Robotic surgery has enabled surgeons to offer more patients a minimally invasive surgical option in the management of their complex diseases. While renal transplantation is associated with significant improvements in quantity and quality of life for most end-stage renal disease (ESRD) patients, it is also not devoid of its surgical risks and potential morbidities. Robotic-assisted kidney transplantation is a recently described, innovative application of the robotic surgery platform, and early experiences suggest that it is associated with comparable graft function and lower rates of complications.Urinary tract obstruction, though less common than ESRD, can be a serious threat to renal function. Severe ureteric stricture disease can represent a clinically complex problem requiring major reconstructive surgery. Completely intra-corporeal robotic renal auto-transplantation is another innovative application of the robotic surgery platform and represents a significant advancement in urologic surgery. Initial reports of this procedure demonstrate safety, feasibility, and excellent renal function outcomes. Robotic-assisted laparoscopic surgery has gained widespread adoption globally within the field of urology. Technologic advancements have led to improved surgical dexterity and vision and, combined with a more facile learning curve than pure laparoscopic surgery, the robotic surgery platform has enabled more surgeons to perform minimally invasive surgical (MIS) procedures. Moreover, not only has robotic surgery afforded more patients access to a MIS option, it has also enabled urologists to perform extremely complex extirpative and reconstructive procedures, traditionally performed in an open fashion due to their technically challenging nature, with a minimally invasive approach.1 and since that time renal transplantation has changed the lives of millions of patients with end-stage renal disease (ESRD) through improved quantity as well as quality of life2. With the success of living donor renal transplantation programs, outcomes for patients with ESRD have improved even further.The first successful kidney transplant was reported in 19565, there are tangible peri-operative risks for ESRD patients undergoing transplantation that need to be weighed against these long-term benefits.Unlike healthy living donors, most kidney transplant recipients are, by the very nature of their disease, high-risk surgical candidates. While the long-term benefits of renal transplantation have been clearly documented8. Level 1 evidence supports a minimally invasive approach for donor nephrectomy9 in a patient population that is healthy and highly screened and selected. But for the high-risk ESRD recipients, for whom the peri-operative advantages of MIS would be evermore beneficial, a more morbid open surgical approach to renal transplantation still remains the gold standard.The advantages of MIS have been well documented in the literature and include less post-operative pain, quicker recovery time, lower rates of incisional complications, less blood loss, and better cosmesisAs robotic surgery has become more pervasive and mainstream, and with improved experience and training, it has allowed highly skilled and innovative surgeons to now offer MIS options for these higher-risk ESRD patients.10; however, this was in essence a hybrid operation whereby the da Vinci®  robotic surgical platform was used to perform a conventional open deceased donor kidney transplant through a large open incision.The first reported use of robotics to perform a kidney transplant was in 200211. Using the da Vinci® robot, Giulianottiet al. performed a deceased donor RAKT utilizing a 7 cm peri-umbilical incision through which the graft was introduced into the peritoneum. Total operative time was 223 minutes, with warm ischemia time of 50 minutes. The following year, Boggiet al. published the first successful European RAKT, but described a slightly different technique12. Rather than a peri-umbilical incision, the authors utilized a 7 cm Pfannenstiel incision. While the vascular anastomoses were performed entirely robotically, the ureterovesical anastomosis was performed in an open fashion through the Pfannenstiel incision. Total operative time was 154 minutes, with 51 minutes of warm ischemia. In 2015, Doumercet al. described another novel approach to RAKT, in which they utilized a vaginal incision to introduce the renal graft into the peritoneum, transvaginally, inside a sterile Endobag13. Similar to the other case reports, mean operative time was 200 minutes and anastomotic time was 55 minutes. While only feasible in female ESRD recipients, this novel transvaginal technique eliminates the need for a larger abdominal incision, further accentuating the minimally invasive nature of RAKT and thereby perhaps further decreasing the morbidity of renal transplantation surgery. Building on their novel technique, later that year, Doumerc and Sallusto reported the first pure robot-assisted approach to living donor kidney transplantation utilizing the transvaginal technique for both the donor and the recipient surgeries14.The first true robotic-assisted laparoscopic kidney transplant (RAKT) was reported by the group from University of Illinois at Chicago in 2010While these pioneering surgeons demonstrated the safety and feasibility of this innovative procedure, the reported RAKT techniques did not involve intra-corporeal cold perfusion of the graft. In addition, these early reports had longer warm ischemic times than commonly seen with open kidney transplantation. How this slight increase in warm ischemia truly impacts long-term graft function is yet unknown, but clearly delineates an opportunity for improvement.16. The authors used a peri-umbilical incision as well, but described the use of a novel gauze-jacket filled with ice-slush. This served to minimize warm ischemia and also allowed for atraumatic handling of the graft. Additional ice-slush was introduced into the peritoneum to cover the graft once it had been placed into the peritoneum. Utilizing this technique, the mean operative time in the case series was 214 minutes, with a mean “warm” ischemia time of 47 minutes15. The authors cited that while there was clear evidence for the feasibility and safety of this innovative procedure, comparative studies were still required to determine the cost-effectiveness of RAKT over conventional open kidney transplantation.In 2014, two separate publications from the same authorship group reported on a RAKT case series that utilized a new technique allowing for intracorporeal regional hypothermia of the graft18. With comparative ischemia times and the ability to cool the graft intracorporeally, graft function outcomes also seem to be at least equivalent to traditional open renal transplantation17.In line with existing comparative literature evaluating laparoscopic and open surgery, initial experiences at various institutions around the world have now demonstrated lower complication rates for RAKT in comparison to similar open renal transplant cohortset al. demonstrated that RAKT was associated with better outcomes in comparison to conventional open kidney transplantation. The authors pointed out that with improved outcomes after RAKT in the morbidly obese patient population, it may result in increased access to life-saving transplantation surgery for patients who may have otherwise been deemed unsuitable candidates due to their increased peri-operative risks17.Among a cohort of morbidly obese ESRD patients, OberholzerThese initial RAKT cohorts still represent a small sample size, and as such comparative assessment with larger cohorts will be necessary in time to further support the initial findings, which are very encouraging.17.One of the most common criticisms regarding the role of RAKT is the fact that an open incision, at least the size of the graft, is required regardless in order to introduce the allograft kidney into the peritoneal space, so why not simply use a slightly larger incision and do the surgery in a more conventional manner. RAKT supporters argue that any minimization of incision length, particularly in the immunosuppressed, ESRD patient population, can decrease the not-insignificant surgical incision-related complication rates seen in these patients. This is particularly true among obese transplant recipients, who are prone to much higher rates of incisional complications19. While much less commonly performed than allograft renal transplantation, ReATx is a definitive surgical option that allows for the preservation of renal function, whether imperative or elective, while reconstituting normal urinary drainage. The morbidity of ReATx is not inconsequential, however, as it involves two very distinct and complex procedures.Renal autologous transplantation (ReATx) was first reported in 1963 and represents a viable option in the management of long or severe upper ureteric strictures21. This approach has demonstrated excellent outcomes and is considered by many to be the gold standard approach.The laparoscopic approach to the management of many different urologic diseases has now become commonplace. With this, the morbidity of ReATx has significantly improved as well and, currently, the most common approach to ReATx is laparoscopic donor nephrectomy followed by open ReATxUnlike deceased donor or living donor renal transplantation surgery, for patients deemed suitable for ReATx, the allograft kidney is already located intra-corporeally. As such, if one were to be able to perform both distinct stages of the ReATx (i.e. donor nephrectomy and auto-transplantation), utilizing a MIS technique while maintaining the allograft intra-corporeally, the morbidity associated with a large surgical incision would be avoided completely. The ability to do this adds extreme technical complexity and would necessitate not only intra-corporeal preparation of the graft but completely intra-corporeal perfusion, and hypothermia as well.22. This truly innovative application of the robotic surgery platform allowed for the management of severe ureteric stricture disease without the allograft ever having to be removed from the patient. Total operative time was 425 minutes and total ischemic time was 127 minutes. While this pioneering report demonstrated the safety and feasibility of robotic-assisted ReATx, the described technique was associated with longer ischemia than one would encounter with the conventional approach to ReATx: laparoscopic donor nephrectomy,ex vivo preparation, and open auto-transplantation23.Taking their experience with RAKT one step further, Abaza and colleagues reported the first ever completely intra-corporeal robotic-assisted ReATx surgery in 201424 but modified the technique utilized by Abaza and colleagues22 in an attempt to minimize renal ischemia. Similar to their described technique, intra-corporeal renal perfusion with cooled HTK and normal saline solution was initiated immediately after donor nephrectomy using a perfusion cannula inserted through a 12 mm assistant port. By altering the technique from a two-stage to a three-stage procedure, however, we were able to decrease the total operative time to 390 minutes and, more significantly, we were able to complete the surgery with only 79 minutes of ischemia , which is more comparable to what is seen with conventional ReATx surgery.Building on their seminal work, we reported the first completely intra-corporeal robotic-assisted ReATx in Canadaex vivo would necessitate significant alterations in technique. We are currently working on developing such techniques that would allow for the allograft to remain intra-corporeally, while simultaneously minimizing renal ischemia during vascular reconstruction and auto-transplantation.While these two initial reports have demonstrated the safety and feasibility of completely intra-corporeal robotic ReATx, both case reports involved kidneys with relatively straightforward renal vasculature. With any increased complexity , vascular reconstruction without removal of the kidneyRobotic surgery has enabled surgeons to offer more patients a minimally invasive surgical option in the management of their complex diseases. Robotic-assisted kidney transplantation and completely intra-corporeal robotic renal auto-transplantation are recent innovative applications of the robotic surgery platform and represent significant advancements in urologic surgery. These novel applications of robotic surgery will hopefully result in improved patient outcomes while simultaneously achieving lower patient morbidity.


The utility of biomechanical models and simulations to examine clinical problems is currently limited by the need for extensive amounts of experimental data describing how a given procedure or disease affects the musculoskeletal system. Methods capable of predicting how individual biomechanical parameters are altered by surgery are necessary for the efficient development of surgical simulations. In this study, we evaluate to what extent models based on limited amounts of quantitative data can be used to predict how surgery influences muscle moment arms, a critical parameter that defines how muscle force is transformed into joint torque. We specifically examine proximal row carpectomy and scaphoid-excision four-corner fusion, two common surgeries to treat wrist osteoarthritis. Using models of these surgeries, which are based on limited data and many assumptions, we perform simulations to formulate a hypothesis regarding how these wrist surgeries influence muscle moment arms. Importantly, the hypothesis is based on analysis of only the primary wrist muscles. We then test the simulation-based hypothesis using a cadaveric experiment that measures moment arms of both the primary wrist and extrinsic thumb muscles. The measured moment arms of the primary wrist muscles are used to verify the hypothesis, while those of the extrinsic thumb muscles are used as cross-validation to test whether the hypothesis is generalizable. The moment arms estimated by the models and measured in the cadaveric experiment both indicate that a critical difference between the surgeries is how they alter radial-ulnar deviation versus flexion-extension moment arms at the wrist. Thus, our results demonstrate that models based on limited quantitative data can provide novel insights. This work also highlights that synergistically utilizing simulation and experimental methods can aid the design of experiments and make it possible to test the predictive limits of current computer simulation techniques. Orthopaedic surgery imposes substantial geometric changes on the musculoskeletal system. Changing geometry directly affects joint congruence and muscle-tendon paths, which in turn affect joint mechanics, muscle-tendon force-generating parameters, and even post-operative functional outcomes. Ideally, the geometric changes imposed by surgery could be input into biomechanical models, the models would appropriately calculate any changes to the joints and muscles, and computer simulations could then be used to predict functional outcomes. Accurate, predictive simulations would improve treatment decisions by allowing the comparison of multiple surgical procedures and providing insights into the procedures with the best outcomes.To date, biomechanical models and simulations have been successfully used to investigate a wide variety of orthopaedic problems, including tendon transfers –4, nerveDespite the widespread adoption and application of biomechanical simulations, the ability to use the current simulation research paradigm to solve clinical problems is highly dependent on the availability, cost, and ease of obtaining the experimental data necessary to build and validate surgical simulations. Importantly, in the current paradigm, building and validating biomechanical models of a given clinical condition is the rate-limiting step. The most widely used rigid-body musculoskeletal models, which describe asymptomatic individuals, leverage decades of experimental work e.g., , 30). Th. Th30]).This study aims to challenge the status quo research paradigm by evaluating whether models based on the limited quantitative data describing joint kinematics, that are available in the published literature, can predict changes in moment arms. We specifically examine two common wrist surgeries, proximal row carpectomy (PRC) and scaphoid-excision four-corner fusion (SE4CF). These surgeries were studied because they are used to treat the same degenerative conditions , but each imposes substantial changes to the wrist’s geometry. Importantly, the imposed geometric changes are very different between the two procedures . Thus, iTo understand to what extent models based on limited quantitative data could predict moment arms following surgical salvage procedures, predictive simulations were performed using models of PRC and SE4CF wrists that were based on extremely limited data. From these predictive simulations, a hypothesis was formulated regarding how these surgeries influence moment arms , and thiThe musculoskeletal models were developed in SIMM  by adapPRC was modeled using geometric changes described in the surgical literature, reported axes of rotation, and assumed muscle-tendon paths. The geometric changes involved removing the proximal row of carpal bones and translating the distal row and hand to establish an interface between the radius and capitate . Axes ofSE4CF was modeled using geometric changes described in the surgical literature, assumed axes of rotation, and muscle-tendon paths equivalent to those of the nonimpaired wrist. The geometric changes involved removing the scaphoid and fusing the lunate, capitate, hamate, and triquetrum using a weld joint . Axes ofFor the two PRC models (each with different muscle-tendon path definitions) and the two SE4CF models (each with different joint axes of rotation), moment arms were calculated for each wrist degree of freedom  for the primary wrist muscles at a neutral position using two methods: (i) the partial velocity method  and (ii)The moment arm experiment was designed to measure the muscle moment arms of the primary wrist and extrinsic thumb muscles. The data describing the primary wrist muscle moment arms has been previously described . BrieflyIn each specimen, data were collected sequentially for three conditions: nonimpaired, SE4CF, and PRC. To simulate SE4CF, the scaphoid was excised and Kirschner wires were used to fuse the lunate, capitate, hamate, and triquetrum. To simulate PRC, the Kirschner wires were removed and the remaining proximal carpal bones (lunate and triquetrum) were excised. Soft tissue and the finger extensor tendons were imbricated to establish an interface between the radius and capitate. Board-certified hand surgeons performed the surgical procedures.Moment arms, defined as the derivative of tendon excursion with respect to joint angle, were determined for each specimen, condition, muscle, and degree of freedom. Tendon excursions were simultaneously recorded from the five primary wrist muscles and the four extrinsic thumb muscles using potentiometers . Joint angles were calculated as the angle between the long axes of the third metacarpal and radius, which was measured by a motion capture system . All data was collected during passive, planar wrist motion for both flexion-extension and radial-ulnar deviation. Data were smoothed by fitting fourth order polynomials to the moment arm versus joint angle curves for each combination of specimen, muscle, surgical condition, and degree of freedom.Statistically significant differences between the nonimpaired, PRC, and SE4CF moment arm versus joint angle curves measured in the experiment were determined using mixed effects models, including condition and joint angle as fixed factors and specimen as a random factor. A significance level of p<0.05 was used for all tests. Multiple comparisons with a Tukey correction were used when the F-test of the ANOVA was significant.The simulation-based hypothesis was evaluated in two stages that utilized the experimental data from the primary wrist and extrinsic thumb muscles, respectively. In the first stage, the muscle moment arms for the primary wrist muscles were examined to determine if the trends predicted by the models were identified in the experiment. Also in this stage, the moment arms predicted by the model were directly compared to those measured in the experiment. For this direct comparison, moment arms at a neutral position were calculated from the experimental data because the simulation-based hypothesis was based on estimation of moment arms in a neutral position. Moment arms at a neutral position were defined as the average muscle moment arm for each muscle, surgical condition, and degree of freedom at zero degrees flexion-extension and zero degrees radial-ulnar deviation. In the second stage, the muscle moment arms for the extrinsic thumb muscles were examined to determine if the trends predicted by the simulation-based hypothesis were generalizable to other muscles crossing the wrist that were not explicitly modeled. This use of the extrinsic thumb muscle data was a form of cross-validation.The musculoskeletal models suggest that PRC primarily alters flexion-extension moment arms, while SE4CF primarily alters radial-ulnar deviation moment arms. Specifically, when compared to the moment arms estimated by the nonimpaired model, the flexion-extension moment arms estimated by the PRC models demonstrated larger changes in magnitude than those estimated by the SE4CF models for all five primary wrist muscles (cf., The experimental results for the primary wrist muscles support the hypothesis that PRC primarily alters flexion-extension moment arms, while SE4CF primarily alters radial-ulnar deviation moment arms. When comparing the nonimpaired and surgically altered flexion-extension moment arms, more muscles demonstrated statistically significant changes following PRC than SE4CF (c.f., The experimental results for the extrinsic thumb muscles demonstrate that the simulation-based hypothesis is generalizable. Similar trends for both flexion-extension moment arms (c.f., This study demonstrates that musculoskeletal models, even if they are based on extremely limited amounts of quantitative data, can provide important insights. The moment arms estimated by the models and measured in the cadaveric experiment both indicate that a critical difference between PRC and SE4CF is how they alter radial-ulnar deviation versus flexion-extension moment arms at the wrist. Although numerous studies have examined post-operative differences between PRC and SE4CF –58, the This study also demonstrates that musculoskeletal models can be used to inform the design of experiments. Traditionally, surgically altered moment arms are examined through cadaveric experiments by performing a muscle-by-muscle comparison of whether changes in moment arm due to a given surgery are statistically significant. However, incremental changes in individual muscle moment arms for multiple muscles at a given joint are difficult to interpret. In this study, we built upon these traditional analyses by using musculoskeletal models to learn about the system before performing an experiment. The need to synthesize and interpret our modeling analyses directly informed our hypothesis, which then guided our experimental design. Specifically, examining global changes in muscle actions, including changes to groups of muscles and differences between degrees of freedom, led to the decision to examine changes in flexion-extension versus radial-ulnar deviation moment arms in the nonimpaired, SE4CF, and PRC wrists.The simulation-based hypothesis was robust because it accurately predicted clinically relevant, global changes in muscle moment arms for both the primary wrist and extrinsic thumb muscles. However, we were unable to exactly predict the values of moment arms for specific muscles through surgical simulations . To furtIn contrast, the PRC model, which was based on assumed muscle lines of action, closely predicted both flexion-extension and radial-ulnar deviation moment arms for two of five primary wrist muscles (This study illustrates that biomechanical models based on extremely limited data sets provide novel insights that can be used to guide the design of experiments and test the predictive limits of current computer simulation techniques. Challenges remain before we can predict the exact values of moment arms for a specific surgical candidate through surgical simulations. However, models are valuable tools when examining of how the geometric changes imposed by orthopaedic surgery impact clinical outcomes.


Hypoplastic left heart syndrome (HLHS) is a congenital heart defect in which the left ventricle of the heart is severely underdeveloped. Applying patient-specific induced pluripotent stem cells (iPSC) with high-throughput sequencing technology in RNA-seq and whole genome sequencing (WGS) provides an unprecedented opportunity to investigate the disease-specific transcription profiles linked to potential genetic causes in HLHS. Bioengineered HLHS patient-specific iPSCs and differentiated cardiac tissues offer a platform to recapitulate the individual developmental process to study the molecular causes of the disease.In this study we reprogrammed the skin fibroblasts from proband and parents into iPSCs that were subsequently differentiated towards beating cardiomyocytes. We performed the RNA-seq on iPSCs and differentiated cardiomyocytes. Whole genome sequencing was done on blood samples from proband and parents. Combining expression differences between patient-specific cells with genomic mutations, we analyzed and integrated all these data to identify the potential genes related to HLHS.de novo mutations, were identified from whole genome sequencing data. LRP2 and PRTG were low expressed in proband in iPSC and differentiated cells and high expressed in the early heart developmental stages. However, DHCR24 were highly expressed in proband in both iPSC and differentiated beating cells and also highly expressed in the early stage of embryogenesis. Furthermore, MYLK, a later expressed gene in natural cardiogenic roadmap, is also highly expressed in proband.We have identified 4000 and 6000 differential genes between the family members in iPSC and differentiated cells respectively. Most the differential genes show a high expression pattern in iPSC from proband. However, the pattern from differentiated cells showed both high and low expression in proband compared with parent. 40 genes with different types of mutations, including compound heterozygosity, X-linked, By integrating the data from WGS, RNA-seq, and the naturally expressed time-course developmental roadmap, we triangulated a list of prioritized candidate genes that may contribute to HLHS and could be a target for future mechanistic studies for disease-specific clinical applications.


Recent advances were made in analyzing simultaneously different aspects of the cell graft , the ischemic lesion  and the neuronal and vascular network. We focus on optical methods, which permit simple animal preparation, repetitive experimental conditions, relatively medium-cost instrumentation and are performed under mild anesthesia, thus nearly under physiological conditions. A selection of recent examples of optical intrinsic imaging, fluorescence imaging and bioluminescence imaging to characterize the stroke pathology and engrafted stem cells are discussed. Special attention is paid to novel optimal reporter genes/probes for genetic labeling and tracking of stem cells and appropriate transgenic animal models. Requirements, advantages and limitations of these imaging platforms are critically discussed and placed into the context of other non-invasive techniques, e.g., magnetic resonance imaging and positron emission tomography, which can be joined with optical imaging in multimodal approaches.Transplanted stem cells can induce and enhance functional recovery in experimental stroke. Invasive analysis has been extensively used to provide detailed cellular and molecular characterization of the stroke pathology and engrafted stem cells. But post mortem analysis is not appropriate to reveal the time scale of the dynamic interplay between the cell graft, the ischemic lesion and the endogenous repair mechanisms. This review describes non-invasive imaging techniques which have been developed to provide complementary  The stroke pathology and regeneration processes induced by endogenous mechanisms or engrafted stem cells have been studied extensively. Invasive studies – including immunohistochemistry, autoradiography, electrophysiology, and molecular biology – revealed the ischemic cascade of pathological and protective signaling events . New neuFigure 1). We provide a selection of recent publications related to stroke and/or stem cell transplantation in rodents, which we find appropriate to introduce current possibilities and constraints of optical neuroimaging. In addition, selected references – in line with most preclinical optical imaging studies – refer to mice or rats and exclude, e.g., stroke studies in non-human primates  which have been extensively developed in the last decade to meet the criteria of a highly sensitive and minimally invasive set-up  and also by water and lipids . Brain tissue requires continuous blood supply, which implies strong light attenuation by absorption. Efficient light propagation through the brain is provided in a naturally existing window of low absorption in the near-infrared , e.g., extracellular fluids (n= 1.335) and triglycerides  camera. But in addition, the set-up is housed in a light-tight chamber to collect fluorescence emission exclusively from the anesthetized animal at the macroscopic level (in vivo neuroimaging due to the strong attenuation of shorter wavelengths (<700 nm). FLI has been adapted to the mouse brain application in the micro- and macroscale to visualize stem cells or stroke-related functional changes based on fluorescent proteins (FPs), fluorescent dyes, and endogenous chromophores.ic level . To peneic level . New sysAequorea victoria paved the way for a universal marker for cell structures and cellular processes detectable by fluorescence microscopy , the extinction coefficient , the FP maturation rate, photostability, pH stability, and aggregation potential (in vivo neuroimaging will profit from novel – much more efficient – NIR probes (The discovery of the green FP (GFP) from the jellyfish croscopy . The divcroscopy . Smart mcroscopy . Howeverotential . FPs forR probes .in vitro (ex vivo fluorescence microscopy (In vivo data has been acquired with NIR cyanine dyes (Cy) – especially Cy5.5 . Targeted probes have been designed to visualize key components of the stroke pathology. Inflammatory processes are monitored by an fluorescence-labeled antibody against the inflammatory receptor CD40 expressed on immune cells . Numerous probes have the potential to visualize stroke-related pathologies and host reactions, e.g., blood–brain-barrier (BBB) breakdown, infiltrating immune cells and (de-) myelination  and long chain carbocyanine dyes ]. A pulsed infrared laser is used to excite fluorophores by the combined power of two long-wavelength photons  is the method of choice to obtain detailed structural information of neural tissue  photons , which pcroscopy . 2PM hascroscopy . Using 2croscopy . Fluoropcroscopy , or FPs croscopy . Label-fcroscopy . Zhang acroscopy .in vivo is functional magnetic resonance imaging (MRI) on the basis of the hemodynamic response, thus the locally dynamic changes in oxy- and deoxyhemoglobin (HbO2 and HbR). Differences in absorption of HbO2 and HbR can be used by intrinsic imaging to record cortical activity on a sub-second time scale based on the changes in blood oxygenation (2 and HbR concentration. At wavelengths where HbO2 and HbR absorption is the same (isosbestic points), changes in total hemoglobin concentration can be measured  and long-lasting impairments on the processing of sensory stimuli by the forelimb somatosensory cortex .Grinvald and colleagues paved the way for a direct method to optically record neuronal activity: voltage sensitive-dyes VSDs;  and volty cortex . WeerakkIn vivo BLI uses, similar to fluorescence imaging (FLI), a CCD camera housed in a light-tight chamber to collect photon emission from the anesthetized animal. But instead of an excitation source, photons are emitted when the intracellular enzyme luciferase oxidizes its substrate luciferin. As luciferase is only expressed in transgenic mammalian cells, there is only negligible BLI background signal and emitted photons can be detected with surpassing sensitivity through the intact skull  and physical factors .ct skull  – even fct skull . BLI is veral mm . Althougveral mm , quantifhe brain , as it hhe brain . Luciferhe brain  and BBB he brain . In addiRenilla reniformis [Renilla luciferase (Rluc)], and the copepod Gaussia princeps , click beetles [click beetle luciferase (CBR)], the sea pansy sorption . Changessorption . To answsorption , the codto hRluc . Most imto hRluc ] or CBG9to hRluc ].in vivo optical imaging after implantation. By using bicistronic vectors for expression of two transgenes, e.g., a bioluminescent reporter for in vivo BLI together with a fluorescent reporter  provides the opportunity to also detect engrafted cells by invasive methods such as histology, microscopy, RT-PCR and Western blot. Notably, some luciferases, as Fluc, are dependent on ATP, thus serving as a non-invasive viability marker of engrafted stem cells to assess survival in vivo. Using this biochemical relationship In vivo BLI has also been used to assess the recruitment of NSCs in stroke mice after contralateral parenchymal, intra-ventricular  and represent useful non-invasive tools to investigate the therapeutic potential of endogenous stem cells for stroke recovery. Using conditional viral vectors, The second source of stem cells for stroke repair is the pool of endogenous stem cells, e.g., within the subventricular zone. These cells can be either targeted by ess Fluc , or by dess Fluc . Both apin vivo activation of neuronal differentiation by coupling Fluc to the NeuroD promoter which is active in neuronal precursor cells . The combination of optical imaging with MRI data could be used to create 3D reconstructions to relocate cell function back to the anatomical structure, which is highly resolved by MRI .Optical imaging provides best sensitivity of 10n stroke . Among mn stroke  and progn stroke  with onen stroke . PET iman stroke . Sequentn stroke . We haven stroke . There, We believe that the application of the non-invasive imaging tools presented here will be essential to fully understand stroke regeneration and the potential of engrafted stem cells in pre-clinical trials – the prerequisite of an effective clinical therapy.Markus Aswendt, Joanna Adamczak and Annette Tennstaedt wrote the manuscript.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.


To the Editor: Progressive disseminated histoplasmosis (PDH) of infancy occurs most commonly in previously healthy infants <1 year of age, typically after exposure to a large fungal inoculum , low fibrinogen level , elevated triglycerides  and elevated soluble interleukin-2 receptor . An HIV serologic test result was negative. Histologic examination of bone marrow showed many activated macrophages, including some with hemophagocytosis . On the Histoplasma capsulatum , according to treatment guidelines for both conditions (>1 of the genes required for perforin-dependent lymphocyte cytotoxicity; secondary HLH occurs in a person who does not have genetic risk factors (HLH is a rare disease, characterized by impaired function of natural killer and cytotoxic T-lymphocytes that results in an unchecked inflammatory response. If not treated promptly, HLH can progress rapidly to multiorgan failure and death (Diagnosis of HLH is based on clinical assessment ; no defi,Many clinical manifestations of disseminated histoplasmosis, including prolonged fever, hepatosplenomegaly, pancytopenia, and coagulopathy, overlap with those of HLH. Because of the similarity in manifestations, differentiating these 2 conditions is challenging without specialized testing. Furthermore, laboratory tests specific for HLH, such as measuring soluble interleukin-2 receptor, have not been investigated in patients with isolated PDH, so whether they distinguish between the 2 conditions is unclear. To add further complexity, coexisting histoplasmosis and HLH has been described in several adult patients (Histoplasma-endemic regions be assessed for histoplasmosis. In addition, the similarity with the clinical features of PDH and the difficulty in diagnosis of HLH without specialized testing raise the question of whether a large number of infants with PDH would also meet the diagnostic criteria for HLH. If so, currently poor outcomes of PDH might be related to co-existing HLH with failure to control the inflammatory response, and the outcomes could be improved by diagnosis and simultaneous treatment of both conditions. Further research is needed to investigate this phenomenon.On the basis of our investigation of this infant with HLH and PDH, we recommend that all infants exhibiting HLH in Wright-Giemsa stain of the bone marrow biopsy specimen of a 6-month-old girl with hemophagocytic lymphohistiocytosis and progressive disseminated histoplasmosis.


Current strategies for optimizing deep brain stimulation (DBS) therapy involve multiple postoperative visits. During each visit, stimulation parameters are adjusted until desired therapeutic effects are achieved and adverse effects are minimized. However, the efficacy of these therapeutic parameters may decline with time due at least in part to disease progression, interactions between the host environment and the electrode, and lead migration. As such, development of closed-loop control systems that can respond to changing neurochemical environments, tailoring DBS therapy to individual patients, is paramount for improving the therapeutic efficacy of DBS. Evidence obtained using electrophysiology and imaging techniques in both animals and humans suggests that DBS works by modulating neural network activity. Recently, animal studies have shown that stimulation-evoked changes in neurotransmitter release that mirror normal physiology are associated with the therapeutic benefits of DBS. Therefore, to fully understand the neurophysiology of DBS and optimize its efficacy, it may be necessary to look beyond conventional electrophysiological analyses and characterize the neurochemical effects of therapeutic and non-therapeutic stimulation. By combining electrochemical monitoring and mathematical modeling techniques, we can potentially replace the trial-and-error process used in clinical programming with deterministic approaches that help attain optimal and stable neurochemical profiles. In this manuscript, we summarize the current understanding of electrophysiological and electrochemical processing for control of neuromodulation therapies. Additionally, we describe a proof-of-principle closed-loop controller that characterizes DBS-evoked dopamine changes to adjust stimulation parameters in a rodent model of DBS. The work described herein represents the initial steps toward achieving a “smart” neuroprosthetic system for treatment of neurologic and psychiatric disorders. Neurologic and psychiatric disorders can be characterized by motor, behavioral, cognitive, affective, or perceptual traits that affect how individuals move, feel, think, and behave  are adjusted in an attempt to minimize adverse effects while maximizing clinical benefits. Although this strategy has provided significant patient benefit, the results are far from optimal. First, this open loop strategy relies on the subjective experiences of both the patient and clinician, without providing objective feedback to support parameter optimization. Second, the therapeutic response observed in this acute setting does not guarantee sustained therapeutic effects. Disease progression, environmental factors, and behaviorally induced changes in network activity can all render therapeutic stimulation ineffective, requiring additional programming sessions   analysis is an electrophysiological technique for detecting changes in brain activity that offers great potential for understanding the network effects of DBS  is typically measured using far-field sensors located on the scalp (EEG) or directly on the brain surface (ECoG). These global field potentials can be used to identify information regarding high-level sensory processing, perception, and locomotor activity  placed in close proximity to the target cells . While focused on DBS of ascending dopaminergic fibers in the MFB for evoking dopamine release in the rat striatum  DBS. Additionally, this paradigm relies on non-linear regression, computational modeling, and constrained optimization techniques to parameterize stimulation-evoked dopamine responses. The inverse dynamics of stimulation-evoked dopaminergic responses are modeled using artificial neural networks (ANN), which also predict stimulation parameters required for sustaining target dopaminergic concentration levels. The performance of this closed-loop paradigm was evaluated by comparing target dopaminergic responses to ad libitum in conventional housing in accordance with National Institutes of Health (NIH) and US Department of Agriculture guidelines.To quantify the dynamics of stimulation-evoked dopamine release, recording FSCV CFM and bipolar DBS macroelectrodes were implanted into the striatum and MFB, respectively, in four anesthetized rats. All animal procedures were performed according to the guidelines of the Mayo Clinic Institutional Animal Care and Use Committee (IACUC). Animals were kept on a standard 12 h light-dark cycle with access to food and water Animals were anesthetized and the head was fixed in a Kopf stereotactic frame  for electrode targeting. Following brain exposure, one bipolar stimulating electrode, one FSCV recording electrode, and one silver-chloride reference electrode was inserted into the left MFB, striatum, and contralateral cortex, respectively. Recording electrodes were allowed to stabilize within the tissue environment for 20 min. Finally, the electrodes were connected to a wireless stimulator and neurotransmitter sensor for real-time detection of stimulation-evoked dopamine release  to remove signal noise. Additionally, the responses to individual stimuli were characterized using a combination of 7th-degree polynomial and 2nd-order exponential mathematical models. The mathematical model parameters  and corresponding stimuli were presented to a double-layer feedforward ANN with sigmoidal and linear transfer functions . Furthermore, these results show that adjusting stimulation parameter intensity can modulate dopamine concentration, and that we can use ANN to dynamically predict stimulation parameters required to adjust stimulation-evoked dopamine levels (Figure Our preliminary results in four anesthetized rats suggest that mathematical models can be used to describe the relationships between stimulation-evoked extracellular dopamine responses and DBS parameters (s Figure . HoweverFrequent adjustment of stimulation settings has been shown to improve the efficacy of DBS therapy (Rosin et al., Before automated adjustment of stimulation parameters can be clinically implemented, however, several key clinical questions need to be investigated. Specifically, the relationship between neurotransmitter levels and symptoms of neurologic disease needs further elucidation. For example, there is indirect evidence to suggest that dopamine depletion plays a role in the symptoms of PD and that dopaminergic medications have a therapeutic response. However, precise concentration changes that occur with symptom exacerbation and amelioration are unknown. Additionally, multiple neurotransmitters may play a critical role in the disease (Fitzgerald, Similarly, an important technical barrier that needs to be addressed is that chronic recordings are not possible using current electrode technology. CFMs are subject to electrode fouling due to the charge imbalance of the waveforms required for FSCV. Efforts are underway to develop electrochemical-sensing techniques capable of extending electrode longevity by renewing the electrochemically active surface following adsorption of chemical species (Takmakov et al., Conventional neuromodulation systems have been successful at achieving therapeutic outcomes in patients with neurologic and psychiatric disorders. However, limitations in existing technology make ensuring optimal benefits a difficult and expensive endeavor. Correlation of multi-modal electrophysiological and neurochemical recordings may provide new insight into the cellular and molecular mechanisms of therapeutic neuromodulation. Therefore, development of smart DBS controllers that rely on the relationships between neurochemical and electrophysiological recordings with the clinical effects of DBS offers the potential of replacing the trial-and-error process used in clinical programming with a deterministic approach. Furthermore, the versatility and adaptability of such controllers will allow expansion of the clinical indications that can be treated with DBS while tailoring its application to individual patients and symptoms. In turn, these will likely improve clinical outcomes, reduce the time and frequency of patient visits, and lower overall health care costs.J. L. Lujan has intellectual property licensed to Boston Scientific. 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.


Paddy-upland rotation is an unavoidable cropping system for Asia to meet the increasing demand for food. The reduction in grain yields has increased the research interest on the soil properties of rice-based cropping systems. Paddy-upland rotation fields are unique from other wetland or upland soils, because they are associated with frequent cycling between wetting and drying under anaerobic and aerobic conditions; such rotations affect the soil C and N cycles, make the chemical speciation and biological effectiveness of soil nutrient elements varied with seasons, increase the diversity of soil organisms, and make the soil physical properties more difficult to analyze. Consequently, maintaining or improving soil quality at a desirable level has become a complicated issue. Therefore, fully understanding the soil characteristics of paddy-upland rotation is necessary for the sustainable development of the system. In this paper, we offer helpful insight into the effect of rice-upland combinations on the soil chemical, physical, and biological properties, which could provide guidance for reasonable cultivation management measures and contribute to the improvement of soil quality and crop yield. Green Revolution technologies have allowed the food supply of Asia to satisfy the demand of its rapidly growing population in the past decades; however, the pressure on soil and other resources has intensified . The culPaddy-upland rotation is the most important cropping system in southern and eastern Asian countries such as Bangladesh, China, India, Nepal, and Pakistan . This tyMany studies on this issue have been conducted because the sustainability of rice-based cropping systems is important to the food security of Asia. Changes in soil properties caused by cultivation and management and their consequences to soil productivity have generated significant research concern for many years. Evidence indicates that the degradation of soil quality is a key factor for the observed declining yield . As a reRice and upland crops are grown annually in sequence influence each other; however, the soil conditions required by rice growth differ from those required by upland crops. Soil is puddled before rice transplanting and kept flooded to create anaerobic conditions for rice growth. By contrast, upland crops are grown in well-drained soil under tillage and aerobic conditions. Several benefits have been found in rice cultivated under puddled condition. Puddling created a plow layer that reduces hydraulic conductivity to support water ponding, which minimized the water percolation losses and enhanced the water and nutrient use efficiency of rice . PuddlinPaddy-upland rotation could change the soil physical properties of long-term-flooded paddy. Studies have indicated that after the application of paddy-upland rotation, the adverse effects in the long-term-flooded paddy fields have shown slight improvements, such as increased soil granular structure and capillary porosity, improved redox potential of soil, and removed secondary gleization. For instance, Huang and Ding  showed t−3 to 1.8 g cm−3 [There is a large variation in soil BD of paddy-upland rotations, which ranges from 0.9 g cm8 g cm−3 , 19–24  status, which is important for nutrient mineralization, soil structural improvements, and favorable soil-water relations . Soil st4 . During 4 . Kumari 4  posited 4 . Soil ty4 .2O emission mainly occurs during the drying period of soil rather than during the flooding period, indicating that the alternation of wetting and drying could improve N2O emission [With its negative effects on the environment, the use of N fertilizers and its cycle has received research interest for several years. N fertilizer application is the most important source of soil N, and the three major paths of N losses  are all influenced by N application, soil moisture state, and tillage practice; consequently, the paddy-upland rotation significantly affects the soil N cycle because of the contrasting soil and water conditions and different nutrients required by the two crops. Research has confirmed that Nemission . Other femission . Bueno aemission  found thThe interactions between paddy rice and upland crops make the soil C and N cycles more complicated, thereby increasing the difficulty of water and nutrient management of paddy-upland rotation. Paddy-upland rotation is generally harmful for soil C and N storage under the current condition of the cultivation and management practice of farmers. The changes in soil fertilizer before and after experiment under different land utilizing types are shown in Soil microorganisms are involved in various biochemical processes and are vital in maintaining soil fertility and plant yields. The diversity of rhizosphere microorganisms is beneficial to soil health, and the trophic interactions within the rhizosphere affect the aboveground community of plants , 51. Dif Bradyrhizobium sp. and Herbaspirillum sp. colonize the interior of rice roots when grown in rotation with a legume crop, which may promote rice growth and productivity [Several studies have reported a positive effect of crop rotation on the abundance of beneficial microorganisms. Cropping systems could markedly affect the composition, abundance, diversity, and activity of soil bacterial communities; evidence shows an association between crop type and microbial community composition. For instance,uctivity . Furtheructivity . Therefouctivity  indicateclerotinia stem rot of oilseed rape, which is caused by Sclerotinia sclerotiorum, is one of the most common fungal diseases of rapeseed. Ding [ Verticillium wilt, which is caused by soil-borne fungus, is a worldwide disease affecting temperate and subtropical regions that causes vascular wilt in many plant species [ Verticillium wilt of eggplant and strawberry. Marenco and Santos [ Pseudomonas fluorescens [ Burkholderia vietnamensis [Other effects of crop rotation on soil biological properties are diseases, especially soil-borne diseases, and weed suppression. Different crop species with diverse root exudates and plant residues create varying patterns of resource competition, allelopathic interference, soil disturbance, and mechanical disruptions , 56, whied. Ding  indicateed. Ding  showed t species . Ebihara species  demonstrd Santos  showed torescens  and Burknamensis  are wellnamensis .The prevention effect of a specific rotation on pest species proliferation may be mediated by impeded drainage and tillage practices, particularly no-till in which plant residues remain on the field . HoweverPaddy-upland rotation is an unavoidable cropping system for Asia to meet the increasing demand for food. However, conventional practices of growing rice and wheat not only deteriorate soil physical properties and decrease water and fertilizer use efficiencies but also cause a stagnation or even reduction in grain yields. Maintaining soil quality at a desirable level is a very complicated and difficult task, because paddy-upland rotation fields are unique from other wetland or upland soils; they are associated with frequent cycling between wetting and drying under anaerobic and aerobic conditions; such rotations change the soil C and N cycles and make the chemical speciation and biological effectiveness of soil nutrient elements varied with seasons, increase the diversity of soil organisms, and make the change of soil physical properties more complicated . Therefo


The use of genetically isolated populations can empower next-generation association studies. In this review, we discuss the advantages of this approach and review study design and analytical considerations of genetic association studies focusing on isolates. We cite successful examples of using population isolates in association studies and outline potential ways forward. Genome-wide association studies (GWAS) have met with widespread success in identifying common-frequency variants associated with complex diseases and medically relevant quantitative traits. Technological advances in genotyping and sequencing have recently enabled access to low-frequency and rare variant genotypes at the population level. The identification of modest effects at individual low frequency and rare variants requires very large sample sizes. Power can be boosted by using statistical approaches to aggregate rare variants across chromosomal regions or functional units. Power can also be increased by leveraging the unique characteristics of isolated, or founder, populations in genetic association studies. In the past, population isolates have typically been used in family-based genetic studies of Mendelian traits , 2. Advae or the effective number of individuals required to explain the observed genetic variability) [Population isolates can be defined as subpopulations derived from a small number of individuals who became isolated because of a founding event  and have stayed so for many generations. The resulting geographical and/or cultural isolation of these populations has genetic consequences, as, for example, endogamy (within community marriage) along with very restricted gene flow (immigration) from neighbouring populations can often be observed. Thus, genomes tend to show higher homogeneity in isolates compared with cosmopolitan populations, which is reflected by a reduced effective population size (Nability)   was recently replicated in an exome chip-wide association scan for lipid traits in ∼1200 individuals from a Greek population isolate [In population isolates, certain alleles reach fixation or extinction at a particular locus, thus reducing the extent of genetic variability , 28. Som isolate . To achiPopulation choice is an important consideration in designing a genetic association study focusing on isolates. Factors such as the number of founding haplotypes, age of divergence from the parent population, effective sample size and degree of admixture with neighbouring populations, all play a role in the population’s allelic architecture. For initial GWAS as well as rare Mendelian gene discovery, the study of young founder populations with recent expansion (e.g. late-settlement Finland) is a powerful strategy  because GWAS arrays by definition assay primarily variants selected to represent common frequency variation across the genome. Low-frequency and rare variants genome-wide cannot be easily captured on a genotyping array because of their large numbers and low levels of correlation. Recently, a genotyping chip focused on likely functional coding low-frequency and rare variants—the exome chip—has been used successfully in founder populations to associate rare variants with proinsulin  and HDL Whole exome sequencing has the advantage of reduced cost compared with whole-genome sequencing, but does not capture variation in non-genic regions. Previous experience from GWAS strongly indicates that the majority of complex trait signals reside outside of genes. High-depth sequencing is considered necessary to call high-quality variants across the allele frequency spectrum . HoweverWhen not all samples can be sequenced, whole-genome sequencing of a subset of cases and controls following an initial GWAS has proven to be a successful strategy for empowering rare variant association in dichotomous trait studies , 41. VarOne intrinsic consequence of genetic isolation is relatedness among individuals, which can conflict with the assumptions of independence of many commonly used analysis tools and inflate test statistics affecting association signals. An efficient approach is to account for relatedness in the association analysis through the use of a linear mixed model (LMM). Until recently, computation of an exact association test statistic such as the Wald statistic or likelihood ratio (implemented in EMMA ) was comSeveral methods have been proposed to improve on the power of single-point tests for rare variants by combining information across multiple sites in a chromosomal region and testing for association with the trait of interest . Relatedwww.1000genomes.org) and/or the UK10K study data (www.uk10k.org) to enhance the resolution of the study. This approach, where positions that were not genotyped in the sample are added using phase information in the reference set, is also relevant to refining genotype calls for low-depth sequencing data.When performing association based on genotyping arrays, it is common practice to impute untyped variants based on a reference panel , with other phased individuals. Relatedness is helpful for phasing because it increases the likelihood of finding a long IBS string of variants; the more related the samples, the more certain it is that these IBS sequences are actually inherited identical-by-descent (IBD), and the probability that unobserved positions are IBS becomes quantifiable , 59. KonP-value-based tests and effect size-based methods, which can be further subdivided into fixed or random effects models [The synthesis of data through meta-analysis can increase the power of association studies. Two different classes of methods have been typically applied in traditional meta-analysis of GWAS: s models . Fixed es models . In addis models , and a cNDST3 and was replicated in 11 independent cohorts of varying ethnicities. Recently, a Greek isolated population replicated a genome-wide significant association between R19X, a cardioprotective variant in APOC3, and low blood triglyceride levels [Several studies from Iceland have been successful in identifying low- /rare-frequency variants associated with sick sinus syndrome, gout, prostate cancer and Alzheimer’s disease , 66–68. e levels . This ste levels .Population isolates are uniquely positioned to usher in the new era of sequence-based association studies. The relative power advantages afforded by studying isolates have been well-documented and recently exemplified in the literature through successful identification of complex trait loci that replicate in other populations. Not all novel discoveries in isolates can be recapitulated in other populations. However, private variants detected in isolates can importantly point to novel biology, identifying potential pathways and loci involved in the aetiopathogenesis of clinically relevant traits. Large-scale efforts to synthesize genome-wide data across isolated populations are an intuitive next step in the field, and hold the promise of catalysing the discovery of further complex trait-associated variants.The population genetics characteristics of an isolate depend on demographic history and the number of bottlenecks/founding events, total number of founders, levels of endogamy, age and duration of isolation.Genetic consequences of isolation such as reduced haplotype diversity and genetic drift can enhance the power for locus identification in genetic association studies of complex traits.Wellcome Trust (098051) and the European Research Council (ERC-2011-StG 280559-SEPI).This work is supported by the


Large quantities of digital images are now generated for biological collections, including those developed in projects premised on the high-throughput screening of genome-phenome experiments. These images often carry annotations on taxonomy and observable features, such as anatomical structures and phenotype variations often recorded in response to the environmental factors under which the organisms were sampled. At present, most of these annotations are described in free text, may involve limited use of non-standard vocabularies, and rarely specify precise coordinates of features on the image plane such that a computer vision algorithm could identify, extract and annotate them. Therefore, researchers and curators need a tool that can identify and demarcate features in an image plane and allow their annotation with semantically contextual ontology terms. Such a tool would generate data useful for inter and intra-specific comparison and encourage the integration of curation standards. In the future, quality annotated image segments may provide training data sets for developing machine learning applications for automated image annotation.http://www.plantontology.org/software/AISO.We developed a novel image segmentation and annotation software application, “Annotation of Image Segments with Ontologies” (AISO). The tool enables researchers and curators to delineate portions of an image into multiple highlighted segments and annotate them with an ontology-based controlled vocabulary. AISO is a freely available Java-based desktop application and runs on multiple platforms. It can be downloaded at AISO enables curators and researchers to annotate digital images with ontology terms in a manner which ensures the future computational value of the annotated images. We foresee uses for such data-encoded image annotations in biological data mining, machine learning, predictive annotation, semantic inference, and comparative analyses. Annotation of Image Segments with Ontologies (AISO) is an interactive tool which allows users to segment and annotate a digital image – such as those produced with digital photography or from scanned prints – with ontology terms. An ontology is a controlled and structured vocabulary of agreed-upon labels (‘terms’) that represent the knowledge of the types of entities within a given domain . Labelinad hoc editing of an image, but any highlighting and labeling utility requires thorough knowledge of the software’s illustration capabilities  and does not include the structured integration of scientific data. For example, any labels applied to hand-illustrated segments superimposed onto an image would have to be individually constructed and associated with a particular portion of an image. AISO simplifies this functionality and requires only a few input gestures and clicks to identify and label segments. The resulting structured image and ontology annotation allows for consistent extraction techniques, enabling future database storage, active learning, and semantic inference functionalities. Researchers are thus empowered to construct meaningful image data sets drawn from their laboratories, online image archives, and publications.Many photo-editing and illustration software packages enable the AISO is a multi-platform, Java desktop application extending the source code of the Interactive Segmentation Tool (IST) , originaSegmentation algorithms are created with different application domains in mind and the computer vision research community is generally focused on segmenting images of the human body and the built environment. Segmenting anatomical images of biological specimens, such as plants, presents a number of challenges that have received scant attention in the literature. Plants contain curvilinear and asymmetric forms, textures, and spatial orientations that make identification and classification more difficult for computer vision algorithms. We chose the Interactive Graph Cuts (IGC) segmentation algorithm because markups have a local effect, thereby avoiding major global deformations in the segmented area. This has great value in plant images that contain densely grouped features, such as many similar, overlapping leaf structures. The IGC algorithm also is more accurate in extracting foreground objects, and includes a responsiveness that allows the user to iteratively refine the segments. The average time required for a user to attain optimal object and boundary accuracy for an image, and the average total time spent annotating each image are much lower when compared to other algorithms .After opening an image file in AISO Figure B,C, AISOThe user can save annotated image files into a custom ZIP package, and may also re-open previously annotated images for continued editing. The original image is always preserved and viewable. When saving annotations, the user may optionally export an HTML file containing a web-enabled version of the annotated image, which allows the user to easily share their work in other media platforms. For example, manuscript authors could submit annotated images along with other supplementary data, to enhance the collection of ontology-based image data for comparative analyses and machine learning. Annotated images could thereafter be used in online resources and publications, or placed in a file archive or database for future analysis.Galanthus elwesii, commonly known as “Giant Snowdrop”, was segmented and labeled with the Plant Ontology terms plant ovary (PO:0009072), style (PO:0009074), tepal (PO:0009033), anther (PO:0009066) and perianth (PO:0009058) , both as a test of AISO’s capabilities and as the beginnings of a training set of segmented image data for a potential future active-learning system. One such image of the floral structures of PO:000907, style (stamen (PO:0009029), carpel (PO:0009030), and tepal (PO:0009033) were applied to user-identified segments in a cross-section image. This particular example highlights the IGC algorithm’s localized border-detection capabilities when constructing an image segment.Additionally, AISO’s capabilities have been applied to paleo-botanical images of fossilized lauraceous flowers from the Eocene epoch Figure . In thiset al.[et al.[AISO brings together disparate image segmentation and semantic labeling functionalities found in existing software and merges them into a user-friendly, science-focused package. Hollink et al. develope[et al. develope[et al., built a[et al. includesFuture enhancements to AISO include extending web service support for multiple ontologies, such as those developed by OBO Foundry  members AISO allows researchers and curators to interactively segment images and assign semantic annotations to those segments. This annotation capability gives biologists the opportunity to enhance the computational value of their own image data. Data-enriched images can be used to mine biological data sets, train machine learning software, and generate conclusions via semantic inference. We believe that the existing functionality of AISO, combined with our future efforts in active learning, will provide a powerful tool for the biology community and for scientific journals interested adding annotated images and associated metadata to their publication pipeline.Project name: Annotation of Image Segments with Ontologies (AISO).Project home page:http://www.plantontology.org/software/AISO.Operating system(s): Platform-independent .Programming language: Java.Other requirements: An Internet connection, the Java Runtime Environment (JRE).License: Creative Commons .Any restrictions to use by non-academics: No.


Esophageal bronchogenic cysts are extremely rare. Here we report a more rare type of both presence of intra- and paraesophageal bronchogenic cyst that was safely removed via surgical resection. A 31-year-old male patient with space-occupying lesions in the mediastinum suddenly presented with persistent chest pain for 2 days and then transferred to dysphagia >1 week. Preoperative diagnosis is difficult. Endoscopic ultrasonography (EUS) showed a hypoechoic cystic-solid mass arising from the muscularis propria and local hyperechoic area in the deeper portion of cyst, concomitant with a heterogeneous center and tube-like structure lesion in mediastinum. Turbid coffee color paste contents were aspirated inside the tumor under endoscopic ultrasonography guided-fine needle aspiration (EUS-FNA). A subsequent surgery was performed and histologic finding was diagnostic of esophageal bronchogenic cyst. Immunohistochemical staining confirmed the cyst was positive for carbohydrate antigen 199 (CA199) and carbohydrate antigen 125 (CA125). At a follow-up visit 3 months later, the patient had a regular diet and no complaint. This study is to summarize the clinical manifestations and EUS features of esophageal bronchogenic cyst by retrospectively reviewing the literature and simultaneously to provide guide for the correct examination scheme.The appearance of esophageal bronchogenic cyst can be great variation; EUS seems to be a valuable option for diagnosis and surveillance. Sporadic cases concerning intramural esophageal bronchogenic cysts are reported.5 Moreover, despite improved imaging modalities, a definitive preoperative diagnosis is difficult. To our knowledge, this is the first report to share our experience with a very rarely clinical entity of both periesophageal and intramural esophageal bronchogenic cysts with positive expressions of CA199 and CA125 by immunohistochemical study and also review of the literature with an emphasis on endoscopic ultrasonography image characteristics to guide correct examination scheme. (The answer to Reviewer #1[1] and [2].) Patients have signed informed consent and data had been anonymized and deidentified.Bronchogenic cysts are rare congenital malformation originated from the development of ventral foregut, and mostly located in the middle and superior mediastinum.A 31-year-old man who presented to our hospital with suddenly symptoms of persistent chest pain for 2 days and then transferred to dysphagia >1 week. He has no fever, cough, vomit, and weight loss. And there was no obvious positive characteristics on physical exams. With regard to laboratory values, only serum tumor marker CA125  and CA199  levels were remarkable increased. Blood routine test, erythrocyte sedimentation rate (ESR), C-reactive protein, hepatic and renal function were all within normal limits. An esophagography obtained before presentation identified a huge filling defect compressed to esophagus under the aortic arch . Here we reported a more rare type of both presence of intra- and para-esophageal bronchogenic cyst that was safely removed via surgical resection. To our knowledge, a similar case had never been reported.Bronchogenic cysts are broncho-pulmonary foregut abnormalities that usually occur along the tracheobronchial tree in the mediastinum. They may be located in paratracheal, carinal, or hilar.10 Despite improved imaging modalities, a definitive diagnosis remains difficult preoperatively. CT or MRI sometimes can indicate the nature of the cyst. But once bronchogenic cyst is infected or high in protein or calcium content, its density may fall into the same solid mass scope that increases diagnostic uncertainty.12 Moreover, the intramural and extramural relationship of esophageal cysts is also difficult to identification. EUS is sensitive for distinguishing cystic from solid masses and facilitates the diagnosis of intramural esophageal lesions because of its capacity to clearly delineate the size, layer of origin, and the relationship of the cyst to the esophagus.3 The present study first to review of the literature with an emphasis on EUS appearance of esophageal bronchogenic cyst, as only 8 cases have been presented in the literature since 2000s17  is a newly effective and safe procedure to treat submucosal tumors originating from the muscularis propria and has been successful to extract an intraesophageal bronchogenic cyst .23 This treatment would be a less complicated and less risky choice, but long-term follow-up visits and complications are required to evaluated further, EUS seems to be a valuable option for diagnosis and surveillance.With regarding to the management of esophageal bronchogenic cysts, complete surgical removal by thoracotomy or video-assisted thoracoscopy is recommended, even when they are asymptomatic, because of subsequent complication of infection, rupture, intracystic hemorrhage, carcinomatous change.Chaoqun Han: collection and assembly of data, data analysis, manuscript writing, performed the literature search; Rong Lin: collection and assembly of data, manuscript writing and performed the literature search; Jun Yu: provision of pathological data and valuable suggestions; Qin Zhang: provision of pathological data and valuable suggestions; Yang Zhang: provision of gross samples post operation; Jun Liu: administrative support and valuable suggestions; Zhen Ding: conception and design, financial support, revised the manuscript as corresponding author; and Xiaohua Hou: financial support and valuable suggestions.


The periodontal microbiome is known to be altered during pregnancy as well as by smoking. However, despite the fact that 2.1 million women in the United States smoke during their pregnancy, the potentially synergistic effects of smoking and pregnancy on the subgingival microbiome have never been studied. Subgingival plaque was collected from 44 systemically and periodontally healthy non-pregnant nonsmokers (control), non-pregnant smokers, pregnant nonsmokers and pregnant smokers and sequenced using 16S-pyrotag sequencing. 331601 classifiable sequences were compared against HOMD. Community ordination methods and co-occurrence networks were used along with non-parametric tests to identify differences between groups. Linear Discriminant Analysis revealed significant clustering based on pregnancy and smoking status. Alpha diversity was similar between groups, however, pregnant women (smokers and nonsmokers) demonstrated higher levels of gram-positive and gram-negative facultatives, and lower levels of gram-negative anaerobes when compared to smokers. Each environmental perturbation induced distinctive co-occurrence patterns between species, with unique network anchors in each group. Our study thus suggests that the impact of each environmental perturbation on the periodontal microbiome is unique, and that when they are superimposed, the sum is greater than its parts. The persistence of these effects following cessation of the environmental disruption warrants further investigation. The oral cavity plays host to a large and diverse group of bacteria; which form biofilm communities in several habitats within the mouth, including the tooth, subgingival sulcus, tongue, buccal mucosa and tonsils56789et al.Bacteria form biofilms in the subgingival habitat soon after the tooth erupts; and a dynamic equilibrium between the subgingival microbiome and the host immune system is a critical determinant of periodontal health .An example of a pulsed perturbation that affects only females is pregnancy. The association between pregnancy and subgingival bacteria has been examined using cultivation, microscopy, DNA-DNA checkerboard and quantitative real-time PCR11121314Earlier studies from our laboratory and others have highlighted the role of an environmental press - cigarette smoking - in changing the oral microbiome; by decreasing the levels of beneficial species, and promoting a pathogen-rich microbial community within 24-hours of biofilm formation8121718Since there is a robust body of evidence to support the individual impacts of these two perturbations on the subgingival microbiome, the purpose of the present investigation was to examine the combined effects smoking and pregnancy in shaping the subgingival microbiome using high-resolution, high-throughput approaches.Approval for this study was obtained from the Office of Responsible Research Practices at The Ohio State University and the study was conducted in accordance with the approved guidelines. Women who were 18–35 years of age and between 21–24 weeks of gestation were recruited during their regular visits to The Ohio State University Wexner Medical Center Prenatal Clinic between March 2010 and May 2011 and informed consent obtained. Age-matched non-pregnant women were recruited from the Dental Clinics of The Ohio State University and informed consent obtained. Subjects had to have at least 20 teeth, periodontal health , bleeding index (BOP) ≤30%), no antibiotics or professional prophylaxis for at least 3 months. Exclusion criteria were carrying more than one fetus during current pregnancy and previous history of miscarriage and/or preterm delivery. Also, women who had health conditions that affect immune or endocrine functions, including diabetes, hypertension, thyroid disorders and women with heart conditions that would require antibiotic prophylaxis prior to dental visits were excluded. Women with asthma and arthritis who required regular use of anti-inflammatory medications were also excluded. In addition, subjects with a history of alcohol/drug abuse and women who were using mood-altering medications were excluded. Inclusion criteria for smokers were 5 pack years or greater of tobacco exposure, and nonsmokers were defined as individuals who had smoked less than 100 cigarettes in their lifetime and were currently not smoking (CDC guidelines). Clinical examination was conducted by a single calibrated periodontist. Prior to clinical examination, maxillary anterior teeth from right second premolar to left second premolar were isolated and dried. Sterile periodontal paper strips  were gently inserted into buccal interproximal areas of teeth without BOP for 30 seconds. Strips were pooled for each subject and stored at −80 °C until further analysis.Paper strips were separated from waxed portion using sterile scissors, 200 μl of sterile cold phosphate buffered saline added and centrifuged. Bacterial DNA was isolated from 100 μl of eluent with a Qiagen DNA MiniAmp kit  using the tissue protocol according to the manufacturer’s instructions.matplotlib. Phylogenetic tree data was visualized through the Interactive Tree Of Life webserverMultiplexed bacterial tag-encoded FLX amplicon pyrosequencing was performed using the Titanium platform  as previously describedMASS package in R. MASS:lda provided singular value decomposition (SVD) values, which were used to calculate variances in each dimension. Packages scatterplot3d and rgl were used to visualize 3D LDA plots. The input for LDA was a matrix of normalized (arc-sine transformed) relative abundances of species-level OTUs. Single and multiple comparisons of distributions were carried out with the statistical facilities provided by JMP (SAS Institute Inc.), as well as the Python libraries SciPy, pandas, and statsmodels. Statistical significances of s-OTUs within the four groups were determined using paired Wilcoxon’s tests. Significant pairwise correlations  and graph theory were used to compute species-level co-occurrence networks for each group. Network graphs were calculated using Networkx package in Python and visualized in GephiLDA was implemented with Networkx” and s-OTUs were ranked based on this metric. For each group, significantly different  species were identified using JMP (SAS Institute Inc.) and species that were present in at least 75% of the subjects were identified using QIIME’s core_microbiome.py script. Species that demonstrated a high betweenness centrality (top 20% in each network), and were either part of the group’s core microbiome or showed significant differences for the groups were identified as network anchors.Network anchors were identified using an algorithm incorporating betweenness centrality, differential abundances, and frequency of occurrence in a group. Betweenness centrality was calculated using Python package “A total of 44 periodontally and systemically healthy women between the ages of 18 and 25 were recruited . 80% of 331601 classifiable sequences were used for analysis. These sequences represented 218 species belonging to 116 genera. Each individual was colonized by 112 ± 18 species. Statistically significant group separation was evident among all groups . The variance expressed by first, second and third linear discriminant (LD) was 49.83%, 31.37%, and 18.81%, respectively. LD1 discriminated between pregnant and non-pregnant women groups, LD2 discriminated between smokers and control groups and LD3 discriminated between smokers and nonsmokers in the pregnant group. .Alpha diversity ranged from 1.75 to 5.75 (Shannon Diversity Index). Both controls and pregnant women demonstrated a bimodal distribution of values, however, for all groups the median diversity was centered around 4. This was not statistically different between the four groups , with some of these uniquely identified in the pregnancy-associated microbiome. Both smoking and pregnancy were associated with a significant decrease in species belonging to Neisseria and Aggregatibacter when compared to controls. Smokers (non-pregnant) demonstrated significantly greater levels of species belonging to TG5, Filifactor, Fusobacterium, Lactobacillus, Desulfobulbus, Streptococcus, Propionibacterium and Corynebacterium than either pregnant women or controls. Filifactor, Fusobacterium and Desulfobulbus formed the numerical majority of the subgingival microbiome of smokers. Similarly, Neisseria and Aggregatibacter were abundant in controls, but demonstrated significantly lower levels in other groups. When pregnant smokers were compared to controls, 51 species (32% of the species found in controls or pregnant smokers) demonstrated differing abundances. Of these, 31 were also different either between smokers and controls or between pregnant women and controls.The differences in gram staining characteristics and oxygen requirements were reflected at the species level ; severalCardiobacterium spp., Neisseria weaveri, Treponema socranskii and Veillonella dispar as significant , part of core species group and highly ranked in betweenness centrality measure. Cardiobacterium spp. and Neisseria weaveri are also unique species of control group. In pregnant women, Acidovorax spp., Actinomyces spp., Campylobacter spp., Capnocytophaga spp., Catonella spp., Corynebacetrium spp., Kingella spp., Dialister spp., and Methylobacterium organophilum were network anchors, while in smokers Catonella spp., Corynebacetrium spp., Cardiobacterium spp., Granulicatella spp., Lautropia spp., Leptotrichia spp., Neisseria spp., Porphyromonas spp., Tannerella spp., and TG5 spp. were important as anchors. Pregnant smokers demonstrated cluster anchors that were not seen in either pregnant women or in smokers, namely, Bradyrhizobium spp., Herbaspirillum, E.coli, Prevotella melalinogenica, and Prevotella spp., along with Corynebacetrium spp., Dialister spp., and Tannerella spp.Bacteria that demonstrated significantly different abundances in each group also demonstrated differing co-occurrence patterns, Co-occurrence networks in the control group are shown in Bacteroides, particularly Bacteroides melaninogenicus ss. intermedius, during the second trimester of pregnancy141530It has been known for a number of decades that both the prevalence and severity of gingivitis is greater in pregnant women than in non-pregnant controls262728We examined the effects of smoking and pregnancy on subgingival microbial profiles using two strategies. Principal co-ordinate analysis of UniFrac distances was initially used to examine similarities in microbial profiles between individuals. There was statistically significant clustering of individuals based on pregnancy and smoking status , indicating that smoking and pregnancy promote colonization by genetically distinct lineages. We then used Linear Discriminant Analysis (LDA) based on relative abundances of species-level OTUs to increase class separation within data and enable computational efficacy. This supervised machine learning technique identifies linear discriminants that maximize the separation between the means of groups of data while minimizing the variance within each group of dataNeisseria, Veillonella and Actinomyces33Neisseria weaveri and Veillonella dispar were two robust network anchors in non-pregnant nonsmokers; however, they were not influential in pregnant women or in smokers. In these women, theThe subgingival microbiomes of both pregnant women and smokers demonstrated significant commensal depletion. Certain commensals with established associations to periodontal health, notably those belonging to Smoking appears to promote the growth of anaerobes, while pregnancy appears to promote the growth of gram-negative facultatives at the expense of anaerobes. This finding is in contrast to earlier studies; which demonstrated a preponderance of gram-negative anaerobes in pregnancy gingivitis13Filifactor, Fusobacterium, Megasphaera, Aeromicrobium, Dialister and Desulfobulbus, while pregnancy was associated with an enrichment of methylotrophs (Pseudomonas and Methylobacterium), Streptococci and phenotypically similar species . Methylotrophs are organisms that obtain energy for growth by oxidizing one-carbon compounds, such as methanol and methane. Streptococci, Enterococcus and Vagococcus share a common ability to metabolize estrogen35363830Candida are also prolific methylotrophs, and their associations with estrogen-rich environmentsAnother key finding was that smoking enriched the microbiome for pathogens whose growth is promoted by reducing environments, for example, Enterobacteriaceae. It is interesting that these fermentative species utilize methanol as a substratePregnant women who smoked demonstrated not only enrichment of both methylotrophs and reduction-sensitive species, but also species that ferment mixed acids, for example, those belonging to Pseudomonas, Prevotella, Methylobacterium, Streptococcus, Megasphaera, Atopobium, Acinetobacter and Escherichia are highly abundant in estrogen-rich environments4647Vagococcus and Enterobacter are two species that cluster with the estrogen metabolizers in these individuals, and Dialister spp. is a network anchor in pregnant smokers and nonsmokers, however, thus far, little is known about these species. Their strong correlation with species that are known to be affected by estrogen provides a strong indication that these species are also capable of estrogen metabolism. Further studies are warranted to test this hypothesis.Possibly one of the most interesting findings in this study is derived from an examination of the co-occurrence networks. Bacterial colonization of a niche is primarily driven either by nutritional, spatial or metabolic factors that select for these species. The deterministic theory of species abundance suggests that the environment imposes habitat filters for species that possess traits suitable for that environmentPrevotella intermedia and Campylobacter rectus- were not detected either as numerically dominantly members or as significantly different members of the subgingival microbiome of pregnant women. This may be because, as stated previously, our cohorts were periodontally healthy. However, in pregnant women, Campylobacter spp. emerged as a co-occurrence influencer, while in pregnant smokers, Prevotella melaninogenica and Prevotella spp. were strong network anchors. It is possible that their important role as community regulators in pregnant women allows them to emerge as numerically dominant members of the community when inflammation is added to the subgingival milieu.Interestingly, two species that have been famously associated with pregnancy-related periodontal diseases- We are aware that the small sample size as well as the lack of correction for multiple comparisons may have some impact on our findings. However, since this was an exploratory analysis, and the focus was more on community level metrics rather than differences in individual taxa, we did not correct of multiple comparisons. Further studies with larger sample sizes are warranted to validate these findings.In summary, the subgingival microbiome responds differently to smoking and pregnancy. While smoking causes an increase in anaerobic organisms that thrive in reducing environments, pregnancy appears to promote colonization by species capable of estrogen metabolism. The greatest implications of this study are for women who smoke during their pregnancy, since the shift in the microbiome does not appear to be simply additive effects of pregnancy or smoking, but a multiplicative effect of the combined effects of these two events on the microenvironment. The study thus highlights the importance of pulsed events in altering microbial dynamics. It is important to examine shifts in the microbiome in these women following parturition.How to cite this article: Paropkari, A. D. et al. Smoking, Pregnancy and the Subgingival Microbiome. Sci. Rep.6, 30388; doi: 10.1038/srep30388 (2016).


Cell polarity has a variety of biological roles, including morphogenesis during development, the maintenance of tissue integrity, and adult tissue regeneration. In epithelial cells, cell polarity is generally regulated by three evolutionarily conserved protein complexes: the apical PAR and Crumbs complexes and the basolateral Scribble (Scrib) complex. Thus, dysfunction of these polarity proteins causes cell polarity defects and disorganization of tissue architecture, resulting in diseases including cancer. Accumulating evidence has also demonstrated that cell fate decisions in tissue stem cells are likely influenced by the polarized distribution of cell polarity proteins. These then act as potential determinants of asymmetric cell division, which allows a stem cell to generate a daughter cell that self-renews to maintain its own stem cell pool and another cell that undergoes differentiation, therefore ensuring a balance between self-renewal and differentiation .Skeletal muscle tissue stem cells are satellite cells with remarkable regenerative capacity after muscle damage . SatelliCell Reports further demonstrated that the cell polarity protein Scrib is a crucial regulator of satellite cell fate decision [Scrib failed to expand their progeny. Indeed, satellite cell-specific Scrib-null mice exhibited a severe regeneration defect with a significant reduction of myogenic progenitors after cardiotoxin-induced muscle injury.A recent study published in decision . ActivatScrib-inactivated satellite cells lost their proliferative ability, the study next focused on how proliferation was affected by the loss of Scrib [Scrib resulted in a decreased expression of phosphorylated c-Jun that sustains the proliferative state of satellite cells. Moreover, Scrib-deficient cells were less responsive to stimulation by growth factors such as IGF-1. Indeed, Scrib appears to act as a signaling scaffold to modulate growth factor signaling pathways for the maintenance of myogenic progenitor expansion. However, in contrast, the overexpression of Scrib suppressed cell proliferation and promoted myogenic differentiation. It is also of interest to note that satellite cell self-renewal was impaired by the retrovirus-mediated constitutive expression of SCRIB, indicating that the down-regulation of Scrib was a necessary step for the self-renewal of satellite cells. Taken together, these results suggest that satellite cell fate decisions determined by Scrib are dose-dependent, and thus, an appropriate level of Scrib might be crucial for the balance between population expansion, differentiation, and self-renewal in satellite cells.Given that of Scrib . The genConsidering the evidence that Scrib is a key cell polarity protein that prevents the outgrowth of tumor cells in epithelial tissues, Scrib is unlikely to function as a universal anti-proliferative factor, and thus its role is likely to be varied and depend specifically upon its cell and tissue distribution. It remains unclear whether the function of Scrib is associated with other polarity regulators such as PAR for the fine-tuning of satellite cell fate. Further studies using genetically engineered mouse models are needed to uncover how polarity proteins regulate the molecular events involved in cell fate determination. This will hopefully support the development of stem cell-based regenerative medicine for muscle wasting diseases such as muscular dystrophies and age-related sarcopenia, as well as for cancer biology.


Sleep is a restorative circadian process underpinned by numerous interrelated biological processes  face unique challenges relative to their ability to achieve sufficient sleep  indicated that male and female swimmers had the highest incidence of sleep medication use  compared to any other sport assessed, with such medication accounting for 10.3% of miscellaneous substance use across all sports  pre/post competition or post training to reduce arousal (sedative action), (ii) to combat jet-lag , and (iii) for habitually poor sleepers (manage insomnia). Sports medicine/science practitioners and their athletes have various classes of sleep medication to rely upon. Those drugs requiring a prescription and used on-label are ligand γ-aminobutyric acid (GABA) agonists or receptor modulators, benzodiazepine hypnotics (for simplicity z-drugs are included within this class), melatonin receptor agonists  and off-label use of sedative anti-depressants. Over-the-counter sleep medications are typically antihistamine based, with some off-label use of allergy specific drugs seen. Pharmacological review of each of these medications is beyond the scope of this opinion piece, instead, practitioners are directed to recent reviews for this information  the next day (or upon awakening) effects of various sleep medications on psychomotor performance have been reported  day athletic performance  are suggestive of an increased cancer risk  use within elite sport, similar to the NCAA data discussed Rexroat, .Sleep medication use within athletic populations  and their impact upon sleep duration, efficiency, latency and inertia together with perceived sleep quality, with efficacy assessed by polysomnography within a laboratory environment and actigraphy within applied settings  and their impact upon intermittent high-intensity, prolonged endurance and resistance based exercise , and cognitive function, within and next day.The efficacy of sleep hygiene and non-pharmacological sleep enhancing interventions Halson,  relativeTherefore, to summarize the authors believe that the following research questions should be explored, in order to provide evidence for practitioners to (a) utilize within their practice and (b) stimulate further more complex research designs:All authors listed, have made substantial, direct and intellectual contribution to the work, and approved it for publication.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.