Effect of head covering on phototherapy induced hypocalcemia in term neonates with hyperbilirubinemia: A randomised controlled study.

BACKGROUND: Phototherapy is the primary treatment for hyperbilirubinemia in neonates. Hypocalcemia is a lesser known but potential detrimental effect of phototherapy. It has been hypothesized that phototherapy inhibits pineal secretion of melatonin, which blocks the effect of cortisol on bone calcium. Therefore, unchecked cortisol increases bone uptake of calcium and induces hypocalcemia. Covering head during phototherapy in order to prevent light reaching to the pineal gland which eventually leads to the prevention of hypocalcemia is hypothesized to prevent hypocalcemia but it lacks sufficient evidence worldwide. METHOD: It is a prospective, randomized controlled study. 112 neonates were randomized into two groups of 56 neonates. Group A underwent phototherapy without head cover and group B with head covered by a cap. RESULT: The mean decline in serum ionic calcium after 48 hours of phototherapy in group A and group B was 0.57±0.37 mg/dl and 0.34±0.24 mg/dl respectively. This decline in serum ionic calcium was significantly higher in group A. (p < 0.001). 26.8% newborns from group A developed hypocalcemia while in group B only 14.3% developed hypocalcemia however it was not found to be statistically significant. Incidence of symptomatic hypocalcemia between the two groups was also not significant. CONCLUSION: There was significant reduction in serum calcium in neonates undergoing phototherapy without head cover as compared to neonates with head cover but risk of hypocalcemia was not significant. Further studies with larger sample size including preterm are recommended.

Alpha lipoic acid supplementation ameliorates the wrath of simulated tropical heat and humidity stress in male Murrah buffaloes.

A supplement which ameliorates temperature-humidity menace in food producing livestock is a prerequisite to develop climate smart agricultural packages. A study was conducted to investigate the heat stress ameliorative efficacy of alpha lipoic acid (ALA) in male Murrah water buffaloes (Bubalus bubalis). Eighteen animals (293.61 ± 4.66Kg Bwt) were randomly allocated into three groups (n = 6); NHSC (non-heat-stressed control), HS (heat-stressed) and HSLA (heat-stressed-supplemented with ALA@32 mg/kg Bwt orally) based on the temperature humidity index (THI) and ALA supplementation. HS and HSLA were exposed to simulated heat challenge in a climatically controlled chamber (40 °C) for 21 consecutive days, 6 h daily. Physiological responses viz. Respiration rate (RR), Pulse rate (PR) and Rectal temperature (RT) were recorded daily before and after heat exposure. Blood samples were collected at the end of heat exposure on days 1, 6, 11, 16, and 21 and on day 28 (7th day post exposure which is considered as recovery) for peripheral blood mononuclear cells (PBMCs) separation, followed by RNA and Protein extraction for Real time quantitative PCR and Western blot analysis respectively, of heat shock proteins (HSPs). Two-way repeated measure ANOVA was performed between groups at different experimental periods. RR (post exposure) in HS and HSLA was significantly higher (P < 0.05) than NHSC from day 1 onwards but HSLA varied significantly from the HS 8th day onwards. Post exposure RT and PR in both HS and HSLA varied (P < 0.05) from NHSC throughout the study; but between HS and HSLA, RT significantly varied on initial 2 days and last 6 days (from days 16 to 21). HSP70 mRNA expression significantly up regulated in high THI groups with respect to the low THI group throughout the experimental period. During chronic stress (days 16 and 21) HSP70 significantly (P < 0.05) increased in HS but not in HSLA (P > 0.05) with respect to NHSC. ALA supplementation up-regulates and sustains (P < 0.05) the expression of HSP90 in HSLA in comparison to the HS and NHSC. HSP105 expression was significantly up-regulated (P < 0.05) in HS on days 16 and 21 (during long-term exposure) but only on day 21 (P < 0.05) in HSLA. HSP70, HSP90, and HSP105 protein expression dynamics were akin to the mRNA transcript data between the study groups. In conclusion, supplementing ALA ameliorates the deleterious effect of heat stress as reflected by improved physiological and cellular responses. ALA supplementation improved cellular antioxidant status and sustained otherwise easily decaying heat shock responses which concertedly hasten the baton change from a limited window of thermo tolerance to long run acclimatization.

Cryopreservation of Axillary Buds of Cannabis sativa L. by V-Cryoplate Droplet-Vitrification: The Critical Role of Sucrose Preculture.

BACKGROUND: Cryopreservation is the only method allowing the safe and cost-effective long-term conservation of important germplasm. Recent use of the cryo-plate system has proven beneficial in further simplifying the cryopreservation protocols. OBJECTIVE: Developing an efficient protocol for the cryopreservation of axillary buds of Cannabis sativa elite cultivars (MX and V1-20) by the V-cryoplate droplet-vitrification technique. MATERIALS AND METHODS: Stem segments (~5 cm in length) with mature axillary buds collected from indoor-grown plants were surface sterilized and then either precultured on MS basal medium with 0.1 M sucrose (1st step preculture) for 72 h or non-precultured. All mature axillary buds (~1 mm) were aseptically excised from stem segments and precultured for an additional 48 h on MS basal medium with sucrose (0.3 M) and 5% DMSO prior to cryopreservation (2nd step preculture). Biomass samples of fully mature mother plants and regrown cryopreserved plants were analyzed for Δ9-THC and CBD content using gas chromatography-flame ionization detector (GC/FID). RESULTS: The survival and regrowth rates of cryopreserved axillary buds of cultivar MX following this two-step preculture were 45% and 42% respectively, while those of cultivar V1-20 were 47% and 44% respectively. A direct preculture of axillary buds (2nd step preculture) on high sucrose (0.3M sucrose) significantly decreased both the survival and regrowth levels of axillary buds of cultivar MX (5% and 3% respectively) as well as those of cultivar V1-20 (20% and 17% respectively). Δ9-THC and CBD content of mother plants and regrown cryopreserved plants were found to be highly comparable to each other. CONCLUSION: The resulting plants after cryopreservation appeared normal without any callus formation or morphogenetic variation. On maturity, mother plants and re-grown cryopreserved plants were comparable in terms of Δ9-THC and CBD content. This report provides an efficient protocol for cryopreservation of axillary buds of Cannabis sativa cultivars which may be applicable to other important cultivars, plant parts and other related medicinal plants.

Dynamics of antibiotic resistance with special reference to Shiga toxin-producing Escherichia coli infections.

The discovery of antibiotics was paralleled by the evolution of antibiotic resistance which is probably the best example of contemporary evolution in action. The selection pressure, imposed by indiscriminate use of antibiotics, has changed the scale, mode and tempo of antibiotic resistance evolution. The presence of multidrug resistance, wide range of adaptability features and the infectivity make antibiotic resistance of Shiga toxin-producing Escherichia coli (STEC) more dangerous. The characterization, prevalence and the virulence factors of STEC have been profusely reported, whereas, the antibiotic resistance has been largely ignored because the antibiotic use in STEC infections is controversial. Thus, the current review has focussed on the source, evolution, persistence, mechanism, dissemination and control of antibiotic resistance viz-a-viz the STEC infections. The resistance development occurs by the inactivation of antibiotics, regulating the membrane permeability, modification of natural antibiotic targets or the use of efflux pumps against antibiotics. And, the dissemination of resistance genes occurs vertically by DNA replication and horizontally by conjugation, transduction and transformation. The prevention of development and dissemination of antibiotic resistance needs international public health bodies to rationalize the antibiotic use, prevent the flux of antibiotics into the environment, develop the rapid diagnostics tests, undertake proper surveillance of antibiotic resistance, promote the research on antibiotic resistance prevention, promote the research and development of novel alternative antibiotics, and encourage the widespread social awareness campaigns against the inappropriate antibiotic usage.

Foliar anatomy and microscopy of six Brazilian species of Baccharis (Asteraceae).

We report for the first time the presence of cluster crystals of calcium oxalate within the glandular trichomes and oil bodies in the mesophyll for Baccharis species. Moreover, the comparative leaf anatomy and micro-morphology of six species of Baccharis, namely B. illinita, B. microdonta, B. pauciflosculosa, B. punctulata, B. reticularioides, and B. sphenophylla is investigated by light and scanning electron microscopy. The studied species exhibited differences in their leaf anatomical features such as the morphology of the cuticle, type and occurrence of the stomata, presence or absence of glandular trichomes, shape of the flagelliform trichomes, and the arrangement of the mesophyll tissues. These differences can be helpful in the species identification and classification and could represent informative characters for the reconstruction of the evolution of the genus.

Assessment of allelic diversity among drought-resistant cotton genotypes using microsatellite markers.

Drought, in conjunction with high temperature, is an important environmental constraint to cotton production. Development of cotton varieties with increased tolerance against adverse environmental conditions has been proposed as effective strategy for ensuring reliable yields. In the present study, 30 simple sequence repeat (SSR) primers were used to estimate genetic divergence among 22 cotton genotypes for drought stress tolerance. Genetic diversity is a prerequisite for developing drought resistant cotton genotypes. Eleven SSR primers out of 30 were able to discriminate among the cotton genotypes, implying that 37% of the primers were informative. In total, 41 alleles were detected, with an average of 3.72 alleles per primer. The number of alleles per locus ranged from one (JESPR-284) to six (JESSPR-302), and the allelic diversity in the experimental material was 0.40. Genetic similarity coefficients ranged from 0.87-1.00. The result of principal component analysis confirmed the clustering of 21 cotton genotypes in two groups leaving one genotype (CIM-109) ungrouped. Overall, genetic diversity among the 22 cotton genotypes was low. More polymorphic SSR markers are needed to explore the workable genetic variation among the screened cotton genotypes in future studies.

Anionic and Cationic Silver Nanoparticle Binding Restructures Net-Anionic PC/PG Monolayers with Saturated or Unsaturated Lipids.

We have examined the interactions between polymer-coated anionic (Ag-COOH) and cationic (Ag-NH) silver nanoparticles, and net-anionic lipid monolayers using dynamic surface pressure measurements. Monolayers composed of saturated or monounsaturated mixtures of anionic phosphatidylglycerol (PG) and zwitterionic phosphatidylcholine (PC) lipids (3:1 molar ratio) were used to determine how lipid packing and monolayer phase state influence the extent of nanoparticle binding and the monolayer response. Anionic Ag-COOH inserted into saturated dipalmitoyl-PC/PG (DPPC/DPPG) and dioleoyl-PC/PG (DOPC/DOPG) monolayers at a low initial surface pressure (10 mN m-1) and caused lipid condensation at high initial surface pressures (20 and 30 mN m-1). Hydrophobic interactions were responsible for insertion, while electrostatic and charge-dipole interactions with PCs were responsible for condensation. In contrast, cationic Ag-NH inserted only into saturated DPPC/DPPG monolayers and otherwise led to lipid condensation. For Ag-NH, adsorption was driven primarily by electrostatic interactions with PGs. Analysis of the subphase Ag and phosphorus concentrations confirmed that Ag-NH had a higher degree binding compared to Ag-COOH, and that the monolayer response was not due to lipid extraction.

Genetic analysis of drought tolerance with respect to fiber traits in upland cotton.

Cotton germplasm was analyzed to investigate its potential for developing water stress tolerance in varieties in the future. Four tolerant (NIAB-78, CIM-482, BH-121, and VH-142) and four susceptible (CIM-446, FH-1000, FH-900, and FH-901) lines were identified of 50 accessions based on their seedling root length. A complete set of diallel crosses among eight selected genotypes was subjected to genetic analysis for fiber property traits. Additive and non-additive genetic variance was involved in the inheritance of fiber strength, fineness, and length under normal and drought conditions. A large proportion of genetic variance was additive, which was further supported by moderately high narrow-sense heritability estimates for the characters. Graphic representation of variance versus covariance also depicted additive gene activity with partial dominance and the absence of non-allelic interactions in trait inheritance. The results of this study suggest that drought tolerance of cotton genotypes can be improved through crosses among tolerant genotypes using conventional selection procedures in segregating generations.

Chemical composition and biological activities of Artemisia judaica essential oil from southern desert of Jordan.

ETHNOPHARMACOLOGIC RELEVANCE: Artemisia judaica L. (Arabic name: Beithran), is a medicinal and aromatic plant growing in the valley bottoms of desert areas, particularly in the southern desert of Jordan nearest to the Jordan-Saudi Arabia borders and in Wadi Araba in the Southern Badia. In Jordan, A. judaica is widely used in traditional medicine being recommended by aboriginal Bedouins in the North Badia region of Jordan as calmative. Furthermore, it is used for the treatment of stomach ache, heart diseases, sexual weakness, diabetes, gastro-intestinal disorders and external wounding. Additionally, other folk medicines of the Arabic region commonly use this aromatic plant for the treatment of inflammatory-related diseases, for instance fungal infections, diabetes, atherosclerosis, cancer and arthritis. AIM OF THE STUDY: Considering the traditional medicinal uses and the lack of scientific studies addressing the cellular and molecular mechanisms behind A. judaica claimed activities, the present study was designed to validate some of the traditional uses ascribed to this species, specifically the antifungal and anti-inflammatory activities of A. judaica essential oil at doses devoid of cytotoxicity to mammalian cells. MATERIALS AND METHODS: Chemical analysis of A. judaica essential oil isolated by hydrodistillation from aerial parts was carried out by gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS). The antifungal activity (minimal inhibitory concentrations and minimal lethal concentrations) was evaluated against yeasts, dermatophyte and Aspergillus strains. In order to deeply explore the mechanisms behind the anti-fungal effect of the essential oil, the germ tube inhibition assay and the biofilms formation assay were evaluated using Candida albicans. The assessment of cell viability was accomplished using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay in both hepatocytes and macrophages. Furthermore, the in vitro anti-inflammatory potential of A. judaica oil was evaluated by measuring nitric oxide (NO) production using lipopolysaccharide (LPS)-stimulated mouse macrophages. RESULTS: Oxygen containing monoterpenes are a representative group of constituents (68.7%) with piperitone (30.4%), camphor (16.1%) and ethyl cinnamate (11.0%) as main compounds. The highest antifungal activity of the oil was observed against Cryptococcus neoformans, with a MIC value of 0.16µL/mL. The oil revealed an important inhibitory effect on germ tube formation in C. albicans with 80% inhibition of filamentation at a concentration of 0.16µL/mL. Importantly, the oil also interfered with pre-formed biofilms by reducing the amount of the attached biomass. Furthermore, the essential oil significantly inhibited NO production evoked by LPS on macrophages at concentrations with very low toxicity (0.32µL/mL) or without toxicity (0.16µL/mL) to both macrophages and hepatocytes. CONCLUSIONS: The present study revealed that A. judaica essential oil from Jordan significantly inhibited germ tube formation and disrupted preformed biofilms of C. albicans, emphasizing the therapeutic potential for the treatment of disseminated candidiasis. Additionally, safe concentrations of this essential oil significantly inhibited NO production elicited by LPS in macrophages, highlighting its potential anti-inflammatory activity. Overall, A. judaica bears promising therapeutic potential for further drug development. Importantly, this work also validates some of the traditional uses of A. judaica.

ErbB2-dependent downregulation of a pro-apoptotic protein Perp is required for oncogenic transformation of breast epithelial cells.

The ability of breast cancer cells to resist anoikis, apoptosis caused by detachment of the non-malignant epithelial cells from the extracellular matrix (ECM), is thought to be critical for breast tumor growth, invasion and metastasis. ErbB2, an oncoprotein that is often overproduced in breast tumors, can block breast cancer cell anoikis via mechanisms that are understood only in part. In an effort to understand them better we found that detachment of the non-malignant human breast epithelial cells from the ECM upregulates a protein Perp in these cells. Perp is a component of the desmosomes, multiprotein complexes involved in cell-to-cell adhesion. Perp can cause apoptosis via unknown mechanisms. We demonstrated that Perp upregulation by cell detachment is driven by detachment-induced loss of epidermal growth factor receptor (EGFR). We also found that Perp knockdown by RNA interference (RNAi) rescues detached cells from death which indicates that Perp contributes to their anoikis. We observed that ErbB2, when overexpressed in detached breast epithelial cells, causes Perp downregulation. Furthermore, ErbB2-directed RNAi or treatment with lapatinib, an ErbB2/EGFR small-molecule inhibitor used for breast cancer therapy, upregulated Perp in ErbB2-positive human breast and ovarian carcinoma cells. We established that ErbB2 downregulates Perp by activating an ErbB2 effector protein kinase Mek that blocks detachment-induced EGFR loss in a manner that requires the presence of a signaling protein Sprouty-2. Finally, we observed that restoration of the wild-type Perp levels in ErbB2-overproducing breast epithelial cells increases their anoikis susceptibility and blocks their clonogenicity in the absence of adhesion to the ECM. In summary, we have identified a novel mechanism of ErbB2-mediated mechanism of anoikis resistance of ErbB2-overproducing breast epithelial cells. This mechanism allows such cells to grow without adhesion to the ECM and is driven by ErbB2-induced activation of Mek, subsequent EGFR upregulation and further EGFR-dependent Perp loss.

Comparative studies on the chemical and enzymatic stability of alpha- and beta-arbutin.

OBJECTIVE: The aim of this study was to establish a comparative analysis of the chemical and enzymatic stability of α- and ß-arbutins as potential sources of the substance of concern hydroquinone (HQ). The study was performed using an array of techniques including HPLC-PDA, nuclear magnetic resonance (NMR) and optical rotation (OR). Both arbutins are emerging as popular and effective skin whiteners, acting as tyrosinase inhibitors in a fashion similar to the popular whitening agent HQ. Due to their structural similarity to the regulated agent HQ, both arbutins may be regarded as potential sources of the active aglycone after chemical or metabolic conversion. METHODS: Various cosmetic formulations including creams, sera, gels and lotions were analysed by HPLC-PDA for their arbutin and HQ content in freshly opened and aged samples stored for 16 months. Solutions of pure compounds were also aged and periodically checked for degradation products using 1D and 2D NMR experiments and OR measurements. The metabolic stability was investigated using pear peels as a biological model. RESULTS: Both arbutins were found to be stable in water and methanol solutions in the absence of buffer or stabilizers. Their stability in cosmetic formulations, however, was found to depend on the type of formulation and pH. Both compounds were unstable under strong hydrolytic conditions, with consequent release of HQ. Enzymatic instability of both arbutins was also observed, although no formation of HQ was observed under the chosen experimental conditions. CONCLUSION: Both arbutins were found to possess similar stability profiles, and to be more prone to in vivo rather than in chemico degradation, although no HQ was found after enzymatic hydrolysis. Also, no epimerization was observed in any of the tested conditions. Diverse experimental approaches can be applied to analyse the chemical and enzymatic stability of arbutins in regard to the potential release of HQ in different types of preparations. These result showed the potential use of NMR and OR as complementary investigative tools for the stability and safety assessment of arbutin along with more established HPLC methods.

CRISPR/Cas9: molecular tool for gene therapy to target genome and epigenome in the treatment of lung cancer.

Although varied drugs and therapies have been developed for lung cancer treatment, in the past 5 years overall survival rates have not improved much. It has also been reported that lung cancer is diagnosed in most of the patients when it is already in the advanced stages with heterogeneous tumors where single therapy is mostly ineffective. A combination of therapies are being administered and specific genes in specific tissues are targeted while protecting normal cell, but most of the therapies face drawbacks for the development of resistance against them and tumor progression. Therefore, therapeutic implications for various therapies need to be complemented by divergent strategies. This review frames utilization of CRISPR/Cas9 for molecular targeted gene therapy leading to long-term repression and activation or inhibition of molecular targets linked to lung cancer, avoiding the cycles of therapy.

Stem cell intervention ameliorates epigallocatechin-3-gallate/lipopolysaccharide-induced hepatotoxicity in mice.

Stem cells are identified as a novel cell therapy for regenerative medicine because of their ability to differentiate into many functional cell types. We have shown earlier a new model of hepatotoxicity in mice by administering (1500 mg/kg) epigallocatechin-3-gallate (EGCG) intragastric (IG) for 5 days after a single intraperitoneal dose (6 mg/kg) of lipopolysaccharide (LPS). In this study, we aimed to study the effect of intrahepatic (IH) injection of mouse embryonic stem cells (MESCs) on the hepatotoxicity induced by EGCG/LPS in mice. Mice were administered EGCG/LPS and rested for 3 days. MESCs were obtained from American Type Culture Collection and cultured in vitro for 4 days. Stem cells were injected IH. Seven days later, a single dose of LPS (6 mg/kg) followed by daily doses of IG administration of EGCG were re-administered for 5 days. At the end of the experiment, blood samples were collected for analysis of biochemical parameters associated with liver. Results showed that the group of mice that were administered MESCs prior to EGCG/LPS showed lower levels of alanine amino transferase, alkaline phosphatase, and bilirubin, higher albumin/globulin ratio, and less remarkable histopathological lesions. Also, that group of mice showed less expression of oxidative stress biomarkers (oxidized low-density lipoprotein Ox.LDL and chemokine CXCL16), less expression of nuclear protein receptors (retinoic acid receptor and retinoid X receptor), and less expression of inflammatory biomarkers (tumor necrosis factor α and transforming growth factor ß1) compared with other groups of mice that were not given MESCs. In conclusion, MESCs can ameliorate EGCG/LPS-induced hepatotoxicity in mice.

Anoikis of colon carcinoma cells triggered by ß-catenin loss can be enhanced by tumor necrosis factor receptor 1 antagonists.

Detachment of non-malignant epithelial cells from the extracellular matrix causes their apoptosis, a phenomenon called anoikis. By contrast, carcinoma cells are anoikis-resistant, and this resistance is thought to be critical for tumor progression. Many oncogenes trigger not only anti- but also pr-apoptotic signals. The proapoptotic events represent an aspect of a phenomenon called oncogenic stress, which acts as a safeguard mechanism blocking tumor initiation. In cells that become malignant, oncogene-induced antiapoptotic signals outbalance the proapoptotic ones. It is now thought that treatments blocking the antiapoptotic events but preserving the proapoptotic signals can be particularly effective in killing tumor cells. Whether or not oncogenes induce any proanoikis signals that can be used for enhancing the efficiency of approaches aimed at triggering anoikis of cancer cells has never been explored. ß-Catenin is a major oncoprotein that is often activated in colorectal cancer and promotes tumor progression via mechanisms that are understood only in part. We found here that ß-catenin triggers both anti- and proanoikis signals in colon cancer cells. We observed that the antianoikis signals prevail and the cells become anoikis-resistant. We further established that one proanoikis signal in these cells is triggered by ß-catenin-induced downregulation of an apoptosis inhibitor tumor necrosis factor receptor 1 (TNFR1) and subsequent reduction of the activity of a transcription factor NF-κB (nuclear factor-κB), a mediator of TNFR1 signaling. We also found that the effect of ß-catenin on TNFR1 requires the presence of transcription factor TCF1, a ß-catenin effector. We demonstrated that ablation of ß-catenin in colon cancer cells triggers their anoikis and that this anoikis is enhanced even further if low TNFR1 or NF-κB activity is artificially preserved in the ß-catenin-deprived cells. Thus, inhibition of TNFR1 or NF-κB activity can be expected to enhance the efficiency of approaches aimed at blocking ß-catenin-driven anoikis resistance of colon carcinoma cells.

DNA barcoding for species identification in the Palmae family.

DNA barcoding is a promising tool for species identification at the molecular level. The barcoding system is well established for species differentiation in animals, while it is less common in plants. We evaluated 2 barcoding regions, maturase K (matK) and ribulose bisphosphate carboxylase (rbcL), to compare species of Palmae according to amplification success, discrimination power, and inter- and intra-specific divergence. Both regions appear to have potential to discriminate most species of Palmae, but 2 species, Phoenix dactylifera and Phoenix sylvestris, did not show variation in the nucleotides of the barcode genes. P. sylvestris is said to be the sister species of P. dactilyfera according to its morphological and genetic proximity to the cultivated date palm. Thus, the status of these 2 species needs to be re-evaluated considering more genes as barcodes. Furthermore, rbcL has a higher discrimination power (90%) than matK (66.6%) and can thus be potentially used as a standard barcode to discriminate the species of Palmae.

Identification and authentication of Rosa species through development of species-specific SCAR marker(s).

Roses (Rosa indica) belong to one of the most crucial groups of plants in the floriculture industry. Rosa species have special fragrances of interest to the perfume and pharmaceutical industries. The genetic diversity of plants based on morphological characteristics is difficult to measure under natural conditions due to the influence of environmental factors, which is why a reliable fingerprinting method was developed to overcome this problem. The development of molecular markers will enable the identification of Rosa species. In the present study, randomly amplified polymorphic DNA (RAPD) analysis was done on four Rosa species, Rosa gruss-an-teplitz (Surkha), Rosa bourboniana, Rosa centifolia, and Rosa damascena. A polymorphic RAPD fragment of 391 bp was detected in R. bourboniana, which was cloned, purified, sequenced, and used to design a pair of species-specific sequence-characterized amplified region (SCAR) primers (forward and reverse). These SCAR primers were used to amplify the specific regions of the rose genome. These PCR amplifications with specific primers are less sensitive to reaction conditions, and due to their high reproducibility, these species-specific SCAR primers can be used for marker-assisted selection and identification of Rosa species.

Modulation of ethanol toxicity by Asian ginseng (Panax ginseng) in Japanese ricefish (Oryzias latipes) embryogenesis.

Alcohol consumption by women during pregnancy often induces fetal alcohol spectrum disorder (FASD) in children who have serious central nervous system (CNS), cardiovascular, and craniofacial defects. Prevention of FASD, other than women abstaining from alcohol drinking during pregnancy, is not known. A limitation of the use of synthetic anti-alcoholic drugs during pregnancy led us to investigate herbal products. In particular, many plants including Asian ginseng (Panax ginseng) have therapeutic potential for the treatment of alcoholism. We used Japanese ricefish (medaka) (Oryzias latipes), an animal model of FASD, for identifying herbal medicines that can attenuate ethanol toxicity. Fertilized eggs in standard laboratory conditions were exposed to ginseng (PG) root extract (0-2 mg/mL) either 0-2 (group A) or 1-3 (group B) day post fertilization (dpf) followed by maintenance in a clean hatching solution. The calculated IC50 as determined 10 dpf in A and B groups were 355.3±1.12 and 679.7±1.6 µg/mL, respectively. Simultaneous exposure of embryos in sub-lethal concentrations of PG (50-200 µg/mL) and ethanol (300 mM) for 48 h disrupted vessel circulation and enhanced mortality. However, PG (100 µg/mL) may partially protect trabecular cartilage (TC) deformities in the neurocranium in B group embryos induced by ethanol (300 mM). To understand the mechanism, embryonic ethanol concentration was measured at 2 dpf and adh5, adh8, aldh2, aldh9a, catalase, GST, and GR mRNAs were analyzed at 6 dpf. It was observed that although ethanol is able to reduce adh8 and GST mRNA contents, the simultaneous addition of PG was unable to alter ethanol level as well as mRNA contents in these embryos. Therefore, antagonistic effects of PG on ethanol toxicity are mediated by a mechanism which is different from those regulating ethanol metabolism and oxidative stress.

Establishment of an in vitro regeneration system for genetic transformation of selected sugarcane genotypes.

A good culture system provides considerable quantities of highly regenerable target tissues. Embryogenic callus cultures are ideal for micro-projectile-mediated transformation, because regenerable cells are not very stable. Effective exploitation of genetic transformation requires good regeneration systems. We selected three sugarcane genotypes for the establishment and optimization of good in vitro regeneration systems, viz., S-2003-us-359, S-2006-sp-30, and S-2003-us-165. Three callus induction media were investigated. These media were composed of Murashige and Skoog (MS) medium salt plus 1, 2, and 3 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D). Medium with 3 mg/L 2,4-D gave the greatest mass of embryogenic calli. The calli produced on the three callus induction media were transferred to 18 types of regeneration media (RM1-RM18). They varied with respect to plant growth regulators and sucrose levels but the basal medium was MS. Two levels of sucrose (30 and 40 g/L), three levels of 2,4-D (0.1, 0.25, 0.5 mg/L) and three levels of 6-benzylaminopurine (0, 0.25 and 0.5 mg/L) were studied in the regeneration media. The effects of callus age on regeneration were evaluated by transferring the calli to regeneration media after 15, 21, 28, and 35 days of culture. The 21-day-old callus of the genotype S-2003-us-359 on RM3 yielded the largest number of plants and was selected as the best for transformation. Six RAPD DNA primers were used to check genetic stability; this medium did not affect the sugarcane genomes.

Development of a species-specific sequence-characterized amplified region marker for roses.

DNA fingerprints of four rose species, Rosa centifolia, R. Gruss-an-Teplitz, R. bourboniana, and R. damascena, were developed using RAPD-PCR. We identified a unique polymorphic band in R. centifolia. This 762-bp fragment was produced by the random primer GLI-2. The fragment was eluted and directly cloned in a TA cloning vector, pTZ57R/T. Digestion of the plasmid with EcoRI confirmed the cloning of GLI-2(762) in pTZ57R/T. A second enzyme, PstI, used in combination with EcoRI, gave complete digestion of the plasmid, and the 762-bp fragment was confirmed on the gel. Subsequently, the polymorphic amplicon was sequenced with an AB1 373 DNA sequencer system using the PRISM(TM) Ready Reaction DyeDeoxy(TM) Terminator Cycle Sequencing kit. After sequencing, specific primers (23 bp long) were designed based on the sequence of the flanking regions of the original RAPD fragment. These primers will effectively allow fingerprinting for the identification of R. centifolia species. In essence, we developed an SCAR marker to authenticate the identity of R. centifolia species and to distinguish it from its substitutes. Such techniques are required not only to complement conventional parameters in creating the passport data of commercial and medicinal products of rose, but also for routine quality control in commercial and government rosaries and rose nurseries.

Immunohistochemical evaluation of p53, FHIT, and IGF2 gene expression in esophageal cancer.

The aim of the study was to evaluate the expression of tumor suppressor genes p53, fragile histidine triad gene (FHIT), and an oncogene insulin-like growth factor 2 (IGF2) as prognostic markers in the etiology of esophageal cancer. Immunohistochemistry (IHC) was performed in 39 archival tissue samples of different esophageal pathologies for the three genes. Abnormal p53 expression was maximum in all the cases of squamous cell carcinoma, while IGF2 expression was enhanced in squamous cell carcinoma (81%), adenocarcinoma (100%), and dysplasia of squamous epithelium (75%) samples when compared with normals (50%). To our surprise, 75% of normal tissues did not show FHIT expression, which was also not seen in 40% of dysplasias of squamous epithelium, 33.3% of adenocarcinoma, and 41% of squamous cell carcinoma. To the best of our knowledge, this is the first study evaluating IGF2 by IHC, as well as, correlating it with the expression of the two tumor suppressor genes, p53 and FHIT, in esophageal tissue. p53 expression was threefold higher than normal in dysplasias of squamous epithelium and adenocarcinoma, while it was eightfold higher in squamous cell carcinoma. IGF2 expression was low in normal and dysplasia tissue but was increased 1.97-fold in both types of malignancy. FHIT and p53 expression were well correlated in squamous cell carcinoma, supporting the observation that FHIT regulates and stabilizes p53. Altered/lowered FHIT levels may be a result of exposure to various exogenous agents; however, this could not be assessed in the present study as it was carried out on archival samples. A larger prospective study is warranted to establish the role of exogenous factors in FHIT expression.