Chemo-small extracellular vesicles released in cisplatin-resistance ovarian cancer cells are regulated by the lysosomal function.

Chemoresistance is a common problem in ovarian cancer (OvCa) treatment, where resistant cells, in response to chemotherapy, secrete small extracellular vesicles (sEVs), known as chemo-sEVs, that transfer resistance to recipient cells. sEVs are formed as intraluminal vesicles (ILVs) within multivesicular endosomes (MVEs), whose trafficking is regulated by Ras-associated binding (RAB) GTPases that mediate sEVs secretion or lysosomal degradation. A decrease in lysosomal function can promote sEVs secretion, but the relationship between MVEs trafficking pathways and sEVs secretion in OvCa chemoresistance is unclear. Here, we show that A2780cis cisplatin (CCDP) resistant OvCa cells had an increased number of MVEs and ILVs structures, higher levels of Endosomal Sorting Complex Required for Transport (ESCRTs) machinery components, and RAB27A compared to A2780 CDDP-sensitive OvCa cells. CDDP promoted the secretion of chemo-sEVs in A2780cis cells, enriched in DNA damage response proteins. A2780cis cells exhibited poor lysosomal function with reduced levels of RAB7, essential in MVEs-Lysosomal trafficking. The silencing of RAB27A in A2780cis cells prevents the Chemo-EVs secretion, reduces its chemoresistance and restores lysosomal function and levels of RAB7, switching them into an A2780-like cellular phenotype. Enhancing lysosomal function with rapamycin reduced chemo-sEVs secretion. Our results suggest that adjusting the balance between secretory MVEs and lysosomal MVEs trafficking could be a promising strategy for overcoming CDDP chemoresistance in OvCa.

Tumor-derived hypoxic small extracellular vesicles promote endothelial cell migration and tube formation via ALS2/Rab5/ß-catenin signaling.

Tumor hypoxia has been associated with cancer progression, angiogenesis, and metastasis via modifications in the release and cargo composition of extracellular vesicles secreted by tumor cells. Indeed, hypoxic extracellular vesicles are known to trigger a variety of angiogenic responses via different mechanisms. We recently showed that hypoxia promotes endosomal signaling in tumor cells via HIF-1α-dependent induction of the guanine exchange factor ALS2, which activates Rab5, leading to downstream events involved in cell migration and invasion. Since Rab5-dependent signaling is required for endothelial cell migration and angiogenesis, we explored the possibility that hypoxia promotes the release of small extracellular vesicles containing ALS2, which in turn activate Rab5 in recipient endothelial cells leading to pro-angiogenic properties. In doing so, we found that hypoxia promoted ALS2 expression and incorporation as cargo within small extracellular vesicles, leading to subsequent transfer to recipient endothelial cells and promoting cell migration, tube formation, and downstream Rab5 activation. Consequently, ALS2-containing small extracellular vesicles increased early endosome size and number in recipient endothelial cells, which was followed by subsequent sequestration of components of the ß-catenin destruction complex within endosomal compartments, leading to stabilization and nuclear localization of ß-catenin. These events converged in the expression of ß-catenin target genes involved in angiogenesis. Knockdown of ALS2 in donor tumor cells precluded its incorporation into small extracellular vesicles, preventing Rab5-downstream events and endothelial cell responses, which depended on Rab5 activity and guanine exchange factor activity of ALS2. These findings indicate that vesicular ALS2, secreted in hypoxia, promotes endothelial cell events leading to angiogenesis. Finally, these events might explain how tumor angiogenesis proceeds in hypoxic conditions.

The Immunoregulatory and Regenerative Potential of Activated Human Stem Cell Secretome Mitigates Acute-on-Chronic Liver Failure in a Rat Model.

Acute-on-chronic liver failure (ACLF) is a syndrome marked by sudden liver function decline and multiorgan failure, predominantly acute kidney injury (AKY), in patients with chronic liver disease. Unregulated inflammation is a hallmark of ACLF; however, the key drivers of ACLF are not fully understood. This study explores the therapeutic properties of human mesenchymal stem cell (MSC) secretome, particularly focusing on its enhanced anti-inflammatory and pro-regenerative properties after the in vitro preconditioning of the cells. We evaluated the efficacy of the systemic administration of MSC secretome in preventing liver failure and AKI in a rat ACLF model where chronic liver disease was induced using by the administration of porcine serum, followed by D-galN/LPS administration to induce acute failure. After ACLF induction, animals were treated with saline (ACLF group) or MSC-derived secretome (ACLF-secretome group). The study revealed that MSC-secretome administration strongly reduced liver histological damage in the ACLF group, which was correlated with higher hepatocyte proliferation, increased hepatic and systemic anti-inflammatory molecule levels, and reduced neutrophil and macrophage infiltration. Additionally, renal examination revealed that MSC-secretome treatment mitigated tubular injuries, reduced apoptosis, and downregulated injury markers. These improvements were linked to increased survival rates in the ACLF-secretome group, endorsing MSC secretomes as a promising therapy for multiorgan failure in ACLF.

Connexin46 Expression Enhances Cancer Stem Cell and Epithelial-to-Mesenchymal Transition Characteristics of Human Breast Cancer MCF-7 Cells.

Connexins (Cxs) are a family of proteins that form two different types of ion channels: hemichannels and gap junction channels. These channels participate in cellular communication, enabling them to share information and act as a synchronized syncytium. This cellular communication has been considered a strong tumor suppressor, but it is now recognized that some type of Cxs can be pro-tumorigenic. For example, Cx46 expression is increased in human breast cancer samples and correlates with cancer stem cell (CSC) characteristics in human glioma. Thus, we explored whether Cx46 and glioma cells, can set up CSC and epithelial-to-mesenchymal transition (EMT) properties in a breast cancer cell line. To this end, we transfected MCF-7 cells with Cx46 attached to a green fluorescent protein (Cx46GFP), and we determined how its expression orchestrates both the gene-expression and functional changes associated with CSC and EMT. We observed that Cx46GFP increased Sox2, Nanog, and OCT4 mRNA levels associated with a high capacity to form monoclonal colonies and tumorspheres. Similarly, Cx46GFP increased the mRNA levels of n-cadherin, Vimentin, Snail and Zeb1 to a higher migratory and invasive capacity. Furthermore, Cx46GFP transfected in MCF-7 cells induced the release of higher amounts of VEGF, which promoted angiogenesis in HUVEC cells. We demonstrated for the first time that Cx46 modulates CSC and EMT properties in breast cancer cells and thus could be relevant in the design of future cancer therapies.

Connexin-46 Contained in Extracellular Vesicles Enhance Malignancy Features in Breast Cancer Cells.

Under normal conditions, almost all cell types communicate with their neighboring cells through gap junction channels (GJC), facilitating cellular and tissue homeostasis. A GJC is formed by the interaction of two hemichannels; each one of these hemichannels in turn is formed by six subunits of transmembrane proteins called connexins (Cx). For many years, it was believed that the loss of GJC-mediated intercellular communication was a hallmark in cancer development. However, nowadays this paradigm is changing. The connexin 46 (Cx46), which is almost exclusively expressed in the eye lens, is upregulated in human breast cancer, and is correlated with tumor growth in a Xenograft mouse model. On the other hand, extracellular vesicles (EVs) have an important role in long-distance communication under physiological conditions. In the last decade, EVs also have been recognized as key players in cancer aggressiveness. The aim of this work was to explore the involvement of Cx46 in EV-mediated intercellular communication. Here, we demonstrated for the first time, that Cx46 is contained in EVs released from breast cancer cells overexpressing Cx46 (EVs-Cx46). This EV-Cx46 facilitates the interaction between EVs and the recipient cell resulting in an increase in their migration and invasion properties. Our results suggest that EV-Cx46 could be a marker of cancer malignancy and open the possibility to consider Cx46 as a new therapeutic target in cancer treatment.

Small Extracellular Vesicles Released from Ovarian Cancer Spheroids in Response to Cisplatin Promote the Pro-Tumorigenic Activity of Mesenchymal Stem Cells.

Despite the different strategies used to treat ovarian cancer, around 70% of women/patients eventually fail to respond to the therapy. Cancer stem cells (CSCs) play a role in the treatment failure due to their chemoresistant properties. This capacity to resist chemotherapy allows CSCs to interact with different components of the tumor microenvironment, such as mesenchymal stem cells (MSCs), and thus contribute to tumorigenic processes. Although the participation of MSCs in tumor progression is well understood, it remains unclear how CSCs induce the pro-tumorigenic activity of MSCs in response to chemotherapy. Small extracellular vesicles, including exosomes, represent one possible way to modulate any type of cell. Therefore, in this study, we evaluate if small extracellular vesicle (sEV) derived from ovarian cancer spheroids (OCS), which are enriched in CSCs, can modify the activity of MSCs to a pro-tumorigenic phenotype. We show that sEV released by OCS in response to cisplatin induce an increase in the migration pattern of bone marrow MSCs (BM-MSCs) and the secretion interleukin-6 (IL-6), interleukin-8 (IL-8), and vascular endothelial growth factor A (VEGFA). Moreover, the factors secreted by BM-MSCs induce angiogenesis in endothelial cells and the migration of low-invasive ovarian cancer cells. These findings suggest that cisplatin could modulate the cargo of sEV released by CSCs, and these exosomes can further induce the pro-tumorigenic activity of MSCs.

Regulation of Connexin-Based Channels by Fatty Acids.

In this mini-review, we briefly summarize the current knowledge about the effects of fatty acids (FAs) on connexin-based channels, as well as discuss the limited information about the impact FAs may have on pannexins (Panxs). FAs regulate diverse cellular functions, some of which are explained by changes in the activity of channels constituted by connexins (Cxs) or Panxs, which are known to play critical roles in maintaining the functional integrity of diverse organs and tissues. Cxs are transmembrane proteins that oligomerize into hexamers to form hemichannels (HCs), which in turn can assemble into dodecamers to form gap junction channels (GJCs). While GJCs communicate the cytoplasm of contacting cells, HCs serve as pathways for the exchange of ions and small molecules between the intra and extracellular milieu. Panxs, as well as Cx HCs, form channels at the plasma membrane that enable the interchange of molecules between the intra and extracellular spaces. Both Cx- and Panx-based channels are controlled by several post-translational modifications. However, the mechanism of action of FAs on these channels has not been described in detail. It has been shown however that FAs frequently decrease GJC-mediated cell-cell communication. The opposite effect also has been described for HC or Panx-dependent intercellular communication, where, the acute FA effect can be reversed upon washout. Additionally, changes in GJCs mediated by FAs have been associated with post-translational modifications (e.g., phosphorylation), and seem to be directly related to chemical properties of FAs (e.g., length of carbon chain and/or degree of saturation), but this possible link remains poorly understood.

Inhibition of the Hantavirus Fusion Process by Predicted Domain III and Stem Peptides from Glycoprotein Gc.

Hantaviruses can cause hantavirus pulmonary syndrome or hemorrhagic fever with renal syndrome in humans. To enter cells, hantaviruses fuse their envelope membrane with host cell membranes. Previously, we have shown that the Gc envelope glycoprotein is the viral fusion protein sharing characteristics with class II fusion proteins. The ectodomain of class II fusion proteins is composed of three domains connected by a stem region to a transmembrane anchor in the viral envelope. These fusion proteins can be inhibited through exogenous fusion protein fragments spanning domain III (DIII) and the stem region. Such fragments are thought to interact with the core of the fusion protein trimer during the transition from its pre-fusion to its post-fusion conformation. Based on our previous homology model structure for Gc from Andes hantavirus (ANDV), here we predicted and generated recombinant DIII and stem peptides to test whether these fragments inhibit hantavirus membrane fusion and cell entry. Recombinant ANDV DIII was soluble, presented disulfide bridges and beta-sheet secondary structure, supporting the in silico model. Using DIII and the C-terminal part of the stem region, the infection of cells by ANDV was blocked up to 60% when fusion of ANDV occurred within the endosomal route, and up to 95% when fusion occurred with the plasma membrane. Furthermore, the fragments impaired ANDV glycoprotein-mediated cell-cell fusion, and cross-inhibited the fusion mediated by the glycoproteins from Puumala virus (PUUV). The Gc fragments interfered in ANDV cell entry by preventing membrane hemifusion and pore formation, retaining Gc in a non-resistant homotrimer stage, as described for DIII and stem peptide inhibitors of class II fusion proteins. Collectively, our results demonstrate that hantavirus Gc shares not only structural, but also mechanistic similarity with class II viral fusion proteins, and will hopefully help in developing novel therapeutic strategies against hantaviruses.

Acidification triggers Andes hantavirus membrane fusion and rearrangement of Gc into a stable post-fusion homotrimer.

The hantavirus membrane fusion process is mediated by the Gc envelope glycoprotein from within endosomes. However, little is known about the specific mechanism that triggers Gc fusion activation, and its pre- and post-fusion conformations. We established cell-free in vitro systems to characterize hantavirus fusion activation. Low pH was sufficient to trigger the interaction of virus-like particles with liposomes. This interaction was dependent on a pre-fusion glycoprotein arrangement. Further, low pH induced Gc multimerization changes leading to non-reversible Gc homotrimers. These trimers were resistant to detergent, heat and protease digestion, suggesting characteristics of a stable post-fusion structure. No acid-dependent oligomerization rearrangement was detected for the trypsin-sensitive Gn envelope glycoprotein. Finally, acidification induced fusion of glycoprotein-expressing effector cells with non-susceptible CHO cells. Together, the data provide novel information on the Gc fusion trigger and its non-reversible activation involving lipid interaction, multimerization changes and membrane fusion which ultimately allow hantavirus entry into cells.

Hantavirus Gn and Gc glycoproteins self-assemble into virus-like particles.

How hantaviruses assemble and exit infected cells remains largely unknown. Here, we show that the expression of Andes (ANDV) and Puumala (PUUV) hantavirus Gn and Gc envelope glycoproteins lead to their self-assembly into virus-like particles (VLPs) which were released to cell supernatants. The viral nucleoprotein was not required for particle formation. Further, a Gc endodomain deletion mutant did not abrogate VLP formation. The VLPs were pleomorphic, exposed protrusions and reacted with patient sera.

Entrenamiento pliométrico sobre el índice elástico en niños no deportistas / Plyometric training on the elastic index of not athlete children

En el presente estudio se aplicó un entrenamiento pliométrico adaptado a un grupo de niños (n=15) no deportistas (12,8±0,8 años; 1,56±0,09 M; 48,7±8.5 Kg.), durante un período de seis semanas de entrenamiento (dos veces por semana). El objetivo fue demostrar si es posible aumentar la potencia (P) de extremidades inferiores mediante el aumento del índice elástico (IE). Se midieron los saltos Squat Jump (SJ), y Countermovement Jump (CMJ) y se calculó P e IE. Los resultados del pre-entrenamiento (SJ=26,51±5,1 cm; CMJ=28,52±6,5 cm; P=1834±853,04 W; IE 8,17±3,7 %) y post-entrenamiento (SJ=28,46±6,2 cm; CMJ=32,07±6,9 cm; P=2018±871,39 W; IE=13,23±1,9 %) indican que se aumentó significativamente la variable IE (p0,05). Los resultados obtenidos demuestran que este tipo de entrenamiento pliométrico puede estimular el componente elástico y neuromuscular en desmedro del componente contráctil, al obtenerse una ganancia relativa de potencia, situación preponderante en la etapa dedesarrollo de los niños.

This study aims to determine the current state of the relation between these variables, in studentsof the Rafael J. Mejia School, in Sabaneta. A stratified simple of 207 students (99 noys, 44%108 girls, 56%) was randomly selected from a total of 455 students. We applied measurementprocedures and evaluation proposed by J uregui and Ordo¤ez (1993). The results showed that aconsiderable proportion of the sample was above the 75th percentile for anthropometric tests andskinfolds; below the 25th percentile in physical abilities. This suggests that there is a tendency inthese children to obesity and physical inactivity.

Critical role of the first transmembrane domain of Cx26 in regulating oligomerization and function.

To identify motifs involved in oligomerization of the gap junction protein Cx26, we studied individual transmembrane (TM) domains and the full-length protein. Using the TOXCAT assay for interactions of isolated TM α-helices, we found that TM1, a Cx26 pore domain, had a strong propensity to homodimerize. We identified amino acids Val-37-Ala-40 (VVAA) as the TM1 motif required for homodimerization. Two deafness-associated Cx26 mutations localized in this region, Cx26V37I and Cx26A40G, differentially affected dimerization. TM1-V37I dimerized only weakly, whereas TM1-A40G did not dimerize. When the full-length mutants were expressed in HeLa cells, both Cx26V37I and Cx26A40G formed oligomers less efficiently than wild-type Cx26. A Cx26 cysteine substitution mutant, Cx26V37C formed dithiothreitol-sensitive dimers. Substitution mutants of Val-37 formed intercellular channels with reduced function, while mutants of Ala-40 did not form functional gap junction channels. Unlike wild-type Cx26, neither Cx26V37I nor Cx26A40G formed functional hemichannels in low extracellular calcium. Thus the VVAA motif of Cx26 is critical for TM1 dimerization, hexamer formation, and channel function. The differential effects of VVAA mutants on hemichannels and gap junction channels imply that inter-TM interactions can differ in unapposed and docked hemichannels. Moreover, Cx26 oligomerization appears dependent on transient TM1 dimerization as an intermediate step.

Different domains are critical for oligomerization compatibility of different connexins.

Oligomerization of connexins is a critical step in gap junction channel formation. Some members of the connexin family can oligomerize with other members and form functional heteromeric hemichannels [e.g. Cx43 (connexin 43) and Cx45], but others are incompatible (e.g. Cx43 and Cx26). To find connexin domains important for oligomerization, we constructed chimaeras between Cx43 and Cx26 and studied their ability to oligomerize with wild-type Cx43, Cx45 or Cx26. HeLa cells co-expressing Cx43, Cx45 or Cx26 and individual chimaeric constructs were analysed for interactions between the chimaeras and the wild-type connexins using cell biological (subcellular localization by immunofluorescence), functional (intercellular diffusion of microinjected Lucifer yellow) and biochemical (sedimentation velocity through sucrose gradients) assays. All of the chimaeras containing the third transmembrane domain of Cx43 interacted with wild-type Cx43 on the basis of co-localization, dominant-negative inhibition of intercellular communication, and altered sedimentation velocity. The same chimaeras also interacted with co-expressed Cx45. In contrast, immunofluorescence and intracellular diffusion of tracer suggested that other domains influenced oligomerization compatibility when chimaeras were co-expressed with Cx26. Taken together, these results suggest that amino acids in the third transmembrane domain are critical for oligomerization with Cx43 and Cx45. However, motifs in different domains may determine oligomerization compatibility in members of different connexin subfamilies.

Gap-junction channels dysfunction in deafness and hearing loss.

Gap-junction channels connect the cytoplasm of adjacent cells, allowing the diffusion of ions and small metabolites. They are formed at the appositional plasma membranes by a family of related proteins named connexins. Mutations in connexins 26, 31, 30, 32, and 43 have been associated with nonsyndromic or syndromic deafness. The majority of these mutations are inherited in an autosomal recessive manner, but a few of them have been associated with dominantly inherited hearing loss. Mutations in the connexin26 gene (GJB2) are the most common cause of genetic deafness. This review summarizes the most relevant and recent information about different mutations in connexin genes found in human patients, with emphasis on GJB2. The possible effects of the mutations on channel expression and function are discussed, in addition to their possible physiologic consequences for inner ear physiology. Finally, we propose that connexin channels (gap junctions and hemichannels) may be targets for age-related hearing loss induced by oxidative damage.

Environmental factors and management practices controlling oxygen dynamics in agricultural irrigation ponds in a semiarid Mediterranean region: implications for pond agricultural functions.

A water quality study was carried out on 40 irrigation ponds located within the main greenhouse areas on the Almería coast, placing special emphasis on the factors controlling the oxygen dynamics, a relevant aspect with agricultural and environmental implications. Considering chemical, physical and biological water characteristics, agricultural irrigation ponds were satisfactorily classified by cluster analysis in four groups. These were congruently arranged by principal components analysis along four main environmental gradients: trophic status, photosynthetic activity, water mineralisation and presence of submerged aquatic vegetation (SAV). Dissolved oxygen (DO) values differed highly among and within each of the four pond groups. DO dynamics was mainly depended on photosynthetic activity, and the environmental factors and management practices controlling it: seasonal and daily climatic changes, pond management (open vs. covered ponds and presence/absence of aquatic vegetation) and trophic status. Overall, different diurnal DO patterns were found between open and covered ponds. The former usually presented DO values above saturation and increasingly higher from early morning to mid-afternoon due to the photosynthetic activity of algae and macrophytic vegetation. In contrast, covered ponds showed relatively stable DO values during the diurnal period regardless of climatic conditions, with absolute values around or below saturation level. Globally, our results suggest that open ponds, with macrophytes concentrated in the deeper layer, can be an effective and sustainable management method of water oxygen enrichment.