[The Polymorphism Analysis of HLA Class II Alleles Based on Next-Generation Sequencing and Prevention Strategy for Allele Dropout].

OBJECTIVE: To investigate the accuracy of next-generation sequencing technology (NGS) in detecting the polymorphisms of HLA-DRB1, DQB1, DQA1, DRB3, DRB4, DRB5, DPA1 and DPB1 alleles in randomly-selected unrelated healthy individuals from Shenzhen Han population, investigate the potential reason for HLA-DRB1 allele dropout in routine NGS, and establish an internal quality control system. METHODS: NGS-based HLA class II genotyping was performed on 1 012 samples using the MiSeqDxTM platform. The suspected missed alleles indicated by the quality control software and HLA-DRB1 homozygotes were confirmed by PCR-SSOP or PCR-SBT methods. RESULTS: A total of 139 alleles were detected, including HLA-DRB1(45), DRB3(7), DRB4(5), DRB5(7), DQA1(17), DQB1(21), DPA1(10) and DPB1(27). HLA-DRB1*09:01(17.09%),15:01(10.72%); DRB3*02:02(25.99%),03:01(10.18%); DRB4*01:03(36.46%); DRB5*01:01(15.42%); DQA1*01:02(20.01%),03:02(17.19%); DQB1*03:01(19.47%),03:03(17.98%), 05:02(11.66%), 06:01(10.67%); DPA1*02:02(54.45%), 01:03(31.18%) and DPB1*05:01(39.13%), 02:01(16.90%) alleles were the most common alleles in Shenzhen Han population (frequencies >10%). There was no statistical difference between the gene frequencies of HLA-DRB1 and DQB1 loci in our study. The HLA Common and Well-Documented Alleles in China (CWD2.4) (χ2=12.68, P >0.05). 94 cases of HLA-DRB1 homozygous samples detected by NGS were retested by PCR-SSOP or SBT method, and one case of allele dropout at HLA-DRB1 locus was found. SBT method confirmed that the allele of DRB1*04:03 was missed. The laboratory internal quality control system was established. Two cases of new alleles were detected and named by WHO Nomenclature Committee for Factors of the HLA System. CONCLUSION: The HLA genotyping results based on NGS showed a significantly lower ambiguity rate. The HLA class II alleles exhibit genetic polymorphism in the Han population of unrelated healthy individuals in Shenzhen. The independent method based on NGS in clinical histocompatibility testing has limitations and requires internal quality control strategies to avoid allele-dropout events.

Characterization of the novel HLA-A*31:01:53 allele by next generation sequencing in a Chinese individual.

HLA-A*31:01:53 differs from HLA-A*31:01:02:01 by one nucleotide change at nucleotide 900 in exon 5 from G to A.

Two novel HLA class II alleles, HLA-DRB1*09:57 and HLA-DRB1*09:58 in Chinese individuals.

Compared with HLA-DRB1*09:01:02:05, the alleles HLA-DRB1*09:57 and HLA-DRB1*09:58 each show one nucleotide change, respectively.

Characterization of the novel HLA-A*30:211 allele by next generation sequencing in a Chinese individual.

HLA-A*30:211 differs from HLA-A*30:01:01:01 by one nucleotide change at nucleotide 344 in exon 3 from G to C.

Full-length sequence of the novel HLA-A*02:1103 allele by next generation sequencing in a Chinese individual.

HLA-A*02:1103 differs from HLA-A*02:01:01:01 by one nucleotide change at nucleotide 811 in exon 4 from G to A.

The novel HLA-B*13:179 variant detected by next generation sequencing in a Chinese individual.

HLA-B*13:179 differs from HLA-B*13:99 by one nucleotide substitution at position 829(A>G) in exon 4.

The novel HLA-B*15:664 allele, identified by next-generation sequencing in a Chinese individual.

B*15:664 differs from B*15:02:01:01 by one nucleotide change at nucleotide 755 in exon 4 from C to G.

A single nucleotide substitution in exon 5 produced the novel allele, HLA-B*40:01:83.

HLA-B*40:01:83, carrying a single nucleotide substitution in exon 5 is described.

Identification of the novel HLA-B*46:01:33 allele by next generation sequencing in a Chinese individual.

B*46:01:33 differs from B*46:01:01:01 by one nucleotide change at nucleotide 105 in exon 2 from C to T.

The Accurate Imaging of Collective Gold Nanorods with a Polarization-Dependent Dark-Field Light Scattering Microscope.

Anisotropic nanomaterials, such as gold nanorods (AuNRs), could be employed as an orientation platform due to their polarization-dependent surface plasmon resonance. However, a variety of factors would affect the dark-field light scattering imaging of anisotropic nanomaterials, resulting in an unstable signal, which is not advantageous to its further application. In this work, the localized surface plasmon resonance properties of a few AuNRs at different angles were excited by polarization with a conventional dark-field microscope, in which it was found that the ratio of AuNRs' light scattering intensity at different polarization angles (I) to that without a polarizer (I0) reflected the orientation information of AuNRs. Furthermore, the light scattering signal ratio between the parallel polarization (Ip) and that without a polarizer (I0) was closely related with the aspect ratio of AuNRs, which could not be affected by external conditions. To verify this concept, a highly sensitive and selective assay of the alkaline phosphatase activity in human serum was successfully developed based on the chemical etching of AuNRs, resulting in a lower aspect ratio and a lesser Ip/I0. This result holds great promise for polarization-dependent colorimetric nanomaterials and single-particle tracers in living cells.

A novel HLA-DQB1*04 variant, HLA-DQB1*04:90, identified in a Chinese Han individual.

Identification of the novel HLA-DQB1*04:90 allele that differs from DQB1*04:01:01:01 at nucleotide 183 in exon 2 from A to T.

Full-length sequence of the novel HLA-C*03:566 allele by next-generation sequencing in a Chinese individual.

HLA-C*03:566 differs from HLA-C*03:04:01:02 by one nucleotide substitution at nucleotide 92 in exon 2 from A to G.

Full-length sequence of the novel HLA-C*03:04:74 allele by next generation sequencing in a Chinese individual.

C*03:04:74 differs from C*03:04:01:02 by one nucleotide change at nucleotide 1047 in exon 6 from G to A.

In Situ Imaging of Ion Motion in a Single Nanoparticle: Structural Transformations in Selenium Nanoparticles.

Intraparticle ion motions are critical to the structure and properties of nanomaterials, but rarely disclosed. Herein, in situ visualization of ion motions in a single nanoparticle is presented by dark-field microscopy imaging, which shows HgCl2 -induced structural transformation of amorphous selenium nanoparticles (SeNPs) with the main composition of Se8 . Owing to the high binding affinity with selenium and coulomb interactions, Hg2+ ions can permeate into the interior of SeNPs, making the amorphous Se8 turn to polycrystalline Hg3 Se2 Cl2 . As a proof of concept, SeNPs then serve as a highly effective scavenger for selective removal of Hg2+ ions from solution. This new finding offers visual proof for the photophysical process involving intraparticle ion motion, demonstrating that tracking the ion motions is a novel strategy to comprehend the formation mechanism with the purpose of developing new nanostructures like nanoalloys and nano metal compounds.

[Exclusion of HLA-C Genotype with Zero Mismatched PCR-SBT Results by Next Generation Sequencing].

OBJECTIVE: Three cases of rare alleles of HLA-C with zero mismatched PCR-SBT results were analyzed by full-length sequencing to determine the true genotypes. METHODS: Three rare HLA-C alleles with zero mismatched PCR-SBT results were screened from clinical transplant matching samples, and the full-length sequence was detected by next-generation sequencing technology. RESULTS: The results of PCR-SBT typing of 3 samples were: HLA-C*03:04, 12:167; HLA-C*07:291, 15:02; HLA-C*01:43, 08:16. Other alleles were not in the CWD table of common and confirmed HLA alleles in China (version 2.3) except common allele HLA-C*03:04, HLA-C*15:02. NGS full-length sequencing revealed that the HLA-C genotypes of the three samples were a combination of common alleles and novel alleles, and the three novel alleles had a base mutation in exons 6, 2, and 4, respectively. The novel allele sequences have been submitted to the Genbank database (MK629722, MK335474, MK641803), which were officially named HLA-C*03:04:74, HLA-C*15:192, HLA-C*08:01:25 by the WHO HLA Nomenclature Committee. The HLA high-resolution typing results of 3 samples were: HLA-C*03:04:74, HLA-C*12:03; HLA-C*07:02, HLA-C*15:192; HLA-C*01:02, HLA-C*08:01:25. CONCLUSION: HLA typing results containing rare alleles should be treated cautiously, and the full-length sequence should be verified by NGS or cloning. The laboratory finally confirmed that the 3 cases of PCR-SBT zero mismatch HLA-C genotypes are the combination of common alleles and novel alleles by NGS sequencing, which provides an accurate basis for clinical transplantation matching and enriches the human HLA genetic database.

Dual-aptamer-based enzyme linked plasmonic assay for pathogenic bacteria detection.

Development of rapid, sensitive, and selective method for pathogenic bacteria detection is of great importance for food safety, medical diagnostic, and environmental monitoring. Currently, most techniques for low numbers of bacteria detection require advanced instrumentation or skilled operators. Herein, we present a facile colorimetric detection platform for bacterial detection using Ag nanoplates as chromogenic substrate, which takes advantages of the high specificity and affinity of aptamer and the ability of catalase to hydrolyze H2O2 that can etch Ag nanoplates. By introducing catalase to the sandwich structure composed by dual-aptamer recognition strategy, bacteria detection signal is converted to the peak shift of LSPR and colorimetric change. This proposed method allows a fast naked-eye detection of S. aureus at the concentration of 60 CFU/mL based on the combination of streptavidin-biotin system and inherent sensitivity of plasmonic Ag nanoplates. Owing to the high selectivity and sensitivity, as well as the low-cost and good adaptability, this plasmonic assay is expected to be suitable for pathogenic bacteria detection in resource-limited settings.

The restructure of Au@Ag nanorods for cell imaging with dark-field microscope.

The facile and noninjurious image of cells with high resolution and low toxicity is essential since imaging can offer rich and direct information and insights into metabolic activities, clinical diagnosis, drug delivery and cancer therapy. In this contribution, a smart imaging probe was employed as a contrast agent for dark-field cell imaging. Au core/Ag shell nanorods (Au@Ag NRs) that characterized by X-ray diffraction and X-ray photoelectron spectroscopy, formed Au@Ag@AgI NRs when exposed to iodine, which greatly enhanced the light scattering of nanorods. Herein, the silver shell acted as the response element for iodine as well as the protective agent for Au core. When conjugated with folate, the nanorods can be used to image human cervical cancer cells (HeLa cells) under a dark-field microscope. Nanorods were demonstrated with excellent tumor cellular uptake ability without obvious cytotoxicity, making them ideal candidates in biosensing and bioimaging applications.

A high-integrated DNA biocomputing platform for MicroRNA sensing in living cells.

DNA logic computing has captured increasing interest due to its ability to assemble programmable DNA computing elements for disease diagnosis, gene regulation, and targeted therapy. In this work, we developed an aptamer-equipped high-integrated DNA biocomputing platform (HIDBP-A) with a dual-recognition function that enabled cancer cell targeting. Dual microRNAs were the input signals and can perform AND logic operations. Compared to the free DNA biocomputing platform (FDBP), the integration of all computing elements into the same DNA tetrahedron greatly improved logic computing speed and efficiency owing to the confinement effect reflected by the high local concentration of computing elements. As a proof of concept, the utilization of microRNA as the input signal was beneficial for improving the scalability and flexibility of the sequence design of the logic nano-platform. Given that the different microRNAs were over-expressed in cancer cells, this new HIDBP-A has great promise in accurate diagnosis and logic-controlled disease treatment.

Characterization of a novel variant allele, HLA-C*03:587, identified in a Chinese Han individual.

The novel HLA-C*03:587 allele differs from the closest allele C*03:03:01:01 in exon 5.

Plasmonic biosensor for the highly sensitive detection of microRNA-21 via the chemical etching of gold nanorods under a dark-field microscope.

MicroRNAs involved in tumor-related tissues at abnormal expression level present tremendous potential in the early diagnosis of cancers. However, their intrinsic shortcomings, for instance, low abundance and high sequence homology, make it challengeable to quantify them with high sensitivity and selectivity. Herein, a highly sensitive platform with great specificity was developed for microRNA-21 based on the produced-I2 triggered chemical etching of gold nanorods to a smaller size, resulting in a significant blue shift and a great intensity decrease in the localized surface plasmon resonance (LSPR) scattering. The synergism of strand displacement and enzymatic reaction enabled the proposed strategy with a high sensitivity and selectivity toward microRNA-21 in a dynamic range from 0.1 to 10,000 pM and a low limit of detection of 71.22 fM (3σ/k) by dark-field microscope. Additionally, the remarkable discrimination of single nucleotide difference suggested the superior selectivity towards microRNA-21, which presented a satisfactory recovery in human serum samples. The proposed plasmon platform could also serve as a universal and sensitive detection of cancer biomarkers, presenting the amusing application prospects in the early diagnosis of various cancers by adapting the corresponding nucleic acid sequences.