Structural insights and functional characterization of a novel ß-glucosidase derived from Thermotoga profunda.

ß-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 ß-glucosidase (Tp-BGL). Tp-BGL exhibited a Km of 0.3798 mM for p-nitrophenyl-ß-d-glucopyranoside (pNPGlc) and 4.44 mM for cellobiose, with kcat/Km of 1211.16 and 4.18 s-1 mM-1, respectively. In addition, Tp-BGL showed significant pH adaptability and thermal stability, with a Tm of 85.7 °C and retaining >90 % of its activity after incubation at 80 °C for 90 min. The crystal structure of Tp-BGL was resolved at 1.95 Å 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.

Effect of Preoperative Malnutrition Based on Albumin and BMI on Hepatocellular Carcinoma Surgery and Prediction of Risk Factors of Complications.

BACKGROUND: To investigate the correlation between preoperative malnutrition and perioperative variables in patients with hepatocellular carcinoma (HCC) and to analyze the risk factors of complications after HCC resection. METHODS: All patients who underwent hepatectomy because of HCC in the First Affiliated Hospital of Chongqing Medical University from June 1, 2018, to December 1, 2021, were analyzed retrospectively. Preoperative malnutrition was defined as body mass index (BMI) < 18.5 kg/m2 or serum albumin level < 3.5 g/dL within 30 days before operation. RESULTS: A total of 415 patients with HCC hepatectomy were included, and 75 (18.1%) were classified as malnutrition group. In the malnutrition group, blood loss (662.1 ± 748.1 VS 404.6 ± 681.9, P = 0.002), transfusion rate (36.0% VS 13.5%, P < 0.001), postoperative hospital stays (13.3 ± 9.6 VS 10.1 ± 4.2, P < 0.001), 30-day postoperative mortality (4.0 VS 0.6%, P = 0.043), complications rate (68% VS 34.8%, P < 0.001), and severe complication rate (17.3% VS 2.4%, P < 0.001) were significantly higher than those in the well-nourished group. Multivariate analysis showed that age (HR 1.037, 95% CI 1.015-1.059, P = 0.001), preoperative malnutrition (HR 2.933, 95% CI 1.515-5.679, P = 0.001), simultaneous cholecystectomy (HR 2.004, 95% CI 1.168-3.440, P = 0.012), cirrhosis (HR 4.997, 95% CI 2.864-8.718, P < 0.001), and transfusion (HR 5.166, 95% CI 2.272-11.748, P < 0.001) were independent risk factors for postoperative complications. In addition, preoperative malnutrition (HR 8.209, 95% CI 2.711-24.864, P < 0.001) and operation time (HR 1.088, 95% CI 1.003-1.103, P = 0.004) were independent risk factors for severe complications. CONCLUSION: Preoperative malnutrition can adversely affect the outcome of HCC resection. For patients with advanced age, cirrhosis, and malnutrition, preoperative planning is very important, and we should be more careful during the operation to avoid transfusion caused by bleeding and not to carry out preventive cholecystectomy, which are helpful to reduce the occurrence of postoperative complications.

Structural basis of the monkeypox virus mRNA cap N7 methyltransferase complex.

The global outbreak of Mpox, caused by the monkeypox virus (MPXV), has attracted international attention and become another major infectious disease event after COVID-19. The mRNA cap N7 methyltransferase (RNMT) of MPXV methylates the N7 position of the added guanosine to the 5'-cap structure of mRNAs and plays a vital role in evading host antiviral immunity. MPXV RNMT is composed of the large subunit E1 and the small subunit E12. How E1 and E12 of MPXV assembly remains unclear. Here, we report the crystal structures of E12, the MTase domain of E1 with E12 (E1CTD-E12) complex, and the E1CTD-E12-SAM ternary complex, revealing the detailed conformations of critical residues and the structural changes upon E12 binding to E1. Functional studies suggest that E1CTD N-terminal extension (Asp545-Arg562) and the small subunit E12 play an essential role in the binding process of SAM. Structural comparison of the AlphaFold2-predicted E1, E1CTD-E12 complex, and the homologous D1-D12 complex of vaccinia virus (VACV) indicates an allosteric activating effect of E1 in MPXV. Our findings provide the structural basis for the MTase activity stimulation of the E1-E12 complex and suggest a potential interface for screening the anti-poxvirus inhibitors.

A multistage Sendai virus vaccine incorporating latency-associated antigens induces protection against acute and latent tuberculosis.

One-quarter of the world's population is infected with Mycobacterium tuberculosis (Mtb). After initial exposure, more immune-competent persons develop asymptomatic latent tuberculosis infection (LTBI) but not active diseases, creates an extensive reservoir at risk of developing active tuberculosis. Previously, we constructed a novel recombinant Sendai virus (SeV)-vectored vaccine encoding two dominant antigens of Mtb, which elicited immune protection against acute Mtb infection. In this study, nine Mtb latency-associated antigens were screened as potential supplementary vaccine candidate antigens, and three antigens (Rv2029c, Rv2028c, and Rv3126c) were selected based on their immune-therapeutic effect in mice, and their elevated immune responses in LTBI human populations. Then, a recombinant SeV-vectored vaccine, termed SeV986A, that expresses three latency-associated antigens and Ag85A was constructed. In murine models, the doses, titers, and inoculation sites of SeV986A were optimized, and its immunogenicity in BCG-primed and BCG-naive mice were determined. Enhanced immune protection against the Mtb challenge was shown in both acute-infection and latent-infection murine models. The expression levels of several T-cell exhaustion markers were significantly lower in the SeV986A-vaccinated group, suggesting that the expression of latency-associated antigens inhibited the T-cell exhaustion process in LTBI infection. Hence, the multistage quarter-antigenic SeV986A vaccine holds considerable promise as a novel post-exposure prophylaxis vaccine against tuberculosis.

Structural analysis of the housecleaning nucleoside triphosphate pyrophosphohydrolase MazG from Mycobacterium tuberculosis.

The housecleaning enzyme of Mycobacterium tuberculosis (Mtb), MazG, is a nucleoside triphosphate pyrophosphohydrolase (NTP-PPase) and can hydrolyze all canonical or non-canonical NTPs into NMPs and pyrophosphate. The Mycobacterium tuberculosis MazG (Mtb-MazG) contributes to antibiotic resistance in response to oxidative or nitrosative stress under dormancy, making it a promising target for treating TB in latent infection patients. However, the structural basis of Mtb-MazG is not clear. Here we describe the crystal structure of Mtb-MazG (1-185) at 2.7 Å resolution, composed of two similar folded spherical domains in tandem. Unlike other all-α NTP pyrophosphatases, Mtb-MazG has an N-terminal extra region composed of three α-helices and five ß-strands. The second domain is global, with five α-helices located in the N-terminal domain. Gel-filtration assay and SAXS analysis show that Mtb-MazG forms an enzyme-active dimer in solution. In addition, the metal ion Mg2+ is bound with four negative-charged residues Glu119, Glu122, Glu138, and Asp141. Different truncations and site-directed mutagenesis revealed that the full-length dimeric form and the metal ion Mg2+ are indispensable for the catalytic activity of Mtb-MazG. Thus, our work provides new insights into understanding the molecular basis of Mtb-MazG.

Noninvasive Prediction of Immune Rejection After Liver Transplantation with T cell immunoglobulin domain, and mucin domain-3.

BACKGROUND: T cell immunoglobulin domain, and mucin domain-3 (Tim-3) is associated with immunosuppression, immune tolerance, and rejection after organ transplantation, but there are no reports of rejection after human liver transplantation. This study aimed to detect Tim-3 expression after liver transplantation to determine whether Tim-3 can be used as a noninvasive index to predict immune rejection. METHODS: Quantitative real-time polymerase chain reaction and enzyme-linked immunosorbent assay were performed on healthy people and patients with liver transplantation to detect Tim-3 and cytokines. Hepatic biochemical test was used to detect liver function. The study conforms to the provisions of the Declaration of Helsinki and Istanbul. RESULTS: The expression of Tim-3, alanine aminotransferase, and aspartate aminotransferase increased first and then decreased, whereas the cytokines showed upward trend. There was no significant difference in the expression of Tim-3 between preoperative patients and normal people (P > .05), whereas the expression of Tim-3 was significantly higher on the third day after operation than that before operation (P < .05). The expression of Tim-3, alanine aminotransferase, and aspartate aminotransferase peaked only once in most liver transplantation patients, but there were 3 patients that were exceptions. CONCLUSIONS: Tim-3 could be used as a potential biomarker to predict rejection after liver transplantation.

Structural and Catalytic Characterization of TsBGL, a ß-Glucosidase From Thermofilum sp. ex4484_79.

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 ß-glucosidase was isolated and purified from the Thermoprotei Thermofilum sp. ex4484_79 and subjected to enzymatic and structural characterization. The purified ß-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 ß-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/ß)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.