Analytical capability of Raman spectroscopy to detect biochemical changes in red blood cell membrane disorders.

Symmetric metamaterial coupling element-based microwave sensor integrated with microfluidic Chip for cell sorting and concentration detection

Scalable synthesis of self-assembled magneto-plasmonic core-satellite nanoparticles for microfluidic sorting and bioorthogonal sensing of targeted cells.

Protocol for the generation of low-input Hi-C sequencing libraries of FACS-isolated mitotic cells.

Human induced pluripotent stem cells (hiPSC) are an invaluable resource for investigating the molecular mechanisms regulating cell fate specification during brain development. However, most directed differentiation methods exhibit significant cell fate heterogeneity and require several months to become functional. To address this challenge, we developed a green fluorescent protein (GFP) reporter system in hiPSC by targeting the genomic locus of Forebrain Enriched Zinc Finger 2 (FEZF2), which encodes a transcription factor essential for the fate specification of sub-cerebral projection neurons (SCPN) during forebrain development. Using this FEZF2-GFP reporter hiPSC line, we optimized a directed differentiation protocol to rapidly and efficiently generate pallial progenitors and glutamatergic neuronal subgroups after 3 weeks. Through fluorescence activated cell sorting for both GFP and CD200, isolated post-mitotic SCPN immediately displayed electrophysiological properties and formed glutamatergic synapses within 4 additional weeks of in vitro cell culture. Co-culture with hiPSC-derived spinal motor neurons further enhanced these electrophysiological characteristics, improved viability, and increased synapse formation in SCPN. This study presents a streamlined and effective strategy to generate, isolate, and characterize human motor neuron circuits, providing insights into the molecular determinants regulating synaptogenesis and functional maturation.

A CRISPR-based Genome-wide Loss-of-function Screen Defines a Role of Host Metabolism in Regulating Hepatitis B Virus Infection.

Introduction.Candida glabrata is a pathogenic yeast in humans, recognized for its genomic plasticity and increasing prevalence of antifungal resistance, including multidrug-resistant phenotypes, especially in the US and European countries.Hypothesis. This study hypothesizes that the resistance mechanisms in clinically resistant strains of C. glabrata differ from laboratory-generated resistant strains.Aim. This study aims to understand the resistance mechanism in Indian clinical isolates of C. glabrata.Methodology. A total of 240 clinical isolates of C. glabrata were tested for antifungal susceptibility and one resistant strain was artificially synthesized in the laboratory. Both clinical and lab-generated resistant strains were analysed for antifungal resistance using methods such as phenotypic assays, real-time quantitative PCR, Fluorescence-activated cell sorting (FACS) analysis and targeted gene sequencing. Mechanisms involving drug efflux pumps, mismatch repair pathways, ergosterol biosynthesis pathway and biofilm formation were systematically studied.Results. Among clinical isolates, one susceptible-dose dependent strain and three fluconazole-resistant strains were identified. Both clinical and lab-generated resistant strains demonstrated antifungal resistance phenotypically, with increased expression of CDR1. Targeted gene sequencing revealed novel mutations in PDR1, while mutations in MSH2 served as genotypic markers for resistance. Overexpression of ERG11 was seen in a lab-generated resistant strain where a specific mutation was identified. Biofilm activity contributed to resistance in one of the clinical strains.Conclusion. This study reports for the first time the fluconazole resistance mechanism in C. glabrata from India. The findings underscore the diversity of resistance mechanisms among clinical and lab-generated isolates, emphasizing the need for novel antifungal therapies to address these emerging resistance profiles effectively.

Establishing an in vitro screening system for synthetic signal peptides in Chinese hamster ovary (CHO) cells is crucial, as selecting an appropriate signal peptide ensures efficient secretion and enhances therapeutic protein production. However, expanding library sizes to increase peptide diversity and improving screening efficiency by minimizing false positives remain significant challenges. In this study, we optimized a synthetic-biology-driven in vitro screening system by incorporating the Beacon optofluidic system to minimize false positives and generating stable CHO cell pools in serum-free suspension culture to accommodate larger libraries. This platform enabled the high-throughput screening of diverse signal peptide variants at the single-cell level, exceeding the limits of conventional fluorescence-activated cell sorting (FACS)-based methods. Using this system, we successfully identified novel synthetic signal peptides for the heavy chain (HC) and light chain (LC) that enhanced the specific protein productivity (qp) of the monoclonal antibody (mAb). The selected synthetic signal peptide combinations improved qp in both transient and stable gene expression systems, with the best-performing combination increasing qp by up to 2.23-fold compared with the native signal peptide. Additionally, substituting the native signal peptide with a screened synthetic signal peptide did not significantly affect the N-terminal cleavage, N-linked glycosylation, size heterogeneity, and biological activity of the mAb. Overall, the synthetic-biology-based in vitro screening system developed in this study enabled the discovery of novel synthetic signal peptides that significantly improved mAb productivity in CHO cells without compromising product quality and function.

Temporomandibular joint osteoarthritis (TMJ-OA) is a prevalent degenerative joint disease that significantly impairs quality of life. Neurogenesis is considered a key initiating factor in this pain; however, the precise mechanisms remain unclear. This study tests the hypothesis that osteoclast-derived slit guidance ligand 3 (SLIT3) plays an important role in osteoarthritis pain. These findings reveal that in TMJ-OA mice, increased osteoclast activation and SLIT3 expression occur in the subchondral bone of the TMJ condyle, accompanied with pain. Interestingly, results from immunofluorescent co-staining and fluorescence-activated cell sorting support that osteoclasts serve as the primary cellular source of SLIT3 in subchondral bone, and SLIT3 produced by TRAP-positive (TRAP+) osteoclasts significantly promotes the growth of sensory nerves. The results of in vivo models demonstrate that the specific knockdown/knockout of Slit3 in TRAP+ osteoclasts significantly reduces the level of SLIT3. More importantly, Slit3 knockdown/knockout in osteoclasts results in reduced sensory nerve innervation in the osteochondral regions, decreased osteoarthritis pain, and alleviated bone and cartilage degeneration in TMJ-OA. Thus, SLIT3 derived from TRAP+ osteoclasts in the subchondral bone plays a crucial role in the progression of TMJ-OA. This suggests that targeting SLIT3 might represent a promising therapeutic approach to alleviate the pain in TMJ-OA.

Inflammation is a key driver of Alzheimer's disease (AD) and may connect all known AD risk factors. Recently, AD resilience outcomes have been developed which have helped to uncover mechanisms that enable some individuals to withstand significant AD pathology or genetic risk, while retaining cognitive function. We conducted a series of transcriptome-wide association studies (TWAS) focusing on monocytes, key innate immune cells that respond to pathogens and invade the CNS. Monocyte expression data under various immune stimulation states (naive, LPS 2 h, LPS 24 h, IFN 24 h) and corresponding genotype data from 432 individuals (Fairfax et al. [1]) were analyzed. We developed cis-genetic expression models using both elastic-net, and MASH combined with LD-pruning; capturing polygenic structures and independent inflammatory eQTLs across conditions, respectively. These models were applied to GWAS summary statistics of three AD resilience phenotypes: cognitive and global AD-resilience, and Amish cognitive preservation. We identified 180 TWAS results surpassing a suggestive significance threshold of PFDR < 0.20, including 92 unique genes. APP, a well-known AD gene, showed the strongest overall association, which may inform ongoing efforts targeting its action in the brain. Whole-blood RNA-seq data from a separate AD cohort confirmed differential expression between AD cases and controls in 35 putative targets, including: SURF1, ACKR3, LILRA5, FBXO2, ITPR1, and HRH4. We also demonstrate that the regulation of these genes is specific to monocytes. Finally, in-silico cell sorting (CIBERSORTx) revealed differential monocyte abundance by AD status, supporting monocyte-driven inflammation as a distinct, complementary pathway of myeloid cell involvement in AD. Together, these findings highlight monocytes as a critical and understudied cellular component for AD resilience mechanisms, with potential implications for novel immunotherapeutic strategies and precision medicine approaches in AD.

Sorting of desired single cells from a cell population is crucial for many applications in biology and biomedicine that require analysis at the cellular level. Microfluidic dielectrophoresis (DEP)-based single-cell sorting method has been demonstrated as a powerful technology to enable high-throughput and accurate sorting of single cells. However, conventional DEP sorting is mainly performed in the oil phase constrained by solid channels, which restricts the capacity and tunability of the sorting path. Here, we describe an approach to sort single cells on multiple paths in air. Our device allows for tunable ejection of droplets containing single cells in air, which are interrogated and sorted by a microfluidic DEP sorter with a cylindrical electrode, showing a sorting accuracy of >99% for all paths with high survival rates. We demonstrate the utility of our device by isolating multiple subpopulations from a cell sample with three types of cells. Our technology holds the potential to perform sorting on numerous paths, making it applicable for multipurpose sorting from complex heterogeneous cell populations.

The selective enrichment of cell populations based on surface markers is critical for the advancement of gene and cell therapies. Current antibody-based cell isolation methods, such as fluorescence- and magnetic-activated cell sorting (FACS and MACS), offer high specificity but are limited by scalability, cost, and potential adverse effects on cellular physiology, including differentiation or apoptosis. In this study, we present an alternative antibody-free approach for reversible cell isolation using pH-responsive peptides that target the CD38 surface marker. Through in silico design, we developed affinity peptides with pH Sensitivity (APPS) that selectively bind CD38 at physiological pH and release target cells under mildly basic conditions (pH 8). The peptides were conjugated to amine-functionalized magnetic beads at controlled surface densities (1.25-40 equivalents) and evaluated for their performance in isolating CD38+ hematopoietic cells from a mixed population of RPMI 8226 (CD38+) and K562 (CD38-) cells. Compared to antibody-based MACS, APPS-functionalized beads achieved superior CD38+ cell purity (> 80% vs. > 50%) while maintaining high cell viability (~90%). The integration of APPS beads into a microfluidic platform enabled pseudo-continuous cell separation with elution rates exceeding 105 cells·mL-1·min-1. These results demonstrate that APPS beads provide a gentle, scalable, and reversible alternative for cell isolation, with significant potential for analytical and preparative applications in cellular therapy manufacturing.

Investigate the cellular response of human nucleus pulposus (HNP) cells to serum deprivation, focusing on the role of high mobility group box1(HMGB1) in regulating autophagy and apoptosis, and elucidate the time-dependent activation of autophagy shifting toward apoptosis under nutrient stress. Additionally, the study evaluated the impact of autophagy inhibition by chloroquine (CQ) on apoptosis progression. HNP samples were obtained from the human biobank with exemption from IRB screening (IRB number DC25SASI0012) to evaluate the impact of nutritional deprivation. Comprehensive analyses encompassed detailed evaluations of cellular morphology, viability, DNA integrity, and metabolic function, providing an integrated view of cellular status. Western blotting (WB), fluorescence-activated cell sorting (FACS), and immunofluorescence (IF) were used to detect LC3, P62, HMGB1, and cleaved caspase-3. Real-time quantitative polymerase chain reaction (RT-qPCR) further revealed changes in gene expression related to autophagy (LC3, P62) and apoptosis (caspase-3), highlighting cellular stress responses. Serum deprivation markedly reduced HNP cell viability, altered morphology, and suppressed metabolic activity, while inducing a time-dependent increase in autophagy, peaking at 48 h. Furthermore, elevated LC3-II, decreased P62, and increased cytoplasmic translocation of HMGB1 indicate activation of HMGB1-mediated autophagy. Simultaneously, cleaved caspase-3 levels rose, suggesting HMGB1’s involvement in shifting the balance toward apoptosis. IF and RT-qPCR confirmed enhanced LC3 and cleaved caspase-3 expression, while FACS analysis revealed increased apoptotic cell populations with declining serum levels. These findings highlight a crucial interplay between autophagy and apoptosis regulated by HMGB1 under nutrient-deprived conditions. Eventually, CQ treatment inhibited autophagic flux by blocking LC3-II degradation, thereby amplifying apoptosis. Serum deprivation potently induced HMGB1-mediated autophagy-apoptosis interplay in HNP cells, with CQ enhancing apoptosis by inhibiting autophagy. Supplementary InformationThe online version contains supplementary material available at 10.1038/s41598-025-23026-7.

Over the past 6 decades, cytometry has evolved from a niche experimental method into a cornerstone of modern biomedical research. The development of the first cell sorter by Mack Fulwyler laid the groundwork for technologies that now define single-cell analysis. From the early challenges of instrument operation and laser alignment to today's era of automated, high-parameter systems, the field has undergone continual transformation. We now enter what may be termed the "diamond age" of cytometry, an era of exceptional sensitivity, resolution, and analytical depth, driven by innovations such as spectral flow cytometry. Here, we reflect on the historical milestones that shaped the discipline, the cultural and educational shifts within the community, and the future challenges of standardization and quantitative rigor.

Head and neck squamous cell carcinoma (HNSCC) is an aggressive malignancy characterized by high metastatic potential and poor prognosis. Previous studies have focused on the correlation between hypermethylated ZNF582 and HNSCC prognosis, particularly in diagnostic contexts, but the underlying molecular mechanisms remain poorly understood. This study explored the biological function and underlying mechanisms of ZNF582 in HNSCC. ZNF582 expression was analyzed in HNSCC and non-tumor tissues. Functional assays, including colony formation, wound healing, Transwell, TUNEL assays, and xenograft models, were performed in HNSCC cell lines with or without ZNF582 overexpression. To assess cancer stemness, fluorescence-activated cell sorting, tumorsphere formation assay, extreme limiting dilution assay, and an orthotopic model were used. High-throughput RNA sequencing was performed to identify the altered signaling pathways. Bioinformatics analysis revealed that low ZNF582 expression in HNSCC correlated with poor prognosis. Overexpression of ZNF582 suppressed HNSCC proliferation, migration, and invasion, while promoting apoptosis both invitro and invivo. ZNF582 also reduced cancer stem cell activity and metastasis in HNSCC. Mechanically, ZNF582 overexpression inhibited the MAPK and PI3K/AKT signaling pathways. Our findings suggested that ZNF582 overexpression inhibits HNSCC progression via the MAPK and PI3K/AKT signaling pathways, highlighting ZNF582 as a potential therapeutic target gene for HNSCC treatment.

Amastigotes are the replicative intracellular life cycle stage of the protozoan parasite Trypanosoma cruzi, the causative agent of Chagas disease. They have the ability to proliferate in any nucleated mammalian cell. A greater understanding of amastigote biology would greatly aid drug development since this life cycle stage is the principal target of chemotherapy. Reports have linked recrudescence following drug treatment with the possible existence of a quiescent amastigote phenotype. However, the lack of a rapid and straightforward method for isolating amastigotes from infected host cells has limited research progress in this area. This is particularly the case with omics technology, where the current complex fractionation and purification procedures can act to perturb RNA and protein expression. Here, we outline a methodology that largely overcomes these problems. Our protocol exploits MP Bio-Lysing Matrix M tubes to promote the differential lysis of host cells and the release of intact amastigotes in a 1-min time frame. Immediate treatment of the lysate with CellCover reagent then maintains RNA and protein in their native states. Amastigotes stabilized in this way can undergo further manipulations, such as cell sorting, without modification of their proteome or transcriptome profiles. This methodology, which is flexible and widely applicable, should greatly benefit research into the intracellular life cycle of T. cruzi.

The genetic manipulation of the human parasite Trypanosoma cruzi has been significantly improved since the implementation of the CRISPR/Cas9 technology for genome editing in this organism. Initially, the system was successfully used for gene knockout and endogenous C-terminal tagging in T. cruzi. Recently, an updated version of this technology has been used for gene complementation, site-directed mutagenesis, and N-terminal tagging in trypanosomatids. This cloning-free strategy, called CRISPR/T7RNAP/Cas9, is extremely useful for identifying essential genes when null mutants are not viable. Mutant cell lines obtained by this new system have been used for the functional characterization of proteins in different developmental stages of this parasite's life cycle, including infective trypomastigotes and intracellular amastigotes. In this chapter, we describe the methodology to achieve genome editing by CRISPR/T7RNAP/Cas9 in T. cruzi. Our method involves the generation of T. cruzi epimastigotes that constitutively express the T7 RNA polymerase (T7RNAP) and SpCas9, and their co-transfection with an sgRNA template and donor DNA(s) as polymerase chain reaction (PCR) products. Using this strategy, we have generated genetically modified parasites in 2-3weeks without the need for gene cloning, cell sorting, or having to perform several transfection attempts to verify the sgRNA efficiency for targeting the gene of interest. The methodology has been organized according to three main genetic purposes: gene knockout, gene complementation of knockout cell lines, and endogenous (N- or C-terminal) tagging in T. cruzi.

Background: Hypoplastic Left Heart Syndrome (HLHS) remains the most lethal form of congenital heart disease (CHD). The next generation of therapies are predicated on understanding the molecular bases for HLHS. We created kmt2d null zebrafish that recapitulate features of human Kabuki Syndrome (KS), including hypoplastic heart and death from circulatory collapse. Notch signaling is hyperactivated in endocardial cells, with aberrant endocardial-to-mesenchymal transition (EMT) that occludes the ventricle; an effect that is rescued by Notch inhibition. Notch hyperactivation and aberrant EMT are hallmarks of endocardial fibroelastosis (EFE) observed in HLHS. Objective: Define how KMT2D deletion disrupts the epigenetic landscape controlling Notch signaling in zebrafish heart development and human induced pluripotent stem cell (iPSC)-derived cardiac lineages. Methods: We performed immunofluorescence followed by Fluorescence Activated Cell Sorting (FACS) on Tg ( kdrl :GFP) WT and kmt2d -null embryos at 2 dpf (n=3 each group), gating on kdrl+ (endocardial) cells, measuring Rbpj fluorescence as a marker of Notch activation and DAPI for cell cycle analysis. Single-cell histone profiling was performed on WT and KMT2D null human iPSC-derived endocardial cells (n=3 each group), using fluorescently labelled antibodies against H3K4me1, H3K4me3, H3K27ac, H3K27me3, Notch1 and cell-cycle proteins. Results: Notch signaling was preferentially elevated in G2 cell-cycle endocardial cells in kmt2d -null embryos, prior to development of heart hypoplasia (p&lt;0.001, n=62-597 cells each group). KMT2D null human iPSC-derived endocardial cells phenocopied the zebrafish null mutants, with increased Notch1 specifically in G2 cell-cycle state compared to WT (p&lt;2.3E-4). H3K4me1 was increased (p &lt;1.5E-47), while H3K4me3 (p&lt;3.7E-56) and H3K27ac (p&lt;5.3E-58) signals were decreased in KMT2D null endocardial cells (n=529-665 cells each group), indicating a proportion of cells that are poised for activation, but transcriptionally silent. Conclusion: KMT2D functions to control Notch expression in endocardial cells in G2 cell cycle. KMT2D deletion disrupts chromatin states that usually repress Notch signaling in endocardial cells, triggering excessive Notch and aberrant EMT. Ongoing experiments are defining the gene regulatory networks modulated by KMT2D that contribute to heart chamber formation. Our findings are relevant beyond KS alone, by furthering our understanding of EFE pathogenesis in HLHS.

Tracking, isolating, and studying antigen-specific B cells is challenging due to their low frequencies. Here, I describe a protocol to study enriched Trypanosoma cruzi (T. cruzi)-specific B cells by flow cytometry. Compared to alternative strategies (e.g., ELISA and ELISPOT), this approach allows detailed phenotyping and/or cell sorting for downstream analysis of T. cruzi-specific B cells.

Understanding and identifying protein-ligand interactions is essential for elucidating fundamental biological processes and developing translational applications such as vaccine and drug targets. Yeast surface display (YSD) systems expressing genome-wide or combinatorial libraries in Saccharomyces cerevisiae are powerful tools to identify protein-ligand interactions in an unbiased fashion. In the following protocol, we couple the YSD system expressing a genome-wide library of the protozoan pathogen Trypanosoma cruzi with magnetic-activated cell sorting to identify antigen-antibody interactions. We describe the enrichment of pathogen antigens targeted by antibodies of infected patients using the YSD library. We also detailed a DNA sequencing methodology and a data analysis computational pipeline. The approach can be easily adapted to identify protein-protein or protein-drug interactions.