BMP4 in Human Endometrial Stromal Cells Can Affect Decidualization by Regulating FOXO1 Expression.

CONTEXT: Recurrent spontaneous abortion (RSA) is defined as the loss of 2 or more consecutive intrauterine pregnancies with the same sexual partner in the first trimester. Despite its significance, the etiology and underlying mechanisms of RSA remain elusive. Defective decidualization is proposed as one of the potential causes of RSA, with abnormal decidualization leading to disturbances in trophoblast invasion function. OBJECTIVE: To assess the role of bone morphogenetic protein 4 (BMP4) in decidualization and RSA. METHODS: Decidual samples were collected from both RSA patients and healthy controls to assess BMP4 expression. In vitro cell experiments utilized the hESC cell line to investigate the impact of BMP4 on decidualization and associated aging, as well as its role in the maternal-fetal interface communication. Subsequently, a spontaneous abortion mouse model was established to evaluate embryo resorption rates and BMP4 expression levels. RESULTS: Our study identified a significant downregulation of BMP4 expression in the decidua of RSA patients compared to the normal control group. In vitro, BMP4 knockdown resulted in inadequate decidualization and inhibited associated aging processes. Mechanistically, BMP4 was implicated in the regulation of FOXO1 expression, thereby influencing decidualization and aging. Furthermore, loss of BMP4 hindered trophoblast migration and invasion via FOXO1 modulation. Additionally, BMP4 downregulation was observed in RSA mice. CONCLUSION: Our findings highlighted the downregulation of BMP4 in both RSA patients and mice. BMP4 in human endometrial stromal cells was shown to modulate decidualization by regulating FOXO1 expression. Loss of BMP4 may contribute to the pathogenesis of RSA, suggesting potential avenues for abortion prevention strategies.

Activated hedgehog gene pattern correlates with dismal clinical outcome and tumor microenvironment heterogeneity in hepatocellular carcinoma.

Background: Activation of the Hedgehog signaling pathway is linked to the initiation and development of human hepatocellular carcinoma (HCC). However, its impact on clinical outcomes and the HCC microenvironment remains unclear. Methods: We performed comprehensive analyses of Hedgehog pathway genes in a large cohort of HCC patients. Specifically, we utilized univariate Cox regression analysis to identify Hedgehog genes linked to overall survival, and the LASSO algorithm was used to construct a Hedgehog-related gene pattern. We subsequently examined the correlation between the Hedgehog pattern and the HCC microenvironment employing the CIBERSORT and ssGSEA algorithms. Furthermore, Tumor Immune Dysfunction and Exclusion (TIDE) algorithm and the anti-PD-L1 treatment dataset (IMvigor210) are used to evaluate the clinical response of the Hedgehog pattern in predicting immune checkpoint inhibitors. Results: We found that the Hedgehog activation score (HHAS), a prognostic score based on 11 Hedgehog genes, was significantly associated with HCC patient survival. Patients exhibiting high HHAS experienced markedly reduced survival rates compared to those with low HHAS, and HHAS emerged as an independent prognostic factor for HCC. Functional enrichment analysis unveiled the association of the HHAS phenotype with functions related to the immune system, and further investigation demonstrated that HCC patients exhibiting low HHAS displayed elevated levels of anti-tumor immune activation in CD8+ T cells, while high HHAS were linked to immune escape phenotypes and increased infiltration of immune suppressive cells. In addition, in the Immune Checkpoint Inhibitor (ICI) cohort of IMvigor210, patients with higher HHAS had worse ICI treatment outcomes and shortened survival time, indicating that the HHAS is a useful indicator for predicting patient response to immunotherapy. Conclusions: In summary, our study offers valuable insights for advancing research on Hedgehog and its impact on tumor immunity, which provides an opportunity to optimize prognosis and immune therapy for HCC.

Does Topical Vancomycin Powder Use in Fracture Surgery Change Bacteriology and Antibiotic Susceptibilities? An Analysis of the VANCO Trial.

OBJECTIVE: To determine whether intrawound vancomycin changes the bacteriology of surgical site infection pathogens and investigate the emergence of antibiotic-resistant pathogens. DESIGN: Secondary analysis of phase III, prospective, randomized clinical trial. SETTING: Thirty-six US trauma centers. PATIENT SELECTION CRITERIA: Patients who became infected after fixation of tibial plateau or pilon fracture. OUTCOME MEASURES AND COMPARISONS: Pathogen types and bacterial susceptibilities as determined from routine clinical culture in the operating room. RESULTS: Seventy-four patients were studied who were 67.5% male with a mean age of 48.6 years. A lower proportion of gram-positive cocci was observed in the vancomycin powder compared with the standard-of-care group (3.7% vs. 8.0%, P = 0.01). Methicillin-resistant Staphylococcus aureus infection incidence was comparable in both the vancomycin powder and the standard-of-care groups, but rates of methicillin-susceptible S. aureus infections were lower in the treatment group (1.4% vs. 4.8%, P = 0.01). The incidence of coagulase-negative Staphylococci and gram-negative rod infections were similar in both groups. There was no significant difference in susceptibilities between groups in rates of vancomycin-resistant enterococcus. CONCLUSIONS: Topical vancomycin powder decreases the likelihood of gram-positive infections consistent with the biologic activity of vancomycin. Fewer methicillin-susceptible S. aureus and coagulase-negative Staphylococci infections were observed in the group treated with vancomycin powder. An effect of vancomycin powder on methicillin-resistant S. aureus infection risk was not detected given the low incidence in both the intrawound vancomycin and the standard-of-care groups. There was no emergence of gram-negative rod infections or increased resistance patterns observed. Use of topical vancomycin powder does not seem to produce infections in these patients with greater antibiotic resistance than would have occurred without its use. LEVEL OF EVIDENCE: Therapeutic Level II. See Instructions for Authors for a complete description of levels of evidence.

Urinary matrix metalloproteinase-7 is a sensitive biomarker to evaluate renal tubular injury in patients with minimal change disease and focal segmental glomerulosclerosis.

Objective: To explore the advantages of urinary matrix metalloproteinase-7 (MMP-7) in evaluating renal tubular injury in minimal change disease (MCD) and focal segmental glomerulosclerosis (FSGS) patients compared with urinary cystatin C (CysC) and retinol-binding protein (RBP). Methods: Serum and urine samples were collected from 20 healthy volunteers, and 40 MCD and 20 FSGS patients. Serum and urinary MMP-7 levels were measured by enzyme-linked immunosorbent assay. Urinary total protein, CysC and RBP levels were measured by automatic specific protein analyzer and compared with urinary creatinine level for calibration. The renal tissue serial sections were stained by MMP-7 immunohistochemistry and periodic acid-Schiff. Results: Under light microscopy, MMP-7 granular weak positive expression was showed sporadically in the cytoplasm of a few renal tubular epithelial cells without obvious morphological changes in MCD patients, and MMP-7-positive expression was observed in the cytoplasm of some renal tubular epithelial cells in FSGS patients. There was no significant difference in serum MMP-7 level among the three groups. Compared with the control group, the urinary MMP-7 level in MCD patients was higher, but urinary CysC and RBP levels were not increased significantly. Compared with the control group and MCD patients, urinary MMP-7, CysC and RBP levels in FSGS patients were upregulated significantly. Conclusions: Urinary MMP-7 could not only evaluate the mild renal tubular epithelial cells injury in MCD patients with massive proteinuria, but also evaluate the continuous renal tubular epithelial cells injury in FSGS patients.

Real-time monitoring of glucose metabolism and effects of metformin on HepG2 cells using 13C in-cell NMR spectroscopy.

Metformin is currently a strong candidate antitumor agent for multiple cancers, and has the potential to inhibit cancer cell viability, growth, and proliferation. Metabolic reprogramming is a critical feature of cancer cells. However, the effects of metformin which targets glucose metabolism on HepG2 cancer cells remain unclear. In this study, to explore the effects of metformin on glucose metabolism in HepG2 cells, we conducted real-time metabolomic monitoring of live HepG2 cells treated with metformin using 13C in-cell NMR spectroscopy. Metabolic tracing with U-13C6-glucose revealed that metformin significantly increased the production of 13C-G3P and 13C-glycerol, which were reported to attenuate liver cancer development, but decreased the production of potential oncogenesis-supportive metabolites, including 13C-lactate, 13C-alanine, 13C-glycine, and 13C-glutamate. Moreover, the expression levels of enzymes associated with the measured metabolites were carried out. The results showed that the levels of ALT1, MCT4, GPD2 and MPC1 were greatly reduced, which were consistent with the changes of measured metabolites in 13C in-cell NMR spectroscopy. Overall, our approach directly provides fundamental insights into the effects of metformin on glucose metabolism in live HepG2 cells, and highlights the potential mechanism of metformin, including the increase in production of G3P and glycerol derived from glucose, as well as the inhibition of glucose incorporation into lactate, alanine, glutamate, and glycine.

Characteristics of innate and adaptive immune disorder in IgA nephropathy based on integrated bioinformatics.

BACKGROUND: Our study aims to investigate the immunological pathogenesis underlying immunoglobulin A nephropathy (IgAN) and explore potential biomarkers for IgAN diagnosis. MATERIALS AND METHODS: Differentially expressed genes (DEGs) of formalin-fixed and paraffin-embedded (FFPE) samples were screened between IgAN patients and healthy people based on GSE115857. Gene oncology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Set Enrichment Analysis (GSEA) enrichment was performed to identify related biological processes and pathways. CIBERSORT was utilized to seek the relationship of immune cell infiltration with IgAN. Finally, the expression of paraoxonase 2 (PON2) related to innate immune response was verified in FFPE samples of minimal change disease and IgAN patients by immunohistochemistry and PAS staining. RESULTS: 25 down-regulated genes and 12 up-regulated genes were identified in IgAN patients, which mainly responded to endothelial cell proliferation, inflammatory response, and angiogenesis. Toll-like receptor signaling pathway and Epstein-Barr virus (EBV) infection might be involved in IgAN pathogenesis. In addition, the infiltration of macrophages M0, naïve B cells, and follicular helper T (Tfh) cells was positively correlated in IgAN patients. Macrophages M1 and M2 infiltration were up-regulated in IgAN patients, which indicated that innate immune response was closely associated with IgAN. Besides, the results of immunohistochemistry showed that PON2 was obviously positively expressed in acute and chronic lesions of IgAN patients. CONCLUSION: In addition to abnormalities in the adaptive immune response, macrophages M1/M2 and innate immune disorder may participate in IgAN pathogenesis. PON2 may become the feasible targets for further investigation of IgAN.

Integrated metabolomics and transcriptomics analyses reveal metabolic responses to TGEV infection in porcine intestinal epithelial cells.

Transmissible gastroenteritis virus (TGEV) is a coronavirus that infects piglets with severe diarrhoea, vomiting, dehydration, and even death, causing huge economic losses to the pig industry. The underlying pathogenesis of TGEV infection and the effects of TGEV infection on host metabolites remain poorly understood. To investigate the critical metabolites and regulatory factors during TGEV infection in intestinal porcine epithelial cells (IPEC-J2), we performed metabolomic and transcriptomic analyses of TGEV-infected IPEC-J2 cells by LC/MS and RNA-seq techniques. A total of 87 differential metabolites and 489 differentially expressed genes were detected. A series of metabolites and candidate genes from glutathione metabolism and AMPK signalling pathway were examined through combined analysis of metabolome and transcriptome. We found glutathione peroxidase 3 (GPX3) is markedly reduced after TGEV infection, and a significant negative correlation between AMPK signalling pathway and TGEV infection. Exogenous addition of the AMPK activator COH-SR4 significantly downregulates stearoyl coenzyme A (SCD1) mRNA and inhibits TGEV replication; while exogenous GSK-690693 significantly promotes TGEV infection by inhibiting AMPK signalling pathway. In summary, our study provides insights into the key metabolites and regulators for TGEV infection from the metabolome and transcriptome perspective, which will offer promising antiviral metabolic and molecular targets and enrich the understanding of the existence of a similar mechanism in the host.

Cascade catalysis-coordinated nanorobots toward synergistic cancer chemoimmunotherapy.

Cancer has always been the biggest threat to human health, but the effect of traditional treatments such as surgery, chemotherapy, and radiotherapy is not satisfactory. Currently, nanomedicine-based chemoimmunotherapy can improve clinical results through unique synergistic effects. However, it is mainly enriched at tumor sites based on EPR effects, without an active delivery strategy and relatively low tumor targeting distribution. Therefore, nanorobots (Cu@MPS-GOD) with magnetic responsiveness and enzyme-like activity were prepared, which can enrich and move to the tumor site under the action of a 3D magnetic field, and cause tumor cell immunogenic death by cascade catalytic Fenton reactions. Meanwhile, Cu@MPS-GOD can also activate immune cells or induce cancer cells to expose surface antigens, trigger systemic anti-cancer immunity, and have a good inhibitory effect on a breast tumor model in mice with an inhibition rate of 59.3%. This work provides an attractive strategy to expand the therapeutic effect of cancer when chemical dynamic therapy is combined with immunotherapy, which has a potential clinical application prospect.

CD38-Specific CAR Integrated into CD38 Locus Driven by Different Promoters Causes Distinct Antitumor Activities of T and NK Cells.

The robust and stable expression of CD38 in T-cell acute lymphoblastic leukemia (T-ALL) blasts makes CD38 chimeric antigen receptor (CAR)-T/natural killer (NK) a potential therapy for T-ALL. However, CD38 expression in normal T/NK cells causes fratricide of CD38 CAR-T/NK cells. Here a "2-in-1" gene editing strategy is developed to generate fratricide-resistant locus-specific CAR-T/NK cells. CD38-specific CAR is integrated into the disrupted CD38 locus by CRISPR/Cas9, and CAR is placed under the control of either endogenous CD38 promoter (CD38KO/KI ) or exogenous EF1α promoter (CD38KO/KI EF1α). CD38 knockout reduces fratricide and allows the expansion of CAR-T cells. Meanwhile, CD38KO/KI EF1α results in higher CAR expression than CD38KO/KI in both CAR-T and CAR-NK cells. In a mouse T-ALL model, CD38KO/KI EF1α CAR-T cells eradicate tumors better than CD38KO/KI CAR-T cells. Surprisingly, CD38KO/KI CAR-NK cells show superior tumor control than CD38KO/KI EF1α CAR-NK cells. Further investigation reveals that endogenous regulatory elements in NK cells lead to higher expression of CD38 CAR than in T cells, and the expression levels of CAR affect the therapeutic outcome of CAR-T and CAR-NK cells differently. Therefore, these results support the efficacy of CD38 CAR-T/NK against T-ALL and demonstrate that the "2-in-1" strategy can resolve fratricide and enhance tumor eradication, paving the way for clinical translation.

Inserting EF1α-driven CD7-specific CAR at CD7 locus reduces fratricide and enhances tumor rejection.

CAR-T therapies to treat T-cell malignancies face unique hurdles. Normal and malignant T cells usually express the same target for CAR, leading to fratricide. CAR-T cells targeting CD7, which is expressed in various malignant T cells, have limited expansion due to fratricide. Using CRISPR/Cas9 to knockout CD7 can reduce the fratricide. Here we developed a 2-in-1 strategy to insert EF1α-driven CD7-specific CAR at the disrupted CD7 locus and compared it to two other known strategies: one was random integration of CAR by a retrovirus and the other was site-specific integration at T-cell receptor alpha constant (TRAC) locus, both in the context of CD7 disruption. All three types of CD7 CAR-T cells with reduced fratricide could expand well and displayed potent cytotoxicity to both CD7+ tumor cell lines and patient-derived primary tumors. Moreover, EF1α-driven CAR expressed at the CD7 locus enhances tumor rejection in a mouse xenograft model of T-cell acute lymphoblastic leukemia (T-ALL), suggesting great clinical application potential. Additionally, this 2-in-1 strategy was adopted to generate CD7-specific CAR-NK cells as NK also expresses CD7, which would prevent contamination from malignant cells. Thus, our synchronized antigen-knockout CAR-knockin strategy could reduce the fratricide and enhance anti-tumor activity, advancing clinical CAR-T treatment of T-cell malignancies.

Network pharmacology-based analysis of Resinacein S against non-alcoholic fatty liver disease by modulating lipid metabolism.

Background: Ganoderma lucidum is reportedly the best source of traditional natural bioactive constituents. Ganoderma triterpenoids (GTs) have been verified as an alternative adjuvant for treating leukemia, cancer, hepatitis and diabetes. One of the major triterpenoids, Resinacein S, has been found to regulate lipid metabolism and mitochondrial biogenesis. Nonalcoholic fatty liver disease (NAFLD) is a common chronic liver disease that has become a major public health problem. Given the regulatory effects on lipid metabolism of Resinacein S, we sought to explore potential protective effects against NAFLD. Methods: Resinacein S was extracted and isolated from G. lucidum. And mice were fed with high fat diet with or without Resinacein S to detect hepatic steatosis. According to Network Pharmacology and RNA-seq, we analyzed the hub genes of Resinacein S against NAFLD disease. Results: Our results can be summarized as follows: (1) The structure of Resinacein S was elucidated using NMR and MS methods. (2) Resinacein S treatment could significantly attenuate high-fat diet (HFD)-induced hepatic steatosis and hepatic lipid accumulation in mouse. (3) GO terms, KEGG pathways and the PPI network of Resinacein S induced Differentially Expressed Genes (DEGs) demonstrated the key target genes of Resinacein S against NAFLD. (4) The hub proteins in PPI network analysis could be used for NAFLD diagnosis and treatment as drug targets. Conclusion: Resinacein S can significantly change the lipid metabolism in liver cells and yield a protective effect against steatosis and liver injury. Intersected proteins between NAFLD related genes and Resinacein S-induced DEGs, especially the hub protein in PPI network analysis, can be used to characterize targets of Resinacein S against NAFLD.

Novel therapeutic perspectives for crescentic glomerulonephritis through targeting parietal epithelial cell activation and proliferation.

INTRODUCTION: Kidney injury is clinically classified as crescentic glomerulonephritis (CrGN) when ≥50% of the glomeruli in a biopsy sample contain crescentic lesions. However, current strategies, such as systemic immunosuppressive therapy and plasmapheresis for CrGN, are partially effective, and these drugs have considerable systemic side effects. Hence, targeted therapy to prevent glomerular crescent formation and expansion remains an unmet clinical need. AREAS COVERED: Hyperproliferative parietal epithelial cells (PECs) are the main constituent cells of the glomerular crescent with cell-tracing evidence. Crescents obstruct the flow of primary urine, pressure the capillaries, and degenerate the affected nephrons. We reviewed the markers of PEC activation and proliferation, potential therapeutic effects of thrombin and thrombin receptor inhibitors, and how podocytes cross-talk with PECs. These experiments may help identify potential early specific targets for the prevention and treatment of glomerular crescentic injury. EXPERT OPINION: Inhibiting PEC activation and proliferation in CrGN can alleviate glomerular crescent progression, which has been supported by preclinical studies with evidence of genetic deletion. Clarifying the outcome of PEC transformation to the podocyte phenotype and suppressing thrombin, thrombin receptors, and PEC hyperproliferation in early therapeutic strategies will be the research goals in the next ten years.

It is clinically classified as crescentic glomerulonephritis (CrGN) when more than 50% of the glomeruli of the kidney in a biopsy sample contain crescentic lesions (crescent shaped injuries). However, current strategies, such as immunosuppressive therapy and plasmapheresis (the removal, treatment and returning of blood) for CrGN, are partially effective, and these drugs have considerable side effects. In order to seek targeted therapy for CrGN, we reviewed the current research evidences. First, the hyperproliferative parietal epithelial cells (PECs) are the main cells within the glomerular crescent seen with cell-tracing evidence. The activated PECs can express specific markers and altered biological characteristics, such as cell growth and multiplication, migration, and extracellular matrix production. CD44, CD74, CD9, and pERK-1/2 are specific markers for PEC activation, and also as the potential therapeutic targets with evidence of gene knockout and inhibitor. Second, during the formation of glomerular crescents, PECs grow and multiply also through cross-talking with podocyte cells by the AngII/SDF-1/CXCR4/ERK1/2, HB-EGF/EGFR/JAK/STAT3, and PDGF/PDGFR signaling pathways, suggesting that the intervention of key molecules in these disease processes may be promising therapeutic targets for CrGN. Third, thrombin and protease-activated receptors (PARs) participate in the excessive proliferation of PEC through activation of the coagulation cascade reaction, PAR-1 and PAR-2. Therefore, anticoagulation therapy, especially inhibition of PAR-1 and PAR-2, is expected to be an effective strategy for the early prevention and treatment of CrGN. The drug vorapaxar selectively antagonizes PAR-1 and is the most promising candidate. These findings will not only improve the outlook for CrGN treatment, but will also help in the treatment of other glomerular diseases with crescentic lesions. [Figure: see text].

Bioorthogonal surface-enhanced Raman scattering flower-like nanoprobe with embedded standards for accurate cancer cell imaging.

Developing precise and effective strategies for cancer identification and imaging is attractive due to their importance for early cancer detection, prognosis, and subsequent treatment. Herein, we reported a novel bioorthogonal surface-enhanced Raman scattering (SERS) nanoprobe for accurate cancer cell imaging. A novel core-molecule-shell nanoflower (Au@4-MBN@Au) with rich electromagnetic hot spots and enhanced Raman scattering was first synthesized by optimizing the embedded concentrations of 4-mercaptobenzonitrile (4-MBN). Then, Au@4-MBN@Au was further modified with FA-PEG-SH molecules to acquire the bioorthogonal SERS nanoprobe Au@4-MBN@Au-PEG-FA. The SERS nanoprobe illustrated a robust and stable nitrile stretching vibration Raman signal (2223 cm-1) in the cellular silent region, ensuring high sensitivity and ultra-accuracy SERS imaging of cancer cells. Furthermore, cell imaging results demonstrated Au@4-MBN@Au-PEG-FA could recognize FR-positive HeLa cells with high selectivity due to the high affinity between folate receptor and folic acid. More notably, Au@4-MBN@Au-PEG-FA has been applied to identify FR-positive Hela cells from co-cultured cancer cells with similar morphology by SERS imaging for the first time. With improved signal-to-background ratio, high selectivity, and excellent stability, we anticipate the SERS nanoprobe Au@4-MBN@Au-PEG-FA could be applied for FR-related cancer theranostics and clinical detection in the future.

Balancing activation and co-stimulation of CAR tunes signaling dynamics and enhances therapeutic potency.

CD19-targeting chimeric antigen receptors (CARs) with CD28 and CD3ζ signaling domains have been approved by the US FDA for treating B cell malignancies. Mutation of immunoreceptor tyrosine-based activation motifs (ITAMs) in CD3ζ generated a single-ITAM containing 1XX CAR, which displayed superior antitumor activity in a leukemia mouse model. Here, we investigated whether the 1XX design could enhance therapeutic potency against solid tumors. We constructed both CD19- and AXL-specific 1XX CARs and compared their in vitro and in vivo functions with their wild-type (WT) counterparts. 1XX CARs showed better antitumor efficacy in both pancreatic and melanoma mouse models. Detailed analysis revealed that 1XX CAR-T cells persisted longer in vivo and had a higher percentage of central memory cells. With fluorescence resonance energy transfer (FRET)-based biosensors, we found that decreased ITAM numbers in 1XX resulted in similar 70-kDa zeta chain-associated protein (ZAP70) activation, while 1XX induced higher Ca2+ elevation and faster extracellular signal-regulated kinase (Erk) activation than WT CAR. Thus, our results confirmed the superiority of 1XX against two targets in different solid tumor models and shed light on the underlying molecular mechanism of CAR signaling, paving the way for the clinical applications of 1XX CARs against solid tumors.

TNF-α/anti-TNF-α drugs and its effect on pregnancy outcomes.

Pregnancy is a complex biological process. The establishment and maintenance of foetal-maternal interface are pivotal events. Decidual immune cells and inflammatory cytokines play indispensable roles in the foetal-maternal interface. The disfunction of decidual immune cells leads to adverse pregnancy outcome. Tumour necrosis factor (TNF)-α, a common inflammatory cytokine, has critical roles in different stages of normal pregnancy process. However, the relationship between the disorder of TNF-α and adverse pregnancy outcomes, including preeclampsia (PE), intrauterine growth restriction (IUGR), spontaneous abortion (SA), preterm birth and so on, is still indefinite. In this review, we thoroughly reviewed the effect of TNF-α disorder on pathological conditions. Moreover, we summarized the reports about the adverse pregnancy outcomes (PE, IUGR, SA and preterm birth) of using anti-TNF-α drugs (infliximab, etanercept and adalimumab, certolizumab and golimumab) currently in the clinical studies. Overall, IUGR, SA and preterm birth are the most common adverse pregnancy outcomes of anti-TNF-α drugs. Our review may provide insight for the immunological treatment of pregnancy-related complication, and help practitioners make informed decisions based on the current evidences.

Annexin A2: The diversity of pathological effects in tumorigenesis and immune response.

Annexin A2 (ANXA2) is widely used as a marker in a variety of tumors. By regulating multiple signal pathways, ANXA2 promotes the epithelial-mesenchymal transition, which can cause tumorigenesis and accelerate thymus degeneration. The elevated ANXA2 heterotetramer facilitates the production of plasmin, which participates in pathophysiologic processes such as tumor cell invasion and metastasis, bleeding diseases, angiogenesis, inducing the expression of inflammatory factors. In addition, the ANXA2 on the cell membrane mediates immune response via its interaction with surface proteins of pathogens, C1q, toll-like receptor 2, anti-dsDNA antibodies and immunoglobulins. Nuclear ANXA2 plays a role as part of a primer recognition protein complex that enhances DNA synthesis and cells proliferation by acting on the G1-S phase of the cell. ANXA2 reduction leads to the inhibition of invasion and metastasis in multiple tumor cells, bleeding complications in acute promyelocytic leukemia, retinal angiogenesis, autoimmunity response and tumor drug resistance. In this review, we provide an update on the pathological effects of ANXA2 in both tumorigenesis and the immune response. We highlight ANXA2 as a critical protein in numerous malignancies and the immune host response.

Signaling Dynamics of TSHR-Specific CAR-T Cells Revealed by FRET-Based Biosensors.

Although most patients with thyroid cancers have good prognosis and long-term survival, some patients are refractory to traditional therapeutic approaches and face a high risk of mortality. CAR-T therapy provides an attractive strategy to treat these patients. Considering the limited expression in thyroid tissues, thyroid-stimulating hormone receptor (TSHR) has been considered as a promising candidate as CAR-T target. However, it is still a challenge to find the optimal CAR design for the treatment of thyroid cancers. Dynamic signaling cascade is initiated by CAR molecules during CAR-T cell activation. The development of FRET-based biosensors enables us to detect the signaling dynamics of key kinases during CAR-T cell activation with high spatiotemporal resolution. Here using the ZAP70 and ERK biosensors, we visualized the dynamics of ZAP70 and ERK activities in TSHR-specific CAR-T cells upon antigen stimulation. We first constructed several TSHR-targeting CARs for the treatment of advanced thyroid cancers. The TSHR CAR-T cells with CD28 or 4-1BB co-stimulatory signaling domains exhibited potent cytotoxicity in vitro. By FRET imaging, we observed rapid increase of ZAP70 and ERK activities in TSHR CAR-T cells upon target cell binding. Even though CD28-based CAR-T cells had similar ZAP70 activation dynamics as 4-1BB-based CAR-T cells, they displayed slightly enhanced ERK activation, which may contribute to their faster anti-tumor kinetics in vivo. These results demonstrated the efficacy of TSHR CAR-T cells to treat advanced thyroid cancers. Our study indicated the potential of applying FRET biosensors to optimize the design of CAR for effective CAR-T therapy.

Are deep infections that present before and after 90 days from orthopaedic trauma different? An analysis of the validity of the recent change in CDC criteria for infections.

BACKGROUND: In 2016, the Centers for Disease Control and Prevention (CDC) changed the time frame for their definition of deep surgical site infection (SSI) from within 1 year to within 90 days of surgery. We hypothesized that a substantial number of infections in patients who have undergone fracture fixation present beyond 90 days and that there are patient or injury factors that can predict who is more likely to present with SSI after 90 days. METHODS: A retrospective review yielded 452 deep SSI after fracture fixation. These patients were divided into two groups-those infected within 90 days of surgery and those infected beyond 90 days . Data were collected on risk factors for infection. Univariate and multiple logistic regression analyses were performed to compare the two groups. A randomly selected control group was used to build infection prediction models for both outcomes. The two outcomes were then modelled against each other to determine whether differences in predictors for early versus late infection exist. RESULTS: Of the 452 infections, 144 occurred beyond 90 days (32% [95% CI, 28%-36%]). No statistically significant patient factors were found in multivariable analysis between the early and late infection groups. The need for flap coverage was the only injury characteristic that differed significantly between groups, with patients in the late infection group more likely to have needed a flap. When modelled against the control group and directly comparing the two models, predictors for early infection include male sex and fractures of the pelvis, acetabulum, or hip, whereas predictors of late infection include hepatitis C and/or human immunodeficiency virus (HIV) and admission to the intensive care unit (ICU). CONCLUSION: Use of the recent CDC definition will underestimate the rate of actual postoperative infections when applied to orthopaedic trauma patients. Hepatitis C and/or HIV and ICU admission are predictors of late infection, whereas male sex and pelvis, acetabulum, or hip fractures are predictors of early infection. Patients who receive flap coverage may be more likely to present with late infection.

Cas9-Cleavage Sequences in Size-Reduced Plasmids Enhance Nonviral Genome Targeting of CARs in Primary Human T Cells.

T cell genome editing holds great promise to advance a range of immunotherapies but is encumbered by the dependence on difficult-to-produce and expensive viral vectors. Here, small double-stranded plasmid DNA modified to mediate high-efficiency homologous recombination is designed. The resulting chimeric antigen receptor (CAR)-T cells display a similar phenotype, transcriptional profile, and in vivo potency to CAR-T cells generated using adeno-associated viral vector. This method should simplify and accelerate the use of precision engineering to produce edited T cells for research and clinical purposes.

Tuning the ignition of CAR: optimizing the affinity of scFv to improve CAR-T therapy.

How single-chain variable fragments (scFvs) affect the functions of chimeric antigen receptors (CARs) has not been well studied. Here, the components of CAR with an emphasis on scFv were described, and then several methods to measure scFv affinity were discussed. Next, scFv optimization studies for CD19, CD38, HER2, GD2 or EGFR were overviewed, showing that tuning the affinity of scFv could alleviate the on-target/off-tumor toxicity. The affinities of scFvs for different antigens were also summarized to designate a relatively optimal working range for CAR design. Last, a synthetic biology approach utilizing a low-affinity synthetic Notch (synNotch) receptor to achieve ultrasensitivity of antigen-density discrimination and murine models to assay the on-target/off-tumor toxicity of CARs were highlighted. Thus, this review provides preliminary guidelines of choosing the right scFvs for CARs.