Impaired Cellular Function Research Articles

Building a model for the differential diagnosis of non-tuberculous mycobacterial lung disease and pulmonary tuberculosis: A case-control study based on immunological and radiological features

BackgroundAlthough the incidence of non-tuberculous mycobacterial pulmonary disease (NTM-PD) is increasing annually, it is easily misdiagnosed as pulmonary tuberculosis (PTB). This study aimed to screen and identify the immunological and radiological characteristics that differentiate NTM-PD from PTB and to construct a discriminatory diagnostic model for NTM-PD, providing new tools for its differential diagnosis. MethodsHospitalised patients diagnosed with NTM-PD or PTB between January 2019 and June 2023 were included in the study. Immunological and radiological characteristics were compared between the two groups. Based on the selected differential features, a logistic regression algorithm was used to construct a discriminatory diagnostic model for NTM-PD, and its diagnostic performance was preliminarily analysed. ResultsPatients with NTM-PD were significantly older than those with PTB and the tuberculosis-specific interferon-gamma release assay (TB-IGRA) positivity rate was significantly lower in the NTM-PD group. Moreover, the absolute counts of total T lymphocytes, CD4+ T lymphocytes, CD8+ T lymphocytes, NK cells, and B lymphocytes were significantly lower in patients with NTM-PD and PTB than in healthy controls. Additionally, patients with NTM-PD had a significantly lower absolute count of B lymphocytes than the PTB group. Radiological analysis revealed significant differences between patients with NTM-PD and PTB in terms of cavity wall thickness, bronchial dilation, lung consolidation, pulmonary nodule size, pulmonary emphysema, lung bullae, lymph node calcification, pleural effusion, mediastinal and hilar lymphadenopathy, and the tree-in-bud sign. Bronchial dilation was identified as the predominant risk factor of NTM-PD, whereas TB-IGRA positivity, lymph node calcification, pleural effusion, and mediastinal and hilar lymphadenopathies were protective factors. Based on this, we constructed a discriminatory diagnostic model for NTM-PD. Its receiver operating characteristic curve demonstrated good diagnostic performance, with an area under the curve of 0.938. At the maximum Youden index of 0.746, the sensitivity and specificity were 0.835 and 0.911, respectively. ConclusionsPatients with NTM-PD and PTB exhibited impaired humoral and cellular immune functions as well as significant differences in radiological features. The constructed NTM-PD diagnostic model demonstrated good diagnostic performance. This study provides a new tool for the differential diagnosis of NTM-PD.

Early Initiation of Antiretroviral Therapy Preserves the Metabolic Function of CD4+ T Cells in Subtype C Human Immunodeficiency Virus 1 Infection.

Immune dysfunction often persists in people living with human immunodeficiency virus (HIV) who are on antiretroviral therapy (ART), clinically manifesting as HIV-1-associated comorbid conditions. Early ART initiation may reduce incidence of HIV-1-associated immune dysfunction and comorbid conditions. Immunometabolism is a critical determinant of functional immunity. We investigated the effect of HIV-1 infection and timing of ART initiation on CD4+ T cell metabolism and function. Longitudinal blood samples from people living with HIV who initiated ART during hyperacute HIV-1 infection (HHI; before peak viremia) or chronic HIV-1 infection (CHI) were assessed for the metabolic and immune functions of CD4+ T cells. Metabolite uptake and mitochondrial mass were measured using fluorescent analogues and MitoTracker Green accumulation, respectively, and were correlated with CD4+ T cell effector functions. Initiation of ART during HHI prevented dysregulation of glucose uptake by CD4+ T cells, but glucose uptake was reduced before and after ART initiation in CHI. Glucose uptake positively correlated with interleukin-2 and tumor necrosis factor-α production by CD4+ T cells. CHI was associated with elevated mitochondrial mass in effector memory CD4+ T cells that persisted after ART and correlated with PD-1 expression. ART initiation in HHI largely prevented metabolic impairment of CD4+ T cells. ART initiation in CHI was associated with persistently dysregulated immunometabolism of CD4+ T cells, which was associated with impaired cellular functions and exhaustion.

Efficient intervention for pulmonary fibrosis via mitochondrial transfer promoted by mitochondrial biogenesis

The use of exogenous mitochondria to replenish damaged mitochondria has been proposed as a strategy for the treatment of pulmonary fibrosis. However, the success of this strategy is partially restricted by the difficulty of supplying sufficient mitochondria to diseased cells. Herein, we report the generation of high-powered mesenchymal stem cells with promoted mitochondrial biogenesis and facilitated mitochondrial transfer to injured lung cells by the sequential treatment of pioglitazone and iron oxide nanoparticles. This highly efficient mitochondrial transfer is shown to not only restore mitochondrial homeostasis but also reactivate inhibited mitophagy, consequently recovering impaired cellular functions. We perform studies in mouse to show that these high-powered mesenchymal stem cells successfully mitigate fibrotic progression in a progressive fibrosis model, which was further verified in a humanized multicellular lung spheroid model. The present findings provide a potential strategy to overcome the current limitations in mitochondrial replenishment therapy, thereby promoting therapeutic applications for fibrotic intervention.

Rehmannia glutinosa Libosch and Cornus officinalis Sieb herb couple ameliorates renal interstitial fibrosis in CKD rats by inhibiting the TGF-β1/MAPK signaling pathway

Ethnopharmacological relevanceHerb couple Rehmannia glutinosa Libosch and Cornus officinalis Sieb (RC), originated from "Liuwei Dihuang Pill" which recorded in Key to Therapeutics of Children's Diseases. Traditionally, they have been used widely for their ability to nourish yin and energize the kidneys. Our previous study indicated that the RC could protect against adenine induced Chronic kidney disease (CKD) rats. Nevertheless, there is still no clear explanation of the mechanisms by which RC affects renal interstitial fibrosis in CKD rats. Aim of the studyCurrent Work aims to explore the amelioration and potential mechanism of RC on renal interstitial fibrosis in CKD rats. Materials and methodsCKD rats were induced by adenine. Two weeks after administration, blood, urine, and kidney tissue were collected for biochemical analysis. Observing the physiological state of rats through the changes of rat body weight and renal index. The pro-inflammatory cytokines were measured by enzyme linked immunosorbent assay (ELISA), while renal tissue damage and fibrosis were assessed with Hematoxylin-eosin staining (H&E) and Masson's trichrome staining. In order to determine the levels of indicators and proteins associated with fibrosis signaling pathways, real time PCR (Rt-PCR), Western blot (WB), and immunofluorescence were employed. ResultsThe renal interstitial fibrosis led to impaired cellular functions with increased the levels of Blood Urea Nitrogen (BUN), Urine protein (UP), Interleukin-1β (IL-1β), Interleukin-6 (IL-6), and Tumor Necrosis Factor alpha (TNF-α). and simultaneous up-regulated collagenⅠ(COL-1), fibronection (FN), α-smooth muscle actin (a-SMA), transforming growth factor-β1 (TGF-β1), c-Jun N-terminal kinase (JNK), p38 and extracellular regulated protein kinases (ERK), down-regulated the expression of the E-cadherin proteins. RC notably improved renal dysfunction in CKD rats as indicated by decreases in BUN, UP, and renal index. In addition, consistent with the morphological changes of renal tissue, renal interstitial fibrosis in CKD rats after RC intervention was significantly improved, mainly manifested by a decrease in the positive expression of COL-1, FN, and a-SMA, and increased levels of E-cadherin protein. Meanwhile, RC reduced the classical pro-inflammatory cytokines IL-1β, IL-6, and TNF-α in adenine-induced CKD rats. Additionally, RC administration also down-regulated TGF-β1, JNK, p38 and ERK. ConclusionIn conclusion, RC may reduce inflammation in adenine induced CKD rats, improve extracellular matrix (ECM) components deposition, and diminish epithelial-mesenchymal transition (EMT) marker protein levels. Furthermore, RC intervention significantly reduces the release of inflammatory cytokines and inhibits the TGF-β1/MAPK signaling pathway. Based on the results, RC might be useful in the treatment of adenine induced renal fibrosis.

Autophagosomes Defeat Ferroptosis by Decreasing Generation and Increasing Discharge of Free Fe2+ in Skin Repair Cells to Accelerate Diabetic Wound Healing.

Ferroptosis plays an essential role in the development of diabetes and its complications, suggesting potential therapeutic strategies targeting ferroptosis. Secretory autophagosomes (SAPs) carrying cytoplasmic cargoes have been recognized as novel nano-warrior to defeat diseases. Here, it is hypothesized that SAPs derived from human umbilical vein endothelial cells (HUVECs) can restore the function of skin repair cells by inhibiting ferroptosis to promote diabetic wound healing. High glucose (HG)-caused ferroptosis in human dermal fibroblasts (HDFs) is observed in vitro, which results in impaired cellular function. SAPs successfully inhibit ferroptosis in HG-HDFs, thereby improving their proliferation and migration. Further research show that the inhibitory effect of SAPs on ferroptosis resulted from a decrease in endoplasmic reticulum (ER) stress-regulated generation of free ferrous ions (Fe2+ ) in HG-HDFs and an increase in exosome release to discharge free Fe2+ from HG-HDFs. Additionally, SAPs promote the proliferation, migration, and tube formation of HG-HUVECs. Then the SAPs are loaded into gelatin-methacryloyl (GelMA) hydrogels to fabricate functional wound dressings. The results demonstrate the therapeutic effect of Gel-SAPs on diabetic wounds by restoring the normal behavior of skin repair cells. These findings suggest a promising SAP-based strategy for the treatment of ferroptosis-associated diseases.

Yttrium Oxide Nanoparticle-Loaded, Self-Assembled Peptide Gel with Antibacterial, Anti-Inflammatory, and Proangiogenic Properties for Wound Healing.

Chronic wounds are a major healthcare challenge owing to their complex healing mechanism and number of impediments to the healing process, like infections, unregulated inflammation, impaired cellular functions, poor angiogenesis, and enhanced protease activity. Current topical care strategies, such as surgical debridement, absorption of exudates, drug-loaded hydrogels for infection and inflammation management, and exogenous supply of growth factors for angiogenesis and cell proliferation, slow the progression of wounds and reduce patient suffering but suffer from low overall cure rates. Therefore, we have developed a proteolytically stable, multifunctional nanoparticle loaded-peptide gel with inherent anti-inflammatory, antibacterial, and pro-angiogenic properties to provide a favorable wound healing milieu by restoring impaired cellular functions. We have fabricated a self-assembled, lauric acid-peptide conjugate gel, LA-LLys-DPhe-LLys-NH2, loaded with yttrium oxide (Y2O3) nanoparticles (NLG). Gel formed a nanofibrous structure, and nanoparticles were passively entrapped within the network. The surface morphology, stability, viscoelastic, and self-healing characteristics of gels were characterized. It showed a high stability against degradation by proteolytic enzymes and highly potent antibacterial activities against E. coli and S. aureus due to the presence of positively charged side chains of lysine in the peptide chain. It also exhibited an excellent antioxidant activity as well as ability to stimulate cell proliferation in murine fibroblast (L929) cells and human umbilical vein endothelial cells (HUVECs). The incorporation of nanoparticles promoted angiogenesis by upregulating pro-angiogenic genes, vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF2), and epidermal growth factor (EGFR), and the gel caused complete wound closure in cells. In summary, the Y2O3 nanoparticle-loaded lauric acid-peptide conjugate gel is able to elicit the desired tissue regeneration responses and, therefore, has a strong potential as a matrix for the treatment of chronic wounds.

The regulation of transcription elongation in embryonic stem cells.

Transcription elongation is a fundamental molecular process which is accurately regulated to ensure proper gene expression in cellular activities whereas its malfunction is associated with impaired cellular functions. Embryonic stem cells (ESCs) have significant value in regenerative medicine due to their self-renewal ability and their potential to differentiate to almost all types of cells. Therefore, dissection of the exact regulatory mechanism of transcription elongation in ESCs is crucial for both basic research and their clinical applications. In this review, we discuss the current understanding on the regulatory mechanisms of transcription elongation mediated by transcription factors and epigenetic modifications in ESCs.

Targeting Mitochondria to Control Ageing and Senescence

Ageing is accompanied by a progressive impairment of cellular function and a systemic deterioration of tissues and organs, resulting in increased vulnerability to multiple diseases. Here, we review the interplay between two hallmarks of ageing, namely, mitochondrial dysfunction and cellular senescence. The targeting of specific mitochondrial features in senescent cells has the potential of delaying or even reverting the ageing process. A deeper and more comprehensive understanding of mitochondrial biology in senescent cells is necessary to effectively face this challenge. Here, we discuss the main alterations in mitochondrial functions and structure in both ageing and cellular senescence, highlighting the differences and similarities between the two processes. Moreover, we describe the treatments available to target these pathways and speculate on possible future directions of anti-ageing and anti-senescence therapies targeting mitochondria.

A facile one-stone-two-birds strategy for fabricating multifunctional 3D nanofibrous scaffolds.

Local bacterial infections lead to delayed wound healing and in extreme cases, such as diabetic foot ulcers, to non-healing due to the impaired cellular function in such wounds. Thus, many scientists have focused on developing advanced therapeutic platforms to treat infections and promote cellular proliferation and angiogenesis. This study presents a facile approach for designing nanofibrous scaffolds in three dimensions (3D) with enhanced antibacterial activity to meet the need of treating chronic diabetic wounds. Being a cationic surfactant as well as an antimicrobial agent, octenidine (OCT) makes a 2D membrane hydrophilic, enabling it to be modified into a 3D scaffold in a "one stone, two birds" manner. Aqueous sodium borohydride (NaBH4) solution plays a dual role in the fabrication process, functioning as both a reducing agent for the in situ synthesis of silver nanoparticles (Ag NPs) anchored on the nanofiber surface and a hydrogen gas producer for expanding the 2D membranes into fully formed 3D nanofiber scaffolds, as demonstrated by morphological analyses. Various techniques were used to characterize the developed scaffold (e.g., SEM, XRD, DSC, FTIR, and surface wettability), demonstrating a multilayered porous structure and superhydrophilic properties besides showing sustained and prolonged release of OCT (61% ± 1.97 in 144 h). Thanks to the synergistic effect of OCT and Ag NPs, the antibacterial performance of the 3D scaffold was significantly higher than that of the 2D membrane. Moreover, cell viability was studied in vitro on mouse fibroblasts L929, and the noncytotoxic character of the 3D scaffold was confirmed. Overall, it is shown that the obtained multifunctional 3D scaffold is an excellent candidate for diabetic wound healing and skin repair.

Correlating viscosity and molecular crowding with fluorescent nanobeads and molecular probes: in vitro and in vivo.

In eukaryotes, intracellular physico-chemical properties like macromolecular crowding and cytoplasmic viscoelasticity influence key processes such as metabolic activities, molecular diffusion and protein folding. However, mapping crowding and viscoelasticity in living cells remains challenging. One approach uses passive rheology in which diffusion of exogenous fluorescent particles internalized in cells is tracked and physico-chemical properties inferred from derived mean square displacement relations. Recently, the crGE2.3 Förster resonance energy transfer biosensor was developed to quantify crowding in cells, though it is unclear how this readout depends on viscoelasticity and the molecular weight of the crowder. Here, we present correlative, multi-dimensional data to explore diffusion and molecular crowding characteristics of molecular crowding agents using super-resolved fluorescence microscopy and ensemble time-resolved spectroscopy. We firstly characterize in vitro and then apply these insights to live cells of budding yeast Saccharomyces cerevisiae. It is to our knowledge the first time this has been attempted. We demonstrate that these are usable both in vitro and in the case of endogenously expressed sensors in live cells. Finally, we present a method to internalize fluorescent beads as in situ viscoelasticity markers in the cytoplasm of live yeast cells and discuss limitations of this approach including impairment of cellular function.

Insights from DOCK2 in cell function and pathophysiology.

Dedicator of cytokinesis 2 (DOCK2) can activate the downstream small G protein Rac and regulate cytoskeletal reorganization. DOCK2 is essential for critical physiological processes such as migration, activation, proliferation, and effects of immune cells, including lymphocytes, neutrophils, macrophages, and dendritic cells. For example, DOCK2 is involved in the development and activation of T and B lymphocytes by affecting synapse formation and inhibiting the development of the Th2 lineage by downregulating IL-4Rα surface expression. Not only that, DOCK2 may be a molecular target for controlling cardiac transplant rejection and Alzheimer’s disease (AD). Patients with defects in the DOCK2 gene also exhibit a variety of impaired cellular functions, such as chemotactic responses of lymphocytes and reactive oxygen species (ROS) production by neutrophils. To date, DOCK2 has been shown to be involved in the development of various diseases, including AD, pneumonia, myocarditis, colitis, tumors, etc. DOCK2 plays different roles in these diseases and the degree of inflammatory response has a different impact on the progression of disease. In this paper, we present a review of recent advances in the function of DOCK2 in various immune cells and its role in various diseases.

Pharmacotherapeutic potential of astaxanthin: Human and animal targeting roles – A review

Abstract The recent pandemic stress and the impacts of climatic changes on humans’ and animals’ health status and well-being resulted in severe drawbacks. Initially, stress-induced oxidation resulting from the generation of free radicals led to the impairment of cellular function and a high possibility of attack with infection. Astaxanthin is a bioactive material derived from fish, crustaceans, and algae with high antioxidative potential. Astaxanthin is a lipid-soluble carotenoid that can easily cross through the cellular membrane layers to catch the reactive oxygen metabolites. Astaxanthin also has pigmentation properties making it suitable for pharmaceutical, cosmetic, nutraceutical, agriculture, and aquaculture sectors. Recently, astaxanthin is suggested as a natural scavenger for free radicals induced by COVID-19. Besides, using astaxanthin as antioxidative and immunostimulant agents is well-reported in several clinical studies. The output of these investigations should be simplified and presented to the scientific community to utilize the available information and fill the gap of knowledge. Also, it is necessary to update the researchers with the recent recommendations of applying astaxanthin in vivo and in vitro to help in proposing new horizons for engaging natural antioxidative agents to protect human and animal health. Herein, this review article tackled the nature, sources, potential roles, applicable sides, and availability of astaxanthin to fortify the scientific community with the required knowledge for further research efforts.

Therapeutic potential of deuterium-stabilized (R)-pioglitazone-PXL065-for X-linked adrenoleukodystrophy.

X‐linked adrenoleukodystrophy (ALD) results from ABCD1 gene mutations which impair Very Long Chain Fatty Acids (VLCFA; C26:0 and C24:0) peroxisomal import and β‐oxidation, leading to accumulation in plasma and tissues. Excess VLCFA drives impaired cellular functions (e.g. disrupted mitochondrial function), inflammation, and neurodegeneration. Major disease phenotypes include: adrenomyeloneuropathy (AMN), progressive spinal cord axonal degeneration, and cerebral ALD (C‐ALD), inflammatory white matter demyelination and degeneration. No pharmacological treatment is available to‐date for ALD. Pioglitazone, an anti‐diabetic thiazolidinedione, exerts potential benefits in ALD models. Its mechanisms are genomic (PPARγ agonism) and nongenomic (mitochondrial pyruvate carrier—MPC, long‐chain acyl‐CoA synthetase 4—ACSL4, inhibition). However, its use is limited by PPARγ‐driven side effects (e.g. weight gain, edema). PXL065 is a clinical‐stage deuterium‐stabilized (R)‐enantiomer of pioglitazone which lacks PPARγ agonism but retains MPC activity. Here, we show that incubation of ALD patient‐derived cells (both AMN and C‐ALD) and glial cells from Abcd1‐null mice with PXL065 resulted in: normalization of elevated VLCFA, improved mitochondrial function, and attenuated indices of inflammation. Compensatory peroxisomal transporter gene expression was also induced. Additionally, chronic treatment of Abcd1‐null mice lowered VLCFA in plasma, brain and spinal cord and improved both neural histology (sciatic nerve) and neurobehavioral test performance. Several in vivo effects of PXL065 exceeded those achieved with pioglitazone. PXL065 was confirmed to lack PPARγ agonism but retained ACSL4 activity of pioglitazone. PXL065 has novel actions and mechanisms and exhibits a range of potential benefits in ALD models; further testing of this molecule in ALD patients is warranted.

The Effect of Increasing Levels of Hyperthermia on Autophagy in Peripheral Blood Mononuclear Cells from Young Adults

Exposure to an elevated state of hyperthermia is associated with heat‐induced cytotoxicity, thereby resulting in impaired cellular function. Cellular dysfunction during heat stress leads to the initiation of systemic inflammation and apoptotic cell death, which if left unabated, can cause tissue damage and death. To counter heat‐induced cellular injury, cells employ the vital mechanism of autophagy which acts to prevent the accumulation of toxic protein aggregates and dysfunctional organelles to promote cellular survival. In our recent ex vivomodel, we demonstrated that the process of autophagy is activated during exposure to increased temperatures (37‐41°C) in peripheral blood mononuclear cells (PBMCs) from young adults which acts to suppress heat‐induced apoptotic cell death. Despite this, the dose‐dependent autophagic response to heat stress has yet to be evaluated in an in vivo human model and thus the relationship between elevations in core body temperature (Tcore) and autophagy have yet to be established in humans. Therefore, we sought to extend upon our previous work by assessing if in vivorises in Tcore are associated with greater autophagic activation in healthy young adults. We evaluated the hypothesis that in parallel to elevations in Tcore(indexed by esophageal temperature), greater autophagic initiation would occur in a dose‐dependent manner in young adults during immersion in mild to very warm water. To assess this hypothesis, on separate days 6 young adults (mean [SD] 22 [3] years; 3 women) were immersed (up to the clavicle) in water set at a temperature to clamp Tcore at either thermoneutral (TN, 37°C), 38°C, or 39°C for 60‐min. Autophagy was characterized in PBMCs prior to and immediately post‐water immersion, as well as following 3‐h of seated recovery. Western blot was used to assess autophagy proteins microtubule associated protein 1 light chain 3 beta (LC3)‐II and sequesterome‐1/p62 (p62). All proteins were normalized to β‐actin and reported as fold change relative to baseline. Data were compared via a two‐way repeated measures ANOVA with Fisher’s LSD (α=0.05). No changes in either LC3‐II or p62 were observed during TN, indicating no time‐dependent changes in autophagy (all p≥0.28). Immediately following the 38°C clamp, LC3‐II increased (1.56 [0.32]; p=0.02) and p62 decreased (0.52 [0.30]; p=0.049) from baseline, with both proteins returning to basal levels within 3‐h (both, p≥0.22). Similarly, LC3‐II increased (1.90 [0.70]; p=0.04) and p62 decreased (0.58 [0.21]; p=0.03) immediately after clamping Tcore at 39°C, however LC3‐II remained elevated above baseline at 3‐h (2.54 [1.08]; p=0.03) while p62 did not differ from pre‐immersion levels at the end of recovery (p=0.57). Given LC3‐II accumulation and p62 degradation indicate elevated autophagy, our preliminary findings indicate greater autophagy during increasing levels of hyperthermia in healthy young adults. Further, we showed increased reliance on autophagy to restore cellular homeostasis after exposure to an elevated state of hyperthermia of 39°C, although the mechanism of activation is unclear.

Vincamine Modulates the Effect of Pantoprazole in Renal Ischemia/Reperfusion Injury by Attenuating MAPK and Apoptosis Signaling Pathways.

Pantoprazole has an antioxidant function against reactive oxygen species (ROS). Vincamine, a herbal candidate, is an indole alkaloid of clinical use against brain sclerosis. The aim of the present experiment is to evaluate, on a molecular level for the first time, the value of vincamine in addition to pantoprazole in treating experimentally induced renal ischemia/reperfusion injury (IRI). One-hundred-and-twenty-eight healthy male Wistar albino rats were included. Serum creatinine, blood urea nitrogen, and malondialdehyde levels were assessed. ELISA was used to estimate the pro-inflammatory cytokines. The expression of Bcl-2 and Bax genes was assessed by quantitative real-time PCR. ERK1/2, JNK1/2, p38, cleaved caspase-3, and NF-κB proteins expressions were estimated using western blot assay. The kidneys were also histopathologically studied. The IRI resulted in impaired cellular functions with increased creatinine, urea nitrogen, malondialdehyde, TNF-α, IL-6, and IL-1β serum levels, and up-regulated NF-ĸB, JNK1/2, ERK1/2, p38, and cleaved caspase-3 proteins. Furthermore, it down-regulated the expression of the Bcl-2 gene and upregulated the Bax gene. The treatment with vincamine, in addition to pantoprazole multiple doses, significantly alleviated the biochemical and histopathological changes more than pantoprazole or vincamine alone, whether the dose is single or multiple, declaring their synergistic effect. In conclusion, vincamine with pantoprazole multiple doses mitigated the renal IRI through the inhibition of apoptosis, attenuation of the extracellular signaling pathways through proinflammatory cytokines’ levels, and suppression of the MAPK (ERK1/2, JNK, p38)–NF-κB intracellular signaling pathway.

Identification of an endoplasmic reticulum proteostasis modulator that enhances insulin production in pancreatic β cells

Perturbation of endoplasmic reticulum (ER) proteostasis is associated with impairment of cellular function in diverse diseases, especially the function of pancreatic β cells in type 2 diabetes. Restoration of ER proteostasis by small molecules shows therapeutic promise for type 2 diabetes. Here, using cell-based screening, we report identification of a chemical chaperone-like small molecule, KM04794, that alleviates ER stress. KM04794 prevented protein aggregation and cell death caused by ER stressors and a mutant insulin protein. We also found that this compound increased intracellular and secreted insulin levels in pancreatic β cells. Chemical biology and biochemical approaches revealed that the compound accumulated in the ER and interacted directly with the ER molecular chaperone BiP. Our data show that this corrector of ER proteostasis can enhance insulin storage and pancreatic β cell function.

Mitophagy in Traumatic Brain Injury: A New Target for Therapeutic Intervention.

Traumatic brain injury (TBI) contributes to death, and disability worldwide more than any other traumatic insult and damage to cellular components including mitochondria leads to the impairment of cellular functions and brain function. In neurons, mitophagy, autophagy-mediated degradation of damaged mitochondria, is a key process in cellular quality control including mitochondrial homeostasis and energy supply and plays a fundamental role in neuronal survival and health. Conversely, defective mitophagy leads to the accumulation of damaged mitochondria and cellular dysfunction, contributing to inflammation, oxidative stress, and neuronal cell death. Therefore, an extensive characterization of mitophagy-related protective mechanisms, taking into account the complex mechanisms by which each molecular player is connected to the others, may provide a rationale for the development of new therapeutic strategies in TBI patients. Here, we discuss the contribution of defective mitophagy in TBI, and the underlying molecular mechanisms of mitophagy in inflammation, oxidative stress, and neuronal cell death highlight novel therapeutics based on newly discovered mitophagy-inducing strategies.

Abstract 11757: Telomerase Treatment Restores Transcriptional Profile and Epigenetic Landscape in Progeria Vascular Cells

Hutchinson-Gilford progeria syndrome (HGPS) is a model of accelerated vascular aging caused by a de novo mutation of a single nucleotide in the Lamin A gene. The accumulation of abnormal lamin A (progerin) in the nuclear envelope induces a nuclear dysmorphology associated with global gene dysregulation, increased expression of inflammatory cytokines, impaired vascular function, and accelerated telomere erosion. These individuals succumb to myocardial infarction or stroke in their mid-teens. We hypothesized that restoration of telomere length might reverse some of the observed derangements in cell function, transcriptional profile and epigenetic alterations. Accordingly, we generated iPSC-derived endothelial cells (ECs) from HGPS children and their unaffected parents. By comparison to the parental controls, HGPS ECs have severely impaired replicative capacity, cellular functions (e.g. NO synthesis, acLDL uptake, network formation, cytokine secretion) and severe telomere shortening. We observed that transient transfection with mRNA encoding human telomerase (hTERT) increases telomere length, restores cellular replicative capacity and improves HGPS-EC functions. Notably, RNAseq studies revealed that that approximately 1200 genes were dysregulated in Progeria, most of which were improved by hTERT treatment, among which approximately 250+ genes were fully normalized. Intriguingly, hTERT treatment reduces DNA damage signals (γH2Ax) and reverses the aberrant chromatin accessibility observed in HGPS-ECs, as assessed by a reduction in the activating histone mark H3K4me3; an increase in the suppressive mark H3K4me3; and normalizes the level of heterochromatin markers HP1, Histone H1 and H3. Preliminary results from ChIPseq studies targeting histone marks characteristic of activators, repressors and enhancers as well as chromatin remodeling proteins; together with Hi-C analyses indicate that there are global changes in topologically associated domains (TADs) and other features of the chromatin landscape in HGPS ECs, which are partially restored by hTERT treatment. We conclude that hTERT treatment has surprisingly global effects on transcriptional profile and epigenetic landscape in addition to telomere extension.

Selective oxidative stress induces dual damage to telomeres and mitochondria in human T cells.

Oxidative stress caused by excess reactive oxygen species (ROS) accelerates telomere erosion and mitochondrial injury, leading to impaired cellular functions and cell death. Whether oxidative stress‐mediated telomere erosion induces mitochondrial injury, or vice versa, in human T cells—the major effectors of host adaptive immunity against infection and malignancy—is poorly understood due to the pleiotropic effects of ROS. Here we employed a novel chemoptogenetic tool that selectively produces a single oxygen (1O2) only at telomeres or mitochondria in Jurkat T cells. We found that targeted 1O2 production at telomeres triggered not only telomeric DNA damage but also mitochondrial dysfunction, resulting in T cell apoptotic death. Conversely, targeted 1O2 formation at mitochondria induced not only mitochondrial injury but also telomeric DNA damage, leading to cellular crisis and apoptosis. Targeted oxidative stress at either telomeres or mitochondria increased ROS production, whereas blocking ROS formation during oxidative stress reversed the telomeric injury, mitochondrial dysfunction, and cellular apoptosis. Notably, the X‐ray repair cross‐complementing protein 1 (XRCC1) in the base excision repair (BER) pathway and multiple mitochondrial proteins in other cellular pathways were dysregulated by the targeted oxidative stress. By confining singlet 1O2 formation to a single organelle, this study suggests that oxidative stress induces dual injury in T cells via crosstalk between telomeres and mitochondria. Further identification of these oxidation pathways may offer a novel approach to preserve mitochondrial functions, protect telomere integrity, and maintain T cell survival, which can be exploited to combat various immune aging‐associated diseases.

The effect of canakinumab on COVID19-associated cardiomyocyte contractile dysfunction and arrhythmias

Abstract Background Cardiac injury associated with cytokine release occurs in almost 20% of SARS-CoV-2 positive patients during hospitalization and mortality is particularly high in these patients. Cardiac enzyme (e.g. troponin or creatinine kinase (CK)) elevations are a frequently reported finding, indicating myocardial damage and arrhythmias are the cause for ICU transfer in up to 12% of patients. However, the mechanistic role of COVID19 associated cytokine-storm for the concomitant cardiac dysfunction and associated arrhythmias is unclear. In addition, the role of anti-inflammatory therapy approaches to mitigate this cardiac dysfunction remains elusive. Methods We investigated the effects of COVID19-associated inflammatory response on cardiac cellular function as well as its cardiac arrhythmogenic potential in rat and induced pluripotent stem cell derived cardiomyocytes (iPSc-CM). Moreover, we evaluated the therapeutic potential of the IL1-beta antagonist Canakinumab using state of the art in-vitro confocal and ratiometric high-throughput microscopy. Results Isolated rat ventricular cardiomyocytes were exposed to control or COVID19 plasma from intensive care unit patients with severe ARDS and impaired cardiac function (LVEF 41±5%; 1/3 of patients on veno-venous extracorporeal membrane oxygenation; CK 154±43 U/l). Cardiomyocytes showed decreased Ca2+ transient amplitudes and altered baseline Ca2+ concentrations leading to impaired cellular contractile function upon electrical field-stimulation and exposure to patient plasma (n=276 control and 359 COVID19 cells; Fura). In addition, we used iPSc-CM to explore the long-term effect of patient plasma on cardiac electrical and mechanical function in a translational setting (24h incubation; Fluo). In iPSc, spontaneous Ca2+ release events (i.e. Ca2+ waves and Ca2+ sparks) were more likely to occur upon incubation with COVID19 plasma and nuclear as well as cytosolic Ca2+ release were altered. Co-incubation with Canakinumab had no effect on pro-arrhythmogenic Ca2+ release or Ca2+ signaling during excitation-contraction coupling but influenced cellular automaticity upon prolonged electrical stimulation. Conclusion Plasma derived from COVID19 patients exerts acute cardio-depressant and chronic pro-arrhythmogenic effects in rat and iPS-derived cardiomyocytes. Chronic co-incubation with Canakinumab had no beneficial effect on cellular Ca2+ signaling during excitation-contraction coupling. Funding Acknowledgement Type of funding sources: None.