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The three-dimensional (3D) structure of the ductal epithelium and the surrounding extracellular matrix (ECM) are integral aspects of the breast tissue, and they have important roles during mammary gland development, function and malignancy. However, the architecture of the branched mammary epithelial network is poorly recapitulated in the current in vitro models. 3D bioprinting is an emerging approach to improve tissue-mimicry in cell culture. Here, we developed and optimized a protocol for 3D bioprinting of normal and cancerous mammary epithelial cells into a branched Y-shape to study the role of cell positioning in the regulation of cell proliferation and invasion. Non-cancerous cells formed continuous 3D cell networks with several organotypic features, whereas the ductal carcinoma in situ (DCIS) -like cancer cells exhibited aberrant basal polarization and defective formation of the basement membrane (BM). Quantitative analysis over time demonstrated that both normal and cancerous cells proliferate more at the branch tips compared to the trunk region of the 3D-bioprinted cultures, and particularly at the tip further away from the branch point. The location-specific rate of proliferation was independent of TGFβ signaling but invasion of the DCIS-like breast cancer cells was reduced upon the inhibition of TGFβ. Thus, our data demonstrate that the 3D-bioprinted cells can sense their position in the branched network of cells and proliferate at the tips, thus recapitulating this feature of mammary epithelial branching morphogenesis. In all, our results demonstrate the capacity of the developed 3D bioprinting method for quantitative analysis of the relationships between tissue structure and cell behavior in breast morphogenesis and cancer., (© 2024. The Author(s).)

Limb regeneration in salamanders is achieved by a complex coordination of various biological processes and requires the proper integration of new tissue with old. Among the tissues found inside the limb, the skeleton is the most prominent component, which serves as a scaffold and provides support for locomotion in the animal. Throughout the years, researchers have studied the regeneration of the appendicular skeleton in salamanders both after limb amputation and as a result of fracture healing. The final outcome has been widely seen as a faithful re-establishment of the skeletal elements, characterised by a seamless integration into the mature tissue. The process of skeletal integration, however, is not well understood, and several works have recently provided evidence of commonly occurring flawed regenerates. In this Review, we take the reader on a journey through the course of bone formation and regeneration in salamanders, laying down a foundation for critically examining the mechanisms behind skeletal integration. Integration is a phenomenon that could be influenced at various steps of regeneration, and hence, we assess the current knowledge in the field and discuss how early events, such as tissue histolysis and patterning, influence the faithful regeneration of the appendicular skeleton., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Purpose: To analyze the adverse events reported in the pharmacovigilance of the health service provider institutions of the municipality of Monteria in the period 2018-2021., Patients and Methods: Descriptive, cross-sectional, retrospective, and quantitative approach study, the information was analyzed by statistical analysis by multiple correspondence using Orange Data Mining software; the analysis consisted of visual programming to perform interactive data exploration, to identify and differentiate associations or oppositions between different categories in space., Results: The most frequently reported adverse events were allergic reactions, with 28.5%. Female sex and adult age are the groups most prone to present these events; antibiotics were the pharmacological group that produced the most adverse events with 18.3%; the main errors that produce these events are related to prescription., Conclusion: Age and sex increase the risk of adverse drug events; most of these events are the product of erroneous prescriptions. The findings presented in this article are useful for pharmacovigilance programs in health institutions., Competing Interests: The authors report no conflicts of interest in this work., (© 2023 Martínez et al.)

The small GTPase Ras is frequently mutated in cancer and a driver of tumorigenesis. The recent years have shown great progress in drug-targeting Ras and understanding how it operates on the plasma membrane. We now know that Ras is non-randomly organized into proteo-lipid complexes on the membrane, called nanoclusters. Nanoclusters contain only a few Ras proteins and are necessary for the recruitment of downstream effectors, such as Raf. If tagged with fluorescent proteins, the dense packing of Ras in nanoclusters can be analyzed by Förster/ fluorescence resonance energy transfer (FRET). Loss of FRET can therefore report on decreased nanoclustering and any process upstream of it, such as Ras lipid modifications and correct trafficking. Thus, cellular FRET screens employing Ras-derived fluorescence biosensors are potentially powerful tools to discover chemical or genetic modulators of functional Ras membrane organization. Here we implement fluorescence anisotropy-based homo-FRET measurements of Ras-derived constructs labelled with only one fluorescent protein on a confocal microscope and a fluorescence plate reader. We show that homo-FRET of both H-Ras- and K-Ras-derived constructs can sensitively report on Ras-lipidation and -trafficking inhibitors, as well as on genetic perturbations of proteins regulating membrane anchorage. By exploiting the switch I/II-binding Ras-dimerizing compound BI-2852, this assay is also suitable to report on the engagement of the K-Ras switch II pocket by small molecules such as AMG 510. Given that homo-FRET only requires one fluorescent protein tagged Ras construct, this approach has significant advantages to create Ras-nanoclustering FRET-biosensor reporter cell lines, as compared to the more common hetero-FRET approaches., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Background: The genus Borrelia is composed of two well-defined monophyletic groups, the Borrelia burgdorferi sensu lato complex (Bb) and the relapsing fever (RF) group borreliae. Recently, a third group, associated with reptiles and echidnas, has been described. In general, RF group borreliae use rodents as reservoir hosts; although neotropical bats may also be involved as important hosts, with scarce knowledge regarding this association. The objective of this study was to detect the presence of Borrelia spp. DNA in bats from the department of Córdoba in northwest Colombia., Methods: During September 2020 and June 2021, 205 bats were captured in six municipalities of Córdoba department, Colombia. Specimens were identified using taxonomic keys and DNA was extracted from spleen samples. A Borrelia-specific real-time PCR was performed for the 16S rRNA gene. Fragments of the 16S rRNA and flaB genes were amplified in the positive samples by conventional PCR. The detected amplicons were sequenced by the Sanger method. Phylogenetic reconstruction was performed in IQ-TREE with maximum likelihood based on the substitution model TPM3+F+I+G4 with bootstrap values deduced from 1000 replicates., Results: Overall, 10.2% (21/205) of the samples were found positive by qPCR; of these, 81% (17/21) and 66.6% (14/21) amplified 16S rRNA and flaB genes, respectively. qPCR-positive samples were then subjected to conventional nested and semi-nested PCR to amplify 16S rRNA and flaB gene fragments. Nine positive samples for both genes were sequenced, and seven and six sequences were of good quality for the 16S rRNA and flaB genes, respectively. The DNA of Borrelia spp. was detected in the insectivorous and fruit bats Artibeus lituratus, Carollia perspicillata, Glossophaga soricina, Phyllostomus discolor, and Uroderma sp. The 16S rRNA gene sequences showed 97.66-98.47% identity with "Borrelia sp. clone Omi3," "Borrelia sp. RT1S," and Borrelia sp. 2374; the closest identities for the flaB gene were 94.02-98.04% with "Borrelia sp. Macaregua." For the 16S rRNA gene, the phylogenetic analysis showed a grouping with "Candidatus Borrelia ivorensis" and "Ca. Borrelia africana," and for the flaB gene showed a grouping with Borrelia sp. Macaregua and Borrelia sp. Potiretama. The pathogenic role of the Borrelia detected in this study is unknown., Conclusions: We describe the first molecular evidence of Borrelia spp. in the department of Córdoba, Colombia, highlighting that several bat species harbor Borrelia spirochetes., (© 2023. The Author(s).)

The axolotl is a great model for studying cartilage, bone and joint regeneration, fracture healing, and evolution. Stainings such as Alcian Blue/Alizarin Red have become workhorses in skeletal analyses, but additional methods complement the detection of different skeletal matrices. Here we describe protocols for studying skeletal biology in axolotls, particularly Alcian Blue/Alizarin Red staining, microcomputed tomography (μCT) scan and live staining of calcified tissue. In addition, we describe a method for decalcification of skeletal elements to ease sectioning., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Ductal carcinoma in situ (DCIS) is a pre-invasive stage of breast cancer. During invasion, the encapsulating DCIS basement membrane (BM) is compromised, and tumor cells invade the surrounding stroma. The mechanisms that regulate functional epithelial BMs in vivo are poorly understood. Myosin-X (MYO10) is a filopodia-inducing protein associated with metastasis and poor clinical outcome in invasive breast cancer (IBC). We identify elevated MYO10 expression in human DCIS and IBC, and this suggests links with disease progression. MYO10 promotes filopodia formation and cell invasion in vitro and cancer-cell dissemination from progressively invasive human DCIS xenografts. However, MYO10-depleted xenografts are more invasive. These lesions exhibit compromised BMs, poorly defined borders, and increased cancer-cell dispersal and EMT-marker-positive cells. In addition, cancer spheroids are dependent on MYO10-filopodia to generate a near-continuous extracellular matrix boundary. Thus, MYO10 is protective in early-stage breast cancer, correlating with tumor-limiting BMs, and pro-invasive at later stages, facilitating cancer-cell dissemination., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Early events during axolotl limb regeneration include an immune response and the formation of a wound epithelium. These events are linked to a clearance of damaged tissue prior to blastema formation and regeneration of the missing structures. Here, we report the resorption of calcified skeletal tissue as an active, cell-driven, and highly regulated event. This process, carried out by osteoclasts, is essential for a successful integration of the newly formed skeleton. Indeed, the extent of resorption is directly correlated with the integration efficiency, and treatment with zoledronic acid resulted in osteoclast function inhibition and failed tissue integration. Moreover, we identified the wound epithelium as a regulator of skeletal resorption, likely releasing signals involved in recruitment/differentiation of osteoclasts. Finally, we reported a correlation between resorption and blastema formation, particularly, a coordination of resorption with cartilage condensation. In sum, our results identify resorption as a major event upon amputation, playing a critical role in the overall process of skeletal regeneration., Competing Interests: CR, ST, KC, CN, DO, MS, JB, JC, TS No competing interests declared, (© 2022, Riquelme-Guzmán et al.)

Introduction: Although more than half of the world's population is already vaccinated, the appearance of new variants of concern puts public health at risk due to the generation of new immunogens against the virus as a crucial and relevant strategy in the control of these new variants., Methods: A preclinical study used a potential vaccine candidate (RBD, SARS-CoV-2). Four groups of BALB/c mice were used, a control group, an adjuvant group, a group inoculated with one dose of RBD subunit protein, and the fourth group inoculated with two doses of RBD subunit protein., Results: No inflammatory or cellular changes were shown in the mice's anatomopathological evaluation. Higher kinetics and 75% seroconversion were obtained in the mice inoculated with two doses of RBD (P < 0.0001)., Conclusions: The application of two doses of the RBD vaccine candidate in BALB/c mice proved safe and immunogenic against SARS-CoV-2., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2022. Published by Elsevier Ltd.)

Background: The ability of SARS-CoV-2 to remain in asymptomatic individuals facilitates its dissemination and makes its control difficult., Objective: To establish a cohort of asymptomatic individuals, change to the symptomatic status, and determine the most frequent clinical manifestations.  METHODS: Between April 9 and August 9, 2020, molecular diagnosis of SARS-CoV-2 infection was confirmed in 154 asymptomatic people in contact with subjects diagnosed with COVID-19. Nasopharyngeal swabs were performed on these people in different hospitals in Córdoba, the Caribbean area of Colombia. The genes E, RdRp, and N were amplified with RT-qPCR. Based on the molecular results and the Cq values, the patients were subsequently followed up through telephone calls to verify their health conditions., Results: Overall, of 154 asymptomatic individuals, 103 (66.9%) remained asymptomatic, and 51 (33.1%) changed to symptomatic. The most frequent clinical manifestations in young people were anosmia and arthralgia. Adults showed cough, ageusia, and odynophagia; in the elderly were epigastralgia, dyspnea, and headache. Mortality was 8%., Conclusions: A proportion of 33% of presymptomatic individuals was found, of which four of them died. This high rate could indicate a silent transmission, contributing significantly to the epidemic associated with SARS-CoV-2., (© 2022. The Author(s).)

Background: The axolotl is a key model to study appendicular regeneration. The limb complexity resembles that of humans in structure and tissue components; however, axolotl limbs develop postembryonically. In this work, we evaluated the postembryonic development of the appendicular skeleton and its changes with aging., Results: The juvenile limb skeleton is formed mostly by Sox9/Col1a2 cartilage cells. Ossification of the appendicular skeleton starts when animals reach a length of 10 cm, and cartilage cells are replaced by a primary ossification center, consisting of cortical bone and an adipocyte-filled marrow cavity. Vascularization is associated with the ossification center and the marrow cavity formation. We identified the contribution of Col1a2-descendants to bone and adipocytes. Moreover, ossification progresses with age toward the epiphyses of long bones. Axolotls are neotenic salamanders, and still ossification remains responsive to l-thyroxine, increasing the rate of bone formation., Conclusions: In axolotls, bone maturation is a continuous process that extends throughout their life. Ossification of the appendicular bones is slow and continues until the complete element is ossified. The cellular components of the appendicular skeleton change accordingly during ossification, creating a heterogenous landscape in each element. The continuous maturation of the bone is accompanied by a continuous body growth., (© 2021 The Authors. Developmental Dynamics published by Wiley Periodicals LLC on behalf of American Association for Anatomy.)

In processes such as development and regeneration, where large cellular and tissue rearrangements occur, cell fate and behaviour are strongly influenced by tissue mechanics. While most well-established tools probing mechanical properties require an invasive sample preparation, confocal Brillouin microscopy captures mechanical parameters optically with high resolution in a contact-free and label-free fashion. In this work, we took advantage of this tool and the transparency of the highly regenerative axolotl to probe its mechanical properties in vivo for the first time. We mapped the Brillouin frequency shift with high resolution in developing limbs and regenerating digits, the most studied structures in the axolotl. We detected a gradual increase in the cartilage Brillouin frequency shift, suggesting decreasing tissue compressibility during both development and regeneration. Moreover, we were able to correlate such an increase with the regeneration stage, which was undetected with fluorescence microscopy imaging. The present work evidences the potential of Brillouin microscopy to unravel the mechanical changes occurring in vivo in axolotls, setting the basis to apply this technique in the growing field of epimorphic regeneration.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the causative agent of the current COVID-19 pandemic, has evolved to have a wide range of hosts, including non-human primates, wild and domestic animals. The ACE2 protein has a high level of conservation and is the common receptor invertebrate species for a viral infection to occur; this receptor could give rise to anthroponotic events. This article describes the first event of symptomatic transmission in Latin America from a human to a dog by the B.1.625 lineage of SARS-CoV-2. We found 21 shared mutations in the complete genomes of viral sequences from owners and dogs. Further phylogenetic and molecular analysis showed that 100% co-localization of the clade helps to understand human-animal transmission. Prediction of the Spike protein structure of the sequenced virus and docking analyzes showed that the E484K mutation in the receptor-binding domain (RBD) could contribute to the viral affinity of dACE2. Therefore, close contact between SARS-CoV-2-infected humans and pets should be avoided to prevent the emergence of novel mutations of public health importance from anthroponotic events., (© 2022. The Author(s).)

Background: Serological evaluation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an alternative that allows us to determine the prevalence and dynamics of this infection in populations. The goal of this study was to determine the clinical and sociodemographic dynamics of SARS-CoV-2 infection in a region of the Colombian Caribbean., Methods: Between July and November 2020, a cross-sectional observational study was carried out in Córdoba, located in northeast Colombia in the Caribbean area. Eight municipalities with the largest populations were chosen and 2564 blood samples were taken. A commercial enzyme-linked immunosorbent assay was used with the recombinant protein antigen N of SARS-CoV-2. The people included in the study were asked for sociodemographic and clinical data, which were analysed by statistical methods., Results: A seroprevalence of 40.8% was obtained for SARS-CoV-2 in the Córdoba region. In the bivariate analysis, no differences were observed in seropositivity against SARS-CoV-2 for gender or age range (p>0.05). Higher seropositivity was found in low socio-economic status and symptomatic patients (p<0.0001). A total of 30.7% of the asymptomatic patients were seropositive for SARS-CoV-2, which could be linked to the spread of this infection. In the multivariate analysis, seroconversion was related to poverty and clinical manifestations such as anosmia and ageusia (p<0.05)., Conclusions: The high seropositivity in Córdoba is due to widespread SARS-CoV-2 in this population. The relationship between seropositivity and socio-economic status suggests a higher exposure risk to the virus caused by informal economic activities in low-income groups. Clinical manifestations such as anosmia and ageusia could be clinical predictors of infection by the new emergent coronavirus., (© The Author(s) 2021. Published by Oxford University Press on behalf of Royal Society of Tropical Medicine and Hygiene.)

Introduction: Currently, more than 4.5 billion doses of SARS-CoV-2 vaccines have been applied worldwide. However, some developing countries are still a long way from achieving herd immunity through vaccination. In some territories, such as the Colombian Amazon, mass immunization strategies have been implemented with the CoronaVac® vaccine. Due to its proximity to Brazil, where one of the variants of interest of SARS-CoV-2 circulates., Objective: To determine the effectiveness of the CoronaVac® vaccine in a population of the Colombian Amazon., Methods: Between February 24, 2021, and August 10, 2021, a descriptive observational study was carried out in which a population of individuals over 18 years of age immunized with two doses of the CoronaVac® vaccine was evaluated. The study site was in the municipality of Mitú, Vaupés, in southeastern Colombia, a region located in the Amazon bordering Brazil., Results: 99% of the urban population of the Mitú municipality were vaccinated with CoronaVac®. To date, 5.7% of vaccinated individuals have become ill, and only 0.1% of these require hospitalization. One death was attributable to COVID-19 has been reported among vaccinated individuals, and the vaccine has shown 94.3% effectiveness against mild disease and 99.9% against severe infection., Conclusions: The herd immunity achieved through mass vaccination in this population has made it possible to reduce the rate of complicated cases and mortality from COVID-19 in this region of the Colombian Amazon., Highlights: CoronaVac® has shown 94.3% effectiveness against mild disease and 99.9% against severe infection in this indigenous population. CoronaVac® reduces the mortality rate from 2.2% in 2020 to 0.22% in 2021. The herd immunity was achieved through mass vaccination in this region of the Colombian Amazon., (© 2022. The Author(s).)

In this study, we report the synthesis and evaluation of in vitro and in vivo antitrypanosomal activity of styrylquinoline-like compounds (SQ) 3a-h . Synthesis was carried out by using quinaldine and 8- hydroxyquinaldine with a variety of aromatic aldehydes. The structure of SQs was corroborated by one and two-dimension NMR spectroscopy. In vitro antitrypanosomal activity on T. cruzi Talahuen strain was evaluated using β-galactosidase enzymatic method; cytotoxicity on U-937 cells was assessed by using MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] method. On the other hand, in vivo therapeutical response to 3a-f compounds was evaluated in BALB/c mice ( Mus musculus ) experimentally infected with T. cruzi blood trypomastigotes and then orally administered with 100 mg/kg weight day for 20 days. All of the compounds showed in vitro activity with EC 50 values ranging between 4.6 ± 0.1 μg/mL (14.4 μM) and 36.6 ± 6.1 μg/mL (91 μM). Furthermore, treatment with 3a-f compounds for 20 days resulted in improvement in all of the mice, with a 83-96% decrease in parasitic load at day 90 post-treatment. Treatment with benznidazol (BZ) managed to cure 100% of the mice at the end of treatment. None of the treatments affected the weight of the animals or alanine aminotransferase (ALT), blood urea nitrogen (BUN) and creatinine levels in serum. These results suggest a therapeutic potential of 3a-f compounds as treatment for the infection., Competing Interests: The authors declare no conflict of interest., (© 2021 The Authors.)

Background and Aim: In 2016, the tilapia-producing farms in the department of Córdoba, Colombia, had witnessed outbreaks of disease with clinical signs compatible with those caused by the tilapia lake virus (TiLV). This study was conducted to confirm the presence of TiLV in some fish farms in the department of Córdoba., Materials and Methods: A descriptive cross-sectional study was conducted in seven farms using a non-random sampling method from July 2016 to December 2017. A total of 66 fish, including 33 healthy fish and 33 fish with clinical signs, were caught, from which 178 tissue samples of spleen, liver, and brain were collected. RNA was extracted from each organ using TRIzol ® . cDNA was synthesized using a retrotranscriptase and a universal amplification primer. The polymerase chain reaction was performed using primers specific to TiLV, in which the primers were amplified in a 491 bp region in segment 3 of TiLV, and the amplicons were sequenced using the Sanger method., Results: Of the seven farms surveyed, 3 (42.85%) had TiLV in the collected fish. Of the 66 collected fish, 18 (27.27%) were infected with TiLV. The virus was detected in the brain (64.3%, 18/28), spleen (61.9%, 13/21), and liver (35.7%, 10/28). The sequences were recorded in GenBank with the codes MH338228, MH350845, and MH350846. Nucleotide homology analyses revealed that this study's circulating strains exhibited 97% identity with the Israeli strain (GenBank KU751816.1)., Conclusion: This is the first official report of TiLV in the department of Córdoba, Colombia. The circulating strains detected in this study exhibited 97% identity with the Israeli strain., (Copyright: © Contreras, et al.)

Dengue, a mosquito-borne zoonotic disease, is the most common vector-borne disease in tropical and subtropical areas. In this study, we aim to demonstrate biological evidence of dengue virus infection in bats. A cross-sectional study was carried out in the departments of Cordoba and Sucre, Colombia. A total of 286 bats were captured following the ethical protocols of animal experimentation. The specimens were identified and euthanized using a pharmacological treatment with atropine, acepromazine and sodium pentobarbital. Duplicate samples of brain, heart, lung, spleen, liver, and kidney were collected with one set stored in Trizol and the other stored in 10% buffered formalin for histopathological and immunohistochemical analysis using polyclonal antibodies. Brain samples from lactating mice with an intracranial inoculation of DENV-2 were used as a positive control. As a negative control, lactating mouse brains without inoculation and bats brains negative for RT-PCR were included. Tissue sections from each specimen of bat without conjugate were used as staining control. In a specimen of Carollia perspicillata captured in Ayapel (Cordoba) and Phylostomus discolor captured in San Carlos (Cordoba), dengue virus was detected, and sequences were matched to DENV serotype 2. In bats RT-PCR positive for dengue, lesions compatible with viral infections, and the presence of antigens in tissues were observed. Molecular findings, pathological lesions, and detection of antigens in tissues could demonstrate viral DENV-2 replication and may correspond to natural infection in bats. Additional studies are needed to elucidate the exact role of these species in dengue epidemics.

Objectives: We investigated seroreactivity by using a commercial SARS-CoV-2 ELISA test in samples collected from different groups of individuals, including patients diagnosed to have Dengue, Zika, and Chikungunya infection between 2015 and 2019, from an endemic area in the Caribbean Colombian region., Methods: A total of 127 sera samples obtained from six different groups of individuals were included in this study: Group A: patients with confirmed SARS-CoV-2 infection; Group B: patients with symptoms suggestive of COVID-19 or asymptomatic contacts with confirmed patients; Group C: patients with acute or recent dengue virus infection; Group D: patients with acute Zika virus infection; Group E: patients with previous Chikungunya virus infection; and Group F: individuals with exposure to spotted fever group rickettsiae., Results: Overall, group A, group B, and group D showed seroreactivity to SARS-CoV-2 in 92%, 75%, and 26% of samples, respectively; furthermore, group C, group E, and group F showed 100% seronegativity., Conclusions: We found 26% of serological cross-reactivity in patients with acute Zika virus infection by using a commercial SARS-CoV-2 ELISA test. Further studies are needed to evaluate whether serological cross-reaction is maintained with time in nonacute patients with previous exposure to the Zika virus and its effect in SARS-CoV-2 serosurveys in endemic areas for this arbovirus., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

A serological survey was carried out in Monteria (500 000 population), a mid-size city in Colombia. An overall prevalence of 55.3% (95% confidence interval, 52.5%-57.8%) was found among a sample of 1.368 people randomly selected from the population. Test positivity was related to economic characteristics with the highest prevalence found in the most impoverished areas, representing 83.8% of the city's population. We found a prevalence that might be associated with some important level of population immunity., (Published by Oxford University Press on behalf of Infectious Diseases Society of America 2020.)

Fibrillar adhesions are important structural and adhesive components in fibroblasts, and are required for fibronectin fibrillogenesis. While nascent and focal adhesions are known to respond to mechanical cues, the mechanoresponsive nature of fibrillar adhesions remains unclear. Here, we used ratiometric analysis of paired adhesion components to determine an appropriate fibrillar adhesion marker. We found that active α5β1-integrin exhibits the most definitive fibrillar adhesion localization compared to other proteins, such as tensin-1, reported to be in fibrillar adhesions. To elucidate the mechanoresponsiveness of fibrillar adhesions, we designed a cost-effective and reproducible technique to fabricate physiologically relevant stiffness gradients on thin polyacrylamide (PA) hydrogels, embedded with fluorescently labelled beads. We generated a correlation curve between bead density and hydrogel stiffness, thus enabling a readout of stiffness without the need for specialized knowhow, such as atomic force microscopy (AFM). We find that stiffness promotes growth of fibrillar adhesions in a tensin-1-dependent manner. Thus, the formation of these extracellular matrix-depositing structures is coupled to the mechanical parameters of the cell environment and may enable cells to fine-tune their matrix environment in response to changing physical conditions., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2020. Published by The Company of Biologists Ltd.)

The digit tip is an exciting model for studying regeneration in mammals, but the precise mechanisms and the populations of cells involved in the formation and remodeling of the blastema remain unknown. In an exciting new work, Storer et al. take advantage of single-cell RNAseq combined with Pdgfra+ ​lineage-tracing to open the way into the enigmatic world of mammalian tissue regeneration., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Cellular mechanics play a crucial role in tissue homeostasis and are often misregulated in disease. Traction force microscopy is one of the key methods that has enabled researchers to study fundamental aspects of mechanobiology; however, traction force microscopy is limited by poor resolution. Here, we propose a simplified protocol and imaging strategy that enhances the output of traction force microscopy by increasing i) achievable bead density and ii) the accuracy of bead tracking. Our approach relies on super-resolution microscopy, enabled by fluorescence fluctuation analysis. Our pipeline can be used on spinning-disk confocal or widefield microscopes and is compatible with available analysis software. In addition, we demonstrate that our workflow can be used to gain biologically relevant information and is suitable for fast long-term live measurement of traction forces even in light-sensitive cells. Finally, using fluctuation-based traction force microscopy, we observe that filopodia align to the force field generated by focal adhesions.

The link between integrin activity regulation and cellular mechanosensing of tissue rigidity, especially on different extracellular matrix ligands, remains poorly understood. Here, we find that primary mouse mammary gland stromal fibroblasts (MSFs) are able to spread efficiently, generate high forces, and display nuclear YAP on soft collagen-coated substrates, resembling the soft mammary gland tissue. We describe that loss of the integrin inhibitor, SHARPIN, impedes MSF spreading specifically on soft type I collagen but not on fibronectin. Through quantitative experiments and computational modeling, we find that SHARPIN-deficient MSFs display faster force-induced unbinding of adhesions from collagen-coated beads. Faster unbinding, in turn, impairs force transmission in these cells, particularly, at the stiffness optimum observed for wild-type cells. Mechanistically, we link the impaired mechanotransduction of SHARPIN-deficient cells on collagen to reduced levels of collagen-binding integrin α11β1. Thus integrin activity regulation and α11β1 play a role in collagen-specific mechanosensing in MSFs., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background and Aim: Venezuelan equine encephalitis virus (VEEV) is an alphavirus that causes encephalitis with a high impact on public health in Latin America. However, only in Guatemala, Trinidad and Tobago, and Mexico have found antibodies in VEEV in bats, using immunohistochemistry, the sensitivity and specificity are improved; thus, it is better for demonstrating natural infection in bats as potential hosts. This study aimed to determine the presence of VEEV in tissues of frugivorous bats., Materials and Methods: A prospective descriptive cross-sectional study with a non-probabilistic sampling was carried out in 12 localities of Córdoba and Sucre area of the Colombian Caribbean. Two hundred and eighty-six bats were captured using fog nets, and the specimens according to taxonomic keys were classified. According to the Ethics Committee of the University of Córdoba, the bats were treated with analgesics and anesthetics. Blood samples were taken and then euthanized to obtain tissues and organs which were preserved in liquid N2 at -196°C. A portion of each organ was fixed in 10% buffered formalin for the detection of antigens by immunohistochemistry. Several pathological anatomy analyses were performed to determine the histological characteristics of tissue lesions of frugivorous bats naturally infected with the VEEV., Results: Of the 286 bats captured, 23 species were identified. In samples of the brain, spleen, and lung of two frugivorous bats (2/286=0.70%) Artibeus planirostris and Sturnira lilium , the presence of VEEV was confirmed by immunohistochemistry., Conclusion: A fragment of the nsP4 non-structural protein gene corresponding to the alphavirus was amplified. Two samples were positive (2/286=0.70%) in frugivorous bats; A. planirostris (code GenBank: MG820274) and S. lilium (code GenBank: MG820275). The present study showed the first molecular evidence and cellular evidence (histopathology and immunohistochemistry) of natural VEEV infection in frugivorous bats in Colombia; these bats could be a host of this zoonosis., (Copyright: © Guzmán, et al.)

While it is clear that key transcriptional programmes are important for maintaining pluripotency, the requirement for cell adhesion to the extracellular matrix remains poorly defined. Human pluripotent stem cells (hPSCs) form colonies encircled by an actin ring and large stable cornerstone focal adhesions (FA). Using superresolution two-colour interferometric photo-activated localisation microscopy, we examine the three-dimensional architecture of cornerstone adhesions and report vertical lamination of FA proteins with three main structural features distinct from previously studied focal adhesions: 1) integrin β5 and talin are present at high density, at the edges of cornerstone FA, adjacent to a vertical kank-rich protein wall, 2) vinculin localises higher than previously reported, displaying a head-above-tail orientation, and 3) surprisingly, actin and α-actinin are present in two discrete z-layers. Finally, we report that depletion of kanks diminishes FA patterning, and actin organisation within the colony, indicating a role for kanks in hPSC colony architecture.

Natural infection of dengue virus (DENV) in bats is an unexplored field in Colombia. To detect the presence of DENV in bats, a descriptive prospective study using a nonprobabilistic sampling was carried out; 286 bats in 12 sites were caught. Sample tissues of different animals were obtained; the RNA was obtained from tissues and a nested-RT-PCR was carried out and detected amplicons of 143 fragment of the NS5 gene were sequenced by the Sanger method. In nonhematophagous bats Carollia perspicillata and Phyllostomus discolor captured in Ayapel and San Carlos (Córdoba), respectively, an amplicon corresponding to NS5 was detected. The amplicons showed a high similarity with serotype-2 dengue virus (DENV-2). This is the first evidence of the DENV-2 genome in bats in from the Colombian Caribbean.

Background: Bats are an important ecological group within ecosystems. The rabies virus is a Lyssavirus, and haematophagous bats are the principal reservoir; however, the virus has also been detected in non-haematophagous bats. The objective was to determine the rabies virus in non-haematophagous bats in the Colombian Caribbean region., Methods: In 2017, a cross-sectional study was carried out with a base-risk sampling in twelve geographic zones of the Colombian Caribbean area that included the main ecosystems of two departments. 286 bats were captured, which were euthanized with a pharmacological treatment following the ethical protocols of animal experimentation. The taxonomic identification was done with dichotomous keys. The necropsy was carried out at the capture site, and brain samples were kept in liquid nitrogen. The extraction of the RNA was carried out from the frozen brains with Trizol™; a fragment of 914 bp of the glycoprotein G of the rabies virus was amplified with RT-PCR. The amplicons were sequenced with the Sanger method., Results: Twenty-three genera of bats were identified, and, in two frugivorous, Artibeus lituratus and Artibeus planirostris, amplicons were obtained and sequenced as the rabies virus., Conclusions: This is the first evidence of natural infection of the rabies virus in frugivorous bats in the Colombian Caribbean area; this result is important for the surveillance and control of rabies.

Alphavirus infection associated encephalitis is an emerging infectious disease with a high impact on public health in Latin America., Objective: To study the eco-epidemiology of alphaviruses in bats of departments of Córdoba and Sucre, Colombia., Methodology: A prospective descriptive cross-sectional study with a non-probabilistic sampling, in 12 localities of Córdoba and Sucre was carried out. Using mist nets capture of the specimens was carried out. The size of the sample was 286 bats, each specimen captured was taxonomically classified. The bats were immobilized with anesthetic and analgesic treatment according to the ethics committee of the University of Córdoba, morphometric measurements and blood samples were taken, later they were necropsied in the field to obtain a collection of tissues which were preserved in liquid N 2 -190 °C. The averages of the climatic conditions of the sampling sites were extracted from the WorldClim database (http://www.worldclim.org/). The open source software QGIS (Quantum GIS Development Team.2015) was used to map and visualize bioclimatic regions of Córdoba. We used descriptive and retrospective information about the equine population and reports of foci of equine encephalitis., Results: In Córdoba and Sucre, 286 bats were captured and 23 species were classified, Artibeus and Phyllostomus discolor were the most frequent captured genus. The geographic ranges of the captured species were variable, some had a wide distribution and others were restricted to some areas. Venezuelan equine encephalitis virus RNA was detected in Artibeus planirostris and Sturnira lilium (2/286 = 0.70%) from Cordoba - Colombia. The univariate descriptive analysis showed no significant association for any of the analyzed variables climatic., Conclusions: Frugivorous bats from the Caribbean area of Colombia may be involved in the Venezuelan equine encephalitis virus enzootic cycle., (Copyright © 2018. Published by Elsevier B.V.)

Angiogenesis and vascular remodeling are driven by extensive endothelial cell movements. Here, we present in vivo evidence that endothelial cell movements are associated with oscillating lamellipodia-like structures, which emerge from cell junctions in the direction of cell movements. High-resolution time-lapse imaging of these junction-based lamellipodia (JBL) shows dynamic and distinct deployment of junctional proteins, such as F-actin, VE-cadherin and ZO1, during JBL oscillations. Upon initiation, F-actin and VE-cadherin are broadly distributed within JBL, whereas ZO1 remains at cell junctions. Subsequently, a new junction is formed at the front of the JBL, which then merges with the proximal junction. Rac1 inhibition interferes with JBL oscillations and disrupts cell elongation-similar to a truncation in ve-cadherin preventing VE-cad/F-actin interaction. Taken together, our observations suggest an oscillating ratchet-like mechanism, which is used by endothelial cells to move over each other and thus provides the physical means for cell rearrangements.

Integrin transmembrane receptors control a wide range of biological interactions by triggering the assembly of large multiprotein complexes at their cytoplasmic interface. Diverse methods have been used to investigate interactions between integrins and intracellular proteins, and predominantly include peptide-based pulldowns and biochemical immuno-isolations from detergent-solubilised cell lysates. However, quantitative methods to probe integrin-protein interactions in a more biologically relevant context where the integrin is embedded within a lipid bilayer have been lacking. Here, we describe 'protein-liposome interactions by flow cytometry' (denoted ProLIF), a technique to reconstitute recombinant integrin transmembrane domains (TMDs) and cytoplasmic tail (CT) fragments in liposomes as individual subunits or as αβ heterodimers and, via flow cytometry, allow rapid and quantitative measurement of protein interactions with these membrane-embedded integrins. Importantly, the assay can analyse binding of fluorescent proteins directly from cell lysates without further purification steps. Moreover, the effect of membrane composition, such as PI(4,5)P 2 incorporation, on protein recruitment to the integrin CTs can be analysed. ProLIF requires no specific instrumentation and can be applied to measure a broad range of membrane-dependent protein-protein interactions with the potential for high-throughput/multiplex analyses.This article has associated First Person interviews with the first authors of the paper (see doi: 10.1242/jcs.223644 and doi: 10.1242/jcs.223719)., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

Loss of endothelial integrity promotes capillary leakage in numerous diseases, including sepsis, but there are no effective therapies for preserving endothelial barrier function. Angiopoietin-2 (ANGPT2) is a context-dependent regulator of vascular leakage that signals via both endothelial TEK receptor tyrosine kinase (TIE2) and integrins. Here, we show that antibodies against β1-integrin decrease LPS-induced vascular leakage in murine endotoxemia, as either a preventative or an intervention therapy. β1-integrin inhibiting antibodies bound to the vascular endothelium in vivo improved the integrity of endothelial cell-cell junctions and protected mice from endotoxemia-associated cardiac failure, without affecting endothelial inflammation, serum proinflammatory cytokine levels, or TIE receptor signaling. Moreover, conditional deletion of a single allele of endothelial β1-integrin protected mice from LPS-induced vascular leakage. In endothelial monolayers, the inflammatory agents thrombin, lipopolysaccharide (LPS), and IL-1β decreased junctional vascular endothelial (VE)-cadherin and induced actin stress fibers via β1- and α5-integrins and ANGPT2. Additionally, β1-integrin inhibiting antibodies prevented inflammation-induced endothelial cell contractility and monolayer permeability. Mechanistically, the inflammatory agents stimulated ANGPT2-dependent translocation of α5β1-integrin into tensin-1-positive fibrillar adhesions, which destabilized the endothelial monolayer. Thus, β1-integrin promotes endothelial barrier disruption during inflammation, and targeting β1-integrin signaling could serve as a novel means of blocking pathological vascular leak., Competing Interests: The authors declare no conflict of interest., (Copyright © 2018 the Author(s). Published by PNAS.)

Fibrosis is a common feature of several chronic diseases and is characterized by exacerbated accumulation of ECM. An understanding of the cellular and molecular mechanisms involved in the development of this condition is crucial for designing efficient treatments for those pathologies. Connective tissue growth factor (CTGF/CCN2) is a pleiotropic protein with strong profibrotic activity. In this report, we present experimental evidence showing that ECM stimulates the synthesis of CTGF in response to lysophosphatidic acid (LPA).The integrin/focal adhesion kinase (FAK) signaling pathway mediates this effect, since CTGF expression is abolished by the use of the Arg-Gly-Asp-Ser peptide and also by an inhibitor of FAK autophosphorylation at tyrosine 397. Cilengitide, a specific inhibitor of αv integrins, inhibits the expression of CTGF mediated by LPA or transforming growth factor β1. We show that ECM obtained from decellularized myofibroblast cultures or derived from activated fibroblasts from muscles of the Duchenne muscular dystrophy mouse model ( mdx) induces the expression of CTGF. This effect is dependent on FAK phosphorylation in response to its activation by integrin. We also found that the fibrotic ECM inhibits skeletal muscle differentiation. This novel regulatory mechanism of CTGF expression could be acting as a positive profibrotic feedback between the ECM and CTGF, revealing a novel concept in the control of fibrosis under chronic damage.

Cell-type-specific functions and identity are tightly regulated by interactions between the cell cytoskeleton and the extracellular matrix (ECM). Human pluripotent stem cells (hPSCs) have ultimate differentiation capacity and exceptionally low-strength ECM contact, yet the organization and function of adhesion sites and associated actin cytoskeleton remain poorly defined. We imaged hPSCs at the cell-ECM interface with total internal reflection fluorescence microscopy and discovered that adhesions at the colony edge were exceptionally large and connected by thick ventral stress fibers. The actin fence encircling the colony was found to exert extensive Rho-ROCK-myosin-dependent mechanical stress to enforce colony morphology, compaction, and pluripotency and to define mitotic spindle orientation. Remarkably, differentiation altered adhesion organization and signaling characterized by a switch from ventral to dorsal stress fibers, reduced mechanical stress, and increased integrin activity and cell-ECM adhesion strength. Thus, pluripotency appears to be linked to unique colony organization and adhesion structure., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Tight regulation of integrin activity is paramount for dynamic cellular functions such as cell matrix adhesion and mechanotransduction. Integrin activation is achieved through intracellular interactions at the integrin cytoplasmic tails and through integrin-ligand binding. In this study, we identify the metabolic sensor AMP-activated protein kinase (AMPK) as a β1-integrin inhibitor in fibroblasts. Loss of AMPK promotes β1-integrin activity, the formation of centrally located active β1-integrin- and tensin-rich mature fibrillar adhesions, and cell spreading. Moreover, in the absence of AMPK, cells generate more mechanical stress and increase fibronectin fibrillogenesis. Mechanistically, we show that AMPK negatively regulates the expression of the integrin-binding proteins tensin1 and tensin3. Transient expression of tensins increases β1-integrin activity, whereas tensin silencing reduces integrin activity in fibroblasts lacking AMPK. Accordingly, tensin silencing in AMPK-depleted fibroblasts impedes enhanced cell spreading, traction stress, and fibronectin fiber formation. Collectively, we show that the loss of AMPK up-regulates tensins, which bind β1-integrins, supporting their activity and promoting fibrillar adhesion formation and integrin-dependent processes., (© 2017 Georgiadou et al.)

SHARPIN is a widely expressed multifunctional protein implicated in cancer, inflammation, linear ubiquitination and integrin activity inhibition; however, its contribution to epithelial homeostasis remains poorly understood. Here, we examined the role of SHARPIN in mammary gland development, a process strongly regulated by epithelial-stromal interactions. Mice lacking SHARPIN expression in all cells (Sharpin cpdm ), and mice with a stromal (S100a4-Cre) deletion of Sharpin, have reduced mammary ductal outgrowth during puberty. In contrast, Sharpin cpdm mammary epithelial cells transplanted in vivo into wild-type stroma, fully repopulate the mammary gland fat pad, undergo unperturbed ductal outgrowth and terminal differentiation. Thus, SHARPIN is required in mammary gland stroma during development. Accordingly, stroma adjacent to invading mammary ducts of Sharpin cpdm mice displayed reduced collagen arrangement and extracellular matrix (ECM) stiffness. Moreover, Sharpin cpdm mammary gland stromal fibroblasts demonstrated defects in collagen fibre assembly, collagen contraction and degradation in vitro Together, these data imply that SHARPIN regulates the normal invasive mammary gland branching morphogenesis in an epithelial cell extrinsic manner by controlling the organisation of the stromal ECM., (© 2016 The Authors.)

Receptor tyrosine kinases (RTKs) and integrins cooperate to stimulate cell migration and tumour metastasis. Here we report that an integrin influences signalling of an RTK, c-Met, from inside the cell, to promote anchorage-independent cell survival. Thus, c-Met and β1-integrin co-internalize and become progressively recruited on LC3B-positive 'autophagy-related endomembranes' (ARE). In cells growing in suspension, β1-integrin promotes sustained c-Met-dependent ERK1/2 phosphorylation on ARE. This signalling is dependent on ATG5 and Beclin1 but not on ATG13, suggesting ARE belong to a non-canonical autophagy pathway. This β1-integrin-dependent c-Met-sustained signalling on ARE supports anchorage-independent cell survival and growth, tumorigenesis, invasion and lung colonization in vivo. RTK-integrin cooperation has been assumed to occur at the plasma membrane requiring integrin 'inside-out' or 'outside-in' signalling. Our results report a novel mode of integrin-RTK cooperation, which we term 'inside-in signalling'. Targeting integrin signalling in addition to adhesion may have relevance for cancer therapy.

Fluorescence resonance energy transfer (FRET) is widely used to study conformational changes of macromolecules and protein-protein, protein-nucleic acid, and protein-small molecule interactions. FRET biosensors can serve as valuable secondary assays in drug discovery and for target validation in mammalian cells. Fluorescence lifetime imaging microscopy (FLIM) allows precise quantification of the FRET efficiency in intact cells, as FLIM is independent of fluorophore concentration, detection efficiency, and fluorescence intensity. We have developed an automated FLIM system using a commercial frequency domain FLIM attachment (Lambert Instruments) for wide-field imaging. Our automated FLIM system is capable of imaging and analyzing up to 50 different positions of a slide in less than 4 min, or the inner 60 wells of a 96-well plate in less than 20 min. Automation is achieved using a motorized stage and controller (Prior Scientific) coupled with a Zeiss Axio Observer body and full integration into the Lambert Instruments FLIM acquisition software. As an application example, we analyze the interaction of the oncoprotein Ras and its effector Raf after drug treatment. In conclusion, our automated FLIM imaging system requires only commercial components and may therefore allow for a broader use of this technique in chemogenomics projects., (© 2015 Society for Laboratory Automation and Screening.)

Fibrotic disorders are characterized by an increase in extracellular matrix protein expression and deposition, Duchene Muscular Dystrophy being one of them. Among the factors that induce fibrosis are Transforming Growth Factor type β (TGF-β) and the matricellular protein Connective Tissue Growth Factor (CTGF/CCN2), the latter being a target of the TGF-β/SMAD signaling pathway and is the responsible for the profibrotic effects of TGF-β. Both CTGF and TGF are increased in tissues affected by fibrosis but little is known about the regulation of the expression of CTGF mediated by TGF-β in muscle cells. By using luciferase reporter assays, site directed mutagenesis and specific inhibitors in C2C12 cells; we described a novel SMAD Binding Element (SBE) located in the 5' UTR region of the CTGF gene important for the TGF-β-mediated expression of CTGF in myoblasts. In addition, our results suggest that additional transcription factor binding sites (TFBS) present in the 5' UTR of the CTGF gene are important for this expression and that SP1/SP3 factors are involved in TGF-β-mediated CTGF expression., (© 2015 Wiley Periodicals, Inc.)

Hotspot mutations of Ras drive cell transformation and tumorigenesis. Less frequent mutations in Ras are poorly characterized for their oncogenic potential. Yet insight into their mechanism of action may point to novel opportunities to target Ras. Here, we show that several cancer-associated mutations in the switch III region moderately increase Ras activity in all isoforms. Mutants are biochemically inconspicuous, while their clustering into nanoscale signaling complexes on the plasma membrane, termed nanocluster, is augmented. Nanoclustering dictates downstream effector recruitment, MAPK-activity, and tumorigenic cell proliferation. Our results describe an unprecedented mechanism of signaling protein activation in cancer.

Rodent-borne hantaviruses (family Bunyaviridae, genus Hantavirus) cause hantavirus pulmonary syndrome in the Americas and hemorrhagic fever with renal syndrome in Europe and Asia. The viruses are transmitted to humans mainly by inhalation of virus-contaminated aerosols of rodent excreta and secreta. Classic clinical hemorrhagic fever with renal syndrome occurs in five phases: fever, hypotension, oliguria, polyuria, and convalescence. Hantavirus pulmonary syndrome is a severe acute disease that is associated with respiratory failure, pulmonary edema and cardiogenic shock. The diagnosis of hantavirus infections in humans is based on clinical and epidemiological information as well as laboratory tests. We review diagnosis for hantavirus infections based on serology, PCR, immunochemistry and virus culture.

Rab proteins constitute the largest subfamily of Ras-like small GTPases. They are central to vesicular transport and organelle definition in eukaryotic cells. Unlike their Ras counterparts, they are not a hallmark of cancer. However, a number of diseases, including cancer, show a misregulation of Rab protein activity. As for all membrane-anchored signaling proteins, correct membrane organization is critical for Rabs to operate. In this chapter, we provide a detailed protocol for the use of a flow cytometry-based Fluorescence Resonance Energy Transfer (FRET)-biosensors assay, which allows to detect changes in membrane anchorage, subcellular distribution, and of the nanoscale organization of Rab-GTPases in mammalian cell lines. This assay is high-throughput amenable and can therefore be utilized in chemical-genomic and drug discovery efforts.

Solution structures and biochemical data have provided a wealth of mechanistic insight into Ras GTPases. However, information on how much the membrane organization of these lipid-modified proteins impacts on their signaling is still scarce. Ras proteins are organized into membrane nanoclusters, which are necessary for Ras-MAPK signaling. Using quantitative conventional and super-resolution fluorescence methods, as well as mathematical modeling, we investigated nanoclustering of H-ras helix α4 and hypervariable region mutants that have different bona fide conformations on the membrane. By following the emergence of conformer-specific nanoclusters in the plasma membrane of mammalian cells, we found that conformers impart distinct nanoclustering responses depending on the cytoplasmic levels of the nanocluster scaffold galectin-1. Computational modeling revealed that complexes containing H-ras conformers and galectin-1 affect both the number and lifetime of nanoclusters and thus determine the specific Raf effector recruitment. Our results show that mutations in Ras can affect its nanoclustering response and thus allosterically effector recruitment and downstream signaling. We postulate that cancer- and developmental disease-linked mutations that are associated with the Ras membrane conformation may exhibit so far unrecognized Ras nanoclustering and therefore signaling alterations.

The clonogenic or colony formation assay is a widely used method to study the number and size of cancer cell colonies that remain after irradiation or cytotoxic agent administration and serves as a measure for the anti-proliferative effect of these treatments. Alternatively, this assay is used to quantitate the transforming potential of cancer associated genes and chemical agents. Therefore, there is a need for a simplified and standardized analysis of colony formation assays for both routine laboratory use and for parallelized automated analysis. Here we describe the freely available ImageJ-plugin "ColonyArea", which is optimized for rapid and quantitative analysis of focus formation assays conducted in 6- to 24-well dishes. ColonyArea processes image data of multi-well dishes, by separating, concentrically cropping and background correcting well images individually, before colony formation is quantitated. Instead of counting the number of colonies, ColonyArea determines the percentage of area covered by crystal violet stained cell colonies, also taking the intensity of the staining and therefore cell density into account. We demonstrate that these parameters alone or in combination allow for robust quantification of IC50 values of the cytotoxic effect of two staurosporines, UCN-01 and staurosporine (STS) on human glioblastoma cells (T98G). The relation between the potencies of the two compounds compared very well with that obtained from an absorbance based method to quantify colony growth and to published data. The ColonyArea ImageJ plugin provides a simple and efficient analysis routine to quantitate assay data of one of the most commonly used cellular assays. The bundle is freely available for download as supporting information. We expect that ColonyArea will be of broad utility for cancer biologists, as well as clinical radiation scientists.

Fluorescence Recovery After Photobleaching (FRAP) and Raster Image Correlation Spectroscopy (RICS) are two powerful techniques to study the diffusion dynamics of fluorescently labeled proteins. FRAP and RICS can be easily applied on any commercial confocal microscope. In this chapter, we describe the principles of these methods and provide the reader with a detailed guide on how to apply these methods in the study of Ras nanoclustering and diffusion in the plasma membrane of live cells.

We tested sera from 286 agricultural workers and 322 rodents in the department of Córdoba, northeastern Colombia, for antibodies against two hantaviruses. The sera were analysed by indirect ELISA using the lysate of Vero E6 cells infected with Maciel virus (MACV) or the N protein of Araraquara virus (ARAV) as antigens for the detection of antibodies against hantaviruses. Twenty-four human sera were IgG positive using one or both antigens. We detected anti-MACV IgG antibodies in 10 sera (3.5%) and anti-ARAV antibodies in 21 sera (7.34%). Of the 10 samples that were positive for MACV, seven (70%) were cross-reactive with ARAV; seven of the 21 ARAV-positive samples were cross-reactive with MACV. Using an ARAV IgM ELISA, two of the 24 human sera (8.4%) were positive. We captured 322 rodents, including 210 Cricetidae (181 Zygodontomys brevicauda, 28 Oligoryzomys fulvescens and 1 Oecomys trinitatis), six Heteromys anomalus (Heteromyidae), one Proechimys sp. (Echimyidae) and 105 Muridae (34 Rattus rattus and 71 Mus musculus). All rodent sera were negative for both antigens. The 8.4% detection rate of hantavirus antibodies in humans is much higher than previously found in serosurveys in North America, suggesting that rural agricultural workers in northeastern Colombia are frequently exposed to hantaviruses. Our results also indicate that tests conducted with South American hantavirus antigens could have predictive value and could represent a useful alternative for the diagnosis of hantavirus infection in Colombia.