An Inflection Point in High-Throughput Proteomics with Orbitrap Astral: Analysis of Biofluids, Cells, and Tissues.

This Technical Note presents a comprehensive proteomics workflow for the new combination of Orbitrap and Astral mass analyzers across biofluids, cells, and tissues. Central to our workflow is the integration of Adaptive Focused Acoustics (AFA) technology for cells and tissue lysis to ensure robust and reproducible sample preparation in a high-throughput manner. Furthermore, we automated the detergent-compatible single-pot, solid-phase-enhanced sample Preparation (SP3) method for protein digestion. The synergy of these advanced methodologies facilitates a robust and high-throughput approach for cell and tissue analysis, an important consideration in translational research. This work disseminates our platform workflow, analyzes the effectiveness, demonstrates the reproducibility of the results, and highlights the potential of these technologies in biomarker discovery and disease pathology. For cells and tissues (heart, liver, lung, and intestine) proteomics analysis by data-independent acquisition mode, identifications exceeding 10,000 proteins can be achieved with a 24 min active gradient. In 200 ng injections of HeLa digest across multiple gradients, an average of more than 80% of proteins have a CV less than 20%, and a 45 min run covers ∼90% of the expressed proteome. This complete workflow allows for large swaths of the proteome to be identified and is compatible with diverse sample types.

A systematic comparison of FOSL1, FOSL2 and BATF-mediated transcriptional regulation during early human Th17 differentiation.

Th17 cells are essential for protection against extracellular pathogens, but their aberrant activity can cause autoimmunity. Molecular mechanisms that dictate Th17 cell-differentiation have been extensively studied using mouse models. However, species-specific differences underscore the need to validate these findings in human. Here, we characterized the human-specific roles of three AP-1 transcription factors, FOSL1, FOSL2 and BATF, during early stages of Th17 differentiation. Our results demonstrate that FOSL1 and FOSL2 co-repress Th17 fate-specification, whereas BATF promotes the Th17 lineage. Strikingly, FOSL1 was found to play different roles in human and mouse. Genome-wide binding analysis indicated that FOSL1, FOSL2 and BATF share occupancy over regulatory regions of genes involved in Th17 lineage commitment. These AP-1 factors also share their protein interacting partners, which suggests mechanisms for their functional interplay. Our study further reveals that the genomic binding sites of FOSL1, FOSL2 and BATF harbour hundreds of autoimmune disease-linked SNPs. We show that many of these SNPs alter the ability of these transcription factors to bind DNA. Our findings thus provide critical insights into AP-1-mediated regulation of human Th17-fate and associated pathologies.

Autonomous service for managing real time notification in detection of COVID-19 virus.

In today's world, the most prominent public issue in the field of medicine is the rapid spread of viral sickness. The seriousness of the disease lies in its fast spreading nature. The main aim of the study is the proposal of a framework for the earlier detection and forecasting of the COVID-19 virus infection amongst the people to avoid the spread of the disease across the world by undertaking the precautionary measures. According to this framework, there are four stages for the proposed work. This includes the collection of necessary data followed by the classification of the collected information which is then taken in the process of mining and extraction and eventually ending with the process of decision modelling. Since the frequency of the infection is very often a prescient one, the probabilistic examination is measured as a degree of membership characterised by the fever measure related to the same. The predictions are thereby realised using the temporal RNN. The model finally provides effective outcomes in the efficiency of classification, reliability, the prediction viability etc.

Quantitative proteomic characterization and comparison of T helper 17 and induced regulatory T cells.

The transcriptional network and protein regulators that govern T helper 17 (Th17) cell differentiation have been studied extensively using advanced genomic approaches. For a better understanding of these biological processes, we have moved a step forward, from gene- to protein-level characterization of Th17 cells. Mass spectrometry-based label-free quantitative (LFQ) proteomics analysis were made of in vitro differentiated murine Th17 and induced regulatory T (iTreg) cells. More than 4,000 proteins, covering almost all subcellular compartments, were detected. Quantitative comparison of the protein expression profiles resulted in the identification of proteins specifically expressed in the Th17 and iTreg cells. Importantly, our combined analysis of proteome and gene expression data revealed protein expression changes that were not associated with changes at the transcriptional level. Our dataset provides a valuable resource, with new insights into the proteomic characteristics of Th17 and iTreg cells, which may prove useful in developing treatment of autoimmune diseases and developing tumor immunotherapy.

Analysis of the plasma proteome using iTRAQ and TMT-based Isobaric labeling.

Over the past decade, chemical labeling with isobaric tandem mass tags, such as isobaric tags for relative and absolute quantification reagents (iTRAQ) and tandem mass tag (TMT) reagents, has been employed in a wide range of different clinically orientated serum and plasma proteomics studies. In this review the scope of these works is presented with attention to the areas of research, methods employed and performance limitations. These applications have covered a wide range of diseases, disorders and infections, and have implemented a variety of different preparative and mass spectrometric approaches. In contrast to earlier works, which struggled to quantify more than a few hundred proteins, increasingly these studies have provided deeper insight into the plasma proteome extending the numbers of quantified proteins to over a thousand.

ESWL for large pancreatic calculi: Report of over 5000 patients.

INTRODUCTION: The primary aim of this study was to evaluate efficacy, safety and short-term pain relief after ESWL for large pancreatic calculi in over 5000 patients at a single center. METHODS: This is a retrospective analysis of prospectively collected data. Patients with painful calculi >5 mm, located in the head, neck and body region in the MPD, who were not amenable for extraction by the standard procedure of endoscopic pancreatic sphincterotomy were subjected to ESWL using a third generation dual focus lithotripter. Patients were followed up at 6 months for outcome evaluation. RESULTS: A total of 5124 patients (66% males) were subjected to ESWL. Majority of stones (79.2%) were radiopaque. Single calculi were seen in 3851 (75.1%).The majority of stones were located in head region of MPD in 2824 (55.1%) patients. 4386 (85.5%) patients required 3 or less sessions for fragmentation and complete stone clearance was achieved in 3722 (72.6%). EPS was performed in 5022 (98%) while PD stenting was required in 3536 (69%) patients. Of the 4280 patients followed up for 6 months, 3529 (82.6%) patients were pain free. Another 512 (11.9%) patients had significant reduction in VAS score. In 229 (5.3%) there was no decrease in pain intensity. Minor and self-limiting complications were reported in 1153 (22.5%). DISCUSSION: Our study confirms the safety and efficacy and short-term pain relief of ESWL for large calculi in the MPD. In properly selected patients, this should be offered as the first line of therapy for all large MPD calculi not amenable to the standard techniques of stone extraction.

Synthesis of Unsymmetrical α,α-Diarylacetates.

Ethyl bromofluoroacetate has been developed as a precursor for the convenient synthesis of unsymmetrical α,α-diarylacetates featuring indoles, anilines, and other electron-rich aromatics. In conjunction with a mild Lewis acid catalyzed C-N → C-C exchange, intermediate arylglycines can be synthesized and transformed into α,α-diarylacetates in a one-pot protocol, resulting in a net diarylation reaction exhibiting a wide scope. In the context of diarylacetates, the synthetic equivalence of the fluorinated reagent with α-nitro-α-diazo carbonyls was established.

Visible Light Mediated Organocatalytic Activation of Ethyl Bromofluoroacetate: Coupling with Indoles and Anilines.

The activation of ethyl bromofluoroacetate employing a visible light mediated, Eosin Y catalyzed photoredox transformation is reported. Using indoles and anilines as nucleophiles, the reaction leads to the formation of two C(sp(2))-C(sp(3)) bonds resulting in an efficient synthesis of bisindolyl and bisanilinoyl acetate derivatives. Application of this method to the direct synthesis of unsymmetrical diarylacetates featuring indoles and N-substituted anilines was also demonstrated.

Comparative proteomics analysis of the mouse mini-gut organoid: insights into markers of gluten challenge from celiac disease intestinal biopsies.

Introduction: Organoid models enable three-dimensional representation of cellular systems, providing flexible and accessible research tools, and can highlight key biomolecules. Such models of the intestinal epithelium can provide significant knowledge for the study of celiac disease and provide an additional context for the nature of markers observed from patient biopsy data. Methods: Using LC-MS/MS, the proteomes of the crypt and enterocyte-like states of a mouse mini-gut organoid model were measured. The data were further compared with published biopsy data by comparing the changes induced by gluten challenge after a gluten-free diet. Results and discussion: These analyses identified 4,850 protein groups and revealed how 400 putative biomarkers of dietary challenge were differentially expressed in the organoid model. In addition to the extensive changes within the differentiated cells, the data reiterated the disruption of the crypt-villus axis after gluten challenge. The mass spectrometry data are available via ProteomeXchange with the identifier PXD025690.

Development of renewable energy fed three-level hybrid active filter for EV charging station load using Jaya grey wolf optimization.

This work develops a hybrid active power filter (HAPF) in this article to operate in conjunction with the energy storage system (ESS), wind power generation system (WPGS), and solar energy system (SES). It employs three level shunt voltage source converters (VSC) connected to the DC-bus. Optimization of the gain values of the fractional-order proportional integral derivative controller (FOPIDC) and parameter values of the HAPF is achieved using the Jaya grey wolf hybrid algorithm (GWJA). The primary objectives of this study, aimed at enhancing power quality (PQ), include: (1) ensuring swift stabilization of DC link capacitor voltage (DCLCV); (2) reducing harmonics and improving power factor (PF); (3) maintaining satisfactory performance under different combinations of loads like EV charging load, non linear load and solar irradiation conditions. The proposed controller's performance is evaluated through three test scenarios featuring different load configurations and irradiation levels. Additionally, the HAPF is subjected to design using other optimization algorithms such as genetic algorithm (GA), particle swarm optimization (PSO), and ant colony optimization (ACO) to assess their respective contributions to PQ improvement.

Risk Factor Profile for Cardiovascular Diseases Among Farmhouse Residents in Vijayapura District, Karnataka: A Cross-Sectional Study.

Background Cardiovascular illnesses are widely regarded as the leading cause of mortality worldwide, with 17.9 million deaths every year. Rheumatic heart disease, coronary heart disease, and cerebrovascular illness are among the conditions relating to the heart and blood vessels together, known as cardiovascular diseases. The main behavioural risk factors for heart disease and stroke include poor diet, inactivity, tobacco use, and excessive alcohol consumption. In contrast, one of the significant environmental risk factors is air pollution. People may experience high blood pressure and high blood sugar due to obesity and overweight as risk factors. Objectives This study aims to find the risk factor profile for cardiovascular diseases among the farmhouse residents of the Vijayapura district, India. Materials and methods A cross-sectional descriptive study was conducted with the farmhouse inhabitants of the Vijayapura district. Using a prestructured proforma that included details on the sociodemographic profile and risk factors of the farmhouse dwellers, 450 sample participants were questioned. Five families within a chosen primary sampling unit (PSU) were chosen randomly from a pool of PSUs that were picked using a probability proportional to size sampling technique. All characteristics were summarized descriptively. Results The study included 450 participants, 50.9% males and 49.1% females. The age range of 21.4% of the male participants was between 41 and 50, whereas 21.7% of the female participants belonged to the 41 to 50 age group. The study showed a proportion of them had the habit of alcohol consumption (5.8%), chewing tobacco (2%), and smoking (1.6%). Compared to female participants (1.3% vs. 1.3%), the majority of male individuals (4.8% vs. 7%) had hypertension and diabetes mellitus, respectively. Conclusion The findings of the current research indicated that the majority of farm inhabitants in the rural Vijayapura district were illiterate, belonged to a lower socioeconomic class, and had intermediate and behavioural risk factors for cardiovascular illnesses.

Posterior Cruciate Ligament Avulsion Fracture of Tibia: Fixation with Screw and Augmentation Using Ethibond Suture for Improved Clinical Outcomes.

Introduction: The posterior cruciate ligament (PCL) is a vital structure in knee biomechanics, and its avulsion fractures present a unique challenge. This prospective cohort study was conducted at Grant Government Medical College and JJ Hospital, Mumbai, aimed to assess the clinical outcomes of open reduction and internal fixation (ORIF) utilizing cancellous screws and ethibond suture augmentation for PCL avulsion fractures. PCL avulsion fractures often result from traumatic incidents, such as road traffic accidents, and are associated with complications if left untreated. Materials and Methods: Patients aged 20-45 years with isolated PCL avulsion fractures, confirmed by clinical and radiological assessments, were included. Seven eligible patients underwent ORIF, and outcomes were evaluated through clinical assessments, radiological imaging, and the Lysholm knee scoring system. Follow-ups were conducted for a mean time of 13.2 months, assessing stability, range of motion, and complications. Results: The study demonstrated a mean post-operative Lysholm score of 93.8, significantly improved from the pre-operative score of 49.6. Post-operative knee flexion averaged 125.2°. Fracture healing was observed in all cases, and complications were minimal. The study provides evidence of the effectiveness of ORIF with ethibond suture augmentation for PCL avulsion fractures. Conclusion: The study concludes that the proposed surgical technique yields positive outcomes, including enhanced knee functionality, successful fracture healing, and minimal complications. This approach, combining cancellous screws and ethibond suture augmentation, presents a promising option for the management of PCL avulsion fractures, contributing to the diverse landscape of effective treatment modalities.

Serum proteomics of mother-infant dyads carrying HLA-conferred type 1 diabetes risk.

In-utero and dietary factors make important contributions toward health and development in early childhood. In this respect, serum proteomics of maturing infants can provide insights into studies of childhood diseases, which together with perinatal proteomes could reveal further biological perspectives. Accordingly, to determine differences between feeding groups and changes in infancy, serum proteomics analyses of mother-infant dyads with HLA-conferred susceptibility to type 1 diabetes (n = 22), weaned to either an extensively hydrolyzed or regular cow's milk formula, were made. The LC-MS/MS analyses included samples from the beginning of third trimester, the time of delivery, 3 months postpartum, cord blood, and samples from the infants at 3, 6, 9, and 12 months. Correlations between ranked protein intensities were detected within the dyads, together with perinatal and age-related changes. Comparison with intestinal permeability data revealed a number of significant correlations, which could merit further consideration in this context.

An inflection point in high-throughput proteomics with Orbitrap Astral: analysis of biofluids, cells, and tissues.

This technical note presents a comprehensive proteomics workflow for the new combination of Orbitrap and Astral mass analyzers across biofluids, cells, and tissues. Central to our workflow is the integration of Adaptive Focused Acoustics (AFA) technology for cells and tissue lysis, to ensure robust and reproducible sample preparation in a high-throughput manner. Furthermore, we automated the detergent-compatible single-pot, solid-phase-enhanced sample Preparation (SP3) method for protein digestion, a technique that streamlines the process by combining purification and digestion steps, thereby reducing sample loss and improving efficiency. The synergy of these advanced methodologies facilitates a robust and high-throughput approach for cells and tissue analysis, an important consideration in translational research. This work disseminates our platform workflow, analyzes the effectiveness, demonstrates reproducibility of the results, and highlights the potential of these technologies in biomarker discovery and disease pathology. For cells and tissues (heart, liver, lung, and intestine) proteomics analysis by data-independent acquisition mode, identifications exceeding 10,000 proteins can be achieved with a 24-minute active gradient. In 200ng injections of HeLa digest across multiple gradients, an average of more than 80% of proteins have a CV less than 20%, and a 45-minute run covers ~90% of the expressed proteome. In plasma samples including naive, depleted, perchloric acid precipitated, and Seer nanoparticle captured, all with a 24-minute gradient length, we identified 87, 108, 96 and 137 out of 216 FDA approved circulating protein biomarkers, respectively. This complete workflow allows for large swaths of the proteome to be identified and is compatible across diverse sample types.

Fenton and photo-assisted advanced oxidative degradation of ionic liquids: a review.

Ionic liquids (ILs) are the class of materials which are purely ionic in nature and liquid at room temperature. Their remarkable properties like very low vapour pressure, non-inflammable and high heat resistance are responsible for their use as a very appealing solvent in a variety of industrial applications in place of regular organic solvents. Because ILs are water soluble to a certain extent, the industrial wastewater effluents are found to contaminate with their traces. The non-biodegradability of ILs attracts the attention of the researchers for their removal or degradation from wastewater. Numbers of methods are available for the treatment of wastewater. However, it is very crucial to use the most efficient method for the degradation of ILs. Advanced oxidation process (AOP) is one of the most important techniques for the treatment of ILs in wastewater which have been investigated during last decades. This review paper covers the cost-effective Fenton, photochemical and photocatalytic AOPs and their combination that could be applied for the degradation of ILs from the wastewater. Theoretical explanations of these AOPs along with experimental conditions and kinetics of degradation or removal of ILs from water and wastewater have been reported and compared. Finally, future perspectives of IL degradation are presented.

In Vitro Antibacterial, Cytotoxicity and Wound Healing Activities of Methanol and Aqueous Extracts from Achyranthes aspera.

Achyranthes aspera (Amaranthaceae) is a most important plant in the Indian medicinal system of Unani, Ayurveda, and Sidha. It is distributed throughout tropical countries particularly this medicinal herb is found as a weed all over India. The antibiotic-resistant bacteria treatment, many medicinal plants act as alternatives to antibiotics and are considered new resources for producing agents. The aim of this study was to evaluate the antioxidant, antibacterial, cytotoxicity, and wound-healing potential of methanolic and aqueous extracts of A. aspera. Physio-chemical parameters of A. aspera were screened by qualitative and quantitative phytochemicals and it revealed the existence of saponins, alkaloids, glycosides, and flavonoids in each extract. The methanolic extract showed strong scavenging effects against 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical in comparison with the aqueous extract. The methanolic extract of A. aspera was bactericidal at 100 µg/ml concentration against Gram-negative (Escherichia coli, Pseudomonas aeruginosa) and Gram-positive bacteria (Bacillus subtilis, Staphylococcus aureus). In addition, the finding suggests A. aspera extract promotes in vitro wound healing by enhancement of Vero cells and has the potential for the treatment of wounds. The study concludes the strong radical scavenging effect of Achyranthes aspera along with remarkable antibacterial and wound healing actions.

Serum APOC1 levels are decreased in young autoantibody positive children who rapidly progress to type 1 diabetes.

Better understanding of the early events in the development of type 1 diabetes is needed to improve prediction and monitoring of the disease progression during the substantially heterogeneous presymptomatic period of the beta cell damaging process. To address this concern, we used mass spectrometry-based proteomics to analyse longitudinal pre-onset plasma sample series from children positive for multiple islet autoantibodies who had rapidly progressed to type 1 diabetes before 4 years of age (n = 10) and compared these with similar measurements from matched children who were either positive for a single autoantibody (n = 10) or autoantibody negative (n = 10). Following statistical analysis of the longitudinal data, targeted serum proteomics was used to verify 11 proteins putatively associated with the disease development in a similar yet independent and larger cohort of children who progressed to the disease within 5 years of age (n = 31) and matched autoantibody negative children (n = 31). These data reiterated extensive age-related trends for protein levels in young children. Further, these analyses demonstrated that the serum levels of two peptides unique for apolipoprotein C1 (APOC1) were decreased after the appearance of the first islet autoantibody and remained relatively less abundant in children who progressed to type 1 diabetes, in comparison to autoantibody negative children.

HIC1 interacts with FOXP3 multi protein complex: Novel pleiotropic mechanisms to regulate human regulatory T cell differentiation and function.

Transcriptional repressor, hypermethylated in cancer 1 (HIC1) participates in a range of important biological processes, such as tumor repression, immune suppression, embryonic development and epigenetic gene regulation. Further to these, we previously demonstrated that HIC1 provides a significant contribution to the function and development of regulatory T (Treg) cells. However, the mechanism by which it regulates these processes was not apparent. To address this question, we used affinity-purification mass spectrometry to characterize the HIC1 interactome in human Treg cells. Altogether 61 high-confidence interactors were identified, including IKZF3, which is a key transcription factor in the development of Treg cells. The biological processes associated with these interacting proteins include protein transport, mRNA processing, non-coding (ncRNA) transcription and RNA metabolism. The results revealed that HIC1 is part of a FOXP3-RUNX1-CBFB protein complex that regulates Treg signature genes thus improving our understanding of HIC1 function during early Treg cell differentiation.

Cardiovascular-related proteomic changes in ECFCs exposed to the serum of COVID-19 patients.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection significantly affects the cardiovascular system, causing vascular damage and thromboembolic events in critical patients. Endothelial dysfunction represents one of the first steps in response to COVID-19 that might lead to cardiovascular complications and long-term sequelae. However, despite the enormous efforts in the last two years, the molecular mechanisms involved in such processes remain poorly understood. Herein, we analyzed the protein changes taking place in endothelial colony forming cells (ECFCs) after the incubation with the serum from individuals infected with COVID-19, whether asymptomatic or critical patients, by application of a label free-quantitative proteomics approach. Specifically, ECFCs from healthy individuals were incubated ex-vivo with the serum of either COVID-19 negative donors (PCR-/IgG-, n:8), COVID-19 asymptomatic donors at different infective stages (PCR+/ IgG-, n:8and PCR-/IgG+, n:8), or hospitalized critical COVID-19 patients (n:8), followed by proteomics analysis. In total, 590 proteins were differentially expressed in ECFCs in response to all infected serums. Predictive analysis highlighted several proteins like CAPN5, SURF4, LAMP2 or MT-ND1, as highly discriminating features between the groups compared. Protein changes correlated with viral infection, RNA metabolism or autophagy, among others. Remarkably, the angiogenic potential of ECFCs in response to the infected serums was impaired, and many of the protein alterations in response to the serum of critical patients were associated with cardiovascular-related pathologies.

"Zoom-ln"--A targeted database search for identification of glycation modifications analyzed by untargeted tandem mass spectrometry.

Post-translational modifications (PTMs) are very important to biological function, however their identification and characterization is technically challenging. In this study, we have identified glycation modifications by nano LC-MSE, a data independent acquisition work flow, followed by database search using the Protein Lynx Global Server (PLGSJ). PLGS search with a complete human protein database hardly identified glycation modifications in a glycated human serum albumin (HSA), which was detected to be glycated by western blotting with advanced glycation end products (AGE) antibody and fluorescence spectroscopy. To overcome this difficulty, "Zoom-In" approach, a targeted database search was used to identify glycation modifications in a glycated HSA, which were further manually validated. This approach was useful for identification of glycation modifications from untargeted tandem mass spectrometryworkflow such as MSE, but may require the development of a new algorithm or an upgrade of the existing software.