First report of Mortierella wolfii causing fungal keratitis from a tertiary eye hospital in India

A young 33 year old male presented with non-resolving corneal infiltrate for 2 month duration in the right eye. KOH/ Calcoflour wet mount revealed sparsely septate fungal hyphae. Post therapeutic penetrating keratoplasty 3 doses of intracameral voriconazole(100μg/0.1ml) was administered suspecting recurrence. Fungal culture revealed non sporulating mould on SDA. PCR based DNA sequencing targeting the ITS region identified the fungal isolate as Mortierella wolfii (M. wolfii) belonging to zygomycetes. To the best of our knowledge, this is the first report of human fungal keratitis caused by M. wolfii.     

Creation of engineered human embryonic stem cell lines using phiC31 integrase

It has previously been shown that the phage-derived phiC31 integrase can efficiently target native pseudo-attachment sites in the genome of various species in cultured cells, as well as in vivo. To demonstrate its utility in human embryonic stem cells (hESC), we have created hESC-derived clones containing expression constructs. Variant human embryonic stem cell lines BG01v and SA002 were used to derive lines expressing a green fluorescent protein (GFP) marker under control of either the human Oct4 promoter or the EF1alpha promoter. Stable clones were selected by antibiotic resistance and further characterized. The frequency of integration suggested candidate hot spots in the genome, which were mapped using a plasmid rescue strategy. The pseudo-attP profile in hESC differed from those reported earlier in differentiated cells. Clones derived using this method retained the ability to differentiate into all three germ layers, and fidelity of expression of GFP was verified in differentiation assays. GFP expression driven by the Oct4 promoter recapitulated endogenous Oct4 expression, whereas persistent stable expression of GFP expression driven by the EF1alpha promoter was seen. Our results demonstrate the utility of phiC31 integrase to target pseudo-attP sites in hESC and show that integrase-mediated site-specific integration can efficiently create stably expressing engineered human embryonic stem cell clones.     

MicroRNA expression pattern of undifferentiated and differentiated human embryonic stem cells

Many of the currently established human embryonic stem (hES) cell lines have been characterized extensively in terms of their gene expression profiles and genetic stability in culture. Recent studies have indicated that microRNAs (miRNAs), a class of noncoding small RNAs that participate in the regulation of gene expression, may play a key role in stem cell self-renewal and differentiation. Using both microarrays and quantitative PCR, we report here the differences in miRNA expression between undifferentiated hES cells and their corresponding differentiated cells that underwent differentiation in vitro over a period of 2 weeks. Our results confirm the identity of a signature miRNA profile in pluripotent cells, comprising a small subset of differentially expressed miRNAs in hES cells. Examining both mRNA and miRNA profiles under multiple conditions using cross-correlation, we find clusters of miRNAs grouped with specific, biologically interpretable mRNAs. We identify patterns of expression in the progression from hES cells to differentiated cells that suggest a role for selected miRNAs in maintenance of the undifferentiated, pluripotent state. Profiling of the hES cell "miRNA-ome" provides an insight into molecules that control cellular differentiation and maintenance of the pluripotent state, findings that have broad implications in development, homeostasis, and human disease states.     

Intron 4 VNTR of endothelial nitric oxide synthase (eNOS) gene and diabetic retinopathy in type 2 patients in southern India

A 27-bp variable number tandem repeat (VNTR) in intron 4 of endothelial nitric oxide synthase (eNOS) gene has been associated with the risk for developing diabetic retinopathy (DR) in various ethnic populations. Hundred and eighty seven patients with retinopathy (cases; DR+) and 188 patients without retinopathy (controls: DR-) from southern India who had type 2 diabetes mellitus (T2DM) for more than 10 years, were included in the study. We could neither find significant allelic association with clinical severity of DR nor with macular edema. Our results suggest lack of association of intron 4 VNTR of eNOS gene with DR in southern India.     

Stem cell plasticity

The central dogma in stem cell biology has been that cells isolated from a particular tissue can renew and differentiate into lineages of the tissue it resides in. Several studies have challenged this idea by demonstrating that tissue specific cell have considerable plasticity and can cross-lineage restriction boundary and give rise to cell types of other lineages. However, the lack of a clear definition for plasticity has led to confusion with several reports failing to demonstrate that a single cell can indeed differentiate into multiple lineages at significant levels. Further, differences between results obtained in different labs has cast doubt on some results and several studies still await independent confirmation. In this review, we critically evaluate studies that report stem cell plasticity using three rigid criteria to define stem cell plasticity; differentiation of a single cell into multiple cell lineages, functionality of differentiated cells in vitro and in vivo, robust and persistent engraft of transplanted cells.