Scalable pattern formation of skeletal myotubes by synergizing microtopographic cues and chiral nematics of cells.

Topographical cues have been widely used to facilitate cell fusion in skeletal muscle formation. However, an unexpected yet consistent chiral orientation of myotubes deviating from the groove boundaries is commonly observed but has long been unattended. In this study, we report a method to guide the formation of skeletal myotubes into scalable and controlled patterns. By inducing C2C12 myoblasts onto grooved patterns with different widths (from 0.4 to 200µm), we observed an enhanced chiral orientation of cells developing on wide grooves (50 and 100µm width) since the first day of induction. Active chiral nematics of cells involving cell migration and chiral rotation of the cell nucleus subsequently led to a unified chiral orientation of the myotubes. Importantly, these chiral myotubes were formed with enhanced length, diameter, and contractility on wide grooves. Treatment of latrunculin A (Lat A) suppressed the chiral rotation and migration of cells as well as the myotube formation, suggesting the essence of chiral nematics of cells for myogenesis. Finally, by arranging wide grooved/striped patterns with corresponding compensation angles to synergize microtopographic cues and chiral nematics of cells, intricate and scalable patterns of myotubes were formed, providing a strategy for engineering skeletal muscle tissue formation.

Microfluidic immunoassay for detection of serological antibodies: A potential tool for rapid evaluation of immunity against SARS-CoV-2.

In December 2019, coronavirus disease 2019 became a pandemic affecting more than 200 countries and territories. Millions of lives are still affected because of mandatory quarantines, which hamstring economies and induce panic. Immunology plays a major role in the modern field of medicine, especially against virulent infectious diseases. In this field, neutralizing antibodies are heavily studied because they reflect the level of infection and individuals' immune status, which are essential when considering resumption of work, flight travel, and border entry control. More importantly, it also allows evaluating the antiviral vaccine efficacy as vaccines are still known for being the ultimate intervention method to inhibit the rapid spread of virulent infectious diseases. In this Review, we first introduce the host immune response after the infection of SARS-CoV-2 and discuss the latest results using conventional immunoassays. Next, as an enabling platform for detection with sufficient sensitivity while saving analysis time and sample size, the progress of microfluidic-based immunoassays is discussed and compared based on surface modification, microfluidic kinetics, signal output, signal amplification, sample matrix, and the detection of anti-SARS-CoV-2 antibodies. Based on the overall comparison, this Review concludes by proposing the future integration of visual quantitative signals on microfluidic devices as a more suitable approach for general use and large-scale surveillance.

Early Committed Clockwise Cell Chirality Upregulates Adipogenic Differentiation of Mesenchymal Stem Cells.

Cell chirality is observed with diverse forms and coordinates various left-right (LR) asymmetry in tissue morphogenesis. To give rise to such diversity, cell chirality may be coupled with cell differentiation. Here, using micropatterned human mesenchymal stem cells (hMSCs), an early committed clockwise (CW) cell chirality that can itself upregulate the adipogenic differentiation is reported. hMSC chirality enables a positively tilted chiral orientation on micropatterned stripes. When cultured as single cells on circular micropatterns, an anticlockwise (ACW)-biased nucleus rotation and swirling pattern of actin filament are observed. Interestingly, with adipogenic induction for 3-6 days, such chirality is reversed to negative chiral orientation and CW-biased rotation, which is earlier than the maturation of other differentiation markers, and consistently expressed in terminally differentiated adipocytes. Using latrunculin A (LatA), cytochalasin D (CD), and nocodazole (Noco) that forces a CW-biased actin filament and nucleus rotation resembling the early differentiated chirality upon adipogenic induction, an upregulation of adipogenic differentiation is found. The result demonstrates that the early differentiated chirality may serve as a mechanical precursor to engage the lineage commitment, suggesting a feedback mechanism of chiral actin in regulating cell differentiation and LR morphogenesis.

Light field display with ellipsoidal mirror array and single projector.

We present a method to create light field display using a single projector and an array of plane mirrors. Mirrors can reproduce densely arranged virtual projectors regardless of the physical size of the real projector, thus producing a light field display of competitive ray density. We propose an ellipsoidal geometric framework and a design pipeline, and use parametric modelling technique to automatically generate the display configurations satisfying target design parameters. Three units of mirror array light field display systems have been implemented to evaluate the proposed methodologies. More importantly, we have experimentally verified that the high-density light field produced by our method can naturally evoke accommodation of the eyes, thereby reducing the vergence-accommodation conflict. The mirror array approach allows flexible trading between the spatial and angular resolutions for accommodating different applications, thus providing a practical solution to realize projection-based light field display.

Remnant Effects of Culture Density on Cell Chirality After Reseeding.

Proper muscle function requires specific orientation of myotubes. Cell chirality, a mechanical behavior of cells, may participate in myogenesis and give rise to left-right (LR) orientation of muscle tissue. Thus, it is essential to understand the factors effecting the cell chirality. Here, using C2C12 cells as a model system, we report that prior culture condition with high/low density can create remnant effects on cell chirality after reseeding. C2C12 myoblasts were first conditioned by a series of subcultures with plating density at 2200 cells/cm2 (low density) or 22 000 cells/cm2 (high density). After reseeding on micropatterned stripes fabricated on glass or polydimethylsiloxane (PDMS) substrates, we found that the cells after low-density cultures exhibited a reduced cell aspect ratio and intercellular alignment, leading to an attenuated chiral orientation only appearing on glass substrate. In contrast, chiral orientation was observed in cells after high-density culture on both substrates. By comparing it to the original cells without being subcultured with high/low density, we found that the series of low-density cultures disorganized the formation of actin rings in single cells, which is an essential structure for cell chirality. Moreover, by using high-density culture supplemented with inhibitors of actin polymerization, the effect of low-density cultures was recaptured, suggesting that the series of subcultures with high/low density may be an in vitro aging process that modifies the actin cytoskeleton, causing a remnant attenuation of cell chirality even after trypsin digestion and reseeding. Together, our result suggests a mechanistic insight of how cytoskeletal structures "memorize" the previous experience through modification of the actin filament, opening up new possibilities for morphogenesis and mechanobiology.

Outline-etching image segmentation reveals enhanced cell chirality through intercellular alignment.

Cells cultured on micropatterns exhibit a chiral orientation, which may underlie the development of left-right asymmetry in tissue microarchitectures. To investigate this phenomenon, fluorescence staining of nuclei has been used to reveal such orientation. However, for images with high cell density, analysis is difficult because of the overlapping nuclei. Here, we report an image processing method that can acquire cell orientations within dense cell populations. After initial separation based on Boolean addition of binarized images using global and adaptive thresholds, the overlapping nucleus contours in the binarized images were segmented by iteratively etching the outlines of nuclei, which allowed the orientations of each cell to be extracted from densely packed cell clusters. In applying this technique to cultured C2C12 myoblasts in micropatterned stripes on different substrates, we found an enhanced chiral orientation on glass substrate. More important, this enhanced chirality was consistently observed with increased intercellular alignment and independent of cell-cell distance or cell density, suggesting that intercellular alignment plays a role in determining the chiral orientation. By segmenting single cells with intact orientation, this technique offers an automated method for quantitative analysis with improved accuracy, providing an essential tool for studying left-right asymmetry and other morphogenic dynamics in tissue formation.

Discrete Element Model for Suppression of Coffee-Ring Effect.

When a sessile droplet evaporates, coffee-ring effect drives the suspended particulate matters to the droplet edge, eventually forming a ring-shaped deposition. Because it causes a non-uniform distribution of solid contents, which is undesired in many applications, attempts have been made to eliminate the coffee-ring effect. Recent reports indicated that the coffee-ring effect can be suppressed by a mixture of spherical and non-spherical particles with enhanced particle-particle interaction at air-water interface. However, a model to comprehend the inter-particulate activities has been lacking. Here, we report a discrete element model (particle system) to investigate the phenomenon. The modeled dynamics included particle traveling following the capillary flow with Brownian motion, and its resultant 3D hexagonal close packing of particles along the contact line. For particles being adsorbed by air-water interface, we modeled cluster growth, cluster deformation, and cluster combination. We found that the suppression of coffee-ring effect does not require a circulatory flow driven by an inward Marangoni flow at air-water interface. Instead, the number of new cluster formation, which can be enhanced by increasing the ratio of non-spherical particles and the overall number of microspheres, is more dominant in the suppression process. Together, this model provides a useful platform elucidating insights for suppressing coffee-ring effect for practical applications in the future.

Abdominal Adiposity, Not Cardiorespiratory Fitness, Mediates the Exercise-Induced Change in Insulin Sensitivity in Older Adults.

Abdominal obesity and low cardiorespiratory fitness (CRF) are associated with insulin resistance in older adults. Exercise is associated with improvement in insulin sensitivity. Whether this association is mediated by change in CRF and/or abdominal obesity is unclear. The current study is a secondary analysis of data from a randomized controlled trial in Kingston, Ontario. Sedentary older adults (60-80 years) (N = 80) who completed the exercise (N = 59) or control (N = 21) conditions for 6 months were included. CRF was measured using a treadmill test, adipose tissue (AT) by magnetic resonance imaging, and insulin sensitivity by hyperinsulinemic-euglycemic clamp. Waist circumference (WC) was measured at the iliac crest. Mediation analyses were used to assess whether abdominal AT and/or CRF mediated the exercise-induced change in insulin sensitivity. By comparison to controls, reduction (mean ± SD) was observed for visceral (-0.4 ± 0.4 kg) and abdominal subcutaneous (-0.4 ± 0.4) AT depots, WC (-4.1 ± 3.2 cm) and BMI (-0.9 ± 0.8 kg/m2) (p < 0.05). Insulin sensitivity (4.2 ± 5.2 M/I) and CRF (0.2 ± 0.3 L/min) improved in the exercise group (p < 0.05). All AT variables, BMI and WC were mediators of the change in insulin sensitivity (p < 0.05). After adjustment for change in total AT, abdominal AT remained a mediator with an effect ratio of 0.79 (p < 0.05), whereas total AT was not significant when adjusted for abdominal AT (p > 0.05). The effect ratio for change in WC and BMI combined (0.63, p<0.05) was greater than either alone. In conclusion, CRF did not mediate the exercise-induced change in insulin sensitivity in older adults. Abdominal adiposity was a strong mediator independent of change in total adiposity.

Nanowire Magnetoscope Reveals a Cellular Torque with Left-Right Bias.

Cellular force regulates many types of cell mechanics and the associated physiological behaviors. Recent evidence suggested that cell motion with left-right (LR) bias may be the origin of LR asymmetry in tissue architecture. As actomyosin activity was found essential in the process, it predicts a type of cellular force that coordinates the development of LR asymmetry in tissue formation. However, due to the lack of appropriate platform, cellular force with LR bias has not yet been found. Here we report a nanowire magnetoscope that reveals a rotating force-torque-exerted by cells. Ferromagnetic nanowires were deposited and internalized by micropatterned cells. Within a uniform, horizontal magnetic field, the nanowires that initially aligned with the magnetic field were subsequently rotated due to the cellular torque. We found that the torque is LR-biased depending on cell types. While NIH 3T3 fibroblasts and human vascular endothelial cells exhibited counterclockwise torque, C2C12 myoblasts showed torque with slight clockwise bias. Moreover, an actin ring composed of transverse arcs and radial fibers was identified as a major factor determining the LR bias of cellular torque, since the disruption of actin ring by biochemical inhibitors or elongated cell shape abrogated the counterclockwise bias of NIH 3T3 fibroblasts. Our finding reveals a LR-biased torque of single cells and a fundamental origin of cytoskeletal chirality. More broadly, we anticipate that our method will provide a different perspective on mechanics-related cell physiology and force transmission necessary for LR propagation in tissue formation.

Substrate Stiffness Regulates the Development of Left-Right Asymmetry in Cell Orientation.

Left-right (LR) asymmetry of tissue/organ structure is a morphological feature essential for many tissue functions. The ability to incorporate the LR formation in constructing tissue/organ replacement is important for recapturing the inherent tissue structure and functions. However, how LR asymmetry is formed remains largely underdetermined, which creates significant hurdles to reproduce and regulate the formation of LR asymmetry in an engineering context. Here, we report substrate rigidity functioning as an effective switch that turns on the development of LR asymmetry. Using micropatterned cell-adherent stripes on rigid substrates, we found that cells collectively oriented at a LR-biased angle relative to the stripe boundary. This LR asymmetry was initiated by a LR-biased migration of cells at stripe boundary, which later generated a velocity gradient propagating from stripe boundary to the center. After a series of cell translocations and rotations, ultimately, an LR-biased cell orientation within the micropatterned stripe was formed. Importantly, this initiation and propagation of LR asymmetry was observed only on rigid but not on soft substrates, suggesting that the LR asymmetry was regulated by rigid substrate probably through the organization of actin cytoskeleton. Together, we demonstrated substrate rigidity as a determinant factor that mediates the self-organizing LR asymmetry being unfolded from single cells to multicellular organization. More broadly, we anticipate that our findings would pave the way for rebuilding artificial tissue constructs with inherent LR asymmetry in the future.

Effects of exercise amount and intensity on abdominal obesity and glucose tolerance in obese adults: a randomized trial.

BACKGROUND: Exercise reduces obesity and related glucose tolerance, but whether increasing exercise intensity offers additional benefit at fixed exercise amounts is unknown. OBJECTIVE: To determine the separate effects of exercise amount and intensity on abdominal obesity and glucose tolerance. DESIGN: 24-week, single-center, parallel-group trial from 2009 to 2013. (ClinicalTrials.gov: NCT00955071). SETTING: Kingston, Ontario, Canada. PARTICIPANTS: 300 abdominally obese adults. INTERVENTION: Control (no exercise) (n = 75) or 5 weekly sessions of low-amount, low-intensity exercise (LALI) (180 and 300 kcal/session for women and men, respectively, at 50% of maximum oxygen consumption [V̇o2peak]) (n = 73); high-amount, low-intensity exercise (HALI) (360 and 600 kcal/session, respectively, at 50% of V̇o2peak) (n = 76); or high-amount, high-intensity exercise (HAHI) (360 and 600 kcal/session, respectively, at 75% of V̇o2peak) (n = 76). Daily unsupervised physical activity and sedentary time were measured by accelerometer. MEASUREMENTS: Waist circumference and 2-hour glucose level (primary outcomes) and cardiorespiratory fitness and measures of insulin action (secondary measurements). RESULTS: 217 participants (72.3%) completed the intervention. Mean exercise time in minutes per session was 31 (SD, 4.4) for LALI, 58 (SD, 7.6) for HALI, and 40 (SD, 6.2) for HAHI. Daily unsupervised physical activity and sedentary time did not change in any exercise group versus control (P > 0.33). After adjustment for age and sex in a linear mixed model, reductions in waist circumference were greater in the LALI (-3.9 cm [95% CI, -5.6 to -2.3 cm]; P < 0.001), HALI (-4.6 cm [CI, -6.2 to -3.0 cm]; P < 0.001), and HAHI (-4.6 cm [CI, -6.3 to -2.9 cm]; P < 0.001) groups than the control group but did not differ among the exercise groups (P > 0.43). After adjustment for covariates, reductions in 2-hour glucose level were greater in the HAHI group (-0.7 mmol/L [-12.5 mg/dL] [CI, -1.3 to -0.1 mmol/L {-23.5 to -1.5 mg/dL}]; P = 0.027) than the control group but did not differ for the LALI or HALI group versus the control group (P > 0.159). Weight loss was greater in all exercise groups than the control group (P < 0.001); however, reduction in body weight did not differ among the exercise groups (P > 0.182). LIMITATION: The clinical importance of reducing 2-hour glucose level in nondiabetic adults remains undetermined. CONCLUSION: Fixed amounts of exercise independent of exercise intensity resulted in similar reductions in abdominal obesity. Reduction in 2-hour glucose level was restricted to high-intensity exercise.

Optimization of Combinatory Nicking Endonucleases for Accurate Identification of Nucleic Acids in Low Abundance.

Nucleic acid biomarkers embody inherent importance for differentiating disease-causing organisms or environmental pathogens. Identifying unknown nucleic acids in low abundance remains extremely challenging. Previously, we reported a method to identify complementary DNA (cDNA) molecules based on sequence-specific topographical labels measured by atomic force microscopy (AFM). However, the accuracy is limited because only one type of nicking endonuclease was used as the labeling agent. Here we investigate how accuracy is improved using multiple types of nicking endonucleases in combinations. The numerical experiments created cDNA molecules incorporating measurement error or labeling defects, which were later compared with the 29,563 human messenger RNA (mRNA) transcript database with ideal labels. After comparison, the unknown cDNA molecule was identified as the transcript with the highest matching score. Thus, the accuracy was determined by the rate of true positives. We found that the accuracy is positively proportional to the label number. Compared with cases using single nicking endonuclease, which has an average accuracy of 51.2% ± 34.4%, the average accuracy was improved to 97.1% ± 5.6% using an optimized combination of NtBsmAI + NtBstNBI + NtAlwI. This improved accuracy is applicable to more than 85% of human mRNA transcripts. Together, our study suggests an optimization strategy for identifying nucleic acids in low abundance using the AFM-based method, with implications for diseases diagnosis, pathogen identification, and forensics at the single molecule level.

Is that lumbar disc symptomatic? Herniated lumbar disc associated with contralateral radiculopathy.

Herniated lumbar disc may be asymptomatic or associated with lower limb radiculopathy. Most spinal surgeons would offer surgery following a period of conservative measures if the radiological and clinical findings correlate. However, the existing dictum that lumbar radiculopathy should correlate with ipsilateral lumbar disc herniation may not be accurate as it can rarely present with contralateral sciatica. Literature regarding this phenomenon is scarce. Therefore, we report a patient with herniated lumbar disc presenting with predominantly contralateral motor weakness radiculopathy, which resolved after discectomy.

Infusion-line pressure as a real-time monitor of convection-enhanced delivery in pre-clinical models.

BACKGROUND: Acute convection-enhanced delivery (CED) is a neurosurgical delivery technique that allows for precise and uniform distribution of an infusate to a brain structure. It remains experimental due to difficulties in ensuring successful delivery. Real-time monitoring is able to provide immediate feedback on cannula placement, infusate distribution, and if the infusion is proceeding as planned or is failing due to reflux or catheter obstruction. NEW METHOD: Pressure gradient is the driving force behind CED, with the infusion pressure being directly proportional to the flow-rate. The aim of this study was to assess the feasibility of using infusion-line pressure profiling to distinguish in real-time between succeeding and failing CED infusions. To do so we delivered cresyl violet dye at 0.5, 1.0 and 2.0 µl/min via CED in vitro using 0.6% agarose gel and in vivo to the rat striatum. RESULTS: Infusions that failed in agarose gel models could only be differentiated late during the procedures. In the rat in vivo model, the infusion-line profiles of obstructed infusions were not distinctive from those of successful infusions. COMPARISON WITH EXISTING METHOD: Intraoperative magnetic resonance imaging (MRI) is used for real-time visualisation of cannula placement and infusate distribution. Particularly for animal pre-clinical work, it would be advantageous to supplement MRI with a cheap, accessible technique to monitor infusions and provide a real-time measure of infusion success or failure. CONCLUSIONS: Infusion-line pressure monitoring was of limited value in identifying successful CED with small volume infusions, whilst its utility for large volume infusion remains unknown.

The changing prevalence of autism in three regions of Canada.

In 2002/2003, the National Epidemiologic Database for the Study of Autism in Canada started capturing information on children diagnosed with autism in different regions of the country. Based on data collected through 2008 in Newfoundland and Labrador and 2010 in Prince Edward Island and Southeastern Ontario, the estimated average annual percent increases in prevalence among children 2-14 years of age ranged from 9.7 % (95 % CI 7.8-11.6) to 14.6 % (95 % CI 11.3-18.0). Differential in-migration and identification of previously undetected cases may have contributed in part to the increases observed, but we cannot rule out the possibility of a true increase in incidence, particularly given the lack of a leveling-off of prevalence among the 6- to 9-year olds.

Prostate cancer-specific survival differences in patients treated by radical prostatectomy versus curative radiotherapy.

OBJECTIVE: We compared the cause-specific survival of patients who received radiotherapy to those who received surgery for cure of their prostate cancer using a number of design and analytic steps to mitigate confounding by indication. METHODS: This was a case-cohort study of 2213 patients in the Ontario Cancer Registry diagnosed between 1990 and 1998 who were either treatment candidates or received curative radiotherapy or surgery. Cases included patients who died of prostate cancer within 10 years. The study population was restricted to those who were candidates for either treatment (radiotherapy or surgery) based on disease severity (low and intermediate risk using the Genitourinary Radiation Oncologists of Canada risk groups). The median follow-up was 51 months. Cause-specific survival was analyzed using Cox-proportional hazards regression with case-cohort variance adjustment. Results from intent-to-treat analyses were compared to results by treatment received. RESULTS: Adjusted hazard ratios for risk of prostate cancer death for radiotherapy compared to surgery for the entire study population were 1.62 (95%CI 1.00-2.61) and 2.02 (1.19-3.43) analyzing by intent-to-treat and treatment received, respectively. Intent-to-treat hazard ratios for the low- and intermediate-risk groups were 0.87 (0.28-2.76) and 1.57 (0.95-2.61), respectively. CONCLUSION: Overall results were driven by the finding in the intermediate-risk group, which indicated that radiotherapy was not as effective as surgery in this group. Confirmation was needed with special attention paid to risk stratification and the impact of more contemporary delivery of these treatment options.

Dose-response effects of exercise on abdominal obesity and risk factors for cardiovascular disease in adults: study rationale, design and methods.

Although progress has been made to elucidate the effects of exercise as a strategy for reducing obesity and related cardiometabolic risk factors, the specific exercise exposures required to achieve optimal benefit continue to be the source of considerable uncertainty and debate. Despite the inference of a dose-dependent relationship between exercise and health benefit, absent from the literature are randomized trials that, without alteration in caloric intake, examine the separate effects of exercise dose and intensity on obesity and associated cardiometabolic risk. We will perform a randomized, controlled trial designed to study the separate effects of habitual exercise differing in dose (energy expenditure, kcal/session) and intensity (relative to VO(2)peak) on abdominal obesity and selected cardiometabolic risk factors. The primary outcomes are waist circumference and 2-hour glucose. We will randomly assign 320 sedentary, abdominally obese men (N=160) and women (N=160) to one of 4 conditions: 1) no-exercise control, 2) low volume, low intensity exercise, 3) high volume, low intensity exercise, and 4) high volume, high intensity exercise. Duration of all treatments will be 6 months. The findings from this study may help resolve the following unanswered questions, "For a given exercise dose does higher exercise intensity result in greater health benefits?" "For a given exercise intensity does higher exercise dose result in greater health benefits?". Identification of the separate effects of exercise dose and intensity on obesity and related cardiometabolic risk factors under controlled conditions is important for development of optimal, lifestyle-based strategies that can subsequently be tested in long-term effectiveness trials.

Forensic psychiatry in Chile.

Mental disorders are among the most prevalent of chronic disorders, and a high prevalence of these disorders has been consistently found in jails and prisons. This study was a retrospective case series that described the population of adults charged with a criminal offense who were court ordered to undergo a psychiatric assessment within the Medical Legal Service in Santiago, Chile from 2005 to 2006. Characteristics were explored in order to better understand this population in light of the recent reforms in the judicial and health systems of Chile. Ninety percent of sampled individuals were male, primarily between the ages of 18-39 years. Seventy percent of the evaluations came from the pre-reformed judicial system and 30% were from the reformed system. Approximately 63% of evaluated offenders were considered to have a psychiatric pathology, the most common being the personality disorders. Of the evaluated offenders, approximately 84% were considered by a psychiatrist to be criminally responsible for their crime, 7% were regarded as having diminished criminal responsibility, 4% were considered to be not criminally responsible for their crime, and 4% were cases where criminal responsibility was not applicable. Profession status, municipality of residence, type of residence, ICD-10 diagnosis, treatment recommendation, and criminal responsibility were found to be significantly different between male and female evaluated offenders. Results from this investigation will contribute to knowledge about forensic psychiatry and mental health in Latin America, and will hopefully pave the way for more research and international comparisons.