Nucleotide Levels Research Articles

Discrimination of superficial lymph nodes using ultrasonography and tissue metabolomics coupled with machine learning

IntroductionDiagnosing the types of malignant lymphoma could help determine the most suitable treatment, anticipate the probability of recurrence and guide long-term monitoring and follow-up care.MethodsWe evaluated the differences in benign, lymphoma and metastasis superficial lymph nodes using ultrasonography and tissue metabolomics.ResultsOur findings indicated that three ultrasonographic features, blood supply pattern, cortical echo, and cortex elasticity, hold potential in differentiating malignant lymph nodes from benign ones, and the shape and corticomedullary boundary emerged as significant indicators for distinguishing between metastatic and lymphoma groups. Metabolomics revealed the difference in metabolic profiles among lymph nodes. We observed significant increases in many amino acids, organic acids, lipids, and nucleosides in both lymphoma and metastasis groups, compared to the benign group. Specifically, the lymphoma group exhibited higher levels of nucleotides (inosine monophosphate and adenosine diphosphate) as well as glutamic acid, and the metastasis group was characterized by higher levels of carbohydrates, acylcarnitines, glycerophospholipids, and uric acid. Linear discriminant analysis coupled with these metabolites could be used for differentiating lymph nodes, achieving recognition rates ranging from 87.4% to 89.3%, outperforming ultrasonography (63.1% to 75.4%).DiscussionOur findings could contribute to a better understanding of malignant lymph node development and provide novel targets for therapeutic interventions.

Investigation of Exome-Wide Tumor Heterogeneity on Colorectal Tissue-Based Single Cells.

The progression of colorectal cancer is strongly influenced by environmental and genetic conditions. One of the key factors is tumor heterogeneity which is extensively studied by cfDNA and bulk sequencing methods; however, we lack knowledge regarding its effects at the single-cell level. Motivated by this, we aimed to employ an end-to-end single-cell sequencing workflow from tissue-derived sample isolation to exome sequencing. Our main goal was to investigate the heterogeneity patterns by laser microdissecting samples from different locations of a tissue slide. Moreover, by studying healthy colon control, tumor-associated normal, and colorectal cancer tissues, we explored tissue-specific heterogeneity motifs. For completeness, we also compared the performance of the whole-exome bulk, cfDNA, and single-cell sequencing methods based on variation at the level of a single nucleotide.

Glutaminase-2 Expression Induces Metabolic Changes and Regulates Pyruvate Dehydrogenase Activity in Glioblastoma Cells.

Glutaminase controls the first step in glutaminolysis, impacting bioenergetics, biosynthesis and oxidative stress. Two isoenzymes exist in humans, GLS and GLS2. GLS is considered prooncogenic and overexpressed in many tumours, while GLS2 may act as prooncogenic or as a tumour suppressor. Glioblastoma cells usually lack GLS2 while they express high GLS. We investigated how GLS2 expression modifies the metabolism of glioblastoma cells, looking for changes that may explain GLS2's potential tumour suppressive role. We developed LN-229 glioblastoma cells stably expressing GLS2 and performed isotope tracing using U-13C-glutamine and metabolomic quantification to analyze metabolic changes. Treatment with GLS inhibitor CB-839 was also included to concomitantly inhibit endogenous GLS. GLS2 overexpression resulted in extensive metabolic changes, altering the TCA cycle by upregulating part of the cycle but blocking the synthesis of the 6-carbon intermediates from acetyl-CoA. Expression of GLS2 caused downregulation of PDH activity through phosphorylation of S293 of PDHA1. GLS2 also altered nucleotide levels and induced the accumulation of methylated metabolites and S-adenosyl methionine. These changes suggest that GLS2 may be a key regulator linking glutamine and glucose metabolism, also impacting nucleotides and epigenetics. Future research should ascertain the mechanisms involved and the generalizability of these findings in cancer or physiological conditions.

Coat-protein Gene Based Identification and Characterization of Yellow Mosaic Virus Infecting Blackgram in Northern Karnataka, India

Aims: Coat-protein (CP) based identification and characterization of yellow mosaic virus infecting blackgram. Place and Duration of Study: The study was carried out during Kharif 2023 at College of Agriculture, Vijayapur. Methodology: The whole genomic DNA was isolated from the leaf tissues of both healthy blackgram plants and plants infected with the yellow mosaic virus by using a modified CTAB method. Specific primers for YMV were tried to amplify coat protein region of blackgram YMV. Results: Sequence analysis revealed that the CP gene of the begomovirus under study shared 99.8 per cent similarity with MYMV Shivamogga isolate (OM106035.1) at the nucleotide level. When comparing the deduced amino acid sequences of individual proteins from YMV infecting blackgram at Vijayapur with those of other begomoviruses, the highest identity was found with the MYMV Shivamogga isolate (OM106035.1), showing 84.59 per cent similarity. A phylogenetic tree constructed based on the full-length coat protein gene sequence of MYMV, along with 37 other Geminivirus sequences, formed two major clusters in which MYMV-Vijayapur isolate appeared in Cluster I. Conclusion: The results of the current study, based on similarities in coat protein sequence at both the nucleotide and amino acid levels, as well as phylogenetic analysis, confirmed the prevalence of MYMV strain rather than MYMIV, HgYMV and DoYMV in blackgram in northern parts of Karnataka.

Metabolic profiling of patient-derived organoids reveals nucleotide synthesis as a metabolic vulnerability in malignant rhabdoid tumors.

Malignant rhabdoid tumor (MRT) is one of the most aggressive childhood cancers for which no effective treatment options are available. Reprogramming of cellular metabolism is an important hallmark of cancer, with various metabolism-based drugs being approved as a cancer treatment. In this study, we use patient-derived tumor organoids (tumoroids) to map the metabolic landscape of several pediatric cancers. Combining gene expression analyses and metabolite profiling using mass spectrometry, we find nucleotide biosynthesis to be a particular vulnerability of MRT. Treatment of MRT tumoroids with de novo nucleotide synthesis inhibitors methotrexate (MTX) and BAY-2402234 lowers nucleotide levels in MRT tumoroids and induces apoptosis. Lastly, we demonstrate invivo efficacy of MTX in MRT patient-derived xenograft (PDX) mouse models. Our study reveals nucleotide biosynthesis as an MRT-specific metabolic vulnerability, which can ultimately lead to better treatment options for children suffering from this lethal pediatric malignancy.

Multi-omics Combined with Machine Learning Facilitating the Diagnosis of Gastric Cancer.

Gastric cancer (GC) is a highly intricate gastrointestinal malignancy. Early detection of gastric cancer forms the cornerstone of precision medicine. Several studies have been conducted to investigate early biomarkers of gastric cancer using genomics, transcriptomics, proteomics, and metabolomics, respectively. However, endogenous substances associated with various omics are concurrently altered during gastric cancer development. Furthermore, environmental exposures and family history can also induce modifications in endogenous substances. Therefore, in this study, we primarily investigated alterations in DNA mutation, DNA methylation, mRNA, lncRNA, miRNA, circRNA, and protein, as well as glucose, amino acid, nucleotide, and lipid metabolism levels in the context of GC development, employing genomics, transcriptomics, proteomics, and metabolomics. Additionally, we elucidate the impact of exposure factors, including HP, EBV, nitrosamines, smoking, alcohol consumption, and family history, on diagnostic biomarkers of gastric cancer. Lastly, we provide a summary of the application of machine learning in integrating multi-omics data. Thus, this review aims to elucidate: i) the biomarkers of gastric cancer related to genomics, transcriptomics, proteomics, and metabolomics; ii) the influence of environmental exposure and family history on multiomics data; iii) the integrated analysis of multi-omics data using machine learning techniques.

Haploinsufficiency of PDE2A causes in mice increased exploratory behavior associated with upregulation of neural nitric oxide synthase in the striatum.

Phosphodiesterase 2 A (PDE2A) function is stimulated by cGMP to catabolize cAMP. However, neurological and neurochemical effects of PDE2A deficiency are poorly understood. To address this gap, we studied behavioral characteristics and cerebral morpho-chemical changes of adult male heterozygous C57BL/6-PDE2A+/- (HET), and wild type C57BL/6-PDE2A+/+ (WT) mice. Behavioral functions of mice were evaluated by a wide test battery. HET mice exhibited greater tendency to explore novel environments in comparison to WT mice, but spatial working memory, anxiety, and sociability were similar in adult HET and WT mice. In HET mice, PDE2A mRNA, PDE2A protein expression, and cGMP hydrolyzing enzymatic activity were consistently reduced by about 50 %. Consequently, the cyclic nucleotide levels were significantly increased in HET mice, but unexpectedly the mean percentage variation was higher for cGMP equal to 153.23 %, and lower for cAMP equal to 16.41 %. Therefore, to try to explain the preponderant increase of cGMP to cAMP we evaluated other PDE enzymes functionally related to PDE2A. Surprisingly, results were quite contradictory: in HET mice protein levels of the other dual-specificity enzymes PDE3 and PDE10A were reduced, whereas the expressions of PDE5 and PDE9 that selectively hydrolyze cGMP were increased. Therefore, we investigated the involvement of neuronal nitric oxide synthase (nNOS) expression, as determinant of a possible increased synthesis of NO/cGMP signaling. Interestingly, in HET mice the expression level of brain nNOS, measured by western blot and immune-histochemistry was significantly increased, particularly in interneurons from the striatum. In conclusion, the deficiency of PDE2A could be compensated in the striatum by upregulating nNOS/NO/cGMP pathway, which in turn likely upregulates PDE2A-dependent cAMP hydrolysis. The neuroanatomical correlation between striatal nNOS upregulation and the behavioral phenotype of increased exploratory behavior in HET mice is advanced.

Gliocidin is a nicotinamide-mimetic prodrug that targets glioblastoma.

Glioblastoma is incurable and in urgent need of improved therapeutics1. Here we identify a small compound, gliocidin, that kills glioblastoma cells while sparing non-tumour replicative cells. Gliocidin activity targets a de novo purine synthesis vulnerability in glioblastoma through indirect inhibition of inosine monophosphate dehydrogenase 2 (IMPDH2). IMPDH2 blockade reduces intracellular guanine nucleotide levels, causing nucleotide imbalance, replication stress and tumour cell death2. Gliocidin is a prodrug that is anabolized into its tumoricidal metabolite, gliocidin-adenine dinucleotide (GAD), by the enzyme nicotinamide nucleotide adenylyltransferase 1 (NMNAT1) of the NAD+ salvage pathway. The cryo-electron microscopy structure of GAD together with IMPDH2 demonstrates its entry, deformation and blockade of the NAD+ pocket3. In vivo, gliocidin penetrates the blood-brain barrier and extends the survival of mice with orthotopic glioblastoma. The DNA alkylating agent temozolomide induces Nmnat1 expression, causing synergistic tumour cell killing and additional survival benefit in orthotopic patient-derived xenograft models. This study brings gliocidin to light as a prodrug with the potential to improve the survival of patients with glioblastoma.

Integrated transcriptomic and metabolomic analyses reveal that MsSPHK1 - A sphingosine kinase gene negatively regulates drought tolerance in alfalfa (Medicago sativa L.)

Alfalfa is a valuable forage crop but voluntarily affected by drought. Understanding the mechanisms of drought resistance in alfalfa is crucial for improving resilient cultivars. In our study, we used four distinct alfalfa accessions two drought-tolerance (DT) and two drought-sensitive (DS) and identified transcriptional modules and candidate genes associated with the drought tolerance in the DS from transcriptomic analyses. Our metabolic profiling of 520 metabolites revealed significant variations between the DS and DT groups, particularly in the levels of flavonoids and nucleotides and their derivatives. The integrated analysis of transcriptome and metabolome analysis revealed that the glycine, serine, and threonine metabolism and the sphingolipid metabolism are associated with the drought resistance. When drought stress occurs, MsSRR (MsG 0180002649.01) and MsSPHK1 (MsG 0280006618.01) are significantly up-regulated, L-serine and dihydrosphingosine (DHS) significantly down-regulated in DS. By silencing the MsSPHK1 gene we found the drought resistance was significantly improved. This was evidenced by a significant increase in the activity of antioxidant enzymes such as SOD, POD, and CAT, compared to the control group. Additionally, the photosynthetic rate, stomatal conductance, and efficiency of photosystem II measured by Fv/Fm, phi2 and qL, were significantly higher in the silenced plants than in the control group. In conclusion, our results suggest that the increased level of dihydrosphingosine improves alfalfa resistance to drought stress. Moreover, the negative regulatory role of MsSPHK1 in drought tolerance provides a promising target for genetic manipulation to enhance the resilience of alfalfa to drought stress.

Boosted Meat Flavor by the Metabolomic Effects of Nile Tilapia Dietary Inclusion of Zophobas atratus Larval Meal.

Zophobas atratus larval meal (ZLM) is a high-quality feed supplement with potential activities that can improve fish growth performance and promote meat quality. However, there have been limited recent studies investigating the metabolic effects of ZLM. Therefore, this study aims to uncover the metabolomic mechanism through which ZLM improves tilapia meat flavor using metabolomic strategies. In this study, soybean meal in the basal diets was replaced with 15%, 30%, or 60% ZLM, where anti-nutrient factors were destroyed by high temperature treatment. After being fed these ZLM supplements for 30 days, dorsal muscles were collected from tilapia for meat sensory evaluation tests. Liver samples were also collected for metabolomic analysis using the gas chromatography-mass spectrometry (GC-MS) platform and combined with biochemical assays to verify metabolism-related enzyme activities and reveal crucial metabolic pathways and critical biomarkers associated with ZLM's ability to improve meat flavor. In tilapia livers, ZLM enhanced the activity of enzymes involved in energy metabolism including succinate dehydrogenase (SDH), pyruvate dehydrogenase (PDH), α-ketoglutarate dehydrogenase (α-KGDH), NADP-malate dehydrogenase (NAD-MDH) and mitochondrial isocitrate dehydrogenase (ICDHm). This resulted in increased levels of reduced nicotinamide adenine dinucleotide (NADH), acetyl CoA and ATP which led to accumulation of flavor fatty acids such as arachidonic acid, linoleic acid (9,12-Octadecadienoic acid), linolenic acid (9,12,15-Octadecatrienoic acid) and oleic acid (9-Octadecenoic acid). Additionally, there was an increase in flavor nucleotides like guanosine adenosine-5'-monophosphate and uridine-5'-monophosphate while off-flavor metabolites like inosine and hypoxanthine decreased. Furthermore, beneficial metabolomic responses led to a decrease in off-flavor metabolites such as 2-methylisoborneol trimethylamine and geosmin while increasing umami metabolites like 2-methyl-3-furanthiol and nonanal. This metabolomic study demonstrates that inclusion of ZLM diets enhances the flavor profile of tilapia dorsal muscle. The accumulation of flavor compounds, coupled with a reduction in earthy taste and off-flavor metabolites, contributes to an improved meat flavor and freshness. Additionally, there is an increase in the levels of flavor-related amino acids and nucleotides. These previously unidentified metabolic effects highlight the potential significance of ZLM as a dietary supplement for enhancing the biosynthesis of flavor metabolites in tilapia.

The study of the hypoxia markers against the background of prolonged hyperglycemia in experimental animals.

Aim: The aim of article is to investigate the energetic functions of retinal mitochondria under conditions of chronic hyperglycemia to understand the pathogenic mechanisms involved in the development of this diabetic complication. Materials and Methods: The experimental study was carried out on non-linear rats which were divided into 2 groups: intact animals and rats with diabetic retinopathy. We investigated the level of lactate, pyruvate and adenine nucleotides. Results: The increase in lactate and pyruvate levels may indicate a reduction in the energy potential of the retina under conditions of experimental diabetic retinopathy, while the elevation of adenine nucleotides activity suggests the activation of energy processes and the development of adaptation to hypoxia in retinal cells. Conclusions: The analysis of lactate and pyruvate levels, as well as adenine nucleotides, may serve as prognostic markers in predicting the severity of diabetic retinopathy in the context of prolonged hyperglycemia.

The Current Role of Hydroxyurea in the Treatment of Sickle Cell Anemia.

Despite advancements in treatment of sickle cell disease (SCD), hydroxyurea, a ribonucleotide reductase inhibitor, remains the cornerstone of therapy. While its primary effect is the elevation of fetal hemoglobin (HbF), hydroxyurea's mechanisms of action are multifaceted. Hydroxyurea (HU) reduces leukocyte and platelet counts, decreases the expression of endothelial adhesion molecules CD36 and CD49d, and increases nitric oxide and cyclic nucleotide levels, which may facilitate vascular dilation and further HbF induction. Numerous studies have demonstrated that hydroxyurea therapy reduces the frequency of painful episodes, acute chest syndrome, and the need for erythrocyte transfusions and hospitalizations. Long-term use of hydroxyurea leads to reduced morbidity and mortality. Hydroxyurea should be initiated in children from 9 months of age, including asymptomatic individuals, and is recommended for adults experiencing pain crises that significantly interfere with daily activities or quality of life, as well as those with severe or recurrent vaso-occlusive crises, ACS, or severe symptomatic anemia. Hydroxyurea is not recommended during pregnancy or lactation due to potential teratogenic effects and transfer into breast milk. However, its use may be considered in high-risk patients, particularly during the second and third trimesters. Concerns about secondary tumor development have not been substantiated in long-term follow-up studies. Alternative therapies, including L-glutamine, crizanlizumab, and voxelotor, are not presently approved or available for clinical use in Europe.

Phosphodiesterase inhibition and Gucy2C activation enhance tyrosine hydroxylase Ser40 phosphorylation and improve 6-hydroxydopamine-induced motor deficits

BackgroundParkinson’s disease is characterized by a progressive loss of dopaminergic neurons in the nigrostriatal pathway, leading to dopamine deficiency and motor impairments. Current treatments, such as L-DOPA, provide symptomatic relief but result in off-target effects and diminished efficacy over time. This study explores an alternative approach by investigating the activation of tyrosine hydroxylase, the rate-limiting enzyme in dopamine synthesis. Specifically, we explore the effects of phosphodiesterase (PDE) inhibition and guanylate cyclase-C (GUCY2C) activation on tyrosine hydroxylase Ser40 phosphorylation and their impact on motor behavior in a 6-hydroxydopamine (6-OHDA) Parkinson's disease model.ResultsOur findings demonstrate that increasing cyclic nucleotide levels through PDE inhibition and GUCY2C activation significantly enhances tyrosine hydroxylase Ser40 phosphorylation. In a Pitx3-deficient mouse model, which mimics the loss of dopaminergic neurons seen in Parkinson’s disease, Ser40 phosphorylation remained manipulable despite reduced tyrosine hydroxylase protein levels. Moreover, we observed no evidence of tyrosine hydroxylase degradation due to Ser40 phosphorylation, challenging previous reports. Furthermore, both PDE inhibition and GUCY2C activation resulted in improved motor behavior in the 6-OHDA Parkinson’s disease mouse model, highlighting the potential therapeutic benefits of these approaches.ConclusionsThis study underscores the therapeutic potential of enhancing tyrosine hydroxylase Ser40 phosphorylation to improve motor function in Parkinson’s disease. Both PDE inhibition and GUCY2C activation represent promising non-invasive strategies to modulate endogenous dopamine biosynthesis and address motor deficits. These findings suggest that targeting cyclic nucleotide pathways could lead to novel therapeutic approaches, either as standalone treatments or in combination with existing therapies like L-DOPA, aiming to provide more durable symptom relief and potentially mitigate neurodegeneration in Parkinson's disease.

Population genetic dissection of HLA-DPB1 amino acid polymorphism to infer selection

Although allele frequency data for most HLA loci provide strong evidence for balancing selection at the allele level, the DPB1 locus is a notable exception, with allele frequencies compatible with neutral evolution (genetic drift) or directional selection in most populations. This discrepancy is especially interesting as evidence for balancing selection has been seen at the nucleotide and amino acid (AA) sequence levels for DPB1. We describe methods used to examine the global distribution of DPB1 alleles and their constituent AA sequences. These methods allow investigation of the influence of natural selection in shaping DPβ diversity in a hierarchical fashion for DPB1 alleles, all polymorphic DPB1 exon 2-encoded AA positions, as well as all pairs and trios of these AA positions. In addition, we describe how asymmetric linkage disequilibrium for all DPB1 exon 2-encoded AA pairs can be used to complement other methods. Application of these methods provides strong evidence for the operation of balancing selection on AA positions 56, 85–87, 36, 55 and 84 (listed in decreasing order of the strength of selection), but no evidence for balancing selection on DPB1 alleles.

Uracil phosphoribosyltransferase is required to establish a functional cytochrome b6f complex.

Arabidopsis uracil phosphoribosyltransferase (UPP) is an essential enzyme and plants lacking this enzyme are strongly compromised in chloroplast function. Our analysis of UPP amiRNA mutants has confirmed that this vital function is crucial to establish a fully functional photosynthesis as the RIESKE iron sulfur protein (PetC) is almost absent, leading to a block in photosynthetic electron transport. Interestingly, this function appears to be unrelated to nucleotide homeostasis since nucleotide levels were not altered in the studied mutants. Transcriptomics and proteomic analysis showed that protein homeostasis but not gene expression is most likely responsible for this observation and high light provoked an upregulation of protease levels, including thylakoid filamentation temperature-sensitive 1, 5 (FtsH), caseinolytic protease proteolytic subunit 1 (ClpP1), and processing peptidases, as well as components of the chloroplast protein import machinery in UPP amiRNA lines. Strongly reduced PetC amounts were not only detected by immunoblot from mature plants but in addition in a de-etiolation experiment with young seedlings and are causing reduced high light-induced non-photochemical quenching Φ(NPQ) but increased unregulated energy dissipation Φ(NO). This impaired photosynthesis results in an inability to induce flavonoid biosynthesis. In addition, the levels of the osmoprotectants raffinose, proline, and fumarate were found to be reduced. In sum, our work suggests that UPP assists in stabilization PetC during import, processing or targeting to the thylakoid membrane, or protects it against proteolytic degradation.

Phylogenomics of the major lineages of Bembidion and related ground beetles (Coleoptera: Carabidae: Bembidiini)

Abstract Bembidion Latreille (Coleoptera: Carabidae) is a genus of small ground beetles containing about 1,380 species. To test previous phylogenetic hypotheses about deeper lineages of Bembidion and near relatives, we recover and examine over 1,800 nuclear protein-coding loci from 33 species representing the main lineages of Bembidion, 10 species of other bembidiine genera, and 7 outgroups. We find that Bembidion exclusive of subgenus Phyla Motschulsky is monophyletic, and we reclassify Phyla as a separate genus. Within Bembidion we find 2 dominant clades, the Bembidion superseries (containing about 490 species in the subgenera Eupetedromus Netolitzky and Lindrochthus Maddison, the Philochthus Stephens complex, and the Bembidion series), and the Ocydromus Clairville superseries (containing almost all other Bembidion representing about 840 species). The only known lineages within Bembidion outside of these superseries are subgenus Hoquedela Müller-Motzfeld and the Desarmatocillenus Netolitzky complex, which combined contain less than 30 species. Most clades are insensitive to variations in analyses and hold up under different sets of taxa and loci, analyses at the nucleotide or amino acid levels, and different analytical methods (maximum likelihood, including posterior mean site frequency analyses, Bayesian analyses, invariant-based methods, and those that consider incomplete lineage sorting). Despite the clarity achieved in most aspects of the phylogeny, there are several unresolved regions, notably the relationships of Desarmatocillenus, Hoquedela, and Phyla to other bembidiines. A divergence dating analysis suggests that crown Bembidion is about 48 million years old (95% confidence intervals 40–58 Ma), and that the 2 large superseries are about 38 million years old (95% confidence intervals about 29–47 Ma).

Comparative genomics reveals ample evidence to Ganoderma sinense cultivars for molecular identification and new FIP exploration

The first dikaryotic genome of Ganoderma cultivar Zizhi S2 (56.76 Mb, 16,681 genes) has been sequenced recently. 98.15% of complete BUSCOs were recovered in this genome assembly and high-confidence annotation rate improved to 91.41%. Collinearity analysis displayed the nuclear genome were 80.2% and 93.84% similar to reference genome of G. sinense at nucleotide and amino acid levels, which presented 8,521 core genes and 880 unique orthologous gene groups. Among that, at least six functional genes (tef1-α, β-tubulin, rpb2, CaM, Mn-SOD and VeA) and a newly discovered fip gene were highly similar 99.27% ∼100% to those in reference genome. And the mt-LSU, mt-SSU and 13 PCGs in their mitogenome were also highly conserved with 99.27%–99.87% and 99.08%–100% identity, respectively. So that, this cultivar Zizhi S2 is confirmed conspecific with Ganoderma sinense (NCBI: txid1077348). The new fip gene (MN635280.1_336bp) existing a novel mutation which can be reflected on the phylogenetic tree and 3-dimensional model topology structure.

Genetic Strategies and Mechanisms for Improving Ribonucleic Acid Levels of Saccharomyces pastorianus.

Developing microorganisms with a high ribonucleic acid (RNA) content is crucial for the RNA industry. Numerous studies have been conducted to enhance RNA production in yeast cells through genetic engineering, yet precise mechanisms remain elusive. Previously, upregulation of TAL1 or PGM2 and deleting PRS5 or DBP8 individually could increase the RNA content in Saccharomyces pastorianus. In this study, within these genetically modified strains, the intracellular nucleotide levels notably increased following cell fragmentation. Deletion of PRS5 and DBP8 within the strain prompted the upregulation of genes sharing similar functions, consequently augmenting the flow of the gene pathway. Furthermore, the upregulation of genes encoding cell-cycle-dependent protein kinases (CDK) was observed in the G03-△PRS5 strain. The influence of TAL1 and PGM2 on RNA content was attributed to the pentose phosphate pathway (PPP). The RNA content of polygenic recombinant strains, G03-△PRS5+△DBP8 and G03-△PRS5+△DBP8+PGM2, displayed the most significant improvement, increasing by 71.8 and 80.1% when compared to the parental strain. Additionally, the maximum specific growth rate of cells increased in these strains. This study contributes valuable insights into the genetic mechanisms underlying high nucleic acid synthesis in S. pastorianus.

In vivo selection of carbapenem resistance during persistent Klebsiella pneumoniae sequence type 395 bloodstream infection due to OmpK36 deletion.

Non-carbapenemase-producing carbapenem-resistant Enterobacterales (non-CP CRE) may be associated with a grave outcome. The common underlying mechanism is beta-lactamases and mutations in outer membrane porins. We report a case of a deep-seated infection caused by Klebsiella pneumoniae ST395 not amenable to source control, involving recurrent bloodstream infection, resulting in in vivo selection of carbapenem resistance under therapy. Three consecutive K. pneumoniae blood isolates were studied using short- and long-read sequencing. The genomes were subject to resistome and virulome, phylogenetic, and plasmid analyses. ompK36 porins were analyzed at the nucleotide and amino acid levels. Genomes were compared to 297 public ST395 K. pneumoniae genomes using cgMLST, resistome, and porin analyses and the EuSCAPE project. Relevant ompK36 and micF sequences were extracted and analyzed as above. The three sequential K. pneumoniae blood isolates belonged to the same clone. Subsequent CR isolates revealed a new large deletion of the ompK36 gene also involving the upstream region (deletion of micF). Comparison with public ST395 genomes revealed the study isolates belonged to clade B, representing a separate clone. N-terminal large ompK36 truncations were uncommon in both public data sets. In vivo selection of non-CP CRE K. pneumoniae could have substantial clinical implications. Such selection should be scrutinized through repeated cultures and frequent susceptibility testing during antimicrobial treatment, especially in the context of persistent or recurrent bloodstream infections and when adequate source control cannot be achieved. The occurrence of an unusually large deletion involving the ompK36 locus and upstream micF should be further studied.

Population genetic status of endangered whitespotted whipray Maculabatis gerrardi (Gray, 1851) in Tanzania

The whitespotted whipray Maculabatis gerrardi is exploited in Tanzania for its meat, skin and cartilage, and is classified as an endangered species by the IUCN. A mitochondrial COI gene fragment from 105 M. gerrardi individuals obtained from four unprotected and one protected marine area in Tanzania was used to determine the present genetic diversity, demographics, and effective population size of whiprays. Lower levels of nucleotide and haplotype diversity and mean mutational effective population size were apparent in unprotected than in protected areas. Analysis of molecular variance (AMOVA) identified a significant genetic difference between subpopulations of M. gerrardi and hierarchical AMOVA identified separate genetic stocks, indicative of high levels of philopatry or individual sedentarity in M. gerrardi. The importance of marine protected areas to conserve genetic diversity of whiprays is highlighted.