An AS-qPCR-based method for the detection of Alzheimer's disease-related SNPs.

Alzheimer's disease (AD) is one of the most serious neurodegenerative diseases in the world and has a strong genetic predisposition. At present, there is still no effective method for the early diagnosis and prevention of AD. Accumulating evidence shows the association of several loci with AD risk, such as apolipoprotein E (APOE) and translocase of outer mitochondrial membrane 40 (TOMM40). However, for routine disease diagnosis in clinics, genotype detection methods based on gene sequencing technology are time-consuming and excessively costly. Thus, in this study, we developed a high-sensitivity, low-cost, and convenient single nucleotide polymorphism (SNP) detection assay method based on allele-specific quantitative polymerase chain reaction (AS-qPCR) technology, which can be used to determine the SNP genotype in APOE and TOMM40. A total of 40 patients were recruited from the outpatient department of the memory clinic of Dongzhimen Hospital, Beijing University of Chinese Medicine. The SNP detection assay method includes three steps. First, positive plasmids with different genotypes (TT/CC/TC) in APOE rs429358, rs7412, and TOMM40 rs11556505 were prepared. Second, 3'-T/3'-C primers were designed to amplify these positive plasmids for each SNP site. Finally, we calculated the log10 of the copy number ratio for each positive plasmid, and the genotype interpretation interval was established. Based on this method, we investigated whether the SNPs in 40 patients could be accurately calculated using AS-qPCR technology. The accuracy of SNP detection was verified by PCR-Pooling sequencing. The results showed that SNP genotypes assessed by AS-qPCR technology corresponded perfectly to the results obtained by conventional DNA sequencing. We have developed a genotype detection method for AD based on AS-qPCR, which can be performed easily, rapidly, accurately, and at low cost. The method will contribute to the early diagnosis of patients with late-onset Alzheimer's and the detection of large clinical samples in the future.

Dual-energy CT of the pancreas: comparison between virtual non-contrast images and true non-contrast images in the detection of pancreatic lesion.

PURPOSE: To evaluate the image quality and diagnostic performance for pancreatic lesion between true non-contrast (TNC) and virtual non-contrast (VNC) images obtained from the dual-energy computed tomography (DECT). METHODS: One hundred six patients with pancreatic mass underwent contrast-enhanced DECT examinations were retrospectively included in this study. VNC images of the abdomen were generated from late arterial (aVNC) and portal (pVNC) phases. For quantitative analysis, the attenuation differences and reproducibility of abdominal organs were compared between TNC and aVNC/pVNC measurements. Qualitatively image quality was assessed by two radiologists using a five-point scale, and they independently compared the detection accuracy of pancreatic lesions between TNC and aVNC/pVNC images. The volume CT dose index (CTDIvol) and size-specific dose estimates (SSDE) were recorded to evaluate the potential dose reduction when using VNC reconstruction to replace the unenhanced phase. RESULTS: A total of 78.38% (765/976) of the attenuation measurement pairs were reproducible between TNC and aVNC images, and 71.0% (693/976) between TNC and pVNC images. In triphasic examinations, a total of 108 pancreatic lesions were found in 106 patients, and no significant difference in detection accuracy was found between TNC and VNC images (p = 0.587-0.957). Qualitatively, image quality was rated diagnostic (score ≥ 3) in all the VNC images. Calculated CTDIvol and SSDE reduction of about 34% could be achieved by omitting the non-contrast phase. CONCLUSION: VNC images of DECT provide diagnostic image quality and accurate pancreatic lesions detection, which are a promising alternative to unenhanced phase with a substantial reduction of radiation exposure in clinical routine.

Progress and Prospect of Immunotherapy for Triple-Negative Breast Cancer.

Breast cancer is the most commonly diagnosed cancer (estimated 2.3 million new cases in 2020) and the leading cause of cancer death (estimated 685,000 deaths in 2020) in women globally. Breast cancers have been categorized into four major molecular subtypes based on the immunohistochemistry (IHC) expression of classic hormone and growth factor receptors including the estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2), as well as a proliferation marker Ki-67 protein expression. Triple-negative breast cancer (TNBC), a breast cancer subtype lacking ER, PR, and HER2 expression, is associated with a high metastatic potential and poor prognosis. TNBC accounts for approximately only 15%-20% of new breast cancer diagnoses; it is responsible for most breast cancer-related deaths due to the lack of targeted treatment options for this patient population, and currently, systemic chemotherapy, radiation, and surgical excision remain the major treatment modalities for these patients with TNBC. Although breast cancer patients in general do not have a robust response to the immunotherapy, a subset of TNBC has been demonstrated to have high tumor mutation burden and high tumor-infiltrating lymphocytes, resembling the features observed on melanoma or lung cancers, which can benefit from the treatment of immune checkpoint inhibitors (ICIs). Therefore, the immunogenic nature of this aggressive disease has presented an opportunity for the development of TNBC-targeting immunotherapies. The recent US Food and Drug Administration approval of atezolizumab in combination with the chemotherapeutic agent nab-paclitaxel for the treatment of PD-L1-positive unresectable, locally advanced, or metastatic TNBC has led to a new era of immunotherapy in TNBC treatment. In addition, immunotherapy becomes an active research area, both in the cancer biology field and in the oncology field. In this review, we will extend our coverage on recent discoveries in preclinical research and early results in clinical trials from immune molecule-based therapy including cytokines, monoclonal antibodies, antibody-drug conjugates, bi-specific or tri-specific antibodies, ICIs, and neoantigen cancer vaccines; oncolytic virus-based therapies and adoptive immune cell transfer-based therapies including TIL, chimeric antigen receptor-T (CAR-T), CAR-NK, CAR-M, and T-cell receptor-T. In the end, we will list a series of the challenges and opportunities in immunotherapy prospectively and reveal novel technologies such as high-throughput single-cell sequencing and CRISPR gene editing-based screening to generate new knowledges of immunotherapy.

Simultaneous measurement of the antibody responses against SARS-CoV-2 and its multiple variants by a phage display mediated immuno-multiplex quantitative PCR-based assay.

To combat the continued pandemic of COVID-19, multiplex serological assays have been developed to comprehensively monitor the humoral immune response and help to design new vaccination protocols to different SARS-CoV-2 variants. However, multiplex beads and stably transfected cell lines require stringent production and storage conditions, and assays based on flow cytometry is time-consuming and its application is therefore restricted. Here, we describe a phage display system to distinguish the differences of immune response to antigenic domains of multiple SARS-CoV-2 variants simultaneously. Compared with linear peptides, the recombinant antigens displayed on the phage surface have shown some function that requires the correct folding to form a stable structure, and the binding efficiency between the recombinant phage and existing antibodies is reduced by mutations on antigens known to be important for antigen-antibody interaction. By using Phage display mediated immuno-multiplex quantitative PCR (Pi-mqPCR), the binding efficiency between the antibody and antigens of different SARS-CoV-2 variants can be measured in one amplification reaction. Overall, these data show that this assay is a valuable tool to evaluate the humoral response to the same antigen of different SARS-CoV-2 variants or antigens of different pathogens. Combined with high-throughput DNA sequencing technology, this phage display system can be further applied in monitoring humoral immune response in a large population before and after vaccination.