Development of a sensitive molecular diagnostic assay for detecting Borrelia burgdorferi DNA from the blood of Lyme disease patients by digital PCR.

Lyme disease patients would greatly benefit from a timely, sensitive, and specific molecular diagnostic test that can detect the causal agent Borrelia burgdorferi at the onset of symptoms. Currently available diagnostic methods recommended by the Centers for Disease Control and Prevention for Lyme disease involve indirect serological tests that rely on the detection of a host-antibody response, which often takes more than three weeks to develop. With this process, many positive cases are not detected within a timely manner, preventing a complete cure. In this study, we have developed a digital polymerase chain reaction (PCR) assay that detects Lyme disease on clinical presentation with a sensitivity two-fold higher than that of the currently available diagnostic methods, using a cohort of patient samples collected from the Lyme disease endemic state of Connecticut, USA, in 2016-2018. Digital PCR technology was chosen as it is more advanced and sensitive than other PCR techniques in detecting rare targets. The analytical detection sensitivity of this diagnostic assay is approximately three genome copies of B. burgdorferi. The paucity of spirochetes in the bloodstream of Lyme disease patients has hindered the clinical adoption of PCR-based diagnostic tests. However, this drawback was overcome by using a comparatively larger sample volume, applying pre-analytical processing to the blood samples, and implementing a pre-amplification step to enrich for B. burgdorferi-specific gene targets before the patient samples are analyzed via digital PCR technology. Pre-analytical processing of blood samples from acute patients revealed that the best sample type for Lyme disease detection is platelet-rich plasma rather than whole blood. If detected in a timely manner, Lyme disease can be completely cured, thus limiting antibiotic overuse and associated morbidities.

Prevailing Practices for the Management of Dry Cough in India: A Questionnaire Based Survey.

PURPOSE: The aim of the survey was to understand the prevailing practice pattern for the management of dry cough among primary care physicians in Indian clinical setting. MATERIAL AND METHODS: This single visit, cross-sectional, non-interventional, interview based physician survey was conducted over a period of 3 months where 500 registered physicians with at least 6 months of clinical practice and willing to participate in the survey were interviewed in their clinic or hospital from June to August 2015. They were asked to complete a structured questionnaire, consisting of 28 questions, regarding their routine clinical practice ranging from patient demographics to etiology to treatment modalities for management of dry cough. RESULTS: Approximately 40% physicians reported that 11-20% of their patients had dry cough predominantly. 57% and 46% physicians reported acute and chronic dry cough in >30% of their patients, respectively. 68% physicians reported that > 21% of their patients with chronic dry cough were smokers and 61-62% physicians reported that 11-30% of their patients had exposure to pollution. As per physicians, 19.6% of their patients had allergy/asthma followed by respiratory tract infections (17.6% patients), smokers cough (11.4%) and gastroesophageal reflux disease (10.4%). 86.4% physicians recommended that the underlying cause of chronic dry cough should be determined prior to initiating the specific therapy and 13.6% recommended that cough should be suppressed to improve quality of life of the patients. Dextromethorphan (ranked 1 by 68% physicians) and codeine (ranked 2 by 47% physicians) were the most recommended antitussives in patients with dry cough. CONCLUSIONS: Dry cough causes the significant impairment in patient's daily associated activities. An increased awareness of treatment patterns for the management of dry cough among physicians could significantly improve patient's quality of life.

Transgenic expression of an unedited mitochondrial orfB gene product from wild abortive (WA) cytoplasm of rice (Oryza sativa L.) generates male sterility in fertile rice lines.

MAIN CONCLUSION: Over-expression of the unedited mitochondrial orfB gene product generates male sterility in fertile indica rice lines in a dose-dependent manner. Cytoplasmic male sterility (CMS) and nuclear-controlled fertility restoration are widespread developmental features in plant reproductive systems. In self-pollinated crop plants, these processes often provide useful tools to exploit hybrid vigour. The wild abortive CMS has been employed in the majority of the "three-line" hybrid rice production since 1970s. In the present study, we provide experimental evidence for a positive functional relationship between the 1.1-kb unedited orfB gene transcript, and its translated product in the mitochondria with male sterility. The generation of the 1.1-kb unedited orfB gene transcripts increased during flowering, resulting in low ATP synthase activity in sterile plants. Following insertion of the unedited orfB gene into the genome of male-fertile plants, the plants became male sterile in a dose-dependent manner with concomitant reduction of ATPase activity of F1F0-ATP synthase (complex V). Fertility of the transgenic lines and normal activity of ATP synthase were restored by down-regulation of the unedited orfB gene expression through RNAi-mediated silencing. The genetic elements deciphered in this study could further be tested for their use in hybrid rice development.

Salicylic acid-mediated establishment of the compatibility between Alternaria brassicicola and Brassica juncea is mitigated by abscisic acid in Sinapis alba.

This work addresses the changes in the phytohormonal signature in the recognition of the necrotrophic fungal pathogen Alternaria brassicicola by susceptible Brassica juncea and resistant Sinapis alba. Although B. juncea, S. alba and Arabidopsis all belong to the same family, Brassicaceae, the phytohormonal response of susceptible B. juncea towards this pathogen is unique because the latter two species express non-host resistance. The differential expression of the PR1 gene and the increased level of salicylic acid (SA) indicated that an SA-mediated biotrophic mode of defence response was triggered in B. juncea upon challenge with the pathogen. Compared to B. juncea, resistant S. alba initiated enhanced abscisic acid (ABA) and jasmonic acid (JA) responses following challenge with this pathogen, as revealed by monitoring the expression of ABA-related genes along with the concentration of ABA and JA. Furthermore, these results were verified by the exogenous application of ABA on B. juncea leaves prior to challenge with A. brassicicola, which resulted in a delayed disease progression, followed by the inhibition of the pathogen-mediated increase in SA response and enhanced JA levels. Therefore, it seems that A. brassicicola is steering the defence response towards a biotrophic mode by mounting an SA response in susceptible B. juncea, whereas the enhanced ABA response of S. alba not only counteracts the SA response but also restores the necrotrophic mode of resistance by enhancing JA biosynthesis.

Native polyubiquitin promoter of rice provides increased constitutive expression in stable transgenic rice plants.

The rice Ubiquitin1 (Ubi1) promoter was tested to evaluate its capacity to express the heterologous gusA gene encoding ß-glucuronidase in transgenic rice tissue relative to the commonly used Ubi1 corn promoter and the rice gibberellic acid insensitive (GAI) gene promoter element. Experimental results showed increased expression of gusA gene in rice tissue when driven by the native Ubi1 promoter when compared to the use of corn Ubi1 promoter. Results further indicated that the cis-regulatory elements present in the native promoter element might have been responsible for high expression. However, the gusA gene expression level when driven by the rice GAI promoter was notably lower than both Ubi1 promoters. The present study, thus, for the first time helped to demonstrate that the native Ubi1 promoter is a promising genetic element in transgenic approaches for constitutive expression of any gene in rice tissue.

An unedited 1.1 kb mitochondrial orfB gene transcript in the wild abortive cytoplasmic male sterility (WA-CMS) system of Oryza sativa L. subsp. indica.

BACKGROUND: The application of hybrid rice technology has significantly increased global rice production during the last three decades. Approximately 90% of the commercially cultivated rice hybrids have been derived through three-line breeding involving the use of WA-CMS lines. It is believed that during the 21st century, hybrid rice technology will make significant contributions to ensure global food security. This study examined the poorly understood molecular basis of the WA-CMS system in rice. RESULTS: RFLPs were detected for atp6 and orfB genes in sterile and fertile rice lines, with one copy of each in the mt-genome. The RNA profile was identical in both lines for atp6, but an additional longer orfB transcript was identified in sterile lines. 5' RACE analysis of the long orfB transcript revealed it was 370 bp longer than the normal transcript, with no indication it was chimeric when compared to the genomic DNA sequence. cDNA clones of the longer orfB transcript in sterile lines were sequenced and the transcript was determined unedited. Sterile lines were crossed with the restorer and maintainer lines, and fertile and sterile F1 hybrids were respectively generated. Both hybrids contained two types of orfB transcripts. However, the long transcript underwent editing in the fertile F1 hybrids and remained unedited in the sterile lines. Additionally, the editing of the 1.1 kb orfB transcript co-segregated with fertility restoring alleles in a segregating population of F2 progeny; and the presence of unedited long orfB transcripts was detected in the sterile plants from the F2 segregating population. CONCLUSION: This study helped to assign plausible operative factors responsible for male-sterility in the WA cytoplasm of rice. A new point of departure to dissect the mechanisms governing the CMS-WA system in rice has been identified, which can be applied to further harness the opportunities afforded by hybrid vigor in rice.