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Tissue clearing combined with deep imaging has emerged as a powerful technology to expand classical histological techniques. Current techniques have been optimized for imaging sparsely pigmented organs such as the mammalian brain. In contrast, melanin-rich pigmented tissue, of great interest in the investigation of melanomas, remains challenging. To address this challenge, we have developed a CRISPR-based gene editing approach that is easily incorporated into existing tissue-clearing workflows such the PACT clearing method. We term this method CRISPR-Clear. We demonstrate its applicability to highly melanin-rich B16-derived solid tumors, including one made transgenic for HER2, constituting one of very few syngeneic mouse tumors that can be used in immunocompetent models. We demonstrate the utility in detailed tumor characterization by staining for targeting antibodies and nanoparticles, as well as expressed fluorescent proteins. With CRISPR-Clear we have unprecedented access to optical interrogation in considerable portions of intact melanoma tissue for stained surface markers, expressed fluorescent proteins, of subcellular compartments, and of the vasculature.

Records of European catfish Silurus glanis in the Neretva River delta (Adriatic Sea basin) in Croatia are reported. The first occurrence of this non-native species was reported via social network which led to its timely detection by researchers. Origin of introduction is unknown but it is most likely the case of human-mediated introduction. The importance of social networks as a useful tool for the timely detection of non-native fish species, as well as the importance of raising awareness about the impacts of biological invasions, is discussed in this paper.

Cyperus esculentus (yellow nutsedge) is an alien species that started to spread in arable fields in Slovenia and has already become a noxious weed. In this study we present the spatio-temporal pattern of this invasion in Slovenia. Species was first recorded in 1980 and currently two hotspots are evident (Ljubljana Basin and Posočje). Habitat preferences of Cyperus esculentus and the floristic compostion of invaded plant communities were studied. These communites were compared to similar communities in Slovenia and to so far described vegetation types with dominating Cyperus esculentus in Europe. Based on these analyses we described new weed association, namely Digitario sanguinalis-Cyperetum esculenti.

A single specimen of Pterois cf. miles has been recorded in the eastern middle Adriatic Sea. It was observed near the island of Vis at a depth of 15 m. The location of the record is further north than previous Adriatic records and it constitutes the northernmost record of this species in the Mediterranean Sea to date. The record is based solely on photographs and video footage provided by a professional underwater photographer.

The angular rough shark, Oxynotus centrina (Linnaeus, 1758), is a poorly known and rare bathydemersal shark inhabiting continental shelves and upper slopes with a significant lack of data and rarely published records in the Adriatic Sea in this century. In this paper, we present 20 new occurrences recorded from May 2015 to September 2021, of which 19 are in Croatian and one in Montenegrin territorial waters. Records of juveniles, subadults and adults are reported. Higher number of records off the Kornati archipelago in the central Adriatic Sea suggest that this area is of particular importance for this species. Furthermore, habitats in the Southern Adriatic might have lower density. Due to the nonsystematic research and nonprobabilistic data collection, it is difficult to establish whether the greater number of records in the continental shelf is just an ostensible phenomenon.

Today’s teaching and didactical methods are progressively aiming to integrate digital technologies, computational thinking (CT), and basic computer science concepts into other subjects. An innovative and creative way of combining and integrating CT and teaching cross-curricular skills without digital devices is to include the game Poly-Universe (PolyUni). According to previous research, the game is expected to have a positive effect on visual perceptual progress, including isolation, and the development of shape-background skills. So far, however, comparatively few attempts have been made to explore the educational possibilities of PolyUni for different school levels and subjects, besides mathematics. Therefore, this article aims to close this gap by exploring how PolyUni can be used to promote CT in three subjects: physical education (PE), digital education (DGE), and biology (B). Furthermore, it evaluates whether the pre-defined learning objectives in those subjects have been achieved, and examines how PolyUni combines the requirements of the different curricula in Austrian secondary school, based on self-designed tasks. Additionally, further aspects of PolyUni such as engagement and collaboration are discussed. To explore the above-mentioned benefits, a mixed-methods study was implemented, whereas the workshops and accompanying teaching materials (e.g., worksheets) were developed based on the COOL Informatics concept. The participant observation method was employed for qualitative data collection, and a self-designed assessment grid as well as additional picture analysis were used for the quantitative data. PolyUni was introduced in three different workshops at Austrian secondary schools with 80 students observed and analyzed. Based on the present data, it can be assumed that PolyUni supports achieving the requirements of the different curricula and pre-defined teaching and learning objectives in a playful way. Furthermore, the game not only promotes CT in secondary school but also encourages enjoyment and collaboration between peers in biological, digital, and physical education lessons. Version of record

The Dinaric Karst is a global hotspot for subterranean diversity, with two distinct peaks of species richness in the northwest and southeast, and an area of a lower species richness in the central part. In this article, we present a species list and describe the ecological conditions of the Lukina jama–Trojama cave system, located in the central part of the Dinaric Karst. This cave system is the deepest and one of the most logistically challenging cave systems sampled so far in the Dinaric Karst. Repeated sampling resulted in a list of 45 species, including 25 troglobionts, 3 troglophiles, 16 stygobionts, and 1 stygophile. Most of the recorded species are endemic to the Velebit Mountain, while three species are endemic to the Lukina jama–Trojama cave system. Within the system, species richness peaks in the deepest third of the cave, most likely reflecting the harsh ecological conditions in the upper parts, including ice, cold winds, and occasional waterfalls. Milder and more stable deeper parts of the cave contain a rich subterranean species community, part of which is associated with two very distinct aquatic habitats, the cave hygropetric and the phreatic zone. The newly recognized hotspot of subterranean biodiversity in the central Dinaric Karst, which has emerged between the two known centers of biodiversity, further highlights the species richness in large cave systems, but also challenges the diversity patterns in the Dinaric Karst overall.

Objectives: The aim of this study was to analyze clinical manifestations, epidemiology and laboratory parameters of B. henselae infection among children treated at the University Hospital for Infectious Diseases “Dr. Fran Mihaljević”, Zagreb from January 2014 until June 2019. Materials and methods: We retrospectively analyzed the epidemiology, clinical and laboratory characteristics among children with positive indirect immunofluorescence assay for B. henselae IgM and IgG or positive B. henselae polymerase chain reaction from lymph node aspirate. Results: A total of 104 patients, 47 (45,1%) female and 57 (54,8%) male were enrolled. The median age was 9,7 (range, 1,1 to 17,3 years). A history of cat contact was present in 101 (97,1%) children. Acute infection was serologically confirmed in 87 (83,6%), in 5 (4,8%) with PCR while both methods were positive in 12 (11,5%) patients. The presentation on B. henselae infection were regional lymphadenopathy , disseminated disease, encephalopathy and fever of unknown origin. Suppurative inflammation was the most common complication in patients with lymphadenopathy 12/92 (13%). Full recovery was the most frequent outcome (96,1%). Conclusion lesion: B. henselae infection among children is usually a mild disease presented as regional lymphadenopathy. Serology and polymerase chain reaction are useful tests for diagnosis. Treatment duration and choice of therapy depend on clinical manifestation and developed complications.

Acute myeloid leukemia (AML) with rearrangement of the lysine methyltransferase 2a gene (KMT2Ar) has adverse outcomes. However, reports on the prognostic impact of various translocations causing KMT2Ar are conflicting. Less is known about associated mutations and their prognostic impact. In a retrospective analysis, we identified 172 adult patients with KMT2Ar AML and compared them to 522 age-matched patients with diploid AML. KMT2Ar AML had fewer mutations, most commonly affecting RAS and FLT3 without significant impact on prognosis, except for patients with ≥2 mutations with lower overall survival (OS). KMT2Ar AML had worse outcomes compared with diploid AML when newly diagnosed and at relapse, especially following second salvage (median OS of 2.4 vs 4.8 months, P KMT2Ar AML (t-AML) had worse outcomes compared with de novo KMT2Ar AML (median OS of 0.7 years vs 1.4 years, P P KMT2Ar did not predict survival, unlike age and allo-HSCT. In conclusion, KMT2Ar was associated with adverse outcomes regardless of translocation subtype. Therefore, AML risk stratification guidelines should include all KMT2Ar as adverse.

Recent rapid progress in genome analysis and large-scale consortia has made it possible to discover variants with a variety of allele frequencies and effect sizes associated with psychiatric disorders. Among psychiatric disorder-susceptibility variants, rare variants with large effect sizes detected by sequencing analysis or array comparative genomic hybridization would be particularly useful for elucidating pathophysiology by developing disease models, such as genome-edited mouse or induced pluripotent stem cells. In the last decade, investigations of rare variants with large effect size have revealed an important role of neurodevelopment in the pathogenesis of psychiatric disorders. In future research, integration of recent evidence concerning the contribution of the immune system or gut microbiota will enhance our understanding of psychiatric disorders and facilitate novel drug development.

Two new ultraspecialized troglomorphic Leptodirini (Coleoptera, Leiodidae, Cholevinae) from two Croatian caves are described. Hygrodromus (new genus) nikolinae, a new species from Mladenova cave near Orebić, is a very enigmatic taxon of “tetramerous”Leptodirini, markedly distinct for several morphological features in antennae, mouthparts, male genitalia and the peculiar sexually dimorphic apex of elytra. However, because of some characters, it could be assigned to the phyletic lineage of Hadesia and Nauticiella, which so far includes only “pentamerous”species. Leptomeson vuicae from Dragina lipa jama cave near Čelopek in central Dalmatia, owing to its morphological features, is the south-eastern substitutive adelphotaxon of L. dombrowskii, from which it differs by its smaller size, longer elytral pubescence and the different shape of the median lobe of the aedeagus with a stouter apex.

The review paper examines the main risk factors for microbial contamination of meat at different stages of its processing. Particular emphasis has been placed on primary animal processing being the most hazardous in terms of microbial contamination of meat. Carcass cross-contamination most frequently occurs during skinning and evisceration since hides and the digestive tract are the primary sources of microbial pathogens. It is necessary to observe stringent sanitary and hygienic rules when performing these operations. Continuous cold chain management along all following stages of meat processing and control of the sanitary status of cold chambers during meat storage are of extreme importance. An increase in the microbial counts due to the high number of manual operations was observed during meat cutting, boning, and trimming. Subsequent stages of meat processing, including mincing, curing, the addition of spices, also promote significant microbial growth. Strict control regarding detection of dangerous pathogens, especially L. monocytogenes, is needed at this stage. In general, to minimize problems linked with meat and meat product safety, it is necessary to take timely measures on sanitary treatment of meat processing facilities, including the prevention of biofilm formation.

This study provides the first data on the reproductive biology of the spiny gurnard Lepidotrigla dieuzeidei. A total of 1027 individuals were collected monthly from September 2011 to August 2012. The total length of the individuals in the sample ranged from 7.46 cm to 14.62 cm with a female to male ratio of 1 to 0.59. Positive allometric growth was observed for both females (b=3.07) and males (b=3.08). Variations of condition factor were similar in both sexes with mean values of 1.07 for females and 1.01 for males. The fish L. dieuzeidei had a long spawning period extending from November to July based on monthly values of gonadosomatic index. Histological analysis showed that this fish was multiple synchronous developments of ovaries with multiple spawning events. The ovaries were always represented with a group of primary oocytes and a diverse population of vitel-logenic oocytes, each specially recruited during the final stages of maturation in uniform or uneven phases. Batch fecundity was estimated gravimetrically using the hydrated oocyte method and was estimated to be around 558 to 10.230 oocytes per female. L. dieuzeidei is an important by-catch species and data from this study represent a valuable contribution to stock management., Ovo istraživanje donosi prve podatke o reproduktivnoj biologiji kokotića, Lepidotrigla dieuzei-dei. Ukupno je prikupljeno 1027 jedinki, uzorkovano je mjesečno od rujna 2011. do kolovoza 2012. Ukupna duljina jedinki bila je u rasponu od 7,46 cm do 14,62 cm s omjerom spolova ženke:mužjaci 1:0,59. Pozitivan alometrijski rast zabilježen je i kod ženka (b = 3,07) i kod mužjaka (b = 3,08). Promjene indeksa kondicije bile su slične kod oba spola sa srednjim vrijednostima od 1,07 za ženke i 1,01 za mužjake. Temeljem mjesečnih vrijednosti gonadosomatskog indeksa utvrđeno je da kokotić ima dugo razdoblje mriješćenja, od studenog do srpnja. Histološka analiza pokazala je multipli sinkroni razvoj jajnika s više razdoblja mriješćenja. U jajnicima su uvijek bile zastupljene skupine primarnih oocita i raznovrsni stadiji vitelogenih oocita, koji su regrutirani u posljednjim stadijima sazrijevanja u jednoličnim ili neravnomjernim fazama. Fekunditet je procijenjen gravimetrijski primjenom metode hidratiziranih oocita i iznosi oko 558 do 10230 oocita po ženki. Kokotić je važna vrsta u prilovu i podatci iz ovog istraživanja predstavljaju značajan doprinos upravljanja stockom.

Despite common resistance to antimicrobials in Escherichia coli isolates from farm animals in Serbia, no data are currently accessible on its occurrence in E. coli isolated from gulls. Therefore, 67 cloacal swabs and 70 fecal samples from black-headed gulls were investigated for the presence of antibiotic-resistant E. coli isolates. Ninety-nine isolates were obtained during the study. Resistotyping and resistance gene typing has shown that 44 isolates harbor resistance to one or more antibiotics. Multidrug resistance was detected in 24 E. coli isolates. Ten isolates were resistant to extended-spectrum cephalosporin antibiotics and were studied in detail including virulence gene typing, phylogenetic and multilocus sequence typing, and mating. These ten isolates belonged to phylogenetic groups B2 (five isolates), D (four isolates) and B1 (one isolate). Five different sequence types (ST38, ST2307, ST224, ST162 and ST34) were detected in E. coli isolates with AmpC phenotype and genotype. One isolate carried the Inc I2/FIB replicon type plasmid with the blaCTX-M-1 gene. Nine isolates had blaCMY-2 genes, which were detected on conjugative plasmids in seven isolates. The virulence genes hly, iroN, iss, ompT and cvaC were detected in one transconjugant. Ten isolates were found to be resistant to ciprofloxacin, whose MIC ranged from 4 to 32 mg/L. Genotyping revealed single or double mutations in the quinolone resistance determining region (QRDR) of the gyrA or gyrA, parC and parE genes, respectively. So, Black-headed gulls from Serbia may be colonized by multidrug-resistant E. coli, some of which are resistant to critically important antibiotics in medicine.

Distributional range of freshwater blenny Salaria fluviatilis extends to the tributaries along the Mediterranean and Black seas. It is considered endangered in several Mediterranean European countries. Although previously mentioned in the lists of freshwater fish species, its presence has never before been confirmed in Bosnia and Herzegovina. Consequently, this has led to doubts about its distribution there. A sample specimen of freshwater blenny was obtained from the lower Neretva River (Adriatic Sea watershed) in Bosnia and Herzegovina in May 2020, representing the first reliable record of this species. Considering that this record confirms older reports, however, it seems that earlier surveys have failed to locate the fish. Knowledge of the composition of fish species in river basins and updating their list are among the important requirements for managing the biodiversity of each country, as well as water and fish resources.

With a phytosociological analysis of more than 200 relevés on more than 25 localities with Gladiolus palustrisand (or) G. illyricusin western and southwestern Slovenia we identified more than 15 communities of association rank that belong to at least eight vegetation alliances and five classes. We determined that these two species can grow on the same localities and sites, but Gladiolus palustrisflowers at least 14 days after G. illyricus. Another reliable distinguishing characteristic is the fibrous tunic covering the corm. Gladiolus palustris is threatened in most of the examined localities, except for the Banjšice Plateau and the Slavnik range in Čičarija, but the current nature conservation policy in Slovenia does not provide for its preservation. Key words: Gladiolus palustris, G. illyricus, phytosociology, syntaxonomy, Natura 2000, nature conservation, Slovenia Izvleček S fitocenološko analizo več kot 200 popisov na več kot 25 nahajališčih v zahodni in jugozahodni Sloveniji, na katerih uspevata vrsti Gladiolus palustrisin (ali) G. illyricus, smo prepoznali več kot 15 združb na rangu asociacije, ki pripadajo vsaj osmim vegetacijskim zvezam in petim razredom. Ugotovili smo, da obe podobni vrsti lahko uspevata na skupnih nahajališčih in rastiščih, toda vrsta Gladiolus palustriscveti vsaj 14 dni kasneje kot vrsta Gladiolus illyricus. Zanesljiv znak za njuno razlikovanje so vlakna, ki obdajajo njun gomolj. Na večini preučenih nahajališč je močvirski meček ogrožen in mu zdajšnja naravovarstvena politika v Sloveniji ne zagotavlja ohranitve, izjema sta planoti Banjšice in pogorje Slavnika v Čičariji. Ključne besede: Gladiolus palustris, G. illyricus, fitocenologija, sintaksonomija, Natura 2000, varstvo narave, Slovenija

We have summarized results of our research of vegetation and flora in the river-basin of the Gačnik (Vojsko, Dolenja Trebuša) in western Slovenia. We enumerate most frequent and characteristic plant communities, which belong to seven habitat types of European conservation importance. In their stands grow two Natura 2000 species (Primula carniolica, Cypripedium calceolus) and almost 60 vascular plant species, which are protected and (or) on the red list. Among communities and plants are the most threatened fens and moist grasslands and their characteristic species: Drosera rotundifolia, Dactylorhiza lapponicasubsp. rhaetica, D. traunsteineri, Eleocharis quinqueflora,Eriophorum angustifolium, E. latifolium, Pinguicula vulgaris and Trichophorum alpinum. Key words: vegetation, flora, wetlands, fens, Natura 2000, Slovenia Izvleček V članku smo povzeli rezultate naših raziskav rastja in rastlinstva v povodja Gačnika na Vojskem in v Trebuši v zahodni Sloveniji. Naštevamo najbolj pogoste in najbolj značilne rastlinske združbe, zavarovane in redke rastline in naravovarstveno vredne habitatne tipe. Slednjih je vsaj sedem. V njihovih združbah uspevata dve Natura 2000 vrsti (Primula carniolica, Cypripedium calceolus) in skoraj 60 zavarovanih in (ali) redkih cevnic (rdeči seznam). Med združbami so najbolj ogrožena mokrišča (nizka barja in mokrotni travniki) in njihove značilne vrste: Drosera rotundifolia, Dactylorhiza lapponicasubsp. rhaetica, D. traunsteineri, Eleocharis quin­queflora,Eriophorum angustifolium, E. latifolium, Pinguicula vulgaris in Trichophorum alpinum. Ključne besede: vegetacija, flora, mokrišča, nizka barja, Natura 2000, Slovenija

This study presents the results of a Europe-wide training and Quality Control (QC) exercise carried out within the framework of the European Aerobiology Society’s QC Working Group. The main aim of this exercise was to examine the feasibility of carrying out a QC exercise for fungal spore monitoring in Europe, using a similar methodology to the one previously used for pollen. The QC survey was conducted in two parts: (1) Coordinators of national and regional aerobiological networks in Europe involved in the monitoring of atmospheric fungal spores were invited to complete a questionnaire survey related to their network and asked whether they were interested in taking part in an external inter-laboratory QC exercise; (2) Participating networks performed an inter-laboratory ring test with the same sample slide in order to determine the reproducibility of identifying and counting two fungal spore taxa (Alternaria and Epicoccum) in air samples collected by a Hirst-type volumetric spore trap. Participants were instructed to read five separate longitudinal transects in the “effective collecting area” of the slide. Reproducibility of analysis was determined following the method previously used in the European Aerobiology Society’s QC exercises for pollen. Thirty-two counters from 16 national or regional networks in Europe participated in the QC exercise. Coefficients of Variation (CV%) ranged from 23.0 to 22.5 when reading one transect and from 14.0 to 16.0 when reading five transects for Alternaria and Epicoccum, respectively. Considering a CV% of 30 as the limit for fungal spores, no significant differences were observed between the absolute errors from two, three, four and five transects. The only significant difference was between one and five transects. We recommend that fungal spore analysis should be carried out on about 5% of the slide (two transects in this study) because results were not significantly different to five transects.

Trypsin is a serine protease with important applications such as protein sequencing and tissue dissociation. Preserving protein structure and its activity during freeze-thawing and prolonging its shelf life is one of the most interesting tasks in biochemistry. In the present study, trypsin cryoprotection was achieved by altering buffer composition. Sodium phosphate buffer at pH 8.0 led to pH shift-induced destabilization of trypsin and formation of a molten globule, followed by significant activity loss (about 70%). Potassium phosphate and ammonium bicarbonate buffers at pH 8.0 were used with up to 90% activity recovery rate after 7 freeze-thaw cycles. The addition of non-ionic surfactants Tween 20 and Tween 80 led to up to 99% activity recovery rate. Amide I region changes, corresponding to specific secondary structures in the Fourier transform infrared (FTIR) spectrum, were modest in the case of Tween 20 and Tween 80. On the other hand, the addition of Triton X-100 led to the destabilization of α-helicoidal segments of trypsin structure after 7 freeze-thaw cycles but also increased protein substrate availability.

[Image: see text] Fibrosis is a major medical problem caused by excessive synthesis of the extracellular matrix, composed predominantly of type I collagen, in various tissues. There are no approved antifibrotic drugs, and the major obstacle in finding clinically relevant compounds is the lack of specificity of current experimental drugs for type I collagen. Here we describe the discovery of a lead compound that specifically inhibited secretion of type I collagen by fibroblasts in culture at IC(50) = 4.5 μM. The inhibition was specific for type I collagen, because secretion of fibronectin was not affected. In vitro, the compound inhibited binding of LARP6, the master regulator of translation of type I collagen mRNAs, to the 5′ stem-loop sequence element which regulates their translation. Because binding of LARP6 to collagen mRNAs is crucial for the development of fibrosis, this inhibitor represents a promising lead for optimization into specific antifibrotic drugs.

CRISPR-mediated genome editing has undoubtedly revolutionized genetic engineering of animals. With the ability for virtually unlimited modification of almost any genome it is easy to forget which amazing discoveries paved the way for this ground-breaking technology. Here, we summarize the history of genome editing platforms, starting from enhanced integration of foreign DNA by meganuclease-mediated double-strand breaks to CRISPR/Cas9, the leading technology to date, and its re-engineered variants.

Sociocultural changes in the human-animal relationship have led to increasing demands for animal welfare in biomedical research. The 3R concept is the basis for bringing this demand into practice: Replace animal experiments with alternatives where possible, Reduce the number of animals used to a scientifically justified minimum and Refine the procedure to minimize animal harm. The generation of gene-modified sentient animals such as mice and rats involves many steps that include various forms of manipulation. So far, no coherent analysis of the application of the 3Rs to gene manipulation has been performed. Here we provide guidelines from the Committee on Genetics and Breeding of Laboratory Animals of the German Society for Laboratory Animal Science to implement the 3Rs in every step during the generation of genetically modified animals. We provide recommendations for applying the 3Rs as well as success/intervention parameters for each step of the process, from experiment planning to choice of technology, harm-benefit analysis, husbandry conditions, management of genetically modified lines and actual procedures. We also discuss future challenges for animal welfare in the context of developing technologies. Taken together, we expect that our comprehensive analysis and our recommendations for the appropriate implementation of the 3Rs to technologies for genetic modifications of rodents will benefit scientists from a wide range of disciplines and will help to improve the welfare of a large number of laboratory animals worldwide.

Centro de Investigación Forestal (CIFOR), Spruce-fir-beech mixed forests cover a large area in European mountain regions, with high ecological and socio-economic importance. As elevation-zone systems they are highly affected by climate change, which is modifying species growth patterns and productivity shifts among species. The extent to which associated tree species can access resources and grow asynchronously may affect their resistance and persistence under climate change. Intra-specific synchrony in annual tree growth is a good indicator of species specific dependence on environmental conditions variability. However, little attention has been paid to explore the role of the inter-specific growth asynchrony in the adaptation of mixed forests to climate change. Here we used a database of 1790 tree-ring series collected from 28 experimental plots in spruce-fir-beech mixed forests across Europe to explore how spatio-temporal patterns of the intra- and inter-specific growth synchrony relate to climate variation during the past century. We further examined whether synchrony in growth response to inter-annual environmental fluctuations depended on site conditions. We found that the inter-specific growth synchrony was always lower than the intra-specific synchrony, for both high (inter-annual fluctuations) and low frequency (mid- to long-term) growth variation, suggesting between species niche complementarity at both temporal levels. Intra- and inter-specific synchronies in inter-annual growth fluctuations significantly changed along elevation, being greater at higher elevations. Moreover, the climate warming likely induced temporal changes in synchrony, but the effect varied along the elevation gradient. The synchrony strongly intensified at lower elevations likely due to climate warming and drying conditions. Our results suggest that intra- and inter-specific growth synchrony can be used as an indicator of temporal niche complementarity among species. We conclude that spruce-fir-beech mixtures should be preferred against mono-specific forests to buffer climate change impacts in mountain regions., This study was supported by the COST Action CLIMO (Climate-Smart Forestry in Mountain Regions – CA15226) financially supported by the EU Framework Programme for Research and Innovation HORIZON 2020. This publication is also part of a project that has received funding from the European Union’s HORIZON 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No 778322 (CARE4C)., 10 Pág.

Fishes of the genus Abudefduf are increasingly being recorded in Mediterranean waters, with most of these occurrences being based on the sole inspection of underwater images or video footage. Here we present a critical review of these occurrences, taking into account the uncertainties related to the identification of the congeneric A. vaigiensis and A. saxatilis, and testing the validity of the external traits used so far to distinguish these two species. After a careful revision of the Mediterranean literature and based on the analyses of images mostly taken in their natural native environment, we highlight the possibility that major morphological characters previously used to distinguish A. saxatilis from A. vaigiensis are not consistent within the species and thus not sufficiently reliable for their discrimination. This also relates to the possible confusion of these two species with a third species A. troschelii, never reported in the Mediterranean Sea to date. Finally, new records of the genus Abudefduf are reported for the Adriatic Sea and Tunisian waters. (PDF) On the occurrence of Abudefduf spp. (Pisces: Pomacentridae) in the Mediterranean Sea: a critical review with new records. Available from: https://www.researchgate.net/publication/347802 447_On_the_occurrence_of_Abudefduf_spp_Pisces_P omacentridae_in_the_Mediterranean_Sea_a_critica l_review_with_new_records [accessed May 21 2021].

Intergenerational inheritance of immune traits linked to epigenetic modifications has been demonstrated in plants and invertebrates. Here we provide evidence for transmission of trained immunity across generations to murine progeny that survived a sublethal systemic infection with Candida albicans or a zymosan challenge. The progeny of trained mice exhibited cellular, developmental, transcriptional and epigenetic changes associated with the bone marrow-resident myeloid effector and progenitor cell compartment. Moreover, the progeny of trained mice showed enhanced responsiveness to endotoxin challenge, alongside improved protection against systemic heterologous Escherichia coli and Listeria monocytogenes infections. Sperm DNA of parental male mice intravenously infected with the fungus C. albicans showed DNA methylation differences linked to immune gene loci. These results provide evidence for inheritance of trained immunity in mammals, enhancing protection against infections. Transgenerational transmission of acquired immunological traits has been demonstrated in invertebrates and plants but not mammals. Katzmarski et al. demonstrate that trained immunity that protects against heterologous infections can be transmitted to F2 offspring.

Osteosarcoma (OS) is a molecularly heterogeneous, aggressive, poorly differentiated pediatric bone cancer that frequently spreads to the lung. Relatively little is known about phenotypic and epigenetic changes that promote lung metastases. To identify key drivers of metastasis, we studied human CCH-OS-D OS cells within a previously described rat acellular lung (ACL) model that preserves the native lung architecture, extracellular matrix, and capillary network. This system identified a subset of cells—termed derived circulating tumor cells (dCTCs)—that can migrate, intravasate, and spread within a bioreactor-perfused capillary network. Remarkably, dCTCs highly expressed epithelial-to-mesenchymal transition (EMT)-associated transcription factors (EMT-TFs), such as ZEB1, TWIST, and SOX9, which suggests that they undergo cellular reprogramming toward a less differentiated state by coopting the same epigenetic machinery used by carcinomas. Since YAP/TAZ and AXL tightly regulate the fate and plasticity of normal mesenchymal cells in response to microenvironmental cues, we explored whether these proteins contributed to OS metastatic potential using an isogenic pair of human OS cell lines that differ in AXL expression. We show that AXL inhibition significantly reduced the number of MG63.2 pulmonary metastases in murine models. Collectively, we present a laboratory-based method to detect and characterize a pure population of dCTCs, which provides a unique opportunity to study how OS cell fate and differentiation contributes to metastatic potential. Though the important step of clinical validation remains, our identification of AXL, ZEB1, and TWIST upregulation raises the tantalizing prospect that EMT-TF-directed therapies might expand the arsenal of therapies used to combat advanced-stage OS.

Introduction/Objective. Aggressive periodontitis (AP) is a progressive disease that leads damage to periodontal tissues. The aim of the study was the analysis of intracellular enzymes: aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), acid phosphatase (ACP) and electrolytes in the saliva of patients with AP and their correlation with clinical parameters before and after the therapy. Methods. The study included 30 patients with AP (experimental group) and 35 patients with healthy parodontium (control group). Intracellular enzymes and electrolytes were analyzed in an unstimulated saliva of subjects with AP, before and after the therapy and in saliva of the control group. The analysis of biochemical markers was carried out using kinetic methods with commercial reagents. Results. Concentrations of the biochemical markers AST (28.18 ± 25.16), ALT (5.48 ± 5.14), ALP (31.13 ± 37.79), ACP (17.53 ± 14.77), calcium (2.80 ± 1.97), phosphate (4.43 ± 1.92) in the saliva of subjects of the experimental group were statistically significantly higher in relation to the control group (p = 0.000; p = 0.001). Significant correlation was found between AST values, debris index (ρ = -0.444; p = 0.026) and calculus index (ρ = -0.513; p = 0.009), and between the plaque index and ALP level in the saliva after therapy (p = 0.020). Conclusion. The investigation will contribute to a better understanding and standardization of biomarkers in the saliva that mayhelp in diagnosing the AP and evaluation of the applied therapy.

Shiga-toxin-producing Escherichia coli (STEC) and Salmonella enterica serovar Typhimurium (STDT104) are foodborne pathogens of public health significance. It is less known that Shiga-toxin-producing Escherichia coli (with cattle being the most probable natural reservoir) can be isolated from pigs, sheep and wildlife as well. The basic information about detection of Shiga-toxin-producing genes in STEC as well as the origin of Salmonella Typhimurium definite phage type 4 (STDT4) the virulence and resistance mechanisms including their distribution in the world is presented. Due to the foodborne transmission mechanisms we emphasize the role of veterinary scientist in Serbia in implementing good management practice on animal farms and in strengthening laboratory diagnostic capacities.

Objectives Malaria treatment is impeded by increasing resistance to conventional antimalarial drugs. Here we explored the activity of ten novel benzothiophene, thiophene and benzene aminoquinolines. Methods In vitro testing was performed by the lactate dehydrogenase assay in chloroquine (CQ)-sensitive Plasmodium falciparum strain 3D7 and CQ-resistant (CQR) P. falciparum strain Dd2. In vivo activity was evaluated by a modified Thompson test using C57BL/6 mice infected with Plasmodium berghei ANKA strain. Results Nine of the ten compounds had a lower 50% inhibitory concentration (IC50) than CQ against the CQR strain Dd2. Five of these compounds that were available for in vivo evaluation were shown to be non-toxic. All five compounds administered at a dose of 160 mg/kg/day for 3 days prolonged the survival of treated compared with untreated mice. Untreated control mice died by Day 7 with a mean parasitaemia of 15%. Among treated mice, a dichotomous outcome was observed, with a two-third majority of treated mice dying by Day 17 with a low mean parasitaemia of 5%, whilst one-third survived longer with a mean hyperparasitaemia of 70%; specifically, five of these mice survived a mean of 25 days, whilst two even survived past Day 31. Conclusions The significant antimalarial potential of this aminoquinoline series is illustrated by its excellent in vitro activity against the CQR P. falciparum strain and significant in vivo activity. Interestingly, compounds ClAQ7, ClAQ9 and ClAQ11 were able to confer resistance to cerebral malaria and afford a switch to hyperparasitaemia to mice prone to the neurological syndrome.

Two new species of the subterranean and anophthalmous genus Paramaurops Jeannel, 1948 are described and illustrated: P. alenkirini sp. n. and P. struyvei sp. n. from Croatia. The distribution of all Croatian records of the genus is given in the map., U radu su opisane dvije nove vrste podzemnih i slijepih kornjaša roda Paramaurops Jeannel, 1948: P. alenkirini sp. n. i P. struyvei sp. n. iz Hrvatske. Ilustrirani su muški kopulatorni organi obje vrste. Distribucija svih zapisa ovog roda u Hrvatskoj prikazana je na karti.

STUDY QUESTION Are de novo mutations in the human genome associated with male infertility? SUMMARY ANSWER We identified de novo mutations in five candidate genes: SEMA5A, NEURL4, BRD2, CD1D, and CD63. WHAT IS KNOWN ALREADY Epidemiological and genetic studies have consistently indicated contribution of genetic factors to the etiology of male infertility, suggesting that more than 1500 genes are involved in spermatogenesis. STUDY DESIGN, SIZE, DURATION First, we searched for de novo mutations in patients with idiopathic azoospermia with whole-exome sequencing (WES). To evaluate the potential functional impact of de novo identified mutations, we analyzed their expression differences on independent testis samples with normal and impaired spermatogenesis. In the next step, we tested additional group of azoospermic patients for mutations in identified genes with de novo mutations. In addition to the analysis of de novo mutations in patients with idiopathic azoospermia, we considered other models of inheritance and searched for candidate genes harboring rare maternally inherited variants and biallelic autosomal and X-chromosome hemizygous variants. PARTICIPANTS/MATERIALS, SETTING, METHODS We performed WES in 13 infertile males with idiopathic azoospermia and their parents. Potential functional impact of de novo identified mutations was evaluated by global gene expression profiling on 20 independent testis samples. To replicate the results, we performed WES in further 16 independent azoospermic males, which were screened for the variants in the same genes. Library preparation was performed with Nextera Coding Exome Capture Kit (Illumina), with subsequent sequencing on Illumina HiSeq 2500 platform. MAIN RESULTS AND THE ROLE OF CHANCE We identified 11 de novo mutations in 10 genes of which 5 were considered potentially associated with azoospermia: SEMA5A, NEURL4, BRD2, CD1D, and CD63. All candidate genes showed significant differential expression in testis samples composed of patients with severely impaired and normal spermatogenesis. Additionally, we identified rare, potentially pathogenic mutations in the genes previously implicated in male infertility-a maternally inherited heterozygous frameshift variant in FKBPL gene and inframe deletion in UPF2 gene, homozygous frameshift variant in CLCA4 gene, and a heterozygous missense variant NR0B1 gene, which represent promising candidates for further clinical implication. LIMITATIONS OF THE STUDY, REASONS FOR CAUTION We provided limited functional support for involvement of de novo identified genes in pathogenesis of male infertility, based on expression analysis. Additionally, the sample size was limited. WIDER IMPLICATIONS OF THE FINDINGS We provide support that de novo mutations might contribute to male infertility and propose five genes as potentially implicated in its pathogenesis.

Background Rare genetic variants contribute to the etiology of both autism spectrum disorder (ASD) and schizophrenia (SCZ). Most genetic studies limit their focus to likely gene-disrupting mutations because they are relatively easier to interpret their effects on the gene product. Interpretation of missense variants is also informative to some pathophysiological mechanisms of these neurodevelopmental disorders; however, their contribution has not been elucidated because of relatively small effects. Therefore, we characterized missense variants detected in NRXN1, a well-known neurodevelopmental disease-causing gene, from individuals with ASD and SCZ. Methods To discover rare variants with large effect size and to evaluate their role in the shared etiopathophysiology of ASD and SCZ, we sequenced NRXN1 coding exons with a sample comprising 562 Japanese ASD and SCZ patients, followed by a genetic association analysis in 4273 unrelated individuals. Impact of each missense variant detected here on cell surface expression, interaction with NLGN1, and synaptogenic activity was analyzed using an in vitro functional assay and in silico three-dimensional (3D) structural modeling. Results Through mutation screening, we regarded three ultra-rare missense variants (T737M, D772G, and R856W), all of which affected the LNS4 domain of NRXN1α isoform, as disease-associated variants. Diagnosis of individuals with T737M, D772G, and R856W was 1ASD and 1SCZ, 1ASD, and 1SCZ, respectively. We observed the following phenotypic and functional burden caused by each variant. (i) D772G and R856W carriers had more serious social disabilities than T737M carriers. (ii) In vitro assay showed reduced cell surface expression of NRXN1α by D772G and R856W mutations. In vitro functional analysis showed decreased NRXN1α-NLGN1 interaction of T737M and D772G mutants. (iii) In silico 3D structural modeling indicated that T737M and D772G mutations could destabilize the rod-shaped structure of LNS2-LNS5 domains, and D772G and R856W could disturb N-glycan conformations for the transport signal. Conclusions The combined data suggest that missense variants in NRXN1 could be associated with phenotypes of neurodevelopmental disorders beyond the diagnosis of ASD and/or SCZ.