Streptococcus pyogenes (Lancefield group A Streptococcus [GAS]) is a β-hemolytic human-selective pathogen that is responsible for a large number of morbid and mortal infections in humans. For efficient infection, GAS requires different types of surface proteins that provide various mechanisms for evading human innate immune responses, thus enhancing pathogenicity of the bacteria. Many such virulence-promoting proteins, including the major surface signature M protein, are translocated after biosynthesis through the cytoplasmic membrane and temporarily tethered to this membrane via a type 1 transmembrane domain (TMD) positioned near the COOH terminus. In these proteins, a sorting signal, LPXTG, is positioned immediately upstream of the TMD, which is cleaved by the membrane-associated transpeptidase, sortase A (SrtA), leading to the covalent anchoring of these proteins to newly emerging l-Ala–l-Ala cross-bridges of the growing peptidoglycan cell wall. Herein, we show that inactivation of the srtA gene in a skin-tropic pattern D GAS strain (AP53) results in retention of the M protein in the cell membrane. However, while the isogenic AP53 srtA strain is attenuated in overall pathogenic properties due to effects on the integrity of the cell membrane, our data show that the M protein nonetheless can extend from the cytoplasmic membrane through the cell wall and then to the surface of the bacteria and thereby retain its important properties of productively binding and activating fluid-phase host plasminogen (hPg). The studies presented herein demonstrate an underappreciated additional mechanism of cell surface display of bacterial virulence proteins via their retention in the cell membrane and extension to the GAS surface.

IMPORTANCE Group A Streptococcus pyogenes (GAS) is a human-specific pathogen that produces many surface factors, including its signature M protein, that contribute to its pathogenicity. M proteins undergo specific membrane localization and anchoring to the cell wall via the transpeptidase sortase A. Herein, we explored the role of sortase A function on M protein localization, architecture, and function, employing, a skin-tropic GAS isolate, AP53, which expresses a human plasminogen (hPg)-binding M (PAM) Protein. We showed that PAM anchored in the cell membrane, due to the targeted inactivation of sortase A, was nonetheless exposed on the cell surface and functionally interacted with host hPg. We demonstrate that M proteins, and possibly other sortase A-processed proteins that are retained in the cell membrane, can still function to initiate pathogenic processes by this underappreciated mechanism.

The capacity of Listeria monocytogenes to adapt to environmental changes is facilitated by a large number of regulatory proteins encoded by its genome. Among these proteins are the uncharacterized LysR-type transcriptional regulators (LTTRs). LTTRs can work as positive and/or negative transcription regulators at both local and global genetic levels. Previously, our group determined by comparative genome analysis that one member of the LTTRs (NCBI accession no. WP_003734782) was present in pathogenic strains but absent from nonpathogenic strains. The goal of the present study was to assess the importance of this transcription factor in the virulence of L. monocytogenes strain F2365 and to identify its regulons. An L. monocytogenes strain lacking lysR (the F2365lysR strain) displayed significant reductions in cell invasion of and adhesion to Caco-2 cells. In plaque assays, the deletion of lysR resulted in a 42.86% decrease in plaque number and a 13.48% decrease in average plaque size. Furthermore, the deletion of lysR also attenuated the virulence of L. monocytogenes in mice following oral and intraperitoneal inoculation. The analysis of transcriptomics revealed that the transcript levels of 139 genes were upregulated, while 113 genes were downregulated in the F2365lysR strain compared to levels in the wild-type bacteria. lysR-repressed genes included ABC transporters, important for starch and sucrose metabolism as well as glycerolipid metabolism, flagellar assembly, quorum sensing, and glycolysis/gluconeogenesis. Conversely, lysR activated the expression of genes related to fructose and mannose metabolism, cationic antimicrobial peptide (CAMP) resistance, and beta-lactam resistance. These data suggested that lysR contributed to L. monocytogenes virulence by broad impact on multiple pathways of gene expression.

IMPORTANCE Listeria monocytogenes is the causative agent of listeriosis, an infectious and fatal disease of animals and humans. In this study, we have shown that lysR contributes to Listeria pathogenesis and replication in cell lines. We also highlight the importance of lysR in regulating the transcription of genes involved in different pathways that might be essential for the growth and persistence of L. monocytogenes in the host or under nutrient limitation. Better understanding L. monocytogenes pathogenesis and the role of various virulence factors is necessary for further development of prevention and control strategies.

The type VI secretion system (T6SS) is a weapon for delivering effectors into target cells that is widespread in Gram-negative bacteria. The T6SS is a highly versatile machine, as it can target both eukaryotic and prokaryotic cells, and it has been proposed that T6SSs are adapted to the specific needs of each bacterium. The expression of T6SS gene clusters and the activation of the secretion apparatus are therefore tightly controlled. In enteroaggregative Escherichia coli (EAEC), the sci1 T6SS gene cluster is subject to a complex regulation involving both the ferric uptake regulator (Fur) and DNA adenine methylase (Dam)-dependent DNA methylation. In this study, an additional, internal, promoter was identified within the sci1 gene cluster using +1 transcriptional mapping. Further analyses demonstrated that this internal promoter is controlled by a mechanism strictly identical to that of the main promoter. The Fur binding box overlaps the –10 transcriptional element and a Dam methylation site, GATC-32. Hence, the expression of the distal sci1 genes is repressed and the GATC-32 site is protected from methylation in iron-rich conditions. The Fur-dependent protection of GATC-32 was confirmed by an in vitro methylation assay. In addition, the methylation of GATC-32 negatively impacted Fur binding. The expression of the sci1 internal promoter is therefore controlled by iron availability through Fur regulation, whereas Dam-dependent methylation maintains a stable ON expression in iron-limited conditions.

IMPORTANCE Bacteria use weapons to deliver effectors into target cells. One of these weapons, the type VI secretion system (T6SS), assembles a contractile tail acting as a spring to propel a toxin-loaded needle. Its expression and activation therefore need to be tightly regulated. Here, we identified an internal promoter within the sci1 T6SS gene cluster in enteroaggregative E. coli. We show that this internal promoter is controlled by Fur and Dam-dependent methylation. We further demonstrate that Fur and Dam compete at the –10 transcriptional element to finely tune the expression of T6SS genes. We propose that this elegant regulatory mechanism allows the optimum production of the T6SS in conditions where enteroaggregative E. coli encounters competing species.

Shigella species, the causal agents of bacillary dysentery, use a type III secretion system (T3SS) to inject two waves of virulence proteins, known as effectors, into the colonic epithelium to subvert host cell machinery. Prior to host cell contact and secretion of the first wave of T3SS effectors, OspD1, an effector and antiactivator protein, prevents premature production of the second wave of effectors. Despite this important role, regulation of the ospD1 gene is not well understood. While ospD1 belongs to the large regulon of VirB, a transcriptional antisilencing protein that counters silencing mediated by the histone-like nucleoid structuring protein H-NS, it remains unclear if VirB directly or indirectly regulates ospD1. Additionally, it is not known if ospD1 is regulated by H-NS. Here, we identify the primary ospD1 transcription start site (+1) and show that the ospD1 promoter is remotely regulated by both VirB and H-NS. Our findings demonstrate that VirB regulation of ospD1 requires at least one of the two newly identified VirB regulatory sites, centered at –978 and –1270 relative to the ospD1 +1. Intriguingly, one of these sites lies on a 193-bp sequence found in three conserved locations on the large virulence plasmids of Shigella. The region required for H-NS-dependent silencing of ospD1 lies between –1120 and –820 relative to the ospD1 +1. Thus, our study provides further evidence that cis-acting regulatory sequences for transcriptional antisilencers and silencers, such as VirB and H-NS, can lie far upstream of the canonical bacterial promoter region (i.e., –250 to +1).

IMPORTANCE Transcriptional silencing and antisilencing mechanisms regulate virulence gene expression in many important bacterial pathogens. In Shigella species, plasmid-borne virulence genes, such as those encoding the type III secretion system (T3SS), are silenced by the histone-like nucleoid structuring protein H-NS and antisilenced by VirB. Previous work at the plasmid-borne icsP locus revealed that VirB binds to a remotely located cis-acting regulatory site to relieve transcriptional silencing mediated by H-NS. Here, we characterize a second example of remote VirB antisilencing at ospD1, which encodes a T3SS antiactivator and effector. Our study highlights that remote transcriptional silencing and antisilencing occur more frequently in Shigella than previously thought, and it raises the possibility that long-range transcriptional regulation in bacteria is commonplace.

Chitotriosidase (CHIT1) is a highly conserved and regulated chitinase secreted by activated macrophages; it is a member of the 18-glycosylase family (GH18). CHIT1 is the most prominent chitinase in humans, can cleave chitin and participates in the body's immune response and is associated with inflammation, infection, tissue damage and remodelling processes. Recently, CHIT1 has been reported to be involved in the molecular pathogenesis of pulmonary fibrosis, bronchial asthma, COPD and pulmonary infections, shedding new light on the role of these proteins in lung pathophysiology. The potential roles of CHIT1 in lung diseases are reviewed in this article.

Physician-led thoracic ultrasound (TUS) has substantially changed how respiratory disorders, and in particular pleural diseases, are managed. The use of TUS as a point-of-care test enables the respiratory physician to quickly and accurately diagnose pleural pathology and ensure safe access to the pleural space during thoracentesis or chest drain insertion. Competence in performing TUS is now an obligatory part of respiratory speciality training programmes in different parts of the world. Pleural physicians with higher levels of competence routinely use TUS during the planning and execution of more sophisticated diagnostic and therapeutic interventions, such as core needle pleural biopsies, image-guided drain insertion and medical thoracoscopy. Current research is gauging the potential of TUS in predicting the outcome of different pleural interventions and how it can aid in tailoring the optimum treatment according to different TUS-based parameters.

Background

People with pulmonary fibrosis often experience a protracted time to diagnosis, high symptom burden and limited disease information. This review aimed to identify the supportive care needs reported by people with pulmonary fibrosis and their caregivers.

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.

New scanning electron microscope observations of unadulterated calcareous nannofossil assemblages on lamina surfaces of Cretaceous Tanzania Drilling Project sediments reveal high diversity in the <3 µm size-range and high abundances of small and frangible morphologies. These assemblages prompt comparison to modern assemblages, which show similar high diversity and abundance of very small and fragile taxa, although these assemblages are generally not preserved in the fossil record due to taphonomic filtering. Not only are there broad similarities between the general composition of modern assemblages and those of the Tanzanian lagerstätte, but also our discovery of several new Cretaceous taxa provides evidence for greatly extended fossil lineages of extant orders, with implications for both deep-time biodiversity divergence and survival through the end-Cretaceous mass extinction. Our findings include: new species that are the first-recorded Mesozoic representatives of the extant Syracosphaeraceae and Papposphaeraceae; potentially previously unrecorded diversity in the Mesozoic Calciosoleniaceae, another extant order, represented by extant species that have been described already; and new species and unusually high abundances of the Mesozoic Stephanolithiaceae. We also highlight the extended range of an incertae sedis Cenozoic genus, Ellipsolithus, into at least the Turonian.

Here, we describe seven new miniscule to very small Cretaceous species: Syracosphaera antiqua, S. repagula, Pocillithus macleodii, P. crucifer, Stradnerlithus wendleri, S.? haynesiae and Tortolithus foramen.

Assemblages of upper lower through upper Miocene Discoaster spp. have been quantified from Integrated Ocean Drilling Program (IODP) Site U1338 in the eastern equatorial Pacific Ocean. These assemblages can be grouped into five broad morphological categories: six-rayed with bifurcated ray tips, six-rayed with large central areas, six-rayed with pointed ray tips, five-rayed with bifurcated ray tips and five-rayed with pointed ray tips. Discoaster deflandrei dominates the assemblages prior to 15.8 Ma. The decline in abundance of D. deflandrei close to the early–middle Miocene boundary occurs together with the evolution of the D. variabilis group, including D. signus and D. exilis. Six-rayed discoasters having large central areas become a prominent member of the assemblages for a 400 ka interval in the late middle Miocene. Five- and six-rayed forms having pointed tips become prominent in the early late Miocene and show a strong antiphasing relationship with the D. variabilis group. Discoaster bellus completely dominates the Discoaster assemblages for a 400 ka interval in the middle late Miocene. Abundances of all discoasters, or discoasters at the species level, show only (surprisingly) weak correlations to carbonate contents or oxygen and carbon isotopes of bulk sediment when calculated over the entire sample interval.

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:A home-based record (HBR) is a health document kept by the patient or their caregivers, rather than by the health care facility. HBRs are used in 163 countries, but they have not been implemented universally or consistently. Effective implementation maximizes both health impacts and cost-effectiveness. We sought to examine this research-to-practice gap and delineate the facilitators and barriers to the effective implementation and use of maternal and child health HBRs especially in low- and middle-income countries (LMICs).Methods:Using a framework analysis approach, we created a framework of implementation categories in advance using subject expert inputs. We collected information through 2 streams. First, we screened 69 gray literature documents, of which 18 were included for analysis. Second, we conducted semi-structured interviews with 12 key informants, each of whom had extensive experience with HBR implementation. We abstracted the relevant data from the documents and interviews into an analytic matrix. The matrix was based on the initial framework and adjusted according to emergent categories from the data.Results:We identified 8 contributors to successful HBR implementation. These include establishing high-level support from the government and ensuring clear communication between all ministries and nongovernmental organizations involved. Choice of appropriate contents within the record was noted as important for alignment with the health system and for end user acceptance, as were the design, its physical durability, and timely redesigns. Logistical considerations, such as covering costs sustainably and arranging printing and distribution, could be potential bottlenecks. Finally, end users' engagement with HBRs depended on how the record was initially introduced to them and how its importance was reinforced over time by those in leadership positions.Conclusions:This framework analysis is the first study to take a more comprehensive and broad approach to the HBR implementation process in LMICs. The findings provide guidance for policy makers, donors, and health care practitioners regarding best implementation practice and effective HBR use, as well as where further research is required.

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.

ABSTRACTIntroduction:We evaluated a 2-way short message service (SMS) communication platform to improve continuation of pre-exposure prophylaxis (PrEP) for HIV prevention among Kenyan women who initiated PrEP within routine maternal child health (MCH) and family planning clinics.Methods:We adapted an existing SMS platform (Mobile WACh [mWACh]) to send PrEP-tailored, theory-based SMS and allow clients to communicate with a remote nurse. Women who did not have HIV and who were initiating PrEP at 2 MCH/family planning clinics in Kisumu County, Kenya, from February to October 2018, were offered enrollment into the mWACh-PrEP program; SMS communication was free. We evaluated acceptability, satisfaction, and implementation metrics. In a pre/postevaluation, we compared PrEP continuation at 1-month postinitiation among women who initiated PrEP in the period before (n=166) versus after mWACh-PrEP implementation, adjusting for baseline differences.Results:Of the 334 women who were screened for enrollment into the mWACh-PrEP program; 193 (58%) were eligible and of those, 190 (98%) accepted enrollment. Reasons for ineligibility (n=141) included no phone access (29%) and shared SIM cards (25%). Median age was 25 years (interquartile range=22–30), and 91% were MCH clients. Compared to women who initiated PrEP in the month before mWACh-PrEP implementation, women who enrolled in mWACh-PrEP were more likely to return for their first PrEP follow-up visit (40% vs. 53%; adjusted risk ratio [aRR]=1.26; 95% confidence interval [CI]= 1.06, 1.50; P=.008) and more likely to continue PrEP (22% vs. 43%; aRR=1.75; 95% CI=1.21, 2.55; P=.003). Among those who returned, 99% reported successful receipt of SMS through the mWACh-PrEP system and 94% reported that mWACh-PrEP helped them understand PrEP better. Concerns about PrEP use, how it works, and side effects accounted for the majority (80%) of issues raised by participants using SMS.Conclusions:Two-way SMS expanded support for PrEP and opportunities for dialogue beyond the clinic and enabled women to ask and receive answers in real time regarding PrEP, which facilitated its continued use.

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.

BACKGROUND

Continuous glucose monitoring (CGM) provides important information to aid in achieving glycemic targets in people with diabetes.

OBJECTIVE

To investigate factors related to glycemic management among members of a professional cycling team with type 1 diabetes over a 7-day Union Cycliste Internationale World Tour stage race.

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

Nonalbuminuric diabetic kidney disease (DKD) has become the prevailing phenotype in patients with type 2 diabetes. However, it remains unclear whether its prognosis is poorer than that of other DKD phenotypes.

OBJECTIVE

Most individuals with type 2 diabetes also have obesity, and treatment with some diabetes medications, including insulin, can cause further weight gain. No approved chronic weight management medications have been prospectively investigated in individuals with overweight or obesity and insulin-treated type 2 diabetes. The primary objective of this study was to assess the effect of liraglutide 3.0 mg versus placebo on weight loss in this population.

OBJECTIVE

Incident type 2 diabetes is common among patients with recent acute coronary syndrome and is associated with an adverse prognosis. Some data suggest that cholesteryl ester transfer protein (CETP) inhibitors reduce incident type 2 diabetes. We compared the effect of treatment with the CETP inhibitor dalcetrapib or placebo on incident diabetes in patients with recent acute coronary syndrome.

OBJECTIVE

Suboptimal adherence to insulin treatment is a main issue in adolescents with type 1 diabetes. However, to date, there are no available data on adherence to adjunct noninsulin medications in this population. Our aim was to assess adherence to ACE inhibitors and statins and explore potential determinants in adolescents with type 1 diabetes.

OBJECTIVE

Sodium–glucose cotransporter 2 (SGLT2) inhibition causes an increase in endogenous glucose production (EGP). However, the mechanisms are unclear. We studied the effect of SGLT2 inhibitors on EGP in subjects with type 2 diabetes (T2D) and without diabetes (non-DM) in kidney transplant recipients with renal denervation.

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

With rising health care costs and finite health care resources, understanding the population needs of different type 2 diabetes mellitus (T2DM) patient subgroups is important. Sparse data exist for the application of population segmentation on health care needs among Asian T2DM patients. We aimed to segment T2DM patients into distinct classes and evaluate their differential health care use, diabetes-related complications, and mortality patterns.

OBJECTIVE

Type 2 diabetes is characterized by increased death rate. In order to tackle this dramatic event, it becomes essential to discover novel biomarkers capable of identifying high-risk patients to be exposed to more aggressive preventive and treatment strategies. hs-CRP and serum amyloid P component (SAP) are two acute-phase inflammation proteins, which interact physically and share structural and functional features. We investigated their combined role in associating with and improving prediction of mortality in type 2 diabetes.

OBJECTIVE

We aimed to evaluate trends in bone mineral density (BMD) and the prevalence of osteoporosis/osteopenia in U.S. adults with prediabetes and normal glucose regulation (NGR) and further investigate the association among prediabetes, osteopenia/osteoporosis, and fracture.

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.

Pharmacokinetic-based prophylaxis of replacement factor VIII (FVIII) products has been encouraged in recent years, but the relationship between exposure (factor VIII activity) and response (bleeding frequency) remains unclear. The aim of this study was to characterize the relationship between FVIII dose, plasma FVIII activity, and bleeding patterns and individual characteristics in severe hemophilia A patients. Pooled pharmacokinetic and bleeding data during prophylactic treatment with BAY 81-8973 (octocog alfa) were obtained from the three LEOPOLD trials. The population pharmacokinetics of FVIII activity and longitudinal bleeding frequency, as well as bleeding severity, were described using non-linear mixed effects modeling in NONMEM. In total, 183 patients [median age 22 years (range, 1-61); weight 60 kg (11-124)] contributed with 1,535 plasma FVIII activity observations, 633 bleeds and 11 patient/study characteristics [median observation period 12 months (3.1-13.1)]. A parametric repeated time-to-categorical bleed model, guided by plasma FVIII activity from a 2-compartment population pharmacokinetic model, described the time to the occurrence of bleeds and their severity. Bleeding probability decreased with time of study, and a bleed was not found to affect the time of the next bleed. Several covariate effects were identified, including the bleeding history in the 12-month pre-study period increasing the bleeding hazard. However, unexplained inter-patient variability in the phenotypic bleeding pattern remained large (111%CV). Further studies to translate the model into a tool for dose individualization that considers the individual bleeding risk are required. Research was based on a post-hoc analysis of the LEOPOLD studies registered at clinicaltrials.gov identifiers: 01029340, 01233258 and 01311648.

In patients with cancer-associated venous thromboembolism, knowledge of the estimated rate of recurrent events is important for clinical decision-making regarding anticoagulant therapy. The Ottawa score is a clinical prediction rule designed for this purpose, stratifying patients according to their risk of recurrent venous thromboembolism during the first six months of anticoagulation. We conducted a systematic review and meta-analysis of studies validating either the Ottawa score in its original or modified versions. Two investigators independently reviewed the relevant articles published from 1st June 2012 to 15th December 2018 and indexed in MEDLINE and EMBASE. Nine eligible studies were identified; these included a total of 14,963 patients. The original score classified 49.3% of the patients as high-risk, with a sensitivity of 0.7 [95% confidence interval (CI): 0.6-0.8], a 6-month pooled rate of recurrent venous thromboembolism of 18.6% (95%CI: 13.9-23.9). In the low-risk group, the recurrence rate was 7.4% (95%CI: 3.4-12.5). The modified score classified 19.8% of the patients as low-risk, with a sensitivity of 0.9 (95%CI: 0.4-1.0) and a 6-month pooled rate of recurrent venous thromboembolism of 2.2% (95%CI: 1.6-2.9). In the high-risk group, recurrence rate was 10.2% (95%CI: 6.4-14.6). Limitations of our analysis included type and dosing of anticoagulant therapy. We conclude that new therapeutic strategies are needed in patients at high risk for recurrent cancer-associated venous thromboembolism. Low-risk patients, as per the modified score, could be good candidates for oral anticoagulation. (This systematic review was registered with the International Prospective Registry of Systematic Reviews as: PROSPERO CRD42018099506).

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.

To identify genomic alterations contributing to the pathogenesis of high-risk chronic lymphocytic leukemia (CLL) beyond the well-established role of TP53 aberrations, we comprehensively analyzed 75 relapsed/refractory and 71 treatment-naïve high-risk cases from prospective clinical trials by single nucleotide polymorphism arrays and targeted next-generation sequencing. Increased genomic complexity was a hallmark of relapsed/refractory and treatment-naïve high-risk CLL. In relapsed/refractory cases previously exposed to the selective pressure of chemo(immuno)therapy, gain(8)(q24.21) and del(9)(p21.3) were particularly enriched. Both alterations affect key regulators of cell-cycle progression, namely MYC and CDKN2A/B. While homozygous CDKN2A/B loss has been directly associated with Richter transformation, we did not find this association for heterozygous loss of CDKN2A/B. Gains in 8q24.21 were either focal gains in a MYC enhancer region or large gains affecting the MYC locus, but only the latter type was highly enriched in relapsed/refractory CLL (17%). In addition to a high frequency of NOTCH1 mutations (23%), we found recurrent genetic alterations in SPEN (4% mutated), RBPJ (8% deleted) and SNW1 (8% deleted), all affecting a protein complex that represses transcription of NOTCH1 target genes. We investigated the functional impact of these alterations on HES1, DTX1 and MYC gene transcription and found derepression of these NOTCH1 target genes particularly with SPEN mutations. In summary, we provide new insights into the genomic architecture of high-risk CLL, define novel recurrent DNA copy number alterations and refine knowledge on del(9p), gain(8q) and alterations affecting NOTCH1 signaling. This study was registered at ClinicalTrials.gov with number NCT01392079.

B-cell receptor (BCR) signaling pathway components represent promising treatment targets in multiple B-cell malignancies including diffuse large B-cell lymphoma (DLBCL). In in vitro and in vivo model systems, a subset of DLBCLs depend upon BCR survival signals and respond to proximal BCR/phosphoinositide 3 kinase (PI3K) blockade. However, single-agent BCR pathway inhibitors have had more limited activity in patients with DLBCL, underscoring the need for indicators of sensitivity to BCR blockade and insights into potential resistance mechanisms. Here, we report highly significant transcriptional upregulation of C-X-C chemokine receptor 4 (CXCR4) in BCR-dependent DLBCL cell lines and primary tumors following chemical spleen tyrosine kinase (SYK) inhibition, molecular SYK depletion or chemical PI3K blockade. SYK or PI3K inhibition also selectively upregulated cell surface CXCR4 protein expression in BCR-dependent DLBCLs. CXCR4 expression was directly modulated by fork-head box O1 via the PI3K/protein kinase B/forkhead box O1 signaling axis. Following chemical SYK inhibition, all BCR-dependent DLBCLs exhibited significantly increased stromal cell-derived factor-1α (SDF-1α) induced chemotaxis, consistent with the role of CXCR4 signaling in B-cell migration. Select PI3K isoform inhibitors also augmented SDF-1α induced chemotaxis. These data define CXCR4 upregulation as an indicator of sensitivity to BCR/PI3K blockade and identify CXCR4 signaling as a potential resistance mechanism in BCR-dependent DLBCLs.

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.

Autophagy is a genetically regulated process of adaptation to metabolic stress and was recently shown to be involved in the treatment response of chronic myeloid leukemia (CML). However, in vivo data are limited and the molecular mechanism of autophagy regulators in the process of leukemogenesis is not completely understood. Here we show that Beclin-1 knockdown, but not Atg5 deletion in a murine CML model leads to a reduced leukemic burden and results in a significantly prolonged median survival of targeted mice. Further analyses of murine cell lines and primary patient material indicate that active BCR-ABL directly interacts with BECLIN-1 and phosphorylates its tyrosine residues 233 and 352, resulting in autophagy suppression. By using phosphorylation-deficient and phosphorylation-mimic mutants, we identify BCR-ABL induced BECLIN-1 phosphorylation as a crucial mechanism for BECLIN-1 complex formation: interaction analyses exhibit diminished binding of the positive autophagy regulators UVRAG, VPS15, ATG14 and VPS34 and enhanced binding of the negative regulator Rubicon to BCR-ABL-phosphorylated BECLIN-1. Taken together, our findings show interaction of BCR-ABL and BECLIN-1 thereby highlighting the importance of BECLIN-1-mediated autophagy in BCR-ABL⁺ cells.

Although highly effective, BCR-ABL1 tyrosine kinase inhibitors do not target chronic myeloid leukemia (CML) stem cells. Most patients relapse upon tyrosine kinase inhibitor therapy cessation. We reported previously that combined BCR-ABL1 and BCL-2 inhibition synergistically targets CML stem/progenitor cells. p53 induces apoptosis mainly by modulating BCL-2 family proteins. Although infrequently mutated in CML, p53 is antagonized by MDM2, which is regulated by BCR-ABL1 signaling. We hypothesized that MDM2 inhibition could sensitize CML cells to tyrosine kinase inhibitors. Using an inducible transgenic Scl-tTa-BCR-ABL1 murine CML model, we found, by RT-PCR and CyTOF proteomics increased p53 signaling in CML bone marrow (BM) cells compared with controls in CD45⁺ and linage-SCA-1⁺C-KIT⁺ populations. CML BM cells were more sensitive to exogenous BH3 peptides than controls. Combined inhibition of BCR-ABL1 with imatinib and MDM2 with DS-5272 increased NOXA level, markedly reduced leukemic linage-SCA-1⁺C-KIT⁺ cells and hematopoiesis, decreased leukemia burden, significantly prolonged the survival of mice engrafted with BM cells from Scl-tTa-BCR-ABL1 mice, and significantly decreased CML stem cell frequency in secondary transplantations. Our results suggest that CML stem/progenitor cells have increased p53 signaling and a propensity for apoptosis. Combined MDM2 and BCR-ABL1 inhibition targets CML stem/progenitor cells and has the potential to improve cure rates for CML.