The Immunosuppressive Receptor CD32b Regulation of Macrophage Polarization and Its Implications in Tumor Progression.

Macrophages, pivotal components of the immune system, orchestrate host defense mechanisms in humans and mammals. Their polarization into classically activated macrophages (CAMs or M1) and alternatively activated macrophages (AAMs or M2) dictates distinct functional roles in immunity and tissue homeostasis. While the negative regulatory role of CD32b within the FC gamma receptor (FCγR) family is recognized across various immune cell types, its influence on macrophage polarization remains elusive. This study aimed to elucidate the regulatory role of CD32b in macrophage polarization and discern the differential expression markers between the M1 and M2 phenotypes following CD32b siRNA transfection. The results revealed a decrease in the CD32b levels in lipopolysaccharide (LPS)-treated M1 and an increase in interleukin-4 (IL-4)-treated M2 macrophages, as observed in macrophage Raw264.7 cells. Furthermore, CD32b siRNA transfection significantly downregulated the M2 markers (IL-10, VEGF, Arg-1, and STAT6), while upregulating the M1 markers (IL-6, NF-κB, NOS2, and STAT1) in the Raw264.7 cells. Similar findings were recapitulated in macrophage-rich adherent cells isolated from mouse spleens. Additionally, the cytopathological analysis of pleural effusions and ascitic fluids from patients with cancer revealed a positive correlation between advanced tumor stages, metastasis, and elevated CD32b levels. In conclusion, this study highlights the regulatory influence of CD32b in suppressing M1 expression and promoting M2 polarization. Moreover, heightened M2 activation and CD32b levels appear to correlate with tumor progression. A targeted CD32b blockade may serve as a novel therapeutic strategy to inhibit M2 macrophage polarization and is promising for anti-tumor intervention.

Highly-specific aptamer targeting SARS-CoV-2 S1 protein screened on an automatic integrated microfluidic system for COVID-19 diagnosis.

Variants of the severe acute respiratory syndrome coronavirus (SARS-CoV-2) have evolved such that it may be challenging for diagnosis and clinical treatment of the pandemic coronavirus disease-19 (COVID-19). Compared with developed SARS-CoV-2 diagnostic tools recently, aptamers may exhibit some advantages, including high specificity/affinity, longer shelf life (vs. antibodies), and could be easily prepared. Herein an integrated microfluidic system was developed to automatically carry out one novel screening process based on the systematic evolution of ligands by exponential enrichment (SELEX) for screening aptamers specific with SARS-CoV-2. The new screening process started with five rounds of positive selection (with the S1 protein of SARS-CoV-2). In addition, including non-target viruses (influenza A and B), human respiratory tract-related cancer cells (adenocarcinoma human alveolar basal epithelial cells and dysplastic oral keratinocytes), and upper respiratory tract-related infectious bacteria (including methicillin-resistant Staphylococcus aureus, Pseudomonas aeruginosa, Acinetobacter baumannii, and Klebsiella pneumoniae), and human saliva were involved to increase the specificity of the screened aptamer during the negative selection. Totally, all 10 rounds could be completed within 20 h. The dissociation constant of the selected aptamer was determined to be 63.0 nM with S1 protein. Limits of detection for Wuhan and Omicron clinical strains were found to be satisfactory for clinical applications (i.e. 4.80 × 101 and 1.95 × 102 copies/mL, respectively). Moreover, the developed aptamer was verified to be capable of capturing inactivated SARS-CoV-2 viruses, eight SARS-CoV-2 pseudo-viruses, and clinical isolates of SARS-CoV-2 viruses. For high-variable emerging viruses, this developed integrated microfluidic system can be used to rapidly select highly-specific aptamers based on the novel SELEX methods to deal with infectious diseases in the future.

An integrated microfluidic platform for detection of ovarian clear cell carcinoma mRNA biomarker FXYD2.

In this work we developed an integrated microfluidic system for automatically detecting the ovarian clear cell carcinoma (OCCC) biomarker FXYD2. Dealing with ascites from ovarian cancer patients, capture of cancer cells, isolation of messenger RNA, and quantitative reverse-transcription polymerase chain reaction were integrated into a single microfluidic chip and carried out on-chip automatically. OCCC is a subtype of ovarian cancer with a high mortality risk, and a high FXYD2 gene expression level was shown to be closely associated with OCCC. The lowest limit of quantification using a benchtop protocol of this system could be as low as 100 copies per sample. By normalizing the expression to a housekeeping gene, GAPDH, a simple cycle threshold ratio index could distinguish high FXYD2 expression cells from the low-expression ones. This developed platform may therefore facilitate future OCCC diagnosis and/or prognosis.

Analyzing the homeostasis of signaling proteins by a combination of Western blot and fluorescence correlation spectroscopy.

The determination of intracellular protein concentrations is a prerequisite for understanding protein interaction networks in systems biology. Today, protein quantification is based either on mass spectrometry, which requires large cell numbers and sophisticated measurement protocols, or on quantitative Western blotting, which requires the expression and purification of a recombinant protein as a reference. Here, we present a method that uses a transiently expressed fluorescent fusion protein of the protein-of-interest as an easily accessible reference in small volumes of crude cell lysates. The concentration of the fusion protein is determined by fluorescence correlation spectroscopy, and this concentration is used to calibrate the intensity of bands on a Western blot. We applied this method to address cellular protein homeostasis by determining the concentrations of the plasma membrane-located transmembrane scaffolding protein LAT and soluble signaling proteins in naïve T cells and transformed T-cell lymphoma (Jurkat) cells (with the latter having nine times the volume of the former). Strikingly, the protein numbers of soluble proteins scaled with the cell volume, whereas that of the transmembrane protein LAT scaled with the membrane surface. This leads to significantly different stoichiometries of signaling proteins in transformed and naïve cells in concentration ranges that may translate directly into differences in complex formation.

Simultaneous detection of intracellular target and off-target binding of small molecule cancer drugs at nanomolar concentrations.

BACKGROUND AND PURPOSE: In vitro assays that determine activities of drug candidates with isolated targets have only limited predictive value for activities in cellular assays. Poor membrane permeability and off-target binding are major reasons for such discrepancies. However, it still difficult to directly analyse off-target binding at the same time as target binding, on a subcellular level. Here, we present a combination of fluorescence correlation spectroscopy (FCS) and fluorescence cross-correlation spectroscopy (FCCS) as a solution to this problem. EXPERIMENTAL APPROACH: The well-established dihydrofolate reductase inhibitor methotrexate and the kinase inhibitors PD173956 and purvalanol B were conjugated via polyethylene glycol linkers with the fluorophore Cy5. The cellular uptake and subcellular distribution of these compounds in single human cancer-derived cells were investigated by confocal laser scanning microscopy. In addition, molecular interactions inside the cell with the respective target proteins and off-target binding were detected simultaneously in the nanomolar range by FCCS and FCS, respectively, using cells expressing green fluorescent protein fusion proteins of dihydrofolate reductase and Abelson kinase 1. KEY RESULTS: Large differences in the interaction patterns were found for these compounds. For methotrexate-Cy5, drug-target interactions could be detected and dissociation constants determined. In contrast, PD173956-Cy5 showed strong interactions with intracellular high-molecular weight structures, other than its target. CONCLUSIONS AND IMPLICATIONS: The combination of FCS and FCCS provides a powerful means to assess subcellular pharmacokinetics and dynamics of drug candidates at nanomolar concentrations.

Large-scale sequencing analysis of the full-length cDNA library of human hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the human cancers clearly linked to viral infections. Although the major risk factors for HCC development have been elucidated, the hepatocellular carcinogenesis pathway resulting in malignant transformation of liver cells remains to be clarified. Recently, some results of microarray and comparative genomic hybridization analysis have been provided as comprehensive studies of genomic instability in HCC, including mutation, deletion and DNA copy losses. In this work, the full-length cDNA library has been constructed and sequenced, and the sequencing results have been further clustered and analyzed. The results show that 1,342 genes have been found, and about 300 of these genes may be important in e.g. cell proliferation, DNA repair and apoptosis. After further analysis of DNA sequences, the deletion genotypes of at least 24 genes have been found. However, the functional changes of these deletion mutants and their significance in hepatocellular carcinogenesis remain to be clarified. This research may be one of the best to obtain the candidate genes for hepatocellular carcinogenesis.