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Univariate and multivariable Cox regression analysis of overall survival (n=95)

Establishment of the FAM135B transgenic mouse model

Esophageal squamous cell carcinoma (ESCC) is one of the most common and deadly diseases. In our previous comprehensive genomics study, we found that family with sequence similarity 135 member B (FAM135B) was a novel cancer-related gene, yet its biological functions and molecular mechanisms remain unclear. In this study, we demonstrate that the protein levels of FAM135B are significantly higher in ESCC tissues than in precancerous tissues, and high expression of FAM135B correlates with poorer clinical prognosis. Ectopic expression of FAM135B promoted ESCC cell proliferation in vitro and in vivo, likely through its direct interaction with growth factor GRN, thus forming a feedforward loop with AKT/mTOR signaling. Patients with ESCC with overexpression of both FAM135B and GRN had worse prognosis; multivariate Cox model analysis indicated that high expression of both FAM135B and GRN was an independent prognostic factor for patients with ESCC. FAM135B transgenic mice bore heavier tumor burden than wild-type mice and survived a relatively shorter lifespan after 4-nitroquinoline 1-oxide treatment. In addition, serum level of GRN in transgenic mice was higher than in wild-type mice, suggesting that serum GRN levels might provide diagnostic discrimination for patients with ESCC. These findings suggest that the interaction between FAM135B and GRN plays critical roles in the regulation of ESCC progression and both FAM135B and GRN might be potential therapeutic targets and prognostic factors in ESCC.Significance:These findings investigate the mechanisms of FAM135B in promoting ESCC progression and suggest new potential prognostic biomarkers and therapeutic targets in patients with ESCC.

Cholesterol (CHO) in human blood is one of the most frequently and crucially quantified substances in diagnostic laboratories. However, the visual and portable point of care testing (POCT) methods have...

RNA-cleaving DNAzymes and their multicomponent nucleic acid enzymes (MNAzymes) have been successfully used to detect nucleic acids and proteins. The appropriate split of the catalytic cores of DNAzymes is critical to the formation of MNAzymes with high catalytic activities. However, for protein detection, no systematic investigation has been made on the effects of the split locations and secondary structures of MNAzymes on the catalytic activities of the cleavage reaction. We systematically studied how split locations and secondary structures affect the activity of the MNAzymes that catalyze multiple cleavage steps. We engineered the MNAzymes on the basis of the RNA-cleaving DNAzyme 10-23 as a model system. We designed 28 pairs of MNAzymes, representing 14 different split locations and two secondary structures: the three-arm and the four-arm structures. By comparing the multiple turnover numbers (kobs.m) of the 28 MNAzymes, we showed that the split location between the seventh cytosine and the eighth thymine of the catalytic core region and the four-arm structure resulted in optimum catalytic activity. Binding-induced DNA assembly of the optimized MNAzymes enabled sensitive detection of two model protein targets, demonstrating promising potential of the binding-assembled MNAzymes for protein analysis. The strategy of binding-assembled MNAzymes and systematic studies measuring multiple turnover numbers (kobs.m) provide a new approach to studying other partial (split) DNAzymes and engineering better MNAzymes for the detection of specific proteins.

The sensitivity, accuracy, and efficiency of fluorescent particle detection can be improved by purifying the fluorescent-dye-labeled particles. In this study, an in-site model of electrophoretic elution (EE) was developed for the facile and efficient removal of unconjugated fluorescent dyes after labeling reactions, thereby facilitating the sensitive fluorescent imaging of proteins captured by microbeads. First, bovine serum albumin (BSA) and magnetic beads (MBs) were chosen as the model protein and particles, respectively, and an MBs-BSA complex was synthesized by mixing the beads with the BSA solution. Second, excessive fluorescein isothiocyanate (FITC) was added to the EP tube with MBs-BSA suspension for the fluorescent labeling of BSA, and a labeled compound was obtained after 8-h incubation in the dark at 4 ℃. The unpurified MBs-BSA

The trans-cleavage activity of the target-activated CRISPR/Cas12a liberated an RNA crosslinker from a molecular transducer, which facilitated the assembly of gold nanoparticles. Integration of the molecular transducer with isothermal amplification and CRISPR/Cas12a resulted in visual detection of the N gene and E gene of SARS-CoV-2 in 45 min.

We have developed a single-tube assay for SARS-CoV-2 in patient samples. This assay combined advantages of reverse transcription (RT) loop-mediated isothermal amplification (LAMP) with clustered regularly interspaced short palindromic repeats (CRISPRs) and the CRISPR-associated (Cas) enzyme Cas12a. Our assay is able to detect SARS-CoV-2 in a single tube within 40 min, requiring only a single temperature control (62 °C). The RT-LAMP reagents were added to the sample vial, while CRISPR Cas12a reagents were deposited onto the lid of the vial. After a half-hour RT-LAMP amplification, the tube was inverted and flicked to mix the detection reagents with the amplicon. The sequence-specific recognition of the amplicon by the CRISPR guide RNA and Cas12a enzyme improved specificity. Visible green fluorescence generated by the CRISPR Cas12a system was recorded using a smartphone camera. Analysis of 100 human respiratory swab samples for the N and/or E gene of SARS-CoV-2 produced 100% clinical specificity and no false positive. Analysis of 50 samples that were detected positive using reverse transcription quantitative polymerase chain reaction (RT-qPCR) resulted in an overall clinical sensitivity of 94%. Importantly, this included 20 samples that required 30–39 threshold cycles of RT-qPCR to achieve a positive detection. Integration of the exponential amplification ability of RT-LAMP and the sequence-specific processing by the CRISPR-Cas system into a molecular assay resulted in improvements in both analytical sensitivity and specificity. The single-tube assay is beneficial for future point-of-care applications.

Molecular diagnosis of COVID-19 primarily relies on the detection of RNA of the SARS-CoV-2 virus, the causative infectious agent of the pandemic. Reverse transcription polymerase chain reaction (RT-PCR) enables sensitive detection of specific sequences of genes that encode the RNA dependent RNA polymerase (RdRP), nucleocapsid (N), envelope (E), and spike (S) proteins of the virus. Although RT-PCR tests have been widely used and many alternative assays have been developed, the current testing capacity and availability cannot meet the unprecedented global demands for rapid, reliable, and widely accessible molecular diagnosis. Challenges remain throughout the entire analytical process, from the collection and treatment of specimens to the amplification and detection of viral RNA and the validation of clinical sensitivity and specificity. We highlight the main issues surrounding molecular diagnosis of COVID-19, including false negatives from the detection of viral RNA, temporal variations of viral loads, selection and treatment of specimens, and limiting factors in detecting viral proteins. We discuss critical research needs, such as improvements in RT-PCR, development of alternative nucleic acid amplification techniques, incorporating CRISPR technology for point-of-care (POC) applications, validation of POC tests, and sequencing of viral RNA and its mutations. Improved assays are also needed for environmental surveillance or wastewater-based epidemiology, which gauges infection on the community level through analyses of viral components in the community's wastewater. Public health surveillance benefits from large-scale analyses of antibodies in serum, although the current serological tests do not quantify neutralizing antibodies. Further advances in analytical technology and research through multidisciplinary collaboration will contribute to the development of mitigation strategies, therapeutics, and vaccines. Lessons learned from molecular diagnosis of COVID-19 are valuable for better preparedness in response to other infectious diseases.

BACKGROUND Targeted arterial infusion of verapamil combined with chemotherapy (TVCC) is an effective clinical interventional therapy for esophageal squamous cell carcinoma (ESCC), but multidrug resistance (MDR) remains the major cause of relapse or poor prognosis, and the underlying molecular mechanisms of MDR, temporal intratumoral heterogeneity, and clonal evolutionary processes of resistance have not been determined. METHODS To elucidate the roles of genetic and epigenetic alterations in the evolution of acquired resistance during therapies, we performed whole-exome sequencing on 16 serial specimens from 7 patients with ESCC at every cycle of therapeutic intervention from 3 groups, complete response, partial response, and progressive disease, and we performed whole-genome bisulfite sequencing for 3 of these 7 patients, 1 patient from each group. RESULTS Patients with progressive disease exhibited a substantially higher genomic and epigenomic temporal heterogeneity. Subclonal expansions driven by the beneficial new mutations were observed during combined therapies, which explained the emergence of MDR. Notably, SLC7A8 was identified as a potentially novel MDR gene, and functional assays demonstrated that mutant SLC7A8 promoted the resistance phenotypes of ESCC cell lines. Promoter methylation dynamics during treatments revealed 8 drug resistance protein-coding genes characterized by hypomethylation in promoter regions. Intriguingly, promoter hypomethylation of SLC8A3 and mutant SLC7A8 were enriched in an identical pathway, protein digestion and absorption, indicating a potentially novel MDR mechanism during treatments. CONCLUSION Our integrated multiomics investigations revealed the dynamics of temporal genetic and epigenetic inter- and intratumoral heterogeneity, clonal evolutionary processes, and epigenomic changes, providing potential MDR therapeutic targets in treatment-resistant patients with ESCC during combined therapies. FUNDING National Natural Science Foundation of China, Science Foundation of Peking University Cancer Hospital, CAMS Innovation Fund for Medical Sciences, Major Program of Shenzhen Bay Laboratory, Guangdong Basic and Applied Basic Research Foundation, and the third round of public welfare development and reform pilot projects of Beijing Municipal Medical Research Institutes.

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated (Cas) protein systems have transformed the field of genome editing and transcriptional modulation. Progress in CRISPR–Cas technology has also advanced molecular detection of diverse targets, ranging from nucleic acids to proteins. Incorporating CRISPR–Cas systems with various nucleic acid amplification strategies enables the generation of amplified detection signals, enrichment of low-abundance molecular targets, improvements in analytical specificity and sensitivity, and development of point-of-care (POC) diagnostic techniques. These systems take advantage of various Cas proteins for their particular features, including RNA-guided endonuclease activity, sequence-specific recognition, multiple turnover trans-cleavage activity of Cas12 and Cas13, and unwinding and nicking ability of Cas9. Integrating a CRISPR–Cas system after nucleic acid amplification improves detection specificity due to RNA-guided recognition of specific sequences of amplicons. Incorporating CRISPR–Cas before nucleic acid amplification enables enrichment of rare and low-abundance nucleic acid targets and depletion of unwanted abundant nucleic acids. Unwinding of dsDNA to ssDNA using CRISPR–Cas9 at a moderate temperature facilitates techniques for achieving isothermal exponential amplification of nucleic acids. A combination of CRISPR–Cas systems with functional nucleic acids (FNAs) and molecular translators enables the detection of non-nucleic acid targets, such as proteins, metal ions, and small molecules. Successful integrations of CRISPR technology with nucleic acid amplification techniques result in highly sensitive and rapid detection of SARS-CoV-2, the virus that causes the COVID-19 pandemic., Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated (Cas) protein systems revolutionize genome engineering and advance analytical chemistry and diagnostic technology.

Esophageal squamous cell carcinoma (ESCC) is one of the most common and deadly diseases. In our previous comprehensive genomics study, we found that family with sequence similarity 135 member B (FAM135B) was a novel cancer-related gene, yet its biological functions and molecular mechanisms remain unclear. In this study, we demonstrate that the protein levels of FAM135B are significantly higher in ESCC tissues than in precancerous tissues, and high expression of FAM135B correlates with poorer clinical prognosis. Ectopic expression of FAM135B promoted ESCC cell proliferation in vitro and in vivo, likely through its direct interaction with growth factor GRN, thus forming a feedforward loop with AKT/mTOR signaling. Patients with ESCC with overexpression of both FAM135B and GRN had worse prognosis; multivariate Cox model analysis indicated that high expression of both FAM135B and GRN was an independent prognostic factor for patients with ESCC. FAM135B transgenic mice bore heavier tumor burden than wild-type mice and survived a relatively shorter lifespan after 4-nitroquinoline 1-oxide treatment. In addition, serum level of GRN in transgenic mice was higher than in wild-type mice, suggesting that serum GRN levels might provide diagnostic discrimination for patients with ESCC. These findings suggest that the interaction between FAM135B and GRN plays critical roles in the regulation of ESCC progression and both FAM135B and GRN might be potential therapeutic targets and prognostic factors in ESCC.Significance:These findings investigate the mechanisms of FAM135B in promoting ESCC progression and suggest new potential prognostic biomarkers and therapeutic targets in patients with ESCC.

Idiopathic pulmonary fibrosis (IPF) is a progressive interstitial lung disease with unclear etiology and poor prognosis. Despite numerous studies on the pathogenesis of IPF, only scant treatment options are available for the management of IPF. Glaucocalyxin A (GLA), an ent-Kaurane diterpenoid, has been demonstrated to exert anti-inflammatory, anti-neoplastic and anti-platelet activities. In this study, we evaluated the role of GLA as an anti-fibrotic agent in bleomycin-induced pulmonary fibrosis in mice and investigated the underlying mechanisms by which GLA attenuates lung fibrosis. Intraperitoneal administration of GLA (10 mg/kg) significantly reduced collagen deposition and hydroxyproline content in mouse lungs treated with bleomycin. Importantly, GLA significantly improved survival in bleomycin treated mice. In addition, GLA reduced weight loss in mice that reflects cachexia due to pulmonary fibrosis. Mechanistically, GLA alleviated the infiltration of macrophages and neutrophils in lungs, attenuated the increases of proinflammatory cytokines in lung tissue and bronchoalveolar lavage fluid, and inhibited the activation of NF-κB in fibrotic lungs induced by bleomycin. These results provide evidence that GLA can effectively ameliorate pulmonary fibrosis through the antagonism of leukocyte infiltration and proinflammatory cytokine production, suggesting that it may become a potential anti-fibrotic agent in IPF management.

Objective— Accumulating evidence suggests a role of semaphorins in vascular homeostasis. Here, we investigate the role of Sema7A (semaphorin 7A) in atherosclerosis and its underlying mechanism. Approach and Results— Using genetically engineered Sema7A −/− ApoE −/− mice, we showed that deletion of Sema7A attenuates atherosclerotic plaque formation primarily in the aorta of ApoE −/− mice on a high-fat diet. A higher level of Sema7A in the atheroprone lesser curvature suggests a correlation of Sema7A with disturbed flow. This notion is supported by elevated Sema7A expression in human umbilical venous endothelial cells either subjected to oscillatory shear stress or treated with the PKA (protein kinase A)/CREB (cAMP response element-binding protein) inhibitor H89 ( N -[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide·2HCl hydrate). Further studies using the partial carotid artery ligation model showed that disturbed flow in the left carotid artery of Sema7A +/+ ApoE −/− mice promoted the expression of endothelial Sema7A and cell adhesion molecules, leukocyte adhesion, and plaque formation, whereas such changes were attenuated in Sema7A −/− ApoE −/− mice. Further studies showed that blockage of β1 integrin, a known Sema7A receptor, or inhibition of FAK (focal adhesion kinase), MEK1/2 (mitogen-activated protein kinase kinase 1/2), or NF-κB (nuclear factor-κB) significantly reduced the expression of cell adhesion molecules and THP-1 (human acute monocytic leukemia cell line) monocyte adhesion in Sema7A-overexpressing human umbilical venous endothelial cells. Studies using chimeric mice suggest that vascular, most likely endothelial, Sema7A plays a major role in atherogenesis. Conclusions— Our findings indicate a significant role of Sema7A in atherosclerosis by mediating endothelial dysfunction in a β1 integrin–dependent manner.

Background: Inhibition of plaque progression by various anti-atherosclerotic drugs is a canonical aspect underlying their therapeutic efficacy. Although existed animal models of plaque regression are available for drug evaluation, they are either highly invasive or time-consuming. We therefore aimed to establish a practical method to study the mechanism of atherosclerosis regression based on hemodynamic regulation of atherosclerosis. Methods: A carotid plaque regression model in mice was developed based on the previously described model. Briefly, a single slipknot was applied to all three branches of the left carotid artery (LCA) in ApoE -/- mice. After 2 weeks on HFD, the blood flow in LCA was reconstructed by slipknot release. Mice were housed for additional 4 weeks. Carotid ultrasonography was used to verify successful induction of disturbed flow and the reconstruction of the blood flow. Atherosclerotic plaques were stained with Sudan IV. The infiltration of smooth muscle cells and macrophages were determined by immunofluorescence staining. Results: Histology studies demonstrated similar cellular components and lipid deposition between the d-flow induced plaques in the LCA in WT mice and atherosclerotic plaques in aorta of ApoE -/- mice on HFD. However, a significantly higher proportion of SMC (n=5, p Conclusion: We have established a highly reproducible atherosclerosis regression model with less invasiveness and genetic independence that supplies a practical tool for evaluation of anti-atherosclerotic drugs and for the pathophysiology of atherosclerosis regression.

Background and Objectives: Although de-escalation of axillary surgery becomes more popular, axillary lymph node dissection (ALND) is still the standard care for sentinel lymph node(SLN) positive patients not meeting the criteria of ACOSOG Z0011 in many hospitals, and frozen section (FS) of SLN is one of the valuble intra-operative assesments to avoid axilla re-operation although it was controversial due to accuracy and efficiency concerns. This study was to assess the performance of selective use of frozen section evaluation guided by MSKCC lymph node metastasis risk prediction nomogram to optimize the proceudure to be more accurate and cost effective. Methods: Surgical pathology records of consecutive 2582 biopsies in 2552 patients breast cancer patients from 2011 to 2017 were reviewed, intra-operative frozen section diagnosis were compared to post-operative paraffin reports. We calculated the sensitivity, specificity, accuracy and FNR for different MSKCC risk, the axilla re-operation rate with or without FS and the number needed to treat (NNT) to avoid second ALND was also analyzed. Results: The sensitivity, specificity, and FNR of FS were 84.7%, 99.9%, and 15.3% respectively. The axilla re-operation rates were significantly decreased if FS was done(4.7%±0.4% with FS versus 35.8%±5.8% without FS, P Conclusion: Stratified decision-making algorithm based on MSKCC prediction model improved the efficiency of FS to avoid axilla re-operatioin in patients undergoing sentinel lymph node biopsy. We recommend FS be restricted to patients with MSKCC risk higher than 0.5 who do not meet ACOSOG Z0011 criteria. Citation Format: Yang H, Wang S, Shen D, Cao Y, Tong F, Chen D, Liu P, Peng Y. Incorporation of MSKCC nomogram to guide the application of intra-operative sentinel lymph node frozen section evaluation in patients with early breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P3-03-43.

Atherosclerosis is a chronic inflammatory disease of the arterial wall elicited by accumulation of LDL and leucocytes in the subendothelium at predilection sites with disturbed laminar flow. Chemokines and their receptors appear to act as critical players in atherosclerosis as they not only direct atherogenic recruitment of leucocytes but also exert cell hemostatic functions by chemokine ligand-receptor axes and their specific or combined contributions. Chemokine (C-C motif) receptor-like 2 (CCRL2) is an atypical chemokine receptor that cooperates with its ligand chemerin to play a role in cell trafficking and inflammatory response, the processes usually occur in atherosclerosis, but its role in atherosclerosis is not clear. Here we investigated the potential role of CCRL2 in atherogenesis using the classic apolipoprotein E-deficient (ApoE-/-) mouse model of atherosclerosis. Atherosclerosis-prone ApoE-/- mice were crossed with CCRL2-/- mice to obtain ApoE-/-CCRL2+/+ and ApoE-/-CCRL2-/- mice. Male mice of both genotypes were fed a standard chow diet until 8 weeks of age and then switched to a high fat diet for 16 weeks. Aortas were dissected and assessed by en face staining. Although CCRL2 deletion did not change mouse blood lipid profile and body weight, the atherosclerotic plaque area of the total aorta was reduced by 27.2% (P=0.0379) in ApoE-/-CCRL2-/- mice compared with ApoE-/-CCRL2+/+ mice with striking difference occurring in aortic arch. A reduction of lipid deposition by 32.6% (P=0.0089) was also observed in aortic root sections when CCRL2 was deleted. Further studies showed that deletion of CCRL2 reduced macrophage accumulation and polarization during the development of atherosclerosis. CCRL2 not only expresses in high levels in the plaques of ApoE-/- mice on a high fat diet but also co-localizes with macrophages and chemerin. Interestingly, using CMKLR1-/- chimeric mice, we showed that chemerin/CMKLR1/CCRL2 axis is involved in leucocyte infiltration and possibly affects plaque formation. More importantly, partial carotid artery ligation (PCL) model confirmed that disturbed blood flow-induced endothelial expression of CCRL2 modulates plaque formation. In conclusion, our results demonstrate that CCRL2 deficiency delays macrophage accumulation and the formation of atherosclerotic plaques potentially via the disruption of chemerin/CMKLR1/CCRL2 axis and the loss of CCRL2 response to the disturbed flow, which may represent a novel mechanism of atherosclerosis. This work was supported by Natural Science Foundation of China (grant 81370373 to L.Z. and 31300781 to C.T.) Disclosures No relevant conflicts of interest to declare.