Biophysical Characterization of RAS-SOS Complexes by Native Mass Spectrometry.

RAS is regulated by specific guanine nucleotide exchange factors, such as Son of Sevenless (SOS), that activates RAS by facilitating the exchange of inactive, GDP-bound RAS with GTP. The catalytic activity of SOS is known to be allosterically modulated by an active, GTP-bound RAS. However, it remains poorly understood how oncogenic RAS mutants interact with SOS and modulate its activity. In this chapter, we describe the application of native mass spectrometry (MS) to monitor the assembly of the catalytic domain of SOS (SOScat) with RAS and cancer-associated mutants. Results from this approach have led to the discovery of different molecular assemblies and distinct conformers of SOScat engaging KRAS. It was also found that KRASG13D exhibits high affinity for SOScat and is a potent allosteric modulator of its SOScat activity. KRASG13D-GTP can allosterically increase the nucleotide exchange rate of KRAS at the active site by more than twofold compared to the wild-type protein. Furthermore, small-molecule RAS•SOS disruptors fail to dissociate KRASG13D•SOScat complexes, underscoring the need for more potent disruptors targeting oncogenic RAS mutants. Taken together, native MS will be instrumental in better understanding the interaction between oncogenic RAS mutants and SOS, which is of crucial importance for development of improved therapeutics.

Isoptericola luteus sp. nov., a novel yellow bacterium isolated from soil.

An aerobic, non-motile, Gram-stain-positive bacterium, designated strain NEAU-Y5T, was isolated from a soil sample collected from Northeast Agricultural University, Heilongjiang province. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain NEAU-Y5T belonged to the genus and showed high 16S rRNA sequence similarity to Isoptericola variabilis (98.9 %), Isoptericola nanjingensis (98.9 %), Isoptericola cucumis (98.5 %), Isoptericola hypogeus (98.5 %), Isoptericola dokdonensis (98.5 %), Isoptericola jiangsuensis (98.3 %), and Isoptericola halalbus (98.1 %), followed by other members of the genus Isoptericola (<98 %), and phylogenetically clustered with I. dokdonensis and I. jiangsuensis. Strain NEAU-Y5T was found to grow at 4-40 °C (optimum, 28 °C), pH 6.0-12.0 (optimum, pH 7.0), and tolerated 0-6 % NaCl (w/v). The cell-wall peptidoglycan type was l-Lys-d-Asp. The whole-cell hydrolysates contained glucose, galactose, and ribose. The major polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, hydroxyphosphatidylethanolamine, phosphatidylinositol, phosphatidylinositol mannoside, and glucosamine unknown phospholipid. Major fatty acids were anteiso-C15 : 0 and anteiso-C17 : 0. The predominant menaquinone was MK-9(H4). The DNA G+C content was 73.4 mol%. The digital DNA-DNA hybridization and average nucleotide identity values between strain NEAU-Y5T and the type strains of the genus Isoptericola ranged from 18.6 to 23.5 % and from 77.3 to 81.6 %, respectively. Based on morphological, physiological, chemotaxonomic, and phylogenetic data, as well as digital DNA-DNA hybridization and average nucleotide identity values, the novel strain NEAU-Y5T could be differentiated from its closest relatives. Therefore, the strain represents a novel species of the genus Isoptericola, for which the name Isoptericola luteus sp. nov. is proposed. The type strain is NEAU-Y5T (=CCTCC AA 2019087T=DSM 110637T).

Savitreea siamensis sp. nov., an ascomycetous yeast species in the family Saccharomycetaceae discovered in Thailand.

Four yeast strains, representing a novel anamorphic species, were isolated in Thailand. The two strains (ST-3660T and ST-3647) were obtained from two different estuarine water samples in a mangrove forest. Strain DMKU-FW1-37 was derived from a grease sample, and another strain (TSU57) was isolated from a fruiting body of Phallus sp. Pairwise sequence analysis showed that the four strains had identical or differed by only one nucleotide substitution in the D1/D2 domains of the large subunit (LSU) rRNA gene, and differed by one to three nucleotide substitutions in the internal transcribed spacer (ITS) regions. Savitreea pentosicarens is the most closely related species to the four strains, but with 9-10 (1.57-1.72 %) nucleotide substitutions in the D1/D2 domains of the LSU rRNA gene and 29-31 (4.22-4.45 %) nucleotide substitutions in the ITS regions. Phylogenetic analyses based on the concatenated sequences of the ITS regions and the D1/D2 domains of the LSU rRNA gene showed that the four strains form a well-separated lineage from S. pentosicarens with high bootstrap support, confirming that they represent a distinct species. Therefore, the four strains are assigned as representives of a novel species of the genus Savitreea, for which the name Savitreea siamensis sp. nov. is proposed. The holotype is TBRC 4481T and the ex-type is PYCC 9794T (=ST-3660T). The MycoBank number of the novel species is MB 851951.

The Potential Role of Autophagy in Progression of Liver Fibrosis in Chronic Hepatitis B Patients Receiving Antiviral Treatment: A Brief Report.

Despite antiviral treatment, some patients with chronic hepatitis B (CHB) progress to cirrhosis. Enhancement of autophagy was implicated in the proliferation of hepatitis B in hepatocytes. This study aimed to evaluate the potential role of autophagy in the progression of liver fibrosis in patients receiving antiviral treatments and having completely inhibited viral replication. This descriptive-analytical study was designed and conducted in 2020 at Mottahhari Hepatitis Clinic affiliated with Shiraz University of Medical Science (Shiraz, Iran). Patients who were on anti-hepatitis B nucleotide treatments for at least two years, and those who were not cirrhotic at baseline but later progressed to cirrhosis were identified to be included in the case group. Besides, for the control group, patients on the nucleotide regimens who did not have cirrhosis at baseline or during follow-up were randomly selected. Ultimately, 16 cases and 14 controls were included in the study. Data were analyzed using SPSS software, and P<0.05 was considered statistically significant. Serum Beclin-1 and LC3 levels were compared between the two groups using enzyme-linked immunosorbent assays. The t test was used to assess the statistical differences between the case and control groups. Beclin-1 level was significantly higher in cirrhosis patients than the control group (1283±244 vs. 1063±257, P=0.024). However, there was no statistical difference between the level of LC3 in the cirrhotic group (168±31) and the control group (150±16) (P=0.065). Autophagy may have a role in the progression of cirrhosis in patients with CHB. Future larger prospective studies are required to determine the effect of blocking on the progression of liver disease in this population A preprint of this study was published at https://www.researchsquare.com/article/rs-1435490/v1.pdf.

The NLR family of innate immune and cell death sensors.

Nucleotide-binding oligomerization domain (NOD)-like receptors, also known as nucleotide-binding leucine-rich repeat receptors (NLRs), are a family of cytosolic pattern recognition receptors that detect a wide variety of pathogenic and sterile triggers. Activation of specific NLRs initiates pro- or anti-inflammatory signaling cascades and the formation of inflammasomes-multi-protein complexes that induce caspase-1 activation to drive inflammatory cytokine maturation and lytic cell death, pyroptosis. Certain NLRs and inflammasomes act as integral components of larger cell death complexes-PANoptosomes-driving another form of lytic cell death, PANoptosis. Here, we review the current understanding of the evolution, structure, and function of NLRs in health and disease. We discuss the concept of NLR networks and their roles in driving cell death and immunity. An improved mechanistic understanding of NLRs may provide therapeutic strategies applicable across infectious and inflammatory diseases and in cancer.

Detection of ac4C in human mRNA is preserved upon data reassessment.

We recently reported the distribution of N4-acetylcytidine (ac4C) in HeLa mRNA at base resolution through chemical reduction and the induction of C:T mismatches in sequencing (RedaC:T-seq). Our results contradicted an earlier report from Schwartz and colleagues utilizing a similar method termed ac4C-seq. Here, we revisit both datasets and reaffirm our findings. Through RedaC:T-seq reanalysis, we establish a low basal error rate at unmodified nucleotides that is not skewed to any specific mismatch type and a prominent increase in C:T substitutions as the dominant mismatch type in both treated wild-type replicates, with a high degree of reproducibility across replicates. In contrast, through ac4C-seq reanalysis, we uncover significant data quality issues including insufficient depth, with one wild-type replicate yielding 2.7 million reads, inconsistencies in reduction efficiencies between replicates, and an overall increase in mismatches involving thymine that could obscure ac4C detection. These analyses bolster the detection of ac4C in HeLa mRNA through RedaC:T-seq.

[Non-targeted metabolomics of spleen and liver metabolism in mice treated with Pruni Semen processed with different methods].

Ultra performance liquid chromatography-quadrupole time-of-flight mass spectrometry(UPLC-Q-TOF-MS) was employed to investigate the impacts of Pruni Semen processed with different methods(raw and fried) on the liver and spleen metabolism in mice. A total of 24 male mice were randomly assigned to three groups: raw Pruni Semen group, fried Pruni Semen group, and control(deionized water) group. Mice in the three groups were orally administrated with 0.01 g·mL~(-1) Pruni Semen decoction or deionized water for one week. After that, the liver and spleen tissues were collected, and liquid chromatography-mass spectrometry(LC-MS)-based metabolomic analysis was carried out to investigate the impact of Pruni Semen on the liver and spleen metabolism in mice. Compared with thte control group, the raw Pruni Semen group showed up-regulation of 11 metabolites and down-regulation of 57 metabolites in the spleen(P<0.05), as well as up-regulation of 15 metabolites and down-regulation of 58 metabolites in the liver(P<0.05). The fried Pruni Semen group showed up-regulation of 31 metabolites and down-regulation of 10 metabolites in the spleen(P<0.05), along with up-regulation of 26 metabolites and down-regulation of 61 metabolites in the liver(P<0.05). The differential metabolites identified in the raw Pruni Semen group were primarily associated with alanine, aspartate, and glutamate metabolism, purine metabolism, amino sugar and nucleotide sugar metabolism, and D-glutamine and D-glutamate metabolism. The differential metabolites identified in the fried Pruni Semen group predominantly involved riboflavin metabolism, amino sugar and nucleotide sugar metabolism, purine metabolism, alanine, aspartate, and glutamate metabolism, D-glutamine and D-glutamate metabolism, and glutathione metabolism. The findings suggest that both raw and fried Pruni Semen have the potential to modulate the metabolism of the liver and spleen in mice by influencing the glutamine and glutamate metabolism.

Molecular detection and genotyping of Dientamoeba fragilis and Blastocystis sp. in housefly Musca domestica (Diptera: Muscidae): first report for Dientamoeba fragilis.

Dientamoeba fragilis and Blastocystis sp. are single-celled protozoan parasites of humans and animals. Although they are found in the intestines of healthy hosts, the pathogenicity of them is still unclear. To date, there is no report on D. fragilis and only two studies (without subtyping) on the occurrence of Blastocystis sp. in Musca domestica. In this study, fly samples were collected from livestock farms and their surroundings in the Kirsehir province (Central Anatolia Region) of Türkiye from May to August 2023. A total of 150 microscopically identified M. domestica samples were analyzed for the detection of D. fragilis and Blastocystis sp. molecularly. The overall prevalence of Blastocystis sp. and D. fragilis in M. domestica was determined to be 3.3% (5/150) and 8.0% (12/150), respectively. The SSU rRNA gene sequences of the isolates indicated genotype 1 of D. fragilis. Eleven isolates were identical and represented a single isolate (KAU-Dfrag1). BLAST analysis of KAU-Dfrag1 indicated identity with the isolates reported from humans, cattle, sheep, and budgerigars. The other isolate (KAU-Dfrag2) was polymorphic at two nucleotides from KAU-Dfrag1 and three nucleotides from known genotypes from GenBank and represented a variant of genotype 1. The Blastocystis sp. isolates were found to be identical and represent a single genotype (KAU-Blast1). BLAST analysis revealed that the KAU-Blast1 genotype belonged to the potentially zoonotic subtype 5 (ST5) and exhibited the highest genetic identity (ranging from 99.4 to 99.6%) with pigs, cattle, and sheep from different countries. Our study provides the first data on the molecular prevalence, epidemiology, and genotypic characterization of D. fragilis and Blastocystis sp. in M. domestica.

The novel HLA-B*44:48:02 allele characterised by two different sequencing-based typing techniques.

The novel allele HLA-B*44:48:02 differs from HLA-B*44:48:01 by one synonymous nucleotide substitution in exon 3.

The novel HLA-C*07:02:147 allele characterised by two different sequencing-based typing techniques.

The novel allele HLA-C*07:02:147 differs from HLA-C*07:02:01:01 by one synonymous nucleotide substitution in exon 2.

The novel HLA-A*36:14 allele characterised by two different sequencing-based typing techniques.

The novel allele HLA-A*36:14 differs from HLA-A*36:01:01:01 by one non-synonymous nucleotide substitution in exon 4.

Identification of two novel HLA-DRB1 alleles, HLA-DRB1*08:03:13 and HLA-DRB1*08:119.

Compared with HLA-DRB1*08:03:02:01, the alleles HLA-DRB1*08:03:13 and HLA-DRB1*08:119 each show one nucleotide substitution, respectively.

The novel HLA-DRB1*03:210 allele characterised by two different sequencing-based typing techniques.

The novel allele HLA-DRB1*03:210 differs from HLA-DRB1*03:01:01:01 by one non-synonymous nucleotide substitution in exon 3.

Full-spectral genome analysis of natural killer/T cell lymphoma highlights impacts of genome instability in driving its progression.

BACKGROUND: Natural killer/T cell lymphoma (NKTCL) is a clinically and genetically heterogeneous disease with poor prognosis. Genome sequencing and mutation characterization provides a powerful approach for patient stratification, treatment target discovery, and etiology identification. However, previous studies mostly concentrated on base-level mutations in primary NKTCL, whereas the large-scale genomic alterations in NKTCL and the mutational landscapes in relapsed/refractory NKTCL remain largely unexplored. METHODS: Here, we assembled whole-genome sequencing and whole-exome sequencing data from 163 patients with primary or relapsed/refractory NKTCL and compared their somatic mutational landscapes at both nucleotide and structure levels. RESULTS: Our study not only confirmed previously reported common NKTCL mutational targets like STAT3, TP53, and DDX3X but also unveiled several novel high-frequency mutational targets such as PRDM9, DST, and RBMX. In terms of the overall mutational landscape, we observed striking differences between primary and relapsed/refractory NKTCL patient groups, with the latter exhibits higher levels of tumor mutation burden, copy number variants (CNVs), and structural variants (SVs), indicating a strong signal of genomic instability. Complex structural rearrangements such as chromothripsis and focal amplification are also significantly enriched in relapsed/refractory NKTCL patients, exerting a substantial impact on prognosis. Accordingly, we devised a novel molecular subtyping system (i.e., C0-C4) with distinct prognosis by integrating potential driver mutations at both nucleotide and structural levels, which further provides an informative guidance for novel treatments that target these specific driver mutations and genome instability as a whole. CONCLUSIONS: The striking differences underlying the mutational landscapes between the primary and relapsed/refractory NKTCL patients highlight the importance of genomic instability in driving the progression of NKTCL. Our newly proposed molecular subtyping system is valuable in assisting patient stratification and novel treatment design towards a better prognosis in the age of precision medicine.

Global and single-nucleotide resolution detection of 7-methylguanosine in RNA.

RNA modifications, including N-7-methylguanosine (m7G), are pivotal in governing RNA stability and gene expression regulation. The accurate detection of internal m7G modifications is of paramount significance, given recent associations between altered m7G deposition and elevated expression of the methyltransferase METTL1 in various human cancers. The development of robust m7G detection techniques has posed a significant challenge in the field of epitranscriptomics. In this study, we introduce two methodologies for the global and accurate identification of m7G modifications in human RNA. We introduce borohydride reduction sequencing (Bo-Seq), which provides base resolution mapping of m7G modifications. Bo-Seq achieves exceptional performance through the optimization of RNA depurination and scission, involving the strategic use of high concentrations of NaBH4, neutral pH and the addition of 7-methylguanosine monophosphate (m7GMP) during the reducing reaction. Notably, compared to NaBH4-based methods, Bo-Seq enhances the m7G detection performance, and simplifies the detection process, eliminating the necessity for intricate chemical steps and reducing the protocol duration. In addition, we present an antibody-based approach, which enables the assessment of m7G relative levels across RNA molecules and biological samples, however it should be used with caution due to limitations associated with variations in antibody quality between batches. In summary, our novel approaches address the pressing need for reliable and accessible methods to detect RNA m7G methylation in human cells. These advancements hold the potential to catalyse future investigations in the critical field of epitranscriptomics, shedding light on the complex regulatory roles of m7G in gene expression and its implications in cancer biology.

Heat flows enrich prebiotic building blocks and enhance their reactivity.

The emergence of biopolymer building blocks is a crucial step during the origins of life1-6. However, all known formation pathways rely on rare pure feedstocks and demand successive purification and mixing steps to suppress unwanted side reactions and enable high product yields. Here we show that heat flows through thin, crack-like geo-compartments could have provided a widely available yet selective mechanism that separates more than 50 prebiotically relevant building blocks from complex mixtures of amino acids, nucleobases, nucleotides, polyphosphates and 2-aminoazoles. Using measured thermophoretic properties7,8, we numerically model and experimentally prove the advantageous effect of geological networks of interconnected cracks9,10 that purify the previously mixed compounds, boosting their concentration ratios by up to three orders of magnitude. The importance for prebiotic chemistry is shown by the dimerization of glycine11,12, in which the selective purification of trimetaphosphate (TMP)13,14 increased reaction yields by five orders of magnitude. The observed effect is robust under various crack sizes, pH values, solvents and temperatures. Our results demonstrate how geologically driven non-equilibria could have explored highly parallelized reaction conditions to foster prebiotic chemistry.

The novel HLA-DPA1*02:117 allele, identified by Sanger dideoxy nucleotide sequencing in a Chinese individual.

HLA-DPA1*02:117 differs from HLA-DPA1*02:02:02:01 by one nucleotide in exon 2.

The novel HLA-DRB1*11:323 allele characterized by two different sequencing-based typing techniques.

The novel allele HLA-DRB1*11:323 differs from HLA-DRB1*11:01:02:01 by one non-synonymous nucleotide substitution in exon 2.

The novel HLA-A*30:01:23 allele characterised by two different sequencing-based typing techniques.

The novel allele HLA-A*30:01:23 differs from HLA-A*30:01:01:01 by one synonymous nucleotide substitution in exon 2.

The significance of detecting HBV pgRNA and HBcrAg in HBV patients treated with NAs.

The value of detecting hepatitis B virus (HBV), pregenomic RNA (pgRNA), and hepatitis B core-related antigen (HBcrAg), both separately and jointly, in the management of HBV patients undergoing treatment with Nucleotide Analog was investigated. A total of 149 HBV patients who were being treated with Nucleotide Analog were enrolled in this study. The quantitative levels of HBV pgRNA and HBcrAg in the sera of these patients were determined, aiming to comprehend their replication levels and expression during the course of antiviral therapy. The patients were separated into 3 groups based on treatment duration: treatment time ≤ 12 months, treatment time ranging from 12 months to <60 months, and treatment time ≥ 60 months. Significantly different levels of HBcrAg and HBV pgRNA were observed among 3 groups (P < .05). In the group of patients with positive hepatitis B e antigen, both HBcrAg and pgRNA levels were higher compared to the group with negative hepatitis B e antigen, and this difference between the 2 groups was found to be statistically significant. Stratified analysis based on levels of hepatitis B surface antigen (HBsAg) revealed that the group with HBsAg levels < 100 IU/mL had lower levels of both HBcrAg and pgRNA compared to the group with HBsAg levels ≥ 100 IU/mL (P < .001). Following antiviral therapy, various degrees of transcription of covalently closed circular DNA continue to exist within the liver of HBV patients. The levels of serum HBcrAg and HBV pgRNA vary among patients with different treatment durations, indicating their efficacy in evaluating disease conditions during antiviral therapy.