Group sequential design with maximin efficiency robust test for immunotherapy with generalized delayed treatment effect.

The delayed treatment effect is a common feature of immunotherapy, characterized by a gradual onset of action ranging from no effect to full effect. In this study, we propose a generalized delayed treatment effect function to depict the delayed effective process precisely and flexibly. To reduce potential power loss caused by the delayed treatment effect in a group sequential trial, we employ the maximin efficiency robust test, which enhances power robustness across a range of possible delays. We present novel approaches based on the Markov chain method for determining group sequential boundaries, calculating the power function, and estimating the maximum sample size through iterative regressions between the square root of the maximum sample size and the normal quantile of power. Extensive simulation studies validate the effectiveness of our approaches, particularly in balanced trials, demonstrating the validity of group sequential boundaries and the accuracy of maximum sample size estimations. Additionally, we utilize a real trial as an example to compare our considered trial with group sequential trials using the log-rank and generalized piecewise weighted log-rank tests. The results show significantly reduced maximum sample sizes, highlighting the economic advantage of our approach.

Sclerotherapy for the intraoral ranula with bleomycin: technical considerations and preliminary experience.

Ranula is a mucous cyst that occurs in the sublingual gland (SLG) in the floor of the mouth. It can be classified into two types based on origins: One is the the lesser sublingual gland (LSLG) in the anterior segment and the Rivini duct, which is connected to it, and the other is the greater sublingual gland (GSLG) in the posterior segment. Because of the anatomical characteristics, surgical resection of the cysts carries the risk of damaging adjacent tissues and has a high recurrence rate. Intralesional injection of sclerotherapy may be a better alternative treatment. We summarized 65 cases of ranula treated with intralesional injections of bleomycin(BML). According to the origin of the ranula, 60 cases were from the LSLG and the Rivini duct, and 5 cases were from the GSLG. The results showed that 60 cases of ranula from LSLG and Rivini ducts were 100% cured during the follow-up period. The median number of injections for all patients was 1.16. All 5 cases of ranula from the GSLG did not wholly recover. This study confirmed that BLM intralesional injection is a safe and effective treatment modality for cysts from LSLG or the ducts of Rivini rather than GSLG. Therefore, before treatment, it is necessary to determine the type and origin of the cyst by characterizing its morphology to ensure the effectiveness of the treatment.

MIDAS-2: an enhanced Bayesian platform design for immunotherapy combinations with subgroup efficacy exploration.

Although immunotherapy combinations have revolutionised cancer treatment, the rapid screening of effective and optimal therapies from large numbers of candidate combinations, as well as exploring subgroup efficacy, remains challenging. This necessitates innovative, integrated, and efficient trial designs. In this study, we extend the MIDAS design to include subgroup exploration and propose an enhanced Bayesian information borrowing platform design called MIDAS-2. MIDAS-2 enables quick and continuous screening of promising combination strategies and exploration of their subgroup effects within a unified platform design framework. We use a regression model to characterize the efficacy pattern in subgroups. Information borrowing is applied through Bayesian hierarchical modelling to improve trial efficiency considering the limited sample size in subgroups. Time trend calibration is also employed to avoid potential baseline drifts. Simulation results demonstrate that MIDAS-2 yields high probabilities for identifying the effective drug combinations as well as promising subgroups, facilitating appropriate selection of the best treatments for each subgroup. The proposed design is robust against small time trend drifts, and the type I error is successfully controlled after calibration when a large drift is expected. Overall, MIDAS-2 provides an adaptive drug screening and subgroup exploring framework to accelerate immunotherapy development in an efficient, accurate, and integrated fashion.

M&M: A maximum duration design with the Maxcombo test for a group sequential trial of an immunotherapy with a random delayed treatment effect.

A random delayed treatment effect is expected in a confirmatory clinical trial for an immunotherapy due to the individual heterogeneity of physiological conditions. For this reason, the delay time will be assumed to follow a continuous distribution that is difficult to estimate accurately based on the early-phase data, which hinders the specification of the most powerful weighted log-rank test. Therefore, we propose a simulation-based maximum duration design with a robustly powerful Maxcombo test for a group sequential trial for the immunotherapy with the random delayed treatment effect. The design obtains the group sequential boundaries by a simulation procedure and determines the required maximum sample size using a one-dimensional search in which another simulation procedure is used to calculate empirical power. The simulation researches proved the accuracy of the group sequential boundaries and their robustness against the misspecified maximum sample sizes for large samples and revealed their moderate sensitivity against the misspecified survival distributions under the null hypothesis of no difference. The studies investigated whether the type I error rate would inflate under the "inferior" null hypothesis and evaluated the robustness against different distributions of the delay time in terms of the empirical power among the Maxcombo tests and component weighted log-rank tests.

Bayesian two-stage sequential enrichment design for biomarker-guided phase II trials for anticancer therapies.

Biomarker-guided phase II trials have become increasingly important for personalized cancer treatment. In this paper, we propose a Bayesian two-stage sequential enrichment design for such biomarker-guided trials. We assumed that all patients were dichotomized as marker positive or marker negative based on their biomarker status; the positive patients were considered more likely to respond to the targeted drug. Early stopping rules and adaptive randomization methods were embedded in the design to control the number of patients receiving inferior treatment. At the same time, a Bayesian hierarchical model was used to borrow information between the positive and negative control arms to improve efficiency. Simulation results showed that the proposed design achieved higher empirical power while controlling the type I error and assigned more patients to the superior treatment arms. The operating characteristics suggested that the design has good performance and may be useful for biomarker-guided phase II trials for evaluating anticancer therapies.

Fine Mapping and Functional Analysis of Major Regulatory Genes of Soluble Solids Content in Wax Gourd (Benincasa hispida).

Soluble solids content (SSC) is an important quality trait of wax gourd, but reports about its regulatory genes are scarce. In this study, the SSC regulatory gene BhSSC2.1 in wax gourd was mined via quantitative trait locus (QTL) mapping based on high-density genetic mapping containing 12 linkage groups (LG) and bulked segregant analysis (BSA)-seq. QTL mapping and BSA-seq revealed for the first time that the SSC QTL (107.658-108.176 cM) of wax gourd was on Chr2 (LG2). The interpretable phenotypic variation rate and maximum LOD were 16.033% and 6.454, respectively. The QTL interval contained 13 genes. Real-time fluorescence quantitative expression analysis, functional annotation, and sequence analysis suggested that Bch02G016960, named BhSSC2.1, was a candidate regulatory gene of the SSC in wax gourd. Functional annotation of this gene showed that it codes for a NADP-dependent malic enzyme. According to BhSSC2.1 sequence variation, an InDel marker was developed for molecular marker-assisted breeding of wax gourd. This study will lay the foundation for future studies regarding breeding and understanding genetic mechanisms of wax gourd.

An Adaptive Information Borrowing Platform Design for Testing Drug Candidates of COVID-19.

Background: There have been thousands of clinical trials for COVID-19 to target effective treatments. However, quite a few of them are traditional randomized controlled trials with low efficiency. Considering the three particularities of pandemic disease: timeliness, repurposing, and case spike, new trial designs need to be developed to accelerate drug discovery. Methods: We propose an adaptive information borrowing platform design that can sequentially test drug candidates under a unified framework with early efficacy/futility stopping. Power prior is used to borrow information from previous stages and the time trend calibration method deals with the baseline effectiveness drift. Two drug development strategies are applied: the comprehensive screening strategy and the optimal screening strategy. At the same time, we adopt adaptive randomization to set a higher allocation ratio to the experimental arms for ethical considerations, which can help more patients to receive the latest treatments and shorten the trial duration. Results: Simulation shows that in general, our method has great operating characteristics with type I error controlled and power increased, which can select effective/optimal drugs with a high probability. The early stopping rules can be successfully triggered to stop the trial when drugs are either truly effective or not optimal, and the time trend calibration performs consistently well with regard to different baseline drifts. Compared with the nonborrowing method, borrowing information in the design substantially improves the probability of screening promising drugs and saves the sample size. Sensitivity analysis shows that our design is robust to different design parameters. Conclusions: Our proposed design achieves the goal of gaining efficiency, saving sample size, meeting ethical requirements, and speeding up the trial process and is suitable and well performed for COVID-19 clinical trials to screen promising treatments or target optimal therapies.

Fine mapping and identification of the candidate gene BFS for fruit shape in wax gourd (Benincasa hispida).

KEY MESSAGE: Non-synonymous mutations in the BFS gene, which encodes the IQD protein, are responsible for the shape of wax gourd fruits. Fruit shape is an important agronomic trait in wax gourds. Therefore, in this study, we employed bulked segregant analysis (BSA) to identify a candidate gene for fruit shape in wax gourds within F2 populations derived by crossing GX-71 (long cylindrical fruit, fruit shape index = 4.56) and MY-1 (round fruit, fruit shape index = 1.06) genotypes. According to BSA, the candidate gene is located in the 17.18 Mb region on chromosome 2. Meanwhile, kompetitive allele-specific PCR (KASP) markers were used to reduce it to a 19.6 Kb region. Only one gene was present within the corresponding region of the reference genome, namely Bch02G016830 (designated BFS). Subsequently, BFS was sequenced in six wax gourd varieties with different fruit shapes. Sequence analysis revealed two non-synonymous mutations in the round wax gourd and one non-synonymous mutation in the cylindrical wax gourd. Quantitative real­time PCR (qRT-PCR) analysis further showed that the expression of BFS in round fruits was significantly higher than in long cylindrical fruits at the ovary formation stage. Therefore, BFS is a candidate gene for determination wax gourd shape. The predicted protein encoded by the BFS gene belongs to the IQ67-domain protein family, which have the structural characteristics of scaffold proteins and coordinate Ca2+ CaM signaling from the membrane to the nucleus. Ultimately, two derived cleaved amplified polymorphic sequence (dCAPS) markers were developed to facilitate marker-assisted selection for wax gourds breeding.

Dynamic prediction of disease processes based on recurrent history and functional principal component analysis of longitudinal biomarkers: Application for ovarian epithelial cancer.

Ovarian epithelial cancer is a gynecological tumor with a high risk of recurrence and death. In the clinical diagnosis of ovarian epithelial cancer, CA125 has become an important indicator of disease burden. To account for patient recurrence and death, a proper method is needed to integrate information from biomarkers and recurrence simultaneously. In the past 10 years, many methods have been proposed for joint modeling of longitudinal biomarkers and survival data, but few of them are applicable to longitudinal data and disease processes, including recurrence and death. In this article, we proposed a new joint frailty model based on functional principal component analysis for dynamic prediction of survival probabilities on the total time scale, which took recurrent history and longitudinal data into account simultaneously. The estimation of the joint frailty model is achieved by maximizing the penalized log-likelihood function. The simulation results demonstrated the advantages of our method in both discrimination and accuracy under different scenarios. To indicate the method's practicality, it is applied to an actual dataset of patients with ovarian epithelial cancer to predict survival dynamically using longitudinal data of biomarker CA125 and recurrent history data.

Ergosta-7,9(11),22-trien-3ß-ol Rescues AD Deficits by Modulating Microglia Activation but Not Oxidative Stress.

Ergosta-7,9(11),22-trien-3ß-ol (EK100) was isolated from the Taiwan-specific medicinal fungus Antrodia camphorata, which is known for its health-promotion and anti-aging effects in folk medicine. Alzheimer's disease (AD) is a major aging-associated disease. We investigated the efficacy and potential mechanism of ergosta-7,9(11),22-trien-3ß-ol for AD symptoms. Drosophila with the pan-neuronal overexpression of human amyloid-ß (Aß) was used as the AD model. We compared the life span, motor function, learning, memory, oxidative stress, and biomarkers of microglia activation and inflammation of the ergosta-7,9(11),22-trien-3ß-ol-treated group to those of the untreated control. Ergosta-7,9(11),22-trien-3ß-ol treatment effectively improved the life span, motor function, learning, and memory of the AD model compared to the untreated control. Biomarkers of microglia activation and inflammation were reduced, while the ubiquitous lipid peroxidation, catalase activity, and superoxide dismutase activity remained unchanged. In conclusion, ergosta-7,9(11),22-trien-3ß-ol rescues AD deficits by modulating microglia activation but not oxidative stress.

Alterations of plant architecture and phase transition by the phytoplasma virulence factor SAP11.

As key mediators linking developmental processes with plant immunity, TCP (TEOSINTE-BRANCHED, CYCLOIDEA, PROLIFERATION FACTOR 1 and 2) transcription factors have been increasingly shown to be targets of pathogenic effectors. We report here that TB/CYC (TEOSINTE-BRANCHED/CYCLOIDEA)-TCPs are destabilized by phytoplasma SAP11 effectors, leading to the proliferation of axillary meristems. Although a high degree of sequence diversity was observed among putative SAP11 effectors identified from evolutionarily distinct clusters of phytoplasmas, these effectors acquired fundamental activity in destabilizing TB/CYC-TCPs. In addition, we demonstrate that miR156/SPLs and miR172/AP2 modules, which represent key regulatory hubs involved in plant phase transition, were modulated by Aster Yellows phytoplasma strain Witches' Broom (AY-WB) protein SAP11. A late-flowering phenotype with significant changes in the expression of flowering-related genes was observed in transgenic Arabidopsis plants expressing SAP11AYWB. These morphological and molecular alterations were correlated with the ability of SAP11 effectors to destabilize CIN (CINCINNATA)-TCPs. Although not all putative SAP11 effectors display broad-spectrum activities in modulating morphological and physiological changes in host plants, they serve as core virulence factors responsible for the witches' broom symptom caused by phytoplasmas.

Optimization of Mixed Solid-state Fermentation of Soybean Meal by Lactobacillus Species and Clostridium butyricum.

Soybean meal is the main vegetable protein source in animal feed. Soybean meal contains several anti-nutritional factors, which directly affect digestion and absorption of soy protein, thereby reducing growth performance and value in animals. Fermented soybean meal is rich in probiotics and functional metabolites, which facilitates soybean protein digestion, absorption and utilization in piglets. However, the mixed solid-state fermentation (SSF) conditions of soybean meal remain to be optimized. In this study, we investigated the optimal parameters for SSF of soybean meal by Lactobacillus species and Clostridium butyricum . The results showed that two days of fermentation was sufficient to increase the viable count of bacteria, lactic acid levels and degradation of soybean protein in fermented soybean meal at the initial moisture content of 50%. The pH value, lowering sugar content and oligosaccharides in fermented soybean meal, was significantly reduced at the initial moisture content of 50% after two days of fermentation. Furthermore, the exogenous proteases used in combination with probiotics supplementation were further able to enhance the viable count of bacteria, degradation of soybean protein and lactic acid level in the fermented soybean meal. In addition, the pH value and sugar content in fermented soybean meal were considerably reduced in the presence of both proteases and probiotics. Furthermore, the fermented soybean meal also showed antibacterial activity against Staphylococcus aureus and Escherichia coli . These results together suggest that supplementation of both proteases and probiotics in SSF improves the nutritional value of fermented soybean meal and this is suitable as a protein source in animal feed.

Phytoplasma SAP11 alters 3-isobutyl-2-methoxypyrazine biosynthesis in Nicotiana benthamiana by suppressing NbOMT1.

Phytoplasmas are bacterial phytopathogens that release virulence effectors into sieve cells and act systemically to affect the physiological and morphological state of host plants to promote successful pathogenesis. We show here that transgenic Nicotiana benthamiana lines expressing the secreted effector SAP11 from Candidatus Phytoplasma mali exhibit an altered aroma phenotype. This phenomenon is correlated with defects in the development of glandular trichomes and the biosynthesis of 3-isobutyl-2-methoxypyrazine (IBMP). IBMP is a volatile organic compound (VOC) synthesized by an O-methyltransferase, via a methylation step, from a non-volatile precursor, 3-isobutyl-2-hydroxypyrazine (IBHP). Based on comparative and functional genomics analyses, NbOMT1, which encodes an O-methyltransferase, was found to be highly suppressed in SAP11-transgenic plants. We further silenced NbOMT1 through virus-induced gene silencing and demonstrated that this enzyme influenced the accumulation of IBMP in N. benthamiana In vitro biochemical analyses also showed that NbOMT1 can catalyse IBHP O-methylation in the presence of S-adenosyl-L-methionine. Our study suggests that the phytoplasma effector SAP11 has the ability to modulate host VOC emissions. In addition, we also demonstrated that SAP11 destabilized TCP transcription factors and suppressed jasmonic acid responses in N. benthamiana These findings provide valuable insights into understanding how phytoplasma effectors influence plant volatiles.

Transgenic plants that express the phytoplasma effector SAP11 show altered phosphate starvation and defense responses.

Phytoplasmas have the smallest genome among bacteria and lack many essential genes required for biosynthetic and metabolic functions, making them unculturable, phloem-limited plant pathogens. In this study, we observed that transgenic Arabidopsis (Arabidopsis thaliana) expressing the secreted Aster Yellows phytoplasma strain Witches' Broom protein11 shows an altered root architecture, similarly to the disease symptoms of phytoplasma-infected plants, by forming hairy roots. This morphological change is paralleled by an accumulation of cellular phosphate (Pi) and an increase in the expression levels of Pi starvation-induced genes and microRNAs. In addition to the Pi starvation responses, we found that secreted Aster Yellows phytoplasma strain Witches' Broom protein11 suppresses salicylic acid-mediated defense responses and enhances the growth of a bacterial pathogen. These results contribute to an improved understanding of the role of phytoplasma effector SAP11 and provide new insights for understanding the molecular basis of plant-pathogen interactions.

Quantitative Trait Loci Sequencing and Genetic Mapping Reveal Two Main Regulatory Genes for Stem Color in Wax Gourds.

Stem color is an important agronomic trait of wax gourds. However, its regulatory genes have not been identified. In this study, 105 inbred lines constructed from two parents (GX-71 and MY-1) were sequenced and quantitative trait loci sequencing was used to mine the genes that regulate stem color in wax gourds. The results identified two quantitative trait loci related to stem color, qSC5 and qSC12, located on Chr05 (11,134,567-16,459,268) and Chr12 (74,618,168-75,712,335), respectively. The qSC5 had a phenotypic variation rate of 36.9% and a maximum limit of detection of 16.9. And the qSC12 had a phenotypic variation rate of 20.9%, and a maximum limit of detection of 11.2. Bch05G003950 (named BchAPRR2) and Bch12G020400 were identified as candidate genes involved in stem color regulation in wax gourds. The chlorophyll content and expression of BchAPRR2 and Bch12G020400 were significantly higher in green-stemmed wax gourds than in white-stemmed ones. Therefore, BchAPRR2 and Bch12G020400 were considered the main and secondary regulatory genes for wax gourd stem color, respectively. Finally, InDel markers closely linked to BchAPRR2 were developed to validate the prediction of wax gourd stem color traits in 55 germplasm lines, with an accuracy of 81.8%. These findings lay the foundation for exploring the genetic regulation of wax gourd stem color and future research on wax gourd breeding.

Secondary Structure-Dependent Charge Transport in iLOV Protein Junctions.

The composition and structure of proteins are crucial for charge migration in the solid-state charge transport (CTp). Despite much progress, it is still challenging to explore the relationship between conformational change and CTp in the complex protein system. Herein, we design three improved light-oxygen-voltage (iLOV) domains, and efficiently regulate the CTp of the iLOV self-assembled monolayers (SAMs) by pH induced conformation variation. The current density can be controlled in the range of one order of magnitude. Interestingly, the CTp of iLOV displays negative linear relations with the ß-sheet contents. Single-level Landauer fitting and transition voltage spectroscopy analysis suggest that ß-sheet-dependent CTp would be related to the coupling between iLOV and electrodes. This work proposes a new strategy to explore the CTp in complex molecular system. Our findings deepen the understanding on protein structure-CTp relationship, and provide predictive mode of protein CTp responses for the design of functional bioelectronics.

Fine mapping of the major gene BhHLS1 controlling seed size in wax gourd (Benincasa hispida).

Introduction/Background: The seed size of wax gourds is an important agronomic trait; however, the associated genes have not yet been reported. Methods: In this study, we used a high-density genetic map constructed based on F8 recombinant inbred line populations derived from a cross between MY-1 (large seed) and GX-71 (small seed) strains to detect quantitative trait locis (QTLs) for seed-size-related traits in wax gourd over a two-year period. Results: Two stable QTLs (qSL10 and qSW10) for seed length (SL) and seed width (SW) on chromosome 10 were repeatedly detected over two years (2021-2022). qSL10 had a phenotypic variation rate of 75.30% and 80.80% in 2021 and 2022, respectively. Whereas, qSW10 had a phenotypic variation rate of 66.60% and 73.80% in 2021 and 2022, respectively. Further, a single nucleotide polymorphism mutation was found to cause early termination of Bch10G006400 (BhHLS1) translation in GX-71 through sequencing analysis of candidate genes. Based on gene functional annotation and quantitative real-time PCR analyses, BhHLS1 encoded a probable N-acetyltransferase HLS1-like protein and its expression level was significantly different between parents. Therefore, BhHLS1 is a major candidate gene associated with a one-factor polymorphism regulating the SL and SW of wax gourds. Finally, based on variation in the BhHLS1 sequence, a cleaved amplified polymorphic sequence marker was developed for the molecular marker-assisted breeding of wax gourds. Discussion: Overall, this study is of great significance for the genetic improvement of seed size, verification of gene functions, and cultivation of specific germplasm resources for wax gourds.

Comparative Study of Bayesian Information Borrowing Methods in Oncology Clinical Trials.

PURPOSE: With deeper insight into precision medicine, more innovative oncology trial designs have been proposed to contribute to the characteristics of novel antitumor drugs. Bayesian information borrowing is an indispensable part of these designs, which shows great advantages in improving the efficiency of clinical trials. Bayesian methods provide an effective framework when incorporating information. However, the key point lies in how to choose an appropriate method for complex oncology clinical trials. METHODS: We divided the borrowing information scenarios into concurrent and nonconcurrent scenarios according to whether the data to be borrowed are observed at the same time as in the current trial or not. Then, we provided an overview of the methods in each scenario. Performance comparison of different methods is carried out with regard to the type I error and power. RESULTS: As demonstrated by the simulation results in each borrowing scenario, the Bayesian hierarchical model and its extensions are more appropriate for concurrent borrowing. The simulation results demonstrate that the Bayesian hierarchical model shows great advantages when the arms are homogeneous. However, such a method should be adopted with caution when heterogeneity exists. We recommend the other methods, considering heterogeneity. Borrow information from informative priors is more suggested for nonconcurrent borrowing scenarios. Multisource exchangeability models are more suitable for multiple historical trials, while meta-analytic-predictive prior should be carefully applied. CONCLUSION: Bayesian information borrowing is useful and can improve the efficiency of clinical trial designs. However, we should carefully choose an appropriate information borrowing method when facing a practical innovative oncology trial, as an appropriate method is essential to provide ideal design performance.

Development and Application of InDel Markers Linked to Fruit-Shape and Peel-Colour Genes in Wax Gourd.

The wax gourd is commonly grown in many countries because of its high nutritional and economic value. While the genes for the fruit shape and peel colour of wax gourd have been reported, the InDel markers linked to these genes remain undeveloped. In this study, the InDel markers linked to fruit-shape (Bch02G016830) and peel-colour (Bch05G003950) genes were developed from resequenced data. We used 120 inbred lines, 536 isolated populations, and 4 commercial hybrids to evaluate the validity and application value of the InDel markers. The accuracy rates of nine pairs of fruit-shape InDel markers (GX1-GX9) were 84.16-91.66% in 120 inbred lines. The accuracy rates of 27 pairs of peel-colour InDel markers (PS1-PS27) within approximately 3.0 Mb upstream and 3.0 Mb downstream of the peel-colour gene were 100% and those of 6 pairs of peel-colour InDel markers (PS28-PS33) within 3.0-20 Mb upstream and downstream of the peel-colour gene were 55.83-90% in 120 inbred lines. The purity of four commercial hybrids determined using GX1, GX2, PS13, and PS14 was highly consistent with the field results for purity determination. Our results provide important information for genetic linkage map construction, molecular-marker-assisted selective breeding, and purity determination of wax gourd hybrids.

Power prior for borrowing the real-world data in bioequivalence test with a parallel design.

Recently, real-world study has attracted wide attention for drug development. In bioequivalence study, the reference drug often has been marketed for many years and accumulated abundant real-world data. It is therefore appealing to incorporate these data in the design to improve trial efficiency. In this paper, we propose a Bayesian method to include real-world data of the reference drug in a current bioequivalence trial, with the aim to increase the power of analysis and reduce sample size for long half-life drugs. We adopt the power prior method for incorporating real-world data and use the average bioequivalence posterior probability to evaluate the bioequivalence between the test drug and the reference drug. Simulations were conducted to investigate the performance of the proposed method in different scenarios. The simulation results show that the proposed design has higher power than the traditional design without borrowing real-world data, while controlling the type I error. Moreover, the proposed method saves sample size and reduces costs for the trial.