Glycyrrhetinic Acid as a Hepatocyte Targeting Ligand-Functionalized Platinum(IV) Complexes for Hepatocellular Carcinoma Therapy and Overcoming Multidrug Resistance.

Promising targeted therapy options to overcome drug resistance and side effects caused by platinum(II) drugs for treatment in hepatocellular carcinoma are urgently needed. Herein, six novel multifunctional platinum(IV) complexes through linking platinum(II) agents and glycyrrhetinic acid (GA) were designed and synthesized. Among them, complex 20 showed superior antitumor activity against tested cancer cells including cisplatin resistance cells than cisplatin and simultaneously displayed good liver-targeting ability. Moreover, complex 20 can significantly cause DNA damage and mitochondrial dysfunction, promote reactive oxygen species generation, activate endoplasmic reticulum stress, and eventually induce apoptosis. Additionally, complex 20 can effectively inhibit cell migration and invasion and trigger autophagy and ferroptosis in HepG-2 cells. More importantly, complex 20 demonstrated stronger tumor inhibition ability than cisplatin or the combo of cisplatin/GA with almost no systemic toxicity in HepG-2 or A549 xenograft models. Collectively, complex 20 could be developed as a potential anti-HCC agent for cancer treatment.

Novel NF-κB Inhibitor-Conjugated Pt(IV) Prodrug to Enable Cancer Therapy through ROS/ER Stress and Mitochondrial Dysfunction and Overcome Multidrug Resistance.

Although cisplatin has been widely used for clinical purposes, its application is limited due to its obvious side effects. To mitigate the defects of cisplatin, here, six "multitarget prodrugs" were synthesized by linking cisplatin and NF-κB inhibitors. Notably, complex 9 demonstrated a 63-fold enhancement in the activity against A549/CDDP cells with lower toxicity toward normal LO2 cells compared to cisplatin. Additionally, complex 9 could effectively cause DNA damage, induce mitochondrial dysfunction, generate reactive oxygen species, and induce cell apoptosis through the mitochondrial pathway and ER stress. Remarkably, complex 9 effectively inhibited the NF-κB/MAPK signaling pathway and disrupted the PI3K/AKT signaling transduction. Importantly, complex 9 showed superior in vivo antitumor efficiency compared to cisplatin or the combination of cisplatin/4, without obvious systemic toxicity in A549 or A549/CDDP xenograft models. Our results demonstrated that the dual-acting mechanism endowed the complexes with high efficiency and low toxicity, which may represent an efficient strategy for cancer therapy.

Transcriptomic and intervention evidence reveals domestic dogs as a promising model for anti-inflammatory investigation.

Domestic dogs have great potential to expand our understanding of the determinants of aging. To understand the aging pattern of domestic dogs and evaluate whether they can be used as an aging model, we performed RNA sequencing on white blood cells from domestic dogs aged 1-9 years and treated aged dogs with classical antiaging approaches. We obtained 30 RNA sequencing libraries and identified 61 age-associated genes with dynamic changes, the majority of which were related to metabolism and immune function, which may be predominant biomarkers for aging in dogs. We next treated aged dogs with canine mesenchymal stem cells (cMSCs), nicotinamide mononucleotide, and rapamycin to determine whether and how they responded to the antiaging interventions. The results showed that these treatments can significantly reduce the level of inflammatory factors (IL-6 and TNF-α). MSCs effectively improved the heart functions of aged dogs. Three key potential age-related genes (PYCR1, CCRL2, and TOX) were reversed by MSC treatment, two of which (CCRL2 and TOX) are implicated in immunity. Overall, we profiled the transcriptomic pattern of domestic dogs and revealed that they may be a good model of aging, especially in anti-inflammatory investigations.

Characterization of novel PHEX variants in X-linked hypophosphatemic rickets and genotype-PHEX activity correlation.

BACKGROUND: X-linked hypophosphatemia (XLHR) is the most common genetic form of hypophosphatemic rickets (HR), which is caused by phosphate regulating endopeptidase homolog X-linked (PHEX) gene mutation. At present, the genotype-phenotype relationship of XLHR and the pathogenic role of PHEX have not been fully understood. METHODS: In this study, we summarized clinical features in a new cohort of 49 HR patients and detected 16 novel PHEX and 5 novel non-PHEX variants. Subsequently, we studied the pathogenesis of new variants by protein expression, glycosylation analysis, subcellular localization and endopeptidase activity. RESULTS: The results showed that missense variants (Q189H and X750R) slightly reduced protein expression without obviously altering protein length and localization, whereas truncating variants significantly impaired the synthesis of PHEX and produced a shorter immature protein in cells. Interestingly, no evident correlation was observed between mutation types and clinical phenotypes. However, when we analyzed the relationship between PHEX activity and serum phosphorus level, we found that patients with low PHEX activity tended to have severe hypophosphatemia and high rickets severity score (RSS). Following this observation, we established two new knock-in XLHR mouse models with two novel Phex variants (c.T1349C and c.C426G, respectively) using CRISPR/Cas9 technology. Both mouse models demonstrated clinical manifestations of XLHR seen in patients and PhexC426G mice showed more severe phenotype than PhexT1349C mice, which further confirmed the rationality of genotype-PHEX enzymatic activity correlation analysis. CONCLUSION: Therefore, our findings demonstrated that novel PHEX variants could disrupt protein function via affecting protein synthesis, post-translational modification, cellular trafficking and catalytic activity. Our study facilitates a better understanding of XLHR pathogenic mechanism and PHEX activity-phenotype correlation, which is of crucial importance for future diagnosis and treatment of XLHR.

Draining Lymph Node Metastasis Model for Assessing the Dynamics of Antigen-Specific CD8+ T Cells During Tumorigenesis.

Tumor antigen-specific CD8+ T cells from draining lymph nodes gain an accumulating importance in mounting anti-tumor immune response during tumorigenesis. However, in many cases, cancer cells form metastatic loci in lymph nodes before further metastasizing to distant organs. To what extent the local and systematic CD8+ T cell responses were influenced by LN metastasis remains obscure. To this end, we set up a murine LN metastasis model combined with a B16F10-GP melanoma cell line expressing the surrogate neoantigen derived from lymphocytic choriomeningitis virus (LCMV), glycoprotein (GP), and P14 transgenic mice harboring T cell receptors (TCRs) specific to GP-derived peptide GP33-41 presented by the class I major histocompatibility complex (MHC) molecule H-2Db. This protocol enables the study of antigen-specific CD8+ T cell responses during LN metastasis. In this protocol, C57BL/6J mice were subcutaneously implanted with B16F10-GP cells, followed by adoptive transfer with naive P14 cells. When the subcutaneous tumor grew to approximately 5 mm in diameter, the primary tumor was excised, and B16F10-GP cells were directly injected into the tumor draining lymph node (TdLN). Then, the dynamics of CD8+ T cells were monitored during the process of LN metastasis. Collectively, this model has provided an approach to precisely investigate the antigen-specific CD8+ T cell immune responses during LN metastasis.

Dual-targeting tumor cells hybrids derived from Pt(IV) species and NF-κB inhibitors enables cancer therapy through mitochondrial dysfunction and ER stress and overcomes cisplatin resistance.

To ameliorate the defects including serious side effects and drug resistance of Pt(II) drugs (e.g., cisplatin and oxaliplatin), here a novel of "dual-prodrug" by containing Pt(II) drugs and NF-κB inhibitors were synthesized and characterized. Among them, Pt(IV) complex 11 exhibited better cytotoxic activity than other Pt(IV) complexes and the corresponding Pt(II) drugs, with IC50 values ranged from 0.31 to 0.91 µM, respectively, and also displayed low toxicity toward two normal cells HL-7702 and BEAS-2B. More importantly, complex 11 significantly reversed cisplatin resistance in A549/CDDP cells, indicating that complex 11 was able to overcome multidrug resistance. Following mechanism studies demonstrated that complex 11 significantly induced DNA damage and ROS generation, arrest the cell cycle at the G2/M stage, suppressed cell migration and intrusion, and induced cell apoptosis through activated ER stress and mitochondrial apoptosis pathway in A549 cells. Moreover, complex 11 effectively suppressed the IKKß phosphorylation, IκBα phosphorylation and NF-κB p65 phosphorylation and nuclear translocation, leading to blocked the NF-κB signal pathway in A549 cells. In vivo tests showed that the inhibitory rate in the complex 11 reached 69.2 %, which was much higher than that of oxaliplatin (55.6 %), 1a (39.7 %) and the combination of oxaliplatin/1a (65.1 %), without causing loss in the body weight.

ORMDL3­mediated bronchial epithelial pyroptosis leads to lung inflammation in obese mice with asthma.

Asthma associated with obesity is a chronic disease that poses a threat to health in children and results in severe wheezing, earlier airway remodeling and increased insensitivity to hormone therapy compared with those who only have asthma. Despite its clinical importance, knowledge on the underlying mechanisms of this disease is limited. The present study aimed to elucidate the pathogenesis of asthma associated with obesity using a murine model. A total of 30 female BALB/c mice were divided into three groups: Normal, mice with asthma and obese mice with asthma. Obese mice with asthma were fed a high­fat diet to induce obesity. Mice with asthma were sensitized and challenged with ovalbumin (OVA). Obese mice were subjected to OVA sensitization and challenge to develop asthma associated with obesity. Airway remodeling was observed in obese mice with asthma through HE and Masson staining. Proteomic and bioinformatics analyses were conducted on lung tissue from obese mice with asthma and normal mice. A total of 200 proteins were differentially expressed in obese mice with asthma compared with normal mice; of these, 53 and 47% were up­ and downregulated, respectively. Pathway analysis revealed that asthma associated with obesity primarily affected the 'lysosome', 'phagosome', and 'sphingolipid metabolism' pathways. Gene Set Enrichment Analysis demonstrated the presence of pyroptosis in obese asthmatic mice, along with significant increases in pyroptosis­-associated factors such as GSDMD and Caspase. High protein expression of orosomucoid­like 3 (ORMDL3), NOD­like receptor thermal protein domain associated protein 3 (NLRP3) and Gasdermin­D (GSDMD) was observed in obese mice with asthma. In vitro experiments using HBE cells infected with ORMDL3­overexpressing lentivirus demonstrated that the overexpression of ORMDL3 led to increased expression of NLRP3, GSDMD and cathepsin D (CTSD). These findings suggested that ORMDL3 may regulate pyroptosis and subsequent airway remodeling in asthma associated with obesity via the CTSD/NLRP3/GSDMD pathway.

Influence of Mode of Delivery on Children's Attention Deficit Hyperactivity Disorder and Childhood Intelligence.

OBJECTIVE: To investigate whether differences exist in attention deficit hyperactivity disorder (ADHD) and intelligence between children born by cesarean delivery and those born by vaginal delivery. METHODS: This retrospective study included singleton children that were born between January 2013 and December 2014. The Chinese version of the Conners' Parent Rating Scale-Revised (CPRS-48) was required on the probability of psychological and behavioral problems. The China-Wechsler Intelligence Scale for Children (C-WIRS) was used for evaluation of crystallized intelligence and Raven's Standard Progressive Matrices for evaluation of fluid intelligence. RESULTS: A total of 10,568 valid questionnaires were obtained. CPRS-48 ADHD index and detection rate were higher in cesarean delivery group than those in vaginal delivery group. Cesarean delivery groups had a lower performance intelligence quotient score according to C-WISC. CONCLUSION: Children born by cesarean delivery were more likely to have a risk of ADHD and a lower performance intelligence quotient compared with those born by vaginal delivery.

CD16+ monocytes are involved in the hyper-inflammatory state of Prader-Willi Syndrome by single-cell transcriptomic analysis.

Background: Patients with Prader-Willi syndrome (PWS) have a reduced life expectancy due to inflammation-related disease including cardiovascular disease and diabetes. Abnormal activation of peripheral immune system is postulated as a contributor. However, detailed features of the peripheral immune cells in PWS have not been fully elucidated. Methods: Serum inflammatory cytokines were measured in healthy controls (n=13) and PWS patients (n=10) using a 65- multiplex cytokine assays. Changes of the peripheral immune cells in PWS was assessed by single-cell RNA sequencing (scRNA-seq) and high-dimensional mass cytometry (CyTOF) using peripheral blood mononuclear cells (PBMCs) from PWS patients (n=6) and healthy controls (n=12). Results: PWS patients exhibited hyper-inflammatory signatures in PBMCs and monocytes were the most pronounced. Most inflammatory serum cytokines were increased in PWS, including IL-1ß, IL-2R, IL-12p70, and TNF-α. The characteristics of monocytes evaluated by scRNA-seq and CyTOF showed that CD16+ monocytes were significantly increased in PWS patients. Functional pathway analysis revealed that CD16+ monocytes upregulated pathways in PWS were closely associated with TNF/IL-1ß- driven inflammation signaling. The CellChat analysis identified CD16+ monocytes transmitted chemokine and cytokine signaling to drive inflammatory process in other cell types. Finally, we explored the PWS deletion region 15q11-q13 might be responsible for elevated levels of inflammation in the peripheral immune system. Conclusion: The study highlights that CD16+ monocytes contributor to the hyper-inflammatory state of PWS which provides potential targets for immunotherapy in the future and expands our knowledge of peripheral immune cells in PWS at the single cell level for the first time.

Evaluation of immunogenicity of gene-deleted and subunit vaccines constructed against the emerging pseudorabies virus variants.

BACKGROUND: Pseudorabies (PR) (also called Aujeszky's disease, AD) is a serious infectious disease affecting pigs and other animals worldwide. The emergence of variant strains of pseudorabies virus (PRV) since 2011 has led to PR outbreaks in China and a vaccine that antigenically more closely matches these PRV variants could represent an added value to control these infections. METHODS: The objective of this study was to develop new live attenuated and subunit vaccines against PRV variant strains. Genomic alterations of vaccine strains were based on the highly virulent SD-2017 mutant strain and gene-deleted strains SD-2017ΔgE/gI and SD-2017ΔgE/gI/TK, which constructed using homologous recombination technology. PRV gB-DCpep (Dendritic cells targeting peptide) and PorB (the outer membrane pore proteins of N. meningitidis) proteins containing gp67 protein secretion signal peptide were expressed using the baculovirus system for the preparation of subunit vaccines. We used experimental animal rabbits to test immunogenicity to evaluate the effect of the newly constructed PR vaccines. RESULTS: Compared with the PRV-gB subunit vaccine and SD-2017ΔgE/gI inactivated vaccines, rabbits (n = 10) that were intramuscularly vaccinated with SD-2017ΔgE/gI/TK live attenuated vaccine and PRV-gB + PorB subunit vaccine showed significantly higher anti-PRV-specific antibodies as well as neutralizing antibodies and IFN-γ levels in serum. In addition, the SD-2017ΔgE/gI/TK live attenuated vaccine and PRV-gB + PorB subunit vaccine protected (90-100%) rabbits against homologous infection by the PRV variant strain. No obvious pathological damage was observed in these vaccinated rabbits. CONCLUSIONS: The SD-2017ΔgE/gI/TK live attenuated vaccine provided 100% protection against PRV variant challenge. Interestingly, the subunit vaccines with gB protein linked to DCpep and PorB protein as adjuvant may also be a promising and effective PRV variant vaccine candidate.

Relationship between serum fibroblast growth factor 19 and vascular endothelial growth factor and soluble klotho protein in type 1 diabetic children.

BACKGROUND: Fibroblast growth factor 19 (FGF19) takes part in maintaining the balance of glycolipids and may be involved in complications of type 1 diabetes(T1D) in children. This study aimed at at evaluating the relationship among the levels of serum FGF19 and vascular endothelial growth factor(VEGF)and soluble klotho protein(sklotho) in type 1 diabetic children. METHODS: In a cross-section single center study samples were obtained from 96 subjects: 66 T1D and 30 healthy children.Serum FGF19 and VEGF and sklotho concentrations were measured by ELISA. And 66 type 1 diabetes participants were divided into two groups according to T1D duration or three groups according to HbA1c.Furthermore,we compared the serum levels of FGF19 and VEGF and sklotho in different groups. RESULTS: The concentration of FGF19 was lower in T1D than in the controls(226.52 ± 20.86pg/mu vs.240.08 ± 23.53 pg/L, p = 0.03),while sklotho was also lower in T1D than in the controls (2448.67 ± 791.92pg/mL vs. 3083.55 ± 1113.47pg/mL, p = 0.011). In contrast, VEGF levels were higher in diabetic patients than in controls (227.95 ± 48.65pg/mL vs. 205.92 ± 28.27 pg/mL, p = 0.016). In T1D, FGF19 and VEGF and sklotho was not correlated with the duration of diabetes. FGF19 and VEGF and sklotho were correlated with HbA1c (r=-0.349, p = 0.004 and r = 0.302, p = 0.014 and r=-0.342, p = 0.005, respectively), but not with blood glucose and lipid. Among subjects in the T1D group, concentrations of FGF19,VEGF and sklotho protein were different between different groups according to the degree of HbA1c(P < 0.005).Furthermore, there was a positive correlation between the serum FGF19 concentration and sklotho levels (r = 0.247,p = 0.045), and a negative correlation between the serum FGF19 concentration and VEGF level(r=-0.335,P = 0.006). CONCLUSIONS: The serum FGF19 levels have a close relation with serum VEGF levels and sklotho levels among T1D subjects. FGF19 may be involved in the development of complications in children with type 1 diabetes through interaction with VEGF and sklotho.

Characterization and genome analysis of G1 sub-cluster mycobacteriophage Lang.

Phage therapy is revitalized as an alternative to antibiotics therapy against antimicrobials resistant pathogens. Mycobacteriophages are genetically diverse viruses that can specifically infect Mycobacterium genus including Mycobacterium tuberculosis and Mycobacterium smegmatis. Here, we isolated and annotated the genome of a mycobacteriophage Lang, a temperate mycobacteriophage isolated from the soil of Hohhot, Inner Mongolia, China, by using Mycolicibacterium smegmatis mc2 155 as the host. It belongs to the Siphoviridae family of Caudovirales as determined by transmission electron microscopy. The morphological characteristics and certain biological properties of the phage were considered in detail. Phage Lang genomes is 41,487 bp in length with 66.85% GC content and encodes 60 putative open reading frames and belongs to the G1 sub-cluster. Genome annotation indicated that genes for structure proteins, assembly proteins, replications/transcription and lysis of the host are present in function clucters. The genome sequence of phage Lang is more than 95% similar to that of mycobacteriophage Grizzly and Sweets, differing in substitutions, insertions and deletions in Lang. One-step growth curve revealed that Lang has a latent period of 30 min and a outbreak period of 90 min. The short latent period and rapid outbreak mark the unique properties of phage Lang, which can be another potential source for combating M. tuberculosis.

Study on internal structure of casein micelles in reconstituted skim milk powder.

In the current study, the internal structure of casein micelles (CMs), primary casein cluster, has been studied by size-exclusion chromatography (SEC) coupled with small-angle X-ray scattering (SAXS), isothermal titration calorimetry (ITC), transmission electron microscopy (TEM) and molecular dynamics (MD) simulations. The casein cluster featured a hierarchical structure predominately consisting of αs-CN and ß-CN molecules and a trace of κ-CN. ITC profile showed a typical enthalpogram of CMs with a critical micelle concentration (CMC) of ~0.85 µg/mL. The casein cluster exhibited apparent characteristics of intrinsically disordered proteins (IDPs) with a secondary structure content of 24 % in α-helix, 35.4 % in antiparallel-parallel, 20.2 % in ß-turn and 20.4 % in random coil. SAXS results revealed a slightly elongated and tortuous worm-like conformation for the casein cluster in solution with an aspect ratio of 1.36 and an estimated molecular weight of 162.7 kDa. Further scattering data analysis proposed three coexisted species (αs1-ß-αs2-CN, αs1-CN and αs1-ß-αs2-CN dimer) with a volume fraction of 57.4 %, 30.1 %, and 12.5 % respectively in the casein cluster. TEM observation was consistent with the results of spectra, SAXS and MD that calcium sequestration resulted in a more extended and loose structure, instead, EDTA chelation induced a more compact conformation of the casein cluster.

Application of magnetic carbon nanotube composite nanospheres in magnetic solid-phase extraction of trace perfluoroalkyl substances from environmental water samples.

The layer-by-layer assembly technique was used to synthesize novel multiwalled carbon nanotubes (MWCNTs) on magnetic carbon (Fe3O4@C) nanospheres, which were then used to extract six perfluoroalkyl substances (PFAS) in environmental real water samples using ultra high-performance liquid chromatography coupled to tandem mass spectrometry. The as-synthesized sorbent MWCNTs@Fe3O4@C was employed for magnetic solid-phase extraction (MSPE). The as-prepared MWCNTs@Fe3O4@C was characterized by scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR) and vibrating sample magnetometer (VSM). The main extraction parameters were systematically optimized by Box-Behnken design. Under optimal conditions, excellent results were achieved. The synthesized sorbent showed wide linear ranges (0.1-1000 ng L-1), low detection limits (0.03-0.09 ng L-1) and good repeatability (3.80%-9.52%) for extracting and detecting six PFAS. The developed method was also applied to analyze six PFAS from environmental water samples. This study indicated that MWCNTs@Fe3O4@C composites are promising materials for the extraction and determination of PFAS from water samples.

A single-nucleus transcriptomic atlas of the dog hippocampus reveals the potential relationship between specific cell types and domestication.

The process of domestication has led to dramatic differences in behavioral traits between domestic dogs and gray wolves. Whole-genome research found that a class of putative positively selected genes were related to various aspects of learning and memory, such as long-term potentiation and long-term depression. In this study, we constructed a single-nucleus transcriptomic atlas of the dog hippocampus to illustrate its cell types, cell lineage and molecular features. Using the transcriptomes of 105 057 nuclei from the hippocampus of a Beagle dog, we identified 26 cell clusters and a putative trajectory of oligodendrocyte development. Comparative analysis revealed a significant convergence between dog differentially expressed genes (DEGs) and putative positively selected genes (PSGs). Forty putative PSGs were DEGs in glutamatergic neurons, especially in Cluster 14, which is related to the regulation of nervous system development. In summary, this study provides a blueprint to understand the cellular mechanism of dog domestication.

Effects of growth hormone on lipid metabolism and sexual development in pubertal obese male rats.

To investigate the effects of growth hormone (GH) on pubertal obese male rats, a rat model of high-fat diet-induced obesity was established in juvenile male rats. The model rats were divided into the treatment group (GH) and the non-treatment group (physiological saline). After 4 weeks, we measured the levels of alanine transaminase (ALT), aspartate aminotransferase (AST), total cholesterol (TC), triglycerides (TGs), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), estrogen (E2), testosterone (T), and insulin-like growth factor (IGF-1). The morphological changes of the liver and testis were assessed, and the expression of aromatase was detected. The levels of ALT, AST, TC, TG, LDL-C, E2, and IGF-1 in the treatment group were significantly lower than in the non-treated model rats (P < 0.001). The levels of HDL-C and T of GH-treated rats were significantly higher than those of the non-treatment group (P < 0.001). Compared with non-treated model rats, GH-treated model rats showed reduced liver steatosis, improved morphological structure of the testicular seminiferous tubules, and an increased number of spermatogenic cells. The treatment group also showed lower expression of aromatase in the liver and testis compared with the non-treatment group. GH partially protected pubertal male rats from obesity-induced lipid metabolic disorder and sexual retardation.

Facile discovery of red blood cell deformation and compromised membrane/skeleton assembly in Prader-Willi syndrome.

Prader-Willi syndrome (PWS) is a rare congenital disease with genetic alterations in chromosome 15. Although genetic disorders and DNA methylation abnormalities involved in PWS have been investigated to a significant degree, other anomalies such as those in erythrocytes may occur and these have not been clearly elucidated. In the present study, we uncovered slight anemia in children with PWS that was associated with increased red blood cell (RBC) distribution width (RDW) and contrarily reduced hematocrit (HCT) values. Intriguingly, the increased ratio in RDW to HCT allowed sufficient differentiation between the PWS patients from the healthy controls and, importantly, with individuals exhibiting conventional obesity. Further morphologic examinations revealed a significant deformity in erythrocytes and mild hemolysis in PWS patients. Comprehensive mechanistic investigations unveiled compromised membrane skeletal assembly and membrane lipid composition, and revealed a reduced F-actin/G-actin ratio in PWS patients. We ascribed these phenotypic changes in erythrocytes to the observed genetic defects, including DNA methylation abnormalities. Our collective data allowed us to uncover RBC deformation in children with PWS, and this may constitute an auxiliary indicator of PWS in early childhood.

Functional and metabolic alterations of gut microbiota in children with new-onset type 1 diabetes.

Gut dysbiosis has been linked to type 1 diabetes (T1D); however, microbial capacity in T1D remains unclear. Here, we integratively profiled gut microbial functional and metabolic alterations in children with new-onset T1D in independent cohorts and investigated the underlying mechanisms. In T1D, the microbiota was characterized by decreased butyrate production and bile acid metabolism and increased lipopolysaccharide biosynthesis at the species, gene, and metabolite levels. The combination of 18 bacterial species and fecal metabolites provided excellently discriminatory power for T1D. Gut microbiota from children with T1D induced elevated fasting glucose levels and declined insulin sensitivity in antibiotic-treated mice. In streptozotocin-induced T1D mice, butyrate and lipopolysaccharide exerted protective and destructive effects on islet structure and function, respectively. Lipopolysaccharide aggravated the pancreatic inflammatory response, while butyrate activated Insulin1 and Insulin2 gene expression. Our study revealed perturbed microbial functional and metabolic traits in T1D, providing potential avenues for microbiome-based prevention and intervention for T1D.

Identification and Characterization of Two Novel Compounds: Heterozygous Variants of Lipoprotein Lipase in Two Pedigrees With Type I Hyperlipoproteinemia.

Background: Type I hyperlipoproteinemia, characterized by severe hypertriglyceridemia, is caused mainly by loss-of-function mutation of the lipoprotein lipase (LPL) gene. To date, more than 200 mutations in the LPL gene have been reported, while only a limited number of mutations have been evaluated for pathogenesis. Objective: This study aims to explore the molecular mechanisms underlying lipoprotein lipase deficiency in two pedigrees with type 1 hyperlipoproteinemia. Methods: We conducted a systematic clinical and genetic analysis of two pedigrees with type 1 hyperlipoproteinemia. Postheparin plasma of all the members was used for the LPL activity analysis. In vitro studies were performed in HEK-293T cells that were transiently transfected with wild-type or variant LPL plasmids. Furthermore, the production and activity of LPL were analyzed in cell lysates or culture medium. Results: Proband 1 developed acute pancreatitis in youth, and her serum triglycerides (TGs) continued to be at an ultrahigh level, despite the application of various lipid-lowering drugs. Proband 2 was diagnosed with type 1 hyperlipoproteinemia at 9 months of age, and his serum TG levels were mildly elevated with treatment. Two novel compound heterozygous variants of LPL (c.3G>C, p. M1? and c.835_836delCT, p. L279Vfs*3, c.188C>T, p. Ser63Phe and c.662T>C, p. Ile221Thr) were identified in the two probands. The postheparin LPL activity of probands 1 and 2 showed decreases of 72.22 ± 9.46% (p<0.01) and 54.60 ± 9.03% (p<0.01), respectively, compared with the control. In vitro studies showed a substantial reduction in the expression or enzyme activity of LPL in the LPL variants. Conclusions: Two novel compound heterozygous variants of LPL induced defects in the expression and function of LPL and caused type I hyperlipoproteinemia. The functional characterization of these variants was in keeping with the postulated LPL mutant activity.

Comparative proteomic analysis of PK15 swine kidney cells infected with a pseudorabies pathogenic variant and the Bartha-K/61 vaccine strain.

Pseudorabies virus (PRV) is the causative agent of Aujeszky's disease and is communicable across species. In particular, the emergence of PRV variants in 2011 have resulted in serious economic losses to the Chinese pig industry. In this study, we used tandem mass tag (TMT) quantitative protein analysis to identify differentially expressed proteins between the PRV variant strain SD-2017 and the vaccine strain Bartha-K/61 in the swine kidney cell line PK15. Overall, we identified 4690 proteins for SD-2017 infection compared with the mock-infected control cells. We found 162 differentially expressed cellular proteins including 41 up- and 121 down-regulated proteins. SD-2017-infected PK15 cells differential proteins were primarily related to gap junctions, the phagosome, antigen processing and presentation, cell adhesion molecules and peroxisome pathways. Compared to Bartha-K/61-infected PK15 cells, SD-2017-infected cells displayed differentially expressed proteins involved in tryptophan metabolism, mitophagy and Notch signaling. Western blot analysis of MARK2, TSR1 and TMED1 three representative proteins validated the reliability of the TMT data. This study is an initial at-tempt to compare the proteomes of PK15 cells infected by a PRV variant and a vaccine strain using TMT technology to provide new insights into the mechanisms of PRV pathogenesis and immune evasion.