Visual Knowledge Domain of Artificial Intelligence in Computed Tomography: A Review Based on Bibliometric Analysis.

ABSTRACT: Artificial intelligence (AI)-assisted medical imaging technology is a new research area of great interest that has developed rapidly over the last decade. However, there has been no bibliometric analysis of published studies in this field. The present review focuses on AI-related studies on computed tomography imaging in the Web of Science database and uses CiteSpace and VOSviewer to generate a knowledge map and conduct the basic information analysis, co-word analysis, and co-citation analysis. A total of 7265 documents were included and the number of documents published had an overall upward trend. Scholars from the United States and China have made outstanding achievements, and there is a general lack of extensive cooperation in this field. In recent years, the research areas of great interest and difficulty have been the optimization and upgrading of algorithms, and the application of theoretical models to practical clinical applications. This review will help researchers understand the developments, research areas of great interest, and research frontiers in this field and provide reference and guidance for future studies.

A computational approach to estimate postmortem interval using postmortem computed tomography of multiple tissues based on animal experiments.

The estimation of postmortem interval (PMI) is a complex and challenging problem in forensic medicine. In recent years, many studies have begun to use machine learning methods to estimate PMI. However, research combining postmortem computed tomography (PMCT) with machine learning models for PMI estimation is still in early stages. This study aims to establish a multi-tissue machine learning model for PMI estimation using PMCT data from various tissues. We collected PMCT data of seven tissues, including brain, eyeballs, myocardium, liver, kidneys, erector spinae, and quadriceps femoris from 10 rabbits after death. CT images were taken every 12 h until 192 h after death, and HU values were extracted from the CT images of each tissue as a dataset. Support vector machine, random forest, and K-nearest neighbors were performed to establish PMI estimation models, and after adjusting the parameters of each model, they were used as first-level classification to build a stacking model to further improve the PMI estimation accuracy. The accuracy and generalized area under the receiver operating characteristic curve of the multi-tissue stacking model were able to reach 93% and 0.96, respectively. Results indicated that PMCT detection could be used to obtain postmortem change of different tissue densities, and the stacking model demonstrated strong predictive and generalization abilities. This approach provides new research methods and ideas for the study of PMI estimation.

A preliminary study of calcium channel-associated mRNA and miRNA networks in post-traumatic epileptic rats.

The calcium channels are the main pathogenesis and therapeutic target for post-traumatic epilepsy (PTE). However, differentially expressed miRNAs (DEMs) and mRNAs associated with calcium channels in PTE and their interactions are poorly understood. We produced a PTE model in rats and conducted RNA-seq in PTE rats. Gene annotation was used to verify differentially expressed mRNAs related to calcium channels. RNAhybrid, PITA, and Miranda prediction were used to build the miRNA-mRNA pairs. Furthermore, Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were used for the functional enrichment analysis of DEMs. The quantification changes of mRNA and miRNA were verified by RT-qPCR. There were 431 identified differentially expressed genes (DEGs) in PTE rats compared with the sham group, of which five mRNAs and 7 miRNAs were related to calcium channels. The miRNA-mRNA network suggested a negative correlation between 11 pairs of miRNA-mRNA involved in the p53 signaling pathway, HIF-1 signaling pathway. RT-qPCR verified three upregulated mRNAs in PTE rats, associated with 7 DEMs negatively related to them, respectively. This study has revealed the changes in miRNA-mRNA pairs associated with calcium channels in PTE, which might contribute to the further interpretation of potential underlying molecular mechanisms of PTE and the discovery of promising diagnostics.

Mapping the knowledge of traffic collision Reconstruction: A scientometric analysis in CiteSpace, VOSviewer, and SciMAT.

Traffic collisions are incidents with high fatality rate which generate billions of US dollars of loss worldwide each year. Post-collision scene reconstruction, which involves knowledge of multiple disciplines, is an important approach to restore the traffic collision and infer the cause of it. This paper uses software CiteSpace, VOSviewer, and SciMAT to conduct a visualization study of knowledge mapping on the literature of traffic collision scene reconstruction from 2001 to 2021 based on the Web of Science database. Knowledge mapping is a cutting-edge research method in scientometric, which has been widely applied in medicine and informatics. Compared with traditional literature review, knowledge mapping with visual techniques identifies hot keywords and key literature in the field more scientifically, and displays them in schematic diagrams intuitively which allows to further predict potential hotspots. A total of 803 original papers are retrieved to analyze and discuss the evolution of the field in the past 20 years, from macro to micro, in term of background information, popular themes, and knowledge structure. Results indicate the number of publications in this field is limited, and collaborations among authors and among institutions are insufficient. In the meantime, mappings imply the top three hot themes being scene reconstruction, computer technology, and injuries. The introduction of AI related technologies, such as neural networks and genetic algorithms, into collision reconstruction would be a potential research direction.

CiteSpace-Based Visualization Analysis of Forensic Research in China from 2010 to 2019.

OBJECTIVES: To analyze the research status of forensic medicine in China from 2010 to 2019, obtain the development trend of forensic medicine and explore the hotspots and research frontiers. METHODS: The forensic medical academic papers published on China National Knowledge Infrastructure (CNKI) database from 2010 to 2019 were collected. CiteSpace 5.7.R1, an information visualization analysis software, was used to analyze publication organizations, authors, keywords, and other elements. RESULTS: The majority of the research institutions were universities, provincial and ministerial scientific research and forensic institutions. Forensic pathology was still an important branch of forensic medicine and a popular research direction. The "polymorphism" and "Y chromosome" had been the research hotspots in recent years. "Medical damage" and "standard" were the most novel studies. CONCLUSIONS: In order to provide scientific basis and research direction for forensic research, this paper analyzes the cooperation network, research hotspots and research innovation in forensic research.

Research progress in digital pathology: A bibliometric and visual analysis based on Web of Science.

BACKGROUND: The development of whole slide image and deep neural network technologies has contributed to the paradigm shift in diagnostic pathology and has received much attention from researchers, with related publications increasing yearly and "exploding" in recent years. However, few studies have systematically reviewed "digital pathology" using bibliometric tools. In this study, we will use multiple approaches to visualize and analyze "digital pathology" to provide a comprehensive and objective picture of the field's historical evolution and future development. METHODS: We use VOSviewer, CiteSpace, Gephi, and R to analyze the authors, institutional and national collaboration networks, keyword co-occurrence, and co-citation analysis to visualize the current status of global digital pathology research. RESULTS: Digital pathology-related research is mainly active in "molecular, biological, and immunology" journal groups, "pharmaceutical, medical, and clinical" journal groups, and "psychology, education, and health" journal groups; in addition to "digital pathology," "diagnosis," "deep learning," "histopathology," and "surgical pathology" are also active research topics; the U.S. has significant research results in digital pathology, with the top 10 publishing institutions all coming from the U.S. In the past two decades, global digital pathology-related research can be divided into two major research areas. One is about system verification and optimization of WSI, and the other is about the application and development of artificial intelligence technology in digital pathology. Among them, based on the development of computer technology and the update of the machine learning concept, the research results for deep neural network technologies have been more concentrated in recent years. The robust performance of deep neural networks in feature extraction and image analysis provides a new research direction for improving digital pathology-aided diagnosis systems, which is where the research hotspots have been in recent years. CONCLUSIONS: The bibliometric analysis may help better understand the current status of research within the field of digital pathology and provide references and lessons for future related research.

Mapping the Knowledge of Antipsychotics-Induced Sudden Cardiac Death: A Scientometric Analysis in CiteSpace and VOSviewer.

The drugs on the market for schizophrenia are first-generation and second-generation antipsychotics. Some of the first-generation drugs have more side effects than the other drugs, so they are gradually no longer being applied clinically. Years of research have shown that the risk of sudden cardiac death in psychotic patients is associated with drug use, and antipsychotic drugs have certain cardiotoxicity and can induce arrhythmias. The mechanism of antipsychotic-induced sudden cardiac death is complicated. Highly cited papers are among the most commonly used indicators for measuring scientific excellence. This article presents a high-level analysis of highly cited papers using Web of Science core collection databases, scientometrics methods, and thematic clusters. Temporal dynamics of focus topics are identified using a collaborative network (author, institution, thematic clusters, and temporal dynamics of focus topics are identified), keyword co-occurrence analysis, co-citation clustering, and keyword evolution. The primary purpose of this study is to discuss the visual results, summarize the research progress, and predict the future research trends by bibliometric methods of CiteSpace and VOSviewer. This study showed that a research hotspot is that the mechanisms of cardiotoxicity, the safety monitoring, and the assessment of the risk-benefit during clinical use of some newer antipsychotics, clozapine and olanzapine. We discussed relevant key articles briefly and provided ideas for future research directions for more researchers to conduct related research.

A bibliometric analysis of the cause of sudden unexplained death in forensic medicine: Research trends, hot spots and prospects.

To better understand the cause of sudden unexplained death, our group evaluated the scientific results of related studies in a global context. A systematic search of the Web of Science, PubMed and MEDLINE databases identified 2001 studies related to this field published from 1997 to 2020. The studies were analyzed using bibliometric methods, and statistical maps were drawn to explore research trends and research frontiers. Sudden cardiac death and sudden unexpected epilepsy death were the two major causes of sudden unexplained deaths. With the rapid development of high-throughput sequencing technology and bioinformatics in the past 10 years, molecular autopsy has become an effective research method as well as a research hotspot for exploring the cause of sudden unexplained deaths. However, molecular autopsy is underutilized in the investigation of sudden unexpected death in epilepsy. Developing standardized guidelines for diagnostic strategies for the deceased and their families, expanding the screening of mutation spectrum of related diseases, studying the association between variants and diseases in complex genetic diseases, and improving variants interpretation guidelines and disease sequencing databases are future research directions.

Identification of miRNA-mRNA regulatory network associated with the glutamatergic system in post-traumatic epilepsy rats.

Background: Glutamate is one of the most important excitatory neurotransmitters in the mammalian brain and is involved in a variety of neurological disorders. Increasing evidence also shows that microRNA (miRNA) and mRNA pairs are engaged in a variety of pathophysiological processes. However, the miRNA and mRNA pairs that affect the glutamatergic system in post-traumatic epilepsy (PTE) remain unknown. Methods: PTE rats were induced by injecting 0.1 mol/L, 1 µL/min FeCl2 solution. Behavioral scores and EEG monitoring were used to evaluate whether PTE was successfully induced. RNA-seq was used to obtain mRNA and miRNA expression profiles. Bioinformatics analysis was performed to screen differentially expressed mRNAs and miRNAs associated with the glutamatergic system and then predict miRNA-mRNA interaction pairs. Real-time quantitative reverse transcription PCR was used to further validate the expression of the differential miRNAs and mRNAs. The microRNA-mRNA was subject to the Pearson correlation analysis. Results: Eight of the 91 differentially expressed mRNAs were associated with the glutamatergic system, of which six were upregulated and two were downregulated. Forty miRNAs were significantly differentially expressed, with 14 upregulated and 26 downregulated genes. The predicted miRNA-mRNA interaction network shows that five of the eight differentially expressed mRNAs associated with the glutamatergic system were targeted by multiple miRNAs, including Slc17a6, Mef2c, Fyn, Slc25a22, and Shank2, while the remaining three mRNAs were not targeted by any miRNAs. Of the 40 differentially expressed miRNAs, seven miRNAs were found to have multiple target mRNAs associated with the glutamatergic system. Real-time quantitative reverse transcription PCR validation and Pearson correlation analysis were performed on these seven targeted miRNAs-Slc17a6, Mef2c, Fyn, Slc25a22, and Shank2-and six additional miRNAs selected from the literature. Real-time quantitative reverse transcription PCR showed that the expression levels of the mRNAs and miRNAs agreed with the predictions in the study. Among them, the miR-98-5p-Slc17a6, miR-335-5p-Slc17a6, miR-30e-5p-Slc17a6, miR-1224-Slc25a22, and miR-211-5p-Slc25a22 pairs were verified to have negative correlations. Conclusions: Our results indicate that miRNA-mRNA interaction pairs associated with the glutamatergic system are involved in the development of PTE and have potential as diagnostic biomarkers and therapeutic targets for PTE.

Knowledge atlas of post-traumatic epilepsy research: Based on citespace visualization analysis.

The mechanism of posttraumatic epilepsy (PTE) is complicated and the treatment and prognostic effects are not satisfactory. In this study, CiteSpace and VOSviewer are used to analyze the literature related to PTE (January 2000-June 2020). The aspects of the cooperative network (author, institution, and country), keywords co-occurrence, document co-citation clustering, and journal dual-map overlay were analyzed, and the atlas was constructed. The United States, Finland, and other research institutions have frequently published PTE-related articles, thus having richer research results. The relevant research was mostly published in journals, such as Journal of Neurotrauma, Journal of Neuroscience, Brain Research, Neurobiology of Disease. Quantitative diffusion MRI plays a critical role in PTE research. The study on the susceptibility to seizures and the underlying mechanism of PTE received different degrees of attention. The present study provided an in-depth understanding of the research foundation, relevant research results, the current research frontiers, and the main research focus in the PTE field. Herein, we briefly discussed relevant key articles and also provided ideas for future research directions.

α-Helical protein absorption at post-traumatic epileptic foci monitored by Fourier transform infrared mapping.

Tissue analysis by Fourier transform infrared (FTIR) imaging can determine the biodistribution of molecules, without pre-analytical modification. We aimed to study the infrared spectroscopic changes of α-helical proteins at post-traumatic epileptic (PTE) foci by FTIR. FITR mapping was applied to detect α-helical proteins in rat brain tissue samples with post-traumatic epilepsy. Histological examination of brain sections showed that the rat model of PTE was successfully established. At the PTE foci, high α-helical absorption regions were evident, where the color difference and absorption were significantly different from those in the low-absorption regions. This provided a distinctive and characteristic pattern at the site of lesions. The use of FTIR imaging means that it is possible to measure the molecular structural changes resulting from PTE pathologies in tissues, providing a novel adjunct to conventional pathological diagnostic techniques.

Identification of MicroRNA-Potassium Channel Messenger RNA Interactions in the Brain of Rats With Post-traumatic Epilepsy.

Background: Dysregulated expression of microRNAs and potassium channels have been reported for their contributions to seizure onset. However, the microRNA-potassium channel gene interactions in traumatic brain injury-induced post-traumatic epilepsy (PTE) remain unknown. Methods: PTE was induced in male rats by intracranial injection with ferrous chloride (0.1 mol/L, 1 µl/min) at the right frontal cortex. Electroencephalography was recorded at 60 min, as well as day 1, 7, and 30, and the behavioral seizures were assessed before injection and at different time points after injection. Rats were killed on day 30 after injection. The right frontal cortex samples were collected and subjected to high throughput messenger RNA (mRNA) and microRNA sequencing. A network of differentially expressed potassium channel mRNAs and microRNAs was constructed using OryCun2.0 and subjected to Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses. The differential mRNA and microRNA expressions were verified using quantitative real-time-PCR. The microRNA-mRNA was subject to the Pearson correlation analysis. Results: A PTE rat model was successfully established, as evidenced by behavioral seizures and epileptiform discharges on electroencephalography in PTE rats compared with sham rats. Among the 91 mRNAs and 40 microRNAs that were significantly differentially expressed in the PTE rat brain, 4 mRNAs and 10 microRNAs were associated with potassium channels. Except for potassium calcium-activated channel subfamily N member 2, the other three potassium channel mRNAs were negatively correlated with seven microRNAs. These microRNA-mRNA pairs were enriched in annotations and pathways related to neuronal ion channels and neuroinflammation. Quantitative real-time-PCR and correlation analysis verified negative correlations in miR-449a-5p-KCNH2, miR-98-5p-KCNH2, miR-98-5p-KCNK15, miR-19b-3p-KCNK15, and miR-301a-3p-KCNK15 pairs. Conclusion: We identified microRNA-potassium channel mRNA interactions associated with PTE, providing potential diagnostic markers and therapeutic targets for PTE.

Histopathology mapping of biochemical changes in diffuse axonal injury by FTIR micro-spectroscopy.

The diagnosis of diffuse axonal injury (DAI) is an important task in forensic pathology and clinical medicine. This study aimed to explore the use of Fourier transform infrared spectroscopy (FTIR) to detect DAI. The DAI area of the rat model was detected point by point by the FTIR-mapping system. Infrared spectral data of DAI were obtained by selecting the amide A band, CH3 symmetric stretching, collagen triple-helix structure and asymmetric stretching vibrational frequency of nucleic acid and phospholipid PO2 as the target peak positions. The system can automatically draw infrared spectral color pathological images. In the DAI group, the amide A protein secondary amine N-H stretching vibration and the collagen triple-helix structure of the high-absorption area were consistent with the DAI area confirmed by the silver and ß-APP staining. The CH3 symmetric stretching, nucleic acid and phospholipid PO2 symmetric stretching vibration absorption spectra showed no significant differences between the experimental and verification groups. The FTIR-mapping technique can visually express the molecular characteristics of DAI, which is expected to be applied to the pathological diagnosis of DAI.

Modelling of the ICF core sets for chronic ischemic heart disease using the LASSO model in Chinese patients.

BACKGROUND: This study aimed to examine the associations among the International Classification of Functioning, Disability, and Health (ICF) core sets relevant to chronic ischemic heart disease (CIHD) using the least absolute shrinkage and selection operator (LASSO) model based on the ICF core sets scale in Chinese patients. METHODS: This was a prospective study of 120 patients with CIHD selected from January 2013 to June 2014 at the Fada Institute of Forensic Medicine & Science (Beijing, China). Functioning was qualified using the ICF core sets checklist for CIHD (Chinese version). The variables of core set categories of the ICF assessment scale for CIHD were entered into the LASSO model for mining dependencies among those variables. Graphical modeling was applied using LASSO generalized linear models. RESULTS: "Muscle endurance functions", "sensations associated with cardiovascular and respiratory functions", "blood vessel functions", and "heart functions" were the most injured in CIHD status. "Recreation and leisure" and "intimate relationships" were the most affected in CIHD status. "General social support services, systems, and policies" and "acquaintances, peers, colleagues, neighbors, and community members" were important for the outcome of functional status of the CIHD patient. "Economic self-sufficiency" and "family relationships" of the CIHD patient were not undermined in most cases. CONCLUSIONS: Graphical modeling can be used to describe associations between different areas of functioning in CIHD patients. The results suggest that these associations could be used as basis to improve rehabilitation and provide a deeper understanding of functioning in Chinese CIHD patients.

A Study on the Estimation of Postmortem Interval Based on Environmental Temperature and Concentrations of Substance in Vitreous Humor.

A method to determine postmortem interval (PMI) based on environmental temperature and the concentrations of vitreous humor (VH) molecules were explored. Rabbit carcasses were placed in a chamber at 5, 15, 25, or 35°C, and 80-100 µL of VH was collected with the double-eye alternating micro-sampling method every 12 h. A Roche DPPI biochemical analyzer was used to measure the concentrations of six substances in VH samples. The interpolation function model and mixed-effect model were employed for data fitting to establish equations for PMI estimation. The concentrations of K+ , P, Mg2+ , creatinine (CRE), and urea nitrogen (UN) exhibited an upward trend with increasing PMI in all temperature groups, while the concentration of Ca2+ showed a downward trend. Validation results using K+ and Mg2+ ions revealed that the mixed-effect model provided a better estimation than the interpolation function model using the data from our experiment. However, both models were able to estimate PMI using temperature and VH molecule concentrations.

Chemical Analysis in the Corpus Callosum Following Traumatic Axonal Injury using Fourier Transform Infrared Microspectroscopy: A Pilot Study.

Evaluating traumatic axonal injury remains challenging in clinical and forensic sciences as its identification is difficult using routine diagnostic methods. This study used Fourier transform infrared microspectroscopy to detect TAI within the corpus callosum in an animal model. Protein conformational analysis revealed significantly increased ß-sheet and ß-turn contents paralleled by a decrease in α-helix content at 24 h postinjury, while the antiparallel ß-sheet content was decreased at 12 h postinjury. Compared with the control group, the lipid/protein ratio was significantly reduced in all of the injured groups. At 24 h postinjury, there were increases in the olefinic=CH and CH3 group of lipids accompanied by the decreased CH2 group, but the results at 12 and 72 h were contrary to that at 24 h. Our study showed that FTIRM could differentiate injured from normal white matter at different time points following TBI via examination of these infrared spectral parameters.

Interpolation function estimates post mortem interval under ambient temperature correlating with blood ATP level.

OBJECTIVE: We developed a mathematical model using interpolation function, to characterize the correlation between blood ATP levels in the right ventricle of rabbit and post mortem interval (PMI) at different ambient temperatures. METHODS: Forty-eight healthy rabbits were randomly divided into 6 groups of 8 each. The sacrificed rabbits were maintained in calorstats at 10°C, 15°C, 20°C, 25°C, 30°C and 35°C, respectively. Blood from the right ventricle was sampled every 4h until 72h after death. At different time points, ATP concentrations in the blood samples were measured using an ATP fluorescence rapid detector, and then displayed on the detector screen in the form of relative light units (RLU). Relationship between PMI and ATP degradation levels was investigated statistically by SPSS 17.0 and MATLAB 10.0 software. RESULTS: We obtained six regression equations (Ra(2)=0.887-0.929) with RLU values at PMIs of 72h (10°C), 60h (15°C), 56h (20°C), 52h (25°C), 40h (30°C) and 32h (35°C), and an interpolation function (Ra(2)=0.930) was established with PMI as the dependent variable (z), RLU value as independent variable (x) and temperature as independent variable (y). CONCLUSION: Interpolation function is an appropriate choice for PMI estimation by weakening influence of ambient temperature.

Preliminary study on diffuse axonal injury by Fourier transform infrared spectroscopy histopathology imaging.

The objective of this study was to evaluate the application of Fourier transform infrared (FTIR) spectroscopy for detecting diffuse axonal injury (DAI) in a mouse model. Brain tissues from DAI mouse model were prepared with H&E, silver, and ß-amyloid precursor protein (ß-APP) immunohistochemistry stains and were also studied with FTIR. The infrared spectrum images showed high absorption of amide II in the subcortical white matter of the experimental mouse brain, while there was no obvious expression of amide II in the control mouse brain. The areas with high absorption of amide II were in the same distribution as the DAI region confirmed by the silver and ß-APP studies. The result suggests that high absorption of amide II correlates with axonal injury. The use of FTIR imaging allows the biochemical changes associated with DAI pathologies to be detected in the tissues, thus providing an important adjunct method to the current conventional pathological diagnostic techniques.

Sudden unexpected death due to Graves' disease during physical altercation.

We report a case of a 30-year-old woman who suddenly collapsed after having a physical altercation with her husband. Despite immediate resuscitation, she died on arrival at the hospital. The victim's parents requested an autopsy because they believed that their daughter was killed by her husband. Postmortem examination revealed that the victim had a diffusely enlarged thyroid gland and cardiomegaly with left ventricular hypertrophy. There was no evidence of significant trauma on the body. Further postmortem thyroid function tests and review of her medical history indicated that her death was due to Graves' disease. To the best of our knowledge, this is the first case reported of sudden death due to cardiac arrhythmia from Graves' disease induced by physical and emotional stress associated with the criminal activity of another person. The autopsy findings are described. In addition, the literature is reviewed and the significance of postmortem evaluation of thyroid hormones in the cases of sudden death is discussed.

Changes in ATP levels in rabbit blood and its application for estimation of the postmortem interval.

Relationship between ATP changes of rabbit blood and postmortem interval (PMI) was studied. Twenty-four healthy rabbits were sacrificed and randomly divided into 3 groups with 8 rabbits of each group. The bodies of three groups were placed in calorstat at temperature of 15°C, 25°C and 35°C, respectively. The blood from the right ventricle was sampled through indwelling needle each 4 h until 72 h after death. ATP levels in the blood samples were measured by using ATP fluorescence rapid detection technique at different PMIs. Blood ATP levels slightly increased in the early stage after death and then constantly declined at all temperatures (15°C, 25°C, and 35°C). Cubic polynomial regression equations with log[ATP] as dependent variable (y) and PMI as independent variable (x) at different temperatures and the optimal time period were established as followed: Under 15°C and during 16-64 h after death, y=-3.027×10(-5)x(3)+0.003x(2)-0.096x-10.625 (R a (2)=0.992, P<0.001); under 25°C and during 8-56 h after death, y=-2.921×10(-5)x(3)+0.002x(2)-0.059x-11.186 (R a (2)=0.989, P<0.001); under 35dgC and during 4-36 h after death, y=-9.769×10(-5)x(3)+ 0.005x(2)-0.117x-11.166 (R a (2)=0.991, P<0.001). The changes in ATP levels in blood collected from right ventricle of rabbit cadavers showed relatively stable and regular degradation within 72 h after death at different temperatures.