Genetic Variability of Microcystin Biosynthesis Genes in Planktothrix as Elucidated from Samples Preserved by Heat Desiccation during Three Decades

Historic samples of phytoplankton can provide information on the abundance of the toxigenic genotypes of cyanobacteria in dependence on increased or decreased eutrophication. The analysis of a time-series from preserved phytoplankton samples by quantitative PCR (qPCR) extends observation periods considerably. The analysis of DNA from heat-desiccated samples by qPCR can be aggravated by point substitutions or the fragmentation of DNA introduced by the high temperature. In this study, we analyzed whether the heat desiccation of the cellular material of the cyanobacterium Planktothrix sp. introduced potential errors to the template DNA that is used for qPCR within (i) 16S rDNA and phycocyanin genes and (ii) the mcyA gene indicative of the incorporation of either dehydrobutyrine (Dhb) or N-methyl-dehydroalanine (Mdha) in position 7, and (ii) the mcyB gene, which is indicative of homotyrosine (Hty) in position 2 of the microcystin (MC) molecule. Due to high temperature desiccation, the deterioration of the DNA template quality was rather due to fragmentation than due to nucleotide substitutions. By using the heat-desiccated samples of Lake Zürich, Switzerland the abundance of the Dhb, Mdha and Hty genotypes was determined during three decades (1977-2008). Despite major changes in the trophic state of the lake resulting in a major increase of the total Planktothrix population density, the proportion of these genotypes encoding the synthesis of different MC congeners showed high stability. Nevertheless, a decline of the most abundant mcyA genotype indicative of the synthesis of Dhb in position 7 of the MC molecule was observed. This decline could be related to the gradual incline in the proportion of a mutant genotype carrying a 1.8kbp deletion of this gene region. The increase of this mcyA (Dhb) gene deletion mutant has been minor so far, however, and likely did not affect the overall toxicity of the population.     

Southern right whales show no behavioral response to low noise levels from a nearby unmanned aerial vehicle

Unmanned aerial vehicles (UAVs) are increasingly used for wildlife research and monitoring, but little information exists on their potential effect on marine mammals. We assessed the effects of a UAV on the behavior of southern right whales (Eubalaena australis) in Australia. Focal follows of ten right whale mother-calf pairs were conducted using a theodolite. Control data were recorded for 30 min, and then a DJI Inspire 1 Pro was flown above the whales for 10 min at 5 m altitude. Potential changes to horizontal behavior (swim speed and turning angle) and surfacing pattern (interbreath intervals) were investigated by comparing mother-calf behavior before and during UAV approaches. Changes in respiration rate were used to quantify energetic effects. We also explored acoustic cue perceptibility of the UAV at 5, 10, and 30 m altitude, by measuring the received UAV underwater noise level on whales equipped with acoustic tags (DTAGs). The received noise levels were 86.0 ± 3.9 dB re 1 μPa, while the measured ambient noise was 80.7 ± 7.3 dB re 1 μPa in the same frequency band (100–1,500 Hz). No behavioral response to the UAV was observed. This provides support for UAVs as a noninvasive tool to study baleen whale behavior and ecophysiology.     

Rat model of fractionated (2 Gy/day) 60 Gy irradiation of the liver: long-term effects

The liver is considered a radiosensitive organ. However, in rats, high single-dose irradiation (HDI) showed only mild effects. Consequences of fractionated irradiation (FI) in such an animal model have not been studied so far. Rats were exposed to selective liver FI (total dose 60 Gy, 2 Gy/day) or HDI (25 Gy) and were killed three months after the end of irradiation. To study acute effects, HDI-treated rats were additionally killed at several time points between 1 and 48 h. Three months after irradiation, no differences between FI and HDI treatment were found for macroscopically detectable small “scars” on the liver surface and for an increased number of neutrophil granulocytes distributed in the portal fields and through the liver parenchyma. As well, no changes in HE-stained tissues or clear signs of fibrosis were found around the portal vessels. Differences were seen for the number of bile ducts being increased in FI- but not in HDI-treated livers. Serum levels indicative of liver damage were determined for alkaline phosphatase (AP), aspartate aminotransferase (AST), alanine aminotransferase (ALT), gamma-glutamyltransferase (γGT) and lactate dehydrogenase (LDH). A significant increase of AP was detected only after FI while HDI led to the significant increases of AST and LDH serum levels. By performing RT-PCR, we detected up-regulation of matrix metalloproteinases, MMP-2, MMP-9, MMP-14, and of their inhibitors, TIMP-1, TIMP-2 and TIMP-3, shortly after HDI, but not at 3 month after FI or HDI. Overall, we saw punctual differences after FI and HDI, and a diffuse formation of small scars at the liver surface. Lack of “provisional clot”-formation and absence of recruitment of mononuclear phagocytes could be one explanation for scar formation as incomplete repair response to irradiation.     

The development and characterization of a competitive ELISA for measuring active ADAMTS-4 in a bovine cartilage ex vivo model

ADAMTS-4 (aggrecanase1) is believed to play an important role in the degradation of aggrecan during the progression of joint diseases. ADAMTS-4 is synthesized as a latent pro-enzyme that requires the removal of the pro-domain, exposing the N-terminal neoepitope, to achieve activity. We developed a monoclonal antibody against this neoepitope of active ADAMTS-4. Furthermore, we established and characterized a competitive ELISA for measuring active ADAMTS-4 form applying the specific antibody. We used this assay to profile the presence of active ADAMTS-4 and its aggrecan degradation product (NITEGE³⁷³) in a bovine cartilage ex vivo model. We found that after stimulation with catabolic factors, the cartilage initially released high levels of aggrecanase-derived aggrecan fragments into supernatant but subsequently decreased to background levels. The level of active ADAMTS-4 released into the supernatant and retained in the cartilage matrix increased continuously throughout the 21 days of the study. The activity of ADAMTS-4 on the last day of catabolic stimulation was verified in vitro by adding deglycosylated or native aggrecan to the conditioned medium. Samples of human cartilage affected by varying degrees of osteoarthritis stained strongly for active ADAMTS-4 where surface fibrillation and clustered chondrocytes were observed. This assay could be an effective tool for studying ADAMTS-4 activity and for screening drugs regulating ADAMTS-4 activation. Moreover, it could be a potential biomarker for degenerative joint disease.     

Evolution in a Changing Environment

We propose a simple model for genetic adaptation to a changing environment, describing a fitness landscape characterized by two maxima. One is associated with “specialist” individuals that are adapted to the environment; this maximum moves over time as the environment changes. The other maximum is static, and represents “generalist” individuals not affected by environmental changes. The rest of the landscape is occupied by “maladapted” individuals. Our analysis considers the evolution of these three subpopulations. Our main result is that, in presence of a sufficiently stable environmental feature, as in the case of an unchanging aspect of a physical habitat, specialists can dominate the population. By contrast, rapidly changing environmental features, such as language or cultural habits, are a moving target for the genes; here, generalists dominate, because the best evolutionary strategy is to adopt neutral alleles not specialized for any specific environment. The model we propose is based on simple assumptions about evolutionary dynamics and describes all possible scenarios in a non-trivial phase diagram. The approach provides a general framework to address such fundamental issues as the Baldwin effect, the biological basis for language, or the ecological consequences of a rapid climate change.     

Effect of weight loss and exercise on angiogenic factors in the circulation and in adipose tissue in obese subjects

Background:

Vascular growth is a prerequisite for adipose tissue (AT) development and expansion. Some AT cytokines and hormones have effects on vascular development, like vascular endothelial growth factor (VEGF‐A), angiopoietin (ANG‐1), ANG‐2 and angiopoietin‐like protein‐4 (ANGPTL‐4).

Methods:

In this study, the independent and combined effects of diet‐induced weight loss and exercise on AT gene expression and proteins levels of those angiogenic factors were investigated. Seventy‐nine obese males and females were randomized to: 1. Exercise‐only (EXO; 12‐weeks exercise without diet‐restriction), 2. Hypocaloric diet (DIO; 8‐weeks very low energy diet (VLED) + 4‐weeks weight maintenance diet) and 3. Hypocaloric diet and exercise (DEX; 8‐weeks VLED + 4‐weeks weight maintenance diet combined with exercise throughout the 12 weeks). Blood samples and fat biopsies were taken before and after the intervention.

Results:

Weight loss was 3.5 kg in the EXO group and 12.3 kg in the DIO and DEX groups. VEGF‐A protein was non‐significantly reduced in the weight loss groups. ANG‐1 protein levels were significantly reduced 22‐25% after all three interventions (P < 0.01). The ANG‐1/ANG‐2 ratio was also decreased in all three groups (P < 0.05) by 27‐38%. ANGPTL‐4 was increased in the EXO group (15%, P < 0.05) and 9% (P < 0.05) in the DIO group. VEGF‐A, ANG‐1, and ANGPTL‐4 were all expressed in human AT, but only ANGPTL‐4 was influenced by the interventions.

Conclusions:

Our data show that serum VEGF‐A, ANG‐1, ANG‐2, and ANGPTL‐4 levels are influenced by weight changes, indicating the involvement of these factors in the obese state. Moreover, it was found that weight loss generally was associated with a reduced angiogenic activity in the circulation.     

Does AMH Reflect Follicle Number Similarly in Women with and without PCOS?

Context

Increased Anti-Mullerian Hormone in polycystic ovary syndrome, may be due to overactive follicles rather than reflect antral follicle count.

Objective

Does Anti-Mullerian Hormone reflect antral follicle count similarly in women with or without polycystic ovary syndrome or polycystic ovarian morphology?

Design

Cross-sectional, case-control.

Setting

Women who delivered preterm in 1999–2006. For each index woman, a woman with a term delivery was identified.

Patients

Participation rate was 69%. Between 2006–2008, 262 women were included, and diagnosed to have polycystic ovary syndrome, polycystic ovarian morphology or to be normal controls.

Intervention(s)

Blood tests, a clinical examination and vaginal ultrasound.

Main Outcome Measure(s)

Anti-Mullerian Hormone / antral follicle count -ratio, SHBG, androstenedione and insulin, to test potential influence on the Anti-Mullerian Hormone / antral follicle count -ratio.

Results

Mean Anti-Mullerian Hormone / antral follicle count ratio in women with polycystic ovary syndrome or polycystic ovarian morphology was similar to that of the controls (polycystic ovary syndrome: 1,2 p = 0,10 polycystic ovarian morphology: 1,2, p = 0,27 Controls 1,3). Anti-Mullerian Hormone showed a positive linear correlation to antral follicle count in all groups. Multivariate analysis did not change the results.

Conclusions

We confirmed the positive correlation between AMH and follicle count. Anti-Mullerian Hormone seems to be a reliable predictor of antral follicle count, independent of polycystic ovary syndrome diagnosis or ovarian morphology.     

Comparative Genome Analysis Provides Insights into the Pathogenicity of Flavobacterium psychrophilum

Flavobacterium psychrophilum is a fish pathogen in salmonid aquaculture worldwide that causes cold water disease (CWD) and rainbow trout fry syndrome (RTFS). Comparative genome analyses of 11 F. psychrophilum isolates representing temporally and geographically distant populations were used to describe the F. psychrophilum pan-genome and to examine virulence factors, prophages, CRISPR arrays, and genomic islands present in the genomes. Analysis of the genomic DNA sequences were complemented with selected phenotypic characteristics of the strains. The pan genome analysis showed that F. psychrophilum could hold at least 3373 genes, while the core genome contained 1743 genes. On average, 67 new genes were detected for every new genome added to the analysis, indicating that F. psychrophilum possesses an open pan genome. The putative virulence factors were equally distributed among isolates, independent of geographic location, year of isolation and source of isolates. Only one prophage-related sequence was found which corresponded to the previously described prophage 6H, and appeared in 5 out of 11 isolates. CRISPR array analysis revealed two different loci with dissimilar spacer content, which only matched one sequence in the database, the temperate bacteriophage 6H. Genomic Islands (GIs) were identified in F. psychrophilum isolates 950106-1/1 and CSF 259–93, associated with toxins and antibiotic resistance. Finally, phenotypic characterization revealed a high degree of similarity among the strains with respect to biofilm formation and secretion of extracellular enzymes. Global scale dispersion of virulence factors in the genomes and the abilities for biofilm formation, hemolytic activity and secretion of extracellular enzymes among the strains suggested that F. psychrophilum isolates have a similar mode of action on adhesion, colonization and destruction of fish tissues across large spatial and temporal scales of occurrence. Overall, the genomic characterization and phenotypic properties may provide new insights to the mechanisms of pathogenicity in F. psychrophilum.     

Weight loss maintenance in severely obese adults after an intensive lifestyle intervention: 2- to 4-year follow-up

Objective: To evaluate the long‐term weight loss maintenance after 2 to 4 years in severely obese subjects after a lifestyle intervention at a weight loss camp in Denmark. Research Methods and Procedures: In a retrospective follow‐up study, we assessed weight loss after 21 weeks of treatment at a weight loss camp, weight loss maintenance after 2 to 4 years, and numbers of subjects with a weight loss maintenance of ≥10% of a total number of 435 severely obese adults participating in an intensive lifestyle intervention with a primary focus on physical activity. Results: We obtained follow‐up data of 249 subjects (180 women and 69 men) with an initial body weight of 142 ± 32 kg. After 21 weeks at the camp, the subjects had reduced their body weight with a mean of 21.9 ± 13 kg (corresponding to a 15% weight loss). The average weight loss maintenance was 5.3% at a follow‐up after 2 to 4 years, and 28.3% had maintained a weight loss above 10% after 4 years of follow‐up. Discussion: Weight loss camps are a relatively new commercial approach in treating severely obese subjects. However, the results demonstrate that even with a multidisciplinary intensive setting with focus on diet, exercise, and psychological counseling, only 28% had maintained a weight loss above 10% after 4 years. This emphasizes that obesity is a chronic condition that needs additional strategies after a weight loss intervention in the efforts to maintain a sufficient weight loss.     

Energy imbalance underlying the development of childhood obesity

Objective: To develop a model based on empirical data and human energetics to predict the total energy cost of weight gain and obligatory increase in energy intake and/or decrease in physical activity level associated with weight gain in children and adolescents. Research Methods and Procedures: One‐year changes in weight and body composition and basal metabolic rate (BMR) were measured in 488 Hispanic children and adolescents. Fat‐free mass (FFM) and fat mass (FM) were measured by DXA and BMR by calorimetry. Model specifications include the following: body mass (BM) = FFM + FM, each with a specific energy content, cff (1.07 kcal/g FFM) and cf (9.25 kcal/g FM), basal energy expenditure (EE), kff and kf, and energetic conversion efficiency, eff (0.42) for FFM and ef (0.85) for FM. Total energy cost of weight gain is equal to the sum of energy storage, EE associated with increased BM, conversion energy (CE), and diet‐induced EE (DIEE). Results: Sex‐ and Tanner stage–specific values are indicated for the basal EE of FFM (kff) and the fat fraction in added tissue (fr). Total energy cost of weight gain is partitioned into energy storage (24% to 36%), increase in EE (40% to 57%), CE (8% to 13%), and DIEE (10%). Observed median (10th to 90th percentile) weight gain of 6.1 kg/yr (2.4 to 11.4 kg/yr) corresponds at physical activity level (PAL) = 1.5, 1.75, and 2.0 to a total energy cost of weight gain of 244 (93 to 448 kcal/d), 267 (101 to 485 kcal/d), and 290 kcal/d (110 to 527 kcal/d), respectively, and to a total energy intake of 2695 (1890 to 3730), 3127 (2191 to 4335), and 3551 (2487 to 4930) kcal/d, respectively. If weight gain is caused by a change in PAL alone and PAL₀ = 1.5 at baseline t = 0, the model indicates a drop in PAL of 0.22 (0.08 to 0.34) units, which is equivalent to 60 (18 to 105) min/d of walking at 2.5 mph. Discussion: Halting the development or progression of childhood obesity, as observed in these Hispanic children and adolescents, by counteracting its total energy costs will require a sizable decrease in energy intake and/or reciprocal increase in physical activity.