Expression of IFN-Gamma is significantly reduced during severity of covid-19 infection in hospitalized patients.

Cytokines play an important role in SARS-CoV-2 infection progression and severity. A number of inflammatory cytokines have been directly associated with disease severity including IL-6 (interleukin-6), IL-10, TNF-α (tumor necrosis factor alpha), IFN-γ (interferon-gamma). Here, in this study, the aim was to better understand the interplay between host immune response mediated by cytokines and severity of SARS-CoV-2 infection by assessing cytokine expression. Therefore, we measured expression levels of a total of 12 genes (IFNA-1, IFN-γ, IL-1α, IL-1ß, IL-4, IL-6, IL-7, IL-10, IL-11, IL-13, IL-15, and IL-27) encoding inflammatory, anti-inflammatory and regulatory cytokines using QRT-PCR in hospitalized patients with severe infection compared to mildly infected. IFN-γ was identified as a potent marker of disease severity as indicated previously. Moreover, levels of IL-7 were also found to be partially reduced in patients compared to the healthy controls and linked negatively to disease severity. Identification of these cytokines may be helpful in not only understanding disease pathogenesis but also in better management of the patients after covid infection.

Knowledge, Attitudes and Perceptions towards COVID-19 Vaccinations: A Cross-Sectional Survey in Pakistan.

Background and Objectives: Several vaccines have been approved for the prevention of the coronavirus disease, discovered on 31 December in Wuhan, China. Pakistan procured vaccines from various countries. However, the lack of knowledge and reluctance of the general population to embrace the use of the vaccines are considered to be the major determinant of the slow vaccination rate. Hence, it is necessary to evaluate the willingness of the general population about their perception of the COVID-19 vaccination. Materials and Methods: A cross sectional survey based on a self-structured questionnaire comprising 18 questions was conducted (from 21 April-21 June) on 400 Pakistani participants to evaluate their knowledge, attitude, and perception towards the COVID-19 vaccination. Chi-square independent t-test and one-way Anova including a multiple step wise linear regression were used to draw conclusions about the results. p < 0.05 was considered significant. Results: A total of 400 participants responded in the knowledge, attitude, and perception (KAP) survey of which 46.5% were female and 53.5% were male. The mean age of participants was 36.08 years. This survey showed a poor knowledge (50.5%), a fair attitude (75.1%) and a poor perception (58.1%) towards the COVID-19 vaccination. Higher mean knowledge and attitude scores were reported in the age group 21-40, females, and unmarried urban citizens. Regression analysis showed that age, education, residence, and employment status influenced the knowledge and perception score to a considerable extent. Conclusions: The findings reflect an inadequate knowledge and perception on the one hand, but a better attitude towards the COVID-19 vaccination. This knowledge attitude and perception (KAP) survey will help in better understanding the opinion of the general population towards vaccination, and will be useful for policy makers and health care authorities aiming to increase the vaccination rate.

DNA methylation of pck1 might contribute to the programming effects of early high-carbohydrate diets feeding to the glucose metabolism across two generations in zebrafish (Danio rerio).

The purpose of this study is to assess the effects of early high-carbohydrate stimulus on glucose metabolism in zebrafish (Danio rerio) over two generations and explore the mechanisms that explain those nutritional programming effects via epigenetic modifications. The larvae were delivered a high-carbohydrate diet (53.66%) that was used as an early nutritional stimulus from the first feeding to the end of the yolk sac (FF) and 5 days after yolk-sac exhaustion (YE). The larvae (F0) and their offspring (F1) were then both fed the control diet (22.69%) until adulthood (15 weeks), and they were challenged with a high-carbohydrate diet (35.36%) at the 16th week. The results indicated that early stimulus immediately raised the mRNA levels of genes involved in glycolysis and gluconeogenesis. At the end of F0 challenge, both treatment groups decreased the plasma glucose levels, increased the expression levels of glucokinase (gck), and inhibited the mRNA during gluconeogenesis. When challenged in F1, the glucose levels were lower in FF (F1), and the mRNA levels of phosphoenolpyruvate carboxykinase 1 (pck1) were decreased in FF (F1) and YE (F1). Besides, in both experimental groups (F0 and F1), the CpG island of pck1 maintained lower levels of hypermethylated expression from F0 adult, 24 h post-fertilization embryo, to F1 adult. In conclusion, these results indicated that an early high-carbohydrate stimulus could significantly reprogram glucose metabolism in adult zebrafish, that those modifications could be partially transmitted to the next generation, and that the DNA methylation of pck1 might work as a stable epigenetic marker to contribute to those processes.

Deep learning techniques for liver and liver tumor segmentation: A review.

Liver and liver tumor segmentation from 3D volumetric images has been an active research area in the medical image processing domain for the last few decades. The existence of other organs such as the heart, spleen, stomach, and kidneys complicate liver segmentation and tumor identification task since these organs share identical properties in terms of shape, texture, and intensity values. Many automatic and semi-automatic techniques have been presented in recent years, in an attempt to establish a system for the reliable diagnosis and detection of liver illnesses, specifically liver tumors. With the evolution of deep learning techniques and their exceptional performance in the field of medical image processing, medical image segmentation in volumetric images using deep learning techniques has received a great deal of emphasis. The goal of this study is to provide an overview of the available deep learning approaches for segmenting liver and detecting liver tumors, as well as their evaluation metrics including accuracy, volume overlap error, dice coefficient, and mean square distance. This research also includes a detailed overview of the various 3D volumetric imaging architectures, designed specifically for the task of semantic segmentation. The comparison of approaches offered in earlier challenges for liver and tumor segmentation, as well as their dice scores derived from respective site sources, is also provided.

Routine Pediatric Vaccination in Pakistan During COVID-19: How Can Healthcare Professionals Help?

Pakistan is still fighting to overcome vaccine-preventable diseases (VPD). The vaccination coverage in rural children remains unsatisfactory amid various barriers including price, hesitancy, and low level of awareness. COVID-19 has decreased the immunization rate in Pakistan due to restricted movements, shortage of vaccines, and low coverage. During the current pandemic, there are high risks that children may get VPD resulting in another infectious disease catastrophe. There is a dire need to put aggressive measures by the government of Pakistan in time to ensure the optimal vaccine coverage. Public education programs for immunization, telehealth services, the involvement of community pharmacies, and the drive-through vaccination system may help to enhance the vaccination rate during the ongoing health crisis.

Correction: Renal Cells Express Different Forms of Vimentin: The Independent Expression Alteration of these Forms is Important in Cell Resistance to Osmotic Stress and Apoptosis.

[This corrects the article DOI: 10.1371/journal.pone.0068301.].

Adaption of the global test idea to proteomics data with missing values.

MOTIVATION: Global test procedures are frequently used in gene expression analysis to study the relationship between a functional subset of RNA transcripts and an experimental group factor. However, these procedures have been rarely used for the analysis of high-throughput data from other sources, such as proteome expression data. The main difficulties in transferring global test procedures from genomics to proteomics data are the more complicated way of obtaining functional annotations and the handling of missing values in some types of proteomics data. RESULTS: We propose a simple mixed linear model in combination with a permutation procedure and missing values imputation to conduct global tests in proteomics experiments. This new approach is motivated by protein expression data obtained by means of 2-D gel electrophoresis within a mouse experiment of our current research. A simulation study yielded that power and testing level of the mixed model alone can be affected by missing values in the dataset. Imputation of missing values was able to correct for a bias in some simulation settings. Our new approach provides the possibility to rank Gene Ontology (GO) terms associated with protein sets. It is also helpful in the case in which a specific protein is represented by multiple spots on a 2-D gel by considering these spots also as a protein set. Analysis of our data points at correlations between the deficiency of the protein 'calreticulin' and protein sets related to biological processes in the heart muscle. AVAILABILITY AND IMPLEMENTATION: Our proposed approach is included in the R-package 'RepeatedHighDim', which already contains a global test procedure for gene expression data. The package can be retrieved from http://cran.r-project.org/. CONTACT: klaus.jung@ams.med.uni-goettingen.de.

Renal cells express different forms of vimentin: the independent expression alteration of these forms is important in cell resistance to osmotic stress and apoptosis.

Osmotic stress has been shown to regulate cytoskeletal protein expression. It is generally known that vimentin is rapidly degraded during apoptosis by multiple caspases, resulting in diverse vimentin fragments. Despite the existence of the known apoptotic vimentin fragments, we demonstrated in our study the existence of different forms of vimentin VIM I, II, III, and IV with different molecular weights in various renal cell lines. Using a proteomics approach followed by western blot analyses and immunofluorescence staining, we proved the apoptosis-independent existence and differential regulation of different vimentin forms under varying conditions of osmolarity in renal cells. Similar impacts of osmotic stress were also observed on the expression of other cytoskeleton intermediate filament proteins; e.g., cytokeratin. Interestingly, 2D western blot analysis revealed that the forms of vimentin are regulated independently of each other under glucose and NaCl osmotic stress. Renal cells, adapted to high NaCl osmotic stress, express a high level of VIM IV (the form with the highest molecular weight), besides the three other forms, and exhibit higher resistance to apoptotic induction with TNF-α or staurosporin compared to the control. In contrast, renal cells that are adapted to high glucose concentration and express only the lower-molecular-weight forms VIM I and II, were more susceptible to apoptosis. Our data proved the existence of different vimentin forms, which play an important role in cell resistance to osmotic stress and are involved in cell protection against apoptosis.

Secretion of ERP57 is important for extracellular matrix accumulation and progression of renal fibrosis, and is an early sign of disease onset.

Renal fibrosis is characterized by excessive accumulation of extracellular matrix (ECM), which compromises organ function by replacing normal organ tissue. The molecular mechanisms leading to renal fibrosis are not fully understood. Here we demonstrated that TGFß1, AGT or PDGF stimulation of renal cells resulted in endoplasmic reticulum (ER) stress followed by activation of the protective unfolded protein response pathway and a high secretory level of protein disulfide isomerase ERP57 (also known as PDIA3). The TGFß1-induced impairment of ER function could be reversed by treatment with BMP7, suggesting a specific involvement in renal fibrosis. A clear correlation between the degree of fibrosis, ER stress and the level of ERP57 could be seen in fibrosis animal models and in biopsies of renal fibrosis patients. Protein interaction studies revealed that secreted ERP57 exhibits a strong interaction with ECM proteins. Knockdown of ERP57 or antibody-targeted inhibition of the secreted form significantly impaired the secretion and accumulation of ECM. Moreover, ERP57 was excreted in the early stages of chronic kidney disease, and its level in urine correlated with the degree of renal fibrosis, suggesting that the secretion of ERP57 represents one of the first signs of renal fibrosis onset and progression.

Impact of the antiproliferative agent ciclopirox olamine treatment on stem cells proteome.

AIM: To investigate the proteome changes of stem cells due to ciclopirox olamine (CPX) treatment compared to control and retinoic acid treated cells. METHODS: Stem cells (SCs) are cells, which have the ability to continuously divide and differentiate into various other kinds of cells. Murine embryonic stem cells (ESCs) and multipotent adult germline stem cells (maGSCs) were treated with CPX, which has been shown to have an antiproliferative effect on stem cells, and compared to stem cells treated with retinoic acid (RA), which is known to have a differentiating effect on stem cells. Classical proteomic techniques like 2-D gel electrophoresis and differential in-gel electrophoresis (DIGE) were used to generate 2D protein maps from stem cells treated with RA or CPX as well as from non-treated stem cells. The resulting 2D gels were scanned and the digitalized images were collated with the help of Delta 2D software. The differentially expressed proteins were analyzed by a MALDI-TOF-TOF mass spectrometer, and the identified proteins were investigated and categorized using bioinformatics. RESULTS: Treatment of stem cells with CPX, a synthetic antifungal clinically used to treat superficial mycoses, resulted in an antiproliferative effect in vitro, without impairment of pluripotency. To understand the mechanisms induced by CPX treatments which results in arrest of cell cycle without any marked effect on pluripotency, a comparative proteomics study was conducted. The obtained data revealed that the CPX impact on cell proliferation was accompanied with a significant alteration in stem cell proteome. By peptide mass fingerprinting and tandem mass spectrometry combined with searches of protein sequence databases, a set of 316 proteins was identified, corresponding to a library of 125 non-redundant proteins. With proteomic analysis of ESCs and maGSCs treated with CPX and RA, we could identify more than 90 single proteins, which were differently expressed in both cell lines. We could highlight, that CPX treatment of stem cells, with subsequent proliferation inhibition, resulted in an alteration of the expression of 56 proteins compared to non-treated cells, and 54 proteins compared to RA treated cells. Bioinformatics analysis of the regulated proteins demonstrated their involvement in various biological processes. To our interest, a number of proteins have potential roles in the regulation of cell proliferation either directly or indirectly. Furthermore the classification of the altered polypeptides according to their main known/postulated functions revealed that the majority of these proteins are involved in molecular functions like nucleotide binding and metal ion binding, and biological processes like nucleotide biosynthetic processes, gene expression, embryonic development, regulation of transcription, cell cycle processes, RNA and mRNA processing. Proteins, which are involved in nucleotide biosynthetic process and proteolysis, were downregulated in CPX treated cells compared to control, as well as in RA treated cells, which may explain the cell cycle arrest. Moreover, proteins which were involved in cell death, positive regulation of biosynthetic process, response to organic substance, glycolysis, anti-apoptosis, and phosphorylation were downregulated in RA treated cells compared to control and CPX treated cells. CONCLUSION: The CPX treatment of SCs results in downregulation of nucleotide binding proteins and leads to cell cycle stop without impairment of pluripotency.

Calreticulin is crucial for calcium homeostasis mediated adaptation and survival of thick ascending limb of Henle's loop cells under osmotic stress.

The thick ascending limb of Henle's loop (TALH) is normally exposed to variable and often very high osmotic stress and involves different mechanisms to counteract this stress. ER resident calcium binding proteins especially calreticulin (CALR) play an important role in different stress balance mechanisms. To investigate the role of CALR in renal epithelial cells adaptation and survival under osmotic stress, two-dimensional fluorescence difference gel electrophoresis combined with mass spectrometry and functional proteomics were performed. CALR expression was significantly altered in TALH cells exposed to osmotic stress, whereas renal inner medullary collecting duct cells and interstitial cells exposed to hyperosmotic stress showed no significant changes in CALR expression. Moreover, a time dependent downregulation of CALR was accompanied with continuous change in the level of free intracellular calcium. Inhibition of the calcium release, through IP3R antagonist, prevented CALR expression alteration under hyperosmotic stress, whereas the cell viability was significantly impaired. Overexpression of wild type CALR in TALH cells resulted in significant decrease in cell viability under hyperosmotic stress. In contrast, the hyperosmotic stress did not have any effect on cells overexpressing the CALR mutant, lacking the calcium-binding domain. Silencing CALR with siRNA significantly improved the cell survival under osmotic stress conditions. Taken together, our data clearly highlight the crucial role of CALR and its calcium-binding role in TALH adaptation and survival under osmotic stress.

Proteomics analysis identifies PARK7 as an important player for renal cell resistance and survival under oxidative stress.

Renal fibrosis is a process that is characterized by declining excretory renal function. The molecular mechanisms of fibrosis are not fully understood. Oxidative stress pathways were reported to be involved in renal tissue deterioration and fibrosis progression. In order to identify new molecular targets associated with oxidative stress and renal fibrosis, differential proteomics analysis was performed with established renal cell lines (TK173 and HK-2). The cells were treated with oxidative stress triggering factor H(2)O(2) and the proteome alterations were investigated. Two dimensional protein maps were generated and differentially expressed proteins were processed and identified using mass spectrometry analysis combined with data base search. Interestingly the increase of ROS in the renal cell lines upon H(2)O(2) treatment was accompanied by alteration of a large number of proteins, which could be classified in three categories: the first category grouped the proteins that have been described to be involved in fibrogenesis (e.g. ACTA2, VIN, VIM, DES, KRT, COL1A1, COL4A1), the second category, which was more interesting involved proteins of the oxidative stress pathway (PRDX1, PRDX2, PRDX6, SOD, PARK7, HYOU1), which were highly up-regulated under oxidative stress, and the third category represented proteins, which are involved in different other metabolic pathways. Among the oxidative stress proteins the up-regulation of PARK7 was accompanied by a shift in the pI as a result of oxidation. Knockdown of PARK7 using siRNA led to significant reduction in renal cell viability under oxidative stress. Under H(2)O(2) treatment the PARK7 knockdown cells showed up to 80% decrease in cell viability and an increase in apoptosis compared to the controls. These results highlight for the first time the important role of PARK7 in oxidative stress resistance in renal cells.

Proteomics characterization of cell model with renal fibrosis phenotype: osmotic stress as fibrosis triggering factor.

Renal fibroblasts are thought to play a major role in the development of renal fibrosis (RF). The mechanisms leading to this renal alteration remain poorly understood. We performed differential proteomic analyses with two established fibroblast cell lines with RF phenotype to identify new molecular pathways associated with RF. Differential 2-DE combined with mass spectrometry analysis revealed the alteration of more than 30 proteins in fibrotic kidney fibroblasts (TK188) compared to normal kidney fibroblast (TK173). Among these proteins, markers of the endoplasmic reticulum (ER) stress- and the unfolded protein response (UPR) pathway (GRP78, GRP94, ERP57, ERP72, and CALR) and the oxidative stress pathway proteins (PRDX1, PRDX2, PRDX6, HSP70, HYOU1) were highly up-regulated in fibrotic cells. Activation of these stress pathways through long time exposition of TK173, to high NaCl or glucose concentrations resulted in TK188 like phenotype. Parallel to an increase in reactive oxygen species, the stressed cells showed significant alteration of fibrosis markers, ER-stress and oxidative stress proteins. Similar effects of osmotic stress could be also observed on renal proximal tubule cells. Our data suggest an important role of the ER-stress proteins in fibrosis and highlights the pro-fibrotic effect of osmotic stress through activation of oxidative stress and ER-stress pathways.