Growth Kinetics Assay Research Articles

Abstract 4193: Lymphotoxin-alpha-armed oncolytic VSV-GP synergizes with SMAC mimetics to induce enhanced tumor cell death and regression

Abstract Introduction: Oncolytic viruses (OVs), which selectively replicate in and destroy tumor cells, form a highly promising class of cancer therapeutics with a strong immune stimulatory potential. VSV-GP, a chimeric Vesicular Stomatitis Virus (VSV) pseudotyped with the glycoprotein of the lymphocytic choriomeningitis virus (LCMV) can selectively replicate and express therapeutic cargos in the tumor microenvironment. Lymphotoxin alpha (LTα) - a member of the TNF superfamily - may present as a potent therapeutic cargo based on its immune stimulatory and cell death modulatory characteristics.In this study, we explore the immune promoting and tumor cell death inducing properties of VSV-GP-LTα, a next generation variant with an NF-κB facilitated mode of action. We further investigate clinically relevant combination therapies with “second mitochondria-derived activator of caspases” mimetic compounds (SMCs). Experimental Procedures: Viral fitness, cargo expression and test substance quality were assessed using growth kinetic, ELISA, RT-qPCR and titration assays. Therapeutic efficacy and safety of VSV-GP-LTα, with or without SMC co-treatment, were assessed in vivo in syngeneic C57BL/6 mice bearing subcutaneous LLC1-IFNAR-/- or B16F10-OVA tumors. To shed further light into the VSV-GP-LTα treatment effects, we applied immunohistochemistry, flow cytometry, multiplex ELISA and Nanostring® assays. Data Summary: VSV-GP-LTα induced stronger anti-tumor effects and increased overall survival compared to VSV-GP. It was also better tolerated than VSV-GP armed with TNF-α, a therapeutic cargo from the same class. Histological and gene expression analysis confirmed widespread cell killing within the tumor and activation of innate and adaptive immune response markers. However, further characterization of the adaptive immune compartment did not show significant changes induced by the expressed LTα. Building on the observed therapeutic strengths of VSV-GP-LTα, we further explored a treatment combination with SMC. This resulted in a therapeutic synergy with improved treatment-induced tumor cell death, especially in a less permissive tumor setting. Conclusion: Together, here we show that arming VSV-GP with LTα resulted in a strong inflammation of the tumor microenvironment and enhanced levels of tumor cell death induction. This effect could be further enhanced by the co-treatment with SMC. Citation Format: Bart Spiesschaert, Philipp Müller, Katharina Angerer, Krishna Das, Tobias Nolden, Carles Urbiola, Judy Ng, Fabian Heinemann, Birgit Stierstorfer, Patrik Erlmann, Guido Wollmann. Lymphotoxin-alpha-armed oncolytic VSV-GP synergizes with SMAC mimetics to induce enhanced tumor cell death and regression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4193.

Magnetic exposure using Samarium Cobalt (SmCO5) increased proliferation and stemness of human Umbilical Cord Mesenchymal Stem Cells (hUC-MSCs)

Despite the extensive reports on the potential hazard of magnetic field (MF) exposures on humans, there are also concurrently reported on the improved proliferative property of stem cells at optimum exposure. However, the effect on mesenchymal stem cells (MSCs) remains unknown. Therefore, we aimed to investigate the impact of induced static MF (SMF) on human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) using Samarium Cobalt (SmCO5). At passage 3, hUC-MSCs (1 × 104) were exposed to 21.6 mT SMF by a direct exposure (DE) showed a significantly higher cell count (p < 0.05) in the growth kinetics assays with the shortest population doubling time relative to indirect exposure and negative control. The DE group was committed into the cell cycle with increased S phase (55.18 ± 1.38%) and G2/M phase (21.75 ± 1.38%) relative to the NC group [S-phase (13.54 ± 2.73%); G2/M phase (8.36 ± 0.28%)]. Although no significant changes were observed in the immunophenotype, the DE group showed an elevated expression of pluripotency-associated markers (OCT4, SOX2, NANOG, and REX1). These results suggest that the MFs could potentially induce proliferation of MSCs, a promising approach to promote stem cells propagation for clinical therapy and research without compromising the stemness of hUC-MSCs.

Enhancement of antibacterial activity of sinigrin-capped silver nanoparticles in combination with myrosinase

Green synthesis of silver nanoparticles (AgNPs) is a widely accepted approach due to its ecofriendly use of non-toxic materials. AgNPs are extensively used in the agriculture and medicine industry as antioxidant, antibacterial, and antifungal agents. In this study, AgNPs were synthesized using phytochemical sinigrin as both reducing and capping agent. Sinigrin present in cruciferous vegetables can be hydrolyzed by endogenous myrosinase (MYR) to produce allyl isothiocyanate (AITC) which has reported antibacterial properties. We hypothesized that sinigrin-capped AgNPs (SIN-AgNPs) will have enhanced antibacterial activity in the presence of MYR enzyme due to the release of AITC from hydrolysis of sinigrin. The formation of SIN-AgNPs was confirmed by UV-Vis absorption spectra which exhibited a peak around 405 nm. The particle size was determined to be ~20 nm using dynamic light scattering. The size was also confirmed through transmission electron microscopy (TEM) imaging with morphology close to spherical shape. Fourier transform infrared analysis confirmed the functional groups of sinigrin capped on the surface of AgNPs. Myrosinase gene (TGG1) sequence was cloned in pET-20b(+) plasmid and expressed in BL21(DE3) E. coli competent cells. The collected bacterial lysate was purified by immobilized metal affinity chromatography to obtain MYR protein which was characterized by SDS-PAGE, Western blot, and enzyme activity. The antibacterial efficacy was evaluated against E. coli using disc diffusion assay and growth kinetics study. Our results showed that SIN-AgNPs in combination with MYR protein exhibited ⁓25% more zone of inhibition by disk diffusion assay at a concentration of 25 µg/ml and inhibited bacterial growth for a longer time in the range of 0.5–1 µg/ml in growth kinetic assay.

Formation, Transmission, and Dynamic Evolution of a Multidrug-Resistant Chromosomally Integrated Plasmid in Salmonella Spp.

IncHI2 plasmids, possessing high flexibility and genetic plasticity, play a vital role in the acquisition and transmission of resistance determinants. Polymorphic mobile genetic elements (MGEs) generated by a chromosomally integrated IncHI2 plasmid in an individual Salmonella isolate have not yet been detected, and the mechanisms of the formation, excision, and dynamic evolution of a multidrug-resistant chromosomally integrated plasmid (MRCP) have remained obscure. Herein, we identified a 260-kb blaCTX–M–55-qnrS1-bearing IncHI2 plasmid within a Salmonella Muenster strain. Plenty of heterogeneous MGEs (new Escherichia coli chromosomally integrated plasmid or circular plasmids with different profiles) were yielded when this MRCP was conjugated into E. coli J53 with a transfer frequency of 10–4–10–5 transconjugants per donor. A bioinformatic analysis indicated that replicative transposition and homologous recombination of IS26 elements were particularly active, and the truncated Tn1721 also played a vital role in the formation of MRCP offspring. More importantly, when released from the chromosome, MRCP could capture and co-transfer adjacent chromosomal segments to form larger plasmid progeny than itself. Stability and growth kinetics assays showed that the biological characteristics of MRCP progeny were differentiated. This study provides an insight into a flexible existence of MRCP. The conversion between vertical and horizontal transmission endowed MRCP with genetic stability as a chromosomal coding structure and transferability as extra-chromosomal elements. This alternation may accelerate the acquisition and persistence of antibiotic resistance of clinical pathogens and enhance their ability to respond to adverse environments, which poses a great challenge to the traditional antibiotic treatment.

In Vitro and In Vivo Activity of Luliconazole (NND-502) against Planktonic Cells and Biofilms of Azole Resistant Aspergillus fumigatus.

Aspergillus fumigatus has become a significant threat in clinical settings. Cases of invasive infections with azole-resistant A. fumigatus isolates (ARAF) increased recently. Developing strategies for dealing with ARAF has become crucial. We here investigated the in-vitro and in-vivo activity of the imidazole luliconazole (LLCZ) against clinical ARAF. In total, the LLCZ minimum inhibitory concentrations (MICs) were tested for 101 A. fumigatus isolates (84 ARAF and 17 azole-susceptible A. fumigatus as wild-type controls) according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST). Additionally, antifungal activity was assessed in vitro, including an XTT planktonic growth kinetics assay and biofilm assays (crystal violet and XTT assay). Further, a single-dose LLCZ treatment (152 mg/L) was tested for seven days in vivo in a Galleria mellonella infection model. LLCZ showed an MIC50 of 0.002 mg/L and no significant difference was found between triazole-resistant and wild-type isolates. Growth inhibition took place between 6 and 12 h after the start of incubation. LLCZ inhibited biofilm formation when added in the pre-adhesion stages. In vivo, single-dose LLCZ-treated larvae show a significantly higher survival percentage than the control group (20%). In conclusion, LLCZ has activity against planktonic cells and early biofilms of ARAF.

Inhibition of azole-resistant Aspergillus fumigatus biofilm at various formation stages by antifungal drugs, including olorofim.

Interest in aspergillosis has increased over the past decades. An increase in the incidence of azole-resistant Aspergillus fumigatus strains has been reported; therefore, the need for novel therapeutic approaches is urgent. The formation of biofilms contributes to pathogen resistance. We investigated the biofilm formation capabilities of azole-resistant A. fumigatus and analysed the susceptibility of biofilms at various developmental stages to three antifungal agents. Biofilm formation of 19 clinical A. fumigatus strains (3 azole-susceptible and 16 azole-resistant strains) was determined by crystal violet staining and by an XTT assay over a period of 48 h. We measured antibiofilm activity of voriconazole, amphotericin B and olorofim. These agents were added before adhesion, after adhesion, after germination and to mature fungal biofilm. Antibiofilm activity was assessed in an XTT assay and in confocal laser scan microscopy. Additionally, a growth-kinetic assay with planktonic A. fumigatus was performed. Each of the antifungal agents inhibited the metabolic activity of A. fumigatus biofilms when applied at early stages of biofilm formation. The mature biofilms were more resistant. Olorofim and voriconazole showed promising effects against A. fumigatus adhesion and germination, whereas the mature biofilm was not affected by treatment. In contrast, the biofilm of A. fumigatus showed amphotericin B susceptibility throughout the entire developmental process. The planktonic cells were susceptible to all three antifungal drug classes with an inhibition peak at 12 h after incubation. This is the first known study to demonstrate the antibiofilm activity of olorofim, voriconazole and amphotericin B against azole-resistant A. fumigatus.

Insights into the Antibacterial Activity of Prolactin-Inducible Protein against the Standard and Environmental MDR Bacterial Strains.

Background: Prolactin inducible protein (PIP) is a small secretary glycoprotein present in most biological fluids and contributes to various cellular functions, including cell growth, fertility, antitumor, and antifungal activities. Objectives: The present study evaluated the antibacterial activities of recombinant PIP against multiple broad-spectrum MDR bacterial strains. Methods: The PIP gene was cloned, expressed and purified using affinity chromatography. Disk diffusion, broth microdilution, and growth kinetic assays were used to determine the antibacterial activities of PIP. Results: Disk diffusion assay showed that PIP has a minimum and maximum zone of inhibition against E. coli and P. aeruginosa, respectively, compared to the reference drug ampicillin. Furthermore, growth kinetics studies also suggested that PIP significantly inhibited the growth of E. coli and P. aeruginosa. The minimum inhibitory concentration of PIP was 32 µg/mL for E. coli (443), a standard bacterial strain, and 64 µg/mL for Bacillus sp. (LG1), an environmental multidrug-resistant (MDR) strain. The synergistic studies of PIP with ampicillin showed better efficacies towards selected bacterial strains having MDR properties. Conclusion: Our findings suggest that PIP has a broad range of antibacterial activities with important implications in alleviating MDR problems.

GC-MS Characterization, In Vitro Antioxidant, Anti-inflammatory and Potential Antidermatophytic Activity of Citrullus lanatus Seed Oil

Background: Dermatophytes are well-known fungus having the capability to invade kerat-inized tissues of nails, skin, and hair, of humans and other animals, causing dermatophytosis. Citrullus lanatus (watermelon) seed is a rich source of vitamin E, essential fatty acids, and minerals, bearing var-ious health benefits. Despite the presence of many useful components, the oil is not much explored. Accordingly, the study was planned to investigate the fatty acid profiling, in vitro antioxidant, anti-inflammatory, and potential antidermatophytic activity of Citrullus lanatus seed oil against dermato-phytic strains. Methods: The C. lanatus oil was extracted from seeds, and fatty acid profiling was characterized by GC-MS. Further, the oil was subjected to DPPH scavenging activity, and the anti-inflammatory activity was evaluated from Bovine serum albumin assay. The antidermatophytic screening against Microsporum canis, Trichophyton rubrum, and Trichophyton mentagrophytes was carried out by agar well diffusion, broth microdilution assay, and growth kinetics assay. Results: The C. lanatus seed oil exhibited good antioxidant activity close to ascorbic acid, i.e., IC50- 52.22 μg/ml, 35.72 μg/ml, respectively, and the oil exhibited moderate anti-inflammatory ac-tivity (315.2 μg/ml) as that of the standard drug diclofenac sodium (174.3μg/ml). The fatty acid profiling indicates the presence of eight main compounds, out of which ç-tocopherol, butylated hy-droxytoluene, and hexadecanoic acid are predominantly present. The C. lanatus seed oil showed promising activity against T. rubrum with a lower minimum inhibitory concentration value with re-spect to positive controls, Fluconazole and Ketoconazole. The oil also had a strong effect on time-dependent kinetic inhibition against T. rubrum. This was the first report of C. lanatus seed oil against any dermatophyte. Conclusion: In conclusion, the study supports the exploitation of C. lanatus seed oil and its main compounds as a source of natural antioxidants, anti-inflammatory, and as a novel agent against dermatophytes for curing superficial fungal infections in the near future.

Ib-AMP4 antimicrobial peptide as a treatment for skin and systematic infection of methicillin-resistant Staphylococcus aureus (MRSA).

Antimicrobial peptide compounds (AMPs) play important roles in the immune system. They also exhibit significant anti-tumor and antibacterial properties. Most AMPs are cationic and are able to bind bacterial cell membranes through electrostatic affinity. Ib-AMP4 is a plant-derived AMP that exerts rapid bactericidal functions. In the present study, the antibacterial efficiency of the produced recombinant Ib-AMP4 in elimination of Methicillin-resistant Staphylococcus aureus (MRSA) bacterial infection, was investigated under in vitro and in vivo situations. The synthesized Escherichia coli codon-optimized gene sequences of the Ib-AMP4 were expressed in E. coli BL21 (DE3) pLysS. The recombinant Ib-AMP4 was purified and refolding conditions were optimized. The antibacterial efficiency of the refolded peptide against MRSA was tested under in vivo and in vitro situations for treatment of skin and systematic infection of MRSA in a mouse model. Antibacterial assays confirmed the antibacterial function of Ib-AMP4 against MRSA. SEM results proved the destructive effects of applying Ib-AMP4 on MRSA biomembrane. Time-kill curve and growth kinetic assay illustrated rapid antibacterial activity of the produced Ib-AMP4. Moreover, Ib-AMP4 showed significant infection treatment ability in a mouse model and all infected mice receiving Ib-AMP4 protein survived and there was no trace of bacteria in their blood samples. The results confirmed the rapid antibacterial potential of the produced recombinant Ib-AMP4 to be used for efficient treatment of MRSA infection.

Antibacterial Activity and Multi-Targeting Mechanism of Dehydrocorydaline From Corydalis turtschaninovii Bess. Against Listeria monocytogenes.

Listeria monocytogenes is a foodborne pathogen, with relatively low incidence but high case-fatality. Phytochemicals have been recognized as a promising antimicrobial agent as an alternative to synthetic chemicals due to their safety and high efficacy with multi-target sites. This study identified and characterized a novel antibacterial agent, dehydrocorydaline, in the Corydalis turschaninovii rhizome using HPLC-LTQ-Orbitrap-HRMS, and its antibacterial effect with lowest MIC (1 mg/mL) and MBC (2 mg/mL) values. In addition, an in vitro growth kinetic assay, cytoplasmic nucleic acid and protein leakage assay, and observation of morphological changes in bacterial cells supported the strong antibacterial activity. Dehydrocorydaline also displayed effective inhibitory effects on biofilm formation and bacterial motility. In order to investigate the potential antibacterial mechanism of action of dehydrocorydaline against L. monocytogenes, label-free quantitative proteomics was used, demonstrating that dehydrocorydaline has multiple targets for combating L. monocytogenes including dysregulation of carbohydrate metabolism, suppression of cell wall synthesis, and inhibition of bacterial motility. Overall, this study demonstrated that dehydrocorydaline has potential as a natural and effective antibacterial agent with multi-target sites in pathogenic bacteria, and provides the basis for development of a new class of antibacterial agent.

In doping effect on the structural, morphological, optical and enhanced antimicrobial activity of facilely synthesized novel CuS nanostructures

In this paper, we report the impact of Indium on the structural, morphological, optical, and antibacterial properties of copper sulfide (CuS) nanoparticles produced by facile chemical Co-precipitation process. X-ray diffraction, Raman, scanning electron microscope and X-ray photoelectron spectroscopy techniques were used to investigate the phase composition and purity. Antibacterial tests were performed by well diffusion method and growth kinetics assay. The hexagonal structure of CuS nanoparticles is revealed by XRD/Raman analysis, with crystallite size ranging from 6 to 11 nm. SEM pictures also revealed a larger crystallite size with the addition of indium doping. For maximum indium doping (5 wt.%), the morphology was modified from nanoparticle to nanosheet. The existence of indium dopant was also confirmed by XPS analysis. The energy band gap shifted from 2.16 – 2.47 eV with altering indium concentrations in CuS. Indium doped CuS NPs exhibited highest antibacterial effect against Mycobacterium smegmatis (20–24 mm) while Pseudomonas aeruginosa had the lowest antimicrobial activity (12–14 mm). Growth kinetics study delineated that In:CuS had high antibacterial efficacy against all the tested microbial strains and suppress the microbial growth in the logarithmic growth phase. Hence, In:CuS might be applied as antimicrobial coatings to inhibit microbial biofilm development.

Phytochemical composition, antifungal activity, in vitro and in vivo toxicity of Syzygium cumini (L.) Skeels leaves extract

This study determined phytochemical composition, antifungal activity and toxicity in vitro and in vivo of Syzygium cumini leaves extract (Sc). Thus, was characterized by gas chromatography coupled to mass spectrometry and submitted to determination of Minimum Inhibitory (MIC) and Fungicidal concentrations (MFC) on reference and clinical strains of Candida spp. and by growth kinetics assays. Toxicity was verified using in vitro assays of hemolysis, osmotic fragility, oxidant and antioxidant activity in human erythrocytes and by in vivo acute systemic toxicity in Galleria mellonella larvae. Fourteen different compounds were identified in Sc, which showed antifungal activity (MIC between 31.25-125 μg/mL) with fungistatic effect on Candida. At antifungal concentrations, it demonstrated low cytotoxicity, antioxidant activity and neglible in vivo toxicity. Thus, Sc demonstrated a promising antifungal potential, with low toxicity, indicating that this extract can be a safe and effective alternative antifungal agent.

In vitro and in silico approach for characterization of antimicrobial peptides from potential probiotic cultures against Staphylococcus aureus and Escherichia coli.

The focus of the present study was to characterize antimicrobial peptide produced by potential probiotic cultures of Enterococcus durans DB-1aa (MCC4243), Lactiplantibacillus plantarum Cu2-PM7 (MCC4246) and Limosilactobacillus fermentum Cu3-PM8 (MCC4233) against Staphylococus aureus MTCC 96 and Escherichia coli MTCC118. The growth kinetic assay revealed 24h of incubation to be optimum for bacteriocin production. The partially purified compound of all the three selected cultures after ion-exchange chromatography was found to be thermoresistant and stable under a wide range of pH. The compound was sensitive to proteinase-K, but resistant to trypsin, α-amylase and lipase. Comparatively, bacteriocins from L. fermentum Cu3-PM8 and L. plantarum Cu2-PM7 showed higher stability under studied parameter, hence was taken up for further investigation. The apparent molecular weight of bacteriocin from L. fermentum MCC4233 and L. plantarum MCC4246 was found to be 3.5kDa. Further, plantaricin gene from MCC4246 was characterized in silico. The translated partial amino acid sequence of the plnA gene in MCC4246 displayed 48 amino acids showing 100 % similarity with plantaricin A of Lactobacillus plantarum (WP_0036419). The sequence revealed 7 β sheets, 6 α sheets, 6 predicted coils and 9 predicted turns. The predicted properties of the peptide included an isoelectric point of 10.82 and a hydrophobicity of 48.6 %. The molecular approach of using Geneious Prime software and protein prediction data base for characterization of bacteriocin is novel and predicts "KSSAYSLQMGATAIKQVKKLFKKWGW" to be a peptide responsible for antimicrobial activity. The study provides information about a broad spectrum bacteriocin in native probiotic culture and paves a way towards its application in functional foods as a biopreservative agent.

Additive manufactured graphene-based electrodes exhibit beneficial performances in Pseudomonas aeruginosa microbial fuel cells

A commercial polylactic acid/graphene (8 wt%) composite filament was used to additive manufacture (AM) graphene macroelectrodes (AM-GMs). The electrode surfaces were characterised and Pseudomonas aeruginosa was utilised as the exoelectrogen. The MFC was optimised using growth kinetic assays, biofilm formation, and quantification of pyocyanin production (via liquid chromatography-mass spectrometry) in conditions that were representative of the batch-fed MFC configuration utilised. Cell potential and bacterial viability was recorded at 0 h, 24 h, 48 h, 72 h, 96 h and 120 h, power density and current density were calculated. There was no significant difference between P. aeruginosa cell proliferation in either media tested. Interestingly, no accumulation of pyocyanin was evident. Additively manufactured electrodes comprised of graphene (AM-GMs) were successfully applied in a P. aeruginosa MFC configuration and power outputs (110.74 ± 14.63 μW m-2) produced were comparable to that of the ‘benchmark’ electrode, carbon cloth (93.49 ± 5.17 μW m-2). The AM-GMs demonstrated power/current outputs similar to that of the carbon cloth electrodes in both anaerobic LB and glucose-based media over 120 h; the AM-GMs had no significant detrimental effect on P. aeruginosa viability. This study highlights the potential application of additive manufactured electrodes with the incorporation of nanomaterials (e.g., graphene) as one approach to enhance power outputs.

A rapid and accurate method for herpesviral gnome editing

To rapidly and accurately manipulate genome such as gene deletion, insertion and site mutation, the whole genome of a very virulent strain Md5 of Marek's disease virus (MDV) was inserted into bacterial artificial chromosome (BAC) through homogeneous recombination. The recombinant DNA was electroporated into DH10B competent cells and identified by PCR and restriction fragment length polymorphism analysis. An infectious clone of Md5BAC was obtained following transfection into chicken embryo fibroblast (CEF) cells. Furthermore, a lorf10 deletion mutant was constructed by two step Red-mediated homologous recombination. To confirm the specific role of gene deletion, the lorf10 was reinserted into the original site of MDV genome to make a revertant strain. All the constructs were rescued by transfection into CEF cells, respectively. The successful packaging of recombinant viruses was confirmed by indirect immunofluorescence assay. The results of growth kinetics assay and plaques area measurement showed that the lorf10 is dispensable for MDV propagation in vitro. Overall, this study successfully constructed an infectious BAC clone of MDV and demonstrated its application in genome manipulation; the knowledge gained from our study could be further applied to other hepesviruses.

Growth capability of epidemic influenza viruses in Japan since the 2009 H1N1 pandemic.

The correlation of viral growth capability (n = 156) with the viral load in nasopharyngeal swabs (n = 76) was assessed. Epidemic influenza A/H1N1, A/H3N2, and B viruses showed a wide range of growth capability (104–1011 copies/mL) in Madin-Darby canine kidney cells. The growth was correlated with the nasopharyngeal viral load (r = 0.53). Six selected strains showed growth-dependent cell death (r = 0.96) in a growth kinetics assay. Epidemic influenza viruses exhibit a wide range of growth capability. Growth capability should be considered one of the key factors in disease prognosis.

Multi-scale Dynamical Modeling of T Cell Development from an Early Thymic Progenitor State to Lineage Commitment

Intrathymic development of committed progenitor (pro)-T cells from multipotent hematopoietic precursors offers an opportunity to dissect the molecular circuitry establishing cell identity in response to environmental signals. This transition encompasses programmed shutoff of stem/progenitor genes, upregulation of Tcell specification genes, proliferation, and ultimately commitment. To explain these features in light of reported cis-acting chromatin effects and experimental kinetic data, we develop a three-level dynamic model of commitment based upon regulation of the commitment-linked gene Bcl11b. The levels are (1) a core gene regulatory network (GRN) architecture from transcription factor (TF) perturbation data, (2) a stochastically controlled chromatin-state gate, and (3) a single-cell proliferation model validated by experimental clonal growth and commitment kinetic assays. Using RNA fluorescence in situ hybridization (FISH) measurements of genes encoding key TFs and measured bulk population dynamics, this single-cell model predicts state-switching kinetics validated by measured clonal proliferation and commitment times. The resulting multi-scale model provides a mechanistic framework for dissecting commitment dynamics.

Hsp90 is involved in pseudorabies virus virion assembly via stabilizing major capsid protein VP5

Many viruses utilize molecular chaperone heat shock protein 90 (Hsp90) for protein folding and stabilization, however, the role of Hsp90 in herpesvirus lifecycle is obscure. Here, we provide evidence that Hsp90 participates in pseudorabies virus (PRV) replication. Viral growth kinetics assays show that Hsp90 inhibitor geldanamycin (GA) abrogates PRV replication at the post-penetration step. Transmission electron microscopy demonstrates that dysfunction of Hsp90 diminishes the quantity of PRV nucleocapsids. Overexpression and knockdown of Hsp90 suggest that de novo Hsp90 is involved in PRV replication. Mechanismly, dysfunction of Hsp90 inhibits PRV major capsid protein VP5 expression. Co-immunoprecipitation and indirect immunofluorescence assays indicate that Hsp90 interacts with VP5. Interestingly, Hsp70, a collaborator of Hsp90, also interacts with VP5, but doesn't affect PRV growth. Finally, inhibition of Hsp90 results in PRV VP5 degradation in a proteasome-dependent manner. Collectively, our data suggest that Hsp90 contributes to PRV virion assembly and replication via stabilization of VP5.

Antimicrobial activity of enacyloxin IIa and gladiolin against the urogenital pathogens Neisseria gonorrhoeae and Ureaplasma spp.

To determine the antimicrobial activity of enacyloxin IIa and gladiolin against Neisseria gonorrhoeae and Ureaplasma spp. The Burkholderia polyketide antibiotics enacyloxin IIa and gladiolin were tested against 14 N. gonorrhoeae and 10 Ureaplasma spp. isolates including multidrug-resistant N. gonorrhoeae isolates WHO V, WHO X and WHO Z as well as macrolide, tetracycline and ciprofloxacin-resistant ureaplasmas. Susceptibility testing of N. gonorrhoeae was carried out by agar dilution, whereas broth micro-dilution and growth kinetic assays were used for Ureaplasma spp. The MIC range for enacyloxin IIa and gladiolin against N. gonorrhoeae was 0·015-0·06mgl-1 and 1-2mgl-1 respectively. The presence of resistance to front line antibiotics had no effect on MIC values. The MIC range for enacyloxin IIa against Ureaplasma spp. was 4-32mgl-1 with a clear dose-dependent effect when observed using a growth kinetic assay. Gladiolin had no antimicrobial activity on Ureaplasma spp. at 32mgl-1 and limited impact on growth kinetics. Enacyloxin IIa and gladiolin antibiotics have antimicrobial activity against a range of antibiotic susceptible and resistant N. gonorrhoeae and Ureaplasma isolates. This study highlights the potential for a new class of antimicrobial against pathogens in which limited antibiotics are available. Development of these compounds warrants further investigation in the face of emerging extensively drug-resistant strains.

Resistance of a Halobacterium salinarum isolate from a solar saltern to cadmium, lead, nickel, zinc, and copper.

The current study focuses on the tolerance of a strain of Halobacterium salinarum isolated from Sfax solar saltern (Tunisia) towards cadmium (Cd), lead (Pb), nickel (Ni), zinc (Zn), and copper (Cu) by using agar dilution methods in complex and minimal media. The results showed the least inhibitory metals based on Minimum Inhibitory Concentrations (MICs) were lead (MIC = 4.5mM), cadmium (MIC = 4mM), and nickel (MIC = 2.5mM) in complex medium. The MICs of these metals were more inhibitory (MIC < 2mM) in the other tested media. The archaeal strain revealed a high sensitivity for copper and zinc, with MICs below 0.5mM for both metals. Growth kinetics in complex and minimal media showed the strain to be more sensitive to the metals in liquid media than in solid media. The growth kinetic assays indicated the presence of selected heavy metals resulted in a lower growth rate and lower total cell mass relative to the control. Despite that cadmium and lead are nonessential and have no nutrient value, they were the most tolerated metals by H. salinarum strain. In addition, pigment intensity in the strain was inhibited by the presence of the heavy metals relative to the control.