Current scenario and challenges ahead in application of spermatogonial stem cell technology in livestock

Journal of Assisted Reproduction and Genetics - Spermatogonial stem cells (SSCs) are the source for the mature male gamete. SSC technology in humans is mainly focusing on preserving fertility in...     

Evaluation of three analyte-specific reagents for detection and typing of herpes simplex virus in cerebrospinal fluid

The performance of three analyte-specific reagents (ASRs); Cepheid herpes simplex virus (HSV) Typing Primer Probe set (CD), Eragen MultiCode-Rtx HSV-1/2 kit primer mix (ER), and Roche LightCycler HSV-1/2 Primer/Hybridization Probes (RD), was evaluated for detection and typing of herpes simplex virus (HSV-1 and HSV-2) in cerebrospinal fluid (CSF) specimens. Of 68 CSF specimens, HSV-1 was detected in 8 specimens and HSV-2 was detected in 17 specimens. ER detected all 25 HSV-positive specimens, whereas CD and RD detected 24 and 23 HSV-positive specimens, respectively. The results of HSV typing with the 3 ASRs were in complete agreement. The analytical sensitivity of all ASRs was determined to be about 10¹ copies/reaction. Our results demonstrate that the performances of all 3 ASRs are comparable and reliable for routine clinical testing in detection and typing of HSV DNA in CSF.     

A new method for species identification and differentiation of Mycobacterium chelonae complex based on amplified hsp65 restriction analysis (AHSPRA)

Members of the Mycobacterium chelonae complex (MCC), namely M. chelonae, Mycobacterium abscessus and Mycobacterium immunogenum, have been implicated in nosocomial infections and occupational respiratory illnesses like hypersensitivity pneumonitis (HP) associated with contaminated metalworking fluid (MWF) exposures. Close relationship among these member species makes their differentiation cumbersome using the existing methods. Here we report a simple and rapid method for unambiguous identification and differentiation of the three-member species of the MCC group with PCR-restriction analysis targeting a 667-bp segment of a variable region of the 65-kDa-heat shock protein (hsp65) gene. This assay, described as Amplified hsp65 Restriction Analysis (AHSPRA), can discriminate all the three individual species using a one-step restriction digestion using either BbvI or Eco0109I. The enzyme NarI can differentiate M. immunogenum from the other two MCC species (M. chelonae and M. abscessus). The developed method was validated using several non-MCC reference species of other rapidly growing mycobacteria (RGM) and MCC field isolates from MWF samples. Direct cell-lysis was used instead of the conventional DNA template preparation, which improved the rapidity, simplicity and adaptability of the developed method. The results suggest that the developed method can unambiguously differentiate species of the M. chelonae complex from other RGM species and from one another.     

The tyrosine phosphatase PTPRD is a tumor suppressor that is frequently inactivated and mutated in glioblastoma and other human cancers

Tyrosine phosphorylation plays a critical role in regulating cellular function and is a central feature in signaling cascades involved in oncogenesis. The regulation of tyrosine phosphorylation is coordinately controlled by kinases and phosphatases (PTPs). Whereas activation of tyrosine kinases has been shown to play vital roles in tumor development, the role of PTPs is much less well defined. Here, we show that the receptor protein tyrosine phosphatase delta (PTPRD) is frequently inactivated in glioblastoma multiforme (GBM), a deadly primary neoplasm of the brain. PTPRD is a target of deletion in GBM, often via focal intragenic loss. In GBM tumors that do not possess deletions in PTPRD, the gene is frequently subject to cancer-specific epigenetic silencing via promoter CpG island hypermethylation (37%). Sequencing of the PTPRD gene in GBM and other primary human tumors revealed that the gene is mutated in 6% of GBMs, 13% of head and neck squamous cell carcinomas, and in 9% of lung cancers. These mutations were deleterious. In total, PTPRD inactivation occurs in >50% of GBM tumors, and loss of expression predicts for poor prognosis in glioma patients. Wild-type PTPRD inhibits the growth of GBM and other tumor cells, an effect not observed with PTPRD alleles harboring cancer-specific mutations. Human astrocytes lacking PTPRD exhibited increased growth. PTPRD was found to dephosphorylate the oncoprotein STAT3. These results implicate PTPRD as a tumor suppressor on chromosome 9p that is involved in the development of GBMs and multiple human cancers.     

Perianal condylomata acuminata in a male child

A one year old male child presented with a perianal growth of two months' duration. Clinical and histopathological findings confirmed the diagnosis of condylomata acuminata, which regressed completely following topical treatment with podophyllin.     

Evaluation of maize grain and polyunsaturated fatty acid (PUFA) as energy sources for breeding rams based on hormonal, sperm functional parameters and fertility

The objective of the present study was to elucidate the effect of different sources of dietary energy (maize vs polyunsaturated fatty acid (PUFA) on semen functional parameters and fertility of adult rams. Eighteen adult rams were divided into two groups (maize and PUFA, n=9). The main energy source for the rams in the maize group was coarsely ground maize grain, whereas in the PUFA group it was sunflower oil (rich in 18:2 linoleic acid, an omega-6 acid). The ration was fed for a minimum period of 60 days and thereafter semen was collected for evaluation. The proportion of progressive forward motility was significantly (P<0.05) higher in the PUFA group compared with the maize group. Sperm lipid peroxidation as measured by malondialdehyde formation (μM per 1×10(9) spermatozoa) was significantly (P<0.05) higher in the PUFA group compared with the maize group. When the semen was diluted with Tris-egg yolk-citrate buffer and incubated for 24h at 4°C, the proportions of plasmalemma integrity, the sperm subpopulation positive for functional membrane and acrosomal integrities, and mitochondrial membrane potential were significantly (P<0.05) higher in PUFA-fed than in maize-fed animals. The different sources of energy did not influence the serum and seminal plasma IGF-I levels. The cleavage rate (percentage) did not differ significantly between PUFA- (45.4±4.91) and maize- (44.63±6.8) fed animals. In conclusion, PUFA feeding influenced sperm quality by altering or stabilising membrane integrity. The present study indicates that PUFA may improve semen quality but did not improve in vitro fertilisation.     

5-Fluoro-[β-11C]-L-tryptophan is a functional analogue of 5-hydroxy-[β-11C]-L-tryptophan in vitro but not in vivo

Introduction5-Hydroxy-[β-¹¹C]-L-tryptophan ([¹¹C]HTP) is an established positron emission tomography (PET) imaging agent for neuroendocrine tumors (NETs). It has also been used for other clinical research purposes in neurology and diabetes. However, its widespread use is limited by the short physical half-life of the radionuclide and a difficult radiosynthesis. Therefore, a Fluorine-18 labeled analogue, 5-[¹⁸F]Fluoro-L-tryptophan ([¹⁸F]FTRP), has been proposed as a functional analogue. There is no published method for the synthesis of L-[¹⁸F]FTRP. We have therefore developed a synthesis of 5-fluoro-[β-¹¹C]-L-tryptophan ([¹¹C]FTRP), based on the existing chemo-enzymatic method for [¹¹C]HTP and evaluated the potential usefulness of radiolabeled FTRP as a substitute for [¹¹C]HTP.MethodsThe in vitro and in vivo behavior of [¹¹C]FTRP, including the dependence of key enzymes in the serotonergic metabolic pathway, was investigated in NET cell lines, NET xenograft carrying immunodeficient mice, normal rats and in non-human primate. [¹¹C]HTP was used for direct comparison.ResultsUptake of [¹¹C]FTRP in NET cell lines in vitro was mediated by enzymes involved in serotonin synthesis and metabolism, similar to [¹¹C]HTP. In vivo biodistribution, either in rodent or non-human primate, was not affected by selectively inhibiting enzymatic steps in the serotonergic metabolic pathway.Conclusion[¹¹C]FTRP has in vitro biological function similar to that of [¹¹C]HTP. However, this function is not retained in vivo as shown by biodistribution and PET/CT studies. Radiolabeled FTRP is thus not likely to provide an advantage over [¹¹C]HTP in PET imaging in oncology, neurology or diabetes.     

Adenoid cystic carcinoma of nose

Adenoid cystic carcinoma in the nose is very rare. This tumour is generally considered to be radioresistant. A case of adenoid cystic carcinoma of the nose treated successfully by primary irradiation is reported here.     

Mutation analysis of the cathepsin C gene in Indian families with Papillon-Lefèvre syndrome

Background

PLS is a rare autosomal recessive disorder characterized by early onset periodontopathia and palmar plantar keratosis. PLS is caused by mutations in the cathepsin C (CTSC) gene. Dipeptidyl-peptidase I encoded by the CTSC gene removes dipeptides from the amino-terminus of protein substrates and mainly plays an immune and inflammatory role. Several mutations have been reported in this gene in patients from several ethnic groups. We report here mutation analysis of the CTSC gene in three Indian families with PLS.

Methods

Peripheral blood samples were obtained from individuals belonging to three Indian families with PLS for genomic DNA isolation. Exon-specific intronic primers were used to amplify DNA samples from individuals. PCR products were subsequently sequenced to detect mutations. PCR-SCCP and ASOH analyses were used to determine if mutations were present in normal control individuals.

Results

All patients from three families had a classic PLS phenotype, which included palmoplantar keratosis and early-onset severe periodontitis. Sequence analysis of the CTSC gene showed three novel nonsense mutations (viz., p.Q49X, p.Q69X and p.Y304X) in homozygous state in affected individuals from these Indian families.

Conclusions

This study reported three novel nonsense mutations in three Indian families. These novel nonsense mutations are predicted to produce truncated dipeptidyl-peptidase I causing PLS phenotype in these families. A review of the literature along with three novel mutations reported here showed that the total number of mutations in the CTSC gene described to date is 41 with 17 mutations being located in exon 7.