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A progeroid family was found to harbor a pathogenic variant in the CASP5 gene that encodes inflammatory caspase 5. Caspase 5-depleted fibroblasts exhibited hyper-activation of inflammatory cytokines in response to pro-inflammatory stimuli. Long-term intermittent hyper-inflammatory response is likely the cause of the accelerated aging phenotype comprised of earlier onset of common aging diseases, supporting inflammaging as a potential common disease mechanism of progeroid syndromes and possibly normative aging.

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Cellular senescence is a dynamic stress response process that contributes to aging. From initiation to maintenance, senescent cells continuously undergo complex molecular changes and develop an altered transcriptome. Understanding how the molecular architecture of these cells evolve to sustain their non-proliferative state will open new therapeutic avenues to alleviate or delay the consequences of aging. Seeking to understand these molecular changes, we studied the transcriptomic profiles of endothelial replication-induced senescence and senescence induced by the inflammatory cytokine, TNF-α. We previously reported gene expressional pattern, pathways, and the mechanisms associated with upregulated genes during TNF-α induced senescence. Here, we extend our work and find downregulated gene signatures of both replicative and TNF-α senescence were highly overlapped, involving the decreased expression of several genes associated with cell cycle regulation, DNA replication, recombination, repair, chromatin structure, cellular assembly, and organization. We identified multiple targets of p53/p16-RB-E2F-DREAM that are essential for proliferation, mitotic progression, resolving DNA damage, maintaining chromatin integrity, and DNA synthesis that were repressed in senescent cells. We show that repression of multiple target genes in the p53/p16-RB-E2F-DREAM pathway collectively contributes to the stability of the senescent arrest. Our findings show that the regulatory connection between DREAM and cellular senescence may play a potential role in the aging process.

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Older age and underlying conditions such as diabetes/obesity or immunosuppression are leading host risk factors for developing severe complications from COVID-19 infection. The pathogenesis of COVID-19-related cytokine storm, tissue damage, and fibrosis may be interconnected with fundamental aging processes, including dysregulated immune responses and cellular senescence. Here, we examined effects of key cytokines linked to cellular senescence on expression of SARS-CoV-2 viral entry receptors. We found exposure of human umbilical vein endothelial cells (HUVECs) to the inflammatory cytokines, TNF-α + IFN-γ or a cocktail of TNF-α + IFN-γ + IL-6, increased expression of ACE2/DPP4, accentuated the pro-inflammatory senescence-associated secretory phenotype (SASP), and decreased cellular proliferative capacity, consistent with progression towards a cellular senescence-like state. IL-6 by itself failed to induce substantial effects on viral entry receptors or SASP-related genes, while synergy between TNF-α and IFN-γ initiated a positive feedback loop via hyper-activation of the JAK/STAT1 pathway, causing SASP amplification. Breaking the interactive loop between senescence and cytokine secretion with JAK inhibitor ruxolitinib or antiviral drug remdesivir prevented hyper-inflammation, normalized SARS-CoV-2 entry receptor expression, and restored HUVECs proliferative capacity. This loop appears to underlie cytokine-mediated viral entry receptor activation and links with senescence and hyper-inflammation.

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Synacinn is a standardized polyherbal extract formulated for the treatment of diabetes mellitus and its complications. This study aims to assess the mutagenicity potential of Synacinn by Ames assay and in vivo bone marrow micronucleus (MN) test on Sprague Dawley rat. Human ether-a-go-go-related gene (hERG) assay and Functional Observation Battery (FOB) were done for the safety pharmacology tests. In the Ames assay, Dose Range Finding (DRF) study and mutagenicity assays (+/- S9) were carried out. For the MN test, a preliminary and definitive study were conducted. In-life observations and number of immature and mature erythrocytes in the bone marrow cells were recorded. The hERG assay was conducted to determine the inhibitory effect on hERG potassium channel current expressed in human embryonic kidney cells (HEK293). FOB tests were performed orally (250, 750, and 2000 mg/kg) on Sprague Dawley rats. Synacinn is non-mutagenic against all tested strains of Salmonella typhimurium and did not induce any clastogenicity in the rat bone marrow. Synacinn also did not produce any significant inhibition (p ≤ 0.05) on hERG potassium current. Synacinn did not cause any neurobehavioural changes in rats up to 2000 mg/kg. Thus, no mutagenicity, cardiotoxicity and neurotoxicity effects of Synacinn were observed in this study.

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BACKGROUND: Werner syndrome (WS) is an autosomal recessive progeroid syndrome caused by variants in WRN. The International Registry of Werner Syndrome has identified biallelic pathogenic variants in 179/188 cases of classical WS. In the remaining nine cases, only one heterozygous pathogenic variant has been identified. METHODS: Targeted long-read sequencing (T-LRS) on an Oxford Nanopore platform was used to search for a second pathogenic variant in WRN. Previously, T-LRS was successfully used to identify missing variants and analyse complex rearrangements. RESULTS: We identified a second pathogenic variant in eight of nine unsolved WS cases. In five cases, T-LRS identified intronic splice variants that were confirmed by either RT-PCR or exon trapping to affect splicing; in one case, T-LRS identified a 339 kbp deletion, and in two cases, pathogenic missense variants. Phasing of long reads predicted all newly identified variants were on a different haplotype than the previously known variant. Finally, in one case, RT-PCR previously identified skipping of exon 20; however, T-LRS did not detect a pathogenic DNA sequence variant. CONCLUSION: T-LRS is an effective method for identifying missing pathogenic variants. Although limitations with computational prediction algorithms can hinder the interpretation of variants, T-LRS is particularly effective in identifying intronic variants.

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A HPLC method has been validated for identifying five markers (gallic acid, rosmarinic acid, catechin, andrographolide and curcumin) and quantifying curcumin in SynacinnTM formulation. The validation (bracketed strengths of 10 mg/mL and 100 mg/mL) involved assessment of selectivity, precision, Limit of Detection (LOD), Limit of Quantification (LOQ), linearity, accuracy, stability in diluent and formulation stability. Meanwhile, in vivo bone marrow micronucleus test data was presented to evaluate the toxicity potential of Synacinn™ to cause clastogenicity and/or disruption of the mitotic apparatus, as measured by its ability to induce micronucleated polychromatic erythrocytes (MN PCE) in Sprague Dawley rat bone marrow. The test was conducted in two phases viz., Phase I (Dose Range Finding experiment) and Phase II (Definitive experiment). Phase I was conducted to assess general toxicity and bone marrow cytotoxicity of Synacinn™, and to select the doses for the definitive experiment. In-life observations included mortality, clinical signs of toxicity and body weight. Bone marrow samples were collected and extracted from the femur bone using fetal bovine serum. The pellet obtained after the centrifugation was used for preparing bone marrow smears to evaluate the number of immature and mature erythrocytes.

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The present data described the analysis of mutagenicity in SynacinnTM by assessing the point mutations occurring due to Synacinn™ exposure to five tester strains of Salmonella typhimurium (TA1537, TA1535, TA98, TA100 and TA102), in the presence or absence of an exogenous mammalian metabolic activation system (S9). It was conducted in two Phases - Phase I (Dose Range Finding experiment-DRF) and Phase II (Mutagenicity Assay 1 and 2). DRF and Mutagenicity Assay 1 was conducted employing plate incorporation method, while Mutagenicity Assay 2 was performed using pre-incubation method. Formulation analysis pertaining to SynacinnTM was performed for both Mutagenicity Assay 1 and 2. Dose formulations were prepared fresh on each day of the experiment. Adventol 50% v/v in purified water was selected as a suitable vehicle based on the preliminary solubility test. Based on the Phase I analysis, 5 mg/plate was selected as the highest concentration of SynacinnTM followed by lower concentrations of 2.5, 1.25, 0.625 and 0.313 mg/plate for the Mutagenicity Assays. Genetic integrity of all the tester strains used was confirmed by performing genotyping before their use. All the data acceptability criteria were fulfilled confirming the validity of the test.

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SMAD4 encodes a member of the SMAD family of proteins involved in the TGF-ß signaling pathway. Potentially heritable, autosomal dominant, gain-of-function heterozygous variants of SMAD4 cause a rare developmental disorder, the Myhre syndrome, which is associated with a wide range of developmental and post-developmental phenotypes that we now characterize as a novel segmental progeroid syndrome. Whole-exome sequencing of a patient referred to our International Registry of Werner Syndrome revealed a heterozygous p.Arg496Cys variant of the SMAD4 gene. To investigate the role of SMAD4 mutations in accelerated senescence, we generated cellular models overexpressing either wild-type SMAD4 or mutant SMAD4-R496C in normal skin fibroblasts. We found that cells expressing the SMAD4-R496C mutant exhibited decreased proliferation and elevated expression of cellular senescence and inflammatory markers, including IL-6, IFNγ, and a TGF-ß target gene, PAI-1. Here we show that transient exposure to TGF-ß, an inflammatory cytokine, followed by chronic IFNγ stimulation, accelerated rates of senescence that were associated with increased DNA damage foci and SMAD4 expression. TGF-ß, IFNγ, or combinations of both were not sufficient to reduce proliferation rates of fibroblasts. In contrast, TGF-ß alone was able to induce preadipocyte senescence via induction of the mTOR protein. The mTOR inhibitor rapamycin mitigated TGF-ß-induced expression of p21, p16, and DNA damage foci and improved replicative potential of preadipocytes, supporting the cell-specific response to this cytokine. These findings collectively suggest that persistent DNA damage and cross-talk between TGF-ß/IFNγ pathways contribute to a series of molecular events leading to cellular senescence and a segmental progeroid syndrome.

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Segmental progeroid syndromes are groups of genetic disorders with multiple features resembling accelerated aging. The International Registry of Werner Syndrome (Seattle, WA) recruits pedigrees of progeroid syndromes from all over the world. We identified two novel LMNA mutations, p.Asp300Gly in a patient from Myanmar, and p.Asn466Lys, in a patient from Greece. Both were referred to our Registry for the genetic diagnosis because of the accelerated aged-appearance and cardiac complications. LMNA mutations are the second most common genetic cause of progeroid syndromes after WRN mutations in our Registry. As the next generation sequencing becomes readily available, we expect to identify more cases of rare genetic diseases in the developing countries.

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Cellular senescence is a cell fate program that entails essentially irreversible proliferative arrest in response to damage signals. Tumor necrosis factor-alpha (TNFα), an important pro-inflammatory cytokine secreted by some types of senescent cells, can induce senescence in mouse and human cells. However, downstream signaling pathways linking TNFα-related inflammation to senescence are not fully characterized. Using human umbilical vein endothelial cells (HUVECs) as a model, we show that TNFα induces permanent growth arrest and increases p21CIP1, p16INK4A, and SA-ß-gal, accompanied by persistent DNA damage and ROS production. By gene expression profiling, we identified the crucial involvement of inflammatory and JAK/STAT pathways in TNFα-mediated senescence. We found that TNFα activates a STAT-dependent autocrine loop that sustains cytokine secretion and an interferon signature to lock cells into senescence. Furthermore, we show STAT1/3 activation is necessary for cytokine and ROS production during TNFα-induced senescence. However, inhibition of STAT1/3 did not rescue cells from proliferative arrest, but rather suppressed cell cycle regulatory genes and altered TNFα-induced senescence. Our findings suggest a positive feedback mechanism via the STAT pathway that sustains cytokine production and reveal a reciprocal regulatory role of JAK/STAT in TNFα-mediated senescence.

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Hyaluronan is known to act as a filling material of extracellular matrices and as an adhesive substrate for cellular migration. Consequently, it is widely used in aesthetic medicine and surgery, and it would be expected to be used in nanomedicine. Previous clinical case reports associated hyaluronic acid implants to delayed immune-mediated adverse effects. A series of experiments to evaluate immune cell activation supported by this dermal filler and nanomedical biomaterial were performed. The study comprised a total of 12 individuals. Four healthy individuals, none with cosmetically injected dermal filler, were considered as control. Five individuals carried injections of hyaluronic acid dermal filler. Three individuals carried injections of hyaluronic acid dermal filler and presented delayed adverse effects related to the dermal filler. Hyaluronic acid-stimulated peripheral blood mononuclear cells (PBMC) produced low levels of pro-inflammatory cytokines. Phytohemagglutinine (PHA)-stimulated PBMC from patients with hyaluronic implants presenting adverse effects showed a slight increase in the production of interferon (IFN)-gamma and higher expression of CD25, CD69, or CD71. In conclusion, hyaluronic acid administration elicited a laboratory evidence of immune cell activation. Production of low levels of proinflammatory cytokines in vitro could be an observation for low-grade inflammation in vivo resulting in T cell activation.

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For more than 40 years, silicone implants had been employed in aesthetic, cosmetic medicine, and plastic surgery. Although adverse reactions produced by these products are rare, cases of immuno-mediated reactions have been reported. To evaluate the aspects of immuno-reactivity to medical-grade silicone dermal filler, peripheral blood mononuclear cells (PBMC) of 39 individuals were studied. PBMC used include individuals with silicone injection-related delayed adverse reactions, with silicone injections, and healthy control. Silicone induced production of TNF-alpha and IL-6 in all three groups. Notably, elevated production of IL-6 was observed in nonstimulated PBMC and also the percentage of CD4(+)CD69(+) T cells was higher in PHA-stimulated PBMC from individuals with silicone injection-related adverse reactions when compared with other two groups. However, IFN-gamma was not released in silicone-stimulated or silicone+LPS-stimulated PBMC from any group and no production of IL-2 was measured indicating no proliferative response of PBMC. Subsequently, no CD4(+)CD69(+) T cells were observed in these conditions. Finally, the inflammatory response in silicone-stimulated cultures of monocyte-derived macrophages with autologous lymphocytes is lesser than that observed in PBMC. In conclusion, silicone induces a release of proinflammatory cytokines but does not act as a polyclonal activator of CD4(+) T cells. Thus, silicone is mounting an immune response in individuals with silicone-related adverse effects but is not silicone antigen-dependent.

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