Background:Common methods for identification of DNA sequence variants use gel electrophoresis or column separation after PCR. Methods: We developed a method for sequence variant analysis requiring only PCR and amplicon melting analysis. One of the PCR primers was fluorescently labeled. After PCR, the melting transition of the amplicon was monitored by high-resolution melting analysis. Different homozygotes were distinguished by amplicon melting temperature (T m ). Heterozygotes were identified by low-temperature melting of heteroduplexes, which broadened the overall melting transition. In both cases, melting analysis required ϳ1 min and no sample processing was needed after PCR. Results: Polymorphisms in the HTR2A (T102C), ␤-globin [hemoglobin (Hb) S, C, and E], and cystic fibrosis (F508del, F508C, I507del, I506V) genes were analyzed. Heteroduplexes produced by amplification of heterozygous DNA were best detected by rapid cooling (>2°C/s) of denatured products, followed by rapid heating during melting analysis (0.2-0.4°C/s). Heterozygotes were distinguished from homozygotes by a broader melting transition, and each heterozygote had a uniquely shaped fluorescent melting curve. All homozygotes tested were distinguished from each other, including Hb AA and Hb SS, which differed in T m by <0.2°C. The amplicons varied in length from 44 to 304 bp. In place of one labeled and one unlabeled primer, a generic fluorescent oligonucleotide could be used if a 5 tail of identical sequence was added to one of the two unlabeled primers.

Background Malignant transformation and progression of cancer are driven by the co-evolution of cancer cells and their dysregulated tumor microenvironment (TME). Recent studies on immunotherapy demonstrate the efficacy in reverting the anti-tumoral function of T cells, highlighting the therapeutic potential in targeting certain cell types in TME. However, the functions of other immune cell types remain largely unexplored. Results We conduct a single-cell RNA-seq analysis of cells isolated from tumor tissue samples of non-small cell lung cancer (NSCLC) patients, and identify subtypes of tumor-infiltrated B cells and their diverse functions in the progression of NSCLC. Flow cytometry and immunohistochemistry experiments on two independent cohorts confirm the co-existence of the two major subtypes of B cells, namely the naïve-like and plasma-like B cells. The naïve-like B cells are decreased in advanced NSCLC, and their lower level is associated with poor prognosis. Co-culture of isolated naïve-like B cells from NSCLC patients with two lung cancer cell lines demonstrate that the naïve-like B cells suppress the growth of lung cancer cells by secreting four factors negatively regulating the cell growth. We also demonstrate that the plasma-like B cells inhibit cancer cell growth in the early stage of NSCLC, but promote cell growth in the advanced stage of NSCLC. The roles of the plasma-like B cell produced immunoglobulins, and their interacting proteins in the progression of NSCLC are further validated by proteomics data. Conclusion Our analysis reveals versatile functions of tumor-infiltrating B cells and their potential clinical implications in NSCLC.

ABSTRACT:Zoning which is to divide the study area into different zones according to their geographical differences at the global, national or regional level, includes natural division, economic division, geographical zoning of departments, comprehensive zoning and so on. Zoning is of important practical significance, for example, knowing regional differences and characteristics, regional research and regional development planning, understanding the favorable and unfavorable conditions of the regional development etc. Geographical environment is arising from the geographical position linkages. Geographical environment unit division is also a type of zoning. The geographical environment indicators are deeply studied and summed up in the article, including the background, the associated and the potential. The background indicators are divided into four categories, such as the socio-economic, the political and military, the strategic resources and the ecological environment, which can be divided into more sub-indexes. While the sub-indexes can be integrated to comprehensive index system by weighted stacking method. The Jenks natural breaks classification method, also called the Jenks optimization method, is a data classification method designed to determine the best arrangement of values into different classes. This is done by seeking to minimize each class's average deviation from the class mean, while maximizing each class's deviation from the means of the other groups. In this paper, the experiment of Chinese surrounding geographical environment unit division has been done based on the natural breaks (jenks) method, the geographical environment index system and the weighted stacking method, taking South Asia as an example. The result indicates that natural breaks (jenks) method is of good adaptability and high accuracy on the geographical environment unit division.The geographical environment research was originated in the geopolitics and flourished in the geo-economics. The main representatives of the geopolitics are German geographer Friedrich Ratzel, British geographer Mackinder and American geographical politician Nicholas John Spykman etc. The main representative of the geo-economics is American geographical economist Edward Luttwak. China has the most neighboring countries in the world, and its geographical environment is extremely complex. With the continuous development of globalization, China's relations with neighboring countries have become more complex and more closely. So it is very meaningful to have depth research on geographical environment unit division of China.

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