Some lactic acid bacteria, especially
spp., possess adhesive properties enabling colonization of the human gastrointestinal tract. Two probiotic
strains, WCSF1 and 299v, display highly different ...mannose-specific adhesion, with
299v being superior to
WCFS1 based on a yeast agglutination assay. A straightforward correlation between the mannose adhesion capacity and domain composition of the mannose-specific adhesin (Msa) in the two strains has not been demonstrated previously. In this study, we analyzed the promoter regions upstream of the
gene encoding a mannose-specific adhesin in these two strains. The promoter region was mapped by primer extension and DNA sequence analysis, and only a single nucleotide change was identified between the two strains. However, Northern blot analysis showed a stronger
transcript band in 299v than in WCFS1 correlating with the different adhesion capacities. During the establishment of a high-throughput yeast agglutination assay, we isolated variants of WCFS1 that displayed a very strong mannose-specific adhesion phenotype. The region upstream of the
gene in these variants showed an inversion of a 104-bp fragment located between two perfectly inverted repeats present in the untranslated leader region. The inversion disrupts a strong hairpin structure that otherwise most likely would terminate the
transcript. In addition, the ribosome binding site upstream of the
gene, which is also masked within this hairpin structure, becomes accessible upon inversion, thereby increasing the frequency of translation initiation in the variant strains. Furthermore, Northern blot analysis showed a higher abundance of the
transcript in the variants than in the wild type, correlating with a strong-Msa phenotype.
Probiotic strains possess adhesive properties enabling colonization of the human intestinal tract through interactions between molecules present on the probiotic bacteria and components of the epithelial surface. In
, interaction is mediated through bacterial surface proteins like Msa, which binds to mannose residues present on the intestinal cells. Such interactions are believed to be important for the health-promoting effects of probiotics, including displacement of pathogens, immunomodulation, and protective effects on the intestinal barrier function. In this study, we have identified a new molecular switch controlling expression of the
gene in
strain WCFS1. Strains with increased
expression could be valuable in the development and manufacture of improved probiotic products.
The light-focusing capability of metal nanoparticles in a periodic square array on top of an upconverting thin film was modeled using the finite-element-frequency-domain method. The particle geometry ...was optimized for efficient concentration of an incoming plane wave into the upconverting medium, in order to enhance the upconversion process. Specifically, the two-dimensional parameter space, corresponding to the height and diameter of nearly cylindrical nanoparticles, was scanned and the optimal particle dimensions were found. A selected geometry was fabricated and the upconversion yield was measured and compared to the case without nanoparticles. Subsequently, the particle spacing was included as another free parameter, and the multi-start Nelder-Mead method and derivative-free simulated annealing wereused to optimize thisthree-parameter system.
Systems genetics has begun to tackle the complexity of insulin resistance by capitalising on computational advances to study high-diversity populations. 'Diversity Outbred in Australia (DOz)' is a ...population of genetically unique mice with profound metabolic heterogeneity. We leveraged this variance to explore skeletal muscle's contribution to whole-body insulin action through metabolic phenotyping and skeletal muscle proteomics of 215 DOz mice. Linear modelling identified 553 proteins that associated with whole-body insulin sensitivity (Matsuda Index) including regulators of endocytosis and muscle proteostasis. To enrich for causality, we refined this network by focusing on negatively associated, genetically regulated proteins, resulting in a 76-protein fingerprint of insulin resistance. We sought to perturb this network and restore insulin action with small molecules by integrating the Broad Institute Connectivity Map platform and in vitro assays of insulin action using the Prestwick chemical library. These complementary approaches identified the antibiotic thiostrepton as an insulin resistance reversal agent. Subsequent validation in ex vivo insulin-resistant mouse muscle and palmitate-induced insulin-resistant myotubes demonstrated potent insulin action restoration, potentially via upregulation of glycolysis. This work demonstrates the value of a drug-centric framework to validate systems-level analysis by identifying potential therapeutics for insulin resistance.
Metabolic disease is caused by a combination of genetic and environmental factors, yet few studies have examined how these factors influence signal transduction, a key mediator of metabolism. Using ...mass spectrometry-based phosphoproteomics, we quantified 23,126 phosphosites in skeletal muscle of five genetically distinct mouse strains in two dietary environments, with and without acute in vivo insulin stimulation. Almost half of the insulin-regulated phosphoproteome was modified by genetic background on an ordinary diet, and high-fat high-sugar feeding affected insulin signalling in a strain-dependent manner. Our data revealed coregulated subnetworks within the insulin signalling pathway, expanding our understanding of the pathway's organisation. Furthermore, associating diverse signalling responses with insulin-stimulated glucose uptake uncovered regulators of muscle insulin responsiveness, including the regulatory phosphosite S469 on Pfkfb2, a key activator of glycolysis. Finally, we confirmed the role of glycolysis in modulating insulin action in insulin resistance. Our results underscore the significance of genetics in shaping global signalling responses and their adaptability to environmental changes, emphasising the utility of studying biological diversity with phosphoproteomics to discover key regulatory mechanisms of complex traits.
This paper describes a systematic design study of periodic gold-nanostrip arrays placed on a thin film aimed at enhancing the electric field inside the film when irradiated by light. Based on the ...study, a "selection rule" is proposed, which provides optimization-based design methods with an a priori choice between field-enhancement dominated by coupling to guided modes, by plasmonic near-field enhancement or by a mix hereof. An appropriate choice of wavelength and grating period is shown to selectively suppress or include waveguiding effects for the optimized designs. The validity of the selection rule is demonstrated through a numerical topology optimization study in which gold nanostrips are optimized for electric-field enhancement in an erbium-doped TiO
thin film, targeting increased spectral upconversion in the erbium ions. The obtained designs exhibit waveguide excitation within the predicted intervals and, for light polarized perpendicularly to the strips, plasmonic response outside.
Insulin resistance (IR) is a complex metabolic disorder that underlies several human diseases, including type 2 diabetes and cardiovascular disease. Despite extensive research, the precise mechanisms ...underlying IR development remain poorly understood. Previously we showed that deficiency of coenzyme Q (CoQ) is necessary and sufficient for IR in adipocytes and skeletal muscle (Fazakerley et al., 2018). Here, we provide new insights into the mechanistic connections between cellular alterations associated with IR, including increased ceramides, CoQ deficiency, mitochondrial dysfunction, and oxidative stress. We demonstrate that elevated levels of ceramide in the mitochondria of skeletal muscle cells result in CoQ depletion and loss of mitochondrial respiratory chain components, leading to mitochondrial dysfunction and IR. Further, decreasing mitochondrial ceramide levels in vitro and in animal models (mice, C57BL/6J) (under chow and high-fat diet) increased CoQ levels and was protective against IR. CoQ supplementation also rescued ceramide-associated IR. Examination of the mitochondrial proteome from human muscle biopsies revealed a strong correlation between the respirasome system and mitochondrial ceramide as key determinants of insulin sensitivity. Our findings highlight the mitochondrial ceramide-CoQ-respiratory chain nexus as a potential foundation of an IR pathway that may also play a critical role in other conditions associated with ceramide accumulation and mitochondrial dysfunction, such as heart failure, cancer, and aging. These insights may have important clinical implications for the development of novel therapeutic strategies for the treatment of IR and related metabolic disorders.
Abstract
The use of the Gram-positive bacterium Lactococcus lactis in recombinant protein production has several advantages, including the organism's long history of safe use in food production and ...the fact that it does not produce endotoxins. Furthermore the current non-dairy L. lactis production strains contain few proteases and can secrete stable recombinant protein to the growth medium. The P170 expression system used for recombinant protein production in L. lactis utilizes an inducible promoter, P170, which is up-regulated as lactate accumulates in the growth medium. We have optimised the components of the expression system, including improved promoter strength, signal peptides and isolation of production strains with increased productivity. Recombinant proteins are produced in a growth medium with no animal-derived components as a simple batch fermentation requiring minimal process control. The accumulation of lactate in the growth medium does, however, inhibit growth and limits the yield from batch and fed-batch processes. We therefore combined the P170 expression system with the REED™ technology, which allows control of lactate concentration by electro-dialysis during fermentation. Using this combination, production of the Staphylococcus aureus nuclease reached 2.5 g L−1.
This study describes the development of a simple production system for manufacturing recombinant proteins and enzymes in a safe bacterial host.
Quantitative measurements of the electric near-field distribution of star-shaped gold nanoparticles have been performed by femtosecond laser ablation. Measurements were carried out on and off the ...plasmon resonance. A detailed comparison with numerical simulations of the electric fields is presented. Semi-quantitative agreement is found, with slight systematic differences between experimentally observed and simulated near-field patterns close to strong electric-field gradients. The deviations are attributed to carrier transport preceding ablation.
Rapid wound closure is important after arthroplasty procedures to prevent postoperative complications. Platelets are rich in growth factors and leukocytes contribute to innate immunity. We ...hypothesized that topical leukocyte platelet‐rich plasma (L‐PRP) derived from the blood of patients would be beneficial to wound healing. In this randomized controlled trial, patients subjected to elective total hip arthroplasty (THA) were assigned by concealed allocation either L‐PRP application onto the sutured fascia or no application (control) after the THA intervention. In addition, all patients received 1.5 g protein/kg, 5 g L‐arginine, 500 mg vitamin C and 44 mg zinc daily over the 4‐week postoperative period to obtain optimal nutrition. The primary endpoint was complete healing of the skin incision. The secondary endpoints were blood transfusions, length of hospital stay, pain and wound infections. Sixteen patients in the L‐PRP group and 17 patients in the control group completed the trial. L‐PRP treatment accelerated complete wound healing after 3 weeks (seven in the L‐PRP group vs. zero in the control group, p = 0.003) and after 4 weeks (12 in the L‐PRP group vs. six in the control group, p = 0.037). No postoperative superficial wound infections occurred within 4 weeks, and there were no significant differences in the other secondary outcomes. L‐PRP generated in 10 sex‐matched healthy volunteers revealed increased concentrations of platelets (5.8‐fold) and leukocytes (2.3‐fold) compared with those in whole blood. Furthermore, the concentration of keratinocyte mitogen epidermal growth factor in L‐PRP (380 ± 130 pg/ml, mean ± SD) was higher (p < 0.001) than that in serum (130 ± 26 pg/ml). In conclusion, a single intraoperative local application of L‐PRP promoted wound healing after THA, possibly mediated by EGF receptor agonists.
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