Profiling SARS-CoV-2 HLA-I peptidome reveals T cell epitopes from out-of-frame ORFs.

T cell-mediated immunity plays an important role in controlling SARS-CoV-2 infection, but the repertoire of naturally processed and presented viral epitopes on class I human leukocyte antigen (HLA-I) remains uncharacterized. Here, we report the first HLA-I immunopeptidome of SARS-CoV-2 in two cell lines at different times post infection using mass spectrometry. We found HLA-I peptides derived not only from canonical open reading frames (ORFs) but also from internal out-of-frame ORFs in spike and nucleocapsid not captured by current vaccines. Some peptides from out-of-frame ORFs elicited T cell responses in a humanized mouse model and individuals with COVID-19 that exceeded responses to canonical peptides, including some of the strongest epitopes reported to date. Whole-proteome analysis of infected cells revealed that early expressed viral proteins contribute more to HLA-I presentation and immunogenicity. These biological insights, as well as the discovery of out-of-frame ORF epitopes, will facilitate selection of peptides for immune monitoring and vaccine development.

Stable SERS Detection of Lactobacillus fermentum Using Optical Tweezers in a Microfluidic Environment.

Rapid identification of fermented lactic acid bacteria has long been a challenge in the brewing industry. This study combined label-free surface-enhanced Raman scattering (SERS) and optical tweezer technology to construct a test platform within a microfluidic environment. Six kinds of lactic acid bacteria common in industry were tested to prove the stability of the SERS spectra. The results demonstrated that the utilization of optical tweezers to securely hold the bacteria significantly enhanced the stability of the SERS spectra. Furthermore, SVM and XGBoost machine learning algorithms were utilized to analyze the obtained Raman spectra for identification, and the identification accuracies exceeded 95% for all tested lactic acid bacteria. The findings of this study highlight the crucial role of optical tweezers in improving the stability of SERS spectra by capturing bacteria in a microfluidic environment, prove that this technology could be used in the rapid identification of lactic acid bacteria, and show great significance in expanding the applicability of the SERS technique for other bacterial testing purposes.

Dysmorphic Gallbladder Found on Prenatal Ultrasound as a Hint for Biliary Atresia.

Biliary atresia (BA) is a rare but devastating cholangiopathy. We report a case series of dysmorphic gallbladders detected during prenatal ultrasound, which were confirmed as BA after birth. We present the prenatal ultrasound findings as well as integral follow-up, with an aim to raise awareness regarding the association between BA and dysmorphic gallbladder. Although this dysmorphic gallbladder is a strong hint for BA, it is also important to search for other related sonographic features, such as the presence of microcysts at the hepatic hilum, dilated right hepatic artery, and seroperitoneum, which may provide more evidence for the diagnosis of BA.

OsGRETCHENHAGEN3-2 modulates rice seed storability via accumulation of abscisic acid and protective substances.

Seed storability largely determines the vigor of seeds during storage and is significant in agriculture and ecology. However, the underlying genetic basis remains unclear. In the present study, we report the cloning and characterization of the rice (Oryza sativa) indole-3-acetic acid (IAA)-amido synthetase gene GRETCHEN HAGEN3-2 (OsGH3-2) associated with seed storability. OsGH3-2 was identified by performing a genome-wide association study in rice germplasms with linkage mapping in chromosome substitution segment lines, contributing to the wide variation of seed viability in the populations after long periods of storage and artificial ageing. OsGH3-2 was dominantly expressed in the developing seeds and catalyzed IAA conjugation to amino acids, forming inactive auxin. Transgenic overexpression, knockout, and knockdown experiments demonstrated that OsGH3-2 affected seed storability by regulating the accumulation level of abscisic acid (ABA). Overexpression of OsGH3-2 significantly decreased seed storability, while knockout or knockdown of the gene enhanced seed storability compared with the wild-type. OsGH3-2 acted as a negative regulator of seed storability by modulating many genes related to the ABA pathway and probably subsequently late embryogenesis-abundant proteins at the transcription level. These findings shed light on the molecular mechanisms underlying seed storability and will facilitate the improvement of seed vigor by genomic breeding and gene-editing approaches in rice.

Genetic Dissection of Seed Dormancy using Chromosome Segment Substitution Lines in Rice (Oryza sativa L.).

Timing of germination determines whether a new plant life cycle can be initiated; therefore, appropriate dormancy and rapid germination under diverse environmental conditions are the most important features for a seed. However, the genetic architecture of seed dormancy and germination behavior remains largely elusive. In the present study, a linkage analysis for seed dormancy and germination behavior was conducted using a set of 146 chromosome segment substitution lines (CSSLs), of which each carries a single or a few chromosomal segments of Nipponbare (NIP) in the background of Zhenshan 97 (ZS97). A total of 36 quantitative trait loci (QTLs) for six germination parameters were identified. Among them, qDOM3.1 was validated as a major QTL for seed dormancy in a segregation population derived from the qDOM3.1 near-isogenic line, and further delimited into a genomic region of 90 kb on chromosome 3. Based on genetic analysis and gene expression profiles, the candidate genes were restricted to eight genes, of which four were responsive to the addition of abscisic acid (ABA). Among them, LOC_Os03g01540 was involved in the ABA signaling pathway to regulate seed dormancy. The results will facilitate cloning the major QTLs and understanding the genetic architecture for seed dormancy and germination in rice and other crops.

Deep learning-based ultra-fast identification of Raman spectra with low signal-to-noise ratio.

Ensuring the correct use of cell lines is crucial to obtaining reliable experimental results and avoiding unnecessary waste of resources. Raman spectroscopy has been confirmed to be able to identify cell lines, but the collection time is usually 10-30 s. In this study, we acquired Raman spectra of five cell lines with integration times of 0.1 and 8 s, respectively, and the average accuracy of using long-short memory neural network to identify the spectra of 0.1 s was 95%, and the average accuracy of identifying the spectra of 8 s was 99.8%. At the same time, we performed data enhancement of 0.1 s spectral data by real-valued non-volume preserving method, and the recognition average accuracy of long-short memory neural networks recognition of the enhanced spectral data was improved to 96.2%. With this method, we shorten the acquisition time of Raman spectra to 1/80 of the original one, which greatly improves the efficiency of cell identification.

Strongly plasticized gelatin-based hydrogel for flexible encapsulation of complex-shaped electronic devices.

The growth of environmentally sensitive complex-shaped electronic devices (ECEDs) has led to a surging demand for flexible electromagnetic wave (EMW) absorbers. Herein, the water loss property of hydrogel was ingeniously applied for the flexible encapsulation (FE) of ECEDs. To be specific, saturated state (SGT) hydrogels were prepared by chemical cross-linking, and the hydrogen bond dissipation network promoted FE. Additionally, SGT has an effective absorption bandwidth (EAB) of 6.04 GHz at 1.65 mm due to the presence of dipole polarization. With the loss of water, SGT transitions to its natural state (NGT), and the decreasing conductivity leads to better impedance matching. NGT exhibited a broader EAB (9.20 GHz at 2.65 mm) and also strength and lightness (density of 0.3 g cm-3). Furthermore, the semi-automatic reversible cyclic transformation between SGT and NGT gels further broadens application scenarios. GT gel combines self-encapsulation and self-optimized performance as a potential EMW absorber for FE.

Precision isolation and cultivation of single cells by vortex and flat-top laser ejection.

Single-cell isolation stands as a critical step in single-cell studies, and single-cell ejection technology based on laser induced forward transfer technology (LIFT) is considered one of the most promising methods in this regard for its ability of visible isolating single cell from complex samples. In this study, we improve the LIFT technology and introduce optical vortex laser-induced forward transfer (OV-LIFT) and flat-top laser-induced forward transfer (FT-LIFT) by utilizing spatial light modulator (SLM), aiming to enhance the precision of single-cell sorting and the cell's viability after ejection. Experimental results demonstrate that applying vortex and flat-top beams during the sorting and collection process enables precise retrieval of single cells within diameter ranges of 50 µm and 100 µm, respectively. The recovery rates of Saccharomyces cerevisiae and Escherichia coli DH5α single cell ejected by vortex beam are 89 and 78%, by flat-top beam are 85 and 57%. When employing Gaussian beam sorting, the receiving range extends to 400 µm, with cultivation success rates of S. cerevisiae and E. coli DH5α single cell are 48 and 19%, respectively. This marks the first application of different mode beams in the ejection and cultivation of single cells, providing a novel and effective approach for the precise isolation and improving the viability of single cells.

Prenatal ultrasound diagnosis of congenital vertical talus.

OBJECTIVE: This study aimed to analyze the ultrasound characteristics of fetal congenital vertical talus (CVT) to provide a detailed basis for the prenatal diagnosis of CVT. METHODS: We retrospectively analyzed the ultrasonographic findings of fetuses with CVT confirmed by X-ray, surgery, or autopsy from 2010 to 2020. Clinical characteristics and ultrasonographic findings of CVT, including foot morphology, ossification center of the calcaneus and talus, associated deformities, and chromosomal test results, were recorded. RESULTS: Thirteen patients diagnosed with CVT by prenatal ultrasound were confirmed postpartum. Nine cases were bilateral, and four were unilateral. Under two-dimensional ultrasound, 13/13 cases had abnormal foot morphology, and 10 of 13 cases (76.9%) showed that the ossification center of the talus moved downward, and the calcaneus moved laterally. Under three-dimensional ultrasound, 11 cases (84.6%) presented a "rocking chair" appearance, and two cases did not obtain satisfactory three-dimensional image due to oligohydramnios and fetal position. In this group of cases, two cases (15.4%) were isolated CVT, and the other 11 cases (84.6%) were complicated with other abnormalities. Eleven cases of non-isolated CVT and 1 case of isolated CVT were induced, and another patient with isolated CVT had undergone postnatal surgery, which had been followed up for 8 years and recovered well. CONCLUSIONS: The combination of fetal foot morphology, ossification center position of the calcaneus and talus, and three-dimensional ultrasound can provide a reliable diagnosis of CVT. Furthermore, we should pay more attention to the evaluation of other systemic and chromosomal abnormalities in CVT cases.

Single cell multiomic analysis reveals diabetes-associated ß-cell heterogeneity driven by HNF1A.

Broad heterogeneity in pancreatic ß-cell function and morphology has been widely reported. However, determining which components of this cellular heterogeneity serve a diabetes-relevant function remains challenging. Here, we integrate single-cell transcriptome, single-nuclei chromatin accessibility, and cell-type specific 3D genome profiles from human islets and identify Type II Diabetes (T2D)-associated ß-cell heterogeneity at both transcriptomic and epigenomic levels. We develop a computational method to explicitly dissect the intra-donor and inter-donor heterogeneity between single ß-cells, which reflect distinct mechanisms of T2D pathogenesis. Integrative transcriptomic and epigenomic analysis identifies HNF1A as a principal driver of intra-donor heterogeneity between ß-cells from the same donors; HNF1A expression is also reduced in ß-cells from T2D donors. Interestingly, HNF1A activity in single ß-cells is significantly associated with lower Na+ currents and we nominate a HNF1A target, FXYD2, as the primary mitigator. Our study demonstrates the value of investigating disease-associated single-cell heterogeneity and provides new insights into the pathogenesis of T2D.

Analysis of genome and methylation changes in Chinese indigenous chickens over time provides insight into species conservation.

Conservation of natural resources is a vital and challenging task. Numerous animal genetic resources have been effectively conserved worldwide. However, the effectiveness of conservation programmes and the variation information of species have rarely been evaluated. Here, we performed whole-genome and whole-genome bisulfite sequencing of 90 Chinese indigenous chickens, which belonged to the Tibetan, Wenchang and Bian chicken breeds, and have been conserved under different conservation programmes. We observed that low genetic diversity and high DNA methylation variation occurs during ex situ in vivo conservation, while higher genetic diversity and differentiation occurs during in situ conservation. Further analyses revealed that most DNA methylation signatures are unique within ex situ in vivo conservation. Moreover, a high proportion of differentially methylated regions is found in genomic selection regions, suggesting a link between the effects of genomic variation and DNA methylation. Altogether our findings provide valuable information about genetic and DNA methylation variations during different conservation programmes, and hold practical relevance for species conservation.

Genomic insights into the recent chromosome reduction of autopolyploid sugarcane Saccharum spontaneum.

Saccharum spontaneum is a founding Saccharum species and exhibits wide variation in ploidy levels. We have assembled a high-quality autopolyploid genome of S. spontaneum Np-X (2n = 4x = 40) into 40 pseudochromosomes across 10 homologous groups, that better elucidates recent chromosome reduction and polyploidization that occurred circa 1.5 million years ago (Mya). One paleo-duplicated chromosomal pair in Saccharum, NpChr5 and NpChr8, underwent fission followed by fusion accompanied by centromeric split around 0.80 Mya. We inferred that Np-X, with x = 10, most likely represents the ancestral karyotype, from which x = 9 and x = 8 evolved. Resequencing of 102 S. spontaneum accessions revealed that S. spontaneum originated in northern India from an x = 10 ancestor, which then radiated into four major groups across the Indian subcontinent, China, and Southeast Asia. Our study suggests new directions for accelerating sugarcane improvement and expands our knowledge of the evolution of autopolyploids.

Allelic variation in class I HLA determines CD8+ T cell repertoire shape and cross-reactive memory responses to SARS-CoV-2.

Effective presentation of antigens by human leukocyte antigen (HLA) class I molecules to CD8+ T cells is required for viral elimination and generation of long-term immunological memory. In this study, we applied a single-cell, multiomic technology to generate a unified ex vivo characterization of the CD8+ T cell response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) across four major HLA class I alleles. We found that HLA genotype conditions key features of epitope specificity, TCRα/ß sequence diversity, and the utilization of pre-existing SARS-CoV-2-reactive memory T cell pools. Single-cell transcriptomics revealed functionally diverse T cell phenotypes of SARS-CoV-2-reactive T cells, associated with both disease stage and epitope specificity. Our results show that HLA variations notably influence the CD8+ T cell repertoire shape and utilization of immune recall upon SARS-CoV-2 infection.

Laser induced forward transfer isolating complex-shaped cell by beam shaping.

Beam shaping techniques have been widely used in holographic optical tweezers to accurately manipulate tiny particles and hologram optimization algorithms have also been widely reported to improve the optical trapping performance. In this paper, we presented a beam shaping laser induced forward transfer (BS-LIFT) technique to isolate complex-shaped cells. To do this, we built up a BS-LIFT instrument which combined beam shaping methods and laser induced forward transfer using liquid-crystal-on-silicon spatial light modulator. The laser beam was modulated into multiple desired points at the focal plane employing the Gerchberg-Saxton (GS) algorithm. Feasibility was verified through transferring various samples. To our knowledge, this is the first demonstration of BS-LIFT applied to the transfer complex-shaped cells. We successfully transferred cells whose size ranged from 1 µm to 100 µm. Our design will provide a novel approach for the application of this beam shaping technique and the isolation of single cells with variable shapes.

Comparative genome/transcriptome analysis probes Boraginales' phylogenetic position, WGDs in Boraginales, and key enzyme genes in the alkannin/shikonin core pathway.

Boraginales (the forget-me-not order) is a core group within the lamiids clade. However, until now, no genome from Boraginales has been reported, and published transcriptomes are also rare. Here, we report the first Boraginales species de novo genome (i.e. Echium plantagineum genome) and seven other Boraginales species transcriptomes to probe three issues: (i) Boraginales' phylogenetic position within the lamiids clade; (ii) potential whole genome duplications (WGDs) in Boraginales; and (iii) candidate key enzyme genes in the alkannin/shikonin core pathway. The results showed that: (i) Boraginales was most probably closer to the Solanales/Gentianales clade than the Lamiales clade, at least based on the single-copy orthologous genes from genome/transcriptome data; (ii) after the gamma (γ) event, Boraginaceae (classified into the Boraginales I clade) probably underwent at least two rounds of WGD, whereas Heliotropiaceae and Ehretiaceae (classified into the Boraginales II clade) probably underwent only one round of WGD; and (iii) several candidate key enzyme genes in the alkannin/shikonin core pathway were inferred, e.g. genes corresponding to geranyl cyclase, naphthol hydroxylase and O-acyl transferase.

Genetic Dissection of Seed Dormancy in Rice (Oryza sativa L.) by Using Two Mapping Populations Derived from Common Parents.

BACKGROUND: Seed dormancy, a quality characteristic that plays a role in seed germination, seedling establishment and grain yield, is affected by multiple genes and environmental factors. The genetic and molecular mechanisms underlying seed dormancy in rice remain largely unknown. RESULTS: Quantitative trait loci (QTLs) for seed dormancy were identified in two different mapping populations, a chromosome segment substitution line (CSSL) and backcross inbred line (BIL) population, both derived from the same parents Nipponbare, a japonica cultivar with seed dormancy, and 9311, an indica cultivar lacking seed dormancy. A total of 12 and 27 QTL regions for seed dormancy were detected in the CSSLs and BILs, respectively. Among these regions, four major loci (qSD3.1, qSD3.2, qSD5.2 and qSD11.2) were commonly identified for multiple germination parameters associated with seed dormancy in both populations, with Nipponbare alleles delaying the seed germination percentage and decreasing germination uniformity. Two loci (qSD3.1 and qSD3.2) were individually validated in the near-isogenic lines containing the QTL of interest. The effect of qSD3.2 was further confirmed in a CSSL-derived F2 population. Furthermore, both qSD3.1 and qSD3.2 were sensitive to abscisic acid and exhibited a significant epistatic interaction to increase seed dormancy. CONCLUSIONS: Our results indicate that the integration of the developed CSSLs and BILs with high-density markers can provide a powerful tool for dissecting the genetic basis of seed dormancy in rice. Our findings regarding the major loci and their interactions with several promising candidate genes that are induced by abscisic acid and specifically expressed in the seeds will facilitate further gene discovery and a better understanding of the genetic and molecular mechanisms of seed dormancy for improving seed quality in rice breeding programs.

Whole-genome resequencing reveals Brassica napus origin and genetic loci involved in its improvement.

Brassica napus (2n = 4x = 38, AACC) is an important allopolyploid crop derived from interspecific crosses between Brassica rapa (2n = 2x = 20, AA) and Brassica oleracea (2n = 2x = 18, CC). However, no truly wild B. napus populations are known; its origin and improvement processes remain unclear. Here, we resequence 588 B. napus accessions. We uncover that the A subgenome may evolve from the ancestor of European turnip and the C subgenome may evolve from the common ancestor of kohlrabi, cauliflower, broccoli, and Chinese kale. Additionally, winter oilseed may be the original form of B. napus. Subgenome-specific selection of defense-response genes has contributed to environmental adaptation after formation of the species, whereas asymmetrical subgenomic selection has led to ecotype change. By integrating genome-wide association studies, selection signals, and transcriptome analyses, we identify genes associated with improved stress tolerance, oil content, seed quality, and ecotype improvement. They are candidates for further functional characterization and genetic improvement of B. napus.

A Genomic Variation Map Provides Insights into the Genetic Basis of Spring Chinese Cabbage (Brassica rapa ssp. pekinensis) Selection.

Chinese cabbage is the most consumed leafy crop in East Asian countries. However, premature bolting induced by continuous low temperatures severely decreases the yield and quality of the Chinese cabbage, and therefore restricts its planting season and geographic distribution. In the past 40 years, spring Chinese cabbage with strong winterness has been selected to meet the market demand. Here, we report a genome variation map of Chinese cabbage generated from the resequencing data of 194 geographically diverse accessions of three ecotypes. In-depth analyses of the selection sweeps and genome-wide patterns revealed that spring Chinese cabbage was selected from a specific population of autumn Chinese cabbage around the area of Shandong peninsula in northern China. We identified 23 genomic loci that underwent intensive selection, and further demonstrated by gene expression and haplotype analyses that the incorporation of elite alleles of VERNALISATION INSENTIVE 3.1 (BrVIN3.1) and FLOWER LOCUS C 1 (BrFLC1) is a determinant genetic source of variation during selection. Moreover, we showed that the quantitative response of BrVIN3.1 to cold due to the sequence variations in the cis elements of the BrVIN3.1 promoter significantly contributes to bolting-time variation in Chinese cabbage. Collectively, our study provides valuable insights into the genetic basis of spring Chinese cabbage selection and will facilitate the breeding of bolting-resistant varieties by molecular-marker-assisted selection, transgenic or gene editing approaches.