[Porphyromonas gingivalis promotes the occurrence of esophageal squamous cell carcinoma via an inflammatory microenvironment].

Objective: To investigate the role of an inflammatory microenvironment induced by Porphyromonasgingivalis (P. gingivalis) in the occurrence of esophageal squamous cell carcinoma (ESCC) in mice. Methods: A total of 180 C57BL/6 mice were randomly divided into 6 groups, i.e. control group, P. gingivalis group, 4NQO group, 4NQO + P. gingivalis group, 4NQO + P. gingivalis + celecoxib group, and 4NQO + P. gingivalis + antibiotic cocktail (ABC, including metronidazole, neomycin, ampicillin, and vancomycin) group, with 30 mice in each group, using the random number table. All mice were normalized by treatment with ABC in drinking water for 2 weeks. In the following 2 weeks, the mice in the control group and the P. gingivalis group were given drinking water, while the other 4 groups were treated with 30 µg/ml 4NQO in the drinking water. In weeks 11-12, the mice in the P. gingivalis group, the 4NQO + P. gingivalis group, the 4NQO + P. gingivalis + celecoxib group, and the 4NQO + P. gingivalis + ABC group were subjected to ligation of the second molar in oral cavity followed by oral P. gingivalis infection thrice weekly for 24 weeks in weeks 11-34. In weeks 13-34, the mice in 4NQO + P. gingivalis+celecoxib group and 4NQO + P. gingivalis + ABC group were administered with celecoxib and ABC for 22 weeks, respectively. At the end of 34 weeks, gross and microscopic alterations were examined followed by RT-qPCR and immunohistochemistry to examine the expression profiles of inflammatory- and tumor-molecules in esophagi of mice. Results: At 34 weeks, 4NQO treatment alone did not affect the foci of papillary hyperproliferation, diseased area, and the thickness of the esophageal wall, but significantly enhanced the foci of hyperproliferation (median 1.00, P＜0.05) and mild/moderate dysplasia (median 2.00, P＜0.01). In addition, the expression levels of IL-6 [8.35(3.45,8.99)], IL-1ß [6.90(2.01,9.72)], TNF-α [12.04(3.31,14.08)], c-myc [2.21(1.80,3.04)], pSTAT3, Ki-67, and pH2AX were higher than those in the control group. The pathological changes of the esophageal mucosa were significantly more overt in the 4NQO + P. gingivalis group in terms of the foci of papillary hyperproliferation (median 2.00), diseased area (median 2.51 mm2), the thickness of the esophageal wall (median 172.52 µm), the foci of hyperproliferation (median 1.00, P＜0.05), and mild/moderate dysplasia (median 1.00, P＜0.01). In mice of the 4NQO + P. gingivalis group, the expression levels of IL-6 [12.27(5.35,22.08)], IL-1ß [13.89(10.04,15.96)], TNF-α [19.56(6.07,20.36)], IFN-γ [11.37(8.23,20.07)], c-myc [2.62(1.51,4.25)], cyclin D1 [4.52(2.68,7.83)], nuclear pSTAT3, COX-2, Ki-67, and pH2AX were significantly increased compared with the mice in the control group. In mice of the 4NQO + P. gingivalis group, the diseased area, invasive malignant foci as well as pSTAT3 and pH2AX expression were significantly blunted by celecoxib. Treatment with ABC markedly reduced the papillary hyperproliferative foci, invasive malignant foci, and pSTAT3 expression but not pH2AX. Conclusions: P. gingivalis promotes the occurrence of esophageal squamous cell carcinoma in mice by inducing an inflammatory microenvironment primed with 4NQO induced DNA damage. Clearance of P. gingivalis with ABC or anti-inflammatory intervention holds promise for prevention of esophageal squamous cell malignant pathogenesis via blockage of IL-6/STAT3 signaling and amelioration of inflammation.

[A Mendelian randomization study on the relationship between insomnia and osteoporosis].

Objective: To explore the relationship between insomnia and osteoporosis. Methods: Mendelian randomization (MR) analysis were used in this study. The single nucleotide polymorphisms (SNPs) related to insomnia from genome-wide association analysis research data were selected as the instrumental variables by using inverse variance weighted (IVW), MR-Egger regression, weighted median method, maximum likelihood, penalized weighted median estimator, and Mendelian randomization robust adjusted profile score (MR-RAPS) to determine the causal relationship between insomnia and osteoporosis. Odds ratio (OR) and 95% confidence interval (CI) values were used to evaluate the association between insomnia and osteoporosis. Cochran's Q-test was used to detect heterogeneity of SNPs, MR-Egger regression was used to test for level pleiotropy, and the leave-one-out method was used to test sensitivity, MR pleiotropy residual sum and outlier (MR-PRESSO) method and radial MR were used to detect erroneous outliers. Results: The screening criteria were set based on the three major assumptions of MR; finally, 31 SNPs were included in the MR analysis. The results of MR causal effect analysis using the IVW method showed that insomnia increased the risk of osteoporosis by about 0.7% (OR=1.007, 95%CI 1.001-1.014, P=0.044); heterogeneity testing showed heterogeneity between SNPs (Q=57.91, P<0.001); and the MR- Egger intercept test did not indicate horizontal pleiotropy in this study (intercept value=3.807×10-5, P=0.888). Leave-one-out method showed that no single SNP had a significant impact on the overall results. No abnormal SNP was detected according to the MR-PRESSO results (P=0.059), and radial MR did not detect any outliers. Conclusion: Mendelian randomization analysis showed that insomnia can increase the risk of osteoporosis.

[Value of radiomics models based on MRI diffusion weighted imaging and apparent diffusion coefficient in differentiating benign and malignant thyroid nodules].

Objective: To investigate the value of radiomics models based on magnetic resonance imaging (MRI) diffusion weighted imaging (DWI) and apparent diffusion coefficient (ADC) maps in distinguishing benign and malignant thyroid nodules. Methods: A cross-sectional study. Clinical data of 148 thyroid nodules (50 benign, 98 malignant) from 140 patients who underwent thyroid MRI examination in Cancer Hospital & Shenzhen Hospital, Chinese Academy of Medical Sciences between January 2019 and December 2022 were retrospectively analyzed. The nodules were used as the study units, and a leave-one-out method was used to randomly divide the nodules into a training set and a test set at a 7∶3 ratio. Region of interest was segmented and radiomics features were extracted from the DWI and ADC images. In the training set, feature selection was performed using inter-observer agreement analysis, U-test, least absolute shrinkage and selection operator algorithm, and correlation analysis. Four classifiers, including support vector machine (SVM), random forest (RF), k-nearest neighbors (KNN) and logistic regression (LR) were used to build models with the selected features, including the DWI models, ADC models, and combined models. The models were independently tested in the test set. The performance of the radiomics models in distinguishing benign and malignant thyroid nodules was evaluated using the receiver operating characteristic (ROC) curve, with pathological results as the gold standard. Results: Of the 140 patients, there were 40 males and 100 females, with a mean age of (38.4±12.2) years. After feature selection, 11 DWI features and 11 ADC features were used to build the models. In the training set, the AUC values of the combined models were higher than those of the corresponding DWI and ADC models. In the test set, the SVM combined model showed the best predictive performance, with an AUC of 0.873 (95%CI:0.740-0.954), accuracy of 75.6%, sensitivity of 46.7%, specificity of 90.0%, positive predictive value (PPV) of 70.0% and negative predictive value (NPV) of 77.1%, while the RF combined model had an AUC of 0.836 (95%CI:0.695-0.929), accuracy of 77.8%, sensitivity of 40.0%, specificity of 96.7%, PPV of 85.7% and NPV of 76.3%, the KNN combined model had an AUC of 0.832 (95%CI:0.691-0.927), accuracy of 77.8%, sensitivity of 33.3%, specificity of 100%, PPV of 100% and NPV of 75.0%, the LR combined model had an AUC of 0.813 (95%CI:0.669-0.914), accuracy of 77.8%, sensitivity of 60.0%, specificity of 86.7%, PPV of 69.2% and NPV of 81.3%. Conclusions: Radiomics models based on DWI and ADC image features can effectively distinguish benign and malignant thyroid nodules. The SVM combined model had the best prediction performance.

Growth performance, digestibility, blood metabolites, ruminal fermentation, and bacterial communities in response to the inclusion of gallic acid in the starter feed of preweaning dairy calves.

The objective of this study was to evaluate the effects of feeding gallic acid on the growth, nutrient digestibility, plasma metabolites, rumen fermentation, and bacterial community in the rumen fluid and feces of preweaning calves. Thirty-six female Holstein calves with similar ages (means ± SD; 3.1 ± 1.39 d) and body weights (40.8 ± 2.87 kg) were randomly assigned to receive 3 treatments. Calves were fed 1 of 3 treatments as follows: basal diet with no gallic acid (control), 0.5 g/kg gallic acid in starter diet (low), and 1 g/kg gallic acid in starter diet (high). The results showed that feeding gallic acid increased growth by improving the starter intake and average daily gain of the calves. The fecal score tended to decrease in a linear manner with the addition of gallic acid. Total-tract apparent protein digestibility tended to increase linearly with feeding gallic acid. Feeding gallic acid led to a linear increase in the plasma total protein and ß-hydroxybutyrate levels. In addition, feeding gallic acid linearly increased catalase and total antioxidant capacity levels and decreased malondialdehyde and tumor necrosis factor-α concentrations. The concentrations of total volatile fatty acids, propionate, butyrate, and valerate in the rumen fluid increased linearly with the addition of gallic acid, resulting in a linear pH reduction. Feeding gallic acid linearly increased the relative abundances of Prevotella_1, Saccharofermentans, and Prevotellaceae_UCG-001 and linearly decreased the relative abundance of Prevotella_7 in the rumen fluid. The Shannon index of ruminal bacterial communities linearly increased by feeding gallic acid. Feeding gallic acid linearly increased the relative abundances of Ruminococcaceae_UCG-005, Bacteroides, and Christensenellaceae_R-7_group in the feces. In summary, feeding gallic acid improved growth, antioxidant function, and rumen fermentation and altered the bacterial community in the rumen fluid and feces of preweaning dairy calves.

[Standardized management of targeted drugs for rheumatism].

In recent years, with the continuous in-depth research on the pathogenesis of rheumatism and the rapid development of biopharmaceutical technology, the development of targeted drugs for rheumatism is in full swing. In order to better standardize the diagnosis and treatment of rheumatism and the rational application of targeted drugs, the Chinese Rheumatology Association will introduce the targeted drugs for rheumatism that have been approved by the China National Medical Products Administration so far, and provide clinicians with standardized diagnosis and treatment reference.

[A case of atypical iris corneal endothelial syndrome].

Iridocorneal endothelial syndrome is a rare ophthalmic disease, most of which are unilateral and common in women. The rate of misdiagnosis and missed diagnosis is relatively high due to its various clinical manifestations. In this case, the patient presented uncontrollable high intraocular pressure, corneal edema leading to difficult observation of corneal endothelium morphology, and accompanied by a small amount of iris neovascularization. No clearly diagnosis was made before glaucoma surgery. Further examination was performed after corneal clearance, and the final diagnosis was iris corneal endothelial syndrome (Chandler syndrome).

Digestibility, lactation performance, plasma metabolites, ruminal fermentation, and bacterial communities in Holstein cows fed a fermented corn gluten-wheat bran mixture as a substitute for soybean meal.

The purpose of this research was to investigate the effects of replacing soybean meal (SBM) with a fermented corn gluten-wheat bran mixture (FCWM) on nutrient digestibility, lactation performance, plasma metabolites, ruminal fermentation, and bacterial communities in Holstein cows. Nine healthy multiparous (parity = 3) Holstein cows with similar body weights (624 ± 14.4 kg), days in milk (112 ± 4.2), and milk yields (31.8 ± 1.73 kg; all mean ± standard deviation) were used in a replicated 3 × 3 Latin square design with 3 periods of 28 d. Cows were fed 1 of 3 dietary treatments in which FCWM replaced SBM as follows: basal diet with no replacement (0FCWM); 50% replacement of SBM with FCWM (50%FCWM); and 100% replacement of SBM with FCWM (100%FCWM). The diets were formulated to be isocaloric and isonitrogenous. The results showed that the total-tract digestibility of dry matter and crude protein increased linearly with increased dietary FCWM, and we found a trend for increased total-tract neutral detergent fiber and potentially digestible NDF digestibility. Milk yield tended to increase in a linear manner as more FCWM was consumed, and energy-corrected milk production was significantly increased with FCWM supplementation as a result of increased milk protein and lactose yields. Plasma glucose and IgG concentrations increased linearly with increasing FCWM supplementation, but plasma malondialdehyde concentration decreased linearly. Concentrations of total volatile fatty acids and propionate showed a linear increase with increasing FCWM supplementation, leading to a linear decrease in pH. The relative abundance of ruminal Prevotellaceae, Veillonellaceae, and Prevotella 1 increased linearly with increasing FCWM supplementation, and the relative abundance of ruminal Succinivibrionaceae and Muribaculaceae decreased linearly. The relative abundance of fecal Ruminococcaceae, Prevotellaceae, and Ruminococcaceae UCG-005 increased linearly with increasing FCWM supplementation, but the relative abundance of fecal Peptostreptococcaceae decreased linearly. Overall, the replacement of SBM with FCWM altered the composition of the ruminal bacterial community and improved nutrient digestibility, lactation performance, and ruminal fermentation in cows, providing a data reference for the use of FCWM in dairy production.

[Analysis of factors influencing radiation-induced liver injury caused by stereotactic radiotherapy in patients with primary hepatocellular carcinoma].

Objective: To analyze the factors influencing radiation-induced liver injury after receiving Cyberknife stereotactic radiotherapy in patients with primary hepatocellular carcinoma. Methods: 278 cases with primary hepatocellular carcinoma from July 2016 to April 2019 were prospectively enrolled. Stereotactic radiosurgery with a prescription dose of 48-55gy/5-8 times were given. Liver function, coagulation function, Child-Pugh score, and liver imaging changes were dynamically observed before and after treatment to evaluate the occurrence of radiation-induced liver injury. Logistic regression model was used to analyze the factors influencing radiation-induced liver injury. Results: Among 278 cases, 3 cases of tumor progression were excluded, and a total of 275 cases were included for analysis. The overall survival rate after 8 months of treatment was 100%. Among them, 22 cases were diagnosed as radiation-induced liver injury, with an incidence rate of 8%, and all cases were recovered after symptomatic treatment. Multivariate analysis result suggested that the peripheral white blood cell count was factors influencing the occurrence of radiation-induced liver injury. Conclusion: Cyberknife stereotactic radiotherapy has a low incidence of radiation-induced liver injury in patients with liver cancer, and it is a relatively safe treatment method. Patients with low peripheral white blood cell counts before treatment should be closely monitored for early detection and treatment.

Molecular characterization of insulin-like peptides in the brown planthopper, Nilaparvata lugens (Hemiptera: Delphacidae).

Insulin-like peptides (ILPs) including insulin, insulin-like growth factor (IGF) and relaxin are evolutionarily conserved hormones in metazoans, and they are involved in diverse physiological processes. The migratory brown planthopper (BPH), Nilaparvata lugens, encodes four ILP genes (Nlilp1, Nlilp2, Nlilp3 and Nlilp4) but their physiological roles are largely unknown. Sequence analysis showed that NlILP1 contained a relaxin-specific G protein-coupled receptor-binding motif and a variant motif of cysteine residues, and NlILP2 and NlILP4 resembled vertebrate IGFs. RNA interference (RNAi)-mediated gene silencing showed that depletion of each of Nlilp1, 2 and 3 significantly delayed the developmental duration of nymphs, and this effect could be exacerbated by double or triple gene depletion. Depletion of Nlilp1, Nlilp2 or Nlilp3 induces the accumulation of glucose, trehalose and glycogen, which is contradictory to depletion of the insulin receptor (NlInR1) in the BPH. Depletion of Nlilp1 significantly enhanced starvation resistance in both females and males although its extent was smaller than NlInR1 depletion. A parental RNAi assay showed that depletion of each of Nlilp1-4 dramatically impaired female fecundity. These findings indicate that NlILP1-4 have redundant and distinct roles in physiological processes in the BPH, thereby enhancing our understanding of the contribution of each NlILP to the ecological success of this species in natural habitats.

[Deep learning indications for high tibial osteotomy].

High tibial osteotomy (HTO) is an effective treatment for knee osteoarthritis. With the application of bi-planer open wedge osteotomy high tibial osteotomy and new angular stable locking plates, HTO has become more accuracy, minimally invasive and standard, achieved satisfactory long-term treatment outcome. The indications of HTO are expanding. We need to comprehensively consider whether the patient has varus deformity, the location and severity of the deformity, the stage of osteoarthritis, age and the demand of activity, as well as individual factors such as weight, gender, bone condition and joint activity, and strive to give the best individualized treatment to osteoarthritis patients in different stages.

The MTase15 regulates reproduction in the wing-dimorphic planthopper, Nilaparvata lugens (Hemiptera: Delphacidae).

S-Adenosyl-l-methionine-dependent methyltransferases (SAMMTases) modulate important cellular and metabolic activities in both prokaryotes and eukaryotes. Here, we functionally characterized an SAMMTase gene (MTase15) in the migratory brown planthopper (BPH), Nilaparvata lugens, which is the most notorious rice pest in Asia. The cDNA sequence of MTase15 is 2764 nt in length with an open reading frame of 1218 nt encoding 405 amino acid residues. Quantitative real-time PCR analysis showed that MTase15 was readily detected from egg to adult stages and extensively distributed in various body parts of adult females and males, with slightly high levels in ovary and testis, respectively. In addition, MTase15 was transcriptionally regulated by the insulin signalling pathway in BPH. RNA-interference-mediated knockdown of MTase15 (dsMtase15) resulted in deficiencies in vitellogenin synthesis and oogenesis, and female infertility. Males with Mtase15 knockdown retained the capability of producing sperms with normal viability, but less sperm was transferred to wild-type (wt) females during copulation, and eggs laid by these wt females arrested embryogenesis. These findings not only assign a functional role to MTase15, but also provide a link between the insulin signalling pathway and epigenetic regulation in BPH reproduction.

ROR2 modulates neuropathic pain via phosphorylation of NMDA receptor subunit GluN2B in rats.

BACKGROUND: Neuropathic pain, a type of chronic pain as a result of direct central or peripheral nerve damage, is associated with significant quality of life and functional impairment. Its underlying mechanisms remain unclear. We investigated whether ROR2, a member of the receptor tyrosine kinase-like orphan receptor (ROR) family, participates in modulation of neuropathic pain. METHODS: Thermal hyperalgesia and mechanical allodynia were measured using radiant heat and von Frey filament testing. Immunofluorescence staining was used to detect expression of ROR2 in neuronal nuclei. Fos expression was determined by immunocytochemistry. Phosphorylation status was detected by western blot and immunoprecipitation. Small interfering RNA was used to knock down ROR2 expression. RESULTS: ROR2 was upregulated and activated in spinal neurones after chronic constriction injury (CCI) in mice [1.3 (0.1) to 2.1 (0.1)-fold of sham, P<0.01] from Day 1-21. CCI induced significant demethylation of the CpG island in the ROR2 gene promoter [0.37 (0.06) vs 0.12 (0.03)% CpG methylation, P<0.001]. Knockdown of ROR2 in the spinal cord prevented and reversed CCI-induced pain behaviours and spinal neuronal sensitisation [Fos expression: 130 (12) vs 81 (8) cells, P<0.05; 120 (11) vs 70 (7) cells, P<0.05]. In contrast, activation of spinal ROR2 by intrathecal injection of Wnt5a induced pain behaviours and spinal neuronal sensitisation [Fos expression: 11 (1) vs 100 (12) cells, P<0.001] in wild-type mice. Furthermore, ROR2-mediated pain modulation required phosphorylation of N-methyl-D-aspartate receptor 2B subunit (GluN2B) at Ser 1303 and Tyr1472 by pathways involving protein kinase C (PKC) and Src family kinases. Intrathecal injection of GluN2B, PKC, or Src family kinase-specific inhibitors significantly attenuated Wnt5a-induced pain behaviours. CONCLUSIONS: ROR2 in the spinal cord regulates neuropathic pain via phosphorylation of GluN2B, suggesting a potential target for prevention and relief of neuropathic pain.

Identification and functional analysis of a novel chorion protein essential for egg maturation in the brown planthopper.

In insect eggs, the chorion has the essential function of protecting the embryo from external agents during development while allowing gas exchange for respiration. In this study, we found a novel gene, Nilaparvata lugens chorion protein (NlChP), that is involved in chorion formation in the brown planthopper, Nilaparvata lugens. NlChP was highly expressed in the follicular cells of female adult brown planthoppers. Knockdown of NlChP resulted in oocyte malformation and the inability to perform oviposition, and electron microscopy showed that the malformed oocytes had thin and rough endochorion layers compared to the control group. Liquid chromatography with tandem mass spectrometry analysis of the eggshell components revealed four unique peptides that were matched to NlChP. Our results demonstrate that NlChP is a novel chorion protein essential for egg maturation in N. lugens, a hemipteran insect with telotrophic meroistic ovaries. NlChP may be a potential target in RNA interference-based insect pest management.

Ion transport peptide (ITP) regulates wing expansion and cuticle melanism in the brown planthopper, Nilaparvata lugens.

Ion transport peptide (ITP) and its alternatively spliced homologous ITP-like (ITPL) products play important roles in various insect developmental processes. We found for the first time that alternative 5' untranslated regions (5' UTRs) of ITPL (NilluITPLs-1, -2, -3 and -4) control spatiotemporal expression in the brown planthopper, Nilaparvata lugens, as demonstrated by reverse-transcription quantitative PCR. By using an alternative 5' UTR, NilluITPL-1 was expressed exclusively in the male reproductive system, resulting in the production of the NilluITPL seminal fluid protein. Interestingly, NilluITPLs-3 and -4 were expressed exclusively in the integument, indicating a specialized function for NilluITPL during ecdysis and eclosion. We investigated the functions of NilluITP and NilluITPL using RNA interference (RNAi). We did not observe apparent phenotypes when expression of NilluITPLs was suppressed. However, when NilluITP expression was suppressed, the insect exhibited melanism and failed wing expansion, indicating that NilluITP is a neuropeptide associated with wing expansion in addition to bursicon. Additionally, in contrast to bursicon, the insects showed increased melanism when NilluITP was eliminated by RNAi. Unlike previous studies of ITP/ITPL in other species, NilluITP was very important in the control of N. lugens postecdysial behaviours but was not critical during ecdysis. Thus, the functions of ITP and ITPL are more complex in insects than previously thought.

[The application of matrix metalloproteinase-3 and 7 joints ultrasonic score in assessment of disease activity in patients with rheumatoid arthritis].

OBJECTIVE: To evaluate the significance of serum matrix metalloproteinase-3 (MMP-3) and joint ultrasonography in assessing the activity of rheumatoid arthritis (RA) by comparing MMP-3 level and the ultrasonic 7 joints (US7) score in RA patients. METHODS: Serum MMP-3 level and US7 score were measured in 133 RA patients by immune turbidity and Doppler ultrasound. Synchronous 53 healthy subjects were recruited as controls. Clinical data were collected. Erythrocyte sedimentation rate (ESR), serum level of anti-cyclic citrullinated peptide (CCP) antibody, health assessment questionnaire (HAQ) and disease activity score 28 (DAS28) were measured. The level of disease activity is interpreted as remission(DAS28<2.6), low(DAS 28≥2.6-<3.2), moderate(DAS 28≥3.2-<5.1), high(DAS28≥5.1). The discriminating validity of MMP-3 and US7 score in disease was evaluated using receiver operating characteristic (ROC) curve analysis with DAS28 as the reference standard. RESULTS: Compared with that in healthy controls [35.20(25.90, 48.90) µg/L] and remission patients[33.40(22.60, 678.40) µg/L], the MMP-3 level in moderate [105.1(61.70, 172.70) µg/L] and high [363.1(161.50, 475.90) µg/L] groups increased dramatically. US7 score in patients with high disease activity was significantly higher than that in other groups. The level of MMP-3 was significantly correlated with DAS28, HAQ, US7 score, yet did not have correlation with anti-CCP antibody. Serum level of MMP-3 was positively correlated with US7 score(r=0.566, P<0.001). In evaluating the disease activity, US7 score combined with MMP-3 (AUC 0.863 2) was not superior to MMP-3 alone (AUC 0.854 3), but significantly better than single US7 score (AUC 0.764 3, P<0.05). CONCLUSIONS: MMP-3 is an effective and simple index in evaluating RA disease activity. The combination of MMP-3 and US7 score does not further improve the efficacy to evaluate disease activity than MMP-3 alone in patients with RA.

Chitinase-like gene family in the brown planthopper, Nilaparvata lugens.

Chitinases are important enzymes required for chitin degradation and reconstruction in insects. Based on a bioinformatics investigation, we identified 12 genes encoding putative chitinase-like proteins, including 10 chitinases (Cht), one imaginal disc growth factor (IDGF) and one endo-ß-N-acetylglucosaminidase (ENGase) in the genome of the brown planthopper, Nilaparvata lugens (Hemiptera: Delphacidae). These 12 genes were clustered into nine different groups, with 11 in glycoside hydrolase family 18 groups (groups I-VIII) and one in the ENGase group. Developmental and tissue-specific expression pattern analysis revealed that the transcript levels of eight genes peaked periodically during moulting and were mainly expressed in the integument, except NlCht2, NlCht4, NlIDGF and NlENGase. NlCht2, NlIDGF and NlENGase were expressed at all stages with slight periodical changes and mainly expressed in the female reproductive organs in adults, whereas NlCht4 was highly expressed only at the adult stage in the male reproductive organs. Lethal phenotypes were observed in insects challenged by double-stranded RNAs for NlCht1, NlCht5, NlCht7, NlCht9 and NlCht10 during moulting, suggesting their significant roles in old cuticle degradation. NlCht1 was the most sensitive gene, inducing 50% mortality even at 0.01 ng per insect. Our results illustrate the structural and functional differences of chitinase-like family genes and provide potential targets for RNA interference-based rice planthopper management.

First Report of Anthracnose of Malva sylvestris Caused by Colletotrichum trifolii in China.

Common mallow (Malva sylvestris L.) is a perennial medicinal plant in the Malvaceae family, which is native to Asia, Europe, and North Africa. In July 2012, typical symptoms of anthracnose disease, with a disease incidence of ~70%, were observed on common mallow in the Medicinal Herb Garden of Shenyang Pharmaceutical University, Liaoning, China. The fungus mainly infected the stalks and leaves of M. sylvestris. Pinpoint, brownish lesions initially appeared at the flowering stage and the disease spread within the field. The lesions on stems gradually enlarged and became dark brown, elliptical, and slightly concave. Concurrently, acervuli and mucilaginous conidial masses of the pathogen appeared on lesions under moist conditions. Conidia were hyaline, one-celled, cylindrical with both ends rounded, and measured 10.0 to 12.5 × 2.5 to 4.0 µm (mean 11.3 × 3.3 µm). The fungus was isolated from symptomatic tissues. Small pieces from leaves and stems were surface disinfested with 70% ethanol and 1.5% sodium hypochlorite for 1 min, then rinsed three times with sterile distilled water, and cultured on potato dextrose agar (PDA) at 25°C. The colonies on PDA had initially white aerial mycelia, and later became greenish black with regularly whorled rings. To confirm Koch's postulates, five 3-month-old plants of M. sylvestris were inoculated with a conidial suspension (105 conidia/ml) prepared from PDA cultures incubated for 14 days. Five non-inoculated plants served as controls. The plants were maintained in the greenhouse at 22 to 25°C and about 75% relative humidity under natural daylight. Typical symptoms on inoculated plants were reproduced after ~10 to 14 days, whereas control plants remained asymptomatic. The pathogen was successfully recovered from symptomatic tissues and re-identified, completing Koch's postulates. The internal transcribed spacer (ITS) and large subunit -28S (LSU) region of rDNA was amplified with primers ITS1/ITS4 and NL1/NL4, respectively, and sequenced. Phylogenetic trees (ITS and LSU) that were obtained using MEGE3.1 with the neighbor-joining method showed that both of the isolates fall in the Colletotrichum trifolii clade. The representative sequences (ITS and LSU) were deposited in GenBank (Accession Nos. KJ155692 and KJ920935). The fungus isolated from symptomatic tissues was identified as C. trifolii on the basis of morphological, cultural characteristics, and sequence analysis (2). According to previous references, C. orbiculare and C. malvarum on Malvaceae were respectively described in America and Europe (1,3,4). However, the isolate from M. sylvestris significantly differed from those of C. orbiculare and C. malvarum in cultural characteristics and sequence analysis. In this paper, the results showed that M. sylvestris is a new host of C. trifolii. To our knowledge, this is the first report of mallow anthracnose caused by C. trifolii in China. References: (1) J. A. Bailey et al. Phytopathology 86:1076, 1996. (2) U. Damm et al. Fungal Divers. 61:29, 2013. (3) K. Hyde et al. Fungal Divers. 39:147, 2009. (4) L. Tosi et al. Plant Dis. 88:425, 2004.

Genome-wide screening for components of small interfering RNA (siRNA) and micro-RNA (miRNA) pathways in the brown planthopper, Nilaparvata lugens (Hemiptera: Delphacidae).

The brown planthopper (BPH), Nilaparvata lugens, is a major rice pest in Asia, and accumulated evidence indicates that this species is susceptible to RNA interference (RNAi); however, the mechanism underlying RNAi and parental RNAi has not yet been determined. We comprehensively investigated the repertoire of core genes involved in small interfering RNA (siRNA) and micro-RNA (miRNA) pathways in the BPH by comparing its newly assembled transcriptome and genome with those of Drosophila melanogaster, Tribolium castaneum and Caenorhabditis elegans. Our analysis showed that the BPH possesses one drosha and two Dicer (dcr) genes, three dsRNA-binding motif protein genes, two Argonaute (ago) genes, two Eri-1-like genes (eri-1), and a Sid-1-like gene (sid-1). Additionally, we report for first time that parental RNAi might occur in this species, and siRNA pathway and Sid-1 were required for high efficiency of systemic RNAi triggered by exogenous dsRNA. Furthermore, our results also demonstrated that the miRNA pathway was involved in BPH metamorphosis as depletion of the ago1 or dcr1 gene severely impaired ecdysis. The BPH might be a good model system to study the molecular mechanism of systemic RNAi in hemimetabolous insects, and RNAi has potential to be developed to control this pest in agricultural settings.

Functional expression of mouse insulin-like growth factor-I with food-grade vector in Lactococcus lactis NZ9000.

AIMS: To functionally express the recombinant mouse insulin-like growth factor-I (rtmIGF-I) in Lactococcus lactis NZ9000 with a food-grade vector. METHODS AND RESULTS: The rtmIGF-I encoding sequence was inserted into secreted food-grade vector pLEB688 and transformed into L. lactis NZ9000. The expression of the recombinant protein rtmIGF-I was confirmed by tricine-SDS-PAGE analysis and Western blot. The concentration of this recombinant protein was 3 mg l(-1) in the medium fraction. Further experiment demonstrated that the recombinant protein was biologically active and promoted NIH3T3 cell proliferation in a concentration-dependent manner. CONCLUSIONS: The rtmIGF-I was expressed in L. lactis and located into the medium fraction. The optimal final concentration which could promote NIH3T3 cell proliferation after incubation was 100 ng ml(-1) . SIGNIFICANCE AND IMPACT OF THE STUDY: The rtmIGF-I was functionally expressed in L. lactis NZ9000 with a food-grade vector. Thus, the recombinant L. lactis NZ9000 could act as a host for the production of rtmIGF-I for further study. The recombinant strain could serve as an IGF-I delivery system.

First Report of Sclerotinia Rot of Chinese Atractylodes Caused by Sclerotinia nivalis in China.

Chinese atractylodes (Atractylodes japonica Koidz.ez Kitam.) is a perennial herb in the Compositae family, and is widely distributed in China. The dried rhizomes of the plant are used in traditional Chinese medicine. During the summer of 2011, typical signs and symptoms of Sclerotinia rot were observed on Chinese atractylodes in a production field of Liaoning Province of China. Symptoms were observed in plants at the flowering stage, distributed in patches throughout the rows, and with a disease incidence of approximately 10 to 15%. The lower mature leaves of infected plants first became yellow and wilted, basal stem areas showed a black-brown rot at the same time under conditions of high humidity, and white cottony mycelium formed along the basal stem and soil surfaces. Ultimately, the basal stem and roots rotted and the plants wilted and died quickly. Black, irregular sclerotia (average 0.8 to 6.9 mm in diameter) were also observed within the pith cavity of split stems and rotted roots. The pathogen was isolated from symptomatic tissues and sclerotia, surface disinfested with 2% sodium hypochlorite, and cultured on potato dextrose agar (PDA) (1). The fungus was mesophilic, with an optimum temperature for mycelial growth in culture of about 20°C. Colonies on PDA produced masses of white aerial mycelium, with small white flocci distributed among sclerotia. After 2 weeks, sclerotia 0.5 to 4.5 mm in diameter were produced near the margin in a uniform distribution. Sclerotia were spherical, elongated, or fused to form irregular shapes and tightly attached to the agar surface by their under surface, which could be seen through the bottom of the petri dishes. DNA sequences of five replicates were obtained using the TianGen DNA secure plant kit. The internal transcribed spacer (ITS) region of rDNA was amplified with primers ITS1/ITS4 and sequenced. BLAST analysis of the 513-bp segment showed high similarity (99%) with a sequence of Sclerotinia nivalis (GenBank Accession No. AB516670). A representative sequence was deposited in GenBank (Accession No. JX294862). The fungus isolated from symptomatic tissues was identified as S. nivalis Saito on the basis of morphological and cultural characteristics (2,3) and ITS sequence analysis. Symptoms were reproduced in the greenhouse by inoculating the basal stem and roots of 15 atractylodes plants at the 7- to 10-leaf stage. Inoculum was prepared by macerating 14-day-old PDA cultures of the fungus in a blender and placing the mixture (approximately 20 g) into the potting medium of each plant. Sterile PDA was used to inoculate the five control plants. Plants were maintained in a greenhouse at 22 to 25°C and about 75% relative humidity. After 7 to 10 days, symptoms were similar to those in the fields. Lower leaves of inoculated plants became yellow and wilted, and infected plants died 2 weeks after inoculation, whereas control plants remained healthy. The pathogen was successfully recovered from symptomatic tissues, completing Koch's postulates. To our knowledge, this is the first report of Sclerotinia rot of Chinese atractylodes. Given its wide host range, S. nivalis has great potential to become an economically important plant pathogen. References: (1) W. G. Kim and W. D. Cho. Mycobiology 30:41, 2002. (2) G. Q. Li et al. Mycol. Res. 104:232, 2000. (3) I. Saito. Mycoscience 38:227, 1997.