Cellular membranes are not homogenous mixtures of proteins; rather, they are segregated into microdomains on the basis of preferential association between specific lipids and proteins. These microdomains, called lipid rafts, are well known for their role in receptor signaling on the plasma membrane (PM) and are essential to such cellular functions as signal transduction and spatial organization of the PM. A number of disease states, including atherosclerosis and other cardiovascular disorders, may be caused by dysfunctional maintenance of lipid rafts. Lipid rafts do not occur only in the PM but also have been found in intracellular membranes and extracellular vesicles (EVs). Here, we focus on discussing newly discovered functions of lipid rafts and microdomains in intracellular membranes, including lipid and protein trafficking from the ER, Golgi bodies, and endosomes to the PM, and we examine lipid raft involvement in the production and composition of EVs. Because lipid rafts are small and transient, visualization remains challenging. Future work with advanced techniques will continue to expand our knowledge about the roles of lipid rafts in cellular functioning.

Lipid rafts are highly ordered regions of the plasma membrane that are enriched in cholesterol and sphingolipids and play important roles in many cells. In hematopoietic stem and progenitor cells (HSPCs), lipid rafts house receptors critical for normal hematopoiesis. Lipid rafts also can bind and sequester kinases that induce negative feedback pathways to limit proliferative cytokine receptor cycling back to the cell membrane. Modulation of lipid rafts occurs through an array of mechanisms, with optimal cholesterol efflux one of the major regulators. As such, cholesterol homeostasis also regulates hematopoiesis. Increased lipid raft content, which occurs in response to changes in cholesterol efflux in the membrane, can result in prolonged receptor occupancy in the cell membrane and enhanced signaling. In addition, certain diseases, like diabetes, may contribute to lipid raft formation and affect cholesterol retention in rafts. In this review, we explore the role of lipid raft-related mechanisms in hematopoiesis and CVD (specifically, atherosclerosis) and discuss how defective cholesterol efflux pathways in HSPCs contribute to expansion of lipid rafts, thereby promoting myelopoiesis and thrombopoiesis. We also discuss the utility of cholesterol acceptors in contributing to lipid raft regulation and disruption, and highlight the potential to manipulate these pathways for therapeutic gain in CVD as well as other disorders with aberrant hematopoiesis.

ABSTRACT

Human immunodeficiency virus type 1 (HIV-1) establishes lifelong infections in humans, a process that relies on its ability to thwart innate and adaptive immune defenses of the host. Recently, we reported that HIV-1 infection results in a dramatic reduction of the cellular peroxisome pool. Peroxisomes are metabolic organelles that also function as signaling platforms in the innate immune response. Here, we show that the HIV-1 accessory protein Vpu is necessary and sufficient for the depletion of cellular peroxisomes during infection. Vpu induces the expression of four microRNAs that target mRNAs encoding proteins required for peroxisome formation and metabolic function. The ability of Vpu to downregulate peroxisomes was found to be dependent upon the Wnt/β-catenin signaling pathway. Given the importance of peroxisomes in innate immune signaling and central nervous system function, the roles of Vpu in dampening antiviral signaling appear to be more diverse than previously realized. Finally, our findings highlight a potential role for Wnt/β-catenin signaling in peroxisome homeostasis through modulating the production of biogenesis factors.

IMPORTANCE People living with HIV can experience accelerated aging and the development of neurological disorders. Recently, we reported that HIV-1 infection results in a dramatic loss of peroxisomes in macrophages and brain tissue. This is significant because (i) peroxisomes are important for the innate immune response and (ii) loss of peroxisome function is associated with cellular aging and neurodegeneration. Accordingly, understanding how HIV-1 infection causes peroxisome depletion may provide clues regarding how the virus establishes persistent infections and, potentially, the development of neurological disorders. Here, we show that the accessory protein Vpu is necessary and sufficient for the induction of microRNAs that target peroxisome biogenesis factors. The ability of Vpu to downregulate peroxisome formation depends on the Wnt/β-catenin pathway. Thus, in addition to revealing a novel mechanism by which HIV-1 uses intracellular signaling pathways to target antiviral signaling platforms (peroxisomes), we have uncovered a previously unknown link between the Wnt/β-catenin pathway and peroxisome homeostasis.

ABSTRACT

Bacteriophages (phages) have been proposed as alternative therapeutics for the treatment of multidrug-resistant bacterial infections. However, there are major gaps in our understanding of the molecular events in bacterial cells that control how bacteria respond to phage predation. Using the model organism Enterococcus faecalis, we used two distinct genomic approaches, namely, transposon library screening and RNA sequencing, to investigate the interaction of E. faecalis with a virulent phage. We discovered that a transcription factor encoding a LytR family response regulator controls the expression of enterococcal polysaccharide antigen (epa) genes that are involved in phage infection and bacterial fitness. In addition, we discovered that DNA mismatch repair mutants rapidly evolve phage adsorption deficiencies, underpinning a molecular basis for epa mutation during phage infection. Transcriptomic profiling of phage-infected E. faecalis revealed broad transcriptional changes influencing viral replication and progeny burst size. We also demonstrate that phage infection alters the expression of bacterial genes associated with intra- and interbacterial interactions, including genes involved in quorum sensing and polymicrobial competition. Together, our results suggest that phage predation has the potential to influence complex microbial behavior and may dictate how bacteria respond to external environmental stimuli. These responses could have collateral effects (positive or negative) on microbial communities, such as the host microbiota, during phage therapy.

IMPORTANCE We lack fundamental understanding of how phage infection influences bacterial gene expression and, consequently, how bacterial responses to phage infection affect the assembly of polymicrobial communities. Using parallel genomic approaches, we have discovered novel transcriptional regulators and metabolic genes that influence phage infection. The integration of whole-genome transcriptomic profiling during phage infection has revealed the differential regulation of genes important for group behaviors and polymicrobial interactions. Our work suggests that therapeutic phages could more broadly influence bacterial community composition outside their intended host targets.

ABSTRACT

Anti-galactose-α-1,3-galactose (anti-α-Gal) antibody is naturally expressed at a high level in humans. It constitutes about 1% of immunoglobulins found in human blood. Here, we designed a live attenuated influenza virus vaccine that can generate α-Gal epitopes in infected cells in order to facilitate opsonization of infected cells, thereby enhancing vaccine-induced immune responses. In the presence of normal human sera, cells infected with this mutant can enhance phagocytosis of human macrophages and cytotoxicity of NK cells in vitro. Using a knockout mouse strain that allows expression of anti-α-Gal antibody in vivo, we showed that this strategy can increase vaccine immunogenicity and the breadth of protection. This vaccine can induce 100% protection against a lethal heterosubtypic group 1 (H5) or group 2 (mouse-adapted H3) influenza virus challenge in the mouse model. In contrast, its heterosubtypic protective effect in wild-type or knockout mice that do not have anti-α-Gal antibody expression is only partial, demonstrating that the enhanced vaccine-induced protection requires anti-α-Gal antibody upon vaccination. Anti-α-Gal-expressing knockout mice immunized with this vaccine produce robust humoral and cell-mediated responses upon a lethal virus challenge. This vaccine can stimulate CD11b^lo/– pulmonary dendritic cells, which are known to be crucial for clearance of influenza virus. Our approach provides a novel strategy for developing next-generation influenza virus vaccines.

IMPORTANCE Influenza A viruses have multiple HA subtypes that are antigenically diverse. Classical influenza virus vaccines are subtype specific, and they cannot induce satisfactory heterosubtypic immunity against multiple influenza virus subtypes. Here, we developed a live attenuated H1N1 influenza virus vaccine that allows the expression of α-Gal epitopes by infected cells. Anti-α-Gal antibody is naturally produced by humans. In the presence of this antibody, human cells infected with this experimental vaccine virus can enhance several antibody-mediated immune responses in vitro. Importantly, mice expressing anti-α-Gal antibody in vivo can be fully protected by this H1N1 vaccine against a lethal H5 or H3 virus challenge. Our work demonstrates a new strategy for using a single influenza virus strain to induce broadly cross-reactive immune responses against different influenza virus subtypes.

ABSTRACT

Human noroviruses (HuNoVs) are the leading cause of nonbacterial gastroenteritis worldwide. Histo-blood group antigen (HBGA) expression is an important susceptibility factor for HuNoV infection based on controlled human infection models and epidemiologic studies that show an association of secretor status with infection caused by several genotypes. The fucosyltransferase 2 gene (FUT2) affects HBGA expression in intestinal epithelial cells; secretors express a functional FUT2 enzyme, while nonsecretors lack this enzyme and are highly resistant to infection and gastroenteritis caused by many HuNoV strains. These epidemiologic associations are confirmed by infections in stem cell-derived human intestinal enteroid (HIE) cultures. GII.4 HuNoV does not replicate in HIE cultures derived from nonsecretor individuals, while HIEs from secretors are permissive to infection. However, whether FUT2 expression alone is critical for infection remains unproven, since routinely used secretor-positive transformed cell lines are resistant to HuNoV replication. To evaluate the role of FUT2 in HuNoV replication, we used CRISPR or overexpression to genetically manipulate FUT2 gene function to produce isogenic HIE lines with or without FUT2 expression. We show that FUT2 expression alone affects both HuNoV binding to the HIE cell surface and susceptibility to HuNoV infection. These findings indicate that initial binding to a molecule(s) glycosylated by FUT2 is critical for HuNoV infection and that the HuNoV receptor is present in nonsecretor HIEs. In addition to HuNoV studies, these isogenic HIE lines will be useful tools to study other enteric microbes where infection and/or disease outcome is associated with secretor status.

IMPORTANCE Several studies have demonstrated that secretor status is associated with susceptibility to human norovirus (HuNoV) infection; however, previous reports found that FUT2 expression is not sufficient to allow infection with HuNoV in a variety of continuous laboratory cell lines. Which cellular factor(s) regulates susceptibility to HuNoV infection remains unknown. We used genetic manipulation of HIE cultures to show that secretor status determined by FUT2 gene expression is necessary and sufficient to support HuNoV replication based on analyses of isogenic lines that lack or express FUT2. Fucosylation of HBGAs is critical for initial binding and for modification of another putative receptor(s) in HIEs needed for virus uptake or uncoating and necessary for successful infection by GI.1 and several GII HuNoV strains.

ABSTRACT

Human noroviruses (HuNoV) are a leading cause of viral gastroenteritis worldwide and a significant cause of morbidity and mortality in all age groups. The recent finding that HuNoV can be propagated in B cells and mucosa-derived intestinal epithelial organoids (IEOs) has transformed our ability to dissect the life cycle of noroviruses. Using transcriptome sequencing (RNA-Seq) of HuNoV-infected intestinal epithelial cells (IECs), we have found that replication of HuNoV in IECs results in interferon (IFN)-induced transcriptional responses and that HuNoV replication in IECs is sensitive to IFN. This contrasts with previous studies that suggested that the innate immune response may play no role in the restriction of HuNoV replication in immortalized cells. We demonstrated that inhibition of Janus kinase 1 (JAK1)/JAK2 enhanced HuNoV replication in IECs. Surprisingly, targeted inhibition of cellular RNA polymerase II-mediated transcription was not detrimental to HuNoV replication but instead enhanced replication to a greater degree than blocking of JAK signaling directly. Furthermore, we demonstrated for the first time that IECs generated from genetically modified intestinal organoids, engineered to be deficient in the interferon response, were more permissive to HuNoV infection. Taking the results together, our work revealed that IFN-induced transcriptional responses restrict HuNoV replication in IECs and demonstrated that inhibition of these responses mediated by modifications of the culture conditions can greatly enhance the robustness of the norovirus culture system.

IMPORTANCE Noroviruses are a major cause of gastroenteritis worldwide, and yet the challenges associated with their growth in culture have greatly hampered the development of therapeutic approaches and have limited our understanding of the cellular pathways that control infection. Here, we show that human intestinal epithelial cells, which represent the first point of entry of human noroviruses into the host, limit virus replication by induction of innate responses. Furthermore, we show that modulating the ability of intestinal epithelial cells to induce transcriptional responses to HuNoV infection can significantly enhance human norovirus replication in culture. Collectively, our findings provide new insights into the biological pathways that control norovirus infection but also identify mechanisms that enhance the robustness of norovirus culture.

ABSTRACT

While there is no effective vaccine against Chlamydia trachomatis infection, previous work has demonstrated the importance of C. trachomatis-specific CD4⁺ T cells (NR1 T cells) in pathogen clearance. Specifically, NR1 T cells have been shown to be protective in mice, and this protection depends on the host’s ability to sense the cytokine gamma interferon (IFN-). However, it is unclear what role NR1 production or sensing of IFN- plays in T cell homing to the genital tract or T cell-mediated protection against C. trachomatis. Using two-photon microscopy and flow cytometry, we found that naive wild-type (WT), IFN-^–/–, and IFN-R^–/– NR1 T cells specifically home to sections in the genital tract that contain C. trachomatis. We also determined that protection against infection requires production of IFN- from either NR1 T cells or endogenous cells, further highlighting the importance of IFN- in clearing C. trachomatis infection.

IMPORTANCE Chlamydia trachomatis is an important mucosal pathogen that is the leading cause of sexually transmitted bacterial infections in the United States. Despite this, there is no vaccine currently available. In order to develop such a vaccine, it is necessary to understand the components of the immune response that can lead to protection against this pathogen. It is well known that antigen-specific CD4⁺ T cells are critical for Chlamydia clearance, but the contexts in which they are protective or not protective are unknown. Here, we aimed to characterize the importance of gamma interferon production and sensing by T cells and the effects on the immune response to C. trachomatis. Our work here helps to define the contexts in which antigen-specific T cells can be protective, which is critical to our ability to design an effective and protective vaccine against C. trachomatis.

ABSTRACT

Production of metallo-β-lactamases (MBLs), which hydrolyze carbapenems, is a cause of carbapenem resistance in Enterobacteriaceae. Development of effective inhibitors for MBLs is one approach to restore carbapenem efficacy in carbapenem-resistant Enterobacteriaceae (CRE). We report here that sulfamoyl heteroarylcarboxylic acids (SHCs) can competitively inhibit the globally spreading and clinically relevant MBLs (i.e., IMP-, NDM-, and VIM-type MBLs) at nanomolar to micromolar orders of magnitude. Addition of SHCs restored meropenem efficacy against 17/19 IMP-type and 7/14 NDM-type MBL-producing Enterobacteriaceae to satisfactory clinical levels. SHCs were also effective against IMP-type MBL-producing Acinetobacter spp. and engineered Escherichia coli strains overproducing individual minor MBLs (i.e., TMB-2, SPM-1, DIM-1, SIM-1, and KHM-1). However, SHCs were less effective against MBL-producing Pseudomonas aeruginosa. Combination therapy with meropenem and SHCs successfully cured mice infected with IMP-1-producing E. coli and dually NDM-1/VIM-1-producing Klebsiella pneumoniae clinical isolates. X-ray crystallographic analyses revealed the inhibition mode of SHCs against MBLs; the sulfamoyl group of SHCs coordinated to two zinc ions, and the carboxylate group coordinated to one zinc ion and bound to positively charged amino acids Lys224/Arg228 conserved in MBLs. Preclinical testing revealed that the SHCs showed low toxicity in cell lines and mice and high stability in human liver microsomes. Our results indicate that SHCs are promising lead compounds for inhibitors of MBLs to combat MBL-producing CRE.

IMPORTANCE Carbapenem antibiotics are the last resort for control of severe infectious diseases, bloodstream infections, and pneumonia caused by Gram-negative bacteria, including Enterobacteriaceae. However, carbapenem-resistant Enterobacteriaceae (CRE) strains have spread globally and are a critical concern in clinical settings because CRE infections are recognized as a leading cause of increased mortality among hospitalized patients. Most CRE produce certain kinds of serine carbapenemases (e.g., KPC- and GES-type β-lactamases) or metallo-β-lactamases (MBLs), which can hydrolyze carbapenems. Although effective MBL inhibitors are expected to restore carbapenem efficacy against MBL-producing CRE, no MBL inhibitor is currently clinically available. Here, we synthesized 2,5-diethyl-1-methyl-4-sulfamoylpyrrole-3-carboxylic acid (SPC), which is a potent inhibitor of MBLs. SPC is a remarkable lead compound for clinically useful MBL inhibitors and can potentially provide a considerable benefit to patients receiving treatment for lethal infectious diseases caused by MBL-producing CRE.

ABSTRACT

Obesity and associated metabolic disorders are worldwide public health issues. The gut microbiota plays a key role in the pathophysiology of diet-induced obesity. Glycerol monolaurate (GML) is a widely consumed food emulsifier with antibacterial properties. Here, we explore the anti-obesity effect of GML (1,600 mg/kg of body weight) in high-fat diet (HFD)-fed mice. HFD-fed mice were treated with 1,600 mg/kg GML. Integrated microbiome, metabolome, and transcriptome analyses were used to systematically investigate the metabolic effects of GML, and antibiotic treatment was used to assess the effects of GML on the gut microbiota. Our data indicated that GML significantly reduced body weight and visceral fat deposition, improved hyperlipidemia and hepatic lipid metabolism, and ameliorated glucose homeostasis and inflammation in HFD-fed mice. Importantly, GML modulated HFD-induced gut microbiota dysbiosis and selectively increased the abundance of Bifidobacterium pseudolongum. Antibiotic treatment abolished all the GML-mediated metabolic improvements. A multiomics (microbiome, metabolome, and transcriptome) association study showed that GML significantly modulated glycerophospholipid metabolism, and the abundance of Bifidobacterium pseudolongum strongly correlated with the metabolites and genes that participated in glycerophospholipid metabolism. Our results indicated that GML may be provided for obesity prevention by targeting the gut microbiota and regulating glycerophospholipid metabolism.

ABSTRACT

The human gut microbiota (HGM) has far-reaching impacts on human health and nutrition, which are fueled primarily by the metabolism of otherwise indigestible complex carbohydrates commonly known as dietary fiber. However, the molecular basis of the ability of individual taxa of the HGM to address specific dietary glycan structures remains largely unclear. In particular, the utilization of β(1,3)-glucans, which are widespread in the human diet as yeast, seaweed, and plant cell walls, had not previously been resolved. Through a systems-based approach, here we show that the symbiont Bacteroides uniformis deploys a single, exemplar polysaccharide utilization locus (PUL) to access yeast β(1,3)-glucan, brown seaweed β(1,3)-glucan (laminarin), and cereal mixed-linkage β(1,3)/β(1,4)-glucan. Combined biochemical, enzymatic, and structural analysis of PUL-encoded glycoside hydrolases (GHs) and surface glycan-binding proteins (SGBPs) illuminates a concerted molecular system by which B. uniformis recognizes and saccharifies these distinct β-glucans. Strikingly, the functional characterization of homologous β(1,3)-glucan utilization loci (1,3GUL) in other Bacteroides further demonstrated that the ability of individual taxa to utilize β(1,3)-glucan variants and/or β(1,3)/β(1,4)-glucans arises combinatorially from the individual specificities of SGBPs and GHs at the cell surface, which feed corresponding signals to periplasmic hybrid two-component sensors (HTCSs) via TonB-dependent transporters (TBDTs). These data reveal the importance of cooperativity in the adaptive evolution of GH and SGBP cohorts to address individual polysaccharide structures. We anticipate that this fine-grained knowledge of PUL function will inform metabolic network analysis and proactive manipulation of the HGM. Indeed, a survey of 2,441 public human metagenomes revealed the international, yet individual-specific, distribution of each 1,3GUL.

IMPORTANCE Bacteroidetes are a dominant phylum of the human gut microbiota (HGM) that target otherwise indigestible dietary fiber with an arsenal of polysaccharide utilization loci (PULs), each of which is dedicated to the utilization of a specific complex carbohydrate. Here, we provide novel insight into this paradigm through functional characterization of homologous PULs from three autochthonous Bacteroides species, which target the family of dietary β(1,3)-glucans. Through detailed biochemical and protein structural analysis, we observed an unexpected diversity in the substrate specificity of PUL glycosidases and glycan-binding proteins with regard to β(1,3)-glucan linkage and branching patterns. In combination, these individual enzyme and protein specificities support taxon-specific growth on individual β(1,3)-glucans. This detailed metabolic insight, together with a comprehensive survey of individual 1,3GULs across human populations, further expands the fundamental roadmap of the HGM, with potential application to the future development of microbial intervention therapies.

ABSTRACT

The translocation of effectors into the host cell through type 3 secretion systems (T3SS) is a sophisticated strategy employed by pathogenic bacteria to subvert host responses and facilitate colonization. Enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC) utilize the Tir and EspFu (also known as TccP) effectors to remodel the host cytoskeleton, culminating in the formation of attaching and effacing (AE) lesions on enterocytes. While some EPEC strains require tyrosine phosphorylation of Tir and recruitment of the host Nck to trigger actin polymerization, EHEC and certain EPEC strains, whose Tir is not phosphorylated, rely on the effector EspFu for efficient actin remodeling. Here, we investigated the role played by Tir-Nck and Tir-EspFu actin polymerization pathways during the infection of epithelial cells, as well as the host transcriptional response to the AE lesion formation induced by EPEC. We found that EspFu-mediated actin assembly promotes bacterial attachment and epithelial colonization more efficiently than Tir-Nck. Moreover, we showed that both actin polymerization mechanisms can activate inflammatory pathways and reverse the anti-inflammatory response induced by EPEC in epithelial cells. However, this activity is remarkably more evident in infections with EspFu-expressing EPEC strains. This study demonstrates the complex interactions between effector-mediated actin remodeling and inflammation. Different strains carry different combinations of these two effectors, highlighting the plasticity of pathogenic E. coli enteric infections.

IMPORTANCE EPEC is among the leading causes of diarrheal disease worldwide. The colonization of the gut mucosa by EPEC results in actin pedestal formation at the site of bacterial attachment. These pedestals are referred to as attaching and effacing (AE) lesions. Here, we exploit the different molecular mechanisms used by EPEC to induce AE lesions on epithelial cells, showing that the effector EspFu is associated with increased bacterial attachment and enhanced epithelial colonization compared to the Tir-Nck pathway. Moreover, we also showed that actin pedestal formation can counterbalance the anti-inflammatory activity induced by EPEC, especially when driven by EspFu. Collectively, our findings provide new insights into virulence mechanisms employed by EPEC to colonize epithelial cells, as well as the host response to this enteric pathogen.

ABSTRACT

Burkholderia pseudomallei, the founding member of the B. pseudomallei complex (Bpc), is a biothreat agent and causes melioidosis, a disease whose treatment mainly relies on ceftazidime and meropenem. The concern is that B. pseudomallei could enhance its drug resistance repertoire by the acquisition of DNA from resistant near-neighbor species. Burkholderia ubonensis, a member of the B. cepacia complex (Bcc), is commonly coisolated from environments where B. pseudomallei is present. Unlike B. pseudomallei, in which significant primary carbapenem resistance is rare, it is not uncommon in B. ubonensis, but the underlying mechanisms are unknown. We established that carbapenem resistance in B. ubonensis is due to an inducible class A PenB β-lactamase, as has been shown for other Bcc bacteria. Inducibility is not sufficient for high-level resistance but also requires other determinants, such as a PenB that is more robust than that present in susceptible isolates, as well as other resistance factors. Curiously and diagnostic for the two complexes, both Bpc and Bcc bacteria contain distinct annotated PenA class A β-lactamases. However, the protein from Bcc bacteria is missing its essential active-site serine and, therefore, is not a β-lactamase. Regulated expression of a transcriptional penB'-lacZ (β-galactosidase) fusion in the B. pseudomallei surrogate B. thailandensis confirms that although Bpc bacteria lack an inducible β-lactamase, they contain the components required for responding to aberrant peptidoglycan synthesis resulting from β-lactam challenge. Understanding the diversity of antimicrobial resistance in Burkholderia species is informative about how the challenges arising from potential resistance transfer between them can be met.

IMPORTANCE Burkholderia pseudomallei causes melioidosis, a tropical disease that is highly fatal if not properly treated. Our data show that, in contrast to B. pseudomallei, B. ubonensis β-lactam resistance is fundamentally different because intrinsic resistance is mediated by an inducible class A β-lactamase. This includes resistance to carbapenems. Our work demonstrates that studies with near-neighbor species are informative about the diversity of antimicrobial resistance in Burkholderia and can also provide clues about the potential of resistance transfer between bacteria inhabiting the same environment. Knowledge about potential adverse challenges resulting from the horizontal transfer of resistance genes between members of the two complexes enables the design of effective countermeasures.

ABSTRACT

Cold seeps and hydrothermal vents deliver large amounts of methane and other gaseous alkanes into marine surface sediments. Consortia of archaea and partner bacteria thrive on the oxidation of these alkanes and its coupling to sulfate reduction. The inherently slow growth of the involved organisms and the lack of pure cultures have impeded the understanding of the molecular mechanisms of archaeal alkane degradation. Here, using hydrothermal sediments of the Guaymas Basin (Gulf of California) and ethane as the substrate, we cultured microbial consortia of a novel anaerobic ethane oxidizer, "Candidatus Ethanoperedens thermophilum" (GoM-Arc1 clade), and its partner bacterium "Candidatus Desulfofervidus auxilii," previously known from methane-oxidizing consortia. The sulfate reduction activity of the culture doubled within one week, indicating a much faster growth than in any other alkane-oxidizing archaea described before. The dominance of a single archaeal phylotype in this culture allowed retrieval of a closed genome of "Ca. Ethanoperedens," a sister genus of the recently reported ethane oxidizer "Candidatus Argoarchaeum." The metagenome-assembled genome of "Ca. Ethanoperedens" encoded a complete methanogenesis pathway including a methyl-coenzyme M reductase (MCR) that is highly divergent from those of methanogens and methanotrophs. Combined substrate and metabolite analysis showed ethane as the sole growth substrate and production of ethyl-coenzyme M as the activation product. Stable isotope probing demonstrated that the enzymatic mechanism of ethane oxidation in "Ca. Ethanoperedens" is fully reversible; thus, its enzymatic machinery has potential for the biotechnological development of microbial ethane production from carbon dioxide.

IMPORTANCE In the seabed, gaseous alkanes are oxidized by syntrophic microbial consortia that thereby reduce fluxes of these compounds into the water column. Because of the immense quantities of seabed alkane fluxes, these consortia are key catalysts of the global carbon cycle. Due to their obligate syntrophic lifestyle, the physiology of alkane-degrading archaea remains poorly understood. We have now cultivated a thermophilic, relatively fast-growing ethane oxidizer in partnership with a sulfate-reducing bacterium known to aid in methane oxidation and have retrieved the first complete genome of a short-chain alkane-degrading archaeon. This will greatly enhance the understanding of nonmethane alkane activation by noncanonical methyl-coenzyme M reductase enzymes and provide insights into additional metabolic steps and the mechanisms underlying syntrophic partnerships. Ultimately, this knowledge could lead to the biotechnological development of alkanogenic microorganisms to support the carbon neutrality of industrial processes.

ABSTRACT

Ever since the discovery of the first rare earth element (REE)-dependent enzyme, the physiological role of lanthanides has become an emerging field of research due to the environmental implications and biotechnological opportunities. In Pseudomonas putida KT2440, the two pyrroloquinoline quinone-dependent alcohol dehydrogenases (PQQ-ADHs) PedE and PedH are inversely regulated in response to REE availability. This transcriptional switch is orchestrated by a complex regulatory network that includes the PedR2/PedS2 two-component system and is important for efficient growth on several alcoholic volatiles. To study whether cellular responses beyond the REE switch exist, the differential proteomic responses that occur during growth on various model carbon sources were analyzed. Apart from the Ca²⁺-dependent enzyme PedE, the differential abundances of most identified proteins were conditional. During growth on glycerol—and concomitant with the proteomic changes—lanthanum (La³⁺) availability affected different growth parameters, including the onset of logarithmic growth and final optical densities. Studies with mutant strains revealed a novel metabolic route for glycerol utilization, initiated by PedE and/or PedH activity. Upon oxidation to glycerate via glyceraldehyde, phosphorylation by the glycerate kinase GarK most likely yields glycerate-2-phosphate, which is eventually channeled into the central metabolism of the cell. This new route functions in parallel with the main degradation pathway encoded by the glpFKRD operon and provides a growth advantage to the cells by allowing an earlier onset of growth with glycerol as the sole source of carbon and energy.

IMPORTANCE The biological role of REEs has long been underestimated, and research has mainly focused on methanotrophic and methylotrophic bacteria. We have recently demonstrated that P. putida, a plant growth-promoting bacterium that thrives in the rhizosphere of various food crops, possesses a REE-dependent alcohol dehydrogenase (PedH), but knowledge about REE-specific effects on physiological traits in nonmethylotrophic bacteria is still scarce. This study demonstrates that the cellular response of P. putida to lanthanum (La³⁺) is mostly substrate specific and that La³⁺ availability highly affects the growth of cells on glycerol. Further, a novel route for glycerol metabolism is identified, which is initiated by PedE and/or PedH activity and provides a growth advantage to this biotechnologically relevant organism by allowing a faster onset of growth. Overall, these findings demonstrate that lanthanides can affect physiological traits in nonmethylotrophic bacteria and might influence their competitiveness in various environmental niches.

ABSTRACT

All enterococci produce a complex polysaccharide called the enterococcal polysaccharide antigen (EPA). This polymer is required for normal cell growth and division and for resistance to cephalosporins and plays a critical role in host-pathogen interaction. The EPA contributes to host colonization and is essential for virulence, conferring resistance to phagocytosis during the infection. Recent studies revealed that the "decorations" of the EPA polymer, encoded by genetic loci that are variable between isolates, underpin the biological activity of this surface polysaccharide. In this work, we investigated the structure of the EPA polymer produced by the high-risk enterococcal clonal complex Enterococcus faecalis V583. We analyzed purified EPA from the wild-type strain and a mutant lacking decorations and elucidated the structure of the EPA backbone and decorations. We showed that the rhamnan backbone of EPA is composed of a hexasaccharide repeat unit of C2- and C3-linked rhamnan chains, partially substituted in the C3 position by α-glucose (α-Glc) and in the C2 position by β-N-acetylglucosamine (β-GlcNAc). The so-called "EPA decorations" consist of phosphopolysaccharide chains corresponding to teichoic acids covalently bound to the rhamnan backbone. The elucidation of the complete EPA structure allowed us to propose a biosynthetic pathway, a first essential step toward the design of antimicrobials targeting the synthesis of this virulence factor.

IMPORTANCE Enterococci are opportunistic pathogens responsible for hospital- and community-acquired infections. All enterococci produce a surface polysaccharide called EPA (enterococcal polysaccharide antigen) required for biofilm formation, antibiotic resistance, and pathogenesis. Despite the critical role of EPA in cell growth and division and as a major virulence factor, no information is available on its structure. Here, we report the complete structure of the EPA polymer produced by the model strain E. faecalis V583. We describe the structure of the EPA backbone, made of a rhamnan hexasaccharide substituted by Glc and GlcNAc residues, and show that teichoic acids are covalently bound to this rhamnan chain, forming the so-called "EPA decorations" essential for host colonization and pathogenesis. This report represents a key step in efforts to identify the structural properties of EPA that are essential for its biological activity and to identify novel targets to develop preventive and therapeutic approaches against enterococci.

Objective

To describe the clinical and pathologic features of a novel pedigree with heterozygous STUB1 mutation causing SCA48.

Late Devonian–Early Carboniferous rocks at the southern end of the Kintyre Peninsula closely resemble those of the Kinnesswood and Clyde Sandstone formations in more easterly portions of the Firth of Clyde. For example, a previously unrecognized thick marlstone with pedogenic calcretes is present in the Kinnesswood Formation at the south tip of the peninsula and, on the west coast, south of Machrihanish, a striking cliffed exposure includes massive phreatic calcretes developed from cross-bedded sandstones and red mudstones closely resembling those of the Clyde Sandstone on Great Cumbrae. A similar phreatic calcrete unit is present in the lower part of the Ballagan Formation in south Bute. The presence of vadose and phreatic calcrete provides valuable information concerning palaeoclimatic conditions in southwestern Scotland during the Devonian–Carboniferous transition. Overlying thick volcanic rocks are correlative with the Clyde Plateau Volcanic Formation. The sediments accumulated in the South Kintyre Basin on the west side of the Highland Boundary Fault (HBF). Formation of this basin, and the North East Arran and Cumbraes basins in the northeastern part of the Firth of Clyde, is interpreted as a response to development of a ‘locked zone’ in the HBF during an episode of sinistral faulting.

Large un-walled backfilled burrows of the Taenidium type are known from Paleozoic deltaic marine environments worldwide where they are often associated with Diplichnites trackways. The latter are generally attributed to arthropleurid myriapods and it may be that the burrows were also made by these animals. Here we describe a Taenidium burrow from the Limestone Coal Formation of the Isle of Arran, a formation that also hosts a well-known example of Diplichnites, supporting the association of the two types of trace fossil and extending their known co-occurrence upward into the Upper Carboniferous.

Members of the light-harvesting complex protein family participate in multiple processes connected with light sensing, light absorption, and pigment binding within the thylakoid membrane. Amino acid residues of the light-harvesting chlorophyll a/b-binding proteins involved in pigment binding have been precisely identified through x-ray crystallography experiments. In vitro pigment-binding studies have been performed with LIGHT-HARVESTING-LIKE3 proteins, and the pigment-binding ability of cyanobacterial high-light-inducible proteins has been studied in detail. However, analysis of pigment binding by plant high-light-inducible protein homologs, called ONE-HELIX PROTEINS (OHPs), is lacking. Here, we report on successful in vitro reconstitution of Arabidopsis (Arabidopsis thaliana) OHPs with chlorophylls and carotenoids and show that pigment binding depends on the formation of OHP1/OHP2 heterodimers. Pigment-binding capacity was completely lost in each of the OHPs when residues of the light-harvesting complex chlorophyll-binding motif required for chlorophyll binding were mutated. Moreover, the mutated OHP variants failed to rescue the respective knockout (T-DNA insertion) mutants, indicating that pigment-binding ability is essential for OHP function in vivo. The scaffold protein HIGH CHLOROPHYLL FLUORESCENCE244 (HCF244) is tethered to the thylakoid membrane by the OHP heterodimer. We show that HCF244 stability depends on OHP heterodimer formation and introduce the concept of a functional unit consisting of OHP1, OHP2, and HCF244, in which each protein requires the others. Because of their pigment-binding capacity, we suggest that OHPs function in the delivery of pigments to the D1 subunit of PSII.

The current coronavirus 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) infection, raises important questions as to whether pre-morbid use or continued administration of inhaled corticosteroids (ICS) affects the outcomes of acute respiratory infections due to coronavirus. Many physicians are concerned about whether individuals positive for SARS-CoV-2 and taking ICS should continue them or stop them, given that ICS are often regarded as immunosuppressive. A number of key questions arise. Are people with asthma or COPD at increased risk of developing COVID-19? Do ICS modify this risk, either increasing or decreasing it? Do ICS influence the clinical course of COVID-19? (figure 1). Whether ICS modify the risk of developing COVID-19 or the clinical course of COVID-19 in people who do not have lung disease should also be considered (figure 1).

Introduction

Inhaled corticosteroids (ICS) achieve disease control in the majority of asthmatic patients, although adherence to prescribed ICS is often poor. Patients with severe eosinophilic asthma may require treatment with oral corticosteroids (OCS) and/or biologic agents such as mepolizumab. It is unknown if ICS adherence changes on, or alters clinical response to, biologic therapy.

Systemic sclerosis sine scleroderma (ssSSc) is a rare variant of systemic sclerosis, with only one pediatric case reported in the medical literature to date. Pulmonary arterial hypertension as the presenting feature of ssSSc is extremely rare, even in adults, and so far has never been reported in children. We report, for the first time, a case of pediatric ssSSc in a 3-year-old girl, who presented with interstitial lung disease and pulmonary hypertension. The patient was prescribed early aggressive pulmonary vasodilators combined with anti-inflammatory medications. The clinical response was good, and her current condition at 12 years of age is remarkable, considering the high mortality rates reported in adults. We underscore the importance of early aggressive treatment in future cases of similar presentation.

OBJECTIVES:

To determine if in utero selective serotonin reuptake inhibitor (SSRI) or selective serotonin norepinephrine inhibitor (SNRI) exposure is associated with developmental vulnerability in kindergarten among children whose mothers were diagnosed with prenatal mood or anxiety disorder.

Key Points

Gamma interferon (IFN-)-induced innate immune responses play important roles in the inhibition of Toxoplasma gondii infection. It has been reported that IFN- stimulates non-acidification-dependent growth restriction of T. gondii in HeLa cells, but the mechanism remains unclear. Here, we found that -aminobutyric acid (GABA) receptor-associated protein-like 2 (GABARAPL2) plays a critical role in parasite restriction in IFN--treated HeLa cells. GABARAPL2 is recruited to membrane structures surrounding parasitophorous vacuoles (PV). Autophagy adaptors are required for the proper localization and function of GABARAPL2 in the IFN- -induced immune response. These findings provide further understanding of a noncanonical autophagy pathway responsible for IFN--dependent inhibition of T. gondii growth in human HeLa cells and demonstrate the critical role of GABARAPL2 in this response.

In mammalian cells, alphavirus replication complexes are anchored to the plasma membrane. This interaction with lipid bilayers is mediated through the viral methyl/guanylyltransferase nsP1 and reinforced by palmitoylation of cysteine residue(s) in the C-terminal region of this protein. Lipid content of membranes supporting nsP1 anchoring remains poorly studied. Here, we explore the membrane binding capacity of nsP1 with regard to cholesterol. Using the medically important chikungunya virus (CHIKV) as a model, we report that nsP1 cosegregates with cholesterol-rich detergent-resistant membrane microdomains (DRMs), also called lipid rafts. In search for the critical factor for cholesterol partitioning, we identify nsP1 palmitoylated cysteines as major players in this process. In cells infected with CHIKV or transfected with CHIKV trans-replicase plasmids, nsP1, together with the other nonstructural proteins, are detected in DRMs. While the functional importance of CHIKV nsP1 preference for cholesterol-rich membrane domains remains to be determined, we observed that U18666A- and imipramine-induced sequestration of cholesterol in late endosomes redirected nsP1 to these compartments and simultaneously dramatically decreased CHIKV genome replication. A parallel study of Sindbis virus (SINV) revealed that nsP1 from this divergent alphavirus displays a low affinity for cholesterol and only moderately segregates with DRMs. Behaviors of CHIKV and SINV with regard to cholesterol, therefore, match with the previously reported differences in the requirement for nsP1 palmitoylation, which is dispensable for SINV but strictly required for CHIKV replication. Altogether, this study highlights the functional importance of nsP1 segregation with DRMs and provides new insight into the functional role of nsP1 palmitoylated cysteines during alphavirus replication.

IMPORTANCE Functional alphavirus replication complexes are anchored to the host cell membranes through the interaction of nsP1 with the lipid bilayers. In this work, we investigate the importance of cholesterol for such an association. We show that nsP1 has affinity for cholesterol-rich membrane microdomains formed at the plasma membrane and identify conserved palmitoylated cysteine(s) in nsP1 as the key determinant for cholesterol affinity. We demonstrate that drug-induced cholesterol sequestration in late endosomes not only redirects nsP1 to this compartment but also dramatically decreases genome replication, suggesting the functional importance of nsP1 targeting to cholesterol-rich plasma membrane microdomains. Finally, we show evidence that nsP1 from chikungunya and Sindbis viruses displays different sensitivity to cholesterol sequestering agents that parallel with their difference in the requirement for nsP1 palmitoylation for replication. This research, therefore, gives new insight into the functional role of palmitoylated cysteines in nsP1 for the assembly of functional alphavirus replication complexes in their mammalian host.

Upon infection, the highly structured 5' untranslated region (5' UTR) of picornavirus is involved in viral protein translation and RNA synthesis. As a critical element in the 5' UTR, the internal ribosome entry site (IRES) binds to various cellular proteins to function in the processes of picornavirus replication. Foot-and-mouth disease virus (FMDV) is an important member in the family Picornaviridae, and its 5' UTR contains a functional IRES element. In this study, the cellular heterogeneous nuclear ribonucleoprotein L (hnRNP L) was identified as an IRES-binding protein for FMDV by biotinylated RNA pulldown assays, mass spectrometry (MS) analysis, and determination of hnRNP L-IRES interaction regions. Further, we found that hnRNP L inhibited the growth of FMDV through binding to the viral IRES and that the inhibitory effect of hnRNP L on FMDV growth was not due to FMDV IRES-mediated translation, but to influence on viral RNA synthesis. Finally, hnRNP L was demonstrated to coimmunoprecipitate with RNA-dependent RNA polymerase (3D^pol) in an FMDV RNA-dependent manner in the infected cells. Thus, our results suggest that hnRNP L, as a critical IRES-binding protein, negatively regulates FMDV replication by inhibiting viral RNA synthesis, possibly by remaining in the replication complex.

IMPORTANCE Picornaviruses, as a large family of human and animal pathogens, cause a bewildering array of disease syndromes. Many host factors are implicated in the pathogenesis of these viruses, and some proteins interact with the viral IRES elements to affect function. Here, we report for the first time that cellular hnRNP L specifically interacts with the IRES of the picornavirus FMDV and negatively regulates FMDV replication through inhibiting viral RNA synthesis. Further, our results showed that hnRNP L coimmunoprecipitates with FMDV 3D^pol in a viral RNA-dependent manner, suggesting that it may remain in the replication complex to function. The data presented here would facilitate further understanding of virus-host interactions and the pathogenesis of picornavirus infections.

One factor that contributes to the high prevalence of fetal alcohol spectrum disorder (FASD) is binge-like consumption of alcohol before pregnancy awareness. It is known that treatments are more effective with early recognition of FASD. Recent advances in retrospective motion correction for the reconstruction of three-dimensional (3D) fetal brain MRI...

Aerosol–radiation interaction (ARI) plays a significant role in the accumulation of fine particulate matter (PM2.5) by stabilizing the planetary boundary layer and thus deteriorating air quality during haze events. However, modification of photolysis by aerosol scattering or absorbing solar radiation (aerosol–photolysis interaction or API) alters the atmospheric oxidizing capacity, decreases...

Inhaled corticosteroid (ICS)–based therapy is often used for patients with chronic obstructive pulmonary disease (COPD). However, this approach is under scrutiny because of ICS overuse in patients for whom it is not recommended and because of concerns about adverse events, particularly pneumonia, with long-term ICS use. Evidence suggests ICS may be beneficial in specific patients, namely, those with high blood eosinophil counts (eg, ≥300 cells/µL) or who are at a high risk of exacerbations. According to the Global Initiative for Chronic Obstructive Lung Disease (GOLD) 2020 ABCD assessment tool, these patients belong in group D. For these patients, recommended initial treatment includes ICS in combination with long-acting β2-agonists (LABAs) when blood eosinophil counts are ≥300 cells/µL or LABA + long-acting muscarinic antagonist (LAMA) when patients are highly symptomatic, that is, with greater dyspnea and/or exercise limitation. Follow-up treatments for patients with persistent dyspnea and/or exacerbations may include LABA + ICS, LABA + LAMA, or LABA + LAMA + ICS, with use of ICS being guided by blood eosinophil counts. In this review, differences in the inflammatory mechanism underlying COPD and asthma and the role of ICS treatment in COPD are summarized. Furthermore, findings from recent clinical trials where use of ICS-based dual or triple therapy in COPD was compared with LABA + LAMA therapy and trials in which ICS withdrawal was evaluated in patients with COPD are reviewed. Finally, a step-by-step guide for ICS withdrawal in patients who are unlikely to benefit from this treatment is proposed. A video of the author discussing the overall takeaway of the review article could be downloaded from the link provided: https://www.youtube.com/watch?v=Uq7Sr5jqPDI.

Marc E. Deetjen, Diana D. Chin, and David Lentink

Animal flight requires aerodynamic power, which is challenging to determine accurately in vivo. Existing methods rely on approximate calculations based on wake flow field measurements, inverse dynamics approaches, or invasive muscle physiological recordings. In contrast, the external mechanical work required for terrestrial locomotion can be determined more directly by using a force platform as an ergometer. Based on an extension of the recent invention of the aerodynamic force platform, we now present a more direct method to determine the in vivo aerodynamic power by taking the dot product of the aerodynamic force vector on the wing with the representative wing velocity vector based on kinematics and morphology. We demonstrate this new method by studying a slowly flying dove, but it can be applied more generally across flying and swimming animals as well as animals that locomote over water surfaces. Finally, our mathematical framework also works for power analyses based on flow field measurements.

Jorge de Costa, Gustavo Barja, and Pedro F. Almaida-Pagan

Lipid composition of cell membranes is linked to metabolic rate and lifespan in mammals and birds but very little information is available for fishes. In this study, three fish species of the short-lived annual genus Nothobranchius with different maximum lifespan potentials (MLSP) and the longer-lived outgroup species Aphyosemion australe were studied to test whether they conform to the predictions of the longevity-homeoviscous adaptation (LHA) theory of aging. Lipid analyses were performed in whole fish samples and peroxidation indexes (PIn) for every PL class and for the whole membrane, were calculated. Total PL content was significantly lower in A. australe and N. korthausae, the two species with the highest MLSP, and a negative correlation between membrane total PIn and fish MLSP was found, this meaning that the longer-lived fish species have more saturated membranes and therefore, a lower susceptibility to oxidative damage, as the LHA theory posits.

Jack Nguyen and Meir M. Barak

Cortical bone remodeling is an ongoing process triggered by microdamage, where osteoclasts resorb existing bone and osteoblasts deposit new bone in the form of secondary osteons (Haversian systems). Previous studies revealed regional variance in Haversian systems structure and possibly material, between opposite cortices of the same bone. As bone mechanical properties depend on tissue structure and material, it is predicted that bone mechanical properties will vary in accordance with structural and material regional heterogeneity. To test this hypothesis, we analyzed the structure, mineral content and compressive stiffness of secondary bone from the cranial and caudal cortices of the white-tailed deer proximal humerus. We found significantly larger Haversian systems and canals in the cranial cortex but no significant difference in mineral content between the two cortices. Accordingly, we found no difference in compressive stiffness between the two cortices and thus our working hypothesis was rejected. Seeing that the deer humerus is curved and thus likely subjected to bending during habitual locomotion, we expect that similar to other curved long bones, the cranial cortex of the deer humerus is likely subjected primarily to tensile strains and the caudal cortex is likely subject primarily to compressive strains. Consequently, our results suggest that strain magnitude (larger in compression) and sign (compression vs. tension) affect differently the osteoclasts and osteoblasts in the BMU. Our results further suggest that osteoclasts are inhibited in regions of high compressive strains (creating smaller Haversian systems) while osteoblasts’ osteoid deposition and mineralization is not affected by strain magnitude and sign.

Relevant publications related to medicinal and toxic aerosols are discussed in this review. Treatment of COPD includes a combination of long-acting bronchodilators and long-acting muscarinic antagonists. A combination of aclidinium bromide and formoterol fumarate was approved in the United States. The combination was superior to its components alone, as well as tiotropium and a salmeterol-fluticasone combination. Increased risk of an asthma exacerbation was reported in children exposed to electronic nicotine delivery systems. A smart inhaler capable of recording inspiratory flow was approved in the United States. The use of as-needed budesonide-formoterol was reported to be superior to scheduled budesonide and as-needed terbutaline for the treatment of adults with mild-to-moderate asthma. A survey among teens with asthma and their caregivers revealed a disagreement in the number of inhaled controller medications the teen was taking. Treatment with inhaled hypertonic saline resulted in a decreased lung clearance index in infants and preschool children with cystic fibrosis. Surgical masks were well tolerated and significantly decreased the burden of aerosolized bacteria generated by coughing in adults with cystic fibrosis. Inhaled liposomal amikacin in addition to guideline-based therapy was reported to be superior to guideline-based therapy alone in achieving negative sputum cultures in adult subjects with Mycobacterium avium complex pulmonary disease. During 2019, lung injury associated to e-cigarette or vaping was reported, including 60 casualties.

BACKGROUND:Prolonged ventilatory weaning may expose patients to unnecessary discomfort, increase the risk of complications, and raise the costs of hospital treatment. In this scenario, indexes that reliably predict successful liberation can be helpful.OBJECTIVE:To evaluate the intra- and interobserver reproducibility of the timed inspiratory effort index as a weaning predictor.METHODS:This prospective observational study included subjects judged as able to start liberation from mechanical ventilation. For the intra-observer analysis, the same investigator performed 2 measurements in each selected patient with an interval of 30 min a rest. For interobserver analysis, 2 measurements were obtained in another sample of subjects, also with an interval of 30 min rest, but each of one performed by a different investigator. The Bland-Altman diagram, the coefficient concordance of kappa, and the Pearson correlation coefficient were used to compare the measurements. The performance of the timed inspiratory effort index was assessed by receiver operating characteristic curves. Values of P < .05 were considered significant.RESULTS:We selected 113 subjects (43 males; mean ± SD age, 77 ± 14 y). Fifty-six (49.6%) achieved successful liberation, and 33 (29%) died in the ICU. The mean ± SD duration of mechanical ventilation was 14.4 ± 6.7 d. The Bland-Altman diagrams that addressed intra- and interobservers agreement showed low variability between measurements. Values of the concordance coefficients of kappa were 0.82 (0.68–0.95) and 0.80 (0.65–0.94), and of the linear correlation coefficients, 0.86 (0.77–0.91) and 0.89 (0.82–0.93) for the intra- and interobservers measurements, respectively. The mean ± SD values for the area under the curve for each pair of the intra- and interobserver measurements were 0.96 ± 0.07 versus 0.94 ± 0.07 (P = .41) and 0.94 ± 0.05 versus 0.90 ± 0.07 (P = .14), respectively.CONCLUSIONS:The variability of the measurement of the timed inspiratory effort index by intra- and interobservers showed very high reproducibility, which reinforced the index as a sensible, accurate, and reliable outcome predictor of liberation from mechanical ventilation.

BACKGROUND:Assisted coughing via mechanical in-exsufflation (MI-E) is a first-line treatment for secretion management in patients with amyotrophic lateral sclerosis (ALS) with unassisted CPF < 4.25 L/s. Some devices enable oscillations to be added to MI-E (MI-E+O). We sought to determine whether adding oscillations to MI-E enables a reduction in the use of invasive secretion management procedures (ie, bronchoscopy or tracheostomy) in subjects with ALS.METHODS:We conducted a 12-month, prospective, randomized follow-up study of subjects with ALS for whom assisted coughing techniques were indicated. One group was treated with oscillations in addition to MI-E (MI-E+O), and the other group was treated with conventional MI-E.RESULTS:29 subjects were included in the MI-E group and 27 subjects were included in the MI-E+O group. Five subjects (8.9%) required invasive techniques for secretion management (3 in the MI-E group and 2 in the MI-E+O group, P = .70). Treatment with MI-E+O did not alter the risk of invasive procedures (odds ratio 0.69, 95% CI 0.10–4.50, P = .70). The mean number of respiratory infections was 0.58 ± 0.16 in the MI-E group and 0.025 ± 0.08 in the MI-E+O group (P = .10). Survival was 8.96 ± 0.18 months in the MI-E group and 7.70 ± 0.70 months in the MI-E+O group (P = .10).CONCLUSION:Adding oscillations to MI-E did not enable a reduction in the need to perform invasive procedures for secretion management in subjects with ALS.

The arrangement of functionally-related genes in operons is a fundamental element of how genetic information is organized in prokaryotes. This organization ensures coordinated gene expression by co-transcription. Often, however, alternative genetic responses to specific stress conditions demand the discoordination of operon expression. During cold temperature stress, accumulation of the gene encoding the sole Asp–Glu–Ala–Asp (DEAD)-box RNA helicase in Synechocystis sp. PCC 6803, crhR (slr0083), increases 15-fold. Here, we show that crhR is expressed from a dicistronic operon with the methylthiotransferase rimO/miaB (slr0082) gene, followed by rapid processing of the operon transcript into two monocistronic mRNAs. This cleavage event is required for and results in destabilization of the rimO transcript. Results from secondary structure modeling and analysis of RNase E cleavage of the rimO–crhR transcript in vitro suggested that CrhR plays a role in enhancing the rate of the processing in an auto-regulatory manner. Moreover, two putative small RNAs are generated from additional processing, degradation, or both of the rimO transcript. These results suggest a role for the bacterial RNA helicase CrhR in RNase E-dependent mRNA processing in Synechocystis and expand the known range of organisms possessing small RNAs derived from processing of mRNA transcripts.

Detailed interpretation of a 3D seismic data volume reveals the detrimental effect that post-depositional tectonic deformation has had on buried Lower Carboniferous (Dinantian–Namurian) shales and its consequences for shale gas exploration in the SW part (Fylde area) of the Craven Basin in NW England. The structural styles primarily result from Devono-Carboniferous (syn-sedimentary) extension, post-rift subsidence and Variscan inversion, a renewed phase of Permo-Triassic extension, and Cenozoic uplift and basin exhumation. In contrast to the shallow dips and bedding continuity that characterizes productive shale gas plays in other basins (e.g. in the USA and Argentina), our mapping shows that the area is affected by deformation that results in the Bowland Shale Formation targets being folded and dissected into fault-bound compartments defined by SW–NE striking (Lower Carboniferous and Variscan) reverse faults and SSW–NNE to N–S striking (Permo-Triassic) normal faults. The fault networks and the misalignment between the elongate compartments they contain and the present-day minimum horizontal stress orientation limit the length over which long lateral boreholes can remain in a productive horizon, placing an important constraint on optimal well positioning, reducing the size of the shale gas resource and affecting well productivity. Our subsurface mapping using this high-fidelity dataset provides an accurate picture of the Upper Palaeozoic structure and demonstrates that faulting is denser and more complex than apparent from geological mapping of the surface outcrop. That structural complexity has direct and significant consequences for: the location of well pads; the lateral continuity of target shale gas horizons; the evaluation of the risk of inducing seismicity on seismically resolvable (large displacement) fault planes prior to drilling; and the likelihood of faults with small throws (below seismic resolution) being present.

Discovery of the Breagh gas field in the Southern North Sea (SNS) has demonstrated the potential that the Lower Carboniferous (Visean, 346.7–330.9 Ma) Farne Group reservoirs have to contribute to the UK's future energy mix. New biostratigraphic correlations provide a basis to compare Asbian and Brigantian sedimentary cores from the Breagh Field and age-equivalent sediments exposed on the Northumberland Coast, which has proved critical in gaining an understanding of exploration and development opportunities. Thirteen facies associations characterize the mixed carbonate–siliciclastic system, grouped into: marine, delta front, delta shoreface, lower delta plain and upper delta plain gross depositional environments. The facies associations are interpreted as depositing in a mixed carbonate and siliciclastic fluvio-deltaic environment, and are arranged into coarsening- and cleaning-upward cycles (parasequences) bounded by flooding surfaces. Most cycles are characterized by mouth bars, distributary channels, interdistributary bays and common braided rivers, interpreted as river-dominated deltaic deposits. Some cycles include rare shoreface and tidally-influenced deposits, interpreted as river-dominated and wave- or tide-influenced deltaic deposits. The depositional processes that formed each cycle have important implications for the reservoir net/gross ratio (where this ratio indicates the proportion of sandstone beds in a cycle), thickness and lateral extent. The deltaic deposits were controlled by a combination of tectonic and eustatic (allocyclic) events and delta avulsion (autocyclic) processes, and are likely to reflect a changing tectonic regime, from extension within elongate fault-bounded basins (synrift) to passive regional thermal subsidence (post-rift). Deep incision by the Base Permian Unconformity across the Breagh Field has removed the Westphalian, Namurian and upper Visean, to leave the more prospective thicker clastic reservoirs within closure.

Thematic collection: This article is part of the Under-explored plays and frontier basins of the UK continental shelf collection available at: https://www.lyellcollection.org/cc/under-explored-plays-and-frontier-basins-of-the-uk-continental-shelf

Exploration success at Breagh demonstrates that western parts of the Mid North Sea High area are prospective despite the absence of an Upper Permian (Rotliegend Group) Leman Sandstone Formation reservoir and source rocks belonging to the Upper Carboniferous Westphalian Coal Measures Group. Detailed seismic and well interpretation shows that the Breagh trap was a long-lived footwall high, the prospectivity of which was enhanced by Variscan folding and uplift, leading to the truncation (subcrop) of Lower Carboniferous reservoirs beneath the Base Permian Unconformity. Its drape (supra-crop) by Upper Permian (Zechstein Super Group) evaporites creates the seal. The complexity of its overburden means that an accurate picture of the Breagh structure only emerges after accurate depth-conversion that takes the effects of the Mesozoic graben into account. Pronounced easterly tilting during the Cenozoic affected the area and controlled gas migration into the structure from palaeostructures lying to the east. However, evidence that Breagh is not filled to spill point (underfill) suggests that charge limitation remained an issue. The study demonstrates that a poorly-documented and under-explored Lower Carboniferous play exists in Southern North Sea, which relies upon careful structural mapping and basin modelling to be undertaken for the play to be understood and its further potential to be realized.

Thematic collection: This article is part of the Under-explored plays and frontier basins of the UK continental shelf collection available at: https://www.lyellcollection.org/cc/under-explored-plays-and-frontier-basins-of-the-uk-continental-shelf

In the Northern Apennine (Italy), the Internal Ligurian Units consist of Middle–Late Jurassic ophiolites covered by thick sedimentary deposits whose top is represented by the Early Paleocene Bocco Shale. This formation is characterized by mass-transport deposits interlayered with thin-bedded siliciclastic turbidites. The sedimentological and structural features of these mass-transport deposits reveal a long-lived history of recycling of heterogeneous material in a subduction setting. This history started with the frontal accretion of a fragment of oceanic crust into an accretionary prism whose lower slope was subsequently affected by tectonic erosion and consequent instability, leading to the production of mass-transport deposits and the transfer of material to the lower plate. These mass-transport deposits were subsequently underthrust and then again transferred to the base of the accretionary prism by coherent underplating, before their exhumation to the surface. The Bocco Shale is thus representative of a subduction setting where both accretionary and erosive events occurred, depending on changing boundary conditions. The reconstructed history for the Bocco Shale indicates that the sedimentary and gravitational processes both at the prism front and on the prism slope, possibly induced by alternating accretion and erosion events, are the most efficient mechanisms of lithological mixing and recycling in subduction margins.

The Northern Paraguai Belt, at the SE border of the Amazonian Craton, central Brazil, has been interpreted as a Brasiliano–Pan-African (c. 650–600 Ma) belt with a foreland basin, recording collisional polyphase tectonism and greenschist-facies metamorphism extending from the late Precambrian to the Cambrian–Ordovician. New structural investigations indicate that the older metasedimentary rocks of the Cuiabá Group represent a Tonian–Cryogenian basement assemblage deformed in two contemporaneous fault-bounded structural sub-domains of wrench-dominated (rake <10°) and contraction-dominated (rake ~30–40°) sinistral transpression, with tectonic vergence towards the SE. The younger late Cryogenian to early Cambrian sedimentary rocks lying to the NW of the Cuiabá Group are non-metamorphic and display only pervasive brittle transtension characterized by normal-oblique faults, fractures and forced drag folds with no consistent vergence pattern. Our analyses suggest that an unconformity separates the metasedimentary Cuiabá Group basement of the Northern Paraguai Belt from the unmetamorphosed sedimentary cover. It is proposed that the latter units were deposited during a post-glacial marine transgression (after c. 635 Ma) in a post-collisional basin. The Paraguai Belt basement and its post-collisional sedimentary cover share a number of significant geological similarities with sequences in the Bassarides Belt and Taoudéni Basin in the SW portion of the West African Craton.

Current pneumococcal vaccines cover the 10 to 23 most common serotypes of the 92 presently described. However, with the increased usage of pneumococcal-serotype-based vaccines, the risk of serotype replacement and an increase in disease caused by nonvaccine serotypes remains. Serotype surveillance of pneumococcal infections relies heavily on culture techniques, which are known to be insensitive, particularly in cases of noninvasive disease. Pneumococcal-serotype-specific urine assays offer an alternative method of serotyping for both invasive and noninvasive disease. However, the assays described previously cover mainly conjugate vaccine serotypes, give little information about circulating nonvaccine serotypes, and are currently available only in one or two specialist laboratories. Our laboratory has developed a Luminex-based extended-range antigen capture assay to detect pneumococcal-serotype-specific antigens in urine samples. The assay targets 24 distinct serotypes/serogroups plus the cell wall polysaccharide (CWP) and some cross-reactive serotypes. We report that the assay is capable of detecting all the targeted serotypes and the CWP at 0.1 ng/ml, while some serotypes are detected at concentrations as low as 0.3 pg/ml. The analytical serotype specificity was determined to be 98.4% using a panel of polysaccharide-negative urine specimens spiked with nonpneumococcal bacterial antigens. We also report clinical sensitivities of 96.2% and specificities of 89.9% established using a panel of urine specimens from patients diagnosed with community-acquired pneumonia or pneumococcal disease. This assay can be extended for testing other clinical samples and has the potential to greatly improve serotype-specific surveillance in the many cases of pneumococcal disease in which a culture is never obtained.

In December 2019, a novel coronavirus causing an infectious respiratory disease (COVID-19) was identified, which since then has developed into a pandemic with higher rates of mortality in older individuals and those with underlying medical conditions.¹ Multiple sclerosis (MS) is an immune-mediated neurologic disease which requires long-term treatment with immunotherapies that have been shown to increase the risk of infections.² As a result, there is significant anxiety among patients and neurologists during the pandemic regarding the infection outcome in this patient population. We present a patient with MS treated with fingolimod who was diagnosed with COVID-19 and had a favorable outcome.

In the article "Neurologic syndromes related to anti-GAD65: Clinical and serologic response to treatment" by Muñoz-Lopetegi et al.,¹ published online March 2, 2020, the y-axis label for figure 5’s right graph should be "CSF anti-GAD65 concentration (IU/mL)." The editorial office regrets the error.

Leprosy is caused by Mycobacterium leprae, and it remains underdiagnosed in Burkina Faso. We investigated the use of fluorescent in situ hybridization (FISH) for detecting M. leprae in 27 skin samples (skin biopsy samples, slit skin samples, and skin lesion swabs) collected from 21 patients from Burkina Faso and three from Côte d’Ivoire who were suspected of having cutaneous leprosy. In all seven Ziehl-Neelsen-positive skin samples (four skin biopsy samples and three skin swabs collected from the same patient), FISH specifically identified M. leprae, including one FISH-positive skin biopsy sample that remained negative after testing with PCR targeting the rpoB gene and with the GenoType LepraeDR assay. Twenty other skin samples and three negative controls all remained negative for Ziehl-Neelsen staining, FISH, and rpoB PCR. These data indicate the usefulness of a microscopic examination of skin samples after FISH for first-line diagnosis of cutaneous leprosy. Accordingly, FISH represents a potentially useful point-of-care test for the diagnosis of cutaneous leprosy.

Pyrazinamide (PZA) is considered the pivot drug in all tuberculosis treatment regimens due to its particular action on the persistent forms of Mycobacterium tuberculosis. However, no drug susceptibility test (DST) is considered sufficiently reliable for routine application. Although molecular tests are endorsed, their application is limited to known PZA resistance associated mutations. Microbiological DSTs for PZA have been restricted by technical limitations, especially the necessity for an acidic pH. Here, for the first time, MODS culture at neutral pH was evaluated using high PZA concentrations (400 and 800 μg/ml) to determine PZA susceptibility directly from sputum samples. Sputum samples were cultured with PZA for up to 21 days at 37°C. Plate reading was performed at two time points: R1 (mean, 10 days) and R2 (mean, 13 days) for each PZA concentration. A consensus reference test, composed of MGIT-PZA, pncA sequencing, and the classic Wayne test, was used. A total of 182 samples were evaluated. The sensitivity and specificity for 400 μg/ml ranged from 76.9 to 89.7 and from 93.0 to 97.9%, respectively, and for 800 μg/ml ranged from 71.8 to 82.1 and from 95.8 to 98.6%, respectively. Compared to MGIT-PZA, our test showed a similar turnaround time (medians of 10 and 12 days for PZA-sensitive and -resistant isolates, respectively). In conclusion, MODS-PZA is presented as a fast, simple, and low-cost DST that could complement the MODS assay to evaluate resistance to the principal first-line antituberculosis drugs. Further optimization of test conditions would be useful in order to increase its performance.