Messenger RNA transport on lysosomal vesicles maintains axonal mitochondrial homeostasis and prevents axonal degeneration.

In neurons, RNA granules are transported along the axon for local translation away from the soma. Recent studies indicate that some of this transport involves hitchhiking of RNA granules on lysosome-related vesicles. In the present study, we leveraged the ability to prevent transport of these vesicles into the axon by knockout of the lysosome-kinesin adaptor BLOC-one-related complex (BORC) to identify a subset of axonal mRNAs that depend on lysosome-related vesicles for transport. We found that BORC knockout causes depletion of a large group of axonal mRNAs mainly encoding ribosomal and mitochondrial/oxidative phosphorylation proteins. This depletion results in mitochondrial defects and eventually leads to axonal degeneration in human induced pluripotent stem cell (iPSC)-derived and mouse neurons. Pathway analyses of the depleted mRNAs revealed a mechanistic connection of BORC deficiency with common neurodegenerative disorders. These results demonstrate that mRNA transport on lysosome-related vesicles is critical for the maintenance of axonal homeostasis and that its failure causes axonal degeneration.

"Tumor Treating Fields" delivered via electromagnetic induction have varied effects across glioma cell lines and electric field amplitudes.

Previous studies reported that alternating electric fields (EFs) in the intermediate frequency (100-300 kHz) and low intensity (1-3 V/cm) regime - termed "Tumor Treating Fields" (TTFields) - have a specific, anti-proliferative effect on glioblastoma multiforme (GBM) cells. However, the mechanism(s) of action remain(s) incompletely understood, hindering the clinical adoption of treatments based on TTFields. To advance the study of such treatment in vitro, we developed an inductive device to deliver EFs to cell cultures which improves thermal and osmolar regulation compared to prior devices. Using this inductive device, we applied continuous, 200 kHz electromagnetic fields (EMFs) with a radial EF amplitude profile spanning 0-6.5 V/cm to cultures of primary rat astrocytes and several human GBM cell lines - U87, U118, GSC827, and GSC923 - for a duration of 72 hours. Cell density was assessed via segmented pixel densities from GFP expression (U87, U118) or from staining (astrocytes, GSC827, GSC923). Further RNA-Seq analyses were performed on GSC827 and GSC923 cells. Treated cultures of all cell lines exhibited little to no change in proliferation at lower EF amplitudes (0-3 V/cm). At higher amplitudes (> 4 V/cm), different effects were observed. Apparent cell densities increased (U87), decreased (GSC827, GSC923), or showed little change (U118, astrocytes). RNA-Seq analyses on treated and untreated GSC827 and GSC923 cells revealed differentially expressed gene sets of interest, such as those related to cell cycle control. Up- and down-regulation, however, was not consistent across cell lines nor EF amplitudes. Our results indicate no consistent, anti-proliferative effect of 200 kHz EMFs across GBM cell lines and thus contradict previous in vitro findings. Rather, effects varied across different cell lines and EF amplitude regimes, highlighting the need to assess the effect(s) of TTFields and similar treatments on a per cell line basis.

YAP localization mediates mechanical adaptation of human cancer cells during extravasation in vivo.

Biophysical profiling of primary tumors has revealed that individual tumor cells fall along a highly heterogeneous continuum of mechanical phenotypes. One idea is that a subset of tumor cells is "softer" to facilitate detachment and escape from the primary site, a step required to initiate metastasis. However, it has also been postulated that cells must be able to deform and generate sufficient force to exit into distant sites. Here, we aimed to dissect the mechanical changes that occur during extravasation and organ colonization. Using multiplexed methods of intravital microscopy and optical tweezer based active microrheology, we obtained longitudinal images and mechanical profiles of cells during organ colonization in vivo. We determined that cells were softer, more liquid like upon exit of the vasculature but stiffened and became more solid like once in the new organ microenvironment. We also determined that a YAP mediated mechanogenotype influenced the global dissemination in our in vivo and in vitro models and that reducing mechanical heterogeneity could reduce extravasation. Moreover, our high throughput analysis of mechanical phenotypes of patient samples revealed that this mechanics was in part regulated by the external hydrodynamic forces that the cancer cells experienced within capillary mimetics. Our findings indicate that disseminated cancer cells can keep mutating with a continuum landscape of mechano-phenotypes, governed by the YAP-mediated mechanosensing of hydrodynamic flow.

The differential impact of COVID-19 on the psychological stress of post-9/11 veterans: Gender, race, and ethnicity.

The COVID-19 pandemic has led to economic turndowns, social restrictions, and family life alterations. The stress induced by the public health crisis and its consequences are beginning to be explored. This study examined stress experiences since the pandemic'sonset in work, financial, social, and health domainsamong a large sample of post-9/11, United States military veterans. The sample, who separated from active-duty service or deactivated from active status in a reserve component in 2016, completed an online survey (n = 3180) in 2020. Participants were 70% White non-Hispanic, 81% male, and had an average age of 38 years. Frequencies and descriptive statistics were calculated. Female veterans and veterans of colour reported significantly higher levels of stress across most life domains. The results suggest White, male, post-9/11 veterans may be somewhat protected from COVID-19 stress, but that the pandemic is exacerbatinghealth and social disparities experienced by post-9/11 veterans of colour and female veterans. Supports and comprehensive care, particularly targeted towardsat-risk populations, are likely needed to provide sufficient resources for resiliency during and after the pandemic.

Towards a scientific community consensus on designating Vulnerable Marine Ecosystems from imagery.

Management of deep-sea fisheries in areas beyond national jurisdiction by Regional Fisheries Management Organizations/Arrangements (RFMO/As) requires identification of areas with Vulnerable Marine Ecosystems (VMEs). Currently, fisheries data, including trawl and longline bycatch data, are used by many RFMO/As to inform the identification of VMEs. However, the collection of such data creates impacts and there is a need to collect non-invasive data for VME identification and monitoring purposes. Imagery data from scientific surveys satisfies this requirement, but there currently is no established framework for identifying VMEs from images. Thus, the goal of this study was to bring together a large international team to determine current VME assessment protocols and establish preliminary global consensus guidelines for identifying VMEs from images. An initial assessment showed a lack of consistency among RFMO/A regions regarding what is considered a VME indicator taxon, and hence variability in how VMEs might be defined. In certain cases, experts agreed that a VME could be identified from a single image, most often in areas of scleractinian reefs, dense octocoral gardens, multiple VME species' co-occurrence, and chemosynthetic ecosystems. A decision flow chart is presented that gives practical interpretation of the FAO criteria for single images. To further evaluate steps of the flow chart related to density, data were compiled to assess whether scientists perceived similar density thresholds across regions. The range of observed densities and the density values considered to be VMEs varied considerably by taxon, but in many cases, there was a statistical difference in what experts considered to be a VME compared to images not considered a VME. Further work is required to develop an areal extent index, to include a measure of confidence, and to increase our understanding of what levels of density and diversity correspond to key ecosystem functions for VME indicator taxa. Based on our results, the following recommendations are made: 1. There is a need to establish a global consensus on which taxa are VME indicators. 2. RFMO/As should consider adopting guidelines that use imagery surveys as an alternative (or complement) to using bycatch and trawl surveys for designating VMEs. 3. Imagery surveys should also be included in Impact Assessments. And 4. All industries that impact the seafloor, not just fisheries, should use imagery surveys to detect and identify VMEs.

RSV infection in children hospitalised with severe lower respiratory tract infection in a low-middle-income setting: A cross-sectional observational study.

INTRODUCTION: Low- and middle-income countries carry the largest burden of Respiratory syncytial virus (RSV) disease, with most deaths occurring in these settings. This study aimed to investigate the burden of RSV disease in South African children hospitalised with lower respiratory tract infection (LRTI), with specific reference to incidence, risk factors, and co-infections. METHODS: A database from a previous prospective study containing demographic, laboratory and clinical data on children hospitalised with LRTIs in Cape Town, South Africa, was used. A nasopharyngeal swab (NP) and induced sputum (IS) were tested for RSV PCR. Descriptive statistics were used to characterise the study population, and a multivariable analysis of risk factors and co-infections was done. RESULTS: RSV was detected in 142 (30.9%; 95% CI 26.7-35.3) of the included 460 study children with LRTI. The median age of RSV-positive children was 4.6 (IQR 2.4-9.7) months compared to RSV-negative children of 10.5 (IQR 4.4-21.3) months, P = <0.001. Most cases occurred in autumn and winter with 126 (89%) cases over this period. IS demonstrated greater sensitivity for RSV diagnosis with 135 cases (95.1%) detected on IS and 57 cases (40.1%) identified on NP; P<0.001. The median length of hospital stay was 3.3 (SD 4.2) days in the RSV positive group and 2.7 (SD 3.3) days in the RSV negative group; P<0.001. The median number of detected viral pathogens was 1 (IQR 0-2) in RSV-positive children (when RSV was excluded from the count) compared to 2 (IQR 2-3) in RSV negative children; P<0.001. The presence of RSV was independently associated with a reduction in the frequency of most viruses tested for on PCR. CONCLUSIONS: RSV is common in children hospitalised with LRTI and mainly affects younger children. There is an urgent need to find an effective vaccine to prevent RSV pneumonia in children worldwide, especially in LMICs that carry the greatest burden of disease.

Defining "Arts Participation" for Public Health Research.

Arts participation has been linked to positive health outcomes around the globe. As more research is taking place on this topic, there is heightened need for definitions for the complex concepts involved. While significant work to define "arts participation" has taken place in the arts sector, less work has been undertaken for the purpose of researching the arts in public health. This study developed a definition for "arts participation" to guide a national arts in public health research agenda and to advance and make more inclusive previous work to define the term. A convergent mixed-methods study design with sequential elements was used to iteratively develop a definition that integrated the perspectives of field experts as well as the general public. Literature review was followed by four iterative phases of data collection, analysis, and integration, and a proposed definition was iteratively revised at each stage. The final definition includes modes, or ways, in which people engage with the arts, and includes examples of various art forms intended to frame arts participation broadly and inclusively. This definition has the potential to help advance the quality and precision of research aimed at evaluating relationships between arts participation and health, as well as outcomes of arts-based health programs and interventions in communities. With its more inclusive framing than previous definitions, it can also help guide the development of more inclusive search strategies for evidence synthesis in this rapidly growing arena and assist researchers in developing more effective survey questions and instruments.

"Tumor Treating Fields" delivered via electromagnetic induction have varied effects across glioma cell lines and electric field amplitudes.

Previous studies reported that alternating electric fields (EFs) in the intermediate frequency (100 - 300 kHz) and low intensity (1 - 3 V/cm) regime - termed "Tumor Treating Fields" (TTFields) - have a specific, anti-proliferative effect on glioblastoma multiforme (GBM) cells. However, the mechanism(s) of action remain(s) incompletely understood, hindering the clinical adoption of treatments based on TTFields. To advance the study of such treatment in vitro , we developed an inductive device to deliver EFs to cell cultures which improves thermal and osmolar regulation compared to prior devices. Using this inductive device, we applied continuous, 200 kHz electromagnetic fields (EMFs) with a radial EF amplitude profile spanning 0 - 6.5 V/cm to cultures of primary rat astrocytes and several human GBM cell lines - U87, U118, GSC827, and GSC923 - for a duration of 72 hours. Cell density was assessed via segmented pixel densities from GFP expression (U87, U118) or from staining (astrocytes, GSC827, GSC923). Further RNA-Seq analyses were performed on GSC827 and GSC923 cells. Treated cultures of all cell lines exhibited little to no change in proliferation at lower EF amplitudes (0 - 3 V/cm). At higher amplitudes (> 4 V/cm), different effects were observed. Apparent cell densities increased (U87), decreased (GSC827, GSC923), or showed little change (U118, astrocytes). RNA-Seq analyses on treated and untreated GSC827 and GSC923 cells revealed differentially expressed gene sets of interest, such as those related to cell cycle control. Up- and down-regulation, however, was not consistent across cell lines nor EF amplitudes. Our results indicate no consistent, anti-proliferative effect of 200 kHz EMFs across GBM cell lines and thus contradict previous in vitro findings. Rather, effects varied across different cell lines and EF amplitude regimes, highlighting the need to assess the effect(s) of TTFields and similar treatments on a per cell line basis.

The Effects of Adverse Childhood Experiences and Warfare Exposure on Military Sexual Trauma Among Veterans.

Military sexual trauma (MST) is a pervasive problem; this study examined the relationship of the precursory traumas of adverse childhood experiences (ACEs) and warfare exposure with MST. Post-9/11 veterans were surveyed at 3 months and at 24 to 30 months post-military separation. Female veterans who experienced at least 1 ACE but no warfare exposure were significantly more likely to receive unwanted sexual attention. Veterans (males and females) experiencing three or more ACEs but no warfare exposure were significantly more likely to receive unwanted sexual attention and contact. Experiencing only warfare exposure was not related to unwanted sexual attention or contact for females; however, a significant interaction was found between combined warfare exposure, ACEs, and MST for males and females. Veterans who reported warfare exposure and one to two or three or more ACEs were more likely to report unwanted sexual attention and/or contact. Exploration of individual ACEs revealed a significant relationship between childhood sexual abuse and unwanted sexual contact. For females, witnessing domestic violence predicted unwanted sexual contact. There was also a significant interaction between childhood sexual abuse and warfare exposure. Females who experienced both childhood sexual abuse and warfare exposure were significantly more likely to receive unwanted sexual attention and unwanted sexual contact. Albeit a small sample, males who experienced both were also significantly more likely to receive unwanted sexual attention. The findings reveal that precursory traumatic experiences in childhood and the interaction of ACEs and warfare exposure during military service can increase the likelihood of unwanted sexual attention and contact. This research further substantiates the need for screening efforts. It also demonstrates the importance of practitioners engaging in trauma-informed care principles and practices to address the residual effects of previous experiences during sexual trauma or mental health treatment efforts.

Computational Modeling and Imaging of the Intracellular Oxygen Gradient.

Computational modeling can provide a mechanistic and quantitative framework for describing intracellular spatial heterogeneity of solutes such as oxygen partial pressure (pO2). This study develops and evaluates a finite-element model of oxygen-consuming mitochondrial bioenergetics using the COMSOL Multiphysics program. The model derives steady-state oxygen (O2) distributions from Fickian diffusion and Michaelis-Menten consumption kinetics in the mitochondria and cytoplasm. Intrinsic model parameters such as diffusivity and maximum consumption rate were estimated from previously published values for isolated and intact mitochondria. The model was compared with experimental data collected for the intracellular and mitochondrial pO2 levels in human cervical cancer cells (HeLa) in different respiratory states and under different levels of imposed pO2. Experimental pO2 gradients were measured using lifetime imaging of a Förster resonance energy transfer (FRET)-based O2 sensor, Myoglobin-mCherry, which offers in situ real-time and noninvasive measurements of subcellular pO2 in living cells. On the basis of these results, the model qualitatively predicted (1) the integrated experimental data from mitochondria under diverse experimental conditions, and (2) the impact of changes in one or more mitochondrial processes on overall bioenergetics.

The Moral Injury Symptoms Scale-Military Version-Short Form: Further Scale Validation in a U.S. Veteran Sample.

This study assessed the Moral Injury Symptoms Scale - Military Version - Short Form's (MISS-M-SF) factor structure and construct validity. Participants included 3650 combat-deployed U.S. veterans who answered all 10 MISS-M-SF items from the sixth wave of The Veterans Metric Initiative (TVMI). EFA results suggested a two-factor solution, based on item wording, fit best. CFA results indicated a bifactor model (one general factor and two method factors, based on item wording) fit best. Further investigation revealed that a one-factor model could be used despite the data's multidimensionality. Item-level analyses revealed four items represented the general factor exceptionally well, potentially simplifying assessment in research and clinical applications. Construct validity was also demonstrated through moderate to high correlations with conceptually related measures.

The subjective underemployment experience of post-9/11 veterans after transition to civilian work.

BACKGROUND: Underemployment is a challenge for the civilian workforce and a particular risk for veterans as they transition from military service to civilian employment. Workers' economic and demographic characteristics factor into underemployment risk. Veterans may be at greater risk due to specific economic and demographic factors, transitional factors (e.g., geographic relocation), and characteristics of their military service (e.g., military skill alignment with civilian jobs). OBJECTIVES: Describe underemployment experiences in employed post-9/11 veterans three years after their military transition to the civilian workforce. METHODS: The current study uses self-reported underemployment experience data from a longitudinal study of transitioning veterans. This study compares average perceptions of veteran underemployment experiences by specific groups (e.g., by race, gender, and paygrade) using analysis of variance and logistic regression. RESULTS: Veterans reported underemployment in their current jobs based on a perceived mismatch between the skills, education, and/or leadership experience they gained during military service. CONCLUSIONS: Veterans who were enlisted rank, identified as non-White, completed a bachelor's degree, and indicated PTSD symptoms reported higher pervasive underemployment. Intervention implications for the results, such as employer and veteran employment supports, are discussed.

Promoting Social and Emotional Learning in Middle School: Intervention Effects of Facing History and Ourselves.

There are fewer evidence-based social and emotional learning programs for middle school students compared to younger grades. This randomized controlled trial tests the effectiveness of Facing History and Ourselves (hereafter, Facing History) with a sample of 694 (Facing History n = 437; Comparison n = 257) students from a low-resourced school district. Youth self-identified as female (59%), Black/African American (61%), Hispanic/Latinx (18%), White (2%), and multi-racial or some other race/ethnicity (19%). Intervention students perceived their classrooms as more caring and democratic than students in the comparison group. They reported higher levels of empathy, prosocial behavior, and stronger participatory citizenship beliefs. This study expands the evidence-based of effective SEL programs available to schools.

The influence of employment program components upon job attainment during a time of identity and career transition.

This study assessed the effectiveness of employment programs components, which resulted in the identification of content and process components that increase employability. Employment program use was studied among 1172 United States of America military veterans to determine which content (i.e., interviewing skills) and process (i.e., working with a mentor/coach) components influence job attainment during a time of career and identity transition. Components were distilled utilizing a common components analysis approach (Morgan et al., 2018). Associations with finding employment up to 15 months after the military-to-civilian transition were explored. Veterans who engaged with employment programs were primarily accessing the following components: career planning, resume writing, and interviewing skills. However, only a few content components were significantly related to obtaining employment: interviewing (with mentor/coach), resume writing (online tools), translating military to civilian work (with mentor/coach), entrepreneurship (with mentor/coach), and virtual career fairs. Furthermore, not all processes or modes of instruction for content components were associated with success in the job market. Having a mentor/coach was one of the most effective delivery strategies. For example, veterans using programs delivered by a mentor/coach that translated military skills to civilian work were more likely to find a job at 6-9-months (84%) and 12-15-months (91%) post military separation. In addition, risks that predicted lower use of employment program components by veterans were identified such as junior enlisted rank, combat exposure, combat arms occupation, and physical health problems. With these findings, program developers, implementers, and funders can channel efforts towards the utilization of employment programs with effective components.

L'influence des composantes du programme d'emploi sur l'obtention d'un travail au cours d' une période de transition identitaire et professionnelle L'utilisation des programmes d'emploi a été étudiée parmi 1172 vétérans de l'armée américaine afin de déterminer quelles composantes du contenu et du processus influencent l'obtention d'un emploi. Une approche d'analyse des composantes communes (common component analysis, CCA) a été utilisée. Les associations avec l'obtention d'un emploi jusqu'à 15 mois après la transition de l'armée à la société civile ont identifié des composantes significativement liées à l'obtention d'un emploi: l'entretien (avec un mentor/coach), la rédaction d'un CV (outils en ligne), la transposition du travail militaire au travail civil (avec un mentor/coach), l'esprit entrepreneurial (avec un mentor/coach) et les salons de l'emploi virtuels. Le recours à un mentor/coach était l'une des stratégies de prestation les plus efficaces. Les risques prédisant une utilisation moindre des composantes du programme ont été identifiés: grade d'enrôlé junior, exposition au combat, occupation dans les armes de combat et problèmes de santé physique.

La influencia de los componentes del programa de empleo en la consecución del trabajo durante una época de transición de identidad y carrera Se estudió el uso del programa de empleo entre 1,172 veteranos militares de los Estados Unidos para determinar qué contenido y componentes del proceso influyen en la consecución del empleo. Se utilizó un enfoque de análisis de componentes comunes. Las asociaciones con la búsqueda de empleo hasta 15 meses después de la transición de militar a civil identificaron componentes relacionados significativamente con la obtención de empleo: entrevistas (con mentor / entrenador), redacción de currículos (herramientas en línea), traducción de trabajo militar a civil (con mentor / entrenador), emprendimiento (con mentor / coach) y ferias de carreras virtuales. Tener un mentor / entrenador fue una de las estrategias de ejecución más efectivas. Se identificaron los riesgos que predecían un menor uso de los componentes del programa: rango de alistados juveniles, exposición al combate, ocupación de armas de combate y problemas de salud física.

Social prescription in the US: A pilot evaluation of Mass Cultural Council's "CultureRx".

Introduction: As the field of public health strives to address the impacts of social determinants of health, it has seen increasing interest in community-referral practices that expand health care beyond clinical spaces. However, community arts and culture organizations are rarely included in these practices, despite accumulating evidence of associated health benefits. In addition, such inclusion has not been formally studied. In response, this article offers an evaluation of "CultureRx" in Massachusetts (MA): the first US model of arts on prescription. The program is a partnership between 20 healthcare providers and 12 cultural organizations, in which providers can offer "prescriptions" to cultural experiences to support patients' health. Methods: Evaluation was undertaken to illuminate participant experiences, program successes and barriers, and recommendations for further development. The cultural organizations collected participant data (n = 84) and completed surveys about their own experiences (n = 12). Authors conducted semi-structured focus groups and interviews with healthcare providers (n = 33). Data analysis was customized for each dataset. Results: Findings indicate that participants enjoyed and hoped to repeat their prescribed experiences, which they saw as beneficial to wellbeing. Providers identified the program as a new and critical addition to their toolkits; they also indicated it had a positive effect on their own wellbeing. Cultural organizations reported varied challenges, learnings, and recommendations. Conclusion: The CultureRx pilot suggests that integrating arts/culture assets into health and social care approaches can enrich and improve traditional US models of community referral. By including arts/culture resources when addressing social determinants of health, communities will be better positioned to equitably and holistically advance health.

Cyclopentane peptide nucleic acid: Gold nanoparticle conjugates for the detection of nucleic acids in a microfluidic format.

Routine patient testing for viral infections is critical to identify infected individuals for treatment and to prevent spreading of infections to others. Developing robust and reliable diagnostic tools to detect nucleic acids of viruses at the point-of-care could greatly assist the clinical management of viral infections. The remarkable stability and high binding affinity of peptide nucleic acids (PNAs) to target nucleic acids could make PNA-based biosensors an excellent starting point to develop new nucleic acid detection technologies. We report the application of cyclopentane-modified PNAs to capture target nucleic acids in a microfluidic channel, and the use of bioorthogonal PNAs conjugated to gold nanoparticles as probes to semi-quantitatively signal the presence of a target nucleic acid derived from HIV-1. The basic results presented could be used to develop more advanced devices to detect nucleic acids from viruses such as HIV, SARS-CoV-2, and a wide range of other human diseases.

The interaction of exposure to adverse childhood and combat experiences on the current mental health of new post-9/11 veterans.

Military veterans have greater exposure to adverse childhood experiences (ACEs) than civilians and many also encounter warfare exposures, which can increase the likelihood of mental health problems. The purpose of this study was to test an interaction between childhood traumas and warfare exposures on the mental health of a sample of nearly 10,000 new post-9/11 veterans. Results revealed that male veterans exposed to one or two ACEs, but no warfare, were more likely to experience anxiety, depression, suicidal thinking, and angry outbursts than the reference group (i.e., no ACEs and no warfare exposure). Female veterans exposed to one or two ACEs, but no warfare, were only more likely to experience suicidal thinking. Male and female veterans exposed to three or more ACEs and no warfare were more likely to experience probable posttraumatic stress disorder (PTSD), anxiety, depression, suicidality, and angry outbursts. Among those veterans who experienced corollaries of combat only (e.g., seeing someone killed or seriously wounded), male, but not female veterans were more likely to have probable PTSD, anxiety, and depression. Veterans exposed to warfare (i.e., combat and the corollaries of combat), irrespective of ACEs exposure, were the most likely to report mental health symptoms. Implications for community-based mental health services are discussed.

Microfluidic Chip Device for In Situ Mixing and Fabrication of Hydrogel Microspheres via Michael-Type Addition.

Hydrogel microspheres are sought for a variety of biomedical applications, including therapeutic and cellular delivery, sensors, and lubricants. Robust fabrication of hydrogel microspheres with uniform sizes and properties can be achieved using microfluidic systems that rely on droplet formation and subsequent gelation to form microspheres. Such systems work well when gelation is initiated after droplet formation but are not practical for timed gelation systems where gelation is initiated prior to droplet formation; premature gelation can lead to device blockage, variable microsphere diameter due to viscosity changes in the precursor solution, and limited numbers of microspheres produced in a single run. To enable microfluidic fabrication of microspheres from timed gelation hydrogel systems, an in situ mixing region is needed so that various hydrogel precursor components can be added separately. Here, we designed and evaluated three mixing devices for their effectiveness at mixing hydrogel precursor solutions prior to droplet formation and subsequent gelation. The serpentine geometry was found to be the most effective and was further improved with the inclusion of a pillar array to increase agitation. The optimized device was shown to fully mix precursor solutions and enable the fabrication of monodisperse polyethylene glycol microspheres, offering great potential for use with timed gelation hydrogel systems.

Microfabricated Devices for Confocal Microscopy on Biological Samples.

Microfabricated devices have found applications in a range of biomedical research problems in recent years, with thousands of research papers published and multiple commercial devices now available. This chapter is intended to provide an overview of the available options for devices compatible with confocal microscopy, including an overview of fabrication techniques and some examples of device use. Although there are times when off-the-shelf devices are well suited for the problem at hand, in some cases customized devices are necessary or more convenient. Protocols for researchers who wish to make their own devices are outlined below; although fabricating templates for devices requires some specialized equipment, making PDMS or hydrogel devices from templates can be done in a standard laboratory setting.

Creating an Artificial 3-Dimensional Ovarian Follicle Culture System Using a Microfluidic System.

We hypothesized that the creation of a 3-dimensional ovarian follicle, with embedded granulosa and theca cells, would better mimic the environment necessary to support early oocytes, both structurally and hormonally. Using a microfluidic system with controlled flow rates, 3-dimensional two-layer (core and shell) capsules were created. The core consists of murine granulosa cells in 0.8 mg/mL collagen + 0.05% alginate, while the shell is composed of murine theca cells suspended in 2% alginate. Somatic cell viability tests and hormonal assessments (estradiol, progesterone, and androstenedione) were performed on days 1, 6, 13, 20, and 27. Confocal microscopy confirmed appropriate compartmentalization of fluorescently-labeled murine granulosa cells to the inner capsule and theca cells to the outer shell. Greater than 78% of cells present in capsules were alive up to 27 days after collection. Artificially constructed ovarian follicles exhibited intact endocrine function as evidenced by the production of estradiol, progesterone, and androstenedione. Oocytes from primary and early secondary follicles were successfully encapsulated, which maintained size and cellular compartmentalization. This novel microfluidic system successfully encapsulated oocytes from primary and secondary follicles, recapitulating the two-compartment system necessary for the development of the mammalian oocyte. Importantly, this microfluidic system can be easily adapted for sterile, high throughput applications.