Aim: In forensic science, the authentication of biological samples is critical for accurate analysis and evidence validation. To evaluate the authenticity and genetic stability of 91 long-term preserved human cell line samples under cryogenic conditions over 34 years, and to explore the applicability of forensic-grade short tandem repeat (STR) profiling for long-term cell line authentication. Methods: We conducted STR-based authentication using 23 forensic STR markers. STR profiles were analyzed using the Tanabe and Masters algorithms for authentication. The study also examined the effects of prolonged passaging and genetic modification on STR stability, and investigated potential contamination events by comparing profiles with public databases. Results: All uniquely labeled human cell lines were successfully revived and yielded complete STR profiles, confirming the efficacy of long-term cryopreservation. One male cell line showed the Y-indel in the absence of a Y-allele at Amelogenin, indicating that the Y-indel alone is insufficient for accurate sex determination. Authentication using the Tanabe and Masters algorithms revealed that stringent criteria provided more definitive results than scoring-based methods. Instances of relatedness among unauthenticated cell lines to problematic references in existing databases suggest early-stage contamination rather than recent laboratory errors. STR profile stability persisted through routine cell line passages, and genetically edited cell lines retained profiles identical to their parental counterparts, indicating a need for additional differentiation markers for accurate derivation authentication. No significant differences in the number of altered alleles between normal and tumor cell lines, implying cell type alone did not dictate STR stability. Conclusion: This study represented one of the most extensive single-laboratory investigations into cell line preservation using forensic-grade tools, providing new reference alleles and valuable insights into cell line authentication. The findings supported the application of forensic STR kits beyond traditional forensic samples, offering a new perspective on their utility in genetic research and laboratory management.

β-glucosidase (BGL) is a pivotal enzyme with broad implications across diverse industrial sectors, demonstrating robust proficiency in catalyzing glycosidic bond hydrolysis, particularly critical in lignocellulosic biomass conversion. This study explored the structural features and enzymatic activity of Thermoproteus sp. AZ2-derived BGL (TsBGL2). The optimum temperature and pH for TsBGL2 were 95 °C and 5.0, respectively. TsBGL2 exhibited strong thermal stability, retaining >95 % of its activity after incubation at 99 °C for 10 h. Three high-resolution crystal structures of TsBGL2 revealed a canonical (α/β)8-barrel catalytic domain and thermostabilization mechanism. Based on the structure of TsBGL2, Δ(473-495) TsBGL2 was constructed, revealing a significant decrease in thermal stability. Collectively, this study provides enzymatic properties and structural analyses of TsBGL2, laying the groundwork for further studies of β-glucosidase.

The testes are the organs of gamete production and testosterone synthesis. Up to date, no model system is available for mammalian testicular development, and only few studies have characterized the mouse testis transcriptome from no more than three postnatal ages. To describe the transcriptome landscape of the developing mouse testis and identify the potential molecular mechanisms underlying testis maturation, we examined multiple RNA-seq data of mouse testes from 3-week-old (puberty) to 11-week-old (adult). Sperm cells appeared as expected in 5-week-old mouse testis, suggesting the proper sample collection. The principal components analysis revealed the genes from 3w to 4w clustered away from other timepoints, indicating they may be the important nodes for testicular development. The pairwise comparisons at two adjacent timepoints identified 7,612 differentially expressed genes (DEGs), resulting in 58 unique mRNA expression patterns. Enrichment analysis identified functions in tissue morphogenesis (3-4w), regulation of peptidase activity (4-5w), spermatogenesis (7-8w), and antigen processing (10-11w), suggesting distinct functions in different developmental periods. 50 hub genes and 10 gene cluster modules were identified in the testis maturation process by protein-protein interaction (PPI) network analysis, and the miRNA-lncRNA-mRNA, miRNA-circRNA-mRNA and miRNA-circRNA-lncRNA-mRNA competing endogenous RNA (ceRNA) networks were constructed. The results suggest that testis maturation is a complex developmental process modulated by various molecules, and that some potential RNA-RNA interactions may be involved in specific developmental stages. In summary, this study provides an update on the molecular basis of testis development, which may help to understand the molecular mechanisms of mouse testis development and provide guidance for mouse reproduction.

Background: Animal models are commonly utilized by the pharmaceutical industry to evaluate new drugs; however, the results of these studies are not always applicable to humans due to differences between the genetic backgrounds. Methods: Since liver plays a prominent role in drug metabolism, we evaluated the expression profiles of human, mouse, rat, and rhesus monkey liver tissues. The differentially expressed genes were identified in different genders, strains, and species. Results: The primates had fewer differences compared to the rodents, suggesting rhesus monkey could be a preferred model organism. Although the human HGFR (MET), a putative AAV3 receptor, together with its ligand HGF, showed no significant expression differences to the mammal models, the ligand-receptor (HGF/MET) ratio difference between the human (HGF/MET = 0.56) and the mice (HGF/MET = 0.12) might result in the transduction failure of murine hepatocytes using the AAV3 vectors. Our results also showed ACE2 expression was not detected in the human liver, while low expression levels were observed in other species, suggesting that the liver might not be the main target of SARS-CoV2. Conclusion: In summary, even though all model organisms have limitations, non-human primates or humanized animals are indispensable during drug development before proceeding to clinical trials.

beta-Glucosidase is a crucial cellulase, as its activity determines the efficiency of cellulose hydrolysis into glucose. This study addresses the functional and structural characteristics of Thermotoga profunda beta-glucosidase (Tp-BGL). Tp-BGL exhibited a K-m of 0.3798 mM for p-nitrophenyl-beta-d-glucopyranoside (pNPGlc) and 4.44 mM for cellobiose, with k(cat)/K-m of 1211.16 and 4.18 s(-1) mM(-1), respectively. In addition, Tp-BGL showed significant pH adaptability and thermal stability, with a T-m of 85.7 degrees C and retaining >90 % of its activity after incubation at 80 degrees C for 90 min. The crystal structure of Tp-BGL was resolved at 1.95 & Aring; resolution, and reveals a typical TIM barrel structure. Comparative structural analysis highlighted that the major distinction between Tp-BGL and the other glucosidases lies in their loop regions.

Ribonucleases (RNases) are responsible for RNA metabolism. RNase J, the core enzyme of the RNA degradosome, plays an essential role in global mRNA decay. Emerging evidence showed that the RNase J of Mycobacterium tuberculosis (Mtb-RNase J) could be an excellent target for treating Mtb infection. Here, crystal structures of Mtb-RNase J in apo-state and complex with the single-strand RNA reveal the conformational change upon RNA binding and hydrolysis. Mtb-RNase J forms an active homodimer through the interactions between the β-CASP and the β-lactamase domain. Knockout of RNase J slows the growth rate and changes the colony morphologies and cell length in Mycobacterium smegmatis, which is restored by RNase J complementation. Finally, RNA-seq analysis shows that the knockout strain significantly changes the expression levels of 49 genes in metabolic pathways. Thus, our current study explores the structural basis of Mtb-RNase J and might provide a promising candidate in pharmacological treatment for tuberculosis.

Gasdermin-D (GSDMD), the executioner of pyroptotic cell death when it is cleaved by inflammatory caspases, plays a crucial role in host defense and the response to danger signals. So far, there are no known mechanisms, other than cleavage, for regulating GSDMD. Here, we show that tripartite motif protein TRIM21 acts as a positive regulator of GSDMD-dependent pyroptosis. TRIM21 interacted with GSDMD via its PRY-SPRY domain, maintaining GSDMD stable expression in resting cells yet inducing the N-terminus of GSDMD (GSDMD-N) aggregation during pyroptosis. TRIM21-deficient cells displayed a reduced cell death in response to NLRP3 or NLRC4 inflammasome activation. Genetic ablation of TRIM21 in mice conferred protection from LPS-induced inflammation and dextran sulfate sodium-induced colitis. Therefore, TRIM21 plays an essential role in GSDMD-mediated pyroptosis and may be a viable target for controlling and treating inflammation-associated diseases.

Beta-glucosidase is an enzyme that catalyzes the hydrolysis of the glycosidic bonds of cellobiose, resulting in the production of glucose, which is an important step for the effective utilization of cellulose. In the present study, a thermostable β-glucosidase was isolated and purified from the Thermoprotei Thermofilum sp. ex4484_79 and subjected to enzymatic and structural characterization. The purified β-glucosidase (TsBGL) exhibited maximum activity at 90°C and pH 5.0 and displayed maximum specific activity of 139.2μmol/min/mgzne against p-nitrophenyl β-D-glucopyranoside (pNPGlc) and 24.3μmol/min/mgzen against cellobiose. Furthermore, TsBGL exhibited a relatively high thermostability, retaining 84 and 47% of its activity after incubation at 85°C for 1.5h and 90°C for 1.5h, respectively. The crystal structure of TsBGL was resolved at a resolution of 2.14Å, which revealed a classical (α/β)8-barrel catalytic domain. A structural comparison of TsBGL with other homologous proteins revealed that its catalytic sites included Glu210 and Glu414. We provide the molecular structure of TsBGL and the possibility of improving its characteristics for potential applications in industries.

β-葡萄糖苷酶(BGL)是催化纤维素产生葡萄糖过程中的关键酶,在生物能源利用和食品领域发挥重要作用.本研究克隆了来源于Thermoplasma volcanium的β-葡萄糖苷酶(BGL)基因,并在大肠杆菌中进行表达,经纯化获得了Tv-BGL.酶学性质分析发现,Tv-BGL在pH6.0和80℃时发挥最佳催化活力,以对硝基苯基-β-D-吡喃葡萄糖苷(pNPGlu)为底物时的Km和Vmax分别为0.7254 mmol/L和614.60 U/mg,Kcat/Km=7.79×105 m01-1·L·s-1,以纤维二糖(Cellobiose)为底物时的Km和Vmax分别为55.63 mmol/L和636.06 U/mg,Kcat/Km=1.06×104 mol-1 ·L·s-1.另外,该酶具有较好的热稳定性,有机溶剂、离子以及葡萄糖耐受性.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a highly conserved enzyme involved in the ubiquitous process of glycolysis and presents a loop (residues 208–215 of Escherichia coli GAPDH) in two alternative conformations (I and II). It is uncertain what triggers this loop rearrangement, as well as which is the precise site from which phosphate attacks the thioacyl intermediate precursor of 1,3-bisphosphoglycerate (BPG). To clarify these uncertainties, we determined the crystal structures of complexes of wild-type GAPDH (WT) with NAD and phosphate or G3P, and of essentially inactive GAPDH mutants (C150S, H177A), trapping crystal structures for the thioacyl intermediate or for ternary complexes with NAD and either phosphate, BPG, or G3P. Analysis of these structures reported here lead us to propose that phosphate is located in the “new Pi site” attacks the thioester bond of the thioacyl intermediate to generate 1,3-bisphosphoglyceric acid (BPG). In the structure of the thioacyl intermediate, the mobile loop is in conformation II in subunits O, P, and R, while both conformations coexist in subunit Q. Moreover, only the Q subunit hosts bound NADH. In the R subunit, only the pyrophosphate part of NADH is well defined, and NADH is totally absent from the O and P subunits. Thus, the change in loop conformation appears to occur after NADH is produced, before NADH is released. In addition, two new D-glyceraldehyde-3-phosphate (G3P) binding forms are observed in WT.NAD.G3P and C150A+H177A.NAD.G3P. In summary, this paper improves our understanding of the GAPDH catalytic mechanism, particularly regarding BPG formation.

Phosphofructokinase B (PfkB) belongs to the ribokinase family, which uses the phosphorylated sugar as substrate, and catalyzes fructose-6-phosphate into fructose-1,6-diphosphate. However, the structural basis of Mycobacterium marinum PfkB is not clear. Here, we found that the PfkB protein was monomeric in solution, which was different from most enzymes in this family. The crystal structure of PfkB protein from M. marinum was solved at a resolution of 2.21 Å. The PfkB structure consists of two domains, a major three-layered α/β/α sandwich-like domain characteristic of the ribokinase-like superfamily, and a second domain composed of four-stranded β sheets. Structural comparison analysis suggested that residues G236, A237, G238, and D239 could be critical for ATP catalysis and substrate binding of PfkB. Our current work provides new insights into understanding the mechanism of the glycolysis in M. marinum.