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Evaluation of blood culture broths with lysis buffer to directly identify specific pathogens by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry methods

Evaluation of blood culture broths with lysis buffer to directly identify specific pathogens by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry methods

Notes: E. coli with (A) or without (B) lysis buffer, respectively. The mass spectra show the difference in peaks (presence or absence) and their intensities between the sample spectrum and those of bacteria identified at first pass by BioTyper. The upper part of the figure within the inner windows shows the unknown spectrum containing perfectly matching peaks (0–200 ppm) in green, imperfectly matching peaks (200–500 ppm) in yellow, and nonmatching peaks in red. The lower part (blue) shows the dedicated main spectrum included in the database. MALDI-TOF MS score values were 2.315 (A) and 1.7799 (B), respectively. The bioMérieux blood culture system was used in both conditions.

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Mosquito salivary gland protein preservation in the field for immunological and biochemical analysis

Mosquito salivary gland protein preservation in the field for immunological and biochemical analysis

Mosquito salivary proteins are involved in several biological processes that facilitate their blood feeding and have also been reported to elicit an IgG response in vertebrates. A growing number of studies have focused on this immunological response for its potential use as a biological marker of exposure to arthropod bites. As mosquito saliva collection is extremely laborious and inefficient, most research groups prefer to work on mosquito salivary glands (SGs). Thus, SG protein integrity is a critical factor in obtaining meaningful data from immunological and biochemical analysis. Current methodologies rely on an immediate freezing of SGs after their collection. However, the maintenance of samples in a frozen environment can be hard to achieve in field conditions. In this study, SG proteins from two mosquito species (Aedes aegypti and Anopheles gambiae s.s.) stored in different media for 5 days at either +4°C or room temperature (RT) were evaluated at the quantitative (i.e., ELISA) and qualitative (i.e., SDS- PAGE and immunoblotting) levels. Our results indicated that PBS medium supplemented with an anti-protease cocktail seems to be the best buffer to preserve SG antigens for 5 days at +4°C for ELISA analysis. Conversely, cell- lysis buffer (Urea-Thiourea-CHAPS-Tris) was best at preventing protein degradation both at +4°C and RT for further qualitative analysis. These convenient storage methods provide an alternative to freezing and are expected to be applicable to other biological samples collected in the field.

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Prevalence of methicillin resistant Staphylococcus aureus (MRSA) infection and the molecular characteristics of MRSA bacteraemia over a two year period in a tertiary teaching hospital in Malaysia

Prevalence of methicillin resistant Staphylococcus aureus (MRSA) infection and the molecular characteristics of MRSA bacteraemia over a two year period in a tertiary teaching hospital in Malaysia

PFGE was performed according to the Centers for Disease Control and Prevention (CDC) PulseNet proto- col [25] with slight modification. In brief, a single colony of bacteria was streaked onto TSA (BD Difco™) and incubated at 37 °C overnight. An aliquot of 100 μl of bacterial cell suspension (containing bacterial culture in cell suspension buffer) was transferred to a microcentri- fuge tube and added with lysostaphin (1 mg/mL) and lysozyme (10 mg/mL) (Sigma-Aldrich, USA). Following incubation at 37 °C, proteinase K (20 mg/mL) (Promega Corporation, USA) and 1% seakem gold agarose (Lonza, USA) were added into the suspension, mixed and allowed to solidify in a plug mold. The plug was trans- ferred into cell lysis buffer (CLB), incubated at 54 °C for 3 h and washed with sterile distilled water and TE buf- fer. A slice of plug was cut and digested with SmaI (Pro- mega Corporation, USA) followed by separation on CHEF MAPPER in 0.5X TBE at 14 °C for 22 h with pulse time of 5 – 60 s. The Salmonella ser. Bran- derup isolate H9812 was used as the reference strain. The gel was stained in Gel Red dye (Biotium, USA) and visualized under BioRad GelDoc XR. BioNu- merics 6.5 Software package was used for cluster ana- lyses of PFGE profiles based on the unweighted pair group method with arithmetic averages (UPGMA) with a tolerance of 1.5% and optimization of 1%. The PFGE profile was assigned an arbitrary designation and the Dice coefficient of similarity, F defined the differences [11].

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SNX17 protects integrins from degradation by sorting between lysosomal and recycling pathways.

SNX17 protects integrins from degradation by sorting between lysosomal and recycling pathways.

HeLa cells were cultured in 6-well plates for 2 wk in R0K0 control, R6K4, and R6K10 media supplemented with 10% dialyzed FBS (media and FBS were obtained from Dundee Cell Products). 200,000 cells per well of a 6-well plate were reverse transfected with scrambled (R0K0), SNX17 (R6K4), and SNX27 (R10K8) siRNA oligos (ON-TARGETplus SMARTpool; Thermo Fisher Scientific) with reagent (HiPerFect; QIAGEN) according to the man- ufacturer’s reverse transfection protocol. 14 h after the first transfection, cells were transfected again using the manufacturer’s standard protocol. The cells were then incubated another 60 h. For the liquid chromatogra- phy–MS analysis of membrane proteins, the cells were detached from the culture dishes with 5 mM EDTA in PBS for 20 min. To verify the efficacy of the siRNA treatment, a fraction of the cells was lysed in standard lysis buffer and subjected to control Western blotting of SNX17 and SNX27. From the remaining cells, a crude membrane extract was obtained with a sub- cellular fractionation kit (QProteome; QIAGEN). The resulting membrane frac- tions were resolved on precast PAGE gels (NuPAGE 4–12%; Invitrogen) stained with colloidal Coomassie (SimplyBlue SafeStain; Invitrogen) fol- lowed by quantification of the amount of protein with a scanner (Odyssey; LI-COR Biosciences). The samples were then pooled in ratios according to the protein quantification, resolved by SDS-PAGE, stained with colloi- dal Coomassie, and analyzed by liquid chromatography–MS/MS-based quantification. For that, the lane was subjected to “gel walking” and cut into 10 slices, all of which were subsequently subjected to in-gel tryptic digestion using an automated digestion unit (ProGest; Digilab UK). The resulting peptides were fractionated using a nano-HPLC system (UltiMate 3000; Dionex). In brief, peptides in 1% (vol/vol) formic acid were injected onto a C18 nanotrap column (Acclaim PepMap; Dionex). After washing with 0.5% (vol/vol) acetonitrile, 0.1% (vol/vol) formic acid peptides were resolved on a 250 mm × 75–µm C18 reverse-phase analytical column (Acclaim PepMap) over a 120-min organic gradient with a flow rate of 300 nl/min 1 . Peptides were ionized by nanoelectrospray ionization

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E1B 19K Blocks Bax Oligomerization and Tumor Necrosis Factor Alpha-Mediated Apoptosis

E1B 19K Blocks Bax Oligomerization and Tumor Necrosis Factor Alpha-Mediated Apoptosis

As with the Sephacryl S-300 column, TNF/CHX induced the formation of the 500-kDa Bax protein complex (fractions 23 to 29) in the mock- and Ad5dl337-infected cells, while the for- mation of this 500-kDa Bax protein complex was almost com- pletely inhibited in the TNF/CHX-treated Ad5dl309-infected cells in the Sephacryl S-100 column fractionation (Fig. 2A). In the untreated mock-, Ad5dl309-, and Ad5dl337-infected cell extracts, 23-kDa Bid fractionated with a peak with a molecular mass of 30 kDa, consistent with Bid being monomeric (Fig. 2B). Following 4 h of TNF/CHX treatment, a significant pro- portion of Bid was cleaved into 15-kDa tBid which fractionated with a peak corresponding to a molecular mass of 43 kDa, whereas the remaining uncleaved Bid still fractionated at 30 kDa in all cases (Fig. 2B). These findings are consistent with tBid-Bax coimmunoprecipitation from TNF/CHX-treated cells (32). Strikingly, tBid did not cofractionate with the 500-kDa FIG. 1. E1B 19K inhibits formation of the TNF-␣-induced 500-kDa Bax protein complex during adenovirus infection. HeLa cells were mock, Ad5dl309, or Ad5dl337 infected for 24 h and then untreated or treated with TNF/CHX for 4 h. Cell lysates prepared in CHAPS lysis buffer were fractionated on a Sephacryl S-300 gel filtration column. Column fractions 20 to 43 were analyzed by SDS-PAGE followed by Western blotting for Bax or E1B 19K, as indicated. Whole-cell lysates from both the minus- and plus-TNF/CHX samples loaded on the column were included on every Western blot as an internal reference for Bax or E1B 19K levels. The peaks at which the molecular weight markers fractionated are indicated.

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Original Article miR-137 regulates testosterone secretion in Leydig cells of rats by Kiss-1

Original Article miR-137 regulates testosterone secretion in Leydig cells of rats by Kiss-1

HEK293 cells were purchased from Guidechem, China; DMEM medium and 10% FBS from Gibco, USA; Percoll from Wikstroms, Sweden; RIPA lysis buffer was from Solarbio, China; BCA Protein Quantification Kit was from Beyotime Biotechnology, China; goat anti-mouse second- ary antibodies, TRIzol, and RNA extraction kits were from Invitrogen, USA; Reverse transcrip- tion cDNA synthesis kits and PCR reaction kits were from Applied Biosystems, China; dual luciferase reporter assay kits were from Promega, China; UV-visible spectrophotome- ters were from Perkin-Elmer, China; and PCR gene amplifiers were from Bio-Rad, China.

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Prevalence of methicillin resistant Staphylococcus aureus (MRSA) infection and the molecular characteristics of MRSA bacteraemia over a two year period in a tertiary teaching hospital in Malaysia

Prevalence of methicillin resistant Staphylococcus aureus (MRSA) infection and the molecular characteristics of MRSA bacteraemia over a two year period in a tertiary teaching hospital in Malaysia

PFGE was performed according to the Centers for Disease Control and Prevention (CDC) PulseNet proto- col [25] with slight modification. In brief, a single colony of bacteria was streaked onto TSA (BD Difco™) and incubated at 37 °C overnight. An aliquot of 100 μl of bacterial cell suspension (containing bacterial culture in cell suspension buffer) was transferred to a microcentri- fuge tube and added with lysostaphin (1 mg/mL) and lysozyme (10 mg/mL) (Sigma-Aldrich, USA). Following incubation at 37 °C, proteinase K (20 mg/mL) (Promega Corporation, USA) and 1% seakem gold agarose (Lonza, USA) were added into the suspension, mixed and allowed to solidify in a plug mold. The plug was trans- ferred into cell lysis buffer (CLB), incubated at 54 °C for 3 h and washed with sterile distilled water and TE buf- fer. A slice of plug was cut and digested with SmaI (Pro- mega Corporation, USA) followed by separation on CHEF MAPPER in 0.5X TBE at 14 °C for 22 h with pulse time of 5 – 60 s. The Salmonella ser. Bran- derup isolate H9812 was used as the reference strain. The gel was stained in Gel Red dye (Biotium, USA) and visualized under BioRad GelDoc XR. BioNu- merics 6.5 Software package was used for cluster ana- lyses of PFGE profiles based on the unweighted pair group method with arithmetic averages (UPGMA) with a tolerance of 1.5% and optimization of 1%. The PFGE profile was assigned an arbitrary designation and the Dice coefficient of similarity, F defined the differences [11].

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An update on the mouse liver proteome

An update on the mouse liver proteome

Mouse liver proteins. A total of 9600 spots derived from 24 gels were excised and digested with trypsin (Promega). Peptides were loaded on an MTP Anchor Chip Target 600/384 (Bruker Daltonics) previously pre- pared with HCCA and analyzed in a MALDI-TOF-TOF spectrometer (Ultraflex, Bruker Daltonics). Peptide matching and protein searches were performed automatically with the MASCOT software. (MASCOT scores are reported in Additional File 4, see column "Score"). The identi- fied proteins were organized with the ProteinScape™ database (Bruker Daltonics), checked individually, and only mouse proteins or highly homologous sequences from other species were considered. In the table, proteins are sorted by accession numbers, and the Swiss-Prot annotation is given. Protein names and gene names are reported as well. Function, subcellular location, and biological process are given herein. In the column "Spot" the minimum number of spots per gel is given for each protein. The column "Gel" indicates in how many different gels of the total 24 gels cut, each protein was identified. Each experiment consisted of 12 gels run at the same time, and a total of four experiments were carried out (see Figure 2). A comparison of our mouse liver proteome with those of two other research groups is given in the column "Who" (i = Fraunhofer ITEM; f = Fountoulakis et al.; e = Expasy). For example, the protein Aldehyde dehy- drogenase 2, mitochondrial (gi|13529509) was identified in all three laboratories. The column "Lysis buffer (LB)" indicates the buffer in which the protein was identified. The column "M.R." reports which liver proteins are in common between mouse and rat. For the subcellular location the following abbreviations were adopted: C, cytosol; M, mitochondria; N, nucleus; P, peroxisome; S, secreted protein; ER, endoplasmic reticulum; G, golgi; L, lysosome; MEM, membrane; MIM, mitochondrial inner membrane; MM, mitochondrial matrix; OMM, outer mitochondrial membrane.

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Detection of porcine reproductive and respiratory syndrome virus in boar semen by PCR

Detection of porcine reproductive and respiratory syndrome virus in boar semen by PCR

Sample preparation and extraction of PRRSV RNA. Semen samples were processed similarly to the method described by Chomczynski and Sacchi (4), with minor modifications. When either whole semen or seminal plasma fractions were tested, 500 ml was added to an equal volume of lysis buffer (4 M guanidinium thiocyanate, 25 mM sodium citrate [pH 7], 0.5% Sarkosyl [N-lauryl sarcosine], 0.1 M 2-mercaptoethanol). When the cell fraction of the semen was used, 500 ml of lysis buffer without 2-mercaptoethanol was mixed with an equal volume of cells. Fifty microliters of this mixture was then added to an additional 450 ml of lysis buffer without 2-mercaptoethanol for a 1:20 dilution. Siliconized polypro- pylene tubes were used for all extraction procedures (USA/Scientific Plastics, Ocala, Fla.). Five hundred microliters of the lysates was then added to 250 m l of phenol and 250 m l of chloroform-isoamyl alcohol (24:1), and the mixtures were vortexed and centrifuged at 10,000 3 g for 5 min. The upper aqueous phase was retained and extracted again with phenol-chloroform-isoamyl alcohol and once with 500 m l of chloroform-isoamyl alcohol. The volume of the upper phase was estimated, and 1/3 volume of 2 M sodium acetate (pH 4) and 2 volumes of cold 95% ethanol were added. The sample was chilled at 2 70 8 C for 1 h and then centrifuged for 30 min at 16,000 3 g. Ethanol was carefully removed, and the pellet was washed twice with 100 m l of 70% ethanol. The pellet was then air dried and reconstituted in 30 m l of distilled water (Gibco, Grand Island, N.Y.).

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Carbohydrates Facilitate Correct Disulfide Bond Formation and Folding of Rotavirus VP7

Carbohydrates Facilitate Correct Disulfide Bond Formation and Folding of Rotavirus VP7

A reducing milieu prevents aggregation of VP7 with manip- ulated carbohydrates. We have previously shown that a brief exposure of rotavirus-infected cells to the reducing agent DTT inhibits formation of correct intradisulfide bonds on VP7 and that prolonged exposure permanently misfolds VP7 (28). To analyze if a reducing milieu also could prevent aggregation of VP7, DTT (2 mM) was added to methionine-cysteine-deficient medium 20 min before a metabolic pulse and maintained dur- ing a 60-min chase. At the end of each radioactive pulse or after a chase period, cells were washed twice with MEM and incubated with ice-cold PBS containing 40 mM NEM for 2 min to alkylate free thiol groups. Cells were then lysed in ice-cold SDS lysis buffer. As illustrated in Fig. 3A, aggregation of VP7 was prevented, both in TM and in TM plus BFA-treated cells. The dramatic effect of DTT on VP7 folding is furthermore illustrated in mock-treated cells, in which VP7 migrates signif- icantly faster under oxidizing conditions than during reducing conditions. To examine if VP7 becomes permanently protected from aggregation after a 60-min incubation in a reducing mi- lieu, DTT was washed out and cells were incubated in oxidizing media up to 60 min. As illustrated in Fig. 3B, VP7 had already begun to aggregate after 30 min in an oxidizing milieu, indi- cating that a reducing milieu prevents aggregation of rotavirus VP7, while aggregation occurs rapidly after a switch to an oxidizing milieu.

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Virus Detection Protocols for West Nile Virus in Vertebrate and Mosquito Specimens

Virus Detection Protocols for West Nile Virus in Vertebrate and Mosquito Specimens

Specimen collection. Mosquitoes were collected in the field and sorted by species. Pools of 50 or fewer were placed into 2-ml safe-lock microfuge tubes (Eppendorf cat. no. 2236335-2), each containing one 4.5-mm-diameter zinc- plated steel BB (Daisy brand), and shipped to the Arbovirus Laboratories on dry ice (15). For evaluation of RNA purification with the use of a robotic worksta- tion, Culex pipiens mosquitoes were inoculated with 10 PFU of WNV, incubated for 4 days at 27°C, and subsequently frozen at ⫺80°C. Dead vertebrates were necropsied at the Wildlife Pathology Unit of the New York State Department of Environmental Conservation, and tissues (kidneys, brains, livers, hearts, and spleens) were shipped to the Arbovirus Laboratories in individual jars on dry ice. RNA extraction. Mosquito pools were homogenized in 0.5 to 1 ml of mosquito diluent (phosphate-buffered saline supplemented with 20% fetal bovine serum, 100 ␮g of streptomycin/ml, 100 U of penicillin/ml, and 0.25 ␮g of amphotericin B/ml) by using a high-speed mechanical homogenizer (Mixer Mill MM300; Qiagen, Inc., Valencia, Calif.) for 30 s at 24 cycles/s. Each homogenized pool was centrifuged at room temperature for 4 min at 5,796 ⫻ g (Sigma centrifuge 4-15C and plate rotor 2 ⫻ 96; Qiagen), and the supernatant was removed and stored at ⫺70°C for virus isolation. The remaining mosquito pellet was lysed in 0.8 to 1 ml of either guanidine isothiocyanate-containing RNA lysis buffer (RLT from RNeasy mini kit; Qiagen) or 1⫻ lysis buffer (Applied Biosystems, Foster City, Calif.) and homogenized again as described above. RNA was extracted from the lysate by using RNeasy or the ABI Prism 6700 nucleic acid workstation (Applied Biosystems), with some modifications of the procedure described previously (12). For the RNeasy method, a 350-␮l aliquot of the lysed mosquito homogenate was used for extraction of RNA, according to the manufacturer’s directions for animal tissue, with elution in a final volume of 50 ␮l. For ABI robot extraction, 200 ␮l of lysed mosquito homogenate was vacuumed through tissue prefilter plates either in the ABI 6700 workstation or in the ABI Prism 6100 nucleic acid prep station to reduce the viscosity of the homogenized tissue lysate and to remove particles from the sample. Then RNA was purified by robot from a 50-␮l aliquot of the prefiltered material, eluting in 100 ␮l of elution buffer. For comparison of RNeasy and ABI Prism RNA extraction methods, 50 ␮l of RLT- extracted RNA was purified with RNeasy and eluted in a 100-␮l volume. For validation of the 6700 workstation, pools of 50 mosquitoes were spiked with a leg, * Corresponding author. Mailing address: The Arbovirus Laborato-

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Quantitative and Qualitative Chemical Extraction of Deoxyribo Nucleic Acid DNA from Human Cell Organelles

Quantitative and Qualitative Chemical Extraction of Deoxyribo Nucleic Acid DNA from Human Cell Organelles

to 5.2 was performed by glacial acetic acid and distilled water added to make final volume of 1 litre. xi. 70% Alcohol: 70 ml absolute alcohol was mixed in 30 ml distilled water. xii. TE Buffer: 10mM Tris-HCl, pH 8.0: 10 ml 1 M Tris-HCl, pH 8.0, 0.2 ml 0.5 M EDTA and 990 ml distilled water were mixed. xiii. 10mg/ml Ethidium Bromide Solution: 100 mg ethidium bromide was dissolved in 10 ml of deionized water and it was wrapped with aluminum foil to avoid any reaction with light. xiv. 50X TAE Buffer, pH 7.2: 242 grams Tris and 100 ml 0.5M EDTA were dissolved in 500 ml of distilled water. 57.1 ml of glacial acetic acid was added and distilled water added to make final volume of 1 litre. xv. 6X Glycerol Gel loading solution: 0.15 % Bromophenol blue, 0.15% Xylene Cyanol FF, 5mM EDTA, 30% Glycerol. 15 mg Bromophenol blue, 15mg Xylene Cyanol FF, 100 µl 0.5 M EDTA (pH 8.0) and 3 ml glycerol were dissolved in final volume of 10 ml with distilled water. 500µl of 10 blood samples were taken for DNA extraction and dissolved in 500 µl of lysis buffer I and heat shock was given at 65 0 C for 5 min for lysis of the cells. The centrifugation at 10000 rpm for 10 min is given to the cell suspension. The supernatant containing the serum was discarded. The pellet containing the cells were disturbed and dissolved in 500 µl of lysis buffer II. The 1 to 5% SDS, 25 to 125 µg proteinase K and incubation temperature 37 o C for 24 h and 56 o C for 2 h were given to the sample mixtures. 500 µl of tris saturated phenol was added to

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The development of polyester bead based particulate subunit vaccine against Johne's disease : a thesis presented in partial fulfillment of the requirements of the degree of Master of Science in Microbiology, Massey University, Palmerston North, Institute

The development of polyester bead based particulate subunit vaccine against Johne's disease : a thesis presented in partial fulfillment of the requirements of the degree of Master of Science in Microbiology, Massey University, Palmerston North, Institute of Fundamental Science

Western blot analysis of the whole cell sample, intermediate product after lysis buffer treatment, and the end product after caustic buffer treatment showed that the treatment by caustic buffer removed unexpected protein bands below the correct molecular weight of 150 kDa (Figure 40B). However, SDS-PAGE showed that the band corresponding to the antigen fusion protein was significantly reduced by caustic buffer washes (Figure 40A). Quantitatively, densitometry analysis showed that the total amount of fusion protein present on the beads was 280 ng of fusion/mg of wet beads. Taking into account that the antigen is 42% of fusion protein; therefore, each mg of beads is composed of 117.7 ng of antigen. Analysis of antigen fusion protein by BCA also showed consistent result. According to the previous immunisation study of tuberculosis antigen-presenting beads, the maximum volume that can be inoculated at a concentration of 20% (w/v) was 200 µl (unpublished data). In addition, the amount of the antigen required in

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Simultaneous purification of DNA and RNA from microbiota in a single colonic mucosal biopsy

Simultaneous purification of DNA and RNA from microbiota in a single colonic mucosal biopsy

The microbial and human RNA is prone to be degraded by RNases present in the human tissue. In the tis- sue lysis protocol of the AllPrep DNA/RNA Mini Kit, RNases and other proteins are efficiently denatured by β-mercaptoethanol added in the buffer RLT Plus. The finding of bead beating and enzymatic lysis in combina- tion with the AllPrep DNA/RNA Mini Kit being superior in breaking the ‘hard-to-lyse’ cell walls of the Firmicutes phylum (Fig. 4) resulted in development of protocol 3 in an attempt to use enzymatic lysis and bead beating and still preserve microbial and human RNA. Protocol 3 is a combination of protocol 2 and a modified version of the enzymatic lysis procedure for stool samples published by Franzosa and colleagues [23]. The enzymatic lysis step was performed after the first two rounds of mechanical tissue lysis and buffer replacement when RNases present Fig. 2 DNA quality. Gel electrophoresis of genomic DNA (1 %

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Antibiotic induced lysis of enterococci

Antibiotic induced lysis of enterococci

Enterococci are resistant to penicillin killing in vivo and in vitro. Because some bacteria resistant to penicillin killing have reduced autolytic activity, we examined the lysis of clinical enterococcal isolates suspended in buffer (spontaneous lysis), and compared it with their susceptibility to antibiotic-induced lysis and killing. We found significant correlations

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An efficient and simple CTAB based method for total genomic DNA isolation from low amounts of aquatic plants leaves with a high level of secondary metabolites

An efficient and simple CTAB based method for total genomic DNA isolation from low amounts of aquatic plants leaves with a high level of secondary metabolites

DNA extraction from plants with high secondary metabolits is very challenging (30, 31). Some methods for DNA extraction from aquatic plants used CTAB method (12). There is another method for DNA extraction from plants with high concentration of secondary metabolites (32). It is however expensive and time consuming because of using suspension buffer with extraction buffer. There are only few modifications from the CTAB method by Doyle & Doyle (2, 33) methodology. Here we have added up sodium acetate for DNA purification. Our protocol does not need proteinase in isolation step. Some aquatic plant plants have relatively high polyphenol such as Potamogeton spp.and Myriophyllum spp. (34, 35). We can remove the polyphenols by using high levels of β-mercaptoethanol as in other protocols (7). The addition of NaCl with concentration higher than 0.5 M to CTAB is known as removing factor for polysaccharides during extraction (36). In this study we used higher concentration of NaCl (1.4 M). Abu- Romman used PVP in CTAB extraction for removing polysaccharides and polyphenol (37). We used it but in case of our experiment we had not adequate amount of good quality DNA. In our protocol DNA is precipitated by using isopropanol and sodium acetate, because isopropanol gives rise to precipitation of DNA and other phenolic and secondary metabolites

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Novel Pentameric Structure of the Diarrhea-Inducing Region of the Rotavirus Enterotoxigenic Protein NSP4

Novel Pentameric Structure of the Diarrhea-Inducing Region of the Rotavirus Enterotoxigenic Protein NSP4

accession numbers 1G1I and 2O1K) is about 81°, and that between successive chains in the third structure (PDB acces- sion number 2O1J), which is a tetramer in the asymmetric unit, varies between 88° and 95°. The fact that the relation between the individual chains of the tetrameric NSP4 is significantly different from the exact 4-fold rotational symmetry might be an indication that the chains are poised to take up different types of associations. The N-terminal part (residues 95 to 120) of the chains of the tetramer being related by a 5-fold relation indi- cates flexibility within single chains. Similarly, in the present structure, the angles of rotation between successive chains do not correspond to a strict 5-fold rotation but vary between 67° and 77°. Transitions between tetrameric and pentameric struc- tures were observed previously for other proteins with parallel helical arrangements similar to that observed for the present structure. The assembly domain of a cartilage oligomeric ma- trix protein (34) and an E. coli major outer membrane lipo- protein (31, 32) were reported previously to undergo such transitions. Note that these are also membrane proteins. In the case of the E. coli protein, mutations at the a and d positions changed the oligomeric state, whereas in the cartilage matrix protein, the mutation of an Asn residue involved in interhelical H bonds to a Leu residue changed the pentameric structure to a tetramer. In the structure of the peptide reported here, a brief exposure to acidic pH during cell lysis, the presence or absence of metal ions, or high protein concentrations appeared to be responsible for the observed transition. Furthermore, many bacterial enterotoxins, such as the heat-labile entero- toxin of E. coli, cholera toxin, and Shiga toxin, are also known to form pentamers (30, 36, 44).

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Plasma Fibrinogen Is a Natural Deterrent to Amyloid β-Induced Platelet Activation and Neuronal Toxicity

Plasma Fibrinogen Is a Natural Deterrent to Amyloid β-Induced Platelet Activation and Neuronal Toxicity

Platelets contribute to >90% amyloid precursor protein in circulation (44), which, upon proteolytic cleavage by β- and γ-secretases, yields Aβ 40 (45). Despite low plasma levels, high local concentra- tions of Aβ are achieved (28) at the site of thrombus formation owing to release from stimulated platelets (46,47) and at the sites of CAA (48) or atherosclerotic plaques (49,50), which can initiate vi- cious cycles of platelet stimulation and Aβ release and potentially lead to mas- sive thrombosis. Based on observations presented in this study, it is tempting to speculate that fibrinogen acts as a physiological “shield” to preclude Aβ-induced activation of platelets, thus keeping the prothrombotic attributes of amyloid peptide strictly under check and providing steady protection against thrombotic vascular occlusion. It has, however, been reported that fibrin clots are rendered resistant to lysis follow- ing interaction with amyloid peptide (28–30). Thus, even as the probability of Aβ-induced platelet activation is mini- mized in the presence of fibrinogen, Aβ would prolong the half-life of an even- tual clot. This hypothesis is consistent with our previous findings, that although Aβ was not thrombogenic on its own, it exacerbated pulmonary thromboembo- lism induced by collagen-epinephrine in a mouse model (26).

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An Efficient Method for Extraction of Genomic DNA from Insect Gut Bacteria - Culture dependent

An Efficient Method for Extraction of Genomic DNA from Insect Gut Bacteria - Culture dependent

3.3 Molecular validation of the extracted DNA In order to check the efficiency and reliability of the genomic DNA isolated by our proposed method, DNA was subjected to restriction digestion and RAPD-PCR analysis. The electrophoresis results illustrated the complete digestion of genomic DNA by Eco RI (Figure 2). It confirmed that the quality of DNA was good, as there was no inhibitory effect of chemicals used in the extraction of genomic DNA. Further, to evaluate the quality of genomic DNA it was counter checked by RAPD-PCR analysis. The electrophoresis results showed an intact banding pattern analysis ranging from 8000 to 1000 bp (Figure 3). The PCR result clearly suggests that it does not contain any inhibitors. It was very useful to study the genetic polymorphism with high resolution. Hence, the current protocol is cost effective and does not involve the usage of any toxic chemicals or allergen. Moreover, it is adaptable to large scale isolation of genomic DNA. The current method will be useful in the study of the insect gut microbial community, and its interaction with host. On the basis of DNA quality and quantity the GBL buffer method is novel, reliable and versatile method for large scale DNA extraction. This methodology also done in triplicate and the results were observed frequently (data not shown). In addition, an extracted DNA can be revalidated by next generation sequencing (NGS) and its accurate quality can be revalidated by Fluorimeter in near future; hence it will pay the base line to study the genome assembly, genetic diversity and quality of obtained DNA.

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Isolation of dimorphic chloroplasts from the single-cell C4 species Bienertia sinuspersici

Isolation of dimorphic chloroplasts from the single-cell C4 species Bienertia sinuspersici

A reliable protocol for separating the dimorphic chloro- plasts is primarily based on the criteria of no cross-con- tamination. The isolation of C-Chls is relatively effortless due to the confinement of these organelles within the large, dense CCC structures, which can be easily recovered from the cell lysates by low-speed cen- trifugation (Figure 7A). It is worthy of note, however, that the C-Chl preparation might be potentially con- taminated with P-Chls due to incomplete protoplast lysis. Previously, we reported a low-speed centrifuga- tion-based method for floatation of healthy, intact chlor- enchyma protoplasts on a sucrose medium and sedimentation of the stressed protoplasts [12]. In the present study, any protoplasts surviving from the osmo- tically mediated lytic treatment might therefore be co- sedimented with the CCC fraction upon low-speed cen- trifugation leading to contamination of this fraction with P-Chls. Accordingly, we optimized our procedures for osmotic shock treatment and showed that the isolated protoplasts at a suitable cell density were almost exclu- sively bursted under the defined chemical, thermal and osmotic conditions (Figure 6), leading to a homogenous preparation (Figure 7A) and satisfactory purity (Figure 8) of C-Chls. In addition to P-Chls, the potential con- tamination of C-Chl preparation with chloroplasts from immature chlorenchyma cells should also be taken into account. The expression patterns of photosynthetic genes strongly suggested that these chloroplasts from immature chlorenchyma cells, as evident in the young leaf samples, might function in a C 3 default mode of

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