Biology subjects

Rojas, M.

Publications and source records attributed to Rojas, M..

28 records · Page 2Linked to original sources

bioRxiv2021

Cardiomyocyte BRAF and type 1 RAF inhibitors promote cardiomyocyte and cardiac hypertrophy in mice in vivo

The extracellular signal-regulated kinase 1/2 (ERK1/2) cascade promotes cardiomyocyte hypertrophy and is cardioprotective, with the three RAF kinases forming a node for signal integration. Our aims were to determine if BRAF is relevant for human heart failure, if BRAF promotes cardiomyocyte hypertrophy, and if Type 1 RAF inhibitors developed for cancer (that paradoxically activate ERK1/2 at low concentrations: the "RAF paradox") may have the same effect. BRAF was upregulated in heart samples from patients with heart failure compared with normal controls. We assessed the effects of activated BRAF in the heart using mice with tamoxifen-activated Cre for cardiomyocyte-specific knock-in of the activating V600E mutation into the endogenous gene. We used echocardiography to measure cardiac dimensions/function. Cardiomyocyte BRAFV600E induced cardiac hypertrophy within 10 d, resulting in increased ejection fraction and fractional shortening over 6 weeks. This was associated with increased cardiomyocyte size without significant fibrosis, consistent with compensated hypertrophy. The experimental Type 1 RAF inhibitor, SB590885, and/or encorafenib (a RAF inhibitor used clinically) increased ERK1/2 phosphorylation in cardiomyocytes, and promoted hypertrophy, consistent with a "RAF paradox" effect. Both promoted cardiac hypertrophy in mouse hearts in vivo, with increased cardiomyocyte size and no overt fibrosis. In conclusion, BRAF potentially plays an important role in human failing hearts, activation of BRAF is sufficient to induce hypertrophy, and Type 1 RAF inhibitors promote hypertrophy via the "RAF paradox". Cardiac hypertrophy resulting from these interventions was not associated with pathological features, suggesting that Type 1 RAF inhibitors may be useful to boost cardiomyocyte function.

biochemistry↗

bioRxiv2021

TSC2-extracellular matrix crosstalk controls pulmonary vascular proliferation and pulmonary hypertension

Increased proliferation and survival of resident cells in small pulmonary arteries (PA) are important drivers of pulmonary hypertension (PH). Tuberous sclerosis complex 2 (TSC2) is a negative regulator of mTOR complex 1 and cell growth. Here we show that TSC2 is deficient in small remodeled PA/PA vascular smooth muscle cells (PAVSMC) from human PAH and experimental PH lungs. TSC2 deficiency was reproduced in vitro by maintaining PAVSMC on pathologically stiff substrates and was required for stiffness-induced proliferation, accumulation of transcriptional co-activators YAP/TAZ and up-regulation of mTOR. Depletion of TSC2 reproduced PH features in vitro in human PAVSMC and in vivo in SM22-Tsc2+/- mice. TSC2 loss in PAVSMC was supported by YAP and led to the up-regulation of YAP/TAZ and mTOR via modulating the extracellular matrix (ECM) composition. ECM, produced by TSC2-deficient PAVSMC, promoted growth of non-diseased PA adventitial fibroblasts and PAVSMC, which, in turn, was prevented by 5{beta}1 integrin receptor antagonist ATN161. In vitro, molecular and pharmacological (SRT2104) restoration of TSC2 down-regulated YAP/TAZ, mTOR, and ECM production, inhibited proliferation and induced apoptosis in human PAH PAVSMC. In vivo, orally administrated SRT2104 restored TSC2, resolved pulmonary vascular remodeling, PH, and improved right heart in two rodent models of PH. Thus, PAVSMC TSC2 is a critical integrator of ECM composition and stiffness with pro-proliferative signaling and PH, and the restoration of functional TSC2 could be an attractive therapeutic option to treat PH. One Sentence SummaryTSC2 acts as mechanosensor and mechanotransducer, integrating ECM composition and stiffness with pro-proliferative signaling in pulmonary vasculature; its deficiency in PA vascular smooth muscle cells results in ECM remodeling, hyper-proliferation and pulmonary hypertension, which could be reversed by pharmacological restoration of functional TSC2.

molecular biology↗

bioRxiv2021

High CXCL6 drives matrix expression and correlate with markers of poor outcome in IPF

Signaling via G protein-coupled receptors (GPCRs) can modulate levels of cyclic adenosine monophosphate (cAMP) and shape the functions of fibroblasts in idiopathic pulmonary fibrosis (IPF). We have identified Chemokine (C-X-C) Motif Ligand 6 (CXCL6) as a potential pro-fibrotic GPCR ligand. We tested the function of CXCL6 in ex vivo human donor and fibrotic lung fibroblasts and in an animal model of pulmonary fibrosis. We also measured levels of CXCL6 in the blood and bronchoalveolar lavage (BAL) of patients with IPF. CXCL6 decreased cAMP levels in a dose-dependent manner in Donor and IPF Fibroblasts. CXCL6 mRNA and protein were localized to epithelial cells. Administration of mCXCL5 (LIX, murine CXCL6 homologue) to mice increased collagen synthesis with and without bleomycin. CXCL6 increased Collagen I and -SMA levels in Donor and IPF Fibroblasts. Silencing of CXCR1/2 as well as Reparixin, a CXCR1/2 inhibitor, blocked effects of CXCL6. Treprostinil blocked effects of CXCL6 only on levels of -SMA but not on Collagen I. CXCL6 levels in the BAL of two separate cohorts of patients with IPF was associated with poor survival. We conclude that high CXCL6 drives fibroblast function and correlates with poor outcomes in IPF.

cell biology↗

bioRxiv2021

Seronegative MSM at high-risk of HIV-1 acquisition show Immune quiescent profile with normal immune response against common antigens

HIV infection still represents a major public health problem worldwide, and a vaccine remains elusive. The study of HIV-exposed seronegative individuals (HESN) brings important information about the natural resistance to HIV, allows a better understanding of the infection and opens doors for new preventive and therapeutic strategies. Among HESN groups there are some men who have sex with men (MSM) with high-risk sexual behaviors, who represent an adequate cohort for the study of HESN because of their major exposure to HIV in the absence of infection. This study aimed to compare the immunological profile of Colombian seronegative MSM with different risk sexual behaviors. Sixty MSM at high-risk (n=16) and low-risk (n=44) of HIV-1 acquisition were included. No sex worker nor homozygous delta 32 mutation subjects were included. All the participants were negative for anti-HIV-1/2 antibodies and HIV-1 proviral DNA. The high-risk MSM presented a higher frequency of sexual partners in the last 3 months previous to the studys enrollment (Median 30 vs. 2), lifetime sexual partners (Median 1708 vs. 26), and unprotected anal intercourse (Median 12.5 vs. 2) than low-risk MSM. This group also showed a quiescent profile of T cells and NK cells, with a significantly lower percentage of CD4+CD38+, CD4+HLADR-CD38+, CD4+Ki67+ T cells, NKG2D+ NK cells (CD3-CD16+CD56+), a significantly higher percentage of CD4+HLADR-CD38- and a tendency to show a higher percentage of CD8+HLADR+CD38-T cells, than the low-risk group. Likewise, they showed higher mRNA levels of Serpin A1 from PBMCs. The results suggest that this cohort of MSM could be HESN individuals and their resistance would be explained by a quiescent profile of T cells and NK cells, and increased expression of Serpin A1. It is necessary to continue the study of MSM at high-risk of exposure to HIV-1 to better understand the natural resistance to HIV.

immunology↗

bioRxiv2021

ER exit sites in Drosophila display abundant ER-Golgi vesicles and pearled tubes but no megacarriers

Secretory cargos are collected at ER exit sites (ERES) before transport to the Golgi apparatus. Decades of research have provided many details of the molecular events underlying ER-Golgi exchanges. Essential questions, however, remain about the organization of the ER-Golgi interface in cells and the type of membrane structures mediating traffic from ERES. To investigate these, we used transgenic tagging in Drosophila flies, 3D-SIM and FIB-SEM to characterize ERES-Golgi units in collagen-producing fat body, imaginal discs and imaginal discs overexpressing ERES determinant Tango1. We found in front of ERES a pre-cis-Golgi region involved in both anterograde and retrograde transport. This pre-cis-Golgi is continuous with the rest of the Golgi, not a separate intermediate compartment or collection of large carriers, for which we found no evidence. We found, however, many vesicles, as well as pearled tubules connecting ERES and Golgi.

cell biology↗

bioRxiv2021

PECAM-1 preserves cardiac function in pressure overload-induced biomechanical stress

BackgroundHaemodynamic forces play a critical role in proper development of the heart, however much less is known about the mechanisms that regulate cardiac remodelling and function in response to haemodynamic stress in the adult. Platelet endothelial cell adhesion molecule-1 (PECAM-1) is a cell adhesion and signalling molecule that has important roles in regulation of junctional integrity, transendothelial migration and mechanotransduction in response to fluid shear stress. Our previous work identified a role for PECAM-1 in regulating baseline cardiac function via regulation of endothelial-cardiomyocyte communication. MethodsThis study investigates the role of PECAM-1 in cardiac remodelling in response to biomechanical stress due to pressure overload induced by transaortic constriction (TAC). ResultsOur data reveal that loss of PECAM-1 is associated with systolic dysfunction that is further accentuated following TAC. Adaptive increases in cardiomyocyte cross-sectional area, capillary density and hypertrophic gene expression were all affected with loss of PECAM-1. In control mice, maintained cardiac function was associated with activation of the c-Jun NH(2)-terminal kinase (JNK) pathway, whereas PECAM-1 deletion significantly decreased JNK activation after pressure overload. Our data suggest that in the absence of PECAM-1 signalling, inadequate remodelling of the heart under increased mechanical strain leads to further deterioration of cardiac function, characterized by reduced cardiomyocyte hypertrophy, capillary density and defects in the JNK signalling pathway. ConclusionsOur study reveals a role for PECAM-1 in preservation of cardiac function in response to biomechanical stress induced by pressure overload.

physiology↗

bioRxiv2020

In vivo stabilization of endogenous chloroplast RNAs by customized artificial pentatricopeptide repeat proteins

Pentatricopeptide repeat (PPR) proteins are helical repeat-proteins that bind RNA in a modular fashion with a sequence-specificity that can be manipulated by the use of an amino acid code. As such, PPR repeats are promising scaffolds for the design of RNA binding proteins for synthetic biology applications. However, the in vivo functional capabilities of artificial PPR proteins built from consensus PPR motifs are just starting to be explored. Here, we report in vivo functions of an artificial PPR protein, dPPRrbcL, made of consensus PPR motifs that were designed to bind a sequence near the 5 end of rbcL transcripts in Arabidopsis chloroplasts. We used a functional complementation assay to demonstrate that this protein bound its intended RNA target with specificity in vivo and that it substituted for a natural PPR protein by stabilizing processed rbcL mRNA. We targeted a second protein of analogous design to the petL 5 UTR, where it substituted for the native stabilizing PPR protein PGR3, albeit inefficiently. These results showed that artificial PPRs can be engineered to functionally mimic the class of native PPR proteins that serve as physical barriers against exoribonucleases.

plant biology↗

bioRxiv2020

Analysis of SARS-CoV-2 ORF3a structure reveals chloride binding sites

SARS-CoV-2 ORF3a is believed to form ion channels, which may be involved in the modulation of virus release, and has been implicated in various cellular processes like the up-regulation of fibrinogen expression in lung epithelial cells, downregulation of type 1 interferon receptor, caspase-dependent apoptosis, and increasing IFNAR1 ubiquitination. ORF3a assemblies as homotetramers, which are stabilized by residue C133. A recent cryoEM structure of a homodimeric complex of ORF3a has been released. A lower-resolution cryoEM map of the tetramer suggests two dimers form it, arranged side by side. The dimers cryoEM structure revealed that each protomer contains three transmembrane helices arranged in a clockwise configuration forming a six helices transmembrane domain. This domains potential permeation pathway has six constrictions narrowing to about 1 [A] in radius, suggesting the structure solved is in a closed or inactivated state. At the cytosol end, the permeation pathway encounters a large and polar cavity formed by multiple beta strands from both protomers, which opens to the cytosolic milieu. We modeled the tetramer following the arrangement suggested by the low-resolution tetramer cryoEM map. Molecular dynamics simulations of the tetramer embedded in a membrane and solvated with 0.5 M of KCl were performed. Our simulations show the cytosolic cavity is quickly populated by both K+ and Cl-, yet with different dynamics. K+ ions moved relatively free inside the cavity without forming proper coordination sites. In contrast, Cl- ions enter the cavity, and three of them can become stably coordinated near the intracellular entrance of the potential permeation pathway by an inter-subunit network of positively charged amino acids. Consequently, the central cavitys electrostatic potential changed from being entirely positive at the beginning of the simulation to more electronegative at the end.

biophysics↗

bioRxiv2020

AKT-like kinase promotes cell survival during nutritional stress in tripanosomatids

Tritryps are protozoan parasites that belong to the Trypanosomatidae family, which encompasses the etiologic agents of leishmaniasis, African and American trypanosomiasis. These parasites undergo different stress conditions across their life cycle, such as nutritional stress, which needs to be deadened in order to guarantee the survival of the parasite inside its vector and mammal hosts. Here we show that the lack of the serine threonine kinase PKB / AKT-like function, either by allosteric inhibition of its Plekstrin domain (PH) in T. cruzi, the reduction of the gene transcripts in T. brucei by RNAi assays, or by AKT-like gene knockout in L. major, reduce the tolerance to nutritional stress of parasites that triggers apoptosis-like events, including DNA fragmentation, mitochondrial damage and loss of plasma membrane integrity. Additionally, we observed that double knockout of Akt-like in L. major impairs its infective capacity. This work confirms some of the previously described functions regarding parasite survival for AKT-like kinases in the Leishmania genus. The present work also provides strong evidence of the probable function of Akt-like in T. cruzi and T. brucei survival and infectivity. Author summaryEndemic countries for neglected tropical diseases are called to play a paramount role in the discovery of new drug candidates through the application of new drug development strategies. Rational drug design method have proven to be compatible with the development of new drug for orphan and neglected diseases since it substantially reduces the costs of discovery and development, a desirable condition for public funded initiatives. Previously we have identified a new parasite protein kinase (AKT-like) as promising new target candidate by means of computational tools and probed its biological role in trypanosomatids. Here we show that inhibition of the AKT-like kinase in trypanosomatids by different approaches (chemical inhibition, interference RNA and gene knockout) decreases the fitness and survival of the parasites in vitro, interfering with the capacity of the parasites to react and survive stress conditions similar to those experienced by the cell in the natural life cycle. Additionally our results strongly supports the potential of a new family of compounds previously described by bioinformatics means as potential trypanocidal agents. Altogether we show that the specific inhibition of the AKT-like is a promising strategy for the further development of anti-trypanosome drugs.

microbiology↗

bioRxiv2018

Using High Throughput DNA Sequencing to Evaluate the Accuracy of Serial Dilution Based Tests of Microbial Activities in Oil Pipelines

Microbial activities have detrimental effects on industrial infrastructure. If not controlled, microbial presence can result in corrosion, biofilm formation, and product degradation. Serial dilution tests are routinely used for evaluating presence and abundance of microorganisms by diluting samples and culturing microbes in specific media designed to support microorganisms with particular properties, such as sulfate reduction.\n\nA high-throughput sequencing approach was used to evaluate changes in microbial composition during four standard serial dilution tests. Analysis of 159 isolates revealed significant differences in the microbial compositions of sequential serial dilution titers and identified several cases where: (a) bacteria known to have a detrimental metabolic function (such as acid production) were lost in the serial dilution medium designed to test for this function; (b) bacteria virtually absent in the original sample became dominant in the serial dilution medium. These observations raise concerns regarding the accuracy and overall usefulness of serial dilution tests.

microbiology↗