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Summary: Background: Oral rehydration solution (ORS) is a form of oral rehydration therapy (ORT) for diarrhoea that has the potential to drastically reduce child mortality; yet, according to UNICEF estimates, less than half of children younger than 5 years with diarrhoea in low-income and middle-income countries (LMICs) received ORS in 2016. A variety of recommended home fluids (RHF) exist as alternative forms of ORT; however, it is unclear whether RHF prevent child mortality. Previous studies have shown considerable variation between countries in ORS and RHF use, but subnational variation is unknown. This study aims to produce high-resolution geospatial estimates of relative and absolute coverage of ORS, RHF, and ORT (use of either ORS or RHF) in LMICs. Methods: We used a Bayesian geostatistical model including 15 spatial covariates and data from 385 household surveys across 94 LMICs to estimate annual proportions of children younger than 5 years of age with diarrhoea who received ORS or RHF (or both) on continuous continent-wide surfaces in 2000–17, and aggregated results to policy-relevant administrative units. Additionally, we analysed geographical inequality in coverage across administrative units and estimated the number of diarrhoeal deaths averted by increased coverage over the study period. Uncertainty in the mean coverage estimates was calculated by taking 250 draws from the posterior joint distribution of the model and creating uncertainty intervals (UIs) with the 2·5th and 97·5th percentiles of those 250 draws. Findings: While ORS use among children with diarrhoea increased in some countries from 2000 to 2017, coverage remained below 50% in the majority (62·6%; 12 417 of 19 823) of second administrative-level units and an estimated 6 519 000 children (95% UI 5 254 000–7 733 000) with diarrhoea were not treated with any form of ORT in 2017. Increases in ORS use corresponded with declines in RHF in many locations, resulting in relatively constant overall ORT coverage from 2000 to 2017. Although ORS was uniformly distributed subnationally in some countries, within-country geographical inequalities persisted in others; 11 countries had at least a 50% difference in one of their units compared with the country mean. Increases in ORS use over time were correlated with declines in RHF use and in diarrhoeal mortality in many locations, and an estimated 52 230 diarrhoeal deaths (36 910–68 860) were averted by scaling up of ORS coverage between 2000 and 2017. Finally, we identified key subnational areas in Colombia, Nigeria, and Sudan as examples of where diarrhoeal mortality remains higher than average, while ORS coverage remains lower than average. Interpretation: To our knowledge, this study is the first to produce and map subnational estimates of ORS, RHF, and ORT coverage and attributable child diarrhoeal deaths across LMICs from 2000 to 2017, allowing for tracking progress over time. Our novel results, combined with detailed subnational estimates of diarrhoeal morbidity and mortality, can support subnational needs assessments aimed at furthering policy makers' understanding of within-country disparities. Over 50 years after the discovery that led to this simple, cheap, and life-saving therapy, large gains in reducing mortality could still be made by reducing geographical inequalities in ORS coverage. Funding: Bill & Melinda Gates Foundation.

Objective: The aim of the study was to determine the incidence of significant weight loss, dehydration, hypernatremia and hyperbilirubinemia in exclusively breast-fed term healthy neonates and compare the incidence of these problems in the warm and cool months.Methods: During the study period 496 neonates were recruited.Results: 157 neonates (31.6%) had significant weight loss (> 10 % cumulative weight loss or per day weight loss > 5%). Clinical dehydration was present in 2.2% of neonates. Of these 157 neonates, 31.8% had hypernatremia and 28 % had hyperbilirubinemia.Conclusion: The incidence of the above mentioned problems were higher in the warm months but the difference was not statistically significant. [ABSTRACT FROM AUTHOR]

Objective: The present study aimed to compare and quantify the surface changes seen in two most commonly used orthodontic miniscrew implants (MSI) materials; titanium and stainless steel after their clinical use., Methods: 40 MSIs (20 titanium and 20 stainless steel) were retrieved from the maxillary arch of 20 subjects (13 females and 7 males) in the age group of 18 - 27 years (mean age=22.4 ± 3.83 years) after their intended use. 40 (20 titanium and 20 stainless steel) asreceived MSIs were used as control. All the MSIs were analyzed under a Scanning Electron Microscope (SEM) for the characterization of their morphological condition (blunting of tip, surface defects and corrosion). Furthermore, Energy Dispersive X-ray (EDX) microanalysis was carried out to study the changes in surface characterization., Results: When imaged using SEM, as-received Titanium and Stainless Steel MSIs demonstrated a relatively smooth surface with no surface defects. However, the retrieved titanium and stainless-steel implants showed increased surface defects (both corrosion and cracks) with the difference being statistically significant. The retrieved Titanium MSIs (115.31±24.38μm) showed 4 times more blunting compared to the retrieved Stainless-steel MSIs (29.74±8.56 μm), with the latter showing 2-3 times more surface corrosion., Conclusion: Clinical usage had pronounced effects on both Titanium and Stainless steel MSI alloys in terms of changes in the surface characteristics. While stainless steel MSIs are more susceptible to surface corrosion, Titanium MSIs exhibit greater alterations in the form of tipblunting and cracks in screw threads.

Arabidopsis lines with loss-of-function mutation in Embryo sac-specific Pectin MethylEsterase Inhibitor ( Atepmei ) gene showed seed sterility with embryo sac cellularization defects. Examination of tissue-cleared mature ovules revealed irregularly positioned nuclei/embryos within the embryo sacs. Egg cell-specific marker (DD45) expression analysis confirmed the presence of multiple egg cells in the mutant embryo sacs. These supernumerary egg cells were functional as evident from the production of twin embryos when supernumerary sperm cells were provided. The results of ruthenium red and tannic acid-ferric chloride staining of developing Atepmei mutant ovules showed that cell wall formation and maintenance were altered around embryo sac nuclei, which also coincided with change in the gamete specification. This report implicates the role of cell walls in gamete cell fate determination by altering cell-cell communication. Our analysis of the twin-embryo phenotype of epmei mutants also sheds light on the boundary conditions for double fertilization in plant reproduction., Competing Interests: The authors declare no competing interests., (© 2024 The Authors.)

Background: Dopamine (D) and serotonin (5-HT) pathways contribute to psoriasis pathobiology. Disruptions incite increased inflammatory mediators, keratinocyte activation and deterioration, and worsening symptoms. Brilaroxazine (RP5063), which displays potent high binding affinity to D 2/3/4 and 5-HT 1A/2A/2B/7 receptors and a moderate affinity to serotonin transporter (SERT), may affect the underlying psoriasis pathology., Methods: An imiquimod-induced psoriatic mouse model (BALB/c) evaluated brilaroxazine's activity in a topical liposomal-aqueous gel (Lipogel) formulation. Two of the three groups (n = 6 per) underwent induction with 5% imiquimod, and one group received topical brilaroxazine Lipogel (Days 1-11). Assessments included (1) Psoriasis Area and Severity Index (PASI) scores (Days 1-12), skin histology for Baker score based on H&E stained tissue (Day 12), and serum blood collection for serum cytokine analysis (Day 12). One-way ANOVA followed by post hoc Dunnett's t-test evaluated significance (p < 0.05)., Results: Imiquimod-induced animal Baker scores were higher versus Sham non-induced control's results (p < 0.001). Brilaroxazine Lipogel had significantly (p = 0.003) lower Baker scores versus the induced Psoriasis group. Brilaroxazine PASI scores were lower (p = 0.03) versus the induced Psoriasis group (Days 3-12), with the greatest effect in the last 3 days. The induced Psoriasis group showed higher Ki-67 and TGF-β levels versus non-induced Sham controls (p = 0.001). The brilaroxazine Lipogel group displayed lower levels of these cytokines versus the induced Psoriasis group, Ki-67 (p = 0.001) and TGF-β (p = 0.008), and no difference in TNF-α levels versus Sham non-induced controls., Conclusion: Brilaroxazine Lipogel displayed significant activity in imiquimod-induced psoriatic animals, offering a novel therapeutic strategy., (© 2024 Reviva Pharmaceuticals Inc. Skin Research and Technology published by John Wiley & Sons Ltd.)

Key Message: Recombinations between the parental genomes produced a novel mitochondrial genome in the cytoplasmic male sterile Brassica juncea cybrid Og1. A mitochondrial stoichiometric shift greatly reduced the molecule containing male-sterility-inducing orf138 gene leading to reversion to male fertility. An improved, chlorosis-corrected, cytoplasmic male sterile Brassica juncea cybrid Og1 derived from Ogura cytoplasm shows frequent reversion to male fertility. To determine the nature of mitochondrial recombination in the cybrid and to uncover the molecular mechanism of male fertility reversion, we sequenced the mitochondrial genomes of Og1, its isonuclear parental lines (OgRLM and Brassica juncea RLM198) and the revertant line (Og1-rt). Assembly of Og1 mitochondrial genome gave two circular molecules, Og1a (250.999 kbp) and Og1b (96.185 kbp) sharing two large direct repeat regions capable of recombining to form a single circular molecule. Og1a contains all essential mitochondrial genes, but the male-sterility-causing orf138 was uniquely present in Og1b along with 16 other complete or partial genes already represented in Og1a. Eleven and four recombinations between the parental mitochondrial genomes produced the Og1a and the Og1b molecules, respectively. Five genes were duplicated within Og1a, of which trnfM was inherited from both the parents while the other four genes, atp4, cox1 nad4L and trnM, were inherited from RLM198. RFLP analysis revealed that orf138-containing molecules were less abundant than Og1a in the male-sterile plants while og1b bearing molecules were undetectable in the revertant line. However, orf138 transcripts were amplified in RT-PCR and were also detected in northern blots revealing that Og1b molecules are not completely lost in the revertant plants. This is the first report where the mitochondrial genome of a cybrid is compared with its actual parents. The findings are discussed in the light of previous reports on mitochondrial genome recombination in cybrids.

Various pharmacological agents are known to create an imbalance in the normal physiology of bone remodeling. Cyclosporine-A (Cs-A) is one of the drugs that is widely used in transplantation and has its main side effect as gingival hyperplasia and alveolar bone loss by their action on the inflammatory mediators. Bisphosphonates are a new class of drugs that inhibit bone resorption by decreasing the osteoclast activity and number. The aim of this study was to evaluate the effect of concomitant administration of alendronate on Cs-A-induced alveolar bone loss in a rat model., Competing Interests: There are no conflicts of interest., (Copyright: © 2020 Journal of Pharmacy and Bioallied Sciences.)

Objective: To investigate the safety and efficacy of goat lung surfactant extract (GLSE) compared with bovine surfactant extract (beractant; Survanta®, AbbVie, USA) for the treatment of neonatal respiratory distress syndrome (RDS)., Study Design: We conducted a double-blind, non-inferiority, randomized trial in seven Indian centers between June 22, 2016 and January 11, 2018. Preterm neonates of 26 to 32 weeks gestation with clinical diagnosis of RDS were randomized to receive either GLSE or beractant. Repeat dose, if required, was open-label beractant in both the groups. The primary outcome was a composite of death or bronchopulmonary dysplasia (BPD) at 36 weeks postmenstrual age (PMA). Interim analyses were done by an independent data and safety monitoring board (DSMB)., Result: After the first interim analyses on 5% enrolment, the "need for repeat dose(s) of surfactant" was added as an additional primary outcome and enrolment restricted to intramural births at five of the seven participating centers. Following second interim analysis after 98 (10% of 900 planned) neonates were enroled, DSMB recommended closure of study in view of inferior efficacy of GLSE in comparison to beractant. There was no significant difference in the primary outcome of death or BPD between GLSE group (n = 52) and beractant group (n = 46) (50.0 vs. 39.1%; OR 1.5; 95% CI 0.7-3.5; p = 0.28). The need for repeat dose of surfactant was significantly higher in GLSE group (65.4 vs. 17.4%; OR 9.0; 95% CI 3.5-23.3; p < 0.001)., Conclusions: Goat lung surfactant was less efficacious than beractant (Survanta®) for treatment of RDS in preterm infants. Reasons to ascertain inferior efficacy of goat lung surfactant requires investigation and possible mitigating strategies in order to develop a low-cost and effective surfactant.

Pulmonary arterial hypertension (PAH), a condition that is defined by pulmonary vasculature constriction and remodeling, involves dysfunctional signaling of the serotonin (5-HT) receptors, 5-HT 2A/2B/7 . In a rat model of monocrotaline (MCT)-induced PAH, the effectiveness of RP5063 (RP), a dopamine and 5-HT receptor modulator, was evaluated as monotherapy and as an adjunct to standard PAH treatments. After a single 60 mg/kg dose of MCT, rats received vehicle (MCT+Veh; gavage twice-daily [b.i.d.]), RP (10 mg/kg; gavage b.i.d.), bosentan (B; 100 mg/kg; gavage BID), sildenafil (S; 50 mg/kg; gavage, BID), treprostinil (T; 100 ng/kg/min over 24 h intravenous), RP+B, RP+S, and RP+T for 28 days. Single-agent RP limited the functional and structural effects of PAH seen in the MCT+Veh group, with significant improvements in pulmonary hemodynamics, right ventricular (RV) hypertrophy, SO 2 , and pulmonary blood vessel structural changes. These effects appeared comparable with those associated with B, S, and T. Adjunctive RP treatment resulted in significantly lower mean pulmonary arterial pressures, RV systolic pressure. It also improved SO 2 measurements, as compared with MCT+Veh (P < 0.05), and diastolic pulmonary artery pressure (P < 0.05), as compared with single-agent B and S therapy (Bonferroni method adjusting for multiplicity). RP+S appeared to show the most consistent and extensive effects on pulmonary hemodynamics, respiratory parameters, and histopathologic changes. These results corroborate earlier preclinical findings supporting the efficacy of single-agent RP in PAH. RP, as mono and adjunctive therapy compared with induced-control, mitigated the functional and structural effects of MCT-induced PAH., (Copyright © 2018 The Author(s). Published by Elsevier B.V. All rights reserved.)

A promoter trap mutant line of Arabidopsis carrying a promoterless β-glucuronidase (uidA) gene exhibited GUS expression predominantly in all the trichomes. In this mutant, the T-DNA insertion was localized at 147bp upstream of the putative start codon, ATG, of the At5g11190 (SHN2) gene. Transcript profiling of the SHN2 suggested a constitutive expression of the gene in all the tissues. Deletion analysis of the upstream sequences established that a 565bp (-594/-30) region confers trichome-specific gene expression. The trichomes isolated from young, mature and senesced leaf tissues also showed the presence of SHN2 transcript. The occurrence of multiple TSSs on the SHN2 gene sequence, presence of the SHN2 transcript in the homozygous trip mutant, despite an insertional mutation event, and diverse reporter gene expression pattern driven by 5' and 3' promoter deletion fragments, suggest a complex transcriptional regulation of SHN2 gene in Arabidopsis. The promoter sequence -594/-30 showed a conserved functional role in conferring non-glandular trichome-specific expression in other heterologous systems like Brassica juncea and Solanum lycopersicon. Thus, in the present study T-DNA tagging has led to the identification of a trichome-specific regulatory sequence in the upstream region of a constitutively expressed SHN2 gene. The study also suggests a complex regulation of SHN2 gene. Isolated trichome specific region retains its functions in other systems like Brassica and tomato, hence could be effectively exploited in engineering trichome cells in heterologous crop plants to manipulate traits like biopharming and insect herbivory., (Copyright © 2017 Elsevier B.V. All rights reserved.)

In plants, the role of TRAF-like proteins with meprin and the TRAF homology (MATH) domain is far from clear. In animals, these proteins serve as adapter molecules to mediate signal transduction from Tumor Necrosis Factor Receptor to downstream effector molecules. A seed-sterile mutant with a disrupted TRAF-like gene ( At5g26290 ) exhibiting aberrant gametogenesis led us to investigate the developmental role of this gene in Arabidopsis ( Arabidopsis thaliana ). The mutation was semidominant and resulted in pleiotropic phenotypes with such features as short siliques with fewer ovules, pollen and seed sterility, altered Megaspore Mother Cell (MMC) specification, and delayed programmed cell death in megaspores and the tapetum, features that overlapped those in other well-characterized mutants. Seed sterility and reduced transmission frequency of the mutant alleles pointed to a dual role, sporophytic and gametophytic, for the gene on the male side. The mutant also showed altered expression of various genes involved in such cellular and developmental pathways as regulation of transcription, biosynthesis and transport of lipids, hormone-mediated signaling, and gametophyte development. The diverse phenotypes of the mutant and the altered expression of key genes related to gametophyte and seed development could be explained based on the functional similarly between At5g26290 and MATH-BTB domain proteins that modulate gene expression through the ubiquitin-mediated proteasome system. These results show a novel link between a TRAF-like gene and reproductive development in plants., (© 2017 American Society of Plant Biologists. All Rights Reserved.)

RP5063, a multimodal dopamine (DA) and serotonin (5-HT) modulator with high affinity for DA 2/3/4 and 5-HT 2A/2B/7 receptors and moderate affinity for SERT, is a novel therapeutic of special interest in the treatment of pulmonary arterial hypertension (PAH). Evidence indicates that therapeutics targeting the 5-HT 2A/2B receptors can influence the pathogenesis of PAH. However, the therapeutic effect of RP5063 in humans has yet to be investigated. A Sugen 5416-hypoxia (SuHx)-induced PAH model was used to evaluate twice-daily (b.i.d.) RP5063 at 10mg/kg (RP-10) and 20mg/kg (RP-20), as compared with positive (sildenafil 50mg/kg b.i.d.; Sil-50) and negative controls (SuHx+vehicle; SuHx+veh), in 24 adult male Wistar-Kyoto rats. RP5063 showed significantly lower systolic pulmonary arterial (both doses) and systolic right ventricular (RP-10) pressures, and improvement in oxygen saturation (RP-20). It significantly reduced small-vessel wall thickness (RP-20), lowered the percentage of muscular vessels (both doses). Both doses limited arterial obliteration due to endothelial cell proliferation, prevented plexiform lesion formation, and stemmed the release of leukotriene B4. Sildenafil showed statistically greater effects on vessel structure than that seen in both RP5063 groups and improved oxygen saturation. Additionally, Sildenafil did not demonstrate any significant effect on arterial obliteration, plexiform lesion development, or pulmonary arterial or right ventricular pressure. As PAH gains in severity, the impact of RP5063 inhibition of 5HT 2B increases, preventing arterial constriction and improving pulmonary hemodynamics. Due to its functional, structural, and chemokine effects, RP5063 represents a promising candidate for investigation in late-phase PAH., (Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.)

Pulmonary arterial hypertension (PAH), a condition characterized by pulmonary vasculature constriction and remodeling, involves dysregulation of the serotonin (5-HT) receptors 5-HT 2A and 5-HT 2B . A rat model of monocrotaline (MCT)-induced PAH was used to examine the potential beneficial effects of RP5063, a 5-HT receptor modulator. After a single 60mg/kg dose of MCT, rats were gavaged twice-daily (b.i.d.) with vehicle, RP5063 (1, 3, or 10mg/kg), or sildenafil (50mg/kg) for 28 days. RP5063 at a dose as low as 1mg/kg, b.i.d. reduced pulmonary resistance and increased systemic blood oxygen saturation. The highest dose of RP5063 (10mg/kg, b.i.d.) reduced diastolic, systolic, and mean pulmonary pressure, right systolic ventricular pressure, ventilatory pressure, and Fulton's index (ratio of right to left ventricular weight). Doses as low as 3mg/kg RP5063, b.i.d. also increased weight gain and body temperature, suggesting an improvement in overall health of MCT-treated animals. Similar reductions in pulmonary, right ventricular, and ventilatory pressure, pulmonary resistance, and Fulton's index as well as increased systemic blood oxygen saturation were observed in animals treated with the reference agent sildenafil at a higher dose (50mg/kg, b.i.d.). Histological examination revealed that RP5063 produced dose-dependent reductions in pulmonary blood vessel wall thickness and proportion of muscular vessels, similar to sildenafil. RP5063 completely blocked MCT-induced increases in the plasma cytokines TNFα, IL-1β, and IL-6 at all doses. In summary, RP5063 improved pulmonary vascular pathology and hemodynamics, right ventricular pressure and hypertrophy, systemic oxygen saturation, and overall health of rats treated with MCT., (Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.)

The Arabidopsis thaliana promoter trap mutant Bitrap-112 expressing green fluorescent protein (GFP) gene in the ovules was found to carry transferred DNA (T-DNA) insertion at -309 position of the APETALA2 (AP2) gene. Bitrap-112 line did not show phenotype associated with the AP2 mutation, suggesting that T-DNA insertion did not interrupt the AP2 promoter. Further, head-to-head orientation of GFP and AP2 genes indicated that the AP2 promoter could be bidirectional. A detailed deletion analysis of the upstream sequences of the AP2 gene was done to identify the promoter. GUS assay of transgenic A. thaliana plants carrying various AP2 upstream fragments fused to the uidA gene showed that ~200-bp 5' UTR sequences are capable of driving gene expression at low levels in vegetative tissues whereas inclusion of further upstream sequences (~300 bp) enhanced uidA expression comparable to native AP2 expression levels in various tissues including ovules. In the reverse orientation, the 519-bp AP2 upstream fragment was found to drive gene expression in immature ovules and pollen. Absence of antisense transcripts corresponding to the sequences upstream of AP2 gene in wild-type A. thaliana plants suggests that promoter trapping has uncovered a cryptic promoter, which in reverse orientation is capable of driving gene expression in ovules and anthers.

Intergenic regions of divergent gene pairs show bidirectional promoter activity but whether regulatory sequences for gene expression in opposite directions are shared is not established. In this study, promoters of divergently arranged gene pair At4g35640-At4g35650 (SERAT3;2-IDH-III) of Arabidopsis thaliana were analyzed to identify overlapping regulatory regions. Both genes showed the highest expression in flower buds and flowers. 5' RACE experiments extended the intergenic region from 161 bp shown in TAIR annotation to 512 bp. GUS analysis of transgenic A. thaliana plants carrying the 691 bp fragment (512 bp intergenic region plus 5' UTR of both the genes) linked to uidA gene revealed that SERAT3;2 promoter drives gene expression in the tapetum, whereas IDH-III promoter functions specifically in microspores/pollen. Serial 5' deletion of the 691 bp fragment showed SERAT3;2 promoter extends up to -355 position, whereas IDH-III promoter encompasses the 512 bp intergenic region. In transgenics, uidA transcript levels were lower than native SERAT3;2 and IDH-III transcripts indicating presence of additional cis regulatory elements beyond the 691 bp fragment. The present study demonstrated for the first time occurrence of a nested promoter in plants and identified a novel bidirectional promoter capable of driving gene expression in tapetum and microspores/pollen.

Tetrapyrrole biosynthesis is one of the most essential metabolic pathways in almost all organisms. Coproporphyrinogen III oxidase (CPO) catalyzes the conversion of coproporphyrinogen III into protoporphyrinogen IX in this pathway. Here, we report that mutation in the Arabidopsis ( Arabidopsis thaliana ) CPO-coding gene At5g63290 ( AtHEMN1 ) adversely affects silique length, ovule number, and seed set. Athemn1 mutant alleles were transmitted via both male and female gametes, but homozygous mutants were never recovered. Plants carrying Athemn1 mutant alleles showed defects in gametophyte development, including nonviable pollen and embryo sacs with unfused polar nuclei. Improper differentiation of the central cell led to defects in endosperm development. Consequently, embryo development was arrested at the globular stage. The mutant phenotype was completely rescued by transgenic expression of AtHEMN1 Promoter and transcript analyses indicated that AtHEMN1 is expressed mainly in floral tissues and developing seeds. AtHEMN1-green fluorescent protein fusion protein was found targeted to mitochondria. Loss of AtHEMN1 function increased coproporphyrinogen III level and reduced protoporphyrinogen IX level, suggesting the impairment of tetrapyrrole biosynthesis. Blockage of tetrapyrrole biosynthesis in the AtHEMN1 mutant led to increased reactive oxygen species (ROS) accumulation in anthers and embryo sacs, as evidenced by nitroblue tetrazolium staining. Our results suggest that the accumulated ROS disrupts mitochondrial function by altering their membrane polarity in floral tissues. This study highlights the role of mitochondrial ROS homeostasis in gametophyte and seed development and sheds new light on tetrapyrrole/heme biosynthesis in plant mitochondria., (© 2017 American Society of Plant Biologists. All Rights Reserved.)

Haploids and doubled haploids are invaluable for basic genetic studies and in crop improvement. A novel method of haploid induction through genetic engineering of the Centromere Histone Protein gene, CENH3 , has been demonstrated in Arabidopsis. The present study was undertaken to develop haploid inducer (HI) lines of Brassica juncea based on the principles elaborated in Arabidopsis. B. juncea was found to carry three copies of CENH3 which generated five different transcripts, of which three transcripts resulted from alternative splicing. Unlike Arabidopsis thaliana where native CENH3 gene was knocked out for constructing HI lines, we used RNAi approach to knockdown the native CENH3 genes. Further, to rescue CENH3 silenced cells, a GFP-CENH3-tailswap construct having N terminal GFP fused to H3.3 tail sequences and synthetic CENH3 histone fold domain sequences was devised. A total 38 transgenic B. juncea plants were regenerated following co-transformation with both silencing and rescue cassettes and transgenics carrying either or both the constructs were obtained. Transgenic status was confirmed through PCR, Southern and qRT-PCR analyses. Co-transformed lines were crossed to untransformed B. juncea or a line expressing only GFP-tailswap. FACS and cytological analyses of progenies revealed partial or complete elimination of B. juncea chromosomes thereby giving rise to aneuploids and haploid. This is the first report in a polyploid crop demonstrating that CENH3 engineering could be used to develop HI lines.

Centromeres are epigenetically specified by the centromeric histone H3 protein (CENH3). The timing and level of expression of CENH3 is tightly regulated to match the demands of the host cell. So far in plants, only CENH3 promoter of Arabidopsis thaliana (L.) Heynh. has been characterized. However, whether CENH3 promoters retain their characteristic mode of regulation in other species remains to be established. In the present study, activity of AtCENH3 promoter was investigated using reporter gene assay in Brassica juncea (L.) Czem. A 1156 bp promoter fragment of AtCENH3 gene (At1g01370) including the first 111 nucleotides of the coding sequence was amplified and cloned into the pORE-R2 binary vector to ensure translation fusion with the uidA coding sequences. The Agrobacteriun tiunefaciens strain GV3101 harbouring the recombinant construct was used to transform B. juncea cv. RLM198 hypocotyl explants. Histochemical assay of To and T, transgenics showed GUS expression in shoot apical meristem, leaf, sepal, flower pedicel and root tip. Intense GUS expression was observed in meristematic tissues, particularly at shoot and root apices. However, mature leaves, flowers, pollen and ovules exhibited very low or no GUS expression. Our results showed that AtCENH3 promoter regulates cognate gene expression in Brassica juncea as it does in A. thaliana, and hence a suitable candidate for developing haploid inducer line in B. juncea.

Steroidogenic acute regulatory related transfer (StART) proteins that are involved in transport of lipid molecules, play a myriad of functions in insects, mammals and plants. These proteins consist of a modular START domain of approximately 200 amino acids which binds and transfers the lipids. In the present study we have performed a genome-wide search for all START domain proteins in chickpea. The search identified 36 chickpea genes belonging to the START domain family. Through a phylogenetic tree reconstructed with Arabidopsis, rice, chickpea, and soybean START proteins, we were able to identify four transmembrane START (TM-START) proteins in chickpea. These four proteins are homologous to the highly conserved mammalian phosphatidylcholine transfer proteins. Multiple sequence alignment of all the transmembrane containing START proteins from Arabidopsis, rice, chickpea, and soybean revealed that the amino acid residues to which phosphatidylcholine binds in mammals, is also conserved in all these plant species, implying an important functional role and a very similar mode of action of all these proteins across dicots and monocots. This study characterizes a few of the not so well studied transmembrane START superfamily genes that may be involved in stress signaling. Expression analysis in various tissues showed that these genes are predominantly expressed in flowers and roots of chickpea. Three of the chickpea TM-START genes showed induced expression in response to drought, salt, wound and heat stress, suggesting their role in stress response.

Introduction: Lactate clearance has been implicated as a predictor of mortality among emergency department (ED) patients with severe sepsis or septic shock. We aimed to validate prior studies showing that lactate clearance during the ED stay is associated with decreased mortality., Methods: Retrospective dual-centered cross-sectional study using patients identified in the Yale-New Haven Hospital Emergency Medicine sepsis registry with severe sepsis or septic shock who had initial lactate levels measured in the ED and upon arrival (<24 hours) to the hospital floor. Lactate clearance was calculated as percent of serum lactate change from ED to floor measurement. We compared mortality and hospital interventions between patients who cleared lactate and those who did not., Results: 207 patients (110 male; 63.17±17.9 years) were included. Two reviewers extracted data with 95% agreement. One hundred thirty-six patients (65.7%) had severe sepsis and 71 patients (34.3%) had septic shock. There were 171 patients in the clearance group and 36 patients in the non-clearance group. The 28-day mortality rates were 15.2% in the lactate clearance group and 36.1% in the non-clearance group (p<0.01). Vasopressor support was initiated more often in the non-clearance group (61.1%) than in the clearance group (36.8%, p<0.01) and mechanical ventilation was used in 66.7% of the non-clearance group and 36.3% of the clearance group (p=0.001)., Conclusion: Patients who do not clear their lactate in the ED have significantly higher mortality than those with decreasing lactate levels. Our results are confirmatory of other literature supporting that lactate clearance may be used to stratify mortality-risk among patients with severe sepsis or septic shock.

Background: External thermal support is critical for preterm or ill infants due to altered thermoregulation. Incubators are the gold standard for long-term support and have been adopted successfully in many countries. Alternatives such as radiant warmers, blankets and others are often used as standard of care (SoC) in resource-limited settings when infants are otherwise not in Kangaroo Mother Care (KMC)., Methods: In this pilot study, we evaluate the feasibility of a conductive thermal mattress (CTM) using phase change materials as a low-cost warmer. We conducted a prospective multicentre open-label randomised controlled trial to determine non-inferiority of this CTM to SoC warming practices in low birthweight infants. The primary outcome was maintenance of axillary temperature., Results: We equally randomised 160 infants to CTM or SoC. The latter cohort continued to receive warmth by radiant warmers (n=48), blankets (n=18), warmed cradles (n=7) or KMC (n=7) before, during and subsequent to the study. CTM was deemed non-inferior since warmed babies had higher axillary temperature compared with SoC (mean increase 0.11±0.03°C SEM; p<0.001). Post hoc comparison to radiant warmers alone showed that CTM led to a higher axillary temperature (mean increase by 0.14±0.03°C SEM; p<0.001)., Conclusions: Short-term use of CTM compared with radiant warmers and other modes of warming is non-inferior to SoC and efficacious in maintaining body temperature. No adverse effects were reported. An extended multinational trial, preferably one that demonstrates longer-term thermoregulation, is warranted., Trial Registration Number: Clinical Trials Registry of India (CTRI/2010/091/002916 and CTRI/2011/04/001696)., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.)

Introduction: In the United States, there are limited studies regarding use of prehospital ultrasound (US) by emergency medical service (EMS) providers. Field diagnosis of life-threatening conditions using US could be of great utility. This study assesses the ability of EMS providers and students to accurately interpret heart and lung US images., Methods: We tested certified emergency medical technicians (EMT-B) and paramedics (EMT-P) as well as EMT-B and EMT-P students enrolled in prehospital training programs within two California counties. Participants completed a pre-test of sonographic imaging of normal findings and three pathologic findings: pericardial effusion, pneumothorax, and cardiac standstill. A focused one-hour lecture on emergency US imaging followed. Post-tests were given to all EMS providers immediately following the lecture and to a subgroup one week later., Results: We enrolled 57 prehospital providers (19 EMT-B students, 16 EMT-P students, 18 certified EMT-B, and 4 certified EMT-P). The mean pre-test score was 65.2%±12.7% with mean immediate post-test score of 91.1%±7.9% (95% CI [22%-30%], p<0.001). Scores significantly improved for all three pathologic findings. Nineteen subjects took the one-week post-test. Their mean score remained significantly higher: pre-test 65.8%±10.7%; immediate post-test 90.5%±7.0% (95% CI [19%-31%], p<0.001), one-week post-test 93.1%±8.3% (95% CI [21%-34%], p<0.001)., Conclusion: Using a small sample of EMS providers and students, this study shows the potential feasibility for educating prehospital providers to accurately identify images of pericardial effusion, pneumothorax, and cardiac standstill after a focused lecture.

Unlabelled: The objectives were to estimate the incidence of inadequate placement of the endotracheal tube (ET) using Tochen's formula (6 + birth weight) and to correlate optimum ET length with anthropometric measurements in neonates. A cross-sectional analytical study was conducted in 50 neonates. Neonates requiring intubation for ventilation, with a confirmatory chest radiograph, were intubated using Tochen's formula, after which tube placement was verified by auscultation. The incidence of inadequate placement and optimum length of ET insertion were estimated from chest radiographs. Anthropometric parameters were measured and correlated with the optimum length and regression equations generated. The incidence of inadequate placement of the ET was 40 % (20 of 50). The incidence of inadequate placement was higher (5 of 6, 83 %) in extremely low birth weight (ELBW) infants, and in extreme preterm infants (5 of 5, 100 %). It was found that all the anthropometric parameters correlated well (r between 0.71 and 0.84) with the optimum ET length., Conclusion: The incidence of inadequate placement was high, especially in the ELBWs' and extreme preterm infants. Birth weight, sternal length, and shoulder umbilical length correlated significantly with optimum ET length and may guide optimal ET placement.

Brassicaceae crops display strong hybrid vigor, and have long been subject to F1 hybrid breeding. Because the most reliable system of F1 seed production is based on cytoplasmic male sterility (CMS), various types of CMS have been developed and adopted in practice to breed Brassicaceae oil seed and vegetable crops. CMS is a maternally inherited trait encoded in the mitochondrial genome, and the male sterile phenotype arises as a result of interaction of a mitochondrial CMS gene and a nuclear fertility restoring (Rf) gene. Therefore, CMS has been intensively investigated for gaining basic insights into molecular aspects of nuclear-mitochondrial genome interactions and for practical applications in plant breeding. Several CMS genes have been identified by molecular genetic studies, including Ogura CMS from Japanese radish, which is the most extensively studied and most widely used. In this review, we discuss Ogura CMS, and other CMS systems, and the causal mitochondrial genes for CMS. Studies on nuclear Rf genes and the cytoplasmic effects of alien cytoplasm on general crop performance are also reviewed. Finally, some of the unresolved questions about CMS are highlighted.

Brassica mustard species represent one of the most important oilseed crops in India, nevertheless, their genetic diversity is barely known. A better understanding on this topic is essential for the proper utilization of genotypes in breeding programmes. We evaluated the genetic diversity among 44 Indian mustard (Brassica juncea) genotypes including varieties/purelines from different agro-climatic zones of India and few exotic genotypes (Australia, Poland and China). For this, we used A and B genome specific SSR markers and phenotypic data on 12 yield and yield contributing traits. Out of the 143 primers tested, 134 reported polymorphism and a total of 355 alleles were amplified. Dendrograms based on Jaccard's similarity coefficients and Manhattan dissimilarity coefficients were generated based on an average linkage algorithm (UPGMA) using marker data and phenotypic data. Genotypes were grouped into four clusters based on genetic distances. Both the clustering patterns based on Jaccard's similarity and Manhattan dissimilarity coefficients, independently, discriminated the genotypes effectively as per their pedigree and origin. PCoA revealed that, the grouping of genotypes based on SSR marker data is more convincing than phenotypic data, however, the correlation between phenotypic and genetic distance matrices was observed to be very low (r = 0.11). Hence, for diversity studies reliability on molecular markers is worth proving and SSR markers are the stronger tools than quantitative traits in discriminating B. juncea genotypes.

Investigation of the transgenic Arabidopsis promoter trap line GFP-868 that showed GFP expression only in anthers revealed the T-DNA insertion at 461bp upstream to the hypothetical gene At4g10596 with the GFP reporter gene in head-to-head orientation to the At4g10596 gene. The expression of the At4g10596 gene in wild type and in GFP-868 plant homozygous for T-DNA insertion was comparable and found in all tissues tested, while the GFP expression was restricted to anthers of the GFP-868 plants suggesting that the 461bp fragment separating the two genes in the GFP-868 line is functioning as bi-directional promoter. This 461bp fragment was cloned upstream to the GUS gene in two orientations to test for bi-directional promoter activity. Transgenic Arabidopsis plants carrying either of these constructs showed GUS activity in anthers indicating that this fragment behaves as bi-directional promoter specific to anthers. These results were also supported by the presence of cis-acting motifs such as TATA box and POLLEN1LELAT52 (AGAAA) within the 461bp sequence in both orientations. However, transcripts corresponding to the upstream sequences beyond -461 nucleotides were not detected in the wild type suggesting that this 461bp fragment is a cryptic promoter. The significance of the promoter trap approach and the usefulness of this type of promoter are discussed., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Background: The childhood burden of disease attributable to Streptococcus pneumoniae is particularly high in India. The immunogenicity and safety of 13-valent pneumococcal conjugate vaccine (PCV13) were compared with 7-valent pneumococcal conjugate vaccine (PCV7) in a randomized, active-controlled, double-blind trial conducted at 12 sites in India., Methods: Healthy infants received PCV13 or PCV7 at 6, 10, and 14 weeks of age (infant series) and at 12 months of age (toddler dose), along with routine pediatric vaccinations. Immunoglobulin G responses against the 13 pneumococcal serotypes were evaluated 1 month after the infant series and after the toddler dose. Pertussis and poliomyelitis immune responses were assessed 1 month after the infant series. Safety and tolerability also were assessed., Results: The immunogenicity results for the 7 common serotypes and the concomitant vaccines (whole-cell pertussis and oral poliovirus) were similar for subjects receiving PCV13 and subjects receiving PCV7. Immune responses to the 6 additional serotypes were higher in the PCV13 group compared with the PCV7 group. PCV13 and PCV7 had similar safety and tolerability profiles., Conclusions: PCV13 has immunogenicity similar to PCV7 in response to the 7 common serotypes, and has generally higher immunogenicity in response to the 6 additional serotypes. PCV13 may provide added protection against pneumococcal disease caused by the additional 6 serotypes and does not interfere with immune responses to whole-cell pertussis and oral poliovirus vaccines. PCV13 has an acceptable safety profile in both infants and toddlers, comparable with that of PCV7.

Introduction: Various ligation techniques and materials have been shown to affect the frictional resistance and the rate of tooth movement with sliding mechanics for space closure. The aim of this study was to evaluate the clinical efficiency of nonconventional elastomeric ligatures and conventional elastomeric ligatures during the canine retraction phase by comparing the rates of canine retraction., Methods: The 20 patients (12 female, 8 male) in our sample had individual canine retraction (in the first premolar extraction space) in each quadrant (2 maxillary, 2 mandibular) with nonconventional elastomeric ligatures and conventional elastomeric ligatures on either side of the arch. The amount of canine retraction in each interval of 1 month was determined. The rate of canine retraction was calculated and subjected to statistical calculations., Results: The rates of canine retraction were higher with the nonconventional elastomeric ligatures. However, no statistically significant difference was observed in relation to the maxillary arch. Clinically, in most instances, canine retraction was completed in the same interval in both groups., Conclusions: No significant difference in the rate of canine retraction was observed between the nonconventional elastomeric ligature and conventional elastomeric ligature groups in the maxillary arch. Clinically, the nonconventional elastomeric ligature group showed no reduction in time required for complete canine retraction in the maxillary and mandibular arches., (Copyright © 2012 American Association of Orthodontists. Published by Mosby, Inc. All rights reserved.)

An Arabidopsis mutant line T90, exhibiting a stem-specific and wound-responsive GUS expression was identified from a population of Arabidopsis thaliana tagged with a promoterless β-glucuronidase (GUS) in the T-DNA. Sequence flanking the insertion from the right border was amplified by TAIL PCR and cloned. The insertion was located in the third chromosome, 57 bp upstream of the ATG start codon in 5' untranslated region (UTR) of the fatty acyl-CoA reductase 6 (FAR6) gene. RT-PCR analysis of the FAR6 gene revealed that the gene is expressed predominantly in stem tissue. Semi-quantitative RT-PCR showed that the expression is also induced by wounding in the epidermal layer of mature stem internodes. The transcription initiation site (TSS) was identified by 5' RACE PCR. Different 5' deletion fragments of the promoter sequences were developed and linked to the GUS reporter gene as transcriptional fusions and the expression patterns of GUS were histochemically analyzed in transgenic Arabidopsis plants. Sequences from -510 bp upstream to the transcriptional start site were sufficient to exhibit wound-inducible GUS expression in the stems. The addition of further upstream sequences (-510 to -958, -1,400 or -1,456) enhanced and extended the wound-inducible GUS expression throughout the mature stem.

Nuclear-mitochondrial gene interactions governing cytoplasmic male sterility (CMS) in angiosperms have been found to be unique to each system. Fertility restoration of three diverse alloplasmic CMS lines of Brassica juncea by a line carrying the fertility-restorer gene introgressed from Moricandia arvensis prompted this investigation to examine the molecular basis of CMS in these lines. Since previous studies had found altered atpA transcription associated with CMS in these lines, the atpA genes and transcripts of CMS, fertility-restored, and euplasmic lines were cloned and compared. atpA coding and downstream sequences were conserved among CMS and euplasmic lines but major differences were found in the 5' flanking sequences of atpA. A unique open reading frame (ORF), orf108, co-transcribed with atpA, was found in male sterile flowers of CMS lines carrying mitochondrial genomes of Diplotaxis berthautii, D. catholica, or D. erucoides. In presence of the restorer gene, the bicistronic orf108-atpA transcript was cleaved within orf108 to yield a monocistronic atpA transcript. Transgenic expression of orf108 with anther-specific Atprx18 promoter in Arabidopsis thaliana gave 50% pollen sterility, indicating that Orf108 is lethal at the gametophytic stage. Further, lack of transmission of orf108 to the progeny showed for the first time that mitochondrial ORFs could also cause female sterility. orf108 was found to be widely distributed among wild relatives of Brassica, indicating its ancient origin. This is the first report that shows that CMS lines of different origin and morphology could share common molecular basis. The gametic lethality of Orf108 offers a novel opportunity for transgene containment.

Hypercalcemia is rare in neonates but may be associated with hypophosphatemia in Extremely low birth weight (ELBW) neonates who are on parenteral nutrition without adequate phosphate supplementation.

One of the many indications for dental arch expansion in treating malocclusion is to achieve arch compatibility, especially in surgical cases with severe Bolton discrepancies or collapsed arches due to congenitally missing anterior teeth. These cases usually require expansion in both the sagittal and transverse plane to achieve normal arch compatibility. Arch compatibility can be achieved by either dentoalveolar or skeletal expansion or both. Orthodontically, dentoalveolar expansion can be achieved by means of expanded arch form, vertical loops, or an added assembly such as a quad helix or Ni-Ti expander from the palatal or lingual aspect of the arch. However, these modalities normally provide expansion along transverse plane. If any expansion along sagittal plane is required, then additional appliances such as TransForce or modifications in the appliance system (eg, a quad helix with extension on anterior teeth) are necessary. Vertical loops do overcome these drawbacks to a certain extent; however, at the expense of generating moments during preactivation, which may lead to tipping of segments adjacent to the loop and precludes its use for larger changes of arch dimension. This article describes a new loop design--the KD loop--that increases the arch perimeter by sagittal and transverse expansion without generating significant moments along vertical plane.

The size of the envelope of tooth movements using fixed mechanotherapy has been increased with the use of temporary anchorage devices (TADs). Orthodontic mini-implants, a form of TADs, have been successfully used for achieving a variety of tooth movements, such as bodily retraction, extrusion, protraction, and even intrusion of maxillary molars. However, the use of orthodontic mini-implants for intruding mandibular molars is questionable due to anatomical constraints. Skeletal anchorage systems (SASs), another form of TADs, overcome these limitations to give promising results for mandibular molar intrusion. The following case report shows the use of unilateral SAS for intruding two mandibular molars and extruding a maxillary molar of the same side to establish a stable occlusal plane. The amount of intrusion achieved in relation to mandibular molars was evaluated by comparing panoramic images. The mandibular left first and second molars were intruded by approximately 1.6 and 2.5 mm, respectively, in relation to the occlusal plane.

Aim: To check the reliability of panoramic radiographs in assessing mesiodistal angulations while considering the true long axes of teeth in relation to a horizontal reference archwire., Method: A clear anatomical typodont with removable teeth was used with 0.8-mm chromium steel balls glued over the incisal/occlusal and apical/furcal portions of teeth to serve as reference markers for representing true long axes of teeth on radiographs and photographs. A photograph of each tooth was taken with a particular technique to serve as a medium through which to measure true mesiodistal angulations of teeth., Results: Only overall maxillary teeth angulations significantly correlated to true mesiodistal angulations. Radiographic relationship of the long axes of adjacent teeth (convergence or divergence) in the mandibular as well as the maxillary arch did not show any significant correlation to true degree of convergence or divergence and displayed a tendency to accentuate the maxillary canine to premolar divergence and mandibular lateral incisor to canine convergence., Conclusion: Panoramic radiographs provide a poor representation of the mesiodistal angulations of teeth and require cautious use with clinical judgment and adjunctive procedures to ascertain root angulations.

Starting from HTS hit 1a, X-ray co-crystallization and molecular modeling were used to design potent and selective inhibitors of PI3-kinase. Bioavailablity in this series was improved through careful modulation of physicochemical properties. Compound 12 displayed in vivo knockdown of PI3K pharmacodynamic markers such as pAKT, pPRAS40, and pS6RP in a PC3 prostate cancer xenograft model., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Molecular markers linked to QTL contributing to agronomic and fibre quality traits would be useful for cotton improvement. We have attempted to tag yield and fibre quality traits with AFLP and SSR markers using F(2) and F(3) populations of a cross between two Gossypium hirsutum varieties, PS56-4 and RS2013. Out of 50 AFLP primer combinations and 177 SSR primer pairs tested, 32 AFLP and four SSR primers were chosen for genotyping F(2) individuals. Marker-trait associations were studied for eight agronomic and five fibre quality traits through simple and multiple regression analysis (MRA) using a set of 92 AFLP polymorphic loci and four SSR markers. Simple linear regression analysis (SLRA) identified 23 markers for eight different traits whereas multiple regression analysis identified 30 markers for at least one of the 13 traits. SSR marker BNL 3502 was consistently identified to be associated with fibre strength. While all the markers identified in SLRA were also detected in MRA, as many as 16 of the 30 markers were identified to be associated with respective traits in both F2 and F3 generations. The markers explained up to 41 per cent of phenotypic variation for individual traits. A number of markers were found to be associated with multiple traits suggesting clustering of QTLs for fibre quality traits in cotton.

Mitochondrial atpA transcripts were examined in cytoplasmic male sterile (CMS) and fertility restorer lines of CMS (Moricandia arvensis) Brassica juncea. Male sterile flowers had longer atpA transcripts than male fertiles. The mitochondrial atpA region of the CMS line was cloned and sequenced. The 5' and 3' ends of the atpA transcripts of the CMS and the fertility restorer lines were mapped and full-length transcripts were cloned and sequenced. A novel orf108 (open reading frame 108) co-transcribed with the atpA gene was found in the male sterile flowers. In the fertility restorer line, the transcript was cleaved within orf108 to yield monocistronic atpA transcripts.

A series of substituted 4,5,6,7-tetrahydrothieno[3,2-c]pyridines (THTPs) was synthesized and evaluated for their human phenylethanolamine N-methyltransferase (hPNMT) inhibitory potency and affinity for the alpha(2)-adrenoceptor. The THTP nucleus was suggested as an isosteric replacement for the 1,2,3,4-tetrahydroisoquinoline (THIQ) ring system on the basis that 3-thienylmethylamine (18) was more potent as an inhibitor of hPNMT and more selective toward the alpha(2)-adrenoceptor than benzylamine (15). Although the isosterism was confirmed, with similar influence of functional groups and chirality in both systems on hPNMT inhibitory potency and selectivity, the THTP compounds proved, in general, to be less potent as inhibitors of hPNMT than their THIQ counterparts, with the drop in potency being primarily attributed to the electronic properties of the thiophene ring. A hypothesis for the reduced hPNMT inhibitory potency of these compounds has been formed on the basis of molecular modeling and docking studies using the X-ray crystal structures of hPNMT co-crystallized with THIQ-type inhibitors and S-adenosyl-L-homocysteine as a template.

A cytoplasmic male sterile (CMS) line of Brassica juncea was derived by repeated backcrossing of the somatic hybrid (Diplotaxis catholica + B. juncea) to B. juncea. The new CMS line is comparable to euplasmic lines for almost all characters, except for flowers which bear slender, needle-like anthers with aborted pollen. Detailed Southern analysis revealed two copies of coxI gene in the CMS line. One copy, coxI-1 is similar to the coxI gene of B. juncea, whereas the second copy, coxI-2 is present in a novel rearranged region. Northern analysis with eight mitochondrial gene probes showed altered transcript pattern only for the coxI gene. Two transcripts of 2.0 and 2.4 kb, respectively, were detected in the CMS line. The novel 2.4 kb transcript was present in floral bud tissue but absent in the leaf tissue. In plants where male sterility broke down under high temperature during the later part of the growing season, the 2.4 kb coxI transcript was absent, which suggested its association with the CMS. The two coxI genes from the CMS line showed two amino acid changes in the coding region. The novel coxI gene showed unique repeats in the 5' region suggesting recombination of mitochondrial genomes of the two species. The possible role of the duplicated coxI gene in causing male sterility is discussed.

We have developed a cytoplasmic male sterile (CMS) line of Brassica juncea through somatic hybridization with Moricandia arvensis and introgressed the fertility restorer gene into B. juncea. This fertility restorer locus is unique in that it is capable of restoring male fertility to two other alloplasmic CMS systems of B. juncea. As a first step toward cloning of this restorer gene we attempted molecular tagging of the Rf locus using the amplified fragment length polymorphism (AFLP) technique. A BC(1)F(1) population segregating for male sterility/fertility was used for tagging using the bulk segregant analysis method. Out of 64 primer combinations tested in the bulks, 5 combinations gave polymorphic amplification patterns. Further testing of these primers in individual plants showed four amplicons associated with the male fertility trait. Polymorphic amplicons were cloned and used for designing SCAR primers. One of the SCAR primers generated amplicons mostly in the fertile plants. Linkage analysis using MAPMAKER showed two AFLP and one SCAR markers linked to the male fertility gene with a map distance ranging from 0.6 to 2.9 cM. All the markers are located on one side of the Rf locus.