news V Sunday of Easter. Eucharistic Liturgy, online at 8pm (GMT 2) from the church of Sant'Egidio May 9 2020 - ROME,...

Ambassador Anwarul K. Chowdhury is a former Under-Secretary-General and High Representative of the UN (2002-2007), Permanent Representative of Bangladesh to UN (1996-2001) and Chairman of the UN General Assembly’s Administrative and Budgetary Committee (1997-1998)

The post UN Faces Financial & Liquidity Crisis as Global Pandemic Rages appeared first on Inter Press Service.

Assistance includes returning a portion of students' tuition fees and giving cash for equipment and living expenses.

Chinese ride-hailing giant Didi Chuxing’s core business has been profitable, president Jean Liu Qing said for the first time, as ride orders in its domestic market recover from the impact of the coronavirus pandemic.In a rare interview, Liu told the CNBC that Didi’s core business has been profitable, without providing specific figures or explaining how or when the company had measured its profitability. The company has no plans for job cuts or raising capital, Liu said in the interview that…

LAHORE: Pakistan Muslim League-N Secretary Information Marriyum Aurangzeb has termed the allegations, levelled by National Accountability Bureau Multan, and the notice issued to Shahbaz Sharif...

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ISLAMABAD: The Universal Service Fund awarded contract worth PKR 588 million to Telenor Pakistan for providing hi-speed broadband in Sanghar Lot .The Federal Minister IT& Telecom, Syed Amin ul...

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LAHORE: The Pakistan Medical Association has expressed serious concern over the increasing number of coronavirus patients and rising death toll of Covid-19 patients in the country.“We are very...

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Oprah Winfrey has reportedly arranged for the Duke and Duchess to move into Tyler Perry's mansion

Expressing serious concern over dozens of unexplained deaths of asymptomatic COVID-19 patients in home isolation in Sindh, local and foreign health experts have advised the government to provide tele-consultation to the patients in home isolation on a regular basis.The experts also asked the...

Next-Generation Miniaturized Rubidium Atomic Clock Improves Performance and adds Features without Increasing Size

Life returned to normal Saturday in the Rashidiyeh Palestinian refugee camp, south of Tyre, after a night of clashes that left one person dead and five others injured.

Tata Value Homes Limited (TVHL), a 100% subsidiary of Tata Housing Development Company Limited, today strengthened its presence in South India by developing Spanish themed residential township at Sriperumbudur, near Chennai. The company plans to Invest around Rs 300 crore in setting up this township. Inspired by Spanish living, Santorini is designed by architects F+A. The township is spread across lush greenery at Sriperumbudur, one of the fastest growing corridors in Chennai. Speaking on the project Brotin Banerjee, MD & CEO, Tata Housing Development Co. Ltd., said, with rapid urbanisation and influx of global lifestyle trends more and more affluent home buyers are looking for homes to reflect the financial […]

NEW DELHI: Real estate developers and property consultants today welcomed the RBI decision to cut statutory liquidity ratio (SLR), saying this would enable banks to lend more to industry, including cash-starved realty sector. However, developers demanded easing of interest rates to boost housing sales. RBI today decided to keep the key policy rate unchanged but slashed statutory liquidity ratio (SLR) by 0.5 per cent to unlock about Rs 40,000 crore into the system. “The RBI’s move to cut SLR by 50 basis points to 22 per cent will give banks more headroom to lend and thereby spur lending in future. However, the much anticipated cut in rates would have been […]

Four decades ago, we introduced a standard way of encoding digital sound. Its first ever upgrade could lead to new genres of music and ways of experiencing sound

Four decades ago, we introduced a standard way of encoding digital sound. Its first ever upgrade could lead to new genres of music and ways of experiencing sound

Four decades ago, we introduced a standard way of encoding digital sound. Its first ever upgrade could lead to new genres of music and ways of experiencing sound

In July 2019, a sophisticated backdoor trojan in Google Play was reported. We conducted an inquiry of our own, discovering a long-term campaign, which we dubbed “PhantomLance”, its earliest registered domain dating back to December 2015.

Title: Hypothyroidism
Category: Diseases and Conditions
Created: 12/31/1997 12:00:00 AM
Last Editorial Review: 9/18/2019 12:00:00 AM

Title: Hyperthyroidism
Category: Diseases and Conditions
Created: 12/31/1997 12:00:00 AM
Last Editorial Review: 11/20/2019 12:00:00 AM

Title: Focus on Healthy Foods, Not Avoiding 'Bad' Ones, for Heart Health: Study
Category: Health News
Created: 4/25/2016 12:00:00 AM
Last Editorial Review: 4/25/2016 12:00:00 AM

Title: Pepcid Ingredient Famotidine Being Tested as COVID-19 Treatment
Category: Health News
Created: 4/27/2020 12:00:00 AM
Last Editorial Review: 4/27/2020 12:00:00 AM

Title: Thrush (Oral Candidiasis)
Category: Diseases and Conditions
Created: 1/31/2005 12:00:00 AM
Last Editorial Review: 3/27/2020 12:00:00 AM

Title: Xiaflex (collagenase clostridium histolyticum)
Category: Medications
Created: 3/3/2020 12:00:00 AM
Last Editorial Review: 3/3/2020 12:00:00 AM

Title: High Heat, Humidity Could Affect More Than 1.2 Billion People by End of Century
Category: Health News
Created: 3/24/2020 12:00:00 AM
Last Editorial Review: 3/25/2020 12:00:00 AM

ABSTRACT

Cryptosporidium parvum and Cryptosporidium hominis have emerged as major enteric pathogens of infants in the developing world, in addition to their known importance in immunocompromised adults. Although there has been recent progress in identifying new small molecules that inhibit Cryptosporidium sp. growth in vitro or in animal models, we lack information about their mechanism of action, potency across the life cycle, and cidal versus static activities. Here, we explored four potent classes of compounds that include inhibitors that likely target phosphatidylinositol 4 kinase (PI4K), phenylalanine-tRNA synthetase (PheRS), and several potent inhibitors with unknown mechanisms of action. We utilized monoclonal antibodies and gene expression probes for staging life cycle development to define the timing of when inhibitors were active during the life cycle of Cryptosporidium parvum grown in vitro. These different classes of inhibitors targeted different stages of the life cycle, including compounds that blocked replication (PheRS inhibitors), prevented the segmentation of daughter cells and thus blocked egress (PI4K inhibitors), or affected sexual-stage development (a piperazine compound of unknown mechanism). Long-term cultivation of C. parvum in epithelial cell monolayers derived from intestinal stem cells was used to distinguish between cidal and static activities based on the ability of parasites to recover from treatment. Collectively, these approaches should aid in identifying mechanisms of action and for designing in vivo efficacy studies based on time-dependent concentrations needed to achieve cidal activity.

IMPORTANCE Currently, nitazoxanide is the only FDA-approved treatment for cryptosporidiosis; unfortunately, it is ineffective in immunocompromised patients, has varied efficacy in immunocompetent individuals, and is not approved in infants under 1 year of age. Identifying new inhibitors for the treatment of cryptosporidiosis requires standardized and quantifiable in vitro assays for assessing potency, selectivity, timing of activity, and reversibility. Here, we provide new protocols for defining which stages of the life cycle are susceptible to four highly active compound classes that likely inhibit different targets in the parasite. We also utilize a newly developed long-term culture system to define assays for monitoring reversibility as a means of defining cidal activity as a function of concentration and time of treatment. These assays should provide valuable in vitro parameters to establish conditions for efficacious in vivo treatment.

ABSTRACT

Frequent and excessive use of antibiotics primes patients to Clostridioides difficile infection (CDI), which leads to fatal pseudomembranous colitis, with limited treatment options. In earlier reports, we used a drug repurposing strategy and identified amoxapine (an antidepressant), doxapram (a breathing stimulant), and trifluoperazine (an antipsychotic), which provided significant protection to mice against lethal infections with several pathogens, including C. difficile. However, the mechanisms of action of these drugs were not known. Here, we provide evidence that all three drugs offered protection against experimental CDI by reducing bacterial burden and toxin levels, although the drugs were neither bacteriostatic nor bactericidal in nature and had minimal impact on the composition of the microbiota. Drug-mediated protection was dependent on the presence of the microbiota, implicating its role in evoking host defenses that promoted protective immunity. By utilizing transcriptome sequencing (RNA-seq), we identified that each drug increased expression of several innate immune response-related genes, including those involved in the recruitment of neutrophils, the production of interleukin 33 (IL-33), and the IL-22 signaling pathway. The RNA-seq data on selected genes were confirmed by quantitative real-time PCR (qRT-PCR) and protein assays. Focusing on amoxapine, which had the best anti-CDI outcome, we demonstrated that neutralization of IL-33 or depletion of neutrophils resulted in loss of drug efficacy. Overall, our lead drugs promote disease alleviation and survival in the murine model through activation of IL-33 and by clearing the pathogen through host defense mechanisms that critically include an early influx of neutrophils.

IMPORTANCE Clostridioides difficile is a spore-forming anaerobic bacterium and the leading cause of antibiotic-associated colitis. With few therapeutic options and high rates of disease recurrence, the need to develop new treatment options is urgent. Prior studies utilizing a repurposing approach identified three nonantibiotic Food and Drug Administration-approved drugs, amoxapine, doxapram, and trifluoperazine, with efficacy against a broad range of human pathogens; however, the protective mechanisms remained unknown. Here, we identified mechanisms leading to drug efficacy in a murine model of lethal C. difficile infection (CDI), advancing our understanding of the role of these drugs in infectious disease pathogenesis that center on host immune responses to C. difficile. Overall, these studies highlight the crucial involvement of innate immune responses, as well as the importance of immunomodulation as a potential therapeutic option to combat CDI.

ABSTRACT

Clostridioides difficile is an important nosocomial pathogen that causes approximately 500,000 cases of C. difficile infection (CDI) and 29,000 deaths annually in the United States. Antibiotic use is a major risk factor for CDI because broad-spectrum antimicrobials disrupt the indigenous gut microbiota, decreasing colonization resistance against C. difficile. Vancomycin is the standard of care for the treatment of CDI, likely contributing to the high recurrence rates due to the continued disruption of the gut microbiota. Thus, there is an urgent need for the development of novel therapeutics that can prevent and treat CDI and precisely target the pathogen without disrupting the gut microbiota. Here, we show that the endogenous type I-B CRISPR-Cas system in C. difficile can be repurposed as an antimicrobial agent by the expression of a self-targeting CRISPR that redirects endogenous CRISPR-Cas3 activity against the bacterial chromosome. We demonstrate that a recombinant bacteriophage expressing bacterial genome-targeting CRISPR RNAs is significantly more effective than its wild-type parent bacteriophage at killing C. difficile both in vitro and in a mouse model of CDI. We also report that conversion of the phage from temperate to obligately lytic is feasible and contributes to the therapeutic suitability of intrinsic C. difficile phages, despite the specific challenges encountered in the disease phenotypes of phage-treated animals. Our findings suggest that phage-delivered programmable CRISPR therapeutics have the potential to leverage the specificity and apparent safety of phage therapies and improve their potency and reliability for eradicating specific bacterial species within complex communities, offering a novel mechanism to treat pathogenic and/or multidrug-resistant organisms.

IMPORTANCE Clostridioides difficile is a bacterial pathogen responsible for significant morbidity and mortality across the globe. Current therapies based on broad-spectrum antibiotics have some clinical success, but approximately 30% of patients have relapses, presumably due to the continued perturbation to the gut microbiota. Here, we show that phages can be engineered with type I CRISPR-Cas systems and modified to reduce lysogeny and to enable the specific and efficient targeting and killing of C. difficile in vitro and in vivo. Additional genetic engineering to disrupt phage modulation of toxin expression by lysogeny or other mechanisms would be required to advance a CRISPR-enhanced phage antimicrobial for C. difficile toward clinical application. These findings provide evidence into how phage can be combined with CRISPR-based targeting to develop novel therapies and modulate microbiomes associated with health and disease.

ABSTRACT

The recent discovery of complete ammonia oxidizers (comammox) contradicts the paradigm that chemolithoautotrophic nitrification is always catalyzed by two different microorganisms. However, our knowledge of the survival strategies of comammox in complex ecosystems, such as full-scale wastewater treatment plants (WWTPs), remains limited. Analyses of genomes and in situ transcriptomes of four comammox organisms from two full-scale WWTPs revealed that comammox were active and showed a surprisingly high metabolic versatility. A gene cluster for the utilization of urea and a gene encoding cyanase suggest that comammox may use diverse organic nitrogen compounds in addition to free ammonia as the substrates. The comammox organisms also encoded the genomic potential for multiple alternative energy metabolisms, including respiration with hydrogen, formate, and sulfite as electron donors. Pathways for the biosynthesis and degradation of polyphosphate, glycogen, and polyhydroxyalkanoates as intracellular storage compounds likely help comammox survive unfavorable conditions and facilitate switches between lifestyles in fluctuating environments. One of the comammox strains acquired from the anaerobic tank encoded and transcribed genes involved in homoacetate fermentation or in the utilization of exogenous acetate, both pathways being unexpected in a nitrifying bacterium. Surprisingly, this strain also encoded a respiratory nitrate reductase which has not yet been found in any other Nitrospira genome and might confer a selective advantage to this strain over other Nitrospira strains in anoxic conditions.

IMPORTANCE The discovery of comammox in the genus Nitrospira changes our perception of nitrification. However, genomes of comammox organisms have not been acquired from full-scale WWTPs, and very little is known about their survival strategies and potential metabolisms in complex wastewater treatment systems. Here, four comammox metagenome-assembled genomes and metatranscriptomic data sets were retrieved from two full-scale WWTPs. Their impressive and—among nitrifiers—unsurpassed ecophysiological versatility could make comammox Nitrospira an interesting target for optimizing nitrification in current and future bioreactor configurations.

ABSTRACT

Patients with COVID-19 infection are at risk of acute respiratory disease syndrome (ARDS) and death. The tissue receptor for COVID-19 is ACE2, and higher levels of ACE2 can protect against ARDS. Angiotensin receptor blockers and statins upregulate ACE2. Clinical trials are needed to determine whether this drug combination might be used to treat patients with severe COVID-19 infection.

ABSTRACT

Clostridium saccharoperbutylacetonicum is a mesophilic, anaerobic, butanol-producing bacterium, originally isolated from soil. It was recently reported that C. saccharoperbutylacetonicum possesses multiple cellulosomal elements and would potentially form the smallest cellulosome known in nature. Its genome contains only eight dockerin-bearing enzymes, and its unique scaffoldin bears two cohesins (Cohs), three X2 modules, and two carbohydrate-binding modules (CBMs). In this study, all of the cellulosome-related modules were cloned, expressed, and purified. The recombinant cohesins, dockerins, and CBMs were tested for binding activity using enzyme-linked immunosorbent assay (ELISA)-based techniques. All the enzymes were tested for their comparative enzymatic activity on seven different cellulosic and hemicellulosic substrates, thus revealing four cellulases, a xylanase, a mannanase, a xyloglucanase, and a lichenase. All dockerin-containing enzymes interacted similarly with the second cohesin (Coh2) module, whereas Coh1 was more restricted in its interaction pattern. In addition, the polysaccharide-binding properties of the CBMs within the scaffoldin were examined by two complementary assays, affinity electrophoresis and affinity pulldown. The scaffoldin of C. saccharoperbutylacetonicum exhibited high affinity for cellulosic and hemicellulosic substrates, specifically to microcrystalline cellulose and xyloglucan. Evidence that supports substrate-dependent in vivo secretion of cellulosomes is presented. The results of our analyses contribute to a better understanding of simple cellulosome systems by identifying the key players in this minimalistic system and the binding pattern of its cohesin-dockerin interaction. The knowledge gained by our study will assist further exploration of similar minimalistic cellulosomes and will contribute to the significance of specific sets of defined cellulosomal enzymes in the degradation of cellulosic biomass.

IMPORTANCE Cellulosome-producing bacteria are considered among the most important bacteria in both mesophilic and thermophilic environments, owing to their capacity to deconstruct recalcitrant plant-derived polysaccharides (and notably cellulose) into soluble saccharides for subsequent processing. In many ecosystems, the cellulosome-producing bacteria are particularly effective "first responders." The massive amounts of sugars produced are potentially amenable in industrial settings to further fermentation by appropriate microbes to biofuels, notably ethanol and butanol. Among the solvent-producing bacteria, Clostridium saccharoperbutylacetonicum has the smallest cellulosome system known thus far. The importance of investigating the building blocks of such a small, multifunctional nanomachine is crucial to understanding the fundamental activities of this efficient enzymatic complex.

ABSTRACT

Hepatitis C virus (HCV) infects millions of people worldwide, causing chronic liver disease that can lead to cirrhosis, hepatocellular carcinoma, and liver transplant. In the last several years, the advent of direct-acting antivirals, including NS3/4A protease inhibitors (PIs), has remarkably improved treatment outcomes of HCV-infected patients. However, selection of resistance-associated substitutions and polymorphisms among genotypes can lead to drug resistance and in some cases treatment failure. A proactive strategy to combat resistance is to constrain PIs within evolutionarily conserved regions in the protease active site. Designing PIs using the substrate envelope is a rational strategy to decrease the susceptibility to resistance by using the constraints of substrate recognition. We successfully designed two series of HCV NS3/4A PIs to leverage unexploited areas in the substrate envelope to improve potency, specifically against resistance-associated substitutions at D168. Our design strategy achieved better resistance profiles over both the FDA-approved NS3/4A PI grazoprevir and the parent compound against the clinically relevant D168A substitution. Crystallographic structural analysis and inhibition assays confirmed that optimally filling the substrate envelope is critical to improve inhibitor potency while avoiding resistance. Specifically, inhibitors that enhanced hydrophobic packing in the S4 pocket and avoided an energetically frustrated pocket performed the best. Thus, the HCV substrate envelope proved to be a powerful tool to design robust PIs, offering a strategy that can be translated to other targets for rational design of inhibitors with improved potency and resistance profiles.

IMPORTANCE Despite significant progress, hepatitis C virus (HCV) continues to be a major health problem with millions of people infected worldwide and thousands dying annually due to resulting complications. Recent antiviral combinations can achieve >95% cure, but late diagnosis, low access to treatment, and treatment failure due to drug resistance continue to be roadblocks against eradication of the virus. We report the rational design of two series of HCV NS3/4A protease inhibitors with improved resistance profiles by exploiting evolutionarily constrained regions of the active site using the substrate envelope model. Optimally filling the S4 pocket is critical to avoid resistance and improve potency. Our results provide drug design strategies to avoid resistance that are applicable to other quickly evolving viral drug targets.

ABSTRACT

The Escherichia coli microcin C (McC) and related compounds are potent Trojan horse peptide-nucleotide antibiotics. The peptide part facilitates transport into sensitive cells. Inside the cell, the peptide part is degraded by nonspecific peptidases releasing an aspartamide-adenylate containing a phosphoramide bond. This nonhydrolyzable compound inhibits aspartyl-tRNA synthetase. In addition to the efficient export of McC outside the producing cells, special mechanisms have evolved to avoid self-toxicity caused by the degradation of the peptide part inside the producers. Here, we report that histidine-triad (HIT) hydrolases encoded in biosynthetic clusters of some McC homologs or by standalone genes confer resistance to McC-like compounds by hydrolyzing the phosphoramide bond in toxic aspartamide-adenosine, rendering them inactive.

IMPORTANCE Uncovering the mechanisms of resistance is a required step for countering the looming antibiotic resistance crisis. In this communication, we show how universally conserved histidine-triad hydrolases provide resistance to microcin C, a potent inhibitor of bacterial protein synthesis.

ABSTRACT

Selectable markers are indispensable for genetic engineering, yet their number and variety are limited. Most selection procedures for prototrophic cells rely on the introduction of antibiotic resistance genes. New minimally invasive tools are needed to facilitate sophisticated genetic manipulations. Here, we characterized three endogenous genes in the human fungal pathogen Aspergillus fumigatus for their potential as markers for targeted genomic insertions of DNAs of interest (DOIs). Since these genes are involved in uptake and metabolization of pyrimidines, resistance to the toxic effects of prodrugs 5-fluorocytosine and 5-fluorouracil can be used to select successfully integrated DOIs. We show that DOI integration, resulting in the inactivation of these genes, caused no adverse effects with respect to nutrient requirements, stress resistance, or virulence. Beside the individual use of markers for site-directed integration of reporter cassettes, including the 17-kb penicillin biosynthetic cluster, we demonstrate their sequential use by inserting three genes encoding fluorescent proteins into a single strain for simultaneous multicolor localization microscopy. In addition to A. fumigatus, we validated the applicability of this novel toolbox in Penicillium chrysogenum and Fusarium oxysporum. Enabling multiple targeted insertions of DOIs without the necessity for exogenous markers, this technology has the potential to significantly advance genetic engineering.

IMPORTANCE This work reports the discovery of a novel genetic toolbox comprising multiple, endogenous selectable markers for targeted genomic insertions of DNAs of interest (DOIs). Marker genes encode proteins involved in 5-fluorocytosine uptake and pyrimidine salvage activities mediating 5-fluorocytosine deamination as well as 5-fluorouracil phosphoribosylation. The requirement for their genomic replacement by DOIs to confer 5-fluorocytosine or 5-fluorouracil resistance for transformation selection enforces site-specific integrations. Due to the fact that the described markers are endogenously encoded, there is no necessity for the exogenous introduction of commonly employed markers such as auxotrophy-complementing genes or antibiotic resistance cassettes. Importantly, inactivation of the described marker genes had no adverse effects on nutrient requirements, growth, or virulence of the human pathogen Aspergillus fumigatus. Given the limited number and distinct types of selectable markers available for the genetic manipulation of prototrophic strains such as wild-type strains, we anticipate that the proposed methodology will significantly advance genetic as well as metabolic engineering of fungal species.

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

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.

Legionella pneumophila, the etiological agent of Legionnaires’ disease, employs an arsenal of hundreds of Dot/Icm-translocated effector proteins to facilitate replication within eukaryotic phagocytes. Several effectors, called metaeffectors, function to regulate the activity of other Dot/Icm-translocated effectors during infection. The metaeffector Lpg2505 is essential for L. pneumophila intracellular replication only when its cognate effector, SidI, is present. SidI is a cytotoxic effector that interacts with the host translation factor eEF1A and potently inhibits eukaryotic protein translation by an unknown mechanism. Here, we evaluated the impact of Lpg2505 on SidI-mediated phenotypes and investigated the mechanism of SidI function. We determined that Lpg2505 binds with nanomolar affinity to SidI and suppresses SidI-mediated inhibition of protein translation. SidI binding to eEF1A and Lpg2505 is not mutually exclusive, and the proteins bind distinct regions of SidI. We also discovered that SidI possesses GDP-dependent glycosyl hydrolase activity and that this activity is regulated by Lpg2505. We have therefore renamed Lpg2505 MesI (metaeffector of SidI). This work reveals novel enzymatic activity for SidI and provides insight into how intracellular replication of L. pneumophila is regulated by a metaeffector.

Vitamin A has diverse biological functions and is essential for human survival at every point from embryogenesis to adulthood. Vitamin A and its derivatives have been used to treat human diseases including vision diseases, skin diseases, and cancer. Both insufficient and excessive vitamin A uptake are detrimental, but how its...

ABSTRACT

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

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

Meike Stumpp, Inga Petersen, Femke Thoben, Jia-Jiun Yan, Matthias Leippe, and Marian Y. Hu

Larval stages of the abulacraria superphylum including echinoderms and hemichordates have highly alkaline midguts. To date the reason for the evolution of such extreme pH conditions in the gut of these organisms remains unknown. Here, we test the hypothesis that analogous to the acidic stomachs of vertebrates, these alkaline conditions may represent a first defensive barrier to protect from environmental pathogens.

pH-optimum curves for five different species of marine bacteria demonstrated a rapid decrease in proliferation rates by 50-60% between pH 8.5 and 9.5. Using the marine bacterium Vibrio diazotrophicus which elicits a coordinated immune response in the sea urchin larva of Strongylocentrotus purpuratus, we studied the physiological responses of the midgut pH regulatory machinery to this pathogen. Gastroscopic microelectrode measurements demonstrate a stimulation of midgut alkalization upon infection with V. diazotrophicus accompanied by an upregulation of acid-base transporter transcripts of the midgut. Pharmacological inhibition of midgut alkalization resulted in an increased mortality rate of larvae during Vibrio infection. Reductions in seawater pH resembling ocean acidification (OA) conditions lead to moderate reductions in midgut alkalization. However, these reductions in midgut pH do not affect the immune response and resilience of sea urchin larvae to a Vibrio infection under OA conditions.

Our study addressed the evolutionary benefits of the alkaline midgut of ambulacraria larval stages. The data indicate that alkaline conditions in the gut may serve as a first defensive barrier against environmental pathogens and that this mechanism can compensate for changes in seawater pH.

Flemming Dahlke, Magnus Lucassen, Ulf Bickmeyer, Sylke Wohlrab, Velmurugu Puvanendran, Atle Mortensen, Melissa Chierici, Hans-Otto Pörtner, and Daniela Storch

The vulnerability of fish embryos and larvae to environmental factors is often attributed to a lack of adult-like organ systems (gills) and thus insufficient homeostatic capacity. However, experimental data supporting this hypothesis are scarce. Here, by using Atlantic cod (Gadus morhua) as a model, the relationship between embryo vulnerability (to projected ocean acidification and warming) and homeostatic capacity was explored through parallel analyses of stage-specific mortality and in vitro activity and expression of major ion pumps (ATP-Synthase, Na⁺/K⁺-ATPase, H⁺-ATPase) and co-transporters (NBC1, NKCC1). Immunolocalization of these transporters was used to study ionocyte morphology in newly-hatched larvae. Treatment-related embryo mortality until hatch (+20% due to acidification and warming) occurred primarily during an early period (gastrulation) characterized by extremely low ion transport capacities. Thereafter, embryo mortality decreased in parallel with an exponential increase in activity and expression of all investigated ion transporters. Significant changes in transporter activity and expression in response to acidification (+15% activity) and warming (-30% expression) indicate some potential for short-term acclimatization, although likely associated with energetic trade-offs. Interestingly, whole-larvae enzyme capacities (supported by abundant epidermal ionocytes) reached levels similar to those previously measured in gill tissue of adult cod, suggesting that early-life stages without functional gills are better equipped in terms of ion homeostasis than previously thought. This study implies that the gastrulation period represents a critical transition from inherited (maternal) defenses to active homeostatic regulation, which facilitates enhanced resilience of later stages to environmental factors.

Endogenous viral elements (EVEs) are found in many eukaryotic genomes. Despite considerable knowledge about genomic elements such as transposons (TEs) and retroviruses, we still lack information about nonretroviral EVEs. Aedes aegypti mosquitoes have a highly repetitive genome that is covered with EVEs. Here, we identified 129 nonretroviral EVEs in the AaegL5 version of the A. aegypti genome. These EVEs were significantly associated with TEs and preferentially located in repeat-rich clusters within intergenic regions. Genome-wide transcriptome analysis showed that most EVEs generated transcripts although only around 1.4% were sense RNAs. The majority of EVE transcription was antisense and correlated with the generation of EVE-derived small RNAs. A single genomic cluster of EVEs located in a 143 kb repetitive region in chromosome 2 contributed with 42% of antisense transcription and 45% of small RNAs derived from viral elements. This region was enriched for TE-EVE hybrids organized in the same coding strand. These generated a single long antisense transcript that correlated with the generation of phased primary PIWI-interacting RNAs (piRNAs). The putative promoter of this region had a conserved binding site for the transcription factor Cubitus interruptus, a key regulator of the flamenco locus in Drosophila melanogaster. Here, we have identified a single unidirectional piRNA cluster in the A. aegypti genome that is the major source of EVE transcription fueling the generation of antisense small RNAs in mosquitoes. We propose that this region is a flamenco-like locus in A. aegypti due to its relatedness to the major unidirectional piRNA cluster in Drosophila melanogaster.

BACKGROUND:High-flow nasal cannula (HFNC) therapy may reduce the re-intubation rate compared with conventional oxygen therapy. However, HFNC has not been sufficiently compared with conventional oxygen therapy with a heated humidifier, even though heated humidification is beneficial for facilitating airway clearance.METHODS:This study was a single-center, open-label, randomized controlled trial. We randomized subjects with respiratory failure after extubation to either HFNC group or to a large-volume humidified nebulization-based nebulizer. The primary end point was the re-intubation rate within 7 d after extubation.RESULTS:We could not recruit enough subjects for the sample size we designed, therefore, we analyzed 69 subjects (HFNC group, 30 subjects; nebulizer group, 39 subjects). The re-intubation rate within 7 d was not significantly different between the HFNC and nebulizer groups (5/30 subjects [17%] and 6/39 subjects [15%], respectively; P > .99). PaO2/set FIO2 at 24 h after extubation was also not significantly different between the respective groups (264 ± 105 mm Hg in the HFNC group vs 224 ± 53 mm Hg in the nebulizer group; P = .07).CONCLUSIONS:Compared with a large-volume nebulization-based humidifier, HFNC may not reduce the re-intubation rate within 7 d. However, because of insufficient statistical power, further studies are needed to reach a conclusion.

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

Routine identification of fungal pathogens from positive blood cultures by culture-based methods can be time-consuming, delaying treatment with appropriate antifungal agents. The GenMark Dx ePlex investigational use only blood culture identification fungal pathogen panel (BCID-FP) rapidly detects 15 fungal targets simultaneously in blood culture samples positive for fungi by Gram staining. We aimed to determine the performance of the BCID-FP in a multicenter clinical study. Blood culture samples collected at 10 United States sites and tested with BCID-FP at 4 sites were compared to the standard-of-care microbiological and biochemical techniques, fluorescence in situ hybridization using peptide nucleic acid probes (PNA-FISH) and matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS). Discrepant results were analyzed by bi-directional PCR/sequencing of residual blood culture samples. A total of 866 clinical samples, 120 retrospectively and 21 prospectively collected, along with 725 contrived samples were evaluated. Sensitivity and specificity of detection of Candida species (C. albicans, C. auris, C. dubliniensis, C. famata, C. glabrata, C. guilliermondii, C. kefyr, C. krusei, C. lusitaniae, C. parapsilosis, and C. tropicalis) ranged from 97.1 to 100% and 99.8 to 100%, respectively. For the other organism targets, sensitivity and specificity were as follows: 100% each for Cryptococcus neoformans and C. gattii, 98.6% and 100% for Fusarium spp., and 96.2% and 99.9% for Rhodotorula spp., respectively. In 4 of the 141 clinical samples, the BCID-FP panel correctly identified an additional Candida species, undetected by standard-of-care methods. The BCID-FP panel offers a faster turnaround time for identification of fungal pathogens in positive blood cultures that may allow for earlier antifungal interventions and includes C. auris, a highly multidrug-resistant fungus.

Clostridioides difficile infection (CDI) is one of the most common health care-associated infections that can cause significant morbidity and mortality. CDI diagnosis involves laboratory testing in conjunction with clinical assessment. The objective of this study was to assess the performance of various C. difficile tests and to compare clinical characteristics, Xpert C. difficile/Epi (PCR) cycle threshold (C_T), and Singulex Clarity C. diff toxins A/B (Clarity) concentrations between groups with discordant test results. Unformed stool specimens from 200 hospitalized adults (100 PCR positive and 100 negative) were tested by cell cytotoxicity neutralization assay (CCNA), C. diff Quik Chek Complete (Quik Chek), Premier Toxins A and B, and Clarity. Clinical data, including CDI severity and CDI risk factors, were compared between discordant test results. Compared to CCNA, PCR had the highest sensitivity at 100% and Quik Chek had the highest specificity at 100%. Among clinical and laboratory data studied, prevalences of leukocytosis, prior antibiotic use, and hospitalizations were consistently higher across all subgroups in comparisons of toxin-positive to toxin-negative patients. Among PCR-positive samples, the median C_T was lower in toxin-positive samples than in toxin-negative samples; however, C_T ranges overlapped. Among Clarity-positive samples, the quantitative toxin concentration was significantly higher in toxin-positive samples than in toxin-negative samples as determined by CCNA and Quik Chek Toxin A and B. Laboratory tests for CDI vary in sensitivity and specificity. The quantitative toxin concentration may offer value in guiding CDI diagnosis and treatment. The presence of leukocytosis, prior antibiotic use, and previous hospitalizations may assist with CDI diagnosis, while other clinical parameters may not be consistently reliable.

Glucagon-like peptide-2 (GLP-2) agonists have therapeutic potential in clinical indications in which the integrity or absorptive function of the intestinal mucosa is compromised, such as in short bowel syndrome (SBS). Native hGLP-2, a 33–amino acid peptide secreted from the small intestine, contributes to nutritional absorption but has a very short half-life because of enzymatic cleavage and renal clearance and thus is of limited therapeutic value. The GLP-2 analog teduglutide (Revestive/Gattex; Shire Inc.) has been approved for use in SBS since 2012 but has a once-daily injection regimen. Pharmacokinetic (PK) and pharmacodynamic studies confirm that apraglutide, a novel GLP-2 analog, has very low clearance, long elimination half-life, and high plasma protein binding compared with GLP-2 analogs teduglutide and glepaglutide. Apraglutide and teduglutide retain potency and selectivity at the GLP-2 receptor comparable to native hGLP-2, whereas glepaglutide was less potent and less selective. In rat intravenous PK studies, hGLP-2, teduglutide, glepaglutide, and apraglutide had clearances of 25, 9.9, 2.8, and 0.27 ml/kg per minute, respectively, and elimination half-lives of 6.4, 19, 16, and 159 minutes, respectively. The unique PK profile of apraglutide administered via intravenous and subcutaneous routes was confirmed in monkey and minipig and translated into significantly greater in vivo pharmacodynamic activity, measured as small intestinal growth in rats. Apraglutide showed greater intestinotrophic activity than the other peptides when administered at less-frequent dosing intervals because of its prolonged half-life. We postulate that apraglutide offers several advantages over existing GLP-2 analogs and is an excellent candidate for the treatment of gastrointestinal diseases, such as SBS.

A multidisciplinary endocrinologist-led shared medical appointment (SMA) model showed statistically significant reductions in A1C from baseline over 3 years that were not significantly different from appointments with endocrinologists or primary care providers alone within a resource-poor population. Similarly, the SMA model achieved clinical outcomes on par with endocrinologist-only visits with the added benefit of improving endocrine provider productivity and specialty access for patients. Greater patient engagement with the SMA model was associated with significantly lower A1C.

ABSTRACT

Bezlotoxumab is a human monoclonal antibody against Clostridium difficile toxin B, indicated to prevent recurrence of C. difficile infection (rCDI) in high-risk adults receiving antibacterial treatment for CDI. An exploratory genome-wide association study investigated whether human genetic variation influences bezlotoxumab response. DNA from 704 participants who achieved initial clinical cure in the phase 3 MODIFY I/II trials was genotyped. Single nucleotide polymorphisms (SNPs) and human leukocyte antigen (HLA) imputation were performed using IMPUTE2 and HIBAG, respectively. A joint test of genotype and genotype-by-treatment interaction in a logistic regression model was used to screen genetic variants associated with response to bezlotoxumab. The SNP rs2516513 and the HLA alleles HLA-DRB1*07:01 and HLA-DQA1*02:01, located in the extended major histocompatibility complex on chromosome 6, were associated with the reduction of rCDI in bezlotoxumab-treated participants. Carriage of a minor allele (homozygous or heterozygous) at any of the identified loci was related to a larger difference in the proportion of participants experiencing rCDI versus placebo; the effect was most prominent in the subgroup at high baseline risk for rCDI. Genotypes associated with an improved bezlotoxumab response showed no association with rCDI in the placebo cohort. These data suggest that a host-driven, immunological mechanism may impact bezlotoxumab response. Trial registration numbers are as follows: NCT01241552 (MODIFY I) and NCT01513239 (MODIFY II).

IMPORTANCE Clostridium difficile infection is associated with significant clinical morbidity and mortality; antibacterial treatments are effective, but recurrence of C. difficile infection is common. In this genome-wide association study, we explored whether host genetic variability affected treatment responses to bezlotoxumab, a human monoclonal antibody that binds C. difficile toxin B and is indicated for the prevention of recurrent C. difficile infection. Using data from the MODIFY I/II phase 3 clinical trials, we identified three genetic variants associated with reduced rates of C. difficile infection recurrence in bezlotoxumab-treated participants. The effects were most pronounced in participants at high risk of C. difficile infection recurrence. All three variants are located in the extended major histocompatibility complex on chromosome 6, suggesting the involvement of a host-driven immunological mechanism in the prevention of C. difficile infection recurrence.