Nonlinearities in plant RNA virus fitness

Una de las mayores amenazas tanto para la salud humana y animal, como para la agronomía es la emergencia de nuevas enfermedades infecciosas, la mayoría de las cuales están causadas por los virus de RNA. La emergencia viral es un problema complejo que consista en la adquisición de la variación genética, por mutación o recombinación, dentro de la población viral en el huésped reservorio la cual podría facilitar la capacidad de infectar de manera eficiente nuevos huéspedes. Los virus de RNA presentan a una evolucionabilidad extraordinaria por sus grandes tamaños poblacionales, cortos tiempos de generación y altas tasas de mutación y recombinación. Comprender los mecanismos evolutivos que podrían dar lugar a un virus emergente es imprescindible para hacer un diseño racional de las estrategias del control y las terapias antivirales. ¿Podríamos predecir la emergencia de un virus y su salto a un huésped nuevo sabiendo el fenotipo del virus, es decir, su eficacia biológica, en su huésped natural? Si es así, la arquitectura de la eficacia biológica del virus estaría determinada solamente por su genotipo (la componente G, o en otras palabras, la mutación) y el ambiente (la componente E o, en otras palabras, el huésped). Sin embargo, podrían existir interacciones entre estos componentes y comprometer la predictibilidad del fenotipo del virus en un huésped alternativo. Saber cómo las mutaciones afectan la eficacia biológica de las poblaciones virales es esencial para llegar a entender la emergencia viral y la posterior adaptación del nuevo virus a su nuevo hospedador. La observación común de que la mayoría de las mutaciones son deletéreas coincide con la predicción teorética de que un organismo ya está adaptado a su ambiente en particular, por lo cual, cualquier cambio genético supondría apartarse del fenotipo optimo. En realidad, este punto de vista es demasiado simplificado; los efectos mutacionales sobre la eficacia biológica constituyen un continuo y están condicionados tanto por el ambiente como por el fondo genético. Estos efectos se nombran comúnmente como interacción genotipo-ambiente (G×E), interacción genética (G×G o epistasia) e interacción G×G×E (epistasia-huésped). En esta Tesis, la contribución de estos componentes a la arquitectura de la eficacia biológica de un virus de RNA de la cadena positiva que infecta plantas, TEV. Los virus de RNA forman el grupo más grande de los virus de las plantas y causan muchas enfermedades económicamente importantes. En primer lugar, para inferir G×E, hemos caracterizado la distribución de los efectos mutacionales sobre la eficacia biológica (DMFE) de TEV utilizando una colección de veinte mutantes simples que tenían una única sustitución nucleotídica. Los efectos mutacionales se midieron en un conjunto de ambientes representados por ocho huéspedes distintos. Cinco de estos huéspedes pertenecían a la familia Solanaceae y se infectaban con TEV de la forma natural. Otros tres huéspedes pertenecían a otras tres familias de las plantas y eran parcialmente susceptibles a la infección con TEV. Primero, hemos encontrado una interacción significativa entre el genotipo del virus y la especie del huésped, generada por diferencias en la varianza genética de la eficacia biológica y los efectos pleiotrópicos de las mutaciones entre huéspedes. Segundo, las DMFE eran profundamente distintos entre los huéspedes que pertenecían a Solanaceae y los que no. La exposición de los genotipos de TEV a las no-solanáceas resultó en una gran reducción en la eficacia biológica, mientras que la varianza permanecía constante y la asimetría de la distribución era positiva. Una asimetría positiva implica que hay más valores mayores que la media, es decir, la cola derecha de la distribución contiene una proporción significativa de mutaciones beneficiosas. Entre solanáceas, la cola izquierda de la distribución tenía mayor peso, indicando un exceso de las mutaciones deletéreas. Conjuntamente, este resultado muestra que TEV puede fácilmente extender su rango de huéspedes y mejorar su eficacia biológica en los nuevos huéspedes, y que conocer la eficacia biológica de un mutante en un huésped no nos permite extrapolar que su eficacia se mantenga en otro(s) huésped(es). En segundo lugar, para inferir la epistasia, hemos generado 53 genotipos de TEV los cuales llevaban pares de únicas sustituciones nucleotídicas y hemos medido sus efectos deletéreos sobre la eficacia biológica por separado y en combinación en el huésped primario: Nicotiana tabacum. Hemos encontrado que el 38% de los pares mostraban epistasia significativa sobre la eficacia biológica. Las desviaciones de la hipótesis nula de efectos multiplicativos eran tanto positivas como negativas, aunque predominaban los casos de epistasia de signo reciproco, lo cual indica de que el paisaje adaptativo de TEV debe de ser muy rugoso. Por último, hemos observado que la epistasia de magnitud correlaciona negativamente con el efecto promedio de las mutaciones, lo cual revela baja robustez genética de los genomas compactos de los virus de RNA. Estas observaciones son malas noticias con respeto a la predictibilidad de cuales genotipos virales podrían ser más propensos a emerger. En tercer lugar, con el objetivo de caracterizar el grado de variación de los efectos epistaticos con los huéspedes en los que estos se miden, hemos cuantificado la eficacia de diez genotipos de TEV que llevaban pares de únicas sustituciones nucleotídicas a través de cuatro huéspedes que variaban en su proximidad taxonómica con el huésped primario. Hemos observado que la epistasia entre pares de mutaciones era dependiente del huésped y, en promedio, positiva en el huésped natural, haciéndose más débil a medida que el parentesco genético de estos huéspedes disminuía respecto al primario. La existencia de la epistasia y su variación entre huéspedes hace que los efectos de las mutaciones individuales sean aun más imprevisibles. Por último, hemos caracterizado empíricamente el paisaje de la eficacia del TEV adaptado a A. thaliana reconstruyendo la mayor parte de los genotipos intermediarios posibles durante el proceso de adaptación y hemos medido dos componentes de la eficacia biológica en el nuevo huésped. En nuestro conjunto de datos predominaba la epistasia de magnitud entre las mutaciones beneficiosas, especialmente en los primeros pasos de la adaptación. La epistasia era pequeña en los genotipos más adaptados. Las interacciones epistaticas consistían en desviaciones de los valores esperados que eran ambos positivos y negativos. La topografía del paisaje era predominantemente neutral y consistía de un único pico. La suavidad del paisaje de TEV en A. thaliana sugiere que el virus se haya adaptado fácilmente a su nuevo huésped. Este resultado es importante desde la perspectiva de las enfermedades virales emergentes y en concordancia con la observación común de que la mayoría de los virus emergentes son efectivamente los virus de RNA.One of the major threats to human and animal health as well as to agronomy is the emergence of new infectious diseases, most of which are caused by RNA viruses. It is a complex, multilevel problem that consists in acquisition of genetic variation by mutation or recombination within a virus population in the reservoir host that would enable the host-switch. RNA viruses show a remarkable evolvability owed to their large population sizes, short generation times and high mutation and recombination rates. Understanding the underlying evolutionary mechanisms by which a virus may become an emergent one is pivotal for the rational design of control strategies and antiviral therapies. Thus, can virus emergence and jump to a new host species be predicted by knowing its phenotype, i.e., fitness, in its natural host? If so, then the architecture of virus fitness would be determined only by its genotype (the G component, i.e., mutation) and environment (the E component, i.e., host). Still, interactions between these components may exist and compromise the predictability of virus phenotype in an alternative host. How mutations affect the fitness of viral populations is essential to understanding viral emergence and adaptation to a new host. The widespread observation that the majority of mutations are deleterious coincides with the theoretical prediction that an organism will be well adapted to its particular environment, so that any genomic change would represent a move-away from the optimal phenotype. In reality, this view is overly simplified; mutational fitness effects constitute a continuum and are conditional upon the environment and genetic background; effects commonly referred to as genotype-by-environment (G×E) genetic (G×G or epistasis) and G×G×E (epistasis-by-host) interactions. In this Thesis the contribution of these components to the architecture of viral fitness was addressed using a plant positive sense RNA virus, TEV. RNA viruses form the largest group of plant viruses and cause many economically important diseases. First, to infer G×E, we characterized the distribution of mutational fitness effects (DMFE) for a collection of twenty single-nucleotide substitution mutants of TEV across a set of eight environments represented by different hosts. Five of these host species were naturally infected by TEV, all belonging to family Solanaceae, whereas other three were partially susceptible hosts belonging to three other plant families. First, we found a significant virus genotype-by-host species interaction, which was sustained by differences in genetic variance for fitness and the pleiotropic effect of mutations among hosts. Second, we found that the DMFEs were markedly different between Solanaceae and non-Solanaceae hosts. Exposure of TEV genotypes to non-Solanaceae hosts led to a large reduction of mean viral fitness, while the variance remained constant and skewness increased towards the right tail, thus containing a significant proportion of beneficial mutations. Within Solanaceae the tail of the distribution was drawn out more to the left side, thus comprising an excess of deleterious mutations. All together, this result suggests that TEV may easily broaden its host range and improve fitness in new hosts, and that knowledge about the DMFE in the natural host does not allow for making predictions about its properties in an alternative host. Secondly, to infer epistasis, we generated 53 TEV genotypes carrying pairs of single nucleotide substitutions and measured their separated and combined deleterious fitness effects in its primary host: Nicotiana tabacum. We found up to 38% of pairs had significant epistasis for fitness, including both positive and negative deviations from the null hypothesis of multiplicative effects. Moreover, we found the predominance of cases of reciprocal sign epistasis, indicating that adaptive landscapes for RNA viruses maybe highly rugged. Finally, we found that the magnitude of epistasis correlated negatively with the average effect of mutations. Negative correlation observed between epistasis and deleterious fitness effects indicates low genetic robustness of the compact RNA virus genomes. These observations are bad news regarding predictability of which viral genotypes may be more prone to emerge. Thirdly, in order to characterize the degree to which epistatic effects vary across hosts, we quantified the fitness of ten TEV genotypes carrying pairs of single nucleotide substitutions across four hosts that differ from the primary host in taxonomic proximity. We found that epistasis among a particular pair of mutations is host-dependent and positive, on average, in natural host and weaker in more distant ones. The existence of epistasis and its variation across hosts makes the effect of individual mutations unpredictable. Finally, we empirically characterized the fitness landscape of TEV adapted to A. thaliana by reconstructing the major part of intermediate and final genotypes of adaptation and measuring their fitness components in the new host. We found prevalent magnitude epistatic effects among the beneficial mutations, especially in the first steps of adaptation. Epistasis is small in the more adapted genotypes. Epistatic interactions included both positive and negative deviations from the expected values. The landscape topography is predominately neutral and single-peaked. The smoothness of TEV landscape in A. thaliana suggests that virus easily adapted to its new host. This is an important result from the perspective of emerging viral diseases and in concordance to common observation that majority of emerging viruses are indeed RNA viruses

The Evolution of Viruses in Multi-Host Fitness Landscapes

Provided that generalist viruses will have access to potentially unlimited hosts, the question is why most viruses specialize in few hosts. It has been suggested that selection should favor specialists because there are tradeoffs limiting the fitness of generalists in any of the alternative hosts or because evolution proceeds faster with narrower niches. Here we review experiments showing that virus adaptation to a specific host is often coupled with fitness losses in alternative ones. In most instances, mutations beneficial in one host are detrimental in another. This antagonistic pleiotropy should limit the range of adaptation and promote the evolution of specialization. However, when hosts fluctuate in time or space, selective pressures are different and generalist viruses may evolve as well

Comparison of Zn Content in Rapid-acting Insulin and Biphasic Suspension by FAAS

Various insulin analogs (rapid-acting and intermediate-acting insulin) have been investigated for the determination of zinc content by flame atomic absorption spectrometry. This paper presents the validation of a method and comparison of zinc content in the insulin samples studied. The method was linear (r)(^2) = 0.9997), the limit of detection was 0.0098 mg L(^{–1}) and the limit of quantification was 0.0296 mg L(^{–1}), the precision (as relative standard deviation) was up to 7.4 %, and the accuracy was within a range of 95.6 % to 100.1 % for the recovery of fortified insulin samples. The zinc content in the insulin samples ranged from 14.9 mg L(^{–1}) to 16.3 mg L(^{–1}) for rapid-acting insulin and 18.7 mg L(^{–1}) to 19.9 mg L(^{–1}) for intermediate-acting insulin. A higher zinc content was found in the intermediate-acting insulins than in the rapid-acting insulin analogs ((p) < 0.05). The obtained results should be considered in the establishment of new or improvement of currently available procedures used to assure the quality, safety and efficacy of insulin products. This work is licensed under a Creative Commons Attribution 4.0 International License

Epistasis between mutations is host-dependent for an RNA virus

How, and to what extent, does the environment influence the way mutations interact? Do environmental changes affect both the sign and the magnitude of epistasis? Are there any correlations between environments in the variability, sign or magnitude of epistasis? Very few studies have tackled these questions. Here, we addressed them in the context of viral emergence. Most emerging viruses are RNA viruses with small genomes, overlapping reading frames and multifunctional proteins for which epistasis is abundant. Understanding the effect of host species in the sign and magnitude of epistasis will provide insights into the evolutionary ecology of infectious diseases and the predictability of viral emergence.Lalic ., J.; Elena Fito, SF. (2012). Epistasis between mutations is host-dependent for an RNA virus. Biology Letters. 9(1):20120396-20120396. doi:10.1098/rsbl.2012.0396S20120396201203969

Continental-Mediterranean and rural-urban differences in cardiovascular risk factors in Croatian population

AIM: To compare the distribution of cardiovascular disease (CVD) factors between continental and Mediterranean areas and urban and rural areas of Croatia, as well as to investigate the differences in achieving treatment goals by the general practitioners (GP) in different settings. ----- METHODS: A multicenter prospective study was performed on 2467 participants of both sexes ≥40 years old, who visited for any reason 59 general practices covering the whole area of Croatia (May-July 2008). The study was a part of the Cardiovascular Risk and Intervention Study in Croatia-family medicine (CRISIC-fm) study. Patients were interviewed using a 140-item questionnaire on socio-demographics and CVD risk factors. We measured body mass index (BMI) and waist circumference and determined biochemical variables including blood pressure, total, high-density lipoprotein-, and low-density lipoprotein-cholesterol, triglycerides, glycemia, and uric acid. ----- RESULTS: Participants from continental rural areas had significantly higher systolic and diastolic blood pressure (P<0.001), obesity (P=0.001), increased waist circumference (P<0.001), and more intense physical activity (P=0.020). Participants from coastal rural areas had higher HDL-cholesterol, participants from continental rural and coastal urban areas had higher LDL-cholesterol, and participants from rural continental had significantly higher BMI and waist circumference. ----- CONCLUSION: Prevalence of CVD risk factors in Croatian population is high. Greater burden of risk factors in continental region and rural areas may be partly explained by lifestyle differences

Disruption of macrodomain protein SCO6735 increases antibiotic production in Streptomyces coelicolor

ADP-ribosylation is a post-translational modification that can alter the physical and chemical properties of target proteins and controls many important cellular processes. Macrodomains are evolutionarily conserved structural domains that bind ADP-ribose derivatives and are found in proteins with diverse cellular functions. Some proteins from the macrodomain family can hydrolyze ADP- ribosylated substrates and therefore reverse this post-translational modification. Bacteria and Streptomyces, in particular, are known to utilize protein ADP-ribosylation, yet very little is known about their enzymes that synthesise and remove this modification. We have determined the crystal structure and characterized, both biochemically and functionally, the macrodomain protein SCO6735 from Streptomyces coelicolor. This protein is a member of an uncharacterised subfamily of macrodomain proteins. Its crystal structure revealed a highly conserved macrodomain fold. We showed that SCO6735 possesses the ability to hydrolyse PARP-dependent protein ADP- ribosylation. Furthermore, we showed that expression of this protein is induced upon DNA damage and that deletion of this protein in S. coelicolor increases antibiotic production. Our results provide the first insights into the molecular basis of its action and impact on Streptomyces metabolism

Effect of Host Species on the Distribution of Mutational Fitness Effects for an RNA Virus

Knowledge about the distribution of mutational fitness effects (DMFE) is essential for many evolutionary models. In recent years, the properties of the DMFE have been carefully described for some microorganisms. In most cases, however, this information has been obtained only for a single environment, and very few studies have explored the effect that environmental variation may have on the DMFE. Environmental effects are particularly relevant for the evolution of multi-host parasites and thus for the emergence of new pathogens. Here we characterize the DMFE for a collection of twenty single-nucleotide substitution mutants of Tobacco etch potyvirus (TEV) across a set of eight host environments. Five of these host species were naturally infected by TEV, all belonging to family Solanaceae, whereas the other three were partially susceptible hosts belonging to three other plant families. First, we found a significant virus genotype-by-host species interaction, which was sustained by differences in genetic variance for fitness and the pleiotropic effect of mutations among hosts. Second, we found that the DMFEs were markedly different between Solanaceae and non-Solanaceae hosts. Exposure of TEV genotypes to non-Solanaceae hosts led to a large reduction of mean viral fitness, while the variance remained constant and skewness increased towards the right tail. Within the Solanaceae hosts, the distribution contained an excess of deleterious mutations, whereas for the non-Solanaceae the fraction of beneficial mutations was significantly larger. All together, this result suggests that TEV may easily broaden its host range and improve fitness in new hosts, and that knowledge about the DMFE in the natural host does not allow for making predictions about its properties in an alternative host

Cystic Fibrosis – results of CFTR modulators in Croatia

Cistična fibroza najčešća je nasljedna bolest, koja skraćuje životni vijek, a uzrokuje je defekt u genu za transmembranski regulator provodljivosti cistične fibroze (eng. cystic fibrosis transmembrane regulator – CFTR). Poremećena je homeostaza elektrolita, što se očituje simptomima u više organskih sustava. Plućne manifestacije, s kroničnim infekcijama, upalom i, na kraju, respiratornim zatajenjem, ostaju i dalje najvažnija prijetnja životnom vijeku bolesnika. Do prije jednog desetljeća bilo je dostupno samo simptomatsko liječenje. Od 2012. g. dostupno je liječenje tzv. modulatorima CFTR-proteina i njihovim kombinacijama za osobe s cističnom fibrozom koje nose različite varijante CFTR-gena. Pojavom tih lijekova uvelike se promijenila perspektiva i kvaliteta života ljudi s cističnom fibrozom, ali postavljeni i novi izazovi u vezi s dugoročnim komplikacijama, pitanje eventualnog smanjenja konvencionalnog liječenja, ali i financiranja terapije, koja je mnogim bolesnicima nedostupna. Iznesene su bazične spoznaje o cističnoj fibrozi i funkciji CFTR-proteina, klasifikaciji varijanata CFTR-gena, mogućnostima liječenja CFTR-modulatorima te osnovni ishodi liječenja bolesnika s cističnom fibrozom u Hrvatskoj, gdje se ta terapija primjenjuje od jeseni 2021. godine.Cystic fibrosis, the most frequent lifespan shortening hereditary disease in Caucasians, is caused by a defect in the CFTR (cystic fibrosis transmembrane regulator) gene. Disturbed electrolyte homeostasis leads to the development of different symptoms in multiple organs. Pulmonary manifestations with chronic infections and inflammation result in respiratory failure and remain the most important life-shortening factor. Until recently only symptomatic treatment was available. In year 2012. a new treatment approach with small molecules that modulate the CFTR protein was introduced. Different combinations of CFTR modulators are applicable to certain patients carrying different variants of the CFTR gene. CFTR modulators made a huge difference in the quality of life and perspectives of people with cystic fibrosis. At the same time, new challenges emerged regarding long term complications and possible reduction of conventional treatment options, as well as financial issues that are an obstacle to the use of these drugs for many patients. This paper brings basic insight into cystic fibrosis, the function of CFTR protein, the classification of CFTR gene variants and possibilities of treatment with CFTR modulators as well as basic outcomes of CFTR modulators treatment in Croatia, where this therapy was introduced in autumn 2021

Characteristics of teamwork, self-efficacy and work engagement among preschool teachers

Namen magistrskega dela je bil ugotoviti, kako se med seboj povezujejo značilnosti timskega dela tandema vzgojitelj – pomočnik vzgojitelja, njuna samoučinkovitost in delovna zavzetost. V raziskavi je sodelovalo 127 tandemov vzgojiteljev in pomočnikov vzgojiteljev, zaposlenih v vrtcih. Povprečna starost udeležencev je bila 39,9 leta (SD = 9,9), povprečna delovna doba v vrtcih pa 15,3 leta (SD = 10,6). Udeleženci so v parih izpolnili vprašalnik ETS (Klassen, Yerdelen in Durksen, 2013), NTSES (Skaalvik in Skaalvik, 2007), Lestvico zaznane kolektivne učinkovitosti učiteljev (Skaalvik in Skaalvik, 2007), Vprašalnik spretnosti timskega dela (Polak, 2012) in Vprašalnik zaupanja (Polak, 2003) ter poročali o demografskih spremenljivkah. Rezultati so pokazali, da se spretnosti timskega dela vzgojiteljev (ne pa tudi pomočnikov vzgojiteljev) pozitivno povezujejo s starostjo udeležencev. Pri spretnostih timskega dela, samoučinkovitosti in delovni zavzetosti ni razlik med vzgojitelji in njihovimi pomočniki. Pri spretnostih timskega dela in samoučinkovitosti ni razlik med tandemi, ki delajo z otroki prve oz. druge starostne skupine, majhna razlika v prid strokovnim delavcem, ki delajo z otroki prve starostne skupine, pa je pri dimenziji socialne zavzetosti do učencev. Rezultati kažejo na pozitivno povezanost samoučinkovitosti in zaznane kolektivne učinkovitosti ter samoučinkovitosti in delovne zavzetosti sodelavcev znotraj tandema, kar kaže na to, da imajo odnosi med sodelavci potencial za socialno okužbo in da so sodelavci drug drugemu delovni vir. Poleg tega so rezultati pokazali, da so samoučinkovitost in spretnosti timskega dela visoko pozitivno povezane z delovno zavzetostjo, razlike v spretnostih timskega dela med sodelavcema v tandemu pa so negativno povezane z njuno delovno zavzetostjo. Pozitivna povezanost obstaja tudi med spretnostmi timskega dela in samoučinkovitostjo. Ugotovitve so skladne s predpostavkami modela JD-R, ki poudarja pomen delovnih virov kot najpomembnejšega napovednika delovne zavzetosti, in so izhodišče za oblikovanje intervencij, usmerjenih na zviševanje delovne zavzetosti, in tako tudi za povečanje kakovosti pedagoškega dela ter opozarjajo na pomen intervencij, usmerjenih na celotni kolektiv.The aim of the Master’s Thesis was to find the relations between self-efficacy and work engagement of a kindergarten teacher and a teaching assistant, who work together in tandem, with the characteristics of their teamwork. The research was conducted on 127 such tandems of kindergarten employees, whose average age was 39,9 years (SD = 9,9), while the average length of their employment was 15,3 years (SD = 10,6). In the survey, the respondents were asked to answer several questionnaires, i.e. ETS (Klassen, Yerdelen in Durksen, 2013), NTSES (Skaalvik in Skaalvik, 2007), Perceived Collective Teacher Efficacy Scale (Skaalvik in Skaalvik, 2007), Teamwork Skills Questionnaire (Polak, 2012) and The Trust Questionnaire (Polak, 2003), while also reporting their demographic variables. The results indicate that the teacher’s teamwork skills (but not necessarily that of teaching assistants) are positively related to the age of the respondent. With respect to the position of the employee, there is no apparent difference shown in teamwork skills, self-efficacy or work engagement. There is also no difference in teamwork skills and self-efficacy according to the age of the children in the group, whereas there is, however, a small distinction in the aspect of social engagement towards the pupils, in favour of the staff who work with children of the first year group. The results show a positive correlation between self-efficacy and the perceived collective efficacy, as well as between self-efficacy and the work engagement of staff within the tandem. This suggests that relations between colleagues have the potential for social contagion, i.e. co-workers can serve as job resources for one another. Furthermore, the results indicate that self-efficacy and teamwork skills are highly positively associated with work engagement, whereas the differences in teamwork skills among colleagues are negatively related to their work engagement. There is also a positive connection between teamwork skills and self-efficacy. The conclusions are consistent with the assumptions of the JD-R model, which accentuates the importance of job resources as the vital prediction of successful work engagement and represents the basis for creating interventions, aimed at increasing work engagement and consequently increasing the quality of pedagogical work, and also indicates the importance of interventions directed at the entire staff in the kindergarten