Extracellular Vesicle Preparation and Analysis: A State-of-the-Art Review.

In recent decades, research on Extracellular Vesicles (EVs) has gained prominence in the life sciences due to their critical roles in both health and disease states, offering promising applications in disease diagnosis, drug delivery, and therapy. However, their inherent heterogeneity and complex origins pose significant challenges to their preparation, analysis, and subsequent clinical application. This review is structured to provide an overview of the biogenesis, composition, and various sources of EVs, thereby laying the groundwork for a detailed discussion of contemporary techniques for their preparation and analysis. Particular focus is given to state-of-the-art technologies that employ both microfluidic and non-microfluidic platforms for EV processing. Furthermore, this discourse extends into innovative approaches that incorporate artificial intelligence and cutting-edge electrochemical sensors, with a particular emphasis on single EV analysis. This review proposes current challenges and outlines prospective avenues for future research. The objective is to motivate researchers to innovate and expand methods for the preparation and analysis of EVs, fully unlocking their biomedical potential.

Distinguishing high-metastasis-potential circulating tumor cells through fluidic shear stress in a bloodstream-like microfluidic circulatory system.

Circulating tumor cells (CTCs) play a critical role as initiators in tumor metastasis, which unlocks an irreversible process of cancer progression. Regarding the fluid environment of intravascular CTCs, a comprehensive understanding of the impact of hemodynamic shear stress on CTCs is of profound significance but remains vague. Here, we report a microfluidic circulatory system that can emulate the CTC microenvironment to research the responses of typical liver cancer cells to varying levels of fluid shear stress (FSS). We observe that HepG2 cells surviving FSS exhibit a marked overexpression of TLR4 and TPPP3, which are shown to be associated with the colony formation, migration, and anti-apoptosis abilities of HepG2. Furthermore, overexpression of these two genes in another liver cancer cell line with normally low TLR4 and TPPP3 expression, SK-Hep-1 cells, by lentivirus-mediated transfection also confirms the critical role of TLR4 and TPPP3 in improving colony formation, migration, and survival capability under a fluid environment. Interestingly, in vivo experiments show SK-Hep-1 cells, overexpressed with these genes, have enhanced metastatic potential to the liver and lungs in mouse models via tail vein injection. Mechanistically, TLR4 and TPPP3 upregulated by FSS may increase FSS-mediated cell survival and metastasis through the p53-Bax signaling pathway. Moreover, elevated levels of these genes correlate with poorer overall survival in liver cancer patients, suggesting that our findings could offer new therapeutic strategies for early cancer diagnosis and targeted treatment development.

Validation and Feasibility of Ultrafast Cervical Spine MRI Using a Deep Learning-Assisted 3D Iterative Image Enhancement System.

Purpose: This study aimed to evaluate the feasibility of ultrafast (2 min) cervical spine MRI protocol using a deep learning-assisted 3D iterative image enhancement (DL-3DIIE) system, compared to a conventional MRI protocol (6 min 14s). Patients and Methods: Fifty-one patients were recruited and underwent cervical spine MRI using conventional and ultrafast protocols. A DL-3DIIE system was applied to the ultrafast protocol to compensate for the spatial resolution and signal-to-noise ratio (SNR) of images. Two radiologists independently assessed and graded the quality of images from the dimensions of artifacts, boundary sharpness, visibility of lesions and overall image quality. We recorded the presence or absence of different pathologies. Moreover, we examined the interchangeability of the two protocols by computing the 95% confidence interval of the individual equivalence index, and also evaluated the inter-protocol intra-observer agreement using Cohen's weighted kappa. Results: Ultrafast-DL-3DIIE images were significantly better than conventional ones for artifacts and equivalent for other qualitative features. The number of cases with different kinds of pathologies was indistinguishable based on the MR images from ultrafast-DL-3DIIE and conventional protocols. With the exception of disc degeneration, the 95% confidence interval for the individual equivalence index across all variables did not surpass 5%, suggesting that the two protocols are interchangeable. The kappa values of these evaluations by the two radiologists ranged from 0.65 to 0.88, indicating good-to-excellent agreement. Conclusion: The DL-3DIIE system enables 67% spine MRI scan time reduction while obtaining at least equivalent image quality and diagnostic results compared to the conventional protocol, suggesting its potential for clinical utility.

Active Constituents with Tyrosinase Inhibitory Activities from Waste Tobacco Leaves.

One novel compound, (R)-3, 6-diethoxy-4-hydroxycyclohex-3-en-1-one (1) and thirteen known compounds were isolated from the waste tobacco leaves. The structures of two compounds (1-2) were confirmed and attributed firstly by the extensive spectroscopic data, including 1D/2D NMR, IR, HR-ESI-MS, CD, and ECD spectra. Notably, seven compounds (2, 3, 9, 10, 11, 12, and 13) exhibited better tyrosinase inhibitory activity than the positive control kojic acid. The binding modes of these compounds revealed that their structure formed strong hydrogen bonds and van der Waals forces with the active sites of tyrosinase. These results indicated that waste tobacco leaves are good resources for developing tyrosinase inhibitors.

Mapping structural covariance networks of emotional withdrawal symptoms in males with methamphetamine use disorder during abstinence.

Individuals with methamphetamine use disorder (MUD) often experience anxiety and depressive symptoms during abstinence, which can worsen the likelihood of relapse. Thus, it is essential to understand the neuro-mechanism behind methamphetamine use and its associated emotional withdrawal symptoms in order to develop effective clinical strategies. This study aimed to evaluate associations between emotional withdrawal symptoms and structural covariance networks (SCNs) based on cortical thickness (CTh) across the brain. The CTh measures were obtained from Tl-weighted MRI data from a sample of 48 males with MUD during abstinence and 48 male healthy controls. The severity of anxiety and depressive symptoms was assessed by the Hamilton Anxiety Scale (HAMA) and depression (HAMD) scales. Two important nodes belonging to the brain reward system, the right rostral anterior cingulate cortex (rACC) and medial prefrontal cortex (medPFC), were selected as seeds to conduct SCNs and modulation analysis by emotional symptoms. MUDs showed higher structural covariance between the right rACC and regions in the dorsal attention, right frontoparietal, auditory, visual and limbic networks. They also displayed higher structural covariance between the right medPFC and regions in the limbic network. Moreover, the modulation analysis showed that higher scores on HAMA were associated with increased covariance between the right rACC and the left parahippocampal and isthmus cingulate cortex in the default mode network. These outcomes shed light on the complex neurobiological mechanisms underlying methamphetamine use and its associated emotional withdrawal symptoms and may provide new insights into the development of effective treatments for MUD.

Incorporation of the 99TcO4- Anion within the Ag24(C≡CtBu)204+ Cluster Unveiling the Unique Shell-to-Core Charge Transfer.

We present the first example of an 99TcO4- anion entrapped within the cavity of a silver cluster, revealing an unprecedented photoinduced charge transfer phenomenon. [Ag24(C≡CtBu)20(99TcO4)]·(BF4)3 (denoted as 99TcO4-@Ag24) was successfully synthesized and structurally characterized. Single-crystal X-ray diffraction and Raman spectroscopy reveal that the tetrahedral structure of the 99TcO4- anion sustains significant symmetry breaking with weakened Tc-O bond strength under confinement within the Ag24(C≡CtBu)204+ cluster. Notably, 99TcO4-@Ag24 exhibits a broadband electronic absorption spectrum in the visible region, which was absent for the other 99TcO4--containing compounds. Density functional theory calculations elucidate that host-guest electrostatic interactions result in an electron polarization effect between the 99TcO4- anion core and the Ag24 cationic shell. The emergence of an absorption band in 99TcO4-@Ag24 is rationalized by intermolecular charge transfer from the Ag24 electronic states to the lowest unoccupied molecular orbitals of 99TcO4- instead of the intramolecular electron transition observed in other 99TcO4--containing compounds.

A High-Throughput Microdroplet-Based Single Cell Transfection Method for Gene Knockout Based on the CRISPR/Cas9 System.

The efficient application of the newly developed gene-editing method CRISPR/Cas9 requires more accurate intracellular gene delivery. Traditional delivery approaches, such as lipotransfection and non-viral delivery methods, must contend with major problems to overcome the drawbacks of low efficiency, high toxicity, and cell-type dependency. The high-throughput microdroplet-based single-cell transfection method presented herein provides an alternative method for delivering genome-editing reagents into single living cells. By accurately controlling the number of exogenous plasmids in microdroplets, this method can achieve high-efficiency delivery of nucleic acids to different types of single cells. This paper presents a high-throughput quantitative DNA transfection method for single cells and explores the optimal DNA transfection conditions for specific cell lines. The transfection efficiency of cells at different concentrations of DNA in microdroplets is measured. Under the optimized transfection conditions, the method is used to construct gene-knockout cancer cell lines to determine specific gene functions through the CRISPR/Cas9 knockout system. In a case study, the migration ability of TRIM72 knockout cancer cells is inhibited, and the tumorigenicity of cells in a zebrafish tumor model is reduced. A single-cell microfluidic chip is designed to achieve CRISPR/Cas9 DNA transfection, dramatically improving the transfection efficiency of difficult-to-transfect cells. This research demonstrates that the microdroplet method developed herein has a unique advantage in CRISPR/Cas9 gene-editing applications.

The impact of type 2 diabetes mellitus on the clinical profile, myocardial fibrosis, and prognosis in non-ischemic dilated cardiomyopathy: a prospective cohort study.

BACKGROUND: The impact of the coexistence of type 2 diabetes mellitus (T2DM) in patients with non-ischemic dilated cardiomyopathy (DCM) on clinical profiles, myocardial fibrosis, and outcomes remain incompletely understood. METHOD: A total of 1152 patients diagnosed with non-ischemic DCM were prospectively enrolled from June 2012 to October 2021 and categorized into T2DM and non-T2DM groups. Clinical characteristics, cardiac function, and myocardial fibrosis evaluated by CMR were compared between the two groups. The primary endpoint included both all-cause mortality and heart transplantation. Cause of mortality was classified into heart failure death, sudden cardiac death, and non-cardiac death. Cox regression analysis and Kaplan-Meier analysis were performed to identify the association between T2DM and clinical outcomes. Propensity score matching (PSM) cohort including 438 patients was analyzed to reduce the bias from confounding covariates. RESULTS: Among the 1152 included DCM patients, 155 (13%) patients had T2DM. Patients with T2DM were older (55 ± 12 vs. 47 ± 14 years, P < 0.001), had higher New York Heart Association (NYHA) functional class (P = 0.003), higher prevalence of hypertension (37% vs. 21%, P < 0.001), atrial fibrillation (31% vs. 16%, P < 0.001), lower left ventricular (LV) ejection fraction (EF) (23 ± 9% vs. 27 ± 12%, P < 0.001), higher late gadolinium enhancement (LGE) presence (55% vs. 45%, P = 0.02), and significantly elevated native T1 (1323 ± 81ms vs. 1305 ± 73ms, P = 0.01) and extracellular volume fraction (ECV) (32.7 ± 6.3% vs. 31.3 ± 5.9%, P = 0.01) values. After a median follow-up of 38 months (interquartile range: 20-57 months), 239 patients reached primary endpoint. Kaplan-Meier analysis showed that patients with T2DM had worse clinical outcomes compared with those without T2DM in the overall cohort (annual events rate: 10.2% vs. 5.7%, P < 0.001). T2DM was independently associated with an increased risk of primary endpoint in the overall (Hazard ratio [HR]: 1.61, 95% CI: 1.13-2.33, P = 0.01) and PSM (HR: 1.54, 95% CI: 1.05-2.24, P = 0.02) cohorts. Furthermore, T2DM was associated with a higher risk of heart failure death (P = 0.006) and non-cardiac death (P = 0.02), but not sudden cardiac death (P = 0.16). CONCLUSIONS: Patients with T2DM represented a more severe clinical profile and experienced more adverse outcomes compared to those without T2DM in a large DCM cohort. TRIAL REGISTRATION: Trial registration number: ChiCTR1800017058; URL: https://www. CLINICALTRIALS: gov .

Comparative study of stretched-exponential and kurtosis models of diffusion-weighted imaging in renal assessment to distinguish patients with primary aldosteronism from healthy controls.

PURPOSE: To compare the ability of diffusion parameters obtained by stretched-exponential and kurtosis models of diffusion-weighted imaging (DWI) to distinguish between patients with primary aldosteronism (PA) and healthy controls (HCs) in renal assessment. MATERIALS AND METHODS: A total of 44 participants (22 patients and 22 HCs) underwent renal MRI with an 11 b-value DWI sequence and a 3 b-value diffusion kurtosis imaging (DKI) sequence from June 2021 to April 2022. Binary logistic regression was used to construct regression models combining different diffusion parameters. Receiver-operating characteristic (ROC) curve analysis and comparisons were used to evaluate the ability of single diffusion parameters and combined diffusion models to distinguish between the two groups. RESULTS: A total of six diffusion parameters (including the cortical anomalous exponent term [α_Cortex], medullary fractional anisotropy [FA_Medulla], cortical FA [FA_Cortex], cortical axial diffusivity [Da_Cortex], medullary mean diffusivity [MD_Medulla] and medullary radial diffusivity [Dr_Medulla]) were included, and 10 regression models were studied. The area under the curve (AUC) of Dr_Medulla was 0.855, comparable to that of FA_Cortex and FA_Medulla and significantly higher than that of α_Cortex, Da_Cortex and MD_Medulla. The AUC of the Model_all parameters was 0.967, comparable to that of Model_FA (0.946) and Model_DKI (0.966) and significantly higher than that of the other models. The sensitivity and specificity of Model_all parameters were 87.2% and 95%, respectively. CONCLUSION: The Model_all parameters, Model_FA and Model_DKI were valid for differentiating between PA patients and HCs with similar differentiation efficacy and were superior to single diffusion parameters and other models.

The Effect of Hearing Loss and Working Memory Capacity on Context Use and Reliance on Context in Older Adults.

OBJECTIVES: Older adults often complain of difficulty in communicating in noisy environments. Contextual information is considered an important cue for identifying everyday speech. To date, it has not been clear exactly how context use (CU) and reliance on context in older adults are affected by hearing status and cognitive function. The present study examined the effects of semantic context on the performance of speech recognition, recall, perceived listening effort (LE), and noise tolerance, and further explored the impacts of hearing loss and working memory capacity on CU and reliance on context among older adults. DESIGN: Fifty older adults with normal hearing and 56 older adults with mild-to-moderate hearing loss between the ages of 60 and 95 years participated in this study. A median split of the backward digit span further classified the participants into high working memory (HWM) and low working memory (LWM) capacity groups. Each participant performed high- and low-context Repeat and Recall tests, including a sentence repeat and delayed recall task, subjective assessments of LE, and tolerable time under seven signal to noise ratios (SNRs). CU was calculated as the difference between high- and low-context sentences for each outcome measure. The proportion of context use (PCU) in high-context performance was taken as the reliance on context to explain the degree to which participants relied on context when they repeated and recalled high-context sentences. RESULTS: Semantic context helps improve the performance of speech recognition and delayed recall, reduces perceived LE, and prolongs noise tolerance in older adults with and without hearing loss. In addition, the adverse effects of hearing loss on the performance of repeat tasks were more pronounced in low context than in high context, whereas the effects on recall tasks and noise tolerance time were more significant in high context than in low context. Compared with other tasks, the CU and PCU in repeat tasks were more affected by listening status and working memory capacity. In the repeat phase, hearing loss increased older adults' reliance on the context of a relatively challenging listening environment, as shown by the fact that when the SNR was 0 and -5 dB, the PCU (repeat) of the hearing loss group was significantly greater than that of the normal-hearing group, whereas there was no significant difference between the two hearing groups under the remaining SNRs. In addition, older adults with LWM had significantly greater CU and PCU in repeat tasks than those with HWM, especially at SNRs with moderate task demands. CONCLUSIONS: Taken together, semantic context not only improved speech perception intelligibility but also released cognitive resources for memory encoding in older adults. Mild-to-moderate hearing loss and LWM capacity in older adults significantly increased the use and reliance on semantic context, which was also modulated by the level of SNR.

Prognostic value of mid-term cardiovascular magnetic resonance follow-up in patients with non-ischemic dilated cardiomyopathy: a prospective cohort study.

BACKGROUND: The prognostic value of follow-up cardiovascular magnetic resonance (CMR) in dilated cardiomyopathy (DCM) patients is unclear. We aimed to investigate the prognostic value of cardiac function, structure, and tissue characteristics at mid-term CMR follow-up. METHODS: The study population was a prospectively enrolled cohort of DCM patients who underwent guideline-directed medical therapy with baseline and follow-up CMR, which included measurement of biventricular volume and ejection fraction, late gadolinium enhancement, native T1, native T2, and extracellular volume. During follow-up, major adverse cardiac events (MACE) were defined as a composite endpoint of cardiovascular death, heart transplantation, and heart-failure readmission. RESULTS: Among 235 DCM patients (median CMR interval: 15.3 months; interquartile range: 12.5-19.2 months), 54 (23.0%) experienced MACE during follow-up (median: 31.2 months; interquartile range: 20.8-50.0 months). In multivariable Cox regression, follow-up CMR models showed significantly superior predictive value than baseline CMR models. Stepwise multivariate Cox regression showed that follow-up left ventricular ejection fraction (LVEF; hazard ratio [HR], 0.93; 95% confidence interval [CI], 0.91-0.96; p < 0.001) and native T1 (HR, 1.01; 95% CI, 1.00-1.01; p = 0.030) were independent predictors of MACE. Follow-up LVEF ≥ 40% or stable LVEF < 40% with T1 ≤ 1273 ms indicated low risk (annual event rate < 4%), while stable LVEF < 40% and T1 > 1273 ms or LVEF < 40% with deterioration indicated high risk (annual event rate > 15%). CONCLUSIONS: Follow-up CMR provided better risk stratification than baseline CMR. Improvements in the LVEF and T1 mapping are associated with a lower risk of MACE.

Liver T1 Mapping Derived From Cardiac Magnetic Resonance Imaging: A Potential Prognostic Marker in Idiopathic Dilated Cardiomyopathy.

BACKGROUND: Hepatic alterations are common aftereffects of heart failure (HF) and ventricular dysfunction. The prognostic value of liver injury markers derived from cardiac MRI studies in nonischemic dilated cardiomyopathy (DCM) patients is unclear. PURPOSE: Evaluate the prognostic performance of liver injury markers derived from cardiac MRI studies in DCM patients. STUDY TYPE: Prospective. POPULATION: Three hundred fifty-six consecutive DCM patients diagnosed according to ESC guidelines (age 48.7 ± 14.2 years, males 72.6%). FIELD STRENGTH/SEQUENCE: Steady-state free precession, modified Look-Locker inversion recovery T1 mapping and phase sensitive inversion recovery late gadolinium enhancement (LGE) sequences at 3 T. ASSESSMENT: Clinical characteristics, conventional MRI parameters (ventricular volumes, function, mass), native myocardial and liver T1, liver extracellular volume (ECV), and myocardial LGE presence were assessed. Patients were followed up for a median duration of 48.3 months (interquartile range 42.0-69.9 months). Primary endpoints included HF death, sudden cardiac death, heart transplantation, and HF readmission; secondary endpoints included HF death, sudden cardiac death, and heart transplantation. Models were developed to predict endpoints and the incremental value of including liver parameters assessed. STATISTICAL TESTS: Optimal cut-off value was determined using receiver operating characteristic curve and Youden method. Survival analysis was performed using Kaplan-Meier and Cox proportional hazard. Discriminative power of models was compared using net reclassification improvement and integrated discriminatory index. P value <0.05 was considered statistically significant. RESULTS: 47.2% patients reached primary endpoints; 25.8% patients reached secondary endpoints. Patients with elevated liver ECV (cut-off 34.4%) had significantly higher risk reaching primary and secondary endpoints. Cox regression showed liver ECV was an independent prognostic predictor, and showed independent prognostic value for primary endpoints and long-term HF readmission compared to conventional clinical and cardiac MRI parameters. DATA CONCLUSIONS: Liver ECV is an independent prognostic predictor and may serve as an innovative approach for risk stratification for DCM. EVIDENCE LEVEL: 1 TECHNICAL EFFICACY: Stage 2.

Reference Ranges of Ventricular Morphology and Function in Healthy Chinese Adults: A Multicenter 3 T MRI Study.

BACKGROUND: Magnetic resonance imaging (MRI) reference ranges for ventricular morphology and function in the Chinese population are lacking. PURPOSE: To establish the MRI reference ranges of left and right ventricular (LV and RV) morphology and function based on a large multicenter cohort. STUDY TYPE: Prospective. POPULATION: One thousand and twelve healthy Chinese Han adults. FIELD STRENGTH/SEQUENCE: Balanced steady-state free procession cine sequence at 3.0 T. ASSESSMENT: Biventricular end-diastolic, end-systolic, stroke volume, and ejection fraction (EDV, ESV, SV, and EF), LV mass (LVM), end-diastolic and end-systolic dimension (LVEDD and LVESD), anteroseptal wall thickness (AS), and posterolateral wall thickness (PL) were measured. Body surface area (BSA) and height were used to index biventricular parameters. Parameters were compared between age groups and sex. STATISTICAL TESTS: Independent-samples t-tests or Mann-Whitney U test to compare mean values between sexes; ANOVA or Kruskal-Wallis test to compare mean values among age groups; linear regression to assess the relationships between cardiac parameters and age (correlation coefficient, r). A P value <0.05 was considered statistically significant. RESULTS: The biventricular volumes, LVM, LVEDD, RVEDV/LVEDV ratio, LVESD, AS, and PL were significantly greater in males than in females, even after indexing to BSA or height, while LVEF and RVEF were significantly lower in males than in females. For both sexes, age was significantly negatively correlated with biventricular volumes (male and female: LVEDV [r = -0.491; r = -0.373], LVESV [r = -0.194; r = -0.184], RVEDV [r = -0.639; r = -0.506], RVESV [r = -0.270; r = -0.223]), with similar correlations after BSA normalization. LVEF (r = 0.043) and RVEF (r = 0.033) showed a significant correlation with age in females, but not in males (P = 0.889; P = 0.282). DATA CONCLUSION: MRI reference ranges for biventricular morphology and function in Chinese adults are presented and show significant associations with age and sex. LEVEL OF EVIDENCE: 2 TECHNICAL EFFICACY: Stage 2.

The diagnostic value of sST2 for myocardial fibrosis in idiopathic inflammatory myopathies in subclinical stage of cardiac involvement.

OBJECTIVE: Myocardial fibrosis occurs in the early subclinical stage of cardiac involvement in idiopathic inflammatory myopathies (IIMs). Soluble suppression of tumorigenicity 2 (sST2) is known to have an immunomodulatory impact during autoimmune disease development. The current study investigated the diagnostic value of sST2 for myocardial fibrosis during early stage of cardiac involvement in IIM. METHODS: A total of 44 IIM patients with normal heart function and 32 age- and gender-matched healthy controls (HCs) were enrolled. Serum sST2 levels were measured by ELISA and cardiac magnetic resonance (CMR) parameters for myocardial fibrosis [native T1, extracellular volume (ECV), late-gadolinium enhancement (LGE)] and oedema (T2 values) were analysed. RESULTS: IIM patients had significantly higher sST2 levels than HCs [67.5 ng/ml (s.d. 30.4)] vs 14.4 (5.5), P < 0.001] and levels correlated positively with diffuse myocardial fibrosis parameters, native T1 (r = 0.531, P = 0.000), ECV (r = 0.371, P = 0.013) and focal myocardial fibrosis index and LGE (r = 0.339, P = 0.024) by Spearman's correlation analysis. sST2 was an independent predictive factor for diffuse and focal myocardial fibrosis after adjustment for age, gender, BMI and ESR. Risk increased ≈15.4% for diffuse [odds ratio (OR) 1.154 (95% CI 1.021, 1.305), P = 0.022] and 3.8% for focal [OR 1.038 (95% CI 1.006, 1.072), P = 0.020] myocardial fibrosis per unit increase of sST2. Cut-off values for diagnosing diffuse and focal myocardial fibrosis were sST2 ≥51.3 ng/ml [area under the curve (AUC) = 0.942, sensitivity = 85.7%, specificity = 98.9%, P < 0.001] and 53.3 ng/ml (AUC = 0.753, sensitivity = 87.5%, specificity = 58.3%, P < 0.01), respectively. CONCLUSION: sST2 showed a marked elevation during the subclinical stage of cardiac involvement in IIM and has potential as a biomarker for predicting diffuse and focal myocardial fibrosis in IIM.

An integrated and multi-functional droplet-based microfluidic platform for digital DNA amplification.

Digital DNA amplification is a powerful method for detecting and quantifying rare nucleic acids. In this study, we developed a multi-functional droplet-based platform that integrates the traditional digital DNA amplification workflow into a one-step device. This platform enables efficient droplet generation, transition, and signal detection within a 5-min timeframe, distributing the sample into a uniform array of 4 × 104 droplets (variation <2%) within a chamber. Subsequent in-situ DNA amplification, fluorescence detection, and signal analysis were carried out. To assess the platform's performance, we quantitatively detected the human epidermal growth factor receptor (EGFR) mutation and human papillomavirus (HPV) mutation using digital polymerase chain reaction (dPCR) and digital loop-mediated isothermal amplification (dLAMP), respectively. The fluorescence results exhibited a positive, linear, and statistically significant correlation with target DNA concentrations ranging from 101 to 105 copies/µL, demonstrating the capability and feasibility of the integrated device for dPCR and dLAMP. This platform offers high-throughput droplet generation, eliminates droplet fusion and transition, is user-friendly, reduces costs compared to current methods, and holds potential for thermocycling and isothermal nucleic acid quantification with high sensitivity and accuracy.

Multiple Hypointense Vessels are Associated with Cognitive Impairment in Patients with Single Subcortical Infarction.

We aimed to explore the relationship between multiple hypointense vessels and cognitive function in patients with single subcortical infarction (SSI) and the role of SSI with different etiological mechanisms in the above relationship. Multiple hypointense vessels were measured by the number of deep medullary veins (DMVs), DMVs score, and cortical veins (CVs) score. The Montreal Cognitive Assessment (MoCA), the Shape Trail Test (STT), and the Stroop Color and Word Test (SCWT) were assessed to evaluate cognitive function. SSI was dichotomized as branch atheromatous disease (BAD) and cerebral small vessel disease (CSVD)-related SSI by whole-brain vessel-wall magnetic resonance imaging. We included a total of 103 acute SSI patients. After adjustments were made for related risk factors of cognitive function, the SSI patients with higher DMVs score were more likely to have longer STT-B (P = 0.001) and smaller STT-B-1 min (P = 0.014), and the SSI patients with higher CVs score were more likely to have shorter STT-A (P = 0.049). In subgroup analysis, we found that the negative relationship between DMVs scores and cognitive function and the positive relationship between CVs scores and cognitive function were significantly stronger in BAD patients. We provided valuable insights into the associations between DMVs, CVs, and multi-domain cognitive impairment in SSI patients, which underscored the necessity to further study the dynamic alterations of venules and their specific influence on post-stroke cognitive impairment.

Prevalence and Predictors of Hemorrhagic Foci on Long-term Follow-up MRI of Recent Single Subcortical Infarcts.

Hemorrhagic foci surrounding the lacune in the long-term evolution of recent single subcortical infarcts (RSSIs) remains largely unexplored. We aimed to determine the prevalence, characteristics, and predictors of hemorrhagic foci in patients with RSSI. From a prospective, longitudinal study of RSSIs, we recruited patients who underwent multimodal MRI assessments both at baseline and approximately one year after the stroke onset. Hemorrhagic foci were identified using susceptibility-weighted imaging (SWI). Among 101 patients with RSSI, nearly half (n = 45, 44.6%) had hemorrhagic foci within the index RSSI lesions on follow-up SWI. RSSIs with hemorrhagic foci formation were associated with a longer time to follow-up imaging (median 449 versus 401 days, P = 0.005) and higher likelihood of being located in the anterior circulation compared to those without hemorrhagic foci (88.9% versus 64.3%, P = 0.003). Hemorrhagic foci were also associated with larger lesion size (P < 0.001), a higher proportion of cavitation formation (P = 0.003), higher baseline NIHSS scores (P = 0.004), and poorer functional outcomes (P = 0.001). In the subset of RSSIs in the lenticulostriate artery (LSA) territory, after adjustment for covariates, larger initial lesion volume (OR 1.80, 95% CI 1.13-2.87; P = 0.014) and greater decreases in LSA total length (OR 0.59, 95% CI 0.36-0.96; P = 0.035) were independently associated with hemorrhagic foci formation. The extent of ischemia in the initial infarct is predictive of the presence of hemorrhagic residues. Our findings contribute to the current understanding of the mechanisms underlying the evolution of RSSIs.

Pyroptosis-related gene signature: A predictor for overall survival, immunotherapy response, and chemosensitivity in patients with pancreatic adenocarcinoma.

Background: Pancreatic adenocarcinoma (PAAD) is a lethal malignancy with high levels of heterogeneity. Pyroptosis is thought to influence the development of various tumors. Nevertheless, the role of pyroptosis-related genes (PRGs) in prognostic risk stratification and therapeutic guidance for PAAD remains ambiguously. Methods: Transcriptome profile and clinical information of PAAD patients were retrieved from The Cancer Genome Atlas (TCGA) as well as Gene Expression Omnibus (GEO) databases, followed by differential analysis. Patients were divided into distinct pyroptosis phenotype subtypes based on the characteristic of differently expressed PRGs (DEPRGs). Then a PRG signature was established through univariate analysis and LASSO algorithm in the training set to assess the prognostic risk, and its reliability was verified in the validation set using receiver operating characteristic(ROC) curve. The correlation of risk score with tumor microenvironment(TME), TMB and chemotherapeutic drug sensitivity were also analyzed. In addition, a nomogram was constructed to promote better clinical application. Results: A total of 28 DEPRGs were determined in the integrated TCGA-GEO datasets. Patients were divided into three pyroptosis phenotype subtypes, Kaplan-Meier curve suggested patients in cluster B had a worse prognosis than those in cluster A and C. Then a price signature comprised of 8 PRGs was generated. TME analysis suggested that the low-risk subgroup displayed potential stronger antitumor immune effect and might respond better to immune checkpoint inhibitors (ICIs) therapy. Furthermore, PRG signature exhibited favorable discriminatory ability for TMB status and the sensitivity of multiple conventional chemotherapeutic agents including paclitaxel. Ultimately, we constructed a promising nomogram according to the risk score and N stage with good predictive accuracy compared with the actual overall survival (OS) probabilities. Conclusion: We established an 8-gene signature that could be regarded as an independent prognostic risk factor for PAAD patients. The 8-gene signature could provide rationale for immunotherapy and chemotherapy, which might help clinicians make precise individualized treatment regimens.

Pulmonary Transit Time Derived from First-Pass Perfusion Cardiac MR Imaging: A Potential New Marker for Cardiac Involvement and Prognosis in Light-Chain Amyloidosis.

BACKGROUND: First-pass perfusion cardiac MR imaging could reflect pulmonary hemodynamics. However, the clinical value of pulmonary transit time (PTT) derived from first-pass perfusion MRI in light-chain (AL) amyloidosis requires further evaluation. PURPOSE: To assess the clinical and prognostic value of PTT in patients with AL amyloidosis. STUDY TYPE: Prospective observational study. POPULATION: 226 biopsy-proven systemic AL amyloidosis patients (age 58.62 ± 10.10 years, 135 males) and 43 healthy controls (age 42 ± 16.2 years, 20 males). FIELD STRENGTH/SEQUENCE: SSFP cine and phase sensitive inversion recovery late gadolinium enhancement (LGE) sequences, and multislice first-pass myocardial perfusion imaging with a saturation recovery turbo fast low-angle shot (SR-TurboFLASH) pulse sequence at 3.0T. ASSESSMENT: PTT was measured as the time interval between the peaks of right and left ventricular cavity arterial input function curves on first-pass perfusion MR images. STATISTICAL TESTS: Independent-sample t test, Mann-Whitney U test, Chi-square test, Fisher's exact test, analysis of variance, or Kruskal-Wallis test, as appropriate; univariable and multivariable Cox proportional hazards models and Kaplan-Meier curves, area under receiver operating characteristic curve were used to determine statistical significance. RESULTS: PTT could differentiate AL amyloidosis patients with (N = 188) and without (N = 38) cardiac involvement (area under the curve [AUC] = 0.839). During a median follow-up of 35 months, 160 patients (70.8%) demonstrated all-cause mortality. After adjustments for clinical (Hazard ratio [HR] 1.061, confidence interval [CI]: 1.021-1.102), biochemical (HR 1.055, CI: 1.014-1.097), cardiac MRI-derived (HR 1.077, CI: 1.034-1.123), and therapeutic (HR 1.063, CI: 1.024-1.103) factors, PTT predicted mortality independently in patients with AL amyloidosis. Finally, PTT could identify worse outcomes in patients demonstrating New York Heart Association class III, Mayo 2004 stage III, and transmural LGE pattern. DATA CONCLUSION: PTT may serve as a new imaging predictor of cardiac involvement and prognosis in AL amyloidosis. LEVEL OF EVIDENCE: 2 TECHNICAL EFFICACY: Stage 2.

Reference ranges of myocardial T1 and T2 mapping in healthy Chinese adults: a multicenter 3T cardiovascular magnetic resonance study.

BACKGROUND: Although reference ranges of T1 and T2 mapping are well established for cardiovascular magnetic resonance (CMR) at 1.5T, data for 3T are still lacking. The objective of this study is to establish reference ranges of myocardial T1 and T2 based on a large multicenter cohort of healthy Chinese adults at 3T CMR. METHODS: A total of 1015 healthy Chinese adults (515 men, age range: 19-87 years) from 11 medical centers who underwent CMR using 3T Siemens scanners were prospectively enrolled. T1 mapping was performed with a motion-corrected modified Look-Locker inversion recovery sequence using a 5(3)3 scheme. T2 mapping images were acquired using T2-prepared fast low-angle shot sequence. T1 and T2 relaxation times were quantified for each slice and each myocardial segment. The T1 mapping and extracellular volume standardization (T1MES) phantom was used for quality assurance at each center prior to subject scanning. RESULTS: The phantom analysis showed strong consistency of spin echo, T1 mapping, and T2 mapping among centers. In the entire cohort, global T1 and T2 reference values were 1193 ± 34 ms and 36 ± 2.5 ms. Global T1 and T2 values were higher in females than in males (T1: 1211 ± 29 ms vs. 1176 ± 30 ms, p < 0.001; T2: 37 ± 2.3 ms vs. 35 ± 2.5 ms, p < 0.001). There were statistical differences in global T2 across age groups (p < 0.001), but not in global T1. Linear regression showed no correlation between age and global T1 or T2 values. In males, positive correlation was found between heart rate and global T1 (r = 0.479, p < 0.001). CONCLUSIONS: Using phantom-validated imaging sequences, we provide reference ranges for myocardial T1 and T2 values on 3T scanners in healthy Chinese adults, which can be applied across participating sites. Trial registration URL: http://www.chictr.org.cn/index.aspx . Unique identifier: ChiCTR1900025518. Registration name: 3T magnetic resonance myocardial quantitative imaging standardization and reference value study: a multi-center clinical study.