Current advances in microbiome sciences within the US Department of Defense: part 2 - enabling technologies and environmental microbiomes.

Microbiomes involve complex microbial communities wherein the micro-organisms interact with one another as well as their associated hosts or environmental niches. Much of the characterisation of these communities and the associations have been achieved through 'omics' technologies, such as metagenomics, metaproteomics and metametabolomics, and model systems. Recent research in host-associated microbiomes has been aimed at understanding the role microbes may play in host fitness or conversely how host activities/conditions may perturb the microbial community, which can further affect host health. These studies have led to the investigation of detection, intervention or modulation methods, which may serve to provide benefits to the host and advance our understanding of microbiome associations. With the clear implications on human health and disease, the US Department of Defense (DoD) has made microbiome research a priority, with the founding of the Tri-Service Microbiome Consortium (TSMC) to enhance collaboration, coordination,and communication of microbiome research among DoD organisations and partners in academia and industry. DoD microbiome research focuses mainly on the following themes: (1) human health and performance, (2) environmental microbiomes and (3) enabling technologies. This review provides an update of current DoD microbiome research efforts centred on enabling technologies and environmental microbiomes and highlights innovative research being done in academia and industry that can be leveraged by the DoD. These topics were also communicated and further discussed in the Fifth Annual TSMC Symposium. This paper forms part of the special issue of BMJ Military Health dedicated to personalised digital technology for mental health in the Armed Forces.

Current advances in microbiome sciences within the US Department of Defense-part 1: microbiomes for human health and performance.

Microbiomes involve complex microbial communities where the microorganisms interact with one another as well as their associated hosts or environmental niches. The characterisation of these communities and associations have largely been achieved through 'omics' technologies, such as metagenomics, metaproteomics and metametabolomics, and model systems. Recent research in host-associated microbiomes have been aimed at understanding the roles microbes may play in host fitness or conversely how host activities/conditions may perturb the microbial community, which can further affect host health. These studies have led to the investigation of detection, intervention or modulation methods, which may serve to provide benefits to the host and advance our understanding of microbiome associations. With the clear implications on human health and disease, the US Department of Defense (DoD) has made microbiome research a priority, with the founding of the Tri-Service Microbiome Consortium (TSMC) to enhance collaboration, coordination and communication of microbiome research among DoD organisations and partners in academia and industry. DoD microbiome research focuses mainly on the following themes: (1) Human health and performance; (2) Environmental microbiomes; and (3) Enabling technologies. This review provides an update of current DoD microbiome research efforts centred on human health and performance and highlights innovative research being done in academia and industry that can be leveraged by the DoD. These topics were also communicated and further discussed during the fifth Annual TSMC Symposium. This paper forms part of the special issue of BMJ Military Health dedicated to Personalised Digital Technology for Mental Health in the Armed Forces.

Resynthesis of sternal dehiscence with autologous bone graft and autologous platelet gel.

Postoperative management of sternal dehiscence requires the organised effort of a multidisciplinary medical team, including orthopaedic surgeons, plastic surgeons, microbiologists, critical care nurses and rehabilitation experts. Clinical care of this complication impacts heavily on health-care costs, length of hospital stay, and the time to full recovery and return to regular work activity. There are various surgical approaches to sternal resynthesis, but they are often unsuccessful. In this paper, we describe the case of a 67-year-old male complaining of chronic pain due to sternal dehiscence after coronary artery bypass grafting surgery. We first report a technique for sternal resynthesis, performed in the cardiac surgery setting, using a combination of autologous bone graft and autologous platelet-derived gel (APG), and describe its postoperative management and outcome. The four-month follow-up was uneventful and a CT scan confirmed full healing of the nonunion site with solid bridging bone.

The quenching of photosystem II fluorescence does not protect the D1 protein against light induced degradation in thylakoids.

In spinach thylakoids, the quenching of the singlet excited state in the photosystem II antenna by m-dinitrobenzene does not change the rate of the light induced degradation of the D1 reaction centre protein and offers only limited protection against photoinhibition itself. These results are discussed in terms of the role of non-photochemical quenching as a photoprotective strategy.

Responses to bleaching herbicides by leaf chloroplasts of maize plants grown at different temperatures.

The effects of growth temperature on chloroplast responses to norflurazon and amitrole, two herbicides inhibiting carotenogenesis, at phytoene desaturation and lycopene cyclization, respectively, were studied in leaves of maize plants grown at 20 degrees C and 30 degrees C in light. At the lower temperature both chemicals caused severe photo-oxidative damage to chloroplasts. In organelles of norflurazon-treated leaves neither carotenoids nor chlorophylls were detectable and the thylakoid system was dismantled. In organelles of amitrole-treated leaves lycopene was accumulated, but small quantities of beta-carotene and xanthophylls were also produced. Moreover, some chlorophyll and a few inner membranes still persisted, although these latter were disarranged, lacking essential protein components and devoid of photosynthetic function. The increase in plant growth temperature to 30 degrees C did not change the norflurazon effects on carotenoid synthesis and the photo-oxidative damage suffered by chloroplasts. By contrast, in organelles of amitrole-treated leaves a large increase in photoprotective carotenoid biosynthesis occurred, with a consequent recovery of chlorophyll content, ultrastructural organization and thylakoid composition and functionality. This suggests that thermo-modulated steps could exist in the carotenogenic pathway, between the points inhibited by the two herbicides. Moreover it shows that, unlike C(3) species, C(4) species, such as maize, can express a strong tolerance to herbicides like amitrole, when supplied to plants growing at their optimum temperature conditions.

The primary structure of globin and linker chains from the chlorocruorin of the polychaete Sabella spallanzanii.

Annelid hemoglobins are organized in a very complex supramolecular network of interacting polypeptides, the structure of which is still not wholly resolved. We have separated by two-dimensional electrophoresis the 4-MDa chlorocruorin of Sabella spallanzanii and identified its components by amino-terminal sequencing. This work reveals a high rate of heterogeneity of constituent chains in a single animal as well as in the Sabella population. Using a cDNA library prepared from the hematopoietic tissue of this worm, we have isolated and fully sequenced most globin and linker cDNAs. The primary structure features of these polypeptides have been characterized by comparison with model globin and linker sequences.

The evolution of extracellular hemoglobins of annelids, vestimentiferans, and pogonophorans.

The evolution of extracellular hemoglobins of annelids, vestimentiferans, and pogonophorans was investigated by applying cladistic and distance-based approaches to reconstruct the phylogenetic relationships of this group of respiratory pigments. We performed this study using the aligned sequences of globin and linker chains that are the constituents of these complex molecules. Three novel globin and two novel linker chains of Sabella spallanzanii described in an accompanying paper (Pallavicini, A., Negrisolo, E., Barbato, R., Dewilde, S., Ghiretti-Magaldi, A., Moens, L., and Lanfranchi, G. (2001) J. Biol. Chem. 276, 26384--26390) were also included. Our results allowed us to test previous hypotheses on the evolutionary pathways of these proteins and to formulate a new most parsimonious model of molecular evolution. According to this novel model, the genes coding for the polypeptides forming these composite molecules were already present in the common ancestor of annelids, vestimentiferans, and pogonophorans.

Photoinhibition of Chlamydomonas reinhardtii in State 1 and State 2: damages to the photosynthetic apparatus under linear and cyclic electron flow.

The relationship between state transitions and photoinhibition has been studied in Chlamydomonas reinhardtii cells. In State 2, photosystem II activity was more inhibited by light than in State 1. In State 2, however, the D1 subunit was not degraded, whereas a substantial degradation was observed in State 1. These results suggest that photoinhibition occurs via the generation of an intermediate state in which photosystem II is inactive but the D1 protein is still intact. The accumulation of this state is enhanced in State 2, because in this State only cyclic photosynthetic electron transport is active, whereas there is no electron flow between photosystem II and the cytochrome b(6)f complex (Finazzi, G., Furia, A., Barbagallo, R. P., and Forti, G. (1999) Biochim. Biophys. Acta 1413, 117-129). The activity of photosystem I and of cytochrome b(6)f as well as the coupling of thylakoid membranes was not affected by illumination under the same conditions. This allows repairing the damages to photosystem II thanks to cell capacity to maintain a high rate of ATP synthesis (via photosystem I-driven cyclic electron flow). This capacity might represent an important physiological tool in protecting the photosynthetic apparatus from excess of light as well as from other a-biotic stress conditions.

Minor head injuries: one year experience according to the new Italian guideline.

BACKGROUND: The data concerning a consecutive series of 4,536 adult patients suffering from minor head injuries treated at the Department of Neurosurgery over a period of one year are reported. METHOD: The patients' age, sex and the circumstances of the injury have been taken into consideration. The patients, according to the new method, were divided into four groups. Group 0 (3,864 patients) included all patients with Glasgow Coma Scale (GCS) score 15. They did not present any clinical features such as loss of consciousness (LOC), post-traumatic amnesia (PTA), headache or vomiting. No risk factors (RF) such as coagulopaties, alcoholism, drug abuse, epilepsy, previous neurological treatment or disabled elderly patients were detected. Group 1 (600 patients) included patients with GCS score 15. The patients presented one or more clinical features (LOC, PTA, headache, vomiting). No RF were presented. Group 2 (24 patients) included patients with GCS score 14 with or without clinical features (LOC, PTA, headache, vomiting) and with or without RF. Group 0-1R (48 patients) included patients with GCS score 15 with or without clinical features (LOC, PTA, headache, vomiting). All of them presented RF. The presence of focal neurological signs, open injury and GCS score < or = 13 were considered criteria for exclusion. FINDINGS: All the patients from groups 1, 0-1R, 2 and 187 patients from group 0 underwent CT scan for a total of 859 exams which are analyzed and discussed. 458 patients were admitted and are divided as follows: 216 from group 0, 192 from group 1, 26 from group 0-1R and all the 24 belonging to group 2. Six patients were treated surgically (3 extradural haematomas, 2 lobe contusions, 1 acute subdural haematoma) and one of them (0.02% of the total) died (extradural haematoma). The patients who were not admitted were sent home with an information sheet after at least a six hour observation period. INTERPRETATION: The authors draw the conclusion that they have evaluated the applicability and efficacy of guidelines, developed by the study group on head injury of the Italian Society of Neurosurgery. A critical part of our guidelines is not only to identify all the intracranial lesions, but to identify patients harbouring relevant intracranial mass before clinical deterioration.

Cab gene expression in bleached leaves of carotenoid-deficient maize.

The chloroplast photo-oxidation and the expression of the Cab gene Lhcb1, encoding the Lhcb1 light-harvesting chlorophyll a/b-protein of PS II, have been studied in leaf cells of maize treated with the two bleaching herbicides norflurazon and amitrole and of the two carotenoid-free mutants vp9 and vp2 grown under high photodamaging light. Both herbicides and mutations caused severe photo-oxidation of organelles. However, the plastids of norflurazon-treated and vp2 leaves were totally devoid of thylakoids and did not contain any chlorophyll, while the organelles of amitrole-treated and vp9 leaves still had a few altered and photosynthetically unfunctional membranes and very small quantities of chlorophylls. Despite the dramatic photodamage undergone by the plastids over several days, the cells of amitrole-treated and vp9 leaves maintained a certain expression of the Lhcb1 gene which, on the contrary, was completely blocked in the cells of norflurazon-treated and vp2 leaves. The experimental results, obtained by integrating biochemical and molecular analyses with ultrastructural observations, show that the maintainance of Cab gene expression does not strictly depend on intact and functional chloroplasts. The transcription of these genes, still maintained in cells with greatly photo-oxidized organelles, seems to be inversely related to the degree of thylakoid demolition, which can affect the last steps of chlorophyll biosynthesis.

Ultraviolet B exposure of whole leaves of barley affects structure and functional organization of photosystem II.

This study examines the effects of ecologically important levels of ultraviolet B radiation on protein D1 turnover and stability and lateral redistribution of photosystem II. It is shown that ultraviolet B light supported only limited synthesis of protein D1, one of the most important components of photosystem II, whereas it promoted significant degradation of proteins D1 and D2. Furthermore, dephosphorylation of photosystem II subunits was specifically elicited upon exposure to ultraviolet B light. Structural modifications of photosystem II and changes in its lateral distribution between granum membranes and stroma-exposed lamellae were found to be different from those observed after photoinhibition by strong visible light. In particular, more complete dismantling of photosystem II cores was observed. Altogether, the data reported here suggest that ultraviolet B radiation alone fails to activate the photosystem II repair cycle, as hypothesized for visible light. This failure may contribute to the toxic effect of ultraviolet B radiation, which is increasing as a consequence of depletion of stratospheric ozone.

Effects of ultraviolet-B light on photosystem II phosphoproteins in barley wild type and its chlorophyll b-less mutant chlorina f2.

The effects of ultraviolet-B light on the level and steady-state phosphorylation of photosystem II proteins have been studied in barley wild type and its chlorophyll b-less mutant chlorina f2. In the wild type, ultraviolet-B radiation is found to promote dephosphorylation of all thylakoid phosphoproteins. In addition, for reaction-centre proteins D1 and D2, dephosphorylation is paralleled by degradation. Photosystem II core proteins in the mutant are not found to be significantly phosphorylated in any experimental conditions, and loss of D1 and D2 reaction-centre proteins is slightly faster than in the wild type. These results are consistent with the possibility that phosphorylation of reaction-centre proteins affects their stability, possibly by slowing down the rate of degradation, as in the case of visible light.

Extradural low cervical chordoma. Case report.

Chordoma of the mobile segments of the spine are infrequent lesions and especially rare are those located in the lower part of the cervical spine. We present the case of a cervical chordoma located in the C6 vertebral body diagnosed by means of magnetic resonance imaging and operated on by an anterior approach. The authors discuss the clinical and neuroradiological features of this disease also analysing some controversial therapeutic aspects.

Cytochrome b6/f complex from the cyanobacterium Synechocystis 6803: evidence of dimeric organization and identification of chlorophyll-binding subunit.

Fractionation of photosynthetic membranes from the cyanobacterium Synechocystis 6803 by polyacrylamide gel electrophoresis in the presence of Deriphat-160 allowed the isolation of a number of pigmented bands. Two of them, with molecular masses of 240+/-20 and 110+/-15 kDa respectively, showed peroxidase activity and, by means of polypeptide composition, immunoblotting and N-terminal sequencing, were identified as dimeric and monomeric cytochrome b6/f complexes, containing 1.3+/-0.35 chlorophyll molecules per cytochrome f. Further fractionation of monomeric complexes by mild gel electrophoresis in the presence of sodium dodecyl sulfate indicated that it is the cytochrome b6 polypeptide which provides the actual binding site for the chlorophyll molecule observed in the complex.

Effects of ultraviolet-B radiation on photosystem II of the cyanobacterium Synechocystis sp. PCC 6083.

The effects of ultraviolet-B radiation (280-320 nm) on photosystem II of Synechocystis sp. PCC 6303 were investigated at the functional and structural levels. Loss of oxygen-evolving and electron-transport activity, measured by various techniques including Clark electrode polarography, fluorescence induction and fluorescence relaxation after a single turnover flash, are discussed in terms of two types of damage caused by ultraviolet-B radiation: (a) depletion of the plastoquinone pool; (b) perturbation and degradation of the D1 protein, with cleavage in the second transmembrane segment. These findings are in full agreement with those obtained, both in vivo and in vitro for higher plants for which a donor-side mechanism involving the water-splitting Mn cluster has been proposed for the main cleavage of the D1 protein. At the structural level, complete disruption of the photosystem II core is documented as a consequence of (or in parallel with) degradation of the D1 protein. From this point of view, ultraviolet-B-induced photoinhibition is unlike the visible-induced type and less susceptible to repair by synthesis and reinsertion of new D1 protein.

Binding of cytosolic proteins to myofibrils in ischemic rat hearts.

Myofibrillar proteins (MPs) were extracted from isolated and perfused rat hearts subjected to different periods of ischemia to investigate the occurrence of protein degradation and/or the association of cytosolic proteins with the myofibrillar pellet. A 23-kD band was detected by SDS-PAGE of MPs after 5 minutes of ischemia, with its density gradually increasing to a plateau after 20 minutes. Longer periods of ischemia were associated with the appearance of a 39-kD band. Irrespective of the duration of ischemia, both these bands persisted during reperfusion. A partial proteolytic degradation of troponin T (TnT) and troponin I (TnI) has been claimed to be responsible for the generation of these peptides. However, the N-terminal sequence of the 39-kD band was identical to that of GAPDH, whereas Edman sequencing after pepsin digestion showed that the 23 kD is alpha B-crystallin. The binding of the two cytosolic proteins to myofibrils was confirmed by immunofluorescence analysis on cryosections of ischemic hearts. In vitro studies showed that acidosis was sufficient to induce the binding of alpha B-crystallin, whereas the inhibition of ATP depletion prevented the binding of GAPDH. Thiol oxidation is unlikely to promote GAPDH binding, since perfusion with iodoacetate under aerobic conditions or treatment of homogenates with N-ethylmaleimide or diamide failed to induce GAPDH association with the myofibrils. These changes of the myofibrillar proteins could be considered as intracellular markers of the evolution of the ischemic damage. In addition, the binding of the 23-kD peptide might be involved in alterations of contractility.

Carnitine-related alterations in patients with intermittent claudication: indication for a focused carnitine therapy.

BACKGROUND: Carnitine metabolism is altered in peripheral arterial disease. L-carnitine supplementation may correct these alterations and improve walking performance. METHODS AND RESULTS: Plasma levels of carnitine and its esters were measured at rest and after maximally tolerated exercise in 22 claudicant patients and 8 normal subjects. One week later, this protocol was repeated in patients after random administration of placebo or L-carnitine (500 mg IV as a single bolus). Two groups of patients emerged. In 10 patients (group IC1), the plasma level of acetylcarnitine at rest was 3.7 +/- 0.2 micromol/L and increased significantly (P<.01) at maximally tolerated exercise. In 12 patients (group IC2), the resting level of plasma acetylcarnitine was elevated (7.9 +/- 0.7 micromol/L, P<.01) and decreased with exercise. Furthermore, group IC2 patients had a significantly lower walking capacity than group IC1 patients. In both groups, placebo did not affect the metabolic profile, nor did it improve exercise performance. Conversely, after L-carnitine administration, all but one patient in group IC2 (n=7) showed an increase in plasma acetylcarnitine concentration during exercise versus the decrease observed without L-carnitine. This metabolic effect was accompanied by a significant increase (P<.01) in walking capacity. Interestingly, in group IC1 patients (n=5), L-carnitine neither improved walking capacity nor modified the metabolic profile. Statistical analysis showed that changes in walking capacity with L-carnitine treatment were influenced exclusively by exercise-induced changes in plasma acetylcarnitine. CONCLUSIONS: In patients with intermittent claudication, assessment of plasma acetylcarnitine at rest and after exercise may be a means to select a target population for L-carnitine therapy.

Pigment-protein complexes from the photosynthetic membrane of the cyanobacterium Synechocystis sp. PCC 6803.

Photosystem I and II core complexes were resolved in a single step from the thylakoid membrane of Synechocystis sp. PCC 6803 by using a mild solubilization procedure in dodecyl beta-D-maltoside and Deriphat/PAGE. For each photosystem, two green bands were obtained containing oligomeric and monomeric forms of the core complexes of either photosystem. The oligomers are likely to be trimers in the case of photosystem I and dimers for photosystem II. The absorption spectra, polypeptide and pigment composition of green bands corresponding to either photosystem I or photosystem II were identical for monomeric and oligomeric forms. The cytochrome b-559 content of photosystem II was evaluated to be one cytochrome b-559/reaction centre both in the monomeric and dimeric forms. Two new 15-kDa and 22-kDa carotenoid-binding protein were isolated and their polypeptides purified to homogeneity.

Characterization of the light-induced cross-linking of the alpha-subunit of cytochrome b559 and the D1 protein in isolated photosystem II reaction centers.

Illumination of the isolated reaction center of photosystem II generates a protein of 41 kDa molecular mass. Using immunoblotting, it is confirmed that the protein is an adduct of the D1 protein and the alpha-subunit of cytochrome b559. Its formation seems to be photochemically induced, being independent of temperature between 4 and 20 degrees C and unaffected by a mixture of protease inhibitors. The maximum levels are detected when the pH is in the region 6.5-8.5 and when illumination intensities are moderate. Although higher light intensities induce a higher rate of formation, the accumulation of elevated levels of the 41-kDa protein does not occur due to light-induced degradation. This degradation is also unaffected by the presence of protease inhibitors. Proteolytic mapping and N-terminal sequencing indicates that the cross-linking process involves the N-terminal serine of the alpha-subunit of cytochrome b559 and D1 residues in the 239-244 FGQEEE motif close to the QB binding site. In conclusion, the results indicate that the N terminus of the alpha-subunit is exposed on the stromal side of photosystem II in such a way as to undergo light-induced cross-linking in the QB region of the D1 protein. They also suggest that the 41-kDa adduct may be an intermediate before the light-induced cleavage of the D1 protein in the FGQEEE region.