A review of approaches to 18F radiolabelling affinity peptides and proteins

Affinity peptide and protein‐ (APP) based radiotracers are an increasingly popular class of radiotracer in positron emission tomography (PET), which was once dominated by the use of small molecule radiotracers. Radiolabelled monoclonal antibodies (mAbs) are important examples of APPs, yet a preference for smaller APPs, which exhibit fast pharmacokinetics and permit rapid PET aided diagnosis, has become apparent. ¹⁸F exhibits favourable physical characteristics for APP radiolabelling and has been described as an ideal PET radionuclide. Notwithstanding, ¹⁸F radiolabelling of APP is challenging, and this is echoed in the literature where a number of diverse approaches have been adopted. This review seeks to assess and compare the approaches taken to ¹⁸F APP radiolabelling with the intention of highlighting trends within this expanding field. Generic themes have emerged in the literature, namely the use of mild radiolabelling conditions, a preference of site‐specific methodologies with an impetus for short, automated procedures which produce high‐yielding [¹⁸F]APPs.     

Investigation of non-linear Mate1-mediated efflux of trimethoprim in the mouse kidney as the mechanism underlying drug-drug interactions between trimethoprim and organic cations in the kidney

The purpose of this study was to elucidate the involvement of Mate1 in the tubular secretion of trimethoprim and saturation of Mate1-mediated efflux to address the mechanisms underlying the pharmacokinetic drug interactions with trimethoprim. Trimethoprim is a more potent inhibitor of MATE2-K than MATE1 with K_i values (μM) of 0.030–0.28 and 2.4–5.9, respectively. Trimethoprim is a substrate of human MATE1 and MATE2-K with K_m values of 2.3 ± 0.9 and 0.018 ± 0.004 μM, and mouse Mate1, but not human OCT2, mouse Oct1 and Oct2. Pyrimethamine significantly reduced the renal clearance (CL_R) of trimethoprim (mL/min/kg) from 40.0 ± 5.1 to 20.1 ± 3.7 (p < 0.05). Trimethoprim was given to mice at three infusion rates (150, 500, and 1500 nmol/min/kg). Together with an increase in the plasma concentrations of trimethoprim, the CL_R (mL/min/kg) of trimethoprim decreased to 25.9 ± 3.2, 13.5 ± 5.7, and 8.92 ± 1.50 at the respective rates. Trimethoprim decreased the CL_R of rhodamine 123 in an infusion rate-dependent manner: 11.5 ± 1.3 (control), 5.17 ± 1.55, 1.31 ± 0.50, and 0.532 ± 0.180. These results suggest that Mate1 mediates the tubular secretion of trimethoprim, and at therapeutic doses, MATEs-mediated efflux can be saturated, and thereby, cause drug interactions with other MATE substrates.     

An update in pathologic diagnosis of uterine mesenchymal tumours

Recent discovery of new disease-defining molecular alterations and development of novel targeted therapies has dramatically changed the classification and management of uterine mesenchymal neoplasms. This review discusses diagnostic updates in endometrial stromal sarcoma, PEComa, uterine tumor resembling ovarian sex cord tumor (UTROSCT), inflammatory myofibroblastic tumor, NTRK fusion uterine sarcoma, COL1A1-PDGFB fusion sarcoma, and SMARC-deficient uterine sarcoma. Key clinical, morphologic, immunophenotypic, and molecular features are reviewed, with emphasis on common differential diagnoses and pitfalls, and their impact on prognosis or management. Where applicable, the role of novel targeted therapies is discussed. A stepwise approach to uterine mesenchymal neoplasms can achieve a proper diagnosis and guide appropriate clinical management in most cases. Nonetheless, given the rarity of these tumors, their overlapping pathologic features, and rapid evolution in their classification and management, we advocate a low threshold for diagnostic consultation.     

基于规律成簇的间隔短回文重复序列及其相关蛋白技术检测乙型肝炎病毒共价闭合环状DNA方法的建立

目的建立一种基于规律成簇的间隔短回文重复序列及其相关蛋白(CRISPR/Cas13a)的乙型肝炎病毒(HBV)共价闭合环状DNA(HBV cccDNA)检测方法。方法提取2017年6月至2020年10月于首都医科大学附属北京佑安医院就诊的4例乙型肝炎患者肝脏总DNA后,使用HindⅢ内切酶和质粒安全性ATP依赖DNA酶(PSAD)分别进行酶切;根据松弛环状DNA(rcDNA)和cccDNA的结构差异,设计特异性扩增HBV cccDNA的引物,对酶切后的产物进行滚环扩增(RCA)和PCR扩增;并筛选crRNA,建立基于CRISPR/Cas13a技术的HBV cccDNA检测新方法。结果利用α-1-抗胰蛋白酶(A1AT)和HBV表面抗原(HBsAg)引物对双重酶切后的产物进行扩增,验证产物中HBV基因组的存在;利用HBV cccDNA和HBV rcDNA引物对PSDA酶切后的产物扩增,验证了产物中只存在HBV cccDNA;利用RCA后的阳性样本作为模板梯度稀释,然后进行PCR扩增转录后使用CRISPR/Cas13a检测,计算出检测下限为10拷贝/μl。结论本研究建立了RCA-PCR-CRISPR-Cas13a的新型检测方法,可对HBV cccDNA进行高灵敏度和高特异性检测,为乙型肝炎患者抗病毒治疗评估、治疗终点的确定以及调整治疗方案提供了有效的监测手段。     

Down‐regulation of KIF2A inhibits gastric cancer cell invasion via suppressing MT1‐MMP

Gastric cancer accounts for a sizeable proportion of global cancer mortality with high morbidity and poor prognosis. Kinesin superfamily proteins (KIFs) are microtubule‐dependent motor proteins that function as oncogenes in cancer cells, it has been discovered in recent years. Kinesin family member 2a (KIF2A), a member of the KIFs, has received attention for its role in carcinogenesis and its prognostic value in several human cancers such as breast cancer, colorectal cancer, and squamous cell carcinoma. However, the role of KIF2A in human gastric cancer remains unknown. In this study we aimed to explore the expression and biological functions of KIF2A in human gastric cancer cells, as well as to reveal its potential action mechanism. First, we found that KIF2A was markedly increased in gastric cancer cells (MKN‐28, MKN‐45, NCI‐N87 and SGC‐7901) compared to normal gastric mucosa epithelial cells (GES‐1). Then KIF2A was successfully silenced in MKN‐45 and SGC‐7901 cells to facilitate further research into its function. We discovered that KIF2A silencing can significantly inhibit the growth and invasion of MKN‐45 and SGC‐7901 cells in a time‐independent manner, accompanying a decreased expression of Membrane type 1‐matrix metalloproteinase (MT1‐MMP). When MT1‐MMP was reintroduced into MKN‐45 and SGC‐7901 cells in the KIF2A‐siRNA group, only invasion inhibition effects on MKN‐45 and SGC‐7901 cells induced by KIF2A silencing can be reversed. In conclusion, our study reveals that down‐regulation of KIF2A can inhibit gastric cancer cell invasion by suppressing MT1‐MMP.     

Identification of two abundant Aerococcus urinae cell wall-anchored proteins

Aerococcus urinae is an emerging pathogen that causes urinary tract infections, bacteremia and infective endocarditis. The mechanisms through which A. urinae cause infection are largely unknown. The aims of this study were to describe the surface proteome of A. urinae and to analyse A. urinae genomes in search for genes encoding surface proteins. Two proteins, denoted Aerococcal surface protein (Asp) 1 and 2, were through the use of mass spectrometry based proteomics found to quantitatively dominate the aerococcal surface. The presence of these proteins on the surface was also shown using ELISA with serum from rabbits immunized with the recombinant Asp. These proteins had a signal sequence in the amino-terminal end and a cell wall-sorting region in the carboxy-terminal end, which contained an LPATG-motif, a hydrophobic domain and a positively charged tail. Twenty-three additional A. urinae genomes were sequenced using Illumina HiSeq technology. Six different variants of asp genes were found (denoted asp1-6). All isolates had either one or two of these asp-genes located in a conserved locus, designated Locus encoding Aerococcal Surface Proteins (LASP). The 25 genomes had in median 13 genes encoding LPXTG-proteins (range 6–24). For other Gram-positive bacteria, cell wall-anchored surface proteins with an LPXTG-motif play a key role for virulence. Thus, it will be of great interest to explore the function of the Asp proteins of A. urinae to establish a better understanding of the molecular mechanisms by which A. urinae cause disease.     

凋亡抑制蛋白的研究进展

近年来新发现的凋亡抑制蛋白(inhibitor of apoptosis proteins,IAPs),是一类高度保守的内源性抗凋亡基因家族表达产物,广泛存在于许多物种如病毒、真核生物、哺乳动物中,起着抑制细胞凋亡的作用.IAPs主要通过抑制caspase,参与TNFR介导的信号转导等途径发挥抗凋亡作用,与恶性肿瘤、神经系统病变等密切相关.该文就IAPs的结构、抑制凋亡的机制及其在临床疾病中的研究现状作一综述.     

Potential application of small myristoylated protein-3 evaluated as recombinant antigen and a synthetic peptide containing its linear B-cell epitope for the serodiagnosis of canine visceral and human tegumentary leishmaniasis

Serological tests are important tools for the diagnosis of Leishmania infection. However, they are not effective markers to diagnose asymptomatic cases of visceral leishmaniasis (VL) and patients developing tegumentary leishmaniasis (TL), since antileishmanial antibodies can be encountered in low levels resulting in false-negative results in the serological trials. In this context, antigens able to be recognized by antibodies in sera from both VL and TL patients will be desirable to be employed in a more sensitivity and specific diagnosis of disease. In the present study, a conserved Leishmania protein, small myristoylated protein-3 (SMP-3), which was showed to be conserved in different Leishmania species and an effective vaccine candidate against Leishmania infantum infection in a murine model, was cloned and the recombinant protein was evaluated as a serological marker for the diagnosis of human TL and canine VL. In addition, a linear B cell-specific epitope (MQKDEESGEFKCEL) was identified, synthetized and also investigated as a serological marker. As antigen controls, rA2 protein and antigenic Leishmania extracts (SLA) were used. Results showed that ELISA-rSMP-3 and ELISA-Peptide presented sensitivity and specificity values higher than 90% in both diseases in humans and canids, having identified all asymptomatic cases and did not present cross-reaction with cross-reactivity diseases in both mammalian hosts. On the other hand, sensitivity and specificity values were worst when rA2 or SLA were used as antigens in humans and dogs. In conclusion, results showed the efficacy and Leishmania SMP-3 protein, employed as a recombinant antigen or a B cell epitope, for the improvement of the serodiagnosis of human TL and canine VL. This candidate can be tested in other diagnostic platforms, such as rapid immunochromatographic dipstick tests, aiming its use in epidemiological studies in remote areas where laboratories are not readily accessible for conventional assays.