Recent Achievements in the Development of Biomaterials Improved with Platelet Concentrates for Soft and Hard Tissue Engineering Applications.

Platelet concentrates such as platelet-rich plasma, platelet-rich fibrin or concentrated growth factors are cost-effective autologous preparations containing various growth factors, including platelet-derived growth factor, transforming growth factor ß, insulin-like growth factor 1 and vascular endothelial growth factor. For this reason, they are often used in regenerative medicine to treat wounds, nerve damage as well as cartilage and bone defects. Unfortunately, after administration, these preparations release growth factors very quickly, which lose their activity rapidly. As a consequence, this results in the need to repeat the therapy, which is associated with additional pain and discomfort for the patient. Recent research shows that combining platelet concentrates with biomaterials overcomes this problem because growth factors are released in a more sustainable manner. Moreover, this concept fits into the latest trends in tissue engineering, which include biomaterials, bioactive factors and cells. Therefore, this review presents the latest literature reports on the properties of biomaterials enriched with platelet concentrates for applications in skin, nerve, cartilage and bone tissue engineering.

Endophytic bacteria isolated from Urtica dioica L.- preliminary screening for enzyme and polyphenols production.

Endophytes, especially those isolated from herbal plants, may act as a reservoir of a variety of secondary metabolites exhibiting biological activity. Some endophytes express the ability to produce the same bioactive compounds as their plant hosts, making them a more sustainable industrial supply of these substances. Urtica dioica L. (common stinging nettle) is a synanthropic plant that is widely used in herbal medicine due to the diversity of bioactive chemicals it contains, e.g., polyphenols, which demonstrate anti-inflammatory, antioxidant, and anti-cancerous capabilities. This study aimed at isolating endophytic bacteria from stinging nettles for their bioactive compounds. The endophytic isolates were identified by both biochemical and molecular methods (16S rRNA) and investigated for enzymes, biosurfactants, and polyphenols production. Each of the isolated bacterial strains was capable of producing biosurfactants and polyphenols. However, three of the isolated endophytes, identified as two strains of Bacillus cereus and one strain of Bacillus mycoides, possessed the greatest capacity to produce biosurfactants and polyphenols. The derivatized extracts from culture liquid showed the 1.633 mol l-1 (9.691 mg l-1) concentration of polyphenol compounds. Therefore, the present study signifies that endophytic B. cereus and B. mycoides isolated from Urtica dioica L. could be a potential source of biosurfactants and polyphenols. However, further study is required to understand the mechanism of the process and achieve efficient polyphenol production by endophytic bacteria.

Toxicity of Carlina Oxide-A Natural Polyacetylene from the Carlina acaulis Roots-In Vitro and In Vivo Study.

There are several reports indicating that the roots of the Carlina acaulis L. used to be commonly applied as a treatment measure in skin diseases and as an antiparasitic agent, starting from antiquity to the 19th century; however, nowadays, it has lost its importance. Currently, numerous studies are being conducted assessing the possibility of reintroducing C. acaulis-derived extracts to phytotherapy. Determining the safety profile of the main constituents of the plant material is crucial for achieving this goal. Here, we aimed to determine the toxicity profile of carlina oxide, one of the most abundant components of the C. acaulis root extract. We obtained the carlina oxide by distillation of C. acaulis roots in the Deryng apparatus. The purity of the standard was evaluated using GC-MS, and the identity was confirmed by IR, Raman, and NMR spectroscopy. In vitro cytotoxicity was assessed using a panel of human cell lines of skin origin, including BJ normal fibroblasts and UACC-903, UACC-647, and C32 melanoma cells. This was accompanied by an in vivo zebrafish acute toxicity test (ZFET). In vitro studies showed a toxic effect of carlina oxide, as demonstrated by an induction of apoptosis and necrosis in both normal and melanoma cells. Decreased expression of AKT kinase and extracellular signal-regulated kinase 1/2 (ERK1/2) was noted in the UACC-647 melanoma cell line. It was also observed that carlina oxide modified the expression of programmed cell death-ligand 1 (PD-L1) in tested cell lines. Carlina oxide exhibited high in vivo toxicity, with LC50 = 10.13 µg/mL upon the 96 h of exposure in the ZFET test. Here, we demonstrate that carlina oxide displays toxic effects to cells in culture and to living organisms. The data indicate that C. acaulis-based extracts considered for therapeutic use should be completely deprived of carlina oxide.

Fishing for a deeper understanding of nicotine effects using zebrafish behavioural models.

Nicotine, the primary psychoactive component of tobacco, is the most widely used drug of abuse. Although the substance is well-known, there is still a lack of information concerning its long-term neurological and physiological effects and its mechanisms of action. In order to search for new, effective drugs in the therapy of nicotinism, as well as to design new drugs that exert positive nicotine-like effects, further experiments are needed, ideally also using new behavioural models and paradigms. A wide range of complex behaviours - including aggression, anxiety, long- and short-term memory, object discrimination and colour preference - have recently been comprehensively classified and characterized in the zebrafish model. Zebrafish offer an attractive experimental platform, based on a microscale in vivo bioassays, which can be used to investigate psychoactive drugs, their effects on the central nervous system and potential treatments of drug addictions. In this review, we present recent data revealing the potential of the zebrafish model to evaluate the effects and molecular mechanisms of nicotine by taking into consideration its impact on anxiety, learning and memory, addiction and social behaviours.

3-Furan-2-yl-N-p-tolyl-acrylamide, a highly selective positive allosteric modulator of α7 nicotinic receptors, produces anxiolytic-like activity in mice.

BACKGROUND: Several lines of investigations support the idea that nicotinic acetylcholine receptors modulate neuronal pathways involved in anxiety and depression. AIMS: The purpose of this study was to determine whether 3-furan-2-yl-N-p-tolyl-acrylamide, a highly selective positive allosteric modulator of α7 nicotinic acetylcholine receptors, influences anxiety-like behaviour in mice, and to determine the modulatory activity of 3-furan-2-yl-N-p-tolyl-acrylamide on mice pretreated with either nicotine or selective α7-agonists (i.e. PNU-282987 or (2.4)-dimethoxybenzylidene anabaseine dihydrochloride). METHODS: The elevated plus maze and novelty suppressed feeding tests were selected to evaluate 3-furan-2-yl-N-p-tolyl-acrylamide and other nicotinic ligands on anxiety-like behaviour in mice. RESULTS: The results indicated that: (a) 3-furan-2-yl-N-p-tolyl-acrylamide induces anxiolytic-like activity at 0.5 (elevated plus maze) and 1.0 (novelty suppressed feeding) mg/kg, respectively, after acute treatment, whereas its efficacy is increased after chronic treatments (i.e. active at 0.1 mg/kg; elevated plus maze). This is the first time showing anxiolytic-like activity elicited by 3-furan-2-yl-N-p-tolyl-acrylamide, contrary to the lack of activity for PNU-120596 (0.1 mg/kg); (b) the anxiolytic-like activity of 0.5 mg/kg 3-furan-2-yl-N-p-tolyl-acrylamide is inhibited by methyllycaconitine, a selective α7-antagonist, suggesting that α7 nicotinic acetylcholine receptors are involved in this process; (c) 0.5 mg/kg 3-furan-2-yl-N-p-tolyl-acrylamide reverses the anxiogenic effects induced by 0.1 mg/kg nicotine but not by 10.0 mg/kg PNU-282987; and (d) inactive doses of both 3-furan-2-yl-N-p-tolyl-acrylamide (0.1 mg/kg) and (2.4)-dimethoxybenzylidene anabaseine dihydrochloride (1.0 mg/kg) produce anxiolytic-like effects, suggesting drug interactions, probably synergistic. CONCLUSIONS: Our findings indicated that anxiolytic-like activity is mediated by α7 nicotinic acetylcholine receptors, supporting the concept that these receptors modulate anxiety processes. The results indicating that the chronic treatment with 3-furan-2-yl-N-p-tolyl-acrylamide is more efficient than the acute treatment in eliciting anxiolytic-like activity, and that 3-furan-2-yl-N-p-tolyl-acrylamide reverses the anxiogenic effects induced by nicotine, might be of therapeutic importance during smoking cessation.

Synthesis and biological assay of new 2'-deoxyuridine dimers containing a 1,2,3-triazole linker. Part I.

We describe a simple method for the synthesis of modified dinucleosides containing pyrimidine nucleoside analogues (2'-deoxyuridine, thymidine and 5-fluoro-2'-deoxyuridine). Six different dimers with a 1,2,3-triazole linkage were obtained by azide-alkyne 1,3-dipolar cycloaddition (click reaction), starting from propargylated 2'-deoxyuridine and 5'-azido-nucleoside derivatives. Their cytotoxic activity was tested in five human cancer cell lines: cervical (HeLa), high grade gliomas (U-118 MG, U-87 MG, T98G), liver (HepG2), and normal human fibroblast cell line (MRC-5) using the sulforhodamine B (SRB) assay. The experiment showed that the obtained dimers with a 1,2,3-triazole moiety were very stable compounds, also in the physiological-like media, and had no anticancer activity.

Impacts of cannabinoid receptor ligands on nicotine- and chronic mild stress-induced cognitive and depression-like effects in mice.

Taking into account the rather frequent concomitance of nicotine abuse and stress, we aimed to research memory- and depression-related effects of nicotine administration in combination with chronic mild unpredictable stress (CMUS) in mice and an involvement of the endocannabinoid system through CB1 and CB2 receptors. Mice were submitted to the CMUS for 4 weeks. Effects on depression-like behaviors and cognition, exerted by a combined administration of CB1, i.e., Oleamide (2.5, 5.0 mg/kg), AM 251 (0.1, 0.25 mg/kg) and CB2, i.e., JWH 133 (0.5, 2.0 mg/kg), AM 630 (0.25, 2.0 mg/kg) receptor ligands and nicotine (0.05, 0.1, 0.2 and 0.5 mg/kg), were then studied in stressed and unstressed mice by the forced swimming test and the passive avoidance paradigm, respectively. The results revealed that the CMUS-exposed mice exhibited depression-like behaviors and memory disturbances, while both effects were alleviated by nicotine. CB1 receptor ligands decreased antidepressive and cognitive (the latter for CB1 receptor antagonist only) effects of subchronic nicotine administration in stressed mice. CB1 and CB2 receptor antagonists exerted themselves some procognitive effects in those mice. Regarding the unstressed mice, CB1 and CB2 receptor ligands reversed the antidepressive effects of subchronic nicotine administration, while nicotine, in an ineffective dose, co-administered with CB2 receptor ligands, improved cognition. We confirmed the role of the two main subtypes of cannabinoid receptors, termed CB1 and CB2, on stress- and nicotine-related behavioral changes in mice. Our study has contributed to the understanding of the mechanisms involved in stress- and nicotine-induced disorders, such as anhedonia and memory disturbances.

New antiglioma zwitterionic pronucleotides with an FdUMP framework.

We have designed and synthesized new 5-fluoro-2'-deoxyuridine 5'-phosphate pronucleotides which can function as potential agents against the glioblastoma multiforme tumor. Their anti-malignant potency has been tested against T98G, U-118 MG, U-87 MG gliomas, HeLa, and Caco-2 cancer cell lines, using MRC-5 healthy cells as a reference. Five of the sixteen compounds (4c, 4f-i) exhibited significant anticancer potency and high selectivity indices (SI 12-66). It is likely that these zwitterionic pronucleotides may function in a similar manner to zwitterionic phospholipids, by inducing cell membrane charge disorder, making the cell permeable to bioactive agents. The most promising therapeutic pronucleotides 4c, 4f-h, have high intestinal-blood uptake potency (Caco-2 cell line), and may be considered as potential, orally administrated, anticancer drugs.

Calcium homeostasis and protein kinase/phosphatase balance participate in nicotine-induced memory improvement in passive avoidance task in mice.

Long-term potentiation (LTP) and long-term depression (LTD) depend on specific postsynaptic Ca2+/calmodulin concentration. LTP results from Ca2+ influx through the activated NMDA receptors or voltage-gated calcium channels (VGCCs) and is linked with activation of protein kinases including mitogen-activated protein kinase (MAPK). Weaker synaptic stimulation, as a result of low Ca2+ influx, leads to activation of Ca2+/calmodulin-dependent phosphatase (calcineurin - CaN) and triggers LTD. Interestingly, both memory formation and drug addiction share similar neuroplastic changes. Nicotine, which is one of the most common addictive drugs, manifests its memory effects through nicotinic acetylcholine receptors (nAChRs). Because nAChRs may also gate Ca2+, it is suggested that calcium signaling pathways are involved in nicotine-induced memory effects. Within the scope of the study was to evaluate the importance of calcium homeostasis and protein kinase/phosphatase balance in nicotine-induced short- and long-term memory effects. To assess memory function in mice passive avoidance test was used. The presented results confirm that acute nicotine (0.1mg/kg) improves short- and long-term memory. Pretreatment with L-type VGCC blockers (amlodipine, nicardipine verapamil) increased nicotine-induced memory improvement in the context of short- and long-term memory. Pretreatment with FK-506 (a potent CaN inhibitor) enhanced short- but not long-term memory effects of nicotine, while SL-327 (a selective MAPK/ERK kinase inhibitor) attenuated both nicotine-induced short- and long-term memory improvement. Acute nicotine enhances both types of memory via L-type VGCC blockade and via ERK1/2 activation. Only short- but not long-term memory enhancement induced by nicotine is dependent on CaN inhibition.

Searching for anti-glioma activity. Ribonucleoside analogues with modifications in nucleobase and sugar moieties.

Several ribonucleoside analogues with modifications in the nucleobase and sugar moiety have been screened for anti-glioma activity in the T98G glioma cell line using cervical (HeLa) cell line as reference human malignant cells, and lung fibroblast (MCR-5) cell line as non-cancerous reference cells. Among the investigated compounds, ribonucleosides containing 6-chloropurine (3), 7-guanine (5) and a pyrrolopyrimidine (18) as nucleobases, show promising anti-glioma activity with good selectivity indices, and can be considered as lead structures for further anti-cancer studies.

ALCOHOL DEPENDENCE--NEUROBIOLOGY AND TREATMENT.

The consequences of alcohol dependence concern serious health care, social and economic problems. The scope of many studies is to better understand mechanisms underlying alcohol addiction in order to work out new, more effective treatment strategies. Alcohol affects many neurotransmission systems within the brain. In general, acute alcohol enhances inhibitory transmission, up-regulating the GABAergic system and impairing glutamatergic function, therefore interfering the balance between excitatory and inhibitory synaptic inputs. Chronic alcohol consumption, meanwhile, in order to restore equilibrium leads to neuroadaptive changes caus- ing both decreased GABAergic and increased glutamatergic activity. Also function of other neurotransmitters and modulators is modified by the presence of alcohol, including glycine, adenosine, serotonin and dopamine. Moreover, a significant impact of alcohol on the endogenous opioid system, nicotinic cholinergic transmission and the endocannabinoids system has been also established. At present, only four medications are approved for the treatment of alcohol dependence in Europe, that is naltrexone, acamprosate, disulfiram and the most recent nalmefene. Among other promising strategies the following drugs are mentioned: baclofen, topiramate, ondansetron, aripiprazole, rimonabant and varenicline. Additionally, the role of appetite-regulating hormones, neuroimmune modulators or the body's stress-response system modulators in reducing alcohol consumption is currently of great interest, however, further investigations are needed.

New 3'-O-aromatic acyl-5-fluoro-2'-deoxyuridine derivatives as potential anticancer agents.

New aromatic and aliphatic 3'-O-acyl-5-fluoro-2'-deoxyuridine derivatives were synthesized and evaluated as candidates for prodrugs against various cancer cell lines. As the most promising candidate for antimalignant therapeutics was found a dual-acting acyl derivative 7h, which apparently released not only the known anticancer nucleoside, 5-fluoro-2'-deoxyuridine (FdU), but also an additional active metabolite, acetylsalicylic acid, reinforcing thus therapeutic effect of FdU. Promising therapeutic indices showed also some aromatic dicarboxylic acids derivatives decorated with FdU esters (11 and 12).

Antidepressant-like effects of the cannabinoid receptor ligands in the forced swimming test in mice: mechanism of action and possible interactions with cholinergic system.

The purpose of the experiments was to explore the role of the endocannabinoid system, through cannabinoid (CB) receptor ligands, nicotine and scopolamine, in the depression-related responses using the forced swimming test (FST) in mice. Our results revealed that acute injection of oleamide (10 and 20 mg/kg), a CB1 receptor agonist, caused antidepressant-like effect in the FST, while AM 251 (0.25-3 mg/kg), a CB1 receptor antagonist, did not provoke any effect in this test. Moreover, acute administration of both CB2 receptor agonist, JWH 133 (0.5 and 1 mg/kg) and CB2 receptor antagonist, AM 630 (0.5 mg/kg), exhibited antidepressant action. Antidepressant effects of oleamide and JWH 133 were attenuated by acute injection of both non-effective dose of AM 251, as well as AM 630. Among the all CB compounds used, only the combination of non-effective dose of oleamide (2.5 mg/kg) with non-effective dose of nicotine (0.5 mg/kg) caused an antidepressant effect. However, none of the CB receptor ligands, had influence on the antidepressant effects provoked by nicotine (0.2 mg/kg) injection. In turn, the combination of non-effective dose of oleamide (2.5 mg/kg); JWH (2 mg/kg) or AM 630 (2 mg/kg), but not of AM 251 (0.25 mg/kg), with non-effective dose of scopolamine (0.1 mg/kg), exhibited antidepressant properties. Indeed, all of the CB compounds used, intensified the antidepressant-like effects induced by an acute injection of scopolamine (0.3 mg/kg). Our results provide clear evidence that the endocannabinoid system participates in the depression-related behavior and through interactions with cholinergic system modulate these kind of responses.

Influence of calcium channel antagonists on nonsomatic signs of nicotine and D-amphetamine withdrawal in mice.

BACKGROUND: Nonsomatic signs of psychostimulant withdrawal, difficult to demonstrate in animal paradigms, may appear to promote drug seeking and drug relapse in humans; thus, it is important to understand the mechanisms that mediate this kind of behaviors. The present study was undertaken to examine the calcium-dependent mechanism of negative nonsomatic and anhedonia-related symptoms of acute and protracted withdrawal of nicotine and D-amphetamine. METHODS: Mice were chronically treated with nicotine (seven days, three times daily, 3.35 mg/kg, sc) or D-amphetamine (14 days, once daily, 2.5mg/kg, ip). Then, at the first, seventh or 14th day of withdrawal, anxiety- or depression-related effects, as well as cognition or nociception were studied. RESULTS: Our results demonstrated that, at the seventh or 14th day of D-amphetamine or nicotine withdrawal, respectively, mice exhibited increased anxiety and depression-like effects, memory impairment and hyperalgesia. Further, major findings showed that calcium channel antagonists, i.e., nimodipine, verapamil and flunarizine (10 and 20mg/kg, ip), injected before the test, attenuated above-mentioned signs of drug withdrawal. CONCLUSIONS: As an outcome, these findings support the hypothesis that similar calcium-dependent mechanisms are involved in an aversive nonsomatic component, associated with nicotine or d-amphetamine withdrawal. We can suggest that calcium channel blockers have potential to alleviate drug withdrawal and may thus be beneficial as pharmacotherapy of drug cessation and relapse.

A comparison of mecamylamine and bupropion effects on memory-related responses induced by nicotine and scopolamine in the novel object recognition test in mice.

BACKGROUND: The aim of the present study was to evaluate the involvement of the cholinergic receptors ligands in the memory-related responses in mice, using the novel object recognition (NOR) test. METHODS: The NOR test is based on natural, exploratory abilities of animals exposed to a new environment. In the first session, two copies of the same object were presented. In the next sessions (30min and 24h after), one of the familiar object and a new object were presented. RESULTS: The mice injected with nicotine (0.035 and 0.175mg/kg, free base, sc) before the first session spent more time exploring the new object than the familiar one at the second and third session, indicating that nicotine improved cognition. In turn, the mice injected with scopolamine (0.3 and 1mg/kg, ip) before the first session spent less time exploring the new object than the familiar one at the second and third trial, indicating that scopolamine impaired the memory performance. Additionally, the acute injection of drugs used in smoking cessation in humans: mecamylamine (0.5 and 1mg/kg) and bupropion (5 and 10mg/kg), prior to injections of nicotine (0.035mg/kg) or scopolamine (1mg/kg), significantly prevented nicotine-induced memory improvement or scopolamine-induced memory impairment, at the second and third session. CONCLUSIONS: The results of our studies unveiling neuronal mechanisms for cholinergic system of memory processes, via both nicotinic and muscarinic cholinergic receptors, will be useful for development of more effective pharmacotherapies for memory impairment-like treatment of human disorders in which cholinergic pathways have been implicated.

Effects of imperatorin on nicotine-induced anxiety- and memory-related responses and oxidative stress in mice.

The purpose of the reported experiments was to examine the effects of imperatorin [9-[(3-methylbut-2-en-1-yl)oxy]-7H-furo[3,2-g]chromen-7-one] on anxiety and memory-related responses induced by nicotine in mice and their relation to the level of nicotine-induced oxidative stress in brain as well as in the hippocampus and the prefrontal cortex. Male Swiss mice were tested for anxiety in the elevated plus maze test (EPM), and for cognition using passive avoidance (PA) procedures. Imperatorin, purified by high-speed counter-current chromatography from methanol extract of fruits of Angelica officinalis, acutely administered at the doses of 10 and 20mg/kg impaired the anxiogenic effect of nicotine (0.1mg/kg, s.c.). Furthermore, acute injections of subthreshold dose of imperatorin (1mg/kg, i.p.) improved processes of memory acquisition when co-administered with nicotine used at non-active dose of 0.05 mg/kg, s.c. Additionally, repeated administration of imperatorin (1mg/kg, i.p., twice daily, for 6 days) improved different stages of memory processes (both acquisition and consolidation) when injected in combination with non-active dose of nicotine (0.05 mg/kg, s.c.) in the PA task. Oxidative stress was assessed by determination of antioxidant enzymes (glutathione peroxidases (GPx), superoxide dismutase (SOD), glutathione reductase (GR)) activities as well as of malondialdehyde (MDA) concentration in the whole brain, the hippocampus and the prefrontal cortex after repeated administration of imperatorin (1mg/kg, 6 days) and single nicotine injection (0.05 mg/kgs.c.) on the seventh day. The results of our research suggest strong behavioural interaction between imperatorin and nicotine at the level of anxiety- and cognitive-like processes. Furthermore, imperatorin inhibited nicotine-induced changes in examined indicators of oxidative stress, especially in the hippocampus and the cortex.

Aryl nucleoside H-phosphonates. Part 16: synthesis and anti-HIV-1 activity of di-aryl nucleoside phosphotriesters.

Di-aryl nucleoside phosphotriesters have been explored as a new type of pronucleotides for the purpose of anti-HIV-1 therapy and efficient synthetic protocols, based on H-phosphonate chemistry, have been developed for the preparation of this class of compounds. It was found that anti-HIV-1 activity of the phosphotriesters bearing an antiviral nucleoside moiety (AZT, ddA) and also ddU was due, at least partially, to intracellular conversion into the corresponding nucleoside 5'-monophosphates, and their efficiency correlated well with the pK(a) values of the aryloxy groups present.