A mini-Tn5-derived transposon with reportable and selectable markers enables rapid generation and screening of insertional mutants in Gram-negative bacteria

We re-engineered a classic tool for mutagenesis and gene expression studies in Gram-negative bacteria. Our modified Tn5-based transposon contains multiple features that allow rapid selection for mutants, direct quantification of gene expression and straightforward cloning of the inactivated gene. The promoter-less gfp-km cassette provides selection and reporter assay depending on the activity of the promoter upstream of the transposon insertion site. The cat gene facilitates positive antibiotic selection for mutants, while the narrow R6Kγ replication origin forces transposition in recipient strains lacking the pir gene and enables cloning of the transposon flanked with the disrupted gene from the chromosome. The suicide vector pCKD100, a plasmid that could be delivered into recipient cells through biparental mating or electroporation, harbours the modified transposon. We used the transposon to mutagenize Pectobacterium versatile KD100, Pseudumonas coronafaciens PC27R and Escherichia coli 35150N. The fluorescence intensities of mutants expressing high GFP could be quantified and detected qualitatively. Transformation efficiency from conjugation ranged from 1600 to 1900 CFU per ml. We sequenced the upstream flanking regions, identified the putative truncated genes and demonstrated the restoration of the GFP phenotype through marker exchange. The mini-Tn5 transposon was also utilized to construct mutant a library of P. versatile for forward genetic screens.     

Variability in host plant resistance of Sorghum to Striga Hermonthica infestation in West Africa

Fourteen elite sorghum lines were evaluated for their resistance to Striga hermonthica at three locations in Nigeria and Mali. Results showed that many of the lines especially MALISOR 84-1, SAMSORG 41, 97-SB-F5DT-64 (Keninkédié) and the check SRN 39 remained resistant to Striga in all locations with low emerged Striga counts, while SAMSORG 14 had the highest Striga infestation in all locations. Considerable variation in reaction to Striga infestation was observed on Séguètana, 97-SB-F5DT-63 (Wasa), 97-SB-F5DT-65, CMDT 38, CMDT 39 and CMDT 45 which were susceptible to Striga at Samaru, Nigeria but were resistant to Striga at both locations in Mali. Based on low Striga resistance and high grain yield, lines MALISOR 84-1, SAMSORG 41, 97-SB-F5DT-64, 97-SB-F5DT-65, CMDT 39 and SAMSORT 14 have been nominated for wider evaluation across more West African countries.     

Synthesis and structure-activity relationships of M(2)-selective muscarinic receptor ligands in the 1-[4-(4-arylsulfonyl)-phenylmethyl]-4-(4-piperidinyl)-piperazine family

The synthesis and muscarinic binding properties of compounds based on the 1-[4-(4-arylsulfonyl)phenylmethyl]-4-(1-aroyl-4-piperidinyl)-piperazine skeleton are described. For compounds, substituted with appropriately configured methyl groups at the benzylic center and at the piperazine 2-position, high levels of selective, M(2) subtype affinity could be obtained, particularly when the terminal N-aroyl residue was ortho-substituted.     

Two common nonsynonymous paraoxonase 1 (<Emphasis Type="Italic">PON1</Emphasis>) gene polymorphisms and brain astrocytoma and meningioma

Background  

Human serum paraoxonase 1 (PON1) plays a major role in the metabolism of several organophosphorus compounds. The enzyme is encoded by the polymorphic gene PON1, located on chromosome 7q21.3. Aiming to identify genetic variations related to the risk of developing brain tumors, we investigated the putative association between common nonsynonymous PON1 polymorphisms and the risk of developing astrocytoma and meningioma.     

Long-term effects of graduated compression stockings on cardiorespiratory performance

The use of graduated compression stockings (GCS) in sport has been increasing in the last years due to their potential positive effects for athletes. However, there is little evidence to support whether these types of garments actually improve cardiorespiratory performance. The aim of this study was to examine the cardiorespiratory responses of GCS during running after three weeks of regular use. Twenty recreational runners performed three tests on different days: test 1) – a 5-min maximal effort run in order to determine the participants’ maximal aerobic speed; and tests 2) and 3) – a fatigue running test of 30 minutes at 80% of their maximal aerobic speed with either GCS or PLACEBO stockings at random. Cardiorespiratory parameters (minute ventilation, heart rate, relative oxygen consumption, relative carbon dioxide production, ventilatory equivalents for oxygen and carbon dioxide, and oxygen pulse) were measured. Before each test in the laboratory, the participants trained with the randomly assigned stockings (GCS or PLACEBO) for three weeks. No significant differences between GCS and PLACEBO were found in any of the cardiorespiratory parameters. In conclusion, the present study provides evidence that running with GCS for three weeks does not influence cardiorespiratory parameters in recreational runners.     

Effect of different C3-aryl substituents on the antioxidant activity of 4-hydroxycoumarin derivatives

The antioxidant activity of 4-hydroxycoumarin synthetic derivatives and 4-methylumbelliferone were determined taking 4-hydroxycoumarin as the reference compound. Six 3-aryl-4-hydroxycoumarin derivatives were synthesized from 4-hydroxycoumarin as precursor in order to evaluate changes in their antioxidant properties due to C3-aryl substituent nature. Free radical scavenging capacities of these compounds against two different species DPPH(·) and ABTS(·+) and the protecting ability towards the β-carotene-linoleic acid co-oxidation enzymatically induced by lipoxygenase were measured. In addition, the relationship between the activities of these molecules against DPPH radical and the bond dissociation energy of O-H (BDE) calculated using methods of computational chemistry was evaluated.     

Discriminating formation of HNO from other reactive nitrogen oxide species

Nitroxyl (HNO) exhibits unique pharmacological properties that often oppose those of nitric oxide (NO), in part due to differences in reactivity toward thiols. Prior investigations suggested that the end products arising from the association of HNO with thiols were condition-dependent, but were inconclusive as to product identity. We therefore used HPLC techniques to examine the chemistry of HNO with glutathione (GSH) in detail. Under biological conditions, exposure to HNO donors converted GSH to both the sulfinamide [GSONH2] and the oxidized thiol (GSSG). Higher thiol concentrations generally favored a higher GSSG ratio, suggesting that the products resulted from competitive consumption of a single intermediate (GSNHOH). Formation of GSONH2 was not observed with other nitrogen oxides (NO, N2O3, NO2, or ONOO(-)),indicating that it is a unique product of the reaction of HNO with thiols. The HPLC assay was able to detect submicromolar concentrations of GSONH2. Detection of GSONH2 was then used as a marker for HNO production from several proposed biological pathways, including thiol-mediated decomposition of S-nitrosothiols and peroxidase-driven oxidation of hydroxylamine (an end product of the reaction between GSH and HNO) and NG-hydroxy-l-arginine (an NO synthase intermediate). These data indicate that free HNO can be biosynthesized and thus may function as an endogenous signaling agent that is regulated by GSH content.     

Insulinoma diagnosed in the postpartum: clinical and immunohistochemical features

Insulinomas are rare pancreatic β-cell tumors with an estimated incidence of 1:250.000 persons/year. We present a novel case of insulinoma manifesting immediately after childbirth. Eight days after delivery, a 21-year-old, previously healthy woman presented paresthesia in hands, upper and lower limbs muscle weakness with difficult walking, which worsened during breastfeeding sessions. Laboratory tests showed blood glucose levels between 37 and 55 mg/dL with inappropriately normal insulin levels (7.78 μUI/mL; normal range: 5-29). An abdominal computed tomography showed a nodular lesion measuring 2 cm at the head of the pancreas. Tumor enucleation resulted in complete resolution of hypoglycemia. Histopathological and immunohistochemical analysis were consistent with an insulinoma. About 27 cases of insulinoma associated with pregnancy have been reported to date, mostly diagnosed before the 16th week. The beginning of symptoms soon after delivery is less common. Understanding the interactions between pancreatic β-cell function and all the physiological metabolic and hormonal adaptations associated with gestation is essential for the adequate management of hypoglycemic disorders in pregnant women.