CRISPR/Cas mediated epigenome editing for cancer therapy.

The understanding of the relationship between epigenetic alterations, their effects on gene expression and the knowledge that these epigenetic alterations are reversible, have opened up new therapeutic pathways for treating various diseases, including cancer. This has led the research for a better understanding of the mechanism and pathways of carcinogenesis and provided the opportunity to develop the therapeutic approaches by targeting such pathways. Epi-drugs, DNA methyl transferase (DNMT) inhibitors and histone deacetylase (HDAC) inhibitors are the best examples of epigenetic therapies with clinical applicability. Moreover, precise genome editing technologies such as CRISPR/Cas has proven their efficacy in epigenome editing, including the alteration of epigenetic markers, such as DNA methylation or histone modification. The main disadvantage with DNA gene editing technologies is off-target DNA sequence alteration, which is not an issue with epigenetic editing. It is known that cancer is linked with epigenetic alteration, and thus CRISPR/Cas system shows potential for cancer therapy via epigenome editing. This review outlines the epigenetic therapeutic approach for cancer therapy using CRISPR/Cas, from the basic understanding of cancer epigenetics to potential applications of CRISPR/Cas in treating cancer.

Validation of an on-line solid-phase extraction method coupled to liquid chromatography-tandem mass spectrometry detection for the determination of Indacaterol in human serum.

Indacaterol has been recently approved in Europe for the treatment of chronic obstructive pulmonary disease (COPD). In the present study, we have developed and validated a rapid and sensitive on-line solid phase extraction (SPE) method coupled to liquid chromatography-tandem mass spectrometry (LC-MS/MS) detection for the determination of Indacaterol in human serum. The sample preparation involves the serum dilution with a 0.2% acetic acid solution prior to the on-line SPE on a mixed-mode cationic (MCX) polymer based sorbent. The samples were then eluted on a reversed phase column with a mobile phase made of acidified water and methanol and detection was performed by MS using electrospay ionization in positive mode. The analysis time between 2 samples was 7.0 min. Standard curves were linear over the range of 10.0 pg/mL (LLOQ) to 1000 pg/mL with correlation coefficient (r(2)) greater than 0.990. The method specificity was demonstrated in six different batches of human serum. Intra-run and inter-run precision and accuracy within ± 20% (at the LLOQ) and ± 15% (other levels) were achieved during a 3-run validation for quality control samples (QCs). The stability at room temperature (38 h) was determined and reported. In addition, the comparison between an off-line SPE procedure and our method gave equivalent results. The results of the present work demonstrated that our on-line SPE-LC-MS/MS method is rapid, sensitive, specific and could be applied to the quantitative analysis of Indacaterol in human serum samples. Our method effectively eliminated the tedious conditioning and rinsing steps associated with conventional off-line SPE and reduced the analysis time. The on-line SPE approach appears attractive for supporting the analysis of several hundreds of clinical samples.

Vibriocidal activity of certain medicinal plants used in Indian folklore medicine by tribals of Mahakoshal region of central India.

OBJECTIVES: Screening of the medicinal plants and determination of minimum inhibitory concentration (MIC) against Vibrio cholerae and Vibrio parahaemolyticus. MATERIALS AND METHODS: A simple in vitro screening assay was employed for the standard strain of Vibrio cholerae, 12 isolates of Vibrio cholerae non-O1, and Vibrio parahaemolyticus. Aqueous and organic solvent extracts of different parts of the plants were investigated by using the disk diffusion method. Extracts from 16 medicinal plants were selected on account of the reported traditional uses for the treatment of cholera and gastrointestinal diseases, and they were assayed for vibriocidal activities. RESULTS: The different extracts differed significantly in their vibriocidal properties with respect to different solvents. The MIC values of the plant extracts against test bacteria were found to be in the range of 2.5-20 mg/ml. CONCLUSIONS: The results indicated that Lawsonia inermis, Saraca indica, Syzygium cumini, Terminalia belerica, Allium sativum, and Datura stramonium served as broad-spectrum vibriocidal agents.

Population dynamics of vibrio species in the river Narmada at Jabalpur.

Several studies on the presence and ecology of various Vibrio sp have been reported in coastal and estuarine waters throughout the world, but there is trifling information available on the distribution of this organism of colossal pathogenic potential in the fresh water riverine environment. Thus, we conducted a multiyearenvironmental study to scrutinize the occurrence of members of genus Vibrio in the largest west flowing river of the Indian subcontinent, which is also the largest river of central India, the Narmada. Statistical analysis was done to reveal major environmental factors controlling the presence of Vibrio sp in the river Narmada. Monthly field samplings were conducted between January 2002 and December 2003 at four different sites in Jabalpur (MP), India. At each site, water samples were taken and physicochemical and bacteriological parameters were measured. The identity of the isolates was confirmed by employing 16S rRNA analysis. The organisms were found to be widely distributed in the river with regular seasonal variations. The density of Vibrio was found to be correlated with temperature, coliforms and other heterotrophic bacteria. Water temperature accounted for most of the variability in the concentration of Vibrio spAs typical fecal pollution indicators may not access public health risk from potential pathogens such as vibrios, hence special monitoring programme for vibrios may adequately be included in the water quality management.

Prevalence of virulence genes (ctxA, stn, OmpW and tcpA) among non-O1 Vibrio cholerae isolated from fresh water environment.

The virulence of a pathogen is reliant on the presence of a discrete set of genetic determinants and their expression in the host. The virulence of Vibrio spp. is regulated by the ctxAB and tcpA genes. These genes are alleged to be exclusively associated with clinical strains of O1 and O139 serogroups. In the present study, we examined the presence of virulence genes viz. stn, OmpW, ctxA and tcpA of classical and ElTor variants, in environmental strains of non-O1 Vibrio cholerae cultured seasonally from four sampling stations of the river Narmada at Jabalpur (MP), India. Unexpectedly, the PCR analysis of the strains revealed the presence of these genes among environmental V. cholerae. The strains harboring the tcpA gene also carried the ctxA gene. Sequencing of the tcpA gene and ctxA gene carried by an environmental strain showed approximately 97% homology with the previously sequenced genes submitted in the GenBank. We report here the prevalence of cholera toxin gene and the gene for toxin co-regulated pilus among non-O1 V. cholerae strains isolated from fresh water environment. This study supports the idea that cholera toxin has an environmental derivation and that the intricate aquatic environment can give rise to pathogenic Vibrio organisms.