High-Level Production of Amorpha-4,11-Diene, a Precursor of the Antimalarial Agent Artemisinin, in Escherichia coli

BACKGROUND: Artemisinin derivatives are the key active ingredients in Artemisinin combination therapies (ACTs), the most effective therapies available for treatment of malaria. Because the raw material is extracted from plants with long growing seasons, artemisinin is often in short supply, and fermentation would be an attractive alternative production method to supplement the plant source. Previous work showed that high levels of amorpha-4,11-diene, an artemisinin precursor, can be made in Escherichia coli using a heterologous mevalonate pathway derived from yeast (Saccharomyces cerevisiae), though the reconstructed mevalonate pathway was limited at a particular enzymatic step. METHODOLOGY/ PRINCIPAL FINDINGS: By combining improvements in the heterologous mevalonate pathway with a superior fermentation process, commercially relevant titers were achieved in fed-batch fermentations. Yeast genes for HMG-CoA synthase and HMG-CoA reductase (the second and third enzymes in the pathway) were replaced with equivalent genes from Staphylococcus aureus, more than doubling production. Amorpha-4,11-diene titers were further increased by optimizing nitrogen delivery in the fermentation process. Successful cultivation of the improved strain under carbon and nitrogen restriction consistently yielded 90 g/L dry cell weight and an average titer of 27.4 g/L amorpha-4,11-diene. CONCLUSIONS/ SIGNIFICANCE: Production of >25 g/L amorpha-4,11-diene by fermentation followed by chemical conversion to artemisinin may allow for development of a process to provide an alternative source of artemisinin to be incorporated into ACTs

THE IMPACT OF DIETARY PROTEIN OR AMINO ACID SUPPLEMENTATION ON MUSCLE MASS AND STRENGTH IN ELDERLY PEOPLE: INDIVIDUAL PARTICIPANT DATA AND META-ANALYSIS OF RCT’S

Objectives Increasing protein or amino acid intake has been promoted as a promising strategy to increase muscle mass and strength in elderly people, however, long-term intervention studies show inconsistent findings. Therefore, we aim to determine the impact of protein or amino acid supplementation compared to placebo on muscle mass and strength in older adults by combining the results from published trials in a metaanalysis and pooled individual participant data analysis. Design We searched Medline and Cochrane databases and performed a meta-analysis on eight available trials on the effect of protein or amino acid supplementation on muscle mass and strength in older adults. Furthermore, we pooled individual data of six of these randomized double-blind placebo-controlled trials. The main outcomes were change in lean body mass and change in muscle strength for both the meta-analysis and the pooled analysis. Results The meta-analysis of eight studies (n=557) showed no significant positive effects of protein or amino acid supplementation on lean body mass (mean difference: 0.014 kg: 95% CI -0.152; 0.18), leg press strength (mean difference: 2.26 kg: 95% CI -0.56; 5.08), leg extension strength (mean difference: 0.75 kg: 95% CI: -1.96, 3.47) or handgrip strength (mean difference: -0.002 kg: 95% CI -0.182; 0.179). Likewise, the pooled analysis showed no significant difference between protein and placebo treatment on lean body mass (n=412: p=0.78), leg press strength (n=121: p=0.50), leg extension strength (n=121: p=0.16) and handgrip strength (n=318: p=0.37). Conclusions There is currently no evidence to suggest that protein or amino acid supplementation without concomitant nutritional or exercise interventions increases muscle mass or strength in predominantly healthy elderly people

Novel essential amino acid supplements enriched with L-leucine facilitate increased protein and energy intakes in older women: a randomised controlled trial

Background: Inadequate protein intake (PI), containing a sub-optimal source of essential amino acids (EAAs), and reduced appetite are contributing factors to age-related sarcopenia. The satiating effects of dietary protein per se may negatively affect energy intake (EI), thus there is a need to explore alternative strategies to facilitate PI without compromising appetite and subsequent EI. Methods: Older women completed two experiments (EXP1 and EXP2) where they consumed either a Bar (565 kJ), a Gel (477 kJ), both rich in EAAs (7.5 g, 40% L-leucine), or nothing (Control). In EXP1, participants (n=10, 68±5 years, mean±SD) consumed Bar, Gel or Control with appetite sensations and appetite-related hormonal responses monitored for one hour, followed by consumption of an ad libitum breakfast (ALB). In EXP2, participants (n=11, 69±5 years) ingested Bar, Gel or Control alongside an ALB. Results: In EXP1, EI at ALB was not different (P=0.674) between conditions (1179±566, 1254±511, 1206±550 kJ for the Control, Bar, and Gel respectively). However, total EI was significantly higher in the Bar and Gel compared to the Control after accounting for the energy content of the supplements (P<0.0005). Analysis revealed significantly higher appetite Area under the Curve (AUC) (P<0.007), a tendency for higher acylated ghrelin AUC (P=0.087), and significantly lower pancreatic polypeptide AUC (P=0.02) in the Control compared with the Bar and Gel. In EXP2, EI at ALB was significantly higher (P=0.028) in the Control (1282±513 kJ) compared to the Bar (1026±565 kJ) and Gel (1064±495 kJ). However, total EI was significantly higher in the Bar and Gel after accounting for the energy content of the supplements (P<0.007). Conclusions: Supplementation with either the Bar or Gel increased total energy intake whether consumed one hour before or during breakfast. This may represent an effective nutritional means for addressing protein and total energy deficiencies in older women

De novo sequencing, assembly and analysis of the genome of the laboratory strain Saccharomyces cerevisiae CEN.PK113-7D, a model for modern industrial biotechnology

Saccharomyces cerevisiae CEN.PK 113-7D is widely used for metabolic engineering and systems biology research in industry and academia. We sequenced, assembled, annotated and analyzed its genome. Single-nucleotide variations (SNV), insertions/deletions (indels) and differences in genome organization compared to the reference strain S. cerevisiae S288C were analyzed. In addition to a few large deletions and duplications, nearly 3000 indels were identified in the CEN.PK113-7D genome relative to S288C. These differences were overrepresented in genes whose functions are related to transcriptional regulation and chromatin remodelling. Some of these variations were caused by unstable tandem repeats, suggesting an innate evolvability of the corresponding genes. Besides a previously characterized mutation in adenylate cyclase, the CEN.PK113-7D genome sequence revealed a significant enrichment of non-synonymous mutations in genes encoding for components of the cAMP signalling pathway. Some phenotypic characteristics of the CEN.PK113-7D strains were explained by the presence of additional specific metabolic genes relative to S288C. In particular, the presence of the BIO1 and BIO6 genes correlated with a biotin prototrophy of CEN.PK113-7D. Furthermore, the copy number, chromosomal location and sequences of the MAL loci were resolved. The assembled sequence reveals that CEN.PK113-7D has a mosaic genome that combines characteristics of laboratory strains and wild-industrial strains

Proteomic analysis of Artemisia annua – towards elucidating the biosynthetic pathways of the antimalarial pro-drug artemisinin

Background: MS-based proteomics was applied to the analysis of the medicinal plant Artemisia annua, exploiting a recently published contig sequence database (Graham et al. (2010) Science 327, 328–331) and other genomic and proteomic sequence databases for comparison. A. annua is the predominant natural source of artemisinin, the precursor for artemisinin-based combination therapies (ACTs), which are the WHO-recommended treatment for P. falciparum malaria. Results: The comparison of various databases containing A. annua sequences (NCBInr/viridiplantae, UniProt/ viridiplantae, UniProt/A. annua, an A. annua trichome Trinity contig database, the above contig database and another A. annua EST database) revealed significant differences in respect of their suitability for proteomic analysis, showing that an organism-specific database that has undergone extensive curation, leading to longer contig sequences, can greatly increase the number of true positive protein identifications, while reducing the number of false positives. Compared to previously published data an order-of-magnitude more proteins have been identified from trichome-enriched A. annua samples, including proteins which are known to be involved in the biosynthesis of artemisinin, as well as other highly abundant proteins, which suggest additional enzymatic processes occurring within the trichomes that are important for the biosynthesis of artemisinin. Conclusions: The newly gained information allows for the possibility of an enzymatic pathway, utilizing peroxidases, for the less well understood final stages of artemisinin’s biosynthesis, as an alternative to the known non-enzymatic in vitro conversion of dihydroartemisinic acid to artemisinin. Data are available via ProteomeXchange with identifier PXD000703

Nutritional, Health, and Technological Functionality of Lupin Flour Addition to Bread and Other Baked Products: Benefits and Challenges

Lupin is an undervalued legume despite its high protein and dietary fiber content and potential health benefits. This review focuses on the nutritional value, health benefits, and technological effects of incorporating lupin flour into wheat-based bread. Results of clinical studies suggest that consuming lupin compared to wheat bread and other baked products reduce chronic disease risk markers; possibly due to increased protein and dietary fiber and bioactive compounds. However, lupin protein allergy has also been recorded. Bread quality has been improved when 10% lupin flour is substituted for refined wheat flour; possibly due to lupin-wheat protein cross-linking assisting bread volume and the high water-binding capacity (WBC) of lupin fiber delaying staling. Above 10% substitution appears to reduce bread quality due to lupin proteins low elasticity and the high WBC of its dietary fiber interrupting gluten network development. Gaps in understanding of the role of lupin flour in bread quality include the optimal formulation and processing conditions to maximize lupin incorporation, role of protein cross-linking, antistaling functionality, and bioactivity of its γ-conglutin protein

Sequence-specific antimicrobials using efficiently delivered RNA-guided nucleases

Current antibiotics tend to be broad spectrum, leading to indiscriminate killing of commensal bacteria and accelerated evolution of drug resistance. Here, we use CRISPR-Cas technology to create antimicrobials whose spectrum of activity is chosen by design. RNA-guided nucleases (RGNs) targeting specific DNA sequences are delivered efficiently to microbial populations using bacteriophage or bacteria carrying plasmids transmissible by conjugation. The DNA targets of RGNs can be undesirable genes or polymorphisms, including antibiotic resistance and virulence determinants in carbapenem-resistant Enterobacteriaceae and enterohemorrhagic Escherichia coli. Delivery of RGNs significantly improves survival in a Galleria mellonella infection model. We also show that RGNs enable modulation of complex bacterial populations by selective knockdown of targeted strains based on genetic signatures. RGNs constitute a class of highly discriminatory, customizable antimicrobials that enact selective pressure at the DNA level to reduce the prevalence of undesired genes, minimize off-target effects and enable programmable remodeling of microbiota.National Institutes of Health (U.S.) (New Innovator Award 1DP2OD008435)National Centers for Systems Biology (U.S.) (Grant 1P50GM098792)United States. Defense Threat Reduction Agency (HDTRA1-14-1-0007)Massachusetts Institute of Technology. Institute for Soldier Nanotechnologies (W911NF13D0001)National Institute of General Medical Sciences (U.S.) (Interdepartmental Biotechnology Training Program 5T32 GM008334)Fonds de la recherche en sante du Quebec (Master's Training Award

Nutrient Administration and Resistance Training

Skeletal muscle tissue is tightly regulated throughout our bodies by balancing its synthesis and breakdown. Many factors are known to exist that cause profound changes on the overall status of skeletal muscle, some of which include exercise, nutrition, hormonal influences and disease. Muscle hypertrophy results when protein synthesis is greater than protein breakdown. Resistance training is a popular form of exercise that has been shown to increase muscular strength and muscular hypertrophy. In general, resistance training causes a stimulation of protein synthesis as well as an increase in protein breakdown, resulting in a negative balance of protein. Providing nutrients, specifically amino acids, helps to stimulate protein synthesis and improve the overall net balance of protein. Strategies to increase the concentration and availability of amino acids after resistance exercise are of great interest and have been shown to effectively increase overall protein synthesis. [1-3] After exercise, providing carbohydrate has been shown to mildly stimulate protein synthesis while addition of free amino acids prior to and after exercise, specifically essential amino acids, causes a rapid pronounced increase in protein synthesis as well as protein balance.[1,3] Evidence exists for a dose-response relationship of infused amino acids while no specific regimen exists for optimal dosing upon ingestion. Ingestion of whole or intact protein sources (e.g., protein powders, meal-replacements) has been shown to cause similar improvements in protein balance after resistance exercise when compared to free amino acid supplements. Future research should seek to determine optimal dosing of ingested intact amino acids in addition to identifying the cellular mechanistic machinery (e.g. transcriptional and translational mechanisms) for causing the increase in protein synthesis

Aging Skin: Nourishing from the Inside Out, Effects of Good Versus Poor Nitrogen Intake on Skin Health and Healing

Skin is the outermost defense organ which protects us from the environment, constituting around 8 % of an adult’s body weight. Healthy skin contains one-eighth of the body’s total proteins. The balance of turnover and synthesis of skin proteins is primarily dependent on the availability of sufficient nitrogen-containing substrates, namely, amino acids, essential for protein metabolism in any other tissue and body organs. The turnover of skin proteins has been shown to be rapid, and the mobilization of amino acids at the expense of skin proteins is relevant in experimental models of protein malnutrition. As a result, alterations in nutritional status should be suspected, diagnosed, and eventually treated for any skin lesions. Protein malnutrition has a dramatic prevalence in patients aged >70 or more, independent of the reason for hospitalization. The quality of nutrition and content of essential amino acids are strictly connected to skin health and integrity of its protein components. Collagen fiber deposition is highly and rapidly influenced by alterations in the essential to nonessential amino acid ratios. The most relevant nutritional factor of skin health is the prevalence of essential amino acids

Parasympathetic Activity and Blood Catecholamine Responses Following a Single Partial-Body Cryostimulation and a Whole-Body Cryostimulation

The aim of this study was to compare the effects of a single whole-body cryostimulation (WBC) and a partial-body cryostimulation (PBC) (i.e., not exposing the head to cold) on indices of parasympathetic activity and blood catecholamines. Two groups of 15 participants were assigned either to a 3-min WBC or PBC session, while 10 participants constituted a control group (CON) not receiving any cryostimulation. Changes in thermal, physiological and subjective variables were recorded before and during the 20-min after each cryostimulation. According to a qualitative statistical analysis, an almost certain decrease in skin temperature was reported for all body regions immediately after the WBC (mean decrease±90% CL, -13.7±0.7°C) and PBC (-8.3±0.3°C), which persisted up to 20-min after the session. The tympanic temperature almost certainly decreased only after the WBC session (-0.32±0.04°C). Systolic and diastolic blood pressures were very likely increased after the WBC session, whereas these changes were trivial in the other groups. In addition, heart rate almost certainly decreased after PBC (-10.9%) and WBC (-15.2%) sessions, in a likely greater proportion for WBC compared to PBC. Resting vagal-related heart rate variability indices (the root-mean square difference of successive normal R-R intervals, RMSSD, and high frequency band, HF) were very likely increased after PBC (RMSSD: +54.4%, HF: +138%) and WBC (RMSSD: +85.2%, HF: +632%) sessions without any marked difference between groups. Plasma norepinephrine concentrations were likely to very likely increased after PBC (+57.4%) and WBC (+76.2%), respectively. Finally, cold and comfort sensations were almost certainly altered after WBC and PBC, sensation of discomfort being likely more pronounced after WBC than PBC. Both acute cryostimulation techniques effectively stimulated the autonomic nervous system (ANS), with a predominance of parasympathetic tone activation. The results of this study also suggest that a whole-body cold exposure induced a larger stimulation of the ANS compared to partial-body cold exposure