TCF1-LEF1 co-expression identifies a multipotent progenitor cell (TH2-MPP) across human allergic diseases.

Repetitive exposure to antigen in chronic infection and cancer drives T cell exhaustion, limiting adaptive immunity. In contrast, aberrant, sustained T cell responses can persist over decades in human allergic disease. To understand these divergent outcomes, we employed bioinformatic, immunophenotyping and functional approaches with human diseased tissues, identifying an abundant population of type 2 helper T (TH2) cells with co-expression of TCF7 and LEF1, and features of chronic activation. These cells, which we termed TH2-multipotent progenitors (TH2-MPP) could self-renew and differentiate into cytokine-producing effector cells, regulatory T (Treg) cells and follicular helper T (TFH) cells. Single-cell T-cell-receptor lineage tracing confirmed lineage relationships between TH2-MPP, TH2 effectors, Treg cells and TFH cells. TH2-MPP persisted despite in vivo IL-4 receptor blockade, while thymic stromal lymphopoietin (TSLP) drove selective expansion of progenitor cells and rendered them insensitive to glucocorticoid-induced apoptosis in vitro. Together, our data identify TH2-MPP as an aberrant T cell population with the potential to sustain type 2 inflammation and support the paradigm that chronic T cell responses can be coordinated over time by progenitor cells.

Dynamic Failure Experimental Study of a Gravity Dam Model on a Shaking Table and Analysis of Its Structural Dynamic Characteristics.

Investigating the dynamic response patterns and failure modes of concrete gravity dams subjected to strong earthquakes is a pivotal area of research for addressing seismic safety concerns associated with gravity dam structures. Dynamic shaking table testing has proven to be a robust methodology for exploring the dynamic characteristics and failure modes of gravity dams. This paper details the dynamic test conducted on a gravity dam model on a shaking table. The emulation concrete material, featuring high density, low dynamic elastic modulus, and appropriate strength, was meticulously designed and fabricated. Integrating the shaking table conditions with the model material, a comprehensive gravity dam shaking table model test was devised to capture the dynamic response of the model under various dynamic loads. Multiple operational conditions were carefully selected for in-depth analysis. Leveraging the dynamic strain responses, the progression of damage in the gravity dam model under these diverse conditions was thoroughly examined. Subsequently, the recorded acceleration responses were utilized for identifying dynamic characteristic parameters, including the acceleration amplification factor in the time domain, acceleration response spectrum characteristics in the frequency domain, and modal parameters reflecting the inherent characteristics of the structure. To gain a comprehensive understanding, a comparative analysis was performed by aligning the observed damage development with the identified dynamic characteristic parameters, and the sensitivity of these identified parameters to different levels of damage was discussed. The findings of this study not only offer valuable insights for conducting and scrutinizing shaking table experiments on gravity dams but also serve as crucial supporting material for identifying structural dynamic characteristic parameters and validating damage diagnosis methods for gravity dam structures.

Application of omics in Sjögren's syndrome.

OBJECTIVE: The pathogenesis, diagnosis, and treatment of Sjögren's syndrome (SS) face many challenges, and there is an urgent need to develop new technologies to improve our understanding of SS. METHODS: By searching the literature published domestically and internationally in the past 20 years, this artical reviewed the research of various omics techniques in SS. RESULTS: Omics technology provided valuable insights into the pathogenesis, early diagnosis, condition and efficacy evaluation of SS. It is helpful to reveal the pathogenesis of the disease and explore new treatment schemes, which will open a new era for the study of SS. CONCLUSION: At present, omics research has made some gratifying achievements, but there are still many uncertainties. Therefore, in the future, we should improve research techniques, standardize the collection of samples, and adopt a combination of multi-omics techniques to jointly study the pathogenesis of SS and provide new schemes for its treatment.

Dynamic changes in the migratory microbial components of colon tissue during different periods of sepsis in an LPS-induced rat model.

Previous studies have shown that bacterial translocation may play an important role in worsening gastrointestinal injury during sepsis. However, the dynamics of specific microbiota components in intestinal tissues at different sepsis stages remain unclear. Rats receiving intraperitoneal lipopolysaccharide (LPS) were sacrificed at 12 h and 48 h post-injection. Routine blood, serum cytokines, and microbiota in colon tissue, colonic contents, and lung tissue at different time points were assessed. Migratory microbial components in colonic tissue at 12 h and 48 h post-LPS were identified using source tracking, characteristic component identification, and abundance difference analyses. Colonic tissue microbiota changed dynamically over time after LPS injection, involving translocation of microbial components from colon contents and lung tissue at different time points. Bacteria migrating to colon tissue at 12 h sepsis were mainly from colonic contents, while those at 48 h were predominantly from the lung tissue. The migratory microbial components in colon tissue were widely associated with blood indicators and colonizing genus abundance and microbiota functionality in colon tissue. In this study, the temporal dynamics of bacterial translocation from various sources into colon tissues at different sepsis progression stages were characterized for the first time, and the species composition of these migrating microbes was delineated. These bacterial migrants may contribute to the pathophysiological processes in sepsis through direct interactions or indirectly by modulating colonic microbiota community structure and function.

Complete biosynthesis of a sulfated chondroitin in Escherichia coli.

Sulfated glycosaminoglycans (GAGs) are a class of important biologics that are currently manufactured by extraction from animal tissues. Although such methods are unsustainable and prone to contamination, animal-free production methods have not emerged as competitive alternatives due to complexities in scale-up, requirement for multiple stages and cost of co-factors and purification. Here, we demonstrate the development of single microbial cell factories capable of complete, one-step biosynthesis of chondroitin sulfate (CS), a type of GAG. We engineer E. coli to produce all three required components for CS production-chondroitin, sulfate donor and sulfotransferase. In this way, we achieve intracellular CS production of ~27 µg/g dry-cell-weight with about 96% of the disaccharides sulfated. We further explore four different factors that can affect the sulfation levels of this microbial product. Overall, this is a demonstration of simple, one-step microbial production of a sulfated GAG and marks an important step in the animal-free production of these molecules.

N-glycolyl chondroitin synthesis using metabolically engineered E. coli.

N-glycolyl chondroitin (Gc-CN) is a metabolite of N-glycolylneuraminic acid (Neu5Gc), a sialic acid that is commonly found in mammals, but not humans. Humans can incorporate exogenous Neu5Gc into their tissues from eating red meat. Neu5Gc cannot be biosynthesized by humans due to an evolutionary mutation and has been implicated in causing inflammation causing human diseases, such as cancer. The study Neu5Gc is important in evolutionary biology and the development of potential cancer biomarkers. Unfortunately, there are several limitations to detecting Neu5Gc. The elimination of Neu5Gc involves a degradative pathway leading to the incorporation of N-glycolyl groups into glycosaminoglycans (GAGs), such as Gc-CN. Gc-CN has been found in humans and in animals including mice, lamb and chimpanzees. Here, we present the biosynthesis of Gc-CN in bacteria by feeding chemically synthesized N-glycolylglucosamine to Escherichia coli. A metabolically engineered strain of E. coli K4, fed with glucose supplemented with GlcNGc, converted it to N-glycolylgalactosamine (GalNGc) that could then be utilized as a substrate in the chondroitin biosynthetic pathway. The final product, Gc-CN was converted to disaccharides using chondroitin lyase ABC and analyzed by liquid chromatography-tandem mass spectrometry with multiple reaction monitoring detection. This analysis showed the incorporation of GalNGc into the backbone of the chondroitin oligosaccharide.

Expression of enzymes for 3'-phosphoadenosine-5'-phosphosulfate (PAPS) biosynthesis and their preparation for PAPS synthesis and regeneration.

The synthesis of sulfated polysaccharides involves the sulfation of simpler polysaccharide substrates, through the action sulfotransferases using the cofactor, 3'-phosphoadenosine-5'-phosphosulfate (PAPS). Three enzymes are essential for the in vitro synthesis of PAPS, namely, pyrophosphatase (PPA), adenosine 5'-phosphosulfate kinase (APSK), and ATP sulfurylase (ATPS). The optimized enzyme expression ratio and effect on PAPS synthesis were evaluated using ePathBrick, a novel synthetic biology tool that assemble multiple genes in a single vector. The introduction of multiple promoters and stop codons at different location enable the bacterial system to fine tune expression level of the genes inserted. Recombinant vectors expressing PPA (U39393.1), ATPS (CP021243.1), and PPA (CP047127.1) were used for fermentations and resulted in volumetric yields of 400-1380 mg/L with accumulation of 34-66% in the soluble fraction. The enzymes from soluble fraction, without any further purification, were used for PAPS synthesis. The PAPS was used for the chemoenzymatic synthesis of a heparan sulfate polysaccharide and coupled with a PAPS-ASTIV regeneration system. ASTIV catalyzes the regeneration of PAPS. A recombinant vector expressing the enzyme ASTIV (from Rattus norvegicus) was used for fermentations and resulted in volumetric yield of 1153 mg/L enzyme with accumulation of 48% in the soluble fraction. In conclusion, we have successfully utilized a metabolic engineering approach to optimize the overall PAPS synthesis productivity. In addition, we have demonstrated that the ePathBrick system could be applied towards study and improvement of enzymatic synthesis conditions. In parallel, we have successfully demonstrated an autoinduction microbial fermentation towards the production of mammalian enzyme (ASTIV). KEY POINTS : • ePathBrick used to optimize expression levels of enzymes. • Protocols have been used for the production of recombinant enzymes. • High cell density fed-batch fermentations with high yields of soluble enzymes. • Robust fermentation protocol successfully transferred to contract manufacturing and research facilities.

Cloning and Expression of Recombinant Chondroitinase ACII and Its Comparison to the Arthrobacter aurescens Enzyme.

Chondroitin sulfates are the glycosaminoglycan chains of proteoglycans critical in the normal development and pathophysiology of all animals. Chondroitinase ACII, a polysaccharide lyase originally isolated from Arthrobacter aurescens IAM 110 65, which is widely used in the analysis and study of chondroitin structure, is no longer commercially available. The aim of the current study is to prepare recombinant versions of this critical enzyme for the glycobiology research community. Two versions of recombinant chondroitinase ACII are prepared in Escherichia coli, and their activity, stability, specificity, and action pattern are examined, along with a non-recombinant version secreted by an Arthrobacter strain. The recombinant enzymes are similar to the enzyme obtained from Arthrobacter for all examined properties, except for some subtle specificity differences toward uncommon chondroitin sulfate substrates. These differences are believed to be due to either post-translational modification of the Arthrobacter-secreted enzyme or other subtle structural differences between the recombinant and natural enzymes. The secreted chondroitinase can serve as a suitable replacement for the original enzyme that is currently unavailable, while the recombinant ones can be applied generally in the structural determination of most standard chondroitin sulfates.

Expression of chondroitin-4-O-sulfotransferase in Escherichia coli and Pichia pastoris.

Chondroitin sulfates are linear sulfated polysaccharides called glycosaminoglycans. They are important nutraceutical and pharmaceutical products that are biosynthesized through the action of chondroitin sulfotransferases on either an unsulfated chondroitin or a dermatan polysaccharide precursor. While the enzymes involved in the biosynthesis of chondroitin sulfates are well known, the cloning end expression of these membrane-bound Golgi enzymes continue to pose challenges. The major chondroitin-4-sulfotransferase, Homo sapiens C4ST-1, had been previously cloned and expressed from mammalian CHO, COS-7, and HEK 293 cells, and its activity was shown to require glycosylation. In the current study, a C4ST-1 construct was designed and expressed in both Escherichia coli and Pichia pastoris in its non-glycosylated and glycosylated forms. Both constructs showed similar activity albeit different kinetic parameters when acting on a microbially prepared unsulfated chondroitin substrate. Moreover, the glycosylated form of C4ST-1 showed lower stability than the non-glycosylated form.

Construction and functional characterization of truncated versions of recombinant keratanase II from Bacillus circulans.

There is a need for degradative enzymes in the study of glycosaminoglycans. Many of these enzymes are currently available either in their natural or recombinant forms. Unfortunately, progress in structure-activity studies of keratan sulfate (KS) have been impeded by the lack of a commercially available endo-ß-N-acetylglucosaminidase, keratantase II. The current study uses a recently published sequence of a highly thermostable keratanase II identified in Bacillus circulans to clone and express a series of truncation mutants in Escherichia coli BL21. The resulting truncated forms of keratanase II exhibit activity and excellent storage and thermal stability making these useful tools for glycobiology research.

High Dose of Atorvastatin for the Treatment of Contrast-Induced Nephropathy After Carotid Artery Stenting.

Statins have been used to prevent contrast-induced nephropathy (CIN). However, the optimal dose of statins is still under controversy. This study aimed to investigate the optimal dose of atorvastatin for the treatment of CIN after carotid artery stenting (CAS). Seventy-six patients receiving selective CAS were randomized to receive 3 different dose of atorvastatin (low dose, 20 mg, n = 30; intermediate dose, 40 mg, n = 24; high dose, 60 mg, n = 22). Preoperatively and on day 3 postoperatively, the levels of serum creatinine, blood urea nitrogen, high-sensitivity C-reactive protein (hs-CRP), alanine aminotransferase (ALT), aspartate aminotransferase (AST), and creatine kinase (CK) were measured. Creatinine clearance (Ccr) and CIN incidence were calculated. In patients treated with high-dose atorvastatin, no significant change was observed in levels of serum creatinine (Scr), blood urea nitrogen (BUN), creatinine clearance, and high-sensitivity C-reactive protein after the CAS procedure (P > 0.05). The CIN incidence in the high-dose group (0%) was significantly lower than the low-dose (13.3%) and intermediate (8.3%) groups (P < 0.05). In the high-dose group, levels of alanine aminotransferase, aspartate aminotransferase, and creatine kinase were significantly increased after CAS (P < 0.05). Pretreatment with 40 mg of atorvastatin is both effective and safe in preventing CIN after CAS. Adverse events of the live and heart should be closely monitored during atorvastatin treatment.

Efficacy of Solitaire Stent Arterial Embolectomy in Treating Acute Cardiogenic Cerebral Embolism in 17 Patients.

BACKGROUND Thrombolysis with rtPA is the only accepted drug therapy for acute ischemic stroke. Since acute cerebral stroke is so pervasive, newly developed recanalization methods have the potential for wide-ranging impacts on patient health and safety. We explored the efficacy and safety of Solitaire stent arterial embolectomy in the treatment of acute cardiogenic cerebral embolism. MATERIAL AND METHODS Between October 2012 and June 2015, 17 patients underwent Solitaire stent arterial embolectomy, either alone or in combination with rtPA intravenous thrombolysis, to treat acute cardiogenic cerebral embolism. Sheath placement time, vascular recanalization time, number of embolectomy attempts, and IV rtPA dose and time were recorded. Success and safety of the recanalization procedure, as well as clinical outcomes, were assessed. These results were compared to 16 control patients who were treated using only rtPA IV thrombolysis. RESULTS Full recanalization of the occluded arteries was achieved in 15 (88.2%) of the Solitaire stent patients. NIH Stroke Scale scores of embolectomy patients improved by an average of 12.59 ± 8.24 points between admission and discharge, compared to 5.56 ± 5.96 in the control group (P<0.05). Glasgow Coma Score improvement between admission and discharge was also significantly higher in the embolectomy group (P<0.05). There was no significant difference in symptomatic intracerebral hemorrhage, high perfusion encephalopathy, incidence of hernia, or mortality between the 2 groups (P>0.05). CONCLUSIONS Solitaire stent embolectomy is a safe and effective alternative to simple venous thrombolytic therapy, and it can significantly improve short-term neurological function and long-term prognosis in acute cardiogenic cerebral embolism.

Keratan sulfate glycosaminoglycan from chicken egg white.

Keratan sulfate (KS) was isolated from chicken egg white in amounts corresponding to ∼0.06 wt% (dry weight). This KS had a weight-average molecular weight of ∼36-41 kDa with a polydispersity of ∼1.3. The primary repeating unit present in chicken egg white KS was →4) ß-N-acetyl-6-O-sulfo-d-glucosamine (1 → 3) ß-d-galactose (1→ with some 6-O-sulfo galactose residues present. This KS was somewhat resistant to depolymerization using keratanase 1 but could be depolymerized efficiently through the use of reactive oxygen species generated using copper (II) and hydrogen peroxide. Of particular interest was the presence of substantial amounts of 2,8- and 2,9-linked N-acetylneuraminic acid residues in the form of oligosialic acid terminating the non-reducing ends of the KS chains. Most of the KS appears to be N-linked to a protein core as evidenced by its sensitivity to PNGase F.

[Study on the Wavelength Selection Based on VIP Analysis in Noninvasive Measurement of Blood Components].

Selection of independent variable is a hotspot in the field of quantitative spectral analysis. Efficient and easy-to-use method for wavelength selection can not only reduce the computation and improve the accuracy of analysis, but also can reduce dependency on the spectral resolution of instruments and cut cost. Wavelength selection is also an important part of the research about noninvasive measurement of blood components by spectrum technology. Dynamic Spectrum theory provides excellent ideas for researchers, but only broadband light source and high-resolution spectrograph were used in correlational studies for a long time. The large number of wavelengths needed for analysis limits the further development of Dynamic Spectrum method. In order to remove redundancy information and make the devices low-cost and integrated, the method of Wavelength selection on the basis of Variable Importance in Projection (VIP) analysis was proposed. Variables with less importance were removed and wavelengths with great explanation power were retained after VIP analysis the number of wavelengths was reduced from 586 to 64. PLS model with 64 wavelengths get a satisfactory predict result that the MREP is 1.82%. The significance test through Bootstrap method validate the selected wavelengths' explanation power. Moreover, the study pointed out the sensitive wavelengths in Dynamic Spectrum method for the first time. Study also took the first step toward the practical application of Dynamic Spectrum and laid the foundation of low-cost on-line analysis, and it also provided valuable references and new ideas for spectral analysis in other areas.

Heparin and related polysaccharides: synthesis using recombinant enzymes and metabolic engineering.

Glycosaminoglycans are linear anionic polysaccharides that exhibit a number of important biological and pharmacological activities. The two most prominent members of this class of polysaccharides are heparin/heparan sulfate and the chondroitin sulfates (including dermatan sulfate). These polysaccharides, having complex structures and polydispersity, are biosynthesized in the Golgi of most animal cells. The chemical synthesis of these glycosaminoglycans is precluded by their structural complexity. Today, we depend on food animal tissues for their isolation and commercial production. Ton quantities of these glycosaminoglycans are used annually as pharmaceuticals and nutraceuticals. The variability of animal-sourced glycosaminoglycans, their inherent impurities, the limited availability of source tissues, the poor control of these source materials, and their manufacturing processes suggest a need for new approaches for their production. Over the past decade, there have been major efforts in the biotechnological production of these glycosaminoglycans. This mini-review focuses on the use of recombinant enzymes and metabolic engineering for the production of heparin and chondroitin sulfates.

Production of chondroitin in metabolically engineered E. coli.

Chondroitin sulfates, widely used in the treatment of arthritis, are glycosaminoglycans extracted from food animal tissues. As part of our ongoing efforts to separate the food chain from the drug chain, we are examining the possibility of using metabolic engineering to produce chondroitin sulfate in Escherichia coli. Chondroitin is a valuable precursor in the synthesis of chondroitin sulfate. This study proposes a safer and more feasible approach to metabolically engineer chondroitin production by expressing genes from the pathogenic E. coli K4 strain, which natively produces a capsular polysaccharide that shares the similar structure with chondroitin, into the non-pathogenic E. coli BL21 Star™ (DE3) strain. The ePathBrick vectors, allowing for multiple gene addition and expression regulatory signal control, are used for metabolic balancing needed to obtain the maximum potential yield. The resulting engineered strain produced chondroitin, as demonstrated by (1)H NMR and disaccharide analysis, relying on chondrotinase treatment followed by liquid chromatography-mass spectrometry. The highest yield from shake flask experiment was 213mg/L and further increased to 2.4g/L in dissolved oxygen-stat fed batch bioreactor.

Masquerading microbial pathogens: capsular polysaccharides mimic host-tissue molecules.

The increasing prevalence of antibiotic-resistant bacteria portends an impending postantibiotic age, characterized by diminishing efficacy of common antibiotics and routine application of multifaceted, complementary therapeutic approaches to treat bacterial infections, particularly multidrug-resistant organisms. The first line of defense for most bacterial pathogens consists of a physical and immunologic barrier known as the capsule, commonly composed of a viscous layer of carbohydrates that are covalently bound to the cell wall in Gram-positive bacteria or often to lipids of the outer membrane in many Gram-negative bacteria. Bacterial capsular polysaccharides are a diverse class of high molecular weight polysaccharides contributing to virulence of many human pathogens in the gut, respiratory tree, urinary tract, and other host tissues, by hiding cell surface components that might otherwise elicit host immune response. This review highlights capsular polysaccharides that are structurally identical or similar to polysaccharides found in mammalian tissues, including polysialic acid and glycosaminoglycan capsules hyaluronan, heparosan, and chondroitin. Such nonimmunogenic coatings render pathogens insensitive to certain immune responses, effectively increasing residence time in host tissues and enabling pathologically relevant population densities to be reached. Biosynthetic pathways and capsular involvement in immune system evasion are described, providing a basis for potential therapies aimed at supplementing or replacing antibiotic treatment.