From Gigabyte to Kilobyte: A Bioinformatics Protocol for Mining Large RNA-Seq Transcriptomics Data

RNA-Seq techniques generate hundreds of millions of short RNA reads using next-generation sequencing (NGS). These RNA reads can be mapped to reference genomes to investigate changes of gene expression but improved procedures for mining large RNA-Seq datasets to extract valuable biological knowledge are needed. RNAMiner—a multi-level bioinformatics protocol and pipeline—has been developed for such datasets. It includes five steps: Mapping RNA-Seq reads to a reference genome, calculating gene expression values, identifying differentially expressed genes, predicting gene functions, and constructing gene regulatory networks. To demonstrate its utility, we applied RNAMiner to datasets generated from Human, Mouse, Arabidopsis thaliana, and Drosophila melanogaster cells, and successfully identified differentially expressed genes, clustered them into cohesive functional groups, and constructed novel gene regulatory networks. The RNAMiner web service is available at http://calla.rnet.missouri.edu/rnaminer/index.html.     

Mutations in CSPP1 Cause Primary Cilia Abnormalities and Joubert Syndrome with or without Jeune Asphyxiating Thoracic Dystrophy

Joubert syndrome (JBTS) is a recessive ciliopathy in which a subset of affected individuals also have the skeletal dysplasia Jeune asphyxiating thoracic dystrophy (JATD). Here, we have identified biallelic truncating CSPP1 (centrosome and spindle pole associated protein 1) mutations in 19 JBTS-affected individuals, four of whom also have features of JATD. CSPP1 mutations explain ∼5% of JBTS in our cohort, and despite truncating mutations in all affected individuals, the range of phenotypic severity is broad. Morpholino knockdown of cspp1 in zebrafish caused phenotypes reported in other zebrafish models of JBTS (curved body shape, pronephric cysts, and cerebellar abnormalities) and reduced ciliary localization of Arl13b, further supporting loss of CSPP1 function as a cause of JBTS. Fibroblasts from affected individuals with CSPP1 mutations showed reduced numbers of primary cilia and/or short primary cilia, as well as reduced axonemal localization of ciliary proteins ARL13B and adenylyl cyclase III. In summary, CSPP1 mutations are a major cause of the Joubert-Jeune phenotype in humans; however, the mechanism by which these mutations lead to both JBTS and JATD remains unknown.     

DO MALES MATTER? TESTING THE EFFECTS OF MALE GENETIC BACKGROUND ON FEMALE MEIOTIC CROSSOVER RATES IN DROSOPHILA MELANOGASTER

Meiotic recombination is a critical genetic process as well as a pivotal evolutionary force. Rates of crossing over are highly variable within and between species, due to both genetic and environmental factors. Early studies in Drosophila implicated female genetic background as a major determinant of crossover rate and recent work has highlighted male genetic background as a possible mediator as well. Our study employed classical genetics to address how female and male genetic backgrounds individually and jointly affect crossover rates. We measured rates of crossing over in a 33 cM region of the Drosophila melanogaster X chromosome using a two‐step crossing scheme exploiting visible markers. In total, we measured crossover rates of 10 inbred lines in a full diallel cross. Our experimental design facilitates measuring the contributions of female genetic background, male genetic background, and female by male genetic background interaction effects on rates of crossing over in females. Our results indicate that although female genetic background significantly affects female meiotic crossover rates in Drosophila, male genetic background and the interaction of female and male genetic backgrounds have no significant effect. These findings thus suggest that male‐mediated effects are unlikely to contribute greatly to variation in recombination rates in natural populations of Drosophila.     

High resolution <Superscript>13</Superscript>C-detected solid-state NMR spectroscopy of a deuterated protein

High resolution ¹³C-detected solid-state NMR spectra of the deuterated beta-1 immunoglobulin binding domain of the protein G (GB1) have been collected to show that all ¹⁵N, ¹³C′, ¹³Cα and ¹³Cβ sites are resolved in ¹³C–¹³C and ¹⁵N–¹³C spectra, with significant improvement in T ₂ relaxation times and resolution at high magnetic field (750 MHz). The comparison of echo T ₂ values between deuterated and protonated GB1 at various spinning rates and under different decoupling schemes indicates that ¹³Cα T ₂′ times increase by almost a factor of two upon deuteration at all spinning rates and under moderate decoupling strength, and thus the deuteration enables application of scalar-based correlation experiments that are challenging from the standpoint of transverse relaxation, with moderate proton decoupling. Additionally, deuteration in large proteins is a useful strategy to selectively detect polar residues that are often important for protein function and protein–protein interactions.     

Intramuscular adaptations to eccentric exercise and antioxidant supplementation

Prophylactic supplementation of N-acetyl-cysteine (NAC) and epigallocatechin gallate (EGCG) was studied for physiological and cellular changes in skeletal muscle after eccentric muscle contractions. Thirty healthy, active males (20.0 ± 1.8 years, 160 ± 7.1 cm, 76.1 ± 17.0 kg) ingested for 14 days either 1,800 mg of NAC, 1,800 mg of EGCG, or 1,000 mg of fiber (glucomannan) placebo (PLC) in a double blind, prophylactic fashion. Subjects completed one eccentric exercise bout (100 repetitions at 30°/s) using the dominant knee extensors. Strength and soreness were assessed, and blood and muscle samples obtained before and 6, 24, 48, and 72 h with no muscle sample being collected at 72 h. Separate mixed factorial repeated measures ANOVA (P < 0.05) were used for all statistical analysis. All groups experienced significantly reduced peak torque production after 6 and 24 h, increased soreness at all time points from baseline [with even greater soreness levels 24 h after exercise in PLC when compared to EGCG and NAC (P < 0.05)], increased lactate dehydrogenase at 6 h, and increased creatine kinase 6, 24 and 48 h after exercise. No significant group × time interaction effects were found for serum cortisol, neutrophil counts, and the neutrophil:lymphocyte ratio; although, all values experienced significant changes 6 h after exercise (P < 0.05), but at no other time points. At 48 h after the exercise bout the Neu:Lym ratio in EGCG was significantly less than NAC (P < 0.05), whereas there was a trend (P = 0.08) for the EGCG values to be less when compared to PLC at this time point. Markers of intramuscular mitochondrial and cytosolic apoptosis were assessed (e.g., bax, bcl-2, cytochrome C, caspase-3 content/enzyme activity, and total DNA content). Significant increases (P < 0.05) in muscle levels of bax and bcl-2 were observed in all groups with no significant differences between groups, whereas no changes (P > 0.05) were reported for cytochrome C, caspase-3 content, caspase-3 enzyme activity, and total DNA. Caspase-3 enzyme activity was significantly greater in all groups 48 h after exercise when compared to baseline (P < 0.05) and 6 h (P < 0.05) after exercise. An eccentric bout of muscle contractions appears to significantly increase muscle damage, markers of mitochondrial apoptosis, apoptotic enzyme activity, and whole-blood cell markers of inflammation with no changes in oxidative stress. While soreness ratings were blunted in the two supplementation groups 24 h after exercise when compared to PLC values, more research is needed to determine the potential impact of EGCG and NAC supplementation on changes related to oxidative stress, apoptosis, and eccentric exercise.     

Determination of protein complex stoichiometry through multisignal sedimentation velocity experiments

Determination of the stoichiometry of macromolecular assemblies is fundamental to an understanding of how they function. Many different biophysical methodologies may be used to determine stoichiometry. In the past, both sedimentation equilibrium and sedimentation velocity analytical ultracentrifugation have been employed to determine component stoichiometries. Recently, a method of globally analyzing multisignal sedimentation velocity data was introduced by Schuck and coworkers. This global analysis removes some of the experimental inconveniences and inaccuracies that could occur in the previously used strategies. This method uses spectral differences between the macromolecular components to decompose the well-known c(s) distribution into component distributions c_k(s); that is, each component k has its own c_k(s) distribution. Integration of these distributions allows the calculation of the populations of each component in cosedimenting complexes, yielding their stoichiometry. In our laboratories, we have used this method extensively to determine the component stoichiometries of several protein-protein complexes involved in cytoskeletal remodeling, sugar metabolism, and host-pathogen interactions. The overall method is described in detail in this work, as are experimental examples and caveats.     

Phenothiazine-mediated rescue of cognition in tau transgenic mice requires neuroprotection and reduced soluble tau burden

Background

It has traditionally been thought that the pathological accumulation of tau in Alzheimer's disease and other tauopathies facilitates neurodegeneration, which in turn leads to cognitive impairment. However, recent evidence suggests that tau tangles are not the entity responsible for memory loss, rather it is an intermediate tau species that disrupts neuronal function. Thus, efforts to discover therapeutics for tauopathies emphasize soluble tau reductions as well as neuroprotection.

Results

Here, we found that neuroprotection alone caused by methylene blue (MB), the parent compound of the anti-tau phenothiaziazine drug, Rember?, was insufficient to rescue cognition in a mouse model of the human tauopathy, progressive supranuclear palsy (PSP) and fronto-temporal dementia with parkinsonism linked to chromosome 17 (FTDP17): Only when levels of soluble tau protein were concomitantly reduced by a very high concentration of MB, was cognitive improvement observed. Thus, neurodegeneration can be decoupled from tau accumulation, but phenotypic improvement is only possible when soluble tau levels are also reduced.

Conclusions

Neuroprotection alone is not sufficient to rescue tau-induced memory loss in a transgenic mouse model. Development of neuroprotective agents is an area of intense investigation in the tauopathy drug discovery field. This may ultimately be an unsuccessful approach if soluble toxic tau intermediates are not also reduced. Thus, MB and related compounds, despite their pleiotropic nature, may be the proverbial "magic bullet" because they not only are neuroprotective, but are also able to facilitate soluble tau clearance. Moreover, this shows that neuroprotection is possible without reducing tau levels. This indicates that there is a definitive molecular link between tau and cell death cascades that can be disrupted.

     

Direct and indirect food web regulation of microbial decomposers in headwater streams

The direct and indirect regulation of primary productivity has been well established in autotrophic‐based ecosystems; however, less is known about the processes affecting decomposers in detrital‐based ecosystems. Because, small headwater, woodland streams are a dominate feature in most ecosystems and are tightly linked to terrestrial detritus, understanding decomposer‐mediated functions in these systems is critical for understanding carbon processes across the landscape. In this light, we conducted a microcosm and mesocosm experiment to test the direct and indirect food web effects on decomposers in small stream ecosystems. The results from the microcosm experiment supported an existing literature, demonstrating that nutrients directly stimulate decomposers and that microbivores directly reduce decomposers. Based on well‐founded food web theory in autotrophic systems, we predicted that fishes from different trophic‐functional guilds would indirectly stimulate decomposers by enhancing dissolved nutrients and by reducing microbivore densities. Our mesocosm experiment partially supported these predictions. Specifically, we found that fishes that consumed mostly terrestrial foods increased decomposers from the bottom–up by enhancing allochthonous nutrient loading into the stream ecosystems. Contrary to our predictions, however, predatory fishes that consume microbivores did not increase decomposers from the top–down. Rather, in streams with the predatory fish species, microbivores increased (rather than decreased) on leaf litter. This may have resulted from an experimental artifact associated with refuge provided by leaf packs. In conclusion, our data demonstrate that decomposers are regulated by similar direct and indirect processes important in autotrophic‐based ecosystems. This provides further evidence that food web processes can regulate leaf decomposition and flux of detrital carbon through ecosystems.     

DnaJ/Hsc70 chaperone complexes control the extracellular release of neurodegenerative‐associated proteins

It is now known that proteins associated with neurodegenerative disease can spread throughout the brain in a prionlike manner. However, the mechanisms regulating the trans‐synaptic spread propagation, including the neuronal release of these proteins, remain unknown. The interaction of neurodegenerative disease‐associated proteins with the molecular chaperone Hsc70 is well known, and we hypothesized that much like disaggregation, refolding, degradation, and even normal function, Hsc70 may dictate the extracellular fate of these proteins. Here, we show that several proteins, including TDP‐43, α‐synuclein, and the microtubule‐associated protein tau, can be driven out of the cell by an Hsc70 co‐chaperone, DnaJC5. In fact, DnaJC5 overexpression induced tau release in cells, neurons, and brain tissue, but only when activity of the chaperone Hsc70 was intact and when tau was able to associate with this chaperone. Moreover, release of tau from neurons was reduced in mice lacking the DnaJC5 gene and when the complement of DnaJs in the cell was altered. These results demonstrate that the dynamics of DnaJ/Hsc70 complexes are critically involved in the release of neurodegenerative disease proteins.