Transient expression of hemagglutinin antigen from low pathogenic avian influenza A (H7N7) in Nicotiana benthamiana

The influenza A virus is of global concern for the poultry industry, especially the H5 and H7 subtypes as they have the potential to become highly pathogenic for poultry. In this study, the hemagglutinin (HA) of a low pathogenic avian influenza virus of the H7N7 subtype isolated from a Swedish mallard Anas platyrhynchos was sequenced, characterized and transiently expressed in Nicotiana benthamiana. Recently, plant expression systems have gained interest as an alternative for the production of vaccine antigens. To examine the possibility of expressing the HA protein in N. benthamiana, a cDNA fragment encoding the HA gene was synthesized de novo, modified with a Kozak sequence, a PR1a signal peptide, a C-terminal hexahistidine (6×His) tag, and an endoplasmic retention signal (SEKDEL). The construct was cloned into a Cowpea mosaic virus (CPMV)-based vector (pEAQ-HT) and the resulting pEAQ-HT-HA plasmid, along with a vector (pJL3:p19) containing the viral gene-silencing suppressor p19 from Tomato bushy stunt virus, was agro-infiltrated into N. benthamiana. The highest gene expression of recombinant plant-produced, uncleaved HA (rHA0), as measured by quantitative real-time PCR was detected at 6 days post infiltration (dpi). Guided by the gene expression profile, rHA0 protein was extracted at 6 dpi and subsequently purified utilizing the 6×His tag and immobilized metal ion adsorption chromatography. The yield was 0.2 g purified protein per kg fresh weight of leaves. Further molecular characterizations showed that the purified rHA0 protein was N-glycosylated and its identity confirmed by liquid chromatography-tandem mass spectrometry. In addition, the purified rHA0 exhibited hemagglutination and hemagglutination inhibition activity indicating that the rHA0 shares structural and functional properties with native HA protein of H7 influenza virus. Our results indicate that rHA0 maintained its native antigenicity and specificity, providing a good source of vaccine antigen to induce immune response in poultry species.     

Specific detection and quantification of culturable and non-culturable mycobacteria in metalworking fluids by fluorescence-based methods

Aims: To optimize and evaluate fluorescence microscopy assays for specific assessment of mycobacteria and co‐contaminants, including culturable and non‐culturable sub‐populations, in metalworking fluids (MWF). Methods and Results: Auramine‐O‐rhodamine (AR) staining and LIVE/DEAD BacLight? Bacterial Viability staining (L/D staining) were adapted and evaluated for detection/quantification and differentiation (viable vs non‐viable) of the MWF‐associated mycobacteria and the background bacterial flora, respectively. The AR staining method was found to be specific to MWF mycobacteria with a minimum detection limit of 10 cells ml^?1 and was comparable to the QPCR in quantification efficiency in MWF matrix. The L/D staining‐based microscopy allowed differential quantification of viable vs non‐viable cells. In general, a 3‐log difference was observed between the L/D microscopy count and culture count accounting for the presence of non‐culturable fraction in the bacterial population in in‐use MWF. Conclusions: The optimized AR staining‐ and the L/D staining‐based microscopy methods have the potential for rapid, specific and differential assessment (viable vs non‐viable) of MWF‐associated mycobacteria and co‐contaminants in field MWF. Significance and Impact of the study: Early detection of MWF mycobacteria by rapid, low‐cost, less‐skill intensive and culture‐independent fluorescence‐based microscopy methods will facilitate timely intervention to protect the machine workers from occupational hazards.     

Phycoremediation resultant lipid production and antioxidant changes in green microalgae Chlorella Sp.

In this investigation, we report on the treatment of tannery wastewater using microalgae Chlorella species to produce lipid and fatty acid as well as changes in antioxidant metabolism during the treatment. The variation in growth, production of pigments, antioxidant metabolism, lipid and fatty acids, and nutrient removal from wastewater during the remediation were observed. Surprisingly, a profuse growth was found in 50% diluted tannery wastewater (TW), which supported to accumulate high yield of lipid (18.5%) and unsaturated fatty acids (50.05%). The antioxidant activity of microalgae in both the concentrations (50% and 100% TW) were viz., lipid peroxidation 1.6 ± 0.1 and 2.3 ± 0.02nmol MDA mg^?1 protein, SOD 10.3 ± 0.4 and 15.7 ± 0.9 U mg^?1 protein, CAT 0.17 ± 0.036 and 0.52 ± 0.06 U mg^?1 protein, and APX 7.2 ± 0.8 and 11.2 ± 09 U mg^?1 protein respectively, which point out that the free radical scavenging mechanism against heavy metal stress. Maximum phycoremediation of heavy metals observed from both concentrations during the healthy growth period were Cr – 73.1, 45.7%, Cu – 90.4, 78.1%, Pb – 92.1, 52.2%, and Zn – 81.2, 44.6%, respectively. This study proved the potential use of Chlorella for heavy metal and nutrient removal from tannery wastewater. Moreover, an unaffected growth with high antioxidant activity of this species promises a sustainable lipid and fatty acid contents for biofuel production.     

Biocidal Activity of Formaldehyde and Nonformaldehyde Biocides toward Mycobacterium immunogenum and Pseudomonas fluorescens in Pure and Mixed Suspensions in Synthetic Metalworking Fluid and Saline

The microbicidal activity of four different biocides was studied in synthetic metalworking fluid (MWF) against Mycobacterium immunogenum, a suspected causative agent for hypersensitivity pneumonitis, and Pseudomonas fluorescens, a representative for the predominant gram-negative bacterial contaminants of MWF. The results indicated that M. immunogenum is more resistant than P. fluorescens to the tested formaldehyde-releasing biocides (Grotan and Bioban), isothiazolone (Kathon), and phenolic biocide (Preventol). Kathon was effective against mycobacteria at lower concentrations than the other three test biocides in MWF. In general, there was a marked increase in biocidal resistance of both the test organisms when present in MWF matrix compared to saline. Increased resistance of the two test organisms to biocides was observed when they were in a mixed suspension (1:1 ratio). The results indicate the protective effect of the MWF matrix against the action of commonly used biocides on the MWF-colonizing microbial species of occupational health significance, including mycobacteria.     

Influence of IGF-I on buffalo (Bubalus bubalis) spermatozoa motility, membrane integrity, lipid peroxidation and fructose uptake in vitro

The objective of the present experiment was to examine the influence of mean physiological concentration of insulin-like growth factor-I (IGF-I) on frozen-thawed Surti buffalo (Bubalus bubalis) spermatozoa functional parameters, i.e., motility, plasmalemma integrity, acrosomal integrity, functional membrane integrity, lipid peroxidation and fructose uptake in vitro. Frozen-thawed semen samples (n=6) were washed in tris buffer and divided into two equal parts (control and IGF-I groups). Only in the IGF-I group, IGF-I (rhIGF-I analogue) was added to a final concentration of 100 ng/ml. The samples were incubated at 37 degrees C for 2h and the assessments were made at 0, 30, 60, 90 and 120 min of incubation. The mean concentration of the buffalo seminal plasma (n=17) IGF-I was 116.83+/-28.34 ng/ml (range 41.4-198.95). IGF-I had significant effect on the total motility (P<0.01), progressive forward motility (P<0.01), functional membrane integrity (P<0.05) and lipid peroxidation levels (P<0.05) during the 120-min study period as assessed by area under curve. Treatment with IGF-I increased (P<0.01) the total spermatozoa motility at 30, 60 and 90 min as compared to the control. The progressive forward motility was significantly (P<0.01) higher at 60 and 90 min of incubation. The addition of IGF-I resulted in significant (P<0.01) increase in straight-line velocity (VSL, microm/s) as compared to the control at 60 and 90 min of incubation. The linearity (%) was significantly (P<0.01) higher in IGF-I treated semen as compared to control at 60 min of incubation. Plasmalemma integrity in IGF-I group was significantly (P<0.05) higher than control at 30 and 60 min of incubation. The functional membrane integrity differed significantly (P<0.01) between groups (control and IGF-I) at 60 and 90 min of incubation. The percentage of acrosomal intact spermatozoa decreased continuously over a period of time in both the groups. As compared to 0 min of incubation, the significant (P<0.05) loss of acrosome was observed at 60 and 90 min of incubation in control (63.87+/-3.17 vs. 58.52+/-2.54) and IGF-I (61.60+/-2.26 vs. 56.11+/-2.12) groups, respectively. Lipid peroxidation levels were significantly lower in IGF-I group at 90 min (P<0.05) and 120 min (P<0.01) of incubation than the control group. Fructose utilization was significantly higher in IGF-I group as compared to control at 30 min (P<0.05) and 60 min (P<0.01) of incubation. The present study suggests that addition of IGF-I improve spermatozoa functional parameters by reducing lipid peroxidation levels.     

Correction to: Dietary Supplementation of Walnut Partially Reverses 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine Induced Neurodegeneration in a Mouse Model of Parkinson’s Disease

Neurochemical Research - The original version of this article unfortunately contains an error in Fig. 2a (4th image for walnut). This has been corrected by publishing this erratum.     

Long-Term Dietary Supplementation of Pomegranates,Figs and Dates Alleviate Neuroinflammation in a Transgenic Mouse Model of Alzheimer’s Disease

Alzheimer’s disease (AD) is a devastating age-related neurodegenerative disease with no specific treatment at present. The APPsw/Tg2576 mice exhibit age-related deterioration in memory and learning as well as amyloid-beta (Aβ) accumulation, and this mouse strain is considered an effective model for studying the mechanism of accelerated brain aging and senescence. The present study was aimed to investigate the beneficial effects of dietary supplements pomegranate, figs, or the dates on suppressing inflammatory cytokines in APPsw/Tg2576 mice. Changes in the plasma cytokines and Aβ, ATP, and inflammatory cytokines were investigated in the brain of transgenic mice. Significantly enhanced levels of inflammatory cytokines IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, TNF-α and Eotaxin activity were decreased by administration of the diet supplements containing pomegranates, figs, or dates. In addition, putative delays in the formation of senile plaques, as indicated by a decreasing tendency of brain Aβ1–40 and Aβ1–42 contents, were observed. Thus, novel results mediated by reducing inflammatory cytokines during aging may represent one mechanism by which these supplements exert their beneficial effects against neurodegenerative diseases such as AD.     

A facile solid-state synthesis and in vitro antimicrobial activities of some 2,6-diarylpiperidin/tetrahydrothiopyran and tetrahydropyran-4-one oximes

Some 2,6-diarylpiperidin/tetrahydrothiopyran/tetrahydropyran-4-one oximes were synthesized in dry media under microwave irradiation and were evaluated for their in vitro antibacterial activity against clinically isolated bacterial strains i.e. S.aureus, β-H.Streptococcus, E.coli, P.aeruginosa, S.typhii and in vitro antifungal activities against fungal strains i.e. C.albicans, Rhizopus, A.niger and A.flavus. Structure-activity relationships for the synthesized compounds showed that compounds 12 and 15 exerted excellent antibacterial activity against all the tested bacterial strains except 15 against S.aureus and β-H.streptococcus. Against C.albicans and A.flavus, compound 15 exerted potent antifungal activities while against Rhizopus, compound 16 showed promising activity.