Delivery strategies for CRISPR/Cas genome editing tool for retinal dystrophies: challenges and opportunities.

CRISPR/Cas, an adaptive immune system in bacteria, has been adopted as an efficient and precise tool for site-specific gene editing with potential therapeutic opportunities. It has been explored for a variety of applications, including gene modulation, epigenome editing, diagnosis, mRNA editing, etc. It has found applications in retinal dystrophic conditions including progressive cone and cone-rod dystrophies, congenital stationary night blindness, X-linked juvenile retinoschisis, retinitis pigmentosa, age-related macular degeneration, leber's congenital amaurosis, etc.

Clinical characteristics, hospital course, and disposition of patients with nontraumatic spinal cord injury in a large private health care system in the United States.

Objectives: To evaluate the clinical characteristics, hospital courses, outcomes after hospitalization, and factors associated with outcomes in patients with nontraumatic spinal cord injuries (NTSCI).

Design: Retrospective analysis.

Setting: A large for-profit United States health care system.

Feasibility of opportunistic screening for oral cancers in a dental outpatient department of a secondary care hospital in Northern India.

Background: Oral cancer is one of the most common forms of cancer in India. Majority of the patients are diagnosed at advanced stages, resulting in poor outcomes. Most of the oral cancers are preceded by visible lesions known as potentially malignant disorders (PMDs), which are amenable to early detection by screening.

Effect of intravenous iron sucrose on hemoglobin level, when administered in a standard-dose, to anemic pregnant women in rural Northern India.

Introduction: To combat anemia among pregnant women, in Haryana, Northern India, administration of intravenous iron sucrose (IVIS) was initiated in 2014 as a public-health measure. We assessed the effect of IVIS on hemoglobin (Hb) levels among the pregnant anemic women.

Methods: Pregnant women identified as moderately or severely anemic (Hb <10.

The calcium binding protein EhCaBP6 is a microtubular-end binding protein in Entamoeba histolytica.

The genome of Entamoeba histolytica encodes several calcium binding proteins and those characterized thus far have been shown to participate predominantly in phagocytosis and endocytosis. Our study showed that EhCaBP6 has two EF-hand domains EFI and EFIII; it can bind Ca(2+) in vitro and undergoes conformational transition on binding Ca(2+) suggesting that it can serve as a calcium signal sensor. EhCaBP6 is localized in the nucleus, cytoplasm and plasma membrane and is sensitive to heat stress.

Polyunsaturated fatty acids induce polarized submembranous F-actin aggregates and kill Entamoeba histolytica.

We have recently identified a novel galacto-glycerolipid (GGL) from the plant Oxalis corniculata that killed the human pathogen Entamoeba histolytica. In this study, we show that the anti-amoebic activity of GGL was due to the polyunsaturated fatty acid α-linolenic acid (C18:3 ) side chain. Treatment of α-linolenic acid to E.

Entamoeba histolytica sirtuin EhSir2a deacetylates tubulin and regulates the number of microtubular assemblies during the cell cycle.

We have discovered four sirtuin genes in Entamoeba histolytica, two of which are similar to eukaryotic sirtuins and two to bacterial and archaeal sirtuins. The eukaryotic sirtuin homologue, EhSir2a, showed NAD(+)-dependent deacetylase activity and was sensitive to class III HDAC inhibitors. Localization of EhSir2a at different cellular sites suggested that this deacetylase could have multiple targets.

Inter-cellular variation in DNA content of Entamoeba histolytica originates from temporal and spatial uncoupling of cytokinesis from the nuclear cycle.

Accumulation of multiple copies of the genome in a single nucleus and several nuclei in a single cell has previously been noted in Entamoeba histolytica, contributing to the genetic heterogeneity of this unicellular eukaryote. In this study, we demonstrate that this genetic heterogeneity is an inherent feature of the cell cycle of this organism. Chromosome segregation occurs on a variety of novel microtubular assemblies including multi-polar spindles.

Entamoeba shows reversible variation in ploidy under different growth conditions and between life cycle phases.

Under axenic growth conditions, trophozoites of Entamoeba histolytica contain heterogenous amounts of DNA due to the presence of both multiple nuclei and different amounts of DNA in individual nuclei. In order to establish if the DNA content and the observed heterogeneity is maintained during different growth conditions, we have compared E. histolytica cells growing in xenic and axenic cultures.

Bipolar spindle frequency and genome content are inversely regulated by the activity of two N-type kinesins in Entamoeba histolytica.

Bipolar microtubular spindles are seen infrequently in Entamoeba histolytica trophozoites while monopolar or radial microtubular assemblies are common. Additionally, heterogeneity in nuclear DNA content and multi-nucleation is found in amoeba cells growing in axenic culture. Taken together these observations indicate that genome segregation is irregular in these cells.

Entamoeba histolytica encodes unique formins, a subset of which regulates DNA content and cell division.

The formin family of proteins mediates dynamic changes in actin assembly in eukaryotes, and therefore it is important to understand the function of these proteins in Entamoeba histolytica, where actin forms the major cytoskeletal network. In this study we have identified the formin homologs encoded in the E. histolytica genome based on sequence analysis.

Structure and content of the Entamoeba histolytica genome.

The intestinal parasite Entamoeba histolytica is one of the first protists for which a draft genome sequence has been published. Although the genome is still incomplete, it is unlikely that many genes are missing from the list of those already identified. In this chapter we summarise the features of the genome as they are currently understood and provide previously unpublished analyses of many of the genes.

Genome re-duplication and irregular segregation occur during the cell cycle of Entamoeba histolytica.

Heterogeneity of genome content is commonly observed in axenic cultures of Entamoeba histolytica. Cells with multiple nuclei and nuclei with heterogenous genome contents suggest that regulatory mechanisms that ensure alternation of DNA synthesis and mitosis are absent in this organism. Therefore, several endo-reduplicative cycles may occur without mitosis.

EhRho1, a RhoA-like GTPase of Entamoeba histolytica, is modified by clostridial glucosylating cytotoxins.

Clostridial glucosylating cytotoxins inactivate mammalian Rho GTPases by mono-O glucosylation of a conserved threonine residue located in the switch 1 region of the target protein. Here we report that EhRho1, a RhoA-like GTPase from the protozoan parasite Entamoeba histolytica, is glucosylated by clostridial cytotoxins. Recombinant glutathione S-transferase-EhRho1 and EhRho1 from cell lysate of Entamoeba histolytica were glucosylated by Clostridium difficile toxin B and Clostridium novyi alpha-toxin.

Eh Klp5 is a divergent member of the kinesin 5 family that regulates genome content and microtubular assembly in Entamoeba histolytica.

Earlier studies have established two unusual features in the cell division cycle of Entamoeba histolytica. First, microtubules form a radial assembly instead of a bipolar mitotic spindle, and second, the genome content of E. histolytica cells varied from 1x to 6x or more.

The genome of the protist parasite Entamoeba histolytica.

Entamoeba histolytica is an intestinal parasite and the causative agent of amoebiasis, which is a significant source of morbidity and mortality in developing countries. Here we present the genome of E. histolytica, which reveals a variety of metabolic adaptations shared with two other amitochondrial protist pathogens: Giardia lamblia and Trichomonas vaginalis.

Constitutive association of Mcm2-3-5 proteins with chromatin in Entamoeba histolytica.

Eukaryotic cells duplicate their genome once and only once per cell cycle. Our earlier studies with the protozoan parasite, Entamoeba histolytica, have shown that genome reduplication may occur several times without nuclear or cellular division. The Mcm2-7 protein complex is required for licensing of DNA replication.

Entamoeba histolytica DNA methyltransferase (Ehmeth) is a nuclear matrix protein that binds EhMRS2, a DNA that includes a scaffold/matrix attachment region (S/MAR).

The protozoan parasite Entamoeba histolytica express a cytosine-5 DNA methyltransferase (Ehmeth) that belongs to the DNMT2 protein family. The biological function of members of this DNMT2 family is unknown. In the present study, we have demonstrated that Ehmeth is a nuclear matrix protein.

Inhibition of gene expression with double strand RNA interference in Entamoeba histolytica.

In order to inhibit gene expression in Entamoeba histolytica, we have developed a method based on expressing double strand RNA interference constructs in stable transformants. The 5' end of Eh Dia was cloned head to head with an intervening non-specific stuffer fragment in the E. histolytica expression vector pJST4.

The Iml3 protein of the budding yeast is required for the prevention of precocious sister chromatid separation in meiosis I and for sister chromatid disjunction in meiosis II.

The mitotic kinetochore of the budding yeast contains a number of proteins which are required for chromosome transmission but are non-essential for vegetative growth. We show that one such protein, Iml3, is essential for meiosis, in that the absence of this protein results in reduced spore viability, precocious sister chromatid segregation of artificial and natural chromosomes in meiosis I and chromosome non-disjunction in meiosis II.

Sequence divergence of Entamoeba histolytica tubulin is responsible for its altered tertiary structure.

Atypical microtubular structures of the protozoan parasite Entamoeba histolytica (Eh) have been attributed to amino acid sequence divergence of Eh tubulin. To investigate if this sequence divergence leads to significant differences in the tertiary structure of the Eh alphabeta-tubulin heterodimer, we have modeled alphabeta-tubulin heterodimer of Eh based on the crystal structure of mammalian tubulin. The predicted 3D homology model exhibits an overall resemblance with the known crystal structure of mammalian tubulin except for the 16 residue long carboxy terminal region of Eh beta-tubulin.

The cell cycle of Entamoeba histolytica.

Entamoeba histolytica, is a microaerophilic protist, which causes amoebic dysentery in humans. This unicellular organism proliferates in the human intestine as the motile trophozoite and survives the hostile environment outside the human host as the dormant quadri-nucleate cyst. Lack of organelles--such as mitochondria and Golgi bodies--and an unequal mode of cell division, led to the popular belief, that this organism preceded other eukaryotes during evolution.