A simplified tempo-spatial model to predict airborne pathogen release risk in enclosed spaces: An Eulerian-Lagrangian CFD approach.

COVID19 pathogens are primarily transmitted via airborne respiratory droplets expelled from infected bio-sources. However, there is a lack of simplified accurate source models that can represent the airborne release to be utilized in the safe-social distancing measures and ventilation design of buildings. Although computational fluid dynamics (CFD) can provide accurate models of airborne disease transmissions, they are computationally expensive. Thus, this study proposes an innovative framework that benefits from a series of relatively accurate CFD simulations to first generate a dataset of respiratory events and then to develop a simplified source model. The dataset has been generated based on key clinical parameters (i.e., the velocity of droplet release) and environmental factors (i.e., room temperature and relative humidity) in the droplet release modes. An Eulerian CFD model is first validated against experimental data and then interlinked with a Lagrangian CFD model to simulate trajectory and evaporation of numerous droplets in various sizes (0.1 µm-700 µm). A risk assessment model previously developed by the authors is then applied to the simulation cases to identify the horizontal and vertical spread lengths (risk cloud) of viruses in each case within an exposure time. Eventually, an artificial neural network-based model is fitted to the spread lengths to develop the simplified predictive source model. The results identify three main regimes of risk clouds, which can be fairly predicted by the ANN model.

Nutritive value of treated Quercus infectoria and Quercus libani leaves with the tannin-degrading bacterium Klebsiella pneumoniae for ruminant feeding in vitro.

AIMS: This study was conducted to evaluate the chemical composition and in vitro gas production (GP) and fermentation parameters of Quercus infectoria and Quercus libani leaves following treatment with the Klebsiella pneumoniae, a tannin-degrading bacterium. METHODS AND RESULTS: This isolate was isolated on medium containing tannic acid as the sole source of carbon and energy, and identified based on 16S rRNA sequencing analysis. In both oak leaf species (i.e. Q. infectoria and Q. libani), inoculation with Klebsiella pneumoniae significantly increased (P < 0·05) dry matter (DM) loss. For Q. libani, crude protein content was increased (P = 0·02) by bacterial treatment vs. control. In both oak leaves, total phenolic content and total tannins were decreased (P < 0·05) as a consequence of bacterial treatment. However, bacterial processing didn't changed (P > 0·05) organic matter (OM), neutral detergent fibre, acid detergent fibre or acid detergent lignin content of treated leaves. In both oak leaves the measuring parameters including GP volume, in vitro digestibility of DM and OM, estimated metabolizable energy, total volatile fatty acids, acetate, ammonia nitrogen concentration, total protozoal population and the subfamily Isotricha in treatments were higher (P < 0·05) than control. CONCLUSIONS: It can be concluded that biological treatment of Q. infectoria and Q. libani leaves with K. pneumoniae represents a useful approach to decrease their phenolic compound content and improve their nutritive value as ruminant feed. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrated that biologically processing of tannin-containing by-products with K. pneumoniae could increase their nutritive value as ruminant feeds and increase animal productivity.

Isolation and identification of cellulolytic bacteria from gastrointestinal tract of Arabian horse and investigation of their effect on the nutritional value of wheat straw.

AIMS: This study was conducted to isolate and identify the cellulolytic bacteria from gastrointestinal tract of Arabian horse and investigate their effect on the nutritional value of wheat straw (WS). METHODS AND RESULTS: Fresh faeces were collected from four Arabian horses. The cellulose-hydrolytic bacteria were isolated by using a medium amended with carboxymethyl cellulose (CMC). The activity of CMC was determined by measuring the release of reducing sugars from CMC. Among the isolates, four isolates (L11, L12, L2 and Z2) showed maximum hydrolysis capacity. 16S rRNA sequence analysis showed that these isolates possessed 99, 99, 99 and 98% similarity with Paenibacillus polymyxa, Paenibacillus polymyxa, Enterobacter cloacae and Escherichia coli respectively. Wheat straw was incubated with the isolated bacteria in liquid medium. Disappeared dry matter (DM) and crude protein (CP), neutral detergent fibre (NDF), indigestible NDF (iNDF) and acid detergent fibre (ADF) contents of WS were affected by microbial treatments (P < 0·05). The highest disappearance of DM and CP contents and the lowest NDF, iNDF and ADF contents of WS were observed by treating with P. polymyxa L11. The values of NDF, iNDF and ADF were the highest and CP was the lowest in control. Gas production parameters and digestibility of processed samples were determined. Bacterial treatments enhanced (P < 0·05) DM, OM, CP, NDF and ADF digestibility of WS. The highest and lowest (P < 0·05) DM, OM, CP, NDF and ADF digestibilities were observed for WS treated with isolates L12 and control respectively. Potential of gas production (b), truly degraded organic matter (TDOM) and microbial biomass of bacteria treatments were significantly higher (P < 0·05) compared to control. CONCLUSIONS: Generally, the results of this study showed that the isolated bacteria from horse faeces are capable of changing the chemical composition, increasing digestibility as well as enhancing nutrition value of WS. SIGNIFICANCE AND IMPACT OF THE STUDY: Wheat straw is a major agro-residue fed to ruminants. However, high fibre contents reduce digestibility and limit animal productivity. It seems that enhancement of ruminal degradation of these by-products is necessary. The results of this study revealed that cellulolytic bacteria isolated from gastrointestinal tract of horse can be used for biological treatment of WS.

Purification and optimization of production conditions of a marine-derived antibiotic and ultra-structural study on the effect of this antibiotic against MRSA.

OBJECTIVES: In this study we have attempted to partially purify, characterize and optimize the fermentation condition for the antimicrobial compound production with anti-MRSA (Methicillin Resistant Staphylococcus aureus) activity produced by Pseudoalteromonas piscicida PG-02 bacterium, isolated from the Persian Gulf, and finally understand the morphological changes in MRSA due to this antibiotic. MATERIALS AND METHODS: Optimization process of antibacterial compound production was studied based on the sources of carbon and nitrogen, optimum temperature, optimum pH and optimum incubation time. The purification of intended antibiotic was done using TLC and also thermostability and enzymatic stability treatment was studied. Ultrastructural study on the effect of intended antibacterial compound on MRSA was done using a Transmission Electron Microscopy (TEM). RESULTS: The optimized bioprocess conditions for the maximum production were at temperature 28 degrees C, pH 7, NaCI 0.5% (w/v), 96 hrs (incubation time), glucose and tryptone as carbon source and nitrogen source, respectively. The antibacterial component showed thermal sensitivity but it was sensitive to proteinase K, so this compound may have proteinaceous nature. The results of sonication revealed that this compound is accumulated in both intra- and extra-cellular locations. TEM pictures showed disorganization of cytoplasmic membrane upon the extract treatment comparing to control so, it can be said that this antibacterial compound can be considered as a bactericidal agent against MRSA. CONCLUSION: On the basis of obtained results, this bacterium can be regarded as a valuable strain for discovery of new weapon as bactericidal agent in fighting against multi-drug resistant bacteria especially MRSA.

Integrative vectors for heterologous gene expression in Streptomyces spp.

Integrative expression vectors for heterologous expression of the genes in Streptomyces were developed. The vectors are comprised of a strong constitutive promoter, PE, a synthetic ribosome-binding site, ATG start codon, multiple cloning site, transcription terminator and hygromycin-resistance-encoding gene. The vectors also contain a ColE1 replicon for propagation in Escherichia coli and a wide-host-range Streptomyces integration element, the mini-circle, to direct the insertion of the vectors into the Streptomyces genome at the mini-circle attachment site. HyR transformants are stable in the absence of drug selection. Conjugative derivatives were also constructed by incorporating oriT, the origin of transfer of the IncP plasmid RK2, into these vectors, and conjugal transfer was demonstrated from an appropriate E. coli donor to Steptomyces lividans (Sl). Derivatives of these vectors potentially useful for gene disruption, as well as complementation, are also described. Replicative forms of the constructed mini-circle-based vectors in Sl, that co-exist with the integrated copy of the vector, were also present without any apparent instability problems. The utility of the vectors was demonstrated by expression of the gene encoding 31-O-methyltransferase, which is involved in methylation at position 31 of the immunosuppressive drug FK506, in Sl.

Identification of Streptomyces olivaceus Tü 2353 genes involved in the production of the polyketide elloramycin.

The genes for the production of elloramycin (ELM) from Streptomyces olivaceus (So) Tü2353 were cloned using a polyketide synthase gene probe from the tetracenomycin pathway. A cosmid clone (16F4) isolated from a gene library of So Tü2353 conferred tetracenomycin C and ELM resistance to S. lividans TK64 and complemented a mutation in So Tü2353R. Introduction of cosmid 16F4 into S. lividans TK64 resulted in the production of 8-demethyl-tetracenomycin C, an intermediate of ELM biosynthesis.

Chemical and biological characterization of two FK506 analogs produced by targeted gene disruption in Streptomyces sp. MA6548.

Two genetically engineered mutant strains of Streptomyces sp. MA6548 produced two FK506 analogs, 9-deoxo-31-O-demethylFK506 and 31-O-demethylFK506. The structures were determined by a combination of NMR and mass spectrometry. These compounds exhibited immunosuppressive and antifungal activities, albeit reduced, compared to FK506. Both compounds contain a free hydroxyl group at C-31 for the synthesis of novel FK506 derivatives.

Antibacterial activity of the fruits of Iranian Torilis leptophylla against some clinical pathogens.

The aim of this study was to examine the antimicrobial activity of the methanolic extract of Torilis leptophylla was tested on eleven bacteria (Bacillus anthracis, Bacillus subtilis, Bacillus pumilus, Staphylococcus aureus, Bacillus licheniformis, Brucella melitensis, Escherichia coli, Salmonella typhi, Proteus mirabilis, Bordetella bronshiseptica and Pseudomonas aeruginosa). Tested extract was effective against all bacteria but not B. subtilis. Consequently, the ethanolic extract had antibacterial activity on some pathogens thus confirming their use in folk medicine.

The biosynthetic gene cluster for the macrolactone ring of the immunosuppressant FK506.

Biosynthesis of the macrolactone ring of FK506 involves 10 elongation cycles that mechanistically resemble the steps in fatty acid synthesis. Sequencing of a 40-kb DNA segment of the FK506 gene cluster from Streptomyces sp. MA6548 has revealed two additional polyketide synthases (PKS) genes fkbB and fkbC which lie upstream of fkbA, a PKS gene recently shown to be responsible for the last four condensation steps of the FK506 biosynthesis [Motamedi, H., Cai, S. J., Shafiee, A. & Elliston, K. O. (1997) Eur. J. Biochem. 244, 74-80]. fkbB and fkbC are contiguous and encode respectively, the first (790129 Da) and the second (374438 Da) components of the FK506 polyketide synthase, a complex of three multidomain polypeptides. The predicted domain structures of FkbB and FkbC are analogous to that of FkbA and comprise 30 fatty-acid-synthase(FAS)-like domains arranged in 6 modules. Each module performs a specific extension cycle in the assembly of the carbon skeleton of the FK506 macrolactone ring. The component activities for the initiation of the polyketide chain consisting of a dihydrocyclohexenylcarbonyl coenzyme A (CoA) synthetase and a dihydrocyclohexenylcarbonyl CoA reductase required for the formation of the dihydrocyclohexylcarbonyl CoA starter unit and an acyl-carrier-protein to which the starter unit is anchored and translocated to the appropriate site on the PKS multienzyme are located at the N-terminal region of the FkbB polypeptide. A third gene, fkbL, lies at one end of the cluster and encodes lysine cyclodeaminase which catalyzes alpha-deamination and cyclization of the lysine into pipecolate. A fourth gene fkbP located at the other end of the sequence reported here encodes a peptide synthetase required for the activation and incorporation of the pipecolate moiety into the completed acyl chain. Finally the cluster carries a gene, fkbO, whose product is presumed to carry out a post-polyketide oxidation step of the FK506 marocycle.

Cloning and heterologous expression of a gene cluster for the biosynthesis of tetracenomycin C, the anthracycline antitumor antibiotic of Streptomyces glaucescens.

Through complementation of mutations specifically blocking the biosynthesis of tetracenomycin C by Streptomyces glaucescens and selecting for resistance to tetracenomycin C in Streptomyces lividans, all of the genes for the production of tetracenomycin C were inserted in pIJ702, a high copy-number Streptomyces gene cloning vector. The tcm biosynthetic and resistance genes occur as a single cluster in the S. glaucescens genome and are expressed in heterologous streptomycete hosts like S. lividans, resulting in the overproduction of pigmented intermediates of the tetracenomycin C biosynthetic pathway.

Variations in serum protein fractions following a continuous long term intake of eugynon and lyndiol by Iranian women.

PIP: Serum total protein and protein fractions were estimated in 150 Iranian subjects, aged 18-41, who had used lyndiol (.15 mg mestranol plus 2.5 mg lynestrenol) and eugynon (.05 mg ethinyl estradiol plus .5 mg norgestrel) for 1-72 months. The control group consisted of 32 Iranian women who were age- and socioeconomic status-matched but had never used oral contraceptives before. A decrease was observed in albumin fraction and increases in alpha 1-alpha 2, and beta globulins were statistically significant (P .01 or P .02). Serum albumin decreased in 100% of patients, whereas the alpha and beta globulins increased in over 95% of the subjects. No statistically significant variation in gamma globulin was observed after intake of either eugynon or lyndiol. No dose response correlation was evident.^ieng

Enzymology of FK-506 biosynthesis. Purification and characterization of 31-O-desmethylFK-506 O:methyltransferase from Streptomyces sp. MA6858.

FK-506 is a macrolide antibiotic with immunosuppressant activity. Structurally, this compound contains three methylated hydroxyl groups at C13, C15 and C31. Previous biosynthetic studies using stable isotope-feeding experiments have established methionine as the source of the methyl for these methylated hydroxyl groups. Based on this information and also the availability of the 31-O-desmethylFK-506, a metabolic precursor for the biosynthesis of FK-506, a S-adenosyl-L-methionine-dependent enzyme assay was developed and the enzyme 31-O-desmethylFK-506 O:methyl-transferase was isolated from an extract of Streptomyces sp. MA 6858 and purified to near homogeneity. 31-O-DesmethylFK-506 O:methyltransferase is a monomeric protein with an apparent molecular mass of 30,000 Da and a pI of 4.4. The first 38 N-terminal amino acids have been sequenced and are H2N-SDVVETLRLPNGATVAHVNAGEAQFLYREIFTDRXYLRH. Functionally, This enzyme has a requirement for Mg2+ with an optimum temperature of 34 degrees C and a pH of 7.4 for full activity. Moreover, it catalyses the methylation of 31-O-desmethylimmunomycin as efficiently as its own natural substrate, 31-O-desmethylFK-506. Additionally, FKMT catalyzes the C31 transmethylation reaction of 13,31-O-bis-desmethyl-, 15,31-O-bisdesmethyl-, 13,15,31-O-trisdesmethyl- and 31-O-19,22-cyclic-hemiketalimmunomycins, which are all structural analogues of FK-506. The reaction is, however, completely blocked if the vicinal hydroxyl which is present at the C-32 position of the 31-O-desmethylFK-506 structure is replaced with azide, phosphate or other substituents. Finally, evidence is presented indicating the close similarity of FKMT and DIMT, a 31-O-desmethyl-immunomycin: O methyltransferase, previously isolated from a cell-free extract of Streptomyces hygroscopicus var ascomyceticus, an immunomycin (ascomycin/FK-520) producer.

Tandem promoters preceding the gene for the M1 RNA component of Escherichia coli ribonuclease P.

The nucleotide sequence of a cloned gene for the RNA component of Escherichia coli ribonuclease P, M1 RNA, is presented. The sequence determined extends 320 nucleotides upstream of the 377-base-pair (bp) structural gene and includes three sequences homologous to the consensus E. coli promoter sequence. Two nucleotides found in the M1 RNA structural gene sequence were not found in a previously determined gene sequence of another M1 RNA clone [Reed, R. E., Baer, M. F., Guerrier-Takeda, C., Donis-Keller, H. & Altman, S. (1982) Cell 30, 627-636]. In vitro transcription of supercoiled plasmid DNA containing the M1 RNA gene resulted in a major transcript arising from the strong promoter nearest to the mature M1 RNA. RNAs encoded by the M1 RNA clone in vivo were examined by S1 nuclease mapping. The results indicated that in vivo transcripts originate from all three promoters preceding the M1 RNA gene. These transcripts are apparently processed in a multistep pathway to generate the 5' end of mature M1 RNA.

Nucleotide sequences and heterologous expression of tcmG and tcmP, biosynthetic genes for tetracenomycin C in Streptomyces glaucescens.

The nucleotide sequence of the tcmIII, tcmIc, and tcmVII region of the tetracenomycin (TCM) C gene cluster of Streptomyces glaucescens ETH 22794 (GLA.0) revealed the presence of two genes, tcmP and tcmG. The deduced product of tcmG resembles flavoprotein hydroxylases found in several other bacteria, whereas the predicted amino acid sequence of tcmP is not significantly similar to those of any known proteins in the available data bases. Southern blot hybridization revealed an approximately 180-bp deletion in a tcmIII (tcmG) mutant and a 1,800-bp insertion in a tcmVII (tcmP) mutant. Heterologous expression of tcmG and tcmP in Streptomyces lividans and tcmP in Escherichia coli established that tcmP encodes an O-methyltransferase, catalyzing the methylation of the C-9 carboxy group of TCM E to yield TCM A2, and that tcmG is responsible for the hydroxylation of TCM A2 at positions C-4, C-4a, and C-12a to give TCM C. These are the final two steps of TCM C biosynthesis.

Isolation of tetracenomycin C-nonproducing Streptomyces glaucescens mutants.

We analyzed the properties of tetracenomycin C (TcmC)-nonproducing mutants of Streptomyces glaucescens to establish the nature of pathway intermediates and to provide some information about the genetics of antitumor anthracycline antibiotic production. Using cosynthesis properties and metabolite accumulation data, we classified a collection of 34 TcmC-nonproducing strains into seven different groups. From this information, we deduced the positions of the tcm mutations in relation to a hypothetical TcmC biosynthetic pathway and suggest which pathway enzymes are affected by the different mutations.

A high-throughput assay to identify compounds that can induce dimerization of the erythropoietin receptor.

Erythropoietin induces dimerization of the erythropoietin receptor on the surface of erythroid progenitor cells, promoting the differentiation of these cells into mature red blood cells. To facilitate screening of large chemical collections for identification of compounds that can dimerize erythropoietin receptor, we have developed a novel, high-throughput in vitro assay to detect compounds that can cause dimerization of the erythropoietin receptor in solution. To develop this assay, amino acid sequences corresponding to the extracellular domain of erythropoietin receptor were expressed in Escherichia coli as erythropoietin-binding protein (rEBP). A modified version of this protein ((33)P-rEBP) containing a protein kinase A substrate site incorporated into the rEBP was also expressed in E. coli and labeled in vitro using protein kinase A and ¿gamma-(33)PATP. An erythropoietin mimetic peptide (EMP-1), that induces dimerization of rEBP in solution was used to demonstrate dimerization of (33)P-rEBP and rEBP in a 96-well microtiter plate format. EMP-1 induced dimerization of rEBP in this assay with an EC(50) of approximately 245 nM and had a maximal effect at 0.5-2 microM and required the presence of rEBP immobilized on the plate capable of binding EMP-1. EMP-1-induced dimerization of (33)P-rEBP and rEBP was reversed by excess unlabeled rEBP and was not masked by complex mixtures such as whole cell fungal extracts. These data demonstrate the ability of (33)P-rEBP to dimerize with rEBP in vitro in a format that is fully compatible with high-throughput screening.

Analysis of the nucleotide sequence of the Streptomyces glaucescens tcmI genes provides key information about the enzymology of polyketide antibiotic biosynthesis.

Key information about the biosynthesis of polyketide metabolites has been uncovered by sequence analysis of the tetracenomycin C polyketide synthase genes (tcml) from Streptomyces glaucescens GLA.0. The sequence data revealed the presence of three complete open reading frames (ORFs). ORF1 and ORF2 appear to be translationally coupled and would encode proteins containing 426 and 405 amino acids, respectively. The two deduced proteins are homologous to known beta-ketoacyl synthases. ORF3 begins 70 nucleotides after the stop codon of ORF2 and would code for an 83 amino acid protein with a strong resemblance to known bacterial, animal and plant acyl-carrier proteins (ACP). The presence of an ACP gene within the tcm gene cluster suggests that different ACPs are used in fatty acid and polyketide biosynthesis in Streptomyces. We conclude from these data and earlier information that polyketide biosynthesis in S. glaucescens, and most likely in other bacteria, involves a multienzyme complex consisting of at least five types of enzymes: acylCoA transferases that load the acyl and 2-carboxyacyl precursors onto the ACP; a beta-ketoacyl synthase that, along with the acylated ACP, forms the poly-beta-ketoacyl intermediates; a poly-beta-ketone cyclase that forms carbocyclic structures from the latter intermediates; a beta-ketoacyl oxidoreductase that forms beta-hydroxyacyl intermediates or reduces ketone groups in fully formed polyketides; and a thioesterase that releases the assembled polyketide from the enzyme.

Structural organization of a multifunctional polyketide synthase involved in the biosynthesis of the macrolide immunosuppressant FK506.

The immunosuppressant FK506 is a 23-membered macrocyclic polyketide produced by several Streptomyces species. Sequencing of a 19.5-kb contiguous segment of DNA from the FK506 gene cluster of Streptomyces sp. MA6548 revealed the presence of a single 19.3-kb open reading frame designated fkbA. fkbA encodes a component of the FK506 polyketide synthase, a complex enzyme system which catalyzes synthesis of the polyketide portion of FK506. The predicted product of gene fkbA is a 630,660-Da protein (6420 amino acids) that contains 19 independent domains with a high degree of amino acid sequence similarity to the catalytic activities of known fatty acid synthases. The identified domains are arranged into four repeated modules with a linear organization precisely as that of animal fatty acid synthase and type I polyketide synthase. Each module participates in one round of chain extension and subsequent processing and thus FkbA polypeptide catalyzes four of the ten condensation steps required for synthesis of the FK506 macrolactone ring. Disruption of fkbA results in the generation of an FK506 non-producing mutant demonstrating direct involvement of fkbA in the biosynthesis of FK506.