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Alternative pre-mRNA splicing increases the complexity of the proteome that can be generated from the available genomic coding sequences. Dysregulation of the splicing process has been implicated in a vast repertoire of diseases. However, splicing has recently been linked to both the aging process itself and pro-longevity interventions. This review focuses on recent research towards defining RNA splicing as a new hallmark of aging. We highlight dysfunctional alternative splicing events that contribute to the aging phenotype across multiple species, along with recent efforts toward deciphering mechanistic roles for RNA splicing in the regulation of aging and longevity. Further, we discuss recent research demonstrating a direct requirement for specific splicing factors in pro-longevity interventions, and specifically how nutrient signaling pathways interface to splicing factor regulation and downstream splicing targets. Finally, we review the emerging potential of using splicing profiles as a predictor of biological age and life expectancy. Understanding the role of RNA splicing components and downstream targets altered in aging may provide opportunities to develop therapeutics and ultimately extend healthy lifespan in humans.

Precursor mRNA splicing homeostasis is a biomarker and predictor of life expectancy in Caenorhabditis elegans and defects in global pre-mRNA splicing associated with age are reduced by dietary restriction via splicing factor 1. Splicing factor 1 overexpression extends lifespan The collapse of protein homeostasis and increasing error rates in transcription are key risk factors for various chronic diseases, and are associated with ageing. William Mair and colleagues demonstrate that pre-mRNA splicing homeostasis is a biomarker and predictor of life expectancy in Caenorhabditis elegans , and that defects in global pre-mRNA splicing associated with age are reduced by dietary restriction via splicing factor 1 (SFA-1). SFA-1 is specifically required for lifespan extension both by dietary restriction, and modulation of TORC1 pathway components. Overexpression of SFA-1 is sufficient to extend lifespan. This work suggests that modulation of specific spliceosome components can promote healthy ageing. Ageing is driven by a loss of transcriptional and protein homeostasis 1 , 2 , 3 and is the key risk factor for multiple chronic diseases. Interventions that attenuate or reverse systemic dysfunction associated with age therefore have the potential to reduce overall disease risk in the elderly. Precursor mRNA (pre-mRNA) splicing is a fundamental link between gene expression and the proteome, and deregulation of the splicing machinery is linked to several age-related chronic illnesses 4 , 5 . However, the role of splicing homeostasis in healthy ageing remains unclear. Here we demonstrate that pre-mRNA splicing homeostasis is a biomarker and predictor of life expectancy in Caenorhabditis elegans . Using transcriptomics and in-depth splicing analysis in young and old animals fed ad libitum or subjected to dietary restriction, we find defects in global pre-mRNA splicing with age that are reduced by dietary restriction via splicing factor 1 (SFA-1; the C. elegans homologue of SF1, also known as branchpoint binding protein, BBP). We show that SFA-1 is specifically required for lifespan extension by dietary restriction and by modulation of the TORC1 pathway components AMPK, RAGA-1 and RSKS-1/S6 kinase. We also demonstrate that overexpression of SFA-1 is sufficient to extend lifespan. Together, these data demonstrate a role for RNA splicing homeostasis in dietary restriction longevity and suggest that modulation of specific spliceosome components may prolong healthy ageing.

Geroscience aims to target the aging process to extend healthspan. However, even isogenic individuals show heterogeneity in natural aging rate and responsiveness to pro-longevity interventions, limiting translational potential. Using RNAseq analysis of young, isogenic, subpopulations of Caenorhabditis elegans selected solely on the basis of the splicing pattern of an in vivo minigene reporter that is predictive of future life expectancy, we find a strong correlation in young animals between predicted life span and alternative splicing of mRNAs related to lipid metabolism. The activity of two RNA splicing factors, Reversed Polarity-1 (REPO-1) and Splicing Factor 1 (SFA-1), early in life is necessary for C. elegans response to specific longevity interventions and leads to context-specific changes to fat content that is mirrored by knockdown of their direct target POD-2/ACC1. Moreover, POD-2/ACC1 is required for the same longevity interventions as REPO-1/SFA-1. In addition, early inhibition of REPO-1 renders animals refractory to late onset suppression of the TORC1 pathway. Together, we propose that splicing factor activity establishes a cellular landscape early in life that enables responsiveness to specific longevity interventions and may explain variance in efficacy between individuals.

Customer churn reduction remains a critical challenge for the Indian telecommunications industry, where intense competition and low switching costs fuel high churn rates. While customer engagement has been recognized as a key driver of loyalty, limited research has examined whether digital or human engagement is more effective in reducing churn. This study investigates the comparative influence of these two modalities on customer churn reduction, using Structural Equation Modeling (SEM) with data collected from 800 telecom users across India. The findings reveal that both digital and human engagement significantly reduce churn; however, digital engagement exerts a stronger influence on churn reduction outcomes, particularly through personalized and continuous interactions via apps, websites, and social media platforms. Human engagement, while comparatively weaker in preventing churn, remains crucial for building trust and relational satisfaction. This research contributes to engagement theory by clarifying the distinct and complementary roles of digital and human interactions in churn reduction. For practitioners, the results highlight the need to prioritize digital engagement strategies while maintaining human touchpoints to strengthen customer trust and loyalty.  

We have generated a single-copy knock-in loci for defined gene expression (SKI LODGE) system to insert any DNA by CRISPR/Cas9 at defined safe harbors in the Caenorhabditis elegans genome. Utilizing a single crRNA guide, which also acts as a Co-CRISPR enrichment marker, any DNA sequence can be introduced as a single copy, regulated by different tissue-specific promoters. The SKI LODGE system provides a fast, economical, and effective approach for generating single-copy ectopic transgenes in C. elegans.

Background Plasmodium enolase is a target for the growth neutralizing antibodies. Interestingly, the three invasive stages i.e. sporozoites, merozoites, and ookinetes express this protein on their cell surface. Polyclonal anti- Plasmodium falciparum enolase (Pfeno) antibodies disrupt traversal of ookinete through mosquito mid-gut wall as well as have inhibitory effect on parasite growth at erythrocytic stage. In a recent study, it was observed that immunization with a unique epitope of parasite enolase (EWGWS) could confer partial protection against mouse malaria. Further validation is needed for the protective potential of this unique epitope in otherwise highly conserved enolase. Methods In order to investigate the efficacy of growth inhibitory potential of the epitope of P falciparum enolase, a monoclonal antibody specific to EWGWS is generated. In vitro parasite growth inhibition assays and passive immunization of Plasmodium yoelii (or Plasmodium berghei ) infected mice were used to assess the parasite growth neutralizing activity of the antibody. Results Screening a panel of monoclonal antibodies raised against recombinant Pfeno that were specific to EWGWS resulted in isolation of H12E1. This antibody recognized only EWGWS epitope containing enolases. H12E1 strongly inhibited parasite growth in culture. This inhibition was strain transcending. Passive infusion of this antibody in P. yoelii or P. berghei infected mice showed significant reduction in parasitemia as compared to controls (p < 0.001). Surface Plasmon Resonance measurements indicated high affinity binding of H12E1 to P. falciparum enolase (K D  ~ 7.6 × 10 −9 M). Conclusions A monoclonal antibody directed against EWGWS epitope of Pfeno was shown to inhibit the growth of blood stage malarial parasites. This inhibition was species/strain transcending and is likely to arise due to blockade of enolase on the surface of merozoites, functionally implicating Pfeno in invasion related events. Presence of enolase on the cell surface of merozoites and ookinetes could potentially result in inhibition of host cell invasions at erythrocytic and transmission stages in the parasite life cycle. It is suggested that antibodies against EWGWS epitope have the potential to confer dual stage, species and strain transcending protection against malaria.

Plasmodium enolase localizes to several sub-cellular compartments viz. cytosol, nucleus, cell membrane, food vacuole (FV) and cytoskeleton, without having any organelle targeting signal sequences. This enzyme has been shown to undergo multiple post-translational modifications (PTMs) giving rise to several variants that show organelle specific localization. It is likely that these PTMs may be responsible for its diverse distribution and moonlighting functions. While most variants have a MW of ~50 kDa and are likely to arise due to changes in pI, food vacuole (FV) associated enolase showed three forms with MW~50, 65 and 75 kDa. Evidence from immuno-precipitation and western analysis indicates that the 65 and 75 kDa forms of FV associated enolase are ubiquitinated. Using mass spectrometry (MS), definitive evidence is obtained for the nature of PTMs in FV associated variants of enolase. Results showed several modifications, viz. ubiquitination at K147, phosphorylation at Y148 and acetylation at K142 and K384. MS data also revealed the conjugation of three ubiquitin (Ub) molecules to enolase through K147. Trimeric ubiquitin has a linear peptide linkage between the NH2-terminal methionine of the first ubiquitin (Ub1) and the C-terminal G76 of the second (Ub2). Ub2 and third ubiquitin (Ub3) were linked through an atypical isopeptide linkage between K6 of Ub2 and G76 of Ub3, respectively. Further, the tri-ubiquitinated form was found to be largely associated with hemozoin while the 50 and 65 kDa forms were present in the NP-40 soluble fraction of FV. Mass spectrometry results also showed phosphorylation of S42 in the cytosolic enolase from P. falciparum and T337 in the cytoskeleton associated enolase from P. yoelii. The composition of food vacuolar proteome and likely interactors of enolase are also being reported.

Plasmodium falciparum enolase (Pfeno) is a dimeric enzyme with multiple moonlighting functions. This enzyme is thus a potential target for anti‐malarial treatments. A unique feature of Pfeno is the presence of a pentapeptide insert 104EWGWS108. The functional role of tryptophan residues in this insert was investigated using site‐directed mutagenesis. Replacement of these two Trp residues with alanines (or lysines) resulted in a near complete loss of enolase activity and dissociation of the normal dimeric form into monomers. Molecular modeling indicated that 340R forms π‐cation bonds with the aromatic rings of 105W and 46Y. Mutation induced changes in the interactions among these three residues were presumably relayed to the inter‐subunit interface via a coil formed by 46Y : 59Y, resulting in the disruption of a salt bridge between 11R : 425E and a π‐cation interaction between 11R : 59Y. This led to a drop of ~ 4 kcal·mole−1 in the inter‐subunit docking energy in the mutant, causing a ~ 103 fold decrease in affinity. Partial restoration of the inter‐subunit interactions led to reformation of dimers and also restored a significant fraction of the lost enzyme activity. These results suggested that the perturbations in the conformation of the surface loop containing the insert sequence were relayed to the interface region, causing dimer dissociation that, in turn, disrupted the enzyme's active site. Since Plasmodium enolase is a moonlighting protein with multiple parasite‐specific functions, it is likely that these functions may map on to the highly conserved unique insert region of this protein. Enzymes Enolase(EC4.2.1.11). Plasmodium falciparum enolase (Pfeno), a multifaceted moonlighting protein, has a unique insert 104EWGWS108 that is absent in host enolases. Site‐directed mutagenesis studies reveal that the tryptophan residues are critical for the enzymatic activity and oligomeric stability of Pfeno. The effect of perturbation of insert residues is transmitted to the active site and dimer interface via the intervening structural elements.

This study investigated arsenic (As) concentrations in diverse environmental components and their potential impact on the health risks faced by residents of the arsenic (As)-contaminated Nadia district in West Bengal, India. A random selection of 182 cattle and 255 goats from 40 livestock farmers in the district revealed that both animals and humans were naturally exposed to elevated arsenic levels through contaminated drinking water, foods, grasses, concentrate feeds, various fodder tree leaves, and other food/feed resources. The mean As concentration in roughages (483.18 µg/kg DM) was significantly higher ( p  < 0.001) than in tree leaves (391.53 µg/kg DM), and concentrate feed/ingredients (186.66 µg/kg DM). Pond water exhibited higher arsenic levels (106.11 µg/L) compared to shallow tube well water (47.96 µg/L) and deep tube well water/tap water (10.64 µg/L and 10.04 µg/L, respectively). The mean arsenic concentration in soils DM of fodder fields, crop fields, and grassland was 10.25, 10.58, and 10.20 mg/kg, respectively. It was observed that protein-rich feeds had lower levels of arsenic accumulation ( p  < 0.048), while fiber-rich feeds containing more cellulose, hemicellulose, and lignin had higher arsenic levels ( p  < 0.017). Goats consumed 73.46% more arsenic per kg body weight compared to dairy cows. Although chronic and sub-chronic arsenic exposure in the district did not typically manifest symptoms or visible signs in ruminant animals, concentrations in the hair and feces of both cattle and goats exceeded normal values. Cattle feces had significantly higher arsenic (410.43 µg/kg DM) levels ( p  < 0.001) than goat feces (227.00 µg/kg DM), and arsenic concentration in cattle hair (1917.74 µg/kg DM) was also significantly greater ( p  < 0.001) than goat hair (1435.74 µg/kg DM). Arsenic levels in milk samples from both species were below 10 µg/kg. Liver (356.02 µg/kg DM) and kidney (317.22 µg/kg DM) contained significantly higher ( p  < 0.001) levels of arsenic compared to muscle (204.23 µg/kg DM), and bone (161.98 µg/kg DM) in local meat-type adult male goats. The skin accumulated the highest amount of arsenic (576.24 µg/kg DM) among the non-edible parts of the goat carcass. The cumulative cancer risk value for adults was 4.96 × 10 −3 , exceeding the threshold value (1 × 10 −6 ). This suggests a significant risk of cancer development for the population in arsenic-affected areas. Non-cancer risks (hazard indexes) were estimated at 11.01 for adults. Our observations revealed that the highest bioaccumulation of arsenic occurred in the hair of cows, and goats in the examined localities. The biotransformation factor (BTF) for hair was much higher compared to other excreted samples from both species. The calculated BTF followed the order: hair > feces > milk for cows and goats. Livestock farmers in Nadia district are advised to carefully select feed resources, prioritizing those high in crude protein and low in neutral detergent fiber, and they should provide drinking water from deep aquifers to ensure the safety of milk and meat for human consumption.

Geroscience aims to target the aging process to extend healthspan. However, the efficacy of pro- longevity interventions is highly heterogeneous, limiting their translational potential. In my dissertation research, I discovered that activity of RNA splicing factors REPO-1 and SFA-1 early in life, modulates effectiveness of known longevity interventions in C. elegans. Inhibition of REPO-1 and SFA-1 during development blocks lifespan extension in genetic mutants of dietary restriction, reduced TORC1 signaling and reduced electron transport chain function. However, they are not required for longevity in the insulin signaling mutants. Strikingly, this is mediated through the regulation of lipid utilization via a POD- 2/ACC1 dependent mechanism. The effect on lifespan is seen only when REPO-1 and SFA-1 are inhibited early in life and in the nervous system. Finally, early inhibition of REPO-1 and SFA-1 blocks lifespan extension by late onset suppression of the TORC1 pathway. Together these data highlight how early RNA splicing changes impacts response to longevity interventions and may explain variance in efficacy between individuals.