Gene Deficiency Research Articles

8509 Molecular and Reproductive Assessment in Ames strain under or not Hormonal Replacement

Abstract Disclosure: B.V. Azevedo: None. N. Trigueiro: None. J.M. Marques: None. V. Yariwake: None. L. Tamashiro: None. R. Cruz: None. M.M. Veras: None. L. Carvalho: None. Introduction: The Ames lineage, a mouse model with a mutation in the Prop1 gene, exhibits GH, TSH, and PRL deficiencies along with low levels of gonadotropins. This has prompted an investigation into aging and reproductive restoration after hormone replacement reported in the literature. Aims: The present study aims to analyze the pathophysiologic mechanisms in the reproductive system and fertility restoration. Methods: This is a prospective study approved by the ethical committee for animal use at the University of Sao Paulo Medical School. An isogenic Ames lineage was categorized into four groups: 1) Ames wild-type mice, 2) untreated Ames dwarf mice with sexual maturity, 3) untreated Ames dwarf mice without sexual maturity, and 4) Ames dwarf mice under hormonal treatment. Male Ames dwarf mice received treatment with levothyroxine and human recombinant GH, starting at 30 days postnatal (dp) for 40 days, followed by weekly applications until 90 dp. At the time of euthanasia, a set of analyses were conducted on animals from each group to characterize sexual maturation, fertility, and tissue collection for reproductive parameter analyses, such as seminiferous tubule morphology by histology, testicular weight, gonadosomatic index, and spermogram analysis. Additionally, pituitary RNAseq was performed using the poly-A tail capture method with the Illumina stranded mRNA prep kit and sequenced on the NextSeq 2000. Results: Treated dwarf mice exhibited fertility restoration, an increase in testicular volume with an improvement of testicular function, and complete spermatogenesis. Untreated Ames dwarf mice were infertile, but those that reached sexual maturity without hormonal replacement showed improvements in reproductive parameters, such as enhanced testicular development, maturation of some seminiferous tubules, increased testicular weight, and improvement in the spermogram compared to immature Ames dwarf mice. Transcriptome analyses revealed distinct patterns of all Ames dwarf mice groups and the wild-type. It was observed a decrease in Fshb expression in the group without sexual maturation compared to the wild-type group. Additionally, Prl and Slc6a12 were upregulated in the spontaneously sexually mature Ames dwarf compared to the non-restored Ames dwarf mice. Conclusion: GH and levothyroxine treatment in the Ames lineage are effective in restoring fertility and improving reproductive parameters in male Ames dwarf mice. However, sexually mature Ames dwarf mice without treatment presented improvements in reproductive and molecular parameters despite remaining infertile. These findings provide valuable insights into the complex interaction among hormonal replacement, modulating mechanisms, and sexual maturity. Presentation: 6/2/2024

8509 Molecular and Reproductive Assessment in Ames strain under or not Hormonal Replacement

Abstract Disclosure: B.V. Azevedo: None. N. Trigueiro: None. J.M. Marques: None. V. Yariwake: None. L. Tamashiro: None. R. Cruz: None. M.M. Veras: None. L. Carvalho: None. Introduction: The Ames lineage, a mouse model with a mutation in the Prop1 gene, exhibits GH, TSH, and PRL deficiencies along with low levels of gonadotropins. This has prompted an investigation into aging and reproductive restoration after hormone replacement reported in the literature. Aims: The present study aims to analyze the pathophysiologic mechanisms in the reproductive system and fertility restoration. Methods: This is a prospective study approved by the ethical committee for animal use at the University of Sao Paulo Medical School. An isogenic Ames lineage was categorized into four groups: 1) Ames wild-type mice, 2) untreated Ames dwarf mice with sexual maturity, 3) untreated Ames dwarf mice without sexual maturity, and 4) Ames dwarf mice under hormonal treatment. Male Ames dwarf mice received treatment with levothyroxine and human recombinant GH, starting at 30 days postnatal (dp) for 40 days, followed by weekly applications until 90 dp. At the time of euthanasia, a set of analyses were conducted on animals from each group to characterize sexual maturation, fertility, and tissue collection for reproductive parameter analyses, such as seminiferous tubule morphology by histology, testicular weight, gonadosomatic index, and spermogram analysis. Additionally, pituitary RNAseq was performed using the poly-A tail capture method with the Illumina stranded mRNA prep kit and sequenced on the NextSeq 2000. Results: Treated dwarf mice exhibited fertility restoration, an increase in testicular volume with an improvement of testicular function, and complete spermatogenesis. Untreated Ames dwarf mice were infertile, but those that reached sexual maturity without hormonal replacement showed improvements in reproductive parameters, such as enhanced testicular development, maturation of some seminiferous tubules, increased testicular weight, and improvement in the spermogram compared to immature Ames dwarf mice. Transcriptome analyses revealed distinct patterns of all Ames dwarf mice groups and the wild-type. It was observed a decrease in Fshb expression in the group without sexual maturation compared to the wild-type group. Additionally, Prl and Slc6a12 were upregulated in the spontaneously sexually mature Ames dwarf compared to the non-restored Ames dwarf mice. Conclusion: GH and levothyroxine treatment in the Ames lineage are effective in restoring fertility and improving reproductive parameters in male Ames dwarf mice. However, sexually mature Ames dwarf mice without treatment presented improvements in reproductive and molecular parameters despite remaining infertile. These findings provide valuable insights into the complex interaction among hormonal replacement, modulating mechanisms, and sexual maturity. Presentation: 6/2/2024

A Risk Model Developed based on Homologous Recombination Deficiency Genes for Evaluating the Drug Sensitivity and Prognostic Prediction of Lung Adenocarcinoma.

This study was designed to construct a risk model based on homologous recombination deficiency (HRD) to evaluate the prognosis and drug sensitivity for patients with lung adenocarcinoma (LUAD). LUAD is a subtype of lung cancer with unfavorable overall survival (OS) and prognosis. HRD has been widely studied in various tumors, but its role in LUAD has not been fully understood. We aimed to construct an HRD-related risk model for predicting the prognosis and drug sensitivity of patients with LUAD. Gene expression data of the LUAD samples were collected from the Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) database. We extracted HRD genes from previous literature and performed univariate COX analysis to select those closely associated with LUAD prognosis. ConsensusClusterPlus was employed to stratify the samples in the TCGA-LUAD cohort into different subtypes. A RiskScore model was established applying random forest method. Furthermore, immunotherapy response and drug sensitivity were predicted using Tumor Immune Dysfunction and Exclusion (TIDE) software and pRRophytic R package, respectively. Finally, the clinical features between High- and Low- RiskScore groups were compared. A total of 16 HRD genes relevant to LUAD prognosis were selected and used to classify 3 LUAD clusters (C1, C2, and C3). Specifically, C1, with a lower TIDE score displayed higher immune infiltration and immunotherapy benefit and the optimal OS, while C2 was closely correlated with tumor-relevant pathways and had the worst OS. Finally, 4 HRD genes (RAD51AP1, BRCA1, H2AFX, and FANCL) were determined to develop a RiskScore signature. It was found that a higher RiskScore was related to more advanced stages, worse OS, and tumor development pathways. Additionally, the High-RiskScore group with a higher TIDE score was sensitive to 44 traditional chemotherapy drugs. A nomogram combined with RiskScore exhibited an accurate survival prediction ability. The HRD-based RiskScore played a crucial role in LUAD development, showing a strong potential to serve as a prognostic indicator for LUAD. Our findings contributed to the diagnosis of LUAD and its treatment.

Homologous recombination deficiency gene panel analysis results in synchronous endometrial and ovarian cancers.

The objective of this study was to analyze the genetic alterations of tumors within the scope of the homologous recombination deficiency gene panel in patients diagnosed with synchronous endometrial ovarian cancer who have been followed for over 5 years using next-generation sequencing. DNA was isolated from the patient's formalin-fixed, paraffin-embedded tissue blocks. Next-generation sequencing was performed using the Illumina capture-based sequencing method. Samples were sequenced using the Sophia HR Solution DNA Kit. Seven patients were included in this study. The ratios of likely pathogenic (LP)/pathogenic (P) somatic mutations in ATM (serine/threonine kinase or Ataxia-telangiectasia mutated gene), BRCA2 (breast cancer type 2 susceptibility gene), BARD1 (BRCA1 associated RING domain 1), TP53 (tumor protein p53), BIRP1 (BRCA1-interacting helicase 1 gene), PALB2 (partner and localizer of BRCA2), and CHECK2 were 21 (48.8%), 8 (18.6%), 5 (11.6%), 3 (6.9%), 2 (4.6%), 2 (4.6%), and 2 (4.6%), respectively, in endometrium, and the ratios of somatic mutations in ATM, BRCA2, TP53, BARD1, RAD54L (DNA repair/recombination protein like), BIRP1, and RAD51D (RAD51 recombinase paralog D) were 24 (60%), 6 (15%), 5 (12.5%), 2 (5%), 2 (5%), 1 (2.5%), and 1 (2.5%), respectively, in ovary. In endometrioid-synchronous endometrial ovarian cancer cases, P/LP mutations were observed in ATM and CHECK2 genes in endometrium and ATM, BRCA2, and TP53 genes in ovary. In two non-endometrioid-synchronous endometrial ovarian cancer cases, CHEK2 (checkpoint kinase 2) mutations were observed in endometrium and ATM and TP53 mutations in ovary, whereas in one case, P/LP mutations in ATM and TP53 genes were common in both tissues. Pathogenic variations confirming the diagnosis of synchronous endometrial ovarian cancer with genetic alterations were identified in all but one case. ATM gene mutation emerged as the most common alteration and has a potential association with a favorable prognosis.

Beta cell specific ZnT8 gene deficiency and resulting loss in zinc content significantly improve insulin secretion

Zinc transporter 8 (ZnT8) is highly expressed in pancreatic beta cells, localizes to insulin secretory granules (ISG), and regulates zinc content. ZnT8 gene polymorphisms have revealed a relationship between ZnT8 activity and type 2 diabetes (T2D) risk, however, the role of beta-cell ZnT8 is not well understood.A beta cell specific ZnT8 knockout (ZnT8 BKO) mouse model was investigated. ZnT8 BKO islets showed significantly reduced ZnT8 gene expression and reduced zinc content. Compared to controls, ZnT8 BKO mice displayed significantly elevated plasma insulin levels and improved glucose tolerance following acute insulin resistance induced via S961. Glucose stimulated insulin secretion from isolated ZnT8 BKO pancreatic islets revealed enhanced insulin secretion capacity. The difference in insulin secretion between ZnT8 BKO and control islets was negated upon zinc supplementation, and the inhibitory effect of zinc on insulin secretion was confirmed in human islets.These results indicate that the loss of ZnT8 activity and accompanying reduced cellular zinc are associated with increased insulin secretion capacity. The reduction in secreted insulin content upon zinc supplementation in ZnT8 BKO islets suggests that ISG-released zinc normally tempers insulin secretion.

Estrogen, via ESR2 receptor, prevents oxidative stress-induced Müller cell death and stimulates FGF2 production independently of NRF2, attenuating retinal degeneration

In this study, we aimed to investigate the effects of the deficient antioxidative gene, nuclear factor-erythroid 2-related factor 2 (Nrf2), on 17β-estradiol (E2)-mediated oxidative stress response, with a specific focus on growth factor production and cell death in Müller cells and retinal tissue. Administration of hydrogen peroxide (H2O2) reduced the viability of Müller cells derived from Nrf2 wild-type (WT) and knockout (KO) mice. However, this effect was more significant in the KO cells than in the WT cells. Pretreatment with E2 inhibited H2O2-induced cell death in both Nrf2 WT and KO Müller cell genotypes. Small interfering RNA-mediated gene silencing of estrogen receptor 2 (Esr2) attenuated the cell survival-promoting activity of E2 in Nrf2 KO Müller cells, while other identified estrogen receptors, Esr1 or G protein-coupled estrogen receptor 1 (Gper1), had no effect. Western blotting revealed higher ESR2 expression levels in Nrf2 KO cells than in WT Müller cells. Conditioned media from E2-and H2O2-treated Nrf2 WT or KO Müller cells enhanced the dissociated retinal cell viability compared with H2O2-treated cells. Both quantitative reverse-transcription polymerase chain reaction assay (qRT-PCR) and enzyme-linked immunosorbent assay exhibited a significant increase in fibroblast growth factor 2 (FGF2) expression levels in E2-and H2O2-treated Nrf2 WT and KO Müller cells compared to those in E2-treated cells. In vivo, administration of N-methyl-N-nitrosourea (MNU) reduced the thickness and cell density of the outer nuclear layer (ONL) in Nrf2 KO mice and enhanced the number of terminal deoxynucleotidyl transferase dUTP nick-end labeling-positive cells in the ONL. However, E2 administration attenuated these defects in MNU-treated mice. Concomitant administration of MNU and E2 enhanced FGF2 protein levels in retinal lysates of Nrf2 KO mice. In conclusion, E2 demonstrated a significant role in preventing oxidative stress-induced retinal cell death by stimulating FGF2 production in Müller cells, independent of the Nrf2 gene. Based on these findings, we anticipate that exogenous administration of estrogens or ESR2-selective agonists could aid in treating patients with oxidative stress-related retinal degenerative diseases such as age-related macular degeneration and retinitis pigmentosa.

Investigation of the Relationship Between DNA Mismatch Repair Genes and Microsatellite Instability in Solid Tumors

Aim: In this study, we aimed to detect the MSI status and somatic mutations in MMR genes (MSH2, MSH6, MLH1, PMS2) of a total of 55 solid tumors diagnosed with colorectal, endometrium and ovarian cancer by NGS method and to reveal the relationship between them. Material and Method: DNA isolation was performed by taking 10-micron sections from paraffin-embedded tissue samples of 55 patients diagnosed with kolorektal, endometrium ve ovarian solid tümörlerinin and Kapa NGS DNA extraction kit was used for sequence analysis. The purity and concentration of the DNA obtained was measured by Qubit fluoremeter, and NadPrep DNA Universal Library Preparation Kit was used for high quality library preparation. Bioinformatics analyses were performed on the Genomize Seq platform. The MSI value was analysed by Roche Navify Mutation Caller software and percentage MSI values were determined using the MSIsensor2 pipeline for secondary analysis of NGS. Results: All ovarian tumors were in the MSI-Stable category. The average MSI value was 2.01. One sample had an MSI of zero. In addition, no mutations in the MSH2 and MLH1 genes were detected in any of the ovarian tumors. 3 of 4 endometrial tumors were in the MSI-Stable category, and 1 tumor was in the MSI-low (MSI value: 13.2) category. No variants were detected in the MSH2 and PMS2 genes in endometrial tumors. 2 of the 41 colorectal tumors (Case4, Case16) were in the MSI-High category. No variants were detected in the MMR genes in 19 tumors. Conclusion: Although frame-shift and stop-gain mutations were detected in 23 tumors that would cause protein deficiency in MMR genes, MSI-H was not detected, as expected, except for two colorectal tumors. Therefore, the results of our study emphasise the need to define new predictive biomarkers clinically within the framework of algorithms to predict response to immunotherapy and determine prognosis.

Abstract P205: Single-nucleus Sequencing Reveals Endothelial Cell-specific Dysregulation in the Kidney in Multiple Models of Hypertension

The kidney plays a crucial role in the onset and progression of hypertension. To investigate the roles of different renal cell types in hypertension, we applied single-nucleus sequencing and spatial transcriptome sequencing to kidney samples from three well-established hypertension models: C57BL/6 mice treated with Ang II, the Dahl salt-sensitive (SS) rat on high-salt diets, and the spontaneously hypertensive rat (SHR), along with their respective controls including Sprague-Dawley (SD) and Wistar Kyoto (WKY) rats. Our dataset comprised 179,637 nuclei from 24 samples across 12 experimental conditions, identifying 17 major cell types including various epithelial cells, endothelial cells, vascular smooth muscle cells, fibroblasts, and immune cells. At baseline, differentially expressed genes between the SS and SD groups were specifically enriched in endothelial cell pathways related to vascular development. This included expression deficiencies in genes involved in angiogenesis in SS rats, such as Arhgap24 and Nox4. Compensatory expression changes in these genes were observed in the SS rats after a high-salt diet and in 26-week-old SHR, but not in their respective controls. These genes were also associated with multiple blood pressure-associated single-nucleotide polymorphisms (SNPs). Furthermore, changes in endothelial cell communication were most prominent upon stimuli, with impaired PTPRM (Protein Tyrosine Phosphatase Receptor Type M) signaling being a robust feature in hypertension models, involving angiogenic endothelial cells in the SS model and proliferating capillary endothelial cells in the SHR model. Our study highlights the critical role of renal endothelial cell angiogenic dysfunction in the pathogenesis of hypertension and suggests a link between specific renal endothelial cell subtype dysregulation and hypertension development.

Abstract 36: Ablation of T-Lymphocyte β-Arrestins Reduces Inflammation and Increases Salt Retention in the Absence of Blood Pressure Changes Following Angiotensin II Infusion

Introduction: T-cells contribute to hypertension (HT) development. Among several G-protein coupled receptors regulating T-cell function, it has been hypothesized that some subpopulations use the angiotensin II (ANG II) type 1 receptor (AT 1 R) to modulate immune responses in HT. Selective ablation of AT 1 Rs in T-cells paradoxically increases hypertensive end-organ damage (EOD). While AT 1 Rs typically induces classical G-protein signaling, recent studies have uncovered a protective signaling cascade via β-arrestin ( ARRB ) proteins. We hypothesize that T-cell ARRB s mediate an anti-hypertensive and anti-inflammatory role in response to ANG II, decreasing EOD in HT. Methods: Breeding CD4-Cre mice with Arrb1 -Flox or Arrb2 -Flox mice generated lines with T-cell-specific deficiency of Arrb1 or Arrb2 genes. Hypertensive conditions modeling kidney damage consisted of unilateral nephrectomy, 4% salt diet, and continuous infusion of 1 mg/kg/min ANG II (UNX/HSD/ANG). Age/sex-matched Cre - littermates were used as controls (WT). Blood pressure (BP) was measured at baseline and for the following three weeks using radiotelemetry. Results: At baseline, there were no significant differences in systolic BP between groups (WT:124.1±8.0 mmHg; CD4 Arrb1-null :125.4±8.1; CD4 Arrb2-null :128.7±3.1; p =0.37; n=12/10/8). Three weeks of UNX/HSD/ANG equally increased systolic BP in all groups (WT:178.0±14.0 mmHg; CD4 Arrb1-null :174.8±26.3; CD4 Arrb2-null :179.9±11.1; p =0.86; n=7/9/7). Despite similar BPs, CD4 Arrb2-null mice demonstrated less renal inflammation than WT after four weeks of UNX/HSD/ANG, with a reduction in interleukin-1α protein (3.8±3.2 vs. 8.3±2.8 p <0.05; n=4/6) and a trend towards decreased tumor necrosis factor-α transcript (0.63-fold change vs. 1.9, p =0.051; n=10/8). Furthermore, CD4 Arrb1-null mice increased 24-hour voluntary water intake compared to WT (15.7±3.4 mL vs. 12.2±1.6; p <0.05; n=12/7), and 24-hour urine output (10.0±3.7 mL/d vs. 6.8±1.5; p <0.05; n=11/8). However, there was no difference in sodium excretion during this period (1.9±0.5 eq/d vs. 1.7±0.1; p =0.14; n=11/7). Interestingly, 4 hours after intraperitoneal saline injection, CD4 Arrb1-null mice excreted significantly less sodium compared to WT (5.6±3.5% vs . 31.4±10.6%; p <0.05; n=4/5). Conclusion: Contrary to our hypothesis, these data suggest that T-cell Arrb s do not protect against the hypertensive response to ANG II, but instead play a complicated role in the modulation of hydromineral balance and inflammation.

(p)ppGpp interacts with TrmE and regulates the PcoI/PcoR quorum-sensing system of Pseudomonas fluorescens 2P241

The efficient colonization of plant-beneficial Pseudomonas spp. is a prerequisite for their biocontrol capacity. Prior work revealed that the PcoI/PcoR quorum-sensing (QS) system plays a pivotal role in the root colonization of P. fluorescens 2P24. During the colonization, strain 2P24 has faced diverse impacts from plant-derived reactive oxygen species and other environmental stress. However, the molecular mechanism by which the PcoI/PcoR QS system is regulated under unfavored conditions remains unclear. Thus, in this study, the role of the (p)ppGpp synthetase RelA and the bifunctional (p)ppGpp synthase/hydrolase SpoT in the PcoI/PcoR QS system of P. fluorescens was investigated. Our data indicated that the deficiency of relA and spoT genes remarkably improved the expression of the pcoI gene, whereas the mutation of the spoT gene significantly repressed the expression of the pcoI gene. We further demonstrated that the regulation of the PcoI/PcoR QS system by (p)ppGpp was dependent on the function of the trmE gene, which encodes a tRNA modification GTPase. Furthermore, the mutation of relA, spoT, or both significantly influenced the motility, biofilm formation, oxidative stress, osmotic tolerance, and rhizosphere colonization. Collectively, our data indicated that the (p)ppGpp signaling pathway mediated by the relA gene and spoT gene was important to the function of the PcoI/PcoR QS system and had important implications for the understanding of the molecular mechanism of (p)ppGpp in epiphytic fitness via TrmE of P. fluorescens.

Decoding plant-induced transcriptomic variability and consistency in two related polyphagous mites differing in host ranges.

The diet breadth of generalist herbivores when compared to specialists tends to be associated with greater transcriptional plasticity. Here, we consider whether it may also contribute to variation in host range among two generalists with different levels of polyphagy. We examined two related polyphagous spider mites with different host ranges, Tetranychus urticae (1200 plants) and Tetranychus truncatus (90 plants). Data from multiple populations of both species domesticated on common beans and transferred to new plant hosts (cotton, cucumber, eggplant) were used to investigate transcriptional plasticity relative to population-based variation in gene expression. Compared to T. truncatus, T. urticae exhibited much higher transcriptional plasticity. Populations of this species also showed much more variable expression regulation in response to a plant host, particularly for genes related to detoxification, transport, and transcriptional factors. In response to the different plant hosts, both polyphagous species showed enriched processes of drug/xenobiotics metabolism, with T. urticae orchestrating a relatively broader array of biological pathways. Through co-expression network analysis, we identified gene modules associated with host plant response, revealing shared hub genes primarily involved in detoxification metabolism when both mites fed on the same plants. After silencing a shared hub CYP gene related to eggplant exposure, the performance of both species on the original bean host improved, but the fecundity of T. truncatus decreased when feeding on eggplant. The extensive transcriptomic variation shown by T. urticae might serve as a potential compensatory mechanism for a deficiency of hub genes in this species. This research points to nuanced differences in transcriptomic variability between generalist herbivores.

Identifying a Gene Deficiency in the Antiviral Innate Immune Signaling Pathway.

Innate immunity is an important defense barrier for the human body. After viral pathogen-associated molecular patterns (PAMPs) are detected by host-pathogen recognition receptors (PRRs), the associated signaling pathways trigger the activation of the interferon (IFN) regulatory factor (IRF) family members and the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). However, any gene defects among the signaling adaptors will compromise innate immune efficiency. Therefore, investigating genetic defects in the antiviral innate immune signaling pathway is important. We summarize the commonly used research methods related to antiviral immune gene defects and outline the relevant research protocols, which will help investigators study antiviral innate immunity.

The Effect of High-Salt Diet on Oxidative Stress Production and Vascular Function in Tff3−/−/C57BL/6N Knockout and Wild Type (C57BL/6N) Mice

Introduction: It is well documented that high-salt (HS) diet increases systemic and vascular oxidative stress in various animal models and in humans, leading to impairment of vascular reactivity. The present study examined the interaction of genotype and HS diet intake and the potential effects of oxidative stress – antioxidative system balance on the flow-induced dilation (FID) in pressurized carotid arteries of normotensive Tff3−/−/C57BL/6N knockout mice and their wild-type (WT) controls. Methods: Male, ten-week-old transgenic Tff3−/−/C57BL/6N (Tff3−/−) knockout mice and WT/C57BL/6N (WT) (parental strain) healthy mice were divided in LS (0.4% NaCl in rodent chow) and HS (4% NaCl in rodent chow fed for 1 week) groups. Additionally, LS and HS groups were treated with 1 mmol/L 4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl (TEMPOL) dissolved in the drinking water. After anesthesia with ketamine chloride (100 mg/kg) and midazolam (5 mg/kg), blood pressure was measured, carotid arteries and aortas were isolated, and blood samples were collected. Results: FID was decreased in WT_HS mice and restored by superoxide scavenger TEMPOL in vivo. On the other hand, attenuated FID of Tff3−/− mice was not further affected by HS diet or TEMPOL in vivo treatment. Vascular superoxide/reactive oxygen species levels were increased with HS diet in both strains and restored by TEMPOL. HS upregulated glutathione peroxidase 1 (GPx1) gene expression in WT_HS and Tff3−/−_HS mice, while GPx activity was significantly decreased only in WT_HS group. Systemic (serum) markers of oxidative stress (oxLDL and AOPP) and arterial blood pressure were similar among groups. Conclusion: HS diet increases vascular oxidative stress and impairs vasodilation in WT mice. Tff3 gene deficiency attenuates vasodilation per se, without further effects of HS intake. This can be attributed to vascular upregulation of antioxidative enzyme GPx1 in Tff3−/−/C57BL/6N mice conferring protection from oxidative stress.

Cfhr1 gene deficiency exacerbates Staphylococcus aureus-induced sepsis and acute lung injury through complement alternative pathway hyperactivation

ObjectiveTo explore the role of excessive activation of the complement alternative pathway (AP) in acute lung injury (ALI) and sepsis induced by Staphylococcus aureus. Subsequently, we aimed to define the effects of Cfhr gene deletion on Factor H expression, AP activation, and the development of sepsis-induced ALI. MethodsA sepsis-induced ALI model was established in Cfhr1-knockout mice by tail vein injection of S. aureus. Sepsis scores, bacterial load in lungs, and cytokine and complement factor levels in blood and lung tissues were evaluated at 6, 12, and 24 h after model establishment. Real-time quantitative PCR and RNA sequencing (RNA-seq) were employed to assess the expression of complement pathway-associated molecules and identify differentially expressed genes (DEGs) related to immune responses. ResultsCompared to wild-type mice, Cfhr1-knockout mice exhibited significantly increased C3a formation in lung tissues following S. aureus infection, indicating enhanced terminal complement pathway activation. Notably, these mice also had higher bacterial colony counts in the lungs, suggesting impaired S. aureus clearance. Transcriptome analysis provided further insights into the impact of Cfhr1 deletion on biological processes and signalling pathways involved in immune response regulation. ConclusionCfhr1 deletion leads to excessive AP activation, exacerbating S. aureus-induced sepsis and ALI.

Alterations in coenzyme Q10 status in a cybrid line harboring the 3243A>G mutation of mitochondrial DNA is associated with abnormal mitochondrial bioenergetics and dysregulated mitochondrial biogenesis

Mitochondrial DNA (mtDNA) mutations, including the m.3243A>G mutation that causes mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS), are associated with secondary coenzyme Q10 (CoQ10) deficiency. We previously demonstrated that PPARGC1A knockdown repressed the expression of PDSS2 and several COQ genes. In the present study, we compared the mitochondrial function, CoQ10 status, and levels of PDSS and COQ proteins and genes between mutant cybrids harboring the m.3243A>G mutation and wild-type cybrids. Decreased mitochondrial energy production, defective respiratory function, and reduced CoQ10 levels were observed in the mutant cybrids. The ubiquinol-10:ubiquinone-10 ratio was lower in the mutant cybrids, indicating blockage of the electron transfer upstream of CoQ, as evident from the reduced ratio upon rotenone treatment and increased ratio upon antimycin A treatment in 143B cells. The mutant cybrids exhibited downregulation of PDSS2 and several COQ genes and upregulation of COQ8A. In these cybrids, the levels of PDSS2, COQ3-a isoform, COQ4, and COQ9 were reduced, whereas those of COQ3-b and COQ8A were elevated. The mutant cybrids had repressed PPARGC1A expression, elevated ATP5A levels, and reduced levels of mtDNA-encoded proteins, nuclear DNA-encoded subunits of respiratory enzyme complexes, MNRR1, cytochrome c, and DHODH, but no change in TFAM, TOM20, and VDAC1 levels. Alterations in the CoQ10 level in MELAS may be associated with mitochondrial energy deficiency and abnormal gene regulation. The finding of a reduction in the ubiquinol-10:ubiquinone-10 ratio in the MELAS mutant cybrids differs from our previous discovery that cybrids harboring the m.8344A>G mutation exhibit a high ubiquinol-10:ubiquinone-10 ratio.

Deficiency of multiple RNA silencing-associated genes may contribute to the increased susceptibility of Nicotiana benthamiana to viruses

Key messageRecently published high-quality reference genome assemblies indicate that, in addition to RDR1-deficiency, the loss of several key RNA silencing-associated genes may contribute to the hypersusceptibility of Nicotiana benthamiana to viruses.

Systemic inflammation biomarkers during angioedema attacks in hereditary angioedema.

Hereditary angioedema (HAE) is a rare disease characterized by localized and self-limited angioedema (AE) attacks. A local increase of bradykinin (BK) mediates AE attacks in HAE, however the role of inflammation in HAE has been poorly explored We aim to analyze the role of inflammatory mediators in HAE patients during AE attacks. Patients with a confirmed HAE diagnosis due to C1 inhibitor deficiency (HAE-C1INH) or patients F12 gene mutations (HAE-FXII) attending to our outpatient clinic between November-2019 and May-2022 were included. Demographic and clinical characteristics were analyzed. Blood samples were collected both during symptom-free periods (baseline) and during HAE attacks, and acute phase reactants (APR), such as serum amyloid A (SAA), erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), D-Dimer and white blood cells were measured. Seventy-eight patients were enrolled in the study, with a predominant representation of women (76%, n=59), and a mean age of 47.8 years (range 6-88). Among them, 67% (n=52) of patients had HAE-C1INH (46 classified as type 1 and 6 as type 2) while 33% (n=26) had HAE-FXII. During attack-free periods, the majority of patients exhibited normal levels of SAA, ESR, D-dimer, ACE and WCC. However, in a subset of patients (16% for SAA, 18% for ESR, and 14.5% for D-dimer), elevations were noted at baseline. Importantly, during HAE attacks, significant increases were observed in SAA in 88% of patients (p< 0.0001 vs. baseline), in ESR in 65% (p= 0.003 vs. baseline) and D-dimer in 71% (p=0.001 vs. baseline) of the patients. A comparison between baseline and acute attack levels in 17 patients revealed significant differences in SAA AA (p<0. 0001), ESR (p<0.0001) and D-dimer (p= 0.004). No significant differences were observed in CRP (p=0.7), ACE (p=0.67) and WCC (p=0.54). These findings remained consistent regardless of HAE type, disease activity or location of angioedema. The systemic increase in APR observed during HAE attacks suggests that inflammation extends beyond the localized edematous area. This finding underscores the potential involvement of inflammatory pathways in HAE and highlights the need for further investigation into their role in the pathophysiology of HAE.

Selonsertib, an ASK1 Inhibitor, Ameliorates Ovalbumin-Induced Allergic Asthma during Challenge and Sensitization Periods.

Apoptosis signal-regulating kinase 1 (ASK1) is an upstream signaling molecule in oxidative stress-induced responses. Because oxidative stress is involved in asthma pathogenesis, ASK1 gene deficiency was investigated in animal models of allergic asthma. However, there is no study to investigate whether ASK1 inhibitors could be applied for asthma to date. Selonsertib, a potent and selective ASK1 inhibitor, was applied to BALB/c mice of an ovalbumin (OVA)-induced allergic asthma model. Selonsertib suppressed antigen-induced degranulation of RBL-2H3 mast cells in a concentration-dependent manner. The administration of selonsertib both before OVA sensitization and OVA challenge significantly reduced airway hyperresponsiveness, and suppressed eosinophil numbers and inflammatory cytokine levels in the bronchoalveolar lavage fluid. Histopathologic examination elucidated less inflammatory responses and reduced mucin-producing cells around the peribronchial regions of the lungs. Selonsertib also suppressed the IgE levels in serum and the protein levels of IL-13 in the bronchoalveolar lavage fluid. These results suggest that selonsertib may ameliorate allergic asthma by suppressing immune responses and be applicable to allergic asthma.

Association between homologous recombination deficiency (HRD) gene mutations and the efficacy of venadaparib in combination with irinotecan as third- or fourth-line treatment in patients with metastatic gastric cancer (mGC).

e16057 Background: Tumors with homologous recombination deficiency (HRD) have been suggested to be associated with a favorable response to poly (adenosine diphosphate [ADP]–ribose) polymerase (PARP) inhibitors. The aim of this exploratory analysis was to evaluate the association between the presence of HRD gene mutations and the efficacy of venadaparib, a novel PARP inhibitor, plus irinotecan in patients with mGC who had failed at least at least 2 lines of therapy. Methods: This was an exploratory analysis from a multi-national, phase 1b trial of venadaparib plus irinotecan in patients with mGC (NCT04725994). Tumor response was evaluated according to RECIST v. 1.1. Genomic analysis was conducted using ctDNA (GuardantOMNI Gene Panel version 1.0, Redwood City, CA) on Day 1 pre-dose. Results of the exploratory genomic analysis, which includes pathogenic or deleterious mutations of following genes: BRCA1/2, ATM, ATR, PALB2, RAD51, RAD51B/C/D, CHEK1/2, POLD1/2/3/4, BARD1, BRIP1, were analyzed with the efficacy outcomes. Results: In the dose-finding part of the clinical study, 26 patients were enrolled:13 had received two prior treatments and 13 had received three prior treatments, respectively. Of the 26 patients enrolled, 7 (26.9%) had mutations involved in HRD (4 with ATM, 1 with BRCA2, 1 with BARD1, and 1 with ATR mutations, respectively), and were treated with varying doses of venadaparib plus irinotecan. Among the 7 patients with a mutation in HRD genes, an objective response rate (ORR) was 57.1%, and median progression free survival (mPFS) was 5.6 months (95% confidence interval [CI] 1.2–not reached). For those without a mutation in HRD genes (n = 19), 1 patient achieved partial response and 13 achieved stable disease as best response with a mPFS of 4.0 (95% CI 2.9-5.5) months. Conclusions: Venadaparib in combination with irinotecan showed promising efficacy outcomes in patients with mGC, especially for those with mutations of HRD-related genes. Further development of this combination may consider a biomarker-based approach. Clinical trial information: NCT04725994 . [Table: see text]

Vitamin D deficiency and VDR gene polymorphism FokI (rs2228570) are associated with diabetes mellitus in adults: COVID-inconfidentes study

HighlightsThe prevalence of vitamin D deficiency was higher in patients with diabetes mellitus;The FokI polymorphism was associated with DM and, in synergism with vitamin D deficiency, increased the likelihood of DM in adults.The FokI polymorphism was the most frequent in individuals with diabetes;Individuals with VDD were more predisposed to have DM regardless of phenotype, meaning that vitamin D deficiency is a relevant predictor for developing DM, regardless of whether or not there is an alteration in the VDR receptor.