Smart palm-size optofluidic hematology analyzer for automated imaging-based leukocyte concentration detection

A critical function of flow cytometry is to count the concentration of blood cells,which helps in the diagnosis of certain diseases.However,the bulky nature of commercial flow cytometers makes such tests only available in hospitals or laboratories,hindering the spread of point-of-care testing(POCT),especially in underdeveloped areas.Here,we propose a smart Palm-size Optofluidic Hematology Analyzer based on a miniature fluorescence microscope and a microfluidic platform to lighten the device to improve its portability.This gadget has a dimension of 35×30×80 mm and a mass of 39 g,less than 5%of the weight of commercially available flow cytometers.Additionally,automatic leukocyte concentration detection has been realized through the integration of image processing and leukocyte counting algorithms.We compared the leukocyte concentration measurement between our approach and a hemocytometer using the Passing-Bablok analysis and achieved a correlation coefficient of 0.979.Through Bland-Altman analysis,we obtained the relationship between their differences and mean measurement values and established 95%limits of agreement,ranging from−0.93×10^(3)to 0.94×10^(3)cells/μL.We anticipate that this device can be used widely for monitoring and treating diseases such as HIV and tumors beyond hospitals.

Live-cell superresolution pathology reveals different molecular mechanisms of pelizaeus-merzbacher disease

Modern cellular pathology relies on conventional microscopes to study the etiology and pathogenesis of diseases,which suffer from limited spatial resolution,little dynamic information due to sample fixation,and the lack of molecular information.For example,Pelizaeus-Merzbacher disease(PMD)is a genetic disorder of the central nervous system caused by different duplications or mutations of the proteolipid protein 1 gene(PLP1)in oligodendrocytes,which leads to hypomyelination and leukodystrophy in patients classified into either classical,transitional,or connatal phenotype[1].However,the correlations between genotypes and phenotypes at cellular level remain elusive.

Molecular regulation of myocardial proliferation and regeneration

Heart regeneration is a fascinating and complex biological process. Decades of intensive studies have revealed asophisticated molecular network regulating cardiac regeneration in the zebrafish and neonatal mouse heart. Here,we review both the classical and recent literature on the molecular and cellular mechanisms underlying heartregeneration, with a particular focus on how injury triggers the cell-cycle re-entry of quiescent cardiomyocytes toreplenish their massive loss after myocardial infarction or ventricular resection. We highlight several importantsignaling pathways for cardiomyocyte proliferation and propose a working model of how these injury-inducedsignals promote cardiomyocyte proliferation. Thus, this concise review provides up-to-date research progresses onheart regeneration for investigators in the field of regeneration biology.

Quantitatively mapping local quality of super-resolution microscopy by rolling Fourier ring correlation

In fluorescence microscopy,computational algorithms have been developed to suppress noise,enhance contrast,and even enable super-resolution(SR).However,the local quality of the images may vary on multiple scales,and these differences can lead to misconceptions.Current mapping methods fail to finely estimate the local quality,challenging to associate the SR scale content.Here,we develop a rolling Fourier ring correlation(rFRC)method to evaluate the reconstruction uncertainties down to SR scale.To visually pinpoint regions with low reliability,a filtered rFRC is combined with a modified resolution-scaled error map(RSM),offering a comprehensive and concise map for further examination.We demonstrate their performances on various SR imaging modalities,and the resulting quantitative maps enable better SR images integrated from different reconstructions.Overall,we expect that our framework can become a routinely used tool for biologists in assessing their image datasets in general and inspire further advances in the rapidly developing field of computational imaging.

A long non-coding RNA LncSync regulates mouse cardiomyocyte homeostasis and cardiac hypertrophy through coordination of miRNA actions

Dear Editor,Maintenance of cardiomyocyte(CM)homeostasis is essential for normal heart function.Long-term imbalance in heart homeostasis could elicit irreversible adaptive change in cell structure and function and tissue architecture,exemplified as cardiac hyper-trophy and fibrosis,and eventually develop into heart failure(Shiojima et al.,2005).Therefore,identifying new genes and path-ways regulating CM homeostasis may help better understand the cause of cardiac hypertrophy.

Superresolution live-cell imaging reveals that the localization of TMEM106B to filopodia in oligodendrocytes is compromised by the hypomyelination-related D252N mutation

Hypomyelination leukodystrophies constitute a group of heritable white matter disorders exhibiting defective myelin development.Initially identified as a lysosomal protein,the TMEM106B D252N mutant has recently been associated with hypomyelination.However,how lysosomal TMEM106B facilitates myelination and how the D252N mutation disrupts that process are poorly understood.We used superresolution Hessian structured illumination microscopy(Hessian-SIM)and spinning discconfocal structured illumination microscopy(SD-SIM)to find that the wild-type TMEM106B protein is targeted to the plasma membrane,filopodia,and lysosomes in human oligodendrocytes.The D252N mutation reduces the size of lysosomes in oligodendrocytes and compromises lysosome changes upon starvation stress.Most importantly,we detected reductions in the length and number of filopodia in cells expressing the D252N mutant.PLP1 is the most abundant myelin protein that almost entirely colocalizes with TMEM106B,and coexpressing PLP1 with the D252N mutant readily rescues the lysosome and filopodia phenotypes of cells.Therefore,interactions between TMEM106B and PLP1 on the plasma membrane are essential for filopodia formation and myelination in oligodendrocytes,which may be sustained by the delivery of these proteins from lysosomes via exocytosis.

Meningeal lymphatic vasculature,a general target for glioblastoma therapy?

Glioblastoma(GBM)causes nearly universal mortality as a result of the failure of conventional therapies including surgical resection,targeted radiation therapy,and chemotherapy.An increasingly important treatment option is combining immunotherapy with other therapies in both preclinical and clinical studies.The central nervous system(CNS)has been historically considered an immune privileged area,but increasing evidence,including the recent rediscovery of meningeal lymphatic vessels(MLVs),has overturned this notion.MLVs are populated by multiple immune cells and connect the CNS to the periphery by draining cerebrospinal fluid with soluble CNS antigens and immune cells into cervical lymph nodes.In the past few years,more and more studies have indicated that MLVs are involved in the regulation of inflammation and the immune response in the pathogenesis of various CNS diseases including GBM.Here,we explore the critical interlinkages between MLVs and GBM therapies including chemotherapy,radiotherapy and immunotherapy,and propose the meningeal lymphatic vasculature as a general target for GBM therapy.

Infection with SARS-CoV-2 can cause pancreatic impairment

Evidence suggests associations between COVID-19 patients or vaccines and glycometabolic dysfunction and an even higher risk of the occurrence of diabetes.Herein,we retrospectively analyzed pancreatic lesions in autopsy tissues from 67 SARS-CoV-2 infected non-human primates(NHPs)models and 121 vaccinated and infected NHPs from 2020 to 2023 and COVID-19 patients.Multi-label immunofluorescence revealed direct infection of both exocrine and endocrine pancreatic cells by the virus in NHPs and humans.Minor and limited phenotypic and histopathological changes were observed in adult models.Systemic proteomics and metabolomics results indicated metabolic disorders,mainly enriched in insulin resistance pathways,in infected adult NHPs,along with elevated fasting C-peptide and C-peptide/glucose ratio levels.Furthermore,in elder COVID-19 NHPs,SARS-CoV-2 infection causes loss of beta(β)cells and lower expressed-insulin in situ characterized by islet amyloidosis and necrosis,activation ofα-SMA and aggravated fibrosis consisting of lower collagen in serum,an increase of pancreatic inflammation and stress markers,ICAM-1 and G3BP1,along with more severe glycometabolic dysfunction.In contrast,vaccination maintained glucose homeostasis by activating insulin receptorαand insulin receptorβ.Overall,the cumulative risk of diabetes post-COVID-19 is closely tied to age,suggesting more attention should be paid to blood sugar management in elderly COVID-19 patients.

Regulation of superoxide flashes by transient and steady mitochondrial calcium elevations

The mitochondria play essential roles in both intracellular calcium and reactive oxygen species signaling.As a newly discovered universal and fundamental mitochondrial phenomenon,superoxide flashes reflect transient bursts of superoxide production in the matrix of single mitochondria.Whether and how the superoxide flash activity is regulated by mitochondrial calcium remain largely unknown.Here we demonstrate that elevating mitochondrial calcium either by the calcium ionophore ionomycin or by increasing the bathing calcium in permeabilized HeLa cells increases superoxide flash incidence,and inhibition of the mitochondrial calcium uniporter activity abolishes the flash response.Quantitatively,the superoxide flash incidence is correlated to the steady-state mitochondrial calcium elevation with 1.7-fold increase per 1.0?F/F0 of Rhod-2 signal.In contrast,large mitochondrial calcium transients(e.g.,peak△F/F0~2.8,duration^2 min)in the absence of steady-state elevations failed to alter the flash activity.These results indicate that physiological levels of sustained,but not transient,mitochondrial calcium elevation acts as a potent regulator of superoxide flashes,but its mechanism of action likely involves a multi-step,slow-onset process.

Super-resolution fluorescence-assisted diffraction computational tomography reveals the threedimensional landscape of the cellular organelle interactome

The emergence of super-resolution(SR)fluorescence microscopy has rejuvenated the search for new cellular substructures.However,SR fluorescence microscopy achieves high contrast at the expense of a holistic view of the interacting partners and surrounding environment.Thus,we developed SR fluorescence-assisted diffraction computational tomography(SR-FACT),which combines label-free three-dimensional optical diffraction tomography(ODT)with two-dimensional fluorescence Hessian structured illumination microscopy.The ODT module is capable of resolving the mitochondria,lipid droplets,the nuclear membrane,chromosomes,the tubular endoplasmic reticulum,and lysosomes.Using dual-mode correlated live-cell imaging for a prolonged period of time,we observed novel subcellular structures named dark-vacuole bodies,the majority of which originate from densely populated perinuclear regions,and intensively interact with organelles such as the mitochondria and the nuclear membrane before ultimately collapsing into the plasma membrane.This work demonstrates the unique capabilities of SR-FACT,which suggests its wide applicability in cell biology in general.

Endothelial mechanosensors: the gatekeepers of vascular homeostasis and adaptation under mechanical stress

Endothelial cells(ECs)not only serve as a barrier between blood and extravascular space to modulate the exchange of fluid,macromolecules and cells,but also play a critical role in regulation of vascular homeostasis and adaptation under mechanical stimulus via intrinsic mechanotransduction.Recently,with the dissection of microdomains responsible for cellular responsiveness to mechanical stimulus,a lot of mechanosensing molecules(mechanosensors)and pathways have been identified in ECs.In addition,there is growing evidence that endothelial mechanosensors not only serve as key vascular gatekeepers,but also contribute to the pathogenesis of various vascular disorders.This review focuses on recent findings in endothelial mechanosensors in subcellular microdomains and their roles in regulation of physiological and pathological functions under mechanical stress.

Ligand binding and conformational changes of SUR1 subunit in pancreatic ATP-sensitive potassium channels

ATe-sensitive potassium channels （KATP） are energy sensors on the plasma membrane. By sensing the intracellular ADP/ATP ratio of β-cells, pancreatic KATe channels control insulin release and regulate metabo- lism at the whole body level. They are implicated in many metabolic disorders and diseases and are there- fore important drug targets. Here, we present three structures of pancreatic KATe channels solved by cryo- electron microscopy （cryo-EM）, at resolutions ranging from 4.1 to 4.5 A. These structures depict the binding site of the antidiabetic drug glibenclamide, indicate how Kir6.2 （inward-rectifying potassium channel 6.2） N-ter- minus participates in the coupling between the periph- eral SUR1 （sulfonylurea receptor 1） subunit and the central Kir6.2 channel, reveal the binding mode of acti- vating nucleotides, and suggest the mechanism of how Mg-ADP binding on nucleotide binding domains （NBDs） drives a conformational change of the SUR1 subunit.

Dgcr8 deletion in the primitive heart uncovered novel microRNA regulating the balance of cardiac-vascular gene program

Primitive mammalian heart transforms from a single tube to a four-chambered muscular organ during a short developmental window.We found that knocking out global microRNA by deleting Dgcr8 microprocessor in Mespl cardiovascular progenitor cells lead to the formation of extremely dilated and enlarged heart due to defective cardiomyocyte(CM)differentiation.Transcriptome analysis revealed unusual upregulation of vascular gene expression in Dgcr8 cKO hearts.Single cell RNA sequencing study further confirmed the increase of angiogenesis genes in single Dgcr8 cKO CM.We also performed global microRNA profiling of E9.5 heart for the first time,and identified that miR-541 was transiently highly expressed in E9.5 hearts.Interestingly,introducing miR-541 back into microRNA-free CMs partially rescued their defects,downregulated angiogenesis genes and significantly upregulated cardiac genes.Moreover,miR-541 can target Ctgf and inhibit endothelial function.Our results suggest that micro-RNAs are required to suppress abnormal angiogenesis gene program to maintain CM differentiation.

Nuclear peripheral chromatin-lamin B1 interaction is required for global integrity of chromatin architecture and dynamics in human cells

The eukaryotic genome is folded into higher-order conformation accompanied with constrained dynamics for coordinated genome functions.However,the molecular machinery underlying these hierarchically organized three-dimensional(3D)chromatin architecture and dynamics remains poorly understood.Here by combining imaging and sequencing,we studied the role of lamin B1 in chromatin architecture and dynamics.We found that lamin B1 depletion leads to detachment of lamina-associated domains(LADs)from the nuclear periphery accompanied with global chromatin redistribution and decompaction.Consequently,the interchromosomal as well as inter-compartment interactions are increased,but the structure of topologically associating domains(TADs)is not affected.Using live-cell genomic loci tracking,we further proved that depletion of lamin B1 leads to increased chromatin dynamics,owing to chromatin decompaction and redistribution toward nucleoplasm.Taken together,our data suggest that lamin B1 and chromatin interactions at the nuclear periphery promote LAD maintenance,chromatin compaction,genomic compartmentalization into chromosome territories and A/B compartments and confine chromatin dynamics,supporting their crucial roles in chromatin higher-order structure and chromatin dynamics.

Acidic pH transiently prevents the silencing of self-renewal and dampens microRNA function in embryonic stem cells

Enhanced glycolysis is a distinct feature associated with numerous stem cells and cancer cells.However,little is known about its regulatory roles in gene expression and cell fate determination.Here,we confirm that glycolytic metabolism and lactate production decrease during the differentiation of mouse embryonic stem cells(mESCs).Importantly,acidic pH due to lactate accumulation can transiently prevent the silencing of mESC self-renewal in differentiation conditions.Furthermore,acidic pH partially blocks the differentiation of human ESCs(hESCs).Mechanistically,acidic pH downregulates AGO1 protein and de-represses a subset of mRNA targets of miR-290/302 family of microRNAs which facilitate the exit of naive pluripotency state in mESCs.Interestingly,AGO1 protein is also downregulated by acidic pH in cancer cells.Altogether,this study provides insights into the potential function and underlying mechanism of acidic pH in pluripotent stem cells(PSCs).

Critical role of zebrafish dnajb5 in myocardial proliferation and regeneration

Myocardial infarction is a devastating disease worldwide.At present,nearly 40 million patients suffer from heart failure.Owing to a lack of adequate blood supply,25%of cardiomyocytes are subjected to apoptosis and necrosis within a few hours after infarction.It remains challenging to find effective therapeutic methods for heart failure.On the other hand,zebrafish and neonatal mouse hearts have a strong ability to regenerate(Poss et al.,2002;Raya et al.,2003;Porrello et al.,2011),and their regeneration derives from existing cardiomyocytes(Jopling et al.,2010;Kikuchi et al..

Mitochondria determine the sequential propagation of the calcium macrodomains revealed by the super-resolution calcium lantern imaging

Despite the wide application of super-resolution(SR)microscopy in biological studies of cells,the technology is rarely used to monitor functional changes in live cells.By combining fast spinning disc-confocal structured illumination microscopy(SD-SIM)with loading of cytosolic fluorescent Ca2+indicators,we have developed an SR method for visualization of regional Ca2+dynamics and related cellular organelle morphology and dynamics,termed SR calcium lantern imaging.In COS-7 cells stimulated with ATP,we have identified various calcium macrodomains characterized by different types of Ca2+release from endoplasmic reticulum(ER)stores.Finally,we demonstrated various roles of mitochondria in mediating calcium signals from different sources;while mitochondria can globally potentiate the Ca2+entry associated with store release,mitochondria also locally control Ca2+release from the neighboring ER stores and assist in their refilling processes.

Structure of an ATP-sensitive potassium channel(K_(ATP))

Subject Code:C05 With the support by the National Natural Science Foundation of China,the collaborative research team led by Prof.Chen Lei(陈雷)at the State Key Laboratory of Membrane Biology,Institute of Molecular Medicine,Peking-Tsinghua Center for Life Sciences,Beijing Key Laboratory of Cardiometabolic

Significant differences of function and expression of microRNAs between ground state and serum-cultured pluripotent stem cells

Serum- and 2i-cultured embryonic stem cells （ESCs） show different epigenetic landscapes and tran- scriptomic profiles. The difference in the function and expression of microRNAs （miRNAs） between these two states remains unclear. Here, we showed that 2i- and serum-cultured ESCs exhibited distinctive miRNA expression profiles with 〉100 miRNAs differentially expressed, and the expression changes were largely due to transcriptional regulation. We further characterized the function of miRNAs differentially expressed under two conditions and found that ESCs exhibited higher degree of dependency on miRNAs for rapid proliferation; since Dgcr8-/- or Dicerl-I- but not wild-type ESCs showed slower growth rate and more accumulation in the G1 phase under 2i than serum condition. More interestingly, introduction of various self-renewal-silencing miRNAs in wild-type or Dgcr8/- ESCs failed to silence the self-renewal in 2i medium, but regained the ability to silence the self-renewal upon the addition of serum. Our findings reveal significant differences in the expression and function of miRNAs between serum- and 2i- cultured ESCs.

Chemical screening links disulfiram with cardiac protection after ischemic injury

Ischemia-reperfusion injury occurs after reperfusion treatment for patients suffering myocardial infarction,however the underlying mechanisms are incompletely understood and effective pharmacological interventions are limited.Here,we report the identification and characterization of the FDA-approved drug disulfiram(DSF)as a cardioprotective compound.By applying high-throughput chemical screening,we found that DSF decreased H_(2)O_(2)-induced cardiomyocyte death by inhibiting Gasdermin D,but not ALDH1,in cardiomyocytes.Oral gavage of DSF decreased myocardial infarct size and improved heart function after myocardial ischemia-reperfusion injury in rats.Therefore,this work reveals DSF as a potential therapeutic compound for the treatment of ischemic heart disease.