Protein O-Mannosylation in the Murine Brain: Occurrence of Mono-O-Mannosyl Glycans and Identification of New Substrates

Protein O-mannosylation is a post-translational modification essential for correct development of mammals. In humans, deficient O-mannosylation results in severe congenital muscular dystrophies often associated with impaired brain and eye development. Although various O-mannosylated proteins have been identified in the recent years, the distribution of O-mannosyl glycans in the mammalian brain and target proteins are still not well defined. In the present study, rabbit monoclonal antibodies directed against the O-mannosylated peptide YAT(α1-Man)AV were generated. Detailed characterization of clone RKU-1-3-5 revealed that this monoclonal antibody recognizes O-linked mannose also in different peptide and protein contexts. Using this tool, we observed that mono-O-mannosyl glycans occur ubiquitously throughout the murine brain but are especially enriched at inhibitory GABAergic neurons and at the perineural nets. Using a mass spectrometry-based approach, we further identified glycoproteins from the murine brain that bear single O-mannose residues. Among the candidates identified are members of the cadherin and plexin superfamilies and the perineural net protein neurocan. In addition, we identified neurexin 3, a cell adhesion protein involved in synaptic plasticity, and inter-alpha-trypsin inhibitor 5, a protease inhibitor important in stabilizing the extracellular matrix, as new O-mannosylated glycoproteins.     

Application of a zona pellucida binding assay (ZBA) in the domestic cat benefits from the use of in vitro matured oocytes

Background

Zona pellucida binding assays (ZBAs) have proven useful in determining the fertilising ability of spermatozoa in several species. Most ZBAs use fresh or salt-stored oocytes collected from fresh ovaries but because ovaries are not easy to obtain on a regular basis, chilled and frozen-thawed ovaries have been tested, with varying results. The present study tested the hypothesis that cat spermatozoa, either fresh or frozen-thawed, can bind to homologous zona pellucida of oocytes retrieved from frozen-thawed queen ovaries to a similar extent as they can bind to the zona pellucida of fresh, in vitro matured oocytes.

Methods

Ovaries were collected from queens after routine ovario-hysterectomy and either stored in NaCl at -20°C until use (treatment animals), or used fresh (controls). Cumulus-oocyte complexes (COCs) were retrieved by ovarian slicing from either source and used directly (immature oocytes from frozen-thawed ovaries; treatment animals) or after in vitro maturation (IVM) (fresh ovaries; controls) for 24 hours in TCM 199, supplemented with 1 IU hCG/mL and 0.5 IU eCG/mL and 0.5% bovine serum albumin (BSA). The oocytes were incubated for 4 hours in 5% CO₂ in air at 38°C and 100% humidity in the presence of 5 × 10⁶ fresh or frozen-thawed spermatozoa/mL. Representative samples of oocytes were processed for scanning electron microscopy (SEM).

Results

Both fresh and frozen-thawed spermatozoa bound to the in vitro matured zona pellucida but significantly fewer, or no, spermatozoa bound to frozen-thawed, immature zona pellucida (P < 0.001). Also, more fresh spermatozoa than frozen-thawed spermatozoa bound to the zona pellucida (P < 0.001). The zona pellucida surface differed in morphology (SEM), with in vitro matured oocytes showing a dense surface with few fenestrations in contrast to their frozen-thawed, immature counterparts, where fenestrations were conspicuously larger.

Conclusion

In conclusion, under the conditions of the present study, immature oocytes recovered from ovaries frozen immersed in NaCl at -20°C are less suitable for use in feline ZBA.     

An increase in pH boosts olfactory communication in sticklebacks

Human-induced eutrophication is a serious environmental problem that constrains visual communication and influences the mate choice process in fishes. Eutrophication also changes the chemical environment and the pH of the water, which could influence the use of olfactory cues in mate choice. Here, we show that an increase in pH enhances the use of male olfactory cues in mate choice in three-spined sticklebacks, Gasterosteus aculeatus. In a laboratory choice experiment, gravid females were more attracted to male olfactory cues when pH was raised. This could compensate for impaired visual communication in eutrophied waters and facilitate adaptive mate choice.     

Selection for novel metabolic capabilities in Salmonella enterica

Bacteria are known to display extensive metabolic diversity and many studies have shown that they can use an extensive repertoire of small molecules as carbon‐ and energy sources. However, it is less clear to what extent a bacterium can expand its existing metabolic capabilities by acquiring mutations that, for example, rewire its metabolic pathways. To investigate this capability and potential for evolution of novel phenotypes, we sampled large populations of mutagenized Salmonella enterica to select very rare mutants that can grow on minimal media containing 124 low molecular weight compounds as sole carbon sources. We found mutants growing on 18 of these novel carbon sources, and identified the causal mutations that allowed growth for four of them. Mutations that relieve physiological constraints or increase expression of existing pathways were found to be important contributors to the novel phenotypes. For the remaining 14 novel phenotypes, whole genome sequencing of independent mutants and genetic analysis suggested that these novel metabolic phenotypes result from a combination of multiple mutations. This work, by virtue of identifying the genetic and mechanistic basis for new metabolic capabilities, sheds light on the properties of adaptive landscapes underlying the evolution of novel phenotypes.     

Sexual selection for bright females prevails under light pollution

The functions of sexually selected traits are particularly sensitive to changes in the environment because the traits have evolved to in-crease mating success u...     

Assessment of Metabolomic and Proteomic Biomarkers in Detection and Prognosis of Progression of Renal Function in Chronic Kidney Disease

Chronic kidney disease (CKD) is part of a number of systemic and renal diseases and may reach epidemic proportions over the next decade. Efforts have been made to improve diagnosis and management of CKD. We hypothesised that combining metabolomic and proteomic approaches could generate a more systemic and complete view of the disease mechanisms. To test this approach, we examined samples from a cohort of 49 patients representing different stages of CKD. Urine samples were analysed for proteomic changes using capillary electrophoresis-mass spectrometry and urine and plasma samples for metabolomic changes using different mass spectrometry-based techniques. The training set included 20 CKD patients selected according to their estimated glomerular filtration rate (eGFR) at mild (59.9±16.5 mL/min/1.73 m²; n = 10) or advanced (8.9±4.5 mL/min/1.73 m²; n = 10) CKD and the remaining 29 patients left for the test set. We identified a panel of 76 statistically significant metabolites and peptides that correlated with CKD in the training set. We combined these biomarkers in different classifiers and then performed correlation analyses with eGFR at baseline and follow-up after 2.8±0.8 years in the test set. A solely plasma metabolite biomarker-based classifier significantly correlated with the loss of kidney function in the test set at baseline and follow-up (ρ = −0.8031; p<0.0001 and ρ = −0.6009; p = 0.0019, respectively). Similarly, a urinary metabolite biomarker-based classifier did reveal significant association to kidney function (ρ = −0.6557; p = 0.0001 and ρ = −0.6574; p = 0.0005). A classifier utilising 46 identified urinary peptide biomarkers performed statistically equivalent to the urinary and plasma metabolite classifier (ρ = −0.7752; p<0.0001 and ρ = −0.8400; p<0.0001). The combination of both urinary proteomic and urinary and plasma metabolic biomarkers did not improve the correlation with eGFR. In conclusion, we found excellent association of plasma and urinary metabolites and urinary peptides with kidney function, and disease progression, but no added value in combining the different biomarkers data.     

Influence of chronic and acute spinal cord injury on skeletal muscle Na+-K+-ATPase and phospholemman expression in humans

Na(+)-K(+)-ATPase is an integral membrane protein crucial for the maintenance of ion homeostasis and skeletal muscle contractibility. Skeletal muscle Na(+)-K(+)-ATPase content displays remarkable plasticity in response to long-term increase in physiological demand, such as exercise training. However, the adaptations in Na(+)-K(+)-ATPase function in response to a suddenly decreased and/or habitually low level of physical activity, especially after a spinal cord injury (SCI), are incompletely known. We tested the hypothesis that skeletal muscle content of Na(+)-K(+)-ATPase and the associated regulatory proteins from the FXYD family is altered in SCI patients in a manner dependent on the severity of the spinal cord lesion and postinjury level of physical activity. Three different groups were studied: 1) six subjects with chronic complete cervical SCI, 2) seven subjects with acute, complete cervical SCI, and 3) six subjects with acute, incomplete cervical SCI. The individuals in groups 2 and 3 were studied at months 1, 3, and 12 postinjury, whereas individuals with chronic SCI were compared with an able-bodied control group. Chronic complete SCI was associated with a marked decrease in [(3)H]ouabain binding site concentration in skeletal muscle as well as reduced protein content of the α(1)-, α(2)-, and β(1)-subunit of the Na(+)-K(+)-ATPase. In line with this finding, expression of the Na(+)-K(+)-ATPase α(1)- and α(2)-subunits progressively decreased during the first year after complete but not after incomplete SCI. The expression of the regulatory protein phospholemman (PLM or FXYD1) was attenuated after complete, but not incomplete, cervical SCI. In contrast, FXYD5 was substantially upregulated in patients with complete SCI. In conclusion, the severity of the spinal cord lesion and the level of postinjury physical activity in patients with SCI are important factors controlling the expression of Na(+)-K(+)-ATPase and its regulatory proteins PLM and FXYD5.     

Effects of ammonium sulphate application on the rhizosphere,fine-root and needle chemistry in aPicea abies (L.) Karst. stand

Rhizosphere, fine-root and needle chemistry were investigated in a 28 year old Norway spruce stand in SW Sweden. The uptake and allocation pattern of plant nutrients and aluminium in control plots (C) and plots repeatedly treated with ammonium sulphate (NS) were compared. Treatments started in 1988. Current year needles, one-year-old needles and cylindrical core samples of the LFH-layer and the mineral soil layers were sampled in 1988, 1989 and 1990. Compared to the control plots, pH decreased significantly in the rhizosphere soil in the NS plots in 1989 and 1990 while the SO₄-S concentration increased significantly. Aluminium concentration in the rhizosphere soil was generally higher in the NS plots in all soil layers, except at 0–10 cm depths, both in 1989 and 1990. Calcium, Mg and K concentrations also increased after treatment with ammonium sulphate. Ammonium ions may have replaced these elements in the soil organic matter. The NS treatment significantly reduced Mg concentrations in fine roots in all layers in 1990. A similar trend was found in the needles. Ca concentrations in fine roots were significantly lower in the NS plots in the LFH layer in 1990 and the same pattern was found in the current needles. The N and S concentrations of both fine roots and needles were significantly higher in the NS plots. It was suggested that NS treatment resulted in displacement of Mg, Ca and K from exchange sites in the LFH layer leading to leaching of these cations to the mineral soil. Further application of ammonium sulphate may damage the fine roots and consequently adversely affect the water and nutrient uptake of root systems.     

The complex barnacle perfume: identification of waterborne pheromone homologues in Balanus improvisus and their differential expression during settlement

A key question in barnacle biology is the nature of cues that induce gregarious settlement. One of the characterised cues is the waterborne settlement pheromone (WSP). This study aimed to identify WSP homologues in Balanus improvisus and to investigate their expression during settlement. Six WSP homologues were identified, all containing an N-terminal signal peptide, a conserved core region, and a variable C-terminus comprising several -GR- and -HDDH- motifs. The B. improvisus WSP homologues were expressed in all settlement stages but showed different expression patterns. The homologue most similar to the B. amphitrite WSP was the most abundant and was constantly expressed during settlement. In contrast, several of the other WSP homologues showed the greatest expression in the juvenile stage. The presence of several WSP homologues suggests the existence of a pheromone mix, where con-specificity might be determined by a combination of sequence characteristics and the concentration of the individual components.     

Type 2 Endoleaks: The Diagnostic Performance of Non-Specialized Readers on Arterial and Venous Phase Multi-Slice CT Angiography

PurposeTo define the diagnostic precision of non-specialized readers in the detection of type 2 endoleaks (T2EL) in arterial versus venous phase acquisitions, and to evaluate an approach for radiation dose reduction.MethodsThe pre-discharge and final follow-up multi-slice CT angiographies of 167 patients were retrospectively analyzed. Image data were separated into an arterial and a venous phase reading set. Two radiology residents assessed the reading sets for the presence of a T2EL, feeding vessels, and aneurysm sac size. Findings were compared with a standard of reference established by two experts in interventional radiology. The effective dose was calculated.ResultsOverall, experts detected 131 T2ELs, and 331 feeding vessels in 334 examinations. Persistent T2ELs causing aneurysm sac growth > 5 mm were detected in 20 patients. Radiation in arterial and venous phases contributed to a mean of 58.6% and 39.0% of the total effective dose. Findings of reader 1 and 2 showed comparable sensitivities in arterial sets of 80.9 versus 85.5 (p = 0.09), and in venous sets of 73.3 versus 79.4 (p = 0.15), respectively. Reader 1 and 2 achieved a significant higher detection rate of feeding vessels with arterial compared to venous set (p = 0.04, p < 0.01). Both readers correctly identified T2ELs with growing aneurysm sac in all cases, independent of the acquisition phase.ConclusionArterial acquisitions enable non-specialized readers an accurate detection of T2ELs, and a significant better identification of feeding vessels. Based on our results, it seems reasonable to eliminate venous phase acquisitions.