Clostridium thermocellum: A microbial platform for high-value chemical production from lignocellulose.

Second generation biorefining, namely fermentation processes based on lignocellulosic feedstocks, has attracted tremendous interest (owing to the large availability and low cost of this biomass) as a strategy to produce biofuels and commodity chemicals that is an alternative to oil refining. However, the innate recalcitrance of lignocellulose has slowed progress toward economically viable processes. Consolidated bioprocessing (CBP), i.e., single-step fermentation of lignocellulose may dramatically reduce the current costs of 2nd generation biorefining. Metabolic engineering has been used as a tool to develop improved microbial strains supporting CBP. Clostridium thermocellum is among the most efficient cellulose degraders isolated so far and one of the most promising host organisms for application of CBP. The development of efficient and reliable genetic tools has allowed significant progress in metabolic engineering of this strain aimed at expanding the panel of growth substrates and improving the production of a number of commodity chemicals of industrial interest such as ethanol, butanol, isobutanol, isobutyl acetate and lactic acid. The present review aims to summarize recent developments in metabolic engineering of this organism which currently represents a reference model for the development of biocatalysts for 2nd generation biorefining.

Enterococcus faecium NCIMB10415 responds to norepinephrine by altering protein profiles and phenotypic characters.

The long-term established symbiosis between gut microbiota and humans is based upon a dynamic equilibrium that, if unbalanced, could lead to the development of diseases. Despite the huge amount of data concerning the microbiota-gut-brain-axis, little information is available on what happens at the molecular level in bacteria, when exposed to human signals. In the present study, the physiological effects exerted by norepinephrine (NE), a human hormone present in significant amounts in the host gut, were analyzed using the commensal/probiotic strain Enterococcus faecium NCIMB10415 as a target. The aim was to compare the protein profiles of treated and untreated bacteria and relating these proteome patterns to some phenotypic modifications important for bacteria-host interaction. Actually, to date, only pathogens have been considered. Combining a gel-free/label-free proteomic analysis with the evaluation of bile salts resistance, biofilm formation and autoaggregation ability (as well as with the bacterial growth kinetics), allowed to detect changes induced by NE treatment on all the tested probiotic properties. Furthermore, exposure to the bioactive molecule increased the abundance of proteins related to stress response and to host-microbe interaction, such as moonlight proteins involved in adhesion and immune stimulation. The results of this investigation demonstrated that, not only pathogens, but also commensal gut bacteria are affected by host-derived hormones, underlining the importance of a correct cross-signalling in the maintenance of gut homeostasis. SIGNIFICANCE: The crucial role played by the human gut microbiota in ensuring host homeostasis and health is definitively ascertained as suggested by the holobiome concept. The present research was intended to shed light on the endocrinological perturbations possibly affecting microbiota. The microbial model used in this study belongs to Enterococcus faecium species, whose controversial role as gut commensal and opportunistic pathogen in the gut ecosystem is well recognized. The results obtained in the present investigation clearly demonstrate that E. faecium NCIMB10415 can sense and respond to norepinephrine, a human hormone abundant at the gut level, by changing protein profiles and physiology, inducing changes that could favor survival and colonization of the host tissues. To our knowledge, this is the first proteomic report concerning the impact of a human hormone on a commensal/probiotic bacterium, since previous research has focused on exploring the effects of neuroendocrine molecules on growth and virulence of pathogenic species.

Construction of lactic acid overproducing Clostridium thermocellum through enhancement of lactate dehydrogenase expression.

Rapid expansion of global market of lactic acid (LA) has prompted research towards cheaper and more eco-friendly strategies for its production. Nowadays, LA is produced mainly through fermentation of simple sugars or starchy biomass (e.g. corn) and its price is relatively high. Lignocellulose could be an advantageous alternative feedstock for LA production owing to its high abundance and low cost. However, the most effective natural producers of LA cannot directly ferment lignocellulose. So far, metabolic engineering aimed at developing microorganisms combining efficient LA production and cellulose hydrolysis has been generally based on introducing designer cellulase systems in natural LA producers. In the present study, the approach consisted in improving LA production in the natural cellulolytic bacterium Clostridium thermocellum DSM1313. The expression of the native lactate dehydrogenase was enhanced by functional replacement of its original promoter with stronger ones resulting in a 10-fold increase in specific activity, which resulted in a 2-fold increase of LA yield. It is known that eliminating allosteric regulation can also increase lactic acid production in C. thermocellum, however we were unable to insert strong promoters upstream of the de-regulated ldh gene. A strategy combining these regulations and inactivation of parasitic pathways appears essential for developing a homolactic C. thermocellum.

Influence of ethanol, malate and arginine on histamine production of Lactobacillus hilgardii isolated from an Italian red wine.

Wine, like other fermented foods, may contain biogenic amines produced by lactic acid bacteria via amino acids decarboxylation. The most relevant amines from the toxicological standpoint are histamine and tyramine. The complexity of fermented substrates makes it difficult to suggest a priori how variables can modulate amine production. Lactobacillus hilgardii ISE 5211 was isolated from an Italian red wine. Besides producing lactate from malate, this strain is also able to convert arginine to ornithine and histidine to histamine. In the present investigation we studied the influence of malate, arginine and ethanol on histamine accumulation by L. hilgardii ISE 5211. Ethanol concentrations above 13% inhibit both histamine accumulation and bacterial growth; concentrations below 9% affect neither growth nor histamine production. However, an ethanol concentration of 11% allows a low but continuous accumulation of histamine to occur. Arginine also delays histamine accumulation, while malate appears to have no effect on histidine-histamine conversion.

Ten years of subproteome investigations in lactic acid bacteria: A key for food starter and probiotic typing.

The definition of safety and efficacy of food-employed bacteria as well as probiotic strains is a continuous, often unattended, challenge. Proteomic techniques such as 2DE, DIGE and LC/LC-MS/MS are suitable and powerful tools to reveal new aspects (positive and negative) of "known" and "unknown" strains that can be employed in food making and as nutraceutical supplements for human health. Unfortunately, these techniques are not used as extensively as it should be wise. The present report describes the most significant results obtained by our research group in 10years of study on subproteomes in bacteria, chiefly lactic acid bacteria. Production of desired and undesired metabolites, differences between strains belonging to same species but isolated from different ecological niches, the effect of cryoprotectants on survival to lyophilization as well as the adhesive capability of strains, were elucidated by analysis of cytosolic, membrane-enriched, surface and extracellular proteomes. The present review opens a window on a yet largely underexplored field and highlights the huge potential of subproteome investigations for more rational choice of microbial strains as food starters, probiotics and for production of nutraceuticals. These analyses will hopefully contribute to manufacturing safer and healthier food and food supplements in the near future. This article is part of a Special Issue entitled: HUPO 2014.

Eye injuries from merchandise display hooks.

PURPOSE/METHODS: Four patients had ocular or ocular adnexal injuries, which resulted from store merchandise display hooks. RESULTS/CONCLUSIONS: Ocular and periocular injuries from display hooks include conjunctival, eyelid, and canalicular lacerations, as well as penetrating brain injury. Display hooks are commonly used in retail establishments and pose a high risk when placed below eye level.

Anophthalmic socket and orbital implants. Role of CT and MR imaging.

The role of CT and MR imaging examination of orbital implants and the anophthalmic socket is expanding constantly. As the imaging techniques gain refinement and resolution, the list of potential clinical application grows. Frequent clinical-radiologic queries regarding the anophthalmic socket include neuro-radiologic findings with congenital anophthalmia or bilateral microphthalmia, the vascularization or position of an orbital implant following enucleation, or orbital recurrence of tumor following enucleation. Common clinical-radiologic questions regarding orbital implants relate to the associated findings of trauma, infection, bleeding, or recurrence of tumor in relationship to the existing orbital implant.

Characteristics of Okihiro syndrome.

PURPOSE: To describe Okihiro syndrome, a syndrome consisting of Duane's syndrome, radial ray anomaly, and sensorineural deafness. METHODS: The incidence of anomalies in 41 individuals from 9 affected families was assessed by literature review and patient examination, and the frequency of the characteristics occurring together was determined. RESULTS: Of all the individuals with at least one of the above characteristics, only 12% had all three characteristics. However, 44% had both Duane's syndrome and radial ray anomaly, whereas sensorineural deafness alone occurred in only 17% of patients. CONCLUSION: Okihiro syndrome is consistent with autosomal dominance with variable penetrance, manifesting primarily with Duane's syndrome and radial ray anomaly, and less frequently, sensorineural deafness.

Chorioretinitis sclopetaria from BB ex memoria.

Chorioretinitis sclopetaria presents a characteristic pattern of choroidal and retinal changes caused by a high velocity projectile passing into the orbit, in close proximity to the globe. While it is unlikely that a patient should completely forget the trauma causing such damage, preserved or compensated visual function may blur the patient's memory of these events over time. Characteristic physical findings help to clarify the antecedent history. Despite the lack of an acknowledged history of ocular trauma or surgery, in our case, the characteristic ocular findings discovered at presentation allowed for recognition of the underlying etiology. Because of good visual function, the patient had completely forgotten about the trauma that occurred 12 years earlier. Strabismus surgery was performed for treatment of the presenting symptomatic diplopia. The pathognomonic findings in chorioretinitis sclopetaria are invaluable in correctly diagnosing this condition, especially when a history of ocular trauma is unavailable.

Ocular anesthesia for cataract surgery: a direct sub-Tenon's approach.

We describe a new technique in which ocular anesthesia is obtained by dissection of the superior quadrants, as in strabismus or retinal surgery, followed by direct infusion of the retrobulbar space with local anesthetic agent, using a blunt 19-gauge cannula. In 112 cases of cataract surgery in which we used this direct sub-Tenon's approach, we found it to be a safe, simple, and effective method of achieving rapid ocular anesthesia.

Purification and catalytic properties of two catechol 1,2-dioxygenase isozymes from benzoate-grown cells of Acinetobacter radioresistens.

Two catechol 1,2-dioxygenase (C1,2O) isozymes (IsoA and IsoB) have been purified to homogeneity from a strain of Acinetobacter radioresistens grown on benzoate as the sole carbon and energy source. IsoA and IsoB are both homodimers composed of a single type of subunit with molecular mass of 38,600 and 37,700, Da respectively. In conditions of low ionic strength, IsoA can aggregate as a trimer, in contrast to IsoB, which maintains the dimeric structure, as also supported by the kinetic parameters (Hill numbers). IsoA is identical to the enzyme previously purified from the same bacterium grown on phenol, whereas the IsoB is selectively expressed using benzoate as carbon source. This is the first evidence of the presence of differently expressed C1,2O isozymes in A. radioresistens or more generally of multiple C1,2O isozymes in benzoate-grown Acinetobacter cells. Purified IsoA and IsoB contain approximately 1 iron(III) ion per subunit and both show electronic absorbance and EPR features typical of Fe(III) intradiol dioxygenases. The kinetic properties of the two enzymes such as the specificities toward substituted catechols, the main catalytic parameters, and their behavior in the presence of different kind of inhibitors are, unexpectedly, very similar, in contrast to most of the previously known dioxygenase isozymes.

[Ventricular pre-excitation syndrome: anesthesiologic aspects]. / Sindrome da preeccitazione ventricolare: aspetti anestesiologici.

Two cases of pre-excitation syndrome are reported and the anaesthesiological management adopted is discussed. After a brief overview of the pathophysiology of the syndrome relative indications and contraindications of the main anaesthetic drugs and volatile agents are analyzed. The high incidence of perioperative atrial fibrillation, atrial flutter or supraventricular tachycardia and the importance of maintaining sinus rhythm is recalled. The task confronting the anaesthetist is to ensure an adequate level of anaesthesia during induction and maintenance while carefully selecting specific drugs associated with minor cardiovascular side effects. In the Authors' experience benzodiazepines, fentanyl, vecuronium and isoflurane have proved to be the most adequate choice for this type of patient.

Benign mixed tumor arising from an accessory lacrimal gland of Wolfring.

We report a 59-year-old man who had a slowly enlarging mass of the central upper eyelid that proved histopathologically to be a benign mixed tumor (pleomorphic adenoma) arising from an accessory lacrimal gland of Wolfring. The tumor was totally separate from the main lacrimal gland, and its deep location in the lid excluded origin from dermal sweat glands. Benign mixed tumors of the accessory lacrimal glands are exceedingly rare tumors of the ocular adnexa.

Gaze-induced amaurosis from central retinal artery compression.

Color Doppler imaging was used to evaluate a patient with gaze-induced amaurosis caused by an intraconal orbital mass. The time-velocity waveform demonstrated abnormally high vascular resistance in the central retinal artery of the affected eye in the primary position. Abduction of the affected eye resulted in transient visual loss with an unreactive pupil. This same maneuver during color Doppler imaging resulted in a dramatic reduction of blood flow in the central retinal artery. Two months after surgical excision of the mass, the gaze-evoked amaurosis was no longer present, and color Doppler imaging demonstrated normal blood flow in the central retinal artery. This suggests that impaired retinal and optic nerve blood flow are responsible for gaze-induced amaurosis from compressive orbital lesions.

Monocanalicular intubation for dacryostenosis in oculo-auriculo-vertebral dysplasia (hemifacial microsomia) with congenital corneal anesthesia.

PURPOSE: To present a case of oculo-auriculo-vertebral dysplasia associated with corneal anesthesia and ipsilateral dacryostenosis that was successfully treated with monocanalicular lacrimal intubation. METHODS: Case report. RESULTS: Previous neurotrophic corneal ulcers in a child with hemifacial microsomia had become secondarily infected from a stagnant tear lake, resulting in significant corneal scarring and visual loss. A single monocanalicular stent maintained nasolacrimal patency without causing further corneal trauma, despite constant medial gaze because of wide lateral tarsorrhaphy and contralateral occlusion therapy for amblyopia. CONCLUSIONS: Monocanalicular stents may aid the treatment of dacryostenosis in the face of compromised corneal sensation.

The diagnosis and management of orbital blowout fractures: update 2001.

A cogent update of orbital blowout history, anatomy, and management are included with a retrospective study of 59 pure orbital blowout fractures which occurred between 1994 and 1998. Our goal is to provide a better understanding of this frequently encountered entity and to help augment the confidence of nonophthalmologists who will often evaluate patients with suspected orbital blowout fractures.

The catechol 1,2 dioxygenase system of Acinetobacter radioresistens: isoenzymes, inductors and gene localisation.

Two different isozymes (Iso A and Iso B) of catechol 1,2 dioxygenase (C1,2O) were isolated from cultures of A. radioresistens grown in two different media, containing phenol and benzoate respectively. In the phenol medium the bacteria expressed about 90% of Iso A, whereas in the benzoate medium the Iso A/Iso B ratio was 40:60. The two proteins have different molecular masses, isoelectric points and N-terminal sequences that are not consistent with simple post-translational modifications. Furthermore, their behaviour differs at high temperatures (42 degrees C-47 degrees C) and at moderately acidic pH (pH 6.0): Iso A proved to be the more stable under conditions of environmental stress. Hybridisation analysis with an A. calcoaceticus catA-derived probe revealed that A. radioresistens C1,2O proteins are encoded by two chromosomally located genes. Bidimensional electrophoresis (2DE) maps of crude extracts of cells grown in different carbon sources (phenol, benzoate and acetate) clearly demonstrated a differential induction pattern for the two proteins. The hypothesis of a double set of genes, one for benzoate catabolism and the other for phenol catabolism, is discussed, and analogies are drawn with other known C1,2Os.

Media containing aromatic compounds induce peculiar proteins in Acinetobacter radioresistens, as revealed by proteome analysis.

An Acinetobacter radioresistens strain able to grow on phenol or benzoate as sole carbon and energy source through the beta-ketoadipate pathway was isolated in our laboratories. In previous research, we found a different expression of catechol-1,2-dioxygenase isoenzymes (C-1,2-O) depending on the growth substrate (phenol or benzoate). In the present study, we used proteome techniques to extend our investigation to other enzymes involved in the aromatic degradation pathway. Since the first nontoxic metabolite in this route is cis,cis-muconic acid, we focused our attention on the enzymes leading to this compound, chiefly phenol hydroxylase (PH), benzoate dioxygenase (BD), cis-1,2-dihydroxycyclohexa-3,5-diene-1-carboxylate dehydrogenase (D) and C-1,2-O. In particular, the A. radioresistens proteome was monitored under different growth substrate conditions, using acetate, benzoate, or phenol as sole carbon source. We compared the protein maps by software image analysis and detected marked differences, suggesting the inducibility of most enzymes. This research also sought to evaluate the conditions allowing the best expression of enzymes to be used in immobilized systems suitable for bioremediation. The experimental data indicate that benzoate is the best carbon source to gain the highest amount of C-1,2-O and D, while phenol is the best growth substrate to obtain PH.