Identification of biomarkers of chronic kidney disease among kidney-derived proteins

Background Chronic kidney disease (CKD) has few objective symptoms, and it is difficult to make an early diagnosis by using existing methods. Therefore, new biomarkers enabling diagnosis of renal dysfunction at an early stage need to be developed. Here, we searched for new biomarkers of CKD by focusing on kidney-derived proteins that could sensitively reflect that organ’s disease state. Methods To identify candidate marker proteins, we performed a proteomics analysis on renal influx and efflux blood collected from the same individual. Results Proteomics analysis revealed 662 proteins in influx blood and 809 in efflux. From these identified proteins, we selected complement C1q as a candidate; the plasma C1q level was significantly elevated in the renal efflux of donors. Moreover, the plasma concentration of C1q in a mouse model of diabetic nephropathy was significantly increased, in association with increases in blood glucose concentration and urinary protein content. Importantly, we demonstrated that the tendency of C1q to increase in the plasma of CKD patients was correlated with a decrease in their estimated glomerular filtration rate. Conclusion Overall, our results indicate that our approach of focusing on kidney-derived proteins is useful for identifying new CKD biomarkers and that C1q has potential as a biomarker of renal function.

In silico Molecular Docking Analysis of Imatinib to Target the Marker Proteins of Breast Cancer

Cancer is an uncontrolled over growth of abnormal cells elsewhere in the body. It is the second leading cause of death globally due to non communicable disease. Among the various types of cancers, the incidence of breast cancer is next to lung cancer. The most commonly used drugs to treat breast cancer are namely, Anastrozole, Arimidex, Letrozol, Imatinib, Tamoxifen, Raloxifene, Toremifene and so on. The hope is to establish the specificity of the drug Imatinib towards the selective potential breast cancers such as mammalian target of rapamycin, (mTOR), human epidermal growth factor receptor 2 (HER2), estrogen receptor (ER), poly (ADP-Ribose) polymerase (PARP) and phosphoprotein 53 (p53). To identify the promising target, the Schrodinger software was utilized for the study. The study helped to evaluate the pharmacokinetic properties and binding efficiency of Imatinib towards the breast cancer proteins. The results of study showed that the Imatinib exhibited better binding affinities to mTOR and HER2 as compared to ER, PARP and P53 proteins. The present study will be more useful to rationalize the anticancer therapy based on the expression levels of the target protein in the cancer microenvironment.

RFPlasmid: predicting plasmid sequences from short-read assembly data using machine learning

Antimicrobial-resistance (AMR) genes in bacteria are often carried on plasmids and these plasmids can transfer AMR genes between bacteria. For molecular epidemiology purposes and risk assessment, it is important to know whether the genes are located on highly transferable plasmids or in the more stable chromosomes. However, draft whole-genome sequences are fragmented, making it difficult to discriminate plasmid and chromosomal contigs. Current methods that predict plasmid sequences from draft genome sequences rely on single features, like k-mer composition, circularity of the DNA molecule, copy number or sequence identity to plasmid replication genes, all of which have their drawbacks, especially when faced with large single-copy plasmids, which often carry resistance genes. With our newly developed prediction tool RFPlasmid, we use a combination of multiple features, including k-mer composition and databases with plasmid and chromosomal marker proteins, to predict whether the likely source of a contig is plasmid or chromosomal. The tool RFPlasmid supports models for 17 different bacterial taxa, including Campylobacter , Escherichia coli and Salmonella , and has a taxon agnostic model for metagenomic assemblies or unsupported organisms. RFPlasmid is available both as a standalone tool and via a web interface.

Agrobacterium tumefaciens Type IV and Type VI Secretion Systems Reside in Detergent-Resistant Membranes

Cell membranes are not homogenous but compartmentalized into lateral microdomains, which are considered as biochemical reaction centers for various physiological processes in eukaryotes and prokaryotes. Due to their special lipid and protein composition, some of these microdomains are resistant to treatment with non-ionic detergents and can be purified as detergent-resistant membranes (DRMs). Here we report the proteome of DRMs from the Gram-negative phytopathogen Agrobacterium tumefaciens. Using label-free liquid chromatography-tandem mass spectrometry, we identified proteins enriched in DRMs isolated under normal and virulence-mimicking growth conditions. Prominent microdomain marker proteins such as the SPFH (stomatin/prohibitin/flotillin/HflKC) proteins HflK, HflC and Atu3772, along with the protease FtsH were highly enriched in DRMs isolated under any given condition. Moreover, proteins involved in cell envelope biogenesis, transport and secretion, as well as motility- and chemotaxis-associated proteins were overrepresented in DRMs. Most strikingly, we found virulence-associated proteins such as the VirA/VirG two-component system, and the membrane-spanning type IV and type VI secretion systems enriched in DRMs. Fluorescence microscopy of the cellular localization of both secretion systems and of marker proteins was in agreement with the results from the proteomics approach. These findings suggest that virulence traits are micro-compartmentalized into functional microdomains in A. tumefaciens.

Mutation in OsFWL7 Affects Cadmium and Micronutrient Metal Accumulation in Rice

Micronutrient metals, such as Mn, Cu, Fe, and Zn, are essential heavy metals for plant growth and development, while Cd is a nonessential heavy metal that is highly toxic to both plants and humans. Our understanding of the molecular mechanisms underlying Cd and micronutrient metal accumulation in plants remains incomplete. Here, we show that OsFWL7, an FW2.2-like (FWL) family gene in Oryza sativa, is preferentially expressed in the root and encodes a protein localized to the cell membrane. The osfwl7 mutation reduces both the uptake and the root-to-shoot translocation of Cd in rice plants. Additionally, the accumulation of micronutrient metals, including Mn, Cu, and Fe, was lower in osfwl7 mutants than in the wildtype plants under normal growth conditions. Moreover, the osfwl7 mutation affects the expression of several heavy metal transporter genes. Protein interaction analyses reveal that rice FWL proteins interact with themselves and one another, and with several membrane microdomain marker proteins. Our results suggest that OsFWL7 is involved in Cd and micronutrient metal accumulation in rice. Additionally, rice FWL proteins may form oligomers and some of them may be located in membrane microdomains.

Effect of a novel piperazine compound on cancer cells

Many drugs have been developed for anticancer chemotherapy. However, more anti-cancer drugs should be developed from potential chemicals to circumvent the disadvantages of existing drugs. Most anti-cancer chemicals induce apoptosis in cancer cells. This study tested the efficiency of a new chemical, the piperazine derivative 1-[2-(Allylthio) benzoyl]-4-(4-methoxyphenyl) piperazine (CB01), on glioblastoma (U87) and cervix cancer (HeLa) cells. CB01 was highly cytotoxic to these cells (IC50S < 50 nM) and induced the traditional apoptotic symptoms of DNA fragmentation and nuclear condensation at 40 nM. Western-blot analysis of the cell lysates revealed that the intracellular apoptotic marker proteins, such as cleaved caspase-3, cytochrome c, and Bax, were highly upregulated in the CB01-treated cells. Furthermore, increased activities of caspase-3 and -9, but not caspase-8, were observed. Therefore, these results suggest that CB01 can act as an anticancer chemotherapeutic by stimulating the intrinsic mitochondrial signaling pathway to induce cytotoxicity and apoptosis in cancer cells.

Inter- and Intrapopulational Heterogeneity of Characteristic Markers in Adult Human Neural Crest-derived Stem Cells

Adult human neural crest-derived stem cells (NCSCs) are found in a variety of adult tissues and show an extraordinary broad developmental potential. Despite their great differentiation capacity, increasing evidence suggest a remaining niche-dependent variability between different NCSC-populations regarding their differentiation behavior and expression signatures. In the present study, we extended the view on heterogeneity of NCSCs by identifying heterogeneous expression levels and protein amounts of characteristic markers even between NCSCs from the same niche of origin. In particular, populations of neural crest-derived inferior turbinate stem cells (ITSCs) isolated from different individuals showed significant variations in characteristic NCSC marker proteins Nestin, S100 and Slug in a donor-dependent manner. Notably, increased nuclear protein amounts of Slug were accompanied by a significantly elevated level of nuclear NF-κB-p65 protein, suggesting an NF-κB-dependent regulation of NCSC-makers. In addition to this interpopulational genetic heterogeneity of ITSC-populations from different donors, single ITSCs also revealed a strong heterogeneity regarding the protein amounts of Nestin, S100, Slug and NF-κB-p65 even within the same clonal culture. Our present findings therefor strongly suggest ITSC-heterogeneity to be at least partly based on an interpopulational genetic heterogeneity dependent on the donor accompanied by a stochastic intrapopulational heterogeneity between single cells. We propose this stochastic intrapopulational heterogeneity to occur in addition to the already described genetic variability between clonal NCSC-cultures and the niche-dependent plasticity of NCSCs. Our observations offer a novel perspective on NCSC-heterogeneity, which may build the basis to understand heterogeneous NCSC-behavior. Graphical Abstract

Comparison of MicroRNA Profiles in Extracellular Vesicles from Small and Large Goat Follicular Fluid

Extracellular vesicles (EVs), which exist in the follicular fluid of ruminant ovaries, are considered as cargo carriers for the transfer of biomolecules to recipient cells. However, the functions and changes in EVs in antral follicles remain ambiguous. In the present study, we isolated and characterized EVs from goat follicular fluid by means of differential ultracentrifugation and Western blotting of marker proteins. Bioinformatics tools were used to detect miRNA expression levels in EVs. Different miRNA expression patterns of EVs exist in small to large follicles. Thirteen differentially expressed miRNAs (seven upregulated and six downregulated) were identified and used for analysis. A total of 1948 predicted target genes of 13 miRNAs were mapped to signaling pathways, and three significantly enriched pathways (FoxO, MAPK, and PI3K-AKT signaling pathways) were involved in follicular development, as revealed by KEGG enrichment analysis. Our findings suggest that EVs in follicular fluid play biofunctional roles during follicular development in goats.

Adaptive responses of Pseudomonas aeruginosa to treatment with antibiotics

Pseudomonas aeruginosa is among the highest priority pathogens for drug development, because of its resistance to antibiotics, extraordinary adaptability, and persistence. Anti-pseudomonal research is strongly encouraged to address the acute scarcity of innovative antimicrobial lead structures. In an effort to understand the physiological response of P. aeruginosa to clinically relevant antibiotics, we investigated the proteome after exposure to ciprofloxacin, levofloxacin, rifampicin, gentamicin, tobramycin, azithromycin, tigecycline, polymyxin B, colistin, ceftazidime, meropenem, and piperacillin/tazobactam. We further investigated the response to CHIR-90, which represents a promising class of lipopolysaccharide biosynthesis inhibitors currently under evaluation. Radioactive pulse-labeling of newly synthesized proteins followed by 2D-PAGE was used to monitor the acute response of P. aeruginosa to antibiotic treatment. The proteomic profiles provide insights into the cellular defense strategies for each antibiotic. A mathematical comparison of these response profiles based on upregulated marker proteins revealed similarities of responses to antibiotics acting on the same target area. This study provides insights into the effects of commonly used antibiotics on P. aeruginosa and lays the foundation for the comparative analysis of the impact of novel compounds with precedented and unprecedented modes of action.

Transformation of Seed Non-Transmissible Hop Viroids in Nicotiana benthamiana Causes Distortions in Male Gametophyte Development

Viroids are small, non-coding, parasitic RNAs that promote developmental distortions in sensitive plants. We analyzed pollen of Nicotiana benthamiana after infection and/or ectopic transformation with cDNAs of citrus bark cracking viroid (CBCVd), apple fruit crinkle viroid (AFCVd) and potato spindle tuber viroid (PSTVd) variant AS1. These viroids were seed non-transmissible in N. benthamiana. All viroids propagated to high levels in immature anthers similar to leaves, while their levels were drastically reduced by approximately 3.6 × 103, 800 and 59 times in mature pollen of CBCVd, AFCVd and PSTVd infected N. benthamiana, respectively, in comparison to leaves. These results suggest similar elimination processes during male gametophyte development as in the Nicotiana tabacum we presented in our previous study. Mature pollen of N. benthamiana showed no apparent defects in infected plants although all three viroids induced strong pathological symptoms on leaves. While Nicotiana species have naturally bicellular mature pollen, we noted a rare occurrence of mature pollen with three nuclei in CBCVd-infected N. benthamiana. Changes in the expression of ribosomal marker proteins in AFCVd-infected pollen were detected, suggesting some changes in pollen metabolism. N. benthamiana transformed with 35S-driven viroid cDNAs showed strong symptoms including defects in pollen development. A large number of aborted pollen (34% and 62%) and a slight increase of young pollen grains (8% and 15%) were found in mature pollen of AFCVd and CBCVd transformants, respectively, in comparison to control plants (3.9% aborted pollen and 0.3% young pollen). Moreover, pollen grains with malformed nuclei or trinuclear pollen were found in CBCVd-transformed plants. Our results suggest that “forcing” overexpression of seed non-transmissible viroid led to strong pollen pathogenesis. Viroid adaptation to pollen metabolism can be assumed as an important factor for viroid transmissibility through pollen and seeds.