Gene diversity and degree of hybridization in water frogs of the genus Pelophylax Fitzinger, 1843 (Anura: Ranidae) from Crna Mlaka

Zelene žabe roda Pelophylax Fitzinger, 1843 jedna su od najistraživanijih životinjskih skupina zbog prirodne hibridizacije nekih vrsta unutar roda. Posljednjih godina razvijene su molekularno biološke metode koje su se pokazale uspješnima u determinaciji vrsta unutar ovog roda, budući da između njih postoji određena genska raznolikost. Velika genska raznolikost zelenih žaba Hrvatske rezultat je njezine geološke prošlosti. U ovom istraživanju korištenjem molekularnih biljega citokrom b i serum albumin intronske regije 1 na području Crne Mlake utvrđena je prisutnost već ranije otkrivenih vrsta Pelophylax lessonae i Pelophylax ridibundus te njihovog hibrida Pelophyax kl. esculentus. Osim njih, utvrđena je i prisutnost dosad neotkrivenih vrsta na području Crne Mlake Pelophylax kurtmuelleri i Pelophylax shqipericus te hibrida P. kurtmuelleri x P. shqipericus. Također, zabilježeni su i dosad u literaturi neopisani hibridi P. shqipericus x P. ridibundus, P. ridibundus x P. kurtmuelleri te P. lessonae x P. kurtmuelleri. „Čiste“ vrste su učestalije od hibrida, a frekvencija „čistih“ vrsta i hibrida je podjednaka i u proljeće i u jesen. Molekularno-filogenetičke metode pokazuju jasno odvajanje P. ridibundus i P. kurtmuelleri. Alohtone vrste zbog svog brzog širenja predstavljaju potencijalnu opasnost za nativne vrste te je potrebno provesti daljnja istraživanja za utvrđivanje stupnja hibridizacije.Water frogs of the genus Pelophylax Fitzinger, 1843 are one of the most studied animal groups because some of the species of this genus naturally hybridise. During last few years, methods in molecular biology have been developed that are succesful in determination of the genus species, when there is some gene variability among them. Croatia is located on a large green frogs gene variability area, which is a result of its geological past. In this study, by using molecular markers citochrome b and serum albumin intron region 1, the results have shown the presence of previously known species Pelophylax lessonae and Pelophylax ridibundus and their hybrid Pelophyax kl. esculentus from Crna Mlaka area. The results have also shown the presence of previously unknown species for that area Pelophylax kurtmuelleri and Pelophylax shqipericus and hybrid P. kurtmuelleri x P. shqipericus. Also, until now unknown hybrids P. shqipericus x P. ridibundus, P. ridibundus x P. kurtmuelleri and P. lessonae x P. kurtmuelleri have been recorded. „Pure“ species are numerous than hybrids, but they are equally frequent both in spring and autumn. Molecular-phylogenetic methods show clear separation of P. ridibundus and P. kurtmuelleri. Allochtonous species represent potential danger for native species because of their rapid spreading and it is important to carry out further studies for establishing the degree of hybridisation

A novel time-lapse imaging method for studying developing bacterial biofilms

In nature, bacteria prevailingly reside in the form of biofilms. These elaborately organized surface‑bound assemblages of bacterial cells show numerous features of multicellular organization. We recently showed that biofilm growth is a true developmental process, which resembles developmental processes in multicellular eukaryotes. To study the biofilm growth in a fashion of eukaryotic ontogeny, it is essential to define dynamics and critical transitional phases of this process. The first step in this endeavor is to record the gross morphological changes of biofilm ontogeny under standardized conditions. This visual information is instrumental in guiding the sampling strategy for the later omics analyses of biofilm ontogeny. However, none of the currently available visualizations methods is specifically tailored for recording gross morphology across the whole biofilm development. To address this void, here we present an affordable Arduino‑based approach for time‑lapse visualization of complete biofilm ontogeny using bright field stereomicroscopy with episcopic illumination. The major challenge in recording biofilm development on the air–solid interphase is water condensation, which compromises filming directly through the lid of a Petri dish. To overcome these trade‑offs, we developed an Arduino microcontroller setup which synchronizes a robotic arm, responsible for opening and closing the Petri dish lid, with the activity of a stereomicroscope‑mounted camera and lighting conditions. We placed this setup into a microbiological incubator that maintains temperature and humidity during the biofilm growth. As a proof‑of‑principle, we recorded biofilm development of five Bacillus subtilis strains that show different morphological and developmental dynamics

SUODNOS FILOGENIJE I ONTOGENIJE BIOFILMOVA BAKTERIJE Bacillus subtilis

Links between ontogeny and phylogeny in animals have been discussed for more than two centuries. With the uprising of molecular biology and bioinformatics, several studies have revealed the presence of the phylogeny-ontogeny correlation on molecular level in developmental transcriptomes of eukaryotic clades with complex multicellularity. These findings open a possibility to test the phylogeny-ontogeny correlation in more basal organisms, with more obscure development and multicellularity characteristics. Using time-resolved transcriptome and proteome profiles, this study showed that Bacillus subtilis biofilm ontogeny correlates with the evolutionary measures through recapitulation pattern, in a way that evolutionary younger and more diverged genes were increasingly expressed towards the later timepoints of the biofilm growth. Molecular and morphological signatures also revealed that biofilm growth is highly regulated and organized into discrete ontogenetic stages. Together, this suggests that the biofilm formation in Bacillus subtilis is a true developmental process comparable to organismal development in animals, plants and fungiKorelacija između filogenije i ontogenije životinja je predmet rasprave među znanstvenicima od početka 19. stoljeća. Razvojem molekularne biologije i bioinformatike te analizama transkriptoma razvojnih stadija, uočena je prisutnost korelacije filogenije i ontogenije na molekularnoj razini u eukariotskim evolucijskim granama sa složenom višestaničnosti. Ova saznanja potaknula su nas da testiramo korelaciju filogenije i ontogenije u bazalnim organizmima s nejasnim razvojnim i višestaničnim karakteristikama. Analizom transkriptomskih i proteomskih podataka, pokazali smo da ontogenija Bacillus subtilis biofilmova korelira s evolucijskim mjerama u obliku rekapitulacijskog profila, odnosno evolucijski mlađi i divergentniji geni su sve više eksprimirani prema kasnijim stadijima rasta biofilma. Dodatne molekularne analize pokazale su da je rast biofilma reguliran i organiziran u jasno odvojene stadije ontogenije. Zaključno, rezultati pokazuju da je rast Bacillus subtilis biofilmova istinski razvojni proces usporediv s razvojem životinja, biljaka i gljiva

SUODNOS FILOGENIJE I ONTOGENIJE BIOFILMOVA BAKTERIJE Bacillus subtilis

Links between ontogeny and phylogeny in animals have been discussed for more than two centuries. With the uprising of molecular biology and bioinformatics, several studies have revealed the presence of the phylogeny-ontogeny correlation on molecular level in developmental transcriptomes of eukaryotic clades with complex multicellularity. These findings open a possibility to test the phylogeny-ontogeny correlation in more basal organisms, with more obscure development and multicellularity characteristics. Using time-resolved transcriptome and proteome profiles, this study showed that Bacillus subtilis biofilm ontogeny correlates with the evolutionary measures through recapitulation pattern, in a way that evolutionary younger and more diverged genes were increasingly expressed towards the later timepoints of the biofilm growth. Molecular and morphological signatures also revealed that biofilm growth is highly regulated and organized into discrete ontogenetic stages. Together, this suggests that the biofilm formation in Bacillus subtilis is a true developmental process comparable to organismal development in animals, plants and fungiKorelacija između filogenije i ontogenije životinja je predmet rasprave među znanstvenicima od početka 19. stoljeća. Razvojem molekularne biologije i bioinformatike te analizama transkriptoma razvojnih stadija, uočena je prisutnost korelacije filogenije i ontogenije na molekularnoj razini u eukariotskim evolucijskim granama sa složenom višestaničnosti. Ova saznanja potaknula su nas da testiramo korelaciju filogenije i ontogenije u bazalnim organizmima s nejasnim razvojnim i višestaničnim karakteristikama. Analizom transkriptomskih i proteomskih podataka, pokazali smo da ontogenija Bacillus subtilis biofilmova korelira s evolucijskim mjerama u obliku rekapitulacijskog profila, odnosno evolucijski mlađi i divergentniji geni su sve više eksprimirani prema kasnijim stadijima rasta biofilma. Dodatne molekularne analize pokazale su da je rast biofilma reguliran i organiziran u jasno odvojene stadije ontogenije. Zaključno, rezultati pokazuju da je rast Bacillus subtilis biofilmova istinski razvojni proces usporediv s razvojem životinja, biljaka i gljiva

SUODNOS FILOGENIJE I ONTOGENIJE BIOFILMOVA BAKTERIJE Bacillus subtilis

Links between ontogeny and phylogeny in animals have been discussed for more than two centuries. With the uprising of molecular biology and bioinformatics, several studies have revealed the presence of the phylogeny-ontogeny correlation on molecular level in developmental transcriptomes of eukaryotic clades with complex multicellularity. These findings open a possibility to test the phylogeny-ontogeny correlation in more basal organisms, with more obscure development and multicellularity characteristics. Using time-resolved transcriptome and proteome profiles, this study showed that Bacillus subtilis biofilm ontogeny correlates with the evolutionary measures through recapitulation pattern, in a way that evolutionary younger and more diverged genes were increasingly expressed towards the later timepoints of the biofilm growth. Molecular and morphological signatures also revealed that biofilm growth is highly regulated and organized into discrete ontogenetic stages. Together, this suggests that the biofilm formation in Bacillus subtilis is a true developmental process comparable to organismal development in animals, plants and fungiKorelacija između filogenije i ontogenije životinja je predmet rasprave među znanstvenicima od početka 19. stoljeća. Razvojem molekularne biologije i bioinformatike te analizama transkriptoma razvojnih stadija, uočena je prisutnost korelacije filogenije i ontogenije na molekularnoj razini u eukariotskim evolucijskim granama sa složenom višestaničnosti. Ova saznanja potaknula su nas da testiramo korelaciju filogenije i ontogenije u bazalnim organizmima s nejasnim razvojnim i višestaničnim karakteristikama. Analizom transkriptomskih i proteomskih podataka, pokazali smo da ontogenija Bacillus subtilis biofilmova korelira s evolucijskim mjerama u obliku rekapitulacijskog profila, odnosno evolucijski mlađi i divergentniji geni su sve više eksprimirani prema kasnijim stadijima rasta biofilma. Dodatne molekularne analize pokazale su da je rast biofilma reguliran i organiziran u jasno odvojene stadije ontogenije. Zaključno, rezultati pokazuju da je rast Bacillus subtilis biofilmova istinski razvojni proces usporediv s razvojem životinja, biljaka i gljiva

EFFECT OF HIGH TEMPERATURE ON THE METABOLISM AND REPRODUCTION OF DAIRY COW

Visoka okolišna temperatura, vrlo često u kombinaciji s visokom relativnom vlagom u zraku rezultiraju toplinskim stresom, a organizam se u takvim uvjetima ne može ohladiti te potiče različite prilagodbene mehanizme. Kod mliječnih krava se u okviru odgovora na toplinski stres mijenja metabolizam koji postaje usporen. Smanjena je konzumacija hrane te proizvodnja mlijeka, izmijenjen je metabolizam minerala, povećan je unos vode te je smanjen njezin gubitak putem urina i fecesa, a povećani su i znojenje, slinjenje te rektalna temperatura. Toplinski stres kod mliječnih krava ima najznačajniji utjecaj na reproduktivni sustav. U radu je opisan utjecaj visokih temperatura, odnosno toplinskog stresa na organizam mliječnih krava, s naglaskom na reprodukciju i metabolizam. Prikazani su tipični simptomi koje krava pokazuje te koji se procesi u organizmu javljaju kao odgovor na visoke temperature, kako dolazi do toplinskog stresa, na koje načine se krava bori s nepovoljnim utjecajima okoliša te kako pokušava umanjiti štetne učinke stresa te im se prilagoditi. Nadalje, ukratko su navedene osnovne metode za ublažavanje učinaka visokih temperatura na organizam mliječnih krava te osnovni principi hranidbe u uvjetima visokih okolišnih temperatura. .High environmental temperatures, often in combination with high relative humidity resulting thermal stress, and the body in such conditions can not be cool and encourages different adaptive mechanisms. In dairy cows in the response to heat stress alters the metabolism to become sluggish. Decreased feed intake and milk production, altered metabolism of minerals, increased water intake and decreased its loss in the urine and feces, and increased sweating, salivation, and rectal temperature. Heat stress in dairy cows has the most significant effect on the reproductive system. This paper describes the effect of high temperature and thermal stress on the body of dairy cows, with an emphasis on reproduction and metabolism. Shown are typical symptoms that show cows and processes that occur in the body in response to high temperatures, as it comes to heat stress, the ways in which the cow is struggling with adverse impacts to the environment and trying to reduce the harmful effects of stress. Furthermore, brief basic methods to mitigate the effects of heat on the body of dairy cows, and the basic principles of nutrition in the conditions of high environmental temperature are shown

Embryo-Like Features in Developing Bacillus subtilis Biofilms

Correspondence between evolution and development has been discussed for more than two centuries. Recent work reveals that phylogeny-ontogeny correlations are indeed present in developmental transcriptomes of eukaryotic clades with complex multicellularity. Nevertheless, it has been largely ignored that the pervasive presence of phylogeny-ontogeny correlations is a hallmark of development in eukaryotes. This perspective opens a possibility to look for similar parallelisms in biological settings where developmental logic and multicellular complexity are more obscure. For instance, it has been increasingly recognized that multicellular behavior underlies biofilm formation in bacteria. However, it remains unclear whether bacterial biofilm growth shares some basic principles with development in complex eukaryotes. Here we show that the ontogeny of growing Bacillus subtilis biofilms recapitulates phylogeny at the expression level. Using time-resolved transcriptome and proteome profiles, we found that biofilm ontogeny correlates with the evolutionary measures, in a way that evolutionary younger and more diverged genes were increasingly expressed toward later timepoints of biofilm growth. Molecular and morphological signatures also revealed that biofilm growth is highly regulated and organized into discrete ontogenetic stages, analogous to those of eukaryotic embryos. Together, this suggests that biofilm formation in Bacillus is a bona fide developmental process comparable to organismal development in animals, plants, and fungi. Given that most cells on Earth reside in the form of biofilms and that biofilms represent the oldest known fossils, we anticipate that the widely adopted vision of the first life as a single-cell and free-living organism needs rethinking.CC BY-NC 4.0</p

DataSheet_1_CD9- and CD81-positive extracellular vesicles provide a marker to monitor glioblastoma cell response to photon-based and proton-based radiotherapy.docx

Glioblastoma multiforme (GBM) is the most aggressive tumor of the central nervous system with a poor prognosis. In the treatment of GBM tumors, radiotherapy plays a major role. Typically, GBM tumors cannot be cured by irradiation because of intrinsic resistance machanisms. An escalation of the irradiation dose in the GBM tumor is difficult due to the high risk of severe side effects in the brain. In the last decade, the development of new irradiation techniques, including proton-based irradiation, promised new chances in the treatment of brain tumors. In contrast to conventional radiotherapy, irradiation with protons allows a dosimetrically more confined dose deposition in the tumor while better sparing the normal tissue surrounding the tumor. A systematic comparison of both irradiation techniques on glioblastoma cells has not been performed so far. Despite the improvements in radiotherapy, it remains challenging to predict the therapeutical response of GBM tumors. Recent publications suggest extracellular vesicles (EVs) as promising markers predicting tumor response. Being part of an ancient intercellular communication system, virtually all cells release specifically composed EVs. The assembly of EVs varies between cell types and depends on environmental parameters. Here, we compared the impact of photon-based with proton-based radiotherapy on cell viability and phenotype of four different glioblastoma cell lines. Furthermore, we characterized EVs released by different glioblastoma cells and correlated released EVs with the cellular response to radiotherapy. Our results demonstrated that glioblastoma cells reacted more sensitive to irradiation with protons than photons, while radiation-induced cell death 72 h after single dose irradiation was independent of the irradiation modality. Moreover, we detected CD9 and CD81-positive EVs in the supernatant of all glioblastoma cells, although at different concentrations. The amount of released CD9 and CD81-positive EVs increased after irradiation when cells became apoptotic. Although secreted EVs of non-irradiated cells were not predictive for radiosensitivity, their increased EV release after irradiation correlated with the cytotoxic response to radiotherapy 72 h after irradiation. Thus, our data suggest a novel application of EVs in the surveillance of anti-cancer therapies.</p