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Multiple sclerosis (MS) is a multifocal and progressive inflammatory disease of the central nervous system (CNS). However, the compartmentalized pathology of the disease affecting various anatomical regions including gray and white matter and lack of appropriate disease models impede understanding of the disease. Utilizing single-nucleus RNA-sequencing and multiplex spatial RNA mapping, we generated an integrated transcriptomic map comprising leukocortical, cerebellar and spinal cord areas in normal and MS tissues that captures regional subtype diversity of various cell types with an emphasis on astrocytes and oligodendrocytes. While we found strong cross-regional diversity among glial subtypes in control tissue, regional signatures become more obscure in MS. This suggests that patterns of transcriptomic changes in MS are shared across regions and converge on specific pathways, especially those regulating cellular stress and immune activation. In addition, we found evidence that a subtype of white matter oligodendrocytes appearing across all three CNS regions adopt pro-remyelinating gene signatures in MS. In summary, our data suggest that cross-regional transcriptomic glial signatures overlap in MS, with different reactive glial cell types capable of either exacerbating or ameliorating pathology.

Glutamine synthetase (GS) is a key enzyme that metabolizes glutamate into glutamine. While GS is highly enriched in astrocytes, expression in other glial lineages has been noted. Using a combination of reporter mice and cell type-specific markers, we show that GS is expressed in myelinating oligodendrocytes (OL) but not oligodendrocyte progenitor cells of the mouse and human ventral spinal cord. To investigate the role of GS in mature OL, we used a conditional knockout (cKO) approach to selectively delete GS-encoding gene (Glul) in OL, which caused a significant decrease in glutamine levels on mouse spinal cord extracts. GS cKO mice (CNP-cre+ :Glulfl/fl ) showed no differences in motor neuron numbers, size or axon density; OL differentiation and myelination in the ventral spinal cord was normal up to 6months of age. Interestingly, GS cKO mice showed a transient and specific decrease in peak force while locomotion and motor coordination remained unaffected. Last, GS expression in OL was increased in chronic pathological conditions in both mouse and humans. We found a disease-stage dependent increase of OL expressing GS in the ventral spinal cord of SOD1(G93A) mouse model of amyotrophic lateral sclerosis. Moreover, we showed that GLUL transcripts levels were increased in OL in leukocortical tissue from multiple sclerosis but not control patients. These findings provide evidence towards OL-encoded GS function in spinal cord sensorimotor axis, which is dysregulated in chronic neurological diseases.

Glutamine synthetase (GS) is a key enzyme that metabolizes glutamate into glutamine. While GS is expressed by astrocytes of the central nervous system (CNS), expression in other glial lineages has been noted. Using a combination of reporter mice and cell typespecific markers, we show that GS is expressed in myelinating oligodendrocytes (OL) but not oligodendrocyte progenitor cells (OPC) of the mouse spinal cord abutting ventral horn motor neurons. To investigate the role of GS in mature OL, we used a conditional knockout (cKO) approach to selectively delete GS-encoding gene (Glul) in OL, which caused a significant decrease in glutamine levels on spinal cord extracts. We evaluated the effect on ventral spinal cord sensorimotor circuits and observed that GS cKO mice (CNP-cre+: Glulfl/fl) showed no differences in motor neuron numbers, size or axon density; OL differentiation and myelination in the ventral spinal cord at 1- and 6-months of age was normal. Interestingly, GS cKO mice showed an early and specific decrease in peak force while motor function remained otherwise unaffected. These findings provide evidence OL-encoded GS functions in spinal cord sensorimotor circuit.Main pointsGlutamine synthetase (GS) is highly expressed in oligodendrocytes (OL) of the mouse spinal cord.OL-specific GS loss of function causes transient decrease in peak force, but other substantial neurological dysfunction was not detected.

U ovom radu su opisani mehanizmi biosinteze, transporta i konjugacije indol-3-octene kiseline (IAA), kao dominantnog oblika auksina, i njen učinak na razvojne i fiziološke procese, s posebnim osvrtom na spoznaje o detaljima tih procesa kod eksperimentalne biljke Arabidopsis thaliana. Tehnologijom in-fusion sintetiziran je binarni vektor pHOV:AtILL2-GFP, te su metodom floral dip regenerirane transgenične biljke A. thaliana ekotip WS i Col-0 s pojačanom ekspresijom gena AtILL2 i rekombinantnog gena AtILL2-GFP. Lokalizacija fuzijskog proteina AtILL2-GFP, praćena fluorescencijskom mikroskopijom, sugerira endoplazmatski retikulum kao mjesto djelovanja hidrolaze ILL2, što je u skladu s dosadašnjim rezultatima istraživanja. Analizirane su mutante s utišanom ekspresijom gena AtILR1, AtILL2 i AtIAR3, te je metodama PCR-a i elektroforeze izdvojena jednostruka nul mutanta ill2-1 u kojoj je identificirana insercijska mutacija u genu AtILL2 koja uzrokuje pomak u okviru čitanja i sintezu krnjeg, nefunkcionalnog proteina. Metodama PCR-a i sekvenciranja izdvojene su jednostruke mutante ilr1-1 i iar3-2 u kojima je identificirana mutacija Gly224Glu u genu AtIAR3 (mutanta iar3-2) i mutacija Gly139Asp u genu AtILR1 (mutanta ilr1-1) koje uzrokuju potpuni ili djelomični gubitak funkcije hidrolaza ILR1 i IAR3. Kombinacijom PCR-a, elektroforeze i sekvenciranja izdvojena je dvostruka mutanta ill2-1/iar3-2 koja ima mutaciju Gly224Glu u genu AtIAR3 i insercijsku mutaciju u genu AtILL2, i trostruka mutanta ilr1-1/ill2-1/iar3-2 koja ima mutaciju Gly139Asp u genu AtILR1, insercijsku mutaciju u genu AtILL2 i mutaciju Gly224Glu u genu AtIAR3. Mutante dobivene i analizirane u ovom radu dalje će se koristiti za istraživanja metabolizam IAA naročito tijekom izloženosti biljaka abiotskom stresu. Indole acetic acid (IAA) is the most dominant form of the naturally occurring auxin in Arabidopsis thaliana. Here, the mechanisms of biosynthesis, transport and conjugation of IAA in A. thaliana are reviewed, and mutants involved in IAA-aminoacid hydrolyze analyzed and regenerated. Binary vector pHOV:AtILL2-GFP was synthesized by using in-fusion technology, and transgenic A. thaliana plants, ecotypes WS and Col-0, with overexpression of AtILL2 and AtILL2-GFP transgenes were regenerated after floral dip transformation. Localization of protein AtILL2-GFP, tracked by fluorescence microscopy, suggest endoplasmic reticulum as the site of action of hydrolase ILL2. Mutants with reduced expression of AtILR1, AtILL2 and AtIAR3 genes were analyzed. Using PCR and electrophoresis mutant ill2-1 with insertional mutation in gene AtILL2, which causes synthesis of truncated nonfunctional protein, was confirmed. Mutants ilr1-1 and iar3-2 were analysed using PCR and sequencing. The mutations Gly224Glu, and Gly139Asp were confirmed in AtIAR3 (iar3-2 mutant) and AtILR1 gene (ilr1-1 mutant), respectively. The double mutant ill2-1/iar3-2 contained mutation Gly224Glu in gene AtIAR3 and insertional mutation in gene AtILL2, and the triple mutant ilr1-1/ill2-1/iar3-2 contained mutation Gly139Asp in gene AtILR1, insertional mutation in gene AtILL2 and mutation Gly224Glu in gene AtIAR3. All mutants will be used further to investigate IAA metabolism, especially during abiotic stress.

U ovom preglednom radu su opisani mehanizmi nastanka tumora odnosno onkogeni, tumor – supresorski geni i microRNA te mutacije u njima koje dovode do nastanka tumora. Opisane su pojedine sastavnice hrane, propolis i pripadni polifenoli te njihov preventivni kao i antitumorski učinak na različitim animalnim modelima. Ispitivanjem flavanoida pokazalo se da oni također mogu djelovati preventivno kao i izravno pokazujući antitumorski učinak, također se mogu kombinirati sa standardnim kemoterapeuticima pri čemu znatno poboljšavaju krajnji ishod liječenja malignih oboljenja. Opisan je kvercetin i njegov neuroprotektivni učinak na kulturama stanice neurona P19. Krizin, naringenin i njihov pozitivni učinak na oksidativni stres kao i sprječavanje osteoporoze su također opisani. In this review are described mechanisms of tumor formation or oncogenes, tumor – suppressor genes and microRNA genes and mutations in them which lead to occurrence of tumors. Here are also described the various components of food, propolis and associated polyphenols and their preventive as well as antitumor effect on various animal models. Flavanoids examinations showed that they may also act preventively and curatively demostrating the antitumor effect and may be also combaining with standard chemotherapeutics wherby considerably improves the outcome of treatment of malgnant disease. Quercetin has been described and its neuroprotective effect to the cell cultures of neurons P19. Chrysin, naringenin and their possitive effect on oxidative stress as well as prevention of osteoporosis are also described.

We present the design of a variable temperature setup that uses a pulse tube cryocooler to perform break-junction experiments at variable temperatures ranging from 12 K to room temperature. The use of pulse tube coolers is advantageous because they are easy to use, can be highly automatized, and used to avoid wastage of cryogenic fluids. This is the reason why dry cryostats are conquering more and more fields in cryogenic physics. However, the main drawback is the level of vibration that can be up to several micrometers at the cold-head. The vibrations make the operation of scanning probe-based microscopes challenging. We implemented vibration-damping techniques that allow obtaining a vibration level of 12 pm between the tip and sample. With these adaptations, we show the possibility to perform break junction measurements in a cryogenic environment and keep in place atomic chains of a few nanometers between the two electrodes.