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This paper covers considerations in using criterion measures based on administrative data. We begin with a conceptual framework for understanding and evaluating administrative criterion measures as “objective” rather than (ratings-based) assessments of job performance. We then describe the associated advantages (e.g., availability) and disadvantages (e.g., contamination) of using administrative data for criterion-related validation purposes. Best practices in the use of administrative data for validation purposes, including procedures for (a) handling missing data, (b) performing data checks, and (c) reporting detailed decision rules so future researchers can replicate the analyses are also described. Finally, we discuss “modern data management” approaches for improving administrative data for supporting organizational decision-making.

A series of 6-amidinobenzothiazoles, linked via phenoxymethylene or directly to the 1,2,3-triazole ring with a p-substituted phenyl or benzyl moiety, were synthesised and evaluated in vitro against four human tumour cell lines and the protozoan parasite Trypanosoma brucei. The influence of the type of amidino substituent and phenoxymethylene linker on antiproliferative and antitrypanosomal activities was observed, showing that the imidazoline moiety had a major impact on both activities. Benzothiazole imidazoline 14a, which was directly connected to N-1-phenyl-1,2,3-triazole, had the most potent growth-inhibitory effect (IC50 = 0.25 µM) on colorectal adenocarcinoma (SW620), while benzothiazole imidazoline 11b, containing a phenoxymethylene linker, exhibited the best antitrypanosomal potency (IC90 = 0.12 µM). DNA binding assays showed a non-covalent interaction of 6-amidinobenzothiazole ligands, indicating both minor groove binding and intercalation modes of DNA interaction. Our findings encourage further development of novel structurally related 6-amidino-2-arylbenzothiazoles to obtain more selective anticancer and anti-HAT agents., Graphical Abstract

Tissue engineering of skeletal muscle aims to replicate the parallel alignment of myotubes on the native tissue. Directional topography gradients allow the study of the influence of topography on cellular orientation, proliferation, and differentiation, resulting in yield cues and clues to develop a proper in vitro environment for muscle tissue engineering. In this study, we used a polydimethylsiloxane-based substrate containing an aligned topography gradient with sinusoidal features ranging from wavelength (lambda) = 1,520 nm and amplitude (A) =176 nm to lambda = 9,934 nm and A = 2,168 nm. With this topography gradient, we evaluated the effect of topography on human myoblasts distribution, dominant orientation, cell area, nuclei coverage, cell area per number of nuclei, and nuclei area of myotubes. We showed that human myoblasts aligned and differentiated irrespective of the topography section. In addition, aligned human myotubes showed functionality and maturity by contracting spontaneously and nuclei peripheral organization resembling natural myotubes.

Background It remains controversial whether weight change could influence the risks of colorectal cancer (CRC) and mortality. This study aimed to quantify the associations between full-spectrum changes in body mass index (BMI) and the risks of colorectal cancer (CRC) incidence, cancer-related and all-cause mortality among midlife to elder population. Methods A total of 81,388 participants who were free of cancer and aged 55 to 74 years from the Prostate, Lung, Colorectal, and Ovarian (PLCO) screening program were involved. The percentage change of BMI was calculated as (BMI in 2006 - BMI at baseline)/BMI at baseline, and was categorized into nine groups: decrease (≥ 15.0%, 10.0–14.9%, 5.0–9.9%, 2.5–4.9%), stable (decrease/increase

Many invasive bacterial diseases are caused by organisms that are ordinarily harmless components of the human microbiome. Effective interventions against these microbes require an understanding of the processes whereby symbiotic or commensal relationships transition into pathology. Here, we describe bacterial genome-wide association studies (GWAS) of Neisseria meningitidis, a common commensal of the human respiratory tract that is nevertheless a leading cause of meningitis and sepsis. An initial GWAS discovered bacterial genetic variants, including single nucleotide polymorphisms (SNPs), associated with invasive meningococcal disease (IMD) versus carriage in several loci across the meningococcal genome, encoding antigens and other extracellular components, confirming the polygenic nature of the invasive phenotype. In particular, there was a significant peak of association around the fHbp locus, encoding factor H binding protein (fHbp), which promotes bacterial immune evasion of human complement by recruiting complement factor H (CFH) to the meningococcal surface. The association around fHbp with IMD was confirmed by a validation GWAS, and we found that the SNPs identified in the validation affected the 5’ region of fHbp mRNA, altering secondary RNA structures, thereby increasing fHbp expression and enhancing bacterial escape from complement-mediated killing. This finding is consistent with the known link between complement deficiencies and CFH variation with human susceptibility to IMD. These observations demonstrate the importance of human and bacterial genetic variation across the fHbp:CFH interface in determining IMD susceptibility, the transition from carriage to disease., Author summary The human bacterial pathogen Neisseria meningitidis (the meningococcus) causes sepsis and meningitis worldwide. Paradoxically, it is carried harmlessly in the nose and throat far more commonly than it causes invasive disease, raising the question of whether the bacteria causing disease are genetically the same as those that do not. We looked for systematic differences in the DNA of the bacteria from invasive disease and those that were carried harmlessly. Whilst controlling for population structure as a confounder, we identified multiple genes apparently contributing to invasion and identified a signal around the gene encoding an important component of some meningococcal vaccines, called factor H binding protein (fHbp). This protein helps the meningococcus evade killing by the human immune response by binding to a human defence protein called complement factor H. We validated this result in an independent population of meningococci and identified that DNA variants that control the amount of fHbp produced by the bacteria were important, with high levels of fHbp expression associated with invasion. Given that human DNA variation in complement factor H increases the risk of meningococcal invasion, this highlights the important connection between human and bacterial genetic variation and helps explain why some people infected with meningococci suffer meningococcal disease whilst others do not.

Plant derived cysteine proteinases (CPs) have long been known to possess anthelmintic properties but have attracted renewed attention recently because of the acute need to discover novel methods for controlling helminth infections as a result of increasing drug resistance. However, surprisingly little is known about the stability of these proteins under typical storage and in vivo exposure conditions. We found that CPs in a supernatant preparation from papaya latex (PLS) were stable during the initial refinement process and when stored under low temperatures, but lost activity during dialysis and within 7 days of storage when kept at ambient temperature (18–20 °C). The enzyme activity in PLS was not affected by repeated freeze-thaw cycles and was also stable under typical in vitro assay conditions at 37 °C used for quantifying effects on helminths. Active enzyme activity was still detectable in the colon 3–4 h after oral administration in rodent models., Papaya latex supernatant; Cysteine proteinase; Anthelmintic; Enzyme activity.

Digestive Chagas disease (DCD) is an enteric neuropathy caused by Trypanosoma cruzi infection. The mechanism of pathogenesis is poorly understood and the lack of a robust, predictive animal model has held back research. We screened a series of mouse models using gastrointestinal tracer assays and in vivo infection imaging systems to discover a subset exhibiting chronic digestive transit dysfunction and significant retention of faeces in both sated and fasted conditions. The colon was a specific site of both tissue parasite persistence, delayed transit and dramatic loss of myenteric neurons as revealed by whole-mount immunofluorescence analysis. DCD mice therefore recapitulated key clinical manifestations of human disease. We also exploited dual reporter transgenic parasites to home in on locations of rare chronic infection foci in the colon by ex vivo bioluminescence imaging and then used fluorescence imaging in tissue microdomains to reveal co-localisation of infection and enteric nervous system lesions. This indicates that long-term T. cruzi-host interactions in the colon drive DCD pathogenesis, suggesting that the efficacy of anti-parasitic chemotherapy against chronic disease progression warrants further pre-clinical investigation., Author summary Chagas disease (American trypanosomiasis) is caused by the protozoan parasite Trypanosoma cruzi. Chagas disease has two types, the cardiac form and the digestive form; some patients have symptoms of both. How the parasite causes digestive disease is poorly understood. It is known that damage to the gut’s nervous system is an important factor, but it has been unclear exactly where and when this damage occurs during the course of an infection and also why only a subset of infected people suffer from this outcome. We studied infections in mice and found certain combinations of strains of parasites and mice that exhibited symptoms similar to human digestive Chagas patients, including a problem with peristalsis that localised specifically to the colon. Using parasites that were genetically engineered to emit both bioluminescent and fluorescent light, we tracked infections over time and were able to analyse rare infected cells deep within the muscle tissue of the wall of the colon. We found evidence of damaged neurons in the same location as these infection foci over 6 months after initial infection. Our results show that digestive Chagas disease probably develops as a result of chronic infection and inflammation, which potentially changes approaches to treatment.

INTRODUCTION: Fat grafting is an established clinical intervention to promote tissue repair. The role of the fat's extracellular matrix (ECM) in regeneration is largely neglected. We investigated in vitro the use of human adipose tissue-derived ECM hydrogels as release platform for factors secreted by adipose derived stromal cells (ASC) MATERIAL & METHODS: Lipoaspirates from non-diabetic and diabetic donors were decellularized. Finely powdered acellular ECM was evaluated for cell remainders and DNA content. Acellular ECM was digested and hydrogels were formed at 37 °C and their viscoelastic relaxation properties investigated. Release of ASC-released factors from hydrogels was immune-assessed and bio-activity was determined by fibroblast proliferation and migration and endothelial angiogenesis.RESULTS: Acellular ECM contained no detectable cell remainders and negligible DNA contents. Viscoelastic relaxation measurements yielded no data for diabetic-derived hydrogels due to gel instability. Hydrogels released several ASC-released factors concurrently in a sustained fashion. Functionally, released factors stimulated fibroblast proliferation and migration as well as angiogenesis. No difference between non-diabetic and diabetic hydrogels in release of factors was measured.CONCLUSION: Adipose ECM hydrogels incubated with released factors by ASC are a promising new therapeutic modality to promote several important wound healing related processes by releasing factors in a controlled way.

Insulin sensitizers and incretin mimetics are antidiabetic agents with vastly different mechanisms of action. Thiazolidinedione (TZD) insulin sensitizers are associated with weight gain, whereas glucagon-like peptide-1 receptor agonists can induce weight loss. We hypothesized that combination of a TZD insulin sensitizer and the glucagon-like peptide-1 receptor agonist liraglutide would more significantly improve mouse models of diabetes and nonalcoholic steatohepatitis (NASH). Diabetic db/db and MS-NASH mice were treated with the TZD MSDC-0602K by oral gavage, liraglutide (Lira) by s.c. injection, or combination 0602K+Lira. Lira slightly reduced body weight and modestly improved glycemia in db/db mice. Comparatively, 0602K-treated and 0602K+Lira-treated mice exhibited slight weight gain but completely corrected glycemia and improved glucose tolerance. 0602K reduced plasma insulin, whereas Lira further increased the hyperinsulinemia of db/db mice. Surprisingly, 0602K+Lira treatment reduced plasma insulin and C-peptide to the same extent as mice treated with 0602K alone. 0602K did not reduce glucose-stimulated insulin secretion in vivo, or in isolated islets, indicating the reduced insulinemia was likely compensatory to improved insulin sensitivity. In MS-NASH mice, both 0602K or Lira alone improved plasma alanine aminotransferase and aspartate aminotransferase, as well as liver histology, but more significant improvements were observed with 0602K+Lira treatment. 0602K or 0602K+Lira also increased pancreatic insulin content in both db/db and MS-NASH mice. In conclusion, MSDC-0602K corrected glycemia and reduced insulinemia when given alone, or in combination with Lira. However, 0602K+Lira combination more significantly improved glucose tolerance and liver histology, suggesting that this combination treatment may be an effective therapeutic strategy for diabetes and NASH.

Background This study examined current physical activity levels and preferences for exercise settings and activities among adult survivors of childhood cancers as a strategy to inform the feasibility and design of such programs. Methods A mixed-methods design was used to investigate current activity levels as well as barriers to and preferences for physical activity among 20 adult survivors of pediatric cancer. Results One-half of participants reported engaging in regular physical activity, although the frequency, intensity, and duration varied. Overall, 17 of the 20 participants (85%) stated they would be interested in participating in a structured exercise intervention, and they expressed a strong interest in walking (76%), bicycling (53%), and weight training (53%). Common barriers to participation in a potential structured exercise program were insufficient time, current health issues, and program location/distance. Nearly all participants agreed that information on nutrition and diet should be included as part of an exercise intervention. Conclusions These findings will help inform the design and implementation of future exercise programs to enhance physical activity among this high-risk group of cancer survivors.

Background Electronic Health Record Systems (EHRs) are being rolled out nationally in many low- and middle-income countries (LMICs) yet assessing actual system usage remains a challenge. We employed a nominal group technique (NGT) process to systematically develop high-quality indicators for evaluating actual usage of EHRs in LMICs. Methods An initial set of 14 candidate indicators were developed by the study team adapting the Human Immunodeficiency Virus (HIV) Monitoring, Evaluation, and Reporting indicators format. A multidisciplinary team of 10 experts was convened in a two-day NGT workshop in Kenya to systematically evaluate, rate (using Specific, Measurable, Achievable, Relevant, and Time-Bound (SMART) criteria), prioritize, refine, and identify new indicators. NGT steps included introduction to candidate indicators, silent indicator ranking, round-robin indicator rating, and silent generation of new indicators. 5-point Likert scale was used in rating the candidate indicators against the SMART components. Results Candidate indicators were rated highly on SMART criteria (4.05/5). NGT participants settled on 15 final indicators, categorized as system use (4); data quality (3), system interoperability (3), and reporting (5). Data entry statistics, systems uptime, and EHRs variable concordance indicators were rated highest. Conclusion This study describes a systematic approach to develop and validate quality indicators for determining EHRs use and provides LMICs with a multidimensional tool for assessing success of EHRs implementations.

Background Health facilities in developing countries are increasingly adopting Electronic Health Records systems (EHRs) to support healthcare processes. However, only limited studies are available that assess the actual use of the EHRs once adopted in these settings. We assessed the state of the 376 KenyaEMR system (national EHRs) implementations in healthcare facilities offering HIV services in Kenya. Methods The study focused on seven EHRs use indicators. Six of the seven indicators were programmed and packaged into a query script for execution within each KenyaEMR system (KeEMRs) implementation to collect monthly server-log data for each indicator for the period 2012–2019. The indicators included: Staff system use, observations (clinical data volume), data exchange, standardized terminologies, patient identification, and automatic reports. The seventh indicator (EHR variable Completeness) was derived from routine data quality report within the EHRs. Data were analysed using descriptive statistics, and multiple linear regression analysis was used to examine how individual facility characteristics affected the use of the system. Results 213 facilities spanning 19 counties participated in the study. The mean number of authorized users who actively used the KeEMRs was 18.1% (SD = 13.1%, pobservations) captured in the EHRs was 3363 (SD = 4259). Only a few facilities(14.1%) had health data exchange capability. 97.6% of EHRs concept dictionary terms mapped to standardized terminologies such as CIEL. Within the facility EHRs, only 50.5% (SD = 35.4%, p< 0.001) of patients had the nationally-endorsed patient identifier number recorded. Multiple regression analysis indicated the need for improvement on the mode of EHRs use of implementation. Conclusion The standard EHRs use indicators can effectively measure EHRs use and consequently determine success of the EHRs implementations. The results suggest that most of the EHRs use areas assessed need improvement, especially in relation to active usage of the system and data exchange readiness.

Chronic Trypanosoma cruzi infections are typically lifelong, with small numbers of parasites surviving in restricted tissue sites, which include the gastrointestinal tract. There is considerable debate about the replicative status of these persistent parasites and whether there is a role for dormancy in long-term infection. Here, we investigated T. cruzi proliferation in the colon of chronically infected mice using 5-ethynyl-2′deoxyuridine incorporation into DNA to provide ‘snapshots’ of parasite replication status. Highly sensitive imaging of the extremely rare infection foci, at single-cell resolution, revealed that parasites are three times more likely to be in S-phase during the acute stage than during the chronic stage. By implication, chronic infections of the colon are associated with a reduced rate of parasite replication. Despite this, very few host cells survived infection for more than 14 days, suggesting that T. cruzi persistence continues to involve regular cycles of replication, host cell lysis and re-infection. We could find no evidence for wide-spread dormancy in parasites that persist in this tissue reservoir.

Many photosynthetic organisms employ a CO2 concentrating mechanism (CCM) to increase the rate of CO2 fixation via the Calvin cycle. CCMs catalyze ≈50% of global photosynthesis, yet it remains unclear which genes and proteins are required to produce this complex adaptation. We describe the construction of a functional CCM in a non-native host, achieved by expressing genes from an autotrophic bacterium in an Escherichia coli strain engineered to depend on rubisco carboxylation for growth. Expression of 20 CCM genes enabled E. coli to grow by fixing CO2 from ambient air into biomass, with growth in ambient air depending on the components of the CCM. Bacterial CCMs are therefore genetically compact and readily transplanted, rationalizing their presence in diverse bacteria. Reconstitution enabled genetic experiments refining our understanding of the CCM, thereby laying the groundwork for deeper study and engineering of the cell biology supporting CO2 assimilation in diverse organisms.

Background The ability to report complete, accurate and timely data by HIV care providers and other entities is a key aspect in monitoring trends in HIV prevention, treatment and care, hence contributing to its eradication. In many low-middle-income-countries (LMICs), aggregate HIV data reporting is done through the District Health Information Software 2 (DHIS2). Nevertheless, despite a long-standing requirement to report HIV-indicator data to DHIS2 in LMICs, few rigorous evaluations exist to evaluate adequacy of health facility reporting at meeting completeness and timeliness requirements over time. The aim of this study is to conduct a comprehensive assessment of the reporting status for HIV-indicators, from the time of DHIS2 implementation, using Kenya as a case study. Methods A retrospective observational study was conducted to assess reporting performance of health facilities providing any of the HIV services in all 47 counties in Kenya between 2011 and 2018. Using data extracted from DHIS2, K-means clustering algorithm was used to identify homogeneous groups of health facilities based on their performance in meeting timeliness and completeness facility reporting requirements for each of the six programmatic areas. Average silhouette coefficient was used in measuring the quality of the selected clusters. Results Based on percentage average facility reporting completeness and timeliness, four homogeneous groups of facilities were identified namely: best performers, average performers, poor performers and outlier performers. Apart from blood safety reports, a distinct pattern was observed in five of the remaining reports, with the proportion of best performing facilities increasing and the proportion of poor performing facilities decreasing over time. However, between 2016 and 2018, the proportion of best performers declined in some of the programmatic areas. Over the study period, no distinct pattern or trend in proportion changes was observed among facilities in the average and outlier groups. Conclusions The identified clusters revealed general improvements in reporting performance in the various reporting areas over time, but with noticeable decrease in some areas between 2016 and 2018. This signifies the need for continuous performance monitoring with possible integration of machine learning and visualization approaches into national HIV reporting systems.

A simple method was developed for evaluating the efficacy of face masks to reduce respiratory droplet emission during speaking., Mandates for mask use in public during the recent coronavirus disease 2019 (COVID-19) pandemic, worsened by global shortage of commercial supplies, have led to widespread use of homemade masks and mask alternatives. It is assumed that wearing such masks reduces the likelihood for an infected person to spread the disease, but many of these mask designs have not been tested in practice. We have demonstrated a simple optical measurement method to evaluate the efficacy of masks to reduce the transmission of respiratory droplets during regular speech. In proof-of-principle studies, we compared a variety of commonly available mask types and observed that some mask types approach the performance of standard surgical masks, while some mask alternatives, such as neck gaiters or bandanas, offer very little protection. Our measurement setup is inexpensive and can be built and operated by nonexperts, allowing for rapid evaluation of mask performance during speech, sneezing, or coughing.

Graft-versus-host disease (GVHD) is a major cause of morbidity and mortality in hematopoietic stem cell transplantation (HSCT). Donor T cells are key mediators in pathogenesis, but a contribution from host T cells has not been explored, as conditioning regimens are believed to deplete host T cells. To evaluate a potential role for host T cells in GVHD, the origin of skin and blood T cells was assessed prospectively in patients after HSCT in the absence of GVHD. While blood contained primarily donor-derived T cells, most T cells in the skin were host derived. We next examined patient skin, colon, and blood during acute GVHD. Host T cells were present in all skin and colon acute GVHD specimens studied, yet were largely absent in blood. We observed acute skin GVHD in the presence of 100% host T cells. Analysis demonstrated that a subset of host T cells in peripheral tissues were proliferating (Ki67(+)) and producing the proinflammatory cytokines IFN-γ and IL-17 in situ. Comparatively, the majority of antigen-presenting cells (APCs) in tissue in acute GVHD were donor derived, and donor-derived APCs were observed directly adjacent to host T cells. A humanized mouse model demonstrated that host skin-resident T cells could be activated by donor monocytes to generate a GVHD-like dermatitis. Thus, host tissue-resident T cells may play a previously unappreciated pathogenic role in acute GVHD.

Background Chronic obstructive pulmonary disease (COPD) is an underdiagnosed condition sharing risk factors with lung cancer. Lung cancer screening may provide an opportunity to improve COPD diagnosis. Using Pan-Canadian Early Detection of Lung Cancer (PanCan) study data, the present study sought to determine the following: 1) What is the prevalence of COPD in a lung cancer screening population? 2) Can a model based on clinical and screening low-dose CT scan data predict the likelihood of COPD? Methods The single arm PanCan study recruited current or former smokers age 50–75 who had a calculated risk of lung cancer of at least 2% over 6 years. A baseline health questionnaire, spirometry, and low-dose CT scan were performed. CT scans were assessed by a radiologist for extent and distribution of emphysema. With spirometry as the gold standard, logistic regression was used to assess factors associated with COPD. Results Among 2514 recruited subjects, 1136 (45.2%) met spirometry criteria for COPD, including 833 of 1987 (41.9%) of those with no prior diagnosis, 53.8% of whom had moderate or worse disease. In a multivariate model, age, current smoking status, number of pack-years, presence of dyspnea, wheeze, participation in a high-risk occupation, and emphysema extent on LDCT were all statistically associated with COPD, while the overall model had poor discrimination (c-statistic = 0.627 (95% CI of 0.607 to 0.650). The lowest and the highest risk decile in the model predicted COPD risk of 27.4 and 65.3%. Conclusions COPD had a high prevalence in a lung cancer screening population. While a risk model had poor discrimination, all deciles of risk had a high prevalence of COPD, and spirometry could be considered as an additional test in lung cancer screening programs. Trial registration (Clinical Trial Registration: ClinicalTrials.gov, number NCT00751660, registered September 12, 2008)

Background Efforts to reduce unnecessary antibiotic prescribing have coincided with increasing awareness of sepsis. We aimed to estimate the probability of sepsis following infection consultations in primary care when antibiotics were or were not prescribed. Methods and findings We conducted a cohort study including all registered patients at 706 general practices in the United Kingdom Clinical Practice Research Datalink, with 66.2 million person-years of follow-up from 2002 to 2017. There were 35,244 first episodes of sepsis (17,886, 51%, female; median age 71 years, interquartile range 57–82 years). Consultations for respiratory tract infection (RTI), skin or urinary tract infection (UTI), and antibiotic prescriptions were exposures. A Bayesian decision tree was used to estimate the probability (95% uncertainty intervals [UIs]) of sepsis following an infection consultation. Age, gender, and frailty were evaluated as association modifiers. The probability of sepsis was lower if an antibiotic was prescribed, but the number of antibiotic prescriptions required to prevent one episode of sepsis (number needed to treat [NNT]) decreased with age. At 0–4 years old, the NNT was 29,773 (95% UI 18,458–71,091) in boys and 27,014 (16,739–65,709) in girls; over 85 years old, NNT was 262 (236–293) in men and 385 (352–421) in women. Frailty was associated with greater risk of sepsis and lower NNT. For severely frail patients aged 55–64 years, the NNT was 247 (156–459) in men and 343 (234–556) in women. At all ages, the probability of sepsis was greatest for UTI, followed by skin infection, followed by RTI. At 65–74 years, the NNT following RTI was 1,257 (1,112–1,434) in men and 2,278 (1,966–2,686) in women; the NNT following skin infection was 503 (398–646) in men and 784 (602–1,051) in women; following UTI, the NNT was 121 (102–145) in men and 284 (241–342) in women. NNT values were generally smaller for the period from 2014 to 2017, when sepsis was diagnosed more frequently. Lack of random allocation to antibiotic therapy might have biased estimates; patients may sometimes experience sepsis or receive antibiotic prescriptions without these being recorded in primary care; recording of sepsis has increased over the study period. Conclusions These stratified estimates of risk help to identify groups in which antibiotic prescribing may be more safely reduced. Risks of sepsis and benefits of antibiotics are more substantial among older adults, persons with more advanced frailty, or following UTIs., In this cohort study, Martin Gulliford and colleagues investigate probability of sepsis in primary care patients with or without prescribed antibiotics., Author summary Why was this study done? Sepsis is a severe reaction to an infection that may lead to life threatening damage to organ systems. Sepsis is an increasingly recognised concern for health professionals and patients in primary care. Inappropriate and unnecessary antibiotic prescribing is a widespread problem in primary care that may be contributing to antimicrobial resistance. This study aimed to estimate the probability of a patient developing sepsis after an infection consultation in primary care if antibiotics are or are not prescribed. What did the researchers do and find? We analysed the electronic health records of all registered patients at 706 general practices, with 66.2 million person-years of follow-up from 2002 to 2017 and 35,244 first episodes of sepsis. We found that the probability of sepsis was lower if an antibiotic was prescribed, but the number of antibiotic prescriptions required to prevent one episode of sepsis (number needed to treat [NNT]) decreased with age. Frailty was associated with greater risk of sepsis and lower NNT. At all ages, the probability of sepsis was greatest for urinary tract infection, followed by skin infection, followed by respiratory tract infection. What do these findings mean? These results show that risks of sepsis and benefits of antibiotics are more substantial among older adults, persons with more advanced frailty, or following urinary tract infections. Antibiotic use may be more safely reduced in groups with lower probability of sepsis. We caution that our results represent averages over diverse localities and years of study, and lack of random allocation to antibiotic therapy might have caused bias.

A protein motif mediates targeting of proteins to the pyrenoid and appears to link the pyrenoid’s three subcompartments., Approximately one-third of the Earth’s photosynthetic CO2 assimilation occurs in a pyrenoid, an organelle containing the CO2-fixing enzyme Rubisco. How constituent proteins are recruited to the pyrenoid and how the organelle’s subcompartments—membrane tubules, a surrounding phase-separated Rubisco matrix, and a peripheral starch sheath—are held together is unknown. Using the model alga Chlamydomonas reinhardtii, we found that pyrenoid proteins share a sequence motif. We show that the motif is necessary and sufficient to target proteins to the pyrenoid and that the motif binds to Rubisco, suggesting a mechanism for targeting. The presence of the Rubisco-binding motif on proteins that localize to the tubules and on proteins that localize to the matrix–starch sheath interface suggests that the motif holds the pyrenoid’s three subcompartments together. Our findings advance our understanding of pyrenoid biogenesis and illustrate how a single protein motif can underlie the architecture of a complex multilayered phase-separated organelle.

GBS is a leading cause of mortality in newborn babies in high- and low-income countries worldwide. Different strains of GBS are characterized by different degrees of virulence, where some are harmlessly carried by humans or animals and others are much more likely to cause disease. The genome sequences of almost 2,000 GBS samples isolated from both animals and humans in high- and low- income countries were analyzed using a pan-genome-wide association study approach. This allowed us to identify 279 genes which are associated with different lineages of GBS, characterized by a different virulence and preferred host. Additionally, we propose that the GBS now carried in humans may have first evolved in animals before expanding clonally once adapted to the human host. These findings are essential to help understand what is causing GBS disease and how the bacteria have evolved and are transmitted., Streptococcus agalactiae (group B streptococcus; GBS) is a colonizer of the gastrointestinal and urogenital tracts, and an opportunistic pathogen of infants and adults. The worldwide population of GBS is characterized by clonal complexes (CCs) with different invasive potentials. CC17, for example, is a hypervirulent lineage commonly associated with neonatal sepsis and meningitis, while CC1 is less invasive in neonates and more commonly causes invasive disease in adults with comorbidities. The genetic basis of GBS virulence and the extent to which different CCs have adapted to different host environments remain uncertain. We have therefore applied a pan-genome-wide association study (GWAS) approach to 1,988 GBS strains isolated from different hosts and countries. Our analysis identified 279 CC-specific genes associated with virulence, disease, metabolism, and regulation of cellular mechanisms that may explain the differential virulence potential of particular CCs. In CC17 and CC23, for example, we have identified genes encoding pilus, quorum-sensing proteins, and proteins for the uptake of ions and micronutrients which are absent in less invasive lineages. Moreover, in CC17, carriage and disease strains were distinguished by the allelic variants of 21 of these CC-specific genes. Together our data highlight the lineage-specific basis of GBS niche adaptation and virulence. The genome sequences of almost 2,000 GBS samples isolated from both animals and humans in high- and low- income countries were analyzed using a pan-genome-wide association study approach. This allowed us to identify 279 genes which are associated with different lineages of GBS, characterized by a different virulence and preferred host. Additionally, we propose that the GBS now carried in humans may have first evolved in animals before expanding clonally once adapted to the human host. These findings are essential to help understand what is causing GBS disease and how the bacteria have evolved and are transmitted.

Background Timely vaccination is key to prevent unnecessary childhood mortality from vaccine-preventable diseases. Despite the substantial efforts to improve vaccination completeness, the effort towards timeliness of vaccination is limited with non-attendance and delays to vaccination appointments remaining a big challenge in developing countries. There is also a limited evidence on timeliness of vaccination. Therefore, this study aimed to determine the magnitude and associated factors for timely completion of vaccination among children in Gondar city administration, north-west, Ethiopia. Method This study employed a community-based cross sectional study design. A sample size of 821 children aged 12 to 23 months were considered. Two stages random sampling technique was used to select study subjects. To account the effect of clustering, bivariable and multivariable multilevel logistic regression analysis were applied. The measures of association estimates were expressed as adjusted odds ratio (AOR) with their 95% confidence intervals (CIs). Results Of the 774 children included for analysis, 498 (64.3%) were fully vaccinated while 247 (31.9%) were fully vaccinated on-time. Caregivers who had secondary education and above (AOR = 2.391; 95% CI: 1.317–4.343), from richest households (AOR = 2.381; 95% CI: 1.502–3.773), children whose mother attended four or more ante natal care visits (AOR = 2.844; 95% CI: 1.310–6.174) and whose mother had two or more post natal care visits (AOR = 2.054; 95%CI:1.377–3.063) were positively associated with on-time full vaccination. In contrary, caregivers aged above 35 years (AOR = 0.469; 95 % CI: 0.253-0.869], being vaccinated at health post (AOR = 0.144; 95%CI: 0.048–0.428) and travelling more than 30 min to the vaccination site (AOR = 0.158; 95%CI: 0.033–0.739) were negatively associated with on-time full vaccination. The random effects indicated that 26% of the variability in on-time full vaccination was attributable to differences between communities. Conclusion In this study, untimely vaccination was found to be high. Different individual and contextual factors were found to be associated with on-time full vaccination. Therefore, tailored strategies have to be designed and implemented to address people and the communities where they live. Moreover, timeliness of vaccination should be considered as important indicator of the immunization program performance in Ethiopia.

Background Constipation is often self-managed by patients and guidelines are available to aid healthcare professionals in the counseling of patients for self-management. Therefore, we have explored the knowledge and attitude of pharmacy personnel towards guidelines for the management of acute and functional chronic constipation and how they affects their recommendations. Methods An online survey was conducted among 201 pharmacists and pharmacy technicians from an existing panel. They were presented with two typical cases, a 62-year old woman with functional chronic constipation and a 42-year old woman with travel plans. For each case, they were asked about their treatment recommendations and the underlying rationale. Thereafter, they were provided with contents from an applicable national guideline and asked again about their recommendations and the underlying rationale. In line with the exploratory nature, data were analyzed in a descriptive manner only. Results Before exposure to guideline content, the most frequent recommendations for chronic constipation were macrogol, fiber and lactulose and for acute constipation sodium picosulfate, bisacodyl and enemas. Following guideline exposure, the most frequent recommendations for chronic constipation were macrogol, bisacodyl and sodium picosulfate and for acute constipation bisacodyl, sodium picosulfate and macrogol (all three equally recommended by the guideline for the management of acute and chronic constipation). Correspondingly, the rationale behind the recommendations shifted with guideline conformity becoming a leading reason. Conclusions Awareness of the content of an applicable guideline on the management of constipation was poor among pharmacy personnel. Accordingly, recommendations in many cases were not in line with the guideline. Greater awareness of guideline content is desirable to enable more evidence-based recommendations in the management of constipation.

BackgroundThe exact onset of brain injury in infants with congenital heart disease (CHD) is unknown. Our aim was, therefore, to assess the association between prenatal Doppler flow patterns, postnatal cerebral oxygenation and short-term neurological outcome.MethodsPrenatally, we measured pulsatility indices of the middle cerebral (MCA-PI) and umbilical artery (UA-PI) and calculated cerebroplacental ratio (CPR). After birth, cerebral oxygen saturation (r(c)SO(2)) and fractional tissue oxygen extraction (FTOE) were assessed during the first 3 days after birth, and during and for 24 hours after every surgical procedure within the first 3 months after birth. Neurological outcome was determined preoperatively and at 3 months of age by assessing general movements and calculating the Motor Optimality Score (MOS).ResultsThirty-six infants were included. MOS at 3 months was associated with MCA-PI (rho 0.41, P = 0.04), UA-PI (rho -0.39, P = 0.047, and CPR (rho 0.50, P = 0.01). Infants with abnormal MOS had lower MCA-PI (P = 0.02) and CPR (P = 0.01) and higher UA-PI at the last measurement (P = 0.03) before birth. In infants with abnormal MOS, r(c)SO(2) tended to be lower during the first 3 days after birth, and FTOE was significantly higher on the second day after birth (P = 0.04). Intraoperative and postoperative r(c)SO(2) and FTOE were not associated with short-term neurological outcome.ConclusionIn infants with prenatally diagnosed CHD, the prenatal period may play an important role in developmental outcome. Additional research is needed to clarify the relationship between preoperative, intra-operative and postoperative cerebral oxygenation and developmental outcome in infants with prenatally diagnosed CHD.

Many bacterial pathogens coexist with host organisms, rarely causing disease while adapting to host responses. Neisseria meningitidis, a major cause of meningitis and septicemia, is a frequent persistent colonizer of asymptomatic teenagers/young adults. To assess how genetic variation contributes to host persistence, whole-genome sequencing and hypermutable sequence analyses were performed on multiple isolates obtained from students naturally colonized with meningococci. High frequencies of gene transfer were observed, occurring in 16% of carriers and affecting 51% of all nonhypermutable variable genes. Comparative analyses showed that hypermutable sequences were the major mechanism of variation, causing 2-fold more changes in gene function than other mechanisms. Genetic variation was focused on genes affecting the outer membrane, with directional changes in proteins responsible for bacterial adhesion to host surfaces. This comprehensive examination of genetic plasticity in individual hosts provides a significant new platform for rationale design of approaches to prevent the spread of this pathogen., Host persistence of bacteria is facilitated by mutational and recombinatorial processes that counteract loss of genetic variation during transmission and selection from evolving host responses. Genetic variation was investigated during persistent asymptomatic carriage of Neisseria meningitidis. Interrogation of whole-genome sequences for paired isolates from 25 carriers showed that de novo mutations were infrequent, while horizontal gene transfer occurred in 16% of carriers. Examination of multiple isolates per time point enabled separation of sporadic and transient allelic variation from directional variation. A comprehensive comparative analysis of directional allelic variation with hypermutation of simple sequence repeats and hyperrecombination of class 1 type IV pilus genes detected an average of seven events per carrier and 2:1 bias for changes due to localized hypermutation. Directional genetic variation was focused on the outer membrane with 69% of events occurring in genes encoding enzymatic modifiers of surface structures or outer membrane proteins. Multiple carriers exhibited directional and opposed switching of allelic variants of the surface-located Opa proteins that enables continuous expression of these adhesins alongside antigenic variation. A trend for switching from PilC1 to PilC2 expression was detected, indicating selection for specific alterations in the activities of the type IV pilus, whereas phase variation of restriction modification (RM) systems, as well as associated phasevarions, was infrequent. We conclude that asymptomatic meningococcal carriage on mucosal surfaces is facilitated by frequent localized hypermutation and horizontal gene transfer affecting genes encoding surface modifiers such that optimization of adhesive functions occurs alongside escape of immune responses by antigenic variation.

Integrons play a major role in the evolution and spread of antimicrobial resistance in human pathogens, including Escherichia coli. This study describes the occurrence of class 1 integrons in human pathogenic E. coli, in three isolate collections involving three periods from the last 100 years (i) the Murray collection (n = 58 bacteria isolated from the 1910s to 1940s); (ii) the E. coli reference (ECOR) collection (n = 37 isolates mainly from the 1980s); and (iii) a recently assembled collection (n = 88 isolates obtained in 2016). High-quality whole genome sequences (WGSs) were available for all isolates. Integrons were detected in the WGSs with the program IntegronFinder and the results compared with three established methods: (i) polymerase chain reaction detection of the integrase gene; (ii) BLAST searching using draft genomes; and (iii) mapping of short reads. No integrons were found in any of the Murray Collection isolates; however, integrons were present in 3% of the isolates from ECOR collection, assembled in the 1980s, and 26% of the isolates from the 2010s. Similarly, antimicrobial resistance determinants were not present in the Murray Collection isolates, whereas they were present in 19% of the ECOR Collection isolates and in 55% of the isolates obtained in during the 2010s.

Background In our clinic, a substantial number of patients present with transtibial residual limb pain of no specific somatic origin. Silicone liner induced tissue compression may reduce blood flow, possibly causing residual limb pain. Thus, as a first step we investigated if the liner itself has an effect on transcutaneous oxygen pressure (TcPO2). Methods Persons with unilateral transtibial amputation and residual limb pain of unknown origin were included. Medical history, including residual limb pain, was recorded, and the SF-36 administered. Resting TcPO2 levels were measured in the supine position and without a liner at 0, 10, 20 and 30 minutes using two sensors: one placed in the Transverse plane over the tip of the Tibia End (= TTE), the other placed in the Sagittal plane, distally over the Peroneal Compartment (= SPC). Measurements were repeated with specially prepared liners avoiding additional pressure due to sensor placement. Statistical analyses were performed using SPSS. Results Twenty persons (9 women, 11 men) with a mean age of 68.65 years (range 47–86 years) participated. The transtibial amputation occurred on average 43 months prior to study entry (range 3–119 months). With liner wear, both sensors measured TcPO2 levels that were significantly lower than those measured without a liner (TTE: p < 0.001; SPC: p = 0.002) after 10, 20 and 30 minutes. No significant differences were found between TcPO2 levels over time between the sensors. There were no significant associations between TcPO2 levels and pain, smoking status, age, duration of daily liner use, mobility level, and revision history. Conclusion Resting TcPO2 levels decreased significantly while wearing a liner alone, without a prosthetic socket. Further studies are required to investigate the effect of liner wear on exercise TcPO2 levels., PLoS ONE, 15 (9), ISSN:1932-6203

Providing prognostic information at the time of cancer diagnosis has important implications for treatment and monitoring. Although cancer staging, histopathological assessment, molecular features, and clinical variables can provide useful prognostic insights, improving risk stratification remains an active research area. We developed a deep learning system (DLS) to predict disease specific survival across 10 cancer types from The Cancer Genome Atlas (TCGA). We used a weakly-supervised approach without pixel-level annotations, and tested three different survival loss functions. The DLS was developed using 9,086 slides from 3,664 cases and evaluated using 3,009 slides from 1,216 cases. In multivariable Cox regression analysis of the combined cohort including all 10 cancers, the DLS was significantly associated with disease specific survival (hazard ratio of 1.58, 95% CI 1.28–1.70, p

Background The role of cement-augmented screw fixation for calcaneal fracture treatment remains unclear. Therefore, this study was performed to biomechanically analyze screw osteosynthesis by reinforcement with either a calcium phosphate (CP)-based or polymethylmethacrylate (PMMA)-based injectable bone cement. Methods A calcaneal fracture (Sanders type IIA) including a central cancellous bone defect was generated in 27 synthetic bones, and the specimens were assigned to 3 groups. The first group was fixed with four screws (3.5 mm and 6.5 mm), the second group with screws and CP-based cement (Graftys (R) QuickSet; Graftys, Aix-en-Provence, France), and the third group with screws and PMMA-based cement (Traumacem (TM) V+; DePuy Synthes, Warsaw, IN, USA). Biomechanical testing was conducted to analyze peak-to-peak displacement, total displacement, and stiffness in following a standardized protocol. Results The peak-to-peak displacement under a 200-N load was not significantly different among the groups; however, peak-to-peak displacement under a 600- and 1000-N load as well as total displacement exhibited better stability in PMMA-augmented screw osteosynthesis compared to screw fixation without augmentation. The stiffness of the construct was increased by both CP- and PMMA-based cements. Conclusion Addition of an injectable bone cement to screw osteosynthesis is able to increase fixation strength in a biomechanical calcaneal fracture model with synthetic bones. In such cases, PMMA-based cements are more effective than CP-based cements because of their inherently higher compressive strength. However, whether this high strength is required in the clinical setting for early weight-bearing remains controversial, and the non-degradable properties of PMMA might cause difficulties during subsequent interventions in younger patients.

Access to efficient laboratory services is critical to patient care. Turnaround Time (TAT) is one of the most important measures when judging the efficiency of any laboratory and care system. Few studies on TAT exist for inpatient care settings within low- and middle-income countries (LMICs). Methods We evaluated therapeutic TAT for a tertiary hospital in Western Kenya, using a time-motion study focusing specifically on common hematology and biochemistry orders. The aim was to determine significant bottlenecks in diagnostic testing processes at the institution. Results A total of 356 (155 hematology and 201 biochemistry) laboratory tests were fully tracked from the time of ordering to availability of results to care providers. The total therapeutic TAT for all tests was 21.5 ± 0.249 hours (95% CI). The therapeutic TAT for hematology was 20.3 ± 0.331 hours (95% CI) while that for biochemistry tests was 22.2 ± 0.346 hours (95% CI). Printing, sorting and dispatch of the printed results emerged as the most significant bottlenecks, accounting for up to 8 hours of delay (Hematology—8.3 ± 1.29 hours (95% CI), Biochemistry—8.5 ± 1.18 hours (95% CI)). Time of test orders affected TAT, with orders made early in the morning and those in the afternoon experiencing the most delays in TAT. Conclusion Significant inefficiencies exist at multiple steps in the turnaround times for routine laboratory tests at a large referral hospital within an LMIC setting. Multiple opportunities exist to improve TAT and streamline processes around diagnostic testing in this and other similar settings. publishedVersion

Background Tandem repeats are highly mutable and contribute to the development of human disease by a variety of mechanisms. It is difficult to predict which tandem repeats may cause a disease. One hypothesis is that changeable tandem repeats are the source of genetic diseases, because disease-causing repeats are polymorphic in healthy individuals. However, it is not clear whether disease-causing repeats are more polymorphic than other repeats. Methods We performed a genome-wide survey of the millions of human tandem repeats using publicly available long read genome sequencing data from 21 humans. We measured tandem repeat copy number changes using . Length variation of known disease-associated repeats was compared to other repeat loci. Results We found that known Mendelian disease-causing or disease-associated repeats, especially CAG and 5′UTR GGC repeats, are relatively long and polymorphic in the general population. We also show that repeat lengths of two disease-causing tandem repeats, in ATXN3 and GLS, are correlated with near-by GWAS SNP genotypes. Conclusions We provide a catalog of polymorphic tandem repeats across a variety of repeat unit lengths and sequences, from long read sequencing data. This method especially if used in genome wide association study, may indicate possible new candidates of pathogenic or biologically important tandem repeats in human genomes.

Investigations into intracellular replication and differentiation of Trypanosoma cruzi within the mammalian host have been restricted by limitations in our ability to detect parasitized cells throughout the course of infection. We have overcome this problem by generating genetically modified parasites that express a bioluminescent/fluorescent fusion protein. By combining in vivo imaging and confocal microscopy, this has enabled us to routinely visualise murine infections at the level of individual host cells. These studies reveal that intracellular parasite replication is an asynchronous process, irrespective of tissue location or disease stage. Furthermore, using TUNEL assays and EdU labelling, we demonstrate that within individual infected cells, replication of both mitochondrial (kDNA) and nuclear genomes is not co-ordinated within the parasite population, and that replicating amastigotes and non-replicating trypomastigotes can co-exist in the same cell. Finally, we report the presence of distinct non-canonical morphological forms of T. cruzi in the mammalian host. These appear to represent transitional forms in the amastigote to trypomastigote differentiation process. Therefore, the intracellular life-cycle of T. cruzi in vivo is more complex than previously realised, with potential implications for our understanding of disease pathogenesis, immune evasion and drug development. Dissecting the mechanisms involved will be an important experimental challenge., Author summary Chagas disease, caused by the protozoan parasite Trypanosoma cruzi, is becoming an emerging threat in non-endemic countries and establishing new foci in endemic countries. The treatment available has not changed significantly in over 40 years. Therefore, there is an urgent need for a greater understanding of parasite biology and disease pathogenesis to identify new therapeutic targets and to maximise the efficient use of existing drugs. We have used genetically modified strains of T. cruzi carrying a bioluminescence/fluorescence dual reporter fusion gene to monitor parasite replication in vivo during both acute and chronic infections in a mouse model. Utilising TUNEL assays for mitochondrial DNA replication and EdU incorporation for total DNA replication, we have found that parasite division within infected cells is asynchronous in all phases of infection. Differentiation also appears to be uncoordinated, with replicating amastigotes co-existing with non-dividing trypomastigotes in the same host cell.

Background Ambroxol relieves cough symptoms based on its secretagogue, anti-inflammatory, anti-oxidant, anti-bacterial, anti-viral, immunomodulatory and local anesthetic effects. The present study was designed to explore differential patient profiles and efficacy against acute respiratory symptoms of four formulations registered as over-the-counter medicines. Methods Nine hundred sixty-five pharmacy customers purchasing one of four branded ambroxol formulations (extended release capsules, adult syrup, pediatric syrup and soft pastilles) filled a questionnaire including a patient-adapted version of the Bronchitis Severity Scale, several questions on degree of impairment by acute cough, time to onset of symptom relief and duration of treatment. Data on pediatric syrup users were entered by their parents. Based on the exploratory character of the study, no hypothesis-testing statistical analysis was applied. Results Users of the pediatric syrup and the pastilles reported somewhat less severe baseline symptoms. The patient-adapted Bronchitis Severity Scale proved feasible as a self-administered tool. Among BSS items, ambroxol formulations improved chest pain while coughing to the largest and sputum to smallest degree (− 75% vs. -40%). Reported efficacy was comparable among formulations with minor differences in favor of the pediatric syrup. Time to onset of symptom relief was less than 60 min in more than 90% of patients and occurred prior to known systemic tmax. Time to onset was the parameter with the greatest differences between formulations, being reported fastest with pastilles and pediatric syrup and, as expected, slowest with extended release capsules. All ambroxol formulations were well tolerated. Conclusions We conclude that over-the-counter formulations of ambroxol exhibit comparable user profiles and efficacy. Differences in speed of onset of symptom relief may involve not only those in systemic pharmacokinetics but also local anesthetic effects of immediate release formulations. Differences between pediatric and adult syrup may in part reflect reporting bias.

Yeast and fast-growing human tumor cells share metabolic similarities in that both cells use fermentation of glucose for energy and both are highly sensitive to the glucose analog 2-deoxyglucose. Spontaneous mutations in S. cerevisiae that conferred resistance to 2-deoxyglucose were identified by whole genome sequencing. Missense alleles of the HXK2, REG1, GLC7 and SNF1 genes were shown to confer significant resistance to 2-deoxyglucose and all had the potential to alter the activity and or target selection of the Snf1 kinase signaling pathway. All three missense alleles in HXK2 resulted in significantly reduced catalytic activity. Addition of 2DG promotes endocytosis of the glucose transporter Hxt3. All but one of the 2DG-resistant strains reduced the 2DG-mediated hexose transporter endocytosis by increasing plasma membrane occupancy of the Hxt3 protein. Increased expression of the DOG (deoxyglucose) phosphatases has been associated with resistance to 2-deoxyglucose. Expression of both the DOG1 and DOG2 mRNA was elevated after treatment with 2-deoxyglucose but induction of these genes is not associated with 2DG-resistance. RNAseq analysis of the transcriptional response to 2DG showed large scale, genome-wide changes in mRNA abundance that were greatly reduced in the 2DG resistant strains. These findings suggest the common adaptive response to 2DG is to limit the magnitude of the response. Genetic studies of 2DG resistance using the dominant SNF1-G53R allele in cells that are genetically compromised in both the endocytosis and DOG pathways suggest that at least one more mechanism for conferring resistance to this glucose analog remains to be discovered., Author summary Yeast and fast-growing human tumor cells share metabolic similarities in that both cells use fermentation of glucose for energy and both are highly sensitive to the glucose analog 2-deoxyglucose. Another similarity between yeast cells and human tumor cells is that both cells can acquire resistance to 2-deoxyglucose, an outcome that can limit the usefulness of some cancer therapeutics. In this study, we used bakers’ yeast as a model organism to better understand the mechanism of toxicity and acquisition of resistance to 2-deoxyglucose. Spontaneous mutations in S. cerevisiae that conferred resistance to 2-deoxyglucose were isolated and identified by whole genome sequencing, a technology that was not available until recently. Our studies indicate that 2-deoxyglucose becomes toxic after it is phosphorylated by an enzyme called hexokinase. One important route to resistance is to reduce hexokinase activity. Other parallel pathways to resistance include increased expression of a hydrolase that degrades the toxic metabolite, altered localization of glucose transporters and altered glucose signal transduction pathways.

Background:Expression of a capsule from one of serogroups A, B, C, W, X or Y is usually required forNeisseria meningitidis(Nme) to cause invasive meningococcal disease. The capsule is encoded by the capsule locus,cps, which is proposed to have been acquired by a formerly capsule null organism by horizontal genetic transfer (HGT) from another species. Following identification of putative capsule genes in non-pathogenicNeisseriaspecies, this hypothesis is re-examined.Methods:Whole genome sequence data fromNeisseriaspecies, includingNmegenomes from a diverse range of clonal complexes and capsule genogroups, and non-Neisseriaspecies, were obtained from PubMLST and GenBank. Sequence alignments of genes from the meningococcalcps, and predicted orthologues in other species, were analysed using Neighbor-nets, BOOTSCANing and maximum likelihood phylogenies.Results:The meningococcalcpswas highly mosaic within regions B, C and D. A subset of sequences within regions B and C were phylogenetically nested within homologous sequences belonging toN. subflava, consistent with HGT event in whichN. subflavawas the donor. In thecpsof 23/39 isolates, the two copies of region D were highly divergent, withrfbABC’sequences being more closely related to predicted orthologues in the proposed speciesN. weixii (GenBank accession numberCP023429.1) than the same genes inNmeisolates lacking a capsule. There was also evidence of mosaicism in therfbABC’sequences of the remaining 16 isolates, as well asrfbABCfrom many isolates.Conclusions:Data are consistent with theen blocacquisition ofcpsin meningococci fromN. subflava, followed by further recombination events with otherNeisseriaspecies. Nevertheless, the data cannot refute an alternative model, in which native meningococcal capsule existed prior to undergoing HGT withN. subflavaand other species. Within-genus recombination events may have given rise to the diversity of meningococcal capsule serogroups.

Pseudomonas aeruginosa (P. aeruginosa) is an important cause of healthcare-associated infections, particularly in immunocompromised patients. Understanding how this multi-drug resistant pathogen is transmitted within intensive care units (ICUs) is crucial for devising and evaluating successful control strategies. While it is known that moist environments serve as natural reservoirs for P. aeruginosa, there is little quantitative evidence regarding the contribution of environmental contamination to its transmission within ICUs. Previous studies on other nosocomial pathogens rely on deploying specific values for environmental parameters derived from costly and laborious genotyping. Using solely longitudinal surveillance data, we estimated the relative importance of P. aeruginosa transmission routes by exploiting the fact that different routes cause different pattern of fluctuations in the prevalence. We developed a mathematical model including background transmission, cross-transmission and environmental contamination. Patients contribute to a pool of pathogens by shedding bacteria to the environment. Natural decay and cleaning of the environment lead to a reduction of that pool. By assigning the bacterial load shed during an ICU stay to cross-transmission, we were able to disentangle environmental contamination during and after a patient’s stay. Based on a data-augmented Markov Chain Monte Carlo method the relative importance of the considered acquisition routes is determined for two ICUs of the University hospital in Besançon (France). We used information about the admission and discharge days, screening days and screening results of the ICU patients. Both background and cross-transmission play a significant role in the transmission process in both ICUs. In contrast, only about 1% of the total transmissions were due to environmental contamination after discharge. Based on longitudinal surveillance data, we conclude that cleaning improvement of the environment after discharge might have only a limited impact regarding the prevention of P.A. infections in the two considered ICUs of the University hospital in Besançon. Our model was developed for P. aeruginosa but can be easily applied to other pathogens as well., Author summary Understanding the transmission dynamics of multi-drug resistant pathogens in intensive-care units is essential for designing successful infection control strategies. We developed a method that estimates the relative importance of several transmission routes of Pseudomonas aeruginosa (P. aeruginosa), a bacterium intrinsically resistant to multiple antibiotics and known to be a major contributor to hospital-acquired infections. Our model includes three different routes: background transmission, cross-transmission and environmental contamination. Since moist environments may serve as natural reservoirs for P. aeruginosa, we focused our study on environmental contamination. Patients contribute to a pool of pathogens by shedding bacteria to the environment. Natural decay and cleaning of the environment lead to a reduction of that pool. By assigning the bacterial load shed during an ICU stay to cross-transmission, we disentangled environmental contamination during and after a patient’s stay. Previous studies were able to assess the role of environmental contamination for specific hospitals using laborious and costly genotyping methods. In contrast, we only used surveillance screening data to estimate the relative contributions of the considered transmission routes. Our results can be used to tailor or assess the effect of interventions. Our model was developed for P. aeruginosa but can be easily applied to other pathogens as well.

Previous studies of adaptation to the glucose analog, 2-deoxyglucose, by Saccharomyces cerevisiae have utilized haploid cells. In this study, diploid cells were used in the hope of identifying the distinct genetic mechanisms used by diploid cells to acquire drug resistance. While haploid cells acquire resistance to 2-deoxyglucose primarily through recessive alleles in specific genes, diploid cells acquire resistance through dominant alleles, haploinsufficiency, gene duplication and aneuploidy. Dominant-acting, missense alleles in all three subunits of yeast AMP-activated protein kinase confer resistance to 2-deoxyglucose. Dominant-acting, nonsense alleles in the REG1 gene, which encodes a negative regulator of AMP-activated protein kinase, confer 2-deoxyglucose resistance through haploinsufficiency. Most of the resistant strains isolated in this study achieved resistance through aneuploidy. Cells with a monosomy of chromosome 4 are resistant to 2-deoxyglucose. While this genetic strategy comes with a severe fitness cost, it has the advantage of being readily reversible when 2-deoxyglucose selection is lifted. Increased expression of the two DOG phosphatase genes on chromosome 8 confers resistance and was achieved through trisomies and tetrasomies of that chromosome. Finally, resistance was also mediated by increased expression of hexose transporters, achieved by duplication of a 117 kb region of chromosome 4 that included the HXT3, HXT6 and HXT7 genes. The frequent use of aneuploidy as a genetic strategy for drug resistance in diploid yeast and human tumors may be in part due to its potential for reversibility when selection pressure shifts., Author summary Yeast and fast-growing human tumor cells share metabolic similarities in that both cells use fermentation of glucose for energy and both are highly sensitive to the glucose analog 2-deoxyglucose. In an earlier study, we examined the genetic mechanism used by haploid yeast cells to acquire resistance to 2-deoxyglucose. Here, we conducted the same genetic selection on diploid cells. Diploid cells use dominant alleles, haploinsufficiency, gene duplication and aneuploidy to achieve drug resistance. The frequent use of aneuploidy as a genetic strategy for drug resistance in diploid yeast and human tumors may be in part due to its potential for reversibility when selection pressure shifts.

The two-tank water system is common practice for the storage and distribution of water in many homes. Water is transported via a pipeline network from the storage tank (lower tank) to the distribution tank (overhead tank) using an electric pumping machine. Due to limited control in the existing pumping system, water wastage becomes inevitable. Determining the quality of water in the overhead tank before supply in the home is still unaddressed. In this work, an integrated Android mobile App and a control system were developed to assess the water quality, perform level check in the overhead tank, and activate intelligent pumping control. An ultrasonic pulse-echo technique was used for water level checks, while the water turbidity and pH signals were used for water quality checks. Three-level control conditions (LC_1, LC_2, LC_3) and two water quality check conditions (QC_1 and QC_2) were devised and used in the intelligent control algorithm of the system. Control valve1 regulates the flushable poor water quality while valve2 regulates the house's supply of good water quality. The absolute relative error between the expected time and the system time of filling the tank level was observed to be less than 10% when the water volume is less than 81%. Hence, distortion in the sensory signals increases and worsen as the water level approaches the ultrasonic sensor position. The poor internet signal network was observed to affect the real-time monitoring and automation of the system control through delay in system responses to commands. However, the average recorded response time of the system is 3 s, and it could be less in the situation of good internet network services., Firebase, Pitch-catch, Pulse-echo, Time-of-flight, Turbidity, pH scale.

Pseudomonas aeruginosa is a highly versatile, antibiotic-resistant Gram-negative bacterium known for causing opportunistic infections and contamination of industrial products. Despite extensive genomic analysis of clinical P. aeruginosa strains, no genomes exist for preservative-tolerant industrial strains. A unique collection of 69 industrial isolates was assembled and compared to clinical and environmental strains; 16 genetically distinct industrial strains were subjected to array tube genotyping, multilocus sequence typing and whole-genome sequencing. The industrial strains possessed high preservative tolerance and were dispersed widely across P. aeruginosa as a species, but recurrence of strains from the same lineage within specific industrial products and locations was identified. The industrial P. aeruginosa genomes (mean=7.0 Mb) were significantly larger than those of previously sequenced environmental (mean=6.5 Mb; n=19) and clinical (mean=6.6 Mb; n=66) strains. Complete sequencing of the P. aeruginosa industrial strain RW109, which encoded the largest genome (7.75 Mb), revealed a multireplicon structure including a megaplasmid (555 265 bp) and large plasmid (151 612 bp). The RW109 megaplasmid represented an emerging plasmid family conserved in seven industrial and two clinical P. aeruginosa strains, and associated with extremely stress-resilient phenotypes, including antimicrobial resistance and solvent tolerance. Here, by defining the detailed phylogenomics of P. aeruginosa industrial strains, we show that they uniquely possess multireplicon, megaplasmid-bearing genomes, and significantly greater genomic content worthy of further study.

Pump-probe microscopy identifies metacinnabar and metallic mercury as possible degradation product of vermilion (α-HgS)., Here, we demonstrate the use of pump-probe microscopy for high-resolution studies of vermilion degradation. Vermilion (mostly α-HgS), an important red pigment used in historical paintings, blackens over time, and metallic Hg and β-HgS have been implicated as possible degradation products. Conventional analysis techniques have trouble differentiating α- and β-HgS with sufficiently high spatial resolution. However, pump-probe microscopy can differentiate metallic mercury, α- and β-HgS, and map each distribution on the microscopic scale. We studied artificial degradation of α-HgS; femtosecond-pulsed laser irradiation induces an irreversible phase shift of α- to β-HgS, in which the initial presence of β-HgS grains can increase the rate of conversion in their vicinity. Continuous ultraviolet exposure instead generates both liquid Hg and β-HgS, with a conversion rate that increases with elevated temperatures. Last, we reveal the presence of β-HgS as a natural degradation product in discolored vermilion layers in a 14th century Italian painting.

Infections of humans and livestock with African trypanosomes are treated with drugs introduced decades ago that are not always fully effective and often have severe side effects. Here, the trypanosome haptoglobin-haemoglobin receptor (HpHbR) has been exploited as a route of uptake for an antibody-drug conjugate (ADC) that is completely effective against Trypanosoma brucei in the standard mouse model of infection. Recombinant human anti-HpHbR monoclonal antibodies were isolated and shown to be internalised in a receptor-dependent manner. Antibodies were conjugated to a pyrrolobenzodiazepine (PBD) toxin and killed T. brucei in vitro at picomolar concentrations. A single therapeutic dose (0.25 mg/kg) of a HpHbR antibody-PBD conjugate completely cured a T. brucei mouse infection within 2 days with no re-emergence of infection over a subsequent time course of 77 days. These experiments provide a demonstration of how ADCs can be exploited to treat protozoal diseases that desperately require new therapeutics., Author summary Here we show that antibody-drug conjugates (ADCs) can be re-purposed from cancer immunotherapeutics to anti-protozoals by changing the specificity of the immunoglobulin to target a trypanosome cell surface receptor. Trypanosomes were used as a model system due to the availability of receptor null cell lines that allowed the unambiguous demonstration that ADCs targeted to a parasite surface receptor could be specifically internalised via receptor-mediated endocytosis. A single low dose of the resulting ADC was able to cure a stage 1 mouse model of trypanosome infection. We have used toxins and conjugation chemistry that are identical to anti-cancer ADCs demonstrating the ability to piggy-back onto the huge research efforts and resources that are being invested in the development of such ADCs. The potential for development of ADCs against a wide range of human pathogens is vast, where only epitope binding sites need vary in order to provide selectivity. This provides a far-reaching opportunity for the rapid development of novel anti-protozoals for the targeted killing of a wide range of pathogens that cause disease worldwide, especially in developing countries.

Summary Many eukaryotic green algae possess biophysical carbon‐concentrating mechanisms (CCMs) that enhance photosynthetic efficiency and thus permit high growth rates at low CO 2 concentrations. They are thus an attractive option for improving productivity in higher plants. In this study, the intracellular locations of ten CCM components in the unicellular green alga Chlamydomonas reinhardtii were confirmed. When expressed in tobacco, all of these components except chloroplastic carbonic anhydrases CAH3 and CAH6 had the same intracellular locations as in Chlamydomonas. CAH6 could be directed to the chloroplast by fusion to an Arabidopsis chloroplast transit peptide. Similarly, the putative inorganic carbon (Ci) transporter LCI1 was directed to the chloroplast from its native location on the plasma membrane. CCP1 and CCP2 proteins, putative Ci transporters previously reported to be in the chloroplast envelope, localized to mitochondria in both Chlamydomonas and tobacco, suggesting that the algal CCM model requires expansion to include a role for mitochondria. For the Ci transporters LCIA and HLA3, membrane location and Ci transport capacity were confirmed by heterologous expression and H14 CO 3 ‐ uptake assays in Xenopus oocytes. Both were expressed in Arabidopsis resulting in growth comparable with that of wild‐type plants. We conclude that CCM components from Chlamydomonas can be expressed both transiently (in tobacco) and stably (in Arabidopsis) and retargeted to appropriate locations in higher plant cells. As expression of individual Ci transporters did not enhance Arabidopsis growth, stacking of further CCM components will probably be required to achieve a significant increase in photosynthetic efficiency in this species.

Introduction and aimsSince 2013 MenC and MenW disease incidence and associated mortality rates have increased in the Republic of Ireland. From 2002/2003 to 2012/2013, the average annual MenC incidence was 0.08/100,000, which increased to 0.34/100,000 during 2013/2014 to 2017/18, peaking in 2016/17 (0.72/100,000) with an associated case fatality rate (CFR) of 14.7%. MenW disease incidence has increased each year from 0.02/100,000 in 2013/2014, to 0.29/100,000 in 2017/18, with an associated CFR of 28.6%. We aimed to characterise and relate recent MenC isolates to the previously prevalent MenC:cc11 ET-15 clones, and also characterise and relate recent MenW isolates to the novel 'Hajj' clones.MethodsUsing WGS we characterised invasive (n = 74, 1997/98 to 2016/17) and carried (n = 16, 2016/17) MenC isolates, and invasive (n = 18, 2010/11 to 2016/17) and carried (n = 15, 2016/17) MenW isolates. Genomes were assembled using VelvethOptimiser and stored on the PubMLST Neisseria Bacterial Isolate Genome Sequence Database. Isolates were compared using the cgMLST approach.ResultsMost MenC and MenW isolates identified were cc11. A single MenC:cc11 sub-lineage contained the majority (68%, n = 19/28) of recent MenC:cc11 disease isolates and all carried MenC:cc11 isolates, which were interspersed and distinct from the historically significant ET-15 clones. MenW:cc11 study isolates clustered among international examples of both the original UK 2009 MenW:cc11, and novel 2013 MenW:cc11clones.ConclusionsWe have shown that the majority of recent MenC disease incidence was caused by strain types distinct from the MenC:cc11 ET-15 clone of the late 1990s, which still circulate but have caused only sporadic disease in recent years. We have identified that the same aggressive MenW clone now established in several other European countries, is endemic in the RoI and responsible for the recent MenW incidence increases. This data informed the National immunisation Advisory Committee, who are currently deliberating a vaccine policy change to protect teenagers.

Introduction. Progenitor cells cultured on biomaterials with optimal physical-topographical properties respond with alignment and differentiation. Stromal cells from connective tissue can adversely differentiate to profibrotic myofibroblasts or favorably to smooth muscle cells (SMC). We hypothesized that myogenic differentiation of adipose tissue-derived stromal cells (ASC) depends on gradient directional topographic features. Methods. Polydimethylsiloxane (PDMS) samples with nanometer and micrometer directional topography gradients (wavelength w=464-10, 990 nm; amplitude a=49-3, 425 nm) were fabricated. ASC were cultured on patterned PDMS and stimulated with TGF-β1 to induce myogenic differentiation. Cellular alignment and adhesion were assessed by immunofluorescence microscopy after 24 h. After seven days, myogenic differentiation was examined by immunofluorescence microscopy, gene expression, and immunoblotting. Results. Cell alignment occurred on topographies larger than w=1758 nm/a=630 nm. The number and total area of focal adhesions per cell were reduced on topographies from w=562 nm/a=96 nm to w=3919 nm/a=1430 nm. Focal adhesion alignment was increased on topographies larger than w=731 nm/a=146 nm. Less myogenic differentiation of ASC occurred on topographies smaller than w=784 nm/a=209 nm. Conclusion. ASC adherence, alignment, and differentiation are directed by topographical cues. Our evidence highlights a minimal topographic environment required to facilitate the development of aligned and differentiated cell layers from ASC. These data suggest that nanotopography may be a novel tool for inhibiting fibrosis.

The spread of antibiotic resistance within and between different bacterial populations is a major health problem on a global scale. The identification of genetic transformation in genomic data from Neisseria meningitidis, the meningococcus (Mc), and other bacteria is problematic, since similar or even identical alleles may be involved. A particular challenge in naturally transformable bacteria generally is to distinguish between common ancestry and true recombined sites in sampled genome sequences. Furthermore, the identification of recombination following experimental transformation of homologous alleles requires identifiable differences between donor and recipient, which in itself influences the propensity for homologous recombination (HR). This study identifies the distribution of HR events following intraspecies and interspecies Mc transformations of rpoB alleles encoding rifampicin resistance by whole-genome DNA sequencing and single nucleotide variant analysis. The HR events analysed were confined to the genomic region surrounding the single nucleotide genetic marker used for selection. An exponential length distribution of these recombined events was found, ranging from a few nucleotides to about 72 kb stretches. The lengths of imported sequences were on average found to be longer following experimental transformation of the recipient with genomic DNA from an intraspecies versus an interspecies donor (P

The meningococcal antigen typing system (MATS) is an enzyme-linked immunosorbent assay (ELISA) that measures both the levels of expression and the immune reactivity of the three recombinant 4CMenB antigens. Together with PorA variable-region sequence data, this system provides an estimation of how susceptible MenB isolates are to killing by 4CMenB vaccine-induced antibodies. Assays based on subcapsular antigen phenotype analyses, such as MATS, are important in situations where conventional vaccine coverage estimations are not possible. Subcapsular antigens are typically highly diverse across strains, and vaccine coverage estimations would require unfeasibly large efficacy trials and screening of an exhaustive strain panel for antibody functional activity. Here, MATS was applied to all invasive meningococcal serogroup B (MenB) strains isolated over four consecutive epidemiological years (n = 105) and predicted reasonably high 4CMenB vaccine coverage in the Republic of Ireland., Neisseria meningitidis is a common cause of bacterial meningitis in children and young adults worldwide. The 4CMenB vaccine (Bexsero), developed to combat meningococcal serogroup B (MenB) disease, contains subcapsular antigens that may induce immunity against strains of N. meningitidis, regardless of serogroup. Owing to differential levels of expression and peptide diversity in vaccine antigens across meningococcal strains, the meningococcal antigen typing system (MATS) was developed to estimate the potential MenB strain coverage of 4CMenB. Prior to introducing the 4CMenB vaccine into routine use, we sought to estimate the potential 4CMenB coverage against invasive MenB strains isolated in the Republic of Ireland (RoI) over four consecutive epidemiological years. MATS was applied to a panel of 105 invasive MenB strains isolated during July 2009 to June 2013. Sequence data characterizing the multilocus sequence typing (MLST) alleles and the major 4CMenB target peptides were extracted from isolate genome sequence data, hosted in the Bacterial Isolate Sequencing database (BIGSdb). MATS data indicated that 4CMenB may induce protective immunity against 69.5% (95% confidence interval [CI95%], 64.8% to 84.8%) of circulating MenB strains. Estimated coverage was highest against the most prevalent disease-causing lineage, cc41/44, where the most frequently observed sequence types, ST-154 and ST-41 (21% of isolates, collectively), were typically covered by three antigens. No significant temporal trends were observed. Overall, these data provide a baseline of strain coverage prior to the introduction of 4CMenB and indicate that a decrease in invasive meningococcal disease (IMD) is predicted following the introduction of 4CMenB into the routine infant immunization schedule in the RoI. IMPORTANCE The meningococcal antigen typing system (MATS) is an enzyme-linked immunosorbent assay (ELISA) that measures both the levels of expression and the immune reactivity of the three recombinant 4CMenB antigens. Together with PorA variable-region sequence data, this system provides an estimation of how susceptible MenB isolates are to killing by 4CMenB vaccine-induced antibodies. Assays based on subcapsular antigen phenotype analyses, such as MATS, are important in situations where conventional vaccine coverage estimations are not possible. Subcapsular antigens are typically highly diverse across strains, and vaccine coverage estimations would require unfeasibly large efficacy trials and screening of an exhaustive strain panel for antibody functional activity. Here, MATS was applied to all invasive meningococcal serogroup B (MenB) strains isolated over four consecutive epidemiological years (n = 105) and predicted reasonably high 4CMenB vaccine coverage in the Republic of Ireland.

The Arctic Ocean is an early warning system for indicators and effects of climate change. We use a novel combination of experimental and time-series data on effects of ocean warming and acidification on the commercially important Northeast Arctic cod (Gadus morhua) to incorporate these physiological processes into the recruitment model of the fish population. By running an ecological-economic optimization model, we investigate how the interaction of ocean warming, acidification and fishing pressure affects the sustainability of the fishery in terms of ecological, economic, social and consumer-related indicators, ranging from present day conditions up to future climate change scenarios. We find that near-term climate change will benefit the fishery, but under likely future warming and acidification this large fishery is at risk of collapse by the end of the century, even with the best adaptation effort in terms of reduced fishing pressure.

Background The long term and complex nature of Chagas disease in humans has restricted studies on vaccine feasibility. Animal models also have limitations due to technical difficulties in monitoring the extremely low parasite burden that is characteristic of chronic stage infections. Advances in imaging technology offer alternative approaches that circumvent these problems. Here, we describe the use of highly sensitive whole body in vivo imaging to assess the efficacy of recombinant viral vector vaccines and benznidazole-cured infections to protect mice from challenge with Trypanosoma cruzi. Methodology/Principal findings Mice were infected with T. cruzi strains modified to express a red-shifted luciferase reporter. Using bioluminescence imaging, we assessed the degree of immunity to re-infection conferred after benznidazole-cure. Those infected for 14 days or more, prior to the onset of benznidazole treatment, were highly protected from challenge with both homologous and heterologous strains. There was a >99% reduction in parasite burden, with parasites frequently undetectable after homologous challenge. This level of protection was considerably greater than that achieved with recombinant vaccines. It was also independent of the route of infection or size of the challenge inoculum, and was long-lasting, with no significant diminution in immunity after almost a year. When the primary infection was benznidazole-treated after 4 days (before completion of the first cycle of intracellular infection), the degree of protection was much reduced, an outcome associated with a minimal T. cruzi-specific IFN-γ+ T cell response. Conclusions/Significance Our findings suggest that a protective Chagas disease vaccine must have the ability to eliminate parasites before they reach organs/tissues, such as the GI tract, where once established, they become largely refractory to the induced immune response., Author summary Chagas disease, which is caused by the protozoan parasite Trypanosoma cruzi, is a major public health problem throughout Latin America. Attempts to develop a vaccine have been hampered by technical difficulties in monitoring the extremely low parasite burden during the life-long chronic stage of infection. To circumvent these issues, we used highly sensitive bioluminescence imaging to assess the ability of recombinant viral vector vaccines and drug-cured infections to confer protection against experimental challenge in mice. We observed that drug-cured infections were much more effective than subunit vaccines. Efficacy was independent of the route of infection or size of the challenge inoculum, and was undiminished after almost a year. In addition, drug-cured infections conferred a high level of cross-strain protection. The highly sensitive imaging procedures enabled us to visualise parasite distribution in mice where full protection was not achieved. This suggested that to confer protection, vaccines must prevent the infection of organs/tissues that act as parasite reservoirs during the chronic stage. Once established at these sites, parasites become largely refractory to vaccine-induced elimination.

While tuberculosis (TB) represents a significant disease burden worldwide, low-incidence countries strive to reach the WHO target of pre-elimination by 2035. Screening for TB in immigrants is an important component of the strategy to reduce the TB burden in low-incidence settings. An important option is the screening and preventive treatment of latent TB infection (LTBI). Whether this policy is worthwhile depends on the extent of transmission within the country, and introduction of new cases through import. Mathematical transmission models of TB have been used to identify key parameters in the epidemiology of TB and estimate transmission rates. An important application has also been to investigate the consequences of policy scenarios. Here, we formulate a mathematical model for TB transmission within the Netherlands to estimate the size of the pool of latent infections, and to determine the share of importation-either through immigration or travel- versus transmission within the Netherlands. We take into account importation of infections due to immigration, and travel to the country of origin, focusing on the three ethnicities most represented among foreign-born TB cases (after exclusion of those overrepresented among asylum seekers): Moroccans, Turkish and Indonesians. We fit a system of ordinary differential equations to the data from the Netherlands Tuberculosis Registry on (extra-)pulmonary TB cases from 1995-2013. We estimate that about 27% of Moroccans, 25% of Indonesians, and 16% of Turkish, are latently infected. Furthermore, we find that for all three foreign-born communities, immigration is the most important source of LTBI, but the extent of within-country transmission is much lower (about half) for the Turkish and Indonesian communities than for the Moroccan. This would imply that contact investigation would have a greater yield in the latter community than in the former. Travel remains a minor factor contributing LTBI, suggesting that targeting returning travelers might be less effective at preventing LTBI than immigrants upon entry in the country.

The endoplasmic reticulum (ER) supports biosynthesis of proteins with diverse transmembrane domain (TMD) lengths and hydrophobicity. Features in transmembrane domains such as charged residues in ion channels are often functionally important, but could pose a challenge during cotranslational membrane insertion and folding. Our systematic proteomic approaches in both yeast and human cells revealed that the ER membrane protein complex (EMC) binds to and promotes the biogenesis of a range of multipass transmembrane proteins, with a particular enrichment for transporters. Proximity-specific ribosome profiling demonstrates that the EMC engages clients cotranslationally and immediately following clusters of TMDs enriched for charged residues. The EMC can remain associated after completion of translation, which both protects clients from premature degradation and allows recruitment of substrate-specific and general chaperones. Thus, the EMC broadly enables the biogenesis of multipass transmembrane proteins containing destabilizing features, thereby mitigating the trade-off between function and stability.