Newborn Mice Research Articles

P300 Maintains Primordial Follicle Activation by Repressing VEGFA Transcription.

During the reproductive life, most primordial follicles remain dormant for years or decades, while some are progressively activated for development. Misactivation of primordial follicles can cause ovarian diseases, for example, premature ovarian insufficiency (POI). Our results show that p300 expression increased with primordial follicle activation. Using a p300 inhibitor resulted in premature activation of primordial follicles in cultured mouse ovaries. Conversely, the ratio of primordial follicle activation was markedly decreased upon culturing with the p300 agonist. Furthermore, p300 regulated primordial follicle activation by inhibiting Vegfa transcription in granulosa cells. Additionally, this study was extended to potential clinical applications, showing that short-term treatment with a p300 inhibitor in vitro significantly increased primordial follicle activation in newborn mouse ovaries after dorsal kidney membrane transplantation in female NSG mice. Our results revealed that p300 controls the activation of primordial follicles in mammalian ovaries.

Evaluation of Postoperative Outcomes Following Early and Late Palate Repair: A Preclinical Study.

To quantitatively assess the impact of early versus late surgical intervention on midfacial growth using a mouse model. A full-thickness mucoperiosteal flap surgery was performed on newborn (P17) mice and on neonatal (P30) mice. High-resolution micro-computed tomographic imaging coupled with histomorphometric analyses was used to assess craniomaxillofacial growth. Histology and immunohistochemical analyses were used to assess cellular and molecular responses postsurgery. Early surgical intervention at P17 resulted in significant midfacial growth arrest, with pronounced maxillary hypoplasia. Histomorphometric analyses revealed significant (P < 0.05) growth disruptions in the mid-palatal suture complex, including premature removal of the cartilaginous growth plate and its replacement by bone. In the suture itself, cell proliferation was significantly reduced (P < 0.05) compared with controls. The same surgical intervention performed in mice at P30 did not lead to significant midfacial growth arrest. Early surgical intervention in a mouse model mirrors the adverse growth outcomes in children undergoing early cleft repair. Molecular and cellular observations accompanying this midfacial growth arrest may inform therapeutic strategies to mitigate midfacial growth disturbances in patients and highlight the need for refined surgical techniques to minimize adverse growth outcomes.

Loss of Surfactant Protein A Alters Perinatal Lung Morphology and Susceptibility to Hyperoxia-Induced Bronchopulmonary Dysplasia

Bronchopulmonary dysplasia (BPD) is a condition of poor alveolar formation that causes chronic breathing impairment in infants born prematurely. Preterm lungs lack surfactant and are vulnerable to oxidative injuries driving the development of BPD. Our recent studies reported that surfactant protein A (SP-A) genetic variants influence susceptibility to neonatal lung disease. SP-A modulates activation of alveolar macrophages and parturition onset in late gestation. We asked whether a lack of SP-A alters alveolarization in a mouse model of hyperoxia-induced BPD. SP-A-deficient and control newborn mice were exposed to either clinically relevant 60% O2 hyperoxia or normoxia for 5–7 days. Alveolar formation was then assessed by mean linear intercept (MLI) and radial alveolar count (RAC) measurements in lung tissue sections. We report that the combination of SP-A deficiency and hyperoxia reduces alveolar growth compared to WT mice. The morphometric analysis of normoxic SP-A-deficient lungs showed lower RAC compared to controls, indicating reduced alveolar number. In the presence of hyperoxia, MLI was higher in SP-A-deficient lungs compared to controls. Differences were statistically significant for female pups. Spatial proteomic profiling of lung tissue sections showed that hyperoxia caused a 4-fold increase in the DNA damage marker γH2Ax in macrophages of SP-A-deficient lungs compared to normoxia. Our short report suggests an important role for SP-A in perinatal lung development and the protection of lung macrophages from oxidant injury. These studies warrant future investigation to discern the temporal interaction of SP-A, gender, oxidant injury, and lung macrophages in perinatal alveolar formation and development of BPD.

Immune cells promote paralytic disease in mice infected with enterovirus D68.

Enterovirus D68 (EV-D68) is associated with acute flaccid myelitis (AFM), a poliomyelitis-like illness causing paralysis in young children. However, mechanisms of paralysis are unclear, and antiviral therapies are lacking. To better understand EV-D68 disease, we inoculated newborn mice intracranially to assess viral tropism, virulence, and immune responses. Wild-type (WT) mice inoculated intracranially with a neurovirulent strain of EV-D68 showed infection of spinal cord neurons and developed paralysis. Spinal tissue from infected mice revealed increased levels of chemokines, inflammatory monocytes, macrophages, and T cells relative to controls, suggesting that immune cell infiltration influences pathogenesis. To define the contribution of cytokine-mediated immune cell recruitment to disease, we inoculated mice lacking CCR2, a receptor for several EV-D68-upregulated cytokines, or RAG1, which is required for lymphocyte maturation. WT, Ccr2 -/- , and Rag1 -/- mice had comparable viral titers in spinal tissue. However, Ccr2 -/- and Rag1 -/- mice had significantly less paralysis relative to WT mice. Consistent with impaired T cell recruitment to sites of infection in Ccr2 -/- and Rag1 -/- mice, antibody-mediated depletion of CD4 + or CD8 + T cells from WT mice diminished paralysis. These results indicate that immune cell recruitment to the spinal cord promotes EV-D68-associated paralysis and illuminate new targets for therapeutic intervention.

Employing Multi-Omics Analyses to Understand Changes during Kidney Development in Perinatal Interleukin-6 Animal Model.

Chronic kidney disease (CKD) is a leading cause of morbidity and mortality globally. Maternal obesity during pregnancy is linked to systemic inflammation and elevated levels of the pro-inflammatory cytokine interleukin-6 (IL-6). In our previous work, we demonstrated that increased maternal IL-6 during gestation impacts intrauterine development in mice. We hypothesized that IL-6-induced inflammation alters gene expression in the developing fetus. To test this, pregnant mice were administered IL-6 or saline during mid-gestation. Newborn mouse kidneys were analyzed using mRNA-seq, miRNA-seq and whole-genome bisulfite-seq (WGBS). A multi-omics approach was employed to quantify mRNA gene expression, miRNA expression and DNA methylation, using advanced bioinformatics and data integration techniques. Our analysis identified 19 key genes present in multiple omics datasets, regulated by epigenetics and miRNAs. We constructed a regulatory network for these genes, revealing disruptions in pathways such as Mannose type O-glycan biosynthesis, the cell cycle, apoptosis and FoxO signaling. Notably, the Atp7b gene was regulated by DNA methylation and miR-223 targeting, whereas the Man2a1 gene was controlled by DNA methylation affecting energy metabolism. These findings suggest that these genes may play a role in fetal programming, potentially leading to CKD later in life due to gestational inflammation.

Prenatal Exposure to Herbicide 2,4-Dichlorophenoxyacetic Acid (2,4D) Exacerbates Zika Virus Neurotoxicity In Vitro and In Vivo.

Zika virus (ZIKV) infection during pregnancy can lead to a set of congenital malformations known as Congenital ZIKV syndrome (CZS), whose main feature is microcephaly. The geographic distribution of CZS in Brazil during the 2015-2017 outbreak was asymmetrical, with a higher prevalence in the Northeast and Central-West regions of the country, despite the ubiquitous distribution of the vector Aedes aegypti, indicating that environmental factors could influence ZIKV vertical transmission and/or severity. Here we investigate the involvement of the most used agrochemicals in Brazil with CZS. First, we exposed human neuroblastoma SK-N-AS cells to the 15 frequently used agrochemical molecules or derivative metabolites able to cross the blood-brain barrier. We found that a derived metabolite from a widely used herbicide in the Central-West region, 2,4-dichlorophenoxyacetic acid (2,4D), exacerbates ZIKV neurotoxic effects in vitro. We validate this observation by demonstrating vertical transmission leading to microcephaly in the offspring of immunocompetent C57BL/6J mice exposed to water contaminated with 0.025 mg/L of 2,4D. Newborn mice whose dams were exposed to 2,4D and infected with ZIKV presented a smaller brain area and cortical plate size compared to the control. Also, embryos from animals facing the co-insult of ZIKV and 2,4D exposition presented higher Caspase 3 positive cells in the cortex, fewer CTIP2+ neurons and proliferative cells at the ventricular zone, and a higher viral load. This phenotype is followed by placental alterations, such as vessel congestion, and apoptosis in the labyrinth and decidua. We also observed a mild spatial correlation between CZS prevalence and 2,4D use in Brazil's North and Central-West regions, with R2 = 0.4 and 0.46, respectively. Our results suggest that 2,4D exposition facilitates maternal vertical transmission of ZIKV, exacerbating CZS, possibly contributing to the high prevalence of this syndrome in Brazil's Central-West region compared to other regions.

Isolation and Whole-Cell Patch-Clamp Recording of Hippocampal Microglia from Adult Mice.

Microglia are resident immune cells in the brain that interact with neurons to maintain the homeostasis of the central nervous system (CNS). Studies show that the microglial surface expresses potassium channels that regulate microglial activation, while abnormalities in these potassium channels can lead to neural diseases. Currently, whole-cell patch-clamp recordings of microglia are mostly performed on cultured primary microglia from fetal or newborn mice due to difficulties in conducting electrophysiological evaluations on acutely isolated microglia. This study introduces an easy-to-follow protocol for isolating hippocampal microglia from adult mice and performing whole-cell patch-clamp recordings on the isolated cells. Briefly, the brain was removed from a mouse after decapitation, the hippocampus was dissected bilaterally, and microglia were isolated using an adult mouse brain dissociation kit. The microglia were then purified using a magnetic-activated cell sorting (MACS) method and seeded onto coverslips. Successful microglial isolation was confirmed by immunofluorescent staining with anti-CD11 and anti-Iba1 antibodies. A cover slip was placed in a recording chamber, and the whole-cell potassium currents of the acutely isolated microglia were recorded under voltage-clamp conditions.

Gene therapy of Dent disease type 1 in newborn ClC-5 null mice for sustained transgene expression and gene therapy effects.

Dent disease type 1 is caused by changes in the chloride voltage-gated channel 5 (CLCN5) gene on chromosome X, resulting in the lack or dysfunction of chloride channel ClC-5. Individuals affected by Dent disease type 1 show proteinuria and hypercalciuria. Previously we found that lentiviral vector-mediated hCLCN5 cDNA supplementary therapy in ClC-5 null mice was effective only for three months following gene delivery, and the therapeutic effects disappeared four months after treatment, most likely due to immune responses to the ClC-5 proteins expressed in the treated cells. Here we tried two strategies to reduce possible immune responses: 1) confining the expression of ClC-5 expression to the tubular cells with tubule-specific Npt2a and Sglt2 promoters, and 2) performing gene therapy in newborn mutant mice whose immune system has not fully developed. We found that although Npt2a and Sglt2 promoters successfully drove ClC-5 expression in the kidneys of the mutant mice, the treatment did not ameliorate the phenotypes. However, gene delivery to the kidneys of newborn Clcn5 mutant mice enabled long-term transgene expression and phenotype improvement. Our data suggest that performing gene therapy on Dent disease affected subjects soon after birth could be a promising strategy to attenuate immune responses in Dent disease type 1 gene therapy.

Sialic acid and PirB are not required for targeting of neural circuits by neurotropic mammalian orthoreovirus.

Serotype 3 (T3) strains of mammalian orthoreovirus (reovirus) spread to the central nervous system to infect the brain and cause lethal encephalitis in newborn mice. Although reovirus targets several regions in the brain, susceptibility to infection is not uniformly distributed. The neuronal subtypes and anatomic sites targeted throughout the brain are not precisely known. Reovirus binds several attachment factors and entry receptors, including sialic acid (SA)-containing glycans and paired immunoglobulin-like receptor B (PirB). While these receptors are not required for infection of some types of neurons, reovirus engagement of these receptors can influence neuronal infection in certain contexts. To identify patterns of T3 neurotropism, we used microbial identification after passive tissue clearance and hybridization chain reaction to stain reovirus-infected cells throughout intact, optically transparent brains of newborn mice. Three-dimensional reconstructions revealed in detail the sites targeted by reovirus throughout the brain volume, including dense infection of the midbrain and hindbrain. Using reovirus mutants incapable of binding SA and mice lacking PirB expression, we found that neither SA nor PirB is required for the infection of various brain regions. However, SA may confer minor differences in infection that vary by region. Collectively, these studies indicate that many regions in the brain of newborn mice are susceptible to reovirus and that patterns of reovirus infection are not dependent on reovirus receptors SA and PirB.IMPORTANCENeurotropic viruses invade the central nervous system (CNS) and target various cell types to cause disease manifestations, such as meningitis, myelitis, or encephalitis. Infections of the CNS are often difficult to treat and can lead to lasting sequelae or death. Mammalian orthoreovirus (reovirus) causes age-dependent lethal encephalitis in many young mammals. Reovirus infects neurons in several different regions of the brain. However, the complete pattern of CNS infection is not understood. We found that reovirus targets almost all regions of the brain and that patterns of tropism are not dependent on receptors sialic acid and paired immunoglobulin-like receptor B. These studies confirm that two known reovirus receptors do not completely explain the cell types infected in brain tissue and establish strategies that can be used to understand complete patterns of viral tropism in an intact brain.

Postnatal hypoxic preconditioning attenuates lung damage from hyperoxia in newborn mice.

Preterm infants frequently require oxygen supplementation at birth. However, preterm lung is especially sensible to structural and functional damage caused by oxygen free radicals. The adaptive mechanisms implied in the fetal-neonatal transition from a lower to a higher oxygen environment were evaluated in a murine model using a custom-designed oxy-chamber. Pregnant mice were randomly assigned to deliver in 14% (hypoxic preconditioning group) or 21% (normoxic group) oxygen environment. Eight hours after birth FiO2 was increased to 100% for 60 min and then switched to 21% in both groups. A control group remained in 21% oxygen throughout the study. Mice in the normoxic group exhibited thinning of the alveolar septa, increased cell death, increased vascular damage, and decreased synthesis of pulmonary surfactant. However, lung histology, lamellar bodies microstructure, and surfactant integrity were preserved in the hypoxic preconditioning group after the hyperoxic insult. Postnatal hyperoxia has detrimental effects on lung structure and function when preceded by normoxia compared to controls. However, postnatal hypoxic preconditioning mitigates lung damage caused by a hyperoxic insult. Hypoxic preconditioning, implemented shortly after birth mitigates lung damage caused by postnatal supplemental oxygenation. The study introduces an experimental mice model to investigate the effects of hypoxic preconditioning and its effects on lung development. This model enables researchers to delve into the intricate processes involved in postnatal lung maturation. Our findings suggest that hypoxic preconditioning may reduce lung parenchymal damage and increase pulmonary surfactant synthesis in reoxygenation strategies during postnatal care.

A novel, centrally acting mammalian aminosterol, ENT-03, induces weight loss in obese and lean rodents.

ENT-03, a spermine bile acid we recently discovered in the brain of newborn mice acts centrally to regulate energy and metabolism. Obese, diabetic (ob/ob) mice treated with five doses of ENT-03 over 2 weeks, demonstrated a rapid decrease in blood glucose levels into the range seen in non-obese animals, prior to any significant weight loss. Weight fell substantially thereafter as food intake decreased, and serum biochemical parameters normalized compared with both vehicle and pair-fed controls. To determine whether ENT-03 could be acting centrally, we injected a single dose of ENT-03 intracerebroventricularly to Sprague-Dawley rats. Weight fell significantly and remained below vehicle injected controls for an extended period. By autoradiography, ENT-03 localized to the arcuate nucleus of the hypothalamus, the choroid plexus and cerebrospinal fluid. Significant cFosactivation occurred in multiple anatomical regions within the hypothalamus and brainstem involved in appetite suppression, food-entrained circadian rhythmicity, autonomic function, and growth. These data support a role for ENT-03 in the treatment of type 2 diabetes and obesity. Phase 1 studies in subjects with obesity and diabetes are currently in progress.

PPARγ drives mitochondrial stress signaling and the loss of atrial cardiomyocytes in newborn mice exposed to hyperoxia

Diastolic dysfunction is increasingly common in preterm infants exposed to supplemental oxygen (hyperoxia). Previous studies in neonatal mice showed hyperoxia suppresses fatty acid synthesis genes required for proliferation and survival of atrial cardiomyocytes. The loss of atrial cardiomyocytes creates a hypoplastic left atrium that inappropriately fills the left ventricle during diastole. Here, we show that hyperoxia stimulates adenosine monophosphate-activated kinase (AMPK) and peroxisome proliferator activated receptor-gamma (PPARγ) signaling in atrial cardiomyocytes. While both pathways can regulate lipid homeostasis, PPARγ was the primary pathway by which hyperoxia inhibits fatty acid gene expression and inhibits proliferation of mouse atrial HL-1 cells. It also enhanced the toxicity of hyperoxia by increasing expression of activating transcription factor (ATF) 5 and other mitochondrial stress response genes. Silencing PPARγ signaling restored proliferation and survival of HL-1 cells as well as atrial cardiomyocytes in neonatal mice exposed to hyperoxia. Our findings reveal PPARγ enhances the toxicity of hyperoxia on atrial cardiomyocytes, thus suggesting inhibitors of PPARγ signaling may prevent diastolic dysfunction in preterm infants.

Abstract 61: Protective role of caspase-1 inhibition in experimental model of neonatal hyperoxia-induced cardio pulmonary dysfunction in adults born preterm.

Background: Adults born preterm are at an increased risk of systemic hypertension and cardiopulmonary morbidities. However, little is known of the underlying mechanisms connecting neonatal hyperoxia (O 2 ) exposure to these long-term cardiovascular consequences in adults born preterm. Pro-inflammatory caspase-1 mediates the activation of cytokines IL-1β, and pore-forming protein Gasdermin-D. Objective: Test the hypothesis that caspase-1 inhibition prevents long-term cardiopulmonary consequences of preterm birth. Design/Methods: Newborn mice (n=60) exposed to normoxia (RA) or hyperoxia (O 2 ) from postnatal day (P) 1 to 14 were randomly assigned to receive daily intraperitoneal (IP) injections of caspase-1 inhibitor (VX-765, 50 ug/Kg) or DMSO as placebo. The mouse pups recovered in RA into adulthood until 3 months of age. The effect of caspase-1 inhibition on Blood pressure (BP), exercise endurance, cardiac function, systemic vascular stiffness, pulmonary vascular remodeling was assessed at 3 months. Results: Adult rats exposed to neonatal O 2 had significantly increased BP associated with vascular stiffness, reduced exercise capacity, cardiac hypertrophy, and cardiovascular inflammation. Caspase-1 inhibition prevented systemic hypertension, systemic vascular stiffness, improved exercise tolerance and decreased cardiac hypertrophy (Fig 1A-D) in adult mice exposed to neonatal O 2 . Moreover, caspase -1 inhibition improved pulmonary vascular remodeling and pulmonary hypertension in these mice (Fig 2A-E). Additionally, western blot analysis demonstrated that early supplementation of VX-765 in O 2 exposed mice led to reduced cardiac proinflammatory markers GSDMD and IL-1β in adulthood (Fig 3A-B). Conclusion: Neonatal O 2 exposure predisposes to cardiopulmonary morbidities in adults born preterm. Inhibition of caspase-1 reduces cardiovascular inflammation and cardiopulmonary dysfunction in mice exposed to hyperoxia during the neonatal period. The functional and molecular impact of the use of caspase-1 suggests that it can be used as a novel preventive strategy for long-term cardiopulmonary dysfunction due to neonatal O 2 exposure in preterm survivors.

Metabolic immaturity and breastmilk bile acid metabolites are central determinants of heightened newborn vulnerability to norovirus diarrhea

The pathogenic outcome of enteric virus infections is governed by a complex interplay between the virus, intestinal microbiota, and host immune factors, with metabolites serving as a key mediator. Noroviruses bind bile acid metabolites, which are produced by the host and then modified by commensal bacteria. Paradoxically, bile acids can have both proviral and antiviral roles during norovirus infections. Working in an infant mouse model of norovirus infection, we demonstrate that microbiota and their bile acid metabolites protect from norovirus diarrhea, whereas host bile acids promote disease. We also find that maternal bile acid metabolism determines the susceptibility of newborn mice to norovirus diarrhea during breastfeeding. Finally, targeting maternal and neonatal bile acid metabolism can protect newborn mice from norovirus disease. In summary, neonatal metabolic immaturity and breastmilk bile acids are central determinants of heightened newborn vulnerability to norovirus disease.

Participation of ventricular trabeculae in neonatal cardiac regeneration leads to ectopic recruitment of Purkinje-like cells.

Unlike adult mammals, newborn mice can regenerate a functional heart after myocardial infarction; however, the precise origin of the newly formed cardiomyocytes and whether the distal part of the conduction system (the Purkinje fiber (PF) network) is properly formed in regenerated hearts remains unclear. PFs, as well as subendocardial contractile cardiomyocytes, are derived from trabeculae, transient myocardial ridges on the inner ventricular surface. Here, using connexin 40-driven genetic tracing, we uncover a substantial participation of the trabecular lineage in myocardial regeneration through dedifferentiation and proliferation. Concomitantly, regeneration disrupted PF network maturation, resulting in permanent PF hyperplasia and impaired ventricular conduction. Proliferation assays, genetic impairment of PF recruitment, lineage tracing and clonal analysis revealed that PF network hyperplasia results from excessive recruitment of PFs due to increased trabecular fate plasticity. These data indicate that PF network hyperplasia is a consequence of trabeculae participation in myocardial regeneration.

Effects of 1-oleate-2-palmitate-3-linoleate glycerol supplementation on the small intestinal development and gut microbial composition of neonatal mice

Recent studies have shown that 1-oleo-2-palmito-3-linoleyl glycerol (OPL) is the most abundant triacylglycerol in human breast milk in China. Epidemiologic studies have shown that sn-2 palmitate improves the absorption of fatty acids and calcium in infants. However, there have been few studies of the specific mechanism by which OPL affects intestinal function. In the present study, we have characterized the effects of various levels of OPL supplementation on the development of the intestinal epithelium and the intestinal microbiota of neonatal mice. OPL supplementation increased the body masses and intestinal lengths of weaned mice and promoted defecation. These positive effects were related to the effect of OPL to promote the development of intestinal villi and crypts. OPL increased the expression of the intestinal stem cell markers Olfm4 and Sox9 in the jejunum and ileum, which promoted their differentiation into goblet cells and Paneth cells. It also promoted the integrity of the epithelial barrier by increasing the secretion of mucin 2 and lysozyme 1 and the expression of the tight junction proteins occludin, ZO1, claudin 2, and claudin 3. More importantly, we found that low dose-OPL promotes the transformation of the intestinal microbiota of neonatal mice to the mature state in 3-month-old mice, increases the proportion of Firmicutes, and reduces the proportion of Bacteroidota. The proportions of anaerobic genera of bacteria, such as Lachnospiraceae_NK4A136_group, Lachnoclostridium, Ligilactobacillus, and Bifidobacterium were higher, as were the key producers of short-chain fatty acids, such as Bacteroides and Blautia. OPL also increased the butyric acid content of the feces, which significantly correlated with the abundance of Lactobacillus. High-dose OPL tended to be more effective at promoting defecation and the development of the villi and crypts, but these effects did not significantly differ from those achieved using the lower dose. A low dose of OPL was more effective at increasing the butyric acid content and causing the maturation of microbes. In summary, the OPL supplementation of newborn mice promotes the establishment of the intestinal epithelial layer structure and barrier function, and also promotes the transformation of the intestinal microbiota to a mature state. This study lays a theoretical foundation for the inclusion of OPL in infant formula and provides a scientific basis for the development of intestinal health products.

Plasticity of mitochondrial proton leak in adults and newborn mice as a possible strategy for high altitude adaptation

Plasticity of mitochondrial proton leak in adults and newborn mice as a possible strategy for high altitude adaptation

Transplacental delivery of factor IX Fc-fusion protein ameliorates bleeding phenotype of newborn hemophilia B mice

Hemophilia B is an inherited hemorrhagic disorder characterized by a deficiency of blood coagulation factor IX (FIX) that results in abnormal blood coagulation. The blood coagulation is already evident in hemophiliacs at the fetal stage, and thus intracranial hemorrhage and other bleeding complications can occur at birth, leading to sequelae. Therefore, it is important to develop effective treatments for hemophiliacs in utero. In this study, in order to transplacentally deliver FIX from pregnant mice to their fetuses, an improved adenovirus (Ad) vector expressing human FIX fused with the IgG Fc domain (FIX Fc fusion protein), which plays a crucial role in neonatal Fc receptor (FcRn)-mediated transcytosis across the placenta, was intravenously administered to E13.5 pregnant mice. Significant levels of FIX Fc fusion protein were detected in 0-day-old newborn mice whose mothers were administered an Ad vector expressing FIX Fc fusion protein. Wild-type FIX overexpressed in the pregnant mice was not delivered to the fetuses. Plasma FIX levels in the newborn mice were relatively well correlated with those in their mothers, although transplacental delivery efficiencies of FIX Fc fusion protein were slightly reduced when the FIX Fc fusion protein was highly expressed in the mother mice. Plasma FIX levels in the newborn mice were about 3.6–6.4% of those in their mothers, Transplacental delivery of FIX Fc fusion protein to their fetuses successfully improved the blood clotting ability in the newborn mice.

Use of proteomics to elucidate characteristics of Cronobacter sakazakii under mild heat stress

Cronobacter sakazakii is an opportunistic pathogen known for causing severe diseases. Mild heat treatment is commonly used in food processing, however, the pathogenic characteristics and underlying mechanisms of Cronobacter sakazakii strains remain poorly understood. In this study, we found that mild heat stress (MHS) at 52 °C can induce several deleterious effects in Cronobacter sakazakii, including damage to the cell wall, genomic DNA breakage, and misfolding of cytoplasmic proteins. These conditions lead to a decreased survival ability under acid, desiccation, and osmotic stress; a reduction in biofilm formation; and diminished motility. Notably, surviving C. sakazakii cells retain their pathogenicity, causing significant intestinal damage in newborn mice. This damage is characterized by epithelial sloughing and disruption of the intestinal structure. Tandem mass tag (TMT)-based proteomics identified 736 proteins with differential abundance across C. sakazakii strains subjected to mild heat stress, highlighting adaptations in biofilm formation, motility, and stress tolerance. Key regulatory changes were observed in phospholipid metabolism and protein synthesis, which underpin this complex stress response. This data illustrates a sophisticated balance between environmental adaptability and pathogenic potential. The metabolic and pathogenic responses of C. sakazakii to mild heat stress are closely linked to its phospholipid metabolism and the production of secretory proteins, both crucial for its virulence and reliant on membrane transport. This complex interplay emphasizes the need to understand these mechanisms to develop effective control strategies.

A GCaMP reporter mouse with chondrocyte specific expression of a green fluorescent calcium indicator

One of the major processes occurring during the healing of a fractured long bone is chondrogenesis, leading to the formation of the soft callus, which subsequently undergoes endochondral ossification and ultimately bridges the fracture site. Thus, understanding the molecular mechanisms of chondrogenesis can enhance our knowledge of the fracture repair process. One such molecular process is calciun (Ca++) signaling, which is known to play a critical role in the development and regeneration of multiple tissues, including bone, in response to external stimuli. Despite the existence of various mouse models for studying Ca++ signaling, none of them were designed to specifically examine the skeletal system or the various musculoskeletal cell types. As such, we generated a genetically engineered mouse model that is specific to cartilage (crossed with Col2a1 Cre mice) to study chondrocytes. Herein, we report on the characterization of this transgenic mouse line using conditional expression of GCaMP6f, a Ca++-indicator protein. Specifically, this mouse line exhibits increased GCaMP6f fluorescence following Ca++ binding in chondrocytes. Using this model, we show real-time Ca++ signaling in embryos, newborn and adult mice, as well as in fracture calluses. Further, robust expression of GCaMP6f in chondrocytes can be easily detected in embryos, neonates, adults, and fracture callus tissue sections. Finally, we also report on Ca++ signaling pathway gene expression, as well as real-time Ca++ transient measurements in fracture callus chondrocytes. Taken together, these mice provide a new experimental tool to study chondrocyte-specific Ca++ signaling during skeletal development and regeneration, as well as various in vitro perturbations.