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

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

A bioinformatics approach was employed to identify transcriptome alterations in the peripheral blood mononuclear cells of well-characterized human subjects who were diagnosed with early disseminated Lyme disease (LD) based on stringent microbiological and clinical criteria. Transcriptomes were assessed at the time of presentation and also at approximately 1 month (early convalescence) and 6 months (late convalescence) after initiation of an appropriate antibiotic regimen. Comparative transcriptomics identified 335 transcripts, representing 233 unique genes, with significant alterations of at least 2-fold expression in acute- or convalescent-phase blood samples from LD subjects relative to healthy donors. Acute-phase blood samples from LD subjects had the largest number of differentially expressed transcripts (187 induced, 54 repressed). This transcriptional profile, which was dominated by interferon-regulated genes, was sustained during early convalescence. 6 months after antibiotic treatment the transcriptome of LD subjects was indistinguishable from that of healthy controls based on two separate methods of analysis. Return of the LD expression profile to levels found in control subjects was concordant with disease outcome; 82% of subjects with LD experienced at least one symptom at the baseline visit compared to 43% at the early convalescence time point and only a single patient (9%) at the 6-month convalescence time point. Using the random forest machine learning algorithm, we developed an efficient computational framework to identify sets of 20 classifier genes that discriminated LD from other bacterial and viral infections. These novel LD biomarkers not only differentiated subjects with acute disseminated LD from healthy controls with 96% accuracy but also distinguished between subjects with acute and resolved (late convalescent) disease with 97% accuracy.

IMPORTANCE Lyme disease (LD), caused by Borrelia burgdorferi, is the most common tick-borne infectious disease in the United States. We examined gene expression patterns in the blood of individuals with early disseminated LD at the time of diagnosis (acute) and also at approximately 1 month and 6 months following antibiotic treatment. A distinct acute LD profile was observed that was sustained during early convalescence (1 month) but returned to control levels 6 months after treatment. Using a computer learning algorithm, we identified sets of 20 classifier genes that discriminate LD from other bacterial and viral infections. In addition, these novel LD biomarkers are highly accurate in distinguishing patients with acute LD from healthy subjects and in discriminating between individuals with active and resolved infection. This computational approach offers the potential for more accurate diagnosis of early disseminated Lyme disease. It may also allow improved monitoring of treatment efficacy and disease resolution.

ABSTRACT

The Candida albicans high-affinity phosphate transporter Pho84 is required for normal Target of Rapamycin (TOR) signaling, oxidative stress resistance, and virulence of this fungal pathogen. It also contributes to C. albicans’ tolerance of two antifungal drug classes, polyenes and echinocandins. Echinocandins inhibit biosynthesis of a major cell wall component, beta-1,3-glucan. Cells lacking Pho84 were hypersensitive to other forms of cell wall stress beyond echinocandin exposure, while their cell wall integrity signaling response was weak. Metabolomics experiments showed that levels of phosphoric intermediates, including nucleotides like ATP and nucleotide sugars, were low in pho84 mutant compared to wild-type cells recovering from phosphate starvation. Nonphosphoric precursors like nucleobases and nucleosides were elevated. Outer cell wall phosphomannan biosynthesis requires a nucleotide sugar, GDP-mannose. The nucleotide sugar UDP-glucose is the substrate of enzymes that synthesize two major structural cell wall polysaccharides, beta-1,3- and beta-1,6-glucan. Another nucleotide sugar, UDP-N-acetylglucosamine, is the substrate of chitin synthases which produce a stabilizing component of the intercellular septum and of lateral cell walls. Lack of Pho84 activity, and phosphate starvation, potentiated pharmacological or genetic perturbation of these enzymes. We posit that low substrate concentrations of beta-d-glucan- and chitin synthases, together with pharmacologic inhibition of their activity, diminish enzymatic reaction rates as well as the yield of their cell wall-stabilizing products. Phosphate import is not conserved between fungal and human cells, and humans do not synthesize beta-d-glucans or chitin. Hence, inhibiting these processes simultaneously could yield potent antifungal effects with low toxicity to humans.

IMPORTANCE Candida species cause hundreds of thousands of invasive infections with high mortality each year. Developing novel antifungal agents is challenging due to the many similarities between fungal and human cells. Maintaining phosphate balance is essential for all organisms but is achieved completely differently by fungi and humans. A protein that imports phosphate into fungal cells, Pho84, is not present in humans and is required for normal cell wall stress resistance and cell wall integrity signaling in C. albicans. Nucleotide sugars, which are phosphate-containing building block molecules for construction of the cell wall, are diminished in cells lacking Pho84. Cell wall-constructing enzymes may be slowed by lack of these building blocks, in addition to being inhibited by drugs. Combined targeting of Pho84 and cell wall-constructing enzymes may provide a strategy for antifungal therapy by which two sequential steps of cell wall maintenance are blocked for greater potency.

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

The appressoria that are generated by the rice blast fungus Magnaporthe oryzae in response to surface cues are important for successful colonization. Previous work showed that regulators of G-protein signaling (RGS) and RGS-like proteins play critical roles in appressorium formation. However, the mechanisms by which these proteins orchestrate surface recognition for appressorium induction remain unclear. Here, we performed comparative transcriptomic studies of Morgs mutant and wild-type strains and found that M. oryzae Aa91 (MoAa91), a homolog of the auxiliary activity family 9 protein (Aa9), was required for surface recognition of M. oryzae. We found that MoAA91 was regulated by the MoMsn2 transcription factor and that its disruption resulted in defects in both appressorium formation on the artificial inductive surface and full virulence of the pathogen. We further showed that MoAa91 was secreted into the apoplast space and was capable of competing with the immune receptor chitin elicitor-binding protein precursor (CEBiP) for chitin binding, thereby suppressing chitin-induced plant immune responses. In summary, we have found that MoAa91 is a novel signaling molecule regulated by RGS and RGS-like proteins and that MoAa91 not only governs appressorium development and virulence but also functions as an effector to suppress host immunity.

IMPORTANCE The rice blast fungus Magnaporthe oryzae generates infection structure appressoria in response to surface cues largely due to functions of signaling molecules, including G-proteins, regulators of G-protein signaling (RGS), mitogen-activated protein (MAP) kinase pathways, cAMP signaling, and TOR signaling pathways. M. oryzae encodes eight RGS and RGS-like proteins (MoRgs1 to MoRgs8), and MoRgs1, MoRgs3, MoRgs4, and MoRgs7 were found to be particularly important in appressorium development. To explore the mechanisms by which these proteins regulate appressorium development, we have performed a comparative in planta transcriptomic study and identified an auxiliary activity family 9 protein (Aa9) homolog that we named MoAa91. We showed that MoAa91 was secreted from appressoria and that the recombinant MoAa91 could compete with a chitin elicitor-binding protein precursor (CEBiP) for chitin binding, thereby suppressing chitin-induced plant immunity. By identifying MoAa91 as a novel signaling molecule functioning in appressorium development and an effector in suppressing host immunity, our studies revealed a novel mechanism by which RGS and RGS-like proteins regulate pathogen-host interactions.

ABSTRACT

Viral diseases cause significant losses in aquaculture. Prophylactic measures, such as immune priming, are promising control strategies. Treatment of the Pacific oyster (Crassostrea gigas) with the double-stranded RNA analog poly(I·C) confers long-term protection against infection with ostreid herpesvirus 1, the causative agent of Pacific oyster mortality syndrome. In a recent article in mBio, Lafont and coauthors (M. Lafont, A. Vergnes, J. Vidal-Dupiol, J. de Lorgeril, et al., mBio 11:e02777-19, 2020, https://doi.org/10.1128/mBio.02777-19) characterized the transcriptome of oysters treated with poly(I·C). This immune stimulator induced genes related to the interferon and apoptosis pathways. This response overlaps the response to viral infection, and high expression levels of potential effector genes are maintained for up to 4 months. This work opens the door to characterization of the phenomena of immune priming in a poorly studied invertebrate model. It also highlights the importance of interferon-like responses for invertebrate antiviral immunity.

ABSTRACT

While the basic mechanisms of flavivirus entry and fusion are understood, little is known about the postfusion events that precede RNA replication, such as nucleocapsid disassembly. We describe here a sensitive, conditionally replication-defective yellow fever virus (YFV) entry reporter, YFVSK/Nluc, to quantitively monitor the translation of incoming, virus particle-delivered genomes. We validated that YFVSK/Nluc gene expression can be neutralized by YFV-specific antisera and requires known flavivirus entry pathways and cellular factors, including clathrin- and dynamin-mediated endocytosis, endosomal acidification, YFV E glycoprotein-mediated fusion, and cellular LY6E and RPLP1 expression. The initial round of YFV translation was shown to require cellular ubiquitylation, consistent with recent findings that dengue virus capsid protein must be ubiquitylated in order for nucleocapsid uncoating to occur. Importantly, translation of incoming YFV genomes also required valosin-containing protein (VCP)/p97, a cellular ATPase that unfolds and extracts ubiquitylated client proteins from large complexes. RNA transfection and washout experiments showed that VCP/p97 functions at a postfusion, pretranslation step in YFV entry. Finally, VCP/p97 activity was required by other flaviviruses in mammalian cells and by YFV in mosquito cells. Together, these data support a critical role for VCP/p97 in the disassembly of incoming flavivirus nucleocapsids during a postfusion step in virus entry.

IMPORTANCE Flaviviruses are an important group of RNA viruses that cause significant human disease. The mechanisms by which flavivirus nucleocapsids are disassembled during virus entry remain unclear. Here, we used a yellow fever virus entry reporter, which expresses a sensitive reporter enzyme but does not replicate, to show that nucleocapsid disassembly requires the cellular protein-disaggregating enzyme valosin-containing protein, also known as p97.

ABSTRACT

The profiling of gene expression by RNA sequencing (RNA-seq) has enabled powerful studies of global transcriptional patterns in all organisms, including bacteria. Because the vast majority of RNA in bacteria is rRNA, it is standard practice to deplete the rRNA from a total RNA sample such that the reads in an RNA-seq experiment derive predominantly from mRNA. One of the most commonly used commercial kits for rRNA depletion, the Ribo-Zero kit from Illumina, was recently discontinued abruptly and for an extended period of time. Here, we report the development of a simple, cost-effective, and robust method for depleting rRNA that can be easily implemented by any lab or facility. We first developed an algorithm for designing biotinylated oligonucleotides that will hybridize tightly and specifically to the 23S, 16S, and 5S rRNAs from any species of interest. Precipitation of these oligonucleotides bound to rRNA by magnetic streptavidin-coated beads then depletes rRNA from a complex, total RNA sample such that ~75 to 80% of reads in a typical RNA-seq experiment derive from mRNA. Importantly, we demonstrate a high correlation of RNA abundance or fold change measurements in RNA-seq experiments between our method and the Ribo-Zero kit. Complete details on the methodology are provided, including open-source software for designing oligonucleotides optimized for any bacterial species or community of interest.

IMPORTANCE The ability to examine global patterns of gene expression in microbes through RNA sequencing has fundamentally transformed microbiology. However, RNA-seq depends critically on the removal of rRNA from total RNA samples. Otherwise, rRNA would comprise upward of 90% of the reads in a typical RNA-seq experiment, limiting the reads coming from mRNA or requiring high total read depth. A commonly used kit for rRNA subtraction from Illumina was recently unavailable for an extended period of time, disrupting routine rRNA depletion. Here, we report the development of a "do-it-yourself" kit for rapid, cost-effective, and robust depletion of rRNA from total RNA. We present an algorithm for designing biotinylated oligonucleotides that will hybridize to the rRNAs from a target set of species. We then demonstrate that the designed oligonucleotides enable sufficient rRNA depletion to produce RNA-seq data with 75 to 80% of reads coming from mRNA. The methodology presented should enable RNA-seq studies on any species or metagenomic sample of interest.

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.

This season’s flu vaccine was 45% effective overall and 55% effective among children and teens, the Centers for Disease Control and Prevention reported in February.

I’ve been talking a lot lately about the importance of working across sectors for public health — of not going it alone to tackle the imposing challenges before us. The ideal public health team is broad and includes not only public health professionals representing the essential services, but also professionals from other disciplines, the general public and students of all stripes.

After more than 20 years of minimal funding, the U.S. is opening its purse strings to research on gun violence prevention.

The conversation about how we create and maintain health has evolved. We have now clearly expanded our thinking beyond an exclusive focus on traditional medical care, and philanthropy can play an important role

To better the health of all North Carolinians, policymakers must come together to improve access to care, expand broadband, and close the coverage gap.

With rising costs and below-average outcomes, North Carolina's health care value proposition is upside down. It's time for employers to lead transformative change.

Authentically engaging community residents is necessary to impact social drivers of health. Acknowledging the value of residents' lived experiences in the planning, implementation, and financial decisions of community engagement initiatives is key. Sustainability of community engagement initiatives depends on open communication and follow-through on commitments.

North Carolina has received national attention for its approach to health care payment and delivery reform. Importantly, payment reform alone is not enough to drive systematic changes in care delivery. We highlight the importance of progress in four complementary areas to achieve system-wide payment and care reform.

Decades of rallying cries from professional societies, medical education and training programs, and government stakeholders have distilled the conversation of social determinants of health (SDOH) from theoretical proposals into practical solutions [1-3]. No longer standing on the precipice of change, we are now in the trenches. The nation's health care system recognizes SDOH as important drivers of health and is taking steps to address them in the practice environment.

More widespread action and attention by the health care system drives the need to train the next generation of physicians in the concepts and actions related to SDOH. This includes SDOH as a core part of the medical curriculum, offering clinical and research experiences and service in the community [4-5]. Unfortunately, to date only a handful of programs have brought this vision to fruition. Across the country, most programs offer educational content that is largely didactic and provided in short or one-time sessions [6]. Though a start, such approaches are insufficient to prepare the next generation of physicians for their important work ahead.

In New Orleans, the NOLA Hotspotters are an interdisciplinary group of medical, public health, nursing, and pharmacy students inspired by the work out of Camden, New Jersey, to "hot spot" patients with high utilization, which is often related to social needs [7]. While the results of the Camden program have been widely discussed following publication of their work, we argue the benefit of such a program exists beyond reduced emergency department visits or health care spending [8]. The...

Among the many trends influencing health and health care delivery over the next decade, three are particularly important: the transition to value-based care and increased focus on population health; the shift of care from acute to community-based settings; and addressing the vulnerability of rural health care systems in North Carolina.

In recent years, North Carolina has attracted significant national attention due to numerous health care reforms underway across government and the private sector. These reforms encompass new incentives, new partnerships, and new models of delivering care, and collectively, they have important implications for health care data.

In 2019, the National Academy of Medicine (NAM) turned to the all-important state level to draw insights on the status of health and health care within the context of the NAM Vital Directions for Health and Health Care initiative. The NAM held a two-day symposium in the Research Triangle to bring together various stakeholders to better understand actions that states and localities are taking to achieve—and the barriers they face in pursuing—more affordable, value-driven quality care and health outcomes. The NAM purposefully chose to pivot to the state level with North Carolina given that it has been at the forefront of health care transformation and illustrates the promise but also the challenges facing US health and health care nationally. A 19-member planning committee, cochaired by NAM President Victor Dzau and Secretary Mandy Cohen of the North Carolina Department of Health and Human Services, selected topics that resonate with the state's activities within the context of the Vital Directions framework, ranging from empowering people and connecting care through the integration of social, physical, and behavioral health to payer alignment though the advancement of new payment models (Figure 1). The priorities discussed during the symposium continue to be central to health reform in North Carolina and are further explored in the commentaries in this issue.

Objective

To investigate the pathogenicity of the TGM6 variant for spinocerebellar ataxia 35 (SCA35), which was previously reported to be caused by pathogenic mutations in the gene TGM6.

In order to establish sustainable heat loading (heat removal and storage) in abandoned flooded mine workings it is important to understand the geomechanical impact of the cyclical heat loading caused by fluid injection and extraction. This is particularly important where significantly more thermal loading is planned than naturally occurs. A simple calculation shows that the sustainable geothermal heat flux from abandoned coal mines can provide less than a tenth of Scotland's annual domestic heating demand. Any heat removal greater than the natural heat flux will lead to heat mining unless heat storage options are also considered.

As a first step, a steady-state, fully saturated, 2D coupled hydromechanical model of a generalized section of pillar-and-stall workings has been created. Mine water rebound was modelled by increasing the hydrostatic pressure sequentially, in line with monitored mine water-level data from Midlothian, Scotland. The modelled uplift to water-level rise ratio of 1.4 mm m^–1 is of the same order of magnitude (1 mm m^–1) as that observed through interferometric synthetic aperture radar (InSAR) data in the coalfield due to mine water rebound. The modelled magnitude of shear stress at the pillar corners, as a result of horizontal and vertical displacement, is shown to increase linearly with water level. Mine heat systems are expected to cause smaller changes in pressure than those modelled but the results provide initial implications on the potential geomechanical impacts of mine water heat schemes which abstract or inject water and heat into pillar-and-stall coal mine workings.

Thematic collection: This article is part of the SJG Collection on Early-Career Research available at: https://www.lyellcollection.org/cc/SJG-early-career-research

Studies of the former NE England coalfield in Tyneside demonstrated that heat flow perturbations in boreholes were due to the entrainment and lateral dispersion of heat from deeper in the subsurface through flooded mine workings. This work assesses the influence of historical mining on geothermal observations across Greater Glasgow. The regional heat flow for Glasgow is 60 mW m^–2 and, after correction for palaeoclimate, is estimated as c. 80 mW m^–2. An example of reduced heat flow above mine workings is observed at Hallside (c. 10 km SE of Glasgow), where the heat flow through a 352 m deep borehole is c. 14 mW m^–2. Similarly, the heat flow across the 199 m deep GGC01 borehole in the Glasgow Geothermal Energy Research Field Site is c. 44 mW m^–2. The differences between these values and the expected regional heat flow suggest a significant component of horizontal heat flow into surrounding flooded mine workings. This deduction also influences the quantification of deeper geothermal resources, as extrapolation of the temperature gradient above mine workings would underestimate the temperature at depth. Future projects should consider the influence of historical mining on heat flow when temperature datasets such as these are used in the design of geothermal developments.

Supplementary material: Background information on the chronology of historical mining at each borehole location and a summary of groundwater flow in mine workings beneath Glasgow are available at https://doi.org/10.6084/m9.figshare.c.4681100

Thematic collection: This article is part of the ‘Early Career Research’ available at: https://www.lyellcollection.org/cc/SJG-early-career-research

Thematic collection: This article is part of the ‘Early Career Researchers’ available at: https://www.lyellcollection.org/cc/SJG-early-career-research

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.

Salicinoids form a specific class of phenolic glycosides characteristic of the Salicaceae. Although salicinoids accumulate in large amounts and have been shown to be involved in plant defense, their biosynthesis is unclear. We identified two sulfated salicinoids, salicin-7-sulfate and salirepin-7-sulfate, in black cottonwood (Populus trichocarpa). Both compounds accumulated in high amounts in above-ground tissues including leaves, petioles, and stems, but were also found at lower concentrations in roots. A survey of salicin-7-sulfate and salirepin-7-sulfate in a subset of poplar (Populus sp.) and willow (Salix sp.) species revealed a broader distribution within the Salicaceae. To elucidate the formation of these compounds, we studied the sulfotransferase (SOT) gene family in P. trichocarpa (PtSOT). One of the identified genes, PtSOT1, was shown to encode an enzyme able to convert salicin and salirepin into salicin-7-sulfate and salirepin-7-sulfate, respectively. The expression of PtSOT1 in different organs of P. trichocarpa matched the accumulation of sulfated salicinoids in planta. Moreover, RNA interference-mediated knockdown of SOT1 in gray poplar (Populus x canescens) resulted in decreased levels of sulfated salicinoids in comparison to wild-type plants, indicating that SOT1 is responsible for their formation in planta. The presence of a nonfunctional SOT1 allele in black poplar (Populus nigra) was shown to correlate with the absence of salicin-7-sulfate and salirepin-7-sulfate in this species. Food choice experiments with leaves from wild-type and SOT1 knockdown trees suggest that sulfated salicinoids do not affect the feeding preference of the generalist caterpillar Lymantria dispar. A potential role of the sulfated salicinoids in sulfur storage and homeostasis is discussed.

Plants require a high concentration of ascorbate as a redox buffer for survival under stress conditions, such as high light. Dehydroascorbate reductases (DHARs) are enzymes that catalyze the reduction of DHA to ascorbate using reduced glutathione (GSH) as an electron donor, allowing rapid ascorbate recycling. However, a recent study using an Arabidopsis (Arabidopsis thaliana) triple mutant lacking all three DHAR genes (herein called dhar) did not find evidence for their role in ascorbate recycling under oxidative stress. To further study the function of DHARs, we generated dhar Arabidopsis plants as well as a quadruple mutant line combining dhar with an additional vtc2 mutation that causes ascorbate deficiency. Measurements of ascorbate in these mutants under low- or high-light conditions indicated that DHARs have a nonnegligible impact on full ascorbate accumulation under high light, but that they are dispensable when ascorbate concentrations are low to moderate. Because GSH itself can reduce DHA nonenzymatically, we used the pad2 mutant that contains ~30% of the wild-type GSH level. The pad2 mutant accumulated ascorbate at a wild-type level under high light; however, when the pad2 mutation was combined with dhar, there was near-complete inhibition of high-light–dependent ascorbate accumulation. The lack of ascorbate accumulation was consistent with a marked increase in the ascorbate degradation product threonate. These findings indicate that ascorbate recycling capacity is limited in dhar pad2 plants, and that both DHAR activity and GSH content set a threshold for high-light–induced ascorbate accumulation.

For decades inhaled corticosteroids have been central to the management of asthma and are proven to be effective in maintaining symptom control, reducing exacerbations and preserving quality of life through mediation of airway inflammation. However, a small minority of patients have disease which is refractory to high dose inhaled corticosteroid (ICS) therapy and require additional oral corticosteroids to achieve acceptable control of symptoms and exacerbations. Severe asthma represents less than 10% of the total asthma population [1] but is the most serious, life-affecting and costly form of the condition [2].

The aim of this study was to identify factors associated with the death of patients with COVID-19 pneumonia caused by the novel coronavirus SARS-CoV-2.

All clinical and laboratory parameters were collected prospectively from a cohort of patients with COVID-19 pneumonia who were hospitalised to Wuhan Pulmonary Hospital (Wuhan City, Hubei Province, China) between 25 December 2019 and 7 February 2020. Univariate and multivariate logistic regression was performed to investigate the relationship between each variable and the risk of death of COVID-19 pneumonia patients.

In total, 179 patients with COVID-19 pneumonia (97 male and 82 female) were included in the present prospective study, of whom 21 died. Univariate and multivariate logistic regression analysis revealed that age ≥65 years (OR 3.765, 95% CI 1.146-17.394; p=0.023), pre-existing concurrent cardiovascular or cerebrovascular diseases (OR 2.464, 95% CI 0.755-8.044; p=0.007), CD3⁺CD8⁺ T-cells ≤75 cells·μL^–1 (OR 3.982, 95% CI 1.132-14.006; p<0.001) and cardiac troponin I ≥0.05 ng·mL^–1 (OR 4.077, 95% CI 1.166-14.253; p<0.001) were associated with an increase in risk of mortality from COVID-19 pneumonia. In a sex-, age- and comorbid illness-matched case–control study, CD3⁺CD8⁺ T-cells ≤75 cells·μL^–1 and cardiac troponin I ≥0.05 ng·mL^–1 remained as predictors for high mortality from COVID-19 pneumonia.

We identified four risk factors: age ≥65 years, pre-existing concurrent cardiovascular or cerebrovascular diseases, CD3⁺CD8⁺ T-cells ≤75 cells·μL^–1 and cardiac troponin I ≥0.05 ng·mL^–1. The latter two factors, especially, were predictors for mortality of COVID-19 pneumonia patients.

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.

The World Health Organization (WHO) has listed moxifloxacin and linezolid among the preferred "group A" drugs in the treatment of multidrug-resistant (MDR)-tuberculosis (TB) [1]. Therapeutic drug monitoring (TDM) could potentially optimise MDR-TB therapy, since moxifloxacin and linezolid show large pharmacokinetic variability [1–4]. TDM of moxifloxacin focuses on identifying patients with low drug exposure who are at risk of treatment failure and acquired fluoroquinolone resistance [5, 6]. Alternatively, TDM of linezolid strives to reduce toxicity while ensuring an adequate drug exposure because of its narrow therapeutic index [1, 3, 7].

Ventilatory settings are critical in mechanically ventilated extremely preterm newborn infants due to the risk of ventilation-induced lung injury (VILI) and the subsequent development of bronchopulmonary dysplasia (BPD) [1]. Positive end-expiratory pressure (PEEP) settings usually rely on blood gases, oxygen requirement, lung auscultation, evaluation of chest radiograph and assessment of the pressure/volume curves provided by ventilators. Studies of optimal PEEP settings in the surfactant-treated preterm infant in need of mechanical ventilation are limited and evidence-based clinical guidelines are sparse [2, 3]. A bedside method identifying the PEEP value that comprises maximal lung volume recruitment and minimising tissue overdistension could improve real-time optimisation of PEEP and potentially minimise the risk of VILI and BPD [4, 5].

Background

We sought to determine the cumulative incidence of readmissions after a seizure-related hospitalization and identify risk factors and readmission diagnoses.

Objective

To assess the risk of subsequent stroke among older patients discharged from an emergency department (ED) without a diagnosis of TIA or stroke.

Background

Transient loss of consciousness (TLOC) is a common reason for presentation to primary/emergency care; over 90% are because of epilepsy, syncope, or psychogenic non-epileptic seizures (PNES). Misdiagnoses are common, and there are currently no validated decision rules to aid diagnosis and management. We seek to explore the utility of machine-learning techniques to develop a short diagnostic instrument by extracting features with optimal discriminatory values from responses to detailed questionnaires about TLOC manifestations and comorbidities (86 questions to patients, 31 to TLOC witnesses).

Dr. Sethi raises an excellent point about the term functional neurologic disorder (FND) in his comment on the editorial.¹ It seems clear that reticence to use the term functional creates the ambiguity he mentions. Medically unexplained symptoms, categorized in the international classification of diseases as undifferentiated somatoform disorders, are a diagnosis that many providers are loathed to give. Whether that is because of concern about missing a diagnosis is not clear. Having evaluated and treated more than 400 of these individuals in the FND clinic at the University of Colorado, I can attest to the fact that patients arrive confused about their diagnosis. Multiple incorrect diagnoses, as Dr. Sethi points out, pack the medical histories of patients with FND, leading doctors and patients astray. I believe that the commentary by Perez et al.² gives us the best chance for a way forward, by teaching a new generation of residents and fellows how to approach patients in a nonjudgmental and open-minded fashion. It took 30 years to add Functional Neurologic Disorder to the Diagnostic and Statistical Manual, and it is still parenthetical to the term Conversion.³ Stripping the diagnosis of FND of its stigma and empowering care providers to rule in functional disorders is an actionable step which should be taken.

I read with interest the editorial by Strom¹ about functional neurologic disorders (FNDs). As a treating physician, I have struggled with the multiple diagnostic labels attached to these patients by physicians of different medical specialties during the course of their clinical disease presentation. A neurologist may assign a patient who presents with chronic fatigue the diagnostic labels of narcolepsy, idiopathic hypersomnia, or chronic Lyme disease. A rheumatologist may assign the label of collagen vascular disease, and a psychiatrist may diagnose depression. This diagnostic ambiguity is troublesome for patients and clinicians alike. I contend that even the term FND needs to be revisited. A patient should be broadly labeled as having a functional disorder and only after characterization sublabeled and referred to an appropriate specialty physician.

The American Academy of Pediatrics (AAP) believes that current data show that hospitals and accredited birth centers are the safest settings for birth in the United States. The AAP does not recommend planned home birth, which has been reported to be associated with a twofold to threefold increase in infant mortality in the United States. The AAP recognizes that women may choose to plan a home birth. This statement is intended to help pediatricians provide constructive, informed counsel to women considering home birth while retaining their role as child advocates and to summarize appropriate care for newborn infants born at home that is consistent with care provided for infants born in a medical care facility. Regardless of the circumstances of his or her birth, including location, every newborn infant deserves health care consistent with that highlighted in this statement, which is more completely described in other publications from the AAP, including Guidelines for Perinatal Care and the Textbook of Neonatal Resuscitation. All health care clinicians and institutions should promote communications and understanding on the basis of professional interaction and mutual respect.