Performance and characterization of snail adhesive mucus as a bioflocculant against toxic Microcystis.

Toxic Microcystis blooms are widespread in aquatic bodies, posing major threats to aquatic and human life. Recently, bioflocculants have attracted considerable attention as a promising biomaterial for Microcystis management. In search of a novel organism that can produce an efficient bioflocculant for controlling harmful algae sustainably, the native gastropod Cipangopaludina chinensis was co-cultured continuously with toxic Microcystis under different initial algal cell densities. The bioflocculation effect of snail mucus on toxic Microcystis, microcystin removal, and toxin accumulation in snails was investigated. In addition, the properties of the adhesive mucus were characterized using microscopic, X-ray diffraction, infrared spectroscopy, and polysaccharide and proteome analyses. Microcystis cells were captured and flocculated by the snail mucus; removal efficiencies of up to 89.9% and 84.8% were achieved for microalgae and microcystin-leucine arginine (MC-LR), respectively, when co-cultured with C. chinensis for only one day. After nine-day exposure, less than 5.49 µg/kg DW microcystins accumulated in the snails, indicating safety for human consumption. The snail mucus contained 104.3 µg/mg protein and 72.7 µg/mg carbohydrate, which provide several functional groups beneficial for Microcystis bioflocculation. The main monosaccharide subunits of polysaccharides are galactose, galactosamine, glucosamine, fucose, glucose, and mannose. Most of them are key components of polysaccharides in many bioflocculants. Gene Ontology analysis indicated the protein enrichment in binding processes and catalytic activity, which may account for Microcystis bioflocculation via protein binding or enzymatic reactions. The findings indicate that native C. chinensis secretes adhesive mucus that can act as bioflocculant for toxic Microcystis from ambient water and can be an effective and eco-friendly tool for Microcystis suppression.

Causes of missed MRI diagnosis of radiotherapy-induced temporal lobe injury in nasopharyngeal carcinoma. / MRIæ£€æŸ¥é¼»å’½ç™Œæ”¾å°„æ€§é¢žå¶æŸä¼¤çš„æ¼è¯ŠåŽŸå› .

OBJECTIVES: Radiotherapy is the primary treatment for nasopharyngeal carcinoma, but it frequently leads to radiotherapy-induced temporal lobe injury (RTLI). Magnetic resonance imaging (MRI) is the main diagnostic method for RTLI after radiotherapy for nasopharyngeal carcinoma, but it is prone to missed diagnoses. This study aims to investigate the causes of missed diagnoses of RTLI in nasopharyngeal carcinoma patients undergoing MRI after radiotherapy. METHODS: Clinical and MRI data from nasopharyngeal carcinoma patients diagnosed and treated with radiotherapy at Xiangya Hospital of Central South University, from January 2010 to April 2021, were collected. Two radiologists reviewed all head and neck MRIs (including nasopharyngeal and brain MRIs) before and after radiotherapy of identify cases of late delayed response-type RTLI for the first time. If the original diagnosis of the initial RTLI in nasopharyngeal carcinoma patients did not report temporal lobe lesions, it was defined as a missed diagnosis. The first diagnosis of RTLI cases was divided into a missed diagnosis group and a non-missed diagnosis group. Clinical and imaging data were compared between the 2 groups, and multivariate logistic regression analysis was used to identify independent risk factors for MRI missed diagnoses of initial RTLI. RESULTS: A total of 187 nasopharyngeal carcinoma with post-radiotherapy RTLI were included. The original diagnostic reports missed 120 cases and accurately diagnosed 67 cases, with an initial RTLI diagnosis accuracy rate of 35.8% and a missed diagnosis rate of 64.2%. There were statistically significant differences between the missed diagnosis group and the non-missed diagnosis group in terms of lesion size, location, presence of contralateral temporal lobe lesions, white matter high signal, cystic degeneration, hemorrhage, fluid attenuated inversion recovery (FLAIR), and examination site (all P<0.05). Multivariate logistic regression analysis showed that lesions ≤25 mm, non-enhancing lesions, lesions without cystic degeneration or hemorrhage, lesions located only in the medial temporal lobe, and MRI examination only of the nasopharynx were independent risk factors for missed MRI diagnosis of initial RTLI (all P<0.05). CONCLUSIONS: The missed diagnosis of initial RTLI on MRI is mainly related to lesion size and location, imaging characteristics, and MRI examination site.

Radiomic-Based MRI for Classification of Solitary Brain Metastases Subtypes From Primary Lymphoma of the Central Nervous System.

BACKGROUND: Differential diagnosis of brain metastases subtype and primary central nervous system lymphoma (PCNSL) is necessary for treatment decisions. The application of machine learning facilitates the classification of brain tumors, but prior investigations into primary lymphoma and brain metastases subtype classification have been limited. PURPOSE: To develop a machine-learning model to classify PCNSL, brain metastases with primary lung and non-lung origin. STUDY TYPE: Retrospective. POPULATION: A total of 211 subjects with pathologically confirmed PCNSL or brain metastases (training cohort 168 and testing cohort 43). FIELD STRENGTH/SEQUENCE: A 3.0 T axial contrast-enhanced T1-weighted spin-echo inversion recovery sequence (T1WI-CE), axial T2-weighted fluid-attenuation inversion recovery sequence (T2FLAIR) ASSESSMENT: Several machine-learning models (support vector machine, random forest, and K-nearest neighbors) were built with least absolute shrinkage and selection operator (LASSO) using features from T1WI-CE, T2FLAIR, and clinical. The model with the highest performance in the training cohort was selected to differentiate lesions in the testing cohort. Then, three radiologists conducted a two-round classification (with and without model reference) using images and clinical information from testing cohorts. STATISTICAL TESTS: Five-fold cross-validation was used for model evaluation and calibration. Model performance was assessed based on sensitivity, specificity, accuracy, and area under the receiver operating characteristic curve (AUC). RESULTS: Twenty-five image features were selected by LASSO analysis. Random forest classifier was selected for its highest performance on the training set with an AUC of 0.73. After calibration, this model achieved an accuracy of 0.70 on the testing set. Accuracies of all three radiologists improved under model reference (0.49 vs. 0.70, 0.60 vs. 0.77, 0.58 vs. 0.72, respectively). DATA CONCLUSION: The random forest model based on conventional MRI and clinical data can diagnose PCNSL and brain metastases subtypes (lung and non-lung origin). Model classification can help foster the diagnostic accuracy of specialists and streamline prognostication workflow. EVIDENCE LEVEL: 4 TECHNICAL EFFICACY: Stage 2.

Identification of the Largest SCA36 Pedigree in Asia: with Multimodel Neuroimaging Evaluation for the First Time.

Spinocerebellar ataxias (SCAs) are a large group of hereditary neurodegenerative diseases characterized by ataxia and dysarthria. Due to high clinical and genetic heterogeneity, many SCA families are undiagnosed. Herein, using linkage analysis, WES, and RP-PCR, we identified the largest SCA36 pedigree in Asia. This pedigree showed some distinct clinical characteristics. Cognitive impairment and gaze palsy are common and severe in SCA36 patients, especially long-course patients. Although no patients complained of hearing loss, most of them presented with hearing impairment in objective auxiliary examination. Voxel-based morphometry (VBM) demonstrated a reduction of volumes in cerebellum, brainstem, and thalamus (corrected P < 0.05). Reduced volumes in cerebellum were also found in presymptomatic carriers. Resting-state functional MRI (R-fMRI) found reduced ReHo values in left cerebellar posterior lobule (corrected P < 0.05). Diffusion tensor imaging (DTI) demonstrated a reduction of FA values in cerebellum, midbrain, superior and inferior cerebellar peduncle (corrected P < 0.05). MRS found reduced NAA/Cr values in cerebellar vermis and hemisphere (corrected P < 0.05). Our findings could provide new insights into management of SCA36 patients. Detailed auxiliary examination are recommended to assess hearing or peripheral nerve impairment, and we should pay more attention to eye movement and cognitive changes in patients. Furthermore, for the first time, our multimodel neuroimaging evaluation generate a full perspective of brain function and structure in SCA36 patients.

Lithography-free synthesis of periodic, vertically-aligned, multi-walled carbon nanotube arrays.

Until now, the growth of periodic vertically aligned multi-walled carbon nanotube (VA-MWCNT) arrays was dependent on at least one lithography step during fabrication. Here, we demonstrate a lithography-free fabrication method to grow hexagonal arrays of self-standing VA-MWCNTs with tunable pitch and MWCNT size. The MWCNTs are synthesized by plasma enhanced chemical vapor deposition (PECVD) from Ni catalyst particles. Template guided dewetting of a thin Ni film on a hexagonally close-packed silica particle monolayer provides periodically distributed Ni catalyst particles as seeds for the growth of the periodic MWCNT arrays. The diameter of the silica particles directly controls the pitch of the periodic VA-MWCNT arrays from 600 nm to as small as 160 nm. The diameter and length of the individual MWCNTs can also be readily adjusted and are a function of the Ni particle size and PECVD time. This unique method of lithography-free growth of periodic VA-MWCNT arrays can be utilized for the fabrication of large-scale biomimetic materials.

A Security-Enhanced Image Communication Scheme Using Cellular Neural Network.

In the current network and big data environment, the secure transmission of digital images is facing huge challenges. The use of some methodologies in artificial intelligence to enhance its security is extremely cutting-edge and also a development trend. To this end, this paper proposes a security-enhanced image communication scheme based on cellular neural network (CNN) under cryptanalysis. First, the complex characteristics of CNN are used to create pseudorandom sequences for image encryption. Then, a plain image is sequentially confused, permuted and diffused to get the cipher image by these CNN-based sequences. Based on cryptanalysis theory, a security-enhanced algorithm structure and relevant steps are detailed. Theoretical analysis and experimental results both demonstrate its safety performance. Moreover, the structure of image cipher can effectively resist various common attacks in cryptography. Therefore, the image communication scheme based on CNN proposed in this paper is a competitive security technology method.

Application of genome-wide insertion/deletion markers on genetic structure analysis and identity signature of Malus accessions.

BACKGROUND: Apple (Malus ssp.), one of the most important temperate fruit crops, has a long cultivation history and is economically important. To identify the genetic relationships among the apple germplasm accessions, whole-genome structural variants identified between M. domestica cultivars 'Jonathan' and 'Golden Delicious' were used. RESULTS: A total of 25,924 insertions and deletions (InDels) were obtained, from which 102 InDel markers were developed. Using the InDel markers, we found that 942 (75.3%) of the 1251 Malus accessions from 35 species exhibited a unique identity signature due to their distinct genotype combinations. The 102 InDel markers could distinguish 16.7-71.4% of the 331 bud sports derived from 'Fuji', 'Red Delicious', 'Gala', 'Golden Delicious', and other cultivars. Five distinct genetic patterns were found in 1002 diploid accessions based on 78 bi-allele InDel markers. Genetic structure analysis indicated that M. domestica showed higher genetic diversity than the other species. Malus underwent a relatively high level of wild-to-crop or crop-to-wild gene flow. M. sieversii was closely related to both M. domestica and cultivated Chinese cultivars. CONCLUSIONS: The identity signatures of Malus accessions can be used to determine distinctness, uniformity, and stability. The results of this study may also provide better insight into the genetic relationships among Malus species.

Association between Aldose Reductase Gene C(-106)T Polymorphism and Diabetic Retinopathy: A Systematic Review and Meta-Analysis.

Controversial results regarding the associations between aldose reductase (AR) genetic polymorphisms and diabetic retinopathy (DR) have been reported for many years. The present meta-analysis was performed to clarify the effects of the AR gene C(-106)T polymorphism on DR risk. The PubMed, Web of Sciences, Cochrane library, EMBASE, Chinese National Knowledge Infrastructure, and Wan Fang databases were extensively searched in Chinese to select relevant studies with an updated date of April 25, 2018. The Newcastle-Ottawa Scale (NOS) was applied to assess quality. The random-effects model was applied to calculate the pooled OR and 95% CI. This meta-analysis identified 23 studies with an average score of 7.52 for NOS analysis, including 4,313 DR cases and 5,128 diabetes mellitus (DM) control cases. In the overall analysis, a significant association between the AR gene C(-106)T polymorphism and DR susceptibility was found. In subgroups stratified by DM type and ethnicity, significantly increased risks for DR were found in DM type 1, East Asian populations, and Middle Eastern populations. Compared with DR control cases, the following associations were found: T vs. C: OR 0.91, 95% CI 0.85-0.97, I2 = 72.9%; CT + TT vs. CC: OR 0.75, 95% CI 0.68-0.81, I2 = 86.7%; and CT vs. CC: OR 0.86, 95% CI 0.78-0.94, I2 = 70.5%. The results of this meta-analysis showed a significant association between the AR gene C(-106)T polymorphism and susceptibility to DR in DM patients. DM patients with allele T and CT+TT genotype of the AR gene may have a lower risk of DR.

Redox Recycling-Triggered Peroxidase-Like Activity Enhancement of Bare Gold Nanoparticles for Ultrasensitive Colorimetric Detection of Rare-Earth Ce3+ Ion.

Although it has been demonstrated that rare-earth elements (REEs) disturb and alter the catalytic activity of numerous natural enzymes, their effects on nanomaterial-based artificial enzymes (nanozymes) have been seldom explored. In this work, the influence of REEs on the peroxidase-like activity of bare gold nanoparticles (GNPs) is investigated for the first time, and a new type of Ce3+-activated peroxidase mimetic activity of GNPs is obtained. The introduced Ce3+ can be bound to the bare GNP surface rapidly through electrostatic attraction, after which it donates its electron to the bare GNP. As H2O2 is a good electron scavenger, more •OH radicals are generated on the surfaces of the bare GNPs, which can considerably enhance TMB oxidation. Due to its redox cycling ability, the activation effect of Ce3+ is proved to be more efficient in comparison to those of the other reported metal ion activators (e.g., Bi3+, Hg2+, and Pb2+). In addition, it is determined that Ce3+ should directly contact with the gold core to trigger its activation effect. When the surface states of the bare GNPs are altered, the Ce3+-stimulated effect is strongly inhibited. Furthermore, a novel colorimetric method for Ce3+ is developed, on the basis of its enhancing effect on the peroxidase mimetic activity of bare GNPs. The sensitivity of this newly developed method for Ce3+ is excellent with a limit of detection as low as 2.2 nM. This study not only provides an effective GNP-based peroxidase mimic but also contributes in realizing new applications for nanozymes.

Three vital RNA functions and interactions in the process of silk gland apoptosis in silkworm Bombyx mori.

The Silkworm Bombyx mori is an important insect in terms of economics and a model organism with a complete metamorphosis. The economic importance of silkworms is dependent on the functions of the silkgland, a specialized organ that synthesizes silk proteins. The silk gland undergoes massive degeneration during the larval to pupal stage, which involves in cell apoptosis. In this paper, high throughput sequencing was used to detect the expression of messenger RNA (mRNA), long noncoding RNA (lncRNA), and microRNA (miRNA) from silk glands of Day 3 in the fifth instar larvae (L5D3) and the spinning 36h (sp36h). We analyzed the Gene Ontology (GO) functions of target genes of the differentially expressed lncRNAs and miRNAs. We investigated the regulations of mRNA, lncRNA, and miRNA on silk gland apoptosis in L5D3 and sp36h. In total, 10,947 lncRNAs were detected in the silk gland and the index number TCONS-00021360 lncRNA may be involved in the process of apoptosis. In addition, 344 miRNAs targeted 285 mRNAs were related to the death process under GO entry. The results indicated that miRNAs play an important role in the molecular regulation of the silk gland apoptosis compared with that of lncRNAs. Finally, we screened 746 lncRNAs and 20 miRNAs that might interact with BmDredd, and drew an interaction network among them.

Single-Field Non-Mydriatic Fundus Photography for Diabetic Retinopathy Screening: A Systematic Review and Meta-Analysis.

PURPOSE: Single-field non-mydriatic fundus photography (NMFP) has been used to detect diabetic retinopathy (DR) in many studies; however, its value in a general clinical setting has not been established. Here we performed a meta-analysis to evaluate its diagnostic effectiveness. METHOD: We systematically searched PubMed, EMBASE, and Cochrane databases for candidate studies published through May 19, 2018. A random-effect model was used to calculate the diagnostic indicators including the sensitivity, specificity, positive likelihood ratio (PLR), negative likelihood ratio (NLR), diagnostic odds ratio (DOR), area under the curve (AUC), and 95% confidence intervals. RESULTS: Ten prospective studies were ultimately included. The pooled sensitivity, specificity, PLR, NLR, and DOR were 0.68, 0.94, 11.2, 0.34 and 33, respectively. The AUC was 0.88. Subgroup analysis showed that single-field NMFP had a respective sensitivity and specificity of 0.73 and 0.91 when compared to standard 7-field mydriatic stereoscopic photography (7SF), and 0.54 and 0.98 when compared to slit-lamp biomicroscopy as reference standard. CONCLUSIONS: Single-field NMFP is inadequate to detect DR. Additionally, it showed higher sensitivity and lower specificity when 7SF was used as reference standard, as compared to slit-lamp biomicroscopy, suggesting that different reference standards used in DR screening might have affected the diagnostic results.

Improved enzymatic assay for hydrogen peroxide and glucose by exploiting the enzyme-mimicking properties of BSA-coated platinum nanoparticles.

Platinum nanoparticles (Pt NPs) covered with a bovine serum albumin scaffold and a particle size of 1.5 nm (BSA-PtS NPs) are shown to display enhanced multiple enzyme-mimicking activities including peroxidase, oxidase, and catalase-like activities. The peroxidase-like activity is characterized by robustness and low signal background. BSA-PtS NPs were used to design colorimetric assays for H2O2 and glucose. H2O2 latter reacts with 3,3',5,5'-tetramethylbenzidine in the presence of BSA-PtS NPs to form a blue product with an absorption maximum at 652 nm. The assay works in the 5-250 µM H2O2 concentration range. The glucose assay is based on its glucose oxidase-catalyzed oxidation to produce gluconic acid and H2O2 which then is colorimetrically quantified. Response is linear in the 10-120 µM glucose concentration range, and the detection limit is 2 µM (at S/N = 3). The method correlates well with the glucose standard method (R2 = 0.997 in the 95% confidence interval) which confirms that glucose in human serum has been successfully detected. Graphical abstractImproved enzymatic assay for hydrogen peroxide and glucose by exploiting the enzyme-mimicking properties of BSA-coated platinum nanoparticles.

Dickeya zeae strains isolated from rice, banana and clivia rot plants show great virulence differentials.

BACKGROUND: Dickeya zeae is the causal agent of maize and rice foot rot diseases, but recently it was also found to infect banana and cause severe losses in China. Strains from different sources showed significant diversity in nature, implying complicated evolution history and pathogenic mechanisms. RESULTS: D. zeae strains were isolated from soft rot banana plants and ornamental monocotyledonous Clivia miniata. Compared with D. zeae strain EC1 isolated from rice, clivia isolates did not show any antimicrobial activity, produced less extracellular enzymes, had a much narrow host ranges, but released higher amount of extracellular polysaccharides (EPS). In contrast, the banana isolates in general produced more extracellular enzymes and EPS than strain EC1. Furthermore, we provided evidence that the banana D. zeae isolate MS2 produces a new antibiotic/phytotoxin(s), which differs from the zeamine toxins produced by rice pathogen D. zeae strain EC1 genetically and in its antimicrobial potency. CONCLUSIONS: The findings from this study expanded the natural host range of D. zeae and highlighted the genetic and phenotypic divergence of D. zeae strains. Conclusions can be drawn from a series of tests that at least two types of D. zeae strains could cause the soft rot disease of banana, with one producing antimicrobial compound while the other producing none, and the D. zeae clivia strains could only infect monocot hosts. D. zeae strains isolated from different sources have diverse virulence characteristics.

BMDREDD REGULATES THE APOPTOSIS COORDINATING WITH BMDAXX, BMCIDE-B, BMFADD, AND BMCREB IN BMN CELLS.

The apoptosis mechanisms in mammals were investigated relatively clearly. However, little is known about how apoptosis is achieved at a molecular level in silkworm cells. We cloned a caspase homologous gene named BmDredd (where Bm is Bombyx mori and Dredd is death-related ced-3/Nedd2-like caspase) in BmN cells from the ovary of Bm and analyzed its biological information. We constructed the N-terminal, C-terminal, and overexpression vector of BmDredd, respectively. Our results showed that the transcriptional expression level of BmDredd was increased in the apoptotic BmN cells. Furthermore, overexpression of BmDredd increased the caspase-3/7 activity. Simultaneously, RNAi of BmDredd could save BmN cells from apoptosis. The immunofluorescence study showed that BmDredd located at the cytoplasm in normal cell otherwise is found at the nucleus when cells undergo apoptosis. Moreover, we quantified the transcriptional expressions of apoptosis-related genes including BmDredd, BmDaxx (where Daxx is death-domain associated protein), BmCide-b (where Cide-b is cell death inducing DFF45-like effector), BmFadd (Fadd is fas-associated via death domain), and BmCreb (where Creb is cAMP-response element binding protein) in BmN cells with dsRNA interferences to detect the molecular mechanism of apoptosis. In conclusion, BmDredd may function for promoting apoptosis and there are various regulatory interactions among these apoptosis-related genes.

TRANSCRIPTION FACTOR Bmsage PLAYS A CRUCIAL ROLE IN SILK GLAND GENERATION IN SILKWORM, Bombyx mori.

Salivary gland secretion is altered in Drosophila embryos with loss of function of the sage gene. Saliva has a reduced volume and an increased electron density according to transmission electron microscopy, resulting in regions of tube dilation and constriction with intermittent tube closure. However, the precise functions of Bmsage in silkworm (Bombyx mori) are unknown, although its sequence had been deposited in SilkDB. From this, Bmsage is inferred to be a transcription factor that regulates the synthesis of silk fibroin and interacts with another silk gland-specific transcription factor, namely, silk gland factor-1. In this study, we introduced a germline mutation of Bmsage using the Cas9/sgRNA system, a genome-editing technology, resulting in deletion of Bmsage from the genome of B. mori. Of the 15 tested samples, seven displayed alterations at the target site. The mutagenesis efficiency was about 46.7% and there were no obvious off-target effects. In the screened homozygous mutants, silk glands developed poorly and the middle and posterior silk glands (MSG and PSG) were absent, which was significantly different from the wild type. The offspring of G0 mosaic silkworms had indel mutations causing 2- or 9-bp deletions at the target site, but exhibited the same abnormal silk gland structure. Mutant larvae containing different open-reading frames of Bmsage had the same silk gland phenotype. This illustrated that the mutant phenotype was due to Bmsage knockout. We conclude that Bmsage participates in embryonic development of the silk gland.

RNAi KNOCKDOWN OF BmRab3 LED TO LARVA AND PUPA LETHALITY IN SILKWORM Bombyx mori L.

Rab3 GTPases are known to play key a role in vesicular trafficking, and express highest in brain and endocrine tissues. In mammals, Rab3 GTPases are paralogs unlike in insect. In this study, we cloned Rab3 from the silk gland tissue of silkworm Bombyx mori, and identified it as BmRab3. Our in silico analysis indicated that BmRab3 is an isoform with a theoretical isoelectric point and molecular weight of 5.52 and 24.3 kDa, respectively. Further, BmRab3 showed the C-terminal hypervariability for GGT2 site but having two other putative guanine nucleotide exchange factor/GDP dissociation inhibitor interaction sites. Multiple alignment sequence indicated high similarities of BmRab3 with Rab3 isoforms of other species. The phylogeny tree showed BmRab3 clustered between the species of Tribolium castaneum and Aedes aegypti. Meanwhile, the expression analysis of BmRab3 showed the highest expression in middle silk glands (MSGs) than all other tissues in the third day of fifth-instar larva. Simultaneously, we showed the differential expression of BmRab3 in the early instar larva development, followed by higher expression in male than female pupae. In vivo dsRNA interference of BmRab3 reduced the expression of BmRab3 by 75% compared to the control in the MSGs in the first day. But as the worm grew to the third day, the difference of BmRab3 between knockdown and control was only about 10%. The knockdown later witnessed underdevelopment of the larvae and pharate pupae lethality in the overall development of silkworm B. mori L.

piggyBac transposon-derived targeting shRNA interference against the Bombyx mori nucleopolyhedrovirus (BmNPV).

The Bombyx mori nucleopolyhedrovirus (BmNPV) is one of the most destructive diseases in silkworm, which has caused the main damage to sericulture industry. In this study, we developed a system of RNAi to prevent the BmNPV infection using the piggyBac transposon-derived targeting short hairpin RNA (shRNA) interference. The shRNAs targeting the genes of i.e.-1, lef-1, lef-2 and lef-3 of BmNPV were designed and used to inhibit the intracellular replication or multiplication of BmNPV in Bm cells. The highest activity was presented in the shRNA targeting the i.e.-1c of BmNPV, of which the inhibition rate reached 94.5 % in vitro. Further a stable Bm cell line of piggyBac transposon-derived targeting shRNA interference against BmNPV was established, which has a highly efficacious suppression on virus proliferation. These results indicated that the recombinant shRNA expression system was a useful tool for resistance to BmNPV in vitro. The approach by recombinant shRNAs opens a door of RNAi technology as a strategy that offering technically simpler, cheaper, and quicker gene knockdown for promising research and biotechnology application on silkworm lethal diseases.

Expression and Characterization of an Efficient Alginate Lyase from Psychromonas sp. SP041 through Metagenomics Analysis of Rotten Kelp.

Alginate is derived from brown algae, which can be cultivated in large quantities. It can be broken down by alginate lyase into alginate oligosaccharides (AOSs), which exhibit a higher added value and better bioactivity than alginate. In this study, metagenomic technology was used to screen for genes that code for high-efficiency alginate lyases. The candidate alginate lyase gene alg169 was detected from Psychromonas sp. SP041, the most abundant species among alginate lyase bacteria on selected rotten kelps. The alginate lyase Alg169 was heterologously expressed in Escherichia coli BL21 (DE3), Ni-IDA-purified, and characterized. The optimum temperature and pH of Alg169 were 25 °C and 7.0, respectively. Metal ions including Mn2+, Co2+, Ca2+, Mg2+, Ni2+, and Ba2+ led to significantly increased enzyme activity. Alg169 exhibited a pronounced dependence on Na+, and upon treatment with Mn2+, its activity surged by 687.57%, resulting in the highest observed enzyme activity of 117,081 U/mg. Bioinformatic analysis predicted that Alg169 would be a double-domain lyase with a molecular weight of 65.58 kDa. It is a bifunctional enzyme with substrate specificity to polyguluronic acid (polyG) and polymannuronic acid (polyM). These results suggest that Alg169 is a promising candidate for the efficient manufacturing of AOSs from brown seaweed.

Expression and functions of dopa decarboxylase in the silkworm, Bombyx mori was regulated by molting hormone.

Insect molting is an important developmental process of metamorphosis, which is initiated by molting hormone. Molting includes the activation of dermal cells, epidermal cells separation, molting fluid secretion, the formation of new epidermis and old epidermis shed and other series of continuous processes. Polyphenol oxidases, dopa decarboxylase and acetyltransferase are necessary enzymes for this process. Traditionally, the dopa decarboxylase (BmDdc) was considered as an enzyme for epidermal layer's tanning and melanization. This work suggested that dopa decarboxylase is one set of the key enzymes in molting, which closely related with the regulation of ecdysone at the time of biological molting processes. The data showed that the expression peak of dopa decarboxylase in silkworm is higher during molting stage, and decreases after molting. The significant increase in the ecdysone levels of haemolymph was also observed in the artificially fed silkworm larvae with ecdysone hormone. Consistently, the dopa decarboxylase expression was significantly elevated compared to the control. BmDdc RNAi induced dopa decarboxylase expression obviously declined in the silkworm larvae, and caused the pupae appeared no pupation or incomplete pupation. BmDdc was mainly expressed and stored in the peripheral plasma area near the nucleus in BmN cells. In larval, BmDdc was mainly located in the brain and epidermis, which is consisted with its function in sclerotization and melanization. Overall, the results described that the dopa decarboxylase expression is regulated by the molting hormone, and is a necessary enzyme for the silkworm molting.

Molecular cloning and transcription expression of 3-dehydroecdysone 3α-reductase (3de 3α-reductase) in the different tissues and the developing stage from the silkworm, Bombyx mori L.

Molting in insects is regulated by molting hormones (ecdysteroids), which are also crucial to insect growth, development, and reproduction etc. The decreased ecdysteroid in titre results from enhanced ecdysteroid inactivation reactions including the formation of 3-epiecdyson under ecdysone oxidase and 3-dehydroecdysone 3α-reductase (3DE 3α-reductase). In this paper, we cloned and characterized 3-dehydroecdysone 3α-reductase (3DE 3α-reductase) in different tissues and developing stage of the silkworm, Bombyx mori L. The B. mori 3DE 3α-reductase cDNA contains an ORF 783 bp and the deduced protein sequence containing 260 amino acid residues. Analysis showed the deduced 3DE 3α-reductase belongs to SDR family, which has the NAD(P)-binding domain. Using the Escherichia coli, a high level expression of a fusion polypeptide band of approx. 33 kDa was observed. High transcription of 3DE 3α-reductase was mainly presented in the midgut and hemolymph in the third day of fifth instar larvae in silkworm. The expression of 3DE 3α-reductase at different stages of larval showed that the activity in the early instar was high, and then reduced in late instar. This is parallel to the changes of molting hormone titer in larval. 3DE 3α-reductase is key enzyme in inactivation path of ecdysteroid. The data elucidate the regulation of 3DE 3α-reductase in ecdyteroid titer of its targeting organs and the relationship between the enzyme and metamorphosis.