Anytime-valid inference in N-of-1 trials

App-based N-of-1 trials offer a scalable experimental design for assessing the effects of health interventions at an individual level. Their practical success depends on the strong motivation of participants, which, in turn, translates into high adherence and reduced loss to follow-up. One way to maintain participant engagement is by sharing their interim results. Continuously testing hypotheses during a trial, known as "peeking", can also lead to shorter, lower-risk trials by detecting strong effects early. Nevertheless, traditionally, results are only presented upon the trial's conclusion. In this work, we introduce a potential outcomes framework that permits interim peeking of the results and enables statistically valid inferences to be drawn at any point during N-of-1 trials. Our work builds on the growing literature on valid confidence sequences, which enables anytime-valid inference with uniform type-1 error guarantees over time. We propose several causal estimands for treatment effects applicable in an N-of-1 trial and demonstrate, through empirical evaluation, that the proposed approach results in valid confidence sequences over time. We anticipate that incorporating anytime-valid inference into clinical trials can significantly enhance trial participation and empower participants

Influence of essential oil Helichrysum italicum (Roth) G. Don on the formation of non-tuberculous mycobacterial biofilm

Cilj: Ispitati antimikrobni i antioksidacijski učinak te sposobnost inhibicije biofilma eteričnog ulja (EU) smilja [Helichrysum italicum (Roth) G. Don (H. italicum)] prema netuberkuloznim mikobakterijama M. avium i M. intracellulare. Materijali i metode: Za određivanje minimalne inhibicijske koncentracije (MIK) i minimalne baktericidne koncentracije (MBK) EU H. italicum prema M. avium i M. intracellulare korištena je metoda mikrodilucije u bujonu. Preživljavanje mikobakterija u Middlebrook 7H9 bujonu pod utjecajem EU smilja praćeno je subkultivacijom suspenzija 0, 1, 4 i 8 dan. Za određivanje inhibicije stvaranja biofilma u vodi primjenjena je metoda bojenja biofilma s kristal violetom. Antioksidacijski učinak MIK koncentracije EU određen je DPPH (2,2-difenil-1-pikril-hidrazil) metodom. Rezultati: MIK i MBK vrijednost EU smilja iznosila je 3,2 mg/ml. Koncentracija ½ MIK vrijednosti dovela je nakon prvog dana do redukcije vijabilnosti M. avium za približno 2 log10 odnosno 2,5 log10 za M. intracellulare, dok je četvrti dan zabilježena potpuna inhibicija porasta obje vrste mikobakterija. M. avium je pokazao veću tendenciju stvaranja biofilma. Koncentracija od ¼ i ½ MIK vrijednosti dovela je do statistički značajne inhibicije stvaranja biofilma ispitivanih mikobakterija. MIK koncentracija EU H. italicum je nakon 60 minuta inhibirala 38,2 % DPPH radikala. Zaključak: Subinhibitorne koncentracije EU smilja značajno reduciraju vijabilnost M. avium i M. intracellulare i dovode do značajne inhibicije stvaranja biofilma ispitivanih mikobakterija u vodi što bi omogućilo primjenu niskih efektivnih koncentracija ovog EU, koje nemaju citotoksični učinak, kao prirodnog dezinficijensa u vodi.Objective: To investigate the antimicrobial and antioxidative effect of the essential oil (EO) of immortelle [Helichrysum italicum (Roth) G. Don (H. italicum)] and its ability to inhibit biofilm of nontuberculous mycobacteria, M. avium and M. intracellulare. Materials and methods: a broth microdilution method was used to determine the minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC) of the H. italicum EO. Survival of mycobacteria in Middlebrook 7H9 broth under the influence of H. italicum EO was monitored by subcultivation of suspension of 0, 1, 4 and 8 days. For determination of the inhibition of biofilm formation in water, the biofilm coloring method with crystal violet was applied. The antioxidant effect of MIC concentrations of H. italicum EO was determined by DPPH (2,2-diphenyl-1-picrylhydrazil) method. Results: MIC and MBC value of immortelle EO was 3.2 mg/ml. Treatment of mycobacteria with H. italicum EO at one half times the MIC resulted in approximately 2 log10 reduction of M. avium and 2.5 log10 M. intracellulare respectively over 24 h, and complete reduction of both mycobacteria on the fourth day. M. avium showed a greater tendency to form biofilms. The concentration of one-quarter and one-half of the MIC values resulted in statistically significant inhibition of biofilm production of the tested mycobacteria. MIC concentrations of H. italicum EU after 60 minutes inhibited 38.2% of DPPH radicals. Conclusion: The subinhibitory concentrations of H. italicum EO significantly reduce the viability of M. avium and M. intracellulare and result in substantial inhibition of biofilm production of the tested mycobacteria in water, which would allow the application of low effective concentrations of this EO that do not have a cytotoxic effect as a natural disinfectant in water

Evaluation of the Antioxidant Capacity, Antimicrobial and Antiproliferative Potential of Fir (Abies alba Mill.) Honeydew Honey Collected from Gorski kotar (Croatia)

U radu su ispitani antiproliferativni, antimikrobni i antioksidacijski učinci medljikovaca jele (Abies alba Mill.) prikupljenih s planinskog područja Hrvatske (Gorski kotar) a u svrhu potencijalne primjene umjesto standardnih antibiotika i kemoterapeutika. Preživljavanje stanica, annexin V test i protočna citometrijska analiza poslužili su za procjenu antiproliferativnog učinka na indukciju apoptoze i smrti humanih tumorskih staničnih linija: HeLa, MCF-7, SW620, CFPAC-1, MIA PaCa-2 i normalnih diploidnih humanih fibroblasta (BJ). Antimikrobna je aktivnost testirana na različitim sojevima bakterija Staphylococcus i Acinetobacter uporabom difuzijske i mikrodilucijske metode. DPPH˙ testom određena je sposobnost uklanjanja radikala, dok je matematičkim modelima proučavana kinetika inhibicije DPPH˙. Uočen je antiproliferativni učinak na sve ispitivane stanične linije kao i na normalne diploidne fibroblaste (BJ), kolorektalni adenokarcinom (SW620, metastatski) i adenokarcinom epitela dojke (MCF-7, metastatski). Mehanizmi antiproliferativnog učinka uključuju nakupljanje stanica u sub-G1 fazi u svim ispitivanim stanicama te indukciju apoptoze osobito u SW620 i MCF-7 stanicama. Antibakterijski testovi pokazuju da su rezistentni sojevi obaju bakterija, uključujući i multirezistentni soj A. baumannii ATCC® BAA-1605™, bili osjetljivi na sve ispitane uzorke meda. Proučavanjem kinetike uklanjanja radikala dokazano je da antioksidansi prisutni u medu posjeduju različite sposobnosti hvatanja radikala, da reagiraju s njima različitim brzinama i time uzrokuju dva stupnja reakcije. Rezultati istraživanja pokazuju da hrvatski medljikovac ima terapeutski potencijal zbog snažne biološke aktivnosti te može služiti u zaštiti ljudskog zdravlja.The paper examines the antiproliferative, antimicrobial and antioxidative effects of fir (Abies alba Mill.) honeydew honey from mountain region of Croatia (Gorski kotar) as a potential replacement for standard antibiotics and chemotherapeutic agents. Cell viability, annexin V assay and flow cytometry analysis served to analyse the antiproliferative effect on, apoptosis induction in and cell death of cancer cell lines: HeLa, MCF-7, SW620, CFPAC-1, MIA PaCa-2 and normal diploid human fibroblasts (BJ). Antimicrobial activity was tested against Staphylococcus and Acinetobacter strains by agar well diffusion and microdilution assays. The DPPH˙ assay determined the radical scavenging activity, while mathematical models helped to evaluate the kinetic data of DPPH˙ inhibition. Antiproliferative effect on all tested cell lines and the prominent effect on normal diploid human fibroblasts (BJ), colorectal adenocarcinoma (SW620, metastatic) and breast epithelial adenocarcinoma (MCF-7, metastatic) was observable. The mechanisms of antiproliferative effect included accumulation of cells in the sub-G1 phase in all tested cells and induction of apoptosis in SW620 and MCF-7 cells predominantly. The antibacterial assays showed that antibiotic resistant strains of both bacteria, including multi-resistant strain A. baumannii ATCC® BAA-1605™, were sensitive to all tested honey samples. Radical scavenging assay suggests that antioxidants present in the honey possess different radical suppressing abilities and that they react at different rates with radicals, thereby causing two steps of reaction. The results of the study indicate that Croatian fir honeydew honey has a therapeutic potential due to the strong biological activity and can serve to protect human health

Program for Dental Health Advancement in Children „Dental Passport“

Cilj: Potreba za poboljšanjem oralnog zdravlja u Republici Hrvatskoj temeljila se na zdravstvenim po-kazateljima i lošem stanju oralnog zdravlja te je rezultirala provedbom mjera za poboljšanje korištenja dentalne zdravstvene zaštite školske djece na temelju nacionalnog programa Zubna putovnica s kojim se počelo u školskoj godini 2017./2018. Svrha ovog rada bila je predstaviti sadržaj i rezultate provedbe programa te ih analizirati s naglaskom na održivost i obuhvatnost programa te provedbu preventivnih aktivnosti, odnosno postupaka. Materijal i metode: Podatci su prikupljeni iz ispunjenih obrazaca Zubna putovnica od rujna 2017. Do kolovoza 2018. godine. Pregledi i dijagnostičko-terapijski postupci (DTP) bilježili su se u sklopu Centralnoga zdravstvenog informacijskog ustava Republike Hrvatske. Rezultati: U školskoj godini 2017./2018. Doktora dentalne medicine posjetilo je 24.729 učenika 6. razreda, s odazivom od 68 % za Republiku Hrvatsku. Prema podatcima CEZIH-a u razdoblju od 1. Rujna do 31. Prosinca 2017. Godine kod djece u dobi od 12 godina zabilježeno je povećanje broja prvih pregleda i dijagnostičko-terapijskih postupaka u odnosu prema istom razdoblju 2015. Najrjeđe zabilježeni preventivni postupci kod predškolske djece i učenika 6. Razreda bili su pečaćenje fisura po zubu i pečatni ispuni, a demonstracija čišćenja usta i motiviranje djece na higijenu usta bili su najčešći. Zaključak: Dobra organizacija rada u djelatnosti školske medicine daje osnovu za provedbu dentalnih programa u koje se uključuje školsku djecu, ali su također veća motiviranost pružatelja dentalne zdravstvene zaštite u provedbi preventivnih postupaka te motiviranost roditelja i pacijena-ta za aktivnije uključivanje u program Zubna putovnica, važne odrednice u daljnjoj provedbi i poboljšanju dentalnog zdravlja djece.Objective: The need for improving oral health in the Republic of Croatia was based on health indicators andpoor oral health status and resulted in the implementation of measures to improve the use of dental health care for schoolchildren through the national program called “Dental Passport”, which began in the 2017/2018 school year. Purpose: The aim of this study was to present the content and results of its implementation and to analyse them with emphasis on the sustainability and inclusion of the program and its implementation in preventive activities and procedures. Material and Methods: The data were collected from the completed “Dental Passport” forms from September 2017 to August 2018. The dental examinations and diagnostic-therapeutic procedures were recorded in The Central Health Information System of Croatia. Results: In the school year 2017/2018, 24,729 6th grade elementary school tudents visited a dentist. The response rate in the Republic of Croatia was 68%. According to the CEZIH data, in the period from September 1 to December 31 2017, an increase in the number of first examinations and diagnostic-therapeutic procedures was observed in children aged 12 years, compared to the same peri-od in 2015. The least reported preventive procedures in preschool children and 6th grade students were fissure sealing and sealing restorations, while motivating and instructing children on oral hygiene were the most common reported procedures. Conclusion: Good organization of work in school medicine pro-vides the basis for the implementation of dental programs involving schoolchildren. However, greater motivation of the dental health care provider in the implementation of preventive procedures and motivation of parents and patients for a more active involvement in the “Dental Passport” program are important determinants in further implementation and improvement of oral health in children

Usporedba intraplazmidne rekombinacije u bakterijama Agrobacterium tumefaciens i Escherichia coli

In this work we have constructed a plasmid to compare intraplasmid recombination efficiency in Agrobacterium tumefaciens and Escherichia coli. The plasmid contains two directly repeated copies of spectinomycin resistance gene, one lacking 5’ and the other lacking 3’ end. These two copies share a 570-bp region of homology and are separated by the ampicillin resistance gene. Homologous recombination between repeated copies of incomplete spectinomycin resistance genes results in the restoration of spectinomycin resistance. During this process, ampicillin resistance gene is either deleted or incomplete spectinomycin genes are amplified along with the ampicillin resistance gene. This experimental system enabled us to follow for the first time the generation of deletions and amplifications during intraplasmid recombination in A. tumefaciens. We show here that predominantly RecA-independent mechanism contributes to the formation of deletion and amplification products in both, A. tumefaciens and E. coli. Additionally, deletion and amplification products were detected at similar frequencies, suggesting that amplifications and deletions probably occur by a similar mechanism.U ovom smo radu konstruirali plazmid koji nam je omogućio da usporedimo intraplazmidnu rekombinaciju u bakterijama Agrobacterium tumefaciens i Escherichia coli. Plazmid sadržava dvije istosmjerno ponovljene kopije gena odgovornog za rezistenciju na spektinomicin, pri čemu jednoj kopiji nedostaje 5\u27, a drugoj 3\u27 kraj gena, a međusobno su homologne u duljini od 570 pb. Osim toga, DNA koja se nalazi između ove dvije istosmjerno ponovljene sekvencije sadržava gen koji daje rezistenciju na antibiotik ampicilin. Homolognom rekombinacijom između nepotpunih gena za rezistenciju na spektinomicin nastaje funkcionalni gen, odgovoran za pojavu rezistencije. Pritom može doći do delecije gena za rezistenciju na ampicilin ili njegovog umnožavanja, zajedno s nepotpunim genima za otpornost na spektinomicin. Ovaj eksperimentalni sustav omogućio nam je da po prvi put pratimo pojavu delecija i amplifikacija tijekom intraplazmidne rekombinacije u bakteriji A. tumefaciens. Pokazali smo da delecije i amplifikacije u bakterijama Agrobacterium tumefaciens i Escherichia coli nastaju prvenstveno RecA-neovisnim mehanizmom. Osim toga, ustanovili smo da se delecije i amplifikacije pojavljuju s podjednakom učestalošću, što upućuje na to da je mehanizam oba rekombinacijska događaja sličan

Synergistic potential of Juniperus communis and Helichrysum italicum essential oils against nontuberculous mycobacteria

Objective. The present study evaluated the possible synergistic antimycobacterial interactions of Juniperus communis (J. communis) and Helichrysum italicum (H. italicum) essential oils (EO). Methods. Antimycobacterial potential was tested against Mycobacterium avium and Mycobacterium intracellulare using broth and water dilution method and checkerboard synergy method. Antiadhesion and antibiofilm effect of EOs was evaluated on biotic (HeLa cells) and abiotic surface (polystyrene). To evaluate the possible mechanisms of action, cellular leakage of proteins and DNA was tested and structural changes were visualized with a transmission electron microscope. Results. MIC, MBC and MEC were 1.6 mg/ml for J. communis EO and 3.2 mg/ml for H. italicum EO against both mycobacteria. All combinations of EOs in checkerboard synergy method produced fractional inhibitory concentration index values ranging from 0.501 to 1.5, corresponding to synergistic, additive or indifferent effects. Mycobacterium avium showed a greater tendency to create biofilm but these EO at subinhibitory concentrations (sMIC) effectively blocked the adhesion and the establishment of biofilm. The exposure of both mycobacteria to MICs and sMICs lead to significant morphological changes: acquired a swollen form, ghost-like cell, disorganized cytoplasm detached from the cell wall. OD value of supernatant for both mycobacteria exposed to EOs have confirmed that there is a leakage of cellular material. Conclusion. The leakage of the cellular material is noticeably higher in sMIC, which is probably due to cell wall damage. sMIC of both EOs have an additive or synergistic effect, reducing MICs, limiting adhesion and preventing the formation of biofilms

Antimikobakterijski potencijal eteričnog ulja plodova borovice (Juniperus communis) u pitkoj vodi

Mycobacterium avium complex-related diseases are often associated with poorly maintained hot water systems. This calls for the development of new control strategies. The aim of this study was to investigate the activity of essential oils (EOs) from the Mediterranean plants, common juniper, immortelle, sage, lavandin, laurel, and white cedar against Mycobacterium avium ssp. avium, Mycobacterium intracellulare, and Mycobacterium gordonae in culturing broth and freshwater as their most common habitat. To do that, we developed a new method of water microdilution to determine their minimal effective concentrations (MEC). The most active EO was the one from the common juniper with the MEC of 1.6 mg mL-1. Gas chromatography / mass spectrometry the juniper EO identified monoterpenes (70.54 %) and sesquiterpenes (25.9 %) as dominant component groups. The main monoterpene hydrocarbons were α-pinene, sabinene, and β-pinene. The juniper EO significantly reduced the cell viability of M. intracellulare and M. gordonae at MEC, and of M. avium at 2xMEC. Microscopic analysis confirmed its inhibitory effect by revealing significant morphological changes in the cell membrane and cytoplasm of all three bacteria. The mode of action of the juniper EO on the cell membrane was confirmed by a marked leakage of intracellular material. Juniper EO has a great practical potential as a complementary or alternative water disinfectant in hot water systems such as baths, swimming pools, spa pools, hot tubs, or even foot baths/whirlpools.Ispitali smo aktivnost eteričnih ulja borovice [Juniperus communis (J. communis)], smilja (Helichrysum italicum), kadulje (Salvia officinalis), lavandina (Lavandula hybrida), lovora (Laurus nobilis) i tuje (Thuja occidentalis) prema sojevima Mycobacterium avium ssp. avium (M. avium), Mycobacterium intracellulare (M. intracellulare) i Mycobacterium gordonae (M. gordonae). Za određivanje minimalnih inhibicijskih koncentracija (MIK) i minimalnih baktericidnih koncentracija (MBK) testiranih eteričnih ulja koristili smo se metodom mikrodiluacije u tekućem bujonu (Middlebrook 7H9), a za određivanje minimalne efektivne koncentracije (MEK) umjesto bujona korištena je sterilna voda iz slavine kojoj je dodano 0,05 % Tweena 80. Dvostruka serijska razrjeđenja eteričnih ulja, počevši od 0,1 do 51,2 mg mL-1, u tekućem bujonu ili u sterilnoj vodi iz slavine inokulirana su s mikobakterijskom suspenzijom uz dodatak resazurina. Najučinkovitije eterično ulje prema svim ispitivanim sojevima mikobakterija bilo je eterično ulje J. communis s MBK / MIK / MEK vrijednošću od 1,6 mg mL-1. Metodom plinske kromatografije i masene spektrometrije analizirano je eterično ulje J. communis. Udio monoterpena iznosio je 70,54 %, a seskviterpena 25,9 %. Glavni monoterpenski ugljikovodici bili su α-pinen, sabinen i β-pinen. Eterično ulje J. communis pokazalo je značajan inhibicijski učinak na M. intracellulare i M. gordonae pri MEK i na M. avium pri dvostrukim MEK. Elektronskom mikroskopijom kod svih triju sojeva mikobakterija nakon izlaganja djelovanju eteričnog ulja J. communis otkrivene su značajne morfološke promjene stanične membrane i citoplazme. Učinak eteričnog ulja J. communis na destrukciju stanične membrane mikobakterija potvrđen je značajnim otpuštanjem unutarstaničnog materijala mjerenjem apsorbancije supernatanta pri 260 nm i 280 nm. Zaključno, u našem smo radu razvili novu metodu za ispitivanje antimikrobnog učinka eteričnih ulja ili drugih prirodnih tvari na netuberkulozne mikobakterije koja oponaša uvjete kao u vodenim sustavima. Prirodni proizvodi, osobito eterična ulja, imaju ne samo velik potencijal kao antimikrobni agensi nego i moguću praktičnu primjenu kao alternativni dezinficijensi