CT whole lung radiomic nomogram: a potential biomarker for lung function evaluation and identification of COPD.

BACKGROUND: Computed tomography (CT) plays a great role in characterizing and quantifying changes in lung structure and function of chronic obstructive pulmonary disease (COPD). This study aimed to explore the performance of CT-based whole lung radiomic in discriminating COPD patients and non-COPD patients. METHODS: This retrospective study was performed on 2785 patients who underwent pulmonary function examination in 5 hospitals and were divided into non-COPD group and COPD group. The radiomic features of the whole lung volume were extracted. Least absolute shrinkage and selection operator (LASSO) logistic regression was applied for feature selection and radiomic signature construction. A radiomic nomogram was established by combining the radiomic score and clinical factors. Receiver operating characteristic (ROC) curve analysis and decision curve analysis (DCA) were used to evaluate the predictive performance of the radiomic nomogram in the training, internal validation, and independent external validation cohorts. RESULTS: Eighteen radiomic features were collected from the whole lung volume to construct a radiomic model. The area under the curve (AUC) of the radiomic model in the training, internal, and independent external validation cohorts were 0.888 [95% confidence interval (CI) 0.869-0.906], 0.874 (95%CI 0.844-0.904) and 0.846 (95%CI 0.822-0.870), respectively. All were higher than the clinical model (AUC were 0.732, 0.714, and 0.777, respectively, P < 0.001). DCA demonstrated that the nomogram constructed by combining radiomic score, age, sex, height, and smoking status was superior to the clinical factor model. CONCLUSIONS: The intuitive nomogram constructed by CT-based whole-lung radiomic has shown good performance and high accuracy in identifying COPD in this multicenter study.

Predicting prostate cancer in men with PSA levels of 4-10 ng/mL: MRI-based radiomics can help junior radiologists improve the diagnostic performance.

To develop MRI-based radiomics model for predicting prostate cancer (PCa) in men with prostate-specific antigen (PSA) levels of 4-10 ng/mL, to compare the performance of radiomics model and PI-RADS v2.1, and to further verify the predictive ability of radiomics model for lesions with different PI-RADS v2.1 score. 171 patients with PSA levels of 4-10 ng/mL were divided into training (n = 119) and testing (n = 52) groups. PI-RADS v2.1 score was assessed by two radiologists. All volumes of interest were segmented on T2-weighted imaging, diffusion weighted imaging, and apparent diffusion coefficient sequences, from which quantitative radiomics features were extracted. Multivariate logistic regression analysis was performed to establish radiomics model for predicting PCa. The diagnostic performance was assessed using receiver operating characteristic curve analysis. The radiomics model exhibited the best performance in predicting PCa, which was better than the performance of PI-RADS v2.1 scoring by the junior radiologist in the training group [area under the curve (AUC): 0.932 vs 0.803], testing group (AUC: 0.922 vs 0.797), and the entire cohort (AUC: 0.927 vs 0.801) (P < 0.05). The radiomics model performed well for lesions with PI-RADS v2.1 score of 3 (AUC = 0.854, sensitivity = 84.62%, specificity = 84.34%) and PI-RADS v2.1 score of 4-5 (AUC = 0.967, sensitivity = 98.11%, specificity = 86.36%) assigned by junior radiologist. The radiomics model quantitatively outperformed PI-RADS v2.1 for noninvasive prediction of PCa in men with PSA levels of 4-10 ng/mL. The model can help improve the diagnostic performance of junior radiologists and facilitate better decision-making by urologists for management of lesions with different PI-RADS v2.1 score.

[The Prognostic Value of FOSB Gene in Acute Myeloid Leukemia].

AbstractObjective: To analyze the expression of FOSB in acute myeloid leukemia （AML） and its correlation with prognosis of the patient based on the large sample data. METHODS: The genome, transcriptome, gene chip and clinical information from multiple public databases were statistical analyzed. RESULTS: The expression of FOSB gene in AML patients was significantly higher than that in normal people. The prognostic analysis of the 163 patients showed that the patients with high FOSB expression showed longer OS and EFS than those with FOSB low expression. The patients were further divided into chemotherapy group and allogeneic hematopoietic stem cell transplantation (allo-HSCT) group according to the treatment method, and then each group was divided into two subgroups (FOSBhigh, FOSBlow) according to the median expression level of FOSB. In the allo-HSCT group, the patients with FOSB high expression was longer event-free survival (EFS: P=0.017) and overall survival (OS: P=0029). At the same time, allo-HSCT in patients with high FOSB expression could improve the prognosis of the patients (Chemotherapy vs Allo-HSCT, OS: P<0.001, EFS: P=0.007). Multivariate analysis showed that the high expression of FOSB was an independent favorable prognostic factor for EFS and OS (EFS: HR=0.501， P=0.019; OS: HR=0.461， P=0.009) of the patients. CONCLUSION: The high expression of FOSB indicated a good prognosis for acute myeloid leukemia.

Kindlin-3 in Immune Cells Is Required to Suppress Prostate Cancer Tumor Growth in Mice.

BACKGROUND/AIM: Kindlins are essential integrin activators. Kindlin-1 and kindlin-2 are often concomitantly expressed in epithelial tumor cells and participate in regulating tumor malignancy. However, it remains unclear whether kindlin-3, the one expressed in immune cells, also plays a role in regulating tumor malignancy. MATERIALS AND METHODS: To examine the role of kindlin-3 in different immune cells in regulating solid tumor growth, a xenograft model of prostate cancer tumor growth in genetically modified kindlin-3 mice was employed. RESULTS: Disruption of crosstalk between kindlin-3 and integrins significantly promoted subcutaneous prostate cancer tumor growth in mice. Furthermore, deficiency of kindlin-3 in T cells and NK cells, but not myeloid cells and B cells, significantly enhanced prostate cancer tumor growth. CONCLUSION: Tumor-killing leukocytes require Kindlin-3 for suppressing cancerous tumor growth, thus providing a novel anticancer mechanism.

Magnetic resonance imaging-radiomics evaluation of response to chemotherapy for synchronous liver metastasis of colorectal cancer.

BACKGROUND: Synchronous liver metastasis (SLM) is an indicator of poor prognosis for colorectal cancer (CRC). Nearly 50% of CRC patients develop hepatic metastasis, with 15%-25% of them presenting with SLM. The evaluation of SLM in CRC is crucial for precise and personalized treatment. It is beneficial to detect its response to chemotherapy and choose an optimal treatment method. AIM: To construct prediction models based on magnetic resonance imaging (MRI)-radiomics and clinical parameters to evaluate the chemotherapy response in SLM of CRC. METHODS: A total of 102 CRC patients with 223 SLM lesions were identified and divided into disease response (DR) and disease non-response (non-DR) to chemotherapy. After standardizing the MRI images, the volume of interest was delineated and radiomics features were calculated. The MRI-radiomics logistic model was constructed after methods of variance/Mann-Whitney U test, correlation analysis, and least absolute shrinkage and selection operator in feature selecting. The radiomics score was calculated. The receiver operating characteristics curves by the DeLong test were analyzed with MedCalc software to compare the validity of all models. Additionally, the area under curves (AUCs) of DWI, T2WI, and portal phase of contrast-enhanced sequences radiomics model (Ra-DWI, Ra-T2WI, and Ra-portal phase of contrast-enhanced sequences) were calculated. The radiomics-clinical nomogram was generated by combining radiomics features and clinical characteristics of CA19-9 and clinical N staging. RESULTS: The AUCs of the MRI-radiomics model were 0.733 and 0.753 for the training (156 lesions with 68 non-DR and 88 DR) and the validation (67 lesions with 29 non-DR and 38 DR) set, respectively. Additionally, the AUCs of the training and the validation set of Ra-DWI were higher than those of Ra-T2WI and Ra-portal phase of contrast-enhanced sequences (training set: 0.652 vs 0.628 and 0.633, validation set: 0.661 vs 0.575 and 0.543). After chemotherapy, the top four of twelve delta-radiomics features of Ra-DWI in the DR group belonged to gray-level run-length matrices radiomics parameters. The radiomics-clinical nomogram containing radiomics score, CA19-9, and clinical N staging was built. This radiomics-clinical nomogram can effectively discriminate the patients with DR from non-DR with a higher AUC of 0.809 (95% confidence interval: 0.751-0.858). CONCLUSION: MRI-radiomics is conducive to predict chemotherapeutic response in SLM patients of CRC. The radiomics-clinical nomogram, involving radiomics score, CA19-9, and clinical N staging is more effective in predicting chemotherapeutic response.

Mesenchymal stem cell treatment improves outcome of COVID-19 patients via multiple immunomodulatory mechanisms.

The infusion of coronavirus disease 2019 (COVID-19) patients with mesenchymal stem cells (MSCs) potentially improves clinical symptoms, but the underlying mechanism remains unclear. We conducted a randomized, single-blind, placebo-controlled (29 patients/group) phase II clinical trial to validate previous findings and explore the potential mechanisms. Patients treated with umbilical cord-derived MSCs exhibited a shorter hospital stay (P = 0.0198) and less time required for symptoms remission (P = 0.0194) than those who received placebo. Based on chest images, both severe and critical patients treated with MSCs showed improvement by day 7 (P = 0.0099) and day 21 (P = 0.0084). MSC-treated patients had fewer adverse events. MSC infusion reduced the levels of C-reactive protein, proinflammatory cytokines, and neutrophil extracellular traps (NETs) and promoted the maintenance of SARS-CoV-2-specific antibodies. To explore how MSCs modulate the immune system, we employed single-cell RNA sequencing analysis on peripheral blood. Our analysis identified a novel subpopulation of VNN2+ hematopoietic stem/progenitor-like (HSPC-like) cells expressing CSF3R and PTPRE that were mobilized following MSC infusion. Genes encoding chemotaxis factors - CX3CR1 and L-selectin - were upregulated in various immune cells. MSC treatment also regulated B cell subsets and increased the expression of costimulatory CD28 in T cells in vivo and in vitro. In addition, an in vivo mouse study confirmed that MSCs suppressed NET release and reduced venous thrombosis by upregulating kindlin-3 signaling. Together, our results underscore the role of MSCs in improving COVID-19 patient outcomes via maintenance of immune homeostasis.

Idiopathic mesenteric phlebosclerosis associated with long-term oral intake of geniposide.

BACKGROUND: Idiopathic mesenteric phlebosclerosis (IMP) is a rare disease, and its etiology and risk factors remain uncertain. AIM: To investigate the possible influence of Chinese herbal liquid containing geniposide on IMP. METHODS: The detailed formula of herbal liquid prescriptions of all patients was studied, and the herbal ingredients were compared to identify the toxic agent as a possible etiological factor. Abdominal computed tomography (CT) and colonoscopy images were reviewed to determine the extent and severity of mesenteric phlebosclerosis and the presence of findings regarding colitis. The disease CT score was determined by the distribution of mesenteric vein calcification and colon wall thickening on CT images. The drinking index of medicinal liquor was calculated from the daily quantity and drinking years of Chinese medicinal liquor. Subsequently, Spearman's correlation analysis was conducted to evaluate the correlation between the drinking index and the CT disease score. RESULTS: The mean age of the 8 enrolled patients was 75.7 years and male predominance was found (all 8 patients were men). The patients had histories of 5-40 years of oral Chinese herbal liquids containing geniposide and exhibited typical imaging characteristics (e.g., threadlike calcifications along the colonic and mesenteric vessels or associated with a thickened colonic wall in CT images). Calcifications were confined to the right-side mesenteric vein in 6 of the 8 patients (75%) and involved the left-side mesenteric vein of 2 cases (25%) and the calcifications extended to the mesorectum in 1 of them. The thickening of colon wall mainly occurred in the right colon and the transverse colon. The median disease CT score was 4.88 (n = 7) and the median drinking index was 5680 (n = 7). After Spearman's correlation analysis, the median CT score of the disease showed a significant positive correlation with the median drinking index (r = 0.842, P < 0.05). CONCLUSION: Long-term oral intake of Chinese herbal liquid containing geniposide may play a role in the pathogenesis of IMP.

ß3-Endonexin interacts with ninein in vascular endothelial cells to promote angiogenesis.

Anti-angiogenesis serves as an effective tumor therapy approach. In a previous study, we found that ß3-endonexin expressed in vascular endothelial cells was involved in promoting proliferation and angiogenesis partially by facilitating VEGF expression. However, it still remains unclear if ß3-endonexin in vascular endothelial cells also employs other mechanisms in regulating angiogenesis. In this study, we utilized a ß3-endonexin mutant (M2) carrying a defective nuclear localization sequence to disrupt its nuclear localization and evaluated its ability to promote HUVEC proliferation and formation of tube-like vascular structures. In addition, we performed yeast 2-hybrid assay to identify potential functional effectors of ß3-endonexin. We found that both wild type ß3-endonexin and the M2 mutant could localize to centrosomes in HUVECs and both were able to promote HUVEC proliferation and formation of vascular structures. However, the M2 mutant failed to promote VEGF expression in HUVECs. Further, we found that both wild type ß3-endonexin and the M2 mutant were capable of binding to ninein, a centrosomal protein with a proangiogenic effect. Knockdown of ninein in HUVECs impeded centrosome localization of wild type ß3-endonexin and the M2 mutant and inhibited HUVEC proliferation and formation of vascular structures. Taken together, these findings suggest that ß3-endonexin interacts with centrosome ninein and contributes to HUVEC proliferation and formation of vascular structures.

[Value of SQLE Expression in Prognostic Evaluation of Patients with Acute Myeloid Leukemia].

OBJECTIVE: To analyze the relationship between the expression level of SQLE and the prognosis of patients with acute myeloid leukemia (AML) through large sample data. METHODS: The data of genome, transcriptome, gene chip expression, and clinical information were statistically analyzed in multiple cohorts of AML patients with large samples. RESULTS: It was found that the expression level of SQLE gene in tumor cells of AML patients was significantly higher than that of healthy controls (P=0.001). In the three AML corhort, the SQLE high expression group showed a worse therapeutic outcome (OS, P=0.009, P=0.0001, P=0.006; EFS, P=0.005, P=0.001). The unvariate and multivariate survival prognosis analysis indicated that the high expression of SQLE suggests lower event-free survival rate (EFS, HR=1.551, P<0.05) and overall survival rate (OS, HR=1.484, P<0.05). At the same time, it was also found that among different risk subgroups, the expression of SQLE in high risk group was higher (P<0.001, P=0.01), while the patients with high SQLE expression, who received allogeneic HSCT, had longer overall survival time (P=0.006). CONCLUSION: The up-regulation SQLE expression suggests a poor prognosis for the patients with AML.

Structure basis of the FERM domain of kindlin-3 in supporting integrin αIIbß3 activation in platelets.

Kindlin-3, a protein 4.1, ezrin, radixin, and moesin (FERM) domain-containing adaptor in hematopoietic cells, is essentially required for supporting the bidirectional integrin αIIbß3 signaling in platelets by binding to the integrin ß3 cytoplasmic tail. However, the structural details of kindlin-3's FERM domain remain unknown. In this study, we crystalized the kindlin-3's FERM domain protein and successfully solved its 3-dimensional structure. The structure shows that the 3 kindlin-3's FERM subdomains (F1, F2, and F3) compact together and form a cloverleaf-shaped conformation, which is stabilized by the binding interface between the F1 and F3 subdomains. Interestingly, the FERM domain of kindlin-3 exists as a monomer in both crystal and solution, which is different from its counterpart in kindlin-2 that is able to form a F2 subdomain-swapped dimer; nonetheless, dimerization is required for kindlin-3 to support integrin αIIbß3 activation, indicating that kindlin-3 may use alternative mechanisms for formation of a functional dimer in cells. To evaluate the functional importance of the cloverleaf-like FERM structure in kindlin-3, structure-based mutations were introduced into kindlin-3 to disrupt the F1/F3 interface. The results show that integrin αIIbß3 activation is significantly suppressed in platelets expressing the kindlin-3 mutant compared with those expressing wild-type kindlin-3. In addition, introduction of equivalent mutations into kindlin-1 and kindlin-2 also significantly compromises their ability to support integrin αIIbß3 activation in CHO cells. Together, our findings suggest that the cloverleaf-like FERM domain in kindlins is structurally important for supporting integrin αIIbß3 activation.

[Prognostic Value of SLC25A12 Expression in Patients with Acute Myeloid Leukemia Based on Integrated Analysis of Multi-dimensional Clinical Data].

OBJECTIVE: To investigate the clinical and prognostic value of SLC25A12 in patients with acute myeloid leukemia (AML). METHODS: The expression levels of SLC25A12 in bone marrow or peripheral blood cells of AML patients and healthy people in two independent cohorts (n=46, n=290, respectively) were compared. Then it was assessed that the prognostic value of SLC25A12 expression in two independent AML study cohorts (n=163, n=329, respectively) by mean of integrated analysis of genomic, transcriptome, clinical and prognosis information. RESULTS: The expression of SLC25A12 in AML patients significantly increased as compared with that of healthy people (P=0.0001, P=0.0238, respectively). Univariate and multivariate analyze showed that high SLC25A12 expression was significantly associated with shorter event-free survival (EFS)(HR=1.605, P=0.018) and overall survival (OS)(HR=1.818, P=0.002) of patients. In favorable-risk and intermediate-risk subgroups, patients with high SLC25A12 expression showed shorter EFS and OS than patients with low SLC25A12 expression. CONCLUSION: High SLC25A12 expression significantly associated with poor prognosis of AML patients, which suggests that SLC25A12 aberrant expression can be used as a potential molecular marker for prognosis evaluation of AML patients.

Peptides derived from the integrin ß cytoplasmic tails inhibit angiogenesis.

BACKGROUND: Integrins are essential regulators of angiogenesis. However, the antiangiogenic potential of peptides derived from the integrin cytoplasmic tails (CT) remains mostly undetermined. METHODS: Here we designed a panel of membrane-penetrating peptides (termed as mßCTPs), each comprising a C-terminal NxxY motif from one of the conserved integrin ß CTs, and evaluated their antiangiogenic ability using both in vitro and in vivo approaches. RESULTS: We found that mß3CTP, mß5CTP and mß6CTP, derived respectively from the integrin ß3, ß5 and ß6 CTs, but not others, exhibit antiangiogenic ability. Interestingly, we observed that the integrin ß3, ß5 and ß6 CTs but not others are able to interact with ß3-endonexin. In addition, the antiangiogenic core in mß3CTP is identical to a previously identified ß3-endonexin binding region in the integrin ß3 CT, indicating that the antiangiogenic mßCTPs may function via their binding to ß3-endonexin. Consistently, knockdown of endogenous ß3-endonexin in HUVECs significantly suppresses tube formation, suggesting that ß3-endonexin is proangiogenic. However, neither treatment with the antiangiogenic mßCTPs nor knockdown of endogenous ß3-endonexin affects integrin-mediated HUVEC adhesion and migration, indicating that their antiangiogenic effect may not rely on directly regulating integrin activity. Importantly, both treatment with the antiangiogenic mßCTPs and knockdown of endogenous ß3-endonexin in HUVECs inhibit VEGF expression and cell proliferation, thereby providing mechanistic explanations for the functional consequences. CONCLUSION: Our results suggest that the antiangiogenic mßCTPs can interact with ß3-endonexin in vascular endothelial cells and suppress its function in regulating VEGF expression and cell proliferation, thus disclosing a unique pathway that may be useful for developing novel antiangiogenic strategies.

Kindlin-3 negatively regulates the release of neutrophil extracellular traps.

Neutrophils fight infections by generating reactive oxygen species (ROS) and extracellular traps (NETs). However, how neutrophils modulate ROS/NET generation is mechanistically unclear. Kindlin-3, an essential integrin activator expressed in hematopoietic cells, is required to support integrin-mediated neutrophil recruitment during inflammation. Here, we report a novel role of kindlin-3 in regulating ROS/NET generation in neutrophils. When overexpressing kindlin-3 in neutrophil-like differentiated HL-60 cells (HL-60N), ROS/NET generation from these cells were significantly suppressed. Interestingly, overexpression of a kindlin-3 mutant that is defective for interacting with integrins in HL-60N cells still inhibited ROS/NET generation, suggesting that the role of kindlin-3 in inhibiting ROS/NET signaling may be independent of its binding to integrins. Consistently, knockdown of kindlin-3 in HL-60N cells led to enhanced ROS/NET generation. In addition, bone marrow neutrophils isolated from kindlin-3-deficient mice showed elevated ROS/NET generation when compared with WT counterparts. As expected, overexpression of exogenous kindlin-3 in mouse neutrophils could suppress NET release ex vivo and in vivo. Collectively, these results demonstrate that kindlin-3 in neutrophils is involved in modulating the ROS/NET signaling, providing a novel mechanism for fine-tuning neutrophil behaviors during inflammation.

Kindlin supports platelet integrin αIIbß3 activation by interacting with paxillin.

Kindlins play an important role in supporting integrin activation by cooperating with talin; however, the mechanistic details remain unclear. Here, we show that kindlins interacted directly with paxillin and that this interaction could support integrin αIIbß3 activation. An exposed loop in the N-terminal F0 subdomain of kindlins was involved in mediating the interaction. Disruption of kindlin binding to paxillin by structure-based mutations significantly impaired the function of kindlins in supporting integrin αIIbß3 activation. Both kindlin and talin were required for paxillin to enhance integrin activation. Interestingly, a direct interaction between paxillin and the talin head domain was also detectable. Mechanistically, paxillin, together with kindlin, was able to promote the binding of the talin head domain to integrin, suggesting that paxillin complexes with kindlin and talin to strengthen integrin activation. Specifically, we observed that crosstalk between kindlin-3 and the paxillin family in mouse platelets was involved in supporting integrin αIIbß3 activation and in vivo platelet thrombus formation. Taken together, our findings uncover a novel mechanism by which kindlin supports integrin αIIbß3 activation, which might be beneficial for developing safer anti-thrombotic therapies.

Kindlin-2 regulates hemostasis by controlling endothelial cell-surface expression of ADP/AMP catabolic enzymes via a clathrin-dependent mechanism.

Kindlin-2, a widely distributed cytoskeletal protein, has been implicated in integrin activation, and its absence is embryonically lethal in mice. In the present study, we tested whether hemostasis might be perturbed in kindlin-2(+/-) mice. Bleeding time and carotid artery occlusion time were significantly prolonged in kindlin-2(+/-) mice. Whereas plasma concentrations/activities of key coagulation/fibrinolytic proteins and platelet counts and aggregation were similar in wild-type and kindlin-2(+/-) mice, kindlin-2(+/-) endothelial cells (ECs) showed enhanced inhibition of platelet aggregation induced by adenosine 5'-diphosphate (ADP) or low concentrations of other agonists. Cell-surface expression of 2 enzymes involved in ADP/adenosine 5'-monophosphate (AMP) degradation, adenosine triphosphate (ATP) diphosphohydrolase (CD39) and ecto-5'-nucleotidase (CD73) were increased twofold to threefold on kindlin-2(+/-) ECs, leading to enhanced ATP/ADP catabolism and production of adenosine, an inhibitor of platelet aggregation. Trafficking of CD39 and CD73 at the EC surface was altered in kindlin-2(+/-) mice. Mechanistically, this was attributed to direct interaction of kindlin-2 with clathrin heavy chain, thereby controlling endocytosis and recycling of CD39 and CD73. The interaction of kindlin-2 with clathrin was independent of its integrin binding site but still dependent on a site within its F3 subdomain. Thus, kindlin-2 regulates trafficking of EC surface enzymes that control platelet responses and hemostasis.

Spatial coordination of kindlin-2 with talin head domain in interaction with integrin ß cytoplasmic tails.

Both talin head domain and kindlin-2 interact with integrin ß cytoplasmic tails, and they function in concert to induce integrin activation. Binding of talin head domain to ß cytoplasmic tails has been characterized extensively, but information on the interaction of kindin-2 with this integrin segment is limited. In this study, we systematically examine the interactions of kindlin-2 with integrin ß tails. Kindlin-2 interacted well with ß(1) and ß(3) tails but poorly with the ß(2) cytoplasmic tail. This binding selectivity was determined by the non-conserved residues, primarily the three amino acids at the extreme C terminus of the ß(3) tail, and the sequence in ß(2) was non-permissive. The region at the C termini of integrin ß(1) and ß(3) tails recognized by kindlin-2 was a binding core of 12 amino acids. Kindlin-2 and talin head do not interact with one another but can bind simultaneously to the integrin ß(3) tail without enhancing or inhibiting the interaction of the other binding partner. Kindlin-2 itself failed to directly unclasp integrin α/ß tail complex, indicating that kindlin-2 must cooperate with talin to support the integrin activation mechanism.

Study on the relationship between TagSNPs and haplotype of hCHK2 and esophageal cancer in Kazakh and Han in Xinjiang.

AIM OF THE STUDY: To determine the association of hCHK2 rs2278022, rs2602431, and rs2970077 polymorphisms and haplotypes with susceptibility to esophageal cancer in Kazakh and Han in Xinjiang Uygur Autonomous Region. MATERIAL AND METHODS: Molecular epidemiology was carried out on 239 cases of esophageal cancer (132 Kazakh, 107 Han) and 513 controls (309 Kazakh, 204 Han) of Xinjiang. Polymorphisms of hCHK2 at rs2278022, rs2602431 and rs2970077 were analyzed by polymerase chain reaction-ligase detection reaction (PCR-LDR). Haplotypes were estimated by the SHEsis software. Statistical differences in genotype/haplotype frequencies, and frequencies between the case group and the control group were estimated. RESULTS: 1) No significant difference was observed in the frequency of hCHK2 at rs2278022, rs2602431 and rs2970077 between the cases and controls in Kazakh and Han (P > 0.05); 2) In Kazakh and Han, the distribution of haplotypes was not significantly different between esophageal cancer cases and controls (P > 0.05). CONCLUSIONS: Polymorphisms of hCHK2 at rs2278022, rs2602431 and rs2970077 and haplotypes are unlikely to be associated with the susceptibility to esophageal cancer in Kazakh and Han.

Membrane binding of the N-terminal ubiquitin-like domain of kindlin-2 is crucial for its regulation of integrin activation.

Kindlin-2 belongs to an emerging class of regulators for heterodimeric (α/ß) integrin adhesion receptors. By binding to integrin ß cytoplasmic tail via its C-terminal FERM-like domain, kindlin-2 promotes integrin activation. Intriguingly, this activation process depends on the N terminus of kindlin-2 (K2-N) that precedes the FERM domain. The molecular function of K2-N is unclear. We present the solution structure of K2-N, which displays a ubiquitin fold similar to that observed in kindlin-1. Using chemical shift mapping and mutagenesis, we found that K2-N contains a conserved positively charged surface that binds to membrane enriched with negatively charged phosphatidylinositol-(4,5)-bisphosphate. We show that while wild-type kindlin-2 is capable of promoting integrin activation, such ability is significantly reduced for its membrane-binding defective mutant. These data suggest a membrane-binding function of the ubiquitin-like domain of kindlin-2, which is likely common for all kindlins to promote their localization to the plasma membrane and control integrin activation.

Structural basis of phosphoinositide binding to kindlin-2 protein pleckstrin homology domain in regulating integrin activation.

Kindlins are a subclass of FERM-containing proteins that have recently emerged as key regulators of integrin receptor activation and signaling. As compared with the conventional FERM domain, the kindlin FERM domain contains an inserted pleckstrin homology (PH) domain that recognizes membrane phosphoinositides, including phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidylinositol 3,4,5-trisphosphate (PIP3). Using NMR spectroscopy, we show that PIP3 site-specifically binds to kindlin-2 PH with substantial chemical shift changes that are much larger than PIP2. This suggests an enhanced association of kindlin-2 with membrane as mediated by PIP3 upon its conversion from PIP2 by phosphoinositide-3 kinase, a known regulator of integrin activation. We determined the NMR structure of the kindlin-2 PH domain bound to the head group of PIP3, inositol 1,3,4,5-tetraphosphate (IP4). The structure reveals a canonical PH domain fold, yet with a distinct IP4 binding pocket that appears highly conserved for the kindlin family members. Functional experiments demonstrate that although wild type kindlin-2 is capable of cooperating with integrin activator talin to induce synergistic integrin α(IIb)ß(3) activation, this ability is significantly impaired for a phosphoinositide binding-defective kindlin-2 mutant. These results define a specific PIP3 recognition mode for the kindlin PH domain. Moreover, they shed light upon a mechanism as to how the PH domain mediates membrane engagement of kindlin-2 to promote its binding to integrin and cooperation with talin for regulation of integrin activation.

The integrin coactivator kindlin-2 plays a critical role in angiogenesis in mice and zebrafish.

Kindlin-2, a widely distributed cytoskeletal protein, has been implicated in integrin activation, and its absence is embryonically lethal in mice and causes severe developmental defects in zebrafish. Knockdown of kindlin-2 levels in endothelial cells resulted in defective adhesive and migratory responses, suggesting that angiogenesis might be aberrant even with partial reduction of kindlin-2. This hypothesis has now been tested in the kindlin-2(+/-) mice. RM1 prostate tumors grown in kindlin-2(+/-) mice had fewer blood vessels, which were thinner and shorter and supported less tumor growth compared with wild-type littermates. The vessels that did form in the kindlin-2(+/-) mice lacked smooth muscle cells and pericytes and had thinner basement membranes, indicative of immature vessels. VEGF-induced angiogenesis in matrigel implants was also abnormal in the kindlin-2(+/-) mice. Vessels in the kindlin-2(+/-) mice were leaky, and BM transplantation from kindlin-2(+/-) to WT mice did not correct this defect. Endothelial cells derived from kindlin-2(+/-) mice had integrin expression levels similar to WT mice but reduced αVß3-dependent signaling, migration, adhesion, spreading, and tube formation. Developmental angiogenesis was markedly impaired by kindlin-2 morpholinos in zebrafish. Taken together, kindlin-2 plays an important role in pathologic and developmental angiogenesis, which arises from defective activation of integrin αVß3.