Tools for Screening and Measuring Anxiety Among Women Living with HIV of Reproductive Age: A Scoping Review.

Emerging evidence suggests that women living with HIV (WLWH) may experience higher rates of anxiety than men living with HIV and women living without HIV. To date, relatively little knowledge exists on valid anxiety screening and diagnostic tools and how they are used among WLWH, specifically WLWH of reproductive age. Thus, the purpose of this scoping review was to describe what is known in the published literature about anxiety among WLWH and the tools used to measure and screen for anxiety in clinical and research contexts. The Arksey and O'Malley methodological framework was used to guide a scoping review of published articles in PsycINFO, Scopus, Sociological Abstracts, and PubMed databases. Twenty-one measures of anxiety were used across the 52 articles identified in the search. Most measures used were self-report. Inconsistencies in standardized screening tools and cutoff scores were observed across studies. Further, measures to assess anxiety varied among studies focused on WLWH. Based on the results from this review, there is a need for consistent, valid measures of anxiety to advance research and clinical practice to support the well-being of WLWH.

The Application of 3base™ Technology to Diagnose Eight of the Most Clinically Important Gastrointestinal Protozoan Infections.

Globally, over 3.5 billion people are infected with intestinal parasites each year, resulting in over 200,000 deaths. Three of the most common protozoan pathogens that affect the gastrointestinal tract of humans are Cryptosporidium spp., Giardia intestinalis, and Entamoeba histolytica. Other protozoan agents that have been implicated in gastroenteritis in humans include Cyclospora cayetanensis, Dientamoeba fragilis, Blastocystis hominis, and the microsporidia Enterocytozoon bieneusi and Encephalitozoon intestinalis. Genetic Signatures previously developed a 3base™ multiplexed Real-Time PCR (mRT-PCR) enteric protozoan kit (EP001) for the detection of Giardia intestinalis/lamblia/duodenalis, Cryptosporidium spp., E. histolytica, D. fragilis, and B. hominis. We now describe improvements to this kit to produce a more comprehensive assay, including C. cayetanensis, E. bieneusi, and E. intestinalis, termed EP005. The clinical performance of EP005 was assessed using a set of 380 clinical samples against a commercially available PCR test and other in-house nucleic acid amplification tests where commercial tests were not available. All methods provided at least 90% agreement. EP005 had no cross-reactivity against 82 organisms commonly found in the gut. The EP005 method streamlines the detection of gastrointestinal parasites and addresses the many challenges of traditional microscopic detection, resulting in cost savings and significant improvements in patient care.

Simple to Complex: The Role of Actin and Microtubules in Mitochondrial Dynamics in Amoeba, Yeast, and Mammalian Cells.

Mitochondria are complex organelles that provide energy for the cell in the form of adenosine triphosphate (ATP) and have very specific structures. For most organisms, this is a reticular or tubular mitochondrial network, while others have singular oval-shaped organelles. Nonetheless, maintenance of this structure is dependent on the mitochondrial dynamics, fission, fusion, and motility. Recently, studies have shown that the cytoskeleton has a significant role in the regulation of mitochondrial dynamics. In this review, we focus on microtubules and actin filaments and look at what is currently known about the cytoskeleton's role in mitochondrial dynamics in complex models like mammals and yeast, as well as what is known in the simple model system, Dictyostelium discoideum. Understanding how the cytoskeleton is involved in mitochondrial dynamics increases our understanding of mitochondrial disease, especially neurodegenerative diseases. Increases in fission, loss of fusion, and fragmented mitochondria are seen in several neurodegenerative diseases such as Parkinson's, Alzheimer's, and Huntington's disease. There is no known cure for these diseases, but new therapeutic strategies using drugs to alter mitochondrial fusion and fission activity are being considered. The future of these therapeutic studies is dependent on an in-depth understanding of the mechanisms of mitochondrial dynamics. Understanding the cytoskeleton's role in dynamics in multiple model organisms will further our understanding of these mechanisms and could potentially uncover new therapeutic targets for these neurodegenerative diseases.

Examining the Impact of Structured, Reflective Prebriefing on Student Performance During Simulation.

ABSTRACT: Little is known about the impact of prebriefing on students' experiences of learning with simulation. This mixed-methods study evaluated the impact of prebriefing activities on nursing students' satisfaction, confidence, and performance of nursing skills during a simulation. Findings revealed students who experienced a structured, more robust prebriefing had improved performance during the simulation and reported higher levels of confidence and satisfaction in learning compared to a group that experienced a standard prebriefing. Findings are significant to the profession, they support the incorporation of structured, reflective prebriefing activities in simulation-based experiences.

Follow-up of SARS-CoV-2 positive subgroup from the Asymptomatic novel CORonavirus iNFection study.

A nested longitudinal study within theAsymptomatic novel CORonavirus iNFfection study followed participants with positive nasopharyngeal swab to query for development of symptoms and assess duration of positive reverse transcription-polymerase chain reaction (RT-PCR) test results. Of the 91 participants initially testing positive, 86 participated in follow-up approximately 14 days after study enrollment; of those 86 participants, 19 (22.1%) developed at least one symptom at any time after the initial positive severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) test result. The median number of days to symptom development after their initial positive test result was 6 (range 1-29 days). No participants reported a SARS-CoV-2-related hospitalization. The most frequently reported symptoms were fatigue or muscle aches (10.5%), headache (9.3%), fever (5.8%), and shortness of breath (5.8%). Of the 78 participants who submitted a nasopharyngeal swab for repeat RT-PCR testing, 17 (21.8%) remained positive at Day 14, 4 of which continued to test positive at Day 28. These findings reinforce the probable role of silent SARS-CoV-2 infections in community transmission, and that reliance on symptom development will miss a large proportion of infections. Broad testing programs not limited to individuals presenting with symptoms are critical for identifying persons with SARS-CoV-2 infection and ultimately slowing transmission.

Correction: MicroRNA regulation of persistent stress-enhanced memory.

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

MicroRNA regulation of persistent stress-enhanced memory.

Disruption of persistent, stress-associated memories is relevant for treating posttraumatic stress disorder (PTSD) and related syndromes, which develop in a subset of individuals following a traumatic event. We previously developed a stress-enhanced fear learning (SEFL) paradigm in inbred mice that produces PTSD-like characteristics in a subset of mice, including persistently enhanced memory and heightened cFos in the basolateral amygdala complex (BLC) with retrieval of the remote (30-day-old) stress memory. Here, the contribution of BLC microRNAs (miRNAs) to stress-enhanced memory was investigated because of the molecular complexity they achieve through their ability to regulate multiple targets simultaneously. We performed small-RNA sequencing (smRNA-Seq) and quantitative proteomics on BLC tissue collected from mice 1 month after SEFL and identified persistently changed microRNAs, including mir-135b-5p, and proteins associated with PTSD-like heightened fear expression. Viral-mediated overexpression of mir-135b-5p in the BLC of stress-resilient animals enhanced remote fear memory expression and promoted spontaneous renewal 14 days after extinction. Conversely, inhibition of BLC mir-135b-5p in stress-susceptible animals had the opposite effect, promoting a resilient-like phenotype. mir-135b-5p is highly conserved across mammals and was detected in post mortem human amygdala, as well as human serum samples. The mir-135b passenger strand, mir-135b-3p, was significantly elevated in serum from PTSD military veterans, relative to combat-exposed control subjects. Thus, miR-135b-5p may be an important therapeutic target for dampening persistent, stress-enhanced memory and its passenger strand a potential biomarker for responsivity to a mir-135-based therapeutic.

A cross-sectional community-based observational study of asymptomatic SARS-CoV-2 prevalence in the greater Indianapolis area.

The Asymptomatic novel CORonavirus iNfection (ACORN) study was designed to investigate the prevalence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in the asymptomatic adult population of the Indianapolis metropolitan area, to follow individuals testing positive for the development of symptoms, and to understand duration of positive test results. ACORN is a cross-sectional community-based observational study of adult residents presenting asymptomatic for COVID-like illness, defined as the self-reported absence of the following three symptoms in the last 7 days: fever (≥100°F), new-onset or worsening cough, and new-onset or worsening shortness of breath. SARS-CoV-2 infection was determined by real-time reverse transcription-polymerase chain reaction in nasopharyngeal swab samples. SARS-CoV-2 infection prevalence was expressed as a point estimate with 95% confidence interval (CI). Test results are reported for 2953 participants who enrolled and underwent nasopharyngeal swab testing between 7 April 2020 and 16 May 2020. Among tested participants, 91 (3.1%; 95% CI: 2.5%-3.7%) were positive for SARS-CoV-2. Overall, baseline characteristics, medical history, and infection risk factors were comparable between SARS-CoV-2 positive and negative participants. Within the ongoing 14-day follow-up period for positive participants, 58 (71.6%) of 81 assessed participants remained asymptomatic while others (n = 23, 28.4%) reported one or more symptoms. Indiana had "Stay-at-Home" orders in place during nearly the entire test period reported here, yet 3.1% of asymptomatic participants tested positive for SARS-CoV-2. These results indicate screening questions had limited predictive utility for testing in an asymptomatic population and suggest broader testing strategies are needed. Importantly, these findings underscore that more research is needed to understand the viral transmission and the role asymptomatic and presymptomatic individuals play in this global pandemic.

Expression of a heroin contextually conditioned immune effect in male rats requires CaMKIIα-expressing neurons in dorsal, but not ventral, subiculum and hippocampal CA1.

The physiological and motivational effects of heroin and other abused drugs become associated with environmental (contextual) stimuli during repeated drug use. As a result, these contextual stimuli gain the ability to elicit drug-like conditioned effects. For example, after context-heroin pairings, exposure to the heroin-paired context alone produces similar effects on peripheral immune function as heroin itself. Conditioned immune effects can significantly exacerbate the adverse health consequences of heroin use. Our laboratory has shown that exposure to a heroin-paired context suppresses lipopolysaccharide (LPS)-induced splenic nitric oxide (NO) production in male rats, and this effect is mediated in part by the dorsal hippocampus (dHpc). However, specific dHpc output regions, whose efferents might mediate conditioned immune effects, have not been identified, nor has the contribution of ventral hippocampus (vHpc) been investigated. Here, we evaluated the role of CaMKIIα-expressing neurons in the dHpc and vHpc main output regions by expressing Gi-coupled designer receptors exclusively activated by designer drugs (DREADDs) under a CaMKIIα promoter in the dorsal subiculum and CA1 (dSub, dCA1) or ventral subiculum and CA1 (vSub, vCA1). After context-heroin conditioning, clozapine-N-oxide (CNO, DREADD agonist) or vehicle was administered systemically prior to heroin-paired context (or home-cage control) exposure and LPS immune challenge. Chemogenetic inhibition of CaMKIIα-expressing neurons in dHpc, but not vHpc, output regions attenuated the expression of conditioned splenic NO suppression. These results establish that the main dHpc output regions, the dSub and dCA1, are critical for this context-heroin conditioned immune effect.

microRNA mir-598-3p mediates susceptibility to stress enhancement of remote fear memory.

microRNAs (miRNAs) have emerged as potent regulators of learning, recent memory, and extinction. However, our understanding of miRNAs directly involved in regulating complex psychiatric conditions perpetuated by aberrant memory, such as in posttraumatic stress disorder (PTSD), remains limited. To begin to address the role of miRNAs in persistent memories, we performed small-RNA sequencing on basolateral amygdala (BLA) tissue and identified miRNAs altered by auditory fear conditioning (FC) one month after training. mir-598-3p, a highly conserved miRNA previously unstudied in the brain, was down-regulated in the BLA. Further decreasing BLA mir-598-3p levels did not increase strength of the remote fear memory. Given that stress is a critical component in PTSD, we next assessed the impact of stress and stress-enhanced fear learning (SEFL) on mir-598-3p levels, finding the miRNA is elevated in the BLA of male, but not female, mice susceptible to the effects of stress in SEFL. Accordingly, intra-BLA inhibition of mir-598-3p interfered with expression and extinction of the remote fear memory in male, but not female, mice. This effect could not be attributed to an anxiolytic effect of miRNA inhibition. Finally, bioinformatic analysis following quantitative proteomics on BLA tissue collected 30 d post-SEFL training identified putative mir-598-3p targets and related pathways mediating the differential susceptibility, with evidence for regulation of the actin cytoskeleton, the core mediator of structural plasticity. Taken together, the results suggest BLA mir-598-3p may be recruited by stress to mediate a critical switch from a salient remote fear memory to one that is enhanced and extinction-resistant.

Hippocampal interleukin-1 mediates stress-enhanced fear learning: A potential role for astrocyte-derived interleukin-1ß.

Post-traumatic stress disorder (PTSD) is associated with immune dysregulation. We have previously shown that severe stress exposure in a preclinical animal model of the disorder, stress-enhanced fear learning (SEFL), is associated with an increase in hippocampal interleukin-1ß (IL-1ß) and that blocking central IL-1 after the severe stress prevents the development of SEFL. Here, we tested whether blocking hippocampal IL-1 signaling is sufficient to prevent enhanced fear learning and identified the cellular source of stress-induced IL-1ß in this region. Experiment 1 tested whether intra-dorsal hippocampal (DH) infusions of interleukin-1 receptor antagonist (IL-1RA, 1.25µg per hemisphere) 24 and 48h after stress exposure prevents the development of enhanced fear learning. Experiment 2 used triple fluorescence immunohistochemistry to examine hippocampal alterations in IL-1ß, glial fibrillary acidic protein (GFAP), an astrocyte-specific marker, and ionized calcium binding adaptor molecule -1 (Iba-1), a microglial-specific marker, 48h after exposure to the severe stressor of the SEFL paradigm. Intra-DH IL-1RA prevented SEFL and stress-induced IL-1ß was primarily colocalized with astrocytes in the hippocampus. Further, hippocampal GFAP immunoreactivity was not altered, whereas hippocampal Iba-1 immunoreactivity was significantly attenuated following severe stress. These data suggest that hippocampal IL-1 signaling is critical to the development of SEFL and that astrocytes are a predominant source of stress-induced IL-1ß.

Dorsal hippocampal neural immune signaling regulates heroin-conditioned immunomodulation but not heroin-conditioned place preference.

Repeated pairings of heroin and a context results in Pavlovian associations which manifest as heroin-conditioned appetitive responses and peripheral immunomodulation upon re-exposure to heroin-paired conditioned stimuli (CS). The dorsal hippocampus (DH) plays a key role in the neurocircuitry governing these context-heroin associations. Within the DH, expression of the pro-inflammatory cytokine interleukin-1ß (IL-1ß) is required for heroin-conditioned peripheral immunomodulation to occur. However, the role of signaling via IL-1 receptor type 1 (IL-1R1) has not been examined. Furthermore, it has not been evaluated whether the involvement of IL-1 in associative learning extends to classically conditioned appetitive behaviors, such as conditioned place preference (CPP). The first set of experiments investigated whether DH IL-1R1 signaling during CS re-exposure modulates heroin-conditioned immunomodulation and heroin-CPP. The second set of experiments employed chemogenetic techniques to examine whether DH astroglial signaling during CS re-exposure alters the same Pavlovian responses. This line of investigation is based on previous research indicating that astrocytes support hippocampal-dependent learning and memory through the expression of IL-1ß protein and IL-1R1. Interestingly, IL-1R1 antagonism disrupted heroin-conditioned suppression of peripheral immune parameters but failed to alter heroin-CPP. Similarly, chemogenetic stimulation of Gi-signaling in DH astrocytes attenuated heroin-conditioned peripheral immunomodulation but failed to alter heroin-CPP. Collectively our data show that both IL-1R1 stimulation and astrocyte signaling in the DH are critically involved in the expression of heroin-conditioned immunomodulation but not heroin-CPP. As such these findings strongly suggest hippocampal neuroimmune signaling differentially regulates Pavlovian immunomodulatory and appetitive behaviors.

Population and evolutionary dynamics of Shiga-toxin producing Escherichia coli O157 in a beef herd: A longitudinal study.

Shiga toxin producing Escherichia coli O157:H7 (STEC O157) is naturally found in the gastrointestinal tract of cattle and can cause severe disease in humans. There is limited understanding of the population dynamics and microevolution of STEC O157 at herd level. In this study, isolates from a closed beef herd of 23 cows were used to examine the population turnover in the herd. Of the nine STEC O157 clades previously described, clade 7 was found in 162 of the 169 isolates typed. Multiple locus variable number tandem repeat analysis (MLVA) differentiated 169 isolates into 33 unique MLVA types. Five predominant MLVA types were evident with most of the remaining types containing only a single isolate. MLVA data suggest that over time clonal replacement occurred within the herd. Genome sequencing of 18 selected isolates found that the isolates were divided into four lineages, representing four different 'clones' in the herd. Genome data confirmed clonal replacement over time and provided evidence of cross transmission of strains between cows. The findings enhanced our understanding of the population dynamics of STEC O157 in its natural host that will help developing effective control measures to prevent the spread of the pathogen to the human population.

Interleukin-1 signaling in the basolateral amygdala is necessary for heroin-conditioned immunosuppression.

Heroin administration suppresses the production of inducible nitric oxide (NO), as indicated by changes in splenic inducible nitric oxide synthase (iNOS) and plasma nitrate/nitrite. Since NO is a measure of host defense against infection and disease, this provides evidence that heroin can increase susceptibility to pathogens by directly interacting with the immune system. Previous research in our laboratory has demonstrated that these immunosuppressive effects of heroin can also be conditioned to environmental stimuli by repeatedly pairing heroin administration with a unique environmental context. Re-exposure to a previously drug-paired context elicits immunosuppressive effects similar to heroin administration alone. In addition, our laboratory has reported that the basolateral amygdala (BLA) and medial nucleus accumbens shell (mNAcS) are critical neural substrates that mediate this conditioned effect. However, our understanding of the contributing mechanisms within these brain regions is limited. It is known that the cytokine interleukin-1 (IL-1) plays an important role in learning and memory. In fact, our laboratory has demonstrated that inhibition of IL-1ß expression in the dorsal hippocampus (DH) prior to re-exposure to a heroin-paired context prevents the suppression of measures of NO production. Therefore, the present studies sought to further investigate the role of IL-1 in heroin-conditioned immunosuppression. Blockade of IL-1 signaling in the BLA, but not in the caudate putamen or mNAcS, using IL-1 receptor antagonist (IL-1Ra) attenuated heroin-conditioned immunosuppression of NO production as measured by plasma nitrate/nitrite and iNOS mRNA expression in spleen tissue. Taken together, these findings suggest that IL-1 signaling in the BLA is necessary for the expression of heroin-conditioned immunosuppression of NO production and may be a target for interventions that normalize immune function in heroin users and patient populations exposed to opiate regimens.

Validating the Performance of the Modified Early Obstetric Warning System Multivariable Model to Predict Maternal Intensive Care Unit Admission.

OBJECTIVES: To evaluate the performance of the Modified Early Obstetric Warning System (MEOWS) to predict maternal ICU admission in an obstetric population. DESIGN: Case-control study. SETTING: Two maternity units in Vancouver, Canada, one with ICU facilities, between January 1, 2000, and December 31, 2011. PATIENTS: Pregnant or recently delivered (≤6 weeks) women admitted to the hospital for >24 hours. Three control patients were randomly selected per case and matched for year of admission. MEASUREMENTS AND MAIN RESULTS: Retrospective, observational, case-control validation study investigating the physiologic predictors of admission in the 24-hour period preceding either ICU admission >24 hours (cases) or following admission (control patients). Model performance was assessed based on sensitivity, specificity, and predictive values. Forty-six women were admitted to the ICU for >24 hours (0.51/1000 deliveries); the study included 138 randomly selected control patients. There were no maternal deaths in the cohort. MEOWS had high sensitivity (0.96) but low specificity (0.54) for ICU admission >24 hours, whereas ≥1 one red trigger maintained sensitivity (0.96) and improved specificity (0.73). CONCLUSION: Altering MEOWS trigger parameters may improve the accuracy of MEOWS in predicting ICU admission. Formal modelling of a MEOWS scoring system is required to support evidence-based care.

Acquisition of heroin conditioned immunosuppression requires IL-1 signaling in the dorsal hippocampus.

Opioid users experience increased incidence of infection, which may be partially attributable to both direct opiate-immune interactions and conditioned immune responses. Previous studies have investigated the neural circuitry governing opioid conditioned immune responses, but work remains to elucidate the mechanisms mediating this effect. Our laboratory has previously shown that hippocampal IL-1 signaling, specifically, is required for the expression of heroin conditioned immunosuppression following learning. The current studies were designed to further characterize the role of hippocampal IL-1 in this phenomenon by manipulating IL-1 during learning. Experiment 1 tested whether hippocampal IL-1 is also required for the acquisition of heroin conditioned immunosuppression, while Experiment 2 tested whether hippocampal IL-1 is required for the expression of unconditioned heroin immunosuppression. We found that blocking IL-1 signaling in the dorsal hippocampus with IL-1RA during each conditioning session, but not on interspersed non-conditioning days, significantly attenuated the acquisition of heroin conditioned immunosuppression. Strikingly, we found that the same IL-1RA treatment did not alter unconditioned immunosuppression to a single dose of heroin. Thus, IL-1 signaling is not a critical component of the response to heroin but rather may play a role in the formation of the association between heroin and the context. Collectively, these studies suggest that IL-1 signaling, in addition to being involved in the expression of a heroin conditioned immune response, is also involved in the acquisition of this effect. Importantly, this effect is likely not due to blocking the response to the unconditioned stimulus since IL-1RA did not affect heroin's immunosuppressive effects.

Baseline characteristics and hospitalizations in patients with schizophrenia receiving olanzapine long-acting injection: an interim analysis from a non-interventional, prospective observational safety study.

BACKGROUND: Depot antipsychotics are a treatment option for medication nonadherence in patients with schizophrenia. Nonadherence can lead to increased relapse and hospitalization rates. This article reports hospitalization data before and after initiation of olanzapine long-acting injection (LAI), a depot antipsychotic. METHODS: Data were assessed from an ongoing, multinational, prospective, observational post-authorisation safety study being conducted to evaluate post-injection delirium/sedation syndrome (PDSS), an adverse reaction that can occur following injection of olanzapine LAI. Eligible patients were aged ≥18 years, diagnosed with schizophrenia, were prescribed olanzapine LAI, and lived outside the United States. Psychiatric hospitalization and medication data were collected retrospectively for the 6-month period before study entry and prospectively throughout the study. Paired t-tests and McNemar's tests were used to assess changes in hospitalization incidence and duration. Stepwise Cox proportional hazards models assessed factors associated with hospitalizations. Analyses were based on data from the first 3 years of the continuously enrolling study (N = 668). RESULTS: The average duration of olanzapine LAI exposure for all patients was 0.768 years. Of the 529 patients who received at least 1 injection of olanzapine LAI and were not hospitalized at study entry, 8.1% had at least 1 subsequent psychiatric hospitalization with a mean duration of 2.0 days. Of the 288 patients who had a >6-month follow-up, 8.3% had at least 1 post-baseline psychiatric hospitalization with a mean duration of 2.3 days. The incidence of hospitalizations in the 6-month period after treatment was significantly lower than that in the 6-month period prior to treatment (8.3 vs 32.6%, respectively; P < 0.001). Furthermore, mean hospitalization duration decreased from 11.5 days in the 6-month period before treatment to 2.3 days in the 6-month period after treatment (P < 0.001). Psychiatric hospitalization in the prior 12 months (P < 0.0001) and recreational drug use within 24 h of baseline visit (P = 0.015) were identified as potential predictors of time to first psychiatric hospitalization after beginning to take olanzapine LAI. At the time of interim analysis, 5 PDSS events had occurred, which was too few for a full analysis of those events. CONCLUSIONS: Results indicate a significant reduction in the incidence and days of hospitalization from the 6-month period before to the 6-month period after olanzapine LAI initiation, which suggests reduced relapse and hospitalization during treatment. Results should be interpreted with caution due to the observational nature of the study and use of retrospective baseline data.

Quantitative analysis of spherical microbubble cavity array formation in thermally cured polydimethylsiloxane for use in cell sorting applications.

Microbubbles are spherical cavities formed in thermally cured polydimethylsiloxane (PDMS) using the gas expansion molding technique. Microbubble cavity arrays are generated by casting PDMS over a silicon wafer mold containing arrays of deep etched pits. To be useful in various high throughput cell culture and sorting applications it is imperative that uniform micron-sized cavities can be formed over large areas (in(2)). This paper provides an in-depth quantitative analysis of the fabrication parameters that effect the microbubble cavity formation efficiency and size. These include (1) the hydrophobic coating of the mold, (2) the mold pit dimensions, (3) the spatial arrangement of the pit openings, (4) the curing temperature of PDMS pre-polymer, (5) PDMS thickness, and (6) the presence and composition of residual gas in the PDMS pre-polymer mixture. Results suggest that the principles of heterogeneous nucleation and gas diffusion govern microbubble cavity formation, and that surface tension prevents detachment of the vapor bubble that forms in the PDMS over the pit. Paramerters are defined that enable the fabrication of large format arrays with uniform cavity size over 6 in(2) with a coefficient-of-variation <10 %. The architecture of the microbubble cavity is uniquely advantageous for cell culture. Large format arrays provide a highly versatile system that can be adapted for use in various high-throughput cell sorting applications. Herein, we demonstrate the use of microbubble cavity arrays to dissect the cellular heterogeneity that exists in a tumorigenic cutaneous squamous cell carcinoma cell line at the single cell level.

The draft genome of the transgenic tropical fruit tree papaya (Carica papaya Linnaeus).

Papaya, a fruit crop cultivated in tropical and subtropical regions, is known for its nutritional benefits and medicinal applications. Here we report a 3x draft genome sequence of 'SunUp' papaya, the first commercial virus-resistant transgenic fruit tree to be sequenced. The papaya genome is three times the size of the Arabidopsis genome, but contains fewer genes, including significantly fewer disease-resistance gene analogues. Comparison of the five sequenced genomes suggests a minimal angiosperm gene set of 13,311. A lack of recent genome duplication, atypical of other angiosperm genomes sequenced so far, may account for the smaller papaya gene number in most functional groups. Nonetheless, striking amplifications in gene number within particular functional groups suggest roles in the evolution of tree-like habit, deposition and remobilization of starch reserves, attraction of seed dispersal agents, and adaptation to tropical daylengths. Transgenesis at three locations is closely associated with chloroplast insertions into the nuclear genome, and with topoisomerase I recognition sites. Papaya offers numerous advantages as a system for fruit-tree functional genomics, and this draft genome sequence provides the foundation for revealing the basis of Carica's distinguishing morpho-physiological, medicinal and nutritional properties.

Characterization of cell seeding and specific capture of B cells in microbubble well arrays.

Development of micro-well array systems for use in high-throughput screening of rare cells requires a detailed understanding of the factors that impact the specific capture of cells in wells and the distribution statistics of the number of cells deposited into wells. In this study we investigate the development of microbubble (MB) well array technology for sorting antigen-specific B-cells. Using Poisson statistics we delineate the important role that the fractional area of MB well opening and the cell seeding density have on determining cell seeding distribution in wells. The unique architecture of the MB well hinders captured cells from escaping the well and provides a unique microenvironmental niche that enables media changes as needed for extended cell culture. Using cell lines and primary B and T cells isolated from human peripheral blood we demonstrate the use of affinity capture agents coated in the MB wells to enrich for the selective capture of B cells. Important differences were noted in the efficacy of bovine serum albumin to block the nonspecific adsorption of primary cells relative to cell lines as well as the efficacy of the capture coatings using mixed primary B and T cells samples. These results emphasize the importance of using primary cells in technology development and suggest the need to utilize B cell capture agents that are insensitive to cell activation.