Search

Vancomycin resistance in bacteria has been classified under high priority category by World Health Organization (WHO) and its presence in hospital effluent is reported to be increasing owing to excess antibiotics use. Among various strategies, bacteriophage has been recently considered as a promising biological agent for combating such antimicrobial resistant bacteria (ARB). However, the influence of effluent's properties on phage-ARB interaction in actual hospital effluent is not completely understood. The present works intends to study this influence of hospital effluent and its parameters on the interaction between vancomycin resistant E. coli (VRE) and its host specific bacteriophage. The isolated VRE was identified by 16S rRNA sequencing, matrix-assisted laser desorption/ionization-time of flight (MALDI - TOF) and whole genome sequencing. The infectivity of phage onto host bacteria was investigated using electron microscopic techniques, dynamic light scattering (DLS), spectrofluorophotometer and confirmed using double agar overlay method. The monovalency and polyvalency of isolated phage against various bacterial species were determined. The phage morphology was identical to T7 phage belonging to Podoviridae. The phage lysis was maximum at pH 7 (90.2%), 37 °C (91.6%) and vancomycin concentration of 50 μg/mL in both synthetic media (89.13%) and effluent (100%). At a maximum vancomycin concentration of 100 μg/mL, decrease in Ca, K, Mg and P (up to 19.70, 14.18, 28, and 15.82% respectively) concentration in effluent was observed due to phage infectivity when compared to control. The whole genome sequencing was performed and the bioinformatics analysis presented the role of mdfA gene encoding the efflux pump in causing vancomycin resistance in E. coli. It also depicted the presence of multiple genes responsible for mercury, cobalt, zinc and cadmium resistance in VRE. These results clearly indicate that bacteriophage mediated combating of VRE is possible in actual hospital effluent and can be used as one of the treatment methods., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Dr. R. Selvakumar reports financial support was provided by Science and Engineering Research Board. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

We previously studied 2-aryl-2-(3-indolyl)acetohydroxamates as potential agents against melanoma. These compounds were ineffective in a mouse melanoma xenograft model, most likely due to unfavorable metabolic properties, specifically due to glucuronidation of the N-hydroxyl of the hydoxamic moiety. In the present work, we prepared a series of analogues, 2-aryl-2-(3-indolyl)acetamides and their oxazoline derivatives, which do not contain the N-hydroxyl group. We investigated the structure-activity relationship in both series of compounds and found that the 2-naphthyl is a preferred group at C-2 of the indole in the amide series, whereas the tetralin moiety is favorable in the same location in the oxazoline series. Overall, three compounds in the amide series have GI 50 values as low as 0.2-0.3 µM and the results clearly indicate that the N-hydroxyl group is not necessary for high potency in vitro., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Ltd.)

The rising temperatures and levels of carbon dioxide in the atmosphere are anticipated to have a significant impact on the productivity of agricultural crops. Although, the individual effects of elevated CO 2 and temperature have been extensively studied in C3 and C4 crops, there remains a scarcity of research investigating their interactive effects specifically on maize hybrids. The impact of elevated temperature and its interaction with elevated CO 2 on phenology, physiology, biomass, and grain yield of maize hybrids was assessed in a field experiment using Free Air Temperature Elevation (FATE) facility. The results showed that elevated temperature (eT) increased the anthesis silking interval (ASI), while the presence of elevated CO 2 along with elevated temperature (eT + eCO 2 ) mitigated this effect. The differential expression were observed between hybrids depending on their genetic potential. Furthermore, the net photosynthetic rate (A net ), stomatal conductance (g s ), and transpiration rate (Tr) of hybrids decreased under elevated temperature but eT + eCO 2 condition helped in reverting its impact to some extent. In term of leaf composition, the highest level of total soluble sugars (TSS) and starch was observed under eT + eCO 2 conditions, possibly due to improved A net in the presence of elevated eCO 2 . The negative impact of eT was also evident through increased proline and MDA content, but eT + eCO 2 ameliorated the adverse effect of eT. The biomass and grain yield also responded similarly, among the hybrids 900M GOLD recorded superior performance for grain yield at eT condition exceeding 35 °C. On the other hand, DHM117 experienced a significant reduction in grain yield under eT, but performed better under eT + eCO 2 due to its improved physiological response to eCO 2 . The study indicated that elevated levels of carbon dioxide can actually mitigate the detrimental effects of elevated temperature on maize crop. This positive impact on maize crop can be attributed to an enhanced physiological performance in the presence of eCO 2 which enables the plants to maintain satisfactory yield levels despite the challenging environmental conditions., (© 2024. The Author(s).)

Background: Sexually transmitted infections (STIs) are one of the major public health problems in India with adolescents being a vulnerable section of the affected population. According to the World Health Organization (WHO), one in twenty adolescents contracts an STI every year, excluding the human immunodeficiency virus (HIV) and other viral infections. It is important to assess the knowledge, attitude, and practices related to STIs among adolescents and young adults of non-medical backgrounds to educate them properly about protective and preventive measures and to allay misconceptions, if any., Materials and Methods: This is a cross-sectional study involving 507 engineering students who were asked to fill out the predesigned anonymous questionnaire form. Results were statistically analyzed and tabulated., Results: Of the 507 students, 375 were males and 132 were females. The mean age of the patients was 20.5 years. HIV was the most commonly identified STI (94.2%), followed by herpes genitalis (76.5%). Around 97.4% and 85.7% identified the sexual route and blood transfusion as the modes of transmission of STIs, respectively. A total of 90.9% said that the Internet was their primary source of information regarding STIs. Infertility and cancer were identified as complications by 59.5% and 25% of them, respectively. Only 23.4% were aware of the National STI Control Program. The results with respect to attitudes and practices related to STIs were variable., Conclusion: Our study reflects that with change in time, the attitude of young adults toward sexuality is becoming very casual, but knowledge remains low. A lot of educational activities should be undertaken to educate our youth about various aspects of STIs and thus reduce the burden of the disease in the community., Competing Interests: There are no conflicts of interest., (Copyright: © 2023 Indian Dermatology Online Journal.)

Background: Leber's Hereditary Optic Neuropathy (LHON) is a rare mitochondriopathy causing retinal ganglion cell degeneration resulting in central vision loss. It is caused by mitochondrial DNA (mtDNA) mutations and thus follows maternal inheritance pattern., Methods: We analysed the whole mitochondrial genome in 100 South Indian LHON patients by utilizing Sanger and Next Generation Sequencing approaches. Haplogroup analysis was performed using HaploGrep2 to predict the risk group. Methylation changes in the mtDNA D-loop region were investigated by performing methylation-specific polymerase chain reaction (MSP)., Results: LHON associated mutations were detected in 55% of the patients of which 42% harboured the primary mutations and 13% harboured potentially pathogenic variants that were previously reported to cause LHON. The candidate mutations identified with confirmed pathogenicity are: m.11778G > A (38%), m.14484 T > C (3%), m.4171C > A (1%) and m.11696G > A (1%). MSP results demonstrated that the D-loop region was unmethylated in all the study subjects including mutation-positive patients, mutation-negative patients, asymptomatic carriers, and controls. Haplogroup-M was prevalent (69%) in the study cohort followed by R (14%), U (9%), N (3%), HV (2%), G (2%), and W (1%). The frequency of the predominant mutation m.11778G > A was found lower (̴ 11%) in haplogroup-U., Conclusions: South Indian LHON cohort shows a unique profile of mtDNA mutations and haplogroup association presumably with no role of D-loop methylation. MT-ND4, MT-ND5, and MT-ND1 serve as the hotspot genes in this cohort. The presence of LHON associated mutations in patients lacking the common primary mutations insists on the necessity of mitochondrial genome sequencing in individuals suspected with LHON., (Copyright © 2022 Elsevier B.V. All rights reserved.)

The current study focused on the comparative removal of hexavalent chromium using two magnetically modified hybrid adsorbent composites. Their precursor chars were prepared from bio sludge and plastic waste derivatives. The coating of magnetite on the chars' surface was evident from the SEM micrographs. Infusion of magnetite nanoparticles in the native chars aided in the reduction of the composite particles' sizes, thus, forming high surface area composites. Screening of uptake capacities among various blends of char and magnetite were surveyed. Composites of both kinds with a 1:5 ratio of char: iron salts composition answered well. The pH ZPC and zeta potential values of the composites indicated the neutral charge on the composites' surface. This suggested the need for a highly acidic environment for efficient Cr(VI) removal. Optimum economic conditions for Cr(VI) removal were obtained from the batch studies (solution pH - 1.5; contact time - (a) MPC [magnetic plastic char] - 60 min (b) MBC [magnetic biochar] - 40 min; temperature - 25 °C). The maximum monolayer adsorption capacity of MPC and MBC were found to be 84.67 mg/g and 53.83 mg/g respectively. Isotherm, kinetic and thermodynamic studies revealed the adsorption systems' inclination towards physisorption. From the characterization and modeling results, electrostatic force of attraction and pore filling was anticipated to be the mechanism of adsorption for both MPC and MBC. Thus, in the relative removal studies, MBC was found to compete better than MPC due to its enhanced porosity and surface area., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Pyomyositis is an infective involvement of systemic skeletal muscles. We discuss the case of 43-year-old male who presented with quadriparesis, anemia, and hypercalcemia, leading to suspicion of multiple myeloma, and on FDG PET-CT, incidentally, pyomyositis was found. FDG PET-CT thus helped in diagnosing an occult infection which helped in the treatment of the patient., Competing Interests: There are no conflicts of interest., (Copyright: © 2022 Indian Journal of Nuclear Medicine.)

Competing Interests: There are no conflicts of interest.

Arylative annulation of 2-carbonyl-3-propargyl indoles with boronic acids under sequential palladium/triflic acid catalysis is described. The present strategy to provide di- and triaryl carbazoles in one pot involves benzannulation through difunctionalization of alkynes. The strategy showed a good substrate scope with respect to boronic acids as well as 2-carbonyl-3-propargyl indoles to afford the corresponding carbazoles in decent yields.

A novel strategy for the synthesis of 3-hydroxycarbazoles involving the consecutive propargylation/palladium-catalyzed hydroxylative benzannulation of indole-2-carbonyls with propargylic alcohols has been exploited. This one-pot procedure leads to a wide range of substituted 3-hydroxycarbazoles in high yield with a broad substrate scope. The method was further extended to access furano-carbazole derivatives from dialkynols via tandem annulations.

An efficient acid-catalyzed propargylation/aza-annulation sequence was developed under metal-free reaction conditions, thus leading to a one-pot synthesis of a variety of substituted β-carbolines starting from propargylic alcohols and indole 2-carbonyls. This versatile strategy was further extended to the synthesis of 5-azaindoles and 5-azabenzothiazoles. Optical properties suggested that manipulation of electron donor and acceptor moieties on β-carbolines has an impact on their ground and excited state electronic behavior. This leads to blue or green emission and should facilitate the development of organic light emitting diodes (OLEDs). Electrochemical and stability studies revealed that 4a-6 shows ease of redox activity and photostability during illumination.

Lolium perenne L. (perennial ryegrass) is the most important pasture grass species in temperate regions of the world. However, its growth is restricted in summer dry environments. Germplasm screening can be used to identify accessions or individual plants for incorporation into breeding programs for drought tolerance. We selected nine perennial ryegrass accessions from different global origins and from a range of climatic and environmental conditions. In addition, the perennial ryegrass cultivar 'Grasslands Impact' was chosen as a reference. The accessions were grown for 360 days in a controlled environment through six consecutive drought stress and recovery cycles. We observed intraspecific differences in drought stress responsiveness for shoot biomass and survival from the third stress cycle. An accession from Norway had 50% more shoot dry matter than the next best-performing accession after six drought cycles. Compared with the reference cultivar 'Grasslands Impact', shoot dry matter of the accession from Norway was more than seven times higher after six drought cycles, indicating superior performance of this ecotype under drought stress. Drought tolerance was characterized by osmotic adjustment and higher relative leaf water content at low soil moisture levels. Furthermore, the findings of this study identify solute potential as an early predictor of drought stress tolerance. These intraspecific differences can be used in breeding programs for the development of drought-tolerant perennial ryegrass cultivars.

Background: Although advance techniques were available for diagnosis and prognosis of oral cancer, histopathology was used as major method in clinical routine. Of all oral subsites, buccal mucosa squamous cell carcinoma is aggressive in nature with poor survival. Therefore, the aim of the present study was to evaluate the relation of tumor histopathological grade with disease recurrence of buccal squamous cell mucosa carcinoma., Materials and Methods: A retrospective study was carried out in regional cancer research institute, Tamil Nadu. Demographic, histopathological and participant's follow-up details were collected from medical records., Results: Of 198 participants, high frequently encountered with well-differentiated squamous cell carcinoma ( n = 98, 49.5%). The clinical characteristics of lymphovascular invasion ( P = 0.031), perineural invasion ( P = 0.019), tumor stage ( P = 0.004), tumor depth ( P = 0.048), lymph node ( P = 0.02) and metastasis ( P = 0.043) had significant association with histopathological grade. In addition, the treatment strategies ( P = 0.014) also showed significance at P < 0.05. Further, multivariate revealed cell differentiation ( P = 0.048), tumor size ( P = 0.037) and depth ( P = 0.021) as independent hazard risk of the development of disease using recurrence-free survival of participants at P < 0.05. Of 198 participants, 24 (12.1%) recurrences reported during 34-month follow-up period and the overall estimated recurrence-free survival was 52%. The high frequency of recurrence, 12 (50%), was identified with moderately differentiated tumor cells. However, poorly differentiated tumor showed significantly lower survival (28%) than moderate (54%) and well differentiated (81%) by Kaplan-Meier analysis using log-rank test ( P = 0.004, P < 0.05)., Conclusions: The present study concludes high frequency of recurrence observed in moderately differentiated and also revealed lower survival in poorly differentiated tumor. Hence, further treatment plans should focus on moderate and poorly differentiated tumors to improve survival outcome., Competing Interests: There are no conflicts of interest.

An efficient and practical one-pot [4 + 2] benzannulation method to produce highly substituted indoles and 1-benzothiophenes via sequential acid-catalyzed propargylation/base-mediated cycloisomerization reactions has been developed. This method allows access to differently substituted (mainly on phenyl ring) indoles and 1-benzothiophenes from the reaction of 3-alkenylpyrroles/-thiophenes as C4 synthons with 1-aryl/1-heteroaryl propargylic alcohols as C2 synthons. Interestingly, dialkynyl substrates can undergo tandem benzannulations to give substituted aza[5]helicenes in 82-83% yield.

Three pigeonpea (Cajanus cajan L. Millsp.) genotypes- GT-1, AKP-1 and PRG-158 with varying crop duration, growth habit and flowering pattern were evaluated for variability in their response for drought stress. Drought stress was imposed at initiation of flowering and the observations on biomass and seed yield parameters were recorded at harvest. The magnitude of response of individual component to drought stress was found to be genotype specific. Drought stress significantly decreased photosynthetic rate (PN), transpiration rate (Tr) and relative water content (RWC) in all the genotypes, however the magnitude of reduction differed with genotype. With drought stress, the reduction of PN was highest in GT-1 while reduction in Tr was highest in PRG-158. The genotype AKP-1, accumulated significantly higher concentrations of osmotic solutes especially proline under water deficit stress, this facilitated it to maintain higher relative water content (RWC) and lower malondialdehyde (MDA) content as compared to other genotypes. Drought stress also impacted biomass production and their partitioning to vegetative and reproductive components at harvest. There was significant variability between the genotypes for seed yield under drought stress while it was non-significant under well-watered condition. Drought stress enhanced flower drop and decreased flower to pod conversion resulting in reduced pod number and seed number in PRG-158 and GT-1. The genotype AKP-1 recorded superior performance for seed yield under stress environment due to its ability in maintaining pod and seed number as well as improved test weight (100 seed weight). Under drought stress, significant positive association of seed yield with proline, seed number, pod number and test weight clearly indicating their role in drought tolerance.

HUB1, also known as Ubl5, is a member of the subfamily of ubiquitin-like post-translational modifiers. HUB1 exerts its role by conjugating with protein targets. The function of this protein has not been studied in plants. A HUB1 gene, LpHUB1, was identified from serial analysis of gene expression data and cloned from perennial ryegrass. The expression of this gene was reported previously to be elevated in pastures during the summer and by drought stress in climate-controlled growth chambers. Here, pasture-type and turf-type transgenic perennial ryegrass plants overexpressing LpHUB1 showed improved drought tolerance, as evidenced by improved turf quality, maintenance of turgor and increased growth. Additional analyses revealed that the transgenic plants generally displayed higher relative water content, leaf water potential, and chlorophyll content and increased photosynthetic rate when subjected to drought stress. These results suggest HUB1 may play an important role in the tolerance of perennial ryegrass to abiotic stresses., (© 2014 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.)

The growth of fall dormant/freezing tolerant plants often surpasses the growth of non-fall dormant/non-freezing tolerant types of the same species under water-limited conditions, while under irrigated conditions non-fall dormant types exhibit superior yield performance. To investigate the mechanism behind this phenomenon, we exposed seven diverse alfalfa (Medicago sativa) cultivars to water-limited and fully watered conditions and measured their shoot growth, shoot water potential and gas exchange parameters and the relative abundance of taproot RNA transcripts associated with chilling stress/freezing tolerance. Fall dormant cultivars had greater shoot growth relative to the fully watered controls under a mild water deficit (a cumulative water deficit of 625 mL pot(-1)) and did not close their stomata until lower shoot water potentials compared with the more non-fall dormant cultivars. Several gene transcripts previously associated with freezing tolerance increased in abundance when plants were exposed to a mild water deficit. Two transcripts, corF (encodes galactinol synthase) and cas18 (encodes a dehydrin-like protein), increased in abundance in fall dormant cultivars only. Once water deficit stress became severe (a cumulative water deficit of 2530 mL pot(-1)), the difference between fall dormancy groups disappeared with the exception of the expression of a type 1 sucrose synthase gene, which decreased in fall dormant cultivars. The specific adaptation of fall dormant cultivars to mild water deficit conditions and the increase in abundance of specific genes typically associated with freezing tolerance in these cultivars is further evidence of a link between freezing tolerance/fall dormancy and adaption to drought conditions in this species.

Surface characteristics of complex aluminosilicate minerals like spodumene [LiAl(SiO(3))(2)], jadeite [NaAl(SiO(3))(2)], feldspar [KAlSi(3)O(8)], and muscovite [K(2)Al(4)(Al(2)Si(6)O(20))(OH)(4)]) are modeled. Surface energies are computed for the cleavage planes of these minerals. Adsorption mechanisms of anionic chemisorbing type oleate and cationic physisorbing type dodecylammonium chloride molecules on two different crystal planes, that is (110) and (001), of spodumene and jadeite are studied in terms of the surface-surfactant interaction energies computed using molecular dynamics (MD) simulations. The conclusions drawn from purely theoretical computations match remarkably well with our experimental results., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Defoliation severity affects grass regrowth. The changes to biological processes affecting regrowth induced by severe defoliation are not fully understood, nor have they been investigated at a molecular level in field-grown plants. Field-grown perennial ryegrass (Lolium perenne L.) plants were defoliated to 20, 40 or 60mm during winter. Throughout regrowth, transcript profiles of 17 genes involved in photosynthesis and carbon metabolism or transport were characterised in stubble and lamina tissue. Although defoliation to 20mm reduced residual lamina area and stubble water-soluble carbohydrate reserves compared with plants defoliated to 40 or 60mm, net herbage regrowth was not reduced. Transcript profiles indicated a potential compensatory mechanism that may have facilitated regrowth. At the one-leaf regrowth stage, plants defoliated to 20mm had greater abundance of photosynthesis-related gene transcripts (rca, rbcS1, rbcS2, fba, fbp and fnr) and 20% greater stubble total nitrogen than plants defoliated to 60mm. A greater capacity for photosynthesis in outer leaf sheaths may be one potential mechanism used by severely defoliated plants to compensate for the reduced residual lamina area; however, this premise requires further investigation.

Oxidative stress has been implicated in the pathogenesis and progression of various hepatic disorders and hence screening for a good hepatoprotective and antioxidant agent is the need of the hour. The present study was aimed to investigate the hepatoprotective and antioxidant property of N-acetylcysteine (NAC) against dimethylnitrosamine (DMN) induced oxidative stress and hepatocellular damage in male Wistar albino rats. Administration of single dose of DMN (5mg/kg b.w.; i.p.) resulted in significant elevation in the levels of serum aspartate transaminase and alanine transaminase, indicating hepatocellular damage. Oxidative stress induced by DMN treatment was confirmed by an elevation in the status of lipid peroxidation (LPO) and reduction in the activities of enzymic antioxidants such as superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase and glutathione-S-transferase and in the levels of non-enzymic antioxidants, reduced glutathione, vitamin-C and vitamin-E in the liver tissue. DMN induced oxidative stress and hepatocellular membrane instability was further substantiated by a decline in the status of the membrane bound ATPases in the liver tissue. Post-treatment with NAC (50mg/kg b.w.; p.o.) for 7days effectively protected against the DMN induced insult to liver by preventing the elevation in the status of the serum marker enzymes and LPO, and restoring the activities of both the enzymic and non-enzymic antioxidants and membrane bound ATPases towards normalcy. These results demonstrate that NAC acts as a good hepatoprotective and antioxidant agent in attenuating DMN induced oxidative stress and hepatocellular damage., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Background: Plants, due to their immobility, have evolved mechanisms allowing them to adapt to multiple environmental and management conditions. Short-term undesirable conditions (e.g. moisture deficit, cold temperatures) generally reduce photosynthetic carbon supply while increasing soluble carbohydrate accumulation. It is not known, however, what strategies plants may use in the long-term to adapt to situations resulting in net carbon depletion (i.e. reduced photosynthetic carbon supply and carbohydrate accumulation). In addition, many transcriptomic experiments have typically been undertaken under laboratory conditions; therefore, long-term acclimation strategies that plants use in natural environments are not well understood., Methodology/principal Findings: Perennial ryegrass (Lolium perenne L.) was used as a model plant to define whether plants adapt to repetitive carbon depletion and to further elucidate their long-term acclimation mechanisms. Transcriptome changes in both lamina and stubble tissues of field-grown plants with depleted carbon reserves were characterised using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The RT-qPCR data for select key genes indicated that plants reduced fructan degradation, and increased photosynthesis and fructan synthesis capacities following carbon depletion. This acclimatory response was not sufficient to prevent a reduction (P<0.001) in net biomass accumulation, but ensured that the plant survived., Conclusions: Adaptations of plants with depleted carbon reserves resulted in reduced post-defoliation carbon mobilization and earlier replenishment of carbon reserves, thereby ensuring survival and continued growth. These findings will help pave the way to improve plant biomass production, for either grazing livestock or biofuel purposes.

Background: Perennial ryegrass (Lolium perenne L.) is an important pasture and turf crop. Biotechniques such as gene expression studies are being employed to improve traits in this temperate grass. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) is among the best methods available for determining changes in gene expression. Before analysis of target gene expression, it is essential to select an appropriate normalisation strategy to control for non-specific variation between samples. Reference genes that have stable expression at different biological and physiological states can be effectively used for normalisation; however, their expression stability must be validated before use., Results: Existing Serial Analysis of Gene Expression data were queried to identify six moderately expressed genes that had relatively stable gene expression throughout the year. These six candidate reference genes (eukaryotic elongation factor 1 alpha, eEF1A; TAT-binding protein homolog 1, TBP-1; eukaryotic translation initiation factor 4 alpha, eIF4A; YT521-B-like protein family protein, YT521-B; histone 3, H3; ubiquitin-conjugating enzyme, E2) were validated for qRT-PCR normalisation in 442 diverse perennial ryegrass (Lolium perenne L.) samples sourced from field- and laboratory-grown plants under a wide range of experimental conditions. Eukaryotic EF1A is encoded by members of a multigene family exhibiting differential expression and necessitated the expression analysis of different eEF1A encoding genes; a highly expressed eEF1A (h), a moderately, but stably expressed eEF1A (s), and combined expression of multigene eEF1A (m). NormFinder identified eEF1A (s) and YT521-B as the best combination of two genes for normalisation of gene expression data in perennial ryegrass following different defoliation management in the field., Conclusions: This study is unique in the magnitude of samples tested with the inclusion of numerous field-grown samples, helping pave the way to conduct gene expression studies in perennial biomass crops under field-conditions. From our study several stably expressed reference genes have been validated. This provides useful candidates for reference gene selection in perennial ryegrass under conditions other than those tested here.

Perennial ryegrass (Lolium perenne L.) is a major grass species used for forage and turf throughout the world, and gains by conventional breeding have reached a plateau. Perennial ryegrass is an outcrossing, self-incompatible diploid (2n = 2x = 14) with a relatively large genome (4067 Mbp/diploid genome; Evans, G.M., Rees, H., Snell, C.L. and Sun, S. (1972) The relation between nuclear DNA amount and the duration of the mitotic cycle. Chrom. Today, 3, 24-31). Using tissues sourced from active pastures during the peak of the autumn, winter, spring and summer seasons, we analysed the ryegrass transcriptome employing a Serial Analysis of Gene Expression (SAGE) protocol, with the dual goals of understanding the seasonal changes in perennial ryegrass gene expression and enhancing our ability to select genes for genetic manipulation. A total of 159,002 14-mer SAGE tags was sequenced and mapped to the perennial ryegrass DNA database, comprising methyl-filtered (GeneThresher) and expressed sequence tag (EST) sequences. The analysis of 14,559 unique SAGE tags, which were present more than once in our SAGE library, revealed 964, 1331, 346 and 131 exclusive transcripts to autumn, winter, spring and summer, respectively. Intriguingly, our analysis of the SAGE tags revealed season-specific expression profiles for the small subunit of ribulose-1,5-bisphosphate carboxylase (Rubisco), LprbcS. The transcript level for LprbcS was highest in spring, and then decreased gradually between summer and winter. Five different copies of LprbcS were revealed in ryegrass, with one possibly producing splice variant transcripts. Two highly expressed LprbcS genes were reported, one of which was not active in autumn. Another LprbcS gene showed an inverse expression profile to the autumn inactive LprbcS in a manner to compensate the expression level.

Molecular dynamics simulations incorporating explicit gold atoms in the simulations have been carried out for alkanethiol self-assembled monolayers chemisorbed on the Au(III) surface. The structural properties of the monolayer are evaluated for two force fields: one in which the Au--S--C bond is fixed (FF I), and the other in which it is flexible (FF II). The influence of these force fields on the structural properties of HS(CH2)14CH3 on the structured Au surface is compared at different temperatures. FF I yields greater tilt angles and a smaller film thickness when compared with FF II. Both of the force fields predict that the tilt angles do not follow a monotonic decrease with temperature but show minima around 200 K. Simulations carried out at different chain lengths at 300 K reveal that FF II predicts a greater film thickness than FF I; however, the difference is within 1 A.

A genomic clone for starch branching enzyme (SBE) IIb was isolated from a sorghum bacterial artificial chromosome (BAC) library. The promoter and 5' flanking sequence, the first four exons and introns as well as the last exon and the 3' untranslated region were sequenced. The tentative transcription start site of sorghum sbeIIb was mapped based on alignment with the maize sbeIIb gene. The exon-intron structure of the 5' portion of sorghum sbeIIb was similar to that of maize sbeIIb but differed from that of barley sbeIIb. Specifically, the intronic BbI element involved in the endosperm specific expression of barley sbeIIb was lacking in the sorghum gene. A cDNA clone for sorghum sbeIIb was reverse PCR amplified and found to encode an 803 amino acids long protein. The amino acid sequence of sorghum SBEIIb was compared to that of sorghum SBEI and corresponding enzymes in barley. The overall identity in amino acid sequence was 54% in the central portion of the enzymes. A major difference between the SBEII and SBEI isoforms was a 67 amino acids-long C-terminal extension in the SBEIs. The spatial and temporal expression patterns of sorghum sbeIIb was determined and compared with those of the sorghum gene for SBEI and the barley genes for SBEIIB and SBEI. All four genes exhibited a seed specific expression. However, while barley sbeIIb and sbeI transcripts were detected exclusively in the endosperm, the sorghum genes were expressed also in the embryo. The activity of sorghum sbeIIb and sbeI exhibited a late onset, with a peak of transcription at around 22 days after pollination. This is similar to the pattern of barley sbeI but different from that of barley sbeIIb, which showed a peak of transcription at 12 days after pollination.

The differential display method was used to identify a novel barley gene, Lem1, expressed primarily in the outer organs (lemma and palea) that enclose developing florets and seeds. The promoter was isolated from a BAC genomic clone and used in a translational fusion with a green fluorescent protein gene (Gfp) to produce a transient expression vector. After particle bombardment, Gfp was expressed only in lemmas, paleas and awns of developing spikelets. Lem1 did not promote Gfp expression in vegetative leaves or in mature spikes, although expression of co-bombarded uidA (GUS) occurred under the regulation of a ubiquitin promoter. This reproduced the developmentally regulated pattern of mRNA accumulation. Deletion studies showed that the promoter activity is confined to a cis element within 80 bp of the transcription start site. Upstream from this, the promoter contains putative auxin-, ethylene- and gibberellin-responsive elements or homologues. Lem1 was found to be a single intronless gene encoding an acidic 102 amino acid protein, possibly associated with membranes. In a two-rowed barley, Lem1 mRNA was absent in the lateral spikelets, which fail to develop, and present only in the developing median spikelets. This suggests that Lem1 may play a role in organ development.

Permatins are antifungal thaumatin-like proteins (TLPs) of the PR-5 family of pathogenesis-related proteins. They occur in many cereals, but little is known of their expression and roles. Permatin cDNA clones were produced and used to study expression in developing barley and oat seeds. Actin and CDC48 mRNAs declined rapidly following inoculation of barley spikes with Fusarium graminearum. Despite this, permatin mRNA levels remained constant or increased slightly. Studies of permatin gene expression in healthy plants revealed that developing barley and oat seeds accumulate permatin mRNA in an unusual bimodal pattern. Permatin mRNA and protein are highly abundant around the time of pollination and then decrease rapidly to near-zero. A second peak occurs in the doughy stage of development. Antibody and DNA probe hybridization studies showed that expression initially occurs in the ovary wall and then switches to the aleurone and ventral furrow of developing seeds, reaching a peak in the doughy stage. Small amounts of permatin mRNAs also occur in certain vegetative tissues. The barley and oat permatin sequences provided sufficient comparisons between cereal TLPs to suggest that deletions or additions in specific elements could have led to the divergence of leaf- and seed-specific TLPs.

The notion of debranching enzyme activity as a participant in starch synthesis is gaining acceptance. Inconsistent reports from mutant analyses implicate either isoamylase or pullulanase as a determinant in amylopectin formation and whether wild-type plants utilize one or the other, or both, of these debranching enzymes in starch synthesis is unclear. Recent results on the sul mutant in maize suggest that both forms of debranching enzymes might be involved in amylopectin formation. We wished to find out if isoamylase takes part in starch synthesis by comparing isoamylase gene activity under three conditions: (1) during starch accumulation in developing sink tissues; (2) during starch degradation in germinating seeds; (3) in ectopic expression after applying sucrose, a starch precursor. We isolated the gene for barley isoamylase, isol, and analysed its expression and regulation in germinating seeds, developing endosperm and vegetative tissues, and compared the isoamylase gene expression in sink tissues from three different species. Our results indicate that isoamylase gene activity is involved in starch synthesis in wild-type plants and is modulated by sucrose.

The sbeIIa and sbeIIb genes, encoding starch-branching enzyme (SBE) IIa and SBEIIb in barley (Hordeum vulgare L.), have been isolated. The 5' portions of the two genes are strongly divergent, primarily due to the 2064-nucleotide-long intron 2 in sbeIIb. The sequence of this intron shows that it contains a retro-transposon-like element. Expression of sbeIIb but not sbeIIa was found to be endosperm specific. The temporal expression patterns for sbeIIa and sbeIIb were similar and peaked around 12 d after pollination. DNA gel-blot analysis demonstrated that sbeIIa and sbeIIb are both single-copy genes in the barley genome. By fluorescence in situ hybridization, the sbeIIa and sbeIIb genes were mapped to chromosomes 2 and 5, respectively. The cDNA clones for SBEIIa and SBEIIb were isolated and sequenced. The amino acid sequences of SBEIIa and SBEIIb were almost 80% identical. The major structural difference between the two enzymes was the presence of a 94-amino acid N-terminal extension in the SBEIIb precursor. The (beta/alpha)8-barrel topology of the alpha-amylase superfamily and the catalytic residues implicated in branching enzymes are conserved in both barley enzymes.

Developing caryopses of barley (Hardeum vulgare L. ev. Golf) were monitored for starch-branching enzyme activity for 1 month after anthesis. Homogenized caryopses from grains with the highest specific activity were used as starting material for purification of branching enzyme by FPLC chromatography. Several branching enzyme activity fractions were resolved. We have previously described the isolation of a 51/50 kDa protein with starch branching activity from one of these fractions. From three other fractions, we have now partially purified starch branching enzymes corresponding to forms I, IIa and IIb.

Anthers from rice (Oryza sativa L.) subspecies japonica initiated more callus than their indica or indica x japonica counterparts. A mild stress, either by slow desiccation or heat shock, prior to the plating of anthers enhanced the ability to initiate callus. Slow dessication of anthers enhanced the ability of the japonica anthers to initiate callus even in medium that was supplemented with NaCl. The ability to initiate callus by the anthers plated on NaCl-supplemented medium decreased as the NaCl level in the medium increased. Among the regenerated plants 2.5% were albino and another 2% were haploid. Androclonal variation for tiller numbers, shoot height, plant dry matter and flowering were noticed in the progenies of the regenerated plants.

Pollen plants were obtained from anther-derived calli of the indica rice variety Basmati-370. Anther-response (anthers producing pollen derived calli) and plant regeneration frequency from the pollen derived calli. was very low. Donor plants which flowered at the average max/min. temperature of 34.2°/23.3°C gave a significantly higher anther-response to in vitro techniques, than did those which flowered at 29.1°/16.4°C. Somatic callus induction and subsequent plant regeneration was readily obtained from mature seed embryos. While 2,4-D or 2,4,5-T (1 or 2 mg/l) proved highly efficient for callus induction, tryptophan (50 or 100 mg/l) induced a high frequency of green plants from the calli.