Real-world evidence of avatrombopag for the treatment of immune thrombocytopenia intolerant or ineffective to eltrombopag/hetrombopag.

Due to the limited real-world research on the application of avatrombopag (AVA) for immune thrombocytopenia (ITP) in China, we evaluated the effectiveness and safety of AVA in clinical practice. We included 121 adult ITP patients treated with AVA across three medical centres. Based on the reasons for choosing AVA, these patients were divided into eltrombopag (ELT)/hetrombopag (HET) intolerance group (IG), and ELT/HET unresponsive group (UG). Compared with UG, more patients in IG had a history of liver disease and received fewer treatments before AVA. Amongst all patients, 83% had platelet response (≥30 × 109/L) after AVA and 62% achieved complete response (≥100 × 109/L, CR). Sixty-two percent in IG and 56% in UG were able to discontinue more than one concomitant ITP medication. A total of 17 patients underwent multiple switches of thrombopoietin receptor agonists (TPO-RAs), resulting in an 88% platelet response rate. Sixty-three patients discontinued AVA, 27% were due to unaffordability. AVA was well tolerated in most patients. In the ITP population, AVA proved effective and safe, particularly in patients intolerant or unresponsive to ELT/HET. Patients benefited from TPO-RAs switches, particularly those undergoing multiple switches. However, many patients struggled with the long-term financial burden of AVA.

Transcriptional level of inflammation markers associates with short-term brain structural changes in first-episode schizophrenia.

BACKGROUND: Inflammation has been implicated in the pathology of schizophrenia and may cause neuronal cell death and dendrite loss. Neuroimaging studies have highlighted longitudinal brain structural changes in patients with schizophrenia, yet it is unclear whether this is related to inflammation. We aim to address this question, by relating brain structural changes with the transcriptional profile of inflammation markers in the early stage of schizophrenia. METHODS: Thirty-eight patients with first-episode schizophrenia and 51 healthy controls were included. High-resolution T1-weighted magnetic resonance imaging (MRI) and clinical assessments were performed at baseline and 2 ~ 6 months follow-up for all subjects. Changes in the brain structure were analyzed using surface-based morphological analysis and correlated with the expression of immune cells-related gene sets of interest reported by previous reviews. Transcriptional data were retrieved from the Allen Human Brain Atlas. Furthermore, we examined the brain structural changes and peripheral inflammation markers in association with behavioral symptoms and cognitive functioning in patients. RESULTS: Patients exhibited accelerated cortical thickness decrease in the left frontal cortices, less decrease or an increase in the superior parietal lobule and right lateral occipital lobe, and increased volume in the bilateral pallidum, compared with controls. Changes in cortical thickness correlated with the transcriptional level of monocyte across cortical regions in patients (r = 0.54, p < 0.01), but not in controls (r = - 0.05, p = 0.76). In addition, cortical thickness change in the left superior parietal lobule positively correlated with changes in digital span-backward test scores in patients. CONCLUSIONS: Patients with schizophrenia exhibit regional-specific cortical thickness changes in the prefrontal and parietooccipital cortices, which is related to their cognitive impairment. Inflammation may be an important factor contributing to cortical thinning in first-episode schizophrenia. Our findings suggest that the immunity-brain-behavior association may play a crucial role in the pathogenesis of schizophrenia.

Differential expression of diacylglycerol kinase ζ is involved in inferior parietal lobule-related dysfunction in schizophrenia with cognitive impairments.

BACKGROUND: Cognitive impairment is the main factor in the poor prognosis of schizophrenia, but its mechanism remains unclear. The inferior parietal lobule (IPL) is related to various clinical symptoms and cognitive impairment in schizophrenia. We aimed to explore the relationship between IPL-related functions and cognitive impairment in schizophrenia. METHODS: 136 schizophrenia patients and 146 demographically matched healthy controls were enrolled for a cross-sectional study. High-spatial-resolution structural and resting-state functional images were acquired to demonstrate the alternations of brain structure and function. At the same time, the digit span and digit symbol coding tasks of the Chinese Wechsler Adult Intelligence Test Revised (WAIS-RC) were utilized in assessing the subjects' cognitive function. Patients were divided into cognitive impairment and normal cognitive groups according to their cognitive score and then compared whether there were differences between the three groups in fractional amplitude of low-frequency fluctuation (fALFF). In addition, we did a correlation analysis between cognitive function and the fALFF for the left IPL of patients and healthy controls. Based on the Allen Human Brain Atlas, we obtained genes expressed in the left IPL, which were then intersected with the transcriptome-wide association study results and differentially expressed genes in schizophrenia. RESULTS: Grouping of patients by the backward digit span task and the digit symbol coding task showed differences in fALFF values between healthy controls and cognitive impairment patients (P < 0.05). We found a negative correlation between the backward digit span task score and fALFF of the left IPL in healthy controls (r = - 0.388, P = 0.003), which was not seen in patients (r = 0.203, P = 0.020). In addition, none of the other analyses were statistically significant (P > 0.017). In addition, we found that diacylglycerol kinase ζ (DGKζ) is differentially expressed in the left IPL and associated with schizophrenia. CONCLUSION: Our study demonstrates that the left IPL plays a vital role in cognitive impairment in schizophrenia. DGKζ may act as an essential regulator in the left IPL of schizophrenia patients with cognitive impairment.

The correlation between dynamic functional architecture and response to electroconvulsive therapy combined with antipsychotics in schizophrenia.

Attempts to determine why some patients respond to electroconvulsive therapy (ECT) are valuable in schizophrenia. Schizophrenia is associated with aberrant dynamic functional architecture, which might impact the efficacy of ECT. We aimed to explore the relationship between pre-treatment temporal variability and ECT acute efficacy. Forty-eight patients with schizophrenia and 30 healthy controls underwent functional magnetic resonance imaging to examine whether patterns of temporary variability of functional architecture differ between high responders (HR) and low responders (LR) at baseline. Compared with LR, HR exhibited significantly abnormal temporal variability in right inferior front gyrus (IFGtriang.R), left temporal pole (TPOsup.L) and right middle temporal gyrus (MTG.R). In the pooled patient group, ∆PANSS was correlated with the temporal variability of these regions. Patients with schizophrenia with a distinct dynamic functional architecture appear to reveal differential response to ECT. Our findings provide not only an understanding of the neural functional architecture patterns that are found in schizophrenia but also the possibility of using these measures as moderators for ECT selection.

Magnetic Resonance Imaging throughout the Clinical Course of Schizophrenia: Neurobiological Underpinnings and Clinical Implications.

As a non-invasive detection method and an advanced imaging method, magnetic resonance imaging (MRI) has been widely used in the research of schizophrenia. Although a large number of neuroimaging studies have confirmed that MRI can display abnormal brain phenotypes in patients with schizophrenia, no valid uniform standard has been established for its clinical application. On the basis of previous evidence, we argue that MRI is an important tool throughout the whole clinical course of schizophrenia. The purpose of this commentary is to systematically describe the role of MRI in schizophrenia and to provide references for its clinical application.

[Laparoscopic management of persistent Müllerian duct syndrome: A case report and pedigree investigation].

OBJECTIVE: To investigate the clinical characteristics, diagnosis, treatment and etiology of persistent Müllerian duct syndrome (PMDS). METHODS: A 3-year-old boy was diagnosed with PMDS according to the clinical manifestations and the results of ultrasonography, laboratory examinations and earlier surgical examination. We performed genetic tests for the patient and his family members, removed the infantile uterus by laparoscopic wedge hysterectomy, biopsied and descended the bilateral testes, and ligated the bilateral internal rings, followed by a retrospective analysis and review of relevant literature. RESULTS: The operation was successful. Gonad biopsy revealed testis tissue, and PMDS was confirmed by intraoperative findings and related examinations. Good bilateral testicular blood supply was found during the 6-month follow-up after surgery. Medical exome sequencing showed the AMHR2 gene c.1499G > A (p.Cys500Tyr) mutant homozygote (A/A) in the patient and his sister and mutant heterozygote (G/A) in his parents. CONCLUSIONS: Laparoscopy is definitely effective for the treatment of PMDS. In surgery, the infantile uterus should be removed in case of good blood supply to the testis, and so were the bilateral testes if they cannot be descended. The homozygous mutation in the AMHR2 gene c. 1499G > A (p. Cys500Tyr) can lead to male PMDS. Pedigree investigation may provide some evidence for possible fertility in PMDS patients.

Reconstruction of Transmission Pairs for Novel Coronavirus Disease 2019 (COVID-19) in Mainland China: Estimation of Superspreading Events, Serial Interval, and Hazard of Infection.

BACKGROUND: Knowledge on the epidemiological features and transmission patterns of novel coronavirus disease (COVID-19) is accumulating. Detailed line-list data with household settings can advance the understanding of COVID-19 transmission dynamics. METHODS: A unique database with detailed demographic characteristics, travel history, social relationships, and epidemiological timelines for 1407 transmission pairs that formed 643 transmission clusters in mainland China was reconstructed from 9120 COVID-19 confirmed cases reported during 15 January-29 February 2020. Statistical model fittings were used to identify the superspreading events and estimate serial interval distributions. Age- and sex-stratified hazards of infection were estimated for household vs nonhousehold transmissions. RESULTS: There were 34 primary cases identified as superspreaders, with 5 superspreading events occurred within households. Mean and standard deviation of serial intervals were estimated as 5.0 (95% credible interval [CrI], 4.4-5.5) days and 5.2 (95% CrI, 4.9-5.7) days for household transmissions and 5.2 (95% CrI, 4.6-5.8) and 5.3 (95% CrI, 4.9-5.7) days for nonhousehold transmissions, respectively. The hazard of being infected outside of households is higher for people aged 18-64 years, whereas hazard of being infected within households is higher for young and old people. CONCLUSIONS: Nonnegligible frequency of superspreading events, short serial intervals, and a higher risk of being infected outside of households for male people of working age indicate a significant barrier to the identification and management of COVID-19 cases, which requires enhanced nonpharmaceutical interventions to mitigate this pandemic.

Immunohistochemical basigin expression level in thyroid cancer tissues.

BACKGROUND: Thyroid cancer (TC) is the most common endocrine malignancy; basigin (also known as BSG) plays a crucial role in tumor cell invasion, metastasis, and angiogenesis. This study was designed to identify the change of BSG expression in TC and its possible potential mechanism. METHODS: The BSG expression levels in TC were demonstrated using data collected from in-house immunohistochemical (IHC), RNA-sequencing (RNA-seq), microarrays, and literatures. Integrated analysis was performed to determined BSG expression levels in TC comprehensively. The Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed with the integration of BSG co-expressed genes and differentially expressed genes (DEGs) in TC tissues to explore the potential mechanisms of BSG in TC. RESULTS: The protein expression level of BSG was significantly higher in TC cases based on the IHC experiments. In addition, the combined SMD for BSG expression was 0.39 (p < 0.0001), the diagnostic odds ratio was 3.69, and the AUC of the sROC curve was 0.6986 using 1182 TC cases and 437 non-cancerous cases from 17 independent datasets. Furthermore, BSG co-expressed genes tended to be enriched in gene terms of the extracellular matrix (ECM), cell adhesion, and cell-cell interactions. The expression levels of nine hub BSG co-expressed genes were markedly upregulated in TC cases. CONCLUSION: BSG expression levels were closely correlated with the progression of TC and may affect the signals of the ECM, cell adhesion, and cell-cell interactions.

Transition-Metal-Complex Cationic Dyes Photosensitive to Two Types of 2D Layered Silver Bromides with Visible-Light-Driven Photocatalytic Properties.

With mixed transition-metal (TM) complex, alkali-metal cations, or halogen anions as structure-directing agents, two types of two-dimensional (2D) layered inorganic-organic hybrid silver bromides were prepared and structurally characterized as K[TM(2,2-bipy)3]2Ag6Br11 (TM = Ni (1), Co (2), Zn (3), Fe (4)) and [TM(2,2-bipy)3]2Ag13Br17 (TM = Ni (5), Co (6), Zn (7), Fe (8)). Compounds 1-4 feature 2D microporous anionic [Ag6Br11]5- layers composed of [Ag3Br7] secondary building units based on AgBr4 tetrahedral units, and compounds 5-8 contain 2D [Ag13Br16]3- layers built from the one-dimensional complex [Ag8Br12] and [Ag5Br8] chains. The photosensitization of TM complex dyes led to the narrow semiconducting behaviors with tunable band gaps of 1.73-2.71 eV for the title compounds, which result in excellent and stable photocatalytic degradation activities over organic pollutants under visible-light irradiation. The studies of photocatalytic mechanism based on radical-trapping experiments and electronic band structural calculation show that the TM complex cations play important roles in the photocatalytical activities and photochemical stabilities due to their excellent separating abilities for photogenerated carriers. This technique affords one new type of visible-light-driven photocatalyst and facilitates the integration of 2D layered materials and semiconducting photocatalytic properties into one hybrid d10 TM halogenide.

Interleukin-7 is decreased and maybe plays a pro-inflammatory function in primary immune thrombocytopenia.

Primary immune thrombocytopenia (ITP) is an autoimmune disease with many immune dysfunctions, including over-proliferation and apoptosis resistance of auto-reactive lymphocytes. This study aimed to determine the effects of interleukin (IL)-7 on the cytokine production and survival of peripheral blood mononuclear cells and bone marrow mononuclear cells from ITP patients. We found that the plasma IL-7 levels in peripheral blood from ITP patients were lower than that of the normal controls, and it had positive correlation with platelet counts. However, the levels of IL-7 did not change in bone marrow serum of ITP patients compared with that of normal controls. The result of further stimulation experiments in vitro showed that IL-7 up-regulated the apoptosis of autologous platelets, promoted the proliferation and secretion of interferon-γ, tumor necrosis factor-α as well as IL-10 of lymphocyte both from peripheral blood and bone marrow. As the role of IL-7 in apoptosis-resistance and stimulation of pro-inflammatory cytokines, we speculated that decreased IL-7 in peripheral blood, maybe, is a consequence of the negative feedback of the pro-inflammatory function in ITP patients.

Large-scale mechanism hypothesis and research prospects of cognitive impairment in schizophrenia based on magnetic resonance imaging.

Cognitive impairments in schizophrenia are pivotal clinical issues that need to be solved urgently. However, the mechanism remains unknown. It has been suggested that cognitive impairments in schizophrenia are associated with connectome damage, and are especially relevant to the disrupted hub nodes in the frontal and parietal lobes. Activating the dorsolateral prefrontal cortex (DLPFC) via repetitive transcranial magnetic stimulation (rTMS) could result in improved cognition. Based on several previous magnetic resonance imaging (MRI) studies on schizophrenia, we found that the first-episode patients showed connectome damage, as well as abnormal activation and connectivity of the DLPFC and inferior parietal lobule (IPL). Accordingly, we proposed that DLPFC-IPL pathway destruction might mediate connectome damage of cognitive impairments in schizophrenia. In the meantime, with the help of multimodal MRI and noninvasive neuromodulation tool, we may not only validate the hypothesis, but also find IPL as the potential intervention target for cognitive impairments in schizophrenia.

Imaging Transcriptomics of the Brain for Schizophrenia.

Schizophrenia is a severe mental disorder with a neurodevelopmental origin. Although schizophrenia results from changes in the brain, the underlying biological mechanisms are unknown. Transcriptomics studies quantitative expression changes or qualitative changes of all genes and isoforms, providing a more meaningful biological insight. Magnetic resonance imaging (MRI) techniques play roles in revealing brain structure and function. We give a narrative focused review on the current transcriptome combined with MRI studies related to schizophrenia and summarize the research methodology and content of these studies to identify the research commonalities as well as the implications for future research, in an attempt to provide new insights into the mechanism, clinical diagnosis, and treatments of schizophrenia.

Identification and bioactivity evaluation of twelve previously undescribed depsidone derivatives from Garcinia oligantha.

Phytochemical studies on the leaves and twigs of Garcinia oligantha Merr. led to the isolation of twelve previously undescribed depsidone derivatives (oliganthdepsidones A-L, 1-12). Their structures were elucidated by extensive spectroscopic analysis including 1H and 13C NMR, HSQC, HMBC and NOESY along with HRESIMS. The structures of oliganthdepsidones G and J were finally determined using DFT-NMR chemical shift calculations and DP4+ methods. Cytotoxicity test in four human cancer cell lines indicated that oliganthdepsidone F had relatively strong cytotoxic effect against A375 (melanoma), A549 (lung cancer), HepG2 (liver cancer), and MCF-7 (breast cancer) cell lines with IC50 of 18.71, 15.44, 10.92, and 15.90 µM, respectively. The dose- and time-dependent antiproliferative effects of oliganthdepsidone F on these cell lines were also observed by CCK-8 test. As determined by fluorescent microscopy and flow cytometry in these cell lines, oliganthdepsidone F could promote cell apoptosis, leading to the inhibition of cell proliferation. The results of wound healing assay and transwell assay showed that oliganthdepsidone F could inhibit the migration and invasion of A549 and MCF-7 cell lines in a concentration-dependent manner.

Inhibition of Bcl-6 Expression Ameliorates Asthmatic Characteristics in Mice.

OBJECTIVE: The function of Bcl-6 in T follicular helper (Tfh) cell maturation is indispensable, and Tfh cells play a pivotal role in asthma. This study investigated the impact of Bcl-6 on asthmatic traits. METHODS: The microscopic pathological alterations, airway resistance (AR), and lung compliance (LC) were determined in asthmatic mice and Bcl-6 interference mice. The surface molecular markers of Tfh cells and the Bcl-6 mRNA and protein expression were determined by flow cytometry, RT-qPCR, and Western blotting, respectively. The relationships between the Tfh cell ratio and the IgE and IgG1 concentrations in peripheral blood mononuclear cells (PBMCs) and bronchoalveolar lavage fluid (BALF) were determined. RESULTS: Asthmatic inflammatory changes were observed in the lung tissue and were attenuated by Bcl-6 siRNA and dexamethasone (DXM). Asthmatic mice exhibited an increased AR and a decreased LC, while Bcl-6 siRNA or DXM mitigated these changes. The percentages of Tfh cells and eosinophils were significantly increased in the asthmatic mice, and they significantly decreased after Bcl-6 inhibition or DXM treatment. RT-qPCR and Western blotting analyses revealed that the Bcl-6 expression level in PBMCs was significantly higher in asthmatic mice, and it decreased following Bcl-6 inhibition or DXM treatment. The IgE expression in the serum and BALF and the B cell expression in PBMCs exhibited a similar trend. In asthmatic mice, the ratio of Tfh cells in the peripheral blood showed a strong positive correlation with the IgE levels in the serum and BALF, but not with the IgG1 levels. CONCLUSION: The amelioration of airway inflammation and airway hyper-responsiveness is achieved through Bcl-6 suppression, which effectively hinders Tfh cell differentiation, ultimately resulting in a concurrent reduction in IgE production.

Data challenges for international health emergencies: lessons learned from ten international COVID-19 driver projects.

The COVID-19 pandemic highlighted the importance of international data sharing and access to improve health outcomes for all. The International COVID-19 Data Alliance (ICODA) programme enabled 12 exemplar or driver projects to use existing health-related data to address major research questions relating to the pandemic, and developed data science approaches that helped each research team to overcome challenges, accelerate the data research cycle, and produce rapid insights and outputs. These approaches also sought to address inequity in data access and use, test approaches to ethical health data use, and make summary datasets and outputs accessible to a wider group of researchers. This Health Policy paper focuses on the challenges and lessons learned from ten of the ICODA driver projects, involving researchers from 19 countries and a range of health-related datasets. The ICODA programme reviewed the time taken for each project to complete stages of the health data research cycle and identified common challenges in areas such as data sharing agreements and data curation. Solutions included provision of standard data sharing templates, additional data curation expertise at an early stage, and a trusted research environment that facilitated data sharing across national boundaries and reduced risk. These approaches enabled the driver projects to rapidly produce research outputs, including publications, shared code, dashboards, and innovative resources, which can all be accessed and used by other research teams to address global health challenges.

Chronic thyrotoxic myopathy development is associated with thyroid hormone sensitivity index, predicted by lower-limb fatigue and the squat-up test.

Thyroid hormones (THs) may affect chronic thyrotoxic myopathy (CTM). The relationship between TH sensitivity and CTM is inconsistent. We aimed to investigate the associations between TH sensitivity and the risk of CTM and to screen potential CTMs with strength and function tests. A total of 162 Chinese patients (36.58% men) with Graves' disease were enrolled and divided into CTM and non-CTM groups. TH and sensitivity indices were measured. Muscle power and function were assessed by grip, upper-limb fatigue (ULFT), lower-limb fatigue (LLFT), and squat-up (SUT) tests, and walking pace. Association between sensitivity to TH indices and the risk of developing CTM was assessed via multivariate logistic regression. The diagnostic effectiveness of muscle power and function for predicting CTM was evaluated via receiver operating characteristic (ROC) curves. Thyroid feedback quantile-based index FT3 (TFQIFT3) and the parametric TFQIFT3 (PTFQIFT3), TFQIFT4, and PTFQIFT4 were positively associated with CTM risk by using inverse probability of treatment weighting multivariate logistic regression. For each 1-SD increase in TFQIFT3 and PTFQIFT3, TFQIFT4 and PTFQIFT4, the odds ratios for CTM were 1.67 (95% CI = 1.17-2.48) ,1.64 (95% CI = 1.51-2.93), 1.60 (95%CI = 1.12-2.32), 1.58 (95%CI = 1.11-2.30), respectively. LLFT and SUT best predicted male/female CTM, respectively (AUC = 0.89/0.85). In Graves' disease patients, TH sensitivity is associated with CTM development, which can be predicted by SUT and LLFT results.

[Research of DNA barcode of Teseudinis Carapax et Planstrum and its adulterants based on COI gene sequence].

OBJECTIVE: To use COI gene on the Mauremys reevesii and its adulterants by molecular identification. Search a rapid, accurate method of identification of Teseudinis Carapax et Planstrum and its adulterants. METHOD: We collected 8 species of the authentic and adulterants of teseudinis carapax et planstrum in a nationwide then, extracted DNA, got the COI sequences. Use ContigExpress, Dnaman, Edit Sequence and Mega 5 to analyze the variable site and construct the N-J tree. RESULT: Compare with the authentic Teseudinis Carapax et Planstrum, the adulterant exist lots of variable site. The N-J tree Indicates that the same genus belong together and each species belong to relatively independent branch. CONCLUSION: Based on the COI gene, the technology of DNA bar code can be a excellent identification of Teseudinis Carapax et Planstrum and its adulterants.

COVID-19 spreading patterns in family clusters reveal gender roles in China.

Understanding different gender roles forms part of the efforts to reduce gender inequality. This paper analyses COVID-19 family clusters outside Hubei Province in mainland China during the 2020 outbreak, revealing significant differences in spreading patterns across gender and family roles. Results show that men are more likely to be the imported cases of a family cluster, and women are more likely to be infected within the family. This finding provides new supportive evidence of the 'men as breadwinner and women as homemaker' (MBWH) gender roles in China. Further analyses reveal that the MBWH pattern is stronger in eastern than in western China, stronger for younger than for elder people. This paper offers not only valuable references for formulating gender-differentiated epidemic prevention policies but also an exemplification for studying group differences in similar scenarios.

The shock, the coping, the resilience: smartphone application use reveals Covid-19 lockdown effects on human behaviors.

Human mobility restriction policies have been widely used to contain the coronavirus disease-19 (COVID-19). However, a critical question is how these policies affect individuals' behavioral and psychological well-being during and after confinement periods. Here, we analyze China's five most stringent city-level lockdowns in 2021, treating them as natural experiments that allow for examining behavioral changes in millions of people through smartphone application use. We made three fundamental observations. First, the use of physical and economic activity-related apps experienced a steep decline, yet apps that provide daily necessities maintained normal usage. Second, apps that fulfilled lower-level human needs, such as working, socializing, information seeking, and entertainment, saw an immediate and substantial increase in screen time. Those that satisfied higher-level needs, such as education, only attracted delayed attention. Third, human behaviors demonstrated resilience as most routines resumed after the lockdowns were lifted. Nonetheless, long-term lifestyle changes were observed, as significant numbers of people chose to continue working and learning online, becoming "digital residents." This study also demonstrates the capability of smartphone screen time analytics in the study of human behaviors. Supplementary Information: The online version contains supplementary material available at 10.1140/epjds/s13688-023-00391-9.

Protocol for the automatic extraction of epidemiological information via a pre-trained language model.

The lack of systems to automatically extract epidemiological fields from open-access COVID-19 cases restricts the timeliness of formulating prevention measures. Here we present a protocol for using CCIE, a COVID-19 Cases Information Extraction system based on the pre-trained language model.1 We describe steps for preparing supervised training data and executing python scripts for named entity recognition and text category classification. We then detail the use of machine evaluation and manual validation to illustrate the effectiveness of CCIE. For complete details on the use and execution of this protocol, please refer to Wang et al.2.