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.

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.

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.

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.

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.

[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.

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.

Natural selection on age-specific fertilities in human females: comparison of individual-level fitness measures.

Lifetime reproductive success and timing of reproduction are key components of life-history evolution. To understand the evolution of reproductive schedules, it is important to use a measure of fitness that is sensitive both to reproductive quantity and reproductive timing. There is a contradiction between the theory, which mainly focuses on the rate measures of fitness (r and lambda), and empirical studies, which mainly use lifetime reproductive success (LRS), or some of its correlates, as a fitness measure. We measured phenotypic selection on age-specific fertilities in three pre-modern human populations using individually estimated finite rate of increase, er (lambda). We found that lambda and lifetime reproductive success ranked individuals differently according to their fitness: for example, a female giving birth to four children at a young age may actually have a higher fitness than a female giving birth to six children at a greater age. Increase in fertility at the young age classes (15-19 years) was favoured by selection, but the intensity of selection on fertility was higher in the older age classes (20-30 years), where the variance in fertility was highest. Hence, variation in fertility in the older age classes (20-30) was actually responsible for most of the observed variation in fitness among the individuals. Additionally, more than 90% of variation in fitness (lambda) was attributable to individual differences in LRS, whereas only about 5% of all variation in fitness was due to differences in the reproductive schedule. The rate-sensitive fitness measure did not significantly challenge the importance of total fertility as a component of fitness in humans. However, the rate-sensitive measure clearly allowed more accurate estimation of individual fitness, which may be important for answering some more specific questions.

Predator avoidance and immune defence: costs and trade-offs in snails.

Organisms are often confronted by both predators and pathogens. Defending against such widely divergent enemies requires more than one type of defence. Multiple defences, however, raise the possibility of trade-offs among defences. We tested for such trade-offs by manipulating the level of predator-avoidance behaviour and immune function in the freshwater snail Lymnaea stagnalis (Gastropoda: Pulmonata). Our results show that predator avoidance and immune function had clear costs in terms of reproduction and survival. Further, we show that increased levels of predator-avoidance behaviour reduced the snails' ability to defend against potential pathogens. Predator-avoidance behaviour may thus have the additional indirect cost of reduced immunocompetence and increased susceptibility to pathogens. Our results suggest that ecological factors (e.g. predator density) may considerably modify the expression and costs of immune defences.

Direct and correlative phenotypic selection on life-history traits in three pre-industrial human populations.

Because natural selection acts simultaneously on several correlated traits, can be under both direct and correlative selection simultaneously. Correlative selection may either weaken or magnify the association between a trait and fitness. Direct effect of a single trait on fitness can be assessed by removing the effects of correlative selection with multivariate techniques. We studied the phenotypic selection on demographic life-history traits in three pre-industrial human populations, which experienced different mortality environments. We used path-analysis to estimate direct and correlative selection on different traits in females and males. Our results indicate substantial differences among the sexes in the force of phenotypic selection on key life-history traits. For females, the most important component of reproductive success was the age at first reproduction. Most important component of fitness for males was the quality of mate. In addition, our analysis revealed considerable among-population variation in the selection on life-history traits. These differences are in accord with the historical notes of among-population variation in the lifestyle and harshness of the environment, suggesting that among-population variation in life-history traits may have been a response to the environmental variation.

Organic halogens in the environment: studies of environmental biodegradability and human exposure.

Organic halogens from chlorobleaching of kraft pulp were not as biorecalcitrant as has been assumed. Fifty percent were removed during biotreatment of wastewater, and 50% of the remaining organohalogens faded in fresh water ecosystems in 200 to 400 days. Molecular size seemed not to hinder biodegradation up to sizes of approximately 2000 daltons. Anoxic biodegradation was of prime importance for halomineralization of pulp bleaching organohalogens but could also lead to toxic metabolites such as vinyl chloride from tri- and tetrachloroethene in anoxic soil. Indigenous soil microbes were unable to clean old organohalogen pollution but had converted chlorophenols into polymeric substances, chlorohumus, which were found bioaccumulable by earthworms in spite of the large (up to 5000 g/mole) molecular sizes. Because of the danger of formation of toxic metabolites, the biochemistry of the xenobiotic degradation must be elucidated before active bioremediation is practiced on polluted soil or water. Groundwater pollution by chlorophenols led to increased disease among the exposed population in one well-studied case. Two further cases of potential environmental health impact are described.

Postural balance and self-reported functional ability in 75-year-old men and women: a cross-national comparative study.

OBJECTIVE: To study postural balance in relation to self-reported functional ability (mobility and ADL) and general physical activity in elderly men and women living in three different Nordic environments. PARTICIPANTS: A random sample of 448 men and 556 women from among the 75-year-old residents in Glostrup, Denmark, and Göteborg, Sweden, and all the residents of relevant age (127 men and 261 women) in Jyväskylä, Finland. MEASUREMENTS: Assessment of postural balance with eyes open and closed using a piezoelectric force platform. A structured interview on self-reported functional ability and physical activity. An in-laboratory medical examination. RESULTS: In spite of some differences in balance between the groups studied (better results in women compared with men and, to some extent, better results in the participants from Denmark and Finland than in those from Sweden), the performance in the balance tests was similarly associated with functional ability within all groups. The subjects reporting no need of help in performing the ADL and mobility functions performed significantly better in the balance tests. These differences were seen more clearly in the control of anteroposterior movement of center of forces than in the mediolateral direction. The performance in the balance tests was also significantly better among the subjects reporting a higher level of general physical activity than in their less active counterparts. Physical activity and than in their less active counterparts. Physical activity and certain long standing illnesses modified significantly the relationship between postural balance and ADL-performance. When these factors were analyzed simultaneously, the role of balance as a predictor of ADL-performance largely disappeared. CONCLUSIONS: The results suggest that good balance is one of the prerequisites of performance without difficulty in mobility and ADL functions. Physical exercise may help to maintain balancing abilities in old age; good balance, in turn, may also enable a physically active way of life. The associations of balance with functional ability and physical activity were independent of sex and locality. The results also support the validity of static stabilometry as a tool for evaluating threats to functional limitations in older subjects.

Phenotypic plasticity and priority rules for energy allocation in a freshwater clam: a field experiment.

We studied resource allocation among maintenance, reproduction and growth in the freshwater clam Anodonta piscinalis. Recent theoretical and empirical studies imply that organisms with indeterminate growth may have priority rules for energy allocation. That being so, the traits involved should potentially be capable of considerable phenotypic modulation, as a mechanism to adjust allocation. We tested this hypothesis using a 1-year reciprocal transplant experiment at six sites. Experimental clams were caged at higher than natural densities in order to detect any phenotypic modulation of the traits and discover the putative priority rules in energy allocation. We recorded the survival and shell growth of clams during the experiment, and the reproductive output, somatic mass and fat content of clams at the end of the experiment. Shell growth, somatic mass, and the reproductive output of females varied more among transplant sites than among the populations of origin, suggesting a high capacity for phenotypic modulation. However, the reproductive investment, somatic mass and shell growth were also affected by origin; clams from productive habitats invested more in reproduction and were heavier. In comparison to undisturbed clams, the reproductive output of the experimental clams was similar and their fat content was higher, whereas their shell growth was considerably slower and their somatic mass lower. These results suggests that when resources are limiting (due to high density) reproductive allocation overrides allocation to somatic growth. The highest mortality during the experiment coincided with the period of reproductive stress in the spring. Additionally, the proportion of reproducing females was lower in those transplant groups where the survival rate was lowest, suggesting that maintenance allocation overrides allocation to reproduction when available resources are scarce. The results of this field experiment support theoretical predictions and results of previous laboratory experiments that suggest that there are priority rules for energy allocation in organisms with indeterminate growth.