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BackgroundPseudomonas aeruginosais a ubiquitous, opportunistic bacterium whose highly plastic genome and adaptable phenotype have yielded serious treatment challenges for immunocompromised patients. Antibiotic alternatives, such as anti-virulence therapeutics, have gained interest because they disable bacterial virulence mechanisms, thereby restoring the killing efficacy of host immunity or traditional antibiotics. Identifying successful anti-virulence therapeutics may require a paradigm shift from the decades-old antimicrobial susceptibility testing (AST) in Mueller Hinton broth to media that foster optimal virulence expression. MethodsThis study evaluates the virulence gene expression and activity ofP. aeruginosaPA14 in host-mimicking conditions, represented by Dulbecco’s Modified Eagle’s Medium (DMEM) without serum, with fetal bovine serum (FBS), or with human serum (HuS) in comparison to standard antimicrobial susceptibility testing conditions, represented by Cation-adjusted Mueller Hinton broth (CAMHB). PA14 twitching motility and pyoverdine production were evaluated under these conditions. ResultsFor the first time, our study reveals that culturing the highly virulentP. aeruginosaPA14 in host-mimicking media enhances the expression of multiple virulence therapeutic targets that are critical to host colonization and infection. RNA sequencing showed that multiple Type III Secretion (T3SS), Type I Secretion (T1SS), pyoverdine biosynthesis, uptake and efflux, and Type IV pili (T4P) initiation genes were promoted when PA14 was transitioned into host-mimicking conditions but remained unchanged when transitioned into standard AST conditions. Moreover, qPCR results disclosed that HuS and FBS delivered differential effects on the expression of membrane-associated virulence genes involved in host colonization. Our macroscopic PA14 twitching motility results aligned more closely with PA14 growth patterns than with virulence gene expression patterns. Our microtiter biofilm assay, however, revealed earlier biofilm formation in DMEM 0 than in AST conditions and both showed inhibited twitching motility in serum conditions. UV-Vis spectra showed that pyoverdine production aligned with our gene expression data, revealing higher pyoverdine production in serum conditions for planktonic PA14. DiscussionOverall, our findings support using host-mimicking conditions to improve the expression of candidate targets for anti-virulence therapeutics againstP. aeruginosaPA14 in a planktonic state. These recommendations may be broadly applicable for antivirulence therapeutic screening against multiple bacterial species at large. 

Pseudomonas aeruginosa is a ubiquitous, motile, gram-negative bacterium that has been recently identified as a multi-drug resistant pathogen in critical need of novel therapeutics. Of the approximately 5,000 strains, PAO1 and PA14 are common laboratory reference strains, modeling moderately and hyper-virulent phenotypes, respectively. PAO1 and PA14 have been instrumental in facilitating the discovery of novel drug targets, testing novel therapeutics, and supplying critical genomic information on the bacterium. While the two strains have contributed to a wide breadth of knowledge on the natural behaviors and therapeutic susceptibilities of P. aeruginosa , they have demonstrated significant deviations from observations in human infections. Many of these deviations are related to experimental inconsistencies in laboratory strain environment that complicate and, at times, terminate translation from laboratory results to clinical applications. This review aims to provide a comparative analysis of the two strains and potential methods to improve their clinical relevance. 

Capsules are one of the major solid dosage forms available in a variety of compositions and shapes. Developments in this dosage form are not new, but the production of non-gelatin capsules is a recent trend. In pharmaceutical as well as other biomedical research, alginate has great versatility. On the other hand, the use of inorganic material to enhance material strength is a common research topic in tissue engineering. The research presented here is a combination of qualities of alginate and montmorillonite (MMT). These two materials were used in this research to produce a soft non-gelatin modified-release capsule. Moreover, the research describes a facile benchtop production of these capsules. The produced capsules were critically analyzed for their appearance confirming resemblance with marketed capsules, functionality in terms of drug encapsulation, as well as release and durability. 

The immunopathology of chlamydial diseases is exacerbated by a broad-spectrum of inflammatory mediators, which we reported are inhibited by IL-10 in macrophages. However, the chlamydial protein moiety that induces the inflammatory mediators and the mechanisms by which IL-10 inhibits them are unknown. We hypothesized that Chlamydia major outer membrane protein (MOMP) mediates its disease pathogenesis, and the suppressor of cytokine signaling (SOCS)1 and SOCS3 proteins are mediators of the IL-10 inhibitory actions. Our hypothesis was tested by exposing mouse J774 macrophages to chlamydial stimulants (live Chlamydia muridarum and MOMP) with and without IL-10. MOMP significantly induced several inflammatory mediators (IL-6, IL-12p40, CCL5, CXCL10), which were dose-dependently inhibited by IL-10. Chlamydial stimulants induced the mRNA gene transcripts and protein expression of SOCS1 and SOCS3, with more SOCS3 expression. Notably, IL-10 reciprocally regulated their expression by reducing SOCS1 and increasing SOCS3. Specific inhibitions of MAPK pathways revealed that p38, JNK, and MEK1/2 are required for inducing inflammatory mediators as well as SOCS1 and SOCS3. Chlamydial stimulants triggered an M1 pro-inflammatory phenotype evidently by an enhanced nos2 (M1 marker) expression, which was skewed by IL-10 towards a more M2 anti-inflammatory phenotype by the increased expression of mrc1 and arg1 (M2 markers) and the reduced SOCS1/SOCS3 ratios. Neutralization of endogenously produced IL-10 augmented the secretion of inflammatory mediators, reduced SOCS3 expression, and skewed the chlamydial M1 to an M2 phenotype. Inhibition of proteasome degradation increased TNF but decreased IL-10, CCL5, and CXCL10 secretion by suppressing SOCS1 and SOCS3 expressions and dysregulating their STAT1 and STAT3 transcription factors. Our data show that SOCS1 and SOCS3 are regulators of IL-10 inhibitory actions, and underscore SOCS proteins as therapeutic targets for IL-10 control of inflammation for Chlamydia and other bacterial inflammatory diseases. 

Inflammation, as induced by the presence of cytokines and chemokines, is an integral part of chlamydial infections. The anti-inflammatory cytokine, interleukin (IL)-10, has been reported to efficiently suppress the secretion of inflammatory cytokines triggered by Chlamydia in mouse macrophages. Though IL-10 is employed in clinical applications, its therapeutic usage is limited due to its short half-life. Here, we document the successful encapsulation of IL-10 within the biodegradable polymeric nanoparticles of PLA-PEG (Poly (lactic acid)-Poly (ethylene glycol), to prolong its half-life. Our results show the encapsulated-IL-10 size (~238 nm), zeta potential (−14.2 mV), polydispersity index (0.256), encapsulation efficiency (~77%), and a prolonged slow release pattern up to 60 days. Temperature stability of encapsulated-IL-10 was favorable, demonstrating a heat capacity of up to 89 °C as shown by differential scanning calorimetry analysis. Encapsulated-IL-10 modulated the release of IL-6 and IL-12p40 in stimulated macrophages in a time- and concentration-dependent fashion, and differentially induced SOCS1 and SOCS3 as induced by chlamydial stimulants in macrophages. Our finding offers the tremendous potential for encapsulated-IL-10 not only for chlamydial inflammatory diseases but also biomedical therapeutic applications. 

Exosomes are small extracellular vesicles that have emerged as an important tool for intercellular communication. In the central nervous system, exosomes can mediate glia and neuronal communication. Once released from the donor cell, exosomes can act as discrete vesicles and travel to distant and proximal recipient cells to alter cellular function. Microglia cells secrete exosomes due to stress stimuli of alcohol abuse. The goal of this study was to investigate the effects of alcohol exposure on the biogenesis and composition of exosomes derived from microglia cell line BV-2. The BV-2 cells were cultured in exosome-free media and were either mock treated (control) or treated with 50 mM or 100 mM of alcohol for 48 and 72 h. Our results demonstrated that alcohol significantly impacted BV-2 cell morphology, viability, and protein content. Most importantly, our studies revealed that exosome biogenesis and composition was affected by alcohol treatment.