Characterization of Precursor PfHsp60 in Plasmodium falciparum Cytosol during Its Asexual Development in Human Erythrocytes

Mitochondrial heat shock protein 60 (Hsp60) is a nuclear encoded gene product that gets post-translationally translocated into the mitochondria. Using multiple approaches such as immunofluorescence experiments, isoelectric point analysis with two-dimensional gel electrophoresis, and mass spectrometric identification of the signal peptide, we show that Hsp60 from Plasmodium falciparum (PfHsp60) accumulates in the parasite cytoplasm during the ring, trophozoite, and schizont stages of parasite development before being imported into the parasite mitochondria. Using co-immunoprecipitation experiments with antibodies specific to cytoplasmic PfHsp90, PfHsp70-1, and PfHsp60, we show association of precursor PfHsp60 with cytoplasmic chaperone machinery. Metabolic labeling involving pulse and chase indicates translocation of the precursor pool into the parasite mitochondrion during chase. Analysis of results obtained with Geldanamycin treatment confirmed precursor PfHsp60 to be one of the clients for PfHsp90. Cytosolic chaperones bind precursor PfHsp60 prior to its import into the mitochondrion of the parasite. Our data suggests an inefficient co-ordination in the synthesis and translocation of mitochondrial PfHsp60 during asexual growth of malaria parasite in human erythrocytes.     

Sliding window discretization: A new method for multiple band matching of bacterial genotyping fingerprints

Microbiologists have traditionally applied hierarchical clustering algorithms as their mathematical tool of choice to unravel the taxonomic relationships between micro-organisms. However, the interpretation of such hierarchical classifications suffers from being subjective, in that a variety of ad hoc choices must be made during their construction. On the other hand, the application of more profound and objective mathematical methods—such as the minimization of stochastic complexity—for the classification of bacterial genotyping fingerprints data is hampered by the prerequisite that such methods only act upon vectorized data. In this paper we introduce a new method, coined sliding window discretization, for the transformation of genotypic fingerprint patterns into binary vector format. In the context of an extensive amplified fragment length polymorphism (AFLP) data set of 507 strains from the Vibrionaceae family that has previously been analysed, we demonstrate by comparison with a number of other discretization methods that this new discretization method results in minimal loss of the original information content captured in the banding patterns. Finally, we investigate the implications of the different discretization methods on the classification of bacterial genotyping fingerprints by minimization of stochastic complexity, as it is implemented in the BinClass software package for probabilistic clustering of binary vectors. The new taxonomic insights learned from the resulting classification of the AFLP patterns will prove the value of combining sliding window discretization with minimization of stochastic complexity, as an alternative classification algorithm for bacterial genotyping fingerprints.     

Young and female‐biased irruptions in pygmy owls Glaucidium passerinum in southern Finland

Irruptive migrants often show biased sex and age ratio, and typically young females are the most abundant class participating in irruptions. Several hypotheses have been proposed to explain differential migration in birds. We examined these hypotheses in a sexually size‐dimorphic species (females being larger than males), the pygmy owl Glaucidium passerinum, whose migration behaviour has also been poorly documented. Migration data were collected at the Hanko Bird Observatory, SW Finland during 1979–2010. Pygmy owls showed large fluctuations in migration numbers between years. Most of the migrants were young birds and females migrated in larger numbers than males. Results support the ‘territory defence hypothesis’ since males showed weaker tendency to migrate despite the smaller body size, but the ‘dominance hypothesis’ was also supported (subordinate young outnumber dominant adults in migration numbers). Findings support the idea that in owl species, which are dependent on nesting cavities, males show lower migration behaviour than females, whereas the pattern is opposite in species which normally breed outside cavities. In addition, adults migrated later than young, likely because they need to moult remiges before departure. Early hatched young also migrated earlier than late hatched young, suggesting that more dominant young migrate first.     

Quality of intertrochanteric cancellous bone as predictor of femoral stem RSA migration in cementless total hip arthroplasty

In cementless total hip arthroplasty, osteoporosis may jeopardize the achievement of immediate stability and lead to migration of anatomically shaped femoral stems. Poor quality of proximal cancellous bone per se may also affect the rate of osseointegration. In a selected group of female total hip arthroplasty patients (mean age 64 years) with unremarkable medical history, intertrochanteric cancellous bone biopsy was taken from the site of stem implantation. Local bone quality, determined by structural μCT imaging and destructive compression testing of the biopsy tissue, was used as the predictor of three-dimensional stem migration determined by radiostereometric analysis (RSA) up to 24 months. The patients exhibited major differences in mechanical properties of the intertrochanteric cancellous bone, which were closely related to the structural parameters calculated from μCT data. Unexpectedly, the major differences observed in the quality of trochanteric cancellous bone had only minor reflections in the RSA migration of the femoral stems. In statistical analysis, the μCT-based bone mineral density quartile (low, middle, high) was the only significant predictor for stem translation at 24 months (p=0.022) but only a small portion (R(2)=0.16) of the difference in translation could be explained by changes in bone mineral density quartile. None of the other parameters investigated predicted stem migration in translation or rotation. In conclusion, poor quality of intertrochanteric cancellous bone seems to contribute to the risk of implant migration less than expected. Probably also the importance of surgical preservation of intertrochanteric cancellous bone has been over-emphasized for osseointegration of cementless stem.     

Long-term yield trends of insect-pollinated crops vary regionally and are linked to neonicotinoid use,landscape complexity,and availability of pollinators

Time series data on crop yields for two main wind-pollinated crops (barley and wheat) and for three crops benefitting from insect pollination (turnip rapeseed, caraway, and black currant), were compiled from official agricultural statistics. In Finland, these statistics are available at aggregate national level, and at the level of each of the 15 provinces of the country. Yields of wind-pollinated crops have steadily increased in Finland, while yields of insect-pollinated crops have been highly variable. The largest crop benefitting from insect pollination is turnip rapeseed, which shows first a clear tendency to increased yields from 1980 to 1993, after which there has been a continuous decline in yields at the national average level. Regionally, the trends in turnip rapeseed yield show large variation, so that in six provinces of Finland, the trend has been significantly decreasing; in five provinces, there has been no significant trend; and in two provinces, there has been a significant linear increase in yields. Yield trends in the two other insect-pollinated crops, caraway and black currants, show similar trend variations. However, at the national average level, caraway yields show no significant trend, while black currant yields have increased during the past 6 years. The possible impact on the trends of insect-pollinated crops of three explanatory variables was analyzed. Significant linear correlation was found between the yield trends (slope of the trends) in rapeseed, and the extent of using neonicotinoid seed dressing in the provinces; the magnitude of yield decline in turnip rapeseed increased, as the use of neonicotinoid seed dressing increased. Similar significant linear correlation was found for the magnitude of yield decline in turnip rapeseed and the complexity of the agricultural landscape in each province; yield trend changed from negative to positive as the proportion of agricultural land of the total terrestrial land area declined from 28% to below 10%. The availability of honey bee colonies with respect to the growing area of crops benefitting from insect pollination also had a linear, significant impact on turnip rapeseed yield trends: yields tended to decline in provinces, where the supply of managed pollinators with respect to demand was low, but tended to increase in provinces, where the number of honey bee colonies were over 30% of the estimated demand. As neither the landscape complexity (proportion of arable land of total terrestrial land area), nor the number of honey bee colonies for pollination have changed significantly over the past 10–20 years, these factors cannot explain the observed differences in the yield trends of the examined insect-pollinated crops. It appears that only the uptake of neonicotinoid insecticide seed dressing about 15 years ago can explain the crop yield declines in several provinces, and at the national level for turnip rapeseed, most likely via disruption of pollination services by wild pollinators.     

A novel fed-batch based cultivation method provides high cell-density and improves yield of soluble recombinant proteins in shaken cultures

Background  

Cultivations for recombinant protein production in shake flasks should provide high cell densities, high protein productivity per cell and good protein quality. The methods described in laboratory handbooks often fail to reach these goals due to oxygen depletion, lack of pH control and the necessity to use low induction cell densities. In this article we describe the impact of a novel enzymatically controlled fed-batch cultivation technology on recombinant protein production in Escherichia coli in simple shaken cultures.     

Activated polyamine catabolism leads to low cholesterol levels by enhancing bile acid synthesis

Transgenic mice with activated polyamine catabolism due to overexpression of spermidine/spermine N¹-acetyltransferase (SSAT) have significantly reduced plasma total cholesterol levels. In our study, we show that low cholesterol levels were attributable to enhanced bile acid synthesis in combination with reduced cholesterol absorption. Hepatic cholesterol 7α-hydroxylase (CYP7A1), the rate-limiting enzyme catalyzing the conversion of cholesterol to bile acids, plays an important role in the removal of excess cholesterol from the body. We suggest that by reducing activity of Akt activated polyamine catabolism increased the stability and activity of peroxisome proliferator-activated receptor γ co-activator 1α, the critical activator of CYP7A1. This is supported by our finding that the treatment with SSAT activator, N ¹,N ¹¹-diethylnorspermine, reduced significantly the amount of phosphorylated (active) Akt in HepG2 cells. In summary, activated-polyamine catabolism is a novel mechanism to regulate bile acid synthesis. Therefore, polyamine catabolism could be a potential therapeutic target to control hepatic CYP7A1 expression.     

Trans-spliced Heat Shock Protein 90 Modulates Encystation in Giardia lamblia

Background

Hsp90 from Giardia lamblia is expressed by splicing of two independently transcribed RNA molecules, coded by genes named HspN and HspC located 777 kb apart. The reasons underlying such unique trans-splicing based generation of GlHsp90 remain unclear.

Principle Finding

In this study using mass-spectrometry we identify the sequence of the unique, junctional peptide contributed by the 5′ UTR of HspC ORF. This peptide is critical for the catalytic function of Hsp90 as it harbours an essential “Arg” in its sequence. We also show that full length GlHsp90 possesses all the functional hall marks of a canonical Hsp90 including its ability to bind and hydrolyze ATP. Using qRT-PCR as well as western blotting approach we find the reconstructed Hsp90 to be induced in response to heat shock. On the contrary we find GlHsp90 to be down regulated during transition from proliferative trophozoites to environmentally resistant cysts. This down regulation of GlHsp90 appears to be mechanistically linked to the encystation process as we find pharmacological inhibition of GlHsp90 function to specifically induce encystation.

Significance

Our results implicate the trans-spliced GlHsp90 from Giardia lamblia to regulate an essential stage transition in the life cycle of this important human parasite.     

Insulin resistance is associated with increased cholesterol synthesis and decreased cholesterol absorption in normoglycemic men

Type 2 diabetes has been associated with high synthesis and low absorption of cholesterol independent of weight, indicating that insulin resistance may be a link between glucose and cholesterol metabolism. Therefore, we investigated the relationship of serum cholesterol precursors, reflecting cholesterol synthesis, and serum plant sterols and cholestanol, reflecting cholesterol absorption efficiency, with insulin sensitivity measured with the hyperinsulinemic euglycemic clamp in 72 healthy normoglycemic men. Men in the most insulin-resistant tertile had higher serum cholesterol precursor ratios (P < 0.05), whereas no significant differences in serum absorption sterols were observed. In bivariate analysis, cholesterol synthesis markers correlated with fasting insulin (r = 0.36-0.46, P < 0.01) and the rates of insulin-stimulated whole-body glucose uptake (WBGU; r = -0.37-0.40, P < 0.01). Also, cholesterol absorption markers correlated with fasting insulin and WBGU (P < 0.05). Fasting insulin correlated with desmosterol (r = 0.286, P = 0.015) and lathosterol (r = 0.248, P = 0.037) even when the rates of WBGU and body mass index (BMI) were controlled for. We conclude that insulin resistance is linked to high cholesterol synthesis and decreased cholesterol absorption. Because fasting insulin correlated with cholesterol synthesis independent of the rates of BMI and WBGU, it is possible that regulation of cholesterol synthesis by hyperinsulinemia may be a link between insulin resistance and cholesterol metabolism.