Urinary metabolomic profiling of a cohort of Colombian patients with systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is an autoimmune and multisystem disease with a high public health impact. Lupus nephritis (LN), commonly known as renal involvement in SLE, is associated with a poorer prognosis and increased rates of morbidity and mortality in patients with SLE. Identifying new urinary biomarkers that can be used for LN prognosis or diagnosis is essential and is part of current active research. In this study, we applied an untargeted metabolomics approach involving liquid and gas chromatography coupled with mass spectrometry to urine samples collected from 17 individuals with SLE and no kidney damage, 23 individuals with LN, and 10 clinically healthy controls (HCs) to identify differential metabolic profiles for SLE and LN. The data analysis revealed a differentially abundant metabolite expression profile for each study group, and those metabolites may act as potential differential biomarkers of SLE and LN. The differential metabolic pathways found between the LN and SLE patients with no kidney involvement included primary bile acid biosynthesis, branched-chain amino acid synthesis and degradation, pantothenate and coenzyme A biosynthesis, lysine degradation, and tryptophan metabolism. Receiver operating characteristic curve analysis revealed that monopalmitin, glycolic acid, and glutamic acid allowed for the differentiation of individuals with SLE and no kidney involvement and individuals with LN considering high confidence levels. While the results offer promise, it is important to recognize the significant influence of medications and other external factors on metabolomics studies. This impact has the potential to obscure differences in metabolic profiles, presenting a considerable challenge in the identification of disease biomarkers. Therefore, experimental validation should be conducted with a larger sample size to explore the diagnostic potential of the metabolites found as well as to examine how treatment and disease activity influence the identified chemical compounds. This will be crucial for refining the accuracy and effectiveness of using urine metabolomics for diagnosing and monitoring lupus and lupus nephritis.

Surface-enhanced Raman Spectroscopy in urinalysis of hypertension patients with kidney disease.

Arterial hypertension (AH) is a multifactorial and asymptomatic disease that affects vital organs such as the kidneys and heart. Considering its prevalence and the associated severe health repercussions, hypertension has become a disease of great relevance for public health across the globe. Conventionally, the classification of an individual as hypertensive or non-hypertensive is conducted through ambulatory blood pressure monitoring over a 24-h period. Although this method provides a reliable diagnosis, it has notable limitations, such as additional costs, intolerance experienced by some patients, and interferences derived from physical activities. Moreover, some patients with significant renal impairment may not present proteinuria. Accordingly, alternative methodologies are applied for the classification of individuals as hypertensive or non-hypertensive, such as the detection of metabolites in urine samples through liquid chromatography or mass spectrometry. However, the high cost of these techniques limits their applicability for clinical use. Consequently, an alternative methodology was developed for the detection of molecular patterns in urine collected from hypertension patients. This study generated a direct discrimination model for hypertensive and non-hypertensive individuals through the amplification of Raman signals in urine samples based on gold nanoparticles and supported by chemometric techniques such as partial least squares-discriminant analysis (PLS-DA). Specifically, 162 patient urine samples were used to create a PLS-DA model. These samples included 87 urine samples from patients diagnosed with hypertension and 75 samples from non-hypertensive volunteers. In the AH group, 35 patients were diagnosed with kidney damage and were further classified into a subgroup termed (RAH). The PLS-DA model with 4 latent variables (LV) was used to classify the hypertensive patients with external validation prediction (P) sensitivity of 86.4%, P specificity of 77.8%, and P accuracy of 82.5%. This study demonstrates the ability of surface-enhanced Raman spectroscopy to differentiate between hypertensive and non-hypertensive patients through urine samples, representing a significant advance in the detection and management of AH. Additionally, the same model was then used to discriminate only patients diagnosed with renal damage and controls with a P sensitivity of 100%, P specificity of 77.8%, and P accuracy of 82.5%.

From Cell to Symptoms: The Role of SARS-CoV-2 Cytopathic Effects in the Pathogenesis of COVID-19 and Long COVID.

Severe Acute Respiratory Syndrome CoronaVirus 2 (SARS-CoV-2) infection triggers various events from molecular to tissue level, which in turn is given by the intrinsic characteristics of each patient. Given the molecular diversity characteristic of each cellular phenotype, the possible cytopathic, tissue and clinical effects are difficult to predict, which determines the heterogeneity of COVID-19 symptoms. The purpose of this article is to provide a comprehensive review of the cytopathic effects of SARS-CoV-2 on various cell types, focusing on the development of COVID-19, which in turn may lead, in some patients, to a persistence of symptoms after recovery from the disease, a condition known as long COVID. We describe the molecular mechanisms underlying virus-host interactions, including alterations in protein expression, intracellular signaling pathways, and immune responses. In particular, the article highlights the potential impact of these cytopathies on cellular function and clinical outcomes, such as immune dysregulation, neuropsychiatric disorders, and organ damage. The article concludes by discussing future directions for research and implications for the management and treatment of COVID-19 and long COVID.

Comparative Analysis of In-House RT-qPCR Detection of SARS-CoV-2 for Resource-Constrained Settings.

We developed and standardized an efficient and cost-effective in-house RT-PCR method to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We evaluated sensitivity, specificity, and other statistical parameters by different RT-qPCR methods including triplex, duplex, and simplex assays adapted from the initial World Health Organization- (WHO) recommended protocol. This protocol included the identification of the E envelope gene (E gene; specific to the Sarvecovirus genus), RdRp gene of the RNA-dependent RNA polymerase (specific for SARS-CoV-2), and RNase P gene as endogenous control. The detection limit of the E and the RdRp genes were 3.8 copies and 33.8 copies per 1 µL of RNA, respectively, in both triplex and duplex reactions. The sensitivity for the RdRp gene in the triplex and duplex RT-qPCR tests were 98.3% and 83.1%, respectively. We showed a decrease in sensitivity for the RdRp gene by 60% when the E gene acquired Ct values > 31 in the diagnostic tests. This is associated with the specific detection limit of each gene and possible interferences in the protocol. Hence, developing efficient and cost-effective methodologies that can be adapted to various health emergency scenarios is important, especially in developing countries or settings where resources are limited.

Explorando el poder de los abordajes transcriptómicos para identificar biomarcadores asociados a daño renal en pacientes con lupus eritematoso sistémico / Exploring the power of transcriptomic approaches to identify biomarkers associated to renal damage in Systemic Lupus Erythematosus patients

Resumen El lupus eritematoso sistémico (LES) es una enfermedad compleja y altamente heterogénea que afecta múltiples órganos, incluyendo articulaciones, corazón, sistema hematopoyético, sistema nervioso y riñón, siendo este último el de peor pronóstico y el que conlleva a nefritis lúpica (NL). Aunque la etiopatogénesis del LES aún no se conoce con claridad, se cree que la susceptibilidad genética y las modificaciones epigenéticas aberrantes favorecen su desenlace. Para establecer una terapia precisa es necesario evaluar de manera eficiente y objetiva el compromiso de órganos y la actividad de la enfermedad, lo cual es muy difícil por las pocas pruebas de laboratorio clínico disponibles en la actualidad. En las últimas décadas la búsqueda de nuevos biomarcadores de LES ha sido una tendencia y se han identificado varios promisorios a nivel de genómica, metabolómica, proteómica y transcriptómica. En esta revisión se resume el estado del arte relacionado con estudios transcriptómicos que han identificado diversos transcritos potencialmente útiles como biomarcadores del LES y la NL.

Abstract Systemic Lupus Erythematosus is a complex and highly heterogeneous disease affecting multiple organs such as joints, heart, hematopoietic system, nervous system and kidney, the latter being the worst prognosis and leading to lupus nephritis (LN). While the etiopathogenesis of SLE is still not completely clear, is believed that genetic susceptibility and aberrant epigenetic modifications favor the outcome of the disease. In order to establish an accurate therapy, it is necessary to efficiently and objectively assess organ involvement and disease activity, which is very difficult due to the clinical laboratory tests currently available. In recent decades, the search for new SLE biomarkers has been a trend and many promising biomarkers have been identified at the genomic, metabolomic, proteomic and transcriptomic levels. In this review we summarize the state of the art related to transcriptomic studies that have identified various potentially useful transcripts as biomarkers of SLE and NL.

Trypanin Disruption Affects the Motility and Infectivity of the Protozoan Trypanosoma cruzi.

The flagellum of Trypanosomatids is an organelle that contributes to multiple functions, including motility, cell division, and host-pathogen interaction. Trypanin was first described in Trypanosoma brucei and is part of the dynein regulatory complex. TbTrypanin knockdown parasites showed motility defects in procyclic forms; however, silencing in bloodstream forms was lethal. Since TbTrypanin mutants show drastic phenotypic changes in mammalian stages, we decided to evaluate if the Trypanosoma cruzi ortholog plays a similar role by using the CRISPR-Cas9 system to generate null mutants. A ribonucleoprotein complex of SaCas9 and sgRNA plus donor oligonucleotide were used to edit both alleles of TcTrypanin without any selectable marker. TcTrypanin -/- epimastigotes showed a lower growth rate, partially detached flagella, normal numbers of nuclei and kinetoplasts, and motility defects such as reduced displacement and speed and increased tumbling propensity. The epimastigote mutant also showed decreased efficiency of in-vitro metacyclogenesis. Mutant parasites were able to complete the entire life cycle in vitro; however, they showed a reduction in their infection capacity compared with WT and addback cultures. Our data show that T. cruzi life cycle stages have differing sensitivities to TcTrypanin deletion. In conclusion, additional work is needed to dissect the motility components of T. cruzi and to identify essential molecules for mammalian stages.

CREditing: a tool for gene tuning in Trypanosoma cruzi.

The genetic manipulation of Trypanosoma cruzi continues to be a challenge, mainly due to the lack of available and efficient molecular tools. The CRE-lox recombination system is a site-specific recombinase technology, widely used method of achieving conditional targeted deletions, inversions, insertions, gene activation, translocation, and other modifications in chromosomal or episomal DNA. In the present study, the CRE-lox system was adapted to expand the current genetic toolbox for this hard-to-manipulate parasite. For this, evaluations of whether direct protein delivery of CRE recombinase through electroporation could improve CRE-mediated recombination in T. cruzi were performed. CRE recombinase was fused to the C-terminus of T. cruzi histone H2B, which carries the nuclear localization signal and is expressed in the prokaryotic system. The fusion protein was affinity purified and directly introduced into epimastigotes and tissue culture-derived trypomastigotes. This enabled the control of gene expression as demonstrated by turning on a tandem dimer fluorescent protein reporter gene that had been previously transfected into parasites, achieving CRE-mediated recombination in up to 85% of parasites. This system was further tested for its ability to turn off gene expression, remove selectable markers integrated into the genome, and conditionally knock down the nitroreductase gene, which is involved in drug resistance. Additionally, CREditing also enabled the control of gene expression in tissue culture trypomastigotes, which are more difficult to transfect than epimastigotes. The considerable advances in genomic manipulation of T. cruzi shown in this study can be used by others to aid in the greater understanding of this parasite through gain- or loss-of-function approaches.

Targeting epimastigotes of Trypanosoma cruzi with a peptide isolated from a phage display random library.

Chagas disease, also known as American trypanosomiasis, is a potentially life-threatening illness caused by the protozoan parasite Trypanosoma cruzi, which is transmitted by insects of the family Reduviidae. Since conventional treatments with nitroheterocyclic drugs show serious adverse reactions and have questionable efficiency, different research groups have investigated polypeptide-based approaches to interfere with the parasite cell cycle in other Trypanosomatids. These strategies are supported by the fact that surface players are candidates to develop surface ligands that impair function since they may act as virulence factors. In this study, we used a phage display approach to identify peptides from one library-LX8CX8 (17 aa) (where X corresponds to any amino acid). After testing different biopanning conditions using live or fixed epimastigotes, 10 clones were sequenced that encoded the same peptide, named here as EPI18. The bacteriophage expressing EPI18 binds to epimastigotes from distinct strains of T. cruzi. To confirm these results, this peptide was synthetized, biotinylated, and assayed using flow cytometry and confocal microscopy analyses. These assays confirmed the specificity of the binding capacity of EPI18 toward epimastigote surfaces. Our findings suggest that EPI18 may have potential biotechnological applications that include peptide-based strategies to control parasite transmission.

Engineering a single-chain antibody against Trypanosoma cruzi metacyclic trypomastigotes to block cell invasion.

Trypanosoma cruzi is a flagellate protozoan pathogen that causes Chagas disease. Currently there is no preventive treatment and the efficiency of the two drugs available is limited to the acute phase. Therefore, there is an unmet need for innovative tools to block transmission in endemic areas. In this study, we engineered a novel recombinant molecule able to adhere to the T. cruzi surface, termed scFv-10D8, that consists of a single-chain variable fragment (scFv) derived from mAb-10D8 that targets gp35/50. The synthetic gene encoding scFv-10D8 was cloned and fused to a 6×His tag and expressed in a prokaryotic expression system. Total periplasmic or 6xHis tag affinity-purified fractions of scFv-10D8 retained the capacity to bind to gp35/50, as shown by Western blot analyses. Pre-incubation of metacyclic trypomastigotes with scFv-10D8 showed a remarkable reduction in cell invasion capacity. Our results suggest that scFv-10D8 can be used in a paratransgenic approach to target parasites in insect vectors, avoiding dissemination of infective forms. Such advances in the development of this functional molecule will surely prompt the improvement of alternative strategies to control Chagas disease by targeting mammalian host stages.

Integrated analysis of microRNA regulation and its interaction with mechanisms of epigenetic regulation in the etiology of systemic lupus erythematosus.

The aim of this study was to identity in silico the relationships among microRNAs (miRNAs) and genes encoding transcription factors, ubiquitylation, DNA methylation, and histone modifications in systemic lupus erythematosus (SLE). To identify miRNA dysregulation in SLE, we used miR2Disease and PhenomiR for information about miRNAs exhibiting differential regulation in disease and other biological processes, and HMDD for information about experimentally supported human miRNA-disease association data from genetics, epigenetics, circulating miRNAs, and miRNA-target interactions. This information was incorporated into the miRNA analysis. High-throughput sequencing revealed circulating miRNAs associated with kidney damage in patients with SLE. As the main finding of our in silico analysis of miRNAs differentially expressed in SLE and their interactions with disease-susceptibility genes, post-translational modifications, and transcription factors; we highlight 226 miRNAs associated with genes and processes. Moreover, we highlight that alterations of miRNAs such as hsa-miR-30a-5p, hsa-miR-16-5p, hsa-miR-142-5p, and hsa-miR-324-3p are most commonly associated with post-translational modifications. In addition, altered miRNAs that are most frequently associated with susceptibility-related genes are hsa-miR-16-5p, hsa-miR-374a-5p, hsa-miR-34a-5p, hsa-miR-31-5p, and hsa-miR-1-3p.

Profiling analysis of circulating microRNA in peripheral blood of patients with class IV lupus nephritis.

Renal involvement in Systemic Lupus Erythematous (SLE) patients is one of the leading causes of morbidity and a significant contributor to mortality. It's estimated that nearly 50% of SLE individuals develop kidney disease in the first year of the diagnosis. Class IV lupus nephritis (LN-IV) is the class of lupus nephritis most common in Colombian patients with SLE. Altered miRNAs expression levels have been reported in human autoimmune diseases including lupus. Variations in the expression pattern of peripheral blood circulating miRNAs specific for this class of lupus nephritis could be correlated with the pathophysiological status of this group of individuals. The aim of this study was to evaluate the relative abundance of circulating microRNAs in peripheral blood from Colombian patients with LN-IV. Circulating miRNAs in plasma of patients with diagnosis of LN-IV were compared with individuals without renal involvement (LNN group) and healthy individuals (CTL group). Total RNA was extracted from 10 ml of venous blood and subsequently sequenced using Illumina. The sequences were processed and these were analyzed using miRBase and Ensembl databases. Differential gene expression analysis was carried out with edgeR and functional analysis were done with DIANA-miRPath. Analysis was carried out using as variables of selection fold change (≥2 o ≤-2) and false discovery rate (0.05). We identified 24 circulating microRNAs with differential abundance between LN-IV and CTL groups, fourteen of these microRNAs are described for the first time to lupus nephritis (hsa-miR-589-3p, hsa-miR-1260b, hsa-miR-4511, hsa-miR-485-5p, hsa-miR-584-5p, hsa-miR-543, hsa-miR-153-3p, hsa-miR-6087, hsa-miR-3942-5p, hsa-miR-7977, hsa-miR-323b-3p, hsa-miR-4732-3p and hsa-miR-6741-3p). These changes in the abundance of miRNAs could be interpreted as alterations in the miRNAs-mRNA regulatory network in the pathogenesis of LN, preceding the clinical onset of the disease. The findings thus contribute to understanding the disease process and are likely to pave the way towards identifying disease biomarkers for early diagnosis of LN.

Effective gene delivery to Trypanosoma cruzi epimastigotes through nucleofection.

New opportunities have raised to study the gene function approaches of Trypanosoma cruzi after its genome sequencing in 2005. Functional genomic approaches in Trypanosoma cruzi are challenging due to the reduced tools available for genetic manipulation, as well as to the reduced efficiency of the transient transfection conducted through conventional methods. The Amaxa nucleofector device was systematically tested in the present study in order to improve the electroporation conditions in the epimastigote forms of T. cruzi. The transfection efficiency was quantified using the green fluorescent protein (GFP) as reporter gene followed by cell survival assessment. The herein used nucleofection parameters have increased the survival rates (>90%) and the transfection efficiency by approximately 35%. The small amount of epimastigotes and DNA required for the nucleofection can turn the method adopted here into an attractive tool for high throughput screening (HTS) applications, and for gene editing in parasites where genetic manipulation tools remain relatively scarce.

High-Throughput Sequencing Reveals Circulating miRNAs as Potential Biomarkers of Kidney Damage in Patients with Systemic Lupus Erythematosus.

Renal involvement is one of the most severe manifestations of systemic lupus erythematosus (SLE). Renal biopsy is the gold standard when it comes to knowing whether a patient has lupus nephritis, and the degree of renal disease present. However, the biopsy has various complications, bleeding being the most common. Therefore, the development of alternative, non-invasive diagnostic tests for kidney disease in patients with SLE is a priority. Micro RNAs (miRNAs) are differentially expressed in various tissues, and changes in their expression have been associated with several pathological processes. The aim of this study was to identify changes in the abundance of miRNAs in plasma samples from patients with lupus nephritis that could potentially allow the diagnosis of renal damage in SLE patients. This is an observational case-control cross-sectional study, in which we characterized the differential abundance profiles of miRNAs among patients with different degrees of lupus compared with SLE patients without renal involvement and healthy control individuals. We found 89 miRNAs with changes in their abundance between lupus nephritis patients and healthy controls, and 17 miRNAs that showed significant variations between SLE patients with or without renal involvement. Validation for qPCR of a group of miRNAs on additional samples from lupus patients with or without nephritis, and from healthy individuals, showed that five miRNAs presented an average detection sensitivity of 97%, a specificity of 70.3%, a positive predictive value of 82.5%, a negative predictive value of 96% and a diagnosis efficiency of 87.9%. These results strongly suggest that miR-221-5p, miR-380-3p, miR-556-5p, miR-758-3p and miR-3074-3p are potential diagnostic biomarkers of lupus nephritis in patients with SLE. The observed differential pattern of miRNA abundance may have functional implications in the pathophysiology of SLE renal damage.

Índices de infestación aedica e identificación de conocimientos, actitudes y prácticas sobre dengue en llanterías del Departamento del Atlántico, Colombia / Aedes aegypti larval infestation index and identification of awareness, attitudes and practices related to Dengue in tire shops in Atlántico, Colombia

Objetivo Identificar los conocimientos, actitudes y prácticas sobre dengue en propietarios y trabajadores de llantería, así como los niveles de infestación del vector en llanterías del Departamento del Atlántico. Métodos Se realizó un estudio tipo descriptivo. Las variables se describieron a partir de porcentajes y medidas de tendencia central y dispersión. Se calculó el índice de infestación larvario para llanterías y el índice de depósitos en cada uno de los municipios muestreados. Se visitaron e inspeccionaron 111 llanterías; el 26,1 % (29/111) de estas se encontraron positivas para formas larvarias del vector Ae. aegypti. Los municipios de Piojó, Santo Tomas, Santa Lucia, Sabanagrande y Luruaco presentaron los índices de infestación larvaria en llanterías más altos (IIL: 50-100 %). Resultados Con respecto al dengue, el 90,9 % de los entrevistados lo consideró un problema para ellos y sus familias. El 94,6 % conoce que es transmitido por mosquitos; el 91,1 % conoce a la larva del vector como "sarapico", el 3,6 % como "gusarapo". El 98,2 % consideró que existe una relación entre la larva y el mosquito Ae. aegypti. El 100 % reconoce las llantas como un criadero para el mosquito. El 85,7 % consideró la fiebre como el síntoma más frecuente, el 83% manifestó asistir al puesto de salud para curar la enfermedad. El 90,8 % arroja las llantas no utilizables como basura. Conclusión En la población estudiada existen buenos conocimientos acerca del dengue y su vector; sin embargo, existen problemas de actitudes y prácticas para su prevención.(AU)

Objective To identify the awareness, attitudes and practices related to dengue in owners and workers of tire ships, as well as the levels of mosquito infestation in tire shops in Atlántico department - Colombia. Methods We conducted a descriptive study. The variables were described as percentages and measures of central tendency and dispersion. Index of larval infestation and containers were calculated in each of the municipalities studied. We visited and inspected 111 tire shops. 26.1 % (29/111) of these were found positive for Ae. aegypti larvae. The municipalities of Piojó, Santo Tomás, Santa Lucia, Sabanagrande and Luruaco were characterized by a higher larval infestation index. Results Regarding dengue, 90.9 % of respondents considered it a problem for them and their families. 94.6 % know that is transmitted by mosquitoes. 91.1 % know the vector larvae under the name "sarapico", 3.6 % as "gusarapo". 98.2 % felt that there is a relationship between the larva and Ae. aegypti mosquito. 100 % of participantes recognized tires to be a breeding for mosquitoes. 85.7% believed fever to be the most common symptom. 83 % reported accessing the health post to cure the disease. 90.8 % throw out unusable tires as if they were garbage. Conclusion In the population studied, there is good awareness of dengue and its vector. Nevertheless, there are problems related to attitudes and prevention practices.(AU)

[Aedes aegypti larval infestation index and identification of awareness, attitudes and practices related to Dengue in tire shops in Atlántico, Colombia]. / Índices de infestación aédica e identificación de conocimientos, actitudes y prácticas sobre dengue en llanterías del Departamento del Atlántico, Colombia.

Objective To identify the awareness, attitudes and practices related to dengue in owners and workers of tire ships, as well as the levels of mosquito infestation in tire shops in Atlántico department - Colombia. Methods We conducted a descriptive study. The variables were described as percentages and measures of central tendency and dispersion. Index of larval infestation and containers were calculated in each of the municipalities studied. We visited and inspected 111 tire shops. 26.1 % (29/111) of these were found positive for Ae. aegypti larvae. The municipalities of Piojó, Santo Tomás, Santa Lucia, Sabanagrande and Luruaco were characterized by a higher larval infestation index. Results Regarding dengue, 90.9 % of respondents considered it a problem for them and their families. 94.6 % know that is transmitted by mosquitoes. 91.1 % know the vector larvae under the name "sarapico", 3.6 % as "gusarapo". 98.2 % felt that there is a relationship between the larva and Ae. aegypti mosquito. 100 % of participantes recognized tires to be a breeding for mosquitoes. 85.7% believed fever to be the most common symptom. 83 % reported accessing the health post to cure the disease. 90.8 % throw out unusable tires as if they were garbage. Conclusion In the population studied, there is good awareness of dengue and its vector. Nevertheless, there are problems related to attitudes and prevention practices.

Cholesterol-rich microdomains as docking platforms for respiratory syncytial virus in normal human bronchial epithelial cells.

Respiratory syncytial virus (RSV) is one of the major causes of respiratory infections in children, and it is the main pathogen causing bronchiolitis in infants. The binding and entry mechanism by which RSV infects respiratory epithelial cells has not yet been determined. In this study, the earliest stages of RSV infection in normal human bronchial epithelial cells were probed by tracking virions with fluorescent lipophilic dyes in their membranes. Virions colocalized with cholesterol-containing plasma membrane microdomains, identified by their ability to bind cholera toxin subunit B. Consistent with an important role for cholesterol in RSV infection, cholesterol depletion profoundly inhibited RSV infection, while cholesterol repletion reversed this inhibition. Merger of the outer leaflets of the viral envelope and the cell membrane appeared to be triggered at these sites. Using small-molecule inhibitors, RSV infection was found to be sensitive to Pak1 inhibition, suggesting the requirement of a subsequent step of cytoskeletal reorganization that could involve plasma membrane rearrangements or endocytosis. It appears that RSV entry depends on its ability to dock to cholesterol-rich microdomains (lipid rafts) in the plasma membrane where hemifusion events begin, assisted by a Pak1-dependent process.