In silico analysis of a novel protein in CAR T- cell therapy for the treatment of hematologic cancer through molecular modelling, docking, and dynamics approach.

Cellular therapy has emerged as a key tool in the treatment of hematological malignancies. An advanced cell therapy known as chimeric antigen receptor T cell therapy (CAR T-cell therapy) has been approved by the United States Food and Drug Administration (FDA) as KYMRIAH by Novartis and YESCARTA by Gilead/Kite pharma in the year 2017. A chimeric receptor is composed of an extracellular antigen recognition site along with some co-stimulating and signalling domains. On the whole, it turns out to be one of the most potent receptors on T cells targeting a specific type of cancer cell based on its antigenic marker. CD19 CAR T-cell therapy is the first clinically approved therapy for lymphoma with remarkable results in complete remission of B cell lymphoblastic leukemia up to 90%. The high rate of effectiveness of the CAR T-cell therapy against B-ALL justifies the investigation and application of this therapy for fatal diseases like all types of hematological malignancies. The most critical aspect of chimeric receptor therapy is designing and building an artificial receptor that is specific to a given type of cancer. For this reason, the in silico technique is an appropriate model to investigate the integrity and effectiveness of the engineered chimeric receptor prior to commencing in vitro experiments followed by clinical trials. This computerized experimental study aids in predicting the molecular mechanism of chimeric protein and how it interacts with both ligands. We have anticipated various features of the chimeric protein in terms of qualitative analysis (structure, protein modelling, physiological properties) and functional analysis (antigenicity, allergenicity, its receptor-ligand binding ability, involving signalling pathways). Furthermore, the reliability and validation of the binding mode of the chimeric protein against receptors were performed through a complex molecular dynamics simulation for a 100 ns timeframe in an aqueous environment. The obtained simulation study showed that CD30 was a better approachable marker as compared to CD20 due to its better binding energy score and also binding conformations stability.

Laser-diode-driven high-luminance white light source with sediment phosphors and optimal opto-thermal performance.

High-luminance light sources are challenging to achieve with light-emitting diodes (LEDs) due to power droop. Since laser diodes (LDs) do not suffer from power droop, they can be used as an alternative. A novel, to the best of our knowledge, high-luminance white light source was developed utilizing LDs combined with a sediment silicone/phosphor composite. The deposition of this sediment phosphor inside an aluminum spacer on top of a sapphire backplate ensures optimal thermal management. To enhance the optical performance, the sapphire plate is coated with a custom-designed blue pass filter in order to recycle most of the converted light that is emitted in the backward direction. The maximal obtained luminance of this light source is 103 MCd/m2 at a luminous flux of 3119 lm.

Role of long non­coding RNAs and related epigenetic mechanisms in liver fibrosis (Review).

Liver fibrosis is one of the major liver pathologies affecting patients worldwide. It results from an improper tissue repair process following liver injury or inflammation. If left untreated, it ultimately leads to liver cirrhosis and liver failure. Long non­coding RNAs (lncRNAs) have been implicated in a wide variety of diseases. They can regulate gene expression and modulate signaling. Some of the lncRNAs promote, while others inhibit liver fibrosis. Similarly, other epigenetic processes, such as methylation and acetylation regulate gene transcription and can modulate gene expression. Notably, there are several regulatory associations of lncRNAs with other epigenetic processes. A major mechanism of action of long non­coding RNAs is to competitively bind to their target microRNAs (miRNAs or miRs), which in turn affects miRNA availability and bioactivity. In the present review, the role of lncRNAs and related epigenetic processes contributing to liver fibrosis is discussed. Finally, various potential therapeutic approaches targeting lncRNAs and related epigenetic processes, which are being considered as possible future treatment targets for liver fibrosis are identified.

Differential transcriptome analysis in HPV-positive and HPV-negative cervical cancer cells through CRISPR knockout of miR-214.

In this study we have investigated the effects of a tumour suppressor microRNA, miR-214, on gene expression in HPV-positive (CaSki) and HPV-negative cervical cancer cells (C33A) by RNA sequencing using next generation sequencing. The HPV-positive and HPV-negative cervical cancer cells were either miR-214- knocked-out or miR-214-overexpressed. Gene expression analysis showed that a total of 904 genes were upregulated and 365 genes were downregulated between HPV-positive and HPV-negative cervical cancer cells with a fold change of +/- 2. Furthermore, 11 differentially expressed and relevant genes (TNFAIP3, RAB25, MET, CYP1B1, NDRG1, CD24, LOXL2, CD44, PMS2, LATS1 and MDM1) which showed a fold change of +/-5 were selected to confirm by real-time PCR. This study represents the first report of miR-214 on global gene expression in the context of HPV.

CRISPR-mediated knockdown of miR-214 modulates cell fate in response to anti-cancer drugs in HPV-negative and HPVpositive cervical cancer cells.

Cervical cancer is the fourth most common cause of mortality in women worldwide. In this study we investigated the effect of a tumour suppressor microRNA miR-214 in modulating the cell death against chemotherapeutic drugs like Doxorubicin, Cisplatin and Paclitaxel. CRISPR-facilitated knockdown and plasmid-based overexpression of miR-214 was performed in cervical cancer cell lines HeLa, C33A and CaSki. It was observed that knocking out miR-214 resulted in reduced apoptosis and cell migration upon drug treatments; while overexpression of miR-214 resulted in marginal increase in apoptosis and cell migration when treated with drugs. However, miR-214 had very little effect on production of reactive oxygen species. Our results also indicate that Doxorubicin was least effective and Paclitaxel most effective in inducing cell death. A combination of miR-214 overexpression and Paclitaxel treatment was found to be most effective in inducing cell death in cervical cancer cells. Analysis of cell cycle phases followed by apoptotic markers also showed that miR-214 overexpression along with Paclitaxel treatment caused an increase in PARP and decline of PI-3 kinase/Akt levels. Therefore, miR-214 levels determine the fate of the cancer cell during chemotherapeutic treatment.

Transcriptomic analyses of gene expression by CRISPR knockout of miR-214 in cervical cancer cells.

In this study, we investigate the effect of one such micro RNA, miR-214 which is frequently down-regulated in cervical cancer. In this study, we either CRISPR knocked out or overexpressed miR-214 in cervical cancer cells and analyzed the global mRNA expression by Next Generation Sequencing (NGS) It was observed that a total of 108 genes were upregulated and 178 downregulated between the samples, above and below the baseline respectively. Gene Ontology and KEGG pathway analysis reveal distinct biological processes and pathways. Analysis of gene regulatory networks also gave different network patterns in the two samples. We confirmed the RNA sequencing data for 10 genes; IFIF27, SMAD3, COX11, TP53INP1, ABL2, FGF8, TNFAIP3, NRG1, SP3 and MDM4 by Real-time PCR. This is the first report on the effect of miR-214 on global mRNA profile in cervical cancer cells. This study also reports new biomarkers for cervical cancer prognosis.

CAR T cell therapy: A new era for cancer treatment (Review).

Cancer has recently been identified as the leading cause of mortality worldwide. Several conventional treatments and cytotoxic immunotherapies have been developed and made available to the market. Considering the complex behavior of tumors and the involvement of numerous genetic and cellular factors involved in tumorigenesis and metastasis, there is a need to develop a promising immunotherapy that targets tumors at both the cellular and genetic levels. Chimeric antigen receptor (CAR) T cell therapy has emerged as a novel therapeutic T cell engineering practice, in which T cells derived from patient blood are engineered in vitro to express artificial receptors targeted to a specific tumor antigen. These directly identify the tumor antigen without the involvement of the major histocompatibility complex. The use of this therapy in the last few years has been successful, with a reduction in remission rates of up to 80% for hematologic cancer, particularly for acute lymphoblastic leukemia (ALL) and non­Hodgkin lymphomas, such as large B cell lymphoma. Recently, anti­CD19 CAR therapy, or UCART19, has been shown to be efficacious in treating relapsed/refractory hematologic cancer. Several other cell surface tumor antigens, such as CD20 and CD22, found in the majority of leukemias and lymphomas are considered potential targets by pharmaceutical companies and research organizations, and trials have been ongoing in this direction. Although this therapeutic regimen is currently confined to treating hematologic cancer, the increasing involvement of several auxiliary techniques, such as bispecific CAR, Tan­CAR, inhibitory­CAR, combined antigens, the clustered regularly interspaced short palindromic repeats gene­editing tool and nanoparticle delivery, may substantially improve its overall anticancer effects. CAR therapy has the potential to offer a rapid and safer treatment regime to treat non­solid and solid tumors. The present review presents an insight into the advantages and the advances of CAR immunotherapy and presents the emerging discrepancy of CAR therapy over usual forms of therapy, such as chemotherapy and radiotherapy.

Modulation of DNA methylation by human papillomavirus E6 and E7 oncoproteins in cervical cancer.

Human papillomaviruses (HPVs) are double stranded circular DNA viruses that infect cutaneous and mucosal epithelial cells. Almost 99% of cervical cancer has a HPV infection. The early oncoproteins E6 and E7 are important in this cellular transformation process. Epigenetic mechanisms have long been known to result in decisive alterations in DNA, leading to alterations in DNA-protein interactions, alterations in chromatin structure and compaction and significant alterations in gene expression. The enzymes responsible for these epigenetic modifications are DNA methyl transferases (DNMTs), histone acetylases and deacetylases. Epigenetics has an important role in cancer development by modifying the cellular micro environment. In this review, the authors discuss the role of HPV oncoproteins E6 and E7 in modulating the epigenetic mechanisms inside the host cell. The oncoproteins induce the expression of DNMTs which lead to aberrant DNA methylations and disruption of the normal epigenetic processes. The E7 oncoprotein may additionally directly bind and induce methyl transferase activity of the enzyme. These modulations lead to altered gene expression levels, particularly the genes involved in apoptosis, cell cycle and cell adhesion. In addition, the present review discusses how epigenetic mechanisms may be targeted for possible therapeutic interventions for HPV mediated cervical cancer.

Transcriptomic analyses of genes differentially expressed by high-risk and low-risk human papilloma virus E6 oncoproteins.

Human papilloma virus is the causative agent for cervical cancer with 99 % of cervical cancer cases containing HPV. The high risk HPV-16, 18 and 31 are the major causative agents. The low risk HPV-6, 11 have been reported to cause penile, laryngeal, bronchogenic and oesophageal cancer. Since E6 oncoprotein is frequently over expressed in cancers, we did gene expression studies to compare between the E6 genes of high-risk (HPV18) or low-risk (HPV11)stably transfected in epithelial cell line EPC-2 or mock transfected with the basic vector pCDNA3.1. Microarray studies showed a total of 697 genes showing differential expression between the samples. Genes involved in several key cellular processes such as cell adhesion, angiogenesis, transcription regulation, cell cycle regulation and cell division showed altered expression between the samples. Gene Ontology mapping of 44 genes according cellular pathways revealed 13 pathways namely angiogenesis, alzhemier's, Wnt, p53, interleukin, TGF-ß, cadherin, integrin, PI3-kinase, catennin, insulin, chemokine and G protein signalling pathways. The microarray results were confirmed by quantitative real-time PCR for some representative genes like IFI27, CTNNA1, OSMR, CYP1B1, TNFSF13, LAMA2 and COL5A3. Analysis of differentially expressed genes by high-risk and low-risk HPV E6 proteins might help in identification of potential biomarkers for diagnosis, progression and therapy of oesophageal cancer. The understanding of mechanisms of activation of these genes as well as the function of gene products will give a further insight into their roles in oesophageal cancer.

Human papillomavirus-16 E5 protein: oncogenic role and therapeutic value.

BACKGROUND: Human papillomavirus (HPV) is a non-enveloped, double-stranded DNA virus. HPV infection occurs through sexual route, and the virus infects mucosal and cutaneous epithelial cells. Inside the cell, the viral DNA replicates extrachromosomally. HPV is the major cause of cervical cancer worldwide. HPVs infecting mucosal epithelial cells are sub-grouped into low-risk or high-risk by virtue of them causing benign warts or cancer, respectively. The early oncoproteins of HPV, namely E4, E5, E6 and E7, are known to contribute to tumorigenesis. The roles and functions of HPV E6 and E7 have been thoroughly studied over the years. However, limited studies have been done on E5 regarding its intracellular functions. CONCLUSIONS: This review attempts to discuss the positive role of HPV16 E5 in the form of therapeutic target for cervical cancer, as well as its role in modulation of several intracellular signalling pathways leading to transformed phenotype of the host cell.

Mycobacterium tuberculosis secreted antigen (MTSA-10) inhibits macrophage response to lipopolysaccharide by redox regulation of phosphatases.

The present study was undertaken to investigate the possible role of a 10-kDa, secretory antigenic protein of Mtb (MTSA-10) in regulating macrophase response to lipopolysacchride (LPS). MTSA-10 inhibited the lipopolysaccharide (LPS)-induced oxidant species generation in the macrophage. Treatment of macrophages with MTSA-10 activated their protein tyrosine phosphatases (PTPs) in a redox-regulated fashion. These activated phosphatases then interfered with the early events of LPS signaling and lower the strength and magnitude of the signal generated, thereby preventing macrophages from making an effective immune response. Mycobacterium tuberculosis Region of Deletion-1 (RD-1)-specific secretory antigen MTSA-10 (encoded by ORF Rv3874 of Mtb genome) modulated the macrophage signaling machinery and prevented it from responding to further activation by LPS.

Human papillomavirus E6 and E7 oncoproteins as risk factors for tumorigenesis.

Human papillomavirus (HPV) is small, double-stranded DNA virus that infects mucosal and cutaneous epithelial tissue. HPV is sexually transmitted and the viral DNA replicates extrachromosomally. The virus is non-enveloped and has an icosahedral capsid. There are approximately 118 types of HPV, which are characterized as high-risk or low- risk types. High-risk HPVs cause malignant transformation while the low-risk ones cause benign warts and lesions. The expression of E6 and E7 is normally controlled during the normal viral life cycle when viral DNA replicates extrachromosomally. HPV E6 and E7 oncoproteins are overexpressed when the viral genome integrates into the host DNA.Deregulated overexpression of E6 and E7 oncoproteins can cause several changes in cellular pathways and functions leading to malignant transformation of cells and tumorigenesis. In this review, we focus on several cellular mechanisms and pathways that are altered in the presence of E6 and E7, the target proteins of E6 and E7 inside the host cell and how they contribute to the development of the transformed phenotype.

Role of M. tuberculosis RD-1 region encoded secretory proteins in protective response and virulence.

A gene fragment corresponding to the region of difference-1 (RD-1) of the Mycobacterium tuberculosis genome, spanning open reading frames Rv3871 to Rv3879c, is missing in all bacillus Calmette-Guerin (BCG) vaccine strains of M. bovis, indicating that this was perhaps the primary deletion event responsible for attenuation of virulent M. bovis. The RD-1 locus has, therefore, been considered crucial in the pathogenesis of M. tuberculosis. Two most predominant secretory proteins encoded by this region viz. CFP-10 (Rv3874) and ESAT-6 (Rv3875) are being widely evaluated as candidate vaccine(s) and used as antigens in the diagnosis of tuberculosis. However, several recent reports have implicated their putative role in deactivation of the macrophage and dendritic cell functions. A large body of recent literature provides an inkling of various mechanisms these proteins might use to down regulate normal macrophage functions and their possible contribution to virulence of M. tuberculosis. This review re-emphasizes the suggestion about the dual function of these two secreted mycobacterial proteins, viz., they have both T-cell activation and macrophage deactivation functions.

Mycobacterium tuberculosis secretory proteins CFP-10, ESAT-6 and the CFP10:ESAT6 complex inhibit lipopolysaccharide-induced NF-kappaB transactivation by downregulation of reactive oxidative species (ROS) production.

Mycobacterium tuberculosis (Mtb) causes death of 2-3 million people annually and is considered one of the most successful intracellular pathogens to persist inside the host macrophage. Recent studies have implicated the role of RD-1 region of Mtb genome in the mycobacterial pathogenesis. The role of RD-1-encoded secretory proteins of Mtb in modulation of macrophage function has not been investigated in detail. Here we show that RD-1 encoded two major secretory proteins, namely, culture filtrate protein-10 kDa (CFP-10) and early secreted antigenic target-6 kDa (ESAT-6), and their 1:1 CFP-10:ESAT6 complex inhibit production of reactive oxidative species (ROS) in RAW264.7 cells. These proteins also downregulated the bacterial lipopolysaccharide (LPS)-induced ROS production, which, in turn, downregulated LPS-induced nuclear factor-kappaB (NF-kappaB) p65 DNA-binding activity, as well as inhibited the NF-kappaB-dependent reporter gene (chloramphenicol acetyl transferase) expression in the treated macrophages. Moreover, addition of N-acetyl cysteine, which is a scavenger of ROS, also inhibited LPS-induced reporter gene expression by scavenging the ROS, thereby preventing NF-kappaB transactivation. These studies indicate that the secretory proteins CFP-10, ESAT-6 and the CFP10:ESAT6 complex of Mtb can inhibit LPS-induced NF-kappaB-dependent gene expression via downregulation of ROS production.

Mycobacterium tuberculosis 6-kDa early secreted antigenic target (ESAT-6) protein downregulates lipopolysaccharide induced c-myc expression by modulating the extracellular signal regulated kinases 1/2.

BACKGROUND: Mycobacterium tuberculosis (Mtb) causes death of 2-3 million people every year. The persistence of the pathogenic mycobacteria inside the macrophage occurs through modulation of host cell signaling which allows them, unlike the other non-pathogenic species, to survive inside the host. The secretory proteins of M. tuberculosis have gained attention in recent years both as vaccine candidates and diagnostic tools; they target the immune system and trigger a putatively protective response; however, they may also be involved in the clinical symptoms of the disease. RESULTS: Our studies showed that RD-1-encoded secretory protein ESAT-6 is involved in modulation of the mitogen-activated protein (MAP) kinase-signaling pathway inside the macrophage. ESAT-6 induced phosphorylation of extracellular signal-regulated kinases 1/2 (ERK1/2) in the cytoplasm but not in the nucleus, which normally is the case for MAP kinases. ESAT-6 also antagonized LPS-induced ERK1/2 phosphorylation in the nucleus. Stimulation of cells by ESAT-6 along with sodium orthovanadate (a tyrosine phosphatase inhibitor) restored phosphorylation of ERK1/2 in the nucleus, suggesting active dephosphorylation of ERK1/2 by some putative phosphatase(s) in the nucleus. Further, ESAT-6 was found to down regulate the expression of LPS-inducible gene c-myc in an ERK1/2-dependent manner. CONCLUSION: This study showed the effect of secretory proteins of M. tuberculosis in the modulation of macrophage signaling pathways particularly ERK1/2 MAP kinase pathway. This modulation appears to be achieved by limiting the ERK1/2 activation in the nucleus which ultimately affects the macrophage gene expression. This could be a mechanism by which secretory proteins of Mtb might modulate gene expression inside the macrophages.

Mycobacterium tuberculosis secreted antigen (MTSA-10) modulates macrophage function by redox regulation of phosphatases.

Macrophages are the primary host cells for Mycobacterium tuberculosis (Mtb). Although macrophages can mount a strong inflammatory response to dispose of invading microbial pathogens, the immune dysfunction of the Mtb-infected macrophage constitutes the hallmark of mycobacterial pathogenesis. A 10-kDa, Mtb secretory antigen (MTSA-10), encoded by ORF Rv3874, is one of the predominant members of the 'region of difference 1' locus of Mtb genome that has been strongly implicated in mycobacterial virulence. In this study, we investigated the possible role of MTSA-10 in modulating the macrophage dysfunction in a mouse macrophage cell line J774.1. We found that recombinant MTSA-10 caused extensive protein dephosphorylation in J774.1 cells as revealed by two-dimensional gel electrophoresis analysis. We also observed that MTSA-10 treatment downregulated the reactive oxygen species levels in the cells leading to activation of cellular protein phosphatases putatively responsible for the dephosphorylation phenomenon. This implied a direct role of MTSA-10 in the disruption of host cell signaling, resulting in downregulation of transcription of several genes essential for macrophage function.