Cross-species bacterial artificial chromosome (BAC) library screening via overgo-based hybridization and BAC-contig mapping of a yield enhancement quantitative trait locus (QTL) yld1.1 in the Malaysian wild rice Oryza rufipogon

The construction of BAC-contig physical maps is an important step towards a partial or ultimate genome sequence analysis. Here, we describe our initial efforts to apply an overgo approach to screen a BAC library of the Malaysian wild rice species, Oryza rufipogon. Overgo design is based on repetitive element masking and sequence uniqueness, and uses short probes (approximately 40 bp), making this method highly efficient and specific. Pairs of 24-bp oligos that contain an 8-bp overlap were developed from the publicly available genomic sequences of the cultivated rice, O. sativa, to generate 20 overgo probes for a 1-Mb region that encompasses a yield enhancement QTL yld1.1 in O. rufipogon. The advantages of a high similarity in melting temperature, hybridization kinetics and specific activities of overgos further enabled a pooling strategy for library screening by filter hybridization. Two pools of ten overgos each were hybridized to high-density filters representing the O. rufipogon genomic BAC library. These screening tests succeeded in providing 69 PCR-verified positive hits from a total of 23,040 BAC clones of the entire O. rufipogon library. A minimal tilling path of clones was generated to contribute to a fully covered BAC-contig map of the targeted 1-Mb region. The developed protocol for overgo design based on O. sativa sequences as a comparative genomic framework, and the pooled overgo hybridization screening technique are suitable means for high-resolution physical mapping and the identification of BAC candidates for sequencing.     

Radial glial cells. are they really glia?

During the development of the cerebral cortex, radial glia serve as a scaffold to support and direct neurons during their migration. This view is now changing in the light of emerging evidence showing that these cells have a much more dynamic and diverse role. A recent series of studies has provided strong support for their role as precursor cells in the ventricular zone that generate cortical neurons and glia, in addition to providing migration guidance.     

Identification of four functionally important microRNA families with contrasting differential expression profiles between drought-tolerant and susceptible rice leaf at vegetative stage

Background

Developing drought-tolerant rice varieties with higher yield under water stressed conditions provides a viable solution to serious yield-reduction impact of drought. Understanding the molecular regulation of this polygenic trait is crucial for the eventual success of rice molecular breeding programmes. microRNAs have received tremendous attention recently due to its importance in negative regulation. In plants, apart from regulating developmental and physiological processes, microRNAs have also been associated with different biotic and abiotic stresses. Hence here we chose to analyze the differential expression profiles of microRNAs in three drought treated rice varieties: Vandana (drought-tolerant), Aday Sel (drought-tolerant) and IR64 (drought-susceptible) in greenhouse conditions via high-throughput sequencing.

Results

Twenty-six novel microRNA candidates involved in the regulation of diverse biological processes were identified based on the detection of miRNA*. Out of their 110 predicted targets, we confirmed 16 targets from 5 novel microRNA candidates. In the differential expression analysis, mature microRNA members from 49 families of known Oryza sativa microRNA were differentially expressed in leaf and stem respectively with over 28 families having at least a similar mature microRNA member commonly found to be differentially expressed between both tissues. Via the sequence profiling data of leaf samples, we identified osa-miR397a/b, osa-miR398b, osa-miR408-5p and osa-miR528-5p as being down-regulated in two drought-tolerant rice varieties and up-regulated in the drought-susceptible variety. These microRNAs are known to be involved in regulating starch metabolism, antioxidant defence, respiration and photosynthesis. A wide range of biological processes were found to be regulated by the target genes of all the identified differentially expressed microRNAs between both tissues, namely root development (5.3–5.7 %), cell transport (13.2–18.4 %), response to stress (10.5–11.3 %), lignin catabolic process (3.8–5.3 %), metabolic processes (32.1–39.5 %), oxidation-reduction process (9.4–13.2 %) and DNA replication (5.7–7.9 %). The predicted target genes of osa-miR166e-3p, osa-miR166h-5p*, osa-miR169r-3p* and osa-miR397a/b were found to be annotated to several of the aforementioned biological processes.

Conclusions

The experimental design of this study, which features rice varieties with different drought tolerance and tissue specificity (leaf and stem), has provided new microRNA profiling information. The potentially regulatory importance of the microRNA genes mentioned above and their target genes would require further functional analyses.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1851-3) contains supplementary material, which is available to authorized users.     

Risk Prediction Score for Severe High Altitude Illness: A Cohort Study

Background

Risk prediction of acute mountain sickness, high altitude (HA) pulmonary or cerebral edema is currently based on clinical assessment. Our objective was to develop a risk prediction score of Severe High Altitude Illness (SHAI) combining clinical and physiological factors. Study population was 1017 sea-level subjects who performed a hypoxia exercise test before a stay at HA. The outcome was the occurrence of SHAI during HA exposure. Two scores were built, according to the presence (PRE, n = 537) or absence (ABS, n = 480) of previous experience at HA, using multivariate logistic regression. Calibration was evaluated by Hosmer-Lemeshow chisquare test and discrimination by Area Under ROC Curve (AUC) and Net Reclassification Index (NRI).

Results

The score was a linear combination of history of SHAI, ventilatory and cardiac response to hypoxia at exercise, speed of ascent, desaturation during hypoxic exercise, history of migraine, geographical location, female sex, age under 46 and regular physical activity. In the PRE/ABS groups, the score ranged from 0 to 12/10, a cut-off of 5/5.5 gave a sensitivity of 87%/87% and a specificity of 82%/73%. Adding physiological variables via the hypoxic exercise test improved the discrimination ability of the models: AUC increased by 7% to 0.91 (95%CI: 0.87–0.93) and 17% to 0.89 (95%CI: 0.85–0.91), NRI was 30% and 54% in the PRE and ABS groups respectively. A score computed with ten clinical, environmental and physiological factors accurately predicted the risk of SHAI in a large cohort of sea-level residents visiting HA regions.     

A Simple Intervention to Prevent Cutaneous Larva Migrans among Devotees of the Nallur Temple in Jaffna,Sri Lanka

Background

A cross sectional study conducted during the annual festival at Nallur temple in Jaffna, Sri Lanka, in 2010, showed that the prevalence of cutaneous larva migrants (CLM) among the devotees who performed the side roll ritual was 58.2% (95%CI: 51.2%–65.0%).

Objective

To test the hypothesis that the deworming stray dogs around the temple premises effectively reduces the prevalence of CLM among devotees.

Methodology/Principal Findings

All stray dogs (8) in the vicinity of the temple were treated, with mebendazole (100 mg) crushed and filled into sausages, 10 days before the commencement of festival in 2011. The same procedure was repeated a week later to ensure complete coverage. A cross-sectional study was conducted among 200 systematically selected devotees in August 2011 using an interviewer-administered questionnaire and the clinical examination of the skin. Baermann''s technique was used for the recovery of nematode larvae from 40 soil samples collected from the temple premises. Ten samples of dog faeces collected from the same premises were also examined for nematode eggs. Prevalence of CLM among devotees in 2010 (Pre intervention) and 2011(Post intervention) were compared to test the hypothesis. Prevalence of CLM declined from 58% to 8% (Chi-square = 112.90, p<0.001) following the intervention. None of the subjects practiced new precautionary measures compared to the previous year. Soil and fecal samples were negative for parasites.

Conclusions/Significance

Regular dog deworming is an important and effective method for the prevention of CLM among the devotees doing side roll ritual and represents a pragmatic intervention that municipal authorities could perform on annual basis.     

Plasma anti-α-galactoside antibody binds to serine- and threonine-rich peptide sequence of apo(a) subunit in Lp(a)

Lipoprotein(a) immune complexes [Lp(a) IC] of varying particle density obtained by ultracentrifugation of plasma from normal healthy donors were markedly dominated by IgG. Lp(a) and immunoglobulins were liberated from plasma Lp(a) IC by treatment with melibiose, a sugar specific for circulating anti-α-galactoside antibody (anti-Gal). Upon incubation with plasma lipoprotein fraction anti-Gal but not the α-glucoside-specific antibody from human plasma formed de novo IC with Lp(a). Binding of Lp(a) sugar-reversibly enhanced the fluorescence of FITC-labeled anti-Gal as did binding of α-galactoside-containing glycoproteins. This effect apparently due to conformational shift in the Fc region of the antibody was also produced by apo(a) subunit separated from Lp(a) and de-O–glycosylated apo(a) but not by any other plasma lipoproteins or by Lp(a) pre-incubated with the O–glycan-specific lectin jacalin. O–Glycans and their terminal sialic acid moieties in apo(a) of circulating Lp(a)-anti-Gal IC, in contrast to those in pure Lp(a), were inaccessible to jacalin and anion exchange resin, respectively. Unlike other plasma lipoproteins, Lp(a) inhibited Griffonia simplicifolia isolectin B4 which also accommodates serine- and threonine-rich peptide sequence (STPS) as surrogate ligand to α-galactosides at its binding site. Results suggest that anti-Gal recognizes STPS in the O-glycan-rich regions of apo(a) subunit in Lp(a) which contains no α-linked galactose.     

Computer aided pharmacokinetic profiling and toxicity analysis of naphthalene

Naphthalene is an aromatic hydrocarbon used as room freshner. Therefore, it is of interest to document the computer aided pharmacokinetic profiling and toxicity analysis data of naphthalene.     

Poor performance of serological tests in the diagnosis of pulmonary tuberculosis: evidence from a contact tracing field study

Background

Delayed or missed diagnosis of TB continues to fuel the global TB epidemic, especially in resource limited settings. Use of serology for the diagnosis of tuberculosis, commonly used in India, is another factor. In the present study a commercially available serodiagnostic assay was assessed for its diagnostic value in combination with smear, culture and clinical manifestations.

Methodology/Principal Findings

A total of 2300 subjects were recruited for the study, but 1041 subjects were excluded for various reasons. Thus 1259 subjects were included in the study of which 470 were pulmonary tuberculosis cases (440 of 470 were culture-positive) and 789 were their asymptomatic contacts. A house-to-house survey method was used. Blood samples were tested for IgM, IgA, and IgG antibodies using the Pathozyme Myco M (IgM), Myco A (IgA) and Myco G (IgG) enzyme immunoassay (EIA). Out of 470 PTB cases, BCG scar was positive in 82.34%. The Mantoux test and smear positivity rates in PTB cases were 94.3% (430/456), and 65.32% (307/470), respectively. Among the asymptomatic contacts, BCG scar was positive in 95.3% and Mantoux test was positive in 80.66% (442/548) contacts. No contact was found falsely smear positive. The sensitivity of IgM, IgA, and IgG EIA tests was 48.7%, 25.7% and 24.4%, respectively, while the specificity was 71.5%, 80.5%, 76.6%, respectively. Performance of EIAs was not affected by the previous BCG vaccination. However, prior BCG vaccination was statistically significantly (p = 0.005) associated with Mantoux test positivity in PTB cases but not in contacts (p = 0.127). The agreement between serology and Mantoux test was not significant.

Conclusion

The commercial serological test evaluated showed poor sensitivity and specificity and suggests no utility for detection of pulmonary tuberculosis.     

Chikungunya infection in India: results of a prospective hospital based multi-centric study

Background

Chikungunya (CHIKV) has recently seen a re-emergence in India with high morbidity. However, the epidemiology and disease burden remain largely undetermined. A prospective multi-centric study was conducted to evaluate clinical, epidemiological and virological features of chikugunya infection in patients with acute febrile illness from various geographical regions of India.

Methods and Findings

A total of 540 patients with fever of up to 7days duration were enrolled at Karnataka Institute of Medical Sciences (KIMS), Karnataka (South); Sawai Man Singh Medical College (SMS) Rajasthan (West), and All India Institute of Medical Sciences (AIIMS) New Delhi (North) from June 2008 to May 2009. Serum specimens were screened for chikungunya infection concurrently through RT-PCR and serology (IgM). Phylogenetic analysis was performed using Bioedit and Mega2 programs. Chikungunya infection was detected in 25.37% patients by RT-PCR and/or IgM-ELISA. Highest cases were detected in south (49.36%) followed by west (16.28%) and north (0.56%) India. A difference in proportion of positives by RT-PCR/ELISA with regard to duration of fever was observed (p<0.05). Rashes, joint pain/swelling, abdominal pain and vomiting was frequently observed among chikungunya confirmed cases (p<0.05). Adults were affected more than children. Anti-CHIK antibodies (IgM) were detected for more than 60days of fever onset. Phylogenetic analysis based on E1 gene from KIMS patients (n = 15) revealed ∼99% homology clustering with Central/East African genotype. An amino acid change from lysine to glutamine at position 132 of E1 gene was frequently observed among strains infecting children.

Conclusions

The study documented re-emergence of chikungunya in high frequencies and severe morbidity in south and west India but rare in north. The study emphasizes the need for continuous surveillance for disease burden using multiple diagnostic tests and also warrants the need for an appropriate molecular diagnostic for early detection of chikungunya virus.     

Risk Stratification of Oral Potentially Malignant Disorders in Fanconi Anemia Patients Using Autofluorescence Imaging and Cytology-On-A Chip Assay

Fanconi anemia (FA) is a hereditary genomic instability disorder with a predisposition to leukemia and oral squamous cell carcinomas (OSCCs). Hematopoietic stem cell transplantation (HSCT) facilitates cure of bone marrow failure and leukemia and thus extends life expectancy in FA patients; however, survival of hematologic malignancies increases the risk of OSCC in these patients. We developed a “cytology-on-a-chip” (COC)–based brush biopsy assay for monitoring patients with oral potentially malignant disorders (OPMDs). Using this COC assay, we measured and correlated the cellular morphometry and Minichromosome Maintenance Complex Component 2 (MCM2) expression levels in brush biopsy samples of FA patients’ OPMD with clinical risk indicators such as loss of autofluorescence (LOF), HSCT status, and mutational profiles identified by next-generation sequencing. Statistically significant differences were found in several cytology measurements based on high-risk indicators such as LOF-positive and HSCT-positive status, including greater variation in cell area and chromatin distribution, higher MCM2 expression levels, and greater numbers of white blood cells and cells with enlarged nuclei. Higher OPMD risk scores were associated with differences in the frequency of nuclear aberrations and differed based on LOF and HSCT statuses. We identified mutation of FAT1 gene in five and NOTCH-2 and TP53 genes in two cases of FA patients’ OPMD. The high-risk OPMD of a non-FA patient harbored FAT1, CASP8, and TP63 mutations. Use of COC assay in combination with visualization of LOF holds promise for the early diagnosis of high-risk OPMD. These minimally invasive diagnostic tools are valuable for long-term surveillance of OSCC in FA patients and avoidance of unwarranted scalpel biopsies.