The effects of short and long incubations on DNA fragmentation of testicular

DNA fragmentation in testicular sperm from men with obstructive azoospermia is increased by 4 hr and 24 hr incubations, and after cryopreservation. The effect is intensified by post-thaw incubations. Testicular sperm to be used clinically in ICSI should be injected without delay.

Prevalence of early and late age-related macular degeneration in a rural population in Northern India: The INDEYE Feasibility Study

PURPOSE. To assess the prevalence of age-related macular degeneration (AMD) in a rural population in Northern India. METHODS. In a pilot feasibility study, 1443 people (median age, 60 years; 52% women), were identified from enumeration of the 50+ age group in 11 randomly sampled villages from a rural, periurban district of Haryana, Northern India. Of those identified, 87% attended an eye examination that included digital fundus photography. Fundus images were graded at a single reading center using definitions from the Wisconsin Age-Related Maculopathy Grading System. RESULTS. Fundus photographs were available for 1101 participants. Overall, 28.8% of participants had ungradable fundus images due to cataract. Including all with ungradable images in the denominator, the prevalence of soft drusen was 34.0% (95% confidence interval [CI] 26.1–42.9); of soft indistinct drusen, 2.2% (95% CI, 1.1–4.4); and of pigmentary irregularities, 10.8% (95% CI, 7.1–16.1). There were 15 (1.4%) cases of late-stage AMD (95% CI, 0.8–2.3) with the prevalence rising from 0.4% in the 50- to 59-year age range to 4.6% in those aged 70 years or older. CONCLUSIONS. Drusen and pigmentary irregularities are common among the rural northern Indian population. The prevalence of late AMD is similar to that encountered in Western settings and is likely to contribute significantly to the burden of vision loss in older people in the developing world.

Smooth Muscle Cells Differentiated from Reprogrammed Embryonic Lung Fibroblasts Through DKK3 Signalling are Potent for Tissue Engineering of Vascular Grafts

Rationale: Smooth muscle cells (SMCs) are a key component of tissue-engineered vessels. However, the sources by which they can be isolated are limited.

Objective: We hypothesized that a large number of SMCs could be obtained by direct reprogramming of fibroblasts, that is, direct differentiation of specific cell lineages before the cells reaching the pluripotent state.

Methods and Results: We designed a combined protocol of reprogramming and differentiation of human neonatal lung fibroblasts. Four reprogramming factors (OCT4, SOX2, KLF4, and cMYC) were overexpressed in fibroblasts under reprogramming conditions for 4 days with cells defined as partially-induced pluripotent stem (PiPS) cells. PiPS cells did not form tumors in vivo after subcutaneous transplantation in severe combined immunodeficiency mice and differentiated into SMCs when seeded on collagen IV and maintained in differentiation media. PiPS-SMCs expressed a panel of SMC markers at mRNA and protein levels. Furthermore, the gene dickkopf 3 was found to be involved in the mechanism of PiPS-SMC differentiation. It was revealed that dickkopf 3 transcriptionally regulated SM22 by potentiation of Wnt signaling and interaction with Kremen1. Finally, PiPS-SMCs repopulated decellularized vessel grafts and ultimately gave rise to functional tissue-engineered vessels when combined with previously established PiPS-endothelial cells, leading to increased survival of severe combined immunodeficiency mice after transplantation of the vessel as a vascular graft.

Conclusions: We developed a protocol to generate SMCs from PiPS cells through a dickkopf 3 signaling pathway, useful for generating tissue-engineered vessels. These findings provide a new insight into the mechanisms of SMC differentiation with vast therapeutic potential.

The lipopolysaccharide outer core of Yersinia enterocolitica serotype O:3 is required for virulence and plays a role in outer membrane integrity

Lipopolysaccharide (LPS) of Yersinia enterocolitica O:3 has an inner core linked to both the O-antigen and to an outer core hexasaccharide that forms a branch. The biological role of the outer core was studied using polar and non-polar mutants of the outer core biosynthetic operon. Analysis of O-antigen- and outer core-deficient strains suggested a critical role for the outer core in outer membrane properties relevant in resistance to antimicrobial peptides and permeability to hydrophobic agents, and indirectly relevant in resistance to killing by normal serum. Wild-type bacteria but not outer core mutants killed intragastrically infected mice, and the intravenous lethal dose was approximately 10(4)-fold higher for outer core mutants. After intragastric infection, outer core mutants colonized Peyer's patches and invaded mesenteric lymph nodes, spleen and liver, and induced protective immunity against wild-type bacteria. In mice co-infected intragastrically with an outer core mutant-wild type mixture, both strains colonized Peyer's patches similarly during the first 2 days, but the mutant was much less efficient in colonizing deeper organs and was cleared faster from Peyer's patches. The results demonstrate that outer core is required for Y. enterocolitica O:3 full virulence, and strongly suggest that it provides resistance against defence mechanisms (most probably those involving bactericidal peptides).

Mapping Protein-DNA Interactions Using ChIP-exo and Illumina-Based Sequencing

Chromatin immunoprecipitation (ChIP) provides a means of enriching DNA associated with transcription factors, histone modifications, and indeed any other proteins for which suitably characterized antibodies are available. Over the years, sequence detection has progressed from quantitative real-time PCR and Southern blotting to microarrays (ChIP-chip) and now high-throughput sequencing (ChIP-seq). This progression has vastly increased the sequence coverage and data volumes generated. This in turn has enabled informaticians to predict the identity of multi-protein complexes on DNA based on the overrepresentation of sequence motifs in DNA enriched by ChIP with a single antibody against a single protein. In the course of the development of high-throughput sequencing, little has changed in the ChIP methodology until recently. In the last three years, a number of modifications have been made to the ChIP protocol with the goal of enhancing the sensitivity of the method and further reducing the levels of nonspecific background sequences in ChIPped samples. In this chapter, we provide a brief commentary on these methodological changes and describe a detailed ChIP-exo method able to generate narrower peaks and greater peak coverage from ChIPped material.