Three-dimensional isotropic imaging of live suspension cells enabled by droplet microvortices.

Fast, nondestructive three-dimensional (3D) imaging of live suspension cells remains challenging without substrate treatment or fixation, precluding scalable single-cell morphometry with minimal alterations. While optical sectioning techniques achieve 3D live cell imaging, lateral versus depth resolution differences further complicate analysis. We present a scalable microfluidic method capable of 3D fluorescent isotropic imaging of live, nonadherent cells suspended inside picoliter droplets with high-speed single-cell volumetric readout (800 to 1,200 slices in 5 to 8 s) and near-diffraction limit resolution (~216 nm). The platform features a droplet trap array that leverages flow-induced droplet interfacial shear to generate intradroplet microvortices, which rotate single cells on their axis to enable optical projection tomography (OPT)-based imaging. This allows gentle (~1 mPa shear stress) observation of cells encapsulated inside nontoxic isotonic buffer droplets, facilitating scalable OPT acquisition by simultaneous spinning of hundreds of cells. We demonstrate 3D imaging of live myeloid and lymphoid cells in suspension, including K562 cells, as well as naive and activated T cells-small cells prone to movement in their suspended phenotype. Our fully suspended, orientation-independent cell morphometry, driven by isotropic imaging and spherical harmonic analysis, enabled the study of primary T cells across various immunological activation states. This approach unveiled six distinct nuclear content distributions, contrasting with conventional 2D images that typically portray spheroid and bean-like nuclear shapes associated with lymphocytes. Our arrayed-droplet OPT technology is capable of isotropic, single live-cell 3D imaging, with the potential to perform large-scale morphometry of immune cell effector function states while providing compatibility with microfluidic droplet operations.

"Switching Hats": Insights From Experienced Clinical Interviewers Turned Novice Research Interviewers.

Health professionals/clinicians interview people regularly as part of their role. However, a qualitative research interview differs considerably to a clinical interview. If clinicians approach qualitative research interviewing based on their expertise in clinical interviewing, it could cause insufficiencies in qualitative data generation. In this reflection article, we, a team of four experienced clinical occupational therapists with no previous experience in qualitative research interviewing, share our experiences while learning to become qualitative research interviewers before undertaking our first qualitative research project. We engaged in self-directed reading, formal training on qualitative interviewing, and practice interviews and used peer feedback and reflection to prepare ourselves to conduct qualitative interviews. We drew upon the work-role transitions theory to work through our adjustment to the new role. Although we set out to "switch hats" as the research topic itself was not clinical, interviewing people on health-related topics will mean bringing our clinical instincts into our research role, while still recognizing the difference between a clinical and research interview. This article can inform experienced clinicians/novice qualitative researchers as they develop this new skillset.

Endemic mycoses in children in North America: a review of radiologic findings.

Clinically significant endemic mycoses (fungal infections) in the United States (U.S.) include Blastomyces dermatitidis, Histoplasma capsulatum, and Coccidioides immitis/posadasii. While the majority of infections go clinically unnoticed, symptomatic disease can occur in immunocompromised or hospitalized patients, and occasionally in immune-competent individuals. Clinical manifestations vary widely and their diagnosis may require fungal culture, making the rapid diagnosis a challenge. Imaging can be helpful in making a clinical diagnosis prior to laboratory confirmation, as well as assist in characterizing disease extent and severity. In this review, we discuss the three major endemic fungal infections that occur in the U.S., including mycology, epidemiology, clinical presentations, and typical imaging features with an emphasis on the pediatric population.

Experiences of homeless men in an affluent town in South Africa.

The aim of this study was to explore experiences of homelessness in an affluent university town. Seven homeless men were recruited at a welfare program for homeless people to participate in in-depth, semistructured interviews that explored their experiences of homelessness in the town. The ages of the participants ranged from 36 to 52 years. There were three White participants, two of mixed race, and two Black participants. Thematic analysis was used to analyze the data, from a bottom-up systems perspective. The results revealed some of the participants' drug-related experiences, their structural experiences (e.g., shelter policy, low wages, and the poverty trap), their social experiences (e.g., the loss of their families and the lack of social standards in the homeless community), and their psychological experiences (i.e., fatalism, complacency, and responsibility). Subsidized housing and social grants are unlikely to effectively address the homelessness of men if other components of their experience, such as their drug-related experiences, their structural experiences, their social experiences, and their psychological experiences, are not attended to.

Exploring Factors That Helped Adolescents Adjust and Continue With Life After the Death of a Parent.

The aim of this study was to explore factors that helped adolescents to adjust and continue with life after the death of a parent. A qualitative research design was utilized, in which 12 participants participated in semistructured interviews. Data were analyzed according to thematic analysis. The following categories and themes emerged from the data: family support-supportive remaining parent, parent has a supportive partner and supportive extended family; social support-child has supportive friends and supportive community; religion--religion as a coping mechanism and religion as means of communicating with the deceased parent; and a strong personal sense of coherence (the ability to relate to and make sense of the world) as an intrapersonal coping mechanism. In addition, the following themes also emerged: exercising, allowing time to prepare for the death of a terminally ill parent, tangible reminders of the deceased, and journal writing. The findings can be used to design interventions for adolescents whose parent has passed away.

Ethnic Culture as Related to Sexual and Reproductive Behavior in the Republic of Suriname: The Pervasiveness of Culture.

We examined the effects of culture and ethnicity on life history strategies in terms of sexual and reproductive behaviors. The sample included 500 adults, aged 25-50 years, from the five major ethnic groups in Suriname, i.e., the Maroons, Creoles, Hindustani, Javanese, and Mixed. First, there were strong gender differences: men reported to have had more sex partners and to have had their first sexual experience earlier than women, whereas women had their first child earlier and had more children than men. Second, in general, ethnicity affected life history substantially. The Maroons stood out by a relatively fast life history: they reported to have had more sexual partners, to have had their first sex and first child at an earlier age, and to have more children than all other groups. The Creoles were in general similar to the Maroons, whereas the Hindustani and the Javanese were characterized by a relatively slow life history: they reported to have had the lowest number of sexual partners, to have had their first sex and first child at the latest age, and to have had the lowest number of children. The differences between the ethnic groups were upheld when controlling for income, educational level, and father absence during childhood. A lower education was associated with reporting to have had one's first sex as well as one's first child at a younger age and children who grew up without a father reported to have had their first sex at a younger age.

A Simple Scale for Screening Lower-Extremity Arterial Disease as a Possible Cause of Low Back Pain: a Cross-sectional Study Among 542 Subjects.

BACKGROUND: Epidemiological, imaging, and anatomical studies suggest an association between proximal arterial atherosclerosis and development of low back pain (LBP). OBJECTIVES: We aimed to define (1) the frequency and (2) factors associated with exercise-induced proximal ischemia (EIPI) in individuals with LBP and (3) develop a clinical screening scale. DESIGN: Monocentric cross-sectional study. PARTICIPANTS: All patients with history of ongoing LBP referred to our exercise investigation laboratory for exercise transcutaneous oximetry (ex-tcPO2) between January 2011 and December 2017 (n = 542; mean age, 65.4 ± 10.9; 83.9% men). MAIN MEASURES: EIPI was defined as a decrease from rest of oxygen pressure (DROP) below - 15 mmHg on the lumbar and/or buttock probes. Ex-tcPO2 is a reliable validated tool for diagnosing EIPI in comparison with arteriography and computed tomography angiography. Ex-tcPO2 was performed on a treadmill until symptom manifestation or exhaustion. Clinical data were collected using interview questionnaires, medical file review, and clinical examination. KEY RESULTS: EIPI was diagnosed in 282 patients (52%). Age ≤ 70 years (OR, 2.22; 95% CI, 1.35-3.57; p = 0.002), a history of proximal revascularization (OR, 2.64; 95% CI, 1.50-4.65; p = 0.001), use of antiplatelet medication (OR, 1.71; 95% CI, 0.96-3.06; p = 0.069), a relationship between exercise and LBP (OR, 2.61; 95% CI, 1.49-4.57; p = 0.001), and an abnormal ankle to brachial index (OR, 2.87; 95% CI, 1.77-4.66; p < 0.0001) were identified as EIPI predictors. Using these items, we developed a screening scale that showed an area under the receiver operating characteristics curve of .756. At a score of ≥ 3, the sensitivity, specificity, and accuracy for EIPI were 84%, 55%, and 71%, respectively. CONCLUSIONS: EIPI was common among our patients with LBP undergoing ex-TcPO2. Our screening scale could help better select the patients who require angiography.

Label-Free Metabolic Classification of Single Cells in Droplets Using the Phasor Approach to Fluorescence Lifetime Imaging Microscopy.

Characterization of single cell metabolism is imperative for understanding subcellular functional and biochemical changes associated with healthy tissue development and the progression of numerous diseases. However, single-cell analysis often requires the use of fluorescent tags and cell lysis followed by genomic profiling to identify the cellular heterogeneity. Identifying individual cells in a noninvasive and label-free manner is crucial for the detection of energy metabolism which will discriminate cell types and most importantly critical for maintaining cell viability for further analysis. Here, we have developed a robust assay using the droplet microfluidic technology together with the phasor approach to fluorescence lifetime imaging microscopy to study cell heterogeneity within and among the leukemia cell lines (K-562 and Jurkat). We have extended these techniques to characterize metabolic differences between proliferating and quiescent cells-a critical step toward label-free single cancer cell dormancy research. The result suggests a droplet-based noninvasive and label-free method to distinguish individual cells based on their metabolic states, which could be used as an upstream phenotypic platform to correlate with genomic statistics. © 2018 International Society for Advancement of Cytometry.

Polycomb-mediated repression during terminal differentiation: what don't you want to be when you grow up?

Chromatin-modifying enzymes are known to be critical components for the correct differentiation of embryonic stem cells into specific lineages, such as neurons. Recently, the role of Polycomb group proteins has been studied in the specification and differentiation of muscle stem cells. In this perspective, we review a recent study by Juan and colleagues (pp. 789-794) in Genes & Development of the role of the polycomb group protein Ezh2 in muscle stem cells, and discuss the implications for general lineage restriction.

Lipoplex-Mediated Single-Cell Transfection via Droplet Microfluidics.

While lipoplex (cationic lipid-nucleic acid complex)-mediated intracellular delivery is widely adopted in mammalian cell transfection, its transfection efficiency for suspension cells, e.g., lymphatic and hematopoietic cells, is reported at only ≈5% or even lower. Here, efficient and consistent lipoplex-mediated transfection is demonstrated for hard-to-transfect suspension cells via a single-cell, droplet-microfluidics approach. In these microdroplets, monodisperse lipoplexes for effective gene delivery are generated via chaotic mixing induced by the serpentine microchannel and co-confined with single cells. Moreover, the cell membrane permeability increases due to the shear stress exerted on the single cells when they pass through the droplet pinch-off junction. The transfection efficiency, examined by the delivery of the pcDNA3-EGFP plasmid, improves from ≈5% to ≈50% for all three tested suspension cell lines, i.e., K562, THP-1, Jurkat, and with significantly reduced cell-to-cell variation, compared to the bulk method. Efficient targeted knockout of the TP53BP1 gene for K562 cells via the CRISPR (clustered regularly interspaced short palindromic repeats)-CAS9 (CRISPR-associated nuclease 9) mechanism is also achieved using this platform. Lipoplex-mediated single-cell transfection via droplet microfluidics is expected to have broad applications in gene therapy and regenerative medicine by providing high transfection efficiency and low cell-to-cell variation for hard-to-transfect suspension cells.

Genomic Privacy.

BACKGROUND: Genetic information is unique among all laboratory data because it not only informs the current health of the specific person tested but may also be predictive of the future health of the individual and, to varying degrees, all biological relatives. CONTENT: As DNA sequencing has become ubiquitous with decreasing cost, large repositories of genomic data have emerged from the domains of research, healthcare, law enforcement, international security, and recreational consumer interest (i.e., genealogy). Broadly shared genomic data are believed to be a key element for future discoveries in human disease. For example, the National Cancer Institute's Genomic Data Commons is designed to promote cancer research discoveries by providing free access to the genome data sets of 12000 cancer patients. However, in parallel with the promise of curing diseases, genomic data also have the potential for harm. Genomic data that are deidentified by standard healthcare practices (e.g., removal of name, date of birth) can be reidentified by methods that combine genomic software with publicly available demographic databases (e.g., phone book). Recent law enforcement cases (i.e., Bear Brook Murders, Golden State Killer) in the US have demonstrated the power of combining DNA profiles with genealogy databases. SUMMARY: We examine the current environment of genomic privacy and confidentiality in the US and describe current and future risks to genomic privacy. Reidentification and inference of genetic information of biological relatives will become more important as larger databases of clinical, criminal, and recreational genomic information are developed over the next decade.

Intra-test and test-retest reliability of exercise oximetry in arterial claudication.

BACKGROUND: Transcutaneous oxygen pressure (tcpO2) reliability is blunted by an unpredictable transcutaneous gradient through the skin. We hypothesized that the "Decrease from Rest of Oxygen pressure (DROP: subtraction of limb-changes from chest-changes from the respective starting values) would show a good to excellent reliability during Exercise -tcpO2 investigations. METHODS: In three different experiments we tested: The intra-test variability at the peripheral level (Experiment A: 32 patients, 16 at each location), at the chest level (Experiment B: 45 patients) and the test-retest reproducibility within 3 months (Experiment C: 67 patients). We calculated the intra-class coefficient of correlation (ICC) with 95% confidence interval [Lower/upper limit]. ICC between 0.60 and 0.749 indicate a good agreement. ICC above 0.750 indicates an excellent agreement. RESULTS: ICC values for DROP-min were 0.848 [0.723/0.935] at the buttock and 0.920 [0.846/0.967] at the calf levels, in experiment A; ICC were 0.873 [0.799/0.923] at the buttock and 0.898 [0.790/0.953] at the calf levels, in experiment B; 0.806 [0.716/0.871] at then buttock level (n = 67) and 0.807 [0.722/0.868] at the calf in experiment C. CONCLUSIONS: Intra-test and test-retest reliability is excellent using the DROP calculation for exercise-tcpO2 investigations.

Post-exercise ankle blood pressure and ankle to brachial index after heavy load bicycle exercise.

The American Heart Association (AHA) recommendations for diagnosing peripheral artery disease (PAD) after exercise are a decrease >20% of ankle brachial index (ABI) or >30 mm Hg of ankle systolic blood pressure (ASBP) from resting values. We evaluated ABI and ASBP values during incremental maximal exercise in physically active and asymptomatic patients. Patients (n = 726) underwent incremental bicycle tests with pre- and post-exercise recording of all four limbs arterial pressures simultaneously. Univariate and multivariate analyses were performed to define the correlation between post-exercise ABI with various clinical factors, including age. Thereafter, the population was divided into groups of age: less than 40 (G < 40), from 40 to 44 (G40/44) from 45 to 49 (G45/49), from 50 to 54 (G50/54), from 55 to 59 (G55/59), from 60 to 64 (G60/64), and 65 and above (G ≥ 65) years. Results are mean ± SD. * is two-tailed P < .05 for ANOVA with Dunnett's post-hoc test from G40. Changes from rest in ASBP were -3 ± 22 (G < 40), -2 ± 20 (G40/44), 4 ± 22* (G45/49), 10 ± 25* (G50/54), 18 ± 21* (G55/59), 23 ± 27* (G60/64), and 16 ± 22* (G ≥ 65) mm Hg. Decreases from rest in ABI were 32 ± 9 (G < 40), 33 ± 9 (G40/44), 29 ± 8 (G45/49), 27 ± 10* (G50/54), 24 ± 7* (G55/59), 22 ± 12* (G60/64), and 21 ± 12* (G ≥ 65) % of resting ABI. Maximal incremental exercise results in ABI and ASBP changes are mostly dependent on age. The AHA limits for post-exercise ABI are inadequate following maximal incremental bicycle testing. Future studies detecting PAD in active patients should account for the effect of age.

Narrative approach in understanding the drivers for resilience of military combat medics.

INTRODUCTION: Qualitative insights may demonstrate how combat medics (CM) deal with stressors and identify how resilience can potentially develop. Yet, qualitative research is scant in comparison to the many quantitative studies of health outcomes associated with military service. METHOD: Semistructured qualitative interviews were used to collect personal narratives of US Army CMs who had previously served in Iraq or Afghanistan. RESULTS: Thematic analysis revealed three key driving forces for how resilience develops in the context of combat and war. The first was patriotism, which captures loyalty and full commitment to the military and its missions. The second was commitment to their family, reflecting the balance of responsibility to family of origin with the obligation one feels towards their military family. The last driving force was faith, or the drive to reach towards the transcendent to provide a moral compass and develop empathy in the face of difficult situations. CONCLUSIONS: An individual's commitment to country, military family and faith strengthens their resilience, and this can be used to inform future research efforts as well as current clinical practice.

A high throughput microfluidic platform for size-selective enrichment of cell populations in tissue and blood samples.

Numerous applications in biology and medicine require the efficient and reliable separation of cells for disease diagnosis, genetic analysis, drug screening, and therapeutics. In this work, we demonstrate a novel technology that integrates a passive and an active device to separate, enrich and release cells on-demand from a complex blood sample, or cancer cells derived from a tissue biopsy. We exploit the high throughput (>1 mL min-1), size-based sorting capability of the passive spiral inertial microfluidic (iMF) device to focus particles/cells towards an active lateral cavity acoustic transducer (LCAT) device for size-selective enrichment. We demonstrate that this platform is capable of efficiently (>90%) removing smaller cells, such as RBCs in a blood sample or smaller cancer cells in a heterogeneous cell line, and providing 44 000× enrichment from the remaining sample within 5 min of device operation. Finally, we use this platform for two applications: selective enrichment of the side-population of DU-145 cells from tissue biopsy and isolation of larger monocytes from blood. Our platform integrates the high throughput (processing rate) capacity of spiral iMF with the high selectivity of LCAT, thereby offering a unique route for highly-selective, label-free particle/cell sorting, with potential application in lab-on-chip platforms for liquid biopsy and diagnostics applications.

There is more: Intrasexual competitiveness, physical dominance, and intrasexual collaboration.

It is emphasized that in organizational settings, the responses to same-sex attractive others are enhanced among individuals high in intrasexual competitiveness; that especially attractive rivals who are perceived as unfriendly will induce competition; that among males, physical dominance may induce more competition than physical attractiveness; and that especially males may prefer to associate with attractive same-sex others for intrasexual collaboration.

Evaluation of quantum dot immunofluorescence and a digital CMOS imaging system as an alternative to conventional organic fluorescence dyes and laser scanning for quantifying protein microarrays.

Organic fluorescent dyes are widely used for the visualization of bound antibody in a variety of immunofluorescence assays. However, the detection equipment is often expensive, fragile, and hard to deploy widely. Quantum dots (Qdot) are nanocrystals made of semiconductor materials that emit light at different wavelengths according to the size of the crystal, with increased brightness and stability. Here, we have evaluated a small benchtop "personal" optical imager (ArrayCAM) developed for quantification of protein arrays probed by Qdot-based indirect immunofluorescence. The aim was to determine if the Qdot imager system provides equivalent data to the conventional organic dye-labeled antibody/laser scanner system. To do this, duplicate proteome microarrays of Vaccinia virus, Brucella melitensis and Plasmodium falciparum were probed with identical samples of immune sera, and IgG, IgA, and IgM profiles visualized using biotinylated secondary antibodies followed by a tertiary reagent of streptavidin coupled to either P3 (an organic cyanine dye typically used for microarrays) or Q800 (Qdot). The data show excellent correlation for all samples tested (R > 0.8) with no significant change of antibody reactivity profiles. We conclude that Qdot detection provides data equivalent to that obtained using conventional organic dye detection. The portable imager offers an economical, more robust, and deployable alternative to conventional laser array scanners.

Regulatory T and B lymphocytes in a spontaneous autoimmune polyneuropathy.

B7-2(-/-) non-obese diabetic (NOD) mice develop a spontaneous autoimmune polyneuropathy (SAP) that mimics the progressive form of chronic inflammatory demyelinating polyradiculoneuropathy (CIDP). In this study, we focused on the role of regulatory T cells (Tregs ) and regulatory B cells (Bregs ) in SAP. We found that deletion of B7-2 in female NOD mice led to a lower frequency and number of Tregs and Bregs in spleens and lymph nodes. Tregs but not Bregs suppressed antigen-stimulated splenocyte proliferation, whereas Bregs inhibited the T helper type 1 (Th1) cytokine response. Both Tregs and Bregs induced an increase in CD4(+) interleukin (IL)-10(+) cells, although less effectively in the absence of B7-2. Adoptive transfer studies revealed that Tregs , but not Bregs , suppressed SAP, while Bregs attenuated disease severity when given prior to symptom onset. B cell deficiency in B cell-deficient (muMT)/B7-2(-/-) NOD mice prevented the development of SAP, which would indicate that the pathogenic role of B cells predominates over its regulatory role in this model. We conclude that Bregs and Tregs control the immunopathogenesis and progression of SAP in a non-redundant fashion, and that therapies aimed at expansion of Bregs and Tregs may be an effective approach in autoimmune neuropathies.

Characterization of hepatitis B virus surface antigen variability and impact on HBs antigen clearance under nucleos(t)ide analogue therapy.

For hepatitis B virus (HBV)-related chronic infection under treatment by nucleos(t)ide analogues (NUCs), HBsAg clearance is the ultimate therapeutic goal but very infrequent. We investigated how HBV envelope protein variability could lead to differential HBsAg clearance on NUCs. For 12 HBV genotype D patients receiving NUCs, six resolvers (HBsAg clearance) were compared to six matched nonresolvers (HBsAg persistence). PreS/S amino acid (aa) sequences were analysed with bioinformatics to predict HBV envelope antigenicity and aa covariance. To enrich our analyses on very rare resolvers, these were compared with other HBV genotype D strains in three characterized clinical cohorts including common chronically infected patients. The sT125M+sP127T combination was observed in four nonresolvers of six, corroborated by aa covariance analysis, associated with a lower predicted antigenicity than sT125T+sP127P. Concordant features within this HBV key functional domain, at positions 125 and 127, were reported from two of the three comparative cohorts. In our hands, a lower ELISA reactivity of HBV-vaccinated mice sera was observed against the sT125M mutant. In the S gene, 56 aa changes in minor variants were detected in non-resolvers, mainly in the major hydrophilic region, vs 28 aa changes in resolvers. Molecular features in patients showing HBsAg persistence on NUCs argue in favour of a different aa pattern in the HBV S gene compared to those showing HBsAg clearance. In nonresolvers, a decrease in HBs 'a' determinant antigenicity and more frequent mutations in the S gene suggest a role for the HBV envelope characteristics in HBsAg persistence.