Pre-analytical factors in clinical proteomics investigations: Impact of ex vivo protein modifications for multiple sclerosis biomarker discovery

Serum proteome investigations have raised an incredible interest in the research of novel molecular biomarker, nevertheless few of the proposed evidences have been translated to the clinical practice. One of the limiting factors has been the lack of generally accepted guidelines for clinical proteomics studies and the lack of a robust analytical and pre-analytical ground for the proposed classification models. Pre-analytical issues may results in a deep impact for biomarker discovery campaign. In this study we present a systematic evaluation of sample storage and sampling conditions for clinical proteomics investigations.We have developed and validated a linear MALDI-TOF-MS protein profiling method to explore the low protein molecular weight region (5–20 kDa) of serum samples. Data normalization and processing was performed using optimise peak detection routine (LIMPIC) able to describe each group under investigation. Data were acquired either from healthy volunteers and from multiple sclerosis patients in order to highlight ex vivo protein profile alteration related to different physio-pathological conditions. Our data showed critical conditions for serum protein profiles depending on storage times and temperatures: 23 °C, 4 °C, ? 20 °C and ? 80 °C. We demonstrated that upon a ? 20 °C short term storage, characteristic degradation profiles are associated with different clinical groups. Protein signals were further identified after preparative HPLC separation by peptide sequencing on a nanoLC-Q-TOF TANDEM mass spectrometer. Apolipoprotein A-IV and complement C3 protein fragments, transthyretin and the oxidized isoforms in different apolipoprotein species represent the major molecular features of such a degradation pattern.     

Sampling of Bemisia tabaci adults using a pre-programmed autonomous pest control robot

An autonomous robot (Cabbot) was built to control insect pests in a plastic greenhouse and utilized to sample adult whiteflies (Bemisia tabaci) on paprika plants (Capsicum annum var. angulosum). To accomplish this, a sampling device consisting of an air compressor for pest agitation, a sticky trap (100 mm × 150 mm) and an image processing system for pest identification were installed on the Cabbot. The sampling precision of the Cabbot (D = 0.16) was higher than that of the sticky trap (D = 0.19) when sampling of adult white flies was conducted in caged pots. The Cabbot could also collect a substantial number of individuals within a short duration (3 minutes). The collection efficiency (i.e., percent of samples collected at least n (1 in this case) individuals to the total sample number) of insects by the Cabbot was markedly high at low population size, showing approximately 30% when the population size was ≤ 16 individuals per plant. The sampling precision and collection efficiency suggested that the Cabbot is effective in local, short-term sampling and could be used for early warning of pest occurrences.     

Sulforaphane improves dysregulated metabolic profile and inhibits leptin-induced VSMC proliferation: Implications toward suppression of neointima formation after arterial injury in western diet-fed obese mice

Sulforaphane (SFN), a dietary phase-2 enzyme inducer that mitigates cellular oxidative stress through nuclear factor erythroid 2-related factor 2 (Nrf2) activation, is known to exhibit beneficial effects in the vessel wall. For instance, it inhibits vascular smooth muscle cell (VSMC) proliferation, a major event in atherosclerosis and restenosis after angioplasty. In particular, SFN attenuates the mitogenic and pro-inflammatory actions of platelet-derived growth factor (PDGF) and tumor necrosis factor-α (TNFα), respectively, in VSMCs. Nevertheless, the vasoprotective role of SFN has not been examined in the setting of obesity characterized by hyperleptinemia and insulin resistance. Using the mouse model of western diet-induced obesity, the present study demonstrates for the first time that subcutaneous delivery of SFN (0.5 mg/Kg/day) for ~ 3 weeks significantly attenuates neointima formation in the injured femoral artery [↓ (decrease) neointima/media ratio by ~ 60%; n = 5–8]. This was associated with significant improvements in metabolic parameters, including ↓ weight gain by ~ 52%, ↓ plasma leptin by ~ 42%, ↓ plasma insulin by ~ 63%, insulin resistance [↓ homeostasis model assessment of insulin resistance (HOMA-IR) index by ~ 73%], glucose tolerance (↓ AUC_GTT by ~ 24%), and plasma lipid profile (e.g., ↓ triglycerides). Under in vitro conditions, SFN significantly decreased leptin-induced VSMC proliferation by ~ 23% (n = 5) with associated diminutions in leptin-induced cyclin D1 expression and the phosphorylation of p70S6kinase and ribosomal S6 protein (n = 3–4). The present findings reveal that, in addition to improving systemic metabolic parameters, SFN inhibits leptin-induced VSMC proliferative signaling that may contribute in part to the suppression of injury-induced neointima formation in diet-induced obesity.     

Rhaetian magneto-biostratigraphy from the Southern Alps (Italy): Constraints on Triassic chronology

New Late Triassic–earliest Jurassic magneto-biostratigraphic data have been obtained from three overlapping sections in the Southern Alps, Italy (Costa Imagna, Brumano, Italcementi Quarry), composed of ~ 520 m of shallow-marine carbonates outcropping in stratigraphic continuity. Characteristic magnetic components of presumed depositional age record a sequence of 9 normal and reverse polarity magnetozones (as defined by at least three stratigraphically superposed samples) linked by conodont and palynofloral evidence from this study and the literature to Rhaetian to Triassic–Jurassic boundary age. This represents a significantly larger number of polarity zones than previously recognized in more condensed Rhaetian sections from the literature, and by inference represents more time. These data are placed in a broader Late Triassic temporal framework by means of correlations to published magneto-biostratigraphic data from the Tethyan marine Pizzo Mondello section and the Newark astronomical polarity time scale (APTS). This framework is consistent with a position of the Norian–Rhaetian boundary (as defined at Brumano and Pizzo Mondello by the first appearance of Misikella posthernsteini) within Newark magnetozones E17r–E19r in the ~ 207–210 Ma time interval, in basic agreement with the position originally estimated in the Newark using pollen biostratigraphy (E18 at 208–209 Ma). This framework is also consistent with the position of the Triassic–Jurassic boundary interval (placed at Italcementi Quarry at the acme of Kraeuselisporites reissingeri coincident with a negative carbon isotope excursion) correlative to just above Newark magnetozone E23r and just below the oldest CAMP lavas dated at ~ 202 Ma. Hence, we estimate the duration of the Rhaetian to be ~ 5.5–8.5 Myr (or even longer if the Triassic–Jurassic boundary is instead placed above the negative carbon isotope excursion as at Kuhjoch, which is the designated GSSP for the base of the Hettangian), and encompassing 9 magnetozones. This duration contrasts with a duration of ~ 2 Myr and only ~ 4 magnetozones in several alternative published magneto-biostratigraphic schemes.     

Stability of oxidative stress biomarkers in flathead mullet,Mugil cephalus,serum during short-term storage

The production of reactive oxygen species (ROS) by aquatic organisms in response to stressful factor is a common feature in aquatic environment. Thus, the evaluation of parameters related to oxidative stress in fish, in first instance those showing the redox potential, is notable to characterize the environmental conditions.Short-term storage of samples is essential when blood must be transported from collection sites to laboratories. Therefore, excessive delays in processing might compromise the reliability of results. The aim of the present study was to assess the effect of short-term storage time at +4 °C on total oxidant status (TOS), total antioxidant capacity (TAC) and oxidative stress index (OSI) in flathead mullet (Mugil cephalus) serum. After blood collection, all sample were analyzed and the assays were repeated after 24, 48 and 72 h from sampling. results showed a significant effect of short-term storage on TOS and TAC (P ≤ 0.0001), while the statistical significance of the linear regression study for these parameters was reduced as consequence of storage. These results highlight that the activities of oxidants and antioxidants in flathead mullet serum change during short-term storage at 4 °C and should be assesses as soon as possible from collection.     

Sap flow and water use in African baobab (Adansonia digitata L.) seedlings in response to drought stress

The African baobab (Adansonia digitata L.) is an important multi-purpose fruit tree with high potential for domestication in drier Africa. Although adult individuals are well-known to be drought resistant, only little has been reported on how young baobab trees can survive drought. Therefore, the aim of this study was to examine short-term soil drought effects on water relations of baobab seedlings. Baobab seedlings used a limited amount of stored water to buffer daily water deficits (~ 8.5 g d^− 1), which contributed up to only ~ 17.5% of daily water use and ~ 6% of total plant water. Under drought, a strong reduction in stomatal conductance (~ 85%) resulted in a midday leaf water potential of − 1 MPa and zero stem sap flow followed by significant leaf loss. Plant anatomy evidenced the presence of water storage tissues and the vulnerability to xylem embolism. The taproot was the most important plant part for water storage (68% of total plant water), suggesting root-succulence rather than stem-succulence. When drought intensified, limitation of leaf transpiration and/or root water uptake led to drought-enforced dormancy. Despite the large amounts of water stored in the taproot (~ 90%) and the stem (~ 75%), only a limited amount of stored water appeared to be used to sustain upper leaves and plant metabolism during the dormant period, and to facilitate recovery following water supply. Drought avoidance, conservative water use and the presence of internal stored water allow baobab seedlings to survive drought.     

Environmental and climatic changes in the central Mediterranean Sea (Siculo–Tunisian Strait) during the last 30 ka based on dinoflagellate cyst and planktonic foraminifera assemblages

A high resolution micropalaeontological study of the core MD 04-2797 CQ recovered in the Sicilian–Tunisian Strait provides insights into the paleoclimatic history of the Mediterranean Sea at the transition between the western and eastern basin over the last 30 ka. Using the analysis of dinoflagellate cyst and planktonic foraminiferal assemblages, we reconstruct the paleoenvironmental changes that took place in this region. High abundances of cold temperate dinocyst species (Nematosphaeropsis labyrinthus, Spiniferites elongatus, Bitectatodinium tepikiense) and the polar planktonic foraminifera Neogloboquadrina pachyderma (left coiling) reveal three major cooling events synchronous with North Atlantic Henrich events 1 and 2 (H1 and H2) and the European and North Atlantic Younger Dryas event. During the Holocene, the presence of warm dinocyst species (Spiniferites mirabilis and Impagidinium aculeatum) and planktonic foraminifera (Globorotalia inflata and Globigerinoides ruber), reflects a significant increase of sea surface temperatures in the western Mediterranean basin, but a full warming was not recorded until 1500 years after the onset of the Holocene. Moreover, our results show that the Holocene was interrupted by at least four brief cooling events at ~ 9.2 ka, ~ 8 ka, ~ 7 ka and ~ 2.2 ka cal. BP, which may be correlated to climatic events recorded in Greenland ice cores and in the Atlantic Ocean.     

Position—Specific contribution of interface tryptophans on membrane protein energetics

Interface tryptophans are key residues that facilitate the folding and stability of membrane proteins. Escherichia coli OmpX possesses two unique interface tryptophans, namely Trp76, which is present at the interface and is solvent-exposed, and Trp140, which is relatively more lipid solvated than Trp76 in symmetric lipid membranes. Here, we address the requirement for tryptophan and the consequences of aromatic amino acid substitutions on the folding and stability of OmpX. Using spectroscopic measurements of OmpX-Trp/Tyr/Phe mutants, we show that the specific mutation W76 → Y allows barrel assembly > 1.5-fold faster than native OmpX, and increases stability by ~ 0.4 kcal mol^− 1. In contrast, mutating W140 → F/Y lowers OmpX thermodynamic stability by ~ 0.4 kcal mol^− 1, without affecting the folding kinetics. We conclude that the stabilizing effect of tryptophan at the membrane interface can be position—and local environment—specific. We propose that the thermodynamic contributions for interface residues be interpreted with caution.     

Ensembles from Ordered and Disordered Proteins Reveal Similar Structural Constraints during Evolution

The conformations accessible to proteins are determined by the inter-residue interactions between amino acid residues. During evolution, structural constraints that are required for protein function providing biologically relevant information can exist. Here, we studied the proportion of sites evolving under structural constraints in two very different types of ensembles, those coming from ordered and disordered proteins. Using a structurally constrained model of protein evolution, we found that both types of ensembles show comparable, near 40%, number of positions evolving under structural constraints. Among these sites, ~ 68% are in disordered regions and ~ 57% of them show long-range inter-residue contacts. Also, we found that disordered ensembles are redundant in reference to their structurally constrained evolutionary information and could be described on average with ~ 11 conformers. Despite the different complexity of the studied ensembles and proteins, the similar constraints reveal a comparable level of selective pressure to maintain their biological functions. These results highlight the importance of the evolutionary information to recover meaningful biological information to further characterize conformational ensembles.     

Functional evaluation of tryptophans in glycolipid binding and membrane interaction by HET-C2, a fungal glycolipid transfer protein

HET-C2 is a fungal glycolipid transfer protein (GLTP) that uses an evolutionarily-modified GLTP-fold to achieve more focused transfer specificity for simple neutral glycosphingolipids than mammalian GLTPs. Only one of HET-C2's two Trp residues is topologically identical to the three Trp residues of mammalian GLTP. Here, we provide the first assessment of the functional roles of HET-C2 Trp residues in glycolipid binding and membrane interaction. Point mutants HET-C2^W208F, HET-C2^W208A and HET-C2^F149Y all retained > 90% activity and 80–90% intrinsic Trp fluorescence intensity; whereas HET-C2^F149A transfer activity decreased to ~ 55% but displayed ~ 120% intrinsic Trp emission intensity. Thus, neither W208 nor F149 is absolutely essential for activity and most Trp emission intensity (~ 85–90%) originates from Trp109. This conclusion was supported by HET-C2^W109Y/F149Y which displayed ~ 8% intrinsic Trp intensity and was nearly inactive. Incubation of the HET-C2 mutants with 1-palmitoyl-2-oleoyl-phosphatidylcholine vesicles containing different monoglycosylceramides or presented by lipid ethanol-injection decreased Trp fluorescence intensity and blue-shifted the Trp λ_max by differing amounts compared to wtHET-C2. With HET-C2 mutants for Trp208, the emission intensity decreases (~ 30–40%) and λ_max blue-shifts (~ 12 nm) were more dramatic than for wtHET-C2 or F149 mutants and closely resembled human GLTP. When Trp109 was mutated, the glycolipid induced changes in HET-C2 emission intensity and λ_max blue-shift were nearly nonexistent. Our findings indicate that the HET-C2 Trp λ_max blue-shift is diagnostic for glycolipid binding; whereas the emission intensity decrease reflects higher environmental polarity encountered upon nonspecific interaction with phosphocholine headgroups comprising the membrane interface and specific interaction with the hydrated glycolipid sugar.     

Redox-Optimized ROS Balance and the relationship between mitochondrial respiration and ROS

The Redox-Optimized ROS Balance [R-ORB] hypothesis postulates that the redox environment [RE] is the main intermediary between mitochondrial respiration and reactive oxygen species [ROS]. According to R-ORB, ROS emission levels will attain a minimum vs. RE when respiratory rate (VO₂) reaches a maximum following ADP stimulation, a tenet that we test herein in isolated heart mitochondria under forward electron transport [FET]. ROS emission increased two-fold as a function of changes in the RE (~ 400 to ~ 900 mV·mM) in state 4 respiration elicited by increasing glutamate/malate (G/M). In G/M energized mitochondria, ROS emission decreases two-fold for RE ~ 500 to ~ 300 mV·mM in state 3 respiration at increasing ADP. Stressed mitochondria released higher ROS, that was only weakly dependent on RE under state 3. As a function of VO₂, the ROS dependence on RE was strong between ~ 550 and ~ 350 mV·mM, when VO₂ is maximal, primarily due to changes in glutathione redox potential. A similar dependence was observed with stressed mitochondria, but over a significantly more oxidized RE and ~ 3-fold higher ROS emission overall, as compared with non-stressed controls. We conclude that under non-stressful conditions mitochondrial ROS efflux decreases when the RE becomes less reduced within a range in which VO₂ is maximal. These results agree with the R-ORB postulate that mitochondria minimize ROS emission as they maximize VO₂ and ATP synthesis. This relationship is altered quantitatively, but not qualitatively, by oxidative stress although stressed mitochondria exhibit diminished energetic performance and increased ROS release.     

Oligomerization–function relationship of EGFR on living cells detected by the coiled-coil labeling and FRET microscopy

The epidermal growth factor receptor (EGFR) is a well-studied receptor tyrosine kinase and an important anticancer therapeutic target. The activity of EGFR autophosphorylation and transphosphorylation, which induces several cell signaling pathways, has been suggested to be related to its oligomeric state. However, the oligomeric states of EGFRs induced by EGF binding and the receptor–ligand stoichiometry required for its activation are still controversial. In the present study, we performed Förster resonance energy transfer (FRET) measurements by combining the coiled-coil tag–probe labeling method and spectral imaging to quantitatively analyze EGFR oligomerization on living CHO-K1 cell membranes at physiological expression levels. In the absence of its ligands, EGFRs mainly existed as monomers with a small fraction of predimers (~ 10%), whereas ~ 70% of the EGFRs formed dimers after being stimulated with the ligand EGF. Ligand-induced dimerization was not significantly affected by the perturbation of membrane components (cholesterol or monosialoganglioside GM3). We also investigated both dose and time dependences of EGF-dependent EGFR dimerization and autophosphorylation. The formation of dimers occurred within 20 s of the ligand stimulation and preceded its autophosphorylation, which reached a plateau 90 s after the stimulation. The EGF concentration needed to evoke half-maximum dimerization (~ 1 nM) was lower than that for half-maximum autophosphorylation (~ 8 nM), which suggested the presence of an inactive dimer binding a single EGF molecule.     

Pre-analytical operating procedures for serum Low Molecular Weight protein profiling

Biological specimen collection and storage are an integral component of serum proteomics research. Although many efforts have been posed to address the effects of pre-analytical procedures, standardized protocols for collection and storage of samples for Low Molecular Weight (LMW) proteome profiling are still needed.Here we report a systematic analysis on the influence of pre-analytical factors [clotting times, temperature and time storage, addition of protease inhibitor (PI)] on serum LMW proteome profiling. Moreover, a comparison between manual versus automated peptide purification by functionalized magnetic bead-based MALDI-MS approach was performed. The results demonstrated best serum LMW proteins recovery and stability using a clotting time between 1 and 2 h, with serum stored up to 2 h either at room temperature or at 4 °C, independently of PI addition. PI addition to whole blood resulted in a lower number of LMW peaks detected. Finally, minimal effects on serum proteome profiles were observed after 1-month storage at ? 80 °C, independently of PI addition on whole blood and/or serum.In conclusion, the use of standardized pre-analytical and storage procedures together with an automated peptide purification might minimize potential bias on serum LMW profiling results, thus allowing a better homogeneity and reproducibility in future proteomics studies.     

Monomerization and aggregation of β-lactoglobulin under adverse condition: A fluorescence correlation spectroscopic investigation

β-Lactoglobulin is one of the major components of bovine milk and it remains in a dimeric form under physiological conditions. The present contribution elucidates the structural change of β-lactoglobulin at pH 7.4 under the action of guanidine hydrochloride (GnHCl) and heat at the single molecular level. The only free cysteine (Cys-121) of β-lactoglobulin has been tagged with 7-diethylamino-3-(4-maleimidophenyl)-4-methylcoumarin (CPM) for this purpose. The dimeric structure of β-lactoglobulin found to undergoes a monomerization prior to the unfolding process upon being subjected to GnHCl. The hydrodynamic diameter of the native dimer, native monomer and the unfolded monomer has been estimated as ~ 55 Å, ~ 29 Å and ~ 37 Å, respectively. The free energy change for the monomerization and denaturation are respectively 1.57 kcal mol^− 1 and 8.93 kcal mol^− 1. With change in temperature, development of two types of aggregates (small aggregates and large aggregates) was observed, which is triggered by the formation of the monomeric structure of β-lactoglobulin. The hydrodynamic diameters of the smaller and larger aggregates have been estimated to be ~ 77 Å and ~ 117 Å, respectively. The formation of small aggregates turns out to be reversible whereas that of larger aggregates is irreversible. The free energy associated with these two steps are 0.69 kcal mol^− 1 and 9.09 kcal mol^− 1. Based on the size parameters, the smaller and larger aggregates have been proposed to contain ~twenty and ~sixty monomeric units. It has also been concluded that the monomeric subunits retain their native like secondary structure in these aggregates.     

Multiple sclerosis: Identification and clinical evaluation of novel CSF biomarkers

Multiple sclerosis (MS) is a neuro-inflammatory and neurodegenerative disease that results in damage to myelin sheaths and axons in the central nervous system and which preferentially affects young adults. We performed a proteomics-based biomarker discovery study in which cerebrospinal fluid (CSF) from MS and control individuals was analyzed (n = 112). Ten candidate biomarkers were selected for evaluation by quantitative immunoassay using an independent cohort of MS and control subjects (n = 209). In relapsing–remitting MS (RRMS) patients there were significant increases in the CSF levels of alpha-1 antichymotrypsin (A1AC), alpha-1 macroglobulin (A2MG) and fibulin 1 as compared to control subjects. In secondary progressive MS (SPMS) four additional proteins (contactin 1, fetuin A, vitamin D binding protein and angiotensinogen (ANGT)) were increased as compared to control subjects. In particular, ANGT was increased 3-fold in SPMS, indicating a potential as biomarker of disease progression in MS. In PPMS, A1AC and A2MG exhibit significantly higher CSF levels than controls, with a trend of increase for ANGT. Classification models based on the biomarker panel could identify 70% of the RRMS and 80% of the SPMS patients correctly. Further evaluation was conducted in a pilot study of CSF from RRMS patients (n = 36), before and after treatment with natalizumab.     

Step-wise change of Asian interior climate preceding the Eocene–Oligocene Transition (EOT)

Understanding the global climate change from greenhouse to icehouse conditions at the Eocene–Oligocene Transition (EOT) 34 million years ago requires climatic records from oceanic as well as continental realms of the key Late Eocene “doubthouse” period preceding this switch. Here, we report integrated stratigraphic results from well-dated Late Eocene continental mudflat to saline lake paleoenvironments of the Xining Basin (northeastern Tibetan Plateau, western China) recording regional and global change. Cyclostratigraphic analysis strongly suggests continuous dominance of the 41-kyr obliquity cycle in the whole late Eocene interval down to the base of polarity chron C18n.2n at 39 Ma with additional input of the ~ 100-kyr eccentricity cycle up to the base of chron C13r at ~ 34.7 Ma. This might imply that high-latitude climates dominated the area long before the EOT, probably related to incipient ice-volume fluctuations. Furthermore, our results reveal two paleoenvironmental deterioration steps preceding the Eocene–Oligocene Transition. The first step occurs in the top of chron C17n.1n at ~ 36.6 Ma. This age closely corresponds to (1) the high-altitude pollen appearance in chron C16.2r at ~ 36.4 Ma in the same section, (2) the recently dated final retreat of the Tarim Sea in western China, and (3) a shift from precession to obliquity dominance in the Atlantic Ocean. This near co-occurrence suggests global change at this time. We hypothesize this change is related to an increase in incipient ice sheet volume leading to passing threshold conditions for the high-altitude pollen appearance and Tarim Sea retreat, finally leading to decreased moisture availability in the Xining Basin. At the second step, in the base of chron C13r at ~ 34.7 Ma, a substantial increase in clastic sedimentation rates is observed. This might relate to increased climate variability preceding the greenhouse to icehouse transition at the EOT that prevented landscapes to attain equilibrium configurations.     

A critical evaluation of salivary testosterone as a method for the assessment of serum testosterone

Although salivary testosterone (T) is often used in clinical studies accuracy is mostly questionable. State of the art data for men is sparse and for women absent. Our objective was to perform a critical evaluation of salivary T (Sal-T) as a method for indirect assessment of serum T using state of the art methods. Saliva was collected via ‘Salivette’ and ‘passive drooling’ methods. Sal-T and free T in serum after equilibrium dialysis were measured by LC-MS/MSResultsEvaluation of Sal-T results versus free T by equilibrium dialysis (ED-T) for men gave: ‘Salivette’ Sal-T = 0.05 + 0.88x ED-T, r = 0.43; ‘passive drooling’ Sal-T = 0.17 + 0.91x ED-T r = 0.71. In women, correlation was comparable but values are higher than free T: ‘passive drooling’ Sal-T = 0.12 + 2.32x ED-T, r = 0.70. The higher than expected T values in saliva, appear to be explained by T binding to salivary proteins. Iso-electric focusing of saliva proteins, followed by fractionation and LC–MS/MS assay of T showed marked testosterone peaks at pH 5.3 and 8.4, providing evidence for T binding in saliva to proteins such as albumin and proline rich protein (PRP).ConclusionsPassive drooling is the collection method of choice for testosterone in saliva. Sal-T is not directly comparable to serum free T due to T binding to saliva proteins, which substantially affects the low Sal-T in women but not the higher Sal-T in healthy adult men.     

Structural changes that occur upon photolysis of the Fe(II)a3–CO complex in the cytochrome ba3-oxidase of Thermus thermophilus: A combined X-ray crystallographic and infrared spectral study demonstrates CO binding to CuB

The purpose of the work was to provide a crystallographic demonstration of the venerable idea that CO photolyzed from ferrous heme-a₃ moves to the nearby cuprous ion in the cytochrome c oxidases. Crystal structures of CO-bound cytochrome ba₃-oxidase from Thermus thermophilus, determined at ~ 2.8–3.2 Å resolution, reveal a Fe–C distance of ~ 2.0 Å, a Cu–O distance of 2.4 Å and a Fe–C–O angle of ~ 126°. Upon photodissociation at 100 K, X-ray structures indicate loss of Fe_a3–CO and appearance of Cu_B–CO having a Cu–C distance of ~ 1.9 Å and an O–Fe distance of ~ 2.3 Å. Absolute FTIR spectra recorded from single crystals of reduced ba₃–CO that had not been exposed to X-ray radiation, showed several peaks around 1975 cm^? 1; after photolysis at 100 K, the absolute FTIR spectra also showed a significant peak at 2050 cm^? 1. Analysis of the ‘light’ minus ‘dark’ difference spectra showed four very sharp CO stretching bands at 1970 cm^? 1, 1977 cm^? 1, 1981 cm^? 1, and 1985 cm^? 1, previously assigned to the Fe_a3–CO complex, and a significantly broader CO stretching band centered at ~ 2050 cm^? 1, previously assigned to the CO stretching frequency of Cu_B bound CO. As expected for light propagating along the tetragonal axis of the P4₃2₁2 space group, the single crystal spectra exhibit negligible dichroism. Absolute FTIR spectrometry of a CO-laden ba₃ crystal, exposed to an amount of X-ray radiation required to obtain structural data sets before FTIR characterization, showed a significant signal due to photogenerated CO₂ at 2337 cm^? 1 and one from traces of CO at 2133 cm^? 1; while bands associated with CO bound to either Fe_a3 or to Cu_B in “light” minus “dark” FTIR difference spectra shifted and broadened in response to X-ray exposure. In spite of considerable radiation damage to the crystals, both X-ray analysis at 2.8 and 3.2 Å and FTIR spectra support the long-held position that photolysis of Fe_a3–CO in cytochrome c oxidases leads to significant trapping of the CO on the Cu_B atom; Fe_a3 and Cu_B ligation, at the resolutions reported here, are otherwise unaltered. This article is part of a Special Issue entitled: Respiratory Oxidases.     

Late Quaternary (Weichselian) alluvial history and neotectonic control on fluvial landscape development in the southern Körös plain,Hungary

Four drill cores and a clay pit section have been examined in the southern part of the Körös plain to understand the history and controls on alluvial sedimentation for the last ~ 40 ka. Four facies groups were identified, such as channel, channel margin, floodplain and floodbasin with seven distinctive facies. Magnetic susceptibility and mineralogy have further characterized the sedimentary facies indicating shifts in humidity conditions, variations in sediment flux and pedogenesis. Detailed pollen analysis of a 7.5 m thick clayey succession indicated climatic variability within the MIS 3 period. The spatial distribution of the different facies allowed outlining alluvial architecture of the study area. Three depositional units composed of various facies were identified based on OSL and radiocarbon data. These packages correspond to three major phases of channel activity: (F-I) pre-LGM period (> 30 ka to 24 ka), (F-II) post-LGM interstadial (18–16 ka), and (F-III) Late Glacial < 15 ka to ~ 10 ka). The pre-LGM and post-LGM “interstadial” phases are characterized by meandering river patterns, while the Late Glacial fluvial activity is characterized by a braided system in the area. Higher sediment supply feeding this braided river was probably caused by neotectonic uplift of the southern margin of the basin, documented by a significant stratigraphic gap between 25 and 14 ka.