Optical sensor arrays for the detection and discrimination of natural products.

Covering: up to the end of 2022Natural products (NPs) have found uses in medicine, food, cosmetics, materials science, environmental protection, and other fields related to our life. Their beneficial properties along with potential toxicities make the detection and discrimination of NPs crucial for their applications. Owing to the merits of low cost and simple operation, optical sensor arrays, including colorimetric and fluorometric sensor arrays, have been widely applied in the detection of small molecule NPs and discrimination of structurally similar small molecule NPs or complex mixtures of NPs. This review provides a brief introduction to the optical sensor array and focuses on its progress toward the detection and discrimination of NPs. We summarized the design principle of sensor arrays toward various NPs (i.e., saccharides and polyhydroxy compounds, organic acids, flavonoids, organic sulfur compounds, amines, amino acids, and saponins) based on their functional groups and characteristic chemical properties, along with representative examples. Moreover, the challenges and potential directions for further research of optical sensor arrays for NPs are proposed.

Characterization of melanoidins in thermal hydrolysis sludge and effects on dewatering performance.

Thermal hydrolysis pretreatment (THP) of sludge can form the refractory brown melanoidins due to the occurrence of the Maillard reaction, which adversely involves the subsequent sludge anaerobic digestion (AD) process. However, details of the generation pattern of melanoidins and how they affect the sludge dewaterability remain largely unknown. This work aims to determine an approach to characterize and quantify the melanoidins created by THP of sludge. On this basis, the effect of melanoidins on sludge dewatering performance was revealed by adding synthetic melanoidins to the mixed sludge. Experimental results showed that three-dimensional fluorescence-region integration (3DEEM-FRI) could effectively distinguish melanoidins from other organic substances and achieve semi-quantitative characterization in sludge. The melanoidins significantly deteriorated the sludge dewaterability, and the lowest solids content of the filter cake (TS) was only 17.78% at the addition of 480 mg (g TS)-1, which was a drop of about 20% compared to the control group. The mechanism investigations indicated that the internal structure of sludge becoming particularly complicated and the opportunities for molecules to collide with each other enlarged because of the contribution of melanoidins, resulting in the increment of the sludge apparent viscosity and consistency coefficient (k), a decline of the flow behavior index (n) and a weakening of flowability. Melanoidins could capture massive water molecules and carry negative charges with the decrease of sludge particle size and zeta potential value, which enhanced the electrostatic repulsion between sludge particles and abated the flocculation ability, thus further aggravating the sludge dewatering performance.

Rheological properties and dewaterability of anaerobic co-digestion with sewage sludge and food waste: effect of thermal hydrolysis pretreatment and mixing ratios.

Anaerobic co-digestion (co-AD) of sewage sludge (SS) and food waste (FW) converts municipal organic waste into renewable energy, which plays an important role in achieving carbon emissions reduction. The existing anaerobic digestion (AD) treatment projects often have problems such as low organic conversion and unstable performance. SS and FW were used as raw materials to explore the effects of thermal hydrolysis pretreatment (THP) and mixing ratios on the dewaterability and rheological properties of the digestate. The results showed that co-digestion of FW and SS in a ratio of 1:1 obtained the highest biogas production (255.14 mL/g VS), which was 1.53 times and 14.5 times higher than that of mono-digestion of FW and thermal hydrolysis pretreatment sewage sludge (THSS), respectively. However, the dewaterability of this ratio deteriorated sharply after co-digestion, with a decrease of 54.92%. The groups containing a higher proportion of THSS had improved dewaterability after AD. The apparent viscosity and shear stress were reduced by co-digestion compared with mono-digestion of THSS and FW, indicating a higher flow property of the co-digestion matrix. After the Herschel-Bulkley model fitting, there were linear correlations between rheological indices and soluble chemical oxygen demand (SCOD), and digestate dewaterability.

CSN1201, a subunit of the COP9 signalosome, regulates the virulence in Cryptococcus neoformans infection.

The COP9 signalosome (CSN) is a multisubunit protein complex, and it now has been found to participate in diverse cellular and developmental processes in various eukaryotic organisms. Cryptococcus neoformans (C. neoformans) is an important basidiomycete pathogen that causes life-threatening meningoencephalitis primarily in the immune compromised population. Here, we generated CSN deletion mutants to investigate the role in Cryptococcus infection. Compared to other CSN mutants, we identified a CSN1201 mutant exhibited severely attenuated virulence. Deletion of CSN1201 made cryptococcal cells more susceptible to nearly all in vitro stresses. Furthermore, deletion of CSN1201 obviously impaired survival of C. neoformans. At the same time, in vivo virulence assay of mouse infection models demonstrated that CSN1201 significantly enhanced the virulence of C. neoformans compared with the other CSN subunit strains, while ELISA analysis of C. neoformans infection in innate or adaptive immune response showed that deletion of CSN1201 significantly impaired cytokines and interferon expression. In vitro model of the blood-brain barrier (BBB) analysis indicated that deletion of CSN1201 reduced the invasion efficacy of Cryptococcusto cross BBB. Taken together, our findings reveal a novel mechanism of CSN1201, which plays a critical role for the virulence composite of C. neoformans, and also provides an additional yeast survival and propagation advantage in the host.

Functionalization of Carbonaceous Nanodots from Mn(II) -Coordinating Functional Knots.

Transition metal (TM)-induced functionalization has been expected to be powerful for the modification of carbonaceous nanodots (CDs) for many years. Although some attempts have been made, relevant research evolutions are at a standstill. In this work, a coordination-carbonization protocol to gain Mn(II) -functionalized CDs (MCDs) was innovatively employed, and the modification of CDs based on metal-coordinating functional knots was realized. By investigating the structural and photophysical properties, coordinating conformation of functional knots was confirmed and considered to be correlated with excitation-independent photoluminescence as well as large Stokes shift of MCDs. Moreover, potential applications of MCDs have been extended from common bioimaging and fluorescent inks to new areas, such as chemosensors for volatile organic compounds and anticorrosion fluorescent films.

Extracorporeal membrane oxygenation technology for adults: an evidence mapping based on systematic reviews.

BACKGROUND: Extracorporeal membrane oxygenation (ECMO) is a cutting-edge life-support measure for patients with severe cardiac and pulmonary illnesses. Although there are several systematic reviews (SRs) about ECMO, it remains to be seen how quality they are and how efficacy and safe the information about ECMO they describe is in these SRs. Therefore, performing an overview of available SRs concerning ECMO is crucial. METHODS: We searched four electronic databases from inception to January 2023 to identify SRs with or without meta-analyses. The Assessment of Multiple Systematic Reviews 2 (AMSTAR-2) tool, and the Grading of Recommendations Assessment, Development and Evaluation (GRADE) system were used to assess the methodological quality, and evidence quality for SRs, respectively. A bubble plot was used to visually display clinical topics, literature size, number of SRs, evidence quality, and an overall estimate of efficacy. RESULTS: A total of 17 SRs met eligibility criteria, which were combined into 9 different clinical topics. The methodological quality of the included SRs in this mapping was "Critically low" to "Moderate". One of the SRs was high-quality evidence, three on moderate, three on low, and two on very low-quality evidence. The most prevalent study used to evaluate ECMO technology was observational or cohort study with frequently small sample sizes. ECMO has been proven beneficial for severe ARDS and ALI due to the H1N1 influenza infection. For ARDS, ALF or ACLF, and cardiac arrest were concluded to be probably beneficial. For dependent ARDS, ARF, ARF due to the H1N1 influenza pandemic, and cardiac arrest of cardiac origin came to an inconclusive conclusion. There was no evidence for a harmful association between ECMO and the range of clinical topics. CONCLUSIONS: There is limited available evidence for ECMO that large sample, multi-center, and multinational RCTs are needed. Most clinical topics are reported as beneficial or probably beneficial of SRs for ECMO. Evidence mapping is a valuable and reliable methodology to identify and present the existing evidence about therapeutic interventions.

Rapid discrimination of Alismatis Rhizoma and quantitative analysis of triterpenoids based on near-infrared spectroscopy.

This study developed a rapid, accurate, objective and economic method to identify and evaluate the quality of Alismatis Rhizoma (AR) commodities. Traditionally, the identification of plant species and geographical origins of AR commodities mainly relied on experienced staff. However, the subjectivity and inaccuracy of human identification negatively impacted the trade of AR. Besides, liquid chromatographic methods such as ultra-high-performance liquid chromatography (UPLC) and high-performance liquid chromatography (HPLC), the major approach for the determination of triterpenoid contents in AR was time-consuming, expensive, and highly demanded in manoeuvre specialists. In this study, the combination of near-infrared (NIR) spectroscopy and chemometrics as the method was developed and utilised to address the two common issues of identifying the quality of AR commodities. Through the discriminant analysis (DA), the raw NIR spectroscopy data on 119 batches samples from two species and four origins in China were processed to the best pre-processed data. Subsequently, orthogonal partial least squares-discriminant analysis (OPLS-DA) and random forest (RF) as the major chemometrics were used to analyse the best pre-processed data. The accuracy rates by OPLS-DA and RF were respectively 100% and 97.2% for the two species of AR, and respectively100% and 94.4% for the four origins of AR. Meanwhile, a quantitative correction model was established to rapidly and economically predict the seven triterpenoid contents of AR through combining the partial least squares (PLS) method and NIR spectroscopy, and taking the triterpenoid contents measured by UPLC as the reference value, and carry out spectral pre-processing methods and band selection. The final quantitative model correlation coefficients of the seven triterpenoid contents of AR ranged from 0.9000 to 0.9999, indicating that prediction ability of this model had good stability and applicability.

What interventions do rural doctors think will increase recruitment in rural areas: a survey of 2778 health workers in Beijing.

BACKGROUND: A shortage of health professionals in rural areas is a major problem facing China, as more than 60% of the population lives in such areas. Strategies have been developed by the government to improve the recruitment of rural doctors. However, the inequitable distribution of doctors working in China has not improved significantly. The objective of this study was to explore the reasons for the poor recruitment and to propose possible strategies to improve the situation. METHODS: Between September 2009 and November 2009 data were collected from 2778 rural doctors in Beijing, China. A quantitative survey was used to explore health workers' perceptions as to what factors would have the greatest impact on recruitment and whether access to training had been effective in increasing their confidence, enhancing their interest in practicing medicine and increasing their commitment to recruitment. RESULTS: Rural doctors were generally older than average in China. Of the 2778 participants, only 7.23% had obtained a license as a qualified doctor. For 53% of the rural doctors, the job was part-time work. The survey showed that rural doctors considered the training strategy to be inadequate. In general, the initiatives identified by rural doctors as being of most value in the recruitment of doctors were those targeting retirement pension and income. CONCLUSIONS: From the perspective of rural doctors, specific initiatives that promised a secure retirement pension and an increased income were considered most likely to assist in the recruitment of rural doctors in Beijing.

Efficacy and safety of traditional Chinese medicine adjuvant therapy for severe pneumonia: evidence mapping of the randomized controlled trials, systematic reviews, and meta-analyses.

Background and Objective: Severe pneumonia is a critical respiratory disease with high mortality. There is insufficient evidence on the efficacy and safety of traditional Chinese medicine (TCM) adjuvant therapy for severe pneumonia. This study aims to identify, describe, assess, and summarize the currently available high-quality design evidence on TCM adjuvant therapy for severe pneumonia to identify evidence gaps using the evidence mapping approach. Methods: Systematic searches were performed on English and Chinese online databases (PubMed, EMBASE, Cochrane Library, Web of Science, CNKI, WanFang Data, CQVIP, and SinoMed) to identify papers from inception until August 2023 for inclusion into the review. Randomized controlled trials (RCTs), systematic reviews (SRs), and meta-analyses concerning TCM adjuvant therapy for severe pneumonia or its complications in adults were included. The risk of bias in RCTs was evaluated by using the Cochrane Handbook ROB tool. The Assessment of Multiple Systematic Reviews 2 (AMSTAR-2), the Risk of Bias in Systematic Review (ROBIS) tool, and the Grading of Recommendations Assessment, Development and Evaluation (GRADE) system were used to assess the methodological quality, risk of bias, and evidence quality of SRs or meta-analyses, respectively. Then, a bubble plot was designed to visually display information in four dimensions. Results: A total of 354 RCTs and 17 SRs or meta-analyses met the inclusion criteria. The published RCTs had several flaws, such as unreasonable design, limited sample size, insufficient attention to non-drug therapy studies and syndrome differentiation, improper selection or use of outcome indicators, and failure to provide high-quality evidence. Sixteen SRs or meta-analyses of methodological quality scored "Critically Low" confidence. Twelve SRs or meta-analyses were rated as "High Risk." Most outcomes were rated as "Low" evidence quality. We found that TCM combined with conventional treatment could improve the clinical total effective rate and the TCM syndromes efficacy. The combined approach could also shorten mechanical ventilation time, infection control time, and length of hospital and ICU stay; significantly reduce temperature, respiratory rate, heart rate, white blood cell counts, levels of C-reactive protein, procalcitonin, blood inflammatory factors, bacteriological response, and D-dimer; decrease CPIS, APACHE II score, and PSI score; improve pulmonary imaging features, arterial blood gas indicators (including arterial oxygen pressure, arterial oxygen saturation, and oxygen index), and lung function (including forced vital capacity and forced expiratory volume in the first second) for severe pneumonia compared with conventional treatment only (p < 0.05). There was no significant difference in adverse reactions and incidence of adverse events (p > 0.05). In addition, compared with conventional treatment only, most SRs or meta-analyses concluded that TCM combined with conventional treatment was "Beneficial" or "Probably beneficial." Conclusion: TCM combined with conventional treatment had advantages in efficacy, clinical signs, laboratory results, and life quality outcomes of severe pneumonia, with no difference in safety outcomes compared with conventional treatment only. QingJin Huatan decoction is the most promising target, and Xuanbai Chengqi decoction has a "Probably beneficial" conclusion. XueBiJing injection and TanReQing injection are two commonly used Chinese herbal injections for treating severe pneumonia, and both are "Probably beneficial." However, there was a need for multicenter RCTs with large sample sizes and high methodological quality in the future. In addition, the methodological design and quality of SRs or meta-analyses should be improved to form high-quality, evidence-based medical evidence and provide evidence for the effectiveness and safety of TCM adjuvant therapy for severe pneumonia.

A ratiometric fluorescent sensor based on carbon dots for rapid determination of bisulfite in sugar.

Sulfite is widely used in food and drugs as a preservative; however, excessive intake of exogenous sulfite is harmful to human health. Hence, developing effective methods to detect sulfite and its derivatives is vital. Herein, we present a ratiometric fluorescent sensor for bisulfite based on a dual-emission boron-nitrogen double-doped carbon dots (BNCDs)-Cr(VI) system. After adding bisulfite, the fluorescence intensity at 450 nm was significantly enhanced because of the inhibition of the inner filter effect, while the fluorescence emission at 535 nm decreased. A sensor-based method was developed to determine bisulfite with a linearity range of 0 - 10.0 × 102 µmol/L and recoveries of 97.33% - 109.00%, achieving the quantitative detection of bisulfite in real sugar samples. Moreover, a paper test strip-based visual measuring system for bisulfite was developed, which realized the rapid and semiquantitative detection of bisulfite.

Identification of Chinese teas by a colorimetric sensor array based on tea polyphenol induced indicator displacement assay.

In this work, we developed an optical colorimetric sensor array for the discrimination of Chinese teas. The sensor array was carefully designed based on tea polyphenol induced indicators displacement assay (IDA), using phenylboronic acids with different substituents as the receptors to polyphenols. The accurate identification for polyphenols with different species or concentrations proved the potential of the sensor array. The sensor array successfully distinguished tea samples within different categories, grades and origins, coupling with PLS-DA. This work offered an efficient and rapid method to distinguish teas and tea-related products. Besides, the assay is supposed to be suitable for the identification of other polyphenol-related natural products.

Efficacy and safety of Shenling Atractylodes Powder in the treatment of ulcerative colitis: A protocol for systematic review and meta-analysis.

BACKGROUND: Ulcerative colitis (UC) is a kind of chronic non-specific ulcerative colitis, which is characterized by repeated abdominal pain, diarrhea, and mucus purulent stool. The disease is more recurrent, easy to delay, and canceration, seriously affect the quality of life, increase the economic burden of patients and society, treatment is more difficult, the World Health Organization as one of the modern refractory diseases. Shenling Atractylodes Powder in the treatment of ulcerative colitis showed a strong advantage, the effect is accurate. Therefore, this paper will systematically evaluate and meta-analyze the efficacy and safety of heat-sensitive moxibustion in the treatment of ulcerative colitis. METHODS: Eight electronic databases were searched, including the PubMed, Embase, Web of Science, Cochrane Library, China National knowledge Infrastructure, China Science and Technology Journal Database, Wanfang Database, and China Biomedical Literature Database. We will search in the above electronic database from early 2021 to December without any language restrictions. Outcome indicators, including colonic mucosal symptom score Mayo colonoscopy grading, total effective rate, total incidence of adverse reactions, clinical symptom score, recurrence rate, laboratory indicators: IL-6, IL-9, TNF-α, IL-4, IL-10 inflammation-related factor levels. Rev Man5.3 software will be used for statistical analysis. The efficacy and safety results of Shenling Atractylodes Powder in the treatment of ulcerative colitis will be used as the average difference between the risk ratio of dichotomy data and the 95% co-card interval of continuous data. RESULTS: When this research program is completed, the relevant results can be obtained. ETHICS AND DISSEMINATION: This article does not need to pass the ethics committee review, because this article does not involve the ethics question, only collates the related literature research. The results of this study will be disseminated in the form of a paper to help better guide the clinical practice of heat-sensitive moxibustion in the treatment of ulcerative colitis. REGISTRATION NUMBER: INPLASY202120018.

Efficacy and safety of heat-sensitive moxibustion in the treatment of ulcerative colitis: A protocol for a systematic review and meta-analysis.

BACKGROUND: Ulcerative colitis (UC) is a chronic nonspecific intestinal inflammatory disease with unclear etiology occurring in the colonic mucosa. Its clinical manifestations are characterized by recurrent abdominal pain, diarrhea, mucous pus, and blood stool. The severity of the disease varies, and itis characterized by a high recurrence rate. Because of its long course of disease, easy to relapse, protracted and difficult to recover, seriously affect the quality of life, increase the economic burden of patients and society, and even the risk of developing cancer, it has become one of the hot issues of general concern in the medical field. Heat-sensitive moxibustion therapy has shown strong advantages in the treatment of UC, and the curative effect is accurate. therefore, this paper will carry out a systematic evaluation and meta analysis of the efficacy and safety of heat-sensitive moxibustion in the treatment of UC. METHODS: We will be searching 8 electronic databases, including PubMed, Embase, Web of Science, Cochrane Library, the China National Knowledge Infrastructure, Chinese Science and Technology Periodical Database, Wanfang Database, and Chinese Biomedical Literature Database. We will search above electronic databases from the beginning to December 2020, without any language restriction. Clinical efficacy, including total effective rate or cure rate, clinical symptom integral (abdominal pain, diarrhea, purulent stool), and recurrence rate will be accepted as the primary outcomes. The changes of cytokine Hs-CRP, IL-6, TNF-αlevels in serum, and improvement of colorectal mucosa will be used as secondary outcomes. RevMan 5.3 software will be used for statistical analysis. The result about the curative effect and safety of heat-sensitive moxibustion for UC will be presented as risk ratio for dichotomous data and mean differences with a 95% confidence interval for continuous data. RESULTS: When this research program is completed, the relevant results can be obtained. CONCLUSIONS: The results of this study will provide reliable evidence for the efficacy and safety of heat-sensitive moxibustion in the treatment of UC. INPLASY REGISTRATION NUMBER: INPLASY20201101034.

BODIPY-based self-assembled nanoparticles as fluorescence turn-on sensor for the selective detection of zinc in human hair.

Zinc plays important roles in regulating physiological and pathological processes. Regrettably, mild to moderate zinc deficiency is common worldwide. Hair Zn(2+) concentration, which reflects a zinc storage status, is useful for tracking trends in zinc status within populations. In this work, we report BODIPY-based self-assembled nanoparticles (NPs) as fluorescence turn-on sensor for the selective sensing of Zn(2+) in human hair. The BODIPY monomers (BAN) self-assemble in aqueous medium to form nonfluorescent NPs. In the presence of Zn(2+) ions, the NPs selectively show an obvious turn-on fluorescence change. This selective response of the NPs allows the determination and quantification of Zn(2+) in human hair with a detection limit of 61.3nM. This study demonstrates that the small molecule self-assembled nanoparticle is a versatile and useful tool, and shows great potential for applications in sensing of important analytes in biological systems.

Intraparticle FRET of Mn(ii)-doped carbon dots and its application in discrimination of volatile organic compounds.

To achieve an energy transfer system in emissive nanoparticles, a conventional strategy is to graft an exterior fluorophore onto the surface of the host. In this paper, we report for the first time an intraparticle Förster resonance energy transfer (IPFRET) system formed intrinsically in Mn(ii)-doped carbon dots (MCDs). In virtue of the small particle size of MCDs and the modified band structure, intraparticle energy transfer from a fluorophore-like donor component to a metal-related acceptor component takes place. The IPFRET of MCDs was found to be sensitive to the chemical environment (e.g., polarity) via the effects of external influences on the metal-to-ligand charge transfer (MLCT). Surface enhanced Raman spectroscopy was employed to verify the MLCT-related metal-coordination conformation, and proved capable of collecting bonding information of metal-doped species of carbon dots. Benefitting from the sensitivity of the IPFRET signal, MCDs exhibited high potential in sensing applications.

The calibration of cellphone camera-based colorimetric sensor array and its application in the determination of glucose in urine.

In this work, a novel approach that can calibrate the colors obtained with a cellphone camera was proposed for the colorimetric sensor array. The variations of ambient light conditions, imaging positions and even cellphone brands could all be compensated via taking the black and white backgrounds of the sensor array as references, thereby yielding accurate measurements. The proposed calibration approach was successfully applied to the detection of glucose in urine by a colorimetric sensor array. Snapshots of the glucose sensor array by a cellphone camera were calibrated by the proposed compensation method and the urine samples at different glucose concentrations were well discriminated with no confusion after a hierarchical clustering analysis.

A novel electronic nose based on porous In2O3 microtubes sensor array for the discrimination of VOCs.

We have innovatively developed an electronic nose consisting of only one type of semiconductor metal oxide (SMO) material. The representative SMO material, porous In2O3 microtubes in this work, offered great surface area and large gas penetration channels. By using a solvent casting process, different amounts of porous In2O3 microtubes were coated on Al2O3 substrate, forming a resistometric SMO sensor array-based electronic nose. Each sensing unit in the electronic nose exhibited independent response toward ethanol. We have successfully applied this electronic nose to distinguish four alcohols at the same concentrations (100 ppm), and also utilized the electronic nose for the discrimination of 14 volatile organic compounds (VOCs). Clear differentiation among all the 14 VOCs both at their immediately dangerous to life or health (IDLH) and the permissible exposure limit (PEL) concentrations has been achieved with no errors or misclassifications. We expect that this method will expand the application of SMO sensor array-based electronic nose which has been largely limited by the selection of commercially available SMOs and dopants.

BODIPY-based fluorometric sensor for the simultaneous determination of Cys, Hcy, and GSH in human serum.

Cysteine (Cys), homocysteine (Hcy), and glutathione (GSH) are interconnected and play essential roles for regulating the redox balance of biological processes. However, finding a simple and effective method for the simultaneous determination for these three biothiols in biological systems is always a challenge. In this work, we report a method for the simultaneous quantitative determination of three biothiols in a mixture using a monochlorinated boron dipyrromethene (BODIPY)-based fluorometric sensor. At a specified period of time, after reacting with excess sensor, Hcy and GSH form predominantly sulfur-substituted BODIPY, while Cys generates sulfur-amino-diBODIPY due to a fast substitution-rearrangement-substitution reaction. A significant difference in polarities of these respective major products simplifies their separation by TLC, thus leading to the simultaneous determination of Cys, Hcy, and GSH readily. The sensor was successfully applied for the simultaneous quantitative detection of three biothiols in human serum, and the results were in good agreement with those obtained via high performance liquid chromatography (HPLC).

An array sensor consisting of a single indicator with multiple concentrations and its application in ion discrimination.

Optical sensor arrays typically require a large set of chemically responsive colorants to enhance discrimination capability. Conversely, we have proven that by using multiple concentrations of one indicator, the discrimination of various analytes could be realized.

Critical level setting of continuous air monitor.

Algorithms used to compensate the radon and thoron progeny's interference are one of the key technologies for continuous air monitors (CAMs). In this study, a CAM that can automatically change filter was manufactured, and equations used to calculate the transuranic aerosol concentration and the corresponding critical level were derived. The parameters used in calculation were acquired by continuous measurement in a high radon environment. At last, validation of the calculation was tested in a cave where the radon concentration fluctuated frequently, and the results were analysed.