Morpho-chemical characterization of individual ancient starches retrieved on ground stone tools from Palaeolithic sites in the Pontic steppe.

Despite the extensive literature on the retrieval of digestible starches from archaeological contexts, there are still significant concerns regarding their genuine origin and durability. Here, we propose a multi-analytical strategy to identify the authenticity of ancient starches retrieved from macrolithic tools excavated at Upper Paleolithic sites in the Pontic steppe. This strategy integrates the morphological discrimination of starches through optical microscopy and scanning electron microscopy with single starch chemo-profiling using Fourier transform infrared imaging and microscopy. We obtained evidence of aging and biomineralization in the use-related starches from Palaeolithic sites, providing a methodology to establish their ancient origin, assess their preservation status, and attempt their identification. The pivotal application of this multidisciplinar approach demonstrates that the macrolithic tools, from which starches were dislodged, were used for food-processing across the Pontic Steppe around 40,000 years ago during the earliest colonization of Eurasia by Homo sapiens.

Unexpected silicon localization in calcium carbonate exoskeleton of cultured and fossil coccolithophores.

Coccolithophores, marine calcifying phytoplankton, are important primary producers impacting the global carbon cycle at different timescales. Their biomineral structures, the calcite containing coccoliths, are among the most elaborate hard parts of any organism. Understanding the morphogenesis of coccoliths is not only relevant in the context of coccolithophore eco-physiology but will also inform biomineralization and crystal design research more generally. The recent discovery of a silicon (Si) requirement for crystal shaping in some coccolithophores has opened up a new avenue of biomineralization research. In order to develop a mechanistic understanding of the role of Si, the presence and localization of this chemical element in coccoliths needs to be known. Here, we document for the first time the uneven Si distribution in Helicosphaera carteri coccoliths through three synchrotron-based techniques employing X-ray Fluorescence and Infrared Spectromicroscopy. The enrichment of Si in specific areas of the coccoliths point to a targeted role of this element in the coccolith formation. Our findings mark a key step in biomineralization research because it opens the door for a detailed mechanistic understanding of the role Si plays in shaping coccolith crystals.

Textile microfibers in wild Antarctic whelk Neobuccinum eatoni (Smith, 1875) from Terra Nova Bay (Ross Sea, Antarctica).

Antarctica has been affected directly and indirectly by human pressure for more than two centuries and recently plastic pollution has been recognized as a further potential threat for its unique biodiversity. Global long-range transport as well as local input from anthropogenic activities are potential sources of plastic pollution in both terrestrial and marine Antarctic territories. The present study evaluated the presence of microplastics in specimens of the Antarctic whelk Neobuccinum eatoni, a key species in benthic communities of the Ross Sea, one of the largest marine protected areas worldwide. To this aim, a thermo-oxidative extraction method was applied for microplastic isolation and quantification, and polymer identification was performed by manual µ-FTIR spectroscopy. Textile (semi-)synthetic or composite microfibers (length range: 0.8-5.7 mm) were found in 27.3% of whelk specimens, suggesting a low risk of bioaccumulation along Antarctic benthic food webs in the Ross Sea. Their polymer composition (of polyethylene terephthalate and cellulose-polyamide composites) matched those of outdoor technical clothing in use by the personnel of the Italian "Mario Zucchelli" station near Terra Nova Bay in the Ross Sea. Such findings indicate that sewage from base stations may act as potential local sources of textile microplastic fibers in this remote environment. More in-depth monitoring studies aiming at defining the extent of microplastic contamination related to such sources in Antarctica are encouraged.

Soft X-ray induced radiation damage in thin freeze-dried brain samples studied by FTIR microscopy.

In order to push the spatial resolution limits to the nanoscale, synchrotron-based soft X-ray microscopy (XRM) experiments require higher radiation doses to be delivered to materials. Nevertheless, the associated radiation damage impacts on the integrity of delicate biological samples. Herein, the extent of soft X-ray radiation damage in popular thin freeze-dried brain tissue samples mounted onto Si3N4 membranes, as highlighted by Fourier transform infrared microscopy (FTIR), is reported. The freeze-dried tissue samples were found to be affected by general degradation of the vibrational architecture, though these effects were weaker than those observed in paraffin-embedded and hydrated systems reported in the literature. In addition, weak, reversible and specific features of the tissue-Si3N4 interaction could be identified for the first time upon routine soft X-ray exposures, further highlighting the complex interplay between the biological sample, its preparation protocol and X-ray probe.

15N isotopic labelling for in-cell protein studies by NMR spectroscopy and single-cell IR synchrotron radiation FTIR microscopy: a correlative study.

The ultimate goal of modern structural biology is to probe protein structures and dynamics in their physiological microenvironment. In-cell NMR spectroscopy is an ideal technique for achieving this goal, being able to investigate proteins at atomic-resolution in living cells. The reliability of the results provided by in-cell NMR relies on the selectivity of the labelling methodology coupled with the filtering capabilities offered by heteronuclear NMR experiments. However, solution NMR is not well-suited either for measuring to what extent the non-specific labelling occurs, or to evaluate how it is affected by cell-to-cell variability and, eventually, whether the labelling procedure affects the cellular macromolecular content in general. To answer these questions, we correlated in-cell 1D 1H and 2D 1H-15N NMR experiments on HEK293T cells overexpressing superoxide dismutase 1 (SOD1) with single-cell Synchrotron Radiation FTIR Microscopy (FTIRM) experiments on the same samples. We verified that SOD1 overexpression in 15N-enriched media does not induce modifications in the overall cellular profile, and that the cell-to-cell labelling variability is independent of SOD1 overexpression and is likely cell cycle-related. We concluded that the non-specific incorporation of 15N into cellular components other than the protein of interest is one of the main factors that hinder the possibility of in-cell conformational studies by FTIRM at the single-cell level. Improving labelling selectivity by employing protein insertion approaches, and increasing FTIRM sensitivity by plasmonic enhancement, would open new perspectives for in-cell ultra-sensitive single-protein conformational studies complementing NMR and vibrational analyses.

Perspective: A toolbox for protein structure determination in physiological environment through oriented, 2D ordered, site specific immobilization.

Revealing the structure of complex biological macromolecules, such as proteins, is an essential step for understanding the chemical mechanisms that determine the diversity of their functions. Synchrotron based X-ray crystallography and cryo-electron microscopy have made major contributions in determining thousands of protein structures even from micro-sized crystals. They suffer from some limitations that have not been overcome, such as radiation damage, the natural inability to crystallize a number of proteins, and experimental conditions for structure determination that are incompatible with the physiological environment. Today, the ultra-short and ultra-bright pulses of X-ray free-electron lasers have made attainable the dream to determine protein structures before radiation damage starts to destroy the samples. However, the signal-to-noise ratio remains a great challenge to obtain usable diffraction patterns from a single protein molecule. With the perspective to overcome these challenges, we describe here a new methodology that has the potential to overcome the signal-to-noise-ratio and protein crystallization limits. Using a multidisciplinary approach, we propose to create ordered, two dimensional protein arrays with defined orientation attached on a self-assembled-monolayer. We develop a literature-based flexible toolbox capable of assembling different kinds of proteins on a functionalized surface and consider using a graphene cover layer that will allow performing experiments with proteins in physiological conditions.

Dietary administration of EDC mixtures: A focus on fish lipid metabolism.

Many man-made chemical compounds are recognized as endocrine disruptors and once released into the environment are likely to spread and bioaccumulate in wild species. Due to their lipophilic nature, these substances pass through the cell membrane or bind to specific receptors activating physiological responses that in the long run can cause reproductive impairment, physiological disorders, including the occurrence of metabolic syndromes. One significant source of contamination is represented by the consumption of polluted food. As a consequence, different environmental pollutants, with similar or different modes of action, can accumulate in organisms and biomagnify along the food web, finally targeting humans. The aim of this study was to analyze, under controlled conditions, the effects induced by the consumption of contaminated diets, focusing on the effects exerted at hepatic level. Juvenile seabream were fed for 21days a diet enriched with different combinations of pollutants, nonylphenol (NP), tert-octylphenol (t-OP) and bisphenol A (BPA). The different diets containing 5mg/kg bw of each contaminant, were formulated as follows: NP+tOP, BPA+NP, BPA+tOP and NP+BPA+tOP (NBO). EDCs, at the doses administered, showed low biomagnification factor (BMF), suggesting that these pollutants hardly accumulate in muscles. The results obtained at hepatic level pinpointed the steatotic effect of all the administered diets, associated to a modulation of the expression of genes involved in lipid metabolism (ppars, fas, lpl, and hsl). Results were compared to those obtained in previous studies in which fish were fed single pollutants evidencing that the administration of mixture of contaminants exerts a milder lipogenic effect, highlighting the contrasting/antagonistic interaction establishing among chemicals. Noteworthy was the setup of a new chromatographic method to detect the presence of the selected chemical in fish muscle and the application of Fourier Transform Infrared (FT-IR) analysis to evaluate pollutant-induced changes in the liver macromolecular building.

Soft X-Ray Microscopy Radiation Damage On Fixed Cells Investigated With Synchrotron Radiation FTIR Microscopy.

Radiation damage of biological samples remains a limiting factor in high resolution X-ray microscopy (XRM). Several studies have attempted to evaluate the extent and the effects of radiation damage, proposing strategies to minimise or prevent it. The present work aims to assess the impact of soft X-rays on formalin fixed cells on a systematic manner. The novelty of this approach resides on investigating the radiation damage not only with XRM, as often reported in relevant literature on the topic, but by coupling it with two additional independent non-destructive microscopy methods: Atomic Force Microscopy (AFM) and FTIR Microscopy (FTIRM). Human Embryonic Kidney 293 cells were exposed to different radiation doses at 1 keV. In order to reveal possible morphological and biochemical changes, the irradiated cells were systematically analysed with AFM and FTIRM before and after. Results reveal that while cell morphology is not substantially affected, cellular biochemical profile changes significantly and progressively when increasing dose, resulting in a severe breakdown of the covalent bonding network. This information impacts most soft XRM studies on fixed cells and adds an in-depth understanding of the radiation damage for developing better prevention strategies.

Vibrational characterization of female gametes: a comparative study.

In the last few years, vibrational spectroscopies have been widely applied in biology and medicine, as a synergic support to commonly used analytical and diagnostic techniques. This review summarizes the relevant researches carried out by using FTIR and Raman spectroscopy on oviparous and mammalian gametes, including human ones.

Infrared microspectroscopy of live cells in microfluidic devices (MD-IRMS): toward a powerful label-free cell-based assay.

Until nowadays most infrared microspectroscopy (IRMS) experiments on biological specimens (i.e., tissues or cells) have been routinely carried out on fixed or dried samples in order to circumvent water absorption problems. In this paper, we demonstrate the possibility to widen the range of in-vitro IRMS experiments to vibrational analysis of live cellular samples, thanks to the development of novel biocompatible IR-visible transparent microfluidic devices (MD). In order to highlight the biological relevance of IRMS in MD (MD-IRMS), we performed a systematic exploration of the biochemical alterations induced by different fixation protocols, ethanol 70% and formaldehyde solution 4%, as well as air-drying on U937 leukemic monocytes by comparing their IR vibrational features with the live U937 counterpart. Both fixation and air-drying procedures affected lipid composition and order as well as protein structure at a different extent while they both induced structural alterations in nucleic acids. Therefore, only IRMS of live cells can provide reliable information on both DNA and RNA structure and on their cellular dynamic. In summary, we show that MD-IRMS of live cells is feasible, reliable, and biologically relevant to be recognized as a label-free cell-based assay.

Congenital defects in assisted reproductive technology pregnancies.

AIM: The aim of this study was to evaluate the prevalence and kind of congenital malformations in assisted reproductive technology (ART) pregnancies. METHODS: This study included pregnancies conceived by in-vitro fertilization (IVF) or intra cytoplasmic sperm injection (ICSI), evaluated in a referral center for prenatal diagnosis between January 2008 and December 2009. The control group included all the pregnancies examined in the same centre during in the same period of time. A computerized database was used to retrospectively identify the two study groups. RESULTS: The study evaluated 225 IVF or ICSI pregnancies (88 IVF and 137 ICSI). A congenital malformation was diagnosed in 13 pregnancies and the malformation rate was 5.8%. The number of malformed fetuses was similar in the ICSI (5.8%) or the IVF (5.7%) pregnancies. The anatomic districts more frequently involved by malformations were the encephalic one (38.5%) and the limbs (23.1%). In the control group, represented by 5,884 pregnancies, the malformation rate was 2.7%; encephalic (22.2%), urogenital (18.4%), cardiac (13.9%) CONCLUSION: In our experience on congenital defects in ART pregnancies, the encephalic district and the limbs are more frequently involved by fetal malformation. No significant difference was observed in the malformation prevalence between the IVF and the ICSI pregnancies.

Crystallization and preliminary X-ray analysis of two pH-dependent forms of cytochrome c2 from Rhodopseudomonas palustris.

Cytochrome c(2) from Rhodopseudomonas palustris has been crystallized in two different crystal forms: a monoclinic form I at pH 4.4 from both reduced and oxidized protein solution and a trigonal form II at pH 9.0 from reduced protein solution. Complete 1. 7 and 1.4 A resolution data sets were collected from the oxidized form I and from the form II, respectively. The preliminary structures show an important change in the iron coordination environment in the trigonal form obtained at basic pH arising from the substitution of the Met ligand by an ammonia molecule.

Evaluating psychiatric morbidity in a general hospital: a two-phase epidemiological survey.

BACKGROUND: This study aimed to assess psychiatric morbidity and to collect information on disability, life events and family support in a representative sample of patients admitted to a general hospital. METHODS: On the basis of information collected in a pilot study a systematic sample of patients consecutively admitted to seven general medical and seven surgical wards of the Academic General Hospital of Verona was selected and interviewed using a two-phase screening procedure and standardized instruments (GHQ-12, HADS, BDQ and CIDI-PHC). All data were analysed using appropriately weighted logistic regression procedures. RESULTS: A total of 1039 patients completed the GHQ-12 and 298 (28.7%) were high-scorers: 363 patients were interviewed with CIDI-PHC. The prevalence of ICD-10 cases was 26.1%. The most common psychiatric diagnoses were current depression (12.8%) and generalized anxiety disorder (10.8%), followed by alcohol related disorders (5 %). A higher prevalence of ICD-10 cases was found in medical wards, among females, patients older than 24 years, unemployed and separated/divorced people. Life events were associated with psychopathology, and so was the number of disability days. Although 49.8% of ICD-10 cases were identified by the hospital doctors as having a psychological disorder, 23.1% of ICD-10 cases were referred to the liaison psychiatric service. CONCLUSION: The results of the present study stress the need to collect epidemiologically-based data on psychological disorders and their recognition not only in general practice, but also in general hospital settings, in order to have a more complete picture of the pathways to specialist care.

The inhibition of a leaf proteinase by L-lysine homopolymers.

The role of interlinked positively charged amino acids in the mechanism of inhibition of a monomeric trypsin-like proteinase has been investigated using high molecular mass L-lysine homopolymers ranging from 3.8 to 109 kDa. The data show that the degree of polymerization enhances the inhibitory efficiency which is maximal for homopolymers with more than eighteen interlinked lysine residues. The inhibition is cooperative and, under the maximal inhibition conditions, nine lysine residues of the polymer are involved in the electrostatic binding to the enzyme. A limited conformational change of the protein molecule accompanies the transition from a fully active to a fully inactivated enzyme.

Clinical significance of mitral regurgitation in patients with recent myocardial infarction.

The prognostic implications of the presence of mitral regurgitation (MR) in patients with recent myocardial infarction has not been clarified yet. In March 1983, we undertook a prospective study in patients surviving a first episode of acute myocardial infarction. Over a 4-year period, 266 patients entered the study. Left ventriculography documented the presence of MR in 51 patients, while 215 did not have angiographic evidence of MR. The presence of MR was associated with larger infarcts, as shown by greater values of peak CK (P less than 0.05) and by the prevalence of Q-wave vs non-Q-wave infarctions (P less than 0.05). Transient left ventricular failure during hospitalization was more frequent in patients with MR (P less than 0.05), while the occurrence of early post-infarction angina was similar in the two groups of patients. No difference was found in the extent of coronary disease, yet patients with MR had higher values of left ventricular end diastolic pressure (LVEDP) (P less than 0.005) and a lower ejection fraction (EF) (P less than 0.001). Patients with MR had a reduced exercise capacity (P less than 0.005), but signs of myocardial ischaemia were similarly distributed in the two groups. Patients with anterior infarcts and MR had higher left ventricular volumes than patients without MR, while no difference was found between patients with and patients without MR and inferior infarction, suggesting that left ventricular dilatation may play an important role in the pathogenesis of MR in patients with anterior but not in those with inferior infarction.(ABSTRACT TRUNCATED AT 250 WORDS)

Clinical and angiographic data in early post-infarction angina.

The preliminary results of a prospective study undertaken in patients with a first episode of acute myocardial infarction are presented. The clinical, electrocardiographic and angiographic characteristics of 34 patients who developed early post-infarction angina (group I) were compared with those of 144 patients who remained asymptomatic after the acute infarct (group II). No difference was found between the two groups as to age, sex prevalence, risk factors, presence of stable angina before infarction, severe ventricular arrhythmias or transient congestive heart failure in CCU and peak CK value. Patients of group I had more frequently ecg signs of myocardial infarction in anterior leads (P less than 0.01) than patients of group II. Exercise testing, performed by 23 patients of group I and by 140 patients of group II, was positive in 14 patients with early post-infarction angina and in 37 who remained asymptomatic after the acute infarct (P less than 0.01). The two groups had similar values of left ventricular end diastolic volume index, left ventricular end diastolic pressure and ejection fraction. Patients with early post-infarction angina however had more frequently double or triple vessel disease, while single vessel disease prevailed in group II patients. Although the follow-up period was limited (average 9 months), a higher incidence of cardiac complications was found in group I patients (P less than 0.01). These data show that early post-infarction angina defines a high-risk subset of patients among those with recent myocardial infarction. A more aggressive approach, including thrombolytic therapy and coronary angioplasty, seems warranted.

[Effects of medical and surgical therapy on the long-term survival of patients with variant angina]. / Effetti della terapia medica e chirurgica sulla sopravvivenza a distanza nell'angina variante.

To determine the effects on survival of the medical and surgical treatment of variant angina, we compared the prognosis of 75 surgically treated subjects with that of 75 medically treated patients, selected from a series of 340 consecutive patients observed between January 1969 and December 1982. The patients were selected on the basis of a developed computer program to match each medically treated patient with one surgically treated patient so that each pair was similar according to the following clinical and angiographic variables: sex, age, previous myocardial infarction, severe ventricular arrhythmias during pain, site of ST elevation (anterior or inferior), coronary artery disease (single or multivessel), left ventricular function (normal or abnormal). Patients who were considered unoperable because of poor ventricular function or distal vessel disease were not included in this study. Mantel-Haenszel log-rank analysis demonstrated a significantly better prognosis in surgically treated patients, particularly in those with multivessel disease as well as in those with ST elevation in anterior leads. However survival in 63 medical patients who were treated with calcium-antagonists was not significantly different from that of their surgical matched patients. During the follow-up period, anginal symptoms were more frequently found in medically treated patients (p less than 0.05). We conclude that in patients with variant angina surgical treatment does not improve survival as compared to medical treatment with calcium blocking drugs. Coronary artery bypass surgery can be carried out at low risk and is particularly indicated in those patients with angina refractory to medical treatment.