Proteomics of Penicillium chrysogenum for a Deeper Understanding of Lead (Pb) Metal Bioremediation.

Penicillium chrysogenum (P. chrysogenum), a ubiquitous filamentous fungus, has demonstrated remarkable potential in the bioremediation of lead-contaminated environments. Its inherent tolerance and bioaccumulation capacity for lead (Pb), coupled with its relatively rapid growth rate, make it an attractive candidate for bioremediation applications. This study aims to identify the proteomic changes in P. chrysogenuminduced by Pb metal stress and unravel the roles of identified proteins in molecular mechanisms and cellular responses. Untargeted proteomic analysis was carried out using a two-dimensional difference in gel electrophoresis (2D-DIGE) coupled with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). This study reported the identification of 43 statistically significant proteins (24 upregulated and 19 downregulated, ANOVA, p ≤ 0.05; fold change ≥1.5) in P. chrysogenum as a consequence of Pb treatment. Proteins were grouped according to their function into 18 groups from which 13 proteins were related to metabolism, 11 were related to cellular process and signaling, and 19 proteins were related to information storage and processing. The current study is considered the first report about the proteomics study of P. chrysogenum under Pb stress conditions, where upregulated proteins could better explain the mechanism of tolerance and Pb toxicity removal. Our research has provided a thorough understanding of the molecular and cellular processes involved in fungal-metal interactions, paving the way for the development of innovative molecular markers for heavy metal myco-remediation. To the best of our knowledge, this study of P. chrysogenum provides valuable insights toward growing research in comprehending the metal-microbe interactions. This will facilitate development of novel molecular markers for metal bioremediation.

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.

Hemp Seed Oil Inhibits the Adipogenicity of the Differentiation-Induced Human Mesenchymal Stem Cells through Suppressing the Cannabinoid Type 1 (CB1).

Central and peripheral mechanisms of the endocannabinoid system (ECS) favor energy intake and storage. The ECS, especially cannabidiol (CBD) receptors, controls adipocyte differentiation (hyperplasia) and lipid accumulation (hypertrophy) in adipose tissue. In white adipose tissue, cannabidiol receptor 1 (CB1) stimulation increases lipogenesis and inhibits lipolysis; in brown adipose tissue, it decreases mitochondrial thermogenesis and biogenesis. This study compared the availability of phytocannabinoids [CBD and Δ9-tetrahydrocannabinol (THC)] and polyunsaturated fatty acids [omega 3 (ω3) and omega 6 (ω6)] in different hemp seed oils (HSO). The study also examined the effect of HSO on adipocyte lipid accumulation by suppressing cannabinoid receptors in adipogenesis-stimulated human mesenchymal stem cells (hMSCs). Most importantly, Oil-Red-O' and Nile red tests showed that HSO induced adipogenic hMSC differentiation without differentiation agents. Additionally, HSO-treated cells showed increased peroxisome proliferator-activated receptor gamma (PPARγ) mRNA expression compared to controls (hMSC). HSO reduced PPARγ mRNA expression after differentiation media (DM) treatment. After treatment with HSO, DM-hMSCs had significantly lower CB1 mRNA and protein expressions than normal hMSCs. HSO treatment also decreased transient receptor potential vanilloid 1 (TRPV1), fatty acid amide hydrolase (FAAH), and monoacylglycerol lipase (MGL) mRNAs in hMSC and DM-hMSCs. HSO treatment significantly decreased CB1, CB2, TRPV1, and G-protein-coupled receptor 55 (GPCR55) protein levels in DM-hMSC compared to hMSC in western blot analysis. In this study, HSO initiated adipogenic differentiation in hMSC without DM, but it suppressed CB1 gene and protein expression, potentially decreasing adipocyte lipid accumulation and lipogenic enzymes.

Plasma Proteomic Signature of Endometrial Cancer in Patients with Diabetes.

The incidence and mortality of endometrial cancer (EC) have increased in recent years. There is mounting evidence that diabetes may play a role in the greater incidence of EC. The molecular mechanisms of the interaction between type 2 diabetes and EC are not yet clearly understood yet. The present study was undertaken to investigate the plasma proteomics of EC patients with diabetes in comparison to those of EC patients without diabetes. Plasma samples were obtained from age-matched patients (EC diabetic and EC nondiabetic). Untargeted proteomic analysis was carried out using a two-dimensional differential gel electrophoresis coupled with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Of the 33 proteins identified, which significantly differed in the plasma abundance between groups, 17 were upregulated and 16 were downregulated. The majority of the altered proteins are involved in the acute phase reaction, cholesterol metabolism, scavenging of heme from plasma, and plasma lipoprotein assembly and mobilization. α-2-macroglobulin, Ras association domain-containing protein 3, apolipoprotein A-I, α-1B-glycoprotein, and zinc-α-2-glycoprotein were significantly upregulated. The significantly downregulated proteins included haptoglobin, apolipoprotein A-IV, hemopexin, and α-1-antichymotrypsin. The differential expression of proteins found in patients who had EC and diabetes indicated severe disease and a poor prognosis. The protein interaction analysis showed dysregulation of cholesterol metabolism and heme scavenging pathways in these patients.

Proteomic Profiling Identifies Distinct Regulation of Proteins in Obese Diabetic Patients Treated with Metformin.

Background: Obesity and type 2 diabetes mellitus (T2DM) are characterized by underlying low-grade chronic inflammation. Metformin has been used as the first line of therapy in T2DM as it decreases hepatic glucose production and glucose intestinal absorption, enhances insulin sensitivity and weight loss, and is known to ameliorate inflammation. The mechanisms through which metformin exerts its effect remain unclear. Proteomics has emerged as a unique approach to explore the biological changes associated with diseases, including T2DM. It provides insight into the circulating biomarkers/mediators which could be utilized for disease screening, diagnosis, and prognosis. Methods: This study evaluated the proteomic changes in obese (Ob), obese diabetics (OD), and obese diabetic patients on metformin (ODM) using a 2D DIGE MALDI-TOF mass spectrometric approach. Results: Significant changes in sixteen plasma proteins (15 up and 1 down, ANOVA, p ≤ 0.05; fold change ≥ 1.5) were observed in the ODM group when compared to the Ob and OD groups. Bioinformatic network pathway analysis revealed that the majority of these altered plasma proteins are involved in distinct pathways involving acute-phase response, inflammation, and oxidative response and were centered around HNF4A, ERK, JNK, and insulin signaling pathways. Conclusions: Our study provides important information about the possible biomarkers altered by metformin treatment in obese patients with and without T2DM. These altered plasma proteins are involved in distinct pathways involving acute-phase response, inflammation, and oxidative response and were centered around HNF4A, ERK, JNK, and insulin signaling pathways. The presented proteomic profiling approach may help in identifying potential biomarkers/mediators affected by metformin treatment in T2DM and inform the understanding of metformin's mechanisms of action.

Synthesis of Gingerol-Metals Complex and in-vitro Cytotoxic Activity on Human Colon Cancer Cell Line.

Introduction: Herbs are excellent sources of medicinal substances, and their curative abilities have been recognized to treat many ailments and are used for example as antioxidants, analgesics, anti-inflammatories, antipyretics, and many other medicinal uses. The properties of natural compounds and their health effects have been studied extensively, especially those that originate from plant sources such as ginger. The ginger plant contains many chemical compounds, such as 6-gingerol, which is characterized by containing active groups such as carbonyl and hydroxide, which can be attached to metal molecules. This is what was done in this study, where the formation of complexes with a group of metals was studied and their effect on cancer cells was investigated. These complexes will open new horizons for further study of medicinal uses. Methods: The synthesis of gingerol-metal complexes was carried out by conjugating gingerol molecules with Ag, Au, Cd, Co, Cu, Ni, and Zn metal ions. The extracted gingerol was transferred to culture tubes and deionized water-DMSO were added followed by sonication. The tubes were incubated at 90°C for two days as well as the control sample. The samples were then filtered and the complex solutions were transferred into new tubes for further studies. Different characterization techniques such as FT-IR, UV-vis spectroscopy, FESEM, and EDX are used to confirm the formation of the complexes. The in vitro of the complexes was tested by the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay against the human colorectal cancer cell lines HCT116 and HT29 which exhibited strong cytotoxicity. Results: The gingerol-metal complexes showed an enhancement as an anticancer agent compared to the control. The in vitro anticancer activity showed that the Ag-gingerol complex showed the most activity among the other complexes. Discussion: Gingerol-metal complexes can inhibit cancer cells, noting that the potency of the complex depends on the type of metal used.

Tissue-Based Proteomic Profiling in Patients with Hyperplasia and Endometrial Cancer.

Uterine cancers are among the most prevalent gynecological malignancies, and endometrial cancer (EC) is the most common in this group. This study used tissue-based proteomic profiling analysis in patients with endometrial cancer and hyperplasia, and control patients. Conventional 2D gel electrophoresis, followed by a mass spectrometry approach with bioinformatics, including a network pathway analysis pipeline, was used to identify differentially expressed proteins and associated metabolic pathways between the study groups. Thirty-six patients (twelve with endometrial cancer, twelve with hyperplasia, and twelve controls) were enrolled in this study. The mean age of the participants was 46-75 years. Eighty-seven proteins were significantly differentially expressed between the study groups, of which fifty-three were significantly differentially regulated (twenty-eight upregulated and twenty-five downregulated) in the tissue samples of EC patients compared to the control (Ctrl). Furthermore, 26 proteins were significantly dysregulated (8 upregulated and 18 downregulated) in tissue samples of hyperplasia (HY) patients compared to Ctrl. Thirty-two proteins (nineteen upregulated and thirteen downregulated) including desmin, peptidyl prolyl cis-trans isomerase A, and zinc finger protein 844 were downregulated in the EC group compared to the HY group. Additionally, fructose bisphosphate aldolase A, alpha enolase, and keratin type 1 cytoskeletal 10 were upregulated in the EC group compared to those in the HY group. The proteins identified in this study were known to regulate cellular processes (36%), followed by biological regulation (16%). Ingenuity pathway analysis found that proteins that are differentially expressed between EC and HY are linked to AKT, ACTA2, and other signaling pathways. The panels of protein markers identified in this study could be used as potential biomarkers for distinguishing between EC and HY and early diagnosis and progression of EC from hyperplasia and normal patients.

Proteomic Analysis of Endometrial Cancer Tissues from Patients with Type 2 Diabetes Mellitus.

Endometrial cancer (EC) is the most common form of gynecological cancer. Type 2 diabetes mellitus is associated with an increased risk of EC. Currently, no proteomic studies have investigated the role of diabetes in endometrial cancers from clinical samples. The present study aims to elucidate the molecular link between diabetes and EC using a proteomic approach. Endometrial tissue samples were obtained from age-matched patients (EC Diabetic and EC Non-Diabetic) during surgery. Untargeted proteomic analysis of the endometrial tissues was carried out using a two-dimensional difference in gel electrophoresis (2D-DIGE) coupled with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF). A total of 53 proteins were identified, with a significant difference in abundance (analysis of variance (ANOVA) test, p ≤ 0.05; fold-change ≥ 1.5) between the two groups, among which 30 were upregulated and 23 downregulated in the EC Diabetic group compared to EC Non-Diabetic. The significantly upregulated proteins included peroxiredoxin-1, vinculin, endoplasmin, annexin A5, calreticulin, and serotransferrin. The significantly downregulated proteins were myosin regulatory light polypeptide 9, Retinol dehydrogenase 12, protein WWC3, intraflagellar transport protein 88 homolog, superoxide dismutase [Cu-Zn], and retinal dehydrogenase 1. The network pathway was related to connective tissue disorder, developmental disorder, and hereditary disorder, with the identified proteins centered around dysregulation of ERK1/2 and F Actin signaling pathways. Cancer-associated protein alterations such as upregulation of peroxiredoxin-1, annexin 5, and iNOS, and downregulation of RDH12, retinaldehyde dehydrogenase 1, SOD1, and MYL 9, were found in the EC tissues of the diabetic group. Differential expression of proteins linked to cancer metastasis, such as the upregulation of vinculin and endoplasmin and downregulation of WWC3 and IFT88, was seen in the patients with diabetes. Calreticulin and alpha-enolase, which might have a role in the interplay between diabetes and EC, need further investigation.

Splice-disrupt genomic variants in prostate cancer.

BACKGROUND: Splice-disrupt genomic variants are one of the causes of cancer-causing errors in gene expression. Little is known about splice-disrupt genomic variants. METHODS AND RESULTS: Here, pattern of splice-disrupt variants was investigated using 21,842,764 genomic variants in different types of prostate cancer. A particular attention was paid to genomic locations of splice-disrupt variants on target genes. HLA-A in prostate cancer, MSR1 in familial prostate cancer, and EGFR in both castration-resistant prostate cancer and metastatic castration-resistant had the highest allele frequencies of splice-disrupt variations. Some splice-disrupt variants, located on coding sequences of NCOR2, PTPRC, and CRP, were solely present in the advanced metastatic castration-resistant prostate cancer. High-risk splice-disrupt variants were identified based on computationally calculated Polymorphism Phenotyping (PolyPhen), Sorting Intolerant From Tolerant (SIFT), and Genomic Evolutionary Rate Profiling (GERP) + + scores as well as the recorded clinical significance in dbSNP database of NCBI. Functional annotation of damaging splice-disrupt variants highlighted important cancer-associated functions, including endocrine resistance, lipid metabolic process, steroid metabolic process, regulation of mitotic cell cycle, and regulation of metabolic process. This is the first study that profiles the splice-disrupt genomic variants and their target genes in prostate cancer. Literature mining based variant analysis highlighted the importance of rs1800716 variant, located on the CYP2D6 gene, involved in a range of important functions, such as RNA spicing, drug interaction, death, and urotoxicity. CONCLUSIONS: This is the first study that profiles the splice-disrupt genomic variants and their target genes in different types of prostate cancer. Unravelling alternative splicing opens a new avenue towards the establishment of new diagnostic and prognostic markers for prostate cancer progression and metastasis.

Association of HER1 and HER2 Gene Variants in the Predisposition of Colorectal Cancer.

BACKGROUND: Colorectal cancer (CRC) is a major health concern worldwide. A series of sequential accumulation of genetic and epigenetic changes are responsible for the initiation and progression of diseases via the normal > adenoma > carcinoma sequence. Genetic variants in crucial cancer-causing genes are known to mediate the risk of cancer. OBJECTIVE: In this case-control study, we examined single nucleotide polymorphism (SNP) in HER1 (rs763317 and rs3752651) and HER2 (rs1136201 and rs1058808) genes to assess their role in the susceptibility of CRC in a Saudi population. METHODS: TaqMan allelic discrimination assay was utilized to identify the genotypes in 163 normal and 143 CRC patients. RESULTS: In the overall analysis, the rs3752651 and rs1136201 were significantly associated with the risk of CRC. Although none of the examined SNPs had any impact on the age at which CRC was diagnosed, interestingly, three SNPs showed a significant association based on gender. The rs3752651 conferred significant protection only in men, whereas rs1136201 diminished the risk and rs1058808 considerably increased the susceptibility of CRC only in women. CONCLUSIONS: Our result suggests that these SNPs in HER1 and HER2 after validation in larger cohorts of different ethnicities may be utilized as genetic screening markers for predicting colorectal cancer predisposition.

Advances in understanding the specificity function of transporters by machine learning.

Understanding the underlying molecular mechanism of transporter activity is one of the major discussions in structural biology. A transporter can exclusively transport one ion (specific transporter) or multiple ions (general transporter). This study compared categorical and numerical features of general and specific calcium transporters using machine learning and attribute weighting models. To this end, 444 protein features, such as the frequency of dipeptides, organism, and subcellular location, were extracted for general (n = 103) and specific calcium transporters (n = 238). Aliphatic index, subcellular location, organism, Ile-Leu frequency, Glycine frequency, hydrophobic frequency, and specific dipeptides such as Ile-Leu, Phe-Val, and Tyr-Gln were the key features in differentiating general from specific calcium transporters. Calcium transporters in the cell outer membranes were specific, while the inner ones were general; additionally, when the hydrophobic frequency or Aliphatic index is increased, the calcium transporter act as a general transporter. Random Forest with accuracy criterion showed the highest accuracy (88.88% ±5.75%) and high AUC (0.964 ± 0.020), based on 5-fold cross-validation. Decision Tree with accuracy criterion was able to predict the specificity of calcium transporter irrespective of the organism and subcellular location. This study demonstrates the precise classification of transporter function based on sequence-derived physicochemical features.

Serum Proteomic Analysis of Cannabis Use Disorder in Male Patients.

Cannabis use has been growing recently and it is legally consumed in many countries. Cannabis has a variety of phytochemicals including cannabinoids, which might impair the peripheral systems responses affecting inflammatory and immunological pathways. However, the exact signaling pathways that induce these effects need further understanding. The objective of this study is to investigate the serum proteomic profiling in patients diagnosed with cannabis use disorder (CUD) as compared with healthy control subjects. The novelty of our study is to highlight the differentially changes proteins in the serum of CUD patients. Certain proteins can be targeted in the future to attenuate the toxicological effects of cannabis. Blood samples were collected from 20 male individuals: 10 healthy controls and 10 CUD patients. An untargeted proteomic technique employing two-dimensional difference in gel electrophoresis coupled with mass spectrometry was employed in this study to assess the differentially expressed proteins. The proteomic analysis identified a total of 121 proteins that showed significant changes in protein expression between CUD patients (experimental group) and healthy individuals (control group). For instance, the serum expression of inactive tyrosine protein kinase PEAK1 and tumor necrosis factor alpha-induced protein 3 were increased in CUD group. In contrast, the serum expression of transthyretin and serotransferrin were reduced in CUD group. Among these proteins, 55 proteins were significantly upregulated and 66 proteins significantly downregulated in CUD patients as compared with healthy control group. Ingenuity pathway analysis (IPA) found that these differentially expressed proteins are linked to p38MAPK, interleukin 12 complex, nuclear factor-κB, and other signaling pathways. Our work indicates that the differentially expressed serum proteins between CUD and control groups are correlated to liver X receptor/retinoid X receptor (RXR), farnesoid X receptor/RXR activation, and acute phase response signaling.

NOTCH Single Nucleotide Polymorphisms in the Predisposition of Breast and Colorectal Cancers in Saudi Patients.

Breast cancer (BC) is a heterogeneous disease and is one of the most common malignancy affecting women worldwide while colorectal cancer (CRC) is estimated to be the third common cancer and second leading cause of cancer related death globally. Both BC and CRC involve multiple genetic and epigenetic alterations in genes belonging to various signaling pathways including NOTCH that has been implicated in the development of these cancers. We investigated four single nucleotide polymorphisms, each in genes encoding NOTCH1-4 receptors for their role in susceptibility to breast and colorectal cancers in Saudi population. In this case-control study, TaqMan genotypic analysis of rs3124591 in NOTCH1 and rs3820041 in NOTCH4 did not exhibit association with breast as well as colorectal cancers. However, a strong association of rs11249433 which is in close proximity to NOTCH2 was observed with breast cancer susceptibility especially with those having an early onset of the disease. Interestingly, the rs1043994 located in NOTCH3 showed gender preference and was found to be significantly associated with colorectal cancers in males. Validation of these findings in bigger populations of different ethnicities may prove beneficial in identifying rs11249433 and rs1043994 as genetic screening markers for early detection of breast and colorectal carcinomas, respectively.

Novel insights of waterborne human rotavirus A in Riyadh (Saudi Arabia) involving G2 predominance and emergence of a thermotolerant sequence.

The routine evaluation of water environments is necessary to manage enteric virus-mediated fecal contamination and the possible emergence of novel variants. Here, we detected human rotavirus A (HRVA) circulating in two wastewater treatment plants, two lakes, irrigation water and a wastewater landfill located in Riyadh. VP7-derived surface protein sequences were assessed by phylogenetic analyses and inspection of thermotolerance-mediated secondary structure and seasonal variation. HRVA was most prevalent at An-Nazim wastewater landfill (AN-WWLF; 63.89%). Phylogenetic analyzes revealed the predominance of HRVA G2 lineage for the first time in Saudi Arabia. Moreover, a single HRVA sequence (2B64I-ANLF3/2018) was recovered at 45 °C from AN-WWLF; secondary structure prediction indicated that this sequence was thermotolerant with a high hydrophobicity, an absence of Ramachandran outliers, and a higher content of proline patches on the protein surface. Varied relationships were significantly observed between sampling areas influenced by temperature ranges (p < 0.05). HRVA prevalence was influenced by seasonal variations, favoring moderate temperatures in late autumn and early winter in all locations. However, a significant temperature impact was detected in Wadi-Hanifah Lake (p = 0.01). Our study extends the knowledge of currently circulating HRVA genotypes, and indicates the probable emergence of thermotolerant strains and seasonally mediated HRVA prevalence.

A Transcription Regulatory Sequence in the 5' Untranslated Region of SARS-CoV-2 Is Vital for Virus Replication with an Altered Evolutionary Pattern against Human Inhibitory MicroRNAs.

Our knowledge of the evolution and the role of untranslated region (UTR) in SARS-CoV-2 pathogenicity is very limited. Leader sequence, originated from UTR, is found at the 5' ends of all encoded SARS-CoV-2 transcripts, highlighting its importance. Here, evolution of leader sequence was compared between human pathogenic and non-pathogenic coronaviruses. Then, profiling of microRNAs that can inactivate the key UTR regions of coronaviruses was carried out. A distinguished pattern of evolution in leader sequence of SARS-CoV-2 was found. Mining all available microRNA families against leader sequences of coronaviruses resulted in discovery of 39 microRNAs with a stable thermodynamic binding energy. Notably, SARS-CoV-2 had a lower binding stability against microRNAs. hsa-MIR-5004-3p was the only human microRNA able to target the leader sequence of SARS and to a lesser extent, also SARS-CoV-2. However, its binding stability decreased remarkably in SARS-COV-2. We found some plant microRNAs with low and stable binding energy against SARS-COV-2. Meta-analysis documented a significant (p < 0.01) decline in the expression of MIR-5004-3p after SARS-COV-2 infection in trachea, lung biopsy, and bronchial organoids as well as lung-derived Calu-3 and A549 cells. The paucity of the innate human inhibitory microRNAs to bind to leader sequence of SARS-CoV-2 can contribute to its high replication in infected human cells.

A matrix-assisted laser desorption/ionization imaging mass spectrometric approach to study weight-related changes within thyroid tissue.

Obesity is associated with numerous comorbidities along with abnormalities of the endocrine system, more commonly manifesting as dysfunctions of the thyroid gland such as goiter. Changes in weight, especially an increase, could lead to an increase in the incidence of thyroid dysfunction; however, its pathophysiology remains to be elucidated. In the present study, we aimed to interrogate the changes in the protein distribution and abundance between the lean patients and patients with obesity thyroid tissue sections through utilizing this technique. The FFPE-fixed thyroid tissue blocks from the selected cases and controls were identified and targeted for matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) analysis. Patients in the 30 to 75 years age group and undergoing total thyroidectomy for benign thyroid disease were recruited. Patients with thyroid cancers, autoimmune disorders, and other inflammatory conditions were excluded from the study. The selected patients were divided into two groups according to their BMIs: lean (BMI < 25) and obese (BMI > 35). An initial trial set was used as a pilot study for the optimization of the MALDI IMS protocol that was next applied to the selected thyroid tissues. MALDI IMS data from all the samples were aligned and statistical analysis carried out by k-means and linear discriminant analysis (LDA) classification model using principle component analysis (PCA) results were evaluated between the two groups: controls (lean) and cases (obese). Receiver operator characteristic (ROC) curves were alternatively used to calculate the variability of the identified peptides. The discriminating peptides were also independently identified and related to their corresponding proteins by using liquid chromatography and tandem mass spectrometry (LC-MS/MS) analyses. Eight peptides mainly from thyroglobulin were found to be upregulated whereas 10 others were found to be downregulated in the lean compared to the obese group. Through this technique, we will be able to better understand the relationship between the disease entity and obesity.

Antimicrobial Mechanism and Identification of the Proteins Mediated by Extracts from Asphaltum punjabianum and Myrtus communis.

Myrtus communis ("myrtle") and Asphaltum punjabianum ("shilajeet") are a medicinal plant and a long-term-humified dead plant material, respectively. We studied their antibacterial and anticandidal activities against Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, and Candida albicans. The activities of the aqueous extracts of the studied materials were measured using agar-well diffusion methods. Furthermore, proteomic analysis of treated microbial cells was conducted to identify affected proteins. The results showed both antibacterial and anticandidal activities for the myrtle extract (ME), while the shilajeet extract (SE) showed antibacterial activity only. The highest antimicrobial activity was observed against E. coli among the microbes tested; therefore, it was taken as the model for the proteomic analysis to identify the antimicrobial mechanism of ME and SE using two-dimensional electrophoresis. Upregulation of expression of 42 proteins and downregulation of expression of 6 proteins were observed in E. coli treated with ME, whereas 12 upregulated and 104 downregulated proteins were detected in E. coli treated with SE, in comparison with the control. About 85% of identified expressed proteins were from the cytoplasm and 15% from microbial cell walls, indicating the penetration of extracts inside cells. A higher percentage of expressed proteins was recorded for enzymatic activity. Our findings suggest that the major targets of the antibacterial action were proteins involved in the outer membrane, oxidative stress, and metabolism. Our data might reveal new targets for antimicrobial agents.

Serum-Based Proteomics Profiling in Adult Patients with Cystic Fibrosis.

Cystic fibrosis (CF), the most common lethal autosomal recessive disorder among Caucasians, is caused by mutations in the CF transmembrane conductance regulator (CFTR) chloride channel gene. Despite significant advances in the management of CF patients, novel disease-related biomarkers and therapies must be identified. We performed serum proteomics profiling in CF patients (n = 28) and healthy subjects (n = 10) using the 2D-DIGE MALDI-TOF proteomic approach. Out of a total of 198 proteins identified, 134 showed a statistically significant difference in abundance and a 1.5-fold change (ANOVA, p < 0.05), including 80 proteins with increased abundance and 54 proteins with decreased abundance in CF patients. A multiple reaction monitoring-mass spectrometry analysis of six differentially expressed proteins identified by a proteomic approach (DIGE-MALD-MS) showed a significant increase in C3 and CP proteins and a decrease in APOA1, Complement C1, Hp, and RBP4proteins compared with healthy controls. Fifteen proteins were identified as potential biomarkers for CF diagnosis. An ingenuity pathway analysis of the differentially regulated proteins indicates that the central nodes dysregulated in CF subjects involve pro-inflammatory cytokines, ERK1/2, and P38 MAPK, which are primarily involved in catalytic activities and metabolic processes. The involved canonical pathways include those related to FXR/RXR, LXR/RXR, acute phase response, IL12, nitric oxide, and reactive oxygen species in macrophages. Our data support the current efforts toward augmenting protease inhibitors in patients with CF. Perturbations in lipid and vitamin metabolism frequently observed in CF patients may be partly due to abnormalities in their transport mechanism.

MicroRNA-3148 acts as molecular switch promoting malignant transformation and adipocytic differentiation of immortalized human bone marrow stromal cells via direct targeting of the SMAD2/TGFß pathway.

MicroRNAs (miRs/miRNAs) play a key role in posttranscriptional regulation of gene expression and are implicated in a number of physiological and pathological conditions, including cellular malignant transformation. In the current study, we investigated the role of miR-3148 in regulating human stromal (mesenchymal) stem cell (hMSC) differentiation and transformation. Stable expression of miR-3148 in telomerized hMSC (hMSC-miR-3148) led to significant increase in in vitro adipocytic differentiation and suppression of osteoblastic differentiation. Concordantly, global gene expression profiling revealed significant enrichment in cholesterol biosynthesis pathway, and pathways related to enhanced cell movement and survival, whereas processes related to bone and connective tissue developments, cell death, apoptosis, and necrosis were downregulated. Global proteomic analysis using 2D-DIGE followed by mass spectrometry (MS) revealed significant changes in protein expression in hMSC-miR-3148 and enrichment in protein networks associated with carcinogenesis. Functional studies revealed that hMSC-miR-3148 exhibited enhanced in vitro cell proliferation, colony formation, migration, invasion, sphere formation, doxorubicin resistance, and increased active number of cells in S and G2/M cell cycle phases and formed sarcoma-like tumors with adipocyte infiltration when implanted into immunocompromised mice. SMAD2 was identified as bone fide gene target for miR-3148 using qRT-PCR, Western blotting, and UTR-based reporter assay. In agreement with our data, SMAD2 expression was downregulated in 47% of patients with soft tissue sarcoma. Bioinformatics analysis revealed that elevated miR-3148 expression correlates with poor prognosis in several human cancer types, including sarcoma. Our study identified miR-3148 as factor regulating hMSC differentiation and is involved in promoting malignant transformation of telomerized hMSC.

Serum proteomic profiling of patients with amphetamine use disorder.

BACKGROUND: Amphetamine use disorder has been recently classified as an epidemic condition. Amphetamine use/abuse has been associated with several neurological and inflammatory effects. However, the exact mechanism involved in these effects warrants further investigation. The aim of this study was to determine any alterations in the serum proteome of individuals classified as patients with amphetamine use disorder compared to that of control subjects. METHODS: An untargeted proteomic approach employing two-dimensional difference in gel electrophoresis coupled with mass spectrometry was used to identify the patterns of differentially expressed proteins. Serum samples were collected from 20 individuals (males) including 10 subjects with amphetamine use disorder and 10 healthy controls for the present study. RESULTS: The analysis revealed 78 proteins with a significant difference in protein abundance between the amphetamine-addicted subjects and controls. Among them, 71 proteins were upregulated while 7 proteins remained downregulated in the amphetamine-addicted group. These proteins were further analyzed by ingenuity pathway analysis (IPA) to investigate their correlation with other biomarkers. IPA revealed the correlation of altered proteins with mitogen-activated protein kinase (MAP2K1/K2), p38MAPK, protein kinase-B (PKB; Akt), extracellular signal-regulated kinase (ERK1/2), and nuclear factor-κB signaling pathways. Importantly, these pathways are highly involved in neurological diseases, inflammatory responses, and cellular compromise. CONCLUSIONS: Our data suggest that the changes in the levels of serum proteins between amphetamine and control groups might affect cellular compromise, inflammatory response, and neurological diseases.