Transcript Abundance Research Articles

Genome-Wide Identification and Characterization of the PPPDE Gene Family in Rice

Protein ubiquitination is common and crucial in cellular functions, however, little is known about how deubiquitinating enzymes (DUBs) reverse regulate the ubiquitination signaling process. PPPDE family proteins are a novel class of deubiquitinating peptidases with demonstrated deubiquitination/deSUMOylating activities. In this study, we identified 10 PPPDE genes from the rice (Oryza sativa L.) genome unevenly distributed on five chromosomes, where most of these members have not been reported to date. Based on the gene structure, the OsPPPDE family consists of three distinct subgroups within the phylogenetic tree. Cis-element analysis identified light/phytohormone-responsive, development, and abiotic stress-related elements in the promoters of OsPPPDE. Furthermore, we conducted and analyzed the transcript abundance of OsPPPDE under various tissues and stresses using the transcriptome data of 352 samples from the Rice Expression Database and GEO datasets. Moreover, OsPPPDE5 showed differential regulation of its transcript abundance during Cd and drought stress. Collinearity and syntenic analysis of 101 PPPDEs and PPPDE-like proteins in 10 plant genomes indicated that this family is evolutionarily conserved. Domestication analysis suggests that OsPPPDEs may contribute to indica–japonica divergence using the data from the 3K Rice Genome Project. Our study provides a foundation for further study on the function and molecular mechanism of the OsPPPDE gene family.

The lack of EphB3 receptor prevents bone loss in mouse models of osteoporosis.

Bone homeostasis is a complex process in which some Eph kinase receptors and their ephrin ligands appear to be involved. In the present study, we address this issue by examining, both in vitro and in vivo, the role of EphB2 and EphB3 in mesenchymal stromal/stem cell (MSC) differentiation into bone tissue. This was first evaluated by quantitative reverse transcription PCR (RT-qPCR) and histological staining in MSCs cultured in specific mediums revealing that although EphB2-/- MSCs mainly expressed pro-adipogenic transcription factors, EphB3-/- MSCs showed abundant osteogenic transcripts, such as Runx2, Msx2, and Sp7. To clarify the underlying molecular mechanisms, we found that the lack of EphB3 signaling alters the genetic profile of differentiating MSCs, reducing the expression of many inhibitory molecules and antagonists of the BMP signaling pathway, and increasing Bmp7 expression, a robust bone inductor. Then, to confirm the osteogenic role of EphB3 in vivo, we studied the condition of 2 mouse models of induced bone loss (ovariectomy or long-term glucocorticoid treatment). Interestingly, in both models, both WT and EphB2-/- mice equally developed the disease but EphB3-/- mice did not exhibit the typical bone loss, nor an increase in urine Ca2+ or blood serum CTX-1. This phenotype in EphB3-KO mice could be due to their significantly higher proportions of osteoprogenitor cells and preosteoblasts, and their lower number of osteoclasts, as compared with WT and EphB2-KO mice. Thus, we conclude that EphB3 acts as a negative regulator of the osteogenic differentiation, and its absence prevents bone loss in mice subjected to ovariectomy or dexamethasone treatment.

Identification of the mechanistic basis of nitrogen responsiveness in two contrasting Setaria italica accessions.

Nitrogen (N) is a macronutrient limiting crop productivity with varied requirements across species and genotypes. Understanding the mechanistic basis of N responsiveness by comparing contrasting genotypes could inform the development and selection of varieties with lower N demands, or inform agronomic practices to sustain yields with lower N inputs. Given the established role of millets in ensuring climate-resilient food and nutrition security, we investigated the physiological and genetic basis of nitrogen responsiveness in foxtail millet (Setaria italica L.). We had previously identified genotypic variants linked to N responsiveness, and here we dissect the mechanistic basis of the trait by examining the physiological and molecular behaviour of N responsive (NRp-SI58) and non-responsive (NNRp-SI114) accessions at high and low N. Under high N, NRp-SI58 allocates significantly more biomass to nodes, internodes and roots, more N to developing grains, and is more effective at remobilizing flag leaf N compared with NNRp-SI114. Post-anthesis flag leaf gene expression suggests that differences in N induce much higher transcript abundance in NNRp-SI114 than NRp-SI58, a large proportion of which is potentially regulated by APETALA2 (AP2) transcription factors. Overall, the study provides novel insights into the regulation and manipulation of N responsiveness in S. italica.

VEEV TC-83 Triggers Dysregulation of the Tryptophan-Kynurenine Pathway in the Central Nervous System That Correlates with Cognitive Impairment in Tg2576 Mice.

Neurodegenerative diseases are chronic conditions affecting the central nervous system (CNS). Alzheimer's disease (AD) is a neurodegenerative disorder characterized by the accumulation of amyloid beta in the limbic and cortical brain regions. AD is presumed to result from genetic abnormalities or environmental factors, including viral infections, which may have deleterious, long-term effects. In this study, we demonstrate that the Venezuelan equine encephalitis virus (VEEV) commonly induces neurodegeneration and long-term neurological or cognitive sequelae. Notably, the effects of VEEV infection can persistently influence gene expression in the mouse brain, suggesting a potential link between the observed neurodegenerative outcomes and long-term alterations in gene expression. Additionally, we show that alphavirus encephalitis exacerbates the neuropathological profile of AD through crosstalk between inflammatory and kynurenine pathways, generating a range of metabolites with potent effects. Using a mouse model for β-amyloidosis, Tg2576 mice, we found that cognitive deficits and brain pathology were more severe in Tg2576 mice infected with VEEV TC-83 compared to mock-infected controls. Thus, during immune activation, the kynurenine pathway plays a more active role in the VEEV TC-83-infected cells, leading to increases in the abundance of transcripts related to the kynurenine pathway of tryptophan metabolism. This pathway generates several metabolites with potent effects on neurotransmitter systems as well as on inflammation, as observed in VEEV TC-83-infected animals.

Precision mapping and expression analysis of recessive bacterial blight resistance gene xa-45(t) from Oryza glaberrima.

Bacterial blight, caused by Xanthomonas oryzae pv. oryzae (Xoo), is one of the most devastating diseases of rice leading to huge yield losses in Southeast Asia. The recessive resistance gene xa-45(t) from Oryza glaberrima IRGC102600B, mapped on rice chromosome 8, spans 80Kb with 9 candidate genes on Nipponbare reference genome IRGSP-1.0. The xa-45(t) gene provides durable resistance against all the ten Xanthomonas pathotypes of Northern India, thus aiding in the expansion of recessive bacterial blight resistance gene pool. Punjab Rice PR127, carrying xa-45(t), was released for wider use in breeding programs. This study aims to precisely locate the target gene among the 9 candidates conferring resistance to bacterial blight disease. Sanger sequencing of all nine candidate genes revealed seven SNPs and an Indel between the susceptible parent Pusa 44 and the resistant introgression line IL274. The genotyping with polymorphic markers identified three recombinant breakpoints for LOC_Os08g42370, and LOC_Os08g42400, 15 recombinants for LOC_Os08g423420 and 26 for LOC_Os08g42440 out of 190 individuals. Relative expression analysis across six time intervals (0, 8, 24, 48, 72, and 96h) after bacterial blight infection showed over expression of LOC_Os08g42410-specific transcripts in IL274 compared to Pusa 44, with a significant 4.46-fold increase observed at 72h post-inoculation. The Indel marker at the locus LOC_Os08g42410 was found co-segregating with the phenotype, suggesting its candidacy towards xa-45(t). The transcript abundance assay provides strong evidence for the involvement of LOC_Os08g42410 in the resistance conferred by the bacterial blight gene xa-45(t).

Changes in characteristics of spermatogonial stem cells in response to heat stress in stallions

Spermatogonial stem cells (SSCs) are essential for the maintenance of male fertility and survival of species. Environmental conditions, notably heat stress, have been identified as important causes of male infertility and have a negative impact on SSCs. Animals with cryptorchid testes (CT) are optimal models for the study of long-term heat stress-related changes in germ cells. The effect of heat stress on germ cells differs depending on the spermatogenesis stage. Thus, verifying whether the specific phase of spermatogenesis is dependent or independent of heat stress in stallions is important. We evaluated the heat stress-related response of SSCs by comparing the relative abundance of mRNA transcripts and expression patterns of the undifferentiated embryonic cell transcription factor 1 (UTF-1) and deleted in azoospermia-like (DAZL) in the seminiferous tubules of CT and normal testes (NT) of stallions using reverse transcription-quantitative polymerase chain reaction (RT-qPCR), immunofluorescence, and western blotting. We also analyzed the relative abundance of mRNA of different proliferative markers, including minichromosome maintenance 2 (MCM2), marker of proliferation Ki-67 (MKI-67), and proliferating cell nuclear antigen (PCNA). Testicular tissues from four Thoroughbred unilateral cryptorchid postpubertal stallions were used in this study during the breeding season. The relative abundance of the mRNA transcripts of UTF-1 and MCM2 was significantly upregulated in the CT group than that of those in the NT group. In contrast, the relative abundance of the mRNA transcripts of DAZL was significantly downregulated in the CT group than that of those in the NT group. Western blot quantification showed that the relative intensity of UTF-1 protein bands was significantly higher, while that of DAZL protein bands was significantly lower in the CT group than in the NT group. Immunofluorescence studies showed that the number of germ cells immunostained with UTF-1 was significantly higher while immunostained with DAZL was significantly lower in the CT group than that in the NT group. The higher expression level of UTF-1 in the CT group shows that undifferentiated SSCs are not affected by long-term exposure to heat stress. These results also indicate that germ cells after differentiation phase are directly affected by heat-stress conditions, such as cryptorchidism, in stallions.

Integrative transcriptome analysis uncovers common components containing CPS2 regulated by maize lncRNA GARR2 in gibberellin response.

The combined transcriptome outcome provides an important clue to the regulatory cascade centering on lncRNA GARR2 and CPS2 gene in GA response. Long non-coding RNAs (lncRNAs) serve as regulatory components in transcriptional hierarchy governing multiple aspects of biological processes. Dissecting regulatory mechanisms underpinning tetracyclic diterpenoid gibberellin (GA) cascade holds both theoretical and applied significance. However, roles of lncRNAs in transcriptional modulation of GA pathway remain largely elusive. Gypsy retrotransposon-derived GIBBERELLIN RESPONSIVE lncRNA2 (GARR2) has been reported as GA-responsive maize lncRNA. Here a novel GARR2-edited line garr2-1 was identified, characteristic of GA-induced phenotype of increased seedling height and elongated leaf sheath. Transcriptome analysis indicated that transcriptional abundance of five genes [ent-copalyl diphosphate synthase2 (CPS2), ent-kaurene synthase4 (KS4), ent-kaurene synthase6 (KS6), ent-kaurene oxidase2 (KO2), and ent-kaurenoic acid oxidase1/Dwarf3 (KAO1/D3)] was elevated in garr2-1 for early steps of GA biosynthesis. Five GA biosynthetic genes as hub regulators were interlaced to shape regulatory network of GA response. Different transcriptome resources were integrated to discover common differentially expressed genes (DEGs) in the independent GARR2-edited lines GARR2KO and garr2-1. A total of 320 common DEGs were retrieved. These common DEGs were enriched in diterpenoid biosynthetic pathway. Integrative transcriptome analysis revealed the common CPS2 encoding the CPS enzyme that catalyzes the conversion of the precursor trans-geranylgeranyl diphosphate to ent-copalyl diphosphate. The up-regulated CPS2 supported the GA-induced phenotype of slender seedlings observed in the independent GARR2-edited lines GARR2KO and garr2-1. Our integrative transcriptome analysis uncovers common components of the GA pathway regulated by lncRNA GARR2. These common components, especially for the GA biosynthetic gene CPS2, provide a valuable resource for further delineating the underlying mechanisms of lncRNA GARR2 in GA response.

Evaluation of the Expression Level of Some Coding and Non-coding Genes in Breast Cancer Samples Based on Bioinformatics and Laboratory Studies

Background: Breast cancer (BC) is the leading cause of cancer-associated mortality in women worldwide. However, the molecular mechanism underlying the process is still unclear. In this regard, bioinformatics studies play a decisive role in facilitating the path of biological investigations and can ultimately lead to the identification of better molecular candidates for further study. Objectives: Due to the abnormal expression of many coding and non-coding genes in all types of cancers and their relationship with various mechanisms of carcinogenesis, this study aimed at evaluating the expression levels of certain coding and non-coding genes involved in BC based on bioinformatics findings and laboratory investigations. Methods: Gene expression dataset, module extraction, functional enrichment analysis, protein-protein interaction network construction, and RT-qPCR were performed based on bioinformatics methods and laboratory investigations. Additionally, the promoter region mutations of these genes were investigated, using sequencing of extracted DNAs from formalin-fixed paraffin-embedded (FFPE) tumor tissues. Results: A module was selected as a candidate for further investigation. Estrogen receptor 1 (ESR1) and forkhead box A1 (FOXA1) showed the highest degrees in the PPI network with 9 and 7 links, respectively. Furthermore, the expression levels of the FOXA1 gene, RNA component of mitochondrial RNA processing endoribonuclease (RMRP), and nuclear enriched abundant transcript 1 (NEAT1) were significantly upregulated in the tumor group compared to the control group (in order, P = 0.044, P = 0.014, and P = 0.0004). The tumors of patients with positive metastasis displayed significantly higher levels of NEAT1 and RMRP expression compared to those of negative metastasis samples (P < 0.05). Moreover, the expression level of RMRP dramatically decreased in HER2-positive patients compared to negative samples (P = 0.011). Finally, no mutations were observed in the promoter sequencing of positive metastasis samples compared to normal samples. Conclusions: The upregulation levels of all three examined genes may correlate with BC progression. Therefore, they could potentially be used as biomarkers for detecting BC development.

Autism patient-derived SHANK2BY29X mutation affects the development of ALDH1A1 negative dopamine neuron

Autism spectrum disorder (ASD) encompasses a range of neurodevelopmental conditions. Different mutations on a single ASD gene contribute to heterogeneity of disease phenotypes, possibly due to functional diversity of generated isoforms. SHANK2, a causative gene in ASD, demonstrates this phenomenon, but there is a scarcity of tools for studying endogenous SHANK2 proteins in an isoform-specific manner. Here, we report a point mutation on SHANK2, which is found in a patient with autism, located on exon of the SHANK2B transcript variant (NM_133266.5), hereby SHANK2BY29X. This mutation results in an early stop codon and an aberrant splicing event that impacts SHANK2 transcript variants distinctly. Induced pluripotent stem cells (iPSCs) carrying this mutation, from the patient or isogenic editing, fail to differentiate into functional dopamine (DA) neurons, which can be rescued by genetic correction. Available SMART-Seq single-cell data from human midbrain reveals the abundance of SHANK2B transcript in the ALDH1A1 negative DA neurons. We then show that SHANK2BY29X mutation primarily affects SHANK2B expression and ALDH1A1 negative DA neurons in vitro during early neuronal developmental stage. Mice knocked in with the identical mutation exhibit autistic-like behavior, decreased occupancy of ALDH1A1 negative DA neurons and decreased dopamine release in ventral tegmental area (VTA). Our study provides novel insights on a SHANK2 mutation derived from autism patient and highlights SHANK2B significance in ALDH1A1 negative DA neuron.

PSIII-11 Impacts of late gestational in utero heat stress on small intestine dynamics of neonatal dairy calves

Abstract Late gestation heat stress in dairy cows has been shown to negatively affect offspring growth and development. In utero heat stressed calves have altered small intestine morphology and an impaired ability to absorb colostral immunoglobulin G (IgG). Thus, the objective of this study was to characterize potential cellular and molecular alterations in the small intestine of neonatal in utero heat stressed calves. In the last ~56 d of gestation pregnant dams were either heat stressed (shade; n = 41) or cooled (shade, fans, soakers; n = 41) resulting in heifers that were in utero heat stressed (IUHS, n = 15) or in utero cooled (IUCL, n = 15). Heifers were fed 3.78 L of high-quality colostrum within 2 h of birth. Apparent efficiency of absorption (AEA) of IgG was calculated by measuring IgG content fed in colostrum and in 24 h serum. Within 4.6 ± 2.3 h of birth a subset of calves (n = 8/treatment) not fed colostrum were euthanized to collect samples from the ileum. Tissue for histological analysis was placed in 10% neutral buffered formalin before being dehydrated, embedded in paraffin, cut into 5 μm thicknesses, and mounted to glass slides. The TUNEL method was used to identify apoptotic cells using the Keyence BZ-X800 Microscope. Tissue for gene expression analysis was snap-frozen and kept at -80°C before RNA extraction and cDNA synthesis. The transcript abundance of endosomal (FCGRT, DAB2, MAMDC4), tight junction (ZO1, ZO2, ZO3, OCLN, CLDN1), and heat shock (HSP90, HSF1) related genes were measured by qPCR using the CFX96 Real-Time PCR detection System (BIO-RAD). Data were analyzed using PROC MIXED or PROC GLIMMIX depending on the response variable. TUNEL results are expressed as the proportion of positive cells to total cells. Quantitative PCR data were analyzed by assessing the estimates of the model relative to the geometric mean of 3 reference genes (ACTB, GADPH, RSP9; ΔΔCT). The concentration of colostrum IgG fed to calves was not different (136.1 vs. 122.5 g/L; IUHS vs. IUCL, respectively; P = 0.32), but AEA of IgG was reduced in IUHS relative to IUCL calves (23.5 vs. 33.3%; P < 0.01). The proportion of apoptotic cells within the villi of the ileum was increased in IUHS calves compared with IUCL (2.11 vs. 1.57%; P < 0.01). Ileum gene transcript abundance of DAB2 and MAMDC4 were increased (P < 0.01), while FCGRT transcripts tended to increase in IUHS calves (P = 0.08). All tight junction and heat shock-related gene transcripts were increased within the ileum of IUHS calves (P ≤ 0.02) with the exceptions of CLDN1 and HSP90, which were not different between treatments (P ≥ 0.22).These results suggest that exposure to heat stress in utero impact small intestine integrity after birth and affect key IgG transporters, providing insight into underlying mechanisms responsible for reduced IgG absorption in IUHS calves.

Abstract PO5-15-07: Integrative proteomics, phosphoproteomics, and RNA profiling revealed novel timeline-dependent vulnerabilities in HER2-positive breast cancer

Abstract Background: Most personalized breast cancer therapies leverage biomarkers identified in the pre-treatment setting. They do not always reflect the dynamic nature of early indicators of treatment response/resistance. Herein, we have a unique cohort of newly diagnosed HER2-positive breast tumors treated with a single low dose of trastuzumab (50mg), and the corresponding residual tumors that did not achieve pathologic complete response (pCR) to HER2-targeted neoadjuvant therapy. We hypothesize that integrative molecular profiling with proteomics and transcriptomics of these breast tumors could reveal novel time-course dependent contributors of therapy response and resistance. Method: This correlative biomarker study was a secondary objective of a parent study that evaluated 64uc-DOTA-trastuzumab PET imaging to predict response to trastuzumab and pertuzumab-based neoadjuvant therapy in HER2-positive breast cancer patients. Eighteen newly diagnosed breast cancer patients were enrolled, and pCR was achieved in 13 patients (72%). This correlative biomarker study was performed on breast tumors collected within 72 hours following administering a low dose trastuzumab (50mg) to the subjects prior to imaging and neoadjuvant therapy. Deep expression proteomics and phosphoproteomics data acquisition were performed on a Thermo Orbitrap Eclipse mass spectrometer. RNA profiling was performed using Nanostring breast cancer profiler. Results: Our multi-omic approach identified increased abundance or activity in the pCR group on the following (p < 0.05): Her2 protein and ERBB2 transcript levels, increased phosphorylation of ERBB2 (T1052), increase in Golgi trafficking activities, posttranslational modification with protein glycosylation, and lipid biosynthesis. Interestingly, compared to the non-pCR group, the pCR group showed lower activity level in ERBB2’s downstream pathways, such as MAPK, SRC, and PI3K. In the non-pCR group, the residual tumor showed decrease in the following (p < 0.05): Golgi trafficking, glycosylation, antigen processing, and Her2 protein and phosphorylation (T1166). The pCR group had a negative enrichment for SGK1, a kinase which mediates PI3K pathway independently of AKT. SGK1 abundance was highest in residual tumors following neoadjuvant therapy. Conclusion: Using integrative proteomic and RNA profiling, we identified several in-vivo biological pertubations following administration of low dose trastuzumab. Increased Her2 abundance and ERBB2 transcript levels are associated with pCR, in keeping with previous reports of known pCR predictors. Decreased abundance of ERBB’s downstream signaling proteins in the pCR group suggests that early treatment response to trastuzumab may be a predictor of pCR. Our integrative approach identified other dynamic biomarkers. For example, compared to non-pCR group, Golgi trafficking is more active in pCR group tumors, and lowest in residual tumors following neoadjuvant therapy. This suggests that novel Golgi-targeting agents may be best utilized in neoadjuvant setting. Further, we identified SGK1 may be an important kinase for mediating therapy resistance early in treatment course, since its level is higher in non-pCR group following early exposure to trastuzumab compared to pCR group, with highest level detected in the residual tumors. These findings warrant orthogonal validation using in-vitro functional assays. Citation Format: Christina Wei, Lixin Yang, Krystine Mansfield, Jessica Liu, Ritin Sharma, Russell Rockne, Raju Pillai, Patrick Pirrotte, Joanne Mortimer. Integrative proteomics, phosphoproteomics, and RNA profiling revealed novel timeline-dependent vulnerabilities in HER2-positive breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-15-07.

Abstract PO3-14-01: Heterogeneity of tumor immune microenvironment to predict everolimus efficacy in premenopausal women with hormone receptor-positive/human epidermal growth factor receptor 2-negative adavanced breast cancer

Abstract Objective Everolimus (EVE), a mammalian target of rapamycin (mTOR) inhibitor, has been shown to prolong survival of patients with hormone receptor-positive/human epidermal growth factor receptor 2-negative (HR+/HER2-), advanced breast cancer (ABC) with endocrine resistance. Preclinical studies have suggested that mTOR is closely associated with the tumor immune microenvironment (TME). We aimed to explore the tumor heterogeneity of TME between patients with different response to everolimus (EVE), and identify potential markers to predict efficacy of EVE in premenopausal patients with HR+/HER2−, advanced breast cancer (ABC) suffering endocrine resistance. Methods Patients were divided into two groups according to the best response to EVE: the responsive subgroup (RS) and the non-responsive subgroup (NRS). Postoperative tumor tissue from patients who received EVE was collected for whole exome sequencing. Transcript abundance of 126 distinct tumor regions of interest (ROIs) were quantitated using digital spatial profiling, including 42 tumor cell zones (TCZs), 42 immune cell zones (ICZs), and 42 stroma cell zones (SCZs). Based on investigation in multiple discrete areas, differences in cell components, function and pathway, gene expression and immune cell infiltration between RS and NRS were investigated. Results Totally, 93 differentially expressed genes (DEGs) were identified in the ICZs, 174 DEGs in the TCZs, and 197 DEGs in the SCZs, using p< 0.05 and |log2FC >1| as the screening criterion. Among all the subgroup analyses on different ROIs, the statistical significance of PIP gene was the highest (ICZs: p=2.6E-12, TCZs: p=1.25E-20, SCZs: p=2.0E-18) in the comparison of RS and NRS. Moreover, we found that PIP was highly expressed in NRS and patients with a progression-free survival < 1 year. The receptor-legend analysis showed that, the intra-and inter-cellular communication in RS was mainly mediated by interaction between CCL-CCR family, while NRS mainly by COL family complex. Comparing all cell components extracted from each ROI, no statistical differences were found between RS and NRS, only a statistical trend in the SCZs (p=0.098). Further cell classification analysis showed fibroblasts highest in TCZs and SCZs, macrophages in ICZs. Compared to NRS, in TCZs, fibroblasts were significantly increased (p=0.01), and plasma cells (p=0.02) and neutrophils (p=0.03) were decreased in RS. In SCZs, the downregulated fibroblasts (p=0.04) and enhanced plasma cells (p=0.01) were observed in NRS. In ICZs, pDCs (p=0.048) and neutrophils (p=0.006) were significantly elevated in NRS compared with RS. Conclusion The utility of digital spatial gene expression profiling might provide some references to predict the efficacy of EVE in premenopausal patients with HR+/HER2- ABC with endocrine resistance. Citation Format: Yujing Tan, Fei Ma, Jiayu Wang, Pin Zhang, Binghe Xu, Liyan Xue, Ying Fan. Heterogeneity of tumor immune microenvironment to predict everolimus efficacy in premenopausal women with hormone receptor-positive/human epidermal growth factor receptor 2-negative adavanced breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-14-01.

Effect of duration of estradiol exposure on embryo survival and endometrial gene expression in anestrous embryo recipient mares

Previous studies indicate a positive correlation between the duration of estrus prior to ovulation and likelihood of pregnancy in embryo recipient mares. However, the mechanisms by which the duration of estrus before may affect fertility remains unclear. This study aimed to determine the effect of different durations of estradiol exposure, prior to progesterone administration, on embryo viability in anestrous recipient mares, and endometrial expression of genes thought to influence embryo survival. Three groups of anestrous recipient mares treated with different duration of estradiol were used: long (LE), short (SE) and no treatment (NE). Day 8 embryos were transferred into recipient mares four days after long-acting progesterone administration and recovered 48h later to examine embryo growth and viability. The endometrial gene expression profile of selected genes was also investigated. The likelihood of recovering an embryo 48h after transfer was 46.1% (6/13), 62.5% (5/8) and 85.7% (6/7) for recipient mares from the NE, SE and LE groups, respectively (P = .09). Embryos recovered from the different groups of recipients did not, however, differ in size, morphology or the proportion of nuclei undergoing mitosis (P > .05). Abundance of mRNA for uterocalin (P19) and insulin-like growth factor 1 (IGF1) were increased in the LE compared to the NE group, while fibroblast growth factor 2 (FGF2), progesterone receptor (PGR) and insulin-like growth factor 1 receptor (IGF1R) transcript abundances were increased (P < 0.05) in the NE group compared to both SE and LE groups. In conclusion, a longer exposure of the endometrium to estradiol before progesterone tended to improve embryo survival within 48h of transfer. However, the grade, growth rate, and proportion of mitotic cells in surviving embryos did not differ among groups. If embryos are destined to fail in a suboptimal endometrial environment, they die and disappear quickly. Moreover, a more adequately estradiol-primed uterus, before the progesterone rise, seems to create a uterine environment, in terms of P19, IGF1, FGF2 and PGR gene expression, more conducive to embryo survival and further development.

HDAC4 Genomic Methylation and Subsequent DNA Binding Following Joint Injury Confers a Sustained Epigenetic Memory of Disrupted Muscle Plasticity

Joint injury is associated with protracted atrophy and weakness that poorly recovers; our objective was to define molecular effectors of lost muscle size, strength, and plasticity following joint injury. We hypothesized that epigenetic imprinting of joint injury in muscle impairs muscle plasticity and contributes to poor functional recovery following surgical repair. We recruited a total of sixteen ACL-injured participants from whom we obtained vastus lateralis muscle biopsies Pre-, 7-days post-, and 4-months post- reconstruction surgery (Sx) and strength measures up to 6-months post-Sx. We performed immunohistochemistry, RNA-seq, reduced representation bisulfite sequencing (RRBS), and HDAC4 chromatin immunoprecipitation sequencing (HDAC4 ChIP-seq) to determine the impact of epigenetics and gene expression on histological profiles, muscle size and muscle strength following injury. Binding and Expression Target Analysis (BETA) of DNA methylation revealed a conserved 985bp regulatory genomic region that was differentially methylated upstream of the HDAC4 promoter. We observed 108 (Pre-Sx), 111 (7d post-Sx), and 110 (4-months post-Sx) differentially methylated promoter sites of which 95 sites were common across all timepoints in the injured limb. Additionally, we observed 1293 differentially methylated intragenic sites within the HDAC4 locus that were also common across all timepoints following injury. Altered HDAC4 methylation was associated with a 2.2 and 4.3 fold change in HDAC4 transcript abundance Pre-Sx and 7d post-Sx, respectively (FDR &lt; 0.05). Notably, HDAC4 binding to critical contractile genes coincided with their downregulation 7d post-Sx (Log2 fold change: ACTA1: -2.02; CKM: -0.97; ATP2A1: -2.34; HOMER2: -1.09; all FDR&lt;0.05). Altered transcript abundance of these genes coincided with lower mean muscle fiber cross-sectional area in the injured limb compared to Healthy (Healthy: 4845 ± 213.4μm2; pre-Sx: 4313 ± 190.6μm2, p&lt;0.05; 7d post-Sx: 3685 ± 228.1μm2, p&lt;0.05; 4-months post-Sx: 3422 ± 142.8μm2, p&lt;0.05) and lower peak torque compared to Healthy (Healthy: 174.8 ± 10.72nm; pre-Sx: 135.2 ± 10.19nm, p&lt;0.05; 4-months post-Sx: 82.64 ± 6.80nm, p&lt;0.05; 6-months post-Sx: 105.9 ± 8.26nm, p&lt;0.05) that was sustained throughout follow-up. Our results suggest that joint injury confers HDAC4 promoter-proximal and intragenic epigenetic imprinting by DNA methylation that increases HDAC4 transcript abundance and HDAC4-DNA binding to represses expression of genes regulating muscle contractile function. This HDAC4 gene regulatory program likely contributes to prolonged deficits in muscle size and strength following joint injury. This work was supported by NIH-NIAMS R01 AR072061. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Deleting the Ribosomal Prolyl Hydroxylase OGFOD1 Leads to Protection in Pressure Overload-Induced Cardiac Hypertrophy in Mice

Molecular changes occurring in heart failure have largely focused on the transcriptional landscape. However, disparities between transcript and protein abundances emphasize the need to understand proteomic changes occurring in heart failure as well the mechanisms governing these changes. Post-translational modifications (PTMs) are a well-known mechanism for regulating protein turnover and activity. A lesser-known enzyme that catalyzes the addition of PTMs on the translational machinery is 2-oxoglutarate- and Fe2+-dependent oxygenase domain-containing protein 1 (OGFOD1). Our published evidence shows OGFOD1 RNA and protein accumulate in human failing hearts, indicating a potential role for this enzyme in heart disease. Because heart failure can develop from many pathologies, including hypertrophy, we investigated the role of OGFOD1 in cardiac hypertrophy. We induced hypertrophy via pharmacological β-adrenergic stimulation by treating wildtype (WT) and OGFOD1-knockout (KO) mice with isoproterenol (ISO), where we found that KO mice were protected from ISO-induced hypertrophy. Based on our findings that OGFOD1-KO mice are protected in ISO-induced cardiac hypertrophy, we tested the hypothesis that KO mice were protected in pressure overload-induced cardiac hypertrophy mediated via transverse aortic constriction (TAC). WT and KO mice were subjected to sham or TAC surgery, and heart weight-to-tibia length (HW/TL) and heart weight-to-body weight (HW/BW) ratios were used to determine extent of hypertrophy. Additionally, hearts were subjected to histological analysis and stained with Masson’s trichrome to assess fibrosis. KO mouse hearts showed significantly less hypertrophy than WT hearts after 2 weeks of TAC according to both HW/TL and HW/BW ratios. KO mice were also protected against cardiac fibrosis. To identify translational changes mediating this protection, future studies will focus on utilizing ribosome profiling to identify actively-translated transcripts whose synthesis is regulated by OGFOD1-mediated prolyl hydroxylation. Results from these studies will provide important mechanistic insight into the therapeutic potential of OGFOD1 in human disease. NHLBI-NIH Intramural Research Program (ZO1-HL002066). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Single Cell RNA-Sequencing Uncover the Molecular Determinants of Sexual Dimorphism in Renal Potassium Regulation

Background: Women are more prone to hypokalemia than men, which exposes them to a higher risk of hypokalemia-induced disorders. We have previously shown the response to potassium (K+) deficiency in mice parallels the human condition, with females exhibiting more severe hypokalemia than males upon dietary K+ deficiency. Here, we investigated the molecular underpinnings of this sexual dimorphism. Methods: The renal adaptations to K+ deficiency were analyzed in Wild-type (WT) male and female mice. Animals were randomized to a control or K+-free diet for 8 days. The transcriptional landscape of control and K+-deprived kidneys was investigated using single nucleus RNA-Seq (snRNA-Seq). At least 5000 nuclei/sample were analyzed. Results: We found female susceptibility to hypokalemia originates from renal K+ loss. Cell type-dependent transcriptome profiling of whole kidneys revealed that all nephron segments undergo transcriptional reprogramming in response to K+ deprivation, with generally more differentially expressed genes (DEGs) in females than males. The proximal tubules (PTs) exhibit major transcriptional reprogramming with 747 DEGs in females compared to 213 DEGs in males. Pathway enrichment analysis of the DEGs in the PT revealed the response to K+ deficiency involves several regulatory mechanisms common to both sexes, such as monocarboxylic acid metabolic pathway and purine-containing compound metabolism. Among all the nephron segments, the connecting tubules (CNTs) exhibit striking sex-dependent changes in response to K+ deprivation with more DEGs in males (100) compared to females (67) despite the less severe hypokalemia in males. Pathway enrichment analysis of the DEGs in the CNT indicated the cytoskeleton and actin organization were mostly reprogrammed in females. In contrast, in male CNT, ion channel regulating pathways exhibited a more active reprogramming. In the CNT, transcript abundance of the Kl gene encoding for Klotho protein, a positive regulator of the K+ exit channel ROMK, was significantly decreased in K+-deprived males only. Similarly, Klotho protein was significantly reduced in response to K+ deprivation, specifically in males but not females. Confocal imaging confirmed decreased ROMK and Klotho in male CNT, consistent with more effcient K+ conservation in males than females. Conclusion: All nephron segments undergo transcriptional reprogramming in response to K+ deprivation, indicating the K+ response is generalized and not restricted to the distal nephron, as previously described. Among all the segments, the CNT exhibits a striking sex-dependent transcriptional reprogramming in response to K+ deficiency. The female susceptibility to hypokalemia results mainly from a less effcient reduction of ROMK-dependent K+ secretion in the CNT, leading to urinary K+ loss. DK054231, DK137331 and CNVA00060589. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Overexpression of the plastidial pseudo-protease AtFtsHi3 enhances drought tolerance while sustaining plant growth.

With climate change, droughts are expected to be more frequent and severe, severely impacting plant biomass and quality. Here, we show that overexpressing the Arabidopsis gene AtFtsHi3 (FtsHi3OE) enhances drought-tolerant phenotypes without compromising plant growth. AtFtsHi3 encodes a chloroplast envelope pseudo-protease; knock-down mutants (ftshi3-1) are found to be drought tolerant but exhibit stunted growth. Altered AtFtsHi3 expression therefore leads to drought tolerance, while only diminished expression of this gene leads to growth retardation. To understand the underlying mechanisms of the enhanced drought tolerance, we compared the proteomes of ftshi3-1 and pFtsHi3-FtsHi3OE (pFtsHi3-OE) to wild-type plants under well-watered and drought conditions. Drought-related processes like osmotic stress, water transport, and abscisic acid response were enriched in pFtsHi3-OE and ftshi3-1 mutants following their enhanced drought response compared to wild-type. The knock-down mutant ftshi3-1 showed an increased abundance of HSP90, HSP93, and TIC110 proteins, hinting at a potential downstream role of AtFtsHi3 in chloroplast pre-protein import. Mathematical modeling was performed to understand how variation in the transcript abundance of AtFtsHi3 can, on the one hand, lead to drought tolerance in both overexpression and knock-down lines, yet, on the other hand, affect plant growth so differently. The results led us to hypothesize that AtFtsHi3 may form complexes with at least two other protease subunits, either as homo- or heteromeric structures. Enriched amounts of AtFtsH7/9, AtFtsH11, AtFtsH12, and AtFtsHi4 in ftshi3-1 suggest a possible compensation mechanism for these proteases in the hexamer.

Ecological intensification strategies increase abundance of denitrifying functional genes in a greenhouse agricultural soil

Understanding the denitrification process in agriculture is key to preventing nitrogen (N) losses and reducing emissions of the greenhouse gas nitrous oxide (N2O) to the atmosphere. Since the type of fertilization influences this process, in this study we evaluated the comparative effect of conventional (CON) versus ecological intensification (EI) soil management under greenhouse conditions on the abundance and expression of the denitrifying functional genes nitrite reductase (nirK, nirS) and nitrous oxide reductase (nosZ). For this purpose, quantitative PCR (qPCR) was performed in three phenological stages of eggplant (Solanum melongena L var. Telma). The results showed that the addition of organic amendments tripled the copies of nirK DNA, doubled the copies of nosZ DNA and increased the copies of nirS, albeit by 2 %, compared to conventionally managed soils. The abundance of mRNA transcripts also increased for all three genes studied: nirK increased 15-fold, followed by nirS, which tripled its expression, and nosZ, which doubled its expression. Ammonium ion (NH4-N) correlated positively with the three genes analyzed, total organic carbon (TOC) with nirK and nosZ, and pH correlated negatively with nirK and nosZ. Regardless of the type of fertilization (CON or EI), a decreasing trend in gene abundance was observed from the initial growth phase of the crop (t0) until the crop was removed and crop residues incorporated into the soil (t2). The results suggest that the dynamics and composition of the denitrifying community varied in response to the type of fertilization and the phenological stage of the crop. Further studies based on metagenomics and transcriptomics, including N2O flux measurements, are needed to better understand the denitrification process in these soils.

Expressing banana transcription factor MaERFVII3 in Arabidopsis confers enhanced waterlogging tolerance and root growth.

Waterlogging poses a significant threat to plant growth and yield worldwide. Identifying the genes responsible for mitigating waterlogging stress is crucial. Ethylene-responsive factors (ERFs) are transcriptional regulators that respond to various biotic and abiotic stresses in plants. However, their roles and involvement in responding to waterlogging stress remain largely unexplored. Hence, this study aimed to elucidate the role of ERFs in enhancing banana plant resilience to waterlogging. We hypothesized that introducing a group VII ERF transcription factor in Arabidopsis could enhance waterlogging stress tolerance. To test this hypothesis, we isolated MaERFVII3 from banana roots, where it exhibited a significant induction in response to waterlogging stress. The isolated MaERFVII3 was introduced into Arabidopsis plants for functional gene studies. Compared with wild-type plants, the MaERFVII3-expressing Arabidopsis showed increased survival and biomass under waterlogging stress. Furthermore, the abundance of transcripts related to waterlogging and hypoxia response showed an elevation in transgenic plants but a decrease in wild-type and empty vector plants when exposed to waterlogging stress. Our results demonstrate the significant contribution of MaERFVII3 to waterlogging tolerance in Arabidopsis, providing baseline data for further exploration and potentially contributing to crop improvement programs.

Differential expression and global analysis of miR156/SQUAMOSA promoter binding-like proteins (SPL) module in oat

SQUAMOSA promoter binding-like proteins (SPLs) are important transcription factors that influence growth phase transition and reproduction in plants. SPLs are targeted by miR156 but the SPL/miR156 module is completely unknown in oat. We identified 28 oat SPL genes (AsSPLs) distributed across all 21 oat chromosomes except for 4C and 6D. The oat- SPL gene family represented six of eight SPL phylogenetic groups, with no AsSPLs in groups 3 and 7. A novel oat miR156 (AsmiR156) family with 21 precursors divided into 7 groups was characterized. A total of 16 AsSPLs were found to be targeted by AsmiR156. Intriguingly, AsSPL3s showed high transcript abundance during early inflorescence (GS-54), as compared to the lower abundance of AsmiR156, indicating their role in reproductive development. Unravelling the SPL/miR156 regulatory hub and alterations in expression patterns of AsSPLs could provide an essential toolbox for genetic improvement in the cultivated oat.