Heterologous protein display on the cell surface of lactic acid bacteria mediated by the s-layer protein

<p>Abstract</p> <p>Background</p> <p>Previous studies have revealed that the C-terminal region of the S-layer protein from <it>Lactobacillus </it>is responsible for the cell wall anchoring, which provide an approach for targeting heterologous proteins to the cell wall of lactic acid bacteria (LAB). In this study, we developed a new surface display system in lactic acid bacteria with the C-terminal region of S-layer protein SlpB of <it>Lactobacillus crispatus </it>K2-4-3 isolated from chicken intestine.</p> <p>Results</p> <p>Multiple sequence alignment revealed that the C-terminal region (LcsB) of <it>Lb. crispatus </it>K2-4-3 SlpB had a high similarity with the cell wall binding domains S_Aand CbsA of <it>Lactobacillus acidophilus </it>and <it>Lb. crispatus</it>. To evaluate the potential application as an anchoring protein, the green fluorescent protein (GFP) or beta-galactosidase (Gal) was fused to the N-terminus of the LcsB region, and the fused proteins were successfully produced in <it>Escherichia coli</it>, respectively. After mixing them with the non-genetically modified lactic acid bacteria cells, the fused GFP-LcsB and Gal-LcsB were functionally associated with the cell surface of various lactic acid bacteria tested. In addition, the binding capacity could be improved by SDS pretreatment. Moreover, both of the fused proteins could simultaneously bind to the surface of a single cell. Furthermore, when the fused DNA fragment of <it>gfp:lcsB </it>was inserted into the <it>Lactococcus lactis </it>expression vector pSec:Leiss:Nuc, the GFP could not be secreted into the medium under the control of the <it>nisA </it>promoter. Western blot, in-gel fluorescence assay, immunofluorescence microscopy and SDS sensitivity analysis confirmed that the GFP was successfully expressed onto the cell surface of <it>L. lactis </it>with the aid of the LcsB anchor.</p> <p>Conclusion</p> <p>The LcsB region can be used as a functional scaffold to target the heterologous proteins to the cell surfaces of lactic acid bacteria <it>in vitro </it>and <it>in vivo</it>, and has also the potential for biotechnological application.</p

Pro-inflammatory Effect of Downregulated CD73 Expression in EAE Astrocytes

CD73, an ectonucleotidase, participates in the regulation of immune responses by controlling the conversion of extracellular AMP to adenosine. In this study, we investigated whether any type of brain cells, especially neuroglia cells, exhibit altered CD73 expression, localization or activity upon experimental autoimmune uveitis (EAU) induction and whether altered CD73 manipulates the activation of effector T cells that interact with such cell types. First, the amount of cell membrane-exposed CD73 was detected by flow cytometry in various types of brain cells collected from either naïve or EAE mice. Compared to that in astrocytes from naïve control mice, the amount of membrane-bound CD73 was significantly decreased in astrocytes from EAE mice, while no significant differences were detected in other cell types. Thereafter, wild-type and CD73-/- astrocytes were used to study whether CD73 influences the function of inflammatory astrocytes, such as the production of cytokines/chemokines and the activation of effector T cells that interact with astrocytes. The results indicated that the addition of exogenous AMP significantly inhibited cytokine/chemokine production by wild type astrocytes but had no effect on CD73-/- astrocytes and that the effect of AMP was almost completely blocked by the addition of either a CD73 inhibitor (APCP) or an adenosine receptor A1 subtype (ARA1) antagonist (DPCPX). Although the addition of AMP did not affect CD73-/- astrocytes, the addition of adenosine successfully inhibited their cytokine/chemokine production. The antigen-specific interaction of astrocytes with invading CD4 cells caused CD73 downregulation in astrocytes from mice that underwent EAE induction. Collectively, our findings support the conclusion that, upon EAE induction, likely due to an interaction with invading CD4+ cells, astrocytes lose most of their membrane-localized CD73; this inhibits the generation of adenosine in the local microenvironment. As adenosine has anti-inflammatory effects on astrocytes and CNS-infiltrating effector T cells in EAE, the downregulation of CD73 in astrocytes may be considered a pro-inflammatory process for facilitating the pathogenesis of EAE

Higher systemic immune-inflammation index and systemic inflammation response index levels are associated with stroke prevalence in the asthmatic population: a cross-sectional analysis of the NHANES 1999-2018

BackgroundSignificant evidence suggests that asthma might originate from low-grade systemic inflammation. Previous studies have established a positive association between the systemic immune-inflammation index (SII) and the systemic inflammation response index (SIRI) levels and the risk of stroke. However, it remains unclear whether SII, SIRI and the prevalence of stroke are related in individuals with asthma.MethodsThe present cross-sectional study used data from the National Health and Nutrition Examination Survey (NHANES) conducted between 1999 and 2018. SII was calculated using the following formula: (platelet count × neutrophil count)/lymphocyte count. SIRI was calculated using the following formula: (neutrophil count × monocyte count)/lymphocyte count. The Spearman rank correlation coefficient was used to determine any correlation between SII, SIRI, and the baseline characteristics. Survey-weighted logistic regression was employed to calculate odds ratios (ORs) and 95% confidence intervals (CIs) to determine the association between SII, SIRI, and stroke prevalence. The predictive value of SII and SIRI for stroke prevalence was assessed through receiver operating characteristic (ROC) curve analysis, with the area under the ROC curve (AUC) being indicative of its predictive value. Additionally, clinical models including SIRI, coronary heart disease, hypertension, age, and poverty income ratio were constructed to evaluate their clinical applicability.ResultsBetween 1999 and 2018, 5,907 NHANES participants with asthma were identified, of which 199 participants experienced a stroke, while the remaining 5,708 participants had not. Spearman rank correlation analysis indicated that neither SII nor SIRI levels exhibited any significant correlation with the baseline characteristics of the participants (r&lt;0.1). ROC curves were used to determine the optimal cut-off values for SII and SIRI levels to classify participants into low- and high-level groups. Higher SII and SIRI levels were associated with a higher prevalence of stroke, with ORs of 1.80 (95% CI, 1.18-2.76) and 2.23 (95% CI, 1.39-3.57), respectively. The predictive value of SIRI (AUC=0.618) for stroke prevalence was superior to that of SII (AUC=0.552). Furthermore, the clinical model demonstrated good predictive value (AUC=0.825), with a sensitivity of 67.1% and specificity of 87.7%.ConclusionIn asthmatics, higher levels of SII and SIRI significantly increased the prevalence of stroke, with its association being more pronounced in individuals with coexisting obesity and hyperlipidaemia. SII and SIRI are relatively stable novel inflammatory markers in the asthmatic population, with SIRI having a better predictive value for stroke prevalence than SII

CYP-omega-hydroxylation-dependent metabolites of arachidonic acid inhibit the basolateral 10pS chloride channel in the rat thick ascending limb

Metabolites of arachidonic acid influence sodium chloride (NaCl) transport in the thick ascending limb. Because a 10pS Cl channel is the major type of chloride channel in the basolateral membrane of this nephron segment, we explored the effect of arachidonic acid on this channel in cell-attached patches. Addition of 5μmol arachidonic acid significantly decreased channel activity (a product of channel number and open probability) while linoleic acid had no effect. To determine if this was mediated by acachidonic acid per se or by its metabolites, we measured channel activity in the presence of the cyclooxygenase inhibitor indomethacin, the selective lipoxygenase inhibitor nordihydroguaiaretic acid, and the cytochrome P-450 (CYP)-ω-hydroxylation inhibitor 17-octadecynoic acid. Neither cyclooxygenase nor lipoxygenase inhibition had an effect on basal chloride channel activity; further they failed to abolish the inhibitory effect of arachidonate on the 10pS channel. However, inhibition of CYP-ω-hydroxylation completely abolished the effect of arachidonic acid. The similarity of the effects of 20-hydroxyeicosatetraenoic acid (20-HETE) and arachidonic acid suggests that the effect of arachidonic acid was mediated by CYP-ω-hydroxylation-dependent metabolites. We conclude that arachidonic acid inhibits the 10pS chloride channel in the basolateral membrane of the medullary thick ascending limb, an effect mediated by the CYP-ω-hydroxylation-dependent metabolite 20-HETE

Chromosome 2p14 Is Linked to Susceptibility to Leprosy

BACKGROUND: A genetic component to the etiology of leprosy is well recognized but the mechanism of inheritance and the genes involved are yet to be fully established. METHODOLOGY: A genome-wide single nucleotide polymorphism (SNP) based linkage analysis was carried out using 23 pedigrees, each with 3 to 7 family members affected by leprosy. Multipoint parametric and non-parametric linkage analyses were performed using MERLIN 1.1.1. PRINCIPAL FINDINGS: Genome-wide significant evidence for linkage was identified on chromosome 2p14 with a heterogeneity logarithm of odds (HLOD) score of 3.51 (rs1106577) under a recessive model of inheritance, while suggestive evidence was identified on chr.4q22 (HLOD 2.92, rs1349350, dominant model), chr. 8q24 (HLOD 2.74, rs1618523, recessive model) and chr.16q24 (HLOD 1.93, rs276990 dominant model). Our study also provided moderate evidence for a linkage locus on chromosome 6q24-26 by non-parametric linkage analysis (rs6570858, LOD 1.54, p = 0.004), overlapping a previously reported linkage region on chromosome 6q25-26. CONCLUSION: A genome-wide linkage analysis has identified a new linkage locus on chromosome 2p14 for leprosy in Pedigrees from China

Fine Tuning of the Lactate and Diacetyl Production through Promoter Engineering in Lactococcus lactis

Lactococcus lactis is a well-studied bacterium widely used in dairy fermentation and capable of producing metabolites with organoleptic and nutritional characteristics. For fine tuning of the distribution of glycolytic flux at the pyruvate branch from lactate to diacetyl and balancing the production of the two metabolites under aerobic conditions, a constitutive promoter library was constructed by randomizing the promoter sequence of the H2O-forming NADH oxidase gene in L. lactis. The library consisted of 30 promoters covering a wide range of activities from 7,000 to 380,000 relative fluorescence units using a green fluorescent protein as reporter. Eleven typical promoters of the library were selected for the constitutive expression of the H2O-forming NADH oxidase gene in L. lactis, and the NADH oxidase activity increased from 9.43 to 58.17-fold of the wild-type strain in small steps of activity change under aerobic conditions. Meanwhile, the lactate yield decreased from 21.15±0.08 mM to 9.94±0.07 mM, and the corresponding diacetyl production increased from 1.07±0.03 mM to 4.16±0.06 mM with the intracellular NADH/NAD+ ratios varying from 0.711±0.005 to 0.383±0.003. The results indicated that the reduced pyruvate to lactate flux was rerouted to the diacetyl with an almost linear flux variation via altered NADH/NAD+ ratios. Therefore, we provided a novel strategy to precisely control the pyruvate distribution for fine tuning of the lactate and diacetyl production through promoter engineering in L. lactis. Interestingly, the increased H2O-forming NADH oxidase activity led to 76.95% lower H2O2 concentration in the recombinant strain than that of the wild-type strain after 24 h of aerated cultivation. The viable cells were significantly elevated by four orders of magnitude within 28 days of storage at 4°C, suggesting that the increased enzyme activity could eliminate H2O2 accumulation and prolong cell survival

Large expert-curated database for benchmarking document similarity detection in biomedical literature search

Document recommendation systems for locating relevant literature have mostly relied on methods developed a decade ago. This is largely due to the lack of a large offline gold-standard benchmark of relevant documents that cover a variety of research fields such that newly developed literature search techniques can be compared, improved and translated into practice. To overcome this bottleneck, we have established the RElevant LIterature SearcH consortium consisting of more than 1500 scientists from 84 countries, who have collectively annotated the relevance of over 180 000 PubMed-listed articles with regard to their respective seed (input) article/s. The majority of annotations were contributed by highly experienced, original authors of the seed articles. The collected data cover 76% of all unique PubMed Medical Subject Headings descriptors. No systematic biases were observed across different experience levels, research fields or time spent on annotations. More importantly, annotations of the same document pairs contributed by different scientists were highly concordant. We further show that the three representative baseline methods used to generate recommended articles for evaluation (Okapi Best Matching 25, Term Frequency-Inverse Document Frequency and PubMed Related Articles) had similar overall performances. Additionally, we found that these methods each tend to produce distinct collections of recommended articles, suggesting that a hybrid method may be required to completely capture all relevant articles. The established database server located at https://relishdb.ict.griffith.edu.au is freely available for the downloading of annotation data and the blind testing of new methods. We expect that this benchmark will be useful for stimulating the development of new powerful techniques for title and title/abstract-based search engines for relevant articles in biomedical research.Peer reviewe

Towards Lightweight Neural Networks with Few Data via Knowledge Distillation

The advancement of deep learning technology has been concentrating on deploying end-to-end solutions using high dimensional data, such as images. With the rapid increase in benchmark performance comes the significant resource requirements to train the network and make inferences with it. Deep learning models that achieve state-of-the-art benchmark result may require a huge amount of computing resources and data. To alleviate this problem, knowledge distillation with teacher-student learning has drawn much attention in compressing neural networks on low-end edge devices, such as mobile phones and wearable watches. However, current teacher-student learning algorithms mainly assume that the complete dataset for the teacher network is also available for the training of the student network. However, for real-world scenarios, users may only access to part of training examples due to commercial profits or data privacy, and severe over-fitting issues would happen as a result. In this study, we tackle the challenge of learning student networks with few data by investigating the ground-truth data-generating distribution underlying these few data. Taking Wasserstein distance as the measurement, we assume that this ideal data distribution lies in a neighborhood of the discrete empirical distribution induced by the training examples. Thus we propose to safely optimize the worst-case cost within this neighborhood to boost the generalization. Furthermore, with theoretical analysis, we derive a novel and easy-to-implement loss for training the student network in an end-to-end fashion. Our analysis is empirically validated through experiments using benchmark datasets, and the result indicates the effectiveness of our proposed method

Characterization of a Novel LysM Domain from Lactobacillus fermentum Bacteriophage Endolysin and Its Use as an Anchor To Display Heterologous Proteins on the Surfaces of Lactic Acid Bacteria▿

The endolysin Lyb5, from Lactobacillus fermentum temperate bacteriophage φPYB5, showed a broad lytic spectrum against Gram-positive as well as Gram-negative bacteria. Sequence analysis revealed that the C terminus of the endolysin Lyb5 (Ly5C) contained three putative lysin motif (LysM) repeat regions, implying that Ly5C was involved in bacterial cell wall binding. To investigate the potential of Ly5C for surface display, green fluorescent protein (GFP) was fused to Ly5C at its N or C terminus and the resulting fusion proteins were expressed in Escherichia coli. After being mixed with various cells in vitro, GFP was successfully displayed on the surfaces of Lactococcus lactis, Lactobacillus casei, Lb. brevis, Lb. plantarum, Lb. fermentum, Lb. delbrueckii, Lb. helveticus, and Streptococcus thermophilus cells. Increases in the fluorescence intensities of chemically pretreated L. lactis and Lb. casei cells compared to those of nonpretreated cells suggested that the peptidoglycan was the binding ligand for Ly5C. Moreover, the pH and concentration of sodium chloride were optimized to enhance the binding capacity of GFP-Ly5C, and high-intensity fluorescence of cells was observed under optimal conditions. All results suggested that Ly5C was a novel anchor for constructing a surface display system for lactic acid bacteria (LAB). To demonstrate the applicability of the Ly5C-mediated surface display system, β-galactosidase (β-Gal) from Paenibacillus sp. strain K1, replacing GFP, was functionally displayed on the surfaces of LAB cells via Ly5C. The success in surface display of GFP and β-Gal opened up the feasibility of employing the cell wall anchor of bacteriophage endolysin for surface display in LAB