SHORT Syndrome with Partial Lipodystrophy Due to Impaired Phosphatidylinositol 3 Kinase Signaling

The phosphatidylinositol 3 kinase (PI3K) pathway regulates fundamental cellular processes such as metabolism, proliferation, and survival. A central component in this pathway is the p85α regulatory subunit, encoded by PIK3R1. Using whole-exome sequencing, we identified a heterozygous PIK3R1 mutation (c.1945C>T [p.Arg649Trp]) in two unrelated families affected by partial lipodystrophy, low body mass index, short stature, progeroid face, and Rieger anomaly (SHORT syndrome). This mutation led to impaired interaction between p85α and IRS-1 and reduced AKT-mediated insulin signaling in fibroblasts from affected subjects and in reconstituted Pik3r1-knockout preadipocytes. Normal PI3K activity is critical for adipose differentiation and insulin signaling; the mutated PIK3R1 therefore provides a unique link among lipodystrophy, growth, and insulin signaling.     

Baboon phylogeny as inferred from complete mitochondrial genomes

Baboons (genus Papio) are an interesting phylogeographical primate model for the evolution of savanna species during the Pleistocene. Earlier studies, based on partial mitochondrial sequence information, revealed seven major haplogroups indicating multiple para‐ and polyphylies among the six baboon species. The most basal splits among baboon lineages remained unresolved and the credibility intervals for divergence time estimates were rather large. Assuming that genetic variation within the two studied mitochondrial loci so far was insufficient to infer the apparently rapid early radiation of baboons we used complete mitochondrial sequence information of ten specimens, representing all major baboon lineages, to reconstruct a baboon phylogeny and to re‐estimate divergence times. Our data confirmed the earlier tree topology including the para‐ and polyphyletic relationships of most baboon species; divergence time estimates are slightly younger and credibility intervals narrowed substantially, thus making the estimates more precise. However, the most basal relationships could not be resolved and it remains open whether (1) the most southern population of baboons diverged first or (2) a major split occurred between southern and northern clades. Our study shows that complete mitochondrial genome sequences are more effective to reconstruct robust phylogenies and to narrow down estimated divergence time intervals than only short portions of the mitochondrial genome, although there are also limitations in resolving phylogenetic relationships. Am J Phys Anthropol, 2013. © 2012 Wiley Periodicals, Inc.     

Aerobic Training in Patients with Congenital Myopathy

Introduction

Congenital myopathies (CM) often affect contractile proteins of the sarcomere, which could render patients susceptible to exercise-induced muscle damage. We investigated if exercise is safe and beneficial in patients with CM.

Methods

Patients exercised on a stationary bike for 30 minutes, three times weekly, for 10 weeks at 70% of their maximal oxygen uptake (VO_2max). Creatine kinase (CK) was monitored as a marker of muscle damage. VO_2max, functional tests, and questionnaires evaluated efficacy.

Results

Sixteen patients with CM were included in a controlled study. VO_2max increased by 14% (range, 6–25%; 95% CI 7–20; p < 0.001) in the seven patients who completed training, and tended to decrease in a non-intervention group (n = 7; change -3.5%; range, -11–3%, p = 0.083). CK levels were normal and remained stable during training. Baseline Fatigue Severity Scale scores were high, 4.9 (SE 1.9), and tended to decrease (to 4.4 (SE 1.7); p = 0.08) with training. Nine patients dropped out of the training program. Fatigue was the major single reason.

Conclusions

Ten weeks of endurance training is safe and improves fitness in patients with congenital myopathies. The training did not cause sarcomeric injury, even though sarcomeric function is affected by the genetic abnormalities in most patients with CM. Severe fatigue, which characterizes patients with CM, is a limiting factor for initiating training in CM, but tends to improve in those who train.

Trial Registration

The Regional Committee on Health Research Ethics of the Capital Region of Denmark H-2-2013-066 and ClinicalTrials.gov H2-2013-066     

Demonstration of pyruvate recycling in primary cultures of neocortical astrocytes but not in neurons

Pyruvate recycling was studied in primary cultures of mouse cerebrocortical astrocytes, GABAergic cerebrocortical interneurons, and co-cultures consisting of both cell types by measuring production of [4-¹³C]glutamate from [3-¹³C]glutamate by aid of nuclear magnetic resonance spectroscopy. This change in the position of the label can only occur by entry of [3-¹³C]glutamate into the tricarboxylic acid (TCA) cycle, conversion of labeled -ketoglutarate to malate or oxaloacetate, malic enzyme-mediated decarboxylation of malate to pyruvate or phosphoenolpyruvate carboxykinase-mediated conversion of oxaloacetate to phosphoenolpyruvate and subsequent hydrolysis of the latter to pyruvate, and introduction of the labeled pyruvate into the TCA cycle, i.e., after exit of the carbon skeleton of pyruvate from the TCA cycle followed by re-entry of the same pyruvate molecules via acetyl CoA. In agreement with earlier observations, pyruvate recycling was demonstrated in astrocytes, indicating the ability of these cells to undertake complete oxidative degradation of glutamate. The recycled [4-¹³C]glutamate was not further converted to glutamine, showing compartmentation of astrocytic metabolism. Thus, absence of recycling into glutamine in the brain in vivo cannot be taken as indication that pyruvate recycling is absent in astrocytes. No recycling could be demonstrated in the cerebrocortical neurons. This is consistent with a previously demonstrated lack of incorporation of label from glutamate into lactate, and it also indicates that mitochondrial malic enzyme is not operational. Nor was there any indication of pyruvate recycling in the co-cultures. Although this may partly be due to more rapid depletion of glutamate in the co-cultures, this observation at the very least indicates that pyruvate recycling is not up-regulated in the neuronal-astrocytic co-cultures.     

Rhizopogon spore bank communities within and among California pine forests

In this study we examine the distribution of Rhizopogon species in spore banks from five California pine forests. Four of the forest sites were discontinuous populations of Pinus muricata and a fifth was a Pinus ponderosa stand in Sierra National Forest. Rhizopogon species were retrieved by bioassaying the soils with pine seedlings followed by isolation of axenic cultures from individual root tips with typical Rhizopogon ectomycorrhizal morphology. The cultures were screened by ITS-RFLP and all unique patterns were sequenced. These sequences then were compared with those derived from identified sporocarp material. Bioassaying proved to be an efficient way to bring Rhizopogon species into culture. Approximately 50% of the pots contained ectomycorrhizal tips with Rhizopogon-like morphology, and axenic Rhizopogon cultures were obtained from half these pots. Our results showed that Rhizopogon spores usually are well distributed within local forest areas, while there is significant structuring of species at the regional scale. Spore longevity and homogenization by soil and water movement might explain their distribution within local forest areas, while the regional pattern might be explained by limited long distance dispersal or climatic and edaphic differences.     

A trypsin-like protease with apparent dual function in early Lepeophtheirus salmonis (Kr?yer) development

Background  

Trypsin-like serine proteases are involved in a large number of processes including digestive degradation, regulation of developmental processes, yolk degradation and yolk degradome activation. Trypsin like peptidases considered to be involved in digestion have been characterized in Lepeophtheirus salmonis. During these studies a trypsin-like peptidase which differed in a number of traits were identified.     

Interrelationships of glycosylation and aggregation kinetics for Peniophora lycii phytase

The kinetics of thermally induced aggregation of the glycoprotein Peniophora lycii phytase (Phy) and a deglycosylated form (dgPhy) was studied by dynamic (DLS) and static (SLS) light scattering. This provided a detailed insight into the time course of the formation of small aggregates ( approximately 10-100 molecules) of the enzyme. The thermodynamic stability of the two forms was also investigated using scanning calorimetry (DSC). It was found that the glycans strongly promoted kinetic stability (i.e., reduced the rate of irreversible denaturation) while leaving the equilibrium denaturation temperature, T(d), defined by DSC, largely unaltered. At pH 4.5-5.0, for example, dgPhy aggregated approximately 200 times faster than Phy, even though the difference in T(d) was only 1-3 degrees C. To elucidate the mechanism by which the glycans promote kinetic stability, we measured the effect of ionic strength and temperature on the aggregation rate. Also, the second virial coefficients (B(22)) for the two forms were measured by SLS. These results showed that the aggregation rate of Phy scaled with the concentration of thermally denatured protein. This suggested first-order kinetics with respect to the concentration of the thermally denatured state. A similar but less pronounced correlation was found for dgPhy, and it was suggested that while the aggregation process for the deglycosylated form is dominated by denatured protein, it also involves a smaller contribution from associating molecules in the native state. The measurements of B(22) revealed that dgPhy had slightly higher values than Phy. This suggests that dgPhy interacts more favorably with the buffer than Phy and hence rules out strong hydration of the glycans as the origin of their effect on the kinetic stability. On the basis of this and the effects of pH and ionic strength, we suggest that the inhibition of aggregation is more likely to depend on steric hindrance of the glycans in the aggregated form of the protein.     

Detecting flying insects using car nets and DNA metabarcoding

Monitoring insects across space and time is challenging, due to their vast taxonomic and functional diversity. This study demonstrates how nets mounted on rooftops of cars (car nets) and DNA metabarcoding can be applied to sample flying insect richness and diversity across large spatial scales within a limited time period. During June 2018, 365 car net samples were collected by 151 volunteers during two daily time intervals on 218 routes in Denmark. Insect bulk samples were processed with a DNA metabarcoding protocol to estimate taxonomic composition, and the results were compared to known flying insect richness and occurrence data. Insect and hoverfly richness and diversity were assessed across biogeographic regions and dominant land cover types. We detected 15 out of 19 flying insect orders present in Denmark, with high proportions of especially Diptera compared to Danish estimates, and lower insect richness and diversity in urbanized areas. We detected 319 species not known for Denmark and 174 species assessed in the Danish Red List. Our results indicate that the methodology can assess the flying insect fauna at large spatial scales to a wide extent, but may be, like other methods, biased towards certain insect orders.     

Complete 1H, 13C and 15N NMR assignments and secondary structure of the 269-residue serine protease PB92 from Bacillus alcalophilus

Summary The ¹H, ¹³C and ¹⁵N NMR resonances of serine protease PB92 have been assigned using 3D tripleresonance NMR techniques. With a molecular weight of 27 kDa (269 residues) this protein is one of the largest monomeric proteins assigned so far. The side-chain assignments were based mainly on 3D H(C)CH and 3D (H)CCH COSY and TOCSY experiments. The set of assignments encompasses all backbone carbonyl and CH_n carbons, all amide (NH and NH₂) nitrogens and 99.2% of the amide and CH_n protons. The secondary structure and general topology appear to be identical to those found in the crystal structure of serine protease PB92 [Van der Laan et al. (1992) Protein Eng., 5, 405–411], as judged by chemical shift deviations from random coil values, NH exchange data and analysis of NOEs between backbone NH groups.Abbreviations 2D/3D/4D two-/three-/four-dimensional - HSQC heteronuclear single-quantum coherence - HMQC heteronuclear multiple-quantum coherence - COSY correlation spectroscopy - TOCSY total correlation spectroscopy - NOE nuclear Overhauser enhancement (connectivity) - NOESY 2D NOE spectroscopy Experiment nomenclature (H(C)CH, etc.) follows the conventions used elsewhere [e.g. Ikura et al. (1990) Biochemistry, 29, 4659–4667].     

Discovery of new PPARγ agonists based on arylopeptoids

In this study we present the design, synthesis and biological evaluation of a small, first-generation library of small molecule aromatic amides based on the arylopeptoid skeleton. The compounds were efficiently synthesized using a highly convenient submonomer solid-phase methodology which potentially allows for access to great product diversity. The synthesized compounds were tested for their ability to activate peroxisome proliferator-activated receptors (PPARs) and they all acted as PPARγ agonists in the μM range spanning from 2.5- to 14.7-fold activation of the receptor. This is the first discovery of bioactive molecules based on the arylopeptoid architecture.