Cellular Responses Research Articles

The diverse functions of DYRK2 in response to cellular stress.

To maintain microenvironmental and cellular homeostasis, cells respond to multiple stresses by activating characteristic cellular mechanisms consisting of receptors, signal transducers, and effectors. Dysfunction of these mechanisms can trigger multiple human diseases as well as cancers. Dual-specificity tyrosine-regulated kinases (DYRKs) are members of the CMGC group and are evolutionarily conserved from yeast to mammals. Previous studies revealed that DYRK2 has important roles in the regulation of the cell cycle and survival in cancer cells. On the other hand, recent studies show that DYRK2 also exhibits significant functions in multiple cellular stress responses and in maintaining cellular homeostasis. Hence, the further elucidation of mechanisms underlying DYRK2's diverse responses to various stresses helps to promote the advancement of innovative clinical therapies and pharmacological drugs. This review summarizes the molecular mechanisms of DYRK2, particularly focusing on cellular stress responses.

Virus-like Particles vaccine based on co-expression of G5 Porcine rotavirus VP2-VP6-VP7 induces a powerful immune protective response in mice

Porcine rotavirus (PoRV), a member of the Reoviridae family, constitutes a principal etiological agent of acute diarrhea in piglets younger than eight weeks of age, and it is associated with considerable morbidity and mortality within the swine industry. The G5 genotype rotavirus strain currently predominates in circulation. To develop a safe and effective porcine rotavirus vaccine, we generated an insect cell-baculovirus expression system, and successfully expressed these three viral proteins and assembled them into virus-like particles (VLPs) co-displaying VP2, VP6, and VP7. Transmission electron microscopy (TEM) analysis revealed that the VP2-VP6-VP7 VLPs exhibited a "wheeled" morphology resembling that of native rotavirus particles, with an estimated diameter of approximately 65nm. To evaluate the immunogenicity and protective efficacy of these VP2-VP6-VP7 VLPs, we immunized BALB/C mice with four escalating doses of the VLPs, ranging from 5 to 40μg of VLP protein per dose. ELISA-based assessments of PoRV-specific antibodies and T cell cytokines, including IL-4, IL-2, and IFN-γ, demonstrate that immunization with VP2-VP6-VP7 VLPs can effectively elicit both humoral and cellular immune responses in mice, resulting in a notable induction of neutralizing antibodies. On days 4, 6, 8, and 10 post-infection (dpi), the VLP-vaccinated group exhibited significantly reduced levels of PoRV RNA copy numbers when compared to the PBS controls. Histological examination of the duodenum, ileum, and kidneys revealed that VP2-VP6-VP7 VLPs provided effective protection against PoRV induced intestinal injury. Collectively, these findings indicate that the VLPs generated in this study possess strong immunogenicity and suggest the considerable promise of the VLP-based vaccine candidate in the prevention and containment of Porcine Rotavirus infections.

CRH upregulates supervillin through ERK and AKT pathways to promote bladder cancer cell migration.

Corticotropin-releasing hormone (CRH) has been well documented playing a role in the regulation of cellular processes, immune responses, and inflammatory processes that can influence the occurrence and development of tumors. Supervillin (SVIL) is a membrane-associated and actin-binding protein, which is actively involved in the proliferation, spread, and migration of cancer cells. This work investigated CRH's influence on bladder cancer cells'migration and relevant mechanisms. By using human bladder cancer cells T24 and RT4 in wound healing experiments and transwell assay, we found that the migration ability of the T24 cells was significantly increased after CRH treatment. In vivo experiments showed that CRH significantly promoted the metastases of T24 cells in cell line-derived xenograft (CDX) mouse model. Interestingly, downregulation of SVIL by SVIL-specifc small hairpin RNAs significantly reduced the promoting effect of CRH on bladder cancer cell migration. Furthermore, CRH significantly increased SVILmessenger RNA and protein expression in T24 cells, accompanied with AKT andERK phosphorylation in T24 cells. Pretreatment with AKT inhibitor (MK2206) blocked the CRH-induced SVIL expression and ERK phosphorylation. Also, inhibition of ERK signaling pathway by U0126 significantly reduced the CRH-induced SVIL expression and AKT phosphorylation. It suggested that cross-talking between AKT and ERK pathways was involved in the effect of CRH on SVIL. Taken together, we demonstrated that CRH induced migration of bladder cancer cells, in which AKT andERK pathways -SVIL played a key role.

The effect of intensive praziquantel administration on vaccine-specific responses among schoolchildren in Ugandan schistosomiasis-endemic islands (POPVAC A): an open-label, randomised controlled trial

Vaccine responses differ between populations and are often impaired in rural and low-income settings. The reasons for this are not fully understood, but observational data suggest that the immunomodulating effects of parasitic helminths might contribute. We hypothesised that Schistosoma mansoni infection suppresses responses to unrelated vaccines, and that suppression could be reversed-at least in part-by intensive praziquantel administration. We conducted an open-label, randomised controlled trial of intensive versus standard intervention against S mansoni among schoolchildren aged 9-17 years from eight primary schools in Koome islands, Uganda. Children were randomly allocated to either an intensive group or a standard group with a computer-generated 1:1 randomisation using permuted blocks sizes 4, 6, 8, and 10. Participants in the intensive group received three praziquantel doses (approximately 40 mg/kg) 2 weeks apart before first vaccination at week 0, and every 3 months thereafter. Participants in the standard group were given one dose of approximately 40 mg/kg praziquantel after the week 8 primary endpoint. Participants in both groups received the BCG vaccine (Serum Institute of India, Pune, India) at week 0; the yellow fever (Sanofi Pasteur, Lyon, France), oral typhoid (PaxVax, London, UK), and first human papillomavirus (HPV) vaccination (Merck, Rahway, NJ, USA) at week 4; and the HPV booster and tetanus-diphtheria vaccine (Serum Institute of India) at week 28. The primary outcome was vaccine response at week 8 (except for tetanus and diphtheria, which was assessed at week 52). The primary analysis population was participants who were infected with S mansoni at baseline, determined retrospectively using either plasma circulating anodic antigen (CAA) or stool PCR. The safety population comprised all randomly allocated participants. The trial was registered at the ISRCTN Registry (ISRCTN60517191) and is complete. Between July 9 and Aug 14, 2019, we enrolled 478 participants, with 239 children per group. 276 (58%) participants were male and 202 (42%) participants were female. Among participants who were positive for S mansoni at baseline (171 [72%] in the intensive group and 164 [69%] in the standard group) intensive praziquantel administration significantly reduced pre-vaccination infection intensity (to median 30 CAA pg/mL [IQR 7-223] vs 1317 [243-8562], p<0·001) compared with standard treatment. Intensive praziquantel administration also reduced week 8 HPV-16-specific IgG response (geometric mean ratio 0·71 [95% CI 0·54-0·94], p=0·017), but had no effect on other primary outcomes. Among all participants (regardless of S mansoni status at baseline) intensive praziquantel administration significantly improved week 8 BCG-specific IFNγ ELISpot response (1·20 [1·01-1·43], p=0·038). Recognised adverse effects of praziquantel were reported more frequently in the intensive group. There were no recorded serious adverse events in either group. We show evidence suggesting that praziquantel administration improves the BCG-specific cellular response, but not humoral responses to other vaccines. Despite observational evidence that helminths impair vaccine response, these results show minimal immediate benefits of reducing helminth burden. The effect of longer-term helminth control should be investigated. UK Medical Research Council. For the Luganda translation of the abstract see Supplementary Materials section.

Anti-fibrotic effects of the sirt6-activator mdl-800 in cardiac stromal cells

Abstract Background Epigenetic changes are among the molecular substrates of cellular stress responses and tissue remodeling in heart failure progression. The sirtuin family of NAD+-dependent histone and non-histone protein deacetylases exert a protective anti-fibrotic role by negatively modulating the inflammatory response and by positive regulation of autophagy-related genes. Sirtuin6 (SIRT6) stands out as a key cardiac protector that can mitigate aging-induced cardiomyocyte senescence and cardiac hypertrophy. Cardiac stromal cells (CSCs) play a pivotal role in fibrosis and remodeling, regulating the composition and stiffness of the extracellular matrix (ECM). Autophagy enhancement has anti-fibrotic effects in CSCs, but the specific role of SIRT6 modulation in CSCs remains unexplored. Purpose To investigate the biological effects of MDL-800, a synthetic allosteric SIRT6 activator, on the stress response and fibrotic activation of CSCs. Methods CSCs were isolated from 4-week-old C57Bl6J mice by explant culture and stimulated with 10ng/ml of TGF-beta1 (TGFb1) up to 72h to induce fibrotic activation. CSCs were treated with MDL-800 up to 10µM and up to 72h, analyzed for cell viability, gene and protein expression levels. Results TGFb1 treatment led to a 0.64-fold reduction in SIRT6 gene expression after 24h. The MTS assay was used to assess a viability dose-response to MDL-800 up to 72h of treatment. The non-toxic concentrations of 2.5µM and 5µM were selected for further experiments (2.5µM vs CTR 0.97-fold and 5µM vs CTR 0.92-fold, normalized OD vs t0). Co-treatment of 5µM MDL-800 with TGFb1 for 72h resulted in a dose-dependent decrease in the expression of TGFb1-induced fibroblast activation markers, as confirmed by immunofluorescence staining for aSMA (TGFb1 vs CTR 2.21-fold, p&amp;lt;0.05; 5µM vs TGFb1 0.46-fold, p&amp;lt;0.05, N=4; mean fluorescence intensity). The anti-fibrotic effect mediated by MDL-800 was further validated by real-time PCR indicating a reduction in the mRNA expression of multiple fibrotic markers, including ACTA2 (TGFb1 vs CTR 1.67-fold, 5µM vs TGFB 0.29-fold; N=2), COL1a1 (TGFb1 vs CTR 1.32-fold, 5µM vs TGFb1 0.87-fold; N=2) COL3a1 (TGFb1 vs CTR 1.06-fold, 5µM vs TGFb1 0.43-fold; N=2) and POSTN (TGFb1 vs CTR 6.37-fold, 5µM vs TGFb1 0.23-fold; N=2). Treatment with 5µM MDL-800 demonstrated an increase in SIRT6 gene expression, suggesting a transcriptional modulation (TGFb1 vs CTR 0.95-fold, 5µM vs TGFb1 1.68-fold; N=2). Western Blot analyses showed an increase in protein levels of the autophagosome marker LC3-II following treatment with 5µM MDL-800 (TGFb1 vs CTR 0.95-fold; 5µM vs TGFb1 3.01-fold), suggesting that autophagy enhancement may be a potential mechanism for the observed anti-fibrotic effects. Conclusion MDL-800 treatment in CSCs is associated with a dose-dependent reduction of TGFb1-induced fibrotic markers and to increased levels of the autophagy marker, suggesting its potential use as an anti-fibrotic molecule for the heart.

Comparative study of lysine acetylation in Vesicomyidae clam Archivesica marissinica and the manila clam Ruditapes philippinarum: adaptation mechanisms in cold seep environments

BackgroundThe deep-sea cold seep zone is characterized by high pressure, low temperature, darkness, and oligotrophy. Vesicomyidae clams are the dominant species within this environment, often forming symbiotic relationships with chemosynthetic microbes. Understanding the mechanisms by which Vesicomyidae clams adapt to the cold seep environment is significant. Acetylation modification of lysine is known to play a crucial role in various metabolic processes. Consequently, investigating the role of lysine acetylation in the adaptation of Vesicomyidae clams to deep-sea environments is worthwhile. So, a comparative study of lysine acetylation in cold seep clam Archivesica marissinica and shallow water shellfish Ruditapes philippinarum was conducted.ResultsA total of 539 acetylated proteins were identified with 1634 acetylation sites. Conservative motif enrichment analysis revealed that the motifs -KacR-, -KacT-, and -KacF- were the most conserved. Subsequent gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) enrichment analyses were conducted on significantly differentially expressed acetylated proteins. The GO enrichment analysis indicated that acetylated proteins are crucial in various biological processes, including cellular response to stimulation, and other cellular processes ( p < 0.05 and false discovery rate (FDR) < 0.25). The results of KEGG enrichment analysis indicated that acetylated proteins are involved in various cellular processes, including tight junction, motor proteins, gap junction, phagosome, cGMP-PKG signaling pathways, endocytosis, glycolysis/gluconeogenesis, among others (p < 0.05 and FDR < 0.25). Notably, a high abundance of lysine acetylation was observed in the glycolysis/glycogenesis pathways, and the acetylation of glyceraldehyde 3-phosphate dehydrogenase might facilitate ATP production. Subsequent investigation into acetylation modifications associated with deep-sea adaptation revealed the specific identification of key acetylated proteins. Among these, the adaptation of cold seep clam hemoglobin and heat shock protein to high hydrostatic pressure and low temperature might involve an increase in acetylation levels. Acetylation of arginine kinase might be related to ATP production and interaction with symbiotic bacteria. Myosin heavy chain (Ama01085) has the most acetylation sites and might improve the actomyosin system stability through acetylation. Further validation is required for the acetylation modification from Vesicomyidae clams.ConclusionA novel comparative analysis was undertaken to investigate the acetylation of lysine in Vesicomyidae clams, yielding novel insights into the regulatory role of lysine acetylation in deep-sea organisms. The findings present many potential proteins for further exploration of acetylation functions in cold seep clams and other deep-sea mollusks.

Perturbation-specific transcriptional mapping for unbiased target elucidation of antibiotics

The rising prevalence of antibiotic resistance threatens human health. While more sophisticated strategies for antibiotic discovery are being developed, target elucidation of new chemical entities remains challenging. In the postgenomic era, expression profiling can play an important role in mechanism-of-action (MOA) prediction by reporting on the cellular response to perturbation. However, the broad application of transcriptomics has yet to fulfill its promise of transforming target elucidation due to challenges in identifying the most relevant, direct responses to target inhibition. We developed an unbiased strategy for MOA prediction, called perturbation-specific transcriptional mapping (PerSpecTM), in which large-throughput expression profiling of wild-type or hypomorphic mutants, depleted for essential targets, enables a computational strategy to address this challenge. We applied PerSpecTM to perform reference-based MOA prediction based on the principle that similar perturbations, whether chemical or genetic, will elicit similar transcriptional responses. Using this approach, we elucidated the MOAs of three molecules with activity against Pseudomonas aeruginosa by comparing their expression profiles to those of a reference set of antimicrobial compounds with known MOAs. We also show that transcriptional responses to small-molecule inhibition resemble those resulting from genetic depletion of essential targets by clustered regularly interspaced short palindromic repeats interference (CRISPRi) by PerSpecTM, demonstrating proof of concept that correlations between expression profiles of small-molecule and genetic perturbations can facilitate MOA prediction when no chemical entities exist to serve as a reference. Empowered by PerSpecTM, this work lays the foundation for an unbiased, readily scalable, systematic reference-based strategy for MOA elucidation that could transform antibiotic discovery efforts.

The Mysterious World of Non-Canonical Caps - What We Know and Why We Need New Sequencing Techniques.

It was long believed that viral and eukaryotic mRNA molecules are capped at their 5' end solely by the N7-methylguanosine cap, which regulates various aspects of the RNA life cycle, from its biogenesis to its decay. However, the recent discovery of a variety of non-canonical RNA caps derived from metabolites and cofactors - such as NAD, FAD, CoA, UDP-glucose, UDP-N-acetylglucosamine, and dinucleoside polyphosphates - has expanded the known repertoire of RNA modifications. These non-canonical caps are found across all domains of life and can impact multiple aspects of RNA metabolism, including stability, translation initiation, and cellular stress responses. The study of these modifications has been facilitated by sophisticated methodologies such as liquid chromatography-mass spectrometry, which have unveiled their presence in both prokaryotic and eukaryotic organisms. The identification of these novel RNA caps highlights the need for advanced sequencing techniques to characterize the specific RNA types bearing these modifications and understand their roles in cellular processes. Unravelling the biological role of non-canonical RNA caps will provide insights into their contributions to gene expression, cellular adaptation, and evolutionary diversity. This review emphasizes the importance of these technological advancements in uncovering the complete spectrum of RNA modifications and their implications for living systems.

Interaction of RECQL4 with poly(ADP-ribose) is critical for the DNA double-strand break response in human cells.

To overcome genotoxicity, cells have evolved powerful and effective mechanisms to detect and respond to DNA lesions. RecQ Like Helicase-4 (RECQL4) plays a vital role in DNA damage responses. RECQL4 is recruited to DNA double-strand break (DSB) sites in a poly(ADP-ribosyl)ation (PARylation)-dependent manner, but the mechanism and significance of this process remain unclear. Here, we showed that the domain of RECQL4 recruited to DSBs in a PARylation-dependent manner directly interacts with poly(ADP-ribose) (PAR) and contains a PAR-binding motif (PBM). By replacing this PBM with a PBM of hnRNPA2 or its mutated form, we demonstrated that the PBM in RECQL4 is required for PARylation-dependent recruitment and the roles of RECQL4 in the DSB response. These results suggest that the direct interaction of RECQL4 with PAR is critical for proper cellular response to DSBs and provide insights to understand PARylation-dependent control of the DSB response and cancer therapeutics using PARylation inhibitors.

Identification and Functional Analysis of Git3 G Protein-Coupled Receptors in Ganoderma boninense PER71

G protein-coupled receptors (GPCRs) are integral components of eukaryotic heterotrimeric G proteins, playing crucial roles in detecting extracellular signals and initiating the activity of signaling proteins within cells to activate cellular responses to these signals. The objectives of this study are to identify and characterize the function of Git3, a Class III GPCR protein, in the oil palm pathogen Ganoderma boninense. To identify the potential genes encoded for GPCR in this fungus, intensive data mining on the genome and transcriptome data has been carried out. A total of six classes of GPCRs have been identified. These include Class II pheromone detectors, Class III carbon detectors, Class IV nitrogen detectors, Class VII proteins similar to glycosyltransferase, Class VIII proteins similar to hemolysin, and Class X protein receptors. Among these, the Class III protein Git3, postulated to be involved in glucose sensing and fungal pathogenicity, was selected for gene knockdown using RNA interference (RNAi). A plasmid, designated pUChph-GIT3, was constructed, to target git3 silencing by incorporating a hygromycin resistance gene cassette and antisense sequences of git3. Transformation of G. boninense PER71 with pUChph-GIT3 produced five potential Δgit3 gene-silenced mutants. PCR analysis confirmed the integration of the RNAi expression cassette into the fungal genome. Quantitative PCR (qPCR) analysis revealed significant reductions in git3 expression in three G. boninense mutants, M42, M66, and M5 by 47%, 23%, and 13%, respectively. The Disease Severity Index (DSI) indicated slower disease progression in oil palm plantlets infected with Δgit3 mutants compared to those infected with wild-type G. boninense PER71. In conclusion, this study successfully isolated and characterized the git3 GPCR from G. boninense and demonstrated that it might play a role during the early stages of infection, as the mutants were able to slow the progression of infection in oil palm plantlets.

Exploring the role of HIF-1α on pathogenesis in Alzheimer's disease and potential therapeutic approaches.

Hypoxia-inducible factor 1α (HIF-1α) is a crucial transcription factor that regulates cellular responses to low oxygen levels (hypoxia). In Alzheimer's disease (AD), emerging evidence suggests a significant involvement of HIF-1α in disease pathogenesis. AD is characterized by the accumulation of amyloid-beta (Aβ) plaques and neurofibrillary tangles (NFTs), leading to neuronal dysfunction and cognitive decline. HIF-1α is implicated in AD through its multifaceted roles in various cellular processes. Firstly, in response to hypoxia, HIF-1α promotes the expression of genes involved in angiogenesis, which is crucial for maintaining cerebral blood flow and oxygen delivery to the brain. However, in the context of AD, dysregulated HIF-1α activation may exacerbate cerebral hypoperfusion, contributing to neuronal damage. Moreover, HIF-1α is implicated in the regulation of Aβ metabolism. It can influence the production and clearance of Aβ peptides, potentially modulating their accumulation and toxicity in the brain. Additionally, HIF-1α activation has been linked to neuroinflammation, a key feature of AD pathology. It can promote the expression of pro-inflammatory cytokines and exacerbate neuronal damage. Furthermore, HIF-1α may play a role in synaptic plasticity and neuronal survival, which are impaired in AD. Dysregulated HIF-1α signaling could disrupt these processes, contributing to cognitive decline and neurodegeneration. Overall, the involvement of HIF-1α in various aspects of AD pathophysiology highlights its potential as a therapeutic target. Modulating HIF-1α activity could offer novel strategies for mitigating neurodegeneration and preserving cognitive function in AD patients. However, further research is needed to elucidate the precise mechanisms underlying HIF-1α dysregulation in AD and to develop targeted interventions.

Differential sensitivity of hemocyte subpopulations (Mytilus edulis) to aged polyethylene terephthalate micro- and nanoplastic particles

Bivalve hemocytes, particularly granulocytes and hyalinocytes, play a crucial role in cell-mediated immunity. However, their interactions with aged plastic particles, exhibiting altered properties that more closely resemble those in natural environments, remain largely underexplored. This study assesses the differential responses of hemocyte subpopulations (Mytilus edulis) to chemically aged polyethylene terephthalate (PET) microplastic (MPs) and nanoplastic (NPs) particles across multiple cellular effect endpoints. Particle characteristics were analyzed using Single Particle Extinction and Scattering, Raman Spectroscopy, Scanning Electron Microscopy, and Dynamic Light Scattering. In vitro experiments with aged PET MPs (1.9 µm) and NPs (0.68 µm) were conducted at three internally relevant concentrations: 10 (C1), 10³ (C2), and 10⁵ particles/mL (C3). Cellular responses were assessed by measuring lysosomal content stability, reactive oxygen species (ROS) production, cellular mortality, and morphological parameters using flow cytometry at 6, 12, 24, and 48 hours. Our findings provide mechanistic insights into the differential sensitivities of granulocytes and hyalinocytes to aged PET, influenced by particle size and concentration. Specifically, aged PET MPs and NPs induce distinct size and concentration-dependent patterns of lysosomal destabilization, coinciding with the loss of functional integrity. Elevated ROS levels were observed only in granulocytes and hyalinocytes exposed to high concentrations of aged PET NPs, underscoring the effects on oxidative stress. Both aged PET MPs and NPs induce significant increases in cellular mortality, particularly after 24 h of exposure at high concentrations. These findings reveal the complex cellular mechanisms underlying hemocyte functional impairment following exposure to aged PET particles under environmentally and biologically relevant conditions.

An integrative network-based approach to identify driving gene communities in chronic obstructive pulmonary disease

Chronic obstructive pulmonary disease (COPD) is an etiologically complex disease characterized by acute exacerbations and stable phases. We aimed to identify biological functions modulated in specific COPD conditions, using whole blood samples collected in the AERIS clinical study (NCT01360398). Considered conditions were exacerbation onset, severity of airway obstruction, and presence of respiratory pathogens in sputum samples. With an integrative multi-network gene community detection (MNGCD) approach, we analyzed expression profiles to identify communities of correlated genes. The approach combined different layers of gene interactions for each explored condition/subset of samples: gene expression similarity, protein-protein interactions, transcription factors, and microRNAs validated regulons. Heme metabolism, interferon-alpha, and interferon-gamma pathways were modulated in patients at both exacerbation and stable-state visits, but with the involvement of distinct sets of genes. An important gene community was enriched with G2M checkpoint, E2F targets, and mitotic spindle pathways during exacerbation. Targets of TAL1 regulator and hsa−let−7b − 5p microRNA were modulated with increasing severity of airway obstruction. Bacterial infections with Moraxella catarrhalis and, particularly, Haemophilus influenzae triggered a specific cellular and inflammatory response in acute exacerbations, indicating an active reaction of the host to infections. In conclusion, COPD is a complex multifactorial disease that requires in-depth investigations of its causes and features during its evolution and whole blood transcriptome profiling can contribute to capturing some relevant regulatory mechanisms associated with this disease. In this work, we explored multi-network modeling that integrated diverse layers of regulatory gene networks and enhanced our comprehension of the biological functions implicated in the COPD pathogenesis.

Mitigation of Cisplatin-Induced Nephrotoxicity and Augmentation of Anticancer Potency via Tea Polyphenol Nanoparticles' Codelivery of siRNA from CRISPR/Cas9 Screened Targets.

Cisplatin, a frontline chemotherapeutic agent against cancer, faces challenges in clinical application due to significant toxicities and suboptimal efficacy. Renal toxicity, a dose-limiting factor of cisplatin, results from multifactorial processes including cisplatin-induced cellular pyroptosis, oxidative damage, and inflammatory responses. Our findings reveal that Tea Polyphenols Nanoparticles (TPNs) derived from Epigallocatechin gallate (EGCG) effectively could address these diverse mechanisms, comprehensively alleviating cisplatin-induced nephrotoxicity. Leveraging TPNs as carriers, chemical conjugation enables the encapsulation of tetravalent cisplatin prodrug, extending its systemic half-life, enhancing tumor tissue accumulation, while simultaneously mitigating renal toxicity. Concurrently, employing a CRISPR/Cas9 kinase library, we identified CSNK2A1 as a target sensitizing tumor cells to cisplatin, enabling specific siRNA sequences to augment cisplatin susceptibility, thereby minimizing the dosage requirement. Benefiting from the versatile carrier properties of TPNs to codeliver cisplatin prodrug and anti-CSNK2A1 siRNA, we developed a codelivery system, Pt-TPNs/siRNA. Pt-TPNs/siRNA not only enhances the anticancer effects but also mitigates cisplatin-induced renal toxicity, achieving efficacy while reducing toxicity. Mechanistic and safety assessments of these nanoparticles were conducted at both cellular and animal levels, opening new avenues for improved clinical utilization of cisplatin.

Cell‐Permeable Nicotinamide Adenine Dinucleotides for Exploration of Cellular Protein ADP‐Ribosylation

AbstractPosttranslational modifications (PTMs) greatly enhance the functional diversity of proteins, surpassing the number of gene‐encoded variations. One intriguing PTM is ADP‐ribosylation, which utilizes nicotinamide adenine dinucleotide (NAD+) as a substrate and is essential in cell signaling pathways regulating cellular responses. Here, we report the first cell‐permeable NAD+ analogs and demonstrate their utility for investigating cellular ADP‐ribosylation. Using a desthiobiotin‐labelled analog for affinity enrichment of proteins that are ADP‐ribosylated in living cells under oxidative stress, we identified protein targets associated with host‐virus interactions, DNA damage and repair, protein biosynthesis, and ribosome biogenesis. Most of these targets have been noted in various literature sources, highlighting the potential of our probes for cellular ADP‐ribosylome studies.

Differential redox sensitivity of tRNA dihydrouridylation.

Various transfer RNA (tRNA) modifications have recently been shown to regulate stress-dependent gene expression by modulating messenger RNA translation. Among these modifications, dihydrouridine stands out for its increase of tRNA structural flexibility. However, whether and how dihydrouridine synthesis reacts to environmental stimuli is largely unknown. In this study, we manipulated the intracellular redox state of Escherichia coli using paraquat, revealing differential sensitivities of the three tRNA-dihydrouridine synthases towards oxidative stress. Using liquid chromatography-mass spectrometryquantification of dihydrouridine in various knockout strains, we validated the use of a specific RNA sequencing method, namely AlkAnilineSeq, for the precise mapping of dihydrouridines throughout E. coli tRNAs. We found DusA showing high activity, followed by DusB and DusC, whose activity was decreased under paraquat treatment. The relative sensitivity is most plausibly explained by a paraquat-dependent drop of NADPH availability. These findings are substantiated by in vitro kinetics, revealing DusA as the most active enzyme, followed by DusB, while DusC showed little activity, likely related to the efficacy of the redox reaction of the flavin coenzyme with NADPH. Overall, our study underscores the intricate interplay between redox dynamics and tRNA modification processes, revealing a new facet of the regulatory mechanisms influencing cellular responses to oxidative stress.

Glycogen Storage Disease Type I and Bone: Clinical and Cellular Characterization.

Glycogen storage disease (GSD) is the most prevalent inherited disorder of glycogen metabolism for which no causal treatment is available. In recent years, thanks to the improved clinical management, the life expectancy of these patients extended, disclosing previously unidentified adverse conditions in other organs. In this study, we evaluated the clinical bone complications and the cellular responses in 20 patients (aged 14.1 ± 3.4years) affected by GSD type I. Fragility fractures were reported in 35% of the patients, which were older than unfractured patients. They involved appendicular skeletal segments, while no vertebral deformity was detected. 60% of the patients had a bone mineral density (BMD) "below the expected range for age", and lumbar spine (LS) BMD Z-scores positively correlated with muscle strength. Circulating mineral and bone markers showed reduction in the older subjects, with no increase in the pubertal age. Significant correlations could not be detected between circulating markers and LS BMD Z-scores, except for sclerostin levels, which also correlated with muscle strength. The osteoclasts differentiated from patients' peripheral blood mononuclear cells did not show cell-autonomous alterations. However, circulating osteoclast precursors from healthy individuals cultured in the presence of patients' sera exhibited increased osteoclastogenesis compared to control sera suggesting that GSD type I serum factors could affect osteoclast function in a non-autonomous manner. In contrast, circulating osteoprogenitors were unremarkable.

Evaluation effect of alginate hydrogel containing losartan on wound healing and gene expression.

Skin tissue engineering has become an increasingly popular alternative to conventional treatments for skin injuries. Hydrogels, owing to their advantages have become the ideal option for wound dressing, and they are extensively employed in a mixture of different drugs to accelerate wound healing. Sodium alginate is a readily available natural polymer with advantages such as bio-compatibility and a non-toxicological nature that is commonly used in hydrogel form for medical applications such as wound repair and drug delivery in skin regenerative medicine. Losartan is a medicine called angiotensin receptor blocker (ARB) that can prevent fibrosis by inhibiting AT1R (angiotensin II type 1 receptor). In this research, for the first time, three-dimensional scaffolds based on cross-linked alginate hydrogel with CaCl2 containing different concentrations of losartan for slow drug release and exudate absorption were prepared and characterized as wound dressing. Alginate hydrogel was mixed with 10, 1, 0.1, and 0.01mg/mL of losartan, and their properties such as morphology, chemical structure, water uptake properties, biodegradability, stability assay, rheology, blood compatibility, and cellular response were evaluated. In addition, the therapeutic efficiency of the developed hydrogels was then assessed in an in vitro wound healing model and with a gene expression. The results revealed that the hydrogel produced was very porous (porosity of 47.37 ± 3.76µm) with interconnected pores and biodegradable (weight loss percentage of 60.93 ± 4.51% over 14days). All hydrogel formulations have stability under various conditions. The use of CaCl2 as a cross-linker led to an increase in the viscosity of alginate hydrogels. An in vitro cell growth study revealed that no cytotoxicity was observed at the suggested dosage of the hydrogel. Increases in Losartan dosage, however, caused hemolysis. In vivo study in adult male rats with a full-thickness model showed greater than 80% improvement of the primary wound region after 2weeks of treatment with alginate hydrogel containing 0.1mg/mL Losartan. RT-PCR and immunohistochemistry analysis showed a decrease in expression level of TGF-β1 and VEGF in treatment groups. Histological analysis demonstrated that the alginate hydrogel containing Losartan can be effective in wound repair by decreasing the size of the scar and tissue remodeling, as evidenced by future in vivo studies.

Effect of RTS,S/AS01E vaccine booster dose on cellular immune responses in African infants and children

RTS,S/AS01E, the first approved malaria vaccine, demonstrated moderate efficacy during the phase 3 pediatric trial. We previously investigated cell-mediated immune (CMI) responses following the primary 3-dose immunization and now report responses to the booster dose given 18 months later. Thirty CMI markers were measured by Luminex in supernatants of peripheral blood mononuclear cells from 709 children and infants after RTS,S/AS01E antigen stimulation, and their associations with malaria risk and antibodies one month post-booster and one year later were assessed. IL-2, IFN-γ, IL-17, IL-5, and IL-13 were associated with RTS,S/AS01E booster vaccination, and IL-2 responses to the circumsporozoite protein (CSP) remained higher after one year. IL-2 was associated with reduced malaria risk in one site, and IL-10 was associated with increased risk in infants. Anti-CSP IgG and IL-2 were moderately correlated one year after booster. This study highlights the moderate cell-mediated immunogenicity of the RTS,S/AS01E booster dose that aligns with partial recovery of RTS,S/AS01E vaccine efficacy.

Paraptosis—A Distinct Pathway to Cell Death

Cell death is a critical biological process necessary for development, tissue maintenance, and defense against diseases. To date, more than 20 forms of cell death have been identified, each defined by unique molecular pathways. Understanding these different forms of cell death is essential for investigating the pathogenesis of diseases such as cancer, neurodegenerative disorders, and autoimmune conditions and developing appropriate therapies. Paraptosis is a distinct form of regulated cell death characterized by cytoplasmic vacuolation and dilatation of cellular organelles like the mitochondria and endoplasmic reticulum (ER). It is regulated by several signaling pathways, for instance, those associated with ER stress, calcium overload, oxidative stress, and specific cascades such as insulin-like growth factor I receptor (IGF-IR) and its downstream signaling pathways comprising mitogen-activated protein kinases (MAPKs) and Jun N-terminal kinase (JNK). Paraptosis has been observed in diverse biological contexts, including development and cellular stress responses in neuronal, retinal, endothelial, and muscle cells. The induction of paraptosis is increasingly important in anticancer therapy, as it targets non-apoptotic stress responses in tumor cells, which can be utilized to induce cell death. This approach enhances treatment efficacy and addresses drug resistance, particularly in cases where cancer cells are resistant to apoptosis. Combining paraptosis-inducing agents with traditional therapies holds promise for enhancing treatment efficacy and overcoming drug resistance, suggesting a valuable strategy in anticancer therapy.