Transportation of oil through road has history of safety issues in developing nations, which in some instances resulted in heavy toll and economic loss. Recent such incident took place in Pakistan in June, 2017 which resulted in death of more than 200 people. There is need to analyze oil transportation mechanism through roads in developing countries to address the issue of safety in order to control such deteriorations which result in heavy losses in terms of human lives and property damages. The guidelines will be helpful in preventing road crashes of oil tankers and if a case of an accident will be helpful in minimizing the loss. These guidelines have been formulated on the basis of Hiddon’s Matrix and recommendations of Word Report of Injury Prevention 2004. Moreover; evidence based best practices on the subject issue have also been searched and incorporated in the report. Multifaceted recommendations have been formulated based upon various factors and elements such as on the basis of transportation planning, the burden of oil transportation on road need to be reduced and segregated to railways and pipeline on safety and economy basis; enforcement mechanisms need to be strengthened by provision of special authorities for regulations of transportation companies, vehicles safety design, fitness and driving licensing; special trainings for first responders and drivers for handling hazardous materials crises; provision of enforcement of driver’s working hours and establishment of rest areas to address issue of drowsiness and fatigue ; and lastly establishment of firefighting center and burn centers at divisional units for handling post-crash issues such a s fire and burn injuries. The report addresses wide range of issues pertaining to road transportation of oil in developing countries and recommendations can be helpful in prevention of related road crashes, post-crash management and addressing associated consequences. Implementation of recommendations will improve safety of oil carriers and will be helpful in reducing related road crashes. Oil Transportation safety


Field‐based course work has been foundational to Ecology and Evolutionary Biology curricula. However, opportunities for these experiences gradually have decreased over the past few decades and are being replaced with technology in the college learning environment. The coronavirus disease 2019 pandemic facilitated a rapid transition of all field‐based courses to online only delivery, which we argue has forced us to reconsider how to deliver course content to retain field experiences in a manner that is safe during the pandemic but robust to ever changing constraints in the college classroom. Here, we propose pairing an intensive laboratory experience with an otherwise online delivery. We discuss several advantages of intensive laboratory experiences that occur in the field over a short but intensive time period over that of the traditional low‐intensity weekly laboratory structure. In particular, intensive laboratory experiences are safer during the pandemic because they allow the group to be tested and isolated, allow more flexibility for students with competing interests for their time, and also enhance student interpersonal skills while still providing strong reinforcement of the skills typically honed through experiential learning. We present case studies for how we intend to apply our proposed model to two courses that heavily rely on field‐based experiential learning to facilitate adoption. We propose an intensive lab experience can safely retain experiential learning in the field during the pandemic and provide a number of advantages to the traditional lab model. Here we provide two examples of how we plan to apply our proposed model to field based courses. It is important for students to experience the processes and concepts they are learning about nature in nature. Experiential learning provides students ample time and space to engage in this process of learning through the senses. This hands‐on approach allows students to contextualize information by experiencing nature and to build knowledge by assimilating new experiences through self‐reflection  curricula because of the undisputed value of learning by doing. We realize that it is difficult to appreciate the evolutionary significance of the enlarged anal scent gland in skunks (Mephitidae) without having smelled it or the effectiveness of epizoochory as a dispersal strategy until removing beggar's lice  during regularly scheduled class laboratories  outdoor laboratory experiences  to apply the knowledge to a real‐world issue . A variety of approaches to information delivery could be adopted but in general, we suggest 1‐ to 3‐hr long field activities 2–3 times per day with 15 min—1‐hr long intermissions. Providing opportunities to work in groups, especially in research related projects, can be particularly effective to enhance experiential learning by strengthening student–student relationships and can even provide a tangible outcome to those students interested in following through with publication of their work  and Wildlife Techniques (WIS 4945C) have been taught at the University of Florida as typical semester long courses with in‐person lectures or discussion and weekly field‐based laboratories. We have reformatted both of these courses to reflect our recommendations above. The courses will have an online synchronous and asynchronous delivery with a paired ILE at the end of the spring semester Table . This stFor both classes, the structure of online content will include approximately 10‐min prerecorded lectures using voice recorded over PowerPoint slides ; Investigation ; Writing‐original draft ; Writing‐review & editing . Robert McCleery: Conceptualization ; Resources ; Writing‐original draft ; Writing‐review & editing .


Bottlenecks reduce the size of the gene pool within populations of all life forms with implications for their subsequent survival. Here, we examine the effects of bottlenecks on bacterial commensal‐pathogens during transmission between, and dissemination within, hosts. By reducing genetic diversity, bottlenecks may alter individual or population‐wide adaptive potential. A diverse range of hypermutable mechanisms have evolved in infectious agents that allow for rapid generation of genetic diversity in specific genomic loci as opposed to the variability arising from increased genome‐wide mutation rates. These localised hypermutable mechanisms include multi‐gene phase variation (PV) of outer membrane components, multi‐allele PV of restriction systems and recombination‐driven antigenic variation. We review selected experimental and theoretical  models pertaining to the hypothesis that localised hypermutation (LH) compensates for fitness losses caused by bottlenecks and discuss whether bottlenecks have driven the evolution of hypermutable loci. Bottlenecks produce dramatic reductions in the genetic diversity of bacterial populations. Bottleneck events can be selective or non‐selective and occur frequently, particularly during transmission between hosts. Multiple mutational strategies have evolved in bacteria that can regenerate genetic diversity within surviving populations following a major reduction in population size. A key on‐going paradigm shift in our understanding of genetic variability in pathogenic bacteria was the demonstration that localised hypermutation (LH) due to hypermutable DNA elements was responsible for the phase variation (PV) phenomenon of rapid ON and OFF switches in surface antigens and restriction‐modification systems (RM). We now recognise that LH and PV are widely distributed across bacterial pathogens and commensals but with significant diversity in the numbers of loci and in the types of functions encoded by these loci Figure . The imp2Bottlenecks are a short‐hand to describe the phenomenon of a rapid reduction in the number of organisms in a population leading to loss of genetic variants Figure . Concept2.1Early evidence of infection‐associated bottlenecks came from studies of pneumococcal pathogenesis and the observation that intravenous injection was followed by a major reduction in population size prior to establishment of high‐level bacteraemia Wright, . Gerlini2.2Neisseria meningitidis to a bactericidal monoclonal antibody specific for a phase‐variable LOS epitope  in a population has an independent probability to initiate an infection without any cooperative or synergistic interactions from other individuals in the population so that infections become progressively more likely to occur for every increase in the infecting population size.Using  Meynell  first prSalmonella cells have inferred the presence of multiple within‐host bottlenecks. Han Lim and Colleagues ; stochastic, reversible, high frequency alterations in the flagella phenotypes of A key early observation was that the majority of contingency loci encode bacterial surface antigens or indirect modifiers of these antigens Figure . SubsequA final generic observation is that LH is not only widespread but exhibits variations in prevalence between and within species Figure . Some sp4In order to support the generic view of the previous section, we review some recent findings on distributions of LH and how hypermutable switches contribute to within‐host evolution.4.1It is a new programme , TRD switching is facilitated by conserved regions flanking the TRD sequences , had evolved longer SSRs as compared to the parental clone during expansion and persistence within the UK of a serogroup W cc11 sub‐clone of 4.7N. meningitidis was associated with reductions in the combined expression states of OMP‐encoding PV loci. High levels of serum antibodies to the capsule and one of these OMPs were present in the carriers studied suggesting that antibody‐mediated selection drives these changes. Using WGS of multiple longitudinal non‐typeable Haemophilus influenzae (NTHi) isolates from 61 persistently chronic obstructive pulmonary disease (COPD) patients, Pettigrew et al. found that increases in repeat number were positively correlated with length of persistence  but also niche specialisation limiting the adaptive benefits of hypermutability. Despite these advances, studies of how different types of bottlenecks are impacted by LH are still a rarity and have yet to explicitly demonstrate that LH enables adaptation to, or survival of, both the initial bottleneck and of a subsequent, alternative selective event.Despite the uncertainties about real‐world bottlenecks and the new findings on LH diversity, mathematical modelling has established a strong basis for unravelling the evolutionary forces acting on LH. The key roles of selection strength, epoch length, bottleneck frequency, bottleneck stringency and compartmentalisation have firm foundations with models that can be applied or adapted to specific situations. There are, of course, still many gaps in our understanding of key processes that mainly stem from a lack of real‐world knowledge; how do we measure selection strength?; are real‐world bottlenecks highly selective for variation in a single genetic locus or multiple loci?; are some bottlenecks completely, or evolutionarily, non‐selective? We also need to be aware of whether specific events are of evolutionary importance or are accidental effects of the existence of certain virulence attributes and LH mechanisms. This latter point is a key concern as theoretical modelling of evolutionary processes needs to focus on attributes that contribute to evolution and not on evolutionary dead ends such as bacterial meningitis. Perhaps unsurprisingly, we still await a definitive theoretically underpinned experimental demonstration that differences in mutability of an LH locus, or even loss of mutability, provide a competitive advantage during multiple cycles of transmission of an infectious agent.The authors declare no conflicts of interest.MDSC, JH, JJW, ERM, MRO and CDB have all made major contributions to the drafting of the manuscript. MDSC, JJW and CDB made and refined figures.


As the prognosis of patients who undergo resection for pancreatic cancer has improved, reports of remnant pancreatic cancer after pancreatic cancer resection have been increasing. Previous studies regarding early‐stage pancreatic cancer showed a high incidence of remnant pancreatic cancer in these patients. The aim of this study was to investigate the incidence of remnant pancreatic cancer according to the degree of progression of the initial pancreatic cancer.Patients who underwent partial pancreatic resection for primary pancreatic cancer were retrospectively reviewed and divided into an early‐stage group and an advanced‐stage group according to the stage of the initial cancer. Patient characteristics and long‐term outcomes, including development of remnant pancreatic cancer, were compared between the two groups.P = .1827).This study included 321 patients who underwent partial pancreatectomy for pancreatic cancer; 32 patients in the early‐stage group and 289 patients in the advanced‐stage group. Remnant pancreatic cancer developed in 19 patients (5.9%); seven patients (21.9%) in the early‐stage group and 12 patients (4.5%) in the advanced‐stage group. The cumulative incidence of remnant pancreatic cancer according to the Kaplan–Meier method was comparable between the two groups (5‐year cumulative incidence: 20.6% vs 9.9%, early‐stage group vs advanced‐stage group; Our results suggested that the potential for developing remnant pancreatic cancer was comparable between the early‐stage and the advanced‐stage groups. Therefore, the incidence of remnant pancreatic cancer may increase along with improved pancreatic cancer treatment. Remnant pancreatic cancer occasionally develops after pancreatic resection for pancreatic cancer. Although the proportion of patients who develop remnant pancreatic cancer was larger in early‐stage pancreatic cancer than advanced‐stage cancer, the potential for developing remnant pancreatic cancer was comparable between early‐stage cancer and advanced‐stage cancer. Patients who underwent pancreatic resection for primary pancreatic cancer at the Department of Surgery and Oncology, Kyushu University, between 1992 and 2013 were retrospectively reviewed. Patients who underwent neoadjuvant therapy for pancreatic cancer and those who underwent total pancreatectomy were excluded. This study was focused on conventional pancreatic ductal adenocarcinoma (PDAC), PDAC derived from intraductal papillary mucinous neoplasm (IPMN), PDAC derived from intraductal tubulopapillary neoplasm, and PDAC derived from mucinous cystic neoplasm; acinar cell carcinomas were excluded. Cases of PDAC concomitant with IPMN were included in the study. Clinicopathological data including age, sex, tumor location, existence of concomitant IPMN, date of surgery, surgical procedures, pathological outcomes . Continuous variables were compared by the Student's t‐test or the Mann–Whitney U‐test. Categorical variables were compared by the chi‐square test or Fischer's exact test. The disease‐free interval was defined as the duration between the date of the initial operation and the date of diagnosis of extrapancreatic recurrence or remnant pancreatic cancer. Overall survival (OS) was defined as the duration between the date of initial surgery and the date of death or last follow‐up. The Kaplan–Meier method was used to estimate OS and the cumulative incidence of extrapancreatic recurrence or remnant pancreatic cancer, and the log‐rank test was used for comparisons. A two‐sided 3P < .0001). OS was also significantly longer in the early‐stage group than in the advanced‐stage group : four patients in the early‐stage group and 223 patients in the advanced‐stage group. Compared with the early‐stage group, the advanced‐stage group showed a higher cumulative incidence of extrapancreatic recurrence . Thirteen of the 19 patients underwent total pancreatectomy for remnant pancreatic cancer. In all 13 patients, the pathological diagnosis of the remnant pancreatic cancer was conventional PDAC. Our indication for surgical resection of remnant pancreatic cancer was the same as for primary pancreatic cancer, and the reasons for non‐surgical treatment were metastatic disease in two patients, locally advanced disease in one patient, advanced age (90 years old) in one patient, and patient refusal in two cases. The resection rates of remnant pancreatic cancer were comparable between the two groups . The prognosis after both the initial pancreatic resection and the diagnosis of remnant pancreatic cancer in the early‐stage group tended to be more favorable compared with the prognosis in the advanced‐stage group, although the difference was not significant : seven patients in the early‐stage group and 12 patients in the advanced‐stage group  the cumulative incidence of remnant pancreatic cancer was comparable between the early‐stage group and the advanced‐stage group, and (b) surgical resection of remnant pancreatic cancer was associated with favorable long‐term outcomes.P = .0011). The significantly smaller proportion of patients who developed remnant pancreatic cancer in the advanced‐stage group is likely attributable to shorter observation periods because of the lower rate of survival in the advanced‐stage group. In other words, if the two groups had shown similar survival rates and observation periods, the proportion of patients who would have developed remnant pancreatic cancer would have been comparable between the two groups. Another possible reason for the larger proportion of patients who developed remnant pancreatic cancer in the early‐stage group is the larger proportion of patients with cancer in the pancreatic body‐tail in the early‐stage group. Generally, pancreatic cancer occurs more frequently in the pancreatic head,The comparable cumulative incidence between the two groups suggested that the remnant pancreas of patients in the advanced‐stage group had the same risk of developing metachronous cancer as in the early‐stage group. It is reasonable to consider that the degree of progression of the initial pancreatic cancer does not influence cancer susceptibility in the remnant pancreas. However, the proportion of patients who developed remnant pancreatic cancer in the early‐stage group was similar to the proportions in previous studies regarding early‐stage pancreatic cancer,Two potential mechanisms underlying the development of remnant pancreatic cancer after partial pancreatic resection for pancreatic cancer have been proposed. One is the recurrence of the initial pancreatic cancer, and the other is the metachronous development of a new primary cancer.P = .968). Whether the stage of the initial cancer influences the prognosis of the remnant pancreatic cancer is unclear. Further analyses, such as a multicenter study or genetic analyses, are necessary to address this question.Although the difference was not statistically significant, the survival time after both the initial pancreatic resection and the diagnosis of remnant pancreatic cancer tended to be longer in the early‐stage group than in the advanced‐stage group. One possible explanation may be that the remnant pancreatic lesions in the advanced‐stage group included more recurrent lesions than those in the early‐stage group. If a remnant pancreatic lesion is due to recurrence, then the prognosis might be poorer than in the case of multifocal lesions.As with primary pancreatic cancer, the treatment options for remnant pancreatic cancer include surgical resection, chemotherapy, and chemoradiotherapy. Our results suggested that surgical resection provides a favorable outcome, if indicated. Suzuki et al analyzed 23 cases of remnant pancreatic cancer after resection of PDAC, and the results showed a significantly better prognosis for patients who underwent surgical resection compared with those who received non‐surgical treatment.The current study has several limitations. Because pathological findings were collected from pathology reports in this study, the status of pancreatic intraepithelial neoplasia (PanIN) in the background pancreas was not assessed. Matsuda et al reported that PDAC with concomitant IPMN was an independent risk factor for remnant pancreatic cancer and that concomitant PDAC and IPMN showed higher PanIN density in the background pancreas than PDAC without concomitant IPMN.In conclusion, this was the first study to analyze the incidence of remnant pancreatic cancer according to the degree of progression of the initial cancer. The potential for developing remnant pancreatic cancer was comparable between the early‐stage and the advanced‐stage groups. The lower incidence of remnant pancreatic cancer in the advanced‐stage group may be attributable to shorter observation periods secondary to the poor prognosis of this group. Therefore, we expect that the incidence of remnant pancreatic cancer will increase if long‐term outcomes of patients who undergo surgery for pancreatic cancer are improved owing to progress in multidisciplinary treatment. In the near future, long‐term surveillance after pancreatic resection for pancreatic cancer will be more important to detect remnant pancreatic cancer in a resectable state.Funding: This study was supported by Grants‐in‐Aid for Scientific Research .Conflict of Interest: The authors declare no conflict of interests related to this article.Ethical Approval: This study was approved by an institutional review board of Kyushu University.


An open label, multicenter 16‐week trial of cryopreserved human umbilical cord (TTAX01) was previously undertaken in 32 subjects presenting with a Wagner grade 3 or 4 diabetic foot ulcer, with 16 (50%) of these having confirmed closure following a median of one product application (previous study). All but two subjects  consented to participate in this follow‐up study to 1‐year postexposure. No restrictions were placed on treatments for open wounds. At 8‐week intervals, subjects were evaluated for adverse events (AEs) and wound status (open or closed). Average time from initial exposure to end of follow‐up was 378 days (range 343‐433), with 29 of 30 (97%) subjects completing a full year. AEs were all typical for the population under study, and none were attributed to prior exposure to TTAX01. One previously healed wound re‐opened, one previously unconfirmed closed wound remained healed, and nine new wound closures occurred, giving 25 of 29 (86.2%) healed in the ITT population. Three of the new closures followed the use of various tissue‐based products. Three subjects whose wounds were healed required subsequent minor amputations due to osteomyelitis, one of which progressed to a major amputation . One additional subject underwent two minor amputations prior to healing. Overall, the study found TTAX01 to be safe in long‐term follow‐up and associated with both a low rate of major amputation and a higher than expected rates of healing. Unlike acute cutaneous injuries in otherwise healthy individuals, diabetic foot ulcers (DFU) typically exhibit delayed healing, a downstream consequence of prolonged hyperglycemia, which triggers a wide range of molecular events leading to neural and vascular damage.The short‐term goals of treatment of DFU complicated by osteomyelitis, but not ischemia, are to remove devitalized bone and tissue, identify and treat infection, and achieve wound healing with the least disruption to anatomy. The longer term goals are maintainence of healing over a meaningful length of time, and preservation of the limb, each of which is challenging given historically high rates of ulcer recurrence and amputation.The present study was designed as an observational extension study from a previous interventional trialThe regulation of birth tissue products used in the treatment of DFU is undergoing a transition from the application of a combined set of rules  to the application of rules governing tissue based biological products.22.1This was a multicenter, noninterventional follow‐up study conducted at healthcare facilities in the United States (11 centers), where each center had previously enrolled at least one patient into a preceding interventional trial of TTAX01 for the treatment of Wagner 3‐4 DFU (NCT03230175).2.22), depth (cm), and volume (cm3). Wound status  was recorded at each visit. New wound closures were recorded based on a single observation of closure rather than the more conservative regulatory requirement of closure confirmation at two subsequent visits each 2 weeks apart, which was followed during the preceding treatment study. Subjects who achieved closure and remained closed at the final visit were considered to have healed their wound.Safety was evaluated by frequency, expectedness, and relationship of adverse events (AEs) calculated for each body system, by preferred terminology, by the preceding treatment, for number of subjects and percentage reporting the event. Wounds were evaluated using the eKare inSight measuring device  by capturing an image for electronic measurement via automatic tracing of area , a concordance of opinion between the Investigator and a single independent expert reviewer of the eKare images in cases where the Investigator reported the wound as healed , or the opinion of a second independent “tie breaker” reviewer in the event that the Investigator and first reviewer had discordant opinions. The primary efficacy calculation was performed for the modified ITT (mITT) population.AEs, including index wound AEs and systemic AEs, were coded for preferred terminologies using Medical Dictionary for Regulatory Activities (MedDRA). AE were grouped into pre‐enrollment and post‐enrollment categories based on the enrollment date and event start date. New and ongoing therapies for the index wound were reported.2.4All continuous data were expressed as mean ± SD (range), while categorical variables were expressed as frequency and percentages. The primary efficacy analysis was performed for the mITT population, which included all enrolled subjects having a wound  at baseline.3Demographics and baseline characteristics for all 30 subjects are summarized in Table Twenty‐four (80%) of 30 subjects experienced one or more AE during the study. All AEs were expected within the demographic although not necessarily expected for individual subjects. None of them was related to prior exposure to TTAX01. Nine subjects experienced one or more serious AE, defined as requiring hospitalization; none of these events was considered related to prior exposure to TTAX01. There were no deaths.Four subjects (13%) of 30 underwent one or more minor (below the ankle) amputations, with one of them (3.3%) eventually undergoing a major below the knee amputation. That subject was one of 17 who had undergone minor amputation at the initial baseline surgical resection in the previous trial, giving a rate of 1 of 17 (5.9%) for progression from minor to major amputation across both studies. Three subjects had healed their target ulcer prior to the amputations, while one had minor amputations unrelated to the target ulcer, which eventually healed. It was an intriguing observation that cutaneous healing was not always indicative of resolution of underlying infection, as seen in the cases where the wound healed but surgical removal of bone and tissue was later required due to osteomyelitis, which was either ongoing or recurrent.There were no disagreements between the Investigator and the expert reviewer regarding final wound status. A high proportion of subjects maintained or achieved persistent closure of their index wound by the end of this long‐term follow‐up study. One of the 16 previously confirmed closed wounds re‐opened. One wound that was previously coded as open, simply because of missing a confirmation of closure visit in the previous treatment study, enrolled into this study with the wound closed, giving 16 with closed wounds at the start of this follow‐up. Fifteen (93.8%) of these subjects experienced a persistent closure through 1 year from initial treatment. Across the two studies, 27 wounds achieved initial closure, one of which failed confirmation of closure and remained open, and one of which was confirmed closed but re‐opened during the observational period, giving 25 persistently closed wounds.Nine subjects who had not achieved confirmed healing in the prior study had a final evaluation of wound closure in this study Table . Among tAmong 20 with biopsy confirmed osteomyelitis at baseline in the previous study, 16 (80%) achieved confirmed closure, and 15 (75%) maintained persistent closure, as did 14 (87.5%) of 16 who had undergone a baseline minor amputation in the prior study and enrolled into this follow‐up. At the intersection of these subsets, 10 (90.9%) of 11 subjects with both confirmed osteomyelitis and a baseline minor amputation went on to achieve complete wound closure across the two studies.Among the nine who achieved closure in follow‐up, three required no advanced therapies, three were treated with silver‐based products in addition to standard care, and three were treated with tissue‐based products  before closing. One subject with a persistently open wound received negative pressure wound therapy, without achieving closure, while another received a different amniotic tissue product (Affinity) without success.4The findings in this multicenter, long‐term follow‐up study are encouraging. No safety signals were detected with respect to prior exposure to the human cryopreserved umbilical cord product (TTAX01), with AEs being unrelated and typical for the population under study.Not only were healing rates high in the previous and present follow‐up study, healing times among subjects with minor baseline amputations in the previous study were much shorter than those reported by Svensson et al, who saw a median time from primary amputation to wound healing of 26 weeks (range 2‐250).Published experience in treating DFU with a range of amnion, chorion and umbilical cord products has been universally positive, although the majority of studies have been with shallow Wagner 1 ulcers, which have no exposure of muscle, fascia, joint capsule or bone.A cryopreserved product showed superiority over standard care in published studies of Wagner 1 DFU, with efficacy over 12 weeks in the range of 62 to 71% in both prospective and retrospective studies.One trial of a dehydrated umbilical cord productWound recurrence rates have not been routinely reported. Publications where recurrence is mentioned give rates of 14%The patients who enrolled in the prior interventional study, TTCRNE‐1501, presented with high grade Wagner 3‐4 DFU which literature and database searches reveal to be particularly difficult to heal. The achievement of a very high rate of healing over the course of this and the previous trial is unexpected, and requires confirmation. Another unexpected finding was the discordance between the primary efficacy endpoint of a healed cutaneous wound, and the expected clinical benefit of a healed foot. Surprisingly, three subjects who achieved and maintained cutaneous healing ended up requiring various degrees of amputation during follow‐up as a result of recurrent or persistent osteomyelitis.It is important to acknowledge that the previous interventional study was open label without concurrent control, and that additional therapies during this follow‐up were unrestricted. In particular, the benefit of prior TTAX01 treatment is unknown in the three subjects who healed following use of a tissue‐based product during this observational study. Consequently, it remains to be seen whether the use of TTAX01 in treating high grade, complex DFU may be superior to standard care when studied in a multicenter randomized trial.Available evidence suggests that standard care alone is unlikely to produce the healing rates seen in this study. In a series of cases of diabetic foot osteomyelitis managed with limb‐sparing surgery, Johnson et al reported that 59% had healed by 1 year,The results of this long‐term follow‐up study continue to be encouraging given the unmet medical need of a treatment for foot wounds complicated with osteomyelitis, in a diabetic population exhibiting high morbidity and mortality. We believe that the healing outcomes and favorable safety data generated in this initial study support proceeding to a prospective multicenter randomized study of TTAX01 in treating Wagner grade 3‐4 wounds.


Dear Editors:Pneumocystis pneumonia (PCP) that were initially managed as suspect cases of COVID‐19, and in whom HIV was not initially considered.As the COVID‐19 pandemic continues to spread [The first case is of a 62‐year‐old male with a history of stage III undifferentiated nasopharyngeal carcinoma and hypothyroidism. He reported no recent travel outside of Singapore in the previous month and no contact with confirmed COVID‐19 cases. He presented with complaints of shortness of breath and diarrhoea for two weeks with generalized lethargy for one month. On admission, he required supplemental oxygen via nasal cannula at 2 L per minute, but his vital signs were otherwise stable.The second case is of a 20‐year‐old female who recently returned from the United Kingdom (UK). She has a history of childhood asthma for which she was not on treatment. She reported cough of three months duration prior to admission with intermittent fever and shortness of breath. However, she developed persistent fever and worsening breathlessness one week prior to admission. Her vital signs were stable on admission.Both cases were admitted and managed with the provisional diagnosis of COVID‐19 infection, and followed very similar courses throughout their stay. Both had lymphopenia and raised lactate dehydrogenase (LDH) levels with radiological features suggestive of COVID‐19 pneumonia. In view of this, both patients had three sets of SARS‐CoV‐2 real‐time polymerase chain reaction performed on nasopharyngeal swab samples 24 hours apart, all of which returned negative. Both patients subsequently deteriorated on the third inpatient day, requiring increasing supplemental oxygen.A computed tomography (CT) of the chest for the first case showed widespread patchy ground glass changes seen in both lungs with upper lobe predominance and peripheral consolidation in the right lower lobe. The CT chest scan of the second case showed extensive ground glass opacities in both lungs with multi‐lobar involvement, worst in the perihilar regions, sparring the subpleural regions and the lung bases.On focused re‐examination, both cases were found to have oral thrush. An HIV antigen‐antibody screening test was then performed for both patients, and both returned positive. On the basis of their clinical and radiologic findings, both patients were started on trimethoprim‐sulfamethoxazole (TMP‐SMX) with prednisolone for severe presumptive PCP. Both patients improved dramatically and were subsequently discharged after 16 days for case 1 and 18 days for case 2 with plans for outpatient follow up for HIV care.PCP and COVID‐19 share numerous characteristics. Both diseases present with fever, fatigue, dry cough and dyspnoea , 3. In tHowever, PCP generally follows a more subacute course than COVID‐19, particularly when considering milder forms of the disease, with 33% of patients having symptoms for at least two to four weeks prior to presentation in a case series from early in the HIV epidemic , 8. DyspAlthough the similarities between the two diseases might explain why the diagnosis of PCP was initially missed, there are also other reasons for the delay in HIV diagnosis in these two patients. Firstly, PCP with HIV infection is less commonly seen in settings with access to antiretroviral therapy (ART), with the majority of cases previously occurring in young children within the first year of life and male adults above the age of 50 years in the pre‐ART era , 19. CasIt is understandable that the first differential for a patient with respiratory symptoms is COVID‐19 during this outbreak. However, there are other respiratory pathogens which may mimic the presentation of COVID‐19. As illustrated in this letter, PCP shares numerous characteristics with COVID‐19, although there are some key differences. We hope to highlight the importance of considering this diagnosis in individuals presenting with suggestive symptoms, so that cases of potential HIV infection are not missed during this outbreak.All authors have no conflict of interest to declare.CYC and CSW contributed to the design of the manuscript. CYC drafted the manuscript with input from CSW. All authors have read and approved the final manuscript.


The relationship between biodiversity and productivity has stimulated an increasing body of research over the past decades, and this topic still occupies a central place in ecology. While most studies have focused on biomass production in quadrats or plots, few have investigated the scale‐dependent relationship from an individual plant perspective. We present an analysis of the effects of biodiversity  on individual tree growth with a data set of 16,060 growth records from a 30‐ha temperate forest plot using spatially explicit individual tree‐based methods. A significant relationship between species diversity and tree growth was found at the individual tree level in our study. The magnitude and direction of biodiversity effects varies with the spatial scale. We found positive effects of species diversity on tree growth at scales exceeding 9 m. Individual tree growth rates increased when there was a greater diversity of species in the neighborhood of the focal tree, which provides evidence of a niche complementarity effect. At small scales (3–5 m), species diversity had negative effects on tree growth, suggesting that competition is more prevalent than complementarity or facilitation in these close neighborhoods. The results also revealed many confounding factors which influence tree growth, such as elevation and available sun light. We conclude that the use of individual tree‐based methods may lead to a better understanding of the biodiversity‐productivity relationship in forest communities. We present an analysis of the effects of biodiversity  on individual tree growth with a data set of 16,060 growth records from a 30‐ha temperate forest plot using spatially explicit individual tree‐based methods. A significant relationship between species diversity and tree growth was found at the individual tree level in our study. The magnitude and direction of biodiversity effects varies with the spatial scale. The goodness of fit of the allometric model was evaluated using the coefficient of determination (R2). The significance of coefficients was calculated for each regression. The fit was evaluated by analyzing the residuals and using root mean square error (RMSE).All woody plants with a DBH larger than 5 cm in the first census were included in this study. The aboveground biomass (AGB) of each tree was estimated using existing allometric regression equations based on the measurement of tree diameter. A logarithmic model was used to fit allometric relationships between the aboveground woody biomass (AGB) and tree DBH  which incorporates species richness and evenness was calculated as follows:ni is the number of individuals of species i and N is the number of all neighbors within a chosen circle around the focal tree.Species diversity was calculated using species richness and Shannon index within the neighborhood of a variety of radii  for each focal tree. Only tree and shrub species were included. Species richness (SR) represents the number of species in the neighborhood. The Shannon index , and specific leaf area (leaf area/dry matter) were obtained. Leaf carbon and nitrogen concentrations were assessed using an elemental analyzer . Leaf carbon‐nitrogen ratios were calculated by dividing the leaf carbon concentrations by the leaf nitrogen concentrations , a wood trait (wood density), and five leaf traits . We measured maximum height using an altimeter pole and a laser telemeter . For each species, wood and leaf traits were collected from 10 to 30 individuals. Wood cores were extracted from the cortex to the pith at 1.3 m height using an increment borer  to determine the wood density, by dividing the wood core dry weight  by its fresh volume. Leaf traits were measured on individuals with DBH between 10 and 20 cm. We took five fresh leaf samples on the highest parts of the tree crown from each individual. Following the standard methods proposed by Cornelissen et al. , we scanns Table .To calculate the functional diversity for multiple traits, we followed the concept of functional trait space based on a geometrical point of view  is a function of the control variables selected from abiotic variables, competition, and neighborhood size structure covariates, and β is a vector of coefficients. The elasticity of substitution θ represents the degree to which species can substitute each other in contributing to productivity  radii (with 1 m increment) for the neighborhood circle around the focal tree. We fitted the models using “lmer” function in the R package “lme4”  kg/yr. The individual tree productivity was relatively low for shrub species, such as Euonymus phellomanus and Acer barbinerve.A total of 16,060 individual trees were alive, both in 2010 and 2015. Comparison of the productivity between species showed that individual tree growth rates varied significantly among species in terms of AGB. The canopy species The mean number of species within the neighborhood radius from 1 m to 20 m for each focal tree ranged from 0.2 to 13.6. The mean value of Shannon index increased with the scale, from 0.1 to 2.14. The functional diversity values were also different among spatial scales: Functional dispersion (FDis) increased with increasing scale at first  was nonsignificant at all scales . However, this was not the case for the functional diversity model . The interaction between competition and functional diversity did not show up in the best‐fit functional diversity model  and Norway spruce (Picea abies), which revealed a positive effect of local neighborhood mixing on biomass production  parameter described in the methods section. An alternative explanation for the lack of a positive effect of functional diversity may be that the trait dataset that we used might not reflect niche complementarity adequately. It seems necessary to consider additional traits in future investigations, such as photosynthetic capacity, and crown plasticity.We did not find a strong positive effect of functional diversity on tree growth in this study, which is in line with other studies in tropical , species diversity had negative effects on tree growth. This result was not surprising because at this scale, competitive exclusion may be the most likely result of species interactions. In mixed‐species forests, competition is a complex phenomenon and an important source of variation in individual tree growth Gadow, . We detePinus koraiensis and Tilia amurensis, see Ni et al., The significant interaction between species diversity and competition index provides further evidence of the necessity to consider competition when analyzing biodiversity‐productivity relationships. Fichtner et al.  claimed Positive (facilitation) and negative (competition) interactions often occur simultaneously. Net biodiversity effect is the combined result of facilitation and competition interactions in response to neighborhood biodiversity ; Formal analysis (lead); Investigation ; Methodology (lead); Software (lead); Writing‐original draft (lead). Lingzhao Tan: Software (supporting). Chunyu Zhang: Funding acquisition ; Methodology . Xiuhai Zhao: Conceptualization ; Funding acquisition . Lushuang Gao: Funding acquisition ; Investigation . Klaus von Gadow: Writing‐original draft (supporting).Appendix S1Click here for additional data file.


Drought causes important declines in crop yield, negatively impacting plant growth, physiology and reproduction. Plants have adopted a wide range of strategies to confront the negative effects of drought, including increasing water uptake by optimizing the root system, closing stomata to limit the water loss caused by transpiration, accumulating osmoprotectants, or producing the hormone abscisic acid (ABA). This intricate scenario makes drought resistance a highly complex trait, with polygenic nature, low heritability, and vastly influenced by genotype‐environment interactions  is a main causal agent of Tomato yellow leaf curl disease (TYLCD), one of the most devastating viral diseases affecting tomato crops in tropical and temperate areas worldwide. Interestingly, infection by TYLCV has been shown to alleviate heat stress responses in tomato  from a 35S promoter to drought treatment and evaluated their performance. Constitutive expression of Rep renders plants non‐viable; hence, this protein was not included in this study. Drought did not affect expression of the transgenes (data not shown). As shown in Figure et al., G2A, which accumulates exclusively in chloroplasts  promoter but display xylem patterning defects similar to those observed in 35S:C4 plants  evaluating the ability of 35S:C4 and G2A35S:C4 plants to respond to exogenously applied ABA, compared with the wild‐type control. Interestingly, C4‐expressing plants did not differ from wild‐type plants in any of the readouts measured  quantifying ABA accumulation and (ii) measuring the expression of ABA‐responsive genes in basal and drought conditions in wild‐type, et al., Considering the current prospects of growth in world population, increasing crop yield is a cornerstone to guarantee food security. In a context of climate change, boosting agricultural productivity will necessarily entail generating not only high‐yielding, but also stress‐resistant crops. While traditional breeding has provided valuable solutions in the past, the current scenario makes it essential to explore alternative approaches, which may allow to narrow the temporal gap between identification of a beneficial trait and its implementation in the field. The study of extremophile species has been suggested as having the potential to unlock novel sources of resistance  signalling (Aguilar In summary, our findings demonstrate that C4 from TYLCV increases plant drought tolerance in an ABA‐independent manner through a mechanism that relies on the presence of this protein at the plasma membrane. Considering the dramatic increase in survival upon water withholding mediated by C4 and the fact that this effect is independent of well‐known hormonal pathways linked to drought stress, we believe this discovery to entail great biotechnological potential. Future efforts will focus on unravelling the molecular events underpinning the C4‐triggered enhanced tolerance to limited water availability: the identification of the viral drought tolerance‐promoting strategy could open up new avenues to explore in the engineering of the highly sought‐after drought‐tolerant crops.ERB, AGC, and RL‐D conceived and supervised the project; MC‐G, LM‐P, and YY generated and analyzed data; LP and XD generated materials; APL, ACG, and RL‐D wrote the manuscript with input from all the authors.The authors declare no conflict of interest.


Surgery can lead to significant muscle loss, which increases recovery time and associates with increased mortality. Muscle loss is not uniform, with some patients losing significant muscle mass and others losing relatively little, and is likely to be accompanied by marked changes in circulating metabolites and proteins. Determining these changes may help understand the variability and identify novel therapeutic approaches or markers of muscle wasting.To determine the association between muscle loss and circulating metabolites, we studied 20 male patients  undergoing aortic surgery. Muscle mass was determined before and 7 days after surgery and blood samples were taken before surgery, and 1, 3, and 7 days after surgery. The circulating metabolome and proteome were determined using commercial services (Metabolon and SomaLogic).CSA) and were defined as wasting. Metabolomic analysis showed that 557 circulating metabolites were altered following surgery (q < 0.05) in the whole cohort and 104 differed between wasting and non‐wasting patients (q < 0.05). Weighted genome co‐expression network analysis, identified clusters of metabolites, both before and after surgery, that associated with muscle mass and function . These clusters were mainly composed of acyl carnitines and dicarboxylates indicating that pre‐existing mitochondrial dysfunction contributes to muscle loss following surgery. Surgery elevated cortisol to the same extent in wasting and non‐wasting patients, but the cortisol:cortisone ratio was higher in the wasting patients (Day 3 P = 0.043 and Day 7 P = 0.016). Wasting patients also showed a greater increase in circulating nucleotides 3 days after surgery. Comparison of the metabolome with inflammatory markers identified by SOMAscan® showed that pre‐surgical mitochondrial dysfunction was associated with growth differentiation factor 15 (GDF‐15)  and that GDF‐15, interleukin (IL)‐8), C‐C motif chemokine 23 (CCL‐23), and IL‐15 receptor subunit alpha (IL‐15RA) contributed to metabolic changes in response to surgery.Ten patients lost more than 10% of the cross‐sectional area of the rectus femoris (RFWe show that pre‐existing mitochondrial dysfunction and reduced cortisol inactivation contribute to muscle loss following surgery. The data also implicate GDF‐15 and IL‐15RA in mitochondrial dysfunction. Both chronic disease and acute injury lead to a loss of muscle mass that impacts both quantity and quality of life. It has been suggested that muscle constitutes a reservoir for amino acids, as both a source of building blocks and energy for the inflammatory response where repair is needed in non‐muscle tissue . In a model of muscle wasting after aortic surgery, we showed that patients who wasted had higher levels of GDF‐15 and lower levels of insulin‐like growth factor 1 (IGF‐1) in their circulation over the 7 days following surgery.CSA)] with those who did not waste significantly using an unbiased approach. Our data set also included significant physiological data allowing us to use the statistical methods employed in the R package weighted genome co‐expression network analysis (WGCNA) to identify clusters of metabolites and their association with physiology and with the levels of GDF‐15 measured in the circulation. Whilst this approach was developed for the unbiased identification of clusters of co‐expressed genes that form networks,It seems likely that the circulating metabolome of patients who waste significantly will differ from those who do not. Analysis of these differences may highlight both potential biomarkers of wasting as well as offer mechanistic insight into muscle loss. Consequently, in this study, we analysed the metabolic profile of patients undergoing aortic surgery in the model we have previously published,et al.CSA between Days 0 and 7 after surgery, determined by ultrasound as previously described.CSA, which we have previously shown is a robust detectable difference in RFCSA determined by ultrasound.FigureThe patients included in this study are a subset from a previously reported cohort.Blood samples were taken before the induction of anaesthesia on the day of surgery (Day 0) and on Days 1, 3, and 7 after surgery into EDTA tubes, and plasma was prepared and stored at −80°C until required. The plasma samples were sent to Metabolon for metabolomic analysis and to SomaLogic for the SOMAscan® assay of 1300 proteins. All samples passed the appropriate quality control measurements.GDF‐15 was quantified separately by enzyme linked immunosorbent assay (ELISA) according to the manufacturer's instructions (R and D Systems).https://www.metaboanalyst.ca/). Missing values were imputed by replacement with the minimum positive value for the metabolite then log transformed. Principle component analysis (PCA) was performed (Aabel™ 3.0 Gigawiz Ltd) and one sample shown to be an outlier. This sample was removed and the PCA repeated.Raw counts were taken from the Metabolon output and entered into MetaboAnalyst 4.0 , the data for each day from all patients except the outlier removed from the PCA were analysed using the R package WGCNA separately. Correlation coefficients between both modules and individual metabolites with physiological parameters were determined using robust biweight‐midcorrelation.CSA by ultrasound allowing us to divide patients into wasting and non‐wasting groups.CSA and 10 patients who did not lose a significant amount of muscle. The demographics and surgical parameters of this set of patients are shown in Table TableFigureWe have previously shown that we can reproducibly detect a loss of 10% RFFigurePrincipal component analysis readily separated samples from the Days 0 and 1 with samples from Days 3 and 7 forming a large third group for the bulk of the data. However, one patient was identified as a significant outlier in the data set with their Days 0 and 1 samples associating more closely with the Day 3/7 clusters than the Days 0 and 1 cluster. Subsequently, it was confirmed that this patient required renal replacement therapy and so their samples were removed from the data set. PCA of the remaining samples showed a similar pattern with three major groups  with time and 104 were different between the wasting and non‐wasting patients with 69 of these metabolites common to both data sets  whereas although the tetrahydrocortisone:cortisone ratio was higher in wasting patients than non‐wasting patients on Days 1 and 3, these differences did not reach statistical significance  and the acyl carnitine derivatives of fatty acid dicarboxylates of all chain lengths. However, with the exception of octadecanedioyl carnitine all the long‐chain acyl carnitines (C ≥ 14) were reduced on the first day after surgery ( and the TablesTablesTableAmino acids were the class of the metabolite that showed the most significant difference in profile between wasting and non‐wasting patients Tables and S2. CSA), RFCSA loss, strength (hand‐grip), strength loss, and GDF‐15 level]. WGCNA grouped the metabolites into 22 clusters on Day 0, 19 clusters on Day 1, 12 clusters on Day 3, and 12 clusters on Day 7  had the strongest associations with muscle mass and function and with circulating GDF‐15 , and cyan0 most tightly associated with circulating GDF‐15 .Comparison of the clusters present on Day 0 with physiology and pre‐surgical GDF‐15 levels indicated that two clusters  was strongly associated with the loss of hand‐grip strength ; again, this module was enriched for amino acids, nucleotides, and their metabolites, and GDF‐15 was associated with circulating levels of these types of metabolites , which was enriched for amino acids and nucleotides , C‐C motif chemokine 23 (CCL‐23) , and IL‐15 receptor subunit alpha (IL‐15RA) , whereas interleukin‐5 (IL‐5) had a negative association with the same module . Together, these data suggest that a range of inflammatory‐signalling factors may contribute to increased muscle loss in this condition either directly stimulating muscle loss, promoting the release of factors that promote muscle wasting, or promoting the recruitment of inflammatory cells to promote muscle catabolism in response to surgery. However, in the Day 0 samples, GDF‐15 showed the strongest association with the turquoises0  from which we obtained the metabolic data was part of a larger study investigating the circulating proteome response to surgery using SOMAscan® that we will report elsewhere. However, as inflammation forms a significant part of the response to surgery, we investigated whether any inflammatory markers from the CC or CXC chemokine families identified in the analysis were stronger associates with the circulating metabolome on Day 1 after surgery. Consistent with our analysis of GDF‐15 by ELISA, GDF‐15 was strongly associated with the turquoisees Table. Severale Figure; these i0 Figure module, Our analysis identifies both general trends in the metabolic response of individuals to surgery  as well as particular differences associated with either the loss of muscle mass or function. Together, they provide a picture where there is a significant loss of lipids from the circulation, and that differences in cortisol metabolism and mitochondrial function prior to surgery contribute to the differences in the muscle response of individuals to this insult. We will deal briefly with the general consequences and then focus on changes associated with changes in muscle mass and function as these were the focus of this study.There are several likely drivers of changes in metabolites that will occur in all patients. The first is the potential for an early starvation response as the patients starved prior to surgery (mostly overnight) and remain starved for a significant period after the operation. The second is a direct response in the level of metabolites released from damaged tissue. Third, there is the potential for the effects of the range of pharmacological agents used to anaesthetise and stabilize the physiology of the patients during surgery as well as for post‐surgical analgesia. Finally, there are potential effects of the cardiopulmonary bypass process itself, including inflammation, the ischemia‐reperfusion response that will arise from the application and subsequent release of the aortic cross clamp and any interactions of the blood with oxygenator membranes and other exogenous surfaces. These factors will drive the early changes in metabolome and will contribute to the later metabolic profile as will the inactivity and other pharmacological interventions required by the period of time spent in the ICU and high‐dependency unit. Other than the presence of drugs in the circulation, the most striking general trend is a reduction in the levels of many circulating lipids including lysophospholipids and NEFA. The early response of mammals to starvation is characterized by glycogenolysis and gluconeogenesis, with lipids released from stores increasing plasma NEFA. This response was demonstrated in a recent metabolomic study of starvation in healthy human subjects that showed a marked increase in circulating NEFA in the first 2 days of starvation.The metabolic changes that differentiate wasting and non‐wasting patients can be classified into those that identify potential biomarkers of wasting and those that provide mechanistic information.The metabolites that are potential circulating biomarkers of a wasting process include amino acids and nucleotides that most likely represent the breakdown of muscle tissue and loss of ATP. Consistent with these molecules representing increased muscle breakdown, rather than reflecting tissue damage directly caused by the surgery, they were increased following surgery to a greater extent in the patients who wasted than in those who did not. Prominent in the amino acids were the methyl derivatives of histidine, consistent with the use of 3‐methyl‐histidine as a marker of elevated muscle breakdown. Furthermore, circulating creatinine was higher in wasting patients than in those who did not waste. Similarly, there is evidence of increased plasma purines and other nucleotide derivatives. The increased purines  are often used as markers of oxidative stress.Two sets of metabolites that differed between wasting and non‐wasting patients perhaps provide some mechanistic information with one set suggesting differences in cortisol metabolism and the other indicating a role for mitochondrial dysfunction in muscle wasting.Unsurprisingly, in all patients, there was a marked increase in circulating concentrations of cortisol following surgery. Circulating cortisol did not differ between wasting and non‐wasting patients, but there was no significant increase in cortisone in the plasma of patients who wasted whereas cortisone increased markedly in non‐wasting patients. Cortisol is a potent signal for muscle wasting,Two of the major sets of metabolites (fatty acid dicarboxylates and the acyl carnitine derivatives) were (i) elevated by surgery and (ii) associated with reduced muscle mass and loss of strength. Conversion of fatty acids into their acetyl carnitine derivatives allow them to be transported into mitochondria for β‐oxidation. As the generation of the acetyl carnitine derivatives is not rate limiting in the oxidation of fatty acids and occurs outside mitochondria, acyl carnitines can accumulate in cells with reduced mitochondrial activity and from there enter the circulation. Consequently, increased circulating acyl‐carnitine levels have been used as a marker of mitochondrial dysfunction.et al. showed a greater reduction in muscle ATP levels and loss of mitochondrial function in patients with significant co‐morbidities prior to the period of critical illness and these patients lost more muscle than those with better preserved mitochondrial function.CO) in COPD patients provides a potential mechanism by which the primary disease may promote muscle mitochondrial dysfunction to reduce energy usage in the muscle.The observation that mitochondrial dysfunction is associated with an increased potential for muscle loss in patients with critical illness is consistent with a number of other studies both in animals and in man. In mouse models of sepsis, there is a marked reduction in mitochondrial mass and function as demonstrated by a reduction in metabolic rate.Comparing the circulating proteome with the metabolome both before and after surgery suggests potential regulators of the changes in metabolism. This analysis suggests a role for both GDF‐15, IL‐15, and IL‐8 in the metabolic response to surgery. GDF‐15 is an anorexogenic hormone that inhibits appetite by activating the GFRAL receptor in the hippocampus.Our study identifies significant changes in the metabolism of patients following aortic surgery some of which appear to distinguish patients who lose muscle from those who do not. Furthermore, it suggests that mitochondrial dysfunction and differential cortisol metabolism are important components of this process. However, the study is correlative in nature and although it is supported by other studies, this study does not establish causation. Our model of muscle loss associated with critical illness is specific to the patient group we have investigated who spend a relatively short time in intensive care. Extrapolation of our findings to a wider critical illness population therefore requires significant care.et al. showed that loss of ATP was a critical component of muscle loss and that this preferentially occurred in patients with pre‐existing chronic disease.The are many factors that lead to muscle loss on the ICU, added to which patients are in a critical condition and have probably started to lose muscle mass prior to their entry to the unit, making quantifying absolute muscle loss difficult. The extent of muscle loss is likely to be dependent on the cause of critical illness , the severity of the illness, duration of inactivity, and associated pharmacotherapy. For example, differences in the size or type of the event can be seen in the greater loss of strength in patients that have an open rather than laparoscopic cholecystectomy.In conclusion, our data indicate that in response to surgery, there is a complex metabolic response with marked proteolysis leading to an increase in free amino acids and nucleotides that are detectable in the circulation. The extent of loss of muscle is associated with this increase in free amino acids. More importantly, our data suggest that differences in cortisol metabolism and in pre‐surgical mitochondrial function are important factors in determining the extent of muscle loss in response to this inflammatory insult.This work was supported by GSK and the National Institute for Health Research (NIHR) Respiratory Disease Biomedical Research Unit at the Royal Brompton and Harefield NHS Foundation Trust and Imperial College London who wholly funded RP.P.K. reports personal fees from GSK, outside the submitted work. M.G. reports grants, personal fees, and non‐financial support from GSK, personal fees from BI, personal fees from Silence therapeutics, personal fees from Cell catapult, outside the submitted work; all other authors have no conflicts of interest.Each clinical study was approved by the appropriate ethics committee. The appropriate study number is 07/Q0204/68. Written informed consent was obtained from all participants in the studies.The overall study was designed by P.K., A.R., and M.G. Patients were recruited and samples were collected by R.P. The laboratory studies were performed by R.P. P.K. wrote the first draft of the paper, and all authors provided critical appraisal and input into the manuscript.Figure S1. Flow diagram showing the selection of samples from the entire cohortFigure S2. The effect of surgery on GDF‐15 in the circulation of wasting and non‐wasting patients.Figure S3. Whole genome co‐expression network analysis (WGCNA) of plasma metabolites before surgery.Figure S4. Whole genome co‐expression network analysis (WGCNA) of plasma metabolites 1 day after surgery.Figure S5. Whole genome co‐expression network analysis (WGCNA) of plasma metabolites 3 days after surgery.Figure S6. Whole genome co‐expression network analysis (WGCNA) of plasma metabolites 7 days after surgery.Figure S7. Whole genome co‐expression network analysis (WGCNA) of plasma metabolites 1 day after surgery compared to plasma cytokines on the same day and their receptors.Figure S8. Whole genome co‐expression network analysis (WGCNA) of plasma metabolites before surgery compared to plasma cytokines on the same day and their receptors.Click here for additional data file.Data S1. Excel file containing the following tables:Table S1. Metabolites that differ (p<0.05) with time identified by 2‐way ANOVATable S2. Metabolites that differ (p<0.05) with phenotype identified by 2‐way ANOVATable S3. Metabolites in light green and cyan clusters day 0Table S4. Metabolites in plasma before surgery that associate with RFCSA before surgeryTable S5. Metabolites in plasma before surgery that associate with GDF‐15 before surgeryTable S6. Metabolites in the red and turquoise modules on day 1Table S7. Metabolites in the turquoise modules on day 3Table S8. Metabolites correlated with GDF‐15 on day 3Table S9. Metabolites correlated with GDF‐15 on day 7Table S10. Metabolites in the turquoise module on day 1 in the subset of samples analysed by SOMAscan® assayTable S11. Metabolites in the turquoise module on day 0 in the subset of samples analysed by SOMAscan® assayClick here for additional data file.Data S2. Normalised metabolite levels for all samplesClick here for additional data file.


Cities are uniquely complex systems regulated by interactions and feedbacks between nature and human society. Characteristics of human society—including culture, economics, technology and politics—underlie social patterns and activity, creating a heterogeneous environment that can influence and be influenced by both ecological and evolutionary processes. Increasing research on urban ecology and evolutionary biology has coincided with growing interest in eco‐evolutionary dynamics, which encompasses the interactions and reciprocal feedbacks between evolution and ecology. Research on both urban evolutionary biology and eco‐evolutionary dynamics frequently focuses on contemporary evolution of species that have potentially substantial ecological—and even social—significance. Still, little work fully integrates urban evolutionary biology and eco‐evolutionary dynamics, and rarely do researchers in either of these fields fully consider the role of human social patterns and processes. Because cities are fundamentally regulated by human activities, are inherently interconnected and are frequently undergoing social and economic transformation, they represent an opportunity for ecologists and evolutionary biologists to study urban “socio‐eco‐evolutionary dynamics.” Through this new framework, we encourage researchers of urban ecology and evolution to fully integrate human social drivers and feedbacks to increase understanding and conservation of ecosystems, their functions and their contributions to people within and outside cities. Researchers recognize CHANS by explicitly acknowledging linked reciprocal interactions between humans and nature—often characterized by flows of material, energy, and information  and other animals. Today, evidence suggests that some of the pigmentation patterns originally favoured by fancy pigeon breeders confer an adaptive advantage for urban pigeons , which have undergone thousands of years of artificial selection, still commonly interbreed with wild coyote , which became more cryptic and less subject to predation  from four cities have convergently evolved novel adaptations which confer resistance to toxins in response to pollution in urban estuaries  has shown repeated phenotypic convergence in the loss of cyanogenesis in response to urbanization  Rattus norvegicus) have coinhabited with humans for centuries by exploiting food and built structures  Aedes aeqypti and Culpex pipiens) are ubiquitous across the globe and are prominent vectors for human disease  and human resistance to those diseases  Trifolium repens), a perennial, herbaceous plant common in lawns and other human‐modified habitats, has been well studied in an urban context. Clover exhibits a Mendelian polymorphism for hydrogen cyanide production (cyanogenesis), which both defends against herbivores and reduces freezing tolerance. White clover repeatedly evolves decreased cyanogenesis in cities, due to putative selection from colder night‐time winter temperatures  Daphnia are common zooplankton species in urban, rural and natural freshwater ponds and lakes across the globe. They vary in several intraspecific life‐history, behavioural, and physiological traits that can elicit strong ecosystem‐level effects. D. magna show reduced body size, higher heat tolerance, faster pace of life, and altered stress physiology in urban populations compared to rural populations, which are most likely adaptations to warmer temperatures  and disservices  that consider urbanization as a continuous gradient  in low‐income neighbourhoods tend to have larger wing and body sizes—traits linked to increased fecundity, survival and ultimately spread of disease  that sustain resource flows within and across cities . The better fit of the simple model suggests that additional unmeasured variables account for the differences in life‐history along urban–rural gradients, and thus many ecological, social and evolutionary factors likely need to be included to accurately predict traits changes associated with urbanization  will require understanding the role of evolution in socio‐ecological systems over various timescales.A complete understanding of urban eco‐evolutionary dynamics will require an explicit consideration of social patterns and processes.The world is increasingly urbanized and the effects of cities extend beyond their borders. Hence, understanding ecological responses to global change will depend on our ability to address #1 & 2.Urban ecosystems are fundamentally regulated, transformed and interconnected by human activity. Thus, integrating human social patterns and processes in urban evolution studies not only presents an opportunity for novel research, but is also imperative for accurately understanding contemporary ecological and evolutionary dynamics in cities. As we move forward, we argue that more fully integrating evolutionary ecology research with the social sciences to address socio‐eco‐evolutionary questions is critical because:Studies of cities as coupled human and natural systems (CHANS) and of eco‐evolutionary dynamics have already provided insights into how urban ecosystems are likely to change over time. We now have the opportunity to leverage these existing bodies of work to create an integrative framework that more fully resembles the simultaneous social, ecological and evolutionary dynamics in urban ecosystems. We encourage a new collaboration among social scientists, ecologists and evolutionary biologists to develop more sophisticated questions and increasingly accurate models of urban systems, and garner a greater understanding of dynamics both within and beyond city boundaries. Understanding urban evolutionary biology will have vast implications for socio‐ecological policies such as those relating to biodiversity management and ecological restoration as well as human health, well‐being and equity. Additionally, we suggest specific, important and timely questions that can be addressed with an integrated socio‐eco‐evolutionary framework (Questions: Box 4).BOX 4Integrating insights from social sciences, ecology and evolutionary biology can help us address critical questions about urban systems. This understanding will feed back to improve our knowledge and predictions about how ecosystems respond to global change. Here, we propose ten key questions to inform an integrated socio‐eco‐evolutionary framework.1. How can incorporating methods from the social sciences improve our understanding of eco‐evolutionary dynamics?2. How do socio‐eco‐evolutionary dynamics scale with the spatial redistribution and generation lengths of humans and associated organisms across space and time?3. What is the relevance and magnitude of evolutionary feedbacks to ecological and social patterns and processes in different urban contexts?4. Can we predict the ways that interspecific interactions will influence eco‐evolutionary dynamics in cities and the ways in which social drivers will modify these dynamics and patterns?5. How important are local dynamics and species identities to eco‐evolutionary dynamics in cities? What are the components of a cohesive theory that is relevant to all or most urban systems, and when do local ecology, culture and politics idiosyncratically shape outcomes?6. How can eco‐evolutionary dynamics feed back to influence social processes in cities? In what ways can social systems change in response to evolutionary changes that are induced by urbanization?7. How can this multidimensional framework help us better understand the resilience of urban ecosystems to pulse disturbances, such as extreme weather events, and ramping disturbances, such as climate change?8. What elements of human social constructs  are likely to impact socio‐eco‐evolutionary dynamics?9. Under what circumstances are eco‐evolutionary processes stronger or weaker in urban compared to nonurban areas?10. How do socio‐eco‐evolutionary changes in cities affect the influences of cities on surrounding landscapes?Cities provide exciting systems to expand our knowledge of eco‐evolutionary dynamics and their social causes and consequences. Studying the social dimensions of eco‐evolutionary dynamics in cities will improve our understanding of the complexity of urban biological communities, which will be increasingly crucial for conserving and maximizing ecosystem functions and contributions to people within and outside cities. Research on urban socio‐eco‐evolutionary dynamics provides a unique opportunity to study evolving metacommunities, the interplay between local and regional responses, and the presence and strength of eco‐evolutionary feedbacks across multiple taxonomic groups. Just as urban ecology grew to consider the social complexity of cities and eco‐evolutionary dynamics integrated the rapid pace of evolution, socio‐eco‐evolutionary research must recognize the dynamism resulting from the interplay of social, ecological and evolutionary dimensions within urban systems.None declared.


OrbiSIMS is a recently developed instrument for label‐free imaging of chemicals with micron spatial resolution and high mass resolution. We report a cryogenic workflow for OrbiSIMS (Cryo‐OrbiSIMS) that improves chemical detection of lipids and other biomolecules in tissues. Cryo‐OrbiSIMS boosts ionization yield and decreases ion‐beam induced fragmentation, greatly improving the detection of biomolecules such as triacylglycerides. It also increases chemical coverage to include molecules with intermediate or high vapor pressures, such as free fatty acids and semi‐volatile organic compounds (SVOCs). We find that Cryo‐OrbiSIMS reveals the hitherto unknown localization patterns of SVOCs with high spatial and chemical resolution in diverse plant, animal, and human tissues. We also show that Cryo‐OrbiSIMS can be combined with genetic analysis to identify enzymes regulating SVOC metabolism. Cryo‐OrbiSIMS is applicable to high resolution imaging of a wide variety of non‐volatile and semi‐volatile molecules across many areas of biomedicine. Cryogenic OrbiSIMS decreases molecular fragmentation and expands the chemical space that is amenable to mass spectrometry imaging with high spatial and mass resolution. Semi‐volatile and non‐volatile molecules can now be imaged simultaneously in biological tissues. Numerous semi‐volatile organic compounds (SVOCs) such as aldehydes, esters and hydrocarbons are synthesized by plants, humans and other animals. SVOCs perform many different biological functions, with several acting as signals that convey pheromonal communication between individuals of the same or other species.m/z 200) with <2 ppm mass accuracy, enabling peaks to be assigned with a higher confidence, albeit at a slower data acquisition rate than for a ToF analyzer. Hence, the OrbiSIMS allows fast image acquisition and high sensitivity using the ToF analyzer to be combined with high mass resolution via the Orbitrap analyzer. We therefore set out to develop a cryogenic workflow for OrbiSIMS that would allow SVOCs to be imaged with high chemical and spatial resolution . As a test case, we chose hydrocarbons because they are biologically important SVOCs but many are volatile under high vacuum at ambient temperature . Hydrocarbons are extensively fragmented by many commonly used ion sources,3++ liquid metal ion gun (LMIG). Analysis of (Z)‐9‐tricosene, an insect pheromone hydrocarbon,3500+ GCIB tends to generate [M+N]+ ions, which may be formed from direct interactions with nitrogen gas in the instrument or via multiple hydrogen loss from [M+NH4]+ adducts . In contrast, the 60 keV Bi3++ LMIG primarily yields [M−2 H]+. ions, as previously reported for GC‐MS,M+N]+ ions . Hydrocarbon peak assignments were validated by comparing MS and MS/MS spectra from the OrbiSIMS Orbitrap with those from direct infusion HESI‐Orbitrap and GC‐MS . These findings show that a cryogenic OrbiSIMS workflow reliably detects hydrocarbons and they also identify the major adducts formed.The OrbiSIMS is a new hybrid SIMS instrument that combines ToF and Orbitrap detectors to allow chemical imaging with micron spatial resolution.3500+‐Orbitrap OrbiSIMS analysis of latent fingerprints at ambient, compared to cryogenic, temperature shows decreased TAG and increased diacylglyceride signals (Supplementary Table 1). Molecular fragmentation occurs because of excess internal energy. This can be decreased by either decreasing the vibrational energy of the molecule or the amount of energy transferred via a series of collisions from the incoming primary ion.n+ primary ion beam. Triolein is detected as [M+Na]+ at m/z 907.77 and its fragmentation can be measured by the ratio of this molecular ion to the total ion counts in the spectrum. At cryogenic temperatures, triolein fragmentation is decreased for all energies per atom with an order of magnitude decrease at 5.7 eV per atom (n=3500) . In addition, cryogenic temperatures increase the absolute yield of the [M+Na]+ molecular ion by a factor of ca. 4.5 . These findings indicate that, in contrast to lowering the energy per atom of the primary ion, cryogenic analysis temperatures decrease fragmentation without sacrificing ionization yield. We conclude that cryogenic sample analysis, together with low energy per atom of the primary ion, improves SIMS detection of biologically important lipids in their intact state by an order of magnitude.Chemical imaging of human latent fingerprints has emerged as an important area of forensic and biomedical science. Several imaging technologies including ToF‐SIMS have previously been used to localize non‐volatile drugs, poisons, antibiotics and other molecules to the ridges and sweat pores of fingerprints.3N2O2]+ (m/z 128.97) specifically localizes to the vicinity of sweat (eccrine) pores, whereas [NaP2O6]+ (m/z 180.91) is in a complementary pattern, largely excluded from sweat pores  and palmitic acid   and the volatile hydrocarbon nonacosane (m/z 403.09), which we validated with GC‐MS alongside a commercial standard (Supplementary Table 1). Importantly, these three molecular ions could be reliably detected on the surface of pine needles using cryogenic but not ambient temperatures. Analysis of a variety of different plant leaf and fruit samples illustrates the broad diversity of hydrocarbons and other SVOCs that can be detected cryogenically (Supplementary Table 1). We also utilized the depth‐profiling mode of the Cryo‐OrbiSIMS in positive polarity to demonstrate that the semi‐volatile hydrocarbon octacosane is enriched at the surface of the cuticle of the gooseberry fruit . Depth profiling of the fruit in negative polarity mode also detected a uniformly distributed putative wax ester, and a free fatty acid enriched in a layer below the surface of the fruit cuticle . The fingerprint and plant cuticle analyses together demonstrate that the new cryogenic workflow expands the chemical space amenable to SIMS imaging to encompass hydrocarbons and other SVOCs, as well as molecules with intermediate vapor pressures like free fatty acids.Hydrocarbons, particularly long‐chain alkanes, are a major component of the cuticular lipid barrier to water loss in both plants and insects.s Figure  b. This Drosophila melanogaster is known to be rich in hydrocarbons, wax esters and other lipids.Drosophila cuticle showed a dramatic increase in the number of features that could be detected via cryogenic rather than ambient OrbiSIMS  resulted in a dramatic decrease in alkanes and alkenes, including tricosene  and a [C7H11]+ (m/z 95.07) fragment are detected across the cuticle of the abdomen and also the wing  or in the sternites (flexible regions) of the abdominal cuticle  should be addressed to the authors.SupplementaryClick here for additional data file.SupplementaryClick here for additional data file.


Growth factor receptors are known to be involved in the process of viral infection. Many viruses not only use growth factor receptors to physically attach to the cell surface and internalize, but also divert receptor tyrosine kinase signaling in order to replicate. Thus, repurposing drugs that have initially been developed to target growth factor receptors and their signaling in cancer may prove to be a fast track to effective therapies against emerging new viral infections, including the coronavirus disease 19 (COVID‐19). ACE2angiotensin‐converting enzyme 2COVID‐19coronavirus disease 19EBVEpstein‐Barr virusEGFRepidermal growth factor receptorFGFRfibroblast growth factor receptorGFRsgrowth factor receptorsHBVhepatitis B virusHCVhepatitis C virusHER2human epidermal growth factor receptor 2HIVhuman immunodeficiency virusHSheparan sulfatesmAbsmonoclonal antibodiesMERS‐CoVmiddle east respiratory syndrome coronavirusNGFnerve growth factorRBDreceptor binding domainSARS‐CoVsevere acute respiratory syndrome coronavirusSARS‐CoV‐2severe acute respiratory syndrome coronavirus 2TGFbRtransforming growth factor beta receptorTMPRSS2transmembrane protease, serine 2VACVvaccinia virus1The recent and rapid worldwide spread of the severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2), that gives rise to the coronavirus disease 19 (COVID‐19),Viruses are cellular parasites that infect eukaryotic and prokaryotic cells and “hijack” their cellular machinery to replicate themselves before being released to further infect neighboring cells and eventually other organisms. The first step in viral infection is the attachment of the virus to the plasma membrane and its entry into the cell, which is followed by intracellular viral replication and finally the release of the newly formed viruses. With regard to COVID‐19, it is known that the angiotensin‐converting enzyme 2 (ACE2) ACE2 provides the cell membrane receptor entry point for SARS‐CoV‐2.Growth factor receptors (GFRs), are transmembrane proteins expressed in eukaryotic cells and whose primary function is to bind to extracellular polypeptide growth factors. The binding of specific growth factors to GFRs results in the activation of their intracellular protein kinase domain that initiates a cascade of signaling events ultimately leading to the regulation of cell growth. Interestingly, GFRs have also been identified as necessary for the entry of some viruses, including coronaviruses, and GFR signaling is involved in viral replication in many instances. Drugs targeting GFRs and their signaling are currently used in clinical practice for the treatment of cancer and given the role of GFRs in virus entry and replication, these drugs could potentially be repurposed against viral infections. In this article, the evidence for the link between viral infection and GFRs will be reviewed, and the value of repurposing current oncologic drugs targeting GFRs in order to expand effective antiviral strategies, including in the fight against COVID‐19, will be discussed.2In multicellular organisms such as the human body, growth factors and GFRs are essential during embryogenesis and post‐natal development because they orchestrate cell survival, proliferation, migration and differentiation in all tissues and organs. In the adult body, GFRs are involved in the control of cellular turnover and homeostasis, as well as in tissue repair and regeneration, and their deregulation can lead to cancer. Nerve growth factor (NGF) and epidermal growth factor (EGF) were the first growth factors to be identified, leading to the award of the 1986 Nobel prize in Physiology and Medicine to Stanley Cohen and Rita Levi‐Montalcini.There are three common characteristics shared by most GFRs: a virtually ubiquitous localization throughout tissues and organs; the existence of multiple GFRs genes/proteins within the same family; and the wide range of cellular effects induced by the same GFRs. This is well illustrated by the receptors for the fibroblast growth factors (FGFRs). There are four different FGFRs (FGFRs1‐4) genes generating seven isoforms. All are expressed in most human cell types and, upon ligand binding, the resultant stimulation of their tyrosine kinases induces a wide range of cellular effects including cell proliferation in epithelial cells and fibroblasts, the migration of endothelial cells, as well as the differentiation of neurons and their subsequent neurite outgrowth. Notably, the activity of FGFRs is also co‐regulated by a class of unbranched glycosaminoglycans, the heparan sulfates (HS), which are abundant on both the cell surface and within the extracellular matrix. HSs make many FGF/FGFR interactions much more efficient by cross‐binding them into trimeric complexes, thus facilitating the activation of FGFRs and their downstream signaling pathways.3The evidence for GFR involvement in viral infections is listed in Table 3.1As early as 1978, it was reported that the growth of cytomegalovirus and herpes simplex virus type 1 in human fibroblasts could be altered by EGF,The overactivation of EGFRs could also result in other long‐term issues for patients affected by respiratory viruses, as was shown with the severe acute respiratory syndrome (SARS) coronavirus; EGRF overactivation leads to increased pulmonary fibrosis.3.2The first study demonstrating a role for FGFRs in viral infection reported that they were a portal of cellular entry for the herpes simplex virus type 1, and presumed it was through the binding of FGF to the viral particle and presentation to the receptors.Similar to the case of EGFRs, FGFR activation may trigger long‐term consequences for virus infected patients. The activation of the FGFR1 signaling pathway by Epstein‐Barr virus‐encoded LMP1 promotes aerobic glycolysis and ultimately the cancerous transformation of human nasopharyngeal epithelial cells.3.3As well as facilitating FGFR activation, the HS complexed into proteoglycan complexes to form HSPGs represent a separate entry point for viruses into human cells. This was first shown for the herpes simplex virus, whose initial interaction with cells is via binding to cell surface HSPGs.3.4TGFbRs have not been reported as a cellular entry point for viruses; however, TGFbR stimulation appears to regulate viral replication. It was initially shown that TGFbR stimulation resulted in the suppression of the human immunodeficiency virus (HIV) expression and replication.Of note, the MERS coronavirus induces apoptosis in lung cells by upregulating SMAD7, a key protein involved in TGFbR signaling.3.5There is less information on the role of other classes of GFRs in virus infection. The hepatocyte growth factor receptor (HGFR) is a known co‐receptor for cell entry of adeno‐associated virus type 2 infection.4The evidence described above points to the extensive involvement of GFRs in the processes of virus entry and replication. It is not only that some viruses can use GFRs as attachment receptor for cellular internalization, but it also appears that their downstream tyrosine kinase activity promotes virus replication. Importantly there is a range of drugs that are already in use in oncology to target GFRs and their tyrosine kinase activities in the treatment of cancer patients. The currently used drugs can be classified into two categories: blocking monoclonal antibodies (mAbs) and tyrosine kinase inhibitors. In addition, clinical heparin can also regulate GFR activation and virus entry.4.1These have been developed specifically to bind to the extracellular domain of GFRs to block ligand binding and thereby inhibit receptor dimerization, autophosphorylation and downstream signaling.4.2Tyrosine kinase inhibitors (TKIs) bind to ATP‐binding pockets located within the intracellular catalytic kinase domain of tyrosine kinase GFRs, so blocking the activation of downstream signaling. They are also frontline targeted therapies for the treatment of several cancer types. They thus hold potential interest for drug repurposing against viruses. TKIs were first developed to target EGFR family members and they were the first to enter clinical use in oncology.4.3HSs found on the cell surface and as part of the extracellular matrix are essential to the activation of several GFRs, mostly by virtue of their facilitation of growth factor binding. Structurally very close to the native ECM glycosaminoglycan HS is the variant heparin, one of the most widely used drugs in the world. Heparin and its derivatives are clinically approved as anticoagulants/anti‐thrombotics, with well‐known bioavailability albeit with a tight safety window. Not all heparin derivatives have anti‐coagulant activity. Importantly, heparin has been shown to have a broad spectrum of activity against many viruses, including Zika5The literature described herein emphasizes the pivotal involvement of GFRs, and co‐receptors such as HSPGs, in viral entry and replication. In the face of the current lack of a vaccine or other effective therapies for the treatment of emerging viral diseases such as COVID‐19, with their dramatic health and economic consequences, the targeting of GFRs using existing oncologic drugs represents an intriguing opportunity. Given the rapidity of COVID‐19 spread throughout the world, drug repurposing offers a fast track for the introduction of therapies using known clinically approved molecules. The value of drug repurposing to fight SARS‐CoV‐2 is well illustrated with chloroquine/hydroxychloroquine, an inexpensive drug with low toxicity that has been used for more than 60 years for the treatment of malaria. It has shown promising effects in reducing the viral load and the duration of infection in initial clinical trials involving patients with COVID‐19.The authors declare no conflicts of interest.