A systematic evaluation of computation methods for cell segmentation.

Cell segmentation is a fundamental task in analyzing biomedical images. Many computational methods have been developed for cell segmentation, but their performances are not well understood in various scenarios. We systematically evaluated the performance of 18 segmentation methods to perform cell nuclei and whole cell segmentation using light microscopy and fluorescence staining images. We found that general-purpose methods incorporating the attention mechanism exhibit the best overall performance. We identified various factors influencing segmentation performances, including training data and cell morphology, and evaluated the generalizability of methods across image modalities. We also provide guidelines for choosing the optimal segmentation methods in various real application scenarios. We developed Seggal, an online resource for downloading segmentation models already pre-trained with various tissue and cell types, which substantially reduces the time and effort for training cell segmentation models.

The functions of exosomes targeting astrocytes and astrocyte-derived exosomes targeting other cell types.

Astrocytes are the most abundant glial cells in the central nervous system; they participate in crucial biological processes, maintain brain structure, and regulate nervous system function. Exosomes are cell-derived extracellular vesicles containing various bioactive molecules including proteins, peptides, nucleotides, and lipids secreted from their cellular sources. Increasing evidence shows that exosomes participate in a communication network in the nervous system, in which astrocyte-derived exosomes play important roles. In this review, we have summarized the effects of exosomes targeting astrocytes and the astrocyte-derived exosomes targeting other cell types in the central nervous system. We also discuss the potential research directions of the exosome-based communication network in the nervous system. The exosome-based intercellular communication focused on astrocytes is of great significance to the biological and/or pathological processes in different conditions in the brain. New strategies may be developed for the diagnosis and treatment of neurological disorders by focusing on astrocytes as the central cells and utilizing exosomes as communication mediators.

Reperfusion status and postoperative blood pressure in acute stroke patients after endovascular treatment.

Background and purpose: An aggressive lowering of blood pressure (BP) could lead to neurological worsening, particularly of the area that has not been reperfused in acute stroke patients with large vessel occlusion (LVO). We sought to investigate the association of reperfusion status and BP course following mechanical thrombectomy (MT) with outcomes in LVO. Materials and methods: Consecutive patients with LVO treated with MT between Jan 2020 to Jun 2021 were enrolled in a retrospective cohort study. Hourly systolic BP (SBP) and diastolic BP (DBP) were recorded for 72 h following MT and maximum SBP and DBP levels were identified. The Extended Thrombolysis in Cerebral Infarction (eTICI) scale was used to assess reperfusion extent. LVO patients were stratified in 2 groups based on reperfusion status: complete reperfusion (eTICI 3) and incomplete reperfusion (eTICI 2b/c). Three-month functional independence was defined as a modified Rankin Scale score of 0-2. Results: A total of 263 acute ischemic stroke patients with LVO were retrospectively evaluated. Complete reperfusion was achieved in 210 patients (79.8%). Post-MT maximum SBP over 160 mmHg was significantly related to worse functional outcome (38.1% vs. 55.7%, p = 0.006), higher likelihood of in-hospital mortality and 3-month mortality (19.0% vs. 6.9%, p = 0.004, 27.4% vs. 14.3%, p = 0.012). No statistical correlation was found between reperfusion status and blood pressure level (p > 0.05). In patients with complete reperfusion, patients with an average BP 120-140 mmHg tends to have worse functional outcome compared with 100-120 mmHg (OR = 1.77, 95%CI: 0.97-3.23, p = 0.061). Conclusion: High maximum SBP levels following MT are associated with an increased likelihood of 3-month functional dependence and mortality. An average BP of 100-120 mmHg tends to have better functional independence in completely reperfused patients. The effect of intensive BP control on incomplete reperfusion still warrants further investigations.

Association between blood pressure and different antihypertensive drugs with outcome after ischemic stroke: A Mendelian randomization study.

BACKGROUND: Observational studies suggest an association between blood pressure (BP) and functional outcomes in ischemic stroke patients but whether this is causal or due to confounding is uncertain. We used Mendelian randomization (MR) to assess causality, and also explore whether particular classes of anti-hypertensives were associated with a better outcome after ischemic stroke. METHODS: We selected genetic variants associated with systolic and diastolic BP and BP-lowering variants in genes encoding antihypertensive drugs from genome-wide association studies (GWAS) on 757,601 individuals. The primary outcome was 3-month dependence or death defined as a modified Rankin Scale (mRS) of 3-6. The secondary outcome was disability or death after 90 days defined as mRS 2-6. Cochran's Q statistic in the inverse variance weighted (IVW) model, the weighted median, MR-Egger regression, leave-one-single-nucleotide polymorphism (SNP)-out analysis, MR-Pleiotropy Residual Sum and Outlier methods were adopted as sensitivity analyses. To validate our primary results, we performed independent repeat analyses and Bi-directional MR analyses. RESULTS: Genetic predisposition to higher systolic and diastolic BP was associated with disability or death after ischemic stroke in univariable IVW MR analysis (odds ratio (OR) 1.29, 95% confidence interval (CI): 1.05-1.59, p = 0.014; OR 1.27, 95% CI: 1.07-1.51, p = 0.006, respectively). Pulse pressure was associated with both dependence or death and disability or death after ischemic stroke (OR = 1.05, 95% CI: 1.02-1.08, p = 0.002; OR = 1.04, 95% CI = 1.01-1.07, p = 0.009, respectively). Angiotensin-converting enzyme inhibitor (ACEI) and calcium channel blocker (CCB) were significantly associated with improved functional outcomes (dependence or death, OR 0.76, 95% CI: 0.62-0.94, p = 0.009; OR 0.89, 95% CI: 0.83-0.97, p = 0.005). Proxies for ß-blockers, angiotensin receptor blockers (ARB), and thiazides failed to show associations with functional outcomes (p > 0.05). CONCLUSION: We provide evidence for an association of genetic predisposition to higher BP with a higher risk of 3-month functional dependence after ischemic stroke. Our findings support ACEI and CCB as promising antihypertensive drugs for improving functional outcomes in ischemic stroke.

Microglia Exhibit Distinct Heterogeneity Rather than M1/M2 Polarization within the Early Stage of Acute Ischemic Stroke.

The classification of microglial M1/M2 polarization in the acute phase of ischemic stroke remains controversial, which has limited further advances in neuroprotective strategy. To thoroughly assess the microglial phenotypes, we made the middle cerebral artery occlusion model in mice to simulate the acute pathological processes of ischemic stroke from normal conditions to acute cerebral ischemia and then to the early reperfusion period. The temporal changes in gene profiles, cell subtypes, and microglial function were comprehensively analyzed using single-cell RNA sequencing. We identified 37,614 microglial cells and divided them into eight distinct subpopulations. Mic_home, Mic_pre1, and Mic_pre2 subpopulations were three clusters mainly composed of cells from the control samples, in which Mic_home was a homeostatic subpopulation characterized by high expression of Hpgd and Tagap, and Mic_pre1 and Mic_pre2 were two clusters with preliminary inflammatory activation characteristics marked by P2ry13 and Wsb1 respectively. Mic_M1L1 and Mic_M1L2 subpopulations exhibited M1-like polarization manifested by the upregulation of inflammatory genes after ischemic stroke, while the intrinsic heterogeneity on the level of inflammatory responses and neurotrophic support properties was observed. Moreover, we identified three unique clusters of cells with low inflammation levels. Mic_np1, Mic_np2, and Mic_np3 were characterized by high expression of Arhgap45, Rgs10, and Pkm respectively. However, these cells did not show significant M2-like characteristics and their classic microglia function was also attenuated. These subpopulations exhibited higher activation of neuropeptide functional pathways. At last, we performed cell-cell communication analysis and identified major couplings contributing to the interaction between microglia and other cell populations. In summary, our study elucidated the temporal heterogeneity of microglia in the acute phase of ischemic stroke, which may facilitate the identification of effective neuroprotective targets to curb ischemic damage at an early stage.

Outcome of Renal Function after Laparoscopic Pyeloplasty in Children with High-Grade Hydronephrosis.

INTRODUCTION: This study aimed to analyze the changes in differential renal function (DRF) after laparoscopic pyeloplasty in children with high-grade hydronephrosis and factors influencing DRF improvement. METHODS: We reviewed the clinical data of unilateral ureteropelvic junction obstruction (UPJO) patients with SFU grade IV who underwent laparoscopic pyeloplasty between February 2018 and October 2020. The patients were divided into two groups: DRF improvement ≥5% (group 1) and DRF improvement <5% (group 2). Preoperative, operative, and postoperative parameters were included in the statistical analysis in both groups. RESULTS: A total of 29 patients with a median age of 6 months were included. The preoperative DRF in group 1 was significantly lower than that in group 2 (36.97 ± 8.47% vs. 45.19 ± 5.22%, p = 0.004). Logistic regression and receiver operating characteristic analysis showed the preoperative DRF was the predictor for renal function improvement after pyeloplasty (p = 0.021) and had a significant predictive value (p = 0.004). CONCLUSION: Nearly half of the patients with high-grade UPJO had improved renal function at 1 year follow-up after laparoscopic pyeloplasty. Preoperative DRF was a predictor of renal function improvement, and postoperative functional recovery was superior in children with lower preoperative DRF.

Preliminary experience with recanalization of large vessel occlusion due to underlying long-segment dissection using a standby microwire technique.

Purpose: This study aimed at investigating a novel standby microwire technique to facilitate revascularization of large vessel occlusion due to underlying long-segment dissection. Methods: Patients with acute ischemic stroke with emergent large vessel occlusion (ELVO) due to underlying long-segment dissection were screened from the prospectively established database between January 2021 and May 2022. The clinical and radiological data of eligible patients who underwent endovascular treatment by using a standby microwire technique were investigated. Results: Of the 165 acute ischemic stroke patients who underwent mechanical thrombectomy, the standby microwire technique was used in five patients aged 33-55 years old with occlusion due to underlying long-segment dissection. Of them, three patients were diagnosed with tandem lesions and three were located at the anterior circulation. A 300 cm exchange microwire was used as the standby microwire. Stent deployment was performed in all five patients. Groin puncture to reperfusion time ranged from 10-68 min. Technical success and favorable clinical outcomes were achieved in all five patients (100%). No technique-related complication was observed. Conclusion: Our preliminary experience showed that the standby microwire technique was a useful ancillary approach to facilitate the revascularization of large vessel occlusion due to underlying long-segment dissection.

In vitro machine learning-based CAR T immunological synapse quality measurements correlate with patient clinical outcomes.

The human immune system consists of a highly intelligent network of billions of independent, self-organized cells that interact with each other. Machine learning (ML) is an artificial intelligence (AI) tool that automatically processes huge amounts of image data. Immunotherapies have revolutionized the treatment of blood cancer. Specifically, one such therapy involves engineering immune cells to express chimeric antigen receptors (CAR), which combine tumor antigen specificity with immune cell activation in a single receptor. To improve their efficacy and expand their applicability to solid tumors, scientists optimize different CARs with different modifications. However, predicting and ranking the efficacy of different "off-the-shelf" immune products (e.g., CAR or Bispecific T-cell Engager [BiTE]) and selection of clinical responders are challenging in clinical practice. Meanwhile, identifying the optimal CAR construct for a researcher to further develop a potential clinical application is limited by the current, time-consuming, costly, and labor-intensive conventional tools used to evaluate efficacy. Particularly, more than 30 years of immunological synapse (IS) research data demonstrate that T cell efficacy is not only controlled by the specificity and avidity of the tumor antigen and T cell interaction, but also it depends on a collective process, involving multiple adhesion and regulatory molecules, as well as tumor microenvironment, spatially and temporally organized at the IS formed by cytotoxic T lymphocytes (CTL) and natural killer (NK) cells. The optimal function of cytotoxic lymphocytes (including CTL and NK) depends on IS quality. Recognizing the inadequacy of conventional tools and the importance of IS in immune cell functions, we investigate a new strategy for assessing CAR-T efficacy by quantifying CAR IS quality using the glass-support planar lipid bilayer system combined with ML-based data analysis. Previous studies in our group show that CAR-T IS quality correlates with antitumor activities in vitro and in vivo. However, current manually quantified IS quality data analysis is time-consuming and labor-intensive with low accuracy, reproducibility, and repeatability. In this study, we develop a novel ML-based method to quantify thousands of CAR cell IS images with enhanced accuracy and speed. Specifically, we used artificial neural networks (ANN) to incorporate object detection into segmentation. The proposed ANN model extracts the most useful information to differentiate different IS datasets. The network output is flexible and produces bounding boxes, instance segmentation, contour outlines (borders), intensities of the borders, and segmentations without borders. Based on requirements, one or a combination of this information is used in statistical analysis. The ML-based automated algorithm quantified CAR-T IS data correlates with the clinical responder and non-responder treated with Kappa-CAR-T cells directly from patients. The results suggest that CAR cell IS quality can be used as a potential composite biomarker and correlates with antitumor activities in patients, which is sufficiently discriminative to further test the CAR IS quality as a clinical biomarker to predict response to CAR immunotherapy in cancer. For translational research, the method developed here can also provide guidelines for designing and optimizing numerous CAR constructs for potential clinical development. Trial Registration: ClinicalTrials.gov NCT00881920.

Circ_ORC2 enhances the regulatory effect of miR-19a on its target gene PTEN to affect osteosarcoma cell growth.

Osteosarcoma is a highly malignant and aggressive bone tumor. Its occurrence and development involve many factors and multiple signaling pathways. Some studies have shown that circular RNAs (circRNAs) play important roles in the development of various tumors. This research showed that circ_ORC2 was generally up-regulated in various osteosarcoma cell lines, and mainly distributed in the cytoplasm. Circ_ORC2 had the binding site of miR-19a, and its expression was positively correlated with miR-19a expression. RIP experiments showed that circ_ORC2 could bind to Ago2 protein. RNA pull-down using biotinylated circ_ORC2 or miR-19a showed that circ_ORC2 could directly interact with miR-19a, and dual luciferase reporter gene assay also confirmed that miR-19a could bind to circ_ORC2. After circ_ORC2 knockdown, miR-19a expression was down-regulated, but the downstream target gene PTEN expression was up-regulated, and the phosphorylation level of Akt was reduced, which indicated that circ_ORC2 enhanced the inhibition of miR-19a on PTEN expression by combining miR-19a. Further functional experiments showed that after circ_ORC2 knockdown, cell proliferation and invasion decreased, while the apoptosis level increased. When co-transfected with circ_ORC2 siRNA and miR-19a mimics or PTEN siRNA, the above cell biological behaviors did not change significantly. Therefore, circ_ORC2 binds with miR-19a and enhances its expression, thereby inhibiting downstream PTEN expression and activating Akt pathway to promote osteosarcoma cell growth and invasion. These findings enrich the circRNA molecular regulation mechanism, and provide more reference ideas for the research and application of circRNAs in tumors and other diseases.

[Distributions of H3K27me3 and its modification enzymes in different tissues of mice].

Objective To investigate the levels of trimethylated histone 3 at lysine residue 27 (H3K27me3) and its modification enzymes Zeste gene enhancer homolog 2 (EZH2), lysine-specific demethylase 6B (Kdm6B/JMJD3) and lysine-specific demethylase 6A (Kdm6A/UTX) in tissues and organs of 7-day and 2-month postnatal mice. Methods Immunohistochemistry was used to detect the expressions of H3K27me3 and its modification enzymes EZH2, JMJD3 and UTX in the brain, salivary glands, back fat, thymus, lung, heart, stomach, intestines, liver, testes, and skin of 7-day and 2-month mice. Real-time quantitative PCR was used to confirm the results. The relationships between H3K27me3 and its modification enzymes were analyzed statistically. Results Immunohistochemistry showed H3K27me3 persistently present in all examined tissues of 7-day and 2-month mice. EZH2 was persistently expressed in the brain, heart, liver, and skin of 7-day and 2-month mice, but only expressed in the salivary glands, adipose tissues, thymus, lung, intestines, and testes of 2-month mice. JMJD3 was expressed in the brain, salivary glands, adipose tissues, lung, heart, stomach, intestines, testes, skin of 7-day mice, but was not expressed in the lung, adipose tissues and stomach of 2-month mice. UTX was expressed in the brain, salivary glands, adipose tissues, lung, heart, testes, skin of 7-day mice, but only expressed in the testes of 2-month mice. Most mRNA of H3K27 modification enzymes were moderately or highly expressed as their immunohistochemical results were positive. Conclusion There was H3K27me3 persistently present in the all examined tissues at different stages. EZH2 was mostly expressed in the brain, salivary glands, adipose tissues, thymus, lung, heart, intestines, liver, testes and skin of 2-month-old mice. JMJD3 and UTX were mostly expressed in the brain, salivary glands, adipose tissues, lung, heart, skin and testes of 7-day-old mice. No significant association was found between the distribution of H3K27me3 and the expression of EZH2. There was also no obvious inverse distribution relationship between H3K27me3 and JMJD3 or UTX. Moreover, there was no negative relationship between the distribution of EZH2, JMJD3 and UTX. These results suggest that EZH2, JMJD3 and UTX may play important roles in many tissues of mice after birth. The levels of H3K27me3 and its modified enzymes may be controlled by multiple factors in vivo to fulfill complex physiological functions.