Detection and identification of phytoplasma associated with witches’ broom and little leaf disease in Arundo donax: first report from India

During the survey of two successive years 2012–2013, in nearby places of Gorakhpur districts, Uttar Pradesh, India, Arundo donax plants were found to be exhibiting witches’ broom, excessive branching accompanied with little leaf symptoms with considerable disease incidence. Nested PCR carried out with universal primers pair R16F2n/R16R2 employing the PCR (P1/P7) product as a template DNA (1:20) resulted in expected size positive amplification ~1.2 kb in all symptom-bearing plants suggested the association of phytoplasma with witches’ broom disease of Narkat plants. BLASTn analysis of the 16S rRNA gene sequence showed the highest (99%) sequence identity with Candidatus phytoplasma asteris (16SrI group). In phylogenetic analysis, the sequence data showed close relationships with the members of 16SrI phytoplasma and clustered within a single clade of 16SrI group and closed to B subgroup representatives. This is a first report of 16Sr I-B group phytoplasma associated with witches’ broom accompanied with little leaf disease of Narkat in India.     

Haemonchus contortus: The in vitro effects of dl-tetramisole and rafoxanide on glycolytic enzymes

Kaur R. and Sood M. L. 1982. Haemonchus contortus: the in vitro effects of dl-tetramisole and rafoxanide on glycolytic enzymes. International Journal for Parasitology 12: 585–588. Various enzymes of glycolysis (hexokinase, phosphoglucomutase, phosphoglucoisomerase, adolase, glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase, phosphoglyceromutase-enolase-pyruvate kinase and lactate dehydrogenase) have been detected in adult Haemonchus contortus. Low pyruvate kinase and lactate dehydrogenase activities suggested an alternate pathway from phosphoenolpyruvate. In vitro incubation had no significant effects on these enzymes and the worm was able to maintain normal metabolism for 12 h. Varying degrees of inhibition of glycolytic enzymes were observed with 50 μg/ml of dl-tetramisole and rafoxanide. The enzymes were inhibited to a greater extent by dl-tetramisole. These effects may block the glycolytic pathway and deprive the parasite of its ATP production.     

Associations between Disease Awareness and Health-Related Quality of Life in a Multi-Ethnic Asian Population

Background

Health related quality of life (HRQoL) is an important dimension of individuals'' well-being, and especially in chronic diseases like diabetes and hypertension. The objective of this study was to evaluate the contributions of disease process, comorbidities, medication or awareness of the disease to HRQoL in diabetes mellitus, hypertension and dyslipidemia.

Methods

This was a cross-sectional study of 3514 respondents from the general community in Singapore, assessed for HRQoL, disease and comorbid conditions through self-report, clinical and laboratory investigations. HRQoL was assessed using SF-36 health survey version 2. For each condition, participants were categorized as having 1) no disease, 2) undiagnosed, 3) diagnosed, not taking medication, and 4) diagnosed, taking medication. Analysis used one-way ANOVA and multiple linear regression.

Results

Diagnosed disease was associated with lower physical health component summary (PCS) scores across all three conditions. After adjustment for comorbidities, this association remained significant only for those not on medication in diabetes (−2.7±1.2 points, p = 0.03) and dyslipidemia (−1.3±0.4 points, p = 0.003). Diagnosed hypertension (no medication −2.6±0.9 points, p = 0.002; medication −1.4±0.5 points, p = 0.004) and dyslipidemia (no medication −0.9±0.4 points, p = 0.03; medication −1.9±0.5 points, p<0.001) were associated with lower mental health component summary (MCS) scores. Undiagnosed disease was associated with higher MCS in diabetes (2.4±1.0 points, p = 0.01) and dyslipidemia (0.8±0.4 points, p = 0.045), and PCS in hypertension (1.2±0.4 points, p = 0.004).

Conclusions

Disease awareness was associated with lower HRQoL across the diseases studied, with PCS associations partially mediated by comorbidities. Equally importantly, undiagnosed disease was not associated with HRQoL deficits, which may partly explain why these individuals do not seek medical care.     

CRISPR-STAT: an easy and reliable PCR-based method to evaluate target-specific sgRNA activity

CRISPR/Cas9 has emerged as a versatile genome-engineering tool that relies on a single guide RNA (sgRNA) and the Cas9 enzyme for genome editing. Simple, fast and economical methods to generate sgRNAs have made targeted mutagenesis routine in cultured cells, mice, zebrafish and other model systems. Pre-screening of sgRNAs for target efficacy is desirable both for successful mutagenesis and minimizing wasted animal husbandry on targets with poor activity. Here, we describe an easy, quick and cost-effective fluorescent polymerase chain reaction (PCR)-based method, CRISPR Somatic Tissue Activity Test (CRISPR-STAT), to determine target-specific efficiency of sgRNA. As a proof of principle, we validated our method using 28 sgRNAs with known and varied levels of germline transmission efficiency in zebrafish by analysis of their somatic activity in injected embryos. Our data revealed a strong positive correlation between the fluorescent PCR profiles of the injected embryos and the germline transmission efficiency. Furthermore, the assay was sensitive enough to evaluate multiplex gene targeting. This method is easy to implement by laboratories with access to a capillary sequencer. Although we validated the method using CRISPR/Cas9 and zebrafish, it can be applied to other model systems and other genome targeting nucleases.     

Maternal footprints of Southeast Asians in North India

We have analyzed 7,137 samples from 125 different caste, tribal and religious groups of India and 99 samples from three populations of Nepal for the length variation in the COII/tRNA(Lys) region of mtDNA. Samples showing length variation were subjected to detailed phylogenetic analysis based on HVS-I and informative coding region sequence variation. The overall frequencies of the 9-bp deletion and insertion variants in South Asia were 1.9 and 0.6%, respectively. We have also defined a novel deep-rooting haplogroup M43 and identified the rare haplogroup H14 in Indian populations carrying the 9-bp deletion by complete mtDNA sequencing. Moreover, we redefined haplogroup M6 and dissected it into two well-defined subclades. The presence of haplogroups F1 and B5a in Uttar Pradesh suggests minor maternal contribution from Southeast Asia to Northern India. The occurrence of haplogroup F1 in the Nepalese sample implies that Nepal might have served as a bridge for the flow of eastern lineages to India. The presence of R6 in the Nepalese, on the other hand, suggests that the gene flow between India and Nepal has been reciprocal.     

Identification of novel ERK2 substrates through use of an engineered kinase and ATP analogs

The mitogen-activated protein kinases are key regulators of cellular organization and function. To understand the mechanisms(s) by which these ubiquitous kinases affect specific cellular changes, it is necessary to identify their diverse and numerous substrates in different cell contexts and compartments. As a first step in achieving this goal, we engineered a mutant ERK2 in which a bulky amino acid residue in the ATP binding site (glutamine 103) is changed to glycine, allowing this mutant to utilize an analog of ATP (cyclopentyl ATP) that cannot be used by wild-type ERK2 or other cellular kinases. The mutation did not inhibit ERK2 kinase activity or substrate specificity in vitro or in vivo. This method allowed us to detect only ERK2-specific phosphorylations within a mixture of proteins. Using this ERK2 mutant/analog pair to phosphorylate ERK2-associated proteins in COS-1 cells, we identified the ubiquitin ligase EDD (E3 identified by differential display) and the nucleoporin Tpr (translocated promoter region) as two novel substrates of ERK2, in addition to the known ERK2 substrate Rsk1. To further validate the method, we present data that confirm that ERK2 phosphorylates EDD in vitro and in vivo. These results not only identify two novel ERK2 substrates but also provide a framework for the future identification of numerous cellular targets of this important signaling cascade.