Protein Activity Research Articles

Two amino acid residues in the N-terminal region of the polymerase acidic protein determine the virulence of Eurasian avian-like H1N1 swine influenza viruses in mice.

Reassortant Eurasian avian-like H1N1 (rEA H1N1) viruses carrying the internal genes of H1N1/2009 virus have been circulating in pigs for more than 10 years and have caused sporadic human infections. The enhanced virulence phenotype of the rEA H1N1 viruses highlights potential risks to public health. However, the molecular mechanism underlying the viral pathogenicity of the currently circulating rEA H1N1 viruses remains unclear. In this study, we found that two naturally isolated rEA H1N1 swine influenza viruses, A/swine/Liaoning/FX38/2017 (FX38) and A/swine/Liaoning/SY72/2018 (SY72), possessed similar genetic characteristics but exhibited significantly different pathogenicity in a mouse model. Using reverse genetics, we demonstrated that amino acid mutations at positions 100 and 122 in the polymerase acidic (PA) protein had individual and synergistic effects on the polymerase activity and viral replication capacity in vitro, as well as the viral pathogenicity in mice. Furthermore, we revealed that amino acid residue 100 in PA influenced the transcription of viral RNA (vRNA) by altering the endonuclease activity, and amino acid residue 122 affected the synthesis of complementary RNA and messenger RNA by altering the RNA-binding ability and endonuclease activity of the PA protein. Taken together, we identified that two naturally occurring amino acid mutations in PA derived from H1N1/2009 virus are crucial determinants of the virulence of rEA H1N1 viruses and revealed the differential mechanism by which these two mutations affect the transcription and replication of vRNA. These findings will extend our understanding of the roles of PA in the virulence of influenza A viruses.IMPORTANCEMultiple genetic determinants are involved in the virulence of influenza A viruses. In this study, we identified two naturally occurring amino acid mutations, located at residues 100 and 122 in the polymerase acidic (PA) protein, which are associated with viral polymerase activity, replication competence, and pathogenicity in mice. In particular, we clarified the specific mechanism by which the two residues play an important role in viral transcription and replication. These findings will help to improve understanding the functions of amino acid residues in the N-terminal region of the PA protein involved in the pathogenicity of influenza A viruses.

Multiplexed profiling of intracellular protein abundance, activity, interactions and druggability with LABEL-seq.

Here we describe labeling with barcodes and enrichment for biochemical analysis by sequencing (LABEL-seq), an assay for massively parallel profiling of pooled protein variants in human cells. By leveraging the intracellular self-assembly of an RNA-binding domain (RBD) with a stable, variant-encoding RNA barcode, LABEL-seq facilitates the direct measurement of protein properties and functions using simple affinity enrichments of RBD protein fusions, followed by high-throughput sequencing of co-enriched barcodes. Measurement of ~20,000 variant effects for ~1,600 BRaf variants revealed that variation at positions frequently mutated in cancer minimally impacted intracellular abundance but could dramatically alter activity, protein-protein interactions and druggability. Integrative analysis identified networks of positions with similar biochemical roles and enabled modeling of variant effects on cell proliferation and small molecule-promoted degradation. Thus, LABEL-seq enables direct measurement of multiple biochemical properties in a native cellular context, providing insights into protein function, disease mechanisms and druggability.

Comparative Study of Cutaneous Squamous Cell Carcinogenesis in Different Hairless Murine Models

Background: In recent decades, a significant global increase in the incidence of non-melanoma skin cancer has been observed. To explore the pathogenesis of and potential therapeutic approaches for squamous cell carcinoma, various in vivo studies using mouse models have been conducted. However, investigations comparing different hairless mouse models, with or without melanin, as well as models with hypercholesterolemia and immunosuppression, in terms of their ability to induce squamous cell carcinoma have yet to be undertaken. Methods: Four mouse strains, namely SKH-hr1, SKH-hr2, SKH-hr2+ApoE, and immunodeficient Nude (Foxn1 knockout), were exposed to UVA and UVB radiation three times per week, initially to 1 Minimal Erythemal Dose (MED), incrementally increased weekly to a maximum dose of 3 MED. Clinical evaluation, photodocumentation, and biophysical parameters were monitored, along with proteasome protein activity and histopathological assessments. Results: The SKH-hr1 model primarily developed actinic keratosis without significant progression to invasive squamous cell carcinoma (SCC), while the SKH-hr2 and SKH-hr2+ApoE models exhibited a higher likelihood and intensity of papilloma and aggressive SCC formation, with the latter showing upregulated proteasome activity. Histopathological analysis confirmed the presence of poorly differentiated, invasive SCCs in the SKH-hr2 and SKH-hr2+ApoE models, contrasting with the less aggressive SCCs in the Nude mice and the mixed lesions observed in the SKH-hr1 mice. Conclusions: The SKH-hr2+ApoE and SKH-hr2 mice were identified as the most suitable for further exploration of squamous cell carcinogenesis. In contrast, the SKH-hr1 mice were found to be the least suitable, even though they are albino. Notably, proteasome analysis revealed a potential role of proteasome activity in squamous cell carcinogenesis.

Nondestructively Assemble Cell Membrane-Coated Nanoparticles by Host-Guest Interactions for Efficient Capture of Bioactive Compounds.

Cell membrane-coated nanoparticles (CNPs) have emerged as an attractive nanomedical tool. The basic premise is that the surface properties of natural cells can be integrated with the physical and chemical properties of nanoparticles by coating them with cell membranes. However, the degree of preservation of membrane proteins on nanoparticles, a key indicator related to the biomedical function of these biomimetic systems, is largely affected by the coating process. Herein, we report a supramolecular cell membrane conjugation strategy mediated by host-guest interactions to assemble CNPs without compromising protein activities. β-cyclodextrin (β-CD) was rapidly and stably inserted into the cell membrane by a lipid anchor without affecting the function of membrane proteins, thus attaching host-guest sites to the membrane surface. By harnessing the excellent binding affinity between β-CD attached to the membrane surface and adamantane, a supramolecular cell membrane-magnetic nanoparticle conjugate (CDM@AMNPs) was synthesized. Thanks to the nondestructive assembly of this strategy, CDM@AMNPs were endowed with a greater number of active binding sites, exhibiting efficient adsorption performance. This supramolecular conjugation strategy mediated by nonreceptor site-based host-guest interactions proposes a scalable and cell-friendly strategy for the development of highly efficient CNPs.

Lower activity of cholesteryl ester transfer protein (CETP) and the risk of dementia: a Mendelian randomization analysis

BackgroundElevated concentrations of low-density lipoprotein cholesterol (LDL-C) are linked to dementia risk, and conversely, increased plasma concentrations of high-density lipoprotein cholesterol (HDL-C) and apolipoprotein-A1 (Apo-A1) associate with decreased dementia risk. Inhibition of cholesteryl ester transfer protein (CETP) meaningfully affects the concentrations of these blood lipids and may therefore provide an opportunity to treat dementia.MethodsDrug target Mendelian randomization (MR) was employed to anticipate the on-target effects of lower CETP concentration (μg/mL) on plasma lipids, cardiovascular disease outcomes, autopsy confirmed Lewy body dementia (LBD), as well as Parkinson’s dementia.ResultsMR analysis of lower CETP concentration recapitulated the blood lipid effects observed in clinical trials of CETP-inhibitors, as well as protective effects on coronary heart disease (odds ratio (OR) 0.92, 95% confidence interval (CI) 0.89; 0.96), heart failure, abdominal aortic aneurysm, any stroke, ischemic stroke, and small vessel stroke (0.90, 95%CI 0.85; 0.96). Consideration of dementia related traits indicated that lower CETP concentrations were associated higher total brain volume (0.04 per standard deviation, 95%CI 0.02; 0.06), lower risk of LBD (OR 0.81, 95%CI 0.74; 0.89) and Parkinson’s dementia risk (OR 0.26, 95%CI 0.14; 0.48). APOE4 stratified analyses suggested the LBD effect was most pronounced in APOE-ε4 + participants (OR 0.61 95%CI 0.51; 0.73), compared to APOE-ε4- (OR 0.89 95%CI 0.79; 1.01); interaction p-value 5.81 × 10− 4.ConclusionsThese results suggest that inhibition of CETP may be a viable strategy to treat dementia, with a more pronounced effect expected in APOE-ε4 carriers.

Neutrophil Extracellular Traps in Pediatric Inflammatory Bowel Disease: A Potential Role in Ulcerative Colitis

Inflammatory bowel disease (IBD), encompassing Crohn’s disease (CD) and ulcerative colitis (UC), is a chronic inflammatory condition of the gut affecting both adults and children. Neutrophil extracellular traps (NETs) are structures released by activated neutrophils, potentially contributing to tissue damage in various diseases. This study aimed to explore the presence and role of NETs in pediatric IBD. We compared intestinal biopsies and peripheral blood from 20 pediatric IBD patients (UC and CD) to controls. Biopsy staining and techniques for neutrophil activation were used to assess neutrophil infiltration and NET formation. We also measured the enzymatic activity of key NET proteins and evaluated NET formation in UC patients in remission. Both UC and CD biopsies showed significantly higher levels of neutrophils and NETs compared to controls (p < 0.01), with UC exhibiting the strongest association. Peripheral blood neutrophils from UC patients at diagnosis displayed increased NET formation compared to controls and CD patients. Interestingly, NET formation normalized in UC patients following remission-inducing treatment. This pilot study suggests a potential role for NETs in pediatric IBD, particularly UC. These findings warrant further investigation into the mechanisms of NET involvement and the potential for targeting NET formation as a therapeutic strategy.

Unlocking the Potential of Circulating miRNAs as Biomarkers in Glioblastoma

Using microRNAs (miRNAs) as potential circulating biomarkers in diagnosing and treating glioblastoma (GBM) has garnered a lot of scientific and clinical impetus in the past decade. As an aggressive primary brain tumor, GBM poses challenges in early detection and effective treatment with significant current diagnostic constraints and limited therapeutic strategies. MiRNA dysregulation is present in GBM. The intricate involvement of miRNAs in altering cell proliferation, invasion, and immune escape makes them prospective candidates for identifying and monitoring GBM diagnosis and response to treatment. These miRNAs could play a dual role, acting as both potential diagnostic markers and targets for therapy. By modulating the activity of various oncogenic and tumor-suppressive proteins, miRNAs create opportunities for precision medicine and targeted therapies in GBM. This review centers on the critical role and function of circulating miRNA biomarkers in GBM diagnosis and treatment. It highlights their significance in providing insights into disease progression, aiding in early diagnosis, and potential use as targets for novel therapeutic interventions. Ultimately, the study of miRNA would contribute to improving patient outcomes in the challenging landscape of GBM management.

Immune protective, stress indicators, antioxidant, histopathological status, and heat shock protein gene expression impacts of dietary Bacillus spp. against heat shock in Nile tilapia, Oreochromis niloticus

This research evaluated the efficacy of mixed Bacillus strains probiotic supplements in mitigating acute thermal-induced stress in Nile tilapia (Oreochromis niloticus). Three experimental fish groups involved 135 Nile tilapia (49 ± 2 g); one control (no added probiotics), 0.5, and 1% of selected Bacillus strains (B. subtilis, B. licheniformis, and B. pumilus) for 58 days. After the feeding period, growth parameters, immunological parameters, stress biochemical markers, and antioxidant parameters in addition to genes related to stress and histopathological changes in fish, were assessed; subsequently subjected to heat shock at 36 ± 0.5 ◦C for 2 h. Before the heat challenge, our results exhibited a marked increase in the growth efficacy (P < 0.05), lower marked serum levels of triglycerides and cholesterol, and tissue malondialdehyde (MDA) levels along with significantly increased superoxide dismutase (SOD) and catalase (CAT) enzymes activity in fish-fed Bacillus probiotic at 0.5% concerning the control group (P < 0.05). There were no significant changes in the serum levels of glucose, cortisol, lactate, phagocytic activity, respiratory burst (ROS), total immunoglobulin Ig, alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), total protein, albumin, globulin, uric acid, urea, creatinine, as well as HSP70, GST, and GPx mRNA expression in most of the probiotic groups compared to the control group (P > 0.05). When Nile tilapia was exposed to heat stress, supplementation with Bacillus probiotic in the diet significantly decreased most of the indices related to serum biochemical (ALT (P < 0.01; P < 0.001), AST (P < 0.01), LDH (P < 0.01), urea (P < 0.05), and creatinine (P < 0.01)), triglycerides (P < 0.001; (P < 0.01)), cholesterol (P < 0.01; (P < 0.05)), glucose (P < 0.001), and cortisol (P < 0.01; (P < 0.05)), with tissue oxidative stress MDA levels (P < 0.05), and HSP70 mRNA expression (P < 0.01; P < 0.001), aligned with the stressed control group. In addition, a notable upsurge in the total protein, albumin, globulin, phagocytic and ROS activities, and total Ig, as well as the enzymatic antioxidant ability (SOD, CAT) (P < 0.01), with GST and GPx mRNA expression (P < 0.05; P < 0.01), were shown in fish-fed Bacillus spp. post-exposure compared with the stressed control group. Besides, no histopathological alterations were revealed in the spleen and brain of fish pre- and post-heat exposure. According to our findings, diet supplementation of Bacillus species has the potential to combat the suppressive effects of heat shock in Nile tilapia.

Value of Functional Protein S Activity Assays for Detection of Type II Protein S Deficiency

Abstract Background Protein S deficiency is a hereditary thrombophilia which occurs in three forms: Type I (quantitative), type II (qualitative) and type III (qualitative due to a gain of function). Literature suggests that of these, type II is the rarest and most difficult to diagnose. Type II protein S deficiency is only detectable by assaying protein S activity with a functional, clot-based test. However, this test is susceptible to interference and has a lower specificity when compared to antigen assays used to detect free and total protein S levels. Subsequently, the Choosing Wisely Guidelines released by the ABIM Foundation recommends against using protein S activity assays entirely. Instead, they suggest using only free protein S antigen assays. Although the rationale is to avoid false positive results from the protein S activity assay, it ultimately may result in a failure to detect type II protein S deficiency. Objective This study, performed at a tertiary medical center in the Midwestern region of the United States, aims to determine the proportion of false positives in functional assays as well as the percent of true type II protein S deficiency cases within this testing population. Methods All cases with abnormal protein S function within the institution were evaluated from July 2018 through July 2023 and classified as acquired or hereditary deficiencies. Cases were classified as acquired if there was evidence of liver disease, vitamin K deficiency, ongoing thrombosis, consumption, disseminated intravascular coagulopathy, or pregnancy. Possible hereditary deficiency cases were classified as type I, type II and type III: Type I if all assays were low, type III if both active and free protein S assays were low with normal total protein S, and type II if only protein S activity was low. The cutoff defined for normal total and free protein S levels was greater than 70% activity, and the low protein S activity cutoff was defined as less than 50%. Patients with low protein S activity but normal immunologic protein S and an elevated factor VIII were considered falsely low values. Chart review will be performed for remaining cases with low protein S activity to rule out confounders such as vitamin K deficiency, warfarin use, acute phase reaction, or pregnancy. Results From a total of 8805 panels performed to assess for possible thrombophilia, 1580 (18%) were found to have decreased protein S activity. Of these cases, 599 (38%) were considered falsely decreased due to elevated factor VIII levels. 322 cases (3.7%) were considered possible hereditary deficiencies: 217 type III (2.5%), 27 type II (0.3%), and 80 type I (0.9%). Discussion Our data indicates the prevalence of subtypes may be differently distributed than previously believed, which may call for current guidelines to be reevaluated.

Evaluation of Soumbara (Parkia biglobosa) Quality During Storage Using Mid-Infrared Spectroscopy and Physicochemical Methods

The fermented seed of néré (Parkia biglobosa), known as Soumbara, is a condiment widely consumed by the population in West Africa and Guinea in particular. The present study aims to use physicochemical, colorimetric methods and mid-infrared (MIR) spectroscopy to evaluate the evolution of the quality of Soumbara during storage (30 days). Thus, 5 kg of Soumbara were taken, subdivided into 7 batches of samples, and analysed every 5 days for 30 days. Physicochemical analyses (water activity, moisture, proteins, and total ash) would show that the storage duration induced a variation during storage; for example, the total ash contents on days 1, 5, 10, 15, 20, 25 and 30 are respectively 3.96 ± 0.25, 3.89 ± 0.43, 3.97 ± 0, 23, 3.99 ± 0.12, 4.54 ± 0.14, 4.57 ± 0.2 and 4.8 ± 0.21%. During the first 15 days of storage, no significant differences (p &lt; 0.05) were observed between samples. The colorimetric measurements showed that the storage duration did not significantly affect the values of a* and b*, except the L* value which decreased significantly (p&lt;0.05) (42.16 ± 0.015 on day 1 to 40.02 ± 0.015 on day 30). Applying statistical methods to mid-infrared spectroscopy allowed a clear distinction between the Soumbara samples during storage for 30 days. The partial least squares regression (PLSR) applied to MIR spectra made it possible to predict water, moisture and protein activity levels, since R2 greater than 0.99 were obtained. The results demonstrated that mid-infrared spectroscopy coupled with chemometric tools could be used as a rapid screening tool to assess and monitor the quality of Soumbara during storage.

Prognostic Significance of Markers of Endothelial Dysfunction in Case of Severe Combined Blunt Abdominal Trauma

INTRODUCTION. In case of polytrauma, an imbalance occurs between substances produced by the endothelium, the rheological properties of the blood are disrupted, the content of procoagulants increases, the permeability of the vascular wall increases, which contributes to thrombus formation and increased tissue hypoxia, and subsequently to the development of multiple organ failure, and, of course, affects the course of traumatic disease, the development of complications and mortality. However, specific markers of endothelial dysfunction and their levels, by which it is possible to reliably predict the course of traumatic disease in severe combined closed abdominal trauma for the appropriate pathogenetic treatment have not yet been determined.AIM OF THE STUDY. To determine the significance of some markers of endothelial dysfunction (C-reactive protein, von Willebrand factor, the number of desquamated endothelial cells in the blood) in combined blunt abdominal trauma to predict the likelihood of complications and adverse outcomes.MATERIAL AND METHODS. The main group consisted of 31 patients with severe combined blunt abdominal trauma, the comparison group consisted of 5 patients operated on for large ventral hernias. All patients underwent blood tests for C-reactive protein, von Willebrand factor, the number of desquamated endothelial cells.RESULTS. The level of all three considered markers of endothelial dysfunction in severe abdominal trauma significantly differs from that in operated non-traumatized patients, which allows differential diagnostics to be made between abdominal wall contusion and damage to internal organs; extremely high values suggest an unfavorable course of traumatic disease and a high probability of a fatal outcome.CONCLUSION. Taking into account that the development of traumatic disease is based on damage to the vascular endothelium, it is necessary to consider as prognostic indicators changes in the level of activity of C-reactive protein, von Willebrand factor, desquamated endothelial cells in patients with severe combined blunt abdominal trauma.

A Novel Secreted Protein of Fusarium oxysporum Promotes Infection by Inhibiting PR-5 Protein in Plant.

Fusarium oxysporum, an important soilborne fungal pathogen that causes serious Fusarium wilt disease, secretes diverse effectors during the infection. In this study, we identified a novel secreted cysteine-rich protein, FolSCP1, which contains unknown protein functional domain. Here, we characterized FolSCP1 as a secreted virulence factor that promotes the pathogen infection of host plants by inhibiting diverse plant defence responses. FolSCP1 interacted with the pathogenesis-related 5 (PR-5) protein SlPR5, a positive regulator of tomato plant immunity against multiple tomato pathogens, and effectively attenuated the antifungal activity of the tomato PR-5 protein. FoSCP1, a homologue of FolSCP1, was secreted by a F. oxysporum isolate from infected tobacco and targeted the tobacco PR-5 protein NtPR5 to suppress plant defence for further infection. In summary, our study revealed a fungal virulence strategy in which F. oxysporum secrete effectors that interfere with plant immunity by binding to the PR-5 protein of the host plant and inhibiting its biological activity, thereby promoting fungal infection.

Opioid receptors reveal a discrete cellular mechanism of endosomal G protein activation.

Many GPCRs initiate a second phase of G protein-mediated signaling from endosomes, which inherently requires an increase in G protein activity on the endosome surface. G s -coupled GPCRs are thought to achieve this by internalizing and allosterically activating cognate G proteins again on the endosome membrane. Here we demonstrate that the μ-opioid receptor (MOR), a G i -coupled GPCR, increases endosomal G protein activity in a different way. Leveraging conformational biosensors, we resolve the subcellular activation dynamics of endogenously expressed MOR and G i/o -subclass G proteins. We show that MOR activation triggers a transient increase of active-state G i/o on the plasma membrane that is followed by a prolonged increase on endosomes. Contrary to the G s -coupled GPCR paradigm, however, we show that the MOR-induced increase of active-state G i/o on endosomes requires neither internalization of MOR nor activation of MOR in the endosome membrane. We propose a distinct and additional cellular mechanism for GPCR-triggered elevation of G protein activity on endosomes that is mediated by regulated trafficking of the activated G protein rather than its activating GPCR.

Genetic knock-in of EIF2AK3 variants reveals differences in PERK activity in mouse liver and pancreas under endoplasmic reticulum stress

Common single-nucleotide variants (SNVs) of eukaryotic translation initiation factor 2 alpha kinase 3 (EIF2AK3) slightly increase the risk of disorders in the periphery and the central nervous system. EIF2AK3 encodes protein kinase RNA-like endoplasmic reticulum kinase (PERK), a key regulator of ER stress. Three exonic EIF2AK3 SNVs form the PERK-B haplotype, which is present in 28% of the global population. Importantly, the precise impact of these SNVs on PERK activity remains elusive. In this study, we demonstrate that PERK-B SNVs do not alter PERK expression or basal activity in vitro and in the novel triple knock-in mice expressing the exonic PERK-B SNVs in vivo. However, the kinase activity of PERK-B protein is higher than that of PERK-A in a cell-free assay and in mouse liver homogenates. Pancreatic tissue in PERK-B/B mice also exhibit increased susceptibility to apoptosis under acute ER stress. Monocyte-derived macrophages from PERK-B/B mice exhibit higher PERK activity than those from PERK-A/A mice, albeit with minimal functional consequences at acute timepoints. The subtle PERK-B-driven effects observed in liver and pancreas during acute stress implicate PERK as a contributor to disease susceptibility. The novel PERK-B mouse model provides valuable insights into ER stress-induced PERK activity, aiding the understanding of the genetic basis of disorders associated with ER stress.

Targeting USP11 regulation by a novel lithium-organic coordination compound improves neuropathologies and cognitive functions in Alzheimer transgenic mice.

Alzheimer's Disease (AD), as the most common neurodegenerative disease worldwide, severely impairs patients' cognitive functions. Although its exact etiology remains unclear, the abnormal aggregations of misfolded β-amyloid peptide and tau protein are considered pivotal in its pathological progression. Recent studies identify ubiquitin-specific protease 11 (USP11) as the key regulator of tau deubiquitination, exacerbating tau aggregation and AD pathology. Thereby, inhibiting USP11 function, via either blocking USP11 activity or lowering USP11 protein level, may serve as an effective therapeutic strategy against AD. Our research introduces IsoLiPro, a unique lithium isobutyrate-L-proline coordination compound, effectively lowers USP11 protein level and enhances tau ubiquitination in vitro. Additionally, long-term oral administration of IsoLiPro dramatically reduces total and phosphorylated tau levels in AD transgenic mice. Moreover, IsoLiPro also significantly lessens β-amyloid deposition and synaptic damage, improving cognitive functions in these animal models. These results indicate that IsoLiPro, as a novel small-molecule USP11 inhibitor, can effectively alleviate AD-like pathologies and improve cognitive functions, offering promise as a potential multi-targeting therapeutic agent against AD.

Reusable Microfluidic Chambers for Single-Molecule Microscopy.

Maintaining a consistent environment in single-molecule microfluidic chambers containing surface-bound molecules requires laborious cleaning and surface passivation procedures. Despite such efforts, variations in nonspecific binding and background signals commonly occur across different chambers. Being able to reuse the chambers without degrading the surface promises significant practical and fundamental advantages; however, this necessitates removing the molecules attached to the surface, such as DNA, proteins, lipids, or nanoparticles. Biotin-streptavidin attachment is widely used for such attachments, as biotin can be readily incorporated into these molecules. In this study, we present single-molecule fluorescence experiments that demonstrate effective resetting and recycling of the chambers at least 10 times by using photocleavable biotin (PC-biotin) and UV-light exposure. This method differs from alternatives as it does not utilize any harsh chemical treatment of the surface. We show that all bound molecules (utilizing various PC-biotin attachment chemistries) can be removed from the surface by a 5 min UV exposure of a specific wavelength. Nonoptimal wavelengths and light sources showed varying degrees of effectiveness. Our approach does not result in any detectable degradation of surface quality as assessed by the nonspecific binding of fluorescently labeled DNA and protein samples and the recovery of the DNA secondary structure and protein activity. The speed and efficiency of the resetting process, the cost-effectiveness of the procedure, and the widespread use of biotin-streptavidin attachment make this approach adaptable for a wide range of single-molecule applications.

Plant protein, fibre and physical activity solutions to address poor appetite and prevent undernutrition in older adults: study protocol for the APPETITE randomised controlled trial.

Reduced appetite with ageing is a key factor that may increase risk of undernutrition. The objective of this study is to determine the impact of innovative plant protein fibre (PPF) products within a personalised optimised diet (PD), a physical activity (PA) programme, and their combination on appetite, and other nutritional, functional and clinical outcomes in community-dwelling older adults in a multi-country randomised controlled intervention trial. One hundred and eighty community-dwelling adults (approximately sixty per trial centre in Germany, Ireland and Italy) aged 65 years and over will be recruited to participate in a 12-week, parallel-group, controlled trial. Participants will be randomised into one of four groups: 1, PD (incorporating two PPF products): 2, PA; 3, PD + PA; and 4, no intervention (control). The primary outcome is appetite measured by visual analogue scales and energy intake from an ad libitum test meal. Secondary outcomes include fasting and postprandial appetite-related gut hormones, Simplified Nutritional Appetite Questionnaire score, body composition, cardiorespiratory fitness, muscle strength, physical function and PA. In addition, self-efficacy, cognitive status, dietary restraint, depressive symptoms and compliance and acceptability of the intervention will be assessed. Metabolomic profiles, RMR, muscle motor unit properties and gut microbiome will also be assessed to explore potential underlying mechanisms. This multi-centre randomised controlled trial will advance knowledge on how PD (incorporating PPF products), PA and their combination influence appetite, nutritional status and related health outcomes in community-dwelling older adults and contribute to the prevention of undernutrition. Trial registration: Clinical Trials.gov Registry NCT05608707 (registered on 2 November 2022). Protocol Version: NCT05608707 Version 4 (registered on 29 September 2023).

Pelargonic acid’s interaction with the auxin transporter PIN1: A potential mechanism behind its phytotoxic effects on plant metabolism

Pelargonic acid (PA) is a saturated fatty acid commonly found in several organisms, that is known for its phytotoxic effect and its use as bioherbicide for sustainable weed management. Although PA is already commercialised as bioherbicide, its molecular targets and mode of action is unknown according to the Herbicide Resistance Action Committee. Therefore, the aim of this work was focusing on the way this natural active substance impacts the plant metabolism of the model species Arabidopsis thaliana. PA caused increase of secondary and adventitious roots, as well as torsion, loss of gravitropism and phytotoxic effects. Moreover, PA altered the cellular arrangement and the PIN proteins activity. Computational simulations revealed that the intermolecular interactions between PA and the polar auxin transporter protein PIN1 are very similar to those established between the natural auxin IAA and PIN1. However, under intracellular conditions, the PA-PIN1 binding is more energetically stable than the IAA-PIN1. These results suggest that PA could act as an auxin-mimics bioherbicide. The exogenous application of PA would be responsible for the alterations observed both at structural and ultrastructural levels, which would be caused by the alteration on the transport of auxins into the plant, inducing root inhibition and ultimately total stop of root growth.

Multi-omics analyses were combined to construct ubiquitination-related features in colon adenocarcinoma and identify ASNS as a novel biomarker

BackgroundAs one of the malignant tumors with the highest incidence and fatality in the world, colon adenocarcinoma (COAD) has a very complex pathogenic mechanism, which has not yet been fully elucidated. Ubiquitin can regulate cell proliferation, cell cycle, apoptosis, DNA damage repair, and other processes by changing the activity of substrate proteins or causing ubiquitin-proteasome degradation. These are the key links in the pathogenesis of COAD, and ubiquitin plays an important role in the occurrence and development of COAD.MethodsWe integrated transcriptomics, single-cell and clinical omics, and TCGA and GEO databases of COAD patient data. Cox and Lasso regression was employed to assess ubiquitination genes in COAD for generating ubiquitination-related features. The aim was to evaluate the prognostic value of these features for tumors and their impact on the immune microenvironment. At the same time, the expression level of model genes was further analyzed using single-cell data. Finally, the expression and function of ASNS, a key gene for this trait, were detected in vitro.ResultsIn our study, based on identifiable changes in the expression of marker genes, this feature can be used to classify patients with COAD. Kaplan-Meier survival analysis indicated that those with elevated risk scores in each cohort experienced inferior outcomes. There is good validation in both the training queue and the validation queue. The results of the immune infiltration analysis showed that the immune infiltration rate was significantly increased in the high-risk group. After the knockdown of ASNS, an important gene in the signature, the activity and migration capacity of SW620 and RKO cell lines and colony formation capacity were dramatically reduced in cell tests.ConclusionWe screened ubiquitination-related genes and constructed ubiquitination-related features, which can be used as reliable prognostic indicators of COAD. ASNS was identified as a possible biomarker for COAD.

X-ray crystal structure of proliferating cell nuclear antigen 1 from Aeropyrum pernix.

Proliferating cell nuclear antigen (PCNA) plays a critical role in DNA replication by enhancing the activity of various proteins involved in replication. Inthis study, the crystal structure of ApePCNA1, one of three PCNAs from thethermophilic archaeon Aeropyrum pernix, was elucidated. ApePCNA1 was cloned and expressed in Escherichia coli and the protein was purified and crystallized. The resulting crystal structure determined at 2.00 Å resolution revealed that ApePCNA1 does not form a trimeric ring, unlike PCNAs from other domains of life. It has unique structural features, including a long interdomain-connecting loop and a PIP-box-like sequence at the N-terminus, indicating potential interactions with other proteins. These findings provide insights into the functional mechanisms of PCNAs in archaea and their evolutionary conservation across different domains of life. A modified medium and protocol were used to express recombinant protein containing the lac operon. The expression of the target protein increased and the total incubation time decreased when using this system compared with those of previous expression protocols.