Evaluation of Sample Recovery Efficiency for Bacteriophage P22 on Fomites

Fomites are known to play a role in the transmission of pathogens. Quantitative analysis of the parameters that affect sample recovery efficiency (SRE) at the limit of detection of viruses on fomites will aid in improving quantitative microbial risk assessment (QMRA) and infection control. The variability in SRE as a function of fomite type, fomite surface area, sampling time, application media, relative humidity (rH), and wetting agent was evaluated. To quantify the SRE, bacteriophage P22 was applied onto fomites at average surface densities of 0.4 ± 0.2 and 4 ± 2 PFU/cm². Surface areas of 100 and 1,000 cm² of nonporous fomites found in indoor environments (acrylic, galvanized steel, and laminate) were evaluated with premoistened antistatic wipes. The parameters with the most effects on the SRE were sampling time, fomite surface area, wetting agent, and rH. At time zero (the initial application of bacteriophage P22), the SRE for the 1,000-cm² fomite surface area was, on average, 40% lower than that for the 100-cm² fomite surface area. For both fomite surface areas, the application medium Trypticase soy broth (TSB) and/or the laminate fomite predominantly resulted in a higher SRE. After the applied samples dried on the fomites (20 min), the average SRE was less than 3%. A TSB wetting agent applied on the fomite improved the SRE for all samples at 20 min. In addition, an rH greater than 28% generally resulted in a higher SRE than an rH less than 28%. The parameters impacting SRE at the limit of detection have the potential to enhance sampling strategies and data collection for QMRA models.     

Intracellular Synthesis of Gold Nanoparticles Using an Ectomycorrhizal Strain EM-1083 of <Emphasis Type="Italic">Laccaria fraterna</Emphasis> and Its Nanoanti-quorum Sensing Potential Against <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis>

In this research work different shapes and sizes of gold nanoparticles (AuNPs) were synthesized through an intracellular biogenic approach, exploiting the chloroauric acid reducing and Au⁰ stabilizing potential of Laccaria fraterna EM-1083 mycelia. The intracellularly synthesized AuNPs exhibits anti-quorum sensing inhibitory potential against Pseudomonas aeruginosa. The synthesized AuNPs were characterized using UV–visible spectroscopy; transmission electron microscopy, X-ray diffraction, energy dispersive X-ray spectroscopy, and Fourier transform infrared spectroscopy. The characterization proved that the successful synthesis of highly stable crystalline AuNPs with various shapes. Here we tested inhibitory activity of AuNPs on QS-regulated biofilm development and pyocyanin production traits of P. aeruginosa. The qualitative and quantitative data demonstrated that AuNPs significantly inhibited the biofilm formation and pyocyanin production. In summary, our results signify the future use of intracellularly synthesized AuNPs in P. aeruginosa mediated diseases.     

Molecular profiling of fungal assemblages in the healthy and infected roots of <Emphasis Type="Italic">Decalepis arayalpathra</Emphasis> (J. Joseph &amp; V. Chandras) Venter,an endemic and endangered ethnomedicinal plant from Western Ghats,India

Decalepis arayalpathra, an endangered, endemic ethnomedicinal plant from southern Western Ghats, India, is targeted for its aromatic and medicinal properties. This study aimed at to identify fungal endophyte populations associated with healthy and diseased roots of this perennial shrub. Healthy and rotted root samples of D. arayalpathra were collected, fungal endophytes assemblages were identified both by culture-dependent and culture-independent approaches, further sequenced and the retrieved sequences were analysed with the reference sequences in GenBank to know their phylogenetic relationships. Analysis of the ITS rDNA region generated 24 different Ascomycota and three Basidiomycota taxa. Trichoderma sp. was most abundant in healthy and diseased root samples, while Penicillium and Aspergillus were confined to healthy roots. Furthermore, Fusarium solani, Fusarium oxysporum and Mucor velutinosus were found to be the most frequent fungi identified from the rotted root samples, thus substantiated to be the cause for D. arayalpathra decline in the wild. Interestingly, the strains assigned to Fusarium sp. were isolated from diseased roots showing typical clearly visible symptoms, such as a severe brown discolouration on the taproot. Molecular profiling of all the pure fungal isolates, viz., Trichoderma, Penicillium, Aspergillus, Fusarium and Mucor, revealed high sequence similarities (≥ 98 %) to corresponding reference sequences. Sequencing of Trichoderma pure cultures isolated from healthy and diseased roots revealed sequence similarities to Trichoderma harzianum, T. hamatum, T. koningiopsis, T. asperellum, T. pubescens and Hypocrea sp. This confirms the morphological examinations, as Hypocrea is the teleomorph stage of Trichoderma sp. This study signifies the first work pertaining to the taxonomy of the fungal endophytic community of D. arayalpathra, and the results reported in this work may help to ascertain the cause of root rot disease often perceived in D. arayalpathra. Also, it could be useful to identify the promising endophytic communities against the root rot diseases occurring in D. arayalpathra.     

Enrichment and identification of glycoproteins in human saliva using lectin magnetic bead arrays

Aberrant glycosylation of proteins is a hallmark of tumorigenesis and could provide diagnostic value in cancer detection. Human saliva is an ideal source of glycoproteins due to the relatively high proportion of glycosylated proteins in the salivary proteome. Moreover, saliva collection is noninvasive and technically straightforward, and the sample collection and storage is relatively easy. Although differential glycosylation of proteins can be indicative of disease states, identification of differential glycosylation from clinical samples is not trivial. To facilitate salivary glycoprotein biomarker discovery, we optimized a method for differential glycoprotein enrichment from human saliva based on lectin magnetic bead arrays (saLeMBA). Selected lectins from distinct reactivity groups were used in the saLeMBA platform to enrich salivary glycoproteins from healthy volunteer saliva. The technical reproducibility of saLeMBA was analyzed with liquid chromatography–tandem mass spectrometry (LC–MS/MS) to identify the glycosylated proteins enriched by each lectin. Our saLeMBA platform enabled robust glycoprotein enrichment in a glycoprotein- and lectin-specific manner consistent with known protein-specific glycan profiles. We demonstrated that saLeMBA is a reliable method to enrich and detect glycoproteins present in human saliva.     

Non structural protein of avian influenza A (H11N1) virus is a weaker suppressor of immune responses but capable of inducing apoptosis in host cells

ABSTRACT: BACKGROUND: The Non-Structural (NS1) protein of Influenza A viruses is an extensively studied multifunctional protein which is commonly considered as key viral component to fight against host immune responses. Even though there has been a lot of studies on the involvement of NS1 protein in host immune responses there are still ambiguities regarding its role in apoptosis in infected cells. Interactions of NS1 protein with host factors, role of NS1 protein in regulating cellular responses and apoptosis are quiet complicated and further studies are still needed to understand it completely. RESULTS: NS1 genes of influenza A/Chicken/India/WBNIV2664/2008 (H5N1) and A/Aquatic bird/India/NIV-17095/2007(H11N1) were cloned and expressed in Human embryonic kidney (293T) cells. Microarray based approach to study the host cellular responses to NS1 protein of the two influenza A viruses of different pathogenicity showed significant differences in the host gene expression profile. NS1 protein of H5N1 resulted in suppression of IFN-beta mediated innate immune responses in 293T cells, leading to down-regulation of the components of JAK-STAT pathway like STAT1 which further suppressed the expression of pro-inflammatory cytokines like CXCL10 and CCL5. The degree of suppression of host immune genes was found considerable with NS1 protein of H11N1 but was not as prominent as with H5N1-NS1. TUNEL assay analyses were found to be positive in both the NS1 transfected cells indicating both H5N1 as well as H11N1 NS1 proteins were able to induce apoptosis in transfected cells. CONCLUSIONS: We propose that NS1 protein of both H5N1 and H11N1 subtypes of influenza viruses are capable of influencing host immune responses and possess necessary functionality to support apoptosis in host cells. H11N1, a low pathogenic virus without any proven evidence to infect mammals, contains a highly potential NS1 gene which might contribute to greater virus virulence in different gene combinations.     

Co-composting of physic nut (Jatropha curcas) deoiled cake with rice straw and different animal dung

To address the dispensing of this growing volume, a study on utilization of jatropha (Jatropha curcas) deoiled cake through compost production was carried out. The deoiled cake was composted with rice straw, four different animal dung (cow dung, buffalo dung, horse dung and goat dung) and hen droppings in different proportions followed by assessment, and comparison of biochemical characteristics among finished composts. Nutrient content in finished compost was within the desired level whereas metals such as copper, lead and nickel were much below the maximum allowable concentrations. Although a few finished material contained phorbol ester (0.12 mg/g), but it was far below the original level found in the deoiled cake. Such a study indicates that a huge volume of jatropha deoiled cake can be eliminated through composting.     

Effect of arbuscular mycorrhizal fungi and Pseudomonas fluorescens on root-rot and wilt,growth and yield of Coleus forskohlii

Root-rot and wilt caused by Fusarium chlamydosporum affects the cultivation of Coleus forskohlii, a medicinal plant grown for its roots, which contain a pharmaceutically important compound called forskolin. In this study, management of this disease under low and high inoculum levels was assessed with four arbuscular mycorrhizal (AM) fungi and a strain of Pseudomonas fluorescens. The AM fungus Glomus fasciculatum and P. fluorescens were the most effective treatments that reduced the severity of root-rot and wilt of C. forskohlii by 56–65% and 61–66%, respectively, under lower and higher levels of pathogen F. chlamydosporum. G. fasciculatum increased the dry shoot and root weight by 108–241% and 92–204%, respectively, while in plants treated with P. fluorescens, an increase of 97–223% and 97–172% in dry shoot and root weight, respectively, was observed. Although P. fluorescens was effective, it gave higher root yields only under lower inoculum level of the pathogen. G. fasciculatum performed equally well under both lower and higher inoculum levels. Increase in yields with both the biocontrol agents was accompanied by increase in P uptake (230–303%) and in K uptake (270–335%). The forskolin content of the roots was significantly increased (14–21%) by G. fasciculatum, P. fluorescens or G. mosseae under lower inoculum level of pathogen.     

Nano-sized titanium(IV) ternary and quaternary complexes with electron-rich oxygen-based bidentate ligands

Some novel ternary and quaternary complexes of titanium(IV) of general formula [Ti(acac)Cl_3−n(OOCR)_n] (R = C₁₅H₃₁ or C₁₇H₃₅ and n = 1-3) have been synthesized by stepwise substitution of chloride ions of [Ti(acac)Cl₃] by straight chain carboxylic acid anions. The complexes are characterized by their elemental analyses, spectral (infrared, FAB mass, ¹H NMR and powder XRD) studies, molecular weight determination and molar conductance measurements. Infrared spectra suggested bidentate chelating nature of both acetylacetonate and carboxylate anions in the complexes. Monomeric nature of the complexes was confirmed by their molecular weight determination and FAB mass spectra. Molar conductance values indicated the complexes to be non-electrolytes in DMF. The complexes exhibited high resistance to hydrolysis. Their powder XRD data indicated the nano-size for the complexes. The coordination number of titanium(IV) in these complexes were found to be six, seven and eight which has been discussed in detail.     

Activation of sucrose-phosphate synthase from Prosopis juliflora in light: Effects of protein kinase and protein phosphatase inhibitors

Sucrose‐phosphate synthase (SPS) activity measured under limiting substrate and in the presence of inorganic phosphate as an allosteric inhibitor (V_lim activity) from the leaves of Prosopis juliflora was earlier observed to respond rapidly and reversibly to light/dark transitions ( Sinha et al. 1997b,c ). The experiments therefore, were conducted to study the potential regulation of the enzyme by a mechanism of phosphorylation/dephosphorylation. The desalted extract of the enzyme prepared from irradiated leaves showed a time‐dependent spontaneous inactivation of the V_lim activity when the extract was preincubated and an additional inactivation when incubated with ATP. The spontaneous inactivation is not inhibited by phosphatase inhibitors but the ATP‐dependent inactivation was abolished when either 5′‐p‐fluorosulphonylbenzoadenosine (FSBA) or glucose‐6‐phosphate (G6P), (both reported as inhibitors for the SPS‐protein kinase from spinach) was included during preincubation. FSBA also prevented the dark inactivation of SPS in the leaves of P. juliflora when fed through the transpiration stream. The activity of SPS measured under the V_max condition remained relatively unaffected by ATP or FSBA. The desalted extract prepared from darkened leaves on the other hand, when preincubated at 25°C showed a time‐dependent increase in the V_lim activity and the activation state of the enzyme. The spontaneous activation observed during preincubation appears to be due to the dephosphorylation of the enzyme and is strongly inhibited by okadaic acid, a potent protein phosphatase inhibitor. Alternately, feeding okadaic acid to excised leaves in the dark also blocked the subsequent light activation of V_lim activity. These results are consistent with the assumption that the light/dark regulation of V_lim activity observed in the leaves of P. juliflora was mediated through a dephosphorylation/phosphorylation mechanism.