In Situ Complexation of sgRNA and Cas12a Improves the Performance of a One-Pot RPA-CRISPR-Cas12 Assay.

Due to their ability to selectively target pathogen-specific nucleic acids, CRISPR-Cas systems are increasingly being employed as diagnostic tools. "One-pot" assays that combine nucleic acid amplification and CRISPR-Cas systems (NAAT-CRISPR-Cas) in a single step have emerged as one of the most popular CRISPR-Cas biosensing formats. However, operational simplicity comes at a cost, with one-pot assays typically being less sensitive than corresponding two-step NAAT-CRISPR-Cas assays and often failing to detect targets at low concentrations. It is thought that these performance reductions result from the competition between the two enzymatic processes driving the assay, namely, Cas-mediated cis-cleavage and polymerase-mediated amplification of the target DNA. Herein, we describe a novel one-pot RPA-Cas12a assay that circumvents this issue by leveraging in situ complexation of the target-specific sgRNA and Cas12a to purposefully limit the concentration of active Cas12a during the early stages of the assay. Using a clinically relevant assay against a DNA target for HPV-16, we show how this in situ format reduces competition between target cleavage and amplification and engenders significant improvements in detection limit when compared to the traditional one-pot assay format, even in patient-derived samples. Finally, to gain further insight into the assay, we use experimental data to formulate a mechanistic model describing the competition between the Cas enzyme and nucleic acid amplification. These findings suggest that purposefully limiting cis-cleavage rates of Cas proteins is a viable strategy for improving the performance of one-pot NAAT-CRISPR-Cas assays.

Synergistic Effects of Weight Loss and Catheter Ablation: Can microRNAs Serve as Predictive Biomarkers for the Prevention of Atrial Fibrillation Recurrence?

In atrial fibrillation (AF), multifactorial pathologic atrial alterations are manifested by structural and electrophysiological changes known as atrial remodeling. AF frequently develops in the context of underlying cardiac abnormalities. A critical mechanistic role played by atrial stretch is played by abnormal substrates in a number of conditions that predispose to AF, including obesity, heart failure, hypertension, and sleep apnea. The significant role of overweight and obesity in the development of AF is known; however, the differential effect of overweight, obesity, cardiovascular comorbidities, lifestyle, and other modifiable risk factors on the occurrence and recurrence of AF remains to be determined. Reverse remodeling of the atrial substrate and subsequent reduction in the AF burden by conversion into a typical sinus rhythm has been associated with weight loss through lifestyle changes or surgery. This makes it an essential pillar in the management of AF in obese patients. According to recently published research, microRNAs (miRs) may function as post-transcriptional regulators of genes involved in atrial remodeling, potentially contributing to the pathophysiology of AF. The focus of this review is on their modulation by both weight loss and catheter ablation interventions to counteract atrial remodeling in AF. Our analysis outlines the experimental and clinical evidence supporting the synergistic effects of weight loss and catheter ablation (CA) in reversing atrial electrical and structural remodeling in AF onset and in recurrent post-ablation AF by attenuating pro-thrombotic, pro-inflammatory, pro-fibrotic, arrhythmogenic, and male-sex-associated hypertrophic remodeling pathways. Furthermore, we discuss the promising role of miRs with prognostic potential as predictive biomarkers in guiding approaches to AF recurrence prevention.

Atrial fibrillation in the setting of cardiac amyloidosis - A review of the literature.

Cardiac amyloidosis (CA) is related to the aggregation of insoluble fibrous deposits of misfolded proteins within the myocardium. Transthyretin amyloidosis (ATTR) and immunoglobulin light-chain amyloidosis are the main forms of CA. Atrial fibrillation (AF) is a common arrhythmia in CA patients, especially in those with ATTR amyloidosis. Increased atrial preload and afterload, atrial enlargement, enhanced atrial wall stress, and autonomic dysfunction are the main mechanisms of AF in CA patients. CA is associated with the formation of endocardial thrombi and systemic embolism. The promoters of thrombogenesis include endomyocardial damage, blood stasis, and hypercoagulability. The prevalence of thrombi in patients with AF remains elevated despite long-term anticoagulation. Consequently, transesophageal ultrasound examinations before cardioversion should be performed to exclude endocardiac thrombi despite anticoagulation. Furthermore, the CHA2DS2-VASc score should not be used to assess the thromboembolic risk in CA patients with AF. Rate control is challenging in patients with CA, while rhythm control is the preferred treatment option, especially in the early stages of the disease process. Although catheter ablation is an effective treatment option, more data are needed to explore the role of the procedure in CA patients.

Ultrahigh-Throughput, Real-Time Flow Cytometry for Rare Cell Quantification from Whole Blood.

We present an ultrahigh-throughput, real-time fluorescence cytometer comprising a viscoelastic microfluidic system and a complementary metal-oxide-semiconductor (CMOS) linear image sensor-based detection system. The flow cytometer allows for real-time quantification of a variety of fluorescence species, including micrometer-sized particles and cells, at analytical throughputs in excess of 400,000 species per second. The platform integrates a custom C++ control program and graphical user interface (GUI) to allow for the processing of raw signals, adjustment of processing parameters, and display of fluorescence intensity histograms in real time. To demonstrate the efficacy of the platform for rare event detection and its utility as a basic clinical tool, we measure and quantify patient-derived circulating tumor cells (CTCs) in peripheral blood, realizing that detection has a sensitivity of 6 CTCs per million blood cells (0.000006%) with a volumetric throughput of over 3 mL/min.

Direct isolation of small extracellular vesicles from human blood using viscoelastic microfluidics.

Small extracellular vesicles (sEVs; <200 nm) that contain lipids, nucleic acids, and proteins are considered promising biomarkers for a wide variety of diseases. Conventional methods for sEV isolation from blood are incompatible with routine clinical workflows, significantly hampering the utilization of blood-derived sEVs in clinical settings. Here, we present a simple, viscoelastic-based microfluidic platform for label-free isolation of sEVs from human blood. The separation performance of the device is assessed by isolating fluorescent sEVs from whole blood, demonstrating purities and recovery rates of over 97 and 87%, respectively. Significantly, our viscoelastic-based microfluidic method also provides for a remarkable increase in sEV yield compared to gold-standard ultracentrifugation, with proteomic profiles of blood-derived sEVs purified by both methods showing similar protein compositions. To demonstrate the clinical utility of the approach, we isolate sEVs from blood samples of 20 patients with cancer and 20 healthy donors, demonstrating that elevated sEV concentrations can be observed in blood derived from patients with cancer.

Atrial Myopathy and Ischemic Stroke in Heart Failure With Preserved Ejection Fraction.

Recent studies suggested an association between atrial myopathy and stroke independent of atrial fibrillation (AF). We examined the hypothesis that atrial myopathy may be associated with ischemic stroke in patients with heart failure with preserved ejection fraction. This is an exploratory, post hoc analysis of the Treatment of Preserved Cardiac Function Heart Failure with an Aldosterone Antagonist (TOPCAT) trial. Patients with sinus rhythm documented at baseline electrocardiogram and without known AF were included in this analysis. Atrial myopathy was defined by echocardiographic evidence of left atrial (LA) enlargement (LA diameter >46 mm or LA volume index >36 ml/m2) or elevated natriuretic peptides (brain natriuretic peptide >100 pg/ml or N-terminal Pro-B-type natriuretic peptide >400 pg/ml). We used Cox regression to investigate the effect of atrial myopathy on incident ischemic stroke over the study period. In 3,445 patients in the TOPCAT trial, 2,225 (mean age 67.5 ± 4.9 years; female 54.8%) had normal sinus rhythm at baseline and no history of AF. Atrial myopathy was present in 756 patients (34.0%). During a median follow-up of 2 years, 56 patients (2.5%) developed ischemic stroke, including 25 with atrial myopathy. Atrial myopathy was associated with increased risk of stroke (hazard ratio = 1.74, 95% confidence interval 1.01 to 2.98, p = 0.04) in multivariate analysis. Diabetes mellitus (hazard ratio = 2.02, 95% confidence interval 1.19 to 3.43 p = 0.01) was the only other independent predictor of stroke. In patients with heart failure with preserved ejection fraction, atrial myopathy increases the risk of ischemic stroke, in the absence of AF. Further investigations are needed to better characterize this association and implement stroke prevention strategies.

Safety and efficacy of colchicine for the prevention of post-operative atrial fibrillation in patients undergoing cardiac surgery: a meta-analysis of randomized controlled trials.

BACKGROUND AND AIMS: Colchicine is an anti-inflammatory drug that may prevent post-operative atrial fibrillation (POAF). The effect of this drug has been inconsistently shown in previous clinical trials. We aimed to compare the efficacy and safety of colchicine vs. placebo to prevent POAF in patients undergoing cardiac surgery. METHODS AND RESULTS: A systematic search of EMBASE, MEDLINE, SCOPUS, ClinicalTrials.gov, and the Cochrane Library for randomized controlled trials (RCTs) was conducted from inception till April 2023. The primary outcome was the incidence of POAF after any cardiac surgery. The secondary outcome was the rate of drug discontinuation due to adverse events and adverse gastrointestinal events. Risk ratios (RR) were reported using the Mantel Haenszel method. A total of eight RCTs comprising 1885 patients were included. There was a statistically significant lower risk of developing POAF with colchicine vs. placebo (RR: 0.70; 95% CI: 0.59-0.82; P < 0.01, I2 = 0%), and this effect persisted across different subgroups. There was a significantly higher risk of adverse gastrointestinal events (RR: 2.20; 95% CI: 1.38-3.51; P < 0.01, I2 = 55%) with no difference in the risk of drug discontinuation in patients receiving colchicine vs. placebo (RR: 1.33; 95% CI: 0.93-1.89; P = 0.11, I2 = 0%). CONCLUSION: This meta-analysis of eight RCTs shows that colchicine is effective at preventing POAF, with a significantly higher risk of adverse gastrointestinal events but no difference in the rate of drug discontinuation. Future studies are required to define the optimal duration and dose of colchicine for the prevention of POAF.

Association of atrial fibrillation and outcomes in patients undergoing bone marrow transplantation.

AIMS: Haematopoietic stem cell transplantation (HSCT) is a potentially curative therapy for several malignant and non-malignant haematologic conditions. Patients undergoing HSCT are at an increased risk of developing atrial fibrillation (AF). We hypothesized that a diagnosis of AF would be associated with poor outcomes in patients undergoing HSCT. METHODS AND RESULTS: The National Inpatient Sample (2016-19) was queried with ICD-10 codes to identify patients aged >50 years undergoing HSCT. Clinical outcomes were compared between patients with and without AF. A multivariable regression model adjusting for demographics and comorbidities was used to calculate the adjusted odds ratio (aOR) and regression coefficients with corresponding 95% confidence intervals and P-values. A total of 50 570 weighted hospitalizations for HSCT were identified, out of which 5820 (11.5%) had AF. Atrial fibrillation was found to be independently associated with higher inpatient mortality (aOR 2.75; 1.9-3.98; P < 0.001), cardiac arrest (aOR 2.86; 1.55-5.26; P = 0.001), acute kidney injury (aOR 1.89; 1.6-2.23; P < 0.001), acute heart failure exacerbation (aOR 5.01; 3.54-7.1; P < 0.001), cardiogenic shock (aOR 7.73; 3.17-18.8; P < 0.001), and acute respiratory failure (aOR 3.24; 2.56-4.1; P < 0.001) as well as higher mean length of stay (LOS) (+2.67; 1.79-3.55; P < 0.001) and cost of care (+67 529; 36 630-98 427; P < 0.001). CONCLUSION: Among patients undergoing HSCT, AF was independently associated with poor in-hospital outcomes, higher LOS, and cost of care.

Cx3cr1 controls kidney resident macrophage heterogeneity.

Kidney macrophages are comprised of both monocyte-derived and tissue resident populations; however, the heterogeneity of kidney macrophages and factors that regulate their heterogeneity are poorly understood. Herein, we performed single cell RNA sequencing (scRNAseq), fate mapping, and parabiosis to define the cellular heterogeneity of kidney macrophages in healthy mice. Our data indicate that healthy mouse kidneys contain four major subsets of monocytes and two major subsets of kidney resident macrophages (KRM) including a population with enriched Ccr2 expression, suggesting monocyte origin. Surprisingly, fate mapping data using the newly developed Ms4a3Cre Rosa Stopf/f TdT model indicate that less than 50% of Ccr2+ KRM are derived from Ly6chi monocytes. Instead, we find that Ccr2 expression in KRM reflects their spatial distribution as this cell population is almost exclusively found in the kidney cortex. We also identified Cx3cr1 as a gene that governs cortex specific accumulation of Ccr2+ KRM and show that loss of Ccr2+ KRM reduces the severity of cystic kidney disease in a mouse model where cysts are mainly localized to the kidney cortex. Collectively, our data indicate that Cx3cr1 regulates KRM heterogeneity and niche-specific disease progression.

Consumer-Led Screening for Atrial Fibrillation: Frontier Review of the AF-SCREEN International Collaboration.

The technological evolution and widespread availability of wearables and handheld ECG devices capable of screening for atrial fibrillation (AF), and their promotion directly to consumers, has focused attention of health care professionals and patient organizations on consumer-led AF screening. In this Frontiers review, members of the AF-SCREEN International Collaboration provide a critical appraisal of this rapidly evolving field to increase awareness of the complexities and uncertainties surrounding consumer-led AF screening. Although there are numerous commercially available devices directly marketed to consumers for AF monitoring and identification of unrecognized AF, health care professional-led randomized controlled studies using multiple ECG recordings or continuous ECG monitoring to detect AF have failed to demonstrate a significant reduction in stroke. Although it remains uncertain if consumer-led AF screening reduces stroke, it could increase early diagnosis of AF and facilitate an integrated approach, including appropriate anticoagulation, rate or rhythm management, and risk factor modification to reduce complications. Companies marketing AF screening devices should report the accuracy and performance of their products in high- and low-risk populations and avoid claims about clinical outcomes unless improvement is demonstrated in randomized clinical trials. Generally, the diagnostic yield of AF screening increases with the number, duration, and temporal dispersion of screening sessions, but the prognostic importance may be less than for AF detected by single-time point screening, which is largely permanent, persistent, or high-burden paroxysmal AF. Consumer-initiated ECG recordings suggesting possible AF always require confirmation by a health care professional experienced in ECG reading, whereas suspicion of AF on the basis of photoplethysmography must be confirmed with an ECG. Consumer-led AF screening is unlikely to be cost-effective for stroke prevention in the predominantly young, early adopters of this technology. Studies in older people at higher stroke risk are required to demonstrate both effectiveness and cost-effectiveness. The direct interaction between companies and consumers creates new regulatory gaps in relation to data privacy and the registration of consumer apps and devices. Although several barriers for optimal use of consumer-led screening exist, results of large, ongoing trials, powered to detect clinical outcomes, are required before health care professionals should support widespread adoption of consumer-led AF screening.

Quantifying Antibody Binding Kinetics on Fixed Cells and Tissues via Fluorescence Lifetime Imaging.

We present a method for monitoring spatially localized antigen-antibody binding events on physiologically relevant substrates (cell and tissue sections) using fluorescence lifetime imaging. Specifically, we use the difference between the fluorescence decay times of fluorescently tagged antibodies in free solution and in the bound state to track the bound fraction over time and hence deduce the binding kinetics. We make use of a microfluidic probe format to minimize the mass transport effects and localize the analysis to specific regions of interest on the biological substrates. This enables measurement of binding constants (kon) on surface-bound antigens and on cell blocks using model biomarkers. Finally, we directly measure p53 kinetics with differential biomarker expression in ovarian cancer tissue sections, observing that the degree of expression corresponds to the changes in kon, with values of 3.27-3.50 × 103 M-1 s-1 for high biomarker expression and 2.27-2.79 × 103 M-1 s-1 for low biomarker expression.

Autonomic Neuromodulation for Atrial Fibrillation Following Cardiac Surgery: JACC Review Topic of the Week.

Autonomic neuromodulation therapies (ANMTs) (ie, ganglionated plexus ablation, epicardial injections for temporary neurotoxicity, low-level vagus nerve stimulation [LL-VNS], stellate ganglion block, baroreceptor stimulation, spinal cord stimulation, and renal nerve denervation) constitute an emerging therapeutic approach for arrhythmias. Very little is known about ANMTs' preventive potential for postoperative atrial fibrillation (POAF) after cardiac surgery. The purpose of this review is to summarize and critically appraise the currently available evidence. Herein, the authors conducted a systematic review of 922 articles that yielded 7 randomized controlled trials. In the meta-analysis, ANMTs reduced POAF incidence (OR: 0.37; 95% CI: 0.25 to 0.55) and burden (mean difference [MD]: -3.51 hours; 95% CI: -6.64 to -0.38 hours), length of stay (MD: -0.82 days; 95% CI: -1.59 to -0.04 days), and interleukin-6 (MD: -79.92 pg/mL; 95% CI: -151.12 to -8.33 pg/mL), mainly attributed to LL-VNS and epicardial injections. Moving forward, these findings establish a base for future larger and comparative trials with ANMTs, to optimize and expand their use.

Neuromodulation of Inflammation to Treat Heart Failure With Preserved Ejection Fraction: A Pilot Randomized Clinical Trial.

Background A systemic proinflammatory state plays a central role in the development of heart failure with preserved ejection fraction. Low-level transcutaneous vagus nerve stimulation suppresses inflammation in humans. We conducted a sham-controlled, double-blind, randomized clinical trial to examine the effect of chronic low-level transcutaneous vagus nerve stimulation on cardiac function, exercise capacity, and inflammation in patients with heart failure with preserved ejection fraction. Methods and Results Patients with heart failure with preserved ejection fraction and at least 2 additional comorbidities (obesity, diabetes, hypertension, or age ≥65 years) were randomized to either active (tragus) or sham (earlobe) low-level transcutaneous vagus nerve stimulation (20 Hz, 1 mA below discomfort threshold), for 1 hour daily for 3 months. Echocardiography, 6-minute walk test, quality of life, and serum cytokines were assessed at baseline and 3 months. Fifty-two patients (mean age 70.4±9.2 years; 70% female) were included (active, n=26; sham, n=26). Baseline characteristics were balanced between the 2 arms. Adherence to the protocol of daily stimulation was >90% in both arms (P>0.05). While the early mitral inflow Doppler velocity to the early diastolic mitral annulus velocity ratio did not differ between groups, global longitudinal strain and tumor necrosis factor-α levels at 3 months were significantly improved in the active compared with the sham arm (-18.6%±2.5% versus -16.0%±2.4%, P=0.002; 8.9±2.8 pg/mL versus 11.3±2.9 pg/mL, P=0.007, respectively). The reduction in tumor necrosis factor-α levels correlated with global longitudinal strain improvement (r=-0.73, P=0.001). Quality of life was better in the active arm. No device-related side effects were observed. Conclusions Neuromodulation with low-level transcutaneous vagus nerve stimulation over 3 months resulted in a significant improvement in global longitudinal strain, inflammatory cytokines, and quality of life in patients with heart failure with preserved ejection fraction. Registration URL: https://www.clinicaltrials.gov; Unique identifier: NCT03327649.

Hybrid Microfluidic Device for High Throughput Isolation of Cells Using Aptamer Functionalized Diatom Frustules.

Circulating tumor cells (CTCs), secreted from primary and metastatic malignancies, hold a wealth of essential diagnostic and prognostic data for multiple cancers. Significantly, the information contained within these cells may hold the key to understanding cancer metastasis, both individually and fundamentally. Accordingly, developing ways to identify, isolate and interrogate CTCs plays an essential role in modern cancer research. Unfortunately, CTCs are typically present in the blood in vanishingly low titers and mixed with other blood components, making their isolation and analysis extremely challenging. Herein, we report the design, fabrication and optimization of a microfluidic device capable of automatically isolating CTCs from whole blood. This is achieved in two steps, via the passive viscoelastic separation of CTCs and white blood cells (WBCs) from red blood cells (RBCs), and subsequent active magnetophoretic separation of CTCs from WBCs. We detail the specific geometries required to balance the elastic and inertial forces required for successful passive separation of RBCs, and the use of computational fluid dynamics (CFD) to optimize active magnetophoretic separation. We subsequently describe the use of magnetic biosilica frustules, extracted from Chaetoceros sp. diatoms, to fluorescently tag CTCs and facilitate magnetic isolation. Finally, we use our microfluidic platform to separate HepG2-derived CTCs from whole blood, demonstrating exceptional CTC recovery (94.6%) and purity (89.7%).

Impact of low-level electromagnetic fields on the inducibility of atrial fibrillation in the electrophysiology laboratory.

BACKGROUND: Atrial fibrillation (AF) is the most common sustained arrhythmia in adults. Research suggests that autonomic nervous (ANS) system dysfunction contributes to AF pathophysiology. Animal studies have shown that low-level electromagnetic fields (LL-EMF) are potentially capable of AF suppression. This study evaluated the safety and efficacy of LL-EMF in suppressing AF in humans. OBJECTIVE: To investigate the impact of LL-EMF on AF inducibility in humans. METHODS: Patients presenting for ablation of paroxysmal AF were randomized to a sham protocol or LL-EMF (3.2 × 10-8 G at 0.89 Hz) applied via a Helmholtz coil around the head. AF was induced via atrial pacing, and was cardioverted if duration was greater than 15 minutes. The protocol was then run for 60 minutes, followed by reinduction of AF. The primary endpoint was the duration of pacing-induced AF after protocol completion compared between groups. RESULTS: Eighteen patients completed the study protocol (n = 10 sham, n = 8 LL-EMF). Pacing-induced AF duration in the LL-EMF group was 11.0 ± 3.43 minutes shorter than control after protocol completion (CI 3.72-18.28 minutes, P = .03). A smaller proportion of LL-EMF patients experienced spontaneous firing initiating an AF episode (0/7 vs 5/6, P = .0047). A significantly greater proportion of patients in the control group required direct current cardioversion after 1 hour (0.78 vs 0.13, P = .02). CONCLUSION: In patients with paroxysmal AF, LL-EMF stimulation results in shorter episodes of pacing-induced AF and a reduced likelihood of spontaneous firing initiating an episode of AF.

In-vitro and in-vivo characterization of CRANAD-2 for multi-spectral optoacoustic tomography and fluorescence imaging of amyloid-beta deposits in Alzheimer mice.

The abnormal deposition of fibrillar beta-amyloid (Aß) deposits in the brain is one of the major histopathological hallmarks of Alzheimer's disease (AD). Here, we characterized curcumin-derivative CRANAD-2 for multi-spectral optoacoustic tomography and fluorescence imaging of brain Aß deposits in the arcAß mouse model of AD cerebral amyloidosis. CRANAD-2 showed a specific and quantitative detection of Aß fibrils in vitro, even in complex mixtures, and it is capable of distinguishing between monomeric and fibrillar forms of Aß. In vivo epi-fluorescence microscopy and optoacoustic tomography after intravenous CRANAD-2 administration demonstrated higher cortical retention in arcAß compared to non-transgenic littermate mice. Immunohistochemistry showed co-localization of CRANAD-2 and Aß deposits in arcAß mouse brain sections, thus verifying the specificity of the probe. In conclusion, we demonstrate suitability of CRANAD-2 for optical detection of Aß deposits in animal models of AD pathology, which facilitates mechanistic studies and the monitoring of putative treatments targeting Aß deposits.

Screening for Atrial Fibrillation in American Indian Adults in a Tribal Primary Care Clinic.

Background American Indian adults have a higher risk of atrial fibrillation (AF) compared with other racial groups. We implemented opportunistic screening to detect silent AF in American Indian adults attending a tribal health system using a mobile, single-lead ECG device. Methods and Results American Indian patients aged ≥50 years followed in a tribal primary care clinic with no history of AF underwent a 30-second ECG. A cardiologist overread all tracings to confirm the diagnosis of AF. After AF was confirmed, patients were referred to their primary care physician for initiation of anticoagulation. Patients seen over the same time period, who were not undergoing screening, served as controls. A total of 1019 patients received AF screening (mean age, 61.5±8.9 years, 62% women). Age and sex distribution of those screened was similar to the overall clinic population. New AF was diagnosed in 15 of 1019 (1.5%) patients screened versus 4 of 1267 (0.3%) patients who were not screened (mean difference, 1.2%; 95% CI, 0.3%-2.2%, P=0.002). Eight of 15 with new screen-detected AF were aged <65 years. Those with screen-detected AF were slightly older and had a higher CHA2DS2-VASc score than those without AF. Fourteen of 15 patients diagnosed with new AF had a CHA2DS2-VASc score ≥1 and initiated anticoagulation. Conclusions Opportunistic, mobile single-lead ECG screening for AF is feasible in tribal clinics, and detects more AF than usual care, leading to appropriate initiation of anticoagulation. AF develops at a younger age in American Indian adults who would likely benefit from earlier AF screening. Registration URL: https://www.clinicaltrials.gov; Unique identifier: NCT03740477.

A weakly supervised deep learning approach for label-free imaging flow-cytometry-based blood diagnostics.

The application of machine learning approaches to imaging flow cytometry (IFC) data has the potential to transform the diagnosis of hematological diseases. However, the need for manually labeled single-cell images for machine learning model training has severely limited its clinical application. To address this, we present iCellCnn, a weakly supervised deep learning approach for label-free IFC-based blood diagnostics. We demonstrate the capability of iCellCnn to achieve diagnosis of Sézary syndrome (SS) from patient samples on the basis of bright-field IFC images of T cells obtained after fluorescence-activated cell sorting of human peripheral blood mononuclear cell specimens. With a sample size of four healthy donors and five SS patients, iCellCnn achieved a 100% classification accuracy. As iCellCnn is not restricted to the diagnosis of SS, we expect such weakly supervised approaches to tap the diagnostic potential of IFC by providing automatic data-driven diagnosis of diseases with so-far unknown morphological manifestations.

Non-invasive vagus nerve stimulation attenuates proinflammatory cytokines and augments antioxidant levels in the brainstem and forebrain regions of Dahl salt sensitive rats.

The anti-inflammatory effects of vagus nerve stimulation are well known. It has recently been shown that low-level, transcutaneous stimulation of vagus nerve at the tragus (LLTS) reduces cardiac inflammation in a rat model of heart failure with preserved ejection fraction (HFpEF). The mechanisms by which LLTS affect the central neural circuits within the brain regions that are important for the regulation of cardiac vagal tone are not clear. Female Dahl salt-sensitive rats were initially fed with either low salt (LS) or high salt (HS) diet for a period of 6 weeks, followed by sham or active stimulation (LLTS) for 30 min daily for 4 weeks. To study the central effects of LLTS, four brainstem (SP5, NAb, NTS, and RVLM) and two forebrain sites (PVN and SFO) were examined. HS diet significantly increased the gene expression of proinflammatory cytokines in the SP5 and SFO. LLTS reversed HS diet-induced changes at both these sites. Furthermore, LLTS augmented the levels of antioxidant Nrf2 in the SP5 and SFO. Taken together, these findings suggest that LLTS has central anti-inflammatory and antioxidant properties that could mediate the neuromodulation of cardiac vagal tone in the rat model of HFpEF.

Deformation of leukaemia cell lines in hyperbolic microchannels: investigating the role of shear and extensional components.

The mechanical properties of cells are of enormous interest in a diverse range of physio and pathological situations of clinical relevance. Unsurprisingly, a variety of microfluidic platforms have been developed in recent years to study the deformability of cells, most commonly employing pure shear or extensional flows, with and without direct contact of the cells with channel walls. Herein, we investigate the effects of shear and extensional flow components on fluid-induced cell deformation by means of three microchannel geometries. In the case of hyperbolic microchannels, cell deformation takes place in a flow with constant extensional rate, under non-zero shear conditions. A sudden expansion at the microchannel terminus allows one to evaluate shape recovery subsequent to deformation. Comparison with other microchannel shapes, that induce either pure shear (straight channel) or pure extensional (cross channel) flows, reveals different deformation modes. Such an analysis is used to confirm the softening and stiffening effects of common treatments, such as cytochalasin D and formalin on cell deformability. In addition to an experimental analysis of leukaemia cell deformability, computational fluid dynamic simulations are used to deconvolve the role of the aforementioned flow components in the cell deformation dynamics. In general terms, the current study can be used as a guide for extracting deformation/recovery dynamics of leukaemia cell lines when exposed to various fluid dynamic conditions.