Y chromosomal noncoding RNAs regulate autosomal gene expression via piRNAs in mouse testis.

BACKGROUND: Deciphering the functions of Y chromosome in mammals has been slow owing to the presence of repeats. Some of these repeats transcribe coding RNAs, the roles of which have been studied. Functions of the noncoding transcripts from Y chromosomal repeats however, remain unclear. While a majority of the genes expressed during spermatogenesis are autosomal, mice with different deletions of the long arm of the Y chromosome (Yq) were previously also shown to be characterized by subfertility, sterility and sperm abnormalities, suggesting the presence of effectors of spermatogenesis at this location. Here we report a set of novel noncoding RNAs from mouse Yq and explore their connection to some of the autosomal genes expressed in testis. RESULTS: We describe a set of novel mouse male-specific Y long arm (MSYq)-derived long noncoding (lnc) transcripts, named Pirmy and Pirmy-like RNAs. Pirmy shows a large number of splice variants in testis. We also identified Pirmy-like RNAs present in multiple copies at different loci on mouse Y chromosome. Further, we identified eight differentially expressed autosome-encoded sperm proteins in a mutant mouse strain, XYRIIIqdel (2/3 Yq-deleted). Pirmy and Pirmy-like RNAs have homology to 5'/3'UTRs of these deregulated autosomal genes. Several lines of experiments show that these short homologous stretches correspond to piRNAs. Thus, Pirmy and Pirmy-like RNAs act as templates for several piRNAs. In vitro functional assays reveal putative roles for these piRNAs in regulating autosomal genes. CONCLUSIONS: Our study elucidates a set of autosomal genes that are potentially regulated by MSYq-derived piRNAs in mouse testis. Sperm phenotypes from the Yq-deleted mice seem to be similar to that reported in inter-specific male-sterile hybrids. Taken together, this study provides novel insights into possible role of MSYq-derived ncRNAs in male sterility and speciation.

A novel method for immobilization of proteins via entrapment of magnetic nanoparticles through epoxy cross-linking.

A method for immobilization of functional proteins by chemical cross-linking of the protein of interest and uncoated iron oxide nanoparticles in the presence of Epichlorohydrin is described. As a result of the cross-linking, the proteins form a matrix in which the particles get entrapped. The optimum concentration of Epichlorohydrin that facilitates immobilization of protein without affecting the functional properties of the protein was determined. This method was used to immobilize several functional proteins and the development and functional activity of Protein A-magnetic nanoparticles (MNPs) is described here in detail. The Protein A-MNPs possess high binding capacity due to the increased surface area of uncoated nanoparticles and robust magnetic separation due to the absence of polymeric coating materials. Protein A-MNPs were successfully used for purification of antibodies and also for immunoprecipitation. We also immobilized enzymes such as horse radish peroxidase and esterase and found that by providing the optimum incubation time, temperature and protein to nanoparticle ratio, we can retain the activity and improve the stability of the enzyme. This study is the first demonstration that Epichlorohydrin can be used to entrap nanoparticles in a cross-linked matrix of protein without impairing the activity of immobilized protein.

Evaluation of Non-vascular Fibula Graft for Mandibular Reconstruction.

INTRODUCTION: Functional and cosmetic defects in maxillofacial region are caused by various ailments like trauma, neoplasm, developmental, infections and iatrogenic causes. Reconstruction of these defects with free flaps remains the gold standard but demerits like need for surgical expertise and equipment, prolonged duration of surgery, compliance of the patient and increased cost are associated with microvascular reconstruction. Hence reconstruction with nonvascular bone grafts can be considered when defect is nonirradiated and <9 cm and with sufficient soft tissue cover available. PURPOSE: To retrospectively evaluate clinical, radiological outcome and complications encountered with mandibular reconstruction using non vascular fibula graft. PATIENTS AND METHODS: This retrospective study included 7 patients who were treated in the Department of Oral and Maxillofacial Surgery, Narayana Dental College and Hospital, Nellore, AP between 2011 and 2013 with histologically proven benign osteolytic lesions of mandible that require a segmental mandibulectomy and primary reconstruction using autogenous non-vascularised fibular graft. The clinical case records of the patients and personal patient assessment forms (Quality of Life Assessment Forms) were analysed. They were recalled every 3rd, 6th and 9th month after surgery for evaluation of clinical, radiological outcome of the graft and complications occurring at recipient and donor sites. RESULTS: In all the 7 patients, the lower border continuity was maintained except in one where the graft was dislodged. Tongue movements in all the patients were unrestricted. Jaw movements were affected in cases of ramus defects with slight deviation to operated side and reduced mouth opening. Radiological observations revealed no significant changes in 3 months except for slight reduction in graft height. The radioopaque bridging with continuity of lower border of mandible was noticed in 6th month indicating the take of the graft. This was achieved in every case except in one where the graft was lost due to dislodged reconstruction plate. In 9th month the edges of the graft i.e., graft to native mandible junction showed more resorption (3 mm) especially where there is >2 mm of gap. Whereas increase in height of graft in other areas especially in graft to graft junction was seen. Significant graft resorption was seen in two cases. There were no major complications associated with the donor site. CONCLUSION: Avascular fibula graft although a second choice to vascularised fibula, is a favourable option for mandible defects of 6-10 cm under optimum conditions especially in developing countries where financial and/or surgical resources are limited. An attempt for primary reconstruction with this is never futile as it prevents aesthetic deformity even in the event of failure and thus makes secondary reconstruction easy. However in order to confirm the results a prospective study with large scale of patients is necessary.

yciM is an essential gene required for regulation of lipopolysaccharide synthesis in Escherichia coli.

The outer membrane of Gram-negative bacteria is an asymmetric lipid bilayer consisting of an essential glycolipid lipopolysaccharide (LPS) in its outer leaflet and phospholipids in the inner leaflet. Here, we show that yciM, a gene encoding a tetratricopeptide repeat protein of unknown function, modulates LPS levels by negatively regulating the biosynthesis of lipid A, an essential constituent of LPS. Inactivation of yciM resulted in high LPS levels and cell death in Escherichia coli; recessive mutations in lpxA, lpxC or lpxD that lower the synthesis of lipid A, or a gain of function mutation in fabZ that increases the formation of membrane phospholipids, alleviated the yciM mutant phenotypes. A modest increase in YciM led to significant reduction of LPS and increased sensitivity to hydrophobic antibiotics. YciM was shown to regulate LPS by altering LpxC, an enzyme that catalyses the first committed step of lipid A biosynthesis. Regulation of LpxC by YciM was contingent on the presence of FtsH, an essential membrane-anchored protease known to degrade LpxC, suggesting that FtsH and YciM act in concert to regulate synthesis of lipid A. In summary, this study demonstrates an essential role for YciM in regulation of LPS biosynthesis in E. coli.

Rhodestrin: a novel indole terpenoid phytohormone from Rhodobacter sphaeroides.

A new phytohormone was isolated as a metabolite of anthranilate photobiotransformation by Rhodobacter sphaeroides OU5. It was identified by MS and NMR ((1)H, (13)C) as an indole terpenoid ester [(2E,4E,6E,8E,10E,12E,14E,16E,18E)24-hydroxy-2,6,10,14,19 pentamethyl tetrecosa-2,4,6,8,10,12,14,16,18 nonenyl-2(hydroxy methyl)-1H-indole-3-carboxylate] and is named as rhodestrin. Rhodestrin at 50 nM: gave positive test in auxin bioassay and initiated more profuse and early rooting in tissue-cultured plants than other auxins at 5 microM.

Production of a novel indole ester from 2-aminobenzoate by Rhodobacter sphaeroides OU5.

Culture supernatants of Rhodobacter sphaeroides OU5 grown in the presence of 2-aminobenzoate gave an orange-red color-reaction with Salper's reagent, suggesting the presence of an indole derivative. This production was light-dependent and inducible only with 2-aminobenzoate. Replacement of 2-aminobenzoate with other 2-substituted benzoates did not result in the formation of indole. Fumarate appeared to be the conjugating molecule with 2-aminobenzoate, resulting in the formation of an indole derivative. The purified indole derivative was orange-brown in color, with a yields 0.34 mM from 1 mM 2-aminobenzoate. Infrared analysis suggested an indole ester and (1)H NMR analysis indicated an indole carboxylate, esterified with a terpenoid alcohol. The indole ester has a mass of 441 with the molecular formula C(27)H(39)NO(4). The IUPAC name of the compound is (3 E,5 E)-14-hydroxy-3,7,11-trimethyl-3,5-tetradecadienyl 2-(hydroxymethyl)-1 H-indole-3-carboxylate; and the common name given to this compound is sphestrin.