Optical sensing and control of T cell signaling pathways.

T cells regulate adaptive immune responses through complex signaling pathways mediated by T cell receptor (TCR). The functional domains of the TCR are combined with specific antibodies for the development of chimeric antigen receptor (CAR) T cell therapy. In this review, we first overview current understanding on the T cell signaling pathways as well as traditional methods that have been widely used for the T cell study. These methods, however, are still limited to investigating dynamic molecular events with spatiotemporal resolutions. Therefore, genetically encoded biosensors and optogenetic tools have been developed to study dynamic T cell signaling pathways in live cells. We review these cutting-edge technologies that revealed dynamic and complex molecular mechanisms at each stage of T cell signaling pathways. They have been primarily applied to the study of dynamic molecular events in TCR signaling, and they will further aid in understanding the mechanisms of CAR activation and function. Therefore, genetically encoded biosensors and optogenetic tools offer powerful tools for enhancing our understanding of signaling mechanisms in T cells and CAR-T cells.

Decreased FAK activity and focal adhesion dynamics impair proper neurite formation of medium spiny neurons in Huntington's disease.

Huntington's disease (HD) is a neurodegenerative disorder caused by a polyglutamine expansion in the protein huntingtin (HTT) [55]. While the final pathological consequence of HD is the neuronal cell death in the striatum region of the brain, it is still unclear how mutant HTT (mHTT) causes synaptic dysfunctions at the early stage and during the progression of HD. Here, we discovered that the basal activity of focal adhesion kinase (FAK) is severely reduced in a striatal HD cell line, a mouse model of HD, and the human post-mortem brains of HD patients. In addition, we observed with a FRET-based FAK biosensor [59] that neurotransmitter-induced FAK activation is decreased in HD striatal neurons. Total internal reflection fluorescence (TIRF) imaging revealed that the reduced FAK activity causes the impairment of focal adhesion (FA) dynamics, which further leads to the defect in filopodial dynamics causing the abnormally increased number of immature neurites in HD striatal neurons. Therefore, our results suggest that the decreased FAK and FA dynamics in HD impair the proper formation of neurites, which is crucial for normal synaptic functions [52]. We further investigated the molecular mechanism of FAK inhibition in HD and surprisingly discovered that mHTT strongly associates with phosphatidylinositol 4,5-biphosphate, altering its normal distribution at the plasma membrane, which is crucial for FAK activation [14, 60]. Therefore, our results provide a novel molecular mechanism of FAK inhibition in HD along with its pathological mechanism for synaptic dysfunctions during the progression of HD.

Optical regulation of endogenous RhoA reveals selection of cellular responses by signal amplitude.

How protein signaling networks respond to different input strengths is an important but poorly understood problem in cell biology. For example, RhoA can promote focal adhesion (FA) growth or disassembly, but how RhoA activity mediates these opposite outcomes is not clear. Here, we develop a photoswitchable RhoA guanine nucleotide exchange factor (GEF), psRhoGEF, to precisely control endogenous RhoA activity. Using this optical tool, we discover that peak FA disassembly selectively occurs upon activation of RhoA to submaximal levels. We also find that Src activation at FAs selectively occurs upon submaximal RhoA activation, identifying Src as an amplitude-dependent RhoA effector. Finally, a pharmacological Src inhibitor reverses the direction of the FA response to RhoA activation from disassembly to growth, demonstrating that Src functions to suppress FA growth upon RhoA activation. Thus, rheostatic control of RhoA activation by psRhoGEF reveals that cells can use signal amplitude to produce multiple responses to a single biochemical signal.

Retraction fibers produced by fibronectin-integrin α5ß1 interaction promote motility of brain tumor cells.

Glioblastoma (GBM) is a refractory disease that has a highly infiltrative characteristic. Over the past decade, GBM perivascular niche (PVN) has been described as a route of dissemination. Here, we investigated that trailed membrane structures, namely retraction fibers (RFs), are formed by perivascular extracellular matrix (ECM) proteins. By using the anatomical GBM database, we validated that the ECM-related genes were highly expressed in the cells within the PVN where fibronectin (FN) induced RF formation. By disrupting candidates of FN-binding integrins, integrin α5ß1 was identified as the main regulator of RF formation. De novo RFs were produced at the trailing edge, and focal adhesions were actively localized in RFs, indicating that adhesive force makes RFs remain at the bottom surface. Furthermore, we observed that GBM cells more frequently migrated along the residual RFs formed by preceding cells in microfluidic channels in comparison to those in the channels without RFs, suggesting that the infiltrative characteristics GBM could be attributed to RFs formed by the preceding cells in concert with chemoattractant cues. Altogether, we demonstrated that shedding membrane structures of GBM cells are maintained by FN-integrin α5ß1 interaction and promoted their motility .

Spatiotemporal Characterization of GPCR Activity and Function during Endosomal Trafficking Pathway.

G protein-coupled receptor (GPCR) is activated by extracellular signals. After their function at plasma membrane, GPCRs are internalized to be desensitized, while emerging evidence suggests that some GPCRs maintain their activity even after internalization. The endosomal trafficking pathway of a prototypic GPCR, ß adrenergic receptor 2 (B2AR), is in the range of several hours, however, spatiotemporal B2AR activity during this long-term endosomal trafficking pathway has not been characterized yet. Here, we analyze an agonist-induced real-time B2AR activity and its downstream function at the level of individual vesicles, utilizing a fluorescence resonance energy transfer (FRET)-based B2AR biosensor and cAMP reporters tethered at different trafficking stages of endosomes. Our results report that the internalized B2ARs sustain the activity and maintain the production of cAMP for several hours during the endosomal trafficking pathway. Temporal kinetics of B2AR activity is mathematically well explained by our active-vesicle population model modified from the Ricker model. Therefore, our GPCR monitoring system and a new kinetics model can be applied to understand the spatiotemporal GPCR activity and its downstream function during the endosomal trafficking pathway.

Genetically Encoded Biosensors Based on Fluorescent Proteins.

Genetically encoded biosensors based on fluorescent proteins (FPs) allow for the real-time monitoring of molecular dynamics in space and time, which are crucial for the proper functioning and regulation of complex cellular processes. Depending on the types of molecular events to be monitored, different sensing strategies need to be applied for the best design of FP-based biosensors. Here, we review genetically encoded biosensors based on FPs with various sensing strategies, for example, translocation, fluorescence resonance energy transfer (FRET), reconstitution of split FP, pH sensitivity, maturation speed, and so on. We introduce general principles of each sensing strategy and discuss critical factors to be considered if available, then provide representative examples of these FP-based biosensors. These will help in designing the best sensing strategy for the successful development of new genetically encoded biosensors based on FPs.

Creating Exercise Habits Using Incentives: The Tradeoff between Flexibility and Routinization.

Habits involve regular, cue-triggered routines. In a field experiment, we tested whether incentivizing exercise routines-paying participants each time they visit the gym within a planned, daily two-hour window-leads to more persistent exercise than offering flexible incentives-paying participants each day they visit the gym, regardless of timing. Routine incentives generated fewer gym visits than flexible incentives, both during our intervention and after incentives were removed. Even among sub-groups that were experimentally induced to exercise at similar rates during our intervention, recipients of routine incentives exhibited a larger decrease in exercise after the intervention than recipients of flexible incentives.

Physical Properties and Biofunctionalities of Bioactive Root Canal Sealers In Vitro.

Calcium silicate-based bioactive glass has received significant attention for use in various biomedical applications due to its excellent bioactivity and biocompatibility. However, the bioactivity of calcium silicate nanoparticle-incorporated bioactive dental sealer is not much explored. Herein, three commercially available bioactive root canal sealers (Endoseal MTA (EDS), Well-Root ST (WST), and Nishika Canal Sealer BG (NBG)) were compared with a resin-based control sealer (AH Plus (AHP)) in terms of physical, chemical, and biological properties. EDS and NBG showed 200 to 400 nm and 100 to 200 nm nanoparticle incorporation in the SEM image, respectively, and WST and NBG showed mineral deposition in Hank's balanced salt solution after 28 days. The flowability and film thickness of all products met the ISO 3107 standard. Water contact angle, linear dimensional changes, and calcium and silicate ion release were significantly different among groups. All bioactive root canal sealers released calcium ions, while NBG released ~10 times more silicon ions than the other bioactive root canal sealers. Under the cytocompatible extraction range, NBG showed prominent cytocompatibility, osteogenecity, and angiogenecity compared to other sealers in vitro. These results indicate that calcium silicate nanoparticle incorporation in dental sealers could be a potential strategy for dental periapical tissue regeneration.

Aß modulates actin cytoskeleton via SHIP2-mediated phosphoinositide metabolism.

Emerging evidences suggest that phospholipid metabolism is altered in Alzheimer's disease (AD), but molecular mechanisms on how this affects neurodegeneration in AD is poorly understood. SHIP2 is a phosphoinositide-metabolizing enzyme, which dephosphorylates PI(3,4,5)P3 resulting to PI(3,4)P2, and it has been recently shown that Aß directly increases the activity of SHIP2. Here we monitored, utilizing fluorescent SHIP2 biosensor, real-time increase of PI(3,4)P2-containing vesicles in HT22 cells treated with Aß. Interestingly, PI(3,4)P2 is accumulated at late endosomes and lysosomal vesicles. We further discovered that ARAP3 can be attracted to PI(3,4)P2-positive mature endosomes via its PH domain and this facilitates the degradation of ARAP3. The reduced level of ARAP3 then causes RhoA hyperactivation and filamentous actin, which are critical for neurodegeneration in AD. These results provide a novel molecular link between Aß and actin disruption through dysregulated phosphoinositide metabolism, and the SHIP2-PI(3,4)P2-ARAP3-RhoA signaling pathway can be considered as new therapeutic targets for synaptic dysfunctions in Alzheimer's disease.

Identification of crizotinib derivatives as potent SHIP2 inhibitors for the treatment of Alzheimer's disease.

SH2 domain-containing inositol 5'-phosphatase 2 (SHIP2) is a lipid phosphatase that produce phosphatidylinositol 3,4-bisphosphate (PI(3,4)P2) from phosphatidylinositol 3,4,5-triphosphate (PI(3,4,5)P3), and is involved in many diseases such as neurodegenerative diseases. A recent report demonstrating that SHIP2 inhibition decreased tau hyperphosphorylation induced by amyloid ß and rescued memory impairment in a transgenic Alzheimer's disease mouse model indicates SHIP2 can be a promising therapeutic target for Alzheimer's disease. In the present study, we have developed novel, potent SHIP2 inhibitors by extensive structural elaboration of crizotinib discovered from a high-throughput screening. Our representative compound 43 potently inhibited SHIP2 activity as well as GSK3ß activation in HT22 neuronal cells. It was also shown that 43 has favorable physicochemical properties, especially high brain penetration. Considering SHIP2 is one of key signal mediators for tau hyperphosphorylation, our potent SHIP2 inhibitor 43 may function as a promising lead compound for the treatment of Alzheimer's disease.

Anti­inflammatory effect of quercetin and galangin in LPS­stimulated RAW264.7 macrophages and DNCB­induced atopic dermatitis animal models.

Flavonols are compounds that have been shown to possess potent anti­inflammatory effects in cellular and animal models of inflammation. In the present study, the anti­inflammatory effects and mechanisms of two natural flavonols, quercetin and galangin, in lipopolysaccharide (LPS)­stimulated RAW264.7 macrophages were investigated. It was identified that quercetin and galangin markedly reduced the production of nitric oxide (NO), inducible NO synthase and interleukin­6, and the nuclear translocation of nuclear factor­κB (NF­κB). In addition, LPS­induced activation of extracellular signal­regulated kinase 1/2 (Erk1/2) and c­Jun N­terminal kinase (JNK) was suppressed by quercetin and galangin. Taken together, these data implied that NF­κB, Erk1/2 and JNK may be potential molecular targets of quercetin and galangin in an LPS­induced inflammatory response. Subsequently, the effects of oral administration of quercetin or galangin, either alone or in combination, in a 2,4­dinitrochlorobenzene­induced atopic dermatitis (AD) mouse model were investigated. As a result, measurements of ear thickness and the levels of serum immunoglobulin E, and histological analysis revealed that the two flavonols led to a decrease in inflammation, whereas, in combination, they were even more effective. These results suggested that quercetin and galangin may be promising therapeutic agents for AD. Additionally, their combination may be a novel therapeutic strategy for the prevention of AD.

Nitidine chloride acts as an apoptosis inducer in human oral cancer cells and a nude mouse xenograft model via inhibition of STAT3.

Nitidine chloride (NC) is a natural alkaloid compound derived from the plant Zanthoxylum nitidum and is known for its therapeutic anticancer potential. In this study, we investigated the effects of NC on growth and signaling pathways in human oral cancer cell lines and a tumor xenograft model. The apoptotic effects and related molecular targets of NC on human oral cancer were investigated using trypan blue exclusion assay, DAPI staining, Live/Dead assay, Western blotting, Immunohistochemistry/Immunofluorescence and a nude mouse tumor xenograft. NC decreased cell viability in both HSC3 and HSC4 cell lines; further analysis demonstrated that cell viability was reduced via apoptosis. STAT3 was hyper-phosphorylated in human oral squamous cell carcinoma (OSCC) compared with normal oral mucosa (NOM) and dephosphorylation of STAT3 by the potent STAT3 inhibitor, cryptotanshinone or NC decreased cell viability and induced apoptosis. NC also suppressed cell viability and induced apoptosis accompanied by dephosphorylating STAT3 in four other oral cancer cell lines. In a tumor xenograft model bearing HSC3 cell tumors, NC suppressed tumor growth and induced apoptosis by regulating STAT3 signaling without liver or kidney toxicity. Our findings suggest that NC is a promising chemotherapeutic candidate against human oral cancer.

Ixeris dentata (Thunb. Ex Thunb.) Nakai Extract Inhibits Proliferation and Induces Apoptosis in Breast Cancer Cells through Akt/NF-κB Pathways.

Ixeris dentata (Thunb. Ex Thunb.) Nakai (ID) exhibits various physiological activities, and its related plant derived-products are expected to represent promising cancer therapeutic agents. However, the anticancer effects of ID extract on breast cancer cells classified as estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) are still unknown. In this study, we investigated the anti-cancer effects and analyzed the molecular mechanism of ID extract in T47D, MCF-7 (ER-, PR-positive, HER2-negative), SK-BR-3(ER-, PR-negative, HER2-positive), and MDA-MB-231 (Triple-negative) through in vitro studies. Additionally, we examined its anti-tumor effects through in vivo studies. Our findings indicated that ID extract-induced apoptosis was mediated via various survival pathways on four breast cancer cells by identifying the factors including Bcl-2 family, phospho-Akt and phospho-nuclear factor-κB (NF-κB). Based on in vitro findings that induced apoptosis via Akt-NF-κB signaling, we investigated the effects of ID extract on mice bearing MDA-MB-231 cells. The results showed that ID extract significantly decreased MDA-MB-231 tumor volume and weight via inducing apoptosis by suppressing phospho-Akt. Overall, these results indicate that ID extract induces apoptosis through the Akt-NFκB signaling pathway in MDA-MB-231 breast cancer cells and tumors, and it may serve as a therapeutic agent for triple-negative human breast cancer.

Anticancer effects of Ixeris dentata (Thunb. ex Thunb.) nakai extract on human melanoma cells A375P and A375SM.

ETHNOPHARMACOLOGICAL RELEVANCE: The plant species Taraxacum coreanum (TC), Youngia sonchifolia (YS), and Ixeris dentata (ID) belong to the family Compositae and are used for medicinal purposes in traditional medicine. However, the anticancer effects of TC, YS, and ID extracts and the underlying molecular mechanisms in melanoma cells have not been elucidated. AIM OF THE STUDY: To investigate the potential anticancer effects of TC, YS, and ID extracts on human melanoma cells and explore the potential pharmacological mechanisms in vitro and in vivo. MATERIALS AND METHODS: In this comparative study, we investigated the effects of TC, YS, and ID extracts on cell proliferation in human melanoma A375P and A375SM cells using MTT[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assays. Apoptotic cells were detected by 4',6-diamidino-2-phenylinodole (DAPI) staining. We also investigated whether the growth-inhibitory effects were associated with the induction of apoptosis and whether the mechanisms of cell death were the result of signaling molecules such as p53, Bax, Bcl-2, caspase-9, Poly-ADP ribose polymerase (PARP), and Erk (Extracellular signal-regulated protein kinase) 1/2. The in vivo antitumor effects were evaluated by measuring the tumor volume and weight and performing Terminal deoxynucleotidyl transferase (TdT) dUTP Nick End Labeling (TUNEL) assay and immunohistochemistry (IHC) in tumor xenograft models. RESULTS: TC, YS, and ID extracts effectively inhibited the growth of A375P and A375SM cells. In addition, several apoptotic events were observed following treatment, including DNA fragmentation and chromatin condensation by DAPI staining. The extracts increased p53, Bax, cleaved-caspase-9 and cleaved-PARP expression, whereas the expression of Bcl-2 was decreased in both cell lines. Furthermore, ID extract significantly inhibited the activation of Erk1/2 in both cell lines. Among the three extracts, ID had the strongest apoptotic effects. The administration of ID extract to mice inhibited tumor growth without any toxicity following 4 weeks of treatment. This extract increased the expression of apoptotic cells and p53 protein and decreased phospho-Erk1/2 protein. CONCLUSION: TC, YS, and ID extracts suppress the growth of human melanoma cells through apoptosis. Among these extracts, ID has the strongest anticancer and apoptotic effects. It induces apoptosis through the inhibition of Erk1/2 in A375P and A375SM human melanoma cells and in tumor xenograft models and may be a potential chemotherapeutic agent against melanoma.

Anticancer Effect of Fucoidan on DU-145 Prostate Cancer Cells through Inhibition of PI3K/Akt and MAPK Pathway Expression.

In this study, we showed that PI3K/Akt signaling mediates fucoidan's anticancer effects on prostate cancer cells, including suppression of proliferation. Fucoidan significantly decreased viability of DU-145 cancer cells in a concentration-dependent manner as shown by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. The drug also significantly increased chromatin condensation, which indicates apoptosis, in a concentration-dependent manner as shown by DAPI (4',6-diamidino-2-phenylindole) staining. Fucoidan increased expression of Bax, cleaved poly-ADP ribose polymerase and cleaved caspase-9, and decreased of the Bcl-2, p-Akt, p-PI3K, p-P38, and p-ERK in a concentration-dependent manner. In vivo, fucoidan (at 5 and 10 mg/kg) significantly decreased tumor volume, and increased apoptosis as assessed by the TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) assay, confirming the tumor inhibitory effect. The drug also increased expression of p-Akt and p-ERK as shown by immunohistochemistry staining. Therefore, fucoidan may be a promising cancer preventive medicine due to its growth inhibitory effects and induction of apoptosis in human prostate cancer cells.

Antitumor and apoptosis-inducing effects of α-mangostin extracted from the pericarp of the mangosteen fruit (Garcinia mangostana L.)in YD-15 tongue mucoepidermoid carcinoma cells.

α-mangostin is a dietary xanthone which has been shown to have antioxidant, anti-allergic, antiviral, antibacterial, anti-inflammatory and anticancer effects in various types of human cancer cells. In the present study, we aimed to elucidate the molecular mechanisms responsible for the apoptosis-inducing effects of α-mangostin on YD-15 tongue mucoepidermoid carcinoma cells. The results from MTT assays revealed that cell proliferation significantly decreased in a dose-dependent manner in the cells treated with α-mangostin. DAPI staining illustrated that chromatin condensation in the cells treated with 15 µM α-mangostin was far greater than that in the untreated cells. Flow cytometric analysis indicated that α-mangostin suppressed YD-15 cell viability by inducing apoptosis and promoting cell cycle arrest in the sub-G1 phase. Western blot analysis of various signaling molecules revealed that α-mangostin targeted the extracellular signal­regulated kinase 1/2 (ERK1/2) and p38 mitogen-activated protein kinase (MAPK) signaling pathways through the inhibition of ERK1/2 and p38 phosphorylation in a dose­dependent manner. α-mangostin also increased the levels of Bax (pro-apoptotic), cleaved caspase-3, cleaved caspase-9 and cleaved-poly(ADP-ribose) polymerase (PARP), whereas the levels of the anti-apoptotic factors, Bcl-2 and c-myc, decreased in a dose-dependent manner. The anticancer effects of α-mangostin were also investigated in a tumor xenograft mouse model. The α-mangostin-treated nude mice bearing YD-15 tumor xenografts exhibited a significantly reduced tumor volume and tumor weight due to the potent promoting effects of α-mangostin on cancer cell apoptosis, as determined by TUNEL assay. Immunohistochemical analysis revealed that the level of cleaved caspase-3 increased, whereas the Ki-67, p-ERK1/2 and p-p38 levels decreased in the α-mangostin­treated mice. Taken together, the findings of our study indicate that α-mangostin induces the apoptosis of YD-15 tongue carcinoma cells through the ERK1/2 and p38 MAPK signaling pathways.

Targeting ERK1/2-bim signaling cascades by BH3-mimetic ABT-737 as an alternative therapeutic strategy for oral cancer.

To date, many different chemotherapeutic agents have been widely used as common treatments for oral cancers. However, their therapeutic effects have been disappointing, and these agents may have unwanted side effects. Among the many regulatory factors, overexpression of pro-survival Bcl-2 family members may promote resistance to chemotherapeutic drugs in many tumors. The BH3 domain-only proteins effectively antagonize their apoptotic activities. Therefore, there is substantial interest in developing chemotherapeutic drugs that directly target pro-survival Bcl-2 proteins by mimicking the BH3 domain and unleashing pro-apoptotic molecules in tumor cells. Among the numerous available small molecule BH3 mimetics, ABT-737, a potent small molecule that binds to Bcl-2/Bcl-xL with high affinity, has anti-tumor activity in a wide variety of cancer cells. However, the effects of ABT-737 on human oral cancers and the underlying molecular mechanisms have not previously been elucidated. In the present study, we observed that inactivation of the ERK1/2 signaling pathway using ABT-737 dramatically increased the expression of pro-apoptotic protein Bim via transcriptional and/or posttranslational regulation, in a cell type-dependent manner, inducing mitochondria-mediated apoptosis of human oral cancer cells. To the best of our knowledge, this is the first demonstration of the antitumor effects of ABT-737 on human oral cancers.

Risk and preventive factors for type 2 diabetes and heart disease among foreign-born older Vietnamese Americans.

The 2009 California Health Interview Survey (CHIS) data was used to examine associations of bodyweight, lifestyles, and demographic variables with type 2 diabetes (T2DM) and heart disease among foreign-born older Vietnamese adults. CHIS consisted of 709 Vietnamese Americans aged 50 to 85. Thirteen percent reported T2DM and 11% had heart disease. Using logistic regression, body mass index ≥ 24, age ≥ 65, and female were significantly associated with T2DM. There was significant interaction effect of alcohol consumption and psychological distress with T2DM. The interaction of vegetable consumption, poverty, and length of living in the U.S. was significantly associated with heart disease.

Mental health and migration: depression, alcohol abuse, and access to health care among migrants in Central Asia.

One-fifth of Kazakhstan's population is labor migrants working in poor conditions with limited legal rights. This paper examines self-rated health, mental health and access to health care among migrant workers. Using geo-mapping, a random sample of internal and external migrant market workers was selected in Almaty (N = 450). We used survey logistic regression adjusted for clustering of workers within stalls. Almost half of participants described their health as fair or poor and reported not seeing a doctor when needed, 6.2% had clinical depression and 8.7% met criteria for alcohol abuse. Female external migrants were at higher risk for poor health and underutilization of health services. High mobility was associated with depression among internal migrants and with alcohol abuse among female migrant workers. This study demonstrates the urgent need to address health and mental health needs and improve access to health care among labor migrants in Central Asia.

Prior incarceration and barriers to receipt of services among entrants to alternative to incarceration programs: a gender-based disparity.

The high levels of health and psychosocial needs among correctional populations strongly shape the well-being of the urban communities from which a large number of criminal justice-involved individuals come or to which they return. The benefits of providing services to correction-involved individuals and linking them to providers such as with alternative to incarceration (ATI) programs may be limited if they encounter difficulties accessing such services. This study identified the types of barriers that have prevented entrants into ATI programs from receiving health and psychosocial services. We then tested the association between number of prior incarcerations and number of barriers by gender. From a random sample of adults (N = 322; 83 women and 239 men) entering ATI programs in New York City, data were collected via structured interviews that elicited self-reported sociodemographics, substance use, prior incarcerations, and barriers that had actually prevented a participant from visiting or returning to a service provider. Participants reported an average of 3.0 barriers that have prevented them from receiving health and psychosocial services. The most prevalent barriers predominantly concerned service providers' inability to accommodate constraints on participants' time availability or flexibility, transportation, and money. Compared to women, men had a significantly different association that was in the adverse direction--i.e., more prior incarcerations was associated with more barriers--between prior incarcerations and encountering service barriers. Findings indicate that ATI program entrants experience many barriers that have prevented them from receiving health and/or psychosocial services. Furthermore, men with more extensive incarceration histories particularly are disadvantaged. ATI programs can improve the public health of urban communities if such programs are prepared and resourced to facilitate the receipt of services among program participants, especially men who have more extensive incarceration histories.