Characteristic Volatile Organic Compound Analysis of Different Cistanches Based on HS-GC-IMS.

Cistanche is a medicinal and food homologous substance with a long history of consumption and medicinal use in China. In order to further understand the volatile organic compound differences between different cistanches, this study selected oil cistanche, blood cistanche and cistanche tubulosa in Xinjiang for HS-GC-IMS volatile organic compounds, and established the characteristic fingerprints of different cistanches for organic content and characteristic organic compound analysis. PCA and cluster analysis were used to study the similarity between different cistanches. After qualitative analysis, a total of 32 volatile organic compounds were identified, covering aldehydes (17), ketones (5), furans (1), alcohols (5), lactones (1) and esters (3), and the volatile organic compounds between samples a, b and c could be significantly distinguished, affecting the flavor of cistanche itself. It provides a basic theoretical basis for the study of cistanche flavor.

Correction: Feng et al. Nicotinamide Phosphoribosyltransferase (Nampt)/Nicotinamide Adenine Dinucleotide (NAD) Axis Suppresses Atrial Fibrillation by Modulating the Calcium Handling Pathway. Int. J. Mol. Sci. 2020, 21, 4655.

The authors wish to make the following corrections to this paper [...].

Neutrophil Elastase Deficiency Ameliorates Myocardial Injury Post Myocardial Infarction in Mice.

Neutrophils are recruited into the heart at an early stage following a myocardial infarction (MI). These secrete several proteases, one of them being neutrophil elastase (NE), which promotes inflammatory responses in several disease models. It has been shown that there is an increase in NE activity in patients with MI; however, the role of NE in MI remains unclear. Therefore, the present study aimed to investigate the role of NE in the pathogenesis of MI in mice. NE expression peaked on day 1 in the infarcted hearts. In addition, NE deficiency improved survival and cardiac function post-MI, limiting fibrosis in the noninfarcted myocardium. Sivelestat, an NE inhibitor, also improved survival and cardiac function post-MI. Flow cytometric analysis showed that the numbers of heart-infiltrating neutrophils and inflammatory macrophages (CD11b+F4/80+CD206low cells) were significantly lower in NE-deficient mice than in wild-type (WT) mice. At the border zone between intact and necrotic areas, the number of terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive apoptotic cells was lower in NE-deficient mice than in WT mice. Western blot analyses revealed that the expression levels of insulin receptor substrate 1 and phosphorylation of Akt were significantly upregulated in NE-knockout mouse hearts, indicating that NE deficiency might improve cardiac survival by upregulating insulin/Akt signaling post-MI. Thus, NE may enhance myocardial injury by inducing an excessive inflammatory response and suppressing Akt signaling in cardiomyocytes. Inhibition of NE might serve as a novel therapeutic target in the treatment of MI.

Programmed Death-Ligand 2 Deficiency Exacerbates Experimental Autoimmune Myocarditis in Mice.

Programmed death ligand 2 (PD-L2) is the second ligand of programmed death 1 (PD-1) protein. In autoimmune myocarditis, the protective roles of PD-1 and its first ligand programmed death ligand 1 (PD-L1) have been well documented; however, the role of PD-L2 remains unknown. In this study, we report that PD-L2 deficiency exacerbates myocardial inflammation in mice with experimental autoimmune myocarditis (EAM). EAM was established in wild-type (WT) and PD-L2-deficient mice by immunization with murine cardiac myosin peptide. We found that PD-L2-deficient mice had more serious inflammatory infiltration in the heart and a significantly higher myocarditis severity score than WT mice. PD-L2-deficient dendritic cells (DCs) enhanced CD4+ T cell proliferation in the presence of T cell receptor and CD28 signaling. These data suggest that PD-L2 on DCs protects against autoreactive CD4+ T cell expansion and severe inflammation in mice with EAM.

MAIR-II deficiency ameliorates cardiac remodelling post-myocardial infarction by suppressing TLR9-mediated macrophage activation.

Macrophages are fundamental components of inflammation in post-myocardial infarction (MI) and contribute to adverse cardiac remodelling and heart failure. However, the regulatory mechanisms in macrophage activation have not been fully elucidated. Previous studies showed that myeloid-associated immunoglobulin-like receptor II (MAIR-II) is involved in inflammatory responses in macrophages. However, its role in MI is unknown. Thus, this study aimed to determine a novel role and mechanism of MAIR-II in MI. We first identified that MAIR-II-positive myeloid cells were abundant from post-MI days 3 to 5 in infarcted hearts of C57BL/6J (WT) mice induced by permanent left coronary artery ligation. Compared to WT, MAIR-II-deficient (Cd300c2-/- ) mice had longer survival, ameliorated cardiac remodelling, improved cardiac function and smaller infarct sizes. Moreover, we detected lower pro-inflammatory cytokine and fibrotic gene expressions in Cd300c2-/- -infarcted hearts. These mice also had less infiltrating pro-inflammatory macrophages following MI. To elucidate a novel molecular mechanism of MAIR-II, we considered macrophage activation by Toll-like receptor (TLR) 9-mediated inflammation. In vitro, we observed that Cd300c2-/- bone marrow-derived macrophages stimulated by a TLR9 agonist expressed less pro-inflammatory cytokines compared to WT. In conclusion, MAIR-II may enhance inflammation via TLR9-mediated macrophage activation in MI, leading to adverse cardiac remodelling and poor prognosis.

Cohort selection for clinical trials using multiple instance learning.

Identifying patients eligible for clinical trials using electronic health records (EHRs) is a challenging task usually requiring a comprehensive analysis of information stored in multiple EHRs of a patient. The goal of this study is to investigate different methods and their effectiveness in identifying patients that meet specific eligibility selection criteria based on patients' longitudinal records. An unstructured dataset released by the n2c2 cohort selection for clinical trials track was used, each of which included 2-5 records manually annotated to thirteen pre-defined selection criteria. Unlike the other studies, we formulated the problem as a multiple instance learning (MIL) task and compared the performance with that of the rule-based and the single instance-based classifiers. Our official best run achieved an average micro-F score of 0.8765 which was ranked as one of the top ten results in the track. Further experiments demonstrated that the performance of the MIL-based classifiers consistently yield better performance than their single-instance counterparts in the criteria that require the overall comprehension of the information distributed among all of the patient's EHRs. Rule-based and single instance learning approaches exhibited better performance in criteria that don't require a consideration of several factors across records. This study demonstrated that cohort selection using longitudinal patient records can be formulated as a MIL problem. Our results exhibit that the MIL-based classifiers supplement the rule-based methods and provide better results in comparison to the single instance learning approaches.

Nicotinamide Phosphoribosyltransferase (Nampt)/Nicotinamide Adenine Dinucleotide (NAD) Axis Suppresses Atrial Fibrillation by Modulating the Calcium Handling Pathway.

Aging and obesity are the most prominent risk factors for onset of atrial fibrillation (AF). Nicotinamide phosphoribosyltransferase (Nampt) is the rate-limiting enzyme that catalyzes nicotinamide adenine dinucleotide (NAD) activity. Nampt and NAD are essential for maintenance of cellular redox homeostasis and modulation of cellular metabolism, and their expression levels decrease with aging and obesity. However, a role for Nampt in AF is unknown. The present study aims to test whether there is a role of Nampt/NAD axis in the pathogenesis of obesity-induced AF. Male C57BL/6J (WT) mice and heterozygous Nampt knockout (NKO) mice were fed with a normal chow diet (ND) or a high-fat diet (HFD). Electrophysiological study showed that AF inducibility was significantly increased in WT+HFD, NKO+ND, and NKO+HFD mice compared with WT+ND mice. AF duration was significantly longer in WT+HFD and NKO+ND mice and further prolonged in NKO+HFD mice compared with WT+ND mice and the calcium handling pathway was altered on molecular level. Also, treatment with nicotinamide riboside, a NAD precursor, partially restored the HFD-induced AF perpetuation. Overall, this work demonstrates that partially deletion of Nampt facilitated HFD-induced AF through increased diastolic calcium leaks. The Nampt/NAD axis may be a potent therapeutic target for AF.

Quercetin reverses tamoxifen resistance in breast cancer cells.

PURPOSE: To investigate the effect of quercetin on the reversal of tamoxifen resistance in breast cancer cells, and explore the underlying mechanism. METHODS: We established a tamoxifen-resistant breast cancer cell line (MCF-7Ca/TAM-R), and exposed it to different concentrations of quercetin (experimental group 1: 10 µM, group 2: 25 µM, and group 3: 50µM). Each group was further subdivided into 2 subgroups: 1) simultaneous administration of quercetin and 4-hydroxytamoxifen (OHT); 2) sequential administration of quercetin (12-h induction) followed by OHT. No drug exposure and OHT alone were used as controls. We determined cell survival, apoptosis, and expression of ERα (estrogen receptor α) and Her-2 (human epidermal growth factor receptor 2). RESULTS: With increasing dosage of quercetin, significant decrease in proliferation and increase in apoptosis was observed. Low concentrations of quercetin (10 µM) had no effects. We found no significant difference between simultaneous and sequential mode of drug administration. Further, with increasing dosage of quercetin, we observed a gradual reduction in Her-2 expression and upregulation of ERα. Again, no difference in Her-2 and ERα protein levels between simultaneous and sequential drug administration was noticed. CONCLUSIONS: Proliferation inhibition and apoptosis in MCF-7Ca/TAM-R cells increase with increasing dosage of quercetin. This suggests that quercetin can reverse tamoxifen resistance in breast cancer cells. The underlying mechanism likely involves upregulation of ERα combined with downregulation of Her-2. However, this effect is independent of whether quercetin and tamoxifen are administered simultaneously or sequentially.

Research Progress on New Functions of Animal and Plant Proteins.

Protein is composed of peptides, essential nutrients for human survival and health, and the easy absorption of peptides further promotes human health. According to the source of the protein, it can be divided into plants, animals, and micro-organisms, which have important physiological effects on the health of the body, especially in enhancing immunity. The most widely used raw materials are animal protein and plant protein, and the protein composition formed by the two in a certain proportion is called "double protein". In recent years, China's State Administration for Market Regulation has issued an announcement on the "Implementation Rules for the Technical Evaluation of New Functions and Products of Health Foods (Trial)", which provides application conditions and listing protection for the research and development of new functions of health foods. At present, some researchers and enterprises have begun to pay attention to the potential of animal and plant proteins to be used in new functions. In this article, the research progress of animal and plant proteins in the new functions of Chinese health food is reviewed in detail, and suggestions for future research on animal and plant proteins are put forward.

The important role of starch fine molecular structures in starch gelatinization property with addition of sugars/sugar alcohols.

The relationship between the fine structure of starch and its gelatinization properties is not well studied, particularly in relation to the influence of sugar or sugar alcohol. In this study, seven starches with distinct molecular structures were investigated to determine how different sugars and sugar alcohols affect their gelatinization properties. The inclusion of sugars and sugar alcohols resulted in a significant elevation of starch gelatinization temperatures (∼ 8 °C), especially with sucrose, isomaltose and isomalt. Nevertheless, the influence of these sugars/ sugar alcohols on the gelatinization temperature range and enthalpy change varied depending on the particular starch varieties. According to the correlation analysis, sugars and sugar alcohols mainly exert their impact on the starch gelatinization temperature range and enthalpy change by possibly interacting with amylose chains possessing a degree of polymerization ranging from 100 to 1000 (p < 0.05) and inhibiting the amylose leaching during gelatinization. These findings help a better understanding of the complex relationship between starch fine structure and gelatinization properties under the influence of sugars and sugar alcohols.

Therapeutic Effects of Natural Products on Liver Cancer and Their Potential Mechanisms.

Liver cancer ranks third globally among causes of cancer-related deaths, posing a significant public health challenge. However, current treatments are inadequate, prompting a growing demand for novel, safe, and effective therapies. Natural products (NPs) have emerged as promising candidates in drug development due to their diverse biological activities, low toxicity, and minimal side effects. This paper begins by reviewing existing treatment methods and drugs for liver cancer. It then summarizes the therapeutic effects of NPs sourced from various origins on liver cancer. Finally, we analyze the potential mechanisms of NPs in treating liver cancer, including inhibition of angiogenesis, migration, and invasion; regulation of the cell cycle; induction of apoptosis, autophagy, pyroptosis, and ferroptosis; influence on tumor metabolism; immune regulation; regulation of intestinal function; and regulation of key signaling pathways. This systematic review aims to provide a comprehensive overview of NPs research in liver cancer treatment, offering a foundation for further development and application in pharmaceuticals and functional foods.

Metformin inhibits proliferation and promotes apoptosis of HER2 positive breast cancer cells by downregulating HSP90.

PURPOSE: To investigate the effects and the possible molecular mechanisms of metformin on HER2 positive breast cancer cells. METHODS: SK-BR-3 HER2 positive breast cancer cells were treated with different concentrations of metformin. The growth inhibitory rate of the cells was calculated by MTT assay, apoptosis was detected by flow cytometry, and the expression level of heat shock protein 90 (HSP90) was performed by Western blot analysis. A control group consisted of cells treated with PBS. RESULTS: With increased concentrations of metformin, cell growth inhibitory rates increased. The growth inhibitory rates with 0.5 mM, 2mM or 8mM metformin were significantly higher compared with the control group (p<0.05). Apoptosis in the metformin treated cells was also significantly higher compared with the control group (p=0.003). The expression level of HSP90 in the metformin group was significantly lower than that in the control group. CONCLUSION: Metformin can inhibit the proliferation and promote apoptosis of HER2 positive breast cancer cells,which is maybe related to inhibition of HSP90.

Formation, Structural Characterization, and Functional Properties of Corn Starch/Zeaxanthin Composites.

Zeaxanthin is a natural xanthophyll carotenoid and the main macular pigment that protects the macula from light-initiated oxidative damage, but it has poor stability and low bioavailability. Absorption of this active ingredient into starch granules as a carrier can be used to improve both zeaxanthin stability and controlled release. Optimization using three variables judged important for optimizing the system (reaction temperature of 65 °C, starch concentration of 6%, and reaction time of 2 h) was conducted for incorporation of zeaxanthin into corn starch granules, aiming for high zeaxanthin content (2.47 mg/g) and high encapsulation efficiency (74%). Polarized-light microscopy, X-ray diffraction, differential scanning calorimetry, and Fourier transform infrared spectroscopy showed that the process partially gelatinized corn starch; additionally, it showed the presence of corn starch/zeaxanthin composites, with the zeaxanthin successfully trapped in corn starch granules. The half-life time of zeaxanthin in corn starch/zeaxanthin composites increased to 43 days as compared with that of zeaxanthin alone (13 days). The composites show a rapid increase in zeaxanthin release with in vitro intestinal digestion, which is favorable for possible use in living systems. These findings could have application in designing effective starch-based carriers of this bioactive ingredient with enhanced storage stability and improved intestines-targeted controlled-release delivery.

Analysis of the active ingredients and health applications of cistanche.

Cistanche is a tonic Chinese medicine commonly used in traditional Chinese medicine, with 2016, CFSA through the alxa desert cistanche safety evaluation, cistanche began to officially enter the food field. At present, the research on cistanche mainly focuses on the extraction, isolation and purification and pharmacological effects, and its pharmacological effects such as neuroprotective effects, immunomodulation, antioxidant anticancer and hepatoprotective liver protection have attracted the attention of researchers. This review mainly reviews the research status, chemical composition and health benefits, analyzes its application prospects in food, and aims to provide certain theoretical support for the safe application of cistanche in functional food.

Effect of total glycosides of Cistanche deserticola on the energy metabolism of human HepG2 cells.

To study the anti-tumor effect of Cistanche deserticola Y. Ma, HepG2 cells were treated with 0, 3.5, 10.5, 21, 31.5, and 42 µg/ml of total glycosides (TG) from Cistanche deserticola. The HepG2 cell survival rate and 50% inhibition concentration (IC50) were detected using the CCK-8 method, and the level of reactive oxygen species (ROS) was detected by using a DCFH-DA fluorescence probe. Finally, a Seahorse XFe24 energy analyzer (Agilent, United States) was used to detect cell mitochondrial pressure and glycolytic pressure. The results showed that TG could reduce the survival rate of HepG2 cells and that the IC50 level was 35.28 µg/ml. With increasing TG concentration, the level of ROS showed a concentration-dependent upward trend. Energy metabolism showed that each dose group of TG could significantly decline the mitochondrial respiratory and glycolytic functions of HepG2 cells. In conclusion, TG could significantly inhibit the mitochondrial respiration and glycolysis functions of HepG2 cells, increase the level of ROS, and inhibit cell proliferation. Thus, this experiment pointed out that Cistanche deserticola can be used as a source of anti-cancer foods or drugs in the future. However, further studies on its mechanisms and clinical applications are needed.

The Emulsifying Properties, In Vitro Digestion Characteristics and Storage Stability of High-Pressure-Homogenization-Modified Dual-Protein-Based Emulsions.

The droplet size, zeta potential, interface protein adsorption rate, physical stability and microrheological properties of high-pressure-homogenization (HPH)-modified, dual-protein-based whey-soy (whey protein isolate-soy protein isolate) emulsions containing different oil phase concentrations (5%, 10% and 15%; w/w) were compared in this paper. The in vitro digestion characteristics and storage stability of the dual-protein emulsions before and after HPH treatment were also explored. The results show that with an increase in the oil phase concentration, the droplet size and interface protein adsorption rate of the untreated dual-protein emulsions increased, while the absolute value of the zeta potential decreased. When the oil phase concentration was 10% (w/w), HPH treatment could significantly reduce the droplet size of the dual-protein emulsion, increase the interface protein adsorption rate, and improve the elasticity of the emulsion. Compared with other oil phase concentrations, the physical stability of the dual-protein emulsion containing a 10% (w/w) oil phase concentration was the best, so the in vitro digestion characteristics and storage stability of the emulsions were studied. Compared with the control group, the droplet size of the HPH-modified dual-protein emulsion was significantly reduced after gastrointestinal digestion, and the in vitro digestibility and release of free amino groups both significantly increased. The storage stability results show that the HPH-modified dual-protein emulsion showed good stability under different storage methods, and the storage stability of the steam-sterilized dual-protein emulsion stored at room temperature was the best. These results provide a theoretical basis for the development of new nutritional and healthy dual-protein liquid products.

The Effect of High Pressure Homogenization on the Structure of Dual-Protein and Its Emulsion Functional Properties.

It has been proven that high-pressure homogenization (HPH) could improve the functional properties of proteins by modifying their structure. This study researched the effect of HPH on the structural and functional properties of whey-soy dual-protein (Soy Protein Isolation-Whey Protein Isolation, SPI-WPI). Different protein solution samples were treated with HPH at 30, 60, 90, 120 and 150 MPa, and the structure changed under different pressures was analyzed by measuring particle size, zeta potential, Fourier infrared spectrum (FTIR), fluorescence spectrum and scanning electron microscope (SEM). The results showed that HPH significantly reduced the particle size of SPI-WPI, changed the secondary and tertiary structures and improved the hydrophobic interaction between molecules. In addition, HPH significantly improved the solubility and emulsification of all proteins, and the improvement effect on SPI-WPI was significantly better than SPI and WPI. It was found that SPI-WPI treated with 60 MPa had the best physicochemical properties. Secondly, we researched the effect of HPH by 60 MPa on the emulsion properties of SPI-WPI. In this study, the SPI-WPI had the lowest surface tension compared to a single protein after HPH treatment. The emulsion droplet size was obviously decreased, and the elastic properties and physical stability of SPI-WPI emulsion were significantly enhanced. In conclusion, this study will provide a theoretical basis for the application of HPH in modifying the structure of dual-protein to improve its development and utilization in liquid specialty food.

Contrast-enhanced ultrasound of granular cell tumor in breast: A case report with review of the literature.

Granular cell tumor is an infrequent, predominantly benign tumor originating from Schwann cells. Granular cell tumor of the breast (GCTB) can simulate breast malignant carcinoma on the clinical assessment. We herein present a rare case of GCTB which recurred in the contralateral breast. We believe the contrast-enhanced ultrasound (CEUS) findings of GCTB have never been described. The high similarity of breast malignant carcinoma makes its differential diagnosis difficult on the clinical and radiological features. In this report, we present the CEUS findings from a rare case of GCTB, explore the possible value of CEUS in differential diagnosis between benign breast lesions and malignant ones, and briefly review the literature.

Safety and immunogenicity of 2009 pandemic influenza A H1N1 vaccines in China: a multicentre, double-blind, randomised, placebo-controlled trial.

BACKGROUND: The current influenza pandemic calls for a safe and effective vaccine. We assessed the safety and immunogenicity of eight formulations of 2009 pandemic influenza A H1N1 vaccine produced by ten Chinese manufacturers. METHODS: In this multicentre, double-blind, randomised trial, 12 691 people aged 3 years or older were recruited in ten centres in China. In each centre, participants were stratified by age and randomly assigned by a random number table to receive one of several vaccine formulations or placebo. The study assessed eight formulations: split-virion formulation containing 7.5 microg, 15 microg, or 30 microg haemagglutinin per dose, with or without aluminium hydroxide adjuvant, and whole-virion formulation containing 5 microg or 10 microg haemagglutinin per dose, with adjuvant. All formulations were produced from the reassortant strain X-179A (A/California/07/2009-A/PR/8/34). We analysed the safety (adverse events), immunogenicity (geometric mean titre [GMT] of haemagglutination inhibition antibody), and seroprotection (GMT >or=1:40) of the formulations. Analysis was by per protocol. Two sites registered their trial with ClinicalTrials.gov, numbers NCT00956111 and NCT00975572. The other eight studies were registered with the State Food and Drug Administration of China. FINDINGS: 12 691 participants received the first dose on day 0, and 12 348 participants received the second dose on day 21. The seroprotection rate 21 days after the first dose of vaccine ranged from 69.5% (95% CI 65.9-72.8) for the 7.5 microg adjuvant split-virion formulation to 92.8% (91.9-93.6) for the 30 microg non-adjuvant split-virion formulation. The seroprotection rate was 86.5% (796 of 920; 84.1-88.7) in recipients of one dose of the 7.5 microg non-adjuvant split-virion vaccine compared with 9.8% (140 of 1432; 8.3-11.4) in recipients of placebo (p<0.0001). One dose of the 7.5 microg non-adjuvant split-virion vaccine induced seroprotection in 178 of 232 children (aged 3 years to <12 years; 76.7%, 70.7-82.0), 211 of 218 adolescents (12 years to <18 years; 96.8%, 93.5-98.7), 289 of 323 adults (18-60 years; 89.5%, 85.6-92.6), and 118 of 147 adults older than 60 years (80.3%, 72.9-86.4), meeting the European Union's licensure criteria for seroprotection in all age-groups. In children, a second dose of the 7.5 microg formulation increased the seroprotection rate to 97.7% (215 of 220, 94.8-99.3). Adverse reactions were mostly mild or moderate, and self-limited. Severe adverse effects occurred in 69 (0.6%, 0.5-0.8) recipients of vaccine compared with one recipient (0.1%, 0-0.2) of placebo. The most common severe adverse reaction was fever, which occurred in 25 (0.22%; 0.14-0.33) recipients of vaccine after the first dose and four (0.04%; 0.01-0.09) recipients of vaccine after the second dose compared with no recipients of placebo after either dose. INTERPRETATION: One dose of non-adjuvant split-virion vaccine containing 7.5 microg haemagglutinin could be promoted as the formulation of choice against 2009 pandemic influenza A H1N1 for people aged 12 years or older. In children (aged <12 years), two 7.5 mug doses might be needed. FUNDING: Sinovac Biotech, Hualan Biological Bacterin, China National Biotec Group, Beijing Tiantan Biological Products, Changchun Institute of Biological Products, Changchun Changsheng Life Sciences, Jiangsu Yanshen Biological Technology Stock, Zhejiang Tianyuan Bio-Pharmaceutical, Lanzhou Institute of Biological Products, Shanghai Institute of Biological Products, and Dalian Aleph Biomedical.

HS-GC-IMS detection of volatile organic compounds in Acacia honey powders under vacuum belt drying at different temperatures.

Honey is a commodity of great nutritional value, but deep-processed honey products are uncommon. Herein, we used vacuum belt dryer to dry Acacia honey at 60°C, 70°C, and 80°C, prepared it into powder, and analyzed its volatile compound differences. We established HS-GC-IMS method to detect the volatile organic compounds (VOCs) of these three Acacia honey powders (AHPs). In total, 77 peaks were detected, and 23 volatile compounds were identified, including eight aldehydes, six ketones, three furans, one alcohol, one phenol, one lactone, one ester, one acid, and one nitrile. Moreover, principal component analysis (PCA) and fingerprint similarity analysis based on the Euclidean distance distinguished the three heating temperature treatments. Clearly, it was concluded that there are significant differences in volatile substances at different tested temperatures, and when the AHP was incubated at 80°C, more volatile compounds were detected.