text:
Characterization of the Enzyme Phospholipase A1 and Its Role in Virulence in the Opportunistic Pathogen Acinetobacter Baumannii
10942 words (44 pages) Dissertation
Tags: MicrobiologyBiomedical Science
SPECIFIC AIMS.
Septicemia and pneumonia are the two most severe consequences of Acinetobacter baumannii infection which have accounted for approximately 50% mortality rate. A. baumannii has recently gained importance due to its ability to form biofilms on abiotic surfaces commonly found in healthcare settings, extreme resistance to antimicrobial agents, and ability to tolerate desiccation, disinfection to persist for a long time in adverse hospital environment. While extensive study has been done on the epidemiology and antibiotic resistance of this pathogen, little information is known so far about its virulence factors or their regulation. A recent study to characterize growth-phase dependent and serum-responsive transcriptomes of A. baumannii has identified a putative Phospholipase A1 (A1S_1919) belonging to the acyl hydrolase family of the phospholipase upregulated during exponential phase in the human serum. Phospholipase A1 in other bacteria has shown to be previously upregulated in the late exponential/stationary phase as other bacterial exoenzymes, however, upregulation of A1S_1919 in the exponential phase in serum may indicate the role of this enzyme in acute infections e.g. septicemia or early stages of biofilm formation.
Phospholipases (PL) are the group of enzymes that cleave membrane phospholipids at a specific ester bond leading to membrane lysis. They belong to two general sets depending on the cleavage of ester bond of the PL; the acyl hydrolases and phosphodiesterases; the latter have been extensively studied and shown to have a role in A. baumannii virulence. However PLAs belonging to the acyl hydrolase family have not been studied in any detail. Thus our hypothesis is that, A1S_1919 in A. baumannii encodes for a true Phospholipase A1 (PLA1) and is a key virulence factor for this bacterium. We plan to test our hypothesis by achieving the following two specific aims:
Aim 1: Determine the sub-cellular location and specific phospholipase A1 activity of A. baumannii A1S_1919 grown in 100% human serum.
Determine the sub-cellular location of A. baumannii A1S_1919 grown in 100% human serum.
Determine the phospholipase enzymatic activity and specific phospholipase A1 activity of A1S_1919.
Aim 2: Determine the role of A1S_1919 in A. baumannii virulence.
Study the ability of A1S_1919 to cause cytolysis and hemolysis of HeLa cells and RBCs respectively.
Determine the delivery of A. baumannii A1S_1919 into the host cells through OMVs.
Summary. PLAs including other phospholipases from other bacteria have been shown to be involved in virulence; however they have not been studied for their biochemical function and activity. Though the phosphodiesterase family of phospholipases has been studied previously, this study on putative phospholipase A1 will for the first time shed light on the novel enzyme belonging to the acyl hydrolase family which has never been characterized before in A. baumannii. This enzyme will also be studied for its role in virulence of this opportunistic pathogen. Understanding of more molecular virulence components may provide us with novel strategies for therapeutic intervention of A. baumannii infections.
I. Significance:
Septicemia and pneumonia are the two most severe consequences of Acinetobacter baumannii infection which have approximately a 50% mortality rate in critically ill-patients [1, 2]. A. baumannii is an emerging Gram-negative pathogen that has been associated with wound infections and bacteremia in wounded soldiers during military operations in Iraq and Afghanistan [1, 3]. It also accounts for about 10% of all nosocomial infections including ventilator associated pneumonia, skin and soft tissue infections, secondary meningitis and bacteremia [4]. According to data available at the Centers for Disease Control and Prevention (CDC), healthcare-associated infections (HAIs) in hospitals impose significant economic consequences on the nation's healthcare system [5]. A recent survey at the World Health Organization (WHO) estimates that A. baumannii accounts for 2%-10% of all Gram-negative bacterial infections in intensive care units in Europe and the United States of America making it an important pathogen to study. Its ability to form biofilms on abiotic surfaces commonly found in healthcare settings, extreme resistance to antimicrobial agents, and ability to tolerate desiccation, disinfection and to persist for a long time in the hospital environment, increases the threat posed by this pathogen to critically ill patients in the intensive care units [1, 3]. A. baumannii infections are difficult to treat because of an alarming increase in the number of multi-drug resistant strains identified in recent years. While extensive research has been done on the epidemiology and antibiotic resistance of this pathogen, little information is known about its virulence factors or their regulation [6, 7].
A. baumannii virulence factors are poorly characterized, however a recent study using a combined approach of proteomic and phenotypic analysis has identified several virulence factors namely exoproteases, phospholipase C, hemolysins, iron-acquisition systems and phenotypic factors such as biofilm formation and resistance to human serum, which are known to cause pathogenesis in other opportunistic pathogens [8]. Clinical isolates of A. baumannii have been shown to produce outer membrane vesicles (OMVs) that harbor a variety of virulence factors like OmpA, which has been shown to be previously involved in biofilm formation, along with proteases and hemolysins [3, 9]. These OMVs are considered important virulence factors for this pathogen as they have been shown to to induce host cell death by delivering cytotoxic proteins. Understanding the factors that contribute to virulence is important in order to control the spread of A. baumannii infections and to develop effective treatment against this harmful pathogen.
Recent studies have targeted the study of the expression properties of growth-phase dependent and serum-responsive transcriptomes in A. baumannii. These data will help future researchers understand the organism's expression of various virulence factors during laboratory culture conditions and in response to human serum. In laboratory culture media, factors that contribute to protein secretion and initial stages of biofilm formation are upregulated during the exponential growth phase, however, during stationary phase growth expression of systems that promote biofilm maturation are found to be upregulated. The organism's ability to colonize and survive in both the host and hospital niche may be mediated by the coordinated regulation of these growth phase-dependent processes [8]. As septicemia is one of the severe consequences of A. baumannii infection, it is important to study the mechanisms that allow for bacterial survival and persistence in blood [10]. Several other virulent determinants such as phospholipase D and OmpA were shown to increase the ability of this pathogen to survive in human serum. Determining additional factors that mediate the ability of these bacteria to survive in human serum would be helpful in designing novel drugs for treatment of A. baumannii infections. Recent study on A. baumannii cells grown in human serum identified a putative Phospholipase A1 (A1S_1919) belonging to the acyl hydrolase family of the phospholipase and was upregulated during exponential phase. Phospholipase A1 in other bacteria have been shown to be previously upregulated in the late exponential/stationary phase along with other bacterial exoenzymes. However, upregulation of A1S_1919 in the exponential phase in serum may indicate the role of this enzyme in acute infections e.g. septicemia or early stages of biofilm formation [8].
Phospholipase (PL) are the group of enzymes that cleave membrane phospholipids at a specific ester bond into lysophospholipids, lysophosphatides and free fatty acids leading to membrane lysis [11]. They belong to two general sets depending on the cleavage of ester bond of the PL; the acyl hydrolases (PLA1, PLA2, PLB and lysophospholipase) and phosphodiesterases (PLC and PLD); the latter have been extensively studied and shown to have a role in A. baumannii virulence [11, 12].
Fig 1. : Ester bond specificity of the Phospholipases. (Smith et.al 2011.)
PLAs have been detected in many pathogens, but only a limited number of PLAs have been studied for their function, activity and substrates specificity. PLA1 enzymes have not been well understood and no crystal structure exists for any true PLA1 so far [13]. Studies on Serratia liquefaciens, Legionella pneumophila and Campylobacter coli have shown PLA1 to be a virulence factor for these pathogens [14].
Innovation:
PLAs in A. baumannii have not been studied in any detail. Our group proposes to biochemically characterize this enzyme in A. baumannii. The gene encoding this enzyme will be cloned into E. coli cells and His-tag purification of this protein will be carried out. Our group has previous experience in cloning and purifying proteins. Many groups have studied the role of Phospholipase C and D in virulence of A. baumannii. However, this is the first study on biochemically characterizing phospholipases in A. baumannii.
Studies on other pathogenic bacteria have identified PLA1 to be a virulence factor. Here we also study the role of this enzyme in the virulence of this pathogen. A. baumannii OMVs are known to consists a variety of virulence factors and responsible for delivery of some of these virulence factors to the host cells, however, A. baumannii OMVs have never been studied previously for the presence of phospholipases. Our group is the first to propose that the A. baumannii OMVs contains phospholipase A1 and thus are involved in the virulence of this pathogen.
(c) APROACH
Published studies globally pertaining to the project.
Transcriptomic data of A. baumannii strain grown in 100% human serum vs. LB. Advances are being made in the identification of novel virulence factors of this pathogen; using transcriptomic studies, Dunman and colleagues comprehensively assessed the gene expression profiles of A. baumannii cells grown under exponential and stationary phase growth conditions in the laboratory media and human serum [8]. Comparison of the expression profiles of A. baumannii strain 98-37-09, identified gene A1S_1919 which was upregulated 2.7 fold in 100% human serum in the exponential phase as compared to cells grown in LB medium .This gene encodes for putative phospholipase A1, which hydrolyses phospholipids leading to cell membrane lysis [8].
Characterization of specific enzymatic activity of A1S_1919 and study its role in virulence. Phospholipase (PL) are the group of enzymes that cleave membrane phospholipids at a specific ester bond into lysophospholipids, lysophosphatides and free fatty acids leading to membrane lysis [11]. They belong to two general sets depending on the cleavage of ester bond of the PL; the acyl hydrolases (PLA1, PLA2, PLB and lysophospholipase) and phosphodiesterases (PLC and PLD). Research on A. baumannii phospholipases has shown that secreted phospholipase C (PLC) causes cytotoxicity of the epithelial cells [12], and phospholipase D is required for serum survival, epithelial cell invasion, and pathogenesis in a murine model of pneumonia [4]. Septicemia is characterized by presence of bacteria in the blood and is one of the severe consequences of A. baumannii infection, leading to high mortality rates [8, 15]. Previous studies on A. baumannii identified factors that contribute to the survival of this pathogen in the human blood to cause septicemia, which include OmpA [15], phospholipase D [4], lipopolysaccharide [16] and capsule [17] and contribute to A. baumannii virulence. Further characterization of additional biological factors that allow survival in human serum of A. baumannii is required to develop novel antimicrobials for infections like septicemia that contribute to high mortality rates worldwide. A recent transcriptomic study on A. baumanni identified gene encoding for a putative phospholipase A1 (PLA1) upregulated during exponential phase when grown in presence of human serum [8]. PLAs have been detected in many pathogens, but only limited PLAs have been studied for their function, activity and substrates specificity. PLA1 enzymes have not been well understood and no crystal structure exists for any PLA1 so far [13]. Studies on Serratia marcescens [18], Legionella pneumophila [19], Campylobacter coli [11] and Helicobacter pylori [20] have shown PLA1 to be a virulence factor. However PLAs in A. baumannii have not been studied in any detail. Thus our hypothesis is that, A1S_1919 in A. baumannii encodes for Phospholipase A1 (PLA1) and is involved in virulence.
Specific aim 1. Determine the sub-cellular location and specific phospholipase A1 activity of A. baumannii A1S_1919 grown in 100% human serum.
Working hypothesis. A1S_1919 grown in 100% human serum is located in the outer membrane and encodes for phospholipase A1.
A. Determine the sub-cellular location of A. baumannii A1S_1919 grown in 100% human serum.
Rationale: Proteomics studies include characterization of in vivo localization and abundance of proteins which are an important basis for development of vaccines and antimicrobial drug against pathogenic bacteria. Identification of the sub-cellular localization of proteins will also shed light on their probable function in the cell. Gram-negative bacteria cell envelope is complex and is composed of outer membrane (OM), the periplasm consisting of the peptidoglycan layer and the cytoplasmic membrane. Phospholipases are class of enzymes that hydrolyze various phospholipid substrates at specific ester bonds and lead to cell membrane lysis [13]. Research has shown that both membrane and secreted phospholipases are involved in bacterial pathogenesis [4, 7, 18, 19]. Gram-negative bacteria like Escherichia coli and Yersinia entercolitica are known to produce cell membrane-associated phospholipases, however Serratia liquefaciens, Serratia marcescens and Legionella pneumophila secrete phosholipases into their extracellular environment [18, 21]. Host–pathogen interactions leading to A. baumannii infection are mediated by a variety of enzymes like phospholipase C and D which are secreted to the extracellular environment and play key roles in the host cell invasion, virulence, pathogenesis and survival inside host cells [3, 4, 7, 12]. Research on pathogenic bacteria's cell envelope outer membrane is important as it the point of contact for the pathogen to its environment, to host cells and to the immune system. Many gram-negative bacteria have specialized secretion systems that are designed to allow extracellular localization of proteins, including the localization of the proteins into the host cell membrane or cytosol contributing to pathogenesis. Previous studies have shown that most virulence factors of pathogenic bacteria are outer membrane associated or secreted outside of the cell. Identification of sub-cellular localization of A. baumannii A1S_1919 (Aim 1.a) will be important in determining the probable desired function of the protein, development of vaccine and/or drug target against this pathogen if this enzyme proves to be involved in virulence.
The proposed approach involves obtaining the sub-cellular fractions of A. baumannii strain 98-37-09 and ΔA1S_1919 grown in 100% human serum. The whole-cell lysate will be obtained by cell lysis and the secreted fraction will be obtained by collecting the supernatant after cell growth. The outer membrane fractions will be obtained by French press lysis of cells followed by chaotropic reagents treatment. Extensive method of Density gradient centrifugation will be used for isolation of all subcellular compartments such as the cytoplasmic and outer membrane fractions, the periplasmic and cytosolic fractions. Recent work by Thein et al. have shown that these two methods have shown the best results for obtaining the outer membrane and cellular sub-fractions respectively. These fractions will be then subsequently probed for the presence of A1S_1919 by immunoblotting using antibody specific to phospholipase A1. Results will indicate if the protein is associated with the inner or outer membrane, cytoplasmic, periplasmic or secreted to the extracellular environment.
Experimental design:
The sub-cellular localization of A. baumannii A1S_1919 will be analyzed by established protocols developed to separate the sub-cellular fractions and subsequently probed for the presence of A1S_1919 by immunoblotting using antibody specific to phospholipase A1.
1.1.1 Bacterial strains and culture conditions: The bacterial strains and plasmids used in this study are listed in Table 1. Bacterial strains will be grown and maintained in Luria-Bertani (LB) broth on a rotary shaker at 200 rpm or on LB agar at 37°C.
Strains Description Source
ATCC 17978 A. baumannii Clinical isolate ATCC
ATCC 17978.OR blsA deletion mutant; Kmr [22]
ATCC 17978.BSP (bSP deletion mutant), Kmr This work
ATCC 17978.BSPc 17978.BSP harboring pWHBSP; Kmr Ampr This work
ATCC 17978.BSPp ATCC 17978.BSP harboring pWH1266 ; Kmr Ampr Tetr This work
E. coli strains
BL21 (DE3) Used for recombinant protein expression Invitrogen
TransforMax™ EC100D™ (pir+) Electrocompetent cells used for rescue cloning and host for pKNOCK-Amp maintenance Epicentre
Plasmids Description Source
pWH1266 E. coli-A. baumannii shuttle vector; Ampr,Tetr [23]
pCR II-TOPO PCR cloning vector; Ampr, Kanr Invitrogen
pWHBSP pWH1266 carrying wild-type copy of bSP expressed under its own promoter, Ampr, Tetr This work
pUC4K Source of Kmr cassette; Kmr Ampr Pharmacia Biotech
pKNOCK-Amp Suicide vector for allelic exchange; Ampr [24]
Table 1. Bacterial strains and plasmids used in this study
1.1.2 A1S_1919 gene deletion: Mutagenesis by gene deletion has been shown to be an effective way to determine the action(s) of a specific of protein in vivo. The deletion of A1S_1919 will be performed using method as described in [25]. The A1S_1919 protein coding gene fragment from the A. baumannii strain 98-37-09 chromosomal DNA, along with flanking sequences (1 kb each upstream and downstream) will be PCR amplified. Briefly, primer pairs will be designed with the forward primer at a natural restriction site within the neighboring up-stream gene, A1S_1918 and the reverse primer will have the addition of an artificial restriction cut site, such as KpnI, at the start site of A1S_1919. Primer pairs for the down-stream gene, A1S_1920, and the termination site for A1S_1919 will be created with the addition of an artificial restriction cut site, such as PstI on the forward primer and the reverse primer at a natural restriction enzyme cut site within the A1S_1919 sequence. PCR amplification of these fragments will be performed with a high-fidelity DNA polymerase such as Phusion (New England Biolabs). The amplicon will be cloned into pCR II-TOPO using the PCR cloning kit, thus incorporating the desired A1S_1919 protein encoding gene. This gene fragment will be excised using KpnI and PstI restriction enzymes, and the excised fragment will be cloned into the suicide vector pEX18Amp plasmid, a gene replacement suicide vector for Acinetobacter spp. This will be done by inserting a Kanamycin resistance cassette from a pUC4K restriction fragment, obtained by PCR with the kanamycin specific primers. To intoduce the desired mutation in the wild-type chromosomal DNA of A. baumannii strain 98-37-09,the recombinant knockout plasmids will be introduced into electrocompetent cells parental strains by electroporation. To confirm if the mutation occurred due to a double crossover event, the total DNA from kanamycin resistant and ampicillin sensitive mutants will be isolated for PCR using the same FW and RV primers used above.
1.1.3 Isolation of sub-cellular fractions: The protocol for isolation of sub-cellular fractions for identifying the sub-cellular localization of A. baumannii A1S_1919 is adapted from [26]. For isolation, A. baumannii strain 98-37-09 and and ΔA1S_1919 cells will be cultured overnight (OD600=0.3) in 100% human serum on a shaker at 37°C. Cellular subfractionation was performed according to two different methods, the first method focused on the fast enrichment of OMs, and thus, of outer membrane proteins based on French press cell lysis followed by treatment with chaotropic reagents., and method two focused on the fractionation of proteins from cytoplasm, cytoplasmic membrane, periplasm and OM from the cell culture. The schematic for the isolation of these sub-cellular fractions is illustrated in Fig 1.
Fig 2. – Flow-chart of the different subfractionation methods. Chaotropic reagents can be used for the fast enrichment of outer membrane proteins, and density gradient centrifugation facilitates the separation of all subcellular fractions from the cell culture.
1.1.4 Cloning, expression, and purification of A1S_1919: The DNA sequence coding for A1S_1919 (1074 bp), lacking the N-terminal signal peptide will be generared using high-fidelity DNA polymerase such as Phusion (New England Biolabs, Ipswich, MA) from A. baumannii strain 98-37-09 genomic DNA [8] using standard PCR amplification techniques. Appropriate PCR primers will be constructed to amplify in-frame A1S_1919 lacking the N-terminal signal peptide, the 5' primer containing GAGA, an XbaI restriction endonuclease site (TCTAGA) and 15 bases from the beginning of the coding sequence. The 3' primer will consist of GAGA, the BamHI restriction endonuclease site (GGATCC) and 14 bases from the 3' end of the sequence. The resulting PCR fragment cleaved by these restriction enzymes will be inserted into XbaI/ BamHI digested pET15b, which has a histidine tag site at the N-terminus and then transformed into E. coli NovaBlue Singles™ cells (Novagen, Madison, WI). Transformants will be screened for the presence of an insert by PCR with vector specific primers. Clones with the correct construct will be propagated in LB broth supplemented with 100µg/ml ampicillin and plasmids will be harvested using plasmid purification midi prep for low copy number plasmids (Qiagen, Hilden, Germany). Harvested plasmids will be sequenced verified to determine the integrity of the plasmid and insert by the Oklahoma State University Recombinant DNA/Protein Resource Facility (Stillwater, OK). The resulting plasmid will used to transform E. coli BL21 (DE3) which will be incubated in LB broth containing 100µg/ml ampicillin, at 37°C for over-expression of the A1S_1919.
1.1.5 Purification of recombinant A1S_1919 and antiserum production: E. coli containing recombinant A1S_1919 will be lysed by sonication and lysates will be subjected to immobilized nickel- chelation affinity chromatography for purification of the 6X-His tagged A1S_1919 using nitrilotriacetic acid (NTA) sepharose beads charged with nickel (Sigma, Steinheim, Germany). Purified recombinant A1S_1919 will be visualized by SDS-PAGE and Coomassie blue staining for assessment of protein purity and expected band size of 40.5kDa to determine the optimized conditions [27]. Recombinant protein concentration will be determined by using a bicinchoninic acid (BCA) protein assay kit (Pierce). To limit the generation of antiserum against the N-terminal 6x-His tag amino acids, purified A1S_1919 will be treated with the Thrombin CleanCleave kit (Sigma-Aldrich) to ensure that only the native protein without N-terminal 6x-His sequence is produced. Polyclonal antibodies against recombinant A1S_1919 will be produced commercially by Thermo Scientific Open Biosystems Custom Antibody and Proteomics Services (Rockford, IL). Briefly, 1.5 mg of recombinant A1S_1919 will be submitted for the immunization of two specific pathogen free New Zealand white rabbits. Immunizations will follow a 90-day protocol and result in up to 200ml of serum. A complete description of the service and protocol can be found online at www.thermo.com/openbiosystems. Antibody specificity will also be tested by immunoblots using recombinant A1S_1919.
1.1.6 Sub-cellular location of A. baumannii A1S_1919 using Western blot: Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) will be performed on the sub-cellular fractions (Figure.2) using the using the Laemmli gel system [28]. In detail, the two sub-cellular protein fractions will boiled for 10 min in 3× SDS sample buffer before loading the gel and then separated by 12% SDS-PAGE under denaturing conditions. Western blot will be performed using standard semi dry protocol [29] by electrotransferring the proteins onto a 0.45 μm polyvinylidene difluoride (PVDF) membrane (EMD Millipore, MA) for protein immobilization as recommended (Bio-Rad, CA). Additional protein sites were blocked by incubating the membrane in Odyssey blocking buffer (Licor, NE). The PVDF membrane will be incubated with primary rabbit polyclonal antibodies raised against recombinant A. baumannii A1S_1919 as mentioned in 1.2.4 at dilutions of 1:5,000 to probe for A1S_1919 on the PVDF membrane. The protein labeled with primary antibody will be detected by labelling it with secondary infrared red labeled Alexa Fluor® 680 goat anti-rabbit IgG and visualized using Odyssey® Infrared imaging system (Licor, NE). The presence of A. baumannii A1S_1919 with a theoretical predicted molecular mass of 43.3 kDa (Expasy sever) in the sub-cellular fractions will be compared to the respective controls which include recombinant A1S_1919 and ΔA1S_1919 cell lysate.
1.1.7 Anticipated Results: Sub-cellular fractionation and subsequent immunoblotting analyses are expected to provide information on the sub-cellular localization of A1S_1919. It is expected that A1S_1919 will be located either in the outer membrane or secreted into the extracellular environment as known for other bacterial phosholipases. The deletion of A1S_1919 is anticipated to produce a viable mutant and reveal an altered phenotype compared to wild-type and A. baumannii ΔA1S_1919:A1S_1919. Over all, these findings would establish the sub-cellular localization of phospholipase A1 encoding A1S_1919 in A. baumannii and would lead towards further characterization of A1S_1919 and the role it plays in pathogenesis.
1.1.8 Potential problems and alternative strategies: The successful cloning of a gene can pose problems starting at amplification of the desired gene region. By changing the genomic DNA concentrations and PCR cycling conditions could result in the amplification of the desired amplicon. Additional problems could arise due to intolerance and stability of the introduced gene in the given expression system. Moreover, the over expression of recombinant proteins can result in the formation of recombinant protein aggregates versus soluble protein. Ways to circumvent these constraints are possible through the use of alternative expression strains that tightly regulate expression of the introduced gene such as BL21(DE3)pLysS and BL21(DE3)pLysE (Novagen, Darmstadt, Germany). Also, if the protein is seen to be localized in the outer membrane, the above mentioned protocol for obtaining recombinant protein will not give desired results. In such case, the proteins have to be purified using protocols available for purification of outer membrane proteins in bacteria [30].
The production of highly reactive and specific antiserum is dependent on the individual animal selected for immunization and also the antigenicity of the protein. Any problems encountered during the production of polyclonal antibodies against A1S_1919 will be expected to be addressed by the Thermo Scientific Open Biosystems Custom Antibody and Proteomics Services. If on recovery the serum obtained has a low titer, determined by Western blot, the IgG will be purified using Protein A (Pierce) and subsequently concentrated.
B. Determine the phospholipase enzymatic activity and specific phospholipase A1 activity of A1S_1919.
1.2.9 Rationale: Since the in vivo action of PLA1 results mainly in the formation of lysophospholipids and free fatty acid by hydrolysis of phospholipids in the membrane, our hypothesis is that A1S_1919 should be able to degrade various phospholipid substrates in vitro. Thus the enzyme activity and specific substrate specificity of A1S_1919 will be determined in this section which has never been studied before in any detail in A. baumannii. The activity of the enzyme will be crucial in determining its function and role in A. baumannii pathogenesis.
The proposed approach will be achieved by measuring the formation of fatty acids which are the products of phospholipase activity after the hydrolysis of the membrane phospholipids. The specific PLA1 activity which is characterized by hydrolyzing the ester bond at the sn-1 position, and not sn-2 position will be measured using various fluorescent phospholipid substrates. Results will indicate if A1S_1919 encodes for phospholipase A1.
1.2.10 Release of free fatty acids from phospholipids: Non-esterified fatty acids (NEFA) released from phospholipids (PLs) will be quantitated by an in vitro enzymatic colorimetric method using a NEFA-C kit (Wako chemical, Japan).The release of free fatty acids after incubation of A1S_1919 with various PLs like phosphatidylcholine (PC), cardiolipin (CL), L-3-phosphatidylinositol (PI), L-α-phosphatidylethanolamine (PE), and sphingomyelin (SPM) will be measured colorimetrically at 550 nm. These various substrate solutions will be prepared according to the protocol mentioned with slight modifications [18]. Briefly, 5 mg of various phospholipids substrates mentioned above will be added to 1 ml of a solution of 2% taurocholic acid and 10 mM CaCl2. 1 μl recombinant A1S_1919 (from 1.2.5) will be added to 29 μl sample of each substrate solution and mixed and incubated at 37°C for 1 hour. Non-enzymatic control will be estimated using heat killed or just buffer alone with the PLs substrates. The release Non-esterified fatty acids (NEFA) of concentrations will be calculated from a calibration curve determined using oleic acid as a standard.
1.2.11 Detection of specific PLA1 activity of A1S_1919: To analyze the specific enzymatic activity of A1S_1919, an fluorometric assay previously described in [18] will be employed. Phospholipase A substrates labeled with BODYIPY FL fluorogenic dyes, 1,2-bis-(4,4-difluoro-5,7-dimethyl-4-bora-3a, 4a-diaza-sindacene-3-undecanoyl)-sn-glycero- 3-phosphocholine (bis-BODIPY®FL C11-PC; Invitrogen, New York, NY). The bis-BODIPY®FL C11-PC is a glycerophosphocholine with the BODYIPY FL fluorogenic dye labeled at the sn-1 and sn-2 acyl chain position. In this the bis-BODIPY®FL C11-PC forms acyl micelles with the acyl-chains including the BODYIPY FL moiety sequestered at the centre. The resulting high localized concentration of BODYIPY FL intermolecular self quenching of its fluorescence. The release of the soluble BODYIPY FL moiety, by acyl chain cleavage by either PLA1 or PLA2 will result in enhanced fluorescence which will be measured at 485 nm excitation and 530 nm emission uisng Biotek Synergy Mx Monochromator-Based Multi-Mode Microplate Reader (Biotek, Winooski, VT). bis-BODIPY®FL C11-PC substrate solution will be prepared at 45nM in 10mM Tris-HCl (pH 8.0), 100mM NaCl and 10mM CaCl2. A 90µl volume of the prepared substrate solution will be incubated with various concentrations of the enzyme solutions (10µl) in 96-well plate for 6 min and increase in fluorescence will be measured over time.
Because either PLA1 or PLA2 activities could lead to an increased fluorescence with BODIPY®FL C11-PC, substrates specific for PLA1 and PLA2, N-((6-(2,4-DNP)amino)hexanoyl)-1-(BODIPY®FL C5)-2-hexyl-sn-glycero-3-phosphoethanolamine (PED-A1, specific for PLA1 activity) and N-((6-(2,4-dinitrophenyl) amino)hexanoyl)-2-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-1-hexadecanoyl- sn-glycero-3-phosphoethanolamine, triethylammonium salt (PED6, specific for PLA2 activity) will be used. PED-A1 and PED6 contain BODYIPY FL fluorogenic dye labeled at the sn-1 and sn-2 acyl chain position respectively, whose fluorescence is quenched by a dinitrophenyl group attached to the polar head group. In this case, with PED-A1 only PLA1 activity and with PED6 only PLA2 activity will be able to cause fluorescence enhancement by cleaving at the sn-1 and sn-2 acyl chain position respectively. Appropriate controls like the commercially available purified PLA1 will be used as a positive control. Non-enzymatic controls like heat inactivated enzyme, buffer alone or non-PL enzyme will be used as negative controls for all enzymatic measurements.
1.2.12 Anticipated Results: The in vitro enzymatic colorimetric method using a NEFA-C kit for the measurement of the free-fatty acids is expected to confirm that A1S_1919 encodes for the phospholipase enzyme. Further, by using the fluorometric assay using three different fluorometric substrates; bis-BODIPY®FL C11-PC, PED-A1and PED6, it is expected that it will be further confirmed that A1S_1919 encodes for Phospholipase A1. Recombinant A1S_1919 is expected to cleave bis-BODIPY®FL C11-PC and PED-A1, however not PED6, thus indicating that A1S_1919 encodes for Phospholipase A1.
1.2.13 Potential problems and alternative strategies: The in vitro enzymatic colorimetric method using a NEFA-C kit for the measurement of the release of free fatty acids is expected to produce reliable results. However, if any technical difficulties arise in the measurement an alternate colorimetric assay using 4-nitro-3-(octanoyloxy)-benzoic acid (Sigma Chemical Co.) as a substrate will be employed. Cleavage of the ester bond of this substrate will lead to increased absorbance at 410 nm, which can be monitored over time using Biotek Synergy Mx Monochromator-Based Multi-Mode Microplate Reader (Biotek, Winooski, VT). The specific enzyme activity measured using fluorescent substrates is very sensitive and reliable method for determining the substrate specificity and enzymatic activity of the enzyme. However, in case of further verification more number of fluorescent substrates like NBD-C6HPC and Red/Green BODIPY®PC-A2 (Invitrogen, New York, NY) specific to PLA1 and PLA2 activity can be used.
Specific aim 2. Determine the role of A1S_1919 in A. baumannii virulence.
Working hypothesis. A1S_1919 induces host cell membrane damage and is delivered into the host cells via the Outer membrane vesicles (OMVs)
Rationale: Research has shown that host cell invasion by most pathogens requires penetration and damage of the cell membrane. This process has seen to be mediated by either enzymes or physical means, or involves combination of these two processes. The host cell envelope comprises of phospholipids and proteins, which are seen to the major constituents of the cell membrane. It is therefore speculate that phospholipases are thought to be involved in disrupting the cell membrane during the host cell invasion by releasing products that destabilize the membranes [14, 31]. All the types of phospholipases known so far are considered virulence factors for most of the bacteria, from Vibrio parahaemolyticus PLA involved in hemolysis [14], Pseudomonas aeruginosa PLC involved in tissue destruction and hemolysis [14] to A. baumannii PLD required for serum survival, epithelial cell invasion, and pathogenesis in a murine model of pneumonia [4]. Studies have shown that PLA1 plays an important role in virulence of some bacteria and fungi. PLA1 activity has been previously linked to lecithin-dependent hemolysis and promotion of colonization, in the human pathogen Yersinia enterocolitica [14]. The importance of bacterial phospholipases in pathogenesis has been reported the exact mechanism of the phospholipase action in vivo has not been exactly determined. Previous studies have linked phospholipase toxicity to cytolytic activity, which results from the accumulation of membrane-destabilizing products or by the massive destruction of membrane phospholipids by the enzyme [14, 31]. Cell lysis can occur via highly active bacterial phospholipase with broad specificity, which depends on its ability to interact directly with and hydrolyse phospholipids in the host cell membrane. Most of the studies for convenience associate the lytic activity as hemolysis, despite the ability of particular bacteria to cause systemic infection or a bacterial phospholipase entering the bloodstream [31].
Septicemia is one of the severe consequences of A. baumannii infection, which have approximately a 50% mortality rate in critically ill-patients [1, 2]. Recent transcriptomic study in A. baumannii strain 98-37-09, identified gene A1S_1919 which was upregulated 2.7 fold in 100% human serum in the exponential phase as compared to cells grown in LB medium .This gene encodes for putative phospholipase A1, which hydrolyses phospholipids leading to cell membrane lysis [8]. The proposed approach aims to study the ability of A1S_1919 to cause cytolysis and hemolysis of HeLa cells and RBCs respectively, which are representative examples of eukaryotic host cell models. Host cell membrane destruction a common mechanism of action of phospholipases represented by cytolysis and hemolysis is an excellent way to demonstrate the role of A1S_1919 in A. baumannii virulence. Also most of the phospholipases for e.g. PLA of S. marcescens have been shown to possess both cytotoxic and hemolytic properties [18]. Cytotoxic activity will be measured as amount of lactate dehydrogenase released and hemolysis will be measured as contact dependent (Absorbance at 405 nm of the hemoglobin released) and contact independent (zone of clearing on the blood agar plates) [18]. Results will indicate if A1S_1919 like other known phospholipases possess both the ability to lyse host cells and mediate hemolysis both of which are considered to be virulence determinants of a pathogen.
2.1 Experimental design:
A. Study the ability of A1S_1919 to cause cytolysis and hemolysis of HeLa cells and RBCs respectively.
2.2.2 Bacterial strains, plasmids, and culture conditions: Bacterial strains and plasmids that will be used are listed in Table1. Bacterial strains will be propagated and maintained in Luria-Bertani (LB) broth on a rotary shaker at 200 rpm or LB agar at 37°C.
2.2.3 Determination of cytotoxicity of A1S_1919: The cytotoxicity experiments will be performed as mentioned in [18] with slight modifications. Briefly, HeLa, HEp-2 and 5637 cells (derived from a human cervical cancer, epidermoid carcinoma and bladder carcinoma, respectively) will be grown in Dulbecco's Modified Eagle's Medium (DMEM) and 1640 RPMI medium (HyClone Laboratories, Logan, UT), respectively, plus fetal calf serum (10% v/v) supplemented with 2 mM L-glutamine, 1000 U/ml penicillin G, and 50 µg/ml streptomycin at 37°C in 5% CO2. 24 hours before the beginning of cytolysis experiments, sterile 96-well culture plates will be seeded with 1.0 × 104 cells per well. After seeding the ce lls, the wells will be washed with medium, and the cells will be further incubated with various concentrations
Fig 3:Measuring cell viability using the CytoTox-One™ Assay (Promega)
of His-A1S_1919 in 100 μl lecithin solution in DMEM at 37°C for
1 hour.Cytolysis will be measured as the amount of lactate dehydrogenase (LDH) released which will be measured with CytoTox-ONE™ Homogeneous Membrane Integrity Assay (Promega) [18] Complete (100%) cytolysis will be measured by quantifying the LDH release after cell lysis with 1% Triton X-100. Heat- inactivated His-A1S_1919 and Bovine ovalbumin (Albumin from chicken egg white) will be used as negative controls. Purified PLA1 will be requested from Dr. Watanabe's lab (NIID, Tokyo, Japan) [18]and used as a positive control in the experiments. Average values will be plotted from three independent experiments (technical and biological replicates).
2.2.4 Determination of hemolytic activity of A1S_1919: The assay forhemolytic activity of purified His-A1S_1919 will be performed as described previously with slight modifications [18]. Briefly, RBC suspension containing will be incubated with various concentrations of His-PhlA (0.1µg to 10 µg) at 37°C for 1 hour. After centrifuging for 5000 rpm, 10 min at room temperature, the supernatant obtained will be assayed spectrophotometrically for measuring the release of hemoglobin form the lysed RBCs. RBCs will be incubated with distilled water under the same conditions mentioned above, and this osmotic lysis will be considered as 100% hemolysis. Heat- inactivated His-A1S_1919 will be used as negative control. Purified PLA1 will be requested from Dr. Watanabe's lab (NIID, Tokyo, Japan) [18] and used as a positive control in the experiment. Average values will be plotted from three independent experiments (technical and biological replicates).
2.2.5 Anticipated Results: The cytotoxicity assay using the CytoTox-ONE™ Homogeneous Membrane Integrity Assay (Promega) for the measurement of the release of LDH is expected to confirm that A1S_1919 possess the ability to lyse the host cell membrane as observed for other known phospholipases. The hemolysis assay is expected to prove the hypothesis that A1S_1919 possess the ability to lyse the host RBCs as seen with the other known phospholipases known so far.
2.2.6 Potential problems and alternative strategies: The cytotoxicity assay using the CytoTox-ONE™ Homogeneous Membrane Integrity Assay (Promega) for the measurement of the release of LDH is expected to produce reliable results. However, if any technical difficulties arise in the measurement an alternate assay by staining the cells with FITC-conjugated Annexin V and PI (BD Pharmingen) will be employed. PI will be added to determine alterations in cell membrane integrity. The samples will be analyzed by the flow cytometry and CellQuest Pro software (BD Biosciences) as mentioned in [9]. The hemolysis experiment is expected to prove the hypothesis that A1S_1919 possesses hemolytic properties. However, if the colorimetric assay doesn't not provide satisfactory results an alternate plate assay will be employed. Briefly, various concentrations of purified His_A1S_1919 (0.1µg to 10 µg) will be inoculated on various blood agar plates such as human, horse and sheep blood agar purchased commercially from (Hardy diagnostics, Santa Maria, CA). These plates will be incubated at 37°C for overnight and the zone of hemolysis (diameter in cm) will be recorded. Average values will be plotted from three independent experiments (technical and biological replicates).
Rationale: Previous studies have shown that Gram-negative bacteria are known to secrete outer membrane vesicles (OMVs) which have been implicated to deliver of virulence factors to host cells [9]. A. baumannii has been shown to secrete OMVs when the cells were cultured in vitro. These OMVs contained virulence factors that include outer membrane protein A (OmpA) and tissue-degrading enzymes. These A. baumannii OMVs are known to deliver virulence factors like OmpA to host cells. It has been shown that the OMV-mediated delivery of virulence factors to host cells contributes to pathogenesis during infection with A. baumannii [9]. The proposed research aims to study if A. baumannii OMVs contain A1S_1919, which encodes for phospholipase known to be a virulence factor in many pathogens and if this enzyme is delivered to the host cells via OMVs. Western blot analysis using purified His-A1S_1919 will be used to study the localization of the enzyme in the OMVs and confocal microscopy using His-A1S_1919 to study the delivery of A1S_1919 into the host cell.
2.2.8 Purification of OMVs from culture supernatants: OMVs will be purified from A. baumannii culture supernatants as described previously [32]. Briefly, A. baumannii will be grown in LB broth under shaking conditions until the optical density at 600 nm (OD600) reaches 1.0. The culture will be centrifuged at 6000 rpm for 15 min for the removal of the bacterial cells. The supernatant will be filtered using a QuixStand Benchtop System (GE Healthcare, Piscataway, NJ) through a 0.2-μm fibre membrane (GE Healthcare) to remove residual bacteria cells and cellular debris. The supernatents will be then concentrated by ultrafiltration with a QuixStand Benchtop System using a 100-kDa hollow fiber membrane (GE Healthcare). The collected OMVs will be further purified by ultracentrifugation at 150 000 g for 3 hours at 4 °C. Purified OMVs will be resuspended in 20mM Tris pH 7.5, and the protein concentration will be determined using the Bradford assay (Bio-Rad Laboratories, Hercules, CA).
2.2.8 Determine the presence of A1S_1919 in the A. baumannii OMVs using Western blot: Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) will be performed on the purified OMVs obtained in 2.2.8 using the using the Laemmli gel system [28]. In detail, the two sub-cellular protein fractions will boiled for 10 min in 3× SDS sample buffer before loading the gel and then separated by 12% SDS-PAGE under denaturing conditions. Western blot will be performed using standard semi dry protocol [29] by electrotransferring the proteins onto a 0.45 μm polyvinylidene difluoride (PVDF) membrane (EMD Millipore, MA) for protein immobilization as recommended (Bio-Rad, CA). Additional protein sites were blocked by incubating the membrane in Odyssey blocking buffer (Licor, NE). The PVDF membrane will be incubated with primary rabbit polyclonal antibodies raised against recombinant A. baumannii A1S_1919 as mentioned in 1.2.4 at dilutions of 1:5,000 to probe for A1S_1919 on the PVDF membrane. The protein labeled with primary antibody will be detected by labelling it with secondary infrared red labeled Alexa Fluor® 680 goat anti-rabbit IgG and visualized using Odyssey® Infrared imaging system (Licor, NE). The presence of A. baumannii A1S_1919 with a theoretical predicted molecular mass of 43.3 kDa (Expasy sever) in the OMVs indicates that OMVs contain virulence factors to mediate pathogenesis.
2.2.9 Delivery of A1S_1919 to host cells via OMVs: Based on previous studies on proteome analysis on A. baumannii OMVs, virulence factors are contained within OMVs and are delivered into the host cells [9]. To test the hypothesis that A1S_1919 is also delivered into the host cells via OMVs we will use the three human cell lines, HeLa cell, HEp-2 cells, and U937 cells. These cell lines will be treated with OMVs acquired from A. baumannii strain 98-37-09 for 24 hours and the cellular distribution of A1S_1919 will be analyzed via confocal microscopy. The cells will be treated with 4′,6-diamidino-2-phenyllindole dihydrochloride (DAPI) for nuclear staining and anti-A1S_1919 polyclonal antibody, followed by Alexa Fluor® 488 or 568 for A1S_1919.
2.2.10 Anticipated Results: As seen with other outer membrane protein (OmpA) in A. baumannii, the Western blot analysis on the purified OMVs from A. baumannii is expected to show that A1S_1919 is localized in the OMVs. Confocal microscopy on the human cell lines used in 2.2.9 treated with OMVs acquired from A. baumannii strain 98-37-09 is expected to show that A1S_1919 is delivered into the host cell cytoplasm via the A. baumannii OMVs as seen with OmpA in A. baumannii [9].
2.2.11 Potential problems and alternative strategies: It has been shown previously that virulence factors of
A. baumannii including the outer membrane protein A (OmpA) is associated with OMVs. However, it is possible that A1S_1919 will not be associated with OMVs. Further if it is true then A1S_1919 will not be delivered into the host cells via OMVs. In this case it could be possible that A1S_1919 is delivered into the host cells via any other secretory pathway. According to the results obtained experiments could be designed to find out alternate mechanism of delivery of A1S_1919 in A. baumannii.
Aims Year 1 Year 2 Year 3
Aim1: Determine the sub-cellular location and specific phospholipase A1 activity of A. baumannii A1S_1919 grown in 100% human serum. >>>>>>>>>>>>>>>>>
Aim2: A1S_1919 induces host cell membrane damage and is delivered into the host cells via the Outer membrane vesicles (OMVs) >>>>>>>>>>>>>>>>>>>>>>>
Literature cited:
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2. Nwugo, C.C., et al., Deciphering the iron response in Acinetobacter baumannii: A proteomics approach. Journal of Proteomics, 2011. 74(1): p. 44-58.
3. Gaddy, J.A., A.P. Tomaras, and L.A. Actis, The Acinetobacter baumannii 19606 OmpA Protein Plays a Role in Biofilm Formation on Abiotic Surfaces and in the Interaction of This Pathogen with Eukaryotic Cells. Infection and Immunity, 2009. 77(8): p. 3150-3160.
4. Jacobs, A.C., et al., Inactivation of Phospholipase D Diminishes Acinetobacter baumannii Pathogenesis. Infection and Immunity, 2010. 78(5): p. 1952-1962.
5. Centers for Disease, C. and Prevention, Direct Medical Costs of Healthcare-Associated Infections in U.S. Hospitals and the Benefits of Prevention. 2009.
6. Jacobs, A.C., et al., Characterization of the Acinetobacter baumannii growth phase-dependent and serum responsive transcriptomes. FEMS Immunology & Medical Microbiology, 2012: p. n/a-n/a.
7. Antunes, L.C.S., et al., Deciphering the Multifactorial Nature of <italic>Acinetobacter baumannii</italic> Pathogenicity. PLoS ONE, 2011. 6(8): p. e22674.
8. Jacobs, A.C., et al., Characterization of the Acinetobacter baumannii growth phase-dependent and serum responsive transcriptomes. FEMS Immunology & Medical Microbiology, 2012. 64(3): p. 403-412.
9. Jin, J.S., et al., <italic>Acinetobacter baumannii</italic> Secretes Cytotoxic Outer Membrane Protein A via Outer Membrane Vesicles. PLoS ONE, 2011. 6(2): p. e17027.
10. Munoz-Price, L.S. and R.A. Weinstein, Acinetobacter Infection. New England Journal of Medicine, 2008. 358(12): p. 1271-1281.
11. Grant, K.A., et al., Molecular characterization of pldA, the structural gene for a phospholipase A from Campylobacter coli, and its contribution to cell-associated hemolysis. Infection and Immunity, 1997. 65(4): p. 1172-1180.
12. Camarena, L., et al., Molecular Mechanisms of Ethanol-Induced Pathogenesis Revealed by RNA-Sequencing. PLoS Pathog, 2010. 6(4): p. e1000834.
13. Richmond, G.S. and T.K. Smith, Phospholipases A1. International Journal of Molecular Sciences, 2011. 12(1): p. 588-612.
14. Schmiel, D.H. and V.L. Miller, Bacterial phospholipases and pathogenesis. Microbes and Infection, 1999. 1(13): p. 1103-1112.
15. Kim, S.W., et al., Serum resistance of Acinetobacter baumannii through the binding of factor H to outer membrane proteins. FEMS Microbiology Letters, 2009. 301(2): p. 224-231.
16. Luke, N.R., et al., Identification and Characterization of a Glycosyltransferase Involved in Acinetobacter baumannii Lipopolysaccharide Core Biosynthesis. Infection and Immunity, 2010. 78(5): p. 2017-2023.
17. Russo, T.A., et al., The K1 Capsular Polysaccharide of Acinetobacter baumannii Strain 307-0294 Is a Major Virulence Factor. Infection and Immunity, 2010. 78(9): p. 3993-4000.
18. Shimuta, K., et al., The hemolytic and cytolytic activities of Serratia marcescens phospholipase A (PhlA) depend on lysophospholipid production by PhlA. BMC Microbiology, 2009. 9(1): p. 261.
19. Flieger, A., B. Neumeister, and N.P. Cianciotto, Characterization of the Gene Encoding the Major Secreted Lysophospholipase A of Legionella pneumophila and Its Role in Detoxification of Lysophosphatidylcholine. Infection and Immunity, 2002. 70(11): p. 6094-6106.
20. Dorrell, N., et al., Characterization of Helicobacter pylori PldA, a phospholipase with a role in colonization of the gastric mucosa. Gastroenterology, 1999. 117(5): p. 1098-1104.
21. Givskov, M. and S. Molin, Expression of extracellular phospholipase from Serratia liquefaciens is growth-phase-dependent, catabolite-repressed and regulated by anaerobiosis. Molecular Microbiology, 1992. 6(10): p. 1363-1374.
22. Mussi, M.A., et al., The opportunistic human pathogen Acinetobacter baumannii senses and responds to light. Journal of Bacteriology, 2010. 192(24): p. 6336-45.
23. Hunger, M., et al., Analysis and Nucleotide-Sequence of an Origin of DNA-Replication in Acinetobacter-Calcoaceticus and Its Use for Escherichia-Coli Shuttle Plasmids. Gene, 1990. 87(1): p. 45-51.
24. Alexeyev, M.F., The pKNOCK series of broad-host-range mobilizable suicide vectors for gene knockout and targeted DNA insertion into the chromosome of Gram-negative bacteria. Biotechniques, 1999. 26(5): p. 824-+.
25. Arroyo, L.A., et al., The pmrCAB Operon Mediates Polymyxin Resistance in Acinetobacter baumannii ATCC 17978 and Clinical Isolates through Phosphoethanolamine Modification of Lipid A. Antimicrobial Agents and Chemotherapy, 2011. 55(8): p. 3743-3751.
26. Thein, M., et al., Efficient Subfractionation of Gram-Negative Bacteria for Proteomics Studies. Journal of Proteome Research, 2010. 9(12): p. 6135-6147.
27. Wilkins, M.R., et al., Protein Identification and Analysis Tools in the ExPASy Server2-D Proteome Analysis Protocols, A.J. Link, Editor. 1999, Humana Press. p. 531-552.
28. Laemmli, U.K., Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4. Nature, 1970. 227(5259): p. 680-685.
29. Mehta, P., Semi-Dry Protein Transfer and Immunodetection of P-Selectin Using an Antibody to its C-terminal TagProtein Blotting and Detection, B.T. Kurien and R.H. Scofield, Editors. 2009, Humana Press. p. 229-235.
30. Beis, K., et al., Two-step purification of outer membrane proteins. International Journal of Biological Macromolecules, 2006. 39(1–3): p. 10-14.
31. Istivan, T.S. and P.J. Coloe, Phospholipase A in Gram-negative bacteria and its role in pathogenesis. Microbiology, 2006. 152(5): p. 1263-1274.
32. Lee, J.C., et al., Klebsiella pneumoniae secretes outer membrane vesicles that induce the innate immune response. FEMS Microbiology Letters, 2012: p. n/a-n/a.
All Answers Ltd. (November 2018). Characterization of the Enzyme Phospholipase A1 and Its Role in Virulence in the Opportunistic Pathogen Acinetobacter Baumannii. Retrieved from https://ukdiss.com/examples/enzyme-phospholipase-a1-acinetobacter-baumannii.php?vref=1
"Characterization of the Enzyme Phospholipase A1 and Its Role in Virulence in the Opportunistic Pathogen Acinetobacter Baumannii." ukdiss.com. 11 2018. All Answers Ltd. 01 2021 <https://ukdiss.com/examples/enzyme-phospholipase-a1-acinetobacter-baumannii.php?vref=1>.
"Characterization of the Enzyme Phospholipase A1 and Its Role in Virulence in the Opportunistic Pathogen Acinetobacter Baumannii." All Answers Ltd. ukdiss.com, November 2018. Web. 28 January 2021. <https://ukdiss.com/examples/enzyme-phospholipase-a1-acinetobacter-baumannii.php?vref=1>.
All Answers Ltd. November 2018. Characterization of the Enzyme Phospholipase A1 and Its Role in Virulence in the Opportunistic Pathogen Acinetobacter Baumannii. [online]. Available from: https://ukdiss.com/examples/enzyme-phospholipase-a1-acinetobacter-baumannii.php?vref=1 [Accessed 28 January 2021].
All Answers Ltd. Characterization of the Enzyme Phospholipase A1 and Its Role in Virulence in the Opportunistic Pathogen Acinetobacter Baumannii [Internet]. November 2018. [Accessed 28 January 2021]; Available from: https://ukdiss.com/examples/enzyme-phospholipase-a1-acinetobacter-baumannii.php?vref=1.
{{cite web|last=Answers |first=All |url=https://ukdiss.com/examples/enzyme-phospholipase-a1-acinetobacter-baumannii.php?vref=1 |title=Characterization of the Enzyme Phospholipase A1 and Its Role in Virulence in the Opportunistic Pathogen Acinetobacter Baumannii |publisher=ukdiss.com |date=November 2018 |accessdate=28 January 2021 |location=Nottingham, UK}}
All Answers ltd, 'Characterization of the Enzyme Phospholipase A1 and Its Role in Virulence in the Opportunistic Pathogen Acinetobacter Baumannii' (ukdiss.com, January 2021) <https://ukdiss.com/examples/enzyme-phospholipase-a1-acinetobacter-baumannii.php?vref=1> accessed 28 January 2021


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redpajama_set_name: RedPajamaStackExchange


The Lafayette, Indiana community of dance is a beautiful thing. With so many different personalities and backgrounds, the people who love dance find meaning in another's arms as they rhythmically move across the dance floor of life.
I'm looking for a place that my wife and I can take swing dancing lessons. We both love to dance and find it difficult to find a location near our home in Lafayette, IN to take lessons.
Had signed up for dance lessons with my wife a few years ago, and promptly blew out my knee days later playing tennis. Was looking to give it another go.
Single girl just wanting to learn how to dance and have some fun.
I want to overcome my insecurities of not being able to dance certain styles.
It will be for me and my soon to be husband we get married on the 13th. And i want us to have a dance for our reception i know its last minute but we both know how just don't do it. And i want it to be prefect.


The US was the highest revenue generator in the market, primarily driven by the rising demand for cosmetic corrections among people, increasing dental deformity cases, and increasing prevalence of maxillofacial deformities or trauma. This was followed by EMEA with technological advancement as the most important market driver. In APAC, the emerging market conditions and high population have contributed to the growth of the dental syringes market.
According to this study, over the next five years, the Dental Needles market will register a 6.3% CAGR in terms of revenue, the global market size will reach US$ 72 million by 2024, from US$ 53 million in 2019. In particular, this report presents the global market share sales and revenue of key companies in the Dental Needles business, shared in Chapter 3.
This report presents a comprehensive overview, market shares, and growth opportunities of Dental Needles market by product type, application, key manufacturers and key regions and countries.
To study and analyze the global Dental Needles consumption value & volume by key regions/countries, product type and application, history data from 2014 to 2018, and forecast to 2024.
To understand the structure of Dental Needles market by identifying its various sub-segments.
Focuses on the key global Dental Needles manufacturers, to define, describe and analyze the sales volume, value, market share, market competition landscape, SWOT analysis, and development plans in the next few years.
To analyze the Dental Needles with respect to individual growth trends, future prospects, and their contribution to the total market.
To project the consumption of Dental Needles submarkets, with respect to key regions along with their respective key countries.


text:
Meta-Synthesizing Qualitative Research in Information Systems
Hossana Twinomurinzi University of South Africa
Roy Johnson Gordon Institute of Business Studies, South Africa
Keywords: Qualitative meta-synthesis, secondary data analysis, meta-triangulation, Information Systems, IS methodology, IS research
The beauty of qualitative research is in its appreciation of context, pluralism and diversity. However, this appreciation creates a problem; the results from such studies are often dissonant or appear to be disconnected. On the other hand, there is a growing acceptance and appeal for the rich insights gained from qualitative studies in Information Systems. In this paper, we propose the Qualitative Meta-Synthesis as a credible method to create substantive Information Systems theories from qualitative studies. We reflect on how Qualitative Meta-Synthesis has been used in other fields before proposing a set of guidelines. The paper makes a contribution to practice and theory. To theory, the paper offers emergent fields in Information Systems, especially those that depend a great deal on qualitative research (such as community informatics, e-government and ICT for development) a tool with which to create micro-, meso- and macro- level theories. For practice, the paper offers an approach that could assist policy makers to make sense of the dissonant findings from qualitative studies towards the creation of policy.
Hossana Twinomurinzi, University of South Africa
Associate Professor | School of Computing
Roy Johnson, Gordon Institute of Business Studies, South Africa
Roy Johnson is a visiting professor at Gordon Institute of Business Studies, South Africa. Email roy_d_johnson@hotmail.com
Responses to reviewer comments - table
Twinomurinzi, H., & Johnson, R. (2015). Meta-Synthesizing Qualitative Research in Information Systems. The Journal of Community Informatics, 11(3). Retrieved from http://ci-journal.net/index.php/ciej/article/view/1167

redpajama_set_name: RedPajamaCommonCrawl


Justin Bieber's Florida DUI Trial Set for May - South Carolina DUI Defense Lawyers | Strom Law Firm, L.L.C.
On Tuesday, March 11th, a trial date for Justin Bieber's Florida DUI charge was set for May 5th.
Bieber was formally charged with driving under the influence of drugs, resisting arrest, and possessing an invalid driver's license.
No plea deal has been offered to the young Canadian pop star, who has officially pleaded not guilty to the DUI charges. Often, first offenders receive a plea deal to avoid potential jail time.
"All we're going to say is, we're going to prepare for a trial and that Mr. Bieber has pleaded not guilty," Bieber's attorney told reporters.
The DUI trial had initially been scheduled for March, but Bieber and his attorneys had to focus on the public release of some of the police videos of the singer's DUI arrest.
Bieber was released after a judge set his bail for $2,500. If convicted on the DUI charges, Bieber faces up to 6 months in prison, but will likely receive a shorter sentence because it is his first offense.
While waiting for his Florida DUI trial, Bieber also faces other criminal charges. He was charged in Toronto for assault on a limo driver after a hockey game on December 30th. His house in California was also raided as part of an investigation into a neighbor's complaints about a January 9th "egging incident," in which Bieber reportedly threw several eggs at a neighbor's home, causing $20,000 in damages. Police reportedly found more drugs in Bieber's home, which police initially reported to be cocaine but now investigators believe to be MDMA, aka Molly or ecstasy.


text:
CNNMoney.com > Small Business > Innovation Nation
Forget India, outsource to Arkansas
Onshore Technology Services CEO Shane Mayes, who launched an IT outsourcing business in Missouri By Jennifer Alsever, contributing writerJuly 8, 2010: 3:56 AM ET
(CNNMoney.com) -- Looking for skilled, low-cost labor? Forget about India and China. How about Jonesboro, Ark.?
As the national unemployment rate hovers near 10%, some companies are starting to eye job-hungry areas of the country as prime candidates for the kind of outsourced work that once would have gone overseas.
Onshore Technology's IT center in Macon, Mo.
Dubbed "ruralsourcing," "rural outsourcing" and "onshoring," the practice relies on two simple premises: Smaller towns need jobs, and they offer a cheaper cost of living than urban centers. So businesses that outsource work to these areas can expect to pay less -- rates are often as much as 25% to 50% lower -- than if they were hiring urbanites with comparable skills.
In response, a new crop of outsourcing startups are popping up with development centers in places like Joplin, Mo., and Eveleth, Minn., where hundreds of employees crank out software code or offer IT support for large corporate clients.
"It's extremely timely given our economic climate," says Mary Lacity, an information systems professor at the University of Missouri-St. Louis who has written 12 books on outsourcing. "And I think there's a demand for it."
Compared with the estimated $60-billion-a-year offshoring industry, rural outsourcing remains just a blip on the radar. Yet the strategy is becoming a more popular option for businesses that are trying to stretch their budgets.
One rural outsourcer, Onshore Technology Services, recruits workers from minimum-wage jobs and gives them intensive training in IT specialties. Sixty-five people work in IT centers in the rural Missouri towns of Macon, Lebanon, and Joplin.
"They're moving people from fast-food to IT jobs and letting them use their brains," Lacity says.
Meanwhile, CrossUSA in Burnsville, Minn., recruits experienced, older IT workers who are nearing retirement for its 100-employee operations in Sebeka, Minn., (population 700) and Eveleth, Minn., (population 3,000).
The draw for workers is the chance to make their money stretch as far as possible prior to retirement. "They're trying to figure out the best way to finish their careers, and some people want a small-town quality of life," says John Beasley, CrossUSA's director of business development.
CrossUSA is growing at a 7% annual clip, with $9 million in sales last year. Its turnover rate is low -- in a town of 3,000 people, who can woo away your employees?
For some companies, the thought of outsourcing work to countries with different laws and business practices feels risky. Take Human Genome Sciences, a Rockville, Md., biotech company. The 900-employee company got bids from several outsourcers to handle the technical support for its back-office software. One of those bidders, AnswerThink, would have offshored the work to India. But the idea of sending confidential company information overseas, outside the reach of American intellectual property law, didn't sit well with David Evans, an IT director at Human Genome Sciences.
Instead, Evans hired Atlanta-based Rural Sourcing Inc. and its team of software experts in Jonesboro, Ark.
"There's a real desire to keep things onshore," Evans says. "There's a backlash against offshoring. There are a lot of horror stories, a lot of jokes."
Evans had his own stories of struggling to work on projects outsourced overseas by Human Genome Sciences' vendors. The outsourcers were in drastically different time zones, concepts were hard to communicate, and cultural differences created misunderstandings.
Human Genome Sciences now pays about $55 an hour for technical support from Rural Sourcing. That's about 15% higher than the rates quoted by the Indian outsourcing firm Evans considered, but half of what it would cost for him to hire a software development firm locally in the Washington, D.C., metro area.
"Now when we have a problem, we can get right on the phone and talk through the issue real-time. That right there is worth the extra cost," Evans says.
Launched in 2004, Rural Sourcing Inc. sets up shop in mid-size cities that are near universities -- places like Jonesboro, where the average IT salary is $35,000, versus $65,000 in a large metro area. The cost of living in Jonesboro is also 23% less than the U.S. average.
Rural Sourcing now counts GlaxoSmithKline (GSK) and R.J. Reynolds as clients, and its revenues are on track to triple this year to $4 million, says CEO Monty Hamilton. The company plans to expand beyond a single center in Jonesboro and open two new IT development centers, including one in Greenville, N.C., by the year's end.
"Companies are stumbling upon us, and it's growing gangbusters," Hamilton says. "There's no reason why we can't put a lot of people to work."
That includes people like Zachariah Carlson, 27, who expected he would have to move to California to get a job after he graduated last fall from Arkansas State University with a computer science degree. (Jonesboro's biggest employers are the university, retailers and hospitals.)
Carlson says he and his girlfriend were grateful he got a job locally customizing back-end software systems for corporate clients. He works out of Rural Sourcing's 60-person development center, not too far from Booneville, the 5,000-person town where he grew up. "This is where we are from. Our whole lives have been here," Carlson says.
But don't call Carlson "cheap labor." He bristles at the idea. After all, he says, he's doing what he wants, where he wants, and he's proud of his work, even if it doesn't come with a Silicon Valley salary.
"The reason we're low-cost is because we're in a more rural area with a lower cost of living," he says. "I didn't have to sacrifice anything to get where I am."
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text:
ACR Bivy Stick Survival Story: David Woolsey
August 24, 2021 August 25, 2021 Inspiration, Survivor Stories
The climb to West Lake was harder than David expected. Despite a decent night's sleep, a solid hot breakfast and decent morning weather, he started losing the mental game that backpacking demands we play. The elevation was affecting his breathing and tiring him out. His pack felt heavier than it should, he lost his appetite and didn't force new calories in to replace the ones he spent. David was drinking water, but not enough. The snow drifts he encountered were deeper and softer than he cared for. But he was getting closer to his destination, so he pushed ahead.
David's GPS guided him when he lost the trail through snow and trees. The area was all scree and without a clear trail to follow, every step demanded caution and focus. Eventually he crested the last rise and looked down into the basin for West Lake. The far slope had several feet of snow all the way to the water's edge. The area was all snow, rocks and trees. There weren't any obvious places to camp. The temperature was dropping. The skies had darkened, like his mood. David was disappointed and tired.
It was still early in the afternoon, but David didn't want to go back down to Green Lake. So he found a patch of rocky somewhat level ground just big enough for his tent and set it. There was little dirt, so David used rocks and sticks to anchor the tent and fly. Just as he finished, it happened. Snow. At first soft flakes that melted as soon as they hit his jacket. Then larger round pieces. They were like hail, but with softer edges. Like Dippin' Dots. Dippin' Dots were falling from the sky, but without all the cool flavors. David later learned this kind of snow has a name – Graupel. Kinda neat reading about it in a warm hotel room. Not kinda neat living in it, not eager to be snow camping. David was prepared for low temps or even rain. He wasn't (mentally) prepared for snow or freezing rain.
Still, David had warm clothes, a rain jacket, rain pants, 20 degree bag and a dry tent all set up. David put all of his clothes in the tent so he had plenty of layers, secured his bear can, covered his pack and hunkered down. His plan was to shelter in place and hike out in the morning. It was all downhill, all the way to the trailhead. No problem…or so he thought.
Oh wait, problem: David had no appetite; the thought of food made him want to vomit. He had a liter of water, and was too tired and angry at his own situation to get more, so he started rationing it, knowing it would have to last him until sometime the next morning. Thankfully he had enough mental energy to throw his Sawyer water filtration system in his sleeping bag with him, so it wouldn't freeze, along with his fuel can. David pulled an updated weather forecast from his Bivy Stick and knew what tomorrow looked like. Snow until midnight, low of 29 degrees, 26% chance of snow around 6am, but temps would rise above freezing around 9am. No big deal, he thought. He had warm clothes and it's all downhill. Just get through the night, stay warm and dry, and all is good.
And mostly, it was. His Nemo Hornet 2P tent was probably not designed to carry the weight of snow, but it did just fine. The fly sagged under its weight, but not so much that it touched the walls or collapsed. At one point he got out for nature's call, and saw it was holding a few inches of snow rather well. Impressed, he climbed back inside and watched a movie to pass the time. After the lightning and thunder (1-1000, 2-1000, 3-1000, that one was pretty close!) passed, the graupel turned to regular silent falling flakes. The sun set somewhere in the world and night fell. David drifted into a fitful sleep, dry and not freezing so things were looking okay!
The next morning he woke to a frigid 28 degrees, but the snow had stopped. While David still had no appetite, he forced a few bites of an RX Bar down. It tasted like paste. He drank some water and didn't vomit, and contemplated getting more water, but the edge of the lake was ringed with ice. He knew if he fell through the ice trying to get water, he'd be in a very bad situation with no one to help him. David packed everything and started carefully making his way across the snow-covered rocks. It was unnerving not being able to see the terrain under the snow and David was in constant worry of twisting an ankle, or worse, falling and breaking a bone.
David's GPS app guided him because there was no sign of the trail. Everything was covered in several inches of fresh snow, but all he really needed to know was to go down. He had been telling himself all night "shelter in place, tomorrow is all downhill, just get below the snow line and walk to the car." But as he made his way through the fluffy white, he looked out at the valley and saw that everything had been covered in snow. Green Lake was covered in white. From his vantage point, his fuddled brain saw only snow and determined there was no snow line. It didn't dawn on him that the valley only received a dusting, and it would melt by the time he got there. David only saw white, and just like that, the seed of doubt was planted.
David kept sinking as he walked, and more than the few inches that had fallen in the night. He wasn't following the trail; he had wandered into areas he had seen off the trail on his way up, areas of snow in perpetual shade that wouldn't melt for maybe another month. On his way up they weren't a concern. They were "over there somewhere." Well, now he was "over there somewhere" too.
He was either sliding on the snow or sinking into it up to his ankles. Then mid-calf. Then to his knees. Eventually to his hips. David was expending huge amounts of energy to go 20 feet, crawling out of snow pockets.
As he made his way down, back towards Green Lake and eventually the trailhead, he noticed a theme to making his way across a steep slope of snow: sink or slide. His feet either sank into the snow or slid out from under him. As he slid, disturbing the top snow, chunks would go sliding down the slope. In his calorie and sleep deprived mind he started thinking of a mini avalanche. I knew the snow wasn't that deep, yet somehow, he didn't know the snow wasn't that deep. Struggling mentally and physically, going 20, 30, 50 feet, stop, catch his breath, exert himself again. David looked up and the peak above him was shrouded in cloud. He looked ahead, down the valley towards Bridgeport, his ultimate destination, and the sky was not blue. It was dark gray and moving towards him. He saw more snow in those clouds. He saw vanilla Dippin' Dots in those clouds. And as hard as it is to admit it, being an ice cream lover, David did not want any more Dippin' Dots.
David was tired, dehydrated, nauseous, short of breath, calorie deficient, and not thinking clearly. He had about a quarter of a liter of water and plenty of food on his back but lacked the will to eat it. Having a soaking wet tent on his back but lacking the will to stop and set it up. All he could think of was "I need to get off this mountain. I do not want to spend another night on this mountain." Never mind it wasn't even 9:00am, his head wasn't clear enough to register that he had TEN hours of daylight left. David had plenty of time to move bit by bit down the hill but didn't know that. He had mental tunnel vision. He was so focused on the perception that he needed to get off this mountain that he couldn't see that he had plenty of time, and all the resources he needed, to get off the mountain. Instead, David was stuck in a loop. "Get off the mountain. But I don't have the energy to get off the mountain. But I need to get off the mountain." Nothing else mattered. His mind had shut down. The feedback loop had started. The seed of doubt was growing. And as he crawled himself out of another waist deep snow pocket, made his way to a stretch of rocky trail before another snow drift, he got lightheaded and this time the tunnel vision wasn't mental. David sat on a rock, chest heaving, and looked up at the sky and everything narrowed in. He felt like he was going to faint, or maybe did but only briefly, because then it opened back up and he hadn't fallen off the rock. And the very next thought was, "Oh shit."
Up until that point, David believed he would make it. It was difficult, it was a challenge, he didn't want to deal with it, but he knew he had the knowledge and tools to make it. He had warm dry clothes and protective outer clothing. He had food. He had a stove. He had a water filter between his shirts and belly, keeping it from freezing with body heat. There was no reason David couldn't make it off that rock, until he almost passed out on that rock.
That bit of tunnel vision changed everything. It was the first time the situation went from "not ideal" to "potentially dangerous." David thought if he fell and hit his head on a rock, or broke a leg, that would be a game changer. And that thought grew and grew. In a matter of moments, it was all he could think about, "Do I call for help now, while things are still actually not life threatening, or do I push on and risk making the situation worse?" On the one hand, David could probably make it out with nothing more than chapped lips and a good story. On the other hand, he could push on, and put himself in a life-threatening position with a broken bone (or worse). And being solo, he didn't have anyone to bounce ideas off of, or catch him if he slipped, or go for help if things turned truly bad.
A few years ago David took a two-day Wilderness First Aid class. Of the many things taught, one of them is that being in the woods, 911 doesn't work. There's no six-minute emergency response time. There aren't any Paramedics a few minutes away, a hospital a few minutes more. Backcountry rescues are measured in hours, not minutes, and in bad cases days. David had a dilemma: request help now, under good conditions, but not dire need, or wait until there was dire need, but conditions were potentially no longer good.
David looked at the gathering clouds and was concerned they could bring more snow. More snow would make potential rescue operations more difficult, and likely more dangerous. The longer he waited, the less daylight there would be. Nighttime rescues are inherently more risky than daytime rescues. They would have his GPS coordinates, but even with that information, finding him would be easier in daylight. David was actually on the trail, in a place where he could wait on rocks instead of directly on snow, an arguably better location to hunker down. While these thoughts floated through his exhausted, clouded mind, hiding in the background was that mantra: "I need to get off the mountain, but I don't have the energy to get myself off the mountain."
Months ago, David had started looking into a GPS locator beacon for the backcountry. Last summer (2020) there were massive wildfires all over California, Oregon, and Colorado. In California, a group of backpackers were caught in the blaze; thankfully they were near a resort, where they were able to take shelter until helicoptered out. David thought if he were caught in a wildfire while backpacking, what would he do if he couldn't get to the trailhead, and his car, and escape? A satellite locator device seemed like a pretty good insurance policy in that kind of situation. After researching half a dozen options, he settled on a device with two-way text communication capabilities. A satellite tracker would allow for critical information about his situation to be communicated to rescue teams, so they know what they were getting into. It allowed up to date localized weather forecasts to be pulled while backpacking. It also allowed for two-way text messages to be sent to anyone, so if something came up worth noting, like wanting to spend an extra day somewhere, people wouldn't worry. David picked the Bivy Stick.
David took the Bivy Stick on this trip to test its features. He had owned it for less than a month and did not expect to be in a situation where he would even remotely consider using the SOS feature of his satellite emergency device. But that's why Bivy Stick is worth having – in expected situations, it's a luxury, but in unexpected situations, it could be a life saver. Out of everything that had happened so far, the tunnel vision is what pushed David over the edge, figuratively. Rather than wait for it to push him over the edge literally, David decided to push the button and ask for help.
With the satellite texting device, David was able to do two important things. First, he was able to alert his wife to the predicament. David knew from the Bivy Stick manual and sign-up process that if he activated the SOS feature, the monitoring service, Global Rescue, would likely call his emergency contact, and so he didn't want that to be a surprise to her. Second, he could let Global Rescue know exactly what his situation was, so they could arrange the best assistance. Some personal locator beacons cost less, have cheaper plans, and don't require a cell phone for communication. David had looked into them, and yes, they can alert someone that there is an emergency at some set of coordinates, but without additional information, authorities have to mobilize for a worst-case scenario, potentially activating resources that aren't truly needed for your rescue, which could perhaps make them unavailable for someone else who truly does need them. Also, it can cause rescuers to take unnecessary risks (think: flying in bad weather) just to determine the nature of the SOS. With a two-way communication device, authorities would know how best to manage a rescue while using the least number of resources necessary, keeping additional rescuers and equipment ready for a different emergency, or lowering the risk involved in getting you the help you need.
In David's case, help came swiftly, more swiftly than he expected. Due to the two-way communication capability of the Bivy Stick , they knew he was conscious, breathing, uninjured and not in imminent life-threatening danger. They knew that getting to him by foot was a completely acceptable, low risk option. Within about four hours, four rescuers from the Mono County Search and Rescue Team found David on the trail. That means they got the call, rallied, drove out the dirt road to the trailhead, and hiked about four miles uphill very quickly. David admired their stamina and appreciated their help. They asked him questions, gave him Gatorade and a dried banana. They took the load off his back and redistributing his gear to carry it down the mountain. They let David know much of the snow between their current location and the parking lot had melted, and the way was now mostly clear of snow drifts. Most of all, they gave David confidence. They were friendly, non-judgmental, and professional. And they were all volunteers. They helped David walk off the mountain on his own two feet, and while still exhausted and short of breath and needing frequent stops on the way down, they reminded David how much of being in the wilderness and hitting "the wall" is in your head. It's a mental game with physical consequences. It's important to fuel the body with calories and hydration, but it's equally important to fuel the mind with positive energy.
David admits that he doesn't like camping in the snow. It's not for him and he's known that for a long time. So, when faced with a snowstorm on the top of that mountain, David was already losing the mental game and saw the rest of the weekend as the thing of dread. Remember, David had everything he needed; warm clothes, waterproof layers, a 20-degree bag, shelter, food, a stove with fuel, and access to water. He also had the know-how to protect his gear, keep his filter from freezing, keep his fuel can warm for better performance, and keep his water bottle from freezing at night. All David was missing was a positive attitude telling himself to view the experience in a positive light. But a negative attitude towards snow camping, and preconceived dread shaped everything that came after. It was a key factor behind the nausea and loss of appetite, which led to fatigue, which led to not having the energy to get down on his own.
And that's the key lesson David takes away from this experience: a negative attitude breeds negative outcomes. That and take a piece of technology with you into the backcountry – just in case you find yourself at 10,000 feet with Dippin' Dots falling from the sky, or worse.
How a Satellite Communication Device Proves Vital to this User's Emotional & Mental Health
ACR Bivy Stick Proves Vital to this Survivor's Cautionary Tale
Bivy Acquired By ACR Electronics
App Updates (1)
Survivor Stories (2)
© 2022 Bivy Hub & Knowledgebase.


From wings to rings, veggies to chips, you'll want to try dipping everything in our scrumptious line-up of Taste Weavers dips.
As delicious as the cookies themselves, this Snickerdoodle Cookie dip is heavenly! Just the right blend of cinnamon, sugar and vanilla – dive right into this dip and enjoy!
Perfect to dip fresh fruit in – we love it with Granny Smith apples.
Warm the dip and drizzle over coffee cake, cinnamon rolls or ice cream!
Dollop on top of spice cake or pound cake for a wonderful dessert.
Serving Size: 2 Tbsp (30 g) Servings Per Container About 9. Calories 100 Calories from Fat 45, Total Fat 5 g (8% DV), Sat. Fat 3 g (16% DV),Trans Fat 0 g, Cholest. 15 mg (5% DV), Sodium 50 mg (2% DV), Total Carb. 14 g (5% DV), Dietary Fiber 0 g (0% DV), Sugars 14 g, Protein 1 g, Vitamin A (4% DV), Vitamin C (0% DV), Calcium (0% DV), Iron (0% DV). Percent Daily Values (DV) are based on a 2,000 calorie diet.
Cream Cheese (Pasteurized Milk and Cream, Palm Oil, Cheese Culture, Salt, Stabilizers [Carob Bean and/or Xanthan Gum and/or Guar Gums], Mono and Diglycerides, Apocarotenal [color]), Granulated Sugar, Water, Cinnamon, Vanilla, Sodium Benzoate and Potassium Sorbate (as preservatives) and Sodium Acid Phosphate. Contains: Milk.


text:
How to Convert From BTU to Fahrenheit
••• Smileus/iStock/GettyImages
How to Calculate Heat Flux
By Christopher Raines
BTU, or the British Thermal Unit, is the amount of heat required to raise one pound of water one degree Fahrenheit. The British Thermal Unit measures the amount of heat, or thermal energy. Temperature is the level rather than amount of heat. Therefore, there is no formula to convert a British Thermal Unit to Fahrenheit. Instead, the British Thermal Unit indicates the ability of a stove, heating system, grill, water heater and other appliances to increase temperature.
Find the British Thermal Unit rating for the appliance or equipment. Consult the specifications page of the owner's or operator's manual or the appliance's manual.
Determine the amount of space or water to be heated. Calculate the space in cubic feet by measuring the length, width and distance from floor to ceiling of the building or space to be heated and multiplying the three figures. For water, multiply the number of gallons to be heated or used by 8.3453, since one gallon of water equals 8.3453 pounds.
Multiply the cubic feet of the area to be heated by 0.133. Divide the British Terminal Unit of the heating unit by the factor of cubic feet and 0.133 to obtain the change in temperature. For heating water, divide the British Thermal Unit by the pounds of water to be heated.
Appliance operator's or owner's manual
Appliance label
To determine the temperature of water after it is heated, add the water temperature change to the temperature of the water before it is heated.
Consult your local or state health department for the required temperatures of water and food for restaurants, child care centers and other public establishments and facilities. The standards for temperatures will determine the British Thermal Unit rating required for the heating element.
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Christopher Raines enjoys sharing his knowledge of business, financial matters and the law. He earned his business administration and law degrees from the University of North Carolina at Chapel Hill. As a lawyer since August 1996, Raines has handled cases involving business, consumer and other areas of the law.