Reduction in animal abundance and oxygen availability during and after the end-Triassic mass extinction

The end-Triassic biodiversity crisis was one of the most severe mass extinctions in the history of animal life. However, the extent to which the loss of taxonomic diversity was coupled with a reduction in organismal abundance remains to be quantified. Further, the temporal relationship between organismal abundance and local marine redox conditions is lacking in carbonate sections. To address these questions, we measured skeletal grain abundance in shallow-marine limestones by point counting 293 thin sections from four stratigraphic sections across the Triassic/Jurassic boundary in the Lombardy Basin and Apennine Platform of western Tethys. Skeletal abundance decreased abruptly across the Triassic/Jurassic boundary in all stratigraphic sections. The abundance of skeletal organisms remained low throughout the lower-middle Hettangian strata and began to rebound during the late Hettangian and early Sinemurian. A two-way ANOVA indicates that sample age (p < .01, η² = 0.30) explains more of the variation in skeletal abundance than the depositional environment or paleobathymetry (p < .01, η² = 0.15). Measured I/Ca ratios, a proxy for local shallow-marine redox conditions, show this same pattern with the lowest I/Ca ratios occurring in the early Hettangian. The close correspondence between oceanic water column oxygen levels and skeletal abundance indicates a connection between redox conditions and benthic organismal abundance across the Triassic/Jurassic boundary. These findings indicate that the end-Triassic mass extinction reduced not only the biodiversity but also the carrying capacity for skeletal organisms in early Hettangian ecosystems, adding to evidence that mass extinction of species generally leads to mass rarity among survivors.     

A study of intermediates involved in the folding pathway for recombinant human macrophage colony-stimulating factor (M-CSF): evidence for two distinct folding pathways.

The folding pathway for a 150-amino acid recombinant form of the dimeric cytokine human macrophage colony-stimulating factor (M-CSF) has been studied. All 14 cysteine residues in the biologically active homodimer are involved in disulfide linkages. The structural characteristics of folding intermediates blocked with iodoacetamide reveal a rapid formation of a small amount of a non-native dimeric intermediate species followed by a slow progression via both monomeric and dimeric intermediates to the native dimer. The transition from monomer to fully folded dimer is complete within 25 h at room temperature at pH 9.0. The blocked intermediates are stable under conditions of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and thus represent various dimeric and folded monomeric species of the protein with different numbers of disulfide bridges. Peptide mapping and electrospray ionization mass spectrometry revealed that a folded monomeric species of M-CSF contained three of the four native disulfide bridges, and this folded monomer also showed some biological activity in a cell-based assay. The results presented here strongly suggest that M-CSF can fold via two different pathways, one involving monomeric intermediates and another involving only dimeric intermediates.     

Determination of amide hydrogen exchange by mass spectrometry: a new tool for protein structure elucidation.

A new method based on protein fragmentation and directly coupled microbore high-performance liquid chromatography-fast atom bombardment mass spectrometry (HPLC-FABMS) is described for determining the rates at which peptide amide hydrogens in proteins undergo isotopic exchange. Horse heart cytochrome c was incubated in D2O as a function of time and temperature to effect isotopic exchange, transferred into slow exchange conditions (pH 2-3, 0 degrees C), and fragmented with pepsin. The number of peptide amide deuterons present in the proteolytic peptides was deduced from their molecular weights, which were determined following analysis of the digest by HPLC-FABMS. The present results demonstrate that the exchange rates of amide hydrogens in cytochrome c range from very rapid (k > 140 h-1) to very slow (k < 0.002 h-1). The deuterium content of specific segments of the protein was determined as a function of incubation temperature and used to indicate participation of these segments in conformational changes associated with heating of cytochrome c. For the present HPLC-FABMS system, approximately 5 nmol of protein were used for each determination. Results of this investigation indicate that the combination of protein fragmentation and HPLC-FABMS is relatively free of constraints associated with other analytical methods used for this purpose and may be a general method for determining hydrogen exchange rates in specific segments of proteins.     

Primary structure of a photoactive yellow protein from the phototrophic bacterium Ectothiorhodospira halophila,with evidence for the mass and the binding site of the chromophore.

The complete amino acid sequence of the 125-residue photoactive yellow protein (PYP) from Ectothiorhodospira halophila has been determined to be MEHVAFGSEDIENTLAKMDDGQLDGLAFGAIQLDGDGNILQYNAAEGDITGRDPKEVIGKNFFKDVAP+ ++ CTDSPEFYGKFKEGVASGNLNTMFEYTFDYQMTPTKVKVHMKKALSGDSYWVFVKRV. This is the first sequence to be reported for this class of proteins. There is no obvious sequence homology to any other protein, although the crystal structure, known at 2.4 A resolution (McRee, D.E., et al., 1989, Proc. Natl. Acad. Sci. USA 86, 6533-6537), indicates a relationship to the similarly sized fatty acid binding protein (FABP), a representative of a family of eukaryotic proteins that bind hydrophobic molecules. The amino acid sequence exhibits no greater similarity between PYP and FABP than for proteins chosen at random (8%). The photoactive yellow protein contains an unidentified chromophore that is bleached by light but recovers within a second. Here we demonstrate that the chromophore is bound covalently to Cys 69 instead of Lys 111 as deduced from the crystal structure analysis. The partially exposed side chains of Tyr 76, 94, and 118, plus Trp 119 appear to be arranged in a cluster and probably become more exposed due to a conformational change of the protein resulting from light-induced chromophore bleaching. The charged residues are not uniformly distributed on the protein surface but are arranged in positive and negative clusters on opposite sides of the protein. The exact chemical nature of the chromophore remains undetermined, but we here propose a possible structure based on precise mass analysis of a chromophore-binding peptide by electrospray ionization mass spectrometry and on the fact that the chromophore can be cleaved off the apoprotein upon reduction with a thiol reagent. The molecular mass of the chromophore, including an SH group, is 147.6 Da (+/- 0.5 Da); the cysteine residue to which it is bound is at sequence position 69.     

聚苯乙烯纳米塑料-植物蛋白冠的形成与特征

为探究聚苯乙烯纳米塑料-植物蛋白冠的形成过程以及蛋白冠的形成对植物可能造成的影响,本研究选用3种平均粒径为200nm不同表面修饰的聚苯乙烯纳米塑料微球和新几内亚凤仙(Impatiens hawkeri)为对象,将3种聚苯乙烯纳米塑料分别与新几内亚凤仙的叶蛋白提取物进行反应,反应时间分别为2、4、8、16、24、36 h。利用扫描电镜(scanning electron microscopy, SEM)观察其形貌变化,原子力显微镜(atomic force microscopy, AFM)进行表面粗糙度测定,使用纳米粒度和zeta电位分析仪测定水合粒径及zeta电位,液相色谱-串联质谱(liquid chromatography-tandem mass spectrometry, LC-MS/MS)鉴定蛋白冠的蛋白成分。从生物学过程、细胞组分以及分子功能3个方面对蛋白进行分类,研究不同表面修饰的纳米塑料对蛋白的吸附选择,探究聚苯乙烯纳米塑料-植物蛋白冠的形成与特征,预测蛋白冠对植物造成的可能影响。结果表明：随着反应时间增加,纳米塑料的形貌变化越发明显,表现为尺寸和粗糙度的增加和稳定性的增...     

Measurement of trace elements in murine liver tissue samples: Comparison between ICP-MS/MS and TXRF

BackgroundTrace elements exhibit essential functions in many physiological processes. Thus, for research focusing on trace element homeostasis and metabolism analytical methods allowing for multi-element analyses are fundamental. Small sample amounts may be a big challenge in trace element analyses especially if also other end points want to be addressed in the same sample. Therefore, the aim of the present study was to examine trace elements (iron, copper, zinc, and selenium) in murine liver tissue prepared by a RIPA buffer-based lyses method.Methods and resultsAfter centrifugation, lysates and pellets were obtained and trace elements were analyzed with TXRF in liver lysates. The results were compared to that obtained by a standard microwave-assisted acidic digestion with subsequent ICP-MS/MS analysis of the same liver tissue, liver lysates, and remaining pellets. In addition, trace element concentrations, determined in murine serum with both methods, were compared. For serum samples, both TXRF and ICP-MS/MS provide similar and highly correlating results. Furthermore, in liver lysate samples prepared with RIPA buffer, comparable trace element concentrations were measured by TXRF as with the standard digestion technique and ICP-MS/MS. Only marginal amounts of trace elements were detected in the pellets.ConclusionTaken together, the results obtained by the present study indicate that the RIPA buffer-based method is suitable for sample preparation for trace element analyses via TXRF, at least for the here investigated murine liver samples.     

Protein structure dynamics by crosslinking mass spectrometry

Crosslinking mass spectrometry captures protein structures in solution. The crosslinks reveal spatial proximities as distance restraints, but do not easily reveal which of these restraints derive from the same protein conformation. This superposition can be reduced by photo-crosslinking, and adding information from protein structure models, or quantitative crosslinking reveals conformation-specific crosslinks. As a consequence, crosslinking MS has proven useful already in the context of multiple dynamic protein systems. We foresee a breakthrough in the resolution and scale of studying protein dynamics when crosslinks are used to guide deep-learning-based protein modelling. Advances in crosslinking MS, such as photoactivatable crosslinking and in-situ crosslinking, will then reveal protein conformation dynamics in the cellular context, at a pseudo-atomic resolution, and plausibly in a time-resolved manner.     

Phosphorylation states greatly regulate the activity and gating properties of Cav3.1 T-type Ca2+ channels

Ca_v3.1 T-type Ca²⁺ channels play pivotal roles in neuronal low-threshold spikes, visceral pain, and pacemaker activity. Phosphorylation has been reported to potently regulate the activity and gating properties of Ca_v3.1 channels. However, systematic identification of phosphorylation sites (phosphosites) in Ca_v3.1 channel has been poorly investigated. In this work, we analyzed rat Ca_v3.1 protein expressed in HEK-293 cells by mass spectrometry, identified 30 phosphosites located at the cytoplasmic regions, and illustrated them as a Ca_v3.1 phosphorylation map which includes the reported mouse Ca_v3.1 phosphosites. Site-directed mutagenesis of the phosphosites to Ala residues and functional analysis of the phospho-silent Ca_v3.1 mutants expressed in Xenopus oocytes showed that the phospho-silent mutation of the N-terminal Ser18 reduced its current amplitude with accelerated current kinetics and negatively shifted channel availability. Remarkably, the phospho-silent mutations of the C-terminal Ser residues (Ser1924, Ser2001, Ser2163, Ser2166, or Ser2189) greatly reduced their current amplitude without altering the voltage-dependent gating properties. In contrast, the phosphomimetic Asp mutations of Ca_v3.1 on the N- and C-terminal Ser residues reversed the effects of the phospho-silent mutations. Collectively, these findings demonstrate that the multiple phosphosites of Ca_v3.1 at the N- and C-terminal regions play crucial roles in the regulation of the channel activity and voltage-dependent gating properties.     

The challenge of estimating kelp production in a turbid marine environment

Coastal kelp forests produce substantial marine carbon due to high annual net primary production (NPP) rates, but upscaling of NPP estimates over time and space remains difficult. We investigated the impact of variable underwater photosynthetically active radiation (PAR) and photosynthetic parameters on photosynthetic oxygen production of Laminaria hyperborea, the dominant NE-Atlantic kelp species, throughout summer 2014. Collection depth of kelp had no effect on chlorophyll a content, pointing to a high photoacclimation potential of L. hyperborea towards incident light. However, chlorophyll a and photosynthesis versus irradiance parameters differed significantly along the blade gradient when normalized to fresh mass, potentially introducing large uncertainties in NPP upscaling to whole thalli. Therefore, we recommend a normalization to kelp tissue area, which is stable over the blade gradient. Continuous PAR measurements revealed a highly variable underwater light climate at our study site (Helgoland, North Sea) in summer 2014, reflected by PAR attenuation coefficients (K_d) between 0.28 and 0.87 m⁻¹. Our data highlight the importance of continuous underwater light measurements or representative average values using a weighted K_d to account for large PAR variability in NPP calculations. Strong winds in August increased turbidity, resulting in a negative carbon balance at depths >3–4 m over several weeks, considerably impacting kelp productivity. Estimated daily summer NPP over all four depths was 1.48 ± 0.97 g C · m⁻² seafloor · d⁻¹ for the Helgolandic kelp forest, which is in the range of other kelp forests along European coastlines.     

Recovery of aSasa tsuboiana population after mass flowering and death

The recovery process of aSasa tsuboiana population after a mass flowering and death in 1977 was investigated by 15 years of observation in the Hira Mountains, Kinki district, western Japan. Seed production was high (6600–13 800 seeds m⁻² inSasa plots and 3900 seeds m⁻² in a forest plot) but emergent seedling density was low (14–21 seedlings m⁻²), probably because of seed predation byMicrotus montebelli occurring between seed shedding and the next spring. The seedling density had decreased further by the next year and theS. tsuboiana population recovered from only a limited number of seedlings. In spite of such a low initial density, theS. tsuboiana population was able to regenerate successfully and attained the previous full stand height in 7–16 years.Miscantbus sinensis invaded and delayed the recovery ofS. tsuboiana in one plot, butS. tsuboiana became dominant as it caught up with the height ofM. sinensis. Seedling growth patterns, such as frequent tillering, the onset of rhizome extension in the early stage of seedling growth and frequent culm production from rhizomes, played important roles in the successful regeneration ofS. tsuboiana.