Targeted editing of tomato carotenoid isomerase reveals the role of 5' UTR region in gene expression regulation.

KEY MESSAGE: A deletion created by CRISPR/Cas9 system in the 5' UTR of the carotenoid isomerase gene in tomato leads to downregulation of the gene resulting in the low conversion of prolycopene to lycopene. CRISPR/Cas9 based genome editing is an effective and useful tool adopted from the bacterial immune response system for altering specific, pre-determined DNA sequences in eukaryotes. Such targeted changes are finding wide application in human health as well as in precision breeding of crop plants for improved traits. Mutations in the coding and regulatory regions can have varying impacts on the function of the gene. In the current study, we demonstrate this on tomato carotenoid isomerase, a key gene in the carotenoid biosynthesis pathway. Mutations were generated in the 5' UTR and exon 1 of the carotenoid isomerase gene using CRISPR/Cas9 expression via Agrobacterium-mediated transformation of tomato variety Periyakulam 1 (PKM1). Molecular and biochemical studies demonstrate that CRISPR-mediated point mutations in the exon sequence lead to complete knockout of protein function whereas deletion in 5' UTR region lowers the expression of the gene leading to changes in plant phenotype.

Comparative assessment of genetic diversity among Indian bamboo genotypes using RAPD and ISSR markers.

Bamboo is one of the important plant for pulp, paper and charcoal industries. After China, India is the second largest bamboo reserve in Asia. Around the globe, wide genetic diversity of bamboo is present which serves as the base for selection and improvement. DNA based molecular markers appears to be a striking substitute for systematic assessment of the genetic diversity in conservation and genetic improvement of plants. DNA based molecular markers such as RAPD and ISSR were used to assess the genetic diversity in 13 bamboo genotypes. Total 120 RAPD and 63 ISSR primers were tested, of which only 42 polymorphic primers (30 RAPD and 12 ISSR), gave reproducible amplification profile and were used in this study. 30 RAPD primers yielded total 645 amplified fragments, of which 623 were polymorphic, and 20.76 polymorphic bands per primer were observed across 13 genotypes. 12 ISSR primers produced 246 amplified fragments, of which 241 were polymorphic, and 20.08 polymorphic bands per primer was observed across 13 different genotypes. The Jaccard's coefficient of RAPD, ISSR and pooled RAPD and ISSR dendrograms ranged from 0.26 to 0.83, 0.23 to 0.86 and 0.26 to 0.84 respectively. The present study found the large genetic diversity present between different elite genotypes of bamboo. Such investigation can deliver a well understanding of the available genotypes, which might be further exploited for the paper industry.

Transcriptional profiling of genes involved in steviol glycoside biosynthesis in Stevia rebaudiana bertoni during plant hardening.

BACKGROUND: Stevioside is a diterpene glycoside found in Stevia rebaudiana Bertoni (Asteraceae) and is 200-300 times sweeter than sucrose. It is synthesized through a plastid localized 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway. Fifteen genes are involved in the formation of steviol glycosides (stevioside and rebaudioside A). In the present investigation, micropropagated plants were allowed to harden for one month during which transcriptional profiling of candidate genes was carried out. Sampling from all the plants was carried out during hardening at different time intervals (day 10, 20, and 30) along with control plants (day 0). Stevioside content was also measured. RESULTS: Of 15 genes, 9 were up-regulated two-fold or greater. Nine genes were expressed at higher levels after 30 days than in the untreated controls. Moreover, these transcriptional differences were correlated with a significant enhancement in stevioside content from 0- (11.48%) to 30- (13.57%) day-old plants. CONCLUSIONS: MEP pathway genes in stevia are expressed at higher levels during hardening, a change to vegetative growth from reproductive growth. Although there were higher transcript levels of candidate genes at the initial phase of hardening, the highest stevioside content was found after 30 days of hardening, suggesting translational/posttranslational regulatory mechanisms.

Bimodal multispectral imaging system with cloud-based machine learning algorithm for real-time screening and detection of oral potentially malignant lesions and biopsy guidance.

SIGNIFICANCE: Screening and early detection of oral potentially malignant lesions (OPMLs) are of great significance in reducing the mortality rates associated with head and neck malignancies. Intra-oral multispectral optical imaging of tissues in conjunction with cloud-based machine learning (CBML) can be used to detect oral precancers at the point-of-care (POC) and guide the clinician to the most malignant site for biopsy. AIM: Develop a bimodal multispectral imaging system (BMIS) combining tissue autofluorescence and diffuse reflectance (DR) for mapping changes in oxygenated hemoglobin (HbO2) absorption in the oral mucosa, quantifying tissue abnormalities, and guiding biopsies. APPROACH: The hand-held widefield BMIS consisting of LEDs emitting at 405, 545, 575, and 610 nm, 5MPx monochrome camera, and proprietary Windows-based software was developed for image capture, processing, and analytics. The DR image ratio (R610/R545) was compared with pathologic classification to develop a CBML algorithm for real-time assessment of tissue status at the POC. RESULTS: Sensitivity of 97.5% and specificity of 92.5% were achieved for discrimination of OPML from patient normal in 40 sites, whereas 82% sensitivity and 96.6% specificity were obtained for discrimination of abnormal (OPML + SCC) in 89 sites. Site-specific algorithms derived for buccal mucosa (27 sites) showed improved sensitivity and specificity of 96.3% for discrimination of OPML from normal. CONCLUSIONS: Assessment of oral cancer risk is possible by mapping of HbO2 absorption in tissues, and the BMIS system developed appears to be suitable for biopsy guidance and early detection of oral cancers.

Phoenix phylogeny, and analysis of genetic variation in a diverse collection of date palm (Phoenix dactylifera) and related species.

Date palm (Phoenix dactylifera), one of the most ancient crops, is grown commercially in >30 countries. Using whole plastome assemblies, phylogenetic analyses revealed that cultivated date palm accessions share the same clade with P hoenix sylvestris, P hoenix pusilla and P hoenix acaulis, which are native to the Indian subcontinent, and Phoenix caespitosa that is native to the Arabian Peninsula and the deserts of Somalia. Analysis of genetic diversity and genetic relationships among date palm accessions from 13 producing countries involved 195 date palm accessions that were genotyped at 19 microsatellite loci. Extensive genetic diversity was observed, with many accessions heterozygous for most markers in this clonally propagated crop. The average number of alleles per locus (42.1), expected heterozygosity (0.8), observed heterozygosity (0.47) and fixation indices (FST = 0.42) demonstrated substantial genetic diversity and population structure. Iraqi accessions were found to have the richest allelic diversity, and the most private alleles. The model-based Bayesian method indicated that these accessions could be broadly divided into two structure groups, one group with predominantly African accessions and another predominantly Asian. Some germplasm, especially from Tunisia and Iraq, deviated from this generalization. Many accessions in the STRUCTURE-derived groups were found to be genetic admixtures, with gene flow between Asian and African groups. Indian and Pakistani date palms were found to be most closely related to North African germplasm.

Is Antimicrobial Photodynamic Therapy Effective as an Adjunct to Scaling and Root Planing in Patients with Chronic Periodontitis? A Systematic Review.

The aim of this systematic review was to investigate whether antimicrobial photodynamic therapy (aPDT) as either a primary mode of treatment or an adjunct to non-surgical treatment was more effective than scaling and root planing (SRP) alone in treating chronic periodontitis in terms of clinical attachment level (CAL) gain and probing depth (PD) reduction. The focused question was developed using the Patient, Intervention, Comparison, and Outcome (PICO) format, and two authors independently searched the Medline, EMBASE, Cochrane Library, Web of Science, Google Scholar, and Scopus databases for relevant studies from January 2008 to December 2016. Twenty studies included in this systematic review were randomized clinical trials (RCTs) or quasi-RCTs of aPDT compared to placebo, no intervention, or non-surgical treatment in an adult population. Basic study characteristics, photosensitizing agents and wavelengths used in aPDT, frequency of aPDT application, effect of aPDT on clinical parameters, antimicrobial effect of aPDT in chronic periodontitis, effect of immunological parameters following aPDT and patient-based outcome measures were collected from the studies. Although there was a wide range of heterogeneity in the included studied, they all indicated that aPDT has the potential to be an effective adjunct in the treatment of chronic periodontitis. Long-term, multicenter studies with larger sample sizes are needed before aPDT can be recommended as an effective treatment modality.

Transcript Quantification of Genes Involved in Steviol Glycoside Biosynthesis in Stevia rebaudiana Bertoni by Real-Time Polymerase Chain Reaction (RT-PCR).

Stevia (Stevia rebaudiana Bertoni) is a medicinal plant having sweet, diterpenoid glycosides known as steviol glycosides which are 200-300 times sweeter than sucrose (0.4 % solution). They are synthesized mainly in the leaves via plastid localized 2-C-methyl-D-erythrose-4-phosphate pathway (MEP pathway). Fifteen genes are involved in the formation of these glycosides. In the present protocol, a method for the quantification of transcripts of these genes is shown. The work involves RNA extraction and cDNA preparation, and therefore, procedures for the confirmation of DNA-free cDNA preparation have also been illustrated. Moreover, details of plant treatments are not mentioned as this protocol may apply to relative gene expression profile in any medicinal plant with any treatment. The treatments are numbered as T0 (Control), T1, T2, T3, and T4.

De novo Transcriptome Sequencing to Dissect Candidate Genes Associated with Pearl Millet-Downy Mildew (Sclerospora graminicola Sacc.) Interaction.

Understanding the plant-pathogen interactions is of utmost importance to design strategies for minimizing the economic deficits caused by pathogens in crops. With an aim to identify genes underlying resistance to downy mildew, a major disease responsible for productivity loss in pearl millet, transcriptome analysis was performed in downy mildew resistant and susceptible genotypes upon infection and control on 454 Roche NGS platform. A total of ~685 Mb data was obtained with 1 575 290 raw reads. The raw reads were pre-processed into high-quality (HQ) reads making to ~82% with an average of 427 bases. The assembly was optimized using four assemblers viz. Newbler, MIRA, CLC and Trinity, out of which MIRA with a total of 14.10 Mb and 90118 transcripts proved to be the best for assembling reads. Differential expression analysis depicted 1396 and 936 and 1000 and 1591 transcripts up and down regulated in resistant inoculated/resistant control and susceptible inoculated/susceptible control respectively with a common of 3644 transcripts. The pathways for secondary metabolism, specifically the phenylpropanoid pathway was up-regulated in resistant genotype. Transcripts up-regulated as a part of defense response included classes of R genes, PR proteins, HR induced proteins and plant hormonal signaling transduction proteins. The transcripts for skp1 protein, purothionin, V type proton ATPase were found to have the highest expression in resistant genotype. Ten transcripts, selected on the basis of their involvement in defense mechanism were validated with qRT-PCR and showed positive co-relation with transcriptome data. Transcriptome analysis evoked potentials of hypersensitive response and systemic acquired resistance as possible mechanism operating in defense mechanism in pearl millet against downy mildew infection.

Diffuse reflection spectroscopy: an alternative to autofluorescence spectroscopy in tongue cancer detection.

Laser-induced autofluorescence (LIAF) and diffuse reflection spectroscopy (DRS) are two emerging noninvasive optical tools that have shown immense potential to detect oral cavity pre-cancer. In a recent study, we have used spectral ratio reference standards (SRRS) of LIAF intensity ratios F500/F635, F500/F685, and F500/F705 for grading of tissues belonging to sites other than dorsal side of tongue (DST), lateral side of tongue (LST), and vermillion border of lip (VBL) that exhibited similar spectral shape for normal and abnormal tissues. This led to dismal diagnostic accuracies, and for the three LIAF-SRRS, normal tissue values were often misclassified as squamous cell carcinoma (SCC), which means that the true negatives were being wrongly identified as true positives. This study examines the applicability of the site-specific diffuse reflection spectral intensity ratio (R545/R575) of the oxygenated hemoglobin bands to classify different DST lesions and compares the results obtained with those obtained using LIAF-SRRS. DRS-SRRS of R545/R575 differentiated benign hyperplastic DST tissues from normal tissue with a sensitivity of 86% and specificity of 80%, which were indistinguishable using LIAF-SRRS. Further, in distinguishing hyperplastic tissues from premalignant dysplastic lesions, DRS-SRRS gave a sensitivity of 90% and a specificity of 86%, as compared to sensitivity of 89% and specificity of 72% shown by the three LIAF-SRRS together. The diagnostic accuracy and statistical adequacy of the two techniques were assessed by receiver operating characteristic curve (ROC-Curve) analysis. Three LIAF ratios gave a low overall ROC area under curve (ROC-AUCs) of 0.521, whereas the DR ratio (R545/R575) has shown an improved accuracy of 0.970 in differentiating different tissue types. While distinguishing hyperplastic from dysplastic tissues, the DR ratio gave a higher discrimination accuracy of 0.9. Based on these findings, it can be concluded that the DRS-SRRS technique by virtue of its low cost and higher diagnostic accuracies could be a viable alternate to LIAF-SRRS for in vivo screening of tongue pre-cancers and grading of different tissue types.