CRISPR-Cas9 mediated d3GHR knockout in HEK293 cells: Revealing the longevity associated isoform stress resilience.

The Growth Hormone Receptor (GHR) gene encodes a protein that is essential for mediating the biological effects of growth hormone (GH). A series of molecular events are set off when GH binds to its receptor, resulting in a variety of physiological reactions linked to development, growth, and metabolism. Recently a particular genetic variation, within the GHR gene that is labeled as the "d3GHR," which lacks exon 3 was associated with longevity. This specific deletion isoform was connected to changes in the structure of the GHR protein, which may have an impact on the GHR's function. To test in vitro the advantage of the d3 carrier that may link to longevity, we employed the CRISPR/Cas9 technique to produce two isoforms: the homozygotes isoform (d3/d3) and the heterozygotes isoform (d3/fl) using HEK293 cell line. The CRISPR editing effectiveness was >85 %, indicating that we had successfully built the Cas9-gRNA complex that is appropriate for the GHR gene. The viability of the resulted isoform cells was examined under three environmental stressors that mimic some aging processes. In addition, we examined the GHR signaling pathway by selecting potential downstream genes in the GHR signaling cascade. The results show that heterozygotes cells demonstrated higher survival rates under UV radiation compared with the WT cells (87 % compared with 67 % for the WT cells when exposed to 2 min of UV radiation), and in fasting conditions, the d3GHR cells showed a 15 % greater viability than the WT cells. Moreover, the baseline expression levels (without intervention) of the IGF1 and JAK/STAT genes signaling pathways significantly declined in the homozygotes cells compared with the WT (p < 0.05). This noteworthy finding might offer a practical approach to test illness prevention and give the scientific community critical new insights on mechanism associated with lifespan.

The d3GHR carrier epigenome in Druze clan longevity.

The Druze are a distinct group known for their close community, traditions, and consanguineous marriages, dating back to the eleventh century. This practice has led to unique genetic variations, impacting both pathology and gene-associated phenotypes. Some Druze clans, particularly those with exceptional long-lived family heads (ELLI), attracted attention. Given that the bulk of these ELLI were men, the d3GHR polymorphism was the first obvious possibility. Among the 73 clan members, 8.2% carried the d3GHR isoform, with nearly 11% being males. There was a significant age-related increase (p = 0.04) in this isoform among males, leading to examination of potential environmental mediators affecting gene regulation among these carriers during life (namely epigenetic). We focused on DNA methylation due to its crucial role in gene regulation, development, and disease progression. We analyzed DNA samples from 14 clan members with different GHR genotypes, finding a significant (p < 0.05) negative correlation between DNA methylation levels and age. Employing a biological age clock, we observed a significant + 4.229 years favoring the d3GHR group over the WT and heterozygous groups. In conclusion, this study highlights the advantage of d3GHR carriers among this unique Druze clan and underscores the importance of genotype-environment interaction in epigenetic regulation and its impact on health.

Genome integrity as a potential index of longevity in Ashkenazi Centenarian's families.

The aging process, or senescence, is characterized by age-specific decline in physical and physiological function, and increased frailty and genomic changes, including mutation accumulation. However, the mechanisms through which changes in genomic architecture influence human longevity have remained obscure. Copy number variants (CNVs), an abundant class of genomic variants, offer unique opportunities for understanding age-related genomic changes. Here we report the spectrum of CNVs in a cohort of 670 Ashkenazi Jewish centenarians, their progeny, and unrelated controls. The average ages of these groups were 97.4 ± 2.8, 69.2 ± 9.2, and 66.5 ± 7.0 respectively. For the first time, we compared different size classes of CNVs, from 1 kB to 100 MB in size. Using a high-resolution custom Affymetrix array, targeting 44,639 genomic regions, we identified a total of 12,166, 22,188, and 10,285 CNVs in centenarians, their progeny, and control groups, respectively. Interestingly, the offspring group showed the highest number of unique CNVs, followed by control and centenarians. While both gains and losses were found in all three groups, centenarians showed a significantly higher average number of both total gains and losses relative to their controls (p < 0.0327, 0.0182, respectively). Moreover, centenarians showed a lower total length of genomic material lost, suggesting that they may maintain superior genomic integrity over time. We also observe a significance fold increase of CNVs among the offspring, implying greater genomic integrity and a putative mechanism for longevity preservation. Genomic regions that experienced loss or gains appear to be distributed across many sites in the genome and contain genes involved in DNA transcription, cellular transport, developmental pathways, and metabolic functions. Our findings suggest that the exceptional longevity observed in centenarians may be attributed to the prolonged maintenance of functionally important genes. These genes are intrinsic to specific genomic regions as well as to the overall integrity of the genomic architecture. Additionally, a strong association between longer CNVs and differential gene expression observed in this study supports the notion that genomic integrity could positively influence longevity.

The Exon 3-Deleted Growth Hormone Receptor (d3GHR) Polymorphism-A Favorable Backdoor Mechanism for the GHR Function.

Growth hormone (GH) is a peptide hormone that plays a crucial role in controlling growth, development, and lifespan. Molecular regulation of GH is accomplished via the GH receptor (GHR), which is the main factor influencing human development and is essential to optimal functioning of the GH/IGF-I axis. Two GHR isoforms have been studied, according to the presence (flGHR) or absence (d3GHR) of exon 3. The d3GHR isoform, which lacks exon 3 has recently been related to longevity; individuals carrying this isoform have higher receptor activity, improved signal transduction, and alterations in the treatment response and efficacy compared with those carrying the wild type (WT) isoform (flGHR). Further, studies performed in patients with acromegaly, Prader-Willi syndrome, Turner syndrome, small for gestational age (SGA), and growth hormone deficiency (GHD) suggested that the d3GHR isoform may have an impact on the relationship between GH and IGF-I levels, height, weight, BMI, and other variables. Other research, however, revealed inconsistent results, which might have been caused by confounding factors, including limited sample sizes and different experimental methods. In this review, we lay out the complexity of the GHR isoforms and provide an overview of the major pharmacogenetic research conducted on this ongoing and unresolved subject.

Copy number variation as a tool for implementing pregnancy as an aging model.

Copy number variations (CNV) are a major contributor to genome variability and have been linked to aging and other degradable phenotypes such as pregnancy physiology. To demonstrate how pregnancy can be used as a model of aging, we used CNVs from pregnant mice. Candidate CNVs were selected by applying case-control analysis in human centenarians compared with control groups. These CNVs were aligned with the mouse genome and their copy variation was assessed using qRT-PCR in liver and blood tissue samples from pregnant mice throughout pregnancy (baseline; first, second, and third trimester; post-partum). Eight of the ten selected CNVs demonstrated a significant decline/increase trend throughout the pregnancy followed by opposite direction soon after delivery in the liver and blood of the mouse tissues. Furthermore, significant differential expression was detected among the candidate CNVs' close vicinity genes (APA2A, LSS, RBDHF1, PLAAT1, and SCL17A2), but not in the WSCD2 gene. Establishing a genetic link between longevity and pregnancy is a significant step toward implementing the pregnancy process as a model for aging. These results in pregnant mice highlight the mechanism and similarities between pregnancy and aging. Investigating the mechanisms that cause such rejuvenation after labor could change our aging treatment paradigm.

Can Epigenetics Predict Drug Efficiency in Mental Disorders?

Psychiatric disorders affect millions of individuals and their families worldwide, and the costs to society are substantial and are expected to rise due to a lack of effective treatments. Personalized medicine-customized treatment tailored to the individual-offers a solution. Although most mental diseases are influenced by genetic and environmental factors, finding genetic biomarkers that predict treatment efficacy has been challenging. This review highlights the potential of epigenetics as a tool for predicting treatment efficacy and personalizing medicine for psychiatric disorders. We examine previous studies that have attempted to predict treatment efficacy through epigenetics, provide an experimental model, and note the potential challenges at each stage. While the field is still in its infancy, epigenetics holds promise as a predictive tool by examining individual patients' epigenetic profiles in conjunction with other indicators. However, further research is needed, including additional studies, replication, validation, and application beyond clinical settings.

Damage-Free Shortening of Telomeres Is a Potential Strategy Supporting Blind Mole-Rat Longevity.

Telomere shortening or loss of shelterin components activates DNA damage response (DDR) pathways, leading to a replicative senescence that is usually coupled with a senescence-associated secretory phenotype (SASP). Recent studies suggested that telomere aberration that activates DDR may occur, irrespective of telomere length or loss of shelterin complex. The blind mole-rat (Spalax) is a subterranean rodent with exceptional longevity, and its cells demonstrate an uncoupling of senescence and SASP inflammatory components. Herein, we evaluated Spalax relative telomere length, telomerase activity, and shelterin expression, along with telomere-associated DNA damage foci (TAFs) levels with cell passage. We show that telomeres shorten in Spalax fibroblasts similar to the process in rats, and that the telomerase activity is lower. Moreover, we found lower DNA damage foci at the telomeres and a decline in the mRNA expression of two shelterin proteins, known as ATM/ATR repressors. Although additional studies are required for understanding the underling mechanism, our present results imply that Spalax genome protection strategies include effective telomere maintenance, preventing early cellular senescence induced by persistent DDR, thereby contributing to its longevity and healthy aging.

Exposure to Juvenile Stress Induces Epigenetic Alterations in the GABAergic System in Rats.

Epigenetics is a gene-environment interaction mechanism, manifested mostly through changes in regulatory gene expression. Stress is an established environmental factor known to induce epigenetic changes. This study aimed to assess the long-term effect of stress as juveniles, or juvenile and adult stress, on alterations in glutamic acid decarboxylase genes (GAD65, GAD67). We assessed DNA methylation and RNA expression in four rat groups: (1) control group, (2) juvenile stress group sacrificed two days following stress exposure (JSe) (RNA only), (3) juvenile stress group sacrificed as adults (JS), and (4) juvenile and adult stress group (JS + AS). Three different areas of the brain were examined in each group: the dorsal dentate gyrus (dDG), the dorsal CA1 (dCA1), and the basolateral amygdala (BLA). A significantly low methylation level of GAD65 in the BLA was observed among the JS group, followed by almost complete recovery among the JS + AS group. However, in dDG, an opposite trend was captured, and higher GAD65 methylation was found in JS. In addition, RNA levels were found to be decreased in JS compared to JSe and JS + AS. These findings can point to a possible mechanism: while juvenile stress may enhance a better coping strategy with life challenges, additional stress in adulthood may trigger a contradictory response, either beneficial or harmful.

Epigenetics and Pregnancy: Conditional Snapshot or Rolling Event.

Epigenetics modification such as DNA methylation can affect maternal health during the gestation period. Furthermore, pregnancy can drive a range of physiological and molecular changes that have the potential to contribute to pathological conditions. Pregnancy-related risk factors include multiple environmental, behavioral, and hereditary factors that can impact maternal DNA methylation with long-lasting consequences. Identification of the epigenetic patterns linked to poor pregnancy outcomes is crucial since changes in DNA methylation patterns can have long-term effects. In this review, we provide an overview of the epigenetic changes that influence pregnancy-related molecular programming such as gestational diabetes, immune response, and pre-eclampsia, in an effort to close the gap in current understanding regarding interactions between the environment, the genetics of the fetus, and the pregnant woman.

Striatal Isolated from Cyathus striatus Extracts Induces Apoptosis in Human Pancreatic Cancer Cells.

The aim of the present study was to identify the structure of active compounds in Cyathus stratus that previously demonstrated anti-pancreatic cancer activity. The active compounds were purified from a crude extract by a series of RP-18 preparative chromatography using homemade octadecyl silica gel column. HPLC injection of the crude extract revealed a chromatogram with three main peaks with retention times (RT) 15.6, 18.2, and 22.5 min. Each fraction that exhibited promising activity in vitro was further separated using various available chromatographic techniques. The purified compound with the ultimate anti-cancer activity appeared at RT of 15.8 in the HPLC chromatogram with more than 90% purity. The main peak at the mass spectra appeared at m/z = 446.2304 with the calculated molecular formula of C25H34O7. One- and two-dimensional NMR analyses indicated that the structure of the active molecule (peak 15.8 min in HPLC) was identified as striatal C. Exposure of human pancreatic cancer cells to purified striatal C resulted in induction of apoptosis. Further studies are needed in order to develop a method for the synthesis of striatal in order to use it in clinical studies for treatment of cancer.

Cyathus striatus Extract Induces Apoptosis in Human Pancreatic Cancer Cells and Inhibits Xenograft Tumor Growth In Vivo.

Pancreatic cancer is a highly lethal disease with limited options for effective therapy and the lowest survival rate of all cancer forms. Therefore, a new, effective strategy for cancer treatment is in need. Previously, we found that a culture liquid extract of Cyathus striatus (CS) has a potent antitumor activity. In the present study, we aimed to investigate the effects of Cyathus striatus extract (CSE) on the growth of pancreatic cancer cells, both in vitro and in vivo. The proliferation assay (XTT), cell cycle analysis, Annexin/PI staining and TUNEL assay confirmed the inhibition of cell growth and induction of apoptosis by CSE. A Western blot analysis demonstrated the involvement of both the extrinsic and intrinsic apoptosis pathways. In addition, a RNAseq analysis revealed the involvement of the MAPK and P53 signaling pathways and pointed toward endoplasmic reticulum stress induced apoptosis. The anticancer activity of the CSE was also demonstrated in mice harboring pancreatic cancer cell line-derived tumor xenografts when CSE was given for 5 weeks by weekly IV injections. Our findings suggest that CSE could potentially be useful as a new strategy for treating pancreatic cancer.

The effects of environmental stressors on candidate aging associated genes.

BACKGROUND: Aging is defined as a biological and physical complex process that is characterized by the increase in susceptibility to diseases and eventually death. Aging may occur at different rates between and within species, especially or (it varies) among the long-lived ones. Here, we ask whether this diversity (e.g. aging phenotype) stems from genetic or environmental factors or as a combination between the two (epigenetics). Epigenetics play a central role in controlling changes in gene expression during aging. DNA methylation is the most abundant epigenetic modification among vertebrates and is essential to mammalian development. MATERIALS AND METHODS: In this study, we utilized the HELPtag assay to identify five candidate genes that were significantly hyper- or hypo-methylated across four different age groups in mice. The candidate genes were annotated using ensemble and their expression was further tested in vitro using the murine RAW 264.7 cell line to examine the effect of three environmental stressors (UV radiation, Hypoxia and fasting) on their expression. RNA was extracted at different time points followed by cDNA synthesis. Changes in gene expression were evaluated using qRT-PCR. RESULTS: We show that fasting and UV radiation reduced the viability of RAW264.7 cells. We also found a significant change in three candidate genes' expression levels during fasting (TOP2B, RNF13 and MRPL4). Furthermore, we found a significant change in the four candidate genes' expression levels following UVC treatment (TOP2B, RNF13, PKNOX1 and CREB5) and yet no changes were recorded in hypoxic conditions. CONCLUSION: Our results suggest that the model we used was a fitting model for the assessment of environmental stressors on candidate gene expression. In addition, we established a cellular response to the environment via changes in gene expression.

Exceptionally Long-Lived Individuals (ELLI) Demonstrate Slower Aging Rate Calculated by DNA Methylation Clocks as Possible Modulators for Healthy Longevity.

Exceptionally long-lived individuals (ELLI) who are the focus of many healthy longevity studies around the globe are now being studied in Israel. The Israeli Multi-Ethnic Centenarian Study (IMECS) cohort is utilized here for assessment of various DNA methylation clocks. Thorough phenotypic characterization and whole blood samples were obtained from ELLI, offspring of ELLI, and controls aged 53-87 with no familial exceptional longevity. DNA methylation was assessed using Illumina MethylationEPIC Beadchip and applied to DNAm age online tool for age and telomere length predictions. Relative telomere length was assessed using qPCR T/S (Telomere/Single copy gene) ratios. ELLI demonstrated juvenile performance in DNAm age clocks and overall methylation measurement, with preserved cognition and relative telomere length. Our findings suggest a favorable DNA methylation profile in ELLI enabling a slower rate of aging in those individuals in comparison to controls. It is possible that DNA methylation is a key modulator of the rate of aging and thus the ELLI DNAm profile promotes healthy longevity.

Telomeres and Longevity: A Cause or an Effect?

Telomere dynamics have been found to be better predictors of survival and mortality than chronological age. Telomeres, the caps that protect the end of linear chromosomes, are known to shorten with age, inducing cell senescence and aging. Furthermore, differences in age-related telomere attrition were established between short-lived and long-lived organisms. However, whether telomere length is a "biological thermometer" that reflects the biological state at a certain point in life or a biomarker that can influence biological conditions, delay senescence and promote longevity is still an ongoing debate. We cross-sectionally tested telomere length in different tissues of two long-lived (naked mole-rat and Spalax) and two short-lived (rat and mice) species to tease out this enigma. While blood telomere length of the naked mole-rat (NMR) did not shorten with age but rather showed a mild elongation, telomere length in three tissues tested in the Spalax declined with age, just like in short-lived rodents. These findings in the NMR, suggest an age buffering mechanism, while in Spalax tissues the shortening of the telomeres are in spite of its extreme longevity traits. Therefore, using long-lived species as models for understanding the role of telomeres in longevity is of great importance since they may encompass mechanisms that postpone aging.

Inula Viscosa Extract Inhibits Growth of Colorectal Cancer Cells in vitro and in vivo Through Induction of Apoptosis.

Colorectal cancer (CRC) is the second most common cancer in females and the third in males worldwide. Conventional therapy of CRC is limited by severe side effects and by the development of resistance. Therefore, additional therapies are needed in order to combat the problem of selectivity and drug resistance in CRC patients. Inula viscosa (IV) is a well-known medicinal perennial herb in traditional medicine. It is used for different therapeutic purposes, such as; topical anti-inflammatic, diuretic, hemostatic, antiseptic, antiphlogistic, and in the treatment of diabetes. Several studies attempted to reveal the anti-cancer activity of different extracts prepared by different organic solvents from different parts of the IV plant. The aim of the present study is to examine the potential beneficial effects of IV leaf aqueous extract on the growth of colon cancer cells in vitro and in vivo. The results indicated that exposure of colorectal cancer cells to IV extract, significantly reduced cell viability in a dose and time dependent manner. Moreover, treatment of cells with 300 µg/ml of IV extract induced apoptosis, as it was detected by Annexin V/FITC/PI, TUNEL assay, and the activation of caspases. In vivo studies revealed that treatment with 150 or 300 mg/kg IV extract inhibited tumor growth in mice transplanted with MC38 cells. Tumors' weight and volume were significantly (P < 0.001) reduced when compared to untreated-control group. Staining of the paraffin section of tumors revealed that IV treatment inhibited cell proliferation and induced apoptosis. Additionally, no side effects such as; weight loss, behavior changes, ruffled fur or changes in kidney, and liver functions were observed. These results may indicate that active doses of IV extract are not toxic. Further studies are needed in order to identify the structure of the active compounds. Results from this study may contribute to the development of new and efficient strategies for treatment of human colon cancer.

Sex-Dependent Sensory Phenotypes and Related Transcriptomic Expression Profiles Are Differentially Affected by Angelman Syndrome.

Angelman syndrome (AS) is a genetic disorder which entails autism, intellectual disability, lack of speech, motor deficits, and seizure susceptibility. It is caused by the lack of UBE3A protein expression, which is an E3-ubiquitin ligase. Despite AS equal prevalence in males and females, not much data on how sex affects the syndrome was reported. In the herein study, we thoroughly characterized many behavioral phenotypes of AS mice. The behavioral data acquired was analyzed with respect to sex. In addition, we generated a new mRNA sequencing dataset. We analyzed the coding transcriptome expression profiles with respect to the effects of genotype and sex observed in the behavioral phenotypes. We identified several neurobehavioral aspects, especially sensory perception, where AS mice either lack the male-to-female differences observed in wild-type littermates or even show opposed differences. However, motor phenotypes did not show male-to-female variation between wild-type (WT) and AS mice. In addition, by utilizing the mRNA sequencing, we identified genes and isoforms with expression profiles that mirror the sensory perception results. These genes are differentially regulated in the two sexes with inverse expression profiles in AS mice compared to WT littermates. Some of these are known pain-related and estrogen-dependent genes. The observed differences in sex-dependent neurobehavioral phenotypes and the differential transcriptome expression profiles in AS mice strengthen the evidence for molecular cross talk between Ube3a protein and sex hormone receptors or their elicited pathways. These interactions are essential for understanding Ube3a deletion effects, beyond its E3-ligase activity.

Genomic Instabilities, Cellular Senescence, and Aging: In Vitro, In Vivo and Aging-Like Human Syndromes.

As average life span and elderly people prevalence in the western world population is gradually increasing, the incidence of age-related diseases such as cancer, heart diseases, diabetes, and dementia is increasing, bearing social and economic consequences worldwide. Understanding the molecular basis of aging-related processes can help extend the organism's health span, i.e., the life period in which the organism is free of chronic diseases or decrease in basic body functions. During the last few decades, immense progress was made in the understanding of major components of aging and healthy aging biology, including genomic instability, telomere attrition, epigenetic changes, proteostasis, nutrient sensing, mitochondrial dysfunction, cellular senescence, stem cell exhaustion, and intracellular communications. This progress has been made by three spear-headed strategies: in vitro (cell and tissue culture from various sources), in vivo (includes diverse model and non-model organisms), both can be manipulated and translated to human biology, and the study of aging-like human syndromes and human populations. Herein, we will focus on current repository of genomic "senescence" stage of aging, which includes health decline, structural changes of the genome, faulty DNA damage response and DNA damage, telomere shortening, and epigenetic alterations. Although aging is a complex process, many of the "hallmarks" of aging are directly related to DNA structure and function. This review will illustrate the variety of these studies, done in in vitro, in vivo and human levels, and highlight the unique potential and contribution of each research level and eventually the link between them.

The GH receptor exon 3 deletion is a marker of male-specific exceptional longevity associated with increased GH sensitivity and taller stature.

Although both growth hormone (GH) and insulin-like growth factor 1 (IGF-1) signaling were shown to regulate life span in lower organisms, the role of GH signaling in human longevity remains unclear. Because a GH receptor exon 3 deletion (d3-GHR) appears to modulate GH sensitivity in humans, we hypothesized that this polymorphism could play a role in human longevity. We report a linear increased prevalence of d3-GHR homozygosity with age in four independent cohorts of long-lived individuals: 841 participants [567 of the Longevity Genes Project (LGP) (8% increase; P = 0.01), 152 of the Old Order Amish (16% increase; P = 0.02), 61 of the Cardiovascular Health Study (14.2% increase; P = 0.14), and 61 of the French Long-Lived Study (23.5% increase; P = 0.02)]. In addition, mega analysis of males in all cohorts resulted in a significant positive trend with age (26% increase; P = 0.007), suggesting sexual dimorphism for GH action in longevity. Further, on average, LGP d3/d3 homozygotes were 1 inch taller than the wild-type (WT) allele carriers (P = 0.05) and also showed lower serum IGF-1 levels (P = 0.003). Multivariate regression analysis indicated that the presence of d3/d3 genotype adds approximately 10 years to life span. The LGP d3/d3-GHR transformed lymphocytes exhibited superior growth and extracellular signal-regulated kinase activation, to GH treatment relative to WT GHR lymphocytes (P < 0.01), indicating a GH dose response. The d3-GHR variant is a common genetic polymorphism that modulates GH responsiveness throughout the life span and positively affects male longevity.