Premanifolds

The tangent hyperplanes of the "manifolds" of this paper equipped a so-called Minkowski product. It is neither symmetric nor bilinear. We give a method to handing such an object as a locally hypersurface of a generalized space-time model and define the main tools of its differential geometry: its fundamental forms, its curvatures and so on. In the case, when the fixed space-time component of the embedding structure is a continuously differentiable semi-inner product space, we get a natural generalization of some important semi-Riemann manifolds as the hyperbolic space, the de Sitter sphere and the light cone of a Minkowski-Lorenz space, respectively

Progress, challenges and perspectives on fish gamete cryopreservation: A mini-review

Protocols for the cryopreservation of fish gametes have been developed for many different fish species, in special, freshwater salmonids and cyprinids. Methods for sperm freezing have progressed during the last decades due to the increasing number of potential applications: aquaculture (genetic improvement programs, broodstock management, helping with species having reproductive problems), biotechnology studies using model fish species (preservation of transgenic or mutant lines), cryobanking of genetic resources from endangered species, etc. This mini-review tries to give an overview of the present situation of this area of research, identifying the main challenges and perspectives, redirecting the reader to more in-depth reviews and papers. (C) 2016 Elsevier Inc. All rights reserved.European Training Network IMPRESS (Marie Sklodowska-Curie Actions) from PROMAR programme [642893]COST Office (Food and Agriculture COST Action FA1205: AQUAGAMETE) from PROMAR programmeResearch Centre of Excellence from PROMAR programme [9878-3/2016/FEKUT, KMR_12-1-2012-0436]NKFIH (OTKA) from PROMAR programme [109847]KLING from PROMAR programme [31-03-05FEP-73]CRIOBIV from PROMAR programme [31-03-05-FEP-59]REPLING from PROMAR programme [31-03-05-FEP-69]info:eu-repo/semantics/publishedVersio

Transplantacija spermatogonija kao nova metoda u akvakulturi i konzervaciji riba

Poslednjih godina, primena primordijalnih germinativnih ćelija (primordial germ cells - PGCs) i spermatogonijalnih stem ćelija (spermatogonial stem cells - SSCs) riba je postala veoma značajna zbog razvoja metode transplantacije ovih ćelija. Od kada su Brinster i Avarbock (1994) razvili ovu metodu, ona se uspešno koristi za čuvanje genetskog materijala ugroženih vrsta i u stvaranju novih transgenih linija kod miševa i domaćih životinja. Uvođenje ove metode kod riba predstavlja značajan napredak u oblasti reproduktivne biotehnologije, akvakulture, konzervacione biologije, kao i u razvoju novih transgenih linija različitih vrsta riba. Osnovu ove metode predstavlja transplantacija germinativnih ćelija (PGC, SSC) iz donorskog organizma u organizam primaoca. Najinteresantnija u tom smislu je upotreba nediferenciranih spermatogonija A tipa (Aund) koje imaju sposobnost samoobnavljanja ali i proizvodnje ćelija kasnijih faza spermatogeneze. Postoji takođe nekoliko specifičnih osobina SSC koje ih čine pogodnim za transplantaciju: (1) sposobnost da kolonizuju testis primaoca odakle produkuju donorsku spermu, (2) mogućnost da se nakon transplantacije u primaocu muškog pola razviju u spermatogonije, a u primaocu ženskog pola u oogonije i (3) mogućnost genske manipulacije sa ciljem produkcije transgenih riba (Lacerda i sar., 2010). Prilikom transplantacije SSC, posebna pažnja mora biti usmerena ka izboru vrste donora i primaoca. Najbolje bi bilo da donor i primalac ne budu filogenetski previše udaljeni, kao i da primalac ima kratak reproduktivni ciklus i manje dimenzije tela kako bi ekonomski bio pogodniji za gajenje. Donorska vrsta je obično vrsta za koju postoji određneni interes, bilo ekonomski, naučni ili konzervacioni. Tokom transplantacije, kompatibilnost između primaoca i donora može biti ograničavajući faktor u uspehu samog procesa. U najgorem slučaju, primalac, usled imune reakcije, može u potpunosti odbaciti transplantirano tkivo ili ćelije. To je najčešće slučaj ukoliko se vrši transplantacija SSC iz odraslog donora u odraslog primaoca. Kako bi se izbegao problem izazvan transplantacijom između dve odrasle jedinke, koristi se prednost ontogenije primaoca, posebno ontogenije njegovog imunog sistema, tako što se za primaoca koriste embrioni ili larve. Ovi stadijumi kod riba nemaju razvijen imuni sistem niti diferencirane T-ćelije (Takeuchi et al., 2003; Yoshizaki et al., 2011) te s toga nemaju mehanizam pomoću kojeg bi odbacili donorsko tkivo. Takođe, lakše je blokirati razvoj endogenih primordijalnih germinativnih ćelija kod larvi, nego ukloniti SSC iz već razvijenih gonada kod odraslog donora. Pored odabira odgovarajuće vrste donora i primaoca, neophodno je na pravi način izolovati specifične ćelije koje treba da budu transplantirane. Ovaj proces je donekle jednostavniji kada je u pitanju tansplantacija PGC s obzirom na njihov daleko manji broj u odnosu na spermatogonije i na to da one još uvek nisu potpuno razvijene u gonadama. S druge strane, SSC su dobro razvijene u gonadama i najčešće zauzimaju karakteristično mesto unutar pojedinačnih niša u testisu specifičnih za tu vrstu ćelija. Prilikom izolacije nediferenciranih spermatogonija A tipa iz testisa odrasle jedinke, veome je bitno voditi računa od morfologiji tih ćelija kao i specifičnim markerima pomoću kojih ih je moguće razlikovati od ostalih tipova spermatogonija (Adiff, B), spermatocita i spermatida. Osnovne histološke metode u kombinaciji sa imunohistohemijom, in situ hibridizacijom ili in situ PCR metodom se mogu koristiti za identifikaciju spefifičnih molekularnih markera (proteina ili RNK) u ćelijama unutar ćelijskih niša i koji se u daljem toku rada mogu koristiti za izolaciju određenih ćelija. Pre transplantacije PGC ili SSC, neophodno je izolovati željene ćelije iz donorskog tkiva. Nakon multienzimske razgradnje tkiva testisa, ćelije se izoluju na osnovu njihove morfologije i veličine i/ili specifičnih molekularnih markera zbog kojih čitav proces može biti species-specifičan. Kombinacija transplantacije PGS i SSC sa krioprezervacijom daje dodatni značaj ovoj metodi s obzirom da još uvek ne postoji optimizovan protokol za uspešnu krioprezervaciju jaja i embriona riba, pre svega zbog prisustva velike količine žumanceta i masti. Krioprezervacija ćelija kao što su PGS i SSC, koje imaju mogućnost da produkuju spermatozoide ili oocite u zavisnosti od pola jedinke primaoca, ima izuzetno veliku perspektivu primene u konzervacionoj biologiji i akvakulturi. Istraživanja su pokazala da krioprezervirane SSC nakon odmrzavanja i transplantacije u telo primaoca mogu proizvesti spermatozoide i oocite donorske vrste (Kobayashi et al., 2007). Na taj način, čuvanje gameta nije neophodno jer krioprezervacijom germinativnih ćelija i njihovom transplantacijom, moguće je dobiti gamete oba pola.In recent years, the importance of manipulations of primordial germ cells (PGCs) and spermatogonial stem cells (SSCs) in fish has drastically increased due to development of transplantation method of these cells. Since its development by Brinster and Avarbock (1994), this method has been successfully used in the preservation of genetic material of endangered species and in the creation of new transgenic lines of mice and farm animals. Introduction of this method in fish leads to advances in reproductive biotechnology, aquaculture, development of new transgenic lines and conservation biology of fish. The base of this method lies in the transplantation of the germinative cells (PGCs, SSCs) from donor organism into recipient organism. Undifferentiated spermatogonia type A (Aund) which have the ability of self-renewal are the most interesting for transplantation since they have the ability of self-renewal, but can also produce later stage cells. There are several advantages of using SSCs in transplantation process: (1) the capability of SSCs to colonize the testis of the recipients where they are able to produce donor-derived sperm, (2) plasticity in development since SSCs can develop into spermatogonia in male recipients and oogonia in female recipients and (3) the possibility of genetic manipulation in SSCs in order to produce transgenic fish (Lacerda et al., 2010). When transplanting SSCs, special attention must be given to the choice of donors and recipients species. It is best that donor and recipient organisms are phylogenetically not too distant, that recipient organisms have a short reproductive cycle and that they are small for a more economic rearing. Donor species are usually species which attract certain interest, whether its an economic, scientific or conservation interest. During transplantation, compatibility between recipient and donor may be a very limiting factor in transplantation success. In the worst-case scenario, recipients may completely reject the transplanted tissue or cells due to immunological reaction. This is especially the case when transplanting SSCs isolated from adult donors into adult recipients. In order to evade the problems caused by adult-adult transplantations, scientists have taken advantage of the ontogeny of recipients, mainly the ontogeny of their immune system, and used embryos and larvae as recipients. Embryos and larvae do not have a developed immune system nor differentiated T-cells (Takeuchi et al., 2003; Yoshizaki et al., 2011), therefore they do not have mechanisms to reject the donor tissue. Furthermore, it is easier to knock-out larval endogenous PGCs than to deplete SSCs from already developed gonad. Apart from choosing the right donor and recipient organisms, it is necessary to isolate specific cells that need to be transplanted. This is to some extent easier when transplanting PGCs, since there are fewer of them than SSCs, and they have not yet fully developed inside the gonads. On the other hand, SSCs are well developed inside the gonads and usually take their specific place within the spermatogonial stem niche. When isolating undifferentiated spermatogonia type A from adult testis, special attention must be given to their morphology and specific markers that distinguish them from other types of spermatogonia (Adiff, B), spermatocytes and spermatids. Basic histology may be coupled with immunohistochemistry, in situ hybridization or in situ PCR which would enable the identification of specific molecular markers within the cells of the niche (proteins or RNA). All this data can be further used in isolation of particular cells. Prior to transplantation, PGCs and spermatogonia need to be isolated from the donor tissue. After multi-enzymatic digestion it is possible to isolate cells based on their morphology and size, and/or specific molecular markers and the whole process can be species-specific. A great advantage of transplantation of PGCs and SSCs is that this method can be very well combined with cryopreservation. There are still no optimized protocols for cryopreservation of fish eggs and embryos, mostly due to presence of large amount of yolk and fat. Since PGCs and SSCs can develop into both sperm and eggs, cryopreservation of these cells could have a great perspective in conservation biology but also in aquaculture. Studies have shown that frozen/thawed SSCs transplanted into recipients give rise to potent donor sperm and eggs in the recipients (Kobayashi et al., 2007). In this way, there is no need to conserve both sperm and eggs since successful cryopreservation of germ cells can give rise to both sperm and eggs after transplantation

3D-Cell-Annotator : an open-source active surface tool for single-cell segmentation in 3D microscopy images

aSummary: Segmentation of single cells in microscopy images is one of the major challenges in computational biology. It is the first step of most bioimage analysis tasks, and essential to create training sets for more advanced deep learning approaches. Here, we propose 3D-Cell-Annotator to solve this task using 3D active surfaces together with shape descriptors as prior information in a semi-automated fashion. The software uses the convenient 3D interface of the widely used Medical Imaging Interaction Toolkit (MITK). Results on 3D biological structures (e.g. spheroids, organoids and embryos) show that the precision of the segmentation reaches the level of a human expert.Peer reviewe

Parental behavior and reproductive output in male-only cared and female-only cared clutches in the Eurasian Penduline Tit (<i>Remiz pendulinus</i>)

Diverse patterns of parental care, including uniparental care by either the male or the female, provide excellent opportunities to investigate how variation in social traits is maintained in wild populations. Coexistence of different parental strategies within the same population is expected when they exhibit similar cost-benefit ratios. We investigated one of the most diverse avian breeding systems and compared parental behavior and reproductive output between nests that are uniparentally cared for by male or female Eurasian Penduline Tits (Remiz pendulinus). In this small passerine bird, full care (incubation and brood care) is provided by the male only (7-18% of nests) or the female only (48-65% of nests). Additionally, a third of all nests are deserted by both parents as a result of intense sexual conflict over care. We show that parental effort is similar between male and female parents, and they achieve similar offspring survival after controlling for clutch-size differences. Females care for larger clutches than males (5.7 +/- 0.1 [SE] and 3.5 +/- 0.2 eggs, respectively), and they produce more nestlings (3.6 +/- 0.2 and 2.4 +/- 0.3 nestlings, respectively), although this difference is not significant at fledging of the chicks (3.1 +/- 0.2 and 2.4 +/- 0.3 young, respectively). Therefore, sex difference in care does not explain the bias toward female-only care in Eurasian Penduline Tits. We propose that the differential benefits of desertion and/or the differential costs of care for males and females facilitate the existence of both uniparental care types in the population. Received 18 April 2012, accepted 2 July 2012

SpheroidPicker for automated 3D cell culture manipulation using deep learning

Recent statistics report that more than 3.7 million new cases of cancer occur in Europe yearly, and the disease accounts for approximately 20% of all deaths. High-throughput screening of cancer cell cultures has dominated the search for novel, effective anticancer therapies in the past decades. Recently, functional assays with patient-derived ex vivo 3D cell culture have gained importance for drug discovery and precision medicine. We recently evaluated the major advancements and needs for the 3D cell culture screening, and concluded that strictly standardized and robust sample preparation is the most desired development. Here we propose an artificial intelligence-guided low-cost 3D cell culture delivery system. It consists of a light microscope, a micromanipulator, a syringe pump, and a controller computer. The system performs morphology-based feature analysis on spheroids and can select uniform sized or shaped spheroids to transfer them between various sample holders. It can select the samples from standard sample holders, including Petri dishes and microwell plates, and then transfer them to a variety of holders up to 384 well plates. The device performs reliable semi- and fully automated spheroid transfer. This results in highly controlled experimental conditions and eliminates non-trivial side effects of sample variability that is a key aspect towards next-generation precision medicine.Peer reviewe

A quantitative metric for the comparative evaluation of optical clearing protocols for 3D multicellular spheroids

3D multicellular spheroids quickly emerged as in vitro models because they represent the in vivo tumor environment better than standard 2D cell cultures. However, with current microscopy technologies, it is difficult to visualize individual cells in the deeper layers of 3D samples mainly because of limited light penetration and scattering. To overcome this problem several optical clearing methods have been proposed but defining the most appropriate clearing approach is an open issue due to the lack of a gold standard metric. Here, we propose a guideline for 3D light microscopy imaging to achieve single-cell resolution. The guideline includes a validation experiment focusing on five optical clearing protocols. We review and compare seven quality metrics which quantitatively characterize the imaging quality of spheroids. As a test environment, we have created and shared a large 3D dataset including approximately hundred fluorescently stained and optically cleared spheroids. Based on the results we introduce the use of a novel quality metric as a promising method to serve as a gold standard, applicable to compare optical clearing protocols, and decide on the most suitable one for a particular experiment. (C) 2021 The Authors. Published by Elsevier B.V. on behalf of Research Network of Computational and Structural Biotechnology.Peer reviewe

Cell lines and clearing approaches : a single-cell level 3D light-sheet fluorescence microscopy dataset of multicellular spheroids

Nowadays, three dimensional (3D) cell cultures are widely used in the biological laboratories and several optical clearing approaches have been proposed to visualize individual cells in the deepest layers of cancer multicellular spheroids. However, defining the most appropriate clearing approach for the different cell lines is an open issue due to the lack of a gold standard quantitative metric. In this article, we describe and share a single-cell resolution 3D image dataset of human carcinoma spheroids imaged using a light-sheet fluorescence microscope. The dataset contains 90 multicellular cancer spheroids derived from 3 cell lines (i.e. T-47D, 5-8F, and Huh-7D12) and cleared with 5 different protocols, precisely Clear(T) , Clear(T2) , CUBIC, ScaleA2, and Sucrose. To evaluate image quality and light penetration depth of the cleared 3D samples, all the spheroids have been imaged under the same experimental conditions, labelling the nuclei with the DRAQ(5) stain and using a Leica SP8 Digital LightSheet microscope. The clearing quality of this dataset was annotated by 10 independent experts and thus allows microscopy users to qualitatively compare the effects of different optical clearing protocols on different cell lines. It is also an optimal testbed to quantitatively assess different com putational metrics evaluating the image quality in the deepest layers of the spheroids. (C) 2021 The Author(s). Published by Elsevier Inc.Peer reviewe

Poreklo i genetička struktura nekoliko mađarskih divljih i domestifikovanih populacija potočne pastrmke na osnovu PCR-RFLP i mikrosatelitskih markera

U Evropi je, na osnovu studija mitohondrijalne DNK, identifikovano pet evolutivnih linija potočne pastrmke (Salmo trutta m. fario L. 1758): Atlanska, Dunavska, Mediteranska, Jadranska i Mramorna. Mađarske linije bi teorijski trebalo da pripadaju Dunavskoj liniji na osnovu hidrogeografije zemlje, ipak, ovo nije potvrđeno genetičkim studijama. Korišćeni su molekularni markeri da bi se ispitalo genetička pozadina populacije potočne pastrmke u Mađarskoj. Istraživanja su uključila jedini matični nasad potočne pastrmke u Mađarskoj, kao i po jedna populacije u planinskim vencima Bükk, Aggtelek, Börzsöny i Visegrádi, po jedna populacija u svakom. Genetička analiza je do sada sprovedena na 533 individue, isečak peraja je uzorkovan sa svake ribe i PCR-RFLP (kontrolni region mitohondrijalne DNK, laktat dehidrogenaza i somatolaktin geni), kao i analiza mikrosatelitnih markeri (BFRO002, OMM1064, Ssa408uos, SsoSL417, SsoSL438) su bili korišćeni da bi se razlikovale Dunavska i Atlanska linija potočne pastrmke. Na osnovu genetičke analize mitohondrijalne DNK divljih populacija, udeo Dunavskog haplotipa je nizak (< 10 %), sa izuzetkom potoka Apátkúti u planinama Visegrádi, gde je nađena relativno visok udeo Dunavskog haplotipa (34%). 401 analizirani primerak matičnog jata farme je skoro u potpunosti Atlanski haplotip, što ukazuje na efekat osnivača. Iako su kasnije prikupljeni primerci iz obližnjeg potoka i pridodati matičnom jatu, njihov broj je bio ograničen jer su uglavnom svi bili mužjaci. Budući da je jedino matično jato u Mađarskoj, ribe sa ove farme se koriste za poribljavanje od strane ribolovaca, što može dovesti do značajnog uticaja na prirodne populacije. Na osnovu analize nuklearnih markera sve populacije su veoma heterogene. Veliki udeo (60-80%) Atlanskih alela primećen za ove markere na svim lokacijama gde je obavljano uzorkovanje ukazuje na efekat intenzivnog poribljavanja mađarskih salmonidnih regiona. Analize mikrosatelitskih markera su ukazale na visoku heterozigotnost i Hardy-Weinberg-ovu ravnotežu svih populacija

Vitrifikacija mleča grgeča (perca fluviatilis)

Vitrifikacija je proces dovođenja vode ili rastvora u čvrsto stanje, odnosno u amorfno ili staklasto stanje koje može da se dostigne veoma brzom hlađenjem (106-1010 °C/s). Nedavno je objavljeno nekoliko istraživanja o vitrifikaciji mleča različitih vrsta riba, međutim nema dostupnih informacija o vitrifikaciji mleča grgeča (Perca fluviatilis). Mužjaci grgeča su uzorkovani 6 dana posle hormonske injekcije (250 IU kg-1 hCG). Evaluirana je pokretljivost spermatozoida pomoću sistema kompjuterske analize sperme CASA. Za process vitrifikacije mleč je razblažen modifikovanim Tanaka ekstenderom na finalni odnos 1:5 (sa krioprotektantima). Posle preliminarnih testova sa kombinacijom metanola i propilen glikola (PG) u različitim koncentracijama, odlučeno je da se koristi 15% metanola i 15 % PG (ukupno 30% krioprotektanata). Suspenzija mleča je ubačena direktno u tečni azot bez prethodnog hlađenja u njegovoj pari. Za sve eksperimente vitrifikacije za hlađenje su korišćene cevčice Cryotop (Kitazato-Dibimed, za 2 µl rastvora). Za fertilizacioni test su prikupljena jaja ženki grgeča. Vitrifikovane Cryotop cevčice otopljene su direktno u 10 µl rastvora za aktivaciju (50 mm NaCl) u petri šoljama koje su sadržale jaja. Svež mleč služio je za kontrolu. Oplođena jaja su inkubirana u plivajućem sistemu. Izvedena su 3 ogleda da bi se utvrdio odgovarajući broj cevčica Cryotop za svaku seriju jaja: 1, 6 i 18 cevčica Cryotop je isprobano za svaku seriju jajnih ćelija. U 2 µl rastvora mleča jedne Cryotop cevčice bilo je oko 0,33 µl mleča. Na osnovu stepena oplođenja u tri ogleda može se zaključiti da povećanje broja Cryotop cevčica pojačava stepen oplođenja. Dalja sitraživanja su neophodna da bi se razvio metod vitrifikacije sa većim preživljavanjem larvi posle oplođenja vitrifikovanim mlečom. Takođe je potrebno ispitati stepen izvaljenih embriona iz ogleda sa vitrifikovanim mlečom, kao i potencijalni uticaj vitrifikacije na larve, pre svega na deformitete i morfološke promene