Expression and characterization of <Emphasis Type="Italic">Trichoderma virens</Emphasis> UKM-1 endochitinase in <Emphasis Type="Italic">Escherichia</Emphasis> <Emphasis Type="Italic">coli</Emphasis>

A gene encoding endochitinase from Trichoderma virens UKM-1 was cloned and expressed in E. coli BL21 (DE3). Both the endochitinase gene and its cDNA sequences were obtained. The endochitinase gene encodes 430 amino acids from an open reading frame comprising of 1,690 bp nucleotide sequence with three introns. The endochitinase was expressed as soluble and active enzyme at 20°C when induced with 1 mM IPTG. Maximum activity was observed at 4 h of post-induction time. SDS-PAGE showed that the purified endochitinase exhibited a single band with molecular weight of 42 kDa. Biochemical characterization of the enzyme displayed a near neutral pH characteristic with an optimum pH at 6.0 and optimum temperature at 50°C. The enzyme is stable between pH 3.0–7.0 and is able to retain its activity from 30 to 60°C. The presence of Mg²⁺ and Ca²⁺ ions increased the enzyme activity up to 20%. The purified enzyme has a strong affinity towards colloidal chitin and low effect on ethyl cellulose and D-cellubiose which are non-chitin related substrates. HPLC analysis from the chitin hydrolysis showed the release of (GlcNAc)₃, (GlcNAc)₂ and GlcNAc, in which (GlcNAc)₂ was the main product.     

Screening of Trace Metals in the Plasma of Breast Cancer Patients in Comparison with a Healthy Population

The plasmas of breast cancer patients and healthy donors were analyzed for selected trace metals by a flame atomic absorption spectrophotometric method. In the plasma of breast cancer patients, mean concentrations of macronutrients/essential metals, Na, K, Ca, Mg, Fe, and Zn were 3584, 197.0, 30.80, 6.740, 5.266, and 6.170 ppm, respectively, while the mean metal levels in the plasma of healthy donors were 3908, 151.0, 72.40, 17.70, 6.613, and 2.461 ppm, respectively. Average concentrations of Cd, Cr, Cu, Mn, Ni, Pb, Sb, Sr, and Zn were noted to be significantly higher in the plasma of breast cancer patients compared with healthy donors. Very strong mutual correlations (r > 0.70) in the plasma of breast cancer patients were observed between Cd–Pb, Cr–Li, Li–K, Li–Cd, K–Cr, Li–Pb, Cr–Co, Cu–Ni, Co–K, Cd–K, and K–Pb, whereas, Al–Cr, Ca–Zn, Cd–Sb, Cd–Zn, Ca–Mg, Fe–Zn, and Na–Mn exhibited strong relationships (r > 0.60) in the plasma of healthy donors. The cluster analysis revealed considerably different apportionment of trace metals in the two groups of donors. The average metal concentrations of different age groups of the two donor categories were also evaluated, which showed the build-up of Al, Cd, Co, Cr, Mn, Li, Pb, Sb, and Zn in the plasma of breast cancer patients. The role of some trace metals in carcinogenesis is also discussed. The study indicated appreciably different patterns of metal distribution and correlation in the plasma of breast cancer patients in comparison with the healthy population.     

IFN-γ-Inducible Irga6 Mediates Host Resistance against Chlamydia trachomatis via Autophagy

Chlamydial infection of the host cell induces Gamma interferon (IFNγ), a central immunoprotector for humans and mice. The primary defense against Chlamydia infection in the mouse involves the IFNγ-inducible family of IRG proteins; however, the precise mechanisms mediating the pathogen''s elimination are unknown. In this study, we identify Irga6 as an important resistance factor against C. trachomatis, but not C. muridarum, infection in IFNγ-stimulated mouse embryonic fibroblasts (MEFs). We show that Irga6, Irgd, Irgm2 and Irgm3 accumulate at bacterial inclusions in MEFs upon stimulation with IFNγ, whereas Irgb6 colocalized in the presence or absence of the cytokine. This accumulation triggers a rerouting of bacterial inclusions to autophagosomes that subsequently fuse to lysosomes for elimination. Autophagy-deficient Atg5−/− MEFs and lysosomal acidification impaired cells surrender to infection. Irgm2, Irgm3 and Irgd still localize to inclusions in IFNγ-induced Atg5−/− cells, but Irga6 localization is disrupted indicating its pivotal role in pathogen resistance. Irga6-deficient (Irga6−/−) MEFs, in which chlamydial growth is enhanced, do not respond to IFNγ even though Irgb6, Irgd, Irgm2 and Irgm3 still localize to inclusions. Taken together, we identify Irga6 as a necessary factor in conferring host resistance by remodelling a classically nonfusogenic intracellular pathogen to stimulate fusion with autophagosomes, thereby rerouting the intruder to the lysosomal compartment for destruction.     

BMI Independently Predicts Younger Age at Hip and Knee Replacement

Obesity has been identified as a risk factor for the development of hip and knee osteoarthritis (OA) and may play a role in exacerbating existing disease. Therefore, we hypothesized that obese patients would present for hip and knee replacement surgery at a younger age than nonobese patients. From our registry, we performed a cross‐sectional study of 841 hip and 804 knee replacement patients. Patients were categorized by BMI ≤25 kg/m², 25.1–29.9 kg/m², 30–34.9 kg/m², and ≥35 kg/m². Linear regression modeling was used to examine the relationship between BMI and age at surgery. Hip and knee replacement patients' mean age at surgery was 7.1 and 7.9 years younger, respectively, if their BMI was ≥35 kg/m² when compared to patients with a BMI ≤25 kg/m² (P = 0.002). BMI was a significant independent (of gender, Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) score, surgeon, and comorbidity) predictor of age at knee replacement (P < 0.05). WOMAC scores were significantly worse preoperatively in patients with a BMI ≥35 kg/m² compared to those with a BMI ≤25 kg/m² (P < 0.05). Our study indicates that obese patients, especially those with a BMI ≥35 kg/m², presented for and underwent joint replacement surgery at a younger age as compared to nonobese patients.     

Phylogenetic diversity and mutational analysis of New Delhi Metallo-β-lactamase (NDM) producing E. coli strains from pediatric patients in Pakistan

The evolution of NDM genes (bla_NDM) in E. coli is accounted for expansive multidrug resistance (MDR), causing severe infections and morbidities in the pediatric population. This study aimed to analyze the phylogeny and mutations in NDM variants of E. coli recovered from the pediatric population. Carbapenem-resistant clinical strains of E. coli were identified using microbiological phenotypic techniques. PCR technique used to amplify the bla_NDM genes, identified on agarose gel, and analyzed by DNA sequencing. The amino acid substitutions were examined for mutations after aligning with wild types. Mutational and phylogenetic analysis was performed using Lasergene, NCBI blastn, Clustal Omega, and MEGA software, whereas PHYRE2 software was used for the protein structure predictions. PCR amplification of the bla_NDM genes detected 113 clinical strains of E. coli with the contribution of bla_NDM-1 (46%), bla_NDM-4 (3.5%), and bla_NDM-5 (50%) variants. DNA sequencing of bla_NDM variants showed homology to the previously described bla_NDM-1, bla_NDM-4, and bla_NDM-5 genes available at GenBank and NCBI database. In addition, the mutational analysis revealed in frame substitutions of Pro60Ala and Pro59Ala in bla_NDM-4 and bla_NDM-5, respectively. The bla_NDM-1 was ortholog with related sequences of E. coli available at GenBank. The phylogenetic analysis indicated that the NDM gene variants resemble other microbes reported globally with some new mutational sites.     

Solutes in native plants in the Arabian Gulf region and the role of microorganisms: future research

Aims One of the outstanding challenges facing humankind is increasing crop production under various types of severe environmental conditions. Many measures have been taken to adopt molecular and biotechnological approaches that lead to the development of transgenic plants able to deal with such harsh and polluted environments. However, such solutions could be very expensive and require considerable efforts and time to achieve these objectives. The main objective of this review is to discuss the new biological solutions that have emerged in the last decade, as environmentally friendly approaches, perhaps to support and/or replace the present efforts. These solutions based on plant–microbe interactions could be a lifeline and promising alternative strategy to create plants with a high resistance to the extreme environments.     

Metal complexes of a new class of polydentate Mannich bases: Synthesis and spectroscopic characterisation

A new class of polydentate Mannich bases featuring an N₂S₂ donor system, bis((2-mercapto-N-phenylacetamido)methyl)phosphinic acid H₃L¹ and bis((2-mercapto-N-propylacetamido)methyl)phosphinic acid H₃L², has been synthesised from condensation of phosphinic acid and paraformaldehyde with 2-mercaptophenylacetamide W1 and 2-mercaptopropylacetamide W2, respectively. Monomeric complexes of these ligands, of general formula K₂[Cr^III(Lⁿ)Cl₂], K₃[M′^II(Lⁿ)Cl₂] and K[M(Lⁿ)] (M′ = Mn(II) or Fe(II); M = Co(II), Ni(II), Cu(II), Zn(II), Cd(II) or Hg(II); n = 1, 2) are reported. The structures of new ligands, mode of bonding and overall geometry of the complexes were determined through IR, UV–Vis, NMR, and mass spectral studies, magnetic moment measurements, elemental analysis, metal content, and conductance. These studies revealed octahedral geometries for the Cr(III), Mn(II) and Fe(II) complexes, square planar for Ni(II) and Cu(II) complexes and tetrahedral for the Co(II), Zn(II), Cd(II) and Hg(II) complexes. Complex formation studies via molar ratio in DMF solution were consistent to those found in the solid complexes with a ratio of (M:L) as (1:1).     

Immobilization of Aspergillus oryzae β galactosidase on zinc oxide nanoparticles via simple adsorption mechanism

The present study demonstrates the immobilization of Aspergillus oryzae β galactosidase on native zinc oxide (ZnO) and zinc oxide nanoparticles (ZnO-NP) by simple adsorption mechanism. The binding of enzyme on ZnO-NP was confirmed by Fourier transform-infrared spectroscopy and atomic force microscopy. Native ZnO and ZnO-NP showed 60% and 85% immobilization yield, respectively. Soluble and immobilized enzyme preparations exhibited similar pH-optima at pH 4.5. ZnO-NP bound β galactosidase retained 73% activity at pH 7.0 while soluble and ZnO adsorbed enzyme lost 68% and 53% activity under similar experimental conditions, respectively. There was a marked broadening in temperature-activity profile for ZnO-NP adsorbed β galactosidase; it showed no difference in temperature-optima between 50 °C and 60 °C. Moreover, ZnO-NP adsorbed β galactosidase retained 53% activity after 1 h incubation with 5% galactose while the native ZnO- and soluble β galactosidase exhibited 35% and 28% activity under similar exposure, respectively. Native ZnO and ZnO-NP adsorbed β galactosidase retained 61% and 75% of the initial activity after seventh repeated use, respectively. It was noticed that 54%, 63% and 71% milk lactose was hydrolyzed by soluble, ZnO adsorbed and ZnO-NP adsorbed β galactosidase in batch process after 9 h while whey lactose was hydrolyzed to 61%, 68% and 81% under similar experimental conditions, respectively. In view of its easy production, improved stability against various denaturants and excellent reusability, ZnO-NP bound β galactosidase may find its applications in constructing enzyme-based analytical devices for clinical, environmental and food technology.     

<Emphasis Type="Italic">Botrytis cinerea</Emphasis>-resistant marker-free <Emphasis Type="Italic">Petunia hybrida</Emphasis> produced using the MAT vector system

The presence of marker genes conferring antibiotic or herbicide resistance in transgenic plants has been a controversial issue and a serious problem for their public acceptance and commercialization. The MAT (multi-auto-transformation) vector system has been one of the strategies developed to excise the selection marker gene and produce marker-free transgenic plants. In an attempt to produce transgenic marker-free Petunia hybrida plants resistant to Botrytis cinerea (gray mold), we used the ipt gene as a selectable marker gene and the wasabi defensin (WD) gene, isolated from Wasabia japonica (a Japanese horseradish which has been a potential source of antimicrobial proteins), as a gene of interest. The WD gene was cloned from the binary vector, pEKH-WD, to an ipt-type MAT vector, pMAT21, by gateway cloning technology and transferred to Agrobacterium tumefaciens strain EHA105. Infected leaf explants of P. hybrida were cultured on hormone- and antibiotic-free MS medium. Extreme shooty phenotype (ESP)/ipt shoots were produced by the explants infected with the pMAT21-WD. The same antibiotic- and hormone-free MS medium was used in subcultures of the ipt shoots. Ipt shoots subsequently produced morphologically normal shoots. Molecular analyses of genomic DNA from the transgenic plants confirmed the integration of the gene of interest and excision of the selection marker. Expression of the WD gene was confirmed by northern blot and western blot analyses. A disease resistance assay of the marker-free transgenic plants exhibited enhanced resistance against B. cinerea strain 40 isolated from P. hybrida.     

Modeling leaflet correction techniques in aortic valve repair: A finite element study

In aortic valve sparing surgery, cusp prolapse is a common cause of residual aortic insufficiency. To correct cusp pathology, native leaflets of the valve frequently require adjustment which can be performed using a variety of described correction techniques, such as central or commissural plication, or resuspension of the leaflet free margin. The practical question then arises of determining which surgical technique provides the best valve performance with the most physiologic coaptation. To answer this question, we created a new finite element model with the ability to simulate physiologic function in normal valves, and aortic insufficiency due to leaflet prolapse in asymmetric, diseased or sub-optimally repaired valves. The existing leaflet correction techniques were simulated in a controlled situation, and the performance of the repaired valve was quantified in terms of maximum leaflets stress, valve orifice area, valve opening and closing characteristics as well as total coaptation area in diastole. On the one hand, the existing leaflet correction techniques were shown not to adversely affect the dynamic properties of the repaired valves. On the other hand, leaflet resuspension appeared as the best technique compared to central or commissural leaflet plication. It was the only method able to achieve symmetric competence and fix an individual leaflet prolapse while simultaneously restoring normal values for mechanical stress, valve orifice area and coaptation area.