Study on the Role of Cytc in Response to BmNPV Infection in Silkworm, Bombyx mori (Lepidoptera).

Bombyx mori nucleopolyhedrovirus (BmNPV) is one of the primary pathogens of the silkworm. Cytochrome c (cytc) showed a significant response to BmNPV infection in our previous transcriptome study. However, little is known about the role of Bombyx mori cytc (Bmcytc) in resistance to BmNPV infection. In this study, the expression levels analysis of Bmcytc showed stable expression levels in selected tissues of the resistant strain AN following BmNPV infection, while there was downregulation in the susceptible strain p50, except in the malpighian tubule. To further study the role of Bmcytc in viral infection, Bmcytc was knocked down with siRNA in vitro, resulting in significant downregulation of selected downstream genes of the mitochondrial pathway, including Bmapaf, Bmcaspase-Nc, and Bmcaspase-1; this was also confirmed by overexpression of Bmcytc using the pIZT/V5-His-mCherry insect vector, except Bmcaspase-1. Moreover, knockdown of Bmcytc significantly promoted the infection process of BmNPV in vitro, while the infection was inhibited by overexpression of Bmcytc at the early stage and subsequently increased rapidly. Based on these results, we concluded that Bmcytc plays a vital role in BmNPV infection by regulating the mitochondrial apoptosis pathway. Our work provides valuable data for the clarification of the mechanism of silkworm resistance to BmNPV infection.

Quantification of Water, Protein and Soluble Sugar in Mulberry Leaves Using a Handheld Near-Infrared Spectrometer and Multivariate Analysis.

Mulberry (Morus alba L.) leaves are not only used as the main feed for silkworms (Bombyx mori) but also as an added feed for livestock and poultry. In order to rapidly select high-quality mulberry leaves, a hand-held near-infrared (NIR) spectrometer combined with partial least squares (PLS) regression and wavelength optimization methods were used to establish a predictive model for the quantitative determination of water content in fresh mulberry leaves, as well as crude protein and soluble sugar in dried mulberry leaves. For the water content in fresh mulberry leaves, the R-square of the calibration set (R2 C), R-square of the cross-validation set (R2 CV) and R-square of the prediction set (R2 P) are 0.93, 0.90 and 0.91, respectively, the corresponding root mean square error of calibration set (RMSEC), root mean square error of cross-validation set (RMSECV) and root mean square error of prediction set (RMSEP) are 0.96%, 1.13%, and 1.18%, respectively. The R2 C, R2 CV and R2 P of the crude protein prediction model are 0.91, 0.83 and 0.92, respectively, and the corresponding RMSEC, RMSECV and RMSEP are 0.71%, 0.97% and 0.61%, respectively. The soluble sugar prediction model has R2 C, R2 CV, and R2 P of 0.64, 0.51, and 0.71, respectively, and the corresponding RMSEC, RMSECV, and RMSEP are 2.33%, 2.73%, and 2.36%, respectively. Therefore, the use of handheld NIR spectrometers combined with wavelength optimization can fastly detect the water content in fresh mulberry leaves and crude protein in dried mulberry leaves. However, it is a slightly lower predictive performance for soluble sugar in mulberry leaves.

Identification and functional analysis of the cathepsin D gene promoter of Bombyx mori.

The gene encoding cathepsin D of silkworm, Bombyx mori (BmCatD) is specifically expressed in the larval fat body and pupal gut, and plays an important role in the programmed cell death during metamorphosis. To identify element involved in this transcription-dependent spatial restriction, truncation and deletion of the 5' terminal from the BmCatD promoter were conducted in vivo. The recombinant AcMNPV vector (Autographa californica multiple nucleopolyhedrovirus) with a dual-luciferase quantitative assay system was used as the transfer. A 289 bp DNA sequence (-1,214 to -925) upstream of the transcriptional start site is found to be responsible for promoting tissue-specific transcription. Further analysis of a series of deletion within the 289 bp region of overlapping deletion showed that a 33 bp region (-1,071 to -1,038) sequence suppresses the ectopic expression of the BmCatD promoter. These results suggest that this 33 bp region could function as a promoter element in the tissue-specificity expression.

BRWMC: Predicting lncRNA-disease associations based on bi-random walk and matrix completion on disease and lncRNA networks.

Many experiments have proved that long non-coding RNAs (lncRNAs) in humans have been implicated in disease development. The prediction of lncRNA-disease association is essential in promoting disease treatment and drug development. It is time-consuming and laborious to explore the relationship between lncRNA and diseases in the laboratory. The computation-based approach has clear advantages and has become a promising research direction. This paper proposes a new lncRNA disease association prediction algorithm BRWMC. Firstly, BRWMC constructed several lncRNA (disease) similarity networks based on different measurement angles and fused them into an integrated similarity network by similarity network fusion (SNF). In addition, the random walk method is used to preprocess the known lncRNA-disease association matrix and calculate the estimated scores of potential lncRNA-disease associations. Finally, the matrix completion method accurately predicts the potential lncRNA-disease associations. Under the framework of leave-one-out cross-validation and 5-fold cross-validation, the AUC values obtained by BRWMC are 0.9610 and 0.9739, respectively. In addition, case studies of three common diseases show that BRWMC is a reliable method for prediction.

Identification of ecdysone response elements (EcREs) in the Bombyx mori cathepsin D promoter.

Bombyx mori Cathepsin D (BmCatD) is specifically expressed in the fat body, and plays a critical role for the programmed cell death of the larval fat body and pupal gut during metamorphosis. To better understand the transcriptional control of BmCatD expression, we conducted this study to identify the ecdysone response elements (EcREs) in the BmCatD promoter and clarify their regulational functions. We inserted EcREs into a recombinant AcMNPV (Autographa californica multiple nucleopolyhedrovirus) vector and performed luciferase assay with a dual-luciferase quantitative assay system. Three putative EcREs were located at positions -109 to -99, -836 to -826 and -856 to -846 relative to the transcription start site. Overlapping deletion studies of this EcRE region showed that the three EcREs could suppress the ectopic expression of the BmCatD promoter. EcRE mutations resulted in the loss of the fat body-specific expression of the BmCatD gene. These results suggest that the EcREs are vital for activation of the promoter by 20-hydroxyecdysone (20E) in the larval fat body and further support the crucial role of ecdysone signaling to control cathepsin D gene transcription. It may suggest that the heterodimeric complex EcR/USP mediates the activation of ecdysone-dependent BmCatD transcription in the larval fat body of B. mori.

The discovery approaches and detection methods of microRNAs.

MicroRNAs (miRNAs) are small, highly conserved, non-coding RNAs that regulate gene expression of target mRNAs through cleavage or translational inhibition. Computer-based approaches for miRNA gene identification are being considered as indispensable in miRNAs research. Similarly, experimental approaches for detection of miRNAs are crucial to the testing and validating of computational algorithms. The detection of miRNAs in tissues or cells can supply valuable information for investigating the biological function of these molecules. Selective and highly sensitive detection methods will pave the way for extended understanding of miRNA function within organisms. In this review, we summarize the various computational methods for identification of miRNAs as well as the methodologies that have been developed to detection miRNAs.

Maternal, placental and neonatal outcomes after asymptomatic SARS-CoV-2 infection in the first trimester of pregnancy: A case report.

The effects of SARS-CoV-2 infection in the first trimester on the pregnant woman and the fetus remain unclear. We describe the complete follow-up of a pregnant woman with asymptomatic SARS-CoV-2 infection in the first trimester. The woman tested positive for SARS-CoV-2 viral RNA in nasopharyngeal swabs in her seventh week of gestation and was admitted to a local hospital for treatment. Although the woman had a BMI above 28 and a total gestational weight gain of 21 kg, no pregnancy complications or severe complications related to SARS-CoV-2 were reported. An ultrasound scan identified no fetal abnormalities at 22 weeks. The pregnancy ended at term (37 weeks), and the newborn's birth weight was 3100 g. Placental insufficiency was revealed by placental histology examination but this appeared not to be related to the SARS-CoV-2 infection. In-situ hybridisation and immunohistochemical tests for SARS-CoV-2 RNA, spike protein 1, and nucleocapsid proteins were negative. However, ACE-2 was positive in samples of the placenta, umbilical cord and fetal membrane. The baby was followed up through to 10 days after birth and grew normally. Our results suggest that asymptomatic SARS-CoV-2 infection in the first trimester of pregnancy might not have significant harmful effects on the mother and the developing fetus. This finding may be of interest to the general public, midwives and general practitioners. However, large population studies are needed to confirm our findings.

Molecular cloning and characterization of hatching enzyme-like gene in the silkworm, Bombyx mori.

Hatching is the important process for the life of the metazoan, in which hatching enzyme (HE) plays a key role. In this paper, we cloned the full-length sequence of hatching enzyme-like cDNA from bluish-silkworm-eggs of Bombyx mori (BmHEL) by the method of in silico cloning, SMART cDNA synthesis and RACE-PCR technique. The BmHEL is 974 bp in length, and contains an ORF of 885 bp, encoding 294 amino acids residues. The deduced amino acid sequence of BmHEL has 30.3-47.1% identities to that of HE identified in the other species. Two similar signature sequences of HE gene family harbor in the BmHEL. The BmHEL gene structure is 6-exon-5-intron, and a promoter region with high scores has been predicted, which harbors some basal elements and some embryo-development related transcription factor binding sites. In the silkworm eggs at different developmental stages during incubation, the BmHEL transcripts can be detected and keep at a low level during the early stages, increase dramatically since 7th day of incubation, and reach to the maximum on 9th day. Change of BmHEL transcripts is in accordance with the process of embryo development and hatching, indicated that it plays an important role in these processes. Moreover, BmHEL transcript can be detected in the midgut and testis at larval stage, suggested that BmHEL may have other biological functions. To the best of our knowledge, this is the first report on HE gene in the Lepidoptera insects and will be helpful to provide a molecular basis for understanding the complicated mechanism underlying silkworm hatching.

BmSd gene regulates the silkworm wing size by affecting the Hippo pathway.

Insect wings are developed from the wing disc during metamorphosis. Bombyx mori, a model lepidopteran insect, loses flight ability after long-term domestication from the wild silkworm, Bombyx mandarina. The mw mutant (u11 strain) shows minute wings compared to wild type (e.g., p50 strain) wings. RNA sequencing analysis previously revealed differential Hippo-pathway-related gene expression between the u11 and p50 strains. The Hippo pathway is an evolutionarily conserved signaling cascade that controls organ size during development in animals. In this study, the function of BmSd which has been characterized as one of the Hippo-pathway-related genes was analyzed for silkworm wing development. We found that mats, warts, and hippo expression levels were higher in u11 compared to p50 wing discs. BmSd (scalloped) expression, which encodes a prominent transcriptional partner to Yorkie (Yki), gradually decreased during the wandering stage in u11, but exhibited the opposite expression pattern in p50. When BmSd was knocked down by small interfering RNA during the wandering stage in the p50 strain, 57.9% of the individuals showed minute wings. Additionally, ex, kibra, and wingless expression levels decreased in the BmSd knockdown mutant. Further, BmSd deletion mediated by clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR-associated protein 9 induced 50% of individuals with minute wings, a phenotype similar to the mw mutant. This result demonstrates that BmSd plays pivotal roles in silkworm wing development. Our results show that the Hippo signaling pathway participates and plays crucial roles in the regulation of silkworm wing development, and our findings provide a basis for further research on B. mori wing development.

Hand-Held Near-Infrared Spectroscopy for Authentication of Fengdous and Quantitative Analysis of Mulberry Fruits.

Recently, miniaturization of Raman, mid-infrared (MIR) and near-infrared (NIR) spectrometers have made substantial progress, and marketing companies predict this segment of instrumentation a significant growth rate within the next few years. This increase will be based on a more frequent implementation for industrial quality and process control and a broader adoption of spectrometers for in-the-field testing, on-site measurements, and every-day-life consumer applications. The reduction in size, however, must not lead to compromises in measurement performance and the hand-held instrumentation will only have a real impact if spectra of comparable quality to laboratory spectrometers can be obtained. The present communication will, on the one hand, explain the instrumental reasons why NIR spectroscopy is presently the most advanced technique regarding miniaturization and on the other hand, it will emphasize the impact of NIR spectroscopy for plant analysis by discussing in some detail a qualitative and a quantitative application example.

lncRNA CASC2/miR­18a­5p axis regulates the malignant potential of nasopharyngeal carcinoma by targeting RBBP8.

Nasopharyngeal carcinoma (NPC) is a prevalent head and neck tumor which has a high mortality rate in Southeast Asia, especially in Southern China. Cancer susceptibility candidate 2 (CASC2) is a newly identified long non­coding RNA (lncRNA) that has been found to play a suppressive role in several types of tumors. However, the expression and functional role of CASC2 in NPC are still unclear. In the present study, using NPC tissues, cells and transplanted mice, we investigated the mechanism of CASC2­mediated regulation of NPC. We showed that the CASC2 level is reduced in NPC tissues and cells. CASC2 downregulation promoted proliferation and inhibited apoptotic cell death in NPC cells. In contrast, CASC2 upregulation inhibited proliferation and increased apoptosis. There were putative binding sites of microRNA (miR)­18a­5p in the promoter of CASC2. The level of miR­18a­5p was upregulated in NPC tissues and cells. We further confirmed that CASC2 could directly bind with miR­18a­5p and inhibit miR­18a­5p expression, using reporter gene and RNA immunoprecipitation assays. miR­18a­5p suppressed CASC2 upregulation­mediated decrease in proliferation and increase in apoptotic cell death. Bioinformatics predicted the putative binding site of miR­18a­5p in the 3' untranslated region of C­terminal binding protein interacting protein (CtIP)/RBBP8. It was further confirmed that miR­18a­5p could directly bind with RBBP8 and inhibit RBBP8 expression. Downregulation of RBBP8 inhibited the anti­miR­18a­5p­mediated increase in apoptosis and decrease in proliferation. Downregulation of CASC2 increased tumor growth, increased the level of miR­18a­5p and decreased RBBP8 expression in vivo. In summary, CASC2 regulates NPC malignancy through modulation of RBBP8 via sponging miR­18a­5p. Our findings highlight the CASC2/miR­18a­5p/RBBP8 axis in NPC pathogenesis and provide new biomarkers and potential targets for the therapy of NPC.

Characterization of CIb1 gene promoter from silkworm, Bombyx mori.

The hemolymph chymotrypsin inhibitor b1 (CIb1) of silkworm, Bombyx mori, plays an important role in innate immunity. In order to study its encoding gene CIb1, five heterogeneous promoter fragments of 844 bp, 682 bp, 516 bp, 312 bp and 82 bp in length were cloned from genomic DNA of the p50 silkworm strain. Characterization of the CIb1 promoter was performed in vitro using the firefly luciferase gene as reporter. The results showed that CIb1 promoter fragments have transcription activities in the B. mori ovary-derived BmN cell line. The 82 bp fragment (-72 to +10 nt) containing the eukaryotic core promoter elements revealed a basic transcription activity. The Bm1 element, upstream the transcription initiation site, showed a positive regulation function to the CIb1 promoter. CIb1 promoter-like fragments from the genomic DNA of the tetra hybrid silkworm SujuxMinghu provided a natural deletion model for the study of the CIb1 promoter. In vitro analysis indicated that the 132 bp fragment from -517 nt to -386 nt upstream of the transcription initiation site strongly suppressed the transcription activity of the CIb1 promoter, suggesting that the 132 bp fragment harbours strong negative cis-acting elements. Infection of Bombyx mori nucleopolyhedrovirus (BmNPV) increased the activity of the CIb1 promoter, having provided another evidence to the function of CIb1 in the innate immunity of silkworm.

N-linked glycosylation of a beetle (Apriona germari) cellulase Ag-EGase II is necessary for enzymatic activity.

We previously reported that the beta-1,4-endoglucanase (EGase) belonging to glycoside hydrolase family (GHF) 45 of the mulberry longicorn beetle, Apriona germari (Ag-EGase II), has three potential N-linked glycosylation sites; these sites are located at amino acid residues 56-59 (NKSG), 99-102 (NSTF), and 237-239 (NYSstop). In the present study, we analyze the functional role of these potential N-linked glycosylation sites. Tunicamycin treatment completely abolished the enzymatic activity of Ag-EGase II. To further elucidate the functional role of the N-linked glycosylation sites in Ag-EGase II, we have assayed the cellulase enzyme activity in Ser58Gln, Thr101Gln, or Ser239Gln mutants. Lack of N-linked glycosylation site at residues 99-102 (NSTF), the site of which is conserved in known beetle GHF 45 cellulases, showed loss of enzyme activity and reduced the molecular mass of the enzyme. In contrast, mutations in Ser58Gln or Ser239Gln affected neither the activity nor the apparent molecular mass of the enzyme, indicating that these sites did not lead to N-linked glycosylation. The present study demonstrates that N-linked glycosylation at residues 99-102 (NSTF), while not essential for secretion, is required for Ag-EGase II enzyme activity.

Molecular cloning, expression, and enzymatic activity of a novel endogenous cellulase from the mulberry longicorn beetle, Apriona germari.

A novel endogenous beta-1,4-endoglucanase (Ag-EGase III) gene belonging to the glycoside hydrolase family (GHF) 5 was cloned from the mulberry longicorn beetle, Apriona germari. The Ag-EGase III gene spans 1061 bp and consists of a single exon coding for 325 amino acid residues. The Ag-EGase III showed 89% protein sequence identity to another beetle, Psacothea hilaris, cellulase belonging to GHF 5. The Ag-EGase III has the potential proton donor and nucleophile amino acids conserved in GHF 5 and two putative N-glycosylation sites. Northern blot and Western blot analyses showed that Ag-EGases were expressed in the gut; Ag-EGase III and Ag-EGase I were expressed in three gut regions, and no Ag-EGase II was found in hindgut, indicating that the foregut and midgut are the prime sites for cellulase synthesis in A. germari larvae. The cDNA encoding Ag-EGase III was expressed as a 47-kDa polypeptide in baculovirus-infected insect Sf9 cells and the enzyme activity of the purified recombinant Ag-EGase III was approximately 1037 U per mg of recombinant Ag-EGase III. The enzymatic property of the purified recombinant Ag-EGase III showed the highest activity at 55 degrees C and pH 6.0, and was stable at 60 degrees C at least for 10 min. In addition, the N-glycosylation of Ag-EGase III was revealed by treatment with tunicamycin of recombinant virus-infected insect Sf9 cells and with endoglycosidase F of purified recombinant Ag-EGase III, demonstrating that the carbohydrate moieties are not necessary for enzyme activity.

Elevated expression of Nrf2 mediates multidrug resistance in CD133+ head and neck squamous cell carcinoma stem cells.

Enhanced expression of the ATP-binding cassette (ABC) transporter protein ABC sub-family G member 2 (ABCG2) in cancer stem cells (CSCs) plays a major role in chemotherapeutic drug efflux, which results in therapy failure and tumor relapse. In addition to downregulating apoptosis in CSCs, it has been reported that the transcriptional upregulation of the redox sensing factor Nrf2 is involved in the upregulation of ABCG2 expression and consequent chemoresistance. The current study investigated the presence of cancer stem-like side population (SP) cells from head and neck squamous cell carcinoma (HNSCC) samples, and evaluated the Nrf2 expression profile and multidrug resistance properties of HNSCC stem cells. Fluorescence-activated cell sorting was used for SP cells detection, while reverse transcription-polymerase chain reaction was used for the analysis of Nrf2 expression. The present study identified ~2.1% SP cells present in HNSCC specimens, which were positive for cluster of differentiation (CD)133 expression and displayed significantly elevated messenger RNA expression of Nrf2, compared with non-SP cells. These data suggest that the ABC transporter ABCG2 is highly upregulated in SP cells, and this results in multidrug resistance. In addition, these CD133+ cells underwent rapid proliferation and exhibited high self-renewal and tumorigenic properties. Taken together, the present findings suggest that elevated expression of Nrf2 mediated drug resistance in HNSCC CSCs, which may be one of the causative factors for cancer treatment failure. Therefore, novel anti-cancer drugs that downregulate the Nrf2 signaling pathway could effectively improve the treatment and survival rate of patients with HNSCC.

Risk for Gestational Diabetes Mellitus and Adverse Birth Outcomes in Chinese Women with Polycystic Ovary Syndrome.

Objective. To examine the association of polycystic ovary syndrome (PCOS) in early pregnancy with gestational diabetes mellitus (GDM) and adverse birth outcomes. Methods. In this retrospective cohort study including 2389 pregnant women, the medical records of 352 women diagnosed with PCOS were evaluated. Outcomes included GDM, preterm birth, low birth weight, macrosomia, and being small and large for gestational age. Multivariable logistic regression models were used to examine the association of the risk for GDM and adverse birth outcomes with PCOS after adjusting for confounders. Results. Women previously diagnosed with PCOS had a higher risk of GDM (adjusted odds ratio [OR] 1.55, 95% confidence interval [CI]: 1.14-2.09). A strong association was seen between PCOS and preterm birth (adjusted OR 1.69, 95% CI: 1.08-2.67). On stratified analysis, the adjusted OR for GDM among women with PCOS undergoing assisted reproductive technology was 1.44 (95% CI: 1.03-1.92) and among women with PCOS who conceived spontaneously was 1.60 (1.18-2.15). No increased risk for other adverse birth outcomes was observed. Conclusions. Women with PCOS were more likely to experience GDM and preterm birth.

Molecular cloning, expression, purification and characterization of a novel cellulase gene (Bh-EGaseI) in the beetle Batocera horsfieldi.

Wood-feeding insects depend heavily on the secretion of a combination of cellulases, mainly endoglucanases and other glucanases such as exoglucanases and xylanases, to achieve efficient digestion of the cellulose of cellulosic materials. In this paper, we report a novel cellulose Bh-EGaseI belonging to the glycoside hydrolase family 45(gh45-1) obtained from the beetle Batocera horsfieldi. The Bh-EGaseI gene spans 714 bp and consists of three exons coding 237 amino acid residues. The cDNA encoding Bh-EGaseI was expressed as 25 KDa in baculovirus-infected Bombyx mori larvae. The expression products of Bh-EGaseI from larval hemolymph showed a specific enzymatic activity of approximately 1030.87 IU per mg. The enzyme was active over a wide range of pH and temperatures; optimal activity was observed at 40 °C and pH 4.0. The effects of ions on Bh-EGaseI activity were also studied, and results indicated that activity decreased to different extents upon addition of ions. Investigations on Bh-EGaseI facilitate their potential application in the production of bioenergy and biomaterials from cellulosic biomass in the future.

Improved 1-Deoxynojirimycin (DNJ) production in mulberry leaves fermented by microorganism.

DNJ, an inhibitor of α-glucosidase, is used to suppress the elevation of postprandial hyperglycemia. In this study, we focus on screening an appropriate microorganism for performing fermentation to improve DNJ content in mulberry leaf. Results showed that Ganoderma lucidum was selected from 8 species and shown to be the most effective in improvement of DNJ production from mulberry leaves through fermentation. Based on single factor and three factor influence level tests by following the Plackett-Burman design, the optimum extraction yield was analyzed by response surface methodology (RSM). The extracted DNJ was determined by reverse-phase high performance liquid chromatograph equipped with fluorescence detector (HPLC-FD). The results of RSM showed that the optimal condition for mulberry fermentation was defined as pH 6.97, potassium nitrate content 0.81% and inoculums volume 2 mL. The extraction efficiency reached to 0.548% in maximum which is 2.74 fold of those in mulberry leaf.

Core promoter regulates the expression of cathepsin B gene in the fat body of Bombyx mori.

Bombyx mori cathepsin B (BmCatB) is involved in the programmed cell death of the fat body during B. mori metamorphosis. For a better understanding of the functional regulatory mechanism, the promoter region of BmCatB in the transcriptional regulation has been identified and analyzed in the present study. BmCatB promoter region performed by the 5' truncation or mutagenesis of EcREs was inserted in the pFA3Luc-A3RL double fluorescence expression vector to activate the fireflies luciferase (FLuc) gene. The results indicated that the dual-luciferase activity of BmCatB gene in the silkworm larval fat body is regulated by the length of promoter. Site-directed mutagenesis of EcRE experiment has shown that the EcREs are up-regulated significantly in the regulation of the BmCatB promoter. A 142bp region (-1165 to -1023) and EcREs are the mainly fat-body tissue-specificity related region and could function as a core promoter element.

Risk factors for proton pump inhibitor refractoriness in Chinese patients with non-erosive reflux disease.

AIM: To analyze risk factors for refractoriness to proton pump inhibitors (PPIs) in patients with non-erosive reflux disease (NERD). METHODS: A total of 256 NERD patients treated with the PPI esomeprazole were enrolled. They were classified into symptom-free and residual symptoms groups according to Quality of Life in Reflux and Dyspepsia (QolRad) scale. All subjects completed questionnaires on psychological status (self-rating anxiety scale; self-rating depression scale) and quality of life scale (Short Form 36). Multivariate analysis was used to determine the predictive factors for PPI responses. RESULTS: According to QolRad, 97 patients were confirmed to have residual reflux symptoms, and the remaining 159 patients were considered symptom free. There were no significant differences between the two groups in lifestyle factors (smoking and alcohol consumption), age, Helicobacter pylori infection, and hiatal hernia. There were significant differences between the two groups in relation to sex, psychological distress including anxiety and depression, body mass index (BMI), and irritable bowel syndrome (IBS) (P < 0.05). Logistic regression analysis found that BMI < 23, comorbid IBS, anxiety, and depression were major risk factors for PPI resistance. Symptomatic patients had a lower quality of life compared with symptom-free patients. CONCLUSION: Some NERD patients are refractory to PPIs and have lower quality of life. Residual symptoms are associated with psychological distress, intestinal disorders, and low BMI.