Molecular characterization, cloning and sequencing of coat protein gene of a Pakistani potato leaf roll virus isolate and its phylogenetic analysis

Total RNA was extracted from potato leaf roll virus (PLRV) positive potato plants and complementary DNA were synthesized. Reverse transcriptase polymerase chain reaction (RT-PCR) based detection conditions were optimized by using coat protein (CP) gene specific primers. A 346 bp amplicon of PLRV- coat protein (CP) gene was amplified. Amplified CP-gene of PLRV was cloned in TA cloning vector, PCR ® 2.1. The clone was confirmed subsequently through restriction digestion analysis. PCR amplification used cDNA clone as a template and nucleotide sequencing. Expected nucleotide sequence of amplified PLRV-CP gene show homology of 94 to 97% when compared to the sequences already reported in GenBank database. This explored novel PLRV-CP gene was submitted at NCBI GenBank for characterization of PLRV Pakistani isolate (Accession No. JN039286). Phylogenetic analysis was also carried out and tree was made by using MEGA 4.0.Key words: Potato, potato leaf roll virus (PLRV), coat protein (CP) gene, reverse transcriptase polymerase chain reaction (RT-PCR), PLRV-CP Pakistani isolate

In-vitro Production of Cabbage and Cauliflower

Background: An efficient method of artificial mass propagation was optimized for two very nutritious vegetables of Pakistan, the cabbage and cauliflower. Being an agrarian economy, Pakistan’s more than half of the population depends directly or indirectly on agricultural products.Methodology: Hypocotyls of germinating of seeds (5-7 days old seedlings) were used as explants. Murashige and Skoog basal medium was supplemented with different concentrations of auxin (2,4 Dichlorophenoxy acetic  acid) and cytokinin (benzyl amino purine) in combination to study the callus forming tendency of cabbage and cauliflower; and found better if used in ratio of 2:1 (at least) respectively.Results: For evaluation of regeneration potential Kinetin, Zeatin, Gibrellic acid-3, and Indole acetic acid were found good in combination with benzyl amino purine. Higher (than auxin) concentration of cytokinin was found essential to obtain good regeneration response of callus.Conclusion:  Effectively concluded that these techniques can be used to raise the disease free stock of cabbage and cauliflower for genetic improvement of in-vitro bulk of varieties on commercial scale in a very brief time span

Errors in surgical pathology reports: a study from a major center in Pakistan

Background: Errors in surgical pathology diagnosis can have serious consequences for the patient. Since the final product of a surgical pathology lab is the report, errors can be picked by reviewing reports of cases.AIM: To determine the frequency and types of error in surgical pathology reports of cases signed out in 2014 in a laboratory in Karachi, Pakistan. Material and Methods: All surgical pathology reports in which changes were made in the original report after sign out and an amended report was issued were included. Errors included: (1) misinterpretations; (2) missing critical information; (3) erroneous critical information; (4) misidentification; and (5) typographic errors. Results: Errors were identified in 210 cases (0.37%). These comprised 199 formalin fixed specimens and 11 frozen sections. The latter represented 3.8% of a total of 2,170 frozen sections. Of the 11 frozen section errors, 10 were misinterpretations. Of the 199 permanent specimens, 99 (49.7%) were misinterpretations, 65 (32.7%) belonged to missing critical information category, 8 (4%) belonged to erroneous critical information category, 8(4%) were misidentifications, 16(8%) were typographic errors while 3 cases (1.5%) were other errors. Most misinterpretations occurred in the gastro intestinal, liver and pancreato biliary tract (23.2%) and breast (13.1%). Another 87 cases were reviewed on the clinicians\u27 request. However diagnosis after review remained the same as the original diagnosis. In 49 out of these (56.3%), additional workup was performed at the time of the review.CONCLUSIONS: Our findings were similar to other published studies. We need to develop documented procedures for timely review of cases to detect errors

Gene Profiling for Invertase Activity: Assessment of Potato Varieties for Resistance towards Cold Induced Sweetening

Background: Potato is the most important staple food in the world. Cold-induced sweetening occurs when potatoes are stored at low temperature for longer period of time. Due to non- enzymatic Millard reaction it causes unwanted changes in colour, taste and in flavor when fried and roasted at high temperature. However, long-term cold storage is mandatory to keep an adequate supply of potatoes throughout the year. The cause of cold-induced sweetening is invertase enzyme. Methods: Five potato varieties (Hermes (A) Lady Rosetta (B) Oscar (C) Kuroda (D) and Multa (E))   were investigated for invertase activity during two month cold storage at 4°C. Crude protein was extracted by PD Midi Trap G25 column technique. Quantification of mRNA expression was employed through QPCR. Determination of sucrose, reducing sugars and organic acids was simply done by 80% ethanol method and concentration were find out by using HPLC with already set standards. The correlation between invertase enzyme, sugar content and mRNA expression was calculated through Statistical methods.Results: Significant activity of invertase was observed at 4ºC with up to 6.3 nmol/min/mg of protein in the type-1 & 4 (cv. Hermes and Kuroda); 2.5 times less in type 2 (Rosetta) and 3.5 times less in type 3 (Multa) when compared with same at 4ºC. In addition, malic acid concentration was found positively correlated with invertase activity at 4ºC as compared with its concentration at harvesting time. However, citric acid and oxalic acid concentrations were independent of invertase enzyme activity. The transcript level of invertase enzyme was found significantly high in potato tubers stored at 4ºC in result 1 & 4 type, less in result type 2(C) and negligible in result 3(E) potato variety when revealed through reverse transcription PCR.Conclusions: In conclusion, Oscar (C) and Multa (E) were found more resistant to CIS at 4ºC storage and may be used for future variety improvement programs for CIS resistant through breeding and molecular approaches

Evaluation of two cotton varieties CRSP1 and CRSP2 for genetic transformation efficiency, expression of transgenes Cry1Ac+Cry2A, GT gene and insect mortality

AbstractExpression of the transgene with a desirable character in crop plant is the ultimate goal of transgenic research. Transformation of two Bt genes namely Cry1Ac and Cry2A cloned as separate cassette under 35S promoter in pKHG4 plant expression vector was done by using shoot apex cut method of Agrobacterium. Molecular confirmation of putative transgenic cotton plants for Cry1Ac, Cry2A and GT gene was done through PCR and ELISA. Transformation efficiency of CRSP-1 and CRSP-2 was calculated to be 1.2 and 0.8% for Cry1Ac while 0.9 and 0.6% for Cry2A and 1.5 and 0.7% for GTG respectively. CRSP-1 was found to adopt natural environment (acclimatized) earlier than CRSP-2 when exposed to sunlight for one month. Expression of Cry1Ac, Cry2A and GTG was found to be 1.2, 1 and 1.3ng/μl respectively for CRSP-1 as compared to CRSP-2 where expression was recorded to be 0.9, 0.5 and 0.9ng/μl respectively. FISH analysis of the transgenic CRSP-1 and CRSP-2 demonstrated the presence of one and two copy numbers respectively. Similarly, the response of CRSP-1 against Glyphosate @1900ml/acre was far better with almost negligible necrotic spot and efficient growth after spray as compared to CRSP-2 where some plants were found to have necrosis and negative control where the complete decay of plant was observed after seven days of spray assay. Similarly, almost 100% mortality of 2nd instar larvae of Heliothis armigera was recorded after three days in CRSP-1 as compared CRSP-2 where insect mortality was found to be less than 90%. Quantitatively speaking non transgenic plants were found with 23–90% leaf damage by insect, while CRSP-1 was with less than 5% and CRSP-2 with 17%. Taken together CRSP1 was found to have better insect control and weedicide resistance along with its natural ability of genetic modification and can be employed by the valuable farmers for better insect control and simultaneously for better production

Gastrointestinal stromal tumors: A clinicopathologic and risk stratification study of 255 cases from Pakistan and Review of Literature

Purpose: To describe the clinicopathological features of gastrointestinal stromal tumors (GIST) diagnosed in our section and to perform risk stratification of our cases by assigning them to specific risk categories and groups for disease progression based on proposals by Fletcher et al and Miettinen and Lasota. Material and Results: We retrieved 255 cases of GIST diagnosed between 2003 and 2014. Over 59% were male. The age range was 16 to 83 years with a mean of 51 years. Over 70% occurred between 40 and 70 years of age. Average diameter of tumors was 10 cms. The stomach was the most common site accounting for about 40%. EGISTs constituted about 16%. On histologic examination, spindle cell morphology was seen in almost of 85% cases. CD117 was the most useful immunohistochemical antibody, positive in 98%. Risk stratification was possible for 220 cases. Based on Fletcher\u27s consensus proposal, 62.3 gastric, 81.8% duodenal, 68% small intestinal, 72% colorectal and 89% EGISTs were assigned to the high risk category; while based on Miettinen and Lasota\u27s algorithm, about 48% gastric, 100% duodenal, 76% small intestinal, 100% colorectal and 100% EGISTs in our study were associated with high risk for disease progression, tumor metastasis and tumor related death. Follow up was available in 95 patients; 26 were dead and 69 alive at follow up. Most of the patients who died had high risk disease and on average death occurred just a few months to a maximum of one to two years after initial surgical resection. Conculsion: Epidemiological and morphologic findings in our study were similar to international published data. The majority of cases in our study belonged to the high risk category

Lymphangioma circumscriptum: clinicopathological spectrum of 29 cases

Objective: To describe the clinicopathological spectrum of Lymphangioma Circumscriptum (LC).Study Design: Observational case series.Place and Duration of Study: Department of Pathology and Microbiology, AKUH, Karachi, from 2002 to 2012.Methodology: All reported cases of LC were retrieved from medical record. Clinical and pathological features were noted. Frequency percentages were determined.Results: There were 29 cases of LC predominantly males (62%). The mean age was 27.17 ±15.5 years. The commonest sites was anal/perianal region (24%) followed by extremities (17%) and tongue, (14%). Vulval LC was seen in 3 patients. Two cases were described on scrotum. The lesions were most commonly suspected as viral warts, mole or polyp (in anal region). Vesicles with erosions and bleeding and localized growth were the usual clinical presentations. Four of the patients presented with swelling since birth. All were treated with surgical excision. Microscopic examination revealed acanthotic squamous epithelium with papillomatosis. The subepithelial region had collections of lymphatic channels composed of ectatic dilated vessels with serum and inflammatory cells in their lumina. The lymphatic channels were seen in deeper layers along with lymphocytic aggregates.Conclusion: Lymphangioma circumscriptum is a malformation of abnormal lymphatic channels with feeding cisterns in subcutaneous tissue. It is a benign lesion usually occurring in anal/perianal region and confused with warts. Surgical excision is preferred mode of treatment

Central, extraventricular and atypical neurocytomas: a clinicopathologic study of 35 cases from Pakistan plus a detailed review of the published literature

Background: Central neurocytomas are rare neuronal neoplasms with a favorable prognosis. They are typically located in the lateral ventricles of the brain and mostly histologically correspond to WHO grade II with a Mib 1 labelling index of 2 % and are designated as atypical neurocytomas.AIM: The aim of our study was to describe the common as well as unusual morphologic features and the role of various immunohistochemical stains in the diagnosis of these rare tumors.MATERIALS AND Methods: We retrieved and reviewed 35 cases diagnosed between 2001 and 2015.Results: Sixty percent of patients were males, and the mean age was 26 years. 31 cases (88.6%) were intraventricular and 4(11.4%) were extraventricular. Histologically, 6 cases (17.1%) were compatible with atypical neurocytomas . All cases showed the classic morphology comprising nests and sheets of uniform, round cells with uniform round to oval nuclei with finely speckled chromatin and perinuclear cytoplasmic clearing (halos). All cases also showed delicate, fibrillary, neuropil-like matrices. Other common histologic features included capillary-sized blood vessels in a branching pattern in 57.1%, foci of calcification in 34.3% and perivascular pseudorosettes in 20%. Rare findings included Homer- Wright or true rosettes in 8.6% and ganglioid cells in 2.9%. Synaptophysin was the most consistent and valuable marker, being positive in almost all cases. GFAP positivity in tumor cells was seen in 25.7% of cases. Follow up was available in 13 patients. Of these 9 had histologically typical and 4 had atypical tumors. Only 1 (with an atypical neurocytoma) died, probably due to complications of surgery within one month, while 12 (including 3 with atypical neurocytomas) remained alive. Recurrence developed in 1 of these 12 patients (histologically consistent with typical morphology) almost 9 years after surgery. Only 4 patients, including 2 with atypical tumors, received postoperative radiotherapy, all with surgery in 2010 or later. Overall, prognosis was excellent with prolonged, recurrence free survival and most patients, even without receiving radiation therapy, were alive and well for many years, even a decade or more after surgery, without developing any recurrence, indicating the benign nature of these neoplasms

Micro Propagation in Advanced Vegetable Production: A Review

Micro propagation is a fast method of plant propagation that has a great potential to develop high quality as well as disease-free plants. Advancements in this field have led to the development of several techniques for rapid multiplication and improvement of a wide range of horticultural crops and their production systems. Micro propagation includes three types of vegetative propagation, 1) somatic embryogenesis, 2) adventitious shoot production and 3) axillary shoot production, which provide excellent opportunities for successful vegetable crops production. In Capsicum genera, annuum-chinense-frutescens complex have been made with white flowers and small yellow seeds. A number of experiments have also been done to demonstrate the influence of hypocotyl explant orientation on shoot bud induction in Capsicum spp.  Sweet potato cuttings when grown in vitro with Florialite gives greater percentage of survival. Similarly, in vitro layering of gourds; a modification in micro propagation methods become more advantageous as it produces single shoot rather than multiple shoots. Likewise, an immobilized culture system of obtaining torpedo-stage embryos of carrots of uniform size and higher tuber yield with rapid multiplication rate for potato were also introduced by micro propagation techniques.