Optimization of ultracentrifugation-based method to enhance the purity and proteomic profiling depth of plasma-derived extracellular vesicles and particles.

Circulating extracellular vesicles and particles (EVPs) are being investigated as potential biomarkers for early cancer detection, prognosis, and disease monitoring. However, the suboptimal purity of EVPs isolated from peripheral blood plasma has posed a challenge of in-depth analysis of the EVP proteome. Here, we compared the effectiveness of different methods for isolating EVPs from healthy donor plasma, including ultracentrifugation (UC)-based protocols, phosphatidylserine-Tim4 interaction-based affinity capture (referred to as "PS"), and several commercial kits. Modified UC methods with an additional UC washing or size exclusion chromatography step substantially improved EVP purity and enabled the detection of additional proteins via proteomic mass spectrometry, including many plasma membrane and cytoplasmic proteins involved in vesicular regulation pathways. This improved performance was reproduced in cancer patient plasma specimens, resulting in the identification of a greater number of differentially expressed EVP proteins, thus expanding the range of potential biomarker candidates. However, PS and other commercial kits did not outperform UC-based methods in improving plasma EVP purity. PS yielded abundant contaminating proteins and a biased enrichment for specific EVP subsets, thus unsuitable for proteomic profiling of plasma EVPs. Therefore, we have optimized UC-based protocols for circulating EVP isolation, which enable further in-depth proteomic analysis for biomarker discovery.

Proteomics profile in encapsulated follicular patterned thyroid neoplasms.

Diagnosing encapsulated follicular-patterned thyroid tumors like Invasive Encapsulated Follicular Variant of Papillary Thyroid Carcinoma (IEFVPTC), Non-invasive Follicular Thyroid Neoplasm with Papillary-like Nuclear Features (NIFTP), and Well-Differentiated Tumor of Uncertain Malignant Potential (WDT-UMP) remains challenging due to their morphological and molecular similarities. This study aimed to investigate the protein distinctions among these three thyroid tumors and discover biological tumorigenesis through proteomic analysis. We employed total shotgun proteome analysis allowing to discover the quantitative expression of over 1398 proteins from 12 normal thyroid tissues, 13 IEFVPTC, 11 NIFTP, and 10 WDT-UMP. Principal component analysis revealed a distinct separation of IEFVPTC and normal tissue samples, distinguishing them from the low-risk tumor group (NIFTP and WDT-UMP). IEFVPTC exhibited the highest number of differentially expressed proteins (DEPs) compared to the other tumors. No discriminatory proteins between NIFTP and WDT-UMP were identified. Moreover, DEPs in IEFVPTC were significantly associated with thyroid tumor progression pathways. Certain hub genes linked to the response of immune checkpoint inhibitor therapy, revealing the potential predictor of prognosis. In conclusion, the proteomic profile of IEFVPTC differs from that of low-risk tumors. These findings may provide valuable insights into tumor biology and offer a basis for developing novel therapeutic strategies for follicular-patterned thyroid neoplasms.

Data-independent LC-MS/MS analysis of ME/CFS plasma reveals a dysregulated coagulation system, endothelial dysfunction, downregulation of complement machinery.

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a debilitating chronic condition that is characterized by unresolved fatigue, post-exertion symptom exacerbation (PESE), cognitive dysfunction, orthostatic intolerance, and other symptoms. ME/CFS lacks established clinical biomarkers and requires further elucidation of disease mechanisms. A growing number of studies demonstrate signs of hematological and cardiovascular pathology in ME/CFS cohorts, including hyperactivated platelets, endothelial dysfunction, vascular dysregulation, and anomalous clotting processes. To build on these findings, and to identify potential biomarkers that can be related to pathophysiology, we measured differences in protein expression in platelet-poor plasma (PPP) samples from 15 ME/CFS study participants and 10 controls not previously infected with SARS-CoV-2, using DIA LC-MS/MS. We identified 24 proteins that are significantly increased in the ME/CFS group compared to the controls, and 21 proteins that are significantly downregulated. Proteins related to clotting processes - thrombospondin-1 (important in platelet activation), platelet factor 4, and protein S - were differentially expressed in the ME/CFS group, suggestive of a dysregulated coagulation system and abnormal endothelial function. Complement machinery was also significantly downregulated, including C9 which forms part of the membrane attack complex. Additionally, we identified a significant upregulation of lactotransferrin, protein S100-A9, and an immunoglobulin variant. The findings from this experiment further implicate the coagulation and immune system in ME/CFS, and bring to attention the pathology of or imposed on the endothelium. This study highlights potential systems and proteins that require further research with regards to their contribution to the pathogenesis of ME/CFS, symptom manifestation, and biomarker potential, and also gives insight into the hematological and cardiovascular risk for ME/CFS individuals affected by diabetes mellitus.

Application of multi-omics techniques to androgenetic alopecia: Current status and perspectives.

The rapid advancement of sequencing technologies has enabled the generation of vast datasets, allowing for the in-depth analysis of sequencing data. This analysis has facilitated the validation of novel pathogenesis hypotheses for understanding and treating diseases through ex vivo and in vivo experiments. Androgenetic alopecia (AGA), a common hair loss disorder, has been a key focus of investigators attempting to uncover its underlying mechanisms. Abnormal changes in mRNA, proteins, and metabolites have been identified in individuals with AGA, and future developments in sequencing technologies may reveal new biomarkers for AGA. By integrating multiple omics analysis datasets such as genomics, transcriptomics, proteomics, and metabolomics-along with clinical phenotype data-we can achieve a comprehensive understanding of the molecular underpinnings of AGA. This review summarizes the data-mining studies conducted on various omics analysis datasets as related to AGA that have been adopted to interpret the biological data obtained from different omics layers. We herein discuss the challenges of integrative omics analyses, and suggest that collaborative multi-omics studies can enhance the understanding of the complete pathomechanism(s) of AGA by focusing on the interaction networks comprising DNA, RNA, proteins, and metabolites.

Regulation of the DLC3 tumor suppressor by a novel phosphoswitch.

Deleted in liver cancer 3 (DLC3) is a Rho GTPase-activating protein (RhoGAP) that plays a crucial role in maintaining adherens junction integrity and coordinating polarized vesicle transport by modulating Rho activity at the plasma membrane and endomembranes. By employing bioinformatical sequence analysis, in vitro experiments, and in cellulo assays we here identified a polybasic region (PBR) in DLC3 that facilitates the association of the protein with cellular membranes. Within the PBR, we mapped two serines whose phosphorylation can alter the electrostatic character of the region. Consequently, phosphomimetic mutations of these sites impaired the membrane association of DLC3. Furthermore, we found a new PBR-dependent localization of DLC3 at the midbody region, where the protein locally controlled Rho activity. Here, the phosphorylation-dependent regulation of DLC3 appeared to be required for proper cytokinesis. Our work thus provides a novel mechanism for spatiotemporal termination of Rho signaling by the RhoGAP protein DLC3.

TMT-based proteomics analysis of the blood enriching mechanism of the total Tannins of Gei Herba in mice.

Lanbuzheng (LBZ) is the traditional seedling medicine in Guizhou, which has the effect of tonifying blood. It has been found that the main active ingredient is tannin, however, the blood-replenishing effect of tannin and its mechanism are still unclear. The study was to explore the mechanisms underlying the therapeutic effects of the total Tannins of Lanbuzheng (LBZT) against anemia in mice. Anemia mice was induced by cyclophosphamide, the effect of LBZT against anemia was determined by analyzing peripheral blood and evaluating organs indexes. Tandem mass tag (TMT)-based quantitative proteomics technology coupled with bioinformatics analysis was then used to identify differentially expressed proteins (DEPs) in spleen. Compared to the model, number of RBCs, PLTs and WBCs, HCT ratio and HGB content were increased, the indexes of thymus, spleen and liver were also increased, after LBZT intervention. A total of 377 DEPs were identified in LBZT group, of which 206 DEPs were significantly up-regulated and 171 DEPs were significantly down-regulated. Bioinformatics analysis showed that hematopoietic function has been restored by activating the complement and coagulation cascade signaling pathways. Results suggest that LBZT exerts it therapeutic effects against anemia by regulating complement and coagulation cascade signaling pathways and provides scientific basis for further mechanistic studies for LBZT.

iTRAQ-based quantitative proteomics revealing the therapeutic mechanism of a medicinal and edible formula YH0618 in reducing doxorubicin-induced alopecia by targeting keratins and TGF-ß/Smad3 pathway.

YH0618, a medicinal and edible formulation, has demonstrated the potential to alleviate doxorubicin-induced alopecia in animal studies and clinical trials. However, the mechanisms underlying its therapeutic effects remain unexplored. The objective of this study was to ascertain possible therapeutic targets of YH0618 in the treatment of doxorubicin-induced alopecia. The assessment of hair loss was conducted through the measurement of the proportion of the affected area and the examination of skin histology. Isobaric tags for relative and absolute quantification (iTRAQ) in quantitative proteomics was employed to discern proteins that exhibited variable expressions. The major proteins associated with doxorubicin-induced alopecia were identified using gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. The interaction network of the differentially expressed proteins was constructed using the STRING database and the Python software. The study analyzed a total of 3894 proteins extracted from the skin tissue of mice. Doxorubicin treatment resulted in the upregulation of 18 distinct proteins, whereas one differential protein was found to be downregulated. The above effects were reinstated after the administration of the YH0618 therapy. The bioinformatic study revealed that the identified proteins exhibited enrichment in many biological processes, including staphylococcus aureus infection, estrogen signaling route, pyruvate metabolism, chemical carcinogenesis, and PPAR signaling pathway. The results of Western blot revealed that the levels of keratin 81 (Krt81), keratin 34 (Krt34), keratin 33a (Krt33a), and Sma and MAD-related protein 3 (Smad3) were upregulated in response to doxorubicin treatment, and were attenuated by the administration of YH0618. These four proteins are likely to correlate with DOX-induced alopecia and serve as promising therapeutic targets for YH0618. This work presents significant insights and empirical evidence for comprehending the process underlying chemotherapy-induced alopecia, paving the way for exploring innovative therapeutic or preventive strategies employing herbal items.

Proteomics and its application in the research of acupuncture: An updated review.

As a complementary and alternative therapy, acupuncture is widely used in the prevention and treatment of various diseases. However, the understanding of the mechanism of acupuncture effects is still limited due to the lack of systematic biological validation. Notably, proteomics technologies in the field of acupuncture are rapidly evolving, and these advances are greatly contributing to the research of acupuncture. In this study, we review the progress of proteomics research in analyzing the molecular mechanisms of acupuncture for neurological disorders, pain, circulatory disorders, digestive disorders, and other diseases, with an in-depth discussion around acupoint prescription and acupuncture manipulation modalities. The study found that proteomics has great potential in understanding the mechanisms of acupuncture. This study will help explore the mechanisms of acupuncture from a proteomic perspective and provide information to support future clinical decisions.

Applications of single-cell multi-omics in liver cancer.

Primary liver cancer, more specifically hepatocellular carcinoma (HCC), remains a significant global health problem associated with increasing incidence and mortality. Clinical, biological, and molecular heterogeneity are well-known hallmarks of cancer and HCC is considered one of the most heterogeneous tumour types, displaying substantial inter-patient, intertumoural and intratumoural variability. This heterogeneity plays a pivotal role in hepatocarcinogenesis, metastasis, relapse and drug response or resistance. Unimodal single-cell sequencing techniques have already revolutionised our understanding of the different layers of molecular hierarchy in the tumour microenvironment of HCC. By highlighting the cellular heterogeneity and the intricate interactions among cancer, immune and stromal cells before and during treatment, these techniques have contributed to a deeper comprehension of tumour clonality, hematogenous spreading and the mechanisms of action of immune checkpoint inhibitors. However, major questions remain to be elucidated, with the identification of biomarkers predicting response or resistance to immunotherapy-based regimens representing an important unmet clinical need. Although the application of single-cell multi-omics in liver cancer research has been limited thus far, a revolution of individualised care for patients with HCC will only be possible by integrating various unimodal methods into multi-omics methodologies at the single-cell resolution. In this review, we will highlight the different established single-cell sequencing techniques and explore their biological and clinical impact on liver cancer research, while casting a glance at the future role of multi-omics in this dynamic and rapidly evolving field.

Ceramide-induced cleavage of GPR64 intracellular domain drives Ewing sarcoma.

Ewing sarcoma is a cancer of bone and soft tissue in children and young adults primarily driven by the EWS-FLI1 fusion oncoprotein, which has been undruggable. Here, we report that Ewing sarcoma depends on secreted sphingomyelin phosphodiesterase 1 (SMPD1), a ceramide-generating enzyme, and ceramide. We find that G-protein-coupled receptor 64 (GPR64)/adhesion G-protein-coupled receptor G2 (ADGRG2) responds to ceramide and mediates critical growth signaling in Ewing sarcoma. We show that ceramide induces the cleavage of the C-terminal intracellular domain of GPR64, which translocates to the nucleus and restrains the protein levels of RIF1 in a manner dependent on SPOP, a substrate adaptor of the Cullin3-RING E3 ubiquitin ligase. We demonstrate that both SMPD1 and GPR64 are transcriptional targets of EWS-FLI1, indicating that SMPD1 and GPR64 are EWS-FLI1-induced cytokine-receptor dependencies. These results reveal the SMPD1-ceramide-GPR64 pathway, which drives Ewing sarcoma growth and is amenable to therapeutic intervention.

Protein research in millets: current status and way forward.

MAIN CONCLUSION: Millets' protein studies are lagging behind those of major cereals. Current status and future insights into the investigation of millet proteins are discussed. Millets are important small-seeded cereals majorly grown and consumed by people in Asia and Africa and are considered crops of future food security. Although millets possess excellent climate resilience and nutrient supplementation properties, their research advancements have been lagging behind major cereals. Although considerable genomic resources have been developed in recent years, research on millet proteins and proteomes is currently limited, highlighting a need for further investigation in this area. This review provides the current status of protein research in millets and provides insights to understand protein responses for climate resilience and nutrient supplementation in millets. The reference proteome data is available for sorghum, foxtail millet, and proso millet to date; other millets, such as pearl millet, finger millet, barnyard millet, kodo millet, tef, and browntop millet, do not have any reference proteome data. Many studies were reported on stress-responsive protein identification in foxtail millet, with most studies on the identification of proteins under drought-stress conditions. Pearl millet has a few reports on protein identification under drought and saline stress. Finger millet is the only other millet to have a report on stress-responsive (drought) protein identification in the leaf. For protein localization studies, foxtail millet has a few reports. Sorghum has the highest number of 40 experimentally proven crystal structures, and other millets have fewer or no experimentally proven structures. Further proteomics studies will help dissect the specific proteins involved in climate resilience and nutrient supplementation and aid in breeding better crops to conserve food security.

A proteomic study of the downregulation of TRIM37 on chondrocytes: Implications for the MULIBREY syndrome.

MULIBREY nanism which results from autosomal recessive mutations in TRIM37 impacts skeletal development, leading to growth delay with complications in multiple organs. In this study, we employed a combined proteomics and qPCR screening approach to investigate the molecular alterations in the CHON-002 cell line by comparing CHON-002 wild-type (WT) cells to CHON-002 TRIM37 knockdown (KD) cells. Our proteomic analysis demonstrated that TRIM37 depletion predominantly affects the expression of extracellular matrix proteins (ECM). Specifically, nanoLC-MS/MS experiments revealed an upregulation of SPARC, and collagen products (COL1A1, COL3A1, COL5A1) in response to TRIM37 KD. Concurrently, large-scale qPCR assays targeting osteogenesis-related genes corroborated these dysregulations of SPARC at the mRNA level. Gene ontology enrichment analysis highlighted the involvement of dysregulated proteins in ECM organization and TGF-ß signaling pathways, indicating a role for TRIM37 in maintaining ECM integrity and regulating chondrocyte proliferation. These findings suggest that TRIM37 deficiency in chondrocytes change ECM protein composition and could impairs long bone growth, contributing to the pathophysiology of MULIBREY nanism.

Shotgun proteomics of thyroid carcinoma exosomes - Insight into the role of exosomal proteins in carcinogenesis and thyroid homeostasis.

BACKGROUND: Transport of molecules via exosomes is one of the factors involved in thyroid cancer development, and transported molecules may serve as cancer biomarkers. The aim of the study was to characterize protein content of thyroid-derived exosomes and their functional effect exerted on recipient cells. METHODS: LC-MS/MS proteomics of exosomes released by FTC and 8305C thyroid carcinoma cell lines, and Nthy-ori 3-1 normal thyroid follicular cells was performed, followed by bioinformatic analysis and functional tests (wound healing and Alamar Blue assays). RESULTS: Exosomes from Nthy-ori 3-1 cells had the highest number of 1504 proteins, while in exosomes from thyroid carcinoma FTC and 8305C cells 730 and 1304 proteins were identified, respectively. For proteins uniquely found in FTC- and 8305C-derived exosomes, enriched cancer-related gene ontology categories included cell adhesion, positive regulation of cell migration, N-glycosylation, drug resistance, and response to NK/T cell cytotoxicity. Furthermore, through label-free quantification (that identified differentially expressed proteins) and comparison with The Human Protein Atlas database several potential diagnostic and/or prognostic biomarkers were indicated. Finally, exosomes from FTC and 8305C cells displayed ability to stimulate migratory properties of recipient Nthy-ori 3-1 cells. Additionally, 8305C-derived exosomes increased recipient cell viability. CONCLUSIONS: Multiple proteins identified in thyroid cancer-derived exosomes have a direct link to thyroid cancer progression. Also, in functional tests exosomes enhanced growth and dissemination of non-transformed thyroid cells. GENERAL SIGNIFICANCE: The obtained results expands the knowledge concerning the role of exosomal proteins in thyroid cancer and indicate potential biomarkers for further evaluation in clinical settings.

Selection Across the Three-Dimensional Structure of Venom Proteins from North American Scolopendromorph Centipedes.

Gene duplication followed by nucleotide differentiation is one of the simplest mechanisms to develop new functions for genes. However, the evolutionary processes underlying the divergence of multigene families remain controversial. We used multigene families found within the diversity of toxic proteins in centipede venom to test two hypotheses related to venom evolution: the two-speed mode of venom evolution and the rapid accumulation of variation in exposed residues (RAVER) model. The two-speed mode of venom evolution proposes that different types of selection impact ancient and younger venomous lineages with negative selection being the predominant form in ancient lineages and positive selection being the dominant form in younger lineages. The RAVER hypothesis proposes that, instead of different types of selection acting on different ages of venomous lineages, the different types of selection will selectively contribute to amino acid variation based on whether the residue is exposed to the solvent where it can potentially interact directly with toxin targets. This hypothesis parallels the longstanding understanding of protein evolution that suggests that residues found within the structural or active regions of the protein will be under negative or purifying selection, and residues that do not form part of these areas will be more prone to positive selection. To test these two hypotheses, we compared the venom of 26 centipedes from the order Scolopendromorpha from six currently recognized species from across North America using both transcriptomics and proteomics. We first estimated their phylogenetic relationships and uncovered paraphyly among the genus Scolopendra and evidence for cryptic diversity among currently recognized species. Using our phylogeny, we then characterized the diverse venom components from across the identified clades using a combination of transcriptomics and proteomics. We conducted selection-based analyses in the context of predicted three-dimensional properties of the venom proteins and found support for both hypotheses. Consistent with the two-speed hypothesis, we found a prevalence of negative selection across all proteins. Consistent with the RAVER hypothesis, we found evidence of positive selection on solvent-exposed residues, with structural and less-exposed residues showing stronger signal for negative selection. Through the use of phylogenetics, transcriptomics, proteomics, and selection-based analyses, we were able to describe the evolution of venom from an ancient venomous lineage and support principles of protein evolution that directly relate to multigene family evolution.

Spatial Proteomic Profiling of Colorectal Cancer Revealed Its Tumor Microenvironment Heterogeneity.

Colorectal cancer is a predominant malignancy with a second mortality worldwide. Despite its prevalence, therapeutic options remain constrained and surgical operation is still the most useful therapy. In this regard, a comprehensive spatially resolved quantitative proteome atlas was constructed to explore the functional proteomic landscape of colorectal cancer. This strategy integrates histopathological analysis, laser capture microdissection, and proteomics. Spatial proteome profiling of 200 tissue section samples facilitated by the fully integrated sample preparation technology SISPROT enabled the identification of more than 4000 proteins on the Orbitrap Exploris 240 from 2 mm2 × 10 µm tissue sections. Compared with normal adjacent tissues, we identified a spectrum of cancer-associated proteins and dysregulated pathways across various regions of colorectal cancer including ascending colon, transverse colon, descending colon, sigmoid colon, and rectum. Additionally, we conducted proteomic analysis on tumoral epithelial cells and paracancerous epithelium from early to advanced stages in hallmark rectum cancer and sigmoid colon cancer. Bioinformatics analysis revealed functional proteins and cell-type signatures associated with different regions of colorectal tumors, suggesting potential clinical implications. Overall, this study provides a comprehensive spatially resolved functional proteome landscape of colorectal cancer, serving as a valuable resource for exploring potential biomarkers and therapeutic targets.

Impact of mineral and non-mineral sources of iron and sulfur on the metalloproteome of Methanosarcina barkeri.

Methanogens often inhabit sulfidic environments that favor the precipitation of transition metals such as iron (Fe) as metal sulfides, including mackinawite (FeS) and pyrite (FeS2). These metal sulfides have historically been considered biologically unavailable. Nonetheless, methanogens are commonly cultivated with sulfide (HS-) as a sulfur source, a condition that would be expected to favor metal precipitation and thus limit metal availability. Recent studies have shown that methanogens can access Fe and sulfur (S) from FeS and FeS2 to sustain growth. As such, medium supplied with FeS2 should lead to higher availability of transition metals when compared to medium supplied with HS-. Here, we examined how transition metal availability under sulfidic (i.e., cells provided with HS- as sole S source) versus non-sulfidic (cells provided with FeS2 as sole S source) conditions impact the metalloproteome of Methanosarcina barkeri Fusaro. To achieve this, we employed size exclusion chromatography coupled with inductively coupled plasma mass spectrometry and shotgun proteomics. Significant changes were observed in the composition and abundance of iron, cobalt, nickel, zinc, and molybdenum proteins. Among the differences were alterations in the stoichiometry and abundance of multisubunit protein complexes involved in methanogenesis and electron transport chains. Our data suggest that M. barkeri utilizes the minimal iron-sulfur cluster complex and canonical cysteine biosynthesis proteins when grown on FeS2 but uses the canonical Suf pathway in conjunction with the tRNA-Sep cysteine pathway for iron-sulfur cluster and cysteine biosynthesis under sulfidic growth conditions.IMPORTANCEProteins that catalyze biochemical reactions often require transition metals that can have a high affinity for sulfur, another required element for life. Thus, the availability of metals and sulfur are intertwined and can have large impacts on an organismismal biochemistry. Methanogens often occupy anoxic, sulfide-rich (euxinic) environments that favor the precipitation of transition metals as metal sulfides, thereby creating presumed metal limitation. Recently, several methanogens have been shown to acquire iron and sulfur from pyrite, an abundant iron-sulfide mineral that was traditionally considered to be unavailable to biology. The work presented here provides new insights into the distribution of metalloproteins, and metal uptake of Methanosarcina barkeri Fusaro grown under euxinic or pyritic growth conditions. Thorough characterizations of this methanogen under different metal and sulfur conditions increase our understanding of the influence of metal availability on methanogens, and presumably other anaerobes, that inhabit euxinic environments.

Proteomic predictors of individualized nutrient-specific insulin secretion in health and disease.

Population-level variation and mechanisms behind insulin secretion in response to carbohydrate, protein, and fat remain uncharacterized. We defined prototypical insulin secretion responses to three macronutrients in islets from 140 cadaveric donors, including those with type 2 diabetes. The majority of donors' islets exhibited the highest insulin response to glucose, moderate response to amino acid, and minimal response to fatty acid. However, 9% of donors' islets had amino acid responses, and 8% had fatty acid responses that were larger than their glucose-stimulated insulin responses. We leveraged this heterogeneity and used multi-omics to identify molecular correlates of nutrient responsiveness, as well as proteins and mRNAs altered in type 2 diabetes. We also examined nutrient-stimulated insulin release from stem cell-derived islets and observed responsiveness to fat but not carbohydrate or protein-potentially a hallmark of immaturity. Understanding the diversity of insulin responses to carbohydrate, protein, and fat lays the groundwork for personalized nutrition.

Longitudinal analysis of the lung proteome reveals persistent repair months after mild to moderate COVID-19.

In order to assess homeostatic mechanisms in the lung after COVID-19, changes in the protein signature of bronchoalveolar lavage from 45 patients with mild to moderate disease at three phases (acute, recovery, and convalescent) are evaluated over a year. During the acute phase, inflamed and uninflamed phenotypes are characterized by the expression of tissue repair and host defense response molecules. With recovery, inflammatory and fibrogenic mediators decline and clinical symptoms abate. However, at 9 months, quantified radiographic abnormalities resolve in the majority of patients, and yet compared to healthy persons, all showed ongoing activation of cellular repair processes and depression of the renin-kallikrein-kinin, coagulation, and complement systems. This dissociation of prolonged reparative processes from symptom and radiographic resolution suggests that occult ongoing disruption of the lung proteome is underrecognized and may be relevant to recovery from other serious viral pneumonias.

Interaction proteomics analysis to provide insight into TFAMoplex-mediated transfection.

In an earlier investigation, our group introduced the TFAMoplex, a transfection agent based on the mitochondrial transcription factor A (TFAM) protein, which complexes DNA into nanoparticles. The original TFAMoplex further contained a bacterial phospholipase to achieve endosomal escape, and the vaccinia-related kinase 1 (VRK1), which significantly boosted the transfection efficiency of the system by an unknown mechanism. This study aims at replacing VRK1 within the TFAMoplex with dynein light chain proteins, specifically RP3, to directly tether the complexes to the dynein motor complex for enhanced cytosolic transport. To confirm the interaction between the dynein complex and the resulting fusion protein, we examined the binding kinetics of TFAM-RP3 to the dynein intermediate chains 1 and 2. Furthermore, we established a proteomics-based assay to compare cytosolic protein interactions of different TFAMoplex variants, including the RP3-modified version and the original VRK1-containing system. In the group of the VRK1-containing TFAMoplex, significant shifts of protein interactors were observed, especially for nucleolar proteins. Leveraging this knowledge, we incorporated one of these nuclear proteins, leucine-rich repeat-containing protein 59 (LRRC59), into the TFAMoplex, resulting in a significant improvement of transfection properties compared to the RP3-modified system and comparable levels versus the original, VRK1-containing version. This study not only advances our comprehension of the TFAMoplex system but also offers broader insights into the potential of protein engineering for designing effective gene delivery systems.

An optimized protocol for pig intestinal mucosa proteomics.

The overall well-being of organisms is widely recognized to be closely intertwined with their intestinal health. The intestinal mucosal layer plays a pivotal role in ensuring the proper functioning of the intestine, a fact observed not only in humans but also in animals like pigs. Any alterations to the mucosal layer of a pig's intestine can potentially disrupt its functionality, thereby impacting the animal's health and productivity. Mass spectrometry-based proteome analysis serves as a valuable tool in investigating the intricate dynamics of the proteome within the intestinal mucosa. Such studies hold promise in uncovering causal relationships between mucosal changes and overall health outcomes in pigs. It is anticipated that insights gathered from proteome studies will inform future strategies aimed at enhancing the health and productivity of pigs. However, the research field lacks a standardized and detailed method to extract proteins from pig intestinal mucosa and prepare proteins for proteome analysis. In the present study, we evaluated three alternative S-Trap-based protocols for analyzing ileal mucosal scrapings from pigs. Samples were either freeze-dried and treated as solid samples or ground in liquid nitrogen, categorized as either solid or liquid samples. In our analysis, a total of 2840 proteins were identified across all samples. Through statistical analysis and gene ontology examinations, we investigated potential differences between the three approaches. Even though our findings revealed no significant differences among the three methods, we propose the use of the protocol wherein samples are freeze-dried and treated as solid for protein extraction. This protocol stands out as the most convenient and practical option, offering ease of use and ensuring consistent and reliable results. By establishing a standardized approach, we aim to advance research efforts in understanding pig intestinal health. SIGNIFICANCE: The development of an optimized protocol for protein extraction of intestinal mucosal scrapings in pigs addresses a gap in the field and enhances future research on pig intestinal health. By use of the protocol and mass spectrometry-based proteome analysis, valuable insights for improving the health and productivity of pigs can be presented. Studying the complex dynamics of the proteome within the intestinal mucosa, potentially identifying links between mucosal changes and health outcomes, provides us with information about the critical connection between intestinal health and the overall well-being and productivity of pigs. By creating a standardized approach, consistent, reliable, and reproducible results can be obtained for this type of research.