Guoheng Xu

The use of experimental animals with unified quality standards is an important condition for ensuring the effectiveness of scientific research. Ectromelia virus (ECTV), murine hepatitis virus (MHV), reovirus type 3 (Reo-3), and murine parvoviruses (MUV) are the four pathogens that need to be eliminated from SPF (Specific Pathogen-Free) level mice. These four pathogens present fast transmission and high pathogenicity, making it difficult to control. The previously described detection methods present substantial limitations in efficiency and accuracy. Thus, there is an urgent need for rapid and precise diagnostic methods to improve prevention and diagnosis efforts. In this study, we developed a one-step multiplex real-time PCR (mrt-PCR) detection method that can simultaneously detect four key viral pathogens causing diseases in laboratory mice without cross-reactivity with other mouse susceptible pathogens. We tested 128 suspected diseased mouse tissue samples collected from Beijing, and the results showed that this new method has higher sensitivity and specificity than ordinary PCR. The detection limit for ECTV, MHV, and MUV was determined to be 1.08 × 10¹ copies/μL, 1.14 × 10¹ copies/μL, 2.38 ×10¹ copies/μL, and 1.08 × 10¹ copies/μL, respectively. In addition, the assay showed excellent reproducibility, with a coefficient of variation below 1.5%, strong linear correlation (R² > 0.996), and amplification efficiency between 90% and 100%. In summary, the mrt-PCR serves not only as a rapid and accurate detection and early prevention method for laboratory mice but also constitutes a robust tool for microbial quality control in laboratory mice.

Background: Previous studies demonstrated that mast cells with their degranulated component heparin are the major endogenous factors that stimulate preadipocyte differentiation and promote fascial adipogenesis, and this effect is related to the structure of heparin. Regarding the structural and physiological properties of the negatively charged polymers, hexasulfonated suramin, a centuries-old medicine that is still used for treating African trypanosomiasis and onchocerciasis, is assumed to be a heparin-related analog or heparinoid. This investigation aims to elucidate the influence of suramin on the adipogenesis. Methods: To assess the influence exerted by suramin on adipogenic differentiation of primary white adipocytes in rats, this exploration was conducted both in vitro and in vivo. Moreover, it was attempted to explore the role played by the sulfonic acid groups present in suramin in mediating this adipogenic process. Results: Suramin demonstrated a dose- and time-dependent propensity to stimulate the adipogenic differentiation of rat preadipocytes isolated from the superficial fascia tissue and from adult adipose tissue. This stimulation was concomitant with a notable upregulation in expression levels of pivotal adipogenic factors as the adipocyte differentiation process unfolded. Intraperitoneal injection of suramin into rats slightly increased adipogenesis in the superficial fascia and in the epididymal and inguinal fat depots. PPADS, NF023, and NF449 are suramin analogs respectively containing 2, 6, and 8 sulfonic acid groups, among which the last two moderately promoted lipid droplet formation and adipocyte differentiation. The number and position of sulfonate groups may be related to the adipogenic effect of suramin. Conclusions: Suramin emerges as a noteworthy pharmaceutical agent with the unique capability to significantly induce adipocyte differentiation, thereby fostering adipogenesis.

High altitude (HA) has become one of the most challenging environments featuring hypobaric hypoxia, which seriously threatens public health, hence its gradual attraction of public attention over the past decade. The purpose of this study is to investigate the effect of HA hypoxia on iron levels, redox state, inflammation, and ferroptosis in adipose tissue. Here, 40 mice were randomly divided into two groups: the sea-level group and HA hypoxia group (altitude of 5000 m, treatment for 4 weeks). Total iron contents, ferrous iron contents, ROS generation, lipid peroxidation, the oxidative enzyme system, proinflammatory factor secretion, and ferroptosis-related biomarkers were examined, respectively. According to the results, HA exposure increases total iron and ferrous iron levels in both WAT and BAT. Meanwhile, ROS release, MDA, 4-HNE elevation, GSH depletion, as well as the decrease in SOD, CAT, and GSH-Px activities further evidenced a phenotype of redox imbalance in adipose tissue during HA exposure. Additionally, the secretion of inflammatory factors was also significantly enhanced in HA mice. Moreover, the remarkably changed expression of ferroptosis-related markers suggested that HA exposure increased ferroptosis sensitivity in adipose tissue. Overall, this study reveals that HA exposure is capable of inducing adipose tissue redox imbalance, inflammatory response, and ferroptosis, driven in part by changes in iron overload, which is expected to provide novel preventive targets for HA-related illness.

As a novel origin of adipocytes, the superficial fascia, a typical soft connective tissue, has abundant adipocytes and preadipocytes, accompanied by numerous mast cells. Blood vessels pass through the fascia to form a network structure. The more reasonable statistical analysis methods can provide a new method for in-depth study of soft connective tissue by clarifying the spatial distribution relation between cells (point structure) and blood vessels (linear structure). This study adopted the Guidolin et al. statistical analysis methods used by epidemiology and ecology to quantitatively analyze the distribution pattern and correlations among blood vessels, adipocytes, and mast cells. Image-processing software and self-written computer programs were used to analyze images of whole-mounted fascia, and the relevant data were measured automatically. Voronoi’s analysis revealed that the vascular network was non-uniformly distributed. In fascia with average area of 3.75 cm², quantitative histological analysis revealed 81.16% of mast cells and 74.74% of adipocytes distributed within 60 μm of blood vessels. A Spearman’s correlation coefficient (rs) of >0.7 showed the co-distribution of the two types of cells under different areas. Ridge regression analysis further revealed the spatial correlation among blood vessels, adipocytes and mast cells. The combination of classical epidemiological analysis and extended computer program analysis can better analyze the spatial distribution relation between cells and vessels and should provide an effective analysis method for study of the histology and morphology of fascia and related connective tissues.

Background and Aims: Hydrogen sulfide (H₂S) plays a protective role in NAFLD. However, whether cystathionine γ lyase (CSE), a dominant H₂S generating enzyme in hepatocytes, has a role in the pathogenesis of NAFLD is currently unclear. Approach and Results: We showed that CSE protein expression is dramatically downregulated, especially in fibrotic areas, in livers from patients with NAFLD. In high‐fat diet (HFD)‐induced NAFLD mice or an oleic acid‐induced hepatocyte model, the CSE/H₂S pathway is also downregulated. To illustrate a regulatory role for CSE in NAFLD, we generated a hepatocyte‐specific CSE knockout mouse (CSE ^LKO). Feeding an HFD to CSE ^LKO mice, they showed more hepatic lipid deposition with increased activity of the fatty acid de novo synthesis pathway, increased hepatic insulin resistance, and higher hepatic gluconeogenic ability compared to CSE ^Loxp control mice. By contrast, H₂S donor treatment attenuated these phenotypes. Furthermore, the protection conferred by H₂S was blocked by farnesoid X receptor (FXR) knockdown. Consistently, serum deoxycholic acid and lithocholic acid (FXR antagonists) were increased, and tauro‐β‐muricholic acid (FXR activation elevated) was reduced in CSE^LKO. CSE/H₂S promoted a post‐translation modification (sulfhydration) of FXR at Cys138/141 sites, thereby enhancing its activity to modulate expression of target genes related to lipid and glucose metabolism, inflammation, and fibrosis. Sulfhydration proteomics in patients' livers supported the CSE/H₂S modulation noted in the CSE ^LKO mice. Conclusions: FXR sulfhydration is a post‐translational modification affected by hepatic endogenous CSE/H₂S that may promote FXR activity and attenuate NAFLD. Hepatic CSE deficiency promotes development of nonalcoholic steatohepatitis. The interaction between H₂S and FXR may be amenable to therapeutic drug treatment in NAFLD.

The organoid is a 3D cell architecture formed by self-organized tissues or cells in vitro with similar cell types, histological structures, and biological functions of the native organ. Depending on the unique organ structures and cell types, producing organoids requires individualized design and is still challenging. Organoids of some tissues, including adipose tissue, remain to generate to be more faithful to their original organ in structure and function. We previously established a new model of the origin of adipose cells originating from non-adipose fascia tissue. Here, we investigated superficial fascia fragments in 3D hydrogel and found they were able to transform into relatively large adipocyte aggregates containing mature unilocular adipocytes, which were virtually “fat organoids”. Such fascia-originated fat organoids had a typical structure of adipose tissues and possessed the principal function of adipose cells in the synthesis, storage, hydrolysis of triglycerides and adipokines secretion. Producing fat organoids from superficial fascia can provide a new approach for adipocyte research and strongly evidences that both adipose tissues and cells originate from fascia. Our findings give insights into metabolic regulation by the crosstalk between different organs and tissues and provide new knowledge for investigating novel treatments for obesity, diabetes and other metabolic diseases. Abbreviations: 3D: three dimensional; ASC: adipose-derived stromal cells; C/EBP: CCAAT-enhancer-binding protein; EdU: 5-ethynyl-2-deoxyuridine; FABP4: fatty acid-binding protein 4; FAS: fatty acid synthase; FSCs: fascia-derived stromal cells; Plin1: perilipin-1; Plin2: perilipin-2; PPARγ: peroxisome proliferator-activated receptor γ; WAT: white adipose tissue

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Background: Hydrogen sulfide (H ₂ S) has antihypertension and anti-inflammatory effects, and its endogenous-generation key enzyme cystathionine γ lyase (CSE) is expressed in CD4 ⁺ T cells. However, the role of CD4 ⁺ T-cell endogenous CSE/H ₂ S in the development of hypertension is unclear. Methods: Peripheral blood lymphocytes were isolated from hypertensive patients or spontaneously hypertensive rats, then H ₂ S production and expression of its generation enzymes, cystathionine β synthase and CSE, were measured to determine the major H ₂ S generation system changes in hypertension. Mice with CSE-specific knockout in T cells (conditional knockout, by CD4 ^cre mice hybridization) and CD4 null mice were generated for investigating the pathophysiological relevance of the CSE/H ₂ S system. Results: In lymphocytes, H ₂ S from CSE, but not cystathionine β synthase, responded to blood pressure changes, supported by lymphocyte CSE protein changes and a negative correlation between H ₂ S production with systolic blood pressure and diastolic blood pressure, but positive correlation with the serum level of interleukin 10 (an anti-inflammatory cytokine). Deletion of CSE in T cells elevated BP (5–8 mm Hg) under the physiological condition and exacerbated angiotensin II–induced hypertension. In keeping with hypertension, mesenteric artery dilation impaired association with arterial inflammation, an effect attributed to reduced immunoinhibitory T regulatory cell (Treg) numbers in the blood and kidney, thus causing excess CD4 ⁺ and CD8 ⁺ T cell infiltration in perivascular adipose tissues and kidney. CSE knockout CD4 ⁺ T cell transfer into CD4 null mice, also showed the similar phenotypes’ confirming the role of endogenous CSE/H ₂ S action. Adoptive transfer of Tregs (to conditional knockout mice) reversed hypertension, vascular relaxation impairment, and immunocyte infiltration, which confirmed that conditional knockout–induced hypertension was attributable, in part, to the reduced Treg numbers. Mechanistically, endogenous CSE/H ₂ S promoted Treg differentiation and proliferation by activating AMP-activated protein kinase. In part, it depended on activation of its upstream kinase, liver kinase B1, by sulfhydration to facilitate its substrate binding and phosphorylation. Conclusion: The constitutive sulfhydration of liver kinase B1 by CSE-derived H ₂ S activates its target kinase, AMP-activated protein kinase, and promotes Treg differentiation and proliferation, which attenuates the vascular and renal immune-inflammation, thereby preventing hypertension.