Kawalpreet K Aneja - Academia.edu (original) (raw)

Papers by Kawalpreet K Aneja

Acta Scientific Microbiology

The microbiome is composed of bacteria, archaea, fungi, protists, and viruses. The human microbio... more The microbiome is composed of bacteria, archaea, fungi, protists, and viruses. The human microbiome is well known but still unfolding in various sites like skin, saliva, mucus, placenta, seminal and ovarian fluids, respiratory, gastrointestinal, and urogenital tract. The mucus lining of our body plays a major role in first-line defense against pathogens. Dietary fiber or plant-based diet and microbial foods strengthen our mucus lining and modulate the innate, adaptive, and regulatory immune system. Perhaps probiotics or microbial foods, personalized nutrition, and supplementation to support gut microbiota should be part of a natural or prescribed regimen to fight against COVID-19 like diseases. Recent studies have found a link between COVID-19 and perturbed gut microbiota. The reduced immunomodulatory bacteria like Faecalibacterium prausnitzii, Eubacterium rectale, and Bifidobacteria were concordant with a surge in inflammatory cytokines like CXCL10, CCL2, IL-10 and TNFα and blood markers such as C reactive protein, lactate dehydrogenase, aspartate aminotransferase, and gamma-glutamyl transferase in patients up to 30 days after COVID-19 resolution. Strong and healthy gut microbiota provide multiple defense systems such as a thick mucus layer that promotes the growth of commensal bacteria, intestinal epithelial cells with tight junctions, and regulate various host factors like antimicrobial peptides, immunoglobulin A, proteases, etc. Can we stop COVID-19 like diseases by improving gut microbiota?

Type II NADH-quinone oxidoreductase (NDH-2) catalyzes the transfer electrons from NADH to the qui... more Type II NADH-quinone oxidoreductase (NDH-2) catalyzes the transfer electrons from NADH to the quinone pool and plays an essential role in the oxidative phosphorylation system of <i>Mycobacterium tuberculosis</i> (Mtb). The absence of NDH-2 in the mammalian mitochondrial electron transport chain makes this enzyme an attractive target for antibiotic development. To fully establish the kinetic properties of this enzyme, we studied the interaction of Mtb NDH-2 with substrates, NADH, and various quinone analogues and their products in both membrane and soluble environments. These studies, and comparative analyses of the kinetics with thio-NAD⁺ and quinone electron acceptors, provided evidence that Mtb NDH-2 catalyzes the transfer electrons from NADH to quinone substrates by a nonclassical, two-site ping-pong kinetic mechanism whereby substrate quinones bind to a site that is distinct from the NADH-binding site. Furthermore, the effects of quinols on Mtb...

Can RNA interference be used as a diagnostic and therapeutic for COVID-19? Can host or viral enco... more Can RNA interference be used as a diagnostic and therapeutic for COVID-19? Can host or viral encoded miRNA or siRNA be used as a vaccine against SARS-CoV-2? RNAi has been used as a platform to make attenuated viral vaccines where the viral genome is engineered and modified to contain miRNA or siRNA binding sites [50]. One of these examples was the creation of self-attenuating Influenza A virus strain that expressed an siRNA from the NS segment (for wild type nonstructural protein NS1) that targets the ORF of the nucleoprotein [NP] segment just at a single site [51]. Intranasal administration of five chemically modified miRNA mimics corresponding to highly expressed miRNAs in respiratory epithelial cells synergistically suppressed H1N1 replication in mice. MicroRNA 122 is another most common example in RNAi literature, antimiR against mir-122 is effective to lower the hepatitis C virus and miR-122 inhibition by anti-miR122 also reduces serum cholesterol levels [40,52]. RNAi patents a...

Frontiers in Microbiology, 2020

Cullen et al. Emerging Priorities for Microbiome Research composition and interactions to develop... more Cullen et al. Emerging Priorities for Microbiome Research composition and interactions to develop engineering solutions, and (5) interventional approaches and engineered microbiota that may be enabled by selectively altering microbial composition. As such, this review seeks to arm the reader with a broad understanding of the priorities and challenges in microbiome research today and provide inspiration for future investigation and multidisciplinary collaboration.

Biocatalysis and Agricultural Biotechnology, 2018

Celem artykułu jest przedstawienie istoty i przejawów oraz ogólna ocena skutków katastrof ekologi... more Celem artykułu jest przedstawienie istoty i przejawów oraz ogólna ocena skutków katastrof ekologicznych (środowiskowych), głównie w kontekście zmian klimatycznych. Autorzy zaprezentowali najpierw rodzaje katastrof i klasyfikację katastrof ekologicznych oraz sposoby ich oceny według przejawów i skutków gospodarczych. Następnie dokonali oceny wpływu ekstremalnych zjawisk klimatycznych na gospodarkę wodną i tendencji zmian w tej dziedzinie, w tym w Polsce. Na zakończenie scharakteryzowali zasady zapobiegania katastrofom ekologicznym oraz likwidacji ich skutków.

Blood, 2009

227 RUNX1/CBFA2 (Core binding factor A2) is a major transcription factor involved in hematopoiesi... more 227 RUNX1/CBFA2 (Core binding factor A2) is a major transcription factor involved in hematopoiesis. RUNX1 mutations are associated with mild thrombocytopenia, platelet dysfunction, and predisposition to acute leukemia. Several patients with mutations in RUNX1 have been shown to have alpha granule deficiency characterized by decreased platelet factor 4 (PF4) content. The mechanisms leading to PF4 deficiency remain unclear in most patients with α-granule abnormalities or the Gray platelet Syndrome (GPS). GPS is a heterogeneous disorder, and defective granule formation and targeting of proteins to the granule have been postulated. Previous studies from our group have documented a patient with mild thrombocytopenia, impaired platelet aggregation, secretion, phosphorylation of pleckstrin and myosin light chain (MLC), and GPIIb-IIIa activation, which was associated with a heterozygous mutation in transcription factor RUNX1. Platelet expression profiling of this patient showed decreased ex...

Frontiers in Microbiology, 2017

The life cycle of Kaposi's sarcoma-associated herpesvirus (KSHV) consists of two phases, latent a... more The life cycle of Kaposi's sarcoma-associated herpesvirus (KSHV) consists of two phases, latent and lytic. The virus establishes latency as a strategy for avoiding host immune surveillance and fusing symbiotically with the host for lifetime persistent infection. However, latency can be disrupted and KSHV is reactivated for entry into the lytic replication. Viral lytic replication is crucial for efficient dissemination from its long-term reservoir to the sites of disease and for the spread of the virus to new hosts. The balance of these two phases in the KSHV life cycle is important for both the virus and the host and control of the switch between these two phases is extremely complex. Various environmental factors such as oxidative stress, hypoxia, and certain chemicals have been shown to switch KSHV from latency to lytic reactivation. Immunosuppression, unbalanced inflammatory cytokines, and other viral co-infections also lead to the reactivation of KSHV. This review article summarizes the current understanding of the initiation and regulation of KSHV reactivation and the mechanisms underlying the process of viral lytic replication. In particular, the central role of an immediate-early gene product RTA in KSHV reactivation has been extensively investigated. These studies revealed multiple layers of regulation in activation of RTA as well as the multifunctional roles of RTA in the lytic replication cascade. Epigenetic regulation is known as a critical layer of control for the switch of KSHV between latency and lytic replication. The viral non-coding RNA, PAN, was demonstrated to play a central role in the epigenetic regulation by serving as a guide RNA that brought chromatin remodeling enzymes to the promoters of RTA and other lytic genes. In addition, a novel dimension of regulation by microPeptides emerged and has been shown to regulate RTA expression at the protein level. Overall, extensive investigation of KSHV reactivation and lytic replication has revealed a sophisticated regulation network that controls the important events in KSHV life cycle.

Journal of Thrombosis and Haemostasis, 2011

Background-Platelet factor 4 (PF4) is an abundant protein stored in platelet α-granules. Several ... more Background-Platelet factor 4 (PF4) is an abundant protein stored in platelet α-granules. Several patients have been described with platelet PF4 deficiency, including the Gray platelet syndrome (GPS), characterized by deficiency of α-granule proteins. Defective granule formation and protein-targeting are considered the predominant mechanisms. We have reported on a patient with thrombocytopenia and impaired platelet aggregation, secretion, and protein phosphorylation, associated with a mutation in transcription factor RUNX1. Platelet expression profiling showed decreased transcript expression of PF4 and its nonallelic variant PF4V1. Objectives-To understand the mechanism leading to PF4 deficiency associated with RUNX1 haplodeficiency, we addressed the hypothesis that PF4 is a transcriptional target of RUNX1. Methods/Results-Chromatin immunoprecipitation and gel-shift assays using PMA-treated human erythroleukemia (HEL) cells revealed RUNX1 binding to RUNX1 consensus sites at-1774/-1769 and-157/-152 on PF4 promoter. In luciferase reporter studies in HEL cells mutation of each site markedly reduced activity. PF4 promoter activity and protein were decreased by siRNA RUNX1 knockdown and increased by RUNX1 overexpression. Conclusions-Our results provide the first evidence that PF4 is regulated by RUNX1 and that impaired transcriptional regulation leads to the PF4 deficiency associated with RUNX1 haplodeficiency. Because our patient had decreased platelet albumin and IgG (not synthesized by megakaryocytes), we postulate additional defects in RUNX1-regulated genes involved in vesicular trafficking. These studies advance understanding of the mechanisms in α-granule deficiency.

Biotechnology Letters, 2009

The class III poly(hydroxyalkanoate) synthase (PHAS) genes (phaC and phaE) of a photosynthetic ba... more The class III poly(hydroxyalkanoate) synthase (PHAS) genes (phaC and phaE) of a photosynthetic bacterium, Allochromatium vinosum ATCC 35206, were cloned, sequenced and expressed in a heterologous host. PCR coupled with a chromosomal gene-walking method was used to clone and subsequently sequence the contiguous phaC (1,068 bps) and phaE (1,065 bps) genes of A. vinosum ATCC 35206. BLASTP search of protein databases showed that the gene-products of phaC and phaE are different (&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt;66% identities) from the previously reported class III PHASs such as those of A. vinosum DSM180. Domain analysis revealed the presence of a conserved alpha/beta-hydrolase fold in PhaC, the putative gene-product of phaC. Upon electroporation of a poly(hydroxybutanoate) (PHB)-negative mutant of Ralstonia eutropha PHB(-)4 with a shuttle plasmid pBHR1 containing the newly cloned phaC and phaE genes, the bacteria resumed the synthesis of PHB, albeit at a low level (4-5% of the cell dry wt) due to kanamycin selection pressure. We further showed that the recombinant strain grown in kanamycin-containing culture medium synthesized a blend of PHA that also contains a high content of 3-hydroxyoctanoate and 3-hydroxydecanoate as its repeat-unit monomers. Genomic analysis suggested the existence of two PHA synthase genes in R. eutropha. The results of this study not only make available a phylogenetically diverse type III phaC and phaE genes, but also confirm through kanamycin selection pressure the existence of multiple PHA biosynthesis systems in R. eutropha.

Archives of Biochemistry and Biophysics, 2009

We have recently identified two promoters, distal and proximal for rat mitochondrial glycerophosp... more We have recently identified two promoters, distal and proximal for rat mitochondrial glycerophosphate acyltransferase (mtGPAT). Here we are reporting further characterization of the promoters. Insulin and epidermal growth factor (EGF) stimulated while leptin and glucagon inhibited the luciferase activity of the distal promoter and the amounts of the distal transcript. Conversely, luciferase activity of the proximal promoter and proximal transcript remained unchanged due to these treatments. Only the distal promoter has binding sites for carbohydrate response element binding protein (ChREBP) and sterol regulatory element binding protein-1 (SREBP-1). Electromobility shift assays and chromatin immunoprecipitation assays demonstrated that ChREBP and SREBP-1 bind to the mtGPAT distal promoter. Insulin and EGF increased while glucagon and leptin decreased the binding of SREBP-1 and ChREBP to the distal promoter. Thus, the distal promoter is the regulatory promoter while the proximal promoter acts constitutively for rat mtGPAT gene under the influence of hormones and growth factor.

Archives of Biochemistry and Biophysics, 2008

Sequence analysis using the Promoser program predicted two promoter like regions for rat mtGPAT: ... more Sequence analysis using the Promoser program predicted two promoter like regions for rat mtGPAT: a distal promoter ∼30 kb upstream and a proximal promoter near the first translational codon. Rat liver cells transfected with pGL3-basic vector containing the distal and proximal promoter resulted in 10.8 and 4.8 fold increase in the luciferase activity, respectively. Results of electromobility shift assay and chromatin immunoprecipitation suggested binding of transcription factors to the distal and proximal promoter regions. 5′RACE PCR showed two transcripts with different transcriptional start sites. When transfected rat liver cells were starved and refed, there was about 2.7 fold increase in the luciferase activity with cells transfected with the distal promoter while the proximal promoter showed no change. Thus, the two promoters could be functionally distinguished. Taken together, we conclude that there are two promoters for rat mtGPAT gene and that the transcriptional regulation is mediated through the distal promoter.

Biochemistry, 2014

Type II NADH-quinone oxidoreductase (NDH-2) catalyzes the transfer electrons from NADH to the qui... more Type II NADH-quinone oxidoreductase (NDH-2) catalyzes the transfer electrons from NADH to the quinone pool and plays an essential role in the oxidative phosphorylation system of Mycobacterium tuberculosis (Mtb). The absence of NDH-2 in the mammalian mitochondrial electron transport chain makes this enzyme an attractive target for antibiotic development. To fully establish the kinetic properties of this enzyme, we studied the interaction of Mtb NDH-2 with substrates, NADH, and various quinone analogues and their products in both membrane and soluble environments. These studies, and comparative analyses of the kinetics with thio-NAD + and quinone electron acceptors, provided evidence that Mtb NDH-2 catalyzes the transfer electrons from NADH to quinone substrates by a nonclassical, two-site pingpong kinetic mechanism whereby substrate quinones bind to a site that is distinct from the NADH-binding site. Furthermore, the effects of quinols on Mtb NDH-2 catalytic activity demonstrate the presence of two binding sites for quinone ligands, one favoring the reduced form and the other favoring the oxidized form.

Journal of Eye and Ophthalmology, 2020

CRISPR-Cas is a novel tool that may pinpoint the underlying cause of eye diseases. Here, we intro... more CRISPR-Cas is a novel tool that may pinpoint the underlying cause of eye diseases. Here, we introduce the different types of CRISPR-Cas systems with an emphasis on type II and present its applications in eye diseases based on previous reports. The CRISPR microbiome is perhaps bigger than the universe. Bacteria contain a self-defense adaptive immune system known as CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated genes) which inactivates invading phages, plasmids, or foreign DNA sequences. CRISPR is an array of repeats separated by spacers along with cas genes. In bacteria CRISPR-array and cas genes together form a cascade complex to destroy the invading foreign DNA. The two important components, the Cas9 nuclease, and the short guide RNA are introduced for a targeted genetic event. The short guide-RNA is the fingerprint of the target DNA. CRISPR-Cas has been successfully demonstrated in RHO, CRYAA, tyr, gol, ddx19, and mitfa genes in zebrafish, mice, and rabbits. The nuclear homeoprotein Six3 mutations by CRISPR-Cas showed eye and anterior head defects in zebrafish. Moreover, CRISPR-Cas has been successfully shown in Crygc gene repair in the cataract mice model carrying 1bp deletion in exon3 of Crygc. The transcription factors like Rx, Six3, Pax6, Sox2, and signaling factors involved in eye formation can be further studied by CRISPR-Cas. The first patient ever to receive CRISPR-Cas9 construct for fixing mutations in a centrosomal protein of 290 kDa, CEP290, is for the blindness caused by a rare disorder in the retina, Leber's congenital amaurosis 10 (LCA10). It is injected directly into the eye near photoreceptor cells and is a landmark clinical trial. The present CRISPR-Cas technology opens a new perspective for new treatments of cataract, retinitis pigmentosa, refractive index error, and any other eye diseases.

Acta Scientific Microbiology

Gene editing depends upon the cell's endogenous DNA repair pathways. Upon DNA double-strand break... more Gene editing depends upon the cell's endogenous DNA repair pathways. Upon DNA double-strand break, two known repair pathways are enacted: nonhomologous end-joining (NHEJ) and homologous recombination (HDR), among others. The HDR pathway works only in the presence of an exogenous homologous template.

Acta Scientific Microbiology

The microbiome is composed of bacteria, archaea, fungi, protists, and viruses. The human microbio... more The microbiome is composed of bacteria, archaea, fungi, protists, and viruses. The human microbiome is well known but still unfolding in various sites like skin, saliva, mucus, placenta, seminal and ovarian fluids, respiratory, gastrointestinal, and urogenital tract. The mucus lining of our body plays a major role in first-line defense against pathogens. Dietary fiber or plant-based diet and microbial foods strengthen our mucus lining and modulate the innate, adaptive, and regulatory immune system. Perhaps probiotics or microbial foods, personalized nutrition, and supplementation to support gut microbiota should be part of a natural or prescribed regimen to fight against COVID-19 like diseases. Recent studies have found a link between COVID-19 and perturbed gut microbiota. The reduced immunomodulatory bacteria like Faecalibacterium prausnitzii, Eubacterium rectale, and Bifidobacteria were concordant with a surge in inflammatory cytokines like CXCL10, CCL2, IL-10 and TNFα and blood markers such as C reactive protein, lactate dehydrogenase, aspartate aminotransferase, and gamma-glutamyl transferase in patients up to 30 days after COVID-19 resolution. Strong and healthy gut microbiota provide multiple defense systems such as a thick mucus layer that promotes the growth of commensal bacteria, intestinal epithelial cells with tight junctions, and regulate various host factors like antimicrobial peptides, immunoglobulin A, proteases, etc. Can we stop COVID-19 like diseases by improving gut microbiota?

Type II NADH-quinone oxidoreductase (NDH-2) catalyzes the transfer electrons from NADH to the qui... more Type II NADH-quinone oxidoreductase (NDH-2) catalyzes the transfer electrons from NADH to the quinone pool and plays an essential role in the oxidative phosphorylation system of <i>Mycobacterium tuberculosis</i> (Mtb). The absence of NDH-2 in the mammalian mitochondrial electron transport chain makes this enzyme an attractive target for antibiotic development. To fully establish the kinetic properties of this enzyme, we studied the interaction of Mtb NDH-2 with substrates, NADH, and various quinone analogues and their products in both membrane and soluble environments. These studies, and comparative analyses of the kinetics with thio-NAD⁺ and quinone electron acceptors, provided evidence that Mtb NDH-2 catalyzes the transfer electrons from NADH to quinone substrates by a nonclassical, two-site ping-pong kinetic mechanism whereby substrate quinones bind to a site that is distinct from the NADH-binding site. Furthermore, the effects of quinols on Mtb...

Can RNA interference be used as a diagnostic and therapeutic for COVID-19? Can host or viral enco... more Can RNA interference be used as a diagnostic and therapeutic for COVID-19? Can host or viral encoded miRNA or siRNA be used as a vaccine against SARS-CoV-2? RNAi has been used as a platform to make attenuated viral vaccines where the viral genome is engineered and modified to contain miRNA or siRNA binding sites [50]. One of these examples was the creation of self-attenuating Influenza A virus strain that expressed an siRNA from the NS segment (for wild type nonstructural protein NS1) that targets the ORF of the nucleoprotein [NP] segment just at a single site [51]. Intranasal administration of five chemically modified miRNA mimics corresponding to highly expressed miRNAs in respiratory epithelial cells synergistically suppressed H1N1 replication in mice. MicroRNA 122 is another most common example in RNAi literature, antimiR against mir-122 is effective to lower the hepatitis C virus and miR-122 inhibition by anti-miR122 also reduces serum cholesterol levels [40,52]. RNAi patents a...

Frontiers in Microbiology, 2020

Cullen et al. Emerging Priorities for Microbiome Research composition and interactions to develop... more Cullen et al. Emerging Priorities for Microbiome Research composition and interactions to develop engineering solutions, and (5) interventional approaches and engineered microbiota that may be enabled by selectively altering microbial composition. As such, this review seeks to arm the reader with a broad understanding of the priorities and challenges in microbiome research today and provide inspiration for future investigation and multidisciplinary collaboration.

Biocatalysis and Agricultural Biotechnology, 2018

Celem artykułu jest przedstawienie istoty i przejawów oraz ogólna ocena skutków katastrof ekologi... more Celem artykułu jest przedstawienie istoty i przejawów oraz ogólna ocena skutków katastrof ekologicznych (środowiskowych), głównie w kontekście zmian klimatycznych. Autorzy zaprezentowali najpierw rodzaje katastrof i klasyfikację katastrof ekologicznych oraz sposoby ich oceny według przejawów i skutków gospodarczych. Następnie dokonali oceny wpływu ekstremalnych zjawisk klimatycznych na gospodarkę wodną i tendencji zmian w tej dziedzinie, w tym w Polsce. Na zakończenie scharakteryzowali zasady zapobiegania katastrofom ekologicznym oraz likwidacji ich skutków.

Blood, 2009

227 RUNX1/CBFA2 (Core binding factor A2) is a major transcription factor involved in hematopoiesi... more 227 RUNX1/CBFA2 (Core binding factor A2) is a major transcription factor involved in hematopoiesis. RUNX1 mutations are associated with mild thrombocytopenia, platelet dysfunction, and predisposition to acute leukemia. Several patients with mutations in RUNX1 have been shown to have alpha granule deficiency characterized by decreased platelet factor 4 (PF4) content. The mechanisms leading to PF4 deficiency remain unclear in most patients with α-granule abnormalities or the Gray platelet Syndrome (GPS). GPS is a heterogeneous disorder, and defective granule formation and targeting of proteins to the granule have been postulated. Previous studies from our group have documented a patient with mild thrombocytopenia, impaired platelet aggregation, secretion, phosphorylation of pleckstrin and myosin light chain (MLC), and GPIIb-IIIa activation, which was associated with a heterozygous mutation in transcription factor RUNX1. Platelet expression profiling of this patient showed decreased ex...

Frontiers in Microbiology, 2017

The life cycle of Kaposi's sarcoma-associated herpesvirus (KSHV) consists of two phases, latent a... more The life cycle of Kaposi's sarcoma-associated herpesvirus (KSHV) consists of two phases, latent and lytic. The virus establishes latency as a strategy for avoiding host immune surveillance and fusing symbiotically with the host for lifetime persistent infection. However, latency can be disrupted and KSHV is reactivated for entry into the lytic replication. Viral lytic replication is crucial for efficient dissemination from its long-term reservoir to the sites of disease and for the spread of the virus to new hosts. The balance of these two phases in the KSHV life cycle is important for both the virus and the host and control of the switch between these two phases is extremely complex. Various environmental factors such as oxidative stress, hypoxia, and certain chemicals have been shown to switch KSHV from latency to lytic reactivation. Immunosuppression, unbalanced inflammatory cytokines, and other viral co-infections also lead to the reactivation of KSHV. This review article summarizes the current understanding of the initiation and regulation of KSHV reactivation and the mechanisms underlying the process of viral lytic replication. In particular, the central role of an immediate-early gene product RTA in KSHV reactivation has been extensively investigated. These studies revealed multiple layers of regulation in activation of RTA as well as the multifunctional roles of RTA in the lytic replication cascade. Epigenetic regulation is known as a critical layer of control for the switch of KSHV between latency and lytic replication. The viral non-coding RNA, PAN, was demonstrated to play a central role in the epigenetic regulation by serving as a guide RNA that brought chromatin remodeling enzymes to the promoters of RTA and other lytic genes. In addition, a novel dimension of regulation by microPeptides emerged and has been shown to regulate RTA expression at the protein level. Overall, extensive investigation of KSHV reactivation and lytic replication has revealed a sophisticated regulation network that controls the important events in KSHV life cycle.

Journal of Thrombosis and Haemostasis, 2011

Background-Platelet factor 4 (PF4) is an abundant protein stored in platelet α-granules. Several ... more Background-Platelet factor 4 (PF4) is an abundant protein stored in platelet α-granules. Several patients have been described with platelet PF4 deficiency, including the Gray platelet syndrome (GPS), characterized by deficiency of α-granule proteins. Defective granule formation and protein-targeting are considered the predominant mechanisms. We have reported on a patient with thrombocytopenia and impaired platelet aggregation, secretion, and protein phosphorylation, associated with a mutation in transcription factor RUNX1. Platelet expression profiling showed decreased transcript expression of PF4 and its nonallelic variant PF4V1. Objectives-To understand the mechanism leading to PF4 deficiency associated with RUNX1 haplodeficiency, we addressed the hypothesis that PF4 is a transcriptional target of RUNX1. Methods/Results-Chromatin immunoprecipitation and gel-shift assays using PMA-treated human erythroleukemia (HEL) cells revealed RUNX1 binding to RUNX1 consensus sites at-1774/-1769 and-157/-152 on PF4 promoter. In luciferase reporter studies in HEL cells mutation of each site markedly reduced activity. PF4 promoter activity and protein were decreased by siRNA RUNX1 knockdown and increased by RUNX1 overexpression. Conclusions-Our results provide the first evidence that PF4 is regulated by RUNX1 and that impaired transcriptional regulation leads to the PF4 deficiency associated with RUNX1 haplodeficiency. Because our patient had decreased platelet albumin and IgG (not synthesized by megakaryocytes), we postulate additional defects in RUNX1-regulated genes involved in vesicular trafficking. These studies advance understanding of the mechanisms in α-granule deficiency.

Biotechnology Letters, 2009

The class III poly(hydroxyalkanoate) synthase (PHAS) genes (phaC and phaE) of a photosynthetic ba... more The class III poly(hydroxyalkanoate) synthase (PHAS) genes (phaC and phaE) of a photosynthetic bacterium, Allochromatium vinosum ATCC 35206, were cloned, sequenced and expressed in a heterologous host. PCR coupled with a chromosomal gene-walking method was used to clone and subsequently sequence the contiguous phaC (1,068 bps) and phaE (1,065 bps) genes of A. vinosum ATCC 35206. BLASTP search of protein databases showed that the gene-products of phaC and phaE are different (&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt;66% identities) from the previously reported class III PHASs such as those of A. vinosum DSM180. Domain analysis revealed the presence of a conserved alpha/beta-hydrolase fold in PhaC, the putative gene-product of phaC. Upon electroporation of a poly(hydroxybutanoate) (PHB)-negative mutant of Ralstonia eutropha PHB(-)4 with a shuttle plasmid pBHR1 containing the newly cloned phaC and phaE genes, the bacteria resumed the synthesis of PHB, albeit at a low level (4-5% of the cell dry wt) due to kanamycin selection pressure. We further showed that the recombinant strain grown in kanamycin-containing culture medium synthesized a blend of PHA that also contains a high content of 3-hydroxyoctanoate and 3-hydroxydecanoate as its repeat-unit monomers. Genomic analysis suggested the existence of two PHA synthase genes in R. eutropha. The results of this study not only make available a phylogenetically diverse type III phaC and phaE genes, but also confirm through kanamycin selection pressure the existence of multiple PHA biosynthesis systems in R. eutropha.

Archives of Biochemistry and Biophysics, 2009

We have recently identified two promoters, distal and proximal for rat mitochondrial glycerophosp... more We have recently identified two promoters, distal and proximal for rat mitochondrial glycerophosphate acyltransferase (mtGPAT). Here we are reporting further characterization of the promoters. Insulin and epidermal growth factor (EGF) stimulated while leptin and glucagon inhibited the luciferase activity of the distal promoter and the amounts of the distal transcript. Conversely, luciferase activity of the proximal promoter and proximal transcript remained unchanged due to these treatments. Only the distal promoter has binding sites for carbohydrate response element binding protein (ChREBP) and sterol regulatory element binding protein-1 (SREBP-1). Electromobility shift assays and chromatin immunoprecipitation assays demonstrated that ChREBP and SREBP-1 bind to the mtGPAT distal promoter. Insulin and EGF increased while glucagon and leptin decreased the binding of SREBP-1 and ChREBP to the distal promoter. Thus, the distal promoter is the regulatory promoter while the proximal promoter acts constitutively for rat mtGPAT gene under the influence of hormones and growth factor.

Archives of Biochemistry and Biophysics, 2008

Sequence analysis using the Promoser program predicted two promoter like regions for rat mtGPAT: ... more Sequence analysis using the Promoser program predicted two promoter like regions for rat mtGPAT: a distal promoter ∼30 kb upstream and a proximal promoter near the first translational codon. Rat liver cells transfected with pGL3-basic vector containing the distal and proximal promoter resulted in 10.8 and 4.8 fold increase in the luciferase activity, respectively. Results of electromobility shift assay and chromatin immunoprecipitation suggested binding of transcription factors to the distal and proximal promoter regions. 5′RACE PCR showed two transcripts with different transcriptional start sites. When transfected rat liver cells were starved and refed, there was about 2.7 fold increase in the luciferase activity with cells transfected with the distal promoter while the proximal promoter showed no change. Thus, the two promoters could be functionally distinguished. Taken together, we conclude that there are two promoters for rat mtGPAT gene and that the transcriptional regulation is mediated through the distal promoter.

Biochemistry, 2014

Type II NADH-quinone oxidoreductase (NDH-2) catalyzes the transfer electrons from NADH to the qui... more Type II NADH-quinone oxidoreductase (NDH-2) catalyzes the transfer electrons from NADH to the quinone pool and plays an essential role in the oxidative phosphorylation system of Mycobacterium tuberculosis (Mtb). The absence of NDH-2 in the mammalian mitochondrial electron transport chain makes this enzyme an attractive target for antibiotic development. To fully establish the kinetic properties of this enzyme, we studied the interaction of Mtb NDH-2 with substrates, NADH, and various quinone analogues and their products in both membrane and soluble environments. These studies, and comparative analyses of the kinetics with thio-NAD + and quinone electron acceptors, provided evidence that Mtb NDH-2 catalyzes the transfer electrons from NADH to quinone substrates by a nonclassical, two-site pingpong kinetic mechanism whereby substrate quinones bind to a site that is distinct from the NADH-binding site. Furthermore, the effects of quinols on Mtb NDH-2 catalytic activity demonstrate the presence of two binding sites for quinone ligands, one favoring the reduced form and the other favoring the oxidized form.

Journal of Eye and Ophthalmology, 2020

CRISPR-Cas is a novel tool that may pinpoint the underlying cause of eye diseases. Here, we intro... more CRISPR-Cas is a novel tool that may pinpoint the underlying cause of eye diseases. Here, we introduce the different types of CRISPR-Cas systems with an emphasis on type II and present its applications in eye diseases based on previous reports. The CRISPR microbiome is perhaps bigger than the universe. Bacteria contain a self-defense adaptive immune system known as CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated genes) which inactivates invading phages, plasmids, or foreign DNA sequences. CRISPR is an array of repeats separated by spacers along with cas genes. In bacteria CRISPR-array and cas genes together form a cascade complex to destroy the invading foreign DNA. The two important components, the Cas9 nuclease, and the short guide RNA are introduced for a targeted genetic event. The short guide-RNA is the fingerprint of the target DNA. CRISPR-Cas has been successfully demonstrated in RHO, CRYAA, tyr, gol, ddx19, and mitfa genes in zebrafish, mice, and rabbits. The nuclear homeoprotein Six3 mutations by CRISPR-Cas showed eye and anterior head defects in zebrafish. Moreover, CRISPR-Cas has been successfully shown in Crygc gene repair in the cataract mice model carrying 1bp deletion in exon3 of Crygc. The transcription factors like Rx, Six3, Pax6, Sox2, and signaling factors involved in eye formation can be further studied by CRISPR-Cas. The first patient ever to receive CRISPR-Cas9 construct for fixing mutations in a centrosomal protein of 290 kDa, CEP290, is for the blindness caused by a rare disorder in the retina, Leber's congenital amaurosis 10 (LCA10). It is injected directly into the eye near photoreceptor cells and is a landmark clinical trial. The present CRISPR-Cas technology opens a new perspective for new treatments of cataract, retinitis pigmentosa, refractive index error, and any other eye diseases.

Acta Scientific Microbiology

Gene editing depends upon the cell's endogenous DNA repair pathways. Upon DNA double-strand break... more Gene editing depends upon the cell's endogenous DNA repair pathways. Upon DNA double-strand break, two known repair pathways are enacted: nonhomologous end-joining (NHEJ) and homologous recombination (HDR), among others. The HDR pathway works only in the presence of an exogenous homologous template.