A method to overcome the waxy surface, cell wall thickening and polyphenol induced necrosis at wound sites - the major deterrents to Agrobacterium mediated transformation of bamboo, a woody monocot

The method is the first successful report of Agrobacterium mediated genetic transformation of the commercially important bamboo, Dendrocalamus hamiltonii. It shows how the resistance provided by the somatic embryos of this woody monocot can be overcome using a simple and effective method. The method thus standardized can be also used for the genetic transformation of other important bamboos. Identification of the factors responsible for the resistance of the somatic embryos to Agrobacterium infection was an absolute requirement for devising a successful method. Necrosis due to polyphenol oxidation, lack of differentiation due to cell wall thickening at wound sites, waxy surfaces of somatic embryos with anti-microbial properties were found to prevent Agrobacterium attachment and infection. Therefore, the somatic embryos were transformed with fresh overnight grown Agrobacterium culture containing 500 mg/l polyvinylpyrrolidone (PVP) and 0.01 % Tween-20 as surfactant followed by co-cultivation on Murashige and Skoog (MS) medium containing the vir gene inducer acetosyringone (100 μM) and 1 mg/l 6-Benzylaminopurine BAP for 2 days. Persistent GUS expression and strong positive signals in PCR, slot blot and Southern hybridization confirmed successful genetic transformation.     

Investigation of protein-tyrosine phosphatases by in-gel assays

Renaturation permits the detection of protein-tyrosine phosphatase (PTP) activities subsequent to separation by SDS-PAGE in the presence of the (32)P-labeled poly(Glu(4)Tyr) as a macromolecular substrate. An efficient corresponding method has been developed by Burridge and Nelson [Anal. Biochem. 232 (1995) 56]. We describe here the modification of the basic method, its extension to two-dimensional gel electrophoresis, and applications to identify PTPs in signaling complexes and reversibly oxidized PTPs. Particular attention is given to the preparation of samples, to interpretation of the results as well as to advantages and limitations of the technique. Immunodepletion and the use of cells from knockout animals have been successful in the identification of individual PTPs. Readily detectable in cell lysates are PTP-PEST, SHP-2, SHP-1, PTP1B, and T-cell PTP. A much greater complexity of activity bands is, to a large extent, due to the generation of active fragments of PTPs. In-gel detection of PTPs can be employed to monitor cell fractionations and potential PTP activity changes.     

Detection of Mycobacterium tuberculosis GlcB or HspX Antigens or devR DNA Impacts the Rapid Diagnosis of Tuberculous Meningitis in Children

Background

Tuberculous meningitis (TBM) is the most common form of neurotuberculosis and the fifth most common form of extrapulmonary TB. Early diagnosis and prompt treatment are the cornerstones of effective disease management. The accurate diagnosis of TBM poses a challenge due to an extensive differential diagnosis, low bacterial load and paucity of cerebrospinal fluid (CSF) especially in children.

Methodology/Principal Findings

We describe the utility of ELISA and qPCR for the detection of Mycobacterium tuberculosis (M. tb) proteins (GlcB, HspX, MPT51, Ag85B and PstS1) and DNA for the rapid diagnosis of TBM. CSF filtrates (n = 532) derived from children were classified as ‘Definite’ TBM (M. tb culture positive, n = 29), ‘Probable and Possible’ TBM (n = 165) and ‘Not-TBM’ including other cases of meningitis or neurological disorders (n = 338). ROC curves were generated from ELISA and qPCR data of ‘Definite’ TBM and Non-Tuberculous infectious meningitis (NTIM) samples and cut-off values were derived to provide ≥95% specificity. devR qPCR, GlcB, HspX and PstS1 ELISAs showed 100% (88;100) sensitivity and 96–97% specificity in ‘Definite’ TBM samples. The application of these cut-offs to ‘Probable and Possible’ TBM groups yielded excellent sensitivity (98%, 94;99) and specificity (98%, 96;99) for qPCR and for GlcB, HspX and MPT51 antigen ELISAs (sensitivity 92–95% and specificity 93–96%). A test combination of qPCR with GlcB and HspX ELISAs accurately detected all TBM samples at a specificity of ∼90%. Logistic regression analysis indicated that these tests significantly added value to the currently used algorithms for TBM diagnosis.

Conclusions

The detection of M. tb GlcB/HspX antigens/devR DNA in CSF is likely to improve the utility of existing algorithms for TBM diagnosis and also hasten the speed of diagnosis.     

Risk Factors Associated with Death in In-Hospital Pediatric Convulsive Status Epilepticus

Objective

To evaluate in-patient mortality and predictors of death associated with convulsive status epilepticus (SE) in a large, multi-center, pediatric cohort.

Patients and Methods

We identified our cohort from the KID Inpatient Database for the years 1997, 2000, 2003 and 2006. We queried the database for convulsive SE, associated diagnoses, and for inpatient death. Univariate logistic testing was used to screen for potential risk factors. These risk factors were then entered into a stepwise backwards conditional multivariable logistic regression procedure. P-values less than 0.05 were taken as significant.

Results

We identified 12,365 (5,541 female) patients with convulsive SE aged 0–20 years (mean age 6.2 years, standard deviation 5.5 years, median 5 years) among 14,965,571 pediatric inpatients (0.08%). Of these, 117 died while in the hospital (0.9%). The most frequent additional admission ICD-9 code diagnoses in addition to SE were cerebral palsy, pneumonia, and respiratory failure.Independent risk factors for death in patients with SE, assessed by multivariate calculation, included near drowning (Odds ratio [OR] 43.2; Confidence Interval [CI] 4.4–426.8), hemorrhagic shock (OR 17.83; CI 6.5–49.1), sepsis (OR 10.14; CI 4.0–25.6), massive aspiration (OR 9.1; CI 1.8–47), mechanical ventilation >96 hours (OR9; 5.6–14.6), transfusion (OR 8.25; CI 4.3–15.8), structural brain lesion (OR7.0; CI 3.1–16), hypoglycemia (OR5.8; CI 1.75–19.2), sepsis with liver failure (OR 14.4; CI 5–41.9), and admission in December (OR3.4; CI 1.6–4.1). African American ethnicity (OR 0.4; CI 0.2–0.8) was associated with a decreased risk of death in SE.

Conclusion

Pediatric convulsive SE occurs in up to 0.08% of pediatric inpatient admissions with a mortality of up to 1%. There appear to be several risk factors that can predict mortality. These may warrant additional monitoring and aggressive management.     

An epidemiologically significant epitope of a 1998 human influenza virus neuraminidase forms a highly hydrated interface in the NA-antibody complex

The crystal structure of the complex between neuraminidase (NA) of influenza virus A/Memphis/31/98 (H3N2) and Fab of monoclonal antibody Mem5 has been determined at 2.1A resolution and shows a novel pattern of interactions compared to other NA-Fab structures. The interface buries a large area of 2400 A2 and the surfaces have high complementarity. However, the interface is also highly hydrated. There are 33 water molecules in the interface>or=95% buried from bulk solvent, but only 13 of these are isolated from other water molecules. The rest are involved in an intricate network of water-mediated hydrogen bonds throughout the interface, stabilizing the complex. Glu199 on NA, the most critical side-chain to the interaction as previously determined by escape mutant analysis and site-directed mutation, is located in a non-aqueous island. Glu199 and three other residues that contribute the major part of the antigen buried surface of the complex have mutated in human influenza viruses isolated after 1998, confirming that Mem5 identifies an epidemiologically important antigenic site. We conclude that antibody selection of NA variants is a significant component of recent antigenic drift in human H3N2 influenza viruses, supporting the idea that influenza vaccines should contain NA in addition to hemagglutinin.     

Evaluation of nested PCR in detection of Helicobacter pylori targeting a highly conserved gene: HSP60

Objective: To comparatively evaluate a new nested set of primers designed for the detection of Helicobacter pylori targeting a highly conserved heat shock protein gene (Hsp60). Methods: A total of 60 subjects having peptic ulcer diseases were tested for the detection of H. pylori using rapid urease test (RUT), histology, culture, and polymerase chain reaction (PCR) in their antral biopsy specimens. A newly designed Hsp60 gene‐based primer set was evaluated against commonly used PCR primers for detection of H. pylori. Results: Forty‐six of the 60 study subjects were found positive for culture isolation and all the 46 culture‐positive specimens were also positive with Hsp60 gene PCR. Of the 46 culture‐positive specimens, 44 were positive for 16S rRNA gene, ureC gene, RUT, and histology whereas only 29 were positive with ureA gene PCR. Of the 14 culture‐negative subjects, 10 were positive with 16S rRNA gene, 4 were positive with ureC (glmM) gene PCR, and 2 were positive with RUT and 1 was positive on histology. Conclusion: This study shows that nested amplification targeting Hsp60 gene is the most sensitive and specific with LR⁺ and LR^? values of ∝ and 0, respectively, when compared with the other three PCR methods. Also, HSP60 gene‐specific nested protocol was the most appropriate for detection of H. pylori in clinical specimens. This is particularly valuable because it can be used as a noninvasive method for detecting H. pylori infection in young children and also, in follow‐up studies with peptic ulcer patients, on samples like feces and saliva.     

Stress-Tolerant <Emphasis Type="Italic">Viridibacillus arenosi</Emphasis> Strain IHB B 7171 from Tea Rhizosphere as a Potential Broad-Spectrum Microbial Inoculant

Viridibacillus arenosi strain IHB B 7171 identified based on 16S rRNA gene sequence produced colony forming units (cfu/ml) ranging from 3.3 × 10⁴ to 1.2 × 10¹⁰ under pH 5–11, 2.2 × 10² to 1.4 × 10¹⁰ for temperature 5–40 °C, 2.4 × 10² to 1.1 × 10¹⁰ for PEG 6000 10–30%, 2.2 × 10² to 1.4 × 10¹⁰ for 2.5–10% NaCl, 3.1 × 10³ to 1.7 × 10⁹ for 2.5–7.5 mM CaCl₂, 2.2 × 10² to 1.4 × 10⁷ for 2.5–7.5 mM AlCl₃, and 3.2 × 10² to 1.2 × 10⁷ for 2.5–7.5 mM FeCl₃. The activities of plant growth-promoting attributes with the increasing acidity, desiccation and salinity ranged from 408 to 101, 20 to 8, 14 to 5 µg/ml P-liberated from tri-calcium phosphate, aluminium phosphate and iron phosphate, 20–9% siderophore units, 14–4 µg/ml IAA and 190–16 α-ketobutyrate h/mg protein ACC-deaminase activity. Plant height, leaf number, and leaf weight on treatment with bacterial inoculum showed an increment of 9.5, 17.6, 54.5 and 31.0% in tea seedlings, respectively. The bacterium also enhanced plant height and yield by 10 and 13% in pea and 2.8 and 13.9% in wheat. The results exhibited stress-tolerance and plant growth-promoting activities by the strain under stressed growth-conditions with potential as a broad-spectrum plant growth-promoting rhizobacterium.     

Cost of reproduction in selected species of zooplankton (rotifers and cladocerans)

Reproduction is an energetically costly biological process. Among the freshwater zooplankton, rotifers and cladocerans reproduce parthenogenetically and the cost of reproduction can be estimated using the life table data from demographic studies. Reduced probability of future survival or future reproduction as a result of current investment in offspring production (trade-off) is the central theme of the cost hypothesis. Correlations using present reproduction vs. future reproduction (called the reproductive costs) or future survival (called the survival costs) can be used to evaluate the cost hypothesis. In this work sets of correlations were made: (1) between present reproduction (m _x) vs. future survival (l _x+1, l _x+2, l _x+3 etc. for the entire lifespan) (survival costs), and (2) present reproduction (m _x) vs. future reproduction (m _x+1, m _x+2, m _x+3 etc. for the entire reproductive span) (reproductive costs). These correlations were plotted against the cohort age-classes in order to quantify survival and reproductive costs in rotifers (Asplanchna girodi, Brachionus macracanthus, B. variabilis and Platyias quadricornis) and cladocerans (Ceriodaphnia cornuta – one strain maintained on Chlorella and another strain adapted to Microcystis), Daphnia carinata, D. laevis, Moina macrocopa, Pleuroxus aduncus, Scapholeberis kingi and Simocephalus vetulus). All the tested rotifer species showed negative tendency in correlation coefficients (when the data of current reproduction vs. future reproduction and future survival were plotted) for both reproductive and survival costs. However, from the total survival and reproductive costs derived, 84% of the former and 42% of the latter were statistically significant. In cladocerans about 80% of the costs (correlations between current reproduction vs. future survival or future reproduction) were negative suggesting that present reproduction had negatively affected both the further survival and reproduction of test populations. In terms of statistically significant survival costs, the cladocerans showed a trend slightly lower (72%) but comparable to rotifers. The reproductive costs were significant in 45% cases. In our study, the simple statistical correlations detected the trade-offs between reproduction and survival. Thus, in more than 60% cases of both survival and reproductive costs in zooplankton were negative, and our data supported the cost hypothesis, in the majority of cases where reproduction by zooplankton of a given age class caused reduced survival and reproduction of the next age class.