In silico genome wide identification of long non-coding RNAs differentially expressed during Candida auris host pathogenesis.

Candida auris is an invasive fungal pathogen of high concern due to acquired drug tolerance against antifungals used in clinics. The prolonged persistence on biotic and abiotic surfaces can result in onset of hospital outbreaks causing serious health threat. An in depth understanding of pathology of C. auris is highly desirable for development of efficient therapeutics. Non-coding RNAs play crucial role in fungal pathology. However, the information about ncRNAs is scanty to be utilized. Herein our aim is to identify long noncoding RNAs with potent role in pathobiology of C. auris. Thereby, we analyzed the transcriptomics data of C. auris infection in blood for identification of potential lncRNAs with regulatory role in determining invasion, survival or drug tolerance under infection conditions. Interestingly, we found 275 lncRNAs, out of which 253 matched with lncRNAs reported in Candidamine, corroborating for our accurate data analysis pipeline. Nevertheless, we obtained 23 novel lncRNAs not reported earlier. Three lncRNAs were found to be under expressed throughout the course of infection, in the transcriptomics data. 16 of potent lncRNAs were found to be coexpressed with coding genes, emphasizing for their functional role. Noteworthy, these ncRNAs are expressed from intergenic regions of the genes associated with transporters, metabolism, cell wall biogenesis. This study recommends for possible association between lncRNA expression and C. auris pathogenesis.

Targeted HBx gene editing by CRISPR/Cas9 system effectively reduces epithelial to mesenchymal transition and HBV replication in hepatoma cells.

BACKGROUND AND AIMS: Hepatitis B virus X protein (HBx) play a key role in pathogenesis of HBV-induced hepatocellular carcinoma (HCC) by promoting epithelial to mesenchymal transition (EMT). In this study, we hypothesized that inhibition of HBx is an effective strategy to combat HCC. METHODOLOGY AND RESULTS: We designed and synthesized novel HBx gene specific single guide RNA (sgRNA) with CRISPR/Cas9 system and studied its in vitro effects on tumour properties of HepG2-2.15. Full length HBx gene was excised using HBx-CRISPR that resulted in significant knockdown of HBx expression in hepatoma cells. HBx-CRISPR also decreased levels of HBsAg and HBV cccDNA expression. A decreased expression of mesenchymal markers, proliferation and tumorigenic properties was observed in HBx-CRISPR treated cells as compared to controls in both two- and three- dimensional (2D and 3D) tumour models. Transcriptomics data showed that out of 1159 differentially expressed genes in HBx-CRISPR transfected cells as compared to controls, 70 genes were upregulated while 1089 genes associated with cell proliferation and EMT pathways were downregulated. CONCLUSION: Thus, targeting of HBx by CRISPR/Cas9 gene editing system reduces covalently closed circular DNA (cccDNA) levels, HBsAg production and mesenchymal characteristics of HBV-HCC cells. We envision inhibition of HBx by CRISPR as a novel therapeutic approach for HBV-induced HCC.

Comparative response of Spodoptera litura challenged per os with Serratia marcescens strains differing in virulence.

Host plays an important role in influencing virulence of a pathogen and efficacy of a biopesticide. The present study was aimed to characterize the possible factors present in Spodoptera litura that influenced pathogenecity of orally ingested S. marcescens strains, differing in their virulence. Fifth instar larvae of S. litura responded differently as challenged by two Serratia marcescens strains, SEN (virulent strain, LC50 7.02 103 cfu/ml) and ICC-4 (non-virulent strain, LC50 1.19 1012 cfu/ml). Considerable increase in activity of lytic enzymes protease and phospholipase was recorded in the gut and hemolymph of larvae fed on diet supplemented with S. marcescens strain ICC-4 as compared to the larvae treated with S. marcescens strain SEN. However, a significant up-regulation of antioxidative enzymes SOD (in foregut and midgut), CAT (in the midgut) and GST (in the foregut and hemolymph) was recorded in larvae fed on diet treated with the virulent S. marcescens strain SEN in comparison to larvae fed on diet treated with the non-virulent S. marcescens strain ICC-4. Activity of defense related enzymes lysozyme and phenoloxidase activity were also higher in the hemolymph of larvae fed with diet treated with S. marcescens strain SEN as compared to hemolymph of S. marcescens strain ICC-4 treated larvae. More number of over-expressed proteins was observed in the gut and hemolymph of S. marcescens strains ICC-4 and SEN treated larvae, respectively. Identification of the selected differentially expressed proteins indicated induction of proteins involved in insect innate immune response (Immunoglobulin I-set domain, Apolipophorin III, leucine rich repeat and Titin) in S. marcescens strain SEN treated larvae. Over-expression of two proteins, actin related protein and mt DNA helicase, were noted in S. marcescens treated larvae with very high levels observed in the non-virulent strain. Up-regulation of homeobox protein was noted only in S. marcescens strain ICC-4 challenged larvae. This study indicated that ingestion of non-virulent S. marcescens strain ICC-4 induced strong immune response in insect gut while there was weak response to the virulent S. marcescens strain SEN which probably resulted in difference in their virulence.

A convenient and robust protocol for preparation of ODAP-free Lathyrus sativus protein.

The present study aimed to develop a protocol for easy removal of ß-ODAP neurotoxin by converting it into its isomer α-ODAP (reported to be less toxic) followed by its separation from the protein fraction in pH dependent manner. Use of ß-mercaptoethanol prevented aggregate formation and increased solubility of the prepared Lathyrus sativus protein. Validation of ODAP removal by paper chromatography and mass spectrometry indicated the robustness of the protocol. Removal of ODAP and presence of high antioxidants and homoarginine content can enable Lathyrus sativus to be an alternate source of protein, as well as have other health benefits, including benefits for patients with cardiovascular diseases.

Designed gRNAs for CRISPR-Cas9 based antifungal resistance in eggplant.

Eggplant is an important vegetable crop and is a good source of antioxidants, minerals, and vitamins. It has been used in ancient medicines for the treatment of multiple diseases. However, the cultivated varieties of eggplant are susceptible to numerous pathogens and pests that have a negative impact on vegetable crops. Increased resistance achieved through resistance genes (R genes) is limited in eggplant breeding due to the fact that R genes are typically specific to a pathogen race and can be quickly surpassed by pathogen evolution. The susceptibility genes (S genes) in plants facilitate pathogen entry and proliferation, thus disabling these genes might be beneficial for providing a broad range and durable resistance against pathogens. Reports on crops such as Arabidopsis, rice, wheat, citrus, and tomatoes have highlighted that the knockout mutants of the S genes are tolerant to multiple different pathogens. The CRISPR/Cas9 system facilitates plant genome editing that can be utilized efficiently for crop improvement. In the current work, we have identified the homologs of candidate S genes DMR1, DMR6, EDR1, and PMR4/5/6 in the eggplant genome and designed and screened putative gRNAs against the identified target loci. The gRNAs were screened and selected on the basis of recognition of the PAM sequence, the MIT score, their minimum free energy, and the secondary structure. Five gRNAs for each gene homolog were selected after an in-depth analysis of all the predicted gRNAs using the above-mentioned criterion.

Molecular docking and dynamic simulation analysis of Hepatitis E virus protease in complexing with the E64 inhibitor.

The unavailability of a suitable treatment for human Hepatitis E virus (HEV) infection necessitate the development of anti HEV drugs. The HEV papain-like cysteine proteases (HEV PCP) is a crucial target to prevent viral replication and progression. E64 is a known HEV PCP inhibitor; however, its molecular mechanism of inhibition is not yet known. Since the crystal structure of HEV PCP is not available, the primary focuses of the present study was to refine the predicted HEV PCP structural model by molecular dynamics (MD) simulation. Further, we performed a 200 ns MD simulation to understand the structural complexity of HEV PCP and the effect of E64 binding with HEV PCP. The E64 binding with active site residues Gln48, Thr51, Gln55, Cys52, Ser81, Gln 98, Cys 132, Arg158, His159, Asn 160 and Ala96 leads to reduced fluctuations in the residue at N-terminal (18-41) that include the CHC motif (26-28). However, most of the other non interacting residues, including the inter-domain linker region (46-87), showed increased fluctuations in the HEV PCP-E64 complex. The residue Asp21 and Ala96 are involved in the formation of interdomain interactions in the HEV PCP apo enzyme. While in the PCP-E64 complex, E64 binds to Ala96 and creates a steric hindrance to prevent interdomain interactions. Thus, the E64 binding reduces interdomain interactions and restrict domain movements in the HEV PCP-E64 complex. This information will be important for the chemically designing more effective derivatives of E64 developing HEV PCP specific inhibitors.Communicated by Ramaswamy H. Sarma.

Rapamycin and Torin2 inhibit Candida auris TOR: Insights through growth profiling, docking, and MD simulations.

The fungus Candida auris is a pathogen of utmost concern due to its rapid emergence across the globe, acquired antifungal drug tolerance, thermotolerance, and ability to survive in hospital settings and preserved foods. Recent incidences of comorbidity of corona patients with its infection in hospital settings highlighted the importance of understanding the pathobiology and drug tolerance of this fungus on priority. The Target of rapamycin (TOR) is a central regulator of growth across eukaryotes with an illustrated role in fungal pathology. The role of the TOR signalling pathway in the growth of C. auris is yet to be described. In-silico, analysis revealed the presence of highly conserved Tor kinase, components of TORC, and key downstream components in C. auris. Rapamycin and Torin2, the specific inhibitors of Tor reduce the growth of C. auris. An inhibition of Tor leads to cell cycle arrest at the G1 phase with a defect in cytokinesis. Interestingly, with an insignificant difference in growth at 30 and 37 °C, a sharp decline in growth is seen with Torin2 at 37 °C. The heterogeneous response emphasizes the importance of physiology-based differential cellular response at different temperatures. In addition, the inhibition of Tor suppresses the biofilm formation. In silico studies through docking and simulations showed rapamycin and torin2 as specific inhibitors of C. auris Tor kinase (CauTor kinase) and hence can be exploited for a thorough understanding of the TOR signalling pathway in pathobiology and drug tolerance of C. auris. HIGHLIGHTSConservation of TOR signalling pathway in Candida aurisRapamycin and torin2 are specific inhibitors of Cau TorUnderstanding of the role of TOR signalling pathway through the use of inhibitors rapamycin and torin2.Heterogenous response of C. auris to torin2 at different physiological conditions.Communicated by Ramaswamy H. Sarma.

VEGF-mediated tumour growth and EMT in 2D and 3D cell culture models of hepatocellular carcinoma.

The purpose of the present study was to evaluate the effects of vascular endothelial growth factor (VEGF) on tumorigenic properties in two-dimensional (2D) and three-dimensional (3D) cultures of hepatoma cells. The proliferation and invasion of hepatoma cells was assessed using wound healing, chemotaxis Transwell, invasion, tube-forming and hanging drop assays in both 2D and 3D cultures. The expression levels of epithelial-mesenchymal transition (EMT) and stemness markers were analysed using reverse transcription-quantitative PCR (RT-qPCR) for mRNA expression and immunofluorescence assay for protein expression. To validate the role of VEGF in tumour growth, a VEGF receptor (VEGFR) inhibitor (sorafenib) was used. The results demonstrated that the hepatoma cells formed 3D spheroids that differed in size and density in the absence and presence of the growth factor, VEGF. In all spheroids, invasion and angiogenesis were more aggressive in 3D cultures in comparison to 2D conditions following treatment with VEGF. Mechanistically, the VEGF-mediated increase in the levels of EMT markers, including Vimentin, N-cadherin 2 (Cadherin 2) and Thy-1 Cell Surface Antigen was observed in the 2D and 3D cultures. Sorafenib treatment for 24 h culminated in a marked reduction in cell migration, cell-cell adhesion, spheroid compaction and EMT gene expression in 3D models as compared to the 2D models. On the whole, the findings of the present study suggested that as compared to the 2D cell cultures, 3D cell cultures model may be used as a more realistic model for the study of tumour growth and invasion in the presence of angiogenic factors, as well as for tumour inhibitor screening.

Towards the diagnosis of dengue virus and its serotypes using designed CRISPR/Cas13 gRNAs.

Dengue Virus (DENV) is a mosquito-borne virus that is prevalent in the world's tropical and subtropical regions. Therefore, early detection and surveillance can help in the management of this disease. Current diagnostic methods rely primarily on ELISA, PCR, and RT-PCR, among others, which can only be performed in specialized laboratories and require sophisticated instruments and technical expertise. CRISPR-based technologies on the other hand have field-deployable viral diagnostics capabilities that could be used in the development of point-of-care molecular diagnostics. The first step in the field of CRISPR-based virus diagnosis is to design and screen gRNAs for high efficiency and specificity. In the present study, we employed a bioinformatics approach to design and screen DENV CRISPR/Cas13 gRNAs for conserved and serotype-specific variable genomic regions in the DENV genome. We identified one gRNA sequence specific for each of the lncRNA and NS5 regions and identified one gRNA against each of DENV1, DENV2, DENV3, and DENV4 to distinguish the four DENV serotypes. These CRISPR/Cas13 gRNA sequences will be useful in diagnosing the dengue virus and its serotypes for in vitro validation and diagnostics.

In silico identification and validation of natural antiviral compounds as potential inhibitors of SARS-CoV-2 methyltransferase.

The novel Coronavirus disease 2019 (COVID-19) is potentially fatal and caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Due to the unavailability of any proven treatment or vaccination, the outbreak of COVID-19 is wreaking havoc worldwide. Hence, there is an urgent need for therapeutics targeting SARS-CoV-2. Since, botanicals are an important resource for several efficacious antiviral agents, natural compounds gaining significant attention for COVID-19 treatment. In the present study, methyltranferase (MTase) of the SARS-CoV-2 is targeted using computational approach. The compounds were identified using molecular docking, virtual screening and molecular dynamics simulation studies. The binding mechanism of each compound was analyzed considering the stability and energetic parameter using in silico methods. We have found four natural antiviral compounds Amentoflavone, Baicalin, Daidzin and Luteoloside as strong inhibitors of methyltranferase of SARS-CoV-2. ADMET prediction and target analysis of the selected compounds showed favorable results. MD simulation was performed for four top-scored molecules to analyze the stability, binding mechanism and energy requirements. MD simulation studies indicated energetically favorable complex formation between MTase and the selected antiviral compounds. Furthermore, the structural effects on these substitutions were analyzed using the principles of each trajectories, which validated the interaction studies. Our analysis suggests that there is a very high probability that these compounds may have a good potential to inhibit Methyltransferase (MTase) of SARS-CoV-2 and to be used in the treatment of COVID-19. Further studies on these natural compounds may offer a quick therapeutic choice to treat COVID-19.Communicated by Ramaswamy H. Sarma.

Targeting delta-endotoxin (Cry1Ac) of Bacillus thuringiensis to subcellular compartments increases the protein expression, stability, and biological activity.

Evolving insect resistance to delta-endotoxins can be delayed by using a few strategies like high dosage, refugia, and gene stacking which require the expression of delta-endotoxins at sufficiently high levels to kill the resistant insects. In this study, we comparatively analyzed the efficacy of targeting truncated cry1Ac protein to the cytoplasm, endoplasmic reticulum (ER), and chloroplast to obtain high protein expression. mRNA and protein profiling of cry1Ac showed that both ER and chloroplast are efficient targets for expressing high levels of truncated cry1Ac. A maximum of 0.8, 1.6, and 2.0% cry1Ac of total soluble protein were obtained when the truncated cry1Ac was expressed in the cytoplasm, routed through ER, and targeted to the chloroplast. We further showed that not only the protein content but also the biological activity of truncated cry1Ac increases by sub-cellular targeting and the biological activity is slightly greater in the ER routed transgenic lines by conducting different bioassays on Helicoverpa armigera. Using native Western analysis, we demonstrated that the truncated cry1Ac protein could exist as oligomers in plant cells and this oligomerization capability is low in the cytoplasm. In conclusion, routing of delta endotoxins through ER is the first choice to obtain high protein expression and bioactivity.

Rifampicin Increases Expression of Plant Codon-Optimized Bacillus thuringiensis δ-Endotoxin Genes in Escherichia coli.

Transgenic crops expressing Cry δ-endotoxins of Bacillus thuringiensis for insect resistance have been commercialized worldwide with increased crop productivity and spectacular socioeconomic gains. To attain the enhanced level of protein expression, the cry genes have to be extensively modified for RNA stability and translation efficiency in the plant systems. However, such modifications in nucleotide sequences make it difficult to express the cry genes in Escherichia coli because of the presence of E. coli rare codons. Induction of gene expression through the T7 promoter/lac operator system results in high levels of transcription but limits the availability of activated tRNA corresponding to rare codons that leads to translation stalling at ribosomes. In the present study, an Isopropyl ß-D-1-thiogalactopyranoside (IPTG)/rifampicin combination-based approach was adopted to induce transcription of cry genes through T7 promoter/lac operator while simultaneously inhibiting the transcription of host genes through rifampicin. The results show that the IPTG/rifampicin combination leads to high-level expression of four plant codon-optimized cry genes (cry2Aa, cry1F, cry1Ac, and cry1AcF). Northern blot analysis of the cry gene expressing E. coli samples showed that the RNA expression level in the IPTG-induced samples was higher as compared to that in the IPTG/rifampicin-induced samples. Diet overlay insect bioassay of IPTG/rifampicin-induced Cry toxins with Helicoverpa armigera larvae showed bioactivity (measured as LC50) similar to the previous studies. The experiment has proved that recombinant synthetic gene (plant codon-optimized gene) with the combination of Rifampicin which inhibits DNA-dependent bacterial RNA polymerase and reduces the excessive baggage of translational machinery of the bacterial cell triggers the production of synthetic protein. Purification of protein using high pH buffer increases the solubility of the protein. Further, LC50 analysis shows no reduction of protein activity leads to protein stability. Further, purified cry toxin protein can be used for crop protection against pests and a purified form of the synthetic protein can be used for antibody production and perform the immunoassay for the identification of the transgenic plant. The crystallographic structure of synthetic protein could be used for interaction study with another insect to see insecticidal activity.

Binding of Bacillus thuringiensis Cry1A toxins with brush border membrane vesicles of maize stem borer (Chilo partellus Swinhoe).

Maize stem borer (Chilo partellus) is a major insect pest of maize and sorghum in Asia and Africa. Bacillus thuringiensis (Bt) δ-endotoxins have been found effective against C. partellus, both in diet-overlay assay and in transgenic plants. Gene stacking as one of the resistance management strategies in Bt maize requires an understanding of receptor sharing and binding affinity of δ-endotoxins. In the present study, binding affinity of three fluorescein isothiocyanate labeled Cry1A toxins showed high correlation with the toxicity of respective δ-endotoxins. Competitive binding studies showed that Cry1Ab toxins share some of the binding sites with Cry1Aa and Cry1Ac with low affinity and that Cry1Ab may have additional binding sites that are unavailable to the other two toxins tested.

Screening of CRISPR/Cas9 gRNA for mimicking Powdery Mildew resistant MLO ol-2 mutant.

Powdery Mildew (PM) caused by fungal pathogen Oidium neolycopersici (O. neolycopersici) affects both greenhouse and field-grown tomato production. Resistance to PM disease can be achieved by selective inactivation of Mildew Resistance Locus O (MLO) genes encoding heptahelical transmembrane domains, which confer susceptibility to fungal pathogens. Natural loss-of-function mutation is a 19 base pair (bp) deletion in the SlMLO1 gene locus responsible for fungal resistance in S. lycopersicum var. cerasiforme. Introgression of these resistance alleles through breeding into elite varieties is possible. However, this is a long and labour-intensive process and has limitations due to linkage drag. Nonetheless, recent developments in the field of genome editing technology particularly CRISPR/Cas9 systems allows quick, effective and accurate genome modification at the target gene locus. Therefore, it is of interest to determine the efficacy and exact deletion that mimics the natural ol-2 (Slmlo1) mutation present in wild tomatoes using CRISPR/Cas9. 947 putative guide RNAs (gRNAs) were designed using Cas9 variants to broaden Protospacer Adjacent Motif (PAM) compatibility and to enhance DNA specificity against the SlMLO1 locus. 60 out of 947 gRNAs were selected based on the recognition of the PAM sequence, the MIT specificity ranking, the off-target sites, their distance from the 19bp natural ol-2 mutation, the secondary structure of the gRNAs, and their minimum free energy. In depth analysis of these 60 gRNAs helped in the selection of the top five gRNAs based on the above-mentioned criteria. These gRNAs are useful for introducing deletions identical to natural ol-2 mutants and impart resistance against fungal pathogen O. neolycopersici in cultivated tomato crops.

TALENs-an indispensable tool in the era of CRISPR: a mini review.

BACKGROUND: Genome of an organism has always fascinated life scientists. With the discovery of restriction endonucleases, scientists were able to make targeted manipulations (knockouts) in any gene sequence of any organism, by the technique popularly known as genome engineering. Though there is a range of genome editing tools, but this era of genome editing is dominated by the CRISPR/Cas9 tool due to its ease of design and handling. But, when it comes to clinical applications, CRISPR is not usually preferred. In this review, we will elaborate on the structural and functional role of designer nucleases with emphasis on TALENs and CRISPR/Cas9 genome editing system. We will also present the unique features of TALENs and limitations of CRISPRs which makes TALENs a better genome editing tool than CRISPRs. MAIN BODY: Genome editing is a robust technology used to make target specific DNA modifications in the genome of any organism. With the discovery of robust programmable endonucleases-based designer gene manipulating tools such as meganucleases (MN), zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats associated protein (CRISPR/Cas9), the research in this field has experienced a tremendous acceleration giving rise to a modern era of genome editing with better precision and specificity. Though, CRISPR-Cas9 platform has successfully gained more attention in the scientific world, TALENs and ZFNs are unique in their own ways. Apart from high-specificity, TALENs are proven to target the mitochondrial DNA (mito-TALEN), where gRNA of CRISPR is difficult to import. This review talks about genome editing goals fulfilled by TALENs and drawbacks of CRISPRs. CONCLUSIONS: This review provides significant insights into the pros and cons of the two most popular genome editing tools TALENs and CRISPRs. This mini review suggests that, TALENs provides novel opportunities in the field of therapeutics being highly specific and sensitive toward DNA modifications. In this article, we will briefly explore the special features of TALENs that makes this tool indispensable in the field of synthetic biology. This mini review provides great perspective in providing true guidance to the researchers working in the field of trait improvement via genome editing.

In silico identification and evaluation of Bacillus subtilis cold shock protein B (cspB)-like plant RNA chaperones.

Cold shock domain (CSD) proteins with nucleic acid binding properties are well conserved from bacteria to higher organisms. In bacteria, the cold shock proteins (CSPs) are single domain RNA chaperones, whereas in animals and plants, CSDs are accompanied by additional domains with roles in transcription regulation. Bacterial CSPs (Escherischia coli-cspA and Bacilus subtilis-cspB) have successfully imparted drought tolerance in transgenic plants; however, these cannot be deployed in food crops due to their low public acceptance of transgenics with bacterial genes. Therefore, this study aimed to identify CSPB-like proteins from plants that can be used for developing drought tolerant transgenic crops. Twelve single domain plant CSPs presenting >40% sequence identity with CSPB were identified. All 12 plant CSPs were modeled by homology modeling and refined by molecular dynamics simulation for 10 ns. Selected plant CSPs and CSPB exhibited high structural similarity (Tm-score: 0.63-0.86). Structure based phylogenetic analysis revealed that Triticum aestivum-csp1 and Aegilops tauschii-cspE are structurally closer to CSPB compared to their orthologs and paralogs. Molecular docking with three RNA molecules (5U, UC3U, and C2UC) indicates that Ricinus communis-csd1 and T. aestivum-csp1 have a binding pattern and docking scores similar to those of CSPB. Furthermore, MD simulations for 20 ns and analysis of RMSD, RMSF, Rg as well as the number of hydrogen bonds in all the three complexes revealed that plant CSP-RNA complexes behave in a similar manner to that of the CSPB-RNA complex, making them highly potential candidate genes for developing drought tolerance in transgenic plants. Communicated by Ramaswamy H. Sarma.

CPP-ZFN: a potential DNA-targeting anti-malarial drug.

BACKGROUND: Multidrug-resistant Plasmodium is of major concern today. Effective vaccines or successful applications of RNAi-based strategies for the treatment of malaria are currently unavailable. An unexplored area in the field of malaria research is the development of DNA-targeting drugs that can specifically interact with parasitic DNA and introduce deleterious changes, leading to loss of vital genome function and parasite death. PRESENTATION OF THE HYPOTHESIS: Advances in the development of zinc finger nuclease (ZFN) with engineered DNA recognition domains allow us to design and develop nuclease of high target sequence specificity with a mega recognition site that typically occurs only once in the genome. Moreover, cell-penetrating peptides (CPP) can cross the cell plasma membrane and deliver conjugated protein, nucleic acid, or any other cargo to the cytoplasm, nucleus, or mitochondria. This article proposes that a drug from the combination of the CPP and ZFN systems can effectively enter the intracellular parasite, introduce deleterious changes in its genome, and eliminate the parasite from the infected cells. TESTING THE HYPOTHESIS: Availability of a DNA-binding motif for more than 45 triplets and its modular nature, with freedom to change number of fingers in a ZFN, makes development of customized ZFN against diverse target DNA sequence of any gene feasible. Since the Plasmodium genome is highly AT rich, there is considerable sequence site diversity even for the structurally and functionally conserved enzymes between Plasmodium and humans. CPP can be used to deliver ZFN to the intracellular nucleus of the parasite. Signal-peptide-based heterologous protein translocation to Plasmodium-infected RBCs (iRBCs) and different Plasmodium organelles have been achieved. With successful fusion of CPP with mitochondrial- and nuclear-targeting peptides, fusion of CPP with 1 more Plasmodium cell membrane translocation peptide seems achievable. IMPLICATIONS OF THE HYPOTHESIS: Targeting of the Plasmodium genome using ZFN has great potential for the development of anti-malarial drugs. It allows the development of a single drug against all malarial infections, including multidrug-resistant strains. Availability of multiple ZFN target sites in a single gene will provide alternative drug target sites to combat the development of resistance in the future.

A regulatory circuit between lncRNA and TOR directs amino acid uptake in yeast.

Long non coding RNAs (lncRNAs) have emerged as crucial players of several central cellular processes across eukaryotes. Target of Rapamycin (TOR) is a central regulator of myriad of fundamental cellular processes including amino acid transport under diverse environmental conditions. Here we investigated the role of lncRNA in TOR regulated amino acid uptake in S. cerevisiae. Transcription of lncRNA regulates local gene expression in eukaryotes. In silico analysis of many genome wide studies in S. cerevisiae revealed that transcriptome includes conditional expression of numerous lncRNAs in proximity to amino acid transporters (AATs). Considering regulatory role of these lncRNAs, we selected highly conserved TOR regulated locus of a pair of AATs present in tandem BAP2 and TAT1. We observed that the expression of antisense lncRNA XUT_2F-154 (TBRT) and AATs BAP2 and TAT1 depends on activities of TOR signaling pathway. The expression of TBRT is induced, while that of BAP2 TAT1 is repressed upon TOR inhibition by Torin2. Notably, upon TOR inhibition loss of TBRT contributed to enhanced activities of Bap2 and Tat1 leading to improved growth. Interestingly, nucleosome scanning assay reveal that TOR signaling pathway governs chromatin remodeling at BAP2 biphasic promoter to control the antagonism of TBRT and BAP2 expression. Further TBRT also reprograms local chromatin landscapes to decrease the transcription of TAT1. The current work demonstrates a functional correlation between lncRNA production and TOR governed amino acid uptake in yeast. Thus this work brings forth a novel avenue for identification of potential regulators for therapeutic interventions against TOR mediated diseases.

MYOD and HAND transcription factors have conserved recognition sites in mTOR promoter: insights from in silico analysis.

mTOR regulates multiple cellular processes that are critical for proper maintenance of cell growth and development. However, mechanisms and factors responsible for transcriptional regulation of mTOR are partially known. To identify different transcription factor binding sites in promoter region of mTOR, we performed in silico phylogenetic foot printing analysis of diverse set of human orthologs. Phylogenetic tree for the orthologs was generated to establish the evolutionary relationships among them. Conserved binding sites among the species were predicted by tool MEME. The predicted conserved sites were further analyzed for binding of transcription factors by MatInspector program. Predicted TFs were then integrated with known physical interactions and coexpression data to decipher the important transcriptional regulators of mTOR signaling. Our study suggests that motifs AGGCGGG (+ 15 to + 21) and GGCGGC (+ 60 to + 65) are highly conserved across the species and are recognition sequence for HAND and MYOD transcription factors, respectively. Also these two transcription factors show direct physical interaction in protein-protein interaction map, indicating their regulatory role on expression of mTOR for control of myogenesis. Our study provides novel clues on differential regulation of mTOR under diverse environmental conditions.

Endothelial Cell-Derived TGF-ß Promotes Epithelial-Mesenchymal Transition via CD133 in HBx-Infected Hepatoma Cells.

Background: Hepatitis B-X Protein (HBx) encoded in Hepatitis B virus (HBV) is known to play a critical role in development and progression of HBV induced hepatocellular carcinoma (HCC). HBx interacts with and activates various cells in HCC microenvironment to promote tumor initiation, progression and invasion. In this study, we investigated how surrounding stromal cells interact with HBx-infected hepatoma cells by a series of in vitro co-culture studies. Methods: Huh7 hepatoma cells were cultured and transfected with the mammalian expression vector pGFP-HBx. Co-culture assays were performed between HBx-transfected Huh7 cells and conditioned media (CM) from stromal cells [endothelial cell lines (HUVECs) and hepatic stellate cell lines (LX2 cells)]. The effect of these interactions was studied by a series of functional assays like chemotaxis, invasion, and wound healing scratch assays. Also, quantitative real time (RT)-PCRs of the mesenchymal genes was performed in the hepatoma cells with and without the co-cultures. Hep3B cells with an integrated HBV genome were taken as positive controls. Results: HBx-transfected Huh7 cells cultured in presence of CM from HUVECs illustrated enhanced migration and tube formation as compared to HBx-transfected cells cultured alone or co-cultured with LX2 cells. HBx-transfected hepatoma cells incubated with CM from HUVECs also expressed mesenchymal genes including Thy1, CDH2, TGFßR1, VIM, and CD133. ELISAs revealed increased levels of TGF-ß in CM from HUVECs. In comparison to unstimulated HBx-transfected Huh7 cells, TGF-ß stimulated cells displayed increased invasive properties and mesenchymal gene expression. RT-PCR and flow cytometry analysis further demonstrated that incubation with either CM from HUVECs or TGF-ß significantly increased the expression of a stemness marker, CD133 in HBx-infected hepatoma cells. Gene inhibition experiments with CD133 siRNA showed a downregulation of mesenchymal gene expression and properties in TGF-ß induced HBx-infected hepatoma cells as compared to that observed in control siRNA treated cells, indicating CD133 as one of the key molecules affecting epithelial to mesenchymal transition (EMT) in HBx-infected cells. Conclusion: The study indicates that secretory factors like TGF-ß from neighboring endothelial cells may enhance expression of CD133 and impart an aggressive EMT phenotype to HBx-infected hepatoma cells in HBV induced HCC.