The membrane asymmetry regulated by P4-ATPases is crucial for the functioning of eukaryotic cells. The underlying spatial translocation or flipping of specific lipids is usually assured by respective P4-ATPases coupled to conforming non-catalytic subunits. Our previous work has identified five P4-ATPases (TgP4-ATPase1-5) and three non-catalytic partner proteins (TgLem1-3) in the intracellular protozoan pathogen, Toxoplasma gondii. However, their flipping activity, physiological relevance and functional coupling remain unknown. Herein, we demonstrate that TgP4-ATPase1 and TgLem1 work together to translocate phosphatidylserine (PtdSer) during the lytic cycle of T. gondii. Both proteins localize in the plasma membrane at the invasive (apical) end of its acutely-infectious tachyzoite stage. The genetic knockout of P4-ATPase1 and conditional depletion of Lem1 in tachyzoites severely disrupt the asexual reproduction and translocation of PtdSer across the plasma membrane. Moreover, the phenotypic analysis of individual mutants revealed a requirement of lipid flipping for the motility, egress and invasion of tachyzoites. Not least, the proximity-dependent biotinylation and reciprocal immunoprecipitation assays demonstrated the physical interaction of P4-ATPase1 and Lem1. Our findings disclose the mechanism and significance of PtdSer flipping during the lytic cycle and identify the P4-ATPase1-Lem1 heterocomplex as a potential drug target in T. gondii.

Heart failure is caused by various factors, making the underlying pathogenic mechanisms difficult to identify. Since cardiovascular disease tends to worsen over time, early diagnosis is key for treatment. In addition, understanding the qualitative changes in the heart associated with aging, where information on the direct influences of aging on cardiovascular disease is limited, would also be useful for treatment and diagnosis. To fill these research gaps, the focus of our study was to detect the structural and functional molecular changes associated with the heart over time, with a focus on glycans, which reflect the type and state of cells.
METHODS: We investigated glycan localization in the cardiac tissue of normal mice and their alterations during aging, using evanescent-field fluorescence-assisted lectin microarray, a technique based on lectin-glycan interaction, and lectin staining.
RESULTS: The glycan profiles in the left ventricle showed differences between the luminal side (medial) and wall side (lateral) regions. The medial region was characterized by the presence of sialic acid residues. Moreover, age-related changes in glycan profiles were observed at a younger age in the medial region. The difference in the age-related decrease in the level of α-galactose stained with Griffonia simplicifolia lectin-IB4 in different regions of the left ventricle suggests spatiotemporal changes in the number of microvessels.
CONCLUSIONS: The glycan profile, which retains diverse glycan structures, is supported by many cell populations, and maintains cardiac function. With further research, glycan localization and changes have the potential to be developed as a marker of the signs of heart failure.

The use of computer-aided methods have continued to propel accelerated drug discovery across various disease models, interestingly allowing the specific inhibition of pathogenic targets. Chloride Intracellular Channel Protein 4 (CLIC4) is a novel class of intracellular ion channel highly implicated in tumor and vascular biology. It regulates cell proliferation, apoptosis and angiogenesis; and is involved in multiple pathologic signaling pathways. Absence of specific inhibitors however impedes its advancement to translational research. Here, we integrate structural bioinformatics and experimental research approaches for the discovery and validation of small-molecule inhibitors of CLIC4. High-affinity allosteric binders were identified from a library of 1615 Food and Drug Administration (FDA)-approved drugs via a high-performance computing-powered blind-docking approach, resulting in the selection of amphotericin B and rapamycin. NMR assays confirmed the binding and conformational disruptive effects of both drugs while they also reversed stress-induced membrane translocation of CLIC4 and inhibited endothelial cell migration. Structural and dynamics simulation studies further revealed that the inhibitory mechanisms of these compounds were hinged on the allosteric modulation of the catalytic glutathione (GSH)-like site loop and the extended catalytic β loop which may elicit interference with the catalytic activities of CLIC4. Structure-based insights from this study provide the basis for the selective targeting of CLIC4 to treat the associated pathologies.

UNASSIGNED: Although Staphylococcus aureus is the leading cause of biofilm-related infections, the lipidomic distributions within these biofilms is poorly understood. Here, lipidomic mapping of S. aureus biofilm cross-sections was performed to investigate heterogeneity between horizontal biofilm layers.
UNASSIGNED: S. aureus biofilms were grown statically, embedded in a mixture of carboxymethylcellulose/gelatin, and prepared for downstream matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS). Trapped ion mobility spectrometry (TIMS) was also applied prior to mass analysis.
UNASSIGNED: Implementation of TIMS led to a ∼ threefold increase in the number of lipid species detected. Washing biofilm samples with ammonium formate (150 mM) increased signal intensity for some bacterial lipids by as much as tenfold, with minimal disruption of the biofilm structure. MALDI TIMS IMS revealed that most lipids localize primarily to a single biofilm layer, and species from the same lipid class such as cardiolipins CL(57:0) - CL(66:0) display starkly different localizations, exhibiting between 1.5 and 6.3-fold intensity differences between layers (n = 3, p < 0.03). No horizontal layers were observed within biofilms grown anaerobically, and lipids were distributed homogenously.
UNASSIGNED: High spatial resolution analysis of S. aureus biofilm cross-sections by MALDI TIMS IMS revealed stark lipidomic heterogeneity between horizontal S. aureus biofilm layers demonstrating that each layer was molecularly distinct. Finally, this workflow uncovered an absence of layers in biofilms grown under anaerobic conditions, possibly indicating that oxygen contributes to the observed heterogeneity under aerobic conditions. Future applications of this workflow to study spatially localized molecular responses to antimicrobials could provide new therapeutic strategies.

UNASSIGNED: Ulcerative colitis (UC) is an inflammatory bowel disease characterized by repeated remissions and relapses. Immunosuppressive drugs have facilitated the induction and maintenance of remission in many patients with UC. However, immunosuppressive drugs cannot directly repair impaired intestinal mucosa and are insufficient for preventing relapse. Therefore, new treatment approaches to repair the damaged epithelium in UC have been attempted through the transplantation of intestinal organoids, which can be differentiated into mucosa by embedding in Matrigel, generated from patient-derived intestinal stem cells. The method, however, poses the challenge of yielding sufficient cells for UC therapy, and patient-derived cells might already have acquired pathological changes. In contrast, human induced pluripotent stem (iPS) cells generated from healthy individuals are infinitely proliferated and can be differentiated into target cells. Recently developed human iPS cell-derived intestinal organoids (HIOs) aim to generate organoids that closely resemble the adult intestine. However, no study till date has reported HIOs injected into in vivo inflammatory models, and it remains unclear whether HIOs with cells that closely resemble the adult intestine or with intestinal stem cells retain the better ability to repair tissue in colitis.
UNASSIGNED: We generated two types of HIOs via suspension culture with and without small-molecule compounds: HIOs that include predominantly more intestinal stem cells [HIO (A)] and those that include predominantly more intestinal epithelial and secretory cells [HIO (B)]. We examined whether the generated HIOs engrafted in vivo and compared their ability to accelerate recovery of the damaged tissue.
UNASSIGNED: Findings showed that the HIOs expressed intestinal-specific markers such as caudal-type homeobox 2 (CDX2) and villin, and HIOs engrafted under the kidney capsules of mice. We then injected HIOs into colitis-model mice and found that the weight and clinical score of the mice injected with HIO (A) recovered earlier than that of the mice in the sham group. Further, the production of mucus and the expression of cell proliferation markers and tight junction proteins in the colon tissues of the HIO (A) group were restored to levels similar to those observed in healthy mice. However, neither HIO (A) nor HIO (B) could be engrafted into the colon.
UNASSIGNED: Effective cell therapy should directly repair tissue by engraftment at the site of injury. However, the difference in organoid property impacting the rate of tissue repair in transplantation without engraftment observed in the current study should be considered a critical consideration in the development of regenerative medicine using iPS-derived organoids.

Fabry disease is an X-linked glycolipid storage disorder caused by mutations in the GLA gene which result in a deficiency in the lysosomal enzyme alpha galactosidase A (AGA). As a result, the glycolipid substrate Gb3 accumulates in critical tissues and organs producing a progressive debilitating disease. In Fabry disease up to 80% of patients experience life-long neuropathic pain that is difficult to treat and greatly affects their quality of life. The molecular mechanisms by which deficiency of AGA leads to neuropathic pain are not well understood, due in part to a lack of in vitro models that can be used to study the underlying pathology at the cellular level. Using CRISPR-Cas9 gene editing, we generated two clones with mutations in the GLA gene from a human embryonic stem cell line. Our clonal cell lines maintained normal stem cell morphology and markers for pluripotency, and showed the phenotypic characteristics of Fabry disease including absent AGA activity and intracellular accumulation of Gb3. Mutations in the predicted locations in exon 1 of the GLA gene were confirmed. Using established techniques for dual-SMAD inhibition/WNT activation, we were able to show that our AGA-deficient clones, as well as wild-type controls, could be differentiated to peripheral-type sensory neurons that express pain receptors. This genetically and physiologically relevant human model system offers a new and promising tool for investigating the cellular mechanisms of peripheral neuropathy in Fabry disease and may assist in the development of new therapeutic strategies to help lessen the burden of this disease.

UNASSIGNED: Transcatheter aortic valve implantation (TAVI) is rapidly replacing cardiac surgery due to its minimal invasiveness and practicality. Midterm immunological studies on the biocompatibility of galactose-alpha-1,3-galactose (α-Gal)-carrying bioprosthetic heart valves for TAVI are not available. In this study we investigated whether bioprosthetic heart valves employed for TAVI augment an α-Gal-specific antibody-dependent and antibody-independent immune response 3 months after TAVI implantation.
UNASSIGNED: This prospective observational study included 27 patients with severe aortic valve stenosis undergoing TAVI and 10 patients with severe mitral valve regurgitation treated with a transcatheter MitraClip (Abbott Laboratories, Abbott Park, Ill) procedure. Blood samples were drawn before and 90 days after treatment at a routine checkup. Serum samples were analyzed using enzyme-linked immunosorbent assay. Serum concentrations of α-Gal-specific immunoglobulin (Ig) G, IgG subclasses and IgE, complement factor 3a, NETosis-specific citrullinated H3, and the systemic inflammation markers soluble suppression of tumorigenicity and interleukin 33 were evaluated.
UNASSIGNED: Three months after TAVI, we found significantly increased serum concentrations of α-Gal-specific IgG3, complement factor complement factor 3a, citrullinated H3 levels, and soluble suppression of tumorigenicity (P = .002, P = .001, P = .025, and P = .039, respectively). Sensitization of α-Gal-specific IgE antibodies occurred in 55% of all patients after TAVI.
UNASSIGNED: Our results indicate that TAVI elicits a midterm, specific humoral immune response against α-Gal and causes an unspecific humoral inflammation compared with patients undergoing MitraClip implantation. This observation will lead to a better understanding of postintervention morbidity and the long-term durability of bioprostheses and indicates that caution is appropriate when designing implantation strategies for younger patients.

It has been previously shown that 40% of murine cortical interneurons are eliminated via apoptosis during the first two weeks of postnatal development [1], [2], [3]. Here, we report data on the effect of ethanol exposure on this process in a mouse model of binge-like alcohol exposure during last trimester of human pregnancy (equivalent to the first postnatal week in mice). We used transgenic mice that express the Venus fluorescent protein in GABAergic interneurons under the control of the vesicular GABA transporter promoter (VGAT-Venus mice) [4]. Mice were exposed to air (controls) or ethanol for 4 hr/day on postnatal days 4 to 9 using vapor inhalation chambers [5]. This exposure paradigm produces peak blood ethanol concentrations between 300 and 400 mg/dl. Transcardial perfusions were performed under anesthesia at postnatal days 5, 7, 10 and 30. Cryostat-prepared floating sections were stained with the fluorescent DNA dye, 4\'6-diamidino-2-phenylindole (DAPI). We then quantified the density of Venus-positive GABAergic interneurons in layers I, II-IV and V of the retrosplenial cortex, which is part of the limbic memory system [6], and is sensitive to ethanol-induced apoptosis during the first postnatal week in mice [7], [8], [9], [10], [11]. The data show that density of interneurons decreases in the retrosplenial cortex layers during the first week of life and that ethanol exposure does not significantly alter this process. These data may be of interest to investigators who are studying the effect of ethanol and other teratogenic agents on developing interneurons in the cerebral cortex.

In the acute phase of spinal cord injury, the initial injury triggers secondary damage due to neuroinflammation, leading to the formation of cavities and glial scars that impair nerve regeneration. Following injuries to the central nervous system, early mobilization promotes the recovery of physical function. Therefore, in the present study, we investigated the effects of early mobilization on motor function recovery and neuroinflammation in rats. Early mobilization of rats with complete spinal cord transection resulted in good recovery of hindlimb motor function after 3 weeks. At 1 week after spinal cord injury, the early-mobilized rats expressed fewer inflammatory M1 microglia/macrophages and more anti-inflammatory M2 microglia. In addition, significantly more matrix metalloproteinase 2 (MMP2)-positive cells were observed at the lesion site 1 week after injury in the early-mobilized rats. Multiple labeling studies suggested that many MMP2-positive cells were M2 microglia. MMP9-positive cells that highly co-expressed GFAP were also observed more frequently in the early-mobilized rats. The density of growth-associated protein-positive structures in the lesion center was significantly higher in the early-mobilized rats at 3 weeks after spinal cord injury. The present results suggest that early mobilization after spinal cord injury reduced the production of M1 microglia/macrophages while increasing the production of M2 microglia at the lesion site. Early mobilization might also activate the expression of MMP2 in M2 microglia and MMP9 in astrocytes. These cellular dynamics might suppress neuroinflammation at the lesion site, thereby inhibiting the progression of tissue destruction and promoting nerve regeneration to recover motor function.

Multiple sclerosis (MS) is an inflammatory autoimmune disease associated with genetic and environmental factors. Cigarette smoking is harmful to health and may be one of the risk factors for MS. However, there have been no systematic investigations under controlled experimental conditions linking cigarette smoke (CS) and MS. The present study is the first inhalation study to correlate the pre-clinical and pathological manifestations affected by different doses of CS exposure in a mouse experimental autoimmune encephalomyelitis (EAE) model. Female C57BL/6 mice were whole-body exposed to either fresh air (sham) or three concentrations of CS from a reference cigarette (3R4F) for 2 weeks before and 4 weeks after EAE induction. The effects of exposure on body weight, clinical symptoms, spinal cord pathology, and serum biochemicals were then assessed. Exposure to low and medium concentrations of CS exacerbated the severity of symptoms and spinal cord pathology, while the high concentration had no effect relative to sham exposure in mice with EAE. Interestingly, the clinical chemistry parameters for metabolic profile as well as liver and renal function (e.g. triglycerides and creatinine levels, alkaline phosphatase activity) were lower in these mice than in naïve controls. Although the mouse EAE model does not fully recapitulate the pathology or symptoms of MS in humans, these findings largely corroborate previous epidemiological findings that exposure to CS can worsen the symptoms and pathology of MS. Furthermore, the study newly highlights the possible correlation of clinical chemistry findings such as metabolism and liver and renal function between MS patients and EAE mice.