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Astrocytes are the main homeostatic cells in the central nervous system, with the unique ability to transform from quiescent into a reactive state in response to pathological conditions by reacquiring some precursor properties. This process is known as reactive astrogliosis, a compensatory response that mediates tissue damage and recovery. Although it is well known that SOX transcription factors drive the expression of phenotype-specific genetic programs during neurodevelopment, their roles in mature astrocytes have not been studied extensively. We focused on the transcription factors SOX2 and SOX9, shown to be re-expressed in reactive astrocytes, in order to study the reactivation-related functional properties of astrocytes mediated by those proteins. We performed an initial screening of SOX2 and SOX9 expression after sensorimotor cortex ablation injury in rats and conducted gain-of-function studies in vitro using astrocytes derived from the human NT2/D1 cell line. Our results revealed the direct involvement of SOX2 in the reacquisition of proliferation in mature NT2/D1-derived astrocytes, while SOX9 overexpression increased migratory potential and glutamate uptake in these cells. Our results imply that modulation of SOX gene expression may change the functional properties of astrocytes, which holds promise for the discovery of potential therapeutic targets in the development of novel strategies for tissue regeneration and recovery.

Comprehensive research, particularly in evaluating drug efficacy, still heavily relies on the results obtained by the utilization of cell monolayers and animals. However, the inherent limitations of these models such as their physiological disparities from humans pose significant obstacles to acquiring reliable results thus impeding further scientific progression. To address this challenge, 3D in vitro cell culture models emerged as physiologically relevant models having the potential to enhance research and drug discovery. Our study aimed to develop a 3D in vitro cell culture model based on bone-like scaffolds in conjunction with a perfusion bioreactor (“3D Perfuse”, Innovation Center FTM, Belgrade, Serbia) for studying both physiological and pathological (i.e. tumors) bone conditions.

The slow advance in anticancer drug development can be attributed to the limitations of conventional models, predominantly monolayer cell (2D) cultures and animal models, which inadequately recapitulate the complex nature of human malignant tumors. Three-dimensional (3D) in vitro models are invaluable tools in drug screening; however, creating a universal model for all cancer types poses challenges due to the diverse nature of cancers. The aim of this work was to develop a single, versatile model using alginate microfibers to accommodate cultivation of various cancer cells.

Nonsteroidal anti-inflammatory drugs (NSAIDs) alleviate inflammation and pain through the inhibition of cyclooxygenase (COX) enzymes. Besides these widely recognized therapeutic utilizations, NSAIDs have been reported to display moderate antimicrobial activity and enhance antimicrobial efficacy when administered in combination with commercial antimicrobial drugs. In the present study, we designed novel potential antimicrobial agents by linking some NSAIDs (ibuprofen, flurbiprofen, and naproxen) to various azole rings (pyrazole, imidazole, triazole, and benzimidazole) via hydrazone functionality. The hydrazone linker was introduced into the chemical scaffold of the title molecules by the reaction between hydrazides obtained from NSAIDs and in-house synthesized azole-carrying benzaldehydes. The structures of the target compounds were elucidated by a combination of spectral methods. The NOESY spectra and stereochemical analyses performed using DFT method confirmed the presence of the target molecules as a mixture of E(C=N)-E(N-N)-synperiplanar and E(C=N)-E(N-N)-antiperiplanar conformers in DMSO-d6 solution. 1H and 13C NMR chemical shift values in DMSO were calculated using the GIAO method and compared with the experimental NMR data. Finally, some derivatives were demonstrated to inhibit Candida albicans filamentation and/or bacterial communication system known as quorum sensing. For COX inhibitor-azole hybrids with antimicrobial potency, naproxen appeared to be the most appropriate NSAID, while bulky benzimidazole was not found as a preferable azole ring.

The application of enzymes is expanding across diverse industries due to their nontoxic and biodegradable characteristics. Another advantage is their cost-effectiveness, reflected in reduced processing time, water, and energy consumption. Although Gram-positive bacteria, Bacillus, and Streptomyces spp. are successfully used for production of industrially relevant enzymes, they still lag far behind Escherichia coli as hosts for recombinant protein production. Generally, proteins secreted by Bacillus and Streptomyces hosts are released into the culture medium; their native conformation is preserved and easier recovery process enabled. Given the resilience of both hosts in harsh environmental conditions and their spore-forming capability, a deeper understanding and broader use of Bacillus and Streptomyces as expression hosts could significantly enhance the robustness of industrial bioprocesses. This mini-review aims to compare two expression hosts, emphasizing their specific advantages in industrial surroundings such are chemical, detergent, textile, food, animal feed, leather, and paper industries. The homologous sources, heterologous hosts, and molecular tools used for the production of recombinant proteins in these hosts are discussed. The potential to use both hosts as biocatalysts is also evaluated. Undoubtedly, Bacillus and Streptomyces spp. as production hosts possess the potential to take on a more substantial role, providing superior (bio-based) process robustness and flexibility.

INTRODUCTION: Tissue engineering strives for innovative solutions in addressing challenges associated with contemporary bone tissue implants. This study focuses on the electrophoretic deposition (EPD) of hydroxyapatite-based bioceramic composites containing antibacterial agents onto titanium surfaces. Two composite coatings, hydroxyapatite/ chitosan (HAP/CS) and hydroxyapatite/chitosan/gentamicin (HAP/CS/Gent) were developed to combat issues such as poor adhesion, limited antibacterial potential, limited bioactivity, and potential toxicity of implant materials [1]. EXPERIMENTAL: EPD was performed at constant voltage (5 V, 12 min) on pure Ti plates from aqueous (HAP/CS and HAP/CS/Gent) suspensions. The uniformity and functionality of the deposited coatings were assessed through comprehensive physico-chemical characterization using X-ray diffraction (XRD) (Philips PW 1710, Netherlands) and scanning electron microscopy (SEM, Hitachi S-4700, J) equipped with energy dispersive X-Ray spectroscopy (EDS, X-Max, Oxford Instruments, UK). Antibacterial activity was evaluated against Staphylococcus aureus TL and Escherichia coli ATCC 25922 by quantitatively monitoring changes in the viable number of bacterial cells in suspension. Cytotoxicity against MRC-5 and L929 cell lines was investigated using trypan blue dye-exclusion test (DET) and MTT assay for assessing cell metabolic activity. Statistical significance was determined for antibacterial and cytotoxicity results by one-way analysis of variance (ANOVA), followed by multiple comparisons post-hoc test. RESULTS AND DISCUSSION: XRD revealed broadened diffraction maximums corresponding to fine HAP crystallites. Porous surface with homogeneously distributed spherical HAP agglomerates embedded in wax-like polymers' matrix of CS was observed for both coatings by SEM. The addition of gentamicin significantly enhanced the antibacterial activity of the HAP/CS/Gent coating – complete reduction of S. aureus bacterial cells was achieved

INTRODUCTION: Following orthopedic surgery, bacterial infection may lead to significant complications related to inflammation in the peri-implant region, including the potential for implant loosening. To address this concern, the surface of metallic implants was modified through the application of bioactive and antibacterial coatings, aiming to mitigate these issues. EXPERIMENTAL: HAP/PVA/CS/Gent coatings was single-step electrophoretically deposited from four-component aqueous suspension containing 1 wt.% hydroxyapatite powder (HAP, particles < 200 nm particle size, Sigma-Aldrich), 0.1 wt.% poly(vinyl alcohol) (PVA, medium molecular weight 89 to 98 kDa, 99 % hydrolysed, Sigma-Aldrich), 0.05 wt% chitosan powder (CS, medium molecular weight, 190 to 310kDa with 75 to 85 % deacetylation degree, Sigma-Aldrich), and aqueous gentamicin sulfate solution (Gent, concentration 50 mg/ml, Sigma-Aldrich) on titanium plates (Sigma-Aldrich). Antibacterial activity of HAP/PVA/CS/Gent coating was evaluated against Staphylococcus aureus TL (culture collection- FTM, University of Belgrade, Serbia) and Escherichia coli ATCC 25922, while kinetics of antibacterial activity was monitored according to our previously published data [1]. In vitro cytotoxicity assay was performed towards two fibroblast cell lines of different origin-mouse origin cell line (L929 (ATCC CRL-6364)) and human lung origin cell line (MRC-5 (ATCC CCL-171)) [2]. To evaluate the statistical significance of the biological assay results (antibacterial activity, cytotoxicity and ALP activity), one-way analysis of variance (ANOVA) followed by a multiple comparisons posthoc test was used. RESULTS AND DISCUSSION: HAP/PVA/CS/Gent coating exhibited strong antibacterial effect against both S. aureus and E. coli, especially pronounced against S. aureus, causing bactericidal effect. Cytotoxic effect of HAP/PVA/CS/Gent coating was not pronounced in investigated MRC-5 and L929 cell lines. MRC-5 fibroblast cells in contact with HAP

INTRODUCTION: Electrochemical methods were employed for synthesizing composites intended for applications in medicine as antibacterial coatings on titanium bone implants or as highly efficient antibacterial hydrogels for accelerated wound healing. EXPERIMENTAL: Hydroxyapatite-based coatings were single-step electrophoretically deposited from fourcomponent aqueous suspension containing 1 wt% hydroxyapatite powder (HAP, particles < 200 nm particle size, Sigma- Aldrich), 0.1 wt% poly(vinyl alcohol) (PVA, medium molecular weight, Sigma-Aldrich), 0.05 wt% chitosan powder (CS, medium molecular weight, Sigma-Aldrich), and aqueous gentamicin sulfate solution (Gent, concentration 50 mg/ml, Sigma-Aldrich) on titanium plates (Sigma-Aldrich). Antibacterial activity of HAP/PVA/CS/Gent coating was evaluated against Staphylococcus aureus TL (culture collection-FTM, University of Belgrade, Serbia) and Escherichia coli ATCC 25922. For poly(vinyl alcohol)/chitosan (PVA/CHI) hydrogel synthesis poly(vinyl alcohol) powder (PVA, Sigma Aldrich, USA), chitosan powder (CHI, Sigma Aldrich, USA), graphene (Graphene Supermarket, USA), glacial acetic acid (Beta Hem), silver nitrate (MP Hemija), potassium nitrate (Centrohem, Serbia) were used. For antibacterial properties evaluation, monobasic and dibasic (Sigma Aldrich, USA) potassium phosphates were used. Cell culture suspensions for cytotoxicity tests were prepared using MTT tetrazolium salt, EDTA, fetal calf serum and antibiotic-antimycotic solution (Sigma Aldrich, USA). RESULTS AND DISCUSSION: Surface modification of titanium with innovative bioactive coatings enhances biomineralization and reduces post-operation pain and infection risk. Using powerful single-step electrophoretic deposition (EPD) at a constant voltage we have produced various hydroxyapatite-based composite bioceramic coatings in combination with polymers (lignin, chitosan), graphene, and antibacterial agents (silver, gentamicin). Electrochemical methods enable in situ synth

Osteosarcoma (OS) is a highly aggressive primary malignant bone tumor that most commonly affects children, adolescents, and young adults. The standard treatment for OS consists of surgical resection and chemotherapy, whereas radiation therapy is recommended for the unresectable tumor. Due to its easy metastasis and recurrence, the 5-year overall survival rate is only 66.5 %. Thus, there is a critical need to recognize the molecular mechanisms underlying OS development and pathogenesis. Traditionally, two-dimensional (2D) cells are widely used in cancer biology and pre-clinical studies. However, 2D models are unable to mimic cell–cell and cell-extracellular matrix interactions which are crucial for adequate cellular function. Three-dimensional (3D) model systems are able to recapitulate key features of human cancer and are recognized as a promising platform for fundamental and translational research. In the present work, we established an osteosarcoma 3D model based on alginate microbeads and studied the effect of combined treatment with doxorubicin (Doxo), widely used chemotherapeutic, and quercetin (Quer), a plant pigment with anticancer properties, on OS model systems.

U cilju poboljšanja bioaktivnosti i antibakterijskih svojstava metalnog implantata (titana), primenom elektroforetskog taloženja u jednom stupnju, iz četvorokomponentne vodene suspenzije dobijene su biokeramičke, kompozitne miltifunkcionalne prevlake na bazi hidroksiapatita, poli(vinil alkohola) i hitozana uz dodatak antibakterijskog agensa, antibiotika gentamicina. Na osnovu rezultata dobijenih FTIR, XRD i TG analizom pokazano je prisustvo karbonatno-supstituisanog hidroksiapatita (prisutnog i u prirodnom koštanom tkivu) i potvrđena je uspešna inkorporacija gentamicina u prevlaku, što ovu kompozitnu prevlaku čini pogodnom za potencijalnu primenu u biomedicini.

Površine titanskih implantata se mogu unaprediti primenom biokeramičkih, multifunkcionalnih prevlaka koristeći proces elektroforetskog taloženja (EPD) u cilju sprečavanja korozije, poboljšanja biokompatibilnosti i antibakterijske aktivnosti, kao i unapređenja osteointegracije. Materijali poput hidroksiapatita (HAP), hitozana (CS), poli(vinil-alkohola) (PVA) i antibiotika gentamicina (Gent) su korišćeni u ovom procesu kako bi se razvile poboljšane antibakterijske kompozitne prevlake. Nakon taloženja, prevlake su potapane u simuliranu telesnu tečnost (SBF) na 37 °C, a zatim je izvršena karakterizacija skenirajućom elektronskom mikroskopijom sa emisijom polja (FE-SEM) i rendgenskom difrakcionom analizom (XRD). Bioaktivnost istaloženih prevlaka je pokazana i analizom aktivnosti alkalne fosfataze prema MRC-5 i L929 ćelijskim linijama. Koncentracija otpuštenog gentamicina je određena korišćenjem tečne hromatografije visokih performansi (HPLC) sa masenom detekcijom (MS).

Plastic waste has become a serious global challenge that calls for sustainable solutions and requires rapid actions. Biocatalysis could present an adequate answer to this problem by providing different enzymes capable of degrading plastic polymers. Streptomyces strains as predominant soil inhabitants have also adapted to the presence of variety of plastic waste in natural environments, so they have been examined for the plastic degrading capabilities. The aim of this work was to improve the biocatalytic properties of Streptomyces strains for their use in biodegradation of plastic polymers and develop a system for heterologous expression of polyethylene therephtalate (PET) degrading enzymes in Streptomyces spp. Well studied Streptomyces lividans TK24 and S. albus NRRL B-1335, as well as two newly isolated Streptomycetes were used for expression of benchmark PETases and cutinases. Enzymes were cloned into pGM1202 Escherichia coli–Streptomyces shuttle vector and subsequently introduced into Streptomyces hosts either by polyethylene glycol-mediated protoplasts transformation or by electroporation. Cell-free extracts and supernatants of transformed cells were tested on different plastics using bis(2-hydroxyethyl) terephthalate (BHET), polycaprolactone (PCL) and Impranil as substrates in plate assays. Expression of leaf-branch compost cutinase in S. albus and S. lividans resulted in an 8.5- and 2.5- times increase in esterase activities, respectively. Introduction of the enzyme into newly isolated strains that already showed some plastic degrading activity resulted in synergistic activity of the recombinant strains.

Nowadays, it is a great challenge to develop new medicines for treating various infectious diseases. The treatment of these diseases is of utmost interest to further prevent the development of multi-drug resistance in different pathogens. Carbon quantum dots, as a new member of the carbon nanomaterials family, can potentially be used as a highly promising visible-light-triggered antibacterial agent. In this work, the results of antibacterial and cytotoxic activities of gamma-rayirradiated carbon quantum dots are presented. Carbon quantum dots (CQDs) were synthesized from citric acid by a pyrolysis procedure and irradiated by gamma rays at different doses (25, 50, 100 and 200 kGy). Structure, chemical composition and optical properties were investigated by atomic force microscopy, transmission electron microscopy, X-ray photoelectron spectroscopy, Fourier transform infrared spectroscopy, Raman spectroscopy, UV-Vis spectrometry and photoluminescence. Structural analysis showed that CQDs have a spherical-like shape and dose-dependent average diameters and heights. Antibacterial tests showed that all irradiated dots had antibacterial activity but CQDs irradiated with dose of 100 kGy had antibacterial activity against all seven pathogenreference bacterial strains. Gamma-ray-modified CQDs did not show any cytotoxicity toward human fetal-originated MRC-5 cells. Moreover, fluorescence microscopy showed excellent cellular uptake of CQDs irradiated with doses of 25 and 200 kGy into MRC-5 cells.

Mitohondrijska DNK (mtDNK) se odlikuje nizom osobina koje je čine pogodnom za istraživanja evolutivne istorije ljudskih populacija koja se zasniva na molekularnim markerima ženske linije nasleđivanja. Tokom poslednje decenije publikovano je više naučnih radova u kojima je analizirana varijabilnost mtDNK u populaciji Srbije primenom markera različite rezolucije uključujući i kompletne genome. U skladu sa očekivanjima zasnovanim na istorijskim, arheološkim i drugim izvorima koji govore u prilog veoma kompleksne istorije populacija na Balkanskom poluostrvu, mtDNK podaci su potvrdili da se srpska populacija odlikuje visokim nivoom raznovrsnosti mtDNK koji je posledica izuzetno složene dinamike ove populacije tokom vremena. Današnji mtDNK profil populacije Srbije ne odstupa od matrilinealnog profila karakterističnog za druge evropske populacije, a genetičke distance pokazuju da ova populacija zauzima centralnu poziciju unutar grupe južnoslovenskih populacija koje se odlikuju visokom heterogenošću. Srpska populacija deli najveći procenat mtDNK haplotipova sa geografski bliskim populacijama Balkanskog poluostrva koje pripadaju južnoslovenskoj grupi, gde su uočeni i potencijalno privatni haplotipovi. Na osnovu filogenetske i filogeografske analize kompletnih mitogenoma u srpskoj populaciji detektovane su retke mtDNK linije, karakteristične za druge regione, poput Bliskog istoka (N1b, HV2), istočne Azije (D4) i Afrike (L2a1), kao i one koje su potencijalno specifične za Balkansko poluostrvo, poput K1a13a1, U4c1b1 i H6a2b. Pored toga, srpska populacija deli određeni broj mtDNK podhaplogrupa sa istočno- i zapadnoslovenskim populacijama kao i sa germanskim populacijama severne i srednje Evrope. Istraživanja varijabilnosti mtDNK su pokazala da se izuzetno velika raznovrsnost mtDNK savremene populacije Srbije može objasniti genetičkim doprinosom kako slovenskih i germanskih, tako i pre-slovenskih populacija koje su naseljavale Balkansko poluostrvo pre Velike seobe naroda., The mitochondrial DNA (mtDNA) is characterized by a number of features that make it suitable for studying the evolutionary history of human populations based on molecular markers with the female-specific line of inheritance. During the last decade, several scientific papers were published in which the mtDNA variability in the population of Serbia was analyzed using markers of different resolution including complete mitogenomes. In accordance with expectations based on historical, archaeological and other sources that speak in favor of a very complex history of populations on the Balkan Peninsula, mtDNA data confirmed that Serbian population is characterized by a high level of mtDNA diversity, which is a consequence of the exceptionally complex dynamics of this population over time. Today’s mtDNA profile of the Serbian population does not differ from the matrilineal landscape characteristic of other European populations, and according to genetic distances, this population occupies a central position within the group of South- Slavic populations characterized by high heterogeneity. The Serbian population shares the highest percentage of mtDNA haplotypes with the geographically close populations of the Balkan Peninsula belonging to the South-Slavic group, where potentially private haplotypes were also observed. Phylogenetic and phylogeographic analysis of complete mitogenomes in the Serbian population revealed rare mtDNA lineages, characteristic of other regions, such as the Middle East (N1b, HV2), East Asia (D4) and Africa (L2a1), as well as those that are potentially specific for Balkan Peninsula, like K1a13a1, U4c1b1 and H6a2b. In addition, Serbian population shares a certain number of mtDNA subhaplogroups with East- and West-Slavic populations as well as with the Germanic populations of Northern and Central Europe. Studies of mtDNA variability have shown that the exceptionally high mtDNA diversity in contemporary Serbian population may be associated with the geneti

Brain trauma leads to the induction of neural stem cell proliferation and the migration of young neurons to injured areas. However, these neurons are insufficient to fully restore neuronal function due to the limited potential of adult neurogenesis. This study aimed to investigate the effect of hypoxia, a condition that underlines a wide spectrum of brain pathologies, on pluripotency and the capacity of stem cells to differentiate into neural progenitors. We analyzed the expression of SOX genes and microRNAs as they control a variety of cellular processes during neuronal differentiation, including cell proliferation and cell fate determination. In vitro neuronal differentiation of human embryonal carcinoma cell line NT2/D1 and induced pluripotent stem cells were used as a model system of adult neurogenesis. Cobalt chloride was used to induce hypoxia. The results of the analysis showed that, following hypoxia, the efficiency of neuronal induction was significantly decreased, that coincident with decline in mRNA expression levels of SOXB and SOXC genes. In contrast to that, the expression level of miR-21 was significantly increased. Our findings advance the study of SOX TFs, miR-21, and their possible interplay in ischemia-related pathologies, establishing them as prospective biomarkers and possible targets for future diagnostic and therapeutic approaches.

The ambigols are cyanobacterial natural products characterized by three polychlorinated aromatic building blocks connected by biaryl and biaryl ether bridges. All ambigols known to date possess promising biological activities. Most significantly, ambigol A was reported to have antibacterial activity against Gram-positive bacteria, such as Bacillus megaterium and B. subtilis. We established a diverse compound library for in-depth biological evaluation building on our previous bio- and total synthetic research on this natural product family. To explore the antimicrobial potential in detail and to determine initial structure–activity relationships of this product class, a large set of dimeric and trimeric compounds were screened against selected bacterial and Candida target strains. Our results reveal exceptional antibiotic activity of the ambigols, especially against challenging clinical isolates.

The discovery of new antimicrobial agents as a means of treating drug-resistant microbial pathogens is of utmost significance to overcome their immense risk to human well-being. The current investigation involves the development, synthesis, and assessment of the antimicrobial efficacy of novel quinoline derivatives incorporating a thiosemicarbazide functionality. To design the target compounds (QST1–QST14), we applied the molecular hybridization approach to link various thiosemicarbazides to the quinoline core with a sulfonyl group. Upon the synthesis and completion of structural characterization via spectroscopic techniques (1H NMR, 13C NMR, 15N NMR, IR, and HRMS), the title molecules were extensively evaluated for their potential antitubercular, antibacterial, and antifungal activities. N-(3-Chlorophenyl)-2-(quinolin-8-ylsulfonyl)hydrazine-1-carbothioamide (QST4), the most effective compound against Mycobacterium tuberculosis H37Rv, was also tested on isoniazid-resistant clinical isolates with katG and inhA promoter mutations. Based on molecular docking studies, QST4 was also likely to demonstrate its antimycobacterial activity through inhibition of the InhA enzyme. Furthermore, three derivatives (QST3, QST4, and QST10) with preferable antimicrobial and drug-like profiles were also shown to be nontoxic against human embryonic kidney (HEK) cells. All compounds were optimized by the density functional theory method using B3LYP with the 6-31+G(d,p) basis set. Structural analysis, natural bond orbital calculations of donor–acceptor interactions, molecular electrostatic potential analysis, and frontier molecular orbital analysis were carried out. Quantum chemical descriptors and charges on the atoms were determined to compare the strengths of the intramolecular hydrogen bonds formed and their stabilities. We determined that the sulfur atom forms a stronger intramolecular hydrogen bond than the nitrogen, oxygen, and fluorine atoms in these sulfonyl thiosemicarbazide der

The SOX genes encode a group of transcription factors showing diverse expression patterns during de- velopment and acting as key regulators of diverse cellular processes. SOX transcription factors are in- volved in multiple events from the maintaining of stem cells pluripotency and cell fate decision to driving terminal differentiation of cells into specialized cell types. During adulthood SOX transcription factors control various physiological processes. Mutations in SOX genes have been associated with severe clin- ical disorders, while deregulation of their expressions cause a broad range of pathological condition. Ac- cumulating evidence suggests that SOX proteins act as oncogenes and recent evidence points toward pro-proliferative, pro-survival and/or anti-differentiation roles of the SOX proteins. The results of long-term research of the structure, regulation of expression and the function of selected SOX genes will be presented. It will include data obtained by studying the roles of SOX genes in in vitro neural differentiation of pluripotent embryonal carcinoma cells, as well as interaction of SOX transcrip- tion factors with signalling pathways active during neurogenesis and oncogenesis. Special focus will be made on ongoing research focused on the roles of SOX genes in promotion of malignant phenotype of cancer cells and maintaining of cancer stem cells. The interplay of SOX transcription factors and mi- croRNAs in the brain under physiological and pathological conditions, along with crosstalk between SOX genes and long non-coding RNAs in glioblastoma will be discussed. The role of SOX transcription factors in ageing and age-related diseases will be outlined.

Introduction: Neurodevelopmental disorders (NDDs), such as autism spectrum disorders (ASD), intellectual disability (ID),schizophrenia, and bipolar disorder, are caused by the alterationsin early brain development. They affect approximately 4% of the European population and represent a high socio-economic impact and financial burden. Treatments of NDDs are focused on symptoms since molecular mechanisms underlying NDDs are still unknown. One of the syndromes with a high risk for NDDs is 22q11.2 Deletion Syndrome (22q11.2DS) caused by microdeletion 22q11.2. 22q11.2 microdeletion is the most common microdeletion in humans; it is one of the strongest known risk factorsfor development of psychiatric illness and the highest known genetic risk for schizophrenia (approximately, 25% of patients with 22q11.2DS develop schizophrenia compared to 1% in the general population). Methods: Genomic and clinical findings in 35 patients with 22q11.2DS were analyzed and peripheral blood mononuclear cells of patients with 22q11.2DS and healthy controls were reprogrammed. Results: The majority of patients have 3 Mb deletion and nine of them have inherited 22q11.2 microdeletion from parents. Twenty-one different clinical presentations are revealed in the cohort with developmental delay detected in about 50% of patients. iPSCs were generated from four patients with 22q11.2 microdeletion and five healthy controls. Conclusion: Cohort of patients with 22q11.2DS isform and iPSCs were generated which enable research of molecular mechanisms underlying NDDs.

Introduction: Prolonged exposure to sunlight, has a harmful effect on skin cells encompassing reduced viability, morphological changes, and altered gene expression. The two most prevalent types ofskin cancer,squamous cell carcinoma (cSCC) and basal cell carcinoma (BCC), arise from malignant transformation of keratinocytes. UV radiation, among other factors, serves as the primary cause of these tumors. Previous data hasshown that changesin different SOX genes expression in these cancer types correlates with disease progression, suggesting their role as oncogenes/tumor suppressors. The presented work is focused on examining the impact of UVB radiation on the expression of SOX2 and SOX9 genesin HaCaT cells derived from human keratinocytes. Methods: Using a custom-made UV solarsimulator for the irradiation of HaCaT cells with 150 mJ/cm2 or 300 mJ/cm2 , we analyzed SOX2 and SOX9 gene expression. In order to determine the protective effects of quercetin, anti-inflammatory bioflavonoid, we treated irradiated HaCaT with quercetin, and analyzed SOX gene expression. Results: Our resultsindicate that UVB radiation induces a dose dependent decrease of SOX2 expression while expression of SOX9 was increased at the dose of 150 mJ/cm2 in HaCaT. Treatment of cells with quercetin increased the expression of both SOX2 and SOX9 genesin HaCaT cellsfollowing UVB radiation at both doses compared to irradiated cells. Conclusions: Further research is needed to understand the molecular mechanisms and significance of SOX2 and SOX9 in UVB-induced cellular responses, in the context of nonmelanoma cancers with potential implications for targeted therapeutic strategies for nonmelanoma cancers

Neurodevelopmental disorders (NDDs), such as autism spectrum disorders (ASD), intellectual disability (ID), schizophrenia, and bipolar disorder, are caused by disruption of brain development. They affect approximately 4% of the European population. However, molecular mechanisms underlying NDDs are still unknown. One of the syndromes with a high risk for NDDs is 22q11.2 Deletion Syndrome (22q11.2DS) caused by microdeletion 22q11.2. 22q11.2DS is the most common microdeletion in humans; approximately, 25% of patients with 22q11.2DS develop schizophrenia compared to 1% in the general population, while an ID is detected in approximately 45% of patients and ASD in 14-50% of cases. We analyzed genomic and clinical findings in our cohort of 35 patients with 22q11.2DS. The majority of patients have 3 Mb deletion and nine patients have inherited 22q11.2 microdeletion from their parents. Twenty-one different clinical presentations are revealed in the cohort with developmental delay detected in about 50% of patients. Approximately 80% of patients have heart malformations, palatal clefts/velopharyngeal insufficiency was detected in about 30% of them, facial dysmorphism in approximately 80% and hypocalcemia was seen in about 20% of patients. Here we presented a cohort of patients with 22q11.2DS which represents a good system for modeling NDDs in vitro.

Astrocytes are the main homeostatic cells in the brain with important roles both in physiological and pathological conditions. They have a unique ability to become reactivated in response to different types of brain pathologies, which serves as a compensatory response that modulates tissue damage and recovery. Also, senescent astrocytes have profound implications in age-related neurodegenerative disorders. The molecular mechanisms underlying astrocyte reactivation and senescence are still not well understood. To investigate the roles of SOX2 and SOX9 transcription factors and miR-21 in these phenotypic alternations of astroglia, astrocytes derived from NT2/D1 cell line (NT2/A) were used as a model system. Western blot analyses showed that the expression of both SOX2 and SOX9 decreases during the maturation of NT2/A and they are re-expressed upon in vitro induced injury. Further modulation of the SOX2 and SOX9 expression will reveal their roles in the regulation of astrocytes reactivation. Down-regulation of mir-21 in both immature and mature NT2/A by using the antisense technology, induced the decline in cell proliferation revealed by Ki67 proliferation marker. Also the premature cellular senescence was induced as indicated by increase in SA-ß-gal activity and the expression of p21 and p53. Additionally, in silico analysis predicted many of the genes, previously shown to be upregulated in senescent astrocytes, as miR-21 targets. Clarifying the roles of SOX genes and miRNAs in astrocyte reactivation and senescence would contribute to better understanding of the functions of these cells at the molecular level, which holds promise for development of new therapeutic strategies.

Neurodevelopmental disorders (NDDs), such as autism spectrum disorders (ASD), schizophrenia, and intellectual disability, are caused by disruption of early brain development. NDDs represent important public health challenge in modern societies with prevalence of about 10 to 15% of all births and the tendency of increasing worldwide. On the other side, treatments of NDDs are focused on symptoms due to limited understanding of underlying pathophysiological mechanisms. Individuals with the 22q11.2 duplication syndrome (22q11.2dup), caused by heterozygous 22q11.2 microduplication, have an elevated risk of developing NDDs. Literature data revealed that ASD is detected in 14-25% of patients with 22q11.2dup while schizophrenia is less common in these patients than in the general population, suggesting that 22q11.2dup might be protective against schizophrenia. We investigated genomic and clinical findings in cohort of 8 patients with 22q11.2dup. The majority of patients have 3Mb duplication. Five patients have 22q11.2 microduplication inherited from their parents. Other CNVs or SNVs are detected in 5 out of 8 patients. Common medical anomalies in our cohort of patients include developmental delay, facial dysmorphism, heart malformations, anomalies of the skeletal system, and anomalies affecting the eye. Characterization of a cohort of patients with 22q11.2dup is important since 22q11.2dup represents a powerful model to get insights into the molecular mechanisms underlying NDDs.

Data obtained with the use of massive parallel sequencing (MPS) can be valuable in population genetics studies. In particular, such data harbor the potential for distinguishing samples from different populations, especially from those coming from adjacent populations of common origin. Machine learning (ML) techniques seem to be especially well suited for analyzing large datasets obtained using MPS. The Slavic populations constitute about a third of the population of Europe and inhabit a large area of the continent, while being relatively closely related in population genetics terms. In this proof-of-concept study, various ML techniques were used to classify DNA samples from Slavic and non-Slavic individuals. The primary objective of this study was to empirically evaluate the feasibility of discerning the genetic provenance of individuals of Slavic descent who exhibit genetic similarity, with the overarching goal of categorizing DNA specimens derived from diverse Slavic population representatives. Raw sequencing data were pre-processed, to obtain a 1200 character-long binary vector. A total of three classifiers were used—Random Forest, Support Vector Machine (SVM), and XGBoost. The most-promising results were obtained using SVM with a linear kernel, with 99.9% accuracy and F1-scores of 0.9846–1.000 for all classes.

Background/Objectives: Genetic tests may facilitate rapid and effective diagnostics but unfortunately their high costs usually limit their application in all patients (1). We aimed to investigate the utility of rapid, cost effective and high sensitive Multiplex ligation probe amplification analysis (MLPA) for detection copy number variants (CNV) in newborns with critical CHD, admitted to the Neonatal Intensive Care Unit (NICU). Methods: Study included 100 consecutive newborns admitted to the NICU, University Children’s Hospital in Belgrade from August 2014 to September 2019. Patients with viable trisomies (21, 18 and 13) were excluded. All participants were tested by MLPA analysis using SALSA MLPA P250-B2 Di George and SALSA MLPA P311-B1 Congenital Heart Disease probemixes (MRC Holland, The Netherland). Results: Pathogenic CNVs were identified in ten (10%) patients. Nine of them had 22q11.2 deletion detected by both kits while one patient had 3p25 deletion detected by P311 kit. Conclusion: Genetic evaluation of all newborns with critical CHD admitted to the NICU by rapid and inexpensive MLPA analysis using combination P250 and P311 SALSA probemixes could contribute to high detection rate of pathogenic variants.

Introduction: The NT2/D1 embryonal carcinoma cell line represents a well-established in vitro model of human neurogenesis. It’s widely used for studying neurodevelopmental processes, neurotoxicity, and neurodegenerative disorders. The utilization of alginate fibers as a 3D cell culture system offers a biocompatible and structurally supportive environment for neural differentiation and maturation of cells, making it a suitable tool for investigating neurodevelopmental processes. Methods: In thisstudy, we evaluated the alginate microfibers as a 3D modelsystem for in vitro neural differentiation of NT2/D1 cells.We described the immobilization of NT2/D1 cellsin alginate microfibers and the effect of propagation in this 3D model on morphological features, viability, and proliferation of immobilized cells. We also assessed the RA-induced initiation of neural differentiation of NT2/D1 cellsin alginate microfibers by comparison with the initiation of neural differentiation in adherent 2D cell culture. Results: Our results showed that immobilized NT2/D1 acquired morphological features characteristic of cells propagated in 3D model systems and retain viability, proliferative capacity, and ability to attach to adherent surfaces. In addition, immobilized NT2/D1 cells preserved neural differentiation capacity. Upon RA induction we detected a marked decrease in the expression of specific pluripotency-maintaining markers, SOX2, OCT4, and NANOG. Consecutively, the expression of early neural markers, SOX3, PAX6, and miR219 was significantly increased. Conclusion: Neural differentiation of NT2/D1 cellsimmobilized within alginate fibersrepresents a highly promising 3D modelsystem forstudying human neurogenesis and offers a valuable platform forscreening the effect of drugs and bioactive compounds on human neural differentiation.

Introduction: Neurodevelopmental disorders (NDDs) are a group of complex and heterogeneous disorders that include autism spectrum disorders, intellectual disability, schizophrenia and bipolar disorder. However, underlying pathophysiological mechanisms are mostly unknown. In order to get better understanding of the underlying mechanisms and to discover potential therapeutics we have focused our research on 22q11.2 Deletion Syndrome (22q11.2DS), caused by microdeletion of the region q11.2 of chromosome 22 and associated with a high risk for NDDs. Methods: To study molecular mechanisms underlying intrafamilial phenotypic variability, we have identified families with the inherited form of 22q11.2DS with the aim of conducting the following analyses: whole genome sequencing in order to detect additional genetic variation(s) present in the affected child; generation of induced pluripotent stem cells (iPSCs) from peripheral blood mononuclear cells; analysis of the effects of 22q11.2 microdeletion on neural differentiation including organoids as 3D model system; transcriptome analysis of iPSC-derived neurons and astrocytesto determine differentially expressed gene sets and dysregulated pathways; and testing the metabolic changes and drug responsiveness of neurons and astrocytes by high-throughput cell-based assays. Results: Peripheral blood mononuclear cells of the families with inherited form of 22q11.2DS were reprogrammed and established iPSCs were characterized. Generated iPSCs will be subjected to the further analyses. Conclusion: Currently, most of the treatments of NDDs are symptom-based due to limited understanding of underlying pathophysiological mechanisms. It is expected that patient-derived iPSCs will enable a deeper understanding of unique disease mechanisms and may also provide a significant contribution in preclinical drug development.

Background/Objectives: 22q11.2 microdeletion, detected in patients with 22q11.2 Deletion Syndrome (22q11.2DS), is the most common microdeletion syndrome in humans. 22q11.2DS has high risk for neurodevelopmental disorders and is associated with more than 180 malformations. Many investigations of the 22q11.2 microdeletion applying different recruitment criteria, revealed detection rate ranging from zero to 34.7%. Here we analyzed the frequency of 22q11.2 microdeletion among children having at least two out of five major characteristics of 22q11.2DS: congenital heart malformations (CHM), facial dysmorphism, immunological problems, palatal clefts and hypocalcemia. Methods: Children with clinical characteristics of 22q11.2DS were analyzed. Fluorescence in situ hybridization and multiplex ligation-dependent probe amplification analysis were applied for detection of 22q11.2 microdeletion. Results: 22q11.2 microdeletion was detected in approximately 40% of children. CHM was found in all patients with 22q11.2 microdeletion. Dysmorphic facial features were present in about 45%, immunological problems in 30%, overt cleft palate in about 4% and hypocalcemia in approximately 60% of patients with 22q11.2 microdeletion. Conclusion: When at least two major features of 22q11.2DS are taking into consideration higher detection rate is obtained compared to one-feature criterion. These criteria could be considered by centers in low-income countries.

Kompozitna prevlaka hidroksiapatita (HAP) sa prirodnim polimerima hitozanom (CS) i polivinil-alkoholom (PVA), kao i sa antibiotikom gentamicinom (Gent) je elektroforetski taložena na titanu, pri konstantnom naponu. Formiranje nove kompozitne HAP/PVA/CS/Gent prevlake je potvrđeno infracrvenom spektroskopijom sa Furijeovom transformacijom. Necitotoksični efekat istaložene HAP/PVA/CS/Gent prevlake je dokazan MTT testom prema dve ćelijske linije (MRC-5 i L929). Sposobnost HAP/PVA/CS/Gent prevlake da indukuje i promoviše osteointegraciju je pokazana ALP testom., Composite coating of hydroxyapatite (HAP) with natural polymers chitosan (CS) and poly(vinyl alcohol) (PVA) with the antibiotic gentamicin (Gent) was electrophoretically deposited on titanium from an aqueous suspension, at a constant voltage. Formation of a new composite HAP/PVA/CS/Gent coating was confirmed by Fourier transform infrared spectroscopy. Non-cytotoxicity of deposited HAP/PVA/CS/Gent coating was demonstrated by MTT assay towards two types of cell lines (MRC-5 and L929). HAP/PVA/CS/Gent coating ability to induce and promote the osseointegration process was proved by the ALP assay.

Introduction: Neurodevelopmental disorders (NDDs), such as autism spectrum disorders (ASD), schizophrenia, and intellectual disability, represent important public health challenge in modern societies with a prevalence of about 10 to 15% of all births and the tendency of increasing worldwide. They are caused by disruption of early brain development. Treatments of NDDs are focused on symptoms due to a limited understanding of underlying pathophysiological mechanisms. Individuals with the 22q11.2 Duplication Syndrome (22q11.2dup), caused by heterozygous 22q11.2 microduplication, have an elevated risk of developing NDDs. Literature data revealed that ASD is detected in 14-25% of patients with 22q11.2dup while schizophrenia is less common in these patients than in the general population, suggesting that 22q11.2 duplication might be protective against schizophrenia. Methods: Genomic and clinical findingsin patients with 22q11.2dup were analyzed and peripheral blood mononuclear cells of patients with 22q11.2dup were reprogrammed. Results: We formed a cohort of 8 patients with 22q11.2dup. The majority of patientsin our cohort have microduplication of approximately 3Mb (80%). Also, the majority of them are familial cases and in 67% of cases, the 22q11.2 microduplication is inherited from the mother. Congenital heart defects were detected in 25% of our patients, while all tested patients have facial dysmorphism. iPSCs were generated from three patients with a familial form of 22q11.2dup and their mothers. Conclusion: A cohort of patients with 22q11.2dup is formed and iPSCs were generated which can be used as a model system for studying NDDs.

Figure S1. TEM micrographs a) CQD_25, b) CQD_50,c) CQD_100, and d) CQD_200 samples, respectively.

Electrophoretic deposition (EPD) was employed to fabricate composite coatings aimed for orthopedic implants. Hydroxyapatite (HAP), poly(vinyl alcohol) (PVA) and chitosan (CS) coatings, with or without antibiotic gentamicin (Gent) were deposited in a single-step process from aqueous suspension, using EPD. Polymer/mineral composite coatings were formed with the aim to mimic the natural composition of bone tissue, along with improved coatings antibacterial properties, at the same time serving as a drug carrier, allowing local administration of antibiotic. Structural and morphological characterization of HAP/PVA/CS and HAP/PVA/CS/Gent coatings was investigated by Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), thermogravimetric analysis (TG/DTG), and scanning electron microscopy (SEM) proving carbonate-substituted HAP. The presence of gentamicin contributed to the bactericidal activity of composite HAP/PVA/CS/Gent coating against Staphylococcus aureus and Escherichia coli. Cytotoxicity studies were performed towards human MRC-5 and mice L929 fibroblast cell lines, and both investigated samples (HAP/PVA/CS and HAP/PVA/CS/Gent) expressed good biocompatibility, without causing toxic effect on tested cells. Biomineralization potential was investigated by measuring the alkaline phosphatase activity, demonstrating high potential of HAP/PVA/CS/Gent to induce the growth of new HAP layer. Therefore, the present investigation proposes the production of nents, along with antibiotic gentamicin, improving the antibacterial coatings’ properties.

Uvođenje antibiotika u polimer/mineralnu kompozitnu prevlaku poboljšava antibakterijska svojstva prevlaka. Kompozitna prevlaka (hidroksiapatit - HAP, poli(vinilalkohol), hitozan, gentamicin) dobijena je elektroforetskim taloženjem iz četvorokomponentne vodene suspenzije. Primenom termogravimetrijske i rendgenske difrakcione analize pokazano je prisustvo karbonatno-supstituisanog HAP. Antibakterijska aktivnost kompozitne prevlake sa gentamicinom potvrđena je protiv soja S. aureus i E. coli, čineći ovu prevlaku pogodnom za potencijalnu primenu u biomedicini., Antibiotic introduction in polymer/mineral coating can improve the antibacterial properties of coatings. Composite coating (hydroxyapatite, poly(vinyl alcohol), chitosan, gentamicin) was fabricated from a four-component aqueous suspension using electrophoretic deposition. Structural and morphological characterization of the coating was investigated by X-ray diffraction and thermogravimetric analysis proving carbonate-substituted HAP. The presence of gentamicin contributed to the antibacterial activity of composite coating against S. aureus and E. coli strains, representing a suitable material for possible application in biomedicine.

Arranged patterns obtained via ultrafast laser processing on the surface of Ti/Cu/Ti/Si and Ti/Zr/Ti/Si thin-film systems are reported. Two differently designed multilayer thin films Ti/Cu/Ti/Si and Ti/Zr/Ti/Si were deposited on silicon using the ion sputtering method. The bioactive surfaces of these systems involve the formation of laser-induced periodic surface structures (LIPSS) in each of the laser-written lines of mesh patterns on 5 × 5 mm areas. The formation of nano- and micro-patterns with an ultra-thin oxide film on the surfaces was used to observe the effects of morphology and proliferation of the MRC-5 cell culture line. To determine whether Ti-based thin films have a toxic effect on living cells, an MTT assay was performed. The relative cytotoxic effect, as a percentage of surviving cells, showed that there was no difference in cell number between the Ti-based thin films and the control cells. There was also no difference in the viability of the MRC-5 cells, except for the Ti/Cu/Ti/Si system, where there was a slight 10% decrease in cell viability. © 2023 by the authors.