Relationship between sarcopenia and fatty liver in middle-aged and elderly patients with type 2 diabetes mellitus.

OBJECTIVE: In this study, we investigated the relationship between sarcopenia and fatty liver in middle-aged and elderly patients diagnosed with type 2 diabetes mellitus (T2DM) to provide a theoretical foundation for the prevention and treatment of sarcopenia. METHODS: A total of 282 patients diagnosed with T2DM aged 50 and older and were admitted to the Endocrinology Department of Xin Medical University First Affiliated Hospital between December 2021 and February 2023, were selected. Body mass index (BMI), and limb and trunk muscle mass of the patients were measured, and data were collected. Patients were grouped based on the sarcopenia diagnostic criteria. All study participants underwent the same physical examinations and laboratory tests. The relationship between the onset of sarcopenia and fatty liver in middle-aged and elderly patients diagnosed with T2DM was then investigated using statistical analysis. RESULTS: Comparing the sarcopenia group to the non-sarcopenia group revealed statistically significant variations in gender, BMI, fatty liver prevalence rate, uric acid (UA), alanine aminotransferase (ALT), blood glucose, blood lipid associated indicators, and limb skeletal muscle content. There were, however, no statistically significant differences in age, disease duration, hypertension, smoking, or alcohol intake. There was a positive correlation between BMI, UA, fasting c-peptide, and Appendicular Skeletal Muscle Index (ASMI). Higher levels of BMI, ASMI, and UA were identified as protective variables against sarcopenia by multifactorial logistic regression analysis. CONCLUSION: Higher levels of BMI, ASMI, and UA can greatly reduce skeletal muscle atrophy in patients with T2DM. Patients with a fatty liver may be less vulnerable to sarcopenia. There is little evidence, however, that a fatty liver works as a preventive factor against sarcopenia.

Comparison of the effect of tea shoots during different seasons in Arma chinensis (Hemiptera: Pentatomidae) reared on Ectropis grisescens (Lepidoptera: Geometridae) pupae.

In this study, we compared the growth, development, and fecundity of Arma chinensis (Fallou) reared on pupae of the geometrid Ectropis grisescens Warren fed on tea shoots during different seasons of the year. The raw data on life history were analyzed using the age-stage, 2-sex life table. When reared on spring or winter geometrid pupae, the duration of the immature stage of A. chinensis was significantly longer than in those produced during the summer or autumn. The survival rate of immature A. chinensis reared on autumn geometrid pupae was significantly lower compared to other treatments. Reproductive diapause was observed in adult A. chinensis reared on winter geometrid pupae. The adult preoviposition period (APOP), total preoviposition period (TPOP), and total longevity were significantly longer in A. chinensis reared on winter pupae than in the other treatments. The fecundity of A. chinensis reared on spring geometrid pupae was significantly lower than in the other treatments. The higher intrinsic rate of increase of the A. chinensis reared on summer pupae (r = 0.0966 day-1) and autumn pupae (r = 0.0983 day-1) resulted in higher fecundity, shorter immature duration, and shorter TPOP compared to the winter and spring populations. These findings can be utilized to enhance and sustain biological control of E. grisescens in tea plantations.

[Blocking Effects of Foliar Conditioners on Cadmium, Arsenic, and Lead Accumulation in Wheat Grain in Compound-contaminated Farmland].

Heavy metal contamination of soil has become a hot issue of social concern due to its impact on the safety of agricultural products in recent years. Wheat is one of the most dominant staple food crops worldwide and has become a major source of toxic metals in human diets. Foliar application was considered to be a more efficient and economical method of heavy metal remediation. Field experiments were carried out in Cd-, As-, and Pb-contaminated farmland soils. The effects of foliar conditioners on the accumulation of Cd, As, and Pb in wheat grains were investigated after being sprayed with Zn (0.2% ZnSO4), Mg (0.4% MgSO4), and Mn (0.2% MnSO4) separately and in combination. Thus, the effective foliar conditioners were selected to block the accumulation of Cd, As, and Pb in wheat grains grown in combined heavy metal-contaminated farmland in north China. The results showed that, compared with that in the control, the Cd, As, and Pb contents in wheat grains of the Zn+Mg+Mn foliar treatment were significantly decreased by 18.96%, 23.87%, and 51.31%, respectively, and TFgrain/straw decreased by 14.62%, 27.73%, and 47.70%, respectively. Thus, spraying the compound foliar conditioner of Zn+Mg+Mn could effectively reduce heavy metal accumulation in wheat grains through inhibition translocation of those metals from stem leaves to grain. In addition, the results indicated that Cd and As were mainly distributed at the central endosperm (34.08%-37.08%), whereas Pb was primarily distributed at the pericarp and seed coat (27.78%) of the wheat grain. Compared with that in the control, spraying the compound foliar conditioner of Zn+Mg+Mn extremely decreased Cd and As accumulation in the aleurone layer of the wheat grain by 81.10% and 82.24%, respectively. Except for the pericarp, seed coat, and central endosperm layers, the Pb content in each grain layer was dramatically decreased by 42.85% to 91.15%. There was only a significant negative correlation between heavy metal content and Zn content in the aleurone layer (P2) of wheat flour. In summary, the accumulation of Cd, As, and Pb in wheat grains, especially in the aleurone layer, could be effectively reduced by foliar conditioner application at the jointing, booting, and early filling stages of wheat, separately. Furthermore, besides the foliar treatment, removing wheat bran to reduce Cd contamination in wheat grains is highly recommended to ensure the safe production of wheat.

Bifunctional Electrocatalysts for Overall and Hybrid Water Splitting.

Electrocatalytic water splitting driven by renewable electricity has been recognized as a promising approach for green hydrogen production. Different from conventional strategies in developing electrocatalysts for the two half-reactions of water splitting (e.g., the hydrogen and oxygen evolution reactions, HER and OER) separately, there has been a growing interest in designing and developing bifunctional electrocatalysts, which are able to catalyze both the HER and OER. In addition, considering the high overpotentials required for OER while limited value of the produced oxygen, there is another rapidly growing interest in exploring alternative oxidation reactions to replace OER for hybrid water splitting toward energy-efficient hydrogen generation. This Review begins with an introduction on the fundamental aspects of water splitting, followed by a thorough discussion on various physicochemical characterization techniques that are frequently employed in probing the active sites, with an emphasis on the reconstruction of bifunctional electrocatalysts during redox electrolysis. The design, synthesis, and performance of diverse bifunctional electrocatalysts based on noble metals, nonprecious metals, and metal-free nanocarbons, for overall water splitting in acidic and alkaline electrolytes, are thoroughly summarized and compared. Next, their application toward hybrid water splitting is also presented, wherein the alternative anodic reactions include sacrificing agents oxidation, pollutants oxidative degradation, and organics oxidative upgrading. Finally, a concise statement on the current challenges and future opportunities of bifunctional electrocatalysts for both overall and hybrid water splitting is presented in the hope of guiding future endeavors in the quest for energy-efficient and sustainable green hydrogen production.

Trimethylamine N-oxide impairs ß-cell function and glucose tolerance.

ß-Cell dysfunction and ß-cell loss are hallmarks of type 2 diabetes (T2D). Here, we found that trimethylamine N-oxide (TMAO) at a similar concentration to that found in diabetes could directly decrease glucose-stimulated insulin secretion (GSIS) in MIN6 cells and primary islets from mice or humans. Elevation of TMAO levels impairs GSIS, ß-cell proportion, and glucose tolerance in male C57BL/6 J mice. TMAO inhibits calcium transients through NLRP3 inflammasome-related cytokines and induced Serca2 loss, and a Serca2 agonist reversed the effect of TMAO on ß-cell function in vitro and in vivo. Additionally, long-term TMAO exposure promotes ß-cell ER stress, dedifferentiation, and apoptosis and inhibits ß-cell transcriptional identity. Inhibition of TMAO production improves ß-cell GSIS, ß-cell proportion, and glucose tolerance in both male db/db and choline diet-fed mice. These observations identify a role for TMAO in ß-cell dysfunction and maintenance, and inhibition of TMAO could be an approach for the treatment of T2D.

Activation of GPR81 by lactate drives tumour-induced cachexia.

Cachexia affects 50-80% of patients with cancer and accounts for 20% of cancer-related death, but the underlying mechanism driving cachexia remains elusive. Here we show that circulating lactate levels positively correlate with the degree of body weight loss in male and female patients suffering from cancer cachexia, as well as in clinically relevant mouse models. Lactate infusion per se is sufficient to trigger a cachectic phenotype in tumour-free mice in a dose-dependent manner. Furthermore, we demonstrate that adipose-specific G-protein-coupled receptor (GPR)81 ablation, similarly to global GPR81 deficiency, ameliorates lactate-induced or tumour-induced adipose and muscle wasting in male mice, revealing adipose GPR81 as the major mediator of the catabolic effects of lactate. Mechanistically, lactate/GPR81-induced cachexia occurs independently of the well-established protein kinase A catabolic pathway, but it is mediated by a signalling cascade sequentially activating Gi-Gßγ-RhoA/ROCK1-p38. These findings highlight the therapeutic potential of targeting GPR81 for the treatment of this life-threatening complication of cancer.

Contrast-enhanced ultrasound for the preoperative prediction of pathological characteristics in breast cancer.

Objective: We aimed to investigate the value of contrast-enhanced ultrasound (CEUS) in the preoperative prediction of the histological grades and molecular subtypes of breast cancer. Methods: A total of 183 patients with pathologically confirmed breast cancer were included. Contrast enhancement patterns and quantitative parameters were compared in different groups. The receiver operating characteristic (ROC) curve was used to analyze the efficacy of CEUS in the preoperative prediction of pathological characteristics, including histologic grade and molecular subtypes. Results: Heterogeneous enhancement, perfusion defects, and peripheral radial vessels were mostly observed in higher histologic grade (grade III) breast cancer. Heterogeneous enhancement and perfusion defect were the most effective indicators for grade III breast cancer, with the areas under the ROC curve of 0.768 and 0.756, respectively. There were significant differences in the enhancement intensity, post-enhanced margin, perfusion defects, and peripheral radial vessel among the different molecular subtypes of breast cancer (all P < 0.01). Perfusion defects and clear edge after enhancement were the best qualitative criteria for the diagnosis of HER-2 overexpressed and triple-negative breast cancers, and the corresponding areas under the ROC curves were 0.804 and 0.905, respectively. There were significant differences in PE, WiR, WiPI, and WiWoAUC between grade III vs grade I and II breast cancer (P < 0.05). PE, WiR, WiPI, and WiWoAUC had good efficiency in the diagnosis of high-histologic-grade breast cancer. PE had the highest diagnostic efficiency in Luminal A, while WiPI had the highest diagnostic efficiency in Luminal B subtype breast cancer, and the areas under the ROC curve were 0.825 and 0.838, respectively. WiWoAUC and WiR were the most accurate parameters for assessing triple-negative subtype breast cancers, and the areas under the curve were 0.932 and 0.922, respectively. Conclusion: Qualitative and quantitative perfusion analysis of contrast-enhanced ultrasound may be useful in the non-invasive prediction of the histological grade and molecular subtypes of breast cancers.

Ultrasound contrast-enhanced diagnosis of testicular Leydig cell tumor: A case report and literature review.

Leydig cell tumor (LCT) is a rare testicular tumor. We report a case of an elderly male patient who discovered a left testicular mass during a regular health examination four years ago. The patient did not experience any significant discomfort and opted for regular follow-up visits. During the most recent visit, we performed routine ultrasound and contrast-enhanced ultrasound (CEUS) examinations. By observing the lesion's location, echogenicity, margins, vascular distribution, as well as the rapid enhancement and slow washout characteristics on contrast-enhanced ultrasound, we arrived at a diagnosis of LCT. Subsequently, the patient underwent left inguinal orchiectomy. Postoperative pathology and immunohistochemistry confirmed the diagnosis of LCT. Additionally, we conducted a comprehensive review of LCT-related literature from PubMed and SCOPUS, summarizing the clinical features, follow-up duration, prognosis, and ultrasound characteristics associated with LCT.

Depression, anxiety, lower sleep quality and social support in square cabin hospitals during Shanghai's COVID-19 lockdown, China.

Objectives: To investigate and compare the associated factors of depression, anxiety, and other psychological differences between patients with Corona Virus Disease 2019 quarantined in square cabin hospitals (SCH) and isolation wards (IW) in China. Methods: Cluster sampling method was performed during Shanghai's Two-Month Lockdown in 2022. Hospital Anxiety and Depression Scale Depression subscale (HADS-D), 7-tiem Generalized Anxiety Disorder Scale (GAD-7), Pittsburgh sleep quality index (PSQI), and Perceived Social Support Scale (PSSS) were used to investigate psychological differences. Results: The HADS-D and GAD-7 scores of SCH patients were significantly higher than those in IW (p < 0.001; p = 0.0295). Sleep latency (SCH-IW = -3.76, p < 0.001), sleep duration (SCH-IW = -2.22, p < 0.05), habitual sleep efficiency (SCH-IW = -4.11, p < 0.001), sleep disturbance (SCH-IW = -3.59, p < 0.001) and use of sleep medication (SCH-IW = -5.18, p < 0.001) of SCH patients were significantly worse. Depression was the main emotional problem of quarantined patients. Patients in SCH had lower social support. Sleep disorders and the lowest oxygen saturation ≤ 93% were risk factors for depression, while social support and child status were protective factors. Myalgia and constipation were risk factors for anxiety, while marital status was the protective factor. Conclusion: Patients quarantined in SCH had higher risks of depression and anxiety, lower sleep quality and social support. Somatic discomfort and sleep disorders exacerbated depression and anxiety, which could be ameliorated by social support and taken into consideration in future SCH construction.

HaMYBA-HabHLH1 regulatory complex and HaMYBF fine-tune red flower coloration in the corolla of sunflower (Helianthus annuus L.).

Sunflowers are well-known ornamental plants, while sunflowers with red corolla are rare and the mechanisms underlying red coloration remain unclear. Here, a comprehensive analysis of metabolomics and transcriptomics on flavonoid pathway was performed to investigate the molecular mechanisms underlying the differential color formation between red sunflower Pc103 and two yellow sunflowers (Yr17 and Y35). Targeted metabolomic analysis revealed higher anthocyanin levels but lower flavonol content in Pc103 compared to the yellow cultivars. RNA-sequencing and phylogenetic analysis identified multiple genes involved in the flavonoid pathway, including series of structural genes and three MYB and bHLH genes. Specifically, HaMYBA and HabHLH1 were up-regulated in Pc103, whereas HaMYBF exhibited reduced expression. HaMYBA was found to interact with HabHLH1 in vivo and in vitro, while HaMYBF does not. Transient expression analysis further revealed that HabHLH1 and HaMYBA cooperatively regulate increased expression of dihydroflavonol 4-reductase (DFR), leading to anthocyanin accumulation. On the other hand, ectopic expression of HaMYBF independently modulates flavonol synthase (FLS) expression, but hindered anthocyanin production. Collectively, our findings suggest that the up-regulation of HaMYBA and HabHLH1, as well as the down-regulation of HaMYBF, contribute to the red coloration in Pc103. It offers a theoretical basis for improving sunflower color through genetic engineering.

Diazo-carboxyl Click Derivatization Enables Sensitive Analysis of Carboxylic Acid Metabolites in Biosamples.

Carboxylic acids are central metabolites in bioenergetics, signal transduction, and post-translation protein regulation. However, the quantitative analysis of carboxylic acids as an indispensable part of metabolomics is prohibitively challenging, particularly in trace amounts of biosamples. Here we report a diazo-carboxyl/hydroxylamine-ketone double click derivatization method for the sensitive analysis of hydrophilic, low-molecular-weight carboxylic acids. In general, our method renders a 5- to 2000-fold higher response in mass spectrometry along with improved chromatographic separation. With this method, we presented the near-single-cell analysis of carboxylic acid metabolites in 10 mouse egg cells before and after fertilization. Malate, fumarate, and ß-hydroxybutyrate were found to decrease after fertilization. We also monitored the isotope labeling kinetics of carboxylic acids inside adherent cells cultured in 96-well plates during drug treatment. Finally, we applied this method to plasma or serum samples (5 µL) collected from mice and humans under pathological and physiological conditions. The double click derivatization method paves a way toward single-cell metabolomics and bedside diagnostics.

Paraboeazunyiensis (Gesneriaceae), a new species from north Guizhou, China.

A new lithophytic species, Paraboeazunyiensis T.Deng, F.Wen & R.B.Zhang (Gesneriaceae), inhabiting Karst rocks in northern Guizhou, China, is introduced and depicted in this study. It bears a resemblance to P.crassifolia (Hemsl.) B.L. Burtt, yet is distinguishable by its shorter filaments and staminodes, triangular ovate calyx segments, and ovaries surpassing the styles in length. Moreover, the phylogenetic tree constructed from nuclear DNA (ITS) and plastid DNA (trnL-F) data firmly support the differentiation of this novel species from P.crassifolia.

Succinate dehydrogenase is essential for epigenetic and metabolic homeostasis in hearts.

A hallmark of heart failure is a metabolic switch away from fatty acids ß-oxidation (FAO) to glycolysis. Here, we show that succinate dehydrogenase (SDH) is required for maintenance of myocardial homeostasis of FAO/glycolysis. Mice with cardiomyocyte-restricted deletion of subunit b or c of SDH developed a dilated cardiomyopathy and heart failure. Hypertrophied hearts displayed a decrease in FAO, while glucose uptake and glycolysis were augmented, which was reversed by enforcing FAO fuels via a high-fat diet, which also improved heart failure of mutant mice. SDH-deficient hearts exhibited an increase in genome-wide DNA methylation associated with accumulation of succinate, a metabolite known to inhibit DNA demethylases, resulting in changes of myocardial transcriptomic landscape. Succinate induced DNA hypermethylation and depressed the expression of FAO genes in myocardium, leading to imbalanced FAO/glycolysis. Inhibition of succinate by α-ketoglutarate restored transcriptional profiles and metabolic disorders in SDH-deficient cardiomyocytes. Thus, our findings reveal the essential role for SDH in metabolic remodeling of failing hearts, and highlight the potential of therapeutic strategies to prevent cardiac dysfunction in the setting of SDH deficiency.

Bromine-Enhanced Generation and Epoxidation of Ethylene in Tandem CO2 Electrolysis Towards Ethylene Oxide.

The indirect electro-epoxidation of ethylene (C2 H4 ), produced from CO2 electroreduction (CO2 R), holds immense promise for CO2 upcycling to valuable ethylene oxide (EO). However, this process currently has a mediocre Faradaic efficiency (FE) due to sluggish formation and rapid dissociation of active species, as well as reductive deactivation of Cu-based electrocatalysts during the conversion of C2 H4 to EO and CO2 to C2 H4 , respectively. Herein, we report a bromine-induced dual-enhancement strategy designed to concurrently promote both C2 H4 -to-EO and CO2 -to-C2 H4 conversions, thereby improving EO generation, using single-atom Pt on N-doped CNTs (Pt1 /NCNT) and Br- -bearing porous Cu2 O as anode and cathode electrocatalysts, respectively. Physicochemical characterizations including synchrotron X-ray absorption, operando infrared spectroscopy, and quasi in situ Raman spectroscopy/electron paramagnetic resonance with theoretical calculations reveal that the favorable Br2 /HBrO generation over Pt1 /NCNT with optimal intermediate binding facilitates C2 H4 -to-EO conversion with a high FE of 92.2 %, and concomitantly, the Br- with strong nucleophilicity protects active Cu+ species in Cu2 O effectively for improved CO2 -to-C2 H4 conversion with a FE of 66.9 % at 800 mA cm-2 , superior to those in the traditional chloride-mediated case. Consequently, a single-pass FE as high as 41.1 % for CO2 -to-EO conversion can be achieved in a tandem system.

Er:YAG Laser Physical Etching and Ultra-High-Molecular-Weight Cross-Linked Sodium Polyacrylate Chemical Etching for a Reliable Dentin Dry Bonding.

Dentin bond interface stability is the key issue of dental adhesion in present clinical dentistry. The concept of selective extrafibrillar demineralization has opened a new way to maintain intrafibrillar minerals to prevent interface degradation. Here, using ultra-high-molecular-weight sodium polyacrylate [Carbopol (Carbo) > 40 kDa] as a calcium chelator, we challenge this concept and propose a protocol for reliable dentin dry bonding. The results of high-resolution transmission electron microscopy revealed periodic bands of 67 nm dentin collagen fibrils after Carbo etching, and the hydroxyproline concentration increasing with prolonged chelating time denied the concept of extrafibrillar demineralization. The results that wet and dry bonding with Carbo-based demineralization produced a weaker bond strength than the traditional phosphoric acid wet adhesion suggested that the Carbo-based demineralization is an unreliable adhesion strategy. A novel protocol of Er:YAG laser physical etching followed by Carbo chemical etching for dentin adhesion revealed that a micro-/nano-level rough, rigid, and non-collagen exposed dentin surface was produced, the micro-tensile bond strength was maintained after aging under dry and wet bonding modes, and in situ zymography and nanoleakage within the hybrid layers presented lower signals after aging. Cell culture in vitro and a rabbit deep dentin adhesion model in vivo proved that this protocol is safe and biocompatible. Taken together, the concept of extrafibrillar demineralization is limited and insufficient to use in the clinic. The strategy of Er:YAG laser physical etching followed by Carbo chemical etching for dentin adhesion produces a bonding effect with reliability, durability, and safety.

[Impacts of Climate Change and Human Activities on NDVI Change in Eastern Coastal Areas of China].

Based on the datasets of normalized difference vegetation index (NDVI), temperature, precipitation, and solar radiation and the methods of trend, partial correlation, and residual analyses, this study explored the spatiotemporal variation in NDVI and its response to climate change from 1982 to 2019 in eastern coastal areas of China. Then, the effects of climate change and non-climatic factors (e.g., human activities) on NDVI trends were analyzed. The results showed that:① the NDVI trend varied greatly in different regions, stages, and seasons. On average, the growing season NDVI increased faster during 1982-2000 (stage I) than that during 2001-2019 (stage Ⅱ) in the study area. Moreover, NDVI in spring showed a more rapid increase than that in other seasons in both stages. ② For a given stage, the relationships between NDVI and each climatic factor varied in different seasons. For a given season, the major climatic factors associated with NDVI change were different between the two stages. The relationships between NDVI and each climatic factor showed great spatial differences in the study period. In general, the increase in growing season NDVI in the study area from 1982 to 2019 was closely related to the rapid warming. The increase in precipitation and solar radiation in stage Ⅱ also played a positive role. ③ In the past 38 years, climate change played a greater role in the change in growing season NDVI than non-climatic factors, including human activities. Whereas non-climatic factors dominated the increase in growing season NDVI during stage I, climate change played a major role during stage Ⅱ. We suggest that more attention should be paid to the impacts of various factors on vegetation cover variation during different periods to promote the understanding of terrestrial ecosystem changes.

A novel N6-Deoxyadenine methyltransferase METL-9 modulates C. elegans immunity via dichotomous mechanisms.

N6-Methyldeoxyadenine (6mA) has been rediscovered as a DNA modification with potential biological function in metazoans. However, the physiological function and regulatory mechanisms regarding the establishment, maintenance and removal of 6mA in eukaryotes are still poorly understood. Here we show that genomic 6mA levels change in response to pathogenic infection in Caenorhabditis elegans (C. elegans). We further identify METL-9 as the methyltransferase that catalyzes DNA 6mA modifications upon pathogen infection. Deficiency of METL-9 impairs the induction of innate immune response genes and renders the animals more susceptible to pathogen infection. Interestingly, METL-9 functions through both 6mA-dependent and -independent mechanisms to transcriptionally regulate innate immunity. Our findings reveal that 6mA is a functional DNA modification in immunomodulation in C. elegans.

Oxidized Dopamine Acrylamide Primer to Achieve Durable Resin-Dentin Bonding.

The durability of the resin-dentin bonding interface is a key issue in clinical esthetic dentistry. Inspired by the extraordinary bioadhesive properties of marine mussels in a wet environment, we designed and synthetized N-2-(3,4-dihydroxylphenyl) acrylamide (DAA) according to the functional domain of mussel adhesive proteins. DAA's properties of collagen cross-linking, collagenase inhibition, inducing collagen mineralization in vitro, and as a novel prime monomer for clinical dentin adhesion use, its optimal parameters, and effect on the adhesive longevity and the bonding interface's integrity and mineralization, were evaluated in vitro and in vivo. The results showed that oxide DAA can inhibit the activity of collagenase and cross collagen fibers to improve the anti-enzymatic hydrolysis of collagen fibers and induce intrafibrillar and interfibrillar collagen mineralization. As a primer used in the etch-rinse tooth adhesive system, oxide DAA can improve the durability and integrity of the bonding interface by anti-degradation and mineralization of the exposed collagen matrix. Oxidized DAA (OX-DAA) is a promising primer for improving dentin durability; using 5% OX-DAA ethanol solution and treating the etched dentin surface for 30 s is the optimal choice when used as a primer in the etch-rinse tooth adhesive system.