Multicomponent Co-Occurring Disorders Treatment and Wraparound Services for Individuals Experiencing Chronic Homelessness.

Individuals experiencing chronic homelessness have high rates of persistent co-occurring mental health and substance use disorders (COD), and they often have difficulty with service engagement and retention, resulting in symptom exacerbation and housing loss. This study pilot tested Maintaining Independence and Sobriety Through Systems Integration, Outreach and Networking (MISSION), a multicomponent wraparound treatment approach to improve COD symptoms and housing stability among individuals experiencing chronic homelessness and persistent COD. This open pilot study enrolled and assessed 109 individuals with a COD experiencing chronic homelessness and offered one year of MISSION. Statistically significant improvements were observed in behavioral health symptoms and functioning, days of illicit drug use, and housing stability. By treatment completion, 85% of participants were referred to social and behavioral supports. This pilot study demonstrates that MISSION helped to successfully engage participants in treatment, reduce substance use and mental health symptoms, and improve housing outcomes.

A Novel Loading Method for Doxycycline Liposomes for Intracellular Drug Delivery: Characterization of In Vitro and In Vivo Release Kinetics and Efficacy in a J774A.1 Cell Line Model of Mycobacterium smegmatis Infection.

Doxycycline (doxy) is used in treating intracellular and extracellular infections. Liposomal (LE) antibiotics allow low-frequency dosing and extended efficacy compared with standard (STD) formulations. We developed a novel sulfuric acid-loading method for doxycycline liposomes (LE-doxy). We hypothesized that a single s.c. injection of LE-doxy would be detectable in serum for at least 2 weeks at concentrations equal to or better than STD-doxy and would be bactericidal in an in vitro Mycobacterium smegmatis infection of J774A.1 macrophage cells. Liposomes were encapsulated by sulfuric acid gradient loading, and release kinetics were performed in vitro and in vivo. LE-doxy made using 8.25 mg/ml doxycycline loaded for 24 hours achieved 97.77% capture in 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 43.87% in sphingomyelin (sphing). Rats were injected s.c. with 50 mg/kg LE-doxy or 5 mg/kg STD-doxy, and serial blood samples were collected. Pharmacokinetics were analyzed using high-performance liquid chromatography. Liver and injection site skin samples were collected at euthanasia (4 weeks postinjection). Minimal histologic tissue reactions occurred after injection of STD (nonliposomal), DPPC, or sphing-doxy. DPPC-doxy had slightly faster in vitro leakage than sphing liposomes, although both were detectable at 264 hours. The mean residence time for DPPC was the highest (111.78 hours), followed by sphing (56.00 hours) and STD (6.86 hours). DPPC and sphing-doxy were detectable at 0.2 µg/ml in serum at 336 hours postadministration. LE-doxy was not toxic to J774A.1 cells in vitro and produced inhibition of viable Mycobacterium smegmatis at 24 and 48 hours. LE-doxy will require further testing in in vivo infection models.

Characterization of a novel heat shock protein (Hsp22.5) involved in the pathogenesis of Mycobacterium tuberculosis.

Tuberculosis is a worldwide health problem, given that one-third of the world's population is currently infected with Mycobacterium tuberculosis. Understanding the regulation of virulence on the molecular level will provide a better understanding of how M. tuberculosis can establish chronic infection. Using in vivo microarray analysis (IVMA), we previously identified a group of genes that are activated in BALB/c mouse lungs compared to in vitro cultures, including the rv0990c gene. Our analysis indicated that this gene is a member of the heat shock regulon and was activated under other stress conditions, including survival in macrophages or during the late phase of chronic tuberculosis in the murine lungs. Deletion of rv0990c from the genome of M. tuberculosis strain H37Rv affected the transcriptional profiles of many genes (n = 382) and operons involved in mycobacterial survival, including the dormancy regulon, ATP synthesis, respiration, protein synthesis, and lipid metabolism. Comparison of the proteomes of the mutant to those of the wild-type strain further confirmed the differential expression of 15 proteins, especially those involved in the heat shock response (e.g., DnaK and GrpE). Finally, the rv0990c mutant strain showed survival equivalent to that of the isogenic wild-type strain during active tuberculosis in guinea pigs, despite showing significant attenuation in BALB/c mice during the chronic phase of the disease. Overall, we suggest that rv0990c encodes a heat shock protein that plays an important role in mycobacterial virulence. Hence, we renamed rv0990c heat shock protein 22.5 (hsp22.5), reflecting its molecular mass.

mosR, a novel transcriptional regulator of hypoxia and virulence in Mycobacterium tuberculosis.

Latent tuberculosis represents a high-risk burden for one-third of the world population. Previous analysis of murine tuberculosis identified a novel transcriptional regulator encoded by Rv0348 that could control the establishment of persistent tuberculosis. Disruption of the Rv0348 gene from the genome of the virulent H37Rv strain of Mycobacterium tuberculosis revealed a global impact on the transcriptional profiles of 163 genes, including induction of the mammalian cell entry (mce1) operon and the repression of a significant number of genes involved in hypoxia and starvation responses. Nonetheless, gel shift assays did not reveal direct binding between Rv0348 and a set of regulated promoters, suggesting an indirect regulatory role. However, when expressed in Mycobacterium smegmatis, the Rv0348 transcripts were significantly responsive to different levels of hypoxia and the encoded protein was shown to regulate genes involved in hypoxia [e.g., Rv3130c (tgs1)] and intracellular survival (e.g., mce1), among other genes. Interestingly, the colonization level of the DeltamosR mutant strain was significantly lower than that of the wild-type strain of M. tuberculosis, suggesting its attenuation in the murine model of tuberculosis. Taken together, our analyses indicated that the Rv0348 gene encodes a novel transcriptional factor that regulates several operons involved in mycobacterial survival, especially during hypoxia; hence, we propose that Rv0348 be renamed mosR for regulator of mycobacterial operons of survival.

Congenital Trypanosoma cruzi transmission in Santa Cruz, Bolivia.

BACKGROUND: We conducted a study of congenital Trypanosoma cruzi infection in Santa Cruz, Bolivia. Our objective was to apply new tools to identify weak points in current screening algorithms, and find ways to improve them. METHODS: Women presenting for delivery were screened by rapid and conventional serological tests. For infants of infected mothers, blood specimens obtained on days 0, 7, 21, 30, 90, 180, and 270 were concentrated and examined microscopically; serological tests were performed for the day 90, 180, and 270 specimens. Maternal and infant specimens, including umbilical tissue, were tested by polymerase chain reaction (PCR) targeting the kinetoplast minicircle and by quantitative PCR. RESULTS: Of 530 women, 154 (29%) were seropositive. Ten infants had congenital T. cruzi infection. Only 4 infants had positive results of microscopy evaluation in the first month, and none had positive cord blood microscopy results. PCR results were positive for 6 (67%) of 9 cord blood and 7 (87.5%) of 8 umbilical tissue specimens. PCR-positive women were more likely to transmit T. cruzi than were seropositive women with negative PCR results (P < .05). Parasite loads determined by quantitative PCR were higher for mothers of infected infants than for seropositive mothers of uninfected infants P < .01). Despite intensive efforts, only 58% of at-risk infants had a month 9 specimen collected. CONCLUSIONS: On the basis of the low sensitivity of microscopy in cord blood and high rate of loss to follow-up, we estimate that current screening programs miss one-half of all infected infants. Molecular techniques may improve early detection.

Neuroinflammation-induced lymphangiogenesis near the cribriform plate contributes to drainage of CNS-derived antigens and immune cells.

There are no conventional lymphatic vessels within the CNS parenchyma, although it has been hypothesized that lymphatics near the cribriform plate or dura maintain fluid homeostasis and immune surveillance during steady-state conditions. However, the role of these lymphatic vessels during neuroinflammation is not well understood. We report that lymphatic vessels near the cribriform plate undergo lymphangiogenesis in a VEGFC - VEGFR3 dependent manner during experimental autoimmune encephalomyelitis (EAE) and drain both CSF and cells that were once in the CNS parenchyma. Lymphangiogenesis also contributes to the drainage of CNS derived antigens that leads to antigen specific T cell proliferation in the draining lymph nodes during EAE. In contrast, meningeal lymphatics do not undergo lymphangiogenesis during EAE, suggesting heterogeneity in CNS lymphatics. We conclude that increased lymphangiogenesis near the cribriform plate can contribute to the management of neuroinflammation-induced fluid accumulation and immune surveillance.

Publisher Correction: Genomic analysis of the emergence of drug-resistant strains of Mycobacterium tuberculosis in the Middle East.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Genomic analysis of the emergence of drug-resistant strains of Mycobacterium tuberculosis in the Middle East.

Tuberculosis (TB) represents a significant challenge to public health authorities, especially with the emergence of drug-resistant (DR) and multidrug-resistant (MDR) isolates of Mycobacterium tuberculosis. We sought to examine the genomic variations among recently isolated strains of M. tuberculosis in two closely related countries with different population demography in the Middle East. Clinical isolates of M. tuberculosis from both Egypt and Saudi Arabia were subjected to phenotypic and genotypic analysis on gene and genome-wide levels. Isolates with MDR phenotypes were highly prevalent in Egypt (up to 35%) despite its relatively stable population structure (sympatric pattern). MDR-TB isolates were not identified in the isolates from Saudi Arabia despite its active guest worker program (allopatric pattern). However, tuberculosis isolates from Saudi Arabia, where lineage 4 was more prevalent (>65%), showed more diversity than isolates from Egypt, where lineage 3 was the most prevalent (>75%). Phylogenetic and molecular dating analyses indicated that lineages from Egypt were recently diverged (~78 years), whereas those from Saudi Arabia were diverged by over 200 years. Interestingly, DR isolates did not appear to cluster together or spread more widely than drug-sensitive isolates, suggesting poor treatment as the main cause for emergence of drug resistance rather than more virulence or more capacity to persist.

Volatilization and precipitation of tellurium by aerobic, tellurite-resistant marine microbes.

Microbial resistance to tellurite, an oxyanion of tellurium, is widespread in the biosphere, but the geochemical significance of this trait is poorly understood. As some tellurite resistance markers appear to mediate the formation of volatile tellurides, the potential contribution of tellurite-resistant microbial strains to trace element volatilization in salt marsh sediments was evaluated. Microbial strains were isolated aerobically on the basis of tellurite resistance and subsequently examined for their capacity to volatilize tellurium in pure cultures. The tellurite-resistant strains recovered were either yeasts related to marine isolates of Rhodotorula spp. or gram-positive bacteria related to marine strains within the family Bacillaceae based on rRNA gene sequence comparisons. Most strains produced volatile tellurides, primarily dimethyltelluride, though there was a wide range of the types and amounts of species produced. For example, the Rhodotorula spp. produced the greatest quantities and highest diversity of volatile tellurium compounds. All strains also produced methylated sulfur compounds, primarily dimethyldisulfide. Intracellular tellurium precipitates were a major product of tellurite metabolism in all strains tested, with nearly complete recovery of the tellurite initially provided to cultures as a precipitate. Different strains appeared to produce different shapes and sizes of tellurium containing nanostructures. These studies suggest that aerobic marine yeast and Bacillus spp. may play a greater role in trace element biogeochemistry than has been previously assumed, though additional work is needed to further define and quantify their specific contributions.

Atrazine exposure affects longevity, development time and body size in Drosophila melanogaster.

Atrazine is the one of the most widely used herbicides in the United States and non-target organisms may encounter it in the environment. Atrazine is known to affect male reproduction in both vertebrates and invertebrates but less is known about its effects on other fitness traits. Here we assessed the effects of five different chronic exposure levels on a variety of fitness traits in Drosophila melanogaster. We measured male and female longevity, development time, proportion pupated, proportion emerged, body size, female mating rate, fertility and fecundity. Atrazine exposure decreased the proportion pupated, the proportion emerged and adult survival. Development time was also affected by atrazine and exposed flies pupated and emerged earlier than controls. Although development time was accelerated, body size was actually larger in some of the exposures. Atrazine exposure had no effect on female mating rate and the effects on female fertility and fecundity were only observed in one of the two independent experimental blocks. Many of the traits showed non-monotonic dose response curves, where the intermediate concentrations showed the largest effects. Overall this study shows that atrazine influences a variety of life history traits in the model genetic system, D. melanogaster, and future studies should aim to identify the molecular mechanisms of toxicity.

CsoR Is Essential for Maintaining Copper Homeostasis in Mycobacterium tuberculosis.

Mycobacterium tuberculosis, a pathogen infecting one third of the world population, faces numerous challenges within the host, including high levels of copper. We have previously shown that M. tuberculosis CsoR is a copper inducible transcriptional regulator. Here we examined the hypothesis that csoR is necessary for maintaining copper homeostasis and surviving under various stress conditions. With an unmarked csoR knockout strain, we were able to characterize the role of csoR in M. tuberculosis as it faced copper and host stress. Growth under high levels of copper demonstrated that M. tuberculosis survives copper stress significantly better in the absence of csoR. Yet under minimal levels of copper, differential expression analysis revealed that the loss of csoR results in a cell wide hypoxia-type stress response with the induction of the DosR regulon. Despite the stress placed on M. tuberculosis by the loss of csoR, survival of the knockout strain was increased compared to wild type during the early chronic stages of mouse infection, suggesting that csoR could play an active role in modulating M. tuberculosis fitness within the host. Overall, analysis of CsoR provided an increased understanding of the M. tuberculosis copper response with implications for other intracellular pathogens harboring CsoR.

Protection by novel vaccine candidates, Mycobacterium tuberculosis ΔmosR and ΔechA7, against challenge with a Mycobacterium tuberculosis Beijing strain.

Mycobacterium tuberculosis, the etiological agent of tuberculosis (TB), infects over two billion people, claiming around 1.5 million lives annually. The only vaccine approved for clinical use against this disease is the Bacillus Calmette-Guérin (BCG) vaccine. Unfortunately, BCG has limited efficacy against the adult, pulmonary form of tuberculosis. This vaccine was developed from M. bovis with antigen expression and host specificity that differ from M. tuberculosis. To address these problems, we have designed two novel, live attenuated vaccine (LAV) candidates on an M. tuberculosis background: ΔmosR and ΔechA7. These targeted genes are important to M. tuberculosis pathogenicity during infection. To examine the efficacy of these strains, C57BL/6 mice were vaccinated subcutaneously with either LAV, BCG, or PBS. Both LAV strains persisted up to 16 weeks in the spleens or lungs of vaccinated mice, while eliciting minimal pathology prior to challenge. Following challenge with a selected, high virulence M. tuberculosis Beijing strain, protection was notably greater for both groups of LAV vaccinated animals as compared to BCG at both 30 and 60 days post-challenge. Additionally, vaccination with either ΔmosR or ΔechA7 elicited an immune response similar to BCG. Although these strains require further development to meet safety standards, this first evidence of protection by these two new, live attenuated vaccine candidates shows promise.

Virtual Screening for UDP-Galactopyranose Mutase Ligands Identifies a New Class of Antimycobacterial Agents.

Galactofuranose (Galf) is present in glycans critical for the virulence and viability of several pathogenic microbes, including Mycobacterium tuberculosis, yet the monosaccharide is absent from mammalian glycans. Uridine 5'-diphosphate-galactopyranose mutase (UGM) catalyzes the formation of UDP-Galf, which is required to produce Galf-containing glycoconjugates. Inhibitors of UGM have therefore been sought, both as antimicrobial leads and as tools to delineate the roles of Galf in cells. Obtaining cell permeable UGM probes by either design or high throughput screens has been difficult, as has elucidating how UGM binds small molecule, noncarbohydrate inhibitors. To address these issues, we employed structure-based virtual screening to uncover new inhibitor chemotypes, including a triazolothiadiazine series. These compounds are among the most potent antimycobacterial UGM inhibitors described. They also facilitated determination of a UGM-small molecule inhibitor structure, which can guide optimization. A comparison of results from the computational screen and a high-throughput fluorescence polarization (FP) screen indicated that the scaffold hits from the former had been evaluated in the FP screen but missed. By focusing on promising compounds, the virtual screen rescued false negatives, providing a blueprint for generating new UGM probes and therapeutic leads.

Bulk elastic moduli and solute potentials in leaves of freshwater, coastal and marine hydrophytes. Are marine plants more rigid?

Bulk modulus of elasticity (ɛ), depicting the flexibility of plant tissues, is recognized as an important component in maintaining internal water balance. Elevated ɛ and comparatively low osmotic potential (Ψπ) may work in concert to effectively maintain vital cellular water content. This concept, termed the 'cell water conservation hypothesis', may foster tolerance for lower soil-water potentials in plants while minimizing cell dehydration and shrinkage. Therefore, the accumulation of solutes in marine plants, causing decreases in Ψπ, play an important role in plant-water relations and likely works with higher ɛ to achieve favourable cell volumes. While it is generally held that plants residing in marine systems have higher leaf tissue ɛ, to our knowledge no study has specifically addressed this notion in aquatic and wetland plants residing in marine and freshwater systems. Therefore, we compared ɛ and Ψπ in leaf tissues of 38 freshwater, coastal and marine plant species using data collected in our laboratory, with additional values from the literature. Overall, 8 of the 10 highest ɛ values were observed in marine plants, and 20 of the lowest 25 ɛ values were recorded in freshwater plants. As expected, marine plants often had lower Ψπ, wherein the majority of marine plants were below -1.0 MPa and the majority of freshwater plants were above -1.0 MPa. While there were no differences among habitat type and symplastic water content (θsym), we did observe higher θsym in shrubs when compared with graminoids, and believe that the comparatively low θsym observed in aquatic grasses may be attributed to their tendency to develop aerenchyma that hold apoplastic water. These results, with few exceptions, support the premise that leaf tissues of plants acclimated to marine environments tend to have higher ɛ and lower Ψπ, and agree with the general tenets of the cell water conservation hypothesis.

Robustness of Helicobacter pylori infection conferred by context-variable redundancy among cysteine-rich paralogs.

Deletion of single genes from expanded gene families in bacterial genomes often does not elicit a phenotype thus implying redundancy or functional non-essentiality of paralogous genes. The molecular mechanisms that facilitate evolutionary maintenance of such paralogs despite selective pressures against redundancy remain mostly unexplored. Here, we investigate the evolutionary, genetic, and functional interaction between the Helicobacter pylori cysteine-rich paralogs hcpG and hcpC in the context of H. pylori infection of cultured mammalian cells. We find that in natural H. pylori populations both hcpG and hcpC are maintained by positive selection in a dual genetic relationship that switches from complete redundancy during early infection, whereby ΔhcpC or ΔhcpG mutants themselves show no growth defect but a significant growth defect is seen in the ΔhcpC,ΔhcpG double mutant, to quantitative redundancy during late infection wherein the growth defect of the ΔhcpC mutant is exacerbated in the ΔhcpC,ΔhcpG double mutant although the ΔhcpG mutant itself shows no defect. Moreover, during early infection both hcpG and hcpC are essential for optimal translocation of the H. pylori HspB/GroEL chaperone, but during middle-to-late infection hcpC alone is necessary and sufficient for HspB/GroEL translocation thereby revealing the lack of functional compensation among paralogs. We propose that evolution of context-dependent differences in the nature of genetic redundancy, and function, between hcpG and hcpC may facilitate their maintenance in H. pylori genomes, and confer robustness to H. pylori growth during infection of cultured mammalian cells.

Transcriptional profiling of mycobacterium tuberculosis during infection: lessons learned.

Infection with Mycobacterium tuberculosis, the causative agent of tuberculosis, is considered one of the biggest infectious disease killers worldwide. A significant amount of attention has been directed toward revealing genes involved in the virulence and pathogenesis of this air-born pathogen. With the advances in technologies for transcriptional profiling, several groups, including ours, took advantage of DNA microarrays to identify transcriptional units differentially regulated by M. tuberculosis within a host. The main idea behind this approach is that pathogens tend to regulate their gene expression levels depending on the host microenvironment, and preferentially express those needed for survival. Identifying this class of genes will improve our understanding of pathogenesis. In our case, we identified an in vivo expressed genomic island that was preferentially active in murine lungs during early infection, as well as groups of genes active during chronic tuberculosis. Other studies have identified additional gene groups that are active during macrophage infection and even in human lungs. Despite all of these findings, one of the lingering questions remaining was whether in vivo expressed transcripts are relevant to the virulence, pathogenesis, and persistence of the organism. The work of our group and others addressed this question by examining the contribution of in vivo expressed genes using a strategy based on gene deletions followed by animal infections. Overall, the analysis of most of the in vivo expressed genes supported a role of these genes in M. tuberculosis pathogenesis. Further, these data suggest that in vivo transcriptional profiling is a valid approach to identify genes required for bacterial pathogenesis.