Microbial cells serve as efficient and environmentally friendly biocatalysts, but their stability and reusability in practical applications must often be improved through immobilization. Acinetobacter sp. Tol 5 shows high adhesiveness to materials due to its large cell surface protein AtaA, which consists of 3630 amino acids (aa). Previously, we developed a method for immobilizing bacteria using AtaA. Herein, we investigated the cell immobilization ability of in-frame deletion (IFD) mutants of AtaA with different sizes in Tol 5. Mini-AtaA, which consists of 775 aa and is functional in Escherichia coli, was produced and present on the cell surface; however, mini-AtaA showed no immobilization ability in Tol 5. A cell immobilization assay was performed with cells expressing 16 IFD mutants of AtaA with different sizes, revealing that a length of at least 1417 aa was required for the sufficient immobilization of Tol 5 cells; thus, the minimum length needed to achieve the adhesive function of AtaA varies among bacterial species. The constructed mutant library of AtaA ranging from 3630 to 775 aa will allow researchers to quickly and easily explore the optimal size of AtaA, even for bacteria newly introduced to AtaA.

OBJECTIVE: Tumor response to radiation is thought to depend on the direct killing of tumor cells. Our laboratory has called this into question. Firstly, we showed that the biology of the host, specifically the endothelial expression of acid sphingomyelinase (ASMase), was critical in determining tumor radiocurability. Secondly, we have shown that the immune system can enhance radiation response by allowing a complete tumor control in hemi-irradiated tumors. In this paper, we focus on the integration of these two findings.
METHODS: We used Lewis Lung Carcinoma (LLC) cells, injected in the flank of either: (i) ASMase knockout or (ii) WT of matched background (sv129xBl/6) or (iii) C57Bl/6 mice. Radiation therapy (RT) was delivered to 50% or 100% of the LLC tumor volume. Tumor response, immune infiltration (CD8+ T cells), ICAM-1, and STING activation were measured. Radiotherapy was also combined with methyl-cyclodextrin, to inhibit the ASMase-mediated formation of ceramide-enriched lipid rafts.
RESULTS: We recapitulated our previous finding, namely that tumor hemi-irradiation was sufficient for tumor control in the LLC/C57Bl/6 model. However, in ASMase KO mice hemi-irradiation was ineffective. Likewise, pharmacological inhibition of ASMase significantly reduced the tumor response to hemi-irradiation. Further, we demonstrated elevated ICAM-1 expression, increased levels of CD8+ T cells, ICAM-1, and STING activation in tumors growing in C57Bl/6 mice, as well as the ASMase WT strain. However, no such changes were seen in tumors growing in ASMase KO mice.
CONCLUSIONS: ASMase and ceramide generation are necessary to mediate a radiation-induced anti-tumor immune response via STING activation.

This study investigated the mechanism by which NR4A1 regulates mitochondrial fission factor (Mff)-related mitochondrial fission and FUN14 domain 1 (FUNDC1)-mediated mitophagy following cardiac ischemia-reperfusion injury(I/R). Our findings showed that the damage regulation was positively correlated with the pathological fission and pan-apoptosis of myocardial cell mitochondria. Compared with wild-type mice (WT), NR4A1-knockout mice exhibited resistance to myocardial ischemia-reperfusion injury and mitochondrial pathological fission, characterized by mitophagy activation. Results showed that ischemia-reperfusion injury increased NR4A1 expression level, activating mitochondrial fission mediated by Mff and restoring the mitophagy phenotype mediated by FUNDC1. The inactivation of FUNDC1 phosphorylation could not mediate the normalization of mitophagy in a timely manner, leading to an excessive stress response of unfolded mitochondrial proteins and an imbalance in mitochondrial homeostasis. This process disrupted the normalization of the mitochondrial quality control network, leading to accumulation of damaged mitochondria and the activation of pan-apoptotic programs. Our data indicate that NR4A1 is a novel and critical target in myocardial I/R injury that exertsand negative regulatory effects by activating Mff-mediated mito-fission and inhibiting FUNDC1-mediated mitophagy. Targeting the crosstalk balance between NR4A1-Mff-FUNDC1 is a potential approach for treating I/R.

Mitophagy selectively eliminates damaged or dysfunctional mitochondria, playing a crucial role in maintaining mitochondrial quality control. However, it remains unclear whether mitophagy can be fully activated and how it evolves after SCI. Our RNA-seq analysis of animal samples from sham and 1, 3, 5, and 7 days post-SCI indicated that mitophagy was indeed inhibited during the acute and subacute early stages. In vitro experiments showed that this inhibition was closely related to excessive production of reactive oxygen species (ROS) and the downregulation of BNIP3. Excessive ROS led to the blockage of mitophagy flux, accompanied by further mitochondrial dysfunction and increased neuronal apoptosis. Fortunately, ligustilide (LIG) was found to have the ability to reverse the oxidative stress-induced downregulation of BNIP3 and enhance mitophagy through BNIP3-LC3 interaction, alleviating mitochondrial dysfunction and ultimately reducing neuronal apoptosis. Further animal experiments demonstrated that LIG alleviated oxidative stress and mitophagy inhibition, rescued neuronal apoptosis, and promoted tissue repair, ultimately leading to improved motor function. In summary, this study elucidated the state of mitophagy inhibition following SCI and its potential mechanisms, and confirmed the effects of LIG-enhanced mitophagy through BNIP3-LC3, providing new therapeutic targets and strategies for repairing SCI.

Membrane protein-mediated resistance is a multidisciplinary challenge that spans fields such as medicine, agriculture, and environmental science. Understanding its complexity and devising innovative strategies are crucial for treating diseases like cancer and managing resistant pests in agriculture. This paper explores the dual nature of resistance mechanisms across different organisms: On one hand, animals, bacteria, fungi, plants, and insects exhibit convergent evolution, leading to the development of similar resistance mechanisms. On the other hand, influenced by diverse environmental pressures and structural differences among organisms, they also demonstrate divergent resistance characteristics. Membrane protein-mediated resistance mechanisms are prevalent across animals, bacteria, fungi, plants, and insects, reflecting their shared survival strategies evolved through convergent evolution to address similar survival challenges. However, variations in ecological environments and biological characteristics result in differing responses to resistance. Therefore, examining these differences not only enhances our understanding of adaptive resistance mechanisms but also provides crucial theoretical support and insights for addressing drug resistance and advancing pharmaceutical development.

Mismatch repair deficient (dMMR)/microsatellite instability-high (MSI-H) gastric cancer (GC) exhibits an immune-active tumor microenvironment (TME) compared to MMR proficient (pMMR)/microsatellite stable/Epstein-Barr virus-negative [EBV (-)] GC. The tumor cell-intrinsic cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway has been considered a key regulator of immune cell activation in the TME. However, its significance in regulating the immune-active TME in dMMR/MSI-H GC remains unclear. Here, we demonstrated that tumor cell-intrinsic cGAS-STING was highly expressed in dMMR GC compared to pMMR/EBV (-) GC. The expression of tumor cell-intrinsic STING was significantly and positively associated with the number of CD8+ tumor-infiltrating lymphocytes in GC. Analysis of TCGA datasets revealed that the expression of interferon-stimulated genes and STING downstream T-cell attracting chemokines was significantly higher in MSI-H GC compared to other subtypes of GC with EBV (-). These results suggest that tumor cell-intrinsic STING signaling plays a key role in activating immune cells in the dMMR/MSI-H GC TME and might serve as a novel biomarker predicting the efficacy of immunotherapy for GC treatment.

Migration of endothelial and many other cells requires spatiotemporal regulation of protrusive and contractile cytoskeletal rearrangements that drive local cell shape changes. Unexpectedly, the small GTPase Rho, a crucial regulator of cell movement, has been reported to be active in both local cell protrusions and retractions, raising the question of how Rho activity can coordinate cell migration. Here, we show that Rho activity is absent in local protrusions and active during retractions. During retractions, Rho rapidly activated ezrin-radixin-moesin proteins (ERMs) to increase actin-membrane attachment, and, with a delay, nonmuscle myosin 2 (NM2). Rho activity was excitable, with NM2 acting as a slow negative feedback regulator. Strikingly, inhibition of SLK/LOK kinases, through which Rho activates ERMs, caused elongated cell morphologies, impaired Rho-induced cell contractions, and reverted Rho-induced blebbing. Together, our study demonstrates that Rho activity drives retractions by sequentially enhancing ERM-mediated actin-membrane attachment for force transmission and NM2-dependent contractility.

BACKGROUND: Most primary cutaneous melanomas have pathogenesis driven by ultraviolet exposure and genetic mutations, whereas acral lentiginous melanoma (ALM) and metastatic melanoma are much less, if at all, linked with the former. Thus, we evaluated both ultraviolet related and non-ultraviolet related melanomas. Mutations in the MUC16 and TTN genes commonly occur concurrently in these melanoma patients, but their combined prognostic significance stratified by gender and cancer subtype remains unclear.
METHODS: The cBioPortal database was queried for melanoma studies and returned 16 independent studies. Data from 2447 melanoma patients were utilized including those with ALM, cutaneous melanoma (CM), and melanoma of unknown primary (MUP). Patients were grouped based on the presence or absence of MUC16 and TTN mutations. Univariate Cox regression and Student\'s t-tests were used to analyze hazard ratios and total mutation count comparisons, respectively.
RESULTS: TTN mutations, either alone or concurrently with MUC16 mutations, significantly correlated with worse prognosis overall, in both genders, and in CM patients. ALM patients with both mutations had better prognoses than CM patients, while ALM patients with neither mutation had worse prognosis than CM patients. For MUP patients, only MUC16 mutations correlated with worse prognosis. ALM patients with neither MUC16 nor TTN mutations had significantly more total mutations than MUP patients, followed by CM patients.
CONCLUSIONS: TTN mutations are a potential marker of poor prognosis in melanoma, which is amplified in the presence of concurrent MUC16 mutations. ALM patients with neither gene mutations had worse prognosis, suggesting a protective effect of having both MUC16 and TTN mutations. Only MUC16 mutations conferred a worse prognosis for MUP patients. Comprehensive genetic profiling in melanoma patients may facilitate personalized treatment strategies to optimize patient outcomes.

Background: Identification of the unknown pathogenic factor driving atherosclerosis not only enhances the development of disease biomarkers but also facilitates the discovery of new therapeutic targets, thus contributing to the improved management of coronary artery disease (CAD). We aimed to identify causative protein biomarkers in CAD etiology based on proteomics and 2-sample Mendelian randomization (MR) design. Methods: Serum samples from 33 first-onset CAD patients and 31 non-CAD controls were collected and detected using protein array. Differentially expressed analyses were used to identify candidate proteins for causal inference. We used 2-sample MR to detect the causal associations between the candidate proteins and CAD. Network MR was performed to explore whether metabolic risk factors for CAD mediated the risk of identified protein. Vascular expression of candidate protein in situ was also detected. Results: Among the differentially expressed proteins identified utilizing proteomics, we found that circulating Golgi protein 73 (GP73) was causally associated with incident CAD and other atherosclerotic events sharing similar etiology. Network MR approach showed low-density lipoprotein cholesterol and glycated hemoglobin serve as mediators in the causal pathway, transmitting 42.1% and 8.7% effects from GP73 to CAD, respectively. Apart from the circulating form of GP73, both mouse model and human specimens imply that vascular GP73 expression was also upregulated in atherosclerotic lesions and concomitant with markers of macrophage and phenotypic switching of vascular smooth muscle cells (VSMCs). Conclusions: Our study supported GP73 as a biomarker and causative for CAD. GP73 may involve in CAD pathogenesis mainly via dyslipidemia and hyperglycemia, which may enrich the etiological information and suggest future research direction on CAD.

Background: Cancer-associated fibroblasts (CAFs) are the key components of the immune barrier in liver cancer. Therefore, gaining a deeper understanding of the heterogeneity and intercellular communication of CAFs holds utmost importance in boosting immunotherapy effectiveness and improving clinical outcomes. Methods: A comprehensive analysis by combing single-cell, bulk, and spatial transcriptome profiling with multiplexed immunofluorescence was conducted to unravel the complexities of CAFs in liver cancer. Results: Through an integrated approach involving 235 liver cancer scRNA-seq samples encompassing over 1.2 million cells, we found that CAFs were particularly increased in hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (ICC). FAP + fibroblasts were identified as the dominant subtype of CAFs, and which were mainly involved in extracellular matrix organization and angiogenesis. These CAFs were enriched in the tumor boundary of HCC, but diffusely scattered within ICC. The DAB2 + and SPP1 + tumor-associated macrophages (TAMs) reinforce the function of FAP + CAFs through signals such as TGF-β, PDGF, and ADM. Notably, the interaction between DAB2 + TAMs and FAP + CAFs promoted the formation of immune barrier and correlated with poorer patient survival, non-response to immunotherapy in HCC. High FAP and DAB2 immunohistochemical scores predicted shorter survival and higher serum AFP concentration in a local clinical cohort of 90 HCC patients. Furthermore, this communication pattern might be applicable to other solid malignancies as well. Conclusions: The interaction between DAB2 + TAMs and FAP + CAFs appears crucial in shaping the immune barrier. Strategies aimed at disrupting this communication or inhibiting the functions of FAP + CAFs could potentially enhance immunotherapy effectiveness and improve clinical outcomes.