Decoding the complexity of on-target integration: characterizing DNA insertions at the CRISPR-Cas9 targeted locus using nanopore sequencing.

BACKGROUND: CRISPR-Cas9 technology has advanced in vivo gene therapy for disorders like hemophilia A, notably through the successful targeted incorporation of the F8 gene into the Alb locus in hepatocytes, effectively curing this disorder in mice. However, thoroughly evaluating the safety and specificity of this therapy is essential. Our study introduces a novel methodology to analyze complex insertion sequences at the on-target edited locus, utilizing barcoded long-range PCR, CRISPR RNP-mediated deletion of unedited alleles, magnetic bead-based long amplicon enrichment, and nanopore sequencing. RESULTS: We identified the expected F8 insertions and various fragment combinations resulting from the in vivo linearization of the double-cut plasmid donor. Notably, our research is the first to document insertions exceeding ten kbp. We also found that a small proportion of these insertions were derived from sources other than donor plasmids, including Cas9-sgRNA plasmids, genomic DNA fragments, and LINE-1 elements. CONCLUSIONS: Our study presents a robust method for analyzing the complexity of on-target editing, particularly for in vivo long insertions, where donor template integration can be challenging. This work offers a new tool for quality control in gene editing outcomes and underscores the importance of detailed characterization of edited genomic sequences. Our findings have significant implications for enhancing the safety and effectiveness of CRISPR-Cas9 gene therapy in treating various disorders, including hemophilia A.

AntDAS-DDA: A New Platform for Data-Dependent Acquisition Mode-Based Untargeted Metabolomic Profiling Analysis with Advantage of Recognizing Insource Fragment Ions to Improve Compound Identification.

Data-dependent acquisition (DDA) mode in ultra-high-performance liquid chromatography-high-resolution mass spectrometry (UHPLC-HRMS) can provide massive amounts of MS1 and MS/MS information of compounds in untargeted metabolomics and can thus facilitate compound identification greatly. In this work, we developed a new platform called AntDAS-DDA for the automatic processing of UHPLC-HRMS data sets acquired under the DDA mode. Several algorithms, including extracted ion chromatogram extraction, feature extraction, MS/MS spectrum construction, fragment ion identification, and MS1 spectrum construction, were developed within the platform. The performance of AntDAS-DDA was investigated comprehensively with a mixture of standard and complex plant data sets. Results suggested that features in complex sample matrices can be extracted effectively, and the constructed MS1 and MS/MS spectra can benefit in compound identification greatly. The efficiency of compound identification can be improved by about 20%. AntDAS-DDA can take full advantage of MS/MS information in multiple sample analyses and provide more MS/MS spectra than single sample analysis. A comparison with advanced data analysis tools indicated that AntDAS-DDA may be used as an alternative for routine UHPLC-HRMS-based untargeted metabolomics. AntDAS-DDA is freely available at http://www.pmdb.org.cn/antdasdda.

Dynamics and competition of CRISPR-Cas9 ribonucleoproteins and AAV donor-mediated NHEJ, MMEJ and HDR editing.

Investigations of CRISPR gene knockout editing profiles have contributed to enhanced precision of editing outcomes. However, for homology-directed repair (HDR) in particular, the editing dynamics and patterns in clinically relevant cells, such as human iPSCs and primary T cells, are poorly understood. Here, we explore the editing dynamics and DNA repair profiles after the delivery of Cas9-guide RNA ribonucleoprotein (RNP) with or without the adeno-associated virus serotype 6 (AAV6) as HDR donors in four cell types. We show that editing profiles have distinct differences among cell lines. We also reveal the kinetics of HDR mediated by the AAV6 donor template. Quantification of T50 (time to reach half of the maximum editing frequency) indicates that short indels (especially +A/T) occur faster than longer (>2 bp) deletions, while the kinetics of HDR falls between NHEJ (non-homologous end-joining) and MMEJ (microhomology-mediated end-joining). As such, AAV6-mediated HDR effectively outcompetes the longer MMEJ-mediated deletions but not NHEJ-mediated indels. Notably, a combination of small molecular compounds M3814 and Trichostatin A (TSA), which potently inhibits predominant NHEJ repairs, leads to a 3-fold increase in HDR efficiency.

SNHG16 lncRNAs are overexpressed and may be oncogenic in human gastric cancer by regulating cell cycle progression.

The small nucleolar RNA host gene 16 (SNHG16) has recently been shown to be a putative oncogene in gastric cancer (GC) and other cancer types, but how its four lncRNA variants are expressed in any physiological and pathological situation remains unknown. To investigate the expression and function of the four lncRNA variants of SNHG16, mainly the variant 1, in GC, we performed quantitative PCR to determine the RNA levels of the four variants in 60 GC tissue samples and several cell lines. We also studied how knocking down of SNHG16 with siRNA affected proliferation, apoptosis, cell cycle progression, as well as migration and invasion of GC cells. Our results showed that variants 1 and 4 were overexpressed in GC tissues compared with adjacent uninvolved tissues. Knockdown of the four variants, mainly the variant 1, enhanced apoptosis and inhibited cell cycle progression of a GC cell line by arresting the cells at the G1 phase. These cellular effects were associated not only with decreased protein levels of c-Myc, PCNA, cyclins D1, E1, A2 and B, as well as CDKs 2 and 6, but also with increased protein levels of the p21, p27 and p53. Knockdown of total SNHG16 lncRNAs also inhibited invasion and migration of the GC cells in vitro. These results collectively suggest that SNHG16 may be oncogenic in GC by regulating cell cycle progression and may serve as a GC biomarker.

[Antibiotic use in very low birth weight/extremely low birth weight infants in 15 hospitals in Jiangsu Province of China: a multicenter survey]. / 江苏省15家医院极低/è¶ ä½Žå‡ºç”Ÿä½“é‡å„¿æŠ—ç”Ÿç´ ä½¿ç”¨çŽ°çŠ¶è°ƒæŸ¥.

OBJECTIVES: To investigate the current status of antibiotic use in very low birth weight/extremely low birth weight infants in Jiangsu Province of China, and to provide a clinical basis for the quality and improvement of antibiotic management in the neonatal intensive care unit (NICU). METHODS: A retrospective analysis was performed on the data on general conditions and antibiotic use in the very low birth weight/extremely low birth weight infants who were admitted to 15 hospitals of Jiangsu Province from January 1, 2019 to December 31, 2020. A questionnaire containing 10 measures to reduce antibiotic use was designed to investigate the implementation of these intervention measures. RESULTS: A total of 1 920 very low birth weight/extremely low birth weight infants were enrolled, among whom 1 846 (96.15%) were treated with antibiotic, and the median antibiotic use rate (AUR) was 50/100 patient-days. The AUR ranged from 24/100 to 100/100 patient-days in the 15 hospitals. After adjustment for the confounding factors including gestational age, birth weight, and neonatal critical score, the Poisson regression analysis showed that there was a significant difference in the adjusted AUR (aAUR) among the hospitals (P<0.01). The investigation results showed that among the 10 measures to reduce antibiotic use, 8 measures were implemented in less than 50% of these hospitals, and the number of intervention measures implemented was negatively correlated with aAUR (rs=-0.564, P=0.029). CONCLUSIONS: There is a high AUR among the very low birth weight/extremely low birth weight infants in the 15 hospitals of Jiangsu Province, with a significant difference among hospitals. The hospitals implementing a relatively few measures to reduce antibiotic use tend to have a high AUR. It is expected to reduce AUR in very low birth weight/extremely low birth weight infants by promoting the quality improvement of antibiotic use management in the NICU.

Glucose screening within six months postpartum among Chinese mothers with a history of gestational diabetes mellitus: a prospective cohort study.

BACKGROUND: Gestational diabetes mellitus (GDM) is a risk factor for diabetes mellitus. The 75-g, 2-h oral glucose tolerance test is recommended for mothers with a history of GDM to screen for diabetes in the postnatal period. The aim of this study was to investigate the rate of glucose screening within 6 months postpartum among Chinese mothers with a history of GDM, and to identify its predictors. METHODS: A prospective cohort study was conducted in a regional teaching hospital in Guangzhou, China, between July 2016 and June 2017. The participants were Chinese mothers (n = 237) who were diagnosed with GDM, were aged 18 years or older with no serious physical or mental disease and had not been diagnosed with type 1 or type 2 diabetes prior to their pregnancy. The revised Chinese version of the Champion's Health Belief Model Scale and social-demographic and perinatal characteristics factors were collected and used to predict postpartum glucose screening (yes or no). Adjust odds ratio (AOR) and 95% confidence interval (95% CI) were calculated. RESULTS: The mean age of the 237 mothers was 32.70 years (range from 22 to 44). Almost half of the mothers (45.6%) were college graduates or higher. Chinese mothers reported a high level of perceived benefits, self-efficacy, and health motivation towards postpartum glucose screening, with a mean score above 3.5. Chinese mothers were more likely to undertake postpartum glucose screening if they were a first-time mother [AOR 2.618 (95% CI: 1.398-4.901)], had a high perceived susceptibility score [AOR 2.173 (95% CI: 1.076-4.389)], a high perceived seriousness score [AOR 1.988 (95%CI: 1.020-3.875)] and high perceived benefits score [AOR 2.978 (95%CI: 1.540-5.759)]. CONCLUSION: The results of this study will lead to better identification of mothers with a history of GDM who may not screen for postpartum glucose abnormality. Health care professionals should be cognizant of issues that may affect postpartum glucose screening among mothers with a history of GDM, including parity, perceived susceptibility, perceived seriousness and perceived benefits.

Rhizobium oryziradicis sp. nov., isolated from rice roots.

Two Gram-stain-negative, aerobic, rod-shaped endophytic bacterial strains, N19T and N11-2, were isolated from fresh rice (Oryza sativa) roots during investigation of the rice endophytic bacterial diversity. The 16S rRNA gene sequence results indicated that the similarity between strains N19T and N11-2 was 100 %. Both of them belong to the genus Rhizobium, with close similarity to Rhizobium taibaishanense CCNWSX 0483T (97.7 %), followed by Rhizobium vitis NCPPB 3554T (97.5 %). The sequence similarities of the housekeeping genes recA, gyrB and glnA between the novel isolates and members of the established species of the genus Rhizobium were less than 87 %. The DNA-DNA hybridization rates between strains N19T and N11-2 were 87.9 % using the initial renaturation rate method. Based on draft genome sequences, strain N19T showed 18.2 % and 19.6 % DNA-DNA hybridization values to R. taibaishanense CCNWSX 0483T and R. vitis S4, which demonstrated that these new isolates represent a novel species in the genus Rhizobium. The main cellular fatty acids were summed feature 8 (C18 : 1ω7c and/or C18 : 1ω6c). The DNA G+C content of strain N19T was 58.7 mol% (Tm). The polar lipid profile of N19T consisted of phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine, an unknown lipid, two unknown aminolipids and an unidentified aminophospholipid. According to physiological and biochemical characteristics and genotypic data, strains N19T and N11-2 are considered to represent a novel species of the genus Rhizobium, for which the name Rhizobium oryziradicis sp. nov. is proposed, with N19T (=ACCC 19962T=KCTC 52413T) as the type strain.

Serratia oryzae sp. nov., isolated from rice stems.

A novel endophytic bacterium, strain J11-6T, was isolated from rice stems. Its taxonomic position was investigated using a polyphasic approach. The novel strain was Gram-staining-negative, facultatively anaerobic, motile and rod-shaped. Although the results of phylogenetic analysis based on 16S rRNA gene sequences indicated that J11-6T represented a member of the genus Rahnella, multilocus sequence analysis (MLSA) on the basis of concatenated partial atpD, gyrB, rpoB and infB gene sequences showed a clear distinction of J11-6T from the type strains of species of the genus Rahnella but indicated that it lay within the clade of the genus Serratia. The phylogenetically closest species were Serratia fonticola and Serratia aquatilis on the basis of the results of the MLSA phylogenetic analysis. The predominant cellular fatty acids were C16 : 1ω7c (38.7 %) and C16 : 0 (25.0 %). The DNA G+C content was 53.2 mol%. The DNA-DNA relatedness was 17.4 % between J11-6T and Rahnella aquatilis CIP 78.65T, and 29.2 % between J11-6T and S. fonticola LMG 7882T which indicates that this strain represents a novel species of the genus Serratia. Characterization by genotypic and phenotypic analysis indicated that J11-6T (=ACCC 19934T=KCTC 52529T) represents a novel species of the genus Serratia, for which the name Serratia oryzae sp. nov. is proposed.

Rhizobium rhizosphaerae sp. nov., a novel species isolated from rice rhizosphere.

Two novel, Gram-negative, motile, rod-shaped, aerobic bacterial strains, MH17T and RD15, were isolated from the sterilized root and rhizosphere soil of rice, respectively. Phylogenetic analysis based on 16S rRNA gene sequences showed that the similarity between strains MH17T and RD15 was 100%. The isolates exhibit high sequence similarities to Rhizobium oryzae CGMCC 1.7048T (98.7%) and Rhizobium petrolearium SL-1T (97.0% and 97.1%), which supports that they belong to a novel species in the genus Rhizobium. Strains MH17T and RD15 exhibited growth at 15-45 °C, pH 5.0-11.0, 0-2.0% sodium chloride (w/v). Sequence analysis of housekeeping genes gyrB, recA, atpD, ropB, gltA showed that these two novel strains had less than 94% similarity with the known species, indicating the distinct position of MH17T and RD15 in the genus Rhizobium. The major cellular fatty acids were identified as summed feature 8 (C18:1 ω7c and/or C18:1 ω6c). Type strain MH17T had 87.5% DNA-DNA relatedness with RD15 by using the initial renaturation rate method. Based on draft genome sequences, strain MH17T showed 30.1% DNA-DNA hybridization values to R. oryzae CGMCC 1.7048T, the closely related strain, which supported that MH17T represents a novel species in the genus Rhizobium. Average nucleotide identity (ANI) between strains MH17T and RD15 were 97.8%, and strain MH17T showed 82.2% ANI value with R. oryzae CGMCC 1.7048T. The DNA G+C content was 60.4 mol% (Tm). Based on physiological, biochemical characteristic, genotypic data, strains MH17T and RD15 are concluded to represent a new species within the genus Rhizobium, for which the name Rhizobium rhizosphaerae sp. nov. is proposed. The type strain is MH17T (=ACCC 19963T = KCTC 52414T).

Paenibacillus oryzisoli sp. nov., isolated from the rhizosphere of rice.

A novel bacterium, strain 1ZS3-15T, was isolated from rhizosphere of rice. Its taxonomic position was investigated using a polyphasic approach. The novel strain was observed to be Gram-stain positive, spore-forming, aerobic, motile and rod-shaped. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain 1ZS3-15T was recovered within the genus Paenibacillus. It is closely related to Paenibacillus pectinilyticus KCTC 13222T (97.9 % similarity), Paenibacillus frigoriresistens CCTCC AB 2011150T (96.8 %), Paenibacillus alginolyticus JCM 9068T (96.4 %) and Paenibacillus chondroitinus DSM 5051T (95.5 %). The fatty acid profile of strain 1ZS3-15T, which showed a predominance of anteiso-C15:0 and iso-C16:0, supported the allocation of the strain into the genus Paenibacillus. The predominant menaquinone was found to be MK-7. The polar lipids profile of strain 1ZS3-15T was found to consist of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, one unidentified lipid and two unidentified aminophospholipids. The cell wall peptidoglycan contains meso-diaminopimelic acid. Based on draft genome sequences, the DNA-DNA relatedness between strain 1ZS3-15T and the closely related species P. pectinilyticus KCTC 13222T are 24.2 ± 1.0 %, and the Average Nucleotide Identity values between the strains are 78.9 ± 0.1 %, which demonstrated that this isolate represents a new species in the genus Paenibacillus. The DNA G+C content was determined to be 45.3 mol%, which is within the range reported for Paenibacillus species. Characterisation by genotypic, chemotaxonomic and phenotypic analysis indicated that strain 1ZS3-15T represents a novel species of the genus Paenibacillus, for which the name Paenibacillus oryzisoli sp. nov. is proposed. The type strain is 1ZS3-15T (= ACCC 19783T = JCM 30487T).

Paenibacillusoryzae sp. nov., isolated from rice roots.

A novel endophytic bacterium, strain 1DrF-4T, isolated from rice roots, was characterized on the basis of its phenotypic characteristics and genotypic information. The novel strain was Gram-positive-staining, endospore-forming, facultatively anaerobic, motile and rod-shaped. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain 1DrF-4T formed a monophyletic clade within the genus Paenibacillus. The most phylogenetically related species was Paenibacillus pinesoli KACC 17472T, with which strain 1DrF-4T showed 16S rRNA gene sequence similarity of 95.2 %. 16S rRNA gene sequence similarities with type strains of other species of the genus Paenibacillus were less than 95 %. The predominant cellular fatty acids were anteiso-C15 : 0 (61.1 %) and C16 : 0 (11.1 %), which is one of the characteristic traits of the genus Paenibacillus. The quinone system contained exclusively menaquinone MK-7. The polar lipid profile contained diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, glycolipid and an unknown phospholipid. The DNA G+C content was 50.16 mol%, which was within the range reported for species of the genus Paenibacillus. Characterization by genotypic, chemotaxonomic and phenotypic analysis indicated that strain 1DrF-4T (=ACCC 19927T=JCM 30486T) represents a novel species of the genus Paenibacillus, for which the name Paenibacillusoryzae sp. nov. is proposed.

[Study on Chemical Constituents and Content Determination of Feifukang Mistura].

Objective: To identify the main chemical constituents and to determine the content in Feifukang mistura. Methods: HPLC-MS technique was used to profile and identify the chemical constituents by comparing the retention time,MS data with the reference standard. The content determination of all the chemical constituents were carried on a HPLC system. Results: Five compounds were separated from Feifukang mistura,which were identified as neomangiferin,mangiferin,calycosin-7-O-glucoside,calycosin,and schizandrol A. The standard curves of them showed good linearity on the range of 2. 08 ~ 104. 0 µg/m L,2. 00 ~ 100. 0 µg/m L,2. 00 ~ 100. 0µg/m L,2. 09 ~ 104. 5 µg/m L,and 1. 98 ~ 99. 0 µg/m L,respectively. The average recoveries were all in the range of 91. 3 ~ 103. 8%. Conclusion: The methods of chemical constituents identification and content determination were established,which may offer better revealing the material basis and controlling quality of Feifukang mistura.

Specific cells in the primary salivary glands of the whitefly Bemisia tabaci control retention and transmission of begomoviruses.

UNLABELLED: The majority of plant viruses are vectored by arthropods via persistent-circulative or noncirculative transmission. Previous studies have shown that specific binding sites for noncirculative viruses reside within the stylet or foregut of insect vectors, whereas the transmission mechanisms of circulative viruses remain ambiguous. Here we report the critical roles of whitefly primary salivary glands (PSGs) in the circulative transmission of two begomoviruses. The Middle East Asia Minor 1 (MEAM1) species of the whitefly Bemisia tabaci complex efficiently transmits both Tomato yellow leaf curl China virus (TYLCCNV) and Tomato yellow leaf curl virus (TYLCV), whereas the Mediterranean (MED) species transmits TYLCV but not TYLCCNV. PCR and fluorescence in situ hybridization experiments showed that TYLCCNV efficiently penetrates the PSGs of MEAM1 but not MED whiteflies. When a fragment of the coat protein of TYLCCNV was exchanged with that of TYLCV, mutated TYLCCNV accumulated in the PSGs of MED whiteflies, while mutant TYLCV was nearly undetectable. Confocal microscopy revealed that virion transport in PSGs follows specific paths to reach secretory cells in the central region, and the accumulation of virions in the secretory region of PSGs was correlated with successful virus transmission. Our findings demonstrate that whitefly PSGs, in particular the cells around the secretory region, control the specificity of begomovirus transmission. IMPORTANCE: Over 75% of plant viruses are transmitted by insects. However, the mechanisms of virus transmission by insect vectors remain largely unknown. Begomoviruses and whiteflies are a complex of viruses and vectors which threaten many crops worldwide. We investigated the transmission of two begomoviruses by two whitefly species. We show that specific cells of the whitefly primary salivary glands control viral transmission specificity and that virion transport in the glands follows specific paths to reach secretory cells in the central region and then to reach the salivary duct. Our results indicate that the secretory cells in the central region of primary salivary glands determine the recognition and transmission of begomoviruses. These findings set a foundation for future research not only on circulative plant virus transmission but also on other human and animal viruses transmitted by arthropod vectors.

Antisense oligonucleotides against microRNA-21 reduced the proliferation and migration of human colon carcinoma cells.

BACKGROUND: Colon carcinoma is one of the commonly tumors that threaten human beings as its highly morbidity and mortality. Recent evidences suggested that microRNA-21 (miR-21) played an important role in the development of colon carcinoma and might be a potential biological marker for the diagnosis and prognosis of colon carcinoma. However, the potential effect of miR-21 based therapeutic studies in colon carcinoma remains to be fully elucidated. METHODS: In present study, we constructed an eukaryotic expression vector encoding antisense oligonucleotides against miR-21 (termed as p-miR-21-ASO) and the expression of miRNA-21 in human colon cancer was detected by Real-time PCR. To assess its possible effect on the proliferation and migration capacity of human colon carcinoma cells in vitro, CCK-8 assay, colony formation assay and cell invasion, as well as migration assay, were performed respectively. Moreover, PTEN, one of target molecules of miRNA-21, was analyzed by Western blot and Fluorescence activated cell sorter assay. Finally, the transduction of AKT and ERK pathways in human colon carcinoma cells was determined by Western blot. RESULTS: We found that transiently transfection of p-miR-21-ASO could efficiently decrease the relative expression of miR-21 in human colon carcinoma HCT116 cells, accompanied by impaired proliferation and clone formation. Furthermore, we found that down-regulation of miR-21 also could significantly abrogate the invasion and migration capacity in vitro, as well as the expression of vascular endothelial growth factor which is critical for the metastatic capacity of colon carcinoma cells. Mechanistic evidence showed that down-regulation of miR-21 increased the expression of its target molecule PTEN in HCT116 cells. Finally, we revealed that the expression level of both phosphor-ERK1/2 and phosphor-AKT also were altered. CONCLUSIONS: Therefore, our data suggested miR-21 ASO against miR-21 might be a useful strategy to alter the expression of miR-21 in colon carcinoma cells, which was helpful for the development of miR-21-based therapeutic strategies against clinical colon carcinoma.

[Determination of the derivatization reactivity between α/ß-dicarbonyl compounds and standard citrullinated peptides based on matrix-assisted laser desorption ionization-time-of-flight mass spectrometry].

Protein citrullination is an irreversible post-translational modification process regulated by peptidylarginine deiminases (PADs) in the presence of Ca2+. This process is closely related to the occurrence and development of autoimmune diseases, cancers, neurological disorders, cardiovascular and cerebrovascular diseases, and other major diseases. The analysis of protein citrullination by biomass spectrometry confronts great challenges owing to its low abundance, lack of affinity tags, small mass-to-charge ratio change, and susceptibility to isotopic and deamidation interferences. The methods commonly used to study the protein citrullination mainly involve the chemical derivatization of the urea group of the guanine side chain of the peptide to increase the mass-to-charge ratio difference of the citrullinated peptide. Affinity-enriched labels are then introduced to effectively improve the sensitivity and accuracy of protein citrullination by mass spectrometry. 2,3-Butanedione or phenylglyoxal compounds are often used as derivatization reagents to increase the mass-to-charge ratio difference of the citrullinated peptide, and the resulting derivatives have been observed to contain α-dicarbonyl structures. To date, however, no relevant studies on the reactivity of dicarbonyl compounds with citrullinated peptides have been reported. In this study, we determined whether six α-dicarbonyl and two ß-dicarbonyl compounds undergo derivatization reactions with standard citrullinated peptides using matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS). Among the α-dicarbonyl compounds, 2,3-butanedione and glyoxal reacted efficiently with several standard citrullinated peptides, but yielded a series of by-products. Phenylglyoxal, methylglyoxal, 1,2-cyclohexanedione, and 1,10-phenanthroline-5,6-dione also derivated efficiently with standard citrullinated peptides, generating a single derivative. Thus, a new derivatization method that could yield a single derivative was identified. Among the ß-dicarbonyl compounds, 1,3-cyclohexanedione and 2,4-pentanedione successfully reacted with the standard citrullinated peptides, and generated a single derivative. However, their reaction efficiency was very low, indicating that the ß-dicarbonyl compounds are unsuitable for the chemical derivatization of citrullinated peptides. The above results indicate that the α-dicarbonyl structure is necessary for realizing the efficient and specific chemical derivatization of citrullinated peptides. Moreover, the side chains of the α-dicarbonyl structure determine the structure of the derivatives, derivatization efficiency, and generation (or otherwise) of by-products. Therefore, the specific enrichment and precise identification of citrullinated peptides can be achieved by synthesizing α-dicarbonyl structured compounds containing affinity tags. The proposed method enables the identification of citrullinated proteins and their modified sites by MS, thereby providing a better understanding of the distribution of citrullinated proteins in different tissues. The findings will be beneficial for studies on the mechanism of action of citrullinated proteins in a variety of diseases.

Genomic and transcriptomic analyses reveal metabolic complementarity between whiteflies and their symbionts.

Nutritional mutualism between insects and symbiotic bacteria is widespread. The various sap-feeding whitefly species within the Bemisia tabaci complex associate with the same obligate symbiont (Portiera) and multiple secondary symbionts. It is often assumed that some of the symbionts residing in the whiteflies play crucial roles in the nutritional physiology of their insect hosts. Although effort has been made to understand the functions of the whitefly symbionts, the metabolic complementarity offered by these symbionts to the hosts is not yet well understood. We examined two secondary symbionts, Arsenophonus and Wolbachia, in two species of the B. tabaci whitefly complex, provisionally named as Asia II 3 and China 1. Genomic sequence analyses revealed that Arsenophonus and Wolbachia retained genes responsible for the biosynthesis of B vitamins. We then conducted transcriptomic surveys of the bacteriomes in these two species of whiteflies together with that in another species named MED of this whitefly complex previously reported. The analyses indicated that several key genes in B vitamin syntheses from the three whitefly species were identical. Our findings suggest that, similar to another secondary symbiont Hamiltonella, Arsenophonus and Wolbachia function in the nutrient provision of host whiteflies. Although phylogenetically distant species of symbionts are associated with their respective hosts, they have evolved and retained similar functions in biosynthesis of some B vitamins. Such metabolic complementarity between whiteflies and symbionts represents an important feature of their coevolution.

A new platform for untargeted UHPLC-HRMS data analysis to address the time-shift problem.

Substantial deviations in retention times among samples pose a great challenge for the accurate screening and identifying of metabolites by ultrahigh-performance liquid chromatography high-resolution mass spectrometry (UHPLC-HRMS). In this study, a coarse-to-refined time-shift correction methodology was proposed to efficiently address this problem. Metabolites producing multiple fragment ions were automatically selected as landmarks to generate pseudo-mass spectra for a coarse time-shift correction. Refined peak alignment for extracted ion chromatograms was then performed by using a moving window-based multiple-peak alignment strategy. Based on this novel coarse-to-refined time-shift correction methodology, a new comprehensive UHPLC-HRMS data analysis platform was developed for UHPLC-HRMS-based metabolomics. Original datasets were employed as inputs to automatically extract and register features in the dataset and to distinguish fragment ions from metabolites for chemometric analysis. Its performance was further evaluated using complex datasets, and the results suggest that the new platform can satisfactorily resolve the time-shift problem and is comparable with commonly used UHPLC-HRMS data analysis tools such as XCMS Online, MS-DIAL, Mzmine2, and Progenesis QI. The new platform can be downloaded from: http://www.pmdb.org.cn/antdas2tsc.

Improved and Flexible HDR Editing by Targeting Introns in iPSCs.

Highly efficient gene knockout (KO) editing of CRISPR-Cas9 has been achieved in iPSCs, whereas homology-directed repair (HDR)-mediated precise gene knock-in (KI) and high-level expression are still bottlenecks for the clinical applications of iPSCs. Here, we developed a novel editing strategy that targets introns. By targeting the intron before the stop codon, this approach tolerates reading frameshift mutations caused by nonhomologous end-joining (NHEJ)-mediated indels, thereby maintaining gene integrity without damaging the non-HDR-edited allele. Furthermore, to increase the flexibility and screen for the best intron-targeting sgRNA, we designed an HDR donor with an artificial intron in place of the endogenous intron. The presence of artificial introns, particularly an intron that carries an enhancer element, significantly increased the reporter expression levels in iPSCs compared to the intron-deleted control. In addition, a combination of the small molecules M3814 and trichostatin A (TSA) significantly improves HDR efficiency by inhibiting NHEJ. These results should find applications in gene therapy and basic research, such as creating reporter cell lines.

Modulation of Immune Reaction in Hydrodynamic Gene Therapy for Hemophilia A.

Hemophilia A (HA) is a monogenic disease characterized by plasma clotting factor 8 (F8) deficiency due to F8 mutation. We have been attempting to cure HA permanently using a CRISPR-Cas9 gene-editing strategy. In this study, we induced targeted integration of BDDF8 (B-domain-deleted F8) gene into the albumin locus of HA mice by hydrodynamic tail vein injection of editing plasmid vectors. One week after treatment, a high F8 activity ranging from 70% to 280% of normal serum levels was observed in all treated HA mice but dropped to background levels 3-5 weeks later. We found that the humoral immune reaction targeting F8 is the predominant cause of the decreased F8 activity. We hypothesized that hydrodynamic injection-induced liver damage triggered the release of large quantities of inflammatory cytokines. However, coinjection of plasmids expressing a dozen immunomodulatory factors failed to curtail the immune reaction and stabilize F8 activity effectively. The spCas9 plasmid carrying a miR-142-3p target sequence alleviated the cellular immune response but could not deliver therapeutic efficacy. Strikingly, immunosuppressant cyclophosphamide virtually abolished the immune response, leading to a year-long stable F8 level. Our findings should have important implications in developing therapies in mouse models using the hydrodynamic gene delivery approach, highlighting the necessity of modulating the innate immune response triggered by liver damage.

A chemometric strategy to automatically screen selected ion monitoring ions for gas chromatography-mass spectrometry-based pseudotargeted metabolomics.

The pseudotargeted metabolomics based on gas chromatography-mass spectrometry (GC-MS) has the advantage of filtering out artifacts originating from sample treatment and accurately quantifying underlying compounds in the analyzed samples. However, this technique faces the problem of selecting high-quality selective ions for performing selected ion monitoring (SIM) on instruments. In this work, we proposed AntDAS-SIMOpt, an automatic untargeted strategy for SIM ion optimization that was accomplished on the basis of an experimental design combined with advanced chemometric algorithms. First, a group of diluted quality control samples was used to screen underlying compounds in samples automatically. Ions in each of the resolved mass spectrum were then evaluated by using the developed algorithms to identify the SIM ion. A Matlab graphical user interface (GUI) was designed to facilitate routine analysis, which can be obtained from http://www.pmdb.org.cn/antdassimopt. The performance of the developed strategy was comprehensively investigated by using standard and complex plant datasets. Results indicated that AntDAS-SIMOpt may be useful for GC-MS-based metabolomics.