Genetic bottlenecks and population passages cause profound fitness differences in RNA viruses.

Repeated clone-to-clone (genetic bottleneck) passages of an RNA phage and vesicular stomatitis virus have been shown previously to result in loss of fitness due to Muller's ratchet. We now demonstrate that Muller's ratchet also operates when genetic bottleneck passages are carried out at 37 rather than 32 degrees C. Thus, these fitness losses do not depend on growth of temperature-sensitive (ts) mutants at lowered temperatures. We also demonstrate that during repeated genetic bottleneck passages, accumulation of deleterious mutations does occur in a stepwise (ratchet-like) manner as originally proposed by Muller. One selected clone which had undergone significant loss of fitness after only 20 genetic bottleneck passages was passaged again in clone-to-clone series. Additional large losses of fitness were observed in five of nine independent bottleneck series; the relative fitnesses of the other four series remained close to the starting fitness. In sharp contrast, when the same selected clone was transferred 20 more times as large populations (10(5) to 10(6) PFU transferred at each passage), significant increases in fitness were observed in all eight passage series. Finally, we selected several clones which had undergone extreme losses of fitness during 20 bottleneck passages. When these low-fitness clones were passaged many times as large virus populations, they always regained very high relative fitness. We conclude that transfer of large populations of RNA viruses regularly selects those genomes within the quasispecies population which have the highest relative fitness, whereas bottleneck transfers have a high probability of leading to loss of fitness by random isolation of genomes carrying debilitating mutations. Both phenomena arise from, and underscore, the extreme mutability and variability of RNA viruses.     

Enhanced Growth and Activity of a Biocontrol Bacterium Genetically Engineered To Utilize Salicylate

Plasmid NAH7 was transferred from Pseudomonas putida PpG7 to P. putida R20 [R20(NAH7)], an antagonist of Pythium ultimum. The plasmid did not affect growth or survival of R20(NAH7) and was stably maintained under nonselective conditions in broth and soil and on sugar beet seeds. Plasmid NAH7 conferred to R20(NAH7) the ability to utilize salicylate in culture, agricultural field soil, and on sugar beet seeds. The metabolic activity of R20(NAH7), but not the wild-type R20, was greatly increased in soil by amendment with salicylate (250 μg/g) as measured by induced respiration. Population densities of R20(NAH7) were also enhanced in salicylate-amended soil, increasing from approximately 1 × 10⁵ CFU/g to approximately 3 × 10⁸ CFU/g after 35 h of incubation. In contrast, population densities of R20(NAH7) in nonamended soil were approximately 3 × 10⁶ CFU/g of soil after 35 h of incubation. The concentration of salicylate in soil affected the rate and extent of population increase by R20(NAH7). At 50 to 250 μg of salicylate per g of soil, population densities of R20(NAH7) increased to approximately 10⁸ CFU/g of soil by 48 h of incubation, with the fastest increase at 100 μg/g. A lag phase of approximately 24 h occurred before the population density increased in the presence of salicylate at 500 μg/g; at 1,000 μg/g, population densities of R20(NAH7) declined over the time period of the experiment. Population densities of R20(NAH7) on sugar beet seeds in soils amended with 100 μg of salicylate per g were not increased while ample carbon was present in the spermosphere. However, after carbon from the seed had been utilized, population densities of R20(NAH7) decreased significantly less (P = 0.005) on sugar beet seeds in soil amended with salicylate than in nonamended soil.     

Low species turnover of upland Amazonian birds in the absence of physical barriers

Aim

One of the oldest and most powerful ways for ecologists to explain distinct biological communities is to invoke underlying environmental differences. But in hyper-diverse systems, which often display high species richness and low species abundance, these sorts of community comparisons are especially challenging. The classic view for Amazonian birds posits that riverine barriers and habitat specialization determine local and regional community composition. We test the tacit, complementary assumption that similar bird communities should therefore permeate uniform habitat between major rivers, regardless of distance.

Location

Upland (terra firme) rainforests of central Amazonia.

Methods

We conducted intensive whole-community surveys of birds in three pairs of 100-ha plots, separated by 40–60 km. We then used dissimilarity indices, cluster analysis, and ordination to characterize differences among the six avian communities.

Results

In all, we detected 244 forest-dependent birds, with an average of 190 species (78%) per plot. Species turnover was negligible, no unique indicator species were found among plot pairs, and all documented species were already known from a complete inventory at one of the three sites.

Main Conclusions

Our study corroborates the classic biogeographical pattern and suggests that turnover contributes little to regional avian diversity within upland forests. Using a grain size of 100 ha, this implies that upland birds perceive the environment as uniform, at least over distances of ~60 km. Therefore, to maximize both local species richness and population persistence, our findings support the conservation of very large tracts of upland rainforest. Our analyses also revealed that the avifauna at Reserva Ducke, encroached by urban sprawl from the city of Manaus, shows the hallmarks of a disturbed community, with fewer vulnerable insectivores. This defaunation signals that even an enormous preserve (10 × 10 km) in lowland Amazonia is not insulated from anthropogenic degradation within the surrounding landscape.     

Environmental influences on local amphibian diversity: the role of floods on river basins

We tested two hypotheses about boundary units and seven about environmental control of species diversity in order to explain geographical trends in the richness of amphibian species in the Mediterranean watershed of the southern coast of the Iberian Peninsula. The number of amphibian species tends to decrease from west to east. The longitudinal trend in the richness of amphibian species actually occurs on passing from one basin to another, but there is not any longitudinal trend within the basins. Multivariate analyses confirmed that the disturbances of episodic river-basin floodings were the principal factor which controls the richness of amphibian species. They explained 94.8% of the observed variations in the richness of amphibian species in this area, according to the intermediate disturbance hypothesis.We propose hydrographic basins as suitable geographical units for further biogeographical analysis and for considering the role of disturbances produced by floods in the environmental control of species diversity.     

The importance of methylthio-IMP for methylmercaptopurine ribonucleoside (Me-MPR) cytotoxicity in Molt F4 human malignant T-lymphoblasts

The importance of methyl-thioIMP (Me-tIMP) formation for methylmercaptopurine ribonucleoside (Me-MPR) cytotoxicity was studied in Molt F4 cells. Cytotoxicity of Me-MPR is caused by Me-tIMP formation with concomitant inhibition of purine de novo synthesis. Inhibition of purine de novo synthesis resulted in decreased purine nucleotide levels and enhanced 5-phosphoribosyl-1-pyrophosphate (PRPP) levels, with concurrent increased pyrimidine nucleotide levels. The Me-tIMP concentration increased proportionally with the concentration of Me-MPR. High Me-tIMP concentration also caused inhibition of PRPP synthesis. Maximal accumulation of PRPP thus occurred at low Me-MPR concentrations. As little as 0.2 μM Me-MPR resulted already after 2 h in maximal inhibition of formation of adenine and guanine nucleotides, caused by inhibition of purine de novo synthesis by Me-tIMP. Under these circumstances increased intracellular PRPP concentrations could be demonstrated, resulting in increased levels of pyrimidine nucleotides. So, in Molt F4 cells, formation of Me-tIMP form Me-MPR results in cytotoxicity by inhibition of purine de novo synthesis.     

Treatment-resistant depression: definition,prevalence, detection,management, and investigational interventions

Treatment-resistant depression (TRD) is common and associated with multiple serious public health implications. A consensus definition of TRD with demonstrated predictive utility in terms of clinical decision-making and health outcomes does not currently exist. Instead, a plethora of definitions have been proposed, which vary significantly in their conceptual framework. The absence of a consensus definition hampers precise estimates of the prevalence of TRD, and also belies efforts to identify risk factors, prevention opportunities, and effective interventions. In addition, it results in heterogeneity in clinical practice decision-making, adversely affecting quality of care. The US Food and Drug Administration (FDA) and the European Medicines Agency (EMA) have adopted the most used definition of TRD (i.e., inadequate response to a minimum of two antidepressants despite adequacy of the treatment trial and adherence to treatment). It is currently estimated that at least 30% of persons with depression meet this definition. A significant percentage of persons with TRD are actually pseudo-resistant (e.g., due to inadequacy of treatment trials or non-adherence to treatment). Although multiple sociodemographic, clinical, treatment and contextual factors are known to negatively moderate response in persons with depression, very few factors are regarded as predictive of non-response across multiple modalities of treatment. Intravenous ketamine and intranasal esketamine (co-administered with an antidepressant) are established as efficacious in the management of TRD. Some second-generation antipsychotics (e.g., aripiprazole, brexpiprazole, cariprazine, quetiapine XR) are proven effective as adjunctive treatments to antidepressants in partial responders, but only the olanzapine-fluoxetine combination has been studied in FDA-defined TRD. Repetitive transcranial magnetic stimulation (TMS) is established as effective and FDA-approved for individuals with TRD, with accelerated theta-burst TMS also recently showing efficacy. Electroconvulsive therapy is regarded as an effective acute and maintenance intervention in TRD, with preliminary evidence suggesting non-inferiority to acute intravenous ketamine. Evidence for extending antidepressant trial, medication switching and combining antidepressants is mixed. Manual-based psychotherapies are not established as efficacious on their own in TRD, but offer significant symptomatic relief when added to conventional antidepressants. Digital therapeutics are under study and represent a potential future clinical vista in this population.     

Synthesis and biological activity of tachykinin analogs containing the adamantane moiety

Summary The adamantane moiety was introduced in the tachykinin NK₂ receptor-selective agonist [-Ala⁸]-NKA(4–10) (H-Asp-Ser-Phe-Val--Ala-Leu-Met-NH₂, MEN 10210) and in different positions of the NK₂ receptor antagonist MEN 10376 (H-Asp-Tyr-d-Trp-Val-d-Trp-d-Trp-Lys-NH₂) in order to investigate how this substitution affects their biological activity at tachykinin NK₁, NK₂ and NK₃ receptors. 1-Adamantaneacetic acid (1-Ada-CH₂COOH) was directly conjugated in the solid phase as the preformed OBt active ester to the N-terminal position of MEN 10210, obtaining MEN 10586 (1-Ada-CH₂CO-Asp-Ser-Phe-Val--Ala-Leu-Met-NH₂). The Pfp ester of adamantaneacetic acid (1) was prepared and used for the acylation of the N-terminal position of MEN 10376, yielding MEN 10606 (1-Ada-CH₂CO-Asp-Tyr-d-Trp-Val-d-Trp-d-Trp-Lys-NH₂). Compound 1 was then used to obtain the building block Fmoc-Lys(1-Ada-CH₂CO)-OH as a modified amino acid for the synthesis of MEN 10818 [H-Asp-Tyr-d-Trp-Val-d-Trp-d-Trp-Lys(1-Ada-CH₂CO)-NH₂]. In order to investigate the biological activity of the peptide bearing the adamantane group together with the free N-terminal amino function, we synthesised MEN 10676 [H-Asp(O-2-Ada)-Tyr-d-Trp-Val-d-Trp-d-Trp-Lys-NH₂] using Fmoc-Asp(O-2-Ada)-OH, in which 2-adamantanole was the protecting group of the aspartate -COOH moiety during the peptide synthesis and survived the final peptide cleavage and deprotection carried out under controlled conditions. MEN 10586 showed an agonist activity comparable to that of the parent compound MEN 10210 at NK₁ and NK₂ receptors of guinea pig ileum, rabbit isolated pulmonary artery and hamster isolated trachea preparations, while it showed a 25-fold higher agonist activity at NK₃ receptors of rat isolated portal vein. The three modified antagonist analogs displayed similar or reduced affinity at NK₁, NK₂ and NK₃ receptors as compared to MEN 10376. The drop was particularly evident (>2 log units) at the NK₂ receptors of the rabbit isolated pulmonay artery.