Virološko i serološko istraživanje influence ptica u riječnih galebova uhvaćenih na gradskom odlagalištu otpada u Zagrebu.

A total of 142 black-headed gulls (BHGs), Chroicocephalus ridibundus, were captured during February and March 2009 at the Zagreb city rubbish dump (45.45 N 16.01 E) in order to collect cloacal swabs and serum samples. Cloacal swabs were tested by virus isolation in embryonated chicken eggs, which resulted in isolation of one avian influenza virus (AIV) that was of the H16 subtype. The collected sera were tested by blocking ELISA for avian influenza antibodies, resulting in 28.2% positive samples, which were retested by haemagglutination inhibition (HI) using H5 and H7 subtype antigens. Only one serum sample was positive for H5 and none for H7 antibodies. Statistically, no significant difference was found between the ages of AIV seropositive birds (2 = 2.08, df = 1, P = 0.15). In contrast, regarding seroprevalence in different months of capture, a higher proportion of positive gulls was found during March than during February (2 = 4.53, df = 1, P = 0.03), especially in younger birds (2 = 7.67, df = 1, P = 0.006). This finding suggests that BHGs might contract AIV infection during their aggregations in large numbers on rubbish dumps or similar feeding sites during the winter. Although only one of the 142 tested birds was positive for H5 subtype antibodies, this finding cannot be neglected seeing that apparently healthy BHGs can carry highly pathogenic AIV of the H5N1 subtype. Nevertheless, the results of our study have shown that BHGs are more often infected with other AIV subtypes and therefore are most likely not the primary carriers of H5 AIV. To our knowledge this is the first detection of H16 AIV in Croatia.Tijekom veljače i ožujka 2009. na gradskom odlagalištu otpada u Zagrebu (45.45 N 16.01 E) uhvaćena su 142 riječna galeba (Chroicocephalus ridibundus) u cilju prikupljanja brisova nečisnica i uzoraka krvi. Brisovi nečisnica su pretraženi izdvajanjem virusa u embrioniranim kokošjim jajima što je rezultiralo izdvajanjem virusa influence ptica koji je bio podtipa H16. Prikupljeni uzorci seruma su pretraženi imunoenzimnim testom za protutijela virusa influence ptica od kojih je 28,2% bilo pozitivno. Pozitivni uzorci su dodatno pretraženi inhibicijom hemaglutinacije s antigenima podtipa H5 i H7 među kojima je samo jedan bio pozitivan na H5 i nijedan na H7 protutijela. Statistički nije ustanovljena značajna razlika među skupinama različite dobi seropozitivnih ptica (2 = 2,08, df = 1, P = 0,15). Nasuprot tome, seroprevalencija u različitim mjesecima lova je imala veći udio tijekom ožujka nego tijekom veljače (2 = 4,53, df = 1, P = 0,03), naročito u mlađih ptica (2 = 7,67, df = 1, P = 0,006). Ovakav nalaz ukazuje na moguću infekciju riječnih galebova tijekom njihovoga brojnog okupljanja na odlagalištima otpada ili sličnim mjestima za hranjenje tijekom zime. Iako je samo jedna od 142 ptice bila pozitivna na protutijela za H5 podtip, nalaz se ne smije zanemariti znajući da naizgled zdravi riječni galebovi mogu nositi virus jako patogene influence podtipa H5N1. Unatoč tome, rezultati našeg istraživanja pokazuju da riječni galebovi češće bivaju inficirani drugim podtipovima virusa influence ptica te stoga najvjerojatnije nisu primarni nosioci virusa influence podtipa H5. Sukladno našim spoznajama, ovo je prvi dokaz virusa influence podtipa H16 u Hrvatskoj

Učinci istodobne primjene THC-a i irinotekana na rast tumora i biokemijske markere na singeničnom modelu raka debelog crijeva u miševa

Clinical treatment with the antineoplastic drug irinotecan (IRI) is often hindered by side effects that significantly reduce the quality of life of treated patients. Due to the growing public support for products with Δ9-tetrahydrocannabinol (THC), even though relevant scientific literature does not provide clear evidence of their high antitumour potential, some cancer patients take unregistered preparations containing up to 80 % THC. This study was conducted on a syngeneic colorectal cancer mouse model to test the efficiency and safety of concomitant treatment with IRI and THC. Male BALB/c mice subcutaneously injected with CT26 cells were receiving 60 mg/kg of IRI intraperitoneally on day 1 and 5 of treatment and/or 7 mg/kg of THC by gavage a day for 7 days. Treatment responses were evaluated based on changes in body, brain, and liver weight, tumour growth, blood cholinesterase activity, and oxidative stress parameters. Irinotecan’s systemic toxicity was evidenced by weight loss and high oxidative stress. The important finding of this study is that combining THC with IRI diminishes IRI efficiency in inhibiting tumour growth. However, further studies, focused on more subtle molecular methods in tumour tissue and analytical analysis of IRI and THC distribution in tumour-bearing mice, are needed to prove our observations.Kliničko liječenje antineoplastičnim lijekom irinotekanom (IRI) često je otežano nuspojavama koje značajno smanjuju kvalitetu života liječenih bolesnika. Zbog sve veće javne potpore proizvodima s Δ9-tetrahidrokanabinolom (THC), iako relevantna znanstvena literatura ne daje jasne dokaze o njihovu visokom antitumorskom potencijalu, oboljeli od raka uzimaju neregistrirane pripravke koji sadržavaju i do 80 % THC-a. Ova studija provedena je na modelu singeničnoga tumora debelog crijeva u miševa kako bi se testirala učinkovitost i sigurnost istodobnog tretmana irinotekanom i THC-om. Mužjaci BALB/c miševa kojima su supkutano injicirane CT26 stanice primili su 60 mg/kg IRI-ja intraperitonealno prvi i peti dan i/ili 7 mg/kg THC-a oralno svaki dan tijekom sedam dana. Učinkovitost tretmana procijenjena je na temelju promjena u težini tijela, mozga i jetre, rasta tumora, aktivnosti kolinesteraza u krvi i parametara oksidacijskoga stresa. Sistemska toksičnost irinotekana potvrđena je smanjenjem težine miševa i povećanjem parametara oksidacijskoga stresa. Značaj je rezultata ove studije u smanjenoj učinkovitosti IRI-ja u inhibiciji rasta tumora tijekom istodobnog uzimanja s THC-om. Međutim, potrebna su daljnja istraživanja usmjerena na suptilnije molekularne metode u tumorskom tkivu i analitička analiza distribucije IRI-ja i THC-a u miševa s tumorom kako bi se dokazala naša opažanja

Environmental shaping of codon usage and functional adaptation across microbial communities.

Microbial communities represent the largest portion of the Earth's biomass. Metagenomics projects use high-throughput sequencing to survey these communities and shed light on genetic capabilities that enable microbes to inhabit every corner of the biosphere. Metagenome studies are generally based on (i) classifying and ranking functions of identified genes; and (ii) estimating the phyletic distribution of constituent microbial species. To understand microbial communities at the systems level, it is necessary to extend these studies beyond the species' boundaries and capture higher levels of metabolic complexity. We evaluated 11 metagenome samples and demonstrated that microbes inhabiting the same ecological niche share common preferences for synonymous codons, regardless of their phylogeny. By exploring concepts of translational optimization through codon usage adaptation, we demonstrated that community-wide bias in codon usage can be used as a prediction tool for lifestyle-specific genes across the entire microbial community, effectively considering microbial communities as meta-genomes. These findings set up a 'functional metagenomics' platform for the identification of genes relevant for adaptations of entire microbial communities to environments. Our results provide valuable arguments in defining the concept of microbial species through the context of their interactions within the community

Oksidacijski stres, aktivnost kolinesteraza i primarna oštećenja u jetri, krvi i plazmi Wistar štakora nakon 28-dnevnog izlaganja glifosatu

In this 28 day-study, we evaluated the effects of herbicide glyphosate administered by gavage to Wistar rats at daily doses equivalent to 0.1 of the acceptable operator exposure level (AOEL), 0.5 of the consumer acceptable daily intake (ADI), 1.75 (corresponding to the chronic population-adjusted dose, cPAD), and 10 mg kg-1 body weight (bw) (corresponding to 100 times the AOEL). At the end of each treatment, the body and liver weights were measured and compared with their baseline values. DNA damage in leukocytes and liver tissue was estimated with the alkaline comet assay. Oxidative stress was evaluated using a battery of endpoints to establish lipid peroxidation via thiobarbituric reactive substances (TBARS) level, level of reactive oxygen species (ROS), glutathione (GSH) level, and the activity of glutathione peroxidase (GSH-Px). Total cholinesterase activity and the activities of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) were also measured. The exposed animals gained less weight than control. Treatment resulted in significantly higher primary DNA damage in the liver cells and leukocytes. Glyphosate exposure significantly lowered TBARS in the liver of the AOEL, ADI, and cPAD groups, and in plasma in the AOEL and cPAD group. AChE was inhibited with all treatments, but the AOEL and ADI groups significantly differed from control. Total ChE and plasma/liver ROS/GSH levels did not significantly differ from control, except for the 35 % decrease in ChE in the AOEL and ADI groups and a significant drop in liver GSH in the cPAD and 100xAOEL groups. AOEL and ADI blood GSH-Px activity dropped significantly, but in the liver it significantly increased in the ADI, cPAD, and 100xAOEL groups vs. control. All these findings show that even exposure to low glyphosate levels can have serious adverse effects and points to a need to change the approach to risk assessment of low-level chronic/sub-chronic glyphosate exposure, where oxidative stress is not necessarily related to the genetic damage and AChE inhibition.U okviru 28-dnevnog pokusa istražili smo učinke herbicida glifosata na modelu odraslih mužjaka Wistar štakora koji su oralno dobivali testirani spoj u subletalnim dnevnim dozama: 0,1 od prihvatljive razine izloženosti operatera (0,1xAOEL), 0,5 od prihvatljivog dnevnog unosa za potrošače (0,5xADI), 1,75 (odgovara kroničnoj populacijskoj prilagođenoj dozi, cPAD) i 10 mg kg-1 tjelesne težine na dan (odgovara 100xAOEL). Tijekom pokusa praćeni su sistemski toksični učinci. Nakon završetka svih tretmana svakoj je pokusnoj životinji izmjerena tjelesna težina i težina jetre te su uspoređene s polazišnim vrijednostima. Alkalnim komet-testom izmjerena je razina primarnih oštećenja DNA u leukocitima i jetrenim stanicama. Primjenom metoda za procjenu oksidacijskog stresa izmjerene su razine lipidne peroksidacije (TBARs), reaktivnih kisikovih vrsta (ROS) i glutationa (GSH) te aktivnost enzima glutation peroksidaze (GSH-Px). Izmjerene su i aktivnosti ukupnih kolinesteraza (ChE), acetilkolinesteraze (AChE) i butirilkolinesteraze (BChE). Izloženi štakori imali su manje priraste težine od kontrolnih. Izloženost glifosatu uzrokovala je značajne poraste razine primarnih oštećenja DNA u jetrenim stanicama te malo manje u leukocitima. U svim izloženim skupinama izmjerene su niže vrijednosti TBARs u odnosu na kontrolu, sa značajno nižim vrijednostima u AOEL, ADI i cPAD skupinama u uzorcima jetre te u AOEL i cPAD skupinama u uzorcima plazme. Aktivnost AChE bila je smanjena u svim tretmanima, s najnižom stopom nakon izlaganja dozi ADI. Aktivnost BChE blago je smanjena nakon izlaganja ADI, a povećana nakon izlaganja dozama cPAD i 100xAOEL. Ukupna aktivnost ChE te razine ROS/GSH u plazmi / jetri nisu se značajno razlikovale od kontrole, osim značajnog smanjenja jetrenog GSH nakon izlaganja dozama cPAD i 100xAOEL te 35-postotnog smanjenja aktivnosti ChE nakon izlaganja dozama AOEL i ADI. Aktivnost GSH-Px u krvi značajno je smanjena u AOEL i ADI tretmanu, a aktivnost GSH-Px u uzorcima jetre značajno je povećana u skupinama ADI, cPAD i 100xAOEL prema kontroli. Dobiveni rezultati pokazuju da čak i izloženost vrlo niskim dozama glifosata može izazvati mjerljive toksične učinke te upućuje na potrebu za promjenom pristupa procjeni rizika zbog kronične/subkronične izloženosti niskim dozama glifosata gdje oksidacijski stres ne mora nužno korelirati s razinom oštećenja DNA i inhibicijom acetilkolinesteraz

Splice Variant of Mouse Stam2 mRNA in Nervous and Muscle Tissue Contains Additional Exon with Stop Codon within Region Coding for VHS Domain

AIM: To analyze the alternatively spliced variant of Stam2 mRNA and determine its distribution in mouse tissues. - - - - - METHODS: We identified a novel alternatively spliced exon by cloning and sequencing of Stam2 cDNA obtained from tissue samples of 3-5 months old male C57Bl/6NCrl mice. The sequence of the alternatively spliced exon was analyzed by bioinformatic tools. The tissue distribution of different Stam2 mRNA variants was determined by reverse transcription-polymerase chain reaction, and the consequences of the alternative splicing at the protein level were analyzed by western blot with the polyclonal anti-STAM2 antibody. - - - - - RESULTS: The novel alternatively spliced exon 1A of mouse Stam2 gene was inserted within Stam2 coding region and it contained a stop codon. The exon did not bear similarities to any other cDNA or protein sequence in the mouse, rat, or human databases. Both mRNA variants, with and without exon 1A, were present in the cortex, hippocampus, olfactory bulb, medulla oblongata, spinal cord, cerebellum, and the skeletal and heart muscle, while the other analyzed organs contained only the variant without the additional exon. The mRNA with the included exon did not give rise to a protein form detectable by western blotting with the polyclonal anti-STAM2 antibody. - - - - - CONCLUSION: The alternatively spliced exon 1A was included in mRNA splice variant present in the nervous and muscle tissues. The alternative splicing event did not have major impact on STAM2 production and functionality. It seems that exon 1A is an evolutionary new exon created by exonization of an intronic sequence

Petogodišnje istraživanje (2018 – 2022) ušare Bubo bubo u Hrvatskoj - Rekolonizacija i procjena brojnosti ušare u nizinskoj Hrvatskoj

The Eagle Owl Bubo bubo is one of the largest owl species in Europe and Asia. It mainly inhabits rocky areas with suitable cliffs, where it nests. The global population trend is declining. In Croatia, the largest part of the population breeds in the Mediterranean. The population in the lowland part was extinct in the first half of the 20th century, but recently they have been observed breeding again. The aim of this paper was to present the results of a five-year study of the Eagle Owl in three parts of Croatia: the lowland, the Primorje and Gorski kotar; and Istria. From 2018 to 2022, educated volunteers collected data about the Eagle Owl active territories by listening to spontaneous singing from 1st February until 15th March. A total of 485 visits were made to 154 locations, and 60 active territories of the Eagle Owl were found. The estimated size of the breeding populations in the lowland part is 32 to 45 active territories, while for Primorje and Gorski kotar, as well as Istria, further research ought to be conducted in order to estimate the population size.Ušara Bubo bubo je jedna od najvećih vrsta sova u Europi i Aziji. Uglavnom nastanjuje stjenovita područja te staništa s prikladnim liticama na kojima gnijezdi. Globalni tren populacije je u padu. Strmi pad europske populacije uslijedio je sredinom 19. i početkom 20. stoljeća, a razlozi smanjenja brojnosti su krivolov, izgradnja infrastrukture te trovanje. U Hrvatskoj se najveći dio populacije ušare gnijezdi u mediteranskom dijelu zemlje. Populacija u nizinskom dijelu Hrvatske bila je izumrla u prvoj polovici 20. stoljeća, no recentni podaci pokazali su da ušara ponovno naseljava i taj dio zemlje. Na temelju toga pokrenut je program praćenja stanja ušare u Hrvatskoj. Cilj ovog rada bio je prikazati rezultate petogodišnjeg istraživanja ušare u Hrvatkoj. Područje istraživanja je podijeljeno na tri dijela: nizinski dio,, Primorje i Gorski kotara te Istru. Od 2018. do 2022. istraživanje se provodilo u razdoblju od 1. veljače do 15. ožujka kada je mužjak ušare vokalno najaktivniji. Educirani volonteri na terenu su metodom pasivnog osluškivanja prikupljali podatke o prisutnosti ušare te bilježili podatke o vremenskim prilikama, buci i prijetnjama. Odrađeno je ukupno 485 posjeta na 154 lokacije na kojima su pronađena 60 aktivna teritorija ušara. U 80,51% slučajeva zabilježeno je glasanje samo mužjaka. Istraživanje je većinom odrađeno u dobrim vremenskim uvjetima, što je preduvjet metodologije. Ovo istraživanje pokazalo je da se ušara ponovno gnijezdi u nizinskom dijelu Hrvatske gdje su važnu ulogu imali bolja zakonska zaštita i smanjen progon, a repopulacija je vjerojatno bila moguća zbog snažnih populacija u mediteranskom dijelu Hrvatske i Slovenije. U tom dijelu zemlje važan element staništa predstavljaju kamenolomi koji su svojevrsna zamjena za prirodne stijene. Procijenjena veličina populacija u nizinskom dijelu je 32 do 45 parova, dok za Primorje i Gorski kotar nije moguce procijeniti veličinu populacije zbog nedovoljno istraženog područja. Rezultati pokazuju izostanak ušare na dva područja u nizinskom dijelu s naizgled povoljnim staništem. Ovaj rad upućuje na povratak ušare u nizinskom dijelu zemlje te potrebu za sustavnim monitoringom te detaljnijim istraživanjem u Primorju i Gorskom kotaru te Istri

DNA Damaging Effects, Oxidative Stress Responses and Cholinesterase Activity in Blood and Brain of Wistar Rats Exposed to Δ⁹-Tetrahydrocannabinol

Currently we are faced with an ever-growing use of Δ9-tetrahydrocannabinol (THC) preparations, often used as supportive therapies for various malignancies and neurological disorders. As some of illegally distributed forms of such preparations, like cannabis oils and butane hash oil, might contain over 80% of THC, their consumers can become intoxicated or experience various detrimental effects. This fact motivated us for the assessments of THC toxicity in vivo on a Wistar rat model, at a daily oral dose of 7 mg/kg which is comparable to those found in illicit preparations. The main objective of the present study was to establish the magnitude and dynamics of DNA breakage associated with THC exposure in white blood and brain cells of treated rats using the alkaline comet assay. The extent of oxidative stress after acute 24 h exposure to THC was also determined as well as changes in activities of plasma and brain cholinesterases (ChE) in THC-treated and control rats. The DNA of brain cells was more prone to breakage after THC treatment compared to DNA in white blood cells. Even though DNA damage quantified by the alkaline comet assay is subject to repair, its elevated level detected in the brain cells of THC-treated rats was reason for concern. Since neurons do not proliferate, increased levels of DNA damage present threats to these cells in terms of both viability and genome stability, while inefficient DNA repair might lead to their progressive loss. The present study contributes to existing knowledge with evidence that acute exposure to a high THC dose led to low-level DNA damage in white blood cells and brain cells of rats and induced oxidative stress in brain, but did not disturb ChE activities

Effects of concomitant use of THC and irinotecan on tumour growth and biochemical markers in a syngeneic mouse model of colon cancer

Clinical treatment with the antineoplastic drug irinotecan (IRI) is often hindered by side effects that significantly reduce the quality of life of treated patients. Due to the growing public support for products with Δ9-tetrahydrocannabinol (THC), even though relevant scientific literature does not provide clear evidence of their high antitumour potential, some cancer patients take unregistered preparations containing up to 80 % THC. This study was conducted on a syngeneic colorectal cancer mouse model to test the efficiency and safety of concomitant treatment with IRI and THC. Male BALB/c mice subcutaneously injected with CT26 cells were receiving 60 mg/kg of IRI intraperitoneally on day 1 and 5 of treatment and/or 7 mg/kg of THC by gavage a day for 7 days. Treatment responses were evaluated based on changes in body, brain, and liver weight, tumour growth, blood cholinesterase activity, and oxidative stress parameters. Irinotecan’s systemic toxicity was evidenced by weight loss and high oxidative stress. The important finding of this study is that combining THC with IRI diminishes IRI efficiency in inhibiting tumour growth. However, further studies, focused on more subtle molecular methods in tumour tissue and analytical analysis of IRI and THC distribution in tumour-bearing mice, are needed to prove our observations

Evaluation of Toxic Effects Induced by Sub-Acute Exposure to Low Doses of α-Cypermethrin in Adult Male Rats

To contribute new information to the pyrethroid pesticide α-cypermethrin toxicity profile, we evaluated its effects after oral administration to Wistar rats at daily doses of 2.186, 0.015, 0.157, and 0.786 mg/kg bw for 28 days. Evaluations were performed using markers of oxidative stress, cholinesterase (ChE) activities, and levels of primary DNA damage in plasma/whole blood and liver, kidney, and brain tissue. Consecutive exposure to α-cypermethrin affected the kidney, liver, and brain weight of rats. A significant increase in concentration of the thiobarbituric acid reactive species was observed in the brain, accompanied by a significant increase in glutathione peroxidase (GPx) activity. An increase in GPx activity was also observed in the liver of all α-cypermethrin-treated groups, while GPx activity in the blood was significantly lower than in controls. A decrease in ChE activities was observed in the kidney and liver. Treatment with α-cypermethrin induced DNA damage in the studied cell types at almost all of the applied doses, indicating the highest susceptibility in the brain. The present study showed that, even at very low doses, exposure to α-cypermethrin exerts genotoxic effects and sets in motion the antioxidative mechanisms of cell defense, indicating the potential hazards posed by this insecticide