Search Results (700)

Fast-channel genetic myasthenic syndromes (FCGMSs) are caused by genetic variants in muscle nicotinic acetylcholine receptor (AChR) subunits that reduce channel open times and impair neuromuscular transmission. Among these, the CHRNE p.P141L variant (εP141L) is associated with particularly severe disease. Here, we characterized a knock-in mouse model harboring the homologous p.P141L variant in Chrne (εP141L)—C57BL/6J-Chrne^em1H/H made by the MRC GEMM program. Homozygous mutant mice fail to thrive, with early lethality (median survival of 16 days), closely recapitulating the severity observed in patients. Despite a preserved neuromuscular junction (NMJ) morphology and robust AChR expression, electrophysiological analyses revealed marked reductions in miniature and evoked endplate potential amplitudes and areas, accompanied by prolonged depolarization kinetics (contrary to expectations for AChR with reduced open times) and increased quantal content, indicative of impaired post-synaptic function with compensatory pre-synaptic adaptation. Notably, disease severity exceeded that of Chrne null mice, likely through competition with more functional g-subunit-containing fetal AChRs. Consistent with this, crossing εP141L mice with CHRNG-expressing mice provided little survival benefit. These findings demonstrate that dysfunctional AChR incorporation is more deleterious than receptor absence and highlight the critical role of subunit composition in sustaining neuromuscular transmission. Pharmacological enhancement of pre-synaptic release with 3,4-diaminopyridine partially improved synaptic parameters. In addition, the AChR-positive allosteric modulator DC-98 modestly improved neurotransmission. Thus, this mouse model provides a faithful platform for mechanistic studies and therapeutic development in FCGMS. Full article

Domoic acid (DA) is a well-known seafood toxin produced by some species of marine phytoplankton in the genus Pseudo-nitzschia during harmful algal blooms (HABs). Acute toxic exposures induce overt clinical signs of neuroexcitotoxicity, such as seizures in mammals due to overstimulation of glutamate receptors in the central nervous system (CNS). Acute DA excitotoxicity via the CNS has been well-studied in both field poisoning events and laboratory exposure studies with rodent models, but little is known about the impacts of low-level DA exposures below those that cause outward signs of neurotoxicity; the impacts on other potential target organs, including the heart; or age-related sensitivities. Here, low-level DA exposures in young adult (9 mo) and old (24 mo) mice were conducted over multiple weeks. Mortality, cardiac function, frailty, and protein expression were quantified to assess age-related DA sensitivity and potential impacts on heart function. Echocardiography and proteome data confirm that chronic low-level DA exposure causes irreversible functional cardiomyopathy and protein remodeling in young adult mice that mimics natural cardiac aging. In addition, old mice exhibit higher mortality and frailty than young adult mice with the same low-level DA exposures. These results provide critical information for assessing potential health risks to humans who regularly consume seafood with low levels of DA. Full article

Background: We previously reported that knocking down the ubiquitin E3 ligase LNX2 in bone marrow monocytes by shRNAs attenuated osteoclastogenesis in vitro. However, the role of LNX2 in the regulation of osteoclasts and bone homeostasis in vivo remains unknown. Methods: In this study, we generated myeloid Lnx2 conditional knockout mice by crossing Lnx2-flox mice with LysM-Cre mice. The role of LNX2 was verified through in vitro osteoclast induction experiments using mononuclear macrophages and experiments on estrogen-deficient osteoporosis models. Results: Micro-CT and histological analysis unveiled that loss of Lnx2 in osteoclast precursor cells decreased osteoclast numbers and increased trabecular bone mass in mice. Moreover, Lnx2 deficiency prevented bone loss in an ovariectomized mouse model of postmenopausal osteoporosis. In vitro mechanistic studies identified that the loss of Lnx2 had little effect on cell proliferation but significantly inhibited the formation of osteoclasts and bone resorption. Furthermore, the deletion of Lnx2 decreased the expression of NOTCH2 and its downstream HES1 via enhancing the level of the NOTCH2 inhibitor, NUMB. Conclusions: Our findings elucidate an important role of Lnx2 in the regulation of osteoclasts and bone metabolism and indicate that Lnx2 is a potential therapeutic target for the treatment of osteoporosis. Full article

Background. The highly mutable influenza virus causes severe annual infections worldwide and results in substantial socioeconomic losses. The spread of infection could be effectively controlled by cross-protective vaccines and universal diagnostic test systems based on the nucleoprotein (NP) as one of the most conserved viral antigens. However, NP also undergoes slow evolutionary changes, and little is known about the influence of these mutations on its antigenicity and immunogenicity. Methods. We expressed the full-length recombinant 6xHis-tagged NPs of ten evolutionary distant influenza A strains of different subtypes in E. coli BL21(DE3) cells and purified these proteins by immobilized metal affinity chromatography. The obtained antigens were identified by mass spectrometry and serological methods. NPs served as antigens for three immunizations of BALB/c mice (15 µg/animal at 14-day interval) and as capturing proteins in ELISA at 2 µg/mL, in order to study the effect of adaptive mutations on the antigenic and immunogenic properties of NPs. Results. A pronounced cross-reactivity of anti-NP antibodies induced in mice by immunization with different NPs was revealed. At the same time, we observed the differences in the humoral immunogenicity of NP, which are in line with the accumulation of evolutionarily driven NP mutations. In general, antibody affinity to heterologous NPs was reduced, indicating the differences in the specificity of anti-NP immunoglobulins, which may be caused by evolutionarily determined variability of immunogenic epitopes leading to the emergence of escape mutations. Conclusions. Overall, our results reflect the slightly evolving nature of the NP antigen, which influences the specificity spectrum of anti-NP antibodies and should be considered as a limitation for the development of NP-based cross-protective vaccines and test systems. Full article

Background: Diets composed of various components have been shown to influence inflammatory diseases such as asthma. While most studies have focused on fiber-rich diets to investigate their effects on the immune system and, consequently, on asthma, little is known about the impact of sugar-rich diets, particularly when such diets are consumed over short periods of time. Methods: To investigate the short-term effects of a sugar-rich diet on allergic airway inflammation, A/J mice were fed either a standard diet or a sugar-enriched diet and subsequently sensitized and challenged with ovalbumin or PBS. Airway inflammation was assessed by bronchoalveolar lavage (BAL) cell analysis, including eosinophil counts and cytokine levels (IL-4, TNF-α, IL-33), and by lung histology (H&E for inflammatory infiltrate and PAS for mucus). Serum IgE levels were also measured. In addition, glucose tolerance, visceral and subcutaneous adipose tissue mass, and inflammatory markers in visceral adipose tissue were evaluated. Results: Short-term consumption of a sugar-rich diet induced glucose intolerance and expansion of adipose tissue, particularly visceral fat, independent of ovalbumin sensitization. Gonadal adipose tissue analysis revealed a shift toward M1 macrophage polarization, characterized by elevated TNF-α, IL-6, and IL-1β, increased leptin levels, and reduced adiponectin. In OVA-sensitized mice, the sugar-rich diet significantly exacerbated eosinophil infiltration in BAL, increased IL-4, TNF-α, and IL-33, and enhanced PAS-positive mucus accumulation and inflammatory infiltrates in the lung. Moreover, total serum IgE was significantly higher in allergic mice fed the sugar-rich diet compared with allergic mice on the standard diet. Importantly, in non-sensitized mice fed the sugar-rich diet, no pulmonary inflammation was detected by BAL, demonstrating that HSD alone does not induce asthma but amplifies allergic responses when sensitization is present. Conclusions: Our findings demonstrate that short-term consumption of a sugar-rich diet is sufficient to exacerbate, but not initiate, allergic pulmonary inflammation. From a translational perspective, reducing dietary sugar intake may represent a valuable adjuvant strategy in the management of allergic asthma. Full article

WNT5a is a lipid-modified glycoprotein member of the WNT family of signaling molecules. Two isoforms of WNT5a have been identified that are conserved across mice and humans. These isoforms display specific functions in regulating cancer cell activities. While WNT5a is, indeed, essential for normal lung development and homeostasis, and is dysregulated in multiple lung diseases, little to no information is available regarding the expression or potential function of WNT5a isoforms in normal or diseased lungs. Such information has the potential to help to elucidate the more precise and nuanced functions of WNT5a in various pulmonary conditions. In this study, we characterized the expression of individual Wnt5a isoforms during mouse lung development and compared their expression across major alveolar cell populations. We further investigated the molecular basis of the signaling mechanisms that regulate Wnt5a isoform expression in fibroblasts, the major lung cell type with high-level Wnt5a expression. We present data that reveal a role for the AKT pathway in differentially regulating the expression of Wnt5a isoforms, a novel finding. Furthermore, we demonstrate that Wnt5a isoforms are dysregulated in bleomycin-induced fibrosis and Pseudomonas aeruginosa (PA)-induced acute lung injury and exhibit distinct impacts in Wnt5a isoform expression in response to lung injury. Full article

Background/Objectives: Anthracyclines such as doxorubicin (DOX) are integral to pediatric cancer protocols, yet little is known about how juvenile DOX exposure shapes the long-term trajectory of bone growth, microarchitectural connectivity, and the functional balance of bone turnover after treatment cessation. This study aimed to define how juvenile DOX exposure remodels trabecular architecture and bone homeostasis both acutely and after recovery. Methods: Four-week-old female BALB/c mice were treated with 6 mg/kg DOX or saline once weekly for four weeks. Bone parameters were analyzed immediately after treatment and after a 4-week drug-free recovery period. Assessments included high-resolution µCT for bone structure and connectivity, H&E and TRAP staining for histological evaluation, and ELISA for bone turnover markers (PINP, OC/BGP, TRACP-5b) in both serum and bone marrow. Results: DOX exposure significantly compromised trabecular bone mass and network connectivity, with persistent bone loss extending into the recovery period. Histologically, DOX caused marked degeneration in the epiphyseal growth plate and calcified zones, alongside a marked increase in osteoclast numbers. Functionally, an acute increase in circulating bone formation markers was observed post-treatment. However, during the recovery phase, this transitioned to a significant suppression of these systemic markers, coupled with significantly increased localized bone resorption. Conclusions: Juvenile DOX exposure produces sustained trabecular network impairment and growth plate degeneration. This durable structural deterioration is functionally associated with the establishment of a localized, pathologically uncoupled remodeling environment. Full article

Background/Objectives: Klebsiella pneumoniae (K. pneumoniae) is a leading cause of serious hospital-acquired and community-acquired infections, with limited treatment options, especially for immunocompromised and critically ill patients. No licensed vaccine is currently available. The FimA antigen, a key fimbrial subunit essential for bacterial adhesion and invasion, represents a promising vaccine target. However, little is known about the immunodominant antibody responses against invasive K. pneumoniae. This study aimed to evaluate the immunogenicity and protective efficacy of recombinant FimA protein, to fine-map its immunodominant linear B-cell epitopes, and to assess the individual and combined protective capacity of these epitopes against both standard and clinically isolated K. pneumoniae strains. Methods: A murine model of lethal K. pneumoniae challenge was used. Recombinant FimA protein was administered to evaluate immunogenicity and protective efficacy. Immunodominant linear B-cell epitopes were identified by overlapping peptide ELISA using immune antisera. The identified epitopes were synthesized and conjugated to keyhole limpet hemocyanin (KLH). Mice were immunized with individual epitope-KLH conjugates or a mixture of all four, then challenged with the standard strain ATCC700721 or with multiple clinical isolates of distinct multilocus sequence types (MLST). Epitope-specific antibody responses (total IgG and IgG subclasses) and survival rates were measured. Results: Immunization with full-length recombinant FimA conferred 90% protection against lethal challenge with the standard strain ATCC700721 and induced robust IgG1-dominant antibody responses. Four novel immunodominant linear B-cell epitopes were identified: FimA_97–114, FimA_103–120, FimA_109–126, and FimA_145–160. Structural mapping revealed that the first three epitopes reside within the α-helical region, while FimA_145–160 is located in the β-sheet domain. These epitopes are highly conserved, exhibiting 100% sequence identity across 36 diverse K. pneumoniae strains. Among individual epitope-KLH conjugates, FimA_109–126-KLH induced the highest epitope-specific antibody titers, followed by FimA_103–120-KLH. Immunization with a mixture of all four epitope-KLH conjugates elicited significant cross-protection against multiple clinical isolates, achieving survival rates of 60%, 50%, 50%, and 40% against strains 10CYZ, 13LGY, 19ZXQ, and 22CZY, respectively. Protective immunity was primarily associated with IgG1 subtype responses. Conclusions: This study provides the first fine-mapping and protective evaluation of immunodominant linear B-cell epitopes within K. pneumoniae FimA. The identification of highly conserved, functionally relevant B-cell epitopes and the demonstration of cross-protection conferred by a multi-epitope formulation underscore the potential of FimA-based epitope-driven vaccines. These findings offer a promising strategy for the development of broadly protective vaccines against K. pneumoniae infections. Full article

Background/Objectives: Previous research demonstrates higher sodium retention with increasing levels of dietary salt in some populations. Our objective was to determine whole-body sodium retention and sodium distribution on high and low salt diets using rodent models. Methods: Whole body retention of orally dosed Na-22, a gamma emitter, was measured in female growing and adult Sprague-Dawley rats on high (3.1% by wt. of diet) and low salt (0.13% by wt. of diet) diets. In a second study, whole-body sodium retention was compared between destructive inductively coupled plasma optical emission spectroscopy (ICP-OES) and neutron activation analysis (NAA) in adult male and female C57BL/6 mice. Results: Whole body retention of Na-22 was not different due to the age of rats on a high salt diet, but rats fed the high salt diet excreted Na-22 much more rapidly than rats fed a low salt diet. In mice, neither sodium retention nor tissue distribution was affected by dietary salt. Bland–Altman analysis indicated overall agreement between NAA and ICP-OES measurements, with observed systematic positive bias. Conclusions: Dietary salt had little effect on retention in normotensive rodents and should be studied in hypertensive models. Full article

Mammals and reptiles possess a metanephric kidney as the terminal renal organ for homeostasis of solutes and waste products. The development of the metanephric kidney has primarily been studied in mammalian model systems. Little is known about the conservation of metanephric kidney formation in non-mammalian species such as reptiles. Uniquely, reptiles maintain kidney progenitor cell populations throughout life and continually develop new nephrons, the functional unit of the kidney. The red-eared slider turtle, Trachemys scripta elegans, was utilized to investigate the conservation of reptilian metanephric kidney development. The nephron progenitor cell (NPC) marker, Six2, was detected in whole-mount turtle kidneys in a similar pattern to mammals. However, there were differences in progenitor cell niche morphology where turtle NPC populations formed distinct elongated rows instead of the rosette-like morphology found in the mouse. The pattern of NPC populations in the embryonic turtle kidney was maintained in the adult turtle. Whole-genome bisulfite sequencing was performed on cortical tissue containing the NPC populations from adult turtle kidneys and compared to those of adult mice. Significant conservation of gene methylation was detected in adult cortical tissue between the two species, although unique signatures were detected in turtle samples related to DNA repair and β-catenin signaling. This suggests a high level of conservation of metanephric kidney development at the genetic level. Full article

Aging is associated with changes in intramuscular collagen structure and metabolism, which may impair mechanical adaptability and regenerative capacity. We investigated the effects of aging and repeated passive stretching on intramuscular collagen remodeling in the tibialis anterior muscles of mice. The tibialis anterior muscles of young and aged mice were exposed to repeated passive stretching, and the localization of collagen and collagen-related factors was evaluated. Baseline gene expression of collagens I and IV was significantly reduced in aged muscles and was not restored by stretching. Repeated passive stretching increased the area and intensity of collagen I immunoreactivity in both young and aged mice but produced little change in collagen IV. Stretch-induced dynamic changes in lysyl oxidase-positive cells in the extracellular matrix (ECM) were evident in young mice but were markedly attenuated in aged mice. In addition, matrix metalloproteinases (MMP2 and MMP9) mRNA and protein expressions did not differ between groups. No significant age- or stretch-dependent changes were observed in the localization of advanced glycation end products. These findings suggest that although the increase in fibrillar collagen in response to stretching is maintained with aging, the regulatory mechanisms controlling ECM stabilization, particularly those related to cross-linking dynamics, may be impaired. Full article

Lower urinary tract dysfunction (LUTD) is a multifactorial disease process that encompasses diverse symptoms ranging from issues with storage and sensation to impaired emptying of the bladder. Furthermore, symptoms tend to worsen with age and other comorbidities and in men can also be influenced by changes to the prostate, making diagnosis and treatment difficult to manage. Environmental factors are thought to contribute to disease risk. In mice, previous work has found that developmental exposure to polychlorinated biphenyls (PCBs) is capable of altering voiding function in offspring. However, the effects of PCB exposure in adulthood instead of development are not well known. Whether changes in voiding are a consequence of early or later life exposures remains an important area of study, as environmental chemicals and exposures can occur across the lifespan and can be mitigated. Here, we test whether PCB exposure in adulthood alters voiding or prostate morphology in male mice. C57Bl/6J adult male mice were exposed to the human-relevant MARBLES PCB mixture (0, 0.1, 1, and 6 mg/kg/d) orally daily for two months. Lower urinary tract function was then assessed through urodynamic testing including void spot assay, uroflowmetry, and anesthetized cystometry. Prostate lobes were collected for histology. The only change to voiding function was a reduction in void duration in the 6 versus 1 mg/kg/d PCB group but not to the vehicle control. Prostate, seminal vesicle, and testes wet weights were unchanged. However, PCB exposure reduced the number of Ki67-positive proliferating cells in the anterior and ventral prostate lobes only at the 1 mg/kg/d dose, with no change to caspase 3-positive cells or smooth muscle thickness. Together, these data indicate that 2-month exposure to PCBs in adult mice has little impact on voiding but is a sufficient exposure to produce changes in cell proliferation in the prostate. The mechanistic impacts of these changes remains to be investigated but could help better understand individual risk for LUTD. Full article

Background/Objectives: A high-fat diet disrupts metabolic and neuroimmune balance in the brain, making neural tissue more reactive to inflammatory challenges. However, it is not well understood how this vulnerability varies across brain regions or how natural anti-inflammatory compounds influence it. Methods: In this study, we examined how the garlic-derived molecule L-alliin modulates the inflammatory response triggered by lipopolysaccharide in the frontal cortex, hippocampus, and hypothalamus of mice fed either a standard or high-fat diet. Results: Measurements of cytokine gene expression showed that the high-fat diet greatly increased the inflammatory response in the frontal cortex and hypothalamus, with the hypothalamus displaying the strongest overall activation. Treatment with L-alliin significantly reduced elevated cytokine levels in both regions, with the reductions most pronounced in animals on the high-fat diet. In contrast, the hippocampus showed a distinct pattern: expression of TNF-α and IL-1β changed very little across diets or treatments, whereas IL-6 and CCL2 were selectively altered by L-alliin, depending on the animals’ metabolic state. Conclusions: These findings demonstrate that diet-induced obesity does not affect the entire brain uniformly. Instead, inflammatory pathways are altered region-specifically, and L-alliin modulates these pathways with sensitivity to both brain region and metabolic condition. This work emphasizes the importance of accounting for neuroanatomical differences when developing strategies to reduce inflammation in obesity-associated conditions. Full article

Background/Objectives: Biotin plays important roles in critical metabolic reactions and also contributes to the regulation of gene expression. While its role in regulating gene expression via transcriptional/epigenetic mechanisms is well established, little is known about its ability to regulate expression at the post-transcriptional level. Methods: To address this, we examined how biotin deficiency affects microRNAs (miRNAs) expression in the colon, a tissue that is impacted by deficiency of this micronutrient. Results: We identified (by miRNA sequencing) 26 miRNAs whose expression was significantly altered in the colon of biotin-deficient mice compared with pair-fed controls. Among these, ten miRNAs with known roles in mucosal physiology and inflammation were selected for direct validation, and their altered expression patterns were confirmed by RT-qPCR. In silico analyses further revealed that important proteins involved in maintaining normal colonic function (the tight junction protein ZO1 and the stem cell marker LGR5) and in mediating inflammation (NLRP3 and calprotectin), all of which are dysregulated in biotin deficiency, possess putative binding sites on 3′-UTR for several of the altered miRNAs. Moreover, transient transfection of miR-190a-5p and miR-199a-5p, whose expression was upregulated during biotin deficiency and predicted to target ZO1 and LGR5, respectively, in human colonic NCM460 cells, led to a significant reduction in the level of the corresponding mRNAs, confirming functional regulation of these targets. IPA further showed that the differentially expressed miRNAs are associated with gastrointestinal and inflammatory disease pathways. Conclusions: Findings of this investigation show that biotin deficiency disrupts colonic miRNA expression, potentially contributing to downstream alterations in important physiological and inflammatory protein expression. Full article

Introduction: The pro-inflammatory cytokine interleukin-17A (IL-17A) has a key role in the inflammatory cascade and promotes vascular inflammation and dysfunction. In addition, IL-17A is centrally involved in several autoimmune diseases. IL-17A deficiency has been linked to reduced vascular inflammation associated with attenuated arterial hypertension under long-term angiotensin II (Ang II) exposure for four weeks. This is of interest as IL-17A is one factor linking several autoimmune diseases with cardiovascular comorbidity. So far, little is known about the effects of IL-17A during the early stages of vascular dysfunction development—an interval possibly representing an optimal therapeutic window. Methods: Mice lacking the IL-17A receptor alpha (IL-17RAdel) and wild-type counterparts were treated with Ang II for one week (1 mg/kg bodyweight/week). We assessed systemic oxidative stress formation and vascular function, as well as inflammatory cells in the vessel wall. In parallel, C57BL/6J mice treated with Ang II received anti-IL-17A therapy, to evaluate the same parameters. Results: Both IL-17RA-deficient mice and anti-IL-17A-treated C57BL/6J mice exhibited an attenuated oxidative stress response and mitigated vascular inflammation following one week of Ang II treatment. These effects did not significantly prevent the onset of Ang II-induced vascular dysfunction at that timepoint. Conclusions: After one week of Ang II treatment, antagonizing IL-17RA or IL-17A only partially reduced/attenuated the Ang II-induced effects on the vasculature. In the context of IL-17A-driven autoimmune diseases with associated vascular pathology, our findings suggest that anti-inflammatory therapies alone may not be sufficient to attenuate vascular impairment. A combined approach including agents with direct protective vascular effects may be required for effective intervention for the associated vascular comorbidity. Full article