Does the size of the neuroendocrine-carcinoma component determine the prognosis of gallbladder cancer?

Background: Gallbladder mixed neuroendocrine-non-neuroendocrine neoplasm generally consists of a gallbladder neuroendocrine tumor and a non-neuroendocrine component. The World Health Organization (WHO) in 2019 established a guideline requiring each component, both neuroendocrine and non-neuroendocrine, to account for a minimum of 30% of the tumor mass. Methods: Patients after surgery resection and diagnosed at microscopy evaluation with pure gallbladder neuroendocrine carcinoma (GBNEC), gallbladder mixed adeno-neuroendocrine carcinoma (GBMANEC, GBNEC≥30%), and gallbladder carcinoma mixed with a small fraction of GBNEC (GBNEC <30%) between 2010 and 2022 at West China Hospital of Sichuan University were collated for the analyses. Demographic features, surgical variables, and tumor characteristics were evaluated for association with patients' overall and recurrence-free survival (OS and RFS). Results: The study included 26 GBNEC, 11 GBMANEC, 4 gallbladder squamous-cell carcinoma (GBSCC), and 7 gallbladder adenocarcinoma (GBADC) mixed with a small fraction of GBNEC. All patients had stage III or higher tumors (AJCC8th edition). The majority of included patients (79.17%) underwent curative surgical resection (R0), with only ten patients having tumoral resection margins. In the analysis comparing patients with GBNEC percentage (GBNEC≥30% vs. GBNEC<30%), the basic demographics and tumor characteristics of most patients were comparable. The prognosis of these patients was also comparable, with a median OS of 23.65 months versus 20.40 months (P=0.13) and a median RFS of 17.1 months versus 12.3 months (P=0.24). However, patients with GBADC or GBSCC mixed with GBNEC <30% had a statistically significant decreased OS and RFS (both P<0.0001)) compared with GBNEC and GBMANEC. Patients with GBNEC who exhibited advanced tumor stages and lymphovascular invasion had a higher risk of experiencing worse overall survival (OS) and recurrence-free survival (RFS). However, a 30% GBNEC component was not identified as an independent risk factor. Conclusion: Patients with GBNEC were frequently diagnosed at advanced stages and their prognosis is poor. The 30% percentage of the GBNEC component is not related to the patient's survival.

Differential short-term and long-term metabolic and cytokine responses to infection of severe fever with thrombocytopenia syndrome virus.

INTRODUCTION: Severe fever with thrombocytopenia syndrome (SFTS) is an emerging infectious disease caused by the SFTS virus (SFTSV), which has a wide geographic distribution. The primary clinical manifestations of SFTS are fever and thrombocytopenia, with multiorgan failure being the leading cause of death. While most patients recover with treatment, little is known about the potential long-term metabolic effects of SFTSV infection. OBJECTIVES: This study aimed to shed light on dysregulated metabolic pathways and cytokine responses following SFTSV infection, which pose significant risks to the short-term and long-term health of affected individuals. METHODS: Fourteen laboratory-confirmed clinical SFTS cases and thirty-eight healthy controls including 18 SFTSV IgG-positive and 20 IgG-negative individuals were recruited from Taizhou city of Zhejiang province, Eastern China. Inclusion criteria of healthy controls included residing in the study area for at least one year, absence of fever or other symptoms in the past two weeks, and no history of SFTS diagnosis. Ultrahigh-performance liquid chromatography-mass spectrometry (UHPLC-MS) was used to obtain the relative abundance of plasma metabolites. Short-term metabolites refer to transient alterations present only during SFTSV infection, while long-term metabolites persistently deviate from normal levels even after recovery from SFTSV infection. Additionally, the concentrations of 12 cytokines were quantified through fluorescence intensity measurements. Differential metabolites were screened using orthogonal projections to latent structures discriminant analysis (OPLS-DA) and the Wilcoxon rank test. Metabolic pathway analysis was performed using MetaboAnalyst. Between-group differences of metabolites and cytokines were examined using the Wilcoxon rank test. Correlation matrices between identified metabolites and cytokines were analyzed using Spearman's method. RESULTS AND CONCLUSIONS: We screened 122 long-term metabolites and 108 short-term metabolites by analytical comparisons and analyzed their correlations with 12 cytokines. Glycerophospholipid metabolism (GPL) was identified as a significant short-term metabolic pathway suggesting that the activation of GPL might be linked to the self-replication of SFTSV, whereas pentose phosphate pathway and alanine, aspartate, and glutamate metabolism were indicated as significant long-term metabolic pathways playing a role in combating long-standing oxidative stress in the patients. Furthermore, our study suggests a new perspective that α-ketoglutarate could serve as a dietary supplement to protect recovering SFTS patients.

Transition-Metal-Free Hydrodefluoroamination of Trifluoromethyl Enones for the Synthesis of α-Fluoroenamides.

A three-component strategy was developed to enable hydrodefluoroamination of ß-trifluoromethyl enones by selectively activating two C(sp3)-F bonds in the trifluoromethyl group. The method involved a sequence of carbonyl reduction, hydrodefluorination, and defluoroamination under transition-metal-free conditions. Synthetically useful (E)-stereospecific α-fluoroenamides were obtained in good yields with diverse functional group tolerance, which could be easily transformed into valuable organofluorides and heterocycles. The carbonyl auxiliary exerts both electronic and steric impacts on the CF3-alkenes, allowing for controllable and selective defluorination.

Integrative analyses of bulk and single-cell transcriptomics reveals the infiltration and crosstalk of cancer-associated fibroblasts as a novel predictor for prognosis and microenvironment remodeling in intrahepatic cholangiocarcinoma.

BACKGROUND: Intrahepatic cholangiocarcinoma (ICC) is a highly malignant neoplasm and characterized by desmoplastic matrix. The heterogeneity and crosstalk of tumor microenvironment remain incompletely understood. METHODS: To address this gap, we performed Weighted Gene Co-expression Network Analysis (WGCNA) to identify and construct a cancer associated fibroblasts (CAFs) infiltration biomarker. We also depicted the intercellular communication network and important receptor-ligand complexes using the single-cell transcriptomics analysis of tumor and Adjacent normal tissue. RESULTS: Through the intersection of TCGA DEGs and WGCNA module genes, 784 differential genes related to CAFs infiltration were obtained. After a series of regression analyses, the CAFs score was generated by integrating the expressions of EVA1A, APBA2, LRRTM4, GOLGA8M, BPIFB2, and their corresponding coefficients. In the TCGA-CHOL, GSE89748, and 107,943 cohorts, the high CAFs score group showed unfavorable survival prognosis (p < 0.001, p = 0.0074, p = 0.028, respectively). Additionally, a series of drugs have been predicted to be more sensitive to the high-risk group (p < 0.05). Subsequent to dimension reduction and clustering, thirteen clusters were identified to construct the single-cell atlas. Cell-cell interaction analysis unveiled significant enhancement of signal transduction in tumor tissues, particularly from fibroblasts to malignant cells via diverse pathways. Moreover, SCENIC analysis indicated that HOXA5, WT1, and LHX2 are fibroblast specific motifs. CONCLUSIONS: This study reveals the key role of fibroblasts - oncocytes interaction in the remodeling of the immunosuppressive microenvironment in intrahepatic cholangiocarcinoma. Subsequently, it may trigger cascade activation of downstream signaling pathways such as PI3K-AKT and Notch in tumor, thus initiating tumorigenesis. Targeted drugs aimed at disrupting fibroblasts-tumor cell interaction, along with associated enrichment pathways, show potential in mitigating the immunosuppressive microenvironment that facilitates tumor progression.

Seroprevalence and transmission of severe fever with thrombocytopenia syndrome virus in a coastal endemic area in Southeastern China.

Severe fever with thrombocytopenia syndrome (SFTS) is a newly emerged tick-borne viral zoonosis and widely prevalent in China, Japan and South Korea. Most reported SFTS cases have been identified in mountainous and hilly areas, with a few in island areas. In this study, we conducted a systematic investigation about natural infection of SFTS virus (SFTSV) among humans, animals and ticks in a coastal endemic prefecture, containing island, plains and mountain settings, in Zhejiang Province, Southeastern China. From July 2020 to June 2021, 1117 participants completed a survey with questionnaire interview and serum testing. Meanwhile, 862 serum samples of domestic animals, 275 spleen tissue samples of wild animals and 829 ticks representing five species (predominantly Haemaphysalis longicornis and Rhipicephalus sanguineus sensu lato) were collected. The seroprevalence of anti-SFTSV total antibody and IgM antibody among the participants was 4.8 % (54/1117) and 0.6 % (7/1117), respectively. Multivariate logistic regression analysis indicated that living in the island area (OR=2.66; 95 %CI: 1.04-6.80; P = 0.041) was significantly associated with seropositivity of total antibody to SFTSV. Furthermore, a higher seroprevalence was observed in domestic animals (36.1 %), while the SFTSV-RNA infection rate was 0.4 % in wild animals and the minimum infection rate (MIR) was 0.8 % for all tick species combined. The only tick species infected with SFTSV was H. longicornis. The prevalence of SFTSV infection in the island area, manifested by anti-SFTSV total antibody (P = 0.012) and IgM antibody (P = 0.004) among humans, anti-SFTSV total antibody (P<0.001) among domestic animals, and SFTSV-RNA among ticks (P = 0.022), was significantly higher than that in the mountainous area and the plain area. Furthermore, phylogenetic analysis showed that SFTSV sequences obtained from ticks in the island area were clustered with reported strains in Japan and South Korea. These results suggest that islands in the study area might be an important natural focus of SFTSV.

Laparoscopic versus open liver resection for intrahepatic cholangiocarcinoma: a systematic review of propensity score-matched studies.

Outcomes of laparoscopic liver resection (LLR) versus open LR (OLR) for intrahepatic cholangiocarcinoma (ICCA) are heterogeneous. We aimed to compare LLR and OLR for ICCA based on propensity-score-matched (PSM) studies. Two reviewers independently searched the online databases (PubMed, Embase, and Cochrane Library) for PSM studies that compared LLR and OLR for ICCA. The Ottawa-Newcastle Quality Assessment Scale with a cutoff of ≥ 7 was used to define higher-quality literature. Only 'high-quality' PSM analyses of the English language that met all our inclusion criteria were considered. A total of ten PSM trials were included in the analyses. Compared with OLR, although the lymph node dissection (LND) (RR = 0.67) and major hepatectomy rates were lower in the LLR group (RR = 0.87), higher R0 resections (RR = 1.05) and lower major complications (Clavien-Dindo grade ≥ III) (RR = 0.72) were also observed in the LLR group. In addition, patients in the LLR group showed less estimated blood loss (MD = - 185.52 ml) and shorter hospital stays as well (MD = - 2.75 days). Further analysis found the overall survival (OS) (HR = 0.91), disease-free survival (DFS) (HR = 0.95), and recurrence-free survival (HR = 0.80) for patients with ICCA after LLR were all comparable to those of OLR. LLR for selected ICCA patients may be technically safe and feasible, providing short-term benefits and achieving oncological efficacy without compromising the long-term survival of the patients.

DNA damage repair mutations in pancreatic cancer- prognostic or predictive?

Objective: The efficacy of platinum-based chemotherapy (PtCh) for pancreatic cancer (PC) patients with DNA damage repair gene mutations (DDRm) compared to those without DDRm remains uncertain. Methods: After a thorough database searching in PubMed, Embase, and Web of Science, a total of 19 studies that met all the inclusion criteria were identified. The primary outcomes were overall survival (OS) and progression-free survival (PFS) for PC patients with DDRm versus those without DDRm after PtCh. Results: Patients with advanced-stage PC who have DDRm tend to have longer OS compared to patients without DDRm, regardless of their exposure to PtCh (HR=0.63; I2 = 66%). Further analyses indicated that the effectiveness of PtCh for OS was modified by DDRm (HR=0.48; I2 = 59%). After the first- line PtCh (1L-PtCh), the PFS of advanced-stage PC with DDRm was also significantly improved (HR=0.41; I2 = 0%). For patients with resected PC, regardless of their exposure to PtCh, the OS for patients with DDRm was comparable to those without DDRm (HR=0.82; I2 = 71%). Specifically, for patients with resected PC harboring DDRm who received PtCh (HR=0.85; I2 = 65%) and for those after non-PtCh (HR=0.87; I2 = 0%), the presence of DDRm did not show a significant association with longer OS. Conclusion: 1L-PtCh treatment is correlated with favorable survival for advanced-stage PC patients with DDRm. For resected-stage PC harboring DDRm, adjuvant PtCh had limited effectiveness. The prognostic value of DDRm needs to be further verified by prospective randomized controlled trials. Systematic Review Registration: https://www.crd.york.ac.uk/prospero/, identifier CRD42022302275.

Tumor immune microenvironment and the current immunotherapy of cholangiocarcinoma (Review).

Cholangiocarcinoma (CCA) is a highly heterogeneous malignancy originating from the epithelial system of the bile ducts, and its incidence in recent years is steadily increasing. The immune microenvironment of CCA is characterized by diversity and complexity, with a substantial presence of cancer­associated fibroblasts and immune cell infiltration, which plays a key role in regulating the distinctive biological behavior of cholangiocarcinoma, including tumor growth, angiogenesis, lymphangiogenesis, invasion and metastasis. Despite the notable success of immunotherapy in the treatment of solid tumors in recent years, patients with CCA have responded poorly to immune checkpoint inhibitor therapy. The interaction of tumor cells with cellular components of the immune microenvironment can regulate the activity and function of immune cells and form an immunosuppressive microenvironment, which may cause ineffective immunotherapy. Therefore, the components of the tumor immune microenvironment appear to be novel targets for immune therapies. Combination therapy focusing on immune checkpoint inhibitors is a promising and valuable first­line or translational treatment approach for intractable biliary tract malignancies. The present review discusses the compositional characteristics and regulatory factors of the CCA immune microenvironment and the possible immune escape mechanisms. In addition, a summary of the advances in immunotherapy for CCA is also provided. It is hoped that the present review may function as a valuable reference for the development of novel immunotherapeutic strategies for CCA.

The prognostic nutritional index and tumor pathological characteristics predict the prognosis of elderly patients with early-stage hepatocellular carcinoma after surgery.

The elderly comprises over one-third of hepatocellular carcinoma (HCC) patients, however, they are not adequately represented in prognostic studies. The study aims to determine the prognostic significance of the preoperative prognostic nutritional index (PNI) and develop nomograms for predicting their recurrence-free and overall survival (RFS and OS). The study consisted of 282 elderly patients (aged ≥ 65 years) with early-stage HCC (China Liver Cancer Staging System: I-IIA) after curative resection (R0). They were randomly divided into a training (n = 197) and a test cohort (n = 85). The patients were stratified into two groups: PNI-low (PNI ≤ 49.05) and PNI-high (PNI > 49.05) based on a cut-off value. Most patients' demographics and perioperative outcomes were comparable, while patients in the PNI-high group were younger (P = 0.002), heavier (P < 0.001), and had lower comorbidity rates (P = 0.003). Although the tumor stages were earlier in the PNI-low group (P < 0.001), patients' OS (5-year OS: 48.9% vs. 93.1%) and RFS (5-year RFS: 27.3% vs. 75.7%) were significantly worse compared to the PNI-high group (both P < 0.0001). Patients' OS and RFS nomograms were developed by incorporating independent survival predictors including chronic obstructive pulmonary disease (COPD), age ≥ 75 years, PNI-low, tumor presence of satellite nodules, capsule, and microvascular invasion. The nomograms showed good calibration and discrimination, with all C-indexes ≥ 0.75 and calibration plots essentially coinciding with the diagonal. In conclusion, for elderly HCC patients, COPD, age ≥ 75 years, PNI-low, and tumor presence of satellite nodules, capsule, and microvascular invasion were independent prognostic factors. The nomogram could accurately predict the prognosis of these patients.

Comparison of the clinical features and long-term prognosis of gallbladder neuroendocrine carcinoma versus gallbladder adenocarcinoma: A propensity score-matched analysis.

Gallbladder neuroendocrine carcinoma (GBNEC) is rare and characterized by a low degree of tumor differentiation. The clinical features of GBNEC versus gallbladder adenocarcinoma (GBADC) remain a subject of debate. A total of 201 GBADC and 36 GBNEC cases that underwent surgery resection between January 2010 and 2022 at the Department of Biliary Surgery, West China Hospital, Sichuan University were included. A 1:1 propensity score matching (PSM) was performed based on seven predefined variables: age, sex, the American Joint Committee on Cancer (AJCC) stage, resection status, perineural invasion (PNI), lymphovascular invasion (LVI), and degree of tumor differentiation. Compared with GBADC, GBNEC patients were younger (median age 56.0 vs 64.0 years; P = 0.001), and more patients presented with advanced stages of tumor (P = 0.003). Patients with GBNEC also had a higher rate of PNI (55.6% vs 22.4%; P < 0.001), and LVI (63.9% vs 45.80%; P = 0.658). Before PSM, GBNEC patients had inferior prognoses compared with GBADC patients with a shorter median overall survival (mOS) (15.02 vs 20.11 months; P = 0.0028) and a shorter median recurrence-free survival (mRFS) (10.30 vs 15.17 months; P = 0.0028). However, after PSM analyses, there were no differences in OS (mOS 18.6 vs 18.0 months; P=0.24) or RFS (mRFS 10.98 vs 12.02 months; P = 0.39) between the GBNEC and GBADC cases. After multivariate analysis, tumor diagnosis (GBNEC vs GBADC) was not identified as an independent risk factor for shorter RFS (P = 0.506) or OS (P = 0.731). Unfavorable pathological features, including advanced AJCC tumor stages, poor differentiation, presence of LVI, and positive resection margins (all P < 0.05), were independent risk factors for inferior OS and RFS. GBNEC is difficult to diagnose early and has a prognosis comparable to stage-matched poorly differentiated GBADC. Tumor diagnosis (either GBADC or GBNEC) was not an independent risk factor for the patient's OS. Unfavorable pathological features of the neoplasm are the main determinants.

Alkali-thermal activated persulfate treatment of tetrabromobisphenol A in soil: Parameter optimization, mechanism, degradation pathway and toxicity evaluation.

The continued accumulation of halogenated organic pollutants in soil posed a potential threat to ecosystems and human health. In this study, tetrabromobisphenol A (TBBPA) was used as a typical representative of halogenated organic pollutants in soil, for alkali-thermal activated persulfate (PS) treatment. The results of response surface methodology (RSM) showed a optimal debromination efficiency of TBBPA was 88.99 % under the optimum reaction conditions. Quenching experiments and electron paramagnetic resonance (EPR) confirmed that SO4-•, HO•, O2-• and 1O2 existed simultaneously in the oxidation process. SO4-• played a major role in the initial stage of the reaction, and O2-• played a major role in the the last stage. Based on density functional theory (DFT) and intermediate products, two degradation pathways were proposed, including debromination reaction and ß bond scission. Moreover, the basic physical and chemical properties of the soil were affected to a certain extent, while the soil surface structure, elements and functional group composition rarely changed. In addition, the T.E.S.T. analysis and biotoxicity tests proved that alkali-thermal activated PS can effectively reduce the toxicity of TBBPA-contaminated soil, which is conducive to the subsequent safe secondary utilization of soil.

Combining Hydrodefluorination and Defluorophosphorylation for Chemo- and Stereoselective Synthesis of gem-Fluorophosphine Alkenes.

A chemo-, regio-, and stereoselective reaction of trifluoromethyl enones, phenylsilane, and phosphine oxides through a sequential hydrodefluorination and defluorophosphorylation relay is developed for the synthesis of distinctive gem-fluorophosphine alkenes. This multicomponent reaction occurred under transition-metal-free conditions with good functional group tolerance. Moreover, the preinstalled carbonyl auxiliary is important for tuning the reactivity of ß-trifluoromethyl enones, thereby enabling controllable and selective functionalization of two fluorine atoms in trifluoromethylated enones.

Whole-genome sequencing of diverse wheat accessions uncovers genetic changes during modern breeding in China and the United States.

Breeding has dramatically changed the plant architecture of wheat (Triticum aestivum), resulting in the development of high-yielding varieties adapted to modern farming systems. However, how wheat breeding shaped the genomic architecture of this crop remains poorly understood. Here, we performed a comprehensive comparative analysis of a whole-genome resequencing panel of 355 common wheat accessions (representing diverse landraces and modern cultivars from China and the United States) at the phenotypic and genomic levels. The genetic diversity of modern wheat cultivars was clearly reduced compared to landraces. Consistent with these genetic changes, most phenotypes of cultivars from China and the United States were significantly altered. Of the 21 agronomic traits investigated, 8 showed convergent changes between the 2 countries. Moreover, of the 207 loci associated with these 21 traits, more than half overlapped with genomic regions that showed evidence of selection. The distribution of selected loci between the Chinese and American cultivars suggests that breeding for increased productivity in these 2 regions was accomplished by pyramiding both shared and region-specific variants. This work provides a framework to understand the genetic architecture of the adaptation of wheat to diverse agricultural production environments, as well as guidelines for optimizing breeding strategies to design better wheat varieties.

Optimization of the Methods to Develop Stable Polymer Gels for Water Management in Medium- and Ultra-High-Salinity Reservoirs.

Polymer gels suffer from a serious syneresis issue when exposed to high-temperature and high-salinity (HTHS) conditions, which limits their use as water-treatment agents in this type of reservoir. In this paper, the effects of the polymer type/concentration, deoxidizers, and stabilizers on the long-term stability of polymer gels were systematically studied; thus, the methods to develop stable polymer gels for two typical levels of salinity were optimized. The results show the following: (1) For a medium-salinity condition (TDS: 33,645.0 mg/L) at 125 °C, conventional HPAM gels completely dehydrate within only 1 day, and the addition of a deoxidizer hardly improved their stability. Some special polymers, e.g., AP-P5, MKY, and CPAM, are able to form stable gels if a high concentration of 0.8% is used; the syneresis rate of these gels is about 10% after 30 days. However, the addition of the complexant sodium oxalate significantly improves the stability of gels formed by all five of these different polymers, which behave with a 0% syneresis rate after 30 days pass. Complexants are the most economical and feasible agents to develop stable gels in medium-salinity water. (2) Gels enhanced using the methods above all become unstable in a more challenging ultra-high-saline condition (TDS: 225,068.0 mg/L). In this case, special calcium- and magnesium-resistant polymers are required to prepare stable gels, which show 0% syneresis rates after 30 days, have relatively low strengths, but do produce a good plugging effect in high-permeability cores.

Strength Investigation and Prediction of Superfine Tailings Cemented Paste Backfill Based on Experiments and Intelligent Methods.

The utilization of solid waste for filling mining presents substantial economic and environmental advantages, making it the primary focus of current filling mining technology development. To enhance the mechanical properties of superfine tailings cemented paste backfill (SCPB), this study conducted response surface methodology experiments to investigate the impact of various factors on the strength of SCPB, including the composite cementitious material, consisting of cement and slag powder, and the tailings' grain size. Additionally, various microanalysis techniques were used to investigate the microstructure of SCPB and the development mechanisms of its hydration products. Furthermore, machine learning was utilized to predict the strength of SCPB under multi-factor effects. The findings reveal that the combined effect of slag powder dosage and slurry mass fraction has the most significant influence on strength, while the coupling effect of slurry mass fraction and underflow productivity has the lowest impact on strength. Moreover, SCPB with 20% slag powder has the highest amount of hydration products and the most complete structure. When compared to other commonly used prediction models, the long-short term memory neural network (LSTM) constructed in this study had the highest prediction accuracy for SCPB strength under multi-factor conditions, with root mean square error (RMSE), correlation coefficient (R), and variance account for (VAF) reaching 0.1396, 0.9131, and 81.8747, respectively. By optimizing the LSTM using the sparrow search algorithm (SSA), the RMSE, R, and VAF improved by 88.6%, 9.4%, and 21.9%, respectively. The research results can provide guidance for the efficient filling of superfine tailings.

Organoids: The current status and biomedical applications.

Organoids are three-dimensional (3D) miniaturized versions of organs or tissues that are derived from cells with stem potential and can self-organize and differentiate into 3D cell masses, recapitulating the morphology and functions of their in vivo counterparts. Organoid culture is an emerging 3D culture technology, and organoids derived from various organs and tissues, such as the brain, lung, heart, liver, and kidney, have been generated. Compared with traditional bidimensional culture, organoid culture systems have the unique advantage of conserving parental gene expression and mutation characteristics, as well as long-term maintenance of the function and biological characteristics of the parental cells in vitro. All these features of organoids open up new opportunities for drug discovery, large-scale drug screening, and precision medicine. Another major application of organoids is disease modeling, and especially various hereditary diseases that are difficult to model in vitro have been modeled with organoids by combining genome editing technologies. Herein, we introduce the development and current advances in the organoid technology field. We focus on the applications of organoids in basic biology and clinical research, and also highlight their limitations and future perspectives. We hope that this review can provide a valuable reference for the developments and applications of organoids.