Identifying and targeting key driver genes for collagen production within the 11q13/14 breast cancer amplicon.

Genetic studies indicate that breast cancer can be divided into several basic molecular groups. One of these groups, termed IntClust-2, is characterized by amplification of a small portion of chromosome 11 and has a median survival of only five years. Several cancer-relevant genes occupy this portion of chromosome 11, and it is thought that overexpression of a combination of driver genes in this region is responsible for the poor outcome of women in this group. In this study we used a gene editing method to knock out, one by one, each of 198 genes that are located within the amplified region of chromosome 11 and determined how much each of these genes contributed to the survival of breast cancer cells. In addition to well-known drivers such as CCND1 and PAK1 , we identified two different genes ( SERPINH1 and P4HA3 ), that encode proteins involved in collagen synthesis and organization. Using both in vitro and in vivo functional analyses, we determined that P4HA3 and/or SERPINH1 provide a critical driver function on IntClust-2 basic processes, such as viability, proliferation, and migration. Inhibiting these enzymes via genetic or pharmacologic means reduced collagen synthesis and impeded oncogenic signaling transduction in cell culture models, and a small-molecule inhibitor of P4HA3 was effective in treating 11q13 tumor growth in an animal model. As collagen has a well-known association with tissue stiffness and aggressive forms of breast cancer, we believe that the two genes we identified provide an opportunity for a new therapeutic strategy in IntClust-2 breast cancers.

Unique vulnerability of RAC1-mutant melanoma to combined inhibition of CDK9 and immune checkpoints.

RAC1P29S is the third most prevalent hotspot mutation in sun-exposed melanoma. RAC1 alterations in cancer are correlated with poor prognosis, resistance to standard chemotherapy, and insensitivity to targeted inhibitors. Although RAC1P29S mutations in melanoma and RAC1 alterations in several other cancers are increasingly evident, the RAC1-driven biological mechanisms contributing to tumorigenesis remain unclear. Lack of rigorous signaling analysis has prevented identification of alternative therapeutic targets for RAC1P29S-harboring melanomas. To investigate the RAC1P29S-driven effect on downstream molecular signaling pathways, we generated an inducible RAC1P29S expression melanocytic cell line and performed RNA-sequencing (RNA-seq) coupled with multiplexed kinase inhibitor beads and mass spectrometry (MIBs/MS) to establish enriched pathways from the genomic to proteomic level. Our proteogenomic analysis identified CDK9 as a potential new and specific target in RAC1P29S-mutant melanoma cells. In vitro, CDK9 inhibition impeded the proliferation of in RAC1P29S-mutant melanoma cells and increased surface expression of PD-L1 and MHC Class I proteins. In vivo, combining CDK9 inhibition with anti-PD-1 immune checkpoint blockade significantly inhibited tumor growth only in melanomas that expressed the RAC1P29S mutation. Collectively, these results establish CDK9 as a novel target in RAC1-driven melanoma that can further sensitize the tumor to anti-PD-1 immunotherapy.

Inhibitory to non-inhibitory evolution of the ζ subunit of the F1FO-ATPase of Paracoccus denitrificans and α-proteobacteria as related to mitochondrial endosymbiosis.

Introduction: The ζ subunit is a potent inhibitor of the F1FO-ATPase of Paracoccus denitrificans (PdF1FO-ATPase) and related α-proteobacteria different from the other two canonical inhibitors of bacterial (ε) and mitochondrial (IF1) F1FO-ATPases. ζ mimics mitochondrial IF1 in its inhibitory N-terminus, blocking the PdF1FO-ATPase activity as a unidirectional pawl-ratchet and allowing the PdF1FO-ATP synthase turnover. ζ is essential for the respiratory growth of P. denitrificans, as we showed by a Δζ knockout. Given the vital role of ζ in the physiology of P. denitrificans, here, we assessed the evolution of ζ across the α-proteobacteria class. Methods: Through bioinformatic, biochemical, molecular biology, functional, and structural analyses of several ζ subunits, we confirmed the conservation of the inhibitory N-terminus of ζ and its divergence toward its C-terminus. We reconstituted homologously or heterologously the recombinant ζ subunits from several α-proteobacteria into the respective F-ATPases, including free-living photosynthetic, facultative symbiont, and intracellular facultative or obligate parasitic α-proteobacteria. Results and discussion: The results show that ζ evolved, preserving its inhibitory function in free-living α-proteobacteria exposed to broad environmental changes that could compromise the cellular ATP pools. However, the ζ inhibitory function was diminished or lost in some symbiotic α-proteobacteria where ζ is non-essential given the possible exchange of nutrients and ATP from hosts. Accordingly, the ζ gene is absent in some strictly parasitic pathogenic Rickettsiales, which may obtain ATP from the parasitized hosts. We also resolved the NMR structure of the ζ subunit of Sinorhizobium meliloti (Sm-ζ) and compared it with its structure modeled in AlphaFold. We found a transition from a compact ordered non-inhibitory conformation into an extended α-helical inhibitory N-terminus conformation, thus explaining why the Sm-ζ cannot exert homologous inhibition. However, it is still able to inhibit the PdF1FO-ATPase heterologously. Together with the loss of the inhibitory function of α-proteobacterial ε, the data confirm that the primary inhibitory function of the α-proteobacterial F1FO-ATPase was transferred from ε to ζ and that ζ, ε, and IF1 evolved by convergent evolution. Some key evolutionary implications on the endosymbiotic origin of mitochondria, as most likely derived from α-proteobacteria, are also discussed.

Unique vulnerability of RAC1-mutant melanoma to combined inhibition of CDK9 and immune checkpoints.

RAC1P29S is the third most prevalent hotspot mutation in sun-exposed melanoma. RAC1 alterations in cancer are correlated with poor prognosis, resistance to standard chemotherapy, and insensitivity to targeted inhibitors. Although RAC1P29S mutations in melanoma and RAC1 alterations in several other cancers are increasingly evident, the RAC1-driven biological mechanisms contributing to tumorigenesis remain unclear. Lack of rigorous signaling analysis has prevented identification of alternative therapeutic targets for RAC1P29S-harboring melanomas. To investigate the RAC1P29S-driven effect on downstream molecular signaling pathways, we generated an inducible RAC1P29S expression melanocytic cell line and performed RNA-sequencing (RNA-seq) coupled with multiplexed kinase inhibitor beads and mass spectrometry (MIBs/MS) to establish enriched pathways from the genomic to proteomic level. Our proteogenomic analysis identified CDK9 as a potential new and specific target in RAC1P29S-mutant melanoma cells. In vitro, CDK9 inhibition impeded the proliferation of in RAC1P29S-mutant melanoma cells and increased surface expression of PD-L1 and MHC Class I proteins. In vivo, combining CDK9 inhibition with anti-PD-1 immune checkpoint blockade significantly inhibited tumor growth only in melanomas that expressed the RAC1P29S mutation. Collectively, these results establish CDK9 as a novel target in RAC1-driven melanoma that can further sensitize the tumor to anti-PD-1 immunotherapy.

Monitoring Mitochondrial Function in Aedes albopictus C6/36 Cell Line during Dengue Virus Infection.

Aedes aegypti and Aedes albopictus mosquitoes are responsible for dengue virus (DENV) transmission in tropical and subtropical areas worldwide, where an estimated 3 billion people live at risk of DENV exposure. DENV-infected individuals show symptoms ranging from sub-clinical or mild to hemorrhagic fever. Infected mosquitoes do not show detectable signs of disease, even though the virus maintains a lifelong persistent infection. The interactions between viruses and host mitochondria are crucial for virus replication and pathogenicity. DENV infection in vertebrate cells modulates mitochondrial function and dynamics to facilitate viral proliferation. Here, we describe that DENV also regulates mitochondrial function and morphology in infected C6/36 mosquito cells (derived from Aedes albopictus). Our results showed that DENV infection increased ROS (reactive oxygen species) production, modulated mitochondrial transmembrane potential and induced changes in mitochondrial respiration. Furthermore, we offer the first evidence that DENV causes translocation of mitofusins to mitochondria in the C6/36 mosquito cell line. Another protein Drp-1 (Dynamin-related protein 1) did not localize to mitochondria in DENV-infected cells. This observation therefore ruled out the possibility that the abovementioned alterations in mitochondrial function are associated with mitochondrial fission. In summary, this report provides some key insights into the virus-mitochondria crosstalk in DENV infected mosquito cells.

Low saliva pH can yield false positives results in simple RT-LAMP-based SARS-CoV-2 diagnostic tests.

Diagnosis of any infectious disease is vital for opportune treatment and to prevent dissemination. RT-qPCR tests for detection of SARS-CoV-2, the causative agent for COVID-19, are ideal in a hospital environment. However, mass testing requires cheaper and simpler tests, especially in settings that lack sophisticated machinery. The most common current diagnostic method is based on nasopharyngeal sample collection, RNA extraction, and RT-qPCR for amplification and detection of viral nucleic acids. Here, we show that samples obtained from nasopharyngeal swabs in VTM and in saliva can be used with or without RNA purification in an isothermal loop-mediated amplification (LAMP)-based assay, with 60-93% sensitivity for SARS-CoV-2 detection as compared to standard RT-qPCR tests. A series of simple modifications to standard RT-LAMP published methods to stabilize pH fluctuations due to salivary acidity resulted in a significant improvement in reliability, opening new avenues for efficient, low-cost testing of COVID-19 infection.

Targeting effector pathways in RAC1P29S-driven malignant melanoma.

Malignant melanoma is characterized by mutations in a number of driver genes, most notably BRAF and NRAS. Recent genomic analyses revealed that 4-9% of sun-exposed melanomas bear activating mutations in RAC1, which encodes a small GTPase that is known to play key roles in cell proliferation, survival, and migration. The RAC1 protein activates several effector pathways, including Group A p21-activated kinases (PAKs), phosphoinositol-3-kinases (PI3Ks), in particular the beta isoform, and the serum-response factor/myocardin-related transcription factor (SRF/MRTF). Having previously shown that inhibition of Group A PAKs impedes oncogenic signalling from RAC1P29S, we here extend this analysis to examine the roles of PI3Ks and SRF/MRTF in melanocytes and/or in a zebrafish model. We demonstrate that a selective Group A PAK inhibitor (Frax-1036), a pan-PI3K (BKM120), and two PI3Kß inhibitors (TGX221, GSK2636771) impede the growth of melanoma cells driven by mutant RAC1 but not by mutant BRAF, while other PI3K selective inhibitors, including PI3Kα, δ and γ, are less effective. Using these compounds as well as an SRF/MRTF inhibitor (CCG-203,971), we observed similar results in vivo, using embryonic zebrafish development as a readout. These results suggest that targeting Group A PAKs, PI3Kß, and/or SRF/MRTF represent a promising approach to suppress RAC1 signalling in malignant melanoma.

RAC1 as a Therapeutic Target in Malignant Melanoma.

Small GTPases of the RAS and RHO families are related signaling proteins that, when activated by growth factors or by mutation, drive oncogenic processes. While activating mutations in KRAS, NRAS, and HRAS genes have long been recognized and occur in many types of cancer, similar mutations in RHO family genes, such as RAC1 and RHOA, have only recently been detected as the result of extensive cancer genome-sequencing efforts and are linked to a restricted set of malignancies. In this review, we focus on the role of RAC1 signaling in malignant melanoma, emphasizing recent advances that describe how this oncoprotein alters melanocyte proliferation and motility and how these findings might lead to new therapeutics in RAC1-mutant tumors.

Metabolism, ATP production and biofilm generation by Staphylococcus epidermidis in either respiratory or fermentative conditions.

Staphylococcus epidermidis is a Gram-positive saprophytic bacterium found in the microaerobic/anaerobic layers of the skin that becomes a health hazard when it is carried across the skin through punctures or wounds. Pathogenicity is enhanced by the ability of S. epidermidis to associate into biofilms, where it avoids attacks by the host and antibiotics. To test the effect of oxygen on metabolism and biofilm generation, cells were cultured at different oxygen concentrations ([O2]). As [O2] decreased, S. epidermidis metabolism went from respiratory to fermentative. Remarkably, the rate of growth decreased at low [O2] while a high concentration of ATP ([ATP]) was kept. Under hypoxic conditions bacteria associated into biofilms. Aerobic activity sensitized the cell to hydrogen peroxide-mediated damage. In the presence of metabolic inhibitors, biofilm formation decreased. It is suggested that at low [O2] S. epidermidis limits its growth and develops the ability to form biofilms.

Wolbachia pipientis grows in Saccharomyces cerevisiae evoking early death of the host and deregulation of mitochondrial metabolism.

Wolbachia sp. has colonized over 70% of insect species, successfully manipulating host fertility, protein expression, lifespan, and metabolism. Understanding and engineering the biochemistry and physiology of Wolbachia holds great promise for insect vector-borne disease eradication. Wolbachia is cultured in cell lines, which have long duplication times and are difficult to manipulate and study. The yeast strain Saccharomyces cerevisiae W303 was used successfully as an artificial host for Wolbachia wAlbB. As compared to controls, infected yeast lost viability early, probably as a result of an abnormally high mitochondrial oxidative phosphorylation activity observed at late stages of growth. No respiratory chain proteins from Wolbachia were detected, while several Wolbachia F1 F0 -ATPase subunits were revealed. After 5 days outside the cell, Wolbachia remained fully infective against insect cells.

On the oxidative damage by cadmium to kidney mitochondrial functions.

In the kidney, the accumulation of heavy metals such as Cd2+ produces mitochondrial dysfunctions, i.e., uncoupling of the oxidative phosphorylation, inhibition of the electron transport through the respiratory chain, and collapse of the transmembrane electrical gradient. This derangement may be due to the fact that Cd2+ induces the transition of membrane permeability from selective to nonselective via the opening of a transmembrane pore. In fact, Cd2+ produces this injury through the stimulation of oxygen-derived radical generation, inducing oxidative stress. Several molecules have been used to avoid or even reverse Cd2+-induced mitochondrial injury, for instance, cyclosporin A, resveratrol, dithiocarbamates, and even EDTA. The aim of this study was to explore the possibility that the antioxidant tamoxifen could protect mitochondria from the deleterious effects of Cd2+. Our results indicate that the addition of 1 µmol/L Cd2+ to mitochondria collapsed the transmembrane electrical gradient, induced the release of cytochrome c, and increased both the generation of H2O2 and the oxidative damage to mitochondrial DNA (among other measured parameters). Of interest, these mitochondrial dysfunctions were ameliorated after the addition of tamoxifen.

Response of Ustilago maydis against the Stress Caused by Three Polycationic Chitin Derivatives.

Chitosan is a stressing molecule that affects the cells walls and plasma membrane of fungi. For chitosan derivatives, the action mode is not clear. In this work, we used the yeast Ustilago maydis to study the effects of these molecules on the plasma membrane, focusing on physiologic and stress responses to chitosan (CH), oligochitosan (OCH), and glycol-chitosan (GCH). Yeasts were cultured with each of these molecules at 1 mg·mL-1 in minimal medium. To compare plasma membrane damage, cells were cultivated in isosmolar medium. Membrane potential (Δψ) as well as oxidative stress were measured. Changes in the total plasma membrane phospholipid and protein profiles were analyzed using standard methods, and fluorescence-stained mitochondria were observed. High osmolarity did not protect against CH inhibition and neither affected membrane potential. The OCH did produce higher oxidative stress. The effects of these molecules were evidenced by modifications in the plasma membrane protein profile. Also, mitochondrial damage was evident for CH and OCH, while GCH resulted in thicker cells with fewer mitochondria and higher glycogen accumulation.

Las enfermedades transmitidas por vectores y el potencial uso de Wolbachia, una bacteria endocelular obligada, para erradicarlas / Vector-borne diseases and the potential use of Wolbachia, an obligate endocellular bacterium, to eradicate them

Resumen Según la Organización Mundial de la Salud (OMS), 17% de las enfermedades infecciosas reportadas en el mundo son transmitidas por vectores artrópodos. Una alternativa para bloquear la transmisión es infectar a los vectores con una bacteria endocelular llamada Wolbachia. Diferentes investigaciones han demostrado que Wolbachia acorta la vida del mosquito, aumenta su resistencia ante la infección de algunos virus como dengue, Zika y Chikungunya, y provoca incompatibilidad citoplasmática, por lo que al liberar mosquitos machos infectados con Wolbachia en una población de hembras no infectadas los productos no son viables, disminuyendo drásticamente la población total. En el presente artículo se incluye una descripción general de las enfermedades infecciosas más comunes transmitidas por vectores así como una revisión del uso de Wolbachia como una posible herramienta para controlar su propagación.

Abstract According to the World and Health Organization (WHO), 17% of the worldwide reported infectious diseases are vector-borne. One alternative for blocking the transmission of these infectious agents is to infect the vectors with the endocellular bacterium Wolbachia. Several studies have shown that Wolbachia shortens mosquitos' lifespan and increases their resistance to some virus like Dengue, Zika or Chikungunya. Wolbachia also causes cytoplasmic incompatibility, so, when Wolbachia-infected male mosquitoes are released among an uninfected female population, the production of an offspring is not viable and the mosquito population decreases drastically. This article includes an overview of the most common vector-borne infectious diseases as well as a review of the use of Wolbachia as a possible tool for controlling the spread of vector-borne diseases.

In female rat heart mitochondria, oophorectomy results in loss of oxidative phosphorylation.

Oophorectomy in adult rats affected cardiac mitochondrial function. Progression of mitochondrial alterations was assessed at one, two and three months after surgery: at one month, very slight changes were observed, which increased at two and three months. Gradual effects included decrease in the rates of oxygen consumption and in respiratory uncoupling in the presence of complex I substrates, as well as compromised Ca2+ buffering ability. Malondialdehyde concentration increased, whereas the ROS-detoxifying enzyme Mn2+ superoxide dismutase (MnSOD) and aconitase lost activity. In the mitochondrial respiratory chain, the concentration and activity of complex I and complex IV decreased. Among other mitochondrial enzymes and transporters, adenine nucleotide carrier and glutaminase decreased. 2-Oxoglutarate dehydrogenase and pyruvate dehydrogenase also decreased. Data strongly suggest that in the female rat heart, estrogen depletion leads to progressive, severe mitochondrial dysfunction.

Staphylococcus epidermidis: metabolic adaptation and biofilm formation in response to different oxygen concentrations.

Staphylococcus epidermidis has become a major health hazard. It is necessary to study its metabolism and hopefully uncover therapeutic targets. Cultivating S. epidermidis at increasing oxygen concentration [O2] enhanced growth, while inhibiting biofilm formation. Respiratory oxidoreductases were differentially expressed, probably to prevent reactive oxygen species formation. Under aerobiosis, S. epidermidis expressed high oxidoreductase activities, including glycerol-3-phosphate dehydrogenase, pyruvate dehydrogenase, ethanol dehydrogenase and succinate dehydrogenase, as well as cytochromes bo and aa3; while little tendency to form biofilms was observed. Under microaerobiosis, pyruvate dehydrogenase and ethanol dehydrogenase decreased while glycerol-3-phosphate dehydrogenase and succinate dehydrogenase nearly disappeared; cytochrome bo was present; anaerobic nitrate reductase activity was observed; biofilm formation increased slightly. Under anaerobiosis, biofilms grew; low ethanol dehydrogenase, pyruvate dehydrogenase and cytochrome bo were still present; nitrate dehydrogenase was the main terminal electron acceptor. KCN inhibited the aerobic respiratory chain and increased biofilm formation. In contrast, methylamine inhibited both nitrate reductase and biofilm formation. The correlation between the expression and/or activity or redox enzymes and biofilm-formation activities suggests that these are possible therapeutic targets to erradicate S. epidermidis.

Effects of ubiquinone derivatives on the mitochondrial unselective channel of Saccharomyces cerevisiae.

Ubiquinone derivatives modulate the mammalian mitochondrial Permeability Transition Pore (PTP). Yeast mitochondria harbor a similar structure: the respiration- and ATP-induced Saccharomyces cerevisiae Mitochondrial Unselective Channel ( Sc MUC). Here we show that decylubiquinone, a well-characterized inhibitor of the PTP, suppresses Sc MUC opening in diverse strains and independently of respiratory chain modulation or redox-state. We also found that naturally occurring derivatives such as hexaprenyl and decaprenyl ubiquinones lacked effects on the Sc MUC. The PTP-inactive ubiquinone 5 (Ub5) promoted the Sc MUC-independent activation of the respiratory chain in most strains tested. In an industrial strain however, Ub5 blocked the protection elicited by dUb. The results indicate the presence of a ubiquinone-binding site in the Sc MUC.

In Saccharomyces cerevisiae fructose-1,6-bisphosphate contributes to the Crabtree effect through closure of the mitochondrial unspecific channel.

In Saccharomyces cerevisiae addition of glucose inhibits oxygen consumption, i.e. S. cerevisiae is Crabtree-positive. During active glycolysis hexoses-phosphate accumulate, and probably interact with mitochondria. In an effort to understand the mechanism underlying the Crabtree effect, the effect of two glycolysis-derived hexoses-phosphate was tested on the S. cerevisiae mitochondrial unspecific channel (ScMUC). Glucose-6-phosphate (G6P) promoted partial opening of ScMUC, which led to proton leakage and uncoupling which in turn resulted in, accelerated oxygen consumption. In contrast, fructose-1,6-bisphosphate (F1,6BP) closed ScMUC and thus inhibited the rate of oxygen consumption. When added together, F1,6BP reverted the mild G6P-induced effects. F1,6BP is proposed to be an important modulator of ScMUC, whose closure contributes to the "Crabtree effect".

First evidence of mineralization of petroleum asphaltenes by a strain of Neosartorya fischeri.

A fungal strain isolated from a microbial consortium growing in a natural asphalt lake is able to grow in purified asphaltenes as the only source of carbon and energy. The asphaltenes were rigorously purified in order to avoid contamination from other petroleum fractions. In addition, most of petroporphyrins were removed. The 18S rRNA and ß-tubulin genomic sequences, as well as some morphologic characteristics, indicate that the isolate is Neosartorya fischeri. After 11 weeks of growth, the fungus is able to metabolize 15.5% of the asphaltenic carbon, including 13.2% transformed to CO(2) . In a medium containing asphaltenes as the sole source of carbon and energy, the fungal isolate produces extracellular laccase activity, which is not detected when the fungus grow in a rich medium. The results obtained in this work clearly demonstrate that there are microorganisms able to metabolize and mineralize asphaltenes, which is considered the most recalcitrant petroleum fraction.