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.     

In vitro anti-cancer activities of Job’s tears (Coix lachryma-jobi Linn.) extracts on human colon adenocarcinoma

The whole seed (W), endosperm (E) and hull (H) of five cultivars of Job’s tears (Coix lachryma-jobi Linn. var. ma-yuen Stapf) including Thai Black Phayao, Thai Black Loei, Laos Black Loei, Laos White Loei and Laos Black Luang Phra Bang were processed before solvent extraction by non-cooking, roasting, boiling and steaming Each part of the Job’s tears was extracted by the cold and hot process by refluxing with methanol and hexane. The total of 330 extracts included 150 methanol extracts and 180 hexane extracts were investigated for anti-proliferative activity on human colon adenocarcinoma cell line (HT-29) by the sulforhodamine B (SRB) assay. The extracts which gave high anti-proliferative activity were tested for apoptotic activity by acridine orange and ethidium bromide double staining and anti-oxidative activities including free radical scavenging and lipid peroxidation inhibition activities. The extract from the hull of Thai Black Loei roasted before extracting by hot methanol (M-HTBL-R2) showed the highest anti-proliferative activity on HT-29 with the IC₅₀ values of 11.61 ± 0.95 μg/ml, while the extract from the non-cooked hull of Thai Black Loei by cold methanol extraction (M-HTBL-N1) gave the highest apoptosis (8.17 ± 1.18%) with no necrosis. In addition, M-HTBL-R2 and M-HTBL-N1 indicated free radical scavenging activity at the SC₅₀ values of 0.48 ± 0.12 and 2.47 ± 1.15 mg/ml, respectively. This study has demonstrated the anti-colorectal cancer potential of the M-HTBL-R2 and M-HTBL-N1 extracts.     

New data on Sicilian prehistoric and historic evolution in a mountain context,Vallone Inferno (Scillato,Italy)

Vallone Inferno rock-shelter is an archaeological site located in the Madonie mountain range in Sicily. Archaeological excavation and research have provided a long prehistoric and historic sequence from the Neolithic to the medieval period, this being the most complete work in this area at present. In this paper we present the preliminary data provided by a multidisciplinary study based on pottery, lithic, human, faunal and plant remains. Stratigraphic studies have identified four complexes, of which complex 3 has provided almost all the archaeological remains. ¹⁴C AMS dates, obtained from four samples, place the human activities between 2601 cal BC and 644 cal AD. These dates are coherent with the cultural attribution of the ceramic and lithic remains. Macrofaunal and plant remains show a persistent use of the shelter for pastoral activities. Environmental data, obtained from microvertebrate and archaeobotanical remains, show the aridification and opening of the landscape from the base to the top of the sequence as a consequence of the human impact.     

Proteomics of wine additives: Mining for the invisible via combinatorial peptide ligand libraries

Combinatorial peptide ligand libraries (CPLLs) have been adopted for harvesting and identifying traces of casein (used as a fining agent) present in white wines. Although minute amounts (200 µL) of CPLL beads are added to the entire content of a wine bottle (750 mL), they are able to sequester with high efficiency (up to 80%) residual traces of casein, permitting a signal “amplification” of at least 5000-fold. It is here demonstrated that as little as 1 µg/L of casein can be efficiently detected in white wines, a major improvement over previous investigations in which the lower detection limit had been estimated at 100 µg/L. The fact that such very low levels of fining agents can still be detected in treated white wines should be taken into consideration by winemakers in labelling their products and by EC rulers in issuing proper regulations.     

Small vertebrates (Amphibia,Squamata, Mammalia) from the late Pleistocene-Holocene of the Valdavara-1 cave (Galicia,northwestern Spain)

The Valdavara-1 cave, located in the westernmost part of the Iberian Peninsula, is an archaeological site that has been excavated since 2007. At least two main sedimentary units have been identified: an upper one, 30 cm-thick, that has yielded many fragments of ceramic characteristic of the Recent Prehistory (with a radiocarbon age of 4.490 ± 40 years BP), and a lower one, 90 cm-thick, separated from the upper one by an erosive discontinuity and late Pleistocene in age (radiocarbon ages between 13.770 ± 70 and 15.120 ± 70 years BP). The small-vertebrate assemblages recovered from the sieving-washing of all the sediment from the excavation campaigns include a total of at least 34 taxa (six amphibians, nine squamate reptiles, five insectivores, two chiropterans and 12 rodents). The materials from this locality constitutes the first mention in northwestern Spain for Microtus (Iberomys) cabrerae and Micromys minutus, currently absent from this area, as well as the first mention in the fossil record for Chioglossa lusitanica, Discoglossus galganoi and Chalcides striatus. In both layers, such small-vertebrate associations suggest a patchy landscape dominated by humid meadows and woodland areas, with the existence of water areas in the vicinity of the cave. The climate shows a more continental pattern during the late Pleistocene, as evidenced by the presence of Microtus arvalis, Sorex minutus and Rana temporaria, and was milder during the Recent Prehistory, with the occurrence of typically Mediterranean taxa such as Crocidura russula, Chioglossa lusitanica, Discoglossus galganoi, Rana iberica, Chalcides striatus and Timon lepidus. The amphibian and squamate associations suggest that the climate change recorded at the Valdavara-1 cave between the latest Pleistocene and Holocene levels may correspond to a difference of 0.8 °C in terms of mean annual temperature, although the difference is 2.8 °C for the mean temperature of the coldest month.     

Evaluating the dendroclimatological potential of Taxus baccata (yew) in southwest Ireland

Tree-ring research in Ireland has typically been dominated by Quercus species, particularly Quercus petraea and Quercus robur. Recent years have seen a greater focus on multi-species reconstructions in Ireland but, due to difficulties with the hardness of the wood, missing/pinched rings and fused stems, Taxus baccata has not been included in these investigations. Despite these difficulties, a 31-tree, 204-year T. baccata chronology was successfully constructed from Killarney National Park, southwest Ireland. The chronology exhibits promising dendroclimatological potential, with climatic responsiveness equivalent to that of the other major Irish tree taxa, including Quercus. The chronology shows the strongest relationship with May–June precipitation from Muckross House synoptic station (1970–2007; r = 0.521, p < 0.01) and Valentia Observatory (1941–2007; r = 0.545, p < 0.01). November–April temperatures also exhibited a strong relationship with the chronology post-1970 (r = 0.605, p < 0.01 for Muckross House, r = 0.567, p < 0.01 for Valentia Observatory), but this relationship is not time stable and breaks down for the pre-1970 Valentia Observatory record. The long-lived nature of T. baccata, the exceptional preservation of wood and rings in this hard softwood species, as well as its prominence in Irish archaeology, all point to the potential to expand this chronology both spatially and temporally, and demonstrate T. baccata's potential in multi-site and multi-species tree-ring studies in the region.     

Reconstruction of Late Cretaceous coastal paleotemperature from halite deposits of the Late Cretaceous Nongbok Formation (Khorat Plateau,Laos)

Cretaceous evaporites of the Maha Sarakhan Formation in Thailand (e.g., the Nongbok Formation, Laos) have been studied for almost a century as the huge potash deposits in the world. The consistently high local paleotemperatures should lead to huge salt deposits during the evaporation process. Primary fluid inclusions in halite can provide surface brine water temperatures directly and quantitatively. Until now, there have been no data published from paleotemperature of primary fluid inclusions of Cretaceous halite. The non-marine halite from the Cretaceous Nongbok Formation (Laos) precipitated from shallow brine waters with temperatures of 17.7–42.3 °C.     

Ultrasound-guided transesophageal HIFU exposures for atrial fibrillation treatment: First animal experiment

Atrial fibrillation is the most frequent cardiac arrhythmia. Recently, an ultrasound-guided transesophageal high-intensity focused ultrasound (HIFU) device has been designed to perform minimally invasive pulmonary vein isolation. It was made with a 3 MHz 8-ring HIFU transducer including in its center a 5 MHz 64-element imaging transducer. The HIFU transducer could focus the ultrasound beam over a broad range of depths from 17 to 55 mm to perform lesions in various locations, while preserving intervening tissues. Those characteristics having been demonstrated in vitro, the aim of this study was to perform deep cardiac lesions in pig model under ultrasound guidance. A first ex vivo/in situ experiment gave promising results. Indeed, wide linear lesions were obtained in depth at desired locations. In vivo investigations are ongoing to confirm those preliminary results.     

L’outillage lithique taillé dans le grand gisement paléolithique de Mitoc-Malu Galben

In this Palaeolithic site, at the moment the largest habitation undergoing investigations in Europe, several archaeological layers were discovered: Lower Aurignacian I, Aurignacian I, II, III and Upper Aurignacian III, Gravettian I, II, III, IV and Dispersed Gravettian. The systematic archaeological investigations were conducted between 1978 and 2017 and covered a surface of more than 900 square meters, reaching a depth of approximately 14 m. Stone artefacts worked in non-local raw materials were identified, as well as a survival of Aurignacian elements within the Gravettian medium, as suggested by the presence of carenated pieces, scarpers, etc.     

Analysis of smoking and LPO in ALS

Smoking has been suggested as one of the risk factor for amyotrophic lateral sclerosis (ALS) development. In order to investigate whether adverse effects of cigarette smoke in ALS have any association with increase in oxidative stress, disease severity, lipid hydroperoxides (LPO) and superoxide dismutase-1 (SOD1) levels were measured in biofluids of smoker and never smoker ALS patients and clinically correlated. Serum and CSF from sporadic ALS patients (n = 50) diagnosed with El Escorial criteria were collected in the study. Serum (n = 50) and CSF (n = 42) were also collected from normal healthy controls. The LPO levels were estimated using commercially available kits. Enzyme-linked immunosorbent assays (ELISAs) were used to quantitate SOD1. Their levels were further analyzed among smoker and never smoker subjects. Significantly elevated LPO in sera and CSF of ALS patients were observed (p < 0.05). There was considerably increased LPO in sera and CSF of smoker ALS subjects matched with disease severity as compared to never smoker ALS (p < 0.05). ALS group did not show any alteration in SOD1 when compared to controls (p > 0.05). In addition, no change has been observed in SOD1 levels in ALS subjects who smoke (p > 0.05). Increased LPO and unaltered SOD1 in ALS patients may suggest the neuro-pathological association of LPO with ALS disease independent of SOD1. With current findings, it may be proposed that LPO levels might constitute as probable biomarker for smoker ALS patients, however, it cannot be concluded without larger gender matched studies. Additional investigations are needed to determine whether LPO upregulation is primary or secondary to motor neuron degeneration in ALS.     

Does interhemispheric communication relate to the bilateral function of muscles? A study of scapulothoracic muscles using transcranial magnetic stimulation

Interhemispheric connections have been demonstrated between the motor cortex controlling muscle pairs. However, these investigations have tended to concentrate upon hand muscles. We have extended these investigations to proximal muscles that control the scapula upon the trunk and help to move and stabilise the shoulder. Using a paired pulse transcranial magnetic stimulation protocol, the interhemispheric interactions between different shoulder girdle muscle pairs, serratus anterior, upper trapezius and lower trapezius were investigated. Test motor evoked potentials were conditioned using conditioning pulse intensities of 80% and 120% of active motor threshold at three different condition-test intervals, during three different tasks. Interhemispheric inhibition was observed in upper trapezius using a conditioning intensity of 120% and condition-test interval of 8 ms (17 ± 18%, p < 0.007). A trend towards inhibition was observed in lower trapezius and serratus anterior using a conditioning intensity of 120% and a condition-test interval of 8 ms (13 ± 22%; p < 0.07 and 10 ± 19% respectively; p < 0.07). No interhemispheric facilitation was evoked. The study demonstrates that a low level of interhemispheric inhibition rather than interhemispheric facilitation could be evoked between these muscle pairs.     

Synthesis,thymidine phosphorylase inhibition and molecular modeling studies of 1,3,4-oxadiazole-2-thione derivatives

Thymidine phosphorylase (TP) inhibitors have attracted great attention due to their ability to suppress the tumors formation. In our ongoing research, a series of 1,3,4-oxadiazole-2-thione (1–12) has been synthesized under simple reaction conditions in good to excellent yields (86–98%) and their TP inhibition potential has also been evaluated. The majority of synthesized compounds showed moderate thymidine phosphorylase inhibitory activity with IC50 values ranging from 38.24 ± 1.28 to 258.43 ± 0.43 μM, and 7-deazaxanthine (7DX) was used as a reference compound (IC50 38.68 ± 4.42). The TP activity was very much dependent on the C-5 substituents; among this series the compound 6 bearing 4-hydroxyphenyl group was found to be the most active with IC50 38.24 ± 1.28 μM. Molecular docking studies revealed their binding mode.     

Salivary vasopressin increases following intranasal oxytocin administration

Extant research has documented the effects of intranasal administration of oxytocin (OT), and to a lesser degree Arginine Vasopressin (AVP) – two structurally-related neuropeptides – on brain and behaviour, yet the effects of exogenous manipulation of one on circulating levels of the other remain unknown. Studies have shown that OT administration impacts the peripheral levels of numerous hormones; however, whether OT administration also increases AVP concentrations has not been explored. Utilizing a double-blind placebo-controlled within-subject design, ten male and female subjects provided ten saliva samples over four consecutive hours: at baseline and nine times following OT administration. Results indicate that salivary AVP increased in the first hour following OT manipulation (OT condition: mean AVP = 2.17 pg/ml, SE = 28, placebo condition: mean AVP = 1.42 pg/ml, SE = .18) but returned to baseline levels at the next assessment (80 min) and remained low for the remaining period. Similar to OT, AVP showed high degree of individual stability and baseline levels of AVP correlated with AVP concentrations at the first and second post-administration hours regardless of drug condition (Pearson r = .85–.93). Validity of salivary AVP ELISA measurement was verified by demonstrating individual stability of salivary AVP over a six-month period (r = .70, p < .000) as well correlation with plasma levels over the same period (r = .32, p = .037) in a sample of 45 young adults who did not participate in the current study. Overall, findings suggest a potential crosstalk between OT and AVP and indicate that baseline levels of the two neuropeptides may shape the degree to which these systems respond to exogenous manipulation.     

Clinical characteristics of tumors derived from colorectal cancer patients who harbor the Tumor Necrosis Factor α-1031T/T and NOD2 3020insC polymorphism

Background: Genetic predispositions to disease have focused on highly penetrant causative changes in tumor suppressor genes or genes associated with DNA mismatch repair. New investigations are revealing new genetic associations with disease that are more subtle in their association with disease and require characterization. Methods: In this report we have examined the tumor characteristics in a group of patients who have been shown to harbor two polymorphisms in two genes that are associated with the immune system NOD2 and TNFα. Results: Colorectal cancers from patients with NOD2 3020insC and TNFα-1031T/T constitutional changes are mostly right-sided disease (OR = 2.21, p = 0.03) with a tendency to higher stages (OR = 2.41, p = 0.06), increased number of associated polyps (OR = 1.77, p = 0.16) and later age of average age of disease onset (p = 0.039). Conclusion: The results reveal that there appear to be specific characteristics associated with the tumors that may aid in determining management strategies to reduce the risk of disease.     

Design,synthesis and biological evaluation of a bivalent μ opiate and adenosine A1 receptor antagonist

The cross talk between different membrane receptors is the source of increasing research. We designed and synthesized a new hetero-bivalent ligand that has antagonist properties on both A₁ adenosine and μ opiate receptors with a K_i of 0.8 ± 0.05 and 0.7 ± 0.03 μM, respectively. This hybrid molecule increases cAMP production in cells that over express the μ receptor as well as those over expressing the A₁ adenosine receptor and reverses the antalgic effects of μ and A₁ adenosine receptor agonists in animals.     

Reduced creatine kinase as a central and peripheral biomarker in Huntington's disease

A major goal of current clinical research in Huntington's disease (HD) has been to identify preclinical and manifest disease biomarkers, as these may improve both diagnosis and the power for therapeutic trials. Although the underlying biochemical alterations and the mechanisms of neuronal degeneration remain unknown, energy metabolism defects in HD have been chronicled for many years. We report that the brain isoenzyme of creatine kinase (CK-BB), an enzyme important in buffering energy stores, was significantly reduced in presymptomatic and manifest disease in brain and blood buffy coat specimens in HD mice and HD patients. Brain CK-BB levels were significantly reduced in R6/2 mice by ～ 18% to ～ 68% from 21 to 91 days of age, while blood CK-BB levels were decreased by ～ 14% to ～ 44% during the same disease duration. Similar findings in CK-BB levels were observed in the 140 CAG mice from 4 to 12 months of age, but not at the earliest time point, 2 months of age. Consistent with the HD mice, there was a grade-dependent loss of brain CK-BB that worsened with disease severity in HD patients from ～ 28% to ～ 63%, as compared to non-diseased control patients. In addition, CK-BB blood buffy coat levels were significantly reduced in both premanifest and symptomatic HD patients by ～ 23% and ～ 39%, respectively. The correlation of CK-BB as a disease biomarker in both CNS and peripheral tissues from HD mice and HD patients may provide a powerful means to assess disease progression and to predict the potential magnitude of therapeutic benefit in this disorder.     

Descriptive analysis of kinematics and kinetics of catchers throwing to second base from their knees

In order to decrease the amount of time that it takes the catcher to throw the ball, a catcher may chose to throw from the knees. Upper extremity kinematics may play a significant role in the kinetics about the elbow observed in catchers throwing from the knees. If relationships between kinematics and kinetics exist then the development of training and coaching instruction may help in reduced upper extremity injury risk. Twenty-two baseball and softball catchers (14.36 ± 3.86 years; 165.11 ± 17.54 cm; 65.67 ± 20.60 kg) volunteered. The catchers exhibited a less trunk rotation (5.6 ± 16.2°), greater elbow flexion (87.9 ± 21.4°) and decreased humeral elevation (71.1 ± 12.3°) at the event of maximum shoulder external rotation as compared to what has previously reported in catchers. These variables are important, as they have previously been established as potential injury risk factors in pitchers, however it is not yet clear the role these variables play in catchers’ risk of injury. A positive relationship between elbow varus torque during the deceleration phase and elbow flexion at MIR was observed (r = 0.609; p = 0.003). Throwing from the knees reduces a catcher’s ability to utilize the proximal kinetic chain and this may help to explain why their kinematics and kinetics differ from what has previously been presented in the literature.     

Le Gravettien et l’Épigravettien de l’Est de la Roumanie : une réévaluation

Opening towards the eastern steppe landmass, the Eastern Romania area covers 87,500 km², and shelters the biggest number of Gravettian and Epigravettian sites currently known. Archaeological researches in the area expanded in the 1950's, during the construction of dams on the Prut and Bistrița valleys. The efforts of the multidisciplinary teams involved and the subsequently published papers highlighted the archaeological potential of the region and, with some inevitable interruptions, investigations continued to this day. Although the time and resources spent were not modest, the outcomes regarding the Upper Palaeolithic cultural sequence often proved contradictory and somehow distant from the general European cultural dynamic. Thus, this reassessment tries to put the chronological, palaeoenvironmental, and empirical data in a more coherent framework. A synthesis of the available Gravettian and Epigravettian data east of the Carpathians reveals the following sequence: (1) an Aurignacian stage on the Prut Valley, paralleled by an indefinite Upper Palaeolithic stage, from sites on the Bistrița Valley, between 31 and 28 ka (uncal BP); (2) a quite early Gravettian presence, roughly between 27 and 25.5 ka, which includes backed laminar blanks, schematic decoration of one pendant, and the use of perforated shells as adornments; (3) a scant shouldered points stage, chronologically close to the European similar phases (25–23 ka), for which the feeble use of obsidian points toward contacts with Central European resources/populations; (4) a Gravettian/Epigravettian interface, between 21 and 19 ka, manifested within numerous archaeological layers, mainly through an increase in bladelets and organic artefacts production/use; (5) an Epigravettian stage, between 18 and 16 ka, largely defined through an upturn in raw material choices, backed implements production, and tool types; (6) one last, roughly 14 ka old Epigravettian stage, in which the technological choices recalled those of the previous one. Regional variability elements and radiocarbon chronology limitations considered, apparently, the Gravettian and the Epigravettian of Eastern Romania share quite a lot of traits with the corresponding Central and Eastern European technocomplexes.     

Dwellings and workspaces at Strandvägen, 5600–5000 cal. BC

This paper describes identified workspaces and the manufacture of slotted bone points at the Late Mesolithic settlement Strandvägen in Motala, in eastern central Sweden. Several dwellings were documented, Dwelling 1 being typically round-oval in shape 9 × 5.5 meters, with a floor area covering 49.5 m². Radiocarbon dates fall between approximately 5600-5200 cal BC. A combined archaeological record, with lithics and bone artefacts as well as analyses of the osteological assemblage has shown that slotted bone tools with mounted lithic inserts have been produced adjacent of the dwelling. The spatial distribution of bone flakes, microblades, processed resin and slotted artefacts testify to a clearly and delimited craft area near the shoreline of the river Motala Ström. Analyses of the finds, e.g. birch bark resin and prepared bone preforms by direct percussion, also help in reconstructing the stages of manufacturing composite projectile points in this part of Eurasia.