Optimization of sperm concentration for in vitro production of mouse embryos

[EN] In vitro fertilization (IVF) is an important technique developed from assisted reproductive technologies (ARTs). Although it has been investigated since years, some optimizations should be done in order to determinate the ideal conditions for practicing this technique. In this work IVF was practiced using mice model. One million spermatozoa (sp.) per millilitre and two millions sp/mL were used in IVF in order to determinate an ideal concentration of spermatozoa for obtaining the highest fertilization rate, preimplantation developmental rate and the best blastocyst quality. Pronucleus were stained with DAPI to analyse the fertilization rate. Beside this, blastocyst were stained with DAPI after a hypotonic treatment was done to count the blastomeres for the determination of the blastocyst quality. Significant differences were not found so that using one or the other concentration we obtained similar results. However, using the lowest concentration may reduce the stress of the oocyte obtaining better result in a long-period time

Optimization of sperm concentration for in vitro production of mouse embryos

[EN] In vitro fertilization (IVF) is an important technique developed from assisted reproductive technologies (ARTs). Although it has been investigated since years, some optimizations should be done in order to determinate the ideal conditions for practicing this technique. In this work IVF was practiced using mice model. One million spermatozoa (sp.) per millilitre and two millions sp/mL were used in IVF in order to determinate an ideal concentration of spermatozoa for obtaining the highest fertilization rate, preimplantation developmental rate and the best blastocyst quality. Pronucleus were stained with DAPI to analyse the fertilization rate. Beside this, blastocyst were stained with DAPI after a hypotonic treatment was done to count the blastomeres for the determination of the blastocyst quality. Significant differences were not found so that using one or the other concentration we obtained similar results. However, using the lowest concentration may reduce the stress of the oocyte obtaining better result in a long-period time

Atomistic Insights of Calmodulin Gating of Complete Ion Channels

Intracellular calcium is essential for many physiological processes, from neuronal signaling and exocytosis to muscle contraction and bone formation. Ca2+ signaling from the extracellular medium depends both on membrane potential, especially controlled by ion channels selective to K+, and direct permeation of this cation through specialized channels. Calmodulin (CaM), through direct binding to these proteins, participates in setting the membrane potential and the overall permeability to Ca2+. Over the past years many structures of complete channels in complex with CaM at near atomic resolution have been resolved. In combination with mutagenesis-function, structural information of individual domains and functional studies, different mechanisms employed by CaM to control channel gating are starting to be understood at atomic detail. Here, new insights regarding four types of tetrameric channels with six transmembrane (6TM) architecture, Eag1, SK2/SK4, TRPV5/TRPV6 and KCNQ1–5, and its regulation by CaM are described structurally. Different CaM regions, N-lobe, C-lobe and EF3/EF4-linker play prominent signaling roles in different complexes, emerging the realization of crucial non-canonical interactions between CaM and its target that are only evidenced in the full-channel structure. Different mechanisms to control gating are used, including direct and indirect mechanical actuation over the pore, allosteric control, indirect effect through lipid binding, as well as direct plugging of the pore. Although each CaM lobe engages through apparently similar alpha-helices, they do so using different docking strategies. We discuss how this allows selective action of drugs with great therapeutic potential.The Government of the Autonomous Community of the Basque Country (IT1165–19) and the Spanish Ministry of Economy, Industry and Competitiveness (RTI2018–097839-B-100) provided financial support for this work. E.N. and A.M-M. were supported by predoctoral contracts of the Basque Government

Gure bihotzeko kalmodulina

Calcium is a universal signaling messenger that participates in essential processes such as apoptosis, cell proliferation and muscle contraction. Many of the proteins involved in these processes must interact with a calcium sensor to respond to the changes of concentration of this ion. The best-studied sensor is Calmodulin (CaM). It regulates the activity of hundreds of proteins through its N- and C- lobes, where two EF-hands are located to bind up to four calcium ions. Although the role of CaM in cell signaling is widespread, its mutations are especially recognized through its effects on cardiac function. In fact, disease-causing mutations in any of the three genes that encode the same CaM proteins cause severe cardiac dysfunction, indicating their importance in regulating excitability. Therefore, knowing the mechanisms involved in these diseases can allow a rational approach to clinical manifestations and contribute to the development of therapeutic strategies.; Kaltzioa seinalizazio unibertsaleko mezulari bat da, funtsezko prozesuetan parte hartzen duena, hala nola apoptosian, zelulen proliferazioan eta muskuluen uzkurduran. Prozesu horietan parte hartzen duten proteina askok ioi honen kontzentrazioari erantzuteko kaltzio sentsore batekin, kalmodulinarekin (CaM), elkarreragin behar dute. Horrek ehunka proteinaren aktibitatea erregulatzen du bere N- eta C-lobuluen bidez, non EF-eskuak aurkezten dituen Ca2+-ari lotzeko. Zelulen seinalizazioan CaM-k duen papera hedatuta dagoen arren, haren mutazioek bereziki bihotzari eragiten diote. Izan ere, CaM proteina berdinak kodetzen dituzten hiru geneetako edozeinetan gaixotasunak eragiten dituzten mutazioek bihotz-disfuntzio larriak eragiten dituzte, eta horrek kitzikagarritasunaren erregulazioan duen garrantzia adierazten du. Beraz, esku hartzen duten mekanismoen ezagutzak aukera eman dezake adierazpen klinikoei modu kritikoan heltzeko eta kalmodulinopatietarako estrategia terapeutikoak garatzen laguntzeko

Molecular dynamics simulations of the calmodulin-induced α-helix in the SK2 calcium-gated potassium ion channel

The family of small-conductance Ca2+-activated potassium ion channels (SK channels) is composed of four members (SK1, SK2, SK3, and SK4) involved in neuron-firing regulation. The gating of these channels depends on the intracellular Ca2+ concentration, and their sensitivity to this ion is provided by calmodulin (CaM). This protein binds to a specific region in SK channels known as the calmodulin-binding domain (CaMBD), an event which is essential for their gating. While CaMBDs are typically disordered in the absence of CaM, the SK2 channel subtype displays a small prefolded α-helical region in its CaMBD even if CaM is not present. This small helix is known to turn into a full α-helix upon CaM binding, although the molecular-level details for this conversion are not fully understood yet. In this work, we offer new insights on this physiologically relevant process by means of enhanced sampling, atomistic Hamiltonian replica exchange molecular dynamics simulations, providing a more detailed understanding of CaM binding to this target. Our results show that CaM is necessary for inducing a full α-helix along the SK2 CaMBD through hydrophobic interactions with V426 and L427. However, it is also necessary that W431 does not compete for these interactions; the role of the small prefolded α-helix in the SK2 CaMBD would be to stabilize W431 so that this is the case. In conclusion, our findings provide further insight into a key interaction between CaM and SK channels that is important for channel sensitivity to Ca2+.The authors thank Donostia International Physics Center (DIPC) for providing access to its computational resources. We acknowledge financial support from the Department of Education, Universities, and Research of the Basque Government and the University of the Basque Country (IT1165-19, KK-2020/00110, and IT1707-22), from the Spanish Ministry of Science and Innovation (projects PID2021-128286NB-100, PID2019-105488GB-I00, TED2021-132074B-C32, and RTI2018-097839-B-100) and from FEDER funds

The Crossroad of Ion Channels and Calmodulin in Disease

Calmodulin (CaM) is the principal Ca2+ sensor in eukaryotic cells, orchestrating the activity of hundreds of proteins. Disease causing mutations at any of the three genes that encode identical CaM proteins lead to major cardiac dysfunction, revealing the importance in the regulation of excitability. In turn, some mutations at the CaM binding site of ion channels cause similar diseases. Here we provide a summary of the two sides of the partnership between CaM and ion channels, describing the diversity of consequences of mutations at the complementary CaM binding domains.The Department of Industry, Tourism and Trade of the Government of the Autonomous Community of the Basque Country (Elkartek 2017 bG17 kk-2017/000843M50.17.EK.C6) and the Spanish Ministry of Economy, Industry and Competitiveness (BFU2015-66910 and RTI2018-097839) provided financial support for this work. E.N. is supported by a predoctoral grant of the Basque Government

Proteinen tolestura tunel erribosomikoan

Proteins are synthesised as linear polymers and must fold into their native three-dimensional structure to perform various functions in the cell. Understanding protein folding is crucial because protein misfolding is at the origin of several neurodegenerative diseases. Protein folding can start cotranslationally, i.e. when the emerging peptide is still asso-ciated with the ribosome. Indeed, it has been shown that more than one third of the cell’s proteins fold in the limited space of the ribosome tunnel. Increasing evidence suggests that the ribosome plays a critical role in protein folding. The ribosome can facilitate protein compaction, cause the creation of non-visible media in solution or delay the onset of folding. However, the study of cotranslational folding presents serious difficulties, mainly due to the limitations of the different current techniques. Hence, most studies on protein folding are based on proteins in solution, which are carried out by unfolding and refolding the protein, without taking into account the role of the ribosome in this process. In this article, we summarised the techniques developed in recent years for the study of cotranslational protein folding.; Proteinak polimero lineal gisa sintetizatzen dira eta beren jatorrizko egitura tridimentsionalean tolestu behar dira zelulan hainbat funtzio betetzeko. Proteinen tolespena ulertzea funtsezkoa da, tolespen okerrak hainbat gaixotasun neuro-degeneratiboren jatorria direlako. Proteinen tolespena modu koitzultzailean has daiteke, hau da, sortzen ari den peptidoa erribosomari lotuta dagoenean oraindik. Izan ere, zelularen proteinen heren bat baino gehiago erribosomaren tunelaren espazio mugatuan tolesten direla frogatu da, hau da, erribosomaren gainazalarekiko interakzioek modulatuta eta erribosoma-tunelaren beraren mugen pean. Gero eta ebidentzia gehiagok iradokitzen dute erribosomak funtsezko zeregina duela proteinen tolespenean. Erribosomak proteina trinkotzea erraztu dezake, soluzioan ikusten ez diren bitartekoak sortzea eragin dezake edo tolestearen hasiera atzeratu dezake. Hala ere, proteinen koitzulpenezko tolesdura aztertzeak zailtasun handiak ditu, batik bat, egungo teknikek dituzten mugengatik. Hori dela eta, proteinen tolesteari buruzko ikerketa gehienak soluzioan dauden proteinetan oinarritzen dira, proteina tolestuz eta destolestuz egiten direnak, prozesu horretan erribosomak duen rola kontuan hartu gabe. Artikulu honetan, azken urteotan proteinen koitzulpenezko tolestura ikertzeko garatu diren tekniken laburpena egin da

KV7.2 kanala: estruktura, erregulazioa eta kitzikagarritasun neuronalean duen ekintza

Potasio-kanalak ia zelula guztien mintzean agertzen dira eta funtzio biologiko garrantzitsuak betetzen dituzte; besteak beste, korronte elektrikoak kontrolatzen dituzte zelula kitzikagarrietan. KV7 kanalen familia 5 kidez osatuta dago (KV7.1-KV7.5), eta horiek kodetzen dituzten geneak patologia esanguratsuekin erlazionatzen dira. KV7 kanalen estrukturak zelula-mintzean txertaturiko 6 segmentuz osaturiko ohiko estruktura partekatzen du; N- eta C-muturrak zelula barnekoak dira. Neuronetan, KV7.2 eta KV7.3 kanalak agertzen dira batik bat; M-korrontea sortuz, neuronen kitzikagarritasuna kontrolatzen duena. M-korrontearen erregulazioa konplexua da seinaleztapen-bidezidor desberdinen bidez erregula baitaiteke. Gq/11 proteinari akoplaturiko hartzaileen bidez erregulatzen da eta seinaleztapen-bidezidorra desberdina da aktibatutako hartzailearen arabera. Horrela, azetilkolinaren M1 hartzaile muskarinikoak KV7.2-aren korrontea inhibituko du PIP2-aren agorpenaren ondorioz. Bradikininaren hartzaileak, ordea, IP3-ak eragindako kaltzio-kontzentrazioaren igoeraren bidez inhibituko du. Mekanismo horietan, hainbat proteinak hartzen dute parte, hala nola kalmodulinak, proteina kinasek eta ainguratze-proteinek. Berrikuspen honetan, KV7.2 kanalari erreparatuko diogu, hainbat gaixotasunen partaide izateagatik eta haren erregulazio konplexuagatik, ikuspuntu farmakologiko batetik itu interesgarria izan baitaiteke.; Potassium channels are present in almost all cell membranes and perform important biological functions, including electrical currents control in excitable cells. The KV7 channels familiy consists of 5 members (KV7.1-KV7.5) and the genes that encode them are related to significant pathologies. The structure of KV7 channels shares the usual six transmem-brane segment structure, with intracellular N- and C-termini. In neurons, KV7.2 and KV7.3 are the main channels, which generate the M-current that controls neuronal excitability. The M-current regulation is complex as it can be regulated by different signalling pathways. It is regulated by Gq/11-coupled receptors, and the signaling pathway depends on the activated receptor. Thus, the M1 muscarinic acetylcholine receptor inhibits KV7.2 current by PIP2 depletion. While the bradykinin receptor inhibits it through the calcium concentration increase driven by IP3. Among these mechanisms several proteins are involved, such as calmodulin, protein kinases and anchor proteins. In this review we will focus on KV7.2 channel, as it is involved in several diseases and for its complex regulation it can be an interesting target from a pharmacological point of view

N-aryltetrahydroisoquinoline derivatives as HA-CD44 interaction inhibitors: Design, synthesis, computational studies, and antitumor effect

Hyaluronic acid (HA) plays a crucial role in tumor growth and invasion through its interaction with cluster of differentiation 44 (CD44), a non-kinase transmembrane glycoprotein, among other hyaladherins. CD44 expression is elevated in many solid tumors, and its interaction with HA is associated with cancer and angiogenesis. Despite efforts to inhibit HA-CD44 interaction, there has been limited progress in the development of small molecule inhibitors. As a contribution to this endeavour, we designed and synthesized a series of N-aryltetrahydroisoquinoline derivatives based on existing crystallographic data available for CD44 and HA. Hit 2e was identified within these structures for its antiproliferative effect against two CD44+ cancer cell lines, and two new analogs (5 and 6) were then synthesized and evaluated as CD44-HA inhibitors by applying computational and cell-based CD44 binding studies. Compound 2-(3,4,5-trimethoxybenzyl)-1,2,3,4-tetrahydroisoquinolin-5-ol (5) has an EC50 value of 0.59 μM against MDA-MB-231 cells and is effective to disrupt the integrity of cancer spheroids and reduce the viability of MDA-MB-231 cells in a dose-dependent manner. These results suggest lead 5 as a promising candidate for further investigation in cancer treatment

Redox regulation of KV7 channels through EF3 hand of calmodulin

Neuronal KV7 channels, important regulators of cell excitability, are among the most sensitive proteins to reactive oxygen species. The S2S3 linker of the voltage sensor was reported as a site-mediating redox modulation of the channels. Recent structural insights reveal potential interactions between this linker and the Ca2+-binding loop of the third EF-hand of calmodulin (CaM), which embraces an antiparallel fork formed by the C-terminal helices A and B, constituting the calcium responsive domain (CRD). We found that precluding Ca2+ binding to the EF3 hand, but not to EF1, EF2, or EF4 hands, abolishes oxidation-induced enhancement of KV7.4 currents. Monitoring FRET (Fluorescence Resonance Energy Transfer) between helices A and B using purified CRDs tagged with fluorescent proteins, we observed that S2S3 peptides cause a reversal of the signal in the presence of Ca2+ but have no effect in the absence of this cation or if the peptide is oxidized. The capacity of loading EF3 with Ca2+ is essential for this reversal of the FRET signal, whereas the consequences of obliterating Ca2+ binding to EF1, EF2, or EF4 are negligible. Furthermore, we show that EF3 is critical for translating Ca2+ signals to reorient the AB fork. Our data are consistent with the proposal that oxidation of cysteine residues in the S2S3 loop relieves KV7 channels from a constitutive inhibition imposed by interactions between the EF3 hand of CaM which is crucial for this signaling.Ministerio de Ciencia e Innovacion PID2021-128286NB-100Wellcome Trust 212302/Z/18/ZMedical Research Centre MR/P015727/1Eusko Jaurlaritza IT1707-22 Ekonomiaren Garapen eta Lehiakortasun Saila, Eusko Jaurlaritza BG2019Ministerio de Ciencia e Innovacion RTI2018-097839-B-100Ministerio de Ciencia e Innovacion RTI2018-101269-B-I00Eusko Jaurlaritza IT1165-19 Ekonomiaren Garapen eta Lehiakortasun Saila,Eusko Jaurlaritza KK-2020/00110Eusko Jaurlaritza PRE_2018-2_0082Eusko Jaurlaritza POS_2021_1_0017Eusko Jaurlaritza PRE_2018-2_012