Pojav interseksulanosti pri sladkovodnih ribah v slovenskih rekah: histološka ocena

The aim of this preliminary research was to establish if there are intersex occurrences in wild freshwater fish in Slovenian rivers and streams. In the first study we evaluated all fish species of both sexes obtained from the river Ljubljanica from its source to mouth. In the second study we focused on the rainbow trout (Oncorhynchus mykiss) and brown trout (Salmo trutta m. fario) males from 30 rivers and streams in different parts of Slovenia. The male gonads were histologically assessed for the presence of oocytes to determine the frequency and degree of intersex. Oocytes were found in the testicular tissue of a single grayling (Thymallus thymallus) and in the adipose tissue adjacent to the testis of a single common barbell (Barbus barbus), both from the Ljubljanica. Several cyst-like structures that resemble degenerated presumptive oocytes were also present in several trout testes. This preliminary report is the first of its kind in Slovenia. To gain a better insight into the intersex issue in Slovenia, we plan to regularly biomonitor freshwater pollution by histologically examining fish gonads and, if possible, by determining vitellogenin plasma levels in fish.V naši raziskavi smo v izbranih slovenskih rekah ugotavljali pojav feminizacije rib. V prvi del raziskave so bile vključene ribe različnih vrst in obeh spolov, izlovljene na različnih lokacijah iz reke Ljubljanice, od njenega izvira do izliva. V drugem delu raziskave smo se osredotočili samo na samce šarenke (Oncorhynchus mykiss) in potočne postrvi (Salmo trutta m. fario) iz izbranih rek in potokov po vsej Sloveniji. Prisotnost in pogostnost jajčnih celic v testisih ter stopnjo interseksulanosti smo ugotavljali s histološko analizo. Prisotnost jajčnih celic smo ugotovili v tkivu moda lipana (Thymallus thymallus) ter še v maščobnem tkivu v neposredni bližina moda mrene (Barbus barbus). Poleg tega smo v posameznih primerih ugotovili še cistam podobne tvorbe, ki so domnevno degenerirane jajčne celice. Z opravljeno študijo smo dobili prvi vpogled o pojavnosti interseksualnosti pri ribah v slovenskih vodotokih

Effect of Pig Domestication on Skeletal Muscle Development, Microstructure, and Genetic Mechanism Involved in Myofibre Type Formation

The wild boar and modern highly selected pigs are phenotypically distant European pig breeds reared in contrasting conditions and present ideal model to better understand the mechanisms behind meat quality deterioration related to domestication and selection pressure, which provoked substantial modifications in the ontogenic development as well as contractile and metabolic properties of skeletal muscles. The skeletal muscle of domestic pigs are less mature at birth and contains a lower number of myofibres compared to wild boars; however, expansive myofibre hypertrophy, protein accretion as well as additional myofibre formation are accelerated in the early postnatal period in some muscles in domestic pigs. A comparative view of the cellular and subcellular mechanisms underlying the skeletal myofibre development could help to design a breeding program that would improve the balance between the growth performance, muscularity and meat quality. This chapter therefore outlines the influence of domestication on myofibre formation and differentiation during growth and provides a comparative view on the developmental expression pattern of the MyHC isoforms, the activity of different metabolic enzymes, and the expression of selected genes responsible for the metabolic diversity of the myofibres. Additionally, there is a special emphasis on the type, composition, and histomorphological traits of myofibres

Critical Impact of Different Conserved Endoplasmic Retention Motifs and Dopamine Receptor Interacting Proteins (DRIPs) on Intracellular Localization and Trafficking of the D2 Dopamine Receptor (D2-R) Isoforms

The type 2 dopamine receptor D2 (D2-R), member of the G protein-coupled receptor (GPCR) superfamily, exists in two isoforms, short (D2S-R) and long (D2L-R). They differ by an additional 29 amino acids (AA) in the third cytoplasmic loop (ICL3) of the D2L-R. These isoforms differ in their intracellular localization and trafficking functionality, as D2L-R possesses a larger intracellular pool, mostly in the endoplasmic reticulum (ER). This review focuses on the evolutionarily conserved motifs in the ICL3 of the D2-R and proteins interacting with the ICL3 of both isoforms, specifically with the 29 AA insert. These motifs might be involved in D2-R exit from the ER and have an impact on cell-surface and intracellular localization and, therefore, also play a role in the function of dopamine receptor signaling, ligand binding and possible homo/heterodimerization. Our recent bioinformatic data on potential new interaction partners for the ICL3 of D2-Rs are also presented. Both are highly relevant, and have clinical impacts on the pathophysiology of several diseases such as Parkinson’s disease, schizophrenia, Tourette’s syndrome, Huntington’s disease, manic depression, and others, as they are connected to a variety of essential motifs and differences in communication with interaction partners

Combined in silico and experimental approach to identify the peptide mimetic of the nanobody that stabilize functional conformational state of the beta2 adrenergic receptor (β2AR)

Stabilization of specific G-protein coupled receptor (GPCR) conformation is achieved by ligand binding to orthosteric or allosteric sites on a GPCRs. A crucial unresolved issue in GPCRs activation/signaling is the role of receptor structural conformations in G protein/effector protein selection. One of the possible approaches to get comprehensive depiction of GPCRs activation dynamics are molecular simulations and recently described nanobody-derived intrabodies. Monomeric single-domain antibody (nanobody) from the Camelid family was found to allosterically bind to and stabilizes distinct conformational states of the β2AR. By applying informational spectrum method (ISM), a virtual spectroscopy method for investigation of the protein-protein interactions, we have designed peptide mimetic of the nanobody related to the β2AR (nanobody derived peptide, NDP). Further, interaction between NDP and the ligand-bound β2AR active conformation have been studied by protein-peptide docking, molecular dynamics simulations and metadynamics calculations of free energy binding. Finally, the affinity of selected NDPs towards agonist-activated β2AR was also studied by microscale thermophoresis (MST) and by bioluminescence resonance energy transfer (BRET) based β-arrestin 2 recruitment assay. MST data predicted micromolar range interaction of selected NDPs with the β2AR, while the preliminary β-arrestin 2 recruitment results suggest prospective further modification and optimization of NDPs toward effective modulators of the β2AR.Special Edition of Book of Abstract

Patterns of human and porcine gammaherpesvirus-encoded BILF1 receptor endocytosis

The viral G-protein-coupled receptor (vGPCR) BILF1 encoded by the Epstein–Barr virus (EBV) is an oncogene and immunoevasin and can downregulate MHC-I molecules at the surface of infected cells. MHC-I downregulation, which presumably occurs through co-internalization with EBV-BILF1, is preserved among BILF1 receptors, including the three BILF1 orthologs encoded by porcine lymphotropic herpesviruses (PLHV BILFs). This study aimed to understand the detailed mechanisms of BILF1 receptor constitutive internalization, to explore the translational potential of PLHV BILFs compared with EBV-BILF1

Computational design and characterization of nanobody-derived peptides that stabilize the active conformation of the β2-adrenergic receptor (β2-AR)

This study aimed to design and functionally characterize peptide mimetics of the nanobody (Nb) related to the β2-adrenergic receptor (β2-AR) (nanobody-derived peptide, NDP). We postulated that the computationally derived and optimized complementarity-determining region 3 (CDR3) of Nb is sufficient for its interaction with receptor. Sequence-related Nb-families preferring the agonist-bound active conformation of β2-AR were analysed using the informational spectrum method (ISM) and β2-AR:NDP complexes studied using protein-peptide docking and molecular dynamics (MD) simulations in conjunction with metadynamics calculations of free energy binding. The selected NDP of Nb71, designated P3, was 17 amino acids long and included CDR3. Metadynamics calculations yielded a binding free energy for the β2-AR:P3 complex of ΔG = (−7.23 ± 0.04) kcal/mol, or a Kd of (7.9 ± 0.5) μM, for T = 310 K. In vitro circular dichroism (CD) spectropolarimetry and microscale thermophoresis (MST) data provided additional evidence for P3 interaction with agonist-activated β2-AR, which displayed ~10-fold higher affinity for P3 than the unstimulated receptor (MST-derived EC50 of 3.57 µM vs. 58.22 µM), while its ability to inhibit the agonist-induced interaction of β2-AR with β-arrestin 2 was less evident. In summary, theoretical and experimental evidence indicated that P3 preferentially binds agonist-activated β2-AR. © 2019, The Author(s)

Computational Modeling and Characterization of Peptides Derived from Nanobody Complementary-Determining Region 2 (CDR2) Targeting Active-State Conformation of the β2-Adrenergic Receptor (β2AR)

This study assessed the suitability of the complementarity-determining region 2 (CDR2) of the nanobody (Nb) as a template for the derivation of nanobody-derived peptides (NDPs) targeting active-state β2-adrenergic receptor (β2AR) conformation. Sequences of conformationally selective Nbs favoring the agonist-occupied β2AR were initially analyzed by the informational spectrum method (ISM). The derived NDPs in complex with β2AR were subjected to protein–peptide docking, molecular dynamics (MD) simulations, and metadynamics-based free-energy binding calculations. Computational analyses identified a 25-amino-acid-long CDR2-NDP of Nb71, designated P4, which exhibited the following binding free-energy for the formation of the β2AR:P4 complex (ΔG = −6.8 ± 0.8 kcal/mol or a Ki = 16.5 μM at 310 K) and mapped the β2AR:P4 amino acid interaction network. In vitro characterization showed that P4 (i) can cross the plasma membrane, (ii) reduces the maximum isoproterenol-induced cAMP level by approximately 40% and the isoproterenol potency by up to 20-fold at micromolar concentration, (iii) has a very low affinity to interact with unstimulated β2AR in the cAMP assay, and (iv) cannot reduce the efficacy and potency of the isoproterenol-mediated β2AR/β-arrestin-2 interaction in the BRET2-based recruitment assay. In summary, the CDR2-NDP, P4, binds preferentially to agonist-activated β2AR and disrupts Gαs-mediated signaling

Tff3 Deficiency Protects against Hepatic Fat Accumulation after Prolonged High-Fat Diet

Trefoil factor 3 (Tff3) protein is a small secretory protein expressed on various mucosal surfaces and is involved in proper mucosal function and recovery via various mechanisms, including immune response. However, Tff3 is also found in the bloodstream and in various other tissues, including the liver. Its complete attenuation was observed as the most prominent event in the early phase of diabetes in the polygenic Tally Ho mouse model of diabesity. Since then, its role in metabolic processes has emerged. To elucidate the complex role of Tff3, we used a new Tff3-deficient mouse model without additional metabolically relevant mutations (Tff3-/-/C57BL/6NCrl) and exposed it to a high-fat diet (HFD) for a prolonged period (8 months). The effect was observed in male and female mice compared to wild-type (WT) counter groups (n = 10 animals per group). We monitored the animals’ general metabolic parameters, liver morphology, ultrastructure and molecular genes in relevant lipid and inflammatory pathways. Tff3-deficient male mice had reduced body weight and better glucose utilization after 17 weeks of HFD, but longer HFD exposure (32 weeks) resulted in no such change. We found a strong reduction in lipid accumulation in male Tff3-/-/C57BL/6NCrl mice and a less prominent reduction in female mice. This was associated with downregulated peroxisome proliferator-activated receptor gamma (Pparγ) and upregulated interleukin-6 (Il-6) gene expression, although protein level difference did not reach statistical significance due to higher individual variations. Tff3-/-/C57Bl6N mice of both sex had reduced liver steatosis, without major fatty acid content perturbations. Our research shows that Tff3 protein is clearly involved in complex metabolic pathways. Tff3 deficiency in C57Bl6N genetic background caused reduced lipid accumulation in the liver ; further research is needed to elucidate its precise role in metabolism-related events