Plasma-based proteomic profiling identifies the distinct regulation of proteins in hyperplasia and endometrial cancer.

BACKGROUND: Among gynaecological malignancies, endometrial cancer (EC) is the most prevalent type of uterine cancer affecting women. This study explored the proteomic profiles of plasma samples obtained from EC patients, those with hyperplasia (Hy), and a control group (CO). A combination of techniques, such as 2D-DIGE, mass spectrometry, and bioinformatics, including pathway analysis, was used to identify proteins with modified expression levels, biomarkers and their associated metabolic pathways in these groups. METHODS: Thirty-four patients, categorized into three groups-10 with EC, 12 with Hy, and 12 CO-between the ages of 46 and 75 years old were included in the study. Untargeted proteomic analysis was carried out using two-dimensional difference in gel electrophoresis (2D-DIGE) coupled with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). RESULTS: In all three groups, 114 proteins that were significantly (p ≤ 0.05 and fold change ≥ 1.5) altered were successfully identified using peptide mass fingerprints (PMFs). Compared with those in the control group (CO), the EC samples had 85 differentially expressed proteins (39 upregulated and 46 downregulated), and in the Hy group, 81 proteins were dysregulated (40 upregulated and 41 downregulated) compared to those in the CO group, while 33 proteins exhibited differential regulation (12 upregulated and 21 downregulated) in the EC plasma samples compared to those in the Hy group. Vitamin D binding protein and complement C3 distinguished Hy and EC from CO with the greatest changes in expression. Among the differentially expressed proteins identified, enzymes with catalytic activity represented the largest group (42.9%). In terms of biological processes, most of the proteins were involved in cellular processes (28.8%), followed by metabolic processes (16.7%). STRING analysis for protein interactions revealed that the significantly differentially abundant proteins in the three groups are involved in three main biological processes: signalling of complement and coagulation cascades, regulation of insulin-like growth factor (IGF) transport and uptake by insulin-like growth factor binding proteins (IGFBPs), and plasma lipoprotein assembly, remodelling, and clearance. CONCLUSION: The identified plasma protein markers have the potential to serve as biomarkers for differentiating between EC and Hy, as well as for early diagnosis and monitoring of cancer progression.

Proteome derangement in malignant epithelial cells and its stroma following exposure to 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone.

Discovering novel changes in the proteome of malignant lung epithelial cells and/or the tumor-microenvironment is paramount for diagnostic, prognostic, and/or therapy development. A time-dependent 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced mouse lung tumor model was used to screen the proteome of lung tumors. NNK-transformed human lung epithelial BEAS-2B cells were then established to evaluate the epithelial cell-specific protein changes. A duration-dependent increase of tumor burden was observed in NNK-treated mice, 2/12 (17%), 8/12 (67%), 9/12 (75%), and 10/10 (100%) at weeks 8, 12, 16, and 20 after the NNK exposure, respectively. A total of 25 differentially expressed proteins (≥ twofold change), predominantly structural, signaling, and metabolic proteins, were detected by two-dimensional difference gel electrophoresis and identified by mass spectrometry. Calregulin, ezrin, histamine releasing factor (HRF), and inorganic pyrophosphatase 1 (PPA1) exhibited changes and were further confirmed via immunoblotting. In addition, immunohistochemistry (IHC) analysis indicated upregulated E-cadherin and decreased vimentin expression in epithelial cells of tumor tissues. Acquisition of a neoplastic phenotype in NNK-transformed BEAS-2B cells was demonstrated by enhanced wound closure and increased anchorage independent colony formation. In transformed BEAS-2B cells, protein expression of E-cadherin, ezrin, and PPA1 (but not calregulin and HRF) was upregulated, as was observed in tumor tissues IHC staining using mouse lung tumor tissues further revealed that HRF upregulation was not lung epithelial cell specific. Altogether, tumorigenesis after NNK exposure may be initiated by protein dysregulation in lung epithelial cells together with proteome derangement derived from other cell types existing in the tumor-microenvironment.

Screening Serum Biomarkers for Rats Preconditioned with Hyperbaric Oxygen: Potential of Predicting Prognosis for Stroke.

BACKGROUND: Stroke is a major health concern and a leading cause of mortality and morbidity. We and other groups have documented that hyperbaric oxygen preconditioning could significantly alleviate neuronal damage in ischemia‒reperfusion models through various mechanisms. However, we found that some of the subjects did not benefit from preconditioning with hyperbaric oxygen. The preconditioning phenomenon is similar to vaccination, in which the endogenous survival system is activated to fight against further injuries. However, with vaccine inoculations, we could test for specific antibodies against the pathogens to determine if the vaccination was successful. Likewise, this experiment was carried out to explore a biomarker that can reveal the effectiveness of the preconditioning before neuronal injury occurs. METHODS: Middle cerebral artery occlusion (MCAO) was used to induce focal cerebral ischemia-reperfusion injury. 2D-DIGE-MALDI-TOF-MS/MS proteomic technique was employed to screen the differentially expressed proteins in the serum of rats among the control (Con) group (MCAO model without hyperbaric oxygen (HBO) preconditioning), hyperbaric oxygen protective (HBOP) group (in which the infarct volume decreased after HBO preconditioning vs. Con), and hyperbaric oxygen nonprotective (HBOU) group (in which the infarct volume remained the same or even larger after HBO preconditioning vs. Con). Candidate biomarkers were confirmed by western blot and enzyme linked immunosorbent assay (ELISA), and the relationship between the biomarkers and the prognosis of cerebral injury was further validated. RESULTS: Among the 15 differentially expressed protein spots detected in the HBOP group by Two-dimensional fluorescence difference gel electrophoresis (2D-DIGE), 3 spots corresponding to 3 different proteins (haptoglobin, serum albumin, and haemopexin) products were identified by MALDI-TOF-MS/MS. Serum albumin and haemopexin were upregulated, and haptoglobin was downregulated in the HBOP group (p < 0.05 vs. Con and HBOU groups). After the western blot study, only the changes in haemopexin were validated and exhibited similar changes in subjects from the HBOP group in accordance with MALDI-TOF-MS/MS proteomic analysis and enzyme linked immunosorbent assay (ELISA) analysis. The serum level of the hemopexin (HPX) at 2 h after HBO preconditioning was correlated with the infarct volume ratio after MCAO. CONCLUSIONS: Haemopexin may be developed as a predictive biomarker that indicated the effectiveness of a preconditioning strategy against cerebral ischaemic injury.

Phospho-DIGE Identified Phosphoproteins Involved in Pathways Related to Tumour Growth in Endometrial Cancer.

Endometrial cancer (EC) is the most common gynecologic malignancy of the endometrium. This study focuses on EC and normal endometrium phosphoproteome to identify differentially phosphorylated proteins involved in tumorigenic signalling pathways which induce cancer growth. We obtained tissue samples from 8 types I EC at tumour stage 1 and 8 normal endometria. We analyzed the phosphoproteome by two-dimensional differential gel electrophoresis (2D-DIGE), combined with immobilized metal affinity chromatography (IMAC) and mass spectrometry for protein and phosphopeptide identification. Quantities of 34 phosphoproteins enriched by the IMAC approach were significantly different in the EC compared to the endometrium. Validation using Western blotting analysis on 13 patients with type I EC at tumour stage 1 and 13 endometria samples confirmed the altered abundance of HBB, CKB, LDHB, and HSPB1. Three EC samples were used for in-depth identification of phosphoproteins by LC-MS/MS analysis. Bioinformatic analysis revealed several tumorigenic signalling pathways. Our study highlights the involvement of the phosphoproteome in EC tumour growth. Further studies are needed to understand the role of phosphorylation in EC. Our data shed light on mechanisms that still need to be ascertained but could open the path to a new class of drugs that could hinder EC growth.

Molecular Identification and Acid Stress Response of an Acidithiobacillus thiooxidans Strain Isolated from Rio Tinto (Spain).

Acidithiobacillus thiooxidans is of paramount importance in the development of biomining technologies. Being widely recognized as an extreme acidophile, extensive research has been dedicated to understanding its significant role in the extraction of several ores in recent years. However, there still exist significant molecular uncertainties surrounding this species. This study focuses on developing a taxonomic assignment method based on the sequencing of the 16S-5S rRNA cluster, along with a qPCR-based technology enabling precise growth determination. Additionally, an approach to understanding its response to acid stress is explored through RT-PCR and MALDI-TOF analysis. Our findings indicate that when subjected to pH levels below 1, the cell inhibits central (carbon fixation and metabolism) and energy (sulfur metabolism) metabolism, as well as chaperone synthesis, suggesting a potential cellular collapse. Nevertheless, the secretion of ammonia is enhanced to raise the environmental pH, while fatty acid synthesis is upregulated to reinforce the cell membrane.

2D-DIGE based proteome analysis of wheat-Thinopyrum intermedium 7XL/7DS translocation line under drought stress.

BACKGROUND: Drought stress is the most limiting factor for plant growth and crop production worldwide. As a major cereal crop, wheat is susceptible to drought. Thus, discovering and utilizing drought-tolerant gene resources from related species are highly important for improving wheat drought resistance. In this study, the drought tolerance of wheat Zhongmai 8601-Thinopyrum intermedium 7XL/7DS translocation line YW642 was estimated under drought stress, and then two-dimensional difference gel electrophoresis (2D-DIGE) based proteome analysis of the developing grains was performed to uncover the drought-resistant proteins. RESULTS: The results showed that 7XL/7DS translocation possessed a better drought-tolerance compared to Zhongmai 8601. 2D-DIGE identified 146 differential accumulation protein (DAP) spots corresponding to 113 unique proteins during five grain developmental stages of YW642 under drought stress. Among them, 55 DAP spots corresponding to 48 unique proteins displayed an upregulated expression, which were mainly involved in stress/defense, energy metabolism, starch metabolism, protein metabolism/folding and transport. The cis-acting element analysis revealed that abundant stress-related elements were present in the promoter regions of the drought-responsive protein genes, which could play important roles in drought defense. RNA-seq and RT-qPCR analyses revealed that some regulated DAP genes also showed a high expression level in response to drought stress. CONCLUSIONS: Our results indicated that Wheat-Th. intermedium 7XL/7DS translocation line carried abundant drought-resistant proteins that had potential application values for wheat drought tolerance improvement.

2D-DIGE based urinary proteomics and functional enrichment studies to reveal novel Mycobacterium tuberculosis and human protein biomarker candidates for pulmonary tuberculosis.

In the absence of a sensitive and specific diagnostic modality capable of detecting all forms of tuberculosis (TB), proteomics may identify specific Mycobacterium tuberculosis (M.tb) proteins in urine, with a potential as biomarkers. To identify candidate biomarkers for TB, proteome profile of urine from pulmonary TB patients was compared with non-disease controls (NDC) and disease controls (DC, Streptococcus pneumonia infected patients) using a combination of two-dimensional difference gel electrophoresis (2D-DIGE) and liquid chromatography tandem mass spectrometry (LCMS/MS). Eleven differentially expressed host proteins and Eighteen high abundant M.tb proteins were identified. Protein-protein interactome (PPI) and functional enrichment analyses like Gene Ontologies, Reactome pathway etc. demonstrated that the human proteins mainly belong to extracellular space and show physiological pathways for immune response and hematological disorders. Whereas, M.tb proteins belong to the cell periphery, plasma membrane and cell wall, and demonstrated catalytic, nucleotide binding and ATPase activities along with other functional processes. The study findings provide valuable inputs about the biomarkers of TB and shed light on the probable disease consequences as an outcome of the bacterial pathogenicity.

Proteomic Analysis of Subchronic Furan Exposure in the Liver of Male Fischer F344 Rats.

Furan is a volatile compound formed during the thermal processing of foods. Chronic exposure has been shown to cause cholangiocarcinoma and hepatocellular tumors in rodent models. We conducted a 90 day subchronic study in Fisher 344 rats exposed to various doses by gavage to determine the NOAEL. Previous reports have outlined changes in the liver using gross necropsy examination, histopathology, clinical biochemistry, hematology, immunohistochemistry, and toxicogenomics. The data revealed that males were more sensitive than females. The focus of this study was to evaluate the toxicoproteomic changes by 2-dimensional differential in gel electrophoresis followed by mass spectrometry analysis. To compliment previous studies, protein expression changes were evaluated of male animals after 90 days of exposure to doses of 0, 0.03, 0.5, and 8.0 mg/kg bw/d. Significant statistical treatment-related changes compared to the controls identified 45 protein spots containing 38 unique proteins. Proteins identified are implicated in metabolism, redox regulation, protein folding/proteolysis as well as structural and transport proteins. At lower doses, multiple cytoprotective pathways are activated to maintain a homeostasis but ultimately the loss of protein function and impairment of several pathways could lead to adverse health effects at higher doses of furan administration.

An Extremely Low-Frequency Vortex Magnetic Field Modifies Protein Expression, Rearranges the Cytoskeleton, and Induces Apoptosis of a Human Neuroblastoma Cell Line.

Homogeneous extremely low-frequency electromagnetic fields (ELF-EMFs) alter biological phenomena, including the cell phenotype and proliferation rate. Heterogenous vortex magnetic fields (VMFs), a new approach of exposure to magnetic fields, induce systematic movements on charged biomolecules from target cells; however, the effect of VMFs on living systems remains uncertain. Here, we designed, constructed, and characterized an ELF-VMF-modified Rodin's coil to expose SH-SY5Y cells. Samples were analyzed by performing 2D-differential-gel electrophoresis, identified by MALDI-TOF/TOF, validated by western blotting, and characterized by confocal microscopy. A total of 106 protein spots were differentially expressed; 40 spots were downregulated and 66 were upregulated in the exposed cell proteome, compared to the control cell proteome. The identified spots are associated with cytoskeleton and cell viability proteins, and according to the protein-protein interaction network, a significant interaction among them was found. Our data revealed a decrease in cell survival associated with apoptotic cells without effects on the cell cycle, as well as evident changes in the cytoskeleton. We demonstrated that ELF-VMFs, at a specific frequency and exposure time, alter the cell proteome and structurally affect the target cells. This is the first report showing that VMF application might be a versatile system for testing different hypotheses in living systems, using appropriate exposure parameters.© 2022 Bioelectromagnetics Society.

Fibrinogen isoforms as potential blood-based biomarkers of Alzheimer's disease using a proteomics approach.

Objective: Alzheimer's disease (AD), the commonest form of dementia which is characterized by progressive decline in cognitive function, can only be definitively diagnosed after death. Although biomarkers may aid diagnosis, currently available AD biomarkers, which are predominantly based on cerebrospinal fluid and neuroimaging facilities, are either invasive or costly. Blood-based biomarkers for AD diagnosis are highly sought after due to its practicality at the clinic. This study was undertaken to determine the differential protein expression in plasma amongst Malaysian AD, mild cognitive impairment (MCI) and non-AD individuals. Methods: A proteomic approach which utilized two-dimensional differential in gel electrophoresis (2 D DIGE) was performed for blood samples from 15 AD, 14 MCI and 15 non-AD individuals. Results: Mass spectrometry (MS)-based protein identification via MALDI ToF/ToF showed that fibrinogen-ß-chain (spot 64) and fibrinogen-γ-chain (spot 91) with differential expression ratio >1.5 were significantly upregulated (p < 0.05) in AD patients when compared to non-AD individuals. Further data analysis using Pearson correlation found that the upregulated fibrinogen-γ-chain was weakly but significantly (p < 0.05) and inversely correlated with cognitive decline. Conclusion: Fibrinogen isoforms may play important roles in the vascular pathology of AD as well as neuroinflammation. As such, fibrinogen appears to be a promising blood-based biomarker for AD. Further validation of the present findings in larger population is now warranted.

Gel-Based Proteomic Identification of Suprabasin as a Potential New Candidate Biomarker in Endometrial Cancer.

Endometrial cancer (EC) is the most frequent gynaecologic cancer in postmenopausal women. We used 2D-DIGE and mass spectrometry to identify candidate biomarkers in endometrial cancer, analysing the serum protein contents of 10 patients versus 10 control subjects. Using gel-based proteomics, we identified 24 candidate biomarkers, considering only spots with a fold change in volume percentage ≥ 1.5 or intensity change ≤ 0.6, which were significantly different between cases and controls (p < 0.05). We used Western blotting analysis both in the serum and tissue of 43 patients for data validation. Among the identified proteins, we selected Suprabasin (SBSN), an oncogene previously associated with poor prognosis in different cancers. SBSN principal isoforms were subjected to Western blotting analysis in serum and surgery-excised tissue: both isoforms were downregulated in the tissue. However, in serum, isoform 1 was upregulated, while isoform 2 was downregulated. Data-mining on the TCGA and GTEx projects, using the GEPIA2.0 interface, indicated a diminished SBSN expression in the Uterine Corpus Endometrial Cancer (UCEC) database compared to normal tissue, confirming proteomic results. These results suggest that SBSN, specifically isoform 2, in tissue or serum, could be a potential novel biomarker in endometrial cancer.

Molecular Fingerprint of BMD Patients Lacking a Portion in the Rod Domain of Dystrophin.

BMD is characterized by a marked heterogeneity of gene mutations resulting in many abnormal dystrophin proteins with different expression and residual functions. The smaller dystrophin molecules lacking a portion around exon 48 of the rod domain, named the D8 region, are related to milder phenotypes. The study aimed to determine which proteins might contribute to preserving muscle function in these patients. Patients were subdivided, based on the absence or presence of deletions in the D8 region, into two groups, BMD1 and BMD2. Muscle extracts were analyzed by 2-D DIGE, label-free LC-ESI-MS/MS, and Ingenuity pathway analysis (IPA). Increased levels of proteins typical of fast fibers and of proteins involved in the sarcomere reorganization characterize BMD2. IPA of proteomics datasets indicated in BMD2 prevalence of glycolysis and gluconeogenesis and a correct flux through the TCA cycle enabling them to maintain both metabolism and epithelial adherens junction. A 2-D DIGE analysis revealed an increase of acetylated proteoforms of moonlighting proteins aldolase, enolase, and glyceraldehyde-3-phosphate dehydrogenase that can target the nucleus promoting stem cell recruitment and muscle regeneration. In BMD2, immunoblotting indicated higher levels of myogenin and lower levels of PAX7 and SIRT1/2 associated with a set of proteins identified by proteomics as involved in muscle homeostasis maintenance.

Gel-based fluorescent proteomic tools for investigating cell redox signaling. A mini-review.

The specific chemical reactivity of thiol groups makes protein cysteines susceptible to reactions with reactive oxygen species (ROS) and reactive nitrogen species (RNS) resulting in the formation of various reversible and irreversible oxidative post-translational modifications (oxPTMs). This review highlights a number of gel-based redox proteomic approaches to detect protein oxPTMs, with particular emphasis on S-nitrosylation, which we believe are currently one of the most accurate way to analyze changes in the redox status of proteins. The information collected in this review relates to the recent progress regarding methods for the enrichment and identification of redox-modified proteins, with an emphasis on fluorescent gel proteomics. Gel-based fluorescent proteomic strategies are low-cost and easy-to-use tools for investigating the thiol proteome and can provide substantial information on redox signaling.

Identification of peroxiredoxin 6 as a potential lung-adenocarcinoma biomarker for predicting chemotherapy response by proteomic analysis.

The prognosis of lung cancer remains poor due to the limited biomarker selection for treating patients with optimal chemotherapy. The aim of this study is to discover and identify new biomarkers with the value of predicting chemotherapy responses in a lung adenocarcinoma (AD) specimen. In this study, six pairs of pre-treatment fresh primary lung AD-cancer tumors with varied chemotherapy responses were used to discover new biomarkers by two-dimensional difference gel electrophoresis (2D DIGE). Among the matched protein spots, 19 were up-regulated and 18 were down-regulated in chemo-sensitive tumors versus chemo-resistant tissues. These differentially expressed proteins could be divided into five classes: redox regulation protein, the cytoskeletal protein, cell metabolism enzymes or proteins, apoptosis, signal transduction mediated molecules, and other functional proteins. Proteins of interest, including PRDX2, PRDX6, and Gelsolin, were differentially expressed in chemo-sensitive tumors versus chemo-resistant tissues and these observations were validated by immunohistochemistry in 92 formalin-fixed and paraffin-embedded (FFPE) specimens. Our results demonstrated that PRDX6 protein expression was closely related to tumor response (cc2 = 5.57, P < 0.05), whereas no relationship of PRDX2 and Gelsolin were obtained with tumor response (cc2 = 0.51 P > 0.05, cc2 = 0.41 P > 0.05). This tissue proteomics study provides evidence that PRDX6 may be regarded as a predictive biomarker for poor chemotherapy response, which can be helpful in guiding pretreatment protocols.

Proteomic analysis reveals differentially abundant proteins probably involved in the virulence of amastigote and promastigote forms of Leishmania infantum.

Owing to the importance and clinical diversity of Leishmania infantum, studying its virulence factors is promising for understanding the relationship between parasites and hosts. In the present study, differentially abundant proteins from strains with different degrees of virulence in promastigote and amastigote forms were compared using two quantitative proteomics techniques, differential gel electrophoresis and isobaric mass tag labeling, followed by identification by mass spectrometry. A total of 142 proteins were identified: 96 upregulated and 46 downregulated proteins in the most virulent strain compared to less virulent. The interaction between the proteins identified in each evolutionary form was predicted. The results showed that in the amastigote form of the most virulent strain, there was a large group of proteins related to glycolysis, heat shock, and ribosomal proteins, whereas in the promastigote form, the group consisted of stress response, heat shock, and ribosomal proteins. In addition, biological processes related to metabolic pathways, ribosomes, and oxidative phosphorylation were enriched in the most virulent strain (BH400). Finally, we noted several proteins previously found to play important roles in L. infantum infection, which showed increased abundance in the virulent strain, such as ribosomal proteins, HSP70, enolase, fructose 1,6-biphosphate aldolase, peroxidoxin, and tryparedoxin peroxidase, many of which interact with each other.

The Distribution of Several Genomic Virulence Determinants Does Not Corroborate the Established Serotyping Classification of Bacillus thuringiensis.

Bacillus thuringiensis, commonly referred to as Bt, is an object of the lasting interest of microbiologists due to its highly effective insecticidal properties, which make Bt a prominent source of biologicals. To categorize the exuberance of Bt strains discovered, serotyping assays are utilized in which flagellin serves as a primary seroreactive molecule. Despite its convenience, this approach is not indicative of Bt strains' phenotypes, neither it reflects actual phylogenetic relationships within the species. In this respect, comparative genomic and proteomic techniques appear more informative, but their use in Bt strain classification remains limited. In the present work, we used a bottom-up proteomic approach based on fluorescent two-dimensional difference gel electrophoresis (2D-DIGE) coupled with liquid chromatography/tandem mass spectrometry(LC-MS/MS) protein identification to assess which stage of Bt culture, vegetative or spore, would be more informative for strain characterization. To this end, the proteomic differences for the israelensis-attributed strains were assessed to compare sporulating cultures of the virulent derivative to the avirulent one as well as to the vegetative stage virulent bacteria. Using the same approach, virulent spores of the israelensis strain were also compared to the spores of strains belonging to two other major Bt serovars, namely darmstadiensis and thuringiensis. The identified proteins were analyzed regarding the presence of the respective genes in the 104 Bt genome assemblies available at open access with serovar attributions specified. Of 21 proteins identified, 15 were found to be encoded in all the present assemblies at 67% identity threshold, including several virulence factors. Notable, individual phylogenies of these core genes conferred neither the serotyping nor the flagellin-based phylogeny but corroborated the reconstruction based on phylogenomics approaches in terms of tree topology similarity. In its turn, the distribution of accessory protein genes was not confined to the existing serovars. The obtained results indicate that neither gene presence nor the core gene sequence may serve as distinctive bases for the serovar attribution, undermining the notion that the serotyping system reflects strains' phenotypic or genetic similarity. We also provide a set of loci, which fit in with the phylogenomics data plausibly and thus may serve for draft phylogeny estimation of the novel strains.

Proteomic analysis of liver tissue between fat and lean broiler lines.

1. The liver is the major site of fatty acid synthesis in chickens. Lipid metabolism in the liver correlates with the deposition of triglycerides in adipose tissue. Northeast Agricultural University broilers lines divergently selected for abdominal fat content (NEAUHLF) provide a unique model to study the mechanisms of fat deposition.2. In previous studies, differentially expressed genes (DEGs) in the livers of fat and lean broilers were evaluated across different developmental stages. Whether protein expression differences exist between the livers of fat and lean broilers is largely unknown. The current experiment used 2D fluorescence difference gel electrophoresis (2D-DIGE) to screen expressed protein (DEP) spots in the liver tissues of NEAUHLF at one, four and seven weeks of age.3. Twenty-two DEPs were identified by MALDI-TOF-MS that were involved in lipid, energy, protein and amino acid metabolism, oxidative stress, cytoskeleton, and transport.4. These data furthered the understanding of the fat and lean phenotypes of broiler chickens.

Variant in NHLRC2 leads to increased hnRNP C2 in developing neurons and the hippocampus of a mouse model of FINCA disease.

BACKGROUND: FINCA disease is a pediatric cerebropulmonary disease caused by variants in the NHL repeat-containing 2 (NHLRC2) gene. Neurological symptoms are among the first manifestations of FINCA disease, but the consequences of NHLRC2 deficiency in the central nervous system are currently unexplored. METHODS: The orthologous mouse gene is essential for development, and its complete loss leads to early embryonic lethality. In the current study, we used CRISPR/Cas9 to generate an Nhlrc2 knockin (KI) mouse line, harboring the FINCA patient missense mutation (c.442G > T, p.Asp148Tyr). A FINCA mouse model, resembling the compound heterozygote genotype of FINCA patients, was obtained by crossing the KI and Nhlrc2 knockout mouse lines. To reveal NHLRC2-interacting proteins in developing neurons, we compared cortical neuronal precursor cells of E13.5 FINCA and wild-type mouse embryos by two-dimensional difference gel electrophoresis. RESULTS: Despite the significant decrease in NHLRC2, the mice did not develop severe early onset multiorgan disease in either sex. We discovered 19 altered proteins in FINCA neuronal precursor cells; several of which are involved in vesicular transport pathways and actin dynamics which have been previously reported in other cell types including human to have an association with dysfunctional NHLRC2. Interestingly, isoform C2 of hnRNP C1/C2 was significantly increased in both developing neurons and the hippocampus of adult female FINCA mice, connecting NHLRC2 dysfunction with accumulation of RNA binding protein. CONCLUSIONS: We describe here the first NHLRC2-deficient mouse model to overcome embryonic lethality, enabling further studies on predisposing and causative mechanisms behind FINCA disease. Our novel findings suggest that disrupted RNA metabolism may contribute to the neurodegeneration observed in FINCA patients.

Update on Proteomic approaches to uncovering virus-induced protein alterations and virus -host protein interactions during the progression of viral infection.

INTRODUCTION: Viruses induce profound changes in the cells they infect. Understanding these perturbations will assist in designing better therapeutics to combat viral infection. System-based proteomic assays now provide unprecedented opportunity to monitor large numbers of cellular proteins. AREAS COVERED: This review will describe various quantitative and functional mass spectrometry-based methods, and complementary non-mass spectrometry-based methods, such as aptamer profiling and proximity extension assays, and examples of how each are used to delineate how viruses affect host cells, identify which viral proteins interact with which cellular proteins, and how these change during the course of a viral infection. PubMed was searched multiple times prior to manuscript submissions and revisions, using virus, viral, proteomics; in combination with each keyword. The most recent examples of published works from each search were then analyzed. EXPERT OPINION: There has been exponential growth in numbers and types of proteomic analyses in recent years. Continued development of reagents that allow increased multiplexing and deeper proteomic probing of the cell, at quantitative and functional levels, enhancements that target more important protein modifications, and improved bioinformatics software tools and pathway prediction algorithms will accelerate this growth and usher in a new era of host proteome understanding.

A proteomic approach towards understanding crypoprotective action of Me2SO on the CHO cell proteome.

Chinese hamster ovary (CHO) cell lines are the most widely used in vitro cells for research and production of recombinant proteins such as rhGH, tPA, and erythropoietin. We aimed to investigate changes in protein profiles after cryopreservation using 2D-DIGE MALDI-TOF MS and network pathway analysis. The proteome changes that occur in CHO cells between freshly prepared cells and cryopreserved cells with and without Me2SO were compared to determine the key proteins and pathways altered during recovery from cryopreservation. A total of 54 proteins were identified and successfully matched to 37 peptide mass fingerprints (PMF). 14 protein spots showed an increase while 23 showed decrease abundance in the Me2SO free group compared to the control. The proteins with increased abundance included vimentin, heat shock protein 60 kDa, mitochondrial, heat shock 70 kDa protein 9, protein disulfide-isomerase A3, voltage-dependent anion-selective channel protein 2. Those with a decrease in abundance were myotubularin, glutathione peroxidase, enolase, phospho glyceromutase, chloride intracellular channel protein 1. The main canonical functional pathway affected involved the unfolded protein response, aldosterone Signaling in Epithelial Cells, 14-3-3-mediated signaling. 2D-DIGE MALDI TOF mass spectrometry and network pathway analysis revealed the differential proteome expression of FreeStyle CHO cells after cryopreservation with and without 5% Me2SOto involve pathways related to post-translational modification, protein folding and cell death and survival (score = 56, 22 focus molecules). This study revealed, for the first time to our knowledge the proteins and their regulated pathways involved in the cryoprotective action of 5% Me2SO. The use of 5% Me2SO as a cryoprotectant maintained the CHO cell proteome in the cryopreserved cells, similar to that of fresh CHO cells.