Critical review of single-cell mechanotyping approaches for biomedical applications.

Accurate mechanical measurements of cells has the potential to improve diagnostics, therapeutics and advance understanding of disease mechanisms, where high-resolution mechanical information can be measured by deforming individual cells. Here we evaluate recently developed techniques for measuring cell-scale stiffness properties; while many such techniques have been developed, much of the work examining single-cell stiffness is impacted by difficulties in standardization and comparability, giving rise to large variations in reported mechanical moduli. We highlight the role of underlying mechanical theories driving this variability, and note opportunities to develop novel mechanotyping devices and theoretical models that facilitate convenient and accurate mechanical characterisation. Moreover, many high-throughput approaches are confounded by factors including cell size, surface friction, natural population heterogeneity and convolution of elastic and viscous contributions to cell deformability. We nevertheless identify key approaches based on deformability cytometry as a promising direction for further development, where both high-throughput and accurate single-cell resolutions can be realized.

CCK+ Interneurons Contribute to Thalamus-Evoked Feed-Forward Inhibition in the Prelimbic Prefrontal Cortex.

Interneurons in the medial prefrontal cortex (PFC) regulate local neural activity to influence cognitive, motivated, and emotional behaviors. Parvalbumin-expressing (PV+) interneurons are the primary mediators of thalamus-evoked feed-forward inhibition across the mouse cortex, including the anterior cingulate cortex, where they are engaged by inputs from the mediodorsal (MD) thalamus. In contrast, in the adjacent prelimbic (PL) cortex, we find that PV+ interneurons are scarce in the principal thalamorecipient layer 3 (L3), suggesting distinct mechanisms of inhibition. To identify the interneurons that mediate MD-evoked inhibition in PL, we combine slice physiology, optogenetics, and intersectional genetic tools in mice of both sexes. We find interneurons expressing cholecystokinin (CCK+) are abundant in L3 of PL, with cells exhibiting fast-spiking (fs) or non-fast-spiking (nfs) properties. MD inputs make stronger connections onto fs-CCK+ interneurons, driving them to fire more readily than nearby L3 pyramidal cells and other interneurons. CCK+ interneurons in turn make inhibitory, perisomatic connections onto L3 pyramidal cells, where they exhibit cannabinoid 1 receptor (CB1R) mediated modulation. Moreover, MD-evoked feed-forward inhibition, but not direct excitation, is also sensitive to CB1R modulation. Our findings indicate that CCK+ interneurons contribute to MD-evoked inhibition in PL, revealing a mechanism by which cannabinoids can modulate MD-PFC communication.

A scan-specific quality control acquisition for clinical whole-body (WB) MRI protocols.

Objective.Image quality in whole-body MRI (WB-MRI) may be degraded by faulty radiofrequency (RF) coil elements or mispositioning of the coil arrays. Phantom-based quality control (QC) is used to identify broken RF coil elements but the frequency of these acquisitions is limited by scanner and staff availability. This work aimed to develop a scan-specific QC acquisition and processing pipeline to detect broken RF coil elements, which is sufficiently rapid to be added to the clinical WB-MRI protocol. The purpose of this is to improve the quality of WB-MRI by reducing the number of patient examinations conducted with suboptimal equipment.Approach.A rapid acquisition (14 s additional acquisition time per imaging station) was developed that identifies broken RF coil elements by acquiring images from each individual coil element and using the integral body coil. This acquisition was added to one centre's clinical WB-MRI protocol for one year (892 examinations) to evaluate the effect of this scan-specific QC. To demonstrate applicability in multi-centre imaging trials, the technique was also implemented on scanners from three manufacturers.Main results. Over the course of the study RF coil elements were flagged as potentially broken on five occasions, with the faults confirmed in four of those cases. The method had a precision of 80% and a recall of 100% for detecting faulty RF coil elements. The coil array positioning measurements were consistent across scanners and have been used to define the expected variation in signal.Significance. The technique demonstrated here can identify faulty RF coil elements and positioning errors and is a practical addition to the clinical WB-MRI protocol. This approach was fully implemented on systems from two manufacturers and partially implemented on a third. It has potential to reduce the number of clinical examinations conducted with suboptimal hardware and improve image quality across multi-centre studies.

Clinical Investigation of Large Volume Subcutaneous Delivery up to 25 mL for Lean and Non-Lean Subjects.

PURPOSE: To evaluate the clinical feasibility and tolerability of large volume subcutaneous delivery at different injection depths for lean and non-lean subjects. METHODS: A single-center, randomized, subject-blinded, crossover study in 62 healthy subjects was conducted to evaluate delivery of a 10-cP solution containing hyaluronic acid. Subjects were separated into lean and non-lean cohort by SC thickness. A syringe pump was used to study the effect of different volumes (5, 12, 25 mL) of a viscous placebo solution and needle lengths (6, 9 and 12 mm) delivered at 0.5 mL/min. RESULTS: Across all treatments, injection sites were observed to have negligible leakage, ~34 kPa of back pressure, and VAS of mild pain with higher pain from needle insertion than during injection. While mild to moderate erythema was the most frequently reported ISR and edema was most prominent for 25 mL injections, all ISRs were resolved within 4 hours post injection. Subjects were unbothered by ISRs across all treatments and rated them as low distress scores (average 1.0-1.5 out of 6). CONCLUSION: SC injection of 25 mL is feasible and tolerable using a low-pain formulation for abdomen injection irrespective of subcutaneous thickness and injection depths at a delivery rate of 0.5 mL/min.

Enhanced acoustic streaming effects via sharp-edged 3D microstructures.

Acoustofluidic micromanipulation is an important tool for biomedical research, where acoustic forces offer the ability to manipulate fluids, cells, and particles in a rapid, biocompatible, and contact-free manner. Of particular interest is the investigation of acoustically driven sharp edges, where high tip velocity magnitudes and strong acoustic potential gradients drive rapid motion. Whereas prior devices utilizing 2D sharp edges have demonstrated promise for micromanipulation activities, taking advantage of 3D structures has the potential to increase their performance and the range of manipulation activities. In this work, we investigate high-magnitude acoustic streaming fields in the vicinity of sharp-edged, sub-wavelength 3D microstructures. We numerically model and experimentally demonstrate this in fabricating parametrically configured 3D microstructures whose tip-angle and geometry influence acoustic streaming velocities and the complexity of streaming vortices, finding that the simulated and realized velocities and streaming patterns are both tunable and a function of microstructure shape. These sharp-edge interfaces hold promise for biomedical studies benefiting from precise and targeted micromanipulation.

Quantitative mechanical stimulation of GPR68 using a novel 96 well flow plugin.

Mechanosensitive proteins play a crucial role in a range of physiological processes, including hearing, tactile sensation and regulating blood flow. While previous work has demonstrated the mechanosensitivity of several proteins, the ability to apply precisely defined mechanical forces to cells in a consistent, replicable manner remains a significant challenge. In this work we present a novel 96-well plate-compatible plugin device for generating highly-controlled flow-based mechanical simulation of cells, which enables quantitative assessment of mechanosensitive protein function. The device is used to mechanically stimulate HEK 293T cells expressing the mechanosensitive protein GPR68, a G protein-coupled receptor. By assaying intracellular calcium levels during flow-based cell stimulation, we determine that GPR68 signalling is a function of the applied shear-force. As this approach is compatible with conventional cell culture plates and allows for simultaneous readout in a conventional fluorescence plate reader, this represents a valuable new tool to investigate mechanotransduction.

A multi-cohort genome-wide association study in African ancestry individuals reveals risk loci for primary open-angle glaucoma.

Primary open-angle glaucoma (POAG), the leading cause of irreversible blindness worldwide, disproportionately affects individuals of African ancestry. We conducted a genome-wide association study (GWAS) for POAG in 11,275 individuals of African ancestry (6,003 cases; 5,272 controls). We detected 46 risk loci associated with POAG at genome-wide significance. Replication and post-GWAS analyses, including functionally informed fine-mapping, multiple trait co-localization, and in silico validation, implicated two previously undescribed variants (rs1666698 mapping to DBF4P2; rs34957764 mapping to ROCK1P1) and one previously associated variant (rs11824032 mapping to ARHGEF12) as likely causal. For individuals of African ancestry, a polygenic risk score (PRS) for POAG from our mega-analysis (African ancestry individuals) outperformed a PRS from summary statistics of a much larger GWAS derived from European ancestry individuals. This study quantifies the genetic architecture similarities and differences between African and non-African ancestry populations for this blinding disease.

Micro-Acoustic Holograms for Detachable Microfluidic Devices.

Acoustic microfluidic devices have advantages for diagnostic applications, therapeutic solutions, and fundamental research due to their contactless operation, simple design, and biocompatibility. However, most acoustofluidic approaches are limited to forming simple and fixed acoustic patterns, or have limited resolution. In this study,a detachable microfluidic device is demonstrated employing miniature acoustic holograms to create reconfigurable, flexible, and high-resolution acoustic fields in microfluidic channels, where the introduction of a solid coupling layer makes these holograms easy to fabricate and integrate. The application of this method to generate flexible acoustic fields, including shapes, characters, and arbitrarily rotated patterns, within microfluidic channels, is demonstrated.

Impact of HLA class I functional divergence on HIV control.

Heterozygosity of Human leukocyte antigen (HLA) class I genes is linked to beneficial outcomes after HIV infection, presumably through greater breadth of HIV epitope presentation and cytotoxic T cell response. Distinct allotype pairs, however, differ in the extent to which they bind shared sets of peptides. We developed a functional divergence metric that measures pairwise complementarity of allotype-associated peptide binding profiles. Greater functional divergence for pairs of HLA-A and/or HLA-B allotypes was associated with slower AIDS progression and independently with enhanced viral load control. The metric predicts immune breadth at the peptide level rather than gene level and redefines HLA heterozygosity as a continuum differentially affecting disease outcome. Functional divergence may affect response to additional infections, vaccination, immunotherapy, and other diseases where HLA heterozygote advantage occurs.

Brain magnetic resonance imaging and computed tomography findings in cats and dogs with central nervous system cryptococcosis in Australia: 23 cases (2009-2020).

OBJECTIVE: To describe the imaging findings in Australian cats and dogs with CNS cryptococcosis. ANIMALS: 23 cases (10 cats; 13 dogs) with CNS cryptococcosis and brain MRI or CT studies available to review. METHODS: Retrospective, multi-institutional case series. Brain MRI or CT studies were reviewed by a board-certified radiologist. Imaging findings were described and the differences between cats and dogs explored. RESULTS: Morphologic features were consistent with extra-axial lesions in all (n = 13) dogs and either intra-axial (5/10) or extra-axial (4/10) lesions in cats, with 1 cat having no detectable lesions in low-field brain MRI scans. Meningeal abnormalities were most common, followed by forebrain and cerebellar lesions. Intracranial MRI lesions were typically T2 hyperintense and T1 hypo- to isointense. Four cases had T2 hypointense lesions affecting the brain, sinonasal cavity, or regional lymph nodes. Intracranial CT lesions were mostly soft tissue attenuating. Contrast enhancement was present in all cases with contrast series available, with ring enhancement shown only in cats. Osteolysis was more common in dogs than cats, particularly affecting the cribriform plate. All 13 dogs and many (6/10) cats had at least 1 lesion affecting sinonasal or contiguous tissues, and locoregional lymphadenomegaly was common (7/10 cats; 11/13 dogs). CLINICAL RELEVANCE: Imaging lesions in cryptococcal meningoencephalitis were extra-axial in dogs but could be intra-axial or extra-axial in cats. Careful examination for extracranial lesions (sinonasal, retrobulbar, facial soft tissue, tympanic bullae, or locoregional lymph nodes) is important to provide alternative safe biopsy sites. T2 hypointense lesions, while rare, should prompt consideration of cryptococcosis.

A Data-Driven Framework for Direct Local Tensile Property Prediction of Laser Powder Bed Fusion Parts.

This article proposes a generalizable, data-driven framework for qualifying laser powder bed fusion additively manufactured parts using part-specific in situ data, including powder bed imaging, machine health sensors, and laser scan paths. To achieve part qualification without relying solely on statistical processes or feedstock control, a sequence of machine learning models was trained on 6299 tensile specimens to locally predict the tensile properties of stainless-steel parts based on fused multi-modal in situ sensor data and a priori information. A cyberphysical infrastructure enabled the robust spatial tracking of individual specimens, and computer vision techniques registered the ground truth tensile measurements to the in situ data. The co-registered 230 GB dataset used in this work has been publicly released and is available as a set of HDF5 files. The extensive training data requirements and wide range of size scales were addressed by combining deep learning, machine learning, and feature engineering algorithms in a relay. The trained models demonstrated a 61% error reduction in ultimate tensile strength predictions relative to estimates made without any in situ information. Lessons learned and potential improvements to the sensors and mechanical testing procedure are discussed.

A Rapid Prototyping Approach for Multi-Material, Reversibly Sealed Microfluidics.

Microfluidic organ-on-chip models recapitulate increasingly complex physiological phenomena to study tissue development and disease mechanisms, where there is a growing interest in retrieving delicate biological structures from these devices for downstream analysis. Standard bonding techniques, however, often utilize irreversible sealing, making sample retrieval unfeasible or necessitating destructive methods for disassembly. To address this, several commercial devices employ reversible sealing techniques, though integrating these techniques into early-stage prototyping workflows is often ignored because of the variation and complexity of microfluidic designs. Here, we demonstrate the concerted use of rapid prototyping techniques, including 3D printing and laser cutting, to produce multi-material microfluidic devices that can be reversibly sealed. This is enhanced via the incorporation of acrylic components directly into polydimethylsiloxane channel layers to enhance stability, sealing, and handling. These acrylic components act as a rigid surface separating the multiple mechanical seals created between the bottom substrate, the microfluidic features in the device, and the fluidic interconnect to external tubing, allowing for greater design flexibility. We demonstrate that these devices can be produced reproducibly outside of a cleanroom environment and that they can withstand ~1 bar pressures that are appropriate for a wide range of biological applications. By presenting an accessible and low-cost method, we hope to enable microfluidic prototyping for a broad range of biomedical research applications.

Breaking barriers: exploring mechanisms behind opening the blood-brain barrier.

The blood-brain barrier (BBB) is a selectively permeable membrane that separates the bloodstream from the brain. While useful for protecting neural tissue from harmful substances, brain-related diseases are difficult to treat due to this barrier, as it also limits the efficacy of drug delivery. To address this, promising new approaches for enhancing drug delivery are based on disrupting the BBB using physical means, including optical/photothermal therapy, electrical stimulation, and acoustic/mechanical stimulation. These physical mechanisms can temporarily and locally open the BBB, allowing drugs and other substances to enter. Focused ultrasound is particularly promising, with the ability to focus energies to targeted, deep-brain regions. In this review, we examine recent advances in physical approaches for temporary BBB disruption, describing their underlying mechanisms as well as evaluating the utility of these physical approaches with regard to their potential risks and limitations. While these methods have demonstrated efficacy in disrupting the BBB, their safety, comparative efficacy, and practicality for clinical use remain an ongoing topic of research.

Thomson-Einstein's Tea Leaf Paradox Revisited: Aggregation in Rings.

A distinct particle focusing spot occurs in the center of a rotating fluid, presenting an apparent paradox given the presence of particle inertia. It is recognized, however, that the presence of a secondary flow with a radial component drives this particle aggregation. In this study, we expand on the examination of this "Thomson-Einstein's tea leaf paradox" phenomenon, where we use a combined experimental and computational approach to investigate particle aggregation dynamics. We show that not only the rotational velocity, but also the vessel shape, have a significant influence on a particle's equilibrium position. We accordingly demonstrate the formation of a single focusing spot in a vessel center, as has been conclusively demonstrated elsewhere, but also the repeatable formation of stable ring-shaped particle arrangements.

Bacterial isolates and antibiotic susceptibility among women with abnormal vaginal discharge attending the gynecology clinic at a tertiary hospital in southwestern Uganda: a cross-sectional study.

BACKGROUND: Abnormal vaginal discharge is a common complaint among women of reproductive age, affecting about one- third of all women. In resource-limited settings where access to laboratory services is limited, treatment is usually syndromic. This approach may result in ineffective treatment, with high recurrence rates and a potential of developing antibiotic resistance. This study aimed to determine the bacterial isolates and antibiotic susceptibility among women with an abnormal vaginal discharge attending the gynecology clinic at a tertiary hospital in Southwestern Uganda. METHODS: We conducted a hospital based cross-sectional study among 361 women aged 15-49 years, presenting with abnormal vaginal discharge at the gynecology clinic of Mbarara Regional Referral Hospital from December 2020 to June 2021. Demographic characteristics were collected using a structured questionnaire. We collected cervical and vaginal sterile swabs and subjected them to wet preparation and gram stain. The specimens were cultured for bacterial isolates. Susceptibility testing was performed on samples with bacterial isolates using the Kirby-Bauer disc diffusion method, on the commonly prescribed antibiotics in this setting. We summarized and described the bacterial isolates and antibiotic susceptibility patterns as frequencies and percentages. RESULTS: We enrolled 361 women with abnormal vaginal discharge. Bacteria were isolated in 29.6% (107/361) of the women, and the commonest isolates included; Staphylococcus aureus 48.6% (52/107), Klebsiella pneumoniae 29.9% (32/107) and Enterococcus faecalis 15% (16/107). Yeast cells were found in 17.7% (64/361) of the women with abnormal vaginal discharge. Cefuroxime (90.7%) and Ciprofloxacin (81.3%) had a high level of sensitivity while high levels of resistance were observed for Doxycycline (86.0%) and Azithromycin (67.0%). CONCLUSION: The common bacterial isolates were Staphylococcus aureus, Klebsiella pneumoniae and Enterococcus faecalis. The isolated bacteria were most sensitive to Cefuroxime and Ciprofloxacin but resistant to Doxycycline and Azithromycin. There is need for routine culture and susceptibility testing of women with abnormal vaginal discharge so as to guide treatment, minimize inappropriate antibiotic use and consequently reduce antibiotic resistance.

Deafening Silence of Male Infertility.

Think about 6 loved ones of reproductive age in your life. Now imagine that 1 of these 6 individuals is suffering from infertility. Perhaps they feel alone and isolated, unable to discuss their heartbreak with their closest friends, family, and support network. Suffering in silence. In this editorial, we discuss the infertility journey through the lens of the patients, the providers, and the scientists who struggle with infertility each and every day. Our goal is to open a dialogue surrounding infertility, with an emphasis on dismantling the longstanding societal barriers to acknowledging male infertility as a disease. Through education, communication, compassion, and advocacy, together we can all begin to break the deafening silence of male infertility.

Calculating the Costs of Implementing Integrated Packages of Community Health Services: Methods, Experiences, and Results From 6 sub-Saharan African Countries.

BACKGROUND: Ensuring access to a package of integrated primary health care services is essential for achieving universal health coverage. In many countries, community health programs are necessary for primary health care service provision, but they are generally underfunded, and countries often lack the necessary evidence on costs and resource requirements. We conducted prospective cost analyses of community health programs in 6 countries in sub-Saharan Africa using the Community Health Planning and Costing Tool. METHODS: The Community Health Planning and Costing Tool is a spreadsheet-based tool designed to cost key programmatic elements of community health services packages, including training, equipment, incentives, supervision, and management. In each country, stakeholders defined a package of community health services and corresponding standard treatment guidelines to estimate normative costs, which were applied to program scale-up targets. The data were entered into the tool, and cost models were prepared for different geographical and service utilization scenarios. The results were reviewed and validated with the governments, implementing partners, and expert panels. Additional scale-up scenarios were modeled, taking into account probable constraints to increasing community health service provision and potential funding limitations. RESULTS: The services and scope of community health service packages varied by country, depending on contextual factors and determined health priorities. The package costs also varied significantly depending on the size and contents of the service package, the service delivery approach, the remuneration of the community health workers, and the cost of medicines and supplies. CONCLUSIONS: Community health programs and service packages are different in every country and change over time as they evolve. They should be routinely costed as an integral part of the planning and budgeting process and to ensure that sufficient resources are allocated for their effective and efficient implementation.

National Diet and Nutrition Survey data reveal a decline in folate status in the United Kingdom population between 2008 and 2019.

BACKGROUND: Folate is essential for healthy growth and development. Fortification of foods with folic acid can improve folate status and reduce risk of neural tube defects (NTD). Following concern around folate status in the United Kingdom, the United Kingdom government announced in 2021 the intention to introduce mandatory folic acid fortification. OBJECTIVE: This study aimed to describe folate status in the United Kingdom population prior to the implementation of mandatory folic acid fortification of non-whole wheat (non-wholemeal) flour and to assess trends in folate status, including in females of reproductive age (FRA). METHODS: Data were from the United Kingdom National Diet and Nutrition Survey Rolling Program (2008-2019), a cross-sectional, nationally representative survey of children and adults aged 1.5+ (n = 5792 with folate result). Serum folate concentration was measured by liquid chromatography tandem mass spectrometry (LC-MS/MS) and red blood cell (RBC) folate concentration by microbiological assay. Concentration data were compared against method-specific cut-offs and thresholds, and relationships were explored against demographic and lifestyle characteristics. RESULTS: RBC and serum folate concentration significantly decreased by ∼3 percentage points per year between 2008 and 2019 in all age/sex groups. Prevalence of deficiency (RBC folate < 305 nmol/L) was highest in children aged 11 to 18 y (17% in 2016-2019). The proportion of FRA below the cut-off for increased risk of NTD (RBC folate < 748 nmol/L) increased from 69% to 89% between 2008 and 2019. Ethnicity, smoking status, and income were significant determinants of RBC and serum folate concentrations. CONCLUSIONS: These data reveal a decline in population folate status in the United Kingdom between 2008 and 2019 and a high prevalence of folate deficiency. A high proportion of FRA had RBC folate concentrations below the cut-off for increased risk of NTD. These data provide information on folate status in a population not currently exposed to mandatory folic acid fortification and are essential to model and assess its impact.

Development of Sensitive Methods for the Detection of Minimum Concentrations of DNA on Martian Soil Simulants.

Several methods used for the quantification of DNA are based on UV absorbance or the fluorescence of complexes with intercalator dyes. Most of these intercalators are used in gels to visualize DNA and its structural integrity. Due to many extraterrestrial samples, such as meteorites or comets, which are likely to contain very small amounts of biological material, and because the ability to detect this material is crucial for understanding the origin and evolution of life in the universe, the development of assays that can detect DNA at low limits and withstand the rigors of space exploration is a pressing need in the field of astrobiology. In this study, we present a comparison of optimized protocols used for the fast and accurate quantification of DNA using common intercalator dyes. The sensitivity of assays exceeded that generated by any commercial kit and allowed for the accurate quantification of minimum concentrations of DNA. The methods were successful when applied to the detection and measurement of DNA spiked on soil samples. Furthermore, the impact of UV radiation as a harsh condition on the surface of Mars was assessed by DNA degradation and this was also confirmed by gel electrophoresis. Overall, the methods described provide economical, simple-step, and efficient approaches for the detection of DNA and can be used in future planetary exploration missions as tests used for the extraction of nucleic acid biosignatures.

Developmental Diet Alters the Fecundity-Longevity Relationship and Age-Related Gene Expression in Drosophila melanogaster.

The standard evolutionary theory of aging predicts a negative relationship (trade-off) between fecundity and longevity. However, in principle, the fecundity-longevity relationship can become positive in populations in which individuals have unequal resources. Positive fecundity-longevity relationships also occur in queens of eusocial insects such as ants and bees. Developmental diet is likely to be central to determining trade-offs as it affects key fitness traits, but its exact role remains uncertain. For example, in Drosophila melanogaster, changes in adult diet can affect fecundity, longevity, and gene expression throughout life, but it is unknown how changes in developmental (larval) diet affect fecundity-longevity relationships and gene expression in adults. Using D. melanogaster, we tested the hypothesis that varying developmental diets alters the directionality of fecundity-longevity relationships in adults, and characterized associated gene expression changes. We reared larvae on low (20%), medium (100%), and high (120%) yeast diets, and transferred adult females to a common diet. We measured fecundity and longevity of individual adult females and profiled gene expression changes with age. Adult females raised on different larval diets exhibited fecundity-longevity relationships that varied from significantly positive to significantly negative, despite minimal differences in mean lifetime fertility or longevity. Treatments also differed in age-related gene expression, including for aging-related genes. Hence, the sign of fecundity-longevity relationships in adult insects can be altered and even reversed by changes in larval diet quality. By extension, larval diet differences may represent a key mechanistic factor underpinning positive fecundity-longevity relationships observed in species such as eusocial insects.