Simplified protocol for the micropropagation of selected Eucalyptus and Banksia species

The Australian native plants Eucalyptus microcorys, Banksia serrata and Banksia oblongifolia were evaluated for their potential to produce tissue culture stocks under a simplified regime of growth substance treatment for shoot multiplication and root induction. The use of N⁶-benzyladenine (BA) alone proved to be successful with the three species in producing shoot multiplication, but only if shoot apices were used as explant material. Juvenile material provided best shoot proliferation, and by varying the concentration of BA, shoot elongation was manipulated. A combination of half-strength Murashige and Skoog (MS) medium and 0.25μM BA proved to be the best compromise medium for shoot proliferation combined with shoot elongation. The use of indole-3-butyric acid (IBA) alone was successful in initiating roots from the three species. Root induction was easiest in E. microcorys, at an optimum concentration of 5μM IBA, but the Banksia species were more difficult. The results demonstrate that a simplified protocol for shoot multiplication and root induction can be applied to a diverse group of Australian plants, which should result in more species being introduced into tissue culture programmes.     

Disease inhibition by major histocompatibility complex binding peptide analogues of disease-associated epitopes: more than blocking alone

Peptide analogues of disease-associated epitopes were studied for inhibition of experimental allergic encephalomyelitis (EAE) and adjuvant arthritis (AA) in Lewis rats. EAE- and AA-associated analogues were selected as competitors because of their in vitro inhibitory activity on proliferation of encephalitogenic and arthritogenic T cells. Although the EAE-associated competitor had a superior major histocompatibility complex (MHC) binding affinity, the AA-associated competitor was a better inhibitor of the in vitro proliferation of arthritogenic T cells. Furthermore, although in vivo EAE was inhibited by both competitors, AA was only inhibited by the AA-associated competitor. Remarkably, in contrast to what was expected of a regular MHC competitor peptide, the AA-associated peptide analogue also prevented AA upon immunization before disease induction and appeared to induce T cell responses that crossreacted with the original disease-associated epitope. Therefore, it is concluded that antigen-specific regulatory mechanisms were involved in synergy with MHC competition. The integration of both qualities into a single "competitor-modulator" analogue peptide may lead to the development of novel, more effective, disease-specific immunomodulatory peptides.     

Feasibility,sensitivity, and reliability of laser-induced fluorescence imaging of green fluorescent protein-expressing tumors in vivo

Whole-body imaging of green fluorescent protein (GFP) can be used to test the efficiency of gene carriers for in vivo transduction. The aim of the current study was to determine the sensitivity and the accuracy of a GFP imaging procedure by in vivo investigation of GFP-expressing tumor cells. An improved method of whole-body GFP imaging made use of a laser excitation source and band-pass filters matched specifically to GFP and constitutive tissue fluorescence emission bands. Processing of the primary GFP fluorescence images acquired by the CCD camera subtracted background tissue autofluorescence. Our approach achieved 100% sensitivity and specificity for in vivo detection of 10%-transfected BxPc3 pancreatic tumor after subcutaneous grafting or orthotopical implantation in the pancreas of nude mice. It also detected less transfected tumors (i.e., 1 to 5%) but with a loss in sensitivity (50% of cases). The system was employed over a 5-week period to monitor the persistence of GFP expression in 10%-transfected BxPc3 tumors orthotopically implanted in the pancreas of two nude mice, allowing the direct visualization of tumor progression and spread. In facilitating the temporal–spatial follow-up of GFP expression in vivo, the optimized laser-induced fluorescence imaging device can support preclinical investigations of vectors for therapeutic gene transduction through regular, harmless, real-time monitoring of theirin vivo transductional efficacy and persistence.     

KPC-2-producing Enterobacter cloacae and pseudomonas putida coinfection in a liver transplant recipient

Carbapenemases are among the newest resistance mechanisms to emerge in some gram-negative bacteria. We describe bacteremia in a critically ill liver transplant recipient infected with KPC-2-producing Enterobacter cloacae and Pseudomonas putida. Although this enzyme has been previously described in Enterobacter spp., this is the first report of KPC carbapenemase in P. putida.     

Synthesis of 2,5-furandicarboxylic acid by a TEMPO/laccase system coupled with Pseudomonas putida KT2440

As a useful and renewable chemical building block from biomass, 2,5-furandicarboxylic acid (FDCA) has become an increasingly desirable platform chemical as a terephthalic acid replacement for polymerization. In this work, an efficient and highly selective biocatalytic approach for the synthesis of FDCA from 5-hydroxymethylfurfural (HMF) was successfully developed using a TEMPO/laccase system coupled with Pseudomonas putida KT2440. TEMPO/laccase afforded the selective oxidation of the hydroxymethyl group of HMF to form 5-formyl-2-furancarboxylic acid as a major product, which was subsequently oxidized to FDCA by P. putida KT2440. Manipulating the reaction conditions resulted in a good conversion of HMF (100%) and an excellent selectivity of FDCA (100%) at substrate concentrations up to 150 mM within 50 h. The cascade catalytic process established in this work offers a promising approach for the green production of FDCA.

A novel biological approach for the production of FDCA by a TEMPO/laccase system coupled with Pseudomonas putida KT2440 was established.     

In vitro antimicrobial activity of ceftizoxime against glucose-nonfermentative gram-negative rods

Ceftizoxime, a new cephalosporin, was active against Pseudomonas cepacia, Flavobacterium meningosepticum, Alcaligenes faecalis, and Acinetobacter calcoaceticus and was more potent against Pseudomonas aeruginosa and Pseudomonas putida than was carbenicillin.     

Antifouling Activities of Antagonistic Marine Bacterium <Emphasis Type="Italic">Pseudomonas putida</Emphasis> Associated with an Octopus

Bacterial symbionts associated with an octopus were isolated and their intra and extracellular extracts were tested for the antagonistic activities against biofilm-forming bacteria. Among the three antagonistic bacterial symbionts, both intra and extracellular extracts of the bacterium, Pseudomonas putida exhibited strong antibacterial activity against biofilm bacteria. Results also showed that both intra and extracellular extracts of P. putida significantly reduced the biofilm formation on test surfaces. The antifouling coating developed with bacterial extracts of P. putida significantly reduced the recruitment of biofouling organisms over a period of 50 days in the coastal waters. The bioactive fractions were partially purified using chromatographic methods such as thin-layer chromatography and high performance liquid chromatography. Based on the findings, it is concluded that potential antifouling compounds could be isolated from the antagonistic bacteria isolated from the octopus.     

A Role for Fibroblast Growth Factor 19 and Bile Acids in Diabetes Remission After Roux-en-Y Gastric Bypass

OBJECTIVE

Roux-en-Y gastric bypass (RYGB) in humans can remit type 2 diabetes, but the operative mechanism is not completely understood. In mice, fibroblast growth factor (FGF) 15 (FGF19 in humans) regulates hepatic bile acid (BA) production and can also resolve diabetes. In this study, we tested the hypothesis that the FGF19–BA pathway plays a role in the remission of human diabetes after RYGB surgery.

RESEARCH DESIGN AND METHODS

Cohorts of diabetic and nondiabetic individuals of various body weights were used. In addition, RYGB patients without diabetes (No-Diabetes), RYGB patients with diabetes who experienced remission for at least 12 months after surgery (Diabetes-R), and RYGB patients with diabetes who did not go into remission after surgery (Diabetes-NoR) were studied. Circulating FGF19 and BA levels, hepatic glycogen content, and expression levels of genes regulating the FGF19–BA pathway were compared among these groups of patients using pre- and postoperative serum samples and intraoperative liver biopsies.

RESULTS

Preoperatively, patients with diabetes had lower FGF19 and higher BA levels than nondiabetic patients, irrespective of body weight. In diabetic patients undergoing RYGB, lower FGF19 levels were significantly correlated with increased hepatic expression of the cholesterol 7alpha-hydroxylase 1 (CYP7A1) gene, which modulates BA production. Following RYGB surgery, however, FGF19 and BA levels (particularly cholic and deoxycholic acids) exhibited larger increases in Diabetic-R patients compared with nondiabetic and Diabetic-NoR patients.

CONCLUSIONS

Taken together, the baseline and postoperative data implicate the FGF19–CYP7A1–BA pathway in the etiology and remission of type 2 diabetes following RYGB surgery.Type 2 diabetes is a metabolic disease with significant health implications for which current therapies do not result in long-term resolution. Roux-en-Y gastric bypass (RYGB) surgery is a complex, anatomically disruptive procedure (1) that leads to remission of diabetes for most, but not all, patients (2,3). The mechanism for the reversal of diabetes by RYGB surgery, however, is not well-understood.Work in mice has identified an enterohepatic pathway involving the fibroblast growth factor (FGF) 15 (FGF19 in humans) that reversed dietary diabetes (4) and improved glycogen synthesis in diabetic mice (5). Most of the FGF family members function in a paracrine fashion to regulate processes of development, transformation, angiogenesis, and energy homeostasis (6,7). FGF19- and FGF21-transgenic mice were resistant to diet-induced obesity and had improved insulin sensitivity and glucose disposal (8,9). Injection of recombinant FGF15 protein into diabetic mice resulted in the reduction of serum glucose and insulin levels, improved glucose tolerance, and decreased hepatosteatosis and body weight (10,11).In humans, serum levels of FGF19 were increased postprandially by chenodeoxycholic acid (CDCA) and decreased by bile acid (BA) sequestrants (12). FGF19 is produced primarily in the ileum and signals in hepatocytes through its two receptors, FGF receptor 4 (FGFR4) and βKlotho, to inhibit expression of cholesterol 7alpha-hydroxylase 1 (CYP7A1) (13). In response to FGF19, CYP7A1 regulates the rate-controlling step for the conversion of cholesterol into BAs (14). In turn, the farnesoid X receptor (FXR) responds to BAs and regulates intestinal FGF19 secretion, which makes it an important regulator of glucose metabolism (15). By a feedback mechanism, BAs also regulate hepatic CYP7A1 gene expression through a multicomponent pathway involving hepatic FXR (16).In this study, we tested the hypothesis that the enterohepatic FGF19–BA pathway plays a role in the etiology of diabetes and its remission following RYGB surgery.     

Model system using controlled receptor expression for evaluating targeted ultrasound contrast agents

This report details a model system for evaluating targeted ultrasound (US) contrast agents using adenoviral (Ad) vectors to regulate target receptor expression. Receptor density in vitro was modulated in breast cancer cells by varying the multiplicity of infection (MOI) from 0 to 100. Target receptors were induced using a green fluorescent protein (GFP) reporter Ad vector for gene transfer and expression of the hemagglutinin (HA) tag. These reporter genes were under the control of the ubiquitous cytomegalovirus (CMV) promoter. Subsequently, receptor expression and anti-HA antibody (Ab) binding was examined with flow cytometry. Targeted US contrast agents, or microbubbles (MB), were created by conjugating either biotinylated anti-HA or isotype control Ab to the surface of biotin coated MBs via a streptavidin bridge. Targeted MBs were incubated with Ad infected 2LMP cells to evaluate in vitro MB binding. Experimental results found GFP expression to be directly correlated with Ad MOI (r² = 0.96). Increasing the Ad MOI produced a corresponding increase in binding and accumulation of anti-HA Ab on the cell surface (p < 0.01). However, no difference was found between Cy5-labeled anti-HA Ab exposed cell groups at an MOI of 0 (p > 0.29). Additionally, no difference was found between the isotype control Ab group (p > 0.44) indicating minimal nonspecific binding. No difference was found between cell groups incubated with isotype-targeted MBs (p > 0.42) regardless of receptor density. However, cells exposed to HA-targeted MBs showed increased levels of cell binding proportional to induced receptor expression levels (p < 0.02).     

A Study on Parameters Optimization for Degradation of Endosulfan by Bacterial Consortia Isolated from Contaminated Soil

Endosulfan, a non-systemic organochlorine pesticide used extensively to control the insect pests of a wide range of crops is of environmental concern because of its apparent persistence and toxicity to many non-target organisms. The present study was aimed to find out the capability of microorganisms to degrade endosulfan, singly and/or in consortium and optimization of various growth parameters to achieve optimal degradation. A total of three isolates showing significantly higher degradation potential were selected from eight isolates demonstrating substantial growth. They were identified as Staphylococcus equorum CM5, Enterobacter sp. MF1 and Bacillus subtilis MF2. The effect of various parameters (pH, temperature, inoculum size, endosulfan concentration, incubation conditions and carbon concentration) were also assessed and optimized for degradation of endosulfan. The consortia of Staphylococcus and Bacillus strain showed near disappearance of endosulfan from the medium after 21 days of incubation. The maximum degradation percentage of endosulfan was observed at slightly alkaline pH 8.0 under shaking conditions of 150 rpm at 30 °C at a concentration of 1 g l^?1 of dextrose and irrespective of the inocula size used in this study. The isolates identified in this study present an efficient, economical and ecological alternative remedy for the removal of endosulfan from contaminated sites.     

Isolation of VIM-2-Producing Pseudomonas monteilii Clinical Strains Disseminated in a Tertiary Hospital in Northern Spain

We describe here the occurrence of bla_VIM-2 in 10 carbapenem-resistant Pseudomonas monteilii strains isolated from different clinical samples from patients at the University Hospital Marqués de Valdecilla in northern Spain. All the bla_VIM-2-harboring P. monteilii isolates possessed a class 1 integron, with the cassette array [intI1_bla_VIM-2_aac(6′)-Ib_qacEΔ1_sul1]. Our results show the emergence of VIM-2-producing multidrug-resistant species other than Pseudomonas aeruginosa or Pseudomonas putida in a Spanish hospital. P. monteilii, although sporadically isolated, should also be considered an important metallo-β-lactamase (MBL) reservoir.     

Green Fluorescent Protein Reporter Microplate Assay for High-Throughput Screening of Compounds against Mycobacterium tuberculosis

An optimal assay for high-throughput screening for new antituberculosis agents would combine the microplate format and low cost of firefly luciferase reporter assays and redox dyes with the ease of kinetic monitoring inherent in the BACTEC system. The green fluorescent protein (GFP) of the jellyfish Aequorea victoria is a useful reporter molecule which requires neither substrates nor cofactors due to the intrinsically fluorescent nature of the protein. The gene encoding a red-shifted, higher-intensity GFP variant was introduced by electroporation into Mycobacterium tuberculosis H₃₇Ra and M. tuberculosis H₃₇Rv on expression vector pFPV2. A microplate-based fluorescence assay (GFP microplate assay [GFPMA]) was developed and evaluated by determining the MICs of existing antimycobacterial agents. The MICs of isoniazid, rifampin, ethambutol, streptomycin, amikacin, ofloxacin, ethionamide, thiacetazone, and capreomycin, but not cycloserine, determined by GFPMA were within 1 log₂ dilution of those determined with the BACTEC 460 system and were available in 7 days. Equivalent MICs of antituberculosis agents in the BACTEC 460 system for both the reporter and parent strains suggested that introduction of pFPV2 did not influence drug susceptibility, in general. GFPMA provides a unique tool with which the dynamic response of M. tuberculosis to the existing and potential antituberculosis agents can easily, rapidly, and inexpensively be monitored.     

Variations in Pentacyclic Triterpenoids in Different Parts of Four <Emphasis Type="Italic">Ocimum</Emphasis> Species Using Reverse Phase-High Performance Liquid Chromatography

Betulinic acid (BA), oleanolic acid (OA) and ursolic acid (UA) are the important pentacyclic triterpenoid compounds known for their anti-inflammatory, trypanocidal, antirheumatic, antiviral, antioxidant, antitumoral and many other activities. The quantitative analysis and distribution of BA, OA, and UA in different parts viz leaf, stem, and inflorescence of Ocimum basilicum (OB), O. gratissimum (OG), O. kilimandscharicum (OK), O. tenuiflorum (OT) was performed using reverse phase-high performance liquid chromatography and further confirmed the presence of triterpenoids in OG by high performance thin layer chromatography analysis. Leaf of OG was rich in OA (0.141 ± 0.007 %) and UA (0.296 ± 0.015 %), while BA (0.043 ± 0.011 %) was higher in the inflorescence of OK. The yield of BA, OA and UA in OG and OK was very significant (p < 0.001) as compared with OB and OT. The content of UA varied (0.008 ± 0.004 to 0.296 ± 0.015 %) within different parts and species, but this content was higher as compared to OA and BA. This communication presents for the first time simultaneous quantification of pentacyclic triterpenoids (BA, OA and UA) in OB, OG, OK and OT.     

Exploring the styrene metabolism by aerobic bacterial isolates for the effective management of leachates in an aqueous system

In the present study, the styrene metabolic profile of three aerobic bacterial isolates explored in a batch mode study. The isolates found application in the management of elachates in the waste dump yard. These three bacterial species have different origins and were studied as a single and mixed consortia. The Lysinibacillus strain M01 (from marine sources), Lysinibacillus strain WD03 (from a waste dump yard), and Pseudomonas strain BG07 (from bovine gut) were used in the present study. The styrene concentration was fixed in the range between 0.5 and 1.5 mL L⁻¹. The metabolites obtained upon microbial degradation were assessed using high-performance liquid chromatography (HPLC), UV-visible spectroscopy, and FTIR spectroscopy (Fourier transform infrared spectroscopy). Furthermore, the genes (Sty A, B, C, D, and E) responsible for the degradation of styrene by the three abovementioned isolates were identified using PCR with respective designed primers. Instrumental analyses revealed the presence of phenylacetic acid (PAA) at significant levels in the growth medium after the scheduled experimental period and confirmed the metabolism of styrene by the chosen isolates. Compared to the case of individual cultures, the results of the mixed consortia support the metabolism of styrene at appreciable levels. The present study provides a suitable biological solution for the management of leachates containing styrene and a way to achieve industrially important chemicals (PAA) through a microbially mediated process.

Profile of the three potential aerobic bacterial isolates responsible for the metabolism of styrene.     

Assessment of Mycobacterium tuberculosis Pantothenate Kinase Vulnerability through Target Knockdown and Mechanistically Diverse Inhibitors

Pantothenate kinase (PanK) catalyzes the phosphorylation of pantothenate, the first committed and rate-limiting step toward coenzyme A (CoA) biosynthesis. In our earlier reports, we had established that the type I isoform encoded by the coaA gene is an essential pantothenate kinase in Mycobacterium tuberculosis, and this vital information was then exploited to screen large libraries for identification of mechanistically different classes of PanK inhibitors. The present report summarizes the synthesis and expansion efforts to understand the structure-activity relationships leading to the optimization of enzyme inhibition along with antimycobacterial activity. Additionally, we report the progression of two distinct classes of inhibitors, the triazoles, which are ATP competitors, and the biaryl acetic acids, with a mixed mode of inhibition. Cocrystallization studies provided evidence of these inhibitors binding to the enzyme. This was further substantiated with the biaryl acids having MIC against the wild-type M. tuberculosis strain and the subsequent establishment of a target link with an upshift in MIC in a strain overexpressing PanK. On the other hand, the ATP competitors had cellular activity only in a M. tuberculosis knockdown strain with reduced PanK expression levels. Additionally, in vitro and in vivo survival kinetic studies performed with a M. tuberculosis PanK (MtPanK) knockdown strain indicated that the target levels have to be significantly reduced to bring in growth inhibition. The dual approaches employed here thus established the poor vulnerability of PanK in M. tuberculosis.     

Mathematically gifted adolescents use more extensive and more bilateral areas of the fronto-parietal network than controls during executive functioning and fluid reasoning tasks

The main goal of this study was to investigate the neural substrates of fluid reasoning and visuospatial working memory in adolescents with precocious mathematical ability. The study population comprised two groups of adolescents: 13 math-gifted adolescents and 14 controls with average mathematical skills. Patterns of activation specific to reasoning tasks in math-gifted subjects were examined using functional magnetic resonance images acquired while the subjects were performing Raven's Advanced Progressive Matrices (RAPM) and the Tower of London (TOL) tasks. During the tasks, both groups showed significant activations in the frontoparietal network. In the math-gifted group, clusters of activation were always bilateral and more regions were recruited, especially in the right hemisphere. In the TOL task, math-gifted adolescents showed significant hyper-activations relative to controls in the precuneus, superior occipital lobe (BA 19), and medial temporal lobe (BA 39). The maximum differences between the groups were detected during RAPM tasks at the highest level of difficulty, where math-gifted subjects showed significant activations relative to controls in the right inferior parietal lobule (BA 40), anterior cingulated gyrus (BA 32), and frontal (BA 9, and BA 6) areas. Our results support the hypothesis that greater ability for complex mathematical reasoning may be related to more bilateral patterns of activation and that increased activation in the parietal and frontal regions of math-gifted adolescents is associated with enhanced skills in visuospatial processing and logical reasoning.