Publications by authors named "Tikam Chand Dakal"

23 Publications

  • Page 1 of 1

Super-rapid race for saving lives by developing COVID-19 vaccines.

J Integr Bioinform 2021 Mar 25;18(1):27-43. Epub 2021 Mar 25.

Institute of Bioinformatics, International Technology Park, Bangalore560066, India.

The pandemic of coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has affected millions of people and claimed thousands of lives. Starting in China, it is arguably the most precipitous global health calamity of modern times. The entire world has rocked back to fight against the disease and the COVID-19 vaccine is the prime weapon. Even though the conventional vaccine development pipeline usually takes more than a decade, the escalating daily death rates due to COVID-19 infections have resulted in the development of fast-track strategies to bring in the vaccine under a year's time. Governments, companies, and universities have networked to pool resources and have come up with a number of vaccine candidates. Also, international consortia have emerged to address the distribution of successful candidates. Herein, we summarize these unprecedented developments in vaccine science and discuss the types of COVID-19 vaccines, their developmental strategies, and their roles as well as their limitations.
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http://dx.doi.org/10.1515/jib-2021-0002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8035961PMC
March 2021

Epigenetic Regulatory Enzymes: mutation Prevalence and Coexistence in Cancers.

Cancer Invest 2021 Mar 3;39(3):257-273. Epub 2021 Feb 3.

Department of Integrated Oncology, CIO Bonn, University Hospital Bonn, Bonn, Germany.

Epigenetic regulation is an important layer of transcriptional control with the particularity to affect the broad spectrum of genome. Over the years, largely due to the substantial number of recurrent mutations, there have been hundreds of novel driver genes characterized in various cancers. Additionally, the relative contribution of two dysregulated epigenomic entities (DNA methylation and histone modifications) that gradually drive the cancer phenotype remains in the research focus. However, a complex scenario arises when the disease phenotype does not harbor any relevant mutation or an abnormal transcription level. Although the cancer landscape involves the contribution of multiple genetic and non-genetic factors, herein, we discuss specifically the mutation spectrum of epigenetically-related enzymes in cancer. In addition, we address the coexistence of these two epigenetic entities in malignant human diseases, especially cancer. We suggest that the study of epigenetically-related somatic mutations in the early cellular differentiation stage of embryonic development might help to understand their later-staged footprints in the cancer genome. Furthermore, understanding the co-occurrence and/or inverse association of different disease types and redefining the general definition of "healthy" controls could provide insights into the genome reorganization.
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http://dx.doi.org/10.1080/07357907.2021.1872593DOI Listing
March 2021

Predicting the functional consequences of genetic variants in co-stimulatory ligand B7-1 using in-silico approaches.

Hum Immunol 2021 Feb 25;82(2):103-120. Epub 2020 Dec 25.

Department of Biosciences, Manipal University Jaipur, Jaipur 303007, Rajasthan, India; Genome and Computational Biology Lab, Department of Biotechnology, Mohanlal Sukhadia University, Udaipur 313001, Rajasthan, India. Electronic address:

The purpose of this research is to identify and characterize deleterious genetic variants in the co-stimulatory ligand B7-1, also known as the human cluster of differentiation CD80 marker. The B7-1 ligand and the major histocompatibility complex class II (MHC II) molecules are the main determinants that provide B-cells the required competency to act as antigen presenting cells. For this, participation of both MHC class II molecules and CD80 is required. The interaction of the CD80 ligand with CD28 on the surface 7 of T cells plays a key role in the activation of TH cells and progression of B cells through the S phase, hence, leading to their proliferation in mitosis. A set of 2313 genetic variants in the B7-1 ligand have been mapped and retrieved from dbSNP database. Subsequently, 150 non-synonymous single nucleotide polymorphisms (nsSNPs) were mapped and subjected to the sequence and structural homology based predictions, which were further analyzed for protein stability and the disease phenotypes. Finally, we identified 7 potentially damaging nsSNPs in the B7-1 ligand that may affect its interaction with the cognitive receptor CD28, hence, may also interfere with TH cell activation and B cell proliferation. We propose that subsequent experimental analyses (stability, expression and interactions) on these proteins can provide a deep understanding about the effect of these variants on the structure and function of CD80.
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http://dx.doi.org/10.1016/j.humimm.2020.12.001DOI Listing
February 2021

SARS-CoV-2 attachment to host cells is possibly mediated via RGD-integrin interaction in a calcium-dependent manner and suggests pulmonary EDTA chelation therapy as a novel treatment for COVID 19.

Immunobiology 2021 01 5;226(1):152021. Epub 2020 Nov 5.

Genome and Computational Biology Lab, Department of Biotechnology, Mohanlal Sukhadia University, Udaipur 313001, Rajasthan, India. Electronic address:

SARS-CoV-2 is a highly contagious virus that has caused serious health crisis world-wide resulting into a pandemic situation. As per the literature, the SARS-CoV-2 is known to exploit humanACE2 receptors (similar toprevious SARS-CoV-1) for gaining entry into the host cell for invasion, infection, multiplication and pathogenesis. However, considering the higher infectivity of SARS-CoV-2 along with the complex etiology and pathophysiological outcomes seen in COVID-19 patients, it seems that there may be an alternate receptor for SARS-CoV-2. I performed comparative protein sequence analysis, database based gene expression profiling, bioinformatics based molecular docking using authentic tools and techniques for unveiling the molecular basis of high infectivity of SARS-CoV-2 as compared to previous known coronaviruses. My study revealed that SARS-CoV-2 (previously known as 2019-nCoV) harbors a RGD motif in its receptor binding domain (RBD) and the motif is absent in all other previously known SARS-CoVs. The RGD motif is well known for its role in cell-attachment and cell-adhesion. My hypothesis is that the SARS-CoV-2 may be (via RGD) exploiting integrins, that have high expression in lungs and all other vital organs, for invading host cells. However, an experimental verification is required. The expression of ACE2, which is a known receptor for SARS-CoV-2, was found to be negligible in lungs. I assume that higher infectivity of SARS-CoV-2 could be due to this RGD-integrin mediated acquired cell-adhesive property. Gene expression profiling revealed that expression of integrins is significantly high in lung cells, in particular αvβ6, α5β1, αvβ8 and an ECM protein, ICAM1. The molecular docking experiment showed the RBD of spike protein binds with integrins precisely at RGD motif in a similar manner as a synthetic RGD peptide binds to integrins as found by other researchers. SARS-CoV-2 spike protein has a number of phosphorylation sites that can induce cAMP, PKC, Tyr signaling pathways. These pathways either activate calcium ion channels or get activated by calcium. In fact, integrins have calcium & metal binding sites that were predicted around and in vicinity of RGD-integrin docking site in our analysis which suggests that RGD-integrins interaction possibly occurs in calcium-dependent manner. The higher expression of integrins in lungs along with their previously known high binding affinity (~K = 4.0 nM) for virus RGD motif could serve as a possible explanation for high infectivity of SARS-CoV-2. On the contrary, human ACE2 has lower expression in lungs and its high binding affinity (~K = 15 nM) for spike RBD alone could not manifest significant virus-host attachment. This suggests that besides human ACE2, an additional or alternate receptor for SARS-CoV-2 is likely to exist. A highly relevant evidence never reported earlier which corroborate in favor of RGD-integrins mediated virus-host attachment is an unleashed cytokine storm which causes due to activation of TNF-α and IL-6 activation; and integrins role in their activation is also well established. Altogether, the current study has highlighted possible role of calcium and other divalent ions in RGD-integrins interaction for virus invasion into host cells and suggested that lowering divalent ion in lungs could avert virus-host cells attachment.
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http://dx.doi.org/10.1016/j.imbio.2020.152021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7642744PMC
January 2021

Is Involved in Urinary Tract and Urorectal Development.

Front Cell Dev Biol 2020 7;8:567. Epub 2020 Aug 7.

Department of Medical Sciences, University of Torino, Turin, Italy.

Previous studies in developing and zebrafish reported that the phosphate transporter is expressed in pronephric kidneys. The recent identification of as a monoallelic candidate gene for cloacal exstrophy further suggests its involvement in the urinary tract and urorectal development. However, little is known of the functional role of in urinary tract development. Here, we investigated this using morpholino oligonucleotide knockdown of the zebrafish ortholog . This caused kidney cysts and malformations of the cloaca. Moreover, in morphants we demonstrated dysfunctional voiding and hindgut opening defects mimicking imperforate anus in human cloacal exstrophy. Furthermore, we performed immunohistochemistry of an unaffected 6-week-old human embryo and detected in the urinary tract and the abdominal midline, structures implicated in the pathogenesis of cloacal exstrophy. Additionally, we resequenced in 690 individuals with bladder exstrophy-epispadias complex (BEEC) including 84 individuals with cloacal exstrophy. We identified two additional monoallelic variants. One was identified in a case-parent trio with classic bladder exstrophy, and one additional novel variant was detected in an affected mother who transmitted this variant to her affected son. To study the potential cellular impact of variants, we expressed them in HEK293 cells. Here, phosphate transport was not compromised, suggesting that it is not a disease mechanism. However, there was a tendency for lower levels of cleaved caspase-3, perhaps implicating apoptosis pathways in the disease. Our results suggest is involved in urinary tract and urorectal development and implicate as a disease-gene for BEEC.
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http://dx.doi.org/10.3389/fcell.2020.00567DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7426641PMC
August 2020

Human exome and mouse embryonic expression data implicate ZFHX3, TRPS1, and CHD7 in human esophageal atresia.

PLoS One 2020 5;15(6):e0234246. Epub 2020 Jun 5.

Center of Pediatric Surgery Hannover, Hannover Medical School, Hannover, Germany.

Introduction: Esophageal atresia with or without tracheoesophageal fistula (EA/TEF) occurs approximately 1 in 3.500 live births representing the most common malformation of the upper digestive tract. Only half a century ago, EA/TEF was fatal among affected newborns suggesting that the steady birth prevalence might in parts be due to mutational de novo events in genes involved in foregut development.

Methods: To identify mutational de novo events in EA/TEF patients, we surveyed the exome of 30 case-parent trios. Identified and confirmed de novo variants were prioritized using in silico prediction tools. To investigate the embryonic role of genes harboring prioritized de novo variants we performed targeted analysis of mouse transcriptome data of esophageal tissue obtained at the embryonic day (E) E8.5, E12.5, and postnatal.

Results: In total we prioritized 14 novel de novo variants in 14 different genes (APOL2, EEF1D, CHD7, FANCB, GGT6, KIAA0556, NFX1, NPR2, PIGC, SLC5A2, TANC2, TRPS1, UBA3, and ZFHX3) and eight rare de novo variants in eight additional genes (CELSR1, CLP1, GPR133, HPS3, MTA3, PLEC, STAB1, and PPIP5K2). Through personal communication during the project, we identified an additional EA/TEF case-parent trio with a rare de novo variant in ZFHX3. In silico prediction analysis of the identified variants and comparative analysis of mouse transcriptome data of esophageal tissue obtained at E8.5, E12.5, and postnatal prioritized CHD7, TRPS1, and ZFHX3 as EA/TEF candidate genes. Re-sequencing of ZFHX3 in additional 192 EA/TEF patients did not identify further putative EA/TEF-associated variants.

Conclusion: Our study suggests that rare mutational de novo events in genes involved in foregut development contribute to the development of EA/TEF.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0234246PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7274392PMC
August 2020

Ubiquitin Carboxyl-Terminal Hydrolases (UCHs): Potential Mediators for Cancer and Neurodegeneration.

Int J Mol Sci 2020 May 30;21(11). Epub 2020 May 30.

Department of Ophthalmology, University Hospital Bonn, 53127 Bonn, Germany.

Emerging evidence suggests an inverse association between cancer and neurodegenerative diseases (NDD). Although phenotypically different, both diseases display a significant imbalance in the ubiquitination/deubiquitination processes. Therefore, we particularly investigated the expression of ubiquitin C-terminal hydrolases (UCHs and ) a subfamily of deubiquitinating enzymes (DUBs), using publically available datasets (GTEx, TCGA) and observed altered expression of -, in 17 cancer types. Interestingly, (known to be enriched in neurons and interacting with the Parkinson's disease-associated protein α-synuclein) appeared to be a prognostic indicator of unfavorable outcome in endometrial and urothelial cancer, while increased expression of and was associated with poor survival in liver and thyroid cancer, respectively. In normal tissues, was found to be strongly expressed in the cerebral cortex and hypothalamus, while expression was somewhat higher in the testis. The occurrence of mutation rates in UCHs also suggests that and may play a more dominant role in cancers than and . We also characterized the functional context and configuration of the repeat elements in the promoter of DUBs genes and found that UCHs are highly discriminatory for catabolic function and are mainly enriched with LINE/CR1 repeats. Regarding the thesis of an inverse association between cancer and NDD, we observed that among all DUBs, UCHs are the one most involved in both entities. Considering a putative therapeutic potential based on presumed common mechanisms, it will be useful to determine whether other DUBs can compensate for the loss of UCH activity under physiological conditions. However, experimental evidence is required to substantiate this argument.
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http://dx.doi.org/10.3390/ijms21113910DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7312489PMC
May 2020

Advanced glycation end products and protein carbonyl levels in plasma reveal sex-specific differences in Parkinson's and Alzheimer's disease.

Redox Biol 2020 07 18;34:101546. Epub 2020 May 18.

Department of Neurology, University Clinic Bonn, Bonn, Germany; German Center for Neurodegenerative Diseases (DZNE), Bonn, Germany. Electronic address:

Neurodegenerative diseases (NDD) such as Alzheimer's (AD) and Parkinson's disease (PD) are distinct clinical entities, however, the aggregation of key neuronal proteins, presumably leading to neuronal demise appears to represent a common mechanism. It has become evident, that advanced glycation end products (AGEs) trigger the accumulation of such modified proteins, which eventually contributes to pathological aspect of NDDs. Increased levels of AGEs are found in amyloid plaques in AD brains and in both advanced and early PD (incidental Lewy body disease). The molecular mechanisms by which AGE dependent modifications may modulate the susceptibility towards NDDs, however, remain enigmatic and it is unclear, whether AGEs may serve as biomarker of NDD. In the present study, we examined AGEs (CML: Carboxymethyllysine and CEL: Carboxyethyllysine), markers of oxidative stress and micronutrients in the plasma of PD and AD patients and controls. As compared to healthy controls, AD females displayed lower levels of CEL while higher levels of CML were found in AD and PD patients. A somewhat similar pattern was observed for protein carbonyls (PC), revealing lower values exclusively in AD females, whereas AD males displayed significantly higher values compared to healthy controls and PD. Sex-specific differences were also observed for other relevant markers such as malondialdehyde, 3-nitrotyrosine, γ -tocopherols, retinol, plasma proteins and α-carotene, while α-tocopherols, β-carotene, lutein/zeaxanthin, β-cryptoxanthin and lycopene showed no relevant association. Taken together, our study suggests yet unappreciated differences of the distribution of AGEs among the sexes in NDD. We therefore suggest to make a clear distinction between sexes when analyzing oxidative (AGEs)-related stress and carbonyl-related stress and vitamins.
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http://dx.doi.org/10.1016/j.redox.2020.101546DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7251371PMC
July 2020

Genome organization in proximity to the BAP1 locus appears to play a pivotal role in a variety of cancers.

Cancer Sci 2020 Apr 12;111(4):1385-1391. Epub 2020 Feb 12.

Department of Ophthalmology, University Hospital Bonn, Bonn, Germany.

Cancer studies primarily focus on the characterization of the key driver genes and the underlying pathways. However, the contribution of other cancer-associated genes located in the genomic neighborhood of the driver genes could help to understand further aspects of cancer progression. Given the frequent involvement of chromosome 3 in multiple human cancers, in particular in the form of the prognostically highly relevant monosomy 3 in uveal melanoma (UM), we investigated the cumulative impact of cancer-associated genes on chromosome 3. Our analysis showed that these genes are enriched with repetitive elements with genes surrounded by distinctive repeats (MIR, hAT-Charlie, ERVL-MaLR, LINE-2, and simple/low complexity) in the promoter being more precisely associated with cancer-related pathways than the ones with major transposable elements (SINE/Alu and LINE-1). Additionally, these genes showed strong intrachromosomal chromatin interactions in 3D nuclear organization. Further investigations revealed a genomic hotspot in the vicinity of BAP1 locus, which is affected in 27 types of different cancers and contains abundant noncoding RNAs that are often expressed in a tissue-specific manner. The cross-species comparison of these cancer-associated genes revealed mostly a shared synteny in closer primates. However, near to the BAP1 locus signs of chromosomal inversions were observed during the course of evolution. To our knowledge, this is the first study to characterize the entire genomic neighborhood of cancer-associated genes located on any single chromosome. Based on our results, we hypothesize that monosomy of chromosome 3 will have important clinical and molecular consequences in the respective diseases and in particular in UM.
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http://dx.doi.org/10.1111/cas.14319DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7156870PMC
April 2020

Mechanistic basis of co-stimulatory CD40-CD40L ligation mediated regulation of immune responses in cancer and autoimmune disorders.

Immunobiology 2020 03 17;225(2):151899. Epub 2019 Dec 17.

Department of Neurology, University Clinic Bonn, Bonn, Germany; Department of Ophthalmology, University Clinic Bonn, Bonn, Germany.

Generation of an accurate humoral and a cell mediated adaptive immune responsesare dictated by binding of an antigen to a T- and a B-cell receptor, respectively (first signal) followed by ligation of costimulatory molecules (second signal). CD40, a costimulatory receptor molecule, expressed mainly on antigen presenting cells, some non-immune cells and tumors, binds to CD40 ligand molecule expressed transiently on T-cells and non-immune cells under inflammatory conditions. In the past decade, the CD40-CD40L interaction has emerged as an immune-potentiating system that governs and regulates host immune response against various diseases and pathogens, failing of which results in detrimental patho-physiologies including cancer and autoimmune disorders. CD40-CD40L transduces immune signals intracellularly via TRAF-dependent and independent mechanisms and further downstream by different MAPK pathways and transcription factors such as NF-κB, p38 etc. While CD40 signaling pathway through its cognate interaction between B and T cells promotes activation and proliferation of B-cells, Ig class switching, and generation of B cell memory; however, CD40-CD40L interaction involving other APCs and non-immune cells relay distinct cell signaling resulting in production of a variety of cytokines/chemokines and cell adhesion molecules ultimately conferring host defense against pathogen. In cancer and autoimmune disorders, CD40-CD40L interaction is also responsible for aberrant expression of many disease specific markers, class I/II MHC molecules and other co-stimulatory molecules such as B7 and CD28 in cell- and disease-specific manner. In the present review, the current state of understanding about the CD40-CD40L mediated regulation of immune and non-immune cells is presented. The current paradigm is to target CD40 using agonist anti-CD40 mAbs alone or in synergistic combination with chemotherapy in order to harness or confer anti-tumor and anti-inflammatory immunity.
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http://dx.doi.org/10.1016/j.imbio.2019.151899DOI Listing
March 2020

Common genetic variants associated with Parkinson's disease display widespread signature of epigenetic plasticity.

Sci Rep 2019 12 5;9(1):18464. Epub 2019 Dec 5.

Department of Neurology, University Clinic Bonn, Bonn, Germany.

Parkinson disease (PD) is characterized by a pivotal progressive loss of substantia nigra dopaminergic neurons and aggregation of α-synuclein protein encoded by the SNCA gene. Genome-wide association studies identified almost 100 sequence variants linked to PD in SNCA. However, the consequences of this genetic variability are rather unclear. Herein, our analysis on selective single nucleotide polymorphisms (SNPs) which are highly associated with the PD susceptibility revealed that several SNP sites attribute to the nucleosomes and overlay with bivalent regions poised to adopt either active or repressed chromatin states. We also identified large number of transcription factor (TF) binding sites associated with these variants. In addition, we located two docking sites in the intron-1 methylation prone region of SNCA which are required for the putative interactions with DNMT1. Taken together, our analysis reflects an additional layer of epigenomic contribution for the regulation of the SNCA gene in PD.
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http://dx.doi.org/10.1038/s41598-019-54865-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6895091PMC
December 2019

Mutational Landscape of the BAP1 Locus Reveals an Intrinsic Control to Regulate the miRNA Network and the Binding of Protein Complexes in Uveal Melanoma.

Cancers (Basel) 2019 Oct 19;11(10). Epub 2019 Oct 19.

Department of Ophthalmology, University Hospital Bonn, 53127 Bonn, Germany.

The (BRCA1-associated protein 1) gene is associated with a variety of human cancers. With its gene product being a nuclear ubiquitin carboxy-terminal hydrolase with deubiquitinase activity, acts as a tumor suppressor gene with potential pleiotropic effects in multiple tumor types. Herein, we focused specifically on uveal melanoma (UM) in which mutations are associated with a metastasizing phenotype and decreased survival rates. We identified the ubiquitin carboxyl hydrolase (UCH) domain as a major hotspot region for the pathogenic mutations with a high evolutionary action (EA) score. This also includes the mutations at conserved catalytic sites and the ones overlapping with the phosphorylation residues. Computational protein interaction studies revealed that distant BAP1-associated protein complexes (FOXK2, ASXL1, BARD1, BRCA1) could be directly impacted by this mutation paradigm. We also described the conformational transition related to BAP1-BRCA-BARD1 complex, which may pose critical implications for mutations, especially at the docking interfaces of these three proteins. The mutations affect - independent of being somatic or germline - the binding affinity of miRNAs embedded within the locus, thereby altering the unique regulatory network. Apart from UM, BAP1 gene expression and survival associations were found to be predictive for the prognosis in several ( = 29) other cancer types. Herein, we suggest that although BAP1 is conceptually a driver gene in UM, it might contribute through its interaction partners and its regulatory miRNA network to various aspects of cancer. Taken together, these findings will pave the way to evaluate BAP1 in a variety of other human cancers with a shared mutational spectrum.
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http://dx.doi.org/10.3390/cancers11101600DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6826957PMC
October 2019

Towards a Central Role of in the Bladder Exstrophy⁻Epispadias Complex (BEEC): Computational Characterization of Genetic Variants and Structural Modelling.

Genes (Basel) 2018 Dec 5;9(12). Epub 2018 Dec 5.

Institute of Human Genetics, University Hospital of Bonn, 53127 Bonn, Germany.

Genetic factors play a critical role in the development of human diseases. Recently, several molecular genetic studies have provided multiple lines of evidence for a critical role of genetic factors in the expression of human bladder exstrophy-epispadias complex (BEEC). At this point, (ISL LIM homeobox 1) has emerged as the major susceptibility gene for classic bladder exstrophy (CBE), in a multifactorial disease model. Here, GWAS (Genome wide association studies) discovery and replication studies, as well as the re-sequencing of , identified sequence variants (rs9291768, rs6874700, c.137C > G (p.Ala46Gly)) associated with CBE. Here, we aimed to determine the molecular and functional consequences of these sequence variants and estimate the dependence of ISL1 protein on other predicted candidates. We used: (i) computational analysis of conserved sequence motifs to perform an evolutionary conservation analysis, based on a Bayesian algorithm, and (ii) computational 3D structural modeling. Furthermore, we looked into long non-coding RNAs (lncRNAs) residing within the region, aiming to predict their targets. Our analysis suggests that the ISL1 protein specific N-terminal LIM domain (which harbors the variant c.137C>G), limits its transcriptional ability, and might interfere with ISL1-estrogen receptor α interactions. In conclusion, our analysis provides further useful insights about the gene, which is involved in the formation of the BEEC, and in the development of the urinary bladder.
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http://dx.doi.org/10.3390/genes9120609DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6315746PMC
December 2018

Evaluation of fingerprinting techniques to assess genotype variation among Zygosaccharomyces strains.

Food Microbiol 2018 Jun 1;72:135-145. Epub 2017 Dec 1.

Department of Life Sciences, University of Modena and Reggio Emilia, Via Amendola 2, Reggio Emilia 42122, Italy.

Molecular typing techniques are key tools in surveillance of food spoilage yeasts, in investigations on intra-species population diversity, and in tracing selected starters during fermentation. Unlike previous works on strain typing of Zygosaccharomyces spoilage species, here Zygosaccharomyces mellis and the Zygosaccharoymces rouxii complex yeasts, which include Z. rouxii, Zygosaccharomyces sapae, and a mosaic lineage (ML) of putatively hybrids, were evaluated by three typing methods for intra- and inter-species resolution. Overall these yeasts are relevant for food fermentation and spoilage, but are quite difficult to discriminate at strain and species level as they evolved by reticulation. A pool of 76 strains from different sources were typed by M13 and (GTG) MSP-PCR fingerprinting and PCR-RFLP of ribosomal intergenic spacer region (IGS). We demonstrated that M13 overcame (GTG) fingerprinting to group Z. sapae, Z. rouxii, Z. mellis and the ML isolates in congruent distinct clusters. Even if (GTG) primer yielded a number of DNA fingerprints comparable with those obtained by M13 primer, it failed to discriminate Z. sapae, Z. mellis and Z. rouxii at species level. Clustering of IGS RFLP patterns obtained with three endonucleases produced groups congruent with species assignment and highlighted intra-species diversity similar to that observed by M13 fingerprinting. However, IGS PCR amplification failed for 14 ML and 6 Z. mellis strains under the experimental conditions tested here, indicating that this marker could be less easy to use in fast typing protocol. Finally, our results posit that the genetic diversity within Z. sapae and Z. mellis could be shaped by isolation source. The information generated in this study would facilitate the monitoring of these yeasts during food processing and storage, and provides preliminary evidences about Z. sapae and Z. mellis intra-species diversity.
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http://dx.doi.org/10.1016/j.fm.2017.11.019DOI Listing
June 2018

Predicting the functional consequences of non-synonymous single nucleotide polymorphisms in IL8 gene.

Sci Rep 2017 07 26;7(1):6525. Epub 2017 Jul 26.

Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, NC, 27599, USA.

Here we report an in-silico approach for identification, characterization and validation of deleterious non-synonymous SNPs (nsSNPs) in the interleukin-8 gene using three steps. In first step, sequence homology-based genetic analysis of a set of 50 coding SNPs associated with 41 rsIDs using SIFT (Sorting Intolerant from Tolerant) and PROVEAN (Protein Variation Effect Analyzer) identified 23 nsSNPs to be putatively damaging/deleterious in at least one of the two tools used. Subsequently, structure-homology based PolyPhen-2 (Polymorphism Phenotyping) analysis predicted 9 of 23 nsSNPs (K4T, E31A, E31K, S41Y, I55N, P59L, P59S, L70P and V88D) to be damaging. According to the conditional hypothesis for the study, only nsSNPs that score damaging/deleterious prediction in both sequence and structural homology-based approach will be considered as 'high-confidence' nsSNPs. In step 2, based on conservation of amino acid residues, stability analysis, structural superimposition, RSMD and docking analysis, the possible structural-functional relationship was ascertained for high-confidence nsSNPs. Finally, in a separate analysis (step 3), the IL-8 deregulation has also appeared to be an important prognostic marker for detection of patients with gastric and lung cancer. This study, for the first time, provided in-depth insights on the effects of amino acid substitutions on IL-8 protein structure, function and disease association.
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http://dx.doi.org/10.1038/s41598-017-06575-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5529537PMC
July 2017

Structural modeling of human organic cation transporters.

Comput Biol Chem 2017 06 18;68:153-163. Epub 2017 Mar 18.

Maisonneuve-Rosemont Hospital, Research Center, Université de Montréal, Department of Medicine, 5415 Boul. de L' Assomption, Montréal, Québec H1T 2M4, Canada.

Human organic cation transporters (hOCTs) belong to solute carriers (SLC) 22 family of membrane proteins that play a central role in transportation of chemotherapeutic drugs for several clinical and pathological conditions, including cancer and diabetes. These transporters mediate drug transport; however, the precise mechanism of drug-binding and transport by them is not fully uncovered yet, partly due to unavailability of any crystal structure record. In this work, we performed a multi-phasic approach to compute the 3D structural models of seven human organic cation transporters (hOCTs) starting from primary protein sequence. Our structure modeling approach included 1) I-TASSER based comparative sequence alignment, threading and ab-initio protein modeling; 2) models comparison with PSIPRED secondary structure prediction; 3) loop modeling for incongruent secondary structure in Chimera 1.10.1; 4) high resolution structure simulation, refinement, energy minimization using ModRefiner, and 5) validation of the structure models using PROCHECK at SAVEs. From structural point, the computed 3D structures of hOCTs consist of a typical major facilitator superfamily (MFS) fold of twelve α-transmembrane helix domains arranged in a manner rendering hOCTs a barrel shaped structure with a large cleft that opens in cytoplasm. The modeled 3D structure of all hOCTs closely resemble to human SLC2A3 (GLUT3) transporter (PDB ID: 5c65) and displayed an outward-open confirmation and putative cyclic C1 protein symmetry. In addition, hOCTs has a large (>100 amino acids) unique extracellular loop between TMH1 and TMH2 having potential glycosylation sites (Asn-Xaa-Ser/Thr) and cysteine residues, both features indicative of putative role in drug binding and uptake. There is an intracellular three/four-helix loop between TMH6 and TMH7 containing putative phosphorylation sites for precise regulation of hOCTs function as drug transporters. There are nine loops of 4 to 11 amino acids length that protrude from membrane, both intracellularly and extracellularly, and connect adjacent TMHs. The 2D structure prediction showed N-C topology of all hOCTs. In the unavailability of the crystal structures of hOCTs, the 3D structural models computed in-silico and presented herein can be used for studying the mechanism of drug binding and transport by hOCTs.
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http://dx.doi.org/10.1016/j.compbiolchem.2017.03.007DOI Listing
June 2017

Mechanistic Basis of Antimicrobial Actions of Silver Nanoparticles.

Front Microbiol 2016 16;7:1831. Epub 2016 Nov 16.

Department of Biotechnology, School of Engineering and Technology, Sharda University Greater Noida, India.

Multidrug resistance of the pathogenic microorganisms to the antimicrobial drugs has become a major impediment toward successful diagnosis and management of infectious diseases. Recent advancements in nanotechnology-based medicines have opened new horizons for combating multidrug resistance in microorganisms. In particular, the use of silver nanoparticles (AgNPs) as a potent antibacterial agent has received much attention. The most critical physico-chemical parameters that affect the antimicrobial potential of AgNPs include size, shape, surface charge, concentration and colloidal state. AgNPs exhibits their antimicrobial potential through multifaceted mechanisms. AgNPs adhesion to microbial cells, penetration inside the cells, ROS and free radical generation, and modulation of microbial signal transduction pathways have been recognized as the most prominent modes of antimicrobial action. On the other side, AgNPs exposure to human cells induces cytotoxicity, genotoxicity, and inflammatory response in human cells in a cell-type dependent manner. This has raised concerns regarding use of AgNPs in therapeutics and drug delivery. We have summarized the emerging endeavors that address current challenges in relation to safe use of AgNPs in therapeutics and drug delivery platforms. Based on research done so far, we believe that AgNPs can be engineered so as to increase their efficacy, stability, specificity, biosafety and biocompatibility. In this regard, three perspectives research directions have been suggested that include (1) synthesizing AgNPs with controlled physico-chemical properties, (2) examining microbial development of resistance toward AgNPs, and (3) ascertaining the susceptibility of cytoxicity, genotoxicity, and inflammatory response to human cells upon AgNPs exposure.
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http://dx.doi.org/10.3389/fmicb.2016.01831DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5110546PMC
November 2016

Contrasting Patterns of rDNA Homogenization within the Zygosaccharomyces rouxii Species Complex.

PLoS One 2016 8;11(8):e0160744. Epub 2016 Aug 8.

Department of Life Sciences, University of Modena and Reggio Emilia, Via Amendola 2, 42122, Reggio Emilia, Italy.

Arrays of repetitive ribosomal DNA (rDNA) sequences are generally expected to evolve as a coherent family, where repeats within such a family are more similar to each other than to orthologs in related species. The continuous homogenization of repeats within individual genomes is a recombination process termed concerted evolution. Here, we investigated the extent and the direction of concerted evolution in 43 yeast strains of the Zygosaccharomyces rouxii species complex (Z. rouxii, Z. sapae, Z. mellis), by analyzing two portions of the 35S rDNA cistron, namely the D1/D2 domains at the 5' end of the 26S rRNA gene and the segment including the internal transcribed spacers (ITS) 1 and 2 (ITS regions). We demonstrate that intra-genomic rDNA sequence variation is unusually frequent in this clade and that rDNA arrays in single genomes consist of an intermixing of Z. rouxii, Z. sapae and Z. mellis-like sequences, putatively evolved by reticulate evolutionary events that involved repeated hybridization between lineages. The levels and distribution of sequence polymorphisms vary across rDNA repeats in different individuals, reflecting four patterns of rDNA evolution: I) rDNA repeats that are homogeneous within a genome but are chimeras derived from two parental lineages via recombination: Z. rouxii in the ITS region and Z. sapae in the D1/D2 region; II) intra-genomic rDNA repeats that retain polymorphisms only in ITS regions; III) rDNA repeats that vary only in their D1/D2 domains; IV) heterogeneous rDNA arrays that have both polymorphic ITS and D1/D2 regions. We argue that an ongoing process of homogenization following allodiplodization or incomplete lineage sorting gave rise to divergent evolutionary trajectories in different strains, depending upon temporal, structural and functional constraints. We discuss the consequences of these findings for Zygosaccharomyces species delineation and, more in general, for yeast barcoding.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0160744PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4976873PMC
August 2017

Differential hypersaline stress response in Zygosaccharomyces rouxii complex yeasts: a physiological and transcriptional study.

FEMS Yeast Res 2016 09 3;16(6). Epub 2016 Aug 3.

Department of Life Sciences, University of Modena and Reggio Emilia, via Amandola 2, Besta building, I-42124 Reggio Emilia, Italy.

The Zygosaccharomyces rouxii complex comprises three distinct lineages of halotolerant yeasts relevant in food processing and spoilage, such as Z. sapae, Z. rouxii and a mosaic group of allodiploid strains. They manifest plastic genome architecture (variation in karyotype, ploidy level and Na(+)/H(+) antiporter-encoding gene copy number), and exhibit diverse tolerances to salt concentrations. Here, we investigated accumulation of compatible osmolytes and transcriptional regulation of Na(+)/H(+) antiporter-encoding ZrSOD genes during salt exposure in strains representative for the lineages, namely Z. sapae ABT301(T) (low salt tolerant), Z. rouxii CBS 732(T) (middle salt tolerant) and allodiploid strain ATCC 42981 (high salt tolerant). Growth curve modelling in 2 M NaCl-containing media supplemented with or without yeast extract as nitrogen source indicates that moderate salt tolerance of CBS 732(T) mainly depends on nitrogen availability rather than intrinsic inhibitory effects of salt. All the strains produce glycerol and not mannitol under salt stress and use two different glycerol balance strategies. ATCC 42981 produces comparatively more glycerol than Z. sapae and Z. rouxii under standard growth conditions and better retains it intracellularly under salt injuries. Conversely, Z. sapae and Z. rouxii enhance glycerol production under salt stress and intracellularly retain glycerol less efficiently than ATCC 42981. Expression analysis shows that, in diploid Z. sapae and allodiploid ATCC 42981, transcription of gene variants ZrSOD2-22/ZrSOD2 and ZrSOD22 is constitutive and salt unresponsive.
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http://dx.doi.org/10.1093/femsyr/fow063DOI Listing
September 2016

Adaptive response and tolerance to sugar and salt stress in the food yeast Zygosaccharomyces rouxii.

Int J Food Microbiol 2014 Aug 25;185:140-57. Epub 2014 May 25.

Department of Life Sciences, University of Modena and Reggio Emilia, Via Amendola 2, 42122, Reggio Emilia, Italy. Electronic address:

The osmotolerant and halotolerant food yeast Zygosaccharomyces rouxii is known for its ability to grow and survive in the face of stress caused by high concentrations of non-ionic (sugars and polyols) and ionic (mainly Na(+) cations) solutes. This ability determines the success of fermentation on high osmolarity food matrices and leads to spoilage of high sugar and high salt foods. The knowledge about the genes, the metabolic pathways, and the regulatory circuits shaping the Z. rouxii sugar and salt-tolerance, is a prerequisite to develop effective strategies for fermentation control, optimization of food starter culture, and prevention of food spoilage. This review summarizes recent insights on the mechanisms used by Z. rouxii and other osmo and halotolerant food yeasts to endure salts and sugars stresses. Using the information gathered from S. cerevisiae as guide, we highlight how these non-conventional yeasts integrate general and osmoticum-specific adaptive responses under sugar and salts stresses, including regulation of Na(+) and K(+)-fluxes across the plasma membrane, modulation of cell wall properties, compatible osmolyte production and accumulation, and stress signalling pathways. We suggest how an integrated and system-based knowledge on these mechanisms may impact food and biotechnological industries, by improving the yeast spoilage control in food, enhancing the yeast-based bioprocess yields, and engineering the osmotolerance in other organisms.
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http://dx.doi.org/10.1016/j.ijfoodmicro.2014.05.015DOI Listing
August 2014

Sex-determination system in the diploid yeast Zygosaccharomyces sapae.

G3 (Bethesda) 2014 Jun 17;4(6):1011-25. Epub 2014 Jun 17.

Department of Life Sciences, University of Modena and Reggio Emilia, 42122, Reggio Emilia, Italy.

Sexual reproduction and breeding systems are driving forces for genetic diversity. The mating-type (MAT) locus represents a mutation and chromosome rearrangement hotspot in yeasts. Zygosaccharomyces rouxii complex yeasts are naturally faced with hostile low water activity (aw) environments and are characterized by gene copy number variation, genome instability, and aneuploidy/allodiploidy. Here, we investigated sex-determination system in Zygosaccharomyces sapae diploid strain ABT301(T), a member of the Z. rouxii complex. We cloned three divergent mating type-like (MTL) α-idiomorph sequences and designated them as ZsMTLα copies 1, 2, and 3. They encode homologs of Z. rouxii CBS 732(T) MATα2 (amino acid sequence identities spanning from 67.0 to 99.5%) and MATα1 (identity range 81.5-99.5%). ABT301(T) possesses two divergent HO genes encoding distinct endonucleases 100% and 92.3% identical to Z. rouxii HO. Cloning of MATA: -idiomorph resulted in a single ZsMTLA: locus encoding two Z. rouxii-like proteins MATA: 1 and MATA: 2. To assign the cloned ZsMTLα and ZsMTLA: idiomorphs as MAT, HML, and HMR cassettes, we analyzed their flanking regions. Three ZsMTLα loci exhibited the DIC1-MAT-SLA2 gene order canonical for MAT expression loci. Furthermore, four putative HML cassettes were identified, two containing the ZsMTLα copy 1 and the remaining harboring ZsMTLα copies 2 and 3. Finally, the ZsMTLA: locus was 3'-flanked by SLA2, suggesting the status of MAT expression locus. In conclusion, Z. sapae ABT301(T) displays an aααα genotype missing of the HMR silent cassette. Our results demonstrated that mating-type switching is a hypermutagenic process in Z. rouxii complex that generates genetic diversity de novo. This error-prone mechanism could be suitable to generate progenies more rapidly adaptable to hostile environments.
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http://dx.doi.org/10.1534/g3.114.010405DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4065246PMC
June 2014

Unravelling genomic diversity of Zygosaccharomyces rouxii complex with a link to its life cycle.

FEMS Yeast Res 2013 May 14;13(3):245-58. Epub 2013 Jan 14.

Department of Life Sciences, University of Modena and Reggio Emilia, Reggio Emilia, Italy.

Zygosaccharomyces rouxii and the related species Zygosaccharomyces sapae (hereafter referred to as Z. rouxii complex) are protoploid hemiascomycete yeasts relevant in the elaboration and spoilage of foodstuff. Divergence of Z. rouxii complex before whole genome duplication, leading to the genus Saccharomyces, makes these yeasts very attractive for genome evolution study. Relatively little is known, however, about the diversity in this branch at the genetic and physiological levels. In this work, we investigated Z. rouxii complex, encompassing strains that in other works have been studied separately and comparing them in a comprehensive way. We showed that the majority of strains are unusually heterogeneous in their ribosomal DNA, a signal of relaxation of concerted evolution. Further analysis showed that they have hypervariable karyotypes, different levels of ploidy, and that housekeeping markers vary both in copy number and sequence. Overall, the results provide compelling evidence that the strains considered in this study are a complex of haploid, aneuploid and diploid mosaic lineages. The reproductive mode and life cycle of Zygosaccharomyces could lead to this unsuspected diversity.
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http://dx.doi.org/10.1111/1567-1364.12027DOI Listing
May 2013

Zygosaccharomyces sapae sp. nov., isolated from Italian traditional balsamic vinegar.

Int J Syst Evol Microbiol 2013 Jan 28;63(Pt 1):364-371. Epub 2012 Sep 28.

Department of Life Sciences, via Amendola 2, Padiglione Besta, University of Modena and Reggio Emilia, 42121 Reggio Emilia, Italy.

Fourteen yeast isolates were recovered from two traditional balsamic vinegar (TBV) samples collected in the provinces of Modena and Reggio Emilia, Italy. Microsatellite-primed-PCR (MSP-PCR) was used to de-replicate the isolate collection into two representative strains, ABT301(T) and ABT601. Phylogenetic analysis based on the D1/D2 domains of the 26S rRNA gene indicated that these strains represented a distinct species of the genus Zygosaccharomyces, closely related to Zygosaccharomyces rouxii and Zygosaccharomyces mellis. Physiological and morphological tests supported the recognition of a novel taxon of halotolerant, osmotolerant, non-psychrotolerant and maltose-fermentation-negative yeasts showing a chain or star-shaped pattern of budding cells, which remained attached to each other. Morphological observations offered evidence of ascospore formation. A novel species, Zygosaccharomyces sapae sp. nov., is proposed to accommodate these strains, with strain ABT301(T) (= CBS 12607(T) = MUCL 54092(T)) as the type strain. Based on D1/D2 domain phylogenetic analysis, the novel strains shared the highest sequence similarity (100 %) with Zygosaccharomyces sp. strain NCYC 3042, previously isolated from sugar [James, S. A., Bond, C. J., Stratford, M. & Roberts, I. N. (2005). FEMS Yeast Res 5, 747-755]. However, based on phylogenetic (internal transcribed spacers, ITS), PCR fingerprinting and physiological analyses, marked differences were observed between the novel species and strain NCYC 3042, and these results are discussed in more detail.
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http://dx.doi.org/10.1099/ijs.0.043323-0DOI Listing
January 2013