Discovery of varlaxins, new aeruginosin-type inhibitors of human trypsins.

Low-molecular weight natural products display vast structural diversity and have played a key role in the development of novel therapeutics. Here we report the discovery of novel members of the aeruginosin family of natural products, which we named varlaxins. The chemical structures of varlaxins 1046A and 1022A were determined using a combination of mass spectrometry, analysis of one- and two-dimensional NMR spectra, and HPLC analysis of Marfey's derivatives. These analyses revealed that varlaxins 1046A and 1022A are composed of the following moieties: 2-O-methylglyceric acid 3-O-sulfate, isoleucine, 2-carboxy-6-hydroxyoctahydroindole (Choi), and a terminal arginine derivative. Varlaxins 1046A and 1022A differ in the cyclization of this arginine moiety. Interestingly, an unusual α-D-glucopyranose moiety derivatized with two 4-hydroxyphenylacetic acid residues was bound to Choi, a structure not previously reported for other members of the aeruginosin family. We sequenced the complete genome of Nostoc sp. UHCC 0870 and identified the putative 36 kb varlaxin biosynthetic gene cluster. Bioinformatics analysis confirmed that varlaxins belong to the aeruginosin family of natural products. Varlaxins 1046A and 1022A strongly inhibited the three human trypsin isoenzymes with IC50 of 0.62-3.6 nM and 97-230 nM, respectively, including a prometastatic trypsin-3, which is a therapeutically relevant target in several types of cancer. These results substantially broaden the genetic and chemical diversity of the aeruginosin family and provide evidence that the aeruginosin family is a source of strong inhibitors of human serine proteases.

Non-COVID-19 excess deaths by age and gender in the United States during the first three months of the COVID-19 pandemic.

OBJECTIVES: The first three months of the COVID-19 pandemic has disrupted healthcare systems, creating an environment by which deaths have occurred that are not directly due to COVID-19, but have occurred owing to the healthcare and societal environment resulting from COVID-19. The objective of this research is to quantify such excess deaths, partitioned by age group and gender. STUDY DESIGN: This is a data analysis. METHODS: Excess deaths by age and gender are estimated using provisional death data available from the Centers for disease control and prevention (CDC) over the time period from March 1, 2020 through May 30, 2020. Previous year fatality and population data are used as the benchmark. RESULTS: Several of the eighteen age and gender cohorts experienced statistically significant excess deaths. The results also indicate that COVID-19 has been protective for one of the age and gender cohorts. CONCLUSIONS: There have been more excess deaths in several age group and gender cohorts during the first three months of the pandemic, beyond direct deaths directly attributable to COVID-19. These non-COVID-19 excess deaths are most apparent in the 25- to 44-year age group for women and 15- to 54-year age group for men. Further research is needed to assess the cause of such excess deaths and introduce safeguards to reduce such deaths in the future.

A new species of Atriophallophorus Deblock & Rosé, 1964 (Trematoda: Microphallidae) described from in vitro-grown adults and metacercariae from Potamopyrgus antipodarum (Gray, 1843) (Mollusca: Tateidae).

The adult and metacercaria life stages of a new species of the microphallid genus Atriophallophorus Deblock & Rosé, 1964 are described from specimens collected at Lake Alexandrina (South Island, New Zealand). In addition to molecular analyses of ribosomal and mitochondrial genes, metacercariae of Atriophallophorus winterbourni n. sp. from the snail host Potamopyrgus antipodarum (Gray) were grown in vitro to characterize internal and external morphology of adults using light and scanning electron microscopy and histological techniques. Atriophallophorus winterbourni n. sp. is readily distinguishable from Atriophallophorus coxiellae Smith, 1973 by having a different structure of the prostatic chamber, sub-circular and dorsal to genital atrium, rather than cylindrical, fibrous, elongate and placed between the seminal vesicle and the genital atrium. The new species is most similar to Atriophallophorus minutus (Price, 1934) with regards to the prostatic chamber and the morphometric data, but possesses elongate-oval testes and subtriangular ovary rather than oval and transversely oval in A. minutus. Phylogenetic analyses including sequence data for A. winterbourni n. sp. suggested a congeneric relationship of the new species to a hitherto undescribed metacercariae reported from Australia, both forming a strongly supported clade closely related to Microphallus and Levinseniella. In addition, we provide an amended diagnosis of Atriophallophorus to accommodate the new species and confirm the sinistral interruption of the outer rim of the ventral sucker caused by the protrusion of the dextral parietal atrial scale at the base of the phallus.

Comparing direct and indirect selfing rate estimates: when are population-structure estimates reliable?

The rate of self-fertilization (that is, selfing) is a key evolutionary parameter in hermaphroditic species, yet obtaining accurate estimates of selfing rates in natural populations can be technically challenging. Most published estimates are derived from population-level heterozygote deficiency (that is, FIS) or identity disequilibria (for example, the software RMES (robust multilocus estimate of selfing)). These indirect methods can be applied to population genetic survey data, whereas direct methods using progeny arrays require much larger data sets that are often difficult to collect in natural populations or even require captive breeding. Unfortunately, indirect methods rely on assumptions that can be problematic, such as negating biparental inbreeding, inbreeding disequilibrium and (for FIS) the presence of null alleles. The performance of indirect estimates against progeny-array estimates is still largely unknown. Here we used both direct progeny-array and indirect population-level methods to estimate the selfing rate in a single natural population of the simultaneously hermaphroditic freshwater snail Radix balthica throughout its reproductive lifespan using 10 highly polymorphic microsatellites. We found that even though progeny arrays (n=1034 field-collected embryos from 60 families) did not reveal a single selfed embryo, FIS-based selfing rates (n=316 adults) were significantly positive in all 6 sequential population samples. Including a locus with a high frequency of null alleles further biased FIS-based estimates. Conversely, RMES-based estimates were very similar to progeny-array estimates and proved insensitive to null alleles. The assumptions made by RMES were thus either met or irrelevant in this particular population, making RMES a valid, cost-efficient alternative to progeny arrays.

Faster clonal turnover in high-infection habitats provides evidence for parasite-mediated selection.

According to the Red Queen hypothesis for sex, parasite-mediated selection against common clones counterbalances the reproductive advantage of asexual lineages, which would otherwise outcompete sexual conspecifics. Such selection on the clonal population is expected to lead to a faster clonal turnover in habitats where selection by parasites is stronger. We tested this prediction by comparing the genetic structure of clonal and sexual populations of freshwater snail Potamopyrgus antipodarum between years 2003 and 2007 in three depth-specific habitats in Lake Alexandrina (South Island, New Zealand). These habitats differ in the risk of infection by castrating trematodes and in the relative proportion of sexual individuals. As predicted, we found that the clonal structure changed significantly in shallow and mid-water habitats, where prevalence of infection was high, but not in the deep habitat, where parasite prevalence was low. Additionally, we found that both clonal diversity and evenness of the asexual population declined in the shallow habitat. In contrast, the genetic structure (based on F-statistics) of the coexisting sexual population did not change, which suggests that the change in the clonal structure cannot be related to genetic changes in the sexual population. Finally, the frequency of sexuals had no effect on the diversity of the sympatric clonal population. Taken together, our results show a more rapid clonal turnover in high-infection habitats, which gives support for the Red Queen hypothesis for sex.

Clonal diversity driven by parasitism in a freshwater snail.

One explanation for the widespread abundance of sexual reproduction is the advantage that genetically diverse sexual lineages have under strong pressure from virulent coevolving parasites. Such parasites are believed to track common asexual host genotypes, resulting in negative frequency-dependent selection that counterbalances the population growth-rate advantage of asexuals in comparison with sexuals. In the face of genetically diverse asexual lineages, this advantage of sexual reproduction might be eroded, and instead sexual populations would be replaced by diverse assemblages of clonal lineages. We investigated whether parasite-mediated selection promotes clonal diversity in 22 natural populations of the freshwater snail Melanoides tuberculata. We found that infection prevalence explains the observed variation in the clonal diversity of M. tuberculata populations, whereas no such relationship was found between infection prevalence and male frequency. Clonal diversity and male frequency were independent of snail population density. Incorporating ecological factors such as presence/absence of fish, habitat geography and habitat type did not improve the predictive power of regression models. Approximately 11% of the clonal snail genotypes were shared among 2-4 populations, creating a web of 17 interconnected populations. Taken together, our study suggests that parasite-mediated selection coupled with host dispersal ecology promotes clonal diversity. This, in return, may erode the advantage of sexual reproduction in M. tuberculata populations.

Distinct roles of carbohydrate esterase family CE16 acetyl esterases and polymer-acting acetyl xylan esterases in xylan deacetylation.

Mass spectrometric analysis was used to compare the roles of two acetyl esterases (AE, carbohydrate esterase family CE16) and three acetyl xylan esterases (AXE, families CE1 and CE5) in deacetylation of natural substrates, neutral (linear) and 4-O-methyl glucuronic acid (MeGlcA) substituted xylooligosaccharides (XOS). AEs were similarly restricted in their action and apparently removed in most cases only one acetyl group from the non-reducing end of XOS, acting as exo-deacetylases. In contrast, AXEs completely deacetylated longer neutral XOS but had difficulties with the shorter ones. Complete deacetylation of neutral XOS was obtained after the combined action of AEs and AXEs. MeGlcA substituents partially restricted the action of both types of esterases and the remaining acidic XOS were mainly substituted with one MeGlcA and one acetyl group, supposedly on the same xylopyranosyl residue. These resisting structures were degraded to great extent only after inclusion of α-glucuronidase, which acted with the esterases in a synergistic manner. When used together with xylan backbone degrading endoxylanase and ß-xylosidase, both AE and AXE enhanced the hydrolysis of complex XOS equally.

Administration of Bifidobacterium animalis subsp. lactis BB-12 in early childhood: a post-trial effect on caries occurrence at four years of age.

Probiotic bifidobacteria are widely used in the prevention of childhood diseases. These bacteria are also associated with caries occurrence. The present secondary analysis in a low-caries population evaluated the effect of early administration of Bifidobacterium animalis subsp. lactis BB-12 (BB-12) on caries occurrence and identified markers of dental decay in early childhood. In the original randomized, double-blind, placebo-controlled study (NCT00638677, http://www.clinicaltrials.gov), infants (n = 106) received BB-12, xylitol or sorbitol tablets from the age of 1-2 months to 2 years with a slow-release pacifier or a spoon (daily dose of BB-12 10(10) colony-forming units, polyol 200-600 mg). The present data were collected using clinical examinations and questionnaires at the age of 4 years. The occurrence of dental caries was assessed using the International Caries Detection and Assessment System. Oral hygiene status and mutans streptococci (MS) levels were also determined. No differences were detected between the study groups in the occurrence of enamel caries (p = 0.268) or obvious dentinal caries (p = 0.201). The occurrence of caries was associated with daily consumption of sweet drinks (p = 0.028), visible plaque observed (p = 0.002) and MS detected in the dental plaque (p = 0.002). Administration of BB-12 in infancy does not seem to increase or decrease the occurrence of caries by 4 years of age in a low-caries population.

Bifidobacterium animalis subsp. lactis BB-12 administration in early childhood: a randomized clinical trial of effects on oral colonization by mutans streptococci and the probiotic.

A randomized clinical trial studied the effects of early administration of Bifidobacterium animalis subsp. lactis BB-12 (BB-12) on oral colonization of (1) mutans streptococci (MS), and (2) BB-12. In this double-blind, placebo-controlled study, infants (n = 106) received probiotic bacteria (BB-12 group), xylitol (X group), or sorbitol (S group). Test tablets were administered twice a day (from the age of 1-2 months) with a novel slow-release pacifier or a spoon (daily dose of BB-12 10(10) CFU, polyol 200-600 mg). Samples were collected from mucosa/teeth at the age of 8 months and 2 years for BB- 12 determination (qPCR) and plate culturing of MS (MSB, TYCSB), lactobacilli (Rogosa) and yeasts (Sabouraud). The MS levels of the mothers were determined (Dentocult SM Strip Mutans). The baseline characteristics of the three groups were similar. Mean duration of tablet delivery was 14.9 ± 6.7 months. In all groups, >90% of the mothers showed high MS counts (log CFU ≥5). MS colonization percentages of the children at the age of 2 years were rather low (BB-12 group: 6%; X group: 31%; S group: 10%; p < 0.05). The levels of lactobacilli and yeasts did not differ between the groups. BB-12 cell counts barely exceeding the detection limit were found in three of the oral samples of the 8-month-old children; however, the 2-year samples did not contain BB-12. The early administration of BB-12 did not result in permanent oral colonization of this probiotic or significantly affect MS colonization in the children.

Pathologic findings and toxin identification in cyanobacterial (Nodularia spumigena) intoxication in a dog.

A 3-year-old Cairn Terrier dog that had been in contact with sea water containing cyanobacteria (blue-green algae) was euthanized because of acute hepatic failure and anuria after a 5-day illness. Histologic findings included lytic and hemorrhagic centrilobular hepatocellular necrosis and renal tubular necrosis. The cyanotoxin nodularin was detected in liver and kidney by high-performance liquid chromatography-mass spectrometry. Nodularin is a potent hepatotoxin produced by the algal species Nodularia spumigena. The intensity of algal blooms has increased during the past decades in the Baltic Sea region, thus increasing the risk for intoxications in domestic and wild animals. The authors describe the pathologic findings of cyanobacterial toxicosis in a dog with direct identification of the toxin from organ samples.

Are cryptic host species also cryptic to parasites? Host specificity and geographical distribution of acanthocephalan parasites infecting freshwater Gammarus.

Many parasites infect multiple host species. In coevolving host-parasite interactions, theory predicts that parasites should be adapted to locally common hosts, which could lead to regional shifts in host preferences. We studied the interaction between freshwater Gammarus (Crustacea, Amphipoda) and their acanthocephalan parasites using a large-scale field survey and experiments, combined with molecular identification of cryptic host and parasite species. Gammarus pulex is a common host for multiple species of Acanthocephala in Europe but, in Switzerland, is less common than two cryptic members of the Gammarus fossarum species complex (type A and type B). We found that natural populations of these cryptic species were frequently infected by Pomphorhynchus tereticollis and Polymorphus minutus. Four additional parasite species occurred only locally. Parasites were more common in G. fossarum type B than in type A. Infection experiments using several host and parasite sources confirmed consistently lower infection rates in G. pulex than in G. fossarum type A, suggesting a general difference in susceptibility between the two species. In conclusion, we could show that cryptic host species differ in their interactions with parasites, but that these differences were much less dramatic than differences between G. fossarum (type A) and G. pulex. Our data suggest that the acanthocephalans in Switzerland have adapted to the two most common Gammarus species in this region where host species frequencies differ from near-by regions in Europe.

[Purification of the mitochondrial calcium uniporter from the beef heart and characterization of its properties].

A low-molecular-weight component (LMC) inducing selective transport of calcium across the bilayer lipid membrane has been isolated from mitochondria of the bovine heart by the method developed in our laboratory, which excludes the use of detergents and proteolytic enzymes. It was shown that, in the presence of 10 mM CaCl2, LMC forms conduction channels in the membrane multiples of 5 pS. The specific inhibitor of mitochondrial calcium uniporter, ruthenium red, closes Ca2(+)-induced channels formed in the membrane by LMC. In the absence of calcium or in the presence of potassium ions only, the component is incapable of forming channels of conduction. It was shown using nuclear magnetic resonance that LMC is a complex consisting of lipids, amino acids, and sugars with a molecular weight of 1-2 kDa.

Parasites and deleterious mutations: interactions influencing the evolutionary maintenance of sex.

The restrictive assumptions associated with purely genetic and purely ecological mechanisms suggest that neither of the two forces, in isolation, can offer a general explanation for the evolutionary maintenance of sex. Consequently, attention has turned to pluralistic models (i.e. models that apply both ecological and genetic mechanisms). Existing research has shown that combining mutation accumulation and parasitism allows restrictive assumptions about genetic and parasite parameter values to be relaxed while still predicting the maintenance of sex. However, several empirical studies have shown that deleterious mutations and parasitism can reduce fitness to a greater extent than would be expected if the two acted independently. We show how interactions between these genetic and ecological forces can completely reverse predictions about the evolution of reproductive modes. Moreover, we demonstrate that synergistic interactions between infection and deleterious mutations can render sex evolutionarily stable even when there is antagonistic epistasis among deleterious mutations, thereby widening the conditions for the evolutionary maintenance of sex.

Dissolution of xylitol from a food supplement administered with a novel slow-release pacifier: preliminary results.

AIM: The aim of the study was to monitor the pattern of release and salivary xylitol concentrations during sucking of a slow-release pacifier used to deliver a novel food supplement. METHODS: The food supplement tablet contained 300 mg xylitol and 0.5 x 10(10) colony-forming units of Bifidobacterium lactis Bb-12 (Bb-12). The reference tablet contained 300 mg xylitol and was used by 10 adults (mean age 32 years) in the study. Whole saliva samples were collected with 2.5 min intervals during pacifier sucking. The salivary xylitol concentrations were determined using an enzyme assay kit. RESULTS: All subjects showed salivary xylitol concentrations exceeding 1% at least at one collection point. The xylitol and xylitol-Bb-12 tablets showed similar dissolving with no clear concentration peaks (comparison of saliva collection times; p = 0.139). CONCLUSION: Xylitol released from the food supplement, delivered with the novel pacifier, may result in salivary xylitol concentrations high enough to inhibit mutans streptococci in vivo.

Among- and within-population variation in outcrossing rate of a mixed-mating freshwater snail.

Mixed-mating animals self-fertilize a proportion of their offspring. Outcrossing rate may covary with the ecological and historical factors affecting the population. Theory predicts that outcrossing is favored when inbreeding depression is high and when individual heterozygosity is important. Self-fertilization is predicted to be favored when costs of male function, or mate finding are high, for example, when empty patches are colonized by few individuals. In this study, we assessed primary (after hatching) and secondary (after juvenile mortality) outcrossing rates of two mixed-mating snail populations. Our purpose was to assess the variation in mating-system parameters and estimate significance of inbreeding depression for secondary outcrossing rate (the realized outcrossing rate of parents that produce the next generation). Secondary outcrossing rate was higher than the primary outcrossing rate in one of the two populations, suggesting considerable inbreeding depression. In the other study population, secondary outcrossing rates were found to increase when initially low, or decrease when initially high, depending on the family. Moderate outcrossing rates were found to be more stable. Parental inbreeding coefficients were close to zero in both populations. Outcrossing rate was much more variable among families in the population with the lower average outcrossing rate, suggesting that individuals differed considerably in their mating system. Our results add to recent studies suggesting that populations of mixed-mating animals may differ in their mating system parameters and expression of inbreeding depression.

Ecological divergence of closely related Diplostomum (Trematoda) parasites.

Parasite life-cycles present intriguing model systems to study divergence in resource use and ecology between parasite taxa. In ecologically similar taxa, consistent selective forces may lead to convergence of life-history traits, but resource overlap and similarity of life-cycles may also promote divergence between the taxa in (1) use of host species or (2) specific niche within a host. We studied the life-history characteristics of 2 sympatric species of Diplostomum parasites, D. spathaceum and D. gasterostei, concentrating particularly on differences in intermediate host use and characteristics of the infective stages between the species. This group of trematodes is a notoriously difficult challenge for morphological taxonomy and therefore any information on the ecology of these species can also be helpful in resolving their taxonomy. We observed that these species indeed had diverged as they used mainly different snail and fish species as intermediate hosts and in controlled experiments infected different regions of the eye (lens and vitreous body) of a novel fish host. Interestingly, cercarial characteristics (activity and life-span) were similar between the species and the species were difficult to separate at the cercarial stage unless one observes their swimming behaviour. The release of cercaria from the snail hosts was higher in D. spathaceum, but when cercarial numbers were proportioned to the volume of the snail host, the production was higher in D. gasterostei suggesting differences in the rate of snail host exploitation between the parasite species. These results corroborate the prediction that closely related parasite taxa which are competing for the same resources should have diverged in their life-history characteristics and host use.

Variation in asexual lineage age in Potamopyrgus antipodarum, a New Zealand snail.

Asexual lineages are thought to be subject to rapid extinction because they cannot generate recombinant offspring. Accordingly, extant asexual lineages are expected to be of recent derivation from sexual individuals. We examined this prediction by using mitochondrial DNA sequence data to estimate asexual lineage age in populations of a freshwater snail (Potamopyrgus antipodarum) native to New Zealand and characterized by varying frequency of sexual and asexual individuals. We found considerable variation in the amount of genetic divergence of asexual lineages from sexual relatives, pointing to a wide range of asexual lineage ages. Most asexual lineages had close genetic ties (approximately 0.1% sequence divergence) to haplotypes found in sexual representatives, indicating a recent origin from sexual progenitors. There were, however, two asexual clades that were quite genetically distinct (> 1.2% sequence divergence) from sexual lineages and may have diverged from sexual progenitors more than 500,000 years ago. These two clades were found in lakes that had a significantly lower frequency of sexual individuals than lakes without the old clades, suggesting that the conditions that favor sex might select against ancient asexuality. Our results also emphasize the need for large sample sizes and spatially representative sampling when hypotheses for the age of asexual lineages are tested to adequately deal with potential biases in age estimates.

Hydrolysis rates, methane production and nitrogen solubilisation of grey waste components during anaerobic degradation.

Municipal grey waste (i.e. the remaining fraction in municipal waste management systems in which putrescibles (biowaste) and other recyclables (paper, metals, glass) are source-segregated) was manually sorted into six main fractions on the basis of composition and also separated by sieving (100 mm mesh size) into two fractions, oversized and undersized, respectively. In practice, in waste management plant the oversized fraction is (or will be) used to produce refuse-derived fuel and the undersized landfilled after biological stabilisation. The methane yields and nitrogen solubilisation of the grey waste and the different fractions (all studied samples were first milled to 5 mm particle samples) were determined in a 237-day methane production batch assay and in a water elution test, respectively. The grey waste was found to contained remnants of putrescibles and also a high amount of other biodegradable waste, including packaging, cartons and cardboard, newsprint, textiles and diapers. These waste fractions comprised 41%-w/w of the grey waste and produced 40-210 m3 methane (total solids (TS))(-1) and less than 0.01 g NH4-N kg TS(added)(-1) except diapers which produced 9.8 g NH4-N kg TS(added)(-1) in the batch assays. In the case of the two sieved fractions and on mass bases, most of the methane originated from the oversized fraction, whereas most of the NH4-N was solublised from the undersized fraction. The first-order kinetic model described rather well the degradation of each grey waste fraction and component, showing the different components to be in the range 0.021-0.058 d(-1), which was around one-sixth of the values reported for the source-segregated putrescible fraction of MSW.

Modeling solid waste decomposition.

The hydrolysis rate coefficients of sorted municipal waste were evaluated from the biochemical methane potential tests using non-linear regression. A distributed mathematical model of anaerobic digestion of rich (food) and lean (non-food) solid wastes with greatly different rates of polymer hydrolysis/acidogenesis was developed to describe the balance between the rates of hydrolysis/acidogenesis and methanogenesis. The model was calibrated using previously published experimental data [Biores. Technol. 52 (1995) 245] obtained upon various initial food waste loadings. Simulations of one- and two-stage digestion systems were carried out. The results showed that initial spatial separation of food waste and inoculum enhances methane production and waste degradation in a one-stage solid-bed digester at high waste loading. A negative effect of vigorously mixing at high waste loading reported in some papers was discussed. It was hypothesized that the initiation methanogenic centers developing in time and expanding in space under minimal mixing conditions might be a key factor for efficient anaerobic conversion of solid waste into methane.