Single nucleotide polymorphisms in ADARB1 and TPH2 genes and suicide in Slovenia

V Sloveniji je med letoma 2010 in 2021 zaradi samomora umrlo v povprečju 407 oseb letno, kar nas po številu samomorov uvršča v sam svetovni vrh. Dejavnikov tveganja za samomorilno vedenje ne moremo pripisati zgolj enemu vzroku, saj gre večinoma za preplet hkratnega delovanja več dejavnikov, kot so npr. genetski dejavniki in dejavniki okolja, ki se izražajo v obliki psiholoških, bioloških in socioloških kazalcev. Pri samomorilnem vedenju je že bilo pokazano, da lahko pride do spremenjenega urejanja določenih genov na nivoju RNA. Ker sta v etiologijo samomorilnega vedenja vpletena tako serotoninski sistem kot tudi urejanje RNA, je ključno, da raziskujemo oba sistema hkrati. V okviru raziskave za magistrsko nalogo smo analizirali polimorfizme posameznih nukleotidov gena, ki je vključen v urejanje RNA, in sicer rs9983925 in rs4819035 v genu ADARB1, in pa tudi rs4290270 v genu TPH2, ki zapisuje za triptofan hidroksilazo 2 oz. hitrost določujoči encim sinteze serotonina v osrednjem živčevju. Cilj naše raziskave je bil ugotoviti, kateri izmed alelov izbranih polimorfizmov so različno zastopani v podskupinah oseb, ki so umrle zaradi samomora, v primerjavi s kontrolno skupino in na ta način prispevati k boljšemu poznavanju molekularne genetike samomorilnega vedenja. V raziskavo smo vključili večje število oseb slovenskega porekla, ki so umrle zaradi samomora, in kontrolno skupino – skupaj 690 oseb. Po izolaciji genomske DNA smo z uporabo hidrolizirajočih sond TaqMan in kvantitativnega PCR v realnem času določili, kateri aleli so prisotni v posameznem vzorcu, statistično analizo pa smo izvedli s pomočjo programa PLINK. Za polimorfizme TPH2 rs4290270, ADARB1 rs4819035 in ADARB1 rs9983925 nismo pri primerjavi podskupin umrlih zaradi samomora s kontrolno skupino ugotovili nobene statistično pomembne alelne oz. genotipske povezave s samomorom v slovenski populaciji.Between the years 2010 and 2021, the number of average yearly suicide deaths in Slovenia reached 407, making us one of the countries with the highest suicide rates in the world. The risk factors for suicidal behaviour cannot be attributed to a single cause, as they are usually a combination of multiple factors, such as genetic and environmental factors, which are expressed as psychological, biological and sociological indicators. It has already been shown that in the context of suicidal behaviour altered regulation of certain genes at the RNA level can occur. As both the serotonin system and RNA editing are involved in the aetiology of suicidal behaviour, it is crucial to investigate both systems simultaneously. In this research, we analysed single nucleotide polymorphisms rs9983925 and rs4819035 in the ADARB1 gene, that is involved in the RNA editing process, as well as rs4290270 in the TPH2 gene, which codes for tryptophan hydroxylase 2, the rate-determining enzyme of serotonin synthesis in the central nervous system. The aim of our study was to identify which of the alleles of the selected polymorphisms are differentially represented in the sub-groups of suicide victims compared to the control group and thus contribute to a better understanding of the molecular genetics of suicidal behaviour. Our study included a large number of suicide victims of Slovenian origin as well as a control group - 690 people in total. After isolation of genomic DNA, we used TaqMan hydrolysis probes and real-time quantitative PCR to determine which alleles were present in each sample. The statistical analysis was performed using PLINK program. For the TPH2 rs4290270, ADARB1 rs4819035 and ADARB1 rs9983925 polymorphisms, we did not find any statistically significant differences in genotype/allele frequency distribution between controls and Slovenian suicide victims

Isolation and characterization of annexin A11 mutants D40G and dN1-118

Amiotrofična lateralna skleroza (ALS) je neozdravljiva nevrodegenerativna bolezen, ki jo povzroči progresivna degeneracija motoričnih nevronov v možganski skorji, možganskem deblu in hrbtenjači. Bolezen so do sedaj povezali z mutacijami več kot 100 genov, med drugimi tudi z mutacijami na genu ANXA11. Aneksin A11 (ANXA11) je 505 aminokislinskih ostankov dolg protein, ki spada v naddružino aneksinov in se v odvisnosti od kalcija veže na fosfolipidne membrane. Protein se deli na dva strukturno povsem različna dela. Na C-koncu vsebuje štiri aneksinske ponovitve, sestavljene iz petih α-vijačnic, N-končna regija pa je v primerjavi s C-končno izredno variabilna in neurejena. Struktura N-končnega repa zaradi svojih intrinzičnih lastnosti še ni bila določena. V okviru diplomskega dela smo želeli pridobiti večje količine skrajšane oblike proteina, ki je imel N-končno domeno skrajšano za prvih 118 aminokislin do Ser119 (označili smo ga ANXA11dN1-118), in ANXA11dN-ter, ki je bil skrajšan do Gly179. Zapisa za rekombinantni obliki proteina smo z metodo od ligacije neodvisnega kloniranja LIC vnesli v bakterijska ekspresijska vektorja pMCSG7 in pMCSG7-GST. Zanimalo nas je, ali bo oznaka GST pomagala pri povečanju topnosti proteina in zmanjšala njegovo cepitev, ki se je pojavila v predhodnih poskusih z drugimi vektoji. Obe obliki smo izrazili v bakterijskih celicah E. coli seva BL21 [DE3] pLysS in ju izolirali s postopkom nikljeve afinitetne kromatografije. Uspelo nam je pridobiti 19,2 mg proteina ANXA11dN1-118 in 6,4 mg ANXA11dN-ter, kar je zadostovalo za nadaljnje kristalizacijske poskuse. Med izolacijo ni prihajalo do cepitve proteinov. Prav tako smo v protein ANXA11 divjega tipa s pomočjo mestnospecifične mutageneze uvedli mutacijo D40G, ki je najpogostejši povzročitelj bolezni ALS v evropski populaciji.Amyotrophic lateral sclerosis (ALS) is an untreatable neurodegenerative disease caused by progressive degeneration of motor neurons in the cerebral cortex, brainstem and spinal cord. Up until now, the disease has been associated with mutations in more than 100 genes, including mutations in the ANXA11 gene. Annexin A11 (ANXA11) a Ca2+ regulated phospholipid-binding protein encoded by 505 amino acids that belongs to a larger annexin protein superfamily. ANXA11 is split into two structurally different parts. The C-terminal core contains four annexin domains, which are composed of five alpha helix motifs, in comparison to the extremely variable and disordered N-terminal tail. Because of its intrinsic properties, the structure of the N-terminal tail is yet to be determined. As part of the research work, we prepared shortened versions of the proteinANXA11dN1-118 had a shortened N-terminal domain by 118 amino acids starting with Ser119 and ANXA11dN-ter starting with Gly179. The sequences for the recombinant proteins were incorporated in bacterial expression vectors pMCSG7 and pMCSG7-GST using ligation independent cloning. We wanted to know whether the GST-tag will increase the protein solubility and reduce its fragmentation, which occurred in previous experiments. Both forms were expressed in the E. coli strain BL21 [DE3] pLysS and isolated using nickel affinity chromatography. We managed to isolate 19,2 mg of ANXA11dN1-118 and 6,4 mg of ANXA11dN-ter protein, which was sufficient for further crystallization experiments. There were no proteolytic cleavages during protein isolation. As part of the research, we also implemented the D40G mutation in ANXA11 wild type protein using site-specific mutagenesis. The mutated form of ANXA11 with D40G mutation is the main cause of ALS in the European population

Guidance on protocol development for EFSA generic scientific assessments

Abstract EFSA Strategy 2027 outlines the need for fit‐for‐purpose protocols for EFSA generic scientific assessments to aid in delivering trustworthy scientific advice. This EFSA Scientific Committee guidance document helps address this need by providing a harmonised and flexible framework for developing protocols for EFSA generic assessments. The guidance replaces the ‘Draft framework for protocol development for EFSA's scientific assessments’ published in 2020. The two main steps in protocol development are described. The first is problem formulation, which illustrates the objectives of the assessment. Here a new approach to translating the mandated Terms of Reference into scientifically answerable assessment questions and sub‐questions is proposed: the ‘APRIO' paradigm (Agent, Pathway, Receptor, Intervention and Output). Owing to its cross‐cutting nature, this paradigm is considered adaptable and broadly applicable within and across the various EFSA domains and, if applied using the definitions given in this guidance, is expected to help harmonise the problem formulation process and outputs and foster consistency in protocol development. APRIO may also overcome the difficulty of implementing some existing frameworks across the multiple EFSA disciplines, e.g. the PICO/PECO approach (Population, Intervention/Exposure, Comparator, Outcome). Therefore, although not mandatory, APRIO is recommended. The second step in protocol development is the specification of the evidence needs and the methods that will be applied for answering the assessment questions and sub‐questions, including uncertainty analysis. Five possible approaches to answering individual (sub‐)questions are outlined: using evidence from scientific literature and study reports; using data from databases other than bibliographic; using expert judgement informally collected or elicited via semi‐formal or formal expert knowledge elicitation processes; using mathematical/statistical models; and – not covered in this guidance – generating empirical evidence ex novo. The guidance is complemented by a standalone ‘template’ for EFSA protocols that guides the users step by step through the process of planning an EFSA scientific assessment