Improved dsRNA isolation and purification method validated by viral dsRNA detection using novel primers in Saccharomyces cerevisiae.

Accurate genomic sequencing demands high-quality double-stranded RNA (dsRNA). Existing methods for dsRNA extraction from yeast, fungi, and plants primarily rely on cellulose, suitable only for small volume extractions, or the time-consuming lithium chloride precipitation. To streamline the traditional phenol-chloroform-based dsRNA extraction method, the main challenge is the reduction of mitochondrial DNA (mtDNA) and Single Stranded RNA (ssRNA) to no detectable levels after gel electrophoresis. This challenge is successfully addressed through the modified approach described here, involving phenol extraction at low pH, followed by the addition of ammonium sulfate to the aqueous buffer. The dsRNA isolated using this novel method exhibits comparable quality to that obtained through cellulose purification, and it is readily amenable to RT-PCR. Moreover, a single batch of yeast cell RNA isolation requires only 2-3 h of hands-on time, thus simplifying and expediting the process significantly.•Buffers were redesigned from [32,33,35].•No DNASE, Ribonuclease A or beads were used during the purification.•Simple and inexpensive dsRNA extraction and purification method is described.

Clinico-pathological characteristics and outcome of upper urinary tract urothelial carcinoma presenting to a tertiary care urology unit in Sri Lanka

Introduction: Upper urinary tract urothelial cancers account for 5% of urothelial tumours. In the West, the majority affect the pelvicalyceal system, with pyelocalyceal to ureteric ratio of 3:1. This study aims to describe the clinico-pathological features and outcome of upper urinary tract urothelial cancer treated surgically in a tertiary care unit in Sri Lanka. Methods: A retrospective analysis of all patients who underwent nephroureterectomy for upper urinary tract urothelial cancer at the Urology Unit at National Hospital of Sri Lanka between January1997 and December 2016 was carried out. Results: There were 43 patients. Male: female=1.87. Median age was 65 years (range:42-83). Macroscopic haematuria was the commonest presentation (n=29; 67.4%). Median duration of symptoms was 3 months (range 0.5-6). In the majority (n=20;46.5%) the tumour was confined to the ureter. Thirty-three (75.6%) were papillary tumours. Twenty-one had non-muscle invasive tumours (pTa: n=6(14%), pT1: n=15(34.9%) and others had invasive cancers (pT2: n=11(25.6%), pT3: n=7(16.3%) and pT4: n=4(9.3%)). Majority were low grade tumours (n=23;53.5%). Twelve (27.9%) had preceding urothelial bladder cancer. Nineteen (44.2%) were lost to follow up after surgery. Median follow up duration of the rest was 40 months (range:4-224months). Of them, 9(20.9%) developed metachronous bladder tumours. Nine had recurrence free survival of ≥5years and 15 had overall survival of ≥5 years. Of them, 4 patients survived ≥10 years. Older age (p=0.015) and presence of necrosis(p=0.05) were the only clinico-pathological parameters predictive of tumour recurrence. Conclusions: A relatively higher number females and high number of ureteric tumours were noted compared to similar studies from Asia.

Proteomic analysis of apoplastic fluid of Coffea arabica leaves highlights novel biomarkers for resistance against Hemileia vastatrix.

A proteomic analysis of the apoplastic fluid (APF) of coffee leaves was conducted to investigate the cellular processes associated with incompatible (resistant) and compatible (susceptible) Coffea arabica-Hemileia vastatrix interactions, during the 24-96 hai period. The APF proteins were extracted by leaf vacuum infiltration and protein profiles were obtained by 2-DE. The comparative analysis of the gels revealed 210 polypeptide spots whose volume changed in abundance between samples (control, resistant and susceptible) during the 24-96 hai period. The proteins identified were involved mainly in protein degradation, cell wall metabolism and stress/defense responses, most of them being hydrolases (around 70%), particularly sugar hydrolases and peptidases/proteases. The changes in the APF proteome along the infection process revealed two distinct phases of defense responses, an initial/basal one (24-48 hai) and a late/specific one (72-96 hai). Compared to susceptibility, resistance was associated with a higher number of proteins, which was more evident in the late/specific phase. Proteins involved in the resistance response were mainly, glycohydrolases of the cell wall, serine proteases and pathogen related-like proteins (PR-proteins), suggesting that some of these proteins could be putative candidates for resistant markers of coffee to H. vastatrix. Antibodies were produced against chitinase, pectin methylesterase, serine carboxypeptidase, reticuline oxidase and subtilase and by an immunodetection assay it was observed an increase of these proteins in the resistant sample. With this methodology we have identified proteins that are candidate markers of resistance and that will be useful in coffee breeding programs to assist in the selection of cultivars with resistance to H. vastatrix.