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The London dispersion interactions between systems undergoing bond breaking, twisting, or compression are not well studied due to the scarcity and the high computational cost of methods being able to describe both the dynamic correlation and the multireference character of the system. Recently developed methods based on the Generalized Valence Bond wave function, such as EERPA-GVB and SAPT(GVB) (SAPT = symmetry-adapted perturbation theory), allow one to accurately compute and analyze noncovalent interactions between multireference systems. Here, we augment this analysis by introducing a local indicator for dispersion interactions inspired by Mata and Wuttke's Dispersion Interaction Density [

Most mammalian genes have multiple polyA sites, representing a substantial source of transcript diversity that is governed by the cleavage and polyadenylation (CPA) regulatory machinery. To better understand how these proteins govern polyA site choice we introduce CPA-Perturb-seq, a multiplexed perturbation screen dataset of 42 known CPA regulators with a 3’ scRNA-seq readout that enables transcriptome-wide inference of polyA site usage. We develop a statistical framework to specifically identify perturbation-dependent changes in intronic and tandem polyadenylation, and discover modules of co-regulated polyA sites exhibiting distinct functional properties. By training a multi-task deep neural network (APARENT-Perturb) on our dataset, we delineate acis-regulatory code that predicts responsiveness to perturbation and reveals interactions between distinct regulatory complexes. Finally, we leverage our framework to re-analyze published scRNA-seq datasets, identifying new regulators that affect the relative abundance of alternatively polyadenylated transcripts, and characterizing extensive cellular heterogeneity in 3’ UTR length amongst antibody-producing cells. Our work highlights the potential for multiplexed single-cell perturbation screens to further our understanding of post-transcriptional regulationin vitroandin vivo.

Clinical trials of combined IDO/PD1 blockade in metastatic melanoma (MM) failed to show additional clinical benefit compared to PD1-alone inhibition. We reasoned that a tryptophan-metabolizing pathway other than the kynurenine one is essential. We immunohistochemically stained tissues along the nevus-to-MM progression pathway for tryptophan-metabolizing enzymes (TMEs; TPH1, TPH2, TDO2, IDO1) and the tryptophan transporter, LAT1. We assessed tryptophan and glucose metabolism by performing baseline C11-labeled α-methyl tryptophan (C11-AMT) and fluorodeoxyglucose (FDG) PET imaging of tumor lesions in a prospective clinical trial of pembrolizumab in MM (clinicaltrials.gov,NCT03089606). We found higher protein expression of all TMEs and LAT1 in melanoma cells than tumor-infiltrating lymphocytes (TILs) within MM tumors (n=68). Melanoma cell-specific TPH1 and LAT1 expression are significantly anti-correlated with TIL presence in MM. High melanoma cell-specific LAT1 and low IDO1 expression were associated with worse overall survival (OS) in MM. Exploratory optimal cut-point survival analysis of pretreatment ‘high’ vs. ‘low’ C11-AMT SUVmaxof the hottest tumor lesion per patient revealed that the ‘low’ C11-AMT SUVmaxwas associated with longer progression-free and OS in our clinical trial (n=26). We saw no such trends with pretreatment FDG PET SUVmax. Treatment of melanoma cell lines with telotristat, a TPH1 inhibitor, increased IDO expression and kynurenine production in addition to suppression of serotonin production. High melanoma tryptophan metabolism is a poor predictor of pembrolizumab response and an adverse prognostic factor. Serotoninergic but not kynurenine pathway activation may be significant. Melanoma cells outcompete adjacent TILs, eventually depriving the latter of an essential amino acid.

Background Falls in older adults are a significant and growing public health concern. There are multiple risk factors associated with falls that may be addressed within the scope of chiropractic training and licensure. Few attempts have been made to summarize existing evidence on multimodal chiropractic care and fall risk mitigation. Therefore, the broad purpose of this review was to summarize this research to date. Body Systematic review was conducted following PRISMA guidelines. Databases searched included PubMed, Embase, Cochrane Library, PEDro, and Index of Chiropractic Literature. Eligible study designs included randomized controlled trials (RCT), prospective non-randomized controlled, observational, and cross-over studies in which multimodal chiropractic care was the primary intervention and changes in gait, balance and/or falls were outcomes. Risk of bias was also assessed using the 8-item Cochrane Collaboration Tool. The original search yielded 889 articles; 21 met final eligibility including 10 RCTs. One study directly measured the frequency of falls (underpowered secondary outcome) while most studies assessed short-term measurements of gait and balance. The overall methodological quality of identified studies and findings were mixed, limiting interpretation regarding the potential impact of chiropractic care on fall risk to qualitative synthesis. Conclusion Little high-quality research has been published to inform how multimodal chiropractic care can best address and positively influence fall prevention. We propose strategies for building an evidence base to inform the role of multimodal chiropractic care in fall prevention and outline recommendations for future research to fill current evidence gaps.

Dermatological conditions constituting the group of autoimmune blistering diseases (AIBD) are characterized by loss of immunotolerance and humoral, as well as cellular, autoimmune responses that result in the development of bullae and erosions on the skin and mucous membranes. AIBDs are broadly categorized into pemphigus and pemphigoid classes with several distinct subtypes amongst them. Advances in genetics have allowed for the study and identification of alleles, and even single nucleotide polymorphisms, that harbor increased susceptibility or confer protection for the development of these conditions. The focus of this chapter pertains to a comprehensive review of the known genetic associations with AIBDs, including HLA class I-III, as well as non-HLA genes and non-coding sequences that influence cellular processes and signaling pathways.

Aspergillus fumigatus isolates display significant heterogeneity in growth, virulence, pathology, and inflammatory potential in multiple murine models of invasive aspergillosis. Previous studies have linked the initial germination of a fungal isolate in the airways to the inflammatory and pathological potential, but the mechanism(s) regulating A. fumigatus germination in the airways is unresolved. To explore the genetic basis for divergent germination phenotypes, we utilized a serial passaging strategy in which we cultured a slow germinating strain (AF293) in a murine-lung-based medium for multiple generations. Through this serial passaging approach, a strain emerged with an increased germination rate that induces more inflammation than the parental strain (herein named LH-EVOL for lung homogenate evolved). We identified a potential loss-of-function allele of Afu5g08390 (sskA) in the LH-EVOL strain. The LH-EVOL strain had a decreased ability to induce the SakA-dependent stress pathway, similar to AF293 ΔsskA and CEA10. In support of the whole-genome variant analyses, sskA, sakA, or mpkC loss-of-function strains in the AF293 parental strain increased germination both in vitro and in vivo. Since the airway surface liquid of the lungs contains low glucose levels, the relationship of low glucose concentration on germination of these mutant AF293 strains was examined; interestingly, in low glucose conditions, the sakA pathway mutants exhibited an enhanced germination rate. In conclusion, A. fumigatus germination in the airways is regulated by SskA through the SakA mitogen-activated protein kinase (MAPK) pathway and drives enhanced disease initiation and inflammation in the lungs. IMPORTANCE Aspergillus fumigatus is an important human fungal pathogen particularly in immunocompromised individuals. Initiation of growth by A. fumigatus in the lung is important for its pathogenicity in murine models. However, our understanding of what regulates fungal germination in the lung environment is lacking. Through a serial passage experiment using lung-based medium, we identified a new strain of A. fumigatus that has increased germination potential and inflammation in the lungs. Using this serially passaged strain, we found it had a decreased ability to mediate signaling through the osmotic stress response pathway. This finding was confirmed using genetic null mutants demonstrating that the osmotic stress response pathway is critical for regulating growth in the murine lungs. Our results contribute to the understanding of A. fumigatus adaptation and growth in the host lung environment.

Microbes expertly manipulate hosts to their advantage, but few completely escape detection. In this issue of Cell Host & Microbe, Takaki et al. (2021) describe how Schistosoma mansoni eggs choreograph macrophage behaviors to promote efficient transmission of mature eggs, while immature eggs remain safely hidden in plain sight.

Aspergillus fumigatus isolates display significant heterogeneity in growth, virulence, pathology, and inflammatory potential in multiple murine models of invasive aspergillosis. Previous studies have linked the initial germination of a fungal isolate in the airways to the inflammatory and pathological potential; but the mechanism(s) regulating A. fumigatus germination in the airways are unresolved. To explore the genetic basis for divergent germination phenotypes, we utilized a serial passaging strategy in which we cultured a slow germinating strain (AF293) in a murine lung based medium for multiple generations. Through this serial passaging approach, a strain emerged with an increased germination rate that induces more inflammation than the parental strain (herein named Lung Homogenate Evolved (LH-EVOL)). We identified a potential loss of function allele of Afu5g08390 (sskA) in the LH-EVOL strain. The LH-EVOL strain had a decreased ability to induce the SakA-dependent stress pathway, similar to AF293 ΔsskA and CEA10. In support of the whole genome variant analyses, sskA, sakA, or mpkC loss of function strains in the AF293 parental strain increased germination both in vitro and in vivo. Since the airway surface liquid of the lungs contains low glucose levels, the relationship of low glucose concentration on germination of these mutant AF293 strains was examined; interestingly, in low glucose conditions the sakA pathway mutants exhibited an enhanced germination rate. In conclusion, A. fumigatus germination in the airways is regulated by SskA through the SakA MAPK pathway and drives enhanced disease initiation and inflammation in the lungs.IMPORTANCEAspergillus fumigatus is an important human fungal pathogen particularly in immunocompromised individuals. Initiation of growth by A. fumigatus in the lung is important for its pathogenicity in murine models. However, our understanding of what regulates fungal germination in the lung environment is lacking. Through a serial passage experiment using lung-based medium, we identified a new strain of A. fumigatus which has increased germination potential and inflammation in the lungs. Using this serially passaged strain we found it had a decreased ability to mediate signaling through the osmotic stress response pathway. This finding was confirmed using genetic null mutants demonstrating that the osmotic stress response pathway is critical for regulating growth in the murine lungs. Our results contribute to the understanding of A. fumigatus adaptation and growth in the host lung environment.

Background: Thousands of genetic variants have been associated with hematological traits, though target genes remain unknown at most loci. Also, limited analyses have been conducted in African ancestry and Hispanic/Latino populations; hematological trait associated variants more common in these populations have likely been missed. Methods: To derive gene expression prediction models, we used ancestry-stratified datasets from the Multi-Ethnic Study of Atherosclerosis (MESA, including N=229 African American and N=381 Hispanic/Latino participants, monocytes) and the Depression Genes and Networks study (DGN, N = 922 European ancestry participants, whole blood). We then performed a transcriptome-wide association study (TWAS) for platelet count, hemoglobin, hematocrit, and white blood cell count in African (N = 27,955) and Hispanic/Latino (N = 28,324) ancestry participants. Results: Our results revealed 24 suggestive signals (p < 1×10^(-4)) that were conditionally distinct from known GWAS identified variants and successfully replicated these signals in European ancestry subjects from UK Biobank. We found modestly improved correlation of predicted and measured gene expression in an independent African American cohort (the Genetic Epidemiology Network of Arteriopathy (GENOA) study (N=802), lymphoblastoid cell lines) using the larger DGN reference panel; however, some genes were well predicted using MESA but not DGN. Conclusions: These analyses demonstrate the importance of performing TWAS and other genetic analyses across diverse populations and of balancing sample size and ancestry background matching when selecting a TWAS reference panel.

Microbial populations form intricate macroscopic colonies with diverse morphologies whose functions remain to be fully understood. Despite fungal colonies isolated from environmental and clinical samples revealing abundant intraspecies morphological diversity, it is unclear how this diversity impacts fungal fitness and disease progression. Here we observe a significant impact of oxygen tension on the macroscopic and biofilm morphotypes of the human fungal pathogen Aspergillus fumigatus. A hypoxia-typic morphotype is generated through the expression of a sub-telomeric gene cluster containing genes that alter the hyphal surface and perturb inter-hyphal interactions to disrupt in vivo biofilm and infection site morphologies. Consequently, this morphotype leads to increased host inflammation, rapid disease progression, and mortality in a murine model of invasive aspergillosis. Taken together, these data suggest filamentous fungal biofilm morphology impacts fungal-host interactions and should be taken into consideration when assessing virulence and host disease progression of an isolated strain.

Eosinophils are seen in a number of dermatologic conditions. While the extent of their function in these diseases remains to be fully elucidated, pathogenic activity in bullous pemphigoid suggests a more significant role than previously thought. Several dermatoses have a fairly characteristic histologic morphology of eosinophil infiltration. We hypothesized that epidermal expression of eotaxins and TSLP would differ by disease, perhaps explaining the different histologic morphologies. We performed a retrospective study of eosinophil rich dermatoses to perform immunohistochemistry. We collected 49 specimens composed of bullous pemphigoid (n = 15), atopic dermatitis (n = 12), drug rash (n = 8), arthropod assault (n = 5), and non-bullous pemphigoid eosinophilic spongiosis (n = 5). We used lichen planus (n = 4) as a control for lymphocyte-mediated inflammation. TSLP was diffusely expressed in all epidermal samples, whereas eotaxins demonstrated a weaker staining. Eotaxins and TSLP demonstrated a gradient between basal and spinous keratinocytes. The correlation between overall basal keratinocyte and spinous keratinocyte staining of eotaxins and TSLP with the number of eosinophils demonstrated a significant correlation between eotaxin-1 (R = 0.404, P = 0.004), eotaxin-2 (R = 0.576, P

BACKGROUND Autoimmune blistering diseases are a group of severe mucocutaneous conditions that typically require the use of prolonged corticosteroids and immunosuppression. Properly managing associated comorbidities is an integral part of these patients' care. The frequency of gastrointestinal symptoms, particularly gastrointestinal bleeding in these patients, is not known. Likewise, the effect of diet on disease is unknown. OBJECTIVE To determine the incidence of gastrointestinal comorbidities and the role of diet in patients with autoimmune blistering disease. METHODS We distributed an e-survey to patients with autoimmune blistering disease utilizing the International Pemphigus and Pemphigoid Foundation's listserv. The incidence of gastrointestinal symptoms and gastrointestinal bleeding were recorded, as were foods avoided and those noted to be beneficial in patients' disease. Historical incidences in the general population were used as controls. RESULTS A total of 200 responses were collected. 30.3% of patients experienced gastroesophageal reflux following treatment of their autoimmune blistering disease, with 51.7% utilizing some form of gastrointestinal symptomatic treatment. The incidence of gastrointestinal bleeding following an autoimmune blistering diagnosis was 2.1%, which remained significant despite correction for non-steroidal anti-inflammatory use (NSAID), but not corticosteroid use. 65.2% of patients reported dietary limitations because of their autoimmune blistering disease. Significant intolerances after correction for multiple comparisons included alcohol, citrus and spicy foods. Greater than 10% of patients reported improvements in their disease with vegetables and dairy. CONCLUSIONS Gastrointestinal comorbidities are common in patients with autoimmune blistering diseases, with gastrointestinal bleeding occurring in 2.1% of patients following a diagnosis of autoimmune blistering disease. While further work is needed to determine the relative risk of routine gastrointestinal prophylaxis in this population, gastrointestinal bleeding prophylaxis should be considered in patients receiving corticosteroids, particularly those taking NSAIDs. Dietary limitations are additionally frequent in this population. Patients should be cautious of alcohol, citrus and spicy foods.

Bullous pemphigoid and pemphigus constitute two major autoimmune blistering diseases (AIBD) with complicated disease pathomechanisms involving a multitude of cytokines and immunological pathways. The purpose of our literature review of the cytokines and chemokines involved in these AIBDs was to allow for a meta-analysis of studies detailing differential cytokine and chemokine changes in these conditions. Elucidation of inflammatory pathways could lead to more targeted therapies, several of which specific monoclonal antibodies already exist and are used safely for other autoimmune diseases. A systematic review of the Pubmed/Medline database was performed for articles characterizing cytokines/chemokines involved in BP and pemphigus. Further, a meta-analysis was carried out using standardized methods, including assessment for heterogeneity. The results of our analysis demonstrated numerous inflammatory alterations in these AIBDs. Significant alterations included serum levels of IL-5, IL-6, IL-8, IL-17, CCL-17, and CCL-26 in patients with BP, and increased blister fluids levels of IL-5, IL-6, IL-8, CCL11, and TNF-α. Blister fluid levels of IL-1α are decreased in BP. In pemphigus, we identified significantly increased serum levels of IL-10, IL-17, and CCL17. We have additionally summarized all studies excluded from meta-analysis to provide a comprehensive summary of cytokine/chemokine alterations in these two conditions.

Prurigo nodualris (PN) is a chronic condition with highly pruritic, hyperkeratotic papules or nodules arising in the setting of chronic pruritus. While PN may serve as a phenotypic presentation of several underlying conditions such as atopic dermatitis, chronic kidney disease-related pruritus, and neurological diseases, it represents a distinct clinical entity that may persist despite the removal of the underlying cause, if one is identified. Neuronal proliferation, eosinophils, mast cells, and small-fiber neuropathy play a role in the production of pruritus in PN, although the exact mechanism has not yet been established. Identifying an underlying cause, if present, is essential to prevent recurrence of PN. Due to often present comorbidities, treatment is typically multimodal with utilization of topical and systemic therapies. We performed a PubMed/MEDLINE search for PN and present a review of recent developments in the treatment of PN. Treatment typically relies on the use of topical or intralesional steroids, though more severe or recalcitrant cases often necessitate the use of phototherapy or systemic immunosuppressives. Thalidomide and lenalidomide can both be used in severe cases; however, their toxicity profile makes them less favorable. Opioid receptor antagonists and neurokinin-1 receptor antagonists represent two novel families of therapeutic agents which may effectively treat PN with a lower toxicity profile than thalidomide or lenalidomide.

The genus Aspergillus encompasses human pathogens such as Aspergillus fumigatus and industrial powerhouses such as Aspergillus niger In both cases, Aspergillus biofilms have consequences for infection outcomes and yields of economically important products. However, the molecular components influencing filamentous fungal biofilm development, structure, and function remain ill defined. Macroscopic colony morphology is an indicator of underlying biofilm architecture and fungal physiology. A hypoxia-locked colony morphotype of A. fumigatus has abundant colony furrows that coincide with a reduction in vertically oriented hyphae within biofilms and increased low oxygen growth and virulence. Investigation of this morphotype has led to the identification of the causative gene, biofilm architecture factor A (bafA), a small cryptic open reading frame within a subtelomeric gene cluster. BafA is sufficient to induce the hypoxia-locked colony morphology and biofilm architecture in A. fumigatus Analysis across a large population of A. fumigatus isolates identified a larger family of baf genes, all of which have the capacity to modulate hyphal architecture, biofilm development, and hypoxic growth. Furthermore, introduction of A. fumigatus bafA into A. niger is sufficient to generate the hypoxia-locked colony morphology, biofilm architecture, and increased hypoxic growth. Together, these data indicate the potential broad impacts of this previously uncharacterized family of small genes to modulate biofilm architecture and function in clinical and industrial settings.IMPORTANCE The manipulation of microbial biofilms in industrial and clinical applications remains a difficult task. The problem is particularly acute with regard to filamentous fungal biofilms for which molecular mechanisms of biofilm formation, maintenance, and function are only just being elucidated. Here, we describe a family of small genes heterogeneously expressed across Aspergillus fumigatus strains that are capable of modifying colony biofilm morphology and microscopic hyphal architecture. Specifically, these genes are implicated in the formation of a hypoxia-locked colony morphotype that is associated with increased virulence of A. fumigatus Synthetic introduction of these gene family members, here referred to as biofilm architecture factors, in both A. fumigatus and A. niger additionally modulates low oxygen growth and surface adherence. Thus, these genes are candidates for genetic manipulation of biofilm development in aspergilli.

Aspergillus fumigatus is a filamentous fungus which can cause multiple diseases in humans. Allergic broncho-pulmonary aspergillosis (ABPA) is a disease diagnosed primarily in cystic fibrosis patients caused by a severe allergic response often to long-term A. fumigatus colonization in the lungs. Mice develop an allergic response to repeated inhalation of A. fumigatus spores; however, no strains have been identified that can survive long-term in the mouse lung and cause ABPA-like disease. We characterized A. fumigatus strain W72310, which was isolated from the expectorated sputum of an ABPA patient, by whole-genome sequencing and in vitro and in vivo viability assays in comparison to a common reference strain, CEA10. W72310 was resistant to leukocyte-mediated killing and persisted in the mouse lung longer than CEA10, a phenotype that correlated with greater resistance to oxidative stressors, hydrogen peroxide, and menadione, in vitro. In animals both sensitized and challenged with W72310, conidia, but not hyphae, were viable in the lungs for up to 21 days in association with eosinophilic airway inflammation, airway leakage, serum IgE, and mucus production. W72310-sensitized mice that were recall challenged with conidia had increased inflammation, Th1 and Th2 cytokines, and airway leakage compared to controls. Collectively, our studies demonstrate that a unique strain of A. fumigatus resistant to leukocyte killing can persist in the mouse lung in conidial form and elicit features of ABPA-like disease. IMPORTANCE Allergic broncho-pulmonary aspergillosis (ABPA) patients often present with long-term colonization of Aspergillus fumigatus. Current understanding of ABPA pathogenesis has been complicated by a lack of long-term in vivo fungal persistence models. We have identified a clinical isolate of A. fumigatus, W72310, which persists in the murine lung and causes an ABPA-like disease phenotype. Surprisingly, while viable, W72310 showed little to no growth beyond the conidial stage in the lung. This indicates that it is possible that A. fumigatus can cause allergic disease in the lung without any significant hyphal growth. The identification of this strain of A. fumigatus can be used not only to better understand disease pathogenesis of ABPA and potential antifungal treatments but also to identify features of fungal strains that drive long-term fungal persistence in the lung. Consequently, these observations are a step toward helping resolve the long-standing question of when to utilize antifungal therapies in patients with ABPA and fungal allergic-type diseases.

The molecular mechanisms of microbial virulence and host defense are most often studied using animal models and Koch’s molecular postulates. A common rationale for these types of experiments is to identify therapeutic targets based on the assumption that microbial or host factors that confer extreme animal model survival phenotypes represent critical virulence and host defense factors., The molecular mechanisms of microbial virulence and host defense are most often studied using animal models and Koch’s molecular postulates. A common rationale for these types of experiments is to identify therapeutic targets based on the assumption that microbial or host factors that confer extreme animal model survival phenotypes represent critical virulence and host defense factors. Yet null mutant strains of microbial (or host) factors often yield extreme survival curve phenotypes because they fail to establish an infection. The lack of infection and disease establishment prevents true assessment of the given factor’s role(s) in disease progression. Here, we posit that the emphasis on extreme survival curve phenotypes in fungal infectious disease models is leading to missed opportunities to identify new fungal and host factors critical for disease progression. We simply do not yet have a sufficient understanding of fungal virulence and host defense mechanisms throughout the temporal course of an infection. We propose that there is a need to develop new approaches and to revisit tried and true methods to define infection site biology beyond the analysis of survival curve phenotypes. To stimulate these new approaches, we propose the (new) terms “disease initiation factor” and “disease progression factor” to distinguish functional roles at distinct temporal stages of an infection and give us targets to foster new discoveries.

Infection tissue microenvironments are dynamic, complex, and play a critical role in host-microbe interaction outcomes. A crucial parameter of the infection site microenvironment is oxygen. Both host and microbial cell physiology is significantly impacted by the availability of oxygen. When oxygen tensions drop to levels that do not meet the metabolic demands of the cell, a hypoxia response ensues. In numerous host-microbe studies, it has now been observed that the host and microbial hypoxia response plays a critical role in disease outcomes. However, in most pathosystems, spatial and temporal oxygen dynamics throughout the infection remain ill defined. Here, we detail a protocol for detecting low oxygen environments in tissue in a murine model of invasive pulmonary aspergillosis. The protocol utilizes mice immune compromised with a high dose of steroid and challenged via the aerosol route with conidia of the major human fungal pathogen Aspergillus fumigatus. Qualitative analysis of oxygen levels at the site of infection in the murine lung is accomplished with pimonidazole-mediated adduct detection via immunohistochemistry. The protocol is adaptable to other host-microbe interaction models.

Infection tissue microenvironments are dynamic, complex, and play a critical role in host-microbe interaction outcomes. A crucial parameter of the infection site microenvironment is oxygen. Both host and microbial cell physiology is significantly impacted by the availability of oxygen. When oxygen tensions drop to levels that do not meet the metabolic demands of the cell, a hypoxia response ensues. In numerous host-microbe studies, it has now been observed that the host and microbial hypoxia response plays a critical role in disease outcomes. However, in most pathosystems, spatial and temporal oxygen dynamics throughout the infection remain ill defined. Here, we detail a protocol for detecting low oxygen environments in tissue in a murine model of invasive pulmonary aspergillosis. The protocol utilizes mice immune compromised with a high dose of steroid and challenged via the aerosol route with conidia of the major human fungal pathogen Aspergillus fumigatus. Qualitative analysis of oxygen levels at the site of infection in the murine lung is accomplished with pimonidazole-mediated adduct detection via immunohistochemistry. The protocol is adaptable to other host-microbe interaction models.

The genusAspergillusencompasses human pathogens such asAspergillus fumigatusand industrial powerhouses such asAspergillus niger.In both cases,Aspergillusbiofilms have consequences for infection outcomes and yields of economically important products. Yet, the molecular components influencing filamentous fungal biofilm development, structure, and function remain ill-defined. Macroscopic colony morphology is an indicator of underlying biofilm architecture and fungal physiology. A hypoxia-locked colony morphotype ofA. fumigatushas abundant colony furrows that coincide with a reduction in vertically-oriented hyphae within biofilms and increased low oxygen growth and virulence. Investigation of this morphotype has led to the identification of the causative gene,biofilm architecture factor A (bafA),a small cryptic open reading frame within a subtelomeric gene cluster. BafA is sufficient to induce the hypoxia-locked colony morphology and biofilm architecture inA. fumigatus.Analysis across a large population ofA. fumigatusisolates identified a larger family ofbafgenes, all of which have the capacity to modulate hyphal architecture, biofilm development, and hypoxic growth. Furthermore, introduction ofA. fumigatus bafAintoA. nigeris sufficient to generate the hypoxia-locked colony morphology, biofilm architecture, and increased hypoxic growth. Together these data indicate the potential broad impacts of this previously uncharacterized family of small genes to modulate biofilm architecture and function in clinical and industrial settings.ImportanceThe manipulation of microbial biofilms in industrial and clinical applications remains a difficult task. The problem is particularly acute with regard to filamentous fungal biofilms for which molecular mechanisms of biofilm formation, maintenance, and function are only just being elucidated. Here we describe a family of small genes heterogeneously expressed acrossAspergillus fumigatusstrains that are capable of modifying colony biofilm morphology and microscopic hyphal architecture. Specifically, these genes are implicated in the formation of a hypoxia-locked colony morphotype that is associated with increased virulence ofA.fumigatus. Synthetic introduction of these gene family members, here referred to as biofilm architecture factors, in bothA. fumigatusandA. nigeradditionally modulates low oxygen growth and surface adherence. Thus, these genes are candidates for genetic manipulation of biofilm development in Aspergilli.

Aspergillus fumigatus is a filamentous fungus which can cause multiple diseases in humans. Allergic Broncho-pulmonary Aspergillosis (ABPA) is a disease diagnosed primarily in Cystic Fibrosis patients caused by a severe allergic response often to long-term A. fumigatus colonization in the lungs. Mice develop an allergic response to repeated inhalation of A. fumigatus spores; however, no strains have been identified that can survive long-term in the mouse lung and cause ABPA-like disease. We characterized A. fumigatus strain W72310 by whole genome sequencing and in vitro and in vivo viability assays in comparison to a common reference strain, CEA10. W72310 was resistant to leukocyte-mediated killing and persisted in the mouse lung longer than CEA10, a phenotype that correlated with greater resistance to oxidative stressors, hydrogen peroxide and menadione, in vitro. In animals both sensitized and challenged with W72310, conidia, but not hyphae, were viable in the lungs for up to 21 days in association with eosinophilic airway inflammation, airway leakage, serum IgE, and mucus production. W72310-sensitized mice that were recall-challenged with conidia had increased inflammation, Th1 and Th2 cytokines, and airway leakage compared to controls. Collectively, our studies demonstrate that a unique strain of A. fumigatus resistant to leukocyte killing can persist in the mouse lung in conidial form and elicit features of ABPA-like disease.IMPORTANCEAllergic Broncho-pulmonary Aspergillosis (ABPA) patients often present with long-term colonization of Aspergillus fumigatus. Current understanding of ABPA pathogenesis has been complicated by a lack of long-term in vivo fungal persistence models. We have identified a clinical isolate of A. fumigatus, W72310, which persists in the murine lung and causes an ABPA-like disease phenotype. Surprisingly, while viable, W72310 showed little to no growth beyond the conidial stage in the lung. This indicates that it is possible that A. fumigatus can cause allergic disease in the lung without any significant hyphal growth. The identification of this strain of A. fumigatus can not only be used to better understand disease pathogenesis of ABPA and potential anti-fungal treatments, but also to identify features of fungal strains that drive long-term fungal persistence in the lung. Consequently, these observations are a step toward helping resolve the long-standing question when to utilize antifungal therapies in patients with ABPA and fungal allergic type diseases.

To the Editor,Table 1. Summary of direct immunofluorescence findings of 145 conjunctival biopsies submitted to the University of Illinois at Chicago Skin Immunology lab. Cases with multiple linear ...

Pseudomonas aeruginosa strains with loss-of-function mutations in the transcription factor LasR are frequently encountered in the clinic and the environment. Among the characteristics common to LasR-defective (LasR−) strains is increased activity of the transcription factor Anr, relative to their LasR+ counterparts, in low-oxygen conditions. One of the Anr-regulated genes found to be highly induced in LasR− strains was PA14_42860 (PA1673), which we named mhr for microoxic hemerythrin. Purified P. aeruginosa Mhr protein contained the predicted di-iron center and bound molecular oxygen with an apparent K(d) of ∼1 µM. Both Anr and Mhr were necessary for fitness in lasR+ and lasR mutant strains in colony biofilms grown in microoxic conditions, and the effects were more striking in the lasR mutant. Among genes in the Anr regulon, mhr was most closely coregulated with the Anr-controlled high-affinity cytochrome c oxidase genes. In the absence of high-affinity cytochrome c oxidases, deletion of mhr no longer caused a fitness disadvantage, suggesting that Mhr works in concert with microoxic respiration. We demonstrate that Anr and Mhr contribute to LasR− strain fitness even in biofilms grown in normoxic conditions. Furthermore, metabolomics data indicate that, in a lasR mutant, expression of Anr-regulated mhr leads to differences in metabolism in cells grown on lysogeny broth or artificial sputum medium. We propose that increased Anr activity leads to higher levels of the oxygen-binding protein Mhr, which confers an advantage to lasR mutants in microoxic conditions.

Journal of high energy physics 2004(4), 70 (2020). doi:10.1007/JHEP04(2020)070, Two-particle azimuthal correlations have been measured in neutral current deep inelastic ep scattering with virtuality Q$^{2}$> 5 GeV$^{2}$ at a centre-of-mass energy $ \sqrt{s} $ = 318 GeV recorded with the ZEUS detector at HERA. The correlations of charged particles have been measured in the range of laboratory pseudorapidity −1.5 < η < 2.0 and transverse momentum 0.1 < p$_{T}$< 5.0 GeV and event multiplicities N$_{ch}$ up to six times larger than the average 〈N$_{ch}$〉 ≈ 5. The two-particle correlations have been measured in terms of the angular observables c$_{n{2}}$ = 〈〈cosnΔφ〉〉, where n is between 1 and 4 and ∆φ is the relative azimuthal angle between the two particles. Comparisons with available models of deep inelastic scattering, which are tuned to reproduce inclusive particle production, suggest that the measured two-particle correlations are dominated by contributions from multijet production. The correlations observed here do not indicate the kind of collective behaviour recently observed at the highest RHIC and LHC energies in high-multiplicity hadronic collisions., Published by SISSA, [Trieste]

Background: Thousands of genetic variants have been associated with hematological traits, though target genes remain unknown at most loci. Moreover, limited analyses have been conducted in African ancestry and Hispanic/Latino populations; hematological trait associated variants more common in these populations have likely been missed. Methods: To derive gene expression prediction models, we used ancestry-stratified datasets from the Multi-Ethnic Study of Atherosclerosis (MESA, including n = 229 African American and n = 381 Hispanic/Latino participants, monocytes) and the Depression Genes and Networks study (DGN, n = 922 European ancestry participants, whole blood). We then performed a transcriptome-wide association study (TWAS) for platelet count, hemoglobin, hematocrit, and white blood cell count in African (n = 27,955) and Hispanic/Latino (n = 28,324) ancestry participants. Results: Our results revealed 24 suggestive signals (p < 1 × 10−4) that were conditionally distinct from known GWAS identified variants and successfully replicated these signals in European ancestry subjects from UK Biobank. We found modestly improved correlation of predicted and measured gene expression in an independent African American cohort (the Genetic Epidemiology Network of Arteriopathy (GENOA) study (n = 802), lymphoblastoid cell lines) using the larger DGN reference panel; however, some genes were well predicted using MESA but not DGN. Conclusions: These analyses demonstrate the importance of performing TWAS and other genetic analyses across diverse populations and of balancing sample size and ancestry background matching when selecting a TWAS reference panel.

IntroductionCOVID-19 may cause severe pneumonitis and trigger a massive inflammatory response that requires ventilatory support. The intensive care unit (ICU)-mortality has been reported to be as high as 62%. Dexamethasone is the only of all anti-inflammatory drugs that have been tested to date that has shown a positive effect on mortality. We aim to explore if treatment with hyperbaric oxygen (HBO) is safe and effective for patients with severe COVID-19. Our hypothesis is that HBO can prevent ICU admission, morbidity and mortality by attenuating the inflammatory response. The primary objective is to evaluate if HBO reduces the number of ICU admissions compared with best practice treatment for COVID-19, main secondary objectives are to evaluate if HBO reduces the load on ICU resources, morbidity and mortality and to evaluate if HBO mitigates the inflammatory reaction in COVID-19.Methods and analysisA randomised, controlled, phase II, open label, multicentre trial. 200 subjects with severe COVID-19 and at least two risk factors for mortality will be included. Baseline clinical data and blood samples will be collected before randomisation and repeated daily for 7 days, at days 14 and 30. Subjects will be randomised with a computer-based system to HBO, maximum five times during the first 7 days plus best practice treatment or only best practice treatment. The primary endpoint, ICU admission, is defined by criteria for selection for ICU. We will evaluate if HBO mitigates the inflammatory reaction in COVID-19 using molecular analyses. All parameters are recorded in an electronic case report form. An independent Data Safety Monitoring Board will review the safety parameters.Ethics and disseminationThe trial is approved by The National Institutional Review Board in Sweden (2020-01705) and the Swedish Medical Product Agency (5.1-2020-36673). Positive, negative and any inconclusive results will be published in peer-reviewed scientific journals with open access.Trial registrationNCT04327505. EudraCT number: 2020-001349-37.

Pseudomonas aeruginosastrains with loss-of-function mutations in the transcription factor are frequently encountered in the clinic and the environment. Among the characteristics common to LasR-defective (LasR-) strains is increased activity of the transcription factor Anr, relative to their LasR+ counterparts, in low oxygen conditions. One of the Anr-regulated genes that was highly induced in the LasR-strains encoded a putative oxygen-binding hemerythrin encoded byPA14_42860(PA1673) which we namedmhrfor microoxic hemerythrin. PurifiedP. aeruginosaMhr protein contained the predicted di-iron center and binds oxygen with aKdof 1 µM. Both Anr and Mhr were necessary for fitness inlasR+ andlasRmutant strains in colony biofilms grown in microoxic conditions, and the effects were more striking in thelasRmutant. Among genes in the Anr regulon,mhrwas most closely co-regulated with the Anr-controlled high affinity cytochromecoxidase genes and in the absence of high affinity cytochromecoxidase activity, deletion ofmhrno longer caused a fitness disadvantage suggesting that Mhr works in concert with microoxic respiration. We demonstrate that Anr and Mhr contribute to LasR-strain fitness even in the normoxic biofilm conditions, and metabolomics data indicate that in alasRmutant, expression of Anr-regulatedmhrleads to differences in metabolism in cells grown on LB and artificial sputum medium. Together these data indicate that increased Anr activity in microoxically-grownlasRmutants confers an advantage in part for its regulation of the O2binding protein Mhr.SignificancePseudomonas aeruginosa, a versatile bacterium that both lives in environmental habitats and causes life-threatening opportunistic infections, uses quorum sensing to coordinate gene expression with cell density. ThelasRgene, which encodes a quorum sensing regulator, is often deleteriously mutated in clinical isolates. Interestingly, LasR-strains have high activity of the oxygen-sensitive transcription factor Anr in microoxic conditions. This report identifies and characterizes an Anr-regulated microoxic hemerythrin that reversibly binds oxygen. We showed bothanrandmhrare critical to fitness in microoxia, and these genes uniquely benefit LasR-strains in normoxia. Our findings enrich our understanding of the success ofP. aeruginosaas a pulmonary resident through its propensity to lose LasR functionality in the context of low-oxygen infection environments.

Whole-genome sequencing (WGS) can improve assessment of low-frequency and rare variants, particularly in non-European populations that have been underrepresented in existing genomic studies. The genetic determinants of C-reactive protein (CRP), a biomarker of chronic inflammation, have been extensively studied, with existing genome-wide association studies (GWASs) conducted in >200,000 individuals of European ancestry. In order to discover novel loci associated with CRP levels, we examined a multi-ancestry population (n = 23,279) with WGS (∼38× coverage) from the Trans-Omics for Precision Medicine (TOPMed) program. We found evidence for eight distinct associations at the CRP locus, including two variants that have not been identified previously (rs11265259 and rs181704186), both of which are non-coding and more common in individuals of African ancestry (∼10% and ∼1% minor allele frequency, respectively, and rare or monomorphic in 1000 Genomes populations of East Asian, South Asian, and European ancestry). We show that the minor (G) allele of rs181704186 is associated with lower CRP levels and decreased transcriptional activity and protein binding in vitro, providing a plausible molecular mechanism for this African ancestry-specific signal. The individuals homozygous for rs181704186-G have a mean CRP level of 0.23 mg/L, in contrast to individuals heterozygous for rs181704186 with mean CRP of 2.97 mg/L and major allele homozygotes with mean CRP of 4.11 mg/L. This study demonstrates the utility of WGS in multi-ethnic populations to drive discovery of complex trait associations of large effect and to identify functional alleles in noncoding regulatory regions.

Aspergillus fumigatus causes life-threatening infections, and treatment options remain limited. Thus, there is an urgent need to find new therapeutic targets to treat this deadly disease. Previously, we have shown that SREBP transcription factors and their regulatory components are critical for the pathobiology of A. fumigatus. Here we identify a role for RbdB, a rhomboid protease, as an essential component of SREBP activity. Our results indicate that mutants lacking rbdB have growth defects under hypoxic conditions, are hypersusceptible to voriconazole, lack extracellular siderophore production, and fail to cause disease in a murine model of invasive pulmonary aspergillosis. This study increases our understanding of the molecular mechanisms involved in SREBP activation in pathogenic fungi and provides a novel therapeutic target for future development., SREBP transcription factors play a critical role in fungal virulence; however, the mechanisms of sterol regulatory element binding protein (SREBP) activation in pathogenic fungi remains ill-defined. Screening of the Neurospora crassa whole-genome deletion collection for genes involved in hypoxia responses identified a gene for an uncharacterized rhomboid protease homolog, rbdB, required for growth under hypoxic conditions. Loss of rbdB in Aspergillus fumigatus also inhibited growth under hypoxic conditions. In addition, the A. fumigatus ΔrbdB strain also displayed phenotypes consistent with defective SREBP activity, including increased azole drug susceptibility, reduced siderophore production, and full loss of virulence. Expression of the basic helix-loop-helix (bHLH) DNA binding domain of the SREBP SrbA in ΔrbdB restored all of the phenotypes linking RdbB activity with SrbA function. Furthermore, the N-terminal domain of SrbA containing the bHLH DNA binding region was absent from ΔrbdB under inducing conditions, suggesting that RbdB regulates the protein levels of this important transcription factor. As SrbA controls clinically relevant aspects of fungal pathobiology in A. fumigatus, understanding the mechanisms of SrbA activation provides opportunities to target this pathway for therapeutic development. IMPORTANCE Aspergillus fumigatus causes life-threatening infections, and treatment options remain limited. Thus, there is an urgent need to find new therapeutic targets to treat this deadly disease. Previously, we have shown that SREBP transcription factors and their regulatory components are critical for the pathobiology of A. fumigatus. Here we identify a role for RbdB, a rhomboid protease, as an essential component of SREBP activity. Our results indicate that mutants lacking rbdB have growth defects under hypoxic conditions, are hypersusceptible to voriconazole, lack extracellular siderophore production, and fail to cause disease in a murine model of invasive pulmonary aspergillosis. This study increases our understanding of the molecular mechanisms involved in SREBP activation in pathogenic fungi and provides a novel therapeutic target for future development.

We present an electron-pairs-based method employing a generalized valence bond perfect-pairing (GVB-PP) ansatz that provides a uniformly accurate description of systems where various types of electron correlation play a role and the GVB-PP wave function is a suitable reference. In the proposed EERPA-GVB approach, a GVB-PP energy is amended by adding correlation among electron pairs. The latter is achieved by embedding single pairs or couples of pairs in the environment of the other electron fragments and separately accounting for intra- and interfragment correlation effects. For this purpose, we employ truncated extended random phase approximation equations. Application of EERPA-GVB to systems governed by both short-range (energy barriers) and long-range (molecular interactions) correlation effects proves the good accuracy of the method. Moreover, EERPA-GVB is shown to cure a notorious problem of uncorrelated electron-pair models, namely, spatial symmetry breaking in aromatic molecules, using the example of benzene. We have also successfully applied EERPA-GVB to a challenging problem of a phase transition of the boron chain system, where the correlation changes its character along the reaction path. The accuracy and versatility of EERPA-GVB are accompanied by its attractively low computational cost. By truncation of the extended RPA equations and consideration of only at most two-fragment correlation contributions, the cost of computing the EERPA correlation energy is reduced to scale only quadratically with the number of pairs of electrons.

Charm production in charged current deep inelastic scattering has been measured for the first time in e ± p collisions, using data collected with the ZEUS detector at HERA, corresponding to an integrated luminosity of 358 pb−1. Results are presented separately for e + p and e − p scattering at a centre-of-mass energy of $$ \sqrt{s} $$ s = 318 GeV within a kinematic phase-space region of 200 GeV2 < Q 2 < 60000 GeV2 and y < 0.9, where Q 2 is the squared four-momentum transfer and y is the inelasticity. The measured cross sections of electroweak charm production are consistent with expectations from the Standard Model within the large statistical uncertainties.