The past 20 years have seen major advances in the understanding and treatment of pulmonary arterial hypertension (PAH; group 1 of the pulmonary hypertension (PH) clinical classification) [1]. A strong basis of knowledge has been acquired in: 1) large randomised clinical trials for drug development; 2) national registries for epidemiology and outcome; and 3) smaller studies on the pathophysiological mechanisms of the disease. This knowledge has been reviewed at World Symposia on Pulmonary Hypertension (the most recent in 2018 [2]) and summarised in European Respiratory Society (ERS)/European Society of Cardiology (ESC) clinical guidelines (the most recent in 2015 [3, 4]). We are, however, much less knowledgeable on specific aspects such as 1) the implementation of guidelines and access to therapies in different European countries; 2) the management of PH crises and progressive (acute on chronic) heart failure; and 3) other groups of PH, such as PH due to lung diseases. Therapeutic strategies also need to be optimised, in particular regarding the combination of drugs, the use of anticoagulants, the place for new medications targeting different pathophysiological pathways, etc.

OBJECTIVES:

To test the hypotheses that minority children with long-bone fractures are less likely to (1) receive analgesics, (2) receive opioid analgesics, and (3) achieve pain reduction.

BACKGROUND:

Medicaid plays a critical role during the perinatal period, but pregnancy-related Medicaid eligibility only extends for 60 days post partum. In 2014, the Affordable Care Act’s (ACA’s) Medicaid expansions increased adult Medicaid eligibility to 138% of the federal poverty level in participating states, allowing eligible new mothers to remain covered after pregnancy-related coverage expires. We investigate the impact of ACA Medicaid expansions on insurance coverage among new mothers living in poverty.

OBJECTIVES:

To describe (1) the developmental trajectories of peer victimization from 6 to 17 years of age and (2) the early childhood behaviors and family characteristics associated with the trajectories.

A 16-year-old girl presented to her primary care physician with a one-month history of decreased appetite and abdominal pain. She had normal bowel movements and no vomiting, but her periumbilical pain limited her ability to finish most meals. She had gradual weight loss over the previous 2 years, and during the previous 4 years, she intermittently received counseling for depression after the loss of her mother. Her initial physical examination and laboratory evaluation were unremarkable. She was referred to a nutritionist, adolescent medicine, and pediatric gastroenterology. Her presentation evolved over time, which ultimately led to a definitive diagnosis.

BACKGROUND:

Several studies have investigated the association of breastfeeding status with offspring mortality in Africa, but most studies were from one center only or had limited statistical power to draw robust conclusions.

BACKGROUND AND OBJECTIVES:

Early diagnosis of cerebral palsy (CP) is critical in obtaining evidence-based interventions when plasticity is greatest. In 2017, international guidelines for early detection of CP were published on the basis of a systematic review of evidence. Our study aim was to reduce the age at CP diagnosis throughout a network of 5 diverse US high-risk infant follow-up programs through consistent implementation of these guidelines.

During meiotic prophase, concurrent transcription, recombination, and chromosome synapsis place substantial topological strain on chromosomal DNA, but the role of topoisomerases in this context remains poorly defined. Here, we analyzed the roles of topoisomerases I and II (Top1 and Top2) during meiotic prophase in Saccharomyces cerevisiae. We show that both topoisomerases accumulate primarily in promoter-containing intergenic regions of actively transcribing genes, including many meiotic double-strand break (DSB) hotspots. Despite the comparable binding patterns, top1 and top2 mutations have different effects on meiotic recombination. TOP1 disruption delays DSB induction and shortens the window of DSB accumulation by an unknown mechanism. By contrast, temperature-sensitive top2-1 mutants exhibit a marked delay in meiotic chromosome remodeling and elevated DSB signals on synapsed chromosomes. The problems in chromosome remodeling were linked to altered Top2 binding patterns rather than a loss of Top2 catalytic activity, and stemmed from a defect in recruiting the chromosome remodeler Pch2/TRIP13 to synapsed chromosomes. No chromosomal defects were observed in the absence of TOP1. Our results imply independent roles for Top1 and Top2 in modulating meiotic chromosome structure and recombination.

Age-at-onset is one of the critical traits in cohort studies of age-related diseases. Large-scale genome-wide association studies (GWAS) of age-at-onset traits can provide more insights into genetic effects on disease progression and transitions between stages. Moreover, proportional hazards (or Cox) regression models can achieve higher statistical power in a cohort study than a case-control trait using logistic regression. Although mixed-effects models are widely used in GWAS to correct for sample dependence, application of Cox mixed-effects models (CMEMs) to large-scale GWAS is so far hindered by intractable computational cost. In this work, we propose COXMEG, an efficient R package for conducting GWAS of age-at-onset traits using CMEMs. COXMEG introduces fast estimation algorithms for general sparse relatedness matrices including, but not limited to, block-diagonal pedigree-based matrices. COXMEG also introduces a fast and powerful score test for dense relatedness matrices, accounting for both population stratification and family structure. In addition, COXMEG generalizes existing algorithms to support positive semidefinite relatedness matrices, which are common in twin and family studies. Our simulation studies suggest that COXMEG, depending on the structure of the relatedness matrix, is orders of magnitude computationally more efficient than coxme and coxph with frailty for GWAS. We found that using sparse approximation of relatedness matrices yielded highly comparable results in controlling false-positive rate and retaining statistical power for an ethnically homogeneous family-based sample. By applying COXMEG to a study of Alzheimer’s disease (AD) with a Late-Onset Alzheimer’s Disease Family Study from the National Institute on Aging sample comprising 3456 non-Hispanic whites and 287 African Americans, we identified the APOE 4 variant with strong statistical power (P = 1e–101), far more significant than that reported in a previous study using a transformed variable and a marginal Cox model. Furthermore, we identified novel SNP rs36051450 (P = 2e–9) near GRAMD1B, the minor allele of which significantly reduced the hazards of AD in both genders. These results demonstrated that COXMEG greatly facilitates the application of CMEMs in GWAS of age-at-onset traits.

There is increasing interest in developing diagnostics that discriminate individual mutagenic mechanisms in a range of applications that include identifying population-specific mutagenesis and resolving distinct mutation signatures in cancer samples. Analyses for these applications assume that mutagenic mechanisms have a distinct relationship with neighboring bases that allows them to be distinguished. Direct support for this assumption is limited to a small number of simple cases, e.g., CpG hypermutability. We have evaluated whether the mechanistic origin of a point mutation can be resolved using only sequence context for a more complicated case. We contrasted single nucleotide variants originating from the multitude of mutagenic processes that normally operate in the mouse germline with those induced by the potent mutagen N-ethyl-N-nitrosourea (ENU). The considerable overlap in the mutation spectra of these two samples make this a challenging problem. Employing a new, robust log-linear modeling method, we demonstrate that neighboring bases contain information regarding point mutation direction that differs between the ENU-induced and spontaneous mutation variant classes. A logistic regression classifier exhibited strong performance at discriminating between the different mutation classes. Concordance between the feature set of the best classifier and information content analyses suggest our results can be generalized to other mutation classification problems. We conclude that machine learning can be used to build a practical classification tool to identify the mutation mechanism for individual genetic variants. Software implementing our approach is freely available under an open-source license.

Key Points

Key Points

The translocated actin recruiting phosphoprotein (Tarp) is a multidomain type III secreted effector used by Chlamydia trachomatis. In aggregate, existing data suggest a role of this effector in initiating new infections. As new genetic tools began to emerge to study chlamydial genes in vivo, we speculated as to what degree Tarp function contributes to Chlamydia’s ability to parasitize mammalian host cells. To address this question, we generated a complete tarP deletion mutant using the fluorescence-reported allelic exchange mutagenesis (FRAEM) technique and complemented the mutant in trans with wild-type tarP or mutant tarP alleles engineered to harbor in-frame domain deletions. We provide evidence for the significant role of Tarp in C. trachomatis invasion of host cells. Complementation studies indicate that the C-terminal filamentous actin (F-actin)-binding domains are responsible for Tarp-mediated invasion efficiency. Wild-type C. trachomatis entry into HeLa cells resulted in host cell shape changes, whereas the tarP mutant did not. Finally, using a novel cis complementation approach, C. trachomatis lacking tarP demonstrated significant attenuation in a murine genital tract infection model. Together, these data provide definitive genetic evidence for the critical role of the Tarp F-actin-binding domains in host cell invasion and for the Tarp effector as a bona fide C. trachomatis virulence factor.

Epstein-Barr virus (EBV) causes B cell lymphomas and transforms B cells in vitro. The EBV protein EBNA3A collaborates with EBNA3C to repress p16 expression and is required for efficient transformation in vitro. An EBNA3A deletion mutant EBV strain was recently reported to establish latency in humanized mice but not cause tumors. Here, we compare the phenotypes of an EBNA3A mutant EBV (3A) and wild-type (WT) EBV in a cord blood-humanized (CBH) mouse model. The hypomorphic 3A mutant, in which a stop codon is inserted downstream from the first ATG and the open reading frame is disrupted by a 1-bp insertion, expresses very small amounts of EBNA3A using an alternative ATG at residue 15. 3A caused B cell lymphomas at rates similar to their induction by WT EBV but with delayed onset. 3A and WT tumors expressed equivalent levels of EBNA2 and p16, but 3A tumors in some cases had reduced LMP1. Like the WT EBV tumors, 3A lymphomas were oligoclonal/monoclonal, with typically one dominant IGHV gene being expressed. Transcriptome sequencing (RNA-seq) analysis revealed small but consistent gene expression differences involving multiple cellular genes in the WT EBV- versus 3A-infected tumors and increased expression of genes associated with T cells, suggesting increased T cell infiltration of tumors. Consistent with an impact of EBNA3A on immune function, we found that the expression of CLEC2D, a receptor that has previously been shown to influence responses of T and NK cells, was markedly diminished in cells infected with EBNA3A mutant virus. Together, these studies suggest that EBNA3A contributes to efficient EBV-induced lymphomagenesis in CBH mice.

IMPORTANCE The EBV protein EBNA3A is expressed in latently infected B cells and is important for efficient EBV-induced transformation of B cells in vitro. In this study, we used a cord blood-humanized mouse model to compare the phenotypes of an EBNA3A hypomorph mutant virus (3A) and wild-type EBV. The 3A virus caused lymphomas with delayed onset compared to the onset of those caused by WT EBV, although the tumors occurred at a similar rate. The WT EBV and EBNA3A mutant tumors expressed similar levels of the EBV protein EBNA2 and cellular protein p16, but in some cases, 3A tumors had less LMP1. Our analysis suggested that 3A-infected tumors have elevated T cell infiltrates and decreased expression of the CLEC2D receptor, which may point to potential novel roles of EBNA3A in T cell and NK cell responses to EBV-infected tumors.

The major obstacle to a cure for HIV infection is the persistence of replication-competent viral reservoirs during antiretroviral therapy. HIV-specific chimeric antigen receptor (CAR) T cells have been developed to target latently infected CD4⁺ T cells that express virus either spontaneously or after intentional latency reversal. Whether HIV-specific CAR-T cells can recognize and eliminate the follicular dendritic cell (FDC) reservoir of HIV-bound immune complexes (ICs) is unknown. We created HIV-specific CAR-T cells using human peripheral blood mononuclear cells (PBMCs) and a CAR construct that enables the expression of CD4 (domains 1 and 2) and the carbohydrate recognition domain of mannose binding lectin (MBL) to target native HIV Env (CD4-MBL CAR). We assessed CAR-T cell cytotoxicity using a carboxyfluorescein succinimidyl ester (CFSE) release assay and evaluated CAR-T cell activation through interferon gamma (IFN-) production and CD107a membrane accumulation by flow cytometry. CD4-MBL CAR-T cells displayed potent lytic and functional responses to Env-expressing cell lines and HIV-infected CD4⁺ T cells but were ineffective at targeting FDC bearing HIV-ICs. CD4-MBL CAR-T cells were unresponsive to cell-free HIV or concentrated, immobilized HIV-ICs in cell-free experiments. Blocking intercellular adhesion molecule-1 (ICAM-1) inhibited the cytolytic response of CD4-MBL CAR-T cells to Env-expressing cell lines and HIV-infected CD4⁺ T cells, suggesting that factors such as adhesion molecules are necessary for the stabilization of the CAR-Env interaction to elicit a cytotoxic response. Thus, CD4-MBL CAR-T cells are unable to eliminate the FDC-associated HIV reservoir, and alternative strategies to eradicate this reservoir must be sought.

IMPORTANCE Efforts to cure HIV infection have focused primarily on the elimination of latently infected CD4⁺ T cells. Few studies have addressed the unique reservoir of infectious HIV that exists on follicular dendritic cells (FDCs), persists in vivo during antiretroviral therapy, and likely contributes to viral rebound upon cessation of antiretroviral therapy. We assessed the efficacy of a novel HIV-specific chimeric antigen receptor (CAR) T cell to target both HIV-infected CD4⁺ T cells and the FDC reservoir in vitro. Although CAR-T cells eliminated CD4⁺ T cells that express HIV, they did not respond to or eliminate FDC bound to HIV. These findings reveal a fundamental limitation to CAR-T cell therapy to eradicate HIV.

During human immunodeficiency virus type 1 (HIV-1) entry into cells, the viral envelope glycoprotein (Env) trimer [(gp120/gp41)₃] binds the receptors CD4 and CCR5 and fuses the viral and cell membranes. CD4 binding changes Env from a pretriggered (state-1) conformation to more open downstream conformations. BMS-378806 (here called BMS-806) blocks CD4-induced conformational changes in Env important for entry and is hypothesized to stabilize a state-1-like Env conformation, a key vaccine target. Here, we evaluated the effects of BMS-806 on the conformation of Env on the surface of cells and virus-like particles. BMS-806 strengthened the labile, noncovalent interaction of gp120 with the Env trimer, enhanced or maintained the binding of most broadly neutralizing antibodies, and decreased the binding of poorly neutralizing antibodies. Thus, in the presence of BMS-806, the cleaved Env on the surface of cells and virus-like particles exhibits an antigenic profile consistent with a state-1 conformation. We designed novel BMS-806 analogues that stabilized the Env conformation for several weeks after a single application. These long-acting BMS-806 analogues may facilitate enrichment of the metastable state-1 Env conformation for structural characterization and presentation to the immune system.

IMPORTANCE The envelope glycoprotein (Env) spike on the surface of human immunodeficiency virus type 1 (HIV-1) mediates the entry of the virus into host cells and is also the target for antibodies. During virus entry, Env needs to change shape. Env flexibility also contributes to the ability of HIV-1 to evade the host immune response; many shapes of Env raise antibodies that cannot recognize the functional Env and therefore do not block virus infection. We found that an HIV-1 entry inhibitor, BMS-806, stabilizes the functional shape of Env. We developed new variants of BMS-806 that stabilize Env in its natural state for long periods of time. The availability of such long-acting stabilizers of Env shape will allow the natural Env conformation to be characterized and tested for efficacy as a vaccine.

Members of the flavivirus genus share a high level of sequence similarity and often circulate in the same geographical regions. However, whether T cells induced by one viral species cross-react with other related flaviviruses has not been globally addressed. In this study, we tested pools of epitopes derived from dengue (DENV), Zika (ZIKV), Japanese encephalitis (JEV), West Nile (WNV), and yellow fever (YFV) viruses by intracellular cytokine staining (ICS) using peripheral blood mononuclear cells (PBMCs) of individuals naturally exposed to DENV or immunized with DENV (TV005) or YF17D vaccine. CD8 T cell responses recognized epitopes from multiple flaviviruses; however, the magnitude of cross-reactive responses was consistently severalfold lower than those to the autologous epitope pools and was associated with lower expression of activation markers such as CD40L, CD69, and CD137. Next, we characterized the antigen sensitivity of short-term T cell lines (TCL) representing 29 different individual epitope/donor combinations. TCL derived from DENV monovalent vaccinees induced CD8 and CD4 T cells that cross-reacted within the DENV serocomplex but were consistently associated with >100-fold-lower antigen sensitivity for most other flaviviruses, with no cross-recognition of YFV-derived peptides. CD8 and CD4 TCL from YF17D vaccinees were associated with very limited cross-reactivity with any other flaviviruses and in five out of eight cases >1,000-fold-lower antigen sensitivity. Overall, our data suggest limited cross-reactivity for both CD4 and CD8 T cell responses between flaviviruses and have implications for understanding immunity elicited by natural infection and strategies to develop live attenuated vaccines against flaviviral species.

IMPORTANCE The envelope (E) protein is the dominant target of neutralizing antibodies for dengue virus (DENV) and yellow fever virus (YFV). Accordingly, several DENV vaccine constructs use the E protein in a live attenuated vaccine format, utilizing a backbone derived from a heterologous flavivirus (such as YF) as a delivery vector. This backbone comprises the nonstructural (NS) and capsid (C) antigens, which are dominant targets of T cell responses. Here, we demonstrate that cross-reactivity at the level of T cell responses among different flaviviruses is very limited, despite high levels of sequence homology. Thus, the use of heterologous flavivirus species as a live attenuated vaccine vector is not likely to generate optimal T cell responses and might thus impair vaccine performance.

A novel lytic bacteriophage, ValSw3-3, which efficiently infects pathogenic strains of Vibrio alginolyticus, was isolated from sewage water and characterized by microbiological and in silico genomic analyses. Transmission electron microscopy indicated that ValSw3-3 has the morphology of siphoviruses. This phage can infect four species in the Vibrio genus and has a latent period of 15 min and a burst size of 95 ± 2 PFU/infected bacterium. Genome sequencing results show that ValSw3-3 has a 39,846-bp double-stranded DNA genome with a GC content of 43.1%. The similarity between the genome sequences of ValSw3-3 and those of other phages recorded in the GenBank database was below 50% (42%), suggesting that ValSw3-3 significantly differs from previously reported phages at the DNA level. Multiple genome comparisons and phylogenetic analysis based on the major capsid protein revealed that phage ValSw3-3 is grouped in a clade with five other phages, including Listonella phage phiHSIC (GenBank accession no. NC_006953.1), Vibrio phage P23 (MK097141.1), Vibrio phage pYD8-B (NC_021561.1), Vibrio phage 2E1 (KX507045.1), and Vibrio phage 12G5 (HQ632860.1), and is distinct from all known genera within the Siphoviridae family that have been ratified by the International Committee on Taxonomy of Viruses (ICTV). An in silico proteomic comparison of diverse phages from the Siphoviridae family supported this clustering result and suggested that ValSw3-3, phiHSIC, P23, pYD8-B, 2E1, and 12G5 should be classified as a novel genus cluster of Siphoviridae. A subsequent analysis of core genes also revealed the common genes shared within this new cluster. Overall, these results provide a characterization of Vibrio phage ValSw3-3 and support our proposal of a new viral genus within the family Siphoviridae.

IMPORTANCE Phage therapy has been considered a potential alternative to antibiotic therapy in treating bacterial infections. For controlling the vibriosis-causing pathogen Vibrio alginolyticus, well-documented phage candidates are still lacking. Here, we characterize a novel lytic Vibrio phage, ValSw3-3, based on its morphology, host range and infectivity, growth characteristics, stability under various conditions, and genomic features. Our results show that ValSw3-3 could be a potent candidate for phage therapy to treat V. alginolyticus infections due to its stronger infectivity and better pH and thermal stability than those of previously reported Vibrio phages. Moreover, genome sequence alignments, phylogenetic analysis, in silico proteomic comparison, and core gene analysis all support that this novel phage, ValSw3-3, and five unclassified phages form a clade distant from those of other known genera ratified by the ICTV. Thus, we propose a new viral genus within the Siphoviridae family to accommodate this clade, with ValSw3-3 as a representative member.

Subtle features of common language can imply to young children that scientists are a special and distinct kind of person—a way of thinking that can interfere with the development of children’s own engagement with science. We conducted a large field experiment (involving 45 prekindergarten schools, 130 teachers, and over 1,100...

HIV-1 persists in cellular reservoirs that can reignite viremia if antiretroviral therapy (ART) is interrupted. Therefore, insight into the nature of those reservoirs may be revealed from the composition of recrudescing viremia following treatment cessation. A minor population of macrophage-tropic (M-tropic) viruses was identified in a library of recombinant viruses...

When transitioning from the environment, pathogenic microorganisms must adapt rapidly to survive in hostile host conditions. This is especially true for environmental fungi that cause opportunistic infections in immunocompromised patients since these microbes are not well adapted human pathogens. Cryptococcus species are yeastlike fungi that cause lethal infections, especially in...

Cowen et al. (1) leverage modern gains in data science to describe impressive cross-cultural similarities in the perception of musical affect and do so in unprecedented detail. Their approach is innovative and fundamentally empirical. As such, it should have important applications for prediction in the field of affective computing, which...

ABSTRACT

By modulating the structure, diversity, and trophic outputs of microbial communities, phages play crucial roles in many biomes. In oligotrophic polar deserts, the effects of katabatic winds, constrained nutrients, and low water availability are known to limit microbial activity. Although phages may substantially govern trophic interactions in cold deserts, relatively little is known regarding the precise ecological mechanisms. Here, we provide the first evidence of widespread antiphage innate immunity in Antarctic environments using metagenomic sequence data from hypolith communities as model systems. In particular, immunity systems such as DISARM and BREX are shown to be dominant systems in these communities. Additionally, we show a direct correlation between the CRISPR-Cas adaptive immunity and the metavirome of hypolith communities, suggesting the existence of dynamic host-phage interactions. In addition to providing the first exploration of immune systems in cold deserts, our results suggest that phages actively challenge niche communities in Antarctic polar deserts. We provide evidence suggesting that the regulatory role played by phages in this system is an important determinant of bacterial host interactions in this environment.

IMPORTANCE In Antarctic environments, the combination of both abiotic and biotic stressors results in simple trophic levels dominated by microbiomes. Although the past two decades have revealed substantial insights regarding the diversity and structure of microbiomes, we lack mechanistic insights regarding community interactions and how phages may affect these. By providing the first evidence of widespread antiphage innate immunity, we shed light on phage-host dynamics in Antarctic niche communities. Our analyses reveal several antiphage defense systems, including DISARM and BREX, which appear to dominate in cold desert niche communities. In contrast, our analyses revealed that genes which encode antiphage adaptive immunity were underrepresented in these communities, suggesting lower infection frequencies in cold edaphic environments. We propose that by actively challenging niche communities, phages play crucial roles in the diversification of Antarctic communities.

ABSTRACT

Insights into the interaction between phages and their bacterial hosts are crucial for the development of phage therapy. However, only one study has investigated global gene expression of Clostridioides (formerly Clostridium) difficile carrying prophage, and transcriptional reprogramming during lytic infection has not been studied. Here, we presented the isolation, propagation, and characterization of a newly discovered 35,109-bp phage, JD032, and investigated the global transcriptomes of both JD032 and C. difficile ribotype 078 (RT078) strain TW11 during JD032 infection. Transcriptome sequencing (RNA-seq) revealed the progressive replacement of bacterial host mRNA with phage transcripts. The expressed genes of JD032 were clustered into early, middle, and late temporal categories that were functionally similar. Specifically, a gene (JD032_orf016) involved in the lysis-lysogeny decision was identified as an early expression gene. Only 17.7% (668/3,781) of the host genes were differentially expressed, and more genes were downregulated than upregulated. The expression of genes involved in host macromolecular synthesis (DNA/RNA/proteins) was altered by JD032 at the level of transcription. In particular, the expression of the ropA operon was downregulated. Most noteworthy is that the gene expression of some antiphage systems, including CRISPR-Cas, restriction-modification, and toxin-antitoxin systems, was suppressed by JD032 during infection. In addition, bacterial sporulation, adhesion, and virulence factor genes were significantly downregulated. This study provides the first description of the interaction between anaerobic spore-forming bacteria and phages during lytic infection and highlights new aspects of C. difficile phage-host interactions.

IMPORTANCE C. difficile is one of the most clinically significant intestinal pathogens. Although phages have been shown to effectively control C. difficile infection, the host responses to phage predation have not been fully studied. In this study, we reported the isolation and characterization of a new phage, JD032, and analyzed the global transcriptomic changes in the hypervirulent RT078 C. difficile strain, TW11, during phage JD032 infection. We found that bacterial host mRNA was progressively replaced with phage transcripts, three temporal categories of JD032 gene expression, the extensive interplay between phage-bacterium, antiphage-like responses of the host and phage evasion, and decreased expression of sporulation- and virulence-related genes of the host after phage infection. These findings confirmed the complexity of interactions between C. difficile and phages and suggest that phages undergoing a lytic cycle may also cause different phenotypes in hosts, similar to prophages, which may inspire phage therapy for the control of C. difficile.

Cervical spondylotic myelopathy (CSM) is a neurologic condition that develops insidiously over time as degenerative changes of the spine result in compression of the cord and nearby structures. It is the most common form of spinal cord injury in adults; yet, its diagnosis is often delayed. The purpose of this article is to review the pathophysiology, natural history, diagnosis, and management of CSM with a focus on the recommended timeline for physicians suspecting CSM to refer patients to a spine surgeon. Various processes underlie spondylotic changes of the canal and are separated into static and dynamic factors. Not all patients with evidence of cord compression will present with symptoms, and the progression of disease varies by patient. The hallmark symptoms of CSM include decreased hand dexterity and gait instability as well as sensory and motor dysfunction. magnetic resonance imaging is the imaging modality of choice in patients with suspected CSM, but computed tomography myelography may be used in patients with contraindications. Patients with mild CSM may be treated surgically or nonoperatively, whereas those with moderate-severe disease are treated operatively. Due to the long-term disability that may result from a delay in diagnosis and management, prompt referral to a spine surgeon is recommended for any patient suspected of having CSM. This review provides information and guidelines for practitioners to develop an actionable awareness of CSM.

Background:

In 2015, Vyvanse (lisdexamfetamine) became the first Food and Drug Administration (FDA)-approved treatment for binge-eating disorder (BED), a condition first recognized by the DSM–V in 2013. Because pharmaceutical companies use continuing medical education (CME) to help sell drugs, we explored possible bias in CME modules on BED.

Background

Kidney injury associated with cold storage is a determinant of delayed graft function and the long-term outcome of transplanted kidneys, but the underlying mechanism remains elusive. We previously reported a role of protein kinase C- (PKC) in renal tubular injury during cisplatin nephrotoxicity and albumin-associated kidney injury, but whether PKC is involved in ischemic or transplantation-associated kidney injury is unknown.

Background

Arteriovenous fistulas placed surgically for dialysis vascular access have a high primary failure rate resulting from excessive inward remodeling, medial fibrosis, and thrombosis. No clinically established pharmacologic or perisurgical therapies currently address this unmet need. Statins’ induction of multiple anti-inflammatory and antithrombotic effects suggests that these drugs might reduce arteriovenous fistula failure. Yet, the in vivo physiologic and molecular effects of statins on fistula patency and maturation remain poorly understood.

BACKGROUND:Pneumoperitoneum and Trendelenburg position affect respiratory system mechanics and oxygenation during elective pelvic robotic surgery. The primary aim of this randomized pilot study was to compare the effects of a conventional low tidal volume ventilation with PEEP guided by gas exchange (VGas-guided) versus low tidal volume ventilation tailoring PEEP according to esophageal pressure (VPes-guided) on oxygenation and respiratory mechanics during elective pelvic robotic surgery.METHODS:This study was conducted in a single-center tertiary hospital between September 2017 and January 2019. Forty-nine adult patients scheduled for elective pelvic robotic surgery were screened; 28 subjects completed the full analysis. Exclusion criteria were American Society of Anesthesiologists physical status ≥ 3, contraindications to nasogastric catheter placement, and pregnancy. After dedicated naso/orogastric catheter insertion, subjects were randomly assigned to VGas-guided (FIO2 and PEEP set to achieve SpO2 > 94%) or VPes-guided (PEEP tailored to equalize end-expiratory transpulmonary pressure). Oxygenation (PaO2/FIO2) was evaluated (1) at randomization, after pneumoperitoneum and Trendelenburg application; (2) at 60 min; (3) at 120 min following randomization; and (4) at end of surgery. Respiratory mechanics were assessed during the duration of the study.RESULTS:Compared to VGas-guided, oxygenation was higher with VPes-guided at 60 min (388 ± 90 vs 308 ± 95 mm Hg, P = .02), at 120 min after randomization (400 ± 90 vs 308 ± 81 mm Hg, P = .008), and at the end of surgery (402 ± 95 vs 312 ± 95 mm Hg, P = .009). Respiratory system elastance was lower with VPes-guided compared to VGas-guided at 20 min (24.2 ± 7.3 vs 33.4 ± 10.7 cm H2O/L, P = .001) and 60 min (24.1 ± 5.4 vs 31.9 ± 8.5 cm H2O/L, P = .006) from randomization.CONCLUSIONS:Oxygenation and respiratory system mechanics were improved when applying a ventilatory strategy tailoring PEEP to equalize expiratory transpulmonary pressure in subjects undergoing pelvic robotic surgery compared to a VGas-guided approach. (ClinicalTrials.gov registration NCT03153592).

Drug-induced liver injury (DILI) is a global medical problem. The risk of DILI is often related to expression and activities of drug-metabolizing enzymes, especially cytochrome P450s (P450s). However, changes on expression and activities of P450s after DILI have not been determined. The aim of this study is to fill this knowledge gap. Acetaminophen (APAP) was used as a model drug to induce DILI in C57BL/6J mice at different ages of days 10 (infant), 22 (child), and 60 (adult). DILI was assessed by levels of alanine aminotransferase and aspartate aminotransferase in plasma with a confirmation by H&E staining on liver tissue sections. The expression of selected P450s at mRNA and protein levels was measured by real-time polymerase chain reaction and liquid chromatography–tandem mass spectrometry, respectively. The activities of these P450s were determined by the formation of metabolites from probe drugs for each P450 using ultraperformance liquid chromatography–quadrupole time of flight mass spectrometry. DILI was induced at mild to severe levels in a dose-dependent manner in 200, 300, and 400 mg/kg APAP-treated groups at child and adult ages, but not at the infant age. Significantly decreased expression at mRNA and protein levels as well as enzymatic activities of CYP2E1, 3A11, 1A2, and 2C29 were found at child and adult ages. Adult male mice were more susceptible to APAP-induced liver injury than female mice with more decreased expression of P450s. These results suggest that altered levels of P450s in livers severely injured by drugs may affect the therapeutic efficacy of drugs, which are metabolized by P450s, more particularly for males.

Contact between inflammatory cells and endothelial cells (ECs) is a crucial step in vascular inflammation. Recently, we demonstrated that the cell-surface level of endomucin (EMCN), a heavily O-glycosylated single-transmembrane sialomucin, interferes with the interactions between inflammatory cells and ECs. We have also shown that, in response to an inflammatory stimulus, EMCN is cleared from the cell surface by an unknown mechanism. In this study, using adenovirus-mediated overexpression of a tagged EMCN in human umbilical vein ECs, we found that treatment with tumor necrosis factor α (TNF-α) or the strong oxidant pervanadate leads to loss of cell-surface EMCN and increases the levels of the C-terminal fragment of EMCN 3- to 4-fold. Furthermore, treatment with the broad-spectrum matrix metalloproteinase inhibitor batimastat (BB94) or inhibition of ADAM metallopeptidase domain 10 (ADAM10) and ADAM17 with two small-molecule inhibitors, GW280264X and GI254023X, or with siRNA significantly reduced basal and TNFα-induced cell-surface EMCN cleavage. Release of the C-terminal fragment of EMCN by TNF-α treatment was blocked by chemical inhibition of ADAM10 alone or in combination with ADAM17. These results indicate that cell-surface EMCN undergoes constitutive cleavage and that TNF-α treatment dramatically increases this cleavage, which is mediated predominantly by ADAM10 and ADAM17. As endothelial cell-surface EMCN attenuates leukocyte–EC interactions during inflammation, we propose that EMCN is a potential therapeutic target to manage vascular inflammation.

Arrestin-1 is the arrestin family member responsible for inactivation of the G protein–coupled receptor rhodopsin in photoreceptors. Arrestin-1 is also well-known to interact with additional protein partners and to affect other signaling cascades beyond phototransduction. In this study, we investigated one of these alternative arrestin-1 binding partners, the glycolysis enzyme enolase-1, to map the molecular contact sites between these two proteins and investigate how the binding of arrestin-1 affects the catalytic activity of enolase-1. Using fluorescence quench protection of strategically placed fluorophores on the arrestin-1 surface, we observed that arrestin-1 primarily engages enolase-1 along a surface that is opposite of the side of arrestin-1 that binds photoactivated rhodopsin. Using this information, we developed a molecular model of the arrestin-1–enolase-1 complex, which was validated by targeted substitutions of charge-pair interactions. Finally, we identified the likely source of arrestin's modulation of enolase-1 catalysis, showing that selective substitution of two amino acids in arrestin-1 can completely remove its effect on enolase-1 activity while still remaining bound to enolase-1. These findings open up opportunities for examining the functional effects of arrestin-1 on enolase-1 activity in photoreceptors and their surrounding cells.

Site-specific recombinases, such as Cre, are a widely used tool for genetic lineage tracing in the fields of developmental biology, neural science, stem cell biology, and regenerative medicine. However, nonspecific cell labeling by some genetic Cre tools remains a technical limitation of this recombination system, which has resulted in data misinterpretation and led to many controversies in the scientific community. In the past decade, to enhance the specificity and precision of genetic targeting, researchers have used two or more orthogonal recombinases simultaneously for labeling cell lineages. Here, we review the history of cell-tracing strategies and then elaborate on the working principle and application of a recently developed dual genetic lineage-tracing approach for cell fate studies. We place an emphasis on discussing the technical strengths and caveats of different methods, with the goal to develop more specific and efficient tracing technologies for cell fate mapping. Our review also provides several examples for how to use different types of DNA recombinase–mediated lineage-tracing strategies to improve the resolution of the cell fate mapping in order to probe and explore cell fate–related biological phenomena in the life sciences.

Premature infants have a higher incidence of indirect hyperbilirubinemia than term infants. Management of neonatal indirect hyperbilirubinemia in late preterm and term neonates has been well addressed by recognized, consensus-based guidelines. However, the extension of these guidelines to the preterm population has been an area of uncertainty because of limited evidence. This leads to variation in clinical practice and lack of recognition of the spectrum of bilirubin-induced neurologic dysfunction (BIND) in this population. Preterm infants are metabolically immature and at higher risk for BIND at lower bilirubin levels than their term counterparts. Early use of phototherapy to eliminate BIND and minimize the need for exchange transfusion is the goal of treatment in premature neonates. Although considered relatively safe, phototherapy does have side effects, and some NICUs tend to overuse phototherapy. In this review, we describe the epidemiology and pathophysiology of BIND in preterm neonates, and discuss our approach to standardized management of indirect hyperbilirubinemia in the vulnerable preterm population. The proposed treatment charts suggest early use of phototherapy in preterm neonates with the aim of reducing exposure to high irradiance levels, minimizing the need for exchange transfusions, and preventing BIND. The charts are pragmatic and have additional curves for stopping phototherapy and escalating its intensity. Having a standardized approach would support future research and quality improvement initiatives that examine dose and duration of phototherapy exposure with relation to outcomes.

Virus-like-particle (VLP) influenza vaccines can be given intramuscularly (i.m.) or intranasally (i.n.) and may have advantages over split-virion formulations in the elderly. We tested a plant-made VLP vaccine candidate bearing the viral hemagglutinin (HA) delivered either i.m. or i.n. in young and aged mice. Young adult (5- to 8-week-old) and aged (16- to 20-month-old) female BALB/c mice received a single 3-μg dose based on the HA (A/California/07/2009 H1N1) content of a plant-made H1-VLP (i.m. or i.n.) split-virion vaccine (i.m.) or were left naive. After vaccination, humoral and splenocyte responses were assessed, and some mice were challenged. Both VLP and split vaccines given i.m. protected 100% of the young animals, but the VLP group lost the least weight and had stronger humoral and cellular responses. Compared to split-vaccine recipients, aged animals vaccinated i.m. with VLP were more likely to survive challenge (80% versus 60%). The lung viral load postchallenge was lowest in the VLP i.m. groups. Mice vaccinated with VLP i.n. had little detectable immune response, but survival was significantly increased. In both age groups, i.m. administration of the H1-VLP vaccine elicited more balanced humoral and cellular responses and provided better protection from homologous challenge than the split-virion vaccine.

Objective

To study the immunomodulatory effect of dimethyl fumarate (DF) on granulocyte macrophage colony-stimulating factor (GM-CSF) production in CD4⁺ T cells in experimental autoimmune encephalomyelitis (EAE) and human peripheral blood mononuclear cells (PBMCs).

Most British railway embankments were constructed between 120 and 180 years ago without the benefit of modern design and construction methods. This can result in undesirable load-deformation characteristics and consequent disruption to present-day railway operations, for which there is unprecedented demand. Annual rail passenger kilometres have approximately doubled in the last 20 years and freight has increased by 60% over the same period. Whereas elements such as rails or bridges can be refurbished or replaced to meet increasing demand, the same is not usually feasible for embankments. Development of techniques to assess embankment performance risks posed by operational capacity enhancements is therefore of increasing significance to railway geotechnical asset management. The two case studies presented in this paper demonstrate how geospatial analysis and data management techniques may be applied to this challenge at both strategic (regional or national) and tactical (site-specific) scales for embankments incorporating plastic clay fill. The case studies also demonstrate, in a world of ever more abundant data, the growing need for engineering geologists and geotechnical engineers to augment their traditional knowledge with comprehensive data management and geospatial analysis skills, these being essential for modern infrastructure asset management.

Thematic collection: This article is part of the ‘Ground-related risk to transportation infrastructure’ collection available at: https://www.lyellcollection.org/cc/Ground-related-risk-to-transportation-infrastructure

Transportation infrastructure owners manage an array of different asset types such as bridges, road pavements, earthworks and drainage. Currently, most organization management procedures are siloed by asset type; however, there are important interactions between these asset groups that need to be managed in a cross-asset way. Although these interactions are known, there is little or no quantification of these interactions. For the first time, this paper quantifies that 74% of Highways England's earthwork failures are a result of drainage-related problems, either the lack of drainage infrastructure or the poor performance of it. The analysis undertaken is an important first step not only in moving towards more connected asset management planning for earthworks and drainage, but to also provide guidance for other owners of earthwork infrastructure assets to improve their strategic asset management procedures.

Thematic collection: This article is part of the Ground-related risk to transportation infrastructure collection available at https://www.lyellcollection.org/cc/Ground-related-risk-to-transportation-infrastructure

Strategic geotechnical asset management considers the whole of an organization's earthworks portfolio and is concerned with setting an overall earthworks asset management policy with long-term objectives related to asset performance, safety and condition, and identifying how those objectives can best be met, now and into the future. A risk-based approach is adopted that requires an understanding of the likelihood that any of the earthworks may fail, combined with a knowledge of the consequences should they fail. Procedures are required to identify those earthworks that are most vulnerable to failure under the influence of triggering events, such as extreme weather. The risks are managed through a mix of interventions to reduce the likelihood of failure and mitigations to reduce the impact of failure. Many of the challenges of implementing a strategic earthworks policy have, or are, being met by the main UK transportation infrastructure organizations.

Thematic collection: This article is part of the Ground-related risk to transportation infrastructure collection available at https://www.lyellcollection.org/cc/Ground-related-risk-to-transportation-infrastructure

In this paper, a decision tree is presented, constructed on the basis of hydrogeological characteristics (water table depth, freshwater thickness, surface area required and distance between wells), to choose the optimal groundwater extraction method in the case of a coastal unconfined aquifer. A comparison is made of the groundwater extraction methods in a freshwater aquifer of limited thickness occurring in coastal dunes in the eastern region of the Province of Buenos Aires (Argentina). The negative effects brought about by the wrong use of the groundwater extraction methods are analysed, because, as a result of excessive extraction, such methods lead to the dramatic decrease of the freshwater reserves. The decision tree is a useful tool to assist decision-makers as it suggests the most suitable groundwater extraction method options (vertical wells or wellpoints), as well as identifying areas that are unsuitable for sustainable groundwater extraction.

There are roughly 48,000 deaths caused by influenza annually and an estimated 200,000 people who have undiagnosed human immunodeficiency virus (HIV). These are examples of acute and chronic illnesses that can be identified by employing a CLIA-waived test. Pharmacies across the country have been incorporating CLIA-waived point-of-care tests (POCT) into disease screening and management programs offered in the pharmacy. The rationale behind these programs is discussed. Additionally, a summary of clinical data for some of these programs in the infectious disease arena is provided. Finally, we discuss the future potential for CLIA-waived POCT-based programs in community pharmacies.

Large-scale metagenomic and metatranscriptomic data analyses are often restricted by their gene-centric approach, limiting the ability to understand organismal and community biology. De novo assembly of large and mosaic eukaryotic genomes from complex meta-omics data remains a challenging task, especially in comparison with more straightforward bacterial and archaeal systems. Here, we use a transcriptome reconstruction method based on clustering co-abundant genes across a series of metagenomic samples. We investigated the co-abundance patterns of ~37 million eukaryotic unigenes across 365 metagenomic samples collected during the Tara Oceans expeditions to assess the diversity and functional profiles of marine plankton. We identified ~12,000 co-abundant gene groups (CAGs), encompassing ~7 million unigenes, including 924 metagenomics-based transcriptomes (MGTs, CAGs larger than 500 unigenes). We demonstrated the biological validity of the MGT collection by comparing individual MGTs with available references. We identified several key eukaryotic organisms involved in dimethylsulfoniopropionate (DMSP) biosynthesis and catabolism in different oceanic provinces, thus demonstrating the potential of the MGT collection to provide functional insights on eukaryotic plankton. We established the ability of the MGT approach to capture interspecies associations through the analysis of a nitrogen-fixing haptophyte-cyanobacterial symbiotic association. This MGT collection provides a valuable resource for analyses of eukaryotic plankton in the open ocean by giving access to the genomic content and functional potential of many ecologically relevant eukaryotic species.

Retrospective lineage tracing harnesses naturally occurring mutations in cells to elucidate single cell development. Common single-cell phylogenetic fate mapping methods have utilized highly mutable microsatellite loci found within the human genome. Such methods were limited by the introduction of in vitro noise through polymerase slippage inherent in DNA amplification, which we characterized to be approximately 10–100x higher than the in vivo replication mutation rate. Here, we present RETrace, a method for simultaneously capturing both microsatellites and methylation-informative cytosines to characterize both lineage and cell type, respectively, from the same single cell. An important unique feature of RETrace was the introduction of linear amplification of microsatellites in order to reduce in vitro amplification noise. We further coupled microsatellite capture with single-cell reduced representation bisulfite sequencing (scRRBS), to measure the CpG methylation status on the same cell for cell type inference. When compared to existing retrospective lineage tracing methods, RETrace achieved higher accuracy (88% triplet accuracy from an ex vivo HCT116 tree) at a higher cell division resolution (lowering the required number of cell division difference between single cells by approximately 100 divisions). Simultaneously, RETrace demonstrated the ability to capture on average 150,000 unique CpGs per single cell in order to accurately determine cell type. We further formulated additional developments that would allow high-resolution mapping on microsatellite-stable cells or tissues with RETrace. Overall, we present RETrace as a foundation for multi-omics lineage mapping and cell typing of single cells.

Mutations in X-linked methyl-CpG-binding protein 2 (MECP2) cause Rett syndrome (RTT). To identify functional pathways that could inform therapeutic entry points, we carried out a genetic screen for secondary mutations that improved phenotypes in Mecp2/Y mice after mutagenesis with N-ethyl-N-nitrosourea (ENU). Here, we report the isolation of 106 founder animals that show suppression of Mecp2-null traits from screening 3177 Mecp2/Y genomes. Whole-exome sequencing, genetic crosses, and association analysis identified 22 candidate genes. Additional lesions in these candidate genes or pathway components associate variant alleles with phenotypic improvement in 30 lines. A network analysis shows that 63% of the genes cluster into the functional categories of transcriptional repression, chromatin modification, or DNA repair, delineating a pathway relationship with MECP2. Many mutations lie in genes that modulate synaptic signaling or lipid homeostasis. Mutations in genes that function in the DNA damage response (DDR) also improve phenotypes in Mecp2/Y mice. Association analysis was successful in resolving combinatorial effects of multiple loci. One line, which carries a suppressor mutation in a gene required for cholesterol synthesis, Sqle, carries a second mutation in retinoblastoma binding protein 8, endonuclease (Rbbp8, also known as CtIP), which regulates a DDR choice in double-stranded break (DSB) repair. Cells from Mecp2/Y mice have increased DSBs, so this finding suggests that the balance between homology-directed repair and nonhomologous end joining is important for neuronal cells. In this and other lines, two suppressor mutations confer greater improvement than one alone, suggesting that combination therapies could be effective in RTT.

Cinnamaldehyde (Cin), a bioactive cinnamon essential oil from traditional Chinese medicine herb Cinnamomum cassia, has been reported to have multipharmacological activities including anti-inflammation. However, its role and molecular mechanism of anti-inflammatory activity in musculoskeletal tissues remains unclear. Here, we first investigated the effects and molecular mechanisms of Cin in human synoviocyte cells. Then in vivo therapeutic effect of Cin on collagen-induced arthritis (CIA) also studied. Cell Counting Kit ‎CCK-8 assay was performed to evaluate the cell cytotoxicity. Proinflammatory cytokine expression was evaluated using quantitative polymerase chain reaction and ELISA. Protein expression was measured by western blotting. The in vivo effect of Cin (75 mg/kg per day) was evaluated in rats with CIA by gavage administration. Disease progression was assessed by clinical scoring, radiographic, and histologic examinations. Cin significantly inhibited interleukin (IL)-1β–induced IL-6, IL-8, and tumor necrosis factor-α release from human synoviocyte cells. The molecular analysis revealed that Cin impaired IL-6–induced activation of Janus kinase 2 (JAK2), signal transducer and activator of transcription 1 (STAT1), and STAT3 signaling pathway by inhibiting the phosphorylation of JAK2, STAT1, and STAT3, without affecting NF-B pathway. Cin reduced collagen-induced swollen paw volume of arthritic rats. The anti-inflammation effects of Cin were associated with decreased severity of arthritis, joint swelling, and reduced bone erosion and destruction. Furthermore, serum IL-6 level was decreased when Cin administered therapeutically to CIA rats. Cin suppresses IL-1β–induced inflammation in synoviocytes through the JAK/STAT pathway and alleviated collagen-induced arthritis in rats. These data indicated that Cin might be a potential traditional Chinese medicine–derived, disease-modifying, antirheumatic herbal drug.

Annelies Wouters, Jan-Pieter Ploem, Sabine A. S. Langie, Tom Artois, Aziz Aboobaker, and Karen Smeets

Pluripotent stem cells hold great potential for regenerative medicine. Increased replication and division, such is the case during regeneration, concomitantly increases the risk of adverse outcomes through the acquisition of mutations. Seeking for driving mechanisms of such outcomes, we challenged a pluripotent stem cell system during the tightly controlled regeneration process in the planarian Schmidtea mediterranea. Exposure to the genotoxic compound methyl methanesulfonate (MMS) revealed that despite a similar DNA-damaging effect along the anteroposterior axis of intact animals, responses differed between anterior and posterior fragments after amputation. Stem cell proliferation and differentiation proceeded successfully in the amputated heads, leading to regeneration of missing tissues. Stem cells in the amputated tails showed decreased proliferation and differentiation capacity. As a result, tails could not regenerate. Interference with the body-axis-associated component β-catenin-1 increased regenerative success in tail fragments by stimulating proliferation at an early time point. Our results suggest that differences in the Wnt signalling gradient along the body axis modulate stem cell responses to MMS.