The number of catalytic cycles in an enzyme's lifetime and why it matters to metabolic engineering.

Metabolic engineering uses enzymes as parts to build biosystems for specified tasks. Although a part's working life and failure modes are key engineering performance indicators, this is not yet so in metabolic engineering because it is not known how long enzymes remain functional in vivo or whether cumulative deterioration (wear-out), sudden random failure, or other causes drive replacement. Consequently, enzymes cannot be engineered to extend life and cut the high energy costs of replacement. Guided by catalyst engineering, we adopted catalytic cycles until replacement (CCR) as a metric for enzyme functional life span in vivo. CCR is the number of catalytic cycles that an enzyme mediates in vivo before failure or replacement, i.e., metabolic flux rate/protein turnover rate. We used estimated fluxes and measured protein turnover rates to calculate CCRs for ∼100-200 enzymes each from Lactococcus lactis, yeast, and Arabidopsis CCRs in these organisms had similar ranges (<103 to >107) but different median values (3-4 × 104 in L. lactis and yeast versus 4 × 105 in Arabidopsis). In all organisms, enzymes whose substrates, products, or mechanisms can attack reactive amino acid residues had significantly lower median CCR values than other enzymes. Taken with literature on mechanism-based inactivation, the latter finding supports the proposal that 1) random active-site damage by reaction chemistry is an important cause of enzyme failure, and 2) reactive noncatalytic residues in the active-site region are likely contributors to damage susceptibility. Enzyme engineering to raise CCRs and lower replacement costs may thus be both beneficial and feasible.

Using continuous directed evolution to improve enzymes for plant applications.

Continuous directed evolution of enzymes and other proteins in microbial hosts is capable of outperforming classical directed evolution by executing hypermutation and selection concurrently in vivo, at scale, with minimal manual input. Provided that a target enzyme's activity can be coupled to growth of the host cells, the activity can be improved simply by selecting for growth. Like all directed evolution, the continuous version requires no prior mechanistic knowledge of the target. Continuous directed evolution is thus a powerful way to modify plant or non-plant enzymes for use in plant metabolic research and engineering. Here, we first describe the basic features of the yeast (Saccharomyces cerevisiae) OrthoRep system for continuous directed evolution and compare it briefly with other systems. We then give a step-by-step account of three ways in which OrthoRep can be deployed to evolve primary metabolic enzymes, using a THI4 thiazole synthase as an example and illustrating the mutational outcomes obtained. We close by outlining applications of OrthoRep that serve growing demands (i) to change the characteristics of plant enzymes destined for return to plants, and (ii) to adapt ("plantize") enzymes from prokaryotes-especially exotic prokaryotes-to function well in mild, plant-like conditions.

Genotype-phenotype correlations in valosin-containing protein disease: a retrospective muticentre study.

BACKGROUND: Valosin-containing protein (VCP) disease, caused by mutations in the VCP gene, results in myopathy, Paget's disease of bone (PBD) and frontotemporal dementia (FTD). Natural history and genotype-phenotype correlation data are limited. This study characterises patients with mutations in VCP gene and investigates genotype-phenotype correlations. METHODS: Descriptive retrospective international study collecting clinical and genetic data of patients with mutations in the VCP gene. RESULTS: Two hundred and fifty-five patients (70.0% males) were included in the study. Mean age was 56.8±9.6 years and mean age of onset 45.6±9.3 years. Mean diagnostic delay was 7.7±6 years. Symmetric lower limb weakness was reported in 50% at onset progressing to generalised muscle weakness. Other common symptoms were ventilatory insufficiency 40.3%, PDB 28.2%, dysautonomia 21.4% and FTD 14.3%. Fifty-seven genetic variants were identified, 18 of these no previously reported. c.464G>A (p.Arg155His) was the most frequent variant, identified in the 28%. Full time wheelchair users accounted for 19.1% with a median time from disease onset to been wheelchair user of 8.5 years. Variant c.463C>T (p.Arg155Cys) showed an earlier onset (37.8±7.6 year) and a higher frequency of axial and upper limb weakness, scapular winging and cognitive impairment. Forced vital capacity (FVC) below 50% was as risk factor for being full-time wheelchair user, while FVC <70% and being a full-time wheelchair user were associated with death. CONCLUSION: This study expands the knowledge on the phenotypic presentation, natural history, genotype-phenotype correlations and risk factors for disease progression of VCP disease and is useful to improve the care provided to patient with this complex disease.

Structure and function of aerotolerant, multiple-turnover THI4 thiazole synthases.

Plant and fungal THI4 thiazole synthases produce the thiamin thiazole moiety in aerobic conditions via a single-turnover suicide reaction that uses an active-site Cys residue as sulfur donor. Multiple-turnover (i.e. catalytic) THI4s lacking an active-site Cys (non-Cys THI4s) that use sulfide as sulfur donor have been biochemically characterized -- but only from archaeal methanogens that are anaerobic, O2-sensitive hyperthermophiles from sulfide-rich habitats. These THI4s prefer iron as cofactor. A survey of prokaryote genomes uncovered non-Cys THI4s in aerobic mesophiles from sulfide-poor habitats, suggesting that multiple-turnover THI4 operation is possible in aerobic, mild, low-sulfide conditions. This was confirmed by testing 23 representative non-Cys THI4s for complementation of an Escherichia coli ΔthiG thiazole auxotroph in aerobic conditions. Sixteen were clearly active, and more so when intracellular sulfide level was raised by supplying Cys, demonstrating catalytic function in the presence of O2 at mild temperatures and indicating use of sulfide or a sulfide metabolite as sulfur donor. Comparative genomic evidence linked non-Cys THI4s with proteins from families that bind, transport, or metabolize cobalt or other heavy metals. The crystal structure of the aerotolerant bacterial Thermovibrio ammonificans THI4 was determined to probe the molecular basis of aerotolerance. The structure suggested no large deviations compared with the structures of THI4s from O2-sensitive methanogens, but is consistent with an alternative catalytic metal. Together with complementation data, use of cobalt rather than iron was supported. We conclude that catalytic THI4s can indeed operate aerobically and that the metal cofactor inserted is a likely natural determinant of aerotolerance.

Cerebrovascular disease in patients with COVID-19: neuroimaging, histological and clinical description.

Since the appearance of the first case of coronavirus disease 2019 (COVID-19) a pandemic has emerged affecting millions of individuals worldwide. Although the main clinical manifestations are respiratory, an increase in neurological conditions, specifically acute cerebrovascular disease, has been detected. We present cerebrovascular disease case incidence in hospitalized patients with SARS-CoV-2 infection. Patients were confirmed by microbiological/serological testing, or on chest CT semiology. Available data on co-morbidity, laboratory parameters, treatment administered, neuroimaging, neuropathological studies and clinical evolution during hospitalization, measured by the modified Rankin scale, were analysed. A bivariate study was also designed to identify differences between ischaemic and haemorrhagic subtypes. A statistical model of binary logistic regression and sensitivity analysis was designed to study the influence of independent variables over prognosis. In our centre, there were 1683 admissions of patients with COVID-19 over 50 days, of which 23 (1.4%) developed cerebrovascular disease. Within this group of patients, cerebral and chest CT scans were performed in all cases, and MRI in six (26.1%). Histological samples were obtained in 6/23 cases (two brain biopsies, and four arterial thrombi). Seventeen patients were classified as cerebral ischaemia (73.9%, with two arterial dissections), five as intracerebral haemorrhage (21.7%), and one leukoencephalopathy of posterior reversible encephalopathy type. Haemorrhagic patients had higher ferritin levels at the time of stroke (1554.3 versus 519.2, P = 0.004). Ischaemic strokes were unexpectedly frequent in the vertebrobasilar territory (6/17, 35.3%). In the haemorrhagic group, a characteristic radiological pattern was identified showing subarachnoid haemorrhage, parieto-occipital leukoencephalopathy, microbleeds and single or multiple focal haematomas. Brain biopsies performed showed signs of thrombotic microangiopathy and endothelial injury, with no evidence of vasculitis or necrotizing encephalitis. The functional prognosis during the hospital period was unfavourable in 73.9% (17/23 modified Rankin scale 4-6), and age was the main predictive variable (odds ratio = 1.5; 95% confidence interval 1.012-2.225; P = 0.043). Our series shows cerebrovascular disease incidence of 1.4% in patients with COVID-19 with high morbidity and mortality. We describe pathological and radiological data consistent with thrombotic microangiopathy caused by endotheliopathy with a haemorrhagic predisposition.

Bioinformatic and experimental evidence for suicidal and catalytic plant THI4s.

Like fungi and some prokaryotes, plants use a thiazole synthase (THI4) to make the thiazole precursor of thiamin. Fungal THI4s are suicide enzymes that destroy an essential active-site Cys residue to obtain the sulfur atom needed for thiazole formation. In contrast, certain prokaryotic THI4s have no active-site Cys, use sulfide as sulfur donor, and are truly catalytic. The presence of a conserved active-site Cys in plant THI4s and other indirect evidence implies that they are suicidal. To confirm this, we complemented the Arabidopsistz-1 mutant, which lacks THI4 activity, with a His-tagged Arabidopsis THI4 construct. LC-MS analysis of tryptic peptides of the THI4 extracted from leaves showed that the active-site Cys was predominantly in desulfurated form, consistent with THI4 having a suicide mechanism in planta. Unexpectedly, transcriptome data mining and deep proteome profiling showed that barley, wheat, and oat have both a widely expressed canonical THI4 with an active-site Cys, and a THI4-like paralog (non-Cys THI4) that has no active-site Cys and is the major type of THI4 in developing grains. Transcriptomic evidence also indicated that barley, wheat, and oat grains synthesize thiamin de novo, implying that their non-Cys THI4s synthesize thiazole. Structure modeling supported this inference, as did demonstration that non-Cys THI4s have significant capacity to complement thiazole auxotrophy in Escherichia coli. There is thus a prima facie case that non-Cys cereal THI4s, like their prokaryotic counterparts, are catalytic thiazole synthases. Bioenergetic calculations show that, relative to suicide THI4s, such enzymes could save substantial energy during the grain-filling period.

Isolation and Quantification of Blood Apoptotic Bodies, a Non-invasive Tool to Evaluate Apoptosis in Patients with Ischemic Stroke and Neurodegenerative Diseases.

BACKGROUND: Improper regulation of apoptosis has been postulated as one of the main factors that contributes to the etiology and/or progression of several prevalent diseases, including ischemic stroke and neurodegenerative pathologies. Consequently, in the last few years, there has been an ever-growing interest in the in vivo study of apoptosis. The clinical application of the tissue sampling and imaging approaches to analyze apoptosis in neurological diseases is, however, limited. Since apoptotic bodies are membrane vesicles that are released from fragmented apoptotic cells, it follows that the presence of these vesicles in the bloodstream is likely due to the apoptotic death of cells in tissues. We therefore propose to use circulating apoptotic bodies as biomarkers for measuring apoptotic death in patients with ischemic stroke and neurodegenerative diseases. RESULTS: Since there is no scientific literature establishing the most appropriate method for collecting and enumerating apoptotic bodies from human blood samples. Authors, here, describe a reproducible centrifugation-based method combined with flow cytometry analysis to isolate and quantify plasma apoptotic bodies of patients with ischemic stroke, multiple sclerosis, Parkinson's disease and also in healthy controls. Electron microscopy, dynamic light scattering and proteomic characterization in combination with flow cytometry studies revealed that our isolation method achieves notable recovery rates of highly-purified intact apoptotic bodies. CONCLUSIONS: This easy, minimally time consuming and effective procedure for isolating and quantifying plasma apoptotic bodies could help physicians to implement the use of such vesicles as a non-invasive tool to monitor apoptosis in patients with cerebrovascular and neurodegenerative diseases for prognostic purposes and for monitoring disease activity.

Simultaneous Angioplasty and Mechanical Thrombectomy in Tandem Carotid Occlusions. Incidence of Reocclusions and Prognostic Predictors.

OBJECTIVES: The best revascularization technique for tandem carotid occlusions is not clearly defined. The primary objective of this study is to describe our technical and clinical results, analyzing the main predictors of functional independence. The secondary objective is the analysis of stent reocclusion rate. METHODS: A single-center series of 250 mechanical thrombectomies in the anterior circulation was studied. A subsequent analysis of 40 carotid occlusions was performed. The demographics, etiology, angiographic results, antithrombotic drugs, and 3-month follow-up were registered. A bivariate analysis was performed to establish the association of the study variables with major clinical complications (death, symptomatic hemorrhagic transformation and early recurrence) and the functional prognosis. In addition, the relationship between the preprocedure antiaggregation regimen and the reocclusions was studied, as well as its clinical impact. Independent predictive factors were studied using a multivariate logistic regression model. RESULTS: Complete recanalization was achieved in 30 cases (75%). Simultaneous stent placement was decided in 32 cases (80%). Functional independence was reached in 19 cases (47.5%), and 3 (7.5%) died at 3 months. Seven major complications were reported (17.5%). In patients with satisfactory revascularization where a carotid stent was used, 9 reocclusions (28.1%) were detected during the follow-up, 2 of them symptomatic. The only factor related independently with functional independence was the administration of single antiaggregation (odds ratio = .31; 95% confidence interval .002-.595; P = .021). CONCLUSIONS: Urgent endovascular treatment of tandem carotid occlusions has shown to be effective and safe in our series. The administration of single antiaggregation is a predictor of functional independence. In patients treated with carotid stent, the reocclusion rate is high, but generally asymptomatic.

Rhizobium tropici CIAT 899 copA gene plays a fundamental role in copper tolerance in both free life and symbiosis with Phaseolus vulgaris.

Rhizobium tropici CIAT 899 is a facultative symbiotic diazotroph able to deal with stressful concentrations of metals. Nevertheless the molecular mechanisms involved in metal tolerance have not been elucidated. Copper (Cu2+) is a metal component essential for the heme-copper respiratory oxidases and enzymes that catalyse redox reactions, however, it is highly toxic when intracellular trace concentrations are surpassed. In this study, we report that R. tropici CIAT 899 is more tolerant to Cu2+ than other Rhizobium and Sinorhizobium species. Through Tn5 random mutagenesis we identify a R. tropici mutant strain with a severe reduction in Cu2+ tolerance. The Tn5 insertion disrupted the gene RTCIAT899_CH17575, encoding a putative heavy metal efflux P1B-1-type ATPase designated as copA. Phaseolus vulgaris plants inoculated with the copA::Tn5 mutant in the presence of toxic Cu2+ concentrations showed a drastic reduction in plant and nodule dry weight, as well as nitrogenase activity. Nodules induced by the copA::Tn5 mutant present an increase in H2O2 concentration, lipoperoxidation and accumulate 40-fold more Cu2+ than nodules formed by the wild-type strain. The copA::Tn5 mutant complemented with the copA gene recovered the wild-type symbiotic phenotypes. Therefore, the copA gene is essential for R. tropici CIAT 899 to survive in copper-rich environments in both free life and symbiosis with P. vulgaris plants.

Resveratrol inhibits cancer cell proliferation by impairing oxidative phosphorylation and inducing oxidative stress.

The resveratrol (RSV) efficacy to affect the proliferation of several cancer cell lines was initially examined. RSV showed higher potency to decrease growth of metastatic HeLa and MDA-MB-231 (IC50 = 200-250 µM) cells than of low metastatic MCF-7, SiHa and A549 (IC50 = 400-500 µM) and non-cancer HUVEC and 3T3 (IC50≥600 µM) cells after 48 h exposure. In order to elucidate the biochemical mechanisms underlying RSV anti-cancer effects, the energy metabolic pathways and the oxidative stress metabolism were analyzed in HeLa cells as metastatic-type cell model. RSV (200 µM/48 h) significantly decreased both glycolysis and oxidative phosphorylation (OxPhos) protein contents (30-90%) and fluxes (40-70%) vs. non-treated cells. RSV (100 µM/1-5 min) also decreased at a greater extent OxPhos flux (net ADP-stimulated respiration) of isolated tumor mitochondria (> 50%) than of non-tumor mitochondria (< 50%), particularly with succinate as oxidizable substrate. In addition, RSV promoted an excessive cellular ROS (2-3 times) production corresponding with a significant decrement in the SOD activity (but not in its content) and GSH levels; whereas the catalase, glutahione reductase, glutathione peroxidase and glutathione-S-transferase activities (but not their contents) remained unchanged. RSV (200 µM/48 h) also induced cellular death although not by apoptosis but rather by promoting a strong mitophagy activation (65%). In conclusion, RSV impaired OxPhos by inducing mitophagy and ROS over-production, which in turn halted metastatic HeLa cancer cell growth.

Characterising the phenotype and mode of inheritance of patients with inherited peripheral neuropathies carrying MME mutations.

BACKGROUND: Mutations in the metalloendopeptidase (MME) gene were initially identified as a cause of autosomal recessive Charcot-Marie-Tooth disease type 2 (CMT2). Subsequently, variants in MME were linked to other late-onset autosomal dominant polyneuropathies. Thus, our goal was to define the phenotype and mode of inheritance of patients carrying changes in MME. METHODS: We screened 197 index cases with a hereditary neuropathy of the CMT type or distal hereditary motor neuropathy (dHMN) and 10 probands with familial amyotrophic lateral sclerosis (fALS) using a custom panel of 119 genes. In addition to the index case subjects, we also studied other clinically and/or genetically affected and unaffected family members. RESULTS: We found 17 variants in MME in a total of 20 index cases, with biallelic MME mutations detected in 13 cases from nine families (three in homozygosis and six in compound heterozygosis) and heterozygous variants found in 11 families. All patients with biallelic variants had a similar phenotype, consistent with late-onset axonal neuropathy. Conversely, the phenotype of patients carrying heterozygous mutations was highly variable [CMT type 1 (CMT1), CMT2, dHMN and fALS] and mutations did not segregate with the disease. CONCLUSION: MME mutations that segregate in an autosomal recessive pattern are associated with a late-onset CMT2 phenotype, yet we could not demonstrate that MME variants in heterozygosis cause neuropathy. Our data highlight the importance of establishing an accurate genetic diagnosis in patients carrying MME mutations, especially with a view to genetic counselling.

Biochemistry and Physiology of Heavy Metal Resistance and Accumulation in Euglena.

Free-living microorganisms may become suitable models for removal of heavy metals from polluted water bodies, sediments, and soils by using and enhancing their metal accumulating abilities. The available research data indicate that protists of the genus Euglena are a highly promising group of microorganisms to be used in bio-remediation of heavy metal-polluted aerobic and anaerobic acidic aquatic environments. This chapter analyzes the variety of biochemical mechanisms evolved in E. gracilis to resist, accumulate and remove heavy metals from the environment, being the most relevant those involving (1) adsorption to the external cell pellicle; (2) intracellular binding by glutathione and glutathione polymers, and their further compartmentalization as heavy metal-complexes into chloroplasts and mitochondria; (3) polyphosphate biosynthesis; and (4) secretion of organic acids. The available data at the transcriptional, kinetic and metabolic levels on these metabolic/cellular processes are herein reviewed and analyzed to provide mechanistic basis for developing genetically engineered Euglena cells that may have a greater removal and accumulating capacity for bioremediation and recycling of heavy metals.

Carotid Intima-Media Thickness is Not Associated with Markers of Atherosclerosis in Stroke Patients.

BACKGROUND: It has been argued that carotid intima-media thickness (IMT) could better reflect an adaptive response of the vessel wall rather than being a marker of atherosclerosis. We explore this hypothesis by analyzing the ARTICO data. METHODS: The ARTICO study was designed to evaluate the prognostic value of the pathological ankle-brachial index (ABI) for the emergence of new vascular events in patients who have suffered a noncardioembolic stroke. Collected variables were as follows: vascular risk factors, mean waist perimeter, quantification of carotid IMT, characteristics of carotid plaques, ABI, and presence of microalbuminuria. RESULTS: A total of 591 patients with a complete carotid evaluation were available. There was no correlation between ABI and IMT (Spearman's, p NS). Logistic regression revealed that pathological ABI correlated significantly only with internal carotid artery stenosis greater than or equal to 50% (OR [odds ratio] 2.80, 1.66-4.71, P < .01) and peripheral artery disease (OR 3.33, 1.63-6.78, P < .01). However, multivariate regression analysis demonstrated that carotid IMT was independently associated with age (OR 1.05, 95% confidence interval [CI] 1.02-1.09, P < .01), hypertension (OR 1.83, 95% CI 1.02-3.26, P = .04), waist circumference (OR 1.03, 95% CI 1.01-1.05, P < .01), and microalbuminuria (OR 2.02, 95% CI 1.22-3.35, P < .01). CONCLUSION: In our patients, carotid IMT does not seem to be associated with unequivocal markers of atheromatosis such as the existence of relevant carotid plaques or pathological ABI. These results as well as the association of IMT with age, hypertension, microalbuminuria, and mean waist perimeter support the hypothesis that IMT must be considered a risk factor for general vascular disease rather than a marker of atherosclerotic burden.

[Giant racemose subarachnoid and intraventricular neurocysticercosis: A case report]. / Neurocisticercosis racemosa subaracnoidea gigante y ventricular: a propósito de un caso.

Neurocysticercosis is the most frequent parasitic disease of the central nervous system. It is caused by the larvae of Taenia solium, which can affect different anatomical sites. In Spain there is an increasing prevalence mainly due to immigration from endemic areas. The extraparenchymal forms are less common, but more serious because they usually develop complications. Neuroimaging plays a major role in the diagnosis and follow-up of this disease, supported by serology and a compatible clinical and epidemiological context. First-line treatments are cysticidal drugs such as albendazole and praziquantel, usually coadministered with corticosteroids, and in some cases surgery is indicated. We here report a case of neurocysticercosis with simultaneous intraventricular and giant racemose subarachnoid involvement.

Ultrasonographic study of the orbit as an early diagnostic tool in Vogt Koyanagi Harada disease (VKH): A case report.

In recent years, ultrasound has demonstrated its usefulness in the approach to vascular structures and other tissues such as the orbit, facilitating the early diagnosis of various diseases without having to rely on other more invasive or less available tests. In Vogt Koyanagi Harada syndrome, characterised by bilateral acute uveitis, ocular ultrasound is a clear example of the usefulness of ultrasonography in early diagnosis, facilitating the initiation of specific treatment to change the ominous natural history of this disease. This case shows the usefulness of the echography to make the differential diagnosis with other diseases that clinical onset could be similar than VKH, but with a different diagnostic and therapeutic approach.

Endovascular treatment in Danon disease: a case report.

BACKGROUND: Danon disease is a lysosomal storage disorder with X-linked inheritance. The classic triad is severe hypertrophic cardiomyopathy, myopathy, and intellectual disability, with different phenotypes between both genders. Ischemic stroke is an uncommon complication, mostly cardioembolic, related to intraventricular thrombus or atrial fibrillation, among others. CASE REPORT: We report the case of a 14-year-old Caucasian male patient with Danon disease who suffered from an acute ischemic stroke due to occlusion in the M1 segment of the middle cerebral artery. He underwent mechanical thrombectomy, resulting in successful revascularization with satisfactory clinical outcome. We objectified the intraventricular thrombus in the absence of arrhythmic events. CONCLUSION: To our knowledge, we report the first case of ischemic stroke related to Danon disease treated with endovascular treatment.

Sulfate uptake in photosynthetic Euglena gracilis. Mechanisms of regulation and contribution to cysteine homeostasis.

BACKGROUND: Sulfate uptake was analyzed in photosynthetic Euglena gracilis grown in sulfate sufficient or sulfate deficient media, or under Cd(2+) exposure or Cys overload, to determine its regulatory mechanisms and contribution to Cys homeostasis. RESULTS: In control and sulfate deficient or Cd(2+)-stressed cells, one high affinity and two low affinity sulfate transporters were revealed, which were partially inhibited by photophosphorylation and oxidative phosphorylation inhibitors and ionophores, as well as by chromate and molybdate; H(+) efflux also diminished in presence of sulfate. In both sulfate deficient and Cd(2+)-exposed cells, the activity of the sulfate transporters was significantly increased. However, the content of thiol-metabolites was lower in sulfate-deficient cells, and higher in Cd(2+)-exposed cells, in comparison to control cells. In cells incubated with external Cys, sulfate uptake was strongly inhibited correlating with 5-times increased intracellular Cys. Re-supply of sulfate to sulfate deficient cells increased the Cys, γ-glutamylcysteine and GSH pools, and to Cys-overloaded cells resulted in the consumption of previously accumulated Cys. In contrast, in Cd(2+) exposed cells none of the already elevated thiol-metabolites changed. CONCLUSIONS: (i) Sulfate transport is an energy-dependent process; (ii) sulfate transporters are over-expressed under sulfate deficiency or Cd(2+) stress and their activity can be inhibited by high internal Cys; and (iii) sulfate uptake exerts homeostatic control of the Cys pool.

Beneficial Effects of Daily Consumption of Garlic and Onion Extract Concentrate on Infectious Respiratory Diseases in Elderly Resident Volunteers.

Aging is a biological process with high susceptibility to several infections. This risk increases in older patients in residential care facilities (RCF). Thus, there is a clear demand for developing preventive interventions with new therapeutic compounds that combine efficacy and safety. This could be the case of compounds derived from plants of the genus Allium spp. The purpose of this study was to evaluate the impact of a combination of a garlic and onion extract concentrate standardized in organosulfur compounds derived from propiin on the incidence of respiratory tract infections in elderly patients of RCF. Sixty-five volunteers were selected at random to receive a placebo or a single daily dose of the extract for thirty-six weeks. Different clinical visits were performed to evaluate the main respiratory diseases with an infectious origin, as well as the associated symptoms and their duration. The extract showed a clinical safety profile and significantly reduced the incidence of respiratory infections. Moreover, the treatment decreased the number and duration of the associated symptoms compared with the placebo group. For the first time, we demonstrated the protective effect of Alliaceae extract in respiratory infectious diseases in elderly healthy volunteers, which could be used prophylactically against the most common infectious respiratory diseases.

Directed Evolution of Aerotolerance in Sulfide-Dependent Thiazole Synthases.

Sulfide-dependent THI4 thiazole synthases could potentially be used to replace plant cysteine-dependent suicide THI4s, whose high protein turnover rates make thiamin synthesis exceptionally energy-expensive. However, sulfide-dependent THI4s are anaerobic or microoxic enzymes and hence unadapted to the aerobic conditions in plants; they are also slow enzymes (kcat < 1 h-1). To improve aerotolerance and activity, we applied continuous directed evolution under aerobic conditions in the yeast OrthoRep system to two sulfide-dependent bacterial THI4s. Seven beneficial single mutations were identified, of which five lie in the active-site cleft predicted by structural modeling and two recapitulate features of naturally aerotolerant THI4s. That single mutations gave substantial improvements suggests that further advance under selection will be possible by stacking mutations. This proof-of-concept study established that the performance of sulfide-dependent THI4s in aerobic conditions is evolvable and, more generally, that yeast OrthoRep provides a plant-like bridge to adapt nonplant enzymes to work better in plants.