Neurocognitive Sequelae and Rehabilitation after Subarachnoid Hemorrhage: Optimizing Outcomes.

Subarachnoid hemorrhage (SAH) is a medical emergency that requires immediate intervention. The etiology varies between cases; however, rupture of an intracranial aneurysm accounts for 80% of medical emergencies. Early intervention and treatment are essential to prevent long-term complications. Over the years, treatment of SAH has drastically improved, which is responsible for the rapid rise in SAH survivors. Post-SAH, a significant number of patients exhibit impairments in memory and executive function and report high rates of depression and anxiety that ultimately affect daily living, return to work, and quality of life. Given the rise in SAH survivors, rehabilitation post-SAH to optimize patient outcomes becomes crucial. The review addresses the current rehabilitative strategies to combat the neurocognitive and behavioral issues that may arise following SAH.

Active Hexose-Correlated Compound Restores Gene Expression and Protein Secretion of Protective Cytokines of Immune Cells in a Murine Stress Model during Chlamydia muridarum Genital Infection.

Chlamydia trachomatis genital infection is the most common bacterial sexually transmitted disease worldwide. Previously, we reported that cold-induced stress results in immune suppression of mice that subsequently leads to increased intensity of Chlamydia muridarum genital infection. Furthermore, we demonstrated that stressed mice orally fed with active hexose-correlated compound (AHCC) have reduced shedding of C. muridarum from the genital tract. However, the mechanism of AHCC in reducing the organ load and changing the immune response in the stress model is not well known. This study evaluated infection and changes in immunological parameters of stressed AHCC-fed mice with or without C. muridarum genital infection. We hypothesized that AHCC feeding to stressed mice restores protective immune function and reduces susceptibility to C. muridarum genital infection. The results show that oral feeding of stressed mice with AHCC resulted in decreased shedding of C. muridarum from the genital tract, reduced production of plasma catecholamines, increased expression of T-bet and reduced GATA-3 in CD4+ T cells, increased production of interleukin-12 (IL-12) and interferon gamma (IFN-γ) and reduced production of IL-4 in CD4+ T cells, and enhanced expression of surface markers and costimulatory molecules of CD4+ T cells, bone marrow-derived dendritic cells (BMDCs), and natural killer cells. Coculturing of mature BMDCs with splenic CD4+ T cells led to the increased and decreased production of T helper 1 and T helper 2 cytokines, respectively. Overall, our results show that AHCC fosters the restoration of Th1 cytokine production while reducing Th2 cytokine production, which would promote C. muridarum clearance in the murine stress model.

Culture of Small Colony Variant of Pseudomonas aeruginosa and Quantitation of its Alginate.

Pseudomonas aeruginosa, an opportunistic Gram-negative bacterial pathogen, can overproduce an exopolysaccharide alginate resulting in a unique phenotype called mucoidy. Alginate is linked to chronic lung infections resulting in poor prognosis in patients with cystic fibrosis (CF). Understanding the pathways that regulate the production of alginate can aid in the development of novel therapeutic strategies targeting the alginate formation. Another disease-related phenotype is the small colony variant (SCV). SCV is due to the slow growth of bacteria and often associated with increased resistance to antimicrobials. In this paper, we first show a method of culturing a genetically defined form of P. aeruginosa SCV due to pyrimidine biosynthesis mutations. Supplementation of nitrogenous bases, uracil or cytosine, returns the normal growth to these mutants, demonstrating the presence of a salvage pathway that scavenges free bases from the environment. Next, we discuss two methods for the measurement of bacterial alginate. The first method relies on the hydrolysis of the polysaccharide to its uronic acid monomer followed by derivatization with a chromogenic reagent, carbazole, while the second method uses an ELISA based on a commercially available, alginate-specific mAb. Both methods require a standard curve for quantitation. We also show that the immunological method is specific for alginate quantification and may be used for the measurement of alginate in the clinical specimens.

Generation of In-Frame Gene Deletion Mutants in Pseudomonas aeruginosa and Testing for Virulence Attenuation in a Simple Mouse Model of Infection.

Microorganisms are genetically versatile and diverse and have become a major source of many commercial products and biopharmaceuticals. Though some of these products are naturally produced by the organisms, other products require genetic engineering of the organism to increase the yields of production. Avirulent strains of Escherichia coli have traditionally been the preferred bacterial species for producing biopharmaceuticals; however, some products are difficult for E. coli to produce. Thus, avirulent strains of other bacterial species could provide useful alternatives for production of some commercial products. Pseudomonas aeruginosa is a common and well-studied Gram-negative bacterium that could provide a suitable alternative to E. coli. However, P. aeruginosa is an opportunistic human pathogen. Here, we detail a procedure that can be used to generate nonpathogenic strains of P. aeruginosa through sequential genomic deletions using the pEX100T-NotI plasmid. The main advantage of this method is to produce a marker-free strain. This method may be used to generate highly attenuated P. aeruginosa strains for the production of commercial products, or to design strains for other specific uses. We also describe a simple and reproducible mouse model of bacterial systemic infection via intraperitoneal injection of validated test strains to test the attenuation of the genetically engineered strain in comparison to the FDA-approved BL21 strain of E. coli.

Generation of a highly attenuated strain of Pseudomonas aeruginosa for commercial production of alginate.

Alginate is an important polysaccharide that is commonly used as a gelling agent in foods, cosmetics and healthcare products. Currently, all alginate used commercially is extracted from brown seaweed. However, with environmental changes such as increasing ocean temperature and the increasing number of biotechnological uses of alginates with specific properties, there is an emerging need for more reliable and customizable sources of alginate. An alternative to seaweed for alginate production is Pseudomonas aeruginosa, a common Gram-negative bacterium that can form alginate-containing biofilms. However, P. aeruginosa is an opportunistic pathogen that can cause life-threatening infections in immunocompromised patients. Therefore, we sought to engineer a non-pathogenic P. aeruginosa strain that is safe for commercial production of alginate. Using a homologous recombination strategy, we sequentially deleted five key pathogenicity genes from the P. aeruginosa chromosome, resulting in the marker-free strain PGN5. Intraperitoneal injection of mice with PGN5 resulted in 0% mortality, while injection with wild-type P. aeruginosa resulted in 95% mortality, providing evidence that the systemic virulence of PGN5 is highly attenuated. Importantly, PGN5 produces large amounts of alginate in response to overexpression of MucE, an activator of alginate biosynthesis. The alginate produced by PGN5 is structurally identical to alginate produced by wild-type P. aeruginosa, indicating that the alginate biosynthetic pathway remains functional in this modified strain. The genetic versatility of P. aeruginosa will allow us to further engineer PGN5 to produce alginates with specific chemical compositions and physical properties to meet different industrial and biomedical needs.

Efficacy of Aerosolized Rifaximin versus Tobramycin for Treatment of Pseudomonas aeruginosa Pneumonia in Mice.

Pseudomonas aeruginosa is a Gram-negative opportunistic bacterial pathogen that can cause chronic lung infections in patients with cystic fibrosis (CF). The current preferred treatment for CF lung infections includes inhaled tobramycin (TOB); however, studies suggest TOB cannot effectively inhibit biofilm formation. Using an NIH small compounds drug library approved for safe use in humans, we identified rifaximin (RFX), a semisynthetic, rifamycin family, nonsystemic antibiotic that inhibits alginate production and growth in P. aeruginosa Inhibition of alginate production was further analyzed using the uronic acid carbazole assay and a promoter reporter assay that measures the transcription of the alginate biosynthetic operon. Compared to TOB, RFX significantly reduced alginate production in laboratory and CF sputum isolates of P. aeruginosa In addition, RFX showed a narrow range of MICs when measured with multidrug-resistant bacterial species of clinical relevance, synergistic activities with TOB or amikacin against clinical isolates, as well as reduction toward in vitro preformed biofilms. In C57BL/6 mice, penetration of nebulized TOB into the lungs was shown at a higher level than that of RFX. Further, in vivo assessment using a DBA/2 mouse lung infection model found increased survival rates with a single-dose treatment of nebulized RFX and decreased P. aeruginosa PAO1 bioburden with a multiple-dose treatment of RFX plus TOB. In addition, mice treated with a single exposure to dimethyl sulfoxide (DMSO), a solvent that dissolves RFX, showed no apparent toxicity. In summary, RFX may be used to supplement TOB inhalation therapy to increase efficacy against P. aeruginosa biofilm infections.

Pyrimidine Biosynthesis Regulates the Small-Colony Variant and Mucoidy in Pseudomonas aeruginosa through Sigma Factor Competition.

Mucoidy due to alginate overproduction by the Gram-negative bacterium Pseudomonas aeruginosa facilitates chronic lung infections in patients with cystic fibrosis (CF). We previously reported that disruption in de novo synthesis of pyrimidines resulted in conversion to a nonmucoid small-colony variant (SCV) in the mucoid P. aeruginosa strain (PAO581), which has a truncated anti-sigma factor, MucA25, that cannot sequester sigma factor AlgU (AlgT). Here, we showed that supplementation with the nitrogenous bases uracil or cytosine in growth medium complemented the SCV to normal growth, and nonmucoidy to mucoidy, in these mucA25 mutants. This conversion was associated with an increase in intracellular levels of UMP and UTP suggesting that nucleotide restoration occurred via a salvage pathway. In addition, supplemented pyrimidines caused an increase in activity of the alginate biosynthesis promoter (P algD ), but had no effect on P algU , which controls transcription of algU Cytosolic levels of AlgU were not influenced by uracil supplementation, yet levels of RpoN, a sigma factor that regulates nitrogen metabolism, increased with disruption of pyrimidine synthesis and decreased after supplementation of uracil. This suggested that an elevated level of RpoN in SCV may block alginate biosynthesis. To support this, we observed that overexpressing rpoN resulted in a phenotypic switch to nonmucoidy in PAO581 and in mucoid clinical isolates. Furthermore, transcription of an RpoN-regulated promoter increased in the mutants and decreased after uracil supplementation. These results suggest that the balance of RpoN and AlgU levels may regulate growth from SCV to mucoidy through sigma factor competition for P algDIMPORTANCE Chronic lung infections with P. aeruginosa are the main cause of morbidity and mortality in patients with cystic fibrosis. This bacterium overproduces a capsular polysaccharide called alginate (also known as mucoidy), which aids in bacterial persistence in the lungs and in resistance to therapeutic regimens and host immune responses. The current study explores a previously unknown link between pyrimidine biosynthesis and mucoidy at the level of transcriptional regulation. Identifying/characterizing this link could provide novel targets for the control of bacterial growth and mucoidy. Inhibiting mucoidy may improve antimicrobial efficacy and facilitate host defenses to clear the noncapsulated P. aeruginosa bacteria, leading to improved prognosis for patients with cystic fibrosis.

In vivo time-lapse imaging shows dynamic oligodendrocyte progenitor behavior during zebrafish development.

Myelinating oligodendrocytes arise from migratory and proliferative oligodendrocyte progenitor cells (OPCs). Complete myelination requires that oligodendrocytes be uniformly distributed and form numerous, periodically spaced membrane sheaths along the entire length of target axons. Mechanisms that determine spacing of oligodendrocytes and their myelinating processes are not known. Using in vivo time-lapse confocal microscopy, we show that zebrafish OPCs continuously extend and retract numerous filopodium-like processes as they migrate and settle into their final positions. Process remodeling and migration paths are highly variable and seem to be influenced by contact with neighboring OPCs. After laser ablation of oligodendrocyte-lineage cells, nearby OPCs divide more frequently, orient processes toward the ablated cells and migrate to fill the unoccupied space. Thus, process activity before axon wrapping might serve as a surveillance mechanism by which OPCs determine the presence or absence of nearby oligodendrocyte-lineage cells, facilitating uniform spacing of oligodendrocytes and complete myelination.