the results of other studies carried out on adults with aa . however , yasemi and colon in their studies had observed no difference between case and control groups . despite the fact that aa patients compared to the general population are more suffering from anxiety and depression , the disorder may not confirm the etiological role of psychological factors , it suggests that anxiety and depression are named as comorbid mental disorders . this aspect is matched with the perspective of sadock et al . about the role of psychological factors involved in this disease . they stated that , although the role of stressful events in onset and aggravation of aa is not clear , still comorbid mental disorders have been seen . psychological diagnoses such as substantial depression , generalized anxiety disorder and paranoid disorders have been reported in many cases . in our study , like firooz who reported no relationship between the concerns of patients about their disease and their age , there was no significant difference between prevalence , severity of anxiety and severity of depression in aa patients and the control group when discussing the age . whereas , safa reported that the number of severe cases of depression in 16 - 25 age - group was more than other age - groups . this difference might be due to differences in age groups or lack of control group in safa study . in our study these findings are in accordance with results of other studies that indicated a higher prevalence of psychiatric disorders among females with aa . the researcher suggested that it was due to higher rate of aesthetic stress for women . nevertheless , firooz found no relationship between patients concerns about the outcomes of the disease and sex . definitely , to study the role of sex in development of mental disorders in aa patients , considerable number of cases is required to be studied and investigated . our findings also showed a significant statistical difference between those with primary and secondary education level concerning the prevalence of anxiety and depression . nevertheless , safa and firooz showed that there was not a significant relationship between education level and psychological disorders in the patients . in line with many published studies , our findings revealed a significant difference in average anxiety and depression scores based on extent of the disease . this finding may confirm that stressful events in life can be considered as an accelerating factor for the onset of aa , as manolache in a study on 114 aa patients concluded that trauma and acute anxiety were the most common causes of aa . no significant relationship was observed between neither the onsite location of the disease and anxiety nor the onsite location and depression . in our patients , there was not a significant relationship between average anxiety and depression scores and duration of the disease . however , firooz pointed out the direct relationship between increased patient s concerns with that of the increased duration of disease . although the fact that aa patients compared to the general population are more suffering from anxiety and depression disorders , but it may not confirm the etiological role of psychological factors , it suggests anxiety and depression as comorbid mental disorders .

alopecia areata ( aa ) is a chronic disease which esthetic outcomes may result in deep effects on mental disorders of patients . in this case - control study , we compared the mental health of 68 patients diagnosed with aa with 68 healthy individuals using hamilton anxiety and depression rating scales . there were significant differences between the case and control groups regarding the prevalence of anxiety and depression . the means of anxiety scores in cases and control group were 12.767.21 vs 8.546.37 , p=0.003 . likewise , the means of depression scores for the groups were 12.844.03 vs 6.224.95 , p=0.001 . further - more , patients with aa were exposed to depression approximately five times and to anxiety about three times more than normal people . our study revealed a high prevalence of anxiety and depression in aa patients . dermatologists should pay more attention on psychological effect of the disease on the patients .

synovial sarcoma is a malignant tumor of pluripotent mesenchymal cells,1 2 3 4 most commonly found in periarticular areas , with few cases occurring in the head and neck.1 2 3 4 5 the first synovial sarcoma was described by jernstrom in 1954.6 in this article , we present a case of synovial sarcoma in the posterolateral wall of the oropharynx . the patient , a 23-year - old woman , was admitted to the otorhinolaryngology clinic at our hospital with a history of dysphagia and difficulty of breathing for 8 months , resulting in progressive worsening and emergence of snoring , muffled voice , and local pain . physical examination revealed an oropharyngeal tumor in the left posterolateral wall near the base of the tongue . 1 ) showed a rounded submucosal tumor with intact mucosa , starting at the left posterolateral wall , touching the epiglottis down to the left , narrowing the light of the entire pharynx , preventing full view of the glottis . cervical magnetic resonance imaging ( figs . 2 , 3 , and 4 ) showed a solid cystic lesion in the submucosal left posterolateral wall of the oropharynx of relatively delimited contour , reducing the airway and rejecting the left piriform sinus . the mass measured 5.3 3.9 2.2 cm in its greatest axis . its left lateral region was predominately solid , showing heterogeneous impregnation of paramagnetic contrast , and measured 2.1 2.0 cm , with the cleavage plane delimited by prevertebral muscles . lymph nodes measured up to 1.3 cm in internal high jugular chains and submandibular ( ib and iia ) on the right . preoperative laryngoscopy showing the mass at the hypopharynx . nuclear magnetic resonance , axial view . nuclear magnetic resonance , coronal view . nuclear magnetic resonance , sagittal view . pathologic examination revealed a synovial sarcoma with positive margins , and mohs technique was proposed for edge control . a full pharyngectomy with ipsilateral modified radical neck dissection was proposed , with the possibility of full laryngeal pharyngectomy depending on surgical margins obtained intraoperatively by freezing anatomy . during surgery , the margins came initially committed , and after expansion , the margins were disease - free . we also performed the proposed neck dissection , tracheostomy , and passage of a nasogastric tube for nutrition . postoperatively , the patient 's condition was stable . upon follow - up with an oncologist , a gastrostomy was performed for nutrition , as oral feeding was impossible . at swallowing endoscopy all mobility of the larynx was preserved . within 1 year postoperatively , the patient continues with locoregional control without recurrence and performs pharyngoesophageal dilatations to treat actinic stenosis . the tracheostomy tube is closed and will be removed as soon as oral feeding is stabilized . despite its name , synovial sarcoma rarely originates in the synovial membranes.7 it is most commonly found in the vicinity of large joints . occurrence in the head and neck , a location poor in synovial tissue , is uncommon.8 synovial sarcoma comprises 8 to 10% of all soft tissue sarcoma,1 3 5 7 9 and it is estimated that 3 to 10% of sarcomas occur in the head and neck.4 5 10 these tumors can be located in the prevertebral space from the base of the skull to the hypopharynx , retropharyngeal and parapharyngeal spaces , and anterior neck along the edges of the sternocleidomastoid muscle , as well as sites in the oropharynx and larynx.8 10 synovial sarcomas show a male predominance ( 3:2 ) and predilection for patients between 25 and 36 years of age.4 11 12 this type of tumor usually appears as an asymptomatic mass until it acquires sufficient volume to cause compressive effects on neighboring structures.1 it can be a painless mass or associated with symptoms such as pain , earache , sore throat , and bleeding . it is important to include laryngoscopy and head , neck , and chest computed tomography to define the local extent of disease and metastasis.4 resection with negative margins remains the foundation of therapy,7 12 which is not so easy to achieve in the head and neck.4 5 local excision is followed by high recurrence rates ( 60 to 90% ) , usually within 2 years.4 5 9 metastasis to regional lymph nodes of synovial sarcomas in the head and neck is not seen often , although there can be clinically enlarged lymph nodes.5 most metastasis originate from hematogenous dissemination , although up to 20% spread through the lymphatics to regional lymph nodes.4 8 cervical lymph node dissection is not routinely performed , except in the presence of enlarged lymph nodes.8 10 hematogenous dissemination is the harbinger of death . pre- and postoperative radiotherapy , with or without chemotherapy , may be responsible for the increase in survival statistics over the last 2 to 3 decades , but studies are still controversial and inconclusive.4 7 9 despite relatively slow growth , long - term survival for synovial sarcomas from anywhere , including the head and neck , is not good.5 synovial sarcoma in the head and neck has an aggressive nature and a reserved prognosis.3 8 several factors have been evaluated for prognosis , and the most significant were tumor size and deep extension at the time of primary treatment,9 with survival varying inversely with these two factors.5 the age at diagnosis seems to have involvement with prognosis,9 although the relative youth of patients with synovial sarcoma of the head and neck does not seem to give a significantly better prognosis.5 synovial sarcoma is a rare malignant tumor which treatment is essentially surgical resection with high rates of relapse . although traditional treatment for hypopharynx tumors has been laryngectomy , it can be modified for sarcomas at this area . in these cases , radiation therapy with or without chemotherapy appears to be responsible for the increased survival in recent decades . it is important for the otolaryngologist and head and neck surgeon to be familiar with this aggressive tumor , which carries high mortality and morbidity .

introduction synovial sarcoma is a malignant tumor of mesenchymal pluripotent cells . objectives we present a case of synovial sarcoma in the posterolateral wall of the oropharynx . resumed report the patient , a 23-year - old woman , was admitted with a history of dysphagia and difficulty in breathing for 8 months , resulting in progressive deterioration and onset of snoring , muffled voice , and local pain . an oropharyngeal tumor in the left posterolateral wall touched the base of the ipsilateral tongue . the patient underwent endoscopic pharyngectomy to remove the lesion . pathologic examination revealed synovial sarcoma with positive margins , and mohs technique was proposed for margin control . the margins were disease - free , without the need for total laryngectomy . the pharynx was reconstructed with a microvascular forearm flap . the patient developed postoperative stability . conclusion despite its name , synovial sarcoma is rarely sourced directly from synovial membranes . it is most commonly found in the vicinity of large joints . the location at the head and neck , a location poor in synovial tissue , is unusual . synovial sarcoma in the head and neck has an aggressive nature and poor prognosis . resection with negative margins remains the foundation of therapy , which is not so easily achieved in the head and neck . it is important for the otorhinolaryngologist and head and neck surgeon to be familiar with this aggressive tumor , which carries high mortality and morbidity . the appropriate diagnosis and treatment can improve prognosis and patient survival .

dexmedetomidine , an -2 receptor agonist , induces light to moderate sedation without causing respiratory depression and has emerged as an alternative to standard sedation therapy , especially during weaning from mechanical ventilator support . many of the commonly utilized sedative agents in the intensive care unit ( icu ) , including benzodiazepines and propofol , modulate the gamma - aminobutyric acid system and are known to produce deeper levels of sedation . these agents have also been associated with a number of adverse effects including systemic accumulation , delirium , and respiratory depression . large , randomized trials comparing dexmedetomidine to benzodiazepines have associated dexmedetomidine with increased number of days alive without coma and a larger percentage of time spent at goal sedation . based on these characteristics , dexmedetomidine use has been increasing in the critical care setting , and this trend has been further augmented by recurring propofol shortages . despite the potential clinical benefits , dexmedetomidine is associated with alterations in hemodynamic stability . by agonizing presynaptic -2a receptors , dexmedetomidine produces sedation by decreasing plasma norepinephrine concentrations and may result in clinically significant bradycardia and hypotension . at present , the food and drug administration - approved labeling of dexmedetomidine includes a 1 mcg / kg loading dose given over 10 min followed by continuous infusion of 0.20.7 mcg / kg / h . due to concerns of hypotension and bradycardia , while earlier studies limited infusion rates to those recommended in the package labeling , recent reports have utilized the maximum rates between 1.4 and 4.0 mcg / kg / h , with dosing most frequently limited to 1.5 mcg / kg / h . although numerous studies have used this dosing strategy , it remains unclear if dosages > 0.7 mcg / kg / h are associated with an increased rate of hypotension . the purpose of this study is to determine the predictors of dexmedetomidine - associated hypotension in critically ill patients . a retrospective review of all adult critically ill patients at our academic medical center who received dexmedetomidine from july 16 , 2009 , to july 15 , 2010 , was performed . this study included patients treated in any of the following adult critical care areas : ( a ) medical icu , ( b ) surgical / trauma icu , ( c ) bone marrow transplant unit , and ( d ) cardiothoracic surgery icu . dexmedetomidine has been used in our critical care units since april 2001 , and our clinical dosing protocol has been described previously . the most recent update to our dexmedetomidine dosing guideline , which included an increase in maximum recommended infusion rate of 1.4 the institutional review board approval was obtained from the university office of responsible research practices institutional review board and was carried out with the ethical standards set forth in the helsinki declaration on 1975 . patients who were < 18 or > 89 years of age , incarcerated , pregnant , hypotensive with a baseline mean arterial pressure ( map ) < 65 mmhg , and who received dexmedetomidine in the operating theater or for conscious sedation were excluded . data collected included demographics , medical history , surgical history , indication for hospital and icu admission , blood pressure , heart rate , acute physiology and chronic health evaluation ii ( apache ii ) score , use of end - organ support ( i.e. , renal replacement and ventilatory support ) , dexmedetomidine dosing , and concurrent medications that could contribute to hypotension or treat hypotension ( i.e. , antihypertensives , vasopressors , inotropes , and continuous intravenous sedation sedatives and analgesics ) . the primary endpoint was the development of hypotension defined as map < 60 mmhg while receiving dexmedetomidine . secondary endpoints included the duration of mechanical ventilation , icu and hospital length of stay , mortality , rate of bradycardia , and treatment of hypotension . continuous parametric data are presented as a mean ( standard deviation ) and analyzed using student 's t - test . continuous nonparametric data are presented as median ( 2575% interquartile range ) and were analyzed using mann nominal data are presented as frequency and percentages and were analyzed using the fisher 's exact test or chi - square test as appropriate . following univariate analyses of patients with and without hypotension , multivariable logistic regression was conducted to determine factors independently associated with hypotension . risk factors significant at the p < 0.20 level in the univariate analysis were considered for inclusion into a step - wise backward multiple logistical regression model . two - tailed statistical tests were utilized , with significance level set at p < 0.05 . all data were analyzed using pasw 19 software for windows ( spss for windows version 19 , ibm , armonk , ny , usa ) . a retrospective review of all adult critically ill patients at our academic medical center who received dexmedetomidine from july 16 , 2009 , to july 15 , 2010 , was performed . this study included patients treated in any of the following adult critical care areas : ( a ) medical icu , ( b ) surgical / trauma icu , ( c ) bone marrow transplant unit , and ( d ) cardiothoracic surgery icu . dexmedetomidine has been used in our critical care units since april 2001 , and our clinical dosing protocol has been described previously . the most recent update to our dexmedetomidine dosing guideline , which included an increase in maximum recommended infusion rate of 1.4 the institutional review board approval was obtained from the university office of responsible research practices institutional review board and was carried out with the ethical standards set forth in the helsinki declaration on 1975 . patients who were < 18 or > 89 years of age , incarcerated , pregnant , hypotensive with a baseline mean arterial pressure ( map ) < 65 mmhg , and who received dexmedetomidine in the operating theater or for conscious sedation were excluded . data collected included demographics , medical history , surgical history , indication for hospital and icu admission , blood pressure , heart rate , acute physiology and chronic health evaluation ii ( apache ii ) score , use of end - organ support ( i.e. , renal replacement and ventilatory support ) , dexmedetomidine dosing , and concurrent medications that could contribute to hypotension or treat hypotension ( i.e. , antihypertensives , vasopressors , inotropes , and continuous intravenous sedation sedatives and analgesics ) . the primary endpoint was the development of hypotension defined as map < 60 mmhg while receiving dexmedetomidine . secondary endpoints included the duration of mechanical ventilation , icu and hospital length of stay , mortality , rate of bradycardia , and treatment of hypotension . continuous parametric data are presented as a mean ( standard deviation ) and analyzed using student 's t - test . continuous nonparametric data are presented as median ( 2575% interquartile range ) and were analyzed using mann nominal data are presented as frequency and percentages and were analyzed using the fisher 's exact test or chi - square test as appropriate . following univariate analyses of patients with and without hypotension , multivariable logistic regression was conducted to determine factors independently associated with hypotension . risk factors significant at the p < 0.20 level in the univariate analysis were considered for inclusion into a step - wise backward multiple logistical regression model . two - tailed statistical tests were utilized , with significance level set at p < 0.05 . all data were analyzed using pasw 19 software for windows ( spss for windows version 19 , ibm , armonk , ny , usa ) . a total of 283 patients were evaluated , with 45.6% treated in the medical icu , 36.4% in the surgical / trauma icu , 16.3% in the cardiothoracic surgery icu , and 1.7% in the bone marrow transplant unit [ table 1 ] . the use of concomitant continuous sedative and analgesic infusions was similar between study subgroups , with fentanyl and midazolam being the most commonly administered . hypotension developed in 42.8% ( 121 ) of patients to a median nadir map of 54 ( 5057 ) mmhg , and a median time to first hypotensive episode of 5 ( 2.713.4 ) h. for those who developed hypotension , the median percentage decrease in map from baseline to nadir was 33.3 ( 24.842.5 ) . eighty - seven percent of patients who developed hypotension received at least one intervention to treat hypotension . the most common treatments for hypotension include decreasing the dosage of dexmedetomidine in 47.1% ( 57 ) of patients , administration of an intravenous fluid bolus in 45.5% ( 55 ) of patients , or stopping dexmedetomidine in 35.5% ( 43 ) of patients [ figure 1 ] . among patients who received an intervention for hypotension , 14.8% ( 18 ) of patients had their vasopressor dose increased and 14% ( 17 ) of patients had a vasopressor initiated . an increased mortality was seen in those that experienced dexmedetomidine - associated hypotension ( 20.7% vs. 9.9% , p = 0.016 ) . bradycardia occurred in 7.4% overall and did not differ between groups ( 6.6% that developed hypotension vs. 8% that did not , p = 0.82 ) . there was a trend toward longer median icu length of stay in the dexmedetomidine - associated hypotension group ( 13 [ 621 ] vs. 10 [ 616 ] , p = 0.094 ) . in addition , longer median duration of mechanical ventilation ( 10 [ 417 ] vs. 7 [ 314 ] days ; p = 0.025 ) and longer median hospital length of stay ( 19 [ 1226 ] vs. 16 [ 1023 ] days , p = 0.047 ) were observed in the dexmedetomidine - associated hypotension group . dex : dexmedetomidine patients who experienced dexmedetomidine - associated hypotension were older ( 55.1 15.2 vs. 50.9 15.6 years ; p = 0.03 ) and a higher apache ii score on day of dexmedetomidine initiation ( 22 [ 1825 ] vs. 20 [ 1622 ] ; p = 0.02 ) . medical icu patients were less likely to develop hypotension ( 38.0% vs. 51.2% ; p = 0.03 ) and there was a trend toward more hypotension in those treated in the cardiothoracic icu ( 21.5% vs. 12.3% , p = 0.05 ) . with regard to past medical and surgical history , there were several significant differences observed in those who experienced hypotension . patients who developed hypotension were more likely to have a documented history of coronary artery disease ( 32.2% vs. 16.0% ; p = 0.009 ) and underwent cardiac surgery during this admission ( 18.2% vs. 4.2% ; p = 0.009 ) . patients who developed hypotension during dexmedetomidine administration had lower map at the time of dexmedetomidine initiation ( 82 [ 7090 ] vs. 90 [ 78100 ] mmhg ; p < 0.001 ) . furthermore , more patients in the hypotensive group had a map < 70 mmhg at initiation of dexmedetomidine ( 25.6% vs. 7.4% ; p < 0.001 ) [ table 1 ] . none of the patients received a loading dose and there was no difference in the median maximum dosage of dexmedetomidine between those who developed hypotension and those who did not ( 0.8 [ 0.41.4 ] mcg / kg / h vs. 0.7 [ 0.41.0 ] mcg / kg / h , p = 0.34 ) [ table 2 ] . approximately , half of all patients received a maximum dosage > 0.7 mcg / kg / h with one - third 1 mcg / kg / h , and there were no differences between groups . although our dexmedetomidine protocol discourages titration sooner than every 30 min , titrations quicker than 30 min occurred in 15.4% who developed hypotension and 19.8% who did not ( p = 0.32 ) . dexmedetomidine dosing there was also no difference in the overall concurrent use of antihypertensive agents between those who experienced hypotension and those who did not ( 54.5% vs. 45.7% ; p = 0.15 ) [ table 3 ] . unlike concomitant antihypertensive use , more patients with hypotension were on at least one vasopressor prior to starting dexmedetomidine ( 14.0% vs. 2.5% ; p < 0.001 ) . the use of norepinephrine ( 9.1% vs. 1.8% ; p = 0.01 ) and epinephrine ( 7.4% vs. 1.2% ; p = 0.01 ) were more common in the hypotensive group . concurrent medications at dexmedetomidine initiation factors entered into the multivariable analysis were icu type , history of congestive heart failure , recipient of cardiac surgery during this admission , history coronary artery disease , weight , baseline map , and apache ii score . the multivariable logistic regression analysis identified decreasing baseline map , increasing apache ii score and coronary artery disease as factors independently associated with dexmedetomidine - associated hypotension [ table 4 ] . for every 1 mmhg decrease in baseline map , the odds of dexmedetomidine - associated hypotension increased by 3% . with regards to apache ii score , each one - unit increase in the severity of illness increased the odds of experiencing a hypotensive episode while on dexmedetomidine by 6% . factors entered into the multivariable analysis were icu type , history of congestive heart failure , recipient of cardiac surgery during this admission , history coronary artery disease , weight , baseline map , and apache ii score . the multivariable logistic regression analysis identified decreasing baseline map , increasing apache ii score and coronary artery disease as factors independently associated with dexmedetomidine - associated hypotension [ table 4 ] . for every 1 mmhg decrease in baseline map , the odds of dexmedetomidine - associated hypotension increased by 3% . with regards to apache ii score , each one - unit increase in the severity of illness increased the odds of experiencing a hypotensive episode while on dexmedetomidine by 6% . dexmedetomidine - associated hypotension was common in this study with preexisting low blood pressure , history of coronary artery disease , and greater severity of illness identified as independent risk factors . the use of dexmedetomidine in the icu more than tripled from 2001 to 2007 . since then , numerous shortages of sedative medications ( e.g. , propofol and benzodiazepines ) and the updated 2013 sedation guidelines have further increased the use of dexmedetomidine in the united states . while dexmedetomidine may be an attractive sedative agent due to its lack of respiratory depression and improved delirium profile , it has been associated with hypotension that has ranged from 13% to 98% in clinical studies . we urge caution when initiating dexmedetomidine in patients with low blood pressure or are at the risk of hypotension , especially in those that are undergoing fluid resuscitation . hypotension is independently associated with mortality in icu patients and preventing hypotension may be beneficial to improve outcomes . in the current study , patients who experienced dexmedetomidine - associated hypotension had a higher mortality rate than those who did not . dexmedetomidine is specific for the -2a receptor , especially at lower concentrations , resulting in both vasodilation and a blunting of the sympathetic response . due to these mechanistic considerations , this is especially true in those who are receiving dexmedetomidine in the settings of hypovolemia , traumatic spinal cord injury , or general anesthetic administration . the use of dexmedetomidine in these patient populations may explain the high rate of hypotension ( 98% ) reported in a recent study in trauma patients where almost 50% of patients had a spinal cord injury . patients with congestive heart failure or coronary artery disease may also be at risk for dexmedetomidine - induced hypotension due to the blunting of adrenergic tone . similarly , blood loss and fluid shifts following surgery may place surgical patients at higher risk for hypovolemia , which may explain some of the observed hypotensive events . the observed hypotension may also be another marker of severity of illness . in our study , patients with increased severity of illness and lower baseline map were also at increased risk for the development of hypotension during dexmedetomidine administration . development of a protocol for selective use of dexmedetomidine in these patients may help to reduce the incidence of dexmedetomidine - associated hypotension and potentially mitigate the poor outcomes observed in this population . despite concerns that larger doses of dexmedetomidine may be associated with complications , we observed no significant relationship between the development of hypotension and the administration of dexmedetomidine dosages of > 0.7 mcg / kg / h . as previously described , dexmedetomidine may produce vasodilatory effects at lower concentrations due to its increased affinity for -2a receptors . as the concentration increases , dexmedetomidine loses its selectivity and also agonizes -2b receptors that cause vasoconstriction . previous pharmacokinetic data in healthy men found that plasma concentrations of dexmedetomidine between 0.7 and 1.2 ng / ml resulted in decreased map from baseline and concentrations above 1.9 ng / ml resulted in a return of map to baseline . a study of critically ill patients receiving dexmedetomidine infusions as high as 0.7 mcg / kg / h observed serum concentrations ranging from 0.711.7 ng / ml . another study of doses as high as 2.5 mcg / kg / h demonstrated a linear increase in serum concentration of dexmedetomidine ( range : 16.8 ng / ml ) as the infusion rate was increased . consequently , it is not surprising that increasing dosages above 0.7 mcg / kg / h is not associated with a higher risk of hypotension as it is likely these doses would result in serum concentrations > 1.9 ng / ml . in addition to pharmacokinetic data , recent clinical trials have produced results that suggest similar findings . the dexmedetomidine versus midazolam for continuous sedation in the icu ( midex ) and dexmedetomidine versus propofol for continuous sedation in the icu ( prodex ) studies used infusion rates between 0.2 and 1.4 mcg / kg / h when comparing dexmedetomidine to midazolam or propofol , respectively . the median dexmedetomidine dosage was 0.45 ( 0.270.76 ) mcg / kg / h in midex and 0.925 ( 0.671.2 ) mcg / kg / h in prodex . hypotension was more common in the midex study ( 20.6% vs. 13% ; p = 0.03 ) where the median dosage of dexmedetomidine was < 0.7 mcg / kg / h . our study has a number of strengths and limitations . strengths include a relatively large sample size , inclusion in our analyses of several confounding factors for the development of hypotension , including concurrent antihypertensives , which have been poorly described to date . finally , a large percentage of patients in our study received the treatment with higher doses of dexmedetomidine , allowing evaluation of higher dosing on hypotension . we were unable to determine retrospectively the volume status in our cohort of patients which could influence the risk of hypotension . changes in mechanical ventilator settings that could impact hemodynamic parameters , such as an increase in positive end - expiratory pressure , were not evaluated . further , 17.3% of patients did have the dose of dexmedetomidine increased too quickly , which may have contributed to the overall high rates of hypotension observed . finally , our center uses a dexmedetomidine dosing protocol and our results may not be applicable elsewhere . the current analysis identified a baseline map < 70 mmhg , history of coronary artery disease , and increasing apache ii score as factors independently associated with the development of hypotension . clinicians should use dexmedetomidine cautiously in patients with low baseline blood pressure and increasing the severity of illness . further trials are needed to determine factors associated with the development of hypotension and potential impact of protocols on minimizing the risk of this adverse effect .

background : dexmedetomidine is commonly used for sedation in the intensive care unit ( icu ) , and its use may be associated with hypotension . we sought to determine predictors of dexmedetomidine - associated hypotension.methods:retrospective , single - center study of 283 icu patients in four adults icus over a 12 month period . univariate analyses were performed to determine factors associated with dexmedetomidine - related hypotension . risk factors significant at the 0.20 level in the univariate analysis were considered for inclusion into a step - wise multiple logistical regression model.results:hypotension occurred in 121 ( 42.8% ) patients with a median mean arterial pressure ( map ) nadir of 54 mmhg . univariate analyses showed an association between hypotension and age ( p = 0.03 ) , acute physiology and chronic health evaluation ii ( apache ii ) score ( p = 0.02 ) , baseline map ( < 0.001 ) , admission to the cardiothoracic icu ( p = 0.05 ) , history of coronary artery disease ( p = 0.02 ) , and postcardiac surgery ( p = 0.0009 ) . admission to the medical icu was associated with a decrease in development in hypotension ( p = 0.03 ) . there was a trend for hypotension with weight ( p = 0.09 ) and history of congestive heart failure ( p = 0.12 ) only map prior to initiation ( odds ratio [ or ] 0.97 , 95% confidence interval [ 95% ci ] 0.950.99 ; p < 0.0001 ) , apache ii scores ( or 1.06 , 95% ci 1.011.12 ; p = 0.017 ) , and history of coronary artery disease ( or 0.48 , 95% ci 0.260.90 , p = 0.022 ) were independently associated with hypotension by multivariable analysis.conclusions:dexmedetomidine-associated hypotension is common . preexisting low blood pressure , history of coronary artery disease , and higher acuity were identified as independent risk factors for dexmedetomidine - associated hypotension .

bonded lingual retainers are used principally for long - term retention of treated orthodontic cases and for the permanent splinting of periodontally involved teeth . the duration of retention should be decided for each case specifically in conjunction with the patient taking into consideration future growth . the placement of a bonded retainer is technique sensitive [ 8 , 9 ] . spiral or multistrand wires appear to be the most popular for direct - bonded retainers . the main advantage of the use of multistrand wire is the irregular surface that offers increased mechanical retention for the composite without the need for the placement of retentive loops . moreover another asset is the flexibility of the wire that allows physiologic movement of the teeth , even when several adjacent teeth are bonded . bonded retainers appear to be well accepted by patients and are relatively independent of patient cooperation . although traditional methods are successful , splinting teeth with reinforcement fibers that can be embedded in composites has gained popularity in last years . fiber - reinforced composite ( frc ) containing various fibers such as carbon , polyaramid , polyethylene , and glass has received increasing acceptance as restorative materials . reinforced polyethylene fiber material was successfully used for fixed orthodontic retainers , space maintainers , temporary postorthodontic fixation devices to attach a pontic to abutment teeth during periodontal therapy [ 18 , 19 ] , and posttraumatic stabilization splints . the polyethylene fiber material adapted easily to dental contours and could be manipulated during the bonding process . it also has acceptable strength because of integration of fibers with composite resin that leads to good clinical longevity . the bonded orthodontic splint provides the patient with an efficient esthetic retainer that can be maintained in the long term . the most common failure type is detachment at the wire - composite interface because of insufficient adhesive over the wire or unfavorable occlusal contacts , which results in abrasion of the composite [ 5 , 22 ] . the abrasion of mandibular retainers has been attributed to mechanical forces such as toothbrushing and chewing . when clinical failure of bonded orthodontic retainers is observed relapse can occur . therefore the purpose of this clinical study was to evaluate the reliability of two different orthodontic retainers : multistrand stainless steel wire and polyethylene fiber - reinforced resin composite ( frc ) . it was also analyzed the level of satisfaction of the patient about the esthetic result such as visibility of the retainer while talking and smiling , by means of vas ( visual analogue scale ) . the null hypothesis of the study was that there is no statistically difference in bond failure rate and vas records between the two different retainers . a total of 34 patients ( mean age : 14.3 ) in the finishing phase of orthodontic treatment , attending the orthodontic department s. palazzi of pavia university , were selected . for this randomized clinical study the inclusion criteria were correct dental alignment , need for permanent orthodontic retention in the lower anterior segments , and being free of occlusal interferences in order to eliminate the influence of trauma . institutional approval was achieved from the department . written patient and parental informed consents were obtained . was applied a multistrand stainless steel wire ( ortosmail krugg , milan , italy ) , as showed in figure 1(a ) and in the second one a polyethylene fiber - reinforced resin composite ( infibra tpitalia , gorle , italy ) , as showed in figure 1(b ) . the retainers were applied in the mandibular arch from right to left canine . since bonding of the retainer all patients were followed with periodic monitoring visits at 30 , 60 , 120 , 180 , 360 days to evaluate detachments . specific data were collected : date of bonding , type of splint , position , number of teeth involved , detachment ( position and number ) , followup at 30 , 60 , 120 , 180 , 360 days . 34 patients were enroled : 17 with a multistrand wire retainer ( total number of teeth bonded 102 ) , while 15 with a polyethylene fiber - reinforced resin composite ( total number of teeth bonded 90 ) . 0.0175 multistranded wire and polyethylene fiber reinforced resin preimpregnated with transbond xt primer all teeth were isolated with cheek retractors and cleaned with a mixture of water and fluoride - free pumice using a rubber polishing cup in a low - speed handpiece . the teeth were rinsed up with water and dried with an oil - free syringe . after lingual surfaces cleaning , each tooth was etched with 37% orthophosphoric acid gel ( 3 m unitek , monrovia , calif , usa ) for 20 seconds and then rinsed and air - dried . the retainers were accurately located on the lingual surface and a thin layer of bonding transbond xt primer ( 3 m unitek , monrovia , calif , usa ) was applied and then light cured with a halogen curing unit ( opitlux 501 ; sds kerr , danbury , conn ; light intensity , 930 mw / cm ; wavelength range , 400505 nm ) for 20 seconds , as suggested by the manufacturer . a small amount of trasbond xt resin ( 3 m unitek , monrovia , calif , usa ) was placed to cover both metallic and frc splints and light - cured for 40 seconds , as suggested by the manufacturer . finishing each patient was asked to express an opinion about the esthetic result such as visibility of the retainer while talking and smiling by means of vas ( visual analogue scale ) in which the 0 point means poor esthetic result effect and 10 means excellent esthetic effect . statistical analysis was performed with a computer software ( stata 7 , college station , tex ) . paired t - test was applied to determine differences in detachment existed among the groups . in addition to the simple event of failure , the time of bonding failure was also considered . kaplan - meier estimates of survival plots were constructed , and a log - rank test was carried out to compare the distribution of survival times in the two groups . frcs and stainless steel splints ' vas was analyzed by means of kruskal wallis test . as reported in table 1 , no significant differences in total number of detachments between the two splints were detected ( p > 0.05 ) . at the end of the followup the percentage of total detachment of the two different retainers were 22,54% for multistrand stainless steel wire and 14,45% for polyethylene ribbon - reinforced resin retainer , respectively . kaplan - meier survival plots for the two types of retainers are shown in figure 2 . there was no statistically significant difference in retainers failure risk over the 12 months of followup ( hazard ratio : 0.77 ; confidence interval 95% : 0.311.93 ; log - rank test : p = 0.58 ) . during the 12-month retention period table 2 shows visual analogue scale ( vas ) of the level of satisfaction of the patient about the esthetic result . the visual analogue scale ( vas ) , in which 0 point means very poor esthetic effect and 10 means excellent esthetic effect , showed that patients with multistranded stainless steel wire expressed a mean value of satisfaction of 8.24 , whereas patients with polyethylene fiber reinforced resin retainer for lingual retention expressed a mean value of satisfaction of 9.73 . no significant difference was detected between the percentage of failures of the two types of bonded retainers whereas significant differences in vas records were reported . survival plots show that polyethylene fiber reinforced resin retainer had a survival rate lower than the multistrand stainless steel wire after 30 days . at the subsequent followups , especially at 180 and 360 days , frcs present a survival plot that overtake multistrand stainless steel wires . neverthless the difference between the two groups was not significant ( p = 0.58 ) . a previous investigation evaluated different orthodontic adhesives for frc bonding and transbond xt exhibited higher shear bond strength values than both flow composite and glass ionomer cement . therefore in the present investigation trasbond xt primer and trasbond xt resin ( 3 m unitek , monrovia , california , usa ) were used . there are few clinical investigations that have compared failure rate of multistrand wire retainer with a polyethylene fiber reinforced resin retainer . the reliability of posttreatment canine - to - canine retention with resin composite retainers reinforced with plasma - treated woven polyethylene ribbons was compared to the reliability of directly bonded , multistranded wire retainers by rose et al . . this prospective study , based on an assessment of 20 patients , demonstrated that ribbon - reinforced retainer remained in place for shorter period of time than multistranded wire . similar findings have been described for glass fiber reinforced retainers when compared with multistranded retainers . these are in disagreement with present study , which shows no statistically significant differences between the two types of splint for the percentage of survival after 12 months . the reason of the different results could be ascribed to the use of different materials and different bonding techniques in the studies . zachrisson in a study of 1977 evaluated 43 direct - bonded mandibular canine - to - canine retainers after observation period between 12 to 30 months . results indicate that the bonded retainer has all the advantages of a fixed soldered retainer , in addition to being invisible . the failure rate in terms of loose retainers ( 11,6% ) was similar to that of the present investigation . moreover even with higher flexibility of polyethylene frc versus glass frc in terms of material properties and diameter , some limitations in the clinical use of polyethylene frcs still persist . in fact in marginal areas the fibers may become exposed and come into contact with oral tissues , saliva , and microbes . this roughness can result in a higher retention of bacteria than the other materials tested [ 2931 ] . the reinforcement of polymers with a ribbon layer slightly increases the transverse strength , but the adherence of the polyethylene fibers to the base polymer have been shown to be poor , and this could represent another limitation for long - term stability of polyethylene frcs . finally in the present investigation a visual analogue scale ( vas ) was used for the subjective evaluation to assess the level of patient satisfaction regarding the esthetic outcome of the metallic and frc splints . this kind of investigation was used in the literature specially to evaluate esthetic result after prosthodontic treatment instead is not widely used in orthodontics [ 3335 ] . in the present investigation significant differences in vas records were reported between the two groups . in fact esthetic result of frc retainers was significantly higher than stainless steel multistranded wire . the results showed that there was no statistically significant difference between the failure rate of the two types of bonded retainers . vas records showed that esthetic result was significantly higher for polyethylene ribbon - reinforced resin retainers .

the purpose of this longitudinal prospective randomized study was to evaluate the reliability of two different types of orthodontic retainers in clinical use : a multistrand stainless steel wire and a polyethylene ribbon - reinforced resin composite . moreover the level of satisfaction of the patient about the esthetic result was also analyzed by means of a visual analogue scale ( vas ) . 34 patients ( 9 boys and 25 girls , mean age 14.3 ) , in the finishing phase of orthodontic treatment , were selected for the study . since splints were applied the number , cause , and date of splint failures were recorded for each single tooth over 12 months . statistical analysis was performed using a paired t - test , kaplan meier survival estimates , and the log - rank test . kruskal wallis test was performed to analyze vas recordings . differences between the bond failure rates were not statistically significant . esthetic result of vas was significantly higher for polyethylene ribbon - reinforced resin retainers than for stainless steel wires .

the us national institutes of allergy and infectious diseases ( niaid ) defines food allergy as adverse health effect arising from a specific immune response that occurs reproducibly on exposure to a given food ( boyce et al . , 2010 ) . undeclared allergens in food label represent a risk for consumers , as there is no therapy for food allergies . according to directive 2003/89/ec ( european commission , 2003 ) declaration of all ingredients and derived substances in the food label a systematic review concerning the epidemiology of food allergies has concluded that in the united states food allergies affect more than 1 - 2% but less than 10% of the population . the number of cases of food allergy varies by age , geographic area , diet and other factors that make difficult a reliable evaluation of the prevalence of food allergies ( sicherer , 2011 ) . even in europe , percentages do not change from those found in the united states ( rona et al . , 2007 ) . the most important foods responsible for allergies are : milk , eggs , peanuts , shellfish , fish , soy , nuts and cereals ; allergies to additives are less common ( sicherer and sampson , 2010 ; young et al . , 1994 ) . the only way to protect allergic subject health is to avoid the ingestion of allergenic substances , therefore the label , which shows the presence of all ingredients and allergens , is an essential tool of food safety . the directive 2003/89/ec ( european commission , 2003 ) , implemented in italy by legislative decree 114/2006 ( italian republic , 2006 , 2006 ) , provides information to allergic consumers , making a list of allergens that must be indicated on the label , and requires the declaration of all substances derived from the main allergens . moreover , the directive takes into account the case of a possible unintentional presence of allergenic substances in products and allows to the food business operators the use of phrases such as it may contain traces of ... or produced in a factory where xy are in use ... the substances included in the list of directive 2003/89/ec are : cereals containing gluten , crustaceans , eggs and egg products , fish , peanuts , soy , milk , nuts , celery , mustard , sesame seeds , sulfur dioxide and sulphites at concentrations above 10 mg / kg or 10 mg / l expressed as so2 . the list was supplemented by directive 2006/142/ec ( european commission , 2003 ) , which added the obligation to indicate also lupine , molluscs and products thereof . since 2007 , in piedmont region ( northwestern italy ) a monitoring plan for the detection of allergens in food was issued in order to monitor the presence of any non - compliance on the labels . this paper presents the results of the monitoring plans carried out in the period 2011 - 2012 . in 2011 - 2012 meat products , meat preparations and minced meat , sweet and salty snacks , baby food , ready - to - eat ( rte ) products and other foodstuffs were tested for egg proteins and -lactoglobulin . samples were collected by the veterinary services and food hygiene and nutrition services at food retails of piedmont region and they were selected based on the absence of information about the presence of allergens in the labels . the sampling was performed according to the procedures of the official sampling plans , providing four or five aliquots . the analyses were performed at the food control laboratory of the veterinary epidemiology centre of piedmont , liguria and valle daosta ( istituto zooprofilattico del piemonte , liguria e valle daosta ) , turin , italy . briefly , 1 g of sample and 20 ml of extraction buffer , specific for the allergen target and provided with the kit , were homogenised by stomaker. therefore , an incubation in a water bath at 60c for 10 min was set up and , later , sample was filtrated or centrifuged . an aliquot of 100 l of the supernatant was added into the wells of a plate , in which specific antibodies were immobilised . the complex antibody / antigen / antibody ( sandwich ) was detected by the addition of the chromogenic substrate that determines a colorimetric reaction . the reading of this reaction was performed using a spectrophotometer at 450 nm of absorbance and samples concentration was calculated according to the lambert - beer law with the aid of the specific programme ridawin software . the detection limit for the elisa kit ridascreen fast -lactoglobulin is 0.5 mg / kg and for the elisa kit ridascreen fast ei/ egg protein is 0.6 mg / kg . in the period 2011 - 2012 , 285 food samples for -lactoglobulin and 234 for egg protein were tested ( tables 2 and 3 ) . overall , 19 samples exceeded the limit of detection and were classified as non - compliant : in particular , 8 out of 285 ( 2.8% ) for -lactoglobulin and 11 out of 234 ( 4.7% ) for egg protein . the non - compliance for -lactoglobulin covered the 5.1% ( 4/78 ) of meat preparations and minced meat , 1.7% ( 2/117 ) of meat products , 2.5% ( 1/40 ) of sweet and salty snacks , and 4.3% ( 1/23 ) of baby food . traces of egg protein were detected in 8.9% ( 7/78 ) of meat preparations and minced meat , in 1.9% ( 2/107 ) of meat products and 8.3% ( 2/24 ) of sweet and salty snacks . some allergens play a critical technological role in food industry and their presence in food can result especially from accidental contamination , as well as voluntary use as an ingredient . in some plants it may be difficult to separate the production lines including the use of allergenic ingredients from those that must be free . the presence of -lactoglobulin and egg protein , especially in meat preparations and minced meat , could be probably attributed to an involuntary contamination . for that reason , the food business operators should increase the level of attention with careful application of self - monitoring plan and implement training programmes of staff in order to ensure the health of allergic consumers . the results and the considerations mentioned above emphasise the important role of official monitoring plans to check the appropriate application of the legislation on the labeling of food products .

the us national institutes of allergy and infectious diseases defines food allergy as adverse health effect arising from a specific immune response that occurs reproducibly on exposure to a given food . undeclared allergens in food label represent a risk for consumers , as there is no therapy for food allergies . according to directive 2003/89/ec , declaration of all ingredients and derived substances in the label is mandatory . in 2011 - 2012 , in piedmont region ( north - western italy ) 285 food samples were analysed for -lactoglobulin and 234 for egg proteins . the aim of this work was to analyse 2 years data in order to assess the presence of undeclared milk and egg allergenic proteins in food placed on the market checking the compliance of labeling of food allergens . analyses were carried out with elisa tests , both for the detection of the egg and milk proteins . -lactoglobulin was found in 2.8% ( 8/286 ) of samples , while egg proteins in 4.7% ( 11/234 ) .

the differentiation and maturation of t cells is mediated largely by a diverse array of phenotypically and functionally distinct epithelial cell types ( ritter and boyd , 1993 , nitta et al . , 2008 ) , which comprises a key component of the thymic stroma . thymic epithelial cells ( tecs ) can be broadly categorized into two major sub - types cortical ( c ) and medullary ( m ) tec ( ritter and boyd , 1993)both of which are required for the development of a self - tolerant , self - restricted t cell repertoire . however , the cellular mechanisms that maintain the different tec sub - lineages of the mature thymus and how these are affected by age to cause thymic involution remains poorly understood . the thymus originates from the third pharyngeal pouches ( 3pp ) of the pharyngeal endoderm ( gordon et al . , 2004 , le douarin and jotereau , 1975 ) , which give rise to the epithelial component of the thymic stroma ; transplantation studies in avians and mice have shown that 3pp endoderm is sufficient to initiate formation of a fully functional and properly patterned thymus in an ectopic site ( gordon et al . , 2004 , le douarin and jotereau , 1975 ) . strong evidence suggests that , during fetal development and in the perinatal thymus , a bipotent progenitor exists that can generate both ctec and mtec ( bennett et al . , 2002 , gill et al . , 2002 , bleul et al . , 2006 , rossi et al . , 2006 ) . the existence of mtec sub - lineage - restricted progenitors , that can generate aire mtec ( required for central tolerance ) ( kyewski and klein , 2006 ) , has been demonstrated in the fetal thymus ( hamazaki et al . , 2007 , sekai et al . , 2014 , lopes et al . , 2015 ) and a putative fetal ctec - restricted progenitor has also been identified ( shakib et al . , 2009 ) . regeneration of ctec following early postnatal ctec ablation has also been demonstrated ( rode and boehm , 2012 ) . in the adult thymus , transplantation data indicate that mhc class ii ( mhcii ) ( gray et al . , 2007 ) and cd80 ( rossi et al . , 2007c ) mtec can give rise to mhcii and cd80 mtec , respectively , including aire cells . as mhcii and cd80 expression levels correlate directly in mtec , this suggests that the mhciicd80 population contains mtec progenitors ( gray et al . additionally , transplantation assay of bulk populations has shown that mhcii ctecs contain the potential to generate both ctec and mtec ( wong et al . , 2014 ) . the existence of a common thymic epithelial progenitor cell ( tepc ) , as well as both cortical and medullary epithelial sub - lineage - restricted progenitors , has also been suggested by a limited retrospective clonal analysis of postnatal day 14 tec ( bleul et al . , 2006 ) . however , recent reports demonstrate that podoplanin tecs , which are located predominantly in the cortex and at the cortico - medullary junction ( cmj ) , contribute to postnatal mtec maintenance ( onder et al . , 2015 ) , and although the thymoproteosome subunit 5 t marks both ctec and mtec progenitors in the fetal and at least some ctec progenitors in the early postnatal thymus , early postnatal mtec progenitors are 5t - negative ( ohigashi et al . , 2015 , mayer et al . , consistent with these data , an epithelial stem cell can be derived and clonally propagated from adult rat thymic epithelium and retains the capacity to contribute to the medullary thymic epithelial network , including generation of tecs expressing the autoimmune regulator aire ( bonfanti et al . , 2010 ) . the identity of the cells from which this in vitro stem cell population is established is unclear . similarly , two recent papers have reported that thymic epithelial cultures can be established from individual initiator adult tecs and can make a limited contribution to medullary and cortical tec networks upon transplantation ( wong et al . , 2014 , ucar et al . , 2014 ) . however , although in one case it was demonstrated that the initiating cell was both epcam- and foxn1-negative and had never expressed levels of foxn1 high enough to drive physiologically relevant cre activity ( ucar et al . , 2014 ) , the details of the identity of the initiating cell remained unclear . of note is that the epcamuea1mhcii population identified in the second report comprises almost 20% of all tecs ( wong et al . , 2014 ) . indeed , difficulties associated with isolating and assaying defined , viable subpopulations of adult tecs have been a major factor hampering progress in this area , while lineage tracing in vivo has been precluded by the absence of tec subset - specific markers . the cell surface marker plet1 has been shown via prospective isolation and functional testing by us and others to mark a population of tepcs during early thymus development that is sufficient to generate an organized and functional thymus upon transplantation ( bennett et al . , 2002 , gill et al . , 2002 , rossi et al . plet1 is also expressed by defined epithelial cell populations in other organs and tissues including the skin , where it has been demonstrated to mark a subset of epithelial stem cells ( depreter et al . , 2008 , nijhof et al . , 2006 , frances and niemann , 2012 , raymond et al . , 2010 ) . from day 9.0 of mouse embryonic development ( e9.0)the stage at which the 3pp are formed to e11.5 , plet1 is uniformly expressed by 3pp cells and can therefore be regarded as a marker of the founder cells of the thymic epithelial lineage ( gordon et al . , 2004 , depreter et al . , 2008 , nowell et al . , 2011 ) . from e12.5 to at least e15.5 , purified plet1 tecs are sufficient to generate a properly organized , fully functional thymus upon transplantation ( bennett et al . , 2002 , gill et al . , 2002 , rossi et al . , 2007a ) , and the plet1 tec population of the early fetal thymus is believed to contain a fetal common thymic epithelial progenitor cell ( rossi et al . , 2006 ) however , plet1 tecs present at later stages of thymus organogenesis do not retain the capacity to initiate de novo organogenesis ( rossi et al . , 2007a ) . in the adult thymus , plet1 tecs have been regarded as a minor subpopulation of mtecs based on immunohistochemical analysis . we have identified a subpopulation of adult plet1 tecs , distinct from the major adult medullary plet1 population , that is defined by co - expression of the ctec - restricted marker ly-51 and located at the cortico - medullary junction . here , we show that this ly-51plet1 population can proliferate and differentiate into both ctecs and mtecs in an established assay of tec potency . in contrast , other adult tec populations could generate only cortical sub - lineage tecs , or could not generate any progeny , in the same assay . the ctec- and mtec - generating activity extinguished at the same frequency in limiting dilution analysis , suggesting the presence of a bipotent tepc within plet1ly-51 tecs , while further phenotypic analysis established that this bipotent progenitor also expressed high levels of mhc class ii . progeny of the plet1ly-51 tecs were present for at least 9 months in in vivo grafts , suggesting this population may contain a bipotent adult thymic epithelial stem cell ( tesc ) . collectively , our data provide clonal resolution analysis of a phenotypically defined adult common thymic epithelial progenitor cell population and thus important mechanistic insight for strategies aimed at improving thymus function in patients . we set out to investigate the cellular mechanism through which the adult thymic epithelium is maintained during homeostasis . to identify subpopulations of adult tecs that contained stem or progenitor cells capable of replenishing ctecs and mtecs , we used flow cytometry to identify discrete tec subpopulations ; in these analyses , tecs were positively identified using epcam staining . we first split adult tecs using ly-51 and uea1 , markers believed to define ctecs and mtecs , respectively . each of these subpopulations was further subdivided using cell surface mhc class ii ( mhcii ) , which is thought to correlate positively with tec maturation status ( gray et al . , 2006 ) and also positively correlates with cd80 in mtecs and cd40 expression in mtecs and ctecs ( reiser and schneeberger , 1994 , galy and spits , 1992 ) . we also included plet1 in our analyses , since this protein identifies tepcs in the early fetal thymus ( bennett et al . , 2002 , gill et al . , 2002 , rossi et al . , this population comprised 30% of all uea1 mtecs ( 33.3% 8.8% ) and was predominantly mhcii ( figures 1a1c ) . in addition , we identified a minor , previously undescribed , population of plet1 tecs that co - expressed ly-51 ( figures 1a and 1b ) . in 8-week - old mice , 50% of this ly-51plet1 population was mhcii ( figure 1c ; mean sd , 8 weeks , 46.3% 8.0% mhcii ) , and ly-51plet1 tecs comprised < 1% of total tecs ( figures 1a1c ; ly-51plet1 0.92% 0.37% of all tecs at 8 weeks old ) . most plet1 tecs were found within the medulla , as previously described ( godfrey et al . , 1990 , these medullary plet1 tecs co - expressed cytokeratin 14 ( k14 ) , k5 , and claudin 4 ( cldn4 ; with plet1 mtecs being a subset of cldn4 mtecs ) and also expressed high levels of rac1 , a skin stem cell - associated marker that has also been implicated in tec maintenance ( benitah et al . separate from this plet1 mtecs population and consistent with our flow cytometric data , we identified a population of ly-51plet1 tecs by immunohistochemistry . most ly-51plet1 tecs were k14-negative by immunostaining , but occasional ly-51plet1k14 cells were observed ( figure 1e ) . to gain insight into the potential function of the tec subpopulations defined above by uea1 , ly-51 , mhcii , and plet1 expression , we sorted them by flow cytometry and interrogated their gene expression profiles by qrt - pcr analysis of 50 cells from each population ( figure 2 ) . the expression profiles of plet1 and bp-1 ( enpep , the ly-51 antigen ) were as expected , with bp-1 restricted to ly-51 ctec populations and high plet1 to plet1 populations ( figure 2a ) . the expression profile of foxn1 was also consistent with previous reports of higher expression in ctecs than mtecs and in mhcii than mhcii tecs ( figure 2b ) ( bredenkamp et al . , 2014a , nowell et al . , 2011 , ki et al . , 2014 ) . furthermore , expression of aire and cd80 ( that are known to be mtec - restricted ) , krt5 ( that is highly upregulated in mtecs and expressed in scattered cells in the thymic cortex ) , and pax1 ( that is reported to be ctec - restricted ) also exhibited the expected expression profiles ( figures 2c and 2d ) . dll4 , the obligate notch ligand required for t cell commitment , is also known to be ctec - restricted ( koch et al . , 2008 , figure 2d indicates that , among ctec subsets in 5- to 8-week - old mice , dll4 mrna expression is limited to plet1 and plet1 mhcii ctecs . comparison of the expression profiles of dll4 and foxn1 among adult tec subsets was consistent with the identification of dll4 as a presumptive direct foxn1 target ( nowell et al . kitl was expressed exclusively in ly-51 tecs , consistent with the requirement for signaling through c - kit in immature thymocytes ( figure 2d ) . p63 , a stem / progenitor cell marker in other epithelial lineages that is required to maintain thymus homeostasis ( pellegrini et al . , 2001 , senoo et al . , 2007 ) , was expressed in all of the tec subsets and enriched in mhcii versus mhcii tecs in both cortex and medulla ( figure 2e ) . il7 was also broadly expressed ; within mtecs , il7 was enriched in mhcii cells , while the highest expression level was in plet1mhcii ctecs ( figure 2e ) . furthermore , while ltbr was expressed in all tec subsets , it was substantially enriched in ly-51 ctecs ( figure 2e ) . among the genes analyzed , sca-1 and k14 showed substantial upregulation in mhciily-51 compared to mhciily-51 populations and in plet1mhciily-51 versus plet1mhciily-51 tecs ( figure 2f ) . collectively , these data indicated that the eight populations identified on the basis of uea , ly-51 , plet1 , and mhcii expression represented distinct cell states , exhibiting different transcriptional profiles . to determine the potency of adult tec populations , we used an assay originally developed to test the differentiation potential of fetal tec subpopulations ( rodewald et al . , 2001 , bennett et al . , 2002 , gill et al . , 2002 ) . thus , tecs were isolated from adult mice that constitutively express membrane - bound gfp ( figure 3a ) , aggregated with unfractionated dissociated e12.5 or e13.5 fetal thymus cells and mouse embryonic fibroblasts ( mefs ) at defined ratios for 1216 hr in vitro , then transplanted under the kidney capsule of syngeneic recipient mice ( figure 3a ) . analysis of the cellular aggregates after 1216 hr ( i.e. , prior to grafting ) demonstrated an even distribution of gfp tecs that were present mostly as single cells ( figure s1a ) . analysis of gfp tec - derived cells within grafts recovered after 4 weeks revealed a strong contribution of gfp cells in both cortical and medullary epithelial compartments of all grafts analyzed ( figures s1b and s1c ; n = 7 ) . the gfp cells within the grafts had proliferated , as demonstrated by ki-67 staining ( figure s1c ) . collectively , these data validated this assay for detection of thymic epithelial progenitor / stem cell ( tep / sc ) activity . we then set out to determine the differentiative potential of the adult tec populations defined above . we initially analyzed five tec populations , which encompassed all of the populations identified in figure 1 : ( 1 ) uea1mhciiplet1 ( plet1 mtec ) , ( 2 ) uea1mhciiplet1 ( plet1 mtec ) , ( 3 ) uea1mhciiplet1 ( plet1 mtec ) , ( 4 ) ly-51plet1 ( ctec ) , and ( 5 ) ly-51plet1 ( see figure 3b for details of sorting strategy ) . these populations were sorted to purities of > 95% from the thymi of 4- to 8-week - old mice . in our initial analyses , 10,000 test cells from each population were reaggregated , with the exception of the ly-51plet1 population for which only 1,500 input cells were tested due to the scarcity of these cells within the adult mouse thymus . the grafts were left for 4 weeks before analysis unless otherwise stated . during this time , they increased substantially in size and developed properly structured cortical and medullary areas , as expected . any grafts that displayed large cystic areas or failed to grow were discarded ; only fully formed organoids were taken forward for analyses of the presence and location of gfp tecs . localization of gfp tecs within cortical and/or medullary regions was determined by counterstaining with ctec- or mtec - restricted markers . we tested three uea1 tec populations , as described above . for grafts seeded with plet1 mtec , a single , small , gfp area was detected in one out of five grafts ; these gfp cells co - stained with k14 and were negative for ly-51 ( figure 3c ; table 1 ) . for grafts seeded with plet1 mtec , one small gfp medullary area was present in one out of two grafts analyzed ( not shown ) . no gfpplet1 cells were detected in any of these grafts . in grafts seeded with the third uea1 population , plet1 mtec , no gfp cells were detected in any of five grafts analyzed . collectively , of the three mtec populations analyzed , two were able to make a very limited contribution to the grafts while the third made no contribution . these input cells only contributed to mtec networks , indicating that they were mtec lineage - restricted ( figure 3c ; table 1 ) . it was not possible to determine in these analyses whether the input cells had differentiated but , consistent with a previous report ( wong et al . , 2014 ) , the small size of the gfp foci suggested strongly that they did not contain progenitor tecs . in grafts seeded with 10,000 ly-51plet1 ctec , extensive areas of gfp tecs were detected in all grafts analyzed ( n = 3 , figure 3d ; table 1 ) . these gfp cells displayed characteristic markers of ctecs , including cd205 , ly-51 , and 5 t ( shown for cd205 in figure 3d ) and were found in cortical areas . in all grafts , multiple foci of gfp ctecs were observed , many situated in the periphery of the thymic lobe . of 389 gfp clusters analyzed , a few that comprised single cells or clusters of less than or equal to three cells co - expressed k14 , and one small k14 medullary area was detected ( table 1 ) . in a graft seeded with 400 ly-51plet1 ctecs , we subsequently analyzed two grafts in which the ly-51plet1 population was split on the basis of mhc class ii expression . the graft seeded with 1,300 ly-51uea1plet1mhcii cells showed an extensive contribution to ctecs and also contained a small number of gfpk14 tecs within one medullary area ( table 1 ) . taken together , these data indicate that the ly-51plet1 ctec population contains cells that can contribute efficiently to ctec networks for at least 4 weeks after grafting , consistent with the existence within this population of ctec - restricted progenitors . these ctec progenitors were present within both the mhcii and mhcii fractions of ly-51plet1 ctecs . however , our data do not provide evidence for a frequent or efficient mtec progenitor in the ly-51plet1 population . all grafts seeded with 1,500 ly-51plet1 tec subpopulation contained very extensive clusters of gfp tecs at 4 weeks post - grafting ( n = 3 ; figures 3e and 4 ; table 1 ) . a strong contribution of gfp tecs was observed in both medullary and cortical areas , with similar numbers of gfp foci in each compartment ( see table 1 for quantification ) . the gfp tecs in the cortical areas co - expressed ly-51 and cd205 , while those in medullary areas were negative for ly-51 . gfp medullary tecs were detected that expressed k14 , uea1 , and aire ( figures 3e and 4 ; table 1 ) , clearly indicating that the input population had differentiated . the gfpplet1 cells included cells located in the medulla that lacked co - expression of cortical markers and cd205 cells close to the cortico - medullary junction ( cmj ) ( figure 4a , s183 arrowhead ) , similar to the ly-51plet1 tecs detected by immunohistochemistry in the native thymus . no differences in outcome were observed from ly-51plet1 tecs isolated from 4- or 8-week - old mice . collectively , of all five populations tested in this assay , only ly-51plet1 tecs could efficiently generate both ctecs and mtecs . to determine whether the ly-51plet1 tec population contained a common tepc , able to generate both cortical and medullary tec subtypes , or comprised separate ctec and mtec progenitors with some shared phenotypic characteristics , we next used a limiting dilution approach to establish the frequency within this population of cells able to generate cortical and medullary tecs . for this , we generated grafts containing defined numbers of test cells , such that the grafted reaggregate fetal thymic organ culture ( rftoc ) were initially seeded with 500 , 250 , 125 , 90 , or 60 ly-51plet1gfp tecs . as above , grafts were left for 4 weeks before analysis for the presence of gfp cells . grafts seeded with 500 gfply-51plet1 tecs all contained extensive contribution of gfp cells in both medullary and cortical areas the gfp clusters contained 100200 tecs and more than 20 gfp areas were identified in each graft . the gfp ctecs expressed ly-51 and cd205 , while the gfp mtecs expressed k14 and uea1 , and gfp cells expressing plet1 were also detected ( table 1 ) . all three grafts seeded with 250 ly-51plet1tecs also contained gfp tecs in both cortex and medulla , and these ctecs and mtecs stained with appropriate sub - lineage - restricted markers . there were fewer gfp areas in these grafts than in grafts seeded with 500 ly-51plet1 cells . however , the relative contribution to ctecs and mtecs was consistent with that observed in the grafts seeded with 500 ly-51plet1 test cells ( table 1 ) . analysis of grafts seeded with 125 ly-51plet1gfp tecs also revealed the presence of gfp tecs in both the cortex and medulla of all grafts analyzed . foci containing plet1gfp tecs were detected in addition to foci containing either ly-51plet1 tecs or k14plet1uea1 tecs ( n = 3 ) ( table 1 ) . in these grafts , only three to eight gfp areas were detected per graft . in grafts seeded with 90 gfply-51plet1 tecs , one out of six grafts analyzed contained gfp tecs . again , gfp cells were found in both cortical and medullary regions and expressed region - appropriate markers . in this graft , one gfp mtec cluster was present and spanned 22 contiguous 8-m sections ( figure 4 ; table s1 ) . two gfp ctec clusters were present , one adjacent to the mtec cluster and one separated spatially but in the same region of the graft ( figure 4 ) . gfpplet1 tecs were detected within the medullary cluster and cortical cluster c2 ( figure 4 ; table s1 ) . the spatial relationship of the three clusters detected in this graft strongly suggested they had arisen from a single cell , as a result of proliferation and cell mixing in the early stages of graft development , prior to sub - lineage commitment . similarly dispersed foci were previously shown to arise from a single fetal tepc ( rossi et al . , unpublished data ) , and this distribution pattern of cells originating from a single lineage - committed cell is commonly described in other tissues ( mathis and nicolas , 2002 ) . no gfp tecs were detected in the cortical or medullary compartments of any of the three grafts generated with an input of 60 ly-51plet1 test cells ( table 1 ) . collectively , these data establish that ly-51plet1 tecs can give rise to both ctecs and mtecs in grafts seeded with as few as 90 input test cells . since the ctec- and mtec - generating activities extinguished at the same input cell number , and the distribution of gfp cells within the 90-cell graft is consistent with a clonal origin of all three gfp foci , they further suggest the presence of a common tepc within this population . using limiting dilution analysis ( hu and smyth , 2009 ) , we calculated the frequency of this putative common tepc within the ly-51plet1 population as 1/53.41/233 , with an average of 1/111 , at the 95% confidence limit ( figure 4 ; table 1 ) . the ly-51plet1 tec population contained both mhcii and mhcii cells ( figure 1b ) . the relative proportions of these fractions changed with age , such that at 4 weeks old , ly-51plet1 tecs were predominantly mhcii , with mhciily-51plet1 tecs increasing in proportion and number between 4 and 8 weeks of age ( mhcii : 4 weeks , 77.3 7.0 ; 8 weeks , 46.3 8.0 ; p = 0.001 ) . we therefore tested these subpopulations individually , to determine whether the common progenitor activity was restricted to one or other subset . this revealed that the mhciily-51plet1 tecs could generate both ctecs and mtecs ( figure 5 ; table 1 ; 4/4 grafts , input cell numbers 129 , 178 , 284 , 415 ) , including aire mtec and plet1 cmj tec areas ( figure 5c ) . large clones containing both ctecs and mtecs and spanning the cmj were observed in three out of four grafts seeded with ly-51plet1mhc ii tecs ( figures 5a5c ) , consistent with the presence of a common progenitor cell within this population . in contrast , the mhc ii fraction contained ctec - restricted progenitors but did not contribute to the cmj or medulla ( figures 5a , 5b , and 5d ; table 1 ; 3/3 grafts , input cell numbers , 188 , 226 , 260 ) . it is not yet clear whether the adult thymus is maintained by thymic epithelial stem cells ( tescs ) , or by a population of tepcs with limited self - renewal potential . to test whether the ly-51plet1 tec population identified above as containing a common tepc might contain bona fide tescs thus , two grafts seeded with gfply-51plet1 tecs were established as above and left for 9 months before analysis . we detected gfp tecs in the cortex and medulla of the 1,000 cell - seeded graft recovered at this time point , but no gfp cells in the 200 cell - seeded graft ( figure 6 ) . we also seeded two grafts with ly-51plet1 cells ( input numbers : 5,984 and 2,141 gfp cells , respectively ) ; no gfp cells were present in these grafts after 9 months . since the turnover time of adult tecs is established as 23 weeks ( gray et al . , 2006 ) , and furthermore , we analyzed the grafts well after the onset of age - related thymic involution , these data indicate that at least some tecs in the ly-51plet1 population are capable of generating long - term surviving progeny in contrast , the ly-51plet1 population appears to contain short - term ctec progenitors , rather than a ctec - restricted stem cell activity . the cellular mechanism that maintains homeostasis of the adult thymic epithelium has been of long - standing interest , but phenotypic identification of an adult thymic epithelial stem or progenitor cell ( tes / pc ) has remained elusive . evidence of a common tepc and of unipotent ctec- and mtec - restricted progenitors was found in a retrospective lineage tracing analysis performed in neonatal mice ( bleul et al . , 2006 ) , and each of these activities has been demonstrated in the fetal thymus ( rossi et al . , 2006 , shakib et al . , 2009 , forward lineage tracing data from young ( up to 3 weeks old ) mice are also consistent with the existence of both a common tepc and unipotent ctec- and mtec - restricted progenitors ( ohigashi et al . , 2015 , onder et al . , 2015 , however , to date , prospective isolation and functional testing has narrowed down the adult tec progenitor compartment only to a broad population comprising mhcii ctecs , that comprises 20% of the total adult tec population and contains both ctec- and mtec - generating activities ( wong et al . , 2014 ) . we have investigated the differentiation potential of six defined subpopulations of adult tecs using a validated transplantation - based assay of tec potency . our data show that a previously unidentified epcamuea1ly-51plet1 population , that comprises < 1% of total tecs , can efficiently generate both ctecs and mtecs at limiting dilution , suggesting that this population contains a common tepc . , the mhcii fraction could efficiently generate ctecs and mtecs , including clones spanning cortex , medulla , and cmj , again consistent with the presence of a common tepc , while epcammhciiuea1ly-51plet1 tecs generated only ctecs . we further demonstrated that epcamuea1ly-51plet1 tecs could generate non - medullary plet1 tecs , and input - derived cells could contribute to tec networks for at least 9 months in vivo , suggesting that this population may contain self - renewing thymic epithelial stem cells . our data also revealed the presence of short - term cortical sub - lineage - restricted progenitors within the ly-51plet1 ctec population . , we did not identify an efficient or frequent tepc activity within any uea1 tec population tested . epcam tecs derived from a foxn1 low / negative lineage were shown to initiate clonal spheroid cultures , under conditions similar to those used to generate mammary epithelial spheres ( ucar et al . , 2014 ) . the thymospheres could be passaged several times , although not indefinitely , and could contribute to some extent to ctec and mtec lineages in an rftoc assay similar to that employed herein . the ly-51plet1 population that we identify here as tepcs are clearly distinct from this population , as ly-51plet1 tecs are epcam and express foxn1 . further analysis is required to determine the relationship , if any , between these populations . however , we note that the relatively extended culture period employed during derivation and passage of the thymospheres may have affected cellular potency ( ucar et al . , 2014 ) . in the second report , tepc activity was detected in the epcamuea1mhcii population , which comprised 20% of all tecs ( wong et al . , 2014 ) . although that report hinted at a common tepc , such an activity was not demonstrated due to the high numbers of input cells used ( 7.5 10 to 1 10 test cells per graft ) ( wong et al . , 2014 ) . our studies indicate that a high efficiency , short - term , ctec - restricted progenitor activity exists within the epcamly-51uea1plet1mhcii tec population , which overlaps with the epcamuea1mhcii population identified by wong et al . we additionally detected a low frequency and low efficiency capacity to generate mtecs within the epcamly-51uea1 plet1mhcii subset of epcamuea1mhcii tecs . while this activity does not appear to represent a high efficiency mtec progenitor , it may reconcile our findings with those of wong et al . ( 2014 ) , especially once the different input cell numbers tested in the two studies is considered . furthermore , although a cldn3/4ssea1 mtec - restricted progenitor was recently identified in the fetal thymus and was shown to both sustain the mtec lineage in long - term assays in grafted reaggregates and initiate clonogenic in vitro cultures , cells of this phenotype in the adult thymus had markedly diminished progenitor capacity compared to their fetal counterparts and could not be cultured in vitro ( sekai et al . , 2014 ) . the absence of a robust mtec - restricted progenitor cell in our analyses is therefore consistent with both this study ( sekai et al . , 2014 ) and that of wong et al . in addition to the high frequency ctec progenitor activities detected in epcamly-51uea1 tecs , we identified a high efficiency common tepc within epcamplet1ly-51uea1mhcii tecs and provided evidence that cells within this population could contribute to generation of ctecs and mtecs for at least 9 months , suggesting the population may contain a bipotent tec stem cell . considering all of the above data , we suggest that this common progenitor / stem tec is likely to be upstream of the epcamuea1mhcii tecs progenitor populations discussed above in the tec cellular hierarchy . the presence of a very low frequency , low efficiency mtec - generating activity within epcamly-51uea1mhcii tecs may reflect plasticity of cell potencies , as shown for stem / progenitor cells in many other lineages ( snippert et al . 0.5% of total tecs , our data represent an enrichment for progenitor / stem cell activity of > 30-fold over the previous report ( wong et al . , 2014 ) . furthermore , our immunohistochemical analyses indicate that this population is located at the cmj and consistent with this , cmj - located plet1 tecs were present in grafts derived from epcamplet1ly-51uea1 tecs . our data thus provide the physical location of a phenotypically defined bipotent adult tec progenitor cell population . it is well established that plet1 marks the founder cells of the thymic epithelial lineage ( gordon et al . , 2004 , , 2008 , moore - scott et al . , 2007 , manley et al . , 2011 ) , that is , the earliest cell , which in vivo will generate thymic epithelial but not other lineage fates during organogenesis ( smith , 2006 ) . the status of plet1 as a tepc marker has , however , been disputed ( rossi et al . , 2007a ) . in each of two clonal analyses demonstrating a fetal common tepc ( bleul et al . , 2006 , rossi et al . , 2006 however , while at developmental stages up to and including e16.5 fetal plet1 tecs can initiate de novo thymus organogenesis ( bennett et al . , 2002 , gill et al . , 2002 , rossi et al . , 2007a ) , this capacity is lost by e18.5 ( rossi et al . , 2007a ) . this finding was taken to indicate that plet1 cells do not maintain the thymus beyond the later stages of fetal development ( i.e. , e16.5 ) ( swann and boehm , 2007 ) , but the alternative explanation , that fetal and adult progenitors might exhibit differential capacities with respect to initiation of de novo organogenesis , was not explored . in the present study , the assay of tec potency used did not require the test population to initiate organogenesis , since this property is not required in adult stem / progenitor cells . ( 2014 ) , we found that the majority of plet1 tecs were unable to contribute to tec networks . however , this assay identified a rare subpopulation of ly-51plet1mhcii tecs as the only adult tec population to contain a bipotent tepc , based on clonal resolution analysis demonstrating active differentiation of ly-51plet1ii input tecs . taken together with previous findings , our data therefore suggest that fetal and adult tepcs have overlapping but not identical properties ; specifically , that the adult tep / sc share very similar or identical differentiation potential to the equivalent fetal population but have lost the capacity to initiate organogenesis de novo . detailed exploration of the relationship between fetal and adult tepcs , and in particular of the transition from fetal to adult functionality , will thus be of interest . in sum , our data provide a phenotypically defined adult tep / sc that represents 0.5% of total tecs . they thus overcome the major hurdle to precise cellular and molecular understanding of the mechanisms regulating thymus homeostasis and age - related thymic involution and the remarkable regenerative capacity of the adult thymus ( dudakov et al . bredenkamp et al . , 2014a ) and pave the way for new regenerative and cell replacement approaches to improving thymus function in patients . cbaxc57bl/6 f1 mice were used for isolation of embryonic tecs and mouse embryonic fibroblasts . for timed matings , c57bl/6 females were housed with cba males and noon of the day of the vaginal plug was taken as e0.5 . cbaxc57bl/6 f1 and nude mice were used at 610 weeks of age as graft recipients . all animals were housed and bred at the institute for stem cell research / centre for regenerative medicine ( iscr / crm ) animal facilities and all experimental procedures were conducted in compliance with the home office animals ( scientific procedures ) act 1986 under license number ppl60/4435 . adult throughout the manuscript in line with information on the jackson laboratories website ; this nomenclature is consistent with current understanding of thymus development , where the thymus attains its adult architecture by 4 weeks of age . murine embryonic fibroblasts ( mefs ) were prepared as previously described ( bennett et al . , 2002 ) . adult thymi and grafted rftoc were processed for flow cytometric sorting and analysis as previously described ( nowell et al . , 2011 , the potency of purified adult tec subpopulations was tested by their ability to differentiate within a functional thymus after grafting under the kidney capsule , as previously described ( bennett et al . , 2002 ) . briefly , defined numbers of gfp adult tecs were mixed with 200,000 wild - type e12.5 or e13.5 fetal thymus cells and 200,000 mefs and reaggregated for 1216 hr in dmem f12 ( invitrogen ) supplemented with 10% fcs , 1% hepes , 2% amino acids , 50 u / ml penicillin , and 50 g / ml streptomycin . reaggregates were then grafted under the kidney capsule of 5- to 10-week - old male f1 or nude mice . grafting into syngeneic and nude mice yielded identical results ( tested using gfpplet1 tecs ; not shown ) . for the data shown in table 1 , for most gfp areas compartmental localization was unambiguous and comprised clusters of gfp cells with a burst size of 20 cells that were clearly integrated into the tec network . in some instances , only a small number of individual gfp tecs of the appropriate phenotype were present in a particular area . we also found one or two instances of an atypical gfp cluster , where gfpk14 cells possibly associated with a cyst were located in a clearly cortical area . these staining patterns are indicated in the text and in table 1 . throughout the text , efficient contribution refers to the first type of cluster . where we refer to high or frequency , this describes the number of clusters of a particular type observed under the particular test condition . for analysis every section was scored for the presence of gfp cells , which were identified as cortical or medullary clusters based on the morphology and position of the cells in the graft . every section ( figure 4 ) or every tenth section ( figures 3 , 5 , and 6 ) was then analyzed by immunohistochemistry in order to confirm the cortical or medullary tec sub - lineage identity of the gfp cells . for figures 4 and 5 , all gfp areas were tracked through the sections in order to map their contributions in the graft . appropriate isotype and negative controls were included in all experiments . for detection of immunofluorescence , slides were examined with a leica aobs ( leica microsystem , gmbh ) or spe confocal microscope . qrt - pcr was performed as previously described ( bredenkamp et al . , 2014b ) , on 50 cells per sample . data are shown after normalization to the geometric mean of three control genes ( hprt , ywhaz , hmbs ) . data analysis was carried out using lightcycler 1.5 software and the ct method ( livak and schmittgen , 2001 ) . statistical analysis for table 1 was performed using the one - way anova test ( two - tailed ) , as appropriate for normally distributed data ( normal distribution was tested using goodness of fit ) . sample sizes of at least n = 3 were used for all analyses except where indicated . limiting dilution analysis no statistical method was used to predetermine sample size , the experiments were not randomized , and the investigators were not blinded to allocation during experiments and outcome assessment . no samples were excluded from the analysis except in the transplantation assay , where grafts that displayed large cystic areas or failed to grow were discarded such that only fully formed organoids were taken forward for analyses of the presence and location of gfp tecs . conceived and designed experiments , performed experiments , analyzed the data and contributed to writing the manuscript . c.c.b . conceived the original idea , designed experiments , contributed to analysis of the data , and wrote the manuscript .

summarythymic epithelial cells ( tecs ) are critically required for t cell development , but the cellular mechanisms that maintain adult tecs are poorly understood . here , we show that a previously unidentified subpopulation , epcam+uea1ly-51+plet1+mhc class iihi , which comprises < 0.5% of adult tecs , contains bipotent tec progenitors that can efficiently generate both cortical ( c ) tecs and medullary ( m ) tecs . no other adult tec population tested in this study contains this activity . we demonstrate persistence of plet1+ly-51 + tec - derived cells for 9 months in vivo , suggesting the presence of thymic epithelial stem cells . additionally , we identify ctec - restricted short - term progenitor activity but fail to detect high efficiency mtec - restricted progenitors in the adult thymus . our data provide a phenotypically defined adult thymic epithelial progenitor / stem cell that is able to generate both ctecs and mtecs , opening avenues for improving thymus function in patients .

exaggerated placental site ( eps ) is defined as a non - neoplastic trophoblastic lesion where middle trophoblasts infiltrate exaggeratedly into endometrium and myometrium . it consists of cells showing the same immunophenotypical features as the intermediate trophoblasts in the normal placental implantation site , and is observed as an exaggerated form of the normal physiological process.1 these lesions may occur following normal pregnancy , ectopic pregnancy , abortus or molar pregnancy . they are seen in 1.6% of the first trimester abortuses.23 despite the fact that eps is non - neoplastic , it may infiltrate prominently into myometrium to generate cordons , and it should be histologically distinguished from the neoplastic lesions , particularly the placental site trophoblastic tumours ( pstt ) . it is important to make the differential diagnosis as the pstt cells of the neoplastic proliferation of intermediate trophoblasts and the eps cells have similar cytological and immunophenotypical features.4 a 26-year - old patient , gravida 3 para 1 , applied with delayed menstruation of 6 weeks , and nausea to our clinic . the uterus was found to be 10-week size in pelvic examination , and a 10 8 cm heterogeneous mass with solid and cystic fields was seen in uterine cavity by abdominopelvic ultrasonography . pre - treatment blood beta human chorionic gonadotropin ( -hcg ) level was measured to be 279.000 miu / ml , and no additional signs were found in systemic examination or scanning . trophoblastic cells were positive with pancytokeratin ( panck ) and high molecular weight cytokeratin ( 34be12 ) , but negative with p63 in sites of eps , whereas the ki 67 proliferation index was stated to be 2 - 3% . the -hcg declined to a level of 6700 miu / ml in the first 4 weeks , but in the following three measurements it increased again . thereupon , single agent methotrexate was given weekly , and remission was achieved after 3 weeks of treatment . infiltration of middle trophoblasts into myometrial smooth muscle layer ( haematoxylin and eosin , 10 ) eps is defined as a non - neoplastic trophoblastic lesion where middle trophoblasts infiltrate exaggeratedly into endometrium and myometrium . it consists of cells showing the same immunophenotypical features as the middle trophoblasts in the normal placental implantation site , and is observed as an exaggerated form of the normal physiological process where the number of these cells are increased.1 this lesion was formerly defined as syncytial endometritis , but this is neither an inflammatory process nor it is limited within the endometrium . therefore the term exaggerated placental site has been recommended by the world health organisation.5 eps may occur following normal pregnancy , ectopic pregnancy , abortus , or molar pregnancy . cases were reported in english literature following spontaneous abortus , elective abortus , c - section delivery and along with placenta previa , but this is the first case together with complete hydatidiform mole.1678 the distinction between normal and eps is subjective because there is no exact information about the proportion and coverage of intermediate trophoblast infiltration in the implantation site in different periods of normal pregnancy.5 while most of the cells constituting this lesion are the mononuclear intermediate trophoblasts , there are also multinuclear intermediate trophoblasts infiltrating in various rates . despite the infiltration of intermediate trophoblastic cells , the structure of implantation site is not impaired in most cases . however , necrosis and degeneration may typically be seen in desidual vicinity in cases of spontanous abortus . other signs of pregnancy such as hyalinised spiral arteries , hypersecretory glands and chorionic villi are usually present.5 it is important to make the differential diagnosis as the pstt cells of the neoplastic proliferation of intermediate trophoblasts and the eps cells have similar cytological and immunophenotypical features . beside these morphological similarities , as the trophoblasts in early pregnancy have primitive appearances and invade the spiral arteries intensively , classic histological criteria distinguishing a malignant process from a benign one fail to be valid . in our case eps was seen in areas together with complete hydatidiform mole , and remission in -hcg levels was not provided following the evacuation of mole . therefore , there seemed to be a persistent gestational trophoblastic disease in this patient , and it was important to distinguish this case from pstt . while a concurrent pregnancy and no mass in the myometrium is helpful for the diagnosis of eps , it is in favour of pstt if it occurs months after a term pregnancy or abortus , with presence of a mass in the myometrium.3 whereas absence of chorionic villi , presence of mitoses and trophoblastic cells constituting joined masses microscopically support the diagnosis of pstt , presence of chorionic villi and absence of mitosis in contrast favour the reaction of eps.910 cases in which distinction is hard to make , ki-67 proliferation index is considerably useful . natural killer cells and activated t lymphocytes are ki-67 positive . while ki-67 proliferation index being less than 5% is in favour of eps , levels more than 5% support pstt . ki-67 proliferation index being close to zero despite the excessive trophoblastic infiltration , brings to mind that the number of increasing middle trophoblasts in implantation site is not dependent of de novo proliferation.5 eps and pstt have similar profiles immunohistochemically . interme diate trophoblasts in implantation site are stained positively with cytokeratin and human placental laktogen ( hpl ) , cd 146 ( mel - cam ) , human leucocyte antigen - g ( hla - g ) and e - cadherine , whereas they are negative with epithelial membrane antigen and ber - ep4.591112 intermediate trophoblasts in the implantation site and the pstt cells do not express p63 . the distinction between the trophoblastic diseases can be done using p63 , hla - g , cytokeratin , hpl and ki-67.13 our case was diagnosed with eps with p63 being negative , cytokeratin being positive and ki-67 proliferation index being less than 5% . eps is a benign trophoblastic lesion and do not require a special treatment or follow - up . it is difficult to distinguish it from pstt morphologically and immunohistochemically , and ki-67 proliferation index is recommended for this distinction .

exaggerated placental site ( eps ) is defined as a non - neoplastic trophoblastic lesion , which intermediate trophoblasts infiltrate exaggeratedly into endometrium and myometrium . these lesions may occur following normal pregnancy , ectopic pregnancy , abortus or molar pregnancy . herein we share a case of eps detected after evacuation due to molar pregnancy . we also review the related literature , where only a few reports exist describing the clinical course , histopathology and differential diagnosis of eps .

since the early 1970s , botulinum toxin type - a has been used for medical applications . in 2002 the us food and drug administration ( fda ) granted approval for cosmetic use of botulinum toxin type - a ( boto - a ) ( botox ; botox cosmetic ; allergan , irvine , ca , usa ) in the treatment of glabellar rhytids . in 2009 the fda approved the second boto - a ( dysport ; ipsen limited , berkshire , uk ) for use in glabellar lines as well . the new availability of a similar , competing product reinforces the utility of a contemporary review of applications of botulinum toxin type - a in the glabellar brow region . there are at least 7 subtypes of the neurotoxin produced by the bacterium clostridium botulinum , but type - a is the most commonly used , and the only one currently approved for cosmetic use in the united states . its selection as the primary therapeutic neurotoxin is due to early animal studies that demonstrated it was the most potent and longest lasting of all subtypes.1 boto - a naturally exists as a protein complex , for which an acidic environment is necessary for stabilization . the average onset of clinically evident action is several days for both botox and dysport . this period of onset is related to the need for the process of endocytosis of the toxin into the cells of the nerve terminal with eventual disruption of the snap-25 protein that is responsible for release of acetylcholine into the neuromuscular junction . paralysis therefore results from blockade of the conversion of an electrical signal from the nerves to the chemical signal at the neuromuscular junction.1 recent reports of undesirable distant effects from boto - a which can resemble botulinum poisoning have surfaced . symptoms reported include dysarthria , dysphagia , loss of bladder control , and difficulty breathing.2 it is important to note , however , that these small numbers of reported severe adverse events were in patients receiving very large doses for conditions such as large muscle spasticity related to cerebral palsy and cervical dystonia.3 there have been no reports of distant complications related to cosmetic use of boto - a in the face . a single botox unit is equivalent to the median lethal dose ( ld50 ) in mice when given intraperitoneally . units between botox and dysport are not equivalent and it has been suggested that the ratio equivalents of botox to dysport range from 1:2.5 to 1:5.4 both manufacturers recommend using preservative - free saline with gentle reconstitution utilizing the vacuum in the vial to draw in the saline . aggressive injection of saline resulting in foaming could theoretically denature the neurotoxin protein complex . for botox only , in our experience , a dilution of 4 ml of saline resulting in 2.5 u per 0.1 ml injection results in highly reliable , effective and lasting results in the treatment of glabellar rhytids . the manufacturers for both botox and dysport recommend using the reconstituted product within four hours although botox has been shown to be effective when preserved in a frozen state up to 6 months5 and in our experience for up to one month when refrigerated . initial reports with dysport suggest that the while it is as efficacious and safe as botox , the primary potential benefit is that a small subset of patients may have a very prolonged effect with normal dosage ( eg , greater than 6 months).6 the main potential drawback is an increase in diffusion of dysport owing to its smaller size ( 500 to 900 kda ) compared to botox ( 900 kda).4 while boto - a is used off - label ( that is , for applications not specifically approved by the fda ) in many practices , including for treatment of facial rhytids around the eyes or from the frontalis muscle , the only approved cosmetic indication for either botox or dysport is in the treatment of glabellar rhytids . carruthers and carruthers7 noted that patients being treated with botulinum toxin for blepharospasm also had the added benefit of improved appearance . several studies since have reproduced the safety and efficacy of boto - a for the treatment of glabellar rhytids . mastery of the anatomy of the glabellar complex is paramount for treatment of glabellar rhytids with boto - a . anatomical study has demonstrated that the thickest portion of the corrugator muscle is found consistently at the medial canthus , but with a significant amount of muscle bulk at the midpupillary line . the depth of the corrugator from the surface of the skin is constant from medial canthus to mid - pupil . there does not appear to be a vertical segment of the corrugator nor does there appear to be an anatomically distinct depressor supercilii as suggested by other authors as being a significant contributor.8,9 appropriate therapeutic botox dosing for treatment of the glabellar rhytids has been reported as anywhere from 20 to 50 u over several injection sites . dose - ranging studies for men and women in botox have been performed and they suggest that 40 u total over the area is effective in women and the minimum recommended starting dose for men.10,11 our experience has been that approximately 95% of patients in our practice have good response and no serious complications using only 7.5 u for each corrugator , 5 u medially , 2.5 u mid - pupil and 2.5 u into the procerus . there is no need to inject the obicularis medially or laterally to successfully treat glabellar rhytids as suggested by other authors.7 thus recommendations for injection ( botox ) are as follows ( figure 1 ) . if you have an adverse aesthetic outcome in the use of boto - a , you can take solace in the knowledge that the effects are temporary . if the patient is pleased with the outcome , they must repeat injections to sustain an effect ; however , if a patient experiences any complications they , too , are temporary . there are , however , a few more significant potential complications directly related to the injections that should be reviewed . the severe systemic effects as described in the fda black box warning have not been reported in the cosmetic use of botox or dysport . eyelid ptosis is caused by effects of the toxin on the upper eyelid levator muscle . this can occur if the toxin is directly injected into the muscle or close enough for the toxin to diffuse to the levator . currently the recommendation is to not inject at or under the brow in the mid - pupil line , as this is the area of highest risk . alpha - adrenergic eye drops ( apraclonidine ) can be used as a mydriatic agent if this complication occurs , and can provide temporary improvement in eyelid symmetry . the alpha - adrenergic medication causes contracture of mueller s muscle , located just deep to the levator , and can thus improve the ptosis by a few millimeters . allergan s fda study for approval of cosmetic botox reported eyelid ptosis at 5.4% ; however , this significantly drops once the physician s experience increases.12 with the above treatment regimen described our complication rate of eyelid ptosis is 0% over the past 10 years . minor complications such as pain , bruising and erythema are related to the needle necessary for injection and usually resolve within minutes to a few days ( in the case of ecchymosis ) . boto - a products should never be used in patients with neuromuscular junction disorders such as myasthenia gravis or lambert - eaton , and they are not recommended for pregnant women.13 hypersensitivity or allergic reactions have not been reported , but resistance to the effect of botox due to antibody - mediated immunoresistance to the neurotoxin its prevalence is less than 5% , however , and appears to be related to frequency of treatment , not duration of the regimen.14 boto - a has been proven to be a safe and effective means to treat glabellar rhytids . the new addition of dysport as an alternative to botox requires further experience in its application but early studies show promise that it is a viable alternative . the few feared systemic complications have been reported only when the toxin is used for large muscle disorders and in much higher doses than typically used for cosmetic indications . serious complications such as eyelid ptosis can be avoided through judicious dosage selection and mastery of the underlying muscle anatomy .

the current literature and state of the art on use of botulinum toxin type - a in the treatment of glabellar rhytids was reviewed . botulinum toxin type - a is a safe and effective way to manage glabellar rhytids . mastery of the anatomy of the glabellar region , familiarity with the products currently available on the market and judicious dosing results in excellent cosmetic results with minimal adverse events .

esophageal cancer has a high mortality rate and is rising in incidence in the western world . in 2006 there were an estimated 15,000 new cases of esophageal cancer in the united states with almost 14,000 deaths , representing 4% of all cancer deaths . most patients present with unresectable or stage iv disease and the median survival of those with stage iv disease who undergo palliative therapy is less than one year . if diagnosed at an earlier stage , esophagectomy is potentially curative but is associated with a high morbidity and high rate of distant recurrence . there is evidence that neo - adjuvant chemo - radiotherapy prior to surgery can improve the 3-year survival and radiation can reduce local - regional cancer recurrence . because the most favorable results are in those patients who have a pathologic response to pre - operative chemoradiation therapy , optimal patient selection is critical . positron emission tomography ( pet)/computed tomography ( ct ) can be helpful in identifying the subset of patients who are responders to pre - operative therapy and can also detect distant metastases , thereby excluding those patients who will not benefit from esophagectomy . radiation treatment of distal esophageal cancers may include the adjacent liver parenchyma , which can appear on pet / ct as an area of increased fluorodeoxyglucose ( fdg ) uptake within the liver parenchyma that mimics metastatic disease . the objective of this study was to evaluate the imaging appearance of radiation injury in the liver on pet / ct in patients with distal esophageal cancer who underwent pre - operative chemoradiation therapy . we performed a retrospective review of consecutive patients with distal esophageal cancer , who underwent neoadjuvant chemoradiotherapy prior to surgery between january 2004 and february 2005 after obtaining institutional review board approval . all patients received 50.4 gy of radiation therapy to the distal esophagus using a 3d conformal technique . all patients underwent baseline pet / ct prior to treatment , followed by a repeat pet / ct scan performed 6 weeks following completion of chemoradiotherapy for the purposes of assessing local tumor response and for detection of new distant metastases . patients were excluded if they had liver metastases , fatty liver , liver cirrhosis or had undergone previous hepatic surgery . all pet / ct examinations were performed on an integrated pet / ct scanner ( discovery st-8 , general electric medical systems , milwaukee , wi ) . blood glucose was checked approximately 4 h prior to scanning and was less than 165 g / dl ; no patients in our study had hyperglycemia requiring deferment of their scans . one hour prior to scanning , patients were injected with a mean of 15 mci ( range 1220 mci ) of radioactively - labeled fdg . pet studies were acquired from the skull base to the upper thighs in two - dimensional mode for 3 min per bed position . pet images were reconstructed using standard vendor - provided reconstruction algorithms which incorporated ordered subset expectation maximization ( osem ) . attenuation correction of pet images was performed using attenuation data from the ct component of the exam ; emission data were corrected for scatter , random events and dead - time losses using the manufacturer 's software . the ct component of the study comprised a non - contrast multidetector ct examination from the base of the skull to the upper thighs ( 120 ma , 140 kvp , table speed 13.5 mm / rotation ) . images from the pet / ct scans were reviewed by consensus at a combined interpreting session by two diagnostic radiologists with experience in interpreting pet / ct scans . the ct , pet and fused pet / ct images were reviewed together in multiple planes ( axial , sagittal , and coronal ) on a ge advantage windows workstation . on ct , images of the liver were examined using both soft tissue ( center , + 40 hu , width , 300 hu ) and liver ( center , + 50 hu , width , 150 hu ) windows . in each patient , maximum standard uptake values ( suvmax ) were measured in the liver using 3d region of interest cursors placed over the left and right lobes of the liver on pet / ct scans performed before and after radiotherapy ; the regions of interest were drawn to include the maximum amount of liver tissue in each lobe without including adjacent tissues . on the ct images , changes such as atrophy and decrease in attenuation of the liver were subjectively evaluated and also documented on scans performed before and after radiotherapy . lastly , the results of laboratory liver function tests were documented at baseline and at 6 weeks following completion of radiotherapy . descriptive analyses were used for patient age , sex , time intervals between scans and imaging findings . to test for differences in the detection probability for radiation injury between pet and ct , we used mcnemar 's test for paired binomial observations . descriptive analyses were used for patient age , sex , time intervals between scans and imaging findings . to test for differences in the detection probability for radiation injury between pet and ct , we used mcnemar 's test for paired binomial observations . a total of 26 patients with distal esophageal cancer were included in the study ( 24 men , 2 women ; mean age 54 years , age range 4178 years ) . histology of the esophageal tumors was 24 adenocarcinomas and 2 squamous cell carcinomas . on baseline pet / ct imaging , there was homogenous fdg uptake within both lobes of the liver with a mean suvmax of 3 ( range 26 ) . there were no significant differences in fdg uptake between the left and right lobes of the liver . pet / ct following radiotherapy , 5 ( 19% ) patients demonstrated changes in the pattern and intensity of fdg uptake . in 2 ( 8% ) of these patients , focal areas of increased fdg uptake were found in the left lobe of the liver adjacent to the irradiated esophageal tumor ; in both of these patients , the suvmax increased by over 50% compared to their baseline scan ( by 54% and 133% , respectively ) . no significant change in the level of fdg uptake was seen within the right lobe of the liver in these two patients . on the ct of these two patients , there was no liver abnormality to indicate metastasis , but there was a well defined diffuse low attenuation characteristic of radiation injury . in one patient a focal area of increased fdg uptake was thought to represent radiation injury and a biopsy at the time of esophagectomy confirmed the presence of radiation injury and the absence of metastatic disease . the remaining three patients demonstrated either diffuse increase in fdg uptake ( > 50% over baseline ) in the right lobe of the liver with no abnormality in the left lobe ( n = 2 ) or decreased fdg uptake ( > 50% over baseline ) in both the left and right lobes of the liver ( n = 1 ) . on ct , characteristic findings of decreased attenuation and atrophy were seen within the left lobe of the liver in 15 ( 58% ) patients . two of these patients also had greater than 50% increase in fdg uptake in the left lobe of the liver . abnormalities in liver function tests were seen in only 3 ( 12% ) patients at the time of re - evaluation following chemoradiotherapy , all three of whom demonstrated increases in alkaline phosphatase by over 200% . two of these three patients had corresponding increases in fdg uptake within the left lobe of the liver ( > 50% over baseline ) . the 95% confidence interval for the pet detection probability of radiation injury as manifested by focal changes in suvmax is ( 0.024 , 0.302 ) based on 3/26 detections using the clopper pearson confidence interval . for ct , the 95% clopper pearson interval for detection of the characteristic well - defined low attenuation of radiation injury is ( 0.369 , 0.766 ) , based on 15/26 detections . when comparing the probability of detecting radiation injury on ct versus pet at this time point after radiotherapy , the p - value for mcnemar 's test of no difference in detection probability is less than 0.003 ( i.e. , p < 0.003 ) . clinical radiation injury in the liver may occur in 666% of patients depending upon the volume of hepatic tissue irradiated and the dose used . factors that increase the likelihood of hepatic toxicity include whole - liver irradiation and doses greater than 30 gy . in esophageal cancer patients undergoing neo - adjuvant therapy , three - dimensional ( 3d ) conformal therapy or intensity modulated therapy ( imrt ) is employed to increase the radiation dose to the primary tumor while limiting damage to surrounding healthy tissue . however , given the anatomical location of the lateral segment of the left lobe of the liver adjacent to the distal esophagus , radiation injury to the liver is difficult to avoid . the irradiated liver appears hypodense on non - contrast ct scans and can be seen in patients who receive more than 45 gy to a portion of the liver , regardless of whether they develop symptoms or other signs of radiation - induced hepatotoxicity . in the acute phase of radiation - induced liver injury , ct demonstrates a well - demarcated area of low attenuation within the hepatic parenchyma that corresponds to the radiation ports used , likely due to edema or fatty infiltration . the fdg - pet findings in radiation injury to the liver have been less well described . reported their findings of the utility of pet / ct in the assessment of liver tissue after intraoperative radiation therapy in a pig model . they found that pet / ct showed a decrease in the uptake of fdg in the irradiated field at 2 and at 4 weeks following completion of intraoperative therapy using 20 gy . at 8 weeks the distribution of the tracer in the irradiated pigs did not differ from that in non - irradiated animals . in our study of patients with esophageal cancer , at 6 weeks after completion of therapy , changes in fdg uptake in the irradiated portion of the liver were seen in 12% ( three patients ) and were somewhat variable ( two patients demonstrating increased fdg uptake and one patient showing decreased uptake ) while characteristic ct changes of atrophy and decreased attenuation were seen in 58% ( 15 ) of patients . the variable fdg uptake at 6 weeks suggest that pet imaging may be less sensitive than ct in detection of established liver injury after therapy is completed . metabolic changes in the liver may have been detected more often if pet imaging had been performed earlier . two additional patients in our study were also noted to have alteration in fdg uptake diffusely in the non - irradiated right lobe of the liver without corresponding ct abnormalities and this finding is more difficult to explain . vascular changes resulting from radiotherapy could cause differential flow to the irradiated and non - irradiated portions of the liver resulting in changes in fdg distribution . these more diffuse metabolic changes in the right lobe might relate to systemic changes from the concurrent chemotherapy that was also administered . clinically , acute radiation hepatitis generally occurs between 2 and 6 weeks after therapy and is characterized by ascites and right upper quadrant discomfort with associated elevation of liver function tests , typically doubling of the alkaline phosphatase , which subsequently returns to normal on ct , imaging findings of established liver injury are not usually found in these patients until a later stage , some 23 months after completion of radiation therapy . however , the results of our study suggest that mild sub - clinical forms of radiation hepatitis can be detected by pet / ct earlier ; in the two patients in our group who demonstrated increased fdg uptake in the left lobe of the liver 6 weeks after completion of radiation therapy , there were accompanying abnormalities in alkaline phosphatase . however , both patients were asymptomatic and did not subsequently develop radiation induced liver disease . it is also important to remember that patients with locally advanced esophageal cancer are also at risk for developing distant metastatic disease . when patients present for re - evaluation after neo - adjuvant therapy , it is important to exclude metastatic disease in order to prevent futile surgeries . pre - operative whole body pet / ct is particularly useful in the re - evaluation of esophageal patients to determine treatment response and exclude metastasis . given the propensity of hepatic metastases in these patients , it is important to recognize fdg avid lesions in the liver that may be metastatic . however , any fdg avid areas in the liver should be correlated for associated anatomic abnormality in the liver on ct . the appearance of radiation injury on ct is quite characteristic and generally shows sharp , straight margins that correspond to the portals used ( fig . metastatic lesions , on the other hand , are generally more mass - like and rounded in contour on ct ( fig . 2 ) . as noted in our data , radiation injury to the liver may be fdg avid in a small number of cases and should not be confused with fdg avid hepatic metastatic disease . figure 1a 63-tear - old male with adenocarcinoma of distal esophagus treated with 50.4 gy with 3d conformal technique 6 weeks earlier . baseline pet showed fdg avid primary tumor only . fused pet / ct coronal ( a ) , sagittal ( b ) and axial ( c ) images and coronal mip ( d ) image show increased fdg uptake focally ( arrow ) in the left lobe of the liver and an fdg avid primary distal esophageal tumor ( arrowheads ) . axial non - contrast ct ( e ) shows well demarcated low attenuation ( curved arrow ) compatible with radiation injury subsequently proven by biopsy . figure 2a 71-year - old male status post chemoradiation and esophagectomy 18 months ago , now with fdg avid liver metastasis ( arrow ) to the left lobe of the liver as seen on coronal ( a ) , axial ( b ) fused pet / ct images as well as axial ( c ) pet and axial non - contrast ct ( d ) . a 63-tear - old male with adenocarcinoma of distal esophagus treated with 50.4 gy with 3d conformal technique 6 weeks earlier . baseline pet showed fdg avid primary tumor only . fused pet / ct coronal ( a ) , sagittal ( b ) and axial ( c ) images and coronal mip ( d ) image show increased fdg uptake focally ( arrow ) in the left lobe of the liver and an fdg avid primary distal esophageal tumor ( arrowheads ) . axial non - contrast ct ( e ) shows well demarcated low attenuation ( curved arrow ) compatible with radiation injury subsequently proven by biopsy . a 71-year - old male status post chemoradiation and esophagectomy 18 months ago , now with fdg avid liver metastasis ( arrow ) to the left lobe of the liver as seen on coronal ( a ) , axial ( b ) fused pet / ct images as well as axial ( c ) pet and axial non - contrast ct ( d ) . limitations of our study stem from its retrospective nature and from the fact that histological confirmation of radiation - induced liver damage was not obtained in all cases . however , it seems reasonable to assume that , in the absence of metastatic liver disease ( as confirmed by subsequent follow - up ) and the location of increased fdg uptake in the left lobe of the liver in most cases , the abnormalities observed on pet / ct were indeed due to radiotherapy . another limitation of this study was that the liver was imaged with pet / ct at only one time point ( 6 weeks ) after neoadjuvant therapy and any changes in fdg uptake before this time point would not have been detected . in patients with distal esophageal cancer receiving neoadjuvant chemoradiotherapy , pet / ct can detect metabolic abnormalities in the adjacent liver parenchyma due to its inclusion in the radiation therapy port . such abnormalities should be correlated with other imaging , clinical and laboratory findings in order to avoid confusion with hepatic metastases .

abstractthis paper evaluates the imaging appearance of radiation injury in the liver on positron emission tomography ( pet)/computed tomography ( ct ) in patients with distal esophageal cancer who underwent pre - operative chemoradiation therapy . twenty - six patients with distal esophageal cancer who received chemoradiotherapy before esophagectomy were included . all patients had baseline and follow - up pet / ct . fluorodeoxyglucose ( fdg ) uptake in both left and right lobes of the liver was evaluated . ct findings suggesting radiation damage were documented . abnormal fdg uptake in the liver was observed in 5 ( 19% ) patients after therapy . these abnormalities were in the left lobe ( 12% ) and right lobe ( 12% ) of the liver . in the irradiated left lobe , fdg uptake increased focally greater than 50% over baseline in two patients ( 54% and 133% ) ; in one of these patients , biopsy confirmed radiation injury . in the non - irradiated right lobe , standard uptake values ( suv ) increased diffusely in two different patients . in one patient , suv decreased by at least 50% in both the right and left lobes . in the remaining patients , there were no significant changes in fdg uptake . atrophy and attenuation changes of irradiated liver on ct were found in 15 ( 58% ) patients . in patients receiving chemoradiotherapy , pet / ct may identify metabolic abnormalities in irradiated liver . such abnormalities should be correlated with other imaging , clinical and laboratory findings to avoid confusion with hepatic metastases .

there are several ways how to perform the controlled ovarian hyperstimulation ( coh ) in patients included in the in vitro fertilization program and each one has its advantages and disadvantages . development of suitable gnrh agonists in the 1980s represented the major progress in the field [ 1 , 2 ] . the most important characteristic of gnrh agonists is prevention of premature lh surge in coh through desensitization of pituitary , which helps to increase the number of retrieved oocytes and decrease the number of cancelled cycles . on one side , this is a good property , but , on the other side , it can lead to the ovarian hyperstimulation syndrome ( ohss ) or some other complications and side effects . due to these deficiencies of gnrh agonists , development of gnrh antagonists represented a major breakthrough because they cause less side effects [ 4 , 5 ] . gnrh antagonists also reduce fsh / lh secretion and in this way they prevent lh surges although their mode of action is opposite to that of gnrh agonists . gnrh agonists bind to their receptor on pituitary and with maintaining the signal they cause desensitization of pituitary and consequently the downregulation of gonadotropin secretion after prolonged time . also gnrh antagonists bind to the receptor on a pituitary but they block it almost straight away and consequently cause the suppression of gonadotropin secretion within a few hours . there are several variations in the protocol of coh using each of the gnrh analogue , but , to simplify , in the conventional long protocol the gnrh agonists are applied from 7 days before menstruation , while gnrh antagonists are applied on a fixed day of ovarian stimulation or when the size of the leading follicle is 14 mm . in the last years also so called mild protocol of coh was introduced into clinical practice , in which the exogenous gonadotropins are administered at lower doses for a shorter duration in a combination with gnrh antagonists , antiestrogens , or aromatase inhibitors by definition of the international society for mild approaches in assisted reproduction ( ismaar ) . the advantages of such approach are especially in lower dose of used gonadotropins ( consequently more kind to patients and lower costs ) and less side effects without impairment of cumulative pregnancy rate . in spite of that , the number of retrieved oocytes and proportion of cycles with embryo cryopreservation seem to be lower . although the question about the mechanism of gnrh agonists and gnrh antagonists action is well answered , there is still no clear answer about which analogue gives better results in clinical practice . the reports are contradictory [ 1118 ] and often favor one type of the analogue . in addition , there is still no generally accepted consensus on how to stimulate the ovaries of good prognosis patients at the beginning of their in vitro fertilization treatment . for this reason , we retrospectively analyzed the data from ivf ( classical ivf and icsi cycles together ) carried out at our centre during years 20102013 in good prognosis patients to elucidate which protocol of coh is optimal for these patients . because most of the reports usually include only comparison of two studied coh protocols , we included in our analysis the data obtained from three different protocols : mild protocol ( cotreatment with gnrh antagonist ) , conventional gnrh agonist , and conventional gnrh antagonist protocol of ovarian stimulation . we comparatively analyzed the main outcomes of coh protocols , such as number of retrieved and fertilized oocytes , embryos , cryopreserved embryos , the proportion of cycles with embryo freezing and the number of cryopreserved embryos , and the clinical outcome in terms of pregnancy rate , live birth rate ( lbr ) , and cumulative lbr . in this retrospective study the data from 2373 in vitro fertilization cycles conducted at ivf unit university medical centre ljubljana from january 2010 to december 2013 in good prognosis patients were analyzed . the gnrh antagonist mild protocol of ovarian stimulation was carried out in 166 cycles , gnrh antagonist protocol in 1096 cycles , and gnrh agonist protocol in 1111 cycles . for cumulative live birth rate good prognosis patients were defined as patients aged 38 years who were undergoing the first or the second cycle of in vitro fertilization and with more than 3 oocytes retrieved regardless of their indication of infertility . in the gnrh antagonist group , a starting daily dose of 200225 iu recombinant fsh ( follitropin alfa , gonal f merck serono , or follitropin beta , puregon msd ) was started on day 2 of the menstrual cycle . women received the gnrh antagonist cetrorelix acetate ( cetrotide ; asta medica ag , frankfurt , germany ) at a dose of 0.25 mg per day from the day when the dominant follicle reached a mean diameter 14 mm until the day of hcg administration . in the stimulation period rfsh daily dose mild group , a starting daily dose of 200225 iu ( follitropin alfa , gonal f merck serono , or follitropin beta , puregon msd ) was started on day 5 of the menstrual cycle . women received the gnrh antagonist cetrorelix acetate ( cetrotide ; asta medica ag , frankfurt , germany ) at a dose of 0.25 mg per day from the day when the dominant follicle reached a mean diameter 14 mm until the day of hcg administration . in the gnrh agonist group ovarian stimulation was performed using gnrh analogues ( suprefact ; hoechst ag , frankfurt / main , germany ) administered from day 22 of the cycle in a daily dose of 0.6 ml ( 600 pg ) s.c . after 14 days , pituitary desensitization was checked by e2 determination and b - mode ultrasound scan . once the criteria for desensitization were fulfilled ( e2 0.05 nmol / l , follicles 5 mm in diameter , and endometrial thickness 5 mm ) , ovarian stimulation with a daily dose of 200225 iu rfsh was started ( follitropin alfa , gonal f merck serono , or follitropin beta , puregon msd ) . later in the stimulation period daily dose of rfsh was adjusted individually . in all groups , organon , oss , the netherlands ) in a dose of 10,000 iu was administered when 3 or more follicles reached a diameter of 18 mm . after retrieval of oocytes , classical ivf or icsi was performed regarding the indication of infertility . ivf was performed in female indications of infertility , while icsi was performed in male indications of infertility such as impaired semen quality , azoospermia , or immunological factor . oocytes were fertilized and cultured until the next day in fertilization medium ( cook , australia ) or universal ivf medium ( origio , denmark ) . the fertilization of oocytes ( presence of two pronuclei and two polar bodies ) was checked 1618 hours after oocyte insemination or sperm microinjection . normally fertilized ( 2pn ) zygotes were cultured in the cleavage medium ( cook ) or blast assist system medium-1 ( origio ) on days 2 and 3 . on days 4 and 5 , embryos were further cultured in blastocyst medium ( cook ) or blast assist system medium-2 ( origio ) . in patients with only one embryo or two embryos , one cleavage - stage embryo or two cleavage - stage embryos were transferred on day 3 , while in patients with more embryos one blastocyst or two blastocysts ( or morulae ) were transferred on day 5 after prolonged culturing . in patients younger than 36 years and high quality embryos the surplus blastocysts were cryopreserved using blastfreeze medium ( origio ) and thawed in blastthaw ( origio , denmark ) medium . the blastocysts that survived the freeze - thawing procedure ( > 50% of nondamaged cells ) were transferred into the uterus in a natural cycle or after hormonal preparation . the data from classical ivf cycles and icsi cycles were joined for each coh protocol and the main outcomes of three different coh protocols ( gnrh antagonist , gnrh antagonist mild , and gnrh agonist ) were compared in terms of average number of retrieved and fertilized oocytes , average number of embryos , number of cryopreserved embryos , the proportion of cycles with embryo cryopreservation , and the clinical outcome of in vitro fertilization ( pregnancy , miscarriage , live birth , cumulative live birth , and twin delivery ) . pregnancy was defined as positive hcg test and the cumulative live birth as the sum of births after fresh and frozen - thawed embryo replacements in the same group of cycles ( until september 2014 ) . to determine the differences between the groups pearson 's chi - squared test , based on inclusion criteria , a total of 2373 cycles of in vitro fertilization were included in this study . of all cycles , gnrh antagonist mild protocol of ovarian stimulation was carried out in 166 cycles , gnrh antagonist protocol in 1096 cycles , and gnrh agonist protocol in 1111 cycles . altogether , as the most important outcome , 674 live births were achieved in all cycles : 610 live births after fresh embryo transfers ( et ) ( 56 live births from day 3 et and 554 from day 5 et ) and additional 65 live births after frozen - thawed embryo transfers ( fet ) ( figure 1 ) . the outcome and comparison of different protocols of coh cycles are presented in table 1 and in table 2 . to summarize , the age of the patients and the baseline fsh were comparable in all groups . the number of retrieved oocytes ( 10.8 5.6 ) was the highest and the proportion of immature oocytes was the lowest ( 14.1% ) in gnrh agonist protocol . also the average number of embryos was the highest in gnrh agonist protocol ( 5.4 3.5 ) , but the difference was significant only when compared to gnrh antagonist protocol ( 4.7 3.3 , p < 0.0001 ) . on the contrary , the proportion of fertilized oocytes ( 57.7% ) and embryos ( 56.3% ) was the highest in gnrh antagonist mild protocol . in addition , the average number of frozen embryos per cycle ( 0.5 1.3 ) and the proportion of cycles with embryo freezing ( 18.9% ) were significantly the lowest in gnrh agonist protocol . in the most important category , in the lbr per cycle after fresh et , the results were comparable between all groups , despite the fact that pregnancy rate per cycle ( 41.6% ) was significantly higher in gnrh antagonist mild protocol compared to both other protocols ( table 2 ) . at this point it is worth it to mention that cumulative lbr per cycle was significantly higher in gnrh antagonist mild protocol ( 35.6% ) compared to gnrh agonist protocol ( 27.3% , p = 0.0275 ) . to determine if the day of the et ( day 3 cleavage embryo or day 5 blastocyst et ) has any influence on the outcome of coh , we additionally analyzed the data about the number of ets , pregnancies , miscarriages , births , and twin deliveries according to the day of et and compared them between coh protocols . the results showed ( figure 1 ) that there is no significant difference in lbr , twin deliveries , and miscarriages per et between coh protocols irrespective of the day of et . the only difference was in pregnancy rate per et on day 5 et , when gnrh antagonist mild ( 46.2% ) and gnrh agonist ( 37.4% , p = 0.0455 ) protocols were compared . in day 3 et group because the number of retrieved oocytes is significantly different between studied coh protocols ( table 1 ) and this could influence the cycle outcome , we divided patients based on the number of retrieved oocytes , to analyze the different outcomes according to the 3 different coh protocols . therefore , the patients were divided to quartiles ( patients with 46 oocytes , 79 oocytes , 1013 oocytes , 14 , or more retrieved oocytes ) and pregnancy rates , lbr , and cumulative lbr were compared between coh protocols . results showed that in groups of patients with 79 oocytes and with 1013 retrieved oocytes there is no difference between coh protocols . the difference was observed in group of patients with 46 oocytes since the pregnancy rate per cycle ( 45.8% versus 29.5% , p = 0.0160 ) , lbr ( 33.6% versus 23.0% , p = 0.0444 ) , and cumulative lbr ( 37.3% versus 24.1% , p = 0.0388 ) were significantly higher in gnrh antagonist mild protocol compared to gnrh agonist protocol . similar trend was observed also in a group of patients where 14 or more oocytes were retrieved . despite there being no difference in pregnancy rate , the lbr was significantly higher in gnrh antagonist mild protocol ( 42.9% ) compared to gnrh antagonist protocol ( 23.1% , p = 0.0480 ) . additionally , also the cumulative lbr was significantly higher in gnrh antagonist mild protocol ( 57.1% ) compared to both gnrh antagonist ( 32.3% , p = 0.0032 ) and gnrh agonist protocol ( 33.1% , p = 0.0256 ) . in this study we retrospectively analyzed the data from ivf program carried out at ivf unit of university medical centre in good prognosis patients to elucidate which protocol of coh gives the best results at our condition . we compared gnrh antagonist , gnrh antagonist mild , and gnrh agonist protocol of coh . as already mentioned above , there is still no consensus , in which protocol of coh is optimal in good prognosis patients at the beginning of in vitro fertilization treatment . the results of orvieto et al . advocated in favor of using gnrh agonist protocol in the first ivf cycles performed in young patients , since the clinical pregnancy rate was significantly higher than in gnrh antagonist protocol . contrarily , the meta - analysis conducted in 2011 showed that there were no significant differences between these two protocols of ovarian stimulation regarding the live birth rate . although the reanalysis of these data disclosed significantly higher live birth rate in the gnrh agonist protocol . the most recent study by grow et al . was carried out in a large number of very similar population of patients than in our study and showed that the gnrh agonist protocol decreased the cancellation risk and increased embryo implantation and live birth rate in comparison with gnrh antagonist protocol of ovarian stimulation . on the contrary , in our study we did not observe any difference in live birth rate after fresh et between all the groups of studied coh protocols , although the cumulative live birth rate was significantly higher in gnrh antagonist mild protocol when compared to gnrh agonist protocol . other results showed that the average numbers of retrieved oocytes , embryos , and transferred embryos per et were significantly higher after gnrh agonist than gnrh antagonist protocol , although the proportion of cycles with embryo cryopreservation was significantly higher in gnrh antagonist protocol . . showed similar results , since there were significant differences in the number of retrieved oocytes and pregnancy rate between gnrh agonist and gnrh antagonist protocol , although there were no differences in live birth rate . the reason why some propose that gnrh agonists are more appropriate in good prognosis patients could lay in the endometrial receptivity . . showed that in gnrh agonist protocol the endometrium is thicker , although some other studies show that there is no difference [ 12 , 13 ] . contradictory are also results of studies , where researchers tried to determine biomarkers of endometrial receptivity . simon et al . showed that endometrial gene expression is more similar to the natural cycle , when gnrh antagonist was applied for coh , while in a study by ruan et al . the results oppositely showed that two biomarkers related to the endometrial receptivity , integrin beta-3 and leukaemia - inhibitory factor , were correlated with the higher implantation rate after coh using gnrh agonist . on the other hand , all these explanations are also not in accordance with the results of our study , since the cumulative live birth rate per cycle was significantly higher in gnrh antagonist mild protocol when compared to gnrh agonist protocol , although the number of retrieved oocytes was significantly lower after gnrh antagonist mild protocol of ovarian stimulation . the lower number of oocytes is often linked to poor ovarian response , but in a case of gnrh antagonist mild protocol of coh the lower number of retrieved oocytes is normal and most probably represents a homogenous group of good quality oocytes [ 24 , 25 ] . this explanation could be confirmed also with the results of our study , since in the subgroup of patients with 46 retrieved oocytes the pregnancy rate per cycle , lbr , and cumulative lbr were significantly higher in gnrh antagonist mild protocol compared to gnrh agonist protocol . also the study by baart et al . indirectly indicated this , since the results showed that both mild and conventional protocols of ovarian stimulation generated on average a similar number of chromosomally normal embryos in spite of significantly lower number of oocytes and embryos retrieved after mild protocol of ovarian stimulation . the meta - analysis showed that optimal embryo implantation was achieved when 5 oocytes were retrieved after mild ovarian stimulation and when 10 oocytes were retrieved after gnrh agonist stimulation . in addition , the ongoing pregnancy rate per embryo transfer , as a function of the number of retrieved oocytes , was significantly higher after the mild protocol of ovarian stimulation . similar to our study , also some other studies showed that the outcome of the ivf / icsi cycle is not compromised , if the mild protocol of ovarian stimulation is used in comparison with gnrh agonist protocol [ 25 , 27 , 28 ] . we may conclude that the gnrh antagonist mild protocol of ovarian stimulation could be the method of choice to stimulate the ovaries of good prognosis patients without a risk of compromising the outcome of ivf cycle .

the reports on how to stimulate the ovaries for oocyte retrieval in good prognosis patients are contradictory and often favor one type of controlled ovarian hyperstimulation ( coh ) . for this reason , we retrospectively analyzed data from ivf / icsi cycles carried out at our ivf unit in good prognosis patients ( aged < 38 years , first and second attempts of ivf / icsi , more than 3 oocytes retrieved ) to elucidate which type of coh is optimal at our condition . the included patients were undergoing coh using gnrh agonist , gnrh antagonist or gnrh antagonist mild protocol in combination with gonadotrophins . we found significant differences in the average number of retrieved oocytes , immature oocytes , fertilized oocytes , embryos , transferred embryos , embryos frozen per cycle , and cycles with embryo freezing between studied coh protocols . although there were no differences in live birth rate ( lbr ) , miscarriages , and ectopic pregnancies between compared protocols , pregnancy rate was significantly higher in gnrh antagonist mild protocol in comparison with both gnrh antagonist and gnrh agonist protocols and cumulative lbr per cycle was significantly higher in gnrh antagonist mild protocol in comparison to gnrh agonist protocol . our data show that gnrh antagonist mild protocol of coh could be the best method of choice in good prognosis patients .

micrornas are a group of small noncoding rna ( ncrna ) molecules , distinct from but related to small interfering rnas ( sirnas ) , that have been identified in a variety of organisms ( for reviews see he and hannon 2004 ; bartel 2004 ) . these small 2022-nucleotide ( nt ) rnas are transcribed as parts of longer molecules several kilobases ( kb ) in length that are processed in the nucleus into hairpin rnas of 70100 nt by the double - stranded rna - specific ribonuclease drosha ( cullen 2004 ) . the hairpin rnas are transported to the cytoplasm , via an exportin 5-dependent mechanism , where they are digested by a second , double - stranded specific ribonuclease called dicer . single - stranded microrna binds specific messenger rna ( mrna ) through sequences that are significantly , though not completely , complementary to the target mrna , mainly to the 3 untranslated region ( 3 utr ) . by a mechanism that is not fully characterized , the bound mrna remains untranslated , resulting in reduced levels of the corresponding protein ; alternatively , the bound mrna can be degraded , resulting in reduced levels of the corresponding transcript ( fig . 1 ) . rna polymerases ii and iii are believed responsible for microrna transcription , although a recent publication indicates that polymerase ii is the main rna polymerase for microrna transcription ( lee et al . ( a ) exonic micrornas in sense orientation as a part of annotated host genes are transcribed as parts of longer molecules that are processed in the nucleus into hairpin rnas of 70100 nt by the dsrna - specific ribonuclease drosha . the hairpin rnas are transported to the cytoplasm where they are digested by a second , double - strand specific ribonuclease called dicer . in animals , single - stranded mirna binds specific mrna through sequences that in most of cases are significantly , though not completely , complementary to the target mrna . ( b ) the excision of intronic micrornas out of the precursors is completed through the process of rna splicing , followed by dicer digestion . micrornas are finally incorporated into an rna - induced silencing complex ( risc ) to induce translation suppression or degradation depending of the degree of complementary with the target mrna . ( c , d ) exonic and intronic micrornas in antisense orientation as a part of annotated host genes can be trancribed as independent transcription units . the mature microrna sequence , in our hypothetical mechanisms , can lead to translation or transcription suppression of the host gene of other target mrnas . microrna genes located in intergenic regions or gene deserts are transcribed as independent transcription units and their biogenesis can be described as in a. biogenesis of micrornas and hypothetical mechanisms in regulation of gene expression . rna polymerases ii and iii are believed responsible for microrna transcription , although a recent publication indicates that polymerase ii is the main rna polymerase for microrna transcription ( lee et al . ( a ) exonic micrornas in sense orientation as a part of annotated host genes are transcribed as parts of longer molecules that are processed in the nucleus into hairpin rnas of 70100 nt by the dsrna - specific ribonuclease drosha . the hairpin rnas are transported to the cytoplasm where they are digested by a second , double - strand specific ribonuclease called dicer . in animals , single - stranded mirna binds specific mrna through sequences that in most of cases are significantly , though not completely , complementary to the target mrna . ( b ) the excision of intronic micrornas out of the precursors is completed through the process of rna splicing , followed by dicer digestion . micrornas are finally incorporated into an rna - induced silencing complex ( risc ) to induce translation suppression or degradation depending of the degree of complementary with the target mrna . ( c , d ) exonic and intronic micrornas in antisense orientation as a part of annotated host genes can be trancribed as independent transcription units . the mature microrna sequence , in our hypothetical mechanisms , can lead to translation or transcription suppression of the host gene of other target mrnas . microrna genes located in intergenic regions or gene deserts are transcribed as independent transcription units and their biogenesis can be described as in a. the central dogma of classical biology is that genetic information flows from dna to rna to proteins . therefore , genes are synonymous with proteins and a gene is defined as a protein - coding region with associated regulatory signals ( mattick 2003 ) . micrornas represent new stars in the gene regulation galaxy , and there is a strong interest among researchers in different fields to understand their mechanism of action and identify their targets . the definition of a gene in the genomics era should be expanded to be a transcription unit or a complete chromosomal segment responsible for making a functional product 2002 ) . because of the complex genetic architecture of eukaryotic cells , this definition is still not complete , and additional issues such as overlap , alternative splicing , or transcribed pseudogenes have to be considered ( snyder and gerstein 2003 ) . thus , the term gene could be expanded to include micrornas ( and other functional ncrnas ) . the number of micrornas is growing rapidly , especially after using in silico cloning ( table 1 ) . one such bioinformatics tool is mirscan which ranks predicted hairpins that are conserved in the genome of two related animals based on several criteria ( lim et al . initially , it was estimated that there could be from 200 to 1000 microrna genes in the mammalian genome ( 1%3% of known genes are represented by micrornas ) . today the number of micrornas , including those electronically cloned , is over 1000 and still growing ( berezikov et al . mammalian microrna genes have been distinguished by using the prefix mir followed by a number ; the prefixes lin and let refer to micrornas originally identified in c. elegans ( see below ) . the microrna registry website contains a comprehensive list of micrornas from all species ( table 1 ) . pavel tomancak ( personal communication)drosophilatarget identification targetscanhttp://genes.mit.edu / targetscanvertebrates diana microthttp://www.diana.pcbi.upenn.edu / cgi - bin / micro_t.cgihuman / mouse mirna target predictionhttp://www.russell.embl.de / mirnas / drosophila mirandahttp://www.microrna.org / miranda.htmldrosophila / human rnahybridhttp://bibiserv.techfak.uni - bielefeld.de / rnahybrid / drosophila rnacalibrate rna effective mirnaviewerhttp://cbio.mskcc.org / mirnaviewer / human pictarhttp://pictar.bio.nyu.edu / humanmicrornas database the micrornas registryhttp://www.sanger.ac.uk / software / rfam / mirna / index.shtmlall microrna websites for in silico cloning , target identification , and the registry more than a decade ago , ambros and colleagues discovered that lin-4 , a gene known to control the timing of c. elegans larval development , does not code for any protein but instead codes for a pair of small rnas ( lee et al . one rna is longer ( 70 nt ) and can fold in a stem - loop structure , as the precursor of the shorter rna ( 22 nt ) . the ambros and ruvkun laboratories later discovered that the small lin-4 rnas had an antisense complementarity to multiple sites in the 3 utr of the lin-14 protein - coding gene ( lee et al . 1993 ; wightman et al . the sequence complementarity between the noncoding lin-4 rna and the 3 utr of the protein - coding lin-14 gene suggested that lin-4 regulated translation of lin-14 by binding to the 3 utr . it was later shown that lin-4 regulated the lin-14 gene during early larval development as well as the lin-28 gene during late larval development by a mechanism involving rna - rna binding that led to suppression of translation ( moss et al . the shorter lin-4 rna is now known as the founding member of the class of microrna genes ( lagos - quintana et al . the majority of micrornas ( 70% ) are located in introns and/or exons , and approximately 30% are located in intergenic regions ( rodriguez et al . the first group of micrornas from introns and/or exons is oriented in sense with the exon - coding host gene and , therefore , may be transcribed as part of annotated genes . the second group of micrornas are transcribed from intergenic regions or gene deserts ( lagos quintana et al . 2001 ; lee and ambros 2001 ) , indicating that they form independent transcription units ( lee et al . the third group of micrornas are derived from introns and/or exons of annotated genes but are transcribed in the antisense orientation , suggesting that they too form their own transcription units . tightly linked micrornas may be transcribed as polycistronic messengers ; however , micrornas separated by more than 50 kb tend to represent independent transcription units ( baskerville and bartel 2005 ) . rna polymerases ii and iii are candidates for pri - micrornas ( primary ) transcription ( fig . 1 ) . in animals ( fig . 1 ) , the first step in microrna maturation is the nuclear cleavage of the several - kb - long pri - microrna , which releases an approximately 70-nt , cropped , hairpin - shaped intermediate known as pre - microrna ( precursor ) . the drosha rnase iii endonuclease is responsible for this nuclear processing ( bartel 2004 ; lee et al . drosha can not cleave without a partner , forming a complex with dgcr8 ( a product of the digeorge syndrome critical region gene 8) , which contains two double - stranded rna - binding domains ( han et al . the hairpin precursor is actively transported from the nucleus to the cytoplasm by exportin 5 ( yi et al . 2004 ) . a second enzyme , an rnase iii endonuclease called dicer , is responsible for generating an approximately 21-nt , short , single - stranded rna that is the mature microrna ( lee et al . 2003 ) . dicer was first recognized for its role in generating sirnas that mediate rna interference ( rnai ) ( bernstein et al . , the microrna pathways of plants and animals share some other steps with rna silencing , and the cleavage products become incorporated as single - stranded rnas into the ribonucleoprotein rna - induced silencing complex risc ( hammond et al . the risc has been purified from fruit fly and human cells and in both cases contains a member of the argonaute protein family ( essential for gene silencing in caenorhabditis elegans , neurospora , and arabidopsis ) , which is thought to be a core component of the complex ( hammond et al . 2001 ; mourelatos et al . 2002 ) . once incorporated in the cytoplasmic risc complex , the microrna will specify cleavage if the mrna has sufficient complementarity to the microrna , or it will block translation if the microrna does not have sufficient complementarity , resulting in reduced expression of the corresponding protein . microrna regulation of the microrna : mrna duplex is mediated mainly through multiple complementary sites in the 3 utrs , but there are many exceptions . micrornas may also bind the 5 utr and/or the coding region of mrnas , resulting in a similar outcome ( fig . 1 ) . the functions of microrna are various , such as the control of leaf and flower development in plants ( aukerman and sakai 2003 ) or the modulation of hematopoietic lineage differentiation in mammals ( chen et al . several groups have uncovered roles for micrornas in the coordination of cell proliferation and cell death during development and in stress resistance and fat metabolism ( ambros 2003 ) . for example , the drosophila microrna gene ( mir-14 ) suppresses cell death and is required for normal fat metabolism ( xu et al . 2003 ) , while the bantam locus encodes a developmentally regulated microrna that controls cell proliferation and regulates the proapoptotic gene hid in drosophila ( brennecke et al . it was recently shown that , in addition to their regulatory functions , cellular micrornas mediate antiviral defenses in human cells ( lecellier et al . the antisense single - stranded micrornas can bind specific mrna transcripts through sequences that are significantly , though not completely , complementary to the target mrna . 2005 ) , and it has been speculated that micrornas could regulate approximately 30% of the human genome ( bartel 2004 ) . micrornas seem to be responsible for fine regulation of gene expression , tuning the cellular phenotype during delicate processes like development and differentiation in all organisms , from plants to mammals . many micrornas are conserved in sequence between distantly related organisms ( see the microrna registry in table 1 ) , suggesting that these molecules participate in essential processes ( lagos - quintana et al . one example is the cluster of mir-16 - 1 and mir-15a , which is highly conserved in nine of ten primate species ( berezikov et al . 2005 ) . target identification has been hampered by the fact that in animals , in contrast to plants , micrornas do not bind perfectly to their targets . mammalian genes can have more than one microrna target site in their 3 utrs and one microrna can target more than one mrna . bioinformatics approaches have been developed to search for the most thermodynamically favorable microrna : mrna duplex interactions ( table 1 ) . several computational procedures such as dianamicrot , targetscan , and miranda ( lewis et al . another set of programs developed for target identification includes rnahybrid , rnacalibrate , and rnaeffective ( rehmsmeier et al . 2004 ) , which work by searching for the most energetically favorable hybridization sites for the smaller microrna within the larger mrna ( table 1 ) . only a few target mrnas have been experimentally proven and studied in vitro , but the numbers of such confirmed interactions are expected to sharply increase as prediction tools become more sophisticated . generally , the proven mrna targets are produced from different chromosomes as the corresponding micrornas ( such as mir-19a on chromosome 13q31.3 having as a target the mrna of the pten tumor suppressor gene located on chromosome 10q23.3 ) , but examples of micrornas targeting an important example is that of mir-196 , which leads to the cleavage and degradation of mrna from the homeobox gene hoxb8 ( yekta et al . micrornas are not the only ncrna sequences that have been shown to play a role in the regulation of gene expression . a large set of ncrnas are known as gene regulators and the list in mammals includes air , h19 , ipw , ntt , tsix and xist ( table 2 ) . these ncrnas have a variety of functions , from potential involvement in the imprinting process to x - chromosome inactivation in mammals . it is likely that other ncrnas will be found , albeit with additional effects on critical biological processes . table 2functional noncoding rnasncrna symbolncrna namesize ( kb)organismfunctionreferencesairantisense igfar rna108mousesilencing autosomal imprinted genessleutels et al . ( 2002)h19h19 fetal liver rna1.7human , mouseimprinted embryonic transcript , tumor supppressor gene , and/or associated with tumor progression in breastbrannan et al . ( 2005)ipwimprinted in prader - willi syndrome2.2human , mouseprader - willi syndrome , imprinting processwevrick and francke ( 1997 ) ; yang et al . ( 1998)nttnoncoding transcript in t cells17humantranscript present only in activated cd4 + t - cell clones , regulation of neighboringliu et al . ( 1997)rox1rna on the x13.7fruit flydosage compensationrattner and meller ( 2004)tsixantisense orientation of xist40mammalsregulation in the early steps of x inactivationlee et al . ( 1999)xist / xistx - inactivation specific transcript15 - 17mammalsx - chromosome inactivationbrockdorff et al . ( 1992 ) ; avner and heard ( 2001)additional nc - rnas can be found at http://biobases.ibch.poznan.pl/ncrna ( erdmann et al . functional noncoding rnas additional nc - rnas can be found at http://biobases.ibch.poznan.pl/ncrna ( erdmann et al . several articles have now shown the possibility that small ncrnas play an important role in cancer ( table 3 ) . we show in fig . 2 diagrams of several proposed mechanisms for micrornas as cancer players ( calin et al . inherited or somatic mutations , amplification , deletion , or epigenetic silencing of microrna genes may not only cause certain cancers but may contribute to an individual s risk of developing cancer ( fig . 2 ) . table 3micrornas and human cancercancer typemicrorna alterationsreferenceb - clldeletions and down regulation of mir-15 andmir-16 ( 68% of cases)calin et al . ( 2005)primary or mature levels of micrornas derived from the mir-17 - 92 locus are often increasedhe et al . ( 2005)colorectal cancerreduced mir-143 and mir-145 ( 80% cases)michael et al . ( 2003)glioblastomamir-21 is overexpressed in human malignant cell lines and tumor tissueschan et al . ( 2004a)lung cancerreduced expression of let-7 associated with short postoperative survival ( 40% cases)takamizawa et al . ( 2005)malignant lymphomagenomic amplification of mir-17 - 91 - 18- 19a-20 - 19b-92 clusterota et al . orange rectangles represent promoters of protein - coding genes and blue rectangles represent the actual coding sequences of their corresponding genes . one mechanism for the downregulation of suppressor - micrornas that has been identified is ( a ) homozygous deletion of microrna coding regions , as exemplified by deletion of the mir-15a / mir-16 - 1 cluster in b - cll ( calin et al . hypothetical mechanisms for downregulation of suppressor - micrornas in cancer include ( b ) the combination of deletion plus promoter hypermethylation , and ( c ) deletion plus mutation . mechanisms for the upregulation of onco - micrornas that have been identified are ( d ) amplification and overexpression of pre - micrornas , as exemplified by mir-155/bic in children s 2004 ) , and ( e ) translocations of either protoconcogenes near the promoter of micrornas or translocation of micrornas near the promoters of oncogenes ( modified after calin et al . ( ) 2004 pnas , national academy of sciences , washington , dc , usa ) . orange rectangles represent promoters of protein - coding genes and blue rectangles represent the actual coding sequences of their corresponding genes . one mechanism for the downregulation of suppressor - micrornas that has been identified is ( a ) homozygous deletion of microrna coding regions , as exemplified by deletion of the mir-15a / mir-16 - 1 cluster in b - cll ( calin et al . 2002 ) . hypothetical mechanisms for downregulation of suppressor - micrornas in cancer include ( b ) the combination of deletion plus promoter hypermethylation , and ( c ) deletion plus mutation . mechanisms for the upregulation of onco - micrornas that have been identified are ( d ) amplification and overexpression of pre - micrornas , as exemplified by mir-155/bic in children s burkitt s lymphoma ( metzler et al . 2004 ) , and ( e ) translocations of either protoconcogenes near the promoter of micrornas or translocation of micrornas near the promoters of oncogenes ( modified after calin et al . ( ) 2004 pnas , national academy of sciences , washington , dc , usa ) . the first report linking micrornas and cancer ( calin et al . 2002 ) involved chronic lymphocytic leukemia ( cll ) , the most common form of adult leukemia in the western world . hemizygous and/or homozygous loss at chromosome 13q14 occurs in more than half of cll cases . loss of chromosome 13q14 is also found in more than 50% of mantle cell lymphomas ( stilgenbauer et al . 1998 ) , in approximately 30% of multiple myeloma ( elnenaei et al . 2003 ) , and in approximately 70% of prostate cancers ( dong et al . 2001 ) , suggesting that one or more tumor suppressor genes located at chromosome 13q14 are involved in the pathogenesis of human tumors ( calin et al . 2002 ) . however , detailed genetic analysis , including extensive loss of heterozygosity ( loh ) , mutation , and expression studies failed to demonstrate the consistent involvement of any of the 12 protein - coding genes located in or close to the deleted region . a cluster of two micrornas , mir-15a and mir-16 - 1 , was found within the minimal region of deletion ( 30 kb ) at 13q14 and to be deleted or downregulated in approximately 70% of cll samples . similarly , a t(12;13 ) chromosome translocation in a patient with cll was found to decrease the mir-15a and mir-16 - 1 precursors ( calin et al . a similar cluster of micrornas , named mir-15b and mir-16 - 2 , is located on human chromosome 3 but is expressed at very low levels in lymphoid cells . the first genome - wide systematic search for correlations between the genomic positions of micrornas and cancer - associated genomic regions ( cagrs ) provided a catalog of micrornas possibly involved in cancer ( calin et al . one hundred eighty - six micrornas were mapped to the human genome and compared with the location of previously reported nonrandom genetic alterations identified in human tumors and with cloned fragile sites ( fras ) . minimal regions of loh are suggestive of the presence of tumor suppressor genes , minimal regions of amplification for the presence of oncogenes , while common breakpoint regions are found close to both types of cancer genes . some common fragile sites predispose to dna instability in cancer cells ; indeed they are preferential sites of sister chromatid exchange , translocation , deletion , amplification , or integration of plasmid dna and tumor - associated viruses . this study concluded that 19% ( 35 of 186 ) of micrornas are located inside or near fras and the relative incidence of micrornas inside fras occurred at a rate nine times higher than in non - fras . a significant proportion , 52.5% ( 98 of 186 ) , of micrornas are in cagrs described in a variety of tumors such as lung , breast , ovarian , colon , gastric , and hepatocellular carcinoma , as well as leukemias and lymphomas . little was known about the expression levels of microrna genes in normal and neoplastic cells until 2002 . to assess cancer - specific expression levels for hundreds of micrornas is time - consuming and requires a large amount of total rna and the use of radioactive isotopes . cdna microarrays are useful tools for identifying different patterns of expression in a large number of samples . 2004a ) , containing hundreds of human precursor and mature microrna probes , identified distinct patterns of microrna expression in human and mouse tissues ( tissue - specific microrna expression signatures ) . another method to determine microrna expression levels involves the use of a bead - based flow cytometric technique ( lu et al . since the development of these two methods of assessing global microrna expression , several commercially available platforms have been developed for microrna gene expression profiling . global analysis of microrna gene expression has shown that these profiles can be used to classify the developmental lineage and differentiation state of tumors ; in addition , microrna profiles appear to provide a distinct signature for tumors which is more precise than that provided by protein - coding genes ( lu et al . these findings lead to a unique opportunity to use these microrna profiles for tumor diagnostics . specific differences in microrna expression between cll samples and normal cd5 + b cells , the latter representing normal cells corresponding to cll malignant cells , were found ( calin et al . 2002 ) . two clusters of cll samples were identified that were associated with the presence or absence of zap-70 expression , a predictor of early disease progression . low levels of zap-70 ( identified by both western blots and flow cytometry ) are associated with good prognosis . two microrna signatures were associated with the presence or absence of mutations in the expressed immunoglobulin variable - region genes or with deletions at chromosome 13q14 , respectively . different microrna expression profiles suggested that specific micrornas can have prognostic significance in cll , thus expanding the spectrum of prognostic markers ( calin et al . 2004b ) . a unique microrna expression signature composed of 13 genes can differentiate cases with low or high zap-70 expression and cases with unmutated or mutated igvh . furthermore , both mir-15a and mir-16 - 1 are members of this signature , suggesting an important functional role in the pathogenesis of human cll ( calin et al . the list of micrornas reported to be involved in cancer is growing continuously ( table 3 ) . one report showed that mir-143 and mir-145 exhibited reduced levels of the mature microrna in colon cancer patients , colon cancer cell lines , and precancerous adenomatous polyps when compared to normal tissues by northern blot ( michael et al . 2003 ) . on the contrary , levels of unprocessed hairpin precursors were not altered . it is possible that the reduced accumulation of the mature microrna reflects early changes in neoplastic cells or that this depletion is related to their activity in binding and inactivating target mrnas in the cytoplasm . putative targets ( human mrnas that display imperfect complementarity of 70%100% ) for mir-143 and mir-145 include several genes that encode components of signal transduction pathways ( such as raf , rho , and g - proteins ) and chromatin - mediated control of gene expression were found by computer search ( table 3 ) . in an independent study , it was shown that mir-143 regulates adipocyte differentiation ( esau et al . 2004 ) . when antisense oligonucleotides ( asos ) targeting human micrornas were transfected into cultured human primary subcutaneous preadipocytes , mir-143 was upregulated as adipocytes differentiated . erk5/bmk1 ( which promotes cell growth and proliferation in response to tyrosine kinase signaling ) was predicted to be a mir-143 target and , in fact , erk5 protein levels were upregulated in mir-143 aso - treated adipocytes . however , because each microrna can regulate multiple target genes , mir-143 could directly inhibit erk5 by binding the 3 utr of erk5 , or indirectly by binding to another target gene ( esau et al . another study reported reduced expression of the let-7 microrna in human lung cancer and the correlation with clinicopathologic features ( takamizawa et al . 2004 ) . reduction in the expression ( > 80% ) levels of let-7 was observed in 60% ( 12 of 20 ) of cancer cell lines and 44% ( 7 of 16 ) of lung tumors when compared with normal lung tissues by northern blots . the reduced expression was significantly associated with shortened survival of patients after potentially curative resection . one hundred forty - three cases had been followed for more than five years after surgery and were used to study the prognostic significance of let-7 . one hundred forty - three non - small - cell lung carcinoma ( nsclc ) cases were classified into clusters 1 and 2 ( cluster 1 with low let-7 expression and higher disease stages ; cluster 2 with high let-7 expression and lower disease stages ) . in addition , overexpression of let-7 in the a549 lung adenocarcinoma cell line inhibited lung cancer cell growth in vitro ( takamizawa et al . several observations led to the conclusion that let-7 acts as a tumor suppressor gene in lung tissue . several let-7 family members , including let-7a-2 , let-7c , and let-7 g , have been mapped to minimally deleted regions in lung cancers ( calin et al . let-7 expression is lower in lung tumors than in normal lung tissue , while ras protein is significantly higher in lung tumors ( johnson et al . 2005 ) . finally , expression of the ras oncogene is regulated by let-7 , and overexpression of let-7 has been shown to inhibit lung tumor cell line growth ( johnson et al . high expression of human bic rna has been found in hodgkin s lymphoma ( van den berg 2003 ) . in contrast , an analysis of non - hodgkin s lymphoma ( nhl ) did not reveal any remarkable upregulation of bic expression . the bic locus was originally identified as a common retroviral integration site in avian leukosis virus - induced b - cell lymphomas ( tam et al . the human bic locus also encodes a microrna , mir-155 ( tam 2001 ; lagos - quintana et al . the conditio sine qua non for the development of burkitt s lymphoma is activation of the myc oncogene , mostly by chromosomal translocations in which myc is juxtaposed next to an immunoglobulin enhancer . but activation of myc alone is not sufficient for full malignancy ; in fact , myc cooperates with other oncogenes . further studies involving a human b - cell line that contains a tetracycline - regulated c - myc gene have shown that overexpression of the c - myc oncogene leads to increased expression of six micrornas , two of which are mir-17 - 5p and mir-20a ( odonnell et al . 2005 ) . these micrornas were found to negatively regulate expression of the e2f1 transcription factor . since e2f1 is also a target of c - myc , these results allude to an intricate network that exquisitely regulates gene expression , ultimately controlling cell proliferation . in childhood burkitt s lymphoma , there has been reported a 100-fold upregulation of the hairpin precursor mir-155 ( metzler et al . it could be speculated that mir-155 directly downregulates one of the myc antagonists such as mad1 , mxi1 , or rox / mnt . accumulation of mir-155 and bic rna was also reported in other b - cell lymphomas ( eis et al . it was recently reported that mir-21 is upregulated in both human glioblastomas as well as cultured glioblastoma multiforma cells and cell lines compared to primary astrocyte cultures derived from normal tissue ; inhibition of mir-21 expression led to the activation of caspases and cell death in vitro ( chan et al . these data suggest a role for mir-21 in malignant glioblastoma , most likely by influencing expression of genes involved in apoptosis ( chan et al . overall , the studies to date clearly implicate a causative role for micrornas in human cancer . the intricate network of signals regulating gene expression requires a delicate balance between protein - coding genes and microrna genes . disruption of this delicate relationship by mutation can tip the balance in favor of the development of cancer . several articles have been published showing a probable link between micrornas and other human diseases , but the precise mechanisms are still not known ( table 4 ) . fragile x syndrome is the most common inherited mental retardation disease . in the u.s . , conservative estimates report that fragile x syndrome affects approximately 1 in 4000 males and 1 in 8000 females ; the rate of the female carrier state has been estimated to be as high as 1 in 250 and the fragile x mental retardation protein ( fmrp ) is an rna - binding protein that can function as a translational suppressor . fmrp can form a messenger ribonucleoprotein ( mrnp ) complex , interacting with specific rna transcripts and proteins . the complex is transported out of the nucleus and into the cytoplasm , where it can either associate with the ribosome or interact with risc ; in both cases , it will lead to protein synthesis regulation . fmrp can interact with micrornas and other components of the microrna pathways , including dicer and the mammalian ortholog of argonaute 1 ( ago1 ) , a component of risc ( jin et al . 2004 ; peng et al . 2004 ) . table 4micrornas and other human diseasesdiseaseprotein alterationreferencesfragile x syndromeloss of expression of the fragile x mental retardation protein ( fmrp)peng et al . ( 2004)spinal muscular atrophy ( sma)reduction of expression in survival of motor neurons protein ( smn)mourelatos et al . ( 2004 ) micrornas and other human diseases spinal muscular atrophy ( sma ) is an inherited neurodegenerative disease with neuromuscular symptoms . survival of motor neurons protein ( smn ) is a component of the smn complex that has critical functions in the assembly and restructuring of diverse ribonucleoprotein ( rnp ) complexes . components of the smn complex ( gemin3 and gemin4 ) are also in a separate complex that contains eif2c2 , a member of the argonaute protein family . this novel complex contains numerous micrornas that form novel rnps named mirnps ( mourelatos et al . this link between micrornas and a devastating neurodegenerative disease is intriguing and will be of great interest to determine what effect these micrornas have on mirnps in sma patients . digeorge syndrome is characterized by a few specific cardiac malformations , facial deformity , and certain endocrine and immune anomalies . the dgcr8 ( digeorge syndrome critical region gene 8) protein and drosha interact in human cells . knockdown of the drosophila dmdgcr8 resulted in a 523-fold accumulation of some pri - micrornas , confirming a role in mediating the genesis of mirnas from the pri - microrna transcript ( landthaler et al . overexpression of mir-375 ( a pancreatic islet - specific microrna ) suppresses glucose - induced insulin secretion , and , conversely , inhibition of endogenous mir-375 by asos enhances insulin secretion in murine pancreatic - and -cell lines . bioinformatics approaches predicted several putative targets with a potential role in insulin secretion such as mapk4 , mki1 , vti1a , and mtpn ; the last two proteins are known to be involved in vesicle transport of neurons and in neurotransmitter release . further investigations are likely to reveal the involvement of additional micrornas and their targets in simple and complex genetic diseases . almost all mammalian micrornas are highly conserved and more than 220 mouse micrornas were cloned by conventional methods ( lagos quintana et al . it has been shown that a tissue - specific microrna dominates the population of expressed micrornas , suggesting important roles in tissue differentiation . for example , in mouse liver , mir-122a and mir-122b represent 72% of all cloned micrornas ; these micrornas were not detected in the other tissues analyzed . in spleen , mir-143 was the most abundant microrna . in colon , mir-142 was cloned several times ; however , because of the strong rnase activity in small intestine and pancreas , too few microrna sequences were obtained from these tissues to reach statistical significance . using a microrna microarray approach , the results were confirmed by northern blotting and were consistent with microrna expression data reported in the literature . furthermore , this study is consistent with microrna expression profiling in human tissues ( liu et al . for example , seven of eight micrornas were brain - specific in both mice and humans . another report gave new light on the world of microrna regulation in humans and mice ( seitz et al . 2004 ) . by bioinformatics approaches , 46 potential micrornas located in the human imprinted 14q32 domain have been identified . forty of these micrornas are clustered and most of them are arranged in tandem arrays . the majority of these genes map within an approximately 40-kb region , making it the largest microrna cluster that has been described . in mouse the detected micrornas are expressed in embryos and placenta , whereas in the adult their expression pattern is limited to the brain . expression of these micrornas comes solely from the maternally inherited chromosome , which is imprinted . regulation of their expression is dictated by a region located approximately 200 kb upstream from the microrna cluster ( seitz et al . research has shown how micrornas have a role in fine - tuning specific hox mrna expression patterns during mouse development ( mansfield et al . a novel approach was used to monitor microrna tissue - specific expression patterns during embryogenesis . until now , microrna expression studies were done by northern blots , tissue - specific rna cloning , and microarrays , but all of these methods are not useful for displaying spatiotemporal expression patterns . in situ hybridization methods are still difficult to adapt in vertebrate embryos because of the small size of micrornas . an alternative approach , previously used in drosophila melanogaster , was used where several reporter transgenes ( sensors ) were made to detect micrornas in the embryo . the sensor was a constitutively expressed reporter gene ( lacz ) containing microrna complementary sequences in the 3 utr . in cells lacking the corresponding micrornas , in contrast , cells expressing the corresponding microrna degrade the lacz message by an rna interfering pathway ( rnai ) , thus resulting in absence of b - galactosidase activity . to prove the biological significance of micrornas in human biology and in cancer in particular , the development of mouse models for deleted and/or overexpressed micrornas will be of invaluable interest for deciphering the regulatory networks where micrornas are involved . loss of dicer1 by replacement of exon 21 with a neomycin - resistance cassette leads to lethality early in embryogenesis at day 7.5 of development ; dicer1-null embryos were found to be depleted of pluripotent stem cells ( bernstein et al . another group generated dicerex1/2 mice that have a deletion of the amino acid sequences from the first and second exons of the dicer gene ( yang et al . dicerex1/2 homozygous embryos died between days 12.5 and 14.5 of gestation , again demonstrating that dicer is necessary for normal mouse development . in addition , blood vessel formation and maintenance in dicerex1/2 embryos and yolk sacs were severely compromised , with altered expression of vegf , flt1 , kdr , and tie1 being detected in the mutant embryos . this study suggests that dicer has a role in embryonic angiogenesis , probably through processing of micrornas that regulate expression levels of key angiogenic regulators ( yang et al . 2005 ) . although dicer - null embryonic stem ( es ) cells are viable , they are defective in rna interference ( rnai ) and the generation of micrornas ( kanellopoulou et al . these mutant es cells displayed severe defects in differentiation both in vitro and in vivo . an increase in centromeric repeat transcripts was detected , whereas the expression of homologous small double - stranded rnas was markedly reduced in dicer - deficient es cells ( kanellopoulou et al . these dicer - deficient cells were able to undergo gene silencing when provided with synthetic sirnas ( kanellopoulou et al . 2005 ; murchison et al . 2005 ) , indicating that dicer function is required for processing of micrornas but not required for the action of micrornas once they are produced . taken together , these studies suggest that the essential role of dicer in the production of mature micrornas is central for biological processes in mammalian organisms , ranging from stem cell differentiation to the integrity and function of centromeric heterochromatin ( kanellopoulou et al . 2005 ) . critical insights into the roles of individual proteins of the microrna processing machinery have been provided by in situ hybridization studies of argonaute ( ago1-ago4 ) family members in mouse embryos ( liu et al . studies in embryos at days 9.514.5 of development have shown that argonaute family members are restricted in both their temporal and spatial patterns of expression . however , ago2 shows the broadest expression in mouse embryos and is consistent with the fact that ago2-deficient mice die early in development ( liu et al . thus , the differential expression of ago family members suggests specific roles for these proteins during organogenesis , most likely involving the production of micrornas that are controlled at different times and in specific tissues by the presence of ago proteins . one example of the use of mouse models to study the roles of specific micrornas in cancer was provided by he et al . their initial observation was that the human mir 17 - 92 cluster was overexpressed by more than 5-fold in a majority of human b - cell lymphomas . to determine whether overexpression of this cluster could drive b - cell lymphomagenesis in mouse , the authors derived hematopoeitic stem cells from a susceptible e-myc transgenic mouse and transduced them with a retrovirus carrying the mir 17 - 92 cluster . injection of transduced cells into normal mice resulted in acceleration of the development of b - cell lymphomas ; in contrast , other subsets of micrornas expressed in these cells did not have similar effects ( he et al . the ability to manipulate the genome of mice coupled with the multitude of inbred strain backgrounds provides invaluable tools to genetically dissect the biological functions of individual micrornas . further studies of mouse models will provide significant insights into the roles of different micrornas in development and disease processes . a link between micrornas and human diseases , in particular , with cancer as focused on in this review , has been recently shown . to better understand the biological significance of micrornas and of alterations found in human diseases ( such as the effect of microrna mutations or variations in expression ) the development of mouse models is mandatory . regulation of gene expression in the mammalian genome , during development , differentiation , and disease , is a complex and multitasked system . to make transcripts instead of proteins is energetically less expensive for cells and could be the reason why regulation at the rna level is cheaper and more efficient than at the protein level . penetrance in gene expression requires a fine regulation and micrornas could have a role in different phenotypic expressions of the same gene . the ability to simultaneously regulate large sets of genes by a single microrna appears to be at the heart of control of multiple pathways that include morphogenesis and cell fate decisions , response to infectious organisms , and centromeric heterochromatin structure . micrornas appear to be at the center of the balance between apoptosis and proliferation in cancer cells and thus could provide novel approaches for therapeutics .

the basis of eukaryotic complexity is an intricate genetic architecture where parallel systems are involved in tuning gene expression , via rna - dna , rna - rna , rna - protein , and dna - protein interactions . in higher organisms , about 97% of the transcriptional output is represented by noncoding rna ( ncrna ) encompassing not only rrna , trna , introns , 5 and 3 untranslated regions , transposable elements , and intergenic regions , but also a large , rapidly emerging family named micrornas . micrornas are short 20 - 22-nucleotide rna molecules that have been shown to regulate the expression of other genes in a variety of eukaryotic systems . micrornas are formed from larger transcripts that fold to produce hairpin structures and serve as substrates for the cytoplasmic dicer , a member of the rnase iii enzyme family . a recent analysis of the genomic location of human microrna genes suggested that 50% of microrna genes are located in cancer - associated genomic regions or in fragile sites . this review focuses on the possible implications of micrornas in post - transcriptional gene regulation in mammalian diseases , with particular focus on cancer . we argue that developing mouse models for deleted and/or overexpressed micrornas will be of invaluable interest to decipher the regulatory networks where micrornas are involved .

diabetes insipidus ( di ) is characterized by copious excretion of urine and excessive thirst . this disease is caused by insufficient production and secretion of antidiuretic hormone ( adh ) , or the inability of the kidney tubules to respond to adh . the former condition is referred to as central di and the latter as nephrogenic di . possible causes of central di include head trauma , pituitary surgery , neoplasms , infection , inflammation , vascular disease , and genetic defects . however , 30% to 50% of central di cases are considered idiopathic ( 1 ) . development of this disease after open - heart surgery has been rarely reported in the literatures ( 2 , 3 ) . to the best of our knowledge , the patient presented in this paper represents the first reported case of central di associated with open - heart surgery in the republic of korea . in individuals undergoing cardiopulmonary bypass ( cpb ) , several endocrine changes due to surgical stress can occur ( 4 , 5 ) . there are some conflicting reports regarding the levels of adh after open - heart surgery . adh levels were found to increase in some studies ( 6 , 7 ) whereas adh deficiency developed in other cases ( 2 , 3 ) . in the present report , we describe a case of transient central di that developed after cpb for coronary artery bypass grafting ( cabg ) . we also discuss the etiology , pathogenesis , and differential diagnosis of this disease based on a literature review . on november 17 , 2011 , a 44-yr - old male underwent cpb to treat a 2 vessel disease involving occlusions in the left anterior descending and right coronary arteries . in addition , the patient did not have a familial history of polyuria . on the day of admission , his blood pressure was 127/66 mmhg and heart rate was 79 beats per minute . the cabg operation lasted 6 hr with a cpb time of 115 min and aortic cross clamp time of 69 min . one hour after the operation , the patient 's urinary output increased to about 1,000 ml / hr . serum creatinine levels increased slightly from 1.15 mg / dl to 1.3 mg / dl . serum glucose and sodium concentrations were 137 mg / dl and 140 meq / l ( fig other laboratory findings did not reveal any meaningful changes compared to the patient 's pre - operative data . over - hydration with hypertonic solutions nor large amounts of diuretics , the patient 's urinary output was continued up to 1,050 ml / hr and he was referred to the nephrology clinic . his clinical and laboratory findings revealed a serum osmolality of 333 mosm / kg , urine osmolality of 80 mosm / kg , and serum sodium of 155 meq / l ( fig . 10 mcg of a desmopressin nasal spray was administered for a desmopressin stimulation test ( fig . his urine output declined dramatically to 125 ml / hr 30 min after desmopressin use . at 60 min after nasal desmopressin administration , urine osmolality increased to 518 mosm / kg and urine output declined from 1,050 ml / hr to 80 ml / hr . based on the laboratory data and an increase in urine osmolality of more than 100% measured by the desmopression stimulation test , the patient was diagnosed with complete central di . on the third post - operative day , magnetic resonance imaging ( mri ) urine output was stabilized by intermittent nasal desmopressin administration until the fourth post - operative day at amount of 150 - 200 thereafter , no further therapy was necessary and the patient has remained symptom - free with no complaints of altered urinary output or excessive thirst . cpb can induce some physiologic changes including fluid , electrolyte , and acid - base imbalances ; renal complications , embolic events , and neuro - endocrine changes . despite some conflicting reports ( 2 , 3 , 6 - 8 ) showing variations in adh levels during and after cpb adh is an octapeptide produced in the superoptic and paraventricular nuclei of the hypothalamus , transported along the hypothalamus - pituitary axis , and stored in the pituitary gland . this factor is normally released from the pituitary gland after non - osmotic or osmotic stimulus . non - osmotic stimulus is associated with variation in extracellular fluid ( ecf ) volume . this is mediated through volume receptors located in the left atrium of the heart , aortic arch , and carotid artery . osmotic stimulus that causes the release of adh involves an increase of plasma osmolality mediated through osmoreceptors in the hypothalamus . after adh is released from the posterior pituitary axis in the presence of either osmotic or non - osmotic stimulus , it directly acts on the kidney ( 9 ) . based on our understanding of adh production , stimulation , release , and action , we focused on disturbances of the osmotic or non - osmotic pathway and the hypothalamus - pituitary axis as the causes of central di . we first considered the possibility of osmoregulation or hypothalamus - pituitary axis disturbances as a mechanism of central di . increases in ecf osmolality due to dehydration decrease the volume of osmoreceptor cell , which triggers an electric stimulus leading to membrane depolarization , exocytosis , and the release of adh by hypothalamus - pituitary axis . the osmotic stimulus which triggers adh release is initiated by 1 to 2% changes in ecf ( 10 ) . in our patient , serum osmolality was increased to 333 mosm / kg and sustained in high level , and massive polyuria continued . there were no specific abnormalities suspecting the disturbance of osmotic stimulus or hypothalamus - pituitary axis . sheehan and murdock described postpartum pituitary necrosis in patients who were in hemorrhage shock after parturition ( 11 ) . they postulated that a circulatory collapse due to diminished blood flow would result in vascular thrombosis in the pituitary gland . they later extended this theory to include posterior necrosis as a cause of di ( 12 ) . in the present study , we could not overlook the possibility of transient cerebral ischemia resulting in disturbances of the osmotic stimulus receptor or hypothalamus - pituitary axis although no severe posterior pituitary damage occurred . according to some reports ( 13 - 15 ) , the incidence of cerebral ischemia developing after open - heart surgery is generally between 1% and 6% . most cases of stroke or transient ischemic damage following open - heart surgery occur in patients in whom no carotid stenosis or other clear etiologic factors can be uncovered . however , 6% to 12% of cabg patients have carotid stenosis greater than 50% preoperatively but have no bruit on physical examination ( 16 ) . since we did not perform a carotid doppler study , subclinical carotid stenosis could not be excluded . in addition , cpb itself could cause transient subclinical circulatory problems in the cerebral blood supply . the major non - osmotic stimulus appears to be associated with volume receptors in the left atrium , which communicate with the hypothalamus through the vagus parasympathetic stimulus . a decrease in the left atrial volume is associated with diminished parasympathetic afferent drive and a release in adh , whereas an increase in volume at the receptor is associated with increased parasympathetic afferent drive and suppression of adh release ( 9 ) . we assumed that the left atrial non - osmotic receptor function could be transiently altered by a standstill heart during cpb . and this would result in an increase in parasympathetic afferent drive associated with suppression of adh release . given the above postulations , concomitant disturbances by both stimuli could induce central di development . during our diagnosis , we considered the other possible causes of polyuria for exclusion : iatrogenic fluid overloading , osmotic diuresis , and recovery from acute tubular necrosis . di development after extracorporeal circulation has been rarely reported and is an overlooked postoperative complication . however , di should be considered in individuals who have undergone open - heart surgery and develop polyuria . it was reported that the presence of carotid bruit , history of cerebral ischemic disease , atrial fibrillation , bypass times greater than 2 hr , previous myocardial infarction , left ventricular mural thrombus , air in the bypass system , and micro - aggregate formation increase the risk of transient ischemic attack or stroke following open - heart surgery ( 17 - 20 ) . with these risk factors , an operator must be aware of post - operative cerebral ischemic complications given the possibility of central di development with polyuria . when di is diagnosed , prompt and adequate adh administration may prevent further serious problems . in conclusion

diabetes insipidus ( di ) is characterized by excessive urination and thirst . this disease results from inadequate output of antidiuretic hormone ( adh ) from the pituitary gland or the absence of the normal response to adh in the kidney . we present a case of transient central di in a patient who underwent a cardiopulmonary bypass ( cpb ) for coronary artery bypass grafting ( cabg ) . a 44-yr - old male underwent a cabg operation . an hour after the operation , the patient developed polyuria and was diagnosed with central di . the patient responded to desmopressin and completely recovered five days after surgery . it is probable that transient cerebral ischemia resulted in the dysfunction of osmotic receptors in the hypothalamus or hypothalamus - pituitary axis during cpb . it is also possible that cardiac standstill altered the left atrial non - osmotic receptor function and suppressed adh release . therefore , we suggest that central di is a possible cause of polyuria after cpb .

activator protein 1 ( aka activating protein 1 , ap-1 ) is a critical transcription factor that participates in a wide range of cellular processes including proliferation , apoptosis , differentiation , survival , cell migration , and transformation . ap-1 has emerged as an actively pursued drug discovery target and has received particular attention over the past 2 decades with a resurgence of interest in recent years ( figure 1 ) . accumulating evidence suggests that ap-1 plays an important role in several severe disorders including cancer , fibrosis , and organ injury , as well as inflammatory disorders such as asthma , psoriasis , rheumatoid arthritis , and transplant rejection . despite the great therapeutic potential of this target and the tremendous academic and industrial efforts dedicated to it 3-{5-[4-(cyclopentyloxy)-2-hydroxybenzoyl]-2-[(3-hydroxy-1,2-benzisoxazol-6-yl)methoxy]phenyl}propionic acid ( t-5224 , 51 ) , a novel ap-1 inhibitor co - developed by toyama chemical and kitasato university , has proven to prevent joint destruction , pannus formation , and osteoclastogenesis in collagen - induced arthritis ( cia ) in rats . 51 shows good promise as a new drug for the treatment of arthritis and is currently in phase ii human clinical trials in japan . additionally , 51 is under investigation for other inflammatory diseases in which ap-1 is involved . this review is structured to provide the readers with a brief summary of ap-1 family proteins , structures , functions , ap-1 associated signaling pathways , and their roles in various human diseases , as well as the development of ap-1 inhibitors and hit - to - lead optimizations over the past 2 decades from a medicinal chemistry perspective . number of papers published between 1990 and 2013 according to recent pubmed search using ap-1 the ap-1 family of transcription factors is composed of homodimers and heterodimers of jun ( v - jun , c - jun , junb , and jund ) , fos ( v - fos , c - fos , fosb , fral , and fra2 ) , atf ( atf2 , atf3/lrf1 , b - atf , jdp1 , and jdp2 ) , and maf ( c - maf , mafb , mafa , mafg / f / k , and nrl ) protein families , which are characterized by highly conserved dimeric basic leucine zipper ( bzip ) dna - binding domains . the leucine zipper is a structural motif that forms an extended -helix in which every seventh amino acid is a leucine . the carboxy - terminal regions of -helixes align to form parallel coiled coils , while the amino - terminal regions make base - specific contacts with dna in the major groove ( figure 2 , c - fos / c - jun , pdb code 1fos ) . there are a number of crystal structures of ap-1 protein domains and complexes available from the protein data bank , which have significantly facilitated the understanding of the ap-1 family proteins and their structural diversities . structures of ap-1 ( c - fos / c - jun , pdb code 1fos ) . these multiple family members are expressed in a cell- and stage - dependent manner during development and mediate the transcription of specific genes at different levels . among them , the jun and fos subfamilies are the most studied and the major ap-1 proteins . although members of the jun and fos families share a high degree of structural homology , the individual ap-1 dimers exert significant differences in their dna binding affinity and their capability of activating or suppressing gene expression , suggesting specific functions in gene regulation for individual ap-1 dimers . the jun proteins can both homo- and heterodimerize with members of fos and atf subfamilies , whereas fos protein can only heterodimerize with jun proteins rather than homodimerize among themselves . jun - jun and jun - fos dimers prefer to bind to a heptamer consensus sequence known as the tpa - responsive element ( tre , 5-tga(c / g)tca-3 ) , whereas jun - atf dimers or atf homodimers preferentially bind to a different consensus sequence known as the camp - responsive element ( cre , 5-tgacgtca-3 ) . in addition , ap-1 proteins can also interact with non - bzip proteins , including the p65 subunit of nf-b , cbp / p300 , and rb , further expanding the combinatorial diversity of ap-1 family proteins and the spectrum of regulated genes . a variety of ap-1 associated biological functions in development and disease have been revealed from extensive analyses of mice and cells harboring genetic modifications of distinct fos and jun genes . as summarized in table 1 , many important insights have been provided though gain - of - function and loss - of - function experiments using transgenic and embryonic stem ( es ) cell technology . ap-1 activity is induced by a plethora of physiological stimuli and environmental insults , such as the phorbol ester tumor promoter tpa , growth factors , neurotransmitters , polypeptide hormones , cell matrix interactions , cytokines , uv irradiation , and bacterial and viral infections . ap-1 protein is primarily regulated at the level of both jun and fos gene transcription involving mitogen - activated protein kinases ( mapks ) pathways and by post - translational modification via phosphorylation and dephosphorylation . as depicted in figure 3 , mapk cascades consist of three- or four - tiered signaling modules in which the mapk is activated by a mapk kinase ( mapkk ) , which in turn is activated by a mapk kinase kinase ( mapkkk ) . the mapkkk itself is activated by a small g - protein such as ras , either directly or via another upstream kinase . among these , the jnks and erks , together with the later discovered p38s , constitute three separate groups of mapks . the erks are activated by the mek1 and mek2 , and the p38 is activated by the mkk3 and mkk6 . after mapks are activated , they are able to regulate downstream transcription factors that induce the transcription of fos and jun genes , thereby increasing the expression of ap-1 complexes . the expression of fos is induced by tcfs , which are activated through phosphorylation by the p38 , jnks , and erks . the expression of jun is induced by mef2c , atf2 , and jun , which are activated through phosphorylation by the p38 and jnk . once ap-1 and other associated cellular factors are activated , they can participate in regulating altered gene expression , cellular proliferation , apoptosis , differentiation , and migration in response to cytokines and growth factors , noxious stimuli , and oncogenic transformation . in addition , various kinases via post - translational phosphorylation also regulate ap-1 activity , including its transactivating potential , dna - binding capacity , and the stability of ap-1 components ; for instance , casein kinase ii ( ckii ) , glycogen synthase kinase-3 ( gsk-3 ) , and ribosomal s6 kinase 2 ( rsk2 ) . recently , it has also been shown that the ap-1 protein can be regulated and activated by several other mechanisms , including genetic interaction with other oncoproteins or ancillary proteins , messenger rna turnover , and protein stability . inflammation is a part of the complex physiological response of the human body to harmful stimuli , such as pathogen invasion , irritants , and tissue injury . inflammation is widely recognized as the body s attempt at self - protection and a beginning of the healing process . in the course of inflammation , immune cells of the innate and/or adaptive immune system are activated , and a variety of different cytokines and chemokines , which are predominantly regulated by ap-1 and other transcription factors including nf-b , nfats , and stats , are recruited to the site of inflammation . inappropriate activation of the immune system , such as in the case when inflammatory cells and proteins attack and destroy healthy tissue , can result in the overproduction of immune cells , inflammatory cytokines , and tissue - destructive enzymes , thereby giving rise to inflammatory disorders including osteoarthritis , periodontal disease , rheumatoid arthritis , multiple sclerosis , asthma , inflammatory bowel disease , and psoriasis . interestingly , many cytokine genes are cooperatively regulated by a transcription factor complex consisting of ap-1 and nfat . ap-1 is a proinflammatory element that is believed to directly control the expression of the cytokines such as tumor necrosis factor ( tnf- ) , interleukin 1 ( il-1 ) , interleukin 2 ( il-2 ) , ifn , gm - csf , and matrix - degrading matrix metalloproteases ( mmps ) such as collagenase 1 and stromelysin at the level of mrna synthesis by directly binding their promoter ap-1 binding motifs . moreover , ap-1 also induces many of the genes that are dependently regulated by nfat . increasing evidence supports that ap-1 plays a vital role in the initiative and development of inflammatory disorders . it is believed that ap-1 activation is a necessary step in the chain of events that lead to joint erosion . systematic administration of an ap-1 decoy oligodeoxynucleotide to competitively inhibit ap-1 dna binding was found to reduce arthritic joint destruction in mice with collagen - induced arthritis . moreover , increased activation and expression of ap-1 have been shown in the airway of asthmatic patients . the gene for muc5b , which contains a putative ap-1 consensus site in its promoter , is responsible for the airways mucus production . furthermore , in psoriatic lesions , epidermal keratinocytes have decreased expression of junb ( 19p13.2 ) , a gene localized in the psoriasis susceptibility region psors6 ( 19p13 ) . both human and inducible mouse models have demonstrated that down - regulation of junb in keratinocytes is one initiating event in the etiology of psoriasis that is characterized by increased cell proliferation and deregulated cytokine expression . taken together , inhibition of ap-1 may act as a promising therapeutic strategy to provide the benefit of treating the underlying inflammatory process and reducing the production of inflammatory cytokines and chemokines , which play a key role in inflammatory disorders . in 1982 , murine c - fos proteins were first identified as the viral oncoproteins v - fos in the finkel biskis later , mouse c - jun protein was also first found in its viral counterparts v - jun in the avian sarcoma virus 17 . other jun ( junb and jund ) and fos ( fosb , fra1 and fra2 ) proteins have also been identified as oncoproteins , all of which are components of ap-1 . several ap-1 proteins such as c - fos , fosb , and c - jun have potent transactivation domains , which are identified by their ability to induce target gene transcription and efficiency in transforming cells in culture . other ap-1 proteins have either a weak transforming activity ( fra1 and fra2 ) or no transforming activity ( junb and jund ) due to the lack of potent transactivation domains . nevertheless , all of these ap-1 proteins are actively involved in tumor development and progression . numerous studies have shown that the ap-1 family of transcription factors play a crucial role in proliferation , apoptosis , angiogenesis , oncogene - induced transformation , and invasiveness ( table 2 ) and are involved in many cancers including breast , ovarian , liver , skin , bone , lung , endometrial , and colorectal tumors . when widely overexpressed in mice , c - fos causes osteosarcoma formation by the transformation of chondroblasts and osteoblasts , which identifies these two cell types as cellular targets of c - fos - induced tumorigenesis . it has also been revealed that c - fos is required for breast cancer cell growth . although c - jun is variably expressed in human breast tumors with only approximately 2040% , c - jun overexpression in mcf-7 breast cancer cells can produce highly invasive and hormone - resistant tumors . it is found that blockade of c - jun with the expression of a dominant - negative c - jun ( tam67 ) reduces the expression of cyclin ds , e2fs , and dp1 , resulting in suppressed cell proliferation in vitro and in vivo . conditional inactivation of c - jun by overexpressing tam67 in mammary epithelial cells , basal keratinocytes , or the liver reduces c - jun activity and interferes with the development of transgene- or chemical - induced er - negative mammary tumor , papillomas , and liver tumors , respectively , suggesting that c - jun is an important target in the development of er - negative breast cancer , skin and liver tumors . in addition , overexpression of fra1 and fra2 in transgenic mice can lead to the development of lung tumors and epithelial tumors , respectively . furthermore , loss of the junb transcription factor induces a myeloproliferative disease ( mpd ) arising from the hematopoietic stem cell ( hsc ) compartment.junb inactivation has been observed in a spectrum of human myeloid malignancies including cml , and down - regulation of junb expression has also been found in the hsc compartment of patients with acute myeloid leukemia . therefore , junb protects against myeloid malignancies by limiting hematopoietic stem cell proliferation and differentiation without affecting self - renewal . in summary , accumulating studies support that increased expression of ap-1 is associated with a variety of human cancers , and ap-1 is actively implicated in tumor progression and development . the available findings suggest that ap-1 may act as a promising target for cancer prevention and therapy and that ap-1 inhibitors may have great potential to be developed as effective drugs for oncogene - induced transformation , premalignant lesions , and tumor cell growth . of the several potential therapeutic indications by modulating . moreover , compelling evidence using genetically modified mice has provided novel insights into the fundamental functions of ap-1 and shown that up - regulation or down - regulation of ap-1 expression also has a significant contribution in the progression or initiation of various other human diseases , including hepatitis , pulmonary fibrosis , atherosclerosis , cardiovascular diseases , and parkinson s disease ( pd ) . c - jun and junb are strongly expressed in the liver of human and murine with hepatitis . in hepatocytes , the dimeric transcription factor c - jun is a major mediator of cell survival during hepatitis ( inflammation of the liver ) . mice lacking c - jun in hepatocytes display increased liver cell death and mortality upon administration of con a , which induces liver inflammation and injury . wagner and colleagues found that this phenotype was caused by impaired expression of inducible nitric oxide synthase ( nos2 ) , a direct transcriptional target of c - jun , and reduced production of hepatoprotective nitric oxide ( no ) . moreover , increased hepatotoxicity in mutant mice is likely caused by hypoxia and oxidative stress and can be pharmacologically rescued by liver - specific no delivery . thus , c - jun is hepatoprotective during acute hepatitis by regulating nos2/no expression and thus functionally antagonizes the cytokine - induced cell death - promoting functions of jnk during hepatitis . surprisingly , c - jun does not seem to be critical for immune response in hepatitis . mice specifically lacking junb in hepatocytes are also found to display a mild increase in con a - induced liver damage . using loss - of - function mouse models for junb , wagner and co - workers have subsequently demonstrated that junb promotes cell death during acute hepatitis by regulating ifn- production in nk and nkt cells and thus functionally antagonizes the hepatoprotective function of c - jun in hepatocytes . in transgenic mice , ectopic expression of fra 2 in various organs results in generalized fibrosis with predominant manifestation in the lung , possibly by linking vascular remodeling and fibrogenesis . strong expression of fra 2 was also observed in human samples of idiopathic and autoimmune - mediated pulmonary fibrosis . fra2 has been considered as a contributing pathogenic factor of pulmonary fibrosis in humans . however , different from fra2 , fra1 plays a protective role in lung fibrosis and modulates early profibrotic cellular responses . fra1 mediates antifibrotic effects through the modulation of expression of proinflammatory , profibrotic , and fibrotic gene both in vitro and in vivo . thus , either fra1 or fra2 transcription factors may act as potential targets for pulmonary fibrosis , a progressive disorder with poor prognosis and treatment . in vitro and in vivo animal studies implicate ap-1 as a critical common inflammatory transcription factor correlated in the initiation and progression of vascular dysfunction and atherogenesis . lindeman and colleagues have demonstrated that abundant ap-1 activation is associated with all stages of atherosclerosis by performing systematic histological evaluation . lai and colleagues have revealed that irbesartan , an angiotensin ii receptor antagonist , may modulate inflammation - based atherosclerotic diseases through a cell - mediated mechanism involving suppression of human t - lymphocytes activation via down - regulation of ap-1 activity . hence , ap-1 inhibition may represent a promising strategy to prevent progression of atherosclerotic disease . early immediate up - regulation of ap-1 in response to cardiac hypertrophic stimuli was reported in the 1990s . two members of the ap-1 family of transcription factors , jund and fra1 , have been found to play an important role in regulation of heart growth during hypertrophic response . jund , the only jun protein constitutively and highly expressed in mammalian heart , can attenuate phenylephrine - mediated cardiomyocyte hypertrophy by inhibiting ap-1 transcriptional activity and may effectively function as an endogenous dominant negative regulator , buffering against extensive hypertrophic growth of cardiomyocytes in response to pathophysiological stress . in several in vitro studies , c - jun and c - fos have been suggested to be required for induction of fetal gene expression and cardiomyocyte hypertrophy in response to different stimuli . in fact , both jun and fos are not essential for postnatal cardiac hypertrophy as well as heart growth in response to mechanical pressure overload from a genetic evidence in vivo . remarkably , however , deletion of jun but not fos can result in progressive myocardial fibrosis , cardiomyocyte apoptosis , and changes in sarcomeric organization . additionally , the ap-1 components fosb and junb regulate the intrinsic matrix metalloproteinase 2 ( mmp-2 ) promoter in vivo following ischemia reperfusion injury . mmp-2 , which influences ventricular performance , is a central component of the response to injury in the heart . ap-1 is involved in the neuropathology of pd patients , whose phenotype can be closely mimicked by mpp - induced neurotoxicity in vitro . using an s - type human neuroblastoma cell line ( sh - ep1 ) as a model , feng and colleagues investigated the involvement of nf-b and ap-1 pathways in mpp - induced neurotoxicity . besides nf-b inhibition of c - jun activation , by a dominant negative c - jun or c - jun inhibitor such as curcumin , can significantly attenuate mpp - mediated cell death , suggesting that c - jun activation is proapoptotic . thus , down - regulating the activation of nf-b , jnk , and c - jun may represent a new strategy for the treatment of pd . as ap-1 plays important pathophysiological roles in various human conditions , ap-1 inhibitors are currently under intense development preclinically and clinically in many therapeutic areas , especially for inflammatory disease and cancers . several core molecular scaffolds have been identified to be associated with anti - ap-1 properties and led to development of a number of potent ap-1 inhibitors . in addition , some natural products bearing ap-1 inhibitory activity are also described herein . there appears to be no generally accepted system of classifying such molecules , and this review relies on broad structural similarity and seeks to provide an update on research advances of the past 2 decades in the development of chemical entities as ap-1 inhibitors . sp100030 ( 1 , figure 4 ) is one of the first reported potent small molecule inhibitors of ap-1 and nf-b transcription activation with an ic50 value of 0.05 m . it is about 10-fold more potent than the earlier hit 2 ( ic50 = 0.5 m ) , which has been identified by using automated high - throughput assays with stably transfected human jurkat t - cells . hit 2 was reported to have an inhibitory effect on the production of il-2 and il-8 levels in stimulated cells , and it was found to be efficacious in an animal model of inflammation . in order to increase the potency of hit 2 , hundreds of compounds have been designed and synthesized through the use of solution - phase parallel chemistry and targeted synthesis and 1 was identified as the most potent analogue . 1 has been demonstrated to selectively inhibit cd8(+ ) t - cells and mrna expression of both th1 and th2 cytokines in vivo instead of inhibiting allergen - induced airway eosinophilia and bronchial hyperresponsiveness ( bhr ) in a rat model of asthma . in addition , 1 was confirmed active in a dose - dependent manner in several animal models of inflammation and immunosuppression ( ip , 1020 mg / kg ) . moreover , daily subcutaneous injection of cachectic yoshida ah-130 ascites hepatoma - bearing rats with 1 at a dose of 1 mg / kg resulted in a clear amelioration of the cachectic effect , especially at the level of skeletal muscle . at this dose , 1 acts as an effective inhibitor of ap-1 , while the nf-b transcription factor is not affected , suggesting that the ap-1 signaling cascade plays an important role in the signaling of muscle wasting associated with disease . however , the low aqueous solubility and high lipophilicity of 1 likely gave reason for the lack of poor oral activity in the animal models , consistent with its poor permeability in the gastrointestinal cell line , caco-2 ( apparent permeability coefficient papp = ( 11 4 ) 10 cm / s ) . chemical structures of 1 and its representative analogues 220 . to improve its potential oral bioavailability and caco-2 permeability , a series of analogues of 1 modified at the 2 , 4 , 5 , and 6 positions of the pyrimidine ring as shown in figure 4 have been prepared by using a solution - phase parallel synthesis technique . all these compounds meet the criteria of lipinski s rule of five . the introduction of a fluoro group in the place of 2-chloro of 1 results in compound 3 with a comparable activity ( ic50 of 0.01 vs 0.05 m ) . however , other substitutions at the 2-position lead to a loss of activity , such as compounds 4 and 5 ( table 3 ) . the trifluoromethyl group at the 4-position can be replaced with a methyl ( 6 ) , chloro ( 7 ) , or phenyl ( 8) with slight changes of activity . when it is moved to the 6-position ( compounds 9 and 10 ) , the activity is totally lost . among these , the 2-methyl analogue ( 6 ) shows comparable in vitro activity to 1 ( ic50 of 0.02 vs 0.05 m ) , with improved caco-2 permeability ( papp of ( 62 6 ) 10 vs ( 11 4 ) 10 cm / s ) and potential oral bioavailability . palanki and co - workers also investigated the importance of the pyrimidine by replacement with several other ring systems while retaining the n-(3,5-bis(trifluoromethyl)phenyl)carboxamide framework . phenyl analogue 11 is completely inactive , while pyridazines ( 12 and 13 ) and pyrazine 14 are less potent than 1 . surprisingly , compound 13 with a 5-hydrogen is more potent than the 5-trifluoromethyl compound 12 ( ic50 of 1.7 vs 10 m ) , which is somewhat different from the compounds with a pyrimidine ring system . however , compound 12 is 34-fold less potent than 1 ( ic50 of 1.7 vs 0.05 m ) and exhibits a comparable potency to its 1,4-isomer 14 ( ic50 of 1.7 vs 4 m ) . in addition , replacing the 2-chloro-4-trifluorobenzamide in 11 with 2-halogen-4-hydroxylbenzamide results in compounds 1517 , which display good inhibitory effects on ap-1 mediated transcriptional activation in the order of br > cl > i ( table 4 ) and are more potent than their corresponding 3,5-dichlorophenyl analogues 1820 . on the other hand , in an effort to increase the druglike properties of highly lipophilic 1 , palanki and co - workers have also designed several conformationally restricted analogues in which the pyrimidine ring and the aniline ring are connected through a second bridge . in fact , their original plan was only to simply substitute one or two trifluoromethyl groups in 1 with any other groups , but all initial attempts resulted in a loss of activity . as shown in figure 5 , compounds 2123 by restricting one or two dihedral angles are about 200-fold less potent than 1 ( table 3 ) . the direct bridged analogue 24 , the carbon - bridged 25 , and the nitrogen - bridged 26 display weaker activity and are 120- to 200-fold less potent than 1 . however , the oxygen - bridged analogue 27 is 3- to 5-fold more potent than 2426 . interestingly , the potency of seven - membered sulfur - bridged analogue 28 bearing only one trifluoromethyl group is comparable to that of 1 ( ic50 of 0.1 vs 0.05 m ) , representing the most potent in this series . the seven - membered sulfur - bridged analogues thus provide a new class of inhibitors for ap-1 and nf-b mediated transcriptional activation . chemical structures of 2128 . by screening their diversified compound library using automated high - throughput assays with stably transfected human jurkat t - cells , palanki and colleagues have also identified another two novel hits , 29 and its isomer 39 ( figures 6 and 7 ) , which display a slightly lower potency than their earlier hit 2 ( ic50 of 2.0 and 1.0 vs 0.5 m , respectively ) . both hits 29(117 ) and 39(116 ) were found to inhibit ap-1 and nf-b mediated transcriptional activation without blocking basal transcription driven by the -actin promoter and to exhibit a similar inhibitory effect on the production of il-2 and il-8 levels in stimulated jurkat t - cells . chemical structures of analogues based on modifications of hit 29 . to improve the potency of 29 , different substituents around the 2 , 4 , and 5 positions of the pyrimidine ring were explored . as shown in figure 6 , all these compounds display similar ic50 values in both ap-1 and nf-b assays . the removal of a methyl group or the introduction of a phenyl group in place of methyl group or the introduction of another methyl group on the citraconamido ring of the 2-position of the pyrimidine ring in 29 results in a slight change in activity ( ic50 of 1.63.9 vs 2.0 m , table 5 ) . however , removal of the citraconamido ring leads to a much weaker potency ( ic50 = 30 m ) . when the hydrogen of nh group at the 2-position is replaced with an alkyl , substituted carbonyl , urea , or carbamate group , all these compounds exhibit 2- to 6-fold improvement in potency ( ic50 of 0.30.83 vs 2.0 m ) . among them , n - methylated 2-amino group of 29 as the most potent compound displays an improved potency in comparison with 30 ( ic50 of 0.3 vs 2.0 m ) . at the 4-position of the pyrimidine ring , replacement of the trifluoromethyl with a methyl group results in a compound with a comparable potency , while that with an ethyl ( 31 ) or pentafluoroethyl group leads to an improved potency ( ic50 of 0.20.4 vs 2.0 m ) . moreover , replacement of the trifluoromethyl of 30 with an ethyl group can further improve its potency about another 8-fold ( 32 , ic50 of 0.035 vs 0.3 m ) . substitutions with bulky groups such as phenyl and benzyl are not favorable to improve the potency in general , while 2-(5-methylthienyl ) analogue ( 33 ) displays a better potency with 40-fold improvement with an ic50 value of 0.045 m . the introduction of a bulkier alkyl ester group such as tert - butyl ester ( 34 ) instead of ethyl ester at the 5-position leads to about a 10-fold increase in potency ( ic50 of 0.21 vs 2.0 m ) . other substitution groups such as a carboxylic acid , carboxamide , or n , n - dimethylcarboxamide result in a loss of potency ( ic50 = 30 m ) . compounds 35 and 36 with a methyl ketone or a phenyl ketone at the 5-position exhibit an improved potency with an ic50 value of 4.4 and 0.098 m , respectively . several bioisosteres of ethyl ester such as oxazoline , isoxazole , oxadiazole , tetrazole , or phenyloxazole moieties were found to display a lower potency than 29 ( ic50 of 2.810 m ) , while analogues 37 and 38 with the methyloxazole exhibit a slightly increased potency . chemical structures of analogues based on modifications of hit 39 . upon the basis of hit 39 as the chemical lead , different substituents at the 2 and 4 positions of the pyrimidine ring have also been investigated to improve its potency . as shown in figure 7 , the substitutions with small alkyl groups such as methyl and ethyl instead of trifluoromethyl moiety at the 2-position generally result in a loss of activity , while bulkier groups such as tert - butyl group can retain the activity with a comparable potency . the substituted or unsubstituted heterocyclic rings at 2-position of the hit generally lead to a reduced activity . however , analogues 40 and 41 with a phenyl or 2-thienyl moiety exhibit a 10- to 50-fold improved activity ( ic50 of 0.10.02 vs 1.0 m , table 4 ) . other substituents on the phenyl ring of 40 or the thienyl ring of 41 appear to be less favorable . the removal of a methyl group or the introduction of a phenyl group instead of methyl or the introduction of an additional methyl group into the citraconamido ring , as well as methylation or acetylation of the nh group at the 4-position of the pyrimidine ring in 39 results in a decreased activity . unfortunately , the most potent compound 41 was found to display no oral activity in rat pk studies and poor caco-2 permeability likely due to the existence of the carboxylate moiety . in addition , several analogues of 41 with oxazoline , isoxazole , oxadiazole , or tetrazole as the carboxylate bioisosteres at the 5-position of pyrimidine ring turn out to be less potent . to improve the druglikeness of highly lipophilic lead 41 , a new class of inhibitors without a carboxylate ester function have been designed and synthesized by introducing a fused phenyl ring on the pyrimidine with a methoxy group at its 5-position ( figure 7 ) . by changing the position and number of the methoxy groups on the phenyl of the quinazoline ring , as well as replacement with other electron - withdrawing or -donating groups , palanki and co - workers have identified several improved compounds with a methoxy group at the 5-position ( 42 ) or 6-position ( 43 ) with 2.5- to 6.7-fold improvement compared to 41 ( ic50 of 0.0080.003 vs 0.02 m ) . compound 42 ( spc-839 ) displays higher permeability than 43 ( pc of ( 1.41 0.4 ) 10 vs ( 1.62 0.2 ) 10 cm / s ) . moreover , 42 has also been demonstrated to be more efficacious in an adjuvant - induced arthritis rat model by reducing the swelling by 65% in the noninjected foot . on the basis of compound 42 as the advanced chemical lead and utilizing the bioisosterism and other medicinal chemistry optimization approaches , giri and colleagues have designed and investigated a series of new compounds with the ( 2-(2,4-disubstituted - thiazole-5-yl)-3-aryl-3h - quinazolin-4-one scaffold as potential inhibitors of nf - kb and/or ap-1 mediated transcriptional activation for developing anti - inflammatory agents ( figure 7 ) . 44 ( ic50 = 5.5 m ) and 45 ( ic50 = 5.5 m ) have been identified to be more selective toward inhibiting ap-1 mediated transcriptional activity over nf-b in luciferase reporter assays on hek293 cells . compound 48 turns out to be the most potent dual inhibitors of nf-b and ap-1 mediated transcriptional activation with an ic50 value of 0.2 and 0.5 m , respectively.48 has also been demonstrated to have significant in vivo efficacy in a carrageenan injection - induced inflammation model with 56% inhibition of rat paw edema . additionally , giri and co - workers have also evaluated the potential of the novel scaffold mentioned above for cancer by inhibiting multiple pathways in luciferase reporter assays on hek293 cells . 46 and 47 ( ic50 of 1.23.0 m ) are more selective toward inhibiting ap-1 mediated transcriptional activity , while 49 is a potent dual inhibitor of both nf-b and ap-1 mediated transcriptional activation with ic50 values of 3.3 and 4.3 m , respectively . by replacement of the trisubstituted thiazole moiety with a thiophenyl ring , a series of 2-(2,3-disubstituted - thiophen-5-yl)-3h - quinazolin-4-one analogues have been designed and explored as potential anti - inflammatory and anticancer agents . compound 50 is the most potent dual inhibitor of nf-b and ap-1 mediated transcriptional activation from the entire series , with ic50 values of 10 and 5 m for nf-b and ap-1 , respectively . 51 ( figure 8) , an inhibitor of the c - fos / ap-1 , has been identified by converting cyclic disulfide decapeptides to a series of nonpeptidic benzophenone derivatives using a lead - hopping approach based on a 3d pharmacophore model . 51 specifically inhibits the dna binding activity of c - fos / c - jun without affecting those of other transcription factors including c / ebpa and atf-2 ( bzip domain ) , myod ( basic helix loop helix domain ) , sp-1 ( zinc - finger domain ) , and nf-b / p65 ( rel homology domain ) , as well as the levels of c - fos family protein members themselves . administration of 51 at a dose of 30 mg / kg was found to resolve type ii collagen - induced arthritis ( cia ) in a preclinical model by reducing the amount of inflammatory cytokines including interleukin 1 and matrix - degrading mmps in vivo in sera and joints as well as in vitro in synovial cell and chondrocyte cultures.51 also synergizes with antitumor necrosis factor ( tnf ) , a signaling molecule immediately downstream of c - fos , to inhibit arthritis . in addition , glucuronides were also found to be major metabolites in human urine . by use of human liver microsomes ( hlms ) , human intestinal microsomes ( hims ) , recombinant human cytochrome p450 ( cyp450 ) , and udp - glucuronosyltransferases ( ugts ) isoforms expressed in baculovirus - infected insect cells , it was predicted that 51 was converted to its acyl o - glucuronide by ugt1a1 and ugt1a3 and to its hydroxyl o - glucuronide by several ugts , but it was not metabolized by the p450 . moreover , the glucuronidation of 51 was estimated to predominantly occur in the liver by comparing the intrinsic clearances ( clint ) between hlm and him . on the basis of its promising pharmacological effects in arthritis , 51 has been developed by toyama chemical as a potential therapeutic agent for rheumatoid arthritis and has advanced into human phase ii clinical trials in japan . recently , it has also been reported that 51 not only can ameliorate lipopolysaccharide ( lps ) induced liver injury through decreasing production of proinflammatory cytokines and chemokines in endotoxemic mice but also has a potential inhibitory effect against endotoxin - induced acute kidney injury ( aki ) by suppressing the tnf- inflammatory response and other downstream effectors . discovery of 51 , a selective ap-1 inhibitor in phase ii human clinical trial . the cyclic disulfide decapeptides , especially the most potent inhibitor ac - c[cys - gly - gln - leu - asp - leu - ala - asp - gly - cys]-nh2 ( 52 ) , have been designed and synthesized as inhibitors of the c - fos / ap-1 de novo by three - dimensional pharmacophore modeling based on the x - ray crystal structure of the bzip domain of the ap-1dna complex . a hypothetical 3d pharmacophore model was then constructed for generating new c - fos / ap-1 inhibitors based on an alanine scan experiment , molecular dynamics simulation of the bzip52 complex , and nmr measurement of the peptide in water . the pharmacophore consists of three hydrophobic groups , one hydrogen bond acceptor or donor , and one acidic group . using a lead - hopping strategy based on the 3d pharmacophore model , tsuchida and co - workers discovered new nonpeptidic small - molecule ap-1 inhibitors based on 1-thia-4-azaspiro[4.5]decane and benzophenone derivatives , which were synthetically accessible and easy to optimize . as shown in figure 8 , compounds 53 and 54 bearing the scaffold of 1-thia-4-azaspiro[4.5]decane and compounds 55 and 56 bearing the scaffold of benzophenone display lower inhibitory activity than peptide 52 ( ic50 = 64 m ) on the binding of ap-1 bzip and oligonucleotides containing the ap-1 binding site ( ic50 of 420650 m ) using an elisa - based ap-1 dna - binding assay . additionally , these compounds also inhibit the expression of ap-1-luciferase by tpa - stimulated nih3t3 cell ( ic50 of 5.013.3 m ) . however , 51 can fit the cyclic peptide 52 -derived model well , with an ic50 value of 10 m for most of the in vitro cellular assays including luciferase assays . moreover , in vivo ed50 of 51 is 110 mg / kg , and the cmax is 0.030.5 m ( 15240 ng / ml ) . compound 51 is the only selective ap-1 inhibitor that has been advanced into human clinical trials to date . k1115 a ( 57 , figure 9 ) , a new anthraquinone derivative , was isolated from the culture broth of streptomyces griseorubiginosus ( mer - k1115).57 inhibits the direct binding of ap-1 to ap-1 oligonucleotide ( ic50 = 100 m ) and the production of collagenase in il-1-stimulated rat synovial cells ( ic50 = 60 m ) in vitro . moreover , 57 can attenuate the inflammatory response mediated by ap-1 through decreasing the ornithine decarboxylase ( odc ) activity of phorbol myristate acetate ( pma ) induced mice . given that certain truncated peptides would have the potential to bind the dna ap-1 consensus sequence under the same conditions as the native ap-1 protein , patterson and co - workers have designed a series of peptide truncated ap-1-like peptides , with five to seven residues , bearing a highly conserved sequence motif lysine cysteine arginine ( kcr ) were attached at the n - terminus to an intercalating anthraquinone moiety of 57 through an amino acid type linker . peptides were found to displace ap-1 protein binding from its dna consensus sequence much more effectively than their respective free peptides using the electrophoretic mobility shift assay ( emsa ) . moreover , the peptide conjugates containing the more basic sequences 2-akcrna ( 58 ) , 2-akcrka ( 59 ) , 2-akcrnra ( 60 ) , and 2-akcrkra ( 61 ) were demonstrated to be the most effective inhibitors in this series . curcumin ( 62 , figure 9 ) , a hydrophobic polyphenol derived from curcuma longa , a plant of the ginger family , is well - known to have diverse biological functions such as anti - inflammatory , antitumor , antioxidative , cytotoxic , antifungal , antibacterial , and antihepatotoxic activities . curcumin was reported to inhibit tpa - induced expression of c - fos and c - jun protooncogene mrna and to repress c - jun binding to its cognate motif in nih3t3 mouse fibroblast cells . additionally , suppression of ap-1 binding to dna was also observed in human leukemia cells and transformed keratinocytes . furthermore , curcumin displays an inhibitory effect against the complex formation of the fos jun dimer and the dna consensus sequence with an ic50 value of 6.9 nm and is about 30-fold more potent than dihydroguaiaretic acid ( dhga , 63 ) , isolated from the aryls of myristicafragrans ( ic50 = 0.21 m ) . however , the analogue of dhga , nordihydroguaiaretic acid ( ndga , 64 ) , exhibits a potency comparable to that of curcumin ( ic50 = 7.9 nm ) . both dhga and ndga are capable of suppressing leukemia and lung and colon cancer in the mtt - based bioassay . curcumin has also progressed to human clinical trials , but it is a nonselective ap-1 inhibitor mixed with activities of other targets including various transcription factors , thereby significantly limiting its potential as a targeted therapy . abbreviations in the peptides are the following : a , ala ; k , lys ; c , cys ; n , asp ; r , arg . to improve the potency and selectivity of curcumin - based compounds , yang and colleagues synthesized a series of symmetrical curcumin analogues and evaluated their inhibitory activity on fos curcuminoids 65 and 66 exhibit an ic50 value of 8.98 and 5.40 m , respectively , which are 60- to 100-fold more potent than curcumin ( ic50 = 540 m ) . in addition , through inhibition of ap-1 transcription , thereby down - regulating the expression of angiogenesis - associated genes ( vegf and mmp-9 ) , curcumin analogues 67 and 68 were found to show the angiogenesis inhibitory effect on the developmental neovascularization of chicken embryonic with 6888% inhibition of embryos exposed to 10 g of the compound treatment . their inhibitory effects on angiogenesis have also been confirmed by wound migration , invasion , and tube formation assays . momordin i ( 69 ) , isolated from ampelopsis radix , and its disaccharide and carboxylic acid modified derivatives 70 and 71 ( figure 10 ) have demonstrated a remarkable inhibitory effect on the formation of fos jun dna complex . momordin i derivatives 70 and 71 display an ic50 value of approximately 4.0 m in emsa assay , which is 30-fold more potent than momordin i ( ic50 = 0.13 mm ) and about 125-fold more effective than curcumin ( ic50 = 0.48 mm ) . microbial transformation is an area of great interest for applying biocatalysis to selectively convert synthetic and natural products to biological compounds that are difficult to obtain by conventional chemical methods . special attention has been paid to filamentous fungi because of their capability of catalyzing regio- and stereoselective hydroxylation of a variety of nonfunctionalized hydrocarbon centers of a great variety of substrates . isosteviol ( 72 , figure 11 ) , with a rigid skeleton comprising four fused rings similar to the steroid skeleton , possesses various biological activities . isosteviol lactone ( 73 ) , an ent - beyerane tetracyclic diterpenoid prepared by reacting isosteviol ( 72 ) with m - chloroperbenzoic acid , was investigated for its activity on mitochondrial metabolism . a number of hydroxylated diterpenoids have been generated from the microbial transformation of isosteviol lactone ( 73 ) with mucorrecurvatus mr 36 , aspergillusniger bcrc 31130 , or absidiapseudocylindrospora atcc 24169 . as shown in table 6 , compounds 7380 exhibit significant inhibitory effects on ap-1 activation in lipopolysaccharide - stimulated raw 264.7 macrophages by ap-1-mediated luciferase reporter gene assay , while 79 is more potent than the reference compound of dexamethasone . given that the important biological properties of steroids are dependent upon its d - ring , a series of compounds with modified d ring have been produced by microbial transformation of isosteviol oxime ( 81 ) with aspergillus niger bcrc 32720 and absidia pseudocylindrospora atcc 24169 . among the compounds tested , 8286 significantly inhibit ap-1 activation in lps - stimulated raw 264.7 macrophages , and in particular , 85 displays an inhibitory activity more potent than dexamethasone . the concentration of each test compound was 10 m . all luciferase activities were normalized to renilla luciferase activity . the data were expressed as multiples of luciferase activity compared to the no - treatment ( control ) group . as shown in figure 12 , norditerpenes nagilactone ( 87 ) and inumakilactone ( 88 ) , isolated from an organic solvent extract of the root bark of podocarpus latifolius ( thunb . ) collected in tanzania , have also been demonstrated to be capable of inhibiting phorbol ester tpa - induced activation of ap-1 activity at the concentrations tested , with ic50 values estimated from dose response curves of 1.5 and 4.0 m , respectively . however , both of them also appear to be toxic , with cell survival of < 50% in each case at more than 2.5 m . quassinoids glaucarubinone ( 89 ) and nothospondin ( 90 ) also display potent , dose - dependent ap-1 inhibition at noncytotoxic concentrations by a -lactamase driven reporter assay using fluorescence resonance energy transfer ( fret ) technology , with ec50 values of 1.49 and 0.13 m , respectively . the potent ap-1 inhibitory activity of 89 may be ascribed to its ether bridge between c-17 and c-11 , which is seen in many other ap-1 active quassinoids , such as 6-senecionylchaparrin ( 91 ) and ailanthinone ( 92 ) . however , 90 is the first quassinoid without an ether linkage that can inhibit ap-1 , albeit at a significantly reduced potency . in addition , 89 is noncytotoxic at a high concentration of 80 m by an xtt assay , while 90 shows some cytotoxicity with an ic50 of approximately 10 m . citrifolinin a ( 93 , figure 13 ) , a new unusual iridoid isolated from the leaves of morindacitrifolia , shows significant inhibition of uvb - induced ap-1 activity in cell cultures , with an ic50 of 69.6 m . citrifolinoside ( 94 ) is another iridoid isolated from the leaves of morindacitrifolia and displays a significant inhibitory effect on uvb - induced ap-1 activity and is 2.4-fold more potent than 93 ( ic50 = 29.0 m ) . from an extract of the palauan cyanobacterium lyngbya majuscule , grassypeptolides f ( 95 , figure 13 ) and g ( 96 ) , bis - thiazoline - containing cyclic depsipeptides with a rare -amino acid , extensive n - methylation , and a large number of d - amino acids , were isolated and found to have moderate inhibitory activity against the transcription factor ap-1 ( ic50 of 5.2 and 6.0 m , respectively ) in hek293 t cells . in addition , compound ( )-97 , a cycloadduct synthesized by tandem retro - diels alder / diels alder cycloadditions of o - quinol dimer under microwave irradiation , shows selective inhibition against ap-1 at 4 m in luciferase reporter assays on hek293 cells ( figure 13 ) . moreover , ( )-97 does not display inhibition of hif-2 , nf-b , or sre dependent transcription at the same concentration . however , during the experiments in the nci 60-cell screen at 10 m , ( )-97 did not pass the threshold for further evaluation of cell growth inhibition . retinoic acid is effective for chemotherapy and chemoprevention of cancer mediated either by blocking ap-1 activity or by activating retinoic acid response element ( rare ) . using an ap-1-luciferase transgenic mouse as a carcinogenesis , dong and colleagues provided the first in vivo evidence that 98 ( sr11302 ) , an ap-1 inhibition - specific retinoid , markedly inhibited both 12-o - tetradecanoylphorbol-13-acetate - induced papilloma formation and ap-1 activation in 7,12-dimethylbenzanthracene - initiated mouse skin . interestingly , 98 does not activate transcription from the rare and displays no activity at retinoic acid receptors ( ec50 > 1 m for rar , rar , rar , and rxr ) . as an ap-1 inhibitor that displays antitumor effects in vivo , 98 serves as an important research tool compound that is commercially available from tocris . in addition , some other nonspecific ap-1 inhibitors such as 6-shogaol and 6-gingerol have also been reported to inhibit 12-o - tetradecanoylphorbol-13-acetate - induced tumor promotion in mice . since the discovery of ap-1 as the pivotal transcription factor in 1987 , significant progress has been made by many pioneers in understanding ap-1 biology and function and identifying ap-1 as an important and a valid therapeutic target for various human diseases . there is a great potential for the development of ap-1 inhibitors as preventive or therapeutic agents for cancer and inflammation , as well as a variety of other human conditions including hepatitis , pulmonary fibrosis , atherosclerosis , cardiovascular diseases , and parkinson s disease . although currently available drugs are helpful in alleviating many of the symptoms of these diseases , more targeted therapeutic approaches based upon the underlying mechanisms remain an urgent need for better specificity of the treatment with lower side effects . to this end , targeting ap-1 with potent and specific inhibitors may represent an attractive molecular approach for new medications . the proof of concept by developing ap-1 inhibitors as a novel therapy for inflammation has been confirmed by the investigation of clinical drug candidate 51 , although its fate is dependent on the outcomes of phase ii human clinical trials . other inhibitors , such as analogues of 1 and 42 , also have the potential to be developed as novel therapeutic agents . nevertheless , researchers have spent tremendous efforts on hit - to - lead optimizations improving their druglike properties of the hits from high - throughput screening , especially their dmpk properties in vivo . thus , safety concerns and in vivo pharmacokinetic alerts should be considered early in the drug discovery process . fragment - based drug design ( fbdd ) , by taking advantage of privileged fragments , may also provide an efficient approach to identify more effective and selective ap-1 inhibitors with better druglikeness . on the other hand , it is worth noting that ap-1 may act as a double - edged sword in tumor development . by regulating genes involved in cell proliferation , differentiation , apoptosis , angiogenesis , and tumor invasion , ap-1 activity in cancer seems to depend upon ap-1 dimer composition and tumor type as well as its differentiation state , tumor stage , and the genetic background of tumor . generally , c - jun mainly has oncogenic functions , while junb and jund have antioncogenic effects . as such , it would be quite challenging for medicinal chemists to design potent and specific ap-1 inhibitors as potential therapy for cancer . individualized treatment may provide a solution to this problem by selecting appropriate patient populations . furthermore , if the antioncogenic function of some ap-1 proteins is properly exploited , it may provide a novel , unexpected , and more tractable direction for the discovery of anticancer treatments that can induce the formation of specific dimers for the selective and efficient killing of cancer cells , thereby actively interfering or antagonizing tumor development . despite the advances in high - throughput screening , structure , and ligand - based molecular modeling and hit - to - lead optimizations , which have identified numerous compounds capable of inhibiting ap-1 transcription activation or its dna binding activity , meanwhile , it has to be pointed out that many of the currently available compounds display polypharmacological profiles by targeting various other transcription factors including nf-b . it is the opinion of the authors that because of the combinatorial diversity and expressed dependence of ap-1 family proteins , the multidisciplinary approaches with combined techniques in molecular pharmacology , chemical biology , and structural biology based on x - ray crystallography of protein inhibitor complexes , as well as computational chemistry , will provide new insights into rational drug design and facilitate the search of novel ap-1 family selective inhibitors . although those identified inhibitors of dual or multiple transcription factors may also be of value for treatment or prevention of diseases , discovery and development efforts remain an urgent need for medicinal chemists to yield more potent , efficacious , and specific ap-1 inhibitors as a viable therapeutic strategy for use in the human clinic .

activator protein 1 ( ap-1 ) is a pivotal transcription factor that regulates a wide range of cellular processes including proliferation , apoptosis , differentiation , survival , cell migration , and transformation . accumulating evidence supports that ap-1 plays an important role in several severe disorders including cancer , fibrosis , and organ injury , as well as inflammatory disorders such as asthma , psoriasis , and rheumatoid arthritis . ap-1 has emerged as an actively pursued drug discovery target over the past decade . excitingly , a selective ap-1 inhibitor t-5224 ( 51 ) has been investigated in phase ii human clinical trials . nevertheless , no effective ap-1 inhibitors have yet been approved for clinical use . despite significant advances achieved in understanding ap-1 biology and function , as well as the identification of small molecules modulating ap-1 associated signaling pathways , medicinal chemistry efforts remain an urgent need to yield selective and efficacious ap-1 inhibitors as a viable therapeutic strategy for human diseases .

the myelodysplastic syndrome ( mds ) is a heterogenous group of stem cell disorders usually characterized by progressive refractory cytopenias with possible progression to acute myeloid leukemia . although cutaneous manifestation in mds is not frequent , mds may be associated with a wide spectrum of skin lesions1 ) and unusual rheumatologic manifestations2,3 ) . however , concurrent erythema nodosum and serositis has rarely been reported . we describe a case of mds with erythema nodosum and immune - mediated pericardial effusion . a 34-year - old woman was diagnosed with mds french - american - british ( fab ) subtype refractory anemia in march , 2000 . the patient did not receive any special treatment except intermittent transfusion of packed red cell . in may , 2003 the patient presented with fatigue , myalgia , fever as well as multiple edematous , tender , erythematous to brownish subcutaneous nodular lesions on both upper and lower extremities and trunk ( figure 1 ) . laboratory finding indicated hemoglobin 5.8 g / dl , white blood cell count 6.810/l , platelet 4.810/l , and normal liver and kidney function test . biopsy of skin lesion revealed lobular panniculitis consistent with erythema nodosum ( figure 2 ) . one month later , dyspnea , chest pain and fever developed and the skin lesions were aggravated . the laboratory tests showed hemoglobin 6.0 g / dl , white blood cell count 4.810/l , platelet 8.010/l , uric acid 2.3 mg / dl , ldh 474 u / l ( normal 211 - 423 ) , serum ferritin 1831 ng / ml , serum iron / tibc 145/162 g / dl . chest x - ray and chest ct scan showed cardiomegaly with pulmonary edema ( figure 3 ) . after the dose of prednisone was increased to 40 mg / day , the pericardial effusion and skin lesions had remarkably improved , suggesting that these newly developed symptoms might be attributed to the immune - mediated complications of mds . the repeated bone marrow biopsy performed one month later revealed no evidence of progression to acute leukemia . however , the dysplastic changes in the myeloid , erythroid and megakaryocytes lineages had progressed compared to the examination in the prior month . while gradually tapering prednisone until july 2004 , she was taking 5 mg / day of prednisone without pericardial effusion . mds can be associated with a wide spectrum of skin lesions including neoplastic cell infiltration , sweet 's syndrome , pyoderma gangrenosum , erythema elevatum diutinum , vasculitis , and panniculitis1 ) . erythema nodosum is the most frequent clinico - pathological variant of panniculitis and manifests as a cutaneous reaction consisting of inflammatory , tender , nodular lesions , usually located symmetrically on the extensor surfaces of the lower extremities5 ) . manifestation of the lesions may be associated with a wide variety of conditions including infections , sarcoidosis , rheumatologic diseases , inflammatory bowel diseases , medications , autoimmune disorders , pregnancy and malignancies6 - 8 ) . the appearance of skin lesions in mds may herald its progression to acute myeloid leukemia . although in this case the repeated bone marrow biopsy did not show any findings of progression to acute leukemia , the dysplastic changes in all lineages of hematopoietic cells had progressed with the development of skin lesions . it has been reported that autoimmune manifestations in patients with mds range from asymptomatic serological abnormalities to classic connective tissue disorders such as sjgren 's syndrome , relapsing polychondritis , systemic lupus erythematosus , rheumatoid arthritis and mixed connective tissue disease2,3,9 ) . in particular , abnormal t - cell responses to antigen presentation and abnormal b - cell and t - cell interactions in mds may be important in the pathogenesis of immune dysregulation leading to the development of an autoimmune phenomenon10 - 12 ) . thus , abnormalities in t - cell function might be associated with the development of mds13 ) . although pericardiocentesis was not performed in our case , the pericardial effusion rapidly regressed after treatment with high doses of systemic corticosteroid , suggesting that this lesion might be a reactive inflammatory change involving immunologic mechanisms . enright et al.9 ) reported that aggressive therapy with immunosuppressive agents in selected patients often controls autoimmune features associated with mds and may lead to hematological responses in some patients . the most commonly used immunosuppressive agents are oral prednisone and high - dose intravenous methylprednisolone . although there may be poor prognosis with the onset of autoimmune disease in patients with mds , patients with a hematologic response to immunosuppressive therapy have prolonged survival compared with patient who do not . in this case , we noted that treatment with prednisone improved not only erythema nodosum and pericardial effusion , which may be related to an autoimmune manifestation , but also thrombo - cytopenia . in summary , mds may be associated with erythema nodosum with skin lesions that may be a result of autoimmune dysfunction . the understanding of the pathophysiology of mds may contribute to explain the autoimmune manifestations and skin lesions in mds .

myelodysplastic syndrome ( mds ) is a heterogenous group of stem cell disorders usually characterized by progressive refractory cytopenias , which could progress to acute myeloid leukemia . mds may be associated with a wide spectrum of skin lesions , including neoplastic cell infiltration , sweet 's syndrome , pyoderma gangrenosum , erythema elevatum diutinum , vasculitis , and panniculitis . however , erythema nodosum is rarely associated with mds . unusual rheumatologic manifestations in patients with mds also have been reported , which range from asymptomatic serological abnormalities to classic connective tissue disorders such as sjogren 's syndrome , relapsing polychondritis , systemic lupus erythematosus , rheumatoid arthritis and mixed connective tissue disease . however , concurrent erythema nodosum and serositis has rarely been reported . we describe a case of mds with erythema nodosum and immune - mediated pericardial effusion in a 34-year - old woman .

hepatocellular carcinoma ( hcc ) has become a global health problem , with more than half a million new cases diagnosed worldwide each year.1 hcc is associated with a high mortality rate , and is the leading cause of liver - related death in patients with compensated cirrhosis.2 the incidence of hcc is increasing in several developed countries , including the us . between 1995 and 1998 alone , there was a 25% increase of hcc incidence in the us.3 it has been estimated that the number of cases of hcc will continue to increase by 81% to the year 2020 , predominantly due to hepatitis c ( hcv ) infection.4 nevertheless , the incidence of hcc is decreasing in some developing countries as a result of the implementation of universal hepatitis b vaccination.5 older age ( > 50 years ) , male gender , severity of compensated liver cirrhosis at presentation , and sustained activity of liver disease are important predictors of hcc , independent of etiology of cirrhosis.6 males typically have a two- to four - fold higher risk of developing hcc than females . more than 80% of patients with hcc have cirrhosis , which remains the major risk factor . hcv infection , hepatitis b ( hbv ) infection , and heavy alcohol use remain the most common causes of cirrhosis in western countries . once the diagnosis of hcc is made and confirmed , there are several treatment options depending on the size and number of tumors , underlying liver function , presence or absence of tumor vascular invasion , and the patient s performance status . hepatic resection may be an option for those with hcc in a noncirrhotic liver or those with hcc and cirrhosis but without portal hypertension and with normal bilirubin . portal hypertension is defined as the presence of hepatic venous pressure gradient greater than 10 mmhg , esophageal varices , or splenomegaly with a platelet count less than 100,000/mm . subjects without relevant portal hypertension and normal bilirubin can achieve a 70% five - year survival after hepatic resection , whereas survival is only 50% in those with portal hypertension , and even lower with both adverse factors.7 hepatic resection is associated with a high tumor recurrence rate , ie , up to 70% in five years , either from true recurrence or development of de novo tumors . liver transplantation is the mainstay treatment for those with hcc and cirrhosis . during transplantation , not only the tumor itself but also the cirrhotic liver that provides the background for new tumor development will be removed . for those who meet the milan criteria ( solitary hcc less than 5 cm in size or up to three tumors with each tumor less than 3 cm in size ) , transplantation provides a five - year survival exceeding 70% while maintaining a low recurrence rate of less than 15%.7,8 in spite of being the best option for patients with hcc , transplantation is limited by the current shortage of available donors . to avoid dropout while awaiting transplantation , transplant candidates usually receive locoregional treatments , such as transarterial chemoembolization or percutaneous ablation , especially if the waiting time exceeds six months.9 due to the small risk of tumor seeding in the needle tract associated with percutaneous ablation , transarterial chemoembolization is more favored as a bridging therapy for transplant candidates . for those with small unresectable hcc , percutaneous ablation , such as percutaneous ethanol injection and radiofrequency ablation , offers comparable survival with that of resection.1013 in addition to similar survival compared with surgery , percutaneous ablation therapy is relatively simple , low - cost , repeatable , and safe . compared with percutaneous ethanol injection , radiofrequency ablation has a higher rate of complete tumor necrosis ( radiofrequency ablation 90% versus percutaneous ethanol injection 80% ) , requires fewer treatment sessions ( radiofrequency ablation 1.2 versus percutaneous ethanol injection 4.8 ) , and is associated with better local recurrence - free survival compared with percutaneous ethanol injection . nevertheless , the incidence of adverse events is not significantly different between these two treatment modalities . in addition to being used as a bridging therapy before transplantation , transarterial chemoembolization also serves as a palliative measure for those with unresectable hcc and who are not amenable to percutaneous ablation . in a systematic review of randomized , controlled trials involving various treatment modalities in patients with unresectable hcc , arterial embolization improved two - year survival compared with controls ( odds ratio [ or ] 0.53 ; 95% confidence interval [ ci ] , 0.320.89 ; p = 0.017).14 sensitivity analysis showed a significant benefit of transarterial chemoembolization ( or 0.42 ; 95% ci 0.200.88 ) but none with bland embolization alone ( or 0.59 ; 95% ci 0.291.20 ) . radioembolization with microspheres carrying yttrium 90 injected via the intrahepatic arterial route has been proposed as an alternative approach to transarterial chemoembolization.15 this method can deliver radiation to the hcc - associated capillary bed . radioembolization requires only single - dose administration , and has been shown to be therapeutically equivalent to transarterial chemoembolization.16 patients with advanced hcc who are not candidates for local approaches are considered for systemic therapy . systemic therapy with cytotoxic agents , such as doxorubicin , provides marginal benefits without improvement in overall survival.17 investigation in hepatocarcinogenesis has led to the discovery of key molecular targets in hcc , such as angiogenesis , epidermal growth factor receptor , and mammalian target of rapamycin . a growing number of molecularly targeted therapies for hcc are currently at different stages of clinical development.18 sorafenib ( nexavir , bayer healthcare ag , leverkusen , germany ) , a multikinase inhibitor , is the only such drug that has been approved in the us and europe for treatment of unresectable hcc . in this review , we provide a critical appraisal of sorafenib in the management of hcc . sorafenib inhibits multiple tyrosine and serine / threonine kinases involved in cell proliferation and angiogenesis . these kinases include raf kinase , vascular endothelial growth factor receptor ( vegfr ) kinases , platelet - derived growth factor receptor ( pdgfr ) kinase , c - kit receptor kinase , ret receptor kinase , and fms - like tyrosine kinase 3.19,20 disruption of pdgfr and vegfr signaling pathways inhibits angiogenesis . raf is an essential serine / threonine kinase in the mitogen - activated protein ( map ) kinase signaling pathway , and is a downstream effector of ras , which is activated in many human malignancies.21 when the raf - map kinase pathway is activated , extracellular signal - regulated kinase ( erk ) will become phosphorylated . subsequently , phosphorylated erk ( perk ) can be translocated to the nucleus where it can regulate gene expression by modulating various transcription factors and target genes . preclinical models have demonstrated that hcc exhibits activation of map signaling and overexpression of angiogenesis factors . sorafenib blocks the raf - map kinase and erk pathway , inhibits tumor angiogenesis , and induces tumor cell apoptosis in hcc cell lines.22 pharmacodynamic assay for raf - map kinase inhibition by sorafenib has been investigated in patients peripheral blood lymphocytes collected at different time points in a phase i study.23 lymphocytes were activated with phorbol myristate acetate , and flow cytometric analyses of perk were performed . the phorbol myristate acetate - induced perk in peripheral blood lymphocytes from six patients was almost completely eradicated by sorafenib after 21 days of the maximally tolerated dose . in a phase ii study of sorafenib in advanced hcc , immunohistochemical study in pretreatment tumor biopsies using an antibody against perk was performed in 33 patients ( of 137 enrolled patients ) , and perk staining was more intense in the nuclei of tumor tissues.24 there was a significant difference in time to progression between patients with higher tumor cell perk staining intensity versus those with lower intensity . a similar finding was also noted in a retrospective biomarker analysis of the phase iii sorafenib hcc assessment randomized protocol ( sharp ) trial.25 the biomarker study from the sharp trial has further revealed that low hepatocyte growth factor and high c - kit serum levels at baseline were associated with improved survival in multivariate analysis . the recommended dose of sorafenib is 400 mg orally twice a day , and should be administered to patients who are fasting or with a moderate - fat meal . a high - fat meal reduces sorafenib s bioavailability by about 30% . the plasma protein binding of sorafenib is about 99% in patients with normal renal and hepatic function.26 sorafenib is primarily metabolized in the liver , predominantly via phase i oxidation by cytochrome p450 ( cyp ) 3a4 , and phase ii conjugation by uridine 5-diphosphoglucuronosyltransferase.27,28 approximately 50% of orally administered sorafenib is recovered as unchanged drug in the feces , due to either biliary excretion or lack of absorption . the mean terminal elimination half - life of sorafenib is approximately 2438 hours , and sorafenib exposures reach steady state within seven days of dosing.29 because sorafenib is mainly metabolized by cyp3a4 , its pharmacokinetics may be affected by drugs modulating the function of cyp3a4 . concurrent administration of rifampin , a cyp3a4 inducer , has been shown to decrease the area under the plasma concentration versus time curve ( auc ) of sorafenib by 37% . other cyp3a4 inducers , including carbamazepine , phenobarbital , and dexamethasone , may decrease the auc of sorafenib.30 neomycin decreases the auc of sorafenib by 54% , probably due to eradication of gastrointestinal bacteria and therefore preventing the enterohepatic recycling of sorafenib.26 due to potential drug interaction , patients taking medications such as warfarin , digoxin , and quinidine may require close monitoring during sorafenib treatment.26,31 it has been shown that the auc for sorafenib is 30%45% lower in japanese or chinese than in caucasian populations , and the clinical significance of this finding remains unknown.30,32 the child - pugh scoring system uses clinical and laboratory information to stratify disease severity , surgical risk , and overall prognosis in patients with liver cirrhosis.33 the auc of sorafenib in patients with mild ( child - pugh a ) and moderate ( child - pugh b ) hepatic impairment has been shown to be 23%65% lower than in subjects with normal hepatic function , and there are no significant differences in sorafenib pharmacokinetics between patients with child - pugh a and child - pugh b hepatic impairment.24,34 no significant difference in sorafenib pharmacokinetics has been shown between patients with normal renal function and mild - to - moderate renal insufficiency.30,35 the pharmacokinetics of sorafenib has not been studied in patients with severe hepatic impairment ( child - pugh c ) or severe renal insufficiency ( creatinine clearance less than 30 ml / min ) . in a phase i pharmacokinetic study of sorafenib in patients with hepatic or renal dysfunction , miller et al showed that patients with severe renal or hepatic dysfunction frequently developed intolerance to sorafenib.35 therefore , it is recommended to start at a lower dose in these patient populations , followed by close monitoring and gradual dose escalation . two phase iii randomized studies , ie , sharp and the asia - pacific study , comparing sorafenib with placebo as first - line systemic treatment in patients with advanced hcc , have demonstrated the efficacy of sorafenib.36,37 patient characteristics and outcome measures of these two studies are summarized in tables 1 and 2 . the sharp study was designed for the regulatory approval of sorafenib in the us and europe , and was a randomized , double - blinded , placebo - controlled , phase iii study conducted in europe , the us , and australasia . it enrolled 602 patients with advanced hcc , well - preserved liver function , and no prior systemic therapy . patients were randomized in a 1:1 ratio to receive either oral sorafenib ( 400 mg twice daily ) or placebo . the number of patients needed for this study was calculated on the basis of overall survival , with a power of 90% to detect a 40% improvement in overall survival in the sorafenib group . in the sharp study , patients were well balanced with respect to baseline characteristics ( table 1 ) . as summarized in table 2 , this study demonstrated a significant improvement in median overall survival on sorafenib versus placebo ( 10.7 versus 7.9 months ; hazard ratio [ hr ] in the sorafenib group 0.69 ; 95% ci 0.550.87 ; p < 0.001 ) . there was significant prolongation of median time to radiologic progression in the sorafenib group ( 5.5 versus 2.8 months ; p < 0.001 ) . in the sorafenib group , seven patients ( 2.3% ) had a partial response and 71 ( 71% ) had stable disease ( according to the response evaluation criteria in solid tumors [ recist ] ) , whereas in the placebo group , two patients ( 1% ) had a partial response and 204 patients ( 67% ) had stable disease . the disease - control rate , defined as complete / partial response or stable disease by recist for four weeks from the first demonstration of that rating , was significantly higher in the sorafenib group than in the placebo group ( 43% versus 32% ; p = 0.002 ) . based on the sharp data , sorafenib was approved by the european medicines agency in october 2007 and by the us food and drug administration in november 2007 for the treatment of patients with unresectable hcc . in order to achieve regulatory approval in china , cheng et al conducted a phase iii randomized study comparing sorafenib with placebo in patients with advanced hcc in the asia - pacific region.37 this study had no predefined primary endpoint , and used the same eligibility criteria as the sharp study . two hundred and twenty - six patients in china , south korea , and taiwan were randomized on an intention - to - treat basis in a 2:1 ratio to sorafenib versus placebo . the number of patients needed for this study was calculated with a power of 83.9% to detect a 20% increase in overall survival in the sorafenib group . overall survival , time to radiologic progression , time to symptomatic progression , disease - control rate , and safety were assessed . overall survival was 6.5 months with sorafenib versus 4.2 months in the placebo group , with an hr of 0.68 ( p = 0.014 ) . additionally , there were significant differences in time to radiologic progression and disease - control rate between the sorafenib and placebo groups , but not in time to symptomatic progression . the asia - pacific study has confirmed the results of the sharp study , and the magnitude of benefits in overall survival and time to radiologic progression derived from sorafenib was almost the same between these two studies , ie , a 31%32% of relative risk reduction in death , and a 42%43% relative benefit of increase in time to progression . however , the absolute benefit of the increase in overall survival and time to radiologic progression derived from sorafenib was smaller in the asia - pacific study than in the sharp study . additionally , the median overall survival and time to radiologic progression were shorter in asia - pacific study . there were more patients with hbv and severe disease , such as macroscopic vascular invasion and/or extrahepatic spread in the asia - pacific study , which might have contributed to the shorter overall survival and time to radiologic progression than were seen in the sharp trial . patients with hbv - related hcc were shown to have poorer overall survival than patients with hcv - related hcc.38 hbv - related hcc was noted in 73% of patients in the asia - pacific study versus 18% in the sharp trial . a subset analysis of 165 patients with hbv - related hcc in the asia - pacific study indicated that sorafenib improved overall survival and time to radiologic progression independent of hbv status , and the safety profile of sorafenib in patients with hbv was comparable with that for the overall study patients.39 in the sharp study , 30% of patients were infected with hcv , and 25% had alcoholic liver disease . in a subset analysis of 178 patients with hcv - related hcc in the sharp trial , overall survival was 14.0 months in 93 patients receiving sorafenib versus 7.9 months in 85 patients receiving placebo . the disease - control rate was higher in the sorafenib group compared with placebo ( 44% versus 31% ) . the safety profile of sorafenib treatment in patients with hcv - associated hcc was similar to that in all patients receiving sorafenib in this study.40 the subgroup analysis in patients with alcohol - related hcc in the sharp study demonstrated a similar outcome and safety profile to the overall population.41 taken together , there is a consistent clinical benefit from sorafenib in hcc , irrespective of hbv / hcv status or alcoholic liver disease . combined analysis of the sharp and asia - pacific trials to examine the effects of macroscopic vascular invasion , extrahepatic spread , and eastern cooperative oncology group ( ecog ) performance status on outcome has shown that sorafenib was effective and safe for the treatment of advanced hcc in patients globally , irrespective of baseline ecog performance status and the presence or absence of macroscopic vascular invasion and/or extrahepatic spread.42 in the sharp study , 38% of patients had macroscopic vascular invasion and 52% had extrahepatic spread versus 35% with macroscopic vascular invasion and 68% with extrahepatic spread in the asia - pacific study ( table 1 ) . however , a significant difference in overall survival based on the presence or absence of macroscopic vascular invasion and/or extrahepatic spread was observed in both trials . for patients treated with sorafenib in the sharp study , median overall survival was 14.5 months in patients without macroscopic vascular invasion or extrahepatic spread versus 8.9 months with macroscopic vascular invasion and/or extrahepatic spread . for patients treated with sorafenib in the asia - pacific trial , median overall survival was 14.3 and 5.6 months , respectively . therefore , for advanced hcc patients with macroscopic vascular invasion and/or extrahepatic spread , there is a dire need for treatment options in addition to sorafenib to improve the outcome . as summarized in table 3 , the common drug - related adverse events in patients receiving sorafenib were hand - foot skin reaction , diarrhea , alopecia , fatigue , skin rash , hypertension , nausea , and anorexia . hand - foot skin reaction and diarrhea were the two most frequent severe ( grade 3 or 4 ) toxicities in patients receiving sorafenib , at about 5%10% . for patients on sorafenib , 26%31% required dose reduction , and 20%38% of patients terminated treatment due to toxicities . there seemed to be less treatment discontinuation in the asia - pacific study regardless of whether patients were on sorafenib or placebo than in the sharp study . there might be ethnic differences in hand - foot skin reaction , which occurred more frequently in asian patients . hand - foot skin reaction is manifested by formation of thick hyperkeratotic lesions affecting pressure points and flexure areas.43 these lesions usually develop after 24 weeks on sorafenib treatment and are often painful , which affects patient quality of life.44 a mild hand - foot skin reaction can be treated with lanolin - based or urea - based lotions . protective measures , such as wearing gloves and socks , are recommended to provide pain relief and prevent skin breakdown . dose reduction or interruption of sorafenib is frequently needed in patients with moderate to severe hand - foot skin reaction . sorafenib can be resumed at a lower dose , with gradual escalation when hand - foot skin reaction is resolved . adverse cardiovascular toxicities , including myocardial infarction , hypertension , congestive heart failure , and arrhythmia , are frequently associated with antiangiogenic inhibitors , and are regarded as a class effect.45 hypertension is the most frequent event , and most of the time is clinically manageable . the incidence of hypertension was 5% in the sharp study and 19% in the asia - pacific study , with a 2% incidence of grade 3 or 4 hypertension noted in sorafenibtreated patients in each of the studies . the incidence of severe cardiac ischemia or infarction in sorafenib - treated patients in the sharp study was 3% versus 1% in patients taking placebo.36 sorafenib was associated with hypophosphatemia in 35% of sorafenib - treated patients compared with 11% taking placebo in the sharp study . grade 3 or 4 hypophosphatemia , which can lead to muscle weakness , altered mental status , and cardiac events , was seen in 11% of patients taking sorafenib.32 therefore , sorafenib is not recommended in patients with a recent history of cardiac disease . all patients taking sorafenib should be monitored frequently for symptoms of cardiac disease and have blood levels monitored regularly for hypophosphatemia . hypothyroidism is a common side effect of sorafenib , and the incidence can be as high as 18%.46 sorafenib can enhance t4 and t3 metabolism , which is probably caused by increased type 3 deiodination.47 clinical hypothyroidism occurs about one to seven months after the initiation of sorafenib.48 therefore , it is important to monitor thyroid function periodically in patients receiving sorafenib . in the sharp study , elevated lipase occurred in 40% of patients receiving sorafenib versus 37% for patients on placebo . however , grade 3 or 4 lipase elevation was seen in 9% of patients receiving sorafenib.32 there have been cases reported of acute pancreatitis in patients receiving sorafenib treatment for hcc or renal cell carcinoma , and pancreatitis usually improves after stopping sorafenib.4951 there are limited data on the use of sorafenib in patients with child - pugh b or child - pugh c hepatic impairment . yau et al reported a study involving 15 patients with child - pugh b / c cirrhosis receiving sorafenib for hcc . thirteen patients discontinued treatment prematurely due to treatment toxicities.52 in a phase ii study of sorafenib in hcc , including 98 child - pugh a and 38 child - pugh b patients , there was no significant difference in sorafenib pharmacokinetics between patients with child - pugh a and child - pugh b impairment.53 however , child - pugh b patients had a higher rate of hyperbilirubinemia , encephalopathy , and worsening ascites , and child - pugh b patients did worse , with overall survival of 14 weeks versus 41 weeks for patients with child - pugh a. therefore , it is recommended to reduce the starting dose , perhaps by 50% , with vigilant monitoring of liver function , if sorafenib is to be used in patients with child - pugh b liver cirrhosis.35,54 for hcc patients with child - pugh c , because of their severe underlying disease with limited life expectancy of usually less than three months , sorafenib treatment is not recommended.55,56 in both the sharp and asian - pacific studies , the functional assessment of cancer therapy hepatobiliary symptom index 8 ( fhsi-8 ) was used to assess time to symptomatic progression . fhsi-8 is a patient - oriented outcome instrument designed to assess symptoms and quality of life in hepatobiliary cancer patients in general.57 fhsi-8 analyses in both studies failed to identify any significant difference between the sorafenib and placebo groups . the quality of life of these patients might have been affected by other concurrent diseases , such as liver cirrhosis . additionally , fhsi-8 might not have been sensitive enough to determine small but significant differences in quality of life between these two groups . sorafenib is the first agent that has resulted in a survival benefit in hcc , and has validated the role of targeted therapy in this disease . its role in the management of advanced unresectable cancer is now well proven in all subpopulations of hcc having adequate hepatic reserve , and has been incorporated in the consensus - based treatment algorithm globally ( see figure 1 for a simplified schema).5860 the role of sorafenib in hcc confined to the liver and as part of multimodality hcc treatment is currently under investigation . ongoing studies will incorporate sorafenib with other treatment modalities , including transarterial chemoembolization , cytotoxic chemotherapy , surgery , and transplantation , in hcc patients.61 further studies are needed to define its dosage and role in patients with child - pugh b and child - pugh c liver cirrhosis . identification of a predictive biomarker signature will help to define the ideal patient population for sorafenib treatment in hcc .

sorafenib is an oral multiple kinase inhibitor that blocks raf , vascular endothelial growth factor receptor , and platelet - derived growth factor receptor . it has been approved in the us and europe for the treatment of advanced hepatocellular carcinoma ( hcc ) . sorafenib has demonstrated a 44% increase in survival for advanced hcc patients , compared with best supportive care alone . we have reviewed the pharmacology , pivotal studies , and safety data for this agent . sorafenib is the first systemic drug demonstrating a significant survival benefit , and is the standard of care for patients with advanced hcc for whom no potential curative option is available .

microvascular decompression ( mvd ) has been known to be an effective surgical modality as the treatment of various hyperactive neurovascular compression syndromes . its high success rate ( over 90% long term cure rate ) in the treatment of hemifacial spasm ( hfs ) has made it the treatment of choice5,10 ) . although mvd is known to be a relatively safe procedure , it is rarely reported in the literature to accompany severe disabling complications . common complications of mvd for hfs are also brainstem or cerebellar infarct , hematomas , cerebrospinal fluid leak and cranial nerve palsy , commonly involving 7th and 8th nerve with rare lower cranial nerve palsies are reported1,5,11 ) . we have experienced unilateral soft palate palsy without the involvement of vocal fold after mvd for hfs . so far , to our knowledge , such complication is very rare , following mvd . here , a 33-year - old female was presented to our out - patient clinic with a history of left hemifacial spasm for 5 years . intra - operatively , bilateral facial electromyography and brain stem auditory evoked potential was monitored . after the dura incision , gentle retraction of the cerebellum was applied using greenburg retractor and we have exposed the lower cranial nerves . dissection of the arachnoid was done from the rostral border of the lower cranial nerves . the arachnoid band was very tough and difficult to dissect , but no obvious injury occurred to the lower cranial nerve during the dissection . the left anterior inferior cerebellar artery was the offending vessel compressing the facial nerve from the caudal side and decompression was successfully completed without any adverse events and with no change in the intraoperative monitoring of brainstem auditory evoked potential . patient recovered well from anesthesia with reduced facial spasm , compared to the pre - operative state . postoperatively , neurologic exam revealed no abnormalities and also patient 's swallowing and vocalization function was intact . barium swallowing test was done to evaluate dysphagia , and minimal nasal regurgitation was demonstrated due to unilateral soft palate palsy . on endoscopic exam , performed by an otolaryngologist , the vocal cord was intact in both sides with unilateral left soft palate palsy , which suggested tensor and/or levator veli palatini muscle weakness ( fig . 2 ) . under the impression of possible lower cranial nerve palsy caused by postoperative edema of the nerve , steroid ( prednisolone ) was prescribed and we decided to observe the symptoms . we encouraged her to sleep on her side ; then her symptom improved . despite persistent speech rehabilitation we performed cranial nerve mri to evaluate for the possibility of any structural abnormalities , such as delayed hematoma or possible infection . however , mri showed no abnormal structural lesions or abnormal enhancement , which may suggest infection ( fig . 3 ) . the patient continued to receive speech rehabilitation with subjective improvement of swallowing and speech . on postoperative 25th day , the patient developed grade ii facial palsy by house - brackmann grade on the left side . patient 's facial palsy improved with administration of acyclovir for 5 days , but hoarseness with swallowing difficulty showed little improvement . the patient received prolonged speech and swallowing rehabilitation therapy , and follow up examination was done before discharge showed improvement of nasalization , but persistent hypernasality . endoscopic examination revealed normalized soft palate palsy and no abnormality in the vocal cord mobility ( fig . however , patient 's subjective difficulty of speech still persisted and we decided to observe her symptoms for any changes in the future . cranial nerve palsy has been reported as complication of mvd and can cause symptoms that markedly decrease the quality of life . most commonly , the 7th and 8th cranial nerve are involved with rare lower cranial nerve involvement . among the lower cranial nerves , glossopharyngeal nerve and vagus nerve glossopharyngeal nerve contains both motor and sensory branches that supply the tongue and stylopharyngeus muscle of the pharynx . impairment of glossopharyngeal nerve would cause decreased sensation in the pharynx including taste in the posterior one third of the tongue with difficulty in swallowing and dysphonia2 - 4 ) . our patient developed swallowing difficulty with dysphonia , which suggests possible dysfunction of the 9th nerve . vagus nerve also contains both motor and sensory branches that innervate all striated muscles of the laryngopharynx , except the stylopharyngeus and tensor veli palatini . patients with unilateral high vagal lesions would suffer from breathy dysphonia and aspiration because of impaired vocal cord movement . due to the loss of sensation of the laryngopharynx , causing loss of gag reflex , patients might also suffer severe dysphagia related to the poor coordination of the oro- and hypo - pharyngeal phases of swallowing2 - 4 ) . unilateral soft palate palsy with dysphonia and dysphagia was observed in our patient , which suggests unilateral vagus nerve dysfunction , but the sparing of the vocal cord suggest the function of the vagus nerve is only partially impaired . although the functions and innervations of the 9th and 10th nerve are known , it is difficult to exactly locate which lower cranial nerve is causing the symptom by physical examination . many symptoms are subtle and overlap with other diseases , making the diagnosis elusive2 ) . common injury to the vagus nerve is accompanied by vocal fold palsy , but our patient only presented with soft palate palsy . in addition , partial injury of the vagus nerve with possible involvement of the glossopharyngeal nerve have never been reported as a complication of mvd in prior literature , thus making it difficult to identify the exact cause of such peculiar symptoms . skull base tumors are the most common cause and trauma or surgery above the skull base and from the cerebellopontine angle can also be a cause . rarely , infection and ischemic vascular insult are reported to cause unilateral vagus nerve palsy3,7,8,12 ) . it is important to note that in our patient , the hoarsness and dysphagia developed several days after the surgery . generally , lower cranial palsy caused by trauma would occur immediately after the event , which implies the possibility of other factors , such as infection contributing in the delayed onset of lower cranial nerve palsy . in the literature , some have reported herpes simplex virus ( hsv ) to be a possible cause of unilateral vagus nerve palsy12 ) . in addition , varicella zoster virus is also reported to cause lower cranial nerve palsy6 ) . although we have failed to show such connection in our patient , history of delayed facial palsy also suggests the possibility of concurrent viral infection . others have also suggested that delayed facial palsy after mvd in patients with hfs may be caused by viral infections . as hsv is widely known to cause bell 's palsy , it could likewise cause reversible inflammation of vagus nerve causing such clinical presentations11 ) . the history of relapsing symptom , followed by tapering of steroid , suggests additional evidence that inflammatory reaction contributed in the development of such symptom . antiviral agent , such as acyclovir , is reported to be an effective treatment that we have used for facial palsy of the patient . some reported rare anatomical variants of lower cranial nerves with interconnection between the glossopharyngeal nerve and vagus nerve . such interconnection is so thin and very fragile that it makes them prone to shearing stress14 ) . though they have not suggested its clinical implication , it is possible that such interconnection holds some significance in the nerve conduction . as such , it may have been overlooked and damaged during the retraction of the surgery , causing lower cranial nerve palsy . the clinical course of unilateral lower cranial nerve palsy is variable depending on the cause . initial extensive laboratory work up with ct and mr imaging is necessary to evaluate the cause . if no particular structural abnormalities are noted , conservative treatment , including acyclovir and steroid administration with speech therapy , seems to be reasonable . if symptoms persist , surgical treatments , such as medialization laryngoplasty and laryngeal reinnervation , can be considered9,13 ) . we have experienced a very rare case of unilateral soft palate palsy , following mvd . although the exact cause of this complication had not been identified , we have suggested possible causes for such rare complication . it is important to keep in mind that mvd is performed to improve the quality of life rather than to save life . although it has relatively little complications , rare and debilitating complications must not be overlooked , and caution must be taken to avoid them . mvd is a very effective and relatively safe surgical modality in the treatment of hfs . the lower cranial nerve palsy we are reporting is one of such complication that can markedly decrease the quality of life of a patient and it is important to note that such delayed complication can occur without evident injury to the nerve during the operation . it is important to consider various etiologies as the treatment , and prognosis differs greatly on the cause . additionally , careful surgical technique with minimal retraction with extra care to avoid damage to the lower cranial nerve during exploration may be important to minimize such complication .

microvascular decompression is a very effective and relatively safe surgical modality in the treatment of hemifacial spasm . but rare debilitating complications have been reported such as cranial nerve dysfunctions . we have experienced a very rare case of unilateral soft palate palsy without the involvement of vocal cord following microvascular decompression . a 33-year - old female presented to our out - patient clinic with a history of left hemifacial spasm for 5 years . on postoperative 5th day , patient started to exhibit hoarsness with swallowing difficulty . symptoms persisted despite rehabilitation . various laboratory work up with magnetic resonance image showed no abnormal lesions . two years after surgery patient showed complete recovery of unitaleral soft palate palsy . various etiologies of unilateral soft palate palsy are reviewed as the treatment and prognosis differs greatly on the cause . although rare , it is important to keep in mind that such complication could occur after microvascular decompression .

hesc culture the hesc line h1 ( wa01 ) was kindly provided by dr . saul sharkis from johns hopkins university , under permission from wicell research institute ( 5 , 16 ) , and hues-17 was kindly provided by dr . all hesc experiments were conducted in accordance with the guidelines for research on human embryonic stem cells , jointly issued by the ministry of science and technology and the ministry of health of china ( 20 ) , and approved by the ethical committee of shanghai institutes for biological sciences . hescs were maintained on feeders in hesc medium , which contained 80% dulbecco 's modified eagle 's medium / ham 's f-12 medium ( f12 ) , 20% knock - out serum replacement , 1 mm l - glutamine , 0.1 mm -mercaptoethanol , 1% nonessential amino acids , and 4 ng / ml human basic fgf . hescs cells were passaged approximately once a week by incubation in 1 mg / ml collagenase iv for 30 min at 37 c . protein factors or sb431542 were added directly to the culture in the continued presence of conditioned medium ( cm ) . recombinant human activin a , recombinant human bmp-4 , and human follistatin were purchased from r&d systems inc . rna isolation and real - time reverse transcription - polymerase chain reaction rna was extracted using trizol reagent for total rna isolation according to the manufacturer 's instructions ( invitrogen ) . cdna was synthesized using the revertaid first strand cdna synthesis kit ( fermentas ) . real - time pcr was performed using a synergy brand greeni - based pcr master mixture ( toyobo ) . the expression value of each gene was normalized to the amount of glyceraldehyde-3-phosphate dehydrogenase cdna to calculate a relative amount of rna present in each sample . the expression level of each gene in a single sample was arbitrarily defined as 1 unit . the normalized expression values for all control and treated samples were averaged , and an average -fold change was determined . analysis of variance was conducted between the normalized relative expression values for control and treated samples to determine statistical significance . immunostaining the following antibodies were used : anti - ssea4 ( developmental studies hybridoma bank ) , anti - hcg ( r&d systems ) , and anti - hcg ( abcam ) . western blotting cells were lysed with 1 lysis buffer : 20 mm tris ( ph 7.5 ) , 150 mm nacl , 1% triton x-100 , 1 mm na3vo4 , and complete mini - protease inhibitor mixture ( roche ) . total protein membranes were blocked in tris - buffered saline with 0.1% tween and 5% milk . the following antibodies were used : anti - phospho - smad2/3 ( cell signaling ) , anti - smad2/3 ( cell signaling ) , anti - phospho - smad1 ( santa cruz ) , anti - smad1 ( santa cruz ) , anti - oct4 ( santa cruz ) , and -actin ( abcam ) . primary antibodies were incubated overnight and secondary antibodies for 2 h. proteins were detected with chemiluminescent ( pierce ) . h1 cells were cultured in cm with or without sb431542 for 12 days , and the medium was changed every day . the hcg concentration was analyzed using a hcg elisa kit ( reci ) , which specifically reacts with cg-. the concentration of estradiol and progesterone were analyzed with an elisa kit . activin / nodal signaling has been shown to play a key role in the maintenance of undifferentiated human es cells ( 16 - 18 ) . to further address the function of activin / nodal signaling in the developmental fate of hescs , and to understand the early developmental mechanisms of human embryogenesis , we inhibited activin / nodal signaling in hescs . two hescs lines , h1 and hues-17 , were used in this study , and the results obtained from these two cells lines were very similar . for this reason , hescs were cultured without murine embryonic fibroblast feeder cells in cm , or cm plus different concentrations of activin / nodal signaling inhibitors , sb431542 or follistatin , for 6 days ; gene expression was analyzed by real - time pcr . consistent with previous studies , the conditioned media to the culture system is sufficient for the maintenance of undifferentiated hescs ( 21 ) . sb431542 inhibits the function of activin receptor - like kinase receptors 4/5/7 thereby acting as a selective inhibitor of activin / nodal signaling , but not those of bmps ( 22 , 23 ) . follistatin is an inhibitor of activin by directly binding with activin and preventing the assembly of an active activin - receptor complex ( 24 ) . when hescs were cultured in cm supplied with sb431542 , the expression levels of p - smad2 , and known downstream targets of activin / nodal signaling , namely nodal , lefty - a , and lefty - b , were significantly inhibited ( figs . sb431542 is a very potent inhibitor of activin / nodal signaling ; in hescs cultured with cm plus 10 m sb431542 , the expression of nodal , lefty - a , and lefty - b decreased to less than 0.1% of hescs cultured with cm . we also determined that hescs underwent differentiation when activin / nodal signaling was inhibited , because the treated cells became flattened and enlarged ( fig . 1b ) and that pluripotency markers , such as oct4 , nanog , and ssea4 were significantly down - regulated ( fig . similar results were obtained with follistatin as an inhibitor of activin / nodal signaling in hescs as observed with sb431542 ( fig . 1 ) . these results demonstrate that and the inhibition of activin / nodal signaling promoted differentiation of hescs . inhibition of activin / nodal signaling in hescs initiates trophoblast differentiation to determine lineage commitment or differentiation due to inhibition of activin / nodal signaling , we analyzed the induction of lineage - specific marker expression . unlike what we observed with a standard differentiation by embryoid bodies formation , we did not observe a significant up - regulation in expression of ectoderm ( neurofilament heavy chain ) , mesoderm ( cardiac actin ) , or endoderm ( 1-antitrypsin ) markers ( fig . 2a ) , indicating that inhibition of activin / nodal signaling under the monolayer culture condition did not initiate differentiation of endoderm , mesoderm , or ectoderm in hescs . however , the trophoblast marker gcm1 was specifically up - regulated ( fig . 2b ) , which suggests that hescs might have differentiated into trophoblasts when activin / nodal signaling was inhibited . the notion of trophoblast differentiation was further supported by the up - regulation of other trophoblast markers , such as cdx2 , gata2 , msx2 , cg- , and cg-. cg- and cg- are subunits of human chorionic gonadotropin ( hcg ) , which is secreted by giant cells of trophoblast - derived placenta . we also analyzed another key regulator of trophoblast differentiation in mice , eomesodermin ( eomes ) ( 25 ) . although eomes plays a key role in mouse trophoblast differentiation , it is a downstream target of activin / nodal signaling in mice and xenopus ( 26 , 27 ) . we observed that eomes showed down - regulation when activin / nodal was inhibited . taken together , these data indicate that the inhibition of activin / nodal signaling results in trophoblast differentiation in hescs . notably , we also observed a slight up - regulation of neuroectoderm markers , such as nestin , sox1 , sox3 , and ngn2 , when activin / nodal signaling was inhibited , which supports a recent article by smith et al . the h1 he s cells were cultured under a feeder - free condition and treated with sb 431542 for 6 ( a and b ) or 12 days ( c ) . then cells were harvested for analyses . a , real - time polymerase chain reaction analysis of endoderm ( 1-at ) , mesoderm ( cact ) , and ectoderm ( nfh ) markers . b , real - time polymerase chain reaction analysis of multiple trophoblast markers . sb431542 up - regulates trophoblast marker expression in a dose - dependent manner . c , differentiated cells form syncytial cells after incubation in cm plus 10 mol / liter sb 431542 for 12 days . the expression level of each gene in h1 hescs maintained on murine embryonic fibroblast feeder cells is arbitrarily defined as 1 unit . 1-at , 1-antitrypsin ; cact , cardiac actin ; nfh , neurofilament heavy chain ; dapi , 4,6-diamidino-2-phenylindole . the h1 he s cells were cultured under a feeder - free condition and treated with sb 431542 for 6 ( a and b ) or 12 days ( c ) a , real - time polymerase chain reaction analysis of endoderm ( 1-at ) , mesoderm ( cact ) , and ectoderm ( nfh ) markers . b , real - time polymerase chain reaction analysis of multiple trophoblast markers . sb431542 up - regulates trophoblast marker expression in a dose - dependent manner . c , differentiated cells form syncytial cells after incubation in cm plus 10 mol / liter sb 431542 for 12 days . the expression level of each gene in h1 hescs maintained on murine embryonic fibroblast feeder cells is arbitrarily defined as 1 unit . 1-at , 1-antitrypsin ; cact , cardiac actin ; nfh , neurofilament heavy chain ; dapi , 4,6-diamidino-2-phenylindole . we attempted to differentiate hescs that were growing as embryoid bodies or as a monolayer ; however , results were similar ( supplemental fig . the differentiation of hescs as a monolayer produced higher expressions of trophoblast markers and lower expression of other lineage markers , such as sox3 . therefore , the data presented in this paper pertain to monolayer cultures , unless specifically mentioned . to understand the kinetics of trophoblast differentiation , we performed time course experiments and analyzed marker expression by real - time pcr . the expression of pluripotency markers , namely oct4 and nanog , decreased in a time - dependent manner ( fig . cdx2 has been shown to be the key regulator of trophoblast commitment and subsequent self - renewal in mice ( 29 ) ; inhibition of activin / nodal signaling in hescs initiated cdx2 expression after 2 days , and expression rose to a peak on day 6 and decreased thereafter ( fig . 3c ) . gcm1 expression was induced by inhibition of activin / nodal signaling on day 4 and continued to increase throughout differentiation ( fig . 3c ) . two additional markers that often associated with bmp activation and trophoblast commitment , gata2 and msx2 , were also dramatically up - regulated and reached a peak level at day 10 ( fig . furthermore , cg- and cg- expression significantly increased at day 6 and reached a surprisingly high level on day 12 ( fig . 3c ) , which suggests that eomes might be dispensable in trophoblast differentiation of hescs . the transient expression of cdx2 suggests that its function could be to induce gcm1 and other trophoblast transcriptional factors , and the down - regulation of cdx2 might allow for further trophoblast maturation . although the hesc is the only available model thus far for studying human embryonic development , the human es cell model may not entirely reflect embryonic development in vivo . to explore this the 8-cell stage mouse embryos were cultured with 10 m sb431542 for 3 days . no gross abnormalities were detected at 4.5 days postcoitum ; the inner cell mass and trophoblast formed normally ( data not shown ) . these observations are in accordance with previous reports , demonstrating that activin / nodal signaling is involved in the propagation of mouse embryonic stem cells , but is not involved in the regulation of pluripotency ( 18 , 30 ) . hesc - derived trophoblast cells secrete placental hormones prolonged cultures of hescs in cm plus sb431542 were performed ( 12 days ) ; the cells continued to develop , and numerous differentiated cells contained multiple nuclei ( fig . 2c ) . ( 13 ) reported that syncytial cells were present only among individualized bmp4-treated hescs plated at low density , whereas bmp4-treated hesc colonies form only mononuclear cells . in contrast , the present study demonstrated that the sb431542- or follistatin - treated hesc colonies formed syncytial cells ( fig . 2c ) , which suggests that inhibition of activin / nodal signaling is more efficient than bmp4 in inducing syncytial cell formation . it was not attempted to induce hesc differentiation in individual cells . to further confirm trophoblast differentiation from hescs , both cg- and cg- proteins were detected in a large percentage of differentiated hescs after 12 days treatment with sb431542 or follistatin ( fig . the percentage of the cg--expressing cells was 74 5% ( n = 3 ) when activin / nodal signaling was inhibited by 10 m sb431542 , and 66 3% ( n = 3 ) when activin / nodal signaling was inhibited by follistatin , respectively . in addition , during hesc differentiation , the placental hormones , hcg ( consisting both a and b subunits ) , estradiol , and progesterone , were secreted in the supernatant in a time- and dose - dependent manner ( fig . inhibition of activin / nodal signaling down - regulates fgf and wnt signals , but up - regulates bmp signals fgf signaling has been shown to be important in the maintenance of hesc pluripotency ( 31 , 32 ) , and wnt signaling has been shown to stimulate the proliferation of hescs ( 33 - 35 ) . previously , we have reported that activin / nodal signaling up - regulates fgf and wnt signaling in hescs ( 16 ) . 5a ) was significantly repressed by the inhibition of activin / nodal signaling , but p - smad1 and bmp4 expression was significantly up - regulated ( fig . 5 , b and c ) . these observations further strengthen our previous hypothesis that activin / nodal signaling plays a key role in the complex signaling network that maintains the hesc phenotype and function ( 16 ) . bmp4-induced trophoblast differentiation correlates with inhibition of activin / nodal signaling as reported by xu et al . ( 16 ) , we also observed that hescs differentiated into trophoblasts when cultured in cm plus bmp4 ( 10 - 50 ng / ml ) , as evidenced by the down - regulation of pluripotency markers , such as oct4 and nanog ( fig . 6a ) , and the up - regulation of cdx2 , gcm1 , gata2 , cg- , and cg- ( fig . 6a ) . at the same time , expression of lefty - a , lefty - b , and nodal was largely inhibited in a dose - dependent manner ( fig . taken together , these results indicate that bmp4 was sufficient to inhibit activin / nodal signaling and that bmp4-induced trophoblast differentiation in hescs correlates to the inhibition of activin / nodal signaling . figure 3.trophoblast differentiation in a dose- and time - dependent manner . the h1 he s cells were cultured under a feeder - free condition and treated with sb431542 for 12 days . real - time pcr analyses of the downstream targets of activin / nodal signaling ( a ) , the pluripotent markers ( b ) , and the trophoblast markers ( c ) , during differentiation of h1 cells to trophoblast cells following induction by sb431542 . relative expression levels of each gene were analyzed at 0 , 2 , 4 , 6 , 8 , 10 , and 12 days , respectively , after addition of sb431542 . the expression level of each gene at day 0 ( prior to the addition of sb431542 ) is arbitrarily defined as 1 unit . the h1 he s cells were cultured under a feeder - free condition and treated with sb431542 for 12 days . real - time pcr analyses of the downstream targets of activin / nodal signaling ( a ) , the pluripotent markers ( b ) , and the trophoblast markers ( c ) , during differentiation of h1 cells to trophoblast cells following induction by sb431542 . relative expression levels of each gene were analyzed at 0 , 2 , 4 , 6 , 8 , 10 , and 12 days , respectively , after addition of sb431542 . the expression level of each gene at day 0 ( prior to the addition of sb431542 ) is arbitrarily defined as 1 unit . inhibition of activin / nodal signaling is essential for trophoblast differentiation we further investigated whether inhibition of activin / nodal signaling is essential for hesc trophoblast differentiation . hesc differentiation was induced by incubating the cells in cm supplemented with bmp4 and gradients of activin a. results showed that activin a restored the expression of lefty - a , lefty - b , and nodal , indicating release of the bmp inhibition effect on activin / nodal signaling ( fig . activin a also significantly inhibited cg- and cg- expression , which was induced by bmp4 ( fig . immunostaining methods were utilized to detect cg- and cg- proteins in hescs after 6 days of treatment with 10 ng / ml bmp4 , or 10 ng / ml bmp4 plus 100 ng / ml activin a. bmp4 induced the hescs to produce cg- and cg-. however , the number of cg-- and cg--positive cells was reduced dramatically when activin a was added ( fig . , elisa analyses demonstrated that activin a significantly repressed the placental hormones , hcg , estradiol , and progesterone , in a dose - dependent manner ( fig . therefore , we conclude that inhibition of activin / nodal signaling is essential for trophoblast differentiation of hescs . bmp activation is required for the trophoblast differentiation from hescs we showed that inhibition of activin / nodal signaling induced the expression of bmp4 ( fig . it is interesting to know if the bmp4 induced by inhibition of activin / nodal is required for the trophoblast differentiation . we took advantage of a glycosylphosphatidylinositol - ap deficient hesc line , namely ar1-c1 ( 37 ) . the bmp signaling depends on a co - receptor , dragon . the function of dragon is disrupted due to the lacking of glycosylphosphatidylinositol anchor . therefore , the extracellular bmp can not bind with the receptor well and the bmp signaling is blocked . the trophoblast development induced by bmps in wild type hescs ( g - gfp ) is blocked in ar1-c1 hescs , evidenced by absence of the expression of trophoblast markers like cdx2 , cg - a , cg - b ( fig . the deficiency of bmp signaling can be rescued by transfection of dragon , which indicates that the deficiency of trophoblast development is caused by deficiency of bmp signaling , not any other signal , in the ar1-c1 cell . we expected that if activation of bmp signaling by bmps was not required for trophoblast differentiation when activin / nodal signaling is repressed , the ar1-c1 cells would differentiate into trophoblast when activin / nodal signaling is repressed . if activation of bmp signaling by bmps is required , the ar1-c1 cells would not differentiate into trophoblast when activin / nodal signaling is repressed . when the ar1-c1 cells were treated with sb431542 to inhibit activin / nodal signaling , no evidence of trophoblast differentiation was observed ( fig . therefore , our data indicated that both inhibition of activin / nodal and activation of bmp signaling were required for trophoblast differentiation from hescs . the first cell lineage segregation in human embryonic development takes place at the blastocyst stage , when the trophoblast segregates from the inner cell mass . due to ethical and practical reasons , it has been difficult to determine the key signals in this event ( 1 ) . we , along with others , have previously shown that activin / nodal signaling maintains pluripotency of hescs ( 16 - 18 , 36 ) . in the present study , it is demonstrated that hescs develop into trophoblasts , when activin / nodal signaling is inhibited ( fig . 2 ) . based on these observations , we propose that the segregation of the trophoblast from the inner cell mass is controlled by activin / nodal signaling . in the human morula , the cells that receive active activin / nodal signals form the inner cell mass ; other cells that do not receive sufficient activin / nodal signals develop into the trophoblast . this suggests that activin / nodal signaling regulates the first differentiation event of human embryonic development . xu et al . ( 13 ) showed that bmp4 is able to initiate trophoblast differentiation . to further address the mechanisms that control cell lineage segregation at the human blastocyst stage , the relation of bmp signal to inhibition of activin / nodal signaling was investigated . results showed that the effect of bmp4 correlates to inhibition of activin / nodal signaling . in addition , inhibition of activin / nodal signaling induced trophoblast differentiation ( figs . 2 and 3 ) , whereas activin / nodal signaling inhibited trophoblast differentiation resulting from bmp4 signals ( fig we conclude that inhibition of activin / nodal signaling is essential for trophoblast differentiation of hescs . our data also showed that when activin / nodal was repressed , bmp4 was induced . this raised the possibility that bmp4 induced by activin / nodal repression promotes trophoblast differentiation . we used the bmp co - receptor , dragon , deficient hescs ( 37 ) to investigate if the bmp4 induced by activin / nodal repression is required for trophoblast differentiation . we found that trophoblast differentiation was still blocked when activin / nodal was repressed . our data suggested that bmp signaling is still required for trophoblast development even when activin / nodal is repressed . the observation should not be simply interpreted that they are upstream and downstream . because trophoblast induction of bmp also depends on the inhibition of activin / nodal . activin / nodal inhibition induces the expression of bmp and activates bmp signaling ; bmp signaling further inhibits activin / nodal . both inhibition of activin / nodal and activation of the bmp signal our observation reveals that a novel mechanism in which a critical interaction of two related but antagonizing signals by activin / nodal and bmp regulates the fate determination of hescs in culture , and possibly also true for human embryo in vivo . a and b , immunofluorescence for cg- and cg-. h1 cells were treated with cm , or cm plus 10 mol / liter sb431542 or 300 ng / ml follistatin for 12 days . c , immunoassays of placental hormones . conditioned culture medium from h1 cells cultured in cm ; cm + 1 sb ; cm + 10 sb ; cm + 30 fs ; or cm + 300 fs were collected at the indicated times and subjected to immunoassays for hcg , estradiol ( e2 ) , and progesterone ( prog ) . cm + 1sb , cm plus 1 mol / liter sb431542 ; cm + 10sb , cm plus 10 mol / liter sb431542 ; cm + 3fs , cm plus 3 ng / ml follistatin ; cm + 30fs , cm plus 30 ng / ml follistatin ; cm + 300fs , cm plus 300 ng / ml follistatin . hesc - derived trophoblast cells secrete placental hormones . a and b , immunofluorescence for cg- and cg-. h1 cells were treated with cm , or cm plus 10 mol / liter sb431542 or 300 ng / ml follistatin for 12 days . c , immunoassays of placental hormones . conditioned culture medium from h1 cells cultured in cm ; cm + 1 sb ; cm + 10 sb ; cm + 30 fs ; or cm + 300 fs were collected at the indicated times and subjected to immunoassays for hcg , estradiol ( e2 ) , and progesterone ( prog ) . cm + 1sb , cm plus 1 mol / liter sb431542 ; cm + 10sb , cm plus 10 mol / liter sb431542 ; cm + 3fs , cm plus 3 ng / ml follistatin ; cm + 30fs , cm plus 30 ng / ml follistatin ; cm + 300fs , cm plus 300 ng / ml follistatin . figure 5.inhibition of activin / nodal signaling down - regulates fgf and wnt signals , but up - regulates bmp signals . the h1 he s cells were cultured under a feeder - free condition and treated with sb431542 for 6 days . then cells were harvested for real - time polymerase chain reaction analysis of ligands of the fgf ( a ) , wnt ( a ) , and bmp ( b ) signaling pathways and western analysis of oct4 , smad2 , p - smad2 , smad1 , and p - smad1 ( c ) . inhibition of activin / nodal signaling down - regulates fgf and wnt signals , but up - regulates bmp signals . the h1 he s cells were cultured under a feeder - free condition and treated with sb431542 for 6 days . then cells were harvested for real - time polymerase chain reaction analysis of ligands of the fgf ( a ) , wnt ( a ) , and bmp ( b ) signaling pathways and western analysis of oct4 , smad2 , p - smad2 , smad1 , and p - smad1 ( c ) . figure 6.bmp4-induced trophoblast differentiation correlates with and depends on inhibition of activin / nodal signaling . real - time polymerase chain reaction analysis of pluripotent markers , trophoblast markers , and downstream targets of activin / nodal signaling were performed . a , bmp4 promoted the trophoblast differentiation of hescs ; b , the trophoblast induction effect of bmp4 correlated with the inhibition of activin / nodal signal ; c , activin a restores expression of lefty - a , lefty - b , and nodal ; d , real - time pcr analysis of the expression of cg- and cg- ; e , the h1 he s cells were cultured feeder free with cm plus 10 ng / ml bmp4 and 100 ng / ml activin a , immunofluorescence of cg- and cg- indicates that activin a inhibit the effect of bmp4 ; f , immunoassays of the placental hormones , hcg , estradiol ( e2 ) , and progesterone ( prog ) . cell culture supernatant of hescs cultured on mef , in cm , cm + 10b , cm + 10b + 1a , cm + 10b + 10a , or cm + 10b + 100a were collected at the indicated times and subjected to immunoassays for human chorionic gonadotropin , estradiol , and progesterone . abbreviations : cm+10b , cm plus 10 ng / ml bmp4 ; cm+10b+1a , cm plus 10 ng / ml bmp4 and 1 ng / ml activin a ; cm+10b+10a , cm plus 10 ng / ml bmp4 and 10 ng / ml activin a ; cm+10b+100a , cm plus 10 ng / ml bmp4 and 100 ng / ml activin a ; acta , activin a. bmp4-induced trophoblast differentiation correlates with and depends on inhibition of activin / nodal signaling . the h1 he s cells were cultured feeder free under the labeled conditions . real - time polymerase chain reaction analysis of pluripotent markers , trophoblast markers , and downstream targets of activin / nodal signaling were performed . a , bmp4 promoted the trophoblast differentiation of hescs ; b , the trophoblast induction effect of bmp4 correlated with the inhibition of activin / nodal signal ; c , activin a restores expression of lefty - a , lefty - b , and nodal ; d , real - time pcr analysis of the expression of cg- and cg- ; e , the h1 he s cells were cultured feeder free with cm plus 10 ng / ml bmp4 and 100 ng / ml activin a , immunofluorescence of cg- and cg- indicates that activin a inhibit the effect of bmp4 ; f , immunoassays of the placental hormones , hcg , estradiol ( e2 ) , and progesterone ( prog ) . cell culture supernatant of hescs cultured on mef , in cm , cm + 10b , cm + 10b + 1a , cm + 10b + 10a , or cm + 10b + 100a were collected at the indicated times and subjected to immunoassays for human chorionic gonadotropin , estradiol , and progesterone . abbreviations : cm+10b , cm plus 10 ng / ml bmp4 ; cm+10b+1a , cm plus 10 ng / ml bmp4 and 1 ng / ml activin a ; cm+10b+10a , cm plus 10 ng / ml bmp4 and 10 ng / ml activin a ; cm+10b+100a , cm plus 10 ng / ml bmp4 and 100 ng / ml activin a ; acta , activin a. figure 7.activation of bmp4 is required for trophoblast differentiation . the parental ( g - gfp ) and ar1-c1 he s cells were cultured under a feeder - free condition and treated with bmp4 ( 50 ng / ml ) or sb431542 ( 10 m ) for 10 days . b , immunofluorescent staining for trophoectoderm markers troma - i ( red ) from the differentiated g - gfp and ar1-c1 cells . the parental ( g - gfp ) and ar1-c1 he s cells were cultured under a feeder - free condition and treated with bmp4 ( 50 ng / ml ) or sb431542 ( 10 m ) for 10 days . b , immunofluorescent staining for trophoectoderm markers troma - i ( red ) from the differentiated g - gfp and ar1-c1 cells . in contrast , ( 28 ) reported that inhibition of activin / nodal signaling promotes specification of human embryonic stem cells into neuroectoderm . we did observe a very slight up - regulation of neuroectoderm markers ( supplemental fig . s1 ) ; however , we also observed a dramatic up - regulation of trophoblast markers ( figs . 2 and 3 and supplemental fig . ( 28 ) did not analyze trophoblast marker expression , it is likely they overlooked the dramatic differentiation of trophoblast in their experiments , which led to improper conclusions . little is known about normal human development during the early post - implantation period . although the mouse is the typical model for experimental mammalian embryology , early structures , including the placenta , extraembryonic membranes , and the egg cylinder , all differ substantially from the corresponding human structure . our results display that although the most key transcriptional factors exhibit similar expression between hescs and mouse escs , some genes , such as eomes , are completely different . eomes has been reported to be essential for trophoblast development in mice ( 25 ) ; however , when activin / nodal signaling is inhibited , causing hescs to differentiate into trophoblasts , the expression of eomes is down - regulated . this suggests that eomes are not essential for human trophoblast differentiation , which might imply that there are substantial differences between mouse and human early development . human and mouse es cells are both blastocyst - derived ; however , they are not equivalent . the mechanisms that human and mouse es cells use to maintain stemness differ greatly ( 16 - 18 , 30 - 32 , 38 - 40 ) , as well as their developmental potential , especially the capacity to form cells of the trophoblast lineage ( 8 , 13 , 41 - 43 ) . hescs and the mouse epiblast stem cell use the same signaling pathways to maintain pluripotency ( 44 ) , hescs can differentiate into all embryonic germ layers , as well as trophoblasts ( 5 , 8 , 13 , 41 ) . in contrast , mouse es cells are capable of reconstituting all cell types of the body , but do not routinely exhibit a capacity for trophoblast cell differentiation ( 42 , 43 , 45 ) . these differences highlight the fact that hescs are a unique and irreplaceable model for studying early human developmental events . human es cells will be particularly valuable for studying development and function of tissues that differ between mice and humans . hescs give rise to early human cell types that were previously almost unobtainable , which is a major advantage ; however , ethical considerations , as well as the practicalities , will make it extremely difficult to validate in vitro results with in vivo significance . we demonstrate that combinatorial signals of activin / nodal and bmp regulate lineage segregation of early human embryo stem cells in vitro ; however , a direct role for activin / nodal signaling in early human embryonic lineage segregation has not been demonstrated in vivo . expression profiles , attained by analysis of est counts at the ncbi database , shows that activin a , follistatin , and bmps are all expressed in the human ovary and/or uterus , which implies their function during early development . the challenge for the future will be to determine whether activin / nodal and bmp signals play a role in early lineage segregation of human embryo in vivo , and to establish the key transcriptional factor pathways in human embryo trophoblast differentiation using hescs as a model .

cell fate commitment of pre - implantation blastocysts , to either the inner cell mass or trophoblast , is the first step in cell lineage segregation of the developing human embryo . however , the intercellular signals that control fate determination of these cells remain obscure . human embryonic stem cells ( hescs ) provide a unique model for studying human early embryonic development . we have previously shown that activin / nodal signaling contributes to maintaining pluripotency of hescs , which are derivatives of the inner cell mass . here we further demonstrate that the inhibition of activin / nodal signaling results in the loss of hesc pluripotency and trophoblast differentiation , similar to bmp4-induced trophoblast differentiation from hescs . we also show that the trophoblast induction effect of bmp4 correlates with and depends on the inhibition of activin / nodal signaling . however , the activation of bmp signaling is still required for trophoblast differentiation when activin / nodal signaling is inhibited . these data reveal that the early lineage segregation of hescs is determined by the combinatorial signals of activin / nodal and bmp .

dental resin composites have borne witness to remarkable refinements in recent years , especially due to filler size reduction , which has enhanced the polishability and wear resistance of these materials . an important factor driving the achievements made in advanced resin composite properties has been nanotechnology . however , despite the ongoing improvements in resin composites , a concern remains about their chemical and enzymatic degradation in the oral environment . apart from the degradation caused by saliva , by foodstuffs and by beverages , bleaching procedures can potentially cause softening and increased surface roughness to resin composites , depending on the resin composite and on the bleaching agent used . contradictory data has been reported regarding the effect of bleaching agents on microhardness and surface roughness of resin composites . although the authors of some investigations have noticed a reduction in surface microhardness of resin composites after exposure to bleaching agents , authors of other papers have described no change or increase in surface microhardness of composites after bleaching . in regard to surface roughness , the application of bleaching agents to resin composites has been reported , in previous studies , as not changing or increasing surface roughness . bleached resin composites have been observed to stain more easily than unbleached counterparts , probably as a result of softening and increased surface roughness . although the literature is still conflicting regarding the susceptibility of bleached and unbleached composites to staining , an important issue is that staining solutions can also soften resin composites . in fact , red wine and coffee , commonly used as staining solutions in the literature , have been proven to decrease microhardness of resin composites . therefore , both bleaching agents and beverages with staining capacity can decrease the microhardness of resin composites , including nanostrucutured materials . considering the softening effect produced by staining solutions , it is important to evaluate whether resin composites that had been previously in contact with bleaching agents would be affected by further softening and darkening as a result of being immersed in different staining solutions . this study aimed at elucidating this issue by evaluating the effect of staining solutions on microhardness and on the shade changes of a nanofilled composite resin previously in contact with bleaching agents . table 1 states the main materials used , the staining solutions and their compositions , ph values , manufacturers , batch numbers and manufacturers directions . nanofilled composite resin , bleaching agents and their composition , manufacturers , batch numbers , directions and ph values a total of 135 disk - shaped specimens were prepared with a nanofilled resin composite ( filtek supreme xt - 3 m espe , st . paul , mn , usa ) , which was inserted with a metal spatula ( minelli no . 1 - golgran , so paulo , sp , brazil ) in circular acrylic molds ( 10 mm diameter 2 mm thick ) . a mylar strip ( dentart - polidental , so paulo , brazil ) was placed on top of and pressed flat with a microscope slide plus a 500 g weight for 30 s. the composite was photocured with a led unit ( radii - cal sdi - melbourne , victoria , australia - 1,200 mw / cm ) for 20 s. following preparation , specimens were stored in relative humidity at 37c for 24 h. the finishing and polishing steps were performed with al2o3 abrasive disks ( sof - lex pop on - 3 m espe , st paul , mn , usa ) of decreasing coarseness . the specimens were then stored at 37c for another 24 h. the specimens were randomly divided into three groups ( n = 45 ) to be subjected to one of the bleaching agents . an individual acetate tray was fabricated in a vacuum - forming machine ( p7/bio - art equip odontolgicos ltda . , so carlos , sp , brazil ) for each specimen , which would be then exposed to 10% or 16% carbamide peroxide ( cp ) bleaching agents [ cp - whiteness perfect fgm produtos odontolgicos , joinville , sc , brazil - table 1 ] . these bleaching agents were applied for 4 h / day , for 14 days , simulating the at - home bleaching technique . in regard to the group exposed to the 35% hydrogen peroxide ( hp ) agent ( hp whiteness hp 35% fgm produtos odontolgicos , joinville , sc , brazil ) , the product was applied with a disposable applicator ( microbrush - vigodent , rio de janeiro , rj , brazil ) for 45 min ( three sequential 15-min applications ) once a week for three consecutive weeks . when the bleaching time was completed , the specimens were rinsed thoroughly with distilled water and stored in distilled water at 37c . when the bleaching procedures were completed , specimens of each group were allocated into three subgroups ( n = 15 ) to be immersed daily in 200 ml of either coffee ( melitta , so paulo , brazil ) or red wine ( concha y toro , santiago , chile ) , as described in table 1 . specimens were immersed in staining solutions or distilled water for 3 h / day , at room temperature , over 40 days . after every 3-h period of immersion time , the specimens were rinsed thoroughly with distilled water and stored in distilled water at 37c . color shade was measured using a spectrophotometer ( vita easyshade - vita zahnfabrik , bad sckingen , germany ) and the data were expressed based on the vita shade guide and then converted into scores , as can be seen in table 2 . color shade was recorded on three different occasions : baseline , after bleaching and after staining . vita shade guide scores three knoop microhardness indents were carried out on the top surface of each specimen , using a hvs-1000 microhardness tester ( panambra , so paulo , sp , brazil ) , under a 50-g load , applied for 15 s. measurements were performed at the baseline , following bleaching and after staining procedures . microhardness data were analyzed using an analysis of variance ( anova ) for split - plot design and tukey 's test , whereas the color shade recordings were submitted to kruskal - wallis and dunn 's tests . statistical procedures were performed with sas 6.11 ( sas institute inc . , cary , nc , usa ) . a total of 135 disk - shaped specimens were prepared with a nanofilled resin composite ( filtek supreme xt - 3 m espe , st . paul , mn , usa ) , which was inserted with a metal spatula ( minelli no . 1 - golgran , so paulo , sp , brazil ) in circular acrylic molds ( 10 mm diameter 2 mm thick ) . a mylar strip ( dentart - polidental , so paulo , brazil ) was placed on top of and pressed flat with a microscope slide plus a 500 g weight for 30 s. the composite was photocured with a led unit ( radii - cal sdi - melbourne , victoria , australia - 1,200 mw / cm ) for 20 s. following preparation , specimens were stored in relative humidity at 37c for 24 h. the finishing and polishing steps were performed with al2o3 abrasive disks ( sof - lex pop on - 3 m espe , st paul , mn , usa ) of decreasing coarseness . the specimens were randomly divided into three groups ( n = 45 ) to be subjected to one of the bleaching agents . an individual acetate tray was fabricated in a vacuum - forming machine ( p7/bio - art equip odontolgicos ltda . , so carlos , sp , brazil ) for each specimen , which would be then exposed to 10% or 16% carbamide peroxide ( cp ) bleaching agents [ cp - whiteness perfect fgm produtos odontolgicos , joinville , sc , brazil - table 1 ] . these bleaching agents were applied for 4 h / day , for 14 days , simulating the at - home bleaching technique . in regard to the group exposed to the 35% hydrogen peroxide ( hp ) agent ( hp whiteness hp 35% fgm produtos odontolgicos , joinville , sc , brazil ) , the product was applied with a disposable applicator ( microbrush - vigodent , rio de janeiro , rj , brazil ) for 45 min ( three sequential 15-min applications ) once a week for three consecutive weeks . when the bleaching time was completed , the specimens were rinsed thoroughly with distilled water and stored in distilled water at 37c . when the bleaching procedures were completed , specimens of each group were allocated into three subgroups ( n = 15 ) to be immersed daily in 200 ml of either coffee ( melitta , so paulo , brazil ) or red wine ( concha y toro , santiago , chile ) , as described in table 1 . specimens were immersed in staining solutions or distilled water for 3 h / day , at room temperature , over 40 days . after every 3-h period of immersion time , the specimens were rinsed thoroughly with distilled water and stored in distilled water at 37c . color shade was measured using a spectrophotometer ( vita easyshade - vita zahnfabrik , bad sckingen , germany ) and the data were expressed based on the vita shade guide and then converted into scores , as can be seen in table 2 . color shade was recorded on three different occasions : baseline , after bleaching and after staining . three knoop microhardness indents were carried out on the top surface of each specimen , using a hvs-1000 microhardness tester ( panambra , so paulo , sp , brazil ) , under a 50-g load , applied for 15 s. measurements were performed at the baseline , following bleaching and after staining procedures . microhardness data were analyzed using an analysis of variance ( anova ) for split - plot design and tukey 's test , whereas the color shade recordings were submitted to kruskal - wallis and dunn 's tests . statistical procedures were performed with sas 6.11 ( sas institute inc . , cary , nc , usa ) . means and standard deviations ( sd ) of microhardness values and color shade recordings of the nanofilled resin composite at baseline , after bleaching and after staining , are shown in tables 3 and 4 , respectively . mean microhardness values ( sd ) of nanofilled resin composite at the baseline , after bleaching and after staining medians and color shade ranges of nanofilled resin composite at the baseline , after bleaching and after staining in regard to the microhardness data , anova for split - plot design showed no significant interaction between the bleaching agent and the staining solution ( p = 0.4384 ) . bleaching agents significantly influenced the microhardness values of the nanofilled resin composite ( p = 0.0003 ) . lower microhardness values were noticed when the composite had been in contact with 35% hp ( p < 0.0001 ) . no difference was noticed between the microhardness values produced by the 10% and 16% cp bleaching agent . overall , there was a significant reduction in the microhardness values from the baseline to the post - staining condition ( p < 0.0001 ) . the microhardness values of the nanofilled resin composite that had been previously exposed to the bleaching agents were lower when red wine or coffee were used as staining solutions , when compared to distilled water . in terms of color change , as examined spectrophotometrically , kruskal wallis and dunn tests demonstrated that the nanofilled resin composite was lighter after bleaching with the 35% hp agent ( p < 0.0500 ) . regardless of the bleaching agent , no difference was observed for the color shade recorded for the nanofilled resin composite as a result of being immersed either in red wine or coffee , both of which produced a darker color than distilled water . the results of the present study showed that the bleaching agents significantly influenced nanofilled resin composite microhardness , which was reduced when the resin composite was exposed to the 35% hp bleaching agent . the adverse effects of this agent at high concentrations ( such as 35% or 38% ) have been demonstrated in previous in vitro studies . it has been speculated that hydrogen - peroxide - based bleaching agents may have high oxidation and reduction capability , thus generating free radicals . these radicals have been thought to degrade the resinous matrix and disrupt the filler / matrix interface , effects which cause increasing water sorption and filler detachment and which , in turn , augment surface roughness . in addition , the oxidative capacity of peroxides may cleave the composite polymer - chains . in this context , the effects caused by 10% cp and cp 16% agents on the surface microhardness of the nanofilled resin composite were significantly lower than those produced by the 35% hp . while some studies have shown significant reduction in knoop microhardness after at - home bleaching , others have demonstrated the opposite . differences in protocols of daily use and in bleaching agents may explain such contradictory findings . have reported a significant decrease in nanofilled composite after a 6 h / day bleaching period with 10% cp ( as opposed to the 4-h period used in the current study ) . the authors here cited hypothesized that the bleaching agent had sufficient time to diffuse into the high molecular weight organic matrix of the nanofilled composite . it is worth noting , however , that these investigations were conducted in in vitro conditions , in which specimens are commonly stored in distilled water . an in situ study demonstrated that bleaching with 15% cp had no effect on nanofilled composite microhardness . the specimens immersed in distilled water ( control ) showed decreased microhardness , which probably occurred because of previous exposure to bleaching agents . this finding may be attributed to the composition of the nanofilled resin composite , the inherent characteristics of the staining solutions and the storage protocol adopted . in this study , red wine and coffee produced lower microhardness values in the resin composite than distilled water . no difference was found between the microhardness values obtained as a result of using red wine or coffee . although the red wine had a lower ph ( 4.2 ) than the coffee ( ph 5.5 ) and an alcoholic strength of 12.5% overall factors ( lower ph and average alcohol by volume ) which seem to affect the resin matrix crosslink , the filler / matrix interface and the filler itself the coffee was used at a temperature above 37c , a condition that may have accelerated the resin composite degradation process . color change was evaluated with the vita easyshade spectrophotometer , which allows tooth shade to be determined based on the vita classical scale . the data from the this study shows that no significant difference existed between the color of the specimens at the baseline and in post - bleaching conditions , except when 35% hp was used , which made the nanofilled composite lighter . this may be attributed to a higher proportion of hydrogen and the more acidic ph of the 35% bleaching gel , as compared to the other home - use gels tested in this study . in fact , color changes of composites have not been clinically detectable after the application of 10% cp agents . the assessment of color stability of resin composites following contact with different immersion media has shown that composites are not inert in the oral environment . the results of color evaluation showed that , after immersion in coffee and red wine , specimens which had been previously exposed to bleaching agents were significantly darker than those exposed to distilled water . it has been reported that color changes may occur in different restorative materials after bleaching followed by immersion in staining solutions , including red wine and coffee . apart from the pigments contained in red wine and coffee , the alcoholic content of the red wine ( 12.5% by volume ) and the coffee temperature used in this study may have softened the organic matrix of the composite and allowed dye absorption . in this study , the effect on nanofilled resin caused by bleaching followed by staining was evident , i.e. microhardness decreased after bleaching and the samples became lighter when subjected to 35% hp . resin composite previously in contact with bleaching agents showed softening and darkening as a result of being stained in red wine and coffee .

objectives : the present study aimed to investigate the effect of staining solutions on microhardness and shade changes of a nanofilled resin composite , which had been previously in contact with bleaching agents.materials and methods : a total of 135 disk - shaped specimens ( 10 mm 2 mm ) were fabricated with a nanofilled resin ( filtek supreme ) and photocured with a light emission diode ( led ) unit and then allocated into three groups to be bleached with 10% or 16% carbamide peroxide ( cp ) bleaching agents or a 35% hydrogen peroxide ( hp ) product . following bleaching , specimens within each group were subdivided into three groups to be immersed in coffee , red wine or distilled water . microhardness and color were monitored at baseline , after bleaching and after staining.results:analysis of variance for split - plot design showed lower microhardness values when the composite had been in contact with hp ( p < 0.0001 ) . the specimens immersed in red wine and coffee provided lower microhardness values than those immersed in distilled water , regardless of the bleaching agent to which the composites were previously exposed . kruskal wallis and dunn tests demonstrated that the composite was lighter after bleaching with a 35% hp agent ( p < 0.0500).conclusion : the composite was darker as a result of being immersed either in red wine or coffee , regardless of the bleaching agent .

prostate cancer ( pca ) is the most frequently diagnosed malignancy and the second leading cause of cancer - specific deaths in men in most industrialized western countries . recently , the incidence of this disease has significantly increased in asian countries , including korea , owing to the reported widespread use of prostate - specific antigen ( psa ) testing and downward stage migration . nevertheless , recent reports have shown that up to 15% of contemporary patients still harbor lymph node invasion ( lni ) at extended pelvic lymph node dissection ( plnd ) in pca . many studies have reported preoperative nomograms for predicting lni after radical prostatectomy ( rp ) and plnd , and these predictive models were reconstructed by using an extended plnd procedure . although recent studies have demonstrated a significantly higher rate of lni in patients undergoing extended plnd relative to patients undergoing only limited plnd , it is a common policy to spare plnd or to perform limited plnd in patients with a low risk of lni because of the widely applied early detection of pca and screening programs and the resulting stage migration . therefore , it became necessary to find a preoperative tool for predicting lni before surgery . recently , genome - wide association studies have identified several genetic variants that are associated with risks for pca and have provided additional predictive gain to prostate - related prognosis . these genetic data have been applied to actual clinical situations and have been validated as genetic biomarkers in pca . thus , in the present study , by using 242,186 single - nucleotide polymorphisms ( snps ) in an exome array from 341 korean patients with pca who underwent rp and plnd , we investigated significant genetic variants for lni and showed how additional predictive gain was achieved by reconstructing a clinicogenetic model . after we obtained institutional review board approval ( irb number , b-1312/232 - 302 ) , we enrolled 1,002 patients with pca from november 2003 to july 2013 . blood specimens were prospectively collected from all of the patients , and genomic dna was extracted by use of purelink genomic dna maxi kits ( invitrogen , carlsbad , ca , usa ) . genomic dna was diluted to 1 g / ml and stored at -20. we included patients who underwent rp and plnd , and most of the patients had undergone a limited extent of plnd . we excluded patients who had undergone neoadjuvant hormone or radiation therapy , prostate biopsies at another institution , or prostate biopsies with < 12 cores taken . we also excluded patients who had a postoperative follow - up of less than 1 year . accordingly , 341 patients were enrolled with complete records of clinical data including data on lni and biochemical recurrence ( bcr ) . transrectal ultrasound - guided multicore ( 12 ) biopsies were obtained from all male patients by use of a biopsy gun with an automatic firing mechanism . bcr was defined as two consecutive psa increases of 0.2 ng / ml or higher within at least 2 months after rp . study samples were processed on the humanexome beadchip 12v1 - 1 system ( illumina inc . , san diego , ca , usa ) , which included 242,901 markers focused on protein - altering variants . details concerning snp content and selection strategies can be found at the exome array design webpage ( http://genome.sph.umich.edu/wiki/exome _ chip_design ) . after additional visual inspection of snps with call rates of < 0.99 and snps with minor allele frequencies of < 0.002 , 242,186 of 242,901 attempted markers ( 99.71% ) were successfully genotyped with call rates > 95% ( average call rate , 99.98% ) . in total , 1,001 of 1,002 individuals ( 99.9% ) were successfully genotyped ( call rate > 98% ) . for the 242,186 snps that passed quality control , genotype concordance among one individual per pair of six known twin pairs and six unknown duplicates were excluded . we performed principal components analysis twice , once excluding hapmap samples to identify population outliers and a second time including hapmap samples to interpret outliers . to avoid artifacts due to family relatedness , we computed the principal components by using snp loadings estimated from a subset of 7,304 notclose relatives . we defined close relatives as an estimated genome - wide identical - by - descent proportion of alleles shared of > 0.10 . we estimated identical - by - descent sharing by using plink 's " -genome " option38 and performed principal components analysis by using smartpca37 on a linkage - disequilibrium - pruned set of 22,464 autosomal snps . these were obtained by removing large - scale , high - linkage disequilibrium ( ld ) regions , snps with a minimum allele frequency < 0.01 , or snps with a hardy - weinberg equilibrium p - value<10 and by performing ld pruning by using the plink option " --indep - pairwise 50 5 0.2 . " after inspecting the first 10 principal components , we identified 12 population outliers , 9 of which had self - reported non - finnish ancestry . snp genotype frequencies were examined for hardy - weinberg equilibrium by using the chi - square statistic and all were found to be consistent ( p>0.05 ) . the data were analyzed by using an unconditional logistic regression to calculate an odds ratio ( or ) as an estimate of the relative risk of lni associated with snp genotypes . to determine the association between the genotype and haplotype distributions , a logistic analysis was performed with control for age ( continuous value ) as a covariate to eliminate or reduce any confounding factors that could influence the findings . lewontin 's d ' ( |d'| ) and the ld coefficient r were examined to measure ld between all pairs of biallelic loci . the haplotypes were inferred from the successfully genotyped snps by using the phase algorithm ver . 2.0 , and sas 9.1 ( sas institute inc . , the effective number of independent marker loci was calculated by using snpspd software ( http://www.genepi.qimr.edu.au/general/dalen/snpspd/ ) . a total of 341 patients were stratified into two groups on the basis of lni . comparing patients with and without lni , we assessed the differences among the clinicopathological profiles of patients by using the chi - square test , fisher exact test , and the mann - whitney test . the multivariate analyses on models adjusted for age , initial psa , biopsy gleason scores , clinical stage , percentage of positive core , and maximal tumor length in a core were performed to identify an independent predictor of lni . the predictive accuracy for this multivariate model was assessed by the receiver operating characteristics - derived area under the curve ( auc ) analysis . another multivariate model was created by using the additional genetic information derived from the exome array , and predictive accuracy was assessed by using the same method . the two aucs were subsequently compared by using the mantel - haenszel test . the cox proportional hazard model for predicting bcr analyses was conducted by adjusting the clinicopathologic parameters , such as pathologic gleason scores , extracapsular extension , seminal vesicle invasion , positive surgical margin , and lni , with or without genetic factors from the exome array . bcr - free survival was measured from the day of surgery to the date of the last follow - up period or until the date of bcr . the predictive accuracy for predicting bcr from each model was compared by use of similar methods . the spss ver . 15.0 ( spss inc . , a two - tailed p<0.05 was considered to be statistically significant for all of the analyses . after we obtained institutional review board approval ( irb number , b-1312/232 - 302 ) , we enrolled 1,002 patients with pca from november 2003 to july 2013 . blood specimens were prospectively collected from all of the patients , and genomic dna was extracted by use of purelink genomic dna maxi kits ( invitrogen , carlsbad , ca , usa ) . genomic dna was diluted to 1 g / ml and stored at -20. we included patients who underwent rp and plnd , and most of the patients had undergone a limited extent of plnd . we excluded patients who had undergone neoadjuvant hormone or radiation therapy , prostate biopsies at another institution , or prostate biopsies with < 12 cores taken . we also excluded patients who had a postoperative follow - up of less than 1 year . accordingly , 341 patients were enrolled with complete records of clinical data including data on lni and biochemical recurrence ( bcr ) . transrectal ultrasound - guided multicore ( 12 ) biopsies were obtained from all male patients by use of a biopsy gun with an automatic firing mechanism . bcr was defined as two consecutive psa increases of 0.2 ng / ml or higher within at least 2 months after rp . study samples were processed on the humanexome beadchip 12v1 - 1 system ( illumina inc . , san diego , ca , usa ) , which included 242,901 markers focused on protein - altering variants . details concerning snp content and selection strategies can be found at the exome array design webpage ( http://genome.sph.umich.edu/wiki/exome _ chip_design ) . after additional visual inspection of snps with call rates of < 0.99 and snps with minor allele frequencies of < 0.002 , 242,186 of 242,901 attempted markers ( 99.71% ) were successfully genotyped with call rates > 95% ( average call rate , 99.98% ) . in total , 1,001 of 1,002 individuals ( 99.9% ) were successfully genotyped ( call rate > 98% ) . for the 242,186 snps that passed quality control , genotype concordance among the 104 blind duplicate sample pairs was 99.998% . one individual per pair of six known twin pairs and six unknown duplicates were excluded . we performed principal components analysis twice , once excluding hapmap samples to identify population outliers and a second time including hapmap samples to interpret outliers . to avoid artifacts due to family relatedness , we computed the principal components by using snp loadings estimated from a subset of 7,304 notclose relatives . we defined close relatives as an estimated genome - wide identical - by - descent proportion of alleles shared of > 0.10 . we estimated identical - by - descent sharing by using plink 's " -genome " option38 and performed principal components analysis by using smartpca37 on a linkage - disequilibrium - pruned set of 22,464 autosomal snps . these were obtained by removing large - scale , high - linkage disequilibrium ( ld ) regions , snps with a minimum allele frequency < 0.01 , or snps with a hardy - weinberg equilibrium p - value<10 and by performing ld pruning by using the plink option " --indep - pairwise 50 5 0.2 . " after inspecting the first 10 principal components , we identified 12 population outliers , 9 of which had self - reported non - finnish ancestry . snp genotype frequencies were examined for hardy - weinberg equilibrium by using the chi - square statistic and all were found to be consistent ( p>0.05 ) . the data were analyzed by using an unconditional logistic regression to calculate an odds ratio ( or ) as an estimate of the relative risk of lni associated with snp genotypes . to determine the association between the genotype and haplotype distributions , a logistic analysis was performed with control for age ( continuous value ) as a covariate to eliminate or reduce any confounding factors that could influence the findings . lewontin 's d ' ( |d'| ) and the ld coefficient r were examined to measure ld between all pairs of biallelic loci . the haplotypes were inferred from the successfully genotyped snps by using the phase algorithm ver . 2.0 , and sas 9.1 ( sas institute inc . , the effective number of independent marker loci was calculated by using snpspd software ( http://www.genepi.qimr.edu.au/general/dalen/snpspd/ ) . a total of 341 patients were stratified into two groups on the basis of lni . comparing patients with and without lni , we assessed the differences among the clinicopathological profiles of patients by using the chi - square test , fisher exact test , and the mann - whitney test . the multivariate analyses on models adjusted for age , initial psa , biopsy gleason scores , clinical stage , percentage of positive core , and maximal tumor length in a core were performed to identify an independent predictor of lni . the predictive accuracy for this multivariate model was assessed by the receiver operating characteristics - derived area under the curve ( auc ) analysis . another multivariate model was created by using the additional genetic information derived from the exome array , and predictive accuracy was assessed by using the same method . the cox proportional hazard model for predicting bcr analyses was conducted by adjusting the clinicopathologic parameters , such as pathologic gleason scores , extracapsular extension , seminal vesicle invasion , positive surgical margin , and lni , with or without genetic factors from the exome array . bcr - free survival was measured from the day of surgery to the date of the last follow - up period or until the date of bcr . the predictive accuracy for predicting bcr from each model was compared by use of similar methods . the spss ver . 15.0 ( spss inc . , a two - tailed p<0.05 was considered to be statistically significant for all of the analyses . among 341 patients with pca , 24 patients ( 7.04% ) had lni after rp and plnd ( table 1 ) . patients with lni displayed significantly higher biopsy gleason scores , percentage of positive core , and maximal tumor length in a core before rp . the rates of extracapsular extension , seminal vesicle invasion , positive surgical margin and bcr were higher in the lni group after rp . there was no significant difference in the rate of lni according to surgical method : open rp , 5.3% ( 7/131 ) ; robotic rp , 8.1% ( 17/210 ) . genotype frequencies in patients with and without lni were analyzed by using a logistic regression model ( fig . the results of genotyping 242,186 snps by use of exome arrays showed that five snps ( rs75444444 , rs8055236 , rs2301277 , rs9300039 , and rs6908581 ) were significantly associated with lni in men who underwent rp ( table 2 ) . these significant snps were all positively correlated with lni ; ors and significance levels are shown in table 2 ; however , the aforementioned snps were not significant after multiple testing with false - discovery rate . the initial serum psa levels , biopsy gleason scores , clinical stage , percentage of positive cores , and maximal tumor length in a core were significant factors for lni in the univariate analysis . the multivariate models incorporated these variables and age , including and excluding the snp variants , as shown in table 3 . the biopsy gleason scores and maximal tumor length in a core were significant factors for predicting lni in a multivariate logistic model ; the predictive accuracy for this model was 80.7% ( 95% confidence interval [ ci ] , 0.762 - 0.847 ) . by adding the genetic variant factors , the clinicogenetic multivariate model showed that predictive accuracy was 93.2% ( 95% ci , 0.900 - 0.956 ) . thus , the accuracy was increased within a significant level ( difference between area , 0.124 ; 95% ci , 0.0357 - 0.213 ; p=0.006 ) ( fig . the clinicopathological multivariate models showed that pathologic gleason scores , seminal vesicle invasion , positive surgical margin , and lni were significant factors for predicting bcr . selected snp - related lni was also a significant factor for bcr in the multivariate cox proportional models ( hazard ratio , 2.244 ; 95% ci , 1.030 - 4.888 ; p=0.042 ) . the auc increased from 71.8% in the model without genetic factors to 72.3% in the model with genetic factors ( p=0.346 ) . in the present study , we investigated potential genetic biomarkers for predicting lni among patients with pca who underwent rp and plnd . the logistic regression analysis results suggested that five snps were significantly associated with the risk for lni . moreover , we applied the information derived from the genetic studies to an actual clinical model based on previously established clinical factors and found an additional predictive gain for predicting lni . the first nomogram introduced by briganti et al . showed 76% accuracy by adjusting for psa , clinical stage , and biopsy gleason scores . in another nomogram that included percentage of positive cores as a covariate , afterwards , many studies that contained external and internal validation results of these nomograms showed a higher accuracy level . recent updated nomograms predicting lni showed 87.6% accuracy in 588 patients with clinically localized pca who underwent rp and extended plnd . showed externally validated updated nomograms among 1,282 pca men and showed 82.9% accuracy . in our setting , including biopsy parameters , the multivariate model for predicting lni was 80.7% ; however , with the addition of the genetic parameters to the multivariate model , accuracy was significantly increased to 93.2% . although recently published nomograms related to pca prognosis need to be improved by including new variables , models based on plasma or tissue quantitative biomarkers have recently been proposed . however , although associations between snps and pca have been reported , whether including any of these snps or a combination might improve the classic nomograms has not been explored . huang et al . showed that among 320 patients with pca after rp , 3 of 20 snps were significantly associated with bcr ( p<0.02 ) after adjustment for clinicopathological factors such as age , preoperative psa , pathologic stage , and surgical margin . likewise , we performed a broader investigation by analyzing 242,186 snps among men who underwent rp by use of a custom humanexome beadchip and applied our genetic information from the exome array to the clinical model . as a result because the action of snps is generally understood to be a function of multiple variant combinations and not a single action , it was important to find many potential genetic variant outcomes . the relationship between the five snps selected from our series and pca has not yet been investigated . rs75444444 , which is located in chromosome 19 ( muc16 ) , is known as the largest glycoprotein of the mucin family . it is normally expressed in the epithelial lining of various tissues and is elevated in some organ malignancies . the high rs8055236 ( ier3 ) expression revealed in the prostate and bladder was described as being regulated by p53 and involved in the proapoptotic action by the tumor suppressor . rs2301277 ( pbx2 ) works as a transcriptional activator for valosin - containing protein expression in breast cancer and is known to be an important factor for cell survival . however , an accurate mechanism of action of these snps in pca has not been published ; it is necessary to confirm the genetic variants and lymph node metastasis in pca through molecular work . actually , our selected snp , which is related to lni , had significance to pca - related bcr in the multivariate analysis ; these markers or the combination of these markers had a likelihood of being a potential biomarker for pca . although the ideal candidate for plnd still needs to be defined , general agreement has been reached that if plnd is contemplated , it should be extended to reach an adequate nodal staging accuracy . consequently , if an lni prediction model is developed , it must be based on an extended plnd series . conversely , applying predictive tools developed on patients treated with limited plnd might significantly underestimate the true risk of lni . however , lni was detected even in low - risk patients with pca in whom plnd is not indicated in the current era ; therefore , we need the available nomogram in this real clinical setting . our predictive model had a higher accuracy rate above 90% and consisted of preoperative parameters with genetic information obtained from serum . therefore , by adding a recently published nomogram obtained from an extended plnd setting , the external validation of our selected biomarker exhibited an excellent model for predicting lni . owing to the limited extent of plnd , the small number of positive lymph nodes was a limitation . the sample size was another limitation ; however , all men included in this study were of a homogeneous racial population . pca diagnosed in asian , american , and european men may have innate differences associated with racial and environmental factors . because pca is a hormone - dependent disease , various investigators have suggested that racial variations in the serum levels of testosterone and derivatives may contribute to differences in pca risks and prognoses among different races . differences in the hormonal milieu and a lack of psa screening have been speculated to play a role in the generally more aggressive profile of pca diagnosed in contemporary korean men compared with their western counterparts . therefore , our data from an asian population may differ from the data for a western population . another limitation was that our data should be validated in other independent cohorts for persistent significance . despite these limitations , a potential predictive marker should be judged on the basis of its capacity to improve the preexisting optimized predictive model rather than simply on the basis of its status as an independent variable . we showed that five snps were significant predictors of lni . furthermore , the addition of genetic information from exome arrays effectively enhanced the predictive accuracy of our multivariate model , which incorporated various clinical factors . these results need to be validated in future studies but may lead to the development of an accurate model that predicts lni in the preoperative setting .

purposegenetic variations among prostate cancer ( pca ) patients who underwent radical prostatectomy ( rp ) and pelvic lymph node dissection were evaluated to predict lymph node invasion ( lni ) . exome arrays were used to develop a clinicogenetic model that combined clinical data related to pca and individual genetic variations.materials and methodswe genotyped 242,186 single - nucleotide polymorphisms ( snps ) by using a custom humanexome beadchip v1.0 ( illumina inc . ) from the blood dna of 341 patients with pca . the genetic data were analyzed to calculate an odds ratio as an estimate of the relative risk of lni . we compared the accuracies of the multivariate logistic model incorporating clinical factors between the included and excluded selected snps . the cox proportional hazard models with or without genetic factors for predicting biochemical recurrence ( bcr ) were analyzed.resultsthe genetic analysis indicated that five snps ( rs75444444 , rs8055236 , rs2301277 , rs9300039 , and rs6908581 ) were significant for predicting lni in patients with pca . when a multivariate model incorporating clinical factors was devised to predict lni , the predictive accuracy of the multivariate model was 80.7% . by adding genetic factors in the aforementioned multivariate model , the predictive accuracy increased to 93.2% ( p=0.006 ) . these genetic variations were significant factors for predicting bcr after adjustment for other variables and after adding the predictive gain to bcr.conclusionsbased on the results of the exome array , the selected snps were predictors for lni . the addition of individualized genetic information effectively enhanced the predictive accuracy of lni and bcr among patients with pca who underwent rp .

breast mri accuracy for assessing residual disease is good and surpasses other diagnostic means . diagnostic accuracy of mri seems to be affected by treatment regimen and breast cancer subtype . neoadjuvant chemotherapy ( nac ) is defined as the administration of chemotherapy to treat invasive breast cancer before local treatment ( i.e. surgery ) . although in breast cancer patients nac was primarily used for treatment of locally advanced disease stages , its use has been extended to the treatment of early stage breast cancers in order to enable breast - conserving therapy for patients who would otherwise undergo mastectomy . in a large meta - analysis by mauri et al . , no significant differences in survival or overall disease progression was observed between patients receiving adjuvant or neoadjuvant chemotherapy . some physicians prefer nac instead of the adjuvant chemotherapy because of the ability to assess tumour response in vivo . in this situation , reliable assessment of pathological tumour response to nac is vital in order to select the most appropriate surgical plan . an imaging modality that could assess tumour response to nac would be beneficial , providing it could detect any residual disease present . in addition , pathological complete response ( pcr , i.e. the absence of any residual invasive tumour cells ) on nac has shown to be a prognostic factor for overall better survival , disease - free survival and recurrence - free survival . in the future many examinations have been proposed to evaluate residual disease and/or complete response to therapy ( such as clinical examination , mammography and ultrasound ) , but their accuracy was only of modest degree . parallel to these findings , ( contrast - enhanced ) magnetic resonance imaging ( mri ) of the breast proved to be superior to mammography and ultrasound with respect to assessing tumour extent , presence of additional foci ( i.e. multifocality and/or multicentricity ) and the presence of contralateral breast tumours [ 4 , 5 ] . therefore , mri might be a promising imaging tool to assess therapy response in the nac setting and for assessing pcr . several reviews have been recently published that assessed the ability of breast mri to predict pcr in patients receiving nac [ 68 ] . however , pcr is only achieved by a minority of patients and as a consequence , the role of breast mri in neoadjuvant chemotherapy was not investigated for a substantial number of patients that still have some degree of residual disease after treatment . in this systematic review , we aimed to address the role of breast mri in assessing both residual disease extent and pcr after nac in breast cancer patients . our study purpose was to assess the role of magnetic resonance imaging ( mri ) in the evaluation in all patients receiving neoadjuvant chemotherapy for invasive breast cancer . for this systematic review , embase , the cochrane library , medline and citations as provided by pubmed were searched until 1 july 2012 , using the search terms breast , breast neoplasms , breast neoplasm , breast cancer , breast carcinoma and breast lesion combined with the search terms magnetic resonance imaging , mri , mr mammography and neoadjuvant therapy , neoadjuvant chemotherapy , neoadjuvant systemic therapy , neoadjuvant , chemotherapy , primary therapy and initial therapy . only original articles were considered for inclusion ( i.e. no reviews , brief communications or letters to the editor ) . studies found through these search terms were assessed for potential eligibility by reading the abstracts first and then applying inclusion and exclusion criteria . included were only those in which breast mri was performed at baseline and prior to surgery ( but after completion of neoadjuvant chemotherapy ) . in addition , the ability of mri to assess pcr was one of our study aims . rates of pcr to nac may vary , depending on the treatment regime used : 615 % in antracycline - based therapies , up to 30 % when adding taxanes . therefore , in order to have some reliable information on the ability of mri to assess residual disease and also pcr , eligible studies should have a sufficiently large study population . to be eligible for this review , we decided that a study should consist of at least 25 patients ( in the final analysis ) with newly diagnosed , histologically proven breast cancer undergoing neoadjuvant chemotherapy who were imaged using clinical mri scanners ( i.e. minimum 1.5 t ) . studies were not excluded if other imaging modalities were performed parallel to mri in order to evaluate treatment response . after this initial assessment included : first author , year of publication , study design ( retrospective or prospective ) , blinding procedures , population size , mean patient age and range , magnetic field strength , contrast agent / dose used , breast cancer stage at inclusion , tumour histology , breast cancer subtypes , chemotherapy regimen , imaging response assessment [ world health organisation ( who ) criteria , response evaluation criteria in solid tumours ( recist ) criteria or other ] and histopathological response assessment . while scoring the extraction forms in consensus , some studies were excluded if the study outcome proved not to contain information on residual disease evaluation by mri . all reported p - values 0.05 were considered statistically significant . the large heterogeneity observed in the included studies precluded us from pooling data ( see also discussion section ) , which is why we chose to use descriptive statistics in this review . since this was a systematic review , no approval from our institutional review board was necessary . in the primary literature search , 3,119 potential studies were identified , of which 515 were double in various searches , leaving 2,604 studies after the primary search . after reading the abstracts , 2,444 were excluded from further evaluation , leaving 160 studies to be analysed using the inclusion and exclusion criteria . in these studies , two additional studies were identified by manually searching the references in the manuscripts . in this analysis , another 98 studies did not comply with our eligibility criteria and were subsequently excluded , leaving 64 studies to be reviewed using the extraction form and consensus reading . this led to the exclusion of another 29 studies , because they did not address the topic of residual disease assessment with mri after neoadjuvant chemotherapy . therefore , a total of 35 studies were eligible for this systematic review [ 1045 ] . 1detailed overview of study selection detailed overview of study selection the majority of studies ( 27 ) were prospective in design . a total of 2,359 patients were included in the studies ( mean 65.5 patients per study , range 30216 ) . three studies were performed on a 3-t mri scanner , four on both 1.5- and 3-t scanners , and the remaining studies on 1.5-t mri scanners . in all studies , a commercially available gadolinium - based contrast agent was used for breast mri at regular clinical administration doses . interestingly , there was a remarkable heterogeneity in breast cancer stages and subtypes , neoadjuvant chemotherapy regimens , and methods used for assessing response in both imaging and histopathological analyses ( tables 1 and 2 ) . this heterogeneity precluded us from further pooling of data in a meta-analysis.table 1overview of included studiesauthorsyearstudy designpopulation sizeage in years ( range)breast cancer stage of included patientsbreast cancer typesbreast cancer subtypesabraham et al.1996prospective3950 ( 3173)iia , iib , iiia , iiibidc , ilc , mixed ductal and lobular carcinomaer , presserman et al.2001prospective3346 ( 3275)iiia , iiib , iiic , to large for breast conserving therapyidc , ilc , inflammatoryer , prrieber et al.2002prospective5851 ( 2772)not reportedidc , ilc , dcis , mucinous carcinoma , ductolobular carcinomanot reportedpartridge et al.2002prospective5247 ( 2972)not reportednot reportednot reportedcheung et al.2003prospective3345 ( 2963)not reportedidc , ilc , mucinous carcinomanot reporteddenis et al.2004prospective4048 ( 2964)labcnot reportednot reportedwarren et al.2004retrospective6746 ( 2862)not reportednot reporteder , prmartincich et al.2004prospective3049 ( 3665)ii , iii , inoperable locally - advanced breast canceridc , ilcer , prschott et al.2005prospective4348 ( 2666)iib , iiia , t1n0/1 , t2n0/1idc , ilc , mixed ductal and lobular carcinoma , anaplastic carcinomaeryeh et al.2005prospective3145 ( 3165)iib , iiia , iiib , iiicidc , ilc , mixed ductal and lobular carcinoma , invasvive carcinoma n.o.s.not reportedbelli et al.2006prospective4554 ( 3076)iia , iib , iiia , iiib , idc , ilc , mucinous carcinoma , tubular carcinomaer , prsegara et al.2007prospective6850 ( 2971)clinical stage i , ii , and iiiidc , ilc , mixed ductal and lobular carcinoma ; sometimes dcis or lcis present in lesionser , pr , her2kim et al.2007prospective5042 ( 2568)iib , iiia , iiib , iiicidc , ilcer , prchen et al.2007prospective5150 ( 3177)ii , iii , ividc , ilcher2bhattacharyya et al.2008prospective3242 ( 2460)>4 cm , large tumours in small breasts , node involvementnot reportednot reportedmoon et al.2009prospective19546 ( not reported)not reportednot reporteder , pr , her2wright et al.2010prospective4847 ( 3072)iib , iiia , iiibidc , ilc , invasive micropapillary carcinomaer , pr , her2woodhams et al.2010prospective69not reportednot reportedidc , ilc , dcis , lcis , mucinous carcinoma , othernot reportedpark et al.2010retrospective5344 ( 2465)iia , iib , iiia , iiibidc , mucinous carcinoma , mixed ductal and lobular carcinomaer , pr , her2de los santos et al.2010retrospective8150 ( 2773)not reportedidc , ilc , mixed ductal and lobular carcinoma , carcinoma n.o.s.er , pr , her2straver et al.2010retrospective20846 ( 2376)>3 cm and/or n+idc , ilc , carcinoma n.o.s.er , pr , her2nakahara et al.2010prospective8648 ( 2462)not reportedidc , papillotubular carcinoma , solid - tubular carcinoma , scirrhous carcinomaer , her2wang et al.2010prospective4348 ( 3469)ii , iiiidcnot reporteddongfeng et al.2011prospective6055 ( not reported)i , iia , iib , iiia , iiib , iiicidc , mucinous carcinomaer , her2fangberget et al.2011prospective3151 ( 3772)not reportedidc , ilcer , pr , her2guarneri et al.2011retrospective5948 ( 3070)iia , iib , iiia , iiib , iiicidc , ilc , other n.o.s.er , pr , her2loo et al.2011prospective11846 ( 2376)>3 cmidc , ilc , adenocarcinoma n.o.s.er , her2shin et al.2011prospective4343 ( 2562)labc / inflammatory breast cancer and at least n1 , or unsuitable for bct , or skin / chest wall involvementidc , micropapillary carcinomaer , her2lyou et al.2011retrospective5744 ( 2464)not reportedidc , ilcnot reportedchen et al.2011prospective5049 ( 2882)not reportedidc , ilc , invasive cancer with squamous differentiationer , pr , her2 , ki-67kim et al.2012prospective5549 ( 2882)iia , iib , iiia , iiib , iiic , ividc , ilc , mucinous carcinomanot reportedkuzucan et al.2012retrospective5446 ( 2963)not reportedidc , ilc , mixed ductal carcinoma with lobular featureser , pr , her2 , ki-67takeda et al.2012prospective3751 ( 3078)not reportednot reportednot reportedshin et al.2012retrospective9046 ( 2468)iia , iib , iiia , iiib , iiicidc , ilc , metaplastic carcinoma , micropapillary carcinomaer , pr , her2hylton et al.2012prospective21648 ( 2668)t3 tumour of at least 3 cmidc , ilc , mixed ductolobular carcinoma , mucinous carcinomaer , pr , her2park et al.2012retrospective3444 ( 2760)tumour size > 2idc , mucinous carcinoma , mixed ductolobular carcinomaher2overview of included studies regarding year of publication , study design , size of study population , age of population , breast cancer stages of patients included , breast cancer types observed in the respective studies and their subtypeslabc locally advanced breast cancer , bct breast - conserving therapy , idc invasive ductal carcinoma , ilc invasive lobular carcinoma , dcis ductal carcinoma in situ , n.o.s . not otherwise specified , er estrogen receptor , pr progesterone receptor , and her human epidermal growth factor receptortable 2overview of included studiesauthorchemotherapy regimenresponse assessmentfield strength ( tesla)pathologic assessmentabraham et al.45 cycles of doxorubicinwho1.54 categories : ( 1 ) no residual disease ; ( 2 ) disease in a single quadrant only ; ( 3 ) multiquadrant small residual disease ; ( 4 ) multiquadrant extensive diseaseesserman et al.4 cycles of doxorubicin / cyclophosphamidewho1.5correlation with tumour size measurementsrieber et al.35 cycles of anthracyclines and epirubicine / paclitaxel , or anthracyclines and cyclophosphamideno who or recist1.5correlation with tumour size measurementspartridge et al.4 cycles of doxorubicin and cyclophosphamide followed by up to 12 cycles of paclitaxelno who or recist1.5correlation with tumour size measurementscheung et al.3 cycles of paclitaxel and epirubicinrecist1.5correlation with tumour size measurementsdenis et al.5-fluorouracil / epirubicine , or 6 cycles of docetaxel or 8 cycles of docetaxel / epirubicinerecist1.5correlation with tumour size measurementswarren et al.6 cycles of doxorubicin / cyclophosphamide , or 6 cycles of doxorubicin / docetaxel , or 4 cycles epirubicin then 4 cycles cyclophosphamide , or 4 cycles epirubicin , or docetaxel / herceptinno who or recist1.5uicc response criteria / nhsbsp guidelinesmartincich et al.4 cycles of doxorubicin bolus , followed by paclitaxel ( n = 29 ) . in one case , doxorubicin was omitted because of low baseline left ventricular ejection fractionwho1.55 grades : ( 1 ) some alteration in individual cells but no reduction in overall number of tumour cells ; ( 2 ) mild loss of invasive tumour cells but still high cellularity ; ( 3 ) estimated > 90 % loss of tumour cells ; ( 4 ) only small clusters of disease remaing , or only in situ component , or only tumour stroma remaining ; ( 5 ) pathologic complete responseschott et al.4 cycles of doxorubicin / docetaxelno who or recist1.5definitions of response : pcr : no viable invasive cancer or dcis . . partial response and progressive disease : not definedyeh et al.4 cycles of doxorubicin , then 9 cycles of paclitaxel ( or vice versa)recist1.5equal to pathology if longest tumour diameter was within 30 % of pathology tumour size . not equal if tumour diameter was less than 70 % of pathology tumour size ( underestimation ) or more than 130 % of pathology tumour size ( overestimation)belli et al.3 cycles of cyclophosphamide , methotrexate , and 5-fluorouracil alternated with adriamycine , or 6 cycles of epirubicin and taxolewho1.5correlation with tumour size measurementssegara et al.er/pr/her2 negative : 4 cycles cisplatin . her2 positive : either trastuzumab / vinorelbine or trastuzumab / carboplatin / docetaxelrecist1.55 grades : ( 1 ) some alteration in individual cells but no reduction in overall number of tumour cells ; ( 2 ) mild loss of invasive tumour cells but still high cellularity ; ( 3 ) estimated > 90 % loss of tumour cells ; ( 4 ) only small clusters of disease remaing , or only in situ component , or only tumour stroma remaining ; ( 5 ) pathologic complete responsekim et al.3 cycles of doxorubicine / docetaxelwho1.5equal to pathology if tumour size was within 50 % of pathology tumour size . not equal if tumour size was less than 50 % of pathology tumour size ( underestimation ) or more than 150 % of pathology tumour size ( overestimation)chen et al.2 cycles of doxorubicin and cyclophosphamide , then either 2 cycles of additional cyclophosphamide or taxane - based regimen ( paclitaxel or nab - paclitaxel combined with carboplatin . + also received trastuzumab , her2- also received bevacizumabrecist1.53 categories : ( 1 ) no residual malignancy or cancer cells ; ( 2 ) no residual invasive cancer , but dcis present ; ( 3 ) residual invasive cancer . categories 1 and 2 are considered pcrbhattacharyya et al.6 cycles of doxorubicin or epirubicin and cyclophosphamiderecist1.5correlation with tumour size measurementsmoon et al.taxane/anthracycline regimen , or anthracycline regimen , or trastuzumabno who or recist1.5correlation with tumour size measurementswright et al.epirubicin/taxotere ( n = 30 ) ; doxorubicin / cyclophosphamid ( n = 17 ) ; cyclophosphamide / taxotere / herceptin ( n = 1)no who or recist1.55 grades : ( 1 ) some alteration in individual cells but no reduction in overall number of tumour cells ; ( 2 ) mild loss of invasive tumour cells but still high cellularity ; ( 3 ) estimated > 90 % loss of tumour cells ; ( 4 ) only small clusters of disease remaing , or only in situ component , or only tumour stroma remaining ; ( 5 ) pathologic complete responsewoodhams et al.4 cycles of anthracycline / cyclophosphamide , followed by 4 cycles of paclitaxelnot applicable1.54 categories : ( 1 ) no residual disease ; ( 2 ) disease in single quadrant only ; ( 3 ) multiple residual diseases including eic limited to one quadrant ; ( 4 ) multiple residual disease including eic in 2 or more quadrantspark et al.3 cycles of docetaxel / doxorubicinrecist1.5correlation with tumour size measurementsde los santos et al.4 cycles of doxorubicin , then 4 cycles of paclitaxel , then 4 cycles of cyclophosphamiderecist1.5assessment for pcrstraver et al.her2- : 6 cycles of doxorubicin / cyclophosphamide , 6 cycles of doxorubicin / docetaxel , or 6 cycles of capecitabine / docetaxel , or the combination of 3 cycles of doxorubicin / cyclophosphamide plus 3 cycles of capecitabine / docetaxel . + : doxorubicin / cyclophosphamide and after 2005 paclitaxel , trastuzumab , carboplatinno who or recist1.5/3assessment for pcrnakahara et al.46 cycles of epirubicin / cyclophosphamide , with or without doxifluridine . in 17 cases continuation with 46 cycles docetaxel or 12 cycles paclitaxel . in 15 cases 6 cycles of docetaxel / epirubicin / cyclophosphamide . in her2 + , also taxanes ( n = 7 ) or taxanes / trastuzumab ( n = 4)recist1.5japanese breast cancer society classification systemwang et al.taxol/carboplatin n.o.s.no who or recist1.5correlation with tumour size measurementsdongfeng et al.4 cycles of paclitaxel / pirarubicinrecist3correlation with tumour size measurementsfangberget et al.letrozol therapy ( n = 2 ) ; or 4 cycles 5-fluorouracil / epirubicin / cyclophosphamide , followed by 2 additional cycles ( n = 10 ) , or switch to taxans ( n = 10 ) or taxans / trastuzumab ( n = 8)recist1.5correlation with tumour size measurementsguarneri et al.48 cycles of either epirubicin / paclitaxel or paclitaxel , followed by 5-fluorouacil / epirubicin / cyclophosphamide . some her2 + patients also received trastuzumab ( n = 25)no who or recist1.5correlation with tumour size measurementsloo et al.different regimes : majority of her2-recieved 6 cycles of cyclosphosphamide / doxorubicin , some her2 - 6 cycles of capecitibine / docetaxel , or doxorubicin / docetaxel ; if no response , switch to 3 cycles of cyclophosphamide / doxorubicin , followed by 3 cycles of capecitibine / docetaxel ; her2 + recieved 8 weeks of paclitaxel / trastuzumab / carboplatin , followed by either 2 8 weeks paclitaxel / trastuzumab / carboplatin , or 4 cycles of trastuzumab / fluorouacil / cyclophosphamidetwo approaches : breast response index , and dichotomously ( presence or absence of residual disease ) . pcr and near - pcr were considered on category ( pcr)1.5/3world health organisation grading of tumoursshin et al.8 cycles of adriamycin / cyclophosphamide / docetaxel , or 8 cycles of capecitabine / vinorelbine / docetaxelrecist1.55 grades : ( 1 ) some alteration in individual cells but no reduction in overall number of tumour cells ; ( 2 ) mild loss of invasive tumour cells but still high cellularity ; ( 3 ) estimated > 90 % loss of tumour cells ; ( 4 ) only small clusters of disease remaining , or only in situ component , or only tumour stroma remaining ; ( 5 ) pathologic complete responselyou et al.36 cycles of taxane / anthracycline , or 34 cycles of anthracycline - based regimen , or 6 cycles of trastuzumab / taxane regimenrecist1.5correlation with tumour size measurementschen et al.2 cycles of doxorubicin , followed by cyclophosphamide biweekly , followed by 12 weeks of taxane based regimen ; or only taxane - based regimennot reported33 grades : ( 1 ) no residual cancer cells ; ( 2 ) no residual invasive cancer cells but ductal carcinoma in situ ; ( 3 ) residual invasive cancer . pcr was defined as being category 1 and 2kim et al.taxane/anthracycline based regimen , orrecist1.5/35 grades : ( 1 ) some alteration in individual cells but no reduction in overall number of tumour cells ; ( 2 ) mild loss of invasive tumour cells but still high cellularity ; ( 3 ) estimated > 90 % loss of tumour cells ; ( 4 ) only small clusters of disease remaining , or only in situ component , or only tumour stroma remaining ; ( 5 ) pathologic complete responsekuzucan et al.combination of doxorubicin and cyclophosphamide and taxane based regimen ; or only cyclophosphamide ; or only taxane - based regimennot reported1.5/3pathologic complete response defined as the absence of invasive cancertakeda et al.docetaxel and cyclophosphamide for 36 cyclesno who or recist3pathologic complete response defined as the absence of invasive cancershin et al.4 cycles of doxorubicin / cyclophosphamide ; or 4 cycles of cyclophosphamide followed by 4 cycles of docetaxel ; or doxorubicin / docetaxel ; or 5-fluorouracil / epirubicin / cyclophosphamide ; or 6 cycles of trastuzumab / paclitaxelrecist1.5pcr classified into two categories : ( 1 ) no residual disease , or ( 2 ) absence of invasive cancer , but dcis presenthylton et al.anthracycline-cyclophosphamide regimen alone or followed by a taxanerecist1.5residual cancer burden and pathologic complete response ; pcr defined as the absence of invasive cancerpark et al.3 cycles of doxorubicin / docetaxel ; or ( if her2-positive ) 6 cycles of paclitaxel / gemcitabine / trastuzumabno who or recist1.55 grades : ( 1 ) some alteration in individual cells but no reduction in overall number of tumour cells ; ( 2 ) mild loss of invasive tumour cells but still high cellularity ; ( 3 ) estimated > 90 % loss of tumour cells ; ( 4 ) only small clusters of disease remaining , or only in situ component , or only tumour stroma remaining ; ( 5 ) pathologic complete response pathologic complete response defined as the absence of invasive cancer . pathologic complete response defined as the absence of invasive cancer pcr classified into two categories : ( 1 ) no residual disease , or ( 2 ) absence of invasive cancer , but dcis present . residual cancer burden and pathologic complete response ; pcr defined as the absence of invasive canceroverview of included studies regarding chemotherapy regimen(s ) that were used in the study , method of response evaluation , field strength of the mri scanner used , method of pathologic response assessmentn.o.s . not otherwise specified , er estrogen receptor , pr progesterone receptor , her human epidermal growth factor receptor , recist response evaluation criteria in solid tumours , who world health organisation , pcr pathologic complete response , dcis ductal carcinoma in situ , eic extensive intraductal component , uicc union for international cancer control and nhsbsp national health service breast screening programme overview of included studies overview of included studies regarding year of publication , study design , size of study population , age of population , breast cancer stages of patients included , breast cancer types observed in the respective studies and their subtypes labc locally advanced breast cancer , bct breast - conserving therapy , idc invasive ductal carcinoma , ilc invasive lobular carcinoma , dcis ductal carcinoma in situ , n.o.s . not otherwise specified , er estrogen receptor , pr progesterone receptor , and her human epidermal growth factor receptor overview of included studies overview of included studies regarding chemotherapy regimen(s ) that were used in the study , method of response evaluation , field strength of the mri scanner used , method of pathologic response assessment n.o.s . not otherwise specified , er estrogen receptor , pr progesterone receptor , her human epidermal growth factor receptor , recist response evaluation criteria in solid tumours , who world health organisation , pcr pathologic complete response , dcis ductal carcinoma in situ , eic extensive intraductal component , uicc union for international cancer control and nhsbsp national health service breast screening programme seventeen studies calculated correlation coefficients for the comparison of mri tumour measurements compared with histopathological results [ 13 , 14 , 17 , 21 , 22 , 25 , 27 , 28 , 3135 , 37 , 39 , 40 , 43 ] . correlation coefficients varied from poor to excellent , but the median value was 0.698 ( range 0.210.982 , table 3 ) . nonetheless , two studies reported non - significant correlation coefficients . in the relatively small ( n = 59 ) retrospective study by guarneri et al . , the correlation coefficients were similar for mri ( 0.53 ) and ultrasound ( 0.66 ) when compared to histopathology , and both were non - significant . in the study of 86 women by nakahara , the correlation coefficient of all included patients was remarkably low ( 0.21 ) , but rose to a strikingly high and statistically significant value of 0.92 when only triple - negative breast cancer types were analysed . a weak correlation coefficient ( 0.30 ) was also presented by chen et al . however , when four cases with a size discrepancy larger than 5 cm were excluded ( all her2-negative tumours presenting as non - mass - like enhancement ) , the correlation coefficient increased remarkably to 0.76 ( p < 0.001 ) . furthermore , both overestimation and underestimation were observed by multiple studies [ 10 , 13 , 15 , 16 , 20 , 22 , 24 , 32 , 37].table 3correlation coefficients of mri and histopathological tumour measurementsauthorcorrelation coefficientp - valuepartridge et al.0.89<0.001cheung et al.0.982<0.001martincich et al.0.72<0.001segara et al.0.749<0.0001kim et al.0.645<0.001moon et al.0.584nawright et al.0.49napark et al.0.667nanakahara et al.0.21nswang et al.0.866<0.01dongfeng et al.0.698<0.001fangberget et al.0.87<0.001guarneri et al.0.53nsshin et al.0.97nachen et al.0.300.03kim et al.0.619<0.0001shin et al.0.781nana not available , ns not significant2011 paper , 2012 papertable 4diagnostic accuracies of mri for predicting pathologic complete responseauthorsensitivity ( % ) specificity ( % ) ppv ( % npv ( % ) accuracy ( % ) prevalencerieber et al.42897371720.21schott et al.25975094890.10bhattacharyya et al.80895696na0.13moon et al.3896nanana0.15de los santos et al.92507280na0.40fangberget et al.3896nanana0.37park et al.1007047100770.21shin et al.7770nanana0.3310091nanana5370nananappv positive predictive value , npv negative predictive value , na not available , tcho total choline - containing compoundsthe results are presented for dynamic , contrast - enhanced mri , with the exception of : diffusion - weighted imaging and mr spectroscopy parameters correlation coefficients of mri and histopathological tumour measurements na not available , ns not significant 2011 paper , 2012 paper diagnostic accuracies of mri for predicting pathologic complete response ppv positive predictive value , npv negative predictive value , na not available , tcho total choline - containing compounds the results are presented for dynamic , contrast - enhanced mri , with the exception of : diffusion - weighted imaging and mr spectroscopy parameters although correlation coefficients are useful tools to describe mri s ability to assess response to nac , it could mask the truth , since the same trend in all studies could result in excellent correlation between histopathological results and mri measurements , yet the actual estimation of pcr might not be accurate . bhattacharyya et al . reported an overestimation of > 10 mm in 4 of 32 cases . belli et al . described a mean overestimation and underestimation of 2.1 and 2.0 mm , respectively . these numbers were 20.2 and 13.8 mm in the study by denis et al . . partridge et al . found the smallest deviation with an overestimation of mri measurements of only 0.9 mm . found a mean size difference of 1.6 and 6.0 mm , respectively [ 35 , 38 ] . with respect to pcr prediction with mri , sensitivity was considered to be the proportion of patients with pcr that were correctly classified with mri as complete responders . specificity was considered to be the proportion of patients with non - pcr correctly classified by mri as non - responders . to illustrate , a sensitivity of 62 % in these studies meant that in 62 out of 100 patients , mri was able to correctly identify patients with pcr ( i.e. mri did not show any residual enhancement ) . eight studies calculated the diagnostic accuracies for mri in predicting pcr ( table 4 ) [ 12 , 18 , 24 , 25 , 29 , 34 , 43 , 45 ] . two studies reported diagnostic accuracies for diffusion - weighted mr imaging , and both reported a sensitivity of 100 % [ 43 , 45 ] . only one study evaluated the potential of mr spectroscopy parameters ( total choline - containing compounds , tcho ) and reported a sensitivity of 53 % and a specificity of 70 % . the remaining studies used dynamic , contrast - enhanced mr imaging . median ( and range ) sensitivity and specificity were 42 % ( 2592 % ) and 89 % ( 5097 % ) , respectively . if reported , median ( and range ) ppv and npv were 64 % ( 5073 % ) and 87 % ( 7196 % ) , respectively . interestingly , only three studies compared diagnostic accuracy of mri and ultrasound for assessing residual disease [ 19 , 21 , 25 ] . in these studies , ultrasound was less accurate than mri . mri was also more accurate than mammography in assessing residual disease , but only one study performed this comparison . finally , mri was more accurate than physical examination for assessing residual disease , which was examined by three studies [ 19 , 21 , 44 ] . in this systematic review , we aimed to analyse the available data on mri accuracy for assessing residual disease and pcr after neoadjuvant chemotherapy in breast cancer patients . many studies compared the measured tumour diameter or volumes on mri with pathological results as the gold standard . correlation coefficients for these comparisons were good to excellent , but both overestimation and underestimation of the mri size measurements were frequently observed . although these correlation coefficients were good , it does not necessarily mean that agreement between these measurements is good . the majority of studies did not investigate the agreement between these measurements , for instance by using bland - altman plots . contrast - enhanced breast mri is superior to other imaging modalities to assess breast tumour extent and the presence of multicentricity or multifocality [ 4 , 5 ] . however , overestimation of tumour extent is a well - known phenomenon in ( preoperative ) breast mri [ 47 , 48 ] and was also observed in the nac setting . confounding factors in overestimating tumour size might be : reactive inflammation caused by tumour response and healing , surrounding sclerosis and necrosis , multiple scattered lesions and presence of accompanying ductal carcinoma in situ [ 19 , 20 , 22 ] . in theory , overestimation of tumour size by mri could result in an altered surgical treatment plan for the individual patient , with the risk of achieving wider resection margins ( with poorer cosmetic results ) or performing unnecessary mastectomy ( where breast - conserving therapy would have been possible ) . in contrast to overestimation of tumour size by mri , underestimation was also observed in the nac setting . causes might be antivascular effects of docetaxel ( resulting in less tumour enhancement ) , lack of inflammatory response surrounding the tumour in docetaxel patients , more extensive ductal carcinoma in situ components and partial volume effects in very small foci of residual disease [ 13 , 15 , 21 ] . underestimation of residual disease could lead to positive resection margins with viable residual tumour cells , necessitating re - surgery . in addition , positive resection margins are associated with an increased long - term risk of disease recurrence in patients who have undergone breast - conserving therapy . straver et al . attempted to create an mri - based model that could help in surgical decision making . in this study , mri underestimated tumour size > 20 mm in 17 % of the patients , in 13 % leading to an incorrect decision to perform breast - conserving surgery . from their study , they concluded that baseline tumour size , tumour size reduction and cancer subtype should be taken into account in the optimal selection of patients eligible for breast - conserving therapy . if reported , median ppv and npv were 64 % and 87 % , respectively . in a meta - analysis of 25 studies , yuan et al . showed that pooled weighted estimates of sensitivity and specificity of mri for demonstrating pcr were 63 % ( range 5670 % ) and 91 % ( range 8992 % ) . these findings were concordant with the observations made by wu et al . , who performed a meta - analysis of 34 studies . they concluded that the sensitivity and specificity of contrast - enhanced breast mri to predict pcr were 68 % and 91 % , respectively . the variation in these findings , especially in specificity , might be explained by the differences in included studies , since these recent publications additionally included studies with smaller populations and lower mri field strengths . since pcr is only achieved in up to 30 % of patients , we think that a minimum number of ten patients is too low to accurately use pcr as a study outcome . therefore , we chose a minimum number of 25 patients to be included in our final analysis . despite the differences in these reviews and our current study , the diagnostic accuracy of breast mri to predict pcr seems to have a high specificity and npv versus only moderate sensitivity and ppv . nevertheless , the varying results in the separate studies ( and their range ) of the recent reviews show that mri 's accuracy for assessing pcr is still under debate and that it is too early to use it as a decision - making tool in studies that investigate other treatment strategies after pcr besides surgery . although the number of studies was small , contrast - enhanced mri outperformed physical examination , ultrasound and mammography in accurately assessing residual disease . in physical examination , this is most likely explained by fibrosis surrounding the tumour bed as a result of the therapy . this fibrotic tissue remains hard , and as such it could lead to misinterpretation of residual disease . these fibrotic changes can also be observed in mammography and ultrasound and can not be easily discerned from residual tumour tissue , but can be excluded by mri since this fibrotic tissue does not show any enhancement after contrast administration . in addition , the diagnostic accuracy of mammography is strongly dependent on breast density , being lower in breasts with extremely dense fibroglandular tissue . if performed in the right period of the menstrual cycle ( day 314 in premenopausal women ) , the accuracy of breast mri was less influenced by breast density . almost all papers used contrast - enhanced breast mri for the evaluation of residual disease and pcr after nac . some studies additionally investigated the ability of diffusion - weighted imaging ( dwi ) for assessing pcr after nac . in dwi , mri is used to assess the brownian motions of water molecules within a certain tissue of interest . in the cell - rich environment of tumours , this results in increased signal intensity on so - called diffusion - weigted images , with corresponding low values for the apparent - diffusion coefficient or adc . woodhams et al . demonstrated that the sensitivity , specificity and accuracy of dwi for assessing pcr was higher than contrast - enhanced breast mri , but the differences observed were not significant with p - values of 0.31 , 0.08 and 0.06 , respectively . in the study by park et al . , dwi was compared to positron emission computed tomography ( pet - ct ) and showed a slightly higher auc for predicting pcr when compared to pet - ct , although this difference was not statistically significant in their population of 34 patients , of which 7 achieved pcr after therapy . , three different mri techniques were compared : dynamic contrast - enhanced mri , dwi and mr spectroscopy . they concluded that the change in adc after treatment was the most accurate predictor of pcr . with an auc of 0.96 , they found that the optimal cutoff value for percentage adc change was 40.7 % , yielding a sensitivity of 100 % and a specificity of 91 % . first , publication bias is a study limitation that merits attention in each systematic review . small studies with less favorable results tend to be published less frequently or not at all . with this potential bias in mind , one should realise that the current positive findings of mri accuracy after nac might be overestimated . second , the lack of study uniformity prevented us from performing a meta - analysis . therefore , we chose to perform a systematic review of the selected studies and provide a descriptive presentation of the observed findings instead of performing a meta - analysis that uses statistical models to adjust for this heterogeneity to some extent . variations in study aim , chemotherapy regimens , response assessment criteria in both imaging and pathological analysis , patient populations and breast cancer subtypes precluded us from drawing more definitive conclusions . for example , chen et al . showed in their study that mri can predict pcr accurately in her2-positive patients , but a high false - negative rate was observed in her2-negative patients , especially when they received anti - angiogenic drugs . loo et al . showed in their study of 118 patients that response monitoring after nac is effective in triple - negative or her2-positive breast cancer subtypes , but is inaccurate in er - positive / her2-negative subtypes . in their 2011 publication , chen et al . demonstrated that mr imaging accuracy was higher for her2-positive cancer types than for her2-negative tumours ( 88 % versus 82 % ) . in the same study , they showed that the average size discrepancy in cases with ki-67 staining of < 10 % was greater than in cases with ki-67 staining of > 40 % . denis et al . showed that mri frequently underestimated residual tumour size in taxane - containing treatments , most likely because of the antivascular effects of these drugs , resulting in less enhancement on contrast - enhanced mri . although no significant differences between who and recist criteria in imaging response assessment were observed in other cancer types , multiple response criteria were used in the selected studies [ 52 , 53 ] . similarly , there are no widely accepted response assessment criteria for pathology . the most important issue in this assessment is the extent of residual dcis . whether or not dcis is included in the analysis might partly explain the differences observed in mri over- and underestimation . from a clinical point of view , it would be most interesting to assess mri accuracy if dcis were included in the definition of pcr , since dcis should also be excised during surgery and identification of dcis extension by mri remains challenging . only four studies had a population size > 100 subjects [ 25 , 30 , 36 , 44 ] and most of the studies were single - centre studies . the statistical noise will be smaller if the sample sizes are increased in ( future ) multicentre studies in order to assess the true accuracy of mri in the nac setting with greater confidence . in summary , breast mri accuracy for assessing residual disease after neoadjuvant chemotherapy is good , but multiple factors , such as cancer subtype and treatment regimen , can influence mri accuracy and should be considered in clinical decision making . clinical decision making based on mri results should therefore be made prudently with these limitations in mind . regardless of the many potential confounders described in this review , we feel that assessment of nac response with mri is promising and ready for more multicentre studies that are able to address these shortcomings .

objectivesthis systematic review aimed to assess the role of magnetic resonance imaging ( mri ) in evaluating residual disease extent and the ability to detect pathologic complete response ( pcr ) after neoadjuvant chemotherapy for invasive breast cancer.methodspubmed , the cochrane library , medline , and embase databases were searched for relevant studies published until 1 july 2012 . after primary selection , two reviewers independently assessed the content of each eligible study using a standardised extraction form and pre - defined inclusion and exclusion criteria.resultsa total of 35 eligible studies were selected . correlation coefficients of residual tumour size assessed by mri and pathology were good , with a median value of 0.698 . reported sensitivity , specificity , positive predictive value and negative predictive value for predicting pcr with mri ranged from 25 to 100 % , 5097 % , 4773 % and 71100 % , respectively . both overestimation and underestimation were observed . mri proved more accurate in determining residual disease than physical examination , mammography and ultrasound . diagnostic accuracy of mri after neoadjuvant chemotherapy could be influenced by treatment regimen and breast cancer subtype.conclusionsbreast mri accuracy for assessing residual disease after neoadjuvant chemotherapy is good and surpasses other diagnostic means . however , both overestimation and underestimation of residual disease extent could be observed.main messages breast mri accuracy for assessing residual disease is good and surpasses other diagnostic means. correlation coefficients of residual tumour size assessed by mri and pathology were considered good. however , both overestimation and underestimation of residual disease were observed. diagnostic accuracy of mri seems to be affected by treatment regimen and breast cancer subtype .

seborrheic dermatitis is a very common papulosquamous dermatoses , affecting over 5% of the general population with male predilections ( 1 ) . this disease is characterized by mild to severe erythema , often accompanied by oily thick scales and mild inflammations . it typically affects areas containing sebaceous glands , particularly the face , ears , scalp , and upper part of the trunk ( 2 ) . since seborrheic dermatitis commonly has a chronic and relapsing course with no permanent cure , various types of treatment have been introduced . the mainstays of treating facial seborrheic dermatitis rely on topical corticosteroids , antifungals , or a combination of both ( 3 , 4 ) . although topical steroids rapidly reduce the severity of the disease by anti - inflammatory action , side effects associated with prolonged application and early relapse after discontinuation of treatment limit its wide usage ( 5 ) . topical antifungal agents are also frequently used in order to decrease the colonization of lipophylic yeasts , malassezia ( pityrosporum ovale ) , but they show a relatively slow onset of the anti - inflammatory effect compared to topical steroids ( 4 , 6 ) . pimecrolimus cream 1% is a new topical macrolactam immunomodulator widely used for the treatment of atopic dermatitis via inhibition of t - cell activation and proinflammatory cytokine production ( 7 ) . the cream has also shown to be effective in the treatment of other inflammatory skin disorders . crutchfield ( 8) suggested that pimecrolimus cream 1% could be used as an effective treatment for facial seborrheic dermatitis . rigopoulos et al . ( 9 ) conducted a randomized open - label clinical trial with pimecrolimus cream 1% and betamethasone 17-valerate 0.1% cream , and reported that pimecrolimus may be an excellent alternative modality for treating seborrheic dermatitis . we herein add our experience of rapid and sustained therapeutic response of pimecrolimus cream 1% in the treatment of facial seborrheic dermatitis . this study was a 4-week , open - label , clinical trial to investigate the efficacy of pimecrolimus cream 1% for the treatment of facial seborrheic dermatitis . it was conducted after the notice of purpose and obtaining signed informed consent from all subjects . a total of 20 patients with a clinical diagnosis of facial seborrheic dermatitis were enrolled . they composed of 16 males and 4 females with a mean age of 47.9 yr ( range , 22 - 79 ) and a mean duration of 3.8 yr ( range , 1 - 13 ) of dermatitis . patients who had taken topical or systemic treatment of corticosteroids , retinoids , or immune - modulating agents within 28 days prior to the study were excluded . patients with any significant medical condition that could compromise immune responsiveness ( lymphoma , acquired immunodefiuency syndrome , other immunodeficiency disorders , or a history of malignant diseases ) were excluded . exclusion criteria also included pregnancy , lactating women , and patients with other facial dermatoses . enrolled patients were instructed to stop using systemic and topical antimicrobials and antifungal agents including tar , zinc pyrithione , or selenium sulfate for 14 days before starting the treatment , but the uas of non - medicated shampoo and facial cleansers were allowed the use of the study . moreover , patients were informed to apply pimecrolimus cream 1% ( norvatis , basel , switzerland ) to affected facial areas only in thin layers , twice daily for 4 consecutive weeks . if complete resolution of seborrheic dermatitis occurred early in the study , patients were instructed to use pimecrolimus cream for an additional week . the evaluation of patients were performed at baseline and at week 1 , 2 , and 4 for the severity of seborrheic dermatitis . four different sites ( forehead / eyebrows , nasolabial folds , perioral areas / chin , and posterior aspect of the ears ) were clinically assessed for erythema , scaling , and pruritus using a 4-point scale ( 0 - 3 ) : 0 , absent ; 1 , mild ; 2 , moderate ; and 3 , severe . scores of each clinical symptom and sign at the baseline were compared to those at subsequent visits to assess the efficacy of pimecrolimus cream . in addition , patients themselves and physicians clinically performed the global assessments of the severity of seborrheic dermatitis at each visit as : completely clear ( no evidence of seborrheic dermatitis ) ; almost clear ( > 90% of clearance ) ; marked improvement ( 75 - 90% of clearance ) ; moderate improvement ( 50 - 75% of clearance ) ; slight improvement ( 25 - 50% of clearance ) ; no change compared with baseline ; or worse compared with baseline . local adverse reactions such as burning sensation , tingling , pain or soreness , or signs of viral or bacterial infections were recorded throughout the study as none , mild , moderate , or severe . a follow - up survey for recurrence of any erythema , scalings , or pruritus after the discontinuation of pimecrolimus was done by telephone . to assess the efficacy of pimecrolimus , data from scales of erythema , scaling , and pruritus were statistically analyzed by means of a repeated - measures analysis of variance ( anova ) model . the study patients had commonly been managed with topical corticosteroids , whereas only 1 patient had had a history of using a topical antifungal agent before visiting our clinic . the 4-week duration of clinical study with pimecrolimus cream 1% was completed in 16 patients , whereas 4 patients discontinued treatment before the completion of the study by the reason of early resolution with their symptoms and signs . at the baseline , all patients had erythema , with a mean grade of 5.15 ; 19 ( 95% ) patients had scaling , with a mean grade of 3.70 ; and 17 ( 85% ) patients had pruritus , with a mean grade of 4.12 . 1 shows the significant decrements in the mean severity scores of each clinical feature during the 4-week application of pimecrolimus cream 1% , and the most prominent reduction of the disease severity was observed at the end of the first week . at week 1 , the mean score of scaling improved most rapidly at 73.0% compared to the mean baseline score . this improvement continued over the following week at 85.1% , and the overall scaling score showed a mean reduction of 91.9% at week 4 ( p<0.001 ) . similar decrements of mean scores were also observed with erythema ( p<0.001 ) and pruritus ( p<0.001 ) at reductions of 53.4% , and 68.7% at week 1 , 69.9% and 88.6% at week 2 , and 87.4% and 91.5% at week 4 compared to the mean baseline scores , respectively . these changes were statistically significant ( p<0.001 ) and clinically well correlated with photographs ( fig . 2 , 3 ) . global improvement of severity assessed by investigators and patients at the week 1 and 4 of treatment are shown in table 1 . similar to the symptoms and signs , the patients ' subjective response to pimecrolimus was mostly significant at the end of the first week . investigators noticed above the moderate improvement of severity in all patients after the first week of treatment . at week 4 , 3 patients ( 15% ) reported marked improvement , 6 ( 30% ) almost clear , and 11 ( 55% ) complete clear of the dermatitis . from physician 's point of view , 12 subjects ( 60% ) including 4 prematurely discontinued patients experienced a complete clearance of clinical signs and symptoms within 4 weeks of pimecrolimus treatment . a total of 9 patients ( 45% ) reported mild ( 1 patient ) to moderate ( 8 patients ) burning or tingling sensations lasting for approximately 20 - 30 min and mostly during the first 2 or 3 days of treatment . these local adverse events did not require any further management nor did they lead to premature discontinuation of the therapy . after the completion of the study , 12 patients were available for follow - up telephone surveys 4 to 8 weeks later . three patients had continued to apply pimecrolimus cream 1% once daily and did not notice any relapse of symptoms or signs of the disease for 8 weeks . nine patients not using any medications were aware of recurrence of the disease , mostly between 3 to 8 weeks after the discontinuation of pimecrolimus . five out of 9 patients remained free of the disease for at least 4 weeks after the discontinuation of pimecrolimus , whereas 4 patients experienced relapse within 4 weeks ( table 2 ) . recent studies ( 8 - 11 ) proposed that pimecrolimus cream 1% is an effective and safe modality in controlling the symptoms and signs of seborrheic dermatitis ( table 3 ) . in the study by rigopoulos et al . ( 9 ) to determine the efficacy of pimecrolimus in comparison with potent topical corticosteroid in the treatment of seborrheic dermatitis , the complete resolution of symptoms was rapidly achieved within 9 days in both the pimecrolimus - treated and the topical corticosteroid - treated group . although both agents were highly effective in the treatment of seborrheic dermatitis , the relapses were more common , rapid , and severe in the corticosteroid - treated group . ( 10 ) treated 19 patients of seborrheic dermatitis with pimecrolimus , and clearance was observed after 1 to 3 weeks of treatment . in addition , patients who were previously unresponsive to topical corticosteroid and antifungals also showed clearance of seborrheic dermatitis with pimecrolimus treatment ( 8 , 11 ) . these results indicate that pimecrolimus cream 1% may be an effective nonsteroidal alternative for the treatment of seborrheic dermatitis . similar results were reported in the use of topical tacrolimus for seborrheic dermatitis ( 12 , 13 ) . in our study , consistent with those previous reports , the mean severity scores of erythema , scaling , and pruritus showed statistically significant decrements after a 4-week application of pimecrolimus cream 1% relative to the baseline . the evident therapeutic responses mostly appeared within the first week of treatment and continued to improve until the end of the study . improvements in global assessment of disease severity determined by patients and investigators also showed excellent results during the first week of the application , and they experienced continuous improvements throughout the duration of the study . according to the follow - up survey , improved states were maintained for at least 4 weeks in 5 out of 9 patients who did not use any medications after the completion of the study . previous studies have indicated that seborrheic dermatitis might develop as a result of inflammatory immune reactions to the lipophilic yeast malassezia ( 14 , 15 ) . humoral and cell - mediated immune response to malassezia has also been advocated ( 16 ) . improvement of the disease by antifungal medications supports the possible role of malassezia ( 14 ) . pimecrolimus selectively inhibits t cell activation via inhibition of the calcineurin - dependent nuclear transcription factor of activated t cells . it prevents the release of cytokines and proinflammatory mediators involved in the inflammatory response ( 7 ) . accordingly , the beneficial effect of pimecrolimus on the course of seborrheic dermatitis in our study is thought to be due to its anti - inflammatory and immunomodulatory properties . however , the possibility of direct antifungal activities of pimecrolimus can not be excluded , since a recent report documented that tacrolimus , another topical immunomodulator , has antifungal activities against some of the malassezia species ( 17 ) . the safety of pimecrolimus cream has been well documented , and in contrast to topical corticosteroids it does not induce skin atrophy or other corticosteroid - related adverse events , especially on the face ( 18 , 19 ) . burning sensations and tingling are well - known local side effects of pimecrolimus , and 9 of 20 ( 45% ) patients experienced these events in our study . however , most reactions were minimal and transient , beginning on day 1 and resolving within 3 days of treatment . no pain , soreness , or signs of viral or bacterial infections were detected . in conclusion , our findings further support the previous reports that pimecrolimus cream 1% can be an effective and well - tolerated modality in the treatment of facial seborrheic dermatitis . however , since the most of previous encouraging reports had been studied in small numbers and in short - term , further clinical troals involving controlled , long - term follow - up protocols with a large scale of patients are warranted to assess the precise efficacy and preventive action of pimecrolimus in the management of seborrheic dermatitis .

pimecrolimus cream 1% has shown to be effective in patients with a variety of inflammatory cutaneous disorders . and it might be a useful modality in the treatment of seborrheic dermatitis . this prospective study was aimed at assessing the efficacy and tolerability of pimecrolimus cream 1% in the treatment of facial seborrheic dermatitis . twenty patients were instructed to apply pimecrolimus cream 1% for 4 consecutive weeks . assessment of the disease severity was performed at baseline and at week 1 , 2 , and 4 . clinical assessments of erythema , scaling , and pruritus were measured using a 4-point scale ( 0 - 3 ) . global assessments of the disease severity by patients and investigators were performed at each visit . mean clinical scores of erythema , scaling , and pruritus significantly improved by 87.4% , 91.9% , and 91.5% respectively at week 4 ( p<0.001 ) . improvements in the global assessment of disease severity determined by patients and investigators also showed excellent results . no specific adverse events other than transient burning and tingling sensations were noted . the relapse of facial seborrheic dermatitis was mostly observed between 3 to 8 weeks after the discontinuation of pimecrolimus . we suggest that the topical application of pimecrolimus cream 1% can be an effective and safe alternative for treatment of facial seborrheic dermatitis .

hernias through the foramen of winslow are extremely rare , accounting for 0.1% of all abdominal hernias we present a case of a patient with strangulated ileum herniated through the foramen of winslow . recent literature review was undertaken on pubmed as a search platform using the keywords foramen of winslow and hernia . an emergency computed tomography scan revealed a loop of ileum in the lesser sac . at emergency laparotomy , a herniated loop of ileum that had become strangulated at its entry to the lesser sac via the foramen of winslow was confirmed . the loop of ileum was reduced but was nonviable , which had to be resected with a primary anastomosis . herniation through the foramen of winslow is a difficult diagnosis and must not be missed . early cross - sectional imaging and surgical intervention are advised in order to reduce morbidity . hernias through the foramen of winslow are extremely rare , accounting for 0.1% of all abdominal hernias1 and 8% of all internal hernias . two - thirds of cases reported were herniation of ileum followed by a mobile cecum or ascending colon.2,3 seldomly , the gallbladder or the omentum has herniated through . as the symptoms are so nonspecific , delayed clinical diagnosis is often observed , resulting in bowel strangulation and high mortality rate between 36% and 49%.4 nonviable bowel is often identified only at laparotomy . we present a case of a patient with strangulated ileum herniated through the foramen of winslow . a 66-year - old man , with no known past medical or surgical history , presented acutely with a few hours history of severe colicky epigastric pain and vomiting . he had similar milder and short - lived episodes of pain in the past , but he had put these episodes down to muscular pain , for which he had never sought medical attention . there was no report of alleviation of pain on truncal flexion . on admission , examination was unremarkable except for mild tenderness of the epigastrium , but the patient was in extreme pain . his blood profile showed a mild neutrophilia of 12.4 10/l and a raised c - reactive protein of 130 mg / l . blood lactate and base excess of an arterial gas sample were also within normal limits . an emergency computed tomography scan of the abdomen revealed distended loops of small bowel showing reduced enhancement , which were located within the lesser sac . both afferent and efferent limbs were visualized in the space between the liver hilum and inferior vena cava , which was widened . the appearance was in keeping with an internal hernia resulting in a closed - loop obstruction ( figs . 1 and 2 ) . this revealed a loop of mid - ileum herniating through the foramen of winslow , and the herniated loop of ileum had become strangulated . the reported demographic for bowel herniation through the foramen of winslow is usually men with a manual occupation , aged between 61 and 69 years . some have suggested that cholecystectomy might be a risk factor.5 other postulated risk factors6 include abnormally long bowel mesentery , abnormally enlarged foramen of winslow , and a defect in the gastrohepatic ligament . herniation through the foramen of winslow is rare because the normal peritoneal orifice is kept closed by normal intra - abdominal pressure ( fig . erskine7 has also postulated the failure of the right colon to retroperitonealize along with changes in the intra - abdominal pressure as a contributing factor . the presence of jaundice has been described due to direct compression of the hepatic pedicle.5 the obstruction in our patient was very proximal , rendering minimal nasogastric drainage . our patient had no previous abdominal surgery or trauma , and the cross - sectional imaging suggested small bowel ischemia , and therefore laparotomy was mandated . there are many more common causes of epigastric pain ; however , these conditions can be excluded quickly . unfortunately , many cases of this condition have been identified at autopsy . some have reported that gas - containing intestinal loops are high in abdomen and medial - posterior to stomach associated with small bowel obstruction . cross - sectional imaging is considered the diagnostic modality of choice,8 largely as a consequence of clinical diagnostic uncertainty.9 classical signs include mesenteric vessels stretching anterior to the inferior vena cava and posterior to the portal vein associated with bowel obstruction in the lesser sac . there were no obvious risk factors in this gentleman , except that he is an average manual worker . this condition is rare , difficult to diagnose , and does not always have the obvious risk factors . a wide kocher s maneuver or opening the gastrohepatic ligaments may be required . due to diagnostic uncertainty even with high - resolution computed tomography , open surgery is usually performed . however , this has led some experienced surgeons to investigate initially with laparoscopy . successful laparoscopic management for the foramen of winslow herniation has now been described.10 the debate continues as to whether the foramen of winslow ought to be closed in order to prevent recurrence . to date , there has not been a report of recurrence probably due to adhesions obliterating the foramen and tethering the remainder of the small bowel . experts have also warned of the potential significant negative consequences of closing the defect : portal vein thrombosis or obstructive jaundice . internal herniation through the foramen of winslow is a difficult clinical diagnosis and must not be missed .

introductionhernias through the foramen of winslow are extremely rare , accounting for 0.1% of all abdominal hernias . delayed diagnosis is often observed , resulting in bowel strangulation and high mortality.methodwe present a case of a patient with strangulated ileum herniated through the foramen of winslow . recent literature review was undertaken on pubmed as a search platform using the keywords foramen of winslow and hernia.case summarya 66-year - old man presented acutely with severe epigastric pain and vomiting . an emergency computed tomography scan revealed a loop of ileum in the lesser sac . at emergency laparotomy , a herniated loop of ileum that had become strangulated at its entry to the lesser sac via the foramen of winslow was confirmed . the loop of ileum was reduced but was nonviable , which had to be resected with a primary anastomosis . the patient s postoperative recovery was uneventful.conclusionherniation through the foramen of winslow is a difficult diagnosis and must not be missed . early cross - sectional imaging and surgical intervention are advised in order to reduce morbidity .

intractable nausea and vomiting is a commonly encountered problem in any general medicine or gastroenterology service . several causes from infection to chronic medical conditions , such as diabetic gastroparesis and gastroparesis due to multiple causes , can be listed . neuromyelitis optica ( nmo ) , also known as devic 's disease , is a rare autoimmune disorder in which a patient 's immune system attacks their own nervous system , especially the optic nerves and the spinal cord , leading to loss of vision and spinal cord dysfunction [ 1 , 2 , 3 ] . the likely target of the autoimmune attack , at least in some patients with nmo , has been identified as a protein of the nervous system astrocytes called aquaporin 4 . typically , nmo is diagnosed in patients presenting with a rapid onset of blindness in one or both eyes , followed within days or weeks by varying degrees of paralysis in the arms and legs . in most cases , however , the interval between optic neuritis and transverse myelitis is significantly longer , in many cases can occur after some years of quiescence . after the initial attack , nmo follows a largely unpredictable course . besides muscle weakness and reduced sensation , bladder and bowel incontinence the disease can also occur as clusters of attacks months or years apart , followed by partial recovery during periods of remission . another form of nmo , in which an individual only has a single , severe attack extending over a month or two , is most likely a distinct disease that affects men and women with equal frequency . the onset of nmo varies from childhood to adulthood , with two peaks , one in childhood and the other in adults ( mostly in their 40s ) [ 1 , 2 , 3 ] . for a while , nmo was considered to be a severe variant of multiple sclerosis ( ms ) because both can cause attacks of optic neuritis and myelitis . recent discoveries , however , suggest it is a separate disease [ 2 , 3 ] . nmo is different from ms in the severity of its attacks and its tendency to solely strike the optic nerves and spinal cord at the beginning of the disease . symptoms outside of the optic nerves and spinal cord are rare , although certain symptoms , including uncontrollable vomiting and hiccups , are now recognized as relatively specific symptoms of nmo that are due to brainstem involvement , specifically the area postrema . we present our encounter with a 38-year - old female with no past medical history who presented to the emergency department with complaints of intractable nausea , vomiting , hiccups and abdominal pain . around thanksgiving 2012 , she had had bronchitis and sinusitis . she had been treated with tylenol cold and cough , a z - pak , inhalers and tessalon perles . this had progressively worsened into intractable nausea and vomiting about 4 days following thanksgiving and lasted till presentation , which was about 2 weeks after onset . complete blood count revealed hemoglobin of 10.1 g / dl , a hematocrit of 31.2% , a platelet count of 225 103/l and white blood cell count of 6.9 10/l . serum electrolytes were as follows : na 136 mmol / l , k 3.6 mmol / l , cl 105 mmol / l , hco3 23 mmol / l , bun 10 mg / dl and creatinine 1.3 mg / dl . she had a right upper quadrant ultrasound that showed some stones within the gallbladder , but no findings suggestive of acute cholecystitis . gastroenterology was consulted and she had an egd that showed mild esophagitis for which she was placed on a proton pump inhibitor . serial daily labs including complete blood count and basic metabolic profile did not reveal any abnormalities . while in the hospital , she was noticed to have a positive babinski 's test on daily physical examination , which was a new finding that had been absent on presentation . at this point she had an mri without contrast showing moderate diffuse atrophy , most particularly in the occipital area ( she denied any prior head trauma or problems at birth ) and very few scattered white matter hyperintensities ( she has a history of tobacco abuse ) . she went on to have a brain mri with and without contrast with results showing small foci of linear abnormal t2 signal with questionable associated vague enhancement at the posterior aspect of the brainstem , in the region that anatomically corresponds to the area postrema ( fig . 1 , fig this finding was suspicious for a variant presentation of nmo with intractable nausea / vomiting . following this finding , she was started on 1 g of iv solu - medrol daily . the day after her first dose of solu - medrol , she was symptom - free and asked for a diet , which she tolerated well . however , the patient started feeling better on solu - medrol and deferred the procedure , saying she would consider it if her symptoms recurred . after 5 days on iv solu - medrol and still symptom - free , she was transitioned to oral steroids and discharged with a taper . she was asked to follow up with the neurology clinic and her primary care doctor . other lab work done included tsh , rpr , heavy metals , uds and serum ace , which all came back negative or within normal range . at the clinic , she was started on rituximab and has remained symptom - free till the writing of this article . intractable nausea and vomiting is a commonly encountered problem in any general medicine or gastroenterology service . several causes from infection to chronic medical conditions , such as diabetic gastroparesis and gastroparesis due to multiple causes , can be listed . nmo , also known as devic 's disease , is a rare autoimmune disorder in which a patient 's immune system affects the optic nerves and spinal cord , leading to loss of vision and spinal cord dysfunction [ 1 , 2 , 3 ] . spinal cord dysfunction is mainly manifested as weakness , paralysis , numbness and possibly bowel and bladder incontinence . due to these symptoms , nmo can often be confused with ms . as rare a disease as nmo is , it is even rarer for intractable nausea and vomiting to be the only initial presenting symptoms , and this is sparsely mentioned in reviewed literature . nmo is caused by the development of autoantibodies ( nmo - igg ) against the protein aquaporin 4 found in the cell membranes of the processes of astrocytes that surround and protect the blood brain barrier . like in ms , demyelination of the optic nerve and spinal cord is also known to occur in nmo , but the exact mechanism is still to be described . however , astrocyte destruction is thought to be more severe than demyelination and as such probably plays a bigger role in the pathogenesis of the disease . they proposed that at least two out of three supporting criteria in addition to the above - listed absolute criteria are needed to make a diagnosis of nmo . the three supporting criteria are : ( 1 ) a contiguous spinal cord lesion on mri extending over three or more segments . ( 2 ) initial brain mri not meeting the usual diagnostic criteria for ms . ( 3 ) seropositivity for nmo - igg . conducted a case - control study on the sensitivity and specificity of nmo - igg in the diagnosis of the disease and concluded that sensitivity and specificity were 73% ( 95% ci 6086 ) and 91% ( 95% ci 79100 ) for nmo . of 14 seropositive cases identified incidentally , 12 had nmo or a high - risk syndrome for the disease . different variants of the disease with unique presenting features have been described . in a case series , apiwattanakul et al . reported on 12 aquaporin 4 antibody - positive patients ( 12% of seropositive mayo clinic patients identified since 2005 ) whose initial presenting symptom of nmo was intractable vomiting . vomiting lasted a median of 4 weeks ( range 2 days to 80 weeks ) . optic neuritis or transverse myelitis developed after vomiting onset in 11 patients . at last evaluation ( median 48 months after vomiting onset ) , 7 patients fulfilled the diagnostic criteria for nmo . from their study they suggested that the aquaporin 4-rich area postrema might be a first point of attack in nmo [ 8 , 9 ] . the disease has recently been associated with several comorbid conditions including collagen vascular diseases , autoantibody syndromes , infections with varicella zoster virus , epstein - barr virus and hiv , and exposure to clioquinol and anti - tuberculosis drugs . a high level of suspicion on the part of the clinician is necessary for early diagnosis , especially in cases where the only initial presenting symptoms are nausea and vomiting . it is also pertinent to note that nmo - igg - negative disease has been described as well , making diagnosis in these circumstances quite challenging . in terms of treatment , intravenous glucocorticoid ( typically 1 g of iv solu - medrol daily over 5 days ) is the recommended treatment during acute attacks . azathioprine , mycophenolate mofetil or rituximab is used to achieve immunosuppression on a chronic state , thereby preventing relapse . patients will typically require this for at least 5 years ( especially if they are nmo - igg - seropositive ) even if they have had just one attack [ 12 , 13 ] . it is our hope that our experience and this article highlight a less often considered cause of intractable nausea and vomiting . if this had been considered earlier , this patient could have achieved a quicker turnaround , with improved health outcomes and cost .

neuromyelitis optica , also known as devic 's disease , is a rare autoimmune disorder in which a patient 's immune system affects the optic nerves and the spinal cord , leading to loss of vision and spinal cord dysfunction . we present our experience with a 38-year - old female who presented to our facility with complaints of intractable nausea and vomiting . after extensive evaluation , she was found to have neuromyelitis optica . her symptoms completely resolved following institution of appropriate therapy . she made a significant recovery and has since been placed on chronic immunosuppressive therapy . through this article we hope to bring attention to a significant cause of intractable nausea and vomiting that may often be forgotten in general medicine or gastroenterology services .

service - learning as a teaching methodology has roots in the experiential education movement of the 1950 s and 1960 s . experiential education was based on the concept that the quality of student learning was measured by the meaning students placed on their experience ( 3 ) . developing knowledge through action , specifically through actions that benefitted course - based , credit - bearing educational experience in which students ( a ) participate in an organized service activity that meets identified community needs and ( b ) reflect on the service activity in such a way as to gain further understanding of course content , a broader appreciation of the discipline , and an enhanced sense of civic responsibility ( 2 ) . according to the national service - learning clearinghouse , almost 30% of the 6.7 million students in public and private 4 year institutions of higher education report participating in a course where service is part of the curriculum ( 10 ) . service - learning in the medical and allied health professions has been a critical component of the curriculum with projects aimed at providing a service to the community and hands - on experience for students . bentley and ellison described a service - learning project designed to teach nursing students key elements of delivering community services to pregnant teens ( 1 ) . nursing students ( n = 20 ) partnered with early head start to address the needs of pregnant teenagers and help them transition from pregnancy to parenthood . specific course objectives were outlined in advance to give students , faculty and community partners clear guidelines for evaluation . make an appointment with the adolescent once per week for six weeks to implement nursing interventions identified from the supporting data following the assessments . to attend the adolescent s delivery if she happens to deliver during this semester . bentley and ellison found that students were able to understand the at - risk population at a deeper level , overcoming preconceived ideas of pregnant teens . in addition , the faculty found that student s commitment to community service following the class increased . health education programs have also included service - learning projects in their curricula as a means of enhancing the preparation of health educators . geiger and werner described a service - learning assignment completed by undergraduate health and elementary education students ( n = 27 ) ( 4 ) . the assignment was designed to reflect the competencies outlined in the national commission of health education credentialing . students engaged in learning by attending lectures on relevant topics , assisting health teachers with instructional planning , and presenting information to elementary school students . student evaluations reflected a positive experience and an ability to overcome preconceived barriers relating to teaching in an inner - city school environment . cooperating teacher evaluations provided valuable feedback to both faculty and students relating to future course preparations . freewheelers designed to increase the mobility of wheel - chair bound nursing home clients ( n = 15 ) through a stretching and strength training program facilitated by kinesiology students ( n = 22 ) enrolled in a motor development laboratory class ( 8) . clearly articulated learning objectives relating to motor development education as well as community service needs were provided . in addition , students were challenged to consider and reflect upon their stereotypes around aging adults . each nursing home resident was assigned to three students from the class with one hour sessions scheduled three times a week on monday , wednesday and friday for eight weeks of the 15-week semester . the students reported greater understanding of motor development over the life span and a deeper appreciation for older adults . in this section , a definition of service - learning was proposed , with examples provided of service - learning projects from nursing , health education and kinesiology . while the spectrum of service - learning projects is broad , all contain some structure of objectives , accountability , and partnering . some service - learning projects take place over the course of a 15-week semester , while other projects are event - based with students completing assignments in preparation for a single event . in this section , a brief description of three service - learning projects conducted by faculty and students from westfield state college will be provided . the original homeschooler s field day was a collaborative effort with students enrolled in an analysis of teaching physical education class , sport promotions class and a community - based homeschooler s programming organization . the now annual event has grown to include faculty and students from the biology department , as well as students enrolled in a kinesiology laboratory class . homeschooler s field day was a collaborative event organized and implemented by students and faculty for homeschooler s in the new england area to engage in fun , motor - skilled activities . in this one day event homeschooled children ( n = 162 ) ages 513 pre - registered and participated in an organized field day scheduled from 11:00 a.m.2:00 p.m. homeschooler s divided into younger ( ages 58 ) and older ( ages 913 ) groups progress through a series of eight 1520 minute stations . kinesiology students developed stations relating to biomechanical principles that could be taught and practiced in a 15-minute station . sport promotions students developed objectives and implemented strategies relating to event promotion , logistics and risk management . movement science majors not enrolled in the classes responsible for the field day were invited to participate as volunteers . parents were invited to join in any activities , adding to the complexity of the teaching for the students in charge of each station . homeschooler s , parents , students and faculty reported positive experiences , with all participants providing valuable feedback for future events . although not one of the original objectives , student stereotypes and attitudes about homeschooler s and the structure of education were challenged . in addition , students reported greater appreciation for the level of planning and preparation required for a successful event . hope for hollie provides an example of a service - learning project that not only engaged students and the larger community , but made a meaningful and lasting difference in the world . hope for hollie was a sports promotions class service - learning project that was undertaken to raise funds for a student who was recovering from meningitis . the students in the sports promotion class were offered an opportunity to apply the principles of sport event management by envisioning , initiating , planning , organizing , implementing and evaluating a real event . the upper - level class ( n = 18 ) consisted of movement science , business and communications seniors . because of student internship and work commitments , the event a run / walk was planned for a friday in april , during the scheduled class from 12:30 p.m. participation objectives included the registration of 100 participants and awareness objectives relating to risks associated with meningitis were also developed . because of the interdisciplinary nature of the class , students were able to draw on their strengths to achieve a successful outcome . movement science majors assisted with risk management , developed pre - walk exercise programs for participants , and contacted exercise - related sponsors . communications majors assisted with the development of a logo , news releases and attractive flyers / promotions . students reported learning about sport promotions , but also learning life lessons about the goodness of humankind . the day of the walk was one of my best days all year , actually of my life . i had no idea how good it would feel to be able to give hollie the check . this was the most rewarding class i have ever taken , not only academically but also on a personal level . .. we bonded with our classmates on a level that usually never happens in a class , we were able to work closely and learn from students from different majors and we were able to take what we learned in the classroom and put it to use in the real world . elementary school students ( n = 84 ) in the pioneer valley chinese immersion charter school ( pvcics ) begin their kindergarten and first grade education with 75% of their day taught in mandarin chinese . drawing on the successful experiences from the homeschooler s field day and hope for hollie events , two faculty from the movement science department at westfield state college initiated , organized and implemented a service - learning event to be taught primarily in chinese . balance across boundaries was the collaborative effort of students ( n = 30 ) enrolled in movement science classes in kinesiology , rehabilitation for special populations , supervising sport and fitness programs and a physical science class for teachers preparing to teach physics . a series of four stations were developed to teach balance using simplified chinese characters for numbers and animals . for example , one station , entitled , balance bingo had the simplified chinese characters for numbers 15 marked in large squares with the characters chinese symbol for an animal at the heading . the elementary school students who landed on a specific square called out the name of the animal and number in chinese the college students repeated in chinese and performed a balance activity for the elementary school student to copy . while the pronunciation of the elementary school students was superior to that of the college students , the ability to communicate movement in another language both challenged and simplified the event . several students reported an inability to understand the elementary school students , but an ability to overcome the language barrier by performing a movement . likewise , students reported admiration for the efforts of the kindergarten students to communicate in chinese . while not an objective of the program , students reported a positive shift in attitude toward chinese - speaking individuals . elementary school students expressed pride in their ability to communicate in their immersion language , and their ability to learn new skills relating to balance . some projects have involved only one class , while others have involved several different classes and disciplines . service - learning provides a methodology and structure that embraces interdisciplinary , intergenerational and international experiences . the original homeschooler s field day was a collaborative effort with students enrolled in an analysis of teaching physical education class , sport promotions class and a community - based homeschooler s programming organization . the now annual event has grown to include faculty and students from the biology department , as well as students enrolled in a kinesiology laboratory class . homeschooler s field day was a collaborative event organized and implemented by students and faculty for homeschooler s in the new england area to engage in fun , motor - skilled activities . in this one day event homeschooled children ( n = 162 ) ages 513 pre - registered and participated in an organized field day scheduled from 11:00 a.m.2:00 p.m. homeschooler s divided into younger ( ages 58 ) and older ( ages 913 ) groups progress through a series of eight 1520 minute stations . learning objectives for students were developed relevant to their course . kinesiology students developed stations relating to biomechanical principles that could be taught and practiced in a 15-minute station . sport promotions students developed objectives and implemented strategies relating to event promotion , logistics and risk management . movement science majors not enrolled in the classes responsible for the field day were invited to participate as volunteers . parents were invited to join in any activities , adding to the complexity of the teaching for the students in charge of each station . homeschooler s , parents , students and faculty reported positive experiences , with all participants providing valuable feedback for future events . although not one of the original objectives , student stereotypes and attitudes about homeschooler s and the structure of education were challenged . in addition , students reported greater appreciation for the level of planning and preparation required for a successful event . hope for hollie provides an example of a service - learning project that not only engaged students and the larger community , but made a meaningful and lasting difference in the world . hope for hollie was a sports promotions class service - learning project that was undertaken to raise funds for a student who was recovering from meningitis . the students in the sports promotion class were offered an opportunity to apply the principles of sport event management by envisioning , initiating , planning , organizing , implementing and evaluating a real event . the upper - level class ( n = 18 ) consisted of movement science , business and communications seniors . because of student internship and work commitments , the event a run / walk was planned for a friday in april , during the scheduled class from 12:30 p.m. participation objectives included the registration of 100 participants and awareness objectives relating to risks associated with meningitis were also developed . because of the interdisciplinary nature of the class , students were able to draw on their strengths to achieve a successful outcome . movement science majors assisted with risk management , developed pre - walk exercise programs for participants , and contacted exercise - related sponsors . communications majors assisted with the development of a logo , news releases and attractive flyers / promotions . students reported learning about sport promotions , but also learning life lessons about the goodness of humankind . the day of the walk was one of my best days all year , actually of my life . i had no idea how good it would feel to be able to give hollie the check . this was the most rewarding class i have ever taken , not only academically but also on a personal level . .. we bonded with our classmates on a level that usually never happens in a class , we were able to work closely and learn from students from different majors and we were able to take what we learned in the classroom and put it to use in the real world . elementary school students ( n = 84 ) in the pioneer valley chinese immersion charter school ( pvcics ) begin their kindergarten and first grade education with 75% of their day taught in mandarin chinese . drawing on the successful experiences from the homeschooler s field day and hope for hollie events , two faculty from the movement science department at westfield state college initiated , organized and implemented a service - learning event to be taught primarily in chinese . balance across boundaries was the collaborative effort of students ( n = 30 ) enrolled in movement science classes in kinesiology , rehabilitation for special populations , supervising sport and fitness programs and a physical science class for teachers preparing to teach physics . a series of four stations were developed to teach balance using simplified chinese characters for numbers and animals . for example , one station , entitled , balance bingo had the simplified chinese characters for numbers 15 marked in large squares with the characters chinese symbol for an animal at the heading . the elementary school students who landed on a specific square called out the name of the animal and number in chinese . the college students repeated in chinese and performed a balance activity for the elementary school student to copy . while the pronunciation of the elementary school students was superior to that of the college students , the ability to communicate movement in another language both challenged and simplified the event . several students reported an inability to understand the elementary school students , but an ability to overcome the language barrier by performing a movement . likewise , students reported admiration for the efforts of the kindergarten students to communicate in chinese . while not an objective of the program , students reported a positive shift in attitude toward chinese - speaking individuals . elementary school students expressed pride in their ability to communicate in their immersion language , and their ability to learn new skills relating to balance . some projects have involved only one class , while others have involved several different classes and disciplines . service - learning provides a methodology and structure that embraces interdisciplinary , intergenerational and international experiences . one of the primary purposes of service - learning education is to expose students to diverse individuals and cultures . preparing students to live in a global community enables them to transition from academia to the workplace more smoothly . traditionally , this exposure to different cultures has taken place in the context of study abroad programs . community service programs designed to assist an under - served population have long been a part of campus ministry and study abroad programming . while worthy and useful , many such programs could be defined more as volunteer than service - learning . however , structured , service - learning projects that provide students an opportunity to learn about different cultures have been developed throughout the world . parsi and list described the powerful experiences reported by medical students involved in a service - learning project in nairobi , kenya ( 6 ) . the authors recommended designing service - learning programs that promote cultural sensitivity , individual and community empowerment and provide education about the historical and global forces that shape communities . riner and becklenberg described a partnership with a nicaraguan community and the indiana university school of nursing that provided a rich opportunity for nursing students to learn about working in a multi - cultural , interdisciplinary team of healthcare professionals ( 7 ) . ngai described a service - learning project implemented at the chinese university of hong kong in which students provided services to needy individuals within the context of a general education class ( 5 ) . while service - learning projects are often designed to assist an at - risk population , many provide students an opportunity to move beyond the confines of a classroom or case study to real people in real situations . as exercise science professionals , we have ample opportunities to develop cultural sensitivity and provide diverse experiences for our students to gain exposure to the global community . in this paper , service - learning has been defined and several examples of service - learning have been provided . a challenge of developing effective service - learning projects is the logistics and ongoing evaluation . many colleges and universities have an office dedicated to service - learning education . working in unison with a service - learning coordinator many study - abroad programs provide service - learning opportunities for students . however , not many such programs are discipline specific . educating and collaborating with study abroad coordinators to educate , envision and organize exercise science related service - learning programs may increase the likelihood of students having discipline specific opportunities . the number of international students enrolled at colleges and universities in the united states has grown exponentially . international student organization leaders can make connections for students to participate in collaborative service - learning projects . for example , international students who are active in sports and physical activities not often played in the united states can partner with exercise science students for a series of laboratory activities . the international students will have an opportunity to teach a skill , take pride in their sport , and forge new relationships . exercise science students can analyze the skills necessary to be successful in the sport , learn more about the origins and history of the culture and sport , or develop specific training programs for athletes involved in the sport . in addition to on - campus opportunities , locally - based , culturally diverse communities provide another opportunity to engage students in service - learning projects focused on physical activity and exercise science . language need not be a barrier to program development . some examples of simple , service - learning projects that can be conducted with minimal language skills are : a student - directed , 8-week walking program with individuals from a local international community at a designated time and place ; a 4-week playground project in which students engage caregivers and children from a local international community in motor skill activities ; a stretching and/or strength program designed to increase mobility of a given population , perhaps from a local ethnic church . while examples of effective service - learning projects abound , it is critical that objectives relating to increasing cultural sensitivity and exposing students to diverse cultures be articulated in advance . as advocates for healthy lifestyles and ambassadors of the exercise is medicine campaign , we have a unique opportunity to envision , create and provide service - learning programming that challenges students to be agents of change . service - learning education provides a vehicle for applying theory to practice and engaging students in programs that develop reflective , global citizens prepared to face the relevant issues and challenges of the future . a challenge of developing effective service - learning projects is the logistics and ongoing evaluation . many study - abroad programs provide service - learning opportunities for students . however , not many such programs are discipline specific . educating and collaborating with study abroad coordinators to educate , envision and organize exercise science related service - learning programs may increase the likelihood of students having discipline specific opportunities . the number of international students enrolled at colleges and universities in the united states has grown exponentially . international student organization leaders can make connections for students to participate in collaborative service - learning projects . for example , international students who are active in sports and physical activities not often played in the united states can partner with exercise science students for a series of laboratory activities . the international students will have an opportunity to teach a skill , take pride in their sport , and forge new relationships . exercise science students can analyze the skills necessary to be successful in the sport , learn more about the origins and history of the culture and sport , or develop specific training programs for athletes involved in the sport . in addition to on - campus opportunities , locally - based , culturally diverse communities provide another opportunity to engage students in service - learning projects focused on physical activity and exercise science . language need not be a barrier to program development . some examples of simple , service - learning projects that can be conducted with minimal language skills are : a student - directed , 8-week walking program with individuals from a local international community at a designated time and place ; a 4-week playground project in which students engage caregivers and children from a local international community in motor skill activities ; a stretching and/or strength program designed to increase mobility of a given population , perhaps from a local ethnic church . while examples of effective service - learning projects abound , it is critical that objectives relating to increasing cultural sensitivity and exposing students to diverse cultures be articulated in advance . as advocates for healthy lifestyles and ambassadors of the exercise is medicine campaign , we have a unique opportunity to envision , create and provide service - learning programming that challenges students to be agents of change . service - learning education provides a vehicle for applying theory to practice and engaging students in programs that develop reflective , global citizens prepared to face the relevant issues and challenges of the future .

service - learning provides students , faculty and community partners an opportunity to go beyond classroom and laboratory instruction to the application of theoretical principles , and the development of civic responsibility by forging meaningful relationships with community partners . service - learning also enables faculty to broaden the cultural sensitivity and international exposure of students by engaging students in directed , purposeful projects aimed at supporting the goals of both the college and the international partners . we have a unique opportunity in exercise science to make lasting changes and improve global conditions relating to student learning and the physical inactivity crisis . the purpose of this article is to define service - learning , describe different types of service - learning projects , and discuss service - learning opportunities in international settings .

phenylketonuria is an autosomal recessive metabolic disease which may cause brain insult in the developing brain , consequently , leading to progressive neurodevelopmental delay . in this genetic metabolic disorder , the hepatic enzyme , this enzyme is necessary to break down amino acid phenylalanine to tyrosine . in deficiency of this enzyme , phenylketonuria is classified into classic phenylketonuria ( pku ) which indicates phenylalanine ( phe ) levels higher than 1200 mol / l and mild pku in which phe levels are between 600 mol / l and 1200 mol / l . in mild hyperphenylalaninemia , phe level is higher than normal limits , but below 600 mol [ 1 , 2 ] . in early infants with phenylketonuria , the serum phenylalanine level is in normal limits at birth , but begins to rise within the first few hours of life . excessive phenylalanine is generally believed to be responsible for the brain insult leading to progressive mental retardation and seizure disorder . without treatment , cognitive delay becomes evident within 6 months of age and is progressive [ 1 , 2 ] . although the principal biochemical defect is obvious in inborn errors of metabolism such as pku and other similar disorders , the exact neurodysfunction defect is not clearly definite [ 13 ] . clinical manifestations in untreated infants with pku include growth failure , microcephaly , seizures , dermatitis , and intellectual defect . remarkable clinical findings are light colored skin , hair and eyes , eczematous rash and behavioral disorders [ 14 ] . different studies reported 25% generalized or partial seizures in phenylketonuria . long eeg recording can detect abnormal epileptic discharge with or without clinical seizure or convulsion . eeg background activity is categorized into normal and abnormal background activity which is classified into type 1 and type 2 of continuous and hyperactive rapid tracing and in highly abnormal background activity , inactive , paroxysmal and low voltage plus theta activity . in this study , we evaluated the association between eeg abnormality and developmental delay as well as behavioral disorders in patients diagnosed as phenylketonuria . in this case - control study , 105 pku patients ( 50 male , 55 female ; mean age , 8.5 6.2 years ) who had been referred to the neurometabolic clinic of mofid children hospital , tehran , iran from 2010 to 2011 were evaluated . these patients were diagnosed as pku patients through newborn screening tests or later during follow - up evaluation for neurodevelopmental delay at the hospital . the diagnosis of classic pku was made on the basis of serum phe level higher than 6 mg / dl in untreated infants , ( we used mg / dl for measuring serum phe level . ) , the patients were matched according to sex , age , and the serum phenylalanine level . then all the patients were referred to the eeg laboratory for eeg recording ( after sleep deprivation for 10 to 12 hours ) . pediatric neurologists took the history of seizures and all the patients who had recent seizures were excluded and only patients who were seizure - free for at least six months before the study were enrolled . based on eeg recording , the patients were divided into two groups : the case group in which the patients had an abnormal eeg and the control group which consisted of those with normal eegs . we evaluated the developmental score of the two groups with the asq questionnaire ( age and stage questionnaire ) . after estimation of developmental score in cases and controls , we detected the developmental quotient ( dq ) of patients using the below equation : ( 1)dq= developmental ageactual age 100 . we also used the test of child symptom inventory-4 ( csi-4 ) for evaluation of behavioral disorders in children . this behavior rating scale screens for dsm - iv emotional and behavioral disorders in children between 5 and 14 years of age . we evaluated generalized anxiety disorder , attention deficit , hyperactivity , attention deficit hyperactivity disorder , oppositional defiant disorder , compulsive disorder , obsession , and autistic behavioral disorder in two groups . we considered an eeg with normal background activity and mild epileptic discharge as mild abnormal eeg . eegs with an abnormal background activity and definitely abnormal by showing paroxysmal epileptic discharge were considered as moderately abnormal . severely abnormal eegs were eegs with continuous spikes and wave discharges or the hypsarrhythmia pattern . finally , the data were compared between the patients ( cases and controls ) and anova and chi - square test were applied for statistical analysis . we evaluated 105 patients ( 50 boys , 55 girls ) with the diagnosis of pku . the mean age of the patients was 8.5 6.2 years . of these pku patients , 52.3% ( 55 patients ) had seizure ( more than 6 months before the study ) and 47.7% ( 50 patients ) were clinically seizure - free . the case group consisted of 70 patients ( 66.6% ) with an abnormal eeg and the controls were 35 patients ( 33.4% ) with a normal eeg . in the case group , 37 patients ( 52.8% ) had a mild abnormal eeg , 10 patients ( 14.2% ) had moderate abnormal eeg and 23 patients ( 32.8% ) had severe abnormality in their eeg ( as we considered in our evaluation and mentioned before ) . in the case group , phe levels were 14 mg / dl ( mean , 21.4 9.1 mg / dl ) at diagnosis , 9 mg / dl ( mean , 10.3 1.6 mg / dl ) after 6 months of diagnosis , and 4 mg / dl ( mean , 7.31 4.1 mg / dl ) at the time of our evaluation . the serum phe levels in controls were 12 mg / dl ( mean , 17.3 6.1 mg / dl ) at diagnosis and from 6 mg / dl ( mean , 8.4 2.1 mg / dl ) after six months of treatment to 4 mg / dl ( mean , 7.39 3.6 mg / dl ) at the time of our study . there was no significant difference between mean phenylalanine levels in the abnormal and normal eeg groups at the time of diagnosis , after six months , and at our evaluation . our group was matched according to serum phe level ( p > 0.05 ) ( table 1 ) . in the case group ( patients with an abnormal eeg ) , 47% ( 33 patients out of 70 patients ) had no clinical seizure . there was no significant difference between age in the two groups regarding dq levels ( above and below 80 ) ( p = 0.2 ) and no significant difference in dq levels between genders ( p = 0.7 ) . comparison between head circumference ( hc ) and the dq level showed a significant difference between hc and dq level in pku patients ( p = 0.04 ) ( table 2 ) . distribution of dq levels in the abnormal and normal eeg groups ( cases and controls ) revealed a significant difference ; an abnormal eeg was associated with a higher percentage of low dq levels ( 90% versus 57.1% ) ( p = 0.001 ) ( table 3 ) . patients in the case group ( abnormal eeg ) according to severity of abnormality in eeg were categorized into three classes . distribution of high and low dq levels in these three subgroups showed a significant difference ( p = 0.001 ) ( table 4 ) . in other words , a severe abnormal eeg was associated with a higher percentage of low dq levels . in the behavioral - emotional scale evaluation , the frequency of attention deficit was 85.7% in the case group and 40% in the controls . 85.7% of the cases showed attention deficit hyperactivity disorders in comparison with 40% in the controls . oppositional defiant disorders were reported 84.2% in the case group and 34.2% in the controls . autistic behavior was detected in 57.1% of the cases and 28.5% of the controls . totally , the frequency of behavioral disorder was 85.7% in cases and 42.8% in control patients . there was a significant relationship between behavioral disorder and eeg abnormality in our study ( p < 0.001 ) ( table 5 ) . in neurometabolic diseases , neurodevelopmental delay , seizures and behavioral disturbances are the prominent presentations . according to our knowledge , different previous studies revealed the relationship between phe level and eeg abnormality and also between phe level and developmental delay , but there are limited studies about the relationship between eeg abnormality and developmental delay in pku patients . studied the effects of treatment of phenylalanine level ( special diet ) on the eeg of six pku patients . computerized eeg analysis showed activity appearance in the low frequency band ( 25 cycle / second ) , dominant rhythm frequency changes and synchronous degree changes between previous identified frequencies . this study described the association between phenylalanine level and eeg changes , when phe level increased , the eeg changes intensified and in contrast when the phe level decreased the eeg changes reversed , indicating that phe or its metabolites lead to eeg abnormalities . in our study , the case group consisted of 70 patients ( 66.6% ) with abnormal eegs . in our evaluation , we could not find any correlations between phe level and eeg changes because we matched phe level in the two groups . between patients with abnormal eegs ( the cases ) there was a significant relationship between eeg findings and phe level ( p < 0.001 ) . according to p. t. gross et al . review , in eeg recording of pku patients , about 45% of the patients had an abnormal eeg and nearly 30% had a normal eeg in the beginning which became abnormal later . study showed 25% of the patients had seizure , but more than 50% had an abnormal electroencephalogram , which means some pku patients had an abnormal eeg without any clinical seizure . in our study , 44% ( 28 patients ) of the patients with abnormal eegs ( out of 63 patients in the case group ) had no clinical seizure . karimzadeh and tabarestani reviewing study reported negative effects of this epileptic form discharge on the choice reaction time , verbal and nonverbal communication and behavioral disorder . our study revealed 85.7% of the patients in the case group and 42.8% of the children in the control group had a pattern of behavioral disorders . these behavioral disorders were in accordance with their eeg abnormalities and behavioral disorders in phenylketonuria patients regardless of seizure and phe level . in smith et study , the rutter behavior questionnaire was used for evaluation of common abnormal behaviors in 544 patients with phenylketonuria ( 8-year - old children ) . children had routine screening testing in the early months of screening program ; anxiety , hyperactivity and less responsibility were detected . all patients received treatment before 4 months . they described that these abnormal behaviors may be the result of both psychologic stress and neurologic impairment . rolle - daya et al . also studied the eeg findings of 90 patients with classic pku in one clinic . 73% of these patients who were diagnosed and were treated early had a normal eeg and 23% had mild abnormal background activity . in patients diagnosed after 6 months of age , 31% had normal eegs , 24% had abnormal background activity , and 45% had abnormal paroxysmal discharges . thirty - four children with phenylketonuria with early treatment were studied by pietz et al . they determined the prognostic value of age on the special diet in the first three months of life and iq score and eeg were investigated . higher iq scores were detected in patients with strict dietary control in comparison with lose control . generalized slowing and paroxysmal activity ( with or without spikes ) were seen more frequently compared to the controls . eeg abnormality was increased independent of iq development and eeg abnormality showed no relationship with the onset age or the quality of dietary treatment . in our present study , we found a significant difference between dq levels in the abnormal eeg group . 74.7% of the patients with abnormal eeg had a low dq level ; therefore , we had a significant relationship between abnormal eeg and the low dq levels ( table 3 ) . in addition , we found a significant correlation between severity of abnormal eegs and the level of dq in pku patients ( table 4 ) . according to our study , ( in pku patients matched in phe levels ) , there was a significant relationship between the abnormal eegs and the low dq level as well as the abnormal eegs and behavioral disorders . based on our findings , the prevalence of seizure was less than eeg abnormality in pku patients . there was a significant relevance between eeg abnormality and behavioral disorders and developmental score in phenylketonuria regardless of seizure or phenylalanine level . the authors believe that physicians should pay attention to these paroxysmal epileptic discharges in pku patients more than before and treatment of these eeg abnormalities may affect developmental scores or may lead to correction of some behavioral disorders in these patients .

background . brain defect leading to developmental delay is one of the clinical manifestations of phenylketonuria . the aim of this study was to evaluate the association between eeg abnormality and developmental delay / behavioral disorders in phenylketonuria . patients and methods . 105 phenylketonuria patients , who were diagnosed through newborn screening tests or during follow - up evaluation , were enrolled . patients who were seizure - free for at least six months before the study were included . the developmental score were evaluated by the asq questionnaire ( age - stage questionnaire ) and the test of child symptom inventory-4 ( csi-4 ) , respectively . results . 55 patients had a history of seizure more than 6 months before the study . seventy had abnormal eeg ( cases ) and 35 had normal eeg ( controls ) . there was no significant difference between mean phenylalanine levels in the abnormal and normal eeg groups at the time of diagnosis , after six months and at our evaluation . distribution of dq level in the abnormal and normal eeg groups revealed a significant difference . an abnormal eeg was associated with a higher percentage of low dq levels . conclusion . paroxysmal epileptic discharges in pku patients are important . treatment of these eeg abnormalities may affect developmental scores or may lead to correction of some behavioral disorders in patients .

leishmaniasis is a vector borne disease , affecting 98 countries and territories around the world which is transmitted by the bite of the female sandfly phlebotomine ( who , 2010 ) . this disease causes a considerable morbidity and mortality in tropical and sub - tropical countries . leishmaniasis presents with four major clinical forms : visceral , cutaneous , diffuse cutaneous and mucocutaneous leishmaniasis . 90% of the cases occur in seven countries including iran , afghanistan , peru , saudi arabia , syria , algeria , and brazil ( desjeux , 2004 ) . it has been estimated that each year 1 - 1.5 million new cases of cl and 500,000 cases of visceral leishmaniasis occur . overall , the prevalence is 12 million and the population at risk is 350 million ( desjeux , 2004 ) . the epidemiology of cl has been significantly changed , mainly because of ecological factors , population displacements and individual risk factors ( desjeux , 2001 ) . vector and reservoir control methods are difficult due to the diverse ecology of many species of sandfly vectors and reservoirs . the standard drugs , pentavalent antimonials , have been used for decades with severe side - effects . moreover , prolonged use and resistant forms have been frequently reported ( hadigi et al . , 2006 ) . in iran two epidemiological forms of cl are present : zoonotic cl ( zcl ) caused by l. major and anthroponotic cl ( acl ) caused by leishmania tropica . the latter species are prevalent in kerman province ( sherifi et al . , 2012 and nadim et al . , 1995 ) and have developed resistance to antimonials ( hadigi et al . , 2006 ; pour et al . , 2010 ) . in the present study , we characterized the ultrastructure alteration of different cell organelles in glucantime unresponsive l. tropica isolates compared with standard strains or sensitive isolates . the evaluation of resistant isolates is important because it permits the development of diagnostic tools for early recognition of unresponsiveness , thus avoiding inefficient and often toxic chemotherapy . to the best of our knowledge , there is no previous study that has been done on electron microscopic changes in unresponsive and sensitive isolates of l. tropica in clinical settings . this study is experimental and was done in the city of bam during 2008 - 2012 in kerman province , south - eastern iran . bam is a well - known focus for acl , where vaccine trials of single and multiple doses of l. major against l. tropica were conducted ( noazi et al . , 2008 , 2009 ) . a devastating earthquake had struck bam in 2003 destroying nearly 90% of medical and social infrastructures . ever since , the mean annual incidence of acl has significantly increased in this region ( sharifi et al . , 2011 ) . the samples were taken from 66 patients who were referred to the cl treatment center in bam . a questionnaire was completed for each individual , recording their demographic characteristics and cl status including age , sex , area of residence , number , type and location of cl lesions , and history of the disease . consent of the patient was obtained and all patients received proper medication free of charge . the scraping smears provided from the edge of active lesions with sterile blades were fixed with methanol , stained by giemsa , and examined under a compound light microscope for amastigote form simultaneously . skin scrapings were inoculated for mass production of promastigotes into nnn biphasic medium and incubated at 241c for 7 days . promastigotes were then sub - cultured in rpmi 1640 medium ( gibco ) containing 15% of heat - inactivated fetal calf serum ( gibco ) with streptomycin ( 200 mg / ml ) , and penicillin ( 200 units / ml ) ( gibco ) , and incubated at 241c . the cultures were checked every 7 days for promastigote growth for up to 28 days ( sharifi et al . , 1999 ) . diagnosis was confirmed by smear and culture media , while identification of species was carried out by nested - pcr , as it was previously performed elsewhere ( unpublished data ) based on national guidelines . resistant case was referred to those cl - infected patients who did not heal after receiving two full courses of treatment by glucantime . on the other hand , a sensitive case was an infected individual who often responded to a full course of glucantime . the l. tropica standard strain ( mhom / ir/02/mash2 ) was provided by the center for research and training in skin diseases and leprosy , tehran university of medical sciences , tehran , iran . to prepare the specimens for transmission electron imaging , promastigotes were fixated with 3% cold glutaraldehyde ( merck chemical co. ) for at least half an hour . the samples were centrifuged and resuspended in 0.1 m sodium cacodylate buffer ( ph 7.2 ) . then , it was again centrifuged , the supernatant was discarded , and resuspended in 3% agar 50c . the promastigotes were further fixed in 1% osmium tetroxide for 2 hours , washed with buffer , and dehydrated through series of ethanol concentrations . using an ultramicrotome , the blocks were cut into 1 m sections , then stained by toluidine blue and were examined under a light microscope to confirm the preparation success ( oryan and mehrabani , 2007 ) . the rest of the electron microscopic sample preparation and imaging were done at janelia research campus , howard hughes medical institute ( hhmi ) . the imaging at hhmi was performed with a scanning transmission electron microscope ( stem ) detector . this detector was developed utilizing a carl zeiss , ultra 55 zeiss schottky field emission sem system , resulting a low voltage ( 2030 kv , which is a few times smaller than the voltage used in conventional tem ) high throughput stem ( lvstem ) . the detector is based on a scintillator and photo - multiplier tube technology and the detector 's optics geometry gets the highest signal to noise ratio in an image ( bolorizadeh , 2009 ) . furthermore , the column design of the sem , having no cross over point and having a capability of upgrading to high current modes ( tens of na ) , made it an ideal candidate for this study . electron microscopic studies prior to imaging were prepared with 40 nm pioloform substrates on 0.5 mm tem slot grids . then , 5 nm carbon was thermally evaporated onto the top surface ( the side facing of primary electron beam ) of the tem grids , to eliminate any charging during the electron beam imaging . to make the grids more hydrophilic , the grids were glow discharged ( using nitrogen dc plasma ) prior to laying down the sections on the grid slots . immediately after the grid substrate preparation , the resin blocks were cut into 40 nm thick sections using an ultramicrotome and laid on the prepared tem slot grids . these sections were post - stained with 1% osmium tetroxide at room temperature for 30 minutes . all the images in this paper were taken with a 30 kev , 16 na electron beam at 10 mhz bandwidth ( 0.1 sec per pixel ) without any image noise reduction and alteration . our ultrastructural findings in unresponsive promastigote revealed that the cytoplasmic mitochondrion was mostly circular [ figure 1a ] . the cytoplasmic microtubules were seen in the middle of the promastigote [ figure 1a ] . the nucleus was bigger than normal and located close to the posterior end of the kinetoplast . the kinetoplast , which is enlarged in figure 1c , was not degenerated as in the sensitive strain the plasma membrane was smooth with normal thickness [ figure 1a and b ] . ( b ) nucleus became swollen , nuclear chromatin fragmented and dispensed instead of rimming around nuclear membrane . n , nucleus ; k , kinetoplast ; v , vesicle ; m , mitochondrion ; f , flagellum ; mi , microtubule ; pm , plasma membrane ; v , vacuole ; fp , flagellar pocket . horizontal bars are 3 m while the vertical bar is 1m the ultrastructural finding in sensitive promastigote revealed the cytoplasmic mitochondrion that was swollen , elongated and located at a short distance from the plasma membrane of the promastigote [ figure 2a ] . the nucleus became elongated and the nuclear chromatin was fragmented and dispensed instead of rimming around the nuclear membrane . its location was at a further distance from the kinetoplast at the posterior end of the organism as compared with the standard or unresponsive isolates as compared with the standard [ figure 3 ] or unresponsive isolates [ figure 1 ] . ultrathin sections of sensitive strain of promastigote forms of leishmania tropica revealed different ultrastructural alteration induced by treatment with meglumine antimoniate ( glucantime ) . ( a ) nucleus became elongated ; nuclear chromatin became fragmented and dispensed instead of rimming around nuclear membrane , mitochondrial swelling , loss of its matrix and cristae . bars are 3 m electron microscopy of standard sample showing : ( a ) few vesicles scattered throughout the cytoplasm . ( b ) normal mitochondria , ( c ) mitochondrion with normal matrix and cristae the mitochondrion - kinetoplast 's complex loss , its matrix and cristae , condensation of dna core and disintegration into a complex of thin filaments [ figure 2b and d ] . meglumine treatment induced intense vesicles which could be seen around and under the nucleus , mostly at the posterior end of the promastigote [ figure 2c ] . intense vacuolization of promastigote was observed in sensitive patients [ figure 2c and e ] . the vacuoles which were not seen in the standard stain were visible close to the kinetoplast and flagellum and at the posterior end of the promastigote [ figure 2c ] . the ultrastructural findings of standard sample revealed few vesicles which were located throughout the cytoplasm [ figure 3a ] and also at the posterior end of the promastigote stage [ figure 3b ] . the nucleus had a normal size and was located at a short distance posterior to the kinetoplast . the cytoplasmic mitochondria were circular and located throughout the promastigote [ figure 3a and b ] . the cytoplasmic microtubules were located at a short distance from the cytoplasmic membrane [ figure 3a ] . the results discussed for unresponsive , sensitive , and standard promastigotes are given in table 1 . this study is experimental and was done in the city of bam during 2008 - 2012 in kerman province , south - eastern iran . bam is a well - known focus for acl , where vaccine trials of single and multiple doses of l. major against l. tropica were conducted ( noazi et al . , 2008 , 2009 ) . a devastating earthquake had struck bam in 2003 destroying nearly 90% of medical and social infrastructures . ever since , the mean annual incidence of acl has significantly increased in this region ( sharifi et al . , 2011 ) . the samples were taken from 66 patients who were referred to the cl treatment center in bam . a questionnaire was completed for each individual , recording their demographic characteristics and cl status including age , sex , area of residence , number , type and location of cl lesions , and history of the disease . consent of the patient was obtained and all patients received proper medication free of charge . the scraping smears provided from the edge of active lesions with sterile blades were fixed with methanol , stained by giemsa , and examined under a compound light microscope for amastigote form simultaneously . skin scrapings were inoculated for mass production of promastigotes into nnn biphasic medium and incubated at 241c for 7 days . promastigotes were then sub - cultured in rpmi 1640 medium ( gibco ) containing 15% of heat - inactivated fetal calf serum ( gibco ) with streptomycin ( 200 mg / ml ) , and penicillin ( 200 units / ml ) ( gibco ) , and incubated at 241c . the cultures were checked every 7 days for promastigote growth for up to 28 days ( sharifi et al . , 1999 ) . diagnosis was confirmed by smear and culture media , while identification of species was carried out by nested - pcr , as it was previously performed elsewhere ( unpublished data ) based on national guidelines . resistant case was referred to those cl - infected patients who did not heal after receiving two full courses of treatment by glucantime . on the other hand , a sensitive case was an infected individual who often responded to a full course of glucantime . the l. tropica standard strain ( mhom / ir/02/mash2 ) was provided by the center for research and training in skin diseases and leprosy , tehran university of medical sciences , tehran , iran . to prepare the specimens for transmission electron imaging , promastigotes were fixated with 3% cold glutaraldehyde ( merck chemical co. ) for at least half an hour . the samples were centrifuged and resuspended in 0.1 m sodium cacodylate buffer ( ph 7.2 ) . then , it was again centrifuged , the supernatant was discarded , and resuspended in 3% agar 50c . the promastigotes were further fixed in 1% osmium tetroxide for 2 hours , washed with buffer , and dehydrated through series of ethanol concentrations . using an ultramicrotome , the blocks were cut into 1 m sections , then stained by toluidine blue and were examined under a light microscope to confirm the preparation success ( oryan and mehrabani , 2007 ) . the rest of the electron microscopic sample preparation and imaging were done at janelia research campus , howard hughes medical institute ( hhmi ) . the imaging at hhmi was performed with a scanning transmission electron microscope ( stem ) detector . this detector was developed utilizing a carl zeiss , ultra 55 zeiss schottky field emission sem system , resulting a low voltage ( 2030 kv , which is a few times smaller than the voltage used in conventional tem ) high throughput stem ( lvstem ) . the detector is based on a scintillator and photo - multiplier tube technology and the detector 's optics geometry gets the highest signal to noise ratio in an image ( bolorizadeh , 2009 ) . furthermore , the column design of the sem , having no cross over point and having a capability of upgrading to high current modes ( tens of na ) , made it an ideal candidate for this study . electron microscopic studies prior to imaging were prepared with 40 nm pioloform substrates on 0.5 mm tem slot grids . then , 5 nm carbon was thermally evaporated onto the top surface ( the side facing of primary electron beam ) of the tem grids , to eliminate any charging during the electron beam imaging . to make the grids more hydrophilic , the grids were glow discharged ( using nitrogen dc plasma ) prior to laying down the sections on the grid slots . immediately after the grid substrate preparation , the resin blocks were cut into 40 nm thick sections using an ultramicrotome and laid on the prepared tem slot grids . these sections were post - stained with 1% osmium tetroxide at room temperature for 30 minutes . all the images in this paper were taken with a 30 kev , 16 na electron beam at 10 mhz bandwidth ( 0.1 sec per pixel ) without any image noise reduction and alteration . this study is experimental and was done in the city of bam during 2008 - 2012 in kerman province , south - eastern iran . bam is a well - known focus for acl , where vaccine trials of single and multiple doses of l. major against l. tropica were conducted ( noazi et al . , 2008 , 2009 ) . a devastating earthquake had struck bam in 2003 destroying nearly 90% of medical and social infrastructures . ever since , the mean annual incidence of acl has significantly increased in this region ( sharifi et al . , 2011 ) . the samples were taken from 66 patients who were referred to the cl treatment center in bam . a questionnaire was completed for each individual , recording their demographic characteristics and cl status including age , sex , area of residence , number , type and location of cl lesions , and history of the disease . consent of the patient was obtained and all patients received proper medication free of charge . the scraping smears provided from the edge of active lesions with sterile blades were fixed with methanol , stained by giemsa , and examined under a compound light microscope for amastigote form simultaneously . skin scrapings were inoculated for mass production of promastigotes into nnn biphasic medium and incubated at 241c for 7 days . promastigotes were then sub - cultured in rpmi 1640 medium ( gibco ) containing 15% of heat - inactivated fetal calf serum ( gibco ) with streptomycin ( 200 mg / ml ) , and penicillin ( 200 units / ml ) ( gibco ) , and incubated at 241c . the cultures were checked every 7 days for promastigote growth for up to 28 days ( sharifi et al . , 1999 ) . diagnosis was confirmed by smear and culture media , while identification of species was carried out by nested - pcr , as it was previously performed elsewhere ( unpublished data ) based on national guidelines . resistant case was referred to those cl - infected patients who did not heal after receiving two full courses of treatment by glucantime . on the other hand , a sensitive case was an infected individual who often responded to a full course of glucantime . the l. tropica standard strain ( mhom / ir/02/mash2 ) was provided by the center for research and training in skin diseases and leprosy , tehran university of medical sciences , tehran , iran . to prepare the specimens for transmission electron imaging , promastigotes were fixated with 3% cold glutaraldehyde ( merck chemical co. ) for at least half an hour . the samples were centrifuged and resuspended in 0.1 m sodium cacodylate buffer ( ph 7.2 ) . then , it was again centrifuged , the supernatant was discarded , and resuspended in 3% agar 50c . the promastigotes were further fixed in 1% osmium tetroxide for 2 hours , washed with buffer , and dehydrated through series of ethanol concentrations . the samples were embedded in agar-100 resin and baked at 65c for 24 hours . using an ultramicrotome , the blocks were cut into 1 m sections , then stained by toluidine blue and were examined under a light microscope to confirm the preparation success ( oryan and mehrabani , 2007 ) . the rest of the electron microscopic sample preparation and imaging were done at janelia research campus , howard hughes medical institute ( hhmi ) . the imaging at hhmi was performed with a scanning transmission electron microscope ( stem ) detector . this detector was developed utilizing a carl zeiss , ultra 55 zeiss schottky field emission sem system , resulting a low voltage ( 2030 kv , which is a few times smaller than the voltage used in conventional tem ) high throughput stem ( lvstem ) . the detector is based on a scintillator and photo - multiplier tube technology and the detector 's optics geometry gets the highest signal to noise ratio in an image ( bolorizadeh , 2009 ) . furthermore , the column design of the sem , having no cross over point and having a capability of upgrading to high current modes ( tens of na ) , made it an ideal candidate for this study . electron microscopic studies prior to imaging were prepared with 40 nm pioloform substrates on 0.5 mm tem slot grids . then , 5 nm carbon was thermally evaporated onto the top surface ( the side facing of primary electron beam ) of the tem grids , to eliminate any charging during the electron beam imaging . to make the grids more hydrophilic , the grids were glow discharged ( using nitrogen dc plasma ) prior to laying down the sections on the grid slots . immediately after the grid substrate preparation , the resin blocks were cut into 40 nm thick sections using an ultramicrotome and laid on the prepared tem slot grids . these sections were post - stained with 1% osmium tetroxide at room temperature for 30 minutes . all the images in this paper were taken with a 30 kev , 16 na electron beam at 10 mhz bandwidth ( 0.1 sec per pixel ) without any image noise reduction and alteration . our ultrastructural findings in unresponsive promastigote revealed that the cytoplasmic mitochondrion was mostly circular [ figure 1a ] . the cytoplasmic microtubules were seen in the middle of the promastigote [ figure 1a ] . the nucleus was bigger than normal and located close to the posterior end of the kinetoplast . the kinetoplast , which is enlarged in figure 1c , was not degenerated as in the sensitive strain the plasma membrane was smooth with normal thickness [ figure 1a and b ] . ( b ) nucleus became swollen , nuclear chromatin fragmented and dispensed instead of rimming around nuclear membrane . n , nucleus ; k , kinetoplast ; v , vesicle ; m , mitochondrion ; f , flagellum ; mi , microtubule ; pm , plasma membrane ; v , vacuole ; fp , flagellar pocket . horizontal bars are 3 m while the vertical bar is 1m the ultrastructural finding in sensitive promastigote revealed the cytoplasmic mitochondrion that was swollen , elongated and located at a short distance from the plasma membrane of the promastigote [ figure 2a ] . the nucleus became elongated and the nuclear chromatin was fragmented and dispensed instead of rimming around the nuclear membrane . its location was at a further distance from the kinetoplast at the posterior end of the organism as compared with the standard or unresponsive isolates as compared with the standard [ figure 3 ] or unresponsive isolates [ figure 1 ] . ultrathin sections of sensitive strain of promastigote forms of leishmania tropica revealed different ultrastructural alteration induced by treatment with meglumine antimoniate ( glucantime ) . ( a ) nucleus became elongated ; nuclear chromatin became fragmented and dispensed instead of rimming around nuclear membrane , mitochondrial swelling , loss of its matrix and cristae . bars are 3 m electron microscopy of standard sample showing : ( a ) few vesicles scattered throughout the cytoplasm . ( b ) normal mitochondria , ( c ) mitochondrion with normal matrix and cristae the mitochondrion - kinetoplast 's complex loss , its matrix and cristae , condensation of dna core and disintegration into a complex of thin filaments [ figure 2b and d ] . meglumine treatment induced intense vesicles which could be seen around and under the nucleus , mostly at the posterior end of the promastigote [ figure 2c ] . intense vacuolization of promastigote was observed in sensitive patients [ figure 2c and e ] . the vacuoles which were not seen in the standard stain were visible close to the kinetoplast and flagellum and at the posterior end of the promastigote [ figure 2c ] . the ultrastructural findings of standard sample revealed few vesicles which were located throughout the cytoplasm [ figure 3a ] and also at the posterior end of the promastigote stage [ figure 3b ] . the nucleus had a normal size and was located at a short distance posterior to the kinetoplast . the cytoplasmic mitochondria were circular and located throughout the promastigote [ figure 3a and b ] . the cytoplasmic microtubules were located at a short distance from the cytoplasmic membrane [ figure 3a ] . the results discussed for unresponsive , sensitive , and standard promastigotes are given in table 1 . this electron microscopic study was done to evaluate the effect of drugs on l. tropica parasite due to the development of drug resistance . we demonstrated the effect of glucantime on various l. tropica promastigote organelles to provide a better understanding of drug - resistance . the results indicated that meglumine acted mainly on the mitochondrial physiology of this species which was distributed in branches close to the promastigote membrane and in the region rich in dna called kinetoplast ( de souza , 2009 ) . our ultrastructural observation of the mitochondria and kinetoplast showed disintegration into a complex of thin filaments , condensation of dna core , loss of its matrix and cristae . similar findings were observed for l. amazonesis with different drugs ( rodrigues et al . , 2005 , 2007 , mitochondria are essential organelles in energy metabolism , oxidative phosphorylation , and respiration . they also have a vital role in the survival of the organism and are an exceptionally chemotherapeutic target ( job et al . , 2012 ) . another deleterious effect was also observed on the microtubules , they disappeared in sensitive promastigote . the microtubules play important roles in intracellular transport , morphogenesis , and maintenance of cellular shape , and are involved in mitosis ( bell , 1998 ) . the extensive increase in vacuole size and number in sensitive strains indicated the cessation of the cell cycle which is consistent with previous findings by de macedo and his colleagues ( 2011 ) . other ultrastructural alteration was structural changes in the nucleus including nuclear chromatin which was fragmented and dispensed instead of rimming around the nuclear membrane and became elongated . in a study that was done by de macedo and his colleagues ( 2011 ) , they observed many changes in mitochondria , nucleus , vesicles and mitochondrion - kinetoplast complexes in l. amazonensis treated with amiodarone drugs . a study by rodrigues and colleagues ( 2008 ) showed the effect of drugs on the organelles of l. amazonensis as alteration of mitochondrion such as swelling , loss of cristae and matrix content . they also observed a large vacuole close to the flagellar pocket and small ones inside it . we also observed intense vacuolization of promastigotes , marked swelling of cytoplasmic mitochondrion and its elongation . all of these alterations in sensitive promastigotes are characteristics of cell death : necrosis and apoptosis . similar findings were observed by de macedo and his colleagues ( 2011 ) who showed the effect of amio drugs on the organelles of l. amazonensis . it seems that resistant forms get more amastigote like structure and more atrophic but viable organelles close to each other for better hemostasis of the parasites . kinetoplasts are the most important damageable organelles . with respect to ultrastructural findings in l. tropica , so far , no study has been done on l. tropica isolates unresponsive to glucantime . to the best of our knowledge , this study is the only investigation performed on ultrastructure of unresponsive and sensitive isolates . the significance and relevance of these changes might help understand drug resistance patterns and help localize the best target site for inactivating the organism . further , ultrastructural studies from skin biopsies of different forms of cl are needed to evaluate in vivo changes of amastigotes . kinetoplasts are the most important damageable organellesmicrotubules play important roles in surveillance of leishmania . kinetoplasts are the most important damageable organelles microtubules play important roles in surveillance of leishmania .

background : the control of leishmaniasis faces serious challenges because of resistance to the first - line antimonial drugs . we aimed to evaluate the differences in organelle changes of cultivated promastigotes obtained from skin lesions of sensitive and unresponsive isolates to meglumine antimoniate ( glucantime ) by electron microscopy.material and methods : this study was done in bam city , southeastern iran , in which the incidence of disease has sharply increased since the earthquake in 2003 . the samples were taken from 66 patients who were referred to the cutaneous leishmaniasis ( cl ) treatment center in bam . a questionnaire was completed for each individual , recording their demographic characteristics and cl status . the scraping smears provided from the edge of active lesions with sterile blades were fixed with methanol , stained by giemsa , and examined under a compound light microscope for amastigote form simultaneously . to prepare the specimens for transmission electron imaging , promastigotes were centrifuged and resuspened.results:transmission electron microscopic study of the cultivated promastigotes revealed that there were alterations in the organelles and structures of sensitive isolates compared with unresponsive and standard ones . organelles and structures such as mitochondria , kinetoplast , microtubules , cytoplasmic vacuoles , plasma membrane and vesicles were studied . the alterations such as disintegration of kinetoplast into thin filaments and condensation of kinetoplast dna core , changes in size , number and location of vesicles and microtubules were observed . we noted intense cytoplasmic vacuolization , and considerable swelling of mitochondria.conclusion:the significance and relevance of these changes might help understand drug resistance patterns and help localize the best target site for inactivating the organism .

industrial growth , technological advancements , and higher living standards in today 's society contribute to the ever increasing generation of solid wastes . industrial park is one of the most important manufacturing bases and an indicator of development in each country . however , each manufacturing process usually generates some amount of nonconsumable waste causing adverse effects on environment . geographical information system ( gis ) technologies are effectively used in the process of site selection , which is a spatial problem . despite the consensus of all countries to achieve the goal of zero waste in industries , the accomplishment of this goal is predicted to be difficult and generation of various wastes arising from manufacturing processes is currently inevitable . a focus on sources of solid waste with respect to management is justified by the fact that waste characteristics and composition differ according to source . the appearance of such problems caused some countries to move their waste to developing countries in order to be saved from the probable risks of the hazardous waste . uncontrolled expansion of industries and ignoring environmental principles in industrial development and overuse have caused much environmental problems . a number of countries have ratified state and national legislation for managing and controlling hazardous waste whose main purpose is to minimize the potential risks of industrial hazardous waste for humans and the environment . in iran , despite the excess growth of industries , urbanization , and industrial centralization around major cities particularly tehran , no fundamental effort has been yet made in the field of industrial waste disposal , and subsequently , there are few rules for controlling and disposal of such materials . evidence shows that solid , semisolid , and liquid wastes of the factories are disposed regardless of health issues and effluent of these industries enters the absorbing wells or spread in surrounding land . solid waste is piled up around the factories for a period , and then a part of the waste is incinerated outdoor and some part is transferred out of the factories along with household waste by municipal general service . large scale land use transportation from agricultural to industrial has made tehran a city for a large center for industries centralization and their peripheral activities . because of the importance of industrial hazardous waste minimization in prevention of pollutant emissions in the environment , it is emphasized by current laws in many countries . moreover , establishment and development of minimization programs have been adopted more than ever through legislation of laws and more restrictions in controlling hazardous waste and prevention of leaving waste in the environment . the application of waste minimization techniques does not necessarily mean the use of complicated technologies or investments on expensive machines . many methods require a simple change in the process of production or in transferring the materials . generally , hazardous waste minimization methods can be used for any production process and the mutual factor in all techniques is the reduction of production costs . in this study , basic and technical information required for the waste management were collected and analyzed due to the increasing growth of industries and the need for developing efficient methods in proportion to the conditions of the studied region . in fact , the main objective of this study is to examine the quantity of industrial solid waste regarding the production line of cb province , iran . the population of this study included all operational industries in 19 industrial zones of cb province , iran , which were studied for 8 months from march to october , 2009 . data were collected partly through questionnaires and partly through field investigation and literature review on the subject on matter , which was carried out using various books and articles . industrial zones of the province were identified in order to specify the production resources . in this regard , detailed information on the industrial zones was taken from the iran small industries and industrial parks organization , in cb province . metal industries with 95 units and electricity and electronics industries with 10 units had maximum and minimum numbers of units in the province , respectively . the first stage of the study including separation and classification of the industrial units was done based on their activities and locations according to the available statistics in the province . the questionnaire contained type of industrial group , the number of personnel , the kind of product and raw materials , the amount and type of generated waste , waste physical state , frequency of generation , method of storage and maintenance , recycling methods , final disposal , and the responsible organizations for waste collection in order to identify industrial waste in cb province . various questionnaires presented by different organizations such as recycle organization , environmental protection organization , and ministry of industries were analyzed and finally a questionnaire containing two forms was prepared . according to the investigations , 19 industrial parks with the total area of 2356.10 hectares were under the coverage of industrial parks organization . among these 19 industrial parks , shahrekord , lordegan , saman , faradnbeh , junghan , farokhshahr , ben , and dastgerd industrial parks had 154 , 22 , 16 , 1 , 2 , 3 , 4 , and 3 units , respectively . therefore , shahrekord with 154 units ( or 49.8% ) and faradnbeh with 1 unit ( or 0.3 percent ) had the most and the least units , respectively . a number of 309 questionnaires were completed by the industrial units . generally , these industries performed various activities , so the amount of industrial waste was influenced by the kind of activities . in this regard , these industries were divided according to the classification of ministry of industries . food , metal , chemical , nonmetallic minerals , cellulose , textile , and electrical and electronics industries comprised 21 , 30.4 , 22 , 12.6 , 7.1 , 3.8 , and 2.9 percent of the evaluated industries in this study . the workers of these 14 industrial parks were 6033 people . based on table 1 , the total industrial waste was 1246 tons per month , and semihousehold waste per capita ( for food consumption of the workers ) was 350 gram per day . table 2 shows that the amount of wastes generated in cb province was approximately 32 ton per month . according to table 3 , the total food waste generated in studied industries was an average of 65516 kg per month which depended on the number of workers . herbal waste was 384 tons a month of which 99.4% and 0.6% were generated in food and chemical industries , respectively . herbal waste of food industries encompassed wheat , straw , bran , fruit , vegetable , and dried fruit waste . herbal waste of chemical industries was related to yarn and fabric dyeing industries . in these industries , flour and doughy waste was generated in industries like macaroni manufacturing , noodle making , and industrial bread . the amount of flour and doughy waste was 22.3 tons per month in the province which was totally generated in food industries . sweets , chocolate , and candy waste was 6.5 tons per month which was totally generated and recycled in food industries . meat and bone waste encompassed chicken and fish packaging waste and sausage production waste which was totally generated in food industries . according to investigations , wood waste was generated in metal , chemical , cellulose , and electricity and electronics industries in a form of wooden pallets which entered the factories along with purchase of tools like the engines of various machines and variety of metal ingots . the amount of wood waste was 17 tons per month , of which the maximum amount of 55.2 percent was related to metal industries . the amounts of plastic waste generated in food , metal , nonmetallic minerals , cellulose , textiles , and electrical and electronics industries were 40.8 , 1.9 , 0.8 , 0.3 , 0.3 , and 2.2 percent , respectively . these types of waste were recycled in chemical industries ; however , no action has been yet done for recycling these wastes by other industries . the amount of glass waste in the entire study was 8.2 tons per month of which 91.8 , 6 , and 2.2 percent were generated in nonmetallic minerals , cellulose , and food industries , respectively . the amount of paper and cardboard waste was 84.5 tons per month of which 83.1 percent was generated in cellulose industries and 12.2 percent was generated in food and other industries . ferrous metals waste included iron chips , iron scraps , and snipping which was 160 tons per month of which 94.8 , 6 , and 0.3 percent were generated in metal and cellulose industries , respectively . nonferrous metal waste included aluminum , copper , and cast iron of which the maximum amount was related to electricity and electronics industries for the use of aluminum and copper in wire and cable industry . the amount of nonferrous metal waste in studied samples was 5.7 tons per month of which 52.2 , 31.4 , 8.7 , 1.2 , and 3.5 percent were generated in electricity and electronics , metal , cellulose , nonmetal , and food industries , respectively . gunny waste consisting of sugar and flour sacks generated 4003 kg per month of which 81.9 , 15.6 , and 2.5 percent were generated from food , chemical , and metal industries . it should be noted that 100 percent of these wastes were reused and exploited in the same industries . trash waste included concrete , mosaics , ceramics , and stone body waste which were totally generated in nonmetal industries . the amount of generated pvc waste in the studied zones was 2.5 tons per month of which chemical , and electricity and electronics industries comprised 2300 kg ( 91.6 percent ) and 70 kg ( 2.8 percent ) , respectively . the amount of generated pet waste was 18 tons ( 93.7 percent ) per month of which 200 kg ( 1.1 percent ) and 935 kg ( 5.2 percent ) were generated in metal and food industries , respectively . the amount of polystyrene waste was 5.8 tons per month which was totally generated in chemical industries . the amount of polypropylene waste was 6.2 tons per month of which 96.8 percent and 3.2 percent were generated in chemical and textile industries , respectively . soil waste consisting of wheat soil and bentonite clay was used for vinegar treatment in industrial vinegar - making and stabilization of edible oils . furthermore , burnt sand waste ( co2 silica sand co2 waste ) is contained in soil waste . the amount of soil waste generated in the province was approximately 130 tons per month of which 96.6% and 3.4% were generated in food and metal industries , respectively . as noted above , burnt sand waste was classified in this category and was generated in cast iron foundry industry . the amount of yarn and fabric waste in all studied zones was about 7.4 tons a month . these wastes included yarn and fabrics , wool , fiber , and polymer of which 93.9 percent and 6.1 percent were generated in textile and chemical industries . the amount of chemical and special waste in all studied zones was about 21 tons per month of which 3 , 41 , 36.9 , 16.6 , 2 , and 4 percent were generated in metal , chemical , nonmetal , cellulose , textile , and electricity and electronics industries . the initial chemical waste produced from industries which produced detergents and disinfectants comprised 1 percent of the raw chemical materials . these wastes consisted of sulfuric acid , diethanolamine , coconut fatty acid , betaine , essence , resin , sulfate , salt , and so forth . the amount of these wastes was 1775 kg per month which comprised 8.1 percent of the total chemical waste . the amount of color waste was 581 kg per month which comprised 2.6 percent of the total special waste . it should be noted that this type of waste was found in metal , chemical , cellulose , and textile industries . the amount of adhesives waste was 172 kg per month which was related to chemical and cellulose industries and was most generated in card board industry . a study on the industries of industrial parks in other region of iran showed that 250 kg of waste containing degradable materials and scrap iron was transferred out of the city and incinerated outdoor by municipal service . overestablishment of industries and conversion of agricultural land to industrial land in tehran , iran , have changed the city to a large center for industries centralization and their peripheral activities . in this study , the maximum amount of generated sludge waste was 8 tons per month which was related to nonmetal industries that was generated in a mosaic - building industrial unit in boroujen industrial zone . the tar waste generated in the province was 220 kg per month which was related to the waterproof membrane industries . the zinc oxide waste was 35 kg per month which comprised 0.1 percent of total special waste in the studied industrial parks . moreover , zinc clay waste was generated by a zinc ingot production cooperative in shahrekord . it was 600 kg per month which comprised 2.7 percent of total chemical and special waste . an ideal waste disposal site is that one which is located fairly close to the source of the waste , has easy transportation access , is not located in a low - lying area or floodplain , and is underlain by geologically stable , strong , and competent rock material . the site selection depends on several factors like land use , environmental , hydrology , socioeconomic , and so forth . according to the literature , the following criteria can be applied for site selection : site must be close to at least a street with a buffer of 30 m;site must not be too far from a transfer station;site must be 3 km from residential areas , with the exception of areas with barriers;there should be a minimum distance of 100 m between site and roads;site must be on a suitable soil;site should be constructed in areas which do not have an important economic or ecological value.such kinds of criteria could be combined in a hierarchal structure shown in figure 1 . site must be close to at least a street with a buffer of 30 m ; site must not be too far from a transfer station ; site must be 3 km from residential areas , with the exception of areas with barriers ; there should be a minimum distance of 100 m between site and roads ; site must be on a suitable soil ; site should be constructed in areas which do not have an important economic or ecological value . the combination of gis and multicriteria decision models ( mcdm ) can be useful in this regard . such approach provides flexible methods for exploring relationships among geographic data and assisting experts from diverse fields in pooling their knowledge to solve complex problems . a list of potential sites which generally satisfy the minimum requirements can be identified for the purpose of effective sanitary landfill - type waste disposal . among these areas , the integration of gis in multicriteria decision analysis ( mcda ) is a powerful tool in solving disposal site selection problem , because it provides efficient spatial data manipulation and presentation . one of the most important issues in industrial waste management is its special waste which distinguishes the industrial waste from the general waste . the waste of this category includes chemical raw materials , color , adhesives , tar , zinc oxide , zinc clay , and sludge . the total generated waste in the studied industrial zones was 1246 tons per month , of which 48.2 , 14.9 , 6.7 , 22 , 0.9 , 0.6 , and 6.5 percent were generated in food , metal , chemical , nonmetallic minerals , textile , electrical and electronics , and cellulose industries , respectively . the results achieved in this study are considered the first step toward proper industrial waste management especially in recycling and disposal of waste . according to the results of this study , environmental general department recommended to use rewards and punishments for reduction of hazardous waste . coordination with financial institutions for giving loans with lower interests in order to take environmental actions or imposing large fines for each kilogram of generated hazardous waste gives instances of reward and punishment . collection , delivery , and disposal of hazardous waste should be done by private section and the officials of environmental general department supervise the performance of the private section . costs of monitoring water , soil , and weather analyses in hazardous waste disposal site should be adopted from pollutant industries and the environmental report on the landfill should be submitted to environmental department every three months . regarding fragile ecosystem of cb province , layout and extension of industrial parks in future finally , an approach to the integration of gis and mcdm was introduced as a powerful tool in solving disposal site selection problem considering several criteria .

the aim of this study is the comprehensive planning for integrated management of solid waste at the industrial parks . the share of each industrial group including food , metal , chemical , non - metallic minerals , textile , electrical and electronical , and cellulose industries were 48.2 , 14.9 , 6.7 , 22 , 0.9 , 0.6 , and 6.5 percent , respectively . the results showed that nearly half of total industrial waste produced from the range of biological materials are biodegradable and discharging them without observing environmental regulations leads to short - term pollution and nuisance in the acceptor environment . also some parts of case study waste were recyclable which is considerable from viewpoint of economical and environmental pollution . long - term impacts will appear due to improper site selection of disposal from the spatial standpoint . in this way , an approach for site selection using several socioeconomic , physical , and environmental criteria based on multicriteria decision making model ( mcdm ) is introduced . health risks and environment pollution such as soil and surface water may be done . it is essential to revise the studied industries layout , particularly those units which produce special waste which should be more cautious . also stricter enforcement is required as an effective step in reducing the harmful impacts of it .

radiotherapy ( rt ) in malignancies can be broadly classified into radical and palliative with regards to the intention of treatment . radical rt primarily is delivered with an intention to cure , and associated toxicities can be anticipated and are acceptable as an expected side effect . however , when cure seems unlikely , the intention of rt becomes palliative , wherein the goal is to achieve durable symptom relief at the shortest expense of time and resources , and while inflicting the least possible toxicity.[13 ] given that primum non nocere ( first , do no harm ) is the principle behind palliative rt , the treatment program is designed to cause the least possible adverse impact upon the patient in all possible aspects . while radical rt programs can often last for around half - a - dozen weeks , often with superimposed administration of concurrent chemotherapy , palliative rt most often consists of an overall lesser dose of radiation , delivered in large fraction sizes ( hypofractionated ) and alone without chemotherapy . further , palliative rt is less expensive , since it involves simple planning and treatment without the need for elaborate conformal plans . overall , palliative rt utilizes sub - therapeutic doses since the goal is not to achieve a complete eradication of tumor . as an example , while head and neck squamous cell carcinomas ( hnscc ) often require > 70 gy , delivered concurrently with chemotherapy to achieve a reasonable tumor control probability ( tcp ) , palliation can be achieved at much lesser doses in the order of 3045 gy delivered mostly in hypofractionated regimens ( utilization of more than 2 gy per fraction).[79 ] in very rare instances , it is not impossible to come across patients who have achieved excellent survival after treatment with palliative intent , in spite of overall rt doses being well below the doses required for expectations of a good tcp . thus , recognizing the existence of such outcomes in the clinic , we intended to analyze the frequency of such occurrences of excellent outcomes after palliative radiation doses . this study is a retrospective analysis of hospital records of the regional cancer centre , shimla , india . patients treated with palliative rt between 1 january 2001 and 31 december 2006 were included , provided their hospital records fulfilled the inclusion criteria [ table 1 ] . eligibility criteria for inclusion into analysis we purposefully excluded malignancies of the breast and prostate , given that these malignancies often have a long natural history.[1012 ] we also excluded malignancies of the central nervous system given the poor survival with or without rt . we also excluded salivary gland adenoid cystic malignancies , given that these malignancies too have a very long natural history wherein patients survive for years even after cannon - ball pulmonary metastases . we excluded hematological and lympho - reticular malignancies since these patients often follow - up elsewhere at departments specializing in hematological oncology . we excluded patients who , after initiation of palliative rt , later on received treatment with radical intent , either with the use of additional doses of radiation , chemotherapy , or surgery . overall , 963 patients were eligible for analysis [ table 2 ] , of whom 23 patients ( 2.4% ) were alive for at least 5 years since the initiation of palliative rt . of these patients who had survived at least 5 years since palliative rt , 73.9% ( 17 of 23 patients ) were free of disease without any clinical or radiological evidence of residual or recurrent malignancy . site - specific distribution of patients eligible for analysis interestingly , in spite of palliative rt being delivered after patients were considered unlikely to benefit from a full - course radical therapy , 1.6% , 2.6% , 5.5% , and 3.5% of patients with lung cancer , gynecological cancer , head / neck cancer , and esophageal cancer , respectively , were alive for more than 5 years of follow - up . less than one - fourth of patients surviving for 5 years after treatment had any evidence of residual or recurrence . the individual characteristics of patients who survived at least for 5 years since the initiation of palliative rt with regards to the diagnosis , age at diagnosis , sex , reason for choosing palliative approach , dose and fractionation of palliative rt , and current status have been presented in tables 35 . characteristics of patients of lung cancer surviving 5 years after palliative rt characteristics of patients of gynecological cancer surviving 5 years after palliative rt characteristics of patients of head and neck , and esophageal cancer surviving 5 years after palliative rt palliative rt is based upon the principles of maximizing symptom relief with minimal consumption of time and resources , and causing the least possible concern to the patient with regards to span of treatment and toxicities afforded . lower total time as well as lower total dose is the hallmark of palliative rt.[13 ] as an example , control of symptoms arising from superior vena caval obstruction syndrome with lung carcinoma is effective even with 20 gy delivered over 5 days , with 5 fractions of 4 gy each . however , a radical rt regime for a patient of lung cancer could take anywhere between 5 and 6 weeks.[1519 ] similarly , a patient of bulky neck nodal disease , which is threatening to rupture , may effectively be palliated with doses as less as 30 gy delivered over 10 fractions of 3 gy each . however , a radical regime consists of at least 70 gy delivered over 35 fractions over 67 weeks , often with concurrent chemotherapy . the toxicities suffered from the higher total dose and the cost incurred by the protracted radical rt regimen can be justifiable , given the chance of cure . however , in the case of palliative rt , since there is an assumption of a low or absent probability of cure , the patient is offered a short course of radiation , which delivers higher dose per fraction , which can be expected to palliate the local symptoms , and at the same time not adding to agony by means of treatment - related toxicity.[2024 ] often , a percentage of patients may not enjoy the expected survival outcomes even after a fully radical course of chemo - radiotherapy . conversely , the opposite , that is , expectations of cure or long - term survival can often be kindled ( in the patient and in the clinician ) when a patient initiated on palliative rt demonstrates very rapid tumor shrinkage . however , more often than not , patients succumb either due to disseminated disease , local recurrence with aggressive repopulation , or a combination of distant and local disease progression . a detailed literature search done on literature indexes ( pubmed , pubmed central , scopus , hinari , index copernicus , google scholar , embase , cinahl , and doaj ) with the terms such as long term survival after palliative radiotherapy , cure after palliative therapy , and tumour control sub - therapeutic radiation dose did not yield any study , either prospective or retrospective , regarding long - term survival after sub - therapeutic / palliative radiation doses . we performed a retrospective analysis utilizing hospital records of patients who were treated with palliative rt from 1 january 2001 to 31 december 2006 . we chose december 2006 as the cutoff so as to ensure that every patient included into the analysis had the opportunity to complete at least 5 years of follow - up . though our original intention was to perform a kaplan meier analysis for survival of all eligible patients who had undergone palliative rt , we found it not feasible given the frequent migration of patients to other centers for treatment and a high attrition rate owing to the inherent logistic difficulties arising from the mountainous terrain of this region . undeniably , many patients who died after having received symptom control could indeed have succumbed to intercurrent illnesses such as infectious diseases and other non - oncological causes concerning the cardiovascular and pulmonary systems . nevertheless , we observed that 2.4% of patients had indeed survived for at least 5 years in spite of the treatment intent not having focused on the goal of prolonged survival . though the percentage value ( 2.4% ) of patients surviving 5 years after palliative rt may seem trivial , the absolute numbers are significant given the large sample size . it is very likely that these very patients could not have survived if in case they were transitioned to supportive care instead of palliative rt . this also is striking , given that often patients do not enjoy survival in spite of a full course of radical chemo - radiotherapy , whereas some patients ( as illustrated in our analysis ) very rarely may indeed benefit excellent survival outcomes even with sub - curative doses of palliative radiation . this could be in part due to various biological factors , such as immunological clearance , induction of apoptotic signals by radiation , or possibly a lack of aggressive clonogens capable of accelerated repopulation . while some cancers such as small cell lung cancer ( sclc ) and poorly differentiated cervical carcinomas are more radiosensitive compared to other malignancies , it must also be acknowledged that tumors of the same diagnosis may have different biological behaviors in different patients . further , a prospective multivariate analysis of various potential factors contributing to exquisite radiosensitivity even with palliative doses can confirm or refute the role of parameters such as , but not limited to , histology , grade of differentiation , immunological indicators including circulating cytokines , anti - tumor antibodies , and growth factor receptors such as epidermal growth factor receptor 1 ( egfr1 ) , her-2-neu , and vascular endothelial growth factor receptor ( vegfr).[2729 ] to conclude , we remind the reader that rt in a palliative role has excellent efficacy with regards to the control of pain and bleeding , and also with the relief of obstructive symptoms such as dyspnea and dysphagia . in addition , as our study suggests , there could be a benefit with regards to prolongation of survival . thus , even seemingly unfit patients must be assessed for the potential utility of palliative rt before the decision is made to transition to terminal end - of - life supportive care . the take - home message for oncologists and palliative care professionals would be that patients not given definitive therapy , but having responded exceptionally well to palliative treatment ( e.g. a patient having achieved near - complete response after a course of palliative rt or a patient with markedly improved performance status after completion of palliative therapy , etc . ) must be closely followed up and considered for further therapy , such as the use of additional chemotherapy for sclc , or the use of additional rt and chemotherapy for patients of cervical and head / neck cancers . summing it up , the noble principle of primum non nocere , which translates to first , do not harm should not be interpreted in a way as if it were encouraging therapeutic nihilism , wherein the clinician 's pessimism with regards to the available therapeutic modalities could diminish a patient 's chance of obtaining benefit from therapy .

context : palliative radiotherapy aims at symptom alleviation and improvement of quality of life . it may be effective in conferring a reasonable quantum of local control , as well as possibly prolonging survival on the short term . however , there can be rare instances where long - term survival , or even cure , results from palliative radiotherapy , which mostly uses sub - therapeutic doses.aim:to categorize and characterize the patients with long - term survival and/or cure after palliative radiotherapy.materials and methods : this study is a retrospective analysis of hospital records of patients treated with palliative radiotherapy from 2001 to 2006 at the regional cancer centre , shimla.results:of the analyzed 963 patients who received palliative radiotherapy , 2.4% ( n = 23 ) survived at least 5 years , with a large majority of these surviving patients ( 73.9% , n = 17 ) being free of disease.conclusions:in addition to providing valuable symptom relief , palliative radiotherapy utilizing sub - therapeutic doses may , in a small proportion of patients , bestow long - term survival , and possibly cure . rationally , such a favorable , but rare outcome can not be expected with supportive care alone .

an 82-year - male presented with severe pain in the low back and both buttocks that was exacerbated by lumbar motion for 3 days . the intensity of the pain was 9/10 on a visual analogue scale ( vas ) . the pain was relieved when he rested in bed , and aggravated by weight loading or motion . the physical examination revealed a reduction in all back movements and tenderness over the l4 spinous process . the patient had straight - leg - raising restriction of 70 in his right leg and 75 in his left leg . the laboratory values were normal , including the erythrocyte sedimentation rate and c - reactive protein . plain radiographs showed hyperostosis in the lumbar spine ( fig . 1 ) and magnetic resonance imaging ( mri ) revealed a schmorl 's node of l4 with adjacent marrow edema ( fig . the spinal canal was narrow at the l3 - 4 and l4 - 5 levels . we performed nerve blocks on the l4 ramus communicans nerve . with the patient prone on the operating table , a 22-gauge spinal needle ( spinocan , b.brown , germany ) was directed underneath the pedicle to rest slightly anterior to the superior aspect of the neural foramen . the needle was advanced until it was positioned at the inferior third aspect of the vertebral body under lateral fluoroscopic view . with the needle in the proper position , a mixture of 0.5 ml of 2% mepivacaine and 1.5 ml of contrast medium was injected to confirm the absence of intravascular , somatic nerve root , or epidural placement ( fig . , a mixture of 2 ml of 1% mepivacaine and 10 mg of triamcinolone was injected on each side . his pain improved dramatically immediately after the nerve block ( vas score 2/10 ) ; there were no serious procedure - related complications . at the 1-month follow - up schmorl first described cartilaginous nodal herniation of the disc into an adjacent vertebral body in 1927 . the majority of authors report that typical schmorl 's nodes are usually asymptomatic . however , an acute or inflamed schmorl 's node may be symptomatic and clinically significant . schmorl 's nodes are most commonly located at the middle third of the inferior endplate , near the thoracolumbar junction . schmorl 's nodes occur when the cartilaginous endplate of the vertebral body has been disrupted by an intrinsic abnormality of the endplate itself such as indentations left by the regression of the chorda dorsalis , ossification gaps , vascular channels , scheuermann 's disease or by alterations in the subchondral bone itself such as osteomalacia , hyperparathyroidism , paget 's disease , infection , neoplasm , trauma or mechanical overuse , scheuermann 's disease , and osteoporosis . such weakening of the endplate is not a necessary prerequisite for extrusion and is thought to be present as an underlying cause only in a small percentage of schmorl 's node cases . most schmorl 's nodes form after axial - loading trauma results in the preferential extrusion of nuclear material through the vertebral endplate , rather than through an intact , normal annulus fibrosus . schmorl 's nodes are commonly seen on radiographs or at autopsy . in most cases , it is a radiographic or autopsy diagnosis . the demonstration of disc prolapse by radiography is usually possible only after the bony reaction of the vertebral body has developed an osseous sclerotic bone casting . mri has contributed to rapid improvements in the fundamental understanding of the bone marrow and its anatomy and physiology . in all symptomatic cases , the vertebral body marrow surrounding the schmorl 's node had low signal intensity on t1-weighted images and high signal intensity on t2-weighted images . in our case , the vertebral body marrow surrounding the schmorl 's node was seen as low signal intensity on t1-weighted images and high signal intensity on t2-weighted images , so we made an early diagnosis of a symptomatic schmorl 's node . the principal branches of the lumbar sympathetic trunks are the rami communicantes to the lumbar ventral rami . white rami communicantes are distributed to the l1 and l2 ventral rami , and grey rami communicantes are distributed to every lumbar ventral ramus . the number of rami communicantes to each lumbar nerve varies from one to three , and exceptionally may be as high as five . in general , the rami communicantes reach the ventral rami by passing through the tunnels deep to the psoas muscle that lie along the concave lateral surfaces of the lumbar vertebral bodies . these tunnels direct them to the lower borders of the transverse processes where the rami communicantes join the ventral rami just outside the intervertebral foramina . the source of the nerve endings in the lumbar discs and vertebral bodies , are two extensive microscopic plexuses of nerve that accompany the anterior and posterior longitudinal ligaments . the anterior plexus bridges the two lumbar sympathetic trunks and covers the anterior longitudinal ligament . it is formed by branches of the sympathetic trunks and branches from the proximal ends of the grey rami communicantes . the posterior plexus is derived from the sinuvertebral nerves and accompanies the posterior longitudinal ligament . the anterior and posterior plexuses are connected around the lateral aspects of the vertebral bodies and discs by way of a lateral plexus that is formed by branches of the grey rami communicantes . the anterior and posterior plexuses supply superficial branches that innervate the periosteum of the vertebral bodies , and long penetrating branches that enter the intervertebral discs and vertebral bodies . through these branches this type of nerve block was initiated from the basis of the observation of the course of gray ramus communicans , containing unmyelinated postganglionic fibers which rejoin dorsal and ventral rami , distributing around vertebral body wall and coursing into anterior disc . it is known that gray ramus communicans nerve provides the greatest source of disc innervations and vertebral column . with respect to complications , chandler et al . reported potential risks , including infection , bowel puncture , intravascular injection , somatic nerve root trauma , intrathecal or epidural injection , kidney puncture with a far lateral approach and pneumothorax in thoracic approach . mri of patients with symptomatic schmorl 's nodes has demonstrated inflammation and edema in the vertebral body , localized to the area around the schmorl 's node . inflammatory change in the vertebral body marrow induced by intraosseous fracture and some biological reaction to the intraspongious disc materials might cause pain . after the fracture has healed and the inflammation subsided , the schmorl 's node would be asymptomatic in analogy with an old vertebral compression fracture . symptomatic schmorl 's nodes should be treated similarly to vertebral compression fracture , and conservative treatment is the first choice . if conservative treatment fails , surgery with anterior interbody fusion might be indicated . in our case , the schmorl 's node was treated similarly to a vertebral compression fracture , and we successfully reduced the pain after blocking the ramus communicans nerve . the pain fell to a vas score of 2 , and the patient was discharged 1 week after the block . most schmorl 's nodes are asymptomatic , although acute or inflammatory schmorl 's nodes are painful and clinically significant . some patients suffer from disabling pain due to schmorl 's nodes , despite conservative treatment . a rami communicans nerve block is one treatment modality for patients with back pain due to a schmorl 's node .

histologically , schmorl 's nodes are defined as the loss of nuclear material through the cartilage plate , growth plate , and end plate into the vertebral body . most schmorl 's nodes are asymptomatic , although there are some reports of symptomatic schmorl 's nodes , which should be treated similarly to vertebral compression fractures , with conservative treatment as the first choice . we report the case that we reduced the pain by blocking the ramus communicans nerve in a patient with schmorl 's node .

hepatocellular carcinoma ( hcc ) accounts for more than a million new cases each year worldwide and is the third leading cause of cancer - related death worldwide . its incidence in the united states has shown a dramatic rise over the past few decades and is expected to increase in the coming years . liver transplantation ( lt ) is in many cases the ideal therapy for hcc , providing not only oncologic resection but also replacement of a diseased organ . an important study by mazzaferro and colleagues demonstrated that if lt was limited to those with early hcc , long - term post - lt survival was excellent . based on this study and others , lt has become the standard of care for those hcc that satisfy the eponymous milan criteria ( a single tumor under 5 cm , or 3 tumors each under 3 cm , without evidence of metastatic disease / vascular invasion ) on radiological imaging . however , despite advances in imaging techniques , almost 20% of hccs are understaged on pre - lt radiological studies and are found on explant to exceed milan criteria , an established risk factor for post - lt hcc recurrence . specific predictors of post - lt recurrence are tumor size , multifocality , and vascular invasion . the efficacy of liver transplantation for hcc is therefore limited by tumor recurrence and the lack of effective preventive strategies for those at high recurrence risk . sorafenib is a multiple tyrosine kinase inhibitor , which inhibits tumor angiogenesis by inhibition of the vascular endothelial growth factor ( vegf ) and platelet derived growth factor ( pdgf ) signaling pathway and is the only fda - approved systemic chemotherapeutic agent for the treatment of advanced hcc at this time [ 4 , 5 ] . we therefore undertook this pilot study to examine the preemptive use of sorafenib in the prevention of hcc recurrence post - lt in those with high - risk explant characteristics . we sought to assess the tolerability and safety of sorafenib in this population and examined rates of hcc recurrence and post - lt survival . all explants were sectioned at 5 mm intervals and examined by an experienced hepatopathologist . patients were offered entry if their explant examination demonstrated viable tumor exceeding milan criteria , specifically any of the following . ( 1 ) 3 or more viable tumors ( if 3 tumors , at least one over 3 cm diameter ) , ( 2 ) one viable tumor greater than 5 cm in diameter , or ( 3 ) evidence of micro- or macrovascular invasion . exclusion criteria were as follows : pathologic features of mixed hepatocellular cancer with cholangiocarcinoma , contraindication to sorafenib , absence of informed consent , and use of sirolimus as part of the immunosuppressant regimen . patients were started on sorafenib 200 mg by mouth once daily within 1224 weeks of lt , and the dose was titrated upwards to the target dose of 400 mg twice daily . patients underwent abdominal mri and ct scan of the chest every three months or earlier if clinically indicated , for hcc surveillance . the 7 patients who satisfied entry criteria were maintained on standard immunosuppression , and sirolimus use was prohibited . to identify a comparison group , we reviewed explant pathology of all patients transplanted for hcc from 2003 ( initiation of the liver transplant program at our institution ) onwards . we identified 12 patients who exceeded milan criteria on explant examination and who did not receive sorafenib preemptively after lt . of these , 9 patients had undergone lt prior to protocol initiation , and 3 patients declined treatment with sorafenib when offered entry into this protocol . there were no significant changes in the immunosuppression protocols or the pretransplant treatment of hcc between the two groups . secondary end points included rate of hcc recurrence , time to recurrence , and mortality . demographic characteristics , clinical variables , and transplant outcomes of patients included in the study were compared using means for continuous variables and frequencies for categorical variables . differences in characteristics between the adjuvant therapy group and historical comparison group were compared using the fisher 's exact test and t - test . posttransplant survival and hcc recurrence between the two groups were compared using kaplan - meier ( k - m ) curves . the majority of patients in both groups were male , and hepatitis c virus ( hcv ) represented the primary underlying etiology of liver disease . all patients underwent either contrast - enhanced magnetic resonance imaging ( mri ) or computerized tomographic ( ct ) scan prior to lt . , even though no patients were noted to have > 3 nodules on pre - lt imaging , 50% and 42.8% , respectively , in the historical and the treated group were found to have multifocal disease on explant examination . vascular invasion was noted on microscopic examination of the explants ( i.e. , microvascular invasion ) in 33% and 28% of patients in each group . the mean alpha - fetoprotein ( afp ) level at lt was not significantly different between groups . all patients received locoregional therapy ( lrt ) prior to lt , except 1 patient in the treatment group , with an equivalent number of lrt sessions between groups . five patients with recurrent hcc in the historical comparison group were treated with sorafenib after recurrence was documented . there were no significant differences between the two groups in terms of the number of tumors , presence of microvascular invasion , mean / maximum tumor diameter , tumor differentiation , and percentage of necrosis . the most frequent side effects of sorafenib were skin rash and diarrhea ( 58.3% of treated patients experiencing either or both adverse effects ) . twenty - two percent of patients experienced hand - foot syndrome . nausea and fatigue were also noted as side effects of sorafenib . there were no differences in the adverse effect profile of patients treated thereafter , patients were started at low doses of 200 mg daily and increased to full doses ( 400 mg twice daily ) as tolerated . the mean follow - up for treated patients was longer than that for those in the historical group but this difference did not reach statistical significance ( 1125 versus 840 days , p value = 0.18 ) . the overall rate of hcc recurrence was lower in the adjuvant therapy group compared to historical controls , ( 29% versus 75% , p = 0.07 , table 3 ) . disease - free 1-year survival for sorafenib and the comparison group was 100% and 66% , respectively ; overall 1-year survival was 100% and 92% , respectively . two of 7 sorafenib treated patients suffered hcc recurrence ( at 14 months and 52 months ) , one patient is deceased ; the other is alive 5 months following documented recurrence . of the comparison group , 9 out of 12 patients the median time to recurrence among those treated with adjuvant therapy was longer but not statistically significant ( 1053 days versus 620 days , p = 0.08 ) . the lung represented the most common site of recurrence ( 6 patients ) , followed by the liver ( 3 patients ) ; 2 patients had multiple sites of recurrence . survival rates at study conclusion were higher for the treatment group ( 85.7% ) compared to the historical arm ( 33.3% ) one patient is alive at the current time 4 months from date of documented recurrence . the mortality incidence for the two groups it can be seen that the historical comparison group appears to have a higher mortality ( 75% ) compared to the adjuvant therapy group ( 14% ) . however , because of the small sample size and the large number of censored observation , the sample size was effectively only 9 patients . this number is too small to use the tests of homogeneity over strata that require larger sample sizes in order to be meaningful . lt offers excellent survival outcomes in patients with hcc who satisfy well - established selection criteria . these criteria are however based on pre - lt imaging studies , which have been shown to underestimate tumor stage in approximately 20% of patients . patients with explant characteristics of tumor multifocality and vascular invasion are at high risk of recurrence . we have previously shown that , in those who exceed milan criteria on explant , the hazard ratio for hcc recurrence increased to 3.14 ( p value 0.03 ) compared to those whose hccs on explant satisfied milan criteria . as a consequence , hcc recurrence after lt occurs at a significant rate , ranging from 3.5 to 26% , with median rates of 13% . this represents an important limitation to the efficacy of lt for malignant liver disease and may not be an appropriate use of an increasingly scarce resource . adjuvant chemotherapy with 5-fluorouracil / carboplatin , cisplatin / adriamycin , or doxorubicin has been studied , but published reports document conflicting efficacy rates and an unfavorable toxicity profile [ 6 , 7 ] . the use of conventional chemotherapeutic regimes appears to be constrained by toxicity and difficulty of administration . the recent development of molecularly targeted therapies with acceptable side effect profiles and oral availability may provide an opportunity to reevaluate our approach to adjuvant treatments . sorafenib is the first of these agents to have been fda approved for the treatment of advanced hcc , and we believe it offers an opportunity for use in the prevention of hcc recurrence in high - risk individuals following lt . saab et al . described 8 patients treated with adjuvant sorafenib therapy and reported recurrence rates of 12.5% , as compared to 50% in a control population . as in our population , sorafenib appeared safe . a study from china consisted of a somewhat ill - defined population , but also appeared to confirm overall enhanced survival rates in patients offered sorafenib . our study comprises a homogenous group of patients , carefully monitored for recurrence with a longer period of follow - up than previously reported . as a result , we are able to report on delayed recurrence ( 52 months ) in one of the treated patients . while it is not possible to base mechanistic insights on such a limited sample , our experience raises tantalizing questions about the mechanism of action of sorafenib suggesting that it may render tumor cells studies that have examined the use of sorafenib prior to liver transplantation have shown mixed results , with a suggestion of increased biliary complications and acute cellular rejection following lt [ 10 , 11 ] . several studies have examined sorafenib as treatment for recurrent hcc following lt [ 1214 ] . other therapies have included surgery , systemic chemotherapy , as well as mammalian target of rapamycin ( m - tor ) inhibitors [ 14 , 15 ] . overall results have been disappointing , suggesting that recurrent hcc following lt is invariably a lethal and unresponsive disease . our small pilot study was primarily designed to assess the safety and tolerability of sorafenib . we found that sorafenib was well tolerated in the majority of patients , with increased tolerability if the dose was gradually titrated upward instead of started at a high dose . the main adverse event was a skin rash , which usually responded well to topical therapy and dose adjustment . there were no observed interactions with immunosuppressant regimens and no episodes of acute cellular rejection . however , there was a striking reduction in the rate of recurrence among treated , as compared to a historical comparison group , matched by explant features . all but one patient with hcc recurrence have succumbed to their disease , and hence the overall mortality rate in the historical control group was much higher ( 75% ) compared to treated patients ( 14% ) . both preemptively treated patients who developed recurrence did so at a much later time point than those who were not treated , raising the possibility that sorafenib may delay the time to recurrence . however , the 5 treated patients without recurrence have now been followed for a mean time of 1125 days and there was no statistically significant difference in follow - up times . our study suffers from the limitation of being a single - center experience with a small number of patients . the comparison group was historical and therefore could not be controlled for many variables . in summary , we have demonstrated that the use of sorafenib in patients transplanted for high - risk hcc is a safe , feasible , and well - tolerated intervention . in our small sample , sorafenib use was significantly associated with a lower risk of hcc recurrence and improved survival . larger , prospective controlled trials will be required and have in fact been planned , to further examine the preemptive use of sorafenib and similar agents in this population , so that we may expand the armamentarium of therapies that are available to this challenging group of patients .

the efficacy of liver transplantation ( lt ) for hepatocellular ( hcc ) is limited by tumor recurrence rates of 1015% . we undertook this pilot study to examine the use of sorafenib as adjuvant therapy in high - risk lt recipients . methods . we prospectively enrolled patients transplanted for hcc into a treatment protocol utilizing sorafenib if their explant examination showed evidence of viable tumor exceeding milan criteria . we utilized as historical controls patients transplanted previously , whose explant tumor characteristics exceeded milan criteria , but who were not preemptively treated with sorafenib . wilcoxon two - sample test and fisher 's exact test were used to compare survival and recurrence rates between the two groups . results . seven patients were treated with sorafenib and compared to 12 historical controls . two of 7 treated patients suffered from hcc recurrence . of the comparison group , 9 experienced hcc recurrence and all succumbed to disease . dose reduction improved tolerance of drug . the overall rate of hcc recurrence was decreased in the adjuvant therapy group compared to historical controls ( 29% versus 75% , p = 0.07 ) . disease free 1-year survival for the treated versus untreated group was 100% versus 66% , respectively . conclusion . adjuvant use of sorafenib is safe and decreases risk of hcc recurrence in high - risk lt recipients .

hepatitis c virus ( hcv ) is the major causative pathogen of chronic liver diseases with a risk of progression to cirrhosis and hepatocellular carcinoma . currently , ribavirin is one of the major agents used in combination therapy with interferon for chronic hepatitis c. during this treatment , however , patients sometimes suffer ribavirin - induced hemolytic anemia as an adverse event , necessitating discontinuation of the therapy or reduction of the dosage of anti - viral agents 1 , 2 . the mechanism of the hemolysis induced by ribavirin has not been clearly determined , but oxidative stress and subsequent membrane fragility of erythrocytes have been suggested to play an important role 3,4 . after administration , ribavirin is converted to its phosphorylated form within the cell using adenosine triphosphate ( atp ) . however , phosphorylated ribavirin is not converted back to the dephosphorylated form because erythrocytes lack the enzymes necessary for dephosphorylation . thus , the level of atp in cells is reduced as a result of ribavirin phosphorylation 4 , 5 . the metabolic system in erythrocytes consists of glycolysis and the pentose phosphate cycle , for which atp is essential . the pentose phosphate cycle begins with glucose-6-phosphate ( g6p ) , which is the first intermediate metabolite produced by glycolysis , and thus it interacts with glycolysis closely . it has been hypothesized that deficiency of atp causes impairment of the glycolytic system , thus triggering an energy crisis within erythrocytes , and inducing oxidative stress , leading to membrane fragility through impairment of the pentose phosphate cycle . in the present study , to investigate changes in the intermediate metabolites of glycolysis and the pentose phosphate cycle in erythrocytes , we carried out the metabolomics analysis of erythrocytes from patients with chronic hepatitis c , who had received antiviral therapy with pegylated interferon plus ribavirin . it was anticipated that this approach would help to clarify the mechanism responsible for ribavirin - induced hemolysis . eight patients with chronic hepatitis c ( 4 men and 4 women , aged 54.111.6 yr , meanstandard deviation ) were treated with pegylated interferon plus ribavirin in accordance with the japanese standard guideline for treatment of chronic hepatitis c 6 . erythrocytes were collected from the patients before and one week after the start of treatment , separated from whole blood by centrifugation , and resuspended in normal saline solution at a concentration of 40010/l . for disruption of the erythrocyte membranes , the levels of intermediate metabolites in erythrocytes were measured by capillary electrophoresis - time - of - flight mass spectrometry ( ce - tofms ) as reported previously 7 - 9 . informed consent was obtained from all patients , and the study was approved by the institutional ethics committee . wilcoxon matched - pairs signed rank test was used for statistical analysis , and differences at p < 0.05 were considered to be significant . hemolytic anemia induced by ribavirin is one of the serious adverse events associated with therapy for chronic hepatitis c 1 , 2 . the erythrocyte count observed in this study showed a significant reduction at 2 weeks after the start of ribavirin administration ( 452.043.910/l vs. 421.752.410/l : pretreatment vs. 2 weeks after treatment , meanstandard deviation , p=0.028 ) . the drop in the erythrocyte count progressed and became 388.158.010/l at 4 weeks after the start of ribavirin administration . although the mechanism of this hemolysis has not been precisely clarified , a reduction of atp due to ribavirin phosphorylation has been suggested 10 . a recent study showed that ribavirin - induced anemia is primarily due to the effect of the drug on guanosine triphosphate , consequently leading to a reduction of atp in erythrocytes 11 . to clarify this mechanism , we focused on the levels of intermediate metabolites of glycolysis and the pentose phosphate cycle in erythrocytes . the changes in the levels of intermediate metabolites belonging to the glycolytic system and the pentose phosphate cycle in erythrocytes between samples collected before and after ribavirin administration are shown in table 1 . as ribavirin was taken up by erythrocytes , atp was consumed for its phosphorylation , and as erythrocytes lack the enzymes necessary for ribavirin dephosphorylation , atp deficiency progressed and eventually inhibited the glycolytic system . here we found that the levels of the intermediate metabolites of glycolysis in erythrocytes , including g6p , fructose-6-phosphate , 2 , 3-diphosphoglyceric acid and 3-phosphoglyceric acid , were significantly lower one week after ribavirin administration than before the start of treatment . g6p is the key metabolite that is central to both glycolysis and the pentose phosphate cycle in erythrocytes . g6p is metabolized to 6-phosphogluconic acid , d - ribose-5-phosphate and d - sedoheptulose-7-phosphate to supply nicotinamide adenine dinucleotide phosphate ( nadph ) for the pentose phosphate cycle . the levels of these metabolites in patients ' erythrocytes were all reduced after ribavirin administration , among which that of d - ribose-5-phosphate became significantly lower . this indicated that the decrease in the level of g6p influenced the activity of the pentose phosphate cycle and caused a decrease of nadph . nadph has an important role in the reduction of glutathione ( gsh ) in concert with glutathione reductase . thus it was suggested that the decrease of nadph resulting from impairment of the pentose phosphate cycle led to a decrease in the concentration of reduced gsh , consequently increasing the degree of oxidative stress in erythrocytes . in fact , a previous study has shown that ribavirin treatment causes a significant increase of malondialdehyde and methemoglobin in erythrocytes , both being typical products of oxidative damage 4 . taken together , the data suggest that two major events in erythrocytes - an energy crisis due to atp deficiency in the glycolytic system and consequent oxidative stress due to impairment of the pentose phosphate cycle - are closely related to the occurrence of hemolysis associated with ribavirin . the single nucleotide polymorphisms ( snps ) of inosine triphosphatase ( itpa ) gene are shown to be strongly associated with ribavirin - induced hemolytic anemia in chronic hepatitis c 12 . further studies are needed to investigate a relation between snps of itpa gene and levels of intermediate metabolites for glycolytic system and pentose phosphate cycle in a larger cohort of patients . in the treatment of chronic hepatitis c , adequate doses of ribavirin are closely associated with viral clearance , and prevention of both viral breakthrough and emergence of resistant variants 2 . for achieving the maximum effect of antiviral therapy , the results of this study suggest that some form of medication strategy for both the energy crisis and oxidative stress caused by ribavirin may be important for prevention of erythrocyte hemolysis .

ribavirin is one of the major agents used in combination therapy with interferon for chronic hepatitis c , but is often associated with hemolytic anemia as a serious adverse event . employing metabolome analysis , we demonstrated that the concentrations of intermediate metabolites produced by glycolysis and the pentose phosphate cycle in patients ' erythrocytes were significantly decreased after administration of ribavirin . our findings suggest that hemolysis associated with ribavirin is triggered by an energy crisis and consequent oxidative stress , thus having implications for the prevention of such hemolysis .

opposite to the term fertility , there is another important aspect called infertility , which is defined as inability to conceive for at least 1 year after regular and unprotected sexual intercourse . the causes for infertility can be generally categorized as male factor , female factor , combination , and unexplained or idiopathic factor . the world health organization ( who ) reported mean infertility rate as 1215% globally , with a prevalence of 24.9% in iran in 2004 . an imbalance in any of these systems leads to infertility and predisposes the couples to mental and psychological problems such as anxiety , depression , and mental pressure , which , at times , result in social problems such as domestic violence . in various studies , it has been reported that the stressful experience of infertility is accompanied with a vast spectrum of psychological problems including impulsive behaviors and spread of anger , the feeling of helplessness and rejection , as well as marital problems . meanwhile , couples negative self - concept and low self - esteem drive them to have unreal and early expectations . on couples failure to achieve their unreal expectations ( e.g. , wishing to have a child ) misbehavior is among the important issues associated with public health , which is not only observed in all geographical areas and religions and racial groups , but also in all levels of education , occupation , and economic and social classes . several studies have reported some factors associated with infertility that can predispose women to misbehavior and increase the rate of violence by twofold . as infertility and its effects may vary in difference cultures , the prevalence of violence has been reported differently in various studies . reported the rate of domestic violence incidence among infertile women as 33.6% in turkey , and this rate was reported as 61.8% in iran . meanwhile , leung et al . reported the incidence of violence to be only 1.8% in hong kong . there are controversial results in the context of the effect of interventional factors such as infertility on the incidence of marital reactions . most of the conducted studies investigated physical violence , although non - physical violence can lead to major complications and due to lack of studies in this subject . therefore , the present study aimed at determining the association between infertility factors and non - physical partner abuse in infertile couples referring to infertility centers in isfahan in 2013 . this is a descriptive cross - sectional study conducted on 262 infertile couples ( 131 male and 131 female ) who referred to the infertility centers of isfahan and met the inclusion criteria . inclusion criteria were primary infertility , infertility with a diagnosed cause , and absence of any diagnosed mental and psychological diseases . data collection tool comprised a demographic characteristics questionnaire , and non - physical abuse of partner scale ( npaps ) as well as partner abuse scale : non - physical ( pasnp ) , both of which include 25 items that were separately completed and measured for each subject . npaps ( 25-item tool ) is one of the care scales in which the concept of the abuser from the level of the abuse delivered to his / her partner is measured . it has been designed to measure the level or severity of non - physical abuse delivered upon the partner , based on the abuser 's self report . pasnp is also a 25-item tool to measure the degree or severity of non - physical behavioral abuse delivered to the clients by their spouse or partner . these two questionnaires , designed by james dublio and garner to measure non - physical abuse , enjoy an excellent internal consistency with = 0.90 , and were adopted for the first time in iran . the present research was approved in isfahan university of medical sciences and its related ethics committee . for questionnaire localization , reliability and validity of this questionnaire were established by face and content validity with the help of the academic members in nursing and midwifery school and school of educational sciences of isfahan university . its validity and reliability were confirmed with cronbach alpha = 0.84 . after obtaining an informed consent from the subjects , both questionnaires are scored based on a likert 's seven - point scale ( 17 ) as follows : never ( score 1 ) , seldom ( score 2 ) , sometimes ( score 3 ) , usually ( score 4 ) , often ( score 5 ) , frequently ( score 6 ) , and always ( score 7 ) , with a calculated mean out of 100 . the collected data were analyzed by descriptive statistics ( mean , sd , min , max ) and interventional statistics ( independent t - test ) in spss 16 with a significance level of p < 0.05 . the results are presented in tables 13 . mean of npaps and pasnp in infertile couples the relationship between infertility factors , and npaps and pasnp in women the relationship between infertility factors , and npaps and pasnp in men female and male subjects mean ages were 27.5 and 31.9 years , respectively . about 90.8% of the men and 26.9% of the women were working . the highest education level of the men and women was high school diploma ( 43.5% and 46.6% , respectively ) . the most common cause of infertility ( 31.3% ) was male factor , and the least ( 17.5% ) was combination factor . as shown in table 1 , the mean scores of npaps were 23.1 10.3 in men and 21.3 10.6 in women out of 100 . the mean scores of pasnp were 13.8 1.9 in men and 20.3 13.4 in women out of 100 . independent t - test showed no significant difference in the mean scores of npaps between men and women ( p = 0.165 ) , although the mean scores of pasnp showed a significant difference between men and women ( p < 0.001 ) . the highest scores of npaps and pasnp were 23.3 and 26.5 in female factor , while the lowest were 17 and 16.5 in idiopathic factor among infertile women . one - way analysis of variance ( anova ) showed no significant association between infertility factor and score of npaps , while it showed a significant association between pasnp and female factor of infertility ( p < 0.01 ) [ table 2 ] . the results showed that when the infertility factor was female factor or combination factor , npaps had the highest mean score of 20.2 among infertile men . meanwhile , one - way anova showed no significant association between infertility factor and the score of npaps ( p = 0.85 ) . when the female factors was a cause of infertility the highest score of pasnp ( 16.7 ) in men was observed . meanwhile , one - way anova showed no significant association between the factor of infertility and npaps in men ( p = 0.15 ) [ table 3 ] . the results of the present study showed that the most common cause of infertility was male factor , which is consistent with the studies of el kissi et al . and chiaffarino et al . meanwhile , volgsten et al . , in sweden , reported female factor , idiopathic factor , and male factor as the causes for infertility respectively . li et al . , in a study conducted in china , reported idiopathic factor as the most common cause for infertility ( 43.9% ) , while female factor ( 20.8% ) , male factor ( 20.3% ) , and combined factor ( 15% ) were ranked after that , respectively . direkavand moghadam et al . , in thier meta - analysis study , reported an equal rate for male and female factors of infertility . with regard to the aforementioned studies , it is found that the prevalence of infertility has been reported differently in various studies . it should be noted that in references , the male factor was reported to be 2540% , the female factor 40 - 55% , the combined factor 10% , and the idiopathic factor was reported as 10% . nowadays , there is an ascending trend for the male factor of infertility due to an increase in industrial occupations and more involvement of men in industries , as well as their contact with environmental and industrial pollutants . isfahan infertility center ( from which most of the subjects were selected ) acts as a referral infertility center where couples with a diagnosed infertility factor are referred from other cities to receive professional treatment and undergo fertility assistive techniques . this can be the reason for the high prevalence of male factor infertility found in the present study . there is no doubt that having a child plays a pivotal role in couples happiness in their marital life . when they are deprived from having their biological offspring in their common life , they not only miss the meaning in their life but also develop conflicts in their interpersonal communication , which predisposes them to mental and psychological problems such as anxiety , depression , and mental pressure , consequently leading to domestic violence . the results of the present study showed that npaps was lowered in women , compared to men , although the mean scores of npaps showed no significant difference between men and women . pasnp was also found to be more among women , compared to men , but its mean score showed a significant difference between men and women . several studies showed that infertility and the attitude toward that puts the couples in an emotional crisis , and meantime , couples negative self - concept and low self - esteem drive them toward inappropriate behaviors such as violence to relieve their discomfort . other studies on domestic violence investigated the npaps just from the viewpoint of one of the couples . meanwhile , in the present study , in addition to npaps , the adopted questionnaire investigated pasnp from the viewpoint of both the couples . akyuz et al . , in a study on infertile women , showed a significant association between the score of violence and infertility factor in such a way that the level of violence was higher in infertility with female factor . one study has reported the rate of domestic violence as 61.8% among infertile women , although all the participants were women with female factor infertility . as the results of the present study showed no significant difference in the factor of infertility and npaps , and mean npaps was not high in women , it can be reasoned that as women with female factor infertility felt somehow defective , they manifested a higher pasnp , although they did not really undergo a higher behavioral abuse . they argue that infertile women are more predisposed to depression , anxiety , stress , and stressful events that can make a potential background for behavioral abuse . one of the major goals of marriage in women is fertility , and infertility , in fact , ruins this value . therefore , women accept to undergo difficult fertility techniques to preserve their condition , which may predispose them to many mental diseases , depression , and other psychological disorders . on the other hand , infertile women face other threats such as a divorce or their spouse 's remarriage , which notably affect their pasnp . the results showed that infertility can act as a factor to make the couples closer to each other and to have a mutual cooperation in infertility treatment , and generally improves their marital relationship , as their npaps and pasnp were found lower in this group of couples in the present study , compared to other studies . both couples should be simultaneously considered in fertility counseling sessions , as well as in medical and clinical treatments given by infertility centers and the associated professionals .

background : infertility predisposes the couples to mental and psychological problems such as anxiety , depression , anger , and partner abuse . this study aimed to investigate the association between infertility factors and the non - physical abuse between infertile spouses.materials and methods : this is a descriptive cross - sectional study conducted on 262 infertile couples ( 131 female and 131 male ) , selected through convenient sampling , who referred to infertility centers in isfahan . data were collected by partner abuse scale : non - physical ( pasnp ) , designed to measure the non - physical abuse experienced in relationship with partner and non - physical abuse of partner scale ( npaps ) , designed to measure the non - physical abuse delivered upon the partner . all data were analyzed through spss version 16.results:mean scores of npaps were 23.1% and 21.3% in men and women , respectively . mean scores of pasnp were 13.8% and 20.3% among men and women , respectively . there was a significant difference in the mean scores of perceived non - physical partner abuse between men and women ( p < 0.001 ) . there was also a significant difference in the mean scores of perceived non - physical partner abuse and factor of infertility ( p < 0.01).conclusions : perceived non - physical abuse and delivered non - physical abuse upon the partner were low among infertile couples . women had a higher perception of abuse when the cause of infertility was female factor , compared to men . however , special attention should be paid to infertile couples . marital counseling , besides infertility counseling , should be conducted for these couples .

growth disorder in children is one of the important health problems in the world , especially in developing countries . regardless of the underlying disease , as the main cause of growth disorders , assessment and correction of nutritional status of these children are very important . given the fundamental importance of this issue and ascending tendency to use complementary medicine in the world , this article discusses the traditional iranian philosopher s views on the role of nutrition in child development . this study reviews textbooks of traditional medicine , particularly in the field of pediatric medicine with a focus on canon of medicine of avicenna . temperament is the physiological concept of the human body in traditional medicine and has an important role in health , diagnosis , and treatment of disease . generally , children are born with warm and wet temperament that provides the best condition for growth . however , the personal temperament of each child determines growth , the need for a variety of food groups , and even physical activity . special lifestyle orders are discussed in detail according to these differentiations and nutritional management is the most important factor considered . in spite of advances in classical medicine in the prevention and treatment of many diseases , there are still a lot of therapeutic challenges in many health problems . temperamental approach to the human body in traditional medicine provides a different perspective on the medicine . reflecting on temperamental view in child development may lead to a better understanding of the diagnosis and treatment of diseases . on the other hand , further research studies based on the reform of nutrition with respect to temperament should be considered as a new strategy in the management of developmental disorders .

background : growth and development are the basic science in pediatric medicine . growth disorder in children is one of the important health problems in the world , especially in developing countries . regardless of the underlying disease , as the main cause of growth disorders , assessment and correction of nutritional status of these children are very important . given the fundamental importance of this issue and ascending tendency to use complementary medicine in the world , this article discusses the traditional iranian philosopher s views on the role of nutrition in child development.methods:this study reviews textbooks of traditional medicine , particularly in the field of pediatric medicine with a focus on canon of medicine of avicenna.results:temperament is the physiological concept of the human body in traditional medicine and has an important role in health , diagnosis , and treatment of disease . generally , children are born with warm and wet temperament that provides the best condition for growth . however , the personal temperament of each child determines growth , the need for a variety of food groups , and even physical activity . different appetite and food preferences in children show temperamental variation . therefore , children need special management regarding special temperament . in iranian traditional medicine , special lifestyle orders are discussed in detail according to these differentiations and nutritional management is the most important factor considered.conclusion:in spite of advances in classical medicine in the prevention and treatment of many diseases , there are still a lot of therapeutic challenges in many health problems . temperamental approach to the human body in traditional medicine provides a different perspective on the medicine . reflecting on temperamental view in child development may lead to a better understanding of the diagnosis and treatment of diseases . on the other hand , further research studies based on the reform of nutrition with respect to temperament should be considered as a new strategy in the management of developmental disorders .

rubinstein - taybi syndrome is a congenital malformation disorder characterized by short stature , mental retardation , microcephaly , broad great toe and thumb , and various facial abnormalities ( hypoplastic maxilla , prominent beaked nose , low - set ears , antimongoloid palpebral fissures).1 first described in 1963 by rubinstein and taybi , this condition occurs in approximately one in 100,000 newborns . most patients with rubinstein - taybi syndrome have a mutation of the creb binding protein gene which codes for a regulatory protein in fetal development.2 most cases are isolated , but parental autosomal dominant transmission can be seen.3 various ocular abnormalities linked with this syndrome ( 84% ) include glaucoma , down - slanting palpebral fissures , epicanthus , strabismus , cataract , refractive error , colobomata , highly arched eyebrows , nasolacrimal duct obstruction , and anterior segment dysgenesis.1,4 van genderen et al found a high frequency of electroretinographic abnormalities in 14 of 18 patients ( 78% ) , eight with decreased cone responses and six with decreased cone and rod responses . similarly , 18 of 23 patients examined ( 78% ) showed mild to severe fundus abnormalities including absent macular reflexes , increased reddening of the foveal area , unusual distribution of pigmentation , lacquer cracks , and chorioretinal coloboma.1 while there are numerous publications on rubinstein - taybi syndrome , the literature on related ocular disturbances is limited . to our knowledge , there are no reports of fluorescein angiography evaluation of rubinstein - taybi syndrome . we report fluorescein angiography findings of vascular attenuation , prolonged arteriovenous transition time , delayed venous filling , small vessel leakage , and peripheral avascularity in a child with rubinstein - taybi syndrome . a 6-year - old male , born at 34 weeks gestation with rubinstein - taybi syndrome due to creb binding protein gene mutation underwent fluorescein angiography to characterize better his retinal vasculature attenuation . the patient had characteristic clinical features of rubinstein - taybi syndrome , including microcephaly , down - slanting palpebral fissures , broad thumbs and first toes ( figure 1 ) , bicuspid aortic valve , and an ocular history of high myopia 6.00 diopters , exotropia , epiblepharon , and congenital glaucoma status post trabeculotomy ou and baerveldt glaucoma implant os . anterior segment examination showed clear corneas ou , clear lens od , small nasal cataract os , superonasal tube os , and posterior synechiae os . fundus examination showed sharp discs with significant glaucomatous cupping ou and optociliary shunt vessel os . choroidal vasculature was prominent due to diffuse retinal atrophy with scattered focal retinal pigment epithelial changes ou ( figure 2 ) . fluorescein angiography showed retinal vascular attenuation , prolonged arteriovenous transition time with delayed venous filling od ( figure 3 ) , and late small vessel leakage nasally os ( figure 4 ) . a total of 360 degrees of peripheral avascularity was also evident ou ( figure 5 ) . vital signs , including heart rate , blood pressure , and oxygen saturations were within normal limits throughout the examination . the present case has many of the ocular findings commonly reported in rubinstein - taybi syndrome including down - slanting palpebral fissures , glaucoma , high myopia , strabismus , and retinal dystrophy.5,6 the fluorescein angiography findings of rubinstein - taybi syndrome identified in this case include vascular attenuation , prolonged arteriovenous transition time with delayed venous filling , small vessel leakage , and peripheral avascularity . prolonged transit time has been identified in glaucoma , but the marked delay in this case is unexpected because intraocular pressures were controlled.7 prolonged transit time can also occur in chorioretinal inflammatory disorders.8 an inflammatory process may have contributed to the prolonged transit time in this case . the late small vessel leakage showed that retinal vascular inflammation was present , and recent baerveldt glaucoma implant surgery may have been a contributing factor . peripheral avascularity , common in retinopathy of prematurity , familial exudative vitreoretinopathy , and incontinentia pigmenti , has not been described previously in rubinstein - taybi syndrome . the degree of peripheral avascularity in the present case is not typical for a birth history of 34 weeks gestational age . mutation of the creb binding protein responsible for growth retardation and other skeletal and cardiac abnormalities in rubinstein - taybi syndrome may also affect vascularization of the retinal periphery . further investigation with fluorescein angiography is warranted to characterize better the retinal vascular abnormalities of rubinstein - taybi syndrome .

the purpose of this report is to describe the fluorescein angiography findings in a case of rubinstein - taybi syndrome . fundus photography and fluorescein angiography were performed on a 6-year - old male with rubinstein - taybi syndrome due to creb binding protein gene mutation . fundus photography showed glaucomatous cupping and diffusely attenuated retinal vasculature . choroidal vasculature was prominent due to diffuse retinal atrophy with scattered focal retinal pigment epithelial changes . fluorescein angiography showed retinal vascular attenuation , prolonged arteriovenous transit time with delayed venous filling , late small vessel leakage , and 360 degrees of peripheral avascularity . peripheral retinal avascularity and retinal vascular inflammation evidenced by late small vessel leakage can be demonstrated by fluorescein angiography in the retinal dystrophy of rubinstein - taybi syndrome .

differential migration in males and females has been demonstrated in various bird species ( cristol et al . it has been extensively studied , in relation to spring migration , that when selection favours earlier arrivals of a given sex ( usually males ) at the breeding grounds , due to competition for high - quality territories and mates ( kokko 1999 ) . much less attention has been paid to differential migration in the autumn , although passage to the wintering grounds may also be time - constrained in a different manner for the two sexes . we investigated the autumn passage of molecularly sexed male and female aquatic warblers acrocephalus paludicola , with regard to their age and body condition . the aquatic warbler is a globally threatened passerine ; its population size and range have decreased by 90 % over the last century ( flade and lachmann 2008 ) . during autumn , the birds migrate to a tropical sub - saharan african location ( buchanan et al . 2011 ; flade et al . 2011 ; foucher et al . 2013 ; salewski et al . 2013 ) through western ( de by 1990 ; aquatic warbler conservation team 1999 ) and possibly southern europe ( salewski et al . 2013 ) . earlier studies of the aquatic warbler 's autumn migration based on small samples did not examine sex differences ( de by 1990 ; atienza et al . 2001 ; julliard et al . 2006 ; migulez et al . 2009 ; neto et al . 2010 ; le nev et al . 2011 ) . however , in view of the breeding biology of the species only the female provides care for the offspring ; the male is free of parental duties after the mating period ( dyrcz et al . one might also expect that the two - peak pattern of autumn migration ( neto et al . this study was carried out at donges on the loire estuary ( 47 18 ' 17.19 " n , 2 2 ' 9.51 " w ) , one of the most important sites in france for capturing the aquatic warbler ( le nev et al . 2011 ) . we performed the fieldwork in 2011 , covering the entire autumn migration period of adult aquatic warblers and almost the entire migration period for young birds in this area ( 18 july15 september ; le nev et al . we captured the birds in 25 mist - nets placed in the reedbed habitat , opened just before local sunrise for circa 6 h ( weather permitting ) . we used tape - luring for the whole capturing period ( played from about 1 h before local sunrise until the nets were closed ) . we measured the right wing with a ruler ( 0.5 mm ) to provide a surrogate measure of body size and body mass using an electronic scale ( 0.1 g , my weigh pointscale 5.0 ) . we determined the fat score according to the 4-score scale proposed by the french ringing centre , crbpo - musum paris . we took 15 body feathers from each bird for molecular sexing , including the primer pair p2 and p8 ( according to the method of griffiths et al . we extracted dna from the proximal tip of feathers using the sherlock ax kit for biological tracks ( a&a biotechnology , gdynia , poland ) . we sampled 176 of 179 ( 98 % ) birds captured during the whole study period , sexing all of them successfully . the median capture dates for different sex and age groups ranged from 11 to 20 august ( fig . 1 ) . in adults , the timing of migration differed between sexes ( mann whitney u test , z29,13 = 3.73 , p < 0.001 ) , with the median date for males being 8 days earlier than for females ( fig . 1 ) . immature males and females migrated at a similar time ( z99,35 = 1.30 , p = 0.19 ) , with only a 1-day difference between median dates ( fig . 1 ) . adult males migrated earlier than immature males ( 9-day difference in the median date ; z29,99 = 5.55 , p < 0.001 ; fig . 1 ) , whereas the timing of adult and immature females was similar ( only a 2-day difference in the median date ; z13,35 = 1.02 , p = 0.31 ; fig . 1timing of autumn migration in aquatic warblers grouped by ages and sex at a stopover site in the loire estuary ( donges , france ) . numbers in brackets denote the relevant sample size timing of autumn migration in aquatic warblers grouped by ages and sex at a stopover site in the loire estuary ( donges , france ) . numbers in brackets denote the relevant sample size body mass was not affected by body size ( ancova , wing length f1,170 = 1.72 , p = 0.19 ) or by sex ( f1,170 = 0.64 , p = 0.43 ) . only age was found to affect body mass , with adults being on average 6 % heavier than immature birds ( f1,170 = 14.85 , p < 0.001 ; fig . 2 ) . the age by sex interaction was not significant ( f1,170 = 0.04 , p = 0.84).fig . 2size - adjusted body mass ( mean and 95 % confidence intervals ) in aquatic warblers of both sex and age categories during autumn migration at the loire estuary stopover site . numbers above the bars denote the relevant sample size size - adjusted body mass ( mean and 95 % confidence intervals ) in aquatic warblers of both sex and age categories during autumn migration at the loire estuary stopover site . numbers above the bars denote the relevant sample size adult males had similar fat scores to adult females ( median fat score for both sexes = 2 ; z29,13 = 0.15 , p = 0.88 ) ; immature males and immature females also had similar fat scores ( median fat score for both sexes = 2 ; z99,35 = 0.94 , p = 0.34 ) . a comparison of the fat scores across age groups revealed no differences in males ( z29,99 = 0.20 , p = 0.84 ) or females ( z13,35 = 0.59 , p = 0.55 ) . the sex ratio differed significantly from parity in both adults ( chi - squared test , 1 = 4.13 , p = 0.04 ) and immature birds ( 1 = 20.91 , p < 0.001 ) . the numbers of captured adult and immature males were 2.2 and 2.8 times higher , respectively , than the corresponding female age groups . we found sex differences in the timing of autumn migration in adult aquatic warblers , with males migrating significantly earlier than adult females . however , immature birds of both sexes migrated at a similar time . this pattern of migration , in which non - parenting adults migrate first , followed by parenting adults and young is common in ducks and shorebirds ( newton 2011 ) . the existence of similar patterns in different taxonomic groups with similar breeding biology emphasizes the link between breeding system and migration behaviour . although the studied site may be considered representative ( le nev et al . 2011 ) , we can not exclude the possibility that the differential migration we observed might be specific to this particular site and season . the males may have stopped predominantly in the study site ( perhaps due to tape - luring , see below ) , whilst the females may have stopped preferentially at another site . it is also possible that the 2011 season was an unusual breeding year with a lot of second brood attempts , this would account for the late passage of adult females and immature birds . a further examination of the autumn migration with respect to sex differences in timing , perhaps across several stopover sites , is desirable . the frequency distribution of the capture of adult aquatic warblers of both sexes was more concentrated in time than that of immature birds , indicating differential migration of the age groups . the most likely explanation for the pattern observed is the difference in previous migration experience ( e.g. , ellegren 1990 ) . tegetmeyer and colleagues ( 2012 ) found some intra - specific variation in timing of moulting , which could be related to the age ( or sex ) groups . however , currently , there are no published data on age- or sex - based winter segregation . however , in line with reports on the aquatic warbler from other stopover sites , and on other acrocephalus species ( julliard et al . 2006 ; jakubas and wojczulanis - jakubas 2010 ) , we found a tendency towards higher body masses , but not fat scores in adults compared to immature birds . this might be explained by more efficient foraging in experienced or higher social status adults . the discrepancy between the results for body mass and fat scores might be accounted for by the low resolution of the fat score system used ( 04 ) and/or a nonlinear relationship between fat mass and fat score ( hedenstrm et al . however , sex ratios in the population at nestling stage as well as in wintering areas seem to be balanced ( giessing 2002 ; dyrcz et al . 2004 ; vogel 2009 ) . the most likely explanation for the biased sex ratio we observed is therefore the tape - luring used in the present study . this procedure caused a sixfold increase in the capture rate of the aquatic warbler ( julliard et al . it has also been shown that in some species ( to date , these do not include the aquatic warbler ) , tape - luring may efficiently attract males , but not females ( e.g. lecoq and catry 2003 ) . however , we can not entirely rule out the possibility that the sex ratio observed during migration reflects a sex difference in migration routes . females may have more frequently used an alternative , more direct migration route ( e.g. via southern europe , salewski et al . 2013 ; but note that only males were investigated in this study ) , as they have less time after breeding for migration than males . these uncertainties indicate clearly that further studies investigating the effects of sex on the migration behaviour of aquatic warblers are needed .

relatively little attention has been paid to sex differences in the migration of birds in autumn . we studied the autumn migration strategy of molecularly sexed males and females in the globally threatened aquatic warbler acrocephalus paludicola . we captured 176 birds at a stopover site in the loire estuary at donges , france . the median date for the passage of adults was 8 days earlier in males than females , although the timing of migration in first - year males and females was similar . this indicates that males , who are without parental duties , can start their migration earlier than females and first - year birds . adults were significantly heavier than immature birds but did not have higher fat scores . in both age categories , more males ( two to three times more ) were captured . however , various factors ( including tape - luring ) can affect observed sex ratio .

it is estimated that 382 million people all over the world had diabetes mellitus in 2013 . chronic loss of retinal neurons occurs due to increased frequency of apoptosis and activation of glial cells . vulnerability to neurons exists before any sign of vascular damage [ 2 - 5 ] . retinal barrier , vasoregression , and impairment of neurovascular interaction [ 6 - 10 ] . homocysteine is a by - product of transmethylation reactions and is detoxified by methionine synthetase , which depends on vitamin b12 and folate as coenzymes for proper function . elevated total plasma levels of homocysteine has been found to be an independent risk factor for retinal vascular occlusive disease . homocysteine has been found to be involved in a complex and dynamic way in vascular injury and repair , thus contributing to the development of diabetic microangiophathy . therefore , strategies for controlling the level of homocysteine by supplementation with folic acid or vitamin b12 may be potential treatment strategies to ameliorate neurodegeneration . the present study was conducted to evaluate the status of serum levels of vitamin b12 , folic acid , and homocysteine in diabetic retinopathy and the correlation with retinal nerve fiber layer ( rnfl ) thinning on spectral domain optical coherence tomography ( sd - oct ) . institutional review board approval was obtained , and the study was performed in accordance with the tenets of the declaration of helsinki . sample size was calculated to be 80 according to the standard sample size calculation formula . the study was conducted in adherence to the arvo guidelines regarding the ethical use of human subjects in research . in this tertiary care center based cross - sectional study , 60 consecutive cases of type 2 diabetes mellitus and 20 healthy controls ( presenting for refraction ) aged between 40 and 65 years were included after informed voluntary consent was obtained . the 60 cases of type 2 diabetes mellitus were divided into three groups : patients with diabetes without retinopathy ( n = 20 ) , patients with non - proliferative diabetic retinopathy with macular edema ( npdr ; n = 20 ) , and patients with proliferative diabetic retinopathy with macular edema ( pdr ; n = 20 ) , based on the early treatment diabetic retinopathy study ( etdrs ) classification . level of diabetic retinopathy was graded according to the eye with the more severe form of disease . the duration of illness was defined as the duration from the time of the diagnosis of diabetes mellitus given to the participant until the time of the examination . exclusion criteria included ocular or systemic diseases affecting the retinal vasculature ( hypertension ) , nervous system ( alzheimer disease , peripheral neuropathy , glaucoma , age - related macular degeneration , end stage renal disease ) , previous intravitreal injection(s ) , ophthalmic surgical or laser interventions , cases on medications , vitamin , or antioxidant supplements , and cases with signal strength 5 or below on the optical coherence tomography examination . the examination consisted of an explanation of the study , measurement of the blood pressure , refraction and assessment of the logmar best - corrected visual acuity ( logmar is expressed as the decadic logarithm of the minimum angle of resolution with 20/20 line equivalent to logmar 0.00 and the 20/200 line to logmar 1.0 ) , and slit - lamp biomicroscopy of the anterior segment . further , goldmann applanation tonometry , gonioscopy , and computerized static perimetry using the full threshold 242 program of the humphrey perimeter ( humphrey field analyzer , zeiss / humphrey systems , dublin , ca ) were performed . fundus examination was performed with slit - lamp biomicroscopy with a 90-diopter lens and indirect ophthalmoscopy . fundus photography was performed in all cases using a zeiss fundus camera ff 450 plus with pixel width of 0.0054 and image size 25881958 . subsequently , all the study subjects were evaluated using sd - oct ( cirrus high definition oct ; carl zeiss meditec inc . , every study subject underwent rnfl thickness analysis using the optic disc cube 200200 feature ( figure 1 ) . the average retinal nerve fiber layer thickness was noted . retinal nerve fiber thickness analysis using optic disc cube 200x200 feature depicting thickness map , thickness deviation , tsnit ( temporal , superior , nasal , inferior , temporal ) normative data and extracted tomograph of both eyes . the quadrant map shows thinning in the superior quadrant of both eyes and thinning in inferior quadrant of left eye . blood samples were collected from the study subjects in the morning after an overnight fasting interval of 68 h. the samples were drawn by vein puncture using a 5 ml metal - free plastic syringe fitted with a 24-gauge stainless steel needle ( nirlife ; nirma limited , sachana , india ) under contamination - controlled conditions , and the blood samples were collected in a 4 ml vacutainer ( vaku-8 ; hindustan syringes and medical devices limited ; faridabad , india ) that contained heparin as an anticoagulant . the volume of the samples ranged from 4 ml to 5 ml . for separation of plasma , the citrated blood was centrifuged at 200 g for 10 min , and the supernatant platelet - rich plasma was aspirated out into another plastic tube . fasting and post - prandial blood glucose levels was estimated using the glucose oxidase method . glycated hemoglobin was measured using the bio - rad d-10 ( bio - rad laboratories , inc . , hercules , ca ) that employs the high - power liquid chromatography cation exchange chromatography method . the serum folic acid assay was performed using elecsys folate iii ( roche diagnostics ; indianapolis , in ) assay method that employs a competitive test principle using natural folate binding protein ( fbp ) specific for folate . the serum vitamin b12 assay was performed using the elecsys vitamin b12 ( roche diagnostics ) assay method that employs a competitive test principle using the intrinsic factor specific for vitamin b12 . assay of homocysteine was performed using the human homocysteine enzyme - linked immunosorbent assay ( elisa ) kit ( mybiosource , inc . , san diego , ca ) . the reagents in the kit were prepared following the standard protocol provided with the kit . human homocysteine standard samples were added to corresponding wells ( 100 l for each well ) , and the 0 nmol / ml well was filled with standard diluent . serial dilutions of the homocysteine standard were performed using 50 , 25 , 12.5 , 6.25 , 3.12 , 1.56 , and 0.78 nmol / ml concentrations to the standard curve following the manufacturer s instructions and run in parallel . the serum sample was diluted ten times with the sample dilution for the best detection range of the elisa kit , and further results of the serum levels of homocysteine multiplied by 10 . standard sample diluent ( 300 l ) was added to each of the seven microtiter wells . diluent ( 300 l ) was pipetted out from one tube to another tube sequentially . reaction wells were sealed with adhesive tape and incubated at 37 c for 90 min . biotinylated human homocysteine antibody liquid was prepared 30 min in advance . biotinylated human homocysteine antibody liquid was added to each well ( 100 l for each ) . reaction wells were sealed and incubated at 37 c for 60 min . enzyme - conjugate liquid was prepared 30 min in advance . enzyme - conjugate liquid was added to each well except the blank wells ( 100 l for each ) . color reagent liquid ( 100 l ) was added to individual wells ( also to the blank well ) and incubated at 37 c . when the color of the high concentration of the standard curve became darker and the color gradient appeared , the hatching was stopped . color reagent c ( 100 l ) was added to individual wells ( also to the blank well ) and mixed well . the intensity of the color was read over the elisa plate reader ( synergy ht ; biotek , winooski , vt ) at 450 nm . the calibration curve of the standard homocysteine was plotted against the homocysteine on the y - axis and the concentration on absorbance on the x - axis . the standard curve was drawn using professional curve software ( curveexpert 1.3 ; hyams development ; madison , al ) to analyze and compute the result . the continuous variables of the study groups were compared with one - factor anova ( anova ) . distribution of variables such as visual acuity , homocysteine , and vitamin b12 was skewed . therefore , log and antilog conversions were made to analyze these variables . for pair - wise comparison between the groups , the newman the discrete ( categorical ) variables were compared with a chi - square ( 2 ) test . the logmar vision score of two groups ( npdr and pdr ) was compared with an independent student t test . all analyses were performed using statistica 6.0 software package ( dell , inc . , round rock , tx ) . assay of homocysteine was performed using the human homocysteine enzyme - linked immunosorbent assay ( elisa ) kit ( mybiosource , inc . , san diego , ca ) . the reagents in the kit were prepared following the standard protocol provided with the kit . human homocysteine standard samples were added to corresponding wells ( 100 l for each well ) , and the 0 nmol / ml well was filled with standard diluent . serial dilutions of the homocysteine standard were performed using 50 , 25 , 12.5 , 6.25 , 3.12 , 1.56 , and 0.78 nmol / ml concentrations to the standard curve following the manufacturer s instructions and run in parallel . the serum sample was diluted ten times with the sample dilution for the best detection range of the elisa kit , and further results of the serum levels of homocysteine multiplied by 10 . standard sample diluent ( 300 l ) was added to each of the seven microtiter wells . diluent ( 300 l ) was pipetted out from one tube to another tube sequentially . reaction wells were sealed with adhesive tape and incubated at 37 c for 90 min . biotinylated human homocysteine antibody liquid was prepared 30 min in advance . biotinylated human homocysteine antibody liquid was added to each well ( 100 l for each ) . reaction wells were sealed and incubated at 37 c for 60 min . enzyme - conjugate liquid was prepared 30 min in advance . enzyme - conjugate liquid was added to each well except the blank wells ( 100 l for each ) . color reagent liquid ( 100 l ) was added to individual wells ( also to the blank well ) and incubated at 37 c . when the color of the high concentration of the standard curve became darker and the color gradient appeared , the hatching was stopped . color reagent c ( 100 l ) was added to individual wells ( also to the blank well ) and mixed well . the intensity of the color was read over the elisa plate reader ( synergy ht ; biotek , winooski , vt ) at 450 nm . the calibration curve of the standard homocysteine was plotted against the homocysteine on the y - axis and the concentration on absorbance on the x - axis . the standard curve was drawn using professional curve software ( curveexpert 1.3 ; hyams development ; madison , al ) to analyze and compute the result . the continuous variables of the study groups were compared with one - factor anova ( anova ) . the kolmogorov distribution of variables such as visual acuity , homocysteine , and vitamin b12 was skewed . therefore , log and antilog conversions were made to analyze these variables . for pair - wise comparison between the groups , the newman the discrete ( categorical ) variables were compared with a chi - square ( 2 ) test . the logmar vision score of two groups ( npdr and pdr ) was compared with an independent student t test . all analyses were performed using statistica 6.0 software package ( dell , inc . , round rock , tx ) . table 1 summarizes the mean age , duration of type 2 diabetes mellitus , visual acuity ( logmar ) , fasting and post - prandial blood glucose levels , serum levels of homocysteine , vitamin b12 , and folic acid , average rnfl thickness , and central sub - field thickness in the study groups . no statistically significant difference in the mean age of the study groups was observed ( f = 1.404 , p = 0.248 ) . the chi - square test revealed similar sex proportions among all four groups ( male / female : 12/8 versus 12/8 versus 13/7 versus 10/10 , 2 = 1.152 ; p = 0.334 ) . a statistically significant difference in the mean duration of diabetes mellitus of the study groups was observed ( f = 25.95 , p<0.001 ) . the level of significance was accepted at p<0.05 . * a compared to control group , b compared to nodr group , c compared to npdr group . the mean glycated hemoglobin in the control , nodr , npdr , and pdr groups was 6.211.02% ( 33 to 56 mmol / mol ) , 6.390.41% ( 42 to 51 mmol / mol ) , 8.352.53% ( 40 to 95 mmol / mol ) , and 7.951.69% ( 45 to 82 mmol / mol ) , respectively . the difference in the glycated hemoglobin levels was statistically significant between the study groups ( f = 8.95 ; p<0.001 ) . pearson correlation analysis revealed a statistically significant correlation between glycated hemoglobin and homocysteine ( r = 0.231 ; p=0.039 ) , vitamin b12 ( r = 0.363 ; p = 0.001 ) , folic acid ( r = 0.358 ; p = 0.001 ) , and average rnfl thickness ( r = 0.357 ; p = 0.001 ) . the mean blood pressure ( systolic/ diastolic ) levels in the control , no dr , npdr , and pdr groups were 114/74 , 126/78 , 134/82 , and 144/86 mmhg , respectively . in the comparison of visual acuity , anova revealed a statistically significant difference in visual acuity in each group ( f = 47.192 , p<0.0001 ) . the anova showed that the difference in the serum levels of homocysteine was statistically significant between the study groups ( f = 53.79 ; p<0.001 ; figure 2 ) . the anova also showed that the difference in average rnfl thickness and central sub - field thickness was statistically significant between the study groups ( p<0.001 and p<0.001 , respectively ) . the serum levels of folic acid and vitamin b12 were observed to be within the normal reference range ( 220 ng / ml and 200900 pg / ml , respectively ) . box and whisker plot graphically representing the descriptive statistics for serum levels of homocysteine among the study groups . the horizontal line within the box indicates the median , boundaries of the box indicate the 25th- and 75th -percentile , and the whiskers indicate the highest and lowest values . an increase in serum level of homocysteine with increase in severity of diabetic retinopathy pearson correlation analysis revealed a statistically significant negative correlation between homocysteine with average rnfl thickness ( r = 0.315 ; p = 0.004 ; figure 3 ; table 2 ) and a positive correlation with central sub - field thickness ( r = 0.240 ; p = 0.032 ) . scatter plot showing a negative correlation between the serum levels of homocysteine and the average retinal nerve fiber layer thickness observed on pearson correlation analysis . this study was conducted to determine the correlation between the serum levels of homocysteine , vitamin b12 , and folic acid and alterations in rnfl thickness in cases of diabetes mellitus . a previous study by corra et al . found an association between a risk factor such as the duration of diabetes mellitus and the severity of retinopathy . in the present study , a statistically significant difference existed among the study groups in the comparison of the duration of diabetes . it was concluded that increased duration of diabetes mellitus is associated with the increased severity of diabetic retinopathy . in this study , visual acuity was found to decrease with the increase in the severity of retinopathy . homocysteine is a by - product of transmethylation reactions and is detoxified by methionine synthetase , which depends on vitamin b12 and folate as coenzymes for proper function . the role of homocysteine has been induced in several microvascular complications of diabetes mellitus , including diabetic retinopathy . found significantly raised fasting plasma levels of homocysteine in patients with diabetes mellitus with microalbuminuria or proliferative retinopathy that were not attributable to confounders , such as age , sex , smoking , or dissimilar plasma folate and vitamin b12 concentrations . several other studies also reported a statistically significant increase in the total plasma level of homocysteine and a statistically significant decrease in the serum levels of vitamin b12 and folic acid in diabetic retinopathy [ 18 - 28 ] . our study demonstrated that the difference in the serum level of homocysteine was statistically significant between the study groups . although the mean serum levels of folic acid and vitamin b12 among the cases were found to be within the normal reference range , the values were on the lower side of normal . the mean serum levels of folic acid and vitamin b12 in the controls were found to be on the higher side of the normal reference range . it can be concluded that increased serum levels of homocysteine are associated with increased severity of diabetic retinopathy . the mechanism of action of homocysteine leading to retinal neurodegeneration has been studied in several animal models . several studies suggested that homocysteine induces apoptosis in retinal ganglion cells with the expression of bax ( bcl-2-associated x protein ) , a proapoptotic protein , as it was found in increased levels in diabetic retinas . in addition , increased levels of terminal nick - end labeling positive cells that correlated with increased levels of caspases ( the enzymes involved in apoptosis ) in the neural retinas of diabetic rats have been found . ganapathy et al . concluded that homocysteine - induced ganglion cell loss involves the dysregulation of mitochondrial dynamics , in vivo and in vitro . disruption of glutamate homeostasis in the diabetic retina initiates the development of diabetic retinopathy [ 35 - 37 ] . the major cause of neuronal cell death following glutamate - induced activation of n - methyl d - aspartate ( nmda ) receptors is generation of free radicals that induce apoptosis . therefore , strategies for decreasing the level of extracellular glutamate or inhibiting the activation of nmda receptors may decrease neurotoxicity and cell death . in vitro studies of retinal ganglion cells and in vivo studies in the brain , flash electroretinography , contrast sensitivity , color vision , and short - wavelength automated perimetry have demonstrated functional deficits in the neuronal component of the diabetic retina . our study for the first time provides clinical evidence of a correlation between increased serum levels of homocysteine and rnfl damage in an in vivo human diabetic retina . the difference in the average rnfl thickness levels was statistically significant between the study groups . increased severity of retinopathy was associated with decreased average rnfl thickness that can be attributed to increased levels of homocysteine . the severity of diabetic retinopathy has been reported to correlate with macular thickness parameters on sd - oct . mean macular thickness , retinal thickness , foveal thickness , and central macular thickness have been shown to correlate with the severity of diabetic retinopathy . a recent study we conducted demonstrated that central sub - field thickness is associated with the severity of diabetic retinopathy . results of the present study are also in concordance with the findings of this previous research . the novelty of our study lies in the demonstration of a correlation between increased serum levels of homocysteine and in vivo retinal nerve fiber layer thinning in the human diabetic retina . a correlation between increased serum levels of homocysteine and increased severity of retinopathy was also found . the novelty of our study lies in the demonstration of a correlation between increased serum levels of homocysteine and in vivo retinal nerve fiber layer thinning in the human diabetic retina . a correlation between increased serum levels of homocysteine and increased severity of retinopathy was also found .

purposeto study the correlation between serum levels of vitamin b12 , folic acid , and homocysteine and the severity of diabetic retinopathy and the correlation with retinal nerve fiber layer ( rnfl ) thinning on spectral domain optical coherence tomography ( sd - oct).methodsin a tertiary care center based prospective cross - sectional study , 60 consecutive cases and 20 healthy controls in the age group of 4065 years were included . the eyes of the cases were divided into three groups according to early treatment diabetic retinopathy study ( etdrs ) classification : diabetes mellitus without retinopathy ( n = 20 ) , non - proliferative diabetic retinopathy with macular edema ( n = 20 ) , and proliferative diabetic retinopathy with macular edema ( n = 20 ) . the serum levels of vitamin b12 and folic acid were measured using a standard protocol . the serum homocysteine assay was performed using an enzyme - linked immunosorbent assay ( elisa ) kit . average rnfl thickness was measured using sd - oct . statistical analysis was used to assess the correlations between the study variables.resultsincreased severity of diabetic retinopathy was found to correlate with an increase in the serum levels of homocysteine ( f = 53.79 ; p<0.001 ) . the mean serum levels of vitamin b12 and folic acid were found to be within the normal reference range . a positive correlation was found between retinal nerve fiber layer thinning and serum levels of homocysteine ( p<0.001).conclusionsthis study , for the first time , demonstrated a correlation between increased homocysteine with a decrease in rnfl thickness and increased severity of diabetic retinopathy .

, it was estimated that in the usa there were nearly 210,000 new cases of invasive breast cancer and more than 40,000 deaths . axillary node status is a major prognostic factor in early - stage disease , and this information is important for treatment . axillary involvement is found in 10%30% of patients with t1 ( < 2 cm ) tumours . this rate reaches 45% for small t2 tumours ( 2.13 cm ) and 55%70% for larger tumours routine axillary lymphadenectomy adds the risk of lymphedema , sensory disturbances , and chronic pain . sentinel lymph node biopsy is a minimal invasive method of checking the potential nodal involvement . it is based on the assumption of an orderly progression of lymph node invasion by metastatic cells from tumour site . thus , the nodal basin is free of malignancy if the sentinel lymph node is not involved . patients with metastasis to a sentinel node would undergo either immediate or delayed completion lymph node dissection . randomized trials demonstrated a marked reduction of complications associated with the sentinel lymph node biopsy when compared with axillary lymph node dissection . in the almanac trial , more than 1,000 patients were randomized to undergo either axillary lymphadenectomy or sentinel node biopsy . lymphedema was present in 13% of the axillary lymphadenectomy group and in 5% of the sentinel node group 12 months after surgery . in 2005 , guidelines from the american society of clinical oncology stressed that a multidisciplinary team should aim at a sentinel node identification rate of 85% with a false - negative rate of 5% or less in order to abandon axillary dissection . false - negative rate is the proportion of axillary node dissection , positive cases with a negative sentinel node at biopsy . false - negative cases may result from massive involvement of the real sentinel node , a circumstance that interferes with the uptake of both radiocolloid and dye and lymph flow that goes to a node other than the true sentinel node . a meta - analysis of 69 trials with a total of 8,059 patients in whom sentinel node biopsy was followed by axillary dissection showed substantial variability in the performance of the technique throughout different centers . however , recent results from large multi - institutional trials showed that all have achieved excellent identification rates , ranging from 93% to 97% , but that none achieved a false - negative rate lower than 5% . the lowest false - negative rates were obtained in the 2 studies in which preoperative lymphoscintigraphy and dual mapping during surgery were required . however , if only blue sentinel nodes are considered , the false - negative rate was 9.1% [ 25 ] . clinical indications for this approach have been changing through the years and there is still a debate on some of them . many centers use sentinel node biopsy only in patients with a unifocal tumour smaller than 3 cm , whereas others have extended the application to patients with large t2 or t3 ( > 5 cm ) tumours , multifocal / multicentric carcinomas , or to patients who have received neoadjuvant chemotherapy . currently , the sentinel node biopsy procedure is recognized as the standard treatment for stages i and ii . in these stages , this approach has a positive node rate similar to those observed after lymphadenectomy , a significant decrease in morbidity and similar nodal relapse rates at 5 years . no significant differences on disease - free survival , overall survival , and local control of disease were seen in case of negative sentinel node . ( i ) pregnancy is no contraindication for sentinel node biopsy , but only for blue dye , and it has been demonstrated that the dose to the foetus from this procedure is negligible . ( ii ) the evidence regarding the safety of sentinel node biopsy is mainly based on studies including t1 and small t2 tumours only . however , in patients with larger tumours ( t3-t4 ) , the false negative rate has been similar and no increased axillary recurrence has been reported [ 6 , 7 ] . ( iii ) multifocal breast cancer is defined as separate foci of ductal carcinoma more than 2 cm apart within the same quadrant , while multicentric breast cancer indicates the presence of separate independent foci of carcinoma in different quadrants . the prevalence of axillary metastases and false negative results is higher in multifocal or multicentric tumours . however , the reported axillary recurrence rates are acceptable also in patients with multifocal or multicentric tumours [ 69 ] . however , invasion is missed in up to 40% of patients in the preoperative diagnosis . therefore , sentinel node biopsy is recommended in patients undergoing mastectomy . in patients with breast conservation , sentinel lymph node biopsy can be performed as a second operation if invasion is detected in the surgical specimen [ 6 , 10 ] . ( v ) palpable axillary nodes may be tumour negative in up to 40% of the patients . preoperative axillary ultrasound with fine needle aspiration cytology or core needle biopsy from the suspicious nodes is a widely accepted policy . in many units , sentinel node biopsy is performed also in patients with palpable nodes if negative in the preoperative diagnosis . ( vi ) internal mammary sentinel node detection rate is significantly affected by the depth of radiopharmaceutical injection . it is generally recognized that mapping of inner mammary chain requires deep injection ( peritumoural or intratumoural ) of radiotracer . with this approach , internal mammary chain sentinel nodes have been detected in about 30% of patients with breast cancer , of which about 60%90% could be harvested during surgery and 11%27% of them will have metastases . however , the significance of internal mammary sentinel node biopsy is under debate . there is evidence that mapping it leads to stage migration and modification of treatment planning with respect to radiotherapy and systemic therapy , but more evidence is necessary to support that it will improve the outcome of treatment and survival [ 11 , 12 ] . a second sentinel node biopsy may be performed in patients with a local recurrence after breast conservation and negative axillary sentinel node biopsy . sentinel node biopsy can be performed in patients undergoing breast surgery due to a local recurrence after breast conservation in dcis . furthermore , plastic surgery with breast augmentation or reduction does not contraindicate the procedure . in prior excisional biopsy the lymph drainage is probably changed in patients who have undergone previous breast surgery ( oncologic and nononcologic ) . extra - axillary drainage is identified more frequently in reoperative sentinel node biopsy than in former sentinel node biopsy . however , there are evidences that sentinel node biopsy performed in the area of previous breast biopsy do not affect the accuracy of the procedure [ 6 , 13 ] . ( vii ) before neoadjuvant chemotherapy , sentinel node biopsy gives a more precise axillary staging , with more information about the nodal spread . but it may delay the beginning of the therapy , and two surgeries can be necessary . after neoadjuvant chemotherapy , the sentinel node biopsy may lead to an underestimation of the initial stage . on the other hand , pathologic complete response in the axilla can be achieved in up to 40% of the patients . available data show that there are no significant differences in the success rate of sentinel node biopsy according to clinical tumour size or clinical nodal status , and that the false - negative rate is not affected by tumour response to chemotherapy [ 14 , 15 ] . despite this , the current controversy in this scenario lies on the question of axillary lymph node dissection after a positive sentinel node biopsy , mainly stimulated by the recent publication of the acosog - z0011 data . the sentinel node procedure uses a radiotracer , a blue dye , or both to find the node . these colloids allow sentinel node visualization with a gamma camera before surgery and intraoperative detection with a hand - held gamma probe . controversies exist with regard to the selection of agents , the size of the particles of the radiotracer , the optimal route for injection , time to scintigraphy and intraoperative detection , and whether or not extra - axillary lymph nodes should be considered as well . mariani et al . suggested that tc - labeled colloids with most of the particles in the 100 to 200 nm size range would be ideal for sentinel node biopsy in breast cancer . tc - labeled colloids of human serum albumin are often used in europe , tc - sulfur colloid is used in the united states ( sometimes after filtration through a 0.1 or 0.2 mm membrane ) , and tc - antimony trisulfide in australia . there is no established difference between a 1-day protocol ( same - day imaging and surgery ) and a 2-day protocol . after an experience of almost 20 years , it is generally assumed that both deep and superficial injection approaches are valid techniques and may be complementary . in most early sentinel node studies , the tracer was injected around the tumour and such peritumoral injection was considered the gold standard against which all other mapping techniques were tested . many investigators have reported good results using injection into the breast skin over the tumour or using a periareolar , subareolar , or even intratumoural injection . one clearly established advantage of deep injections ( peritumoral / intratumoural ) is its ability to also reveal extra - axillary drainage . on the other hand , superficial injection techniques ( subdermal / areolar ) provide a faster lymphatic drainage , yield more radioactive counts at the axillary sentinel nodes , and are independent of the palpable or nonpalpable nature of the tumour . hence , the tracer is not always transported to the same axillary node , regardless the injection site . however , if the goal is axillary staging only , a superficial tracer injection is preferable due to better visualization of axillary sentinel nodes . if the aim is to stage also the extra - axillary nodal basins , peri- or intratumoural injection should be applied [ 6 , 17 , 18 ] . lymphoscintigraphy has been an essential component for the preoperative sentinel node identification in breast cancer . lymphoscintigraphy has the potential to both improve accuracy and reduce morbidity relative to gamma probe alone by providing the surgeon with a roadmap of lymphatic drainage and the location of sentinel nodes . to identify all sentinel nodes and avoid confusion with a stasis in a lymphatic vessel , with planar scintigraphy , combining 2 views may help prevent some sentinel nodes from being missed ( figure 1 ) . the functional information from spect can be combined with the morphological information from ct by applying both techniques in one session . the resulting spect / ct fused images depict sentinel nodes in an anatomical landscape providing a helpful roadmap for surgeons . in recent years , spect / ct has been used in breast cancer patients with unusual or complex drainage . spect / ct can also detect hot nodes missed by planar imaging because of shine - through from the injection site or in overweight patients [ 19 , 20 ] ( figure 2 ) . differentiating a true sentinel node from a secondary echelon node also , lymphatics of a tumor site can drain simultaneously to more than one sentinel node . lymphoscintigraphy is able to identify sentinel nodes in a majority of cases by acquiring early and delayed planar images . in current protocols , spect / ct is performed following delayed planar images ( mostly 24 hours after tracer administration ) . however , it is necessary to specify the criteria for sentinel node identification on preoperative images . major criteria to identify lymph nodes as sentinel nodes are the visualization of lymphatic ducts , the time of appearance , the lymph node basin , and the intensity of lymph node uptake . following these criteria visualized radioactive lymph nodes may be classified as followsdefinitively sentinel nodes : this category concerns all lymph nodes draining from the site of the primary tumour through an own lymphatic vessel , or a single radioactive lymph node in a lymph node basin . highly probable sentinel nodes : this category includes lymph nodes appearing between the injection site and a first draining node , or nodes with increasing uptake appearing in other lymph node stationsless probable sentinel nodes : all higher echelon nodes may be included in this category . definitively sentinel nodes : this category concerns all lymph nodes draining from the site of the primary tumour through an own lymphatic vessel , or a single radioactive lymph node in a lymph node basin . highly probable sentinel nodes : this category includes lymph nodes appearing between the injection site and a first draining node , or nodes with increasing uptake appearing in other lymph node stations less probable sentinel nodes : all higher echelon nodes may be included in this category . the use of these categories to characterize radioactive lymph nodes is also helpful for clinical decision making . lymph nodes of the first two categories ( definitively sentinel node or highly probable sentinel node ) are the nodes recognized by the nuclear physician and that must be removed at the operation room by the surgeon . less probable sentinel nodes may sometimes be removed depending on the degree of remaining radioactivity measured by the gamma probe during the control of the surgical field . isosulfan blue is of greater use in the united states , and patent blue v in europe . data from nsabp b-32 and acosog - z0010 with isosulfan , and from almanac with patent blue v , showed that the overall risk of allergic reaction is close to 1% for both dyes , with an approximately 0.1% risk of severe reactions ( grade iii ) . despite a risk of allergic reactions to blue dye , most teams favour the combinative mapping procedure . in one multicentric study , the false - negative rate was 17.7% if only 1 node was resected , 10% if 2 , 6.9% if 3 , 5.5% if 4 , and 1% if 5 or more . these results should not imply removal of multiple nodes for an optimal sentinel node procedure . careful palpation by the surgeon of the operative field is also required to identify any suggestive large , hard nonblue and nonradioactive nodes . during the last decade , intraoperative imaging devices have become available for clinical practice and can be used during surgery as they provide information that can be combined with data obtained with conventional gamma probes . however , since nonimaging probes are still the standard equipment for detection of radiolabeled tissue in the operating room , the role of intraoperative imaging is generally limited and constitutes an additional aid to the surgeon . intraoperative imaging with portable gamma cameras provides real - time imaging with a global overview of all radioactive hot spots in the whole surgical field . its position can be adjusted to also show sentinel nodes near the injection area , which can easily be missed when using the non - imaging probe . some authors have tried to clarify the added value of portable gamma camera in clinical practice . in fact , there is no consensus on the real need for an intraoperative imaging device to help detection of the sentinel lymph node . the usefulness of the portable gamma cameras in breast cancer patients lies on when no conventional gamma camera is available or in particular cases with extra - axillary drainage ( intramammary and internal mammary chain nodes ) or when the sentinel lymph node is located very close to the injection site . although the majority of these cases can be solved with the presurgical information provided by spect / ct , real - time images acquired with a portable gamma camera can be an alternative to hybrid imaging . on the other hand , the preoperative anatomical information obtained by spect / ct appears to lead to a more optimal use of portable devices for sentinel localization in the operation room . using an intraoperative imaging device implies the possibility to better planning the procedure and to monitor the lymphatic basin before and after removal of the hot nodes , so to verify completeness of lymph node excision . after excision of each lymph node , a new image is acquired and compared with the situation before excision . if focal radioactivity remains at the same location , it is concluded that another possible sentinel lymph node is still in place [ 22 , 23 ] . thanks to novel technological possibilities , combining a spatial localization system and two tracking targets to be fixed on a conventional , hand - held gamma probe results in new 3d visualization of the traditional acoustic signal of the gamma probe . in this regard , the most recent development is the system so - called free - hand spect , in which a continuous positioning system installed in the operating room is based on a fix - pointing device , on the patient 's body and , respectively , on the hand - held gamma counting probe , thus permitting a virtual reconstruction in a 3d environment . the surgeon can easily check location and depth of the foci of radioactivity accumulation to be resected , and this 3d information may be further used for precise localization and targeting of the radioactive sentinel lymph node(s ) and of tumour tissue . the device can ensure permanent assistance and transparent documentation of soft tissue removal during the intervention . on the other hand , the possibility of combining the current radiopharmaceuticals with other agents opens new fields to explore . in this regard , a radiolabeled nanocolloid agent has been combined with icg , a fluorescent agent , for sentinel node detection . in contrast to the use of a single - fluorescent agent , this bimodal tracer may allow the surgeons to integrate the standard approach based on radioguided detection with a portable gamma camera with a new optical modality based on fluorescent signal detection . this approach is being successfully applied in several malignancies and to localize sentinel nodes outside the axilla in breast cancer ( figure 3 ) . for all these new intraoperative modalities , the preoperative anatomical spect / ct acquisition remains essential and is the starting point for surgical planning . before sending for histological examination , any lymph node removed should be rechecked by the probe to demonstrate that they are radioactive . histopathological assessment of the sentinel lymph node is the golden standard procedure for the subsequent management of the conservative surgery in breast cancer patients . the sentinel nodes are assessed intraoperatively using imprint cytology , frozen sectioning , or both , and more thoroughly after the operation . the sensitivity of the intraoperative diagnosis in variable and many units do not adopt it at all . some molecular methods have been used previously for sentinel node diagnosis but have shown a lack of reproducibility , a longer time for the intraoperative assessment , and an inability to study the whole lymph node . a new molecular method has been developed recently , based on an one - step nucleic acid amplification ( osna ) method . this procedure is in the phase of validation in many centres , although there are others that routinely apply this method . in summary , after two decades of sentinel lymph node biopsy use in breast cancer , this technique is the current standard of care for locoregional staging . however , some concerns remain as there is no only one standardized technique and many controversies are still unsolved . however , with the recent technologic and histologic developments , a more accurate and refinate technique can be achieved by virtually identifying-100% of sentinel nodes and reduce the false negative rate .

axillary node status is a major prognostic factor in early - stage disease . traditional staging needs levels i and ii axillary lymph node dissection . axillary involvement is found in 10%30% of patients with t1 ( < 2 cm ) tumours . sentinel lymph node biopsy is a minimal invasive method of checking the potential nodal involvement . it is based on the assumption of an orderly progression of lymph node invasion by metastatic cells from tumour site . thus , when sentinel node is free of metastases the remaining nodes are free , too ( with a false negative rate lesser than 5% ) . moreover , randomized trials demonstrated a marked reduction of complications associated with the sentinel lymph node biopsy when compared with axillary lymph node dissection . currently , the sentinel node biopsy procedure is recognized as the standard treatment for stages i and ii . in these stages , this approach has a positive node rate similar to those observed after lymphadenectomy , a significant decrease in morbidity and similar nodal relapse rates at 5 years . in this review , the indications and contraindications of the sentinel node biopsy are summarized and the methodological aspects discussed . finally , the new technologic and histologic developments allow to develop a more accurate and refinate technique that can achieve virtually the identification of 100% of sentinel nodes and reduce the false negative rate .

nonalcoholic fatty liver disease ( nafld ) has become a global health problem in both adults and children , the prevalence of which has doubled during last 20 years in tune with the increasing incidence of obesity and insulin resistance . improper regulation of lipogenesis and lipid oxidation is a prime factor in the genesis of fatty liver . lipogenesis encompasses the processes of fatty acid synthesis and subsequent formation of triglyceride ( tg ) . sterol regulatory elemental binding protein-1c ( srebp-1c ) belongs to a family of transcription factors that are sensitive to cellular availability of cholesterol and promotes fatty acid synthesis and lipid deposition . srebp-1c regulates lipogenesis by increasing the expression of genes involved in fatty acid biosynthesis such as acetyl coa carboxylase , fatty acid synthase , and stearoyl coa desaturase . srebp-1c has been implicated in the development of human metabolic disorders like obesity , type 2 diabetes , dyslipidemia , atherosclerosis , lipodystrophy , and metabolic syndrome . lds , long considered as inert substances , are now recognized for their dynamic role in lipid metabolism . they are structurally similar to circulating lipoproteins , consisting of a core of esterified lipids ( tg , cholesterol ester , retinol esters , or ether lipids , depending on the cell type ) covered by a phospholipid monolayer , free cholesterol , and a coat of specific proteins [ 3 , 4 ] . the ld proteins include the founding member perilipin ( plin1 ) , adipophilin ( plin2 , also known as adipocyte differentiation - related protein ( adrp ) ) , tail - interacting protein of 47 kilo daltons ( tip47 , plin3 ) , s3 - 12 ( plin4 ) , and myocardial lipid droplet protein ( plin5 ) . these are collectively known as the pat family of proteins named after perilipin , adipophilin , and tip47 . another protein termed fat - specific protein 27 ( fsp27 ) , although not a member of pat family proteins , is recognized to be a structural protein present in ld and is specifically involved in diet - induced fatty liver . ld proteins are involved in lipid metabolism and transport , intracellular trafficking , signaling , and cytoskeletal organization [ 79 ] . ld proteins also regulate lipolysis in adipose tissue by modulating the access of hormone sensitive lipase ( hsl ) to lipid surface . the ld proteins also serve to regulate lipolysis of tg rich droplets in response to insulin and adrenergic stimulation . peroxisome proliferator - activated receptor- ( ppar- ) is a nuclear receptor protein and is the first member of ppar family cloned in 1990 . among the three isoforms of ppar family- , , and , ppar- is predominantly present in liver and is involved in the activation of hepatic lipid catabolism by targeting genes involved in cellular fatty acid uptake and transport , mitochondrial and peroxisomal fatty acid uptake , and -oxidation . among the various endogenous ligands for ppar- , long - chain fatty acids are the most studied . thus ppar- serves as a lipid sensor and directs lipid metabolism towards oxidation when upregulated by agonists . grape seed proanthocyanidins ( gsp ) is a complex mixture of oligomeric compounds possessing high antioxidant activity with preventive effects on some forms of cancer and oxidative injury . proanthocyanidin is richly present in red wine , and its content in red wine is reported to be 117.18 96.06 mg the first clue to its lipid - lowering effect came from the french paradox , which refers to the lower mortality rates of cardiovascular disease despite high intake of saturated fats due to red wine consumption . in fact studies have shown that gsp can lower postprandial plasma tgs and apolipoproteins levels in healthy rats . the insulin - sensitizing agent metformin ( met ) is used as a treatment strategy in nafld patients due to the role of insulin resistance in the pathogenesis of nafld . since nafld is a multifactorial disease , combination therapy is recommended to address the individual components like hyperlipidemia and insulin resistance . we hypothesize that gsp may help to increase the oxidation of lipids by reducing the lipogenic pathway and lipid storage while met may improve the hepatic insulin sensitivity . hence , in this study , we compared the effects of gsp and met individually and in combination against diet - induced nafld . proanthocyanidin - rich extract from grape seed ( gsp , gravinol - super ) was kindly provided by kikkoman co. ( chiba , japan ) . the grape seed extract contained 89% proanthocyanidin , 6% monomers , and 5% other materials . the normal rat feed was obtained from sai enterprise , chennai , india , which contained 60% starch , 22.08% protein , and 4.38% fat . this commercial diet provided 382.61 cal/100 g. the high - fat / fructose diet ( hffd ) prepared in our laboratory contained 45% fructose , 20% fat , ( 10% beef tallow , 10% groundnut oil ) , and 22.5% casein and provided 471.25 cal/100 g. this study was conducted in strict accordance with the guidelines of the committee for the purpose of control and supervision on experiments on animals ( cpcsea ) . all procedures were approved and adhered to the guidelines of the institutional animal ethical committee ( iaec ) . six - week - old male albino wistar rats ( n = 6 ) were purchased from rajah muthiah medical college and hospital ( rmmc and h , annamalai university ) and left for acclimatization in the department animal house for a week . obesity was induced in rats by feeding hffd . therapeutic intervention with gsp or met or combination of both was initiated after 30 days of hffd feeding . gsp ( 100 mg / kg b.w / day in water ) and met ( 50 mg / kg b.w / day in water ) were administered orally once a day to the respective groups from the 31st day till the 45th day . for combination treatment , gsp and met were administered at an interval of 4 hours . at the end of the experiment , the rats were fasted for 18 h. after decapitation , blood samples were collected in tubes containing edta and centrifuged to obtain plasma . oral glucose tolerance test ( ogtt ) was carried out on the 44th day after an overnight fast ( 12 hours ) as described elsewhere . for this , animals were given glucose ( 2 g / kg b.w , oral ) after collecting fasting blood samples . additional blood samples were drawn after one hour and two hours by sinoocular puncture in heparinized test tubes and centrifuged at 3000 g for 10 minutes to separate plasma . plasma insulin was assayed using an enzyme - linked immunosorbent assay kit ( accubind , monobind chemicals , ltd . , total lipids , extracted with chloroform - methanol mixture ( 2 : 1 ( v / v ) ) from liver , were evaporated to dryness and used for the estimation . estimation of cholesterol , tg , and ffas in plasma and liver was carried out following procedures described earlier . high density lipoprotein cholesterol ( hdl - c ) was determined following the kit procedure ( agappe diagnostics ) and low density lipoprotein cholesterol ( ldl - c ) and very low density lipoprotein cholesterol ( vldl - c ) were calculated using the following equations : vldl ( mg / dl ) = tg/5 ; ldl ( mg / dl ) = total cholesterol(hdl - c + vldl - c ) . total rna was isolated from rat liver using the trizol reagent ( invitrogen , carlsbad , ca , usa ) . rna concentration was assessed in a biophotometer ( eppendorf , hamburg , germany ) and reverse - transcribed using m - mulv - reverse transcriptase ( fermentas ) and oligo dt primers ( invitrogen ) . cdna was quantified using spectrophotometer ( eppendorf ) and amplified using the maxima sybr green qpcr master mix ( fermentas , pittsburgh , usa ) . amplification of target genes detailed in table 1 was performed using realplex mastercycler ( eppendorf ) using gapdh as endogenous control . the amount of target gene , normalized to gapdh and relative to a calibrator , was determined by the arithmetic formula 2 by the comparative ct method . histologic analyses of liver were performed after liver tissue samples were fixed at room temperature in 4% formaldehyde and embedded in paraffin . five - micrometer sections were mounted on glass slides , deparaffinized in xylol , and stained for hematoxylin and eosin to evaluate steatosis and inflammation . localization of lipids was performed by oil red o staining of frozen - liver sections , followed by counterstaining with hematoxylin for visualization of the nuclei . the statistical significance of differences between groups was determined by one - way anova followed by the duncan 's multiple comparison tests . ogtt results ( figure 1(a ) ) show a rise in glucose levels in hffd - fed rats by 2.8- , 2.9- , and 2.3-fold in plasma samples collected at 0 , 60 , and 120 min , respectively , after an oral glucose challenge compared to con . combination of gsp and met showed a normal response and an additive improvement towards glucose load . isi0,120 ( figure 1(b ) ) , a measure of insulin sensitivity , assessed using the ogtt values , was significantly decreased in hffd - fed rats as compared to con rats . the values of isi0,120 were 2.13- and 2.45-fold , respectively , in gsp and met group compared to hffd . combination of gsp and met showed additive improvement in isi values ( by 2.97-fold ) compared to hffd . plasma and hepatic cholesterol , tg , and ffa levels are given in figures 2(a)2(c ) and figures 3(a)3(c ) , respectively . hffd - fed rats showed increased cholesterol ( 2-fold in plasma and 2.3-fold in liver ) , tg ( 3-fold in plasma and liver ) , and ffa ( 2.6-fold in plasma and 2.5-fold in liver ) compared to con rats . these were reduced effectively and significantly after treatment with gsp in plasma ( by 29% , 49% , and 45% of cholesterol , tg and ffa , resp . ) and liver ( by 46% , 45% , and 45% of cholesterol , tg and ffa , resp . ) compared to hffd - fed rats . met was not much effective compared to gsp although it reduced the lipids levels in the plasma ( by 8% , 10% , and 10% in cholesterol , tg and ffa , resp . ) and liver ( by 25% , 29% , and 15% of cholesterol , tg and ffa , resp . ) compared to hffd group . combination of met with gsp in hffd - fed rats showed marked improvement in plasma ( by 27% , 44% and 41% of cholesterol , tg and ffa , resp . ) and liver ( by 40% , 38% , and 42% of cholesterol , tg and ffa , resp . ) compared to hffd group of rats . plasma lipoproteins , namely , hdl - c , ldl - c , and vldl - c were analyzed , and the values are presented in figures 4(a)4(c ) . hffd - fed rats showed 55% reduction in hdl - c and 5.1- and 3-fold increase in ldl - c and vldl - c values , respectively , compared to control rats . compared to that of hffd - fed rats , 0.9- fold increase in hdl - c and 52% and 47% decrease in the ldl - c and vldl - c were observed during gsp treatment . met showed 37% improvement in hdl - c and 16% and 8% decrease in ldl - c and vldl - c , respectively , whereas the combination of met with gsp showed similar effects to that of gsp alone compared to hffd - fed rats ( 99% increase in hdl - c and 49% and 44% decrease in ldl - c , and vldl - c resp . ) . mrna expression of the lipogenic transcription factor , srebp-1c ( figure 5(a ) ) , and the rate limiting enzyme in cholesterol biosynthesis and hmg coa reductase ( figure 5(b ) ) , was found to be 6.4- and 4.5-fold , respectively , in liver of hffd - fed rats compared to con rats . this increase was attenuated significantly by gsp and met supplementation . with respect to con , the expression levels of srebp1c and hmg coa reductase were 3.7- and 3.2- fold in gsp and 5- and 3.8-fold in met , respectively . combination of met and gsp showed similar effects as that of gsp in mrna expression of srebp-1c and hmg coa reductase ( see figures 5a and 5b in supplementary material available online at http://dx.doi.org/10.1155/2013/153897 ) . mrna expression of the fat sensor and ppar- was found to be markedly lower ( by 68% ) in liver of hffd - fed rats compared to con ( figure 6 ) . + gsp , the expression was improved by 56% compared to hffd indicating improved fatty acid oxidation despite continuous hffd feeding . mrna expression of ppar- in met administered rats was improved by 37% compared to hffd which is less than that induced by gsp alone . in met and gsp treatment group , ppar- expression increased by 54% with respect to hffd group ( see figure 6 in supplementary material available online at http://dx.doi.org/10.1155/2013/153897 ) . increase in the mrna expression of genes specific to ld proteins like perilipin , adipophilin , tip47 , and fsp27 by 3.5- , 7.8- , 4.1- and 6.4-folds , respectively , was observed during hffd feeding compared to con . however , treatment groups showed reduced expression of ld proteins , compared to hffd groups . the expression of perilipin , adipophilin , tip47 , and fsp27 was 1.4- , 3.5- , 1.9- and 2.5-folds in hffd + gsp , and 2.5- , 5.5- , 3.5- and 4.9-folds in hffd + met with respect to con . in gsp + met group , the expression of perilipin , adipophilin , tip47 , and fsp27 was 1.5- , 3.7- , 1.9- and 2.5-folds respectively , with respect to con ( figure 7 ) ( see figures 7a , 7b , 7c and 7d in supplementary material available online at http://dx.doi.org/10.1155/2013/153897 ) . the extent of inflammation and steatosis was examined histologically in liver sections using hematoxylin and eosin staining . the representative photographs show ( figure 8) severe micro- and macrovesicular fatty changes with dense perivascular inflammatory infiltration in tissues from hffd - fed rats ( figure 8(b ) ) . upon gsp administration to hffd - fed rats , the inflammatory infiltration is reduced to a minimum with mild steatosis ( figure 8(c ) ) . hffd + met group of rats show moderate amount of inflammatory infiltration with microvesicular degeneration of hepatocytes ( figure 8(d ) ) . hffd + gsp + met group of rats show hepatocytes with marked reduction of inflammation and steatosis ( figure 8(e ) ) . liver architecture of gsp - treated control rats ( figure 8(f ) ) appears normal as that of control rat liver ( figure 8(a ) ) . figure 9 shows accumulation of lds in liver sections examined by oil red o staining . sections from liver tissue of hffd - fed rats show severe steatosis and ballooning degeneration of hepatocytes ( figure 9(b ) ) . hffd + met showed moderate steatosis ( figure 9(d ) ) , whereas hffd + gsp showed minimal amount of steatosis ( figure 9(c ) ) . hffd + gsp + met showed hepatocytes with occasional areas of steatosis ( figure 9(e ) ) . con ( figure 9(a ) ) and con + gsp ( figure 9(f ) ) show liver sections with normal lipid distribution . the present study examined whether the supplementation of gsp , met , or both could exert protective effects against hffd - induced obesity and nafld in wistar rats . the expression profiles of genes related to lipogenesis and fatty acid oxidation in liver were examined . the two , administration of gsp showed more favorable effect on hepatic steatosis in hffd - induced obese rats as compared to met . . altered plasma lipid profile and lipoprotein abnormalities like decreased hdl - c and increased ldl - c , and vldl - c were observed in hffd - fed rats . accumulation of tg , a chief matrix of the lipid droplets in hepatocytes , is accelerated under conditions such as insulin resistance or hypertriglyceridemia , if left without appropriate treatment . accumulation of intracellular lds in nonadipose tissues is recognized as a strong prognostic factor for the development of insulin resistance in obesity . a recent study has shown that reducing adrp and tip47 in the liver via antisense oligonucleotide treatment attenuated steatosis and improved insulin sensitivity and glucose metabolism in c57bl/6j mice fed with high - fat diet . knockdown of fsp27 in the ob / ob mouse liver partially improved the fatty liver suggesting that fsp27 plays a vital role in the development of liver steatosis . given the importance of pat family proteins in nafld , we investigated their status in hffd - fed rats , and in the treatment groups . in hffd rats we observed that gsp reduces the levels of the ld - specific proteins . also , considering the reduced tg content in liver and plasma and liver histology using oil red o , it is clear that gsp has the ability to reduce tg storage and ld formation . histological examination of liver using hematoxylin and eosin and oil red o staining showed increased accumulation of inflammatory cells and lds , with macrovesicular fatty changes and hepatocyte ballooning in liver of hffd group of rats . gsp administration to nafld rats reduced steatosis to microvesicular fatty changes and inflammatory cell infiltration whereas met showed reduced inflammation . however , in met + gsp group , the expected combined positive effects were not observed , the reason for which is yet to be explored . srebp-1c controls hepatic de novo lipogenesis ( dnl ) primarily by regulation of expression of genes involved in dnl and lipid homeostasis . therefore , this transcription factor is a favored candidate in experiments that investigate the role of hypolipidemic agents in the prevention of fatty liver disease . similarly , hmg coa reductase is an enzyme of much importance in lipogenesis since it catalyzes the rate - limiting step in cholesterol biosynthesis . increase in this enzyme indicates increased production of cholesterol . the observed increase in mrna expression of srebp-1c and hmg coa reductase in hffd - fed rat liver ppar- mediates -oxidation of fatty acids , and agonists of ppar- possess the property of reducing lipid dystrophy and obesity . ppar- activates some of the key enzymes of -oxidation and -oxidation of fatty acids in liver . multitude of studies have postulated that , during diet - induced obesity , fatty acid oxidation is left incomplete accompanied by mitochondrial lipid overload and dysfunction [ 29 , 30 ] . gsp treatment reduced the levels of ffa in plasma and liver and also improved the expression of ppar-. the lipid regulatory mechanism of gsp on lipid metabolism is observed to be at the genetic level . additionally , gsp may improve cellular fatty acid uptake and mitochondrial function which needs to be studied in future . in conclusion , we have shown that gsp protects against hepatic steatosis in obesity by suppressing lipogenesis and promoting -oxidation in liver . gsp reduces ld formation by reducing hepatic tg content , regulates lipid biosynthesis , and promotes -oxidation through its effects on srebp-1c , ld proteins , and ppar- mrna expression . since nafld and insulin resistance are closely associated , we linked and studied the action of gsp along with met , an insulin sensitizer , on lipid abnormalities . although met showed significant restoration of lipid levels , comparative results point out that gsp is more effective . histopathological examination provides good evidence for the lipid - lowering property of gsp whereas met does not show a strong action against lipid abnormalities . combination of met with gsp showed effective improvement in insulin sensitivity and better reduction in hyperlipidemia compared to met alone . the effects of met and gsp on the parameter studied are not additive during combination treatment , the reason for which is unclear at this stage . combination of drugs with different mechanisms of action has always been encouraged by clinicians in order to obtain the best results . further studies are warranted to attest their putative positive effects of gsp and met on other components of nafld and to see if nafld can be dealt using the combination of these two drugs .

nonalcoholic fatty liver disease ( nafld ) , a premorbid condition , lacks proper management owing to multitude of abnormalities . in this study , we compared the effects of a potent antioxidant , grape seed proanthocyanidins ( gsp ) , and an insulin sensitizer , metformin ( met ) , in high - fat - fructose - diet- ( hffd- ) induced albino wistar rat model of nafld . either gsp ( 100 mg / kg b.w ) or met ( 50 mg / kg b.w ) or both were administered as therapeutic options . hffd - fed rats showed abnormal plasma lipid profile , inflammation , and steatosis of the liver when examined by biochemical and histology techniques . increased lipid storage , lipogenesis , and reduced lipolysis were evident from mrna expression studies of hepatic lipid droplets ( ld ) proteins , sterol regulatory element binding 1c ( srebp 1c ) , and peroxisome proliferator activated receptor- ( ppar- ) . gsp administration to hffd - fed rats caused 69% reduction in hepatic tg levels , whereas met caused only 23% . the combination treatment reduced tg levels by 63% . gsp reduced the mrna expression of srebp1c and ld proteins and increased that of ppar- more effectively compared to met in hffd - induced hyperlipidemic rats . combination of met and gsp improved the metabolism of lipids effectively , but the effect was not additive in restoring lipid levels .

while the first reports of mammalian adult neurogenesis in the 1960s were met with little fanfare , the revival of the field in the 1990s incited both excitement and skepticism ; until that time , it was accepted that after birth , the brain could only lose and not gain neurons . however , with the advent of new techniques to label dividing cells and distinguish neuronal from glial populations , evidence for adult neurogenesis accumulated throughout the 1990s and 2000s . it is now well established that the birth of new neurons in the adult brain occurs in two regions : the subventricular zone of the lateral ventricles and the hippocampus . increasingly sophisticated questions have since been asked , refining our image of how these newborn neurons are regulated and how they function . in this review , we will cover several themes that have emerged in the last few years in the study of adult hippocampal neurogenesis . these themes share a common goal of defining the unique contributions of adult - born cells , albeit at different levels of study : learning and memory , physiology , hippocampal anatomy , and comparative biology . both positive and negative alterations in neurogenesis affect discrimination between memories of recalled events and similar present events , also referred to as behavioral pattern separation . ( b ) young adult - born hippocampal neurons have unique physiology and circuitry that may contribute to pattern separation . although links are largely speculative , current hypotheses have attempted to link pattern separation to the period of hyperexcitability in newborn neurons . ( c ) the position of newborn cells within the dentate gyrus may alter their fate and function . differences in proliferation , maturation , and function of newborn neurons have been identified along the longitudinal axis of the dentate gyrus . ( d ) the function of new neurons in humans may be unique when compared with other mammals . differences in maturation rates , newborn neuron proportion , and anatomy between humans and animal models are now being recognized and studied . current inquiries into the functional significance of adult - born neurons have heavily emphasized the concept of pattern separation . pattern separation , a term appropriated from computational models of neural networks , has been loosely used to describe the process by which similar , but not identical , patterns of inputs are classified as distinct [ 1 - 3 ] . at the behavioral level , these inputs reflect both external sensory input and internally generated representations of a previously experienced environment or event . by manipulating the rate of adult neurogenesis , several groups have shown that newborn neurons are critical for making fine discriminations between neighboring spatial locations or highly similar environments in tests of working memory and long - term memory . ablating adult neurogenesis via irradiation impairs performance on a touchscreen task in which mice must discriminate between two adjacent stimuli presented simultaneously in close proximity . the same irradiation treatment interfered with the recall of nearby arm locations after a brief delay ( < 30 s ) on a radial arm maze task . in contrast , an increase in rates of neurogenesis via running improves the performance on the touchscreen task . improving the survival of newborn neurons via a genetic strategy similarly enhances the ability to discriminate between two similar contexts during contextual fear conditioning , a distinction that arises over the course of several days of training . notably , the contribution of adult neurogenesis in each of these cases emerges only on more difficult discriminations . when difficulty is reduced by either increasing the spatial separation or making the contextual conditioning environments more distinct , both impaired and enhanced neurogenesis groups perform comparably to controls . these data support the hypothesis that the advantage provided by adult - born neurons grows as the overlap between two input patterns increases . newborn neurons that are 3 - 4 weeks of age are particularly distinct in function from mature granule cells ( see below ) . optogenetic silencing of newborn cells at 4 weeks of age , but not at 2 or 8 weeks , impairs the retrieval of both previously learned spatial location on the morris water maze and of a previously learned fear - conditioned context . from these studies , it remains unclear whether these 4-week - old cells contribute solely to the retrieval of an existing memory or whether they also play a role in encoding . genetic suppression of mature granule cell activity , while leaving younger granule cells less than 4 weeks of age intact , also improves contextual fear discrimination of two similar contexts . conversely , this young cell - dominated dentate gyrus impairs performance on a water maze task requiring the opposite process pattern completion when navigation must be completed using only a subset of the cues present during training . this study suggests that a dentate gyrus network dominated by young granule cells is biased towards interpreting similar but not identical inputs as distinct , whereas older granule cells are biased towards interpreting similar inputs as equivalent . however , the behaviorally optimal amount of pattern separation versus completion will depend on the relevance of particular stimuli to the demands of the task and motivation of the individual . it is possible that a deficit in pattern separation does not automatically imply an enhancement of pattern completion but rather a global deficit in the detailed recall of a particular context . the physiological properties of newborn neurons and their effects on dentate gyrus circuitry provide some explanation for their time - limited role in pattern separation as described above . newborn neurons have a developmental time window during which they exhibit unique intrinsic and circuit - level properties . physiology and novel circuit tracing studies in mice have revealed details in newborn neuron development . days after birth , newly generated cells respond to ambient -aminobutyric acid ( gaba ) with tonic activation due to a high concentration of intracellular chloride that leads to depolarization . in 1 - 2 weeks after 2 - 3 weeks , they start expressing glutamatergic receptors , and subsequently , the direction of the chloride gradient switches such that gabaergic input hyperpolarizes newborn neurons [ 10 - 12 ] . at around 1 month , new neurons receive synaptic glutamatergic input from the entorhinal cortex , similar to mature cells [ 13 - 16 ] . however , at this time point , new neurons have a lower density of gaba inputs and inhibitory post - synaptic currents ( ipscs ) compared with mature granule neurons . once fully mature ( about 6 weeks after birth ) , newborn neurons are essentially indistinguishable physiologically from developmentally born granule neurons . hence , at 4 weeks after birth , new neurons receive input from the same sources as mature granule cells but , because of their unique properties , they are hyperexcitable and show broader tuning than surrounding mature granule cells ; young neurons are more likely to respond to two separate inputs than mature granule cells . the effect of this developmental time course is a period during which excitation dominates over inhibition . by contrast , inputs to fully mature granule neurons are biased towards inhibition , as they fire very infrequently . adult neurogenesis therefore serves to make the principal cells of the dentate gyrus physiologically heterogeneous and distinct subpopulations are constantly shifting : as one newborn neuron subpopulation matures , another develops to replace it . the physiological differences between mature granule cells and 4-week - old newborn neurons suggest at least two mechanisms for how neurogenesis may affect pattern separation and completion . one mechanism involves interpreting the activity of individual granule cells directly in response to two different inputs as being reflective of overall dentate gyrus activity . in this case , mature granule neurons , which require high input specificity and maintain a high activation threshold , potentially serve as good pattern separators . the hyperexcitability and broad tuning properties of newborn neurons would conversely suggest that they could serve as pattern integrators . however , this hypothesis is inconsistent with the behavioral evidence discussed above , in which increases in the newborn neuron population improve pattern separation . an alternative mechanism is to consider how local dentate gyrus interneurons may influence overall dentate gyrus activity . both young and mature granule cells stimulate local inhibitory interneurons either directly or indirectly via mossy cells in the hilus . those interneurons in turn also send efferents to granule cells but with a higher density of inhibitory inputs to mature as compared with young neurons ( as described above ) . thus , by dampening the activity of mature granule cells , newborn cells increase their influence on the network . by this proposed mechanism , increased numbers of hyperexcitable neurons paradoxically make the dentate gyrus sparser , supporting a role for newborn neurons in enhancing pattern separation function . while the evidence for a role for newborn neurons in pattern separation is accumulating , we are also beginning to appreciate that this function depends on the location of new cells within the dentate gyrus . the dentate gyrus can be anatomically distinguished along the dorsal - ventral ( septo - temporal ) axis , and it is becoming increasingly recognized that newborn neurons arising from the dentate gyrus may be functionally distinguished along this axis . differences in the generation and maturation of adult - born neurons appear along the dorsal - ventral axis . new neurons in the dorsal dentate gyrus may mature significantly faster than in the ventral dentate gyrus because of higher levels of basal local network activity in the former . these distinctions might arise from the region - specific variation in cortical and subcortical connections to the hippocampus . a newborn neuron 's maturation time and therefore its unique properties is linked with its position within the dentate gyrus . an additional distinction of newborn neuron function may be related to the idea that the dorsal hippocampus is involved in spatial learning and memory , whereas the ventral hippocampus has been linked with emotional behavior . with these associations , it has been proposed that a cognitive - specific task or a stimulus with emotional valence would selectively affect neurogenesis in the dorsal and ventral hippocampus , respectively when adult rats learned a spatial water maze task , enhanced neurogenesis was observed in the dorsal , but not ventral , hippocampus , as predicted . in probing the activation of granule cells after water maze learning , c - fos expression was higher in the dorsal than ventral hippocampus when considering the overall granule cell population , but among the adult - born subpopulation alone , c - fos expression was higher in the ventral hippocampus . acute stress or administration of corticosterone in adult rats was predicted to alter neurogenesis in the ventral hippocampus , when , in fact , neurogenesis increases were observed selectively in the dorsal hippocampus . therefore , although there is evidence for cognition primarily involving the dorsal hippocampus and emotional behavior utilizing mainly the ventral hippocampus , the links with neurogenesis require further refinement . collectively , these studies highlight the idea that newborn neurons can be born at the same time yet mature differently , and/or subserve unique functions depending on their position along the long axis of the dentate gyrus . the nature and consequence of having variability within a newborn neuron population along the dorsal - ventral axis are usually not reported and warrant re - evaluation of previous studies and further attention in future studies . while we have known for 15 years that adult neurogenesis takes place in the adult human , most of our understanding of mammalian adult neurogenesis comes from studies of rodents . therefore , it has been tempting to view our knowledge of rodent adult neurogenesis from an anthropocentric viewpoint . however , recent adult neurogenesis studies in primates and humans are reshaping ideas of how new neurons may function within us . as suggested from evaluating non - human primates , the developmental timeline of newborn neurons in humans may be much longer than previously estimated . two - thirds of brdu cells continued to express the immature neuron marker doublecortin ( dcx ) at 6 months in an adult macaque hippocampus , whereas dcx expression in the dentate gyrus neurons in an adult rodent lasts only several weeks . a report by spalding , frisen and colleagues confirmed that neurogenesis continues to occur in the adult human hippocampus and to a much greater extent than previously estimated . they showed that hippocampal neurogenesis in adult humans is robust and continues through at least the fifth decade of life . while these data suggest that the age - related decline in adult neurogenesis is significantly slower in humans than in rodents , a direct comparison requires further validation because of the impoverished and sedentary environment of rodents reared under typical laboratory conditions . an emerging idea is that newborn neurons may be playing significantly larger roles in hippocampal function and human cognition than previously thought . previous models utilized rodent neurogenesis data for proposing computational roles for newborn neurons in learning and memory . given that the study by spalding and colleagues suggests that most human dentate gyrus neurons are subject to turnover compared with 10% of mouse dentate granule neurons , new models may be required for predicting the contribution of adult - born neurons in human hippocampal function . the next leap in understanding the role of newborn neurons in human health and disease will require studying hippocampus - dependent behaviors in humans . development of novel imaging techniques to visualize newborn neuron activity in the dentate gyrus of humans would lead to breakthroughs . one example of a funding initiative that would encourage development of necessary technologies is the recently announced brain research through advancing innovative neurotechnologies ( brain ) funding initiative by the obama administration , which encourages development of these transformational techniques . about 20 years ago , the dogma that no new neurons are born in adulthood began to crumble . the advances in the last few years have shown why neurogenesis may be important ( theme 1 ) , revealed a mechanism by which it may work ( theme 2 ) , suggested that newborn neuron function is heterogeneous along its axes ( theme 3 ) , and hinted at how what we know may be modified in the case of humans ( theme 4 ) . although these themes may at first appear to concern separate aspects of neurogenesis , it could be that the inter - relationships between these themes will be critical for understanding the importance of newborn neurons in humans . for example , the longer developmental time window in humans compared with rodents suggests a relatively prolonged state of hyperexcitability in newborn neurons . this extended period may impact the human dentate gyrus circuitry such that pattern separation may function qualitatively or quantitatively differently in humans than in rodents . does the presence of a different / larger ventral dentate gyrus have implications for whether newborn neurons function differently there ? and , as noted , the larger scale of human adult neurogenesis may mean that we need to modify our computational models . recent work has sharpened our view of the regulation and function of adult hippocampal neurogenesis . new techniques and advancements will literally and figuratively present a progressively higher - resolution image of this striking example of neural plasticity .

the birth of new neurons in the adult mammalian brain once thought impossible is now a well - accepted phenomenon that takes place in the subventricular zone of the lateral ventricles and the hippocampus . this review focuses on the recent work that has sharpened our views of how hippocampal newborn neurons are regulated and function . areas of study include ( a ) how neurogenesis contributes to behavioral pattern separation , ( b ) how pattern separation may be influenced by the properties and circuitry of newborn neurons , ( c ) differences along the dorsal - ventral axis of how neurogenesis is regulated and functions , and ( d ) adult neurogenesis in primates , including new human data . these current avenues of research reveal new details of adult neurogenesis and foreshadow what we may learn about this exciting phenomenon in the near future .

the prevalence of chronic kidney disease in the adult cystic fibrosis ( cf ) population is estimated to be between 27 and 42% . there are a number of cf - associated factors contributing to renal impairment in this population , including aminoglycoside toxicity , diabetic nephropathy , non - steroidal anti - inflammatory drug toxicity , renal amyloidosis , nephrocalcinosis and iga nephropathy . despite the multitude of factors impairing renal function in the cf population , we present a case of a potential novel cf renal phenotype in this cohort of patients . in september 2005 , a 32-year - old male patient with cf ( f508/f508 ) presented to the cork adult cystic fibrosis centre with a 6-month history of peripheral oedema . past medical history was otherwise unremarkable with infrequent previous infective pulmonary exacerbations and no courses of intravenous antibiotics over the previous 10 years . there was no past history of hypertension , smoking , hearing impairment or diabetes mellitus . forced expiratory volume in 1 second ( fev1 ) % predicted was stable at 57% predicted ( 2.36 l / min ) and serum ( creatinine ) was 169 a spot urine protein - to - creatinine ratio revealed severe proteinuria at 691 mg / mmol ( estimated at 6.1 g protein/24 h ) with an associated hypoalbuminaemia ( 24 the patient repeatedly had normal oral glucose tolerance tests ( ogtt ) and normal haemoglobin a1c ( all < 6.2% ) . findings were consistent with a nephrotic syndrome , and treatment with an angiotensin - converting enzyme inhibitor and natriuretic agent was instigated and a renal biopsy performed . renal biopsy findings revealed nodular glomerulosclerosis ( ngs ) ( figure 1a ) , identical to the classic kimmelstiel congo red staining for amyloid was negative , immunofluorescence was negative for immunoglobulin deposition ( polyclonal , igg , iga , igm , c3c ) and electron microscopy excluded fibrillary glomerulonephritis , amyloidosis and monoclonal immunoglobulin deposition disease . ( a ) haematoxylin and eosin - stained section demonstrating increased mesangial matrix with kimmelstiel wilson - like mesangial nodules . ( c ) periodic acid methenamine silver - stained section again highlighting the nodular lesion . ( d ) electron microscopy of glomerulus demonstrating absence of amyloid fibrils and absence of evidence for monoclonal immunoglobulin deposition disease and fibrillary glomerulonephritis . despite standard conservative management , he remained proteinuric , with a progressive deterioration in estimated glomerular filtration rate . an intercurrent growth of a mucoid strain of pseudomonas aeruginosa led to the initiation of alternate monthly nebulized tobramycin 300 mg bis in die ( bid ) in july 2007 at his annual assessment , in keeping with best international standards of care for patients with cf . this led to a sharp decline in his renal function , reverting back to a steady baseline rate of renal function decline on discontinuation of same after september 2007 ( figure 2 ) . no other changes to his therapies were made around this period and he was not on any other nephrotoxic medications . it is anticipated that he will require renal replacement therapy over the next 6 months and has been prepared for pre - emptive renal transplantation . creatinine over time plot demonstrating the progressive decline in renal function and intercurrent reversible dip following the temporary initiation of nebulized tobramycin 300 mg bid in july and september 2007 ( circled area ) . this is the first reported case of ngs in a cf patient without diabetes mellitus in a european population and highlights the importance of multidisciplinary consultation in this cohort of patients . the differential diagnosis for proteinuria in cf has , until recently , been predominantly limited to the identification of diabetic nephropathy , renal amyloidosis , iga nephropathy , fibrillary glomerulonephritis and immunoglobulin deposition disease . historically , the presence of ngs on renal biopsy was considered pathognomonic of diabetic nephropathy , designated the kimmelstiel wilson lesion . ngs - like lesions have subsequently been described in a wide variety of conditions , most prominently amyloidosis and fibrillary glomerulonephritis . our findings are consistent with a previous australian report describing ngs in cf patients in the absence of diabetes mellitus and other recognized causes of ngs , supporting the existence of a cf - related ngs . all patients described to date are male cf patients , > 30 years of age , presenting clinically with nephrotic syndrome in the setting of repeatedly normal ogtts . interestingly , all patients progressed towards end - stage renal disease over the subsequent 4 years ( personal communication with authors ) . progressive renal failure is of increasing importance for patients with cf given that a creatinine clearance < 50 ml / min/1.73 m is frequently viewed as a contraindication to lung transplantation . it is postulated that the pathogenesis of cf - related ngs in normoglycaemic , non - diabetic cf patients is similar to that of classic diabetic ngs , and may be mediated by specific binding to the receptor for advanced glycosylation end products ( rage ) . in cf , chronic pulmonary infection / inflammation , in combination with reduced glutathione levels , contributes to an oxidative state ; this oxidative state could result in the formation of advanced glycosylation end products ( age ) in conditions of normoglycaemia . patients with cf have a number of other potential mechanisms for rage activation in the absence of hyperglycaemia , including an increase in serum levels of the s100/calgranulin pro - inflammatory cytokines . there is also emerging evidence that high - mobility group box 1 ( hmbg-1 ) , a potent inflammatory mediator known to be elevated in the serum of patients with cf , can cause an epithelial to mesenchymal transition ; this has previously been shown to contribute to the renal accumulation of matrix protein by its action on rage . we speculate that the pathogenesis of cf - related ngs is consequent to ligand binding of rage by s100/calgranulin , hmbg-1 and age . additionally , this is the first reported case of nebulized tobramycin - induced toxicity in a patient with cf with pre - existing chronic kidney disease . it is important to highlight the rapid decline in renal function on administration of nebulized tobramycin to our patient ( figure 2 ) . previous studies in non - cf patients with renal impairment describe nephrotoxic serum trough tobramycin levels up to 2.5 g / ml following the administration of nebulized tobramycin 300 mg bd . another study reports acute renal failure secondary to nebulized tobramycin in a patient with cf without pre - existing renal disease . the bioavailability of nebulized tobramycin is known to vary widely between patients , which is related to a number of factors including incomplete delivery of the aerosolized drug to the lung , possible systemic absorption from the oropharynx , the method of nebulization used and the pre - existing renal function . we suggest that trough serum level monitoring should be considered for patients with pre - existent renal disease . in summary , this report highlights the importance of renal biopsy in proteinuric renal disease in adult cf patients . as the management of patients with cf continues to improve , the increasing life spans may lead to an increased identification of proteinuric renal disease , the aetiology of which can include cf - related ngs . in addition , the potential for nephrotoxicity secondary to nebulized tobramycin in patients with pre - existing renal dysfunction is highlighted . it may be that , in these patients , monitoring of serum levels of tobramycin post - nebulization is required .

cystic fibrosis ( cf ) is a multisystemic disease but without a classical disease - specific renal phenotype . a 32-year - old male patient with cf ( f508/f508 ) presented with a nephrotic syndrome . renal biopsy revealed nodular glomerulosclerosis ( ngs ) occurring in the absence of diabetes mellitus , amyloidosis and any other known common cause of ngs . he had a progressive decline in estimated glomerular filtration rate ( egfr ) to chronic kidney disease stage v ( egfr < 15 ml / min/1.73 m2 ) over a 3-year period despite optimal medical management . this is the fourth reported case of ngs in a patient with cf without diabetes and is the first to originate from a european country . this case supports the concept of a cf - related ngs .

different ways exist to conceptualize executive functioning . for research that has applied latent statistical models , the focus is on classification and documenting both the diversity and unity of efs . although related , efs can be classified into three categories , including ( i ) working memory and updating , ( ii ) executive attention or cognitive control , and ( iii ) inhibition ( e.g. , miyake et al . , 2000 ; friedman et al . , 2006 ; miyake and friedman , 2012 ) . but the three aspects also form an integrated mechanism for processing information . this broader mechanism underlies the ability to manipulate and maintain tasks , plans , and goals ( i.e. , their mental representations ) in an active state while monitoring performance and inhibiting distracting stimuli , whether from the environment or internally ( kane and engle , 2002 ) . executive functioning is a central topic in many areas of psychology ( posner and rothbart , 2007 ) . various theoretical frameworks emphasize that while some cognitive processes can run efficiently on limited cognitive resources , many others engage executive functioning . within social psychology , efs are important for a wide array of sub - fields that inform the nature of social behavior , including persuasion , attitudes and prejudice , social perception , self - control , and emotion regulation , for example ( e.g. , smith and decoster , 2000 ; von hippel , 2007 ) . in all these areas of social functioning , automatic processes are thought to help create a first - pass , working model of the event ( e.g. , perceiver readily infers a speaker seems competent due to style of dress ) . assuming the perceiver is motivated and has the requisite cognitive resources ( efs ) , they then can integrate more information about the event to enrich the working model and arrive at a deeper social understanding ( for reviews , see chaiken and trope , 1999 ; smith and decoster , 2000 ) . one central question about efs in day - to - day life is their malleability . except for changes across the lifespan ( e.g. , von hippel , 2007 ) , people s executive functioning has long been viewed as relatively static perhaps because of robust individual differences in efs . however , starting with recent reports of successful cognitive interventions , this view has begun to change and now efs are seen as much more open to both short- and long - term training , warm - up , and exhaustion effects . research has shown , for example , that working memory training not only leads to improvements on distinct measures of executive functioning but also to transfer effects resulting in improvement on measures of fluid intelligence ( e.g. , jaeggi et al . , 2008 , 2011 ) . in young children , cognitive training has been shown to improve subsequent attention control ( e.g. , thorell et al . , 2008 ) . other findings indicate efs benefit from meditation training ( e.g. , tang and posner , 2009 ) . however , some of the most intriguing evidence comes from research showing that social engagement enhances performance on standard ef tests . this is important since the majority of people s life takes place in the social world it has long been argued that communicating with others , taking others perspectives , and following social rules sets the stage for the development and maintenance of efs , thus helping structure general mental functioning ( e.g. , vygotsky , 1978 ; buttelmann et al . , 2009 ) . as previously mentioned , the developmental literature has long linked efs to performance on some aspects of tom ( hughes and ensor , 2007 ) . selection pressures related to the complex , dynamic and mixed - motive nature of social life have also been posited to partly underlie the evolution of primate intelligence ( e.g. , jolly , 1966 ; humphrey , 1976 ; dunbar and shultz , 2007 ) . there also are intriguing suggestions regarding the overlap of the neural underpinnings of ef and social reasoning processes ( von hippel , 2007 ; adolphs , 2009 ) . and there are now several reports of positive correlations of some brain structures with social network size . for example , amygdala size correlates with the size and complexity of real social networks ( bickart et al . , 2011 ) , whereas gray matter correlates with the number of facebook friends ( kanai et al . , 2011 ) . more impressively , it appears that , at least in macaques , social network size causally influences brain structure and function ( sallet et al . , 2011 ) . with humans , much cross - sectional and longitudinal research has found positive relationships between social participation / engagement and executive functioning and related mental health outcomes ( e.g. , fratiglioni et al . , 2004 ) . some findings with distinct populations thus , for example , a vygotskian based curriculum ( tools of the mind ) which requires much social interaction and taking others perspective boosted not only preschoolers social skills but also their executive functioning ( e.g. , diamond et al . , 2007 ) . although most of the available work examining the effects of social engagement relies on correlational designs , a few studies are beginning to show positive causal effects of social processes on executive functioning , and the most direct evidence comes from social psychological research investigating the effect of on - line social interaction on executive functioning . in one experiment , we had participants interact by having a discussion of a social topic ( ybarra et al . , 2008 ) . participants were given 10 min total , with a few minutes to prepare for the discussion . following the interaction the participants evaluated the activity and then completed tests of cognitive functioning , in particular a test of working memory which is a critical component of executive functioning . we also tested participants assigned to either an intellectual activities condition or a control condition that involved watching a 10-min video . another aspect of this mental exercising through simple socializing research that should be highlighted is that participants assigned to the video condition were presented with social content involving several human characters that were engaged in social interaction ( ybarra et al . , 2008 ) . in a sense , the video content represents a rich stimulus compared to many of the stimuli used to study social cognitive processes from a neuroscientific perspective . still , the video condition did not result in any boosts to efs ; it was the real , on - line social interaction that resulted in ef boosts , which were equivalent to those resulting from engaging in difficult intellectual activities . thus , benefits to efs are selectively seen in on - line social cognition , where participants dynamically construct a model of another person , and less in off - line social cognition where participants deal with static or impoverished and less engaging representations of others . regarding the above research , the argument could be made that the discussion that comprised the social interaction was atypical in some way , in that participants had to take positions , make arguments , and discuss their point of view . but as recent theorizing on the evolution of reasoning suggests , this aspect of social interaction evaluating arguments and proffering others may have been an important pressure on the evolution of our distinctive human cognition ( mercier and sperber , 2011 ) . further , as we will discuss later , recent research indicates ef boosts also can result from basic get - to - know - you interactions if structured in a particular , mind - engaging way ( ybarra et al . so the positive effects on efs can occur from other types of on - line social interactions . however , evidence also exists for the detrimental effects of some social interactions on efs . most of these findings come from research on intergroup social interactions ( e.g. , richeson and trawalter , 2005 ) . in one study , for example , participants who underwent an interracial interview compared to a same race interview subsequently exhibited more interference on a stroop task , a measure of ef . further , this effect was greater the stronger participants associations between concepts denoting african - americans and negative personality characteristics , as measured with the iat test . studies found that after interacting with an attractive female , men showed worse performance on a subsequent ef task , and the decline was related to the degree men tried to manage their impression in the interaction ( karremans et al . , 2009 ) . interactions can result in reduction of cognitive functioning ( finkel et al . , 2006 ) . these investigators had participants coordinate on a task with a confederate , who in some cases made scripted errors ( high maintenance ) and in some cases did not ( low maintenance ) . participants in the high maintenance condition performed worse on various subsequent tests , although in this research the tests were indirectly tied to efs . note that most , if not all , studies showing ef reductions invoked self - presentation concerns ( e.g. , about appearing non - prejudiced , unintelligent , etc . ) . first , it constitutes a working memory load ( what was the interaction about ? second , it triggers effortful attempts to self - regulate , which , if extensive , could deplete cognitive resources ( muraven and baumeister , 2000 ) . at a broader level this reasoning also applies to some of the findings dealing with performance decrements under conditions of stereotype threat , for example . in our opinion , however , one key difference between studies showing reductions and boosts in efs has to do with the nature of the interaction . in particular , it is critical whether participants mentally engage with others and attempt to build a rich model of their minds , that they toggle between self and other perspectives , and that they communicate and create meaning during the social interaction versus disengage from the interaction . we argue that this is often , though not always ( see below ) , determined by whether the interaction is cooperative or competitive . however , the default in a cooperative setting is often to engage with the other person , build a model of their mind , figure out whether or not they are trustworthy , and convey to them that they can trust us . in fact , under these trust - building conditions neuroimaging work has reported some of the most robust effects of mentalizing on activity in the medial prefrontal cortex ( for reviews , see frith and singer , 2008 ) . further , children s ability to mentalize ( i.e. , perform tom tasks ) has been shown to be positively correlated to the likelihood of cooperating , for example , in prisoner s dilemma games and also to making fair offers in the ultimatum game ( sally and hill , 2006 ; also see buttelmann et al . , 2009 ) . in contrast , the default under competitive goals , as realized in the above research on intergroup interactions , is often to become self - protective and withdraw from engaging the other person . this may occur because the interaction is ambiguous and not well structured , which inclines people to back away from the situation as a general way of deterring interpersonal costs ( ybarra et al . this may also occur because people have a tendency to dehumanize outgroups and thus attribute less mind to their competitors ( harris and fiske , 2006 ) . finally , it is possible that competition ( but also other high - stake situations ) may sometimes usurp the cognitive resources necessary for careful and effortful mentalizing . so , people focus on themselves and their own interests rather than the other person , and they do not engage in attempts to take perspective and create a rich model of the other and the event . a key aspect then of whether or not social interaction creates subsequent ef boosts rests on people engaging each other . we propose that doing so invokes processes that exercise underlying efs , such as working memory and executive attention . thus , when people engage with others in social interaction , versus withdraw into themselves , they can exercise or warm - up these core cognitive processes , whose influence is then transferred ( far ) to executive functioning tasks ( ybarra et al . , if such social cognitive processes underlie the cognitive boosts , disrupting them should eliminate the cognitive benefits . consistent with this idea , our recent experiments found that interaction goals ( competition ) that disengage participants from perspective taking and mentalizing eliminate the cognitive benefits that can result from social interaction ( ybarra et al . importantly , follow - up work has shown that getting people to engage others during interaction , even when the interaction is competitive , helps counter the loss in cognitive benefits ( ybarra et al . this is consistent with other research in social cognition suggesting that skepticism , suspiciousness , and other competitive approaches can sometimes improve mental performance ( schul et al . , 2004 , 2008 ) . these findings provide evidence that engaging the other during interaction along with concomitant social cognitive processing ( perspective taking , mind - reading ) may partly underlie the boosts to executive functioning following social interaction . the above findings also help inform , at least in some small measure , the assumption that competition in social contexts played an important role in the evolution of primate cognition and the more intense varieties of social cognition and mentalizing ( e.g. , byrne and whiten , 1988 ; whiten and byrne , 1997 ) . dealing with competitors can of course implicate the understanding of others behavioral tendencies and psychological states ( e.g. , tomasello et al . , 2003 ; decety et al . , 2004 ) , but as we have shown in our experiments , people who expect to compete during social interaction , if not given an explicit goal to read the other person and form a model of what they are like , will disengage and behave evasively ( ybarra et al . , 2010 ) and not receive cognitive benefits from the interaction ( ybarra et al . , 2011 ) . these findings suggest that people when competing have diverse options they can undertake , such as to try to hide or foil prediction . only when no , low - risk option is available will they engage or confront the opponent . this , however , does not mean some level of social understanding is not sought or created under competitive circumstances , but it may be that the working model of the other is of a generic , stereotypic nature that relies less on efs , similar to the social perception differences found when people judge members of outgroups versus ingroups ( neuberg and fiske , 1987 ; brewer , 1988 ) . when interdependence is called for meaning you have to interact on - line with a person and that your behavior is to some degree yoked to theirs the situation should instigate more intense social cognition and mentalizing to build a richer model of the other party . the discussion of competition helps highlight the role we assign to mentalizing and understanding others psychological states during social interaction . we propose that mental engagement with others leads to cognitive benefits from social interaction especially when the involved parties are taking perspective and dynamically building a model of what the other person is like . as we noted in the introduction , some social inferential processes can occur quite efficiently with little reliance on efs ( e.g. , winter and uleman , 1984 ; trope , 1986 ) . a similar theme comes up in research on theory of mind , with certain processes ( e.g. , the calculation of what another sees ) thought to be carried out efficiently and automatically ( e.g. , moll and tomasello , 2006 ; onishi and baillargeon , 2005 ; qureshi et al . , 2010 ) . related ideas on the role of efficient social understanding also have been discussed from the point of view of embodied and situated social cognition ( leudar et al . , 2004 ; however , some tom and mentalizing processes such as the selection of information for further processing , though , are thought to require limited cognitive resources or ef ( e.g. , leslie et al . , 2005 ; bull et al . , 2008 ; qureshi et al 2010 ) , and engagement with others in social interaction is considered the real domain in which minds are known ( reddy and morris , 2004 ) . thus , there are important elements of building a model of what another person is like , what they are thinking , and what they might do next that rely on efs . our proposal is that during real social interaction both low - level and high - level mentalizing and behavior prediction processes interact and inform each other . the automatic processes serve as input that feed into richer representations that are shaped and updated by processes requiring limited cognitive resources in real time . as noted with competition , it is not that people who are dealing with antagonistic parties fail to attempt to understand their foes but it is during social interaction when the parties are actively engaged with each other that richer representations of the other and of the interaction are created and dynamically updated , which entails the participation of efs . in addition to the issues discussed above , further work is needed to address the following questions : what specific types of social interactions benefit ef and what are the underlying processes that underlie theses boosts ? given both the unity and diversity of efs , does social interaction affect some ef elements more than others , or does this also depend on the type of interaction ? how long do cognitive boosts last and what is there time course , and does this depend on the task to which the cognitive processes are applied ? are the neural correlates underlying on - line social interaction , in particular those underlying efs , similar or different from the correlates unearthed for off - line social interaction ? how do short - term training effects of social interaction translate into long - term cognitive reserve , which may be captured in the cross - sectional and longitudinal studies on social engagement and cognitive functioning ? and , what are the longer - term consequences of repeated interactions that result in ef reductions ? if people do not avoid others different from the self , can they learn to cope with such challenging interactions ? it has long been argued that communicating with others , taking others perspectives , and following social rules sets the stage for the development and maintenance of efs , thus helping structure general mental functioning ( e.g. , vygotsky , 1978 ; buttelmann et al . , 2009 ) . as previously mentioned , the developmental literature has long linked efs to performance on some aspects of tom ( hughes and ensor , 2007 ) . selection pressures related to the complex , dynamic and mixed - motive nature of social life have also been posited to partly underlie the evolution of primate intelligence ( e.g. , jolly , 1966 ; humphrey , 1976 ; dunbar and shultz , 2007 ) . there also are intriguing suggestions regarding the overlap of the neural underpinnings of ef and social reasoning processes ( von hippel , 2007 ; adolphs , 2009 ) . and there are now several reports of positive correlations of some brain structures with social network size . for example , amygdala size correlates with the size and complexity of real social networks ( bickart et al . , 2011 ) , whereas gray matter correlates with the number of facebook friends ( kanai et al . , 2011 ) . more impressively , it appears that , at least in macaques , social network size causally influences brain structure and function ( sallet et al . , 2011 ) . with humans , much cross - sectional and longitudinal research has found positive relationships between social participation / engagement and executive functioning and related mental health outcomes ( e.g. , fratiglioni et al . , 2004 ) . some findings with distinct populations thus , for example , a vygotskian based curriculum ( tools of the mind ) which requires much social interaction and taking others perspective boosted not only preschoolers social skills but also their executive functioning ( e.g. , diamond et al . , 2007 ) . although most of the available work examining the effects of social engagement relies on correlational designs , a few studies are beginning to show positive causal effects of social processes on executive functioning , and the most direct evidence comes from social psychological research investigating the effect of on - line social interaction on executive functioning . in one experiment , we had participants interact by having a discussion of a social topic ( ybarra et al . , 2008 ) . participants were given 10 min total , with a few minutes to prepare for the discussion . following the interaction the participants evaluated the activity and then completed tests of cognitive functioning , in particular a test of working memory which is a critical component of executive functioning . we also tested participants assigned to either an intellectual activities condition or a control condition that involved watching a 10-min video . another aspect of this mental exercising through simple socializing research that should be highlighted is that participants assigned to the video condition were presented with social content involving several human characters that were engaged in social interaction ( ybarra et al . , 2008 ) . in a sense , the video content represents a rich stimulus compared to many of the stimuli used to study social cognitive processes from a neuroscientific perspective . still , the video condition did not result in any boosts to efs ; it was the real , on - line social interaction that resulted in ef boosts , which were equivalent to those resulting from engaging in difficult intellectual activities . thus , benefits to efs are selectively seen in on - line social cognition , where participants dynamically construct a model of another person , and less in off - line social cognition where participants deal with static or impoverished and less engaging representations of others . regarding the above research , the argument could be made that the discussion that comprised the social interaction was atypical in some way , in that participants had to take positions , make arguments , and discuss their point of view . but as recent theorizing on the evolution of reasoning suggests , this aspect of social interaction evaluating arguments and proffering others may have been an important pressure on the evolution of our distinctive human cognition ( mercier and sperber , 2011 ) . further , as we will discuss later , recent research indicates ef boosts also can result from basic get - to - know - you interactions if structured in a particular , mind - engaging way ( ybarra et al . so the positive effects on efs can occur from other types of on - line social interactions . . however , evidence also exists for the detrimental effects of some social interactions on efs . most of these findings come from research on intergroup social interactions ( e.g. , richeson and trawalter , 2005 ) . in one study , for example , participants who underwent an interracial interview compared to a same race interview subsequently exhibited more interference on a stroop task , a measure of ef . further , this effect was greater the stronger participants associations between concepts denoting african - americans and negative personality characteristics , as measured with the iat test . studies found that after interacting with an attractive female , men showed worse performance on a subsequent ef task , and the decline was related to the degree men tried to manage their impression in the interaction ( karremans et al . , 2009 ) . interactions can result in reduction of cognitive functioning ( finkel et al . , 2006 ) . these investigators had participants coordinate on a task with a confederate , who in some cases made scripted errors ( high maintenance ) and in some cases did not ( low maintenance ) . participants in the high maintenance condition performed worse on various subsequent tests , although in this research the tests were indirectly tied to efs . however , the findings are also puzzling . why do some interactions lead to ef impairments but other interactions to benefits ? note that most , if not all , studies showing ef reductions invoked self - presentation concerns ( e.g. , about appearing non - prejudiced , unintelligent , etc . ) . first , it constitutes a working memory load ( what was the interaction about ? second , it triggers effortful attempts to self - regulate , which , if extensive , could deplete cognitive resources ( muraven and baumeister , 2000 ) . at a broader level this reasoning also applies to some of the findings dealing with performance decrements under conditions of stereotype threat , for example . in our opinion , however , one key difference between studies showing reductions and boosts in efs has to do with the nature of the interaction . in particular , it is critical whether participants mentally engage with others and attempt to build a rich model of their minds , that they toggle between self and other perspectives , and that they communicate and create meaning during the social interaction versus disengage from the interaction . we argue that this is often , though not always ( see below ) , determined by whether the interaction is cooperative or competitive . however , the default in a cooperative setting is often to engage with the other person , build a model of their mind , figure out whether or not they are trustworthy , and convey to them that they can trust us . in fact , under these trust - building conditions neuroimaging work has reported some of the most robust effects of mentalizing on activity in the medial prefrontal cortex ( for reviews , see frith and singer , 2008 ) . further , children s ability to mentalize ( i.e. , perform tom tasks ) has been shown to be positively correlated to the likelihood of cooperating , for example , in prisoner s dilemma games and also to making fair offers in the ultimatum game ( sally and hill , 2006 ; also see buttelmann et al . , 2009 ) . in contrast , the default under competitive goals , as realized in the above research on intergroup interactions , is often to become self - protective and withdraw from engaging the other person . this may occur because the interaction is ambiguous and not well structured , which inclines people to back away from the situation as a general way of deterring interpersonal costs ( ybarra et al . , 2010 ) . this may also occur because people have a tendency to dehumanize outgroups and thus attribute less mind to their competitors ( harris and fiske , 2006 ) . finally , it is possible that competition ( but also other high - stake situations ) may sometimes usurp the cognitive resources necessary for careful and effortful mentalizing . so , people focus on themselves and their own interests rather than the other person , and they do not engage in attempts to take perspective and create a rich model of the other and the event . a key aspect then of whether or not social interaction creates subsequent ef boosts rests on people engaging each other . we propose that doing so invokes processes that exercise underlying efs , such as working memory and executive attention . thus , when people engage with others in social interaction , versus withdraw into themselves , they can exercise or warm - up these core cognitive processes , whose influence is then transferred ( far ) to executive functioning tasks ( ybarra et al . , 2008 ) . if such social cognitive processes underlie the cognitive boosts , disrupting them should eliminate the cognitive benefits . consistent with this idea , our recent experiments found that interaction goals ( competition ) that disengage participants from perspective taking and mentalizing eliminate the cognitive benefits that can result from social interaction ( ybarra et al . , 2011 , study 1 ) . importantly , follow - up work has shown that getting people to engage others during interaction , even when the interaction is competitive , helps counter the loss in cognitive benefits ( ybarra et al . this is consistent with other research in social cognition suggesting that skepticism , suspiciousness , and other competitive approaches can sometimes improve mental performance ( schul et al . these findings provide evidence that engaging the other during interaction along with concomitant social cognitive processing ( perspective taking , mind - reading ) may partly underlie the boosts to executive functioning following social interaction . the above findings also help inform , at least in some small measure , the assumption that competition in social contexts played an important role in the evolution of primate cognition and the more intense varieties of social cognition and mentalizing ( e.g. , byrne and whiten , 1988 ; whiten and byrne , 1997 ) . dealing with competitors can of course implicate the understanding of others behavioral tendencies and psychological states ( e.g. , tomasello et al . , 2003 ; decety et al . , 2004 ) , but as we have shown in our experiments , people who expect to compete during social interaction , if not given an explicit goal to read the other person and form a model of what they are like , will disengage and behave evasively ( ybarra et al . , 2010 ) and not receive cognitive benefits from the interaction ( ybarra et al . , 2011 ) . these findings suggest that people when competing have diverse options they can undertake , such as to try to hide or foil prediction . only when no , low - risk option is available will they engage or confront the opponent . this , however , does not mean some level of social understanding is not sought or created under competitive circumstances , but it may be that the working model of the other is of a generic , stereotypic nature that relies less on efs , similar to the social perception differences found when people judge members of outgroups versus ingroups ( neuberg and fiske , 1987 ; brewer , 1988 ) . when interdependence is called for meaning you have to interact on - line with a person and that your behavior is to some degree yoked to theirs the situation should instigate more intense social cognition and mentalizing to build a richer model of the other party . the discussion of competition helps highlight the role we assign to mentalizing and understanding others psychological states during social interaction . we propose that mental engagement with others leads to cognitive benefits from social interaction especially when the involved parties are taking perspective and dynamically building a model of what the other person is like . as we noted in the introduction , some social inferential processes can occur quite efficiently with little reliance on efs ( e.g. , winter and uleman , 1984 ; trope , 1986 ) . a similar theme comes up in research on theory of mind , with certain processes ( e.g. , the calculation of what another sees ) thought to be carried out efficiently and automatically ( e.g. , moll and tomasello , 2006 ; onishi and baillargeon , 2005 ; qureshi et al . , related ideas on the role of efficient social understanding also have been discussed from the point of view of embodied and situated social cognition ( leudar et al . , 2004 ; barrett and henzi , 2005 ; iacoboni et al . , 2005 ) . however , some tom and mentalizing processes such as the selection of information for further processing , though , are thought to require limited cognitive resources or ef ( e.g. , leslie et al . , 2005 ; bull et al . , 2008 ; qureshi et al . , 2010 ) , and engagement with others in social interaction is considered the real domain in which minds are known ( reddy and morris , 2004 ) . thus , there are important elements of building a model of what another person is like , what they are thinking , and what they might do next that rely on efs . our proposal is that during real social interaction both low - level and high - level mentalizing and behavior prediction processes interact and inform each other . the automatic processes serve as input that feed into richer representations that are shaped and updated by processes requiring limited cognitive resources in real time . as noted with competition , it is not that people who are dealing with antagonistic parties fail to attempt to understand their foes but it is during social interaction when the parties are actively engaged with each other that richer representations of the other and of the interaction are created and dynamically updated , which entails the participation of efs . in addition to the issues discussed above , further work is needed to address the following questions : what specific types of social interactions benefit ef and what are the underlying processes that underlie theses boosts ? given both the unity and diversity of efs , does social interaction affect some ef elements more than others , or does this also depend on the type of interaction ? how long do cognitive boosts last and what is there time course , and does this depend on the task to which the cognitive processes are applied ? are the neural correlates underlying on - line social interaction , in particular those underlying efs , similar or different from the correlates unearthed for off - line social interaction ? how do short - term training effects of social interaction translate into long - term cognitive reserve , which may be captured in the cross - sectional and longitudinal studies on social engagement and cognitive functioning ? and , what are the longer - term consequences of repeated interactions that result in ef reductions ? if people do not avoid others different from the self , can they learn to cope with such challenging interactions ? in addition to theoretical implications regarding the difference between real , on - line social cognition versus off - line social cognition , the processes discussed here have several practical implications . the major one is that certain social interactions can be an effective way of keeping mentally fit . sharpbrains , a company that tracks the mental fitness industry , estimated that worldwide revenue associated with cognitive training programs ( e.g. , computer software ) surged to $ 850 million in 2008 , up from $ 250 million in 2005 . unfortunately , many of these training programs are not only expensive , but few have been scientifically evaluated ( jaeggi et al . , 2011 ) . more germane to this proposal , what is important to emphasize is that what also matters is engagement and taking an active and not a passive role to the technology or the social interaction . as reviewed earlier , some forms of social interaction result in no boosts , whereas those in which the parties were engaged and actively tried to form a working model of the other yielded cognitive benefits . however , we would add that given that social connections are at the core of primate life ( jolly , 1966 ) , are central to the human survival strategy ( barash , 1986 ; dunbar , 1992 , 1998 ; baumeister , 2005 ) , and yield various benefits to health and well - being ( e.g. , house et al . , 1988 ; ybarra et al . , 2008 ) , engaged socializing with others in cooperative interaction may not only strengthen people s brains and minds but possibly their social relations as well , allowing them to reap the various benefits that flow from such bonds . in short , our review highlights the essential role of studying on - line social interactions for understanding the operation of fundamental cognitive processes . although we focused on how executive functioning can change due to task context , especially the on - line social context , it is also important to appreciate the role of people s beliefs and strategies in social navigation . most of us probably know people who seem quite intelligent but still do many dumb things in the cpu is a work of exquisite engineering , but if you try to use such a computer , task performance will be suboptimal and frustrating . the point here is simple : raw executive functioning matters for many social tasks , but so does the content of people s beliefs and strategies their rationality and match to the environment ( both in terms of controlled and automatic mental processes ) . but social life , especially who people interact and associate with , also plays a central role in the beliefs and values that end up populating people s minds . the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest .

a successful social interaction often requires on - line and active construction of an ever - changing mental - model of another person s beliefs , expectations , emotions , and desires . it also requires the ability to maintain focus , problem - solve , and flexibly pursue goals in a distraction - rich environment , as well as the ability to take - turns and inhibit inappropriate behaviors . many of these tasks rely on executive functions ( ef ) working memory , attention / cognitive control , and inhibition . executive functioning has long been viewed as relatively static . however , starting with recent reports of successful cognitive interventions , this view is changing and now efs are seen as much more open to both short- and long - term training , warm - up , and exhaustion effects . some of the most intriguing evidence suggests that engaging in social interaction enhances performance on standard ef tests . interestingly , the latest research indicates these ef benefits are selectively conferred by certain on - line , dynamic social interactions , which require participants to mentally engage with another person and actively construct a model of their mind . we review this literature and highlight its connection with evolutionary and cultural theories emphasizing links between intelligence and sociality .

insulin dependent diabetes mellitus ( iddm ) , type 1 diabetes , is a form of diabetes mellitus that results from autoimmune destruction of insulin - producing -cell of the pancreas . the deficiency or complete lack of insulin secretion leads to elevated blood glucose level [ 1 , 2 ] . patients with type 1 diabetes present lipid disorders or hyperlipidemia , including elevated levels of total serum cholesterol ( tc ) , triglycerides ( tg ) , low - density lipoprotein ( ldl - c ) , apolipoprotein a ( apoa ) , apolipoprotein b ( apob ) , malondialdehyde ( mda ) [ 4 , 5 ] , very low - density lipoprotein ( vldl ) , and low level of high density lipoprotein ( hdl - c ) . it is well known that the liver is a central organ in lipogenesis , gluconeogenesis , and cholesterol metabolism . hepatic lipid metabolism is influenced by the balance between the degradation and synthesis and/or import and export of triglyceride ( tg ) and fatty acids ( fa ) . generally , fatty acids are degraded through -oxidation or esterified and then stored as tg . hepatic tg accumulation finally resulting in hepatic steatosis .moreover , the fa transport process appears to be disturbed in obesity and diabetes mellitus . accumulating evidences proved that free fatty acids are taken up by the hepatocytes in a facilitated fashion rather than by passive processes [ 7 , 11 ] . it is well known that fatty acid translocase is abundantly expressed in tissues with high metabolic capacity for fatty acids . a number of studies have shown that fatty acid translocase ( fat / cd36 ) , fatty acid bounding protein ( fabp ) , and fatty acid transporter ( fatp ) are membrane glycoproteins present on mononuclear phagocytes , adipocytes , and hepatocytes with multiple functions , which have also been identified to facilitate fa uptake and -oxidation [ 1215 ] . several studies have demonstrated that fat / cd36 as a shared transcriptional target is regulated by liver x receptor ( lxr ) , pregnane x receptor ( pxr ) , and aryl hydrocarbon receptor ( ahr ) [ 9 , 1618 ] . overexpression of fat / cd36 results in an increased rate of fa uptake and increased rate of fa metabolism . fatty acids are taken up into the cells and temporarily stored in a triglyceride pool . fa will be finally oxidized in mitochondria by means of carnitine palmitoyl transferase 1 ( cpt1 ) and peroxisomal acyl - coenzyme a oxidase 1 ( acox1 ) . the insulin - mimetic properties and antidiabetic effects of vanadium compounds have been widely documented both in vivo and in vitro [ 2022 ] . vanadium compounds stimulate glycogen synthesis and lipogenesis and inhibit lipolysis [ 24 , 25 ] . recently , various organic vanadium compounds with dipic , dipic - oh , or dipic - nh2 as organic ligand were reported as antidiabetic agents with little side effects and higher absorption than the simple salts [ 22 , 2628 ] . moreover , it was observed that vanadate can restore the altered lipogenic enzyme activities to the normal level . our previous studies showed that vanadium compounds treatment potentially ameliorate lipid metabolism in diabetes [ 2 , 22 , 3033 ] . therefore , the aim of the study was an attempt to elucidate the hypolipidemic effect of v4dipic - cl , if any , in regulating hepatic fat / cd36-induced fa uptake and tg accumulation in stz - induced diabetic rats . moreover , serum biochemical parameters and histopathological examination were used to evaluate the side effect of v4dipic - cl on hepatic functions in diabetic rats . stz was purchased from sigma ( sigma - aldrich , usa ) h2dipic - cl and v4dipic - cl were gifts from dr . tissue triglyceride ( tg ) kit was purchased from pplygen ( pplygen , china ) . rna isolation reagent , ultrasybr mixture , and -actin antibody were purchased from beijing cowin bioscience ( china ) . radio immunoprecipitation assay lysis buffer ( ripa ) , hrp - labeled goat anti - rabbit igg , hrp - labeled goat anti - mouse igg , and electrochemiluminescence ( ecl ) reagent were purchased from beyotime ( china ) . male wistar rats ( 220 10 g ) were purchased from beijing academy of military medical sciences . the animals were maintained under standard conditions ( 12 h light / dark cycle , 22 2c ) and had free access to standard laboratory chow and water . the animals were cared for in accordance with the principles of the guide for care and use of experimental animals . diabetes was induced by a single intravenous injection of freshly prepared stz ( 40 mg / ml ; 55 mg / kg body weight ) in 0.1 mol / l citrate buffer ( ph 4.5 ) . animals with a fasting blood glucose level higher than 13.3 mm were considered to be diabetic rats . normal and diabetic rats were randomly divided into four groups : control group ( c , n = 5 ) , diabetic group ( d , n = 5 ) , h2dipic - cl - treated group ( l , n = 5 ) , and v4dipic - cl - treated group ( v4 , n = 5 ) . v4dipic - cl was orally administrated to diabetic rats in drinking water at a concentration of 50 g v / ml daily for 28 days . we have selected this concentration of vanadium on the basis of earlier reports and the same has also been standardized in our laboratory to exhibit the glucose - lowering effects in stz - induced diabetic animals [ 2 , 30 , 31 , 3336 ] . in this present study , fresh solutions of h2dipic - cl and v4dipic - cl were prepared every day and were given to the animals through drinking bottles . at the end of the treatment schedules , the livers were perfused in situ with saline and then were immediately removed , collected , and stored in liquid nitrogen . biochemical parameters in serum , including tc , tg , hdl - c , ldl - c , alanine transaminase ( alt ) , and aspartate aminotransferase ( ast ) were determined using an olympus au400 chemistry analyzer . sections measuring approximately 0.2 cm 0.2 cm were taken from the liver of each rat . they were dehydrated through graded solutions of alcohol ending in two changes of absolute alcohol for 2 h each . they were cleared in 2 changes of xylene , infiltrated in 2 changes of paraffin wax for 2 h each , and embedded in molten paraffin wax . sections were cut at 4 m with rotary microtome and stained with hematoxylin and eosin ( h&e ) . futher , the stained slides was observed under light microscope at 10x and 40x magnifications for histopathological examination . real - time pcr was carried out using the method described by xue et al . . the cdna was used as a template to examine the mrna levels of fat / cd36 , fatp5 , fabp1 , cpt1 , acox1 , apob , lxr , pxr , and ahr by using ultrasybr mixture . the pcr cycle was as follows : initial denaturation at 95c for 10 min , followed by 40 cycles of denaturation at 95c for 30 s , annealing at 60c for 30 s , and extension at 72c for 30 s. the primers for target genes are shown in table 1 . liver tissues were lysed in 1 ml of radio immunoprecipitation assay lysis buffer ( ripa ) and then centrifuged at 14000 rpm for 5 min . the lysates were subjected to sodium dodecyl sulfate polyacrylaminde gel electrophoresis ( sds - page ) . the gels were transferred to polyvinylidene fluoride ( pvdf ) membrane by semidry electrophoretic transfer at an electric current 1 ma / cm for 90 min . the pvdf membrane was blocked with 5% no - fat milk for 1 h at the room temperature and then incubated with the primary antibody ( 1 : 1000 ) overnight at 4c in a table concentrator . the membrane was washed per 5 min for 4 times prior to incubation in the secondary antibody ( 1 : 1000 ) solution for 1 h at the room temperature . immunoreactive bands were detected with electrochemiluminescence ( ecl ) reagents according to the manufacturer 's instructions . the statistical analysis was performed by one - way anova followed by tukey 's test . as shown in figures 1(a)1(c ) , serum tg , tc , and hdl - c levels in diabetic group were higher than those in control group . however , the level of serum tg was significantly decreased after treatment with v4dipic - cl . the concentration of tc in diabetic rats remained unchanged after treatment with v4dipic - cl . the ldl - c levels were not significantly different among the four groups of rats . moreover , the activities of serum alt and ast were markedly increased in diabetic rats . however , the alt and ast activities were decreased in v4dipic - cl - treated diabetic rats ( figures 1(e ) and 1(f ) ) . the hepatic tg level in diabetic group was higher than that in normal rats , which was significantly decresed after treatment with v4dipic - cl ( figure 2 ) . in comparison with the control group ( figure 3(a ) ) , inflammatory cells infiltrate of liver lobules and dilated congested central vein were observed in figure 3(b ) . however , the pathological alterations were ameliorated after treatment with v4dipic - cl ( figure 3(d ) ) compared to those of h2dipic - cl - treated diabetic rats ( figure 3(c ) ) . the mrna expression levels of fat / cd36 , fabp1 , and fatp5 in diabetes group were higher than those in control group ( figure 4 ) . however , the mrna expression level of fat / cd36 was significantly decreased after treatment with v4dipic - cl . moreover , the mrna expression levels of fat / cd36 , fabp1 , and fatp5 were significantly decreased in the h2dipic - cl - treated group . in contrast , treatment with v4dipic - cl did not affect the mrna expression levels of fabp and fatp in stz - induced diabetes . the mrna expression levels of lxr and pxr in diabetic group were significantly higher than those in control group . moreover , the mrna expression levels of lxr , pxr , and ahr were significantly elevated after treatment with v4dipic - cl ( figure 5 ) . however , the mrna expression levels of lxr , pxr , and ahr were significantly decreased in the h2dipic - cl - treated group as compared with the diabetic group ( figures 5(a)5(c ) ) . carnitine palmitoyltransferase i(cpt1 ) is located on the outer membrane of mitochondria and participates in fatty acid transportation into mitochondria . as shown in figure 6(a ) , the mrna expression level of cpt1 was increased in diabetic group . however , the mrna expression level of cpt1 was significantly decreased after treatment with v4dipic - cl and h2dipic - cl . in contrast , the mrna expression levels of peroxisomal acyl - coenzyme a oxidase 1 ( acox1 ) and apolipoprotein b ( apob ) were not significantly different among the four groups of rats ( figures 6(b ) and 6(c ) ) . accumulating evidences have demonstrated that stz - induced diabetes mellitus and insulin deficiency lead to hyperglycemia and dyslipidemia . it has been previously reported that hyperglycemia and dyslipidemia are associated with specific diabetic complications and disturbances in various tissues , such as diabetic nephropathy and cardiovascular diseases , but only limited data is available on the possible association between diabetic complications and liver function [ 30 , 40 ] . the present study was designed to evaluate the effects of v4dipic - cl on lipid metabolism disorders in the liver of stz - induced diabetic rats . it has been recognized that dyslipidemia is a frequent complication in all types of diabetes which can range from hypercholesterolemia to hyperlipoproteinemia . hyperlipidemia could be a factor for fatty liver formation . in the present study , as expected serum tg and tc as well as hepatic tg levels were elevated in the diabetic group compared to those in the control group , which is consistent with other studies [ 35 ] . however , the elevated level of serum tg in diabetic rats was significantly decreased after treatment with v4dipic - cl . this is in agreement with the evidence that vanadium compounds decrease the high levels of tg in serum and liver [ 29 , 30 ] . moreover , the altered expression of genes involved in lipid biosynthetic pathways in diabetes returned to normal level after treatment with vanadium compounds [ 2 , 42 ] . elevated activities of serum aminotransferases are a common sign of liver diseases [ 30 , 40 ] . typical serum biochemical parameters , such as alt and ast , are often examined to evaluate whether the liver is damaged or diseased . in the present study , our findings of elevated serum alt and ast levels are in agreement with the findings of zafar et al . . the increase in alt and ast activities may be due to the cellular damage in the liver caused by stz - induced diabetes . after 28 days of treatment with v4dipic - cl , the activities of both alt and ast were significantly decreased . the result suggests that v4dipic - cl may be capable of ameliorating the impaired liver function in stz - induced diabetic rats , which is consistent with previously reported results for treatment with vanadium complexes . described the fatty liver and hyperlipidemia in idmm of treated shrews . in the present study , the histopathology of liver showed a development of the lesions which seems to be due to stz treatment . most liver sections showed inflammatory cells infiltrate of liver lobules and dilated congested central vein . zafar et al . who showed dilatation of veins and liver fibrosis in their study [ 41 , 44 ] . however , we found that treatment with v4dipic - cl dramatically improved pathologic lesions seen in the liver . free fatty acids are a major component of blood lipids and plays a key role in regulating blood lipid levels , especially in triglyceride metabolism . in addition , elevated plasma fa is a risk factor for metabolic syndrome , which can lead to hyperlipidemia , fatty liver , and insulin resistance [ 46 , 47 ] . fat / cd36 is a rate - limiting enzyme in high - affinity peripheral fa uptake in the liver . thus , fat / cd36 is an important regulator in the uptake of fatty acids in the liver and the pathogenesis of fatty liver disease . it was reported that fa uptake is reduced in fat / cd36 null mice and is reconstituted when fat / cd36 is reexpressed . luiken et al . reported that fat / cd36 mrna expression is increased in streptozotocin - induced diabetes . thus , fat / cd36 may participate in the pathogenesis of liver ectopic fat deposition . in the present study , we found that the mrna expression level of fat / cd36 in diabetes group was significantly higher than that in control group . however , v4dipic - cl treatment significantly resulted in decrease in the mrna and protein expression levels of fat / cd36 in v4dipic - cl - treated group treatment . in addition to fat / cd36 , goldberg and ginsberg described that fatty acid - binding protein ( fabp ) and fatty acid transport protein ( fatp ) can mediate fatty acid uptake in the liver . in the present study , the mrna expression levels of fabp1 and fafp5 were increased in diabetic group , which is consistent with the previous report that the expression of fabp is increased in stz - induced diabetes . however , treatment with v4dipic - cl did not affect the expression of fabp and fatp in stz - induced diabetes . thus , we propose that v4dipic - cl can mainly regulate fatty acid transporter fat / cd36 in liver . zhou et al . described that nuclear acceptors ahr , pxr , and lxr cooperate to promote hepatic steatosis by increasing the expression of fat / cd36 [ 16 , 17 ] . more recently , cheng et al . demonstrated that the mrna expression level of lxr was markedly increased in diabetic rats . reported that fenofibrate , ppar agonist , dramatically reduced hepatic triglyceride levels by activating expression of acox1 and cpt1 involved in fatty acid turnover . in the present study , we also found that the mrna expression levels of lxr , pxr , and cpt1 were increased in diabetic group . however , treatment with v4dipic - cl did not decrease the mrna expression levels of lxr , pxr , and ahr as well as the fa oxidation - related cpt1 and acox1 as compared to h2dipic - cl - treated diabetic rats . it is possible that some other mechanisms may contribute to regulation of fat / cd36 expression through modulating nuclear receptors by vanadium compounds . this study showed that v4dipic - cl ameliorates stz - induced hepatic inflammatory infiltration , liver disfunction , and hepatic tg accumulation . this effects were likely associated with the modification of lipid metabolism - related fat / cd36 in liver . these results together with the previous observations suggest that v4dipic - cl can be used as a therapeutic agent for treatment of metabolic disorder in diabetes mellitus .

vanadium complexes are potent antidiabetic agents for therapeutical treatment of diabetes . in the present study , we investigated the hypolipidemic effect of vivo(dipic - cl)(h2o)2 ( v4dipic - cl ) in liver of streptozotocin- ( stz-)-induced diabetic rats . we found that diabetic animals exhibited hepatic inflammatory infiltration and impaired liver function along with triglyceride ( tg ) accumulation in the liver . v4dipic - cl treatment not only ameliorated liver pathological state but also reduced hepatic tg level . moreover , the upregulation of fatty acid translocase ( fat / cd36 ) mrna ( 4.0-fold ) and protein ( 8.2-fold ) levels in the liver of diabetic rats were significantly reversed after v4dipic - cl treatment . however , no significant effects of v4dipic - cl on the mrna expression of fatty acid metabolism - related fatty acid bounding protein 1 ( fabp1 ) and fatty acid transporter 5 ( fatp5 ) were observed . these results suggest that the modification of lipid metabolism - related fat / cd36 in the liver of diabetic rats is likely involved in the hypolipidemic effects of v4dipic - cl .

over the last few decades , minimally invasive surgery has been widely applied , particularly in gynaecology , where laparoscopic surgery has assumed an increasingly central role . data regarding minimally invasive surgery have shown more benefits compared to abdominal surgery in terms of surgical outcomes , cost , quality of life , reduced morbidity , shorter hospital stay and a rapid return to daily activities . moreover , recent advances in laparoscopic instruments and techniques have allowed completion of a great number of complex surgical procedures including in gynaecologic oncology surgery . recently , the laparoscopic approach has been revised and laparoendoscopic single - site surgery ( less ) has been introduced . the term single - site describes the use of a single small skin incision instead of the multiple accesses created in conventional laparoscopy . the less port is a multichannel port that allows the insertion of up to three instruments . according to many authors , reducing number of ports on the abdominal wall may improve the cosmetic results and decrease postoperative pain and complications such as hernias , infection and nerve injuries . the first application of less was a tubal sterilisation in 1969 , while the first hysterectomy with salpingo - oophorectomy was performed in 1991 . thereafter , less became effective , safe and feasible for performing complex surgical procedures in gynaecology and urology and in abdominal surgery . the aim of this study was to assess the surgical and oncological outcome in the management of endometrial cancer ( ec ) by less . all patients diagnosed with clinical international federation of gynecology and obstetrics ( figo ) stage i ii ec , both endometrioid and non - endometrioid , and confirmed at the definitive histological examination , who underwent less were included . before surgery , all patients underwent a clinical and instrumental evaluation , including taking medical history , undergoing a physical examination , a vaginal - pelvic examination , a chest x - ray , an ultrasound scan and pelvic magnetic resonance imaging scan . an intraoperative histological examination was required during all the surgeries and a pelvic lymphadenectomy was performed only when risk factors ( myometrial invasion more than 50% , high grading and non - endometrioid histotype ) were detected . para - aortic lymphadenectomy is not routinely performed unless pelvic lymph nodes are confirmed to have metastatic disease on frozen section evaluation in order to determine the field of postoperative radiation . informed consent for less was obtained from all patients in accordance with local and international legislation ( declaration of helsinki ) . patient characteristics were recorded , including age , body mass index ( bmi ) , histotype , figo stage , grading and prior abdominal surgery . subsequently , the intraoperative parameters were recorded : median skin incision , operative time , blood loss , transfusions , conversion rate and intraoperative complications . blood loss was evaluated by the difference in the total amounts of suction and irrigation fluids . postoperative parameters included early postoperative complications ( in the first 30 days after surgery ) and later postoperative complications ( more than 30 days after surgery ) , type of adjuvant therapy ( radiotherapy and/or chemotherapy ) , median follow - up in months , recurrence , time to recurrence and median follow - up . postoperative pain assessment was performed in all patients using a validated numeric rate score ( nrs ) and scored from 0 to 10 ( 0 = no pain and 10 = agonising pain ) . nrs was recorded at regular intervals : t0 ( end of surgery ) , t1 ( after 1 h ) , t2 ( after 6 h ) , t3 ( after 12 h ) , every 24 h starting from the second postoperative day . analgesic therapy with tramadol 100 mg plus ketorolac 60 mg by continuous infusion for the first 24 h after the end of surgery was administered . adjuvant therapy was tailored to the pathologic findings at primary operation after multidisciplinary tumour board ( gynaecologic oncology , pathology , radiation oncology , medical oncology ) discussion . treatment was based on the results of prospective randomised clinical trials and national comprehensive cancer network guidelines . follow - up was performed every 3 - 4 months with physical and gynaecological examination for the first 2 years , and then with a 6-month interval until 5 years . the patient was positioned in the dorsal lithotomic trendelenburg position , with both legs supported by straps . no uterus manipulator devices were used , but the cervix was closed with a modified tenaculum called simple nebs arising incision landmark ( snail , snail is obtained by the modification of an instrument named uterine tenaculum forceps , model schroder , code 32 - 622 - 25 of martin catalogue . currently the snail was patented but not yet marketed ) . a medical - grade silicone balloon , named colpo - pneumo occluder ( cooper surgical , coopersurgical , inc . , 95 corporate drive , trumbull , ct 06611 , usa ) was also emplaced in the vagina in order to preserve an adequate pneumoperitoneum during colpotomy . by , using the hasson technique , it was possible to place the single - incision laparoscopic surgery ( sils ) port ( covidien , 710 medtronic parkway , minneapolis , usa ) through a 2 - 2.5 cm umbilical incision [ figure 1a ] . the sils port is a multichannel port that allows the insertion of up to three instruments . after emplacing the co2 insufflation through one of the three available channels , it was possible to introduce a 5-wmm trocar for the insertion of a 30-degree laparoscope . in the remaining two channels , two 5-mm trocars were placed for the introduction of additional instruments , such as as roticulator endo dissect or mini - shears or grasp - all 5 mm ( covidien ) [ figure 1b ] . a careful inspection of the entire abdominal cavity was performed as the first surgical step in order to identify any suspicious peritoneal lesions that would exclude the patient from having the procedure completed by less . after cauterising the fallopian tubes , the right round ligament was sectioned , entering into the retro - peritoneal space . it was possible to cauterise the uterine vessels . finally , a sufficient margin of vagina was exposed , allowing its section . all patients underwent type a or b1 hysterectomy plus salpingo - oophorectomy ( querleu morrow classification ) . in the end , the uterus and adnexa were extracted through the vagina and the vaginal cuff was sutured through the vagina , while the site of introduction of the port was closed and the fascia of the rectus muscles sutured using vicryl 1/0 ( ethicon endo surgery , somerville nj ) . pelvic lymphadenectomy was performed based on frozen section analysis according to the technique of escobar et al . ( a ) less device inserted into the umbilicus ( b ) less device with three 5-mm trocars all patients diagnosed with clinical international federation of gynecology and obstetrics ( figo ) stage i ii ec , both endometrioid and non - endometrioid , and confirmed at the definitive histological examination , who underwent less were included . before surgery , all patients underwent a clinical and instrumental evaluation , including taking medical history , undergoing a physical examination , a vaginal - pelvic examination , a chest x - ray , an ultrasound scan and pelvic magnetic resonance imaging scan . an intraoperative histological examination was required during all the surgeries and a pelvic lymphadenectomy was performed only when risk factors ( myometrial invasion more than 50% , high grading and non - endometrioid histotype ) were detected . para - aortic lymphadenectomy is not routinely performed unless pelvic lymph nodes are confirmed to have metastatic disease on frozen section evaluation in order to determine the field of postoperative radiation . informed consent for less was obtained from all patients in accordance with local and international legislation ( declaration of helsinki ) . patient characteristics were recorded , including age , body mass index ( bmi ) , histotype , figo stage , grading and prior abdominal surgery . subsequently , the intraoperative parameters were recorded : median skin incision , operative time , blood loss , transfusions , conversion rate and intraoperative complications . blood loss was evaluated by the difference in the total amounts of suction and irrigation fluids . postoperative parameters included early postoperative complications ( in the first 30 days after surgery ) and later postoperative complications ( more than 30 days after surgery ) , type of adjuvant therapy ( radiotherapy and/or chemotherapy ) , median follow - up in months , recurrence , time to recurrence and median follow - up . postoperative pain assessment was performed in all patients using a validated numeric rate score ( nrs ) and scored from 0 to 10 ( 0 = no pain and 10 = agonising pain ) . nrs was recorded at regular intervals : t0 ( end of surgery ) , t1 ( after 1 h ) , t2 ( after 6 h ) , t3 ( after 12 h ) , every 24 h starting from the second postoperative day . analgesic therapy with tramadol 100 mg plus ketorolac 60 mg by continuous infusion for the first 24 h after the end of surgery was administered . adjuvant therapy was tailored to the pathologic findings at primary operation after multidisciplinary tumour board ( gynaecologic oncology , pathology , radiation oncology , medical oncology ) discussion . treatment was based on the results of prospective randomised clinical trials and national comprehensive cancer network guidelines . follow - up was performed every 3 - 4 months with physical and gynaecological examination for the first 2 years , and then with a 6-month interval until 5 years . the patient was positioned in the dorsal lithotomic trendelenburg position , with both legs supported by straps . no uterus manipulator devices were used , but the cervix was closed with a modified tenaculum called simple nebs arising incision landmark ( snail , snail is obtained by the modification of an instrument named uterine tenaculum forceps , model schroder , code 32 - 622 - 25 of martin catalogue . currently the snail was patented but not yet marketed ) . a medical - grade silicone balloon , named colpo - pneumo occluder ( cooper surgical , coopersurgical , inc . , 95 corporate drive , trumbull , ct 06611 , usa ) was also emplaced in the vagina in order to preserve an adequate pneumoperitoneum during colpotomy . by , using the hasson technique , it was possible to place the single - incision laparoscopic surgery ( sils ) port ( covidien , 710 medtronic parkway , minneapolis , usa ) through a 2 - 2.5 cm umbilical incision [ figure 1a ] . the sils port is a multichannel port that allows the insertion of up to three instruments . after emplacing the co2 insufflation through one of the three available channels , it was possible to introduce a 5-wmm trocar for the insertion of a 30-degree laparoscope . in the remaining two channels , two 5-mm trocars were placed for the introduction of additional instruments , such as as roticulator endo dissect or mini - shears or grasp - all 5 mm ( covidien ) [ figure 1b ] . a careful inspection of the entire abdominal cavity was performed as the first surgical step in order to identify any suspicious peritoneal lesions that would exclude the patient from having the procedure completed by less . after cauterising the fallopian tubes , the right round ligament was sectioned , entering into the retro - peritoneal space . then , the ureter and uterine artery were visualised and adnexal resection was performed . subsequently , vescicouterine , vascicovaginal and rectovaginal spaces were developed . in this way , it was possible to cauterise the uterine vessels all patients underwent type a or b1 hysterectomy plus salpingo - oophorectomy ( querleu morrow classification ) . in the end , the uterus and adnexa were extracted through the vagina and the vaginal cuff was sutured through the vagina , while the site of introduction of the port was closed and the fascia of the rectus muscles sutured using vicryl 1/0 ( ethicon endo surgery , somerville nj ) . pelvic lymphadenectomy was performed based on frozen section analysis according to the technique of escobar et al . ( a ) less device inserted into the umbilicus ( b ) less device with three 5-mm trocars between july 2009 and june 2013 , 50 women underwent laparoendoscopic single - site hysterectomy with or without pelvic lymphadenectomy for the treatment of ec at the gynecological oncology unit at the regina elena national cancer institute , rome , italy . median age was 45 years ( range , 39 - 84 years ) and median bmi was 21.8 kg / m ( range , 19 - 48 kg / m ) . in 15 patients ( 29.7% ) , previous abdominal surgical procedures were found . port placement , taking a median time of 1.5 min ( range , 1 - 3 min ) , was successfully performed in all cases without accidental port removal . median operative time was 100 min ( range , 50 - 240 min ) and median blood loss was 90 ml ( range , 10 - 300 ml ) . pelvic lymphadenectomy was performed in 6 patients , and the median numbers of pelvic lymph nodes retrieved was 14 ( range , 5 - 20 ) . no intraoperative complications occurred , but there were 4 postoperative complications : a vaginal cuff dehiscence , an umbilical wound infection and a lymphorrhoea and bladder fistula with the vagina in the same patient who underwent hysterectomy plus pelvic lymphadenectomy on the 13th postoperative day . all procedures were successfully completed but there were 2 patients who required a laparoscopic conversion due to extensive adhesions from previous surgery . it was strongly influenced by the prolonged hospital stay of a patient who had a length of stay of 9 days due to an umbilical wound infection . in fact , without this patient , the median length of stay was 2 days . all patients had a good cosmetic result [ figure 2 ] and postoperative pain control . regarding this last point , the median nrs at t0 was 3.5 ( range , 1 - 5 ) , at t1 was 2 ( range , 1 - 4 ) , at t2 was 1.5 ( range , 1 - 3 ) , and at t3 and during patient discharge was 0.5 ( range , 0 - 1 ) . ( a ) umbilical appearance at the end of surgery ( b ) umbilical appearance 6 months after surgery adjuvant therapy was performed in 10 patients [ table 3 ] : 9 patients underwent to radiotherapy ( 6 patients figo stage ib g3 and 3 figo stage ii ) and only 1 underwent radio - chemotherapy ( figo stage iiia ) . the median follow - up was 36 months ( range , 16 - 62 months ) and only 1 patient died due to the occurrence of a new cancer after 11 months from surgery . our report shows that less could be a safe and reliable technique in terms of surgical and oncological outcomes in patients with ec . during the last decade , minimally invasive surgery has been widely introduced in the management of the gynaecological malignant disease . even though the role of less in gynaecological oncology is well established , it is in constant development . in particular , over the last few years , published studies have shown the excellent application of the less in the treatment of early - stage ec . our results are similar to those reported in the literature , except for a few aspects . undoubtedly , any previous abdominal surgery could limit the application and render the performance of this surgery difficult . moreover , surgery in our cases was performed by a surgeon with extensive skill and experience in laparoscopic surgery , which makes the application of less easier . in a review of literature on laparoendoscopic single - site hysterectomy with or without pelvic lymphadenectomy for the treatment of ec between 2009 and 2014 the operating time was 35 - 342 min , blood loss was 0 - 700 ml , pelvic and para - aortic lymph nodes removed were 2 - 43 and 2 - 28 , respectively , while hospital stay was 1 - 6 days . the number of conversions reported was 21 ( 4.8% ) , with intraoperative and postoperative complications of 22 ( 5% ) and 14 ( 3.2% ) , respectively [ table 4 ] . literature review of less procedure in only ec fagotti et al . reported the widest series of early ec treated with less described in the literature . among the 100 cases , total hysterectomy and bilateral salpingo - oopherectomy were performed , while pelvic and para - aortic lymphadenectomy in 48 and 27 patients , respectively . the median number of pelvic and para - aortic lymph nodes was 16 and 7 , respectively , similar to those reported in studies utilising traditional laparoscopy . two patients undergoing lymphadenectomy required conversion , and 4 intraoperative and 4 postoperative complications occurred overall . overall , less entailed good results in terms of surgical outcomes , postoperative pain and cosmetics . moreover , operative time seemed to decrease with surgeon 's experience , increasing the number of performed surgeries . furthermore , escobar et al . in 2010 described single - port laparoscopic pelvic and para - aortic lymphadenectomy in 21 patients . the median number of pelvic and para - aortic lymph nodes retrieved was 14 and 6 , respectively , and all procedures were successfully completed with only one conversion to traditional laparoscopy . in literature , oncological outcomes are not often investigated , and we actually report a higher median follow - up . in fact , with a median follow - up of 36 months ( range , 16 - 62 months ) , we had only 1 patient die due to the occurrence of a new cancer 11 months after surgery . given the important role of minimally invasive surgery in gynaecological surgery , several studies have compared the perioperative outcomes of less with other , different minimally invasive techniques . escobar et al . compared less , conventional laparoscopic and robotic surgery in the treatment of ec , but no differences were found in terms of operative time , blood loss , hospital stay and complication rates . subsequently , another study compared less and robotic surgery , showing that less had a shorter operative time ( 122 min vs 175 min ) and lower blood loss ( 50 ml vs 80 ml ) than robotic surgery . furthermore , a case - control study compared sils ( 38 patients ) and robotic single - port surgery ( 19 patients ) , showing no clinical relevant differences . in 2012 , fanfani et al . , compared less and mini - laparoscopic surgery in a series of patients , including cases with malignant disease , and did not find any significant differences in terms of surgical outcomes except for operative time , which was longer in less groups . less is widely applied in gynaecological surgery with the same absolute contraindications of conventional laparoscopy : metastatic disease , heart or pulmonary disease , and severe abdominal adhesions . moreover , the surgeon should have advanced skills in laparoscopic and single - port surgery . obesity and previous abdominal surgery should no longer be considered absolute limits in the performance of less . one of the most important technical challenges is the instrument crowding due to the restricted space available to make necessary movements . the lack of triangulation is another important problem that can be easily overcome by applying articulating and flexible instruments or curved instruments . however , extensive experience in laparoscopic surgery might be enough to overcome the challenges in single - port surgery . the latest innovation in minimally invasive surgery is the introduction of the robotic system da vinci ( intuitive surgical inc , 1266 kifer road , building 101 sunnyvale , ca ) . the combination of robot and single port offers new advantages as well as in allowing many challenges to be overcome in single - port surgery . despite being an expensive surgical technique , the robotic single port could be a promising new surgical approach , including in morbidly obese women . in a recent study , robotic surgery was confirmed to be a good surgical approach in the treatment of ec in obese women . the application of the da vinci robotic system in gynaecological surgery may make single - site surgery feasible and safe for many including morbidly obese women . in conclusion , less is a safe and feasible surgical approach in the treatment of ec . further studies are needed to define the long - term outcomes , the disease - free survival and overall survival . robotic single - port surgery is a potential area of development , but it is still not clear whether the benefits of robotic surgery will justify the high costs of this new approach .

background : the aim of this study was to assess the surgical and oncological outcome for the management of endometrial cancer ( ec ) by laparoendoscopic single - site surgery ( less).patients and methods : we performed a retrospective chart review of patients who underwent a less for ec . all the patients were treated by the same surgical team between july 2009 and june 2013 at the gynaecologic oncologic unit , regina elena national cancer institute , rome , italy.results:a total of 50 women were included , with a median age of 45 years ( range , 39 - 84 years ) and a median body mass index ( bmi ) of 21.8 kg / m2 ( range , 19 - 48 kg / m2 ) . median operative time was 100 min ( range , 50 - 240 min ) , median blood loss was 90 ml ( range , 10 - 300 ml ) and median hospital stay was 3 days ( range , 2 - 9 days ) . the median number of pelvic lymph nodes retrieved was 14 ( range , 5 - 20 ) . no intraoperative complications occurred , but there were 4 postoperative complications . two patients required a laparoscopic conversion . the median follow - up was 36 months ( range , 16 - 62 months ) and no recurrence occurred.conclusion:our report showed that the less approach in the treatment of early ec can be a safe and reliable technique in terms of surgical and oncological outcomes .

g - protein - coupled receptors ( gpcrs ) constitute the largest family of membrane proteins and are known to respond to a plethora of different stimuli by activating numerous intracellular signaling pathways . because of their involvement in a variety of biological processes , gpcrs are the target of more than 50% of currently marketed drugs . to date , most of the drugs targeting gpcrs are known to interact with the orthosteric site - that is , the endogenous ligand - binding pocket . these orthosteric ligands need , however , to overcome many limitations , such as decreased selectivity , insufficient clinical efficacy , and undesirable effects on receptor regulation . the past decade witnessed the discovery of new pharmaceutical compounds that modulate receptor function by targeting allosteric sites . allosteric sites are , by definition , domains topographically distinct from the orthosteric binding pocket . these allosteric modulators are very promising pharmaceutical drugs devoid of many of the disadvantages of orthosteric ligands . in fact , these allosteric compounds show higher subtype selectivity ( allosteric sites are generally less conserved than orthosteric sites ) , often have a safer pharmacological profile ( exert their effects only in the presence of endogenous ligands ) and , in some cases , do not induce receptor desensitization . there are three general categories of allosteric modulators ( figure 1a ) : first , modulators affecting the binding affinity of orthosteric ligands , such as amno82 , which is responsible for the incomplete inhibition of orthosteric antagonist binding to the metabotropic glutamate ( mglu ) 7 receptor , mglu7r ; and second , modulators affecting orthosteric ligand efficacy . one example is cgp7930 , which is known to enhance gamma - aminobutyric acid receptor b ( gabab)-mediated gtp s ( guanosine 5-o-[gamma - thio]triphosphate ) binding . third , allosteric agonists and inverse agonists , known to engender a unique gpcr conformation that alters receptor signaling in the absence of orthosteric ligands . for example , mpep ( 2-methyl-6-(phenylethynyl)pyridine ) acts as a negative allosteric modulator of mglu5r by inhibiting inositol phosphate production in the absence of an agonist . another example is ac42 , which was first identified as a selective agonist of the m1 muscarinic acetylcholine receptor ( m1achr ) ; however , mutation studies have implied that this ligand activates the receptor via an allosteric site . ( a ) an allosteric ligand may modulate binding ( 1 ) and signaling ( 2 ) of orthosteric ligands or ( 3 ) induce signal transduction by its own ( allosteric agonists ) ( adapted from langmead and christopoulos ) . ( b , c ) comparison of different binding modes of orthosteric ( red circle ) and allosteric ( red triangle ) ligands as monovalent ligands ( b ) or bivalent bitopic ligands ( c ) . binding of orthosteric ligands induces signal 1 , which can be modulated by allosteric ligands binding to sites that are close to or distant from the orthosteric binding site , generating signal 1 * or signal 1**. alternatively , the signal generated by the orthosteric ligand in one protomer can be allosterically modulated by the other protomer within a gpcr dimer , generating signal 1***. allosteric agonists can induce signaling by their own in the absence of orthosteric ligands ( signal 2 ) . signals 1 * , 1 * * , and 1 * * * can also be generated by bitopic ligands . in addition , bitopic ligands could induce signals ( signals 1 , 1 , and 1 ) that are specific for these ligands and not observed upon simultaneous stimulation with monovalent orthosteric and allosteric ligands . recently , a new class of ligands , termed bitopic or dualsteric ligands , which simultaneously target orthosteric and allosteric sites , emerged ( figure 1b ) . the development of bitopic ligands is based on the idea of combining high affinity ( via orthosteric sites ) with high selectivity ( via allosteric sites ) . until now , muscarinic acetylcholine receptors ( machrs ) have proved to be a particularly fruitful receptor model for the development and characterization of bitopic ligands . in 2004 , tahtaoui et al . synthesized seven fluorescent derivatives of the bodipy - labeled pirenzipine , an m1achr antagonist , and used fluorescent resonance energy transfer technology to study receptor - ligand interactions . the authors showed that these analogs might interact with both the acetylcholine ( orthosteric ) and brucine ( allosteric ) binding domains of the m1achr , therefore behaving as potential bitopic ligands . the literature also reports the design of hybrid molecules from allosteric modulators of w84-type compounds and orthosteric antagonists or agonists of machrs to obtain subtype selective muscarinic ligands with agonistic or antagonistic properties . in a recent publication , christopoulos and colleagues elegantly showed that mcn - a-343 , a selective m2achr partial agonist , is actually a bitopic ligand . using several different binding and functional assays , the authors showed that mcn - a-343 is composed of an orthosteric agonist coupled to an allosteric modulator ( 3-chlorophenylcarbamate moiety ) . truncated derivatives of mcn - a-343 retaining the chlorophenylcarbamate moiety were positive modulators of the orthosteric antagonist n-[3h]methylscopolamine in radioligand binding assays , but in functional assays , such as m2achr - mediated erk1/2 ( extracellular signal - regulated kinase 1/2 ) phosphorylation , they acted as negative modulators of agonist efficacy . finally , in 2009 , antony et al . presented a novel approach to pharmacologically design subtype and signaling selective receptor agonists , once again using the machrs as templates . their paper reports the synthesis of two hybrids fusing a highly potent oxotremorine - like orthosteric activator with m2-selective bis(ammonio)alkane - type allosteric fragments . radioligand binding in wild - type and mutant receptors supplemented by receptor docking simulations showed an m2-selective and true allosteric / orthosteric binding . moreover , g - protein activation , in this context , was mediated by the orthosteric moieties . due to the unique features of bitopic ligands , their number is likely to expand in the near future . this may include the re - examination of already existing ligands , as was the case with mcn - a-343 , and the de novo design of bitopic ligands as shown by antony et al . . where can we expect to see the fastest progress ? the number of already existing ligands that might turn out to be bitopic is difficult to estimate and will strongly depend on the degree of information available for these putative bitopic molecules . the design of new bitopic ligands requires extensive knowledge about orthosteric and allosteric ligands available for a given gpcr as well as its ligand binding sites . this may explain the fact that bitopic ligands have predominantly been described for muscarinic receptors , for which detailed knowledge on allosteric binding sites and ligands is indeed available . once allosteric ligands have been identified , the choice of an optimal linker has to be considered . the extensive literature on the design and synthesis of bivalent ligands intended to target two orthosteric ligand binding sites within a gpcr dimer will be a rich source of inspiration for the synthesis of bitopic ligands . as illustrated by compound mcn - a-343 and hybrid compounds described by antony et al . , the better the structural knowledge about orthosteric and allosteric binding sites , the easier the rational design of the linker ( chemical nature and length of linker , linker attachment point to pharmacophores ) will be . recently solved gpcr structures will assist the design and synthesis of bitopic ligands that bind to identified orthosteric and allosteric binding pockets . such gpcr structures have not only provided detailed knowledge about orthosteric binding pockets , but also revealed the important role of extracellular receptor domains in guiding small molecular weight ligands to their high affinity ligand binding pockets within the transmembrane domain . these ligand entrance channels are interesting new targets for allosteric modulation by bitopic ligands . finally , the concept of bitopic ligands may also be extended to gpcr dimers , where the orthosteric and allosteric pharmacophores bind to two different protomers in the receptor dimer ( figure 1b ) .

natural ligands of g - protein - coupled receptors interact with the orthosteric ligand binding site , as do most of the classical synthetic ligands . the discovery of ligands targeting different , allosteric binding sites considerably expanded the repertoire of g - protein - coupled receptor ligands . more recently , bitopic ligands have been described that target both orthosteric and allosteric sites at the same time .

anisakiasis is one of the fishborne zoonotic diseases , especially in localities , where people have a custom of eating raw marine fish and squids like in japan and korea . the concept of this disease was first raised by van thiel et al . in the netherlands . after then , human infection cases were consequently reported in england , japan , and the republic of korea ( korea ) [ 4 - 7 ] . nowadays , this zoonotic nematode disease is a clinical concern worldwide , including japan , korea , the netherlands , spain , france , italy , germany , and the united states . especially in japan , it has been known that more than 2,000 cases occurred annually . since the first human case was reported by kim et al . in 1971 , until now numerous patients have increasingly occurred in korea . especially the increasing tendency of cases was accelerated by the common use of endoscopy in the diagnosis and treatment of gastric anisakiasis . recently , lee et al . analyzed total 141 cases diagnosed by the endoscopy in a local clinic in jinju - si , gyeongsangnam - do . clarified the etiologic agents of 16 human anisakiasis in korea , 15 of which were confirmed as anisakis pegreffii larvae and only 1 was anisakis simplex larva using a molecular analysis . in this study , we report an additional 15 gastric anisakiasis cases that occurred in gyeongsangnam - do , and then analyzed the epidemiologic and clinical characteristics of korean anisakiasis cases through a literature review . total 15 anisakiasis cases were diagnosed with the gastric endoscopy in the gyeongsang national university hospital and a local clinic located in jinhae - gu , changwon - si , gyeongsangnam - do from october 1999 to january 2010 . most of the cases ( 80% ) were men , and only 3 were women . total a single worm was detected from 12 cases , 2 worms from 1 case , and even 9 larvae were found from 2 cases . epigastric pain was the most common symptom in almost all cases , and nausea and vomiting were accompanied in some cases . symptom manifestations began at 10 - 12 hr after eating fish in most cases ( 73.3% ) . endoscopy was performed in 1 - 2 days after symptom onset in most cases ( 66.7% ) . as the probable source of infection , 7 ( 46.7% ) and 2 ( 13.3% ) cases recalled that they had eaten raw common conger , conger myriaster , and anchovy , respectively . however , 6 cases could not remember the exact species of fish ; several kinds of fish mixed or uncertain . the case information is summarized in table 1 . among the total 32 anisakis type i larvae ( either the 3rd or 4th - stage ) , 7 were morphologically observed and measured with a micrometer ( table 2 ) . the articles of korean anisakiasis cases were basically searched through pubmed site using the internet . the references in all articles were strictly seen to prevent missing cases . until now , a total of 645 cases have been reported in 64 articles since the first case recorded by kim et al . in 1971 . during 1990 - 1999 , a total of 249 cases ( 38.6% ) were reported in 25 papers ( 39.1% ) . the number of anisakiasis cases reported in the korean literature is shown in table 3 . the incidence rate was the highest in jeju - do ( 34.4% ) , followed by in gyeongsangnam - do ( 22.8% ) , seoul ( 20.0% ) , busan ( 10.1% ) , incheon ( 6.5% ) , gyeongsangbuk - do ( 3.2% ) , and other 9 administrative regions ( 0.16 - 0.79% ) ( table 4 ) . among a total of 404 cases which revealed the age and sex of patients , 185 ( 45.8% ) were men and the remaining 219 ( 54.2% ) were women . the age prevalence was the highest in forties ( 34.7% ) , followed by thirties ( 32.9% ) , fifties ( 14.9% ) , twenties ( 9.7% ) , over sixty ( 6.2% ) , and teens ( 1.7% ) ( table 5 ) . as the probable source of human infections , the common conger was most frequently mentioned by patients ( 38.6% ) , followed by croaker ( 11.6% ) , squid ( 10.3% ) , yellowtail ( 9.7% ) , flatfish ( flounder : 5.5% ) , and the other 12 species of marine fish ( table 6 ) . the etiologic larval type was designated in 203 cases in the literatures . among them , 165 cases ( 81.3% ) were due to anisakis type i larvae , 24 ( 11.8% ) were by terranova type a , and 8 cases ( 3.9% ) were by anisakis type ii . the predilection site was the stomach ( 82.4% ) , followed by small intestines ( 11.4% ) , gastro - esophageal junction ( 3.5% ) , large intestines ( 1.5% ) , esophagus ( 0.5% ) , and others including the palatine tonsil , ileo - cecal region , and mesocolic lymph node ( table 8) . in most cases ( 65.7% ) , symptoms were manifested before 12 hr from eating raw flesh of fish ( table 9 ) . the seasonal prevalence was the highest in winter ( 38.8% ) , followed by spring ( 23.1% ) , autumn ( 19.9% ) , and summer ( 18.2% ) ( table 10 ) . the articles of korean anisakiasis cases were basically searched through pubmed site using the internet . the references in all articles were strictly seen to prevent missing cases . until now , a total of 645 cases have been reported in 64 articles since the first case recorded by kim et al . in 1971 . during 1990 - 1999 , a total of 249 cases ( 38.6% ) were reported in 25 papers ( 39.1% ) . the number of anisakiasis cases reported in the korean literature is shown in table 3 . the incidence rate was the highest in jeju - do ( 34.4% ) , followed by in gyeongsangnam - do ( 22.8% ) , seoul ( 20.0% ) , busan ( 10.1% ) , incheon ( 6.5% ) , gyeongsangbuk - do ( 3.2% ) , and other 9 administrative regions ( 0.16 - 0.79% ) ( table 4 ) . among a total of 404 cases which revealed the age and sex of patients , 185 ( 45.8% ) were men and the remaining 219 ( 54.2% ) were women . the age prevalence was the highest in forties ( 34.7% ) , followed by thirties ( 32.9% ) , fifties ( 14.9% ) , twenties ( 9.7% ) , over sixty ( 6.2% ) , and teens ( 1.7% ) ( table 5 ) . as the probable source of human infections , the common conger was most frequently mentioned by patients ( 38.6% ) , followed by croaker ( 11.6% ) , squid ( 10.3% ) , yellowtail ( 9.7% ) , flatfish ( flounder : 5.5% ) , and the other 12 species of marine fish ( table 6 ) . the etiologic larval type was designated in 203 cases in the literatures . among them , 165 cases ( 81.3% ) were due to anisakis type i larvae , 24 ( 11.8% ) were by terranova type a , and 8 cases ( 3.9% ) were by anisakis type ii . the predilection site was the stomach ( 82.4% ) , followed by small intestines ( 11.4% ) , gastro - esophageal junction ( 3.5% ) , large intestines ( 1.5% ) , esophagus ( 0.5% ) , and others including the palatine tonsil , ileo - cecal region , and mesocolic lymph node ( table 8) . in most cases ( 65.7% ) , symptoms were manifested before 12 hr from eating raw flesh of fish ( table 9 ) . the seasonal prevalence was the highest in winter ( 38.8% ) , followed by spring ( 23.1% ) , autumn ( 19.9% ) , and summer ( 18.2% ) ( table 10 ) . by the present study , 15 cases of gastric anisakiasis have been added among the korean anisakiasis cases . like in the previous studies , epigastric pain was the chief complain and most commonly manifested in almost all cases . it has been known that severe bleeding can occur due to anisakid larva infection in patients with gastric ulcer [ 15,17 - 19 ] . time intervals from fish eating to symptom onset and from symptom onset to endoscopy were 10 - 12 hr and 1 - 2 days in most cases . the common conger , c. myriaster , is suspected as the most probable source of infection in 7 patients ( 46.7% ) . until now , in korea , a total of 645 cases have been reported in 64 papers since the first case reported by kim et al . . accordingly , as 15 cases have been added by this study , the total number of anisakiasis cases in korea became 660 cases in the korean literature . among 32 anisakis type i larvae detected , 7 were in the fourth - stage ( l4 ) , which had molted in the stomach of the patients . they were similar in size with the third stage larvae ( l3 ) , but they can be morphologically differentiated from l3 by the following characteristic features : ( 1 ) absence of the boring tooth at the anterior end and the mucron at the posterior end ; ( 2 ) the presence of the 3 well - defined lips ; ( 3 ) the appearance of transverse striations on the cuticular body surface ; ( 4 ) the appearance of altered ventriculus and intestine ( table 2 ) . the number of human anisakiasis cases ( 38.6% ) and papers ( 39.1% ) published in korea were the highest during 1990 - 1999 , while 163 cases ( 25.3% ) were reported in 10 articles ( 15.6% ) during 2000 - 2009 , and only 77 ( 11.9% ) were recorded in 18 papers ( 28.1% ) during 1980 - 1989 . during 1980 - 1989 , each anisakiasis case was meaningful and could be easily published in journals . however , recently , anisakiasis case reports were done after gathering large numbers of cases to demonstrate something different and unique findings [ 15,22 - 24 ] . so , the number of articles published was not so many during the recent years . the higher incidence rate was found in coastal areas , such as jeju - do , jinju , ( gyeongsangnam - do ) , busan , incheon , and pohang ( gyeongsangbuk - do ) , where people favorably consume raw flesh of marine fish . moreover , the presence of medical authorities , who are able to produce manuscripts describing case reports , is also an important factor [ 10 - 15,25,26 ] . among total 404 cases that revealed the age and sex of patients , the number of women was slightly more than that of men , with the ratio of women to men of 1.18:1 . , who analyzed 141 gastrointestinal anisakiasis cases in a local clinic in jinju - si , gyeongsangnam - do . in their study , the number of women ( 91 cases ) was much more than that of men ( 50 ) . . reported total 107 cases in jeju - do , the number of women ( 47 cases ) was lesser than that of men ( 60 ) , with the ratio of women to men of 0.78:1 . regarding the age prevalence , most of the cases ( 67.6% ) were patients in their thirties and forties . various species of marine fish and cephalopods have been known as the sources of human infections . in european countries , especially in the netherlands and spain , raw or pickled fish , such as herring , hake , anchovy , and cod , are known to be important infection sources . in japan , some species of flounders , japanese common squid , the pacific cod , and the bluefin tuna are the main sources in hokkaido , and the spotted sardine , the chub mackerel , the horse mackerel , the oceanic bonito , and the chum salmon are important infection sources in kyushu . however in korea , the common conger was the most frequently mentioned by the patients as the probable source of infections , followed by the croaker , squid , yellowtail , flatfish , and the other 12 species of marine fish . it has been known that the majority of human infections were caused by the larvae of a. simplex ( anisakis type i ) , and rarely or occasionally by those of anisakis physeteris ( anisakis type ii ) , and pseudoterranova decipiens ( terranova type a ) [ 13,14,30 - 33 ] . in the present review , 81.3% cases were due to anisakis type i larvae , and 11.8% and 3.9% were by terranova type a and anisakis type ii larvae , respectively . recently , the etiologic agents of human anisakiasis in korea , which were morphologically anisakis type i larvae , were molecularly confirmed to be a. pegreffii larvae . moreover , most of the anisakis type i larvae detected in 7 fish species from the yellow sea and the south sea in korea were also identified as a. pegreffii . although only 15 human cases were enrolled by lim et al . , it seems that most of the korean ansakiasis cases may be caused by a. pegreffii based on the above 2 molecular studies . molecular methods should be more frequently adopted to identify the causative agent of human anisakiasis in the future . however , anisakid larvae were found rarely in other digestive organs , i.e. , esophagus , small intestines , large intestines , and mesocolic lymph node . in most cases , the seasonal prevalence was the highest in winter , followed by in spring , autumn , and summer . this seasonality seemed directly related to the frequency of eating raw fish flesh in each season . the consumption of raw fish meat is more popular in winter season rather than in summer season in korea because of the anxiety for food poisoning .

the present study was performed to report 15 anisakiasis cases in korea and to review the korean cases reported in the literature . total 32 anisakis type i larvae were detected in the stomach of 15 patients by the endoscopy . single worm was detected from 12 cases , and even 9 larvae were found from 2 cases . epigastric pain was most commonly manifested in almost all cases , and hemoptysis and hematemesis were seen in 1 case each . symptom manifestations began at 10 - 12 hr after eating fish in 73.3% cases . endoscopy was performed 1 - 2 days after the symptom onset in most cases . the common conger , conger myriaster , was the probable infection source in 7 cases . in the review of korean anisakiasis cases , thus far , total 645 cases have been reported in 64 articles . anisakis type i larva was the most frequently detected ( 81.3% ) . the favorable infection site of larvae was the stomach ( 82.4% ) . the common conger was the most probable source of human infections ( 38.6% ) . among the total 404 cases which revealed the age and sex of patients , 185 ( 45.8% ) were males , and the remaining 219 ( 54.2% ) were female patients . the age prevalence was the highest in forties ( 34.7% ) . the seasonal prevalence was highest in winter ( 38.8% ) . by the present study , 15 cases of gastric anisakiasis are added as korean cases , and some epidemiological characteristics of korean anisakiasis were clarified .

the purpose of lowering high blood pressure is to prevent morbidity and mortality from hypertensive complications , such as heart attack , stroke , renal failure , and heart failure . the veterans administration studies in the 1960s proved beyond doubt that treating hypertension with thiazides and -blockers ( the drugs available at that time ) significantly diminished the rates of strokes , renal failure , and heart failure , although the decrease in the rate of myocardial infarcts did not reach statistical significance . it has been estimated that a 1015 mmhg fall in systolic blood pressure should lead to a 15% reduction in relative risk for heart attack and to a 40% reduction for stroke . with the advent of new classes of antihypertensive agents , the emphasis shifted from efficacy in lowering blood pressure , which is taken for granted , to potential to protect against end - organ damage . controlled clinical trials have indicated that drugs from different classes have different neurohumoral and metabolic profiles , which might enhance or partially offset the benefits from blood pressure lowering per se . for example , thiazides and -adrenergic blockers have been reported to further increase insulin resistance , and hence to accentuate the dysmetabolic syndrome that commonly accompanies essential hypertension . on the contrary , 1-adrenergic blockers and angiotensin - converting enzyme ( ace ) inhibitors have been reported to improve insulin sensitivity and the lipid profile , whereas calcium - channel blockers were found to be metabolically neutral . in terms of neurohormonal changes , the stimulation of the renin angiotensin and sympathetic systems associated with the use of diuretics and dihydropyridines should be detrimental to end organs , whereas angiotensin blockers and sympathetic blockers should be beneficial to them . several such effects that are theoretically considered to be beneficial have been used as ' surrogate endpoints ' in the absence of firm data on morbidity and mortality . improvement in surrogate endpoints may be encouraging but is not always predictive of real endpoints , and should not be sufficient to influence clinical decisions . this was shown repeatedly by recent trials ( e.g. with estrogen replacement or various antioxidants ) , where amelioration in various markers did not result in improved cardiovascular outcomes . nevertheless , clinical trials on selected subpopulations as well as meta - analyses of pooled data suggest that , at levels producing a similar blood pressure lowering effect , -blockers were cardioprotective and ace inhibitors were both cardioprotective and nephroprotective , while calcium - channel blockers might offer better protection from stroke . these newer classes have not strictly speaking been proven to reduce morbidity and mortality from hypertension , as they could not ethically be tested against placebo . they could , however , be tested against ' the gold standard ' , a thiazide that has been proven to reduce morbidity and mortality in the placebo - controlled trials . this is what led to the antihypertensive and lipid lowering treatment to prevent heart attack trial ( allhat ) . the double - blind , active - controlled component of the allhat was designed to determine whether the rate of the primary outcome ( a composite of fatal myocardial infarcts and nonfatal coronary events ) would be different in high - risk older patients treated with a drug from each one of three classes of antihypertensives , an ace inhibitor ( lisinopril ) , a calcium blocker ( amlodipine ) , or an 1-adrenergic blocker ( doxazosin ) , compared with patients treated with a thiazide diuretic ( chlorthalidone ) . the trial started in february 1994 and , after an interim analysis in january 2000 , an independent review committee recommended that the doxazosin arm be discontinued . this was because , compared with chlorthalidone , doxazosin had a significantly higher relative risk of stroke ( 1.19 ) and of combined cardiovascular disease ( relative risk = 1.25 ) , and a more than double risk of congestive heart failure ( relative risk = 2.04 ) . there was dismay that , once more , improvement in surrogate endpoints ( blood pressure , lipid profile , and other parameters of the dysmetabolic syndrome ) did not translate into favorable outcomes . there was speculation on what concurrent changes might have overridden the benefits of those improvements . we would like to add our own plausible , although speculative , explanation for these findings . in addition to the renin - angiotensin and the sympathoadrenal systems , arginine vasopressin ( avp ) is the third major systemic pressor hormone . its pressor function is partly offset by its sensitizing influence on baroreflexes , not fully apparent until the other two systems have been impaired . the importance of avp to systemic or regional vascular resistance can not necessarily be predicted from the circulating levels , as it is markedly increased after effective sympathetic inhibition even in the absence of a further increase in plasma levels . it can only be accurately estimated from the response to a selective antagonist of the v1 type receptors of avp . using such a pharmacologic probe , we have found that the pressor action of avp is maximized after 1-adrenergic blockade . under certain conditions , avp becomes an important vasopressor factor in patients with hypertension and/or congestive heart failure . its pressor influence is most apparent in patients with autonomic insufficiency , such as diabetics or elderly individuals . severe coronary constriction in response to avp has been proposed as the mechanism underlying a number of acute ischemic events reported in the earlier literature . although avp does not seem to cause myocardial ischemia under normal conditions , it may well do so under chronic 1-adrenergic receptor blockade , especially in older patients and in those with various degrees of autonomic insufficiency . the experimental evidence suggests that , in the presence of functional baroreflexes , the small elevation in avp following 1-adrenergic receptor blockade produces an increase in systemic resistance with a strong reflex suppression of cardiac output . in the absence of an intact sympathetic system , the vascular sensitivity to the vasoconstrictor effect of avp is enhanced by several orders of magnitude . accordingly , a combination of these factors could explain the increased rates of ischemic cardiomyopathy and/or heart failure in these patients . what are the implications of the allhat findings ? one implication is obviously that 1-adrenergic antagonists should not be first choice antihypertensives , and this message has already been widely disseminated . these agents are , however , used extensively for their urodynamic properties in patients with benign prostatic hypertrophy , many of whom are older hypertensives ( possibly with ischemic heart disease ) who prefer to use one drug as monotherapy for both purposes . indeed , doxazosin and terazosin are probably much more popular for the treatment of prostatic symptoms than for hypertension , and in this setting any cardiovascular adverse effects from their widespread use would most probably go unappreciated . ace = angiotensin - converting enzyme ; avp = arginine vasopressin . allhat = antihypertensive and lipid lowering treatment to prevent heart attack trial .

the lowering of high blood pressure is supposed to protect target organs from hypertensive damage . the antihypertensive and lipid lowering treatment to prevent heart attack trial was designed to compare the cardioprotective properties of three antihypertensives from different classes ( lisinopril , amlodipine and doxazosin ) with chlorthalidone . despite effective blood pressure lowering and a favorable metabolic profile , the doxazosin arm of the trial had a significantly higher relative risk of cardiovascular disease and heart failure compared with the chlorthalidone arm . this article speculates on possible causes for this unexpected result and suggests that the culprit may be accentuation of the vascular effects of vasopressin , which are maximized under -adrenergic blockade . these findings may have implications for the large number of older men who receive monotherapy with -blockers for treatment of prostatic symptoms .

estrogens play crucial roles in the development and maintenance of normal sexual and reproductive function . 17-estradiol ( e2 ) is the predominant and most potent sexual hormone during the reproductive stage in females . its main functions are associated with reproduction , although it is also involved with different pathologies , such as cancer , autoimmune diseases , and infectious processes , in which the innate immune response plays a key function . in bovines , during the period around parturition , cows experience an increased susceptibility to inflammatory disorders in the mammary gland and uterus , for example , mastitis . this increased susceptibility has been correlated with the decreased functionality of the components of the innate immune response and with abrupt changes in the levels of sex steroids . e2 levels rise abruptly in the last week before parturition , peak in the last 3 days before delivery , and fall rapidly after calving and regain basal values [ 3 , 4 ] . accordingly , lavon et al . reported that cows with subclinical mastitis ( without apparent signs ) exhibit low circulating e2 levels . in addition , the immunomodulatory effects of e2 in cows with mastitis are associated with a reduction of neutrophil migration . most of the genomic actions of estrogens , including e2 , are mediated by two estrogen receptors ( ers ) , er and er , and bovine mammary epithelial cells express both . however , there is little information concerning the immunomodulatory effects of e2 on epithelial cells from the bovine mammary gland . it is well known that e2 is required for mammary epithelial cell proliferation and ductal development in the growing animal . bovine mammary epithelial cells ( bmecs ) play a relevant role during intramammary infections because they are in intimate contact with the pathogens responsible for mastitis and are a target for intracellular bacteria causing chronic and subclinical infections , such as staphylococcus aureus [ 8 , 9 ] . this pathogen can persist for long periods of time in cows and is internalized by a zipper - type mechanism depending on the presence of fibronectin - binding proteins ( fnbps ) on the bacterial surface , as well as the interaction of fibronectin and the host - cell 51 integrin , in which integrin is taken up together with its ligands . in addition , bmecs participate in the innate immune response of the bovine mammary gland producing pro- and anti - inflammatory mediators , as well as antimicrobial peptides , nitric oxide , and so forth [ 11 , 12 ] , but the role of e2 on this response has not been explored . considering the immunomodulatory effects of e2 in different tissues and that one of its targets is the mammary epithelium , the aim of this work was to analyze whether this hormone modulates the innate immune response of bmecs and its implications during s. aureus internalization . 17-estradiol was acquired from sigma , and working solutions were dissolved in 1% ethanol ( 1500 pg / ml ) . for all of the experiments , 1% ethanol ( vehicle ) was used as a control . the live / dead baclight bacterial viability kit ( thermo scientific ) the monoclonal blocking antibodies anti-51 integrin ( mab2514 ) and anti - tlr2 ( tlr2.1 ) were obtained from millipore and abcam , respectively . the anti - phospho - er ( 2511s , ser118 ) was obtained from cell signaling and the anti - er ( 517700 ) was acquired from life technologies . the fitc - conjugated secondary antibodies against mouse and rat iggs were purchased from invitrogen and thermo scientific , respectively . this strain was isolated from a case of bovine clinical mastitis and has the capacity to invade bmecs . s. aureus were grown at 37c overnight in luria - bertani broth ( lb bioxon ) , and the cfus were adjusted by measuring the optical density at 600 nm ( od 0.2 = 9.2 10 cfu / ml ) . bmecs were isolated from the alveolar tissue of the udders of healthy lactating cows as previously described . the bmecs were cultured in growth medium ( gm ) that was composed of a dmem medium / nutrient mixture f12 ham ( dmem / f12k , sigma ) supplemented with 10% fetal calf serum ( equitech bio ) , 10 g / ml insulin ( sigma ) , 5 g / ml hydrocortisone ( sigma ) , 100 u / ml penicillin , 100 g / ml streptomycin , and 1 g / ml amphotericin b ( invitrogen ) . the cells were grown in a 5% co2 atmosphere at 37c . to perform the e2 and/or s. aureus challenge , polarized monolayers of bmecs ( dishes covered with 610 g / cm rat - tail type i collagen , sigma ) were cultured in serum - free dmem / f12k without phenol red ( sigma ) and antibiotics ( incomplete medium ) for 24 h , and then they were treated with the hormone and/or infected with the bacteria . for the invasion assays , bmec polarized monolayers were used ( ~10,000 cells were cultured onto 96-well flat - bottom dishes ( corning ) ) , which were incubated with different concentrations of e2 ( 1 , 5 , 10 , 50 , 150 , 300 , and 500 pg / ml ) in dmem / f12k ( sigma ) without antibiotics , serum , and phenol red ( incomplete medium ) for 24 h and then were infected with s. aureus ( moi 30 : 1 bacteria per cell ) . for this , the bmecs were inoculated with bacterial suspensions from a broth of 9.2 10 cfu / ml and incubated for 2 h in 5% co2 at 37c . then , the cells were washed three times with pbs ( ph 7.4 ) and incubated with incomplete medium that was supplemented with 80 g / ml gentamicin for 1 h at 37c to eliminate extracellular bacteria . finally , the bmec monolayers were detached with trypsin- ( 0.05% ) edta ( 0.02% ) ( sigma ) and lysed with 250 l of sterile distilled water . the bmec lysates were diluted 100-fold , plated on lb agar in triplicate , and incubated overnight at 37c . the number of bmecs cultured in each well plate was calculated for each invasion assay using an automated cellular counter ( bio - rad , tc20 ) . the data are presented as the ratio of the cfu recovered per bmec . for the invasion assays in the presence of the blocking antibodies , one hour previous to the addition of s. aureus to the bmecs , the blocking antibodies anti-51 integrin or anti - tlr2 were added separately ( 10 and 5 g / ml , resp . ) to triplicate wells . rat iggs ( purified from normal rat serum with protein a - sepharose beads ( sigma ) ) or mouse iggs ( purified from normal mouse serum and acquired from pierce ) were used as the negative controls . the invasion assays were performed using gentamicin protection assays as previously described . to determine the effect of e2 on bmec viability , 10,000 cells were incubated with different concentrations of the hormone in incomplete medium for 24 h at 37c in 96-well plates . then , 10 l of a 5 mg / ml of 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2h - tetrazolium bromide ( mtt , sigma ) solution in pbs was added to each well and incubated for 4 h at 37c . finally , 100 l of acid isopropanol ( 95% isopropanol and 5% 1 n hcl ) were added to dissolve the formazan crystals . the optical density was measured with a microplate reader ( bio - rad ) at 595 nm . the bmec viability was also tested using the trypan blue exclusion assay and the cells were counted in an automated cell counter ( bio - rad , tc20 ) . to analyze the effect of e2 on s. aureus growth , 9.2 10 cfu / ml were cultured at 37c in lb broth . the bacterial suspensions were treated with different concentrations of the hormone ( 1 , 5 , 10 , 50 , 150 , 300 , and 500 pg / ml ) and growth was monitored turbidimetrically ( 600 nm ) over 24 h ( measuring the absorbance at 2 , 4 , 6 , 8 , 12 , and 24 h ) . to evaluate the s. aureus viability in the presence of e2 , bacteria were grown ( as previously described ) in lb broth and were treated with e2 for 24 h at 37c overnight . later , the bacteria were plated on lb agar in triplicate and were incubated overnight at 37c . to do this , the bmecs were treated with e2 and infected with s. aureus as described above . after 2 h of infection , the media were recovered and the bacteria pellet was obtained by centrifugation at 10,000 rpm for 10 min at 4c and was washed three times with pbs . the s. aureus viability was measured using a live / dead baclight bacterial viability kit ( thermo scientific ) . the pellet was incubated with equal volumes ( 1.5 ml ) of component a ( syto 9 dye , live indicator ) and component b ( propidium iodide , dead indicator ) at room temperature in the dark for 15 min . after staining the bacteria , the fluorescent signals of 10,000 events were measured and evaluated using a bd accuri c6 cytometer . in addition , s. aureus growth was also analyzed using the e2-treated bmec conditioned media . to do this , the bacteria were grown at 37c overnight , and a s. aureus suspension containing 9.2 10 cfu / ml ( od 0.2 at 600 nm ) was incubated at 37c for 2 h with the conditioned media . then , the bacteria were plated on lb agar in triplicate and were incubated overnight at 37c . the bmec polarized monolayers were cultured in 24-well dishes ( corning ) and were treated with e2 ( 50 pg / ml ) for 24 h and/or s. aureus as described above . after the treatment , the cells were washed three times with pbs , detached with trypsin- ( 0.05% ) edta ( 0.02% ) ( sigma ) , centrifuged at 2,500 rpm for 10 min at 4c , and washed with pbs . the bmec pellet was blocked with normal goat serum ( 5% in pbs , pierce ) for 30 min at 4c with shaking , and then the cells were centrifuged and the pellet was incubated with the primary antibodies anti - tlr2 or anti - integrin separately . to analyze the presence of the ers ( er or er ) , nuclei from the bmecs were obtained using the proteojet kit ( fermentas ) , and the procedure continued as described above . to measure the tlr2 membrane abundance ( ma ) or the nuclear presence of the ers , the cells were incubated with the antibody at a dilution of 1 : 50 ( pbs containing bsa 0.1% ) for 1 h at 4c with shaking . for the determination of integrin ma , invasion assays were performed using different infection times : 30 , 60 , and 120 min in control bmecs or e2-treated ( 50 pg / ml , during 24 h ) bmecs . the cells were incubated with the anti-51 integrin at a concentration of 10 g / ml for 2 h at 4c with shaking . in all cases , after the primary antibody incubation , the bmecs were washed three times with pbs and incubated with the respective secondary antibody ( diluted 1 : 50 ) ( fitc - conjugated anti - mouse iggs for tlr2 and er and fitc - conjugated anti - rat iggs for er and 51 integrin ) for 1 h at 4c with shaking in the dark . the pellet was recovered by centrifugation , washed , and fixed with paraformaldehyde ( 4% ) for 10 min at 4c and finally washed three times with pbs . the fluorescent signals of 10,000 events were measured and evaluated using the bd accuri c6 cytometer . the polarized bmec monolayers were cultivated in 96-well flat - bottom plates that were coated ( corning - costar ) with 610 g / cm rat - tail type i collagen ( sigma ) . the bmecs were incubated with e2 ( 50 pg / ml for 24 h ) . prior to the invasion assay ( 30 min , 1 or 2 h ) , pharmacological inhibitors of p38 ( 5 m , sb203580 ) , jnk ( 20 m , sp600125 ) , or erk1/2 ( 2.5 m , u0126 ) were added separately to the bmecs . the cells lysates were plated on lb agar in triplicate and incubated overnight at 37c . the cfu numbers were determined with the standard colony counting technique or s. aureus viability was measured using the live / dead baclight bacterial viability kit as described above . 0.1% dmso ( vehicle ) was used as a control . to evaluate the map kinase activation levels by flow cytometry , the bmecs were treated with e2 ( 50 pg / ml ) , s. aureus , or both , and the samples ( 30 g of protein ) were prepared according to the manufacturer 's protocol for adherent cells ( becton dickinson , germany ) . pp38 ( t180/y182 ) , pjnk1/2 ( t183/185 ) , and perk1/2 ( t202/y204 ) were quantitatively determined using antibodies from a flex set cytometric bead array ( becton dickinson ) according to the manufacturer 's protocol . the flow cytometric analyses were performed using the bd accuri c6 and the cba analysis fcap software ( becton dickinson ) . the minimum detection levels for each phosphoprotein were 0.38 u / ml for pjnk and 0.64 u / ml for pp38 and perk . monolayers of bmecs were cultured in 6-well dishes ( ~5 10 ) and then were incubated with 50 pg / ml of e2 ( 24 h ) and/or s. aureus ( moi 30 : 1 ) for 2 h , as described above . the rna was extracted with trizol reagent ( invitrogen ) according to the manufacturer 's instructions . the rt - qpcr assay was performed using the comparative ct method ( ct ) with a stepone plus real - time pcr system ( applied biosystems ) according to the manufacturer 's instructions . the reactions were carried out with a sybr green pcr master mix ( applied biosystems ) . the specific primer pairs were obtained from invitrogen ; their sequences and pcr conditions used to amplify the different bovine mrnas are detailed in table 1 . for the measurement of the tnf- , il-1 , il-6 , and bovine -defensin 1 ( defb1 ) concentrations in the medium , the conditioned media from bmecs treated with e2 ( 50 pg / ml ) and/or s. aureus were collected . the concentrations of tnf- were measured using the duoset elisa development kit ( r&d systems ) to quantify the bovine tnf- levels according to the manufacturer 's instructions , and the concentrations of il-1 and il-6 were assessed using the bovine il-1 and il-6 screening kits , respectively ( thermo scientific ) . the measurement of bovine defb1 in bmecs culture medium was analyzed using the elisa kit for defb1 ( mybiosource ) . the nitric oxide ( no ) secreted by bmecs into culture medium was evaluated by measuring the nitrite concentration ( no ) in cell - free media using the griess reaction . after the invasion assays , the medium was filtered through 0.22 m membranes ( millipore ) to eliminate bacteria . the data were obtained from three independent experiments , each of which was performed in triplicate ( except for the elisa of defb1 , which was performed in duplicate ) and compared by analysis of variance ( anova ) . the results are reported as the means the standard errors ( se ) and the significance level was set at p < 0.05 , except for rt - qpcr analysis where standard deviations are shown and fold - change values greater than 2 or less than 0.5 were considered as significantly differentially expressed mrnas according to morey et al . . the results from internalization assays , gene expression analyses , and receptor membrane abundance experiments are shown normalized to the cells treated with vehicle ( 1% ethanol ) . to evaluate the effect of e2 on s. aureus growth and bmec viability , we incubated bacteria or bmecs in the presence of different concentrations of this hormone ( 1500 pg / ml ) . the results showed that e2 did not have effect on bmec viability after 24 h of culture ( figures 1(a ) and 1(b ) ) , which was determined by a trypan blue exclusion analysis and the mtt assays . in the same way , we showed that e2 did not affect the bacterial viability and growth ( figures 1(c ) and 1(d ) ) . to determine the effect of e2 on s. aureus internalization into the bmecs bmecs were treated with different concentrations of the hormone ( 1500 pg / ml ) 24 h before the challenge with the bacteria . according to the number of cfus recovered , a concentration of 50 pg / ml of e2 significantly inhibited s. aureus internalization into the bmecs by 50% ( figure 2 ) . the concentrations of 1 , 10 , and 300 pg / ml also reduced internalization by 20% . the other concentrations did not affect the internalization rate of s. aureus into the bmecs . considering these results , in the following experiments related to the regulation of the innate immune response of bmecs by e2 , we analyzed only the concentration of 50 pg / ml . because 51 integrin is one of the main extracellular receptors of epithelial cells employed during s. aureus internalization , we wondered whether the specific functional blocking of 51 integrin would modify the number of bacteria internalized into the bmecs . to test this , primary cultures of bmecs treated for 24 h with e2 were incubated with a specific antibody against 51 integrin ( figure 3(a ) ) . e2 treatment ( 50 pg / ml ) reduced the number of cfus internalized into the bmecs , but the blockade of 51 integrin slightly reduced this number ( ~20% ) . in addition , the bmecs treated with vehicle reduced ~60% of the s. aureus internalization when 51 integrin was blocked . this finding suggests that , during the internalization of s. aureus into the e2-treated bmecs , integrin 51 does not participate . to determine whether this effect is correlated with 51 integrin gene expression s. aureus induced the expression of the 5 subunit ( ~8-fold ) , and e2 upregulated the expression of both subunits . however , the effect of both treatments was similar to the effect of s. aureus ( figure 3(b ) ) . to analyze whether this effect is correlated with the 51 integrin ma in the bmecs the mean fluorescence intensity was used as an indicator of the level of integrin expression per cell in the population . e2 significantly reduces the level of 51 integrin ma ( ~40% ) ; however , after 2 h of challenge with s. aureus , the level of 51 integrin ma was reduced in a similar manner as that of the e2-treated cells . a similar effect was also observed when the e2-treated bmecs were infected , indicating that integrin is taken up together with bacteria . typical population profiles from the flow cytometry analysis are also shown in figure 3(c ) . we additionally evaluated the level of 51 integrin ma at different times of challenge with s. aureus in the e2-treated bmecs . the presence of 51 integrin at the membrane of the bmecs was reduced starting from 30 min of s. aureus challenge , showing a similar behavior both in the control and e2-treated cells . despite the data showing that e2 reduces integrin ma ( 24 h treatment ) , these results suggest that bacteria are able to induce integrin trafficking . thus , 51 integrin ma does not contribute to the reduction in internalization detected in the e2-treated bmecs ( figure 2 ) . to explore whether tlr2 participates in the reduction of s. aureus internalization into the bmecs induced by e2 , we investigated whether the blockade of this receptor with a specific antibody would modify bacterial endocytosis . as shown in figure 4(a ) , the blockade of tlr2 results in a significant reduction in the amount of s. aureus internalized ( ~50% ) ; however , a slight reduction was detected in the e2-treated cells ( ~15% ) . surprisingly , e2 induces tlr2 gene expression ( ~6-fold ) ( figure 4(b ) ) . as we previously reported , s. aureus induces tlr2 mrna expression ( ~3-fold ) ; however , the effect of both treatments was similar to the effect of s. aureus alone . the analysis of the mean fluorescence intensity ( figure 4(c ) ) shows that only s. aureus ( ~1.4-fold ) increased the tlr2 ma , but there is no difference in the tlr2 ma in the presence of e2 and bacteria compared with that obtained with infection alone . thus , tlr2 could not be involved in s. aureus internalization , and it is probably inactivated in e2-treated bmecs . next , we evaluated whether e2 ( 50 pg / ml ) induces the activation of mapks ( p38 , jnk , or erk1/2 ) , which are activated by tlr2 . initially , we investigated whether these kinases participate in s. aureus internalization into bmecs using pharmacological inhibitors prior to bacterial invasion . bmecs that were incubated ( for 30 min or 1 or 2 h ) with pharmacological inhibitors of p38 ( 5 m , sb203580 ) , jnk ( 20 m , sp600125 ) , or erk1/2 ( 2.5 m , u0126 ) showed a considerable reduction in s. aureus internalization ( ~5060% reduction ) , indicating that these kinases are involved in this process ( figure 5(a ) ) . s. aureus internalization was also analyzed by cfu counting and flow cytometry , which obtained similar results ( figure 5(b ) ) . interestingly , these kinases do not participate in the reduction of bacterial internalization induced by e2 because in the presence of inhibitors this reduction persists . furthermore , we evaluated p38 , jnk , and erk1/2 phosphorylation to relate their activation states with the e2-mediated reduction of s. aureus internalization into bmecs . when the bmecs were s. aureus - challenged ( 2 h ) , the basal activation of p38 was not modified , phosphorylated jnk1/2 was augmented , and erk1/2 activation was reduced ( figure 5(c ) ) , as we previously reported . the vehicle employed for the mapk dilutions was 0.1% dmso , which did not have an effect on internalization assays ( data not showed ) , as was previously reported . interestingly , the bmecs that were treated with e2 demonstrated reduced p38 phosphorylation ( ~5-fold ) , and the erk1/2 activation levels were also reduced by ~1-fold , but jnk1/2 remained unchanged in relation to the control cells . when the e2-treated cells were s. aureus - challenged , the p38 activation level was augmented ; however , the jnk1/2 and erk1/2 activation levels were essentially not changed . the mapk results indicated that e2 , prior to bacterial invasion , inhibits the bmec inflammatory response via p38 and erk1/2 inactivation . estrogens act through the estrogen receptors er and er , which belong to the nuclear receptor superfamily of transcription factors . to determine whether these receptors are activated in bmecs treated with e2 and/or infected with s. aureus , we performed a flow cytometry analysis . we used an anti - er antibody , which recognizes its phosphorylated isoform , as an indicator of its activation . to perform these analyses , we isolated bmec nuclei . in figure 6(a ) , we show that e2 slightly induces the activation of er ( ~20% ) , which was returned to basal levels during s. aureus infection . regarding the expression of er mrna , we observed that e2 also induces the expression of its gene ( ~5-fold ) , but after infection this induction is higher ( ~25-fold ) ( figure 6(b ) ) . with regard to er activation , we performed flow cytometry analysis on the bmec nuclei . in figure 6(c ) , we show that only the bmecs infected with s. aureus induce the activation of this receptor in both vehicle- or e2-treated cells in addition , the expression of er mrna was induced both in the e2-treated and the s. aureus - challenged bmecs ( ~3.5-fold ) ( figure 6(d ) ) . the infection of the bmecs treated with the hormone maintained the upregulated levels of er ( ~2.5-fold ) . according to these results , we hypothesized that 50 pg / ml e2 activates the bmecs via er. next , we explored whether e2 modified the inflammatory response of the bmecs before and during s. aureus infection . we focused on ( i ) proinflammatory cytokines , such as tnf- , il-1 , and il-6 ; ( ii ) an anti - inflammatory cytokine ( il-10 ) ; and ( iii ) a chemokine , il-8 ( figure 7 ) . regarding the proinflammatory cytokines , we performed rt - qpcr for mrna expression and elisas for the evaluation of cytokine secretion . the bmecs treated with e2 ( 50 pg / ml , 24 h ) significantly increased tnf- and il-1 mrna expression ( ~7- and 3-fold , resp . ) ( figure 7(a ) ) . s. aureus infection alone also induced the expression of both cytokines ( ~5-fold for tnf- and ~3-fold for il-1 ) . in the presence of s. aureus , interestingly , in the e2-treated cells the secretion of these proinflammatory cytokines was diminished in relation to bmecs treated with vehicle ( figure 7(c ) ) . furthermore , 50 pg / ml of e2 also reduced the secretion of tnf- and il-6 in the infected bmecs . only the secretion of il-1 was increased when the e2-treated cells were infected with s. aureus . in figure 7(b ) , we show that e2 also induces the expression of il-10 and il-8 mrnas ( ~2.5 and 4.5-fold , resp . ) . the infection induced the expression of il-10 but together with the hormone increased its expression ( ~8-fold ) . the expression of il-8 was not modified in the presence of infection both in the vehicle- and e2-treated cells . altogether , these results point to the anti - inflammatory effects of e2 on the bmecs infected with s. aureus . to explore if some antimicrobial mediators are being regulated by e2 , which could explain the reduction in s. aureus internalization , we measured the viability of s. aureus in the infection assays . in figure 8(a ) , we show the viability of s. aureus from the supernatants obtained from the invasion assays ( 2 h ) . s. aureus employed in the invasion assays from the e2-treated bmec cultures showed a reduced viability rate ( 60% of reduction ) compared to the bacteria recovered from the supernatants of the vehicle - treated bmecs . this result suggests that antimicrobial components are present in the culture media from the bmecs treated with 50 pg / ml of e2 . with the purpose of reinforcing these data , we incubated s. aureus for 2 h with 1 ml of conditioned media obtained from the vehicle- or e2-treated bmecs . according to figure 8(b ) , the conditioned medium from the e2-treated bmecs reduces the number of cfu of s. aureus ( a 60% of reduction ) . next , we explored whether the gene expression of some antimicrobial elements was induced in the e2-treated bmecs . figure 8(c ) shows the rt - qpcr analysis of different antimicrobial peptides , including several -defensins and 1 psoriasin ( s100a7 ) . ( defb1 , ~5-fold ) and bovine neutrophil beta defensin 5 ( bnbd5 , ~3-fold ) . in addition , e2 induces the expression of psoriasin s100a7 ( ~6.5-fold ) . in the presence of infection , a similar behavior was observed in the cells treated with e2 and infected with s. aureus , where the expression of psoriasin was upregulated by ~9-fold . considering these results , we measured the accumulation of defb1 in the bmec medium by elisa . in figure 8(d ) , we show that e2 does not induce the secretion of defb1 in the culture medium , which is stimulated by infection . s. aureus infection in the e2-treated cells reduces the concentration of defb1 secreted in relation to infection alone . thus , this is not the mechanism by which bacterial internalization is reduced in the e2-treated cells . in addition , the secretion of no was not modified in the presence of e2 in the bmecs ( figure 8(e ) ) , and thus it is not involved in the reduced internalization detected in the e2-treated bmecs . it is well known that , during the postpartum period , dairy cows experience an increased incidence of mammary infectious diseases , such as mastitis . this augmented frequency has been correlated with a decreased functionality of the innate immune system . during this period , e2 levels rise abruptly in the last week before parturition , peak in the last 3 days before delivery , and fall rapidly after calving and regain basal values [ 3 , 4 ] . it has been described that estrogens have both anti - inflammatory and proinflammatory functions , and their role in the modulation of the innate immune response of epithelial cells during infection has been documented . it has been shown that e2 is required for mammary epithelial cell proliferation and ductal development in the growing animal . the goal of this work was to study the immunomodulatory functions of e2 on bmecs during s. aureus infection , which have not been explored to date . according to previous reports showing immunomodulatory functions of e2 on bovine neutrophils , we evaluated e2 in a range of concentrations between 1 and 500 pg / ml . thus , it is important to analyze whether e2 alters s. aureus viability or growth . at the incubation times and the range of e2 concentrations analyzed we did not detect a bacteriostatic or a bactericidal effect of e2 on s. aureus ( figure 1 ) . in agreement , hosoda et al . showed that different steroid hormones were not bactericidal for s. aureus . in addition , at the range of concentrations tested , e2 was not toxic to the bovine mammary epithelial cells . have reported that similar concentrations of e2 and progesterone exert stimulatory effects on autophagy in bmecs after 48 h , which is implied during the involution of the bovine mammary gland in the dry period . in the present study , we can not discard the possibility that longer incubation times with e2 could be cytotoxic for these cells . in this work , we demonstrated that 50 pg / ml of e2 significantly inhibits ~50% s. aureus internalization into the bmecs ( figure 2 ) . because e2 does not have antibacterial activity , this reduction might be the result of the immunomodulatory effects that have been associated with this hormone . according to our results , the effect of e2 on s. aureus internalization shows a nonmonotonic dose - response curve . this behavior requires further investigation , however ; nonmonotonic effects of e2 have been described on the development of mammary gland in in vivo models , as well as in human breast cancer cells . a possible explanation could be related to the versatility of e2 actions , which include the classic genomic functions , as well as nongenomic or membrane - initiated estrogen - signaling pathway [ 24 , 25 ] . the inhibitory effect of e2 on s. aureus internalization can not be attributable to the reduction in integrin 51 ma because this receptor does not participate in the internalization of s. aureus in e2-treated bmecs . in addition , although e2 reduces 51 ma , the presence of bacteria promotes its traffic at early times of infection ( figure 3 ) , as we reported previously . considering this , the inhibition on s. aureus internalization in the e2-treated bmecs could be a consequence of the activation of immunomodulatory pathways . to explore this possibility accordingly , the results shown in figure 4 suggest that e2 does not induce the ma of this receptor despite the fact that it upregulates the expression of its gene . mammary epithelial cells play an essential role in the surveillance of mammary tissue during infections because they help in immune cell recruitment and bacterial recognition via the tlr signaling pathways [ 26 , 27 ] . we have previously demonstrated that s. aureus induces tlr2 ma and activation in bmecs in the same way that the lactogenic hormone prolactin stimulates s. aureus internalization in the bmecs . e2 shows differential effects on tlr2 expression and/or activation ; for example , in thp-1 cells , it increases the expression of tlr2 mrna , but , in a bovine oviduct epithelial cell culture , it reduces the lps - induced tlr2 mrna expression . thus , the activation of the tlr2 pathways by e2 depends on the tissue and the physiological condition of the organism . in agreement with the absence of tlr2 activation in e2-treated bmecs , we detected that the mapk p38 and erk1/2 are inactivated in these cells ( figure 5 ) . in addition , these kinases are not employed during the inhibition of s. aureus internalization induced by e2 ( figure 5 ) . we have previously reported that these three mapks are required during s. aureus internalization into the bmecs . we next explored whether e2 or infection regulates ers activation because the direct effects of this hormone are exerted via intracellular ers . in figure 6 , we showed that e2 induces the intracellular activation of er , as well as upregulating the expression of its gene . likewise , s. aureus infection increases er mrna expression in the e2-treated bmecs , but this induction is not related with er activation . in agreement , yart et al . reported that in a cell line of bmecs ( mac - t ) the expression of er protein increases in response to e2 treatment . however , the activation of er by infection has not been extensively explored to date . the presence of er in the nuclei from infected bmecs ( figure 6(c ) ) indicates the participation of this receptor during s. aureus infection , which has not been reported . nevertheless , flow cytometry analysis reveals that e2 does not activate er. taken together , these results suggest that e2 activates er in bmecs , which might be directly or indirectly implicated in the reduction of s. aureus internalization . because we did not detect the activation of mapks in the e2-treated bmecs ( figure 5(c ) ) it is probable that e2 triggers the direct activation of er in bmecs , which up- and/or downregulates the transcription of various genes by binding to the estrogen response element of the genes or by interacting with other transcription factors . to address this hypothesis , we analyzed some inflammatory genes in the e2-treated bmecs in the absence or presence of s. aureus infection . we previously reported that , with the exception of tnf- , s. aureus inhibits the innate immune response of bmecs , as it is unable to induce the gene expression of some proinflammatory cytokines , such as il-1 or il-6 . however , we also reported that , in the presence of ethanol as vehicle ( 2% ) , s. aureus slightly induces il-1 mrna expression . interestingly , e2 at 50 pg / ml significantly increases proinflammatory cytokine mrna expression such as tnf- and il-1 , prior to s. aureus infection . however , upon infection , the levels of both cytokine mrnas were downregulated in e2-treated bmecs . the differential influence of e2 on the expression of innate immune response genes depends on multiple factors , such as the hormone concentration or the physiological condition of the individuals . these results coincide with other reports using urinary epithelial cells , where e2 reduces the lps - induced cytokine expression . steroids have the ability to suppress ( and/or resolve ) an inflammatory response . in agreement with the anti - inflammatory function of e2 on bmecs , we detected a significant reduction in the secretion of proinflammatory cytokines ( figure 7(c ) ) in the e2-treated cells . this effect was maintained in the presence of s. aureus , with exception of il-1 , which was induced in the infected e2-treated cells . this effect can be the consequence of the activation of other mechanisms , such as inflammasome activation , which requires further research . interestingly , and in agreement with the anti - inflammatory actions of the hormone , e2 also induces the mrna expression of il-10 , an anti - inflammatory cytokine , which was upregulated by the hormone in the e2-treated cells challenged with s. aureus . in addition to the immunomodulatory role of e2 , it has been reported that this hormone also induces the production of antibacterial compounds in uterine epithelial cells ; a similar effect in the present model could explain the reduction in the internalization of s. aureus . our data suggest that e2-treated bmecs produce an antibacterial compound , which is secreted into the culture medium ( figures 8(a ) and 8(b ) ) . to determine the origin of this activity , we analyzed the mrna expression of different antimicrobial peptides ( -defensins and psoriasin ) . we also analyzed the secretion of defb1 into the culture medium , which was only induced by bacteria and not by e2 . it is necessary to analyze the concentrations of bnbd5 or psoriasin in the culture medium to resolve if they are implicated in the reduction of s. aureus internalization in the e2-treated bmecs . accordingly , fahey et al . determined that in uterine epithelial cells e2 exerts anti - inflammatory effects and enhances the production of antimicrobial peptides , which display direct defense actions during the infection of this tissue . it is possible that a similar effect could be occurring in bmecs , but further research is necessary to corroborate this possibility . in addition , the induction of psoriasin expression by e2 has been demonstrated in the human mammary epithelial cell line mcf-7 . according to our results altogether , these results indicate that e2 at 50 pg / ml reduces s. aureus internalization in the bmecs by modulating the innate immune response through the activation of er. because we did not detect er activation in s. aureus - challenged bmecs , we can hypothesize that 50 pg / ml of e2 during 24 h activates er in bmecs , which promotes a slightly anti - inflammatory response that is enhanced during infection . it is possible that during infection other mechanisms ( i.e. , transcription factors ) are participating because the activation of er is turned off . in this process e2 ( 50 pg / ml ) induces the anti - inflammatory response of the bmecs during s. aureus internalization through the participation of er. this leads to the increased production of antimicrobial molecules , favoring s. aureus elimination .

17-estradiol ( e2 ) , the predominant sexual hormone in females , is associated with the modulation of the innate immune response ( iir ) , and changes in its levels at parturition are related to intramammary infections , such as mastitis . in bovine mammary epithelial cells ( bmecs ) , e2 regulates differentiation and proliferation , but its immunomodulatory functions have not been explored . staphylococcus aureus is the predominant pathogen causing mastitis , which can persist intracellularly in bmecs . the aim of this work was to analyze whether e2 modulates the iir of bmecs during s. aureus internalization . bmecs treated with e2 ( 50 pg / ml , 24 h ) reduced bacteria internalization ( ~50% ) . the host receptors 51 and tlr2 do not participate in this reduction . however , e2 activates er and modulates the iir reducing the s. aureus induced - mrna expression of tnf- ( ~50% ) and il-1 ( 90% ) . e2 also decreased the secretion of these cytokines as well as il-6 production ; however , in infected bmecs , e2 induced the secretion of il-1. furthermore , e2 upregulates the expression of the antimicrobial peptides defb1 , bnbd5 , and psoriasin s100a7 ( ~5- , 3- , and 6-fold , resp . ) . in addition , e2 induced the production of antimicrobial compounds in bmec culture medium , which , together with the modulation of the iir , could be related to the reduction of s. aureus internalization .

renal transplantation is the best available renal replacement therapy for end - stage renal disease . kidney transplant recipients have a better quality of life and consume fewer health care resources compared with patients on dialysis ( 1 ) . the number of patients with end - stage renal disease is rising rapidly , while those who can undergo a kidney graft are limited because of the donor organ shortage . the organs supplied by living donors are superior to those from cadaveric sources ( 2 ) . improvements in the use of immunosuppression and advances in tissue typing have been associated with better patient and graft survivals in recent years ( 3 ) . despite studies that compared the outcome of related and unrelated living donation worldwide , an evaluation of the impact of live unrelated kidney donor ( lurd ) as a source for renal transplantation has not been adequately studied in egypt ( 4 ) . concerning this hypothesis , we decided to study the donor - recipient relationship and its impact on both graft and patient survival among egyptian patients . in egypt , there are no cadaveric kidney transplantations and the only source for renal transplantation is through living donation . so , we tried to encourage all types of living donation participants to answer the question whether unrelated donation is inferior to related donation . this study was comprised of 2,485 kidney transplant recipients who received their grafts between march 1976 and december 2013 at our center . out of this total , 2,075 received their grafts from living related donors ( related group a parent , a child , or a sibling of the recipient ) , while 410 received their grafts from live unrelated kidney donors ( unrelated group - spouses , friends , or altruistic individuals ) . we compared demographic characteristics , acute rejection episodes , chronic rejection , complication rates and long - term graft , and patient survivals among the groups . rejection was diagnosed on the basis of an increase in serum creatinine , confirmed by examination of a graft biopsy sample . all donors and recipients were evaluated by standard biochemical , serological , and radiological evaluation and they received immunosuppressive therapy . a total of 2,075 living related donor ( lrd ) and 410 living unrelated donor ( lurd ) transplants were performed during the period . our results showed high statistical significance regarding both donor and recipient age ( p < 0.001 ) ; the mean age of donors was higher in the related group ( lrd 36.2 10.5 years versus lurd 31.4 6.4 years ) , while the mean age of recipients was higher in the unrelated group ( lurd 34.8 11.1 years versus lrd 28.8 9.8 years ) . the percentage of male donors was significantly higher in the unrelated group ( p < 0.001 ) . hematological workup showed that blood grouping had a high statistical significance ( p = 0.002 ) , however , no significant difference regarding blood transfusion could be observed between both groups ( p = 0.71 ) . the percentage of couples with one dr matched locus was higher in the unrelated group ( lurd 94.9% versus lrd 85.5% ) , while the percentage of couples with two dr matched loci was higher in the related group ( lrd 14.5% versus lurd 5.1% ) . the percentages of couples with zero , one , and two hla matching were higher in the related group ( 8.8% , 12.8% , 64.5% ) , respectively , while the percentages of couples with three and four hla matching were higher in the unrelated group ( 39.8% and 21.9% ) ( table 1 ) . ( % ) or mean sd . the most common causes of end - stage kidney disease ( eskd ) in the lurd group were glomerulonephritis and polycystic kidney disease ( n = 66 , 16.1% and n = 43 , 10.5% ) , while in the lrd were due to unknown causes and obstructive uropathy ( n = 1,406 , 67.7% and n = 98 , 4.7% ) ( table 2 ) . to induction and maintenance immunosuppressive protocols , atg induction had the highest percentage in the unrelated group ( lurd 16.4% versus lrd 6.9% ) ( table 3 ) . the percentages of recipients maintained on steroid - azathioprine or mycophenolate mofteil ( mmf ) and tacrolimus ( tac)-mmf were significantly higher in the related group ( lrd 13.5% versus lurd 7.1% , and lrd 17.1% versus lurd 11.4% ) , respectively , while the percentages of recipients maintained on steroid - cyclosporine azathioprine or mmf were significantly higher in the unrelated group ( lurd 61.2% versus lrd 52.2% ) with comparable percentages in both groups regarding other protocols ( table 4 ) . there were no significant differences between both groups regarding hypertension , diabetes mellitus , hepatic problems , infections , or malignancy ( table 5 ) . the rate of acute vascular rejection was significantly higher in the unrelated group ( lurd n = 26 , 6.3% versus lrd n = 71 , 3.41% ) , while the rate of cases without acute rejection was significantly higher in the related group ( lrd n = 960 , 46.3% versus lurd there was no statistical significance between both groups in regards to creatinine clearance and serum creatinine for one , three , and five years post transplantation ( p = 0.684 , 0.579 , 0.201 , and 0.107 ) , respectively ( table 7 ) . the graft and patient survival of each group is shown in tables 8 and 9 . kaplan - meier graft and patient survival curves for each group are shown in figures 1 and 2 . there were no significant differences regarding graft and patient survival between both groups ( p = 0.071 and p = 0.386 , respectively ) . kidney donation by biologically unrelated persons has been attempted in different areas of the world , including the middle and far east ( 5 ) . these donations have received adverse publicity because of multiple factors , including the following : unresolved ethical issues like donor payment and possible coercion , unacceptably high donor and recipient morbidity and mortality , and poor allograft survival rates ( 6 ) . with these points in mind , our center allows transplantation from living unrelated donors under certain circumstances , like hereditary nephritis , polycystic renal diseases and in the case of re - transplantation . renal transplantation from living unrelated donors is successful , but has been met with some opposition due to poor tissue antigen compatibility and fear of commercialization . in the present study , living unrelated recipients tend to be more elderly with younger donors , and a high percentage of male donors ; however , in the living related group there are a high percentage of female donors , which was observed in live - donor programs in most countries , including the united states and australia ( 7 , 8) . in australia , female donors accounted for 53% and 62% of overall lrd and lurd donors , respectively ; the latter likely reflects the growth in spousal donation ( 9 ) . the reason for the greater proportion of female donors remains unclear , although some contributing factors could be medical ( higher rates of cardiovascular disease in men ) or psychosocial ( financial issues and differing perception towards donation between genders ) ( 10 , 11 ) . our immunological work agrees with fuller tf et al . and humar a et al . ( 12 , 13 ) , since the number of live related transplants ( lrt ) with 3 & 4 hla & 2dr matching are significantly higher than in the live unrelated transplants ( lurt ) . we started our transplantation program in the mansoura urology and nephrology center ( unc ) moving from one immunosuppressive protocol to another by starting with steroid and azathioprine and moving to the use of mmf , tac , and sirolimus . our study revealed no significant differences between lrt and lurt regarding immunosuppressive protocols , apart from the protocols steroid - azathioprine or mmf and tac - mmf where a higher percentage of lrd group ( p < 0.001 ) and ( p = 0.004 ) respectively and this correlated with better hla matching that encouraged less immunosuppressive drugs , like a steroid - free regimen ( tac and mmf protocol ) , while the protocols steroid - cyclosporine azathioprine or mmf were significantly higher in the unrelated group ( p < 0.001 ) . for induction immunosuppression , we considered the poorer hla matching in the unrelated group and used anti - thymocyte globulin ( atg ) and this correlated with the kdigo guidelines that recommend the use of atg , which is a potent immunosuppressive agent , rather than interleukin-2 receptor antibodies principally for groups at high - risk for allograft rejection ( 14 ) . although the incidence of early graft loss because of acute rejection has decreased steadily over the past decades , acute rejection is considered a major risk factor for chronic rejection and a strong predictor of long - term graft survival in both cadaveric and living donor kidney transplants ( 15 , 16 ) . in the present study , the percentages of patients with acute vascular rejection were significantly higher in the unrelated group ( p = 0.005 ) . ( 13 ) , who reported that the incidence of acute rejection was not higher for lurd recipients after comparing 595 lrds with 116 lurds ; these mismatching results could be explained by the difference in immunosuppression protocols or the difference in hla matching . surprisingly in our study , there was no difference in the biopsy proved chronic rejection between both groups ( p = 0.07 ) , despite the higher incidence of acute vascular rejection in lurd . we found a higher incidence of early rejection in lurd compared to lrd and this agrees with fuller et al ( 12 , 16 ) , who reported higher percentages of early and severe rejections in lurt than lrt . there are some important factors that might impede the use of lurd sources , such as the elderly age of donors and the higher number of hla mismatches compared with lrd ( 12 ) . our study is not in agreement with previous studies , since the lurd ages were significantly younger than lrd ages ; this may be due to most lurd recipients in that study being friends and spouses , which is not the same as in our study . we reported no significant differences in regards to creatinine clearance and serum creatinine for one , three , and five years post transplantation between the two groups . the one- , five- , and ten - year graft survival rates were 97% , 86.6% , and 67.9% , respectively , for recipients of lrd , while that for recipients of lurd were 95.4% , 83.6% , and 66.7% , respectively ( figure 1 ) ( tables 8 and 9 ) . the one- , five- , and ten - year patient survival rates were 97.1% , 95.1% , and 80.8% , respectively , for recipients of lrd , while that for recipients of lurd were 95% , 88.8% , and 67% , respectively ( figure 2 ) ( tables 8 and 9 ) . for example , in the 2008 annual report of the scientific registry of transplant recipients , the unadjusted five - year survival of lurd kidneys was the same as that of living related donor kidneys ( approximately 80 % ) ( 17 ) . in italy , graft survival rates of 172 lurt recipients were 87% in one year , 79% in five years , and 69% in nine years ( 18 ) . 19 ) , reported that patient survival in lurd recipients was worse than in lrd recipients ; however , this study included a high percentage of diabetic patients . second , there were many changes in immunosuppressive protocols over the last few decades , but we should consider that the study was comprised of live matched donors and the majority were related donors with insignificant immunological risks in the unrelated group . graft survival is affected by factors like age of the donor , degree of hla compatibility , original kidney disease , number and severity of acute rejection episodes , despite that kidney transplant recipients who received their grafts either from live related donors or live unrelated donors had a comparable patient and graft survival . kidney donation by volunteers who are genetically unrelated to their recipients is medically successful , socially valuable , and ethically acceptable provided that donors are healthy , competent , and well - informed . second , there were many changes in immunosuppressive protocols over the last few decades , but we should consider that the study was comprised of live matched donors and the majority were related donors with insignificant immunological risks in the unrelated group . graft survival is affected by factors like age of the donor , degree of hla compatibility , original kidney disease , number and severity of acute rejection episodes , despite that kidney transplant recipients who received their grafts either from live related donors or live unrelated donors had a comparable patient and graft survival . kidney donation by volunteers who are genetically unrelated to their recipients is medically successful , socially valuable , and ethically acceptable provided that donors are healthy , competent , and well - informed .

backgroundrenal transplantation is the ideal method for management of end - stage renal disease . the use of living donors for renal transplantation was critical for early development in the field and preceded the use of cadaveric donors . most donors are related genetically to the recipients , like a parent , a child , or a sibling of the recipient , but there are an increasing percentage of cases where donors are genetically unrelated like spouses , friends , or altruistic individuals . donor shortages constitute the major barrier for kidney transplantation , and much effort has been made to increase the supply of living donors . the impact of donor source on the outcome of renal transplantation is not adequately studied in our country.objectivesthe aim of the study was to evaluate the impact of donor source on the outcome of live donor kidney transplantation.patients and methodsfrom march 1976 to december 2013 , the number of patients that underwent living renal transplantation sharing at least one hla haplotype with their donors was 2,485 . we divided these patients into two groups : ( 1 ) 2,075 kidney transplant recipients ( 1,554 or 74.9% male and 521 or 25.1% female ) for whom the donors were living related , ( 2 ) 410 kidney transplant recipients ( 297 or 72.4% male and 113 or 27.6% female ) for whom the donors were living unrelated . all patients received immunosuppressive therapy , consisting of a calcineurin inhibitor , mycophenolate mofetil , or azathioprine and prednisolone . we compared acute rejection and complication rates , as well as long - term graft and patient survival of both groups . demographic characteristics were compared using the chi - square test . graft survival and patient survival were calculated using the kaplan - meier method.resultsthe percentages of patients with acute vascular rejection were significantly higher in the unrelated group , while percentages of patients with no rejection were significantly higher in the related group , but there were no significant differences regarding patient and graft survivals between both groups.conclusionskidney transplant recipients who received their grafts either from live related donors or live unrelated donors had comparable patient and graft survival outcomes .

a patient with massive exposure to tetrachloroethylene fumes presented with coma and severe pulmonary edema . sequential blood gases , chest x - rays , and clinical findings showed dramatic improvement with conventional but aggressive management and the patient recovered completely . there was no evidence of permanent renal , hepatic , or central nervous system damage.imagesfigure 1.figure 2.figure 3 .

it covers a vast array of devices that are derived from the areas of engineering , biology , physics and chemistry . these devices include vectors for the targeted delivery of anticancer drugs , imaging contrast agents and arrays for the early detection of tumors ( 1 ) . these , and other nano - devices , may support the future diagnosis and treatment of common conditions such as cancer ( 24 ) . nanotechnology was first proposed in 1867 by james clarke maxwell , a scottish physicist who developed electromagnetic theory and statistical physics . there is plenty of room at the bottom , given at caltech in 1959 , he spoke of building factories that maneuvered things atom by atom . later in 1974 , this concept was defined by professor norio taniguchi of tokyo university of science as the separation , consolidation , and deformation of materials by one atom or one molecule . k. eric drexler , who suggested that it would be possible to build computers and robots far smaller than a single cell ( 5 , 6 ) . nanotechnology typically refers to structures up to 200 nanometers in size . as a system s size diminishes to nano - scale quantum realm. in this realm , particle behavior and processes are governed by quantum and statistical mechanics and atoms are manipulated by covalent , electromagnetic and van der walls forces . structures made up of a monolayer ( a sheet that is one atom thick ) , have properties that differ markedly from conventional experience . opaque and stable metals , such as gold , are transformed into monolayers that are transparent , inflammable catalysts . nanotechnology is being used not only in the development of such catalysts , but additionally in semi - conductor technologies , such as memory storage , construction and household products such as sunglasses ( 7 ) . relating to cancer , nanotechnology is being applied in the following two broad areas : the development of nanovectors - nanoparticles which can be loaded with drugs or imaging agents and then targeted to tumor cells.nanosensors - devices for the detection of cancer cells ( 8 , 9 ) . the development of nanovectors - nanoparticles which can be loaded with drugs or imaging agents and then targeted to tumor cells . nanovectors may eventually become a powerful tool for the delivery of anticancer drugs . usually have a tripartite constitution ; a core constituent material , a therapeutic and / or imaging payload , and biological surface modifiers which enhance the bio - distribution and tumor targeting of the nanoparticle ( 10 ) . through the process of targeted biorecognition , nanovectors have the potential to deliver large amounts of a therapeutic or imaging agent to a tumor . for example , a nanovector may selectively bind to the neovascular endothelium of a tumor and release a penetration enhancer . this may then deploy a track of molecular motor molecules such as actin ( 11 ) . the nanovector , or another , can then release a therapeutic agent , bound to a conjugate molecule , such as myosin . this agent could then travel along the molecular track , reaching deep into the cancer lesion and overcoming opposing oncotic and osmotic pressures . in addition to the delivery of therapeutic agents , nanovector s formulations can be designed to reduce the clearance time of small peptide drugs by providing protection of active agents from enzymatic or environmental degradation and avoiding obstacles to the targeting of the active moiety ( 12 ) . this includes gadolinium or iron oxide - based nanoparticles and multiple - mode imaging contrast nano - agents , which combine magnetic resonance with biological targeting and optical detection . low - density lipid nanoparticles also have been used to enhance ultrasound imaging ( 13 ) . many polymer - based nanovectors have been investigated and some appear more promising for clinical translation . for different imaging modalities , it is possible to develop nanoparticles , which can provide signal enhancement combined with biomolecular targeting capabilities ( 14 ) . these nanovectors have typically been developed as components of a multi - functional unit with generic core components , a nanoplatform . this is coupled to a generic image enhancing agent , a generic therapeutic agent ( such as a cell toxin ) and a generic reporter agent which enables sensors to detect the successful delivery of the therapeutic agent to its target ( 1517 ) . a vector has been engineered at the atomic level to deliver a magnetic core and an optical probe to cells with receptors for the luteinizing hormone ( lh - rh ) ( 18 ) . they consist of three major architectural components : core , branches , and end groups . one of the most appealing aspects of technologies based on dendrimers is that it is relatively easy to precisely control their size , composition , and chemical reactivity . dendrimers can serve as versatile nanoscale platforms for creating multifunctional devices capable of detecting cancer and drug delivery ( 19 , 20 ) . nanoparticles can be designed to extravasate into the tumour stroma through the fenestrations of the angiogenic vasculature and demonstrate targeting by enhanced permeation and retention . the particles carry multiple antibodies , which direct antitumor action by targeting epitopes on cancer cells . when irradiated by external energy , nanoparticles become activated and release their cytotoxic action . through this , they could provide anti - angiogenic therapy by preferentially adhering to cancer neovasculature and causing it to collapse ( 21 , 22 ) . cantilevers are constructed as parts of a larger diagnostic device and can provide rapid and selective detection of cancer - related molecules . cancer specific molecules bind to molecules that act as sensors . this movement is exaggerated as the sensor molecule acts like a single lever cantilever bridge ( 2325 ) . dna - coated gold nanoparticles ( nps ) form the basis of a system that also uses larger magnetic microparticles ( mmps ) to detect femtomolar ( 10 molar ) concentrations of serum proteins . in this case , a monoclonal antibody to prostate specific antigen ( psa ) is attached to the mmp , creating a reagent to capture free psa . a second antibody to psa is attached to the nps and two particles create a sandwich of the captured protein which can be easily separated using a magnetic field . this technique has the potential to quantify an antigen at concentrations that would not be possible using a conventional enzyme linked imunosorbant assay ( elisa ) ( 2628 ) . in an ideal scenario , the onset of the transformational processes leading towards malignancy would be detected early . this could exist as routine screening by non - invasive means such as proteomic pattern analysis from blood samples , or the in vivo imaging of molecular profiles and evolving lesion contours . the biology of the host and the disease would be accurately determined , and dictate choices for targeting and barrier - avoiding strategies for an intervention plan . transforming cellular populations would be eradicated or at least contained , without collateral effects on healthy tissues , in a routine that could be repeated many times . if fully integrated with the established cancer research enterprise , nanotechnology might help this vision become reality . some of the principal challenges along this path are as follows : developing approaches for the in vivo detection and monitoring of cancer markersimprovement of the targeting efficacy of therapeutic or imaging agents to cancer lesions and their microenvironmentrefining technology platforms for early detection of cancer biomarkers ex vivoengineering nanoparticles to avoid biological and biophysical barriers ( 2931 ) . developing approaches for the in vivo detection and monitoring of cancer markers improvement of the targeting efficacy of therapeutic or imaging agents to cancer lesions and their microenvironment refining technology platforms for early detection of cancer biomarkers ex vivo engineering nanoparticles to avoid biological and biophysical barriers ( 2931 ) . nanotechnology is expected to play an important role in the early detection of transforming cell populations by in vivo imaging or ex vivo analysis . this new and highly sophisticated technology allows the appropriate combination of agents , based on accurate tumour specific biological information , targeting them to the cancer cells , whilst avoiding biological barriers . however , their approval perspectives , biodistribution , reliability of production protocols , as well as safety for patients and the health - care workers should not be ignored .

nanotechnology is the engineering of functional systems at the molecular scale which may exert a revolutionary impact on cancer diagnosis and therapy . nanotechnology is being applied to cancer in two broad areas : i ) the development of nanovectors such as nanoparticles which can be loaded with drugs or imaging agents and then targeted to tumours , and ii ) high - throughput nanosensor devices for detecting the biological signatures of cancer . combined , such technologies could lead to earlier diagnosis and better treatment for cancer patients .

using a hierarchical framework , tissue engineering can be subdivided into different strategies or concepts ( fig . 1 ) . one strategy is purely cell - based involving the transplantation of autologous or allogeneic cell suspensions or cell - sheets that are injected and/or transplanted to a defect site or an injured tissue . another strategy utilizes biomolecules ( growth factors , completely lyophilized cell fractions , peptides , polysaccharides , etc . ) aimed at delivering cues to the cells of the host tissue . other methods are based on the use of different types of matrices ( hydrogels , microspheres / beads , etc . ) in combination with cells and/or biomolecules . the fourth and most frequently applied strategy focuses on seeding and culturing specific cell types in 3-d environments that closely mimic natural extracellular matrix . such 3-d environments are specifically configured as cellular solids and referred to as ' scaffolds ' ( fig . a paradigm shift is taking place in orthopaedic and reconstructive surgery from using medical devices and tissue grafts to engineering a tissue engineered construct ( c ) that uses biodegradable scaffolds ( a ) combined with cells ( b ) or biological molecules ( b # ) to repair and/or regenerate tissues . the tissue engineering laboratory at nus did design , fabricate and characterize composite scaffolds made of mpcl- cap by fdm in vitro ( ho st , hutmacher dw . ( zhou yf , chou am , li zm , hutmacher dw , sae - lim v , lim tm . combined marrow stromal cell sheet techniques and high strength biodegradable composite scaffolds for engineered functional bone grafts . biomaterials . 2007 ; 28:81424 ) polymer / cap composites confer favourable mechanical and biochemical properties , including strength viathe ceramic phase , toughness and plasticity viathe polymer phase , more favourable degradation and resorption kinetics , and graded mechanical stiffness . in these scaffolds , ceramic phase was homogenously distributed in the matrix ( arrows , inset left corner ) as well as exposed on the surface ( figure a , arrows highlight cap particles on surface ) . contact angle measurements showed that such composite surfaces were more hydrophilic and that the degradation kinetics were accelerated 3 to 4 times compared to pcl alone . it can be argued that the ' scaffold - based tissue engineering concept ' was introduced in the mid- 1980s when dr joseph vacanti of the children 's hospital approached dr robert langer of mit with an idea to design scaffolds for cell delivery as opposed to seeding cells onto or mixing cells into naturally occurring matrices with physical and chemical properties that are difficult to manipulate . today 's concepts of scaffold- and matrix - based tissue engineering involve the combination of a scaffold with cells and/or biomolecules that promote the repair and/or regeneration of tissues . these tissue- engineered constructs ( tec ) are under intense investigation and various approaches and strategies are continually being developed . however , despite intense efforts , the ideal scaffold / cell or scaffold / neotissue construct ( even for a specific tissue type ) has yet to be developed . certain minimum requirements are essential when developing tecs that must address the biochemical as well as chemical and physical properties of native tissue . of these requirements , biocompatibility , vascularization [ 3 , 4 ] and chemotaxis are vital . the scaffold must also be nonimmunogenic and free from prions involved in disease transmission and it must also possess suitable architectural qualities that are easily reproducible . lastly , one must consider the temporal and spatial variations in some of these factors both in vitro and/or in vivo . despite rapid advancements in the fabrication of tecs , tissue engineering is still a long way off matching natures ' ability to grow and repair tissues and organs . hence , tissue engineering still has room for improvement , nevertheless , from an engineering perspective the past decade has seen significant advances in scaffold design and fabrication . starting with simple foams and fibers , porosity and pore interconnectivity has quickly become a central theme , with pore dimensions and material properties being vital in promoting cell seeding , migration , proliferation and the de novo production of extra - cellular matrix . significant advances have also been made in the incorporation of bioactive molecules into scaffolds , specifically in the area of bone engineering ( figs 3 and 4 ) . constructs with bmsc sheet - scaffolds ( mpcl - tcp ) were implanted into nude rat , harvested after 28 , 56 and 84 days . ( a ) gross appearance of bmsc sheet - scaffolds constructs with osteogenic induction ( right ) and non - induction ( control ) ( left ) after 4 weeks . ( b ) x - ray detected bone like tissue formation in 28 day ( c ) 56 day ( d ) 84 day implants . ( e ) x - ray image of constructs without cell seeding ( control ) . ( f ) micro - ct demonstrated the overall highly mineralized tissue similar to cortical ( golden ) and cancellous ( red ) bone in implanted constructs after 28 day . mineralized tissue with similar density with cancellous bone was detected ( darker areas ) while the lighter colour represented cortical bone . ( h ) fluorescence was detected on the formed bone tissue after 28 days ; the fluorescence came from the cfda labeled bmsc . ( i ) micro - ct quantification of implant tissue compositions depicted substantial bone formation in the induction group , accounting for 40% total volume for the 28 , 56 and 84 day implantation , while the control group formed only connective tissue . ( j ) h&e staining shows lamellar bone - like tissue formed in both outer part and interior of constructs after 28 days . ( k ) high magnification image shows well organized lamellar bone like tissue ( bo ) with distinct osteocytes located within bone tissue . ( l ) the typical osteoblasts ( ob , black arrow ) located on the surface of neo mineralized tissue with marrow cavities and blood vessels in 56 days implants . ( m ) safranin - o staining demonstrated that hypertrophic chondrocytes ( white arrow ) were observed in 56 day implants in very low numbers . high magnification shows the chondrocytes ( white arrow ) with weak safranin - o staining surrounded by the osteocyte ( black arrow ) . ( n ) ocn staining . strong signals ( arrow head ) were detected on neo mineralized tissue while very weak to no - signals were detected on chondrocyte like cells ( o ) . limited signals ( arrow head ) were detected on chondrocyte like cells while no staining for the mineralized tissues . bv : blood vessel + red blood cells ; ma : marrow ; ob : osteoblast ; bo : bone ; oc : osteocyte ; cy : chondrocyte . defects of load - bearing long bones , for instance , require constructs with high mechanical stability whereas initial plasticity is not essential . on the other hand for craniofacial applications , e.g. orbital floor fractures , moldable scaffolds ( a d ) are favorable . depending on the implantation site , initial vascularization is essential for enhanced engraftment and prevention of infections . mechanical stability , osteoconductivity by attracting and stimulating bone - forming cells of the host bone , and ease of handling have been well balanced in the design of mpcl scaffold sheets in order to properly meet the clinician 's needs . the clinical follow up 2.5 years postsurgery ( lower ct image ) of a patient receiving a mpcl scaffold ( defect site shown in upper ct image ) for the reconstruction of a orbital floor fracture defect showed complete bone regeneration of the defect site ( arrow ) . native bone tissue is a major storage site for growth factors , especially for those characterized by their high affinity to heparin ( heparin - binding growth factors [ hbgfs ] ) , such as fibroblast growth factors ( fgfs ) , transforming growth factor-s ( tgf-s ) , and bone morphogenic proteins ( bmps ) . bmps are biologically active molecules capable of inducing new bone formation and have shown significant potential for clinical use in the repair of bone defects [ 7 , 8 ] . although there have been several successful studies using bmps , there remain many unanswered questions such as patient site - specificity , for example , the ideal dose of bmp in an anterior cervical spine fusion may be quite different to that of a cranial defect . carriers for bmp play an equally important role when it comes to the optimal delivery leading to predictable and functional bone regeneration . for instance , a non - compressible scaffold has been shown to promote better fusion in the posterolateral spine than the clinically used collagen sponge . in addition to the delivery of growth factors scaffold design is focusing on supporting the adhesion , growth and function of anchorage - dependent cell types by mimicking the interaction between cell surface receptors and extracellular matrix ( ecm ) molecules . this interaction is vital in regulating cellular functions including adhesion , survival , proliferation , migration and differentiation . the major focus in the literature has been on the integrin family of receptors that interact with a wide variety of ecm proteins . more recently , heparan sulphate proteoglycans ( hspgs ) have been identified as having a role in cell adhesion . these interactions occur mainly through electrostatic interactions between the negatively charged hspg and the cluster of positively charged amino acids within the binding domains of ecm proteins . hspgs are abundant cell surface and ecm molecules that consist of a defining core protein ( such as syndecan , glypican or perlecan ) to which are attached highly sulphated glycosaminoglycan ( gag ) side - chains of heparan sulphate ( hs ) ( fig . 5 ) . a ) heparan sulfate proteoglycans can be broadly classified as glypicans , perlecans and syndecans , depending on their distribution within and around the cell . glypicans are attached to the cell membrane by gpi anchors , whereas syndecans are transmembrane proteins . perlecans are actively secreted into the pericellular matrix and are predominantly found in the basement membrane . ( b ) heparan sulfate is composed of repeating disaccharide units of glucuronic acid and glucosamine of varying length that can be modified at irregular intervals along the chain . these modifications cluster the sulfation patterns on the hs chain into discrete protein - binding domains and ultimately lead to different growth factor - binding specificities of different hs species . copyright permission granted from nature . the importance of hs in mediating cell responses has also been shown with heparinase / heparitinase and sodium chlorate treatments . heparinase cleaves hs chains into inactive disaccharide and tetrasaccharide components that have been shown to inhibit fgf2-mediated smooth muscle cell proliferation in injured carotid arteries . several in vitro studies have also cultured cells in media supplemented with chlorate to study the role of sulphated gags in different cell types . sodium chlorate ( naclo3 ) imparts its effects by inhibiting atp - sulphurylase , the first enzyme in the synthesis of 3-phosphoadenyl 5-phosphosulphate ( paps ) , a high - energy sulphate donor in biological reactions . the mitogenic response of cells to fgf2 is inhibited when cells are cultured in the presence of 30 mm naclo3 . such data has helped establish the idea that hs does not only bind a ligand for delivery to its cognate receptor , but must also itself bind to the receptor , in order to initiate signal transduction ; a powerful molecule indeed . numerous growth and adhesive proteins contain heparin - binding domains that specifically interact with hs . in all cases , the binding to hs modifies their biochemical properties and biological activity . thus understanding the specific nature of the cell surface receptor - ecm interactions may provide a foundation for developing functional biomaterials designed to promote cell adhesion and growth . indeed , the attachment of adhesive - peptide ligands mimicking heparin - binding domains to biomaterial surfaces has been shown to promote cell attachment . based on the above background , the authors of this review began a collaboration in 2004 with the aim of developing a bone engineering platform technology that combines novel composite scaffolds that mimic the host tissue matrix with growth factor potentiating hs sugar isoforms ( fig . the ability of hs to regulate a suite of endogenous factors involved in the repair process of bone makes it an attractive therapeutic agent compared to the application of a single growth factor , e.g. bmp . we studied the bone regeneration potential of mpcl / tcp - col1 scaffolds doped with 5 ( left ) and 30 ( right ) microgram hs in critical sized rat cranial defects . ( a ) mpcl - tcp scaffold ( 5 mm diameter and 1 mm thick , 0 - 90o lay - down pattern , 70% porosity ) before treatment , left , and after lyophilization of 350 mg of rat tail collagen type 1 , right . ( b ) light microscopy showing the scaffold , left , and immunofluorescence showing the distribution of the hs within the lyophilised collagen . ( c ) two full - thickness critical bone defects ( 5 mm in diameter ) were created in the rat parietal bone . implantation of mpcl / tcp - col1 scaffolds doped with 5 and 30 microgram hs into the defects . ( a ) macroscopically observation of the defects sites shows excellent integration of the constructs with the host tissue ( arrows ) . a smooth and non- fibrous integration could be observed for all groups at all time points . ct scanning of the skull defect showing the progressive bone formation at 1 month ( b ) and 2 month ( c ) months with 5 microgram ( hsl ) and 30 microgram hs ( hsh ) . ( d ) antibody staining with a bone specific marker , namely osteocalcin ( arrows show interface between host bone and newly formed bone ) shows a stronger signal from the hsh group when compared to hsl group . as discussed above , a conceptual shift is taking place in orthopaedic and reconstructive surgery from using synthetic implants and tissue grafts to a tissue engineering approach that uses biodegradable scaffolds integrated with biological cells or molecules to regenerate tissues . this new paradigm requires scaffolds that balance temporary mechanical function with morphological properties ( pore architecture , size and interconnectivity ) to aid biological delivery and tissue regeneration . complex scaffold architecture designs generated using hierarchical image - based or cad techniques usually can not readily be built using conventional techniques . instead , such scaffold architectures must be built using layer - by - layer manufacturing processes known collectively as solid free - form fabrication ( sff ) . a number of review articles [ 2 , 6 ] and book chapters have reviewed and compared sff scaffold fabrication methods so this review will concentrate on how scaffolds have per- formed clinically in bone engineering , and future directions for their use in regenerative medicine . melt extrusion - based sff systems provide a powerful instrument for the generation of scaffold platforms . recent advances in both computational scaffold design and sff have made it possible for tissue engineers to design and fabricate a whole range of new types of scaffolds . one of the major benefits is the flexibility to create scaffolds with highly reproducible architecture and compositional morphological variation across the entire matrix , due to the computer controlled fabrication process ( fig . 7 ) . the applications of sff technologies in scaffold fabrication are wide and varied , however , only a small number are have reached the clinical arena . an interdisciplinary group at the national university of singapore has evaluated and patented the parameters necessary to process medical grade polycaprolactone ( mpcl ) and mpcl composites [ pcl / ha , pcl / tcp , etc . ] by fused deposition modelling ( fdm ) . these socalled first - generation scaffolds have been studied for more than 5 years in a clinical setting and gained food & drug administration ( fda ) approval via a 510k application in 2006 . has used mpcl scaffolds as burr whole plugs in a pilot study for cranioplasty . the clinical outcome after 12 months was positive , with all patients tolerating the implants with no adverse side effects reported . more then 200 patients have received burr hole plugs , scaffolds for orbital floor reconstruction ( fig . the majority of current tissue engineering approaches rely on the so called extrinsic mode of neovascularization . the neovascular bed originates from the periphery of the construct which should be implanted into a site of high vascularisation potential . here recites a core limitation for transfer of tissue engineering models from the in vitro to the in vivo environment diffusion is the initial process involved , but it can only provide for cell nutrient supply and waste transport within a maximum range of 200 m into the matrix . the survival of cells in the center of large cell containing constructs is therefore often limited by suboptimal initial vascularization . cell labeling experiments have disclosed a considerable loss of osteoblasts within the first week following transplantation in porous cancellous bone matrices . hence , reconstruction of small to moderate sized bone defects using extrinsic engineered bone tissues is technically feasible , and some of the currently developed concepts may represent alternatives to autologous bone grafts for certain clinical conditions , the reconstruction of large volume defects however remains challenging . hence , reconstructive surgeons aim to generate so called axially vascularized tissues that can be transferred to the defect site using microsurgical techniques of vascular anastomosis . these tissues are immediately vascularized upon implantation into the defect as free flaps do erol and spira were the firast to report prefabrication of skin flaps by using an arteriovenous vessel which introduced this technique to the tissue engineering community . most recently , the horch / kneser laboratory started collaboration with the hutmacher laboratory to use the by av - loop model ( figure c , small arrows ) in combination with a composite scaffold ( figure c , long arrow shows with contrast agent filled loop , highly vascularized composite scaffold is found quadrant ) ) which is manufactured by a melt extrusion based sff technique . micro ct analysis ( figure b ) of the with a construct agent perfused vascular network ( figure d , arrow show perfusion site with microfil filled needle , double line arrow shows with microfil filled vascular loop with caplillary network ) of the engineered construct revealed a highly vascular zed construct . the second generation scaffolds produced by fdm for bone engineering are based on composites and have been evaluated in vitro and in vivo . mpolymer / cap composites confer favourable mechanical and biochemical properties , including strength via the ceramic phase and toughness and plasticity via the polymer phase , they also possess more favourable degradation and resorption kinetics , and graded mechanical stiffness . in these scaffolds , the ceramic phase was homogenously distributed in the matrix as well as exposed on the surface . contact angle measurements have shown that such composite surfaces were more hydrophilic and that the degradation kinetics were seen to be accelerated three to four times compared to pcl alone . biochemical advantages include improved cell seeding , and enhanced control and/or simplification of the incorporation and immobilization of biological factors , such as bmp 's . most recently the group at the national university of singapore have undertaken several studies utilizing mpcl / cap composite scaffolds in large animal models ( fig . implantation of a msc loaded mpcl - cap / pcl scaffold ( long arrow ) into a high load - bearing osteochondral defect ( inset small arrows , defect size in the medial condyle diameter 5 mm , depth 8 mm ) in a pig model . implantation of a msc loaded mpcl - cap / scaffold ( 14125 mm3 ) into a spinal fusion model ( inset , disks were removed on level l1/l2 and l4/l5 and tissue engineered constructs were combined with internal fixation devices ) in a 6 month old domestic pig . orthopaedic , plastic and reconstructive surgery is beginning to use bone engineering as a preferential route to autologous or allogenic bone grafting . one treatment concept involves either doping a scaffold with growth factors ( bmp , fgf , vascular endothelial growth factor [ vegf ] , etc . ) or other biological molecules ( hs , etc ) . in growth factor therapy , the drug delivery system should not only promote effective biological activity but should also limit spatial spread of the factor . many of the growth factors currently being investigated for fracture healing are hbgfs that are produced by the osteoblasts and stored within the matrix of bone . of these , fgf-1 and fgf-2 have been extensively investigated as candidates for fracture healing . fgf-1 has been shown to aid in the bridging of a parietal bone critical defect and to increase the bone - implant interface when used in combination with titanium - based scaffolds . fgf-2 has been more extensively studied for its use in fracture healing , and it has been shown that daily , intravenous injections of fgf-2 for up to 2 weeks can enhance bone formation in rats [ 2426 ] . members of the tgf- superfamily have also been shown to be successful in enhancing osteogenesis . both tgf-1 and 2 are known to increase differentiation of committed osteoprogenitor cells , and are potent stimulators of bone repair in calvarial and long bone defects when administered alone or in combination with either igf - i or igf - i / growth hormone ( gh ) . in addition , growth factors can directly regulate the availability of other growth factors ; fgfs have been shown to regulate the expression of vegf and hgf , factors that are also mitogenic for osteoprogenitor cells . likewise , combined local delivery of igf - i in combination with tgf-1 in a hydrogel scaffold has been shown to significantly enhance bone formation in a rat tibial segmental defect over igf - i alone suggesting that perhaps the mode of delivery also affects the potential for growth factor - mediated bone healing . importantly , these findings clearly demonstrate that a universally applicable combination of growth factors has yet to be established for fracture therapy . hs by virtue of its binding to a multitude of growth factors normally present in the fracture haematoma , may represent an exciting avenue to augment fracture healing . indeed , we have shown that a single application of hs at the time of injury is sufficient to enhance fracture repair , and is responsible for the up - regulation of endogenous growth factors , notably fgf-1 , igf - ii , tgf- 1 and vegf . in addition to effective dose , incompatibility with sterilization procedures , poor thermal and ph tolerance and delivery regimes [ 3234 ] , growth factor efficacy in orthopaedics is complicated by virtue of their inherent instability . growth factors are rapidly cleared in vivo as well as being highly susceptible to proteolytic degradation . as such , large quantities of growth factors furthermore , the number of growth factors that are proto - oncogenic is increasing that is becoming an important translational problem . thus , the long - term use of growth factors as a therapy is still being debated . as such , an alternative , bioactive agent that can protect , localize and/or enhance the effects of exogenously applied growth factors in vivo would be a great advantage in orthopaedics as it would reduce the dosage of exogenous growth factors required . furthermore , an agent that can be used independently to augment in vivo bone formation by harnessing the potential of endogenously produced growth factors would be even more therapeutically desirable . recently , the use of bmp inhibitors together with fgf2 in the culture medium of human embryonic stem cells ( he s ) has been shown to facilitate the longterm maintenance of these cells in a pluripotent state . hence , the balance between fgf- and bmpmediated signalling is thought to impose a primary control over cell - fate decisions . as immunological rejection limits the use of allogenic or xenogenic bone grafting , there has been a significant shift within the field of orthopaedics towards bone engineering and its potentially more sophisticated techniques for promoting osteogenesis . one particular avenue our group is exploring is better harnessing the unique properties of growth factors by presenting them together with specific co - factors , such as the hs molecules together with osteoconductive scaffolds ( fig . 4a and b ) . bmp / heparin mimetics have shown promise when delivered on collagen sponges.now what is needed is the coupling of bioactive molecules to the next generation of scaffolding materials that will provide the key elements mentioned previously in this review.we are thus developing methodologies to engineer scaffolding devices that sustain tissue growth and maturation in the presence of functionalized growth factor / hs combinations . this is an important paradigm shift that we belief is achievable due to the resilience of hs to sterilization techniques , ph change , temperature and chemical solvents required for scaffold processing . therefore , successful bone tissue engineering could be achieved through the delivery of bioactive growth factors in bioresorbable scaffolding matrices functionalized with hs . this method is particularly attractive as it can boost local osteoprogenitor cell recruitment , proliferation and differentiation . hs - like molecules have been tested over the last decade for their ability to augment bone formation , as well as in the healing of other tissue types . dextran derivatives substituted with carboxymethyl benzylamide sulfonate ( cmdbs ) have been shown to mimic heparin / hs by providing a somewhat similar protection and stabilization capability for growth factors as imparted by hs ; albeit with less affinity . otherwise known as ' regenerating agents ' ( rgtas ) , these hs - like molecules have been shown to stimulate tissue repair in skin , bone , muscle and the cornea [ 3844 ] . in all cases , these hslike molecules are thought to exert their effect by increasing both cell proliferation and differentiation within the wound site , presumably by enhancing growth factor - mediated signals . although hs - like molecules have been shown to have the potential to accelerate fracture repair , hs molecules themselves are only now being used as a treatment therapy in bone . ( 2006 ) recently demonstrated that hs harvested from osteoblasts can increase trabecular bone volume by 20% following a once - off application into a rat middiaphyseal femoral fracture . furthermore , hs supplementation was shown to increase the expression of alkaline phosphatase ( alp ) and runx2 , as well as the expression of a number of hbgfs present within the callus .together , these findings show that hs and hs - like molecules appear to have the remarkable potential to create an environment favourable to fracture repair . what is needed now is a better understanding of how best to deliver these potent molecules . due to the temporal pattern of growth factor expression during bone repair , we hypothesize that prolonged localized delivery of hs over the period of healing may further improve the effects of hs . we recently developed two techniques to prolong the delivery of hs [ 45 , 46 ] . in the first study , hs was incorporated into electrospun poly( - caprolactone ) ( pcl ) nanofibre mats . this produced fibres with smooth surfaces and no bead defects and was spun from polymer solutions with 8% w / v pcl in 7:3 dichloromethane : methanol solvent . assessment of hs loading and imaging of fluorescently labelled hs showed a homogenous distribution throughout the fibre mats and a sustained release for up to 14 days . importantly , the hs fibres did not induce an inflammatory response in macrophage cells in vitro and the released hs had sustained biological activity . in the later study , we encapsulated hs by the water - in oil - in water ( w1/o / w2 ) technique using polycaprolactone ( pcl ) microcapsules and achieved similar biocompatibility and bioactivity results with a prolonged delivery of up to 40 days . whilst promising in terms of hs release and bioactivity , these material constructs require extensive in vivo testing . do they induce ectopic bone formation , can they be used to co - deliver important growth factors ? sff techniques are experiencing increasing application in many biomedical fields , including regenerative medicine and tissue engineering . in general , scaffold - based tissue engineering requires a welldefined internal structure with interconnected porosity and sff techniques allow us to design and fabricate such scaffolds . from a biological point of view , the designed scaffold should serve several functions , including ( 1 ) the ability to act as an immobilization site for transplanted cells , ( 2 ) the formation of a protective space to prevent unwanted tissue growth into the wound bed and allow healing with differentiated tissue , ( 3 ) directing the migration or growth of cells via surface properties of the scaffold and ( 4 ) directing migration or growth of cells via release of soluble molecules , such as growth factors , hormones and/or cytokines . future work has to provide further compelling evidence that other sff methods next to fdm offer the right balance of capability and practicality to be suitable for fabrication of materials in sufficient quantity and quality to move holistic tissue engineering technology platforms into a clinical application . in addition to considerations of scaffold performance based on tissue engineering strategies , practical considerations of manufacture also arise . from a clinical point of view , it must be possible to manufacture scaffolds under good manufacturing practice ( gmp ) conditions in a reproducible and quality controlled fashion at an economic cost and speed . to move the current tissue engineering practices to the next frontier , some manufacturing processes will be required to accommodate the incorporation of cells and/or biological molecules during the scaffold fabrication process . these approaches are working towards the enablement of the tissue - engineered construct to not only have a controlled spatial distribution of cells and molecules , but also to possess a versatility of scaffold material and microstructure within one specifically designed and fabricated construct , for implantation in an intended anatomical site . the development of an ' hs approach ' for the control of progenitor phenotype has become more feasible in recent years as our understanding of hs cell biology improves . hss are particularly attractive , as they potentiate the powerful effects of growth factors on cell recruitment , proliferation and differentiation , and do not depend on the synthesis of other co - factors or other specific activating agents . we would anticipate that hs , by virtue of its modulation of the biological activities of the fgfs and the bmps , offers the possibility of intervening not just in embryonic growth , and stem and progenitor cell self - renewal , but also in stem cell - dependent wound healing and scaffold - based tissue engineering . unlike other nucleic acid - based clinical approaches ( e.g. virus and sirna ) , therapeutic intervention may be particularly advantaged because hss are chemically stable . realization of the potential of hs for regenerative medicine will depend , however , on suitable delivery systems with its own intrinsic properties that mimic the host tissue architecture .

abstracta paradigm shift is taking place in orthopaedic and reconstructive surgery from using medical devices and tissue grafts to a tissue engineering approach that uses biodegradable scaffolds combined with cells or biological molecules to repair and/or regenerate tissues . one of the potential benefits offered by solid free - form fabrication technology ( sff ) is the ability to create scaffolds with highly reproducible architecture and compositional variation across the entire scaffold , due to its tightly controlled computer - driven fabrication . in this review , we define scaffold properties and attempt to provide some broad criteria and constraints for scaffold design in bone engineering.we also discuss the application - specific modifications driven by surgeon 's requirements in vitro and/or in vivo . next , we review the current use of sff techniques in scaffold fabrication in the context of their clinical use in bone regeneration . lastly , we comment on future developments in our groups , such as the functionalization of novel composite scaffolds with combinations of growth factors ; and more specifically the promising area of heparan sulphate polysaccaride immobilization within the bone tissue engineering arena .

osseointegrated dental implants have proven to be predictably successful when appropriate guidelines are followed . traditionally , implant treatment of edentulous patients was based on a two - stage surgical protocol , with a healing period of three to six months , during which the implants were submerged to achieve osseointegration . this approach was considered to be an essential step for successful implant treatment , as it was believed that the micromovement of the implants , due to functional forces at the bone - implant interface during wound healing , could induce the formation of fibrous tissue rather than bone , leading to failure . several aspects of the implant morphology have been studied for years . among these , a submerged implant was thought necessary to prevent infection and epithelial downgrowth . more recently , several reports have demonstrated that one - stage surgery , with immediate loading , is possible , with good clinical results . although zdis have been used since the nineties , there are few reports focusing on the clinical outcome of these fixtures . consequently , we decided to perform a retrospective study on a large series of zdis , to identify the variables statistically associated with the clinical outcome . in the period between january 2007 and june 2011 , 125 patients were treatetd with zdis by two surgeons ( cms and ez ) . the last check - up was performed in june 2012 , with a mean follow - up period of 17 9 months ( minimummaximum , 8 - 64 months ) . controlled oral hygiene , the absence of any lesions in the oral cavity , and sufficient residual bone volume to receive implants at least 3.25 mm in diameter and 8.0 mm in length . in addition , the patients had to agree to participate in a post`-operative check - up program . insufficient bone volume , a high degree of bruxism , smoking more than 20 cigarettes / day and excessive consumption of alcohol , localized radiation therapy of the oral cavity , antitumor chemotherapy , liver , blood , and kidney diseases , immunosupressed patients , patients taking corticosteroids , pregnant women , patients with inflammatory and autoimmune diseases of the oral cavity , and poor oral hygiene . before surgery , radiographic examinations were done with the use of an orthopantomograph and computed tomography ( ct ) scans . in each patient , the peri - implant crestal bone levels were evaluated by a calibrated examination of the ortopantomograph x - rays . measurements were recorded before surgery ; after surgery , and at the end of the follow - up period . the measurements were carried out mesially and distally to each implant , calculating the distance between the edge of the implant and the most coronal point of contact between the bone and the implant . the bone level recorded just after the surgical insertion of the implant was the reference point for the following measurements . a peak scale loupe with a magnifying factor of seven times and a scale graduated in 0.1 mm was used . peri - implant probing was not performed because controversy still existed with regard to the correlation between the probing depth and implant success rates . the implant success rate ( scr i.e. , good clinical , radiological , and esthetic outcomes ) was evaluated according to the following criteria : ( 1 ) absence of persisting pain or dysesthesia ; ( 2 ) absence of peri - implant infection with suppuration ; ( 3 ) absence of mobility ; and ( 4 ) absence of persisting peri - implant bone resorption greater than 1.5 mm during the first year of loading and 0.2 mm / year during the following years . in 125 patients a total of 556 plants were inserted : two hundred and ninety - five ( 53.1% ) in the maxilla and 261 ( 46.9% ) in the mandible . there were 480 ( 86.3% ) screw - vents [ figure 1 ] , 51 ( 9.2% ) swiss - plusses [ figure 2 ] , and 25 ( 4.5% ) splines [ figure 3 ] . sixteen , 355 , 34 , 90 , 55 , and 6 fixtures had a diameter of 3.25 , 3.7 , 3.75 , 4.1 , 4.7 , and 4.8 mm , respectively . twenty - eight , 145 , 5 , 217 , 8 , 141 , and 12 implants had a length of 8 , 10 , 11 , 11.5 , 12 , 13 , and 14 mm , respectively . the implants were inserted to replace 136 ( 24.5% ) incisors , 80 ( 14.4% ) cuspids , 198 ( 35.6% ) premolars , and 142 ( 25.5% ) molars . two surgeons inserted the implants : cms 324 ( 58.3% ) and ez 232 ( 41.7% ) . three hundred and twenty - seven ( 58.8.% ) were inserted in females and 229 ( 41.2 % ) in males . seven ( 1.3% ) implants were inserted in diabetic patients and 80 ( 14.4% ) in subjects treated with anti - hypertensive drugs . thirty - four ( 6.1 ) were inclinated as they were used for an all - on - four technique and 179 ( 32.2% ) were inserted in smokers ( less than 20 cigarettes per day ) . an antimicrobial prophylaxis was administered with 1 g amoxycillin twice daily for five days , starting one hour before surgery . local anesthesia was induced by infiltration of articaine / epinephrine and postsurgical analgesic treatment was performed with 100 mg nimesulid , twice daily , for three days . the sutures were removed seven days after surgery . in case of a two stage procedure , following 24 weeks of implant insertion , the provisional prosthesis was provided , and the final restoration was usually delivered within an additional eight weeks . the number of prosthetic units ( i.e. , implant / crown ratio ) was about 0.6 . the mean age was 62 10 years ( minimummaximum 23 - 88 years ) . two hundred and thirty - two ( 41.7% ) implants were insered after the use of piezo , laser , and platelet - rich plasma derivates . fourteen ( 2.5% ) were covered with resorbable membranes and 244 ( 43.9% ) were inserted in post - extractive sockets . as no implants were lost ( i.e. , srv = 100% ) and no statistical differences were detected among the studied variables , no or reduced crestal bone resorption was considered as an indicator of scr , to evaluate the effect of several host- , implant- , and occlusion - related factors . the difference between the implant abutment junction and the bone crestal level was defined as the implant abutment junction ( iaj ) and calculated at the time of operation and during follow - up . delta iaj was the difference between iaj at the last check - up and iaj recorded just after the operation . the pearson chi - sqaure test was used to detect the variables most associated with implant success . in the period between january 2007 and june 2011 , 125 patients were treatetd with zdis by two surgeons ( cms and ez ) . the last check - up was performed in june 2012 , with a mean follow - up period of 17 9 months ( minimummaximum , 8 - 64 months ) . controlled oral hygiene , the absence of any lesions in the oral cavity , and sufficient residual bone volume to receive implants at least 3.25 mm in diameter and 8.0 mm in length . in addition , the patients had to agree to participate in a post`-operative check - up program . insufficient bone volume , a high degree of bruxism , smoking more than 20 cigarettes / day and excessive consumption of alcohol , localized radiation therapy of the oral cavity , antitumor chemotherapy , liver , blood , and kidney diseases , immunosupressed patients , patients taking corticosteroids , pregnant women , patients with inflammatory and autoimmune diseases of the oral cavity , and poor oral hygiene . controlled oral hygiene , the absence of any lesions in the oral cavity , and sufficient residual bone volume to receive implants at least 3.25 mm in diameter and 8.0 mm in length . in addition , the patients had to agree to participate in a post`-operative check - up program . insufficient bone volume , a high degree of bruxism , smoking more than 20 cigarettes / day and excessive consumption of alcohol , localized radiation therapy of the oral cavity , antitumor chemotherapy , liver , blood , and kidney diseases , immunosupressed patients , patients taking corticosteroids , pregnant women , patients with inflammatory and autoimmune diseases of the oral cavity , and poor oral hygiene . before surgery , radiographic examinations were done with the use of an orthopantomograph and computed tomography ( ct ) scans . in each patient , the peri - implant crestal bone levels were evaluated by a calibrated examination of the ortopantomograph x - rays . measurements were recorded before surgery ; after surgery , and at the end of the follow - up period . the measurements were carried out mesially and distally to each implant , calculating the distance between the edge of the implant and the most coronal point of contact between the bone and the implant . the bone level recorded just after the surgical insertion of the implant was the reference point for the following measurements . a peak scale loupe with a magnifying factor of seven times and a scale graduated in 0.1 mm was used . peri - implant probing was not performed because controversy still existed with regard to the correlation between the probing depth and implant success rates . the implant success rate ( scr i.e. , good clinical , radiological , and esthetic outcomes ) was evaluated according to the following criteria : ( 1 ) absence of persisting pain or dysesthesia ; ( 2 ) absence of peri - implant infection with suppuration ; ( 3 ) absence of mobility ; and ( 4 ) absence of persisting peri - implant bone resorption greater than 1.5 mm during the first year of loading and 0.2 mm / year during the following years . in 125 patients a total of 556 plants were inserted : two hundred and ninety - five ( 53.1% ) in the maxilla and 261 ( 46.9% ) in the mandible . there were 480 ( 86.3% ) screw - vents [ figure 1 ] , 51 ( 9.2% ) swiss - plusses [ figure 2 ] , and 25 ( 4.5% ) splines [ figure 3 ] . sixteen , 355 , 34 , 90 , 55 , and 6 fixtures had a diameter of 3.25 , 3.7 , 3.75 , 4.1 , 4.7 , and 4.8 mm , respectively . twenty - eight , 145 , 5 , 217 , 8 , 141 , and 12 implants had a length of 8 , 10 , 11 , 11.5 , 12 , 13 , and 14 mm , respectively . the implants were inserted to replace 136 ( 24.5% ) incisors , 80 ( 14.4% ) cuspids , 198 ( 35.6% ) premolars , and 142 ( 25.5% ) molars . two surgeons inserted the implants : cms 324 ( 58.3% ) and ez 232 ( 41.7% ) . three hundred and twenty - seven ( 58.8.% ) were inserted in females and 229 ( 41.2 % ) in males . seven ( 1.3% ) implants were inserted in diabetic patients and 80 ( 14.4% ) in subjects treated with anti - hypertensive drugs . thirty - four ( 6.1 ) were inclinated as they were used for an all - on - four technique and 179 ( 32.2% ) were inserted in smokers ( less than 20 cigarettes per day ) . an antimicrobial prophylaxis was administered with 1 g amoxycillin twice daily for five days , starting one hour before surgery . local anesthesia was induced by infiltration of articaine / epinephrine and postsurgical analgesic treatment was performed with 100 mg nimesulid , twice daily , for three days . the sutures were removed seven days after surgery . in case of a two stage procedure , following 24 weeks of implant insertion , the provisional prosthesis was provided , and the final restoration was usually delivered within an additional eight weeks . the number of prosthetic units ( i.e. , implant / crown ratio ) was about 0.6 . the mean age was 62 10 years ( minimummaximum 23 - 88 years ) . two hundred and thirty - two ( 41.7% ) implants were insered after the use of piezo , laser , and platelet - rich plasma derivates . fourteen ( 2.5% ) were covered with resorbable membranes and 244 ( 43.9% ) were inserted in post - extractive sockets . as no implants were lost ( i.e. , srv = 100% ) and no statistical differences were detected among the studied variables , no or reduced crestal bone resorption was considered as an indicator of scr , to evaluate the effect of several host- , implant- , and occlusion - related factors . the difference between the implant abutment junction and the bone crestal level was defined as the implant abutment junction ( iaj ) and calculated at the time of operation and during follow - up . delta iaj was the difference between iaj at the last check - up and iaj recorded just after the operation . the pearson chi - sqaure test was used to detect the variables most associated with implant success . the mean peri - implant bone resorption was 0.7 0.6 mm ( minimum - maximum 0 - 5.7 mm ) . twenty - seven fixtures had a peri - implant bone resorption greater than the cut - off values ( scr = 95.1% ) , and therefore , were used to detect those variables statistically related to an augmented peri - implant bone resorption . among the studied variables ( i.e. , implant type , diameter , length , gender , smoke , diabetes , drugs , platelet - rich plasma , sinus augmentation , membrane , immediate loading , fixture inclination , laser , piezo , post - extraction socket , surgeon , tooth position , and jaws ) only jaws was statistically significant , with a better outcome for implants inserted in the maxilla ( p = 0.017 ) . zdis have been used since the nineties , but few reports analyzed the clinical outcome of these fixtures . in 2001 , khayat et al . reported a study on a wide diameter screw - vent . one hundred and eleven implants were evaluated at the recall examination . almost all implants ( 109 ) supported a fixed partial prosthesis . no implants were lost during the loading period . in 2002 , arlin analyzed 435 screw - vent implants . the focus group was compared to a mixed implant design group , with a variety of abutment connections and surfaces from several other manufacturers . the cumulative survival rates were 94.2% ( n = 435 ) for the focus group and 90.1% ( n = 2339 ) for the reference group . in 2005 , minichetti et al . reported the results of implants placed into extraction sites grafted with particulate mineralized bone allograft ( puros ) . a total of 313 extraction sites were grafted with mineralized bone graft during a 36-month period . a total of 252 screw - vent implants were placed into the grafted extraction sites after a four- to seven - month healing period . a total of 244 implants were restored with fixed prosthesis and six with removable overdentures , for a total of 250 loaded implants . a total of six implants failed , which required their removal ( two implants before load and four after loading ) , resulting in a 97.6% implant success rate . in 2007 , khayat et al . a total of 835 implants , with diameters of 3.7 mm ( 9% ) , 4.7 mm ( 76% ) , and 6.0 mm ( 15% ) were placed in 328 patients , using a single - stage , delayed - loading protocol . the implants were restored with a variety of prostheses and monitored over two years of functional loading . cumulative implant survival was 99.4% ( n = 835 ) ; differences between mandibular ( 99.0% , n = 408 ) and maxillary ( 99.8% , n = 427 ) implants were not statistically significant . six implants failed to meet the success criteria by sustaining mesial and distal bone loss below the first implant thread ; however , they remained stable and continued functioning without pain or inflammation . cumulative implant success was 98.6% ( n = 835 ) ; the differences between the maxillary ( 98.6% ) and mandibular ( 98.8% ) implants were not statistically significant . the success rates by implant diameter were 98.6% ( 3.7 mm ) , 98.4% ( 4.7 mm ) , and 100% ( 6 mm ) . the authors concluded that after two years of functional loading , the survival and success rates for screw - vent implants placed in a non - submerged protocol , equaled or surpassed those of single - thread , straight - walled implant historical controls . one year later , minichetti et al . reported the results of implants placed in the maxillary sinuses grafted with particulate mineralized cancellous bone allograft alone or in combination with resorbable hydroxyapatite , over a three - year period . a total of 56 sinuses were grafted , and 136 dental implants were placed into the grafted sites after a four- to eight - month healing period . all re - entries revealed a bony hard structure acceptable for osteotomy preparation . of these implants , 124 had been restored with fixed prosthesis and 12 with removable overdentures for a total of 136 loaded implants . a total of three implants required removal ( failure ) resulting in a 97.7% implant success rate ( 2.3% failure rate ) . the authors concluded that a mineralized human allograft placed in a lateral window sinus elevation , was a clinically predicable method , acceptable for implant placement and restoration . evaluated the immediate loading implant and possible effects correlated with peri - implants . in 2008 , ormianer et al . , performed a non - randomized , uncontrolled , retrospective study to evaluate the clinical outcomes of treatment with tapered , multithreaded implants , with a special emphasis on peri - implant crestal bone status . chart reviews were conducted of 60 patients who had been treated with 267 implants for the placement of one or more missing and/or unsalvageable teeth , and who met the general inclusion criteria for dental implant therapy . in all cases , marginal bone changes were calculated from the cementoenamel junction or the implant neck to the crestal bone level , with standardized radiographs taken at the implant placement ( baseline ) and during annual follow - up . after a mean follow - up of 7.5 years , the implant survival was 98.5% ( 263/267 ) for all implants placed , and the implant success was 96.2% ( 253/263 ) for all surviving implants . crestal bone loss was observed in 25% ( 15/60 ) of the total study subjects and in 12% ( 32/263 ) of all surviving implants : twenty - nine implants exhibited 1 mm of bone loss and three implants lost 2 mm of bone . low - density maxillary jawbone and more extensive bone remodeling , which were required around the implants immediately placed into extraction sockets , were the probable causes of the observed bone loss in this study . the implants exhibited excellent long - term outcomes , with little or no bone loss . most of the studied variables ( i.e. , implant type , diameter , length , gender , smoke , diabetes , drugs , platelet - rich plasma , sinus augmentetion , membrane , immediate loading , fixture inclination , laser , piezo , post - extraction socket , surgeon , and tooth position ) have no impact on the clinical outcome . implants inserted in the mandible have a slight , but a statistically significant worse outcome , with a higher peri - implant bone resorption , compared to those inserted in the maxilla . in conclusion , zdis are reliable devices to use in implantology .

background : one - stage surgery with immediate loading is possible , with good clinical results . many types of dental implants are available in the market . zimmer dental implants ( zdis ) have been used since the nineties , but few reports have analyzed the clinical outcome of these fixtures . we planned a retrospective study on a series 566 zdis , to evaluate their clinical outcome.materials and methods : in the period between january 2007 and june 2011 , 125 patients were treatetd with zdis . the last check - up was performed in june 2012 , with a mean follow - up period of 17 9 months ( minimum maximum , 8 - 4 months ) . zdis were inserted as follows : 295 ( 53.1% ) in the maxilla and 261 ( 46.9% ) in the mandible . there were 480 ( 86.3% ) screw - vents , 51 ( 9.2% ) swiss plusses , and 25 ( 4.5% ) splines . sixteen , 355 , 34 , 90 , 55 , and six fixtures had a diameter of 3.25 , 3.7 , 3.75 , 4.1 , 4.7 , and 4.8 mm , respectively . twenty - eight , 145 , 5 , 217 , 8 , 141 , and 12 implants hade a length of 8 , 10 , 11 , 11.5 , 12 , 13 , and 14 mm , respectively . the implants were inserted to replace 136 ( 24.5% ) incisors , 80 ( 14.4% ) cuspids , 198 ( 35.6% ) premolars , and 142 ( 25.5% ) molars.results:no implants were lost ( i.e. , srv = 100% ) . among the studied variables , only those for the jaws were statistically significant , with a better outcome for implants inserted in the maxilla ( p = 0.017).conclusions : zdis are reliable devices to be used in implantology , althougth a higher marginal bone loss has to be expected when these implants are inserted in mandible .

aneurysm formation after pancreatitis is well documented in the literature , caused by the digestion of arterial walls by the enzymes such as elastase released by the pancreas . rupture is generally considered to be a rare complication after pancreatitis , but the formation of aneurysms themselves is relatively common , occurring in 10% of patients following pancreatitis . endoscopic retrograde cholangiopancreatography ( ercp ) is a common procedure used both as a diagnostic and therapeutic tool in patients with biliary tree obstruction . it is considered a safe procedure and serious complications are rare ; with the most common complications being pancreatits , cholangitis , bowel perforation , and bleeding from the ampulla of vater following sphincterotomy . however , although far rarer than any of the above the following case illustrates life - threatening and therefore significant complication following ercp . we present the case of a 75 year old female admitted with acute pancreatitis , deranged liver function and gram - negative rod septicaemia . she was admitted to intensive care and magnetic resonance cholangiopancreatography carried out to determine the aetiology of the pancreatitis . this showed a 2 cm filling defect in the dilated lower end of the common bile duct , suggestive of an impacted stone . endoscopic retrograde cholangiopancreatography ( ercp ) was performed 10 days later , but showed an undilated biliary system with no filling defects ; despite trawling the common bile duct several times . subsequently the patient had several episodes of haematemesis and malaena , with the patient s haemoglobin repeatedly dropping despite transfusion . oesophogastroduodenal ( ogd ) endoscopy and computerised tomography ( ct ) scan failed to identify a source . the only positive finding on ct scan was pneumobilia , most likely due to sphincterotomy during the ercp . the haematemesis continued and emergency laparotomy was performed . on external examination of the stomach and duodenum pyloroduodenotomy identified a small healed duodenal ulcer and fresh clots in the first part of duodenum . on palpation of the third part of duodenum , a periampullary diverticulum was identified , although it was not bleeding . a second urgent ogd endoscopy showed more blood clots and fresh blood in the fundus , but once again no source for the bleeding was seen . this showed a 1 cm pseudoaneurysm of the right hepatic artery in the right lobe close to the porta hepatis . the pseudoaneurysm arose from the anterior branch of the right hepatic artery and communicated with the bile duct . the 2 part of the duodenum was opacified , showing haemorrhage from the lesion into the duodenum via the bile duct ( figure 1 ) . initially , selective embolisation of the anterior right hepatic artery was performed using small coils ( cook uk ) and small particles of grated gelatin sponge soaked in 0.5ml of 5% ethanolamine . however this proved to be ineffective ( figure 2 ) , and so the posterior branch of the right hepatic artery was embolised , successfully controlling the haemobilia . there were signs of recurrent haemorrhage such as fluctuating serum haemoglobin levels and some ongoing malaena . however , these ceased spontaneously after a few days , and the patient subsequently made an uneventful recovery . the formation of pseudoaneurysms after pancreatitis is very common , although most of these will not rupture and therefore remain asymptomatic . cases of haematemesis following spontaneous fistulation into the biliary tree after pancreatitis are described in the literature ; although often they have presented after a more severe necrotising pancreatitis . similar presentations have also been described with a different pancreatic source , such as a bleeding pancreatic pseudocyst . given that the haematemesis commenced following ercp in this case , it is likely that it was the instrumentation of the common bile duct which led to the fisulation of the aneurysm into the biliary tree . this theory is feasible as the branches of the hepatic artery and portal vein are in very close proximity to the bile duct allowing concurrent injury to occur . we hypothesize that during the ercp procedure the walls of the intra - hepatic bile duct and paralleling artery were injured during steel guide wire maneuvering , causing the false aneurysm . it is important to recognize that iatrogenic causes are the most common cause for haemobilia ( 52% ) . we also assume the bleeding in the acute recovery phase post embolisation was mainly due to the dissolving nature of the gelatin sponge . however , even though a fraction of the particles may have dissolved , the bleeding subsequently stopped as low enough perfusion pressures were maintained to stabilize the lesion . ogd endoscopy is of course the first line investigation in any patient presenting with haematemesis , but in this case was unhelpful because there was no blood discharging from the ampulla of vater at the time of investigation . ct scanning lacked the sensitivity required to detect the 1 cm pseudoaneurysm described in this case , though it has been successfully used in the diagnosis of larger aneurysms in other cases . angiography is considered the gold standard in the diagnosis of this condition , and it is important learning point to remember this as the next line of investigation of haematemesis , particularly in patients with a history of pancreatitis . in this case there was a delay before an angiogram was done , and this delay was very nearly fatal for the patient . although this case report describes a rare complication of pancreatitis , it is a complication that is readily treatable provided the diagnosis is made in time . therefore we publish it in the hope that clinicians will think of it in patients with upper gastrointestinal bleeding of unknown origin .

we present a case of pancreatitis with secondary pseudoaneurysm formation in a branch of the hepatic artery , which fistulated into the common bile duct following endoscopic retrograde cholangiopancreatography . this aneurysm manifested itself clinically with recurrent haematemesis , and was treated by embolisation of the anterior and posterior branches of the right hepatic artery .

oropharyngeal cancers account for a major proportion of cancers of the upper aerodigestive tract . in 1995 , the standard treatments for these cancers can be either surgery followed by radiotherapy eventually with concomitant chemotherapy or exclusive radiotherapy eventually with concomitant chemotherapy . in spite of the lack of methodologically solid comparative studies , no difference in terms of survival between these two options seems to exist . a new challenge recently appeared , certainly less important than survival , but important all the same : the quality of life of patients among whom dysphagia seems to be a very important criterion . some treatments may be too detrimental in particular with regard to swallowing . in the same way , the recently developed concept of therapeutic utility ( measurement of the preference of patients for a health condition ) is raised : will a patient prefer to live for three years with normal eating ability or for four years with exclusive enteral feeding ? the aim of our study was to evaluate the short- , medium- , and long - term ( five to 10 years ) feeding abilities of patients treated for oropharyngeal cancer to determine prognostic factors for feeding difficulties . after consulting the medical information department database of the croix - rousse hospital , 252 consecutive patients treated between january 1st , 1998 and december 31st , 2003 for oropharyngeal squamous cell carcinoma were selected . if patients were operable , they were offered surgical treatment ( tumor and lymphadenectomy ) followed by radiotherapy ( 5054 gy in 5 to 6 weeks ) sometimes potentiated by cisplatin according to the pathology results ( more than 3 metastatic nodes , extracapsular extension , endovascular metastases , or perineural infiltration ) . the surgery procedures [ 4 , 5 ] were transoral approach , submandibular approach ( lateral oropharyngectomy , hyosubglossoepiglottectomy ) , or transmandibular approach ( mandibular swing or pharyngectomy with mandibulectomy ) . during surgery , a nasogastric tube was inserted , and tracheotomy was performed except in the case of small tumors treated by the transoral approach . most of the patients benefited from swallowing rehabilitation during their hospitalization , which in certain cases continued after return home or after admission to a rehabilitation institution . unfortunately , this variable could not be exploited because of the lack of data . if operable patients refused surgery , they received exclusive radiotherapy ( 70 gy in 6 - 7 weeks ) generally potentiated by cisplatin . in the event of an inoperable tumour , and sometimes after induction chemotherapy , the patient received exclusive radiotherapy ( 70 gy in 6 to7 weeks ) generally potentiated by cisplatin . radiotherapy fractionation was standard ( 2 gy per session , 5 sessions a week ) ; 2 lateral ports with 4 to 8 mv x - rays split at 40 gy due to the spinal cord tolerance limit to 2 anterior lateral ports with x - rays and 2 posterior ports with 8 to 10 mev elecron beams . the level iv regions were treated with mixed 46 mv x - rays and 8 to 10 mev elecron beams . additional boosts were given for high - risk nodal areas ( in cases of node involvement with extracapsular extension ) either with 8 to 10 mev electron beams . the following data were retrospectively collected : weight , swallowing ability ( solids , pastes , liquids ) according to doctors ' and speech pathologists ' notes , and the use of a nasogastric tube during the posttherapeutic followup ( five to ten years ) . after exclusion of the patients initially treated with palliative care or palliative chemotherapy ( including metastatic patients ) , functional tolerance to treatments was analyzed in order to determine prognostic factors for post - therapeutic swallowing difficulties and their repercussion on body weight . factors which were thought to have a significant impact on swallowing were the sex , the age , the american society of anaesthesiology ( asa ) score , previous cancer of the head and neck , the tumour , location in the oropharynx , the t stage , the n stage , the use of induction chemotherapy , the type of treatment , the reconstruction type , the existence of a postoperative complication , the dose of radiation to the tumour and the dose of radiation to the nodes . for the study of weight , a linear regression according to time was used with nested mixed models on repeated data . the binary variables of liquids , pastes , and solid foods were studied using a multivariate model of survival censored by intervals . the reference patient was by definition a 55-year - old man , asa 1 , with no past history of cancer who developed t1n0 cancer of the base of tongue who received no induction chemotherapy and was treated using pharyngectomy with mandibulectomy , without postoperative complications or postoperative radiotherapy . such a patient does not exist but changing one ( or more ) parameters of the model gives a hazard ratio that allows comparisons to be made . sixty - two patients had previously at least one cancer of the head and neck ( 24.6% of the patients ) . one hundred and thirty - five patients still drank alcohol at the time of the diagnosis ( 68.5% of the data collected on this item ) , while 133 patients still smoked at the time of the diagnosis ( 65.6% of the data collected on this item ) . four patients presented pulmonary metastases , and one patient had osseous metastases at the time of the diagnosis . eighty - one patients ( 32.1% ) had induction chemotherapy , the majority with 5-fluorouracil / cisplatin because of inoperable tumours . ( i ) surgery for the tumourone hundred and twenty - two patients ( 48.4% of the patients ) did not have tracheotomy , 25 patients ( 9.9% of total patients , 15.8% of operated patients ) underwent prepared tracheotomy , and 70 patients ( 27.7% of total patients , 44.3% of operated patients ) had tracheotomy . the mean duration of the tracheotomy was 2.2 weeks ( es 3.3 weeks ) , the mean time before postsurgical recovery of feeding was 1.8 weeks ( es 1.8 weeks ) , and the mean duration of postsurgical nasogastric tube was 2.7 weeks ( es 1.8 weeks ) . one hundred and twenty - two patients ( 48.4% of the patients ) did not have tracheotomy , 25 patients ( 9.9% of total patients , 15.8% of operated patients ) underwent prepared tracheotomy , and 70 patients ( 27.7% of total patients , 44.3% of operated patients ) had tracheotomy . the mean duration of the tracheotomy was 2.2 weeks ( es 3.3 weeks ) , the mean time before postsurgical recovery of feeding was 1.8 weeks ( es 1.8 weeks ) , and the mean duration of postsurgical nasogastric tube was 2.7 weeks ( es 1.8 weeks ) . ( ii ) reconstructive surgerythe loss of substance due to tumor surgery required a pectoralis major flap for 23 patients ( 18 mandibular swings , 4 pharyngectomies with mandibulectomy , 1 lateral oropharyngectomy ) and a sternocleidomastoid flap for seven patients , all after lateral oropharyngectomy . the loss of substance due to tumor surgery required a pectoralis major flap for 23 patients ( 18 mandibular swings , 4 pharyngectomies with mandibulectomy , 1 lateral oropharyngectomy ) and a sternocleidomastoid flap for seven patients , all after lateral oropharyngectomy . ( iii ) complications of the surgeryforty - one patients ( 25.9% of the operated patients ) had a surgery - related complication : one death from an unknown cause , cervical infection ( 9 ) , bleeding ( 8) , pharyngostoma ( 7 ) , cervical hematoma ( 4 ) , pneumonia ( 3 ) , orostoma ( 2 ) , fever of an undetermined cause ( 2 ) , flap disunion ( 2 ) , hepatic failure ( 1 ) , acute delirium ( 1 ) , and lymphorrhea ( 1 ) . the last nine patients were transferred to the intensive care unit during the postoperative phase : five transfers were scheduled ( two for dialysis for chronic renal failure , three patients with a heavy cardiac past history requiring close surveillance ) , and four whose stay was unscheduled ( one pneumonia , one hemorrhagic hypovolemic shock , one hepatic failure , one cardiac failure ) . forty - one patients ( 25.9% of the operated patients ) had a surgery - related complication : one death from an unknown cause , cervical infection ( 9 ) , bleeding ( 8) , pharyngostoma ( 7 ) , cervical hematoma ( 4 ) , pneumonia ( 3 ) , orostoma ( 2 ) , fever of an undetermined cause ( 2 ) , flap disunion ( 2 ) , hepatic failure ( 1 ) , acute delirium ( 1 ) , and lymphorrhea ( 1 ) . the last nine patients were transferred to the intensive care unit during the postoperative phase : five transfers were scheduled ( two for dialysis for chronic renal failure , three patients with a heavy cardiac past history requiring close surveillance ) , and four whose stay was unscheduled ( one pneumonia , one hemorrhagic hypovolemic shock , one hepatic failure , one cardiac failure ) . table 2 presents the different types of radiotherapy and table 3 the doses of radiation . fifteen patients had local brachytherapy during postoperative external radiotherapy , four during exclusive external radiotherapy , and one patient without external radiotherapy ( postoperative or exclusive ) . 167 patients ( 87.7% ) were operated on by the same surgeon who also ensured the medical followup of 242 patients ( 96.0% ) . the data collecting is therefore very uniform . figure 1 presents the evolution of swallowing ability with time by type of treatment , and figure 2 shows the evolution of the use of a nasogastric tube by type of treatment . the mean weight at diagnosis was 64.9 kg . in the event of a history of cancer of the upper aerodigestive tract , the mean weight at diagnosis was significantly lower ( 3.6 kg ; p = .0173 ) . patients with a post - operative complication were significantly heavier ( + 1.2 kg , p < 10 ) . age , location of the oropharyngeal tumor , t stage , and n stage had no significant impact on weight at the time of diagnosis ( p = .2282,.7536,.4524 , resp . ) . the average weight loss was 1.56 kg / yr ( p < 10 ) throughout the followup , and the type of treatment did not have a significant impact on weight loss ( p = .634 ) . table 4 presents the results of the models ' parameters . because of the small numbers of patients in some categories ( e.g. pharyngectomy with mandibulectomy ) , the confidence interval was large and did not allow us to prove any significant differences . in the international literature , patients ' weight is seldom taken into account in the estimate of the functional results of treatments for cancer of the upper aerodigestive tract . however , denutrition is a predictive factor of tolerance to the treatment . according to our analyses , the treatments for oropharynx cancers , whatever they are and whatever the site of the oropharyngeal lesion is , caused a continuous , considerable ( 1.56 kg / yr ) , significant and long - term ( 60 to 120 months of followup ) weight loss in patients in an often already precarious nutritional state . however , correct feeding was rapidly possible for the majority of patients and remained possible with time . significantly , factors influencing the ability to swallow liquids , pastes , and normal foods were generally the same : the initial t stage , induction chemotherapy , and the type of treatment . because of the absence of anatomophysiological explanations for the harmful impact of induction chemotherapy on swallowing and the fact that this finding has not been described in other studies , it needs to be interpreted with caution , and other studies need to be conducted to rule out or to confirm this hypothesis . putting aside the possible role of induction chemotherapy , the data of our study are in agreement with those already published . indeed , for several authors [ 79 ] , the functional outcomes of surgery followed by radiotherapy and radiochemotherapy were not significantly different except for those reported by allal et al . for whom the functional quality of life and abilities after exclusive radiotherapy were similar for t1t2 but significantly better for t3t4 . other authors showed the existence of predictive factors of good functional performances , but these factors varied from one study to another . for zuydam et al . , swallowing was significantly better in the absence of radiotherapy ( p < .001 ) and in the event of direct closure ( p = .003 ) , and speech was also significantly better in the event of direct closure . for zelefski et al . , the predictive factors of good swallowing were the type of surgery ( in ascending order towards better swallowing : pharyngectomy with mandibulectomy , mandibular swing , and lateral oropharyngectomy ) and location ( in ascending order tonsil then base of tongue ) . this study should probably not be retained because of the illogical physiological character and the fact it is isolated from its conclusions . [ 14 , 15 ] , swallowing depended especially on the volume of tissue removed . , only postoperative radiation decreased swallowing ability . in conclusion of the analysis of the literature , the functional results seemed better in the absence of postoperative radiation and of reconstructive surgery and in the event of small tumour size , which is perfectly logical from a physiological point of view . we could not show that certain treatments ( in particular exclusive radiotherapy and transmandibular approaches ) caused more serious swallowing disorders in the medium and long term . in our everyday clinical experience , the principal factor that has an impact on function , which proved to be relevant for all locations of upper aerodigestive tract cancers , is radiation and there seems to be a threshold level of radiation ( 60 gy ) above which deterioration of functional abilities is accentuated . however , this hypothesis could not be proved in this study probably because of the high doses of postoperative radiation , more often closer to 6064 gy than to 5054 gy ( figure 2 ) . patients had a dose of radiation that was higher than the requested dose , and it is advisable to carefully discuss doses of radiation with the radiotherapists . whatever the possible and selected treatment was , the impact on the functional capacities and , thus , the quality of life of the patients was considerable . even though we could not significantly demonstrate that exclusive radiotherapy caused more long - term undesirable effects than surgery followed by radiotherapy , our daily practice has shown that we should favour the latter . further prospective studies about long - term side effects of treatments of oropharyngeal malignancies should be conducted .

objective . to analyze the functional impact of the various possible treatments of oropharyngeal squamous cell carcinomas to find the main prognostic factors of dysphagia induced by these treatments . patients . clinical data from 254 patients treated for squamous cell carcinoma of the oropharynx between 1998 and 2003 were retrospectively analyzed . a multivariate model enabled us to evaluate the role of each potentially harmful factor on swallowing . main outcome measures . the significant factors influencing the consumption of liquid , pasty , and normal food were the same : the initial t stage and the type of treatment . conclusion . whatever the possible and selected treatment was , the impact on the functional capacities , and thus , the quality of life of the patients was considerable . even though we could not significantly demonstrate exclusive radiotherapy caused more long - term undesirable effects than surgery followed by radiotherapy , our daily practice has shown that we should favour the latter .

the receptor - binding cancer antigen expressed on siso cells ( rcas1 ) was initially recognized by the mouse monoclonal antibody , 22 - 1 - 1 , which was raised by the immunization of mice with the human uterine cervical adenocarcinoma cell line siso . it is a 40-kd type ii membrane protein that forms homo - oligomers through its c - terminal coiled - coil structures , whereas it also exists in the soluble form probably by alternative splicing . rcas1 has currently been considered as a novel tumor - associated antigen that can function as a ligand for a putative receptor that is present on various human cell lines , such as erythroid leukaemia and normal peripheral lymphocytes , inhibiting cell growth and inducing apoptosis [ 3 , 4 ] . thus , rcas1 upregulation is considered to play a crucial role in tumor progression by enabling cancer cells to evade immune surveillance [ 5 , 6 ] . gastric cancer is the second largest cause of cancer - related death worldwide , presenting the higher incidence in japan and china , lower in europe , and the lowest in the usa [ 7 , 8 ] . studies on gastric cancer development suggest that genetic predisposition , infection , and diet are parts of a complex interaction [ 8 , 9 ] . helicobacter pylori ( hp ) infection and to a lesser extent smoking have been identified as the main environmental risk factors for gastric cancer [ 8 , 9 ] . chronic inflammation plays important roles in the development of various cancers of the digestive tract , including hp - associated gastric cancer . notably , several inflammatory mediators have been associated with tumor progression , angiogenesis , and metastasis in gastric cancer [ 11 , 12 ] . in the last few years , rcas1 protein expression has been described in a variety of human malignancies , including oral , esophageal , breast , thyroid , lung , neurological , pancreatic , colorectal , urothelial , endometrial , cervical , and ovarian carcinomas [ 13 , 14 ] . notably , rcas1 expression was shown to be associated with important clinicopathological parameters for patients ' management and prognosis , being considered as an informative biomarker in several types of tumour malignancy [ 13 , 14 ] . however , the available studies so far assessing the clinical significance of the rcas1 expression in gastric neoplasia remain scarce , and they are restricted to t3 gastric carcinoma cases or they do not concern patients ' prognosis [ 1517 ] . in view of the previous considerations , the present study aimed to assess immunohistochemically rcas1 expression in 54 gastric adenocarcinoma specimens and to evaluate the association of rcas1 expression levels with clinicopathological parameters , tumor proliferative capacity , and patients ' survival . fifty - four gastric tumoral samples obtained from an equal number of patients , who underwent surgical resection due to gastric cancer at the 2nd department of propedeutic surgery , medical school , university of athens , greece , were consecutively included in this study . the study was approved by the hospital ethics committee , and informed consent was obtained from all participants . forty - three ( 79.6% ) of the patients were men and 11 ( 20.4% ) were women . the mean age of the patient cohort was 67.33 8.47 years ( median : 67 years , range : 3988 years ) . tumors were typed according to lauren classification as intestinal in 24 ( 44.4% ) and diffuse in 30 ( 55.6% ) gastric adenocarcinoma cases . three levels differentiation were used to classify grading as : well in 1 ( 1.9% ) , moderately in 24 ( 44.4% ) , and poorly differentiated in 29 ( 53.7% ) patients . tumors staging was assessed using the 5th edition of the tumor , node , and metastasis ( tnm ) system according to the union internationale contra la cancrum ( uicc ) and the american joint committee on cancer ( ajcc ) . the resected tumors were staged as : t1 in 6 ( 11.1% ) ; t2 in 15 ( 27.7% ) ; t3 in 27 ( 50.0% ) , and t4 in 6 ( 11.1% ) cases . nineteen ( 35.2% ) patients were lymph node negative ( n0 ) and 35 ( 64.8% ) lymph node positive ; n1 in 32 ( 59.3% ) and n2 in 3 ( 5.6% ) cases . patients were followed up for a time interval between 1 to 148 months ( 28.21 22.63 months ) . rcas1 immunostaining was performed on formalin - fixed , paraffin - embedded gastric adenocarcinoma tissue sections using a mouse monoclonal anti - rcas1 antibody ( mbl international co , nagoya , japan ) . briefly , 4 m thick tissue sections were dewaxed in xylene and were brought to water through graded alcohols . antigen retrieval was performed by microwaving slides in 10 mm citrate buffer ( ph 6.1 ) for 20 minutes ( min ) at high power , according to the manufacturer 's instructions . to remove the endogenous peroxidase activity , sections were treated with freshly prepared 0.3% hydrogen peroxide in methanol in the dark , for 30 min , at room temperature . nonspecific antibody binding was blocked using sniper , a specific blocking reagent for mouse primary antibodies ( sniper , biocare medical , walnut , creek , ca , usa ) for 5 min . the sections were incubated for 1 hour ( h ) , at room temperature , with the primary antibody against rcas1 , diluted 1 : 750 in phosphate buffered saline ( pbs ) . after washing three times with pbs , sections were incubated at room temperature with biotinylated linking reagent ( biocare medical ) for 10 min , followed by incubation with peroxidase - conjugated streptavidin label ( biocare medical ) for 10 min . the resultant immune peroxidase activity was developed using a dab substrate kit ( vector laboratories , usa ) for 10 min . sections were counterstained with harris ' hematoxylin and mounted in entellan ( merck , darmstadt , germany ) . appropriate negative controls were performed by omitting the primary antibody and/or substituting it with an irrelevant antiserum . pancreatic and thyroid cancer tissue sections with known increased rcas1 immunoreactivity were used as positive control [ 20 , 21 ] . immunohistochemical evaluation was performed by counting at least 1000 tumor cells in each case by two independent observers ( s.t . and p.a . the immunoreactivity of the tumor cells for rcas1 was scored according to the percentage of rcas1 positive tumor cells as 0 : < 5% of tumor cell positive ; 1 : 524% of tumor cells positive ; 2 : 2549% of tumor cells positive ; 3 : 5074% of tumor cells positive ; 4 : and 75100% of tumor cells positive and according to its intensity as 0 : negative staining , 1 : mild staining ; 2 : intermediate staining ; and 3 : intense staining . finally , the expression of rcas1 was classified as low ; if the total score was 5 and high ; if the total score was 6 . in this way , we ensure that each group has a sufficient and more homogeneous number of cases in order to be comparable with the other groups [ 2022 ] . chi - square test was used to assess the associations of rcas1 protein expression with clinicopathological variables . survival curves were constructed using the kaplan - meier method and the differences between the curves were compared by the log - rank test . a cox proportional - hazards regression model was developed to evaluate the association between the potential prognostic marker and patients ' survival . cox regression analysis was conducted at both univariate and multivariate levels . a p value less than 0.05 was considered the limit of statistical significance . rcas1 was abundantly expressed in the gastric adenocarcinoma cases examined , presenting both a cytoplasmic and membraneous pattern of staining ( figure 1 ) . all cases were found positive for rcas1 , while the mean percentage rcas1 expression value was 64.5% . the intensity of rcas1 immunostaining was classified as mild in 4 ( 7.4% ) , moderate in 19 ( 35.2% ) , and intense in 21 ( 57.4% ) out of 54 gastric adenocarcinoma cases . by applying the immunohistochemical score , 25 ( 46.3% ) cases were found to present low rcas1 expression , and the remaining 29 ( 53.7% ) cases showed high expression . weak cytoplasmic rcas1 expression was also noted in less than 5% in normal ( nonmalignant ) gastric tissues . in cross - tables , high rcas1 expression was significantly more frequently observed in gastric adenocarcinoma cases with advanced histopathological stage and presence of organ metastasis ( table 1 , p = 0.0327 and p = 0.0084 , resp . ) . rcas1 expression did not show statistical significant associations or trends of correlation with the other clinicopathological variables examined ( table 1 , p > 0.05 ) . univariate analysis was performed to assess the strength of the association of each clinicopathological parameter and rcas1 expression ( low versus high ) with overall patients ' survival . patients ' age , histopathological type , grade of differentiation , disease stage , tumor size , presence of lymph node and organ metastasis , as well as rcas1 expression were identified as significant prognostic factors of overall patients ' survival ( table 2 , p = 0.0031 , p = 0.0003 , p < 0.001 , p = 0.001 , 0.023 , 0.0021 , 0.09 , and p = 0.021 , resp . ) . kaplan - meier survival curves indicated that patients with high rcas1 expressing tumors had significantly shorter survival times compared to those with low rcas1 expression ( figure 2(a ) , log - rank test , p = 0.016 ) . the mean survival time in patients presenting high rcas1 expression was 33.88 months ( 23.19 months ) , whereas in those presenting low rcas1 expression was 23.32 months ( 17.73 months ) . in multivariate analysis , tumor histopathological grade of differentiation , stage , and rcas1 expression proved to be independent prognostic factors of overall patients ' survival ( table 3 , p = 0.003 , p = 0.020 and p = 0.008 , resp . ) . statistical analysis for rcas1 expression was further performed in each gastric adenocarcinoma histopathological type , separately . in diffuse - type gastric adenocarcinoma , rcas1 expression was significantly associated with the presence of organ metastasis ( p = 0.025 ) and borderlined with histopathological stage ( p = 0.078 ) . kaplan - meier survival curves indicated that diffuse - type gastric adenocarcinoma patients with high rcas1 expression presented significantly shorter survival times compared to those with low rcas1 expression ( figure 2(b ) , log - rank test , p = 0.033 ) . histopathological stage and rcas1 expression were identified as significant predicting factors for patients prognosis in multivariate analysis ( cox regression analysis , p = 0.018 and p = 0.048 , resp . ) . in intestinal - type gastric adenocarcinoma , intestinal - type gastric adenocarcinoma patients presenting high rcas1 expression levels showed shorter survival times compared to those with low levels at a no significant level , though ( figure 2(c ) , log - rank test , p = 0.225 ) . rcas1 has been documented to be overexpressed in various tumors , affecting many aspects of cancer biology , such as differentiation , proliferation , invasion , and angiogenesis [ 13 , 14 , 23 ] . elevated rcas1 expression has been associated with the malignant state of several tissue types and may play crucial role in tumor progression by enabling cancer cells to evade immune surveillance [ 13 , 14 , 23 ] . the expression of rcas1 in tumors of the gastrointestinal tract has already been documented ; however , its associations with clinicopathological parameters and patients ' prognosis in gastric neoplasia remains scarce . in the present study moreover , 29 ( 53.7% ) out of the 54 gastric cancer cases showed high rcas1 expression . notably , current substantial evidence supported that the fact rcas1 may be considered as a promising target for future ( gene ) therapeutic approaches [ 13 , 14 , 23 ] . in this aspect , the increased frequency of high rcas1 positivity in the examined gastric adenocarcinoma cases reinforces the therapeutic utility of this receptor in gastric cancer chemoprevention and treatment . importantly , the recent evidence that rcas1 sirna suppressed rcas1 mrna and protein expression and delayed tumor growth , in vivo , has already unfolded new perspectives for the development of future rcas1 receptor target inhibitors . antiestrogens may also employ rcas1 as a molecular switch to activate immune cytotoxicity against tumor cells . the present study further showed that rcas1 expression was significantly elevated in gastric cancer patients with advanced histopathological stage and presence of organ metastasis . elevated rcas1 expression was also significantly associated with poor patients ' prognosis in both univariate and multivariate analysis . these findings are in accordance with previous substantial evidence in carcinomas of other anatomic sites of the gastrointestinal tract , such as oral , esophageal and colorectal carcinomas [ 2633 ] . concerning gastric cancer , nakamura et al . documented that rcas1 staining pattern ( diffuse or not ) correlated with tumor size , depth of tumour invasion , histological type , and lymph node metastasis . however , rcas1 positivity was not associated with the clinicopathological variables examined , while no data concerning the patients ' survival was reported . in a study by fukuda et al , which was performed on 129 t3 gastric carcinoma cases , rcas1 expression was also significantly correlated with histological type and lymph node metastasis , as well as patients ' prognosis . in another study applying a semiquantitative rt - pcr analysis , the perinuclear pattern of rcas1 expression was more frequently observed in well differentiated gastric adenocarcinoma compared to moderate ones . the diffuse pattern of rcas1 expression was more frequently recognized in gastric carcinomas which invaded beyond the submucosa compared to intramucosal carcinomas . in contrast to the previous studies , we did not find any significant association with the presence of lymph node metastasis and the grade of differentiation ; however , diffuse type gastric cancer cases showed an increased frequency of enhanced rcas1 expression compared to intestinal ones without , though , reaching statistical significance . currently , substantial evidence has suggested that rcas1 expression upregulation may play a crucial role in tumor progression by enabling cancer cells to evade immune surveillance [ 3 , 34 ] . interestingly , rcas1 induced apoptosis in both cultured human lymphoma cell lines and normal peripheral lymphocytes , which express rcas1 receptor . this apoptotic effect was supported to be mediated through induction of genes or caspase molecules , which strongly abrogated rcas1-induced apoptosis , in vitro . moreover , rcas1 facilitated tumor cell invasion of connective tissue in uterine cervical cancer via the enhancement of invasive potency by induction of stromal tissue remodeling , as well as through evasion of antitumor immune surveillance by an apoptotic counterattack mechanism against lymphocytes . rcas1 may also modulate surface expression of tumor - associated o - linked glycan structures , which are considered to participate in cell adhesion , invasion , and metastasis of cancer cells . taking into account the previous considerations , further studies should be performed in order to delineate whether rcas1 positive tumor cells may induce apoptosis to their surrounding tumor infiltrating lymphocytes . as rcas1 is easily detectable in biofluids [ 36 , 37 ] further research effort should also be orientated to the determination of rcas1 levels in serum or saliva during and after the treatment of gastric cancer patients with the aim of recognizing patients ' recurrence . rcas1 expression was associated with clinicopathological parameters crucial for patients ' management and prognosis . of even more clinical significance are the data supporting the contribution of rcas1 in the pathophysiological aspects of the disease that affect patients ' survival . these findings may suggest an important potential role of rcas1 in the biological mechanisms underlying the carcinogenic evolution of gastric adenocarcinoma . further research conducted on larger cohorts and focused on each histopathological type separately that additionally concerned more sensitive techniques is strongly recommended in order to assess whether rcas1 may be considered as a useful biomarker in gastric adenocarcinoma , evaluating also its therapeutic utility in gastric cancer chemoprevention and treatment .

background . the receptor - binding cancer antigen expressed on siso cells ( rcas1 ) is a human tumor - associated antigen that has been considered to play a crucial role in tumor progression by enabling cancer cells to evade immune surveillance . the present study aimed to evaluate the clinical significance of the rcas1 expression in gastric adenocarcinoma . material and methods . rcas1 protein expression was assessed immunohistochemically on 54 gastric adenocarcinoma tissue samples and was analyzed in relation to clinicopathological parameters , tumor proliferative capacity , and patients ' survival . results . enhanced rcas1 expression levels were significantly associated with advanced histopathological stage and presence of organ metastasis ( p = 0.0084 and p = 0.0327 ) . gastric cancer patients with elevated rcas1 expression levels showed significantly shorter survival times compared to those with low rcas1 expression ( log - rank test , p = 0.0168 ) . in multivariate analysis , histopathological stage and grade of differentiation as well as the rcas1 expression were identified as independent prognostic factors ( cox regression analysis , p = 0.0204 , p = 0.0035 , and p = 0.0081 ) . conclusions . our data support the evidence that rcas1 upregulation may contribute to gastric malignant progression , representing a useful biomarker to predict the biological behaviour and prognosis in gastric neoplasia .

with the growth of the internet use over the last two decades , there has been an increase in its usages as well as in the frequency of experienced dysfunctions related to its overuse . users report loss of control over their internet use , social problems as well as school and/or occupational difficulties . public health concerns are emerging concerning the propensity of compulsive internet use developing into pathological behaviors . about 20% and 33% of internet users engage in some form of online sexual activity . nearly 80% of online gamers are losing out at least one element of their lives , such as sleep , work , education , socializing with friends , family , and interaction with a partner . the younger the players , the longer the time they dedicated to playing online games , leading to further functional impairment in their lifestyle . poor coping and cognitive expectations also mediate the development of the excessive use of internet if other risk factors are present such as depression , social anxiety , low self - esteem , low self - efficacy , and high stress . depression , social phobia , hostility , and symptoms of adhd are seen as comorbid condition to problematic internet use . individuals with social anxiety reported a greater feeling of comfort and self - disclosure when socializing online compared to face - to - face communication . about 8% of the pathological users used the internet to meet new people for emotional support and to play interactive games . about 9% of the clinical subjects ( n = 300 ) have problematic usage of social networking sites . in previous studies conducted in the indian context has shown problematic to the addictive use of technology . the majority of the subjects had psychological distress as the comorbid condition . users were also using information technology to manage their psychological distress , to avoid a stressful situation , and way of managing boredom . there is a dearth of information about the pattern of technology use among the psychiatric population as well as its relationship with other sociodemographic variables . survey method was used to recruit 75 subjects ( male / female ) from the in - patient and out - patient psychiatric setting of national institute of mental health and neurosciences , bengaluru , karnataka with inclusion criteria of age range of 16 years and above , using internet for the minimum duration of 1 year and ability to read and write english . subjects with active psychopathology , illiterate , and unwillingness to participate were excluded from the study . background data sheet developed by the investigator to record sociodemographic details which covers age , sex , socioeconomic status , education , occupation religion , marital status and type of family , details of the psychiatric illness ( as per file diagnosis as per international classification of diseases-10 [ icd-10 ] or diagnostic and statistical manual of mental disorders criteria ) such as duration of illness , nature and course of illness , treatment taken , and premorbid personality traits . information related to technology use , the age at which individual starts using it , type of information technology used , reason to start using information technology , frequency of use , sites accessed , currently accessed sites , individual / group activities , duration of use , having smart phone with internet , availability at home , purpose of using information technology , situation associated with the use of information technology , any history of attempt to reduce the usage of information technology , perception about the usage , relationship of coping ( to manage boredom , emotional state etc.)/psychiatric condition with technology use as well as for seeking health information , type of activity ; impact of technology use on one 's life , care giver perspective and need for change . internet addiction impairment index is a twenty items questionnaire based on 5-point likert scale to assess addiction to internet . internet addiction impairment index can be utilizing to help classify the behavior regarding mild - moderate and severe impairment . the scale covering the degree to which their internet use affecting their daily routine , social life productivity , sleeping pattern , and feelings video game use patterns , to assess individuals video game use pattern in 9-item scale with two self - reported assessment of video game using pattern , and the emotional distress associated with it . pornography addiction screening tool is a twenty items questionnaire based on 5-point likert scale to assess addiction to pornography and online sexual behavior . screening for mobile phone use evolved screening questions developed for icmr funded behavioral addiction project will be used . subjects were taken from the in - patient / out - patient psychiatric setting of nimhans bengaluru , karnataka . prior consent was obtained from the concerned treating team as well as from the user . the process and objectives of the study were explained to the patients and informed consent was sought . the sociodemographic information were filled as per the information given by the patient and care givers as well as from the case file . the internet addiction questionnaire , video game use pattern questionnaire , facebook intensity questionnaire , pornography addiction test , and screening questionnaire for mobile phone addiction were administered in individual setting . the data were coded for the computer analysis and statistical package for social science 16.0 version ( 2008 ) was used to carry out the analysis of the quantitative data . descriptive statistics such as mean , standard deviation percentage , and frequencies were used to analyze the demographic data as well as the details of psychiatric condition . pearson 's chi - square test was computed to examine the significance of the relation among the variables . all the figures have been rounded off to two decimal places and for the level of significance probability level of 0.05 and 0.01 are used . survey method was used to recruit 75 subjects ( male / female ) from the in - patient and out - patient psychiatric setting of national institute of mental health and neurosciences , bengaluru , karnataka with inclusion criteria of age range of 16 years and above , using internet for the minimum duration of 1 year and ability to read and write english . subjects with active psychopathology , illiterate , and unwillingness to participate were excluded from the study . background data sheet developed by the investigator to record sociodemographic details which covers age , sex , socioeconomic status , education , occupation religion , marital status and type of family , details of the psychiatric illness ( as per file diagnosis as per international classification of diseases-10 [ icd-10 ] or diagnostic and statistical manual of mental disorders criteria ) such as duration of illness , nature and course of illness , treatment taken , and premorbid personality traits . information related to technology use , the age at which individual starts using it , type of information technology used , reason to start using information technology , frequency of use , sites accessed , currently accessed sites , individual / group activities , duration of use , having smart phone with internet , availability at home , purpose of using information technology , situation associated with the use of information technology , any history of attempt to reduce the usage of information technology , perception about the usage , relationship of coping ( to manage boredom , emotional state etc.)/psychiatric condition with technology use as well as for seeking health information , type of activity ; impact of technology use on one 's life , care giver perspective and need for change . internet addiction impairment index is a twenty items questionnaire based on 5-point likert scale to assess addiction to internet . internet addiction impairment index can be utilizing to help classify the behavior regarding mild - moderate and severe impairment . the scale covering the degree to which their internet use affecting their daily routine , social life productivity , sleeping pattern , and feelings video game use patterns , to assess individuals video game use pattern in 9-item scale with two self - reported assessment of video game using pattern , and the emotional distress associated with it . pornography addiction screening tool is a twenty items questionnaire based on 5-point likert scale to assess addiction to pornography and online sexual behavior . screening for mobile phone use evolved screening questions developed for icmr funded behavioral addiction project will be used . subjects were taken from the in - patient / out - patient psychiatric setting of nimhans bengaluru , karnataka . prior consent was obtained from the concerned treating team as well as from the user . the process and objectives of the study were explained to the patients and informed consent was sought . the sociodemographic information were filled as per the information given by the patient and care givers as well as from the case file . the internet addiction questionnaire , video game use pattern questionnaire , facebook intensity questionnaire , pornography addiction test , and screening questionnaire for mobile phone addiction were administered in individual setting . the data were coded for the computer analysis and statistical package for social science 16.0 version ( 2008 ) was used to carry out the analysis of the quantitative data . descriptive statistics such as mean , standard deviation percentage , and frequencies were used to analyze the demographic data as well as the details of psychiatric condition . pearson 's chi - square test was computed to examine the significance of the relation among the variables . all the figures have been rounded off to two decimal places and for the level of significance probability level of 0.05 and 0.01 are used . 17 were married ( 22.67% ) , 57 were unmarried ( 76% ) , and 1 was divorced ( 1.33% ) . 36% were from the rural area and the 64% were from the urban area [ table 1 ] . sociodemographic information of the sample table 2 shows the diagnosis of the sample population and its frequency , 32 different diagnoses in different frequencies were taken . frequencies and percentages of subjects with psychiatric diagnosis according to international classification of diseases-10 ( f - code ) table 3 indicates the presence of addiction for mobile phone ( 18.67% ) , internet addiction ( 16% ) , pornography ( 46.67% ) , and video games ( 14.67% ) . pattern of information technology addiction among sample table 4 shows the duration of illness of the sample ( n = 75 ) , varies from 6 months to 21 years , and the mean is 6.4 years with the standard deviation of 4 . pattern of duration of psychiatric illness and premorbid personality of sample table 5 shows that 58.7% of individuals in the overall sample reported that they were spending more time with information technology to 14.7% were using to avoid any negative emotions , 2.7% ( 2 people ) were using to cope with the situations and the 24% of the total sample spending time for other purposes like to get general information or as the part of career and academics . use of information technology to avoid negative emotions / as a method of coping was more among users who had a 5 h or more usage per day . relationship between average time using for the internet per day and the situations associated with the usage of internet table 6 shows that disturbance of sleep was ( delay in initiation of sleep ) more in moderate to severe category of use . relationship between internet addiction and sleep ( delay in the initiation of sleep ) table 7 shows that age had negative correlation with the duration of the illness , the average time spending on internet , internet addiction , mobile addiction , video game use , and pornography addiction . average time spending per day on the internet showing a positive correlation with the mobile phone , internet , videogame , and pornography addiction . mobile phone addiction had significant positive correlation with internet , video game use , and pornography addiction . this study indicates the trend toward the presence of addiction to the mobile phone ( 18.67% ) , internet addiction ( 16% ) , pornography ( 46.67% ) , and video games ( 14.67% ) among subjects seeking treatment for psychiatry problems [ table 3 ] . age has a negative correlation with internet addiction , video game addiction mobile addiction , and pornography . the mean age of the sample was 26.67 with the standard deviation of 6.5 [ tables 1 and 7 ] . duration of illness of the sample ( n = 75 ) , varies from 6 months to 21 years , and the mean is 6.4 years with the standard deviation of 4 . 49.33% had personality characterized by difficulty in adjustment and personality traits [ table 4 ] . use of information technology was seen to avoid negative emotions / as a method of coping was more among users who had a 5 h or more usage per day [ table 5 ] . moderate to the severe use of information technology was associated with a delay in initiation of sleep [ table 6 ] . age had negative correlation with the duration of the illness , the average time spending on internet , internet addiction , mobile addiction , video game use , and pornography addiction . average time spending per day on the internet showing a positive correlation with the mobile phone , internet , videogame , and pornography addiction ( vii ) . individuals belonging to the age group of 2029 used the internet more , while internet addiction scores of the individuals belonging to the group of 19 and below was higher than other groups and that this situation varied according to gender . problematic internet use showed the correlation of 75% with depression ; 57% with anxiety , 100% with symptoms of adhd ; 60% with obsessive - compulsive symptoms and 66% with hostility / aggression . the adolescents who play more than 1 h of console or internet video games may have more or more intense symptoms of adhd or inattention than those who do not . people with low self - esteem , self - efficacy , and vulnerability to stress are more prone to have a general internet addiction . sleep deprivation seems to be one of the major problematic effect of internet addiction and late night logins . the present work documents the presence of information technology addiction among subjects with psychiatric problems . addiction to internet and pornography is also associated with delay in the initiation of sleep . although the obtained prevalence is low in comparison to international prevalence , it can be addressed in a large sample study . the present communication gave trend toward association of age / average time spent per day with addiction to information technology ; use of information technology as a coping method . the present work has implications in term of screening the technology addiction as comorbid condition among the psychiatric population . the future work can focus on exploring the psychosocial correlates among subjects with psychological problems , caregiver issues related to the handling of addictive use of information technology as well as evolving the intervention for the promotion of the healthy use of technology .

background : technology usage has seen an increase among users . the usage varies from social , personal , and psychological reasons . users are frequently using to overcome mood states as well as to manage the other psychological states . this work is going to explore the information technology use among subjects with a psychiatric disorder.materials and methods : a total of 75 subjects were assessed using background data sheet , internet addiction impairment index , video game use pattern , pornography addiction screening tool and screening for mobile phone use , from in - patient and out - patient setting of tertiary mental health setting.results:it showed the presence of addiction to mobile , internet , video game , and pornography . age was found to be negatively correlated with this addiction . average usage time had been associated with management of mood states . the addiction to information technology had been associated with a delay in initiation of sleep.conclusion:this work has implication for screening technology addiction among subjects seeking treatment for psychological problems and motivate them to develop the healthy use of technology .

for a long time , plants represented one of the most important therapies for different diseases . nowadays , the popular use of plants as a way of treatment is still very important for human beings . pterospartum tridentatum ( l. ) willk , gomphrena globosa l. , and cymbopogon citratus ( dc . ) stapf are examples of those plants that are widely used in folk medicine , mostly as infusions . p. tridentatum ( family : fabaceae ) is a european endemic species and its flowers infusion is used against liver , bladder , kidney , and rheumatism problems ; it is also used for high blood pressure , cough , kidney stones , diabetes , and bronchitis [ 1 , 2 ] . g. globosa ( family : amaranthaceae ) is native from panama and guatemala and its aqueous extract of its purple inflorescences is good to treat bronchial asthma , acute and chronic bronchitis , and whooping cough ; the infusion of the flowers is used to treat oliguria and indigestion , also as expectorant and pertussis [ 3 , 4 ] . c. citratus ( family : poaceae ) is native from the southwest asia and its aqueous extract ( i.e. , in the form of infusion ) is used for the treatment of several inflammation - based pathologies , in digestive disorders , diabetes , nervous disorders , and fever [ 57 ] . according to novais , the infusion is used as gastric analgesic , intestinal anti - inflammatory , and renal antispasmodic and for gall - bladder ailments , sea - sickness , and bladder ailments . there are several reports about the biological activity of the mentioned plants , especially concerning antioxidant activity [ 5 , 8 ] , that have been related to their phenolic composition [ 3 , 9 ] , but studies regarding the composition on primary metabolites and nutrients are scarce . moreover , many of the commercially available samples are wild and require chemical characterization . besides secondary metabolites / nonnutrients such as phenolic compounds , medicinal plants contain primary metabolites and nutrients ( e.g. , as sugars , organic acids , and tocopherols ) that need to be profiled and quantified . mono- and oligosaccharides , with low molecular weight , and their derivatives such as sugar alcohols display a major role in the structure and function of all living cells . organic acids are involved in several biochemical pathways , including energy production and formation of precursors for amino - acid biosynthesis . vitamin e ( including tocopherols ) is known to be an essential micronutrient for maintaining the health and wellbeing of humans and other animals . several studies suggest that the use of vitamin e may contribute to help lowering the risks of specific chronic and degenerative diseases such as alzheimer 's disease , some types of cancer , cataracts , and ischemic heart disease . all these molecules can be determined by high performance liquid chromatography ( hplc ) coupled to different detectors according to their chemical properties , namely , fluorescence for tocopherols , refraction for sugars , and uv absorption for organic acids . therefore , the objective of the present paper was to characterize tocopherols , sugars , and organic acids in three medicinal plants ( p. tridentatum , g. globosa , and c. citratus ) , widely consumed as infusions . plant material of pterospartum tridentatum ( l. ) willk , gomphrena globosa l. , and cymbopogon citratus ( dc . ) stapf was purchased from ervital , a portuguese company in castro daire ( portugal ) . this company , settled in a high diverse mountain region ( montemuro , the natura 2000 site ) , markets several certified plant materials with different origin , such as sustainable wild harvesting of spontaneous local species and organic farming of exogenous species . pterospartum tridentatum flowers were wildly gathered in spring 2012 ( respecting plant phenology and abundance ) and the other studied species were grown , also in 2012 , with organic farming methods . harvested plants were processed using in - storage and low temperature drying methods ( solar heated air , average daily temperature around 3032c in shade conditions , and controlled relative humidity ) . samples for analysis were prepared from dried plant materials provided by the company , and botanical identification was confirmed by ana maria carvalho , responsible for the medicinal plant collection of the herbarium of the escola superior agrria ( bresa ) , of the polytechnic institute of bragana ( trs - os - montes , portugal ) . hplc - grade acetonitrile , ethyl acetate , and n - hexane were purchased from fisher scientific ( lisbon , portugal ) . l - ascorbic acid , tocopherol , sugar , and organic acid standards were purchased from sigma ( st . louis , mo , usa ) . racemic tocol ( 50 mg / ml ) was purchased from matreya ( pleasant gap , pa , usa ) . water was treated in milli - q water purification system ( tgi pure water systems , greenville , sc , usa ) . the equipment consisted of an integrated system with a pump ( knauer , smartline system 1000 , berlin , germany ) , degasser system ( smartline manager 5000 ) , autosampler ( jasco as-2057 , easton , md , usa ) , and a fluorescence detector ( jasco fp-2020 ) programmed for excitation at 290 nm and emission at 330 nm . the chromatographic separation was achieved with a polyamide ii ( 250 mm 4.6 mm i.d . ) normal - phase column from ymc waters ( dinslaken , germany ) operating at 30c ( 7971 r grace oven ) . the mobile phase used was a mixture of n - hexane and ethyl acetate ( 70 : 30 , v / v ) at a flow rate of 1 ml / min , and the injection volume was 20 l . the compounds were identified by chromatographic comparisons with authentic standards . quantification was based on calibration curves obtained from commercial standards of each compound using the internal standard ( is ) methodology ; racemic tocol was used as is . free sugars were determined by high performance liquid chromatography coupled to a refraction index detector ( hplc - ri ) , after an extraction procedure previously described . analysis was performed by hplc ( equipment described above ) using an ri detector ( knauer smartline 2300 , berlin , germany ) . the chromatographic separation was achieved with a eurospher 100 - 5 nh2 column ( 4.6 250 mm , 5 mm , knauer , berlin , germany ) operating at 30c . the mobile phase was acetonitrile / deionized water , 70 : 30 ( v / v ) at a flow rate of 1 ml / min . the analysis was performed using a shimadzu 20a series uflc ( shimadzu corporation , kyoto , japan ) . separation was achieved on a sphereclone ( phenomenex , torrance , ca , usa ) reverse phase c18 column ( 5 m , 250 mm 4.6 mm i.d . ) the elution was performed with sulfuric acid ( 3.6 mm ) using a flow rate of 0.8 ml / min . detection was carried out in a pda ( photodiode array detector ) , using 215 and 245 nm ( for ascorbic acid ) as preferred wavelengths . the organic acids found were quantified by comparison of the area of their peaks recorded at 215 nm with calibration curves obtained from commercial standards of each compound . all the assays were carried out in triplicate , and the results are expressed as mean values and standard deviation ( sd ) . the results were analyzed using one - way analysis of variance ( anova ) followed by tukey 's hsd test with = 0.05 . the chemical composition of the three plant species in tocopherols , free sugars , and organic acids is presented in table 1 . c. citratus gave the highest - and total tocopherols content but did not present -tocopherol that was found in the other two species ( e.g. , figure 1(a ) ) . tocopherols are lipid - soluble antioxidants , being -tocopherol the most active isoform , due to its role in lipid peroxidation inhibition . these molecules are widely used as functional ingredients in food , pharmaceutical , and cosmetic preparations . as far as we know this is the first report on tocopherols composition of c. citratus and g. globosa ; otherwise , the values obtained for p. tridentatum were similar to the ones described by the authors for a wild traditionally shade - dried sample ( 8.8 g / g dw ) . furthermore , tocopherols have also been reported in other species of the fabaceae family , such as cicer arietinum , lathyrus sativus , cytisus multiflorus , cytisus scoparius , and cytisus striatus ; and the amounts found ranged between 6.3 and 23.1 mg/100 g of dw , which is in the range of the sample studied herein , detecting all the isoforms . all isoforms of tocopherols have also been reported in poaceae and amaranthaceae families , but the quantities can not be compared due to the units in which they are expressed . regarding free sugars , p. tridentatum showed the highest levels of fructose and total sugars ; nevertheless , the values obtained were much higher than the concentrations found in a wild sample previously studied ( 0.3 and 49.6 g/100 g dw for fructose and total sugars , resp . ) . this could be due to different growth conditions of the plants ( e.g. , variability of weather and soil characteristics are major factors affecting plant development ) and to different drying processes applied , which influence moisture content and plant material quality . simulated consumers ' traditional conditions of use ( shade - drying , plant material being stored in a dark , dry place and at room temperature for 30 days ) ; dried plant material used for analysis in this study was processed in five days under the best conditions of shade , daily temperature , and relative humidity as well as airflow rate . furthermore , the presence of sugars has also been reported in the poaceae family , namely , sucrose , glucose , fructose , trehalose , and raffinose , but the amounts obtained can not be compared to the ones studied herein . some species belonging to the fabaceae family ( mentioned above ) [ 8 , 18 ] have also shown the presence of sugars , mainly fructose , glucose , sucrose , and trehalose , although some species revealed the presence of other sugars . the amounts found ranged between 2.56 and 18.67 g/100 g of dw ; these results are much lower than the one present in this study . it should be highlighted that fructose can display antioxidant properties due to its reducing capacity . furthermore , sugars are one of the molecules present in plant infusions that contribute to their energetic value . c. citratus gave the highest levels of glucose and sucrose ( table 1 and figure 1(b ) ) . no reports were found considering sugars composition in the mentioned species or in g. globosa . concerning organic acids , g. globosa was the sample with the highest concentration of these compounds , mainly malic and oxalic acids ( table 1 and figure 1(c ) ) . citric and succinic acids were found in higher levels in p. tridentatum and c. citratus , respectively . the latter also presented ascorbic acid , a powerful antioxidant phytochemical [ 22 , 23 ] . besides their important role in the human metabolism , organic acids have other applications ; for example , citric acid is a crystal thickener in bones , succinic acid is known to help in diabetes treatment , and malic acid is reported to have a bactericidal effect . among the three analyzed species , only p. tridentatum was previously studied regarding organic acids composition ; despite the fact that similar total amount was found ( 8.1 mg / g dw ) , the profile then described was slightly different , reporting also the presence of quinic , succinic , and fumaric acids . as mentioned before , differences may be caused by distinct ecological conditions for plant development ( plant material provenance is quite different ) and also by different characteristics of the plant material used for analysis , as a consequence of the drying processes applied to each material . a five - day controlled in - storage process may produce a better quality plant material , in terms of color ( visually confirmed ) , texture , and moisture content , than the traditional shade - drying techniques . all the organic acids , with the exception of quinic acid , have been previously reported in some species ( mentioned above ) belonging to the fabaceae family [ 8 , 18 ] , although the quantities present have a large variation depending on the species . overall , c. citratus possessed the highest content of - and total tocopherols , glucose , sucrose , succinic , and ascorbic acids . otherwise , g. globosa showed the highest organic acids concentration , due to the highest content of oxalic and malic acids . this study is of great importance because it fills an existing void in relation to chemical characterization of these plants , more precisely its composition in sugars , organic acids , and tocopherols , that can be present in the consumed forms ( mostly infusions ) .

pterospartum tridentatum ( l. ) willk , gomphrena globosa l. , and cymbopogon citratus ( dc . ) stapf are medicinal plants that require a more detailed chemical characterization , given the importance of their consumption as infusions . therefore , the individual profiles in tocopherols , free sugars , and organic acids were obtained by high performance liquid chromatography ( hplc ) coupled to different detectors ( fluorescence , refraction index , and photodiode array , resp . ) . c. citratus revealed the highest content of - , and total tocopherols , glucose , sucrose , succinic , and ascorbic acids . p. tridentatum presented the highest fructose and total sugars content . otherwise , g. globosa showed the highest organic acids concentration . as far as we know , this is the first study reporting the mentioned chemical compounds in g. globosa and c. citratus .

the worldwide incidence of cervical cancer is 15.3 / 100,000 women per year ( 1 ) . cancer of the cervix uteri is the second most common cancer among women worldwide , with an estimated 529,409 new cases and 274,883 deaths in 2008 ( 1 ) . about 86% of the cases occur in developing countries , representing 13% of female cancers . the cause of this cancer is attributed to human papillomavirus in 99% of these cases ( 2 ) . worldwide , mortality rates of cervical cancer are substantially lower than incidence with a ratio of mortality to incidence to 52% ( 1 ) . the highest incidence rates are reported from eastern and western africa at 35.4% and 33.7% respectively . in the developing countries , there is however marked variation in the frequency of cervical cancer in the developing countries owing to the differences in screening programmes and the prevalence of risk factors ( 3 ) . the lowest reported incidence rates are from middle east and are among muslims and jews , compared to other religious groups ( 1 , 3 ) . in saudi arabia the most recent estimates from cancer registry 2007 report indicate that cervical cancer ranks as the 5 most frequent cancer among women in between 30 - 44 years of age representing an incidence of 3.1% ( 4 ) . data is not yet available on hpv burden in the general population of saudi arabia . the overall age - standardized incidence rate is 2.2/100,000 with highest incidence of 8.1% in age group of 60 - 64 years . the age - standardized mortality rate is 0.8 /100,000 in women population ( 1 ) . the highest incidence is reported in jouf region with 5.6% followed by hail region 3.5% , najran 3.2% , al baha 2.2 % and the lowest in jazan and mekkah with 2.2% ( 4 ) . there are no reported rates for other regions such as riyadh , eastern and northern region , madinah , qassim , asir , and tabuk ( 4 ) . cervical intraepithelial neoplasia incidence in saudi arabia is not registered because there are no well - developed screening programs nationwide . however recent studies from different regions of the kingdom show slight increase in the prevalence of epithelial cell abnormalities ( eca ) in pap smears ( ps ) ( 5 - 8 ) . a study from maternity and children 's hospital , saudi arabia reported that cervical cancer among saudi nations represents 33.5% of all gynecological malignancies ( 9 ) . the incidence and mortality of cervical cancer has declined dramatically ever since the introduction and wide spread utilization of ps screening test especially in countries with well - established cervical screening programs such as usa where it is considered the sixth most common cancer in women ( 10 ) . cytological evidence of all eca can be easily , safely and economically obtained by the preparation and examination of cytology smears ( 11 ) . identification of preventable , precursor lesions by cytology could prevent further progress of the disease by simple therapeutic procedures and continued surveillance ( 11 ) and as such emphasizes the need for early detection of cervical intraepithelial neoplasia . over the years , the original ps classification system has been revised in response to our growing knowledge of cervical cancer precursors and our understanding of the role of hpv cervical carcinogenesis ( 12 ) . standardization of ps reporting using the bethesda system ( rbs ) has unified various overlapping and confusing terminologies ( 12 ) and has overcome the consequences of inter - observer subjectivity in interpretation and application of these terminologies to some extent . the current study was conducted to explore the changing pattern of epithelial cell abnormalities ( eca ) detected in ps in females of the western region of saudi arabia at king abdulaziz university hospital , jeddah using bethesda system ( rbs ) . a retrospective study targeting 15805 pss performed during january 2000- october 2012 at king abdulaziz university hospital ( kauh ) jeddah was performed . the data was collected by conducting a computerized search through the cytopathology archives at kauh jeddah . this study included both saudi and non saudi females , since kauh is a teaching hospital of king abdulaziz university medical school . it is the only hospital at jeddah providing treatment to all nationalities thereby providing the unique opportunity of studying cross cultural variations and similarities .the data was filtered indicating the following essential parameters : date of undergoing ps , personal identity ( medical record number , age , sex etc ) and relevant clinical information ( indicated for routine or for gynecological symptoms).the study included all pss performed at kauh during the period of study . repeated smears from the same patients with insignificant cytological changes and carrying the same diagnosis and unsatisfactory pss were excluded from this study . the data were rechecked manually to avoid duplications . statistical package for the social sciences , version 15.0 ( spss inc . , chicago , il , usa ) program was used for analysis . descriptive and frequency statistics were obtained for the variables studies . the total number of pap smears examined ( tse ) after excluding unsatisfactory smears and the total number and percentages of abnormal pap smears ( taps ) were analyzed and correlated with the patient 's age . the procedures followed in the present study were approved by and were in accordance with the ethical standards of the hospital ethical committee on human experimentation and with the helsinki declaration of 1975 , as revised in 2000 . a smear was considered sufficient when it was appropriately labeled having adequate sampling of the endocervical transformation zone with optimal preservation of the cells . all sufficient cervical cytology smears were reviewed and reclassified according to the 2001 rbs of ps reporting ( 12 ) ( table 1 ) , into negative for intraepithelial lesions , positive for epithelial cell abnormalities ( eca ) and sub classified into specific categories . quality indicators such as inflammation , hemorrhage and reactive cellular changes associated with repair , squamous metaplasia , follicular cervicitis , atrophy and organisms were also identified . in cases of ambiguous diagnoses , the cytology slides were reviewed on a multi - headed microscope by a senior cytotechnologist and two pathologists to resolve the discrepancy . a total of 15805 cervical pss were retrieved and reported in the department of cytopathology at kauh , jeddah in the period from january 2000 to october 2012 . the study identified 15721 ( 99.46% ) sufficient smears and 84 ( 0.53% ) unsatisfactory smears ( table 1 ) . the latter cases were excluded from further analysis . of the 15721 cervical pss , 12797 ( 81.15% ) smears were reported as negative for eca while 2295 ( 14.52% ) were diagnosed as positive for eca . the smears positive for squamous and glandular cell abnormalities were further sub classified according to the 2001 rbs ( 12 ) as shown in table 2 . unsatisfactory smears were 0.53 % among other quality indicators and were withdrawn for further analysis as they were beyond the scope of this study . we compared our results to the past studies from our institution in table 3 and other recent studies from the saudi arabia , arab world and other international studies in table 4 . in comparison to the previous studies from our institution ( table 3 ) ( 7 , 13 ) there is a significant increase in the number of smears positive for eca from 4.7% in the study that was conducted in year 2006 to 17.3% in the study that was conducted in 2009 . in the present study squamous cell abnormalities have also increased from 3.5% in the year 2006 to 17.3% in 2009 to 11.42 % at the time of this study and glandular cell abnormalities from 1.10 in the year 2006 to 3.35% in 2009 to 3.1% at the time of the study . cervical intraepithelial neoplasia and invasive cervical carcinoma are less common in saudi arabia compared to the western countries ( 1 , 2 , 4 , 8 , 9 ) and early stages of cervical cancer are even less ( 4 - 9 ) . the original ps reporting system contained several overlapping terminologies and in order to present a more comprehensive system it was updated in 2001 ( 12 ) . the observations in the present study imply an overall increase in the number of abnormal ps with a relative and significant increase in squamous cell abnormalities especially abnormal squamous cells of undetermined significance ( asc - us ) . the published data of 2009 ( 13 ) showing a marked increase in eca , squamous and glandular abnormalities raised concerns and led the cytopathologists at our institution to strictly adhere to the criteria for each diagnostic category thereby addressing issues of over interpretation with obvious improvement . the current study , however still seems to revalidate and compliment the previous studies as regards the increase in number of eca . although the comparison of the previous studies are not valid due to overlap of data but it is a crude method to indicate that there is a continuous increase in the number of abnormal pss . the increase in the total number of pss however is explained by a number of reasons , such as the increasing awareness among the females in the western region of saudi arabia regarding the importance of cervical cancer screening through campaigns from cervical cancer scientific chair in jeddah area . if we assess studies from other parts of saudi arabia ( 5 , 7 , 8 , 14 , 15 ) ( table 4 ) using bs in diagnosis of pss , we notice a gradual increase in the incidence of eca especially in the squamous cell category is being reported . furthermore studies from the arab world have also reported a significant increased trend in asc - us and agus over the years ( table 4 ) ( 16 - 18 ) . abul el - all et al study from egypt reported eca of 7.8% , asc - us of 34.4% , low grade squamous intraepithelial lesion ( lgsil ) 41% , high grade squamous intraepithelial lesion ( hgsil ) 5.2% , squamous cell carcinoma ( scc ) of 0.5% and agus of 15.3% ( 18 ) . most of these studies indicate that there has been a definite increase in the prevalence of eca in the current years . it is not clear whether it is due to a high level of awareness of the targeted population within these studies or it is related to the trend of acquiring a more open life style .these issues need further investigation and assessment . however the lowest reported incidence of eca is from the eastern region , al khobar , wherein ascus detected was 0.48% , abnormal squamous cells - high grade ( acs - h ) was 0.06% , lsil was 0.19% , hsil was 0.37% , scc was 0.10% and agus was 0.25% ( 15 ) . other recent studies from asia report an eca between 5 - 10.2% ( 19 - 21 ) ( table 4 ) and although the asc - us category in these studies is within an acceptable range of 0.3 - 1 % but they show a higher incidence of lgsil and hgsil which could be partly explained by the variation in the population under consideration . also it could be partly due to subjective variation in application of asc - us criteria early in these lesions . there are 2 - 3 million asc - us , 1.25 million lgsil and 300,000 hsil in usa ( 22 ) . approximately three fourth of all cervical cancers in usa are squamous cell and the remaining are adenocarcinomas ( 3 ) . hpv 16 and 18 account for approximately 68% of squamous cell carcinomas and 83% of adenocarcinomas . this finding warrants the need for incorporating hpv - dna testing in the screening programme ( 3 , 23 ) . in most developed countries the current prevalence of cytological abnormality is between 5 - 10% ( 23 , 24 ) . hpv l1 capsid protein and hterc gene ( human telomerase rna component ) may serve as markers for the early diagnosis and prediction of cervical lesions . the increase in l1/hterc ratio reflects the progression of cervical lesions to a certain extent ( 24 ) . currently there is no organized cervical screening programme in saudi arabia ( 5 ) so information regarding the rate of ps testing in saudi arabia remains obscure . in the arab world a screening rate of 14.3% ( 25 ) is reported . in a study from usa the prevalence of hpv16 was 13.3% among asc - us , 23.6% among lsil and 60.7% among hsil ( 26 ) . in the developing countries , we , however face an important challenge to apply hpv - based technology ( 3 ) . hpv - dna testing has higher sensitivity but lower specificity than thin layer pap screening ( 3 ) . the age - specific incidence of cervical cancer peaks around the age of 40 years , which suggests that the specificity and efficacy of hpv screening is maximal when it is performed on women between 30 and 40 years of age ( 27 ) . virus - like particles ( vlps ) containing hpv structural proteins are being used as vaccines and can induce genotype specific virus neutralizing antibodies for preventing hpv infections ( 28 , 29 ) . however , hpv infection as the current concept of the etiology of cervical cancer lacks adequate validation in terms of effective screening especially in saudi arabia . no population based data is available to indicate the prevalence of hpv cervical infections or their age specific incidence in saudi arabia . in saudi population , co factors found contributory to cervical cancer are smoking and use of oral contraceptives ( 1 ) . a recent study emphasizes the need to include patients more than 45 years of age for pap screening ( 19 ) in developing countries suggesting that a negative history before the age of 50 years does not necessarily exclude the risk of having cervical cancer . the limitation of the current study is that the diagnosis of asc - us is much over the acceptable range although the asc - us / sil ratio of 2.3 is within the limit ( 30 , 31 ) . the increased trend at our institution is partly explained by the fact that the criterion for asc - us and lsil is quantitative and cytopathologists prefer to use the diagnosis of asc - us rather than lsil . the possible rationale behind this is that patients with asc - us diagnosis are treated for inflammation followed by a repeat ps within 6 months , while patients with lsil diagnosis are subjected to colposcopy and biopsy , especially in the absence of routine hpv testing in asc - us or sil diagnosis . there is evidence indicating increase in the number of abnormal pss over the last decade . it may not be a significant increase to call for nationwide mass cervical cancer screening program , which is a continuous process that requires allocation of huge resources that should be justifiable . it should , however , be obligatory for all women in the age of 35 - 55 to undergo pss and those with abnormal ec findings to have a test for hpv subtype . the data from all provinces should be evaluated thoroughly and if the results indicate considerable health problem with a definite increase in the incidence over successive years , proper documentation of such data should be enforced for more comprehensive analysis and decision on mass screening programme nationwide .

objective(s ) : in developing countries and worldwide cervical cancer is an important cause of female mortality . reports describing the frequency and pattern of abnormal pap smears in saudi arabia , using the revised bethesda system ( rbs ) are very few . the current study was conducted to explore the changing pattern of epithelial cell abnormalities ( eca ) detected in pap smears ( ps ) in females of the western region of saudi arabia at king abdulaziz university hospital , jeddah using the rbs.materials and methods : a retrospective study was designed to review all the pss from the archives of cytopathology department at king abdulaziz university hospital , starting from january 2000 to october 2012 using rbs . cytological aspects of pss were reviewed with age distribution.results : of the 15805 ps , 84 ( 0.53% ) unsatisfactory smears were excluded . there were 2295 cases ( 14.52% ) with eca . in the abnormal squamous cell category the distribution of lesions was as follows : atypical squamous cells of indeterminate significance ( asc - us ) were 7.1% ; atypical squamous cells , can not exclude high squamous intraepithelial lesion ( asc - h ) were 1.08% ; low grade squamous intraepithelial lesion ( lsil ) including human papillomavirus was 2.2% , high grade squamous intraepithelial lesion ( hsil ) was 0.8% and high grade squamous intraepithelial lesion with suspicious invasion was 0.06% smears . the mean age ( ma ) incidence was 39,43,45,46 and 45 years respectively.conclusion : the percentage of abnormal ps is increasing ( 14.52% ) over the last decade . this increase is evident by different studies conducted across saudi arabia . under present circumstances the need for mass screening .

the application of osseointegration , introduced by branemark , was initially limited to edentulous patients but is now widely used in both edentulous patients and patients with missing individual teeth . replacement of missing teeth by dental implant has become an integral part of modern dental health care1 . there has been much technological development to enhance the survival rates of implants , particularly with regard to implant surface treatment . albrektsson et al.2 reported that , when an implant had a rough surface rather than a machined surface , more rapid bone growth and superior physical adhesion were observed , while osteoblasts were better attached to rough surface implants , thus influencing the maturation , differentiation , and bone - implant interface of attached cells . wennerberg et al.3 reported that a rough surface implant had larger bone - implant contact ( bic ) and higher removal torque compared with a machined surface implant . surface treatment methods , by which an implant surface is made rough to increase the bone - implant interface , include acid etching4 , blasting5 , hydroxyapatite coating6 , titanium plasma spray7 and sandblasting with large grit , and acid etching ( sla)8 . buser et al.9 reported that the 10-year survival rate of implants with the sla surface was 98.8% , while the prevalence of peri - implantitis over 10 years was 1.8% . in the present study , the authors evaluated the five - year survival rate and the surrounding bony change of dental implants with the sla surface ( snucone dental implant , daegu , korea ) . the subjects in the study are 54 patients who have been followed - up to date , of the patients who underwent implant surgery at chungnam national university hospital ( daejeon , korea ) from may 1 , 2009 through april 30 , 2011 . medically compromised patients were sent them to physicians to undergo medical treatment prior to implant surgery . in all , 176 implant placements were performed . the implant surgeries were divided into first and the second surgeries and were carried out in two stages . the surgeries and prosthetic treatments were carried out by one skillful oral and maxillofacial surgeon . the first surgeries were performed according to general surgery principles and a protocol provided by the manufacturer . in the case of lateral window opening cases , horizontal and vertical incisions were made on the alveolar ridge and the lateral surface of the maxillary sinus , respectively , for exposure of the lateral surface of the maxillary sinus . in all lateral window opening cases , the bone graft material and the membrane were xenograft ( bio - oss ; geistlich pharma ag , wolhusen , switzerland ) and collagen membrane ( bio - gide ; geistlich pharma ag ) , respectively . after the first surgeries , antibiotics and analgesics were prescribed for all patients for seven to ten days . all the patients were instructed to not apply any stimuli to the surgical site until the removal of the stitches . they were also instructed to perform a 0.12% chlorhexidine mouth rinse three or four times a day . , the second surgery was performed an average of 2.5 months after the first surgery . in the maxilla , however , the second surgery was performed five months after the first . for lateral window opening cases , dental impressions were usually performed two weeks after the second surgery , and temporary prostheses were installed one week after the impressions were created . panoramic radiographs were collected before and immediately after the first surgery , after the second surgery , immediately after the final prosthesis setting , three months after the final prosthesis setting , six months after the final prosthesis setting , and every year after that . bone loss surrounding an implant was evaluated in accordance with the method suggested by romanos and nentwig10 . evaluation of bone loss was graded as follows : group 0 , no bone loss ; group m , minimal bone loss ( less than 2 mm at the crestal aspect of the implant ) ; group 1 , bone loss involving 1/4 of the implant length ; group 2 , progressive bone loss between 1/4 and 1/2 of the implant length . bone loss measurements were made on the mesial and distal sides of the fixture - abutment junction to the cortical bone . the values from the mesial and distal sides were averaged to be used in the evaluation . bone loss was evaluated by calculation of the rates to the known length of the fixture . the radiographs taken right after the final setting of the prosthetic appliance were compared with the follow - up radiographs for evaluation . as the number of implants placed was not large , no statistical analysis was performed . the age range of the men was 24 years to 70 years , with an average of 54.8 years . the age range of the women was 39 years to 75 years , with an average of 57.4 years . among men , those in their 50s were the most common , at 20 patients . among women , those in their 60s were the most common , at 6 patients . of the 176 implants , 106 were placed in the maxilla , while 70 were placed in the mandible ; 122 implants were performed in men and 54 in women.(table 1 ) fixtures 4.3 mm in diameter and 10 mm in length were used most often . the number of the fixtures used is shown in table 2 . in lateral window opening cases , 30 implant placements were performed in 18 cases in 16 men , and 14 implant placements in eight cases in eight women . an osteotome technique with a crestal approach was used for 14 implant placements in 10 men , while no women were treated with an osteotome technique . as for the kinds of prosthetic appliances , the number of the implants manufactured as a bridge was 156 , while the number of single prostheses was 20 . in a total of 54 patients the range of the follow - up periods was four years to six years , with an average of 4.9 years . the bone loss evaluated by the method suggested by romanos and nentwig10 is shown in table 3 . the numbers of implants included in group m were nine for molar teeth in the maxilla , three for molar teeth in a mandible , and two for premolar teeth in the maxilla.(fig . 1 ) of these , five teeth in molar areas in the maxilla and three teeth in molar areas in the mandible were single prostheses . the horizontal positions of the alveolar bone were maintained in a relatively stable state in the rest of the implants.(fig . 2 ) a total of six implants failed - four in the mandible and two in the maxilla ; all of these were single - implant cases . in one case , a fixture placed in the mandible of a male patient experienced fracture . in three cases , the patients were receiving chemotherapy , while two patients were suffering from diabetes or chronic renal failure . in all five cases , except for the patient with the fractured fixture , no abnormalities were found in the radiograph tests performed right before the implant failures occurred . all the failed implants belonged to group 0 , and no failed implants belonged to group m. the implant survival rate was 98.1% in the maxilla and 94.3% in the mandible , resulting in an overall survival of 96.6% . studies are continually being performed to improve the survival rate of implants and to shorten the healing period through development of bone graft material and improvement of surgical methods , implant design , and surface treatment methods . fast healing of peri - implant bone after implant placement and continuous and stable maintenance of osseointegration are important factors . several studies have shown that a rough surface implant has more merits than a machined surface implant211121314 . osteoblasts have greater binding affinity with a rough surface than with a machined surface , while a rough surface influences the maturation , differentiation , and bone - implant interface of attached cells . deposition of an inorganic component in bone tissues and the bone cell alkaline phosphatase activity have been found to be higher on a rough implant surface . the surface treatment method , which is widely used lately , uses either resorbable blasting media ( rbm ) or sla . the rbm method uses resorbable particles , such as calcium phosphate , in order to supplement the shortfall of blasted particles left on the implant surface5 . i m et al.17 reported higher bic in sla implants ( 82.7% ) than in rbm implants ( 78.3% ) 12 weeks after implant placement in adult mutts . elkhaweldi et al.18 , who compared the survival rate of rbm surface implants with that of sla surface implants , reported that the former was 95.2% and the latter 99.1% , with a higher survival rate observed in sla surface implants . they reported that sla surface implants produced better results in the posterior maxilla with poor bone . for long - term success of an implant , stable maintenance of the peri - implant bone is an important indicator . in this study , we evaluated the bone loss in accordance with the method suggested by romanos and nentwig10 . the implants included in group m were placed in areas experiencing high occlusal force , while they occurred between two and three years after the final placement of the prosthetic appliances , and many of them were in the maxilla . considering that most of the failed implants occurred within one year after placement , a somewhat longer follow - up period is thought to be needed . the reason for the higher number of failures in the maxilla is thought to be similar to the reason for the lower implant survival rate in the upper jaw than in the mandible1819 . after about one year , peri - implant bone had become stable , and little subsequent change was observed . the use of panoramic radiographs , which have lower resolution than periapical radiographs , might have resulted in a somewhat low accuracy ; however , it is believed that there were no major errors because we performed the measurements in consideration of the rates to the known length of the fixtures . in the external type of implant , where the abutment and the fixture are connected with a butt joint connection system , sealing was not secured between the abutment and the fixture , allowing the possibility of microleakage of bacteria . in addition , most of the stress caused by occlusal force is concentrated on the fixture top , causing marginal bone loss20 . therefore , in the external type of implant , bone loss of 1.5 mm or less , with exposure of one or two threads , one year after loading and bone loss of 0.2 mm of less per year after that have generally been accepted as normal21 . lazzara and porter22 introduced a platform switching concept and demonstrated the maintenance of healthy soft tissue around an implant , with little marginal bone loss . an internal type of implant was used in this study , where the abutment and the fixture were connected onto a slip joint connection system , similar to the platform switching concept , and where , it is believed , the fixture and the abutment were so closely connected that no microleakage occurred and healthy biologic width was maintained , maintaining the marginal bone . except for the one fractured implant , all five failed implants occurred in patients suffering from serious systemic diseases . there were no abnormalities found in the panoramic view examinations of these patients , and clinical examinations were performed right before the failures . unless demineralization of bone exceeds 30% or more , it is difficult to evaluate the bony lesion ; therefore , we used panorama radiographs , whose resolution is slightly lower than that of periapical radiographs . for this reason , we think radiographs , which are more accurate , are necessary in implant follow - up . there were no failed implants in the group m. the survival rate of sla - treated implants was 98.1% in the maxilla and 94.3% in the mandible , with an overall survival rate of 96.6% . within the limitations of this study , implants with the sla surface have a very superior survival rate . in particular , implants with the sla surface seem to be superior in areas of relatively poor bone such as the maxilla . it is necessary to evaluate marginal bone loss using more accurate radiographs , such as periapical radiographs or cone - beam computed tomography , throughout the treatment periods .

objectivesthe purpose of this study is to evaluate five - year radiographic follow - up results of the korean sandblasting with large grit , and acid etching ( sla)-treated implant system.materials and methodsthe subjects of the study are 54 patients who have been followed - up to date , of the patients who underwent implant surgery from may 1 , 2009 to april 30 , 2011 . in all , 176 implant placements were performed . radiographs were taken before the first surgery , immediately after the first and second surgeries , immediately and six months after the final prosthesis installation , and every year after that . bone loss was evaluated by the method suggested by romanos and nentwig.resultsa total of 176 implant placements were performed-122 in men and 54 in women . these patients have been followed - up for an average of 4.9 years . in terms of prosthetic appliances , there were 156 bridges and 20 single prostheses . nine implants installed in the maxillary molar area , three in the mandibular molar area and two in the maxillary premolar area were included in group m , with bone loss less than 2 mm at the crestal aspect of the implant . of these , eight implants were single prostheses . in all , six implants failed - four in the mandible and two in the maxilla . all of these failures occurred in single - implant cases . the implant survival rate was 98.1% on the maxilla and 94.3% on the mandible , with an overall survival of 96.6%.conclusionwithin the limitations of this study , implants with the sla surface have a very superior survival rate in relatively poor bone environments such as the maxilla .

the advent of next - generation sequencing ( ngs ) technologies has unveiled the entire picture of the cellular transcriptome , wherein various non - coding rna ( ncrna ) are widely expressed from a large part of the genome.1,2 the functional significance of ncrnas is particularly evident for the short 20- to 31-nucleotide ( nt ) ncrnas bound to the family of argonaute proteins.35 micrornas ( mirnas ) , the best - studied class of such rnas , are approximately 22 nt in length and posttranscriptionally regulate the expression of complementary target mrnas . while nearly 2,800 species of human mirnas are currently annotated in the mirbase database,6 computational analyses of massive rna - sequencing ( rna - seq ) data has kept increasing the number of mirna species.7,8 mirnas are estimated to regulate the expression of most protein - coding genes,9 and a wide variety of other short and long functional ncrna species coexist in the cellular transcriptome,10,11 exhibiting a tremendous impact of ncrna - orchestrated regulatory mechanisms on normal developmental and physiological processes and diseases.12,13 ngs studies have accelerated findings on not only novel ncrna repertoires but also well - characterized fundamental rna species with canonical functions , such as transfer rnas ( trnas ) , which stimulated us to revisit their significance and versatility . trnas are 70- to 90-nt ncrnas that fold into a cloverleaf secondary structure and l - shaped tertiary structure . since their discovery in the 1950s , trnas have been best known as fundamental adapter components of translational machinery , where they translate codons in mrna into amino acids in protein.14 the human genome encodes > 500 trna genes,15 along with numerous trna - lookalikes resembling nuclear and mitochondrial trnas.16,17 the multitude of trna genes in the genome , along with high stability,18 places trnas among the most abundant rna molecules in the cellular transcriptome . because of their abundance and well - defined role in translation , rna fragments derived from trnas , found in early ngs studies , were often disregarded as nonfunctional degradation products . however , recent biochemical and bioinformatic evidence has generated a previously unsuspected concept that trnas are not always end products but are further able to serve as a source for short ncrnas . in many organisms , specific trna - derived ncrnas are produced from mature trnas or their precursor transcripts , not as random degradation products , but as functional molecules involved in many biological processes beyond translation.1923 trna - derived ncrnas identified to date can be classified into two groups : trna halves and trna - derived fragments ( trfs).1923 trna halves are composed of 3035 nt fragments derived from either the 5- or the 3-part of mature trnas . trfs are shorter than trna halves , ranging from 13 to 32 nt in length and are currently subclassified into four subgroups : 5-trfs , 3-cca trfs , 3-u trfs , and 5-leader - exon trfs . the 5-trfs and 3-cca trfs correspond to the 5- and 3-parts of mature trnas containing processed mature 5- and 3-cca termini , respectively ( fig . the 3-u trfs are derived from the 3-leader sequence of precursor trnas ( pre - trnas ) that harbor 3-terminal uridine stretches , which result from transcriptional termination by rna polyme rase iii at thymidine tracts ( fig . the 5-leader - exon trfs include the 5-leader sequence of pre - trnas and the 5-part of mature trnas . although several excellent reviews have already described the repertoire , biogenesis , and functional significance of each class of the trna - derived ncrnas,1923 in this review , we particularly focus on the trfs bound to argonaute proteins and summarize their conceivable biogenesis factors and emerging roles in gene expression regulation as mirnas . in humans , the argonaute protein family has eight members : four of the ago clade ( ago14 ) , which are ubiquitously expressed in numerous tissues , and four of the piwi clade , whose expressions are restricted to the germline.35 all four ago members associate with mirnas to form effector complexes for their posttranscriptional regulatory roles , while only ago2 shows endonucleolytic cleavage activity of target mrnas . biochemical purification and identification of mirnas and their target mrnas have been accomplished by purifying ago proteins using immunoprecipitation ( ago - ip ) followed by investigation of the ago - bound rnas in the ip fraction.24,25 similar sizes of trfs with mirnas allowed researchers to reason that trfs may be ago - bound rna species . although early studies have already observed trna - derived sequences from ago - ip fraction , as described below , more recent reports have demonstrated an evident association of the trna - derived ncrnas with ago proteins . hereafter , we will use trf to denote a trf derived from the trna corresponding to the amino acid and anticodon sequences . the first focused detection of ago - bound trna - derived ncrnas in humans was reported in 2009.26 cole et al . identified numerous trfs in rna - seq data from hela cells.26 majority of the identified trfs were 5-trfs , and 5-trf , selected for the analyses , this result suggests the loading of the trf in both of the ago proteins , although 5-trf was also found in the fraction where ago proteins were absent and therefore it seems that not all trfs are loaded onto the ago proteins . detected 3-cca trfs and 3-u trfs in human embryonic kidney 293 ( hek293 ) cells by northern blots.27 interestingly , the identified trf was selectively loaded onto different ago proteins ; 3-u trf was found to be loaded onto ago3 and ago4 but not to ago1 and ago2.27 burroughs and ando et al . performed ago1 - 3 ips using thp-1 cells followed by ngs of the rna contents of the ip fractions , and identified several 5-trfs that were specifically loaded onto ago1 , but slightly or not at all onto ago2 and ago3.28,29 they also observed the presence of 3-cca trfs in their ago - bound rna libraries . li et al . identified numerous 5-trfs and 3-cca trfs in rna - seq libraries from bcp1 and hek293 cells and demonstrated that both of the selected trfs , 3-cca trf and 3-cca trf , were loaded onto ago2 but not onto ago1.30 maute et al . focused on 3-cca trf whose expression levels are regulated by differentiation stages in mature b cells.31 the trf was enriched in the ago4-ip fraction , followed by fractions from ago1- and ago3-ips , but less enriched in the ago2-ip fraction . analyzed many available short rna libraries and observed numerous 5-trfs , 3-cca trfs , and 3-u trfs , whose expression profiles varied in different cell lines and tissues.32 their analyses of hek293 ago - ip fractions obtained from photoactivatable - ribonucleoside - enhanced crosslinking and immunoprecipitation ( par - clip)33 suggested that 5-trfs and 3-cca trfs associate with ago1 , ago3 , and ago4 , but not with ago2,32 whereas 3-u trfs were absent in all four ago - ip fractions . positional analyses of crosslink - induced mutations in par - clip data indicated that trfs were loaded onto ago proteins in a manner similar to mirnas.32 taken together , it could be concluded that the attempts to connect trfs , at least partially , with the mirna pathway have been successful . as mirnas , some trf species of the three classes ( 5-trfs , 3-cca trfs , and 3-u trfs ) are the interacting partners of ago proteins . the presence of trfs in the ago - ip fraction has also been reported for many other organisms such as mouse,34 drosophila,35 and bombyx,36 as well as in plant.37 along the same lines , piwi proteins , the other argonaute family subclade that associates with piwi - interacting rnas ( pir - nas ) for germline development,35 have also been reported to interact with trna - derived ncrnas in various organisms such as human,38 marmoset,39 drosophila,40 trypanosoma,41 and tetrahymena.42,43 functional significance of the piwi - bound trfs in rna metabolism has been shown in tetrahymena.43 these findings suggest that trna - derived ncrnas are widely - conserved functional factors across phyla that interact with argonaute family proteins . asymmetric loading of trfs onto the different ago proteins could be due to coupling of trf biogenesis with ago loading mechanism and/or preferences of the ago proteins and other biogenesis factors for the particular structures of trfs or their precursors , namely mature trnas and pre - trnas . mature trnas are produced from pre - trnas , which undergo several maturation steps44 ( fig . pre - trnas are first transcribed by rna polymerase iii , and their 5-leader and 3-trailer sequences are removed by endonucleolytic cleavage of rnase p and rnase z , respectively . trinucleotides are then attached to the 3-termini by a cca - adding enzyme , and various different non - canonical base modifications are attached by modification enzymes . judging from the regions and sequences that the trfs originate from , both pre - trnas and mature trnas must serve as substrates for the production of trfs . in vertebrates , canonical mirna biogenesis ( fig . 1 ) starts from transcription of the mirna gene by rna polymerase ii to generate a primary transcript ( pri - mirna ) , which is subsequently cleaved by drosha , an endonuclease belonging to the rnase iii superfamily , forming a complex with dgcr8.35 the resultant 70-nt precursor mirna ( pre - mirna ) with a hairpin - shaped structure is further processed by dicer , another rnase iii endonuclease , leading to the production of rna duplex , one strand of which is then loaded onto ago proteins.35 considering the expression of trfs complexed with ago proteins , one may infer drosha and dicer endonucleases are involved in the production of such ago - loaded trfs . indeed , the biogenesis of some trfs belonging to 5-trfs , 3-cca trfs , or 3-u trfs have been reported to be dependent on dicer in humans,26,27,31 mice,45 and drosophila35 ( fig . , dicer is also reported to cleave trna to generate 5-trf in vitro.26 although the cloverleaf secondary structure of trnas does not meet the criteria for canonical dicer substrates , some precursor and mature trna molecules may alternatively form a long hairpin structure , particularly before modifications are attached , which may make trna structures suitable for dicer processing as predicted in the case of mice trna.45 some trfs seem to be generated via dicer - independent biogenesis mechanism.27,32 because several non - canonical mirna biogenesis pathways are independent of drosha or dicer and utilize precursor rnas forming various structures other than canonical hairpin - loop structures , such as single - stranded rnas , ago - bound trfs may be produced through such non - canonical pathways.46,47 alternatively , the other enzymes , which are not involved in mirna biogenesis would be involved in the production of ago - bound trfs . rnase z cleavage has been shown to produce 3-u trfs from pre - trnas in a dicer - independent manner.27,48 moreover , angiogenin ( ang ) , a member of the rnase a superfamily , is the candidate enzyme responsible for ago - bound trfs . in mammalian cells , ang was first discovered to be the enzyme that cleaves the anticodon loops of mature trnas to produce trna halves upon stress stimuli.49,50 however , it has also been shown that ang is able to cleave the t - loop of trnas to produce 3-cca trf in vitro ( fig . it remains to be elucidated how the biogenesis factors are able to access and process the rigid structure of mature trnas , because , in the cytoplasm , mature aminoacylated trnas form an l - shaped tertiary structure and are tightly bound by an elongation factor to be transported to the ribosome . because trfs and trna halves coexist ( e.g. , 5-trfs were purified together with 5-trna halves in dicer - immunoprecipitates),36,5154 the trna halves may be direct precursors of trfs in some instances . because the anticodon loop is exposed and accessible in animoacylated trna elongation factor complexes , mature trnas may first be subjected to anticodon cleavage by ang or other nucleases and then the generated trna halves dissociated from an elongation factor may be further processed for the production of trfs . further analyses are required to fully unveil the biogenesis factors and mechanisms for ago - bound trfs . mirnas deposit ago proteins onto target rnas by recognizing complementary sequences generally located in their 3-utr.35 imperfect mirna base - pairing with target mrnas appears to induce translational silencing , whereas extensive base - pairing triggers exonucleolytic decay of the target mrnas . considering their association with ago proteins and their similar size to mirnas , it is not surprising that ago - bound trfs act as mirnas and silence the expression of their complementary target mrnas . research on viruses has shown the first demonstration of ago - bound trfs functioning as mirnas . it has long been known that retroviruses use host trna as a primer for reverse transcription during the first step of retroviral replication . the 3-part of such trnas is complementary and binds to the primer - binding site ( pbs ) of the virus sequence . in human mt4 t - cells infected with human immunodeficiency virus type 1 ( hiv-1 ) , yeung et al . observed the abundant expression of an 18-nt 3-cca trf which is complementary to the pbs of hiv-1.55 moreover , the 3-cca trf was loaded onto ago2 and induced ago2-mediated cleavage of the pbs , thereby silencing pbs - containing reporter gene and a hiv-1 gene . these results suggest important roles of ago - loaded trfs on repression of virus replication . because the human genome contains many endogenous retroviral sequences and 3-trfs are highly complementary to these,30 trf - directed pathways may also play ubiquitous roles in silencing endogenous viral elements . as another example of the involvement of trfs in viral expression , wang et al . reported that infection with human respiratory syncytial virus ( rsv ) led to accumulation of 5-trfs derived from various cytoplasmic trnas.56 one chosen 5-trf , 5-trf , was shown to be generated by ang - mediated cleavage at sites adjacent to the 5-end of the anticodon loop . because the 5-trf showed activity in repressing complementary reporter gene expression , the trf was further shown to have functional significance in regulating rsv replication.56 in addition to the viral research , maute et al . reported a trf functioning as mirna in mature human b cells.31 a 22-nt ago - bound 3-cca trf showed activity in repressing the expression of target mrnas in a sequence - specific manner . rpa1 , an essential gene for dna dynamics and repair , was identified as one of the endogenous targets containing complementary sequences of the trf in its 3-utr . stable expression of the trf suppressed cell proliferation and modulated the response to dna damage in an rpa1-dependent manner , indicating the clear biological importance of 3-cca trf.31 haussecker et al . used a reporter assay to confirm that ago - bound 3-cca trfs and 3-u trfs repressed complementary target rnas.27 these results suggested that the trfs were able to function as mirnas , although further identification of endogenous trf targets using clip25,33 and degradome rna analysis57 will be required to fully understand their biological significance . one of the trfs used in their analysis , a 3-u trf , was reported to promote cell proliferation in the other study by lee et al.48 , validating that at least this trf has an important cellular role . in addition to their roles in translational repression in the cytoplasm , ago proteins have been reported to play distinct roles in the nucleus , such as modulation of histone methylation , mrna splicing , and dna damage repair , which is guided by mir - nas.58,59 ago - bound trfs may be involved in such nuclear functions of ago proteins . accumulation of further evidence of the functionality and endogenous biological roles of ago - bound trfs would lead to clearcut designation of these trfs as regulatory rna molecules . the number of reports characterizing expression and function of trna - derived ncrnas has been growing , which enforces the conceptual consensus that cells utilize mature and precursor trnas as a source for short , functional ncrnas . although this review detailed argonaute - bound trfs , there are various other classes of trna - derived ncrnas that are functional through mechanisms without involvement of argonaute proteins . a representative example is the trna halves , termed trna - derived stress - induced rnas ( tirnas),49 whose production through ang cleavage of the trna anticodon are triggered by various stress stimuli.1923 tirnas promote the stress response by stress granule formation60 and global inhibition of translation by yb-1-involved pathway,49,61,62 thereby associating with normal biological processes and diseases.1923,52 as another example of functional trna halves , sex hormone - dependent trna - derived rnas ( shot - rnas ) have been recently identified in breast and prostate cancers.63 shot - rnas are also produced by ang cleavage of the trna anticodon , which is promoted by sex hormones and their receptors , and these enhance cell proliferation.63 despite the findings described in this review , information regarding expression profiles , biogenesis pathways , and biological meanings of ago - bound trfs is still fragmented . one of the obstacles for comprehensive identification of the trfs would be the extensive presence of posttranscriptional modifications in trnas . over 100 posttranscriptional modifications are present in trnas , many of which play crucial roles in trna folding and function such as codon recognition.6467 because it is highly plausible that many trna - derived ncrnas are produced from modified trnas and because many modifications inhibit watson crick base paring and thus arrest reverse transcription,68 heavily modified trna - derived ncrnas would not be accurately captured by rt - pcr based sequencing methods . for example , the presence of base - pairing - inhibitory modification inside the ago - bound trf would affect the efficiency of targeting and regulation of mrnas . these points based on trna biological properties should be borne in mind for analyses of trna - derived ncrnas . as another considerable point genomes encode many identical trna genes and their slightly different variants , and identical trna fragment sequences can be present in non - trna regions in the genome.69 very recently , during revising this manuscript , exhaustive analyses of massive rna - seq data have newly identified the fifth class of trf , i - trfs , which are wholly derived from internal regions of mature trnas and are loaded onto ago proteins.70 trfs including i - trfs were reported to be differentially expressed in different cell types , tissues , disease states and human race.70 as in the effort to generate a trna fragment database by kumar et al.71 , cataloging the expression of trna fragments in various cell types and tissues , which should be based on appropriate bioinformatics approach and uniform nomenclatures will accelerate future investigations of trna - derived ncrnas . trna abundance varies greatly among different human cells and tissues , and the translational regulation by trna heterogeneity has been suggested.7274 the expression of trna halves is also variable in different cells and tissues and further regulated by trna modification states , various stressors , and hormones.1923,63 the uniquely regulated mechanism in controlling the quality and quantity of trnas and trna halves may consequently result in the opportunity for cells to gene rate , from these precursor rnas , specific class of ncrnas whose species and abundance would then also be uniquely controlled . in that regards , as shown in the case of various types of ncrnas,11 trna - derived ncrnas may emerge as a source of biomarkers and targets for therapeutics . indeed , use of trna - derived ncrnas as indicators for biological states has been suggested.63,70,75,76 research on trna - derived ncrnas is still in the initial stage and their biological functions may extend beyond our expectations . further efforts to understand the expression profiles , biogenesis , and function of trna - derived ncrnas will advance our knowledge regarding the expanding trna world and may provide significant insights into the pathophysiology of diseases .

the advent of next - generation sequencing technologies has not only accelerated findings on various novel non - coding rna ( ncrna ) species but also led to the revision of the biological significance and versatility of fundamental rna species with canonical function , such as transfer rnas ( trnas ) . although trnas are best known as adapter components of translational machinery , recent studies suggest that trnas are not always end products but can further serve as a source for short ncrnas . in many organisms , various trna - derived ncrna species are produced from mature trnas or their precursor transcripts as functional molecules involved in various biological processes beyond translation . in this review , we focus on the trna - derived ncrnas associated with argonaute proteins and summarize recent studies on their conceivable biogenesis factors and on their emerging roles in gene expression regulation as regulatory rnas .

embolism of the pulmonary artery or one of its branches is the most striking and characteristic appearance of thromboembolic disease ( 1 - 6 ) . according to textbooks and most of literature in most cases the starting point of the embolus is phlebothrombosis or thrombophlebitis of veins of lower extremities ( 1 - 5 ) . pulmonary embolism is very common nowadays , and favoring factors for its occurrence are the chronic diseases and therefore a long lying ( bed rest ) , which leads to the development of phlebothrombosis and a large number of different surgical operations ( 1 - 6 ) phlebothrombosis and thrombophlebitis of lower extremities veins phlebothrombosis of pelvic and abdominal veins usually after surgery thrombophlebitis and phlebothrombosis of veins of lower extremities after trauma and various surgical operations intracardiac thrombosis of the right cardiac ventricle thrombosis of the pulmonary artery or some of its branches in situ there are other , less common types of emboli : e.g. tissue cells , fat , oil , gas embolism and the like ( 1 ) phlebothrombosis and thrombophlebitis of lower extremities veins phlebothrombosis of pelvic and abdominal veins usually after surgery thrombophlebitis and phlebothrombosis of veins of lower extremities after trauma and various surgical operations intracardiac thrombosis of the right cardiac ventricle thrombosis of the pulmonary artery or some of its branches in situ there are other , less common types of emboli : e.g. tissue cells , fat , oil , gas embolism and the like ( 1 ) factors dependent on the patient : age over 40 years obesity immobilization / bed rest/- lying in bed for longer than 4 days deep vein thrombosis or pulmonary embolism in history thrombophilia , deficiency of antithrombin(at ) iii factor , protein c , protein s ; lupus anticoagulant , resistance to activated protein c , hemocistinemia ( 2 , 4 , 5 ) immobilization / bed rest/- lying in bed for longer than 4 days deep vein thrombosis or pulmonary embolism in history thrombophilia , deficiency of antithrombin(at ) iii factor , protein c , protein s ; lupus anticoagulant , resistance to activated protein c , hemocistinemia ( 2 , 4 , 5 ) factors dependent on the type of illness or surgery trauma or surgery : especially the pelvis , hip , leg malignant processes - particularly in the pelvis , abdomen , primary or metastatic heart failure recent myocardial infarction hormonal therapy ( estrogen , progesterone) using the contraceptive pills paraplegia severe infection intestinal inflammations polycythemia paraproteinemia behcet 's disease paroxysmal nocturnal hemoglobinuria ( 2 , 4 , 6 ) trauma or surgery : especially the pelvis , hip , leg malignant processes - particularly in the pelvis , abdomen , primary or metastatic recent myocardial infarction hormonal therapy ( estrogen , progesterone ) using the contraceptive pills intestinal inflammations paroxysmal nocturnal hemoglobinuria ( 2 , 4 , 6 ) although etiology and common risk factors for pulmonary thromboembolism are well known there is little data about frequency of postoperative pulmonary thromboembolism after different surgical operations . phlebothrombosis and thrombophlebitis of lower extremities veins phlebothrombosis of pelvic and abdominal veins usually after surgery thrombophlebitis and phlebothrombosis of veins of lower extremities after trauma and various surgical operations intracardiac thrombosis of the right cardiac ventricle thrombosis of the pulmonary artery or some of its branches in situ there are other , less common types of emboli : e.g. tissue cells , fat , oil , gas embolism and the like ( 1 ) phlebothrombosis and thrombophlebitis of lower extremities veins phlebothrombosis of pelvic and abdominal veins usually after surgery thrombophlebitis and phlebothrombosis of veins of lower extremities after trauma and various surgical operations intracardiac thrombosis of the right cardiac ventricle thrombosis of the pulmonary artery or some of its branches in situ there are other , less common types of emboli : e.g. tissue cells , fat , oil , gas embolism and the like ( 1 ) factors dependent on the patient : age over 40 years obesity immobilization / bed rest/- lying in bed for longer than 4 days deep vein thrombosis or pulmonary embolism in history thrombophilia , deficiency of antithrombin(at ) iii factor , protein c , protein s ; lupus anticoagulant , resistance to activated protein c , hemocistinemia ( 2 , 4 , 5 ) immobilization / bed rest/- lying in bed for longer than 4 days deep vein thrombosis or pulmonary embolism in history thrombophilia , deficiency of antithrombin(at ) iii factor , protein c , protein s ; lupus anticoagulant , resistance to activated protein c , hemocistinemia ( 2 , 4 , 5 ) factors dependent on the type of illness or surgery trauma or surgery : especially the pelvis , hip , leg malignant processes - particularly in the pelvis , abdomen , primary or metastatic heart failure recent myocardial infarction hormonal therapy ( estrogen , progesterone) using the contraceptive pills paraplegia severe infection intestinal inflammations polycythemia paraproteinemia behcet 's disease paroxysmal nocturnal hemoglobinuria ( 2 , 4 , 6 ) trauma or surgery : especially the pelvis , hip , leg malignant processes - particularly in the pelvis , abdomen , primary or metastatic recent myocardial infarction hormonal therapy ( estrogen , progesterone ) using the contraceptive pills intestinal inflammations paroxysmal nocturnal hemoglobinuria ( 2 , 4 , 6 ) although etiology and common risk factors for pulmonary thromboembolism are well known there is little data about frequency of postoperative pulmonary thromboembolism after different surgical operations . our objective is to show the number of patients with postoperative pulmonary thromboembolism ( ppte ) treated in intensive care unit of clinic for pulmonary diseases an tb podhrastovi in three - year period : from june 1 , 2011 - june 1 , 2014 and to indicate the importance of various surgical operations in the development of pulmonary thromboembolism ( pte ) . this is the retrospective study which shows the number of patients with postoperative pulmonary thromboembolism treated in intensive care unit of clinic for pulmonary diseases an tb it represents the number of these patients , per cent of patients with postoperative pulmonary thromboembolism of total patients with pulmonary thromboembolism , age and sex of patients , type of surgery , period expressed in days from surgery to clinical presence of pulmonary thromboembolism , presence of deep venous thrombosis ( dvt ) of lower extremities , massiveness of ppte e.g. level of pulmonary artery with embolus : segmental , lobar , main branches of pulmonary artery ; unilateral , bilateral . in three - year period 232 patients with pte were treated in intensive care unit of clinic podhrastovi . there were 24 males or 40% middle - aged 58.5 years , and 36 females or 60% middle - aged 56.3 years . only one man had dvt ( deep venous phlebothrombosis ) ( after orthopedic surgery ) , and five of them had anamnesis about previous dvt ( one with abdominal , one with vascular , one with orthopedic and two with cardiology surgery ) . only one woman had dvt ( after orthopedic surgery ) , and no one had anamnesis about previous dvt or pte . patients were subjected to different types of surgical operations which are presented on figure 1 . pte developed in 15 patients with abdominal , 11with urologic , 8 with gynecologic , 15 with orthopedic , 4 with cardiologic . 2 with vascular , 3 with neurosurgical , 1 with glandular ( breast ) and 1 with orl operations the average period in days from surgery to the development of pte . pte developed in one to thirty days after operation , on average for abdominal operations it was 19 days , for urologic 5 , for gynecologic 10 , for orthopedic 15.5 , for cardiologic 13 , for vascular 4.5 , for neurosurgical 18.5 , for glandular 10 and for orl operations 10 days after operation . ppte developed on average 10 days in women , and 14 days after operation in men . massiveness of ppte e.g. , the site or level of embolus was different - from segmental to main branches of pulmonary artery which is shown on figure 3 and figure 4 . the level or site of pulmonary embolus according to the type of surgery in women . the level of pulmonary embolism is different , from segmental to main branches of pulmonary artery with different number of patients according to operation . the level or site of pulmonary embolus according to the type of surgery in men . the level of pulmonary embolism is different , from segmental to main branches of pulmonary artery with different number of patients according to operation there is notably increasing number of patients with pulmonary thromboembolism in recent years treated in clinic for pulmonary diseases and tb podhrastovi we are not yet sure whether it is real increasing or it is matter of better diagnostics . in three - year period 60 of them or 25.86% were patients where pte developed after different types of surgical operations . we intended with this study to point out the significance and frequency of postoperative pulmonary thromboembolism , and to indicate that surgical operations of different type are significant etiologic and risk factor for the development of pte . first of all we did not have real data about regularity of preoperative and postoperative prophylaxis with anticoagulants , early rising from bed after surgery ( mechanical prophylaxis ) , we did not have data about intraoperative or early postoperative deaths caused possibly by pte . furthermore , not small number of patients with postoperative pte was treated in corresponding surgical clinic under the control of pulmonologist from our clinic , and released home to continue anticoagulant therapy under the control of pulmonologist in outpatient department . although common risk factors for the development of pte are well known , studied and confirmed in clinical practice there are no enough data in literature about postoperative dvt or pte out of controlled studies ( 7 ) . many authors indicate the significance of preoperative anticoagulant prophylaxis ( 7 , 8 , 9 , 10 ) . one big prospective study was done by group of authors on 75 771 patients with vascular and orthopedic operations from 1996 to 2001 in veteran health administration hospital ( 7 ) . major comorbidities included diabetes mellitus , chronic obstructive pulmonary disease , and congestive heart failure . symptomatic pte was diagnosed in 805 patients ( 0.68% ) and varied significantly with procedures : 0.14% for carotid endarterectomy to 1.34% for total hip arthroplasty . in our study we dealt with patients of both sex ( 60% of them women ) , average age was 58.5 years for men , and aged 56.3 years for women , and patients were subjected to different types of surgery . we dealt with patients not prospectively , but only with surgical treated patients with different operations with diagnosed ppte to indicate the importance of surgery to the development of pte . these authors ( 7 ) indicate the postoperative importance of pneumonia and other infections , including urinary tract infection , myocardial infarction . only two patients in our study had dvt after operation ( one man and one woman ) , but in all others were not be able to find the starting point of embolus . so we think that the preoperative and postoperative prophylaxis ( either anticoagulant , or mechanical - early rise from bed ) is of the most significance , especially in patients with risk factors for dvt or pte , even it is only the expected lying in bed ( bed - rest ) longer than 4 days . there is the need for one long prospective study in different surgical clinic for the assessment of more factors ( age , sex , preoperative diseases , type and site of operation , postoperative complications , duration of bed - rest , anticoagulant and mechanical prophylaxis etc . ) to give the more realistic picture of postoperative pulmonary thromboembolism . there is great significance of anticoagulant prophylaxis before surgery even in patients with no anamnesis of previous dvt or pte .

objective : our objective is to show the number of patients with postoperative pulmonary thromboembolism ( ppte ) treated in intensive care unit of clinic for pulmonary diseases an tb podhrastovi in three - year period : from june 1 , 2011 - june 1 , 2014 and to indicate the importance of various surgical operations in the development of pulmonary thromboembolism ( pte).material and methods : this is the retrospective study which shows the number of patients with ppte treated in intensive care unit of clinic for pulmonary diseases an tb podhrastovi in three - year period : from 01.06.2011.-01.06.2014 . it represents the number of these patients , per cent of patients with ppte of total patients with pte , age and sex of patients , type of surgery , period expressed in days from surgery to diagnosis of pte , presence of deep venous thrombosis ( dvt ) of lower extremities , massiveness of ppte e.g. level of pulmonary artery with embolus.results:in three - year period 232 patients with pte were treated in intensive care unit of clinic podhrastovi . 60 of them or 25.86% were patients with 24 males or 40% middle - aged 58.5 years , and 36 females or 60% middle - aged 56.3 years . ppte developed in 15 patients with abdominal , 11 with urologic , 8 with gynecologic , 15 with orthopedic , 4 with cardiologic , 2 with vascular , 3 with neurosurgical , 1 with glandular and 1 with orl operations . the average period from operation to diagnosis of ppte was 10.5 days for women , and 13.8 days for men . only two patients had acute dvt after operation ( one man and one woman ) , and five had amnesias of previous dvt or pte . the level or the site of pulmonary embolus was different from segmental to main branches of pulmonary artery.conclusion:different surgical operations are the big risk factor for the development of pte . there is great significance of anticoagulant prophylaxis before surgery even in patients with no anamnesis of previous dvt or pte .

systems biology is an emerging academic field aiming at system - level understanding of biological systems . recent progress in molecular biology has enabled us to gain information on the interactions among the underlying molecules from comprehensive experimental data sets . in general , a system - level understanding of a biological system can be derived from insight into four key properties : ( 1 ) the system 's structure , ( 2 ) the system dynamics , ( 3 ) the control method , and ( 4 ) the design method . equivalently , identifying related components and their interactions , gathering qualitative and quantitative information about the system 's evolution under different circumstances , achieving the desired outputs by controlling the input with appropriate definitions of inputs and outputs of the system , and reconstructing analogous systems by eliminating the undesired properties are four essential steps in systems biology done by collaboration among engineers , biologists , and doctors . systems biology is a cross - cutting research area connecting control engineering , biology , and medical science , as shown in figure 2 . it aims at understanding the bare function and integration function of the cell to reconstruct the biological systems with desired features . control and automation play critical roles in this novel field not only by providing new technology and equipment for biologists to design and perform meticulous experiments , to take high - throughput measurements , and to analyze experimental data efficiently , but also by offering doctors new medical applications and improving the precision of medical manipulations . the wide range of aspects which control and automation have been applied to include , but are not limited to , gene regulation [ 3 , 4 ] , drug delivery [ 2 , 5 ] , and neuron networks [ 6 , 7 ] . the equipment provided by control engineers includes , but is not limited to , nanodevices , biochips , cuvettes for electroporation , and gene guns . biologists perform various biological experiments , such as protein synthesis and virus dna modifications , to gather measurements for model revisions and verifications , to conclude theoretical and practical results from evidence , and to help medical practice . doctors use both theoretical and practical results from biologists to perform tissue engineering , such as organ transplants and artificial tissue construction . according to their scales , biological systems can be divided into three levels : the molecular level ( nm ) , cellular level ( m ) , and tissue level ( cm ) , analogous to the part , individual , and group , respectively . molecular - level research focuses on how , when , where , and to what extent a gene is expressed . the essential goal is to sketch a complete blueprint of genes by identifying the control sequences of coding dna segments and their interactions . cellular level research , in general , treats one cell as a plant in classical control theory and investigates the reactions of the cell to the changing environment , for instance , concentration changes of related chemicals . state - of - the - art medical therapies are primarily based on experimental results at the cellular level . tissue - level research mainly concerns tissue reconstruction , artificial tissue substitutes , or tissue function recovery . in contrast , understanding biological systems at the molecular level is crucial , since species have the same basic inheritance , dna macromolecules , and follow a common rule in gene expression , the central dogma in molecular biology . molecular level understanding of biological systems provides instrumental information about radical causes of many diseases and the genetic evidence of evolution . it also helps biologists to gain a better understanding of molecular level interactions , draw a complete blueprint of gene networks , improve existing means , create novel means to cure genetic diseases , and to elaborate on the theory of evolution . recent technology in gene sequencing makes it possible to conquer the difficulty in measurement at the molecular level and to identify the nucleotide sequences of a particular dna segment . targeted sequencing is the most promising step toward maximizing the efficiency of the next - generation sequencing technology using polymerase chain reaction . the availability of dna microarray makes it possible to accomplish tens of thousands of genetic tests for picomoles ( 10 ) of a specific dna sequence . researchers have applied various methods to model , simulate , and control the gene regulation processes . early attempts to model and simulate gene regulatory systems are summarized in , including direct graphs , bayesian networks , boolean networks , ordinary and partial different equations , qualitative differential equations , quantative differential equations , stochastic equations , and role - base formalisms . other approaches include petri nets , transformational grammars [ 12 , 13 ] , and process algebra . three important modeling methods in recent work are gene regulatory units viewed under compound control [ 4 , 1518 ] , logic network models [ 19 , 20 ] , and base - to - base molecular - level formulation [ 21 , 22 ] . the first modeling method quantitatively describes chemical concentration variations corresponding to external environmental changes at the cellular level . the last modeling converts dna segments to discrete vectors . in our paper , we adapt the base - to - base molecular level formulation to express state variables . most existing models are constructed by data - driven or hypothesis - driven methods , with only partial information available . due to the complexity of the systems and incomplete information , the mathematical models are usually formulated by modifying empirical equations or proposing heuristic equations . although those models can disclose significant details of the system 's structure and dynamics , the inconsistency between theoretical and experimental results creates difficulties for control engineers to verify the models , develop optimal controls , and reconstruct systems with desired properties . in this paper , we use a novel approach to build up abstract mathematical models at the molecular level in section 2.2 , based directly on biological theory . with reasonable assumptions different from existing methods , focusing on a gene or changes in chemical concentration , we emphasize the base change in the nucleotide bases . the cost function , a summation of costs for applying mutagens and the off - trajectory penalty , together with the system equations , formulates the optimal control problem . section 3 shows simulation results of the optimal control problem at different scales and is followed by several important propositions . the central dogma of molecular biology , first elaborated in and restated in , illustrates the detailed residue - by - residue transfer of genetic sequential information . nowadays , it describes the genetic information flow among three kinds of biopolymers : dna , rna , and protein . in most living organisms , this process is usually irreversible , thus protein always acts as the sink of information flow . a codon consists of three consecutive nucleotide bases , corresponding to one amino acid according to the genetic codes . since there are only 20 kinds of amino acids and 64 combinations of codons , there exists redundancy in genetic codes . we are particularly interested in mutations that happen during the process of dna replication , as dna serves as long - term genetic information storage and is the basis of genetic inheritance , the accuracy of which is particularly important to ensure the correct expression of genes . dna molecules consist of four kinds of nucleotide acids , adenine ( a ) , thymine ( t ) , guanine ( g ) , and cytosine ( c ) , and a backbone made of sugars and phosphate . in 1953 , james d. watson and francis crick found the double helix structure of dna and the rule of basepairing , known as watson - crick basepairing [ 25 , 26 ] . a always pairs with t , g always pairs with c , and vice versa . in nature , replication errors occur at a very low rate , one error for every 10 nucleotides added . the redundancy of information caused by the double - helix structure ensures the accuracy of dna replication . some dna self - repair mechanisms , listed in , such as proofreading , also help to eliminate errors during the replication process . point mutations can be further divided into transitions ( ag or ct ) and transversions ( a / gc / t ) . transversions are theoretically expected to be twice as frequent as transitions , but transitions may be favored over transversions in coding dna because they usually result in a more conserved polypeptide sequence . in this section , we first give the problem statement in section 2.1 , and then we construct system equations for both deterministic and stochastic mutations in section 2.2 . at last , in section 2.3 , we formulate the optimal control problem and apply dynamic programming algorithm to solve it . figure 3 shows the system diagram of restoring an abnormal dna segment back to a normal sequence by applying mutagens during the process of dna replication . after we obtain a patient 's genome , we compare the coding dna segments with normal dna segments in our database to figure out the possible range of mutated segments . due to the redundancy in genetic codes , as long as any two dna segments can be transcribed and then translated to the same amino acid sequence , the distance reference between them is considered to be zero . therefore , instead of having a predetermined final state or a neighborhood of a final state , our final state lies in a set where the distance reference between any sequence in this set and the desired sequence is zero . the prescription is then determined by comparing the current measurement and every sequence in the desired set . we treat dna sequences as state variables , the on / off controls of all available mutagens at every spot on the given dna segment as inputs , the measurements as the outputs , and one cell cycle as the step increment in our system equations . the objective function is defined as a summation of the costs ( including risks ) of applying mutagens and the off - trajectory penalty . the optimal control sequences are computed beforehand to let doctors make treatment plans according to the patient 's condition . in general , the optimal control sequence and the corresponding optimal trajectory are not unique because the bases mutate independently in most cases and the order of mutating different bases does not matter if the number of medical treatment sessions is not under a tight restriction . additional measurements are taken before and after each treatment , if necessary . in deterministic cases , the purpose of taking additional measurements is to check the current sequence and to eliminate both internal and external disturbances . in stochastic settings , the measurements are taken to conquer the randomness caused by both mutagens and other noises . , our system is a discrete - time dynamic system with finite state space and output space , and a set of on / off switches as controls . our goal is to optimally drive this system from a given initial state to a desired final set at the lowest cost . we mainly focus on applying chemical mutagens and radiation to restore the original amino acid sequence during the process of dna replication . other factors that may affect the gene mutation , including temperature and electroporation , are not within our consideration . in addition , we assume that chemical mutagens or radiation target one and only one nucleotide base at any preset site , despite the technical limitation , and the results of applying chemical mutagens and radiation are independent . for simplicity , we normalize the dose of mutagens to transfer one nucleotide base to another in one step to 1 . in most cases , nucleotide bases mutate independently , therefore there is no chain effect caused by mutagens . to avoid reactions among different mutagens , we require that at most one chemical mutagen and one radiation be applied in each cell cycle . while constructing a generalized model , since the order of applying chemical mutagens and radiation does not affect the results , without loss of generality , we require they be applied in the order shown in figure 4 . that is , chemical mutagens are always applied before the duplication process starts , radiation is always applied in the middle of the cell cycle , and the measurements are taken before every replication starts . lastly , we assume that the measurements are always correct , and dna replication error , background mutation rate , and other random noise can be eliminated from measurements by considering them as spontaneous mutation . denote the targeted dna segment with n nucleotide bases at kth step by a column vector x k , as shown in figure 5 . x k is the ith element of x k. let p be the transfer matrix from x k to x k+1 , for all k , k { 0 } , without mutation . then , the perfect dna replication process can be expressed as proposition 1 p = i . proofas no mutation occurs , x k+1 is completely complementary to x k by watson - crick base pairing rule , and x k+2 is completely complementary to x k+1 . therefore , x k+2 is exactly the same as x k. thus , ( 2)xk+2=pxk+1=p2xkp2=i . since every base mutates independently , every element of x k+1 only depends on the corresponding element of x k , thus p is diagonal . in addition , x k+1 x k , we conclude p = i . as no mutation occurs , x k+1 is completely complementary to x k by watson - crick base pairing rule , and x k+2 is completely complementary to x k+1 . therefore , x k+2 is exactly the same as x k. thus , ( 2)xk+2=pxk+1=p2xkp2=i . since every base mutates independently , every element of x k+1 only depends on the corresponding element of x k , thus p is diagonal . in addition , x k+1 x k , we conclude p = i . based on proposition 1 , we assign values to nucleotide bases set { a , g , c , t , o } , where o is an artificial nonsense base . define an equivalence relationship between { a , g , c , t , o } and { 1,2 , 2 , 1,0 } , that is , { a , g , c , t , o}{1,2 , 2 , 1,0 } , with ( 3)xki={1,if a,2,if g,2,if c,1,if t,0,if o. proposition 2{1,2 , 2 , 1,0 } is a field under proper definitions of addition and multiplication . { 1,2 , 2 , 1,0 } is a field under proper definitions of addition and multiplication . proofdefining the addition table and multiplication table as in tables 1 and 2 , we check if the set { 1,2 , 2 , 1,0 } satisfies the definition of field . defining the addition table and multiplication table as in tables 1 and 2 , we check if the set { 1,2 , 2 , 1,0 } satisfies the definition of field . commutativity of addition and multiplicationsatisfied as tables 1 and 2 are symmetric according to the diagonal . additive and multiplicative inversesadditive inverses pair : 11 , 22 , 00.multiplicative inverses pair : 11 , 22 , 11 . we conclude { 0,1 , 2 , 2 , 1 } is a field under addition and multiplication defined by tables 1 and 2 . from now on , we use to denote the field { 0,1 , 2 , 2 , 1}. and x k is the state vector representing a dna segment with n nucleotide bases , where is the set of -valued vectors of dimension n. we start with the simplest form of mutations , point mutation . suppose there is a point mutation , we write it mathematically as ( 4)xk+1=(i+s)xk+w , where x k+1 , x k , and i reduces to 1 as only one base is involved . the corresponding values of s and w , obtained by reverse engineering with all possible pairs of x k and x k+1 , are listed in table 3 . here , s represents the mutation from four normal nucleotide bases , and w corresponds to mutation from nonsense base , that is , w 0 only if x k = 0 . rewriting ( 4 ) by collecting all values of s and w in table 3 , we get ( 5a)xk+1=(i+j=04ukjsj)xk+j=04ckjwj ( 5b)=(i+uks)xk+ckw , where { s 0 , s 1 , s 2 , s 3 , s 4 } = { w 0 , w 1 , w 2 , w 3 , w 4 } = { 0,1 , 2 , 2 , 1 } , u k , c k { 0,1 } , representing the on / off controls , u k = [ u k u k u k u k u k ] , c k = [ c k c k c k c k c k ] , and s = w = [ 0 1 2 2 1 ] . in ( 5a ) , s j and w j are constants for all k and j. u k and c k , the inputs of the system , are the on / off controls for chemical mutagens or radiation . clearly , j=0 c k = 1 only if x k = 0 . equation ( 5b ) is a simplified version of ( 5a ) as we put u k , s j , c k , w j into vector form u k , s , c k , w. s and w serve as vector basis for base - to - base deterministic model . u k and c k are now multi - input controls ; each of them contains 5 on / off controls , corresponding to all possible transfer patterns . for a particular k , at most one of u k s and c k s can be 1 , as stated in proposition 3 . this is consistent with the fact that every state can be transferred to only one of the five states in the state space with corresponding mutagens available . proposition 3it is always 1 1 transfer when mutation occurs , that is , one nucleotide base can only transfer to another one , thereforeif x k = 0 and c k = 0 , or c k = [ 1 0 0 0 0 ] , then x k+1 = 0,if x k 0 , then c k = 0 and u k is either 0 or a unit row vector , if x k = 0 , then u k = 0 and c k is either 0 or a unit row vector , u k + c k is either 0 or a unit row vector , for all k { 0}. it is always 1 1 transfer when mutation occurs , that is , one nucleotide base can only transfer to another one , thereforeif x k = 0 and c k = 0 , or c k = [ 1 0 0 0 0 ] , then x k+1 = 0,if x k 0 , then c k = 0 and u k is either 0 or a unit row vector , if x k = 0 , then u k = 0 and c k is either 0 or a unit row vector , u k + c k is either 0 or a unit row vector , for all k { 0}. if x k = 0 and c k = 0 , or c k = [ 1 0 0 0 0 ] , then x k+1 = 0 , if x k 0 , then c k = 0 and u k is either 0 or a unit row vector , if x k = 0 , then u k = 0 and c k is either 0 or a unit row vector , u k + c k is either 0 or a unit row vector , for all k { 0}. now , suppose for some reason we need to take an addition , measurement in the middle of the cell cycle , after the completion of the kth duplication and before the start of the ( k + 1)th . we name this kind of measurement an intermediate state , and denote by it x k. then , we have ( 6)xk+1=(i+s ) xk+w , where the values of s and w , listed in table 4 , are obtained in the same way as getting s and w in table 3 . comparing tables 3 and 4 , we find the collection of s and s , w and w , form the same set , respectively . thus , we continue using s and w when rewriting ( 6 ) in the form of ( 5a ) and ( 5b ) , that is , ( 7)xk+1=(i+vks)xk+ckw , where v k , c k are the counterparts of u k , c k , respectively , and s , w are the same as in ( 5b ) . similar to proposition 3 , we get proposition 4 . proposition 4 v k and c k in ( 7 ) need to satisfy the following conditions.if x k = 0 and c k = 0 , or c k = [ 1 0 0 0 0 ] , then x k+1 = 0.if x k 0 , then c k = 0 and v k is either 0 or a unit row vector.if x k = 0 , then v k = 0 and c k is either 0 or a unit row vector . v k + c k is either 0 or a unit row vector , for all k { 0}. v k and c k in ( 7 ) need to satisfy the following conditions.if x k = 0 and c k = 0 , or c k = [ 1 0 0 0 0 ] , then x k+1 = 0.if x k 0 , then c k = 0 and v k is either 0 or a unit row vector.if x k = 0 , then v k = 0 and c k is either 0 or a unit row vector . v k + c k is either 0 or a unit row vector , for all k { 0}. if x k = 0 and c k = 0 , or c k = [ 1 0 0 0 0 ] , then x k+1 = 0 . if x k 0 , then c k = 0 and v k is either 0 or a unit row vector . if x k = 0 , then v k = 0 and c k is either 0 or a unit row vector . v k + c k is either 0 or a unit row vector , for all k { 0}. now take , both chemical mutagens and radiative rays under our consideration and apply them in the order as shown in figure 4 . then , we can express our system equation as ( 8a)xk=(i+uksmutations caused by chemicalmutagens from normal bases)xk + ckwmutations caused by chemicalmutagens from o , ( 8b)xk+1=(i+vksmutations caused by radiativerays from normal bases)xk + ckwmutations caused by radiativerays from o , ( 8c ) yk = xk , where u k and v k are the inputs of the system and y k is the measurement . obviously , ( 8a ) is modified from ( 5b ) and ( 8b ) from ( 7 ) . the two - step mutation and the intermediate state x k avoid the case x k is changed to different bases by radiation and chemical mutagens simultaneously , which causes confusion . substituting ( 8a ) and ( 8b ) , we get ( 9a)xk+1=(i+vks)(i+uks)xk+(i+vks)ckw+ckw , ( 9b)yk = xk . obviously , proposition 3 still holds for u k and c k , and proposition 4 holds for v k and c k for ( 9a ) . for point mutations , we have 20 on / off controls in total for every step k , 10 for chemical mutagens as described before , and the rest for radiation . in general , now , we show how to extend our model to large - scale systems . suppose we have a coding dna segment with length n , then x k . since a coding dna segment usually contains integer number of codons , which is made of three consecutive bases , n is a multiple of 3 . let x k denote the ith component of x k. this notation is consistent with the one in section 2.2.1 . initiated by the base - to - base deterministic model from section 2.2.1 , we write our system equation for large - scale system as ( 10 ) xk=(i+i=1nukiskimutations caused by chemicalmutagens from normal bases)xk + ikckiwkimutations caused by chemicalmutagens from o , xk+1=(i+i=1nvkiqkimutations caused by radiative raysfrom normal bases)xk + ikbkirkimutations caused by radiativerays from o , yk = xk , where u k , v k , c k , b k are on / off controls of the ith element , s k , q k are n n square matrices corresponding to the mutations between normal bases or from normal bases by chemicals and radiation , respectively , w k , r k are n - dimensional column vectors representing mutations from nonsense bases by chemicals and radiation , respectively , and k = { i : x k = 0,1 i n } , k = { i : x k = 0,1 i n}. s k and q k are diagonal matrices since each base mutates independently . the values in the first four rows of tables 3 and 4 correspond to the diagonal elements of s k and q k , respectively . the last rows of tables 3 and 4 are assigned to w k and r k , n - dimensional vectors , at nonsense base 's spots for x k. define = { s j e i e i , i , j , 0 j 4,1 i n } , a collection of n n matrices , where s j is the same as in ( 5a ) and ( 5b ) , e i is the unit column vector of length n with ith component equal to 1 and all other components equal to 0 , and e i e i is the square matrix with only the ith element on the diagonal equals to 1 , and 0 otherwise . then , s k , q k can be written as linear combinations of all elements from , with the coefficient of each element either 0 or 1 corresponding to the on / off control u k and v k , respectively . similarly , define = { w j e i , i , j , 0 j 4,1 i n } , where w j is the same as ( 5a ) and ( 5b ) . w k , r k can be written as linear combinations of all components from , with coefficient of every component either 0 or 1 corresponding to the on / off control c k and c k , respectively . therefore , instead of using step - varying s k , s k , w k , r k , we find matrix basis for those four square matrices to make the controls to be the only variables depending on k , as we did for single - base cases . then , we can , write ( 10 ) as ( 11a)xk=(i+i=1n j=04uk(i , j)sjeieit)xk+ik j=04ck(i , j)wjei , ( 11b)xk+1=(i+i=1n j=04vk(i , j)sjeieit)xk+ik j=04ck(i , j)wjei , ( 11c)yk = xk , where u k , v k , c k , c k { 0,1}. as shown in ( 11a ) , ( 11b ) , and ( 11c ) , multisites mutations contain 20n controls in total for every step k , where n is the number of nucleotide bases on the targeted gene . similar to point mutations , every single site has 20 controls in each step , 10 for chemical mutagens and 10 for radiation . we can view u k , v k , c k , c k as binary matrices of dimension n 5 , and u k , v k c k , c k are the corresponding element of ith row and jth column . use u k , v k , c k , b k , binary row vectors of dimension 5 , to denote the ith row of u k , v k , c k , c k , respectively . again , s = w = [ 0 1 2 2 1 ] . combining ( 11a ) and ( 11b ) , and writing control variables in vector forms , we get ( 12)xk+1=(i+i=1nvkiseieit)(i+i=1nukiseieit)xk + ( i+i=1nvkiseieit)ikckiwei+ikbkiwei , yk = xk . proposition 5for large - scale deterministic system , u k , v k , c k , c k satisfy conditions below.if e i x k = 0 , then i k.if e i x k = 0 , c k = 0 or c k = [ 1 0 0 0 0 ] , then i k.for all i k , u k is either 0 or a row unit vector and c k = 0.for all i k , c k is either 0 a row unit vector and u k = 0.for all i k , v k is either 0 or a row unit vector and b k = 0.for all i k , b k is either 0 or a row unit vector and v k = 0.for all i , k , 1 i n , k { 0 } , u k + c k is either 0 or a unit row vector and v k + b k is either 0 or a unit row vector . for large - scale deterministic system , u k , v k , c k , c k satisfy conditions below.if e i x k = 0 , then i k.if e i x k = 0 , c k = 0 or c k = [ 1 0 0 0 0 ] , then i k.for all i k , u k is either 0 or a row unit vector and c k = 0.for all i k , c k is either 0 a row unit vector and u k = 0.for all i k , v k is either 0 or a row unit vector and b k = 0.for all i k , b k is either 0 or a row unit vector and v k = 0.for all i , k , 1 i n , k { 0 } , u k + c k is either 0 or a unit row vector and v k + b k is either 0 or a unit row vector . if e i x k = 0 , then i k. if e i x k = 0 , c k = 0 or c k = [ 1 0 0 0 0 ] , then i k. for all i k , u k is either 0 or a row unit vector and c k = 0 . for all i k , c k is either 0 a row unit vector and u k = 0 . for all i k , v k is either 0 or a row unit vector and b k = 0 . for all i k , b k is either 0 or a row unit vector and v k = 0 . for all i , k , 1 i n , k { 0 } , u k + c k is either 0 or a unit row vector and v k + b k is either 0 or a unit row vector . the mathematical model ( 12 ) is quite flexible and can be easily extended to many cases , such as transcription process , multiple spot mutations within one - step or broken dna strands . our system equation can represent this phenomenon by dividing the whole system into small subsystems . significant brokage of dna strands is simply eliminated by cell mechanism to ensure the accuracy to dna replication . equation ( 13 ) shows the case of one single dna strand breaking into two segments by chemical mutagens ( 13)(xk(1)xk(2))=(im+i=1mukiseieit00inm+i = m+1nukiseieit ) (xk(1)xk(2))+ ( ik,1imckiweiik , ( m+1)inckiwei),xk+1(1)=(im+i=1mvkiseieit)xk(1)+ik , 1imbkiwei , xk+1(2)=(inm+i = m+1nvkiseieit)xk(2 ) + ik , ( m+1)inbkiwei . in reality , therefore , we need to derive the model for gene - to - gene stochastic mutations . introduce new random variables , h k , l1 , r k , l2 , h k , l3 , r k , l4 { 0,1 } , associated with probability p l1,j , p l2,j , p l3,j , p l4,j , for all i , k , 1 i n , k { 0 } , respectively , where k is the step index , l 1 , l 2 are indices for chemical mutagens inducing mutation from normal bases and from o , respectively , l 3 , l 4 are indices for radiation inducing mutation from normal bases and from o , respectively , i is the index of dna segment , and the value of j corresponds to the transfer pattern , which can be found in tables 3 and 4 . note different mutagens have different probability assignments , the probability assignments are only related to the type of mutagens , and the probability associated with every kind of mutagens sums up to 1 , that is , ( 14)j=04pl1,j(h)=1 , l1 , 1l1l,j=04pl2,j(r)=1 , l2 , 1l2m,j=04pl3,j(h)=1 , l3 , 1l3l,j=04pl4,j(r)=1 , l4 , 1l4m. the controls are u k , l1 , c k , l2 , v k , l3 , b k , l4 { 0,1 } , with the fact that 1 representing mutagen with corresponding index is applied at ith spot of dna segment at kth generation , and 0 representing mutagen with corresponding index is not applied at spot i at kth step , similar to sections 2.2.1 and 2.2.2 . the mutagen indices l 1 , l 2 , l 3 , l 4 can be omitted in deterministic mutations since given the current state and control , the next state is unique . however , they are necessary for stochastic mutations , because there exist multiple possible states for the next stage given the control . in other words , the next state is determined by random variables h k , l1 , r k , l2 , h k , l3 , r k , l4 , given the values of u k , l1 , c k , l2 , v k , l3 , b k , l4 , and x k. suppose we have ( l + m ) kinds of chemical mutagens available , with l kinds to induce mutations from normal bases and m kinds to induce mutations from o. and we have ( l + m ) kinds of radiation available , with l kinds to induce mutations from normal bases and m kinds to induce mutations from o. therefore , we have total ( l + m + l + m ) controls for each spot i at each step k. we can write our system equation as ( 15a)xk=(i+l1=1l i=1nuk , l1ij=04hk , l1(i , j)sjeieitmutations caused by chemical mutagens from normal bases)xk + l2=1 m ikck , l2ij=04rk , l2(i , j)wjeimutations caused by chemicalmutagens from o , ( 15b)xk+1=(i+l3=1l i=1nvk , l3ij=04hk , l3(i , j)sjeieitmutations caused by radiative rays from normal bases)xk + l4=1m ikbk , l4ij=04rk , l4(i , j)wjeimutations caused by radiative rays from o , ( 15c ) yk = xk . again , we define h k , l1 , r k , l2 , h k , l3 , r k , l4 the elements at ith row and jth column of n 5 binary matrices h k , l1 , r k , l2 , h k , l3 , r k , l4 , respectively . h k , l1 , r k , l2 , h k , l3 , r k , l4 , and binary row vectors of dimension 5 denote the ith row of h k , l1 , r k , l2 , h k , l3 , r k , l4 , respectively . then , we can simplify ( 15a ) , ( 15b ) , and ( 15c ) and combine ( 15a ) and ( 15b ) as ( 16)xk+1=(i+l3=1l i=1nvk , l3ihk , l3iseieit ) (i+l1=1l i=1nuk , l1ihk , l1iseieit)xk + ( i+l3=1l i=1nvk , l3ihk , l3iseieit)l2=1 m ikck , il2rk , l2iwei + l4=1m ikbk , l4irk , l4iwei , yk = xk . proposition 6for large - scale stochastic system , u k , l1 , h k , l1 , c k , l2 , r k , l2 , v k , l3 , h k , l3 , b k , l4 , r k , l4 follow the rules below.if e i x k = 0 , then i k.if e i x k = 0 and l2=1 c k , l2 = 0 , then i k.if e i x k = 0 , l2=1 c k , l2 = 1 and r k , l2 = [ 1 0 0 0 0 ] , then i k.for all i , k , l 1 , 1 i n , k { 0},1 l 1 l , if u k , l1 = 1 , then h k , l1 is a unit row vector.for all i , k , l 2 , 1 i n , k { 0 } , 1 l 2 m , if c k , l2 = 1 , then r k , l2 is a unit row vector.for all i , k , l 3 , 1 i n , k { 0 } , 1 l 3 l , if v k , l3 = 1 , then h k , l3 is a unit row vector.for all i , k , l 4 , 1 i n , k { 0 } , 1 l 4 m , if b k , l4 = 1 , then rk , l4 is a unit row vector.for all i k , l1=1 u k , l1 = 0 or 1 and c k , l2 = 0 , for all l 2 , 1 l 2 m.for all i k , l2=1 c k , l2 = 0 or 1 and u k , l1 = 0 , for all l 1 , 1 l 1 l.for all i k , l3=1 v k , l3 = 0 or 1 and b k , l4 = 0 , for all l 4 , 1 l 4 m.for all i k , l4=1 b k , l4 = 0 or 1 and v k , l3 = 0 , for all l 3 , 1 l 3 n , k { 0 } , l1=1 u k , l1 + l2=1 c k , l2 = 0 or 1 and l3=1 v k , l3 + l4=1 b k , l4 = 0 or 1 . for large - scale stochastic system , u k , l1 , h k , l1 , c k , l2 , r k , l2 , v k , l3 , h k , l3 , b k , l4 , r k , l4 follow the rules below.if e i x k = 0 , then i k.if e i x k = 0 and l2=1 c k , l2 = 0 , then i k.if e i x k = 0 , l2=1 c k , l2 = 1 and r k , l2 = [ 1 0 0 0 0 ] , then i k.for all i , k , l 1 , 1 i n , k { 0},1 l 1 l , if u k , l1 = 1 , then h k , l1 is a unit row vector.for all i , k , l 2 , 1 i n , k { 0 } , 1 l 2 m , if c k , l2 = 1 , then r k , l2 is a unit row vector.for all i , k , l 3 , 1 i n , k { 0 } , 1 l 3 l , if v k , l3 = 1 , then h k , l3 is a unit row vector.for all i , k , l 4 , 1 i n , k { 0 } , 1 l 4 m , if b k , l4 = 1 , then rk , l4 is a unit row vector.for all i k , l1=1 u k , l1 = 0 or 1 and c k , l2 = 0 , for all l 2 , 1 l 2 m.for all i k , l2=1 c k , l2 = 0 or 1 and u k , l1 = 0 , for all l 1 , 1 l 1 l.for all i k , l3=1 v k , l3 = 0 or 1 and b k , l4 = 0 , for all l 4 , 1 l 4 m.for all i k , l4=1 b k , l4 = 0 or 1 and v k , l3 = 0 , for all l 3 , 1 l 3 l.for all i , k , 1 i n , k { 0 } , l1=1 u k , l1 + l2=1 c k , l2 = 0 or 1 and l3=1 v k , l3 + l4=1 b k , l4 = 0 or 1 . if e i x k = 0 , then i k. if e i x k = 0 and l2=1 c k , l2 = 0 , then i k. if e i x k = 0 , l2=1 c k , l2 = 1 and r k , l2 = [ 1 0 0 0 0 ] , then i k. for all i , k , l 1 , 1 i n , k { 0},1 l 1 l , if u k , l1 = 1 , then h k , l1 is a unit row vector . for all i , k , l 2 , 1 i n , k { 0 } , 1 l 2 m , if c k , l2 = 1 , then r k , l2 is a unit row vector . for all i , k , l 3 , 1 i n , k { 0 } , 1 l 3 l , if v k , l3 = 1 , then h k , l3 is a unit row vector . for all i , k , l 4 , 1 i n , k { 0 } , 1 l 4 m , if b k , l4 = 1 , then rk , l4 is a unit row vector . for all i k , l1=1 u k , l1 = 0 or 1 and c k , l2 = 0 , for all l 2 , 1 l 2 m. for all i k , l2=1 c k , l2 = 0 or 1 and u k , l1 = 0 , for all l 1 , 1 l 1 l. for all i k , l3=1 v k , l3 = 0 or 1 and b k , l4 = 0 , for all l 4 , 1 l 4 m. for all i k , l4=1 b k , l4 = 0 or 1 and v k , l3 = 0 , for all l 3 , 1 l 3 l. for all i , k , 1 i n , k { 0 } , l1=1 u k , l1 + l2=1 c k , l2 = 0 or 1 and l3=1 v k , l3 + l4=1 b k , l4 = 0 or 1 . we close this section with the definition of controllability to the system equations proposed above . dna replication systems with system equations proposed as ( 9a ) , ( 9b ) , ( 12 ) , and ( 16 ) are completely controllable if and only if for all x 0 , x 2k1 , x 2k2 + 1 , k 1 , k 2 { 0 } , at least one path from x 0 to x 2k1 and at least one path from x 0 to x 2k2 + 1 by applying proper mutagens in the correct order , with k 1 , k 2 finite . we first define our objective function that can be adapted to all kinds of systems proposed in section 2.2 with minor changes . mathematically , in systems where the controllable parameters of interest are discrete , the objective function is usually a weighted sum representing the number of times that a piece of equipment is turned on or off or the number of resources needed to execute certain tasks in the frequent cases . in our case , this summation is the total number of times that different mutagens are applied weighted by the corresponding cost ( including the risk ) . another key factor of objective function is the off - trajectory penalty . designing a trajectory if the measurement indicates that the current state is off the predefined trajectory , we include a distance reference between current state and desired state as penalty and change the treatment plan accordingly . therefore , our objective function can be expressed as ( 17)j0(x0)=minu , c , v , c h , h,r , r[k=0n1l1=1li=1nl1uk , l1i+k=0n1l2=1mi=1nl2ck , l2icost of applying chemical mutagens + k=0n1l3=1li=1nl3vk , l3i+k=0n1l4=1mi=1nl4bk , l4icost of applying radiative rays + k=0nd(xk,{xkd})tracing cost ] , with x 0 , x k , 1 k n , n 0 ( mod 3 ) given . the physical meaning of u k , l1 , c k , l2 , v k , l3 , b k , l4 , l 1 , l 2 , l 3 , l 4 is the same as in section 2.2.3 . l1 , l2 , l3 , l4 , for all l 1 , l 2 , l 3 , l 4 , 1 l 1 1 l 2 m , 1 l 3 l , 1 l 4 m , are the corresponding cost of applying chemical mutagens and radiative rays indexed l 1 , l 2 , l 3 , l 4 , respectively . { x k } : { 0 } denotes the desired set at kth stage , generated by the dna sequences representing the same amino acid sequence as x k , the desired state at kth step . and d(x k , { x k } ) is the distance reference of the current state x k to the desired set { x k } at kth step . the final penalty , the distance reference from the final state to the desired set at k = n , is included in the last term . in general , l2 , l4 l1 , l3 , for all l 1 , l 2 , l 3 , l 4 , 1 l 1 l , 1 l 2 m , 1 l 3 l , 1 l 4 m , because physically o is a set of nonsense bases and more details are necessary to convert an o back to normal bases , for instance , the cost to identify the exact element in the set o. our goal is to drive our system optimally from initial state x 0 to the desired final set { x n } by applying a sequence of mutagens indexed with { l 1 , l 2 , l 3 , l 4 } , at problematic positions i , and in a correct order k. in ( 17 ) , the first four terms inside the expectation do not depend on random variables h k , l1 , r k , l2 , h k , l3 , and r k , l4 , for all i , k , l 1 , l 2 , l 3 , l 4 as the treatment plan is computed based on the initial state x 0 . given y k , the last term inside expectation , k=0 d(x k , { x k } ) , is the only term in summation that depends on the distribution of the random variables . the constraint of the optimal control problem , in general , is the system equation . we choose multidimensional stochastic system equation as the generalized constraints as it can be degenerated to one - dimensional and multidimensional deterministic cases by proper modifications . therefore , we can rewrite our objective function and formulate our optimal control problem as ( 18)j0(x0)=min{u , c , v , c}0,1, ,n1[k=0n1l1=1li=1nl1uk , l1i+k=0n1l2=1mi=1nl2ck , l2i + k=0n1l3=1li=1nl3vk , l3i+k=0n1l4=1mi=1nl4bk , l4i + k=0n{h , r , h,r}0,1, ,n1[d(xk,{xkd } ) ] ] , subject to ( 19)xk+1=(i+l3=1li=1nvk , l3ihk , l3iseieit ) (i+l1=1li=1nuk , l1ihk , l1iseieit)xk + ( i+l3=1li=1nvk , l3ihk , l3iseieit)l2=1mikck , l2irk , l2iwei + l4=1mikbk , l4irk , l4iwei , yk = xk . we need to choose a proper distance reference to quantitatively describe the relationship between dna segments of same length . we first define the distance reference between codons , and the distance reference between dna segments is a weighted sum of distance reference between every pair of codons . the distance reference between codons , d( 1 , 2 ) , 1 , 2 , needs to fulfill the biological requirements as below . mathematically , d : { 0 } , d( 1 , 2 ) 0 . symmetry : the distance reference from codon 1 to codon 2 equals the distance reference from codon 2 to codon 1 , that is , d( 1 , 2 ) = d( 2 , 1 ) . the distance reference between two codons corresponding to different amino acids should reveal the chemical and physical differences between two amino acids . the distance reference from stop codons to all other codons is much larger than those between other codons as early termination of amino acid sequences is more harmful than other forms of mutations . all the existing metric defined on the finite field can not achieve all the requirements above . the second requirement violates the identity of indiscernible , that is , d( 1 , 2 ) = 0 if and only if 1 = 2 . the redundancy in genetic codes implies d( 1 , 2 ) = 0 if those two amino acids , 1 and 2 , are translated into the same amino acids . in addition , the triangular inequality is not necessarily true , according to the underlying physical meanings . we take the assumption that the stop codons are of the same distance reference from and to all other codons . we ignore codons containing o since their chemical and physical properties can not be found in literature . from table 5 , we can see all codons are divided into different sets with each set corresponding to one amino acid . the size and this implies that the costs of driving one codon to the desired final set generated by the desired final state might be different from the costs of driving the complementary codon to the desired final set generated by the complementary of desired final state . more discussions about this issue are presented in section 3 . the distance reference between any two codons can be defined by a weighted sum of the differences between physical and chemical properties or other reasonable functions . and the distance reference between two dna sequences is defined as the sum of distance reference between the corresponding pair of codons . an example of the distance function can be expressed as ( 20)d(1,2)=polarity polarity(1,2 ) + ph ph(1,2)+size size(1,2),polarity(1,2 ) = { 0if 1,2 are both polar or non - polar,1if one of 1,2 is polar , and the other non - polar , ph(1,2 ) = |ph value of 1ph value of 2|,size(1,2 ) = { 0,if 1,2 are both tiny , small , or normal,1,if one of 1,2 is tiny , and the other small,2,if one of 1,2 is tiny , and the other normal,3,if one of 1,2 is small , and the other normal , where 1 , 2 are two amino acids . d( 1 , 2 ) is then assigned to d( 1 , 2 ) with 1 , 2 corresponding to amino acids 1 , 2 , respectively . since the generalized optimal control problem in ( 18 ) and ( 19 ) is a multistage problem that can be broken down into simpler steps at different time points . for dynamic programming , the optimal control policy is constructed backward . and bellman 's principle of optimality states that the optimal policy for x 0 to { x n } is also the optimal policy for the tail problem , from x q to { x n } . the tail problem is defined as ( 21)jq(xq)=min{u , c , v , c}q , q+1, ,n1{k = qn1l1=1li=1nl1uk , l1i+k = qn1l2=1mi=1nl2ck , l2i + k = qn1l3=1li=1nl3vk , l3i+k = qn1l4=1mi=1nl4bk , l4i + k = qn{h , r , h,r}q , q+1, ,n1[d(xk,{xkd})]}. the iterative update equation to find optimal policy can be expressed by ( 22 ) , according to the dynamic programming algorithm in . ( 22)jn(xn)=d(xn,{xnd}),jq(xq)=minuq , cq , vq , cq hq , rq , hq,rq[l1=1li=1nl1uq , l1i+l2=1mi=1nl2cq , l2i+l3=1li=1nl3vq , l3i + l4=1mi=1nl4cq , l4i+d(xq,{xqd})+jq+1(xq+1)]=minuq , cq , vq , cq{l1=1li=1nl1uq , l1i+l2=1mi=1nl2cq , l2i+l3=1li=1nl3vq , l3i + l4=1mi=1nl4cq , l4i + hq , rq , hq,rq[d(xq,{xqd})+jq+1(xq+1 ) ] } , q=0,1, ,n1 . in the following examples , we consider applying chemical mutagens only because the randomness of applying radiation is much larger and more difficult to control . we also omit the mutations between a normal base and o because of high - cost l2 and the unavailable chemical and physical properties for codons containing o. the distance reference between codons used in sections 3.2 and 3.3 is computed by ( 20 ) with polarity = 8 , ph = 3 , size = 1 , 1 = 2 , 2 = 5 and 3 = 3 . we only keep the final penalty but omit the off - trajectory penalty along the trajectory . we define the distance reference between bases as ( 23)d(1,2)={0,if xn = xnd,,if xnxnd , with 1 , 2 {0 } , where {0 } denotes the set excluding the element 0 . our optimal control problem for point mutations is ( 24)j0(x0)=minuk , l1 , 0kn , 1l1l{k=0n1 l1=1ll1uk , l1 } , subject to ( 25)xk+1=(i+l1=1luk , l1s)xk , xn = xnd , with x 0 given , x k {0}. suppose that there are 12 kinds of mutagens ( l 1 = 12 ) , each corresponding to a specific transfer pattern as in table 6 , all available controls and the respective costs can be immediately listed as in tables 7 and 8 . the elements along the antidiagonal of table 7 , u at , u gc , u cg , and u ta , are artificially added , because the complementary transfers naturally happen and no mutagen is necessary . thus , the costs along the antidiagonal of table 8 are all zero , that is , at = gc = cg = tc = 0 . the equivalence relationship between subscription in two nucleotide bases and subscription in integer l 1( 1 2 ) : { a , t , g , c } { a , t , g , c } { integers from 1 to 12 } is defined by table 6 . under the above assumptions , we can write update equation for optimal policy explicitly as ( 26)jq(xq)=minuq , l1{xq+jq+1(),{a , t , g , c}{0}}. proposition 7for the same x n , jq()jq+1( ) , for all q , 0 q n 1 , for all { a , t , g , c } , where denotes the complementary base of . if , in addition , the system is completely controllable , m , s.t . j m( ) is the global minimum and for all q m , jq()=jm( ) if m q 1 ( mod 2 ) , and j q( ) = j m( ) if m q 0 ( mod 2 ) . in our example , m n 6 . for the same x n , jq()jq+1( ) , for all q , 0 q n 1 , for all { a , t , g , c } , where denotes the complementary base of . if , in addition , the system is completely controllable , m , s.t . j m( ) is the global minimum and for all q m , jq()=jm( ) if m q 1 ( mod 2 ) , and j q( ) = j m( ) if m q 0 ( mod 2 ) . in our example , proofthis first part is due to the zero cost for the transfers between complementary bases in the consecutive steps . for any 0 q n 1 , the relationship between minimal costs in consecutive steps is shown in ( 26 ) . since { a , t , g , c } , +jq+1( ) is one of the four elements in the set from which the j q( ) is picked . moreover , =0 . therefore , jq+1( ) is one of the four elements in the set . since j q( ) is the minimum picking for a set containing jq+1( ) , we conclude that jq()jq+1().the m value in our example is proved by brute force method , that is , j n6( ) is a guaranteed global minimum . for completely controllable systems the existence of m implies that for without limitation in the number of steps , we can reach the global optimal in n m steps , 6 steps in our example.suppose that q = m , j m( ) is the global minimum , thus jm-1()jm( ) . jm-1( ) is also a global minimum.by backward induction , suppose for q = q 1 , the statement is true , that is , jq1 - 1()=jq1( ) is the global optimal either from xq1 - 1= or x q1 = to x n . obviously , for q = q 1 1 , the statement is still true . therefore , jq()=jq-2()=jq-1( ) , {a , t , g , c } , for all q , 2 q m. this first part is due to the zero cost for the transfers between complementary bases in the consecutive steps . for any 0 q n 1 , the relationship between minimal costs in consecutive steps is shown in ( 26 ) . since { a , t , g , c } , +jq+1( ) is one of the four elements in the set from which the j q( ) is picked . since j q( ) is the minimum picking for a set containing jq+1( ) , we conclude that jq()jq+1( ) . the m value in our example is proved by brute force method , that is , j n6( ) is a guaranteed global minimum . for completely controllable systems , this m always exists . the existence of m implies that for without limitation in the number of steps , we can reach the global optimal in n m steps , 6 steps in our example . suppose that q = m , j m( ) is the global minimum , thus jm-1()jm( ) . suppose for q = q 1 , the statement is true , that is , jq1 - 1()=jq1( ) is the global optimal either from xq1 - 1= or x q1 = to x n . obviously , for q = q 1 1 , the statement is still true . therefore , jq()=jq-2()=jq-1( ) , {a , t , g , c } , for all q , 2 q m. in the proof of global minimum that can be reached in the finite step in proposition 7 , we also discover proposition 8 . here , j q(x q , x n ) denotes the optimal cost from x q to x n . proposition 8given two single base mutation optimal control problems , with the same fixed n , with and desired final states complementary to each other . if j m( , x n ) is the global minimum , then jm(,xnd ) is also the global minimum , that is , the global minimum of both systems is reach at the same stage m. moreover , for all q , 0 q m , ( 27)jq(,xnd)=jq(,xnd ) , ,xnd{a , t , g , c}. given two single base mutation optimal control problems , with the same fixed n , with and desired final states complementary to each other . if j m( , x n ) is the global minimum , then jm(,xnd ) is also the global minimum , that is , the global minimum of both systems is reach at the same stage m. moreover , for all q , 0 q m , ( 27)jq(,xnd)=jq(,xnd ) , ,xnd{a , t , g , c}. physically , proposition 8 states that the optimal can be achieve at the same step from a pair of complementary bases to another pair of complementary bases at the same cost . however , this fact is true only for base - to - base deterministic mutations , because the distance reference is well defined by ( 23 ) . since we apply mutagens before the replication starts , u aa actually transfer a to t and then to a by replication . for simplicity , we just use the kth and ( k + 1)th step states as subscripts to represent the corresponding control and cost . therefore , ac , ca , gt , tg is smaller than other mutagens , except artificial ones . if we use to denote the costs of mutagens as listed in table 9 , then ( 28)=[5.216.602.3306.158.9503.824.6109.177.2400.645.0910.28]=[aaagacatgagggcgtcacgcccttatgtctt][1230450670890101112 ] . running the dynamic programming for every pair of ( x q , x n ) { a , t , g , c } { a , t , g , c } , n = 9 . here , we sightly modify our notation . we use j q(x q , x n ) to denote the optimal cost from x q to x n . then , ( 29)jq=[jq(a , a)jq(a , g)jq(a , c)jq(a , t)jq(g , a)jq(g , g)jq(g , c)jq(g , t)jq(c , a)jq(c , g)jq(c , c)jq(c , t)jq(t , a)jq(t , g)jq(c , c)jq(t , t ) ] . the path to reach the optimal cost is denoted by ( 30)pq=[pq(a , a)pq(a , g)pq(a , c)pq(a , t)pq(g , a)pq(g , g)pq(g , c)pq(g , t)pq(c , a)pq(c , g)pq(c , c)pq(c , t)pq(t , a)pq(t , g)pq(t , c)pq(t , t ) ] , where p q(x q , x n ) is the ( q + 1)th state from x q to x n , that is , x q+1 = p q(x q , x n ) . the simulation results are shown as below , including optimal costs for all possible transfer pairs ( x q , x n ) { a , t , g , c } { a , t , g , c } , j q , 0 q 8 , graphical representation in figure 6 , and optimal path p q , 0 q 7 . ( 31)j0=[5.215.090.6406.156.7903.823.8206.796.1500.645.095.21],j1=[00.645.095.213.8206.796.156.156.7903.825.215.090.640],j2=[5.215.090.6406.156.7903.823.8206.796.1500.645.095.21],j3=[00.645.095.213.8206.796.156.156.7903.825.215.090.640],j4=[5.215.090.6406.156.7903.823.8206.796.1500.645.095.21],j5=[00.645.095.213.8206.796.156.156.7903.825.215.090.640],j6=[5.215.090.6406.156.7903.823.8207.886.1500.645.095.21],j7=[00.645.095.213.8208.486.156.157.8803.825.215.090.640],j8=[5.216.602.3306.158.9503.824.6109.177.2400.645.0910.28 ] , ( 32)p0=[a , tttta , ca , ccc , tggggaa , ga , ca],p1=[ttta , tc , tca , ca , cggggaa , ca , ga],p2=[a , tttta , ca , ccc , tggggaa , ga , ca],p3=[ttta , tc , tca , ca , cggggaa , ca , ga],p4=[a , tttta , ca , ccc , tggggaa , ga , ca],p5=[ttta , tc , tcaa , cggggaa , ca , ga],p6=[a , tttta , cacc , tggtgaa , ga , ca],p7=[tttatcaagtggacga ] . for simplicity , we use 1 to represent a , 2 to g , 3 to c , and 4 to t in graphical interpretation . from figure 6 , we can see clearly that the optimal cost decreases as q decreases in the first few steps , and then optimal cost remains at the global minimum . this phenomenon obeys proposition 7 . in this example , global optimal is reached at m = 5 for all pairs of initial and final states as j 7 j 5 = j 3 and j 6 j 4 = j 2 . so , the global minimum is achieved before we reach n 5 = 4 in this particular case . this also implies that with n free we can reach desired final state in 4 steps from given initial state . observing closely to j q , 0 q 5 , j q1 equals to j q by exchanging the first and the last columns , and the second and the third columns , which is consistent with proposition 8 . or we can exchange the first and the last rows , and the second and the third rows of j q to obtain j q1 . j q1 and j q2 are the same for q 1 , q 2 m = 5 for q 1 q 2 = 0 ( mod 2 ) . the optimal trajectories are generated from p q(x q , x n ) . for example , given x 2 = t , and the final state x 9 = g , we want to generate the optimal trajectories . x 3 = p 2(t , g ) = a , g. if x 3 = a , x 4 = p 3(a , g ) = t ; if x 3 = g , x 4 = p 3(a , g ) = c. if x 4 = t , x 5 = p 4(t , g ) = a , g ; if x 4 = c , x 5 = p 4(c , g ) = g. if x 5 = a , x 6 = p 5(a , g ) = t ; if x 5 = g , x 6 = p 5(g , g ) = c. if x 6 = t , x 7 = p 6(t , g ) = a , g ; if x 6 = c , x 7 = p 6(c , g ) = g. if x 7 = a , x 8 = p 7(a , g ) = t ; if x 7 = g , x 8 = p 7(g , g ) = c. so , the optimal routes are tatatattgutgg ; tatattgutggcg ; tattgutggcgcg ; ttgutggcgcgcg . consequently , the optimal cost is j 2(t , g ) = 0.64 = tg . optimal trajectories for other pairs of initial and final states can be obtained in the same manner . it takes less than 1 second to generate optimal trajectories for all pairs of initial and final states with n = 9 on a regular desktop . since we have already proven by the brute force method that the global optimal can be achieved with m 6 , the computation time can be further reduced by taking n = 6 with all the results necessary for this example . for codon - to - codon deterministic mutations , we formulate our optimal control problem as ( 33)j0(x0)=minuk , l1i , 0kn,1l1l , 1i3{k=0n1l1=1li=13l1uk , l1i+d(xn,{xnd } ) } , subject to ( 34)xk+1=(i+l1=1li=13uk , l1iseieit)xk , with x 0 , x n {0 } given , x k {0 } , for all k , 0 k n , and d( 1 , 2 ) , 1 , 2 {0 } as defined in section 2.3 . if we take the same assumption on available mutagens as in section 3.1 , then we can write update equation for optimal control policy explicitly as ( 35)jq(xq)=minuq , l1i , 1l1l , 1i3{xq11+jq+1([1xq2xq3 ] ) , xq22+jq+1([xq12xq3]),xq33+jq+1([xq1xq23 ] ) , 1,2,3{a , t , g , c}{0 } } , with x q { a , t , g , c} {0 } , 1 i 3 denotes the ith element of x q {0 } , and xqi denotes the complementary base of x q . the optimal control sequences depend on the numerical values of l1s and d( 1 , 2 ) , 1 , 2 {0 } . though we do not have real values of l1s and d( 1 , 2 ) , we can always obtain simulation results to compare the result differences by assigning different numerical values to those parameters . therefore , we use three different assignments of l1s and the same d( 1 , 2 ) to generate our simulation results . those three assignments of l1s are , 5 , and 0.5 , respectively , with the same as assigned in section 3.1 . in every particular example , it takes approximately 2 seconds on a regular desktop to generate the optimal path table for all pairs of initial and final states with n = 19 , and the dynamic programming algorithm ensures that the optimal control for tail problems is generated at the same time . the graphical interpretation of three assignments are shown in figures 7 , 8 , and 9 , respectively . the x - axis and y - axis denote x q and x n , respectively . for a codon [ 1 2 3 ] , 1 , 2 , 3 { a , t , g , c} {0 } , its index is calculated by ( 36)42(11)+4(21)+3,where i = 1 if a , i = 2 if g , i = 3 if c , and i = 4 if t , 1 i 3 , for the simplicity of graphical interpretation . thus , there are 64 pairs of initial and final desired states , and there are 64 21 pairs of initial state and final desired set . j q is calculated following the same procedure as in base - to - base deterministic cases . the value of optimal cost can be read directly from graphical interpretation , and the optimal path can be generated from path matrix p q , similar to base - to - base deterministic case . both j q and p q , for all q , 0 q n , are of 64 64 dimension . from the graphical interpretation and table 10 , we find that the value of q where the global minimum is reached at the first time decreases as l1 decreases . and j 0 is more similar to j 18 with = 5 than with = or = 0.5. this implies that if d( 1 , 2 ) are the deterministic term in our objective function , then the treatment plan is made to drive the final state as close to the desired set as possible ; if the costs of applying mutagens is the deterministic term in the objective function , then the treatment plan tends to stay in the original state and applies less mutagens ; if they are of equal weight , then the treatment plan deals with this tradeoff . moreover , no matter how the numerical values of final penalty and the costs of applying mutagens changes in our objective function as shown in ( 33 ) , there is always a m , m n 18 , j m(x m ) is global minimum . proposition 7 can be extended to codon - to - codon deterministic mutations as stated in proposition 9 . proposition 9given an optimal control problem with objective function in the form of ( 33 ) , constraints in the form of ( 34 ) , and all available chemical mutagens and their corresponding transfer pairs and costs as listed in tables 6 , 7 , and 8 , jq()jq+1( ) , {0}3 . if , in addition , the system is completely controllable , m , s.t . j m( ) is the global minimum and for all q m , jq()=jm( ) if m q 1 ( mod 2 ) , and j q( ) = j m( ) if m q 0 ( mod 2 ) . in our example , m n 18 . given an optimal control problem with objective function in the form of ( 33 ) , constraints in the form of ( 34 ) , and all available chemical mutagens and their corresponding transfer pairs and costs as listed in tables 6 , 7 , and 8 , jq()jq+1( ) , {0}3 . if , in addition , the system is completely controllable , m , s.t . j m( ) is the global minimum and for all q m , jq()=jm( ) if m q 1 ( mod 2 ) , and j q( ) = j m( ) if m q 0 ( mod 2 ) . in our example , m n 18 n 18 here since the rest is similar to the proof of proposition 7 . the objective function in ( 33 ) can be written as the summation of three separate single - base mutation systems and the distance reference between final states and the final desired set , that is , ( 37)jq(xq)=minn1,n2,n302nn1+n2+n33n1{jn1(xn1)(xq1,1) optimal costs of base - to - basedeterministic optimal controlproblem formed by the 1st base + jn2(xn2)(xq2,2)optimal costs of base - to - base deterministic optimal controlproblem formed by - the 2nd base + jn3(xn3)(xq3,3)optimal costs of base - to - basedeterministic optimal control problemformed by the 3rd base + d([123],{xnd } ) } , where n q = ( n n 1 ) + ( n n 2 ) + ( n n 3).according to proposition 7 , j n6(x n6 ) is guaranteed to be the global optimal for single - base mutations . therefore , optimal costs corresponding to three single - base mutation systems , j n1(x n1)(x q , 1 ) , j n2(x n2)(x q , 2 ) , j n3(x n3)(x q , 3 ) are guaranteed to reach their own global optimal at n 1 = n 2 = n 3 = n 6 with all possible combinations of 1 , 2 , 3 { a , t , g , c}. therefore , q = n 18 is a guaranteed global optimal . n 18 here since the rest is similar to the proof of proposition 7 . the objective function in ( 33 ) can be written as the summation of three separate single - base mutation systems and the distance reference between final states and the final desired set , that is , ( 37)jq(xq)=minn1,n2,n302nn1+n2+n33n1{jn1(xn1)(xq1,1) optimal costs of base - to - basedeterministic optimal controlproblem formed by the 1st base + jn2(xn2)(xq2,2)optimal costs of base - to - base deterministic optimal controlproblem formed by - the 2nd base + jn3(xn3)(xq3,3)optimal costs of base - to - basedeterministic optimal control problemformed by the 3rd base + d([123],{xnd } ) } , where n q = ( n n 1 ) + ( n n 2 ) + ( n n 3 ) . according to proposition 7 , j n6(x n6 ) is guaranteed to be the global optimal for single - base mutations . therefore , optimal costs corresponding to three single - base mutation systems , j n1(x n1)(x q , 1 ) , j n2(x n2)(x q , 2 ) , j n3(x n3)(x q , 3 ) are guaranteed to reach their own global optimal at n 1 = n 2 = n 3 = n 6 with all possible combinations of 1 , 2 , 3 { a , t , g , c}. therefore , q = n 18 is a guaranteed global optimal . indeed , the m value where the first global optimal is reached at earlier stage as listed in table 10 . graphically , the indices of complementary codons 1 = [ 1 2 3 ] and 2=[1 2 3]t sum up to 65 , that is , ( 38 ) ( 16(11)+4(21)+3 ) + ( 16(11)+4(21)+3 ) = ( 16(11)+4(21)+3 ) + ( 16((51)1)+4((52)1)+(53 ) ) = 65 . therefore , j q and j q1 , 1 q m are symmetric about the plane x = 32.5 , j q2 and j q , 2 q m are the same , as shown in figures 7 , 8 , and 9 . however , proposition 8 can not be extended to codon - to - codon deterministic case due to the redundancy of genetic codes , that is , the set of codons translated to the same amino acid varies from one amino acid to another as shown in table 5 . the simulation results show that the costs , a pair of complementary codons , to two final desired set generated by a pair of complementary final desired codons are different , that is , jq(xq,{xnd})jq(xq,{xnd } ) , in general , for any q. graphically , the optimal cost profile j q is not symmetric about the plane y = 32.5 for j q1 , 1 q m. therefore , the doctors need to pick the strand with lower cost to make the treatment plan . this also implies that in large - scale cases , for instance , a gene containing hundreds of nucleotide bases , the doctors should make the treatment plan based on the strand the total cost of which is lower than the other . the optimal control problem of codon - to - codon stochastic mutations can be written as ( 39)j0(x0)=minuk , l1i , 0kn11l1l , 1i3{k=0n1l1=1li=13l1uk , l1i + hk , l1i , 0kn11l1l , 1i3[d(xn,{xnd } ) ] } , subject to ( 40)xk+1=ixk+l1=1li=13uk , l1ihk , l1iseieitxk , with x 0 , x n {0 } given , x k the major difference between deterministic and stochastic systems is that we impose the random binary vector , h k , l1 , in our system equation ( 40 ) . we denote the probability associated with h k , l1 to be p l1,12 with 1 , 2 { a , t , g , c}. the equivalence relationship between j and 1 2 can be found in table 3 . again , we assume that we have l 1 = 12 kinds of mutagens , each corresponding to one major transfer pattern , associated with probability assignments , as listed in table 11 . then , we can write updated formula for optimal control policy explicitly as ( 41)jq(xq)=minuq , l1i , 1l1l , 1i3{xq11+hq , l1(xq11)1[jq+1([xq2xq3 ] ) ] , xq22+hq , l1(xq22)2[jq+1([xq1xq3 ] ) ] , xq33+hq , l1(xq33)3[jq+1([xq1xq2 ] ) ] , 1,2,3{a , t , g , c}{0 } } where ( 42)hq , l1(xq11)1[jq+1([xq2xq3])]=pl1(xq11 ) , xq1a(h)[jq+1([axq2xq3 ] ) ] + pl1(xq11 ) , xq1g(h)[jq+1([gxq2xq3 ] ) ] + pl1(xq11 ) , xq1c(h)[jq+1([cxq2xq3 ] ) ] + pl1(xq11 ) , xq1t(h)[jq+1([txq2xq3 ] ) ] , where x q { a , t , g , c} {0 } , 0 q n 1 , 1 i 3 denotes the ith element of x q {0 } , xqi denotes the complementary base of x q , and l 1 : 1 2 { a , t , g , c } { a , t , g , c } { integers from 1 to 12 } , the mapping from major transfer pattern 1 2 to mutagen index , as shown in table 11 . the mathematical expression of hq , l1(xq22)2[jq+1([xq1 xq3]t ) ] and hq , l1(xq33)3[jq+1([xq1xq2]t ) ] is similar to hq , l1(xq11)1[jq+1([ xq2 xq3]t ) ] as shown above . in order to run the simulation , we assign numerical values to probabilities in table 11 , as illustrated in table 12 . as in section 3.2 , we use three different assignments for l1s , , 5 , and 0.5 , respectively . the optimal cost profile j q with selected q values , for every pair of ( x q , x n ) , is graphically interpreted in figures 10 , 11 , and 12 , respectively , with n = 29 , with computation time of approximately 7 seconds on a regular desktop . the profile of j 0 is more similar to j 29 when l1s are assigned 5 than or 0.5. this implies in codon - to - codon stochastic mutations ; the optimal control sequence behaves as codon - to - codon deterministic cases , that is , the system tends to getting as close as possible to the final desired set if l1s are much smaller than d( 1 , 2 ) , and the system tends to remain in the same state with minor mutations when l1s are relatively larger than d( 1 , 2 ) , 1 , 2 {0 } . and jq()jq+1( ) , {0 } is still valid in codon - to - codon stochastic case . however , for stochastic cases , we can not reach a global minimum because of the randomness caused by mutagens . since , in usual cases , there exists no stationary global minimum , we need to define error tolerance , that is , if |jq()-jq-1()| with the same { x n } , then we can stop at j q(x q ) . however , we can still observe figures 10 , 11 , and 12 to conclude that j 0 and j 2 are almost of the same shape in all three different parameter assignments . higher dimensional optimal control problems , gene - to - gene stochastic mutations , can be solved as a series of cascade codon - to - codon stochastic problems . in this paper , we present a mathematical model to deal with mutations in the process of dna replication in the view of control systems . different from the existing models , our model is constructed directly from the basic biological theories , the central dogma in molecular biology , and the complementary base pair for dna molecules with double helix structure . it precisely describes how the induced mutations affect the targeted dna segments at molecular level . it provides instrumental information of molecule interactions in gene mutation for biologists and doctors to gain a better understanding of cellular and tissue level systems ' behavior . though we emphasize that we target at induced mutations during the process of dna replication in our work , this model can be extended to other biological processes at molecular level , such as transcription process and dna brokage . in our optimal control problem , the objective function includes two factors : the risk / cost of applying mutagens and the off - trajectory penalty . under optimal control policy , the summation of those two factors are minimized , by dynamic programming , to propose a low - risk treatment plan . we define the distance reference following the chemical and physical properties of amino acids , representing the penalty . our objective is to drive the system from given initial state to the final desired set generated by the final desired state at the lowest cost . we define the final desired set since redundancy in genetic codes gives us additional options of final desired state to further lower the cost . we also discuss three different small - scale system , and show the simulation results of examples . the optimal control problems of base - to - base deterministic mutations and codon - to - codon deterministic mutations are of theoretical importance . as shown in the propositions , the global optimal if the step limit is larger than the number of steps that global optimal can achieve , then we have some flexibility in our treatment plan . in addition , there exist multiple optimal paths with the same total cost , given the initial state and the final desired set . the optimal control problem of codon - to - codon stochastic mutations is of practical importance , since codon is the basic component forming long dna sequences . the step limit n is decided by doctors according to patients ' conditions , and the treatment plan is made according to the initial state , the final desired set , and the step limit . since the doctors constantly take measurement to see the result of treatments at current stage , the treatment plan is updated accordingly . solving codon - to - codon stochastic optimal control problem is a key step to realize the optimal control to gene - to - gene stochastic mutations in the real world . the optimal control sequences generated by dynamic programming make it possible for biologists and doctors to mutate certain sections of genes on purpose in laboratory , at a relative low cost and low risk , which is an essential step to identify the functional units , to examine the interactions among different segments , and to find healthy , harmful , and lethal nucleotide sequences . all those results are beneficial in gene network construction . in addition , the fundamental details of gene mutations at molecular level help biologists to elaborate on the biological theories at the cellular and tissue levels , such as the theory of evolution . moreover , by our method , biologists can distinguish the harmful and beneficial mutations and induce beneficial mutations during the evolution process in a proper way , which greatly helps to save rare species in danger . furthermore , our solution to the optimal control problem proposed provides a new medical intervention to genetic diseases . compared to the existing gene therapy , treatments by mutagens are safer by avoiding the side effect of virus infection . calculation errors , the mispairings in the process of two single - stranded dna segments , can be corrected at lowest cost by applying a correct mutagen sequence . further work can be done by extending codon - to - codon stochastic optimal control problem to gene - to - gene stochastic mutations . the distance reference between dna segment with equal length can be defined as a weighted sum of the distance references between codons . since certain combinations of amino acids this goal can be achieved by either defining a preset trajectory or eliminating high - risk sequences in the state space . under this situation , the spontaneous mutations can be modeled as an additional random factor in our state updating equations , and another random noises should be added to the output equation .

we propose a molecular - level control system view of the gene mutations in dna replication from the finite field concept . by treating dna sequences as state variables , chemical mutagens and radiation as control inputs , one cell cycle as a step increment , and the measurements of the resulting dna sequence as outputs , we derive system equations for both deterministic and stochastic discrete - time , finite - state systems of different scales . defining the cost function as a summation of the costs of applying mutagens and the off - trajectory penalty , we solve the deterministic and stochastic optimal control problems by dynamic programming algorithm . in addition , given that the system is completely controllable , we find that the global optimum of both base - to - base and codon - to - codon deterministic mutations can always be achieved within a finite number of steps .

we defined cholera decedents as persons who died of suspected cholera ( acute watery diarrhea in persons > 5 years of age ) with illness onset after october 16 , 2010 , three days before the first case - patients were seen at the hospital ( reflecting the 3-day average incubation period ) . to locate decedents , we obtained reports of cholera - related deaths from 2 sources : admission records from 2 hospitals in artibonite that had cholera treatment centers ( hpital albert schweitzer and hpital charles colimon ) and verbal reports from community health workers ( chws ) . we attempted to locate households of all decedents from hospital records and verbal reports . logistic and time constraints limited case finding to communities within 2 hours travel from the hospitals . we visited decedents households ; obtained informed consent ; and interviewed families about demographics , symptoms , health - seeking behavior , treatment , type of health facility , and knowledge about cholera . we also asked decedents household members and local chws about other cholera - related deaths . the centers for disease control and prevention institutional review board ( atlanta , ga , usa ) and mspp determined that this emergency response activity was nonresearch . of 28 decedents identified from hospital records , we found homes of 22 ( 79% ) ; homes of 6 decedents could not be located or were too remote for inclusion . illness onset ranged from october 16 through november 14 ; a total of 29 ( 33% ) persons died during the first week of the epidemic ( figure 2 ) . median age of decedents was 50 years ( range 5100 years ) ; 58 ( 67% ) were male . forty - eight ( 55% ) decedents died in a health facility ( health facility decedents ) and 39 ( 45% ) died at home or en route to a facility ( community decedents ) . we identified 17 ( 35% ) health facility decedents from hospital records and 31 ( 65% ) from community interviews ; we identified 5 ( 13% ) community decedents from hospital records and 34 ( 87% ) from community interviews . number of persons who died of cholera , artibonite department , haiti , october 16november 14 , 2010 . twenty - three ( 48% ) health facility decedents and 9 ( 23% ) community decedents had used oral rehydration solution ( ors ) at home before seeking care ( table 1 ) . ors use at home was lower for persons who died during the first week of the outbreak ( 7 [ 27% ] ) than during the second ( 8 [ 40% ] ) or third ( 17 [ 46% ] ) weeks . we observed ors sachets in homes of 17 ( 35% ) health facility decedents and 14 ( 36% ) community decedents . health facility decedent , cholera case - patient who died in a health facility ; community decedent , cholera case - patient who died at home or en route to a health facility ; ors , oral rehydration solution . median time from illness onset to death was 20 hours ( range 3 hours7 days ) for health facility decedents and 12 hours ( range 2 hours8 days ) for community decedents . twenty - two ( 46% ) health facility decedents died on day of admission and 26 ( 54% ) died after spending > 1 night in the facility ( table 2 ) . twenty - three ( 59% ) community decedents never sought care , 8 ( 21% ) died en route to care , and 8 ( 21% ) died after discharge . of those who sought care , 29 ( 60% ) health facility decedents and 7 ( 44% ) community decedents waited < 2 hours to visit a health facility . family members of community decedents reported the following reasons for not seeking care : no need for care ( 19 [ 69% ] ) , long distance to the health facility ( 6 [ 26% ] ) , too ill to travel ( 4 [ 17% ] ) , lack of transport ( 3 [ 13% ] ) , unsafe to travel at night ( 3 [ 13% ] ) , and cost of transport ( 1 [ 4% ] ) . * health facility decedent , cholera case - patient who died in a health facility ; community decedent , cholera case - patient who died at home or en route to a health facility ; na , not applicable . of 48 health facility decedents , 38 ( 79% ) were treated in hospital and 10 ( 21% ) at a health center or dispensary . decedents received intravenous fluids ( 35 [ 73% ] ) , ors ( 27 [ 56% ] ) , both ( 20 [ 42% ] ) , or neither ( 3 [ 9% ] ) . household members of 33 ( 69% ) health facility decedents and 30 ( 81% ) community decedents reported receiving information about cholera after the outbreak started . the most common information sources for families of health facility and community decedents , respectively , were radio ( 26 [ 79% ] vs. 26 [ 89% ] ) , friend ( 6 [ 18% ] vs. 8 [ 27% ] ) , cellular telephone text message from mspp ( 4 [ 12% ] vs. 4 [ 13% ] ) , community meeting ( 2 [ 6% ] vs. 2 [ 7% ] ) , and chws ( 1 [ 3% ] vs. 3 [ 10% ] ) . fewer than half of family members of health facility ( 23 [ 48% ] ) and community ( 19 [ 49% ] ) decedents believed cholera was treatable . of these , 16 ( 70% ) health facility decedents and 17 ( 90% ) community decedents knew to seek care at a health facility . our findings suggest that , early in the cholera epidemic in haiti , death occurred rapidly , and care was either inadequate or nonexistent . global deficiencies in the distribution and use of ors in recent years have impeded the ability of chws to initiate treatment ( 7 ) . second , chws probably lacked sufficient information , experience , and resources to provide proper treatment early in the outbreak . deaths in health facilities in haiti might have resulted from problems commonly observed elsewhere : overwhelming patient load , inadequate supplies , and health worker shortages ( 8) . third , decedents relatives identified several commonly observed barriers to care : distance to health facility , lack of transport , and unaffordable transport ( 9 ) . research suggests that the effect of distance and lack of transport on cholera - related death can be mitigated by local treatment with ors by chws ( 10,11 ) . finally , the epidemic strain , which was particularly virulent , might have contributed to deaths ( 12 ) . this finding suggests that cholera - related deaths might have been underreported , particularly in more remote communities . first , time and logistics limited our ability to visit remote communities where more deaths might have occurred . second , our geographically circumscribed convenience sample might not have been representative of all cholera deaths . findings from this assessment suggested several practical actions that could mitigate the risk for death from cholera . chws , particularly in remote settings , should receive training in cholera treatment and referral and adequate supplies of ors ; similar efforts for hiv and tuberculosis in haiti have been promising ( 13,14 ) . longer term efforts to increase health facility staffing and improve access to care should be prioritized . in response to the epidemic , training and supplies have been provided to health workers in all 10 departments of haiti . by april 2011

we evaluated a high ( 6% ) cholera case - fatality rate in haiti . of 39 community decedents , only 23% consumed oral rehydration salts at home , and 59% did not seek care , whereas 54% of 48 health facility decedents died after overnight admission . early in the cholera epidemic , care was inadequate or nonexistent .