Nα-Acetylation of the virulence factor EsxA is required for mycobacterial cytosolic translocation and virulence.

The Mycobacterium tuberculosis virulence factor EsxA and its chaperone EsxB are secreted as a heterodimer (EsxA:B) and are crucial for mycobacterial escape from phagosomes and cytosolic translocation. Current findings support the idea that for EsxA to interact with host membranes, EsxA must dissociate from EsxB at low pH. However, the molecular mechanism by which the EsxA:B heterodimer separates is not clear. In the present study, using liposome-leakage and cytotoxicity assays, LC-MS/MS-based proteomics, and CCF-4 FRET analysis, we obtained evidence that the Nα-acetylation of the Thr-2 residue on EsxA, a post-translational modification that is present in mycobacteria but absent in Escherichia coli, is required for the EsxA:B separation. Substitutions at Thr-2 that precluded Nα-acetylation inhibited the heterodimer separation and hence prevented EsxA from interacting with the host membrane, resulting in attenuated mycobacterial cytosolic translocation and virulence. Molecular dynamics simulations revealed that at low pH, the Nα-acetylated Thr-2 makes direct and frequent "bind-and-release" contacts with EsxB, which generates a force that pulls EsxB away from EsxA. In summary, our findings provide evidence that the Nα-acetylation at Thr-2 of EsxA facilitates dissociation of the EsxA:B heterodimer required for EsxA membrane permeabilization and mycobacterial cytosolic translocation and virulence.

Characterization of interactions between heparin/glycosaminoglycan and adeno-associated virus.

Adeno-associated virus (AAV) is a key candidate in the development of gene therapy. In this work, we used surface plasmon resonance spectroscopy to study the interaction between AAV and heparin and other glycosaminoglycans (GAGs). Surface plasmon resonance results revealed that heparin binds to AAV with an extremely high affinity. Solution competition studies showed that binding of AAV to heparin is chain length-dependent. AAV prefers to bind full chain heparin. All sulfo groups (especially N-sulfo and 6-O-sulfo groups) on heparin are important for the AAV-heparin interaction. Higher levels of sulfo group substitution in GAGs enhance their binding affinities. Atomic force microscopy was also performed to image AAV-2 in a complex with heparin.

Critical access medication for opioid use disorder (MOUD) treatment facilities in the continental United States.

Research objective: Medication opioid use disorder (MOUD) treatment is the first-line approach to the treatment of opioid use disorder (OUD). This analysis seeks to identify "critical access" MOUD facilities that ensure geographic access for MOUD patients. Using public-source data and spatial analysis, we identify the top 100 "critical access" MOUD units across the continental U.S. Study design: We use locational data from SAMHSA's Behavioral Health Treatment Services Locator and DATA 2000 waiver buprenorphine providers. We identify the closest MOUDs to each ZIP Code Tabulation Area (ZCTA)'s geographic centroid. We then construct a difference-in-distance metric by computing the difference in this distance measure between closest and second-closest MOUD, multiplied by ZCTA population, ranking MOUDs by difference-distance scores. Population studied: All listed MOUD treatment facilities and all listed ZCTA's across the continental U.S., and all listed MOUD providers proximate to these areas. Principal findings: We identified the top 100 critical access MOUD units in the continental United States. Many critical providers were in rural areas in the central United States, as well as a band extending east from Texas to Georgia. Twenty-three of the top 100 critical access providers were identified as providing naltrexone. Seventy-seven were identified as providing buprenorphine. Three were identified as providing methadone. Conclusions: Significant areas of the United States are dependent on a single critical access MOUD provider. Implications for policy or practice: Place-based supports may be warranted to support MOUD treatment access in areas dependent upon critical access providers.

Social-spatial network structures among young urban and suburban persons who inject drugs in a large metropolitan area.

Background: It is estimated that there are 1.5% US adult population who inject drugs in 2018, with young adults aged 18-39 showing the highest prevalence. PWID are at a high risk of many blood-borne infections. Recent studies have highlight the importance of employing the syndemic approach to study opioid misuse, overdose, HCV and HIV, along with the social and environmental contexts where these interrelated epidemics occur in already marginalized communities. Social interactions and spatial contexts are important structural factors that are understudied. Methods: Egocentric injection network and geographic activity spaces for young (aged 18-30) PWID and their injection, sexual, and social support network members (i.e., where reside, inject drugs, purchase drugs, and meet sex partners) were examined using baseline data from an ongoing longitudinal study (n=258). Participants were stratified based on the location of all place(s) of residence in the past year i.e., urban, suburban, and transient (both urban and suburban) to i) elucidate geospatial concentration of risk activities within multi-dimensional risk environments based on kernel density estimates; and ii) examine spatialized social networks for each residential group. Results: Participants were mostly non-Hispanic white (59%); 42% were urban residents, 28% suburban, and 30% transient. We identified a spatial area with concentrated risky activities for each residence group on the West side of Chicago where a large outdoor drug market area is located. The urban group (80%) reported a smaller concentrated area (14 census tracts) compared to the transient (93%) and suburban (91%) with 30 and 51 tracts, respectively. Compared to other areas in Chicago, the identified area had significantly higher neighborhood disadvantages (e.g., higher poverty rate, p <0.001). Significant ( p <0.01 for all) differences were observed in social network structures: suburban had the most homogenous network in terms of age and residence, transient participants had the largest network (degree) and more non-redundant connections. Conclusion: We identified concentrated risk activity spaces among PWID from urban, suburban, and transient groups in a large outdoor urban drug market area, which highlights the need for considering the role of risk spaces and social networks in addressing the syndemics in PWID populations.

Social-spatial network structures among young urban and suburban persons who inject drugs in a large metropolitan area.

BACKGROUND: Recent studies underscore the significance of adopting a syndemics approach to study opioid misuse, overdose, hepatitis C (HCV) and HIV infections, within the broader context of social and environmental contexts in already marginalized communities. Social interactions and spatial contexts are crucial structural factors that remain relatively underexplored. This study examines the intersections of social interactions and spatial contexts around injection drug use. More specifically, we investigate the experiences of different residential groups among young (aged 18-30) people who inject drugs (PWID) regarding their social interactions, travel behaviors, and locations connected to their risk behaviors. By doing so, we aim to achieve a more comprehensive understanding of the multidimensional risk environment, thereby facilitating the development of informed policies. METHODS: We collected and examined data regarding young PWID's egocentric injection network and geographic activity spaces (i.e., where they reside, inject drugs, purchase drugs, and meet sex partners). Participants were stratified based on the location of all place(s) of residence in the past year i.e., urban, suburban, and transient (both urban and suburban) to i) elucidate geospatial concentration of risk activities within multidimensional risk environments based on kernel density estimates; and ii) examine spatialized social networks for each residential group. RESULTS: Participants were mostly non-Hispanic white (59%); 42% were urban residents, 28% suburban, and 30% transient. We identified a spatial area with concentrated risky activities for each residential group on the West side of Chicago in Illinois where a large outdoor drug market area is located. The urban group (80%) reported a smaller concentrated area (14 census tracts) compared to the transient (93%) and suburban (91%) with 30 and 51 tracts, respectively. Compared to other areas in Chicago, the identified area had significantly higher neighborhood disadvantages. Significant differences were observed in social network structures and travel behaviors: suburban participants had the most homogenous network in terms of age and residence, transient participants had the largest network (degree) and more non-redundant connections, while the urban group had the shortest travel distance for all types of risk activities. CONCLUSION: Distinct residential groups exhibit varying patterns of network interaction, travel behaviors, and geographical contexts related to their risk behaviors. Nonetheless, these groups share common concentrated risk activity spaces in a large outdoor urban drug market area, underscoring the significance of accounting for risk spaces and social networks in addressing syndemics within PWID populations.

Measuring Cytosolic Translocation of Mycobacterium marinum in RAW264.7 Macrophages with a CCF4-AM FRET Assay.

The CCF4-AM Förster resonance energy transfer (FRET) assay is a sensitive approach to measure bacterial cytosolic translocation in live cells. The FRET pair hydroxycoumarin (donor) and fluorescein (acceptor) are linked by a CCF4-AM ß-lactam ring, the substrate of ß-lactamase. The exogenously added, neutral charged-FRET reagent can diffuse across the membrane and stay in the cytosol only once it is charged in the cytosol. When bacteria translocate from subcellular organelles (e.g., phagosomes) to the cytosol, the bacteria-associated ß-lactamase cleaves the ß-lactam ring, resulting in loss of FRET signal. Here we describe the fluorometer-based approach optimized for direct measurement of cytosolic translocation as a result of the EsxAB complex of Mycobacterium marinum in RAW264.7 cells.

A Fluorescence Dequenching-based Liposome Leakage Assay to Measure Membrane Permeabilization by Pore-forming Proteins.

Pore-forming toxins (PFTs) have been discovered in a wide range of organisms. Their functions are essential to the survival or virulence of many species. PFTs often interact with lipid membranes. Large unilamellar vesicles (LUV), also known as liposomes, have been commonly used as reliable membrane models for testing PFTs activity. Liposomes have great adaptability in size, lipid composition, and loading cargo. Incorporating the fluorescent dye/quencher pair, 8-Aminonaphthalene-1,3,6-Trisulfonic Acid (ANTS) and p-Xylene-Bis-Pyridinium Bromide (DPX), in liposomes is an effective approach for measuring membrane leakage. When ANTS and DPX are encapsulated in a liposome, the fluorescence of ANTS is quenched by DPX. However, disruption of liposome integrity and subsequent leakage result in measurable fluorescence emitted by ANTS. Here, we report our protocol for optimal liposome preparation for measuring liposome leakage by fluorescence dequenching.

Computational Study on the Function of Palmitoylation on the Envelope Protein in SARS-CoV-2.

SARS-CoV-2 that caused COVID-19 has spread since the end of 2019. Its major effects resulted in over four million deaths around the whole world by August 2021. Therefore, understanding virulence mechanisms is important to prevent future outbreaks and for COVID-19 drug development. The envelope (E) protein is an important structural protein, affecting virus assembly and budding. The E protein pentamer is a viroporin, serving as an ion transferring channel in cells. In this work, we applied molecular dynamic simulations and topological and electrostatic analyses to study the effects of palmitoylation on the E protein pentamer. The results indicate that the cation transferring direction is more from the lumen to the cytosol. The structure of the palmitoylated E protein pentamer is more stable while the loss of palmitoylation caused the pore radius to reduce and even collapse. The electrostatic forces on the two sides of the palmitoylated E protein pentamer are more beneficial to attract cations in the lumen and to release cations into the cytosol. The results indicate the importance of palmitoylation, which can help the drug design for the treatment of COVID-19.

Interaction between Pain, Disability, Mechanosensitivity and Cranio-Cervical Angle in Subjects with Cervicogenic Headache: A Cross-Sectional Study.

The relationship between the forward head posture and mechanosensitivity in subjects with a cervicogenic headache (CGH) remains uncertain. The aim of the study was to evaluate if there was a relationship between the tissue mechanosensitivity and cranio-cervical angle (CCA) that was moderated by pain intensity and/or disability in subjects with CGH. A convenience sample of 102 subjects was recruited. The CCA was measured with photographs, using a postural assessment software. The pain intensity was measured with a visual analogue scale (VAS), and the disability was measured with the Northwick Park Questionnaire. The pressure pain threshold (PPT) was measured at the spinous process of C2, the upper trapezius and splenius capitis muscles, and the median nerve. Simple moderation multiple regression analyses were constructed. There was a positive relationship between PPT at C2 and CCA, but a nonsignificant relationship for the PPT measured at the muscles and median nerve. The effect of PPT at C2 over CCA was moderated by pain intensity (R2 = 0.17; R2 change = 0.06; p < 0.05) but not disability. The Johnson-Neyman analysis revealed a cut-off point for the statistical significance of 4.66 cm in VAS. There seems to be a positive relationship between PPT at C2 and CCA, which is positively moderated by pain intensity in subjects with CGH.

Development of an In Vitro Membrane Model to Study the Function of EsxAB Heterodimer and Establish the Role of EsxB in Membrane Permeabilizing Activity of Mycobacterium tuberculosis.

EsxA and EsxB are secreted as a heterodimer and have been shown to play critical roles in phagosome rupture and translocation of Mycobacterium tuberculosis into the cytosol. Recent in vitro studies have suggested that the EsxAB heterodimer is dissociated upon acidification, which might allow EsxA insertion into lipid membranes. While the membrane permeabilizing activity (MPA) of EsxA has been well characterized in liposomes composed of di-oleoyl-phosphatidylcholine (DOPC), the MPA of EsxAB heterodimer has not been detected through in vitro assays due to its negligible activity with DOPC liposomes. In this study, we established a new in vitro membrane assay to test the MPA activity of N-terminal acetylated EsxA (N-EsxA). We established that a dose-dependent increase in anionic charged lipids enhances the MPA of N-EsxA. The MPA of both N-EsxA and EsxAB were significantly increased with this new liposome system and made it possible to characterize the MPA of EsxAB in more physiologically-relevant conditions. We tested, for the first time, the effect of temperature on the MPA of N-EsxA and EsxAB in this new system. Interestingly, the MPA of N-EsxA was lower at 37 °C than at RT, and on the contrary, the MPA of EsxAB was higher at 37 °C than at RT. Surprisingly, after incubation at 37 °C, the MPA of N-EsxA continuously decreased over time, while MPA of EsxAB remained stable, suggesting EsxB plays a key role in stabilizing N-EsxA to preserve its MPA at 37 °C. In summary, this study established a new in vitro model system that characterizes the MPA of EsxAB and the role of EsxB at physiological-relevant conditions.