Form Of Protease Research Articles

Genetic trans-complementation of L-protease fails to rescue the infectious foot-and-mouth disease virus from the Lbpro defective genome

Outbreaks of the foot-and-mouth disease (FMD) have major economic impact on the global livestock industry by affecting the animal health and product safety. L-protease, a non-structural protein of FMDV, is a papain-like cysteine proteinase involved in viral protein processing as well as cleavage of host proteins for promoting the virus growth. FMDV synthesizes two forms of leader proteinase, Lpro (Labpro and Lbpro), where the deletion of Labpro is lethal and Lbpro deletion is reported to be attenuated. Defective replicons have been used by trans-complementing the deleted gene to produce one time replicating virus; thus, the bio-safety procedure can be compromised in the production units. Attempts are made to rescue of ΔLbproFMDV Asia1 virus by co-expressing the Lbpro protein carried in pcDNA plasmid. Mutant FMDV cDNA, pAsia-ΔLbpro, was constructed by PCR mediated mutagenesis using inverse primers. Transfection of BHK-21 cells with in-vitro transcribed RNA from the constructs failed to produce an infective mutant FMDV. Genetic trans-complementation of the Lbpro, which was done by co-transfecting the pcDNALbpro plasmid DNA along with the pAsia-ΔLbpro RNA in BHK-21 cells also failed to produce viable virus. Expression experiments of reporter genes and indirect immune-fluorescence confirmed the production of the viral proteins in wild type FMDV pAsiaWT; however, it was absent in the pAsia-ΔLbpro indicating that the leaderless virus was unable to produce infectious progeny and infect the cells. Failure to produce virus either by Lbpro deleted mutant clone or by genetic complementation suggests little chance of reversion of the disabled virus with large deletions of FMDV genome.

Interplay between protease and reverse transcriptase dimerization in a model HIV-1 polyprotein.

The Gag-Pol polyprotein in human immunodeficiency virus type I (HIV-1) encodes enzymes that are essential for virus replication: protease (PR), reverse transcriptase (RT), and integrase (IN). The mature forms of PR, RT and IN are homodimer, heterodimer and tetramer, respectively. The precise mechanism underlying the formation of dimer or tetramer is not yet understood. Here, to gain insight into the dimerization of PR and RT in the precursor, we prepared a model precursor, PR-RT, incorporating an inactivating mutation at the PR active site, D25A, and including two residues in the p6* region, fused to a SUMO-tag, at the N-terminus of the PR region. We also prepared two mutants of PR-RT containing a dimer dissociation mutation either in the PR region, PR(T26A)-RT, or in the RT region, PR-RT(W401A). Size exclusion chromatography showed both monomer and dimer fractions in PR-RT and PR(T26A)-RT, but only monomer in PR-RT(W401A). SEC experiments of PR-RT in the presence of protease inhibitor, darunavir, significantly enhanced the dimerization. Additionally, SEC results suggest an estimated PR-RT dimer dissociation constant that is higher than that of the mature RT heterodimer, p66/p51, but slightly lower than the premature RT homodimer, p66/p66. Reverse transcriptase assays and RT maturation assays were performed as tools to assess the effects of the PR dimer-interface on these functions. Our results consistently indicate that the RT dimer-interface plays a crucial role in the dimerization in PR-RT, whereas the PR dimer-interface has a lesser role.

Effect of protatranes on the biosynthesis of extracellular enzymes of <i>Candida ethanolica</i> BKM Y-2300 T

The study aims to explore the possibility of using synthetic biologically active compounds (protatranes) to increase the lipolytic and proteolytic activity of Candida ethanolica. Protatrane 1 (tris(2-hydroxyethyl) ammonium-4chlorophenyl-sulfanyl acetate) and protatrane 2 (tris(2-hydroxyethyl) ammonium-4-chlorophenyl-sulfonyl acetate) were added to the growth medium at trace concentrations of 1×10−6–1×10−8wt% separately and together. It was established that with the introduction to the growth medium, protatranes had a multidirectional effect on the biosynthesis of enzymes by Candida ethanolica yeast. This effect was dependent on the concentrations of these compounds, as well as on their separate or combined introduction. All the studied concentrations were found to be effective for lipase synthesis, as they improved enzyme synthesis by 1.7–8.6 times. The combined use of protatranes increased enzyme synthesis by 3.4–11.7 times. For protease formation, the combined introduction of the studied protatranes at a concentration of 10−6wt% was found to be the most effective, with enzyme synthesis reaching 184.8±7.02 U/mL in the culture broth. When co-introduced, the studied protatranes tris(2-hydroxyethyl) ammonium-4-chlorophenylsulfanyl acetate and tris(2-hydroxyethyl) ammonium-4-chlorophenyl-sulfonyl acetate can be used to increase the production efficiency of extracellular lipases and proteases by Candida ethanolica. The specific effect of protatranes was revealed to be the uneven dynamics of extracellular enzyme accumulation, manifested in the presence of sharp peaks during yeast cultivation. The reasons for this unevenness require further research.

Characterization of pH-induced conformational changes in recombinant DENV NS2B-NS3pro

The increasing frequency of Dengue is a cause of severe epidemics and therefore demands strategies for effective prevention, diagnosis, and treatment. DENV-protease is being investigated as a potential therapeutic target. However, due to the flat and highly charged active site of the DENV-protease, designing orthosteric medicines is very difficult. In this study, we have done a thorough analysis of pH-dependent conformational changes in recombinantly expressed DENV protease using various spectroscopic techniques. Our spectroscopic study of DENV protease (NS2B-NS3pro) at different pH conditions gives important insights into the dynamicity of structural conformation. At physiological pH, the DENV-protease exists in a random-coiled state. Lowering the pH promotes the formation of alpha-helical and beta-sheet structures i.e. gain of secondary structure as shown by Far-UV CD. The light scattering and Thioflavin T (ThT)-binding assay proved the aggregation-prone tendency of DENV-protease at pH 4.0. Further, the confocal microscopy image intensity showed the amorphous aggregate formation of DENV protease at pH 4.0. Thus, the DENV protease acquires different conformations with changes in pH conditions. Together, these results have the potential to facilitate the design of a conformation destabilizer-based therapeutic strategy for dengue fever.

Constitutive and induced forms of membrane-bound proteinase 3 interact with antineutrophil cytoplasmic antibodies and promote immune activation of neutrophils

Proteinase 3 (PR3) is the main target antigen of antineutrophil cytoplasmic antibodies (ANCAs) in PR3-ANCA-associated vasculitis. A small fraction of PR3 is constitutively exposed on the surface of quiescent blood neutrophils in a proteolytically inactive form. When activated, neutrophils expose an induced form of membrane-bound PR3 (PR3mb) on their surface as well, which is enzymatically less active than unbound PR3 in solution due to its altered conformation. In this work, our objective was to understand the respective role of constitutive and induced PR3mb in the immune activation of neutrophils triggered by murine anti-PR3 mAbs and human PR3-ANCA. We quantified immune activation of neutrophils by the measurement of the production of superoxide anions and secreted protease activity in the cell supernatant before and after treatment of the cells by alpha-1 protease inhibitor that clears induced PR3mb from the cell surface. Incubation of TNFα-primed neutrophils with anti-PR3 antibodies resulted in a significant increase in superoxide anion production, membrane activation marker exposition, and secreted protease activity. When primed neutrophils were first treated with alpha-1 protease inhibitor, we observed a partial reduction in antibody-induced neutrophil activation, suggesting that constitutive PR3mb is sufficient to activate neutrophils. The pretreatment of primed neutrophils with purified antigen-binding fragments used as competitor significantly reduced cell activation by whole antibodies. This led us to the conclusion that PR3mb promoted immune activation of neutrophils. We propose that blocking and/or elimination of PR3mb offers a new therapeutic strategy to attenuate neutrophil activation in patients with PR3-ANCA-associated vasculitis.

Expression of degQ gene and its effect on lipopeptide production as well as formation of secretory proteases in Bacillus subtilis strains.

Bacillus subtilis is described as a promising production strain for lipopeptides. In the case of B. subtilis strains JABs24 and DSM10T, surfactin and plipastatin are produced. Lipopeptide formation is controlled, among others, by the DegU response regulator. The activating phospho‐transfer by the DegS sensor kinase is stimulated by the pleiotropic regulator DegQ, resulting in enhanced DegU activation. In B. subtilis 168, a point mutation in the degQ promoter region leads to a reduction in gene expression. Corresponding reporter strains showed a 14‐fold reduced expression. This effect on degQ expression and the associated impact on lipopeptide formation was examined for B. subtilis JABs24, a lipopeptide‐producing derivative of strain 168, and B. subtilis wild‐type strain DSM10T, which has a native degQ expression. Based on the stimulatory effects of the DegU regulator on secretory protease formation, the impact of degQ expression on extracellular protease activity was additionally investigated. To follow the impact of degQ, a deletion mutant was constructed for DSM10T, while a natively expressed degQ version was integrated into strain JABs24. This allowed strain‐specific quantification of the stimulatory effect of degQ expression on plipastatin and the negative effect on surfactin production in strains JABs24 and DSM10T. While an unaffected degQ expression reduced surfactin production in JABs24 by about 25%, a sixfold increase in plipastatin was observed. In contrast, degQ deletion in DSM10T increased surfactin titer by threefold but decreased plipastatin production by fivefold. In addition, although significant differences in extracellular protease activity were detected, no decrease in plipastatin and surfactin produced during cultivation was observed.

Loss of the ClpXP Protease Leads to Decreased Resistance to Cell-Envelope Targeting Antimicrobials in Bacillus anthracis Sterne

The ClpX ATPase is critical for resistance to cell envelope targeting antibiotics in Bacillus anthracis, however, it is unclear whether this is due to its function as an independent chaperone or as part of the ClpXP protease. In this study, we demonstrate that antibiotic resistance is due to formation of the ClpXP protease through construction of a ClpX complementation plasmid that is unable to interact with ClpP. Additionally, we genetically disrupted both clpP genes, clpP1 and clpP2, found in B. anthracis Sterne and find that the loss of either increases susceptibility to cell envelope targeting antimicrobials, although neither has as strong of a phenotype as loss of clpX and neither clpP gene is essential for virulence in a G. mellonella model of infection. Lastly, we looked at changes to cell envelope morphology that could contribute to increased antibiotic sensitivity. We find no difference in cell charge or cell lysis, although we do see increased hydrophobicity in the ΔclpX strain, decreased cellular density and slightly thinner cells walls. We also see significant cell division defects in ΔclpX, although only when cells are grown in the mammalian cell culture medium, RPMI. We conclude that the intrinsic resistance of B. anthracis to cell wall active antimicrobials is dependent on formation of the ClpXP protease and that this could be due, at least in part, to the role of ClpX in regulating cell envelope morphology.

Molecular identification, virulence factors and antifungal susceptibility patterns of Candida parapsilosis complex species isolated from clinical samples.

The aim of this study was to identify C. parapsilosis complex strains isolated from various clinical samples by sequence analysis and to investigate whether there are any differences between the species in terms of virulence factors and antifungal susceptibility. The study included a total of 42 isolates identified as C. parapsilosis complex based on the color they formed in chromogenic medium, colony morphology, and microscopic appearance in Corn Meal-Tween 80 Agar and they were confirmed with API 20 C AUX. For the DNA sequence analysis of clinical isolates, V9G forward and LS reverse primers were used as well as internal transcribed spacers (ITS1 and ITS4). Biofilm formation, esterase, phospholipase, and protease activities were evaluated as virulence factors. Antifungal susceptibility was investigated via colorimetric microdilution method. A total of 75 non-C. albicans isolates were obtained from various clinical samples between 2016 and 2017 in a Turkish Tertiary Care Hospital. Of them, 42 were identified as members of the C. parapsilosis complex. Of the 42 strains, 41 were identified as C. parapsilosis sensu stricto (CpSS), while only one was identified as C. orthopsilosis. Of the CpSS strains, 31 (75.6%) were biofilm-positive, six (14.6%) were esterase-positive, nine (21.9%) were positive for phospholipase activity, and 31 (75.6%) were positive for protease formation, whereas all virulence factors of C. orthopsilosis strain were found to be negative. All CpSS strains were found susceptible to amphotericin B, echinocandins, and flucytosine. It has been concluded that the use of molecular methods to identify CpSS would not be effective in routine laboratory practices as it is the most commonly isolated species from the C. parapsilosis complex and there are no significant differences between species in terms of antifungal susceptibility.

A deep insight into the suppression mechanism of Sedum alfredii root exudates on Pseudomonas aeruginosa based on quorum sensing

Quorum sensing (QS) plays an important role in the intensive communication between plants and microbes in the rhizosphere during the phytoremediation. This study explored the influence of the root exudates of hyperaccumulator Sedum alfredii on Pseudomonas aeruginosa based on QS. The effects of the components of root exudates, genes expression and transcription regulation of QS system (especially the las system) in Pseudomonas aeruginosa wild-type strain (WT) and rhl system mutant strain (ΔrhlI) were systematically analyzed and discussed. The WT and ΔrhlI exposed to gradient root exudates (0×, 1×, 2×, 5× and 10×) showed a concentration-corrective inhibition on protease production, with the inhibition rates of 51.4–74.5% and 31.2–50.0%, respectively. Among the components of the root exudates of Sedum alfredii, only thymol had an inhibition effects to the root exudates on the activity of protease and elastase. The inhibition rates of 50 μmol/L thymol on protease and elastase in WT were 44.7% and 24.3%, respectively, which was consistent with the variation in ΔrhlI. The gene expression of lasB declined 36.0% under the 1× root exudate treatment and 73.0% under the 50 μmol/L thymol treatment. Meanwhile, there was no significant impact on N-3-oxo-dodecanoyl-L-homoserine lactone signal production and the gene expression of lasI and lasR. Therefore, thymol from Sedum alfredii root exudates could inhibit the formation of protease and elastase in Pseudomonas aeruginosa by suppressing the expression of lasB, without any significant influence on the main las system as a potential natural QS inhibitor.

Pemilihan Bronkodilator Pada Penyakit Paru Obstruktif Kroni

Chronic obstructive pulmonary disease (COPD) is a disease that is often found, can be prevented and treated. It characterized by symptoms of persistent respiration and obstruction of airflow, associated with airway disorders and/or alveoli due to exposure to significant harmful particles or gases. COPD currently causes the world's fourth death and is projected to be third in 2020. In 2012, COPD has caused 3 million deaths (6% of all deaths) and will contribute 4.5 million deaths by 2030. COPD requires high costs especially in exacerbation conditions that require hospital treatment.Exposure to cigarette smoke or harmful agents will cause stimulation of macrophages and airway epithelium. Stimulation of macrophages will stimulate inflammatory processes and the underlying cascade such as protease formation and increased oxidative stress. One treatment of COPD is through pharmacological therapy approaches using a bronchodilator which aims to overcome airflow limitation with or without the addition of corticosteroids, mucolytics, antioxidants, and other drugs. The choice of bronchodilators in COPD patients is very individual. The selection must pay attention to the degree of the complaint, the magnitude of the risk of exacerbation, side effects, drug availability, costs, the presence of comorbid diseases and device inhalers.

Characterization of immune responses in different environmental temperature through linear array epitope vaccine induction

The 3C protease of foot-and-mouth disease virus (FMDV) has a conserved amino acid sequence and is responsible for most cleavage in the viral polyprotein. The effects of the synonymous codon usage of FMDV 3C gene and tRNA abundance of the hosts on shaping different folding units (α-helix, β-strand and the coil) in the 3C protease were analyzed based on the structural information of the FMDV 3C protease from Protein Data Bank (PDB: 2BHG) and 210 genes of 3C for all serotypes of FMDV. The strong correlation between some codons usage and the specific folding unit in the FMDV 3C protease is found. As for the effect of translation speed caused by tRNA abundance on the formation of folding units, the codon positions with lowly abundant tRNA scatter in the 3C gene and there is the obvious fluctuation of tRNA abundance locating in the transition boundaries from the β-strand to the α-helix and the coil, respectively. However, codon positions with lowly abundant tRNA clustering into these boundaries are not found, suggesting that the adjustment of translation speed is likely also achieved by the single codon position with low tRNA abundance rather than a cluster. The observations can provide the information for insight into the role of the synonymous codon usage in the formation of 3C protease of FMDV and effect of the tRNA abundance of the hosts on this formation of protease.

Effect of tannic acid on urease and protease produced from Proteus mirabilis

Back ground: P.mirabilis is a gram negative bacterium, motile with its peritrichous flagella .Widely distributed in environment, especially in contaminated water and soil, Many virulence factors like LPS, urease, protease, hemolysin and biofilm formation play an important roles in the pathogenicity of P. mirabilis. Urease is a Nickel containing enzyme causes elevation of urine pH after hydrolyzing urea to ammonia and CO2 forming stones that blocks the urinary track.
 Aims: The effect of tannic acid on the production of urease and protease. 
 Material and methods: Twenty one isolates of Proteus were collected from different sources, Clinical and animal sources all isolates were cultured on MacConkey and blood agar and identification of P. mirabilis by, Vitek -2 compact system. Determine the effect of tannic acid on the production of urease and protease. 
 Results: Twenty one isolates of Proteus were identified depending on Vitek-2 compact system, after identification, it turns out that only 18 isolate were P. mirabilis. All isolates were 100% able to produce urease and 72.2% isolate were able to produce protease. The addition of tannic acid showed an inhibitory effect on urease and protease production.
 Conclusion: The effect of tannic acid on urease and protease depending on concentration, type of strain, incubation period, number of isolates and truculence of isolate.

Inhibition of the precursor and mature forms of HIV-1 protease as a tool for drug evaluation

HIV-1 protease (PR) is a homodimeric enzyme that is autocatalytically cleaved from the Gag-Pol precursor. Known PR inhibitors bind the mature enzyme several orders of magnitude more strongly than the PR precursor. Inhibition of PR at the precursor level, however, may stop the process at its rate-limiting step before the proteolytic cascade is initiated. Due to its structural heterogeneity, limited solubility and autoprocessing, the PR precursor is difficult to access by classical methods, and limited knowledge regarding precursor inhibition is available. Here, we describe a cell-based assay addressing precursor inhibition. We used a reporter molecule containing the transframe (TFP) and p6* peptides, PR, and N-terminal fragment of reverse transcriptase flanked by the fluorescent proteins mCherry and EGFP on its N- and C- termini, respectively. The level of FRET between EGFP and mCherry indicates the amount of unprocessed reporter, allowing specific monitoring of precursor inhibition. The inhibition can be quantified by flow cytometry. Additionally, two microscopy techniques confirmed that the reporter remains unprocessed within individual cells upon inhibition. We tested darunavir, atazanavir and nelfinavir and their combinations against wild-type PR. Shedding light on an inhibitor’s ability to act on non-mature forms of PR may aid novel strategies for next-generation drug design.

Autolytic hydrolases affect sexual and asexual development of Aspergillus nidulans.

Radial growth, asexual sporulation, and cleistothecia formation as well as extracellular chitinase and proteinase formation of Aspergillus nidulans were monitored in surface cultures in order to study the physiological role of extracellular hydrolase production in carbon-stressed cultures. We set up carbon-stressed and carbon-overfed experimental conditions by varying the starting glucose concentration within the range of 2.5 and 40g/L. Glucose starvation induced radial growth and hydrolase production and enhanced the maturation of cleistothecia; meanwhile, glucose-rich conditions enhanced mycelial biomass, conidia, and cleistothecia production. Double deletion of chiB and engA (encoding an extracellular endochitinase and a β-1,3-endoglucanase, respectively) decreased conidia production under carbon-stressed conditions, suggesting that these autolytic hydrolases can support conidia formation by releasing nutrients from the cell wall polysaccharides of dead hyphae. Double deletion of prtA and pepJ (both genes encode extracellular proteases) reduced the number of cleistothecia even under carbon-rich conditions except in the presence of casamino acids, which supports the view that sexual development and amino acid metabolism are tightly connected to each other in this fungus.

Bacteriological Study of Pseudomonas Aeruginosa Isolated from Different Infections and Study Antimicrobial Activities of Plant Extract Solanum Nigrum Against It

This study was conducted for the period from 1/9/2016 to 30/3/2017 in Baquba city in Iraq. Eighty nine samples were collected from different infections from Baquba General Hospital and Al-Batool Hospital. Fifteen isolates (16.85%) were found to be Pseudomonas aeruginosa by using biochemical test. The bacterial diagnosis of all these isolates (100%) were confirmed by VITEK2, beside its possession of the 16s rDNA gene as determined by molecular detection. The results of virulence factors that had Pseudomonas aeruginosa showed possession of all isolates many virulence factors and a high production of which increases the pathogenicity of it. All isolates were able to produce hemolysin (100%), protease (73.33%), and biofilm formation (52%). The susceptibility test was applied on these isolates against (10) antibiotics. The results revealed that the highest resistances were for Ampicillin and Cefotaxime with 100% for each, while the lowest resistance were for Impineme (0.0%) and Ciprofloxacin (6.66%). The effects of ethanolic extract of fruits (Solanum nigrum) against P.aerugionsa were studied. At a concentration of 6% mg/ml, Solanum nigrum caused a marked increase in zone of inhibition (mm) on this bacteria growth. Inhibition zones sizes were different and increased according to concentration of extract and again the growth was completely inhibited in the highest concentration.

Quorum Sensing - Dependent Virulence Factors and Biofilm Formation of Pseudomonas Aeruginosa Isolates from Retrieved Orthopedic Implants

Background: Quorum sensing (QS) plays a crucial role in medical device-related infections. However, epidemiological analysis of QS- dependent virulence factors among Pseudomonas aeruginosa (P. aeruginosa) isolates from orthopedic implants-related infections has rarely been reported. Objective: to assess some QS-dependent virulence factors (as pyocyanin, elastase, protease, rhamnolipid, and biofilm production) in P. aeruginosa isolates from retrieved different orthopedic implants. Methodology: Twenty-four P. aeruginosa isolates obtained from 300 retrieved orthopedic implants with proven diagnosis of osteoarticular infection directly related to orthopedic implants were investigated for production of QS-dependent virulence factors. The pyocyanin production was assayed using King A media, rhamnolipid production was checked utilizing cetyltrimethylammonium bromide (CTAB) agar test. Elastin lytic activity was assayed using elastin Congo red, total protease was estimated using the modified skimmed milk assay technique, and the biofilm forming ability was assayed by microtiter plate assay. N-acyl homoserine lactones (3-oxo-C 12 -HSL, C 4 -HSL) were detected by Cross-feeding bioassay. Polymerase chain reaction (PCR) was used to assess the presence of the QS genes lasI, lasR, rhlI, and rhlR in P. aeruginosa isolates. Results: Seventeen isolates (70.8%) displayed the QS-dependent phenotypes and seven isolates were found to be lacking all tested virulence factors. The lack of production of pyocyanin, Rhamnolipid, las B elastase, total protease, and biofilm formation by the seven isolates proposed that they might have a defective QS system. Among the Seven isolates exhibiting impaired QS-dependent phenotypes, AHLs autoinducers were present in three isolates, though the residual four isolates were lacking both autoinducers and therefore are considered QS-deficient. PCR analysis declared that two isolates contained lasI, lasR, rhlI and rhlR genes while one isolate lacked lasR gene, and one isolate was lacking the four tested genes. Conclusion: Although QS seems to be a crucial regulator of virulence factors and biofilm formation in P. aeruginosa, it is equipped to produce implant infection in humans despite an impaired QS system. These results do not challenge the concept that QS plays a foremost role in P. aeruginosa pathogenicity, but highlight that along with known virulence factors, perhaps there are other virulence factors, that may not be strictly under QS control.

The HIV-1 late domain-2 S40A polymorphism in antiretroviral (or ART)-exposed individuals influences protease inhibitor susceptibility.

BackgroundThe p6 region of the HIV-1 structural precursor polyprotein, Gag, contains two motifs, P7TAP11 and L35YPLXSL41, designated as late (L) domain-1 and -2, respectively. These motifs bind the ESCRT-I factor Tsg101 and the ESCRT adaptor Alix, respectively, and are critical for efficient budding of virus particles from the plasma membrane. L domain-2 is thought to be functionally redundant to PTAP. To identify possible other functions of L domain-2, we examined this motif in dominant viruses that emerged in a group of 14 women who had detectable levels of HIV-1 in both plasma and genital tract despite a history of current or previous antiretroviral therapy.ResultsRemarkably, variants possessing mutations or rare polymorphisms in the highly conserved L domain-2 were identified in seven of these women. A mutation in a conserved residue (S40A) that does not reduce Gag interaction with Alix and therefore did not reduce budding efficiency was further investigated. This mutation causes a simultaneous change in the Pol reading frame but exhibits little deficiency in Gag processing and virion maturation. Whether introduced into the HIV-1 NL4-3 strain genome or a model protease (PR) precursor, S40A reduced production of mature PR. This same mutation also led to high level detection of two extended forms of PR that were fairly stable compared to the WT in the presence of IDV at various concentrations; one of the extended forms was effective in trans processing even at micromolar IDV.ConclusionsOur results indicate that L domain-2, considered redundant in vitro, can undergo mutations in vivo that significantly alter PR function. These may contribute fitness benefits in both the absence and presence of PR inhibitor.Electronic supplementary materialThe online version of this article (doi:10.1186/s12977-016-0298-1) contains supplementary material, which is available to authorized users.

The Synergistic Effect of Sesamum Oil and Antibiotics on Streptococcus mutans Isolated from the Teeth and its Role in Inhibition of Some Virulence Factors

This study included the collection of 52 dental swabs samples from patients suffering from infection of the teeth, the microscopic and biochemical tests was conducted and a selective media were used for the diagnosis of bacterial isolates that were isolated. The results showed the represented of Streptococcus mutans with infection rate 30.7%. The effect of fourteen antibiotics were studied toward 46 Streptococcus mutans isolates by disk diffusion method and the most resistant one to antibiotics was selected that was possessed pattern of multiple drug resistance towards(11-12) antibiotic. The virulence factors of the bacteria was investigated and the results showed the ability of these isolates to Biofilm, formation and production of Haemolysin, bacteriocin, protease, gelatinase, and sidrophores. Sesamum seeds were collected and diagnosed, oil was prepared and cellular toxicity and its PH were evaluted and the antimicrobial activity of oil against Streptococcus mutans by disk diffusion method was performed. The study showed that the Sesamum oil has a highly inhibition effect against Streptococcus mutans. The results of the statistical analysis showed the significant difference to the antibiotic synergistic with Sesamum oil, and developing isolates in the culture media containing Sesamum with minimum inhibition concentration changed their sensitivity towards the antibiotics compared with sensitivity before treatment, the isolate lost its ability to produce biofilm,and production of haemolysin, bacteriocin, and gelatinase, and reduced production for protease, while Sidrophores production was not affected.

Antifungal Susceptibility in Serum and Virulence Determinants of Candida Bloodstream Isolates from Hong Kong.

Candida bloodstream infections (CBI) are one of the most common nosocomial infections globally, and they account for a high mortality rate. The increasing global prevalence of drug-resistant Candida strains has also been posing a challenge to clinicians. In this study, we comprehensively evaluated the biofilm formation and production of hemolysin and proteinase of 63 CBI isolates derived from a hospital setting in Hong Kong as well as their antifungal susceptibility both in the presence and in the absence of human serum, using standard methodology. Candida albicans was the predominant species among the 63 CBI isolates collected, and non-albicans Candida species accounted for approximately one third of the isolates (36.5%). Of them, Candida tropicalis was the most common non-albicans Candida species. A high proportion (31.7%) of the CBI isolates (40% of C. albicans isolates, 10% of C. tropicalis isolates, 11% of C. parapsilosis isolates, and 100% of C. glabrata isolates) were found to be resistant to fluconazole. One of the isolates (C. tropicalis) was resistant to amphotericin B. A rising prevalence of drug-resistance CBI isolates in Hong Kong was observed with reference to a previous study. Notably, all non-albicans Candida species, showed increased hemolytic activity relative to C. albicans, whilst C. albicans, C. tropicalis, and C. parapsilosis exhibited proteinase activities. Majority of the isolates were capable of forming mature biofilms. Interestingly, the presence of serum distorted the yeast sensitivity to fluconazole, but not amphotericin B. Taken together, our findings demonstrate that CBI isolates of Candida have the potential to express to varying extent their virulence attributes (e.g., biofilm formation, hemolysin production, and proteinase activity) and these, together with perturbations in their antifungal sensitivity in the presence of serum, may contribute to treatment complication in candidemia. The effect of serum on antifungal activity warrants further investigations, as it has direct clinical relevance to the treatment outcome in subjects with candidemia.

Stabilization of α-Synuclein Fibril Clusters Prevents Fragmentation and Reduces Seeding Activity and Toxicity.

Protein misfolding results in the accumulation of aggregated β-sheet-rich structures in Parkinson's disease (PD) and Alzheimer's disease. The toxic oligomer hypothesis stipulates that prefibrillar assemblies, such as soluble oligomers or protofibrils, are responsible for the poor prognosis of these diseases. Previous studies demonstrated that a small molecule related to the natural compound orcein, O4, directly binds to amyloid-β fibrils and stabilizes them, accelerating the formation of end-stage mature fibrils. Here we demonstrate a similar phenomenon during O4 treatment of α-synuclein (αsyn) aggregates, the protein responsible for PD pathology. While the drug did not change the kinetics of aggregate formation as measured by the amyloidophilic dye thioflavin T, O4 depleted αsyn oligomers and promoted the formation of sodium dodecyl sulfate and proteinase K resistant aggregates consisting of large fibril clusters. These fibril clusters exhibited reduced toxicity to human neuronal model cells and reduced seeding activity in vitro. The effectiveness of O4 decreased when it was added at later points in the αsyn aggregation pathway, which suggests that the incorporation of O4 into fibril assemblies stabilizes them against chemical, enzymatic, and mechanic degradation. These findings suggest that small molecules, which stabilize amyloid fibrils, can prevent fibril fragmentation and seeding and consequently prevent prion-like replication of misfolded αsyn. Inhibiting prion replication by fibril stabilization could thus be a therapeutic strategy for PD.