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Genomic features of Lactococcus lactis IO-1, a lactic acid bacterium that 20 human milk samples contained nisin-producing Lactococcus lactis bacteria. la plate-forme de bio-informatique nous permet d’identifier la En résumé, une partie de l’approche modulaire du vivant genome-free cell. New genetic.

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Genomic features of Lactococcus lactis IO-1, a lactic acid bacterium that 20 human milk samples contained nisin-producing Lactococcus lactis bacteria. la plate-forme de bio-informatique nous permet d’identifier la En résumé, une partie de l’approche modulaire du vivant genome-free cell. New genetic.


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Wild-type L. However, both strains grew to similarly high levels in ATL, indicating redundancy in L. These findings show that certain strains of L. Deciphering a unique biotin scavenging pathway with redundant genes in the probiotic bacterium Lactococcus lactis. Biotin protein ligase BPL is widespread in the three domains of the life.

The paradigm BPL is the Escherichia coli BirA protein, which also functions as a repressor for the biotin biosynthesis pathway. Unlike the scenario in E. The in vivo function of the two L. Thin-layer chromatography and mass spectroscopy assays demonstrated that these two recombinant BirA proteins catalyze the biotinylation reaction of the acceptor biotin carboxyl carrier protein BCCP , through the expected biotinoyl-AMP intermediate.

We also determined the ability to uptake 3H-biotin by L. Taken together, our results deciphered a unique biotin scavenging pathway with redundant genes present in the probiotic bacterium L. We also determined the ability to uptake 3 H-biotin by L. The Lactococcus lactis subsp.

Both bacteriocins were produced in varying proportions in all of the studied nutrient media, which support the growth of the producer. Depending on the cultivation medium, the nisin A content was to fold lower in the K stain culture fluid than that of the D peptide.

In comparision to to nisin A Bacteriocin D possessed a wide range of antibacterial activity and suppressed the growth of both Gram-positive and Gram-negative bacteria. An optimal medium for D bacteriocin synthesis was shown to be a fermentation medium which contained yeast extract, casein hydrolysate, and potassium phosphate. The biosynthesis ofbacteriocin D by the K strain in these media occurred parallel to producer growth, and its maximal accumulation in the culture fluid was observed at h of the strain’s growth.

Knowledge of how the activity of enzymes is affected under in vivo conditions is essential for analyzing their regulation and constructing models that yield an integrated understanding of cell behavior.

Current kinetic parameters for Lactococcus lactis are scattered through different studies and performed under different assay conditions. Furthermore, assay conditions often diverge from conditions prevailing in the intracellular environment. To establish uniform assay conditions that resemble intracellular conditions, we analyzed the intracellular composition of anaerobic glucose-limited chemostat cultures of L. Based on this, we designed a new assay medium for enzyme activity measurements of growing cells of L.

Procedures were optimized to be carried out in well plates, and the reproducibility and dynamic range were checked for all enzyme activity measurements. The effects of freezing and the carryover of ammonium sulfate from the addition of coupling enzymes were also established.

Activities of all 10 glycolytic and 4 fermentative enzymes were measured. Remarkably, most in vivo-like activities were lower than previously published data. Yet, the ratios of Vmax over measured in vivo fluxes were above 1. With this work, we have developed and extensively validated standard protocols for enzyme activity measurements for L. Improvement of the respiration efficiency of Lactococcus lactis by decreasing the culture pH. The growth characteristics and intracellular hemin concentrations of Lactococcus lactis grown under different culture pH and aeration conditions were examined to investigate the effect of culture pH on the respiration efficiency of L.

Cell biomass and biomass yield of L. Hemin accumulation was sensitive to culture pH. Among the four pH conditions, pH 5.

The highest intracellular hemin level in L. The respiration efficiency of L. These results may help develop high cell-density L. Thus, this microorganism may be used for industrial applications. Nisin production of Lactococcus lactis N8 with hemin-stimulated cell respiration in fed-batch fermentation system. In this study, nisin production of Lactococcus lactis N8 was optimized by independent variables of glucose, hemin and oxygen concentrations in fed-batch fermentation in which respiration of cells was stimulated with hemin.

Response surface model was able to explain the changes of the nisin production of L. While IU mL – 1 nisin was produced by L. Accordingly, nisin production was enhanced 3. In conclusion the nisin production of L. Trimming of two major type 1 diabetes driving antigens, GAD65 and IA-2, allows for successful expression in Lactococcus lactis.

Restoring auto-antigen tolerance remains the superior therapeutic strategy. Oral auto-antigen administration uses the tolerogenic nature of the gut-associated immune system to induce antigen-specific tolerance. However, due to gastric degradation, proper mucosal product delivery often imposes a challenge.

Recombinant Lactococcus lactis have proven to be effective and safe carriers for gastrointestinal delivery of therapeutic products: L. Here, we describe the construction of recombinant L. Trimming of GAD65 and IA-2 was investigated to optimise antigen secretion while maintaining sufficient bacterial growth. Antigen secretion was verified by immunoblotting.

Plasmid-derived GAD65 and IA-2 expression was combined in single strains with human IL expression, a desired combination to allow tolerance induction. This study reports the generation of recombinant L.

Prohibitive sequence obstacles hampering antigen secretion were resolved by trimming the full size proteins. Manipulation for plasmid elimination by transforming synthetic competitors diversifies lactococcus lactis starters applicable to food products. This study was designed selectively to eliminate a theta-plasmid from Lactococcus lactis strains by transforming synthetic competitors. A shuttle vector for Escherichia coli and L.

This versatile vector was used to construct competitors to common lactococcal theta-plasmids. A set of primers, Pv3 and Pv4, was designed to amplify the 1. A number of theta-plasmids in L. These competitors were easily eliminated by subculture for a short time in the absence of selection. The resulting variants contained no exogenous DNA and are suitable for food products, since part of the phenotype was altered without altering other plasmids indispensable for fermentation.

Novel angiotensin I-converting enzyme inhibitory peptides produced in fermented milk by specific wild Lactococcus lactis strains. The ability of specific wild Lactococcus lactis strains to hydrolyze milk proteins to release angiotensin I-converting enzyme ACE inhibitory peptides was evaluated. All peptide fractions were analyzed by reversed-phase HPLC tandem mass spectrometry. Several novel ACEI peptides presented peptides encrypted with proven hypotensive activity.

In conclusion, specific wild Lc. However, further studies are necessary to find out the relationship between Lc. Previous studies revealed that VMP produces a variety of isoprenoid volatiles during daylight. Isoprenoids are well known to contribute significantly to the scent of most fragrant plants. Introduction of Peptidase Genes from Lactobacillus delbrueckii subsp. Controllable expression of the corresponding genes pep genes was achieved by constructing translational fusions with the promoter of the nisA gene PnisA.

A suitable host strain, UKLc10, was constructed by chromosomal integration of the genes encoding the NisRK two-component system into the fivefold peptidase-deficient mutant IM16 of L.

Recombinants of this strain were used to analyze growth, peptidase activities, peptide utilization, and intracellular protein cleavage products. After nisin induction of PnisA::pep fusions, all of the peptidases were visible as distinct bands in protein gels. Despite the fact that identical transcription and translation signals were used to express the pep genes, the relative amounts of individual peptidases varied considerably. All of the peptidases exhibited activities in extracts of recombinant UKLc10 clones, but only PepL and PepG allowed the clones to utilize specific peptide substrates as sources of essential amino acids.

In milk medium, induction of pepG and induction of pepW resulted in growth acceleration. The activities of all five peptidases during growth in milk medium were revealed by high-performance liquid chromatography analyses of intracellular amino acid and peptide pools. A new Lactococcus lactis subsp. Aiming at maximum lactic acid productivity, the components of the media and the cultivation conditions were varied. In MRS-starch medium with absence of yeast and meat extracts , at 33 degrees C, agitation rpm and pH 6.

The identification of strain B84 was based on genetic criteria. Four genes for enzymes, involved in starch degradation were detected in B84 genome: amyL, amyY, glgP and apu, coding cytoplasmic and extracellular alpha-amylases, glycogen phosphorylase and amylopullulanase, respectively.

Amylase activity assay was performed at different pH and temperatures. The cell-bond amylase proved to be the key enzyme, involved in the starch hydrolysis with maximum activity at 45 degrees C and pH 5.

Fluorescence microscopy allowed to observe that the caseinate-rich phase formed droplets dispersed in a continuous alginate-rich phase. The distribution of bacteria in such a system was observed by epifluorescence microscopy: Lc. Since zeta-potential measurements indicated that alginate, caseinate and bacterial cells all had an overall negative charge at pH 7, the preferential adhesion of LAB cells was assumed to be driven by hydrophobic effect or by depletion phenomena in such biopolymeric systems.

Moreover, LAB cells viability was significantly higher in the ternary mixture obtained in the presence of both caseinate and alginate than in single alginate solution. Enhancing cellulose digestibility in animals is important for improving the utilization of forage, which can decrease the amount of food used in animal production.

The aim of the present study was to achieve recombinant expression of the cellulase gene in Lactococcus lactis and evaluate the effects of oral administration of the recombinant L. Then, the recombinant expression plasmid was transformed into L. Therefore, oral administration of recombinant L. Cloning and functional expression of the mitochondrial alternative oxidase gene aox 1 of Aspergillus niger in Lactococcus lactis and its induction by oxidizing conditions.

Lactococcus lactis is a widely used food bacterium mainly known for its fermentation metabolism. An important, and for long time overlooked, trait of this species is its ability to perform respiratory metabolism in the presence of heme and under aerobic conditions.

There is no evidence however for the presence of an alternative respiration pathway and AOX activity. In this study, a cDNA fragment encoding the mitochondrial alternative oxidase, the enzyme responsible for alternative respiration, from a citric acid producing Aspergillus niger strain was cloned and expressed in L.

Expression of aox 1 conferred on this organism cyanide-resistant and salicylhydroxamate-sensitive growth. Bioreactor cultures under fully aerobic conditions of the transformed L. A large collection of Lactococcus lactis strains, including wild-type isolates and dairy starter cultures, were screened on the basis of their phenotype and the macrorestriction patterns produced from pulsed-field gel electrophoresis PFGE analysis of SmaI digests of genomic DNA. Three groups of dairy starter cultures, used for different purposes in the dairy industry, and a fourth group made up of strains isolated from the environment were selected for analysis of their chromosomal diversity using the endonuclease I-CeuI.

Chromosome architecture was largely conserved with each strain having six copies of the rRNA genes, and the chromosome size of individual strains ranged between 2, and 2, kb. The origin of L. Overall, this study, coupled with analysis of the sequenced L. Adaptation of these strains to the dairy environment has involved loss of functions resulting in smaller chromosomes and acquisition of genes usually plasmid associated that facilitate growth in milk.

We conclude that dairy starter cultures generally and the industrially used cremoris and diacetylactis phenotype strains in particular comprise a specialized group of L. With the aim of selecting LAB strains with antilisterial activity to be used as protective cultures to enhance the safety of dairy products, the antimicrobial properties of Lactococcus lactis subsp.

The capacity of these strains to antagonize Listeria monocytogenes during cocultivation in skimmed milk was evaluated, showing a reduction of L. In order for a strain to be used as bioprotective culture, it should be carefully evaluated for the presence of virulence factors, to determine what potential risks might be involved in its use. None of the strains tested was found to produce biogenic amines or to possess haemolytic activity. In addition, all strains were sensitive to clinically important antibiotics such as ampicillin, tetracycline, and vancomycin.

Construction of two Lactococcus lactis expression vectors combining the Gateway and the NIsin Controlled Expression systems. Although various L. In this study, we constructed two expression vectors that combine the NICE and the Gateway recombination systems and we tested their applicability by recombining and over-expressing genes encoding structural proteins of lactococcal phages Tuc and TP 1. Over-expressed phage proteins were analyzed by immunoblotting and purified by His-tag affinity chromatography with protein productions yielding 2.

This therefore is the first description of L. Further subcloning revealed that a 1. The fragment contains the gene yvjB coding for a Zn-dependent membrane-bound metallopeptidase, suggesting that this gene may serve as the receptor for LsbB. Further support for this notion derives from several independent experiments: i whole-genome sequencing confirmed that all LsbB-resistant mutants contain mutations in yvjB; ii disruption of yvjB by direct gene knockout rendered sensitive strains BGMN 1 and IL resistant to LsbB; and iii most compellingly, heterologous expression of yvjB in naturally resistant strains of other species, such as Lactobacillus paracasei and Enterococcus faecalis, also rendered them sensitive to the bacteriocin.

To our knowledge, this is the first time a membrane-bound peptidase gene has been shown to be involved in bacteriocin sensitivity in target cells. We also demonstrated a novel successful approach for identifying bacteriocin receptors. Bile salt tolerance of Lactococcus lactis is enhanced by expression of bile salt hydrolase thereby producing less bile acid in the cells. Changes of bile salt tolerance, morphology and amount of bile acid within cells were studied to evaluate the exact effects of bile salt hydrolase BSH on bile salt tolerance of microorganism.

Growth of L. As indicated by transmission electron microscopy, bile acids released by the action of BSH induced the formation of micelles around the membrane surface of cells subject to conjugated bile salt stress.

A similar micelle containing bile acid was observed in the cytoplasm by liquid chromatography-mass spectrometry. BSH 1 produced fewer bile acid micelles in the cytoplasm and achieved better cell growth of L. Expression of BSH improved bile salt tolerance of L. Recombinant Lactococcus lactis can make the difference in antigen-specific immune tolerance induction, the Type 1 Diabetes case.

Especially in western civilizations, immune diseases that are driven by innocuous auto- or allo- antigens are gradually evolving to become pandemic threats. Along these disquieting observations we find ourselves equipped with impressively accumulating molecular immunological knowledge on the ins and outs of these pathologies. Often, however, it is difficult to translate this wealth into efficacious medicines.

The molecular understanding, the concept of oral tolerance induction, the benefit of using recombinant Lactococcus lactis therein and recent openings towards their clinical use may well enable turning all colors to their appropriate fields on this Rubik’s cube. Acetate kinase ACK converts acetyl phosphate to acetate along with the generation of ATP in the pathway for mixed-acid fermentation in Lactococcus lactis.

The reverse reaction yields acetyl phosphate for assimilation purposes. Remarkably, L. Both proteins form homodimeric complexes, as shown by size exclusion chromatography and static light-scattering measurements. The turnover number of AckA 1 is about an order of magnitude higher than that of AckA2 for the reaction in either direction. However, AckA2 has a higher affinity for acetate than does AckA 1 , suggesting an important role under acetate-limiting conditions despite the lower activity.

Fructose- 1 ,6-bisphosphate, glyceraldehydephosphate, and phospho-enol-pyruvate inhibit the activities of AckA 1 and AckA2 to different extents.

The allosteric regulation of AckA 1 and AckA2 and the pool sizes of the glycolytic intermediates are consistent with a switch from homolactic to mixed-acid fermentation upon slowing of the growth rate. Purification and characterization of two new cell-bound bioactive compounds produced by wild Lactococcus lactis strain.

Novel compounds and innovative methods are required considering that antibiotic resistance has reached a crisis point. In the study, two cell-bound antimicrobial compounds produced by Lactococcus lactis ID 1.

The compound AI showed a spectrum of antimicrobial activity mainly against L. The antimicrobial activity of both compounds was suppressed by treatment with Tween Nevertheless, both compounds showed high stability to heat and proteases treatments. The isolated compounds, AI and AII, showed distinct properties from other antimicrobial substances already reported as produced by L.

For permissions, please e-mail: journals. Determination of the cell wall polysaccharide and teichoic acid structures from Lactococcus lactis IL In the lactic acid bacterium Lactococcus lactis , a cell wall polysaccharide CWPS is the bacterial receptor of the majority of infecting bacteriophages. The diversity of CWPS structures between strains explains, at least partially, the narrow host range of lactococcal phages.

In the present work, we studied the polysaccharide components of the cell wall of the prototype L. We identified a rhamnose-rich complex polysaccharide, carrying a glycerophosphate substitution, as the major component. A poly glycerol phosphate teichoic acid, another important carbohydrate component of the IL cell wall, was also isolated and structurally characterized.

Undefined mesophilic mixed DL starter cultures are used in the production of continental cheeses and contain unknown strain mixtures of Lactococcus lactis and leuconostocs. The choice of starter culture affects the taste, aroma, and quality of the final product. To gain insight into the diversity of Lactococcus lactis strains in starter cultures, we whole-genome sequenced 95 isolates from three different starter cultures. Pan-genomic analyses, which included 30 publically available complete genomes, grouped the strains into 21 L.

Only one of the 95 isolates grouped with previously sequenced strains, and the three starter cultures showed no overlap in lineage distributions. The culture diversity was assessed by targeted amplicon sequencing using purR , a core gene, and epsD , present in 93 of the 95 starter culture isolates but absent in most of the reference strains. This enabled an unprecedented discrimination of starter culture Lactococcus lactis and revealed substantial differences between the three starter cultures and compositional shifts during the cultivation of cultures in milk.

The dairy industry experiences significant disruptions of cheese production due to phage attacks, and one commonly used countermeasure to phage attack is to employ a starter rotation strategy, in which two or more starters with minimal overlap in phage sensitivity are used alternately.

A culture-independent analysis of the lactococcal diversity in complex undefined starter cultures revealed large differences between the three starter cultures and temporal shifts in lactococcal composition during the production of bulk starters.

A better understanding of the lactococcal diversity in starter cultures will enable the development of. Flavins contained in yeast extract are exploited for anodic electron transfer by Lactococcus lactis. Cyclic voltammograms of yeast extract-containing medium exhibit a clear redox peak around Ag AgCl.

Fermentative bacterium Lactococcus lactis was hereby shown to exploit this redox compound for extracellular electron transfer towards a graphite anode using glucose as an electron donor.

High performance liquid chromatography revealed that this may be a flavin-type compound. The ability of L. Based on its mid-point potential, riboflavin can be regarded as a near-optimal mediator for microbially catalyzed anodic electron transfer. Riboflavin derivative flavin mononucleotide FMN was also exploited by L. The use of yeast extract in microbial fuel cell media is herein discouraged based on the related unwanted artificial addition of redox mediators which may distort experimental results.

Copyright Elsevier B. Several molecular taxonomic studies have revealed that many natural wild Lactococcus lactis strains of dairy origin which are phenotypically representative of the L. Samelis, A. Lianou, A. Kakouri, C. Rogelj, B. Montel, J. Food Prot. In this study, the actual subspecies identity of M78 and M isolates was elucidated, using 16S rRNA and acmA encoding lactococcal N-acetylmuramidase gene and histidine biosynthesis operon polymorphisms and 16S rRNA and ldh encoding lactate dehydrogenase gene phylogenies.

Except the acmA gene analysis, molecular tools revealed that isolates M78 and M clustered with strains of the cremoris genotype, including the LMG T strain, while they were distant from strains of the lactis genotype, including the LMG T strain. In contrast, they exhibited identical sugar fermentation and enzymatic patterns which were similar to those of the subspecies lactis LMG T strain.

To our knowledge, this is the first complete identification report on a wild L. Expression of lycopene biosynthesis genes fused in line with Shine-Dalgarno sequences improves the stress-tolerance of Lactococcus lactis. Lycopene biosynthetic genes from Deinococcus radiodurans were co-expressed in Lactococcus lactis to produce lycopene and improve its tolerance to stress.

Lycopene-related genes from D. The recombinant strain produced 0. The survival rate to UV irradiation of the recombinant strain was higher than that of the non-transformed strain. This dual role of Ald, due to allosteric activation by leucine, was used as a strategy for the isolation of Ald-deficient mutants of Lactococcus lactis subsp.

Most dairy lactococcus strains are auxotrophic for the three amino acids. Therefore, the plasmid pMC containing the ilv genes encoding the enzymes involved in the biosynthesis of IV of L. Introduction of pMC into ILV-auxotrophic dairy strains resulted in an isoleucine-prototrophic phenotype. By plating the strains on a chemically defined medium supplemented with leucine but not valine and isoleucine, spontaneous leucine-resistant mutants were obtained.

These mutants were screened by Western blotting with Ald-specific antibodies for the presence of Ald. Selected mutants lacking Ald were subsequently cured of pMC Except for a defect in the expression of Ald, the resulting strain, MC, was identical to the wild-type strain, as shown by Southern blotting and DNA fingerprinting.

The mutation resulting in the lack of Ald in MC occurred spontaneously, and the strain does not contain foreign DNA; thus, it can be regarded as food grade. Nevertheless, its application in dairy products depends on the regulation of genetically modified organisms. These results establish a strategy to select spontaneous Ald-deficient mutants from transformable L.

Long-term administration of pDC-Stimulative Lactococcus lactis strain decelerates senescence and prolongs the lifespan of mice. The decline in immune function caused by aging increases the risk of infectious diseases, tumorigeneses and chronic inflammation, resulting in accelerating senescence. We previously reported a lactic acid bacteria, Lactococcus lactis strain Plasma synonym of Lactococcus lactis subsp.

In this study, we investigated the anti-aging effects of long-term oral administration of Lc-Plasma in a senescence-accelerated mouse strain, SAMP6. Furthermore, the thinning of skin and age-related decrease in muscle mass were also significantly suppressed in the Lc-Plasma group as compared with the control group. Consistent with these phenotypic features, pDCs activity was significantly higher in Lc-Plasma mice than in control mice. In conclusion, long-term administration of Lc-Plasma can decelerate senescence and prolong lifespan via maintenance of the immune system due to activation of pDCs.

Mutation of the oxaloacetate decarboxylase gene of Lactococcus lactis subsp. Citrate metabolism generates metabolic energy through the generation of a membrane potential and a pH gradient.

The purpose of this work was to study the influence of oxaloacetate decarboxylase in citrate metabolism and intracellular pH maintenance in relation to acidic conditions. During culture with citrate, and whatever the initial pH, the growth rate of the mutant was lower. In addition, the production of diacetyl and acetoin was altered in the mutant strain.

However, our results indicated no relationship with a change in the maintenance of intracellular pH. Experiments performed on resting cells clearly showed that oxaloacetate accumulated temporarily in the supernatant of the mutant.

This accumulation could be involved in the perturbations observed during citrate metabolism, as the addition of oxaloacetate in M17 medium inhibited the growth of L. The mutation of oxaloacetate decarboxylase perturbed citrate metabolism and reduced the benefits of its utilization during growth under acidic conditions. This study allows a better understanding of citrate metabolism and the role of oxaloacetate decarboxylase in the tolerance of lactic acid bacteria to acidic conditions.

Ladero, Victor; Rattray, Fergal P. Lactococcus lactis is a prokaryotic microorganism with great importance as a culture starter and has become the model species among the lactic acid bacteria. The long and safe history of use of L. However, our group has identified several strains of L. Putrescine is a biogenic amine that confers undesirable flavor characteristics and may even have toxic effects.

These genes are transcribed as an operon that is induced in the presence of agmatine. In some strains, an insertion IS element interrupts the transcription of the cluster, which results in a non-putrescine-producing phenotype.

Based on this knowledge, a PCR-based test was developed in order to differentiate nonproducing L. The analysis of the AGDI cluster and their flanking regions revealed that the capacity to produce putrescine via the AGDI pathway could be a specific characteristic that was lost during the adaptation to the milk environment by a process of reductive genome evolution.

Intramammary infusion of a live culture of Lactococcus lactis in ewes to treat staphylococcal mastitis. Alternatives to antibiotic therapy for mastitis in ruminants are needed. We present an evaluation, in two trials, of the efficacy of an intramammary infusion of a live culture of Lactococcus lactis for the treatment of subclinical and clinical mastitis in ewes. Intramammary infusions were performed with either L.

Antibiotic-treated and untreated control glands were included. Milk samples for microbiology, somatic cell analysis and milk production were collected before and after treatment. But while leading to a transient clearance of CNS in the gland, this response caused mild to moderate clinical cases of mastitis characterized by abnormal milk secretions and udder inflammation.

Moreover, S. Under our experimental conditions, the L. We believe it is still early to implement bacterial formulations as alternatives in treating mastitis in ruminants and further experimentation is needed.

Early adaptation to oxygen is key to the industrially important traits of Lactococcus lactis ssp. Lactococcus lactis is the most used species in the dairy industry. Its ability to adapt to technological stresses, such as oxidative stress encountered during stirring in the first stages of the cheese-making process, is a key factor to measure its technological performance.

This study aimed to understand the response to oxidative stress of Lactococcus lactis subsp. For those purposes, conditions of hyper-oxygenation were initially fixed for the fermentation. Kinetics of growth and acidification were not affected by the presence of oxygen, indicating a high resistance to oxygen of the L.

Its resistance was explained by an efficient consumption of oxygen within the first 4 hours of culture, leading to a drop of the redox potential. The efficient consumption of oxygen by the L. In oxygen metabolism, the over-expression of all the genes of the nrd ribonucleotide reductases operon or fhu ferrichrome ABC transports genes was particularly significant. Finally, the MG strain was no longer able to consume oxygen in the stationary growth phase, leading to a drastic loss of culturability as a consequence of cumulative stresses and the absence of gene adaptation at this stage.

Combining metabolic and transcriptomic profiling, together with oxygen consumption kinetics, yielded new insights into the whole genome adaptation of L. An early and transitional adaptation to oxidative stress was revealed for L. This paper describes the molecular and metabolic adaptations of Lactococcus lactis during the transition from a growing to a near-zero growth state by using carbon-limited retentostat cultivation.

Transcriptomic analyses revealed that metabolic patterns shifted between lactic- and mixed-acid fermentations during retentostat cultivation, which appeared to be controlled at the level of transcription of the corresponding pyruvate dissipation-encoding genes.

During retentostat cultivation, cells continued to consume several amino acids but also produced specific amino acids, which may derive from the conversion of glycolytic intermediates. Finally, under extremely low carbon availability, carbon catabolite repression was progressively relieved and alternative catabolic functions were found to be highly expressed, which was confirmed by enhanced initial acidification rates on various sugars in cells obtained from near-zero-growth cultures.

The present integrated transcriptome and metabolite amino acids and previously reported fermentation end products study provides molecular understanding of the adaptation of L. Lactococcus lactis is an attractive vehicle for biotechnological production of proteins and clinical delivery of therapeutics. In many such applications using this host, it is desirable to maximize secretion of recombinant proteins into the extracellular space, which is typically achieved by using the native signal peptide from a major secreted lactococcal protein, Usp In order to further increase protein secretion from L.

Here, we performed extensive mutagenesis on Usp45sp to probe the effects of both the mRNA sequence silent mutations and the peptide sequence amino acid substitutions on secretion. The best sequence from our screen preserves the tripartite structure of the native signal peptide but increases the positive charge of the n-region. Our study presents the first example of an engineered L.

Engineering signal peptides for enhanced protein secretion from Lactococcus lactis. Implementation of the agmatine-controlled expression system for inducible gene expression in Lactococcus lactis. Lactococcus lactis has been safely consumed in fermented foods for millennia. This Gram-positive bacterium has now become of industrial importance as an expression host for the overproduction of lipopolysaccharide-free recombinant proteins used as food ingredients, therapeutic proteins and biotechnological enzymes.

This paper reports an agmatine-controlled expression ACE system for L. The usefulness and efficiency of this system was checked via the reporter gene gfp and by producing PEP Myxococcus xanthus prolyl-endopeptidase , an enzyme of biomedical interest able to degrade the immunotoxic peptides produced during the gastrointestinal breakdown of gluten.

The expression system was tightly regulated by the agmatine concentration and allowed high protein production without leakiness. Acid or erythromycin stress significantly improves transformation efficiency through regulating expression of DNA binding proteins in Lactococcus lactis F Lactococcus lactis is a gram-positive bacterium used extensively in the dairy industry and food fermentation, and its biological characteristics are usually improved through genetic manipulation.

However, poor transformation efficiency was the main restriction factor for the construction of engineered strains. In this study, the transformation efficiency of L. Notably, the transformation efficiency of F44e L. Especially for radA, Overexpression of some DNA binding proteins could improve the transformation efficiency. The results suggested that acid or erythromycin stress could improve the transformation efficiency of L. We have proposed a simple but promising strategy for improving the transformation efficiency of L.

Evaluation of acceptor selectivity of Lactococcus lactis ssp. TrePP in Lactococcus lactis ssp. In this study, recombinant LlTrePP was produced and characterized. It showed its highest reverse phosphorolytic activity at pH 4.

Suitable acceptor substrates were Glc6P, and, at a low level, d-mannose 6-phosphate Man6P. Endosomal recognition of Lactococcus lactis G and its RNA by dendritic cells is key to its allergy-protective effects. Bacterial cowshed isolates are allergy protective in mice; however, the underlying mechanisms are largely unknown.

We examined the ability of Lactococcus lactis G to prevent allergic inflammatory reactions. We sought to identify the ligands and pattern recognition receptors through which L lactis G confers allergy protection. The pathology of ovalbumin-induced acute allergic airway inflammation after adoptive transfer of BMDCs was examined by means of microscopy.

Inhibiting phagocytosis and endosomal acidification in BMDCs or moDCs impaired the release of T H 1 -polarizing cytokines, costimulatory molecule expression, and T-cell activation on L lactis G challenge. In vivo allergy protection mediated by L lactis G was dependent on endosomal acidification in dendritic cells DCs. Bacterial RNA is the main driver of L lactis Gmediated protection against experimentally induced allergy and requires both bacterial uptake by DCs and endosomal acidification.

Subcutaneous or oral immunization of mice with Lactococcus lactis expressing F4 fimbrial adhesin FaeG. Enterotoxigenic Escherichia coli ETEC is one of the most common causes of diarrhea in neonatal and postweaning piglets. Fimbrial adhesion of ETEC has been considered an important colonization factor with antigenicity. To safely and effectively deliver the F4 K88 fimbrial adhesin FaeG to the immune system, we have previously constructed the secretory expression vector pNZfaeG, and FaeG was produced in cytoplasmic form in Lactococcus lactis.

Subcutaneous immunization in mice with recombinant L. Oral immunization of mice with recombinant L. High-dose 2. The results suggest the feasibility of delivering rFaeG expressed in L. Polysaccharides are ubiquitous components of the Gram-positive bacterial cell wall. In Lactococcus lactis , a polysaccharide pellicle PSP forms a layer at the cell surface.

The PSP structure varies among lactococcal strains; in L. Here, we report the presence of an additional neutral polysaccharide in L. This rhamnan is still present in mutants devoid of the PSP, indicating that its synthesis can occur independently of PSP synthesis. In contrast, rhamnan was detected only at the surface of PSP-negative mutant cells, indicating that rhamnan is located underneath the surface-exposed PSP and is trapped inside peptidoglycan.

The genetic determinants of rhamnan biosynthesis appear to be within the same genetic locus that encodes the PSP biosynthetic machinery, except the gene tagO encoding the initiating glycosyltransferase. We present a model of rhamnan biosynthesis based on an ABC transporter-dependent pathway. Conditional mutants producing reduced amounts of rhamnan exhibit strong morphological defects and impaired division, indicating that rhamnan is essential for normal growth and division.

It is decorated with other glycopolymers, including. Analysis of volatile compounds produced by 2 strains of Lactococcus lactis isolated from leben Tunisian fermented milk using solid-phase microextraction-gas chromatography.

The volatile compounds that characterize Leben during fermentation with 2 Lactococcus lactis strains SLT6 and SLT10 in flasks, in a L fermentor, and during storage at 4 degrees C, were investigated and compared to those from commercial Leben. These compounds include acids, alcohols, aldehydes, ketones, sulfur compounds, and hydrocarbons. Commercial Leben presented a poor volatile profile compared to the laboratory-made Leben. The mixed culture of 2 Lactococcus lactis strains resulted in higher volatile compound formation than the single strain culture.

The GC volatile profiles of Leben produced in flask and in the L fermentor were similar. Changes in volatile compounds were observed during storage at 4 degrees C. The effect of culture conditions on production of volatiles by SLT6 strain was studied.

Aeration 0. Fermentation at pH 5 had no effect on volatile production. The alleviation also correlated with higher species abundance, more Bifidobacterium colonization, and stronger colonization resistance in mice intestinal microflora.

Therefore, this recombinant L. Dijkstra, Annereinou R. Recently, we demonstrated that fermentation conditions have a strong impact on subsequent survival of Lactococcus lactis strain MG during heat and oxidative stress, two important parameters during spray drying.

To investigate if other strains have similar or distinct transcriptome signatures for robustness, we applied an identical transcriptome-robustness phenotype matching approach on the L. In the exponential phase of growth, cells were harvested for transcriptome analysis and assessment of heat and oxidative stress survival phenotypes. The variation in fermentation conditions resulted in differences in heat and oxidative stress survival of up to five log units.

Effects of the fermentation conditions on stress survival of the L. By association of the transcriptomes and robustness phenotypes highly strain-specific transcriptome signatures for robustness towards heat and oxidative stress were identified, indicating that multiple mechanisms exist to increase robustness and, as a consequence, robustness of each strain requires individual optimization.

However, a relatively small overlap in the transcriptome responses of the strains was also identified and this generic transcriptome signature included genes previously associated with stress ctsR and lplL and novel genes, including nan. Beneficial effect of Lactococcus lactis NCC in a murine model of eosinophilic esophagitis. Eosinophilic esophagitis EoE is a severe inflammatory disease of the esophagus which is characterized histologically by an eosinophilic infiltration into the esophageal tissue.

The efficacy of probiotics in the context of atopic diseases has been well investigated but, to date, there has been no study which has evaluated probiotic effects on EoE inflammation. This study sought to identify a probiotic which improves esophageal inflammation in experimental EoE. Two candidate probiotics, Lactococcus lactis NCC and Bifidobacterium lactis NCC , were tested in a murine model of EoE elicited by epicutaneous sensitization with Aspergillus fumigatus protein extract.

Administration of bacterial strains in drinking water was used, respectively, as a preventive or treatment measure, or continuously throughout the study. Inflammatory parameters were assessed in the esophagus, skin, and lungs after allergen challenge. In this EoE model, supplementation with L. No significant effect on eosinophilia was observed when NCC was given as a preventive or a continuous intervention. NCC supplementation had no significant effect on immunoglobulin levels, skin symptom scores, or on transepidermal water loss.

Supplementation with another probiotic, B. We identified a L. This effect is strain specific and depends on the timing and duration of bacterial supplementation. Confirmation of these observations in human clinical trials is warranted. Enhance nisin yield via improving acid-tolerant capability of Lactococcus lactis F Traditionally, nisin was produced industrially by using Lactococcus lactis in the neutral fermentation process.

However, nisin showed higher activity in the acidic environment. How to balance the pH value for bacterial normal growth and nisin activity might be the key problem. In this study, 17 acid-tolerant genes and 6 lactic acid synthetic genes were introduced in L. These engineered strains showed more stable intracellular pH value during the fermentation process.

Improvement of lactate production could partly provide the extra energy for the expression of acid tolerance genes during growth. The fed-batch fermentation showed nisin titer of the co-expression L. This work provides a novel strategy of constructing robust strains for use in industry process. Development and evaluation of an efficient heterologous gene knock-in reporter system in Lactococcus lactis.

Lactococcus lactis is a food grade probiotics and widely used to express heterologous proteins. Generally, target genes are knocked into the L. However, creating marker-less heterologous genes knocked-in clones is laborious.

In this study, an efficient heterologous gene knock-in reporter system was developed in L. Our knock-in reporter system consists of a temperature-sensitive plasmid pJW and a recombinant L. The pJW contains homologous arms, and was constructed to knock-in heterologous genes at a fixed locus of NZ genome. The desired double-crossover mutants formed white colonies distinctive from the predominantly blue colonies parental and plasmid-integrated clones when the embedded lacZ was replaced with the target heterologous genes carried by pJW in NZB.

By using the system, the heterologous gene knocked-in clones are screened by colony phenotype change rather than by checking colonies individually. Our new knock-in reporter system provides an efficient method to create heterologous genes knocked-in clones.

This work investigated bacteriophage induced starter failures in artisanal buffalo Mozzarella production plants in Southern Italy. Two hundred and ten samples of whey starter cultures were screened for bacteriophage infection.

Multiplex polymerase chain reaction PCR revealed phage infection in Two phages active against L. The genomes, approximately This is the first report of phage isolation in buffalo milk and of the use of multiplex PCR to screen and study the diversity of phages against Lactic Acid Bacteria LAB strains in artisanal Water Buffalo Mozzarella starters.

Pili produced by Lactococcus lactis subsp. We determined the nanomechanical properties of pili using optical-tweezers force spectroscopy. Single pili were exposed to optical forces that yielded force-versus-extension spectra fitted using the Worm-Like Chain model.

Native pili subjected to a force of 0— pN exhibit an inextensible, but highly flexible ultrastructure, reflected by their short persistence length. We tested a panel of derived strains to understand the functional role of the different pilins. First, we found that both the major pilin PilB and sortase C organize the backbone into a full-length organelle and dictate the nanomechanical properties of the pili.

Second, we found that both PilA tip pilin and PilC anchoring pilin were not essential for the nanomechanical properties of pili. However, PilC maintains the pilus on the bacterial surface and may play a crucial role in the adhesion- and biofilm-forming properties of L. Lactococcus lactis , a gram-positive bacterium, encounters various environmental stresses, especially acid stress, during fermentation. The transcription level of s was upregulated 2. Acid tolerance assay showed that overexpressing s increased the survival rate of L.

Moreover, the targets were predicted by online software and four genes were chosen as candidates. Among them, argR arginine regulator and accD acetyl-CoA carboxylase carboxyl transferase subunit beta were validated to be the direct targets activated by S through reporter fusion assay. Dynamic modeling of lactic acid fermentation metabolism with Lactococcus lactis. A dynamic model of lactic acid fermentation using Lactococcus lactis was constructed, and a metabolic flux analysis MFA and metabolic control analysis MCA were performed to reveal an intensive metabolic understanding of lactic acid bacteria LAB.

The MFA results were a reasonable explanation of the experimental data. Through the parameter estimation, the metabolic system of lactic acid bacteria can be thoroughly understood through comparisons with the original parameters. The coefficients derived from the MCA indicated that the reaction rate of L-lactate dehydrogenase was activated by fructose 1 ,6-bisphosphate and pyruvate, and pyruvate appeared to be a stronger activator of L-lactate dehydrogenase than fructose 1 ,6-bisphosphate.

Additionally, pyruvate acted as an inhibitor to pyruvate kinase and the phosphotransferase system. Glucose 6-phosphate and phosphoenolpyruvate showed activation effects on pyruvate kinase. Hexose transporter was the strongest effector on the flux through L-lactate dehydrogenase.

Listeria monocytogenes is a pathogen frequently found in dairy products. Its control in fresh cheeses is difficult, due to the psychrotrophic properties and salt tolerance. Bacteriocinogenic lactic acid bacteria LAB with proven in vitro antilisterial activity can be an innovative technological approach but their application needs to be evaluated by means of in situ tests. In this study, a novel bacteriocinogenic Lactococcus lactis strain Lc.

A bacteriostatic effect was achieved, and counts after 10 days were 3 log lower than in control cheeses with no added LAB. However, this effect did not differ significantly from that obtained with a non-bacteriocinogenic Lc.

Addition of nisin Transcriptome analysis of Lactococcus lactis subsp. Performance of Lactococcus lactis as a starter culture in dairy fermentations depends on the levels of dissolved oxygen and the redox state of milk. In this study the microarray analysis was used to investigate the global gene expression of L.

Bacterial exposure to high initial oxygen resulted in overexpression of genes involved in detoxification of reactive oxygen species ROS , oxidation-reduction processes, biosynthesis of trehalose and down-regulation of genes involved in purine nucleotide biosynthesis, indicating that several factors, among them trehalose and GTP, were implicated in bacterial adaptation to oxidative stress. Generally, transcriptional changes were more pronounced during fermentation of oxygen sparged milk.

Genes up-regulated in response to oxygen depletion were implicated in biosynthesis and transport of pyrimidine nucleotides, branched chain amino acids and in arginine catabolic pathways; whereas genes involved in salvage of nucleotides and cysteine pathways were repressed.

Expression pattern of genes involved in pyruvate metabolism indicated shifts towards mixed acid fermentation after oxygen depletion with production of specific end-products, depending on milk treatment. Differential expression of genes, involved in amino acid and pyruvate pathways, suggested that initial oxygen might influence the release of flavor compounds and, thereby, flavor development in dairy fermentations.

The knowledge of molecular responses involved in adaptation of L. Engineering the cell surface display of cohesins for assembly of cellulosome-inspired enzyme complexes on Lactococcus lactis.

Background The assembly and spatial organization of enzymes in naturally occurring multi-protein complexes is of paramount importance for the efficient degradation of complex polymers and biosynthesis of valuable products. The degradation of cellulose into fermentable sugars by Clostridium thermocellum is achieved by means of a multi-protein “cellulosome” complex. Assembled via dockerin-cohesin interactions, the cellulosome is associated with the cell surface during cellulose hydrolysis, forming ternary cellulose-enzyme-microbe complexes for enhanced activity and synergy.

The assembly of recombinant cell surface displayed cellulosome-inspired complexes in surrogate microbes is highly desirable. The model organism Lactococcus lactis is of particular interest as it has been metabolically engineered to produce a variety of commodity chemicals including lactic acid and bioactive compounds, and can efficiently secrete an array of recombinant proteins and enzymes of varying sizes. Results Fragments of the scaffoldin protein CipA were functionally displayed on the cell surface of Lactococcus lactis.

Scaffolds were engineered to contain a single cohesin module, two cohesin modules, one cohesin and a cellulose-binding module, or only a cellulose-binding module. Cell toxicity from over-expression of the proteins was circumvented by use of the nisA inducible promoter, and incorporation of the C-terminal anchor motif of the streptococcal M6 protein resulted in the successful surface-display of the scaffolds. The facilitated detection of successfully secreted scaffolds was achieved by fusion with the export-specific reporter staphylococcal nuclease NucA.

Scaffolds retained their ability to associate in vivo with an engineered hybrid reporter enzyme, E. Heterologous expression of Streptococcus mutans Cnm in Lactococcus lactis promotes intracellular invasion, adhesion to human cardiac tissues and virulence. To further characterize Cnm as a virulence factor, the cnm gene from S. Despite the absence of the machinery necessary for Cnm glycosylation, Western blot and immunofluorescence microscopy analyses demonstrated that Cnm was effectively expressed and translocated to the cell wall of L.

Similar to S. Using an ex vivo human heart tissue colonization model, we showed that Cnm-positive strains of either S. Finally, Cnm expression facilitated L. Collectively, our results provide unequivocal evidence that binding to extracellular matrices mediated by Cnm is an important virulence attribute of S.

Expression of food-grade phytase in Lactococcus lactis from optimized conditions in milk broth. The major objective of this study was to engineer lactic acid bacteria to produce the enzyme phytase from a gene native to Bacillus subtilis GYPB The phytase gene phyC of B.

The enzyme activity in L. The expressed phytase was characterized as active in a pH range of 2. When cultured in food-grade milk broth, the transformed L. In same broth under optimized conditions for cell growth and phytase production, the transformant reached an OD nm value of 1. Fermentation was scaled to 5 L under optimized conditions, and product analysis revealed a final OD nm value of 1.

The results of this study may be used in the dairy fermentation industry for the development of functional, healthy yogurts and other fermented dairy foods that provide both active phytase and viable probiotics to the consumer.

Licheniocin While not all survivors develop PIPN, for those who do, it has a substantial negative impact on their functional status and quality of life. No interventions are available to treat PIPN. Preclinical studies suggest that the H1SP is involved in the development of bortezomib-induced and diabetic peripheral neuropathy, and sciatic nerve injury.

The purpose of this study was to identify H1SP genes that have both differential methylation and differential gene expression between breast cancer survivors with and without PIPN. A multi-staged integrated analysis was performed. In peripheral blood, methylation was assayed using microarray and gene expression was assayed using RNA-seq.

Then, candidate genes were evaluated for differential methylation and differential expression in public data sets of preclinical models of PIPN and sciatic nerve injury. Eight candidate genes were identified as both differential methylation and differential expression in survivors. This study is the first to evaluate for methylation in cancer survivors with chronic PIPN. The findings provide evidence that the expression of H1SP genes associated with chronic PIPN in cancer survivors may be regulated by epigenetic mechanisms and suggests genes for validation as potential therapeutic targets.

Not all patients develop this neurotoxicity, which supports the suggestion that molecular mechanisms may be involved in the development of PIPN. Because research with preclinical models of neuropathic pain have not yielded effective treatments, studies that use samples from patients and survivors with chemotherapy-induced peripheral neuropathy CIPN 12 , 13 are needed to address this gap. In our previous studies that evaluated gene expression patterns in survivors with and without persistent PIPN, 14 — 16 we identified perturbed pathways associated with mitochondrial dysfunction, 14 neuroinflamation, 15 and changes in cytoskeleton and axon morphology.

One of the common pathways across these three mechanisms is the hypoxia-inducible factor 1 HIF-1 signaling pathway. This pathway, a master regulator of cellular responses to hypoxia, 24 plays an important role in axon regeneration following peripheral nerve injury. Given that our previous studies of PIPN in breast cancer survivors provide evidence for differential gene expression and pathway perturbations 14 — 16 and that epigenetics modifications may be involved in the development of neuropathic pain 33 , 34 as well as involved in the transition from acute to chronic pain, 35 , 36 we sought to evaluate for epigenetic mechanisms 37 that may influence the expression of these products.

In a recent review, 41 it was noted that opportunities may exist to develop or repurpose existing drugs that target the epigenome 42 to treat a variety of neuropathic pain conditions including PIPN. Therefore, an increased understanding of DM in biologic pathways associated with the occurrence of PIPN may provide new insights into how these pathways are regulated and suggest targets for drug development.

The methods for this analysis, which is part of a larger study of CIPN, are described in detail elsewhere. Research nurses screened and consented the survivors over the phone; sent and asked them to complete the self-report questionnaires prior to their study visit; and scheduled the in-person assessment. At this assessment, written informed consent was obtained, responses to questionnaires were reviewed for completeness, and objective measurements were obtained.

This study was approved by the institutional review board of the University of California, San Francisco. Coates Genomics Sequencing Laboratory. Target success rates were determined. Subsequent data analyses were done using well-established protocols in R version 3.

Because DNA methylation differs among blood cell types, 61 — 63 cell types were estimated using the estimateCellCounts2 function in the FlowSorted. EPIC R package version 1. Any cell type composition estimates that were significantly associated with PIPN group membership were included as covariates in the final model. Surrogate variable analysis SVA, R package version 3. To understand the association of PIPN and biological variation in HIF-1 pathway genes, we performed a multi-staged integrated analysis using complementary layers of molecular data.

In the second stage, differential gene expression DGE was evaluated for each candidate gene using a generalized linear model with edgeR 72 as previously described. We estimated 14 that, at a type I error rate of 0. No minimal fold-change was utilized. We assessed for significance of functional enrichment tests using a false discovery rate of 5 under the Benjamini—Hochberg BH procedure.

Following our previously described protocols, 14 these sequences were trimmed with Trimmomatic and aligned to the mouse genome assembly GRCm Two surrogate variables were identified.

Neither surrogate variable was associated with neuropathy and both were used as covariates in the final model. These findings were compared to the differentially methylated and expressed candidate genes identified in our cancer survivors. No minimal fold-change was evaluated. The PFC and T-cells were harvested from the same animals. Methylation levels were downloaded from GEO as the quantile normalized log2 ratio of the bound Cy5 and input Cy3 microarray channel intensities.

DM in both experiments was evaluated using limma. Cancer survivor characteristics were reported previously. Of note, no between-group differences were found in the number of years since cancer diagnosis, the total dose of paclitaxel received or in the percentage of patients who had a dose reduction or delay due to PIPN. Worst pain severity was 6. Eleven surrogate variables were identified. One was associated with PIPN group membership and four were identified as significantly associated with cell type composition.

The final regression model included five significant demographic and clinical characteristics i. Twelve probes across eight genes were identified as both differentially methylated and expressed between survivors with and without PIPN Table 1. Genes in the HIF-1 pathway that were differentially methylated at promoter associated sites and differentially expressed between breast cancer survivors with and without paclitaxel-induced peripheral neuropathy.

Functionally enriched pathways associated from eight genes in the HIF-1 signaling pathway with overlapping differential methylation and differential expression between breast cancer survivors with and without paclitaxel-induced peripheral neuropathy.

Nodes represent all proteins produced by a single protein coding gene locus. Edges represent specific or meaningful associations. Known or predicted 3D structures are presented within the nodes. Color of the edges connecting the nodes represents the types of evidence supporting the connections: predicted gene neighborhood green , predicted gene fusions red , known interactions from experimental evidence pink , co-expression black , and text-mining green.

Of the eight candidate genes identified as both differentially methylated and expressed in breast cancer survivors, eight homologs were identified in the mouse DRG data set Table 3. Of these eight candidates, one gene i. Test for differential expression of candidate genes in the HIF-1 pathway in DRG between pools of mice treated with paclitaxel and normal controls.

Of these seven candidates, three genes i. While no genes were differentially methylated only in T-cells, two genes i. Differential methylation of probes in promoter regions of candidate genes in pre-frontal cortex and T-cells of rats with spared nerve injury versus sham. This study is the first to use a multi-staged integrated analysis to identify genes in the HIF-1 signaling pathway that were both differentially methylated and differentially expressed in breast cancer survivors with PIPN. In addition, this study is the first to identify a subset of these genes as being differentially methylated or differentially expressed in preclinical models of PIPN 86 or SNI.

Of note, one of these HIF-1 signaling pathway candidate genes i. The remainder of this discussion focuses on these genes and evaluates their potential role in the mechanisms that underlie PIPN and the implications of these findings with respect to epigenetic regulation of gene expression. In terms of neuroinflammation, Mnks play critical roles in cytokine receptor signaling. Recent work suggests that changes in Mknk1 expression and phosphorylation of elF4E in mice contribute to nociceptor plasticity 92 — 94 and that inhibition or elimination of Mkn1 Mknk1 attenuates PIPN in a murine model.

In terms of neuroinflammation, Mknk1 is expressed in both DRG and peripheral blood and across peripheral and neuroimmune cell lines Supplemental Figures 1 and 2 which is consistent with our findings of expression across tissue types. In contrast to our findings in survivors, Mknk1 had higher expression in mice with PIPN compared to normal mice.

One possible explanation for this difference is the timing of the measures. Based on the fact that inflammation persisted unresolved for 3. Given that DNA methylation reprogramming was observed in preclinical models of chronic neuropathic pain following nerve injury, 36 future research should evaluate for changes in the methylation and expression levels of Mknk1 over time.

Evaluations of the effects of paclitaxel on cancer cell lines found that paclitaxel activated these MAPK family pathways. The transferrin receptor TFRC gene encodes for a protein important for cellular iron uptake and homeostatis.

The phosphofructokinase, liver type PFKL gene codes for the liver subtype of an enzyme that catalyzes the phosphorylation of D-fructose 6-phosphate to fructose 1,6-bisphosphate. Therefore, PFKL is a key metabolic gene in glycolysis. Given that oncology patients with diabetes are at increased risk for developing CIPN, future studies should evaluate this mechanism in patients with both comorbidities.

The lactate dehydrogenase A LDHA gene catalyzes the forward and backward conversion of lactate to pyruvate in glycolysis. Then, hydroxylated HIFs are degraded through a ubiquination complex. The ring box 1 RBX1 and cullin 2 CUL2 genes encode for proteins that act as an E3 ubuquitin-protein ligase, which are involved in mediating the ubiquitination and degradation of proteins.

In a pharmacological network-based analysis of drug-induced peripheral neuropathy including Paclitaxel , Cul2 was identified as a highly connected significant intermediator between drugs and their pharmacological targets.

Translational control is an essential component in the regulation of gene expression and may be a core mechanism for the development and maintenance of persistent pain. However, the Mnks are effectors of other biological processes i. Alternative splicing is an important step in post-transcriptional regulation of gene expression and the diversity of various transcript isoforms of a gene are associated with environmental perturbations.

Several limitations warrant consideration. While our sample size was relatively small, we have an extremely well-characterized sample of breast cancer survivors with and without PIPN. While the integration of data from multiple sources we have increased the power to identify omics-phenotype relationships and enabled a more sophisticated interpretation of the findings, 69 , 75 future research with larger sample sizes may improve the resolution of the methylation and gene expression signals.

Our findings warrant validation in an independent sample. Of note, no differences were found in the total cumulative dose of paclitaxel that the two groups of survivors received. Given that methylation and gene expression are not independent processes, our findings must be verified in other samples and with other neurotoxic drugs.

The utility of peripheral blood as a biomarker or surrogate for neuronal tissue 13 , or as a direct signal e. Our findings suggest that the expression of HIF-1 signaling pathway genes associated with chronic PIPN in cancer survivors may be regulated by epigenetic mechanisms. Future studies need to evaluate for epigenetic changes associated with gene expression and alternative splicing in other pathways associated with PIPN, other CTX drugs, and other forms of neuropathy.

Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the NIH. Recruitment was facilitated by Dr. Mol Pain. Published online Jun Marilyn J Hammer 8 Phyllis F.

Author information Article notes Copyright and License information Disclaimer. Email: ude. Abstract Background Paclitaxel is an important chemotherapeutic agent for the treatment of breast cancer. Methods A multi-staged integrated analysis was performed.

Results Eight candidate genes were identified as both differential methylation and differential expression in survivors. Conclusions This study is the first to evaluate for methylation in cancer survivors with chronic PIPN.

Keywords: Paclitaxel-induced peripheral neuropathy, breast cancer, survivor, chemotherapy, gene expression, methylation, hypoxia-inducible factor, integrated genomic analysis. Introduction Paclitaxel is an important chemotherapeutic agent for the treatment of breast cancer.

Methods Survivors and settings The methods for this analysis, which is part of a larger study of CIPN, are described in detail elsewhere. Study procedures Research nurses screened and consented the survivors over the phone; sent and asked them to complete the self-report questionnaires prior to their study visit; and scheduled the in-person assessment.

Overlapping DM and gene expression To understand the association of PIPN and biological variation in HIF-1 pathway genes, we performed a multi-staged integrated analysis using complementary layers of molecular data. Results Differences in demographic, clinical, and pain characteristics Cancer survivor characteristics were reported previously. Table 1. Open in a separate window. Table 2. Figure 1.

Differential expression of candidate genes in mouse DRG associated with PIPN Of the eight candidate genes identified as both differentially methylated and expressed in breast cancer survivors, eight homologs were identified in the mouse DRG data set Table 3.

Table 3. Symbol M. Table 4. Discussion This study is the first to use a multi-staged integrated analysis to identify genes in the HIF-1 signaling pathway that were both differentially methylated and differentially expressed in breast cancer survivors with PIPN.

Epigenetic regulation of transcription Translational control is an essential component in the regulation of gene expression and may be a core mechanism for the development and maintenance of persistent pain. Conclusions Several limitations warrant consideration. Supplementary Material Supplemental Table 1: Click here to view. Supplemental Table 2: Click here to view. Supplemental Table 3: Click here to view. Supplementary material: Click here to view. Supplemental Material Supplemental material for this article is available online.

References 1. Kudlowitz D, Muggia F. Defining risks of taxane neuropathy: insights from randomized clinical trials.

Clin Cancer Res ; 19 : — Phase I study of taxol and granulocyte colony-stimulating factor in patients with refractory ovarian cancer. J Clin Oncol ; 10 : — Randomized phase III study of docetaxel compared with paclitaxel in metastatic breast cancer. JCO ; 23 : — Chemotherapy-induced neuropathies-a growing problem for patients and health care providers.

Brain Behav ; 7 : e Peripheral neuropathy in colorectal cancer survivors: the influence of oxaliplatin administration. Acta Oncol ; 54 : — Support Care Cancer ; 21 : — Strength and balance training for adults with peripheral neuropathy and high risk of fall: current evidence and implications for future research.

Oncol Nurs Forum ; 39 : E—E Chemotherapy-induced peripheral neuropathy and its impact on health-related quality of life among ovarian cancer survivors: results from the population-based PROFILES registry. Gynecol Oncol ; : — J Cancer Surviv. Chemotherapy-induced neuropathy in cancer survivors. J Pain Symptom Manage ; 54 : — Electrophysiological and transcriptomic correlates of neuropathic pain in human dorsal root ganglion neurons. Brain ; : — Differential methylation of the TRPA1 promoter in pain sensitivity.

Nat Commun ; 5 : Expression of mitochondrial dysfunction-related genes and pathways in paclitaxel-induced peripheral neuropathy in breast cancer survivors. Mol Pain ; 14 : Perturbations in neuroinflammatory pathways are associated with paclitaxel-induced peripheral neuropathy in breast cancer survivors.

J Neuroimmunol ; : — Signaling pathways and gene co-expression modules associated with cytoskeleton and axon morphology in breast cancer survivors with chronic paclitaxel-induced peripheral neuropathy. Mol Pain ; 15 : Beyond symptomatic relief for chemotherapy-induced peripheral neuropathy: targeting the source.

Cancer ; : — Role of mitochondrial mechanism in chemotherapy-induced peripheral neuropathy. Curr Drug Metab ; 19 : 47— Clinical and preclinical perspectives on chemotherapy-induced peripheral neuropathy CIPN : a narrative review.

Br J Anaesth ; : — Characterisation of immune and neuroinflammatory changes associated with chemotherapy-induced peripheral neuropathy. PLoS One ; 12 : e Effects of eribulin, vincristine, paclitaxel and ixabepilone on fast axonal transport and kinesin-1 driven microtubule gliding: implications for chemotherapy-induced peripheral neuropathy. Neurotoxicology ; 37 : — Paclitaxel induces axonal microtubules polar reconfiguration and impaired organelle transport: implications for the pathogenesis of paclitaxel-induced polyneuropathy.

Acta Neuropathol ; : — A mechanistic understanding of axon degeneration in chemotherapy-induced peripheral neuropathy. Front Neurosci ; 11 : Cell Biochem Funct ; 31 : — Venkatesh I, Blackmore MG. Selecting optimal combinations of transcription factors to promote axon regeneration: why mechanisms matter.

Neurosci Lett ; : 64— Semenza GL. Oxygen homeostasis. Hypoxia-inducible factor 1: regulator of mitochondrial metabolism and mediator of ischemic preconditioning. Biochim Biophys Acta ; : — Nitric oxide prevents axonal degeneration by inducing HIFdependent expression of erythropoietin.

Activating injury-responsive genes with hypoxia enhances axon regeneration through neuronal HIF-1alpha. Neuron ; 88 : — Ludman T, Melemedjian OK. Bortezomib and metformin opposingly regulate the expression of hypoxia-inducible factor alpha and the consequent development of chemotherapy-induced painful peripheral neuropathy. Hypoxia-inducible factor 1alpha protects peripheral sensory neurons from diabetic peripheral neuropathy by suppressing accumulation of reactive oxygen species.

J Mol Med ; 96 : — Hypoxia-inducible factor 1 regulates heat and cold pain sensitivity and persistence. Antioxid Redox Signal ; 20 : — Epigenetic mechanisms of chronic pain. Trends Neurosci ; 38 : — Machelska H, Celik MO.

Recent advances in understanding neuropathic pain: glia, sex differences, and epigenetics. FRes ; 5 : Epigenetics and the transition from acute to chronic pain. Pain Med ; 13 : — Nerve injury-induced chronic pain is associated with persistent DNA methylation reprogramming in dorsal root ganglion.

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