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Cells must detect and respond to molecular events such as the presence or absence of specific small molecules. To accomplish this, cells have evolved methods to measure the presence and concentration of these small molecules in their environment and enact changes in gene expression or behavior. However, cells dont usually change their DNA in response to such outside stimuli. In this work, we have engineered a genetic circuit that can enact specific and controlled genetic changes in response to changing small molecule concentrations. Known DNA sequences can be repeatedly integrated into a genomic array such that their identity and order encodes information about past small molecule concentrations that the cell has experienced. To accomplish this, we use catalytically inactive CRISPR-Cas9 (dCas9) to bind to and block attachment sites for the integrase Bxb1. Therefore, through the co-expression of dCas9 and guide RNA, Bxb1 can be directed to integrate one of two engineered plasmids, which correspond to two orthogonal small molecule inducers that can be recorded with this system. We identified the optimal location of guide RNA binding to the Bxb1 attP integrase attachment site, and characterized the detection limits of the system by measuring the minimal small molecule concentration and shortest induction time necessary to produce measurable differences in array composition as read out by Oxford Nanopore long read sequencing technology.
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Objective:To investigate the diagnostic efficacy and clinical application value of 99Tc m-diethylene triamine pentaacetic acid (DTPA) SPECT/CT imaging in cerebrospinal fluid leakage (CSFL). Methods:A total of 23 patients (11 males, 12 females; age (44.2±15.1) years) who underwent endoscopic repair surgery for suspected CSFL in Shanghai Jiao Tong University Affiliated Sixth People′s Hospital between April 2018 and January 2020 were retrospectively reviewed. All patients performed 99Tc m-DTPA SPECT/CT imaging, paranasal sinus high resolution CT (HRCT) and MRI before surgery. The diagnostic efficacies of 3 imaging techniques were calculated according to the result of surgery regarded as the golden standard. χ2 test was used to compare the qualitative and localized diagnostic efficacies of 3 imaging techniques for CSFL. Results:Of 23 patients, 21 were finally confirmed with CSFL and 24 leak locations were identified according to the results of surgery; the other 2 patients had no obvious CSFL and no leak location was found during the operation. The sensitivity and accuracy of 99Tc m-DTPA SPECT/CT, MRI and HRCT for the diagnosis of CSFL were 100%(21/21) and 95.7%(22/23), 85.7%(18/21) and 82.6%(19/23), 76.2%(16/21) and 69.6%(16/23), respectively. The accuracy of 99Tc m-DTPA SPECT/CT, MRI and HRCT for the diagnosis of leak location was 79.2%(19/24), 50.0%(12/24) and 45.8%(11/24), respectively. There was no statistically significant difference of diagnostic efficacies for CSFL among 3 imaging techniques ( χ2 values: 0.451-3.453, all P>0.05). For leak location, the diagnostic efficacy of 99Tc m-DTPA SPECT/CT was significantly better than that of MRI and HRCT ( χ2 values: 4.463, 5.689, both P<0.05). Conclusion:99Tc m-DTPA SPECT/CT imaging shows an excellent diagnosis efficacy not only for CSFL but also for leak location, which is helpful for guiding surgery.
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Hyperinflammation and lymphopenia provoked by SARS-CoV-2-activated macrophages contribute to the high mortality of Coronavirus Disease 2019 (COVID-19) patients. Thus, defining host pathways aberrantly activated in patient macrophages is critical for developing effective therapeutics. We discovered that G9a, a histone methyltransferase that is overexpressed in COVID-19 patients with high viral load, activates translation of specific genes that induce hyperinflammation and impairment of T cell function or lymphopenia. This noncanonical, pro-translation activity of G9a contrasts with its canonical epigenetic function. In endotoxin-tolerant (ET) macrophages that mimic conditions which render patients with pre-existing chronic inflammatory diseases vulnerable to severe symptoms, our chemoproteomic approach with a biotinylated inhibitor of G9a identified multiple G9a-associated translation regulatory pathways that were upregulated by SARS-CoV-2 infection. Further, quantitative translatome analysis of ET macrophages treated progressively with the G9a inhibitor profiled G9a-translated proteins that unite the networks associated with viral replication and the SARS-CoV-2-induced host response in severe patients. Accordingly, inhibition of G9a-associated pathways produced multifaceted, systematic effects, namely, restoration of T cell function, mitigation of hyperinflammation, and suppression of viral replication. Importantly, as a host-directed mechanism, this G9a-targeted, combined therapeutics is refractory to emerging antiviral-resistant mutants of SARS-CoV-2, or any virus, that hijacks host responses.
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Objective:To explore the efficacy of percutaneous osteoplasty (POP) combined with 131I therapy in patients with bone metastases from differentiated thyroid carcinoma (DTC) and assess the survival. Methods:From Januray 2008 to January 2020, 29 DTC patients with bone metastases (16 males, 13 females, age range: 24-64 years) who received POP combined with 131I therapy in Shanghai Sixth People′s Hospital were retrospectively analyzed. Clinical data and characteristics of patients were analyzed. The efficacy and prognosis were evaluated based on the changes of serum thyroglobulin (Tg) and relieving condition of bone pain after the combined treatment. χ2 test was used to determine the association between clinical characteristics and efficacy, and Kaplan-Meier analysis was used to estimate the overall survival (OS) rate. Results:The biochemical response rate of serum Tg was 68.97% (20/29) after the combined treatment. For the influence on changes of serum Tg, whether or not combined with non-osseous distant metastasis, and cumulative dose of 131I treatment(≥22.2 vs <22.2 GBq) were statistically significant ( χ2 values: 5.448 and 4.371, both P<0.05). The rate of bone pain relief was 65.52%(19/29). Age (≥55 vs <55 years) and the cumulative dose of 131I treatment had statistically influences on bone relief ( χ2 values: 7.486 and 5.154, both P<0.05). The 5-years OS rate of patients was 87.68%, while the 10-years OS rate was 65.76%. Conclusion:POP combined with 131I therapy is effective on relieving the pain, reducing the serum Tg to some extent, and improving the long-term survival of DTC patients with bone metastasis.
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Objective To investigate the effects of microRNA (miR)106a on the proliferation, apoptosis, migration and invasion of thyroid cancer cells in vitro.Methods 8505C and CGTH-W3 cell lines were used in the study.Overexpression and inhibition of miR106a were achieved by transfection of lentiviral vectors.The changes of gene expression were detected by quantitative real-time PCR (qRT-PCR) and Western blot analysis.Cell viability and apoptosis were evaluated by MTT assay and flow cytometry analysis, respectively.The caspase-9 activities in parental CGTH-W3 and 8505C cells and transfected sublines were measured.Wound healing and Transwell invasion assays were performed to determine cell migration and invasion.Two-sample t test and one-way analysis of variance were used to analyze the data.Results The level of miR106a in 8505C was up-regulated when compared to that in CGTH-W3 cells (t=10.28, P<0.01).Scrambled control and miR106a(-) were also successfully transfected into cells.Inhibition of miR106a suppressed cell viability, migration and invasion while promoted apoptosis and caspase-9 activity of 8505C cells, with significant differences among 8505C, 8505C-control, 8505C-miR106a(-) cells (F=147.0, 19.2, 100.3, 537.8, 804.3;all P<0.01).Overexpression of miR106a promoted cell viability, migration and invasion while inhibited apoptosis and caspase-9 activity of CGTH-W3 cells, with significant differences among CGTH-W3, CGTH-W3-control, CGTH-W3-miR106a(+) cells(F=9.2, 13.3, 622.8, 12.3, 19.6, all P<0.01).In addition, miR106a may up-regulate the expression of MEKK2 and p-ERK1/2.Conclusion Acting as an onco-miR, miR106a might promote the proliferation, migration and invasion of thyroid cancer cells and inhibit their apoptosis in vitro.
ABSTRACT
DTC is the most common endocrine carcinoma and its routine treatment consists of total thyroidectomy and 131I thyroid remnant ablation.Currently,standard follow-up for DTC comprises Tg measurement and neck ultrasound as well as an additional radioiodine scan when indicated.As thyroid cells are assumed to be the only source of Tg in serum,circulating Tg serves as an excellent marker of persistent or recurrent disease in DTC follow-up.With the development of highly sensitive Tg assays,now it is possible to detect very low Tg concentrations which reflect minimal amounts of thyroid tissue without the need for TSH stimulation.This review is to introduce clinical implications of highly sensitive Tg assays.
