[Optimization of processing technology of bark of Ilicis Rotundae Cortex based on quality by design concept].

Based on the concept of quality by design(QbD), this study optimized the processing technology of Ilicis Rotundae Cortex. According to the processing method and ingredient requirements of Ilicis Rotundae Cortex in the Chinese Pharmacopoeia, the content of syringin and pedunculoside, alcohol extract, fragmentation rate, and moisture content were taken as the critical quality attributes(CQAs). The soaking time, moistening time, and drying time were taken as critical process parameters(CPPs) by single factor tests. The weight coefficients of CQAs were determined by the analytic hierarchy process(AHP)-entropy weighting method, and the comprehensive score was calculated. With the comprehensive score as the response value, Box-Behnken design was employed to establish a mathematical model between CPPs and CQAs, and the design space for the processing of Ilicis Rotundae Cortex was built and verified. The results of ANOVA showed that the mathematical model had the P value below 0.05, the lack of fit greater than 0.05, adjusted R~2=0.910 5, and predicted R~2=0.831 0, which indicated that the proposed model had statistical significance and good prediction performance. Considering the factors in production, the best processing conditions of Ilicis Rotundae Cortex were decoction pieces of about 1 cm soaking for 1 h, moistening for 4 h, and drying at 60-70 ℃ in a blast drier for 2 h. The optimized processing technology of Ilicis Rotundae Cortex was stable and feasible, which can provide a reference for the standardized preparation and stable quality of Ilicis Rotundae Cortex.

Association between sleep duration, sleep quality, bedtime and myopia: A systematic review and meta-analysis.

BACKGROUND: We performed a systematic review and meta-analysis to investigate the links between different sleep characteristics and risk of myopia. METHODS: PubMed, EMBASE, Web of Science, the Cochrane Library, PsycINFO, Wanfang, and CNKI were searched from inception to August 26, 2022, without any language restriction. Cross-sectional, case-control, or cohort studies that explored the association between sleep duration, sleep quality, bedtime, and myopia were included. NIH quality assessment tools were used to assess the methodological quality of included studies. Random-effect or fixed-effect models were used to pool the associations according to whether there is heterogeneity. RESULTS: A total of 31 studies with 205 907 participants were included in the final analysis (25 studies reporting sleep duration; four studies examining sleep quality and six studies evaluating bedtime). Compared to reference sleep duration, sufficient sleep duration (OR = 0.63, 95% CI = 0.51-0.78) was associated with a lower risk of myopia, and short sleep duration (OR = 1.66, 95% CI = 1.14-2.42) was associated with a higher risk of myopia. In addition, poor sleep quality (OR = 1.24, 95% CI = 1.05-1.47) was associated with a higher risk of myopia while late bedtime (OR = 1.30, 95% CI = 0.96-1.75) was not significantly associated with an increased risk of myopia. CONCLUSIONS: Alteration in sleep duration and sleep quality may influence the risk of myopia. Well-designed cohort studies are needed in future investigations to identify a causal relationship between different sleep characteristics and myopia.

Latently and uninfected healthcare workers exposed to TB make protective antibodies against Mycobacterium tuberculosis.

The role of Igs in natural protection against infection by Mycobacterium tuberculosis (Mtb), the causative agent of TB, is controversial. Although passive immunization with mAbs generated against mycobacterial antigens has shown protective efficacy in murine models of infection, studies in B cell-depleted animals only showed modest phenotypes. We do not know if humans make protective antibody responses. Here, we investigated whether healthcare workers in a Beijing TB hospital-who, although exposed to suprainfectious doses of pathogenic Mtb, remain healthy-make antibody responses that are effective in protecting against infection by Mtb. We tested antibodies isolated from 48 healthcare workers and compared these with 12 patients with active TB. We found that antibodies from 7 of 48 healthcare workers but none from active TB patients showed moderate protection against Mtb in an aerosol mouse challenge model. Intriguingly, three of seven healthcare workers who made protective antibody responses had no evidence of prior TB infection by IFN-γ release assay. There was also good correlation between protection observed in vivo and neutralization of Mtb in an in vitro human whole-blood assay. Antibodies mediating protection were directed against the surface of Mtb and depended on both immune complexes and CD4+ T cells for efficacy. Our results indicate that certain individuals make protective antibodies against Mtb and challenge paradigms about the nature of an effective immune response to TB.

MRI-based nomogram estimates the risk of recurrence of primary nonmetastatic pancreatic neuroendocrine tumors after curative resection.

BACKGROUND: Accurate estimation of the recurrence of pancreatic neuroendocrine tumors help with prognosis, guide follow-up, and avoid futile treatments. PURPOSE: To investigate whether MRI features could preoperatively estimate the recurrence of pancreatic neuroendocrine tumors (PNETs) and to refine a novel prognostic model through developing a nomogram incorporating various MRI features. STUDY TYPE: Retrospective. POPULATION: In all, 81 patients with clinicopathologically confirmed nonmetastatic PNETs. FIELD STRENGTH/SEQUENCES: 1.5 T MR, including T1 -weighted, T2 -weighted, and diffusion-weighted imaging sequences. ASSESSMENT: Qualitative and quantitative MRI features of PNET were assessed by three experienced radiologists. STATISTICAL TESTS: Uni- and multivariable analyses for recurrence-free survival (RFS) were evaluated using a Cox proportional hazards model. The MRI-based nomogram was then designed based on multivariable logistic analysis in our study and the performance of the nomogram was validated according to C-index, calibration, and decision curve analyses. RESULTS: MRI features, including tumor size (hazard ratio [HR]: 14.131; P = 0.034), enhancement pattern (HR: 21.821, P = 0.032), and the apparent diffusion coefficient (ADC) values (HR: 0.055, P = 0.038) were significant independent predictors of RFS at multivariable analysis. The performance of the nomogram incorporating various MRI features (with a C-index of 0.910) was improved compared with that based on tumor size, enhancement pattern, and ADC alone (with C-index values of 0.672, 0.851, and 0.809, respectively). The calibration curve of the nomogram exhibited perfect consistency between estimation and observation at 0.5, 1, and 2 years after surgery. The decision curve showed that a nomogram incorporating three features had more favorable clinical predictive usefulness than any single feature. DATA CONCLUSION: MRI features can be considered effective recurrence predictors for PNETs after surgery. The preliminary nomogram incorporating various MRI features could assess the risk of recurrence in PNETs and may be used to optimize individual treatment strategies. LEVEL OF EVIDENCE: 4 Technical Efficacy: Stage 2 J. Magn. Reson. Imaging 2019;50:397-409.

[Purification technology of manninotirose in Rehmanniae Radix Praeparata by D-101 microporous adsorption resin].

This paper was aim to optimize the purification technology of Rehmanniae Radix Praeparata extract with macroporous adsorption resin. With the content of manninotriose as index, the absorptive flow and time were investigated, as well as kinds, amount, flow of eluent. D-101 type macroporous adsorption resin was the best choice for the purification of manninotriose. The optimized parameters were as follows: the content of manninotriose at 161.16-53.72 mg x g(-1), absorption time 240 min, eluting solvent of purified water, volume flow at 1.5 BV x h(-1), and eluant volume at 6 BV. D-101 type macroporous adsorption resin could significantly increase the purity of Rehmanniae Radix Praeparata extract with the advantage of high absorption, remove most part of impurity, and the effect of semi-works production was better.

De novo synthesis of a novel hapten and development of a monoclonal antibody-based immunoassay for the detection of dichlorvos and trichlorfon.

The absence of high-affinity antibodies has hindered the development of satisfactory immunoassays for dichlorvos (DDVP) and trichlorfon (TCP), two highly toxic organophosphorus pesticides. Herein, the de novo synthesis of a novel anti-DDVP hapten was introduced. Subsequently, a specific anti-DDVP monoclonal antibody (Mab) was produced with satisfying affinity to DDVP (IC50: 12.4 ng mL-1). This Mab was highly specific to DDVP, and TCP could readily convert into DDVP under mild alkaline conditions. Leveraging this insight, an indirect competitive ELISA was successfully developed for simultaneous detection of DDVP and TCP. The limit of detection in rice, cabbage and apple for DDVP /TCP was found to be 12.1/14.6 µg kg-1, 7.3/8.8 µg kg-1 and 6.9/8.3 µg kg-1, respectively. This study not only provides an effective strategy for producing a high-quality anti-DDVP Mab but also affords a reliable and cost-effective tool suitable for high-throughput detection of DDVP and TCP in food samples.

Enhanced 2-MIB degradation by UV-LED/chlorine process: reaction kinetics, wavelength dependence, influencing factors and degradation pathways.

The efficient removal of 2-Methylisoborneol (2-MIB), a typical odour component, in water treatment plants (WTPs), poses a great challenge to conventional water treatment technology due to its chemical stability. In this study, the combination of ultraviolet light-emitting diode (UV-LED) and chlorine (UV-LED/chlorine) was exploited for 2-MIB removal, and the role of ultraviolet (UV) wavelength was investigated systematically. The results showed that UV or chlorination alone did not degrade 2-MIB effectively, and the UV/chlorine process could degrade 2-MIB efficiently, following the pseudo-first-order kinetic model. The 275 nm UV exhibited higher 2-MIB degradation efficiency in this UV-LED/chlorine system than 254 nm UV, 265 nm UV and 285 nm UV due to the highest mole adsorption coefficient and quantum yield of chlorine in 275 nm UV. ·OH and ·Cl produced in the 275 nm UV/chlorine system played major roles in 2-MIB degradation. HCO3- and Natural organic matter (NOM), prevalent in water, consumed ·OH and ·Cl, thus inhibiting the 2-MIB degradation by UV-LED/chlorine. In addition, NOM and 2-MIB could form a photonic competition effect. The degradation of 2-MIB by UV-LED/chlorine was done mainly through dehydration and demethylation, and odorous intermediates, such as camphor, were produced. 2-MIB was degraded through the α bond fracture and six-membered ring opening to form saturated or unsaturated hydrocarbons and aldehydes. Four DBPs, chloroform (CF), trichloroacetaldehyde (TCE), trichloroacetone (TCP) and dichloroacetone (DCP), were mainly generated, and CF was the most significant by-product.

Role of a GATA-type transcriptional repressor Sre1 in regulation of siderophore biosynthesis in the marine-derived Aureobasidium pullulans HN6.2.

The GATA-type transcriptional repressor structural gene SRE1 was isolated from both the genomic DNA and mRNA of the marine yeast Aureobasidium pullulans HN6.2 by inverse PCR and RACE. An open reading frame (ORF) of 1,002 bp encoding a 334 amino acid protein (a calculated isoelectric point: 8.6) with a calculated molecular weight of 35.1 kDa was characterized. The corresponding gene had one single intron of 51 bp, and in its promoter two putative 5'-HGATAR-3' sequences could be recognized. The deduced protein from the cloned gene contained two conserved zinc-finger domains [Cys-(X2)-Cys-(X17)-Cys-(X2)-Cys)], nine sequences of Ser(Thr)-Pro-X-X which was characteristics of the regulator, and one cysteine-rich central domain which was located between the two zinc fingers. The SRE1 gene in A. pullulans HN6.2 was disrupted by integrating the hygromycin B phosphotransferase gene into the ORF of the SRE1 gene using homologous recombination. Two hundreds of the disruptants (Δsre1) (one of them was named R6) obtained still synthesized both intracellular and extracellular siderophores in the presence of added Fe(3+) and the expression of the SidA gene encoding L-ornithine N(5)-oxygenase in the disruptant R6 was also partially derepressed in the presence of added Fe(3+). The colonies of the disruptant R6 grown on the iron-replete medium with 1.5 and 2.0 mM Fe(3+) and also with 1.5 mM Fe(2+) became brown. In contrast, A. pullulans HN6.2 could not grow in the iron-replete medium with 1.5 mM and 2.0 mM Fe(3+). The brown-colored colonies of the disruptant R6 also had high level of siderophore and iron.

[Data validation methods and discussion on Chinese materia medica resource survey].

From the beginning of the fourth national survey of the Chinese materia medica resources, there were 22 provinces have conducted pilots. The survey teams have reported immense data, it put forward the very high request to the database system construction. In order to ensure the quality, it is necessary to check and validate the data in database system. Data validation is important methods to ensure the validity, integrity and accuracy of census data. This paper comprehensively introduce the data validation system of the fourth national survey of the Chinese materia medica resources database system, and further improve the design idea and programs of data validation. The purpose of this study is to promote the survey work smoothly.

Gestational diabetes mellitus: The optimal time of delivery.

Gestational diabetes mellitus (GDM) is a common pregnancy complication strongly associated with poor maternal-fetal outcomes. Its incidence and prevalence have been increasing in recent years. Women with GDM typically give birth through either vaginal delivery or cesarean section, and the maternal-fetal outcomes are related to several factors such as cervical level, fetal lung maturity, the level of glycemic control still present, and the mode of treatment for the condition. We categorized women with GDM based on the latter two factors. GDM that is managed without medication when it is responsive to nutrition- and exercise-based therapy is considered diet- and exercise-controlled GDM, or class A1 GDM, and GDM managed with medication to achieve adequate glycemic control is considered class A2 GDM. The remaining cases in which neither medical nor nutritional treatment can control glucose levels or patients who do not control their blood sugar are categorized as class A3 GDM. We investigated the optimal time of delivery for women with GDM according to the classification of the condition. This review aimed to address the benefits and harms of giving birth at different weeks of gestation for women with different classes of GDM and attempted to provide an analytical framework and clearer advice on the optimal time for labor.

Ratiometric Fluorescence Immunoassay Based on MnO2 Nanoflakes for Sensitive and Accurate Detection of Tricaine.

Tricaine is a common anesthetic used in the long-distance transport of live fish. Recently, its negative impact on human health has aroused extensive concern. Thus, rapid and reliable techniques for tricaine residue analysis are essential to ensuring the quality of aquatic products. Herein, a specific anti-tricaine monoclonal antibody (Mab) was prepared. Then, a sensitive and robust ratiometric fluorescence ELISA (RF-ELISA) was constructed for detecting tricaine based on two MnO2 nanoflake-mediated (MnO2 NFs) fluorogenic reactions. In the RF-ELISA protocol, MnO2 NFs with oxidase-like activity can trigger the formation of fluorescent 2,3-diaminophenazine (oxOPD) with an emissive peak at 570 nm from non-fluorescent o-phenylenediamine (OPD), while ascorbic acid (AA) can decompose MnO2 NFs to lose their oxidase-mimicking activity, which is accompanied by the oxidation of AA into dehydroascorbic acid (DHAA). The subsequent reaction between the generated DHAA and OPD will result in the production of 3-(1,2-dihydroxy ethyl)furo[3,4-b]quinoxalin-1(3H)-on (DFQ), which has a potent emission peak at 445 nm. By virtue of the alkaline phosphatase (ALP) labeled on the antibody, which can catalyze the production of AA from ascorbic acid 2-phosphate (AAP), the concentration of tricaine can be linked to the variation of the RF signal (F445/F570) via a competitive immunoreaction. After optimization, RF-ELISA displayed a detection limit (LOD) of 0.28 ng/mL toward tricaine (in buffer solution), which was 376-fold lower than that of the traditional colorimetric ELISA. For practical application, the LODs of RF-ELISA for tricaine detection in shrimp and tilapia samples were determined to be 2.8 and 5.6 ng/g, respectively. Recoveries for spiked shrimp and tilapia samples, as well as the validation data from LC-MS/MS, showed that RF-ELISA exhibited good accuracy, precision, and reliability. This RF-ELISA protocol opened up new ways for tricaine and other-target analyses in food safety detection.

GhSINA1, a SEVEN in ABSENTIA ubiquitin ligase, negatively regulates fiber development in Upland cotton.

Protein ubiquitination is essential for plant growth and responses to the environment. The SEVEN IN ABSENTIA (SINA) ubiquitin ligases have been extensively studied in plants, but information on their roles in fiber development is limited. Here, we identified GhSINA1 in Upland cotton (Gossypium hirsutum), which has a conserved RING finger domain and SINA domain. Quantitative real-time PCR (qRT-PCR) analysis showed that GhSINA1 was preferentially expressed during fiber initiation and elongation, especially during initiation in the fuzzless-lintless cotton mutant. Subcellular localization experiments indicated that GhSINA1 localized to the nucleus. In vitro ubiquitination analysis revealed that GhSINA1 has E3 ubiquitin ligase activity. Ectopic overexpression of GhSINA1 in Arabidopsis thaliana reduced the number and length of root hairs and trichomes. Yeast two-hybrid (Y2H), firefly luciferase complementation imaging (LCI), and bimolecular fluorescence complementation (BiFC) assays demonstrated that the GhSINA1 proteins could interact with each other to form homodimers and heterodimers. Overall, these results suggest that GhSINA1 may act as a negative regulator in cotton fiber development through homodimerization and heterodimerization.

Unraveling genomic regions and candidate genes for multiple disease resistance in upland cotton using meta-QTL analysis.

Verticillium wilt (VW), Fusarium wilt (FW) and Root-knot nematode (RKN) are the main diseases affecting cotton production. However, many reported quantitative trait loci (QTLs) for cotton resistance have not been used for agricultural practices because of inconsistencies in the cotton genetic background. The integration of existing cotton genetic resources can facilitate the discovery of important genomic regions and candidate genes involved in disease resistance. Here, an improved and comprehensive meta-QTL analysis was conducted on 487 disease resistant QTLs from 31 studies in the last two decades. A consensus linkage map with genetic overall length of 3006.59 cM containing 8650 markers was constructed. A total of 28 Meta-QTLs (MQTLs) were discovered, among which nine MQTLs were identified as related to resistance to multiple diseases. Candidate genes were predicted based on public transcriptome data and enriched in pathways related to disease resistance. This study used a method based on the integration of Meta-QTL, known genes and transcriptomics to reveal major genomic regions and putative candidate genes for resistance to multiple diseases, providing a new basis for marker-assisted selection of high disease resistance in cotton breeding.

The Arabidopsis J-protein AtDjB1 facilitates thermotolerance by protecting cells against heat-induced oxidative damage.

AtDjB1 belongs to the J-protein family in Arabidopsis thaliana. Its biological functions in plants are largely unknown. In this study, we examined the roles of AtDjB1 in resisting heat and oxidative stresses in A. thaliana using reverse genetic analysis. AtDjB1 knockout plants (atj1-1) were more sensitive to heat stress than wildtype plants, and displayed decreased concentrations of ascorbate (ASC), and increased concentrations of hydrogen peroxide (H(2)O(2)) and oxidative products after heat shock. Application of H(2)O(2) accelerated cell death and decreased seedling viability in atj1-1. Exogenous ASC conferred much greater thermotolerance in atj1-1 than in wildtype plants, suggesting that a lower concentration of ASC in atj1-1 could be responsible for the increased concentration of H(2)O(2) and decreased thermotolerance. Furthermore, AtDjB1 was found to localize to mitochondria, directly interact with a mitochondrial heat-shock protein 70 (mtHSC70-1), and stimulate ATPase activity of mtHSC70-1. AtDjB1 knockout led to the accumulation of cellular ATP and decreased seedling respiration, indicating that AtDjB1 modulated the ASC concentration probably through affecting the function of mitochondria. Taken together, these results suggest that AtDjB1 plays a crucial role in maintaining redox homeostasis, and facilitates thermotolerance by protecting cells against heat-induced oxidative damage.

The unique role of siderophore in marine-derived Aureobasidium pullulans HN6.2.

The L -ornithine-N (5)-monooxygenase structural gene (SidA gene, accession number: FJ769160) was isolated from both the genomic DNA and cDNA of the marine yeast Aureobasidium pullulans HN6.2 by inverse PCR and RT-PCR. An open reading frame of 1,461 bp encoding a 486 amino acid protein (isoelectric point: 7.79) with calculated molecular weight of 55.4 kDa was characterized. The promoter of the gene (intronless) was located from -1 to -824 and had three HGATAR boxes which were putative binding motifs for the respective DNA-binding motifs and one CATA box. The SidA gene in A. pullulans HN6.2 was disrupted by integrating the hygromycin B phosphotransferase (HPT) gene into Open Reading Frame of the SidA gene using homologous recombination. Of all the disruptants obtained, one strain S6 (∆sidA) did not synthesize both intracellular and extracellular fusigen so that it could not inhibit growth of the pathogenic bacteria Vibrio anguillarum and Vibrio parahaemolyticus. The disruptant S6 did not grow in the iron-deplete medium and seawater medium because cell budding was stopped, but could grow in the iron-replete medium with 10 µM Fe(3+) and Fe(2+). H(2)O(2) in the medium was more toxic to the disruptant S6 than to its wild type HN6.2. Thus, we infer that the fusigen produced by the marine-derived A. pullulans HN6.2 can play a unique role in chelating, uptake and concentration of iron to maintain certain proper physiological functions within the cells and secretion of siderophore may represent an efficient tool to eliminate competitors to compete for limiting nutritional resources in marine environments.

Genetic surface-display of methyl parathion hydrolase on Yarrowia lipolytica for removal of methyl parathion in water.

In this study, the mph gene encoding methyl parathion hydrolase from Pseudomonas sp. WBC-3 was expressed in Yarrowia lipolytica and the expressed methyl parathion hydrolase was displayed on cell surface of Y. lipolytica. The activity of methyl parathion hydrolase displayed on the yeast cells of the transformant Z51 was 59.5 U mg⁻¹ of cell dry cells (450.6 U per mL of the culture) in the presence of 5.0 mM of Co²âº. The displayed methyl parathion hydrolase had the optimal pH of 9.5 and the optimal temperature of 40 °C, respectively and was stable in the pH range of 4.5-11 and up to 40 °C. The displayed methyl parathion hydrolase was also stimulated by Co²âº, Cu²âº, Ni²âº and Mn²âº, and was not affected by Fe²âº, Fe³âº, Na⁺, K⁺, Ca²âº and Zn²âº, but was inhibited by other cations tested. Under the optimal conditions (OD(600 nm) = 2.6, the substrate concentration = 100 mg L⁻¹ and 40 °C), 90.8 % of methyl parathion was hydrolyzed within 30 min. Under the similar conditions, 98.7, 97.0, 96.5 and 94.4 % of methyl parathion in tap water (pH 9.5), tap water (pH 6.8), seawater (pH 9.5) and natural seawater (pH 8.2) were hydrolyzed, respectively, suggesting that the methyl parathion hydrolase displayed on the yeast cells can effectively remove methyl parathion in water.

[An automated method to fit stellar continuum based on statistic windows].

A novel statistic window based method to fit stellar continuum is proposed. First a stellar spectrum is divided into a series of statistic windows in which a certain percent of flux points is selected according to S/N ratio; then low order polynomial iteration fitting is carried out based on the selected flux points to obtain the stellar continuum. Experimental results show that the continuum obtained by the proposed method is more close to the real continuum, compared to other existed methods. This method has a better practical applicability and robustness to all kinds of spectra (except M-type spectrum) in SDSS. It also works well for Guoshoujing Telescope (LAMOST) pilot survey spectra.

Disrupted presynaptic nectin1-based neuronal adhesion in the entorhinal-hippocampal circuit contributes to early-life stress-induced memory deficits.

The cell adhesion molecule nectin3 and its presynaptic partner nectin1 have been linked to early-life stress-related cognitive disorders, but how the nectin1-nectin3 system contributes to stress-induced neuronal, circuit, and cognitive abnormalities remains to be studied. Here we show that in neonatally stressed male mice, temporal order and spatial working memories, which require the medial entorhinal cortex (MEC)-CA1 pathway, as well as the structural integrity of CA1 pyramidal neurons were markedly impaired in adulthood. These cognitive and structural abnormalities in stressed mice were associated with decreased nectin levels in entorhinal and hippocampal subregions, especially reduced nectin1 level in the MEC and nectin3 level in the CA1. Postnatal suppression of nectin1 but not nectin3 level in the MEC impaired spatial memory, whereas conditional inactivation of nectin1 from MEC excitatory neurons reproduced the adverse effects of early-life stress on MEC-dependent memories and neuronal plasticity in CA1. Our data suggest that early-life stress disrupts presynaptic nectin1-mediated interneuronal adhesion in the MEC-CA1 pathway, which may in turn contribute to stress-induced synaptic and cognitive deficits.

Epidermal growth factor receptor gene amplification and protein overexpression in basal-like carcinoma of the breast.

AIMS: Epidermal growth factor receptor (EGFR) is frequently expressed in basal-like breast cancer (BLBC). The aim of this study was to evaluate their correlation as detected by immunohistochemistry (IHC) or fluorescence in-situ hybridization (FISH). METHODS AND RESULTS: IHC for oestrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor (HER) 2, cytokeratin (CK) 5/6 and EGFR, and FISH for EGFR amplification, were performed in 59 cases of BLBC. EGFR IHC results were scored semiquantitatively, and compared with its gene amplification status. ER, PR and HER2 were negative in all cases, whereas 35 and 55 cases were positive for CK5/6 and EGFR. For EGFR IHC, 20, 11, 11 and 17 cases showed a negative, a low, an intermediate or a high staining level, respectively, and seven cases showed gene amplification by FISH, with two, 19, 11 and 20 cases showing balanced monosony, disomy, trisomy, and polysomy respectively. Immunohistochemical expression in gene-amplified tumours was significantly higher than in those without amplification, including balanced polysomy tumours. EGFR immunohistochemical expression also correlated with the EGFR/chromosome 7 ratio. High sensitivity (86%) and negative predictive value (98%) were achieved with high-level immunohistochemical expression as a cut-off to predict gene amplification. CONCLUSIONS: High-level EGFR immunohistochemical expression correlated with and predicted EGFR amplification, and may be used as a screening method to exclude gene amplification.

Abdominal-B regulates structure and development of the Harmonia axyridis cremaster.

The ladybird Harmonia axyridis is an insect that exhibits pupal attachment to plants, which facilitates development and environmental adaptation. The cremaster is highly specialized for this behavior. However, the underlying molecular regulation of the cremaster remains unclear; therefore, we performed experiments to investigate the transcriptional regulation of cremaster development. First, we examined the morphological structure of the cremaster to reveal its function in pupal attachment of H. axyridis. Next, we analyzed the Hox gene Ha-Abd-B using RNA interference (RNAi) to determine its function in regulating cremaster formation; Ha-Abd-B up-regulation promoted effective pupal attachment, whereas successful RNAi caused severe down-regulation of this gene, and pupae were unable to attach. Furthermore, successful RNAi and subsequent Ha-Abd-B down-regulation caused phenotypic changes in cremaster structure, including its complete disappearance from some individuals. Finally, we observed unique development of the cremaster and dynamic expression of Ha-Abd-B during pre-pupal development; consequently, we hypothesized that there was specific pre-pupal development of the cremaster. Overall, based on these results, the specialized cremasteric structure located on the posterior side of H. axyridis was determined to be a key organ for pupal attachment. Cremaster identification in H. axyridis is regulated by Ha-Abd-B and exhibits preferential development. Pupal attachment of H. axyridis reveals an environmental adaptation of this species; thus, this study and future molecular studies will help determine the role of Hox genes in regulation of insect attachment and further our understanding of the multiple functions of Hox genes.