Next-generation sequencing identified that RET variation associates with lymph node metastasis and the immune microenvironment in thyroid papillary carcinoma.

BACKGROUND: To date, although most thyroid carcinoma (THCA) achieves an excellent prognosis, some patients experience a rapid progression episode, even with differentiated THCA. Nodal metastasis is an unfavorable predictor. Exploring the underlying mechanism may bring a deep insight into THCA. METHODS: A total of 108 THCA from Chinese patients with next-generation sequencing (NGS) were recruited. It was used to explore the gene alteration spectrum of THCA and identify gene alterations related to nodal metastasis in papillary thyroid carcinoma (PTC). The Cancer Genome Atlas THCA cohort was further studied to elucidate the relationship between specific gene alterations and tumor microenvironment. A pathway enrichment analysis was used to explore the underlying mechanism. RESULTS: Gene alteration was frequent in THCA. BRAF, RET, POLE, ATM, and BRCA1 were the five most common altered genes. RET variation was positively related to nodal metastasis in PTC. RET variation is associated with immune cell infiltration levels, including CD8 naïve, CD4 T and CD8 T cells, etc. Moreover, Step 3 and Step 4 of the cancer immunity cycle (CIC) were activated, whereas Step 6 was suppressed in PTC with RET variation. A pathway enrichment analysis showed that RET variation was associated with several immune-related pathways. CONCLUSION: RET variation is positively related to nodal metastasis in Chinese PTC, and anti-tumor immune response may play a role in nodal metastasis triggered by RET variation.

Modular metabolic engineering of Bacillus amyloliquefaciens for high-level production of green biosurfactant iturin A.

As a kind of biosurfactants, iturin A has attracted people's wide attentions due to their features of biodegradability, environmentally friendly, etc.; however, high production cost limited its extensive application, and the aim of this research wants to improve iturin A production in Bacillus amyloliquefaciens. Firstly, dual promoter was applied to strengthen iturin A synthetase expression, and its yield was increased to 1.25 g/L. Subsequently, original 5'-UTRs of downstream genes (ituA, ituB, and ituC) in iturin A synthetase cluster were optimized, which significantly increased mRNA secondary stability, and iturin A yield produced by resultant strain HZ-T3 reached 2.32 g/L. Secondly, synthetic pathway of α-glucosidase inhibitor 1-deoxynojirimycin was blocked to improve substrate corn starch utilization, and iturin A yield was increased by 34.91% to 3.13 g/L. Thirdly, efficient precursor (fatty acids, Ser, and Pro) supplies were proven as the critical role in iturin A synthesis, and 5.52 g/L iturin A was attained by resultant strain, through overexpressing yngH, serC, and introducing ocD. Meanwhile, genes responsible for poly-γ-glutamic acid, extracellular polysaccharide, and surfactin syntheses were deleted, which led to a 30.98% increase of iturin A yield. Finally, lipopeptide transporters were screened, and iturin A yield was increased by 17.98% in SwrC overexpression strain, reached 8.53 g/L, which is the highest yield of iturin A ever reported. This study laid a foundation for industrial production and application development of iturin A, and provided the guidance of metabolic engineering breeding for efficient production of other metabolites synthesized by non-ribosomal peptide synthetase. KEY POINTS: • Optimizing 5'-UTR is an effective tactics to regulate synthetase cluster expression. • Blocking 1-DNJ synthesis benefited corn starch utilization and iturin A production. • The iturin A yield attained in this work was the highest yield reported so far.

Vertical Nystagmus Recognition Based on Deep Learning.

Vertical nystagmus is a common neuro-ophthalmic sign in vestibular medicine. Vertical nystagmus not only reflects the functional state of vertical semicircular canal but also reflects the effect of otoliths. Medical experts can take nystagmus symptoms as the key factor to determine the cause of dizziness. Traditional observation (visual observation conducted by medical experts) may be biased subjectively. Visual examination also requires medical experts to have enough experience to make an accurate diagnosis. With the development of science and technology, the detection system for nystagmus can be realized by using artificial intelligence technology. In this paper, a vertical nystagmus recognition method is proposed based on deep learning. This method is mainly composed of a dilated convolution layer module, a depthwise separable convolution module, a convolution attention module, a Bilstm-GRU module, etc. The average recognition accuracy of the proposed method is 91%. Using the same training dataset and test set, the recognition accuracy of this method for vertical nystagmus was 2% higher than other methods.

Systematical analysis of sludge treatment and disposal technologies for carbon footprint reduction.

This study aims to comprehensively analyze the Greenhouse Gases (GHGs) emissions from current sewage sludge treatment and disposal technologies (building material, landfill, land spreading, anaerobic digestion, and thermochemical processes) based on the database of Science Citation Index (SCI) and Social Science Citation Index (SSCI) from 1998 to 2020. The general patterns, spatial distribution, and hotspots were provided by bibliometric analysis. A comparative quantitative analysis based on life cycle assessment (LCA) put forward the current emission situation and the key influencing factors of different technologies. The effective GHG emissions reduction methods were proposed to mitigate climate change. Results showed that incineration or building materials manufacturing of highly dewatered sludge, and land spreading after anaerobic digestion have the best GHG emissions reduction benefits. Biological treatment technologies and thermochemical processes have great potential for reducing GHGs. Enhancement of pretreatment effect, co-digestion, and new technologies (e.g., injection of carbon dioxide, directional acidification) are major approaches to facilitate substitution emissions in sludge anaerobic digestion. The relationship between the quality and efficiency of secondary energy in thermochemical process and GHGs emission still needs further study. Solid sludge products generated by bio-stabilization or thermochemical processes are considered to have a certain carbon sequestration value and can improve the soil environment to control GHG emissions. The findings are useful for future development and processes selection of sludge treatment and disposal facing carbon footprint reduction.

Galangin suppresses RANKL-induced osteoclastogenesis via inhibiting MAPK and NF-κB signalling pathways.

Osteoclasts play a critical role in osteoporosis; thus, inhibiting osteoclastogenesis is a therapeutic strategy for osteoporosis. Galangin, a natural bioflavonoid extracted from a traditional Chinese herb, possesses a variety of biological activities, including anti-inflammation and anti-oxidation. However, its effects on osteoporosis have not been elucidated. In this study, we found that galangin treatment dose-dependently decreased osteoclastogenesis in bone marrow-derived macrophages (BMMs). Moreover, during osteoclastogenesis, osteoclast-specific genes, such as tartrate-resistant acid phosphatase (TRAP), cathepsin K (CtsK), ATPase, H + transporting, lysosomal V0 subunit D2 (V-ATPase d2) and dendritic cell-specific transmembrane protein (DC-STAMP), were down-regulated by galangin treatment. Furthermore, the results of the pit formation assay and F-actin ring staining revealed impaired osteoclastic bone resorption in the galangin-treated group compared with that in the control group. Additionally, galangin treatment also inhibited the phosphorylation of p38 and ERK of MAPK signalling pathway, as well as downstream factors of NFATc1, C-Jun and C-Fos. Consistent with our in vitro results, galangin suppressed lipopolysaccharide (LPS)-induced bone resorption via inhibition of osteoclastogenesis. Taken together, our findings provide evidence that galangin is a promising natural compound for the treatment of osteoporosis and may be associated with the inhibition of MAPK and NF-κB signalling pathways.

Identification and expression of two novel cytochrome P450 genes, CYP6CV1 and CYP9A38, in Cnaphalocrocis medinalis (Lepidoptera: Pyralidae).

Cnaphalocrocis medinalis Güenée can cause severe losses in rice. Cytochrome P450s play crucial roles in the metabolism of allelochemicals in herbivorous insects. Two novel P450 cDNAs, CYP6CV1 and CYP9A38, were cloned from the midgut of C. medinalis. CYP6CV1 encodes a protein of 500 amino acid residues, while CYP9A38-predicted protein has 531 amino acid residues. Both cDNA-predicted proteins contain the conserved functional domains for all P450s. Phylogenetic analyses showed that CYP6CV1 is grouped in the cluster containing CYP6B members, while CYP9A38 is in the cluster including CYP9 members. However, both clusters are contained in the same higher lineage. Homologous analysis revealed that CYP6CV1 is most similar to CYP6B8, CYP6B7, CYP6B6, CYP6B2, and CYP6B4 with the highest amino acid identity of 41%. CYP9A38 is closest to CYP9A17, CYP9A21, CYP9A20, and CYP9A19 with the highest amino acid identity of 66%. Studies of temporal expression profiles revealed that CYP9A38 showed a steady increase in mRNA level during the five instar stages, but a low-expression level in pupae, and then presented at a high-expression level again in adults. Similar expression patterns were obtained with CYP6CV1. In the fifth instar larvae, CYP6CV1 was mainly expressed in midgut and fat bodies, whereas CYP9A38 was mainly expressed in midgut. Expression studies also revealed a 3.20-fold over-expression of CYP6CV1 and 3.54-fold over-expression of CYP9A38 after larval exposure to host rice resistance. Our results suggest that both CYP6CV1 and CYP9A38 may be involved in detoxification of rice phytochemicals.

Molecular cloning and differential expression analysis of a squalene synthase gene from Dioscorea zingiberensis, an important pharmaceutical plant.

Diosgenin is a steroid derived from cholesterol in plants and used as a typical initial intermediate for synthesis of numerous steroidal drugs in the world. Commercially, this compound is extracted mainly from the rhizomes or tubers of some Dioscorea species. Squalene synthase (SQS: EC 2.5.1.21) catalyzes the condensation of two molecules of farnesyl diphosphate to form squalene, the first committed step for biosynthesis of plant sterols including cholesterol, and is thought to play an important role in diosgenin biosynthesis. A full-length cDNA of a putative squalene synthase gene was cloned from D. zingiberensis and designated as DzSQS (Genbank Accession Number KC960673). DzSQS was contained an open reading frame of 1,230 bp encoding a polypeptide of 409 amino acids with a predicted molecular weight of 46 kDa and an isoelectric point of 6.2. The deduced amino acid sequence of DzSQS shared over 70 % sequence identity with those of SQSs from other plants. The truncated DzSQS in which 24 amino acids were deleted from the carboxy terminus was expressed in Escherichia coli, and the resultant bacterial crude extract was incubated with farnesyl diphosphate and NADPH. GC-MS analysis showed that squalene was detected in the in vitro reaction mixture. Quantitative real-time PCR analysis revealed that DzSQS was expressed from highest to lowest order in mature leaves, newly-formed rhizomes, young leaves, young stems, and two-year-old rhizomes of D. zingiberensis.

Enhancement of GABA-activated currents by arginine vasopressin in rat dorsal root ganglion neurons.

A growing number of studies have shown that arginine vasopressin (AVP) plays an analgesia role in the modulation of nociception. Previous studies have focused on the central mechanisms of AVP analgesia. The aim of the present study was to find out whether peripheral mechanisms are also involved. The effect of AVP on GABA-activated currents (IGABA) and GABAA receptor function in freshly isolated dorsal root ganglion (DRG) neurons of rats were studied using whole cell patch clamp technique. The result showed that, IGABA were potentiated by pre-treatment with AVP (1 × 10⁻¹°-1 × 10⁻5 mol/L) in a concentration-dependent manner. Meanwhile, the GABA concentration-response curve was shifted upwards, with an increase of (49.1 ± 4.0)% in the maximal current response but with no significant change in the EC50 values. These results indicate that the enhancing effect is non-competitive. In addition, the effects of AVP on IGABA might be voltage-independent. This potentiation of IGABA induced by AVP was almost completely blocked by the V1a receptor antagonist SR49059 (3 × 10⁻6 mol/L). Also it could be removed by intracellular dialysis of either GDP-ß-S (5 × 10⁻4mol/L), a non-hydrolyzable GDP analog, or GF109203X (2 × 10⁻6 mol/L), a selective protein kinase C (PKC) inhibitor, with the re-patch clamp. These results suggest that AVP up-regulates the function of the GABAA receptor via G protein-coupled receptors and PKC-dependent signal pathways in rat DRG neurons, and this potentiation may underlie the analgesia induced by AVP.

A prediction model for osteonecrosis of femoral head after internal fixation with multiple cannulated compression screws for adult femoral neck fractures.

OBJECTIVES: This study aims to investigate the high-risk factors for osteonecrosis of the femoral head (ONFH) after internal fixation with multiple cannulated compression screws for adult femoral neck fractures and to construct a prediction model. PATIENTS AND METHODS: Between from January 2012 and December 2020, a total of 268 patients (138 males, 130 females; mean age: 53±10 years; range, 23 to 70 years) with ONFH who had complete follow-up data were included. Closed reduction in combination with open reduction were performed. All patients received internal fixation with multiple cannulated compression screws and were assigned to ONFH and non-ONFH groups. Logistic regression model was utilized to identify independent risk factors for postoperative ONFH, followed by constructing a nomogram prediction model. The predictive ability of the model was evaluated by receiver operating characteristic curve, Hosmer-Lemeshow test, and calibration curve. RESULTS: Multivariate analysis revealed that older age (odds ratio [OR]: 2.307, 95% confidence interval [CI]: 1.295-4.108], Charlson Comorbidity Index (CCI) ≥2 (OR: 2.214, 95% CI: 1.035-4.739), fracture displacement (OR: 2.426, 95% CI: 1.122-5.247), unsatisfactory reduction (OR: 2.629, 95% CI: 1.275-5.423), postoperative removal of internal fixation implant (OR: 2.200, 95% CI: 1.051-4.604) were independent risk factors for postoperative ONFH (p<0.05). The nomogram prediction model constructed based on these clinical characteristics showed high predictive value (AUC=0.807) and consistency (p>0.05). CONCLUSION: Age, comorbidity index, fracture type, reduction quality and postoperative removal of internal fixation implant are of utmost importance for postoperative ONFH in patients with femoral neck fractures. The established nomogram prediction model can accurately predict the occurrence of postoperative ONFH.

Immune-Related Colitis Is Associated with Fecal Microbial Dysbiosis and Can Be Mitigated by Fecal Microbiota Transplantation.

Colitis induced by treatment with immune-checkpoint inhibitors (ICI), termed irColitis, is a substantial cause of morbidity complicating cancer treatment. We hypothesized that abnormal fecal microbiome features would be present at the time of irColitis onset and that restoring the microbiome with fecal transplant from a healthy donor would mitigate disease severity. Herein, we present fecal microbiota profiles from 18 patients with irColitis from a single center, 5 of whom were treated with healthy-donor fecal microbial transplantation (FMT). Although fecal samples collected at onset of irColitis had comparable α-diversity to that of comparator groups with gastrointestinal symptoms, irColitis was characterized by fecal microbial dysbiosis. Abundances of Proteobacteria were associated with irColitis in multivariable analyses. Five patients with irColitis refractory to steroids and biologic anti-inflammatory agents received healthy-donor FMT, with initial clinical improvement in irColitis symptoms observed in four of five patients. Two subsequently exhibited recurrence of irColitis symptoms following courses of antibiotics. Both received a second "salvage" FMT that was, again, followed by clinical improvement of irColitis. In summary, we observed distinct microbial community changes that were present at the time of irColitis onset. FMT was followed by clinical improvements in several cases of steroid- and biologic-agent-refractory irColitis. Strategies to restore or prevent microbiome dysbiosis in the context of immunotherapy toxicities should be further explored in prospective clinical trials.

Torsional nystagmus recognition based on deep learning for vertigo diagnosis.

Introduction: Detection of torsional nystagmus can help identify the canal of origin in benign paroxysmal positional vertigo (BPPV). Most currently available pupil trackers do not detect torsional nystagmus. In view of this, a new deep learning network model was designed for the determination of torsional nystagmus. Methods: The data set comes from the Eye, Ear, Nose and Throat (Eye&ENT) Hospital of Fudan University. In the process of data acquisition, the infrared videos were obtained from eye movement recorder. The dataset contains 24521 nystagmus videos. All torsion nystagmus videos were annotated by the ophthalmologist of the hospital. 80% of the data set was used to train the model, and 20% was used to test. Results: Experiments indicate that the designed method can effectively identify torsional nystagmus. Compared with other methods, it has high recognition accuracy. It can realize the automatic recognition of torsional nystagmus and provides support for the posterior and anterior canal BPPV diagnosis. Discussion: Our present work complements existing methods of 2D nystagmus analysis and could improve the diagnostic capabilities of VNG in multiple vestibular disorders. To automatically pick BPV requires detection of nystagmus in all 3 planes and identification of a paroxysm. This is the next research work to be carried out.

Potential Effect of Glutamine in the Improvement of Intestinal Stem Cell Proliferation and the Alleviation of Burn-Induced Intestinal Injury via Activating YAP: A Preliminary Study.

Burn injury is a common form of traumatic injury that leads to high mortality worldwide. A severe burn injury usually induces gut barrier dysfunction, partially resulting from the impairment in the proliferation and self-renewal of intestinal stem cells (ISCs) post burns. As a main energy substance of small intestinal enterocytes, glutamine (Gln) is important for intestinal cell viability and growth, while its roles in ISCs-induced regeneration after burns are still unclear. To demonstrate the potential effects of Gln in improving ISCs proliferation and alleviating burn-induced intestinal injury, in this study, we verified that Gln significantly alleviated small intestine injury in burned mice model. It showed that Gln could significantly decrease the ferroptosis of crypt cells in the ileum, promote the proliferation of ISCs, and repair the crypt. These effects of Gln were also confirmed in the mouse small intestine organoids model. Further research found that Yes-associated protein (YAP) is suppressed after burn injury, and Gln could improve cell proliferation and accelerate the renewal of the damaged intestinal mucosal barrier after burns by activating YAP. YAP is closely associated with the changes in intestinal stem cell proliferation after burn injury and could be served as a potential target for severe burns.

Modular Engineering to Enhance Keratinase Production for Biotransformation of Discarded Feathers.

Biotransformation of wasted feathers via feather-degrading enzyme has gained immense popularity, low conversion efficiency hinders its scale application, and the main purpose of this study is to improve feather-degrading enzyme production in Bacillus licheniformis. Firstly, keratinase from Bacillus amyloliquefaciens K11 was attained with the best performance for feather hydrolysis, via screening several extracellular proteases from Bacillus; also, feather powder was proven as the most suitable substrate for determination of feather-degrading enzyme activity. Then, expression elements, including signal peptides and promoters, were optimized, and the combination of signal peptide SPSacC with promoter Pdual3 owned the best performance, keratinase activity aggrandized by 6.21-fold. According to amino acid compositions of keratinase and feeding assays, Ala, Val, and Ser were proven as critical precursors, and strengthening these precursors' supplies via metabolic pathway optimization resulted in a 33.59% increase in the keratinase activity. Furthermore, keratinase activity reached 2210.66 U/mL, up to 56.74-fold from the original activity under the optimized fermentation condition in 3-L fermentor. Finally, the biotransformation process of discarded feathers by the fermented keratinase was optimized, and our results indicated that 90.94% of discarded feathers (16%, w/v) were decomposed in 12 h. Our results suggested that strengthening precursor amino acids' supplies was an efficient strategy for enhanced production of keratinase, and this research provided an efficient strain as well as the biotransformation process for discarded feather re-utilization.

Actin polymerization inhibition by targeting ARPC2 affects intestinal stem cell homeostasis.

Background: The rapid turnover of the intestinal epithelium is driven by the proliferation and differentiation of intestinal stem cells (ISCs). The dynamics of the F-actin cytoskeleton are critical for maintaining intercellular force and the signal transduction network. However, it remains unclear how direct interference with actin polymerization impacts ISC homeostasis. This study aims to reveal the regulatory effects of the F-actin cytoskeleton on the homeostasis of intestinal epithelium, as well as the potential risks of benproperine (BPP) as an anti-tumor drug. Methods: Phalloidin fluorescence staining was utilized to test F-actin polymerization. Flow cytometry and IHC staining were employed to discriminate different types of intestinal epithelial cells. Cell proliferation was assessed through bromo-deoxyuridine (BrdU) and 5-ethynyl-2'-deoxyuridine (EdU) incorporation assays. The proliferation and differentiation of intestinal stem cells were replicated in vitro through organoid culture. Epithelial migration was evaluated through BrdU pulse labeling and chasing in mice. Results: The F-actin content was observed to significantly increase as crypt cells migrated into the villus region. Additionally, actin polymerization in secretory cells, especially in Paneth cells (PCs), was much higher than that in neighboring ISCs. Treatment with the newly identified actin-related protein 2/3 complex subunit 2 (ARPC2) inhibitor BPP led to a dose-dependent increase or inhibition of intestinal organoid growth in vitro and crypt cell proliferation in vivo. Compared with the vehicle group, BPP treatment decreased the expression of Lgr5 ISC feature genes in vivo and in organoid culture. Meanwhile, PC differentiation derived from ISCs and progenitors was decreased by inhibition of F-actin polymerization. Mechanistically, BPP-induced actin polymerization inhibition may activate the Yes1-associated transcriptional regulator pathway, which affects ISC proliferation and differentiation. Accordingly, BPP treatment affected intestinal epithelial cell migration in a dose-dependent manner. Conclusion: Our findings indicate that the regulation of cytoskeleton reorganization can affect ISC homeostasis. In addition, inhibiting ARPC2 with the Food and Drug Administration-approved drug BPP represents a novel approach to influencing the turnover of intestinal epithelial cells.

Bio-capture of Cr(VI) in a denitrification system: Electron competition, long-term performance, and microbial community evolution.

Chromium is widely applied in industries as an important metal resource, but the discharge of Cr(VI) containing wastewater leads to the loss of chromium resources. This study proposed a bio-capture process of chromium in a denitrification system. The bio-capture potentiality was explored by investigating the electron competition between Cr(VI) and nitrogen compounds reduction, the long-term bio-capture performance, and the microbial community evolution. In the competition utilization of electron donors, both NO3--N and NO2--N took precedence over Cr(VI), and NO2--N reduction was proved to be the rate-limiting step. Under the optimum conditions of 20 mg/L NO3--N and 6 h HRT, 99.95% of 30 mg/L Cr(VI) could be reduced, and 220980 µg Cr/g MLSS was captured by the biofilm, which was fixed in intercellular as Cr(III). Microbiological analysis confirmed that the bio-reduction of Cr(VI) and NO3--N was mediated by synergistic interactions of a series of dominant bacteria, including genera Acidovorax, Thermomonas, and Microbacterium, which contained both the denitrification genes (narG, narZ, nxrA, and nirK) and chromate reduction genes (chrA and chrR). This study proved the feasibility of chromium bio-capture in denitrification systems and provided a new perspective for the Cr(VI) pollution treatment.

Enhanced production of iturin A by strengthening fatty acid synthesis modules in Bacillus amyloliquefaciens.

Iturin A is a biosurfactant with various applications, and its low synthesis capability limits its production and application development. Fatty acids play a critical role in cellular metabolism and target product syntheses, and the relationship between fatty acid supplies and iturin A synthesis is unclear. In this study, we attempted to increase iturin A production via strengthening fatty acid synthesis pathways in Bacillus amyloliquefaciens. First, acetyl-CoA carboxylase AccAD and ACP S-malonyltransferase fabD were overexpressed via promoter replacement, and iturin A yield was increased to 1.36 g/L by 2.78-fold in the resultant strain HZ-ADF1. Then, soluble acyl-ACP thioesterase derived from Escherichia coli showed the best performance for iturin A synthesis, as compared to those derived from B. amyloliquefaciens and Corynebacterium glutamicum, the introduction of which in HZ-ADF1 further led to a 57.35% increase of iturin A yield, reaching 2.14 g/L. Finally, long-chain fatty acid-CoA ligase LcfA was overexpressed in HZ-ADFT to attain the final strain HZ-ADFTL2, and iturin A yield reached 2.96 g/L, increasing by 6.59-fold, and the contents of fatty acids were enhanced significantly in HZ-ADFTL2, as compared to the original strain HZ-12. Taken together, our results implied that strengthening fatty acid supplies was an efficient approach for iturin A production, and this research provided a promising strain for industrial production of iturin A.

Molecular cloning and characterization of an acetylcholinesterase cDNA in the brown planthopper, Nilaparvata lugens.

A full cDNA encoding an acetylcholinesterase (AChE, EC 3.1.1.7) was cloned and characterized from the brown planthopper, Nilaparvata lugens Stål (Hemiptera: Delphacidae). The complete cDNA (2467 bp) contains a 1938-bp open reading frame encoding 646 amino acid residues. The amino acid sequence of the AChE deduced from the cDNA consists of 30 residues for a putative signal peptide and 616 residues for the mature protein with a predicted molecular weight of 69,418. The three residues (Ser242, Glu371, and His485) that putatively form the catalytic triad and the six Cys that form intra-subunit disulfide bonds are completely conserved, and 10 out of the 14 aromatic residues lining the active site gorge of the AChE are also conserved. Northern blot analysis of poly(A)+ RNA showed an approximately 2.6-kb transcript, and Southern blot analysis revealed there likely was just a single copy of this gene in N. lugens. The deduced protein sequence is most similar to AChE of Nephotettix cincticeps with 83% amino acid identity. Phylogenetic analysis constructed with 45 AChEs from 30 species showed that the deduced N. lugens AChE formed a cluster with the other 8 insect AChE2s. Additionally, the hypervariable region and amino acids specific to insect AChE2 also existed in the AChE of N. lugens. The results revealed that the AChE cDNA cloned in this work belongs to insect AChE2 subgroup, which is orthologous to Drosophila AChE. Comparison of the AChEs between the susceptible and resistant strains revealed a point mutation, Gly185Ser, is likely responsible for the insensitivity of the AChE to methamidopho in the resistant strain.

Characterization and induction of two cytochrome P450 genes, CYP6AE28 and CYP6AE30, in Cnaphalocrocis medinalis: possible involvement in metabolism of rice allelochemicals.

Two cDNAs specific for P450 genes, CYP6AE28 and CYP6AE30, have been isolated from the rice leaf folder Cnaphalocrocis medinalis Guenée (Lepidoptera: Pyralidae). Both cDNA-predicted proteins have 504 amino acid residues in length, but with molecular masses of 60177 Dalton for CYP6AE28 and 60020 Dalton for CYP6AE30, and theoretical pI values of 8.49 for CYP6AE28 and 8.56 for CYP6AE30, respectively. Both putative proteins contain the conserved structural and functional domains characteristic of all CYP6 members. CYP6AE28 and CYP6AE30 show 52% amino acid identity to each other; both of them have 49-56% identities with CYP6AE1, Cyp6ae12, and CYP6AE14. Phylogenetic analysis showed that the two P450s are grouped in the lineage containing some of the CYP6AE members, CYP6B P450s and CYP321A1. The transcripts of CYP6AE28 and CYP6AE30 were found to be induced in response to TKM-6, a rice variety with high resistance to C. medinalis. The results suggest that the two P450s may play important roles in adaptation to the host plant rice. This is the first report of P450 genes cloned in C. medinalis.

cDNA cloning, heterogeneous expression and biochemical characterization of a novel trypsin-like protease from Nilaparvata lugens.

A reverse transcription-polymerase chain reaction (RT-PCR) strategy was used to clone diverse trypsin-like protease gene transcripts from midguts of the brown planthopper Nilaparvata lugens Stål (Homoptera: Delphacidae). Six individual trypsin-like protease transcripts were identified. On the basis of one nucleotide sequence of the six clones, a full-length cDNA sequence (1902 bp) was obtained by rapid amplification of cDNA ends (RACE). The cDNA contained an 1128-bp open reading frame encoding a putative protein of 375 amino acids with typical features of the trypsin-like protease. Heterogeneous expression of the coding sequence for the mature peptide in Escherichia coli cells showed that the expressed protease with a molecular weight of 27.0 is active, for its BApNAse activity assayed by using BApNA (N-benzoyl-D,L-arginine-p-nitroanilide) as substrate. The protease had its maximum activity at pH 8.0 and 35 degrees C. A much better stability was observed at pH values above 4.0 and temperatures below 40 degrees C. The enzyme was strongly inhibited by serine protease inhibitor. The trypsin-like protease is therefore likely one of the major digestive proteases responsible for protein hydrolysis in N. lugens gut, and multiple gene families encoding digestive proteases may help in adaptation of this sap-sucker to different rice varieties.

Bioelectrochemical degradation of monoaromatic compounds: Current advances and challenges.

Monoaromatic compounds (MACs) are typical refractory organic pollutants which are existing widely in various environments. Biodegradation strategies are benign while the key issue is the sustainable supply of electron acceptors/donors. Bioelectrochemical system (BES) shows great potential in this field for providing continuous electrons for MACs degradation. Phenol and BTEX (Benzene, Toluene, Ethylbenzene and Xylenes) can utilize anode to enhance oxidative degradation, while chlorophenols, nitrobenzene and antibiotic chloramphenicol (CAP) can be efficiently reduced to less-toxic products by the cathode. However, there still have several aspects need to be improved including the scale, electricity output and MACs degradation efficiency of BES. This review provides a comprehensive summary on the BES degradation of MACs, and discusses the advantages, future challenges and perspectives for BES development. Instead of traditional expensive dual-chamber configurations for MACs degradation, new single-chamber membrane-less reactors are cost-effective and the hydrogen generated from cathodes may promote the anode degradation. Electrode materials are the key to improve BES performance, approaches to increase the biofilm enrichment and conductivity of materials have been discussed, including surface modification as well as composition of carbon and metal-based materials. Besides, the development and introduction of functional microbes and redox mediators, participation of sulfur/hydrogen cycling may further enhance the BES versatility. Some critical parameters, such as the applied voltage and conductivity, can also affect the BES performance, which shouldn't be overlooked. Moreover, sequential cathode-anode cascaded mode is a promising strategy for MACs complete mineralization.