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81.
Platelet-rich plasma, especially when combined with a TGF-ß inhibitor promotes proliferation, viability and myogenic differentiation of myoblasts in vitro
Robi Kelc, Martin Trapečar, Lidija Gradišnik, Marjan Rupnik, Matjaž Vogrin, 2015, izvirni znanstveni članek

Opis: Regeneration of skeletal muscle after injury is limited by scar formation, slow healing time and a high recurrence rate. A therapy based on platelet-rich plasma (PRP) has become a promising lead for tendon and ligament injuries in recent years, however concerns have been raised that PRP-derived TGF-β could contribute to fibrotic remodelling in skeletal muscle after injury. Due to the lack of scientific grounds for a PRP -based muscle regeneration therapy, we have designed a study using human myogenic progenitors and evaluated the potential of PRP alone and in combination with decorin (a TGF-β inhibitor), to alter myoblast proliferation, metabolic activity, cytokine profile and expression of myogenic regulatory factors (MRFs). Advanced imaging multicolor single-cell analysis enabled us to create a valuable picture on the ratio of quiescent, activated and terminally committed myoblasts in treated versus control cell populations. Finally high-resolution confocal microscopy validated the potential of PRP and decorin to stimulate the formation of polynucleated myotubules. PRP was shown to down-regulate fibrotic cytokines, increase cell viability and proliferation, enhance the expression of MRFs, and contribute to a significant myogenic shift during differentiation. When combined with decorin further synergistc effects were identified. These results suggest that PRP could not only prevent fibrosis but could also stimulate muscle commitment, especially when combined with a TGF-β inhibitor.
Ključne besede: muscles, skeletal, injuries, TGF-beta, plasma, thrombocytes, myoblasts, fibrosis, prevention, regeneration
Objavljeno: 19.06.2017; Ogledov: 56; Prenosov: 0
.pdf Polno besedilo (2,07 MB)

82.
Novel insights into the downstream pathways and targets controlled by transcription factors CREM in the testis
Rok Košir, Peter Juvan, Martina Perše, Tomaž Büdefeld, Gregor Majdič, Martina Fink, Paolo Sassone-Corsi, Damjana Rozman, 2012, izvirni znanstveni članek

Opis: The essential role of the Crem gene in normal sperm development is widely accepted and is confirmed by azoospermia in male mice lacking the Crem gene. The exact number of genes affected by Crem absence is not known, however a large difference has been observed recently between the estimated number of differentially expressed genes found in Crem knock-out (KO) mice compared to the number of gene loci bound by CREM. We therefore re-examined global gene expression in male mice lacking the Crem gene using whole genome transcriptomeanalysis with Affymetrix microarrays and compared the lists of differentially expressed genes from Crem-/- mice to a dataset of genes where binding of CREM was determined by Chip-seq. We determined the global effect ofCREM on spermatogenesis as well as distinguished between primary and secondary effects of the CREM absence. We demonstrated that the absence of Crem deregulates over 4700 genes in KO testis. Among them are 101 genes associated with spermatogenesis 41 of which are bound by CREM and are deregulated in Crem KO testis. Absence of several of these genes in mouse models has proven their importance for normal spermatogenesis and male fertility. Our study showed that the absence of Crem plays a more important role on different aspects of spermatogenesis as estimated previously, with itsimpact ranging from apoptosis induction to deregulation of major circadian clock genes, steroidogenesis and the cell-cell junction dynamics. Several new genes important for normal spermatogenesis and fertility are down-regulated inKO testis and are therefore possible novel targets of CREM.
Ključne besede: spermatogenesis, genetics, Crem, mice
Objavljeno: 19.06.2017; Ogledov: 25; Prenosov: 0
.pdf Polno besedilo (1,77 MB)

83.
Malpighian tubule cells in overwintering cave crickets Troglophilus cavicola (Kollar, 1833) and T. neglectus Krauss, 1879 (Rhaphidophoridae, Ensifera)
Saška Lipovšek Delakorda, Tone Novak, Franc Janžekovič, Nina Weiland, Gerd Leitinger, 2016, izvirni znanstveni članek

Opis: During winter, cave cricket larvae undergo dormancy in subterranean habitats; this dormancy is termed diapause in second year Troglophilus cavicola larvae because they mature during this time, and termed quiescence in T. neglectus, because they mature after dormancy. Here we used electron microscopy to analyze ultrastructural changes in the epithelial cells in the Malpighian tubules (MTs) of T. cavicola during diapause, in order to compare them with previous findings on T. neglectus. Moreover, the autophagosomes were studied with immunofluorescence microscopy in both species. Although the basic ultrastructure of the cells was similar, specific differences appeared during overwintering. During this natural starvation period, the nucleus, rER, the Golgi apparatus and mitochondria did not show structural changes, and the spherites were exploited. The abundances of autophagic structures in both species increased during overwintering. At the beginning of overwintering, in both species and sexes, the rates of cells with autophagic structures (phagophores, autophagosomes, autolysosomes and residual bodies) were low, while their rates increased gradually towards the end of overwintering. Between sexes, in T. cavicola significant differences were found in the autophagosome abundances in the middle and at the end, and in T. neglectus at the end of overwintering. Females showed higher rates of autophagic cells than males, and these were more abundant in T. cavicola. Thus, autophagic processes in the MT epithelial cells induced by starvation are mostly parallel in diapausing T. cavicola and quiescent T. neglectus, but more intensive in diapausing females.
Ključne besede: cave cricket, overwintering, Troglophilus cavicola, Troglophilus neglectus
Objavljeno: 19.06.2017; Ogledov: 34; Prenosov: 1
.pdf Polno besedilo (9,29 MB)

84.
Highly divergent Clostridium difficile strains isolated from the environment
Sandra Janežič, Mojca Potočnik, Valerija Tkalec, Maja Rupnik, 2016, izvirni znanstveni članek

Opis: Clostridium difficile is one of the most important human and animal pathogens. However, the bacterium is ubiquitous and can be isolated from various sources. Here we report the prevalence and characterization of C. difficile in less studied environmental samples, puddle water (n = 104) and soil (n = 79). C. difficile was detected in 14.4% of puddle water and in 36.7% of soil samples. Environmental strains displayed antimicrobial resistance patterns comparable to already published data of human and animal isolates. A total of 480 isolates were grouped into 34 different PCR ribotypes. More than half of these (52.9%; 18 of 34) were already described in humans or animals. However, 14 PCR ribotypes were new in our PCR ribotype library and all but one were non-toxigenic. The multilocus sequence analysis of these new PCR ribotypes revealed that non-toxigenic environmental isolates are phylogenetically distinct and belong to three highly divergent clades, two of which have not been described before. Our data suggest that environment is a potential reservoir of genetically diverse population of C. difficile.
Ključne besede: clostridium difficile, environment, isolation
Objavljeno: 19.06.2017; Ogledov: 58; Prenosov: 2
.pdf Polno besedilo (1,13 MB)

85.
Gut microbiota patterns associated with colonization of different clostridium difficile ribotypes
Jure Škraban, Sašo Džeroski, Bernard Ženko, Domen Mongus, Simon Gangl, Maja Rupnik, 2013, izvirni znanstveni članek

Opis: Abstract Introduction Materials and Methods Results Discussion Supporting Information Acknowledgments Author Contributions References Reader Comments (0) Figures Abstract C. difficile infection is associated with disturbed gut microbiota and changes in relative frequencies and abundance of individual bacterial taxons have been described. In this study we have analysed bacterial, fungal and archaeal microbiota by denaturing high pressure liquid chromatography (DHPLC) and with machine learning methods in 208 faecal samplesfrom healthy volunteers and in routine samples with requested C. difficile testing. The latter were further divided according to stool consistency, C. difficile presence or absence and C. difficile ribotype (027 or non-027). Lower microbiota diversity was a common trait of all routine samples and not necessarily connected only to C. difficile colonisation. Differences between the healthy donors and C. difficile positive routine samples were detected in bacterial, fungal and archaeal components. Bifidobacterium longum was the single most important species associated with C. difficile negative samples. However, by machine learning approaches we haveidentified patterns of microbiota composition predictive for C. difficile colonization. Those patterns also differed between samples with C. difficile ribotype 027 and other C. difficile ribotypes. The results indicate that not only the presence of a single species/group is important but that certain combinations of gut microbes are associated with C. difficile carriage and that some ribotypes (027) might be associated with more disturbed microbiota than the others.
Ključne besede: microbiota, clostridium difficile, chromatography
Objavljeno: 19.06.2017; Ogledov: 33; Prenosov: 2
.pdf Polno besedilo (1,01 MB)

86.
Family history as a predictor for disease risk in healthy individuals
Zalika Klemenc-Ketiš, Borut Peterlin, 2013, izvirni znanstveni članek

Opis: Background Family history can be used as a genetic risk predictor for common non-communicable diseases. The aim of this study was to determine the prevalence of healthy individuals at risk of developing these diseases, based on their self-reported family history.Methods and Findings This was a cross-sectional observational study. Data were collected in the three largest occupational practices in primary health care centres in Slovenia, a Central European country. The study population consisted of consecutive individuals who came to occupational practices for their regular preventive check-up from November 2010 to June 2012. We included 1,696 individuals. Data were collected by a self-developed questionnaire. The main outcome was the number of participants at a moderate or high risk for the development of cardiovascular diseases, diabetes, and cancer.The final sample consisted of 1,340 respondents. Moderate or high risk for the development of cardiovascular diseases was present in 280 (20.9%) participants, for the development of diabetes in 154 (11.5%) participants and for cancer in 163 (12.1%) participants. Conclusions In this study, we found a significant proportion of healthy individuals with an increased genetic risk for common non-communicable diseases; consequently further genetic and clinical evaluation and preventive measures should be offered.
Ključne besede: family history, predictor for disease risk, sectional study in Slovenia
Objavljeno: 19.06.2017; Ogledov: 24; Prenosov: 0
.pdf Polno besedilo (161,26 KB)

87.
A co-culture model of the developing small intestine offers new insight in the early immunomodulation of enterocytes and macrophages by Lactobacillus spp. through STAT1 and NF-kB p65 translocation
Martin Trapečar, Aleš Goropevšek, Mario Gorenjak, Lidija Gradišnik, Marjan Rupnik, 2014, izvirni znanstveni članek

Opis: The early establishment of a complete microbiome has been shown to play an integral part in the development and maintenance of an intact intestine and its immune system, although much remains unknown about the specific mechanisms of immune modulation in newborns. In our study we show in a co-culture model of the undeveloped small intestine that members of Lactobacillus spp. influence STAT1 and NF-kB p65 nuclear translocation in both intestinal epithelial cells as well as underlying macrophages. Moreover, by using imaging flow cytometry we were able to monitor each individual cell and create a framework of the percentage of cells in which translocation occurred in challenged versus control cell populations. We also observed a significant difference in baseline translocation in intestinal cells when cultured alone versus those in a co-culture model, underpinning the importance of 3D models over monolayer set-ups in epithelial in vitro research. In conclusion, our work offers new insights into the potential routes by which the commensal microbiome primes the early immune system to fight pathogens, and shows how strain-specific these mechanisms really are.
Ključne besede: microbiome, Lactobacillus, immune system, pathogens
Objavljeno: 19.06.2017; Ogledov: 52; Prenosov: 0
.pdf Polno besedilo (2,86 MB)

88.
Functional connectivity in islets of Langerhans from mouse pancreas tissue slices
Jurij Dolenšek, Matjaž Perc, Marko Marhl, Marjan Rupnik, Dean Korošak, Andraž Stožer, Marko Gosak, 2013, izvirni znanstveni članek

Opis: We propose a network representation of electrically coupled beta cells in islets of Langerhans. Beta cells are functionally connected on the basis of correlations between calcium dynamics of individual cells, obtained by means of confocal laser-scanning calcium imaging in islets from acute mouse pancreastissue slices. Obtained functional networks are analyzed in the light of known structural and physiological properties of islets. Focusing on the temporal evolution of the network under stimulation with glucose, we show thatthe dynamics are more correlated under stimulation than under non-stimulated conditions and that the highest overall correlation, largely independent of Euclidean distances between cells, is observed in the activation and deactivation phases when cells are driven by the external stimulus. Moreover, we find that the range of interactions in networks during activity shows a clear dependence on the Euclidean distance, lending support to previous observations that beta cells are synchronized via calcium waves spreading throughout islets. Most interestingly, the functional connectivity patterns between beta cells exhibit small-world properties, suggesting that beta cells do not form a homogeneous geometric network but are connected in a functionally more efficient way. Presented results provide support for the existing knowledge of beta cell physiology from a network perspective and shedimportant new light on the functional organization of beta cell syncitia whose structural topology is probably not as trivial as believed so far.
Ključne besede: islets of Langerhans, mouse pancreas
Objavljeno: 16.06.2017; Ogledov: 131; Prenosov: 1
.pdf Polno besedilo (798,57 KB)

89.
Intracellular serotonin modulates insulin secretion from pancreatic ß-cells by protein serotonylation
Nils Paulmann, Maik Grohmann, Jörg-Peter Voigt, Bettina Bert, Jakob Vowinckel, Michael Bader, Maša Skelin, Marko Jevšek, Heidrun Fink, Marjan Rupnik, Diego Walther, 2009, izvirni znanstveni članek

Opis: While serotonin (5-HT) co-localization with insulin in granules of pancreatic ß-cells was demonstrated more than three decades ago, its physiological role in the etiology of diabetes is stili unclear. We combined biochemical and electrophysiological analyses of mice selectively deficient in peripheral tryptophan hydroxylase (Tph1-/-) and 5-HT to show that intracellular 5-HT regulates insulin secretion. We found that these mice are diabetic and have an impaired insulin secretion due to the lack of 5-HT in the pancreas. The pharmacological restoration of peripheral 5-HT levels rescued the impaired insulin secretion in vivo. These findings were further evidenced by patch clamp experiments with isolated Tph1-/- ß-cells, which clearly showed that the secretory defect is downstream of Ca2+ -signaling and can be rescued by direct intracellular application of 5-HT via the clamp pipette. In elucidating the underlying mechanism further, we demonstrate the covalent coupling of 5-HT by transglutaminases during insulin exocytosis to two key players in insulin secretion, the small GTPases Rab3a and Rab27a. This renders them constitutively active in a receptor-independent signaling mechanism we have recently termed serotonylation. Concordantly, an inhibition of such activating serotonylation in ß-cells abates insulin secretion. We also observed inactivation of serotonylated Rab3a by enhanced proteasomal degradation, which is in line with the inactivation of other serotonylated GTPases. Our results demonstrate that 5-HT regulates insulin secretion by serotonylation of GTPases within pancreatic ß-cells and suggest that intracellular 5-HT functions in various microenvironments via this mechanism in concert with the known receptor-mediated signaling.
Ključne besede: insulin secretion, serotonin, insulin, glucose, diabetes mellitus, guanosine triphosphatase, exocytosis, pancreas
Objavljeno: 16.06.2017; Ogledov: 95; Prenosov: 0
.pdf Polno besedilo (774,75 KB)

90.
Percutaneous trigeminal ganglion balloon compression rhizotomy: experience in 27 patients
Tadej Strojnik, Tomaž Šmigoc, 2012, izvirni znanstveni članek

Opis: Purpose. Percutaneous ganglion balloon compression (PBC) is a minimally invasive procedure for treatment of trigeminal neuralgia. Materials and Methods. Twenty-seven (19 female and 8 male) patients, who presented with classical symptoms of trigeminal neuralgia, were included. Age ranged from34 to 91 years (median 62 years), 33 procedures were performed. Duration of the symptoms ranged from 1 year to 30 years (median 5 years). Results. After the procedure, pain relief was reported in 25 (93%) patients. In two patients, the pain remained the same. The pain free period ranged from 2 to 74 months (median 15 months). A mean duration of analgesia was longer in patients with ideal pear shape of balloon at the time of the procedure compared to nonideal shape (P = 0.01). No major complications occurred in our group of patients. Conclusions. Percutaneous trigeminal ganglion balloon compression is a safe, simple, and effective method for temporary pain relief in a selective group of trigeminal neuralgia patients.
Ključne besede: balloon compression
Objavljeno: 15.06.2017; Ogledov: 42; Prenosov: 0
.pdf Polno besedilo (2,62 MB)

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