Analysis of the interlink between glucose-6-phosphate dehydrogenase (G6PD) and lung cancer through multi-omics databases.

Glucose-6-Phosphate Dehydrogenase (G6PD) is a crucial enzyme that executes the pentose phosphate pathway. Due to its critical nodal position in the metabolic network, it is associated with different forms of cancer tumorigeneses and progression. Nonetheless, its functional role and molecular mechanism in lung cancer remain unknown. The present study provides intricate information associated with G6PD and Lung Cancer. Varieties of public datasets were retrieved by us, including UALCAN, TCGA, cBioPortal, and the UCSC Xena browser. The data obtained were used to assess the expression of G6PD, its clinical features, epigenetic regulation, relationship with tumour infiltration, tumour mutation burden, microsatellite instability, tumour microenvironment, immune checkpoint genes, genomic alteration, and patient's overall survival rate. The present study revealed that the G6PD expression was correlated with the clinical features of lung cancer including disease stage, race, sex, age, smoking habits, and lymph node metastasis. Moreover, the expression profile of G6PD also imparts epigenetic changes by modulating the DNA promoter methylation activity. Methylation of promoters changes the expression of various transcription factors, genes leading to an influence on the immune system. These events linked with G6PD-related mutational gene alterations (FAM3A, LAG3, p53, KRAS). The entire circumstance influences the patient's overall survival rate and poor prognosis. Functional investigation using STRING, GO, and KEGG found that G6PD primarily engages in hallmark functions (metabolism, immunological responses, proliferation, apoptosis, p53, HIF-1, FOXO, PI3K-AKT signaling). This work provides a wide knowledge of G6PD's function in lung cancer, as well as a theoretical foundation for possible prognostic therapeutic markers.

Assessment of HBV variants and novel viral and immune biomarkers in chronic hepatitis B patients with metabolic dysfunction associated steatotic liver disease.

Co-existing chronic hepatitis B virus (CHB) infection and metabolic dysfunction associated steatotic liver disease (MASLD) can exert complex effects on hepatic metabolism, requiring mechanistic study. CHB participants were assessed for MASLD and the impact of hepatic steatosis/metabolic syndrome (MetS) on novel viral and immunological markers. In this prospective, cohort study, untreated CHB subjects were assessed for liver disease by non-invasive tests (i.e. FibroScan, controlled attenuation parameter, CAP). Subjects were tested for cytokines and IFN-γ ELISPOT assay to HBV Surface (S) and Core (C) proteins. Standard HBV serological, exploratory biomarkers and deep sequencing of HBV S and C genes were performed. In 53 subjects (median age 45 years [SD = 10.6], 35% F, 56% Asian, 20% Black, 3% White), 94% (50) HBeAg negative, 63% genotype B/C, mean HBV DNA 3.2 log10 IU/mL (SD = 1.8), quantitative HBsAg 2.9 log10 IU/mL (SD = 1.2) and HBV pgRNA 2.1 log10 copies/mL (SD = 1.3). In enrolled subjects, the mean ALT was 41.9 U/L (SD = 24.0), FibroScan was 5.7 kPa (SD = 1.9) and CAP was 306.4 dB/m (SD = 49.0). The mean BMI was 28.2 kg/m2 (SD = 4.2), 20% (11/53) had diabetes, 35% (19/53) dyslipidaemia and 24% (13/53) hypertension. Subjects with MetS and steatosis showed lower HBV markers (p < .01), higher HBV S diversity (p = .02) and greater frequency of HBV variants associated with host-anti-viral immune escape. Pro-inflammatory cytokine levels and HBV-specific cellular responses were higher in participants with hepatic steatosis. In CHB, MASLD/hepatic steatosis was associated with HBV variants and systemic immune responses potentially impacting liver disease progression despite low-level viraemia.

Factor-Dependent Internal Ribosome Entry Site and -1 Programmed Frameshifting Signal in the Bemisia-Associated Dicistrovirus 2.

The dicistrovirus intergenic (IGR) IRES uses the most streamlined translation initiation mechanism: the IRES recruits ribosomes directly without using protein factors and initiates translation from a non-AUG codon. Several subtypes of dicistroviruses IRES have been identified; typically, the IRESs adopt two -to three overlapping pseudoknots with key stem-loop and unpaired regions that interact with specific domains of the ribosomal 40S and 60S subunits to direct translation. We previously predicted an atypical IGR IRES structure and a potential -1 programmed frameshift (-1 FS) signal within the genome of the whitefly Bemisia-associated dicistrovirus 2 (BaDV-2). Here, using bicistronic reporters, we demonstrate that the predicted BaDV-2 -1 FS signal can drive -1 frameshifting in vitro via a slippery sequence and a downstream stem-loop structure that would direct the translation of the viral RNA-dependent RNA polymerase. Moreover, the predicted BaDV-2 IGR can support IRES translation in vitro but does so through a mechanism that is not typical of known factorless dicistrovirus IGR IRES mechanisms. Using deletion and mutational analyses, the BaDV-2 IGR IRES is mapped within a 140-nucleotide element and initiates translation from an AUG codon. Moreover, the IRES does not bind directly to purified ribosomes and is sensitive to eIF2 and eIF4A inhibitors NSC1198983 and hippuristanol, respectively, indicating an IRES-mediated factor-dependent mechanism. Biophysical characterization suggests the BaDV-2 IGR IRES contains several stem-loops; however, mutational analysis suggests a model whereby the IRES is unstructured or adopts distinct conformations for translation initiation. In summary, we have provided evidence of the first -1 FS frameshifting signal and a novel factor-dependent IRES mechanism in this dicistrovirus family, thus highlighting the diversity of viral RNA-structure strategies to direct viral protein synthesis.

Development of a single-domain antibody to target a G-quadruplex located on the hepatitis B virus covalently closed circular DNA genome.

To achieve a virological cure for hepatitis B virus (HBV), innovative strategies are required to target the covalently closed circular DNA (cccDNA) genome. Guanine-quadruplexes (G4s) are a secondary structure that can be adopted by DNA and play a significant role in regulating viral replication, transcription, and translation. Antibody-based probes and small molecules have been developed to study the role of G4s in the context of the human genome, but none have been specifically made to target G4s in viral infection. Herein, we describe the development of a humanized single-domain antibody (S10) that can target a G4 located in the PreCore (PreC) promoter of the HBV cccDNA genome. MicroScale Thermophoresis demonstrated that S10 has a strong nanomolar affinity to the PreC G4 in its quadruplex form and a structural electron density envelope of the complex was determined using Small-Angle X-ray Scattering. Lentiviral transduction of S10 into HepG2-NTCP cells shows nuclear localization, and chromatin immunoprecipitation coupled with next-generation sequencing demonstrated that S10 can bind to the HBV PreC G4 present on the cccDNA. This research validates the existence of a G4 in HBV cccDNA and demonstrates that this DNA secondary structure can be targeted with high structural and sequence specificity using S10.

tRNA shape is an identity element for an archaeal pyrrolysyl-tRNA synthetase from the human gut.

Protein translation is orchestrated through tRNA aminoacylation and ribosomal elongation. Among the highly conserved structure of tRNAs, they have distinguishing features which promote interaction with their cognate aminoacyl tRNA synthetase (aaRS). These key features are referred to as identity elements. In our study, we investigated the tRNA:aaRS pair that installs the 22nd amino acid, pyrrolysine (tRNAPyl:PylRS). Pyrrolysyl-tRNA synthetases (PylRSs) are naturally encoded in some archaeal and bacterial genomes to acylate tRNAPyl with pyrrolysine. Their large amino acid binding pocket and poor recognition of the tRNA anticodon have been instrumental in incorporating >200 noncanonical amino acids. PylRS enzymes can be divided into three classes based on their genomic structure. Two classes contain both an N-terminal and C-terminal domain, however the third class (ΔpylSn) lacks the N-terminal domain. In this study we explored the tRNA identity elements for a ΔpylSn tRNAPyl from Candidatus Methanomethylophilus alvus which drives the orthogonality seen with its cognate PylRS (MaPylRS). From aminoacylation and translation assays we identified five key elements in ΔpylSn tRNAPyl necessary for MaPylRS activity. The absence of a base (position 8) and a G-U wobble pair (G28:U42) were found to affect the high-resolution structure of the tRNA, while molecular dynamic simulations led us to acknowledge the rigidity imparted from the G-C base pairs (G3:C70 and G5:C68).

Enzymes known as PylRS offer the remarkable ability to expand the natural genetic code of a living cell with unnatural amino acids. Currently, over 200 unnatural amino acids can be genetically encoded with the help of PylRS and its partner tRNAPyl, enabling us to endow proteins with novel properties, or regulate protein activity using light or inducible cross-linking. One intriguing feature of PylRS enzymes is their ability to avoid cross-reactivity when two PylRS homologs from different organisms-such as those from the archaea Methanosarcina mazei and Methanomethylophilus alvus-are co-expressed in a single cell. This makes it possible to simultaneously encode two unnatural amino acids in a single protein. This study illuminates the elusive mechanism of PylRS specificity by using cryo-electron microscopy, biochemistry and molecular simulations. The interaction of PylRS from M. alvus with its tRNAPyl is best described as two pieces of a jigsaw puzzle; in which PylRS recognizes the unique shape of its cognate tRNA instead of specific nucleotides in the tRNA sequence like other tRNA-binding enzymes. This finding may streamline the rational design of tools for simultaneous genetic incorporation of multiple unnatural amino acids, thereby facilitating the development of valuable proteins for research, medicine, and biotechnology.