Engaging Stakeholders to Develop a Roadmap for Dry Eye and MGD PCORI-Funded Research.

Introduction: Although affecting an estimated 35% of the population, Dry Eye is not well understood by patients and the medical community. As a result, both in research and clinical settings, diagnostic and treatment protocols tend to be non-specific, ad hoc, and inadequate, with a narrow industry-driven focus. The purpose of this convening was to propose a research roadmap that orients Dry Eye researchers toward a comprehensive patient-centered approach to diagnosing and treating Dry Eye, Meibomian gland dysfunction (MGD), and related comorbidities with a goal of improving clinical outcomes for Dry Eye/MGD patients. Methods: Sixteen participants, including Dry Eye/MGD patients, caregivers, and patient advocates together with a group of experts in Dry Eye, MGD and other fields identified gaps in research on Dry Eye and MGD diagnostic and treatment approaches (age range 20-80; male to female ratio of 7:11; patients: 7). During a 2-day virtual convening, participants were assigned to topic-specific focus-group sessions to discuss and develop research questions pertaining to Dry Eye and MGD. The research questions were compiled into a proposed patient-centered roadmap for Dry Eye and MGD research. Two additional participants contributed to the proposed roadmap following the convening. Results: The focus groups identified over 80 patient-centered research questions important to patients and other stakeholders and compiled these into a proposed research roadmap. Conclusion: The convened stakeholders aim to establish a cohesive and comprehensive patient-centered approach to treating Dry Eye, Meibomian Gland Dysfunction, and comorbidities. The research roadmap will serve as a reference for researchers, educational institutions, clinicians, and others evaluating diagnostic and treatment protocols in Dry Eye and MGD.

Dynamic evaluation of blood immune cells predictive of response to immune checkpoint inhibitors in NSCLC by multicolor spectrum flow cytometry.

Introduction: Immune checkpoint inhibitors (ICIs) only benefit a subset of cancer patients, underlining the need for predictive biomarkers for patient selection. Given the limitations of tumor tissue availability, flow cytometry of peripheral blood mononuclear cells (PBMCs) is considered a noninvasive method for immune monitoring. This study explores the use of spectrum flow cytometry, which allows a more comprehensive analysis of a greater number of markers using fewer immune cells, to identify potential blood immune biomarkers and monitor ICI treatment in non-small-cell lung cancer (NSCLC) patients. Methods: PBMCs were collected from 14 non-small-cell lung cancer (NSCLC) patients before and after ICI treatment and 4 healthy human donors. Using spectrum flow cytometry, 24 immune cell markers were simultaneously monitored using only 1 million PBMCs. The results were also compared with those from clinical flow cytometry and bulk RNA sequencing analysis. Results: Our findings showed that the measurement of CD4+ and CD8+ T cells by spectrum flow cytometry matched well with those by clinical flow cytometry (Pearson R ranging from 0.75 to 0.95) and bulk RNA sequencing analysis (R=0.80, P=1.3 x 10-4). A lower frequency of CD4+ central memory cells before treatment was associated with a longer median progression-free survival (PFS) [Not reached (NR) vs. 5 months; hazard ratio (HR)=8.1, 95% confidence interval (CI) 1.5-42, P=0.01]. A higher frequency of CD4-CD8- double-negative (DN) T cells was associated with a longer PFS (NR vs. 4.45 months; HR=11.1, 95% CI 2.2-55.0, P=0.003). ICIs significantly changed the frequency of cytotoxic CD8+PD1+ T cells, DN T cells, CD16+CD56dim and CD16+CD56- natural killer (NK) cells, and CD14+HLDRhigh and CD11c+HLADR + monocytes. Of these immune cell subtypes, an increase in the frequency of CD16+CD56dim NK cells and CD14+HLADRhigh monocytes after treatment compared to before treatment were associated with a longer PFS (NR vs. 5 months, HR=5.4, 95% CI 1.1-25.7, P=0.03; 7.8 vs. 3.8 months, HR=5.7, 95% CI 169 1.0-31.7, P=0.04), respectively. Conclusion: Our preliminary findings suggest that the use of multicolor spectrum flow cytometry helps identify potential blood immune biomarkers for ICI treatment, which warrants further validation.

Full spectrum flow cytometry and mass cytometry: A 32-marker panel comparison.

High-dimensional single-cell data has become an important tool in unraveling the complexity of the immune system and its involvement in homeostasis and a large array of pathologies. As technological tools are developed, researchers are adopting them to answer increasingly complex biological questions. Up until recently, mass cytometry (MC) has been the main technology employed in cytometric assays requiring more than 29 markers. Recently, however, with the introduction of full spectrum flow cytometry (FSFC), it has become possible to break the fluorescence barrier and go beyond 29 fluorescent parameters. In this study, in collaboration with the Stanford Human Immune Monitoring Center (HIMC), we compared five patient samples using an established immune panel developed by the HIMC using their MC platform. Using split samples and the same antibody panel, we were able to demonstrate highly comparable results between the two technologies using multiple data analysis approaches. We report here a direct comparison of two technology platforms (MC and FSFC) using a 32-marker flow cytometric immune monitoring panel that can identify all the previously described and anticipated immune subpopulations defined by this panel.

Stratification of hospitalized COVID-19 patients into clinical severity progression groups by immuno-phenotyping and machine learning.

Quantitative or qualitative differences in immunity may drive clinical severity in COVID-19. Although longitudinal studies to record the course of immunological changes are ample, they do not necessarily predict clinical progression at the time of hospital admission. Here we show, by a machine learning approach using serum pro-inflammatory, anti-inflammatory and anti-viral cytokine and anti-SARS-CoV-2 antibody measurements as input data, that COVID-19 patients cluster into three distinct immune phenotype groups. These immune-types, determined by unsupervised hierarchical clustering that is agnostic to severity, predict clinical course. The identified immune-types do not associate with disease duration at hospital admittance, but rather reflect variations in the nature and kinetics of individual patient's immune response. Thus, our work provides an immune-type based scheme to stratify COVID-19 patients at hospital admittance into high and low risk clinical categories with distinct cytokine and antibody profiles that may guide personalized therapy.

Use of transcranial magnetic stimulation for studying the neural basis of numerical cognition: A systematic review.

Complex numerical cognition is a crucial ability in the human brain. Conventional neuroimaging techniques do not differentiate between epiphenomena and neuronal groups critical to numerical cognition. Transcranial magnetic stimulation (TMS) allows defining causal models of the relationships between specific activated or inhibited neural regions and functional changes in cognition. However, there is insufficient knowledge on the differential effects of various TMS protocols and stimulation parameters on numerical cognition. This systematic review aimed to synthesize the evidence that different TMS protocols provide regarding the neural basis of numerical cognition in healthy adults. We included 21 experimental studies in which participants underwent any transcranial magnetic stimulation such as a single pulse TMS, repetitive TMS, and theta-burst stimulation. The primary outcome measures were any change in numerical cognition processes evidenced by numerical or magnitude tasks, measured with any independent variable like reaction times, accuracy, or congruency effects. TMS applied to regions of the parietal cortex and prefrontal cortex has neuromodulatory effects, which translate into measurable behavioral effects affecting cognitive functions related to arithmetic and numerical and magnitude processing. The use of TMS for the study of the neural bases of numerical cognition allows addressing issues such as localization, timing, lateralization and has allowed establishing site-function dissociations and double site-function dissociations. Moreover, this technique is in a moment of expansion due to the growing knowledge of its physiological effects and the enormous potential of combining TMS with other techniques such as electroencephalography, functional magnetic resonance imaging, or near-infrared spectroscopy to reach a more precise brain mapping.

High-Dimensional Immunophenotyping with 37-Color Panel Using Full-Spectrum Cytometry.

A comprehensive study of the cellular components of the immune system demands both deep and broad immunophenotyping of numerous cell subsets in an effective and practical way. Novel full-spectrum technology reveals the complete emission spectrum of each dye maximizing the amount of information that can be obtained on a single sample regarding conventional flow cytometry and provide an expanded knowledge of biological processes. In this chapter, we describe a 37-color protocol that allows to identify more than 45 different cell populations on whole blood samples of SARS-CoV-2-infected patients.

Panel Optimization for High-Dimensional Immunophenotyping Assays Using Full-Spectrum Flow Cytometry.

Technological advancements in fluorescence flow cytometry and an ever-expanding understanding of the complexity of the immune system have led to the development of large flow cytometry panels reaching up to 43 colors at the single-cell level. However, as panel size and complexity increase, so too does the detail involved in designing and optimizing successful high-quality panels fit for downstream high-dimensional data analysis. In contrast to conventional flow cytometers, full-spectrum flow cytometers measure the entire emission spectrum of each fluorophore across all lasers. This allows for fluorophores with very similar emission maxima but unique overall spectral fingerprints to be used in conjunction, enabling relatively straightforward design of larger panels. Although a protocol for best practices in full-spectrum flow cytometry panel design has been published, there is still a knowledge gap in going from the theoretically designed panel to the necessary steps required for panel optimization. Here, we aim to guide users through the theory of optimizing a high-dimensional full-spectrum flow cytometry panel for immunophenotyping using comprehensive step-by-step protocols. These protocols can also be used to troubleshoot panels when issues arise. A practical application of this approach is exemplified with a 24-color panel designed for identification of conventional T-cell subsets in human peripheral blood. © 2021 Malaghan Institute of Medical Research, Cytek Biosciences. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Preparation and evaluation of optimal spectral reference controls Support Protocol 1: Antibody titration Support Protocol 2: Changing instrument settings Basic Protocol 2: Unmixing evaluation of fully stained sample Basic Protocol 3: Evaluation of marker resolution Support Protocol 3: Managing heterogeneous autofluorescence Basic Protocol 4: Assessment of data quality using expert gating and dimensionality reduction algorithms.

Cell by cell immuno- and cancer marker profiling of non-small cell lung cancer tissue: Checkpoint marker expression on CD103+, CD4+ T-cells predicts circulating tumor cells.

Non-small cell lung cancer (NSCLC) has a poor prognosis. Targeted therapy and immunotherapy in recent years has significantly improved NSCLC patient outcome. In this study, we employed cell-by-cell immune and cancer marker profiling of the primary tumor cells to investigate possible signatures that might predict the presence or absence of circulating tumor cells (CTCs). We performed a comprehensive study on 10 NSCLC patient tissue samples with paired blood samples. The solid tissue biopsy samples were dissociated into single cells by non-enzymatic tissue homogenization and stained with a total 25 immune, cancer markers and DNA content dye and analyzed with high-parameter flow cytometry. CTCs were isolated and analyzed from the paired peripheral blood. We investigated a total of 74 biomarkers for their correlation with CTC number. Strong correlations were observed between CTC number and the frequency of immune checkpoint marker expressing lymphocytes (CTLA-4, LAG3, TIM3, PD-1), within the CD103+CD4+ T lymphocyte subset. CTC number is also correlated with the frequency of PD-L1 expressing cancer cells and cancer cell DNA content. In contrast, CTC number inversely correlated to the frequency of CD44+E-cadherin- cancer cells. Unsupervised clustering analysis based on the biomarker analysis separated the CTC negative patients from the CTC positive patients. Profiling multiple immune and cancer markers on cancer samples with multi-parametric flow cytometry allowed us to obtain protein expression information at the single cell level. Clustering analysis of the proteomic data revealed a signature driven by checkpoint marker expression on CD103+CD4+ T cells that could potentially be predictive of CTCs and targets of therapy.

OMIP-069: Forty-Color Full Spectrum Flow Cytometry Panel for Deep Immunophenotyping of Major Cell Subsets in Human Peripheral Blood.

This 40-color flow cytometry-based panel was developed for in-depth immunophenotyping of the major cell subsets present in human peripheral blood. Sample availability can often be limited, especially in cases of clinical trial material, when multiple types of testing are required from a single sample or timepoint. Maximizing the amount of information that can be obtained from a single sample not only provides more in-depth characterization of the immune system but also serves to address the issue of limited sample availability. The panel presented here identifies CD4 T cells, CD8 T cells, regulatory T cells, γδ T cells, NKT-like cells, B cells, NK cells, monocytes and dendritic cells. For each specific cell type, the panel includes markers for further characterization by including a selection of activation and differentiation markers, as well as chemokine receptors. Moreover, the combination of multiple markers in one tube might lead to the discovery of new immune phenotypes and their relevance in certain diseases. Of note, this panel was designed to include only surface markers to avoid the need for fixation and permeabilization steps. The panel can be used for studies aimed at characterizing the immune response in the context of infectious or autoimmune diseases, monitoring cancer patients on immuno- or chemotherapy, and discovery of unique and targetable biomarkers. Different from all previously published OMIPs, this panel was developed using a full spectrum flow cytometer, a technology that has allowed the effective use of 40 fluorescent markers in a single panel. The panel was developed using cryopreserved human peripheral blood mononuclear cells (PBMC) from healthy adults (Table 1). Although we have not tested the panel on fresh PBMCs or whole blood, it is anticipated that the panel could be used in those sample preparations without further optimization. @ 2020 Cytek Biosciences, Inc. Cytometry Part A published by Wiley Periodicals LLC on behalf of International Society for Advancement of Cytometry.

Panel Design and Optimization for High-Dimensional Immunophenotyping Assays Using Spectral Flow Cytometry.

Technological advances in fluorescence flow cytometry and an ever-expanding understanding of the complexity of the immune system have led to the development of large (20+ parameters) flow cytometry panels. However, as panel complexity and size increase, so does the difficulty involved in designing a high-quality panel, accessing the instrumentation capable of accommodating large numbers of parameters, and analyzing such high-dimensional data. A recent advancement is spectral flow cytometry, which in contrast to conventional flow cytometry distinguishes the full emission spectrum of each fluorophore across all lasers, rather than identifying only the peak of emission. Fluorophores with a similar emission maximum but distinct off-peak signatures can therefore be accommodated within the same flow cytometry panel, allowing greater flexibility in terms of panel design and fluorophore detection. Here, we highlight the specific characteristics of spectral flow cytometry and aim to guide users through the process of building, designing, and optimizing high-dimensional spectral flow cytometry panels using a comprehensive step-by-step protocol. Special considerations are also given for using highly overlapping dyes, and a logical selection process for optimal marker-fluorophore assignment is provided. © 2020 by John Wiley & Sons, Inc.

Prevalencia y factores relacionados de sobrepeso y obesidad en estudiantes de una universidad pública / Prevalence and related factors of overweight and obesity in students of a public university

Fundamentos: El sobrepeso y la obesidad resultantes de hábitos al desarrollo de enfermedades cardiovasculares (ECV), síndromes metabólicos y de otras enfermedades crónicas no transmisibles, constituyen la principal causa de muerte a nivel mundial. El objetivo fue determinar la prevalencia y factores relacionados de sobrepeso y obesidad en estudiantes de una Universidad pública en Popayán, Colombia. Métodos: Estudio cuantitativo, descriptivo de corte transversal; mediante un muestreo aleatorio se seleccionaron 378 estudiantes, aceptando y firmando un consentimiento informado; se aplicó una encuesta indagando aspectos sociodemográficos, conocimiento sobre obesidad, percepción de figura corporal, hábitos y medidas antropométricas. Se calculó la prevalencia, se realizó análisis descriptivo de los factores asociados y se establecieron asociaciones con análisis inferencial. Resultados: Se encontró en el Índice de Masa Corporal una prevalencia de sobrepeso del 19,8%, de obesidad del 2,5%; siendo más frecuente en hombres (28,3%), en mayores de 23 años (36,7%), en quienes tienen pareja (50%); se encontró riesgo de padecer ECV (18,3%) y alto riesgo de síndrome metabólico (9%). Conclusión: La prevalencia de obesidad y sobrepeso encontrada en los estudiantes universitarios es alta, presentando alto riesgo de padecer ECV o Síndrome metabólico (AU)

Background: Overweight and obesity rtesulting from unhealthy lifestyle habits, areconsidered risk factors that predispose to the development of cardiovascular diseases (CVD), metabolic syndromes and other chronic noncommunicable diseases, are the leading cause of death worldwide. The objective was to determine the prevalence and health factors related to overweight and obesity in students of a public university in Popayan, Colombia. Methods: Quantitative, descriptive cross-sectional study; random sampling 378 students were selected, Accepting and signing informed consent; A survey was carried out investigating sociodemographic aspects, knowledge about obesity, perception of body figure, habits and anthropometric measurements. The prevalences were calculated, a descriptive analysis of the associated factors was performed and associations were established with inferential analysis. Results: The prevalence of overweight was found in the students according to the BMI of 19.8% and of obesity of 2.5%; being more frequent in males (28.3%), in those older than 23 years (36.7%), in those with a partner (50%); Presented a risk of CVD (18.3%) and high risk of metabolic syndrome (9%). Conclusion: The prevalence of obesity and overweight found in college students is high, presenting a high risk of CVD or metabolic syndrome (AU)

Actitudes, prácticas y estilos de vida en adolescentes de instituciones de educación secundaria de la ciudad de popayán, 2016

Objetivo: Determinar los estilos de vida de los entornos escolares, sus conocimientos, actitudes y prácticas hacia su salud, en los estudiantes de educación media vocacional en seis instituciones educativas públicas y privadas de la ciudad de Popayán. Materiales y métodos: Estudio descriptivo, observacional de corte transversal, con muestra probabilística seleccionando 135 estudiantes. Se aplicó un instrumento tipo encuesta para el análisis de factores sociodemográficos, actitudes, prácticas y hábitos de vida de los adolescentes. Resultados: De los estudiantes encuestados son de grado noveno (51,9%); han fumado tabaco alguna vez (38,5%); han consumido alcohol (88,9%); han consumido sustancias psicoactivas (29,6%); realizan actividad física (87,4%); han iniciado su vida sexual (59,3%). Conclusiones: Los hábitos no saludables en los adolescentes se ven influenciados por la vulnerabilidad y entorno social, el uso de la tecnología y la vida moderna, los cuales afectan el tiempo libre y los determinantes sociales en salud.

Objective: To determine the lifestyles of school environments, their knowledge, attitudes and practices towards their health, in vocational secondary education students in six public and private educational institutions from Popayán. Materials and methods: Descriptive, observational cross -sectional study with a probabilistic sample, selecting 135 students, a survey- type instrument was used to analyze the socio-demographic factors, attitudes, practices and life customs of teenagers. Results: The respondent students, they were nineth grade (51.9%); have had smoked cigarettes (38.5%); consumed alcohol (88.9%) and psychoactive substances (29.6%); performed physical activity (87.4%); having started their sexual life (59.3%). Conclusions: Unhealthy habits in adolescents are influenced by vulnerability and social environment, the use of technology and modern life, which affect free time and social determinants in health.

Objetivo: Determinar os estilos de vida no ambiente escolar, seus conhecimentos, atitudes e práticas para sua saúde, nos estudantes do ensino meio em seis escolas públicas e privadas na cidade de Popayán. Métodos: Estudo descritivo, observacional de tipo transversal, com amostra probabilística selecionando 135 estudantes. Aplicou-se o instrumento tipo enquete para as análises dos fatores sociodemogra-ficos, atitudes, práticas e estilos de vida dos adolescentes. Resultados: Os alunos enquistados são do nono ano (51,9%), tem fumado alguma vez (38,5%), tem consumido álcool (88,9%), tem consumido substâncias psicoativas (29,6%), praticam atividade física (87,4%), tem vida sexual ativa (59,3%). Conclusões: os estilos de vida não saludáveis nos adolescentes tem influencia pela vulnerabilidade e entorno social, o uso da tecnologia, a vida moderna os quais afetam seu tempo livre e os determinantes sociais na saúde.

Standardizing Flow Cytometry Immunophenotyping Analysis from the Human ImmunoPhenotyping Consortium.

Standardization of immunophenotyping requires careful attention to reagents, sample handling, instrument setup, and data analysis, and is essential for successful cross-study and cross-center comparison of data. Experts developed five standardized, eight-color panels for identification of major immune cell subsets in peripheral blood. These were produced as pre-configured, lyophilized, reagents in 96-well plates. We present the results of a coordinated analysis of samples across nine laboratories using these panels with standardized operating procedures (SOPs). Manual gating was performed by each site and by a central site. Automated gating algorithms were developed and tested by the FlowCAP consortium. Centralized manual gating can reduce cross-center variability, and we sought to determine whether automated methods could streamline and standardize the analysis. Within-site variability was low in all experiments, but cross-site variability was lower when central analysis was performed in comparison with site-specific analysis. It was also lower for clearly defined cell subsets than those based on dim markers and for rare populations. Automated gating was able to match the performance of central manual analysis for all tested panels, exhibiting little to no bias and comparable variability. Standardized staining, data collection, and automated gating can increase power, reduce variability, and streamline analysis for immunophenotyping.

Synthesis of highly substituted γ-butyrolactones by a gold-catalyzed cascade reaction of benzyl esters.

Easily accessible benzylic esters of 3-butynoic acids in a gold-catalyzed cyclization/rearrangement cascade reaction provided 3-propargyl γ-butyrolactones with the alkene and the carbonyl group not being conjugated. Crossover experiments showed that the formation of the new C-C bond is an intermolecular process. Initially propargylic-benzylic esters were used, but alkyl-substituted benzylic esters worked equally well. In the case of the propargylic-benzylic products, a simple treatment of the products with aluminum oxide initiated a twofold tautomerization to the allenyl-substituted γ-butyrolactones with conjugation of the carbonyl group, the olefin, and the allene. The synthetic sequence can be conducted stepwise or as a one-pot cascade reaction with similar yields. Even in the presence of the gold catalyst the new allene remains intact.

Toward development of a comprehensive external quality assurance program for polyfunctional intracellular cytokine staining assays.

The External Quality Assurance Program Oversight Laboratory (EQAPOL) Flow Cytometry Program assesses the proficiency of NIH/NIAID/DAIDS-supported and potentially other interested research laboratories in performing Intracellular Cytokine Staining (ICS) assays. The goal of the EQAPOL Flow Cytometry External Quality Assurance Program (EQAP) is to provide proficiency testing and remediation for participating sites. The program is not punitive; rather, EQAPOL aims to help sites identify areas for improvement. EQAPOL utilizes a highly standardized ICS assay to minimize variability and readily identify those sites experiencing technical difficulties with their assays. Here, we report the results of External Proficiency 3 (EP3) where participating sites performed a 7-color ICS assay. On average, sites perform well in the Flow Cytometry EQAP (median score is "Good"). The most common technical issues identified by the program involve protocol adherence and data analysis; these areas have been the focus of site remediation. The EQAPOL Flow Cytometry team is now in the process of expanding the program to 8-color ICS assays. Evaluating polyfunctional ICS responses would align the program with assays currently being performed in support of HIV immune monitoring assays.

Establishment and maintenance of a PBMC repository for functional cellular studies in support of clinical vaccine trials.

A large repository of cryopreserved peripheral blood mononuclear cells (PBMCs) samples was created to provide laboratories testing the specimens from human immunodeficiency virus-1 (HIV-1) vaccine clinical trials the material for assay development, optimization, and validation. One hundred thirty-one PBMC samples were collected using leukapheresis procedure between 2007 and 2013 by the Comprehensive T cell Vaccine Immune Monitoring Consortium core repository. The donors included 83 human immunodeficiency virus-1 (HIV-1) seronegative and 32 HIV-1 seropositive subjects. The samples were extensively characterized for the ability of T cell subsets to respond to recall viral antigens including cytomegalovirus, Epstein-Barr virus, influenza virus, and HIV-1 using Interferon-gamma (IFN-γ) enzyme linked immunospot (ELISpot) and IFN-γ/interleukin 2 (IL-2) intracellular cytokine staining (ICS) assays. A subset of samples was evaluated over time to determine the integrity of the cryopreserved samples in relation to recovery, viability, and functionality. The principal results of our study demonstrate that viable and functional cells were consistently recovered from the cryopreserved samples. Therefore, we determined that this repository of large size cryopreserved cellular samples constitutes a unique resource for laboratories that are involved in optimization and validation of assays to evaluate T, B, and NK cellular functions in the context of clinical trials.

Highly active phosphite gold(I) catalysts for intramolecular hydroalkoxylation, enyne cyclization and furanyne cyclization.

New and highly active mononuclear phosphite gold(I) catalysts are described. Turn-over numbers up to 37,000 for the furan-yne reaction and up to 28,000,000 for the two-fold hydroalkoxylation of alkynes are reported.

From ynamides to highly substituted benzo[b]furans: gold(I)-catalyzed 5-endo-dig-cyclization/rearrangement of alkylic oxonium intermediates.

A series of arylynamides with alkyloxy groups at the ortho position of the aryl group was prepared through a short alkylation/cross-coupling/amidation sequence. The gold-catalyzed conversion of these substrates combined both C-O and C-C formation steps, thus providing benzofurans with amine functionalities at the 2-position and alkyl groups at the 3-position. Cross-over experiments showed that the alkyl-migration step was an intermolecular process. X-ray crystal-structure analysis of two of the products supported our structural assignment. In some cases, the corresponding benzofurans without the alkyl group at the 3-position were obtained as side-products, which were formed through a competing protodeauration process.