Differences In Gene Expression Research Articles

Water Versus Electrolyte Rehydration on Vocal Fold Osmotic and Oxidative Stress Gene Expression.

Systemic dehydration may induce osmotic and oxidative stress in the vocal folds, but our knowledge of the biology and mitigation with rehydration is limited. The purpose of this experiment was to evaluate whether systemic dehydration induces vocal fold oxidative and osmotic stress and to compare the impact of rehydration by water intake versus electrolyte intake on osmotic and oxidative stress-related gene expression. Four-month-old male Sprague-Dawley rats (N = 32) underwent water restriction. Rehydration was achieved with ad libitum access to water or electrolytes for 24 hours. Rats were divided into four groups: euhydration control, dehydration-only, dehydration followed by either water or electrolyte rehydration (n = 8/group). Gene expression was assessed via RT2 Gene Expression Profiler arrays. With respect to oxidative stress, 10 genes were upregulated and 2 were downregulated after vocal fold dehydration compared with the euhydrated control. Concerning osmotic stress, six genes were upregulated with dehydration only, six genes were upregulated following rehydration with water, whereas a single gene was upregulated with electrolyte rehydration. All genes with significantly different expression between the rehydration groups showed lower expression with electrolytes compared with water. The results support a potential role of oxidative and osmotic stresses in vocal folds related to systemic dehydration. The differences in stress-related gene expression in vocal fold tissue between rehydration with electrolytes or water, albeit modest, suggest that both rehydration options offer clinical utility to subjects experiencing vocal fold dehydration with preliminary evidence that electrolytes may be more effective than water in resolving osmotic stress. NA (prospective animal study) Laryngoscope, 2024.

Identification of new targets for glioblastoma therapy based on a DNA expression microarray

This study provides a comprehensive perspective on the deregulated pathways and impaired biological functions prevalent in human glioblastoma (GBM). In order to characterize differences in gene expression between individuals diagnosed with GBM and healthy brain tissue, we have designed and manufactured a specific, custom DNA microarray. The results obtained from differential gene expression analysis were validated by RT-qPCR. The datasets obtained from the analysis of common differential expressed genes in our cohort of patients were used to generate protein-protein interaction networks of functionally enriched genes and their biological functions. This network analysis, let us to identify 16 genes that exhibited either up-regulation (CDK4, MYC, FOXM1, FN1, E2F7, HDAC1, TNC, LAMC1, EIF4EBP1 and ITGB3) or down-regulation (PRKACB, MEF2C, CAMK2B, MAPK3, MAP2K1 and PENK) in all GBM patients. Further investigation of these genes and enriched pathways uncovered in this investigation promises to serve as a foundational step in advancing our comprehension of the molecular mechanisms underpinning GBM pathogenesis. Consequently, the present work emphasizes the critical role that the unveiled molecular pathways likely play in shaping innovative therapeutic approaches for GBM management. We finally proposed in this study a list of compounds that target hub of GBM-related genes, some of which are already in clinical use, underscoring the potential of those genes as targets for GBM treatment.

Bayesian estimation of gene constraint from an evolutionary model with gene features.

Measures of selective constraint on genes have been used for many applications, including clinical interpretation of rare coding variants, disease gene discovery and studies of genome evolution. However, widely used metrics are severely underpowered at detecting constraints for the shortest ~25% of genes, potentially causing important pathogenic mutations to be overlooked. Here we developed a framework combining a population genetics model with machine learning on gene features to enable accurate inference of an interpretable constraint metric, shet. Our estimates outperform existing metrics for prioritizing genes important for cell essentiality, human disease and other phenotypes, especially for short genes. Our estimates of selective constraint should have wide utility for characterizing genes relevant to human disease. Finally, our inference framework, GeneBayes, provides a flexible platform that can improve the estimation of many gene-level properties, such as rare variant burden or gene expression differences.

Differences in volatile composition and expression of genes involved in terpenoids biosynthesis in Chrysanthemum indicum var. aromaticum

Chrysanthemum indicum var. aromaticum is an important ornamental plant in the genus Chrysanthemum, known for its unique and intense aroma. However, little is known about the composition of aroma components and their molecular regulatory mechanisms, which limits the development and utilization of Ci. var. aromaticum. In this study, headspace solid-phase microextraction (HS-SPME) combined with gas chromatography-mass spectrometry (GC-MS) and gas chromatography-olfactometry (GC-O) were used to analyze the aroma components of different organs (stem, leaf, and flower in different stages) of Ci. var. aromaticum. Meanwhile, the relative expression levels of key genes of terpenoid metabolism in different organs were determined by real-time quantitative PCR (qRT-PCR). Results showed that, a total of 128 volatiles were detected in 5 organs of Ci. var. aromaticum with more than 88.99% of terpenoids proportion, and 38 of the 128 were characteristic aroma compounds. Among the characteristic aroma compounds, 13 of them, including β-eudesmol, β-caryophyllene, zingiberene, α-bergamotene, α-curcumene, germacrene D, δ-cadinene, γ-terpinene, β-bisabolene, α-cubebene, aromandendrene, alloaromadendrene, and eucalyptol, formed the main aroma of Ci. var. aromaticum, and it was suggested that β-caryophyllene, zingiberene, eucalyptol, β-eudesmol, β-pinene, α-muurolene, and isocaryophyllene were responsible for the difference in aroma between the different organs of Ci. var. aromaticum. Combined with metabolomics, it was inferred that terpenoids such as β-eudesmol, cis-sabinol, α-thujone, α-thujene, etc. were involved in mevalonate (MVA) pathway; β-phellandrene, α-copaene, etc. were involved in methylerythritol phosphate (MEP) pathway. This study provided some theoretical guidance for further exploration of the aroma mechanism of Ci. var. aromaticum, which was helpful to the fully development and utilization of Ci. var. aromaticum resources.

Curcumin ameliorates pyroptosis in diabetic seminal vesicles by upregulating TRPV6.

Diabetes damages the seminal vesicle tissues leading to a decrease in seminal fluid secretion, so investigations are ongoing to identify specific therapeutic approaches to address diabetes-induced damage to seminal vesicles. This study investigated the secretory dysfunction of seminal vesicles and how curcumin can ameliorate this dysfunction. First, 40 diabetic males (DM group) and 40 nondiabetic males (control group) underwent seminal vesicle ultrasound evaluation and ejaculate volume measurements. Then, the effects of curcumin on seminal vesicle function were investigated in a diabetic rat model. Fifty 8-week-old SPF-grade SD rats were categorized into five groups: control, DM (diabetes mellitus), low-dose CUR (curcumin 50mg/kg/d), medium-dose CUR (curcumin 100mg/kg/d), and high-dose CUR (curcumin 150mg/kg/d). After a month-long diet with varying curcumin doses, key parameters such as body weight, blood glucose levels, seminal vesicle volume, and seminal fluid secretion were measured. Transcriptome sequencingwas performed to assess differences in gene expression and structural changes in rat seminal vesicle tissues were examined by HE staining. Finally, human seminal vesicle cell lines were cultured and divided into five groups (HG-CON, HG-CUR-5µM, HG-CUR-10µM, HG-CUR-20µM, and HG-CUR-50µM) to measure the fructose levels in the seminal vesicle cell culture fluids and evaluate the expression of CASP1, GSDMD, and TRPV6. Post TRPV6 interference, variations in the gene expression of CASP1, GSDMD, and TRPV6 were monitored. Diabetic patients exhibited a notable reduction in seminal vesicle volume and ejaculate volume compared with the control group, with a direct correlation between the decrease in ejaculate and seminal vesicle volume. Animal studies demonstrated that curcumin supplementation significantly augmented seminal vesicle volume in diabetic rats and notably improved their seminal vesicle secretory dysfunction, particularly in the high-dose curcumin group. Transcriptome sequencing and experimental verification pinpointed the differential expression of TPRV6 and pyroptosis-associated genes (CASP1, GSDMD), with reduced TRPV6 expression but increased markers of pyroptosis (CASP1 and GSDMD) in diabetic rats. Curcumin treatment reversed these effects with an increase in TRPV6 and a decrease in GSDMD and CASP1. Cell transfection experiments indicated that TRPV6 downregulation increased GSDMD and CASP1 gene expression. Curcumin effectively activates TRPV6, thereby diminishing pyroptosis in the seminal vesicle tissues of diabetic rats. This activation not only leads to an increase in the seminal vesicle volume but also significantly ameliorates the seminal vesicle secretory dysfunction in diabetic rats.

Novel Insights into Changes in Gene Expression within the Hypothalamus in Two Asthma Mouse Models: A Transcriptomic Lung–Brain Axis Study

Patients with asthma experience elevated rates of mental illness. However, the molecular links underlying such lung–brain crosstalk remain ambiguous. Hypothalamic dysfunction is observed in many psychiatric disorders, particularly those with an inflammatory component due to many hypothalamic regions being unprotected by the blood–brain barrier. To gain a better insight into such neuropsychiatric sequelae, this study investigated gene expression differences in the hypothalamus following lung inflammation (asthma) induction in mice, using RNA transcriptome profiling. BALB/c mice were challenged with either bacterial lipopolysaccharide (LPS, E. coli) or ovalbumin (OVA) allergens or saline control (n = 7 per group), and lung inflammation was confirmed via histological examination of postmortem lung tissue. The majority of the hypothalamus was micro-dissected, and total RNA was extracted for sequencing. Differential expression analysis identified 31 statistically significant single genes (false discovery rate FDR5%) altered in expression following LPS exposure compared to controls; however, none were significantly changed following OVA treatment, suggesting a milder hypothalamic response. When gene sets were examined, 48 were upregulated and 8 were downregulated in both asthma groups relative to controls. REACTOME enrichment analysis suggests these gene sets are involved in signal transduction metabolism, immune response and neuroplasticity. Interestingly, we identified five altered gene sets directly associated with neurotransmitter signaling. Intriguingly, many of these altered gene sets can influence mental health and or/neuroinflammation in humans. These findings help characterize the links between asthma-induced lung inflammation and the brain and may assist in identifying relevant pathways and therapeutic targets for future intervention.

P-370 The age-associated increase in ovarian stiffness compromises follicle development and induces changes in the follicles’ transcriptome, resulting in poor-quality oocytes

Abstract Study question Does the age-associated increase in ovarian stiffness trigger the decline in oocyte quantity and quality observed in women of advanced maternal age? Summary answer An encapsulated in vitro follicle culture mimicking ovarian biomechanics revealed that age-associated increase in stiffness impacts follicle growth, modulates follicles’ transcriptome and reduces oocyte quality. What is known already Female reproductive aging is characterized by a progressive decline in oocyte quality and quantity, resulting in sub-fertility. We recently discovered that with aging the mouse ovary assumes a pro-fibrotic milieu which induces ovarian stiffness. Moreover, human ovaries also become stiffer with aging; however, it is not known whether this age-associated increase in stiffness impacts ovarian function. The present study aims to examine whether stiffness is a novel mechanism that mediates the age-associated progressive decline in oocyte quality and how stiffness affects the transcriptome of growing follicles, resulting in poor-quality oocytes. Study design, size, duration We used an in vitro alginate-encapsulated follicle culture to mimic young and old environments in which follicles grow. Alginate hydrogel synthesis replicated the soft (0.5% - 1.79±0.08 kPa) and stiff (2% - 4.56±2.03 kPa) environments of young and aged ovaries, respectively. Mouse secondary follicles were cultured in 0.5 or 2% alginate and used for i) long-term culture (day 0-10) to evaluate follicle development and oocyte quality; ii) short-term culture (hour 0-24) to investigate follicles’ transcriptome. Participants/materials, setting, methods During the long-term culture, markers of follicle development were evaluated (n = 31 follicles), including: follicle size, estradiol production (ELISA), cell death (cleaved-caspase3,CC3; immunohistochemistry). At D10, oocyte quality was inspected on isolated oocytes. For the short-term culture experiment, 32 follicles were analyzed by RNAsequencing at 3h (baseline) and 24h. We compared 3h and 24h gene expression (DESeq2,R/Bioconductor) in 0.5% and 2%. Significant differences reported at padj<0.05 and log2FoldChange±2. Two mice strains (CB6F1;CD1) were used in all experiments. Main results and the role of chance Follicles cultured in stiff environment showed a significant reduction in follicle size compared to follicles cultured in soft environment (0.5% 226.9±17.4um, 2% 160.8±9.9um; p < 0.0001). These size differences suggest that granulosa cells are not proliferating in a stiff environment. Estradiol production was reduced in follicles cultured at 2% (0.5% 11.3±15.9ng/ml, 2% 0.3±0.5ng/ml, p = 0.296), while CC3 intensity was increased (CC3 staining intensity/area, 0.5% 20.4±5.8a.u., 2% 27.1±4.3a.u., p < 0.05), suggesting that the stiff environment impacts granulosa cell viability. Oocyte quality significantly declined in follicles cultured in 2%, with 68.9±16.8% of the oocytes degenerated, compared to 23.6±9.2% in 0.5% alginate. The use of two different mice strains yielded comparable results. Using RNAsequencing analysis, we found 1033 differentially expressed genes (DEGs) between 3h and 24h of culture in 2% alginate, mostly involved in apoptosis, inflammation and collagen processes, biological pathways typically associated with aging. The 64% of those DEGs were upregulated at 24h. Conversely, the 1295 DEGs (61% upregulated at 24h) detected comparing 3h and 24h in 0.5% alginate were relate to metabolism, cell cycle and development pathways, biological processes associated to the normal behavior of growing follicles. These differences in gene expression suggest an early-response in the transcriptome of follicles cultured in a stiff environment. Limitations, reasons for caution The stiffness of the alginate gels was based on whole ovary biomechanics; future investigations are needed to measure each follicle-stage-specific stiffness. The changes in follicles’ transcriptome in the stiff environment were evaluated only in short-term culture. This study utilizes in vitro approaches, in vivo modulation of stiffness is currently undergoing. Wider implications of the findings Increased ovarian stiffness is observed in conditions such as aging or ovaries exposed to cancer treatments. Studying how stiffness impacts oocyte quality will allow the development of suitable in vitro systems to support human folliculogenesis and in vivo approaches to modulate biomechanics of the ovary, preserving female fertility. Trial registration number not applicable

P-518 Differentially gene expression identified by RNA seq in aneuploid human blastocysts is more influenced by the phenomenon of aneuploidy than the involved chromosome

Abstract Study question What insights do RNA-Seq profiles provide about gene expression in aneuploid blastocysts with small and large chromosomes compared to euploid blastocysts? Summary answer The difference in gene expression is affected by aneuploidy and is more related to the presence of aneuploidy than the size of the aneuploid chromosomes. What is known already Embryonic aneuploidy is one of the main common causes of implantation failure and miscarriage. RNA-Seq investigations of the embryo in the early stages of preimplantation indicate that with the gradual replacement of the embryonic profile and the reduction of the oocyte residues, the gene expression profile in the blastocyst is more dependent on the embryo’s genome. A recent study by comparing the gene expression profile of euploid blastocysts and specific aneuploidies such as trisomy 21, trisomy 18, XXY and XO syndromes using RNA-Seq, showed how the type of aneuploidy might affect gene expression. Study design, size, duration This study was conducted on surplus embryos of the patients with history of recurrent implantation failure or recurrent miscarriage following PGT by Day-5 trophectoderm biopsy. Inclusion criteria involved maternal age ≤37, normal parental karyotypes, and ovulation by long stimulation protocol. As advanced maternal age and ovarian stimulation method can affect the aneuploidy rate and expression profile, embryos belonging to mothers > 37 years and included only patients with the long stimulation protocol were excluded. Participants/materials, setting, methods The normal embryos and those with single aneuploidy were selected for comparison of gene expression profiles using RNA sequencing. The single aneuploid embryos were classified in two groups: with defined aneuploidy(DA) of small chromosomes (including chromosomes 16, 20, 21, 22), and with undefined aneuploidy(UA) as single aneuploids for other chromosomes. To neutralize the effect of genomic content on gene expression profile in DA group, embryos were pooled reciprocally (trisomy and monosomy for the same chromosome). Main results and the role of chance Principal Component Analysis (PCA) validated embryos classification considering their differentially expressed genes (DEG) clustering into three distinct groups. To minimize the role of chance, only genes with significant adjusted P-values in both STAR and Hisat data analyses were included in consequent interpretations. In addition to comparison between and among the DA, UA and normal groups, the entire set of aneuploid groups was compared with the normal group. The DEG in both aneuploid groups compared to the normal group represented eminent upregulation rather than downregulation. Gene Ontology analyses indicated an upregulation of transcription, ubiquitination, autophagy, mitochondrial, chromosome segregation and DNA repair pathways in both DA and UA compared to normal group. Overexpression of the DNA repair pathway and other validated genes of interest in aneuploid embryos was in agreement with qPCR findings. Translation and protein synthesis pathways also showed overexpression as part of the functional response to aneuploidy. Upregulation of apoptotic process pathway was evident in the UA group. While comparison of gene expression between DA and UA showed overexpression of 143 genes, but cellular lipid synthesis was the only pathway with a significant difference. The distribution of DEG on chromosomes in the DA group was not associated with selected aneuploidies. Limitations, reasons for caution The number of biological replicates in each category was limited in order to provide more confident data by minimizing the time interval between providing the samples and RNA sequencing. Larger studies might change some affected pathways due to differences in identified DEG. Wider implications of the findings This study shed light on the black box of human preimplantation aneuploidy. Considering that the transcriptome is affected by the genomic status and some debates on genomic based current PGT approaches, this study is a step for selection of healthy and viable embryos by revealing the molecular mechanisms. Trial registration number not applicable

P-172 Mitochondria are dysregulated in poor quality oocytes and can be targeted in vitro to alter oocyte quality

Abstract Study question Does mitochondrial gene expression differ between good and poor quality oocytes and can mitochondria be targeted in vitro to alter oocyte quality? Summary answer Mitochondrial-related genes are dysregulated in poor quality oocytes and mitochondrial inhibitors can be used during IVM to alter oocyte quality. What is known already Good oocyte quality is essential for successful pregnancy yet we still have a poor understanding of the optimal composition of a good egg. Mitochondrial number and activity are important for oocyte quality and during maturation the ultrastructure of the mitochondria changes to a hooded or ring form. We have observed differences in the timing of this morphological change between good and poor quality oocytes in a sheep model. Oligomycin treatment of oocytes decreases the proportion of hooded mitochondria and increases mitochondrial membrane potential (MMP) indicating that hooded mitochondria may form during maturation to reduce mitochondrial activity prior to fertilisation. Study design, size, duration Gene expression differences were compared between poorer quality peripubertal lamb oocytes and adult oocytes harvested during estrus from pools of ∼80 abattoir-derived ovaries on three separate dates for each age. Denuded oocytes (GV stage) were frozen in three pools of ∼20 per age and total RNA isolated. Sheep oocytes (∼30 per group) were treated with oligomycin, FCCP or vehicle control in final 2 hours of IVM on five separate dates, fertilised and cultured. Participants/materials, setting, methods RNA sequencing cleaned reads were mapped using HISAT2 on sheep genome version ARS-UI_Ramb_v2. Count matrix were generated using featureCount and differential gene expression was performed using DESeq2. After normalisation, Wald test was used to define genes differentially expressed. Genes were considered significantly altered with a FDR < 5% after Benjamini-Hochberg correction. Mean cleavage rate was compared to vehicle control in five replicate experiments using one-way ANOVA with Benjamini, Krieger and Yekutieli FDR < 5%. Main results and the role of chance Oligomycin (ATP synthase inhibitor) decreased cleavage rate compared to control (P < 0.001) while FCCP (electron transport chain uncoupler) had no effect. There were 163 genes up-regulated and 194 genes down-regulated in lamb GV oocytes compared to adult GV stage (FDR <5%). Metascape pathway analysis of the up-regulated genes in lamb oocytes showed the most enriched pathway was Thermogenesis which included these genes: ACOT9, AJUBA, ATP5ME, ATP5MF, CALML4, CREB3L3, CRPPA, DNAJC15, DPM3, FMC1, LDHA, NDUFA12, NDUFAF2, NDUFS5, NT5C, PHGDH, POLR2G, PPARG, PRPS1L1, SDHAF3, SLIRP, TIMM8B and UBB. Six of these up-regulated genes were in the top 20 most highly expressed genes. Thermogenesis is the process by which the mitochondrial electron transport chain becomes uncoupled to dissipate heat and may also control production of free radicals by mitochondria and their response to oxidants. These genes and other mitochondrial related genes that were up-regulated in lamb GV oocytes (CKS2, DCN, PCP4L1, SLC51B) are involved in formation of the electron transport chain complexes, oxidative phosphorylation, ATP synthesis, mitochondrial organization and regulation of membrane potential. Together these results indicate that mitochondrial function is dysregulated in poorer quality oocytes and that treatment with mitochondrial inhibitors during IVM can alter oocyte quality. Limitations, reasons for caution This study was carried out in sheep oocytes and results would need to be confirmed in human oocytes if possible. Further research is needed to determine if decreasing MMP can improve oocyte quality and embryo development. Wider implications of the findings This animal model study indicates that mitochondria may be targeted during IVM to alter oocyte quality and this knowledge could be applied to develop ways of increasing human IVM and ART success rates. Trial registration number not applicable

Sex differences in intestinal morphology and increase in diencephalic neuropeptide Y gene expression in female but not male Pekin ducks exposed to chronic heat stress.

The impact of heat stress (HS) on production is intricately linked with feed intake. We investigated the effects of HS on intestines and diencephalic genes in Pekin ducks. One hundred and sixty adult ducks were allocated to two treatment rooms. The control room was maintained at 22°C and the HS room at 35°C for the first 10 h of the day then reduced to 29.5°C. After 3 weeks, 10 hens and 5 drakes were euthanized from each room and jejunum and ileum collected for histology. Brains were collected for gene expression analysis using qRT-PCR. Intestinal morphology data were analyzed with two-way ANOVA and diencephalic gene data were analyzed with Kruskal-Wallis test. There was an increase in villi width in the ileum (p = .0136) and jejunum (p = .0019) of HS hens compared to controls. HS drakes showed a higher crypt depth (CD) in the jejunum (p = .0198) compared to controls. There was an increase in crypt goblet cells (GC) count in the ileum (p = .0169) of HS drakes compared to HS hens. There was higher villi GC count (p = .07) in the jejunum of HS drakes compared to controls. There was an increase in the crypt GC density (p = .0054) in the ileum, not jejunum, of HS drakes compared to HS hens. Further, there were no differences in the proopiomelanocortin gene expression in either sex but there was an increase in the expression of neuropeptide Y (NPY) gene in HS hens (p = .031) only and a decrease in the corticotropin releasing hormone gene in the HS drakes (p = .037) compared to controls. These data show that there are sex differences in the effect of HS on gut morphology while the upregulation in NPY gene may suggest a role in mediating response to chronic HS.

P-401 Aging affects endometrial receptivity factors. Can DHEA modulate this age-related effect?

Abstract Study question Identification of age-related gene expression differences of the human endometrium during its receptive period – Dehydroepiandrosterone (DHEA) effects. Summary answer Decrease in expression of OPN and HOXA10, receptivity markers, between increasing female age groups in primary human endometrial cells was presented. DHEA altered gene expression. What is known already The increased infertility noted in the world population, has been related to increasing maternal age. Our previous work points out the role of women’s age in endometrial cell function. Endometrial proliferation and differentiation are two important steps that lead to increased cell receptivity during the window of implantation. Optimal receptivity is achieved also through changes inexpression of several factors throughout the endometrial cycle. It is well known that androgen levels, in the female reproductive system, decrease with increasing age.Therefore, DHEA an androgen precursor, found to be reduced with age is thought to be a candidate for infertility treatment. Study design, size, duration Maternal age has not been previously directly correlated with endometrial receptivity factors. We studied 39 endometrial samples divided in three different women’s age groups (25-35 years, 36-40 years and 41-46years) and changes in the mRNA and protein expression of osteopontin (OPN), CD44 and HOXA10 in human endometrium stromal cells were analyzed, in vitro. DHEA was added on stromal cells (decidualized or not) in order to observe changes in mRNA expression. Participants/materials, setting, methods Pipelle endometrial biopsies (secretory phase) were collected from ovulating, fertile, Caucasian women volunteers aged 25–46 years. Stromal cells were isolated. These samples were divided into three age groups: 25-35 years, 36-40 years and 41-46 years. Protein and mRNA expression of receptivity modulators OPN, CD44 and HOXA10 were analyzed using Real-time PCR and Western blot analysis, respectively. Stromal cells were decidualized in vitrousing bromo-8-cAMP in order to observe DHEA effects. Main results and the role of chance Data presented showed a statistically significant decrease (p ≤ 0.05) in OPN expression levels with increasing age groups. Women included in age group of 41-45y presented a significant downregulation of OPN expression compared to younger age group 25-35y (p ≤ 0.01).CD44 expressiondid not differ with age. Furthermore, a statistically significant decrease (p ≤ 0.05) was evident in HOXA10 expression levels with increasing age. Women belonging to the age group 41-45y expression presented with significant HOXA10 downregulation compared to younger age group 25-35y (p ≤ 0.05). Analysis of OPN protein levels showed that cell-extracted OPN decreased from theage of 33y, while the secreted protein gradually decreased from the age of 38y. CD44 protein expression, similarly to the mRNA expression did not show differences with age. HOXA10 protein expression was found to be decreased mainly after the age of41y. DHEA changed, with statistical significance, the expression of decidualization marker IGFBP and OPN in decidualized stromal cells. Overall, our results support our hypothesis as expected, but we must also consider specific cases where older women have the same results as younger ones and the opposite, so factors that are specific for each individual must be further investigated. Limitations, reasons for caution It must be pointed out that there is a plethora of characterized molecules like cytokines and immune cells that also participate in the optimal receptivity status of the endometrium – leading to a successful implantation procedure – that future studies need to examine, as well, in relation to women’s age. Wider implications of the findings Human endometrial aging can affect embryo receptivity markers and therefore, receptivity. Further investigation of receptivity factors that change with the age could be a useful pool in identifying targets for diagnosis and therapy of human implantation failure. Also, DHEA must be further investigated as a tool for infertility therapy. Trial registration number not applicable

Preliminary Analysis of Genetic Markers for Functional Ethanol Tolerance in Honey Bees (Apis mellifera) Using a Free-Flying Paradigm

Honey bees are a commonly used species for alcohol research due to their genome being fully sequenced, their behavioral changes following consumption, and their preference for alcohol. The purpose of this article is to provide a preliminary examination of the genetic expression of heat shock protein 70 (HSP70) and big potassium ion channel protein (BKP) in honey bees following the consumption of either 0%, 2.5%, 5%, or 10% ethanol (EtOH) solutions. The foraging behaviors of the bees were observed and recorded through their return and drinking times. There were significant differences in the return and drinking times between some of the groups. The bees in the 10% condition took significantly longer to return compared to the other groups. Additionally, the bees in the 5% group spent significantly more time drinking compared to the bees in the control (0%) group. There were no significant differences in HSP70 or BKP between the different ethanol groups. Cumulatively, these findings suggest that, while bees may exhibit behavioral differences, the differences in gene expression may not be observed at the transcriptional level.

Brain regions differences in amyloid-β and gene expression in early APP/PS1 mice and identification of Npas4 as a key molecule in Alzheimer's disease.

Distinct brain regions are differentially affected during the various stages of Alzheimer's disease (AD). While the hippocampus and cortex are known to play significant roles, the involvement of the cerebellum has received less attention. Understanding the changes in diverse brain regions is essential to unravel the neuropathological mechanism in early-stage AD. Our research aimed to explore and compare amyloid-β (Aβ) pathology and gene expression profiles across the hippocampus, cortex, and cerebellum in the early stages of the Amyloid Precursor Protein/Presenilin-1 (APP/PS1) mouse model. By 7 months of age, significant Aβ plaque accumulation was observed in the hippocampus and cortex of APP/PS1 mice, while no such deposits were found in the cerebellum. Gene expression analysis revealed predominant effects on immune response pathways in the hippocampus and cortex. Even in the absence of Aβ deposition, notable gene expression changes were observed in the cerebellum of APP/PS1 mice. Intriguingly, Neuronal PAS Domain protein 4 (Npas4) expression was consistently down-regulated across all brain regions, independent of Aβ plaque presence. Our findings reveal distinct transcriptomic alterations and Aβ pathology in select cerebral regions during the initial phase of AD. Notably, the diminished expression of the Npas4 across three brain regions implies that Npas4 could play a pivotal role in the early pathogenesis of AD.

Combining non-invasive liquid biopsy and a methylation analysis to assess surgical risk for early esophageal cancer.

While the widespread use of endoscopic submucosal dissection (ESD) has significantly reduced the incidence of early esophageal cancer (ESCA), the limited ability of ESD to strip deep infiltrating esophageal lesions results in a considerable risk of intraoperative perforation. Circulating-free DNA (cfDNA) is widely used in modern tumor screening due to its non-invasive detection capabilities. A methylation analysis offers vital insights into the condition and advancement of malignancies due to its unique positioning, such as a marker of cancer. This study investigated the potential of combining a non-invasive liquid biopsy technique, along with a methylation analysis, to assess the surgical perforation risk of ESCA patients. In this study, we conducted an analysis of gene expression differences between stage I esophageal squamous carcinoma samples and healthy tissue samples using data from The Cancer Genome Atlas (TCGA) database. We also identified the genes associated with progression-free survival (PFS) in esophageal squamous carcinoma. Integrating the framework of the methylation analysis, we explored the methylated sites of these distinct genes. To refine this process, we used the Shiny Methylation Analysis Resource Tool (SMART) to conduct a comprehensive analysis of these sites. We then confirmed the stability of the methylation sites in different lesion conditions using methylation-specific quantitative polymerase chain reaction (MS-qPCR) with paraffin tissue samples collected after ESD. We analyzed RNA-sequencing data from 42 early stage ESCA patients and 17 controls, identifying 1,263 up-regulated and 460 down-regulated genes. Functional analyses revealed involvement in key pathways such as cell cycle regulation and immune responses. Furthermore, we identified 38 differentially expressed genes associated with PFS. Using SMART analysis, we found 217 hyper-methylated regions in 38 genes, suggesting potential early markers for ESCA. Validation experiments confirmed the reliability of 29 hyper-methylated regions in FFPE tissue samples and 6 regions in cfDNA. A LunaCAM model showed high accuracy [area under the curve (AUC) =0.89] in discriminating early ESCA. Integrated assessment of six highly methylated regions significantly improved predictive performance, with 90.56% sensitivity, highlighting the importance of combinatorial biomarker evaluation for early cancer detection. This study established a novel approach that integrates non-invasive testing with a methylation analysis to assess the surgical risk of early ESCA patients. The significance of changes in methylation sites in relation to lesion status should not be underestimated, as they have the potential to offer vital insights for proactive risk assessments before surgery.

A machine learning and drug repurposing approach to target ferroptosis in colorectal cancer stratified by sex and KRAS.

The landscape of sex differences in Colorectal Cancer (CRC) has not been well characterized with respect to the mechanisms of action for oncogenes such as KRAS. However, our recent study showed that tumors from male patients with KRAS mutations have decreased iron-dependent cell death called ferroptosis. Building on these findings, we further examined ferroptosis in CRC, considering both sex of the patient and KRAS mutations, using public databases and our in-house CRC tumor cohort. Through subsampling inference and variable importance analysis (VIMP), we identified significant differences in gene expression between KRAS mutant and wild type tumors from male patients. These genes suppress (e.g., SLC7A11 ) or drive (e.g., SLC1A5 ) ferroptosis, and these findings were further validated with Gaussian mixed models. Furthermore, we explored the prognostic value of ferroptosis regulating genes and discovered sex- and KRAS-specific differences at both the transcriptional and metabolic levels by random survival forest with backward elimination algorithm (RSF-BE). Of note, genes and metabolites involved in arginine synthesis and glutathione metabolism were uniquely associated with prognosis in tumors from males with KRAS mutations. Additionally, drug repurposing is becoming popular due to the high costs, attrition rates, and slow pace of new drug development, offering a way to treat common and rare diseases more efficiently. Furthermore, increasing evidence has shown that ferroptosis inhibition or induction can improve drug sensitivity or overcome chemotherapy drug resistance. Therefore, we investigated the correlation between gene expression, metabolite levels, and drug sensitivity across all CRC primary tumor cell lines using data from the Genomics of Drug Sensitivity in Cancer (GDSC) resource. We observed that ferroptosis suppressor genes such as DHODH , GCH1 , and AIFM2 in KRAS mutant CRC cell lines were resistant to cisplatin and paclitaxel, underscoring why these drugs are not effective for these patients. The comprehensive map generated here provides valuable biological insights for future investigations, and the findings are supported by rigorous analysis of large-scale publicly available data and our in-house cohort. The study also emphasizes the potential application of VIMP, Gaussian mixed models, and RSF-BE models in the multi-omics research community. In conclusion, this comprehensive approach opens doors for leveraging precision molecular feature analysis and drug repurposing possibilities in KRAS mutant CRC.

Spatial transcriptomics combined with single-nucleus RNA sequencing reveals glial cell heterogeneity in the human spinal cord.

Glial cells play crucial roles in regulating physiological and pathological functions, including sensation, the response to infection and acute injury, and chronic neurodegenerative disorders. Glial cells include astrocytes, microglia, and oligodendrocytes in the central nervous system, and satellite glial cells and Schwann cells in the peripheral nervous system. Despite the greater understanding of glial cell types and functional heterogeneity achieved through single-cell and single-nucleus RNA sequencing in animal models, few studies have investigated the transcriptomic profiles of glial cells in the human spinal cord. Here, we used high-throughput single-nucleus RNA sequencing and spatial transcriptomics to map the cellular and molecular heterogeneity of astrocytes, microglia, and oligodendrocytes in the human spinal cord. To explore the conservation and divergence across species, we compared these findings with those from mice. In the human spinal cord, astrocytes, microglia, and oligodendrocytes were each divided into six distinct transcriptomic subclusters. In the mouse spinal cord, astrocytes, microglia, and oligodendrocytes were divided into five, four, and five distinct transcriptomic subclusters, respectively.The comparative results revealed substantial heterogeneity in all glial cell types between humans and mice. Additionally, we detected sex differences in gene expression in human spinal cord glial cells. Specifically, in all astrocyte subtypes, the levels of NEAT1 and CHI3L1 were higher in males than in females, whereas the levels of CST3 were lower in males than in females. In all microglial subtypes, all differentially expressed genes were located on the sex chromosomes. In addition to sex-specific gene differences, the levels of MT-ND4, MT2A, MT-ATP6, MT-CO3, MT-ND2, MT-ND3, and MT-CO2 in all spinal cord oligodendrocyte subtypes were higher in females than in males. Collectively, the present dataset extensively characterizes glial cell heterogeneity and offers a valuable resource for exploring the cellular basis of spinal cord-related illnesses, including chronic pain, amyotrophic lateral sclerosis, and multiple sclerosis.

Exosomes from umbilical cord mesenchymal stromal cells promote the collagen production of fibroblasts from pelvic organ prolapse

BACKGROUND Pelvic organ prolapse (POP) involves pelvic organ herniation into the vagina due to pelvic floor tissue laxity, and vaginal structure is an essential factor. In POP, the vaginal walls exhibit abnormal collagen distribution and decreased fibroblast levels and functions. The intricate etiology of POP and the prohibition of transvaginal meshes in pelvic reconstruction surgery present challenges in targeted therapy development. Human umbilical cord mesenchymal stromal cells (hucMSCs) present limitations, but their exosomes (hucMSC-Exo) are promising therapeutic tools for promoting fibroblast proliferation and extracellular matrix remodeling. AIM To investigate the effects of hucMSC-Exo on the functions of primary vaginal fibroblasts and to elucidate the underlying mechanism involved. METHODS Human vaginal wall collagen content was assessed by Masson’s trichrome and Sirius blue staining. Gene expression differences in fibroblasts from patients with and without POP were assessed via RNA sequencing (RNA-seq). The effects of hucMSC-Exo on fibroblasts were determined via functional experiments in vitro . RNA-seq data from fibroblasts exposed to hucMSC-Exo and microRNA (miRNA) sequencing data from hucMSC-Exo were jointly analyzed to identify effective molecules. RESULTS In POP, the vaginal wall exhibited abnormal collagen distribution and reduced fibroblast 1 quality and quantity. Treatment with 4 or 6 μg/mL hucMSC-Exo suppressed inflammation in POP group fibroblasts, stimulated primary fibroblast growth, and elevated collagen I (Col1) production in vitro . High-throughput RNA-seq of fibroblasts treated with hucMSC-Exo and miRNA sequencing of hucMSC-Exo revealed that abundant exosomal miRNAs downregulated matrix metalloproteinase 11 (MMP11) expression. CONCLUSION HucMSC-Exo normalized the growth and function of primary fibroblasts from patients with POP by promoting cell growth and Col1 expression in vitro . Abundant miRNAs in hucMSC-Exo targeted and downregulated MMP11 expression. HucMSC-Exo-based therapy may be ideal for safely and effectively treating POP.

Single-cell transcriptomic analysis identifies downregulated phosphodiesterase 8B as a novel oncogene in IDH-mutant glioma.

Glioma, a prevalent and deadly brain tumor, is marked by significant cellular heterogeneity and metabolic alterations. However, the comprehensive cell-of-origin and metabolic landscape in high-grade (Glioblastoma Multiforme, WHO grade IV) and low-grade (Oligoastrocytoma, WHO grade II) gliomas remains elusive. In this study, we undertook single-cell transcriptome sequencing of these glioma grades to elucidate their cellular and metabolic distinctions. Following the identification of cell types, we compared metabolic pathway activities and gene expressions between high-grade and low-grade gliomas. Notably, astrocytes and oligodendrocyte progenitor cells (OPCs) exhibited the most substantial differences in both metabolic pathways and gene expression, indicative of their distinct origins. The comprehensive analysis identified the most altered metabolic pathways (MCPs) and genes across all cell types, which were further validated against TCGA and CGGA datasets for clinical relevance. Crucially, the metabolic enzyme phosphodiesterase 8B (PDE8B) was found to be exclusively expressed and progressively downregulated in astrocytes and OPCs in higher-grade gliomas. This decreased expression identifies PDE8B as a metabolism-related oncogene in IDH-mutant glioma, marking its dual role as both a protective marker for glioma grading and prognosis and as a facilitator in glioma progression.

Understanding the heterogeneity in liver hepatocellular carcinoma with a special focus on malignant cell through single-cell analysis

IntroductionHepatocellular carcinoma (HCC) is the most common form of liver cancer globally and remains a major cause of cancer-related deaths. HCC exhibits significant intra-tumoral and interpatient heterogeneity, impacting treatment efficacy and patient prognosis.MethodsWe acquired transcriptome data from the TCGA and ICGC databases, as well as liver cancer chip data from the GEO database, and processed the data for subsequent analysis. We also obtained single cell data from the GEO database and performed data analysis using the Seurat package. To further investigate epithelial cell subgroups and their copy number variations, we used the Seurat workflow for subgroup classification and the InferCNV software for CNV analysis, utilizing endothelial cells as a reference. Pseudo-time analysis and transcription factor analysis of epithelial cells were performed using the monocle2 and SCENIC software, respectively. To assess intercellular communication, we employed the CellChat package to identify potential ligand-receptor interactions. We also analyzed gene expression differences and conducted enrichment analysis using the limma and clusterProfiler packages. Additionally, we established tumor-related risk characteristics using Cox analysis and Lasso regression, and predicted immunotherapy response using various datasets.ResultsThe samples were classified into 23 clusters, with malignant epithelial cells being the majority. Trajectory analysis revealed the differentiation states of the malignant epithelial cells, with cluster 1 being in the terminal state. Functional analysis revealed higher aggressiveness and epithelial-mesenchymal transition (EMT) scores in cluster 1, indicating a higher propensity for metastasis. RBP4+ tumor cells were highly enriched with hypoxia process and intensive cell-to-cell communication. A prognostic model was established, and immune infiltration analysis showed increased infiltration in the high-risk group. TP53 demonstrated significant differences in mutation rate between the two risk groups. Validation analysis confirmed the up-regulation of model genes, including AKR1B10, ARL6IP4, ATP6V0B, and BSG in tumor tissues.ConclusionA prognostic model was established based on HCC malignant cell associated gene signature, displaying decent prognosis guiding effectiveness in the multiple cohorts. The study provided comprehensive insights into the heterogeneity and potential therapeutic targets of LIHC.

Altering CO2 Laser Pulse Energy Settings Did Not Influence Differential Gene Transcription in Normal Skin in a Red DurocPig Model.

Fractional ablative CO2 lasers are used clinically to treat cutaneous burn scars with reported varying degrees of effectiveness. It was hypothesized that different laser pulse energy settings may lead to differential gene transcription in a porcine model. Uninjured skin from red Duroc pigs was treated with a fractional ablative CO2 laser set to 70, 100, or 120 mJ across the abdomen (n = 4 areas per treatment). Punch biopsies of both treated and untreated sites were taken before treatment (baseline), at 30 min, and at each hour for 6 h and stored in All-Protect tissue reagent. The biopsies were then used to isolate RNA, which was subsequently used in qRT-PCR for eight genes associated with wound healing and the extracellular matrix: CCL2, IL6, FGF2, TIMP1, TIMP3, COL1A2, MMP2, andDCN. RPL13a was used as a housekeeping gene to normalize the eightgenes of interest. One-way ANOVA tests were used to assess for differences among laser pulse energies and two-way ANOVA tests were used to assess the differences between treated and untreated areas. While six of the eight genes were upregulated after treatment (p < 0.05), there were no significant differences in gene expression between the different laser pulse energies for any of the eight genes. While laser treatment is correlated with a positive and significant upregulation for six of the eight genes 4 h after intervention, the pulse energy settings of the laser did not lead to a statistically significant difference in gene transcription among the treatment areas. Different laser pulse energies may not be required to induce similar cellular responses in a clinical setting.