Repeatability and reproducibility of the Forskolin-induced swelling (FIS) assay on intestinal organoids from people with Cystic Fibrosis.

BACKGROUND: The forskolin-induced swelling (FIS) assay measures CFTR function on patient-derived intestinal organoids (PDIOs) and may guide treatment selection for individuals with Cystic Fibrosis (CF). The aim of this study is to demonstrate the repeatability and reproducibility of the FIS assay following a detailed Standard Operating Procedure (SOP), thus advancing the validation of the assay for precision medicine (theranostic) applications. METHODS: Over a 2-year period, FIS responses to CFTR modulators were measured in four European labs. PDIOs from six subjects with CF carrying different CFTR genotypes were used to assess the repeatability and reproducibility across the dynamic range of the assay. RESULTS: Technical, intra-assay repeatability was high (Lin's concordance correlation coefficient (CCC) 0.95-0.98). Experimental, within-subject repeatability was also high within each lab (CCCs all >0.9). Longer-term repeatability (>1 year) showed more variability (CCCs from 0.67 to 0.95). The reproducibility between labs was also high (CCC ranging from 0.92 to 0.97). Exploratory analysis also found that between-lab percentage of agreement of dichotomized CFTR modulator outcomes for predefined FIS thresholds ranged between 78 and 100 %. CONCLUSIONS: The observed repeatability and reproducibility of the FIS assay within and across different labs is high and support the use of FIS as biomarker of CFTR function in the presence or absence of CFTR modulators.

Malacological, socio-environmental evaluation, and evidence of local transmission and maintenance of schistosomiasis in an urban area of Northeast Brazil.

To present the current epidemiological scenario of schistosomiasis related to urban transmission through an epidemiological risk assessment in Porto de Galinhas, a coastal area of Pernambuco, Brazil. Malacological and parasitological surveys were performed between the years 2018 and 2020. Snails were identified taxonomically and examined to confirm infection by Schistosoma mansoni, and so to identify Schistosomiasis Transmission Foci (STF) by the artificial light exposure technique. Stool samples were examined using the Kato-Katz method to identify schistosomiasis cases. Socioeconomic, environmental, behavioural and health data were collected by a questionnaire applied to participates in the survey and used to predict the schistosomiasis risk occurrence by multivariate logistic regression. In all, a total of 6466 snails of Biomphalaria glabrata were collected and 36 breeding sites were identified, of which 25 % were STF. A total of 2236 individuals took part of the survey which identified 187 cases of schistosomiasis, registering a positivity percentage of 8.36 %. The surveys identified the neighbourhoods with the highest risk for transmission while the socioenvironmental analysis identifies other risk factors for disease occurrence, such as gender, age range, level of education and absence of water drainage. We found that areas with poor sanitation, flooding during winter periods and dwellings located near mangroves should be treated by health authorities as priority areas for health interventions to minimize disease transmission. In addition, efforts to improve the population's educational level could certainly contribute to the adoption of measures to prevent and control this neglected tropical disease.

Personalized medicine: Function of CFTR variant p.Arg334Trp is rescued by currently available CFTR modulators.

Most of the 2,100 CFTR gene variants reported to date are still unknown in terms of their disease liability in Cystic Fibrosis (CF) and their molecular and cellular mechanism that leads to CFTR dysfunction. Since some rare variants may respond to currently approved modulators, characterizing their defect and response to these drugs is essential for effective treatment of people with CF (pwCF) not eligible for the current treatment. Here, we assessed how the rare variant, p.Arg334Trp, impacts on CFTR traffic and function and its response to existing CFTR modulators. To this end, we performed the forskolin-induced swelling (FIS) assay on intestinal organoids from 10 pwCF bearing the p.Arg334Trp variant in one or both alleles of the CFTR gene. In parallel, a novel p.Arg334Trp-CFTR expressing CFBE cell line was generated to characterize the variant individually. Results show that p.Arg334Trp-CFTR does not significantly affect the plasma membrane traffic of CFTR and evidences residual CFTR function. This CFTR variant is rescued by currently available CFTR modulators independently of the variant in the second allele. The study, predicting clinical benefit for CFTR modulators in pwCF with at least one p.Arg334Trp variant, demonstrates the high potential of personalized medicine through theranostics to extend the label of approved drugs for pwCF carrying rare CFTR variants. We recommend that this personalized approach should be considered for drug reimbursement policies by health insurance systems/national health services.

Novel synthetic derivatives of cinnamic and p-coumaric acids with antiproliferative effect on breast MCF-7 tumor cells.

p-Coumaric acid is derived from cinnamic acid and is one of the major compounds in the Brazilian green propolis extract. Studies have shown that both p-coumaric acid and cinnamic acid have promising antiproliferative effects. In this context, aiming to increase the complexity of these active natural products and their activities, we performed coupling reactions with propargylamine and benzylamine, as well as with threonine, phenylalanine and lysine amino acids, aiming to enhance their antiproliferative effects towards the hormone-dependent breast cancer MCF-7 cells. Overall, the p-coumaric acid coupling with L-threonine amino acid (compound 15) had the best selectivity index (SI = 5.1), with half-maximal inhibitory concentration of 39.6 ± 1 µM, showing a high selectivity against hormone-dependent breast cancer cell lines MCF-7 and low cytotoxicity against the normal breast cell lines MCF-10A. Thus, this new natural product derivative may represent a prototype for the future development of antiproliferative agents, especially against hormone-dependent breast cancer.

Additive Potentiation of R334W-CFTR Function by Novel Small Molecules.

The R334W (c.1000C>T, p.Arg334Trp) is a rare cystic fibrosis (CF)-causing mutation for which no causal therapy is currently approved. This mutation leads to a significant reduction of CF transmembrane conductance regulator (CFTR) channel conductance that still allows for residual function. Potentiators are small molecules that interact with CFTR protein at the plasma membrane to enhance CFTR-dependent chloride secretion, representing thus pharmacotherapies targeting the root cause of the disease. Here, we generated a new CF bronchial epithelial (CFBE) cell line to screen a collection of compounds and identify novel potentiators for R334W-CFTR. The active compounds were then validated by electrophysiological assays and their additive effects in combination with VX-770, genistein, or VX-445 were exploited in this cell line and further confirmed in intestinal organoids. Four compounds (LSO-24, LSO-25, LSO-38, and LSO-77) were active in the functional primary screen and their ability to enhance R334W-CFTR-dependent chloride secretion was confirmed using electrophysiological measurements. In silico ADME analyses demonstrated that these compounds follow Lipinski's rule of five and are thus suggested to be orally bioavailable. Dose−response relationships revealed nevertheless suboptimal efficacy and weak potency exerted by these compounds. VX-770 and genistein also displayed a small potentiation of R334W-CFTR function, while VX-445 demonstrated no potentiator activity for this mutation. In the R334W-expressing cell line, CFTR function was further enhanced by the combination of LSO-24, LSO-25, LSO-38, or LSO-77 with VX-770, but not with genistein. The efficacy of potentiator VX-770 combined with active LSO compounds was further confirmed in intestinal organoids (R334W/R334W genotype). Taken together, these molecules were demonstrated to potentiate R334W-CFTR function by a different mechanism than that of VX-770. They may provide a feasible starting point for the design of analogs with improved CFTR-potentiator activity.

Preparation and characterization of cellulose fibers from Meghatyrsus maximus: Applications in its chemical derivatives.

Non-wood lignocellulosic fibers have emerged and are becoming increasingly important as an alternative source of cellulose for derivatives, functional materials, and biofuels. This work aimed, to obtain cellulose from Meghatyrsus maximus grass with adequate properties through an alkaline delignification and alkaline hydrogen peroxide bleaching. Meghatyrsus maximus was chemically characterized as lignocellulosic biomass, which consisted of 45.0 %, cellulose, 35.0 % hemicellulose, and 20.0 % lignin. The obtained cellulose was characterized by Fourier transform infrared spectroscopy, X-ray diffraction analysis, thermogravimetric analysis, and scanning electron microscopy. The alpha-cellulose content was 98.50 % with a crystallinity of 61.0 %. The morphological study by scanning electron microscopy images indicates a clean surface and removal of non-cellulosic components present in the initial raw fibers. These results showed that high-quality cellulose was obtained and is comparable to a commercial alpha-cellulose, highlighting Meghatyrsus maximus as an alternative source of lignocellulosic fibers.

Advances in Preclinical In Vitro Models for the Translation of Precision Medicine for Cystic Fibrosis.

The development of preclinical in vitro models has provided significant progress to the studies of cystic fibrosis (CF), a frequently fatal monogenic disease caused by mutations in the gene encoding the CF transmembrane conductance regulator (CFTR) protein. Numerous cell lines were generated over the last 30 years and they have been instrumental not only in enhancing the understanding of CF pathological mechanisms but also in developing therapies targeting the underlying defects in CFTR mutations with further validation in patient-derived samples. Furthermore, recent advances toward precision medicine in CF have been made possible by optimizing protocols and establishing novel assays using human bronchial, nasal and rectal tissues, and by progressing from two-dimensional monocultures to more complex three-dimensional culture platforms. These models also enable to potentially predict clinical efficacy and responsiveness to CFTR modulator therapies at an individual level. In parallel, advanced systems, such as induced pluripotent stem cells and organ-on-a-chip, continue to be developed in order to more closely recapitulate human physiology for disease modeling and drug testing. In this review, we have highlighted novel and optimized cell models that are being used in CF research to develop novel CFTR-directed therapies (or alternative therapeutic interventions) and to expand the usage of existing modulator drugs to common and rare CF-causing mutations.

Zebrafish optineurin: genomic organization and transcription regulation.

Optineurin (OPTN) is involved in a variety of mechanisms, such as autophagy, vesicle trafficking, and nuclear factor kappa-B (NF-κB) signaling. Mutations in the OPTN gene have been associated with different pathologies, including glaucoma, amyotrophic lateral sclerosis, and Paget's disease of bone. Since the relationship between fish and mammalian OPTN is not well understood, the objective of the present work was to characterize the zebrafish optn gene and protein structure and to investigate its transcriptional regulation. Through a comparative in silico analysis, we observed that zebrafish optn presents genomic features similar to those of its human counterpart, including its neighboring genes and structure. A comparison of OPTN protein from different species revealed a high degree of conservation in its functional domains and three-dimensional structure. Furthermore, our in vitro transient-reporter analysis identified a functional promoter in the upstream region of the zebrafish optn gene, along with a region important for its transcription regulation. Site-directed mutagenesis revealed that the NF-κB motif is responsible for the activation of this region. In conclusion, with this study, we characterize zebrafish optn and our results indicate that zebrafish can be considered as an alternative model to study OPTN's biological role in bone-related diseases.

Measuring cystic fibrosis drug responses in organoids derived from 2D differentiated nasal epithelia.

Cystic fibrosis is caused by genetic defects that impair the CFTR channel in airway epithelial cells. These defects may be overcome by specific CFTR modulating drugs, for which the efficacy can be predicted in a personalized manner using 3D nasal-brushing-derived airway organoids in a forskolin-induced swelling assay. Despite of this, previously described CFTR function assays in 3D airway organoids were not fully optimal, because of inefficient organoid differentiation and limited scalability. In this report, we therefore describe an alternative method of culturing nasal-brushing-derived airway organoids, which are created from an equally differentiated airway epithelial monolayer of a 2D air-liquid interface culture. In addition, we have defined organoid culture conditions, with the growth factor/cytokine combination neuregulin-1<i>ß</i> and interleukin-1<i>ß</i>, which enabled consistent detection of CFTR modulator responses in nasal-airway organoid cultures from subjects with cystic fibrosis.

Absence of EPAC1 Signaling to Stabilize CFTR in Intestinal Organoids.

The plasma membrane (PM) stability of the cystic fibrosis transmembrane conductance regulator (CFTR), the protein which when mutated causes Cystic Fibrosis (CF), relies on multiple interaction partners that connect CFTR to signaling pathways, including cAMP signaling. It was previously shown that activation of exchange protein directly activated by cAMP 1 (EPAC1) by cAMP promotes an increase in CFTR PM levels in airway epithelial cells. However, the relevance of this pathway in other tissues, particularly the intestinal tissue, remains uncharacterized. Here, we used Western blot and forskolin-induced swelling assay to demonstrate that the EPAC1 protein is not expressed in the intestinal organoid model, and consequently the EPAC1 stabilization pathway is not in place. On the other hand, using cell surface biotinylation, EPAC1-mediated stabilization of PM CFTR is observed in intestinal cell lines. These results indicate that the EPAC1 stabilization pathway also occurs in intestinal cells and is a potential target for the development of novel combinatorial therapies for treatment of CF.

Effects of multiple risk factors on upper limb musculoskeletal disorders among workers in inner Brazil.

BACKGROUND: The literature discussing musculoskeletal diseases of inner northeastern workers is scarce, although 67,559 cases were reported in Brazil between 2007 and 2016. OBJECTIVE: This study aimed to evaluate the effect of multiple risk factors that influence the symptoms of work-related musculoskeletal disorders (WMSDs) in wrists, elbows, and shoulders in workers from four different economic sectors. METHODS: A sample included 420 workers from the inner regions of the Brazilian states of Alagoas and Bahia. The Nordic Musculoskeletal Questionnaire was used to capture pain symptoms on both sides of the body (left and right). Sociodemographic variables, items from the biomechanical exposure and organizational conditional, in addition to other questionnaires (JCQ, COPSOQ II, ERI) were used to assess the characteristics and occupational risks of the respondents. Ordinal logistic regression model was using to identify the relationship between symptoms and factors. RESULTS: This study highlights the psychosocial, biomechanical, occupational, and sociodemographic variables contributed to development of WMSDs. Use of hand-vibrating tool increased the likelihood of symptoms manifesting on the body. On the other hand, high job control and high job insecurity reduced the likelihood of developing symptoms. On the other hand, high job control and job satisfaction reduced the likelihood of developing symptoms. Factors such as age, curved spine, high job insecurity and excessive commitment contributed to the development of WMSDs only on one side of the body. CONCLUSIONS: The development of WMSDs is multifactorial. Sociodemographic, occupational, biomechanical, and psychosocial factors may commonly contribute to WMSD manifesting only on one side of the body more than on both sides.

Alfabetización en salud mental: conocimientos y creencias de una población adolescente / Mental Health Literacy: knowledge and beliefs of an adolescent population

Introducción: la alfabetización en salud mental (ASM) abarca los conocimientos y las creencias sobre los trastornos mentales, incluido el reconocimiento, la gestión y la prevención. Varios estudios revelan un nivel inadecuado de la ASM en adolescentes, lo que lleva a un aplazamiento en la búsqueda de ayuda. El objetivo fue evaluar el nivel de ASM en una población de adolescentes. Material y métodos: se aplicó el cuestionario de alfabetización en salud mental (MHLq). El análisis estadístico se realizó en el SPSS®22.0, utilizando la prueba t-Student (significación estadística p <0,05). Resultados: se obtuvieron 179 cuestionarios correctamente completados. En el 24% de los casos hubo una respuesta afirmativa con respecto a la presencia de enfermedad mental en la familia. En cuanto a la puntuación total obtenida en el MHLq, se obtuvo un promedio de 122 puntos (+/- 8) con un valor máximo de 145 puntos. En cuanto a las subescalas definidas en el cuestionario, encontramos 2 relaciones estadísticamente significativas con el género femenino: la primera tiene que ver con tener menos creencias/mitos erróneos sobre la enfermedad mental (p = 0,02) y la segunda, con la presencia de una enfermedad mental en la familia (p = 0,001). Conclusiones: el MHLq demostró ser un método fácil de cribado en ASM. La población considerada presentó un conocimiento razonable en esta área y la presencia de enfermedad mental en la familia influye positivamente en los resultados. Se destaca la importancia de una futura intervención para la educación y la deconstrucción de mitos que, especialmente en este grupo de edad, condicionan actitudes y prejuicios (AU)

Introduction: mental Health Literacy (MHL) encompasses knowledge and beliefs about mental disorders, including aspects related to their recognition, management and prevention. Several studies reveal an inadequate level of MHL in the adolescent population, leading to a delay in seeking help. the objective was to assess the level of MHL in a population of adolescents.Material and methods: we administered the mental health literacy questionnaire (MHLq) to the sample. The statistical analysis was performed with the software SPSS®22.0, using the Student t test (statistical significance p <0.05).Results: we received 179 correctly completed questionnaires, and 24% of respondents reported the presence of mental illness in the family. The mean total score in the MHLq, was 122 points (SD, 8) out of a total maximum of 145 points. When it came to the subscales of the questionnaire, we found a statistically significant lower level of erroneous beliefs/myths about medical illness in female vs male participants (p = 0.02) and respondents who reported mental illness in the family (p = 0.001).Conclusions: the MHLq proved to be an easy method for screening MHL. The population under study exhibited reasonable knowledge in this area, and the presence of mental illness in the family had a favourable impact on the results. The study highlighted the importance of future interventions for education and dispelling myths that condition attitudes and prejudices, especially in this age group. (AU)

New drugs in cystic fibrosis: what has changed in the last decade?

Cystic fibrosis (CF), a life-limiting chronic disease caused by mutations in the cystic fibrosis transmembrane regulator (CFTR) gene, affects more than 90,000 people worldwide. Until recently, the only available treatments were directed to symptom control, but they failed to change the course of the disease. New drugs developed in the last decade have the potential to change the expression, function, and stability of CFTR protein, targeting the basic molecular defect. The authors seek to provide an update on the new drugs, with a special focus on the most promising clinical trials that have been carried out to date. These newly approved drugs that target specific CFTR mutations are mainly divided into two main groups of CFTR modulators: potentiators and correctors. New therapies have opened the door for potentially disease-modifying, personalized treatments for patients with CF.

Confirmed Severe Acute Respiratory Syndrome Coronavirus 2 Reinfections After a Second Wave With Predominance of Lambda in Lima and Callao, Peru.

Background: Coronavirus disease 2019 (COVID-19) infection is a major public health problem in the world and reinfections are becoming more frequent. Our main objective was to describe the epidemiological, clinical, and genomic characteristics of the confirmed cases of reinfection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in the capital of Lima and Callao, Peru. Methods: We searched in the Peruvian laboratory information system from April 2020 up to May 2021, looking for cases having 2 positive molecular tests for SARS-CoV-2 with more than 90 days between them. We performed genomic sequencing to the available pairs of samples and described the clinical characteristics, epidemiological impact, and genomic analysis of the confirmed reinfections. Results: There were 1 694 164 people with a positive diagnostic test for SARS-CoV-2 in Lima/Callao during the study period. Of these, 1695 had 2 positive molecular tests with more than 90 days between them. Two hundred eleven had both samples available for genomic analysis according to our selection criteria, and these were retrieved and submitted to sequencing. Thirty cases were confirmed to be SARS-CoV-2 reinfections with 2 different lineages in the 2 episodes. The variant Lambda (C.37) was the most common during the second infection and accounted for 19 (63.3%) of the 30 cases. Conclusions: We report 30 cases of confirmed SARS-CoV-2 reinfections. The Lambda variant was the most common cause of the second infections, in concordance with its predominant circulation during Peru's second wave. This report describes the largest series of confirmed reinfections by SARS-CoV-2 in Latin America.We describe the epidemiological, clinical, and genomic characteristics of the confirmed cases of reinfection by severe acute respiratory syndrome coronavirus 2 in Lima and Callao, durante la segunda ola en Peru. The Lambda variant (C.37) was the most common cause of the second infections.

Pediatric population with cystic fibrosis in the centre of Portugal: candidates for new therapies

Abstract Objectives: Cystic fibrosis (CF) is a severe autosomal recessive disease that results from mutations in a gene encoding the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein, a chloride channel. This study aims to characterize the clinical and genetic features of a cohort of pediatric people with CF (PwCF) in the center of Portugal and to determine which ones are candidates for the new drugs modulating the CFTR channel. Methods: A review of the demographic, genetic and clinical characteristics of PwCF undergoing follow-up at a CF reference center was carried out. Results: Twenty-three PwCF (12 male), with a median age of 12 years, were followed up. All patients carry the F508del mutation in at least one allele. Fifteen PwCF were F508del-homozygous, median BMI z-score was -0.13, all are pancreatic insufficient and median FEV1 value was 78.1%. These PwCF are eligible for dual therapy (lumacaftor/tezacaftor+ivacaftor) and for triple therapy (tezacaftor+ivacaftor+elexacaftor). PwCF with 711 +1G->T (n = 2), 2184insA (n = 1) mutations and a novel mutation c.3321dup (n = 1) have minimal function mutation and patients with a residual function mutation: R334W (n = 3) and P5L (n = 1) have a less severe phenotype. All these patients, because they also carry F508del mutation, are elegible to triple therapy. Conclusions: Genetic and molecular characterization of PwCF poses an important step not just for CF diagnosis and prognosis which is tightly correlated with the clinical phenotype, but also for the eligibility of CFTR modulator drugs.

Reinfecciones por SARS-CoV-2 durante la segunda ola pandémica en Iquitos, Perú / SARS-CoV-2 reinfections during the second pandemic wave in Iquitos, Peru

Señor editor: La infección por SARS-CoV-2 ha ocasionado gran impacto en todo el mundo estimándose en más de 439 millones de casos y más de 5,9 millones de muertes. El Perú ha sido uno de los países en donde la mortalidad de su población ha descrito cifras muy elevadas llegando hasta una tasa de letalidad de 9.14%. Iquitos ha sido una de las ciudades más afectadas desde el inicio de la pandemia en el Perú, en donde se describió una seroprevalencia COVID-19 de 70% una de las más altas reportadas después de la primera ola pandémica de COVID-19. Es de esperar que esta seroprevalencia haya aumentado luego de la segunda ola. La duración de la inmunidad frente al SARS-CoV-2 ya sea por infección previa o por vacunación efectiva continúa siendo una de las interrogantes más importantes, en ese contexto, reportamos 4 casos de reinfecciones confirmadas en Iquitos Perú.

Dear Editor: SARS-CoV-2 infection has caused great impact worldwide, estimated at more than 439 million cases and more than 5.9 million deaths. Peru has been one of the countries where the mortality of its population has described very high figures reaching a case fatality rate of 9.14%. Iquitos has been one of the most affected cities since the beginning of the pandemic in Peru, where a COVID-19 seroprevalence of 70% was described, one of the highest reported after the first COVID-19 pandemic wave. It is to be expected that this seroprevalence has increased after the second wave. The duration of immunity against SARS-CoV-2 either by previous infection or by effective vaccination continues to be one of the most important questions, in that context, we report 4 cases of confirm reinfections in Iquitos Peru.

Systems Approaches to Unravel Molecular Function: High-content siRNA Screen Identifies TMEM16A Traffic Regulators as Potential Drug Targets for Cystic Fibrosis.

An attractive approach to treat people with Cystic Fibrosis (CF), a life-shortening disease caused by mutant CFTR, is to compensate for the absence of this chloride/bicarbonate channel by activating alternative (non-CFTR) chloride channels. One obvious target for such "mutation-agnostic" therapeutic approach is TMEM16A (anoctamin-1/ANO1), a calcium-activated chloride channel (CaCC) which is also expressed in the airways of people with CF, albeit at low levels. To find novel TMEM16A regulators of both traffic and function, with the main goal of identifying candidate CF drug targets, we performed a fluorescence cell-based high-throughput siRNA microscopy screen for TMEM16A trafficking using a double-tagged construct expressed in human airway cells. About 700 genes were screened (2 siRNAs per gene) of which 262 were identified as candidate TMEM16A modulators (179 siRNAs enhanced and 83 decreased TMEM16A traffic), being G-protein coupled receptors (GPCRs) enriched on the primary hit list. Among the 179 TMEM16A traffic enhancer siRNAs subjected to secondary screening 20 were functionally validated. Further hit validation revealed that siRNAs targeting two GPCRs - ADRA2C and CXCR3 - increased TMEM16A-mediated chloride secretion in human airway cells, while their overexpression strongly diminished calcium-activated chloride currents in the same cell model. The knockdown, and likely also the inhibition, of these two TMEM16A modulators is therefore an attractive potential therapeutic strategy to increase chloride secretion in CF.

Rescue of Mutant CFTR Trafficking Defect by the Investigational Compound MCG1516A.

Although some therapeutic progress has been achieved in developing small molecules that correct F508del-CFTR defects, the mechanism of action (MoA) of these compounds remain poorly elucidated. Here, we investigated the effects and MoA of MCG1516A, a newly developed F508del-CFTR corrector. MCG1516A effects on wild-type (WT) and F508del-CFTR were assessed by immunofluorescence microscopy, and biochemical and functional assays both in cell lines and in intestinal organoids. To shed light on the MoA of MCG1516A, we evaluated its additivity to the FDA-approved corrector VX-661, low temperature, genetic revertants of F508del-CFTR (G550E, R1070W, and 4RK), and the traffic-null variant DD/AA. Finally, we explored the ability of MCG1516A to rescue trafficking and function of other CF-causing mutations. We found that MCG1516A rescues F508del-CFTR with additive effects to VX-661. A similar behavior was observed for WT-CFTR. Under low temperature incubation, F508del-CFTR demonstrated an additivity in processing and function with VX-661, but not with MCG1516A. In contrast, both compounds promoted additional effects to low temperature to WT-CFTR. MCG1516A demonstrated additivity to genetic revertant R1070W, while VX-661 was additive to G550E and 4RK. Nevertheless, none of these compounds rescued DD/AA trafficking. Both MCG1516A and VX-661 rescued CFTR processing of L206W- and R334W-CFTR with greater effects when these compounds were combined. In summary, the absence of additivity of MCG1516A to genetic revertant G550E suggests a putative binding site for this compound on NBD1:NBD2 interface. Therefore, a combination of MCG1516A with compounds able to rescue DD/AA traffic, or mimicking the actions of revertant R1070W (e.g., VX-661), could enhance correction of F508del-CFTR defects.