In the present century both basic research in orchid flower evo-devo and the interest for generating novel horticultural varieties have driven the characterization of many members of the MADS-box family encoding key regulators of flower development. This perspective summarizes the picture emerging from these studies and discusses the advantages and limitations of the comparative strategy employed so far. I address the growing role of natural and horticultural mutants in these studies and the emergence of several model species in orchid evo-devo and genomics. In this context, I make a plea for an increasingly integrative approach.The diverse morphology of orchid flowers and their complex, often deceptive strategies to become pollinated have fascinated researchers for a long time. However, it was not until the 20th century that the ontogeny of orchid flowers, the genetic basis of their morphology and the complex phylogeny of Orchidaceae were investigated. In parallel, the improvement of techniques for  The unique diversification of flower morphology in Orchidaceae has taken place in the framework of a relatively conserved structure. Generally orchid flowers consist of three outer tepals similar to each other, two distinct inner lateral tepals and a highly differentiated inner median tepal or labellum Figure . Female Arabidopsis thaliana and some non-model species like Tulipa gesneriana and Lilium regale.Because of the key role of the gynostemium and labellum in orchid reproduction their origin has been a recurring question in botany and evolutionary biology since the 19th century. The finding that flower organ identity is specified by the genetic and physical interaction of MADS domain transcription factors  are expressed mostly in the gynostemium and in some instances also in the perianth  are reproducibly expressed in the gynostemium and ovary  isolated so far are expressed in all flower organs , DcOAG1 (AG-like) or OMADS6 (SEP3-like)  to actinomorphy   in the outer perianth was associated to the transformation of outer tepals into organs resembling inner-lateral tepals and labellum, thus suggesting this gene is involved in the specification of the inner perianth  and CeMADS2 (DcOAG1-like) showed that while both genes are expressed in the gynostemium and buds of wild-type flowers, CeMADS1 is not expressed in developing buds of the multitepal mutant. This study thus suggests that CeMADS1 is a class C gene and both CeMADS1 and CeMADS2 are not functionally redundant in the specification of gynostemium identity.More recently, the study of floral terata from glyp mutant of Phalaenopsis hyb. “CD1” the inner lateral tepals bear ectopic pollinia and their epidermal cells are morphologically intermediate between those of wild-type tepals and those of the gynostemium  and PeMADS7  was detected exclusively in the column of the wild-type flowers, while only PeMADS1 was detected in the gynostemium-like inner lateral tepals of the glyp mutant. While this study suggests PeMADS1  have been generated for these species  advance ten orchid species as candidate models from each of the five Orchidaceae subfamilies as well as the sister group Hypoxidaceae: Apostasia shenzhenica and Neuwiedia malipoensis (Apostasioideae); Vanilla shenzhenica and Galeola faberi (Vanilloideae); Paphiopedilum armeniacum and Cypripedium singchii (Cypripedioideae); Habenaria delavayi and Hemipilia forrestii (Orchidoideae); Cymbidium sinense as well as the previously mentioned Phalaenopsis equestris (Epidendroideae) and Sinocurculigo taishanica (Hypoxidaceae) . Recently, the chloroplast genome of E. pusilla and a transcriptome have been sequenced  based on Arabidopsis thaliana and Antirrhinum majus as well as from research on other monocot species like Tulipa gesneriana and Lilium regale. On the other hand, this comparative approach and the technical limitations to genetically manipulate orchids have set important challenges to functionally approach the genetic basis of orchid flower development.At its beginnings orchid flower evo-devo greatly profited from knowledge on well- established model species like The systematic morphological and molecular characterization of flower terata offers a way around these limitations and has enabled the formulation of several testable models based on the large amount of information on class B MADS-box genes, the most studied developmental genes in this family.Arabidopsis thaliana (Smyth et al., The growing amount of transcriptomic information in a diverse group of orchid species calls for a second wave of integration and comparative analysis at an unprecedented scale. While the apparent number of “endless forms most beautiful,” the sinking prices of RNA-seq and the competitive nature of scientific endeavor might tempt us to sequence “yet another orchid transcriptome” the most significant advances on this subject will come from systematically integrating all available information and testing our findings experimentally by means of unifying developmental and evolutionary hypotheses and models. This process requires not only sharing and comparing information but also the agreement on common concepts for the developmental processes we are investigating. For example, because most studies describe orchid flowers buds based on their size it is not possible to make an objective comparison of transcriptomes or other patterns of gene expression within and between species. An alternative would be that for every species with a transcriptome a description of discrete stages of its development is generated and considered in the design of future expression studies as it is routinely done for Because of the prevalent occurrence of gene duplication in orchids the value of gene phylogenies and profiles of gene expression strongly depends on considering as many known duplicates as technically possible. By doing so it is possible to objectively compare studies and minimize the artifacts coming from simultaneously measuring the expression of highly similar paralogs.Because orchid evo-devo is a relatively young area there are still many major challenges to overcome. In the near future the vitality of its research program depends on the consolidation of one or several model species amenable to genetic manipulation or with a rapid life cycle that enables the fruitful integration of genetic analysis and transcriptomic resources. In the long run, the scientific relevance and reach of orchid evo-devo will rely on its contribution to understanding orchid ecology and evolution in questions like the interaction between environmental variables, pollinators and the activity of developmental transcription factors.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Plasmodium falciparum infections within the urban extent of Khartoum state in Sudan is investigated using data from cross-sectional surveys undertaken from 1999 to 2008 to inform the Khartoum Malaria Free Initiative (KMFI).Identifying the location and size of residual foci of infections is critical where malaria elimination is the primary goal. Here the spatial heterogeneity of P. falciparum parasites. Residential blocks were mapped. Data were analysed for spatial clustering using the Bernoulli model and the significance of clusters were tested using the Kulldorff scan statistic.From 1999–2008 the KMFI undertook cross-sectional surveys of 256 clusters across 203 random samples of residential blocks in the urban Khartoum state in September of each year. Within sampled blocks, at least five persons, including at least one child under the age of five years, were selected from each household. Blood smears were collected from the sampled individuals to examine the presence of A total of 128,510 malaria slide examinations were undertaken during the study period. In 1999, overall prevalence was 2.5%, rising to 3.2% in 2000 and consistently staying below 1% in subsequent years. From 2006, over 90% of all surveyed clusters reported no infections. Spatial clustering of infections was present in each year but not statistically significant in the years 2001, 2002, 2004 and 2008. Spatial clusters of high infection were often located at the junction of the Blue and White Niles.Persisting foci of malaria infection in Khartoum are likely to distort wide area assessments and disproportionately affect future transmission within the city limits. Improved investments in surveillance that combines both passive and active case detection linked to a geographic information system and a more detailed analysis of the location and stability of foci should be undertaken to facilitate and track malaria elimination in the state of Khartoum. Spatial heterogeneity in risk of malaria infection is regarded as a significant driver of the basic reproduction rate of transmission in any endemic area but becomes increasingly important as the overall intensity of transmission declines Plasmodium falciparum infections across 256 cross-sectional surveys undertaken from 1999 to 2009 within the urban extent of Khartoum state in Sudan is examined to inform the Khartoum Malaria Free Initiative established in 2002.Techniques that detect the presence of statistically significant small-area clusters are often used to assess local heterogeneity of disease 2Anopheles arabiensis is the main vector of malaria Khartoum state is one of the 26 states in Sudan with a total population of more than 5 million people in an area of approximately 28,000 KmMalaria control in Khartoum dates back to 1904 when retained oil was used as the main vector control tool leading to the eradication of the disease in the state Since 1995, the KMFI, led by the state malaria control programme, undertook cross-sectional community surveys during the peak transmission month of September in selected areas in Khartoum. Sample sizes were calculated on an expected prevalence of 10% which was continually revised downwards each year as prevalence decreased and, by 2004, surveys were powered on a prevalence of 0.5% P. falciparum infection rates. A spatial only cluster analysis was undertaken because the cross-sectional surveys relied on a fresh sample each year and consequently few clusters were surveyed continuously throughout the study period. A Bernoulli model was used for the analysis of spatial clustering in the data for several reasons. First, the number of people surveyed varied by location and it was important to adjust for these sampling changes to avoid clusters that are driven by the number of people surveyed rather than the number of people who had infection. Second, this model allowed for locations that are always of high malaria prevalence relative to other locations/years to contribute to the spatial clustering, a particularly important advantage given the generally low prevalence of the survey locations throughout the study period. The Bernoulli model requires the case and control data, represented respectively by P. falciparum positive and negative samples, and the spatial location for each case and control for each survey year P. falciparum infection and the P-value of the Kulldorff scan statistic.The Kulldorff spatial scan statistic Ethical approval for this study was obtained from the National Research Ethics Committee of the Federal Ministry of Health. Formal permission was obtained from the Khartoum malaria control program of the State Ministry of Health. Written consent was sought from all participants and parents/guardians of young children before blood samples were collected.From 1999–2008 a total of 128,510 malaria slide examinations from cross-sectional surveys in 256 sampled clusters in 203 residential blocks were undertaken by the Khartoum national malaria programme . The numP. falciparum prevalence in each year from 1999–2008  The analysis here demonstrates the utility of spatial cluster analysis techniques to help identify possible residual foci of infections in an area of very low malaria transmission. The observed heterogeneity of risk therefore implies that clusters of high prevalence contribute disproportionately to wide area mean estimates of infection prevalence and is likely to be the main reservoirs of continued transmission P. vivax cases was observed across the study period [unpublished data] there is need for a much better understanding of the burden of this parasite. This is particularly important given the challenges to elimination posed by the hypnozoite, liver stage of the parasite life cycle, which stays dormant for long periods and a single infection can lead to a series of relapses Furthermore, although only one case of Surveys of residential blocks and enhanced clinical surveillance should be improved to define progress and possible success in malaria elimination. The declining levels of infection prevalence will render community cross-sectional parasite surveys inefficient in monitoring transmission over time and expanding them to the required sample sizes will become very expensive This study demonstrates the potential of cluster analysis techniques in identifying the location and radius of spatial clusters of malaria infection which are likely to be residual foci of infections. The results provide evidence that the KMFI has reached a level of measurable success however the remaining foci are likely to distort wide area assessments and disproportionately affect future transmission within the city limits. Data of higher spatial and temporal resolution with detailed series of individual, household and cluster level predictors of infections are required to for a comprehensive assessment of the location and stability of residual foci of infections in Khartoum state. Bespoke versions of the cluster analysis techniques could be developed for applications to new forms of clinical case detection data with sufficient investment in health information systems.


Influence of isotropic and anisotropic properties of membrane constituents (nanodomains) on formation of tubular membrane structures in two-component vesicle is numerically investigated by minimization of the free energy functional based on the deviatoric-elasticity model of the membrane. It is shown that the lateral redistribution and segregation of membrane components may induce substantial change in membrane curvature resulting in the growth of highly curved tubular structures. The shape of lipid bilayers, cellular or artificial, strongly depends on composition and lateral distribution of membrane components Except for the sake of simplicity there is no a priori reason to consider membrane constituents/nanodomains to be isotropic [6 7] instead of anisotropic, which actually represents a more general approach Not only proteins and/or protein-lipid complexes but also lipid molecules should be in general considered anisotropic elongated inner stiff supporters, e.g. microtubules external pulling forces, such as, optical tweezers anisotropic properties. As for example, the membrane attached crescent shaped BAR domain proteins have clearly anisotropic shape and therefore their energy depend on their local orientation or statistically averaged local orientation, depending on the local curvature of the membrane Coupling between the cell/liposome shape and non-homogeneous lateral distribution The aim of this work is to study the influence of anisotropy of membrane nanodomains on the shape transformations and lateral segregation of membrane components in two-component axially symmetric vesicles of fixed topology. The special attention is devoted to the stability and growth of tubular membrane structures with thin tubular protrusions having small spherical vesicles at their free tips  induced The model vesicles are built up by two components (A and B) ant their shapes are obtained numerically by the direct minimization of the free energy functional of the membrane under the constraints of constant vesicle surface area In this work, we have investigated under what conditions the formation of thin tubular structures is favorable. The special examples of such systems observed in experiments , i.e. th2. The chosen vesicle radius in the calculations was of the order of 250 nm.The calculations were performed for different values of the bending rigidity for each component, When both components are isotropic, the vesicle is composed of small spherical beads connected by narrow passages, such as the first vesicle in In the membrane systems encountered in the nature a small number of membrane components (minority) is usually strongly anisotropic, while a much larger number of membrane components (majority) is considerably less anisotropic, or isotropic. The anisotropic BAR domain proteins attached to the bilayer membrane The cylindrical protrusions are formed when at least one component is anisotropic. Such behavior is demonstrated in It is interesting to note that very small amount of the anisotropic component is enough to induce the formation of the cylindrical protrusion. Moreover, the length of the protrusion depends on the concentration. This is due to the separation of the components in the membrane, where the anisotropic components are located mainly in the tubular part which has a very small surface area compared to the rest of the vesicle. It is interesting to note, that total component segregation was observed for The calculations presented in The complete mixing was observed for small concentration of the anisotropic component, The anisotropy of one of the components is not however a sufficient condition for the formation of the tubular structures. We have also observed that cylindrical protrusions may be induced by changing the properties of the isotropic component. It is demonstrated in In the systems in which the cylindrical tubules are created when the proteins  are adsorbed at the membrane surface, the radius of the tubule is determined by the intrinsic curvature of the protein stable tubular protrusions. When the components are isotropic such cylindrical structures may be created only when some external force is applied. For example when membrane is pushed by growing microtubules or pulled by molecular motors. The width of the tubes depends on the intrinsic curvatures of anisotropic components. When the membrane is composed of isotropic components the stable protrusions which are created without any external force are built of a series of connected beads.We have shown that accumulation of anisotropic components may lead to the formation of thin tubular protrusions. The anisotropy of components is a necessary condition for creation of the In the model the membrane is composed of two components A and B which can be either isotropic or anisotropic and are characterized by the intrinsic principal curvatures For simplicity we assume linear dependence of the bending rigidity The contour of the vesicle is parametrized by the angle, tion see  [24], [3The ansatz for the local relative concentration of the component A has the form :see also In numerical calculation we have to find both the function


Chondrolysis of the ankle is a very rare condition. We report a case of chondrolysis of the ankle following ankle arthroscopy and microfracture of the osteochondral lesion of the talar dome. The patient's symptoms were relieved after articulated distraction arthroplasty. Chondrolysis is a clinical condition characterized by rapid destruction of articular cartilage on both sides of the joint leading to loss of joint space and joint stiffness. The cause has not truly been identified . ChondroA 32-year-old gentleman had inversion injury to his left ankle on 2007 resulting in persistent medial ankle pain. He was treated with physiotherapy without improvement. Radiographs and magnetic resonance imaging (MRI) of his left ankle showed the presence of osteochondral lesion (OCL) of the medial talar dome . Ankle aChondrolysis is characterized by progressive loss of joint space and increased stiffness resulting from destruction of the articular cartilage on both sides of the joint . It is aThe pain is usually out of proportion to the clinical findings, leading to a misdiagnosis of complex regional pain syndrome. This condition should be suspected in patients complaining of persistent, unrelenting pain, stiffness, and severe diffuse articular cartilage loss, within twelve months after an operation or potential cartilage insult to the ankle joint. The clinical symptoms should generally exceed a comparable amount of joint destruction in an otherwise chronic condition, for which the patient has likely had more time to adapt to the damage of the joint cartilage .Proposed etiologic factors can be classified into four categories including 1) thermal  laser), (2) chemical , 3) mechanical , or 4) other  factors [ other (e thermal  mechanicOptimal treatment of chondrolysis of the ankle is inconclusive because of its rarity. Although total joint arthroplasty remains the gold standard for the treatment of extensive articular cartilage damage, nonarthroplasty options are reasonable for those with chondrolysis of the ankle as the patient is frequently of young age with high physical demands . Nonoper


Nodular thyroid disease affects 500 to 600 million people worldwide. Tumours of the thyroid account for about 1% overall human cancers. Thyroidectomy is the most common endocrine operation. Surgical treatment for benign thyroid nodules is recommended for: progressive increase in nodule size, substernal extension, compressive symptoms in the neck region, the development of thyrotoxicosis and in case of preference of that kind of treatment reported by the patient. In Poland thyroidectomy is the fourth surgical procedure and concerns 25000 operations yearly. Reduction of surgical injury with simultaneous retention of current safety and radical nature of surgical procedure forces the work in a relatively small operating field. Electric devices enabling the achievement of full and lasting haemostasis during thyroidectomy supplant traditional surgical method  with no impact on the incidence of perioperative complications, while at the same time allowing to shorten the duration of the procedure. The haemostatic effect is associated with generation of heat, which apart from the intended result may bring about thermal tissue injury. During the surgical procedure important is to determine the thermal spread around the active tip of electric devices in the operating field during thyroidectomy, and the safe temperature range during the operation to protect important structures of the neck. The mean safe distance of the active tip of an electric device from important anatomic structures is 5mm minimally and depends on the device type, time of using and its power settings. All the modern techniques of vessel sealing are associated with generation of heat and its spherical spread, which causes thermal injury to the surrounding tissues. Their mode of operation through, among others, structural changes in collagen and elastin, leads to lasting joining of sealed vessel walls and tissue structures. These systems enable a safe sealing of vessels of up to 7mm in diameter.In conclusions: In the cases analysed by the author concerning the thyroidectomy techniques, it is recommended to replace to electric devices with ligatures or clips or human fibrinogen in place near the laryngeal nerves, parathyroid glands and the trachea. The decision on the change of the method of haemostasis maintenance in the vicinity of crucial structures belongs to the surgeon.


To control this disease, funding and research should be prioritized on the basis of determined needs. Although Rift Valley fever is a disease that, through its wider societal effects, disproportionately affects vulnerable communities with poor resilience to economic and environmental challenge, Rift Valley fever virus has since its discovery in 1931 been neglected by major global donors and disease control programs. We describe recent outbreaks affecting humans and animals and discuss the serious socioeconomic effects on the communities affected and the slow pace of development of new vaccines. We also discuss the mixed global response, which has largely been fueled by the classification of the virus as a potential bioterrorism agent and its potential to migrate beyond its traditional eastern African boundaries. We argue for a refocus of strategy with increased global collaboration and a greater sense of urgency and investment that focuses on an equity-based approach in which funding and research are prioritized by need, inspired by principles of equity and social justice. Since Rift Valley fever virus (RVFV) was first identified in 1931, after an investigation of an epizootic among sheep on a farm in the Great Rift Valley of Kenya, the understanding of this zoonotic disease has grown considerably (Although the disease disproportionately affects vulnerable communities with low resilience to economic and environmental challenges, RVF has remained largely neglected by major global donors and disease control programs. With high numbers of competent vector species present in disease-free regions, the intensification of international trade in live animals, and the uncertain effects of climate change, RVF is now considered a major challenge in global zoonotic disease control (The potential of RVFV to migrate was established after large outbreaks of RVF occurred among animals and humans in Egypt in 1977, in other geographic zones of Africa, and then outside the African continent in Saudi Arabia and Yemen in 2000 (The Table further demonstrates the spread of the disease; 7 of 9 major outbreaks in the past 15 years resulted in human cases outside the Rift Valley region in East Africa. The Table also highlights the difficulty of developing adequate surveillance systems and therefore the difficulty of accurately estimating morbidity and mortality rates for human populations in resource-poor settings. In the 5 outbreaks for which estimated numbers of human cases have been published, ≈339,000 infections are believed to have occurred. In the 4 outbreaks for which estimated and reported cases are documented, numbers of estimated cases are 78× higher than numbers of reported cases (There is a paucity of studies that have examined the socioeconomic effects of past outbreaks of RVFV, which reflects a lack of research focus on the broader social effects of the disease. One study that did examine the socioeconomic effects of the 2006/2007 RVFV outbreak in Kenya highlighted the concern that the outbreak had tended to disproportionately affect impoverished pastoralist communities, with those in the North Eastern Province of Kenya being hardest hit (The ban of livestock imports to the Middle East from East Africa, instituted after the 1997/1998 RVFV outbreak in Kenya and Somalia, particularly affected the export trade out of Somalia. The ban was variably enforced by several Middle Eastern countries but most notably by Saudi Arabia, which imports large numbers of ruminants for the annual Hajj pilgrimage. In 1997, the year before the onset of the ban, 2.8 million live animals were exported from the Somaliland port of Berbera, making it the single biggest exporting port for ruminants in the world that year. With the livestock trade accounting for 65% of gross domestic product in Somaliland, the export ban had a devastating effect on a region already suffering in the grip of a protracted civil war (The slow pace of development of new vaccines  and diagBefore modern safety standards were instituted in laboratories, RVFV was regularly transmitted between laboratory staff; 47 cases were documented worldwide (Fortunately, with the advent of recombinant genetic technology and the development of reverse transcription PCR techniques obviating the need to handle and store live virus, new vaccines and diagnostic tests in development can now be produced in laboratories of lower BSL (Interest in RVFV and investment in its control were only substantially increased among the global health research and policy community after greater awareness of its potential to migrate beyond its traditional East African boundaries was noted. However, the recognition that much of the industrialized world has animals and arthropod vectors capable of transmitting the virus seems to have focused and accelerated efforts to develop improved tools for outbreak forecasting, monitoring, diagnosis, and prevention.In more recent years, the classification of the virus as a potential bioterrorism/agroterrorism agent has also helped spur investment and activity, particularly in the area of vaccine development and diagnostics (Growing restrictions stemming from biosecurity concerns now affect research activity across a range of infectious diseases and have most recently been highlighted by concerns over the publication of research into the production of genetically engineered variants of the influenza A subtype H5N1 virus (Increased sales costs of vaccines have a variety of negative consequences; in particular, this increase could put at risk well-established mechanisms of international cooperation in global infectious disease surveillance. This risk was dramatically highlighted in 2006 and 2007 when Indonesia refused to share samples of influenza subtype H5N1 isolates with the World Health Organization. The event caused a risk to global health and occurred in direct protest to the inequitable sharing of virus samples and vaccine development technology (Despite some of these challenges, some positive developments have occurred in global collaborative efforts for controlling zoonotic diseases, including RVFV. These include initiatives like the One Health (In recent years, the perceived risk of RVFV becoming established in Europe and North America, and the theoretical risk of it being used as a bioterrorism agent, has brought a welcomed increase in investment to combat the disease yet has skewed priority areas of focus for that investment. The ideal that should be adopted is a more equity-based approach in which funding and research are prioritized on a needs-identified basis for the aid of those most disadvantaged in the global community. This approach would concentrate efforts on those interventions that most positively affect these vulnerable communities and, in addition, prevent or minimize the spread of the disease to previously non–disease-endemic high-income countries.Such an approach would ensure research and policy emphasis on the socioeconomic effects of RVFV outbreaks. Interventions could then address international trade policies and their ramifications on livestock trade and the development of appropriate support systems within exporting countries to mitigate and minimize the risk of bans being instituted. In addition, encouraging farmers to focus their livestock-rearing efforts on breeds more resistant to infection with RVFV and a greater study of the genetic factors that make these breeds resistant should also be promoted as part of this global effort. Developing better surveillance systems is key.Fears of RVFV being used as a bioterrorism agent should not sideline the real security effects of the disease in driving impoverished communities to find other, more dangerous means of income. Did the bans on livestock from Somalia, for instance, and the resulting lost economic opportunities afforded by a well-developed functioning ruminant export market, contribute to the drive of persons and communities to seek alternative sources of income, including taking part as combatants in the civil war in or in the piracy trade that has developed in the region? Are the stringent measures being imposed on laboratories that store or work with the virus serving to concentrate technical expertise and industrial know-how in the hands of scientists in a very few industrialized countries, thus contributing to limited scientific inquiry and collaboration, which further escalates costs? Although these questions are yet to be answered conclusively, exploring the case for lowering current BSL requirements of laboratories and production facilities could be 1 method of mitigating these costs.A greater sense of urgency and investment is required for controlling, better managing, and preventing future large-scale outbreaks of RVFV. Future long-term success lies in building on global collaborative initiatives, the closer integration of multilateral agencies, and a wider participation from livestock-importing countries and emerging economies that are investing in RVFV-endemic countries. A worldwide strategy, both in tune with and inspired by principles of equity and social justice, could ultimately deliver the best outcomes in combating this neglected tropical disease.Rift Valley fever vaccine development.


Our understanding of the mechanisms governing the response to DNA damage in higher eucaryotes crucially depends on our ability to dissect the temporal and spatial organization of the cellular machinery responsible for maintaining genomic integrity. To achieve this goal, we need experimental tools to inflict DNA lesions with high spatial precision at pre-defined locations, and to visualize the ensuing reactions with adequate temporal resolution. Near-infrared femtosecond laser pulses focused through high-aperture objective lenses of advanced scanning microscopes offer the advantage of inducing DNA damage in a 3D-confined volume of subnuclear dimensions. This high spatial resolution results from the highly non-linear nature of the excitation process. Here we review recent progress based on the increasing availability of widely tunable and user-friendly technology of ultrafast lasers in the near infrared. We present a critical evaluation of this approach for DNA microdamage as compared to the currently prevalent use of UV or VIS laser irradiation, the latter in combination with photosensitizers. Current and future applications in the field of DNA repair and DNA-damage dependent chromatin dynamics are outlined. Finally, we discuss the requirement for proper simulation and quantitative modeling. We focus in particular on approaches to measure the effect of DNA damage on the mobility of nuclear proteins and consider the pros and cons of frequently used analysis models for FRAP and photoactivation and their applicability to non-linear photoperturbation experiments. The DNA damage response plays a crucial role in oncogenesis  or the intercalating dye Hoechst, exposure of cells to UVA illumination leads to single and double strand breaks  required for these transitions are delivered via ultrashort pulses (ps to fs) thus limiting the average laser power to levels compatible with cell viability. For non-linear excitation, the sum of the energy of the incoming photons has to match the definite energy gap between two electronic states. For DNA bases, the maximum of linear absorption lies at 260 nm and excitation at this wavelength leads to the formation of UV-photoproducts, as mentioned above. Hence, the same type of lesion can be generated by irradiating cell nuclei with femtosecond pulses at a wavelength of 780 nm, corresponding to the simultaneous absorption of three near-infrared photons  excitation is the tool of choice. First described in theory by Maria Göppert-Mayer in 1931 and demonstrated experimentally by Kaiser and Garrett in 1961, this process relies on the simultaneous absorption of multiple photons at very high photon densities, as they are present within the focus of the objective lens  causes a temperature rise of 0.2 K due to linear absorption , and, to a lesser extent, photoactivation, have yielded important insights into the dynamics and binding properties of histones in intact, native chromatin Kimura, . MobilitFluorescence photoactivation has also been combined with laser microirradiation to study local changes of chromatin structure due to DNA strand breaks . These simplifications have enabled analytical solutions of the reaction-diffusion model, exactly describing the redistribution of the fluorescence signal to equilibrium as a function of time. The basic assumptions that are employed for this type of approach are the following:The system's properties remain close to unperturbed, i.e., the number and the distribution of binding sites do not change during the observation period. In addition, binding sites are assumed to be homogenously distributed within the nucleus. The latter does not hold true for chromatin proteins because chromatin density varies between subnuclear regions .The photobleached/photoactivated volume has a uniform extension along the z-dimension. According to this assumption intensity changes introduced by the photobleaching/-activating laser do not vary along the optical axis, and the system can be described by a two-dimensional model.The finite dimension and the geometry of the cell nucleus can be neglected. This simplification applies only when the photobleached/photoactivated spot is small as compared to the nuclear volume.Numerous studies have undertaken quantitative analysis of FRAP and photoactivation data of nuclear proteins under undisturbed conditions . In general, a reaction-diffusion model is assumed to be the best mathematical description of the dynamic behavior of these proteins. According to our current understanding, nuclear proteins diffuse stochastically in all three dimensions within the nucleoplasm until they collide with binding partners with whom they undergo transient interactions. This model assigns to each protein the following characteristic parameters: the binding and release constants Otherwise, the solution of the reaction-diffusion model has to be found via numerical modeling where changes in fluorescence are approximated consecutively for each time point for a given set of parameters. For both the analytical and the numerical procedure, the obtained solutions are optimized by testing combinations of different parameters iteratively until the simulated behavior fits the experimental data.The power of kinetic modeling consists in its ability to make quantitative predictions about the reaction of the biological system under study. For FRAP models, this ability has been questioned, because different approaches have yielded very different results for the same or similar proteins. Therefore, a cross-validation strategy that compares different models as well as different experimental methods to generate the primary data is highly recommended . Exposure to short laser pulses to induce DNA damage initiates a signal chain that may develop on different time scales for different types of lesions/binding sites. This amplification process is inherent to the biological response to DNA damage and leads to a fast and massive propagation of the damage signal, as best exemplified by the spreading of the phosphorylation of histone H2AX from the initial strand break to chromatin regions of the size of a few megabases   within a significantly larger zone containing the damage . On the other hand, the small spot size minimizes the influence of nuclear geometry which may therefore be neglected without inducing significant errors (point 3).An overview of recently proposed approaches for the analysis of FRAP and photoactivation experiments specifying their most important features is given in Table Numerical solutions of the reaction-diffusion model provide a more accurate approach to describe protein dynamics. In their study, Beaudouin et al. present a method that includes both the real geometry of the nucleus and an inhomogeneous distribution of binding sites and is independent of the shape of the bleached/photoactivated region. The differential equations are solved numerically using a finite difference method. The approach was validated for the photoactivation of five different chromatin proteins (Beaudouin et al., None of the currently available models addresses the issue of the temporal non-equilibrium of the system that is characteristic for mobility measurements performed subsequently to DNA microdamage, either via FRAP or photoactivation. Addressing this issue is a promising avenue for future work and a prerequisite for the proper quantitative description of the response of nuclear proteins to DNA damage.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


There are 16 morphologically defined classes of rat retinal ganglion cells (RGCs). Most commonly, they are classified on the basis of several criteria including: soma size, dendritic field diameter, the dendritic branching pattern and the depth of stratification in the inner plexiform layer. Recently, it has also been shown that the intrinsic physiological properties of each rat RGC type vary enormously. Using multicompartment models of RGC types we investigated whether the location of the axon initial segment (AIS), the site of greatest sodium channel density and lowest voltage threshold, can be predicted by measurements of spike waveform made at the soma.The action potential waveform in many neurons consists of several components, which can be determined by examining the first and second derivatives of the membrane potential. In this study, we focus on this technique as an objective method to analyze the action potential waveform for different morphological RGC types. In addition, we analyze the features of the phase plot, which shows the rate of change of the membrane potential against the membrane potential itself. Phase plot analysis allows the measurement of subtle differences in the action potential waveform such as the initial segment-soma/dendritic break (ISSD), which corresponds to the early rising phase of the action potential. When the recording is made at the soma, the presence of the ISSD in the phase plot indicates that a low threshold region (i.e. the AIS) is further away from the soma.Rat RGCs were characterized electrophysiologically using standard whole cell patch clamp recording techniques. Data were acquired at 20 kHz using custom software developed in LabView . Spontaneous spikes and spikes evoked by just-threshold current were used for analysis. For each of the recordings, the amplitude and time of the trough between the peaks in the second-order derivatives were analyzed. After three dimensional confocal reconstruction of each recorded cell (Zeiss PASCAL) it was classified morphologically into one of the 16 predefined types. Multicompartment models of real retinal ganglion cells were constructed from 3D rendering confocal reconstructions and their physiology was simulated using the Hodgkin-Huxley formalism in the NEURON environment. Sodium channel density in the AIS and its distance from the soma were systematically varied and the effects on the phase plot analyzed.Simulations showed that the further the AIS was from the soma, the more pronounced the ISSD break, resulting in a larger break with a deeper trough between the two peaks in the phase plot. This result allows us to predict the location of the AIS based on recordings of the impulse waveform. In addition, we found that the density of sodium channels in the AIS affects spike propagation into the soma. We observed that decreasing sodium conductance in the AIS, required two spikes to occur in the AIS in order to evoke a somatic spike. This was also observed experimentally, in particular in C4 cells. Further analysis of individual RGC spike waveforms demonstrated that certain RGC types could be reliably identified using their spike waveforms.


For the case of the infinite-sites model, I derive analytical formulas for the expected number of polymorphic sites in sample of DNA sequences, and apply the developed simulation and analytical methods to explore the fit of the model to HIV genetic diversity based on serial samples of HIV DNA sequences from 9 HIV-infected individuals. The results particularly show that the estimates of the ratio of recombination rate over mutation rate can vary over time between very high and low values, which can be considered as a consequence of the impact of selection forces.This paper presents a novel population genetic model and a computationally and statistically tractable framework for analyzing within-host HIV diversity based on serial samples of HIV DNA sequences. This model considers within-host HIV evolution during the chronic phase of infection and assumes that the HIV population is homogeneous at the beginning, corresponding to the time of seroconversion, and evolves according to the Wright-Fisher reproduction model with recombination and variable mutation rate across nucleotide sites. In addition, the population size and generation time vary over time as piecewise constant functions of time. Under this model I approximate the genealogical and mutational processes for serial samples of DNA sequences by a continuous coalescent-recombination process and an inhomogeneous Poisson process, respectively. Based on these derivations, an efficient algorithm is described for generating polymorphisms in serial samples of DNA sequences under the model including various substitution models. Extensions of the algorithm are also described for other demographic scenarios that can be more suitable for analyzing the dynamics of genetic diversity of other pathogens  Recombination has an important role in shaping the dynamics of within-host HIV genetic diversity, particularly making the virus capable of escaping the pressures of antiviral drugs and immune system Early studies Under these models the expected numbers of average pairwise differences in serial samples stay the same From the same data sets, I also observe linear relationships between the dynamics of the numbers of polymorphic sites and the numbers of average of pairwise differences in each individual's case . To makeTo illustrate non-linear relationship between the expected dynamics of the two statistics for the observed sample sizes under the Wright-Fisher model with constant population size, I compute the expected numbers of polymorphic sites under the finite-sites Jukes-Cantor model as well as under the infinite-sites model by using the formulas of Tajima While the standard coalescent as well as the ancestral recombination graph might not be directly applicable for analyzing the dynamics of HIV genetic diversity within a host, the concepts and features of these models had and have great impact on extending coalescent theory for other evolutionary settings. Both models were derived as continuous limits of discreet genealogical and mutational processes under the Wright-Fisher models with constant population size and with and without recombination by applying the time scaling concept that is measuring time proportional to a very large population size. This concept was also applied for other forward in time Wright-Fisher models with variable population size, selection, or migration but without recombination see e.g.  to derivLater studies To overcome the limitations of the previous models and methods mentioned above, I first describe a forward in time population genetic model to represent HIV evolution in HIV-infected individuals in the chronic phase of infection. The population in the model is considered to be homogeneous at the beginning, representing the time of HIV seroconversion, and to evolve according to the Wright-Fisher reproduction model with recombination by allowing the population size and generation time to vary over time. To make this model computationally and statistically tractable for analyzing serial samples of DNA sequences, I apply the time scaling approach at multiple time intervals  to describe a continuous coalescent-recombination process for tracing the lineages of the samples back in time and superimposing mutational events on the lineages according to an inhomogeneous Poisson process. Based on these processes I describe computationally efficient algorithm for generating polymorphisms in serial samples of DNA sequences drawn randomly under this population genetic model. Further extensions of the algorithm are also described for population genetic models that can be more suitable for analyzing the dynamics of genetic diversity of other pathogen populations in vivo and in vitro.Within this framework I consider two substitution models: a finite-sites model with variable mutation rate across nucleotide sites and the infinite-sites model. For the infinite-sites case, I derive analytical formulas for the expected number of polymorphic sites in samples of DNA sequences. For this quantity, Tajima To model within-host HIV evolution, I take into account the following observations: (1) HIV population within HIV-infected individuals usually collapses at seroconversion  after several weeks of infection and recovers quickly as a homogeneous population In this model DNA sequences are represented as a combination of I design the finite-sites model in a such way that it represents the heterogeneity of substitution rate across nucleotide sites and infer some of the parameters in this model based on serial samples of HIV-1 DNA sequences from envelop gene region The population model for Variation in a sample of DNA sequences under the above population model can be described by combining the genealogical history of the sequences with mutations on the lineages of the sequences. The genealogical history traces the ancestral lineages of the sequences back in time before time For each interval For this Markov chain the transition from the state The following algorithm uses the tracing procedure recursively to describe a bottom up process for generating genealogical history of serial samples and a top down process for adding mutation events on the genealogy. The algorithm can be used to generate variation in serial samples under the population model described above.Algorithm 1Set the values of Apply the above procedure for tracing the lineages of the Update the values of As the value of Add mutation events independently on different branches of the genealogical history for time interval Increase the value of For the case of a non-recombining locus  also holds for the case of I apply the models and methods described in the previous section for exploring within-host HIV evolution based on serial samples of HIV DNA sequences from 9 HIV-1-infected individuals studied by First, I fit the population genetic model to data sets under the finite-sites model, in which the values of I implemented Algorithm 1 for this mutation model into a computer program (in C programming language) for generating the polymorphisms in serial samples and applying the Monte Carlo approach to estimate the expected values of summary statistics for the observed sample sizes. To fit the model to the data sets for each individual's case, I consider two summary statistics: the numbers of polymorphic sites and divergences in the serial samples. Divergence in a sample of sequences is defined as the average of the numbers of differences between the founder sequence and the sequences in the sample. The founder sequence is the sequence of the homogeneous population at the beginning; and for the observed samples, the founder sequence is inferred from the alignment of the sequences in the first sample taken  and (3) are used for computing the expected values of the four statistics. In this case the estimated expected values of the four statistics do not show strong qualitative discrepancy with their observed values except in the case of individual Pt9 see  and 4. TThe contrast between the mimicking powers of the two mutation models in the content of the four statistics is more obvious when I fit the population genetic model to the data sets under the infinite-sites model by matching the observed values of the numbers of polymorphic sites and average numbers of pairwise differences in the serial samples to their expected values, and I use the other two statistics as controls. The overall-fit scores in this case are also in I use the estimated models for the case of the finite-sites model (described in the previous section) to explore the signature of recombination on the dynamics of HIV genetic diversity. For this purpose I choose two statistics based on the linkage disequilibrium measure Using the computer program based on Algorithm 1 and Monte Carlo approach, I estimated the expected values of In the case of patient Pt1, I explore further and consider three hypotheses for The purpose of this study was to develop a computationally and statistically tractable framework, including recombination, for analyzing the dynamics of HIV genetic diversity in HIV-infected individuals. To derive this framework, I first designed a population genetic model that carries some of the features of within-host HIV evolution. Particularly, the model includes recombination, variability in population size and generation time, and heterogeneity of mutation rate across nucleotide sites. In addition, I considered the population size and generation time to vary over time as piecewise constant functions of time; these choices were made in order to derive the framework including recombination and without overwhelming the model with parameters that would be difficult to estimate.In spite of these choices, the model and framework can be extended for other evolutionary settings. Particularly, the model can be extended to include various distributions for the breakpoints along HIV genome, as well as for mutation rates across nucleotide sites. As another extension of the framework, I described Algorithm 2 that is applicable for serial samples of non-recombining sequences in a more general demographic scenario by allowing the population size to be a piecewise continuous function of time. Such a demographic scenario may be more suitable for exploring evolutionary dynamics of other pathogens at genomic level in vitro and in vivo. Particularly in vitro experiments in which a bacterial population goes through recurrent bottlenecks by growing or declining exponentially over time. However, in this setting the number of the parameters can increase and can be challenging to estimate them. For example, if the population size Another extension of the model is to replace the assumption of homogeneous population at time Note that the developed framework can be applied to generate HIV transmission chains and HIV epidemics at the genomic level. To be able to accomplish such a task, it is important to have a better understanding of the space of the values of the vectorsThe application of the framework to the serial samples of HIV DNA sequences from nine HIV infected individuals allowed to explore the fit of the model to the data sets and the impact of the recombination on the dynamics of within-host HIV genetic diversity. Particularly, these results show large variability for inferring the e points . These rThe wide spectrum of the inferred values of Since selection forces have impact on shaping within-host HIV genetic diversity ntervals  and 7 caAppendix S1The proof of Lemma 1. This section shows the derivation of the formula (2) by using the developed representation for polymorphisms in samples of DNA sequences under the piecewise constant population size model and the memoryless property of coalescence waiting times in the standard coalescent.(PDF)Click here for additional data file.


Here we review the studies to date that have employed novel optogenetic tools to improve our understanding of the pain pathway at the peripheral, spinal and supraspinal levels.The process of pain perception begins in the periphery by activation of nociceptors. From here nociceptive signals are conveyed via the dorsal horn of the spinal cord to multiple brain regions, where pain is perceived. Despite great progress in pain research in recent years, many questions remain regarding nociceptive circuitry and behavior, in both acute nociception and chronic pain states. Techniques that allow for selective activation of neuronal subpopulations  Chronic pain represents a significant clinical problem affecting up to 20% of the general population  in a mouse model of visceral pain (Crock et al., Another important region involved in the modulation of pain is the PFC. As with the amygdala, this region is particularly associated with the affective component of the pain experience (Tracey and Mantyh, Recently we have applied an optogenetic approach to explore novel brain regions involved in opiate analgesia. Opioids are important clinically, however their use is limited through the development of tolerance and addiction (Ling et al.,  The significance of this study lies in the ability to modulate the pain sensing TRPA1 neurons, without the need for genetic manipulations to confer this sensitivity. This approach may be useful for research and treatment of pain in the future. In particular, such an approach would allow selective optical control of subpopulations of peripheral nociceptors without the need for genetic manipulations.Optogenetic approaches require the expression of an exogenous light sensitive molecule in the target system. Although this has proved a very useful tool in basic neurobiological research, by definition this approach is of limited use clinically. An alternative approach is the use of “optopharmacology”, that is administration of compounds that confer light sensitivity onto a specific cell type (Kramer et al., Another approach has been the recent development of a photoactive MOR, the main receptor mediating the analgesic actions of morphine (Barish et al., The power of optogenetics lies in the ability to achieve regional and cell type specific neuronal activation. These methods provide an unprecedented opportunity to probe the complexities of the pain pathway at the peripheral, spinal and supraspinal levels. Among the most exciting developments in the field to date is the ability to produce pain-like behaviors in transgenic mice by optical stimulation of nociceptors in the skin (Daou et al., Somewhat surprisingly, to date no studies have taken advantage of these tools within the dorsal horn however this may reflect technical challenges in inserting optogenetic fibers into the spinal cord. Considering the importance of this component of the pain pathway, however, it is likely that optogenetics will also provide valuable new insights into this complex circuitry. Interneuron based optogenetic experiments have been performed within the ventral horn of the spinal cord, to investigate the contribution of particular subsets to locomotor activity (Dougherty et al., As described, a small number of studies have explored brain areas in pain using optogenetics, however many other areas of the pain matrix remain to be studied in this way. Both brain studies to date have relied either on electrophysiological recordings (Ji and Neugebauer, The studies described here are only the beginning of what we expect to be fruitful and informative exploration of pain circuits using optogenetic tools. It is likely that subsequent studies will move beyond these initial proof of concept studies, and use optogenetic tools to tackle unanswered questions regarding pain circuitry.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The persistent spread of Rhodesian human African trypanosomiasis (HAT) in Uganda in recent years has increased concerns of a potential overlap with the Gambian form of the disease. Recent research has aimed to increase the evidence base for targeting control measures by focusing on the environmental and climatic factors that control the spatial distribution of the disease.One recent study used simple logistic regression methods to explore the relationship between prevalence of Rhodesian HAT and several social, environmental and climatic variables in two of the most recently affected districts of Uganda, and suggested the disease had spread into the study area due to the movement of infected, untreated livestock. Here we extend this study to account for spatial autocorrelation, incorporate uncertainty in input data and model parameters and undertake predictive mapping for risk of high HAT prevalence in future.Using a spatial analysis in which a generalised linear geostatistical model is used in a Bayesian framework to account explicitly for spatial autocorrelation and incorporate uncertainty in input data and model parameters we are able to demonstrate a more rigorous analytical approach, potentially resulting in more accurate parameter and significance estimates and increased predictive accuracy, thereby allowing an assessment of the validity of the livestock movement hypothesis given more robust parameter estimation and appropriate assessment of covariate effects.Analysis strongly supports the theory that Rhodesian HAT was imported to the study area via the movement of untreated, infected livestock from endemic areas. The confounding effect of health care accessibility on the spatial distribution of Rhodesian HAT and the linkages between the disease's distribution and minimum land surface temperature have also been confirmed via the application of these methods.Predictive mapping indicates an increased risk of high HAT prevalence in the future in areas surrounding livestock markets, demonstrating the importance of livestock trading for continuing disease spread. Adherence to government policy to treat livestock at the point of sale is essential to prevent the spread of sleeping sickness in Uganda. Trypanosoma brucei rhodesiense and Trypanosoma brucei gambiense, cause the fatal disease human African trypanosomiasis (HAT); the clinical progression, as well as the preferred diagnostic and treatment methods differ between the two types. Currently, the two do not overlap, although recent spread of Rhodesian HAT in Uganda has raised concerns over a potential future overlap. A recent study using geo-referenced HAT case records suggested that the most recent spread of Rhodesian HAT may have been due to movements of infected, untreated livestock (the main reservoir of the parasite). Here, the initial analysis has been extended by explicitly accounting for spatial locations and their proximity to one another, providing improved accuracy. The results provide strengthened evidence of the significance of livestock movements for the continued spread of Rhodesian HAT within Uganda, despite the introduction of cattle treatment regulations which were implemented in an effort to curb the disease's spread. The application of predictive mapping indicates an increased risk of HAT in areas surrounding livestock markets, demonstrating the importance of livestock trading for continuing disease spread. This robust evidence can be used for the targeting of disease control efforts within Uganda to prevent further spread of Rhodesian HAT.The tsetse transmitted parasites,  Trypanosoma brucei rhodesiense parasite, and the Gambian form of the disease, caused by Trypanosoma brucei gambiense are not believed to overlap, and Uganda is the only country thought to support transmission of both diseases within its borders Glossina spp), and are fatal if untreated, although the speed of progression to death varies between the two (within approximately six months for Rhodesian HAT compared with years for Gambian HAT). A reservoir of infection is present for T. b. rhodesiense (predominantly livestock in Uganda) in contrast with T. b. gambiense for which no known reservoir exists other than humans. As a result, the most effective control options for the two forms of the disease differ, as do diagnostic procedures and treatment regimes. Currently, treatment is implemented based on knowledge of the areas affected by each type of HAT; Gambian HAT occurs in the north west of Uganda and Rhodesian HAT in the south east. Medical staff in endemic areas will presume infection is caused by the subtype known to exist in that area and implement the appropriate treatment regimen. A definitive diagnostic differentiation between the two parasite subtypes is difficult and requires expensive, complex methods which are not currently available in affected areas. Consequently, spatial concurrence of the two forms of HAT would compromise diagnostic and treatment protocols, resulting in a higher proportion of treatment failures and placing increased pressure on an already stretched health system The geographical ranges of Rhodesian human African trypanosomiasis , caused by the T. b. rhodesiense endemic areas at a local livestock market Recent research has focused attention on the environmental and climatic variables involved in the spatial distribution and spread of Rhodesian HAT Glossina species present across the sub-Saharan fly belt; within the areas of Uganda affected by Rhodesian HAT, the predominant species of tsetse vector is Glossina fuscipes fuscipes, which is restricted to riverine vegetation habitats  It is well documented that the focal distribution of human HAT is determined largely by the ecological and environmental requirements of the tsetse fly vectors The dependence of HAT transmission on the availability of competent vector populations leads to indirect associations between the spatial distribution of HAT and a variety of environmental and climatic factors. Within affected regions, areas with high HAT incidence tend to occur where there is a lot of contact between humans, tsetse flies and animal reservoirs (for Rhodesian HAT), for example, watering points et alBatchelor T. b. rhodesiense to the study area was assessed, given a more robust parameter estimation and appropriate assessment of covariate effects. The application of such methods to epidemiological research for the estimation of covariate effects and predictive mapping has been demonstrated in several recent studies including Diggle et alet alet alet alThe use of non-spatial methods for the analysis of data with a spatial structure can lead to biased regression parameters, underestimated standard errors, falsely narrow confidence intervals and, thus, an overestimation of the significance of covariates No patient names were recorded to maintain patient confidentiality and to adhere to the International Ethical Guidelines for Biomedical Research Involving Human Subjects. The use of these data was approved by the University of Edinburgh Research Ethics Committee.2 and a population of approximately 261,000 T. b. rhodesiense in the reservoir and, thus, altered the epidemiology of HAT in this area in the subsequent year; hence we have excluded from the analysis any cases diagnosed after 2006.The area of study included Kaberamaido and Dokolo districts (in the Eastern and Northern regions respectively) in Uganda, which have been affected by Rhodesian HAT since 2004. The study districts border the northern shore of Lake Kyoga with a combined area of approximately 2,740 kmet alRecords of all patients resident within Kaberamaido and Dokolo districts that received a positive diagnosis  of HAT between January 2004 and December 2006 were obtained from Lwala hospital (Kaberamaido district) and Serere hospital . All villages within Kaberamaido and Dokolo districts were geo-referenced using a handheld global positioning system , with direction from local guides. The HAT records were linked to the geo-referenced village dataset using village of residence and visualised using ArcMap 9.1 . To maintain the anonymity of subjects and patient confidentiality and to adhere to the International Ethical Guidelines for Biomedical Research Involving Human Subjects, no patient names were recorded within the database or as part of the data collection process. Further details regarding the provenance of these data have previously been published by Batchelor Non-spatial logistic regression methods were used to identify a set of environmental, climatic and social variables that were significantly correlated with HAT prevalence et alThe locations of all livestock markets and health centres within the study area were recorded during fieldwork using a handheld GPS. Maps detailing areas of woodland within the study area were obtained from the National Biomass Survey, which was conducted by the Uganda Forest Department between 1995 and 2002 The spatial variation in HAT prevalence within Kaberamaido and Dokolo districts was modelled using model-based geostatistics S(x) as follows:The total number of HAT cases The stochastic spatial component is modelled as a zero mean Gaussian process with variance geoRglmThe model parameters were estimated using a Bayesian framework with a Markov chain Monte Carlo (MCMC) algorithm in the package Priors (in Bayesian inference a prior is a probability distribution expressing uncertainty about a parameter before taking into account data observations) were selected for each parameter to represent prior knowledge of their distributions. Non-informative, uniform priors were selected for the regression parameters, Due to convergence and mixing problems when including all of the covariates listed in th iteration thereafter stored to assess the significance of each explanatory variable. Convergence and mixing of the MCMC algorithms was judged based on traceplots and autocorrelation plots for each model parameter to ensure that convergence had been reached, the chains had mixed adequately and autocorrelation amongst the samples was minimal. The mean values from the posterior distribution and their 95% credible intervals (CI)s were calculated and exponentiated to provide odds ratios (OR)s and their respective uncertainty measures. Only those covariates that were significantly associated with HAT prevalence  were selected for the multivariate spatial regression model.The univariate spatial models were run for 2,000,000 iterations, with the first 1,000,000 discarded and every 100An initial run of the multivariate model was carried out following the optimisation of th iteration thereafter stored, resulting in a total of 5,000 samples from the posterior distributions. The regression parameters and 95% CIs were obtained from the model and exponentiated as above.The fixed A 2 km spatial resolution prediction grid was created for the study area, containing covariate values at each prediction location (grid cell). Samples from the predictive distribution for each prediction location were generated using the MCMC algorithm given the explanatory variables at each grid cell. The posterior medians and lower and upper 95% CI limits from the predictive distributions were extracted to give predicted prevalence and uncertainty estimates at all locations. The predictions were then exported to ArcMap for illustrative purposes.et alA scatter plot of predicted prevalence versus observed prevalence was created to illustrate the relationship between the model predictions and observations, and the correlation between fitted and observed prevalence was calculated. In addition, the mean error, median error and absolute mean error  were calculated based on the difference between observed and predicted prevalence at each location, to give an indication of the prediction bias (mean and median error) and accuracy (absolute mean error). The Pearson residuals were calculated There were a total of 692 villages within the study area (Kaberamaido and Dokolo districts); all but two were geo-referenced . Of the remaining 690 villages, 18 that had recently separated into two were merged for the purpose of the analysis. Within the study period 354 cases of HAT were reported from these two districts, which equates to an overall period prevalence (2004–2006) of 0.14 per 100 population, although this value is very likely to be an underestimate due to complex issues surrounding care seeking behaviour for HAT and the under utilisation of health services From the univariate spatial regression model, five variables which were significantly correlated with HAT prevalence using deterministic, non-spatial logistic regression did not retain their statistical significance , and so was omitted from the final multivariate model. The remaining three covariates retained significance at the 95% level see . Both in2σ there was a slight positive skew. Traceplots and autocorrelation plots for model parameters were examined to assess the mixing and convergence of the MCMC algorithms and each appeared to have reached convergence during the burn-in period and to be mixing well. Autocorrelation amongst samples was minimal. The posterior distribution curves for the final model parameters  analysis as published by Batchelor et alFollowing on from the non-spatial logistic regression methods discussed in Batchelor T. b. rhodesiense to Kaberamaido and Dokolo districts. Previous research has established that the introduction of Rhodesian HAT transmission within Soroti district (which neighbours the study area) was due to movements of untreated cattle from endemic areas through a local livestock market et alT. b. rhodesiense is likely to have been introduced to Dokolo and Kaberamaido via the continued movement of untreated livestock, despite the introduction of a law requiring the treatment of livestock from endemic areas, prior to sale Five covariates did not retain statistical significance during the univariate spatial regression and one  did not retain significance during the multivariate spatial regression, indicating that the non-spatial model may have inflated the significance of covariates and produced inaccurate parameter estimates. The final spatial model included three covariate effects: distance to the closest livestock market, distance to the closest health centre and minimum LST. These results, using a more robust assessment of covariate effects, provide considerable strength to the hypothesis that the movement of infected, untreated livestock from endemic areas resulted in the introduction of Within the study area, it is problematic to separate the effects of differential utilisation of the HAT treatment centre, where those living closer are more likely to travel there for diagnosis and treatment than those living further away, from the purposeful siting of the treatment centre within the area most affected by HAT. Following the detection of a number of cases in Kaberamaido district in 2004, appropriate training and equipment were provided to one hospital within the area. The facility was selected based on a number of criteria, including the location within the affected area. Due to this difficulty, the distance to the closest health centre of any kind was used rather than distance to the HAT treatment centre. The significance of this variable in the spatial regression model highlights the importance of accessibility to health services as has been shown previously Minimum LST was observed to be a risk factor for HAT, with higher prevalence in areas with higher minimum LST. Minimum LST is calculated using measurements of radiance modified by the atmosphere in several spectral wavebands and varies depending on climate and also landcover properties  et alWhen the performance of the spatial regression model was compared with that of the non-spatial model  with only sporadic transmission to humans The research described utilised a variety of data sources providing information relevant to the distribution of the tsetse fly vector and, thus, also the distribution of Rhodesian HAT. However, accurate tsetse distribution or density data were not available for the study area, although the explicit inclusion of information on the spatial distribution of tsetse may have resulted in improved predictive power and provided further information on the determinants influencing the spatial heterogeneity in HAT prevalence within the main focus of disease. Additional factors that may play an important role in the observed spatial heterogeneity of HAT within Uganda include demographic factors, migration and human movement and behaviour patterns, due to their influence on the frequency of interaction between humans, tsetse and livestock. Although human migration has the potential to introduce The current research has demonstrated the application of Bayesian geostatistical modelling to the spatial distribution of HAT within a small area of Uganda. The more robust results provide strengthened evidence of the role of livestock trade in the continued spread of Rhodesian HAT within Uganda and the utility of this methodology for the prediction of HAT prevalence based on external covariates has also been demonstrated. The dataset used in this situation covered a relatively small area (two districts) with as complete a dataset as possible . The predictive power of this model over larger areas  is constrained due to the limited area from which the observed data came. To allow the full exploitation of these methods, future work will focus on a larger study area using a sample of villages. This will allow an investigation of HAT prevalence in relation to wider covariate ranges and will allow extrapolation over larger areas. The Bayesian implementation of model-based geostatistics as described here is computationally expensive and can be time consuming, but the application of such methods to epidemiological research is being assisted by a growing base of knowledge and expertise, along with the creation of more efficient algorithms et alThe research presented here illustrates the importance of spatial autocorrelation in epidemiological variables; the use of non-spatial logistic regression analysis resulted in a model with a large number of covariates, complicating the interpretation of their effects. The use of a generalised linear geostatistical modelling framework, which models the residual autocorrelation after accounting for covariate effects, gave more precise and less biased parameter and significance estimates, with only three covariates retaining significance in the final model. The Bayesian implementation of the method allowed the incorporation of uncertainty in each of the model parameters from the posterior distributions and from the definition of a random variable. By carrying out the spatial-regression analysis, the quantified relationships between HAT prevalence and significant covariates can be more confidently described and interpreted. The predictive accuracy was also increased by using the spatial regression when compared to the non-spatial logistic regression analysis. These results strengthen the evidence in support of the hypothesis generated by the analysis discussed in Batchelor Figure S1Posterior distributions for model parameters.(0.25 MB TIF)Click here for additional data file.Figure S2Traceplots of MCMC output for each parameter.(0.68 MB TIF)Click here for additional data file.Figure S3Autocorrelation plots of MCMC output for each parameter.(0.37 MB TIF)Click here for additional data file.


There was an error in the first sentence of the Funding Statement. The first sentence of the Funding Statement should read: "The funders of Sividion Diagnostics, Cologne, Germany conducted the Endopredict assay on all 34 tissue samples in a blinded way upon request of the authors."


There is a pressing need for further clinical evidence to better inform both patients and clinicians when recommending the optimal type and timing of breast reconstruction. The QUEST trials are unique; the first surgical trials comparing different types (A) and timing (B) of Latissimus dorsi (LD) reconstruction with a primary outcome of quality of life (QoL). Surgical trials are challenging, therefore necessitating a feasibility study to assess the acceptability of randomisation from both the perspectives of the patients and healthcare professionals. It was decided to develop a DVD to compliment the patient information sheet, in order to help patients understand the concepts of the clinical trials, surgical techniques, randomisation and clinical equipoise.All the women filmed in the DVD had a prior breast reconstruction and were invited to participate in a Q&A session with the Chief Investigator (CI) of the QUEST trials. The CI and 2 breast care nurses were also interviewed about the surgical techniques, complications and side-effects and the time taken to return to normal everyday activities after a breast reconstruction. All women will be given a short questionnaire to complete, assessing their level of understanding of randomisation and clinical equivalence irrespective of their entry into the QUEST trial.Filming took place over 1 day in Bristol and editing over a further 2 months. Drawings were produced to pictorially explain clinical equivalence, randomisation and the different types of LD reconstruction. The DVDs and the patient information sheets will be given to all women who are eligible and interested in the QUEST trials. We will be evaluating the impact of this DVD on patient recruitment by both quantitative (questionnaires) and qualitative (interviews) methodology.It proved feasible to develop a patient targeted DVD based on the experience of patients and healthcare professionals to enable potential QUEST participants to make fully informed decisions.


Accurate temporal estimations are essential in order to face the surrounding variety of everyday situations  The evidence of a close relationship, in childhood populations, between temporal accuracy and the performance in tasks involving WM, attention and impulsivity control; (ii) The evidence of age related functional differences comparing the activity of the prefrontal cortex during the execution of timing as well as WM, attentive and impulsivity control tasks.The implications behind this hypothesis are intriguing because they may help to clarify, through the study of cognitive development models, the relationship between the development of the EF and the progression of the level of sophistication of time keeping skills. Moreover, the study of the time keeping functions in childhood populations could represent a potential element of evaluation to qualitatively determine and/or monitor the EF development during the critical phases of brain growth. Finally, one advantage in charting the developmental trajectory of time processing and EF at certain critical moments of development is that this can help to differentiate between experience-dependent versus inborn aspects of time and EF.cognitively controlled” and “automatic” timing processes  mechanisms involved in the formation of three dimensions of EF discussed in this article. However, cognitively controlled timing skills cannot be reduced to these three EF, considering that the representation of time is built also with the active involvement of other processes (e.g., those implied in the representation of space and quantity, see Walsh, Future works devoted to exploring the developmental hypothesis discussed in this paper may wish to combine behavioral measures and brain methods in a longitudinal perspective, which may be recognized as important in addressing the link between cognitive and neural development. This approach would help to clarify whether and how these three domains of EF and cognitively controlled timing skills develop in parallel.


We have reported the risk of chest drain insertion inferior to the diaphragm when using current international guidelines . AnotherWe used the above guidelines to place markers (representing chest drains) in the thoracic wall of 16 cadavers bilaterally (32 sides), 1 cm anterior to the midaxillary line. Subsequent dissection identified the course and termination of the long thoracic nerve, the site of lateral cutaneous branches of intercostal nerves, and their relation to the markers.Grays' Anatomy (40th edition) it terminated before the inferior border of serratus anterior. Most commonly it was found to end by branching in the fourth (right) or fifth (left) intercostal space (range third to sixth). Lateral cutaneous branches of intercostal nerves were found in the fifth intercostal space in 25 of 32 cases. Contrary to the description in Last's Anatomy (12th edition) they always passed anterior to the midaxillary line (and marker).The long thoracic nerve was found in the fifth intercostal space in 16 of 32 cases, always in or posterior to the midaxillary line. Contrary to the description in Placement 1 cm anterior to the midaxillary line minimises risk to the long thoracic nerve and lateral cutaneous branches of intercostal nerves. We therefore conclude that not all areas of the British Thoracic Society safe triangle are indeed safe, and anteroposterior placement should follow the European Trauma Course and ATLS guidelines: just anterior to the midaxillary line .