Adipocytokines and Inflammation in Patients and a Gerbil Model: Implications for Obesity-Related and Nonobese Diabetes.

Background: Obesity is a predisposing risk factor for type 2 diabetes mellitus (T2DM). Actually, not only obese/overweight but also nonobese/lean individuals may be prone to T2DM. This study is aimed at identifying the contribution of adipose tissue to the development of nonobese diabetes (NOD) and obese diabetes (OD). Methods: Serum samples from the nonobese nondiabetes (NOND, n = 47, age = 46.8 ± 8.4, BMI ≤ 23.9 kg/m2) controls, NOD (n = 48, age = 50.7 ± 6.5, BMI ≤ 23.9 kg/m2) and OD (n = 65, age = 49.8 ± 10.2, BMI ≥ 28 kg/m2) patients were utilized to measure the expression of metabolic indicators, adipocytokines, inflammatory factors. Different adipose depots from offspring with corresponding blood glucose and obesity levels of a spontaneously diabetic gerbil line with various degrees of diabetic penetrance and body weights were examined for adipocytokines and inflammation factors detected by ELISA and western blot. Adipose tissue volume and fat cell size of the gerbils were evaluated by magnetic resonance imaging and immunohistochemistry, respectively. Results: The study yielded four key findings. Firstly, in comparison to the NOD group, the OD group exhibited more severe insulin resistance (IR) and metabolic dysfunction in both patients and gerbils, attributed to higher visceral adipose tissue mass and larger fat cell sizes. Secondly, in gerbils, gonadal fat deposition was linked to obesity development, whereas kidney fat deposition correlated with obesity and diabetes occurrence. Thirdly, in both patients and gerbils, the interplay between adiponectin and leptin levels in serum may significantly influence the development of obesity and diabetes. Lastly, heightened expression of MCP3 in gerbils' kidney adipose tissue may serve as a pivotal factor in initiating obesity-associated diabetes. Conclusions: Our study, which may be considered a pilot investigation, suggests that the interaction of adipocytokines and inflammation factors in different adipose depots could play diverse roles in the development of diabetes or obesity.

Overcoming multi-drug resistance in SCLC: a synergistic approach with venetoclax and hydroxychloroquine targeting the lncRNA LYPLAL1-DT/BCL2/BECN1 pathway.

BACKGROUND: Small cell lung cancer (SCLC) stands as one of the most lethal malignancies, characterized by a grim diagnosis and prognosis. The emergence of multi-drug resistance poses a significant hurdle to effective therapy. Although previous studies have implicated the long noncoding RNA LYPLAL1-DT in the tumorigenesis of SCLC, the precise role of the highly expressed LYPLAL1-DT in SCLC chemoresistance and the underlying mechanism remain inadequately understood. METHODS: cDDP-, VP-16- and PTX-resistant SCLC cells lines were established. The viabilities of SCLC cells were assessed by CCK-8 assay in vitro and xenograft tumor formation assay in vivo. Apoptosis was evaluated by FACS, Western blot and JC-1 fluorescence staining, while autophagy was explored via autophagic flux detection under confocal microscopy and autophagic vacuole investigation under transmission electron microscopy (TEM). The functional role and mechanism of LYPLAL1-DT were further investigated by gain- and loss-of-function assays in vitro. Furthermore, the therapeutic efficacy of the combination of venetoclax and HCQ with cDDP, VP-16 or PTX was evaluated by cell line, cell-derived xenograft (CDX) and patient-derived xenograft (PDX) mice model. RESULTS: Our findings revealed that LYPLAL1-DT is upregulated in chemoresistant SCLC cell lines. Gain- and loss-of-function assays demonstrated that LYPLAL1-DT impairs sensitivity to cDDP, VP-16, or PTX both in vitro and in vivo. Overexpression of LYPLAL1-DT significantly enhanced autophagy and inhibited apoptosis in SCLC cells. Further analyses, including RIP and RNA pull-down assays, revealed that LYPLAL1-DT promotes the expression of BCL2 by sponging miR-204-5p and is implicated in the assembly of the autophagy-specific complex (BECN1/PtdIns3K complex). Combining venetoclax and HCQ with cDDP, VP-16, or PTX effectively mitigated chemoresistance in SCLC cells and suppressed tumor growth in CDX and PDX models without inducing obvious toxic effects. CONCLUSIONS: Our findings demonstrate that upregulation of LYPLAL1-DT sequesters apoptosis through the LYPLAL1-DT/miR-204-5p/BCL2 axis and promotes autophagy by facilitating the assembly of the BECN1/PtdIns3K complex, thereby mediating multi-drug resistance of SCLC. The triple combination of venetoclax, HCQ, in conjunction with cDDP, VP-16 or PTX overcomes refractory SCLC, shedding light on a potential therapeutic target for combating SCLC chemoresistance.

Hypoxia exacerbates the malignant transformation of gastric epithelial cells induced by long-term H. pylori infection.

Helicobacter pylori is a microaerophilic Gram-negative bacterium that resides in the human stomach and is classified as a class I carcinogen for gastric cancer. Numerous studies have demonstrated that H. pylori infection plays a role in regulating the function of host cells, thereby contributing to the malignant transformation of these cells. However, H. pylori infection is a chronic process, and short-term cellular experiments may not provide a comprehensive understanding of the in vivo situation, especially when considering the lower oxygen levels in the human stomach. In this study, we aimed to investigate the mechanisms underlying gastric cell dysfunction after prolonged exposure to H. pylori under hypoxic conditions. We conducted a co-culture experiment using the gastric cell line GES-1 and H. pylori for 30 generations under intermittent hypoxic conditions. By closely monitoring cell proliferation, migration, invasion, autophagy, and apoptosis, we revealed that sustained H. pylori stimulation under hypoxic conditions significantly influences the function of GES-1 cells. This stimulation induces epithelial-mesenchymal transition and contributes to the propensity for malignant transformation of gastric cells. To confirm the in vitro results, we conducted an experiment involving Mongolian gerbils infected with H. pylori for 85 weeks. All the results strongly suggest that the Nod1 receptor signaling pathway plays a crucial role in H. pylori-related apoptosis and autophagy. In summary, continuous stimulation by H. pylori affects the functioning of gastric cells through the Nod1 receptor signaling pathway, increasing the likelihood of cell carcinogenesis. The presence of hypoxic conditions further exacerbates this process.IMPORTANCEDeciphering the collaborative effects of Helicobacter pylori infection on gastric epithelial cell function is key to unraveling the development mechanisms of gastric cancer. Prior research has solely examined the outcomes of short-term H. pylori stimulation on gastric epithelial cells under aerobic conditions, neglecting the bacterium's nature as a microaerophilic organism that leads to cancer following prolonged stomach colonization. This study mimics a more genuine in vivo infection scenario by repeatedly exposing gastric epithelial cells to H. pylori under hypoxic conditions for up to 30 generations. The results show that chronic exposure to H. pylori in hypoxia substantially increases cell migration, invasion, and epithelial-mesenchymal transition, while suppressing autophagy and apoptosis. This highlights the significance of hypoxic conditions in intensifying the carcinogenic impact of H. pylori infection. By accurately replicating the in vivo gastric environment, this study enhances our comprehension of H. pylori's pathogenic mechanisms in gastric cancer.

Establishment and Application of a Novel Genetic Detection Panel for SNPs in Mongolian Gerbils.

The Mongolian gerbil is a distinctive experimental animal in China, as its genetic qualities possess significant value in the field of medical biology research. Here, we aimed to establish an economical and efficient panel for genetic quality detection in Mongolian gerbils using single-nucleotide polymorphism (SNP) markers. To search for SNPs, we conducted whole-genome sequencing (WGS) in 40 Mongolian gerbils from outbred populations. Reliable screening criteria were established to preliminarily select SNPs with a wide genome distribution and high levels of polymorphism. Subsequently, a multiple-target regional capture detection system based on second-generation sequencing was developed for SNP genotyping. Based on the results of WGS, 219 SNPs were preliminarily selected, and they were established and optimized in a multiple-amplification system that included 206 SNP loci by genotyping three outbred populations. PopGen.32 analysis revealed that the average effective allele number, Shannon index, observed heterozygosity, expected heterozygosity, average heterozygosity, polymorphism information content, and other population genetic parameters of the Capital Medical University (CMU) gerbils were the highest, followed by those of Zhejiang gerbils and Dalian gerbils. Through scientific screening and optimization, we successfully established a novel, robust, and cost-effective genetic detection system for Mongolian gerbils by utilizing SNP markers for the first time.

Development of a new cerebral ischemia reperfusion model of Mongolian gerbils and standardized evaluation system.

BACKGROUND: The Mongolian gerbil is an excellent laboratory animal for preparing the cerebral ischemia model due to its inherent deficiency in the circle of Willis. However, the low incidence and unpredictability of symptoms are caused by numerous complex variant types of the circle. Additionally, the lack of an evaluation system for the cerebral ischemia/reperfusion (I/R) model of gerbils has shackled the application of this model. METHODS: We created a symptom-oriented principle and detailed neurobehavioral scoring criteria. At different time points of reperfusion, we analyzed the alteration in locomotion by rotarod test and grip force score, infarct volume by triphenyltetrazolium chloride (TTC) staining, neuron loss using Nissl staining, and histological characteristics using hematoxylin-eosin (H&E) straining. RESULTS: With a successful model rate of 56%, 32 of the 57 gerbils operated by our method harbored typical features of cerebral I/R injury, and the mortality rate in the male gerbils was significantly higher than that in the female gerbils. The successfully prepared I/R gerbils demonstrated a significant reduction in motility and grip strength at 1 day after reperfusion; formed obvious infarction; exhibited typical pathological features, such as tissue edema, neuronal atrophy and death, and vacuolated structures; and were partially recovered with the extension of reperfusion time. CONCLUSION: This study developed a new method for the unilateral common carotid artery ligation I/R model of gerbil and established a standardized evaluation system for this model, which could provide a new cerebral I/R model of gerbils with more practical applications.

Sustained exposure to Helicobacter pylori induces immune tolerance by desensitizing TLR6.

Helicobacter pylori (H. pylori, Hp) has been designated a class I carcinogen and is closely associated with severe gastric diseases. During colonization in the gastric mucosa, H. pylori develops immune escape by inducing host immune tolerance. The gastric epithelium acts as the first line of defense against H. pylori, with Toll-like receptors (TLRs) in gastric epithelial cells being sensitive to H. pylori components and subsequently activating the innate immune system. However, the mechanism of immune tolerance induced by H. pylori through the TLR signalling pathway has not been fully elucidated. In this research, we detected the expression of TLRs and inflammatory cytokines in GES-1 cells upon sustained exposure to H. pylori or H. pylori lysate from 1 to 30 generations and in Mongolian gerbils infected with H. pylori for 5 to 90 weeks. We found that the levels of TLR6 and inflammatory cytokines first increased and then dropped during the course of H. pylori treatment in vitro and in vivo. The restoration of TLR6 potentiated the expression of IL-1ß and IL-8 in GES-1 cells, which recruited neutrophils and reduced the colonization of H. pylori in the gastric mucosa of gerbils. Mechanistically, we found that persistent infection with H. pylori reduces the sensitivity of TLR6 to bacterial components and regulates the expression of inflammatory cytokines in GES-1 cells through TLR6/JNK signaling. The TLR6 agonist obviously alleviated inflammation in vitro and in vivo. Promising results suggest that TLR6 may be a potential candidate immunotherapy drug for H. pylori infection.

ELAVL4 promotes the tumorigenesis of small cell lung cancer by stabilizing LncRNA LYPLAL1-DT and enhancing profilin 2 activation.

Small cell lung cancer (SCLC) is one of the most malignant tumors that has an extremely poor prognosis. RNA-binding protein (RBP) and long noncoding RNA (lncRNA) have been shown to be key regulators during tumorigenesis as well as lung tumor progression. However, the role of RBP ELAVL4 and lncRNA LYPLAL1-DT in SCLC remains unclear. In this study, we verified that lncRNA LYPLAL1-DT acts as an SCLC oncogenic lncRNA and was confirmed in vitro and in vivo. Mechanistically, LYPLAL1-DT negatively regulates the expression of miR-204-5p, leading to the upregulation of PFN2, thus, promoting SCLC cell proliferation, migration, and invasion. ELAVL4 has been shown to enhance the stability of LYPLAL1-DT and PFN2 mRNA. Our study reveals a regulatory pathway, where ELAVL4 stabilizes PFN2 and LYPLAL1-DT with the latter further increasing PFN2 expression by blocking the action of miR-204-5p. Upregulated PFN2 ultimately promotes tumorigenesis and invasion in SCLC. These findings provide novel prognostic indicators as well as promising new therapeutic targets for SCLC.

LncRNA LYPLAL1-DT screening from type 2 diabetes with macrovascular complication contributes protective effects on human umbilical vein endothelial cells via regulating the miR-204-5p/SIRT1 axis.

Long noncoding RNAs (lncRNAs) are involved in diabetes related diseases. However, the role of lncRNAs in the pathogenesis of type 2 diabetes with macrovascular complication (DMC) has seldomly been recognized. This study screened lncRNA profiles of leukocytes from DMC patients and explored protective role of lncRNA LYPLAL1-DT in endothelial cells (EC) under high glucose (HG) and inflammatory conditions (IS). Between DMC and healthy controls, 477 differential expression lncRNAs (DE-lncRNAs) were identified. The enrichment and pathway analysis showed that most of the DE-lncRNAs belonged to inflammatory, metabolic, and vascular diseases. A total of 12 lncRNAs was validated as significant DE-lncRNAs in expanding cohorts. Furthermore, these DE-lncRNAs were shown to be significantly related to hypoxia, HG, and IS in EC, especially lncRNA LYPLAL1-DT. LYPLAL1-DT overexpression results in the promotion of the proliferation, and migration of EC, as well as an elevation of autophagy. Overexpressed LYPLAL1-DT reduces the adhesion of monocytes to EC, boosts anti-inflammation, and suppresses inflammatory molecules secreted in the medium. Mechanistically, LYPLAL1-DT acts as competing endogenous RNA (ceRNA) by downregulating miR-204-5p, therefore enhancing SIRT1 and protecting EC autophagy function; thus, alleviating apoptosis. Finally, exosome sequencing revealed LYPLAL1-DT expression was 4 times lower in DMC cells than in healthy samples. In general, we identified LYPLAL1-DT having protective effects on EC as ceRNA mediated through the miR-204-5p/SIRT1 pathway. Therefore, it inhibits the autophagy of EC as well as modulating systemic inflammation. This approach could be regarded as a new potential therapeutic target in DMC.

Establishment of Noninvasive Methods for the Detection of Helicobacter pylori in Mongolian Gerbils and Application of Main Laboratory Gerbil Populations in China.

Identifying Helicobacter pylori (H. pylori, Hp) infection in animals before and after artificial infection influences the subsequent experiment. We established effective and noninvasive detection methods, including the gastric fluid nested polymerase chain reaction (PCR) method and the 13C-urea breath test, which can detect Hp before modeling Hp infection in Mongolian gerbils. We designed a gas collection equipment for gerbils. Hp nested PCR was also performed on gastric fluid, gastric mucosa, duodenal contents, and faeces of gerbils challenged with Hp. Conventional Hp detection methods, including rapid urease assay and immunohistochemistry, were compared. Moreover, we assessed the natural infection of Hp in 135 gerbils that had never been exposed to Hp artificially from the major laboratory gerbil groups in China. In 10 Hp infected gerbils, the positive detection results were 100%, 100%, 90%, and 10% in gastric fluid, gastric mucosa, duodenal contents, and faeces with nested PCR, respectively. A rapid urease test performed on gastric mucosa showed that all animals were infected with Hp. Immunohistochemical detection and bacteria culture of gastric mucosa samples that were positive by the nested PCR method also confirmed the presence of Hp. 9% (3/35) and 6% (2/31) natural infection rates were found in conventional gerbil groups from the Capital Medical University and Zhejiang Laboratory Animal Center. In conclusion, we established two noninvasive Hp detection methods that can be performed before modelingHp infection, including the gastric fluid nested PCR method and the 13C-urea breath test.

The effect of cofilin 1 expression and phosphorylation dynamics on cell fate determination and neuron maturation in neural stem cells.

Previous studies showed that neural stem cells (NSCs) have an ability to differentiate into neurons, astrocytes and oligodendrocytes. However, the mechanisms that govern the fate of neural stem cell determination have not yet been fully clarified. In this study, we demonstrated that expression and activation of cofilin 1, a F-actin depolymerizing factor, are significantly changed during the development of brain, cortex or NSCs. Using Neuro-2a cells as a model, we found that overexpression of cofilin 1 significantly inhibit the cell differentiation and neurite outgrowth, while inhibition of intracellular cofilin 1 phosphorylation was significantly promoted. In cultured NSCs, we observed that cofilin 1 reduced the proportion of neurons derived from NSC due to inhibition of the phosphorylation, while the morphological maturation of neurons was promoted. Together, our findings revealed that cofilin 1 plays dynamic regulatory role on NSC cell fate determination and enhance neuronal maturation through regulating its activity and expression.

Development of Microsatellite Marker System to Determine the Genetic Diversity of Experimental Chicken, Duck, Goose, and Pigeon Populations.

Poultries including chickens, ducks, geese, and pigeons are widely used in the biological and medical research in many aspects. The genetic quality of experimental poultries directly affects the results of the research. In this study, following electrophoresis analysis and short tandem repeat (STR) scanning, we screened out the microsatellite loci for determining the genetic characteristics of Chinese experimental chickens, ducks, geese, and pigeons. The panels of loci selected in our research provide a good choice for genetic monitoring of the population genetic diversity of Chinese native experimental chickens, ducks, geese, and ducks.

Profile Screening of Differentially Expressed lncRNAs of Circulating Leukocytes in Type 2 Diabetes Patients and Differences From Type 1 Diabetes.

Long noncoding RNAs (lncRNAs) have been reported to have multiple functions and can be used as markers of various diseases, including diabetes. This study was conducted to determine the lncRNA profile in leukocytes from patients with type 2 diabetes (T2D). Differential expression of lncRNAs in T2D and type 1 diabetes (T1D) was also examined. RNA sequencing was performed in a critically grouped sample of leukocytes from T2D patients and healthy persons. A total of 845 significantly differentially expressed lncRNAs were identified, with 260 downregulated and 585 upregulated lncRNAs in T2D. The analysis of functions of DE-lncRNA and constructed co-expression networks (CNC) showed that 21 lncRNAs and 117 mRNAs harbored more than 10 related genes in CNC. Fourteen of 21 lncRNAs were confirmed to be significantly differentially expressed was detected by qPCR between the T2D and control validation cohorts. We also identified a panel of 4 lncRNAs showing significant differences in expression between T1D and T2D. Collectively, hundreds of novel DE-lncRNAs we identified in leukocytes from T2D patients will aid in epigenetic mechanism studies. Fourteen confirmed DE-lncRNAs can be regarded as diagnostic markers or regulators of T2D, including 4 lncRNAs that chould distinguish T1D and T2D in clinical practice to avoid misdiagnosis.

The endothelial nitric oxide synthase/cyclic guanosine monophosphate/protein kinase G pathway activates primordial follicles.

In mammals, the well-organized activation of quiescent primordial follicles is pivotal for female reproductive reserve. In the present study, we examined the mechanisms underlying primordial follicle activation in mice. We found that endothelial nitric oxide synthase (eNOS) and its downstream effectors, cyclic guanosine monophosphate (cGMP) and cGMP-dependent protein kinase G (PKG), were expressed in pre-granulosa cells and promoted primordial follicle activation, oocyte growth and granulosa cell proliferation in neonatal ovaries. Mammalian target of rapamycin (mTOR) colocalized with PKG in pre-granulosa cells and was essential for eNOS/cGMP/PKG pathway-induced primordial follicle activation. The eNOS/cGMP/PKG pathway was found to stabilize mTOR protein. The mRNA levels of F-box and WD repeat domain containing 7 (FBXW7), an E3 ubiquitin ligase, correlated negatively with mTOR protein levels in neonatal ovaries. FBXW7 bound to and destabilized mTOR protein in pre-granulosa cells in a ubiquitin/proteasome-dependent manner. However, agonists of the eNOS/cGMP/PKG pathway reduced FBXW7 mRNA levels. FBXW7 overexpression suppressed primordial follicle activation and prevented the eNOS/cGMP/PKG pathway from activating primordial follicles and stabilizing mTOR protein. These findings demonstrate that the eNOS/cGMP/PKG pathway activates primordial follicles by suppressing FBXW7-induced ubiquitination of mTOR in mice.

A Genome-Wide Analysis of Long Noncoding RNAs in Circulating Leukocytes and Their Differential Expression in Type 1 Diabetes Patients.

Long noncoding RNAs (lncRNAs) regulate gene expression at different levels in various diseases, including type 1 diabetes (T1D). However, the expression of circulating lncRNAs in leukocytes in T1D has not been well documented. To identify differentially expressed lncRNAs between T1D patients and healthy controls, RNA sequencing was performed on samples of leukocytes collected from both healthy persons and T1D patients. The categories, enriched pathways, coexpression networks, and the characteristics of novel lncRNAs were analyzed to provide an extensive profile. qPCR was adopted to validate the differential expression of lncRNAs in the validation cohort. A total of 14,930 lncRNAs and 16,063 mRNAs were identified in the peripheral blood leukocyte of T1D patients. After optimization using an adjusted p value (threshold of <0.05), 393 circulating lncRNAs were identified, of which 69 were downregulated and 324 were upregulated in T1D patients. Gene Ontology analysis indicated that these lncRNAs and mRNAs were enriched in the immune system category. Further analysis showed that 61.28% of the novel lncRNAs were conserved in humans. A set of 12 lncRNAs were selected for qPCR validation, and 9 of 12 lncRNAs were confirmed to show significant differential expression between the T1D and control validation cohorts. Among the 9 confirmed lncRNAs, lncRNA MSTRG.128697 and lncRNA MSTRG.128958 were novel and human-specific; however, further validation is required. lncRNA MSTRG.63013 has orthologous sequences in the mouse genome and was identified as a key node for etiology and pathophysiology in animal studies, which will help understand the epigenetic mechanisms of T1D complications.

Sustained Exposure to Helicobacter pylori Lysate Inhibits Apoptosis and Autophagy of Gastric Epithelial Cells.

Helicobacter pylori is designated as a class I carcinogen of human gastric cancer following long-term infection. During this process, H. pylori bacteria persist in proliferation and death, and release bacterial components that come into contact with gastric epithelial cells and regulate host cell function. However, the impact of long-term exposure to H. pylori lysate on the pathological changes of gastric cells is not clear. In this study, we aimed to investigate the regulation and mechanisms involved in gastric cell dysfunction following continuous exposure to H. pylori lysate. We co-cultured gastric cell lines GES-1 and MKN-45 with H. pylori lysate for 30 generations, and we found that sustained exposure to H. pylori lysate inhibited GES-1 cell invasion, migration, autophagy, and apoptosis, while it did not inhibit MKN-45 cell invasion or migration. Furthermore, Mongolian gerbils infected with H. pylori ATCC 43504 strains for 90 weeks confirmed the in vitro results. The clinical and in vitro data indicated that sustained exposure to H. pylori lysate inhibited cell apoptosis and autophagy through the Nod1-NF-κB/MAPK-ERK/FOXO4 signaling pathway. In conclusion, sustained exposure to H. pylori lysate promoted proliferation of gastric epithelial cells and inhibited autophagy and apoptosis via Nod1-NF-κB/MAPK-ERK/FOXO4 signaling pathway. In the process of H. pylori-induced gastric lesions, H. pylori lysate plays as an "accomplice" to carcinogenesis.

Profilin 2 promotes growth, metastasis, and angiogenesis of small cell lung cancer through cancer-derived exosomes.

Small cell lung cancer (SCLC) is highly aggressive and prone to hypervascular metastases. Recently, we found profilin 2 (PFN2) expression in SCLC but not in normal tissues. Furthermore, PFN2 expression had been shown to promote angiogenesis through exosomes. However, it remains unclear whether PFN2 contributes to the progression and metastasis of SCLC through angiogenesis. We report here that overexpression (OE) of PFN2 increased, whereas its knockdown (KD) decreased the proliferation, migration, and invasion of SCLC cell H446. The exosomes from OE-H446 (SCLC-OE-exo) exhibited similar effects on H446 properties. Culturing of endothelial cells (ECs) in SCLC-OE conditioned medium (CM) or SCLC-OE-exo increased the migration and tube formation ability of ECs, whereas SCLC-KD-CM and SCLC-KD-exo had inhibitory effects. Interestingly, both SCLC- and EC-derived exosomes were internalized in H446 more rapidly than in ECs. More importantly, OE-PFN2 dramatically elevated SCLC growth and vasculature formation as well as lung metastasis in tumor xenograft models. Finally, we found that PFN2 activated Smad2/3 in H446 and pERK in ECs, respectively. Taken together, our study revealed the role of PFN2 in SCLC development and metastasis, as well as tumor angiogenesis through exosomes, providing a new molecular target for SCLC treatment.

Exploring the mechanism of cisplatin resistance by transcriptome sequencing and reversing the chemoresistance by autophagy inhibition in small cell lung cancer.

Cisplatin plays a key role in treating small cell lung cancer (SCLC); however, the rapid development of cisplatin resistance limits its treatment effect. The detailed mechanisms of cisplatin-resistance, particularly in SCLC, remain unclear. We analyzed the differentially expressed genes (DEGs) between cisplatin-resistant small cell lung cancer cell line H446/CDDP and its parental cell line H446, using the transcriptome sequencing technique. Gene ontology (GO) analysis and the subsequent tests demonstrated that the functions of protein ubiquitination and autophagy are more active in the H446/CDDP cells. Autophagy plays a protective role in the H446/CDDP cells by using the autophagy inhibitors, 3-methyladenine and bafilomycin A1. Moreover, antimalarial drugs that inhibit autophagy by increasing the pH of lysosomes can also enhance cisplatin-induced cell death.

Generation of Gene-Knockout Mongolian Gerbils via CRISPR/Cas9 System.

The Mongolian gerbil (Meriones unguiculatus), a well-known "multifunctional" experimental animal, plays a crucial role in the research of hearing, cerebrovascular diseases and Helicobacter pylori infection. Although the whole-genome sequencing of Mongolian gerbils has been recently completed, lack of valid gene-editing systems for gerbils largely limited the further usage of Mongolian gerbils in biomedical research. Here, efficient targeted mutagenesis in Mongolian gerbils was successfully conducted by pronuclear injection with Cas9 protein and single-guide RNAs (sgRNAs) targeting Cystatin C (Cst3) or Apolipoprotein A-II (Apoa2). We found that 22 h after human chorionic gonadotropin (hCG) injection, zygote microinjection was conducted, and the injected zygotes were transferred into the pseudopregnant gerbils, which were induced by injecting equine chorionic gonadotropin (eCG) and hCG at a 70 h interval and being caged with ligated male gerbils. We successfully obtained Cst3 and Apoa2 gene knockout gerbils with the knockout efficiencies of 55 and 30.9%, respectively. No off-target effects were detected in all knockout gerbils and the mutations can be germline-transmitted. The absence of CST3 protein was observed in the tissues of homozygous Cst3 knockout (Cst3-KO) gerbils. Interestingly, we found that disruption of the Cst3 gene led to more severe brain damage and neurological deficits after unilateral carotid artery ligation, thereby indicating that the gene modifications happened at both genetic and functional levels. In conclusion, we successfully generated a CRISPR/Cas9 system based genome editing platform for Mongolian gerbils, which provided a foundation for obtaining other genetically modified gerbil models for biomedical research.

Development of an effective microsatellite marker system to determine the genetic structure of Meriones meridianus populations.

Understanding the genetic quality of the gerbil, Meriones meridianus, plays an important role in the study of medical biology. However, no effective system has been established for evaluating a population's genetic diversity to date. In the present study, we established a set of reasonable evaluative systems based on microsatellite markers of the Mongolian gerbil by using the method of cross-amplification of species. Following electrophoresis analysis, short tandem repeat (STR) scanning, and sequencing, 11 microsatellite loci were identified by matching the criteria characteristics and were used to evaluate the genetic diversity of two stocks of Meriones meridianus: Meriones meridianus jei Wang, 1964 (M. m. jei) and Meriones meridianus cryptorhinus Blanford, 1875 (M. m. cryptorhinus) from Xinjiang, China. The microsatellite loci screened were highly polymorphic and were suitable for genetic quality control of Meriones meridianus. In addition, the quality of the non-bred M. m. jei and M. m. cryptorhinus strains in our study is sufficient for them to be promising stocks in the future for the farmed animal industry.

eEF1A2 exacerbated insulin resistance in male skeletal muscle via PKCß and ER stress.

Recent studies raise the possibility that eukaryotic translation elongation factor 1 alpha (eEF1A) may play a role in metabolism. One isoform, eEF1A2, is specifically expressed in skeletal muscle, heart and brain. It regulates translation elongation and signal transduction. Nonetheless, eEF1A2's function in skeletal muscle glucose metabolism remains unclear. In the present study, suppression subtractive hybridisation showed a decrease in Eef1a2 transcripts in the skeletal muscle of diabetic Mongolian gerbils. This was confirmed at mRNA and protein levels in hyperglycaemic gerbils, and in db/db and high-fat diet-fed mice. Further, this downregulation was independent of Eef1a2 promoter methylation. Interestingly, adeno-associated virus-mediated eEF1A2 overexpression in skeletal muscle aggravated fasting hyperglycaemia, hyperinsulinaemia and glucose intolerance in male diabetic gerbils but not in female gerbil models. The overexpression of eEF1A2 in skeletal muscle also resulted in promoted serum glucose levels and insulin resistance in male db/db mice. Up- and downregulation of eEF1A2 by lentiviral vector transfection confirmed its inhibitory effect on insulin-stimulated glucose uptake and signalling transduction in C2C12 myotubes with palmitate (PA)-induced insulin resistance. Furthermore, eEF1A2 bound PKCß and increased its activation in the cytoplasm, whereas suppression of PKCß by an inhibitor attenuated eEF1A2-mediated impairment of insulin sensitivity in insulin-resistant myotubes. Endoplasmic reticulum (ER) stress was elevated by eEF1A2, whereas suppression of ER stress or JNK partially restored insulin sensitivity in PA-treated myotubes. Additionally, eEF1A2 inhibited lipogenesis and lipid utilisation in insulin-resistant skeletal muscle. Collectively, we demonstrated that eEF1A2 exacerbates insulin resistance in male murine skeletal muscle via PKCß and ER stress.