Designing, optimization, and validation of whole blood direct T-ARMS PCR for precise and rapid genotyping of complex vertebral malformation in cattle.

BACKGROUND: DNA testing in the cattle industry undergoes multiple hurdles. Successful genotyping involves the transportation of samples from the field to the laboratory in a chilled environment followed by DNA extraction, and finally, a specific genotyping protocol is followed. Various researches are focused on overcoming these issues. Microcards offer blood transportation at ambient temperature. Direct PCR methods can save the time of DNA extraction but available only for simplex PCR. Tetra Primer-Amplification Refractory Mutation System based Polymerase Chain Reaction (T-ARMS PCR) can make DNA testing faster in a low-cost setting. The present study was aimed to design, optimize, and validate a T-ARMS PCR for faster DNA testing of SNP responsible for Complex Vertebral Malformation (CVM)-an important genetic disease of the cattle industry. Further, a direct T-ARMS PCR from whole blood was developed to avoid the DNA extraction steps. Lastly, using the optimized protocol, genotyping of blood spotted on Microcard eliminates the need for cold chain maintenance in the transportation of samples. RESULTS: The present study demonstrated a novel T-ARMS PCR-based genotyping of the SNP rs438228855, which is responsible for CVM. Here, wild genotypes were recognized by 389 bp and 199 bp bands in agarose gel, while the carrier genotype showed an additional 241 bp band. The developed protocol was validated using PCR-Primer Introduced Restriction Analysis (PCR-PIRA) and sequencing. The present study further established a direct T-ARMS PCR for this SNP from whole blood. Different conditions such as heparin and EDTA treated blood, the need for pre-treatment, and two different DNA Polymerases for the direct PCR were optimized. Finally, our optimized protocol successfully genotyped the whole blood samples dried on Insta™DNA cards. CONCLUSIONS: The present study reported the usefulness of primer modified T-ARMS PCR for detecting CVM for the first time. To the best of our knowledge, direct PCR in T-ARMS PCR has never been reported. Lastly, the use of microcards in the developed protocol can make the assay useful in the DNA testing of field samples.

NHS Breast Screening multidisciplinary working group guidelines for the diagnosis and management of breast lesions of uncertain malignant potential on core biopsy (B3 lesions).

Needle core biopsy is considered the histological diagnostic method of choice for screen-detected breast lesions. Although the majority are definitively diagnosed as normal, benign, or malignant, approximately 7% are categorised as B3, of uncertain malignant potential. These include a wide range of lesions with different risks of associated malignancy from <2% to approaching 40% from literature review in UK practice. Historically, these have typically been surgically excised as a diagnostic procedure but the majority are then proven to be benign. An alternative approach, for many of these lesions, is thorough sampling/excision by vacuum-assisted biopsy techniques to exclude the presence of co-existing carcinoma. This would potentially reduce the benign open biopsy rate whilst maintaining accuracy of cancer diagnosis. A group from the Radiology, Surgery, and Pathology NHS Breast Screening Programme Co-ordinating Committees and an additional co-opted expert were charged with review and development of guidelines for the clinical management of B3 lesions. The guidelines reflect suggested practice as stated by the NHS Breast Screening Programme and approved by the Royal College of Radiologists.

Analysis of genetic relatedness among Indian cattle (Bos indicus) using genotyping-by-sequencing markers.

Genetic relatedness of 24 animals belonging to seven Indian cattle breeds was studied using high throughput genotyping-by-sequencing (GBS) markers. GBS produced 93.6 million reads with an average of about 3.9 million reads per animal. A total of 107 488 SNPs were identified in these individuals. When only one SNP per read was considered, a total of 60 261 SNPs representing independent reads were identified with an average SNP-to-SNP distance of 45 kb across the bovine reference genome. About 24% of the GBS-SNP markers were more than 100 kb apart. Of these, 58 322 SNPs mapped to autosomes, 1645 to the X chromosome and 28 to the Y chromosome. The average SNP-to-SNP distance on the X chromosome was 91.3 kb, whereas on the Y chromosome it was 1546.4 kb. The minor allele frequency within the Indian cattle varied from 0.103 (Ongole) to 0.177 (Siri), whereas Holstein cattle had the lowest value of 0.089. This is the first application of GBS in cattle of South Asia. The baseline information generated in this study might prompt implementation of GBS in breeding of cattle belonging to this region.

Functional proteomic analysis of crossbred (Holstein Friesian × Sahiwal) bull spermatozoa.

Male infertility is one of the prime concerns of dairy cattle production. The study was designed to find out differentially expressed proteins in categorized crossbred (Holstein Friesian × Sahiwal) bull semen to serve as potential biomarkers for male infertility. Frozen crossbred bull semen with satisfactory phenotypic records were defined as "good" and "poor" based on their fertility rates. A total of 1,547 proteins were detected in bull spermatozoa using liquid chromatography-mass spectrometer (LC-MS/MS) analysis. Results revealed that 558 (36.1%) and 653 (42.2%) proteins were expressed to good and poor quality bull spermatozoa, respectively. A total of 336 proteins (21.7%) were reported to be unique for both good and poor quality bull semen, and among the common proteins, 224 (66.7%) and 112 (33.3%) were up- and downregulated in good and poor quality categorized bull semen, respectively. Gene Ontology analysis of global proteomes identified different signalling pathways, and most of them were related to cellular motility, immune systems as well as cellular metabolisms. The distinctive presence of some of the proteins may provide an insight into the molecular mechanistic role played by these proteins in crossbred bull infertility.

Association analysis of novel polymorphisms in 2', 5'-oligoadenylate synthetase gene with reproductive traits in indigenous and cross-bred cattle of Indian Origin.

2', 5'-Oligoadenylate synthetases (OAS) are important components of an interferon-mediated antiviral pathway. No polymorphisms in exonic regions of bovine OAS1 gene have been identified and associated with reproduction traits. The objective of the study was to detect and evaluate the effects of mutations in exonic region of bovine OAS1 gene with reproduction traits in cattle. DNA samples collected from 250 individual cows of two Indian dairy breeds (Sahiwal and Frieswal) of cattle were used in the study. The genetic variants of the OAS1 gene were identified with polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) and sequence analysis using seven set of primer pairs. The PCR-SSCP analysis revealed polymorphism in the fragments comprising of exon 2, exon 5 and first fragment of exon 6 while the fragments of exons 1, 3, 4 and second fragment of exon 6 were monomorphic in Sahiwal and Frieswal cattle. The mutations in the amplified region comprising of exon 2 were found to have significant association with age at first breeding and calving, service period, dry period and pregnancy rate. Significant associations were found between SNPs in the exon 5 and service and dry periods of the animal, whereas the genetic variants in the first fragment of the exon 6 showed significant association with age at first breeding and calving. To our knowledge, this study demonstrated for the first time that the polymorphisms in OAS1 gene were associated with reproductive traits and it can be chosen as a candidate gene for improvement of reproductive performance of cattle.

Bovine Circadian Locomotor Output Cycles Kaput (CLOCK) and Clusterin (CLU) mRNA Quantitation in Ejaculated Crossbred Bull Spermatozoa.

Mammalian circadian locomotor output cycles kaput (CLOCK) gene encodes a transcription factor that affects both the persistence and the period of circadian rhythms. Earlier reports suggested that CLOCK gene might be associated with male infertility in human. Present investigation, for the first time, reports that CLOCK gene expresses differentially between good and poor quality crossbred bull semen. The relative expression of CLOCK was significantly (p < 0.05) higher among good quality bull semen than motility-impaired ones. Clusterins (CLU) are series of genes associated with a variety of physiological activities including spermatogenesis, apoptosis and degenerative disease conditions. In the present context, we also investigated that the expression of CLU gene was significantly (p < 0.05) higher among motility-impaired crossbred bull semen compared to the good quality one.

Primary small bowel liposarcoma (atypical lipomatous tumour) with myogenic differentiation.

Primary small intestinal liposarcomas originating in the small bowel are uncommon with a generally poor prognosis due to the advanced stage at the time of diagnosis. We describe a case of primary small bowel dedifferentiated liposarcoma presenting as a solid mass in the right iliac fossa. The current case is unusual as the tumour seemingly originated from the bowel and the well-differentiated component was seen extensively infiltrating the bowel wall including the small bowel submucosa.

Using PCR-PIRA based genotyping for identifying complex vertebral malformation allele in Frieswal young bulls in India.

Complex vertebral malformation (CVM) has considerable economic impact on dairy cattle breeding due to extensive use of artificial insemination (AI). Identifying the carrier is an important factor to reduce the incidence of the genetic disorder. The study was conducted to identify the carriers of CVM in Frieswal cattle by polymerase chain reaction-primer-introduced restriction analysis (PCR-PIRA) method, which was further confirmed by sequencing. Carrier prevalence of 1% was observed in the Frieswal cattle. The results of the study clearly demonstrated the existence of carriers of CVM among Frieswal bull calves. Due to the widespread use of AI it is recommended to screen young bulls at early stages for this defective allele in order to avoid its rapid spread within the population.

Recombinant 20.8-kDa protein of Mycobacterium avium subsp. paratuberculosis-based sero-diagnosis of paratuberculosis.

Johne's disease or paratuberculosis is a chronic infectious enteric disease of ruminants caused by the intracellular pathogen. The control of the Johne's disease is hampered by lack of specific diagnostic tests. In this study, we have cloned and expressed the N-terminal region of the locus tag Map 1637c encoding 20.8-kDa (r20.8) protein of Mycobacterium avium subsp. paratuberculosis. The recombinant protein r20.8 was expressed in high levels in Escherichia coli. The protein r20.8 was purified by single-step chromatography using Ni-NTA agarose. The protein r20.8 was reacted with anti-r20.8 antibodies as well as cattle sera infected with Map on Western blot. ELISA using well-characterized sera (both positive and negative; n = 60 each) Map-infected and non-infected cattle, respectively, yielded a sensitivity of 73.3% and a specificity of 98.3%. The 20.8 kDa protein expressed in the present study will prove useful as reagent in diagnostic test.

LAMP assay for rapid diagnosis of cow DNA in goat milk and meat samples.

Animal species detection is one of the crucial steps for consumer's food analysis. In the present study we developed an in-house built loop-mediated isothermal amplification (LAMP) assay for rapid detection of adulterated cow DNA in goat milk/meat samples. The cow milk/tissue DNA in goat milk/meat samples were identified in the developed LAMP assay by either naked eye visualizing with SYBR Green I dyes or by detecting the typical ladder pattern on gel electrophoresis. This test can detect up to minimum 5% level of cow components admixed in goat milk/meat samples and can be completed within 1 h 40 min starting from DNA extraction from milk/meat samples and can be performed in a water bath. Developed LAMP methodology is simple; rapid and sensitive techniques that can detect adulterant like cow components in goat milk/meat are more accurate than other existing DNA based technologies.

Expression and purification of a gene encoding a 9.7 kDa PE protein of Mycobacterium avium subsp. paratuberculosis.

Mycobacterium avium subsp. paratuberculosis (Map) contains PE family antigens which are Proline and glutamic acid rich and may play important role as T-cell antigens. In the present study, the Map 1507 ORF encoding 9.7 kDa PE protein was amplified by polymerase chain reaction and cloned into E. coli vector pQE30 UA. The recombinant plasmid designated as pQ PE was transformed into E. coli M15 cells and induced with IPTG revealed the high level expression of 11.9 kDa His-fusion protein as estimated by migration in 15 % sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Recombinant PE protein was purified by Ni-NTA agarose chromatography. Polyclonal antibodies raised against purified recombinant PE protein reacted with expressed PE protein as well as with Map sonicate. The recombinant PE protein was also recognized by serum from goat with clinical paratuberculosis. The protein elicited significant delayed type hypersensitivity (DTH) skin reaction in mice sensitized with Map. The results indicated that the recombinant PE protein of Map was associated with T-cell response.

L-myo-lnositol 1-Phosphate Synthase from Plant Sources (Characteristics of the Chloroplastic and Cytosolic Enzymes).

L-myo-inositol 1-phosphate synthase (EC 5.5.1.4) from cyanobacterial (Spirulina platensis), algal (Euglena gracilis), and higher plant (Oryza sativa, Vigna radiata) sources was purified to electrophoretic homogeneity, biochemically characterized, and compared. Both chloroplastic and cytosolic forms of the enzyme were detected in E. gracilis, O. sativa, and V. radiata, whereas only the cytosolic form was detected in streptomycin-bleached or chloroplastic mutants of E. gracilis and in S. platensis. Both the chloroplastic and cytosolic forms from different sources could be purified following the same three-step chromatographic protocol. L-myo-inositol 1-phosphate synthases purified from these different sources do not differ significantly with respect to biochemical and kinetic parameters except for the molecular mass of the chloroplastic and cytosolic native holoenzymes, which appear to be homotetrameric and homotrimeric associations of their constituent subunits, respectively. Monovalent and divalent cations, sugar alcohols, and sugar phosphates are inhibitory to the enzyme activity. N-ethylmaleimide inhibition of synthase activity could be protected by the combined presence of the substrate glucose-6-phosphate and cofactor NAD+. Antibody raised against the cytosolic enzyme from E. gracilis immunoprecipitates and cross-reacts with both chloroplastic and cytosolic forms from the other sources studied.

[Conservative treatment of facial wrinkles in the hands of the plastic surgeon]. / Konservative Faltenbehandlung in der Hand des Plastischen Chirurgen.

The conservative treatment of facial wrinkles is an integral part of the growing market for aesthetic surgery. Unfortunately, physicians of any specialty and even nonphysicians tried to occupy this lucrative field without providing serious information and knowledge about a holistic plastic surgical concept, which includes second- and third-step aesthetic surgical procedures when conservative treatment does not suffice. The following article outlines and critically evaluates the current state of knowledge.

[New developments in skin replacement materials]. / Aktuelle Trends in der Entwicklung von Hautersatzmaterialien.

Current treatment strategies in intensive care medicine permit survival of patients with burns of more than 80% of the total body surface area (TBSA). Major burns result in extensive skin defects. Thus, burn victims often suffer from scar contractures, altered thermoregulation, and unsatisfactory cosmetic results. In addition to the well-established cultivated epithelial autografts, a number of new composite grafts have been developed in the field of tissue engineering. The combination of synthetic and allogenic matrix structures together with an allogenic or autologous epithelium allows the possibility of mimicking skin structure. The aim is to achieve improved wound healing by regeneration of dermal tissue instead of scarring. This article provides an overview of the currently available products which have already been introduced into clinical routine as well as describing advantages and disadvantages of the individual products and their indications.

[Primary treatment of burn patients]. / Die Akutversorgung von Brandverletzten.

Burn injuries can be caused by thermal, electrical, chemical, or mechanical trauma or radiation and are relatively rare, as they represent only about 1% of all emergencies. They are caused by accidents at home, during recreational activities, or in the occupational environment. Minor burn traumas are much more common than severe burn injuries with their systemic and potentially life-threatening effects. Altogether, these circumstances may result in a lack of routine for treating such injuries properly by physicians and their colleagues in the emergency room or intensive care unit. A clearly outlined concept for preclinical and clinical treatment can be the keystone of successful further clinical progress. The following article summarizes the current guidelines for first medical aid at the injury scene, burn stabilization and assessment in the emergency room, and the interdisciplinary approach for further clinical care. The treatment of dermatologic emergencies (acute epidermolytic syndromes) or caustic injuries by chemical agents is similar to the treatment of burn victims in many aspects but must be adapted in selected cases.

[Secondary plastic surgical reconstruction in severely burned patients]. / Die plastisch-chirurgische Sekundärrekonstruktion von Schwerstbrandverletzten.

Secondary reconstruction following severe burn trauma has improved markedly over the last few decades using all aspects of modern plastic surgery. In surgical reconstruction of burns, it is essential to design comprehensive, clear-cut, and long-term treatment plans. Good patient compliance and thorough follow-ups are imperative regarding the extent of scar and contracture formation, regularly requiring multiple-step surgery. Each treatment site will have to be evaluated separately, taking into account adequate surgical and conservative measures (the "reconstructive ladder"). Aiming at realistic and satisfactory results, surgery does not suffice alone in treating severely burned patients but also requires a well coordinated and seasoned team of occupational and physical therapists, psychologists, and plastic surgeons.

Genetic polymorphism and association of kappa-casein gene with milk production traits among Frieswal (HF × Sahiwal) cross breed of Indian origin.

The aim of the present study was to screen the genotype profile of bovine kappa-casein gene among Frieswal (HF × Sahiwal) crossbred cattle developed in India. A total number of two hundred Frieswal cows were evaluated for HinfI RFLP based genotyping of kappa-casein gene. We observed that only two genotypes (AA and AB) exist among the studied population with the genotype frequency of 0.58 (n=117) and 0.42 (n=83), respectively. The calculated allele frequency for A and B was 0.79 and 0.21, respectively. Association of genotypes with certain milk production traits revealed that AB had significant (P<0.05) effect on total milk yield, peak yield, yield at 300 days and SNF% as compared to AA.

Vascular invasion is not a risk factor in oesophageal cancer recurrence.

AIM: The outcome of the treatment of oesophageal cancer remains poor despite improved treatment modalities and recurrence remains a major problem despite improved staging and treatment. The aim was to identify the independent risk factors responsible in the recurrence of oesophageal cancer. METHODS: The patients who had elective oesophagectomy (n = 244) with curative intent were studied. One hundred and eighty four patients had surgery alone, 44 patients had neo-adjuvant chemotherapy and surgery while 16 patients had neo-adjuvant chemotherapy, surgery and adjuvant chemotherapy. We have analyzed patients who had surgery alone (n = 184). Data was collected for demography, type of operation, histology, staging (TNM), vascular invasion (VI), differentiation of tumour, type of chemotherapy and recurrence of tumour. RESULTS: The median age was 67 years (IQR 60, 71). The T1, T2, T3 distribution was 10%, 24% and 66% respectively. Forty percent had no nodal involvement (N0) and 60% had N1 stage disease. Twenty three percent of patients had vascular invasion. Univariate analysis of histo-pathological factors identified lymph node yield (p = 0.06), curative resection R0 (p = 0.004) and vascular invasion (VI) (P = 0.69) as prognostic indicators of recurrence. Multivariate analysis showed that number of lymph nodes yielded (p = 0.01) and R0 resection remain independent indicators of recurrence of tumour. However, VI (p = 0.2) and age at disease onset (p = 0.8) were not indicators of recurrence in oesophageal cancer patients. CONCLUSION: R0 and lymph node yield may help to predict the recurrence of oesophageal cancer. However the presence of VI may not be a significant risk factor in disease recurrence.

MRI in T staging of rectal cancer: How effective is it?

BACKGROUND: Rectal cancer constitutes about one-third of all gastrointestinal (GI) tract tumors. Because of the high recurrence rates (30%) in rectal cancer, it is vitally important to accurately stage these tumours preoperatively so that appropriate surgical resection can be undertaken. MRI is the ideal technique for the preoperative staging of these tumours. AIM: To determine the accuracy of local T staging of rectal cancer with MRI, using histopathological staging as the gold. MATERIALS AND METHODS: Forty consecutive patients admitted with rectal cancer over a period of 18 months were included in this retrospective study. MRI scans were performed prior to surgery in all patients, on 1.5T scanners. Two radiologists, with a special interest in gastrointestinal imaging reported all images. Two dedicated histopathologists reported the histology slides. The accuracy of preoperative local MRI T staging was assessed by comparison with postoperative histopathological staging. RESULTS: There was agreement between MRI and histopathology (TNM) staging in 12 patients (30%). The sensitivity and specificity of MRI for T staging was 89% and 67% respectively. The circumferential resection margin (CRM) status was accurately staged in 94.1% of the patients. CONCLUSIONS: Preoperative staging with MRI is sensitive in identifying CRM involvement, which is the main factor affecting the outcome of surgery.