Reductase Subunit Research Articles

Abstract 3963: Ribonucleotide reductase M2 subunit switching in hepatoblastoma drug resistance and relapse

Abstract Background and Rationale: Hepatoblastoma is the most common primary liver cancer in infants and young children. Despite being a very rare cancer that accounts for only 0.5-2% if all childhood cancer cases, HB has the largest increase in incidence among childhood cancers in the United States and worldwide. The five-year survival rate of children with the aggressive forms of HB, including those that have developed metastatic or recurrent diseases, is less than 40% due to the lack of effective treatment. We aim to identify targetable mechanisms underlying the progression and drug resistance of high-risk HB. Results: Our recent work on HB mouse and organoid models, patient-derived xenografts (PDX) and primary patient samples revealed a significant upregulation of ribonucleotide reductase (RNR) subunit M2 (RRM2) in high-risk HB. RNR is the sole enzymatic complex in mammal cells that converts ribonucleotides to deoxyribonucleotides and plays a critical role in regulating cell division and DNA repair. We found standard chemotherapy agents as well as two RRM2 inhibitors, triapine and MK1775, were capable of reducing RRM2 expression effectively in vitro. However, we found a significant induction of another RNR subunit M2B (RRM2B) in treated cells in corresponding to RRM2 reduction. While no changes in drug response were noticed in RRM2B knockout (KO) HB cells. RRM2B levels in HB cells showed a strong impact on cells’ ability to recover after chemotherapy. RRM2BOE HB cells showed a significant increase in their colony formation potential after chemotherapy where RRM2BKO cells formed much fewer colonies after treatment compared to the control cells. Interestingly, we noticed a reversed subunit switch from RRM2B to RRM2 during the recovery period when cell proliferation was restored. RRM2, indeed, had a much higher enzymatic activity in converting ribonucleotides to deoxyribonucleotides than RRM2B and promoted cell growth much more efficiently than RRM2B when both were overexpressed in HB cells. Finally, combining the RRM2 inhibitor MK1775 with standard chemotherapy in HB PDX models, although showing no additional benefit in reducing tumor size, significantly delayed tumor relapse after drug withdrawal. Conclusion: In this study, we demonstrated an intriguing switching between two RNR subunits, RRM2 and RRM2B in HB cells undergoing drug treatment and during their recovery afterwards. Our data suggest that RRM2 supports HB growth while its switching to RRM2B is critical to tumor cell survival under drug treatment. When tumor relapses, there is a reversed subunit switch from RRM2B to RRM2 to supports the recurrent growth of the tumor, which can serve as a potential therapeutic target in preventing HB relapse. Citation Format: Anthony Ray Brown, Emilie Indersie, Shaina Porter, Baranda Hansen, Li Fan, Liyuan Li, Cheng Tian, Haiyan Tan, Shondra Miller, Junmin Peng, Stefano Cairo, Liqin Zhu. Ribonucleotide reductase M2 subunit switching in hepatoblastoma drug resistance and relapse [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3963.

Abstract 2605: BH3 mimetics synergize with the Wee1 inhibitor ZN-c3 by activating caspases which induce DNA damage and degrade key proteins

Abstract Background: Wee1 is a crucial cell cycle checkpoint kinase that regulates the G2/M checkpoint in response to DNA damage, and its inhibition can cause mitotic catastrophe and apoptosis in tumor cells. Inhibitors of BCL-2 have been shown to also induce apoptosis. ZN-d5 and ZN-c3, currently in clinical development for the treatment of cancer, are highly selective and potent inhibitors of Bcl-2 and Wee1, respectively. The combination of ZN-d5 with ZN-c3 induced robust tumor regressions in preclinical models and the mechanism of action of this activity is presented. Methods: Cell proliferation in tumor cell lines was measured using CellTiter-Glo® (Promega). Cleaved caspase-3, cleaved PARP, phosphorylated CDK1 (p-CDK1), γH2AX, Wee1, RRM2 and other markers were measured by Western blotting, IHC and/or flow cytometry. Anti-tumor efficacy was determined in several xenograft models. Results: The combination of ZN-c3 and ZN-d5 resulted in additive or synergistic anti-proliferative activity in several tumor cell lines. This enhancement of anti-proliferative activity was also observed at concentrations of ZN-d5 that induced caspase activation, but minimal apoptosis. Treatment of tumor cell lines with ZN-d5 alone at these sublethal concentrations caused induction of γH2AX, a marker of double-stranded DNA breaks, and degradation of caspase-sensitive proteins relevant to the DNA Damage Response (DDR), such as RRM2 (a ribonucleotide reductase subunit) and Wee1. Degradation of Wee1 correlated with decreases in p-CDK1, a marker of Wee1 inhibition. These effects were mainly reversible and were inhibited by pre-treatment with caspase inhibitors. Mechanistically, the combination of ZN-c3 and ZN-d5 resulted in increased levels of γH2AX, enhanced inhibition of p-CDK1, and increased levels of apoptotic markers compared to single agent treatment. Similar results were obtained when ZN-c3 was combined with inhibitors of other BCL-2 family proteins such as inhibitors of MCL-1 and BCL-xL. Finally, the combination of ZN-c3 with ZN-d5 or other BH3 mimetics showed synergistic anti-tumor activity in several xenograft models further supporting the rationale for combining these agents. Conclusions: ZN-d5 or other BH3 mimetics caused caspase-mediated DNA damage and degraded relevant proteins such as Wee1 and RRM2 resulting in additive or synergistic anti-tumor activity when combined with ZN-c3. These results support a novel use of BH3 mimetics to activate caspase activity, independently of apoptosis induction, to significantly enhance the activity of DDR inhibitors such as ZN-c3 Citation Format: Hooman Izadi, Noah Ibrahim, Tiffany Hoang, Jianhui Ma, Petrus R. de Jong, Joseph Pinchman, Kevin D. Bunker, Ahmed A. Samatar, Fernando Doñate. BH3 mimetics synergize with the Wee1 inhibitor ZN-c3 by activating caspases which induce DNA damage and degrade key proteins [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2605.

Requirement and functional redundancy of two large ribonucleotide reductase subunit genes for cell cycle, chloroplast biogenesis and photosynthesis in tomato.

Ribonucleotide reductase (RNR), functioning in the de novo synthesis of deoxyribonucleoside triphosphates (dNTPs), is crucial for DNA replication and cell cycle progression. In most plants, the large subunits of RNR have more than one homologous gene. However, the different functions of these homologous genes in plant development remain unknown. In this study, we obtained the mutants of two large subunits of RNR in tomato and studied their functions. The mutant ylc1 was obtained by ethyl methyl sulfonate (EMS) treatment. Through map-based cloning, complementation and knock-out experiments, it was confirmed that YLC1 encodes a large subunit of RNR (SlRNRL1). The expression level of the genes related to cell cycle progression, chloroplast biogenesis and photosynthesis was assessed by RNA-sequencing. In addition, we knocked out SlRNRL2 (a SlRNRL1 homologue) using CRISPR-Cas9 technology in the tomato genome, and we down-regulated SlRNRL2 expression in the genetic background of slrnrl1-1 using a tobacco rattle virus-induced gene silencing (VIGS) system. The mutant slrnrl1 exhibited dwarf stature, chlorotic young leaves and smaller fruits. Physiological and transcriptomic analyses indicated that SlRNRL1 plays a crucial role in the regulation of cell cycle progression, chloroplast biogenesis and photosynthesis in tomato. The slrnrl2 mutant did not exhibit any visible phenotype. SlRNRL2 has a redundant function with SlRNRL1, and the double mutant slrnrl1slrnrl2 is lethal. SlRNRL1 is essential for cell cycle progression, chloroplast biogenesis and photosynthesis. In addition, SlRNRL1 and SlRNRL2 possess redundant functions and at least one of these RNRLs is required for tomato survival, growth and development.

Acquired chemoresistance drives spatial heterogeneity, chemoprotection and collective migration in pancreatic tumor spheroids.

Tumors display rich cellular heterogeneity and typically consist of multiple co-existing clones with distinct genotypic and phenotypic characteristics. The acquisition of resistance to chemotherapy has been shown to contribute to the development of aggressive cancer traits, such as increased migration, invasion and stemness. It has been hypothesized that collective cellular behavior and cooperation of cancer cell populations may directly contribute to disease progression and lack of response to treatment. Here we show that the spontaneous emergence of chemoresistance in a cancer cell population exposed to the selective pressure of a chemotherapeutic agent can result in the emergence of collective cell behavior, including cell-sorting, chemoprotection and collective migration. We derived several gemcitabine resistant subclones from the human pancreatic cancer cell line BxPC3 and determined that the observed chemoresistance was driven of a focal amplification of the chr11p15.4 genomic region, resulting in over-expression of the ribonucleotide reductase (RNR) subunit RRM1. Interestingly, these subclones display a rich cell-sorting behavior when cultured as mixed tumor spheroids. Furthermore, we show that chemoresistant cells are able to exert a chemoprotective effect on non-resistant cells in spheroid co-culture, whereas no protective effect is seen in conventional 2D culture. We also demonstrate that the co-culture of resistant and non-resistant cells leads to collective migration where resistant cells enable migration of otherwise non-migratory cells.

Metabolic Regulation of Mesophilic Methanosarcina barkeri to Ammonium Inhibition.

Undesirable ammonium concentrations can lead to unstable anaerobic digestion processes, and Methanosarcina spp. are the representative methanogens under inhibition. However, no known work seems to exist for directly exploring the detailed metabolic regulation of pure cultured representative Methanosarcina spp. to ammonium inhibition. We used transcriptomics and proteomics to profile the metabolic regulation of Methanosarcina barkeri to 1, 4, and 7 g N/L of total ammoniacal nitrogen (TAN), where free ammonia concentrations were between 1.5 and 36.1 mg N/L. At the initial stages of ammonium inhibition, the genes participating in the acquisition and assimilation of reduced nitrogen sources showed significant upregulation where the minimal fold change of gene transcription was about 2. Apart from nitrogen metabolism, the transcription of some genes in methanogenesis also significantly increased at the initial stages. For example, the genes encoding alternative heterodisulfide reductase subunits (HdrAB), energy-converting hydrogenase subunit (EchC), and methanophenazine-dependent hydrogenase subunits (VhtAC) were significantly upregulated by at least 2.05 times. For the element translocation at the initial stages, the genes participating in the uptake of ferrous iron, potassium ion, and molybdate were significantly upregulated with a minimal fold change of 2.10. As the cultivation proceeded, the gene encoding the cell division protein subunit (FtsH) was significantly upregulated by 13.0 times at 7 g N/L of TAN; meanwhile, an increment in OD600 was observed at the terminal sampling point of 7 g N/L of TAN. The present study explored the metabolic regulation of M. barkeri in stress response, protein synthesis, signal transduction, nitrogen metabolism, methanogenesis, and element translocation. The results would contribute to the understanding of the metabolic effects of ammonium inhibition on methanogens and have significant practical implication in inhibited anaerobic digestion.

RRM2 Mediates the Anti-Tumor Effect of the Natural Product Pectolinarigenin on Glioblastoma Through Promoting CDK1 Protein Degradation by Increasing Autophagic Flux.

The natural product pectolinarigenin exerts anti-inflammatory activity and anti-tumor effects, and exhibits different biological functions, particularly in autophagy and cell cycle regulation. However, the antineoplastic effect of pectolinarigenin on glioblastoma (GBM) remains unclear. In the present study, we found that pectolinarigenin inhibits glioblastoma proliferation, increases autophagic flux, and induces cell cycle arrest by inhibiting ribonucleotide reductase subunit M2 (RRM2), which can be reversed by RRM2 overexpression plasmid. Additionally, pectolinarigenin promoted RRM2 protein degradation via autolysosome-dependent pathway by increasing autophagic flow. RRM2 knockdown promoted the degradation of CDK1 protein through autolysosome-dependent pathway by increasing autophagic flow, thereby inhibiting the proliferation of glioblastoma by inducing G2/M phase cell cycle arrest. Clinical data analysis revealed that RRM2 expression in glioma patients was inversely correlated with the overall survival. Collectively, pectolinarigenin promoted the degradation of CDK1 protein dependent on autolysosomal pathway through increasing autophagic flux by inhibiting RRM2, thereby inhibiting the proliferation of glioblastoma cells by inducing G2/M phase cell cycle arrest, and RRM2 may be a potential therapeutic target and a prognosis and predictive biomarker in GBM patients.

Association between the 5-methyl tetrahydrofolate reductase (5-MTHFR) and integrin Β3 subunit (ITGB3) genes polymorphism and major depressive disorder

Background and objectivesMajor depression disorder (MDD) is a common condition and syndromic sickness with devastating psychological, social, and economic effects. In general, it is a widespread problem, and the prevalence of MDD varies by country. The genetic contribution to MDD is estimated to be around 35%. This study was done to explore the association between 5-methyl tetrahydrofolate reductase (5-MTHFR) and integrin β3 subunit (ITGB3) genes polymorphism for development of major depressive disorder. Materials and methodsThe current study comprised thirty MDD patients, ranging in age from 18 to 52 years. All of the individuals were diagnosed using the Diagnostic and Statistical Manual of Mental Axis I Disorder in the outpatient departments of Taif Psychiatry Health Hospital (DSM-IV). The three polymorphic markers, C667T and A1298C of the 5-MTHFR gene and T1565C of the ITGB3 gene, were genotyped using the PCR-RFLP method. ResultsThe C667T single nucleotide polymorphism was found to be in heterozygous state in ten patients, while the A1298C single nucleotide polymorphism was not found in any of them. Twenty-one patients had the T1565C single nucleotide polymorphism in the ITGB3 gene, five of whom were homozygous state and 16 of whom were heterozygous state. Seven patients were discovered to have compound mutations in both genes. Interpretation & conclusionThe current findings could point to a link between MDD and the T allele of the C667T polymorphism in the 5-MTHFR gene and the C allele of the T1565C polymorphism in the ITGB3 gene.

A Commensal Streptococcus Dysregulates the Pseudomonas aeruginosa Nitrosative Stress Response.

Chronic infections in the cystic fibrosis (CF) airway are composed of both pathogenic and commensal bacteria. However, chronic Pseudomonas aeruginosa infections are the leading cause of lung deterioration in individuals with CF. Interestingly, oral commensals can translocate to the CF lung and their presence is associated with improved lung function, presumably due to their ability to antagonize P. aeruginosa. We have previously shown that one commensal, Streptococcus parasanguinis, produces hydrogen peroxide that reacts with nitrite to generate reactive nitrogen intermediates (RNI) which inhibit P. aeruginosa growth. In this study, we sought to understand the global impact of commensal-mediated RNI on the P. aeruginosa transcriptome. RNA sequencing analysis revealed that S. parasanguinis and nitrite-mediated RNI dysregulated expression of denitrification genes in a CF isolate of P. aeruginosa compared to when this isolate was only exposed to S. parasanguinis. Further, loss of a nitric oxide reductase subunit (norB) rendered an acute P. aeruginosa isolate more susceptible to S. parasanguinis-mediated RNI. Additionally, S. parasanguinis-mediated RNI inactivated P. aeruginosa aconitase activity. Lastly, we report that P. aeruginosa isolates recovered from CF individuals are uniquely hypersensitive to S. parasanguinis-mediated RNI compared to acute infection or environmental P. aeruginosa isolates. These findings illustrate that S. parasanguinis hinders the ability of P. aeruginosa to respond to RNI, which potentially prevents P. aeruginosa CF isolates from resisting commensal and host-induced RNI in the CF airway.

Oxymatrine induces anti-tumor response in cervical cancer by modulating circ_0008460/miR-197-3p/ribonucleotide reductase subunit M2 (RRM2)

ABSTRACT Oxymatrine (OMT) has exhibited an anti-cancer role in human cancers, including cervical cancer (CC). The dysregulated circular RNAs (circRNAs) are key regulators in cancer biology, and circ_0008460 was upregulated in CC. This study was performed to investigate the circRNA-based molecular mechanism for OMT in CC. RNA detection for circ_0008460, microRNA-197-3p (miR-197-3p), or ribonucleotide reductase subunit M2 (RRM2) was completed using reverse transcription-quantitative polymerase chain reaction assay. Cell behaviors were assessed by Cell Counting Kit-8 assay for cell viability, colony formation assay or Edu assay for cell proliferation, flow cytometry for cell apoptosis, and wound healing assay/transwell assay for migration/invasion. Protein expression examination was conducted using western blot. Dual-luciferase reporter assay and RNA pull-down assay were applied to confirm target binding. Tumor xenograft assay was performed for OMT research in vivo. OMT induced circ_0008460 downregulation in CC cells. OMT-induced inhibitory effects on cell growth, migration, and invasion but promoting effect on cell apoptosis were attenuated by circ_0008460. Circ_0008460 directly interacted with miR-197-3p, and OMT inhibited malignant behaviors of CC cells via mediating circ_0008460/miR-197-3p axis. RRM2 acted as a target for miR-197-3p and circ_0008460 affected the RRM2 level through absorbing miR-197-3p. OMT upregulated miR-197-3p to inhibit RRM2 expression to impede CC cell development. CC tumorigenesis was suppressed by OMT via targeting circ_0008460/miR-197-3p/RRM2 axis in vivo. These results suggested that OMT restrained CC cell progression in vitro and tumor growth in vivo by downregulating circ_0008460 to mediate miR-197-3p/RRM2 axis.

Suppression of methanogenesis in paddy soil increases dimethylarsenate accumulation and the incidence of straighthead disease in rice

Some soil microbes can methylate arsenic (As) and produce dimethylarsenate (DMA) as a main product. Excessive accumulation of DMA by rice plants can cause the straighthead disease, a physiological disorder leading to substantial yield losses. DMA can also be demethylated in soil, but the mechanism and the microbes involved are not well understood. We investigated the dynamics of methylated As species, including monomethylarsenate (MMA), DMA and dimethyl-monothioarsenate (DMMTA), in three paddy soils that produced the straighthead disease. The soils were incubated under flooded conditions with or without the addition of 2-bromoethanesulfonate (BES), a specific inhibitor of methanogenesis. DMA and DMMTA concentrations in porewater increased initially as soil redox potential decreased, and then decreased rapidly coinciding with the production of methane. BES addition largely suppressed methanogenesis and the disappearance of DMA and DMMTA, but not of MMA. BES addition suppressed the transcript levels of archaeal 16S rRNA and, particularly, mcrA gene encoding methyl-coenzyme reductase subunit A. Among the core genera of archaea, the absolute abundances of Methanomassiliicoccus and Methanosarcina were decreased significantly by BES in the three soils. In a pot experiment with two soils, BES addition significantly increased DMA accumulation in rice husks and the incidence of the straighthead disease in rice. The results suggest that DMA and DMMTA demethylation in paddy soil is coupled to methanogenesis with Methanomassiliicoccus and Methanosarcina likely playing an important role.

Cryo-EM structure of the EBV ribonucleotide reductase BORF2 and mechanism of APOBEC3B inhibition.

Viruses use a plethora of mechanisms to evade immune responses. A recent example is neutralization of the nuclear DNA cytosine deaminase APOBEC3B by the Epstein-Barr virus (EBV) ribonucleotide reductase subunit BORF2. Cryo-EM studies of APOBEC3B-BORF2 complexes reveal a large >1000-Å2 binding surface composed of multiple structural elements from each protein, which effectively blocks the APOBEC3B active site from accessing single-stranded DNA substrates. Evolutionary optimization is suggested by unique insertions in BORF2 absent from other ribonucleotide reductases and preferential binding to APOBEC3B relative to the highly related APOBEC3A and APOBEC3G enzymes. A molecular understanding of this pathogen-host interaction has potential to inform the development of drugs that block the interaction and liberate the natural antiviral activity of APOBEC3B. In addition, given a role for APOBEC3B in cancer mutagenesis, it may also be possible for information from the interaction to be used to develop DNA deaminase inhibitors.

Accelerated evolution of Vkorc1 in desert rodent species reveals genetic preadaptation to anticoagulant rodenticides.

Some rodent species living in arid areas show elevated physiological tolerance to anti-vitamin K rodenticides (AVKs), which seems to be due to some unknown selective pressures that rodents may experience in desert habitats. Genes involved in the ϒ-carboxylation of blood coagulation, including vitamin K epoxide reductase complex, subunit 1 (Vkorc1), ϒ-glutamyl-carboxylase (Ggcx) and NAD(P)H quinone one dehydrogenase (Nqo1) are associated with anticoagulant resistance, or some levels of elevated tolerance, in rodents. To detect whether the DNA sequences of the three genes are also under natural selection in the desert rodent species, we analyzed the Vkorc1, Ggcx and Nqo1 genes of the desert rodents and compared them with other rodent species. We found an accelerated evolutionary rate in Vkorc1 of desert rodents, especially in Mus spretus, Nannospalax galili and Psammomys obesus. By contrast, signals of positive selection were absent for Ggcx and Nqo1 in all species. Mapping the amino acid variations on the VKORC1 protein three-dimensional model suggested most interspecific amino acid variations occur on the outer surface of the VKORC1 pocket, whereas most intraspecific amino acid changes and known AVK resistance mutations occurred on the inner surface and endoplasmic reticulum luminal loop regions. Some desert-species-specific amino acid variations were found on the positions where known resistance mutations occurred, indicating these variations might be related to the elevated physical tolerance to AVKs in desert rodents. The evolution of Vkorc1 has been accelerated in some desert rodent species, indicating genetic preadaptation to anticoagulant rodenticides. Positive selection and relaxed selection have been detected in Psammomys obesus and Nannospalax galili, indicating the two rodent species might also show tolerance to AVKs, which needs further verification. © 2022 Society of Chemical Industry.

Neutron scattering maps the higher-order assembly of NADPH-dependent assimilatory sulfite reductase

Precursor molecules for biomass incorporation must be imported into cells and made available to the molecular machines that build the cell. Sulfur-containing macromolecules require that sulfur be in its S2− oxidation state before assimilation into amino acids, cofactors, and vitamins that are essential to organisms throughout the biosphere. In α-proteobacteria, NADPH-dependent assimilatory sulfite reductase (SiR) performs the final six-electron reduction of sulfur. SiR is a dodecameric oxidoreductase composed of an octameric flavoprotein reductase (SiRFP) and four hemoprotein metalloenzyme oxidases (SiRHPs). SiR performs the electron transfer reduction reaction to produce sulfide from sulfite through coordinated domain movements and subunit interactions without release of partially reduced intermediates. Efforts to understand the electron transfer mechanism responsible for SiR’s efficiency are confounded by structural heterogeneity arising from intrinsically disordered regions throughout its complex, including the flexible linker joining SiRFP’s flavin-binding domains. As a result, high-resolution structures of SiR dodecamer and its subcomplexes are unknown, leaving a gap in the fundamental understanding of how SiR performs this uniquely large-volume electron transfer reaction. Here, we use deuterium labeling, in vitro reconstitution, analytical ultracentrifugation (AUC), small-angle neutron scattering (SANS), and neutron contrast variation (NCV) to observe the relative subunit positions within SiR’s higher-order assembly. AUC and SANS reveal SiR to be a flexible dodecamer and confirm the mismatched SiRFP and SiRHP subunit stoichiometry. NCV shows that the complex is asymmetric, with SiRHP on the periphery of the complex and the centers of mass between SiRFP and SiRHP components over 100 Å apart. SiRFP undergoes compaction upon assembly into SiR’s dodecamer and SiRHP adopts multiple positions in the complex. The resulting map of SiR’s higher-order structure supports a cis/trans mechanism for electron transfer between domains of reductase subunits as well as between tightly bound or transiently interacting reductase and oxidase subunits.

Energy-conserving dimethyl sulfoxide reduction in the acetogenic bacterium Moorella thermoacetica.

Moorella thermoacetica is one of the well-studied thermophilic acetogenic bacteria. It grows by oxidation of organic substrates, CO or H2 coupled to CO2 reduction to acetate. Here, we describe that M. thermoacetica can also use dimethyl sulfoxide as terminal electron acceptor. Growth of M. thermoacetica on glucose or H2 +CO2 was stimulated by dimethyl sulfoxide (DMSO). Membranes showed a DMSO reductase activity, that was induced by growing cells in presence of DMSO. The enzyme used reduced anthraquinone-2,6-disulfonate, benzyl- and methyl viologen as electron donor, but not NAD(P)H. Activity was highest at pH5 and 60°C, the Km for DMSO was 2.4 mM. Potential DMSO reductase subunits were identified by peptide mass fingerprinting; they are encoded in a genomic region that contains three potential dmsA genes, three dmsB genes and one dmsC gene. Transcriptome analysis revealed that two different dmsAB gene clusters were induced in the presence of DMSO. The function of these two and their predicted biochemical features are discussed. In addition, the data are in line with the hypothesis that M. thermoacetica can use DMSO alongside CO2 as electron acceptor and DMSO reduction is catalysed by an energy-conserving, membrane-bound electron transport chain with DMSO as final electron acceptor.

Reduced Abundance of Nitrate-Reducing Bacteria in the Oral Microbiota of Women with Future Preeclampsia.

The oral microbiota can contribute to the regulation of blood pressure by increasing the availability of nitric oxide through the reduction of nitrate to nitrite, which can be converted into nitric oxide in the stomach and then enter the circulation. It is unclear if the composition of the oral microbiota is different between women who do and do not develop preeclampsia. This study aimed to compare the composition of the buccal microbiota just prior to the development of symptoms at 36 weeks gestation in 12 women who developed late-onset preeclampsia and 24 matched women who remained normotensive throughout pregnancy by 16S rRNA gene amplicon sequencing. The abundance of the nitrate-reducing Veillonella spp V. parvula and V. dispar and a subunit of nitrate reductase narH was compared using real-time PCR. The abundance of bacteria was correlated with maternal blood pressure and dietary intake of nitrate-containing vegetables. The results showed that the abundance of nitrate-reducing bacteria including Veillonella, specifically V. parvula, and Prevotella was reduced in women who developed preeclampsia. Veillonella but not Prevotella abundance was negatively correlated with maternal blood pressure. The dietary intake of nitrate-containing vegetables did not differ between the groups and was not correlated with the abundance of Veillonella. There was no difference in the abundance of the nitrate reductase subunit narH between the groups. These results suggest that the abundance of nitrate-reducing bacteria is reduced in the oral microbiota of women who later develop preeclampsia, indicating a potential pathway for prevention.

An ultra-rare cause of severe hypotonia mimicking Pompe disease in an infant: RRM2B related mitochon- drial DNA depletion syndrome with a novel mutation

Ribonucleotide-diphosphate reductase subunit M2B(RRM2B)-related mitochondrial disease is one of the ultra-rare mitochondrial depletion syndromes. A-2-months of age girl who had severe hypotonia with absent reflexes, failure to thrive, and developmental delay was hospitalized under our care. The initial diagnosis was Pompe disease with absent reflexes and increased creatine kinase level. Enzyme analysis for Pompe disease was normal and next-generation sequence panel analysis of 450 genes related to metabolic disorders revealed a novel mutation in the RRM2B gene. The patient died at the age of 2.5 months. Up to date, there have been reports of 31 patients with infantile forms of RRM2B. This patient presented with little features to suggest a mitochondrial disorder. In conclusion, RRM2B mutations should be included in the differential diagnosis of the Pompe disease in infants with severe hypotonia. This case report also expands the mutation spectrum of rare infantile form of the RRM2B mutations.

Serum level and tumor tissue expression of Ribonucleotide-diphosphate Reductase subunit M2 B: a potential biomarker for colorectal cancer.

This study explored the applicability of serum level and tissue expression of Ribonucleotide-diphosphate Reductase subunit M2 B (RRM2B) as reliable biomarkers for colorectal cancer (CRC) progression and metastasis. The present descriptive-analytic cohort study was conducted on 50 newly diagnosed CRC patients (stage II, III) and 50 healthy individuals. The new cases had not received any therapeutic intervention and underwent surgery immediately after the initial diagnosis. Tumorous tissues and marginal healthy tissues (as control) were excised to determine the mRNA tissue expression of RRM2B by Real-Time PCR. Serum RRM2B protein was measured using an ELISA method once in the control group. In the patients, serum RRM2B protein was evaluated before, 1 and 3 months after surgery. The tumor metastasis node (TMN) classification system and liver metastasis were evaluated in CRC patients. The results showed significantly lower RRM2B serum levels in 1 and 3 months after surgery compared with the pre-surgery condition (P = 0.014, P < 0.001 respectively). The mean RRM2B gene expression was 51% lower in tumor tissue than its adjacent normal tissue (P < 0.001). No significant relationship was found between serum level of RRM2B and tumor staging and metastasis in patients before surgery (P = 0.373, P = 0.189), 1 month after surgery (P = 0.960, P = 0.088), and 3 months after surgery (P = 0.407, P = 0.724). RRM2B expression in tumor tissue is not associated with tumor staging and metastasis (P = 0.254, P = 0.721). These data suggest measuring serum protein level of RRM2B could have a role in CRC progression, although this study should be considered preliminary due to small sample size and short follow-up duration.

Exhausting hsa_circ_0072088 restrains proliferation, motility and angiogenesis of breast carcinoma cells through regulating miR-1236-3p and RRM2 in a ceRNA pathway

BackgroundHsa_circ_0072088 (circ_0072088) is a novel and informative circular RNA in several human cancers, including breast carcinoma (BRCA). Herewith, we aimed at investigating the role and mechanism of circ_0072088 in BRCA cells. MethodsExpression of circ_0072088, microRNA (miR)-1236-3p, ribonucleotide reductase subunit M2 (RRM2), and epithelial-to-mesenchymal transition (EMT)-related markers E-cadherin, N-cadherin and Vimentin was detected by quantitative PCR and/or western blotting. Cell proliferation was measured by assays using MTS, EdU, colony formation, immunohistochemistry and nude mice tumorigenicity. Migration and/or invasion and angiogenesis were determined by scratch wound assay, transwell assay, and Capillary-like tube formation assay. Intermolecular interdependence was determined by bioinformatics algorithms, dual-luciferase reporter assay, RNA pull-down assay and Pearson's correlation coefficient analysis. ResultsCirc_0072088 was upregulated in BRCA patients’ tumors, and its high expression was associated with clinical metastasis and unfavorable disease-free survival. Interfering circ_0072088 could weaken cell viability, colony formation, EdU incorporation, and tumor growth of BRCA cells; moreover, circ_0072088 interference suppressed wound healing, migration/invasion and EMT process of BRCA cells, and tube formation of endothelial cells. Strikingly, either miR-1236-3p inhibitor or RRM2 overexpression could significantly abate in vitro roles of circ_0072088 blockage. Allied with that, circ_0072088 was a sponge of miR-1236-3p in regulating expression of miR-1236-3p-specific RRM2. ConclusionInhibiting circ_0072088 might treat BRCA via miR-1236-3p/RRM2 axis, suggesting circPHKA2 as a novel molecular inhibitor of RRM2, a Food and Drug Administration (FDA)-approved drug target.

NifH gene analysis of endophytic bacteria of sweet potato under various climatic locations

Beyond the nodule symbiosis process, some endophytic bacteria can convert the atmospheric nitrogen to available form for their own and the inhabiting host plants. These bacteria possess a nitrogenase enzyme complex involved in producing ammonia from atmospheric dinitrogen. The nitrogenase reductase subunit of the enzyme complex is governed by the nifH gene and is being used as a suitable marker. In the present study, the nifH gene containing endophytic bacteria had been identified from two sweet potato cultivars grown in different locations. Both the cultureindependent and culture-dependent methods were used to identify the nitrogen-fixing bacteria in sweet potatoes. The nifH sequences closest to Azospirillum, Bradyrhizobium, Herbaspirillum, Klebsiella, Kluyvera, Paenibacillus, Pseudacidovorax, Rhizobium and Sphingomonas were identified in this study. It was supposed that soils and varieties have more effects than climatic factors on nitrogen-fixing bacteria of sweet potato. These nitrogen-fixing bacteria could be utilized to develop biofertilizers helpful for sustainable agriculture.

Nucleotide biosynthesis links glutathione metabolism to ferroptosis sensitivity.

Nucleotide synthesis is a metabolically demanding process essential for DNA replication and other processes in the cell. Several anticancer drugs that inhibit nucleotide metabolism induce apoptosis. How inhibition of nucleotide metabolism impacts non-apoptotic cell death is less clear. Here, we report that inhibition of nucleotide metabolism by the p53 pathway is sufficient to suppress the non-apoptotic cell death process of ferroptosis. Mechanistically, stabilization of wild-type p53 and induction of the p53 target gene CDKN1A (p21) leads to decreased expression of the ribonucleotide reductase (RNR) subunits RRM1 and RRM2 RNR is the rate-limiting enzyme of de novo nucleotide synthesis that reduces ribonucleotides to deoxyribonucleotides in a glutathione-dependent manner. Direct inhibition of RNR results in conservation of intracellular glutathione, limiting the accumulation of toxic lipid peroxides and preventing the onset of ferroptosis in response to cystine deprivation. These results support a mechanism linking p53-dependent regulation of nucleotide metabolism to non-apoptotic cell death.