Mitochondria is known as powerhouse of cell because they generate most of the cell's supply of adenosine triphosphate (ATP), used as a source of chemical energy. In addition to supplying cellular energy, mitochondria are involved in a range of other processes, such as signaling, cellular differentiation, cell growth, and cell death. Transcription and replication of mitochondrial DNA (mtDNA) are important steps in mitochondrial biogenesis and mitochondrial transcription factor A (Tfam) is essential for mtDNA transcription and replication. However, the functional significance of mitochondria has not been established in osteoclastic bone resorption.There is accumulating evidence that osteoclasts, the primary cells responsible for bone resorption, are involved in bone and joint destruction in rheumatoid arthritis. Bone resorption is highly regulated by mature osteoclast function as well as osteoclastogenesis. The life span of mature osteoclasts is relatively short both fl/fl mice with cathepsin K-Cre transgenic mice, in which the Cre recombinase gene is knocked into the cathepsin K locus and specifically expressed in mature osteoclasts. The in vivo effects of Tfam deficiency on bone metabolism were examined by histological and histomorphometric analysis. The survival and bone-resorbing activity of Tfam cKO osteoclasts were determined by in vitro survival assay and pit formation assay, respectively.To address this question, we generated osteoclast-specific Tfam conditional knock-out (cKO) mice by mating TfamThe expression level of Tfam, mtDNA copy number, and cellular ATP level were markedly reduced in osteoclasts derived from Tfam cKO mice. The body size of Tfam cKO mice was smaller than that of the control mice, although trabecular bone volume remained unchanged by Tfam deficiency. However, histological sections of proximal tibia and lumbar spine of Tfam cKO mice showed significantly decreased osteoclast number. Interestingly, Tfam cKO osteoclasts exhibited increased bone-resorbing activity in spite of their pro-apoptotic tendency.This study demonstrates that Tfam cKO osteoclasts exhibited increased bone resorption with accelerated apoptosis, indicating that there may be an inverse correlation between osteoclast survival vs bone resorption. Further investigation of mitochondria in bone-resorbing osteoclasts will give us new insights into the molecular mechanism regulating bone homeostasis.


In this study, we used high-dimensional pattern regression methods based on structural  and functional  brain data to identify cross-sectional imaging biomarkers of cognitive performance in cognitively normal older adults from the Baltimore Longitudinal Study of Aging (BLSA). We focused on specific components of executive and memory domains known to decline with aging, including manipulation, semantic retrieval, long-term memory (LTM), and short-term memory (STM). For each imaging modality, brain regions associated with each cognitive domain were generated by adaptive regional clustering. A relevance vector machine was adopted to model the nonlinear continuous relationship between brain regions and cognitive performance, with cross-validation to select the most informative brain regions (using recursive feature elimination) as imaging biomarkers and optimize model parameters. Predicted cognitive scores using our regression algorithm based on the resulting brain regions correlated well with actual performance. Also, regression models obtained using combined GM, WM, and PET imaging modalities outperformed models based on single modalities. Imaging biomarkers related to memory performance included the orbito-frontal and medial temporal cortical regions with LTM showing stronger correlation with the temporal lobe than STM. Brain regions predicting executive performance included orbito-frontal, and occipito-temporal areas. The PET modality had higher contribution to most cognitive domains except manipulation, which had higher WM contribution from the superior longitudinal fasciculus and the genu of the corpus callosum. These findings based on machine-learning methods demonstrate the importance of combining structural and functional imaging data in understanding complex cognitive mechanisms and also their potential usage as biomarkers that predict cognitive status.  Aging is associated with declines in neurocognitive functions often characterized by degeneration of brain tissue, changes in brain function, and accelerated cognitive decline, particularly in clinical cases such as Alzheimer's disease (AD). However, despite the connection with neural changes, diagnosis of clinical cognitive impairment to date still primarily depends on cognitive changes measured by neuropsychological assessments. Neuroimaging studies have shown that structural and functional brain changes often precede clinical symptoms of cognitive impairment , which ra priori. As such, characterizations of brain changes in normal or clinical aging have had specific and limited scopes within each study and have been relatively unwieldy in the context of the multi-faceted nature of the issue.Finding specific brain regions involved in a cognitive process has been a recurrent goal in functional neuroimaging in the last two decades -5. In thTo overcome the limitations of univariate and ROI-based approaches, machine learning-based pattern analysis methods have been widely adopted to automatically derive distinctive brain regions related to a hypothesis or a given study objective . The maja priori hypotheses  water. The images were obtained on a GE 4096 scanner, during a resting state scan with eyes open [Details of our image acquisition parameters have been described in . Brieflyyes open . After tAll MR images used in this study were pre-processed using a mass-preserving shape transformation method . Gray ma15O] PET-CBF image pre-processing included normalization for global activity, removal of extraneous signal scatter by thresholding the image intensity at 80% of the gray matter mean, and removal of activity in the skull, nasal sinuses, and cerebellum. PET-CBF images were rigidly registered to the corresponding participants’ MR image then deformed to the same template space based on the spatial normalization transformation parameters determined from the MRI. After registration, PET-CBF images were smoothed using a 12mm Gaussian filter. PET-CBF alone, and one using both MRI and PET. To evaluate model performances, we examined the MSEs and correlation coefficients indexing the strength of agreement between the actual cognitive scores and scores predicted by the models. Since MSEs and correlation coefficients are not normally distributed, differences in cognitive score predictions between models were statistically compared using non-parametric random permutation testing [For step 3 of each cognitive domain, a summary map of brain region contributions to cognitive score prediction was obtained by averaging the spatial difference maps from all leave-k-out cases. To facilitate comparison of contribution maps across the optimized models using single or combined imaging modalities, we then normalized the contribution levels to a range from 0 to 1, indexing lowest to highest predictive contributions respectively. The predicted cognitive scores were also based on averaged optimal predicted scores across all leave-k-out cases for each cognitive domain. Overall, we performed three RVR computations for each of the four cognitive domains: one RVR using MRI (white and gray matter structure) alone, one using [ testing , which wThe study was approved by the local Institutional Review Board of the University of Pennsylvania and the National Institute on Aging. Written informed consent was obtained from all participants at each visit. MSEs were used to describe the performance of machine learning-based regression models  top. As For visualization, We additionally evaluated whether the detected imaging biomarkers were more effective predictors of cognitive performance than another important factor related to cognitive decline, age. For comparison, The top panels of As shown in Several brain regions predicting LTM were also involved in STM . SpecifiFor semantic retrieval, predictive brain regions included left occipital-temporal, bilateral inferior frontal, insula, medial and lateral orbitofrontal areas, as well as right superior longitudinal fasciculus and periventricular areas . MaximumFor manipulation, predictive contributions were observed in the right inferior frontal, and insula regions, left precentral and middle frontal regions, bilateral anterior cingulate and temporal areas, as well as the left uncinate fasciculus and genu of the corpus callosum . MaximumTo validate the performance of our multi-modality pattern regression method, we also applied conventional GLM analysis with the same cognitive components. The GLM analyses revealed positive correlations between GM/WM/PET and cognitive performance, as shown in The GLM analysis also showed that manipulation was significantly associated with WM volumes across several regions. Similarly, the majority of brain regions detected by multi-modality pattern regression were from structural MRI, especially WM, which had the highest contribution to manipulation performance . HoweverWe highlight that GLM maps indicate the directions of associations separately between each imaging modality and each cognitive domain. By contrast, the contribution maps obtained from pattern regression shows the combined power of multi-modality imaging with no sign of association. Thus, the combined associations are less restricted and show simultaneous contributions across imaging modalities that could be due to a positive brain-behavior association in one modality but a negative association in another. In addition, direct comparison of the contributions across modalities is not meaningful with the GLM maps but relatively straightforward with the contribution indices used in our pattern regression method.This study applied a multi-modality pattern regression method to investigate the brain imaging biomarkers related to memory and executive functions. Combined regression models for LTM, STM and semantic retrieval had similar prediction power, with regression rates of 0.41, 0.45 and 0.46 respectively . AlthougOur finding that LTM and STM involved middle and medial frontal regions, along with temporal regions and the hippocampus and amygdala (for LTM) is consistent with the existing literature on brain regions involved in episodic memory. Episodic memory tasks generally involve accessing the representations of the test items including contextual information about when and where the items were encountered. Many studies have shown that the hippocampus is an important region that forms and stores such item-context associations , superior longitudinal fasciculus and genu were associated with semantic retrieval and manipulation performance, with the maximum contribution from white-matter images. Other studies have similarly found that these white-matter regions show structural declines with age that are associated with poorer performance in tests of executive function and working memory -57. ThesThis present study focused on modeling the continuous cross-sectional distribution of cognitive performance across individuals instead of individual longitudinal performance changes over time. It is plausible that longitudinal data may capture distinct important regional contributions toward cognitive performance ,58. We nWe investigated the utility of multi-modality high-dimensional pattern regression for detecting imaging-based biomarkers of cognitive ability in a cohort of cognitively normal older adults. Our findings show that this machine learning-based pattern regression method is able to detect specific brain regions associated with different cognitive processes, while simultaneously assessing the contribution of different imaging modalities. Moreover, the combination of PET and MRI was better than each modality alone. Critically, our results suggest that functional imaging provided slightly better predictive ability than structural MRIs for memory functions, but WM modality outperformed PET when predicting executive functions. The predicted cognitive scores using our methodology could be used to identify individuals with risk of future cognitive decline from baseline images, such as in the case of when the predicted score deviate from the normal range. Future studies will evaluate imaging biomarkers of additional aspects of memory and executive function as well as the predictive ability with respect to longitudinal performance.


Heterotopic transplantation of pluripotent stem cells (PSCs) produces growths known as teratomas (Greek word for monstrous), which consist of a heterogeneous amalgamation of fetal-like tissues. Engraftment of even a few undifferentiated cells is potentially sufficient for teratoma formation. Such an event would have far reaching consequences for the future of this field. Current differentiation protocols produce cultures with unknown degrees of purity and are therefore potentially hazardous. We provide here a succinct overview of current methods and outline challenges for the removal of residual teratoma-initiating cells.OCT4 promoter controls HSV1 thymidine kinase expression, allowing for selective ablation of undifferentiated PSCs upon treatment with of Ganciclovir [Retrospective and prospective approaches for teratoma depletion have been the subject of significant investigation. Retrospective removal includes standard oncologic treatments of formed tumors via radiation, chemotherapy, and surgery . To avoiciclovir . UnfortuProspective depletion of teratoma-initiating cells is advantageous to retrospective removal as such methods prevent initial tumor formation. Two main strategies of prospective removal include treatment with agents specifically cytotoxic to undifferentiated cells and mechanical separation. The first was extensively developed by the lab of Dr. Andre Choo, who produced the cytotoxic antibody mAb 84 which targets PODXL, a protein abundantly expressed by PSCs .Non-cytotoxic cell separation has emerged as a central approach for depletion of teratoma-initiating cells. This method relies on tagging undifferentiated cells either by inducing reporter gene expression or through reversible labeling of surface antigens. Labeled cells are then removed through fluorescence- or magnetic-activated cell sorting (FACS or MACS). Although tagging PSC-specific surface antigens is considered safer than introducing gene reporters , this goA major advantage to utilizing surface markers for prospective separation is that this method relies on the intrinsic properties of the cells and therefore can be applied to all PSC lines and differentiation conditions. A typical protocol may entail removing cells expressing a tailored combination of surface markers such as SSEA-5 and/or CD9 and/or CD90 . Such a In summary, we propose that prospective removal is superior to retrospective approaches since the latter may result in anatomical damage and also risks malignant and/or metastastic transformation. As such, retrospective removal should always be considered a backup rather than first line. We also believe that clinical utilization of PSC derivatives should rely on combination of prospective approaches to provide the highest level of safety. One example is to utilize PSC-specific antibodies for cell separation followed by a subsequent cytotoxic antibody incubation step . Ultimat


Dear editor,Aortocaval Fistula (ACF) is a rare condition of an Abdominal Aorta Aneurysm (AAA). ACF caused by perforation of atherosclerotic infrarenal aortic aneurysm into the adjacent IVC, iliac vein, or left renal vein. Its inciA 63-year-old man presented to Emergency Department with sudden left flank pain.Abdominal physical examination revealed palpable mass, tenderness and self-guarding in left flank area without rebound tenderness. All peripheral pulses were present.Sonography of abdomen displayed infrarenal AAA with 63mm diameter. CT-images demonstrated infrarenal aortic aneurysm with fistula to IVC arising from aneurysm lumen that penetrated to retroaortic renal vein situated beneath the aneurysm .At laparotomy No hematoma or active bleeding existed in peritoneal space. Followed by controlling blood flow of aneurysm neck, distal common iliac arteries were clamped. Therefore, renal veins became noticeable posterior the aorta. IVC was dilated excessively 4 cm and trill was palpable on aneurysmal site. Further investigations revealed that exact site of fistula at the entrance of left renal vein to IVC.IVC was repaired and renal vein was ligated and aneurysm repaired using 20 tubular Dacron graft. There are several causes for ACF including spontaneous rupture of atherosclerotic aneurysm directly into adjacent IVC, penetratEarly diagnosis of ACF prior to surgery is vital and also difficult due to rarity of complication and non-specificity of the signs and symptoms. Standard signs of ACF as a result of aneurysmal disease, include acute abdominal pain, hypotension, and pulsatile abdominal mass. However Specific treatment for ACF is operative transaortic closure of fistula and placement of prosthetic graft or endovascular repair of fistula.


Dear Editor,We enjoyed reading the excellent article by Yilmaz and colleagues on noninvasive assessment of liver fibrosis using aspartate transaminase to platelet ratio index (APRI) in adult patients with chronic liver disease (CLD) . They peA drawback for using APRI is that its performance is not reliable in women, younger patients and in those with non-vertically-transmitted hepatitis C virus infection . Alterna


With the recognition of early trauma coagulopathy, trauma resuscitation has recently shifted towards early and aggressive transfusion of platelets (PLTs). However, the clinical benefits of this strategy remain controversial. This systematic review examined the impact of an aggressive approach (higher PLT:RBC ratios) compared to restrictive PLT transfusions (lower PLT:RBC ratios) in the acute phase of trauma resuscitation.We systematically searched Medline, Embase, Web of Science, Biosis, Cochrane Central and Scopus to identify relevant randomized controlled trials (RCTs) and observational studies comparing the effect of two or more different PLT:RBC ratios in trauma resuscitation. We excluded studies using whole blood or systematically addressing the use of hemostatic products. Two independent reviewers selected the studies, extracted data using a standardized form, and assessed the risk of bias using the Newcastle-Ottawa scale and a checklist of key methodological elements . Disagreements were solved by consensus or a third party. The primary outcome was mortality. Secondary outcomes were multiple organ failure (MOF), lung injury and sepsis. A meta-analysis using random effects models was planned.n = 4,230 patients). No RCT was identified. All studies were considered to be at low risk of bias and addressed confoundings through multivariate regression or propensity scores. Four studies  reported a decrease in mortality with higher PLT:RBC ratios in patients requiring massive transfusion and one study observed no mortality difference  in nonmassively transfused patients. Two studies reported on the implementation of a massive transfusion protocol with higher PLT:RBC ratios; only one revealed a survival benefit (n = 211). Of the three studies accounting for survival bias, two demonstrated a survival benefit . Among two studies reporting on the secondary outcomes (n = 854), one observed an increase in MOF with higher PLT:RBC ratios. Clinical heterogeneity between studies and methodological limitations precluded the use of a meta-analysis.From 6,123 citations, seven observational studies were included (There is insufficient evidence to strongly support the use of a specific PLT:RBC ratio for acute trauma resuscitation, especially considering survival bias and nonmassively transfused patients. RCTs examining both safety and efficacy of liberal PLT transfusions are warranted.


To describe a consensus-based case review development process that applies eligibility and diagnostic criteria for diverse clinical trials of chiropractic care for chronic pain conditions.A multidisciplinary team of investigators and clinical research staff provide iterative feedback to develop case review processes for application across complex clinical trials involving biomechanical testing and/or spinal manipulation. Investigative team members design eligibility criteria and operational definitions consistent with the specific aims of the trial. These parameters are codified in study protocols and programmed into secure, web-based data collection systems that record eligibility decisions. The clinical team develops study-specific procedures, documentation templates, and flow-charts for conducting meetings. Research records are uploaded onto a web server for panel members to view during the meeting.Our case review panel consists of research clinicians, project managers, study coordinators and the senior clinician who convene twice weekly to discuss participants who remain eligible following completion of baseline visits. The research clinician who performed the eligibility examination begins case review with an oral presentation of the case and leads the case review panel through a discussion of safety and compliance issues, eligibility concerns, diagnosis determination, and clinical precautions. Panel members provide clinical recommendations and determine final eligibility status by consensus vote. The senior clinician determines eligibility only in cases where consensus (80%) is not reached. Through this process, our research clinic presented 534 cases, excluded 174 participants, and allocated 291 into two phase II clinical trials and six feasibility studies over a three year period.Our case review process utilizes the combined scientific and clinical experience of panel members for consistent eligibility determination, thereby reducing selection bias and ensuring that participants are appropriate candidates for study procedures. The consensus building process creates a collegial environment that enhances clinical trial management and fosters rigorous clinical research.


Avian malaria is an important cause of the decline of endemic Hawaiian honeycreepers. Because of the complexity of this disease system we used a computer model of avian malaria in forest birds to evaluate how two proposed conservation strategies: 1) reduction of habitat for mosquito larvae and 2) establishment of a low-elevation, malaria-tolerant honeycreeper (Hawaii Amakihi) to mid-elevation forests would affect native Hawaiian honeycreeper populations. We evaluated these approaches in mid-elevation forests, where malaria transmission is seasonal and control strategies are more likely to work. Our model suggests the potential benefit of larval habitat reduction depends on the level of malaria transmission, abundance of larval cavities, and the ability to substantially reduce these cavities. Permanent reduction in larval habitat of >80% may be needed to control abundance of infectious mosquitoes and benefit bird populations. Establishment of malaria-tolerant Amakihi in mid-elevation forests increases Amakihi abundance, creates a larger disease reservoir, and increases the abundance of infectious mosquitoes which may negatively impact other honeycreepers. For mid-elevation sites where bird populations are severely affected by avian malaria, malaria-tolerant Amakihi had little impact on other honeycreepers. Both management strategies may benefit native Hawaiian honeycreepers, but benefits depend on specific forest characteristics, the amount of reduction in larval habitat that can be achieved, and how malaria transmission is affected by temperature. Plasmodium relictum) and its vector, the Southern House Mosquito (Culex quinquefasciatus), are considered primary contributors to population declines of native Hawaiian forest birds More than one-third of all U.S. listed bird species occur in Hawaii, and 71 Hawaiian birds have gone extinct since humans colonized the islands Temporal and spatial dynamics of malaria in Hawaii are driven by seasonal, annual, and elevational weather patterns To reduce future population impacts and potential species extinctions, conservation strategies that reduce the impact of avian malaria on Hawaiian honeycreepers are essential. However, conservation strategies to preserve future populations of Hawaiian birds can be controversial if focused strictly on habitat improvement Sus scrofa) feed on native tree ferns (Cibotium glacum) creating cavities that fill with rainwater. These cavities are an important and ideal habitat for mosquito larvae, especially in mid-elevation forests Common methods for reducing mosquito abundance include reduction of larval habitat, insecticides that increase larval or adult mortality rates, and the introduction of predators on mosquitoes Hemignathus virens) found in low-elevation forests may have developed malaria-tolerance compared to conspecific birds at mid- and high- elevations Although most of the native Hawaiian forest birds are highly susceptible to avian malaria Ecological models can provide an indispensable tool for exploring the consequences of alternative management strategies All necessary access and collection permits for this study were obtained from the State of Hawaii, Department of Land and Natural Resources and Hawaii Volcanoes National Park.2 study sites with mosquito larval habitat  larval mosquito habitat and associated larval mosquitoes  were in Himatione sanguinea sanguinea), and Iiwi (Vestiaria coccinea). The model also includes the Japanese White-eye (Zosterops japonicus) to represent introduced species, which typically have lower prevalence and intensity of infection with malaria Our simulation model Mosquitoes and all bird species are divided into immature and adult stages. Adult mosquitoes are subdivided into susceptible, latent, and infectious disease stages. Native juvenile (susceptible when hatched) and adult birds are subdivided into susceptible, acutely infected, and recovered stages. Acutely infected birds have a high parasitemia Mosquito dynamics are driven by rainfall and temperature 2, and no mosquito larvae. Simulations were then conducted for a 24 year period (1980–2003) using historical weather conditions (see above). Mosquito dynamics typically became stable within a few years regardless of the initial densities or elevation We modeled the potential impact of reducing larval habitat for two study sites where pig-created cavities provide an important habitat for mosquitoes (Cooper and Waiakea). We modeled the potential impact of Amakihi establishment at all four study sites. Initial conditions for our simulations used predicted bird densities in the absence of malaria To assess the potential effects of different weather patterns caused by variation in elevation and topography across the Hawaiian landscape, we modeled both conservation strategies under five climate scenarios derived from our baseline climate (1980–2003) data at study site Cooper . Three dIn mid-elevation forests with feral pigs, our model predicts that density of adult and infectious mosquitoes change in proportion to the carrying capacity for larval mosquitoes and in response to climate . For bas2. Once this threshold was reached, further reductions produced dramatic increases in honeycreeper populations. These simulated population increases were accompanied by large increases in the percentage of susceptible and large decreases in the percentage of chronically-infected individuals  is an important driver of mosquito dynamics, but density of infectious mosquitoes is the primary driver of malaria transmission to susceptible Hawaiian birds.Weather patterns interact with the larval habitat reduction needed to enhance bird populations . Effecti2 after establishment of malaria-tolerant Amakihi. Presence of malaria-tolerant Amakihi in mid-elevation forests increased the predicted density of Amakihi by >900 birds/km2 at Cooper and Waiakea, while Apapane and Iiwi were not additionally affected  in mid-elevation forests, but the density of infectious mosquitoes increased by 25% at Cooper, 26% at Waiakea, and 303% at Crater. At Pu'u, the density of infectious mosquitoes was <1/kmaffected  because affected . At Pu'uaffected . The pre2 threshold, indicating that less tolerant Amakihi populations at these sites were limited by malaria. In these simulations the proportion of chronically-infected Amakihi exceeded 85–89%, indicating high rates of disease transmission.Predicted changes in malaria-tolerant Amakihi depended on the density of infectious mosquitoes . ClimatePredicted Apapane and Iiwi population responses to malaria-tolerant Amakihi depended on the density of infectious mosquitoes. Apapane and Iiwi densities were generally unaffected in forests where malaria transmission was either very low (Pu'u) or high (Waiakea and Cooper). At high transmission sites, malaria infection had already reached a high level so further increasing the reservoir of chronically-infected birds had little effect. At low transmission sites, predicted Amakihi density was not affected because there was little malaria impact and bird communities in these forests were unaffected. Apapane and Iiwi populations declined in forests where the density of infectious mosquitoes was below the threshold needed to enhance native bird populations . In these forests small increases in density of infectious mosquitoes increased rates of disease transmission which benefited malaria-tolerant Amakihi, but were detrimental to susceptible Apapane and Iiwi.Avian malaria has a significant population impact on susceptible Hawaiian honeycreepers and disease transmission by mosquito vectors is likely a primary factor contributing to the decline and restricted distribution of native Hawaiian forest birds 2) before mosquito control benefits mid-elevation honeycreepers. For infectious mosquito densities above this threshold, most surviving birds are predicted to be chronically-infected with malaria, and therefore a disease reservoir for susceptible birds. Although both rainfall and temperature affect the relationship between larval habitat and malaria transmission, model results indicate that the density of infectious mosquitoes was the key factor influencing transmission and bird abundance . Above this threshold, an increase in the density of infectious mosquitoes from malaria-tolerant Amakihi does not significantly increase the impact of avian malaria on bird populations (Our model indicates malaria-tolerant Amakihi could increase the abundance of infectious mosquitoes in some mid-elevation forests and therefore decrease densities of more susceptible honeycreepers. The negative impact of malaria-tolerant Amakihi on other honeycreepers should be highest at sites where density of infectious mosquitoes is below a predicted threshold of 300/kmulations . OverallWhether translocation of Amakihi would succeed depends on many factors In Hawaii, malaria infection patterns are driven by the effects of temperature and rainfall on mosquito dynamics across an elevational gradient, seasonal weather patterns, and availability of larval habitat Even low densities of infectious mosquitoes appear sufficient to produce high rates of malaria infection in native Hawaiian birds. In part, these high rates of malaria transmission likely result from favorable climatic conditions, abundant larval mosquito habitat, and efficient transmission of the malaria parasite between mosquito vectors and avian hosts. Native birds that survive malaria infection acquire immunity, but also become an effective reservoir for malaria transmission to mosquitoes and then to susceptible Hawaiian birds. Reducing the amount of larval habitat for mosquitoes by feral pig management or other alternatives provides one potential strategy to help control malaria impacts and would likely be most beneficial in mid-elevation forests. Establishment of malaria-tolerant Amakihi to mid-elevation forests benefits Amakihi populations, but could negatively impact other honeycreepers depending on the larval habitat and malaria transmission. Our evaluation uses an ecological model that provides a simplified representation of the Hawaiian forest bird malaria system which appears to provide realistic, but somewhat uncertain predictions of mosquito, bird, and malaria patterns in Hawaii


The eyedrop form of rebamipide was approved in Japan for use in the treatment of dry eye diseases, because it up-regulates mucin secretion and production. Others reported that rebamipide, a gastroprotective drug, could not only increase gastric mucus production but also suppressed gastric mucosal inflammation and that it was dominantly distributed in mucosal tissues. In this study we investigated whether rebamipide has anti- inflammatory effects in the ocular surface.We used ELISA and quantitative RT-PCR assay to examine the effects of rebamipide on polyI:C-induced cytokine expression by primary human conjunctival epithelial cells. We studied the effects of rebamipide on ocular surface inflammation in our murine experimental allergic conjunctivitis (EAC) model. Moreover, we also observed their condition of allergic conjunctivitis when we treated the dry eye patients accompanied with allergic conjunctivitis using rebamipide.Rebamipide could suppress polyI:C-induced cytokine mRNA expression and the production of CXCL10, CXCL11, RANTES, MCP-1, and IL-6 in human conjunctival epithelial cells. The topical administration of rebamipide suppressed conjunctival allergic eosinophil infiltration in our EAC model. Furthermore, the allergic conjunctivitis which accompanied by dry eye patients treated with rebamipide tended to be better.The topical application of rebamipide on the ocular surface might suppress ocular surface inflammation by suppressing the production of cytokines by ocular surface epithelial cells.


Long-lasting insecticidal nets (LLINs) and indoor residual sprays (IRS) have decimated malaria transmission by killing indoor-feeding mosquitoes. However, complete elimination of malaria transmission with these proven methods is confounded by vectors that evade pesticide contact by feeding outdoors.For any assumed level of indoor coverage and personal protective efficacy with insecticidal products, process-explicit malaria transmission models suggest that insecticides that repel mosquitoes will achieve less impact upon transmission than those that kill them outright. Here such models are extended to explore how outdoor use of products containing either contact toxins or spatial repellents might augment or attenuate impact of high indoor coverage of LLINs relying primarily upon contact toxicity.LLIN impact could be dramatically enhanced by high coverage with spatial repellents conferring near-complete personal protection, but only if combined indoor use of both measures can be avoided where vectors persist that prefer feeding indoors upon humans. While very high levels of coverage and efficacy will be required for spatial repellents to substantially augment the impact of LLINs or IRS, these ambitious targets may well be at least as practically achievable as the lower requirements for equivalent impact using contact insecticides.Vapour-phase repellents may be more acceptable, practical and effective than contact insecticides for preventing outdoor malaria transmission because they need not be applied to skin or clothing and may protect multiple occupants of spaces outside of treatable structures such as nets or houses. Long-lasting insecticidal nets (LLINs) and indoor residual spraying (IRS) have dramatically reduced malaria transmission by indoor-feeding (endophagic) mosquito populations in recent years -4. HowevWhile mosquitocidal vaccines  and drugHowever, it is crucial to clearly distinguish between alternative modes of action of vector control products and assess their potential comparative value for preventing malaria. Pesticide products either deter insects away from protected houses, sleeping spaces and humans, or they kill those that make physical contact with them . While rh,Ω) compared with that predicted under baseline conditions  where neither measure was in place:Existing models of the interaction between contrasting deterrent and toxic properties ,18 assumh,0,Ω) or as a community-wide average, reflecting the coverage-weighted mean of such non-users and users of one or both measures  [This term was calculated for unprotected individuals lacking either of these measures  . For simAnopheles gambiae, which rarely uses cattle as alternative non-human hosts, and zoophagic Anopheles arabiensis, which does where they are available. These relative exposure outcomes (ψ) were predicted as a function of the mean proportion of malaria transmission exposure of a non-user of any measure which occurs indoors (πi) [Δ) or pre-feed mortality  of mosquitoes attacking users of the product [In all cases, coverage levels of 80% were assumed for both the indoor and outdoor protection measures, consistent with current global targets for LLINs and IRS , and twoors (πi) ,25,26 an product . By comp product . TherefoLLINs depend upon high proportions of human exposure occurring indoors to achieve maximum impact upon malaria transmission Figure . Note thi), spatial repellents applied outdoors consistently supplement the impact of indoor toxins  because mosquitoes diverted from outdoor repellent users may subsequently attack those protected indoors by lethal LLINs  and vectors rarely feed upon non-human blood sources , Syngenta (SJM), Pinnacle Development (SJM) and SC Johnson (SJM).Both authors formulated the research questions and developed the conceptual basis of the model. GFK drafted the model formulation and manuscript, which was then critiqued and edited by SJM. All authors have read and approved the final version of the manuscript.Figure S1. Purely community-level impact of products for outdoor malaria prevention expressed in terms of the mean relative risk of exposure experienced by non-users of any protective measure.Click here for fileFigure S2. Additional incremental community-level impact of outdoor contact toxins or repellents that are exclusively used outdoors or used both indoors and outdoors when combined with indoor LLINs with contact toxins, compared with their direct impact as stand-alone intervention strategies.Click here for fileFigure S3. Progressive community-level impact upon a completely outdoor transmission system of products with increasing efficacy of personal protection achieved by either repelling or killing attacking mosquitoes before they feed upon human users.Click here for fileSupplemental methods S4. Detailed model description.Click here for file


To identify whether axillary US is less accurate in invasive lobular breast cancer than in ductal breast cancer.Randomised cohorts of screening and symptomatic patients were retrospectively identified from histology records of 2010/11. Axillary US of 65 patients with primary breast cancers (BC) from each group of invasive lobular cancer (ILC) and invasive ductal cancer (IDC) were reviewed. Preoperative US-guided needle biopsy sampling was performed on abnormal lymph nodes (LN).See Tables The previous literature on this topic is inconclusive. Some authors have suggested axillary ultrasound in ILC may be less accurate than in IDC, with a higher false-negative axillary assessment rate. Another study concluded that axillary US accuracy rates in ILC were comparable with previous published studies of IDC, used FNA in all cases. We specifically compared accuracy rates of preoperative axillary staging between ILC and IDC in own institution, with 14G needle biopsy as the procedure of choice to sample abnormal nodes. We found that there is no statistical difference in accuracy in US axillary staging between ILC and IDC.


P301S) mice develop tau filamentous inclusions and axonopathy in retinal ganglion neurons (RGCs), in the absence of neuronal loss or alterations in the outer retina. Moreover, we showed that tauP301S transgenic retinal explants do not respond to neurotrophic stimuli in vitro. Here, we investigated the impact of tau pathology on RGC physiology in living animals and neurotrophin signaling pathways in vivo. In anesthetized 5-month old wild type (WT) and tauP301S mice, we measured RGCs activity using pattern electroretinogram (pERG), which selectively detects RGC response upon pattern light stimuli exposure. In transgenic tauP301S mice the amplitude of both P1 positive and N2 negative components of pERG at saturating contrast and spatial frequency was significantly smaller than WT values. Furthermore, retinal acuity was significantly reduced in tauP301S mice. Using uniform flickers of light (flash ERG), we measured the activity of the outer retina and found that outer retina response was preserved in tauP301S mice. Neurotrophins, and especially brain-derived neurotrophic factor (BDNF), are important modulators of neuronal survival and function in the brain and in the visual system. We therefore investigated the BDNF signaling pathway and found that BDNF signalling was altered in tauP301S transgenic retinas. Our results indicate that, in the tauP301S mouse, tau pathology specifically impairs the activity of RGCs, without affecting the outer retina function and is associated with BDNF signalling alterations. Given the role of BDNF in synaptic plasticity, these data suggest that mild levels of tau pathology are sufficient to trigger significant neuronal dysfunction possibly through alteration of neurotrophic signalling. Funded by a grant of Compagnia di San Paolo awarded to LG.Intracellular inclusions made of microtubule-associated tau protein are a defining pathological hallmark of tauopathies, which include Alzheimer disease and familial frontotemporal dementia and parkinsonism linked to chromosome 17. Altered levels of tau protein have been detected in the retina and optic nerve of patients with glaucoma, suggesting that retina degeneration and tauopathies share similar pathogenic mechanisms. We have recently demonstrated that P301S mutant human tau (tau


The insect olfactory system is capable of classifying odorants by encoding and processing the neural representations of chemical stimuli. Odors are transformed into a neuronal representation by a number of receptor classes, each of which encodes a certain combination of chemical features. Those representations resemble a multivariate representation of the stimulus space . The inschannel correlation). In previous work, we demonstrated how lateral inhibition in an olfaction-inspired network reduced channel correlation [Olfactory receptors typically have broad receptive fields, and the odor spectra of individual receptor classes overlap. From the viewpoint of multivariate data processing, overlapping receptive fields cause correlation between input variables  ,5. After


Intrinsic foot muscle weakness has been implicated in a range of foot deformities and disorders. However, to establish a relationship between intrinsic muscle weakness and foot pathology, an objective measure of intrinsic muscle strength is needed. The aim of this review was to provide an overview of the anatomy and role of intrinsic foot muscles, implications of intrinsic weakness and evaluate the different methods used to measure intrinsic foot muscle strength.Literature was sourced from database searches of MEDLINE, PubMed, SCOPUS, Cochrane Library, PEDro and CINAHL up to June 2012.There is no widely accepted method of measuring intrinsic foot muscle strength. Methods to estimate toe flexor muscle strength include the paper grip test, plantar pressure, toe dynamometry, and the intrinsic positive test. Hand-held dynamometry has excellent interrater and intrarater reliability and limits toe curling, which is an action hypothesised to activate extrinsic toe flexor muscles. However, it is unclear whether any method can actually isolate intrinsic muscle strength. Also most methods measure only toe flexor strength and other actions such as toe extension and abduction have not been adequately assessed. Indirect methods to investigate intrinsic muscle structure and performance include CT, ultrasonography, MRI, EMG, and muscle biopsy. Indirect methods often discriminate between intrinsic and extrinsic muscles, but lack the ability to measure muscle force.There are many challenges to accurately measure intrinsic muscle strength in isolation. Most studies have measured toe flexor strength as a surrogate measure of intrinsic muscle strength. Hand-held dynamometry appears to be a promising method of estimating intrinsic muscle strength. However, the contribution of extrinsic muscles cannot be excluded from toe flexor strength measurement. Future research should clarify the relative contribution of intrinsic and extrinsic muscles during intrinsic foot muscle strength testing. Intrinsic foot muscles contribute to the support of the medial longitudinal arch,2 and arThe electronic databases MEDLINE, PubMed, SCOPUS, Cochrane Library and CINAHL were searched between 21 May and 21 June 2012 to locate scientific articles on intrinsic foot muscles and muscle strength measurement. The main search terms and number of articles retrieved are listed in Table Fifty three research articles were identified that related to intrinsic foot muscles and strength measurement. Articles had to meet certain criteria for inclusion. The inclusion criteria were as follows.(i) Research related to the role of intrinsic foot muscles(ii) Research related to the anatomy of intrinsic foot muscles(iii) Research describing the measurement of intrinsic muscles and toe muscle strength or weakness. Papers relating to the intrinsic foot muscle strength were considered initially, but it became apparent that few papers existed. Therefore the search was broadened to include articles relating to the measurement of toe muscles(iv) Publication in peer-reviewed journals(v) Full-text English language articlesplantar aponeurosis and includes the abductor hallucis, flexor digitorum brevis, and the abductor digiti minimi[quadratus plantae and the lumbricals. The third layer consists of adductor hallucis transverse, adductor hallucis oblique, flexor hallucis brevis and flexor digiti minimi brevis. The deepest layer consists of the three plantar interossei. All the plantar intrinsic muscles are innervated by the medial and lateral plantar branches of the tibial nerve[The plantar and dorsal intrinsic muscles of the foot have both their origin and insertion within the foot,27. Intrti minimi. The secextensor hallucis brevis and extensor digitorum brevis. The deep layer consists of the dorsal interossei muscles. The extensor hallucis brevis and extensor digitorum brevis is innervated by the deep fibular nerve while the dorsal interossei are innervated by the lateral plantar nerve with the first and second dorsal interossei also receiving part of their innervation from the deep fibular nerve[extensor hallucis brevis and extensor digitorum brevis during walking varied significantly between participants, with some participants demonstrating no activation of extensor digitorum brevis during gait[. The extensor hallucis brevis and extensor digitorum brevis muscles are now widely used in tissue grafts, such as the island flap to cover soft tissue defects in the distal leg and ankle regions[The dorsal intrinsic muscles of the foot can be divided into two layers. The mosar nerve. The dorar nerve. Early Eing gait. The ext regions. Therefoquadratus plantae disproves this hypothesis. The medial and lateral attachment sites of the quadratus plantae muscle into the calcaneus is unique to humans[quadratus plantae is unique to the foot as there is no analogous muscle in the hand[. Since the flexor digitorum longus tendon enters the foot from the medial side and pulls the toes medially[quadratus plantae allows the toes to flex in the sagittal plane by redirecting the pull of the flexor digitorum longus. This is a necessary development for bipedal ambulation[It has been hypothesised that during human evolution, toe flexor force and function are gradually diminishing and therefore plantar intrinsic muscles are becoming largely redundant in the foot. In simio humans and quadthe hand. Since tmedially, one thebulation. Therefoabductor digiti minimi, abductor hallucis, flexor digitorum brevis, dorsal interossei and lumbrical muscles were all active during the stance phase of gait and continued until toe off[flexor hallucis brevis  and flexor digitorum brevis muscles are able to exert forces approximately 36% and 13% of body weight during the propulsive phase of walking[et al.[A number of studies reveal that intrinsic foot muscles are active as a group during walking,4,36. A  walking. However walking or have  walking. Mann ang[et al. and Goldg[et al. postulatg[et al.,40. Therabductor hallucis, flexor digitorum brevis and the quadratus plantae muscles during relaxed standing with a significant increase in activity with increased postural demands[et al.[The role of intrinsic muscles in the support of the medial longitudinal arch has been investigated in both standing-5 and wa demands. Reeser s[et al. suggestes[et al.. The lacs[et al.. TherefoThe next section will review the influence of intrinsic muscle weakness in the development of pes cavus in Charcot-Marie-Tooth disease, lesser toe deformities, hallux valgus and heel pain.Charcot-Marie-Tooth disease (CMT) is a peripheral neuropathy, where anatomically distal muscles including the intrinsic muscles are preferentially affected. WeaknesMuscle imbalances between the intrinsic and extrinsic foot muscles have been proposed as the possible cause for lesser toe deformity,11,46. Cextensor digitorum longus and brevis are balanced by the flexors forces produced by long and short toe flexors[et al.[et al.[In an unaffected foot, the strong extension forces at the MTP joint by the  flexors. However flexors. The fins[et al. support s[et al.,46, rupts[et al.. These as[et al. in partil.[et al. reportedbunion, describes a foot deformity characterised by lateral deviation of the great toe at the MTP joint away from the midline of the body[abductor hallucis compared to the adductor hallucis transverse and adductor hallucis oblique[abductor hallucis muscle in patients with symptomatic hallux valgus deformity[Hallux valgus, or the body. One pros oblique,13. Whens oblique. This thet al.[The role of the intrinsic muscle weakness in the development of plantar heel pain, or plantar fasciitis, is unclear. One theory proposed by Allen and Gross describeet al. of partiet al.. The atret al.. Hence, The next section will review the ‘direct’ and ‘indirect’ methods of measuring intrinsic muscle strength. The subheading ‘direct methods of assessing intrinsic/extrinsic muscle strength’ reviews the methods that can directly measure a unit of force or power. However, these ‘direct’ methods actually measure toe flexion strength which is a combination of intrinsic and extrinsic muscle strength. The subheading ‘indirect methods of assessing intrinsic muscle strength’ reviews methods that are unable to directly measure force but provide information regarding intrinsic muscle structure and activity.The direct methods reported in the literature include a variety of clinical tests,19,49,50Toe dynamometry is an objective tool used to measure toe flexor strength. Different methods of using toe dynamometry have been reported including hand-held dynamometry, fixed dThe different types of toe dynamometers allow testing of different actions of the toes. The procedure used to measure toe flexor strength with hand-held dynamometry involves the dynamometer being positioned beneath the interphalangeal joint of the hallux to measure either greater toe strength or interphalangeal joints two to five, for lesser toe strength. As suchinterossei and lumbrical muscles, Garth and Miller[interossei and lumbrical muscles can be electrically stimulated to produce flexion at the MTP joint and extension at the interphalangeal joint[interossei and lumbrical in the hand and foot, flexion at the MTP joint and extension interphalangeal joint are likely actions of intrinsic foot muscles. Therefore, hand-held dynamometry might activate intrinsic muscles more effectively than other types of toe dynamometry because it promotes flexion at the MTP joint and extension at the interphalangeal joint.The different types of toe dynamometry may activate intrinsic muscles to varying degrees because each model promotes different actions of the toes. Cuff-based fixed dynamometry, the modified hand grip tester and fixed dynamometry all allow flexion at the MTP joint, but do not provide a way to limit excessive flexion at the interphalangeal joints. A toe curling action can occur during toe flexor testing, an action which is hypothesised to activate the long (extrinsic) toe flexors. Based od Miller postulatd Miller. Studiesal joint. Based oAnother important consideration when measuring intrinsic muscle strength is the position of the ankle. Spink and co-workers hypothesThe Paper Grip Test involves the participant attempting to hold a standard piece of paper, like a business card, underneath the hallux or lesser toes while the examiner attempts to pull the card away,15. The et al.[There are a limited number of validation studies of the Paper Grip test as a measure of intrinsic muscle strength. De Win et al. conducteet al.. Furtheret al.. TherefoPlantar pressure sensors can assess the force beneath the toes. Plantar pressure measurement is generally available in two different forms: (1) in-shoe systems such as Novel Pedar®, TekScan F-Scan®, RS-Scan Insole®, IVB Biofoot ® and; (2)The validity of using plantar pressures to determine intrinsic muscle strength is questionable because the contribution of the extrinsic muscles during the pressure measurement is unknown. Electromyography performed during Paper Grip Test has revealed that some extrinsic toe flexor muscles, particularly the long toe flexors muscles and ankle plantar flexor muscles were active. TherefoThe Intrinsic Positive Test is a qualitative test designed to assess intrinsic muscle function of the lesser toes. The tesThe indirect methods that will be reviewed are: Magnetic resonance imaging (MRI); computerised tomography (CT); ultrasonography; electromyography (EMG) and muscle biopsy. Indirect methods are generally used to estimate muscle structure -24,55-57Magnetic resonance imaging (MRI) is the method of choice for detecting soft tissue structure and abnormalities,59. It het al.[MRI has been used in three main ways to assess intrinsic muscle atrophy: (1) qualitative observation of muscle atrophy,7; (2) fet al. has alsoThe significant limitation of qualitative observations of muscle atrophy and using the five point scale to assess intrinsic muscle atrophy is that conclusions are based on a selected image, which may not be representative of the entire muscle. The MR images of intrinsic foot muscles can be taken in the coronal,7, transThe total volume of intrinsic foot muscles can be calculated by multiplying the total cross sectional area of the muscle, determined from MRI, by the distance between the sections, which is the gap between each MRI slice,57,60. Tet al.[MRI can also be used to estimate physiological cross-sectional area (PCSA) of intrinsic muscles. PCSA haet al. has showet al. and suggAn important advance in MRI is the fast-cine phase contrast (or dynamic MRI), which allows images to be acquired while the participant performs an action. Dynamicet al.[et al.[Computerised tomography (CT) is an imaging technique that uses ionising radiation to generate three dimensional images of musculoskeletal structures. CT scanet al. and Muell.[et al. performel.[et al.. Furtherl.[et al..dorsal interosseus muscle, adductor hallucis muscle, and the first lumbrical muscle[abductor hallucis[abductor digiti minimi[flexor hallucis brevis[quadratus plantae[extensor digitorum brevis[et al.[abductor hallucis muscles.Ultrasonography is a technique that uses mechanically produced longitudinal sound waves to create an image. Ultrasol muscle. More re hallucis,61,69, aum brevis. StudiesUltrasonography has two main limitations; low spatial resolution of the image, and theet al.[Electromyography (EMG) has been assessed by non-invasive surface electrodes and invasive intramuscular electrodes. Surfaceet al. recordedet al.. Therefoabductor hallucis, flexor digitorum brevis, dorsal interossei and quadratus plantae during standing task with increasing postural difficulty. The study revealed increased EMG signal amplitude in all muscles with increasing postural demands of the task, as assessed by deviation of the centre of pressure[Intramuscular EMG involves needle electrodes placed directly in the muscle. Most intramuscular EMG studies of intrinsic foot muscles insert needle electrodes into individual muscle bellies-5,28. Hopressure. The limpressure. Therefoet al.[abductor hallucis muscles in participants with symptomatic hallux valgus and reported histological abnormalities including muscle fibres atrophy, lipid laden fibres and ultrastructural changes in the mitochondria. However, the limitation of muscle biopsies is that findings may not be representative of the whole muscle belly[Muscle biopsy can be used to detect changes in muscle histology and ultrastructure. Biopsy et al. performele belly. TherefoThere is no gold standard method to measure intrinsic muscle strength in the foot. At present methods of measuring intrinsic muscle strength can be categorised as direct and indirect methods. Direct methods are able to quantify,15-19,50However, hand-held dynamometry has some barriers to accurately measure intrinsic muscle strength. First, it is currently unknown which intrinsic muscles are active during the hand-held dynamometry test. Second, it is unclear what action should be performed by the toes to best activate the individual intrinsic muscles during the dynamometry test. Garth and Miller hypothesIndirect methods such as imaging modalities-24,55-57This review suggests that at present, there is no adequately validated method of measuring intrinsic muscle strength. The main challenges are that no direct method can isolate intrinsic muscle strength from extrinsic muscle strength, and indirect methods cannot quantify muscle force. Future research in this field would benefit from using a combination of indirect and direct methods to measure intrinsic muscle force, because both measures have their strengths and limitations. For instance, a study comparing muscle architectural information from MRI, muscle modelling estimates of muscle force and muscle activation data from EMG, with the findings of direct methods such as hand held dynamometry would disentangle each method’s limitation. Also indwelling EMG during hand-held dynamometry would enable both intrinsic and extrinsic muscle activity patterns to be compared, although this approach has numerous practical and analytical considerations to overcome. Nevertheless, a strong correlation would validate the findings of direct methods used to measure intrinsic muscle strength.An accurate and reliable measure of intrinsic muscle strength will enable prospective studies to address the causal relationship questions between intrinsic muscle weakness and foot/toe deformity in conditions like Charcot-Marie-Tooth disease,7, diabeIn conclusion, measuring intrinsic muscle strength poses many challenges. Most studies have focussed on toe flexor strength as a surrogate measure of intrinsic muscle strength, while other actions of toe muscles have not been assessed. Hand-held dynamometry represents a promising method of estimating intrinsic muscle strength. However, the contribution of extrinsic muscles cannot be excluded from toe flexor strength measurement. Future research should clarify the relative contribution of intrinsic and extrinsic muscles during intrinsic foot muscle strength testing.The authors declare that they have no competing interests.AS searched the literature, analysed the articles and drafted the review. JB, CH and KR conceptualised and edited the review. All authors read and approved the final manuscript.


Mesorhizobium metallidurans STM 2683T and Mesorhizobium sp. strain STM 4661 were isolated from nodules of the metallicolous legume Anthyllis vulneraria from distant mining spoils. They tolerate unusually high Zinc and Cadmium concentrations as compared to other mesorhizobia. This work aims to study the gene expression profiles associated with Zinc or Cadmium exposure and to identify genes involved in metal tolerance in these two metallicolous Mesorhizobium strains of interest for mine phytostabilization purposes.Mezorhizobium strains were sequenced and used to map RNAseq data obtained after Zinc or Cadmium stresses. Comparative genomics and transcriptomics allowed the rapid discovery of metal-specific or/and strain-specific genes. Respectively 1.05%  and 0.97%  predicted Coding DNA Sequences (CDS) for STM 2683 and STM 4661 were significantly differentially expressed upon metal exposure. Among these, a significant number of CDS involved in transport  and sequestration  were identified. Thirteen CDS presented homologs in both strains and were differentially regulated by Zinc and/or Cadmium. For instance, several PIB-type ATPases and genes likely to participate in metal sequestration were identified. Among the conserved CDS that showed differential regulation in the two isolates, we also found znuABC homologs encoding for a high affinity ABC-type Zinc import system probably involved in Zinc homeostasis. Additionally, global analyses suggested that both metals also repressed significantly the translational machinery.The draft genomes of the two Mesorhizobium strains. Very few of them were involved in the non-specific metal response, indicating that the approach was well suited for identifying genes that specifically respond to Zinc and Cadmium. Among significantly up-regulated genes, several encode metal efflux and sequestration systems which can be considered as the most widely represented mechanisms of rhizobial metal tolerance. Downstream functional studies will increase successful phytostabilization strategies by selecting appropriate metallicolous rhizobial partners.The comparative RNAseq-based approach revealed a relatively low number of genes significantly regulated in the two  Such a phytostabilization approach is nevertheless challenging since it requires metallicolous plants able to grow in soils where nutrients are most often dramatically scarce. Legume/rhizobia symbioses which transform atmospheric dinitrogen into organic nitrogen is of ecological interest here as it can improve natural soil fertility and thereby allow the colonization of other plant species and the installation of a plant cover ..Strains,Click here for filePrimers used for quantitative PCR assays.Click here for file


Propionic acidaemia (PA) results from deficiency of Propionyl CoA carboxylase, the commonest form presenting in the neonatal period. Despite best current management, PA is associated with severe neurological sequelae, in particular movement disorders resulting from basal ganglia infarction, although the pathogenesis remains poorly understood. The role of liver transplantation remains controversial but may confer some neuro-protection. The present study utilises quantitative magnetic resonance spectroscopy (MRS) to investigate brain metabolite alterations in propionic acidaemia during metabolic stability and acute encephalopathic episodes.Quantitative MRS was used to evaluate brain metabolites in eight children with neonatal onset propionic acidaemia, with six elective studies acquired during metabolic stability and five studies during acute encephalopathic episodes. MRS studies were acquired concurrently with clinically indicated MR imaging studies at 1.5 Tesla. LCModel software was used to provide metabolite quantification. Comparison was made with a dataset of MRS metabolite concentrations from a cohort of children with normal appearing MR imaging.MRI findings confirm the vulnerability of basal ganglia to infarction during acute encephalopathy. We identified statistically significant decreases in basal ganglia glutamate+glutamine and N-Acetylaspartate, and increase in lactate, during encephalopathic episodes. In white matter lactate was significantly elevated but other metabolites not significantly altered. Metabolite data from two children who had received liver transplantation were not significantly different from the comparator group.The metabolite alterations seen in propionic acidaemia in the basal ganglia during acute encephalopathy reflect loss of viable neurons, and a switch to anaerobic respiration. The decrease in glutamine + glutamate supports the hypothesis that they are consumed to replenish a compromised Krebs cycle and that this is a marker of compromised aerobic respiration within brain tissue. Thus there is a need for improved brain protective strategies during acute metabolic decompensations. MRS provides a non-invasive tool for which could be employed to evaluate novel treatments aimed at restoring basal ganglia homeostasis. The results from the liver transplantation sub-group supports the hypothesis that liver transplantation provides systemic metabolic stability by providing a hepatic pool of functional propionyl CoA carboxylase, thus preventing further acute decompensations which are associated with the risk of brain infarction. Propionic acidaemia  results from deficiency of the biotin-dependent mitochondrial enzyme propionyl CoA carboxylase. The commonest and most severe form of PA presents in the neonatal period with acute metabolic acidosis, hyperammonaemia and progressive encephalopathy. Despite optimal management, later neurologic sequelae are a major cause of morbidity, and include neurodevelopmental delay, episodic acute encephalopathy, and movement disorders due to a propensity to basal ganglia infarction . The maiThe pathogenesis of the neurological insult remains poorly understood. Neurological damage may occur secondary to hyperammonaemia; alternatively inhibition of the mitochondrial Krebs cycle and electron transfer chain by accumulating metabolites may lead to a "bioenergetic stroke" . Based oin vivo changes in regional metabolites within the brain, and could be specifically employed to monitor directly alterations in brain energy metabolites including glutamine, glutamate and lactate. Previous MRS reports have demonstrated elevated glutamine+glutamate (Glx) in the basal ganglia of metabolically stable PA patients [Magnetic resonance spectroscopy (MRS) is a non-invasive technique capable of probing patients , while lpatients .The present study utilises quantitative MRS to investigate brain metabolite alterations both during metabolic stability and acute encephalopathic episodes, confirming the basal ganglia as a particular target and demonstrating novel insights in to alterations in neurotransmitter metabolites in particular glutamine and glutamate.Eight children  with propionic acidaemia were studied (median age (range) at first MRS, 36 7-190) months) . During these episodes all children required ventilatory support and continuous veno-venous haemofiltration (CVVH), and only one episode was associated with significant persistent hyperammonaemia. During these episodes three children received intravenous sodium benzoate and two received sodium phenylbutyrate.MRS studies were performed concurrently with clinically indicated MRI scans at 1.5 Tesla  facilitated by general anaesthesia as needed. Five studies from three children were acquired during severe acute episodes requiring intensive care support. Six studies from six children were acquired with elective outpatient MRI studies undertaken during metabolic stability and before any severe acute episode had occurred.MRS was acquired using point resolved spectroscopy (PRESS) technique  with 2 cm-sided voxels located in the left basal ganglia and right parieto-occipital white matter. Voxels were placed in standard positions guided by T1 and T2 weighted image sequences in coronal, sagittal and axial planes. Spectra were quality-assessed to ensure appropriate voxel localisation, shimming and water suppression. Raw data were processed using LCModel [Comparison was made with MRS metabolite data from a cohort of children with normal appearing MRI and similar age profile who had undergone MRI/MRS for investigation of various suspected neurodegenerative and neurocognitive conditions  age 4.7(0.5-16.7) years; basal ganglia n = 63, 4.1(0.5-16.7) years).Mean spectra were generated for each cohort for basal ganglia and white matter from LCModel fitted spectra after baseline subtraction. Spectra were compared by qualitative visual inspection.Metabolite concentrations for each region were compared between groups by Mann Whitney nonparametric U test (SPSS Statistics 17.0), with the p-value determined by the exact 2-tailed significance, with significance level set at p < 0.05.The basal ganglia appeared normal in all children (n = 6) prior to any severe acute episode beyond the neonatal period. MRI obtained during severe acute episodes demonstrated abnormal signal in the basal ganglia variably including the caudate heads, putamen and globus pallidi and in some cases there was also evidence of acute changes in the dentate nuclei and cerebral cortex. One child demonstrated evidence of previous basal ganglia infarction relating to a previous acute encephalopathic episode, with cystic degeneration of caudate, putamen and globus pallidus. Interestingly the hippocampi appeared atrophic in seven cases. Cerebral volume loss was most marked in children with preceding severe acute episodes. The two children who had liver transplantation had normal appearing basal ganglia, cortex and white matter on MRI undertaken more than 10 years post-transplant, with one showing probable temporal mesial sclerosis.Analysis of mean spectra figure  and meanMRS studies undertaken during metabolic stability before any severe acute episodes beyond the neonatal period demonstrated decreased Glx in basal ganglia compared to the normal MRI comparator group but a trend to increase in white matter. Glutamine alone was significantly decreased in basal ganglia during metabolic stability. Mean lactate was not elevated, although two cases had reliably detected lactate in basal ganglia. There was no significant decrease in the total N-acetylaspartate and N-acetylaspartylglutamate (tNAA) in this group.myo-inositol, and no propionic acid was detected in the propionic acidaemia cohort.MRS studies acquired during severe acute episodes demonstrated significantly decreased tNAA, Glx and creatine, and significantly elevated lactate, in the basal ganglia compared to both the normal MRI comparator group and to the metabolically stable propionic acidaemia cohort. In white matter lactate was significantly elevated but other metabolites were not significantly altered. . There were no significant differences between groups in brain Separate analysis of brain metabolites from the two children post-liver transplant demonstrated normal basal ganglia tNAA and slightly decreased Glx. tNAA was elevated in white matter and total choline decreased.The data presented represent the largest series of children with the severe neonatal form of propionic acidaemia to be investigated with MRS, and is to our knowledge the first to study children during severe acute encephalopathic episodes. The selective vulnerability of the basal ganglia in PA is well established, and our data provide further insight in to processes occurring within the basal ganglia. MRI results found that basal ganglia appeared normal in children who had not had a severe acute encephalopathic episode beyond the neonatal period, as similarly reported in glutaric aciduria , but werDecreased tNAA is seen in many pathologies, and is thought to reflect loss of viable neurons  includinThe alterations seen in glutamine and glutamate in basal ganglia are of particular note. Glx was significantly decreased during severe acute episodes, with a smaller (non-significant) decrease noted in basal ganglia in studies acquired during metabolic stability. The ratio of Glx/tNAA was lower in the propionic acidaemia cohort than in cohorts of children with other defined inherited metabolic disorders including a cohort with confirmed respiratory chain disorders, while the Glx/NAA ratio was elevated in a cohort with the urea cycle disorder argininosuccinic aciduria . This suggests that the alterations seen in Glx here were not simply a reflection of neuronal loss represented by lower tNAA.Although the spectral signals from glutamine and glutamate overlap, there is increasing evidence that they can be differentiated in the analysis of cohorts even at 1.5Tesla using software such as LCModel. Such evidence includes Montecarlo simulations of normal brain spectra fitted with LCModel , compariA previous MRS study reported elevated basal ganglia Glx in three metabolically stable propionic acidaemia patients who had normal ammonia and near-normal glutamine and glutamate in plasma and cerebrospinal fluid leading the authors to suggest differential mechanisms regulating brain parenchymal metabolism . HoweverThe administration of the alternative pathway drugs used to control hyperammonaemia depletes plasma glutamine and glycine and may contribute to the decreased brain Glx seen. Furthermore, all children were haemofiltered during severe acute episodes which may further deplete glutamine due to its high sieving coefficient . HoweverThe acute decrease seen in Glx supports the hypothesis that they are consumed to replenish a depleted Krebs cycle , and thaThere is a need for brain protective strategies during acute metabolic decompensations, with potential methods including brain cooling, adequate energy provision, and anaplerotic therapies. MRS could provide a tool to monitor the effect of dietary supplements aimed at increasing glutamine levels, specifically monitoring metabolite levels within the target-organ of interest, namely the brain.The two children who had liver transplant have had no severe acute encephalopathic episodes beyond the neonatal period, and have normal appearing basal ganglia on MRI. MRS demonstrated normal/high tNAA reflecting the absence of major neuronal loss and correlating with their good neurocognitive status, and no reliably detected lactate. The small numbers preclude statistical significance, but Glx was slightly increased in white matter but decreased in basal ganglia compared to the comparator group. These results support the hypothesis that liver transplantation provides systemic metabolic stability by providing a hepatic pool of functional propionyl CoA carboxylase, thus preventing further acute metabolic decompensations which are associated with the risk of brain infarction.The data presented here have yielded some interesting observations, however further prospective data collection and analysis could overcome some of the specific limitations of the present work. In particular, use of higher field strength MRI scanners (3Tesla and above) would provide more robust differentiation of the metabolites of interest, notably glutamine and glutamate. Serial data collection in specific patients could also confirm the alterations in brain metabolites seen before, during and after metabolic decompensations. Finally analysis of a larger cohort of post-liver transplant patients as well as longer follow up with serial studies will provide stronger evidence for the therapeutic value of liver transplantation in PA.in vivo brain metabolic derangements, and can feasibly be employed in children with acute encephalopathy. In propionic acidaemia the use of MRS in conjunction with MRI has confirmed the basal ganglia as a particularly vulnerable target and has demonstrated novel insights in to alterations in neurotransmitter metabolites. Evaluation of patients in long-term follow up after liver transplantation demonstrates the potential for good neurological outcome and near-normal brain metabolism. MRS provides a tool to directly evaluate the tissue-level effects of novel treatment strategies including anaplerotic therapies.We have demonstrated that quantitative MRS is able to offer insight in to The authors declare that they have no competing interests.JED carried out data analysis and collated clinical data and drafted the manuscript.NPD, MW, YS assisted with data processing including software development.AC, PJM and JHW provided clinical management for subjects, assisted with patient recruitment and critically appraised the manuscript.PG and ACP conceived of the study and participated in its design.All authors read and approved the final manuscript.


Implementation of alcohol screening and brief intervention (SBI) is a prevention priority. The Veterans Affairs (VA) Healthcare System uses a clinical reminder (CR) in the electronic medical record to prompt and document results of screening and trigger a subsequent CR for BI when screening is positive. Although screening rates are over 90%, marked variability in screening quality has been documented. Four researchers observed clinician interactions with CRs during alcohol screening at nine primary care clinics in the northwest US to identify barriers and facilitators to using CRs to implement quality screening. Observers took handwritten notes, which were transcribed and analyzed qualitatively using an a priori coding template adapted during analyses. We observed 58 support staff  caring for 166 patients. Alcohol screening prompted by the CR was often uncomfortable and of low quality. Clinicians often offered disclaimers prior to screening or made adjustments to how questions were presented, with some citing the sensitive nature of the questions. Verbal screening typically did not include asking questions verbatim. There was substantial variability in methods of conducting screening across clinics, with some using the CR to facilitate in-person screening by interview and others entering patient responses into the CR after completion of a paper-based screen. Although the CR was designed to trigger a subsequent CR for BI when positive, some clinics used paper encounter forms for this. Findings suggest that VA CRs have important limitations as a method of facilitating effective, high-quality alcohol screening. Barriers observed reflect a combination of limitations of CR technology , ways the CR was implemented, clinical workflow, complexity of patient needs, and alcohol-related stigma. Future research should address these barriers to effectively implement recommended care.


The pressure flow model of phloem transport envisaged by Münch  has gain The pressure flow model of phloem transport Münch,  has gainP) for a given viscosity (η), predicts that variation in flow path geometries of STs , will alter their hydraulic conductances (Lo and see Equation 2) and hence impact axial volume flow rates (Rv) through them.Experimental observations provide persuasive support for phloem sap moving through STs by bulk flow  by variations in sieve pore radii  provided by plasmodesmata interconnecting these cells.Phloem unloading rates are regulated by hydraulic conductances (Equation 2) of the unloading pathway rather than the axial hydrostatic pressure differential Equation 1). In this context, the latter is comparable between a source and an array of sinks irrespective of their rates of phloem unloading  of symplasmic unloading pathways linking SEs with phloem parenchyma cells in the release phloem Figure .Key elements of the high-pressure manifold model of phloem transport articulated by Fisher  and captOur analysis of the high-pressure manifold model of phloem transport primarily rests on evaluating the extent to which the model is consistent with known properties of resource flow through the phloem. Of necessity much of this analysis will focus on phloem transport in herbaceous plants where most experimental studies have been undertaken. However, with appropriate caveats, the analysis is extended to phloem transport in trees.Bulk flow in the Münch model is conceived to be restricted to STs alone in which substantial hydrostatic pressure gradients develop from their source to sink ends. In contrast, the high-pressure manifold model  trees during the photoperiod  and stem (transport) lengths that accounts for phloem translocation by Münch pressure flow in plant heights spanning several orders of magnitude  exceed the potential capacity of phloem loading, phloem sap concentrations of sugars, accompanied by ST hydrostatic pressures, decline until a new equilibrium between loading and unloading rates are reached. This condition could cause a pronounced axial gradient in ST hydrostatic pressure from source to sink to develop. The corollary is sink limitation whereby the potential capacity for phloem loading of sugars exceeds that of phloem unloading. For this condition, any increase in rates of phloem unloading is met by a commensurate increase in phloem loading. This condition would sustain high phloem sap sugar concentrations and hence ST hydrostatic pressures throughout the system.There are no direct measures of rates of phloem loading and unloading. However, there is considerable indirect evidence suggesting that potential capacities of leaf photosynthesis arranged in series with phloem loading exceed those of phloem unloading into expanding and storage sinks. For instance, under optimal environmental conditions for leaf photosynthesis, increasing sink/source ratios by experimentally attenuating sugar export from a portion of leaves  results in elevated rates of sugar export from the untreated leaves of trees  expressed on a cross-sectional area basis i.e., πr2) would vary directly with sieve pore radius raised to the second power. However, across a cohort of test species, estimates of phloem transport velocities were found to exhibit an inverse rather than the predicted direct relationship with their estimated ST hydraulic conductivities  in STs  plants whose STs do not seal upon severance, allowing phloem transport to continue. Hence rates of phloem transport can be estimated by measuring quantities  of phloem sap exuded from cut surfaces across specified times. The estimated transport rates can be expressed as ST fluxes based on measures of their cross-sectional areas through which axial transport takes place  surgical removal of phloem cross-sectional area in stems was found to exert little influence over axial flow rates in both woody plants  or storage  organs. In particular, under high source/sink ratios, net resource flows occur from transport phloem of petioles, stems and roots into storage pools of their ground tissues in which transport phloem is embedded. A striking example of this phenomenon is accumulation of sucrose in storage parenchyma cells of sugarcane stems to concentrations that match those of the phloem sap Patrick, . Thus, cPhloem unloading may be arbitrarily divided into SE unloading and post-SE unloading which extends from phloem parenchyma cells to cellular sites of resource utilization or storage in non-vascular cells. For the purposes of evaluating the high-pressure manifold model of phloem transport, the primary focus is on SE unloading wherein resources reach the phloem parenchyma cells. However, some consideration, where relevant, will be given to post-SE unloading.A central tenant of the high-pressure manifold model is that hydraulic conductances of the symplasmic unloading pathways, from SEs of release phloem, impose a major constraint over bulk flow of resources through the source-path-sink system Figure . This diThe most definitive experimental approach to mapping phloem-unloading pathways has been through the use of membrane-impermeant fluorochromes introduced into phloem sap as membrane-permeant esters or as tags linked to transgenically-expressed foreign molecules under the control of companion cell specific promoters.Axial delivery pathways into apical meristems comprise protophloem SEs giving way to strands of provascular cells more proximal to the apical dome. Distribution of membrane-impermeant fluorochromes, imported through the phloem, have identified putative symplasmic unloading pathways extending from terminal ends of protophloem SEs through provascular stands to reach meristematic cells in root apices of monocots  predicts that any symplasmic bulk flow of phloem sap exiting from SE/CC complexes will encounter considerable hydraulic resistances and hence a proportionate reduction in hydrostatic pressure upon entering recipient cells. These predictions are consistent with the presence of large (1.0 MPa) differences in hydrostatic pressures between release phloem SEs and cortical/epidermal cells of root tips measured directly using aphid stylet micromanometry and pressure probe respectively Table . Similar−1 into a developing wheat grain across a set of microchannel radii (see Table Rv – Equation 1) from which microchannel numbers supporting the observed volume flow rate were derived. From these data, microchannel numbers per plasmodesmata were estimated assuming that a single row of microchannels occupy 50% of the sleeve cross-section. Microchannel numbers per plasmodesma then allow determination of plasmodesmatal numbers required to support the observed volume flow rate  of a solute i are described by Fick's First Law of diffusion as:D is the diffusion coefficient of solute i; A is the cross-sectional area (r = πr2) of the diffusional pathway; ΔC is the concentration difference of solute i between either end of the diffusive pathway; Δx is the length of the diffusive pathway.Since SE/CC complexes and vascular parenchyma cells share identical apoplasmic water potentials, turgor differentials arise from intracellular osmotic differentials and, as a result, concentration differences of phloem sap constituents. Hence diffusion must be a component of phloem unloading and the issue of the relative contributions of symplasmic phloem unloading by diffusion and bulk flow becomes one of degree. Diffusion rates  and bulk flow (Equation 1) in SE unloading, published sucrose concentrations in SEs  with that of 14C-sucrose along elongating pea epicotyls, independent of their differing symplasmic concentration gradients, suggests symplasmic unloading of these disparate solutes is primarily by bulk flow rather than diffusion  and Ficks First Law of diffusion Equation 3). That is, rates of these two processes are proportional to the radius of the conducting pipe raised to the fourth and second powers respectively. Thus, control of pipe radius can exert a four-fold greater control over rates of bulk flow compared to diffusion. This translates into an operational system whereby modest changes in microchannel dimensions can elicit substantial impacts on bulk flow rates of solute unloading from release phloem as illustrated in Tables . That isIn addition to optimizing control over phloem unloading (see above), bulk flow also avoids a dependence upon a very high level of co-ordination of unloading between individual solute species to avoid compromising phloem transport as outlined in the following scenario. If symplasmic unloading were to occur primarily by diffusion, then a mechanism must be present to ensure exit rates of each solute species and water from release phloem SEs match their rates of replenishment by axial transport. This is not a trivial requirement as diffusion rate of each solute moving through the same unloading pathway geometry depends not only on their concentration differences between SEs and vascular parenchyma cells but also on their individual diffusion coefficients (see Equation 3). Moreover phloem-imported water will diffuse at rates dictated by water potential differences between SE-CCs and vascular parenchyma cells. In these circumstances it would be very problematic for axial rates of each solute and water transported into, to match those of their diffusional exit from, release phloem SEs. As a consequence, regulation of ST hydrostatic pressures of release phloem could be compromised. This problem does not apply if SE unloading were to occur by bulk flow since solutes and solvent are transported at identical fluxes .Rv and see Equation 1) and cortical cell turgor points to hydraulic conductances of plasmodesmata, located along the symplasmic unloading pathway, exerting a major regulatory influence over radial fluxes of water and hence solutes travelling from SEs to cortical cells. This explanation also accounts for transport behaviors in developing seeds whereby coat cell turgors were found to be invariant across a three-fold range of seed growth rates exhibited between French bean cultivars  and densities, formed during their development at various cellular interfaces located along phloem unloading pathways set upper limits for symplasmic hydraulic conductances. For example, inferred from Stoke's radii of the permeating molecules, these data yield conservative estimates of plasmodesmal microchannel radii for phloem and non-phloem domains that extend over a two-fold range (see Section Is Symplasmic Sieve Element Unloading Dominated by Bulk Flow?). In relative terms, these radii translate into a 16-fold range of hydraulic conductances. Second, plasmodesmal gating (open versus closed), mediated by callose deposition/hydrolysis around plasmodesmal neck regions, has been demonstrated to play a key role in regulating transmission of macromolecular signals orchestrating cell development (Burch-Smith and Zambryski, Conductance for symplasmic movement between two adjacent cells is a function of their averaged conductance of plasmodesmata summed over the density of plasmodesmata located in their shared wall. Plasmodesmal densities are under both positional and developmental control (Burch-Smith and Zambryski, Within meristematic sinks, plant hormones regulate rates of cell division and hence sink demand. This hormone function is linked with their ability to directly act on transport processes delivering resources to hormone-enriched sites Patrick, . Fully-eFor expansion/storage sinks, some insights into the regulatory mechanisms integrating sink demand with plasmodesmal control of resource flow along phloem unloading pathways has been obtained from observations of developing seeds. Phloem unloading pathways in developing seeds universally contain a symplasmic discontinuity at, or proximal to, their maternal and filial interfaces at which phloem-imported resources are released to, and retrieved from, the seed apoplasmic space (Zhang et al., Crop biomass yield potential is the harvested yield per plant of any cultivar raised under optimal environmental conditions for its development in the absence of any limitation imposed by abiotic or biotic stresses (Evans and Fischer, The elements carbon, hydrogen and oxygen account for ca 90% of crop biomass yield. These elements reach the harvested units as phloem-imported sugars that form the major osmotica generating ST hydrostatic pressures propelling bulk flow from source to sink. The preceding analysis of the high-pressure manifold model of phloem transport identified hydraulic conductances of plasmodesmata, located at the sink-end of phloem pathways, as key regulators of bulk flow rates from source to sink and for partitioning of flow rates between competing sinks Figure . This shZea mays ADP-glucosepyrophosphorylase, under control of an endosperm-specific promoter, in pre-storage grains of wheat (Smidansky et al., The pre-storage phase of harvestable organ development is photoassimilate limited as demonstrated by positive responses of harvestable organ numbers to elevated source/sink ratios (Ruan et al., The storage phase filling rates are characteristically sink-limited (Borrás et al., Overall, the above circumstantial evidence indicates that opportunities may well exist to improve crop biomass yield through manipulating resource transport informed by the high-pressure manifold model of phloem transport.Reviewing current knowledge of phloem transport leads to a strong circumstantial case being made for the high-pressure manifold model of phloem transport. This is considered sufficient to warrant further study of the model. Initially, most effort could be directed to increasing knowledge of the plasmodesmal sub-structures and how these influence resource flow through their plasmodesmal microchannels. In particular, it is crucial to obtain direct evidence that hydraulic conductances of plasmodesmata, linking SEs with surrounding phloem parenchyma cells, support SE unloading of phloem sap by bulk flow. This enterprise could be informed by the exciting findings emerging from investigations of water flows through nanotubes showing that, with radii of several nm, flow velocities exceed those predicted by the Hagen-Poiseuille law by four to five orders of magnitude (Majumber et al., This paper is dedicated to the memory of Donald Fisher, a modest, but passionate, plant scientist. Don's contributions to advancing our conceptual knowledge of phloem transport biology were substantial, unyielding in their rigor and trail blazing in their impact on the field.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.