Over the past decade, our understanding of human diseases has rapidly grown from the rise of single-cell spatial biology. While conventional tissue imaging has focused on visualizing morphological features, the development of multiplex tissue imaging from fluorescence-based methods to DNA- and mass cytometry-based methods has allowed visualization of over 60 markers on a single tissue section. The advancement of spatial biology with a single-cell resolution has enabled the visualization of cell-cell interactions and the tissue microenvironment, a crucial part to understanding the mechanisms underlying pathogenesis. Alongside the development of extensive marker panels which can distinguish distinct cell phenotypes, multiplex tissue imaging has facilitated the analysis of high dimensional data to identify novel biomarkers and therapeutic targets, while considering the spatial context of the cellular environment. This mini-review provides an overview of the recent advancements in multiplex imaging technologies and examines how these methods have been used in exploring pathogenesis and biomarker discovery in cancer, autoimmune and infectious diseases.

With severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) as an emergent human virus since December 2019, the world population is susceptible to coronavirus disease 2019 (COVID-19). SARS-CoV-2 has higher transmissibility than the previous coronaviruses, associated by the ribonucleic acid (RNA) virus nature with high mutation rate, caused SARS-CoV-2 variants to arise while circulating worldwide. Neutralizing antibodies are identified as immediate and direct-acting therapeutic against COVID-19. Single-domain antibodies (sdAbs), as small biomolecules with non-complex structure and intrinsic stability, can acquire antigen-binding capabilities comparable to conventional antibodies, which serve as an attractive neutralizing solution. SARS-CoV-2 spike protein attaches to human angiotensin-converting enzyme 2 (ACE2) receptor on lung epithelial cells to initiate viral infection, serves as potential therapeutic target. sdAbs have shown broad neutralization towards SARS-CoV-2 with various mutations, effectively stop and prevent infection while efficiently block mutational escape. In addition, sdAbs can be developed into multivalent antibodies or inhaled biotherapeutics against COVID-19.

Intrinsic protein flexibility is of overwhelming relevance for intermolecular recognition and adaptability of highly dynamic ensemble of complexes, and the phenomenon is essential for the understanding of numerous biological processes. These conformational ensembles-encounter complexes-lack a unique organization, which prevents the determination of well-defined high resolution structures. This is the case for complexes involving the oncoprotein SET/template-activating factor-Iβ (SET/TAF-Iβ), a histone chaperone whose functions and interactions are significantly affected by its intrinsic structural plasticity. Besides its role in chromatin remodeling, SET/TAF-Iβ is an inhibitor of protein phosphatase 2A (PP2A), which is a key phosphatase counteracting transcription and signaling events controlling the activity of DNA damage response (DDR) mediators. During DDR, SET/TAF-Iβ is sequestered by cytochrome c (Cc) upon migration of the hemeprotein from mitochondria to the cell nucleus. Here, we report that the nuclear SET/TAF-Iβ:Cc polyconformational ensemble is able to activate PP2A. In particular, the N-end folded, globular region of SET/TAF-Iβ (a.k.a. SET/TAF-Iβ ΔC)-which exhibits an unexpected, intrinsically highly dynamic behavior-is sufficient to be recognized by Cc in a diffuse encounter manner. Cc-mediated blocking of PP2A inhibition is deciphered using an integrated structural and computational approach, combining small-angle X-ray scattering, electron paramagnetic resonance, nuclear magnetic resonance, calorimetry and molecular dynamics simulations.

The development of cancer generally occurs as a result of various deregulated molecular mechanisms affecting the genes that can control normal cellular growth. Signal transducer and activator of transcription 3 (STAT3) pathway, once aberrantly activated can promote carcinogenesis by regulating the transcription of a number of oncogenic genes.
Here, we evaluated the impact of fangchinoline (FCN) to attenuate tumor growth and survival through modulation of oncogenic STAT3 signaling pathway using diverse tumor cell lines and a xenograft mouse model.
To evaluate the action of FCN on STAT3 cascade, protein levels were analyzed by Western blot analysis and electrophoretic mobility shift assay (EMSA). Translocation of STAT3 was detected by immunocytochemistry. Thereafter, FCN-induced ROS was measured by GSH/GSSG assay and H2DCF-DA. FCN-induced apoptosis was analyzed using Western blot analysis and flow cytometry for various assays. Finally, anti-cancer effects of FCN in vivo was evaluated in a myeloma model.
We noted that FCN abrogated protein expression levels of STAT3 and upstream signals (JAK1/2 and Src). In addition, FCN also attenuated DNA binding ability of STAT3 and its translocation into the nucleus. It altered the levels of upstream signaling proteins, increased SHP-1 levels, and induced substantial apoptosis in U266 cells. FCN also promoted an increased production of reactive oxygen species (ROS) and altered GSSG/GSH ratio in tumor cells. Moreover, FCN effectively abrogated tumor progression and STAT3 activation in a preclinical myeloma model.
Overall, this study suggests that FCN may have a tremendous potential to alter abnormal STAT3 activation and induce cell death in malignant cells along with causing the suppression of pathogenesis and growth of cancer through a pro-oxidant dependent molecular mechanism.

We report clinical, serologic, and immunogenetic studies of a set of monozygotic male twin patients who develop autoimmune thyroiditis and vitiligo associated with the HLA-DRB1*04-DQB1*03:02 and HLA-DRB1*03-DQB1*0201 haplotypes. The patients had detectable anti-thyroid and anti-melanocyte autoantibodies. A critical review is presented regarding the role of MHC II molecules linked to clinical manifestations of various autoimmune diseases displayed in a single patient, as is the case in the twin patients reported here.

UNASSIGNED: Intractable ulcers may ultimately lead to amputation. To promote wound healing, researchers developed a serum-free ex vivo peripheral blood mononuclear cell quality and quantity culture (MNC-QQc) as a source for cell therapy. In mice, pigs, and even humans, cell therapy with MNC-QQc reportedly yields a high regenerative efficacy. However, the mechanism of wound healing by MNC-QQc cells remains largely unknown. Hence, using an in vitro wound healing model, this study aimed to investigate MNC-QQc cells and the migratory potential of dermal fibroblasts.
UNASSIGNED: After separation from a 50 mL blood sample from healthy individuals, mononuclear cells were cultured for 7 days in a serum-free ex vivo expansion system with five different cytokines (MNC-QQc method). The effects of MNC-QQc cells on human dermal fibroblast migration were observed by scratch assay. An angiogenesis array screened the MNC-QQc cell supernatant for proteins related to wound healing. Finally, fibroblast migration was confirmed by observing the intracellular signal transduction pathways via Western blot.
UNASSIGNED: The migration of fibroblasts co-cultured with MNC-QQc cells increased by matrix metallopeptidase-9 (MMP9) secretion, as suggested by the angiogenesis array. Furthermore, the phosphorylation of signal transducer and activator of transcription 3 (STAT3) in fibroblast/MNC-QQc cell co-culture and fibroblast culture with added recombinant human MMP9 protein increased. When fibroblasts were cultured with either an MMP9 inhibitor or a STAT3 inhibitor, both fibroblast migration and STAT3 phosphorylation were significantly suppressed.
UNASSIGNED: MNC-QQc cells promote wound healing by the secretion of MMP9, which induces fibroblast migration via the STAT3 signaling pathway.

Thoracic aortic aneurysms (TAA) pathogenesis and progression include many mechanisms. The authors investigated the role of autophagy, oxidative stress, and endothelial dysfunction in 36 TAA patients and 23 control patients. Univariable and multivariable analyses were performed. TAA patients displayed higher oxidative stress and endothelial dysfunction then control patients. Autophagy in the TAA group was reduced. The association of oxidative stress and autophagy with aortic disease supports the role of these processes in TAA. The authors demonstrate a putative role of Nox2 and autophagy dysregulation in human TAA. These findings could pinpoint novel treatment targets to prevent or limit TAA progression.

Overactive bladder (OAB) is the most bothersome symptom in lower urinary tract symptoms (LUTS). Current pharmacologic treatment aims to inhibit detrusor contraction; however, shows unsatisfied efficacy and high discontinuation rate. LIM kinases (LIMKs) promote smooth muscle contraction in the prostate; however, their function in the bladder smooth muscle remains unclear. Here, we studied effects of the LIMK inhibitors on bladder smooth muscle contraction and proliferation both in vitro and in vivo experiments. Bladder expressions of LIMKs are elevated in OAB rat detrusor tissues. Two LIMK inhibitors, SR7826 and LIMKi3, inhibit contraction of human detrusor strip, and cause actin filament breakdown, as well as cell proliferation reduction in cultured human bladder smooth muscle cells (HBSMCs), paralleled by reduced cofilin phosphorylation. Silencing of LIMK1 and LIMK2 in HBSMCs resulted in breakdown of actin filaments and decreased cell proliferation. Treatment with SR7826 or LIMKi3 decreased micturition frequency and bladder detrusor hypertrophy in rats with bladder outlet obstruction. Our study suggests that LIMKs may promote contraction and proliferation in the bladder smooth muscle, which could be inhibited by small molecule LIMK inhibitors. LIMK inhibitors could be a potential therapeutic strategy for OAB- related LUTS.

Protein-protein interactions are of paramount importance in regulating normal cardiac physiology. Methodologies to elucidate these interactions in vivo have been limited. Recently, proximity-dependent biotinylation, with the use of BioID, TurboID, and ascorbate peroxidase, has been developed to uncover cellular neighborhoods and novel protein-protein interactions. These cutting-edge techniques have enabled the identification of subcellular localizations of specific proteins and the neighbors or interacting proteins within these subcellular regions. In contrast to classic methods such as affinity purification and subcellular fractionation, these techniques add covalently bound tags in living cells, such that spatial relationships and interaction networks are not disrupted. Recently, these methodologies have been used to identify novel protein-protein interactions relevant to the cardiovascular system. In this review, we discuss the development and current use of proximity biotin-labeling for cardiovascular research.

UNASSIGNED: The development of novel and efficient biomarkers for primary bone cancers is of grave importance.
UNASSIGNED: The expression pattern of osteopontin (OPN) was investigated in the 153 patients with benign (n = 72) and malignant (n = 81) primary bone cancers. Both local and circulating OPN mRNA expression levels and their protein concentration in serum and tumor site were assessed using real-time qRT-PCR, ELISA, and immunohistochemistry techniques, respectively. As a control, 29 healthy individuals were considered. The number of 153 tumor tissue specimens and the 153 paired margins were taken on surgical resection from the patients. 153 blood samples were also drained from all participants, then peripheral blood mononuclear cells (PBMC) and sera were separated.
UNASSIGNED: The mean mRNA expression was significantly higher in all of the cancerous tissues than the paired margins and the PBMC of the patients than the controls. Consistently, the protein concentrations of OPN in serum and tumor tissues were significantly higher in the patients. Furthermore, the malignant cases had significantly elevated the mRNA levels and the protein compared to the benign cases. OPN could potentially differentiate the patients from the controls with 100% sensitivity and specificity in serum. Moreover, OPN could predict some of the malignant cases\' clinicopathological features, including metastasis, recurrence, grade, and response to chemotherapy.
UNASSIGNED: In conclusion, OPN might be involved in the pathogenesis of primary bone tumors and can be considered as a potential biomarker to bone cancer diagnosis.