Cyclic AMP binding to a universal stress protein in Mycobacterium tuberculosis is essential for viability.

Mycobacterial genomes encode multiple adenylyl cyclases and cAMP effector proteins, underscoring the diverse ways these bacteria utilize cAMP. We identified universal stress proteins, Rv1636 and MSMEG_3811 in Mycobacterium tuberculosis and Mycobacterium smegmatis, respectively, as abundantly expressed, novel cAMP-binding proteins. Rv1636 is secreted via the SecA2 secretion system in M. tuberculosis but is not directly responsible for the efflux of cAMP from the cell. In slow-growing mycobacteria, intrabacterial concentrations of Rv1636 were equivalent to the concentrations of cAMP present in the cell. In contrast, levels of intrabacterial MSMEG_3811 in M. smegmatis were lower than that of cAMP and therefore, overexpression of Rv1636 increased levels of "bound" cAMP. While msmeg_3811 could be readily deleted from the genome of M. smegmatis, we found that the rv1636 gene is essential for the viability of M. tuberculosis and is dependent on the cAMP-binding ability of Rv1636. Therefore, Rv1636 may function to regulate cAMP signaling by direct sequestration of the second messenger. This is the first evidence of a "sponge" for any second messenger in bacterial signaling that would allow mycobacterial cells to regulate the available intrabacterial "free" pool of cAMP.

Measuring practice preference variation for quality improvement in neonatal respiratory care.

OBJECTIVES: The authors sought to measure and compare practice preference variation in neonatal respiratory care within and between neonatal intensive care units (NICUs) using the Neonatology Survey of Interdisciplinary Groups in Healthcare Tool (NSIGHT). STUDY DESIGN: Eleven NICUs completed the NSIGHT between 2019 and 2021. Net preference was measured by mean response; agreement was ranked by standard distribution of response values. Heat maps showed comparisons between NICUs and disciplines. RESULTS: NICUs and individuals agreed most often on use of pressure support with mandatory ventilation and on use of non-invasive positive pressure ventilation for apnea. High preference variation surrounded decisions for invasive ventilation versus continuous positive airway pressure for extremely low birth weight infants. Preference difference was most frequent between neonatologists and nurses. CONCLUSIONS: Patterns of practice preference variation in neonatal respiratory care are specific to clinical scenario. Measuring preference variation may inform psychology of change and strengthen quality improvement efforts.

Enhanced sunlight-driven catalysis for hydrogen generation and dye remediation using synergistic p-Co3O4/n-TiO2 nanocomposites.

In this study, p-Co3O4/n-TiO2 nanocomposites were synthesized using different ratios of cobalt and titanium precursors through a hydrothermal method. These nanocomposites demonstrated notable potential in photocatalytic applications for hydrogen production and orange-red dye degradation under sunlight. Various techniques, including XRD, Raman spectroscopy, XPS, FESEM, TEM, and BET analysis, were used to comprehensively characterize their structural, morphological, and optical properties. The nanocomposites exhibited both cubic and tetragonal phases of Co3O4 and TiO2, and their combined effect resulted in a narrowed band gap. Additionally, the presence of Co3O4 induced surface plasmon resonance on the TiO2 surface, effectively impeding electron-hole recombination. The nanocomposites displayed an average particle size of ∼20 to 30 nm with substantial visible light absorption. High crystallinity and well-dispersed nanocomposites were confirmed by XRD and Raman, with BET surface areas ranging between 49 and 106 m2 g-1. Notably, the p-Co3O4/n-TiO2 nanocomposite showed superior photocatalytic activity, achieving a maximum hydrogen generation rate of 1120 µmol h-1 g-1 and an 83% degradation efficiency of the orange-red dye within 6 minutes under sunlight. This study emphasizes the enhanced performance of the p-Co3O4/n-TiO2 nanocomposite, indicating its potential in photocatalytic applications, conforming to a pseudo-first-order kinetics model.

An Endosomal Acid-Regulatory Feedback System Rewires Cytosolic cAMP Metabolism and Drives Tumor Progression.

Although suppressed cAMP levels have been linked to cancer for nearly five decades, the molecular basis remains uncertain. Here, we identify endosomal pH as a novel regulator of cytosolic cAMP homeostasis and a promoter of transformed phenotypic traits in colorectal cancer. Combining experiments and computational analysis, we show that the Na+/H+ exchanger NHE9 contributes to proton leak and causes luminal alkalinization, which induces resting [Ca2+], and in consequence, represses cAMP levels, creating a feedback loop that echoes nutrient deprivation or hypoxia. Higher NHE9 expression in cancer epithelia is associated with a hybrid epithelial-mesenchymal (E/M) state, poor prognosis, tumor budding, and invasive growth in vitro and in vivo. These findings point to NHE9-mediated cAMP suppression as a pseudostarvation-induced invasion state and potential therapeutic vulnerability in colorectal cancer. Our observations lay the groundwork for future research into the complexities of endosome-driven metabolic reprogramming and phenotype switching and the biology of cancer progression. IMPLICATIONS: Endosomal pH regulator NHE9 actively controls cytosolic Ca2+ levels to downregulate the adenylate cyclase-cAMP system, enabling colorectal cancer cells to acquire hybrid E/M characteristics and promoting metastatic progression.

Use of an airway bundle to reduce unplanned extubations in a neonatal intensive care unit.

BACKGROUND: Following the opening of an infant cardiac neonatal intensive care unit, our aim was to determine a baseline UE rate and implement initiatives to target a goal less than 0.5 UEs/100 ventilator days. METHODS: We utilized the Model for Improvement. Key stakeholders included neonatal providers, nurses, and respiratory therapists. We focused on the creation of an airway bundle that addressed securement methods, communication and education. RESULTS: From October 2017 to January 2018, our baseline UE rate was 0.92 UEs/100 ventilator days. Subsequent to the implementation of an airway bundle with high compliance, we observed a significant change in the centerline (0.45 to 0.02 UEs/100 ventilator days) during the spring of 2021, followed by a period of 480 days with no UEs. CONCLUSION: In a unit where UEs were infrequent events, high compliance with an airway bundle led to a significantly sustained decrease in our UE rates.

A High-Protein Diet Promotes Atrial Arrhythmogenesis via Absent-in-Melanoma 2 Inflammasome.

High-protein diets (HPDs) offer health benefits, such as weight management and improved metabolic profiles. The effects of HPD on cardiac arrhythmogenesis remain unclear. Atrial fibrillation (AF), the most common arrhythmia, is associated with inflammasome activation. The role of the Absent-in-Melanoma 2 (AIM2) inflammasome in AF pathogenesis remains unexplored. In this study, we discovered that HPD increased susceptibility to AF. To demonstrate the involvement of AIM2 signaling in the pathogenesis of HPD-induced AF, wildtype (WT) and Aim2-/- mice were fed normal-chow (NC) and HPD, respectively. Four weeks later, inflammasome activity was upregulated in the atria of WT-HPD mice, but not in the Aim2-/--HPD mice. The increased AF vulnerability in WT-HPD mice was associated with abnormal sarcoplasmic reticulum (SR) Ca2+-release events in atrial myocytes. HPD increased the cytoplasmic double-strand (ds) DNA level, causing AIM2 activation. Genetic inhibition of AIM2 in Aim2-/- mice reduced susceptibility to AF, cytoplasmic dsDNA level, mitochondrial ROS production, and abnormal SR Ca2+-release in atrial myocytes. These data suggest that HPD creates a substrate conducive to AF development by activating the AIM2-inflammasome, which is associated with mitochondrial oxidative stress along with proarrhythmic SR Ca2+-release. Our data imply that targeting the AIM2 inflammasome might constitute a novel anti-AF strategy in certain patient subpopulations.

The evolutionary divergence of receptor guanylyl cyclase C has implications for preclinical models for receptor-directed therapeutics.

Mutations in receptor guanylyl cyclase C (GC-C) cause severe gastrointestinal disease, including meconium ileus, early onset acute diarrhea, and pediatric inflammatory bowel disease that continues into adulthood. Agonists of GC-C are US Food and Drug Administration-approved drugs for the treatment of constipation and irritable bowel syndrome. Therapeutic strategies targeting GC-C are tested in preclinical mouse models, assuming that murine GC-C mimics human GC-C in its biochemical properties and downstream signaling events. Here, we reveal important differences in ligand-binding affinity and GC activity between mouse GC-C and human GC-C. We generated a series of chimeric constructs of various domains of human and mouse GC-C to show that the extracellular domain of mouse GC-C contributed to log-orders lower affinity of mouse GC-C for ligands than human GC-C. Further, the Vmax of the murine GC domain was lower than that of human GC-C, and allosteric regulation of the receptor by ATP binding to the intracellular kinase-homology domain also differed. These altered properties are reflected in the high concentrations of ligands required to elicit signaling responses in the mouse gut in preclinical models and the specificity of a GC inhibitor towards human GC-C. Therefore, our studies identify considerations in using the murine model to test molecules for therapeutic purposes that work as either agonists or antagonists of GC-C, and vaccines for the bacterial heat-stable enterotoxin that causes watery diarrhea in humans.

Reprogramming of cis-regulatory networks during skeletal muscle atrophy in male mice.

A comprehensive atlas of cis-regulatory elements and their dynamic activity is necessary to understand the transcriptional basis of cellular structure maintenance, metabolism, and responses to the environment. Here we show, using matched single-nucleus chromatin accessibility and RNA-sequencing from juvenile male C57BL6 mice, an atlas of accessible chromatin regions in both normal and denervated skeletal muscles. We identified cell-type-specific cis-regulatory networks, highlighting the dynamic regulatory circuits mediating transitions between myonuclear types. Through comparison of normal and perturbed muscle, we delineated the reprogramming of cis-regulatory networks in response to denervation, described the interplay of promoters/enhancers and target genes. We further unveil a hierarchical structure of transcription factors that delineate a regulatory network in atrophic muscle, identifying ELK4 as a key atrophy-related transcription factor that instigates muscle atrophy through TGF-ß1 regulation. This study furnishes a rich genomic resource, essential for decoding the regulatory dynamics of skeletal muscle in both physiological and pathological states.

ROCK1 activates mitochondrial fission leading to oxidative stress and muscle atrophy.

Chronic kidney disease (CKD) is often associated with protein-energy wasting (PEW), which is characterized by a reduction in muscle mass and strength. Although mitochondrial dysfunction and oxidative stress have been implicated to play a role in the pathogenesis of muscle wasting, the underlying mechanisms remain unclear. In this study, we used transcriptomics, metabolomics analyses and mouse gene manipulating approaches to investigate the effects of mitochondrial plasticity and oxidative stress on muscle wasting in mouse CKD models. Our results showed that the expression of oxidative stress response genes was increased, and that of oxidative phosphorylation genes was decreased in the muscles of mice with CKD. This was accompanied by reduced oxygen consumption rates, decreased levels of mitochondrial electron transport chain proteins, and increased cellular oxidative damage. Excessive mitochondrial fission was also observed, and we found that the activation of ROCK1 was responsible for this process. Inducible expression of muscle-specific constitutively active ROCK1(mROCK1ca)exacerbated mitochondrial fragmentation and muscle wasting in CKD mice. Conversely, ROCK1 depletion (ROCK1-/-) alleviated these phenomena. Mechanistically, ROCK1 activation promoted the recruitment of Drp1 to mitochondria, thereby facilitating fragmentation. Notably, the pharmacological inhibition of ROCK1 mitigated muscle wasting by suppressing mitochondrial fission and oxidative stress. Our findings demonstrate that ROCK1 participates in CKD-induced muscle wasting by promoting mitochondrial fission and oxidative stress, and pharmacological suppression of ROCK1 could be a therapeutic strategy for combating muscle wasting in CKD conditions.

Neuronal expression in Drosophila of an evolutionarily conserved metallophosphodiesterase reveals pleiotropic roles in longevity and odorant response.

Evolutionarily conserved genes often play critical roles in organismal physiology. Here, we describe multiple roles of a previously uncharacterized Class III metallophosphodiesterase in Drosophila, an ortholog of the MPPED1 and MPPED2 proteins expressed in the mammalian brain. dMpped, the product of CG16717, hydrolyzed phosphodiester substrates including cAMP and cGMP in a metal-dependent manner. dMpped is expressed during development and in the adult fly. RNA-seq analysis of dMppedKO flies revealed misregulation of innate immune pathways. dMppedKO flies showed a reduced lifespan, which could be restored in Dredd hypomorphs, indicating that excessive production of antimicrobial peptides contributed to reduced longevity. Elevated levels of cAMP and cGMP in the brain of dMppedKO flies was restored on neuronal expression of dMpped, with a concomitant reduction in levels of antimicrobial peptides and restoration of normal life span. We observed that dMpped is expressed in the antennal lobe in the fly brain. dMppedKO flies showed defective specific attractant perception and desiccation sensitivity, correlated with the overexpression of Obp28 and Obp59 in knock-out flies. Importantly, neuronal expression of mammalian MPPED2 restored lifespan in dMppedKO flies. This is the first description of the pleiotropic roles of an evolutionarily conserved metallophosphodiesterase that may moonlight in diverse signaling pathways in an organism.

Insulin Resistance and Insulin Handling in Chronic Kidney Disease.

Insulin regulates energy metabolism involving multiple organ systems. Insulin resistance (IR) occurs when organs exhibit reduced insulin sensitivity, leading to difficulties in maintaining glucose homeostasis. IR ensures decades prior to development of overt diabetes and can cause silent metabolic derangements. IR is typically seen very early in the course of chronic kidney disease (CKD) and is evident even when the estimated glomerular filtration rate (eGFR) is within the normal range and IR persists at various stages of kidney disease. In this article, we will discuss insulin handling by the kidneys, mechanisms responsible for IR in CKD, measurements and management of IR in patients with CKD, and recent type 2 diabetic trials with implications for improved cardiovascular outcomes in CKD. © 2023 American Physiological Society. Compr Physiol 13:5069-5076, 2023.

Epidemiology, risk factors, and applicability of CDC definitions for healthcare-associated bloodstream infections at a level IV neonatal ICU.

OBJECTIVES: We studied the epidemiology of primary bloodstream infections (BSIs), secondary BSIs, and central line-associated BSIs (CLABSIs) and applicability of CDC definitions for primary sources of infection causing secondary BSIs in patients in the neonatal ICU. STUDY DESIGN: We classified healthcare-associated BSIs (HABSIs) as primary BSIs, secondary BSIs, and CLABSIs using CDC surveillance definitions and determined their overall incidence and incidence among different gestational age strata. We assessed the applicability of CDC definitions for infection sources causing secondary BSIs. RESULTS: From 2010 to 2019, 141 (32.7%), 202 (46.9%), and 88 (20.4%) HABSIs were classified as primary BSIs, secondary BSIs, and CLABSIs, respectively; all declined during the study period (all p < 0.001). Gestational age <28 weeks was associated with increased incidence of all HABSI types. CDC criteria for site-specific primary sources were met in 137/202 (68%) secondary BSIs. CONCLUSIONS: Primary and secondary BSIs were more common than CLABSIs and should be prioritized for prevention.

Chronic kidney disease promotes atrial fibrillation via inflammasome pathway activation.

Chronic kidney disease (CKD) is associated with a higher risk of atrial fibrillation (AF). The mechanistic link between CKD and AF remains elusive. IL-1ß, a main effector of NLR family pyrin domain-containing 3 (NLRP3) inflammasome activation, is a key modulator of conditions associated with inflammation, such as AF and CKD. Circulating IL-1ß levels were elevated in patients with CKD who had AF (versus patients with CKD in sinus rhythm). Moreover, NLRP3 activity was enhanced in atria of patients with CKD. To elucidate the role of NLRP3/IL-1ß signaling in the pathogenesis of CKD-induced AF, Nlrp3-/- and WT mice were subjected to a 2-stage subtotal nephrectomy protocol to induce CKD. Four weeks after surgery, IL-1ß levels in serum and atrial tissue were increased in WT CKD (WT-CKD) mice versus sham-operated WT (WT-sham) mice. The increased susceptibility to pacing-induced AF and the longer AF duration in WT-CKD mice were associated with an abbreviated atrial effective refractory period, enlarged atria, and atrial fibrosis. Genetic inhibition of NLRP3 in Nlrp3-/- mice or neutralizing anti-IL-1ß antibodies effectively reduced IL-1ß levels, normalized left atrial dimensions, and reduced fibrosis and the incidence of AF. These data suggest that CKD creates a substrate for AF development by activating the NLRP3 inflammasome in atria, which is associated with structural and electrical remodeling. Neutralizing IL-1ß antibodies may be beneficial in preventing CKD-induced AF.

Autonomous face mask detection using single shot multibox detector, and ResNet-50 with identity retrieval through face matching using deep siamese neural network.

The COVID-19 pandemic poses a global health challenge. The World Health Organization states that face masks are proven to be effective, especially in public areas. Real-time monitoring of face masks is challenging and exhaustive for humans. To reduce human effort and to provide an enforcement mechanism, an autonomous system has been proposed to detect non-masked people and retrieve their identity using computer vision. The proposed method introduces a novel and efficient method that involves fine-tuning the pre-trained ResNet-50 model with a new head layer for classification between masked and non-masked people. The classifier is trained using adaptive momentum optimization algorithm with decaying learning rate and binary cross-entropy loss. Data augmentation and dropout regularization are employed to achieve best convergence. During real-time application of our classifier on videos, a Caffe face detector model based on Single Shot MultiBox Detector is used to extract the face regions of interest from each frame, on which the trained classifier is applied for detecting the non-masked people. The faces of these people are then captured, which is passed on to a deep siamese neural network, based on VGG-Face model for face matching. The captured faces are compared with the reference images from the database, by extracting the features and calculating cosine distance. If the faces match, the details of that person are retrieved from the database and displayed on the web application. The proposed method has secured best results where the trained classifier has achieved 99.74% accuracy, and the identity retrieval model achieved 98.24% accuracy.

Next Steps for Health Care-Associated Infections in the Neonatal Intensive Care Unit.

We discuss the burden of health care-associated infections (HAIs) in the neonatal ICU and the role of quality improvement (QI) in infection prevention and control. We examine specific QI opportunities and approaches to prevent HAIs caused by Staphylococcus aureus , multidrug-resistant gram-negative pathogens, Candida species, and respiratory viruses, and to prevent central line-associated bloodstream infections (CLABSIs) and surgical site infections. We explore the emerging recognition that many hospital-onset bacteremia episodes are not CLABSIs. Finally, we describe the core tenets of QI, including engagement with multidisciplinary teams and families, data transparency, accountability, and the impact of larger collaborative efforts to reduce HAIs.

The continued effects of COVID-19 on the lives and livelihoods of vegetable farmers in India.

India experienced a rapid rise in COVID-19 infections from March 2021. States imposed varying levels of lockdowns and curfews to curb the spread of the disease. These restrictions severely affected the functioning of food systems. The objective of this study was to analyze how COVID-19 continues to affect agricultural production, food security and household diets of vegetable farmers. A phone-based survey was conducted with 595 vegetable farmers in the states of Andhra Pradesh, Assam, Jharkhand, Karnataka and Odisha, 60% of whom had been interviewed a year earlier. Overall, 60% of farmers experienced decreased vegetable production; over 80% reported a reduction in consumption of at least one food group; and 45% reported some level of food insecurity between May 2020 and May 2021. Farmers who reported decreased staples production, difficulty accessing seeds/seedlings, or reduced their household spending were more likely to report decreased vegetable production. Vegetable consumption was positively associated with receipt of COVID-19 relief benefits, borrowing money, or having home gardens. Farmers who received public agricultural assistance, or had reduced expenses, were more likely to have lower vegetable consumption. Greater severity of food insecurity was associated with farmers belonging to underprivileged social groups, non-Hindus, or those who experienced decrease in livestock production, weather related disruptions or received COVID-19 assistance. This is one of few studies that have conducted a longitudinal assessment of the impacts across multiple waves of COVID-19. COVID-19 is seen to be one among several shocks experienced by farm households, and exacerbated existing issues within agriculture and food security. There is a need for public policy support to strengthen both production and consumption of vegetables.