Rescue of alveolar wall liquid secretion blocks fatal lung injury due to influenza-staphylococcal coinfection.

Secondary lung infection by inhaled Staphylococcus aureus (SA) is a common and lethal event for individuals infected with influenza A virus (IAV). How IAV disrupts host defense to promote SA infection in lung alveoli, where fatal lung injury occurs, is not known. We addressed this issue using real-time determinations of alveolar responses to IAV in live, intact, perfused lungs. Our findings show that IAV infection blocked defensive alveolar wall liquid (AWL) secretion and induced airspace liquid absorption, thereby reversing normal alveolar liquid dynamics and inhibiting alveolar clearance of inhaled SA. Loss of AWL secretion resulted from inhibition of the cystic fibrosis transmembrane conductance regulator (CFTR) ion channel in the alveolar epithelium, and airspace liquid absorption was caused by stimulation of the alveolar epithelial Na+ channel (ENaC). Loss of AWL secretion promoted alveolar stabilization of inhaled SA, but rescue of AWL secretion protected against alveolar SA stabilization and fatal SA-induced lung injury in IAV-infected mice. These findings reveal a central role for AWL secretion in alveolar defense against inhaled SA and identify AWL inhibition as a critical mechanism of IAV lung pathogenesis. AWL rescue may represent a new therapeutic approach for IAV-SA coinfection.

Implementation of a herpes zoster immunization program in an outpatient hospital-based pharmacy.

BACKGROUND: Administration of the recombinant zoster vaccine (RZV) in an ambulatory clinic visit is not covered by Medicare, and the pharmacy at Tufts Medical Center (TMC) had not previously offered immunizations, leaving clinicians to instruct their patients to receive RZV at outside pharmacies without close follow-up. OBJECTIVE: Improve immunization access by standardizing administration of RZV for eligible patients at TMC. PRACTICE DESCRIPTION: TMC is an academic medical center that serves a vast patient population. The adult primary care clinic offers services for approximately 40,000 patients. Atrium 3 Pharmacy is a 340B hospital-based outpatient pharmacy located in TMC. PRACTICE INNOVATION: Patient referrals for RZV administration were sent from the adult primary care clinic to a designated certified pharmacy technician (CPhT) working within the outpatient pharmacy. The CPhT contacted patients for cost disclosure and scheduling vaccination appointments. EVALUATION METHODS: After the CPhT processed the referrals and scheduled the patient, they were then immunized at the herpes zoster clinic by a pharmacist and asked to complete a survey to assess their experience. RESULTS: Over a 3-month pilot period from January 1, 2022, to March 15, 2022, there were 151 patient referrals placed from the adult primary care clinic. A total of 50 patients received their first RZV dose and 10 patients received their second RZV dose for an overall total of 60 RZV doses. Survey results showed that 96% of patients answered that their overall satisfaction improved, with 57% of patients selecting that they strongly agree and 39% of patients selecting that they agree. CONCLUSION: An outpatient pharmacy-based herpes zoster vaccine immunization program was successfully established. Most patients found the expansion of services in the outpatient pharmacy satisfactory.

Discovery and Structure-Based Design of Potent Covalent PPARγ Inverse-Agonists BAY-4931 and BAY-0069.

The ligand-activated nuclear receptor peroxisome-proliferator-activated receptor-γ (PPARG or PPARγ) represents a potential target for a new generation of cancer therapeutics, especially in muscle-invasive luminal bladder cancer where PPARγ is a critical lineage driver. Here we disclose the discovery of a series of chloro-nitro-arene covalent inverse-agonists of PPARγ that exploit a benzoxazole core to improve interactions with corepressors NCOR1 and NCOR2. In vitro treatment of sensitive cell lines with these compounds results in the robust regulation of PPARγ target genes and antiproliferative effects. Despite their imperfect physicochemical properties, the compounds showed modest pharmacodynamic target regulation in vivo. Improvements to the in vitro potency and efficacy of BAY-4931 and BAY-0069 compared to those of previously described PPARγ inverse-agonists show that these compounds are novel tools for probing the in vitro biology of PPARγ inverse-agonism.

Improving Recruitment for a Newborn Screening Pilot Study with Adaptations in Response to the COVID-19 Pandemic.

Seven months after the launch of a pilot study to screen newborns for Duchenne Muscular Dystrophy (DMD) in New York State, New York City became an epicenter of the coronavirus disease 2019 (COVID-19) pandemic. All in-person research activities were suspended at the study enrollment institutions of Northwell Health and NewYork-Presbyterian Hospitals, and study recruitment was transitioned to 100% remote. Pre-pandemic, all recruitment was in-person with research staff visiting the postpartum patients 1-2 days after delivery to obtain consent. With the onset of pandemic, the multilingual research staff shifted to calling new mothers while they were in the hospital or shortly after discharge, and consent was collected via emailed e-consent links. With return of study staff to the hospitals, a hybrid approach was implemented with in-person recruitment for babies delivered during the weekdays and remote recruitment for babies delivered on weekends and holidays, a cohort not recruited pre-pandemic. There was a drop in the proportion of eligible babies enrolled with the transition to fully remote recruitment from 64% to 38%. In addition, the proportion of babies enrolled after being approached dropped from 91% to 55%. With hybrid recruitment, the proportion of eligible babies enrolled (70%) and approached babies enrolled (84%) returned to pre-pandemic levels. Our experience adapting our study during the COVID-19 pandemic led us to develop new recruitment strategies that we continue to utilize. The lessons learned from this pilot study can serve to help other research studies adapt novel and effective recruitment methods.

Engineering a Proximity-Directed O-GlcNAc Transferase for Selective Protein O-GlcNAcylation in Cells.

O-Linked ß-N-acetylglucosamine (O-GlcNAc) is a monosaccharide that plays an essential role in cellular signaling throughout the nucleocytoplasmic proteome of eukaryotic cells. Strategies for selectively increasing O-GlcNAc levels on a target protein in cells would accelerate studies of this essential modification. Here, we report a generalizable strategy for introducing O-GlcNAc into selected target proteins in cells using a nanobody as a proximity-directing agent fused to O-GlcNAc transferase (OGT). Fusion of a nanobody that recognizes GFP (nGFP) or a nanobody that recognizes the four-amino acid sequence EPEA (nEPEA) to OGT yielded nanobody-OGT constructs that selectively delivered O-GlcNAc to a series of tagged target proteins (e.g., JunB, cJun, and Nup62). Truncation of the tetratricopeptide repeat domain as in OGT(4) increased selectivity for the target protein through the nanobody by reducing global elevation of O-GlcNAc levels in the cell. Quantitative chemical proteomics confirmed the increase in O-GlcNAc to the target protein by nanobody-OGT(4). Glycoproteomics revealed that nanobody-OGT(4) or full-length OGT produced a similar glycosite profile on the target protein JunB and Nup62. Finally, we demonstrate the ability to selectively target endogenous α-synuclein for O-GlcNAcylation in HEK293T cells. These first proximity-directed OGT constructs provide a flexible strategy for targeting additional proteins and a template for further engineering of OGT and the O-GlcNAc proteome in the future. The use of a nanobody to redirect OGT substrate selection for glycosylation of desired proteins in cells may further constitute a generalizable strategy for controlling a broader array of post-translational modifications in cells.

Impact of Hospitalist-Led Interdisciplinary Antimicrobial Stewardship Interventions at an Academic Medical Center.

BACKGROUND: Approximately 20%-50% of antimicrobial use in hospitals is inappropriate. Limited data exist on the effect of frontline provider engagement on antimicrobial stewardship outcomes. METHODS: A three-arm pre-post quality improvement study was conducted on three adult internal medicine teaching services at an urban academic hospital. Data from September through December 2016 were compared to historic data from corresponding months in 2015. Intervention arms were (1) Educational bundle (Ed-only); (2) Educational bundle plus antimicrobial stewardship rounds twice weekly with an infectious disease-trained clinical pharmacist (Ed+IDPharmDx2); and (3) Educational bundle plus internal medicine-trained clinical pharmacist embedded into daily attending rounds (Ed+IMPharmDx5). RESULTS: Total antibiotic use decreased by 16.8% (p < 0.001), 6.8% (p = 0.08), and 33.0% (p < 0.001) on Ed-only, Ed+IDPharmDx2, and Ed+IMPharmDx5 teams, respectively. Broad-spectrum antibiotic use decreased by 26.2% (p < 0.001), 7.8% (p = 0.09), and 32.4% (p < 0.001) on the Ed-only, Ed+IDPharmDx2, and Ed+IMPharmDx5 teams, respectively. Duration of inpatient antibiotic therapy decreased from 4 to 3 days on the Ed+IMPharmDx5 team (p = 0.01). Length of stay for patients who received any antibiotic decreased from 9 to 7 days on the Ed-only team (p < 0.001) and from 9 to 6 days on the Ed+IMPharmDx5 team (p < 0.001). There was no significant change in 30-day readmission to the same facility, transfer to ICU, or in-hospital mortality for any team. CONCLUSION: Multidisciplinary, frontline provider-driven approaches to antimicrobial stewardship may contribute to reduced antibiotic use and length of hospital stay.

A Natural History of Actinic Keratosis and Cutaneous Squamous Cell Carcinoma Microbiomes.

Cutaneous squamous cell carcinoma (SCC) is the second-most-common cancer in Australia. The majority of SCCs progress from premalignant actinic keratosis (AK) lesions that form on chronically sun-exposed skin. The role of skin microbiota in this progression is not well understood; therefore, we performed a longitudinal microbiome analysis of AKs and SCCs using a cohort of 13 SCC-prone immunocompetent men. The majority of variability in microbial profiles was attributable to subject, followed by time and lesion type. Propionibacterium and Malassezia organisms were relatively more abundant in nonlesional photodamaged skin than in AKs and SCCs. Staphylococcus was most commonly associated with lesional skin, in particular, sequences most closely related to Staphylococcus aureus Of 11 S. aureus-like operational taxonomic units (OTUs), six were significantly associated with SCC lesions across seven subjects, suggesting their specific involvement with AK-to-SCC progression. If a causative link exists between certain S. aureus-like OTUs and SCC etiology, therapeutic approaches specifically targeting these bacteria could be used to reduce SCC.IMPORTANCE Actinic keratosis (AK) and cutaneous squamous cell carcinoma (SCC) are two of the most common dermatologic conditions in Western countries and cause substantial morbidity worldwide. The role of human papillomaviruses under these conditions has been well studied yet remains inconclusive. One PCR-based study has investigated bacteria in the etiology of these conditions; however, no study has investigated the microbiomes of AK and SCC more broadly. We longitudinally profiled the microbiomes of 112 AK lesions, profiled cross sections of 32 spontaneously arising SCC lesions, and compared these to matching nonlesional photodamaged control skin sites. We identified commonly occurring strains of Propionibacterium and Malassezia at higher relative abundances on nonlesional skin than in AK and SCC lesions, and strains of Staphylococcus aureus were relatively more abundant in lesional than nonlesional skin. These findings may aid in the prevention of SCC.

Mould meningitis associated with intravenous drug use.

Fungal meningitis is most commonly causes by Cryptococcus species and dimorphic fungi. We present a rare case of mould meningitis, ventriculitis and subependymal nodules in an immunocompetent patient, having likely seeded the meninges and ventricular system through intravenous drug use. The causative mould remains undetermined. The case highlights the poor sensitivity of CSF culture and the need to consider surgical biopsy where there is diagnostic difficulty and fungal infection is being considered.

Twist on a classic: vitamin D and hypercalcaemia of malignancy.

Malignancy is the most common cause of hypercalcaemia in the inpatient setting. Most cases are caused by tumour production of parathyroid hormone-related protein and osseous metastases. In less than 1% of cases, hypercalcaemia is driven by increased production of 1,25-dihydroxyvitamin D (1,25(OH)2D), a mechanism most commonly seen in haematological malignancies. Here, we describe a woman with metastatic small cell cervical carcinoma who developed hypercalcaemia secondary to paraneoplastic overproduction of 1,25(OH)2D, a finding that, to our knowledge, has not been previously associated with this cancer. We also review the current cases of solid tumours reported to have this mechanism of hypercalcaemia and the evidence behind multiple therapeutic approaches.

Altered serum levels of the osteoclast-specific TRACP 5b isoform in Chinese children undergoing orthodontic treatment.

Orthodontic tooth movement is dependent upon the ability of mechanical forces to induce remodelling activity within the tooth-supporting alveolar bone. In view of the importance of bone resorption in mediating tooth movement, the aim of this study was to establish if alterations in the osteoclast-specific bone marker tartrate-resistant acid phosphatase (TRACP) 5b could be detected in the sera of patients undergoing orthodontic treatment. The sample consisted of 14 subjects (10 girls and 4 boys) aged 10.5-16.5 years (mean 12.6 years) being treated with fixed appliances and a distalizing headgear. Venous blood samples (3 ml) were collected from the cubital vein pre-treatment (T0) and 2, 4, and 6 months into treatment (T1-T3); serum TRACP 5b levels were quantified using a solid-phase immunofixed enzyme activity assay. When the data were pooled and treated cross-sectionally, a significant increase in immunoreactive TRACP 5b was detected at 2 months (T1) indicating increased bone resorptive activity. However, when the serum profiles of individual patients were recorded longitudinally, a very different pattern emerged, not all patients following the same trend. This is not surprising given normal anatomical variation and differences between the patients in age, gender, and mechanotherapy. Designed as a pilot to demonstrate 'proof of principle', this study is the first to show that the TRACP 5b isoform can be detected in the sera of patients undergoing orthodontic treatment. It further suggests that serum bone marker measurements offer a simple and minimally invasive method for correlating the findings of laboratory and animal experimentation with clinical data.

Sex differences in the cannabinoid regulation of energy homeostasis.

This review highlights the progress made thus far in characterizing the behavioral and cellular mechanisms through which cannabinoids regulate energy homeostasis. We performed microstructural analysis of feeding behavior in gonadectomized guinea pigs and gonadally intact, transgenic CB1 receptor knockout mice to determine how cannabinoids affect circadian rhythms in food intake and meal pattern. We also implanted data loggers into the abdominal cavity to correlate the appetite-modulating properties of cannabinoids with changes in core body temperature. We then coupled the effects on feeding behavior and temperature regulation with synaptic changes in the hypothalamic feeding circuitry via whole-cell patch clamp electrophysiological recording from neurons in the arcuate nucleus (ARC), in order to gain a more global perspective on the cannabinoid modulation of energy homeostasis. We observed marked sex differences in cannabinoid effects on food intake and core body temperature--with male guinea pigs exhibiting a comparatively greater sensitivity to the hyperphagia and hypophagia, as well as the hypothermia and hyperthermia, produced by CB1 receptor agonists and antagonists, respectively. In addition, male but not female CB1 receptor knockout mice show a diminished nocturnal food intake and average daily body weight relative to their wildtype littermate controls. The disparity in the CB1 receptor-mediated hyperphagia is paralleled by sex differences in the cellular effects of cannabinoids at anorexigenic, guinea pig proopiomelanocortin (POMC) synapses. Postsynaptically, cannabinoids potentiate an A-type K+ current (I(A)) in POMC neurons from female guinea pigs, whereas in males the activation of an inwardly rectifying K+ current is observed. Presynaptically, while cannabinoids inhibit glutamatergic input onto POMC neurons in males and females to similar degrees, males are more refractory to the cannabinoid-induced inhibition of convergent GABAergic input than females. These data reveal pervasive sex differences in the cannabinoid regulation of energy homeostasis that are consistent with changes in the excitability of POMC neurons.

Polyglutamine proteins at the pathogenic threshold display neuron-specific aggregation in a pan-neuronal Caenorhabditis elegans model.

The basis of neuron-specific pathogenesis, resulting from the expression of misfolded proteins, is poorly understood and of central importance to an understanding of the cell-type specificity of neurodegenerative disease. In this study, we developed a new model for neuron-specific polyQ pathogenesis in Caenorhabditis elegans by pan-neuronal expression that exhibits polyQ length-dependent aggregation, neurotoxicity, and a pathogenic threshold at a length of 35-40 glutamines. Analysis of specific neurons in C. elegans revealed that only at the threshold length, but not at shorter or longer lengths, polyQ proteins can exist in a soluble state in certain lateral neurons or in an aggregated state in motor neurons of the same animal. These results provide direct experimental evidence that the expression of a single species of a toxic misfolded protein can exhibit a range of neuronal consequences.

Sex differences in the cannabinoid modulation of an A-type K+ current in neurons of the mammalian hypothalamus.

Cannabinoids regulate biological processes governed by the hypothalamus including, but not limited to, energy homeostasis and reproduction. The present study sought to determine whether cannabinoids modulate A-type K(+) currents (I(A)) in neurons of the hypothalamic arcuate nucleus (ARC). Whole cell patch-clamp recordings were performed in slices through the ARC prepared from castrated female and male guinea pigs. Forty percent of guinea pig ARC neurons exhibited a transient outward current that was antagonized by high (mM) concentrations of 4-aminopyridine and (100 nM) rHeteropodatoxin-2. Five of these neurons also were immunopositive for both beta-endorphin and the Kv4.2 channel subunit. Bath application of the CB1 receptor agonists WIN 55,212-2 (1 microM) or ACEA (1 microM) selectively induced a rightward shift in the inactivation curve for the I(A), significantly increasing the half-maximal voltage without affecting the peak current magnitude, in neurons from female but not male animals. The CB1 receptor antagonist AM251 (1 microM) reversed this action. Collectively, these data reveal that guinea pig ARC neurons, including proopiomelanocortin neurons, express a prominent I(A) that is positively modulated by cannabinoids in a sex-specific way by altering the voltage dependence of its inactivation. The resultant inhibitory effect on this neuronal population may shed some insight into the mechanism(s) by which cannabinoids influence hypothalamic function.