Search Results (54)

Parvoviruses have compact genomic organizations with overlapping open reading frames and thus utilize alternative RNA processing strategies, alternative splicing, alternative polyadenylation, and alternative translation mechanisms to generate a range of diverse proteins encoded within their genome. This comprehensive review provides an update on recent insights into the diverse mRNA processing mechanisms utilized by members of the Parvoviridae family, with emphasis on Bocaparvoviruses and Dependoparvoviruses to expand their protein repertoire and maintain their replicative advantage in infected host cells. It highlights the role of Bocaparvovirus ancillary nonstructural protein NP1; the first parvovirus protein involved in mRNA processing, specifically alternative splicing and alternative polyadenylation. Full article

Background/Objectives: Studies have reported an increased risk of type 2 diabetes among people with higher protein intake. Moreover, branched-chain amino acids (BCAA) are reported to be positively associated with insulin resistance (IR). However, it is not understood whether elevated levels of BCAA are causal to IR development, or if higher BCAA are a marker of IR. The objective of this study was to examine the effects of long-term protein and carbohydrate supplementation on plasma BCAA levels, and the relationship between plasma BCAA and IR in postmenopausal women. Methods: Stored samples and data from 84 postmenopausal women who participated in a protein supplementation trial (SPOON) were included. Exclusion criteria consisted of protein intakes less than 0.6 g/kg or greater than 1.0 g/kg, a body mass index (BMI) greater than 32 kg/m² or less than 19 kg/m² diseases, and conditions and medications known to impact musculoskeletal health. Subjects were randomized to a whey protein (PRO: n = 38) or maltodextrin supplement (CHO: n = 46) for 18 months. Plasma BCAA, homeostatic model assessment of insulin resistance (HOMA-IR) and body composition were analyzed at baseline and 18 months. Results: At baseline, there were no significant associations between plasma BCAA and IR. There were also no significant changes in plasma BCAA or IR by study arm. However, there was a significant positive association between plasma BCAA and IR in both groups at 18 months (CHO: r = 0.35, p = 0.02; PRO: r = 0.35, p = 0.03). Conclusions: Findings from this study warrant future research to examine other diet and lifestyle factors that may mediate the relationship between circulating BCAA and IR in postmenopausal women. Full article

mRNA-based drug development is revolutionizing tumor therapies by enabling precise cancer immunotherapy, tumor suppressor gene restoration, and genome editing. However, the success of mRNA therapies hinges on efficient delivery systems that can protect mRNA from degradation and facilitate its release into the cytoplasm for translation. Despite the emergence of lipid nanoparticles (LNPs) as a clinically advanced platform for mRNA delivery, the efficiency of endo/lysosomal escape still represents a substantial bottleneck. Here, we summarize the intracellular fate of mRNA-loaded LNPs, focusing on their internalization pathways and processing within the endo-lysosomal system. We also discuss the impact of endo-lysosomal processes on mRNA delivery and explore potential strategies to improve mRNA escape from endo-lysosomal compartments. This review focuses on molecular engineering strategies to enhance LNP-mediated endo/lysosomal escape by optimizing lipid composition, including ionizable lipids, helper lipids, cholesterol, and PEGylated lipids. Additionally, ancillary enhancement strategies such as surface coating and shape management are discussed. By comprehensively integrating mechanistic insights into the journey of LNPs within the endo-lysosome system and recent advances in lipid chemistry, this review offers valuable inspiration for advancing mRNA-based cancer therapies by enabling robust protein expression. Full article

Voltage-gated Ca²⁺ (Ca_V) channels are transmembrane proteins comprising the pore-forming subunit Ca_Vα₁ and the ancillary proteins Ca_Vα₂δ and Ca_Vβ. They are expressed in various tissues, including the nervous system, where they regulate Ca²⁺ entry in response to membrane potential changes. The increase in intracellular Ca²⁺ allows for regulating cell excitability and releasing neurotransmitters, among other cellular events. Leucine-rich repeat kinase 2 (LRRK2) is a serine–threonine kinase involved in vesicular mobilization. Previously, it has been shown that LRRK2 regulates neurotransmission by phosphorylating the Ca_Vβ auxiliary subunit of the Ca_V2.1 (P/Q-type) presynaptic channels. However, it is unknown whether the kinase can regulate the activity of other Ca_V channel subtypes, such as Ca_V1.3 (L-type), which play a significant role in the excitability of dopaminergic neurons in the substantia nigra pars compacta (SNc) and whose dysregulation contributes to neurodegeneration in Parkinson’s disease (PD). Here, we found potential phosphorylation sites for LRRK2 in Ca_Vβ₃ and examined how these molecules interact. We used immunoprecipitation and electrophysiology in HEK-293 cells expressing recombinant Ca_V1.3 channels, both with and without wild-type LRRK2 or its LRRK2^G2019S mutation, which plays a role in familial PD through a possible gain-of-toxic-function mechanism. Our results show that LRRK2^G2019S significantly increases current density through Ca_V1.3 channels, and this effect depends on the presence of Ca_Vβ₃. Site-directed mutagenesis revealed that phosphorylation at S152 in the sequence of Ca_Vβ₃ is necessary and sufficient to explain the abnormal regulation of the channels mediated by LRRK2^G2019S. These data provide new insights into the molecular regulation that mutant LRRK2 may exert on L-type Ca_V1.3 channels, which determine pacemaker activity in dopaminergic neurons of the SNc and may, therefore, play a relevant role in the molecular pathophysiology of PD. Full article

The DNA damage response protein p53-binding protein 1 (53BP1) accumulates and forms foci at double-strand DNA breaks, indicating the extent of DNA instability. However, the potential role of 53BP1 as a molecular biomarker for hypopharyngeal squamous cell carcinoma (HPSCC) diagnosis remains unknown. Here, we evaluated the potential of immunofluorescence-based analysis of 53BP1 expression to differentiate the histology of hypopharyngeal neoplasms. A total of 125 lesions from 39 surgically or endoscopically resected specimens from patients with HPSCC was histologically evaluated. 53BP1 expression in the nucleus was examined using immunofluorescence. The number of 53BP1 nuclear foci increased with the progression from non-tumorous to low-grade dysplasia, high-grade dysplasia, and squamous cell carcinoma. Unstable 53BP1 expression served as an independent factor for distinguishing lesions that required intervention. Colocalization of 53BP1 foci in proliferating cells, as assessed by Ki67, was increased in tumors ≥ 1000 µm in depth compared to those <1000 µm in depth at the tumor surface. Hence, the expression patterns of nuclear 53BP1 foci were associated with the progression of hypopharyngeal neoplasms. These findings suggest that 53BP1 could serve as an ancillary marker to support histological diagnosis and predict the factors that influence prognosis in patients with HPSCC. Full article

The (patho)physiological function of the sphingolipids ceramide-1-phosphate (C1P), sphingosine-1-phosphate (S1P), and sphingosylphosphorylcholine (SPC) in articular joints during osteoarthritis (OA) is largely unknown. Therefore, we investigated the influence of these lipids on protein expression by fibroblast-like synoviocytes (FLSs) from OA knees. Cultured human FLSs (n = 7) were treated with 1 of 3 lipid species—C1P, S1P, or SPC—IL-1β, or with vehicle. The expression of individual proteins was determined by tandem mass tag peptide labeling followed by high-resolution electrospray ionization (ESI) mass spectrometry after liquid chromatographic separation (LC-MS/MS/MS). The mRNA levels of selected proteins were analyzed using RT-PCR. The 3sphingolipids were quantified in the SF of 18 OA patients using LC-MS/MS. A total of 4930 proteins were determined using multiplex MS, of which 136, 9, 1, and 0 were regulated both reproducibly and significantly by IL-1β, C1P, S1P, and SPC, respectively. In the presence of IL-1ß, all 3 sphingolipids exerted ancillary effects. Only low SF levels of C1P and SPC were found. In conclusion, the 3 lipid species regulated proteins that have not been described in OA. Our results indicate that charged multivesicular body protein 1b, metal cation symporter ZIP14, glutamine-fructose-6-P transaminase, metallothionein-1F and -2A, ferritin, and prosaposin are particularly interesting proteins due to their potential to affect inflammatory, anabolic, catabolic, and apoptotic mechanisms. Full article

Chronic granulomatous disease (CGD) is a group of rare primary inborn errors of immunity characterised by a defect in the phagocyte respiratory burst, which leads to severe and life-threatening infective and inflammatory complications. Despite recent advances in our understanding of the genetic and molecular pathophysiology of X-linked and autosomal recessive CGD, and growth in the availability of functional and genetic testing, there remain significant barriers to early and accurate diagnosis. In the current review, we provide an up-to-date summary of CGD pathophysiology, underpinning current methods of diagnostic testing for CGD and closely related disorders. We present an overview of the benefits of early diagnosis and when to suspect and test for CGD. We discuss current and historical methods for functional testing of NADPH oxidase activity, as well as assays for measuring protein expression of NADPH oxidase subunits. Lastly, we focus on genetic and genomic methods employed to diagnose CGD, including gene-targeted panels, comprehensive genomic testing and ancillary methods. Throughout, we highlight general limitations of testing, and caveats specific to interpretation of results in the context of CGD and related disorders, and provide an outlook for newborn screening and the future. Full article

Autophagy is a degradative recycling process central to the maintenance of homeostasis in all eukaryotes. By ensuring the degradation of damaged mitochondria, it plays a key role in maintaining mitochondrial health and function. Of the highly conserved autophagy proteins, autophagy-related protein 1 (Atg1) is essential to the process. The involvement of these proteins in intracellular signalling pathways, including those involving mitochondrial function, are still being elucidated. Here the role of Atg1 was investigated in the simple model organism Dictyostelium discoideum using an atg1 null mutant and mutants overexpressing or antisense-inhibiting atg1. When evaluated against the well-characterised outcomes of mitochondrial dysfunction in this model, altered atg1 expression resulted in an unconventional set of phenotypic outcomes in growth, endocytosis, multicellular development, and mitochondrial homeostasis. The findings here show that Atg1 is involved in a tightly regulated signal transduction pathway coordinating energy-consuming processes such as cell growth and multicellular development, along with nutrient status and energy production. Furthermore, Atg1’s effects on energy homeostasis indicate a peripheral ancillary role in the mitochondrial signalling network, with effects on energy balance rather than direct effects on electron transport chain function. Further research is required to tease out these complex networks. Nevertheless, this study adds further evidence to the theory that autophagy and mitochondrial signalling are not opposing but rather linked, yet strictly controlled, homeostatic mechanisms. Full article

Over the past three decades, significant efforts have been focused on unraveling congenital intestinal disorders that disrupt the absorption of dietary lipids and fat-soluble vitamins. The primary goal has been to gain deeper insights into intra-enterocyte sites, molecular steps, and crucial proteins/regulatory pathways involved, while simultaneously identifying novel therapeutic targets and diagnostic tools. This research not only delves into specific and rare malabsorptive conditions, such as chylomicron retention disease (CRD), but also contributes to our understanding of normal physiology through the utilization of cutting-edge cellular and animal models alongside advanced research methodologies. This review elucidates how modern techniques have facilitated the decoding of CRD gene defects, the identification of dysfunctional cellular processes, disease regulatory mechanisms, and the essential role of coat protein complex II-coated vesicles and cargo receptors in chylomicron trafficking and endoplasmic reticulum (ER) exit sites. Moreover, experimental approaches have shed light on the multifaceted functions of SAR1B GTPase, wherein loss-of-function mutations not only predispose individuals to CRD but also exacerbate oxidative stress, inflammation, and ER stress, potentially contributing to clinical complications associated with CRD. In addition to dissecting the primary disease pathology, genetically modified animal models have emerged as invaluable assets in exploring various ancillary aspects, including responses to environmental challenges such as dietary alterations, gender-specific disparities in disease onset and progression, and embryonic lethality or developmental abnormalities. In summary, this comprehensive review provides an in-depth and contemporary analysis of CRD, offering a meticulous examination of the CRD current landscape by synthesizing the latest research findings and advancements in the field. Full article

The aim of this study was to assess the potential value of circulating active and inactive IL-18 levels in distinguishing pseudo and true tumor progression among NSCLC patients receiving immune checkpoint inhibitor treatments (ICIs). Methods: This ancillary study includes 195 patients with metastatic non-small-cell lung cancer (NSCLC) treated with ICI in monotherapy, either pembrolizumab or nivolumab. Plasmatic levels of IL-18-related compounds, comprising the inhibitor IL-18 binding protein (IL-18BP), the inactive IL-18 (corresponding to IL-18/IL-18BP complex), and the active free IL-18, were assayed by ELISA. Objective tumoral response was analyzed by ¹⁸FDG PET-CT at baseline, 7 weeks, and 3 months post treatment induction, using PERCIST criteria. Results: Plasmatic IL-18BP and total IL-18 levels are increased at baseline in NSCLC patients compared with healthy controls, whereas IL-18/IL-18BP complexes are decreased, and free IL-18 levels remain unchanged. Neither of the IL-18-related compounds allowed to discriminate ICI responding to nonresponding patients. However, inactive IL-18 levels allowed to discriminate patients with a first tumor progression, assessed after 7 weeks of treatment, with worse overall survival. In addition, we showed that neutrophil concentration is also a predictive indicator of patients’ outcomes with OS (HR = 2.6, p = 0.0001) and PFS (HR = 2.2, p = 0.001). Conclusions: Plasmatic levels of inactive IL-18, combined with circulating neutrophil concentrations, can effectively distinguish ICI nonresponding patients with better overall survival (OS), potentially guiding rapid decisions for therapeutic intensification. Full article

Peptides possessing antihypertensive attributes via inhibiting the angiotensin-converting enzyme (ACE) were derived through the enzymatic degradation of Trichiurus lepturus (ribbonfish) using alkaline protease. The resulting mixture underwent filtration using centrifugation, ultrafiltration tubes, and Sephadex G-25 gels. Peptides exhibiting ACE-inhibitory properties and DPPH free-radical-scavenging abilities were isolated and subsequently purified via LC/MS-MS, leading to the identification of over 100 peptide components. In silico screening yielded five ACE inhibitory peptides: FAGDDAPR, QGPIGPR, IFPRNPP, AGFAGDDAPR, and GPTGPAGPR. Among these, IFPRNPP and AGFAGDDAPR were found to be allergenic, while FAGDDAPRR, QGPIGPR, and GPTGPAGP showed good ACE-inhibitory effects. IC₅₀ values for the latter peptides were obtained from HUVEC cells: FAGDDAPRR (IC₅₀ = 262.98 μM), QGPIGPR (IC₅₀ = 81.09 μM), and GPTGPAGP (IC₅₀ = 168.11 μM). Peptide constituents derived from ribbonfish proteins effectively modulated ACE activity, thus underscoring their therapeutic potential. Molecular docking and modeling corroborated these findings, emphasizing the utility of functional foods as a promising avenue for the treatment and prevention of hypertension, with potential ancillary health benefits and applications as substitutes for synthetic drugs. Full article

Wound healing is an intricate process involving coordinated interactions among inflammatory cells, skin fibroblasts, keratinocytes, and endothelial cells. Successful tissue repair hinges on controlled inflammation, angiogenesis, and remodeling facilitated by the exchange of cytokines and growth factors. Comorbid conditions can disrupt this process, leading to significant morbidity and mortality. Stem cell therapy has emerged as a promising strategy for enhancing wound healing, utilizing cells from diverse sources such as endothelial progenitor cells, bone marrow, adipose tissue, dermal, and inducible pluripotent stem cells. In this systematic review, we comprehensively investigated stem cell therapies in chronic wounds, summarizing the clinical, translational, and primary literature. A systematic search across PubMed, Embase, Web of Science, Google Scholar, and Cochrane Library yielded 22,454 articles, reduced to 44 studies after rigorous screening. Notably, adipose tissue-derived mesenchymal stem cells (AD-MSCs) emerged as an optimal choice due to their abundant supply, easy isolation, ex vivo proliferative capacities, and pro-angiogenic factor secretion. AD-MSCs have shown efficacy in various conditions, including peripheral arterial disease, diabetic wounds, hypertensive ulcers, bullous diabeticorum, venous ulcers, and post-Mohs micrographic surgery wounds. Delivery methods varied, encompassing topical application, scaffold incorporation, combination with plasma-rich proteins, and atelocollagen administration. Integration with local wound care practices resulted in reduced pain, shorter healing times, and improved cosmesis. Stem cell transplantation represents a potential therapeutic avenue, as transplanted stem cells not only differentiate into diverse skin cell types but also release essential cytokines and growth factors, fostering increased angiogenesis. This approach holds promise for intractable wounds, particularly chronic lower-leg wounds, and as a post-Mohs micrographic surgery intervention for healing defects through secondary intention. The potential reduction in healthcare costs and enhancement of patient quality of life further underscore the attractiveness of stem cell applications in wound care. This systematic review explores the clinical utilization of stem cells and stem cell products, providing valuable insights into their role as ancillary methods in treating chronic wounds. Full article

Hypertrophic scars (HTSs) are pathological structures resulting from chronic inflammation during the wound healing process, particularly in complex injuries like burns. The aim of this work is to propose Biofiber PF (biodegradable fiber loaded with Pirfenidone 1.5 w/w), an electrospun advanced dressing, as a solution for HTSs treatment in complex wounds. Biofiber has a 3-day antifibrotic action to modulate the fibrotic process and enhance physiological healing. Its electrospun structure consists of regular well-interconnected Poly-L-lactide-co-poly-ε-caprolactone (PLA-PCL) fibers (size 2.83 ± 0.46 µm) loaded with Pirfenidone (PF, 1.5% w/w), an antifibrotic agent. The textured matrix promotes the exudate balance through mild hydrophobic wettability behavior (109.3 ± 2.3°), and an appropriate equilibrium between the absorbency % (610.2 ± 171.54%) and the moisture vapor transmission rate (0.027 ± 0.036 g/min). Through its finer mechanical properties, Biofiber PF is conformable to the wound area, promoting movement and tissue oxygenation. These features also enhance the excellent elongation (>500%) and tenacity, both in dry and wet conditions. The ancillary antifibrotic action of PF on hypertrophic scar fibroblast (HSF) for 3 days downregulates the cell proliferation over time and modulates the gene expression of transforming growth factor β1 (TGF-β1) and α-smooth muscle actin (α-SMA) at 48–72 h. After 6 days of treatment, a decrement of α-SMA protein levels was detected, proving the potential of biofiber as a valid therapeutic treatment for HTSs in an established wound healing process. Full article

Purpose: Gene therapy actually seems to have promising results in the treatment of Leber Congenital Amaurosis and some different inherited retinal diseases (IRDs); the primary goal of this strategy is to change gene defects with a wild-type gene without defects in a DNA sequence to achieve partial recovery of the photoreceptor function and, consequently, partially restore lost retinal functions. This approach led to the introduction of a new drug (voretigene neparvovec-rzyl) for replacement of the RPE65 gene in patients affected by Leber Congenital Amaurosis (LCA); however, the treatment results are inconstant and with variable long-lasting effects due to a lack of correctly evaluating the anatomical and functional conditions of residual photoreceptors. These variabilities may also be related to host immunoreactive reactions towards the Adenovirus-associated vector. A broad spectrum of retinal dystrophies frequently generates doubt as to whether the disease or the patient is a good candidate for a successful gene treatment, because, very often, different diseases share similar genetic characteristics, causing an inconstant genotype/phenotype correlation between clinical characteristics also within the same family. For example, mutations on the RPE65 gene cause Leber Congenital Amaurosis (LCA) but also some forms of Retinitis Pigmentosa (RP), Bardet Biedl Syndrome (BBS), Congenital Stationary Night Blindness (CSNB) and Usher syndrome (USH), with a very wide spectrum of clinical manifestations. These confusing elements are due to the different pathways in which the product protein (retinoid isomer-hydrolase) is involved and, consequently, the overlapping metabolism in retinal function. Considering this point and the cost of the drug (over USD one hundred thousand), it would be mandatory to follow guidelines or algorithms to assess the best-fitting disease and candidate patients to maximize the output. Unfortunately, at the moment, there are no suggestions regarding who to treat with gene therapy. Moreover, gene therapy might be helpful in other forms of inherited retinal dystrophies, with more frequent incidence of the disease and better functional conditions (actually, gene therapy is proposed only for patients with poor vision, considering possible side effects due to the treatment procedures), in which this approach leads to better function and, hopefully, visual restoration. But, in this view, who might be a disease candidate or patient to undergo gene therapy, in relationship to the onset of clinical trials for several different forms of IRD? Further, what is the gold standard for tests able to correctly select the patient? Our work aims to evaluate clinical considerations on instrumental morphofunctional tests to assess candidate subjects for treatment and correlate them with clinical and genetic defect analysis that, often, is not correspondent. We try to define which parameters are an essential and indispensable part of the clinical rationale to select patients with IRDs for gene therapy. This review will describe a series of models used to characterize retinal morphology and function from tests, such as optical coherence tomography (OCT) and electrophysiological evaluation (ERG), and its evaluation as a primary outcome in clinical trials. A secondary aim is to propose an ancillary clinical classification of IRDs and their accessibility based on gene therapy’s current state of the art. Material and Methods: OCT, ERG, and visual field examinations were performed in different forms of IRDs, classified based on clinical and retinal conditions; compared to the gene defect classification, we utilized a diagnostic algorithm for the clinical classification based on morphofunctional information of the retina of patients, which could significantly improve diagnostic accuracy and, consequently, help the ophthalmologist to make a correct diagnosis to achieve optimal clinical results. These considerations are very helpful in selecting IRD patients who might respond to gene therapy with possible therapeutic success and filter out those in which treatment has a lower chance or no chance of positive results due to bad retinal conditions, avoiding time-consuming patient management with unsatisfactory results. Full article

The POUNDS Lost trial is a 2-year clinical trial testing the effects of dietary interventions on weight loss. This study included 811 adults with overweight or obesity who were randomized to one of four diets that contained either 15% or 25% protein and 20% or 40% fat in a 2 × 2 factorial design. By 2 years, participants on average lost from 2.9 to 3.6 kg in body weight in the four intervention arms, while no significant difference was observed across the intervention arms. In POUNDS Lost, we performed a series of ancillary studies to detect intrinsic factors particular to genomic, epigenomic, and metabolomic markers that may modulate changes in weight and other cardiometabolic traits in response to the weight-loss dietary interventions. Genomic variants identified from genome-wide association studies (GWASs) on obesity, type 2 diabetes, glucose and lipid metabolisms, gut microbiome, and dietary intakes have been found to interact with dietary macronutrients (fat, protein, and carbohydrates) in relation to weight loss and changes of body composition and cardiometabolic traits. In addition, we recently investigated epigenomic modifications, particularly blood DNA methylation and circulating microRNAs (miRNAs). We reported DNA methylation levels at NFATC2IP, CPT1A, TXNIP, and LINC00319 were related to weight loss or changes of glucose, lipids, and blood pressure; we also reported thrifty miRNA expression as a significant epigenomic marker related to changes in insulin sensitivity and adiposity. Our studies have also highlighted the importance of temporal changes in novel metabolomic signatures for gut microbiota, bile acids, and amino acids as predictors for achievement of successful weight loss outcomes. Moreover, our studies indicate that biochemical, behavioral, and psychosocial factors such as physical activity, sleep disturbance, and appetite may also modulate metabolic changes during dietary interventions. This review summarized our major findings in the POUNDS Lost trial, which provided preliminary evidence supporting the development of precision diet interventions for obesity management. Full article