Gender disparities and woman-specific trends in Barrett’s esophagus in the United States:An 11-year nationwide populationbased study

BACKGROUND Barrett's esophagus(BE)is a known premalignant precursor to esophageal adenocarcinoma(EAC).The prevalence rates continue to rise in the United States,but many patients who are at risk of EAC are not screened.Current practice guidelines include male gender as a predisposing factor for BE and EAC.The population-based clinical evidence regarding female gender remains limited.AIM To study comparative trends of gender disparities in patients with BE in the United States.METHODS A nationwide retrospective study was conducted using the 2009-2019 National Inpatient Sample(NIS)database.Patients with a primary or secondary diagnosis code of BE were identified.The major outcome of interest was determining the gender disparities in patients with BE.Trend analysis for respective outcomes for females was also reported to ascertain any time-based shifts.RESULTS We identified 1204190 patients with BE for the study period.Among the included patients,717439(59.6%)were men and 486751(40.4%)were women.The mean age was higher in women than in men(67.1±0.4 vs 66.6±0.3 years,P<0.001).The rate of BE per 100000 total NIS hospitalizations for males increased from 144.6 in 2009 to 213.4 in 2019(P<0.001).The rate for females increased from 96.8 in 2009 to 148.7 in 2019(P<0.001).There was a higher frequency of obesity among women compared to men(17.4%vs 12.6%,P<0.001).Obesity prevalence among females increased from 12.3%in 2009 to 21.9%in 2019(P<0.001).A lower prevalence of smoking was noted in women than in men(20.8%vs 35.7%,P<0.001).However,trend analysis showed an increasing prevalence of smoking among women,from 12.9%in 2009 to 30.7%in 2019(P<0.001).Additionally,there was a lower prevalence of alcohol abuse,Helicobacter pylori(H.pylori),and diabetes mellitus among females than males(P<0.001).Trend analysis showed an increasing prevalence of alcohol use disorder and a decreasing prevalence of H.pylori and diabetes mellitus among women(P<0.001).CONCLUSION The prevalence of BE among women has steadily increased from 2009 to 2019.The existing knowledge concerning BE development has historically focused on men,but our findings show that the risk in women is not insignificant.

Socioeconomic traits and the risk of Barrett’s esophagus and gastroesophageal reflux disease: A Mendelian randomization study

BACKGROUND Previous observational studies have shown that the prevalence of gastroesophageal reflux disease(GERD)and Barrett’s esophagus(BE)is associated with socioeconomic status.However,due to the methodological limitations of traditional observational studies,it is challenging to definitively establish causality.AIM To explore the causal relationship between the prevalence of these conditions and socioeconomic status using Mendelian randomization(MR).METHODS We initially screened single nucleotide polymorphisms(SNPs)to serve as proxies for eight socioeconomic status phenotypes for univariate MR analysis.The inverse variance weighted(IVW)method was used as the primary analytical method to estimate the causal relationship between the eight socioeconomic status phenotypes and the risk of GERD and BE.We then collected combinations of SNPs as composite proxies for the eight socioeconomic phenotypes to perform multivariate MR(MVMR)analyses based on the IVW MVMR model.Furthermore,a two-step MR mediation analysis was used to examine the potential mediation of the associations by body mass index,major depressive disorder(MDD),smoking,alcohol consumption,and sleep duration.RESULTS The study identified three socioeconomic statuses that had a significant impact on GERD.These included household income[odds ratio(OR):0.46;95% confidence interval(95%CI):0.31-0.70],education attainment(OR:0.23;95%CI:0.18-0.29),and the Townsend Deprivation Index at recruitment(OR:1.57;95%CI:1.04-2.37).These factors were found to independently and predominantly influence the genetic causal effect of GERD.Furthermore,the mediating effect of educational attainment on GERD was found to be mediated by MDD(proportion mediated:10.83%).Similarly,the effect of educational attainment on BE was mediated by MDD(proportion mediated:10.58%)and the number of cigarettes smoked per day(proportion mediated:3.50%).Additionally,the mediating effect of household income on GERD was observed to be mediated by sleep duration(proportion mediated:9.75%)CONCLUSION This MR study shed light on the link between socioeconomic status and GERD or BE,providing insights for the prevention of esophageal cancer and precancerous lesions.

Hybrid argon plasma coagulation for the treatment of Barrett’s esophagus:A prospective,multicenter study

BACKGROUND The incidence of Barrett’s esophagus(BE)in China is lower compared to the Western populations.Hence,studies conducted in the Chinese population has been limited.The current treatment options available for BE treatment includes argon plasma coagulation(APC),radiofrequency ablation and cryoablation,all with varying degrees of success.AIM To determine the efficacy and safety of HybridAPC in the treatment of BE.METHODS The study cohort consisted of patients with BE who underwent HybridAPC ablation treatment.These procedures were performed by seven endoscopists from different tertiary hospitals.The duration of the procedure,curative rate,complications and recurrent rate by 1-year follow-up were recorded.RESULTS Eighty individuals were enrolled for treatment from July 2017 to June 2020,comprising of 39 males and 41 females with a median age of 54 years(range,30 to 83 years).The technical success rate of HybridAPC was 100%and the overall curative rate was 98.15%.No severe complications occurred during the operation.BE cases were classified as short-segment BE and long-segment BE.Patients with short-segment BE were all considered cured without complications.Thirty-six patients completed the one-year follow-up without recurrence.Twenty-four percent had mild dysplasia which were all resolved with one post-procedural treatment.The mean duration of the procedure was 10.94±6.52 min.CONCLUSION Treatment of BE with HybridAPC was found to be a simple and quick procedure that is safe and effective during the short-term follow-up,especially in cases of short-segment BE.This technique could be considered as a feasible alternative ablation therapy for BE.

Barrett’s食管、糜烂性食管炎及非糜烂性反流病发生机制的比较

目的:比较糜烂性食管炎(EE)、非糜烂性反流病(NERD)及Barrett’s食管(BE)患者反流发生特点,探讨不同类型GERD的可能发病机制.方法:105例GERD患者根据胃镜及病理情况分为:EE组(35例)、BE组(34例)及NERD组(36例),对照组30例为健康志愿者.比较4组受检者食管测压与24h食管pH及胆汁反流同步监测结果的差异.结果:与对照组相比,GERD各组LES静息压均有不同程度减低,以EE组最明显(P<0.05).EE与BE组患者远段食管收缩波幅及有效蠕动百分比明显低于NERD组与对照组(均P<0.05).DeMeester评分等酸反流指标在EE组最高.BE组Abs>0.14时间百分比等长时间胆汁反流指标最高.NERD患者中仅52.8%存在病理性反流.各组中混合反流类型占各组病理性反流总人数的比例分别为68.57%(EE组)、84.38%(BE组)及63.15%(NERD组).结论:胃酸与胆汁的混合反流是GERD患者中最常见病理性反流类型,且对食管黏膜的损害较单纯酸或胆汁反流为重,胆汁反流在BE的发病机制中占有重要地位.

食管炎、食管溃疡和Barrett’s食管的胃食管反流与胃内pH值的关系

目的 探讨胃内 pH值与胃食管反流的关系及胃 食管反流的病理生理机制。 方法 采用便携式 pH监测仪对 1 8例食管炎、1 2例食管溃疡、1 0例Barrett’s食管、1 5例健康人行 2 4h食管和胃 pH值同步监测 ,分别计算胃内各段 pH值、胃 食管酸反流次数、长反流次数、最长反流时间以及pH值 <4的时间占总时间的百分比。 结果 ①食管炎组 :上述各项指标在胃内 pH值为 1～ 4时明显高于对照组 (P <0 .0 5 ) ;当胃内 pH值为 1～ 2时 ,酸反流次数明显高于其他pH值段 (P <0 .0 5 ) ;②食管溃疡组 :当胃内 pH值为 1～ 2时 ,酸反流次数、长反流时间明显高于其他 pH值段 (P <0 .0 5 ) ;胃内 pH值为 1～ 3时 ,酸反流次数、长反流时间明显高于对照组 (P <0 .0 5 ) ;③Barrett’s食管组 :胃内pH值为 2～ 3时的酸反流次数明显高于对照组 ;④当胃内 pH值为 1～ 2时 ,食管炎组、食管溃疡组酸反流次数明显高于Barrett’s食管组。结论 当胃内 pH值在 1～ 2之间时 ,食管炎、食管溃疡的胃食管酸反流最为明显 ,食管黏膜损伤的严重程度与食管接触酸的次数及接触酸的时间长短有关 ;Barrett’s食管的胃

洛阳地区Barrett’s食管的发病情况、内镜和病理学特点

目的探讨洛阳地区一组Barrett’s食管（BE）的发病情况、内镜和病理学特点及其与幽门螺杆菌（H．pylori）感染的关系。方法采用普通胃镜检查结合病理检查结果，对洛阳地区有消化道症状的一组BE患者进行分析，同时采用尿素[^14C]呼气试验药盒检查H·pylori感染对BE发病学的影响。结果2006年8月～2007年4月共有593例接受胃镜检查，有22例诊断为BE，BE发病率为3．71％；以短段、舌型发病率最高，分别占81．81％和77．27％；7例（31．8％）有典型反流症状；伴有低度异型性增生3例（13．64％），重度异型性增生1例（4．55％），并发腺癌1例（4．55％），其中伴有重度异型性增生的1例随访1年后并发腺癌；对其中的10例进行H．pylori检查，阳性率为90％。结论洛阳地区BE发病率较高，以短段、舌型为主，有典型反流症状者较少，伴有异型性增生、腺癌者及并发H．pylori感染者常见。

内镜热极技术联用抑酸剂治疗Barrett’s食管的临床观察

目的探讨内镜热极技术联用抑酸剂治疗Barrett’s食管的疗效。方法2006年8月～2007年8月经内镜和病理组织学检查诊断的Barrett’s食管患者100例，其中95例患者在内镜下采用热极治疗，术后口服抑酸剂巩固治疗，另5例患者仅口服抑酸剂治疗进行对照，两组均进行内镜及临床随访。结果95例患者经过1次热极治疗Barrett’s食管完全消除，术后38例（40％）出现胸骨后隐痛；54例患者半年后随访，总有效率为92．59％；对照组3例患者半年后随访，总有效率为0。结论热极联用抑酸剂治疗Barrett’s食管安全有效、短期疗效好。

短节段Barrett’s食管的内镜和病理分析

目的研究内镜和病理下短节段Barrett’s食管(SSBE)的临床症状、内镜分型和病理特征之间的关系。方法对140例内镜和病理诊断为SSBE的患者进行临床症状评估,组织标本HE染色,并对临床症状、内镜分型及病理特征之间的关系进行相关分析。结果SSBE的内镜检出率为9.75%,男女比为1.12∶1,平均发病年龄为51.64岁,有典型胃食管反流病(GERD)症状占70.00%;140例患者中反酸54例,烧心18例,胸骨后痛31例,上腹痛29例,无症状8例。140例患者中病理诊断胃底腺者30例,贲门腺者98例,肠上皮化生者12例;内镜诊断岛型54例,舌型66例,全周型20例。140例患者中贲门腺组反酸、烧心、胸骨后痛、无症状的发生率与其他两组比较明显增高。140例患者中贲门腺组内镜表现为舌型和岛型的检出率明显高于其他两组。主要临床症状的发生率在三种内镜类型之间比较差异无统计学意义。结论SSBE病理特征和胃食管反流症状的发生,以及病理特征和内镜分型之间均密切相关,SSBE临床症状和内镜分型之间无明显关系。

食管腺癌与Barrett’s食管基因表达谱的研究

目的探讨从Barrett’s食管到食管腺癌的癌变过程中基因表达谱的变化。方法取同一食管腺癌患者大体手术标本经病理确诊的腺癌组织、Barrett’s食管组织及正常组织,分别提取组织上皮的总RNA并纯化mRNA;将mRNA逆转录合成以Cy5和Cy3标记的cDNA链做探针,分别混合后在两张基因表达谱芯片上进行杂交。用扫描仪扫描芯片荧光信号,用软件对扫描图像进行分析。结果食管腺癌与正常食管上皮比较差异2倍以上共有214个基因;Barrett’s食管与正常食管上皮比较差异2倍以上共有90个基因。而Barrett’s食管组织和食管腺癌组织均下调的基因有24个,均上调的基因有21个。其中符合从Barrett’s食管到食管腺癌演变趋势的基因中,上调的有9个,下调的有18个。结论这些基因或其产物可作为Barrett’s食管具有癌变高危性的检测指标。

Caspase-3和hTERT在Barrett’s食管中的表达及其相互关系

目的研究半胱天冬蛋白酶-3(Caspase-3)和端粒酶逆转录酶(hTERT)在Barrett’s食管(Barrett’s esophagus,BE)及食管炎伴贲门腺或胃底腺化生组织中的表达,探讨其在BE发病中的作用。方法应用免疫组织化学法(LDP)检测22例BE和38例食管炎伴贲门腺或胃底腺化生组织中Caspase-3和hTERT的表达,比较两者表达的相关性。结果Caspase-3在BE中的阳性表达率为95.45%,明显高于在食管炎伴贲门腺或胃底腺化生组织中的表达(68.42%)(P=0.000);hTERT在BE中的阳性表达率为27.27%,明显低于在食管炎伴贲门腺或胃底腺化生组织中的表达(65.79%)(P=0.000)。BE中Caspase-3与hTERT的表达呈强负相关(r=-1,P=0.000)。结论BE中Caspase-3高表达,hTERT低表达。Caspase-3和hTERT在BE发生过程中起重要作用,可能成为预测BE发生的重要参考指标。

胆汁反流、幽门螺杆菌感染对Barrett’s食管的影响

目的探讨Barrett’s食管(Barrett’s esophagus,BE)与幽门螺杆菌(H.pylori)感染、胆汁反流的关系。方法采用胃镜检查结合病理检查结果对30例BE患者进行分析,同时选取健康对照者30名作为对照组,采用快速尿素酶法及Warthin-Starry银染两种方法检测H.pylori感染;应用便携式胆汁监测仪检测食管24 h胆汁反流情况。结果 BE组中,H.pylori感染率为30.00%,胆汁反流患病率为76.67%。对照组中,H.pylori感染率为60.00%,胆汁反流患病率为6.67%。BE组H.pylori的检出率明显低于对照组(P<0.05)。BE组胆汁反流发生率明显高于对照组(P<0.05)。结论 H.pylori对BE可能有保护作用,胆汁反流与BE关系密切。

HSP27、70、90α和105在Barrett’s食管中的表达及其相互关系

目的研究热休克蛋白27、70、90α、105(HSP27、70、90α、105)在Barrett’s食管(Barrett’s esophagus,BE)和食管炎伴贲门腺或胃底腺化生组织中的表达及相互关系,探讨其在BE发病中的作用。方法应用免疫组织化学法(LDP)检测22例BE和38例食管炎伴贲门腺或胃底腺化生组织中HSP27、70、90α和105的表达,并比较其表达的相关性。结果 HSP70和HSP90α在BE中的阳性表达率分别为31.82%和9.09%,明显低于食管炎伴贲门腺或胃底腺化生组织中的表达(47.37%、42.11%)(P=0.001和P=0.000);HSP105在BE中的阳性表达率为100%,明显高于食管炎伴贲门腺或胃底腺化生组织中的表达(63.16%)(P=0.000);HSP27在BE中的阳性表达率为77.27%,与食管炎伴贲门腺或胃底腺化生组织中的表达率相比无明显差异(50.00%)(P=0.422)。BE中HSP70和HSP90α的表达存在强正相关(r=1,P=0.000),HSP70、HSP90α的表达均与HSP105的表达存在强负相关(r=-1,P=0.000)。结论 BE上皮中的肠化细胞凋亡和增殖失调,HSP70、HSP90α和HSP105在BE发生过程中起重要作用,可能成为预测BE发生的重要指标。

短段Barrett’s食管的不同组织类型、内镜形态、异型程度与Cyclin D1、P53表达的相关性研究

目的研究细胞周期蛋白D1(Cyclin D1)、P53蛋白在短段Barrett’s食管(short segment Barrett’s esophagus,SSBE)不同组织类型、不同内镜形态、不同异型程度分组中的表达,预测不同类型SSBE上皮癌变的风险性,准确筛选高危人群,为指导制定内镜随访计划提供依据。方法选取经电子胃镜、病理检查确诊为SSBE的患者51例,另取32例正常食管黏膜组织作为对照。再根据SSBE不同内镜下形态分为全周型16例、舌型13例、岛型22例,根据不同病理组织类型分为胃底型20例、贲门型21例、特殊肠化型10例,根据BE组织异型增生的程度分为无异型增生组37例、轻度异型增生组14例、重度异型增生组0例。采用免疫组化法检测不同亚组细胞Cyclin D1和P53蛋白的表达,并进行统计学分析。结果 Cyclin D1、P53在SSBE组及正常食管黏膜组中的表达均较低,差异无统计学意义(P>0.05)。Cyclin D1、P53在SSBE不同内镜下形态分型、不同组织学分型各亚组中的表达差异均无统计学意义(P>0.05)。Cyclin D1、P53在合并异型增生组中均表达升高,Cyclin D1在无异型增生、轻度异型增生组中的表达阳性率分别为21.6%、64.3%,组间差异有统计学意义(P=0.007);P53在无异型增生、轻度异型增生组中的表达阳性率分别为27.0%、71.4%,组间差异有统计学意义(P=0.006)。结论 SSBE总体癌变风险低。SSBE的癌变风险与内镜形态分型及组织学分型的不同无直接相关性。对合并异型增生者,Cyclin D1和P53的表达明显升高,提示SSBE伴异型增生者癌变风险明显增加。

高频电治疗Barrett’s食管30例

目的探讨高频电治疗Barrett’s食管的疗效。方法对无异型增生的BE患者行热探针治疗,有异型增生的BE患者行内镜下黏膜切除。结果全部患者肉眼观察均修复为正常的鳞状上皮,组织学仅有3例(10%)残存特殊柱状上皮,随访3～6个月复发4例(13%),并发症轻微短暂。结论高频电治疗BE安全、方便、有效。

Barrett’s食管临床特征

食管癌是所有癌瘤中最致命的癌症之一。Barrett’s食管或Barrett’s肠化是食管腺癌的癌前病变,近年发病率有增高趋势。为了改善预后,提高生存率,手术是最有希望的治疗,但大多数病人仅在发生癌症状后发现,此时病人多有广泛转移,又缺乏有效的化疗和放疗,因此很少使预后改善。因此,提高癌前病变检出率和早期诊断是一个极为现实的临床课题。

肠上皮化生在Barrett’s食管进展为食管腺癌中作用的研究进展

食管腺癌(esophageal adenocarcinoma,EAC)的发病率有逐年增加的趋势,EAC预后差,5年生存率不足20%.Barrett’s食管(barrett’sesophagus,BE)是目前唯一已知的EAC癌前病变,伴肠上皮化生(intestinal metaplasia,IM)的BE有更高的风险进展为EAC,探究IM的发生机制,寻找BE的针对性治疗靶点,成为预防肿瘤的重要措施.胆汁酸反流被认为是IM发生的重要因素,并促进BE向EAC进展,但胆汁反流诱导肠化、癌变的分子调节机制尚不清楚.本文就IM发生的环境、意义、细胞起源学说,胆汁反流的毒性作用以及IM向肿瘤进展的分子改变进行综述,旨在提高临床医师对BE中IM的认识,为早期干预BE和防治EAC提供证据.

MicroRNAs, development of Barrett’s esophagus, and progression to esophageal adenocarcinoma

Barrett's esophagus is a premalignant condition caused by gastroesophageal reflux. Once developed, it can progress through varying grades of dysplasia to esoph-ageal adenocarcinoma. Whilst it is well accepted that Barrett's esophagus is caused by gastroesophageal reflux, the molecular mechanisms of its pathogenesis and progression to cancer remain unclear. MicroRNAs (miRNAs) are short segments of RNA that have been shown to control the expression of many human genes. They have been implicated in most cellular processes, and the role of miRNAs in disease development is be-coming increasingly evident. Understanding altered miRNA expression is likely to help unravel the molecular mechanisms that underpin the development of Barrett's esophagus and its progression to cancer.

Prevalence and risk factors for Barrett’s esophagus in Taiwan

BACKGROUND Barrett’s esophagus(BE)is a pre-malignant condition associated with the development of esophageal adenocarcinoma.The prevalence of BE in the general populations of Asian countries ranges from 0.06%to 1%.However,with lifestyle changes in Asian countries and adoption of western customs,the prevalence of BE might have increased.AIM To determine the current prevalence of BE in Taiwan,and to investigate risk factors predicting the presence of BE.METHODS This retrospective study was conducted at the Health Evaluation Center of Kaohsiung Veterans General Hospital in Taiwan.Between January 2015 and December 2015,3385 subjects undergoing routine esophagogastroduodenoscopy examinations as part of a health check-up at the Health Evaluation Center were included.Patient characteristics and endoscopic findings were carefully reviewed.Lesions with endoscopic findings consistent with BE awaiting histological evaluation were judged as endoscopically suspected esophageal metaplasia(ESEM).BE was defined based on extension of the columnar epithelium≥1 cm above the gastroesophageal junction and was confirmed based on the presence of specialized intestinal metaplasia(IM)in the metaplastic esophageal epithelium.Clinical factors of subjects with BE and subjects without BE were compared,and the risk factors predicting BE were analyzed.RESULTS A total of 3385 subjects(mean age,51.29±11.42 years;57.1%male)were included in the study,and 89 among them were confirmed to have IM and presence of goblet cells via biopsy examination.The majority of these individuals were classified as short segment BE(n=85).The overall prevalence of BE was 2.6%.Multivariate analysis disclosed that old age[odds ratio(OR)=1.033;95%confidence interval(CI):1.012-1.055;P=0.002],male gender(OR=2.106;95%CI:1.145-3.872;P=0.017),ingestion of tea(OR=1.695;95%CI:1.043-2.754;P=0.033),and presence of hiatal hernia(OR=3.037;95%CI:1.765-5.225;P<0.001)were significant risk factors predicting BE.The independent risk factor for the presence of IM in ESEM lesions was old age alone(OR=1.029;95%CI:1.006-1.053;P=0.014).CONCLUSION Current prevalence of BE among the general population in Taiwan is 2.6%.Old age,male gender,ingestion of tea and hiatal hernia are significant risk factors for BE.

Artificial intelligence system for the detection of Barrett’s esophagus

BACKGROUND Barrett’s esophagus(BE),which has increased in prevalence worldwide,is a precursor for esophageal adenocarcinoma.Although there is a gap in the detection rates between endoscopic BE and histological BE in current research,we trained our artificial intelligence(AI)system with images of endoscopic BE and tested the system with images of histological BE.AIM To assess whether an AI system can aid in the detection of BE in our setting.METHODS Endoscopic narrow-band imaging(NBI)was collected from Chung Shan Medical University Hospital and Changhua Christian Hospital,resulting in 724 cases,with 86 patients having pathological results.Three senior endoscopists,who were instructing physicians of the Digestive Endoscopy Society of Taiwan,independently annotated the images in the development set to determine whether each image was classified as an endoscopic BE.The test set consisted of 160 endoscopic images of 86 cases with histological results.RESULTS Six pre-trained models were compared,and EfficientNetV2B2(accuracy[ACC]:0.8)was selected as the backbone architecture for further evaluation due to better ACC results.In the final test,the AI system correctly identified 66 of 70 cases of BE and 85 of 90 cases without BE,resulting in an ACC of 94.37%.CONCLUSION Our AI system,which was trained by NBI of endoscopic BE,can adequately predict endoscopic images of histological BE.The ACC,sensitivity,and specificity are 94.37%,94.29%,and 94.44%,respectively.

Expanding beyond endoscopy: A review of non-invasive modalities in Barrett’s esophagus screening and surveillance

Barrett’s esophagus(BE)is a condition that results from replacement of the damaged normal squamous esophageal mucosa to intestinal columnar mucosa and is the most significant predisposing factor for development of esophageal adenocarcinoma.Current guidelines recommend endoscopic evaluation for screening and surveillance based on various risk factors which has limitations such as invasiveness,availability of a trained specialist,patient logistics and cost.Trans-nasal endoscopy is a less invasive modality but still has similar limitations such as limited availability of trained specialist and costs.Non-endoscopic modalities,in comparison,require minimal intervention,can be done in an office visit and has the potential to be a more ideal choice for mass public screening and surveillance,particularly in patents at low risk for BE.These include newer generations of esophageal capsule endoscopy which provides direct visualization of BE,and tethered capsule endomicroscopy which can obtain high-resolution images of the esophagus.Various cell collection devices coupled with biomarkers have been used for BE screening.Cytosponge,in combination with TFF3,as well as EsophaCap and EsoCheck have shown promising results in various studies when used with various biomarkers.Other modalities including circulatory microRNAs and volatile organic compounds that have demonstrated favorable outcomes.Use of these cell collection methods for BE surveillance is a potential area of future research.