[Molecular epidemiology and antibiotic resistance of Pseudomonas aeruginosa isolated from blood in a hospital in Shandong Province from 2014 to 2021].

Objective: To identify the antibiotic resistance, virulence genes, and sequence types of Pseudomonas aeruginosa (P. aeruginosa) strains isolated from blood. Methods: From November 2014 to December 2021, a total of 94 nonrepetitive P. aeruginosa isolates were obtained from blood samples of patients at the First Affiliated Hospital of Shandong First Medical University in Shandong Province, China. The bacteria were identified using matrix-assisted laser desorption ionization time of flight mass spectrometry. Antibiotic resistance of the P. aeruginosa isolates was detected using Vitek 2 Compact system. Polymerase chain reaction (PCR) was conducted for the 18 virulence genes, and multi locus sequence typing (MLST) was performed to identify the sequence types of the P. aeruginosa strains. The resistance rates and distributions of virulence genes between carbapenem resistant pseudomonas aeruginosa (CRPA) and carbapenem susceptible pseudomonas aeruginosa (CSPA) isolates were compared using the Chi-square test. Results: Among 94 P. aeruginosa isolates, 19 (20.2%) isolates were found to be multidrug resistant (MDR) bacteria, of which 17 were CRPA isolates and 2 were CSPA isolates. All strains contained more than 10 virulence genes. Except for exoU gene, the detection rate of other genes was above 83%. MLST analysis revealed a total of 66 different STs, including 59 existing STs and 7 novel STs. Among them, ST244 (n=11, 11.7%) and ST270 (n=7, 7.4%) were the dominant STs. Although these two types of isolates harbored the same virulence genes, the resistance rates to carbapenem were different. 54.5% (6/11) ST244 isolates were CRPA but all 7 ST270 isolates were CSPA. Conclusion: Although the resistance rates of P. aeruginosa strains isolated from blood were at a low level, some MDR and CRPA isolates were detected. As the high virulence gene detection rates and genetic diversity were found for P. aeruginosa strains isolated from blood, close attention should be paid to avoid transmission and outbreaks.

[Questionnaire survey analysis on the screening of tuberculosis among diabetic patients in general hospitals of Hunan Province].

Objective: To investigate the development of tuberculosis screening-related tests in general public hospitals(GPHs) of different levels in Hunan Province and the"awareness and practice of screening tuberculosis in diabetic patients"by doctors directly involved in diabetes diagnosis and treatment in the hospitals, aiming to provide reference for the formulation of the tuberculosis-diabetes joint prevention and control activity plan based on our national conditions. Methods: Stratified sampling was used to select 43 GPHs at three different levels in Hunan province: 14 tertiary GPHs, 13 secondary GPHs, and 16 primary GPHs. 284 endocrinologists working in enrolled hospitals were invited to participate in the on-site questionnaire-survey and 277 qualified. The study used SPSS 22.0 statistical software to analyze the data. The prevalence rate of tuberculosis screening test among hospitals at all levels was compared by chi-square test, and logistic regression was used to analyze the related factors affecting doctors' screening awareness. P≤0.05 was considered statistically significant. Results: The allocation of digital X-ray cameras, tuberculin skin tests, sputum acid-fast bacillus smears, sputum cultures for Mycobacterium tuberculosis, and interferon-gamma release assays in the 43 GPHs were 90.7% (39/43), 72.1% (31/43), 55.8% (24/43), 34.9% (15/43), 27.9% (12/43) with significant differences between the different hospital levels(P<0.05). 173 endocrinologists considered it necessary to proactively initiate tuberculosis screening for patients at first diagnosis. When admitting patients, 197 endocrinologists chose tuberculosis screening only for diabetes mellitus patients with suspected tuberculosis symptoms. The most possible reasons why diabetes mellitus patients wouldn't undergo tuberculosis screening were"patients refused(76.5%, 212/277)","patients didn't complain of the symptom(46.9%, 130/277)", and"tuberculosis screening-related tests haven't been conducted in the hospital(35.7%, 99/277)". Conclusions: Although endocrinologists displayed some tuberculosis-related knowledge and awareness of the need for proactive tuberculosis screening, the actual screening rate in the clinical setting was low. This may be related to multiple factors, including those of patients, doctors, and medical institutions.

[Application of transvaginal morcellation within disposable extraction bag with traction wire in laparoscopic myomectomy].

To investigate the effect and clinical value of morcellation within disposable extraction bag with traction wire through posterior vaginal fornix in laparoscopic myomectomy. A total of 42 patients who underwent laparoscopic myomectomy and morcellation through posterior vaginal fornix in the Second Affiliated Hospital of Soochow University from June 2019 to June 2021 were retrospectively analyzed. After the uterine fibroids were removed, the fibroids were placed into the extraction bag, tightening the mouth of the bag with a traction wire to make it airtight. After the uterine incisions were sutured, the extraction bag was taken out through the posterior fornix of the vagina, and the fibroids were broken up with a scalpel in the bag and taken out. The fibroids were successfully removed from the 42 patients through the posterior fornix of the vagina. There were no fibroids fragments found in the peritoneal cavity and vagina. There were no malignant cells or spindle cells found in the peritoneal lavage cytology before and after the operation. After filling the extraction bags with water, there was no leakage. There were 39 cases of uterine leiomyoma, 2 cases of cell-rich uterine leiomyoma, and 1 case of smooth muscle tumor of uncertain malignant potential in postoperative pathological diagnosis. Forty-two cases were followed up for 6 to 30 months. The posterior vaginal fornix incision healed well and there was no recurrence or metastasis. Morcellation within disposable extraction bag with traction wire through posterior vaginal fornix in laparoscopic myomectomy is a safe and feasible method for fibroids removal, which may help to reduce the dissemination of iatrogenic tumors.

Cohesive properties of PbBi/Fe3O4 and PbBi/(Fe,Cr)3O4 interfaces.

First principles calculations reveal that the effects of PbBi on the cohesive properties of Fe3O4 and (Fe,Cr)3O4: PbBi can reduce the cohesive strength of the oxides, and the contents of O and Cr on the O-terminated oxide side play a significant role in the cohesive properties of the PbBi/Fe3O4 and PbBi/(Fe,Cr)3O4 interfaces. Specifically, the performance of oxidation decreases more significantly under the conditions of insufficient oxygen, and a high ratio of Cr of the subsurface of oxides can lead to the reduction of the cohesive properties of O-terminated interfaces. Calculations also show that the Pb-O-terminated interfaces are energetically favorable and are more stable than the Bi-O-terminated surfaces due to the strong bond of Pb-O, while the Bi-Cr and Bi-Fe interfaces are more stable than the Pb-Cr and Pb-Fe interfaces. Moreover, it is found that the stability and cohesion of the PbBi/Fe3O4 and PbBi/(Fe,Cr)3O4 interfaces will decrease when the oxygen concentration is insufficient or the degree of wetting of PbBi of oxides is low, and the PbBi/Fe3O4 interface is more sensitive to these conditions. The bond-dissociation energies and electronic structures provide a deep understanding of various interface properties, and the obtained results are in good agreement with experimental measurements in the literature.

ORAOV1-B Promotes OSCC Metastasis via the NF-κB-TNFα Loop.

Metastasis, a powerful prognostic indicator of oral squamous cell carcinoma (OSCC), is chiefly responsible for poor cancer outcomes. Despite an increasing number of studies examining the mechanisms underlying poor outcomes, the development of potent strategies is hindered by insufficient characterization of the crucial regulators. Long noncoding RNAs (lncRNAs) have recently been gaining interest as significant modulators of OSCC metastasis; however, the detailed mechanisms underlying lncRNA-mediated OSCC metastasis remain relatively uncharacterized. Here, we identified a novel alternative splice variant of oral cancer overexpressed 1 (ORAOV1), named as ORAOV1-B, which was subsequently validated as an lncRNA and correlated with OSCC lymph node metastasis; significantly increased invasion and migration were observed in ORAOV1-B-overexpressing OSCC cells. RNA pulldown and mass spectrometry identified Hsp90 as a direct target of ORAOV1-B, and cDNA microarrays suggested TNFα as a potential downstream target of ORAOV1-B. ORAOV1-B was shown to directly bind to and stabilize Hsp90, which maintains the function of client proteins, receptor-interaction protein, and IκB kinase beta, thus activating the NF-κB pathway and inducing TNFα. Additionally, TNFα reciprocally enhanced p-NF-κB-p65 and the downstream epithelial-mesenchymal transition. ORAOV1-B effects were reversed by a TNFα inhibitor, demonstrating that TNFα is essential for ORAOV1-B-regulated metastatic ability. Consistent epithelial-mesenchymal transition in the ORAOV1-B group was demonstrated via an orthotopic model. In the metastatic model, ORAOV1-B significantly contributed to OSCC-related lung metastasis. In summary, the novel splice variant ORAOV1-B is an lncRNA, which significantly potentiates OSCC invasion and metastasis by binding to Hsp90 and activating the NF-κB-TNFα loop. These findings demonstrate the versatile role of ORAOV1 family members and the significance of genes located within 11q13 in promoting OSCC. ORAOV1-B might serve as an attractive OSCC metastasis intervention target.

[Exploration and application of a novel attempt to recruit participants in clinical trials of vaccines under the emergency].

Objective: To evaluate and share the novel method for recruiting participants in clinical trials of vaccines in emergency situations. Methods: To publish recruitment notice in local areas of Wuhan through websites and medium, and guide interested persons to log in to the"Clinical Trials of SARS-CoV-2 Vaccine Reservation and Health Declaration System"to appoint and register their health information. The "Health Declaration System" provides each volunteer evaluation and risk levels to preliminarily exclude those who do not meet the inclusion criteria. Researchers review the qualified volunteers by telephone, organize them to go to the vaccination site, and finally conduct a strict medical screening to determine the final subjects. Results: A total of 4 819 people and 5 132 people registered in the Phase Ⅰ and Phase Ⅱ recruitment system respectively, with men 2 912 (60.43%) and 2 887 (56.25%) more than women 1 907 (39.57%) and 2 245 (43.75%), mostly in the 20-39 age group, with 3 211 (66.63%) and 3 966 (77.28%). All 13 districts in Wuhan have interested residents to participate clinical research.The initial qualified rate of the Phase Ⅱ recruitment system was higher than that of Phase Ⅰ, with men 2 047 (70.28%) and 2 135(73.95%), higher than women 1 083 (56.80%) and 1 472 (65.57%); 440 and 689 people were reviewed by telephone in Phase Ⅰ and Phase Ⅱ respectively, and the number of verified volunteers was about 440 (35.00%) and 689 (67.20%); Of the 201 603 people who arrived at the vaccination site, 12 and 26 of them were positive for the SARS-CoV-2 antibody with an antibody positive rate of 6.00% and 4.31% respectively. Conclusion: The novel method for recruiting subjects in this clinical study is efficient and reliable, and the recruitment situation of Phase Ⅰ had set a good example for Phase Ⅱ but the medium-and long-term compliance of subjects and the separation of willingness and behaviors still need to be further studied.

Translational outcomes in a full gene deletion of ubiquitin protein ligase E3A rat model of Angelman syndrome.

Angelman syndrome (AS) is a rare neurodevelopmental disorder characterized by developmental delay, impaired communication, motor deficits and ataxia, intellectual disabilities, microcephaly, and seizures. The genetic cause of AS is the loss of expression of UBE3A (ubiquitin protein ligase E6-AP) in the brain, typically due to a deletion of the maternal 15q11-q13 region. Previous studies have been performed using a mouse model with a deletion of a single exon of Ube3a. Since three splice variants of Ube3a exist, this has led to a lack of consistent reports and the theory that perhaps not all mouse studies were assessing the effects of an absence of all functional UBE3A. Herein, we report the generation and functional characterization of a novel model of Angelman syndrome by deleting the entire Ube3a gene in the rat. We validated that this resulted in the first comprehensive gene deletion rodent model. Ultrasonic vocalizations from newborn Ube3am-/p+ were reduced in the maternal inherited deletion group with no observable change in the Ube3am+/p- paternal transmission cohort. We also discovered Ube3am-/p+ exhibited delayed reflex development, motor deficits in rearing and fine motor skills, aberrant social communication, and impaired touchscreen learning and memory in young adults. These behavioral deficits were large in effect size and easily apparent in the larger rodent species. Low social communication was detected using a playback task that is unique to rats. Structural imaging illustrated decreased brain volume in Ube3am-/p+ and a variety of intriguing neuroanatomical phenotypes while Ube3am+/p- did not exhibit altered neuroanatomy. Our report identifies, for the first time, unique AS relevant functional phenotypes and anatomical markers as preclinical outcomes to test various strategies for gene and molecular therapies in AS.

Genetic Polymorphism of Y Chromosome Haplogroup D-M174 in East Asian Populations.

ABSTRACT: Objective To explore the genetic polymorphism of Y chromosome D-M174 haplogroup and sub-haplogroups in East Asia. Methods The samples of 1 426 unrelated male individuals from East Asia were collected, and then 7 Y chromosome haplogroup D-M174 and the Y-SNP of its sub-haplogroups were detected with mini-sequencing. The 22 Y-STR genotypes were detected with DNA Typer™ Y26 kit. The haplogroup was analyzed using direct counting method, heatmap, phylogenetic cluster and network graph cluster, and then distribution of genetic polymorphism and the clustering relation between populations and samples of Y chromosome D haplogroup were discussed. Results Haplogroup D-M174 were distributed mostly among Tibetans （40.96%）and Japanese （35.71%）, while less or none were distributed among the surrounding areas of Tibet and other areas. Conclusion The geographical distribution of Y chromosome D-M174 haplogroup in East Asian populations has significant characteristics.

Network Analysis of Y-STR in Six Ethnic Populations in Guangxi and Its Forensic Significances.

ABSTRACT: Objective To explore the distribution of genetic structure of Y-SNP and Y-STR genetic markers in different ethnic groups and its application in forensic science. Methods SNaPshot minisequencing was used to detect the polymorphisms of 12 Y-SNP loci in 439 males from 6 ethnic groups, including Guangxi Han, Guangxi Jing, Guangxi Miao, Guangxi Yao, Guangxi Zhuang and Guangxi Dong. DNATyperTM Y26 kit was used to multiplex-amplify 26 Y-STR loci. The PCR products were analyzed by 3130xl genetic analyzer. The network analysis of Y-STR haplotype under the same Y-SNP haplogroup was analyzed by Network 5.0 software. Results Six haplogroups defined by 12 Y-SNP loci were detected in 6 ethnic groups, and 362 haplotypes were detected in 26 Y-STR loci. The haplotype diversity was 0.996 6. In the C haplogroup, the samples from Guangxi Yao, Guangxi Zhuang and Guangxi Dong were clustered on different branches; in the O1 haplogroup, those from Guangxi Zhuang, Guangxi Miao and Guangxi Jing were relatively independent and clustered separately; in the O2 haplogroup, some samples from Guangxi Miao and Guangxi Yao were gathered in a cluster. Conclusion Based on the Y-STR network analysis of samples with identical haplogroup of Y-SNP, some ethnic groups can be preliminarily distinguished, which could be used to infer male suspects' ethnic group through detecting their genetic markers left in the crime scene.

Snail1-dependent cancer-associated fibroblasts induce epithelial-mesenchymal transition in lung cancer cells via exosomes.

BACKGROUND: Epithelial-mesenchymal transition (EMT) is an essential component of metastasis. Our previous study demonstrated that cancer-associated fibroblasts (CAFs) induce EMT in lung cancer cells. In recent years, many studies have demonstrated that CAFs induce metastasis and drug resistance in cancer cells via exosomes. AIM: We sought to discover the mechanism underlying how CAFs induce EMT in lung cancer cells, unveiling the role of exosomes in lung cancer progression. DESIGN: We cultured lung cancer cell (i) with control medium, normal fibroblasts (NFs) or CAFs; (ii) with SNAI1-transfected or NC (negative control)-transfected CAFs; (iii) with exosomes extracted from NF- or CAF-conditioned medium; (iv) with exosomes released by SNAI1 or NC-transfected CAFs; (v) with CAF-conditioned medium or exosome-depleted CAF-conditioned medium. METHODS: qRT-PCR was conducted to examine the expression of CDH1 (gene of E-cadherin) and VIM (gene of Vimentin), western blotting was conducted to examine E-cadherin and vimentin levels in lung cancer cells. RESULTS: Exosomes released by CAFs-promoted EMT in lung cancer cells. Interestingly, SNAI1 levels in exosomes secreted from CAFs were correlated with SNAI1 expression in CAFs. Furthermore, the level of SNAI1 in exosomes was crucial for inducing EMT in lung cancer cells. Finally, treatment of CAFs with GW4869, an inhibitor of exosome release, noticeably inhibited their EMT-inducing effect on recipient epithelial cells. CONCLUSIONS: The molecular mechanism underlying how CAFs induce EMT in cancer cells may be that CAFs deliver SNAI1 to recipient cancer cells via exosomes.

Functional characterization of ultraspiracle in Leptinotarsa decemlineata using RNA interference assay.

A heterodimer of ultraspiracle (USP) and ecdysone receptor (EcR) mediates 20-hydroxyecdysone (20E) signalling cascade to regulate insect moulting and metamorphosis. However, at least two questions remain to be addressed in terms of the molecular importance of USP in insect species. First, is USP involved in both regulation of ecdysteroidogenesis and mediation of 20E signalling in non-drosophilid insects, as in Drosophila melanogaster? Second, does USP play any role in larval metamorphosis except as the partner of heterodimeric receptor to activate the downstream 20E signalling genes? In this paper, we found that RNA interference (RNAi) of LdUSP in the final (fourth) instar larvae reduced the messenger RNA levels of four ecdysteroidogenesis genes (Ldspo, Ldphm, Lddib and Ldsad) and 20E titre, and repressed the expression of five 20E signal genes (EcRA, HR3, HR4, E74 and E75) in Leptinotarsa decemlineata. The LdUSP RNAi larvae remained as prepupae, with developing antennae, legs and discs of forewings and hindwings. Dietary supplement with 20E restored the expression of the five 20E signal genes, but only partially alleviated the decreased pupation rate in LdUSP RNAi beetles. Knockdown of LdUSP at the penultimate (third) instar larvae did not affect third-fourth instar moulting. However, silencing LdUSP caused similar but less severe impairments on pupation. Accordingly, we propose that USP is undoubtedly necessary for ecdysteroidogenesis, for mediation of 20E signalling and for initiation of metamorphosis in L. decemlineata.

[Regulation mechanism of E2F1 transcription factor on M2 macrophages in full-thickness skin defect wounds of mice].

Objective: To explore the regulatory mechanism of E2F1 transcription factor on M2 macrophages in full-thickness skin defect wounds of mice. Methods: E2F1 gene knockout heterozygotes C57BL/6 mice and wild-type C57BL/6 mice were introduced and self-reproduced. Two weeks after birth, E2F1 gene knockout homozygotes mice and wild-type mice were identified by polymerase chain reaction (PCR). Twelve identified 6-8 weeks old male E2F1 gene knockout homozygotes C57BL/6 mice and wild-type C57BL/6 mice were selected respectively according to the random number table and set as E2F1 gene knockout group and wild-type group. A full-thickness skin defect wound was made on the back of each mouse. On post injury day (PID) 2 and 7, 6 mice in each group were selected according to the random number table and sacrificed, and the wound tissue was excised. The expression of CD68 and CD206 double positive M2 macrophages was observed by immunofluorescence method, and the percentage of CD206 positive cells was calculated. The protein expression of CD206 was detected by Western blotting. The mRNA expression of arginase 1 was detected by real-time fluorescent quantitative reverse transcription PCR (RT-PCR). Wound tissue specimens of the two groups on PID 7 were obtained, and the protein and mRNA expressions of peroxisome proliferator-activated receptor gamma (PPAR-γ) were detected by Western blotting and real-time fluorescent quantitative RT-PCR respectively. The above-mentioned experiments were repeated four times. Three specimens of wound tissue of mice in wild-type group on PID 7 were obtained to detect the relationship between E2F1 and PPAR-γ by co-immunoprecipitation and Western blotting, and this experiment was repeated two times. Data were processed with unpaired t test. Results: The size of PCR products of E2F1 gene knockout homozygotes C57BL/6 mice and wild-type C57BL/6 mice were 227 and 172 bp respectively, which were the same as those of the designed DNA fragments. On PID 2 and 7, the number of CD68 and CD206 double positive M2 macrophages in the wound tissue of mice in E2F1 gene knockout group was more than that of wild-type group, and the percentages of CD206 positive cells in the wound tissue of mice in E2F1 gene knockout group were (0.234±0.032)% and (0.584±0.023)% respectively, which were significantly higher than (0.129±0.017)% and (0.282±0.071)% of wild-type group (t=3.29, 3.54, P<0.05). On PID 2 and 7, the protein expression of CD206 in the wound tissue of mice in E2F1 gene knockout group were 1.00±0.23 and 1.63±0.26 respectively, which were significantly higher than 0.43±0.06 and 0.97±0.08 of wild-type group (t=2.41, 2.45, P<0.05). On PID 2 and 7, the mRNA expressions of arginase 1 in the wound tissue of mice in E2F1 gene knockout group were 0.482±0.105 and 0.195±0.031 respectively, which were significantly higher than 0.163±0.026 and 0.108±0.017 of wild-type group (t=3.04, 2.86, P<0.05). On PID 7, the protein and mRNA expressions of PPAR-γ in the wound tissue of mice in E2F1 gene knockout group were 0.61±0.12 and 0.51±0.13 respectively, which were significantly higher than 0.20±0.04 and 0.20±0.04 of wild-type group (t=3.36, 2.86, P<0.05). On PID 7, detection of the wound tissue of mice in wild-type group showed that PPAR-γ had unidirectional effect on E2F1. Conclusions: E2F1 transcription factor affects the polarization of M2 macrophages by inhibiting the expression of PPAR-γ, thereby inhibiting the healing process of full-thickness skin defect wounds in mice.

Impairment of pupation by RNA interference-aided knockdown of Broad-Complex gene in Leptinotarsa decemlineata (Say).

Dietary delivery of bacterially expressed double-stranded RNA (dsRNA) has a great potential for management of Leptinotarsa decemlineata. An important first step is to discover possible RNA-interference (RNAi)-target genes effective against larvae, especially the old larvae. In the present paper, five putative Broad-Complex (BrC) cDNAs (Z1-Z4, and Z6) were identified in L. decemlineata. The expression of the five LdBrC isoforms was suppressed by juvenile hormone signaling, whereas the transcription was upregulated by 20-hydroxyecdysone signaling at the fourth (final) instar larval stage. Feeding of bacterially expressed dsBrC (derived from a common fragment of the five LdBrC variants) in the third- and fourth-instar larvae successfully knocked down the target mRNAs. For the fourth-instar LdBrC RNAi hypomorphs, they had a higher larval mortality compared with the controls. Moreover, most dsBrC-fed beetles did not pupate normally. After removal of the apolysed larval cuticle, a miniature adult was found. The adult head, compound eyes, prothorax, mesothorax, metathorax were found on the dorsal view. Distinct adult cuticle pigmentation was seen on the prothorax. The mouthparts, forelegs, midlegs, and hindlegs could be observed on the ventral view of the miniature adults. For the third-instar LdBrC RNAi specimens, around 20% moribund beetles remained as prepupae and finally died. Therefore, LdBrC is among the most attractive candidate genes for RNAi to control the fourth-instar larvae in L. decemlineata.

Isoform specific roles of Broad-Complex in larval development in Leptinotarsa decemlineata.

Broad-Complex (BrC) is a downstream target of both 20-hydroxyecdysone and juvenile hormone signalling. BrC regulates morphogenetic changes between nymphal instars in hemimetabolans, whereas it controls pupal commitment, pupal morphogenesis and inhibits adult differentiation in holometabolans. Among five BrC cDNAs (Z1-Z4 and Z6) identified in the Colorado potato beetle, we found in this work that Z1, Z2 and Z6 were mainly expressed at the last (fourth) instar and prepupal stages, whereas the levels of Z3 and Z4 increased during the penultimate (third) instar stage, peaked at the last instar larval phase and gradually decreased at the prepupal and pupal periods. When knocking down all BrC isoforms by RNA interference (RNAi) at the penultimate instar stage, around 20% of the resultant larvae remained as moribund beetles. These moribund BrC RNAi larvae were completely or partially wrapped in old cuticle. Likewise, a portion of larvae treated for a single double-stranded RNA of Z3, Z4 or Z6 displayed a degree of similar aberrancies, increasing in the order of isoforms Z6 < Z3 < Z4. When silencing all BrC isoforms at the last instar period, most of the RNAi larvae did not normally pupate or emerge as adults. Separately silencing each of the five zinc finger domains revealed that approximately 70% of the Z1 RNAi larvae remained as prepupae, around 60% of the Z6 RNAi specimens formed aberrant prepupae or pupae and about 60% of the Z2 RNAi beetles became deformed pupae. After removal of the old exuviae, these deformed larvae in which either Z1, Z2 or Z6 was depleted possessed adult prothorax and mesothorax, developing antenna, mouthparts and wing discs. Moreover, less than 50% of the resultant pupae finally emerged as adults when either of Z1, Z2 or Z6 was knocked down. Therefore, our findings reveal, for the first time, that the two roles of BrC in insect groups (ie directing morphogenetic changes during juvenile development and regulating larval-pupal-adult metamorphosis) are played by different BrC isoforms in Leptinotarsa decemlineata.

Requirement of Leptinotarsa decemlineata gene within the 74EF puff for larval-pupal metamorphosis and appendage growth.

Two Drosophila melanogaster E-twenty-six domain transcription factor isoforms (E74A and E74B) act differentially at the start of the 20-hydroxyecdysone (20E) signalling cascade to regulate larval-pupal metamorphosis. In the present paper, we identified the two isoforms (LdE74A and LdE74B) in Leptinotarsa decemlineata. During the larval development stage, the mRNA transcript levels of the two LdE74 isoforms were correlated with circulating 20E titres. In vitro midgut culture and in vivo dietary supplementation with 20E revealed that the presence of 20E induced expression peaks of both LdE74A and LdE74B, with similar patterns observed for the two isoforms. Moreover, the mRNA transcript levels of both LdE74A and LdE74B isoforms were significantly downregulated in the L. decemlineata ecdysone receptor RNA interference (RNAi) specimens, but not in the LdE75 RNAi beetles. Ingestion of 20E reduced the larval fresh weights and shortened the larval development period, irrespective of knockdown of LdE74 or not. RNAi of LdE74 did not affect 20E-induced expression of the Ecdysone induced protein 75-hormone receptor 3-fushi tarazu factor 1 (E75-HR3-FTZ-F1) transcriptional cascade. Thus, it seems that LdE74 mediates 20E signalling independent of the E75-HR3-FTZ-F1 transcriptional cascade. Furthermore, silencing of both LdE74 isoforms caused failure of ecdysis. Most of the LdE74 RNAi beetles remained as prepupae. The LdE74 RNAi prepupae exhibited adult character-like forms underneath after removal of the apolysed larval cuticle. Their appendages such as antennae, legs and wings were shorter than those of control larvae. Only a few LdE74 RNAi larvae finally became deformed pupae, with shortened antennae and legs. Therefore, LdE74 is required for larval-pupal metamorphosis and appendage growth in L. decemlineata.

A study on natural recovery of tassel fertilization and doubling method in maize haploids.

Doubling method is the technical barriers in maize haploid breeding. It was very important to establish the independent intellectual property rights for doubling method. In this experiment, the maize haploid inducer, TG15, was used for producing maternal haploids. Also, haploids were obtained from two kinds of maternal genotypes involved in the experiment, including high-oil type and common type. Significant differences were observed among offspring of various genotypes in the recovery of haploid fertilization. In 21 hybrid offspring haploids, the average powder rate was 8.28%, and the seed setting rate was 4.98%. The experimental results showed that when the hybrids were treated with 0.08% colchicine, the average powder rate and seed setting rate of offspring haploids were 35.53 and 20.30%, respectively, which were significantly higher than the hybrids with natural recovery ability. This study primarily established the doubling method of haploids called "bud seedling method" in China which was very practicably in maize doubled haploid breeding.

Chemical composition, true nutrient digestibility, and true metabolizable energy of novel pet food protein sources using the precision-fed cecectomized rooster assay.

A wide variety of animal protein-based ingredients is commonly used in the pet food products. The raw ingredients and processing procedures used may greatly affect protein quality. Testing the quality of alternative protein sources is necessary and contributes to the sustainability of pet foods. The objective of this study was to test the chemical composition of 8 protein sources intended for use in dog and cat foods (calamari meal, pork peptone, alligator meal, lamb meal, venison meal, chicken meal, and 2 duck meals), and evaluate their true nutrient digestibility and nitrogen-corrected true ME (TMEn) using the precision-fed cecectomized rooster assay. Calamari meal and pork peptone had lower ash (4.4 and 3.6% of DM, respectively) but greater CP (88.1 and 80.5% of DM, respectively) and either greater or similar GE (5.6 and 5.3 kcal/g of DM, respectively) compared with alligator, lamb, venison, chicken, and duck meals (11.8 to 24.5% ash, 58.7 to 65.9% CP, and 4.6 to 5.3 kcal GE/g). Acid-hydrolyzed fat (AHF) was lower in calamari meal (8.7% of DM) compared with the other proteins tested (15.5-22.1% of DM). True nutrient digestibility was variable among the protein sources (52 to 79% of DM, 60 to 83% of OM, 78 to 92% of AHF, and 70 to 89% of GE) with pork peptone having the highest DM, AHF, and GE digestibility and calamari meal having the highest OM digestibility. True indispensable AA digestibility was highest for calamari meal, with all AA having a digestibility greater than 90%. Except for histidine, all indispensable AA had a digestibility over 85% for pork peptone. In contrast, true indispensable AA digestibility was lowest for lamb meal, with histidine having digestibility less than 70% and the other entire indispensable AA having digestibility between 72 and 88%. The TMEn of calamari meal (4.82 kcal/g DM and 86.9% of GE) was greater ( < 0.05) than the other protein sources. The lamb meal had the lowest TMEn value (3.12 kcal/g DM and 66.9% of GE), with others being intermediate (3.46-4.09 kcal/g DM and 71.2-77.9% of GE). This study demonstrates the considerable variability that exists not only in the chemical composition but also in the true nutrient digestibility among protein sources intended for use in dog and cat foods and justifies further in vivo testing of novel protein sources.

[Using (1)H-nuclear magnetic resonance metabolomics and gene ontology to establish pathological staging model for esophageal cancer patients].

OBJECTIVES: By combining the metabolomics and computational biology, to explore the relationship between metabolic phenotype and pathological stage in esophageal cancer patients, to find the mechanism of metabolic network disturbance and develop a new method for fast preoperative clinical staging. METHODS: A prospective cohort study (from April 2013 to January 2016) was conducted. The preoperative patients from Sichuan Provincial People's Hospital, who were diagnosed with esophageal cancer from May 2013 to April 2014 were included, and their serum samples were collected to detect (1)H-nuclear magnetic resonance (NMR) metabolomics for the purpose of drawing the metabolic fingerprinting in different stages of patients with esophageal cancer. The data were processed with these methods-principal components analysis: partial least squares regression and support vector machine, for the exploration of the enzyme-gene network regulatory mechanism in abnormal esophageal cancer metabolic network regulation and to build the quantitative prediction model of esophageal cancer staging in the end. All data were processed on high-performance computing platforms Matalab. The comparison of data had used Wilcoxon test, variance analysis, χ(2) test and Fisher exact test. RESULTS: Twenty patients with different stages of esophageal cancer were included; and their serum metabolic fingerprinting could differentiate different tumor stages. There were no difference among the five teams in the age (F=1.086, P>0.05), the body mass index (F=1.035, P>0.05), the distance from the incisors to tumor (F=1.078, P>0.05). Among the patients with different TNM stages, there was a significant difference in plasma metabolome. Compared to ⅡB, ⅢA, Ⅳstage patients, increased levels of butanone, ethanol amine, homocysteine, hydroxy acids and estriol, together with decreased levels of glycoprotein, creatine, choline, isobutyricacid, alanine, leucine, valine, were observed inⅠB, ⅡA stage patients. Four metabolic markers (ethanol amine, hydroxy-propionic acid, homocysteine and estriol) were eventually selected. gene ontology analysis showed that 54 enzymes and genes regulated the 4 key metabolic markers. The quantitative prediction model of esophageal cancer staging based on esophageal cancer NMR spectrum were established. Cross-validation results showed that the predicted effect was good (root mean square error=5.3, R(2)=0.47, P=0.036). CONCLUSIONS: The systems biology approaches based on metabolomics and enzyme-gene regulatory network analysis can be used to quantify the metabolic network disturbance of patients with advanced esophageal cancer, and to predict preoperative clinical staging of esophageal cancer patients by plasma NMR metabolomics.

Occidiofungin is an important component responsible for the antifungal activity of Burkholderia pyrrocinia strain Lyc2.

AIMS: To identify the taxonomy of tobacco rhizosphere-isolated strain Lyc2 and investigate the mechanisms of the antifungal activities, focusing on antimicrobials gene clusters identification and function analysis. METHODS AND RESULTS: Multilocus sequence typing and 16S rRNA analyses indicated that strain Lyc2 belongs to Burkholderia pyrrocinia. Bioassay results indicated strain Lyc2 showed significant antifungal activities against a broad range of plant and animal fungal pathogens and control efficacy on seedling damping off disease of cotton. A 55·2-kb gene cluster which was homologous to ocf gene clusters in Burkholderia contaminans MS14 was confirmed to be responsible for antifungal activities by random mutagenesis; HPLC was used to verify the production of antifungal compounds. Multiple antibiotic and secondary metabolized biosynthesis gene clusters predicated by antiSMASH revealed the broad spectrum of antimicrobials activities of the strain. CONCLUSIONS: Our results revealed the mechanisms of antifungal activities of strain Lyc2 and expand our knowledge about production of occidiofungin in the bacteria Burkholderia. SIGNIFICANCE AND IMPACT OF THE STUDY: Understanding the mechanisms of antifungal activities of strain Lyc2 has contributed to discovery of new antibiotics and expand our knowledge of production of occidiofungin in the bacteria Burkholderia.