The arginine methyltransferase Prmt1 coordinates the germline arginine methylome essential for spermatogonial homeostasis and male fertility.

Arginine methylation, catalyzed by the protein arginine methyltransferases (PRMTs), is a common post-translational protein modification (PTM) that is engaged in a plethora of biological events. However, little is known about how the methylarginine-directed signaling functions in germline development. In this study, we discover that Prmt1 is predominantly distributed in the nuclei of spermatogonia but weakly in the spermatocytes throughout mouse spermatogenesis. By exploiting a combination of three Cre-mediated Prmt1 knockout mouse lines, we unravel that Prmt1 is essential for spermatogonial establishment and maintenance, and that Prmt1-catalyzed asymmetric methylarginine coordinates inherent transcriptional homeostasis within spermatogonial cells. In conjunction with high-throughput CUT&Tag profiling and modified mini-bulk Smart-seq2 analyses, we unveil that the Prmt1-deposited H4R3me2a mark is permissively enriched at promoter and exon/intron regions, and sculpts a distinctive transcriptomic landscape as well as the alternative splicing pattern, in the mouse spermatogonia. Collectively, our study provides the genetic and mechanistic evidence that connects the Prmt1-deposited methylarginine signaling to the establishment and maintenance of a high-fidelity transcriptomic identity in orchestrating spermatogonial development in the mammalian germline.

BrBCAT1 mutation resulted in deficiency of epicuticular wax crystal in Chinese cabbage.

KEY MESSAGE: BrBCAT1 encoding a branched-chain amino acid aminotransferase was responsible for the glossy trait, which was verified by allelic mutants in Chinese cabbage. The glossy characteristic, thanks to the epicuticular wax crystal deficiency, is an excellent commodity character for leafy vegetables. Herein, two allelic glossy green mutants, wdm11 and wdm12, were isolated from an ethyl methane sulfonate (EMS)-mutagenized population of Chinese cabbage, and the mutant phenotype was recessive inherited. Cryo-SEM detected that epicuticular wax crystal in the mutant leaves was virtually absent. MutMap and Kompetitive allele-specific PCR analyses demonstrated that BraA06g006950.3C (BrBCAT1), homologous to AtBCAT1, encoding a branched-chain amino acid aminotransferase was the candidate gene. A SNP (G to A) on the fourth exon of BrBCAT1 in wdm11 caused the 233rd amino acid to change from glycine (G) to aspartic acid (D). A SNP (G to A) on the second exon of BrBCAT1 in wdm12 led to the 112th amino acid change from glycine (G) to arginine (R). Both of the allelic mutants had genetic structural variation in the candidate gene, which indicated that the mutant phenotype was triggered by the BrBCAT1 mutation. The expression levels of BrBCAT1 and genes related to fatty acid chain extension were decreased significantly in the mutant compared to the wild-type, which might result in epicuticular wax crystal deficiency in the mutants. Our findings proved that the mutation of BrBCAT1 induced the glossy phenotype and provided a valuable gene resource for commodity character improvement in Chinese cabbage.

Mutation in BrFLS encoding flavonol synthase induced anthocyanin accumulation in Chinese cabbage.

KEY MESSAGE: BrFLS mutation promoted anthocyanin accumulation in Chinese cabbage, which was verified in four allelic mutants. Chinese cabbage is a major vegetable crop in Eastern Asia. Anthocyanin-rich vibrantly colored varieties are increasingly favored by consumers for their higher nutritional and aesthetic value compared to the typical green varieties of Chinese cabbage. Herein, we identified an anthocyanin accumulation mutant aam1 from a mutant library of EMS-mutagenized Chinese cabbage DH line 'FT', which appeared partial purple on leaves, bolting stems and floral buds. This anthocyanin accumulation trait was genetically controlled by a recessive nuclear gene, and through MutMap mapping and KASP genotyping, BraA10g030950.3C was identified as the candidate causal gene with a G202 to A202 non-synonymous SNP variation in exon 1. Three additional mutants allelic to aam1 were obtained via screening of similar-phenotype mutants from the mutant library, namely aam2/3/4, where the causal SNPs reside in the same gene as aam1, corroborating that the mutation of BraA10g030950.3C caused anthocyanin accumulation. BraA10g030950.3C encodes a flavonol synthase that catalyzes dihydroflavonols substrate into flavonols and is homologous to Arabidopsis FLS1 (AT5G08640), named BrFLS. Compared to wildtype, the expression level of BrFLS was significantly reduced in the mutants, while BrDFR, which is involved in the anthocyanin biosynthesis and competes with FLS for the common substrate dihydroflavonols, was increased. The flavonol synthase activity decreased, and dihydroflavonol 4-reductase activity was elevated. Differentially accumulated flavonoid metabolites were detected between wildtype and aam1, which were enriched primarily in flavonol and anthocyanin pathways. Our results revealed that mutations in the BrFLS gene could contribute to anthocyanin accumulation and provide a new target for Chinese cabbage color modification.

Reactivity to conditioned threat cues is distinct from exploratory drive in the elevated plus maze.

Fear and anxiety are adaptive states that allow humans and animals alike to respond appropriately to threatening cues in their environment. Commonly used tasks for studying behaviour akin to fear and anxiety in rodent models are Pavlovian threat conditioning and the elevated plus maze (EPM), respectively. In threat conditioning the rodents learn to associate an aversive event with a specific stimulus or context. The learnt association between the two stimuli (the 'memory') can then be recalled by re-exposing the subject to the conditioned stimulus. The elevated plus maze is argued to measure the agoraphobic avoidance of the brightly lit open maze arms in crepuscular rodents. These two tasks have been used extensively, yet research into whether they interact is scarce. We investigated whether recall of an aversive memory, across contextual, odour or auditory modalities, would potentiate anxiety-like behaviour in the elevated plus maze. The data did not support that memory recall, even over a series of time points, could influence EPM behaviour. Furthermore, there was no correlation between EPM behaviour and conditioned freezing in independent cohorts tested in the EPM before or after auditory threat conditioning. Further analysis found the production of 22 kHz ultrasonic vocalisations revealed the strongest responders to a conditioned threat cue. These results are of particular importance for consideration when using the EPM and threat conditioning to identify individual differences and the possibility to use the tasks in batteries of tests without cross-task interference.

BrKCS6 mutation conferred a bright glossy phenotype to Chinese cabbage.

KEY MESSAGE: BrKCS6 encoding 3-ketoacyl-CoA synthases was cloned through MutMap and KASP analysis, and its function was verified via allelic mutants in Chinese cabbage. Bright and glossy green appearance is an attractive commodity character for leafy vegetables and is mainly caused by the absence of epicuticular wax crystals. In this study, two allelic epicuticular wax crystal deficiency mutants, wdm9 and wdm10, were obtained from an EMS mutagenesis population of Chinese cabbage (Brassica rapa L. ssp. pekinensis). Genetic analysis revealed that the mutant phenotype was controlled by a recessive nuclear gene. BrKCS6 encoding 3-ketoacyl-CoA synthases was identified as the candidate gene by MutMap and KASP analysis. A SNP (G to A) on BrKCS6 in wdm9 led to the amino acid substitution from serine (S) to phenylalanine (F), and another SNP (G to A) in wdm10 resulted in the amino acid substitution from serine (S) to leucine (L). Both SNPs are located in the ACP_syn_III_C conserved domain, corresponding to two highly conserved sites among BrKCS6 and its homologs. These two amino acid substitutions changed the secondary structure and the three-dimensional structure of BrKCS6 protein. qRT-PCR results showed that the relative expression of BrKCS6 significantly decreased in the flower, stem, and leaves in mutant, and the relative expressions of the downstream key genes of BrKCS6 were down-regulated in mutant. We were the first to clone the precious glossy bright gene BrKCS6 which has a great potential for commodity quality breeding in Chinese cabbage.

BrACOS5 mutations induced male sterility via impeding pollen exine formation in Chinese cabbage (Brassica rapa L. ssp. pekinensis).

KEY MESSAGE: BrACOS5 mutations led to male sterility of Chinese cabbage verified in three allelic male-sterile mutants. Chinese cabbage (Brassica rapa L. ssp. pekinensis) is one of the major vegetable crops in East Asia, and the utilization of male-sterile line is an important measure for its hybrid seed production. Herein, we isolated three allelic male-sterile mutants, msm1-1, msm1-2 and msm1-3, from an ethyl methane sulfonate (EMS) mutagenized population of Chinese cabbage double-haploid (DH) line 'FT', whose microspores were completely aborted with severely absent exine, and tapetums were abnormally developed. Genetic analyses indicated that the three male-sterile mutants belonged to allelic mutation and were triggered by the same recessive nuclear gene. MutMap-based gene mapping and kompetitive allele-specific PCR (KASP) analysis demonstrated that three different single-nucleotide polymorphisms (SNPs) of BraA09g012710.3C were responsible for the male sterility of msm1-1/2/3, respectively. BraA09g012710.3C is orthologous of Arabidopsis thaliana ACOS5 (AT1G62940), encoding an acyl-CoA synthetase in sporopollenin biosynthesis, and specifically expressed in anther, so we named BraA09g012710.3C as BrACOS5. BrACOS5 localizes to the endoplasmic reticulum (ER). Mutations of BrACOS5 resulted in decreased enzyme activities and altered fatty acid contents in msm1 anthers. As well as the transcript accumulations of putative orthologs involved in sporopollenin biosynthesis were significantly down-regulated excluding BrPKSA. These results provide strong evidence for the integral role of BrACOS5 in conserved sporopollenin biosynthesis pathway and also contribute to uncovering exine development pattern and underlying male sterility mechanism in Chinese cabbage.

Investigation of the Key Genes Associated with Anthocyanin Accumulation during Inner Leaf Reddening in Ornamental Kale (Brassica oleracea L. var. acephala).

Ornamental kale (Brassica oleracea L. var. acephala) is a popular decorative plant in late autumn and winter. However, only during low-temperature color-changed periods below rough 15 °C can the plant accumulate anthocyanins and exhibit a diverse array of foliar color patterns. In this study, we probed into the potential mechanism of inner leaf reddening in a red-leaf pure line of ornamental kale by physiological, metabolic, and transcriptomic analyses. Determination of anthocyanin contents in the uncolored new white leaves (S0), the light red leaves (S1) in the reddening period and the red leaves (S2) completing color change, and analysis of anthocyanin metabolites at stage S2, revealed that the coloring of red leaves was mainly attributed to the accumulation of cyanidins. We further used transcriptomic sequencing between the pairwise S0, S1, and S2 stages to identify 21 differentially expressed genes (DEGs) involved in anthocyanin biosynthesis, among which the expression level of 14 DEGs was positively correlated with anthocyanin accumulation, and 6 DEGs were negatively correlated with anthocyanin accumulation. A total of 89 co-expressed genes were screened out, from which three DEGs (BoCHI, Bo4CL3, and BoF3H) were identified as hub genes in co-expression DEGs network. BoDFR and BoCHI were the DEGs with the highest expressions at S2. Moreover, two co-expressed DEGs related to stress response (BoBBX17 and BoCOR47) also exhibited upregulated expressions and positive correlations with anthocyanin accumulation. A deep dive into the underlying regulatory network of anthocyanin accumulation comprising these six upregulated DEGs from S0 to S2 was performed via trend, correlation, and differentially co-expression analysis. This study uncovered the DEGs expression profiles associated with anthocyanin accumulation during ornamental kale inner leaf reddening, which provided a basis for further dissecting the molecular mechanisms of leaf color characteristic change in ornamental kale at low temperatures.

BrCWM Mutation Disrupted Leaf Flattening in Chinese Cabbage (Brassica rapa L. ssp. pekinensis).

Leaf flattening plays a vital role in the establishment of plant architecture, which is closely related to plant photosynthesis and, thus, influences the product yield and quality of Chinese cabbage. In this study, we used the doubled haploid line 'FT' of Chinese cabbage as the wild type for ethyl methanesulfonate (EMS) mutagenesis and obtained a mutant cwm with stably inherited compact and wrinkled leaves. Genetic analysis revealed that the mutated trait was controlled by a single recessive nuclear gene, Brcwm. Brcwm was preliminarily mapped to chromosome A07 based on bulked segregant RNA sequencing (BSR-seq) and fine-mapped to a 205.66 kb region containing 39 genes between Indel12 and Indel21 using SSR and Indel analysis. According to the whole-genome re-sequencing results, we found that there was only one nonsynonymous single nucleotide polymorphism (SNP) (C to T) within the target interval on exon 4 of BraA07g021970.3C, which resulted in a proline to serine amino acid substitution. The mutated trait co-segregated with the SNP. Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) revealed that BraA07g021970.3C expression was dramatically higher in 'FT' leaves than that in cwm leaves. BraA07g021970.3C is homologous to AT3G55000 encoding a protein related to cortical microtubule organization. A similar phenotype of dwarfism and wrinkled leaves was observed in the recessive homozygous mutant cwm-f1 of AT3G55000, and its T3 transgenic lines were restored to the Arabidopsis wild-type phenotype through ectopic overexpression of BraA07g021970.3C. These results verified that BraA07g021970.3C was the target gene essential for leaf flattening in Chinese cabbage.

Mutation in BrGGL7 gene encoding a GDSL esterase / lipase causes male sterility in Chinese cabbage (Brassica rapa L. ssp. pekinensis).

KEY MESSAGE: MutMap and KASP analyses revealed that the BrGGL7 gene is responsible for the male-sterile trait of ftms1 in Chinese cabbage, with functional verification in Arabidopsis. The application of a male-sterile line is an ideal approach of hybrid seed production in Chinese cabbage. In this study, we obtained a male-sterile mutant (ftms1) from the double haploid line 'FT' using ethyl methane sulfonate (EMS) mutagenesis. The mutant was completely sterile due to abnormal enlargement and vacuolization of the tapetum cells. A single recessive nuclear gene was found to control male sterility in the mutant, while MutMap and KASP analyses identified BraA05g022470.3C (BrGGL7), which encodes a GDSL esterase / lipase, as the candidate mutant gene. A single nucleotide substitution from C to T occurred within the domain of BrGGL7 in ftms1, resulting in premature translation termination in the fourth exon. Meanwhile, qRT-PCR analysis indicated that BrGGL7 was prominently expressed in the anthers, and expression was greater in the wild-type 'FT' than ftms1. Genetic complementation of the orthologous Arabidopsis ggl7 mutant further confirmed the role of BrGGL7 in pollen development. These findings suggest that BrGGL7 plays a fundamental role in pollen formation, providing important insight into the molecular mechanisms underlying male sterility in Chinese cabbage.

The Role of Klotho Protein Against Sevoflurane-Induced Neuronal Injury.

The effects of general anesthetics on the developing brain have aroused much attention in recent years. Sevoflurane, a commonly used inhalation anesthetic especially in pediatric anesthesia, can induce developmental neurotoxicity. In this study, the differentially expressed mRNAs in the hippocampus of newborn rats exposed to 3% sevoflurane for 6 h were detected by RNA-Sequencing. Those data indicated that the mRNA of Klotho was increased after exposure to sevoflurane. Moreover, the protein expression of Klotho was assayed by Western Blot. Besides over-expression and under-expression of Klotho protein, we also detected changes of cell proliferation, ROS, JC-1, and Bcl-2/Bax ratio in PC12 cells exposed to sevoflurane. After exposure to 3% sevoflurane, the expression of Klotho protein increased in the hippocampus of neonatal rats. In PC12 cells, exposure to sevoflurane could increase cellular ROS level, reduce mitochondrial membrane potential and Bcl-2/Bax ratio. While overexpression of Klotho alleviated the above changes, knockdown of Klotho aggravated the injury of sevoflurane. Klotho protein could reduce oxidative stress and mitochondrial injury induced by sevoflurane in the neuron.

Dental Implant Navigation System Based on Trinocular Stereo Vision.

Traditional dental implant navigation systems (DINS) based on binocular stereo vision (BSV) have limitations, for example, weak anti-occlusion abilities, as well as problems with feature point mismatching. These shortcomings limit the operators' operation scope, and the instruments may even cause damage to the adjacent important blood vessels, nerves, and other anatomical structures. Trinocular stereo vision (TSV) is introduced to DINS to improve the accuracy and safety of dental implants in this study. High positioning accuracy is provided by adding cameras. When one of the cameras is blocked, spatial positioning can still be achieved, and doctors can adjust to system tips; thus, the continuity and safety of the surgery is significantly improved. Some key technologies of DINS have also been updated. A bipolar line constraint algorithm based on TSV is proposed to eliminate the feature point mismatching problem. A reference template with active optical markers attached to the jaw measures head movement. A T-type template with active optical markers is used to obtain the position and direction of surgery instruments. The calibration algorithms of endpoint, axis, and drill are proposed for 3D display of the surgical instrument in real time. With the preoperative path planning of implant navigation software, implant surgery can be carried out. Phantom experiments are carried out based on the system to assess the feasibility and accuracy. The results show that the mean entry deviation, exit deviation, and angle deviation are 0.55 mm, 0.88 mm, and 2.23 degrees, respectively.

Fine Mapping of BoVl Conferring the Variegated Leaf in Ornamental Kale (Brassica oleracea var. acephala)

Ornamental kale, as a burgeoning landscaping plant, is gaining popularity for its rich color patterns in leaf and cold tolerance. Leaf variegation endows ornamental kale with unique ornamental characters, and the mutants are ideal materials for exploring the formation mechanisms of variegated phenotype. Herein, we identified a novel variegated leaf kale mutant 'JC007-2B' with green margins and white centers. Morphological observations and physiological determinations of the green leaf stage (S1), albino stage (S2) and variegated leaf stage (S3) demonstrated that the chloroplast structure and photosynthetic pigment content in the white sectors (S3_C) of variegated leaves were abnormal. Genetic analysis revealed that a single dominant nuclear gene (BoVl) controlled the variegated leaf trait of 'JC007-2B', and three candidate genes for BoVl were fine-mapped to a 6.74 Kb interval on chromosome C03. Multiple sequence alignment among the green-leaf mapping parent 'BS', recombinant individuals, mutant parent 'JC007-2B' and its same originated DH line population established that the mutation sites in Bo3g002080 exhibited a complete consensus. Bo3g002080, homologous to Arabidopsis MED4, was identified as the candidate gene for BoVl. Expression analysis showed that Bo3g002080 displayed a 2158.85-fold higher expression at albino stage than that in green leaf stage. Transcriptome analysis showed that related pathways of photosynthesis and chloroplast development were significantly enriched in the white sectors, and relevant DEGs involved in these pathways were almost down-regulated. Overall, our study provides a new gene resource for cultivar breeding in ornamental kale and contributes to uncovering the molecular genetic mechanism underlying the variegated leaf formation.

Application and evaluation of nucleic acid sequence-based amplification, PCR and cryptococcal antigen test for diagnosis of cryptococcosis.

BACKGROUND: Cryptococcosis is a major opportunistic invasive mycosis in immunocompromised patients, but it is also increasingly seen in immunocompetent patients. In the early stages of cryptococcosis, limitations of the detection method may hinder the diagnosis. A molecular diagnostic technique based on nucleic acid sequence-based amplification (NASBA) method was developed to fulfil the need for efficient diagnosis of cryptococcosis. METHODS: We compared the diagnostic performance of NASBA, PCR and cryptococcal antigen (CrAg) test (colloidal gold method) in clinical samples from 25 cryptococcosis patients (including 8 cryptococcal meningoencephalitis and 17 pulmonary cryptococcosis) who were categorized as proven cases (n = 10) and probable cases (n = 15) according to the revised EORTC/MSG definitions. 10 patients with non-Cryptococcus infection and 30 healthy individuals were categorized as control group. RESULTS: The lowest detection limit of NASBA was 10 CFU/mL, and RNA of non-target bacteria or fungi was not amplified. The sensitivity of NASBA, PCR and colloidal gold method was 92.00% (95% CI 72.50-98.60%), 64.00% (95% CI 42.62-81.29%), 100.00% (95% CI 83.42-100.00%), and the specificity was 95.00% (95% CI 81.79-99.13%), 80.00% (95% CI 63.86-90.39%) and 82.50% (95% CI 66.64-92.11%) respectively. The highest specificity (97.50%), accuracy (95.38%) and k value (0.90) were achieved when both NASBA and colloidal gold results were positive. CONCLUSIONS: NASBA is a new alternative detection method for cryptococcosis which is both accurate and rapid without expensive equipment and specialised personnel. It may be used as a tool for confirming current infection as well as monitoring the effectiveness of antifungal treatment. The use of NASBA to detect Cryptococcus RNA in blood samples is of great significance for the diagnosis of pulmonary cryptococcosis. The combination of NASBA and colloidal gold can improve the diagnostic accuracy of cryptococcosis.

Increased frequency of ß cells with abnormal NKX6.1 expression in type 2 diabetes but not in subjects with higher risk for type 2 diabetes.

BACKGROUND: NKX6.1 is a transcription factor for insulin, as well as a marker for ß cell maturity. Abnormal NKX6.1 expression in ß cells, such as translocation from the nucleus to cytoplasm or lost expression, has been shown as a marker for ß cell dedifferentiation. METHODS: We obtained pancreatic sections from organ donors and immunofluorescence staining with NKX6.1 and insulin was performed to characterize NKX6.1 expression in subjects with or without type 2 diabetes mellitus (T2DM). RESULTS: Our results showed that cells with insulin expression but no nucleic NKX6.1 expression (NKX6.1Nuc-Ins+), and cells with cytoplasmic NKX6.1 expression but no insulin expression (NKX6.1cytIns-) were significantly increased in T2DM subjects and positively correlated with glycated hemoglobin (HbA1c), indicating the elevated ß cell dedifferentiation with NKX6.1 inactivation in T2DM. To investigate whether ß cell dedifferentiation has initiated in subjects with higher risks for T2DM, we next analyzed the association between ß-cell dedifferentiation level in ND subjects with different ages, body mass index, and HbA1c. The results showed the absolute number and percentage of dedifferentiated ß cells with NKX6.1 inactivation did not significantly change in subjects with advanced aging, obesity, or modest hyperglycemia, indicating that the ß cell dedifferentiation might mainly occur after T2DM was diagnosed. CONCLUSION: Our results suggested that NKX6.1 expression in ß cells was changed in type 2 diabetic subjects, evidenced by significantly increased NKX6.1Nuc-Ins+ and NKX6.1cytIns- cells. This abnormality did not occur more frequently in subjects with a higher risk for T2DM, suggesting that ß cell dedifferentiation might be secondary to the pathological changes in T2DM.

High Precision Optical Tracking System Based on near Infrared Trinocular Stereo Vision.

A high precision optical tracking system (OTS) based on near infrared (NIR) trinocular stereo vision (TSV) is presented in this paper. Compared with the traditional OTS on the basis of binocular stereo vision (BSV), hardware and software are improved. In the hardware aspect, a NIR TSV platform is built, and a new active tool is designed. Imaging markers of the tool are uniform and complete with large measurement angle (>60°). In the software aspect, the deployment of extra camera brings high computational complexity. To reduce the computational burden, a fast nearest neighbor feature point extraction algorithm (FNNF) is proposed. The proposed method increases the speed of feature points extraction by hundreds of times over the traditional pixel-by-pixel searching method. The modified NIR multi-camera calibration method and 3D reconstruction algorithm further improve the tracking accuracy. Experimental results show that the calibration accuracy of the NIR camera can reach 0.02%, positioning accuracy of markers can reach 0.0240 mm, and dynamic tracking accuracy can reach 0.0938 mm. OTS can be adopted in high-precision dynamic tracking.

Hypoxia-inducible factor-1α mediates the expression of mature ß cell-disallowed genes in hypoxia-induced ß cell dedifferentiation.

Hypoxia affects the function of pancreatic ß cells, and the molecular mechanism underlying hypoxia-related ß cell dysfunction in human type 2 diabetes mellitus (T2DM) remains to be elucidated. In this study, by comparing the gene expression profiles of islets from nondiabetic and T2D subjects using gene chip array, we aimed to elucidate that hypoxia signaling pathways are activated in human T2DM islets. CoCl2 treatment, which was employed to mimic hypoxic stimulation in human islets, decreased insulin secretion, insulin content, and the functional gene expression of human islets. In parallel, the expression of mature ß cell-disallowed genes was upregulated by CoCl2, including progenitor cell marker NGN3, ß cell differentiation marker ALDH1A3, and genes that are typically inhibited in mature ß cells, namely, GLUT1 and LDHA, indicating that CoCl2-mimicked hypoxia induced ß cell dedifferentiation of human islets. This finding in human islets was confirmed in mouse ß cell line NIT-1. By using Dimethyloxalylglycine (DMOG) to activate hypoxia-inducible factor-1α (HIF-1α) or siRNAs to knockdown HIF-1α, we found that HIF-1α was a key regulator of hypoxia-induced dedifferentiation of ß cells by upregulating mature ß cell-disallowed genes. Our findings suggested that HIF-1α activation might be an important contributor to ß cell dedifferentiation in human T2DM islets, and HIF-1α-targeted therapies may have the potential to reverse ß cell dedifferentiation of human T2DM islets.

Opposing effects of IL-1ß/COX-2/PGE2 pathway loop on islets in type 2 diabetes mellitus.

The cyclooxygenase2 (COX-2) enzyme catalyzes the first step of prostanoid biosynthesis, and is known for its crucial role in the pathogenesis of several inflammatory diseases including type 2 diabetes mellitus (T2DM). Although a variety of studies revealed that COX-2 played a role in the IL-1ß induced ß cell dysfunction, the molecular mechanism remains unclear. Here, using a cDNA microarray and in silico analysis, we demonstrated that inflammatory responses were upregulated in human T2DM islets compared with non-diabetic (ND) islets. COX-2 expression was significantly enhanced in human T2DM islets, correlated with the high inflammation level. PGE2, the catalytic product of COX-2, downregulated the functional gene expression of PDX1, NKX6.1, and MAFA and blunted the glucose induced insulin secretion of human islets. Conversely, inhibition of COX-2 activity by a pharmaceutical inhibitor prevented the ß-cell dysfunction induced by IL-1ß. COX-2 inhibitor also abrogated the IL-1ß autostimulation in ß cells, which further resulted in reduced COX-2 expression in ß cells. Together, our results revealed that COX-2/PGE2 signaling was involved in the regulation of IL-1ß autostimulation, thus forming an IL-1ß/COX-2/PGE2 pathway loop, which may result in the high inflammation level in human T2DM islets and the inflammatory impairment of ß cells. Breaking this IL-1ß/COX-2/PGE2 pathway loop provides a potential therapeutic strategy to improve ß cell function in the treatment of T2DM patients.

Ethylenecarbodiimide-fixed splenocytes carrying whole islet antigens decrease the incidence of diabetes in NOD mice via down-regulation of effector memory T cells and autoantibodies.

Type 1 diabetes mellitus (T1DM) is a syndrome of loss of glucose homeostasis caused by the loss of ß cell chronic autoimmunity against islet cells. Islet-specific epitopes coupled antigen presenting cells by Ethylenecarbodiimide (ECDI) is a promising strategy to induce antigen-specific tolerance. However, single epitope induced tolerance is insufficient to prevent the onset of T1DM. The aim of this study is to evaluate the efficacy of whole islet antigens in preventing the onset and progression of T1DM and identify the underlying immune mechanism in NOD mice. In this study, the whole islet antigens, derived from islet lysate isolated from BALB/c mice, were coupled to splenocytes of BALB/c mice by ECDI fixation (SP-Islet lysate), and then intravenously administrated to NOD mice. The results showed that, compared with control group, SP-Islet lysate group significantly decreased T1DM incidence and improved the survival of NOD mice. SP-Islet lysate treated mice had reduced insulitis score and autoantibody levels, and improved glucose tolerance and insulin/glucagon production. Furthermore, the effector memory T cells (TEMs) were downregulated and regulatory T cells (Tregs) were upregulated by the SP-Islet lysate treatment, with reduced populations of Th1&Th17 cells. In conclusion, ECDI-fixed splenocytes carrying whole islet antigens effectively prevented the onset of T1DM in NOD mice, via suppressing the production of autoantibodies and inducing anergy of autoreactive T cells.

Phenotypic and genotypic characterization of antimicrobial resistance profiles in Streptococcus dysgalactiae isolated from bovine clinical mastitis in 5 provinces of China.

Bovine mastitis is among the most prevalent and costly diseases of dairy animals and is caused by a variety of bacterial pathogens including Streptococcus dysgalactiae. However, comprehensive studies reporting the prevalence and antimicrobial resistance profiles of S. dysgalactiae isolated from bovine mastitis are scarce. Therefore, this study was to investigate the occurrence of S. dysgalactiae associated with bovine clinical mastitis, to assess their antimicrobial resistance profiles, and to analyze the phenotypic and genotypic profiling of resistant isolates. In total, 1,180 milk samples were collected from dairy cows with clinical mastitis belonging to 74 commercial dairy herds located in 14 provinces of China from January 2014 to May 2016. Overall S. dysgalactiae isolates were recovered from 88 (7.5%) of the mastitic milk samples. The antimicrobial susceptibility of these isolates was tested against 8 antimicrobial agents by using minimum inhibitory concentrations. Results showed that 82 (93.2%) isolates expressed resistance to more than one antimicrobial agent. Antimicrobial resistance was highest against kanamycin (89.8%), sulfonamide (83.0%), and streptomycin (58.0%), which can be attributed to the intrinsic resistance for most of Streptococcus spp. against those antimicrobial substances. Strikingly, 30 (34.1%) and 12 (13.6%) isolates were found resistant to cephalexin and ceftriaxone, respectively. BlaTEM, ermB, and tetM were the most prevalent resistance genes. All isolates carried at least one of all tested resistance genes. Also, 1.1, 12.5, 18.2, 36.4, and 31.8% of isolates were positive for at least one tested resistance gene in 1, 2, 3, 4, or 5 classes of antimicrobials. Survival analysis showed a significant association between ermB and survival of the S. dysgalactiae isolates at increasing erythromycin concentrations. No other statistically significant associations were observed between the phenotypic and genotypic resistance profiles. This study concludes a considerable prevalence of S. dysgalactiae associated with bovine mastitis in dairy herds of China and these isolates exhibited high resistance rates to tested antimicrobials, coupled with high occurrence of resistance genes. Both the prevalence of S. dysgalactiae and their antimicrobial resistance profiles strongly varied among dairy herds, demonstrating the need for antimicrobial susceptibility surveillance at the herd level to ensure optimal therapeutic results.