Function analysis and characterisation of a novel chitinase, MdCht9, in Musca domestica.

Insect chitinases have been proposed as potential targets for pest control. In this work, a novel group IV chitinase gene, MdCht9, from Musca domestica was found to have multiple functions in the physiological activity, including chitin regulation, development and antifungal immunity. The MdCht9 gene was cloned and sequenced, its phylogeny was analysed and its expression was determined in normal and 20E treated larvae. Subsequently, RNA interference (RNAi)-mediated MdCht9 knockdown was performed, followed by biochemical assays, morphological observations and transcriptome analysis. Finally, the recombinant protein MdCht9 (rMdCht9) was purified and tested for anti-microbial activity and enzyme characteristics. The results showed that MdCht9 consists of three domains, highly expressed in a larval salivary gland. RNAi silencing of MdCht9 resulted in significant down-regulation of chitin content and expression of 15 chitin-binding protein (CBP) genes, implying a new insight that MdCht9 might regulate chitin content by influencing the expression of CBPs. In addition, more than half of the lethality and partial wing deformity appeared due to the dsMdCht9 treatment. In addition, the rMdCht9 exhibited anti-microbial activity towards Candida albicans (fungus) but not towards Escherichia coli (G-) or Staphylococcus aureus (G+). Our work expands on previous studies of chitinase while providing a potential target for pest management.

Molecular Characterization of 14-3-3 Zeta Gene in Musca domestica (Diptera: Muscidae) and Its Roles in Response to Bacterial Infection.

The 14-3-3 gene plays important role in many biological processes, including cell survival, apoptosis, and signal transduction. However, function of the 14-3-3 homologous gene in Musca domestica remains unclear. Here, we identified and characterized the 14-3-3ζ of M. domestica. We found that Md14-3-3ζ gene was highly homologous with other close insects. The qRT-PCR analysis revealed that the Md14-3-3ζ was highly expressed in adults, and was expressed predominantly in hemocytes and fat body. Meanwhile, the expression of Md14-3-3ζ was up-regulated after injecting Escherichia coli and Staphylococcus aureus. Moreover, the recombinant protein rMd14-3-3ζ strongly inhibits the growth of E. coli and S. aureus. Notably, the rMd14-3-3ζ inhibits E. coli and S. aureus by permeating the cell membrane. Taken together, our findings suggested that Md14-3-3ζ is involved in the immune response against bacteria through damaging the cell membrane.

Immune priming with Candida albicans induces a shift in cellular immunity and gene expression of Musca domestica.

Many insects are capable of developing enhanced resistance in response to repeated infection with the same pathogen, which is defined "immune priming". However, little is known in housefly, an ideal insect model for studying immunity. Here, Candida albicans (C. albicans) was used as the pathogen to explore whether housefly larvae are capable of eliciting immune priming. Firstly, we found that 2nd-instar larvae pre-exposure to heat-killed C. albicans could confer protection upon re-infection with C. albicans, as evidenced by the survival rate was higher in C. albicans primed larvae. Moreover, the hemocyte density was increased by priming, but phenoloxidase (PO) activity was not affected. For this reason, RNA sequencing (RNA-seq) was performed and found that 145 genes were differentially expressed after priming, in which 22 genes were related to immune response. Then, KEGG enrichment showed that Toll signaling pathway and Phagosome signaling pathway, as well as many other signaling pathways were enriched. Finally, qPCR was performed and found that the expression of 2 pattern recognition receptor (PRR) genes (PGRP-SD-like precursor and lectin subunit alpha-like) and 6 immune effector genes (phormicin, cecropin-A2-like, defensin-1, attacin-A-like, sarcotoxin-1C and lysozyme 1-like) in C. albicans primed larvae was significantly up-regulated after challenge. Taken together, our findings suggested that housefly larvae are capable of eliciting immune priming against C. albicans, and cellular immunity as well as the gene expression, especially genes involved in Toll signaling pathway were induced by immune priming with C. albicans.

Identification and function analysis of chitinase 2 gene in housefly, Musca domestica.

Chitinases are hydrolytic enzymes that play important roles in chitin degradation during the insect development process, and thus are considered as the potential targets for pest management. Here, we identified and characterized the group VII chitinase gene from health pest Musca domestica (MdCht2). We found that MdCht2 was 1932 bp in length with an open reading frame of 1530 bp, which encodes a polypeptide of 509 amino acid residues. Phylogenetic analysis showed that MdCht2 gene was homologs with other closed insects, and belong to the group VII chitinases. Moreover, Real-time PCR analysis indicated that MdCht2 mRNA was highly expressed in pupa stage, as well as in integument and trachea. However, RNAi-mediated knockdown of MdCht2 resulted in high mortality rates and abnormal eclosion. Therefore, we hypothesized that MdCht2 was a crucial gene required for housefly development, which was supported by the transcription level of MdCht2 could be induced by 20-hydroxyecdysone (20E), and the dsMdCht2 could resulted in decrease of the chitinase activity and increase of the chitin content. Taken together, our findings suggested that MdCht2 regulated the chitin content via chitinases, thereby leading to abnormal development. Our results provide a potential target for M. domestica management.

The prognostic value of the Barthel Index for mortality in patients with COVID-19: A cross-sectional study.

Objective: This study aimed to analyze the association between the activity of daily living (ADL), coronavirus disease (COVID-19), and the value of the Barthel Index in predicting the prognosis of patients. Methods: This study included 398 patients with COVID-19, whose ADL at admission to hospital were assessed with the Barthel Index. The relationship between the index and the mortality risk of the patients was analyzed. Several regression models and a decision tree were established to evaluate the prognostic value of the index in COVID-19 patients. Results: The Barthel Index scores of deceased patients were significantly lower than that of discharged patients (median: 65 vs. 90, P < 0.001), and its decrease indicated an increased risk of mortality in patients (P < 0.001). After adjusting models for age, gender, temperature, pulse, respiratory rate, mean arterial pressure, oxygen saturation, etc., the Barthel Index could still independently predict prognosis (OR = 0.809; 95% CI: 0.750-0.872). The decision tree showed that patients with a Barthel Index of below 70 had a higher mortality rate (33.3-40.0%), while those above 90 were usually discharged (mortality: 2.7-7.2%). Conclusion: The Barthel Index is of prognostic value for mortality in COVID-19 patients. According to their Barthel Index, COVID-19 patients can be divided into emergency, observation, and normal groups (0-70; 70-90; 90-100), with different treatment strategies.

Establishment of a Faba Bean Banker Plant System with Predator Orius strigicollis for the Control of Thrips Dendrothrips minowai on Tea Plants under Laboratory Conditions.

The stick tea thrip Dendrothrips minowai (Priesner) (Thysanoptera: Thripidae) is a destructive pest in tea plantations in south and southwest China. To control this pest, a non-crop banker plant system was developed using a polyphagous predator Orius strigicollis (Poppius) (Heteroptera: Anthocoridae) with the black bean aphid Aphis fabae (Scopoli) (Hemiptera: Aphididae) as an alternative prey and the faba bean Vicia faba as the banker plant to support the predator in targeting the pest. The fitness of A. fabae on tea plants and faba bean was evaluated to determine its host specificity. Moreover, the control efficacy of the banker plant system on D. minowai on tea plants was tested in the laboratory and compared with that of direct release of O. strigicollis. The experiments showed that faba bean was an excellent non-crop host for A. fabae because, while the aphid population increased quickly on faba bean, it could only survive for up to 9 days on tea plants. Compared with direct release of O. strigicollis, lower densities of pest were observed when introducing the banker plant system. Our results indicate that this banker plant system has the potential to be implemented in the field to improve the control of the pest thrips.

[Preparation of transgenic Musca domestica by microinjection method].

The transposon vector containing enhanced green fluorescent protein (EGFP) was injected into early housefly (Musca domestica L.) eggs by microinjection method to realize stable gene expression in vivo for verification, and to study housefly gene function. A borosilicate glass micro injection needle suitable for microinjection of housefly eggs was made, the softening treatment conditions of housefly egg shells were explored, and a microinjection technology platform suitable for housefly was constructed with a high-precision microsyringe Nanoject Ⅲ as the main body. The recombinant plasmid PiggyBac-[3×P3]-EGFP containing the eye-specific 3×P3 promoter and EGFP and the stable genetic expression helper plasmid pHA3pig helper were microinjected into the treated housefly eggs. After emergence, the eye luminescence was observed, and the expression and transcription level of EGFP were detected. The results showed that the normal hatching rate of housefly eggs was 55% when rinsed in bleaching water for 35 s. The hardness of the egg shell treated for 35 s was suitable for injection and the injection needle was not easy to break. About 3% of the emerged housefly eyes had green fluorescence. Through further molecular detection, EGFP specific fragments with a size of 750 bp were amplified from DNA and RNA of housefly. Through the technical platform, the stable expression of reporter genes in housefly can be conveniently and effectively realized, and a bioreactor with housefly as the main body can be established, which provides certain reference value for subsequent research on housefly gene function.

Molecular epidemiology study of measles viruses in Kunming area of China.

The present study assessed the variation of measles viruses (MV) and its association with clinical manifestations in patients with MV. A total of 38 pediatric patients with MV at the acute infection stage were selected and 2 ml venous blood was collected from each of them. Serum immunoglobulin M antibodies were determined by ELISA. Urine specimens were collected from 30 of the 38 patients and associated genetic structures were detected by reverse-transcription polymerase chain reaction mapping. At the same time, clinical epidemiological manifestations were collected to perform an epidemiological analysis. The MV-positive rate within the cohort determined in serum was 100%. Seven MV strains were isolated from urine specimens of 30 patients and the positive rate was 23.33%. Four MV strains were randomly selected from the 7 strains and the results revealed that they were all of the H1a genotype. In addition, there was no significant correlation between clinical manifestation of pediatric patients with measles and the genotype of the MV. In conclusion, the preponderant genotype of MV in Kunming was H1a and there was obvious nucleotide or amino acid mutation. The clinical manifestation of MV infection in pediatric patients was not associated with the MV genotype.

Transcriptional Responses of Candida albicans to Antimicrobial Peptide MAF-1A.

Candida albicans is a major fungal pathogen in humans. Novel antifungal agents are urgent demanded due to the challenges of the resistance. Antimicrobial peptides (AMPs) are critical components of the innate immune system against pathogenic microorganism infection. MAF-1A is a novel cationic AMP that comes from Musca domestica and is effective against C. albicans, but the antifungal mechanism remains unclear. In this study, we performed a transcriptomics analysis in C. albicans using RNA-seq technique under the treatment of MAF-1A. A total of 5654 genes were identified. Among these, 1032 were differentially expressed genes (DEGs), including 575 up-regulated genes and 457 down-regulated genes. In these DEGs, genes encoding ergosterol metabolism and fatty acid biosynthesis were identified to be significantly down-regulated, while genes associated with oxidative stress response and cell wall were identified to be significantly up-regulated. Using pathway enrichment analysis, 12 significant metabolic pathways were identified, and ribosome, oxidative phosphorylation, citrate cycle were mainly involved. The results revealed that MAF-1A induces complex responses in C. albicans. This study provides evidence that MAF-1A may inhibit the growth through affect multi-targets in C. albicans cells.

Characterization of a new multifunctional beta-glucosidase from Musca domestica.

OBJECTIVE: To engineer Pichia pastoris for heterologous production of cellulase from Musca domestica and explore its potential for industrial applications. RESULTS: A new beta-glucosidase gene (bg), encoding 562 amino acids, was cloned from M. domestica by using rapid amplification of cDNA ends. The gene bg was linked to pPICZαA and expressed in P. pastoris with a yield of 500 mg l-1. The enzyme has the maximum activity with 27.6 U mg-1 towards cellulose. The beta-glucosidase has stable activity from 20 to 70 °C and can tolerate one-mole glucose. It has the maximum activities for salicin (25.9 ± 1.8 U mg-1), cellobiose (40.1 ± 2.3 U mg-1) and cellulose (27.6 ± 3.5 U mg-1). The wide-range substrate activities of the beta-glucosidase were further verified by matrix-assisted laser desorption/ionization mass spectra. Structural analysis shows that the beta-glucosidase belongs to glycoside hydrolase family Ι and possesses O-glycosylation sites. CONCLUSIONS: Thus, a multifunctional beta-glucosidase was expressed from M. domestica and provides a potential tool for industrial application of cellulose.

Efficacy and safety of Laggera pterodonta in children 3-24 months with acute bronchiolitis: a randomized controlled trial.

INTRODUCTION: Laggera pterodonta, a traditional Chinese medicine, has been commonly used in respiratory tract infections for more than hundreds of years without any randomized controlled trials to evaluate its efficacy and safety. OBJECTIVES: To evaluate the efficacy and safety of Laggera pterodonta in hospitalized children aged 3-24 months with acute bronchiolitis. METHODS: A double-blind, randomized-controlled trial was conducted in three tertiary hospitals of Kunming, China. A total of 133 acute bronchiolitis children with an initial episode of wheezing were randomly assigned to a control mixture or Laggera pterodonta mixture. All recruited patients were given three doses of the mixture every 24 h for 5 days. Clinical symptoms and responses including adverse events in both groups were assessed and laboratory tests were done at enrolment and then after 120 h. Analysis was performed based on an intention-to-treat principle. RESULTS: Significantly more hospitalized children fulfilled the discharge criteria at 96 h and 120 h in the Laggera pterodonta mixture group compared to the control group (97% vs 75.8% P < 0.001 and 98.5% vs 89.4% P = 0.03), respectively. Better responses on clinical severity score, respiratory rate, oxygen saturation, wheezing and heart rate were also detected in the Laggera pterodonta mixture group along with lower white blood cell count, platelet count and aspartate aminotransferase. Vomiting and diarrhea were more common in the control group. CONCLUSION: Laggera pterodonta mixture is effective and safe to be prescribed in hospitalized children with acute bronchiolitis.

Satisfaction and Preference for Traditional Chinese Medicine Drugs Among Guardians of Children with Acute Bronchiolitis.

OBJECTIVES: To evaluate the satisfaction, concerns, and preference for Traditional Chinese Medicine (TCM) drugs among guardians of children with acute bronchiolitis, assess the agreement between satisfaction and preference, and explore the factors associated with satisfaction. DESIGN: A cross-sectional study was conducted in three main tertiary hospitals in Kunming, China, from September 2012 to July 2013 by structured questionnaire interviews. The characteristics of children and their guardians and guardians' satisfaction with and preference for TCM drugs were collected and analyzed by using chi-square or Fisher exact test. The agreement of satisfaction and preference was assessed by κ and prevalence-adjusted κ coefficients. Multiple logistic regression was used to identify factors associated with satisfaction. RESULTS: A total of 286 guardians were enrolled in the study. Most children (97.6%) were younger than age 2 years. In total, the rates of satisfaction and preference for TCM drugs were 75.5% and 73.1%, respectively, and preference and satisfaction levels did not significantly differ between TCM and non-TCM hospitals. Guardians with higher socioeconomic status were more satisfied with TCM drugs, and the most common reason for preference for TCM drugs was that they had less toxicity or fewer adverse effects. Guardians were concerned most about safety and least concerned about cost. The agreement between satisfaction and preference was moderate in general (prevalence-adjusted κ=0.42). CONCLUSIONS: Three fourths of the Chinese guardians of children with acute bronchiolitis were satisfied with and preferred the TCM drugs. Moderate agreement between satisfaction and preference was shown.

Elective cesarean delivery as a predisposing factor of respiratory syncytial virus bronchiolitis in children.

OBJECTIVE: To investigate the effect of cesarean delivery and other predisposing factors of respiratory syncytial virus (RSV)-positive acute bronchiolitis in children. MATERIAL AND METHOD: The case-control study was conducted in three main tertiary hospitals in Kunming, China between September 2012 and July 2013. Children with first episode of wheezing diagnosed as bronchiolitis and testedfor RSV were included RSV was detected by real-time reverse transcription polymerase chain reaction. Mode ofdelivery and characteristics of children, parents, and household were interviewed and analyzed with RSV-positive status by multiple logistic regression. RESULTS: Of 265 children, RSV-positive was found in 75.5%, and the majority of children (83.3%) were younger than 12 months. Compared to vaginal delivery, the odds of RSV-positive detection were double in children born by elective cesarean delivery (adjusted odds ratio 2.32; 95% confidence interval 1.19-4.52). Children aged less than 6 months, born in the rainy season, having maternal history of asthma and living in family that smoked more than 20 cigarettes per day were more likely to be RSV-positive. CONCLUSION: Children born by elective cesarean delivery increased the risk of RSV-positive acute bronchiolitis after adjusting for age, birth season, maternal asthma, and family smoking status.

Construction and analysis of gonad suppression subtractive hybridization libraries for the rice field eel, Monopterus albus.

The objective of this study was to investigate gene transcription profiles of the stage IV ovary and the ovotestis of the rice field eel (Monopterus albus) in an attempt to uncover genes involved in sex reversal and gonad development. Suppression subtractive hybridization (SSH) libraries were constructed using mRNA from the stage IV ovary and the ovotestis. In total 100 positive clones from the libraries were selected at random and sequenced, and then expressed sequence tags (ESTs) were used to search against sequences in the GenBank database using the BLASTn and BLASTx search algorithms. High quality SSH cDNA libraries and 90 ESTs were obtained. Of these ESTs, 43 showed high homology with genes of known function and these are associated with energy metabolism, signal transduction, transcription regulation and so on. The remaining 47 ESTs shared no homology with any genes in GenBank and are thus considered to be hypothetical genes. Furthermore, the four genes F11, F63, R11, and R47 from the forward and reverse libraries were analyzed in gonad, brain, heart, spleen, liver, kidney and muscle tissues. The results showed that the transcription of the F11 and F63 genes was significantly increased while the expression of the R11 and R47 genes was significantly decreased from IV or V ovary. In addition, the results also indicated that the four genes' expression was not gonad-tissue specific. This results strongly suggested that they may be involved in the rice field eel gonad development and/or sex reversal.

Molecular characterization of vernalization and response genes in bread wheat from the Yellow and Huai Valley of China.

BACKGROUND: Flowering time greatly influences the adaptation of wheat cultivars to diverse environmental conditions and is mainly controlled by vernalization and photoperiod genes. In wheat cultivars from the Yellow and Huai Valleys, which represent 60%-70% of the total wheat production in China, the large-scale genotyping of wheat germplasms has not yet been performed in terms of vernalization and photoperiod response alleles, limiting the use of Chinese wheat germplasms to a certain extent. RESULTS: In this study, 173 winter wheat cultivars and 51 spring wheat cultivars from China were used to identify allelic variations of vernalization and photoperiod genes as well as copy number variations of Ppd-B1 and Vrn-A1. Two new co-dominant markers were developed in order to more precisely examine Vrn-A1b, Vrn-B1a, and Vrn-B1b. Two novel alleles at the Vrn-B3 locus were discovered and were designated Vrn-B3b and Vrn-B3c. Vrn-B3b had an 890-bp insertion in the promoter region of the recessive vrn-B3 allele, and Vrn-B3c allele had 2 deletions (a 20-bp deletion and a 4-bp deletion) in the promoter region of the dominant Vrn-B3a allele. Cultivar Hemai 26 lacked the Vrn-A1 gene. RT-PCR indicated that the 890-bp insertion in the Vrn-B3b allele significantly reduced the transcription of the Vrn-B3 gene. Cultivars Chadianhong with the Vrn-B3b allele and Hemai 26 with a Vrn-A1-null allele possessed relatively later heading and flowering times compared to those of Yanzhan 4110, which harbored recessive vrn-B3 and vrn-A1 alleles. Through identification of photoperiod genes, 2 new polymorphism combinations were found in 6 winter wheat cultivars and were designated Hapl-VII and Hapl-VIII, respectively. Distribution of the vernalization and photoperiod genes indicated that all recessive alleles at the 4 vernalization response loci, truncated "Chinese Spring" Ppd-B1 allele at Ppd-B1 locus and Hapl-I at the Ppd-D1 locus were predominant in Chinese winter wheat cultivars. CONCLUSION: This study illustrated the distribution of vernalization and photoperiod genes and identified 2 new Vrn-B3 alleles, 1 Vrn-A1-null allele, and two new Ppd-D1 polymorphism combinations, using developed functional markers. Results of this study have the potential to provide useful information for screening relatively superior wheat cultivars for better adaptability and maturity.

Alveograph and Mixolab parameters associated with Puroindoline-D1 genes in Chinese winter wheats.

BACKGROUND: Grain texture is one of the most important characteristics of bread wheat and has a significant influence on end-use qualities. RESULTS: Forty-three Chinese cultivars were tested under three environments and used to characterise kernel hardness, Puroindoline-D1 alleles and Alveograph and Mixolab parameters. The results indicated that SKCS hardness was positively correlated with Alveograph tenacity and P/L and Mixolab protein weakening (C2) and water absorption and negatively correlated with Mixolab starch gelatinisation (C3), amylasic activity (C4) and starch gelling (C5). Variance analysis showed that Puroindoline-D1 had a significant impact on SKCS hardness and most Alveograph and Mixolab parameters. Furthermore, among three Puroindoline-D1 genotypes, PINA-null/Pinb-D1a possessed the highest SKCS hardness, Alveograph tenacity and W and Mixolab stability and water absorption but the lowest Alveograph extensibility and G and Mixolab C3, C4 and C5. Pina-D1a/Pinb-D1a had the lowest SKCS hardness, Alveograph tenacity and W and Mixolab C2, water absorption and stability but the highest Alveograph extensibility and G and Mixolab C3, C4 and C5. Pina-D1a/Pinb-D1b possessed the lowest Mixolab C2 - C1, C3 - C2, C4 - C3 and C5 - C4. CONCLUSION: Pina-D1a/Pinb-D1a was softer and had lower tenacity and water absorption. PINA-null/Pinb-D1a was harder and had higher tenacity and water absorption. Pina-D1a/Pinb-D1b had lower difference values among Mixolab parameters.

[Effects of temperature on the growth and development of Aglossa dimidiata parasitized on Litsea coreana].

In order to reveal the effects of temperature on the growth and development of Aglossa dimidiata parasitized on Litsea coreana, a laboratory experiment was conducted to study the mean development duration, development rate, and survival rate of A. dimidiata at its different growth stages at 31 degrees C, 28 degrees C, 25 degrees C, 22 degrees C, and 19 degrees C, with the development threshold temperature and effective accumulated temperature for different growth stages calculated. Temperature had significant effects on the developmental duration. Except that the development duration of egg was shortened with increasing temperature, the development durations of larva, pupa, and immature A. dimidiata were the shortest at 25 degrees C, being 249.53 +/- 23.83, 12.94 +/- 1.27, and 273.00 +/- 24.19 days, respectively. There existed significant relationships between the development rates of A. dimidiata at its different growth stages and temperature, with positive linear relationship at egg stage, and quadratic relationship at larva, pupa, and immature stages. Temperature also had significant effects on the survival rate of A. dimidiata. The survival rates of A. dimidiata at its different growth stages were all the highest at 25 degrees C, being 94.0%, 73.8%, 91.3%, and 63.4% for the egg, larva, pupa, and immature A. dimidiata, respectively, followed by at 22 degrees C and 19 degrees C, and the lowest at 31 degrees C. No larva and pupa could survive at 31 degrees C, suggesting that A. dimidiata was not resistant to high temperature. The development threshold temperature for egg, larva, pupa, and immature A. dimidiata was 13.21 degrees C, 17.12 degrees C, 14.76 degrees C, and 16.47 degrees C, and the effective accumulated temperature was 117.94, 870.88, 149.70, and 1442.75 day-degree, respectively. The results coincided with the fact that the A. dimidiata reproduced 2 or 3 generations a year in Xifeng area of Guizhou, Southwest China.

Two-dimensional analysis of spurious modes in aluminum nitride film resonators.

In this paper, a hybrid method, which combines the traditional concept of guided waves and the finite element method (FEM), is proposed to analyze the spurious modes of aluminum nitride (AIN) film with electrodes. First, the guided wave modes in the plated area are obtained by 1-D FEM. Second, a mode-match method is used to satisfy the boundary conditions. The vibration of the film resonator is a superposition of all of the guided modes. With respect to an A1N film resonator, which is a thickness-stretch mode resonator, we have identified three families of spurious modes: extension, thickness-stretch, and thickness-shear. The spectrum of spurious modes is calculated and the influence of the spurious modes is discussed.