ABSTRACT

Interspecific hybridization can drive evolutionary adaptation to novel environments. The Saccharomycotina clade of budding yeasts includes many hybrid lineages, and hybridization has been proposed as a source for new pathogenic species. Candida orthopsilosis is an emerging opportunistic pathogen for which most clinical isolates are hybrids, each derived from one of at least four independent crosses between the same two parental lineages. To gain insight into the transcriptomic aftermath of hybridization in these pathogens, we analyzed allele-specific gene expression in two independently formed hybrid strains and in a homozygous strain representative of one parental lineage. Our results show that the effect of hybridization on overall gene expression is rather limited, affecting ~4% of the genes studied. However, we identified a larger effect in terms of imbalanced allelic expression, affecting ~9.5% of the heterozygous genes in the hybrids. This effect was larger in the hybrid with more extensive loss of heterozygosity, which may indicate a tendency to avoid loss of heterozygosity in these genes. Consistently, the number of shared genes with allele-specific expression in the two independently formed hybrids was higher than random expectation, suggesting selective retention. Some of the imbalanced genes have functions related to pathogenicity, including zinc transport and superoxide dismutase activities. While it remains unclear whether the observed imbalanced genes play a role in virulence, our results suggest that differences in allele-specific expression may add an additional layer of phenotypic plasticity to traits related to virulence in C. orthopsilosis hybrids.

IMPORTANCE How new pathogens emerge is an important question that remains largely unanswered. Some emerging yeast pathogens are hybrids originated through the crossing of two different species, but how hybridization contributes to higher virulence is unclear. Here, we show that hybrids selectively retain gene regulation plasticity inherited from the two parents and that this plasticity affects genes involved in virulence.

ABSTRACT

The prevailing paradigm in obstetrics has been the sterile womb hypothesis. However, some are asserting that the placenta, intra-amniotic environment, and fetus harbor microbial communities. The objective of this study was to determine whether the fetal and placental tissues of rhesus macaques harbor bacterial communities. Fetal, placental, and uterine wall samples were obtained from cesarean deliveries without labor (~130/166 days gestation). The presence of bacteria in the fetal intestine and placenta was investigated through culture. The bacterial burden and profiles of the placenta, umbilical cord, and fetal brain, heart, liver, and colon were determined through quantitative real-time PCR and DNA sequencing. These data were compared with those of the uterine wall as well as to negative and positive technical controls. Bacterial cultures of fetal and placental tissues yielded only a single colony of Cutibacterium acnes. This bacterium was detected at a low relative abundance (0.02%) in the 16S rRNA gene profile of the villous tree sample from which it was cultured, yet it was also identified in 12/29 background technical controls. The bacterial burden and profiles of fetal and placental tissues did not exceed or differ from those of background technical controls. By contrast, the bacterial burden and profiles of positive controls exceeded and differed from those of background controls. Among the macaque samples, distinct microbial signals were limited to the uterine wall. Therefore, using multiple modes of microbiologic inquiry, there was not consistent evidence of bacterial communities in the fetal and placental tissues of rhesus macaques.

IMPORTANCE Microbial invasion of the amniotic cavity (i.e., intra-amniotic infection) has been causally linked to pregnancy complications, especially preterm birth. Therefore, if the placenta and the fetus are typically populated by low-biomass microbial communities, current understanding of the role of microbes in reproduction and pregnancy outcomes will need to be fundamentally reconsidered. Could these communities be of benefit by competitively excluding potential pathogens or priming the fetal immune system for the microbial bombardment it will experience upon delivery? If so, what properties (e.g., microbial load and community membership) of these microbial communities preclude versus promote intra-amniotic infection? Given the ramifications of the in utero colonization hypothesis, critical evaluation is required. In this study, using multiple modes of microbiologic inquiry (i.e., culture, quantitative real-time PCR [qPCR], and DNA sequencing) and controlling for potential background DNA contamination, we did not find consistent evidence for microbial communities in the placental and fetal tissues of rhesus macaques.

Molar element ratio analysis of element concentrations consists of four basic tools that provide substantial insight into the lithogeochemistry (and mineralogy) of rocks under examination. These tools consist of: (1) conserved element ratio analysis; (2) Pearce element ratio analysis; (3) general element ratio analysis; and (4) lithogeochemical mineral mode analysis. Conserved element ratio analysis is useful in creating a chemostratigraphic model for the host rocks to mineral deposits, whereas Pearce element ratio analysis and general element ratio analysis are primarily used to identify mineralogical and metasomatic controls on rock compositions and to investigate and quantify the extent of the material transfers that formed the host rocks and mineralization. Lithogeochemical mineral mode analysis converts element concentrations into mineral concentrations using a matrix-based change-of-basis operation, allowing lithogeochemical data to be interpreted in terms of mineral modes. It can be used to provide proper names to rocks, an important activity for an exploration geologist because of the implications that rock names have on genetic processes and mineral deposit models.

This paper provides a review of the theoretical foundations of each of these four tools and then illustrates how these techniques have been used in a variety of exploration applications to assist in the search for, evaluation and planning of, and the mining of mineral deposits. Examples include the evaluation of total digestion lithogeochemical datasets from mineral deposits hosted by igneous and sedimentary rocks and formed by hydrothermal and igneous processes. In addition, this paper illustrates a more recent geometallurgical application of these methods, whereby the mineral proportions determined by lithogeochemical mineral mode analysis are used to predict rock properties and obtain the ore body knowledge critical for resource evaluation, mine planning, mining and mine remediation.

Thematic collection: This article is part of the Exploration 17 collection available at: https://www.lyellcollection.org/cc/exploration-17

Multi-element geochemical surveys of rocks, soils, stream/lake/floodplain sediments and regolith are typically carried out at continental, regional and local scales. The chemistry of these materials is defined by their primary mineral assemblages and their subsequent modification by comminution and weathering. Modern geochemical datasets represent a multi-dimensional geochemical space that can be studied using multivariate statistical methods from which patterns reflecting geochemical/geological processes are described (process discovery). These patterns form the basis from which probabilistic predictive maps are created (process validation). Processing geochemical survey data requires a systematic approach to effectively interpret the multi-dimensional data in a meaningful way. Problems that are typically associated with geochemical data include closure, missing values, censoring, merging, levelling different datasets and adequate spatial sample design. Recent developments in advanced multivariate analytics, geospatial analysis and mapping provide an effective framework to analyse and interpret geochemical datasets. Geochemical and geological processes can often be recognized through the use of data discovery procedures such as the application of principal component analysis. Classification and predictive procedures can be used to confirm lithological variability, alteration and mineralization. Geochemical survey data of lake/till sediments from Canada and of floodplain sediments from Australia show that predictive maps of bedrock and regolith processes can be generated. Upscaling a multivariate statistics-based prospectivity analysis for arc-related Cu–Au mineralization from a regional survey in the southern Thomson Orogen in Australia to the continental scale, reveals a number of regions with a similar (or stronger) multivariate response and hence potentially similar (or higher) mineral potential throughout Australia.

Thematic collection: This article is part of the Exploration 17 collection available at: https://www.lyellcollection.org/cc/exploration-17

In the past decade, significant research efforts have been devoted to mineral chemistry studies to assist porphyry exploration. These activities can be divided into two major fields of research: (1) porphyry indicator minerals (PIMs), which are used to identify the presence of, or potential for, porphyry-style mineralization based on the chemistry of magmatic minerals such as zircon, plagioclase and apatite, or resistate hydrothermal minerals such as magnetite; and (2) porphyry vectoring and fertility tools (PVFTs), which use the chemical compositions of hydrothermal minerals such as epidote, chlorite and alunite to predict the likely direction and distance to mineralized centres, and the potential metal endowment of a mineral district. This new generation of exploration tools has been enabled by advances in and increased access to laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS), short-wave length infrared (SWIR), visible near-infrared (VNIR) and hyperspectral technologies. PIMs and PVFTs show considerable promise for exploration and are starting to be applied to the diversity of environments that host porphyry and epithermal deposits globally. Industry has consistently supported development of these tools, and in the case of PVFTs encouraged by several successful blind tests where deposit centres have successfully been predicted from distal propylitic settings. Industry adoption is steadily increasing but is restrained by a lack of the necessary analytical equipment and expertise in commercial laboratories, and also by the ongoing reliance on well-established geochemical exploration techniques (e.g. sediment, soil and rock chip sampling) that have aided the discovery of near-surface resources over many decades, but are now proving less effective in the search for deeply buried mineral resources and for those concealed under cover.

Thematic collection: This article is part of the Exploration 17 collection available at: https://www.lyellcollection.org/cc/exploration-17

Extractables and leachables (E&L) are identified and quantified so that their impact on patient safety can be established and assessed. The uncertainty in the impact assessment is affected by the uncertainty in the substance's experimentally determined identity and concentration. Thus, these experimentally determined quantities must be reported not only in terms of their absolute result but also in terms of the uncertainty in the result, which is based on the amount and rigor of the information on which the result is based. In this way, the impact assessment can be tempered to account for the uncertainty in its input data. To facilitate the assignment and reporting of uncertainty, classification hierarchies are proposed and discussed for both identification and quantitation. Both hierarchies establish levels or degrees of identification and quantitation based on the uncertainty of the result and contain descriptions of the quality and quantity of information required to achieve a certain level within the hierarchy. The minimal levels that must be achieved to support impact assessment are also established.

The bioprocessing industry uses recombinant mammalian cell lines to generate therapeutic biologic drugs. To ensure consistent product quality of the therapeutic proteins, it is imperative to have a controlled production process. Regulatory agencies and the biotechnology industry consider cell line "clonal origin" an important aspect of maintaining process control. Demonstration of clonal origin of the cell substrate, or production cell line, has received considerable attention in the past few years, and the industry has improved methods and devised standards to increase the probability and/or assurance of clonal derivation. However, older production cell lines developed before the implementation of these methods, herein referred to as "legacy cell lines," may not meet current regulatory expectations for demonstration of clonal derivation. In this article, the members of the IQ Consortium Working Group on Clonality present our position that the demonstration of process consistency and product comparability of critical quality attributes throughout the development life cycle should be sufficient to approve a license application without additional genetic analysis to support clonal origin, even for legacy cell lines that may not meet current day clonal derivation standards. With this commentary, we discuss advantages and limitations of genetic testing methods to support clonal derivation of legacy cell lines and wish to promote a mutual understanding with the regulatory authorities regarding their optional use during early drug development, subsequent to Investigational New Drug (IND) application and before demonstration of product and process consistency at Biologics License Applications (BLA) submission.

Trumenba (MenB-FHbp; bivalent rLP2086), the first meningococcal serogroup B vaccine approved in the United States and subsequently approved in Europe, Canada, and Australia, is well-characterized. Pfizer devised a control strategy approach by using a simplified control strategy wheel for Trumenba based on International Council for Harmonisation (ICH) Q8 (R2), Q9, Q10, and Q11 guidelines, which provide complementary guidance on pharmaceutical development, quality risk management, quality systems, and development and manufacture of drug substances, respectively. These guidelines ensure product quality using a scientific and risk-based approach. Trumenba contains two factor H binding proteins (FHbps), one from each of the two FHbp subfamilies (A and B), adsorbed onto aluminum phosphate. Trumenba manufacturing processes are complicated by the recombinant protein expression of Subfamily A and B proteins and the nature of the drug product (suspension in syringes); the latter also introduces challenges in controlling product critical quality attributes during the development process. In such complex systems, the control strategy is critical to ensuring consistent desired product quality; it also supports the regulatory requirement of continued improvement through continuous process verification and aids regulatory filing. This article describes Pfizer's approach toward robust control strategy development, built on product and process understanding, and links control strategy to regulatory document sections and flow of controls. Specifically, an approach is presented on product quality attribute criticality determination based on safety and efficacy and on an understanding of process parameter criticality. This was achieved by studying the impact of the approach on product quality attributes to define process parameter and in-process controls. This approach is further explained through Trumenba case studies, highlighting specific quality attributes and the associated controls implemented, and provides a holistic view of controls employed for both drug substance and drug product.

Glass is the favorite material for parenteral packaging because of its physico-chemical properties. Type I borosilicate glass is worldwide use at this scope, but it may have some issues related to breakage, corrosion and delamination that might compromise the drug quality, safety and efficacy. These issues can be mitigated and avoided starting from the appropriate selection of the most suitable raw material at the early stage of the glass container design. In this study, Type I borosilicate glass vials manufactured using two glass tubes having different chemical compositions, were studied and compared in terms of their resistance to corrosion. Testing design was applied with the aim to select the best practice approach comparing different storage simulation conditions: ageing treatment through autoclaving and stability testing (real-time and accelerated). Clear differences were found between the different glass types in terms of hydrolytic and corrosion resistance that highlighted the relation between chemical composition and glass chemical durability. Non-negligible differences were also observed using different storage conditions.

While flagella have been studied extensively as motility organelles, with a focus on internal structures such as the axoneme, more recent research has illuminated the roles of the flagellar surface in a variety of biological processes. Parasitic protists of the order Kinetoplastida, which include trypanosomes and Leishmania species, provide a paradigm for probing the role of flagella in host-microbe interactions and illustrate that this interface between the flagellar surface and the host is of paramount importance. An increasing body of knowledge indicates that the flagellar membrane serves a multitude of functions at this interface: attachment of parasites to tissues within insect vectors, close interactions with intracellular organelles of vertebrate cells, transactions between flagella from different parasites, junctions between the flagella and the parasite cell body, emergence of nanotubes and exosomes from the parasite directed to either host or microbial targets, immune evasion, and sensing of the extracellular milieu. Recent whole-organelle or genome-wide studies have begun to identify protein components of the flagellar surface that must mediate these diverse host-parasite interactions. The increasing corpus of knowledge on kinetoplastid flagella will likely prove illuminating for other flagellated or ciliated pathogens as well.

In bacterial populations, quorum sensing (QS) systems participate in the regulation of specialization processes and regulate collective behaviors that mediate interactions and allow survival of the species. In Gram-positive bacteria, QS systems of the RRNPP family (Rgg, Rap, NprR, PlcR, and PrgX) consist of intracellular receptors and their cognate signaling peptides. Two of these receptors, Rap and NprR, have regained attention in Bacillus subtilis and the Bacillus cereus group. Some Rap proteins, such as RapH and Rap60, are multifunctional and/or redundant in function, linking the specialization processes of sporulation and competence, as well as global expression changes in the transition phase in B. subtilis. NprR, an evolutionary intermediate between Rap and RRNPP transcriptional activators, is a bifunctional regulator that modulates sporulation initiation and activates nutrient scavenging genes. In this review, we discuss how these receptors switch between functions and connect distinct signaling pathways. Based on structural evidence, we propose that RapH and Rap60 should be considered moonlighting proteins. Additionally, we analyze an evolutionary and ecological perspective to understand the multifunctionality and functional redundancy of these regulators in both Bacillus spp. and non-Bacillus Firmicutes. Understanding the mechanistic, structural, ecological, and evolutionary basis for the multifunctionality and redundancy of these QS systems is a key step for achieving the development of innovative technologies for health and agriculture.

An aberrant bi-apical Follicucullus specimen (Albaillellaria, Radiolaria) has been discovered from an upper Guadalupian (Middle Permian) chert block of the Kamiaso Unit of the Mino terrane, central Japan. If this specimen was formed with double malformation, it would be the oldest record of this phenomenon in radiolarians and the first record of its kind in Albaillellaria.

Despite the importance of the Bering Sea for subarctic oceanography and climate, relatively little is known of the foraminifera from the extensive Aleutian Basin. We report the occurrence of modern deep-water agglutinated foraminifera collected at seven sites cored during Integrated Ocean Drilling Program (IODP) Expedition 323 in the Bering Sea. Assemblages collected from core-top samples contained 32 genera and 50 species and are described and illustrated here for the first time. Commonly occurring species include typical deep-water Rhizammina, Reophax, Rhabdammina, Recurvoides and Nodulina. Assemblages from the northern sites also consist of accessory Cyclammina, Eggerelloides and Glaphyrammina, whilst those of the Bowers Ridge sites consist of other tubular genera and Martinottiella. Of the studied stations with the lowest dissolved oxygen concentrations, the potentially Bering Sea endemic Eggerelloides sp. 1 inhabits the northern slope, which has the highest primary productivity, and the potentially endemic Martinottiella sp. 3 inhabits Bowers Ridge, which has the lowest oxygen concentrations but relatively low annual productivity. Martinottiella sp. 3, with open pores on its test surface, has previously been reported in Pliocene to Recent material from Bowers Ridge. Despite relatively small sample sizes, ecological constraints may imply that the Bering Sea experienced high productivity and reduced oxygen at times since at least the Pliocene. We note the partially endemic nature of the agglutinated foraminiferal assemblages, which may at least in part be due to basin restriction, the geologically long time period of reduced oxygen, and high organic carbon flux. Our results indicate the importance of gathering further surface sample data from the Aleutian Basin.

The Henry Buckley Collection of Planktonic Foraminifera at the Natural History Museum in London (NHMUK) consists of 1665 single-taxon slides housing 23 897 individuals from 203 sites in all the major ocean basins, as well as a vast research library of Scanning Electron Microscope (SEM) photomicrographs. Buckley picked the material from the NHMUK Ocean-Bottom Deposit Collection and also from fresh tow samples. However, his collection remains largely unused as he was discouraged by his managers in the Mineralogy Department from working on or publicizing the collection. Nevertheless, Buckley published pioneering papers on isotopic interpretation of oceanographic and climatic change and was one of the first workers to investigate foraminiferal wall structure using the SEM technique. Details of the collection and images of each slide are available via the NHMUK Data Portal (http://dx.doi.org/10.5519/0035055). The Buckley Collection and its associated Ocean-Bottom Deposit Collection have great potential for taxon-specific studies as well as geochemical work, and both collections are available on request.

A new larger benthic porcelaneous foraminifer of soritid affinity is described as Tarburina zagrosiana n. gen., n. sp. from the late Maastrichtian of the Tarbur Formation, Zagros Zone, SW Iran. It occurs in foraminiferal–dasycladalean wackestones and packstones, in association with Loftusia ssp., dicyclinids/cuneolinids, Neobalkhania bignoti Cherchi & Schroeder, Gyroconulina columellifera Schroeder & Darmoian, Spirolina? farsiana Schlagintweit & Rashidi, Broeckina cf. dufrenoyi (d'Archiac), other benthic foraminifers, and dasycladalean algae. Due to its elongate test and marginal chamber subdivision by aligned vertical partitions, Tarburina n. gen. can be compared with representatives of the Praerhapydionininae. The interio-marginal slit-like foramina/aperture of Tarburina represents an outstanding feature in complex porcelaneous taxa. The monospecific genus Tarburina is considered a Maastrichtian newcomer within the Late Cretaceous Global Community Maturation cycle of larger benthic foraminifera. A biostratigraphic and palaeobiogeographical restriction seems possible, as reported for many other Late Cretaceous larger benthic foraminifera.

Planktonic foraminifera are a source of important geochemical, palaeoceanographic, and palaeontological data. However, many aspects of their ecology remain poorly understood, including whether or not gross morphology has an ecological function. Here, we measure the force needed to crush multiple planktonic foraminiferal morphotypes from modern core top and tow samples. We find significant differences in the resistance of different morphotypes to compressional force. Three species, Globorotalia tumida (biconvex, keeled), Menardella menardii (discoidal, keeled), Truncorotalia truncatulinoides (conical, keeled), require on average 59% more force (1.07 v. 0.47 N) to crush than the least resistant species (Orbulina universa and Trilobatus sacculifer) in core-top samples. Towed samples of pre-gametogenic individuals also show significant differences of the same magnitude (0.693 v. 0.53 N) between the conical (T. truncatulinoides) and globular/spherical morphologies (Globoconella inflata and O. universa). We hypothesize that the greater compressional strength of certain shapes confers a fitness advantage against predators and could contribute to the repeated, convergent evolution of keeled, conical and bi-convex forms in planktonic foraminifer lineages.

Supplementary material: Raw data for all crushing experiments, wall thickness measurements, and results for all pair-wise Kolmogorov-Smirnov Tests are available at https://doi.org/10.6084/m9.figshare.c.3725236.v1

An exceptionally well-preserved specimen of the articulated rhodophyte Permocalculus, compared with P. tenellus sensu Elliott, 1955, is described from fine-grained Upper Permian limestones of the Khuff Formation of Saudi Arabia. Longitudinal medullary and sheaf-like cortical filaments extend through the uniserial series of elongate-globular, concave- and convex-terminating, interlocking segments for which they are interpreted to have functioned in articulation. The filaments tend to splay and branch laterally into the cortex where they terminate at the pores. At the terminal aperture, the filaments extend as bifurcating and possibly trifurcating branches and may serve as the origin of a new segment. Numerous elongate-globular chambers, up to five in each row and intimately involved with the filaments, are developed in the outer medulla and are considered to represent reproductive sporangia. The specimen is considered to have occupied predominantly low-energy, normal to slightly elevated salinity, shallow conditions within the subtidal regime of a lagoon.

ABSTRACTBackground:Some opportunities to routinely capture and improve respectful maternity care (RMC) during facility-based childbirth include quality improvement (QI) initiatives, community-based monitoring efforts through community score cards (CSC), and performance-based financing (PBF) initiatives. But there is limited guidance on which types of RMC indicators are best suited for inclusion in these initiatives. We sought to provide practical evidence-based recommendations on indicators that may be used for routine measurement of RMC in programs.Methods:We used a rapid review approach, which included (1) reviewing existing documents and publications to extract RMC indicators and identify which have or can be used in facility-based QI, CSCs, and PBF schemes; (2) surveying RMC and maternal health experts to rank indicators, and (3) analyzing survey data to select the most recommended indicators.Results:We identified 49 indicators spanning several domains of RMC and mistreatment including dignified/nondignified care, verbal and physical abuse, privacy/confidentiality, autonomy/loss of autonomy, supportive care/lack thereof, communication, stigma, discrimination, trust, facility environment/culture, responsiveness, and nonevidence-based care. Based on the analysis of the survey data, we recommend 33 indicators (between 2 and 6 indicators for each RMC domain) that may be suited for incorporation in both facility-based QI and CSC-related monitoring efforts.Conclusion:Integrating RMC indicators into QI and CSC initiatives, as well as in other maternal and neonatal health programs, could help improve RMC at the facility and community level. More research is needed into whether RMC can be integrated into PBF initiatives. Integration of RMC indicators into programs to improve quality of care and other health system outcomes will facilitate routine monitoring and accountability around experience of care. Measurement and improvement of women's experiences will increase maternal health service utilization and improve quality of care as a means of reducing maternal and neonatal morbidity and mortality.

ABSTRACTBackground:Female sex workers (FSWs) in Cameroon commonly have unmet need for contraception posing a high risk of unintended pregnancy. Unintended pregnancy leads to a range of outcomes, and due to legal restrictions, FSWs often seek unsafe abortions. Aside from the high burden of HIV, little is known about the broader sexual and reproductive health of FSWs in Cameroon.Methods:From December 2015 to October 2016, we recruited FSWs aged ≥18 years through respondent-driven sampling across 5 Cameroonian cities. Cross-sectional data were collected through a behavioral questionnaire. Modified-robust Poisson regression was used to approximate adjusted prevalence ratios (aPR) for TOP and current use of effective nonbarrier contraception.Results:Among 2,255 FSWs (median age 28 years), 57.6% reported history of unintended pregnancy and 40.0% reported prior TOP. In multivariable analysis, TOP history was associated with current nonbarrier contraceptive use (aPR=1.23, 95% confidence interval [CI]=1.07, 1.42); ever using emergency contraception (aPR=1.34, 95% CI=1.17, 1.55); >60 clients in the past month (aPR=1.29, 95% CI= 1.07, 1.54) compared to ≤30; inconsistent condom use with clients (aPR=1.17, 95% CI=1.00, 1.37); ever experiencing physical violence (aPR=1.24, 95% CI=1.09, 1.42); and older age. Most (76.5%) women used male condoms for contraception, but only 33.2% reported consistent condom use with all partners. Overall, 26.4% of women reported currently using a nonbarrier contraceptive method, and 6.2% reported using a long-acting method. Previous TOP (aPR=1.41, 95%CI=1.16, 1.72) and ever using emergency contraception (aPR=2.70, 95% CI=2.23, 3.26) were associated with higher nonbarrier contraceptive use. Recent receipt of HIV information (aPR=0.72, 95% CI=0.59, 0.89) and membership in an FSW community-based organization (aPR=0.73, 95% CI=0.57, 0.92) were associated with lower use nonbarrier contraceptive use.Conclusions:Experience of unintended pregnancies and TOP is common among FSWs in Cameroon. Given the low use of nonbarrier contraceptive methods and inconsistent condom use, FSWs are at risk of repeat unintended pregnancies. Improved integration of client-centered, voluntary family planning within community-led HIV services may better support the sexual and reproductive health and human rights of FSWs consistent with the United Nations Declaration of Human Rights.

ABSTRACTBackground:The focused assessment with sonography for HIV-associated tuberculosis (TB) (FASH) ultrasound protocol has been increasingly used to help clinicians diagnose TB. We sought to quantify the diagnostic utility of FASH for TB among individuals with HIV in Malawi.Methods:Between March 2016 and August 2017, 210 adults with HIV who had 2 or more signs and symptoms that were concerning for TB (fever, cough, night sweats, weight loss) were enrolled from a public HIV clinic in Lilongwe, Malawi. The treating clinicians conducted a history, physical exam, FASH protocol, and additional TB evaluation (laboratory diagnostics and chest radiography) on all participants. The clinician made a final treatment decision based on all available information. At the 6-month follow-up visit, we categorized participants based on clinical outcomes and diagnostic tests as having probable/confirmed TB or unlikely TB; association of FASH with probable/confirmed TB was calculated using Fisher's exact tests. The impact of FASH on empiric TB treatment was determined by asking the clinicians prospectively about whether they would start treatment at 2 time points in the baseline visit: (1) after the initial history and physical exam; and (2) after history, physical exam, and FASH protocol.Results:A total of 181 participants underwent final analysis, of whom 56 were categorized as probable/confirmed TB and 125 were categorized as unlikely TB. The FASH protocol was positive in 71% (40/56) of participants with probable/confirmed TB compared to 24% (30/125) of participants with unlikely TB (odds ratio=7.9, 95% confidence interval=3.9,16.1; P<.001). Among those classified as confirmed/probable TB, FASH increased the likelihood of empiric TB treatment before obtaining any other diagnostic studies from 9% (5/56) to 46% (26/56) at the point-of-care. For those classified as unlikely TB, FASH increased the likelihood of empiric treatment from 2% to 4%.Conclusion:In the setting of HIV coinfection in Malawi, FASH can be a helpful tool that augments the clinician's ability to make a timely diagnosis of TB.

OBJECTIVE

There is a controversy over the association between obesity and end-stage renal disease (ESRD) in people with or without type 2 diabetes; therefore, we examined the effect of BMI on the risk of ESRD according to glycemic status in the Korean population.

OBJECTIVE

To report U.S. national population-based rates and trends in diabetic ketoacidosis (DKA) and hyperglycemic hyperosmolar state (HHS) among adults, in both the emergency department (ED) and inpatient settings.

OBJECTIVE

The effect of early-life antibiotic treatment on the risk of type 1 diabetes is debated. This study assessed this question, applying a register-based design in children up to age 10 years including a large sibling-control analysis.

OBJECTIVE

Metformin is the first pharmacological option for treating type 2 diabetes. However, the use of this drug is not recommended in individuals with impaired kidney function because of the perceived risk of lactic acidosis. We aimed to assess the efficacy and safety of metformin in patients with type 2 diabetic kidney disease (DKD).

OBJECTIVE

We evaluated the effect of optimizing metformin dosing on glycemia and body weight in type 2 diabetes.

Receptor-mediated endocytosis, which contributes to a wide range of cellular functions, including receptor signaling, cell adhesion, and migration, requires endocytic vesicle release by the large GTPase dynamin 2. Here, the role of dynamin 2 was investigated in platelet hemostatic function using both pharmacological and genetic approaches. Dnm2^fl/fl Pf4-Cre (Dnm2^Plt–/–) mice specifically lacking dynamin 2 within the platelet lineage developed severe thrombocytopenia and bleeding diathesis and Dnm2^Plt–/– platelets adhered poorly to collagen under arterial shear rates. Signaling via the collagen receptor GPVI was impaired in platelets treated with the dynamin GTPase inhibitor dynasore, as evidenced by poor protein tyrosine phosphorylation, including that of the proximal tyrosine kinase Lyn on its activating tyrosine 396 residue. Platelet stimulation via GPVI resulted in a slight decrease in GPVI, which was maintained by dynasore treatment. Dynasore-treated platelets had attenuated function when stimulated via GPVI, as evidenced by reduced GPIbα downregulation, α-granule release, integrin αIIbβ3 activation, and spreading onto immobilized fibrinogen. By contrast, responses to the G-protein coupled receptor agonist thrombin were minimally affected by dynasore treatment. GPVI expression was severely reduced in Dnm2^Plt–/– platelets, which were dysfunctional in response to stimulation via GPVI, and to a lesser extent to thrombin. Dnm2^Plt–/– platelets lacked fibrinogen in their α-granules, but retained von Willebrand factor. Taken together, the data show that dynamin 2 plays a proximal role in signaling via the collagen receptor GPVI and is required for fibrinogen uptake and normal platelet hemostatic function.

Cytogenetic risk stratification at diagnosis has long been one of the most useful tools to assess prognosis in acute lymphoblastic leukemia (ALL). To examine the prognostic impact of cytogenetic abnormalities on outcomes after allogeneic hematopoietic cell transplantation, we studied 1731 adults with Philadelphia-negative ALL in complete remission who underwent myeloablative or reduced intensity/non-myeloablative conditioning transplant from unrelated or matched sibling donors reported to the Center for International Blood and Marrow Transplant Research. A total of 632 patients had abnormal conventional metaphase cytogenetics. The leukemia-free survival and overall survival rates at 5 years after transplantation in patients with abnormal cytogenetics were 40% and 42%, respectively, which were similar to those in patients with a normal karyotype. Of the previously established cytogenetic risk classifications, modified Medical Research Council-Eastern Cooperative Oncology Group score was the only independent prognosticator of leukemia-free survival (P=0.03). In the multivariable analysis, monosomy 7 predicted post-transplant relapse [hazard ratio (HR)=2.11; 95% confidence interval (95% CI): 1.04-4.27] and treatment failure (HR=1.97; 95% CI: 1.20-3.24). Complex karyotype was prognostic for relapse (HR=1.69; 95% CI: 1.06-2.69), whereas t(8;14) predicted treatment failure (HR=2.85; 95% CI: 1.35-6.02) and overall mortality (HR=3.03; 95% CI: 1.44-6.41). This large study suggested a novel transplant-specific cytogenetic scheme with adverse [monosomy 7, complex karyotype, del(7q), t(8;14), t(11;19), del(11q), tetraploidy/near triploidy], intermediate (normal karyotype and all other abnormalities), and favorable (high hyperdiploidy) risks to prognosticate leukemia-free survival (P=0.02). Although some previously established high-risk Philadelphia-negative cytogenetic abnormalities in ALL can be overcome by transplantation, monosomy 7, complex karyotype, and t(8;14) continue to pose significant risks and yield inferior outcomes.

Mixed lineage leukemia (MLL/KMT2A) rearrangements (MLL-r) are one of the most frequent chromosomal aberrations in acute myeloid leukemia. We evaluated the function of Meningioma 1 (MN1), a co-factor of HOXA9 and MEIS1, in human and murine MLL-rearranged leukemia by CRISPR-Cas9 mediated deletion of MN1. MN1 was required for in vivo leukemogenicity of MLL positive murine and human leukemia cells. Loss of MN1 inhibited cell cycle and proliferation, promoted apoptosis and induced differentiation of MLL-rearranged cells. Expression analysis and chromatin immunoprecipitation with sequencing from previously reported data sets demonstrated that MN1 primarily maintains active transcription of HOXA9 and HOXA10, which are critical downstream genes of MLL, and their target genes like BCL2, MCL1 and Survivin. Treatment of MLL-rearranged primary leukemia cells with anti-MN1 siRNA significantly reduced their clonogenic potential in contrast to normal CD34⁺ hematopoietic progenitor cells, suggesting a therapeutic window for MN1 targeting. In summary, our findings demonstrate that MN1 plays an essential role in MLL fusion leukemias and serve as a therapeutic target in MLL-rearranged acute myeloid leukemia.

Mutation and translocation of fibroblast growth factor receptors often lead to aberrant signaling and cancer. This work focuses on the t(8;22)(p11;q11) chromosomal translocation which creates the breakpoint cluster region (BCR) fibroblast growth factor receptor1 (FGFR1) (BCR-FGFR1) fusion protein. This fusion occurs in stem cell leukemia/lymphoma, which can progress to atypical chronic myeloid leukemia, acute myeloid leukemia, or B-cell lymphoma. This work focuses on the biochemical characterization of BCR-FGFR1 and identification of novel therapeutic targets. The tyrosine kinase activity of FGFR1 is required for biological activity as shown using transformation assays, interleukin-3 independent cell proliferation, and liquid chromatography/mass spectroscopy analyses. Furthermore, BCR contributes a coiled-coil oligomerization domain, also essential for oncogenic transformation by BCR-FGFR1. The importance of salt bridge formation within the coiled-coil domain is demonstrated, as disruption of three salt bridges abrogates cellular transforming ability. Lastly, BCR-FGFR1 acts as a client of the chaperonin heat shock protein 90 (Hsp90), suggesting that BCR-FGFR1 relies on Hsp90 complex to evade proteasomal degradation. Transformed cells expressing BCR-FGFR1 are sensitive to the Hsp90 inhibitor Ganetespib, and also respond to combined treatment with Ganetespib plus the FGFR inhibitor BGJ398. Collectively, these data suggest novel therapeutic approaches for future stem cell leukemia/lymphoma treatment: inhibition of BCR oligomerization by disruption of required salt bridges; and inhibition of the chaperonin Hsp90 complex.

Although studies of mixed chimerism following hematopoietic stem cell transplantation in patients with sickle cell disease (SCD) may provide insights into the engraftment needed to correct the disease and into immunological reconstitution, an extensive multilineage analysis is lacking. We analyzed chimerism simultaneously in peripheral erythroid and granulomonocytic precursors/progenitors, highly purified B and T lymphocytes, monocytes, granulocytes and red blood cells (RBC). Thirty-four patients with mixed chimerism and ≥12 months of follow-up were included. A selective advantage of donor RBC and their progenitors/precursors led to full chimerism in mature RBC (despite partial engraftment of other lineages), and resulted in the clinical control of the disease. Six patients with donor chimerism <50% had hemolysis (reticulocytosis) and higher HbS than their donor. Four of them had donor chimerism <30%, including a patient with AA donor (hemoglobin >10 g/dL) and three with AS donors (hemoglobin <10 g/dL). However, only one vaso-occlusive crisis occurred with 68.7% HbS. Except in the patients with the lowest chimerism, the donor engraftment was lower for T cells than for the other lineages. In a context of mixed chimerism after hematopoietic stem cell transplantation for SCD, myeloid (rather than T cell) engraftment was the key efficacy criterion. Results show that myeloid chimerism as low as 30% was sufficient to prevent a vaso-occlusive crisis in transplants from an AA donor but not constantly from an AS donor. However, the correction of hemolysis requires higher donor chimerism levels (i.e. ≥50%) in both AA and AS recipients. In the future, this group of patients may need a different therapeutic approach.

The Diabetes Control and Complications Trial/Epidemiology of Diabetes Interventions and Complications (DCCT/EDIC) study demonstrated that intensive glucose control reduced the risk of developing diabetic peripheral neuropathy (DPN) and cardiovascular autonomic neuropathy (CAN). We evaluated multiple risk factors and phenotypes associated with DPN and CAN in this large, well-characterized cohort of participants with type 1 diabetes, followed for >23 years. DPN was defined by symptoms, signs, and nerve conduction study abnormalities in ≥2 nerves; CAN was assessed using standardized cardiovascular reflex tests. Generalized estimating equation models assessed the association of DPN and CAN with individual risk factors measured repeatedly. During DCCT/EDIC, 33% of participants developed DPN and 44% CAN. Higher mean HbA_1c was the most significant risk factor for DPN, followed by older age, longer duration, greater height, macroalbuminuria, higher mean pulse rate, β-blocker use, and sustained albuminuria. The most significant risk factor for CAN was older age, followed by higher mean HbA_1c, sustained albuminuria, longer duration of type 1 diabetes, higher mean pulse rate, higher mean systolic blood pressure, β-blocker use, estimated glomerular filtration rate <60 mL/min/1.73 m², higher most recent pulse rate, and cigarette smoking. These findings identify risk factors and phenotypes of participants with diabetic neuropathy that can be used in the design of new interventional trials and for personalized approaches to neuropathy prevention.

Inadequate insulin secretion in response to glucose is an important factor for β-cell failure in type 2 diabetes (T2D). Although HMG-CoA reductase degradation 1 (HRD1), a subunit of the endoplasmic reticulum–associated degradation complex, plays a pivotal role in β-cell function, HRD1 elevation in a diabetic setting contributes to β-cell dysfunction. We report in this study the excessive HRD1 expression in islets from humans with T2D and T2D mice. Functional studies reveal that β-cell–specific HRD1 overexpression triggers impaired insulin secretion that will ultimately lead to severe hyperglycemia; by contrast, HRD1 knockdown improves glucose control and response in diabetic models. Proteomic analysis results reveal a large HRD1 interactome, which includes v-maf musculoaponeurotic fibrosarcoma oncogene homolog A (MafA), a master regulator of genes implicated in the maintenance of β-cell function. Furthermore, mechanistic assay results indicate that HRD1 is a novel E3 ubiquitin ligase that targets MafA for ubiquitination and degradation in diabetic β-cells, resulting in cytoplasmic accumulation of MafA and in the reduction of its biological function in the nucleus. Our results not only reveal the pathological importance of excessive HRD1 in β-cell dysfunction but also establish the therapeutic importance of targeting HRD1 in order to prevent MafA loss and suppress the development of T2D.

Genes of the Notch signaling pathway are expressed in different cell types and organs at different time points during embryonic development and adulthood. The Notch ligand Delta-like 1 (DLL1) controls the decision between endocrine and exocrine fates of multipotent progenitors in the developing pancreas, and loss of Dll1 leads to premature endocrine differentiation. However, the role of Delta-Notch signaling in adult tissue homeostasis is not well understood. Here, we describe the spatial expression pattern of Notch pathway components in adult murine pancreatic islets and show that DLL1 and DLL4 are specifically expressed in β-cells, whereas JAGGED1 is expressed in α-cells. We show that mice lacking both DLL1 and DLL4 in adult β-cells display improved glucose tolerance, increased glucose-stimulated insulin secretion, and hyperglucagonemia. In contrast, overexpression of the intracellular domain of DLL1 in adult murine pancreatic β-cells results in impaired glucose tolerance and reduced insulin secretion, both in vitro and in vivo. These results suggest that Notch ligands play specific roles in the adult pancreas and highlight a novel function of the Delta/Notch pathway in β-cell insulin secretion.

Glucagon is classically described as a counterregulatory hormone that plays an essential role in the protection against hypoglycemia. In addition to its role in the regulation of glucose metabolism, glucagon has been described to promote ketosis in the fasted state. Sodium–glucose cotransporter 2 inhibitors (SGLT2i) are a new class of glucose-lowering drugs that act primarily in the kidney, but some reports have described direct effects of SGLT2i on α-cells to stimulate glucagon secretion. Interestingly, SGLT2 inhibition also results in increased endogenous glucose production and ketone production, features common to glucagon action. Here, we directly test the ketogenic role of glucagon in mice, demonstrating that neither fasting- nor SGLT2i-induced ketosis is altered by interruption of glucagon signaling. Moreover, any effect of glucagon to stimulate ketogenesis is severely limited by its insulinotropic actions. Collectively, our data suggest that fasting-associated ketosis and the ketogenic effects of SGLT2 inhibitors occur almost entirely independent of glucagon.

Chalongrat Noree and Naraporn Sirinonthanawech

Previously, we have developed an extramitochondrial assembly system, where mitochondrial targeting signal (MTS) can be removed from a given mitochondrial enzyme, which could be used to characterize the regulatory factors involved in enzyme assembly/disassembly in vivo. Here, we demonstrate that addition of exogenous acetaldehyde can quickly induce the supramolecular assembly of MTS-deleted aldehyde dehydrogenase Ald4p in yeast cytoplasm. Also, by using PCR-based modification of the yeast genome, cytoplasmically targeted Ald4p cannot polymerize into long filaments when key functional amino acid residues are substituted, as shown by N192D, S269A, E290K and C324A mutations. This study has confirmed that extramitochondrial assembly could be a powerful external system for studying mitochondrial enzyme assembly, and its regulatory factors outside the mitochondria. In addition, we propose that mitochondrial enzyme assembly/disassembly is coupled to the regulation of a given mitochondrial enzyme activity.

Sonu Shrestha Baral, Molly E. Lieux, and Patrick J. DiMario

Different stem cells or progenitor cells display variable threshold requirements for functional ribosomes. This is particularly true for several human ribosomopathies in which select embryonic neural crest cells or adult bone marrow stem cells, but not others, show lethality due to failures in ribosome biogenesis or function (now known as nucleolar stress). To determine if various Drosophila neuroblasts display differential sensitivities to nucleolar stress, we used CRISPR-Cas9 to disrupt the Nopp140 gene that encodes two splice variant ribosome biogenesis factors (RBFs). Disruption of Nopp140 induced nucleolar stress that arrested larvae in the second instar stage. While the majority of larval neuroblasts arrested development, the mushroom body (MB) neuroblasts continued to proliferate as shown by their maintenance of deadpan, a neuroblast-specific transcription factor, and by their continued EdU incorporation. MB neuroblasts in wild-type larvae appeared to contain more fibrillarin and Nopp140 in their nucleoli as compared to other neuroblasts, indicating that MB neuroblasts stockpile RBFs as they proliferate in late embryogenesis while other neuroblasts normally enter quiescence. A greater abundance of Nopp140 encoded by maternal transcripts in Nopp140-/- MB neuroblasts of 1­­­–2-day-old larvae likely rendered these cells more resilient to nucleolar stress.

This article has an associated First Person interview with the first author of the paper.

BackgroundA brief intervention whereby GPs opportunistically facilitate an NHS-funded referral to a weight loss programme is clinically and cost-effective.AimTo test the acceptability of a brief intervention and attendance at a weight loss programme when GPs facilitate a referral that requires patients to pay for the service.Design and settingAn observational study of the effect of a GP encouraging attendance at a weight loss programme requiring self-payment in the West Midlands from 16 October 2018 to 30 November 2018, to compare with a previous trial in England in which the service was NHS-funded.MethodSixty patients with obesity who consecutively attended primary care appointments received an opportunistic brief intervention by a GP to endorse and offer a referral to a weight loss programme at the patient’s own expense. Participants were randomised to GPs who either stated the weekly monetary cost of the programme (basic cost) or who compared the weekly cost to an everyday discretionary item (cost comparison). Participants were subsequently asked to report whether they had attended a weight loss programme.ResultsOverall, 47% of participants (n = 28) accepted the referral; 50% (n = 15) in the basic cost group and 43% (n = 13) in the cost comparison group. This was significantly less than in a previous study when the programme was NHS-funded (77%, n = 722/940; P<0.0001). Most participants reported the intervention to be helpful/very helpful and appropriate/very appropriate (78%, n = 46/59 and 85%, n = 50/59, respectively) but scores were significantly lower than when the programme was NHS-funded (92% n = 851/922 and 88% n = 813/922, respectively; P = 0.004). One person (2%) attended the weight loss programme, which is significantly lower than the 40% of participants who attended when the programme was NHS-funded (P<0.0001).ConclusionGP referral to a weight loss programme that requires patients to pay rather than offering an NHS-funded programme is acceptable; however, it results in almost no attendance.

BackgroundUrinary tract infections (UTIs) are one of the most common bacterial infections managed in general practice. Many women with symptoms of uncomplicated UTI may not benefit meaningfully from antibiotic treatment, but the evidence base is complex and there is no suitable shared decision-making resource to guide antibiotic treatment and symptomatic care for use in general practice consultations.AimTo develop an evidence-based, shared decision-making intervention leaflet to optimise management of uncomplicated UTI for women aged <65 years in the primary care setting.Design and settingQualitative telephone interviews with GPs and patient focus group interviews.MethodIn-depth interviews were conducted to explore how consultation discussions around diagnosis, antibiotic use, self-care, safety netting, and prevention of UTI could be improved. Interview schedules were based on the Theoretical Domains Framework.ResultsBarriers to an effective joint consultation and appropriate prescribing included: lack of GP time, misunderstanding of depth of knowledge and miscommunication between the patient and the GP, nature of the consults (such as telephone consultations), and a history of previous antibiotic therapy.ConclusionConsultation time pressures combined with late symptom presentation are a challenge for even the most experienced of GPs: however, it is clear that enhanced patient–clinician shared decision making is urgently required when it comes to UTIs. This communication should incorporate the provision of self-care, safety netting, and preventive advice to help guide patients when to consult. A shared decision-making information leaflet was iteratively co-produced with patients, clinicians, and researchers at Public Health England using study data.

BackgroundIn the context of a variable condition such as asthma, patient recognition of deteriorating control and knowing what prompt action to take is crucial. Yet, implementation of recommended self-management strategies remains poor.AimTo explore how patients with asthma and parents/carers of children with asthma develop and establish recommended self-management strategies for living with asthma, and how clinicians can best support the process.Design and settingA qualitative study in UK primary care.MethodPatients with asthma and parents/carers of children with asthma from 10 general practices were purposively sampled (using age, sex, and duration of asthma) to participate in focus groups or interviews between May 2016 and August 2016. Participants’ experiences of health care, management of asthma, and views on supported self-management were explored. Interviews and focus group sessions were audio-recorded and transcribed verbatim. Iterative thematic analysis was conducted, guided by the research questions and drawing on habit theory in discussion with a multidisciplinary research team.ResultsA total of 49 participants (45 patients; 4 parents/carers) took part in 32 interviews and five focus groups. Of these, 11 reported using an action plan. Patients learnt how to self-manage over time, building knowledge from personal experience and other sources, such as the internet. Some regular actions, for example, taking medication, became habitual. Dealing with new or unexpected scenarios required reflective abilities, which may be supported by a tailored action plan.ConclusionPatients reported learning intuitively how to self-manage. Some regular actions became habitual; dealing with the unexpected required more reflective cognitive skills. In order to support implementation of optimal asthma self- management, clinicians should consider both these aspects of self-management and support, and educate patients proactively.