Bcp1 Is the Nuclear Chaperone of Rpl23 in Saccharomyces cerevisiae.

Eukaryotic ribosomes are composed of rRNAs and ribosomal proteins. Ribosomal proteins are translated in the cytoplasm and imported into the nucleus for assembly with the rRNAs. It has been shown that chaperones or karyopherins responsible for import can maintain the stability of ribosomal proteins by neutralizing unfavorable positive charges and thus facilitate their transports. Among 79 ribosomal proteins in yeast, only a few are identified with specific chaperones. Besides the classic role in maintaining protein stability, chaperones have additional roles in transport, chaperoning the assembly site, and dissociation of ribosomal proteins from karyopherins. Bcp1 has been shown to be necessary for the export of Mss4, a phosphatidylinositol 4-phosphate 5-kinase, and required for ribosome biogenesis. However, its specific function in ribosome biogenesis has not been described. Here, we show that Bcp1 dissociates Rpl23 from the karyopherins and associates with Rpl23 afterward. Loss of Bcp1 causes instability of Rpl23 and deficiency of 60S subunits. In summary, Bcp1 is a novel 60S biogenesis factor via chaperoning Rpl23 in the nucleus.

A review of emerging applications of ultrasonication in Comparison with non-ionizing technologies for meat decontamination.

Meat is highly susceptible to contamination with harmful microorganisms throughout the production, processing, and storage chain, posing a significant public health risk. Traditional decontamination methods like chemical sanitizers and heat treatments often compromise meat quality, generate harmful residues, and require high energy inputs. This necessitates the exploration of alternative non-ionizing technologies for ensuring meat safety and quality. This review provides a comprehensive analysis of the latest advancements, limitations, and future prospects of non-ionizing technologies for meat decontamination, with a specific focus on ultrasonication. It further investigates the comparative advantages and disadvantages of ultrasonication against other prominent non-ionizing technologies such as microwaves, ultraviolet (UV) light, and pulsed light. Additionally, it explores the potential of integrating these technologies within a multi-hurdle strategy to achieve enhanced decontamination across the meat surface and within the matrix. While non-ionizing technologies have demonstrated promising results in reducing microbial populations while preserving meat quality attributes, challenges remain. These include optimizing processing parameters, addressing regulatory considerations, and ensuring cost-effectiveness for large-scale adoption. Combining these technologies with other methods like antimicrobial agents, packaging, and hurdle technology holds promise for further enhancing pathogen elimination while safeguarding meat quality.

Evaluation of ultrasound and pulsed electric field combinations on the cooking Losses, texture Profile, and Taste-Related amino acids of chicken breast meat.

The search to improve the quality of meat while maintaining its nutritional value and flavor profile has driven the investigation of emerging clean-label non-thermal technologies in the field of meat processing. Ultrasound (US) and pulsed electric field (PEF) treatments have emerged as promising tools for producing high-quality meat products. This study investigated the combined effects of ultrasound and PEF on chicken breast meat quality, focusing on cooking loss, texture, and taste-related amino acids. Ultrasound (24.5 kHz, 300 W, 10 min) combined with PEF for 30 s (1.6, 3.3, and 5.0 kV/cm as US + PEF 1, US + PEF 3, and US + PEF 5, respectively) significantly reduced cooking losses (up to 28.78 %), potentially improving the product yield. Although US + PEF significantly (p < 0.05) affected pH, particularly at a higher PEF intensity (5 kV/cm), the overall color appearance of the treated meat remained unchanged. The combined treatments resulted in a tenderizing effect and decreased meat hardness, adhesiveness, and chewiness. Interestingly, US + PEF with increasing PEF intensity (1.6 to 5.0 kV/cm) led to a gradual increase in taste-related amino acids (aspartic acid, glutamic acid, etc.), potentially enhancing flavor. FTIR spectra revealed alterations in protein and lipid structures following treatment, suggesting potential modifications in meat quality. Scanning electron microscopy (SEM) revealed significant changes in the texture and structure of US + PEF-treated meat, depicting structural disruptions. Furthermore, Pearson's correlation analysis and principal component analysis (PCA) revealed a clear relationship between the physicochemical characteristics, free amino acids, color, and texture attributes of chicken meat. By optimizing treatment parameters, US + PEF could offer a novel approach to improve chicken breast meat quality.

Improving resistant starch content of cassava starch by pulsed electric field-assisted esterification.

This study aims to investigate the effect of pulsed electric field (PEF) assisted OSA esterification treatment on the multi-scale structure and digestive properties of cassava starch and structure-digestion relationships. The degree of substitution (DS) of starch dually modified at 1.5-4.5 kV/cm was 37.6-55.3 % higher than that of starch modified by the conventional method. Compared with native starch, the resistant starch (RS) content of esterified starch treated with 3 kV/cm significantly increased by 17.13 %, whereas that of starch produced by the conventional method increased by only 5.91 %. Furthermore, assisted esterification at low electric fields (1.5-3 kV/cm) promotes ester carbonyl grafting on the surface of starch granules, increases steric hindrance and promotes the rearrangement of the amorphous regions of starch, which increases the density of the double-helical structure. These structural changes slow down starch digestion and increase the RS content. Therefore, this study presents a potential method for increasing the RS content of starch products using PEF to achieve the desired digestibility.

Enhancing chicken breast meat quality through ultrasonication: Physicochemical, palatability, and amino acid profiles.

Ultrasonication, a technology that employs high-frequency sound waves, has demonstrated potential for modifying the properties of various food items. However, the effect of ultrasonication on chicken meat, particularly concerning amino acid composition and flavor enhancement, has not been sufficiently investigated. The objective of this research was to bridge the gap in the literature by exploring the impact of various ultrasonic treatments at varying power levels (300, 500, and 800 W) and durations (10 and 30 min) on the physicochemical characteristics, texture, and amino acid profile of chicken breast meat, with a focus on improving its palatability and flavor. The results indicated that ultrasonication reduced the pH and cooking loss, as well as hardness and chewiness while simultaneously increasing lightness and yellowness values of chicken breast meat. Moreover, ultrasonication enhanced the amounts of essential amino acids, including glutamic acid, alanine, and glycine as well as the free amino acid content, which gives meat its savory and umami flavor. Furthermore, the results demonstrated significant changes in the texture and structure, as demonstrated by the scanning electron microscopy (SEM) images, and in chemical makeup of chicken breast meat, as indicated by the FTIR spectra. These modifications in the molecular and microstructural characteristics of meat, as induced by ultrasonication, may contribute to the enhancement of tenderness, juiciness, and overall palatability.

Characterization and application in yogurt of genipin-crosslinked chitosan microcapsules encapsulating with Lactiplantibacillus plantarum DMDL 9010.

Microcapsules could improve the protection of probiotics in the lyophilization and gastrointestinal digestion process. The purpose of this study was to prepare Lactiplantibacillus plantarum DMDL 9010 (LP9010) microcapsules by cross-linking chitosan with genipin and to determine the encapsulation efficiency, morphological characterization, storage stability and the application of the microcapsules in fermentation. The results showed that the LP9010 microcapsules embedded in 1.00 wt% genipin cross-linked chitosan were in a uniform spherical shape with a smooth surface and satisfying agglomeration. The LP9010 microcapsules demonstrated the reasonable thermal stability and persistence of biological activity in the range of -20 °C to 25 °C. Additionally, yogurt obtained from the ST + LB + ELP9010 strain formulation with the addition of microencapsulated LP9010 had smaller particles, better taste, and better stability compared with the yogurt obtained from other strain formulations. As detected by GC-MS, the yogurt formulated with ST + LB + ELP9010 as a strain retained more flavor substances and the content of flavor substances was greater than that of the yogurt obtained from other strain formulations. Therefore, genipin cross-link chitosan could be a suitable microencapsulated material for producing yogurt fermentation strains.

The effects of pulsed electric fields treatment on the structure and physicochemical properties of dialdehyde starch.

The structural and physicochemical properties changes of corn starch oxidized by sodium periodate under the assistance of pulsed electric fields (PEF) were studied. It was found that dialdehyde starch (DAS) particles produced by PEF-assisted oxidation exhibited shrinkage and pits, and had a larger particle size when compared to the control without PEF. The solubility of the DAS (12 kV/cm PEF- assisted oxidation) improved by 70.2% when compared to the native starch. Increment in the strength of the PEF, led to a decrease in the viscosity of the DAS. In addition, the aldehyde group content of the DAS produced by PEF-assisted oxidation exhibited shrinkage and pits, and had a larger particle size when compared to the control increased by 11.6% when compared with the traditional oxidation method. PEF is an effective method to promote oxidation reaction of starch.

Pulsed electric field-assisted esterification improves the freeze-thaw stability of corn starch gel by changing its molecular structure.

The influence of pulsed electric field (PEF) combined with octenyl succinic anhydride (OSA) on the freeze-thaw stability of corn starch gel was investigated. After five freeze-thaw cycles, the syneresis value of OSA starch treated with PEF-assisted esterification for 15 min was lower by 29.5 %, while that of OSA starch without PEF treatment was lower by 10.17 %, compared to that of native starch. Low-field nuclear magnetic resonance data showed that the introduction of OSA groups greatly increased the water-holding capacity of starch. Results from differential scanning calorimetry (DSC) and X-ray diffraction (XRD) showed that the PEF-assisted esterification markedly hindered the re-formation of the helical structure of starch during freeze-thaw cycles. Moreover, PEF-assisted esterification improved the viscoelastic properties of the starch gel. It is found that the freeze-thaw stability of the PEF-modified starch depends not only on the degree of substitution but also on the starch molecular fine structure. PEF-assisted OSA starch with a high degree of substitution, a low content of amylose, and a high content of short amylopectin chains were found to have high freeze-thaw stability. This study shows that PEF-assisted esterification is a promising technique that should be used for preserving the quality of frozen foods.

Preoperative automatic reminder systems and impact on quality and compliance with colonoscopy preparation: A multicenter randomized controlled trial.

OBJECTIVES: Traditional preoperative reminding services have been applied to enhance the quality of bowel preparation for colonoscopy. In this study we aimed to evaluate the effectiveness of an automated electronic reminder system (E-reminder) on improving bowel preparation and the quality of preoperative education before colonoscopy. METHODS: From August 2021 to March 2022, 833 outpatients aged 50-75 years who underwent colonoscopy were included and randomly assigned to the E-reminder group and the control group. While the control group received routine preoperative education. The E-reminder group received automatic phone call, text message reminders and web services regarding the details of bowel preparation before the colonoscopic examination. The quality of bowel preparation was evaluated by the Boston Bowel Preparation Scale (BBPS) score and the previously validated objective evaluation scale of automatic BBPS (e-BBPS). RESULTS: In manual assessment, the rate of adequate bowel preparation was improved in the E-reminder group of intention-to-treat population using BBPS (60.7% vs 54.5%, P = 0.01). The percentage of objective evaluated adequate bowel preparation using e-BBPS in the E-reminder group of per-protocol population was significantly higher than that in the control group (76.9% vs 69.2%, P = 0.02). CONCLUSIONS: E-reminder was an effective tool to improve the quality of bowel preparation and compliance with medical instructions. It may be regarded as an efficient and convenient education tool, improving the quality of medical service.

Shape Dependence of Silver-Nanoparticle-Mediated Synthesis of Gold Nanoclusters with Small Molecules as Capping Ligands.

In this study, differently shaped silver nanoparticles used for the synthesis of gold nanoclusters with small capping ligands were demonstrated. Silver nanoparticles provide a reaction platform that plays dual roles in the formation of Au NCs. One is to reduce gold ions and the other is to attract capping ligands to the surface of nanoparticles. The binding of capping ligands to the AgNP surface creates a restricted space on the surface while gold ions are being reduced by the particles. Four different shapes of AgNPs were prepared and used to examine whether or not this approach is dependent on the morphology of AgNPs. Quasi-spherical AgNPs and silver nanoplates showed excellent results when they were used to synthesize Au NCs. Spherical AgNPs and triangular nanoplates exhibited limited synthesis of Au NCs. TEM images demonstrated that Au NCs were transiently assembled on the surface of silver nanoparticles in the method. The formation of Au NCs was observed on the whole surface of the QS-AgNPs if the synthesis of Au NCs was mediated by QS-AgNPs. In contrast, formation of Au NCs was only observed on the edges and corners of AgNPts if the synthesis of Au NCs was mediated by AgNPts. All of the synthesized Au NCs emitted bright red fluorescence under UV-box irradiation. The synthesized Au NCs displayed similar fluorescent properties, including quantum yields and excitation and emission wavelengths.

Enhanced encapsulation of lutein using soy protein isolate nanoparticles prepared by pulsed electric field and pH shifting treatment.

In this study, soy protein isolate (SPI) was modified by a pulsed electric field (PEF) combined with pH shifting treatment (10 kV/cm, pH 11) to prepare SPI nanoparticles (PSPI11) for efficient loading of lutein. The results showed that when the mass ratio of SPI to lutein was 25:1, the encapsulation efficiency of lutein in PSPI11 increased from 54% to 77%, and the loading capacity increased by 41% compared to the original SPI. The formed SPI-lutein composite nanoparticles (PSPI11-LUTNPs) had smaller, more homogeneous sizes and larger negative charges than SPI7-LUTNPs. The combined treatment favored the unfolding of the SPI structure and could expose its interior hydrophobic groups to bind with lutein. Nanocomplexation with SPIs significantly improved the solubility and stability of lutein, with PSPI11 showing the greatest improvement. As a result, PEF combined with pH shifting pretreatment is an effective method for developing SPI nanoparticles loaded and protected with lutein.

Validation of a real-time biliopancreatic endoscopic ultrasonography analytical device in China: a prospective, single-centre, randomised, controlled trial.

BACKGROUND: Endoscopic ultrasonography (EUS) is a key procedure for the diagnosis of biliopancreatic diseases. However, the performance among EUS endoscopists varies greatly and leads to blind spots during the operation, which can impair the health outcomes of patients. We previously developed an artificial intelligence (AI) device that accurately identified EUS standard stations and significantly reduced the difficulty of ultrasonography image interpretation. In this study, we updated the device (named EUS-IREAD) and validated its performance in improving the quality of EUS procedures. METHODS: In this single-centre, randomised, controlled trial, we updated EUS-IREAD so it consisted of five learning models to identify eight EUS stations and 24 anatomical structures. The trial was done at the Renmin Hospital of Wuhan University (Wuhan, China) and included patients aged 18 years or older with suspected biliopancreatic (pancreas and biliopancreatic duct) lesions due to clinical symptoms, radiological findings, or laboratory findings, and with a high risk of pancreatic cancer. Patients were randomly assigned (1:1) by a dedicated research assistant using a computer-generated random number series (with a block size of four) to undergo the EUS procedure with or without the assistance of EUS-IREAD. Endoscopists in the EUS-IREAD-assisted group were required to observe all standard stations and anatomical structures according to the prompts by the AI device. Data collectors, the independent data anaylsis team, and patients were masked to group allocation. The primary outcome was the missed scanning rate of standard stations between the two groups, which was assessed in patients who underwent EUS procedure in accordance with the assigned intervention (per protocol). This trial is registered with ClinicalTrials.gov, NCT05457101. FINDINGS: Between July 9, 2022, and Feb 28, 2023, 290 patients (mean age 55·93 years [SD 14·06], 152 [52%] male, and 138 [48%] female) were randomly assigned and analysed, including 144 in the EUS-IREAD-assisted group and 146 in the control group. The EUS-IREAD-assisted group had a lower missed scanning rate of stations than the control group (4·5% [SD 0·8] vs 14·3% [1·0], -9·8% [95% CI -12·2 to -7·5]; odds ratio 3·6 [95% Cl 2·6 to 4·9]; p<0·0001). No significant adverse event was found during the study. INTERPRETATION: Our study confirms the capability of EUS-IREAD to monitor the blind spots and reduce the missed rate of stations and structures during EUS procedures. The EUS-IREAD has the potential to play an essential part in EUS quality control. FUNDING: Innovation Team Project of Health Commission of Hubei Province and College-enterprise Deepening Reform Project of Wuhan University.

Diversity-oriented synthesis of marine sponge derived hyrtioreticulins and their anti-inflammatory activities.

Diversity-oriented synthesis is aimed to increase the chemical diversity of target natural products for extensive biological activity evaluation. Indole ring is an important functional group in a large number of drugs and other biologically active agents, and indole-containing natural products have been frequently isolated from marine sources in recent years. In this paper, a series of indole-containing marine natural hyrtioreticulin derivatives, including 19 new ones, were designed, synthesized through a key Pictet-Spengler reaction, and evaluated for their inflammation related activity. Compound 13b displayed the most promising activity by inhibiting TNF-α cytokine release with an inhibitory rate of 92% at a concentration of 20 µmol·L-1. A preliminary structure-activity relationship analysis was also discussed. This research may throw light on the discovery of marine indole alkaloid derived anti-inflammatory drug leads.

Succinylated whey protein isolate-chitosan core-shell composite particles as a novel carrier: Self-assembly mechanism and stability studies.

Single protein [whey protein isolate (WPI) or succinylated whey protein isolate (SWPI)] and composite particles of proteins with chitosan (CS) were tested for their ability to encapsulate and protect curcumin (CUR). Combining protein and CS resulted in changes in zeta-potential and surface hydrophobicity, particularly in the SWPI-H (high degree of succinylation, 90 %) and CS composite particle (H-CS). Furthermore, the secondary and tertiary structures were dramatically altered using Fourier transform infrared (FTIR), circular dichroism (CD), and X-ray diffraction (XRD). Scanning electron microscopy (SEM) and atomic force microscope (AFM) analyses revealed that H-CS exhibited a soft core-rigid shell morphology due to electrostatic interactions, hydrophobic interactions, and H-bond interactions. Fluorescence quenching results demonstrated that H-CS had a higher binding constant (K, 1.69 ×104 M-1) and encapsulation effectiveness (EE, 88.3 %) of CUR. Because of increased binding sites and steric hindrance, CUR was stabilized more effectively in H-CS in photostability and thermostability tests,. These results show that SWPI-CS composite particles can be utilized to build a protection system for water-insoluble nutritional supplements.

Inhibition of Biofilm Formation of Foodborne Staphylococcus aureus by the Citrus Flavonoid Naringenin.

Taking into consideration the importance of biofilms in food deterioration and the potential risks of antiseptic compounds, antimicrobial agents that naturally occurring are a more acceptable choice for preventing biofilm formation and in attempts to improve antibacterial effects and efficacy. Citrus flavonoids possess a variety of biological activities, including antimicrobial properties. Therefore, the anti-biofilm formation properties of the citrus flavonoid naringenin on the Staphylococcus aureus ATCC 6538 (S. aureus) were investigated using subminimum inhibitory concentrations (sub-MICs) of 5~60 mg/L. The results were confirmed using laser and scanning electron microscopy techniques, which revealed that the thick coating of S. aureus biofilms became thinner and finally separated into individual colonies when exposed to naringenin. The decreased biofilm formation of S. aureus cells may be due to a decrease in cell surface hydrophobicity and exopolysaccharide production, which is involved in the adherence or maturation of biofilms. Moreover, transcriptional results show that there was a downregulation in the expression of biofilm-related genes and alternative sigma factor sigB induced by naringenin. This work provides insight into the anti-biofilm mechanism of naringenin in S. aureus and suggests the possibility of naringenin being used in the industrial food industry for the prevention of biofilm formation.

Enhanced synthesis of succinylated whey protein isolate by pulsed electric field pretreatment.

The objective of this study was to investigate the feasibility of pulse electric field (PEF) as a pretreatment for whey protein isolate (WPI) before its succinylation. The degree of succinylation (DS) of WPI increased from 88.31% for native WPI to 93.45% for PEF-pretreated WPI (PWPI, initial pH 10.0) for the same succinic anhydride (SA) to WPI ratio (1:1). Fourier transform infrared spectroscopy and scanning electron microscopy analysis proved the successful succinylation of WPI. For PWPIs, the surface hydrophobicity, exposed sulphydryl, and total sulphydryl decreased, which indicates the occurrence of changes in protein structures with more hydrophilic groups and better protein dispersion. Moreover, PEF may expose more amino acid residues binding sites that are present inside the protein, which is more suitable for succinylation. Therefore, the PEF pretreatment of proteins can improve their efficient use that is expected to play a critical role in succinylation industry.

Silver Nanoparticle-Mediated Synthesis of Fluorescent Thiolated Gold Nanoclusters.

A new strategy using silver nanoparticles (Ag NPs) to synthesize thiolated Au NCs is demonstrated. The quasi-spherical Ag NPs serve as a platform, functioning as a reducing agent for Au (III) and attracting capping ligands to the surface of the Ag NPs. Glutathione disulfide (GSSG) and dithiothreitol (DTT) were used as capping ligands to synthesize thiolated Au NCs (glutathione-Au NCs and DTT-Au NCs). The glutathione-Au NCs and DTT-Au NCs showed red color luminance with similar emission wavelengths (630 nm) at an excitation wavelength of 354 nm. The quantum yields of the glutathione-Au NCs and DTT-Au NCs were measured to be 7.3% and 7.0%, respectively. An electrophoretic mobility assay showed that the glutathione-Au NCs moved toward the anode, while the DTT-Au NCs were not mobile under the electric field, suggesting that the total net charge of the thiolated Au NCs is determined by the charges on the capping ligands. The detection of the KSV values, 26 M-1 and 0 M-1, respectively, revealed that glutathione-Au NCs are much more accessible to an aqueous environment than DTT-Au NCs.

Pulsed electric field assisted modification of octenyl succinylated potato starch and its influence on pasting properties.

The physicochemical properties and structural changes of potato starch esterified with octenyl succinic anhydride (OSA) assisted with pulsed electric field (PEF) were investigated. Results showed that PEF treatment during esterification resulted in a significant modification of pasting properties. The pasting temperature at 2-6 kV/cm reduced by 7.6-15.1 °C for PEF-assisted OSA starches but only by 3 °C for OSA modified starch without PEF treatment as compared to that of native starch. PEF-assisted esterification could reduce the reaction time and improve the reaction efficiency over the control by 6.1-39.1 %. A novel schematic model on structure-functionality relationship for PEF-assisted OSA modified starch was proposed. Structural disorganizations of starch induced lower pasting temperature and paste viscosity. The results suggest that PEF could be a potential eco-friendly and cost-effective physical technique to prepare starch products with desired paste behaviors and to broaden its application area especially in papermaking and textile industries.

A systematic review of clean-label alternatives to synthetic additives in raw and processed meat with a special emphasis on high-pressure processing (2018-2021).

The meat industry is continuously facing challenges with food safety, and quality losses caused by thermal processing. This systematic review reports recent clean label approaches in high-pressure production of meat. A literature search was performed using Scopus, Web of Science, PubMed, and Springer databases for studies published in 2018-2021. In this regard, 69 articles were assessed out of 386 explored research articles in the identified stage. The findings indicate that most of the earlier work on high-pressure processing (HPP) focused on physicochemical and sensorial meat quality rather than providing nutritional aspects and clean-label solutions. However, few advanced studies report effective and innovative solutions to develop low salt/fat, and reduced nitrite for raw and cured meat products. HPP could help on increasing the shell life by five times in meat products; however, it depends on the formulation and packaging, etc. HPP can also preserve nutrients by using this non-thermal technology and reduce food waste as once the shelf life of products is known, it easily reduces the shrinkage in the marketplace. This review explores the latest trend of experimental research in high-pressure processing alone, or multi-hurdle techniques employed to increase the effect of clean-label ingredients for enhanced meat safety/quality.

Purification, characterization, and identification of 3-hydroxy-4-methoxy benzal acrolein-an intermediate of synthesizing advantame.

Advantame is a novel sweetener, and 3-hydroxy-4-methoxy benzal acrolein is an important intermediate to synthesize the sweetener. The aim of this study was to evaluate a new low-cost method to purify 3-hydroxy-4-methoxy benzal acrolein, and the crude intermediate was used as raw material. The intermediate was purified using a low-pressure column chromatography with a C18 column, using a methanol-water (6:4, v/v) at a flow rate of 6.0 ml/min, and the loading amount of the crude intermediate in solution was 10.0 mg in total. A method for the analysis of 3-hydroxy-4-methoxy benzal acrolein was established using high-performance liquid chromatography (HPLC). This method was validated in terms of its linearity, repeatability, accuracy, detection limit, and quantitation limit. The calibration curves of 3-hydroxy-4-methoxy benzal acrolein were linear (r > .999) over a wide concentration range of 0.005-0.08 mg/ml, by comparing the ratio of signal to noise, and the detection limit was 5.0 ng/ml and the quantification limit was 15.0 ng/ml. Good repeatability was obtained (RSD < 2%, n = 6), and the recoveries calculated using mixed sample previously quantified ranged from 94.5% to 106.37%. As long as, this method has been successfully applied to the analysis of 3-hydroxy-4-methoxy benzal acrolein; therefore, the method can be put into practical use during the industrial synthesis and real-time detection of the intermediate.