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.

[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.

GREPore-seq: A robust workflow to detect changes after gene editing through long-range PCR and nanopore sequencing.

To achieve the enormous potential of gene-editing technology in clinical therapies, one needs to evaluate both the on-target efficiency and unintended editing consequences comprehensively. However, there is a lack of a pipelined, large-scale, and economical workflow for detecting genome editing outcomes, in particular insertion or deletion of a large fragment. Here, we describe an approach for efficient and accurate detection of multiple genetic changes after CRISPR/Cas9 editing by pooled nanopore sequencing of barcoded long-range PCR products. Recognizing the high error rates of Oxford nanopore sequencing, we developed a novel pipeline to capture the barcoded sequences by grepping reads of nanopore amplicon sequencing (GREPore-seq). GREPore-seq can assess nonhomologous end-joining (NHEJ)-mediated double-stranded oligodeoxynucleotide (dsODN) insertions with comparable accuracy to Illumina next-generation sequencing (NGS). GREPore-seq also reveals a full spectrum of homology-directed repair (HDR)-mediated large gene knock-in, correlating well with the fluorescence-activated cell sorting (FACS) analysis results. Of note, we discovered low-level fragmented and full-length plasmid backbone insertion at the CRISPR cutting site. Therefore, we have established a practical workflow to evaluate various genetic changes, including quantifying insertions of short dsODNs, knock-ins of long pieces, plasmid insertions, and large fragment deletions after CRISPR editing. GREPore-seq is freely available at GitHub (https://github.com/lisiang/GREPore-seq) and the National Genomics Data Center (NGDC) BioCode (https://ngdc.cncb.ac.cn/biocode/tools/BT007293).

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.

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.

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.

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.

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.

Danshensu attenuates cisplatin-induced nephrotoxicity through activation of Nrf2 pathway and inhibition of NF-κB.

The clinical application of cisplatin was mainly limited by severe nephrotoxicity. Danshensu was the main pharmacological active diterpenoids which extracted from the roots of Salvia milthiorriza Bunge. This study is aimed to investigate the protective effects and potential mechanisms of Danshensu against cisplatin-induced nephrotoxicity. After fasting for 12 h, all mice groups except the control group were administered a single intraperitoneal injection of 25 mg/kg cisplatin. 1 h later, cisplatin (25 mg/kg) + Danshensu (15 mg/kg, 30 mg/kg, 60 mg/kg) groups were treated with corresponding doses of Danshensu once a day for 7 consecutive days. Blood urea nitrogen (BUN), creatinine, reactive oxygen species (ROS), superoxide dismutase (SOD), Glutathione peroxidase (GPx), Catalase (CAT) and malondialdehyde (MDA) were assayed in this study. The expression of inflammatory cytokines TNF-α, IL-6 and IL-1ß were examined by ELISA. The results showed that Danshensu could improve kidney damage, attenuate serum BUN, creatinine, cytokines and oxidative stress markers. Further studies showed that Danshensu can induce Nrf2/HO-1 activation and inhibition of NF-κB pathway. In conclusion, Danshensu exerts the protective effects on cisplatin-induced nephrotoxicity, which may be related to the activation of Nrf2/HO-1 and inhibition of NF-ĸB pathway.

Alterations in thymocyte populations under conditions of endotoxin tolerance.

BACKGROUND: Endotoxin tolerance (ET) is a protective phenomenon in which pre-treatment with a tolerance dose of lipopolysaccharide (LPS) leads to dramatically elevated survival. Accumulating evidence has shown that peripheral T cells contribute to the induction of ET. However, what happens to T cell development in the thymus under ET conditions remains unclear. The purpose of this study was to analyze the alterations in thymocyte populations (double-positive [DP] and single-positive [SP] cells) under ET conditions. METHODS: Mice were intraperitoneally injected with LPS at a concentration of 5 mg/kg to establish an LPS tolerance model and were divided into two groups: a group examined 72 h after LPS injection (72-h group) and a group examined 8 days after LPS injection (8-day group). Injection of phosphate-buffered saline was used as a control (control group). Changes in thymus weight, cell counts, and morphology were detected in the three groups. Moreover, surface molecules such as CD4, CD8, CD44, CD69, and CD62L were analyzed using flow cytometry. Furthermore, proliferation, apoptosis, cytokine production, and extracellular signal-regulated kinase (ERK) pathway signaling were analyzed in thymocyte populations. The polymorphism and length of the T-cell receptor (TCR) ß chain complementarity-determining region 3 (CDR3) were analyzed using capillary electrophoresis DNA laser scanning analysis (ABI 3730). RESULTS: Thymus weight and cell counts were decreased in the early stage but recovered by the late stage in a murine model of LPS-induced ET. Moreover, the proportions of DP cells (control: 72.130 ±â€Š4.074, 72-h: 10.600 ±â€Š3.517, 8-day: 84.770 ±â€Š2.228), CD4+ SP cells (control: 15.770 ±â€Š4.419, 72-h: 44.670 ±â€Š3.089, 8-day: 6.367 ±â€Š0.513), and CD8+ SP cells (control: 7.000 ±â€Š1.916, 72-h: 34.030 ±â€Š3.850, 8-day: 5.133 ±â€Š0.647) were obviously different at different stages of ET. The polymorphism and length of TCR ß chain CDR3 also changed obviously, indicating the occurrence of TCR rearrangement and thymocyte diversification. Further analysis showed that the expression of surface molecules, including CD44, CD69, and CD62L, on thymocyte populations (DP and SP cells) were changed to different degrees. Finally, the proliferation, apoptosis, cytokine production, and ERK pathway signaling of thymocyte populations were changed significantly. CONCLUSION: These data reveal that alterations in thymocyte populations might contribute to the establishment of ET.

A chemometric strategy for accurately identifying illegal additive compounds in health foods by using ultra-high-performance liquid chromatography coupled to high resolution mass spectrometry.

The accurate identification of unknown illegal additive compounds in complex health foods continues to be a challenging task in routine analysis, because massive false positive results can be screened with ultra-high-performance liquid chromatography coupled to high-resolution mass spectrometry-based untargeted techniques and must be manually filtered out. To address this problem, we developed a chemometric-based strategy, in which data analysis was first performed by using XCMS, MS-DIAL, Mzmine2, and AntDAS2, to select those that provided acceptable results to extract common features (CFs), which can be detected by all of the selected methods. Then, CFs whose contents were significantly higher in the suspected illegal additive group were screened. Isotopic, adduct, and neutral loss ions were marked based on the CFs by using a new adaptive ion annotation algorithm. Fragment ions originating from the same compound were identified by using a novel fragment ion identification algorithm. Finally, a respective mass spectrum was constructed for each screened compound to benefit compound identification. The developed strategy was confirmed by using a complex Chinese health food, Goujiya tea. The features of all illegal additive compounds were precisely screened by the developed strategy, and massive false positive features from the current data analysis method were greatly reduced. The constructed respective mass spectra can benefit compound identification and avoid the risk of identifying ions from the same illegal compound as different compounds. Moreover, unknown compounds that are contained in an illegal compound library can be screened.

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.

Transcriptomic analyses of chemosensory genes in Trichogramma japonicum (Hymenoptera: Trichogrammatidae).

Insects perceive dangerous or attractive chemicals in the environment (such as the presence of predators, food or mates) through their olfaction and gustation. This leads to host searching, mate finding and other behaviors that are critical for insect survival. These vital activities are mediated by many chemosensory receptor proteins, including odorant receptors (ORs), gustatory receptors (GRs) and ionotropic receptors (IRs). Trichogramma japonicum Ashmead (Hymenoptera: Trichogrammatidae) is a commonly used egg parasitoid for controlling lepidopteran pests in rice fields. To reveal the roles of its olfactory and gustatory systems in biological control, we systematically analyzed major chemosensory receptor genes from T. japonicum. Through transcriptomic analyses of male and female heads, we found that the differentially expressed genes (DEGs) were mainly related to chemosensory functions, and there were more chemosensory-related genes expressed in female heads than male heads, which may be related to the need for females to search for their hosts or sense male pheromone. Furthermore, we identified 66 chemosensory receptor genes, including 51 ORs, seven IRs and eight GRs, that were most closely relate to those of other hymenopteran species according to the results of both Blast best-hit and phylogenetic analyses. The tissue expression profile showed that 65 of the 66 chemosensory receptors were highly expressed in the heads, suggesting their putative roles in olfaction and gustation. In addition, the sex-specific expression patterns suggested their potential functions in host-seeking or mate sensing behaviors. This study may provide base for further understanding the olfactory and gustatory systems of T. japonicum and increasing its efficiency in pest control in the future.

Specific deletion of CDC42 in pancreatic ß cells attenuates glucose-induced insulin expression and secretion in mice.

Insulin is a key hormone for maintaining glucose homeostasis in organisms. In general, deficiency of insulin synthesis and secretion results in type I diabetes, whereas insulin resistance leads to type 2 diabetes. Cell division cycle 42 (CDC42), a member of Rho GTPases family, has been shown as an essential regulator in the second phase of glucose-induced insulin secretion in pancreatic islets ß cells in vitro. However, the effect of CDC42 on insulin expression has not been explored. Here we reported that the glucose-induced insulin expression and secretion were significantly inhibited in mice lacking CDC42 gene in pancreatic ß cells (Rip-CDC42cKO) in vivo and in vitro. Deletion of CDC42 gene in pancreatic ß cells did not affect survival or reproduction in mice. However, the Rip-CDC42cKO mice showed the systemic glucose intolerance and the decrease of glucose-induced insulin secretion without apparent alterations of peripheral tissues insulin sensitivity and the morphology of islets. Furthermore, we demonstrated that deletion of CDC42 gene in pancreatic ß cells significantly attenuated the insulin expression through inhibiting the ERK1/2-NeuroD1 signaling pathway. Taken together, our study presents novel evidence that CDC42 is an important modulator in glucose-induced insulin expression as well as insulin secretion in pancreatic ß cells.

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.

Effects of fermented Chinese herbal medicines on milk performance and immune function in late-lactation cows under heat stress conditions.

Heat stress (HS) causes significant economic losses and has become a continual challenge in the dairy industry worldwide. The objective of this study was to evaluate the effects of a dietary supplement on milk performance and immune function in late-lactation cows under HS conditions. The supplement was a fermented Chinese herbal medicines (CHMs) mixture consisting of 18 herbs. Forty lactating Holstein cows (560 ± 51.0 kg of initial BW, 230 ± 10.0 DIM, 16 ± 3.0 kg of milk per day) were randomly assigned into 4 treatment groups (10 cows per group). Each group was fed a dietary supplemented with 0, 25, 50, or 100 g CHMs per cow per day. Cows were housed at high ambient temperature-humidity index (average 74.5) for an experimental period of 42 d during the summer months. Milk yield, composition, immune responses involving blood lymphocyte apoptosis rate, serum biochemical parameters, and genes expression in lymphocytes were evaluated on days 14, 28, and 42, respectively. Results showed that milk yield, milk fat, and protein content were greater (all P < 0.05) for 50 or 100 g/d CHMs compared with the group without CHMs supplements throughout the experimental period. On the other hand, increasing CHMs dose demonstrated a greater lymphocyte or leukocyte count (P < 0.01). By flow cytometry analysis, early or late apoptosis rate of the lymphocytes was decreased (P < 0.05) by CHMs supplements. The immunity-related biochemistry and genes transcript responses involving cytokines (IL-1, IL-2, IL-6, and IL-12), apoptosis (Bak, Mcl-1, Bax, Bcl-2, Bcl-xl, and P53), and immunoglobulins (IgA, IgG, and IgM) were investigated. Compared with the unsupplemented group, the serum IL-2 and IL-6 levels, as well as IL-2 mRNA expression, increased (P < 0.05) for 100 g/d. However, the serum IL-1 level tended to decrease (P = 0.08) with increasing CHMs dose, and IL-1 mRNA expression was down-regulated (P = 0.02) by up to 24% for 100 g/d. Additionally, the serum Bax level decreased (P < 0.01) and Bcl-2 level increased (P = 0.01) for 100 g/d. Bax and Bak mRNA expressions were down-regulated (P < 0.05), and Bcl-2 and Bcl-xl expression were up-regulated (P < 0.05) for 50 or 100 g/d. The mRNA expressions of P53 and Mcl-1 were not affected by CHMs (P > 0.10). Besides, serum IgG levels were greater (P < 0.01) for 50 or 100 g/d, compared with unsupplemented group. In conclusion, CHMs supplements may improve milk performance and immune function in dairy cows under HS conditions.

[As2O3 Up-Regulates the Proportion of CD4+CD25+CD127low Tregs in Peripheral Blood of Patients with Severe Aplastic Anemia].

OBJECTIVE: To investigate the effect of ATO on the proportion of Treg in the peripheral blood of patients with severe aplastic anemia (SAA) in vitro. METHODS: The peripheral blood of 20 newlydiagnosed patients were collected, and the peripheral blood monomuclear cells (PBMNC) were extracted. After the PBMNC were treated with ATO of different concentrotions (0, 1, 2.5 and 5 µmol/L) for 96 hours, the proportion of CD44+ CD25+CD127low regulatatory T cells (Treg) were detected by flow cytometry. The expression levels of Foxp3 mRNA were detected by RT-PCR, and the levels of IFN-γ,IL-4,IL-17 and TGF-ß1 were detected by ELTSA to verify the results of flow cytomery. RESULTS: ATO significantly increased the proportion of Treg (P<0.01) at the concentration of 2.5 and 5 µmol/L, and the rising degree of Treg proportion improved with the increasing ATO concentration(r= 0.524). Treg proportion increased at a concentration of 1 µmol/L, but without statistical significance (P>0.05). At 1(P<0.05), 2.5(P<0.01) and 5 µmol/L(P<0.01), ATO significantly up-regulated the expression of Foxp3 mRNA, and the increase of Foxp3 mRNA positively and linearly correlated with the increase of Treg cell-frequency(r=0.523). ATO significantly reduced the levels of IFN-γ (at ATO 1,2.5 and 5 µmol/L, P<0.01), IL-4 (at ATO 2.5 µmol/L, P<0.01; at ATO 5 µmol/L, P<0.01) and IL-17(at ATO 2.5 µmol/L, P<0.05; at ATO 5 µmol/L, P<0.01). ATO had no significant effect on TGF-ß1 at 1(P>0.05) and 2.5 µmol/L (P>0.05), but significantly reduced TGF-ß1 level at 5 µmol/L (P<0.05). CONCLUSION: ATO can mediate the immune regulation through up-regulating the proportion of Treg in peripheral blood of patients with SAA and reducing the levels of IFN-γ, IL-4 and IL-17.

Palmitic Acid Hydroxystearic Acids Activate GPR40, Which Is Involved in Their Beneficial Effects on Glucose Homeostasis.

Palmitic acid hydroxystearic acids (PAHSAs) are endogenous lipids with anti-diabetic and anti-inflammatory effects. PAHSA levels are reduced in serum and adipose tissue of insulin-resistant people and high-fat diet (HFD)-fed mice. Here, we investigated whether chronic PAHSA treatment enhances insulin sensitivity and which receptors mediate PAHSA effects. Chronic PAHSA administration in chow- and HFD-fed mice raises serum and tissue PAHSA levels ∼1.4- to 3-fold. This improves insulin sensitivity and glucose tolerance without altering body weight. PAHSA administration in chow-fed, but not HFD-fed, mice augments insulin and glucagon-like peptide (GLP-1) secretion. PAHSAs are selective agonists for GPR40, increasing Ca+2 flux, but not intracellular cyclic AMP. Blocking GPR40 reverses improvements in glucose tolerance and insulin sensitivity in PAHSA-treated chow- and HFD-fed mice and directly inhibits PAHSA augmentation of glucose-stimulated insulin secretion in human islets. In contrast, GLP-1 receptor blockade in PAHSA-treated chow-fed mice reduces PAHSA effects on glucose tolerance, but not on insulin sensitivity. Thus, PAHSAs activate GPR40, which is involved in their beneficial metabolic effects.