SMN requirement for synaptic vesicle, active zone and microtubule postnatal organization in motor nerve terminals

Low levels of the Survival Motor Neuron (SMN) protein produce Spinal Muscular Atrophy (SMA), a severe monogenetic disease in infants characterized by muscle weakness and impaired synaptic transmission. We report here severe structural and functional alterations in the organization of the organelles and the cytoskeleton of motor nerve terminals in a mouse model of SMA. The decrease in SMN levels resulted in the clustering of synaptic vesicles (SVs) and Active Zones (AZs), reduction in the size of the readily releasable pool (RRP), and the recycling pool (RP) of synaptic vesicles, a decrease in active mitochondria and limiting of neurofilament and microtubule maturation. We propose that SMN is essential for the normal postnatal maturation of motor nerve terminals and that SMN deficiency disrupts the presynaptic organization leading to neurodegeneration.     

Caloric restriction suppresses microglial activation and prevents neuroapoptosis following cortical injury in rats

Traumatic brain injury (TBI) is a widespread cause of death and a major source of adult disability. Subsequent pathological events occurring in the brain after TBI, referred to as secondary injury, continue to damage surrounding tissue resulting in substantial neuronal loss. One of the hallmarks of the secondary injury process is microglial activation resulting in increased cytokine production. Notwithstanding that recent studies demonstrated that caloric restriction (CR) lasting several months prior to an acute TBI exhibits neuroprotective properties, understanding how exactly CR influences secondary injury is still unclear. The goal of the present study was to examine whether CR (50% of daily food intake for 3 months) alleviates the effects of secondary injury on neuronal loss following cortical stab injury (CSI). To this end, we examined the effects of CR on the microglial activation, tumor necrosis factor-α (TNF-α) and caspase-3 expression in the ipsilateral (injured) cortex of the adult rats during the recovery period (from 2 to 28 days) after injury. Our results demonstrate that CR prior to CSI suppresses microglial activation, induction of TNF-α and caspase-3, as well as neurodegeneration following injury. These results indicate that CR strongly attenuates the effects of secondary injury, thus suggesting that CR may increase the successful outcome following TBI.     

Non-linear dynamics in muscle fatigue and strength model during maximal self-perceived elbow extensors training

Our aim was to determine the dynamics in muscle strength increase and fatigue development during repetitive maximal contraction in specific maximal self-perceived elbow extensors training program. We will derive our functional model for m. triceps brachii in spirit of traditional Hill’s two-component muscular model and after fitting our data, develop a prediction tool for this specific training system. Thirty-six healthy young men (21±1.0 y, BMI 25.4±7.2 kg/m²), who did not take part in any formal resistance exercise regime, volunteered for this study. The training protocol was performed on the isoacceleration dynamometer, lasted for 12 weeks, with a frequency of five sessions per week. Each training session included five sets of 10 maximal contractions (elbow extensions) with a 1 min resting period between each set. The non-linear dynamic system model was used for fitting our data in conjunction with the Levenberg–Marquardt regression algorithm. As a proper dynamical system, our functional model of m. triceps brachii can be used for prediction and control. The model can be used for the predictions of muscular fatigue in a single series, the cumulative daily muscular fatigue and the muscular growth throughout the training process. In conclusion, the application of non-linear dynamics in this particular training model allows us to mathematically explain some functional changes in the skeletal muscle as a result of its adaptation to programmed physical activity—training.     

Association of angiotensin-converting enzyme insertion-deletion polymorphism with preeclampsia

The aim of this study was to determine if insertion-deletion polymorphism of angiotensin-converting enzyme is a risk factor for the development of preeclampsia. Sixty women with preeclampsia and 50 normotensive pregnant women were included in this study. Preeclampsia was defined as blood pressure >140/90 mmHg in a previously normotensive women with proteinuria >300 mg/L in a 24-hours. Twelve women also had preeclampsia in previous pregnancy. The genotyping of polymorphism in the intron 16 of the angiotensin-converting enzyme was performed by the polymerase chain reaction followed by the agarose electrophoresis. The patients were divided into three groups according to the presence (I) or absence (D) of insertional polymorphism (II, ID, and DD). Genotype distribution and allele frequencies were compared by Mantel-Haenszel chi2 testing. The frequency of DD genotype was not significantly higher in women with preeclampsia (26/60) than in the control group (14/50, p=0.096). The D allele frequency was significantly higher in 17 women with preeclampsias who required delivery before 34 weeks of pregnancy (0.735), than in 43 women in whom obstetric complications took place after 34 weeks of pregnancy (0.56, p=0.036). The D allele frequency was 0.83 in women having recurrent preeclampsia, i.e. significantly higher compared with women, who were for the first time, experienced preeclampsia (0.57, p=0.013). This study showed a significantly positive association between D allele frequency and risk of recurrent preeclampsia and preterm delivery before 34 weeks of pregnancy. The deletion genotype could be an important contributing factor for an early onset and recurrent preeclampsia.     

Evaluation of Common Methods for Sampling Invertebrate Pollinator Assemblages: Net Sampling Out-Perform Pan Traps

Methods for sampling ecological assemblages strive to be efficient, repeatable, and representative. Unknowingly, common methods may be limited in terms of revealing species function and so of less value for comparative studies. The global decline in pollination services has stimulated surveys of flower-visiting invertebrates, using pan traps and net sampling. We explore the relative merits of these two methods in terms of species discovery, quantifying abundance, function, and composition, and responses of species to changing floral resources. Using a spatially-nested design we sampled across a 5000 km² area of arid grasslands, including 432 hours of net sampling and 1296 pan trap-days, between June 2010 and July 2011. Net sampling yielded 22% more species and 30% higher abundance than pan traps, and better reflected the spatio-temporal variation of floral resources. Species composition differed significantly between methods; from 436 total species, 25% were sampled by both methods, 50% only by nets, and the remaining 25% only by pans. Apart from being less comprehensive, if pan traps do not sample flower-visitors, the link to pollination is questionable. By contrast, net sampling functionally linked species to pollination through behavioural observations of flower-visitation interaction frequency. Netted specimens are also necessary for evidence of pollen transport. Benefits of net-based sampling outweighed minor differences in overall sampling effort. As pan traps and net sampling methods are not equivalent for sampling invertebrate-flower interactions, we recommend net sampling of invertebrate pollinator assemblages, especially if datasets are intended to document declines in pollination and guide measures to retain this important ecosystem service.     

Propofol-induced changes in neurotrophic signaling in the developing nervous system in vivo

Several studies have revealed a role for neurotrophins in anesthesia-induced neurotoxicity in the developing brain. In this study we monitored the spatial and temporal expression of neurotrophic signaling molecules in the brain of 14-day-old (PND14) Wistar rats after the application of a single propofol dose (25 mg/kg i.p). The structures of interest were the cortex and thalamus as the primary areas of anesthetic actions. Changes of the protein levels of the brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF), their activated receptors tropomyosin-related kinase (TrkA and TrkB) and downstream kinases Akt and the extracellular signal regulated kinase (ERK) were assessed by Western immunoblot analysis at different time points during the first 24 h after the treatment, as well as the expression of cleaved caspase-3 fragment. Fluoro-Jade B staining was used to follow the appearance of degenerating neurons. The obtained results show that the treatment caused marked alterations in levels of the examined neurotrophins, their receptors and downstream effector kinases. However, these changes were not associated with increased neurodegeneration in either the cortex or the thalamus. These results indicate that in the brain of PND14 rats, the interaction between Akt/ERK signaling might be one of important part of endogenous defense mechanisms, which the developing brain utilizes to protect itself from potential anesthesia-induced damage. Elucidation of the underlying molecular mechanisms will improve our understanding of the age-dependent component of anesthesia-induced neurotoxicity.     

Absolute Proteome and Phosphoproteome Dynamics during the Cell Cycle of Schizosaccharomyces pombe (Fission Yeast)

To quantify cell cycle-dependent fluctuations on a proteome-wide scale, we performed integrative analysis of the proteome and phosphoproteome during the four major phases of the cell cycle in Schizosaccharomyces pombe. In highly synchronized cells, we identified 3753 proteins and 3682 phosphorylation events and relatively quantified 65% of the data across all phases. Quantitative changes during the cell cycle were infrequent and weak in the proteome but prominent in the phosphoproteome. Protein phosphorylation peaked in mitosis, where the median phosphorylation site occupancy was 44%, about 2-fold higher than in other phases. We measured copy numbers of 3178 proteins, which together with phosphorylation site stoichiometry enabled us to estimate the absolute amount of protein-bound phosphate, as well as its change across the cell cycle. Our results indicate that 23% of the average intracellular ATP is utilized by protein kinases to phosphorylate their substrates to drive regulatory processes during cell division. Accordingly, we observe that phosphate transporters and phosphate-metabolizing enzymes are phosphorylated and therefore likely to be regulated in mitosis.Cell replication involves a complex series of highly regulated and evolutionary conserved events, called the “cell cycle.” Aberrations in the cell cycle have severe implications and can cause cancerous growth. A detailed understanding of the cell cycle and its regulation may identify additional targets for cancer therapy (1–3). The cell cycle has been the subject of previous proteomics studies. Olsen et al. (4) measured the dynamics of thousands of proteins and phosphorylation events across cell cycle phases of HeLa cells, providing insights into the underlying regulatory mechanisms and pointing to a general increase in phosphorylation site occupancy during M phase. In a targeted study, Pagliuca et al. (5) investigated interactors of cyclins E1, A2, and B1 in HeLa cells, revealing key mechanistic links between DNA replication and mitosis.Schizosaccharomyces pombe (fission yeast) is a unicellular organism, which can easily be genetically manipulated and carries many cell cycle features similar to metazoan cells. It is an important model organism to study the cell cycle and its checkpoint controls (6). Recent global proteomics studies of yeasts and their cell cycle (7–13) have mainly focused on Saccharomyces cerevisiae (budding yeast), with only a few studies of fission yeast (14, 15), although the fission yeast cell cycle may be more representative of eukaryotic cell cycles (16). However, attention of the proteomics community toward S. pombe is increasing. Recent proteomics studies covered up to 4087 S. pombe proteins (71% of the predicted proteome) and 1544 phosphoproteins in both asynchronous and synchronized cell cultures (17–22); however, a comprehensive analysis of the S. pombe cell cycle is so far missing.Here, we use high resolution mass spectrometry in combination with stable isotope labeling by amino acids in the cell culture (SILAC)¹ method, termed super-SILAC (23), and intensity-based absolute quantification (iBAQ) (24) to measure relative and absolute dynamics of the proteome and phosphoproteome during the cell cycle of fission yeast. We estimate copy numbers for 3178 S. pombe proteins, and we combine these data with calculated phosphorylation site stoichiometry to estimate the total amount of protein-bound phosphate and its dynamics across the cell cycle. Providing the global absolute dynamics and stoichiometry of proteins and their modifications will be a valuable resource for classical and systems biologists alike.     

Biochemical screening of fetal aneuploidies and neural tube defects by "double-test" in Croatia: a 10 years' experience

The aim of the study is to investigate the efficiency of the second-trimester biochemical screening, with maternal serum alpha-fetoprotein (MS-AFP) and free beta-subunit of human chorionic gonadotropin (free beta-hCG), during the ten-year period. The study included 11,292 of pregnant women between the 15th and 18th gestational week, who underwent screening from November 1996 to December 2006. The risk for trisomy 21 and trisomy 18 were calculated by computer software, based on a model which generated the final risk for fetal aneuploidies from the pregnant woman's a priori age risk and the likelihood ratio of the distribution of the biochemical markers, according to the second-trimester gestation. With the cut-off value of the final risk > or = 1:250, the detection rate for trisomy 21 was 75% (21/28). In women less than or equal to 35, the detection was 57.1% (8/14) and 92.9% (13/14) in those over 35 years, respectively. The detection rate of trisomy 18 was 50% (2/4). The results confirmed that the implementation of double-test, as non-invasive screening for fetal aneuploidies, should be accepted as a complementary method of antenatal care.     

Cheater or mutualist? Novel florivory interaction between nectar‐rich Crotalaria cunninghamii and small mammals

Animals visit flowers to access resources and by moving pollen to conspecific individuals act as pollinators. While biotic pollinators can increase the seed set of plants, other flower visitors can reduce seed set directly by damaging vital reproductive organs and indirectly by affecting the way the plant interacts with subsequent flower visitors. It is, therefore, vital to understand the varied effects of all visitors and not only pollinators on plant fitness, including those visitors that are temporally or spatially rare. We document the first known case of flower visitation by small mammals to Crotalaria cunninghamii (Fabaceae), a plant species morphologically suited to bird pollination. During a rain‐driven resource pulse in the Simpson Desert in 2011, the rodents Mus musculus (Muridae) and Pseudomys hermannsburgensis (Muridae) visited flowers to remove nectar by puncturing the calyx. We investigated the effects of this novel interaction on the reproductive output of C. cunninghamii. Compared with another recent resource pulse in 2007, plants flowering during mammal visitation had five times as many inflorescences per plant, 90% more flowers per inflorescence, and two to three times more nectar per flower, but this nectar was 30% less sugar rich. Concurrently, rodent plagues were up to three times larger during this rain‐driven resource pulse than during a previous pulse in 2007. Up to 75% of flowers had evidence of small mammal florivory, but this was not necessarily destructive, as up to 90% of fruit had the remains of florivory. Through a series of exclusion experiments, we found that small mammal florivory did not directly reduce seed set. We conclude that rain‐driven resource pulses led to a unique combination of events that facilitated the novel florivory interaction. Our findings emphasize the dynamic nature of biotic interactions and the importance of testing the role of all visitors to pollination services.