Uncaria tomentosa in minimizing the side effects of chemotherapy and improving the antioxidant status of colorectal cancer (CRC) patients, a randomized clinical trial was conducted. Patients (43) undergoing adjuvant/palliative chemotherapy with 5-Fluorouracil/leucovorin + oxaliplatin (FOLFOX4) were split into two groups: the UT group received chemotherapy plus 300 mg of Uncaria tomentosa daily and the C group received only FOLFOX4 and served as a control. Blood samples were collected before each of the 6 cycles of chemotherapy, and hemograms, oxidative stress, enzymes antioxidants, immunologic parameters, and adverse events were analyzed. The use of 300 mg of Uncaria tomentosa daily during 6 cycles of FOLFOX4 did not change the analyzed parameters, and no toxic effects were observed.To evaluate the effectiveness of  Howeverecursors  and the  . Howeve+ and CD4+ T cells (Tregs) levels, shows a correlation with survival. The CD4+/CD8+ ratio of the tumor infiltrating lymphocytes is significantly associated with colorectal cancer prognosis [γ production through the action of tumor-specific CD8+ T cells that infiltrate the tumor, and it promotes T cell-dependent antitumor responses in vivo [The immune status of patients, particularly CD8rognosis . Treatme in vivo . Again,  in vivo .Uncaria tomentosa  has antioxidant properties [Ut extract promotes proliferation of myeloid precursors through the increase in serum colony stimulating growth factors (CSFs). Other preclinical experiments have demonstrated the positive effect of aqueous Ut extract on leukocyte counts over a period of eight weeks in healthy animals [Ut could minimize the undesirable effects of chemotherapy and might improve the balance between stress and antioxidants in cancer patients. This clinical study aimed to evaluate the effect of coadjuvant treatment with Ut compared with conventional chemotherapy for colorectal cancer. The investigation evaluated the effect of Ut on oxidative stress and its consequences in relation to neutropenia, other hematological parameters, immune system, safety, and side effects.For these reasons, it is important to search for complementary treatments, including phytotherapic plants, that minimize the neutropenia associated with colon cancer chemotherapy. operties  and can operties  and myeloperties . Eberlinoperties  showed t animals  and afte animals . Given tWe performed a randomized interventional study of colorectal cancer patients who were submitted to chemotherapy treatment. Hospital Universitário de Santa Maria, Brazil. The study was carried out with 43 patients  who had undergone complete resection of their colorectal cancer, which was of histologically scored as stage IIB, III, or IV, and who were going to begin adjuvant/palliative chemotherapy with FOLFOX4 at the Ut, and the control group (C) received only FOLFOX4. Patients remained on study during 6 chemotherapies cycles, of 15 days each. The doses of medication in UT group were as follows: Oxaliplatin, 85 mg/m² on day1; 5FU, 1 g/m² on days 1 and 2; Leucovorin, 200 mg/m² on days 1 and 2; and Ut (Unha de Gato Herbarium), 3 tablets daily from day 3 to day 15. The dose of Ut is similar to that used in previous study, using 250–350 mg C-MED-100, an aqueous extracts of Ut [Patients were randomly grouped into two groups, according to the date of treatment start as follows: the first patient who agreed participating in the study was included into UT group, the second into the C group, and successively until the end. The UT group was treated with FOLFOX4 plus ts of Ut . No chanα) of 5%, and statistical power of 90% (β 10%), using as a reference the study of Sheng et al. [The calculation for estimating the sample size required for a randomized clinical trial was according to Greenberg et al. , with cog et al. .Universidade Federal de Santa Maria approved this study, and informed consent was obtained from all participants (protocol n. 0169.0.243.000-07).The Human Ethics Committee of the Ut extract. Biological materials used in the tablets were derived from plants in their natural habitat. The Ut extract was prepared by ultra-turrax extraction  from ground bark (Centroflora) using 70% ethanol . The HPLC analysis of Ut dry extract presents the content of 2.57% pentacyclic oxindole alkaloids (POAs), which were calculated with reference to external calibration curves of mitraphylline. The analysis of extract showed absence of tetracyclic oxindole alkaloids in the sample, allowing its use for therapeutic and research purposes in accordance with U.S. Pharmacopeia.Each tablet of Unha de Gato Herbarium contained 100 mg dry Blood was collected in citrated, EDTA, heparin, and without anticoagulant Vacutainer tubes, before chemotherapy and after each of the 6 cycles. CAT and SOD activities were determined using whole blood diluted in a 1 : 20 saline solution.A COBAS INTEGRA system was used for the quantitative determination of the chemical constituents of the blood, and data were acquired using a COBAS INTEGRA 400 plus apparatus (USA).The carbonylation of serum proteins was determined through a modification of the Levine method .Lipid peroxidation was estimated by measuring TBARS in plasma samples according to a modification of the method of Jentzsch et al. .The determination of CAT activity level was carried out in accordance with a modification of the method of Nelson and Kiesow . SOD actBlood samples were analyzed using a Pentra apparatus (France). The lowest values were confirmed by observation of slides using May Grünwald-Giemsa stain and optical microscopy.ELISA assays of IL-6 were carried out according to a previously published method  at room Alkaline comet assays were performed as described by Singh et al. . One hun+ cells.Samples were collected in EDTA, and analyses were performed using a three-color fluorescence-activated cell sorter  and Multiset software (Becton Dickinson). FITC-conjugated anti-CD4, PE conjugated anti-CD8 and PerCp conjugated anti-CD3 were used. Immune subpopulations were measured as a percentage of the total number of CD3The Common Terminology Criteria for Adverse Events (AE) v3.0 (CTCAE) from the National Institutes of Health/National Cancer Institute-EUA  has beent-test and were expressed as a mean ± SD. When the variances were not homogenous and ANOVA was not appropriate , Wilcoxon two-sample test was used to evaluate the data. P < 0.05 was considered statistically significant.Data were analyzed using the EpiInfo computer program, version 3.5.1 from the CDC, USA. The data were evaluated using analysis of variance (ANOVA) and The general characteristics of colorectal cancer patients included in this study are shown in One aim of the study was to evaluate the neutropenia, thrombocytopenia, and anemia. The hemograms were analyzed each 15 days, and there were no significant differences in hematological parameters Tables  and 3 beUt supplementation did not change oxidative stress values or activity of the antioxidant enzymes. Similarly, the comet assay, a sensitive technique for the detection of DNA damage at the level of the individual eukaryotic cell, demonstrated no differences between groups  used for the evaluation of the immune status of CRC patients were not statistically different following Ut supplementation , and bilirubin levels, and kidney function is evaluated by dosage of urea, metabolic parameters , and constitutive parameters (weight loss) (data not shown). There was a small difference in creatinine levels between groups before treatment , which remained at the sixth cycle of treatment .The assessment of adverse events (AEs) related to treatment was conducted by interviewing the patients during each cycle of chemotherapy and through analysis of laboratory tests and observation of abnormal symptoms presented by patients. AEs were classified according to the Common Terminology Criteria for Adverse Events v3.0 (CTCAE v3.0) . Since ts Tables  and 7. AComplementary and alternative medicine (CAM) has been used by a large number of cancer patients worldwide. Cultural, socioeconomic, and spiritual differences affect the rate of use. For instance, there are high rates of use in Mexico (97.2%) and China (97%) , 24, an Ut in minimizing the main side effects of chemotherapy, a decrease in neutrophil and platelet counts, hemograms were analyzed before each FOLFOX4 cycle . Treatment with Ut was suspended on the days that patients received chemotherapy, because some antineoplastic drugs and CAMs are metabolized through the cytochrome P450 family, and an interaction could alter the metabolism of patients [Ut did not improve the WBC, RBC, or platelet counts, because there were no differences between groups (UT versus C). In vivo assays in rats undergoing chemotherapy demonstrated that neutrophils recover significantly sooner with Ut supplementation [Ut on WBC is due both to the stimulation of myeloid precursors [Ut. In a human volunteer study, Ut extract was given at 250 or 350 mg/day for 8 consecutive weeks to healthy adult. There were no statistically significant differences among the groups in WBC [Ut daily. In this study, we found significant differences between the group that received Ut and the control group; the Ut group showed higher neutrophils counts compared with the control group (unpublished data). The differences in the drugs used in the treatment of breast cancer  versus CRC  have to be considered, as do the differences in time between cycles of chemotherapy (21 versus 15 days). We must also consider the fact that all CRC patients in the present study underwent colectomy, which could interfere with the absorption of Ut.To evaluate the effectiveness of patients . Treatmeentation . This efecursors  and the ecursors  and inhiecursors . We did s in WBC . Our groUt, and its potent radical scavenger activity was confirmed by several assays including the following: the capacity to reduce the free radical diphenylpycrilhydrazyl (DPPH assay) [Ut extracts was further assayed through determination of TBARS production (using rat liver homogenates and sarcoplasmic reticulum membranes) and by the inhibition of free radical-mediated DNA-sugar damage [in vitro tests with one in vivo test [Ut, as assessed by lipid peroxidation (TBARS) and protein carbonyls. In addition, no differences were observed in the antioxidant enzymes SOD or catalase. Many previous studies have clearly shown the potential of the antioxidant H assay) , 31, theH assay) , and hydH assay)  as well H assay) , 32, andH assay) . The antr damage , 33. TheUt caused a significant decrease in DNA damage and a concomitant increase in DNA repair in volunteers. However, in our study, the comet assay did not demonstrate a significant difference in the group that received Ut. Ut extract was prepared through an extraction of ground bark with 70% ethanol. This process altered the composition of the extract  compared with an aqueous extract . Howeveg et al. . FurtherUt did not show an effect on the analyzed immunologic parameters, the CD4+ T cell-CD8+ T cell count and the IL-6 levels despite the in vitro evidence [Similar to oxidative stress, evidence .Ut extract at a repeated dose of 300 mg/day for 12 consecutive weeks (Unha de Gato Herbarium), when judged in terms of clinical symptoms, serum clinical chemistry, whole blood analysis, and leukocyte differential counts. Similar results have been shown in previous studies with volunteers [There were no drug-related toxic effects observed for lunteers , 36. Natlunteers  has repoAdverse events related to antineoplastic drugs  are well known  and are Ut at dose 300 mg dry extract daily is not effective in reducing the most prevalent adverse events due to treatment with 5FU/Leucovorin and oxaliplatin in patients with advanced CRC. No toxic effects related to Ut were observed in the group that received 300 mg dry extract daily for 12 weeks. Additional studies are needed to evaluate under which conditions, drugs, or types of cancer Ut might have a positive effect on treatment, in decreasing neutropenia and thrombocytopenia, or in improving the immune response.All the authors deny any conflict of interests. This work had a financial support from the government agencies CNPq and CAPES.


Across the first year of life, infants achieve remarkable success in their ability to interact in the social world. The hierarchical nature of circuit and skill development predicts that the emergence of social behaviors may depend upon an infant's early abilities to detect contingencies, particularly socially-relevant associations. Here, we examined whether individual differences in the rate of associative learning at one month of age is an enduring predictor of social, imitative, and discriminative behaviors measured across the human infant's first year. One-month learning rate was predictive of social behaviors at 5, 9, and 12 months of age as well as face-evoked discriminative neural activity at 9 months of age. Learning was not related to general cognitive abilities. These results underscore the importance of early contingency learning and suggest the presence of a basic mechanism underlying the ontogeny of social behaviors. During the first year of life, human infants develop remarkable social skills such as an emerging understanding of others' thoughts and intentions Associative learning via classical eyeblink conditioning is an ideal strategy to examine the relations between contingent learning and later social behavior development in human infants. It has been extensively used to examine learning in early infancy F4,276 = 24.75, P<.001, At one month of age, infant associative learning was measured using a delay eyeblink conditioning paradigm in which infants were presented with several pairings of a tone followed by a puff of air presented to the eye r51 = .39, P<.01, r45 = .36, P<.05). Infants who learned more rapidly at one month of age displayed heightened contingent social positivity at 5 months of age.At five months of age, the puppets and peek-a-boo tasks were administered r50 = .37, P<.01, At 9 months of age, the still-face and modified peek-a-boo tasks were used to examine social contingency detection. Detection of social contingencies in these tasks was measured by examining observed changes in infant behavior following violations of social expectation. A single Social Contingency Detection score was computed, averaged across both tasks. Remarkably, longitudinal stability of the predicted relations between 1-month early associative learning and 9-month social contingency detection was found, with one-month Learning Slope significantly positively correlated with Social Contingency Detection  were recorded while infants viewed images of their mother's face and a stranger's face Ps>.20; Infant cognitive performance on the Mullen Scales of Early Learning (MSEL) The current study is the first to directly assess predictive relations between early associative learning and the emergence of social behaviors over the first year of life. Data reveal that individual differences in associative learning measured at one month of age relate to later social behavior across a variety of tasks and ages. Moreover, we discovered a significant neural correlate by demonstrating a relation between 1-month associative learning rate and neural responses to familiar versus unfamiliar face stimuli. These relations were specific to social behaviors and were not the result of individual differences in general cognition, suggesting that early associative learning may serve as a major building block for the development of social behavior.These behavioral findings are consistent with the basic neurodevelopmental concept that both circuits and skills are built from simple to more complex, the latter being highly dependent upon the former The University of Maryland Institutional Review Board approved all procedures. Prior to data collection, written informed consent was obtained from a parent or caregiver during each visit.Seventy full-term healthy infants  participated in the current study. Families were contacted by mail using commercially available lists of names and addresses compiled from local hospitals and infant registries. Prior to the laboratory visit at one month, parents completed a brief phone survey. Infants were excluded from participating in the study if they were born prior to 38 weeks of gestation, had reported birth complications or injury, serious illness, or diagnosed syndromic disorder.ts<1; Ps>.2).The population of infants used in the current study was representative of the greater Washington, DC area with 51.4% Caucasian, 21.4% African American, 5.7% Hispanic, 4.3% Asian, and 17.1% mixed ethnicity. The infants' mothers were well educated with 41.1% completing a graduate degree, 38.6% completing a college degree, 4.3% completing a professional or trade certificate, and 14.3% completing a high school degree. Of the 70 families that visited the laboratory at one month, 10 did not return for follow-up visits for a permanent attrition rate of 15%. Of the remaining 60 families who continued to participate in the study, 55 returned for the 5-month visit, 51 returned for the 9-month visit, and 48 returned for the 12-month visit for attrition rates of 8%, 15%, and 21% respectively. Learning abilities of infants who participated in the follow-up assessments did not differ significantly from those who did not participate in the follow-up assessments  and auditory conditioned stimulus (CS). The airpuff was presented through tubing that was attached to a flexible plastic arm connected to the left speaker. The arm was positioned approximately 1 inch from the infant's left eyelid.th trial was an airpuff-alone trial to test the somatosensory response and the 10th trial was a tone-alone trial to test for a conditioned response. Stimuli were presented for a total of 15 blocks .Trials consisted of the presentation of a 1000-ms, 1000-Hz tone that overlapped and co-terminated with a 100-ms airpuff, yielding a 900-ms delay interval. In each block of 10 trials, the 6The raw electromyographic (EMG) signal was amplified using a custom bioelectric amplifier  with a gain of 1000 Hz and filtered using high and low pass filters of 1 and 1250 Hz respectively. The amplified signal was digitized at a sampling rate of 512 Hz using a 12-bit A/D converter (±2.5 V input range) and Snap-Master data acquisition software . Prior to recording EMG from each participant, a 50 µV 10 Hz signal was input into the channel and the amplified signal was recorded for calibration purposes. The raw signal was processed and analyzed offline using the EMG Analysis System from James Long Company . The signal was filtered digitally offline with a high-pass filter of 28 Hz, a low-pass filter of 250 Hz, and a digital band-stop filter (50–70 Hz) was used to remove 60-Hz noise. The signal was rectified and smoothed by using moving averages with a 20-ms window. Baseline EMG value was defined as the average activity recorded during the 20 ms prior to CS onset.Tone-alone trials were examined for the occurrence of conditioned responses (CRs). Each trial was visually examined for the occurrence of an eyeblink response that was defined as a rapid deflection in the EMG signal that was at least 1 SD above the mean of the baseline and occurred between 800 and 1500 ms after tone onset. It should be noted that previous studies examining eyeblink conditioning in awake adults have defined CRs as blink responses that are at least 5 SD above a mean baseline The percentage of CRs (%CR) across conditioning trials was used as the primary measure of learning and was computed across the 15 blocks in aggregates of 3 trials for a total of five 3-trial bins. To examine the relation between individual differences in learning and later social behaviors, the individual slope of the learning curve was computed for each infant by regressing %CR on Bin and used as the predictor variable in all subsequent analyses.During the 5-month assessment, two tasks were administered and maternal report on infant temperament was obtained. The tasks included were the peek-a-boo and puppets games The peek-a-boo and puppets tasks were specifically designed to elicit individual differences in displays of social positivity During the puppets game z scores and averaged across both the peek-a-boo and puppets games. Of the 55 infants who participated in these tasks, 4 were unable to be coded due to technical difficulties with the video recording and were not used in the current analysis.During the peek-a-boo and puppets games, behavioral positivity was coded separately including smiling intensity (0–3), intensity of vocalizations (0–3), and intensity of positive motor acts (0–2). The peek-a-boo game was divided into nine 10-s epochs for which the scores of each behavior were recorded. The puppets game was divided into a total of 5 epochs with four epochs equaling the time between each tickle and the last epoch occurring when the puppets were placed in front of the infant. In addition, presence of looking at the mother (0–1) was also coded during the puppets game as an index of social referencing. Two independent coders who were blind to the infants' 1-month learning abilities achieved sound inter-rater reliability on 20% of the data. Kappas ranged between .78 and .97 for behavioral positivity measured during the peek-a-boo game and .67 and .94 for the puppets game. The kappa for frequency of looking at the mother was .86. Individual measures for behavioral positivity were averaged across epochs for each game separately. The frequency of looking at the mother during the 5 epochs of the puppets game was computed. To obtain an overall Social Responsivity Score, behavioral positivity scores and frequency of social referencing were converted to Maternal report of infant temperament was obtained using the IBQ During the 9-month assessment, four tasks were administered in order to obtain individual differences in performance across several aspects of social behavior including social contingency detection, imitation, and social discrimination. These tasks include the still-face task During the still-face task In order to obtain a negativity proportion score for each interaction phase, the frequency of protest and withdrawn behavior was summed separately for each phase and divided by the total duration of each phase. The classic still-face response is a decrease in positivity and increase in negativity from the face-to-face interaction phase to the still-face phase During the modified peek-a-boo game z-scores and averaged separately across the trials in which the mother appeared behind the door and the trials in which the mother did not appear behind the door in order to obtain separate Social Positivity scores during mother-present and mother-absent trials. To examine individual differences in social contingency during the modified peek-a-boo task, a peek-a-boo Social Contingency Detection score was computed and defined as the difference between mother-present Social Positivity scores and mother-absent Social Positivity scores.Behavioral scores were converted to To examine imitation at 9 months, an experimenter presented the infant with four novel age-appropriate toys which were first described in studies conducted by Meltzoff During the mother-stranger face discrimination task Testing occurred while the infant sat on his or her mother's lap in a dimly lit room. Stimuli were presented using E-Prime software . Faces were presented on a black background and in the center of the screen. The computer monitor was 34 cm wide and 27 cm high. Infants viewed images at a distance of 60 cm. A camera mounted above the monitor allowed for simultaneous video recording of the infant's face during the experiment. The experimenter presented images during the experiment only when the infant was attending to the monitor. Trials were marked for deletion if the infant looked away during presentation of an image. Infants were presented 60 images of the mother's face and 60 images of the stranger's face. Faces were presented pseudo-randomly such that within every four presentations, the infant was randomly presented two images of the mother's face and two images of the stranger's face. Stimuli were presented for 500 ms followed by an inter-stimulus interval of at least 1000 ms during which time the screen was black with a white cross in the center.ERPs were recorded using a 64-channel HydroCel Geodesic Sensor Net . Signals were amplified using an EGI NetAmps 200 amplifier and sampled at 250 Hz with a band-pass filter of 0.1–100 Hz. Once the impedance values were reduced below 100 kΩ, data acquisition was started. EEG was recorded continuously and referenced to Cz, and after acquisition, data was re-referenced using an average reference.Data were filtered offline using a 30-Hz lowpass and a 1-Hz highpass filter. Trials consisted of a 400-ms baseline period and 600-ms period following stimulus onset. Data were baseline corrected to the average voltage during the 400 ms prior to stimulus onset. Data were segmented and visually inspected for EOG and motion artifact. Data from individual sensors were rejected if there was artifact resulting from poor contact or movement. The entire trial was excluded if more than 15 sensors had been rejected, or if an eyeblink or other significant movement artifact had occurred. Of the trials that were not rejected, individual channels containing artifact were replaced using spherical spline interpolation. Individual subject averages were constructed separately for the mother and stranger faces.r-values between Learning Slope and the Nc mean amplitude difference at each electrode site were subsequently submitted to EEGLAB software N = 4) or becoming too upset and unable to finish the task (N = 3). These infants were excluded from the current ERP analysis.Of particular interest to the current study was examination of the Nc component, a fronto-central negative deflection that reflects some aspect of visual attention During the 12-month assessment, one task was administered to examine infant joint attention and one task was administered to examine infant cognitive abilities. The tasks included were the Early Social Communication Scale (ESCS) rs were .767 and .921 for IJA and RJA, respectively. A total Joint Attention score was computed by averaging standardized scores (z-scores) of IJA and RJA.The ESCS The MSEL Repeated measures ANOVA with Bin as the within measure was used to determine if infants' learning increased across the course of the experiment. Pearson's correlations were used to determine if there were significant relations between individual differences in 1-month learning and 5-, 9-, and 12-month social outcome measures and 12-month cognition.


Further, performance of transgenic finger millet under drought, salinity and oxidative stress was studied at plant level in T1 generation and in T1 and T2 generation seedlings. Results from these experiments showed that transgenic finger millet had better growth under drought and salinity stress compared to wild-type. At plant level, transgenic plants showed better osmotic adjustment and chlorophyll retention under drought stress compared to the wild-type. However, the overall increase in stress tolerance of transgenics for the three stresses, especially for oxidative stress, was only marginal compared to other mtlD gene expressing plant species reported in the literature. Moreover, the Agrobacterium-mediated genetic transformation protocol developed for finger millet in this study can be used to introduce diverse traits of agronomic importance in finger millet.Finger millet is susceptible to abiotic stresses, especially drought and salinity stress, in the field during seed germination and early stages of seedling development. Therefore developing stress tolerant finger millet plants combating drought, salinity and associated oxidative stress in these two growth stages is important. Cellular protection through osmotic adjustment and efficient free radical scavenging ability during abiotic stress are important components of stress tolerance mechanisms in plants. Mannitol, an osmolyte, is known to scavenge hydroxyl radicals generated during various abiotic stresses and thereby minimize stress damage in several plant species. In this study transgenic finger millet plants expressing the mannitol biosynthetic pathway gene from bacteria, mannitol-1-phosphate dehydrogenase ( Eleusine coracana) constitutes ∼12% of the global millet area and is cultivated in more than 25 countries in Africa and Asia. It is an important food crop in India, Nepal and several African countries Finger millet , from Escherichia coli is known to catalyze mannitol production in various transgenic plants engineered with this gene and also these plants showed enhanced abiotic stress tolerance mtlD gene targeted to chloroplast showed increased hydroxyl radical scavenging capacity and retention of chlorophyll in leaf tissue under methyl viologen-induced oxidative stress Mannitol, a sugar alcohol, is an important osmolyte and scavenger of reactive oxygen species (ROS). This compound has been reported to impart tolerance to abiotic stresses in different plant species Agrobacterium tumefaciens (here after referred as Agrobacterium)-mediated transformation in finger millet. Also, by using this protocol mtlD gene expressing transgenic plants were developed and their tolerance under drought, salinity and oxidative stresses was studied.Finger millet is known to accumulate proline under stress The finger millet (var. GPU 28) seeds were obtained from all India coordinated research project , University of Agricultural Sciences, GKVK Bangalore India. For the seedling level experiments, seeds were germinated on moist filter paper at 30°C and 80% relative humidity. Seedlings (1.5 to 2 cm length) were used for stress experiments. For plant level experiments, plants were grown in plastic pots with the potting mixture consisting red soil: sand: vermiculite in the ratio of 3∶1∶1 by volume (22% water holding capacity). GPU28 was used for all experiments described in this manuscript except for optimization of hormonal concentrations for callus induction and plant regeneration in MS medium.Tobacco (var. KST 19) plants were grown in the greenhouse and ∼30–35 day old plants were used for stress experiments unless otherwise specified.−2 s−1.For both plant species, recommended fertilizers including micro and macronutrients were provided. Prophylactic measures were taken to maintain the plants disease and pest free. Plants were grown in the greenhouse at 22–27°C with 60% relative humidity and a 12 h photoperiod with mild-day light intensity of ∼500 µmol mUidA) mtlDHptII). These constructs were mobilized into Agrobacterium strain EHA105 and used for plant transformation experiments.The binary vectors pCAMBIA 1301 having beta-glucuronidase (Seeds were surface sterilized with 0.1% mercuric chloride for 5 min and then rinsed for four times in sterile de-ionized water. Sterilized seeds were placed on Murashige and Skoog (MS) medium Agrobacterium strains EHA105 harboring the binary constructs were grown on AB minimal medium with 50 mg/L kanamycin for 16 h at 28°C. Cultures (OD600 = 0.1) were resuspended in liquid co-cultivation medium supplemented with 100 µM acetosyringone . Calli (45 day old) were incubated with bacterial (carrying mtlD or UidA gene construct) suspension medium for 10 min. They were then blotted dry on sterile filter paper to remove excess Agrobacterium and co-cultivated for 48 h at 25°C in dark. Subsequently, they were washed using sterile water containing 300 mg/L cefotaxime for four to five times, blotted on sterile filter paper and inoculated on antibiotic selection medium . This step was repeated if Agrobacterium growth occurred.After three to four weeks of incubation the surviving calli were transferred to regeneration medium (MS with 0.5 mg/L BA) with hygromycin for plant-let growth and maintained in light for 20 days. Later, the shoot-lets were sub-cultured to rooting medium (MS with 0.5 mg/L BA and 0.1 mg/L NAA) for two weeks. Rooted plants were then transplanted in to soil mixture for further growth. Overall steps involved in mtlD transgenic plant development are described in Finger millet seeds from T1 generation were germinated on moist filter paper at 30°C for 36 h and uniform seedlings were acclimated with respective stress  for 8 h. Then, the seedlings were transferred to Petri plates with filter paper containing polyethylene glycol  or NaCl (300 and 400 mM) or the naptho-quinone compound, menadione , solution. PEG, NaCl and menadione induces osmotic, salinity and oxidative stresses respectively and solutions were prepared as described in previous literatures Fifteen day old plants were planted in pots filled with potting mixture of known weight. Pots were irrigated until all the soil macro and micro pores were filled and excess water was drained. Based on water holding capacity for this soil mixture, total weight of pot with soil mix for 100% field capacity (FC) was arrived. Plants were maintained at 100% FC (control) until imposition of drought stress. Drought stress was imposed by following gravimetric method as described previously −2 s−1 light for 2 h for stress acclimation. They were then exposed to high light (700–800 µmol m−2 s−1) with 2 µM methyl viologen for 6 h. Methyl viologen accepts electron from the last step of photosynthetic electron transport chain and produces free radicals Leaf segments (1 cm diameter) were taken from 45 day old transgenic and wild-type tobacco or finger millet plants grown under non-stress condition. The leaf segments were initially incubated in lower concentration (0.5 µM) of methyl viologen  with 100 µmol mUidA gene 5′-GGG CAG GCC AGC GTA TCG TG-3′ and 5′-GTC CCG CTA GTG CCT TGT CCA GTT 3′ and primer pairs 5′-ATC CGC TGC CCC TCT CT-3′ and 5′-ACA TGG CCT TTC AGC TGC GTG GTA-3′ that amplify 500 bp fragment of the mtlD gene. Primer pairs 5′- GAG GCT ATT CGG CTA TGA CTG-3′ and 5′-ATC GCG AGG GGC GAT ACC GTA-3′ were used to amplify 750 bp fragment of hptII gene. Further, primer pairs 5′-TCC ATA ATG AAG TGT GAT GT-3′ and 5′-GGA CCT GAC TCG TCA TAC TC-3′ were used to amplify 300 bp fragment of Actin gene.The genomic DNA was isolated from the leaves of putative transformed and wild-type plants. The transformed plants were confirmed by PCR using primers specific to UidA or mtlD genes were analyzed for integration and copy number of the transgene. Genomic DNA from the transgenic and wild-type plants were restrict digested (XbaI), resolved on 0.8% agarose gel and transferred to positively charged nylon membrane. The membrane was exposed to UV (1200 µJ for 60 s) for cross linking. The blot was probed with the respective radio labeled (32P dCTP 3000 Ci mmol) inserts. Pre-hybridization was done at 42°C for 2 h and hybridization was done at 55°C overnight with blocking solution . The hybridized blot was washed in the following sequence with 6× saline sodium citrate (SSC) for 10 min at 37°C, 4× SSC for 10 min at 37°C; 2× SSC for 10 min at 37°C; 2× SSC for 15 min at 55°C; 0.2× SSC for 10 min at 55°C. The hybridization signals were detected following exposure of Kodak X-ray film to the membrane at −70°C for 24 h and developed by autoradiography.Putative finger millet transgenic plants having Hpt) was prepared by labeling it with 32P dCTP as described under Southern blot. The blot was hybridized using the protocol described under Southern blot and developed by autoradiography.Genomic DNA (50 ng) from finger millet plants was spotted on the nitrocellulose membrane and fixed by exposing to UV (1200 µJ for 60 s) in an UV cross linker . Probe  and first strand cDNA was synthesized by oligo (dT) primers using Molony Murine Leukaemia Virus reverse transcriptase  according to manufacturer's instructions. The cDNA pool was used as a template to perform RT- PCR analysis using the following primers 0 generation) from the PCR positive transgenic plants were incubated at 37°C in GUS staining solution  for 8 h. After incubation they were transferred to 80% (v/v) ethanol/water solution to remove the chlorophyll, and later photographed. Similarly, seedlings (T1 generation) were stained with GUS solution and photographed.GUS activity in putative transformed callus was assayed as described in the literature th leaf was excised from stressed and well-watered plants and sampled at midday, quickly sealed in plastic bags, and kept on ice. These leaves were used to determine the relative water content (RWC) and solute potential.From the drought exposed  finger millet plants 5After determining the fresh weight, the leaf segments were floated on deionized water for 5 h to determine their turgid weight. The dry weight was determined after oven-drying to a constant weight. The RWC was calculated using the formula:Leaf samples were frozen in liquid nitrogen, thawed, and centrifuged for 5 min at 20 000 g. The Ψs of the extracted sap was measured by VAPRO vapor pressure osmometer . S100 - control leaf ΨS100From the values of RWC and solute potential of control and stress-grown plants, the osmotic adjustment was calculated using the formula: Osmotic adjustment (OA)  =  drought leaf ΨChlorophyll was extracted from 100 mg of leaf tissue in 1 ml of acetone and dimethyl sulphoxide  mix. The absorbance was recorded at 663 and 645 nm using UV–visible spectrophotometer Model DU800 . Total chlorophyll was estimated as described previously The standard graph was developed using Mannitol  using the previously described method Cell viability was measured by 2,-3,-5-triphenyltetrazolium chloride  as described in the previous literature Leaf segments (1 cm diameter each having dry weight (0.27 mg) were incubated in 1 ml of K-phosphate buffer  containing 500 µM of XTT  along with methyl viologen and exposed to high light stress. XTT is a sodium 3′[1--bis [4 methoxy-6-nitro] benzene sulfonic acid hydrate]. Later, increase in absorbance at 470 nm of incubation medium was measured using a micro titer plate reader  as described in the previous literature P≤0.05) or Duncan's multiple range test or students t-test. Statistical significance of values in graphs was indicated either as asterisks or as letters.Analysis of variance (anova) was performed using MS Excel software, and data points were analyzed for Fisher's least significant difference  seeds were used as explant . Among 1Agrobacterium strain EHA105 harboring pCAMBIA 1301/UidA constructs were co-cultivated for 48 h. The Agrobacterium culture was incubated with 100 µM of acetosyringone for virulence gene induction −2 s−1) for 10 days. The surviving calli were then transferred to regeneration medium (MS+0.5 mg/L BA+30 mg/L hygromycin and 300 mg/L cefotaxime). Later, as per previously standardized protocol  showed single copy integration of UidA gene in the finger millet genome  selected on the hygromycin  putative transgenic plants expressing mtlD were developed. Hygromycin resistant putative transgenic finger millet seedlings  from inde plants . Furtheric lines . Furtherern blot . The expy RT-PCR . The segy RT-PCR . Unlike Drought stress and salinity stress induces higher levels of free radical production in finger millet 1, mtlD-3 T1 and mtlD-5 T1) showed moderate level of stress tolerance under PEG-induced osmotic stress  were grown for 15 days in pots and used for testing their drought tolerance. Initially the plants were subjected to gradual acclimation stress and later maintained at severe stress levels for seven days. Phenotypic observations were made from the stressed plants (mtlD-1 T1) and wild-type. At 50% FC stress for seven days, wild-type and the transgenic plants did not show visible wilting phenotype. At much higher stress levels (20% FC) the transgenic plants showed improved tolerance to drought stress than wild-type plants (1 and mtlD-5 T1) exposed to 50% FC stress maintained higher osmotic adjustment and ∼10% less chlorophyll degradation in all the three transgenic lines than wild-type plants  germinated on hygromycin medium (2 seedlings showed no significant improvement in stress tolerance under PEG stress (2 seedlings showed only slight improvement in growth under PEG-induced osmotic stress (2 generation) were used for mannitol quantification. Results showed that mannitol content was high in all the tested seed lots (The Tn medium  were traG stress . Similarc stress . The trac stress . Stress c stress . In ordeeed lots . Similareed lots . This ca1). Wild-type tobacco plants are more susceptible to stress compared to wild-type finger millet In order to understand the reason for only marginal improvement in oxidative stress tolerance levels in transgenic finger millet, transgenic tobacco plants expressing mtlD was used and compared their stress tolerance increase with finger millet plants  of chlorophyll maintenance over wild-type under oxidative stress (mtlD gene showed only slight improvement (<3%) in oxidative stress tolerance  which were confirmed by PCR and Southern blot were grown in greenhouse and seeds were collected. For further experiments, out of 20 putative transgenic events, four T0 transgenic events were used. These four events were designated as mtlD-1, mtlD-3, mtlD-5, mtlD-16 and taken forward. Seeds obtained from three (T0) events  were used for further experiments. Seeds from each event were bulked and used for seedling experiment  or plant level experiment. Plants obtained from individual seeds representing each (T1) event were assessed for presence of mtlD gene by PCR and then seeds were collected from these plants. These seeds were designated as mtlD-1-2 T2, mtlD-3-1 T2 and mtlD-5-1 T2 and used for mannitol quantification. A sub-set of seedlings obtained from these seeds were used for stress experiments.(PDF)Click here for additional data file.Figure S2Germination of seeds obtained from UidA gene expressing finger millet transgenic plants on hygromycin. Wild-type (var. GPU28) finger millet seeds were germinated in petri dishes on filter paper with different concentration of hygromycin for 5 days at 30°C with 70% relative humidity in dark and germination percentage was recorded at the end of treatment period (A). Similarly, seeds obtained from the GUS positive transgenic finger millet plants  and wild-type (var. GPU28) were germinated on hygromycin (60 mg/L) and number of seedlings survived was recorded (B). Asterisks indicate values are statistically significant  versus corresponding wild-type. Each bar represents the mean of standard error values (n = 10).(PDF)Click here for additional data file.Figure S31 generation) and wild-type seedlings on hygromycin medium.Growth of mtlD expressing finger millet transgenic  and wild-type  were inoculated on the MS medium containing hygromycin (20 mg/L or 40 mg/L) for five days. Seedling growth on antibiotic medium was photographed at the end of treatment period.(PDF)Click here for additional data file.Figure S4Performance of mtlD expressing finger millet transgenic seedlings under osmotic, salinity and oxidative stress. Finger millet transgenic (T1 generation) and wild-type (var. GPU28) seedlings (1.5 cm length) were initially acclimated with lower concentration of corresponding stresses  for 8 h and then subjected to indicated concentrations of respective severe stress levels for 48 h. Seedlings were allowed to recover for two days and recovery growth of osmotic stress (A), salinity stress (B) and menadione induced oxidative stress (C) were measured and percent reduction in growth over corresponding control was calculated. The percent relative water content was measured from the plants exposed to 100%, 50% and 30% field capacity (D). Each bar represents the mean of standard error values (n = 20). Experiments were repeated twice. Alphabets above bar indicates the statistical significance (ANNOVA). Same alphabets indicate no significant difference (p<0.05).(PDF)Click here for additional data file.Figure S52 generation) and wild-type seedlings on hygromycin medium.Growth of mtlD expressing finger millet transgenic (T Seeds from mtlD expressing transgenic finger millet (mtlD-1-2 T2) and wild-type  were incubated on the MS medium containing hygromycin (20 mg/L or 40 mg/L) for five days. Seedling growth on antibiotic medium was photographed at the end of treatment period.(PDF)Click here for additional data file.Figure S6Mannitol accumulation in mtlD gene expressing finger millet germinating seedlings. Mannitol content in the seedlings obtained from T2 generation plants were estimated as described in material and methods. Alphabets above bar indicates the statistical significance (Duncan's multiple range test). Same alphabets indicate no significant difference (p<0.05).(PDF)Click here for additional data file.Figure S7Chlorophyll retention in tobacco and finger millet plants expressing mtlD gene under methyl viologen coupled with high light-induced oxidative stress. Leaf segments were taken from transgenic tobacco, finger millet (mtlD-5) and corresponding wild-type plants grown under non-stress condition. These leaf segments were exposed to high light (800 µmol m−2 s−1) stress with methyl viologen as described in materials and methods section. At the end of stress period, total chlorophyll was measured and percent reduction in total chlorophyll over their corresponding non-stress control was calculated. Values are mean of three replications and the error bar represents standard error. Alphabets above bar indicates the statistical significance (ANNOVA). Same alphabets indicate no significant difference (p<0.05).(PDF)Click here for additional data file.Figure S8Comparison of different features of genetic transformation protocols in finger millet reported in previous literature. In the literature, three research groups have demonstrated genetic transformation in finger millet. First, particle gun-mediated protocol was developed by Latha and co-workers. Second, Agrobacterium-mediated protocol was developed by Antony Ceasar and Ignacimuthu. Both protocol uses shoot apex as explants. Third, Sharma et al., and Jagga-Chugh et al., have demonstrated UidA gene expression. In this pictorial representation, different features of these protocols are compared with the protocols developed from this current study.(PDF)Click here for additional data file.Table S1Standardization of hormonal concentrations for efficient callus induction in finger millet (var. Indaf 9).(PDF)Click here for additional data file.Table S2Standardization of hormonal concentrations for efficient shoot induction from the callus in finger millet (var. Indaf 9).(PDF)Click here for additional data file.Table S3Efficiency of Agrobacterium-mediated finger millet transformation.(PDF)Click here for additional data file.Table S4Comparison of mtlD gene expressing plant species reported in the literature with their corresponding wild-type plants for their abiotic stress tolerance.(PDF)Click here for additional data file.


Clinical handover is an important process for the transition of patient-care responsibility to the next healthcare provider, but it may divert the attention of the team away from active patients. This is challenging in the Emergency Department (ED) because of highly dynamic patient conditions and is likely relevant in conditions that requires time-sensitive therapies, such as sepsis. We aimed to examine the management and outcomes of patients presenting with sepsis and septic shock to the ED during nursing handover.This retrospective cohort study was conducted at a 115-bed ED and more than 200,000 annual ED visits, within a 900-bed academic tertiary care center. Data on Surviving Sepsis Campaign (SSC) bundle elements and hospital mortality were collected for all ≥14-year-old patients who presented to the ED with a diagnosis of sepsis and septic shock between January 1, 2011 and October 30, 2013. Our primary outcome was time to antibiotics, were other SSC bundle elements and mortality counted as secondary outcomes. Patients were divided into two groups: 1) handover time group, comprising patients who presented an hour before or after the start of handover time (6–8 AM/PM), and 2) non-handover time group, comprising patients who presented over the remaining 20 h.P = 0.07), median time to serum lactate result  and median time to obtain blood culture , and hospital mortality rate .During the study period, 1330 patients presented with sepsis or septic shock . No significant differences were found between the handover time and non-handover time groups, respectively, in median time to antibiotic administration (100 [interquartile range (IQR) 57–172] vs. 95 [IQR 50–190] minutes; No significant differences were found in median time of SSC bundle elements or hospital mortality between patients who presented during the handover and non-handover times. Handover is an essential process in the Emergency Department (ED), as patient care is provided in a continuum around the clock manner; it follows, staff must work in shifts . HandoveNursing handover is a complex process that requires effective transfer of all required patient information in the most time-efficient manner. This process needs good communication skills and time management. Trivial miscommunication may lead to delivery of inaccurate or incomplete data, resulting in delayed care or other adverse effects , 4. NursReviewing the literature, we found ED handover studies that focused on evaluating the quality of care transfer, the need to initiate a standardized tool to aid the process, and on reporting handover-related errors and adverse events. Interestingly, we did not find any studies that investigated the effect of handover time on the patients with time-sensitive disorders who visited the ED during the nursing handover time. Sepsis is an important time-sensitive condition in which delays in providing care, such as delays in antibiotic administration, are associated with adverse outcomes. In particular, delays in antibiotic administration have been shown to be associated with a 7.6% decrease in survival for each hour of delay in antimicrobial administration during the 6 h after the first hour of documented hypotension . In thisThe study was performed in a large, urban, tertiary-care ED with an Emergency Medicine Residency Program. The ED is staffed with board-certified emergency medicine physicians, and has 115 beds. The number of annual ED visits range from approximately 200,000 to 214,000 per year at this 900-bed academic tertiary care center. In this study, we used data from the Sepsis Database, collected as part of a quality improvement project conducted by the Intensive Care and Emergency Medicine Departments. The Sepsis Database used the 2008 and 2012 Surviving Sepsis Campaign (SSC) tools.We included all patients aged ≥14 years who were admitted to the ED with a diagnosis of sepsis and septic shock between January 1, 2011 and October 30, 2013. In our institution, nursing shifts are 12-h based in all departments. Nursing handover process is done at the bedside using both verbal and written forms. The form used in our institution is SBAR  , which mWe extracted the following data from the Sepsis Database: ED arrival time, source of infection, physical examination findings, laboratory findings, time-to-antibiotic administration , time-to-lactate results and time-to-obtaining blood culture, mechanical ventilation requirement, and hospital mortality . The prit-test, and categorical values were compared using the chi-square test. All data management and analysis was performed with SAS .Because of the skewed data distribution, we have presented data as median and inter-quartile range for continuous variables, and frequency and proportion for categorical variables. Continuous variables were compared between two groups using the Over the study period, 1330 patients fulfilled the diagnostic criteria for sepsis and septic shock, and were included in the final analysis: 228 patients in the handover time group and 1102 patients in the non-handover time group.The presenting characteristics of patients who arrived during the handover time and those who arrived during the non-handover time are presented in Table P = 0.07). The distribution of median time-to-antibiotic administration by hour of day is presented in Fig. P = 0.33). The median time-to-obtaining blood cultures in the handover time group (54 [IQR 36–119] minutes) was not significantly different from that in the non-handover time group . The hospital mortality rate in the handover time group (29.4%) was not significantly different from that in the non-handover time group .All patients received antibiotics, and median time-to-antibiotic administration showed a tendency of being longer in the handover time group (100 [IQR 57–172] minutes) as compared to the non-handover time group (95 [IQR 50–190] minutes; Handovers are pivotal junctures and integral process in the continuity of care in every patient’s clinical course. To the best of our knowledge, the idea of handover time as a possible distractor that might delay urgent patient care is not addressed in current literature. We aim to shed light on the duration of handover process as a possible time where patient care is affected. In this study, we evaluated the direct effect of nursing handover process on patient care. We used a sepsis database to compare ED processes and outcomes between patients who arrived at the ED during nursing handover time and those who arrived during non-handover time.Our results showed a trend of longer time-to-antibiotic administration in handover group, however this was not clinically nor statistically significant. Nonetheless, the clinical value is unconvincing; as 5 min’ difference, might be minor when it comes to antibiotic delivery. It follows, additional studies with another time sensitive assessment tools are needed to address the clinical outcome of this delay, and how to prevent it. We found no significant association between ED nursing handover and time-to- lactate results or time-to-obtaining blood culture, or hospital mortality in patients admitted with a diagnosis of sepsis and septic shock. This could be explained by our ED nurses are vigilant with alerts and our institution was conducting SSC with constant reminders of early management and septic alerts . ConsequA prospective observational study addressing ED handover problems revealed deficiencies in the handover processes . These dThe main strengths of our study include the numbers of patients included, detailed data collection, the tertiary academic setting with numerous complex and critically ill patients, and standardized data collection using the SSC tools. As a retrospective cohort study, the present study has some important limitations. Foremost, the study aimed to evaluate the direct impact of handover time on time to time-to-antibiotic administration, time-to-lactate results, and time-to-obtaining blood culture, as surrogate indicators of the quality of sepsis and septic shock management. Nonetheless, other important measures in sepsis management were not investigated such as time to effective fluid resuscitation. Additionally, there is inherent variation and subjectivity in the handover process among ED nursing staff might have underpowered our results. Lastly, because of the retrospective nature of this study and the fact that it was conducted in a busy ED, others factors, such as ED overcrowding and boarding patients in ED, could have affected the study results.This is one of the first reports of the impact of ED nursing handover on time-sensitive interventions that involve multiple tasks performed by ED nurses. Due to the retrospective nature, patient population with single pathology, and our structured handover process that might have reflected on the results of this study. Future studies are still needed to explore ED functionality during the handover time.


The effect of swap disorder on the physical properties of the quaternary Heusler alloy PdMnTiAl is described from first principles. Y-type1, in good agreement with the Slater–Pauling rule. From the band structure and the density of states, it is predicted that this Y-type1 configuration is a new gapless semi-metal material. Furthermore, it was discovered that the Pd–Mn swap-disordered structure is more stable than the Y-type1 structure. The present work provides a guide for experiments to synthesize and characterize this Heusler alloy.Heusler alloys crystallize in a close-packed cubic structure, having a four-atom basis forming a face-centred cubic lattice. By selecting different composite elements, Heusler alloys provide a large family of members for frontier research of spintronics and magnetic materials and devices. In this paper, the structural, electronic and magnetic properties of a novel quaternary Heusler alloy, PdMnTiAl, have been investigated using a first-principles computational materials calculation. It was found that the stable ordered structure is a non-magnetic  The Y-type1 structure with space group b), the Y-type2 and Y-type3 configurations are found from our calculations to have relatively higher total energies. Both VASP and AkaiKKR were used to optimize the structures of these three types of alloy to obtain the equilibrium lattice parameters. Similar trends were observed, such that the Y-type1 configuration had the smallest lattice parameter and Y-type3 the largest. A comparison of the two different calculation methods is shown in Table 1VASP and AkaiKKR predictions for the magnetic moments. The Y-type1 configuration of PdMnTiAl was found to have zero magnetization by both methods. To confirm the DFT calculation results, we checked the magnetic moments with the Slater–Pauling (SP) rule , Ti (s2d2), Mn (s2d5) and Al (s2p1). Thus the structure of our Y-type1 configuration complies perfectly with the Slater–Pauling rule.First, the ordered quaternary Heusler alloy PdMnTiAl is considered. Fig. 1VASP and AkaiKKR. Figs. 2AkaiKKR as with VASP. Fig. 2Y-type structures have bands which cross the Fermi level, thus indicating metallic behaviour. The nonmagnetic Y-type1 structure has a pseudo-gap-like DOS at the Fermi level, indicating a semi-metal, whereas the magnetic structures Y-type2 and Y-type3 have Fermi levels located near DOS peaks. This may help to explain the energetic instability of the Y-type2 and Y-type3 configurations. In Fig. 3From the optimized structures, we calculated their density of states (DOS) and band structures using Y-type structures have different total energies and magnetic moments, and PdMnTiAl is more likely to have the Y-type1 structure. The Y-type2 and Y-type3 structures have higher energy, larger lattice parameters and greater magnetic moments than the Y-type1 structure. To investigate the atomistic disordered configurations of these quaternary Heusler alloys further, we carried out advanced calculations using AkaiKKR. Based on the above ordered structures, we considered a number of possible swap disorder types by intermixing between any two of the Pd, Ti, Mn and Al atoms. In the following, we use the numerals I, II, III, IV, V and VI to represent interchanges, or swaps, between Ti–Mn, Pd–Mn, Ti–Al, Pd–Al, Pd–Ti and Mn–Al, respectively, and VII, VIII, IX, X, XI and XII to represent Pd–Mn, Ti–Al, Pd–Ti, Mn–Al, Ti–Mn and Pd–Al swaps, respectively. For example, the M point in Fig. 40.7Mn0.3)Ti(Mn0.7Pd0.3)Al with a Pd–Mn swap. Fig. 4Y-type1. Moreover, the Pd–Mn swap not only lowers the total energy, but also gives rise to significant magnetization. The new ground-state disordered structure (Pd0.7Mn0.3)Ti(Mn0.7Pd0.3)Al has an energy decrease of 0.092 eV per formula unit compared with the ordered Y-type1 PdMnTiAl, with its total magnetic moment enhanced to 0.964 µB. The half-and-half Pd–Mn randomly disordered configuration (Pd0.5Mn0.5)Ti(Mn0.5Pd0.5)Al is 0.047 eV lower in energy per formula unit and has the maximum magnetic moment of 1.132 µB. In the other cases of disorder, the total energies are increased by the disordering effect and the swap disorders tend to introduce finite total magnetization. We also calculated the DOS for all the different disordered structures, as presented in Fig. S1 in the supporting information.From the above calculations the ordered VASP to verify the validity of the corresponding AkaiKKR results. In a double-sized supercell with 32 atoms (eight formula units), 25% swap disorders were simulated by exchanging two of the eight Pd atom positions and two of the eight Mn atom positions. Similarly, 50% swap disorders were simulated by exchanging four of the eight Pd atoms and four of the eight Mn atom positions. Four possible disordered supercell structures for the 50% configurational swap disorder are shown in Fig. S2 in the supporting information. We found the swap-disordered structures to be more stable than the ordered PdMnTiAl Y-type1 structure and have also verified qualitatively the correctness of the magnetic moments. The results, shown in Table S1 in the supporting information, suggest that much larger supercells might be necessary to achieve a better quantitative comparison between AkaiKKR disorder calculations and VASP supercell calculations. This comparison is beyond our current computational resources. However, experimental work is expected to synthesize and characterize the proposed PdMnTiAl quaternary Heusler alloy for eventual confirmation of our prediction.We attempted to construct larger supercells and to use 4.Y-type1 is more stable than those of Y-type2 and Y-type3. The semi-metallic Y-type1 configuration shows zero magnetic moment, in good agreement with the Slater–Pauling rule. Interestingly, we discovered that the Pd–Mn swap-disordered structure is more stable than the ordered Y-type1 configuration, and that the total magnetizations of these disordered (Pdx1−Mnx)Ti(Mnx1−Pdx)Al compounds are dependent on the degree of Pd–Mn swap, 0 < x < 1. We hope these findings will stimulate further investigation into spintronics materials and devices.The structure and electronic and magnetic properties of the quaternary Heusler alloy PdMnTiAl have been investigated by first-principles calculations. In these compounds, the ordered configuration of 10.1107/S205225251700745X/ct5004sup1.pdfAdditional table and figures. DOI:


Prognosis for metastatic melanoma has improved significantly with the use of immune checkpoint inhibitors. Given improvements in survival, aggressive surgical treatment may be considered in patients with life-threatening complications from their disease that would not otherwise be considered in advanced disease. Patients with preexisting autoimmune diseases or prior immune-related adverse events from therapy are largely excluded from clinical trials. Concerns exist that immunotherapy in these patients could worsen autoimmune disease or increase the risk of developing additional immune-related adverse events on therapy. We present a case of a patient with rheumatoid arthritis that presented with obstructive heart failure secondary to melanoma that had metastasized to the right atrium. After aggressive surgical resection to stabilize him from his life-threatening heart failure, he was treated with ipilimumab, which was stopped due to an immune-related adverse event. He was then started on pembrolizumab and had a durable response to therapy. Aggressive surgical treatment should be considered in patients with a cancer that may respond to immunotherapy. Furthermore, some patients with preexisting autoimmune disease may be safely treated with checkpoint inhibition therapy, and patients with a severe immune toxicity from one class may successfully be treated with an alternate class. Melanoma is an aggressive cutaneous malignancy that accounts for 1 to 2 percent of all cancer-related deaths annually [Immune checkpoint inhibition therapy for metastatic melanoma has been shown to improve survival. Monoclonal antibodies targeting the cytotoxic T-lymphocyte antigen 4 (CTLA4) and programmed death-1 (PD-1) pathways inhibit downregulation of the immune system, thereby allowing an enhanced T-cell immune response. These pathways are essential regulators in immune tolerance tissue, and their inhibition could lead to a myriad of autoimmune conditions known as immune-related adverse events (irAEs). Patients with preexisting autoimmune diseases were excluded from clinical trials of these therapies, and only one trial included patients with a prior irAE . Here, wA 54-year-old white male with a past medical history of rheumatoid arthritis on anti-TNFalpha therapy with etanercept was admitted to the hospital with a 3-month history of dyspnea on exertion, fatigue, and lower extremity edema after a transthoracic echocardiogram (TTE) revealed a reduced ejection fraction of 40% with a large right atrial mass. Cardiac magnetic resonance imaging (MRI) identified a 5.4 × 5.3 centimeter lobulated right atrial mass  with extThe patient was stabilized and discharged with outpatient medical oncology follow-up to discuss treatment. However, days prior to his appointment he returned to the Emergency Department with worsening dyspnea due to the right atrial mass. Although he had not received treatment for his metastatic melanoma, heart failure due to obstructive cardiac metastasis is generally a poor prognostic indicator. Consequently, the benefits and risks of the procedure were extensively discussed between the medical oncologists and cardiothoracic surgeons. It was determined to proceed with aggressive measures, given the potential for long-term durable responses from immune checkpoint inhibitor therapy. He underwent a radical resection of the right atrial mass  and recoAfter recovery from surgical resection of the metastatic heart lesion, the patient was started on immunotherapy. The patient's rheumatoid arthritis was previously well controlled with etanercept monotherapy, which was stopped prior to treatment. He was started on 3 mg/kg dose of ipilimumab (anti-CTLA4) every 3 weeks. After 3 doses, he developed grade III acute kidney injury, nephrotic-range proteinuria, and anasarca requiring hospitalization. Renal biopsy demonstrated minimal change disease with acute interstitial nephritis. His creatinine increased to 5.22 mg/dl from a baseline of 1 mg/dl. He was treated with pulse methylprednisolone at 1 gm/day and intravenous diuretics with gradual improvement in his kidney function. He did not require hemodialysis, and his steroids were successfully tapered over the next 3 months. Following his steroid taper, imaging demonstrated tumor progression in his peritoneal cavity despite improvement in his liver lesions and no recurrence in his heart.Given clear evidence of progression outside of the pseudoprogression window, he was started on 2 mg/kg pembrolizumab (anti-PD-1) every 3 weeks. He had an objective response to treatment with reduction in tumor burden in his liver and peritoneal cavity. Before treatment with pembrolizumab, the patient's rheumatoid arthritis was controlled with prednisone 5 mg daily. After pembrolizumab treatment, he began experiencing diffuse arthralgias. Although arthralgias are a known side effect of anti-PD-1 inhibitors, he experienced significant morning stiffness and wrist swelling, indicating that he was having a true flare of his autoimmune disease. He was started on hydroxychloroquine for symptom control. With low-dose prednisone and hydroxychloroquine, his synovitis remained stable and low grade. Despite concurrent ongoing treatment with hydroxychloroquine and low-dose prednisone, his disease continued to respond to immunotherapy. The patient was treated with pembrolizumab for approximately 19 months, and he is currently living nearly 3 years after diagnosis in clinical remission and off therapy. He had no recurrence of kidney injury on PD-1 monotherapy.While metastatic melanoma frequently involves the heart, it is rarely diagnosed because it is typically asymptomatic . PatientMultiple studies have demonstrated the importance of the CTLA4 and PD-1 pathways in immune tolerance. Both play a critical role in immune regulation, and decreased gene expression is associated with an increased risk of autoimmune disease –9. PatieOur patient's durable response to anti-PD-1 therapy with minimal side effects is impressive given his history of rheumatoid arthritis and prior high-grade irAE. Successful treatment of a patient with anti-PD-1 therapy following high-grade irAE from ipilimumab therapy, without recurrence of that irAE, suggests that it may be possible for patients to be safely treated with immune checkpoint inhibitors of an alternate class following resolution of immune toxicity from the first agent.A recent retrospective study evaluated the safety of anti-PD-1 use in 119 patients with either a preexisting autoimmune disease or an irAE related to prior ipilimumab treatment . 38% of Our patient's clinical course is consistent with the emerging evidence that anti-PD-1 therapy is less toxic and yields better outcomes in comparison to anti-CTLA4 therapy . Despite


Many genetic causes of focal segmental glomerulosclerosis (FSGS) have been described. A paradox is that the science in the molecular biology, which generally appears of high quality, is not mirrored by a similarly critical analysis of the renal pathology. FSGS has been applied to such a wide range of conditions that it can reasonably be said to have no useful meaning. Attempts to refine the term have been largely ignored. Study of 252 papers on genetic causes of FSGS found various clinical features. Many papers took the reported diagnosis without question. Few papers reported a pathological review, almost half reported FSGS and up to six other conditions caused by any particular gene, some reported FSGS with recognisable glomerular disorders, over 80% did not apply the Columbia classification, and in nearly all with photomicrographs, the images were not useful for refinement of FSGS. Some workers commented on a lack of genotype-phenotype correlation. One reason is a disregard of the principle that scientific investigation requires an unambiguous definition of the condition studied, to allow others to replicate or refute the findings. Genetic studies of FSGS should use a similarly rigorous approach to renal pathology to that used in molecular biology.The online version of this article (10.1007/s00467-018-4161-6) contains supplementary material, which is available to authorized users. Many genetic causes of focal segmental glomerulosclerosis (FSGS) have been described. As examples, Gast et al.  gave a lThe molecular biology in papers on genetic causes is presumably rigorously considered by referees and editors before acceptance by journals. Mutation of a single base pair in an exon of a gene is commonly reported as a significant finding, although other genetic features such as polymorphisms are also said to be associated with FSGS. The mere finding of an alteration in a gene is no longer enough by itself to justify the conclusion that the name of the gene can be added to the list of those in which a mutation can cause FSGS. Various conditions should be satisfied before this can be done, listed and discussed in detail by Lovric et al.  and otheA scientific paradox considered in this review is the discrepancy between the rigour, precision, close control, reproducibility and generally high quality of the molecular biology and if applicable the experimental work in studies of genetic causes of FSGS and the widespread lack of any of these properties in the selection of cases that are included in reports.FSGS continues to be used as a diagnosis even though virtually every paper or review accepts that the term has no useful, definable meaning applicable to a single condition , 11–13. FSGS is often defined tautologously as the finding on microscopy of sclerosed areas, meaning areas that appear solid and stain black with periodic acid-methenamine silver, that are segmental, meaning in only part of a glomerular tuft, and focal, meaning not in every glomerulus. There is a view that more detailed pathological study does not contribute to understanding of FSGS. As examples, comments can be found that ‘At a practical therapeutic level all FSGS should be considered equal’  and ‘TheEven the common attempt at refinement by the division into primary and secondary types of FSGS is crude, imprecise and unsatisfactory. What do these terms mean? Is FSGS caused by a genetic disorder primary or secondary or neither? FSGS of presumed genetic cause is often said to be not primary, and so is presumably or explicitly secondary , 13, 18,The Columbia classification of FSGS was an attempt by pathologists to reduce the ambiguity of the term FSGS by the introduction of five types or variants, called cellular, collapsing, hilar, tip and not otherwise specified (NOS) . Even thThere was a range of ways in which people were identified and reported to have a genetic cause of FSGS. Most were found by study of groups with features in common, but some were explicitly reports of a single case or more than one case. The distinction between a case report and a series was not always easy to make, especially in papers about families with an apparently inherited condition. Of the 252 papers, 199 were regarded as series in the broadest sense, including most with familial cases, and 53 as case reports. Familial cases were reported in 71 papers, sporadic cases in 25 and both familial and sporadic cases in 56, while 100 papers did not say whether cases were familial or sporadic. There was a range of ages studied. Only children were included in 119 papers, only adults in 50 and both children and adults in 74, while nine papers did not specify the age of subjects.Clinical features of cases varied. There were three reasons why there was difficulty in determining precise numbers of subjects with different clinical features. At least 17 papers included patients reported in previous studies, sometimes without identifying which ones were included. Patients with different features were grouped in some papers. Some papers did not even give clinical details. The nephrotic syndrome was a common clinical finding and a common reason for inclusion in series, but sometimes, this was specifically only steroid-resistant nephrotic syndrome, sometimes both steroid-resistant and steroid-sensitive nephrotic syndrome, sometimes unspecified nephrotic syndrome and sometimes not differentiated from non-nephrotic proteinuria. Congenital nephrotic syndrome was sometimes included with other types of the nephrotic syndrome, sometimes excluded, sometimes the only clinical feature and frequently not mentioned in childhood studies.For the reasons given, precise numbers of these different features cannot be determined, but at least 145 series appear to have had the nephrotic syndrome as the main or only clinical feature. Other clinical features, if they were mentioned, included such things as non-nephrotic proteinuria, microscopic haematuria, renal failure, hypertension and combinations of features. Some of these were the main reason for inclusion in series.A diagnosis of FSGS was a reason for selection of cases for study in 70 series, but this was accompanied by a variety of clinical features, and was not necessarily the only reason for selection. Accordingly, there is an overlap between these 70 papers and many with the nephrotic syndrome. Thirteen papers did not specify any clinical features in those with FSGS. Ten papers selected FSGS specifically with the nephrotic syndrome but not all with the same type of nephrotic syndrome, as described above, while 34 papers selected FSGS with the nephrotic syndrome or other clinical features, eight selected either FSGS or the nephrotic syndrome and five selected FSGS with other glomerular disorders, such as IgA nephropathy or thin glomerular basement membrane disease. In some series, particularly on patients with steroid-resistant nephrotic syndrome, some were said to have FSGS even though a renal biopsy was reported to show something else , or patiWT1 mutations, such as Frasier and Denys-Drash syndromes (14 papers), various disorders associated with mitochondrial DNA abnormalities, such as MELAS syndrome and maternally inherited diabetes mellitus and deafness (10), Alport syndrome (6), Charcot-Marie-Tooth syndrome (5), Galloway-Mowat syndrome (3), Pierson syndrome (3) and Schimke immunoosseous dysplasia (3). Among the others were seven with tubular disorders, particularly Dent syndrome (3) and Bartter syndrome (2). Other reasons for inclusion in series were, as examples, that there was a known genetic mutation or polymorphism, or simply that the patients had been referred for genetic testing.In 62 papers, both series and case reports, the reason for inclusion was a variety of syndromes that included renal disease, not necessarily with the nephrotic syndrome. The most numerous syndromes were those with There were various clinical reasons for exclusion of cases from 44 series, particularly causes of secondary FSGS such as reduced renal mass or obesity  or extrarenal features (8). HIV infection was included in seven other series. The remaining series and case reports either included patients with extrarenal features (59) or did not mention whether there were any exclusions on clinical grounds (142).NPHS1 and NPHS2, a couple of the earliest genes to be described. In theory, every paper should have searched for all reported genetic causes before an apparently new abnormality was described, which may have been a coincidental and irrelevant finding. Similarly, in theory, whenever any new cause is published, authors of every previous paper should review their cases to see whether that cause was missed, and was a more likely cause or contributor to the condition. Bierzynska and Saleem [In the 252 papers, 91 excluded an abnormality in at least one relevant gene, such as mutations in d Saleem  warned tThis overview shows that genetic studies of FSGS had no consistency in the population studied. Although many patients with FSGS had steroid-resistant nephrotic syndrome, some did not. Was the FSGS reported in anyone with steroid-resistant nephrotic syndrome, for example, the same as the FSGS reported in a case of congenital nephrotic syndrome, or an adult with asymptomatic proteinuria, or a child with Alport syndrome or someone with a tubular disorder?GLA, mutated in Fabry disease, which affects podocytes [COQ2, INF2, LAMB2, LMX1B, SMARCAL1 and WT1, and so genes causing FSGS can affect podocytes and cells outside the kidney. Similarly confusing is the finding that several genetic abnormalities that cause tubular disorders [CLCN5, mutated in Dent disease 1 [SLC12A1, mutated in Bartter syndrome type 1 [The concept of phenocopies of genetic causes of FSGS or steroid-resistant nephrotic syndrome was introduced by Warejko et al. , withoutodocytes . The nonodocytes  includedodocytes , such asisorders  have beeisease 1 , and SLCe type 1 , althouge type 1 –39, and e type 1 , 41.WT1 mutations, and Sadowski et al. [NPHS2 mutations. Can genetic abnormalities really cause multiple glomerular disorders, or is a more likely explanation that there was inconsistency between pathologists in these studies?There seemed a readiness of many pathologists to apply the term FSGS and a readiness of those doing the genetic studies to accept the diagnosis. In only 21 papers (8% of 252) was there a mention that there was a review of light microscopic sections on every renal biopsy or every available biopsy or most biopsies by at least one pathologist. This should in theory give consistency in the cases included and excluded e.g. , 42, 43.i et al.  reportedSometimes, among conditions as well as FSGS that were reported in particular mutations, unsatisfactory terms appeared, such as ‘minimal unspecific glomerular changes’, ‘unclear (or no) diagnosis’, ‘mild (or minor) glomerular changes’, ‘nonspecific tubulointerstitial nephropathy’, ‘not classifiable’, ‘unclassifiable glomerulopathy’, ‘nonspecific pathology’ and ‘no findings’. This again emphasises how unreliable is mere copying of reports from a variety of pathologists, especially those who are not experts in renal pathology. Also, FSGS was said to occur in glomeruli with diagnoses such as IgA nephropathy , diabetiApplication of the Columbia classification would be a test of whether a paper attempted to refine the diagnosis of FSGS. Columbia appeared in 2004 , althougPhotomicrographs were common in papers on genetic causes of FSGS, and were included in 86 of 252 papers (34%). A problem is that the figures were almost always just of one glomerulus, and an illustration of one glomerulus in itself generally is of little value. For satisfactory interpretation of a segmental glomerular lesion, the ideal information should be a description of the lesion, its site within the glomerulus, which requires the landmarks of the hilum and tubular origin to be shown, the condition and size of the rest of the glomerulus, the number of glomeruli affected and the condition of the rest of the kidney . Hardly No paper mentioned that the position of segmentally sclerosed glomeruli in the cortex was analysed. Following the work of Rich , FSGS isThese findings on the selection, diagnosis, description and illustration of cases show that the literature on genetic causes of FSGS is in confusion. One reason why this is important is because the first requirement for study of any condition is to have a precise definition of that condition so that as in any scientific study, other workers can investigate and replicate or refute the original findings. At the moment, there can be little certainty that any paper on FSGS is studying the same condition as any other paper. The confused state of genetics of FSGS contrasts sharply with the relatively much more straightforward genetics of membranous nephropathy, a diagnosis that is uncontroversial compared with FSGS . The criAnother reason why this is important is related to understanding of the pathogenesis of segmental lesions. A few papers reporting mitochondrial DNA mutations have shown that the lesions in affected glomeruli are at the vascular pole or hilum and are associated with abnormalities of arterioles –37, 39. DGKE variants causing a glomerular microangiopathy: ‘There were also secondary focal and segmental sclerotic glomeruli, which could be seen in the advanced stage of any glomerulopathy’. Although most genes said to cause FSGS are expressed in podocytes [ACTN4 mutations, even though some biopsies did not seem to show significant loss of renal mass, nor glomerular enlargement. They speculated that podocytes with ACTN4 mutations were less mechanically robust than normal podocytes, and more sensitive to damage by normal glomerular capillary pressures. No papers analysed the distribution of segmentally sclerosed glomeruli in the cortex to see if genetic disorders showed the typical childhood pattern, with juxtamedullary glomeruli affected first [Hardly any other papers suggested a mechanism of development of segmental sclerosing lesions, other than vague ideas that the lesions were due to podocyte disorders, which would be expected to produce lesions randomly distributed in glomeruli . One papodocytes , 29, 30,odocytes , who fouodocytes , were coed first , 55.NPHS1 and NPHS2 mutations’. The importance of phenotyping in genetic studies, including the renal biopsy diagnosis, has been stressed by others [A more scientific approach to all cases of FSGS may reveal more precise genotype-phenotype correlations than are currently known. Until this is done, the study of genetic causes of FSGS will remain unsatisfactory. Some workers have expressed disillusionment with reported renal biopsy findings. These include Bierzynska et al. , who wroy others , 64. They others .ESM 1(DOCX 83 kb)


Risk assessment and therapeutic options are challenges when counselling patients with an atypical ductal hyperplasia (ADH) to undergo either open surgery or follow-up only.We retrospectively analyzed a series of ADH lesions and assessed whether the morphological parameters of the biopsy materials indicated whether the patient should undergo surgery. A total of 207 breast biopsies [56 core needle biopsies (CNBs) and 151 vacuum-assisted biopsies (VABs)] histologically diagnosed as ADH were analyzed retrospectively, together with subsequently obtained surgical specimens. All histological slides were re-analyzed with regard to the presence/absence of ADH-associated calcification, other B3 lesions , extent of the lesion, and the presence of multifocality.p = 0.002) and presence of multifocality in VAB specimens (p = 0.0176) were significant risk factors for the underestimation of the disease . In the multivariate logistic regression model, the absence of calcification (p = 0.0252) and the presence of multifocality  in VAB specimens were significant risk factors for underestimation.The overall underestimation rate for the whole cohort was 39% . In the univariate analysis, the method of biopsy , we could not identify a subgroup that would not require an open biopsy. Atypical ductal hyperplasia (ADH) is a small, mostly unifocal, low-grade intraductal lesion in the breast, which in most cases is detected by the associated calcification seen on mammograms –4. The hIn our study, we collected histological slides from 207 patients diagnosed with ADH with either a preoperative diagnostic core needle biopsy (CNB) or VAB and correlated the histological findings with the biopsy method and the diagnostic outcome after a subsequent open biopsy. Our aim was to identify any histological parameters predictive of outcome in any subgroup of patients.We analyzed the clinicopathological data of 207 patients histologically diagnosed with ADH, classified as B3 , or B4 , by CNB or VAB, who had undergone subsequent open surgery in 2002–2015. Of the 207 diagnostic biopsies, 56 were CNBs and 151 were VABs. All biopsies were performed at the Breast Center Seefeld Zurich and were histologically processed at the Institute of Pathology and Molecular Pathology of the University Hospital Zurich, Switzerland.® , Suros Eviva® , or Mammotome® . In general, 7G or 9G needles were used for the biopsies performed under magnetic resonance imaging (MRI) and ultrasound (US), and 11G needles were used for stereotactic or tomosynthesis-guided biopsies. All procedures were performed under local anesthesia, except in two patients who required general anesthesia because multiple bilateral lesions were removed under US guidance.In 2002–2015, 5213 VABs were performed at the Breast Center Seefeld Zurich, using EncoreOf the 5213 VABs, 1722 were guided by US (handheld), 125 by MRI, and 3366 by stereotactic surgery . The main indication for stereotactic or tomosynthesis-guided VAB was suspicious microcalcifications, and the indications of asymmetric density and architectural distortion were observed in only a few patients. MRI-guided VAB was indicated when a suspicious MRI finding could not be confirmed by mammogram or a second look US. The indication for handheld VAB was a US finding requiring histological clarification. As well as benign findings (71.30%), DCIS (10.5%), and invasive cancers (3.95%), 13.8% of the specimens showed cellular atypia, and 191 (3.7%) of these were classified as ADH.In 2001–2015, 15,528 US-guided CNBs (with 14G needles) were performed, 66 of which indicated a diagnosis of ADH.The decision to perform initially either CNB or VAB depended on the imaging characteristics. CNB was the preferred method in case of a visible, mostly mass lesion on ultrasound while VAB was the preferred method in cases with mammographically detected calcification not visible at ultrasound.We included in this study only patients with ADH who had undergone subsequent open surgery and for whom it was possible to correlate the ADH diagnosis based on VAB or CNB with that based on the surgical specimen.The statistical analyses were performed with frequency tables and univariate and multivariate logistic regression models. All statistical analyses were performed by the Swiss Group for Clinical Cancer Research, Bern, Switzerland.All biopsy materials were fixed in 4% neutral buffered formalin and embedded in paraffin blocks with routine histological processing. Sections (2 µm thick) were prepared from the paraffin blocks; different levels from each block were cut separately and stained routinely with hematoxylin and eosin.For all biopsies, the diagnosis of ADH was made according to the current World Health Organization classification of breast tumors . ADH wasThe presence of calcification and multifocality (as B3 or B4 category) was noted. Findings additional to ADH, including lobular neoplasia (LN), radial scar (RS), flat epithelial atypia (FEA) and intraductal papilloma, were noted in the reports. In many cases, immunohistochemical analyses of estrogen receptors and basal cytokeratins 5/6 were also performed to confirm the ADH diagnosis. The diagnosis of ADH was confirmed if immunohistochemistry showed the diffuse upregulation of estrogen receptors and negativity for basal cytokeratins  Fig. 1)1).The whole cohort underwent a retrospective review, and the histological  slides were reviewed by two pathologists who are specialists in breast pathology . The following features were assessed in the review: the histological diagnosis of ADH, the presence of multifocality in the biopsies, the presence of necrosis and associated calcification, and the extent of the lesion (in millimeters). The presence of FEA, RS, LN, and papilloma was also noted in the histological review.Additionally, an age parameter (cut-off at 50 years) was added to the criteria.‘Upgrade’ was defined as a diagnosis of DCIS or invasive carcinoma based on the subsequently obtained surgical specimens  could be re-analyzed by reviewing the histological slides and classifying ADH according to the histological criteria mentioned above.p = 0.002) and the presence of multifocality in the VAB specimens  were significant risk factors (Tables p = 0.0252) and the presence of multifocality  in VAB specimens as significant risk factors. The other parameters including the age parameter showed no significant association with the rate of disease upgrade based on subsequent surgical specimens. We did not identify large comedo type necrosis, however, smaller necrotic foci in association with calcification were observed. In the surrounding breast tissue, no relevant calcifications were found.The histological findings of additional B3 lesions are listed in Table s Tables . The mulThe upgrade rate in the cohort with the lowest risk  was 16.5% (10 DCIS and 1 invasive cancer in 63 patients). The overall upgrade rate in the whole cohort was 39% .To the best of our knowledge, this is the largest study to show that the biopsy method (CNB or VAB) influences the outcome determined based on surgical specimens, with a greater rate of disease upgrade after diagnosis by CNB than by VAB. In one previous study, Badan et al. reported similar observations in 40 patients . Our casSeveral previous studies have analyzed the relationships between pathological parameters  with the outcome determined from surgical specimens , 19, 27.Our results partly support those of previous studies regarding the importance of calcification. Our study shows that calcification predicts diagnostic upgrade only in cases of ADH diagnosed by VAB, with a significantly lower upgrade rate in patients with calcification. The upgrade rate in cases of ADH diagnosed by CNB was independent of the presence of calcification. A relationship between calcification and diagnostic upgrade has been reported in previous studies , 30. OurIt can be assumed, that ADH cases detected on VAB lacking calcifications were most likely representing a non calcified extension of DCIS, which was not detectable as such in mammography.The data reported in the literature on multifocality and the diagnostic upgrade of ADH are controversial , 20, 25.Interestingly, as in our data, the data in the literature show fairly consistently that there is no association between ADH upgrade and the presence of other B3 lesions, such as LN, papilloma, RS, or FEA , 23, 31.One earlier study by Hartman et al. reported a significant interaction between premenopausal status and histological findings. Breast cancer risk among patients < 45 years at the time of ADH diagnosis, was 6.99 times the expected risk, which was significantly lower (3.37) in postmenopausal patients (> 55 years), as was shown in Fig. 2 in this paper . InteresIn conclusion, we have provided evidence that the biopsy method used (CNB or VAB) influences the diagnostic upgrade rate. Especially in cases of ADH diagnosed by VAB, the presence of calcification in a unifocal lesion is associated with a significantly lower disease upgrade rate than is the absence of calcification and/or the presence of multifocality. These factors should be considered, in addition to imaging characteristics, when deciding whether a patient should undergo open surgery or simply be managed conservatively. These data require validation in further studies.