In Silico Sperm Proteome Analysis to Investigate DNA Repair Mechanisms in Varicocele Patients

Varicocele, a condition associated with increased oxidative stress, negatively affects sperm DNA integrity and reduces pregnancy rates. However, the molecular mechanisms related to DNA integrity, damage, and repair in varicocele patients remain unclear. This study aimed to determine the role of DNA repair molecular mechanisms in varicocele-related infertility by combining an in silico proteomics approach with wet-laboratory techniques. Proteomics results previously generated from varicocele patients (n=50) and fertile controls (n=10) attending our Andrology Center were reanalyzed using bioinformatics tools, including the WEB-based Gene SeT AnaLysis Toolkit, Open Target Platform, and Ingenuity Pathway Analysis (IPA), to identify differentially expressed proteins (DEPs) involved in DNA repair. Subsequently, selected DEPs in spermatozoa were validated using western blotting in varicocele (n = 13) and fertile control (n = 5) samples. We identified 99 DEPs mainly involved in male reproductive system disease (n=66) and male infertility (n=47). IPA analysis identified five proteins [fatty acid synthase (FASN), myeloperoxidase (MPO), mitochondrial aconitate hydratase (ACO2), nucleoporin 93 (NUP93), and 26S proteasome non-ATPase regulatory subunit 14 (PSMD14)] associated with DNA repair deficiency, which showed altered expression in varicocele (P <0.03). We validated ACO2 downregulation (fold change=0.37, change%=-62.7%, P=0.0001) and FASN overexpression (fold change = 4.04, change %= 303.7%, P = 0.014) in men with varicocele compared to controls. This study combined a unique in silico approach with an in vitro validation of the molecular mechanisms that may be responsible for varicocele-associated infertility. We identified ACO2 and FASN as possible proteins involved in DNA repair, whose altered expression may contribute to DNA damage in varicocele pathophysiology.

Effect of Metabolic Regulators and Aeration on Isocitric Acid Synthesis by Yarrowia lipolytica Grown on Ester-Aldehyde Fraction

Isocitric acid (ICA) has found wide application in medicine as a promising compound with powerful antioxidant activity to combat oxidative stress. In the known microbiological processes of ICA production by non-conventional yeast Yarrowia lipolytica, the pure carbon sources are commonly used. ICA can be also synthetized by Y. lipolytica from ester-aldehyde fraction (EAF)-waste of the ethanol production process. A highly effective method of ICA production from EAF based on regulation of key enzymes (aconitate hydratase and isocitrate lyase) by metabolic regulators (iron and itaconic acid) and aeration was developed. It is recommended to cultivate Y. lipolytica VKM Y-2373 under nitrogen deficiency conditions, a high aeration (60% of air saturation), an addition of 15 mM itaconic acid, and 2.4 mg/L iron. Under optimal conditions, Y. lipolytica VKM Y-2373 produced 83 g/L ICA with isocitrate to citrate ratio of 4.1:1 and mass yield of 1.1 g/g. The putative mechanism of ICA overproduction from EAF by Y. lipolytica was suggested.

Iron-sulphur cluster biogenesis factor LYRM4 is a novel prognostic biomarker associated with immune infiltrates in hepatocellular carcinoma

Background LYRM4 is necessary to maintain the stability and activity of the human cysteine desulfurase complex NFS1-LYRM4-ACP. The existing experimental results indicate that cancer cells rely on the high expression of NFS1. However, the role of LYRM4 in liver hepatocellular carcinoma (LIHC) remains unclear. Methods In this study, we combined bioinformatics analysis and clinical specimens to evaluate the mRNA, protein expression, and gene regulatory network of LYRM4 in LIHC. Furthermore, we detected the activity of several classical iron-sulphur proteins in LIHC cell lines through UV-vis spectrophotometry. Results The mRNA and protein levels of LYRM4 were upregulated in LIHC. Subsequent analysis revealed that the LYRM4 mRNA expression was related to various clinical stratifications, prognosis, and survival of LIHC patients. In addition, the mRNA expression of LYRM4 was significantly associated with ALT, tumour thrombus, and encapsulation of HBV-related LIHC patients. IHC results confirmed that LYRM4 was highly expressed in LIHC tissues and showed that the expression of LYRM4 protein in LIHC was significantly correlated with age and serum low-density lipoprotein (LDL) and triglyceride (TG) content. In particular, the mRNA expression of key iron- sulphur proteins POLD1 and PRIM2 was significantly overexpressed and correlated with poor prognosis in LIHC patients. Compared with hepatocytes, the activities of mitochondrial complex I and aconitate hydratase (ACO2) in LIHC cell lines were significantly increased. These results indicated that the iron-sulphur cluster (ISC) biosynthesis was significantly elevated in LIHC, leading to ISC-dependent metabolic reprogramming. Changes in the activity of ISC-dependent proteins may also occur in paracancerous tissues. Further analysis of the biological interaction and gene regulation networks of LYRM4 suggested that these genes were mainly involved in the citric acid cycle and oxidative phosphorylation. Finally, LYRM4 expression in LIHC was significantly positively correlated with the infiltrating levels of six immune cell types, and both factors were strongly associated with prognosis. Conclusion LYRM4 could be a novel prognostic biomarker and molecular target for LIHC therapy. In particular, the potential regulatory networks of LYRM4 overexpression in LIHC provide a scientific basis for future research on the role of the ISC assembly mechanism and LYRM4-mediated sulphur transfer routes in carcinogenesis.

Prospecting Biomarkers for Diagnostic and Therapeutic Approaches in Pythiosis

Pythiosis, whose etiological agent is the oomycete Pythium insidiosum, is a life-threatening disease that occurs mainly in tropical and subtropical countries, affecting several animal species. It is frequently found in horses in Brazil and humans in Thailand. The disease is difficult to diagnose because the pathogen’s hyphae are often misdiagnosed as mucoromycete fungi in histological sections. Additionally, there is no specific antigen to use for rapid diagnosis, the availability of which could improve the prognosis in different animal species. In this scenario, we investigated which P. insidiosum antigens are recognized by circulating antibodies in horses and humans with pythiosis from Brazil and Thailand, respectively, using 2D immunoblotting followed by mass spectrometry for the identification of antigens. We identified 23 protein spots, 14 recognized by pooled serum from horses and humans. Seven antigens were commonly recognized by both species, such as the heat-shock cognate 70 KDa protein, the heat-shock 70 KDa protein, glucan 1,3-beta-glucosidase, fructose-bisphosphate aldolase, serine/threonine-protein phosphatase, aconitate hydratase, and 14-3-3 protein epsilon. These results demonstrate that there are common antigens recognized by the immune responses of horses and humans, and these antigens may be studied as biomarkers for improving diagnosis and treatment.

First report of soft rot of cabbage caused by Pectobacterium wasabiae in Japan

Cabbage (Brassica oleracea var. capitata) is one of the important vegetables in Japan. In the summer of 2019, some cabbages with soft rot were found in commercial fields in Hokkaido, the northern island in Japan. All diseased plants showed grey to brown discoloration and expanding water-soaked lesions on leaves. We obtained two independent strains (NACAB191 and NACAB192) from diseased leaves. DNA from these strains yielded an expected single size amplicon with the primer set of PhF/PhR for P. wasabiae (De Boer et al. 2012) by PCR, but did not yield the expected amplicon with the primer set of BR1f/L1r for P. carotovorum subsp. brasiliense (Duarte et al. 2004) and Eca1f/Eca2r for P. atrosepticum (De Boer et al., 1995) by PCR. These two strains grew at 37°C, and their ability to utilize raffinose and lactose. These bacterial strains were gram-negative and rod-shaped. The bacterium was positive for O-nitrophenyl-beta-D-galactopyranoside, N-acetylglucosaminyl transferase, gelatin liquefaction, and acid production from D-galactose, lactose, melibiose, raffinose, citrate, and trehalose. The bacterium was negative for indole production and acid production from maltose, α-methyl-D-glucoside, sorbitol, D-arabitol, inositol, inulin, and melezitose. All strains exhibited pectolytic activity on potato slices. The sequence analysis of 16S rDNA (LC597897 and LC597898) showed more than 98% identities to P. wasabiae strain (e.g. HAFL01 in Switzerland) by BLAST analysis. In addition, Multi-locus sequence analysis (Ma et al. 2007) was performed by MEGA10 (Kumer et al. 2018) using concatenated DNA sequences of seven housekeeping genes (aconitate hydratase(acnA, LC597923 and LC597924), glyceraldehyde-3-phosphate dehydrogenase A(gapA, LC597970 and LC597971), isocitrate dehydrogenase (icdA, LC597996 and LC597997), malate dehydrogenase(mdh, LC598022 and LC598023), mannitol-1-phosphate dehydrogenase (mtlD, LC598048 and LC598049), glucose-6-phosphate isomerase (pgi, LC598074 and LC598075) and gamma-glutamyl phospate reductase (proA, LC598079 and LC598080)), and all clustered NACAB191 and NACAB192 into a clade containing other confirmed strains of P. wasabiae. As a result, these two strains shared high identity with each other (>98%, E-Values showed 0). The clade containing these two strains was consistently placed in a larger clade with the other P. wasabiae and 100% bootstrap support for its separation from other Pectobacterium species available in GenBank when the consensus tree constructed using Maximum Likelihood method. Pathogenicity of these strains against cabbage (cv. ‘Rakuen’) was confirmed by the field experiments with five weeks growth plants sprayed with bacterial suspension (1×107cfu/ml). Thirty cabbages per strain were used in this study, 12 plants treated the suspension of NACAB191 and 16 plants treated the suspension of NACAB192 which died with the same soft rot symptoms about four weeks after inoculation. Whereas water-inoculated plants remained symptomless. Strains re-isolated from the artificially diseased stems were confirmed as P. wasabiae using the methods as biochemical characterization and multiple genetic analyses. Based on the disease symptoms, the cultural, molecular, and pathological features of the strains, we conclude that the soft rot symptoms of cabbage in Hokkaido in 2019 were caused by P. wasabiae. To our knowledge, this is the first report of P. wasabiae as the soft rot disease agent of cabbage in Japan.

First report of soft rot of onion caused by Pectobacterium wasabiae in Japan

Onion (Allium cepa L.) is one of the important vegetables in Japan. In the summer of 2019, onions with soft rot were found in commercial fields in Hokkaido, the northern island in Japan. Diseased onion showed chlorosis, maceration of leaves, and rotted bulbs. We sampled some diseased onions and isolated three independent isolations (NAONI191, NAONI192 and NAONI193) from infected bulbs on LB medium. These strains were identified as Pectobacterium wasabiae based on their inability to grow at 37°C, and their ability to utilize raffinose and lactose. These bacterial strains were gram-negative, rod-shaped, N-acetylglucosaminyl transferase, gelatin liquefaction. The bacterium was positive for O-nitrophenyl-beta-D-galactopyranoside, N-acetylglucosaminyl transferase, gelatin liquefaction, and acid production from D-galactose, lactose, melibiose, raffinose, citrate, and trehalose. The bacterium was negative for indole production and acid production from maltose, α-methyl-D-glucoside, sorbitol, D-arabitol, inositol, inulin, and melezitose. All the strains exhibited pectolytic activity on potato slices. DNA from these strains yielded a single size amplicon with the primer set of PhF/PhR for P. wasabiae (De Boer et al. 2012) by PCR. However, DNA from these strains did not yield the expected amplicon with the primer set of BR1f/L1r for P. carotovorum subsp. brasiliense (Duarte et al. 2004) and Eca1f/Eca2r for P. atrosepticum (De Boer et al., 1995) by PCR. The sequence analysis of 16S rDNA (LC597917- LC597919) showed more than 98% identities to P. wasabiae strains (e.g. HAFL01 in Switzerland) by BLAST analysis. In addition, Multi-locus sequence analysis (Ma et al. 2007) was performed by MEGA6.06 using concatenated DNA sequences of seven housekeeping genes (aconitate hydratase(acnA, LC597925- LC597927), glyceraldehyde-3-phosphate dehydrogenase A(gapA, LC597972-LC597974), isocitrate dehydrogenase (icdA, LC597998- LC597998LC598000), malate dehydrogenase(mdh, LC598024- LC598026), mannitol-1-phosphate dehydrogenase (mtlD, LC598050- LC598052), glucose-6-phosphate isomerase (pgi, LC598076- LC598078) and gamma-glutamyl phospate reductase (proA, LC598099- LC598101)), and all clustered into a clade containing other confirmed strains of P. wasabiae. As a result, these three strains shared high identity with each other (>98%, E-Values showed 0). The clade containing these three strains was consistently placed in a larger clade with the other P. wasabiae and 100% bootstrap support for its separation from other Pectobacterium species available in GenBank when the consensus tree constructed using Maximum Likelihood method. Pathogenicity of these strains against onion (cv. ‘Hayate’) was confirmed by the field experiments with 5 weeks growth plants sprayed with bacterial suspension (1×107cfu/ml) resulting in soft rot on the plants about four weeks after inoculation, whereas water-inoculated plants remained symptomless. Strains re-isolated from the artificially diseased stems were confirmed as P. wasabiae using the methods as biochemical characterization and multiple genetic analyses. Based on the disease symptoms, the cultural, molecular, and pathological features of the strains, we conclude that the soft rot symptoms of onion in Hokkaido in 2019 were caused by P. wasabiae. To our knowledge, this is the first report of P. wasabiae as the soft rot disease agent of onion in Japan.

Specialization of the Reiterated Copies of the Heterodimeric Integration Host Factor Genes in Geobacter sulfurreducens

Integration host factor (IHF) is a widely distributed small heterodimeric protein member of the bacterial Nucleoid-Associated Proteins (NAPs), implicated in multiple DNA regulatory processes. IHF recognizes a specific DNA sequence and induces a large bend of the nucleic acid. IHF function has been mainly linked with the regulation of RpoN-dependent promoters, where IHF commonly recognizes a DNA sequence between the enhancer-binding region and the promoter, facilitating a close contact between the upstream bound activator and the promoter bound, RNA polymerase. In most proteobacteria, the genes encoding IHF subunits (ihfA and ihfB) are found in a single copy. However, in some Deltaproteobacteria, like Geobacter sulfurreducens, those genes are duplicated. To date, the functionality of IHF reiterated encoding genes is unknown. In this work, we achieved the functional characterization of the ihfA-1, ihfA-2, ihfB-1, and ihfB-2 from G. sulfurreducens. Unlike the ΔihfA-2 or ΔihfB-1 strains, single gene deletion in ihfA-1 or ihfB-2, provokes an impairment in fumarate and Fe(III) citrate reduction. Accordingly, sqRT-PCR experiments showed that ihfA-1 and ihfB-2 were expressed at higher levels than ihfA-2 and ihfB-1. In addition, RNA-Seq analysis of the ΔihfA-1 and ΔihfB-2 strains revealed a total of 89 and 122 differentially expressed genes, respectively. Furthermore, transcriptional changes in 25 genes were shared in both mutant strains. Among these genes, we confirmed the upregulation of the pilA-repressor, GSU1771, and downregulation of the triheme-cytochrome (pgcA) and the aconitate hydratase (acnA) genes by RT-qPCR. EMSA experiments also demonstrated the direct binding of IHF to the upstream promoter regions of GSU1771, pgcA and acnA. PilA changes in ΔihfA-1 and ΔihfB-2 strains were also verified by immunoblotting. Additionally, heme-staining of subcellular fractions in ΔihfA-1 and ΔihfB-2 strains revealed a remarkable deficit of c-type cytochromes. Overall, our data indicate that at least during fumarate and Fe(III) citrate reduction, the functional IHF regulator is likely assembled by the products of ihfA-1 and ihfB-2. Also, a role of IHF controlling expression of multiple genes (other than RpoN-dependent) affects G. sulfurreducens physiology and extracellular electron transfer.

Quest for Novel Preventive and Therapeutic Options Against Multidrug-Resistant Pseudomonas aeruginosa

Pseudomonas aeruginosa is a critical healthcare challenge due to its ability to cause persistent infections and the acquisition of antibiotic resistance mechanisms. Lack of preventive vaccines and rampant drug resistance phenomenon has rendered patients vulnerable. As new antimicrobials are in the preclinical stages of development, mining for the unexploited drug targets is also crucial. Here, we designed a chimeric vaccine against P. aeruginosa using a subtractive proteomics approach and identified nine unique enzymes as novel drug targets in PAO1 proteome. A total of five unique proteins were selected as potential vaccine candidates based on essentiality, extracellular localization, virulence, antigenicity, pathway association, protein-protein interaction analysis, hydrophilicity, and low molecular weight. These include two outer membrane porins OprF (P13794) and OprD (P32722), a protein activator precursor pra (G3XDA9), a probable outer membrane protein precursor PA1288 (Q9I456), and a conserved hypothetical protein PA4874 (Q9HUT9). These proteins were further analyzed using a reverse vaccinology approach to identify immunogenic and antigenic T cell and B cell epitopes. The best scoring epitopes qualifying for all set criteria were then further subjected to the construction of a polypeptide multi-epitope vaccine construct with cholera toxin B (CtxB) subunit as an adjuvant. The identified drug targets qualifying the screening criteria were: UDP-2-acetamido-2-deoxy-d-glucuronic acid 3-dehydrogenase WbpB (G3XD23), aspartate semialdehyde dehydrogenase (Q51344), 2-amino-4-hydroxy-6-hydroxymethyldihydropteridine pyrophosphokinase (Q9HV71), 3-deoxy-D-manno-octulosonic-acid transferase (Q9HUH7), glycyl-tRNA synthetase alpha chain (Q9I7B7), riboflavin kinase/FAD synthase (Q9HVM3), aconitate hydratase 2 (Q9I2V5), probable glycosyltransferase WbpH (G3XD85) and UDP-3-O-[3-hydroxylauroyl] glucosamine N-acyltransferase (Q9HXY6). For druggability and pocketome analysis crystal and homology structures of these proteins were retrieved and developed. A sequence-based search was performed in different databases (ChEMBL, Drug Bank, PubChem and Pseudomonas database) for the availability of reported ligands and tested drugs for the screened targets. These predicted targets may provide a basis for the development of reliable antibacterial preventive and therapeutic options against P. aeruginosa.

Effects of Medium Components on Isocitric Acid Production by Yarrowia lipolytica Yeast

The microbiological production of isocitric acid (ICA) is more preferable for its application in medicine and food, because the resulting product contains only the natural isomer—threo-DS. The aim of the present work was to study ICA production by yeast using sunflower oil as carbon source. 30 taxonomically different yeast strains were assessed for their capability for ICA production, and Y. lipolytica VKM Y-2373 was selected as a promising producer. It was found that ICA production required: the limitation of Y. lipolytica growth by nitrogen, phosphorus, sulfur or magnesium, and an addition of iron, activating aconitate hydratase, a key enzyme of isocitrate synthesis. Another regulatory approach capable to shift acid formation to a predominant ICA synthesis is the use of inhibitors (itaconic and oxalic acids), which blocks the conversion of isocitrate at the level of isocitrate lyase. It is recommended to cultivate Y. lipolytica VKM Y-2373 under nitrogen deficiency conditions with addition of 1.5 mg/L iron and 30 mM itaconic acid. Such optimized nutrition medium provides 70.6 g/L ICA with a ratio between ICA and citric acid (CA) equal 4:1, a mass yield (YICA) of 1.25 g/g and volume productivity (QICA) of 1.19 g/L·h.

Dietary Mg2+ Intake and the Na+/Mg2+ Exchanger SLC41A1 Influence Components of Mitochondrial Energetics in Murine Cardiomyocytes

Cardiomyocytes are among the most energy-intensive cell types. Interplay between the components of cellular magnesium (Mg) homeostasis and energy metabolism in cardiomyocytes is poorly understood. We have investigated the effects of dietary Mg content and presence/functionality of the Na+/Mg2+ exchanger SLC41A1 on enzymatic functions of selected constituents of the Krebs cycle and complexes of the electron transport chain (ETC). The activities of aconitate hydratase (ACON), isocitrate dehydrogenase (ICDH), α-ketoglutarate dehydrogenase (KGDH), and ETC complexes CI–CV have been determined in vitro in mitochondria isolated from hearts of wild-type (WT) and Slc41a1−/− mice fed a diet with either normal or low Mg content. Our data demonstrate that both, the type of Mg diet and the Slc41a1 genotype largely impact on the activities of enzymes of the Krebs cycle and ETC. Moreover, a compensatory effect of Slc41a1−/− genotype on the effect of low Mg diet on activities of the tested Krebs cycle enzymes has been identified. A machine-learning analysis identified activities of ICDH, CI, CIV, and CV as common predictors of the type of Mg diet and of CII as suitable predictor of Slc41a1 genotype. Thus, our data delineate the effect of dietary Mg content and of SLC41A1 functionality on the energy-production in cardiac mitochondria.