Automated stress detection using mobile application and wearable sensors improves symptoms of mental health disorders in military personnel.

Leading causes in global health-related burden include stress, depression, anger, fatigue, insomnia, substance abuse, and increased suicidality. While all individuals are at risk, certain career fields such as military service are at an elevated risk. Cognitive behavioral therapy (CBT) is highly effective at treating mental health disorders but suffers from low compliance and high dropout rates in military environments. The current study conducted a randomized controlled trial with military personnel to assess outcomes for an asymptomatic group (n = 10) not receiving mental health treatment, a symptomatic group (n = 10) using a mHealth application capable of monitoring physiological stress via a commercial wearable alerting users to the presence of stress, guiding them through stress reduction techniques, and communicating information to providers, and a symptomatic control group (n = 10) of military personnel undergoing CBT. Fifty percent of symptomatic controls dropped out of CBT early and the group maintained baseline symptoms. In contrast, those who used the mHealth application completed therapy and showed a significant reduction in symptoms of depression, anxiety, stress, and anger. The results from this study demonstrate the feasibility of pairing data-driven mobile applications with CBT in vulnerable populations, leading to an improvement in therapy compliance and a reduction in symptoms compared to CBT treatment alone. Future work is focused on the inclusion of passive sensing modalities and the integration of additional data sources to provide better insights and inform clinical decisions to improve personalized support.

Barriers and Potential Solutions to Implementing Evidence-Based PTSD Treatment in Military Treatment Facilities.

INTRODUCTION: Prolonged exposure therapy is an effective treatment for posttraumatic stress disorder that is underutilized in health systems, including the military health system. Organizational barriers to prolonged exposure implementation have been hypothesized but not systematically examined. This multisite project sought to identify barriers to increasing the use of prolonged exposure across eight military treatment facilities and describe potential solutions to addressing these barriers. MATERIALS AND METHODS: As part of a larger project to increase the use of prolonged exposure therapy in the military health system, we conducted a needs assessment at eight military treatment facilities. The needs assessment included analysis of clinic administrative data and a series of stakeholder interviews with behavioral health clinic providers, leadership, and support staff. Key barriers were matched with potential solutions using a rubric developed for this project. Identified facilitators, barriers, and potential solutions were summarized in a collaboratively developed implementation plan for increasing prolonged exposure therapy tailored to each site. RESULTS: There was a greater than anticipated consistency in the barriers reported by the sites, despite variation in the size and type of facility. The identified barriers were grouped into four categories: time-related barriers, provider-related barriers, barriers related to patient education and matching patients to providers, and scheduling-related barriers. Potential solutions to each barrier are described. CONCLUSIONS: The findings highlight the numerous organizational-level barriers to implementing evidence-based psychotherapy in the military health system and offer potential solutions that may be helpful in addressing the barriers.

Ivermectin-induced cell death of cervical cancer cells in vitro a consequence of precipitate formation in culture media.

The anthelmintic ivermectin has been reported to possess anticancer and antiviral efficacy. However, the effective concentrations reported in vitro are near the predicted aqueous solubility limit for this hydrophobic drug. We observed that ivermectin-induced cell death in two cervical cancer cell lines correlated with the formation of solid ivermectin aggregates in both serum-free and serum-supplemented culture media. Filtration of ivermectin particles >0.2 µm abolished these cytolytic effects in both cell lines. An inhibitory effect on cell proliferation persisted for filtered solutions, but only for ivermectin concentrations higher than reported to be clinically attainable in humans. In addition to the importance of distinguishing between free and bound drug in solution, our data emphasize the importance of acknowledging the likely solubility limit of hydrophobic drugs when assessing their in vitro cytotoxicity.

KCa3.1-dependent uptake of the cytotoxic DNA-binding dye Hoechst 33258 into cancerous but not healthy cervical cells.

The poor and nonselective penetration of current chemotherapeutics across the plasma membranes of cancer cells, which is necessary for the targeted disruption of the intracellular machinery, remains a major pharmaceutical challenge. In several cell types, including mast cells and macrophages, exposure to extracellular ATP is known to stimulate passive entry of large and otherwise membrane impermeable cationic dyes, which is usually attributed to conduction through ionotropic P2X receptors. Here, we report that elevations in cytosolic Ca2+ stimulate the rapid uptake and nuclear accumulation of a DNA-binding fluorescent cation, Hoechst 33258 (H33258), in cervical cancer cells. The H33258 uptake was dependent on activation of intermediate conductance Ca2+-activated K+ channels (KCa3.1), and direct stimulation of the channel with the activators SKA 31 and DCEBIO was sufficient to induce cellular uptake of H33258 directly. In contrast to the results from cancerous cervical cells, KCa3.1-dependent H33258 uptake was rarely observed in epithelial cells derived from the ectocervix and transformation zone of healthy cervical tissue. Furthermore, whole-cell patch clamp experiments and assessment of membrane potential using the slow voltage-sensitive dye bis-(1,3-diethylthiobarbituric acid)trimethine oxonol revealed a significant difference in functional KCa3.1 activity between cancerous and healthy cervical epithelial cells, which correlated strongly with the incidence of KCa3.1-dependent H33258 uptake. Finally, we show that activation of KCa3.1 channels caused a modest but significant sensitization of cancer cells to the growth suppressant effects of H33258, lending plausibility to the idea of using KCa3.1 channel activators to enhance cell penetration of small cationic toxins into cancer cells expressing these channels.

Targeted Assessment and Context-Tailored Implementation of Change Strategies (TACTICS) to increase evidence based psychotherapy in military behavioral health clinics: Design of a cluster-randomized stepped-wedge implementation study.

BACKGROUND: Despite efforts by the U.S. Department of Defense to train behavioral health (BH) providers in evidence-based psychotherapies (EBPs) for posttraumatic stress disorder (PTSD), numerous barriers limit EBP implementation. A context-tailored implementation approach called TACTICS (Targeted Assessment and Context-Tailored Implementation of Change Strategies) holds promise for increasing the use of EBPs such as prolonged exposure therapy (PE) in military treatment facilities. TACTICS combines a needs assessment, a rubric for selecting implementation strategies based on local barriers, an implementation toolkit, and external facilitation to support local champions and their implementation teams in enacting changes. This paper describes the rationale for and design of a study that will evaluate whether TACTICS can increase implementation of PE for PTSD and improve patient outcomes in military BH clinics relative to provider training in PE alone. METHODS: The study is a multi-site, cluster randomized, stepped-wedge trial, with the military treatment facility as the unit of analysis. Eight facilities undergo a provider-training phase, followed by 5 months of TACTICS implementation. The timing of TACTICS at each facility is randomly assigned to begin 9, 14, or 19 months after beginning the provider-training phase. Primary analyses will compare the proportion of PTSD patients receiving PE and patients' mean improvement in PTSD symptoms before and after the onset of TACTICS. DISCUSSION: TACTICS endeavors to balance standardization of empirically-supported implementation strategies with the flexibility of application necessary for success across varied clinical settings. If successful, TACTICS may represent a systematic and scalable method of promoting and supporting EBP implementation. TRIAL REGISTRATION: Clinicaltrials.gov Identifier: NCT03663452.

Establishment and optimization of epithelial cell cultures from human ectocervix, transformation zone, and endocervix optimization of epithelial cell cultures.

Cervical cancer is a major public health problem and research using cell culture models has improved understanding of this disease. The human cervix contains three anatomic regions; ectocervix with stratified squamous epithelium, endocervix with secretory epithelium, and transformation zone (TZ) with metaplastic cells. Most cervical cancers originate within the TZ. However, little is known about the biology of TZ cells or why they are highly susceptible to carcinogenesis. The goal of this study was to develop and optimize methods to compare growth and differentiation of cells cultured from ectocervix, TZ or endocervix. We examined the effects of different serum-free media on cell attachment, cell growth and differentiation, and cell population doublings in monolayer culture. We also optimized conditions for organotypic culture of cervical epithelial cells using collagen rafts with human cervical stromal cells. Finally, we present a step-by-step protocol for culturing cells from each region of human cervix.

HPV16-Immortalized Cells from Human Transformation Zone and Endocervix are More Dysplastic than Ectocervical Cells in Organotypic Culture.

A major risk factor for cervical cancer is persistent infection with high-risk human papillomaviruses (HPV) which can cause cervical intraepithelial neoplasia. Greater than 90% of cervical cancers develop in the transformation zone (TZ), a small region of metaplastic squamous epithelium at the squamocolumnar junction between endocervix and ectocervix. However, it is unclear why this region is highly susceptible to malignant progression. We hypothesized that cells from TZ were more susceptible to dysplastic differentiation, a precursor to cervical cancer. We used three-dimensional organotypic culture to compare differentiation of HPV16-immortalized epithelial cell lines derived from ectocervix, TZ, and endocervix. We show that immortal cells from TZ or endocervix form epithelia that are more dysplastic than immortal cells from ectocervix. A higher percentage of immortal cells from TZ and endocervix express the proliferation marker Ki-67 and are positive for phospho-Akt. Immortal cells from TZ and endocervix invade collagen rafts and express increased levels of matrix metalloproteinase-1. Inhibition of MMP-1 or Akt activity blocks invasion. We conclude that HPV16-immortalized cells cultured from TZ or endocervix are more susceptible to dysplastic differentiation, and this might enhance their susceptibility to cervical cancer.

Susceptibility of epithelial cells cultured from different regions of human cervix to HPV16-induced immortalization.

Persistent infection with high-risk human papillomavirus (HPV) is a major risk factor for cervical cancer. Greater than 90% of these cancers originate in the cervical transformation zone (TZ), a narrow region of metaplastic squamous epithelium that develops at the squamocolumnar junction between the ectocervix and endocervix. It is unclear why the TZ has high susceptibility to malignant transformation and few studies have specifically examined cells from this region. We hypothesized that cells cultured from TZ are more susceptible to cellular immortalization, an alteration that contributes to malignant development. We cultured primary epithelial cells from each region of human cervix (ectocervix, endocervix and TZ) and measured susceptibility to immortalization after transfection with the complete HPV-16 genome or infection of HPV16 E6/E7 retroviruses. Cells cultured from each cervical region expressed keratin markers (keratin 14 and 18) that confirmed their region of origin. In contrast to our prediction, cells from TZ were equally susceptible to immortalization as cells from ectocervix or endocervix. Thus, increased susceptibility of the TZ to cervical carcinogenesis is not due to increased frequency of immortalization by HPV-16. We developed a series of HPV16-immortalized cell lines from ectocervix, endocervix and TZ that will enable comparisons of how these cells respond to factors that promote cervical carcinogenesis.

Nanoreactors based on DNAzyme-functionalized magnetic nanoparticles activated by magnetic field.

A new biomimetic nanoreactor design, MaBiDz, is presented based on a copolymer brush in combination with superparamagnetic nanoparticles. This cellular nanoreactor features two species of magnetic particles, each functionalized with two components of a binary deoxyribozyme system. In the presence of a target mRNA analyte and a magnetic field, the nanoreactor is assembled to form a biocompartment enclosed by the polymeric brush that enables catalytic function of the binary deoxyribozyme with enhanced kinetics. MaBiDz was demonstrated here as a cellular sensor for rapid detection and imaging of a target mRNA biomarker for metastatic breast cancer, and its function shows potential to be expanded as a biomimetic organelle that can downregulate the activity of a target mRNA biomarker.

Magnetic Field-Activated Sensing of mRNA in Living Cells.

Detection of specific mRNA in living cells has attracted significant attention in the past decade. Probes that can be easily delivered into cells and activated at the desired time can contribute to understanding translation, trafficking and degradation of mRNA. Here we report a new strategy termed magnetic field-activated binary deoxyribozyme (MaBiDZ) sensor that enables both efficient delivery and temporal control of mRNA sensing by magnetic field. MaBiDZ uses two species of magnetic beads conjugated with different components of a multicomponent deoxyribozyme (DZ) sensor. The DZ sensor is activated only in the presence of a specific target mRNA and when a magnetic field is applied. Here we demonstrate that MaBiDZ sensor can be internalized in live MCF-7 breast cancer cells and activated by a magnetic field to fluorescently report the presence of specific mRNA, which are cancer biomarkers.

Selective permeabilization of cervical cancer cells to an ionic DNA-binding cytotoxin by activation of P2Y receptors.

Extracellular ATP is known to permeabilize certain cell types to polyatomic cations like YO-PRO1. Here, we report that extracellularly applied ATP stimulated rapid uptake and accumulation of an otherwise weakly membrane permeable fluorescent DNA-binding cytotoxin, Hoechst 33258, into cervical cancer cells. While ATP stimulated Hoechst 33258 uptake in 20-70% of cells from seven cervical cancer cell lines, it stimulated uptake in less than 8% of cervical epithelial cells obtained from the normal transformation zone and ectocervix tissue of 11 patients. ATP-evoked Hoechst 33258 uptake was independent of ionotropic P2X receptors, but dependent on activation of P2Y receptors. Thus, we show here that cervical cancer cells can be selectively induced to take up and accumulate an ionic cytotoxin by exposure to extracellular ATP.

Towards early detection of cervical cancer: Fractal dimension of AFM images of human cervical epithelial cells at different stages of progression to cancer.

We used AFM HarmoniX modality to analyse the surface of individual human cervical epithelial cells at three stages of progression to cancer, normal, immortal (pre-malignant) and carcinoma cells. Primary cells from 6 normal strains, 6 cancer, and 6 immortalized lines (derived by plasmid DNA-HPV-16 transfection of cells from 6 healthy individuals) were tested. This cell model allowed for good control of the cell phenotype down to the single cell level, which is impractical to attain in clinical screening tests (ex-vivo). AFM maps of physical (nonspecific) adhesion are collected on fixed dried cells. We show that a surface parameter called fractal dimension can be used to segregate normal from both immortal pre-malignant and malignant cells with sensitivity and specificity of more than 99%. The reported method of analysis can be directly applied to cells collected in liquid cytology screening tests and identified as abnormal with regular optical methods to increase sensitivity. FROM THE CLINICAL EDITOR: Despite cervical smear screening, sometimes it is very difficult to differentiate cancers cells from pre-malignant cells. By using AFM to analyze the surface properties of human cervical epithelial cells, the authors were able to accurately identify normal from abnormal cells. This method could augment existing protocols to increase diagnostic accuracy.

Recovery of aging-related size increase of skin epithelial cells: in vivo mouse and in vitro human study.

The size increase of skin epithelial cells during aging is well-known. Here we demonstrate that treatment of aging cells with cytochalasin B substantially decreases cell size. This decrease was demonstrated on a mouse model and on human skin cells in vitro. Six nude mice were treated by topical application of cytochalasin B on skin of the dorsal left midsection for 140 days (the right side served as control for placebo treatment). An average decrease in cell size of 56±16% resulted. A reduction of cell size was also observed on primary human skin epithelial cells of different in vitro age (passages from 1 to 8). A cell strain obtained from a pool of 6 human subjects was treated with cytochalasin B in vitro for 12 hours. We observed a decrease in cell size that became statistically significant and reached 20-40% for cells of older passage (6-8 passages) whereas no substantial change was observed for younger cells. These results may be important for understanding the aging processes, and for cosmetic treatment of aging skin.

Relationship of Sensory Modality to Retention of Episodic Memory.

This study investigated the difference between episodic memory for verbal information presented in an oral format versus equivalent material presented in a written format. The study utilized the Logical Memory subtest of the Wechsler Memory Scales-Fourth Edition and the recently validated Morris Revision-IV Paragraphs. In a sample of 97 normal participants, auditory and visual memory performances were found to be significantly correlated (r = .651, p < .001). Post-hoc analysis revealed a slight though not clinically significant preference for retention in the visual modality. The results demonstrate a high-degree correlation for retention of episodic memory for these two sensory modalities in normal participants.

Fabrication, nanomechanical characterization, and cytocompatibility of gold-reinforced chitosan bio-nanocomposites.

Chitosan, a naturally derived polymer represents one of the most technologically important classes of active materials with applications in a variety of industrial and biomedical fields. Gold nanoparticles (~32 nm) were synthesized via a citrate reduction method from chloroauric acid and incorporated in Chitosan matrix. Bio-nanocomposite films with varying concentrations of gold nanoparticles were prepared through solution casting process. Uniform distribution of gold nanoparticles was achieved throughout the chitosan matrix and was confirmed with SEM. Synthesis outcomes and prepared nanocomposites were characterized using SEM, TEM, EDX, SAED, UV-vis, XRD, DLS, and Zeta potential for their physical, morphological and structural properties. Nanoscale properties of materials under the influence of temperature were characterized through nanoindentation techniques. From quasi-static nanoindentation, it was observed that hardness and reduced modulus of the nanocomposites were increased significantly in direct proportion to the gold nanoparticle concentration. Gold nanoparticle concentration also showed positive impact on storage modulus and thermal stability of the material. The obtained films were confirmed to be biocompatible by their ability to support growth of human cells in vitro. In summary, the results show enhanced mechanical properties with increasing gold nanoparticle concentration, and provide better understanding of the structure-property relationships of such biocompatible materials for potential biomedical applications.

Development of alternate paragraphs for the Logical Memory subtest of the Wechsler Memory Scale-IV.

The purpose of the two studies included in this article was to validate an alternate form, the Morris Revision-Fourth Edition (MR-IV), to the Logical Memory paragraphs of the Wechsler Memory Scale-Fourth Edition (LM-IV) for use when retesting of individuals is desired. Study I demonstrated high correlation with the LM-IV paragraphs. Study II was a replication that again demonstrated high correlation between the original LM-IV and the new MR-IV paragraphs. High interrater reliability also was demonstrated. Consequently, the MR-IV paragraphs can be considered an alternate form to the LM-IV paragraphs. Although other attempts have been made to develop alternate stories, these new paragraphs are the only ones that are equivalent in structure, affective tone, and number of scorable units. They have considerable clinical utility and research potential.

Human papillomavirus type 16 E6 and E 7 proteins alter NF-kB in cultured cervical epithelial cells and inhibition of NF-kB promotes cell growth and immortalization.

The NF-kB family of transcription factors regulates important biological functions including cell growth, survival and the immune response. We found that Human Papillomavirus type 16 (HPV-16) E7 and E6/E7 proteins inhibited basal and TNF-alpha-inducible NF-kB activity in human epithelial cells cultured from the cervical transformation zone, the anatomic region where most cervical cancers develop. In contrast, HPV-16 E6 regulated NF-kB in a cell type- and cell growth-dependent manner. NF-kB influenced immortalization of cervical cells by HPV16. Inhibition of NF-kB by an IkB alpha repressor mutant increased colony formation and immortalization by HPV-16. In contrast, activation of NF-kB by constitutive expression of p65 inhibited proliferation and immortalization. Our results suggest that inhibition of NF-kB by HPV-16 E6/E7 contributes to immortalization of cells from the cervical transformation zone.

Inhibition of the epidermal growth factor receptor by erlotinib prevents immortalization of human cervical cells by Human Papillomavirus type 16.

The Human Papillomavirus type-16 (HPV-16) E6 and E7 oncogenes are selectively retained and expressed in cervical carcinomas, and expression of E6 and E7 is sufficient to immortalize human cervical epithelial cells. Expression of the epidermal growth factor receptor (EGFR) is often increased in cervical dysplasia and carcinoma, and HPV oncoproteins stimulate cell growth via the EGFR pathway. We found that erlotinib, a specific inhibitor of EGFR tyrosine kinase activity, prevented immortalization of cultured human cervical epithelial cells by the complete HPV-16 genome or the E6/E7 oncogenes. Erlotinib stimulated apoptosis in cells that expressed HPV-16 E6/E7 proteins and induced senescence in a subpopulation of cells that did not undergo apoptosis. Since immortalization by HPV E6/E7 is an important early event in cervical carcinogenesis, the EGFR is a potential target for chemoprevention or therapy in women who have a high risk for cervical cancer.

Detection of cancerous cervical cells using physical adhesion of fluorescent silica particles and centripetal force.

Here we describe a non-traditional method to identify cancerous human cervical epithelial cells in a culture dish based on physical adhesion between silica beads and cells. It is a simple optical fluorescence-based technique which detects the relative difference in the amount of fluorescent silica beads physically adherent to surfaces of cancerous and normal cervical cells. The method utilizes the centripetal force gradient that occurs in a rotating culture dish. Due to the variation in the balance between adhesion and centripetal forces, cancerous and normal cells demonstrate clearly distinctive distributions of the fluorescent particles adherent to the cell surface over the culture dish. The method demonstrates higher adhesion of silica particles to normal cells compared to cancerous cells. The difference in adhesion was initially observed by atomic force microscopy (AFM). The AFM data were used to design the parameters of the rotational dish experiment. The optical method that we describe is much faster and technically simpler than AFM. This work provides proof of the concept that physical interactions can be used to accurately discriminate normal and cancer cells.

Towards nonspecific detection of malignant cervical cells with fluorescent silica beads.

To date, the methods for detection of cancer cells are mostly based on traditional techniques used in biology, such as visual identification of malignant changes, cell-growth analysis, specific ligand-receptor labeling, or genetic tests. Despite being well developed, these methods are either insufficiently accurate or require a lengthy complicated analysis. A search for alternative methods for the detection of cancer cells may be a fruitful approach. Proposed here is a novel method for the detection of cancer cells in vitro, which is based on nonspecific adhesion of silica beads to cells. First, atomic force microscopy is used to study the adhesion of single silica beads to malignant and normal cells cultured from human cervix. It is found that adhesion depends on the time of contact, and can be statistically different for malignant and normal cells. Using these data, an optical method utilizing fluorescent silica beads is developed, which is based on detection of the difference in the number of adherent particles. The method is tested using primary cells cultured from cervical tissues of three healthy individuals and three patients with cervical cancer. The method shows sufficiently high sensitivity for cancer to make it interesting to perform further statistical tests.