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Chondroitin sulfate (CS) stands as a pivotal compound in dietary supplements for osteoarthritis treatment, propelling significant interest in the biotechnological pursuit of environmentally friendly and safe CS production. Enzymatic synthesis of CS for instance CSA has been considered as one of the most promising methods. However, the bottleneck consistently encountered is the active expression of chondroitin 4- O -sulfotransferase (C4ST) during CSA biosynthesis. This study meticulously delved into optimizing C4ST expression through systematic enhancements in transcription, translation, and secretion mechanisms via modifications in the 5′ untranslated region, the N-terminal encoding sequence, and the Komagataella phaffii chassis. Ultimately, the active C4ST expression escalated to 2713.1 U/L, representing a striking 43.7-fold increase. By applying the culture broth supernatant of C4ST and integrating the 3′-phosphoadenosine-5′-phosphosulfate (PAPS) biosynthesis module, we constructed a one-pot enzymatic system for CSA biosynthesis, achieving a remarkable sulfonation degree of up to 97.0 %. The substantial enhancement in C4ST expression and the development of an engineered one-pot enzymatic synthesis system promises to expedite large-scale CSA biosynthesis with customizable sulfonation degrees. [Display omitted] [ABSTRACT FROM AUTHOR]

Low-molecular-weight heparins (LMWHs), especially the specific-sized heparin oligosaccharides, are attractive for the therapeutic applications, while their synthesis remains challenging. In the present study, unsaturated even-numbered heparosan oligosaccharides were firstly prepared by cleaving high-molecular-weight heparosan using recombinant heparinase III (HepIII). The conversion rates of the unsaturated disaccharides, tetrasaccharides, hexasaccharides, octasaccharides, and decasaccharides were 33.9 %, 47.9 %, 78.7 %, 71.8 %, and 53.4 %, respectively. After processing the aforementioned heparosan oligosaccharides with the Δ4,5 unsaturated glycuronidase, saturated odd-numbered heparosan trisaccharides, pentasaccharides, heptasaccharides, and nonasaccharides were produced. It was observed that among them, the pentasaccharides were the smallest units of saturated odd-numbered oligosaccharides recognized by HepIII. These oligosaccharides were further catalyzed with bifunctional heparan sulfate N -deacetylase/ N -sulfotransferase (NDST) under optimized reaction conditions. It was found that the tetrasaccharide was defined as the smallest recognition unit for NDST, obtaining the N -sulfonated heparosan tetrasaccharides, pentasaccharides, and hexasaccharides with a single sulfonate group, as well as N -sulfonated heparosan heptasaccharides, octasaccharides, and nonasaccharides with multiple sulfonate groups. These results provide an easy pathway for constructing a library of specific-sized N -sulfonated heparosan oligosaccharides that can be used as the substrates for the enzymatic synthesis of LMWHs and heparin oligosaccharides, shedding new light on the substrate preference of NDST. [ABSTRACT FROM AUTHOR]

Chondroitin sulfate (CS) is widely applied in the medical, clinical, and nutraceutical fields. However, all commercialized CS is extracted from animal tissues, which is associated with many problems, such as heavy reliance on antibiotics, animal excrement waste and long breeding time. Here, we developed a promising new green route for the de novo biosynthesis of non-animal CS from methanol, a one-carbon compound, using the engineered Pichia pastoris cell factory. Firstly, three exogenous genes, namely kfoC and kfoA from Escherichia coli K4 and tuaD from Bacillus subtilis, were introduced into P. pastoris to construct the chondroitin synthesis pathway, which yielded 5.5 mg L−1 chondroitin. After optimizing the expression of committed enzymes by codon optimization, the production of chondroitin improved by approximately 35-fold to 189.8 mg L−1. Thereafter, 6 kinds of Kozak sequences and 10 different endogenous promoters were compared and applied to achieve the expression of active chondroitin-4-O-sulfotransferase (41.3 U L−1). After integrating the chondroitin biosynthesis and sulfonation modules, the engineered strain, Pp006, successfully produced 182.0 mg L−1 CSA [GlcA-GalNAc (4S)] with a sulfation degree of 1.1%. We further improved the sulfation degree to 2.8% by strengthening the 3′-phosphoadenosine-5′-phosphosulfate (PAPS) supply with the overexpression of adenosine-5′-triphosphate sulfurylase and adenosine 5′-phosphosulfate kinase. In the fed-batch cultivation, the final titer of CSA reached 2.1 g L−1 and the sulfation degree increased to 4.0%. Moreover, the results suggest that the P. pastoris cell factory could be used as a green route to produce other sulfated glycosaminoglycans. [ABSTRACT FROM AUTHOR]

Vibrio cholerae causes a severe disease that kills thousands of people annually. The outer membrane protein OmpU is the most abundant outer membrane protein in V. cholerae, and has been identified as an important virulence factor that is involved in host‐cell interaction and recognition, as well as being critical for the survival of the pathogenic V. cholerae in the host body and in harsh environments. The mechanism of these processes is not well understood owing to a lack of the structure of V. cholerae OmpU. Here, the crystal structure of the V. cholerae OmpU trimer is reported to a resolution of 2.2 Å. The protomer forms a 16‐β‐stranded barrel with a noncanonical N‐terminal coil located in the lumen of the barrel that consists of residues Gly32–Ser42 and is observed to participate in forming the second gate in the pore. By mapping the published functional data onto the OmpU structure, the OmpU structure reinforces the notion that the long extracellular loop L4 with a β‐hairpin‐like motif may be critical for host‐cell binding and invasion, while L3, L4 and L8 are crucially implicated in phage recognition by V. cholerae. [ABSTRACT FROM AUTHOR]

Adjuvants can enhance vaccine immunogenicity and induce long-term enhancement of immune responses. Thus, adjuvants are important for vaccine research. Polysaccharides isolated from select Chinese herbs have been demonstrated to possess various beneficial functions and excellent adjuvant abilities. In the present study, the polysaccharides IIP-A-1 and IIP-2 were isolated from Isatis indigotica root and compared with the common vaccine adjuvant aluminum hydroxide via intramuscular co-administration of inactivated rabies virus rCVS-11-G into mice. Blood was collected to determine virus neutralizing antibody (VNA) titers and B and T lymphocyte activation status. Inguinal lymph node samples were collected and used to measure B lymphocyte proliferation. Splenocytes were isolated, from which antigen-specific cellular immune responses were detected via ELISpot, ELISA and intracellular cytokine staining. The results revealed that both types of polysaccharides induce more rapid changes and higher VNA titers than aluminum hydroxide. Flow cytometry assays revealed that the polysaccharides activated more B lymphocytes in the lymph nodes and more B and T lymphocytes in the blood than aluminum hydroxide. Antigen-specific cellular immune responses showed that IIP-2 strongly induced T lymphocyte proliferation in the spleen and high levels of cytokine secretion from splenocytes, whereas aluminum hydroxide induced proliferation in only a small number of lymphocytes and the secretion of only small quantities of cytokines. Collectively, these data suggest that the polysaccharide IIP-2 exhibits excellent adjuvant activity and can enhance both cellular and humoral immunity. [ABSTRACT FROM AUTHOR]

• Polysaccharide PCP-II was first used as adjuvant in rabies vaccine. • PCP-2 could induce rapid and high antibody responses in mice and dogs. • PCP-II could activate lymphocytes and promote cytokines secretion in mice. • PCP-II is a good candidate for adjuvant on inactivated rabies vaccine for animal and human use. Adjuvants are important components of vaccination strategies because they boost and accelerate the immune response. The aim of this study was to investigate the adjuvant activity of PCP-II, a polysaccharide isolated from Poria cocos , together with an inactivated rabies vaccine. The polysaccharide PCP-II was compared with the common veterinary rabies vaccine adjuvant Alhydrogel by co-administration of either adjuvant with the inactivated rabies virus rCVS-11-G to mice via the intramuscular route. Blood samples were collected to determine the virus-neutralizing antibody (VNA) titer and assess activation of B and T lymphocytes. Inguinal lymph node samples were collected, and proliferation of B lymphocytes was measured. Splenocytes were isolated, and antigen-specific cellular immune responses were evaluated by enzyme-linked immunospot and immunosorbent assays (ELISpot assay and ELISA, respectively). The results showed that PCP-II enhanced and promoted an increase in the VNA titer in the mice compared to Alhydrogel. Flow cytometry assays revealed that the polysaccharide activated more B lymphocytes in the lymph nodes and more B and T lymphocytes in the blood. Assessment of antigen-specific cellular immune responses showed that PCP-II strongly induced T lymphocyte proliferation in the spleen and high levels of cytokine secretion from splenocytes. All of these data suggest that PCP-II possesses excellent adjuvant activity and enhances both cellular and humoral immunity in mice. After examining the adjuvant activities of PCP-II in mice, dogs were immunized with rCVS-11-G together with Alhydrogel or PCP-II as an adjuvant; the control group was injected with a commercial rabies vaccine. Serum samples were collected, and the VNA titers were measured. PCP-II caused increases in the VNA titers in both the booster and single-dose immunization tests when co-administered with rCVS-11-G compared with Alhydrogel. The VNA titer of the commercial vaccine group was also significantly lower than that of the PCP-II group. These data indicate that PCP-II is an excellent candidate adjuvant for inactive rabies vaccines in the veterinary setting. [ABSTRACT FROM AUTHOR]

Aims: To explore the roles of apolipoprotein E (APOE) ε4 on the neuropathology and neuroinflammation in Alzheimer's disease (AD) patients. Methods: AD patients were divided into the APOE ε4 carrier and the APOE ε4 non‐carrier groups according to APOE genotype. Demographic information, cognitive function, the levels of neuropathological proteins and neuroinflammatory factors in cerebrospinal fluid (CSF) were compared between the two groups, and their correlations were subsequently analyzed. Results: β amyloid protein (Aβ)1–42 level from the APOE ε4 carrier group was significantly lower than that from the non‐carrier group (p = 0.023), which was associated with worse cognitive function. The nitric oxide (NO) level was significantly elevated in the APOE ε4 carrier group compared to the non‐carrier group (p = 0.016), which was significantly and positively correlated with the Trail Making Test (TMT)‐A‐time (r = 0.21, p = 0.026) and TMT‐B‐time (r = 0.38, p < 0.01). Conclusion: APOE ε4 is associated with poorer cognition, particularly the early symptoms of memory, language, and attention. APOE ε4 is associated with lower Aβ1–42 level, and the more numbers of APOE ε4 are carried, the lower level of Aβ1–42 is measured. APOE ε4 is associated with elevated NO level, which is linked to the impaired attention and executive function. [ABSTRACT FROM AUTHOR]

Aims Methods Results Conclusion To investigate the roles of neurotrophic factors on cognition in patients with Alzheimer's disease (AD) carrying Apolipoprotein E (APOE) ε4.Totals of 173 patients with AD were divided into APOE ε4 carrier and non‐carrier groups, and their demographics, cognition, and neurotrophic factors in cerebrospinal fluid (CSF) were compared. Multiple linear regression analyses were performed to assess correlations among APOE ε4, neurotrophic factors and cognition. Mediation analyses were conducted to assess the sequential associations among APOE ε4, nerve growth factor (NGF), and cognition.Global cognition and multiple domains were impaired in the APOE ε4 carrier group (all p < 0.05). NGF level in the APOE ε4 carrier group was lower than that in the non‐carrier group (p = 0.016). NGF level showed significant correlations with both global and multiple domains cognitions. Specifically, NGF mediated the association between APOE ε4 and Animal Fluency Test score (β, −0.45; 95% CI [−0.96, −0.07]; p < 0.001) and Trail Making Test‐A (time) (β, 0.15; 95% CI [0.01, 0.33]; p < 0.001).APOE ε4 is associated with cognitive impairment, and those carrying APOE ε4 have decreased NGF level in CSF. Declined NGF level is correlated with compromised cognition. NGF mediates APOE ε4‐associated cognitive impairment. [ABSTRACT FROM AUTHOR]

Rabbit hemorrhagic disease virus (RHDV) is the etiological agent behind rabbit hemorrhagic disease (RHD), which is lethal and contagious in rabbits. The virus does not replicate in cell culture and the only commercial inactivated vaccine available is derived from infected rabbit livers. RHDV capsid protein, VP60, is the main antigen comprising the virion. We used a baculovirus–silkworm pupae system to express VP60, which self-assembled into virus-like particles (VLPs) with a similar size and morphology to RHDV. Hemagglutination assays (HAs) showed that VP60 expression levels of VP60 reached as high as 10 7 HA units (HAU) per pupa. A single intramuscular injection with 10 4 HAU of VLPs completely protected rabbits for at least 180 days against RHDV challenge, and for at least 360 days when the VLPs were emulsified with Freund's complete adjuvant. These data suggest that silkworm pupae can be used to develop VLP-based vaccines which confer durable protection against RHD. [ABSTRACT FROM AUTHOR]

Influenza A virus (IAV) and SARS‐CoV‐2 (COVID‐19) cause pandemic infections where cytokine storm syndrome and lung inflammation lead to high mortality. Given the high social and economic cost of respiratory viruses, there is an urgent need to understand how the airways defend against virus infection. Here we use mice lacking the WD and linker domains of ATG16L1 to demonstrate that ATG16L1‐dependent targeting of LC3 to single‐membrane, non‐autophagosome compartments – referred to as non‐canonical autophagy – protects mice from lethal IAV infection. Mice with systemic loss of non‐canonical autophagy are exquisitely sensitive to low‐pathogenicity IAV where extensive viral replication throughout the lungs, coupled with cytokine amplification mediated by plasmacytoid dendritic cells, leads to fulminant pneumonia, lung inflammation and high mortality. IAV was controlled within epithelial barriers where non‐canonical autophagy reduced IAV fusion with endosomes and activation of interferon signalling. Conditional mouse models and ex vivo analysis showed that protection against IAV infection of lung was independent of phagocytes and other leucocytes. This establishes non‐canonical autophagy in airway epithelial cells as a novel innate defence that restricts IAV infection and lethal inflammation at respiratory surfaces. SYNOPSIS: Non‐canonical functions of the autophagy protein ATG16L1 are mediated by its C‐terminal WD domain. Here, mice expressing WD domain‐lacking ATG16L1 are found to be highly sensitive to influenza virus infection, resulting in cytokine storm and lethal pneumonia. The WD domain of ATG16L1 prevents lethal infection by influenza A virus.Infection is controlled by epithelial cells and is independent of phagocytic cells.Non‐canonical ATG16L1 function reduces interferon signalling by slowing virus endocytosis and fusion with endosomal membranes. [ABSTRACT FROM AUTHOR]

Glycosaminoglycans (GAGs) are naturally occurring acidic polysaccharides with wide applications in pharmaceuticals, cosmetics, and health foods. The diverse biological activities and physiological functions of GAGs are closely associated with their molecular weights and sulfation patterns. Except for the non-sulfated hyaluronan which can be synthesized naturally by group A Streptococcus , all the other GAGs such as heparin and chondroitin sulfate are mainly acquired from animal tissues. Microbial cell factories provide a more effective platform for the production of structurally homogeneous GAGs. Enhancing the production efficiency of polysaccharides, accurately regulating the GAGs molecular weight, and effectively controlling the sulfation degree of GAGs represent the major challenges of developing GAGs microbial cell factories. Several enzymatic, metabolic engineering, and synthetic biology strategies have been developed to tackle these obstacles and push forward the industrialization of biotechnologically produced GAGs. This review summarizes the recent advances in the construction of GAGs synthesis cell factories, regulation of GAG molecular weight, and modification of GAGs chains. Furthermore, the challenges and prospects for future research in this field are also discussed. [Display omitted] • Various microbial cell factories have been constructed to efficiently synthesize unsulfured GAGs. • Many strategies have been developed for controlling the molecular weight of GAGs. • Artificial sulfation systems have been designed and constructed for preparing GAGs and its oligosaccharides. • The biosynthesis of GAGs with microbial cell factories has many advantages comparing the traditional extraction methods. [ABSTRACT FROM AUTHOR]

Objective: To evaluate the state of evidence for the beneficial and harmful effects of Tai Chi on non-motor disorders in post-stroke patients. Design: Systematic review and meta-analysis of published studies. Subjects: Stroke survivors who received conventional rehabilitation therapy or Tai Chi training. Data sources: We searched seven electronic literature databases and one clinical registry platform to collect data from randomized controlled trials published up to July 26, 2020. Results: A total of 11 randomized controlled trials with 723 stroke survivors met the inclusion criteria, of which six were included in the meta-analysis. Among the 11 studies, one was assessed as "low", eight were assessed as "moderate", and only two were assessed as "high" for the assessment of methodologic quality. Compared to patients who received conventional rehabilitation therapy, those who received Tai Chi training showed greater improvement in scores of depression (standardized mean difference (SMD) [95% confidence interval (CI)] = 0.36 [0.10, 0.61], Grading of Recommendations Assessment, Development, and Evaluation [GRADE]: very low). There were no differences in the improvements in post-stroke global mental disorders (mean difference (MD [95% CI] = 6.15 [−3.05, 15.36], GRADE: moderate) or sleep disorders (MD [95% CI] = 0.33 [−1.51, 1.81], GRADE: low) between Tai Chi and control groups. Conclusion: Tai Chi may alleviate post-stroke depression in stroke survivors but has no clear effects on post-stroke cognitive and sleep disorders. [ABSTRACT FROM AUTHOR]

• The solubility of csABC I was improved with fusion of the MBP tag. • Mutation of I309 V significantly increased the catalytic activity. • OmpA signal peptide achieved efficient secretion of csABC I. • Deletion of a lipoprotein encoding gene lpp increased csABC I secretion. • Chondroitin sulfate oligosaccharides were produced with engineered csABC I. Chondroitin sulfate ABC lyases (csABCs) have attracted intensive attention because of their wide potential applications in promoting tissue regeneration and generating oligosaccharides. In the present study, three csABC I encoding sequences were analyzed and site-directed mutagenesis results demonstrate that residues Leu125 and Leu322 are essential to activity and mutation of each leucine residue to proline dramatically decreased enzymatic activity. Additionally, our results showed that mutation of I309 V significantly increased the catalytic efficiency. By recruiting OmpA signal peptide and engineering the permeability of cell membrane with deletion of a lipoprotein encoding gene lpp , all recombinant enzymes were secreted and the extracellular activity was finally increased to 2.99 ± 0.1 U/mL in batch fermentation. More importantly, the engineered csABC I with high activity can rapidly degrade chondroitin sulfate to the end tetrasaccharides and disaccharides, demonstrating its applicability for preparation of chondroitin sulfate oligosaccharides. [ABSTRACT FROM AUTHOR]

Middle East respiratory syndrome coronavirus (MERS-CoV) has continued spreading since its emergence in 2012 with a mortality rate of 35.6%, and is a potential pandemic threat. Prophylactics and therapies are urgently needed to address this public health problem. We report here the efficacy of a vaccine consisting of chimeric virus-like particles (VLP) expressing the receptor binding domain (RBD) of MERS-CoV. In this study, a fusion of the canine parvovirus (CPV) VP2 structural protein gene with the RBD of MERS-CoV can self-assemble into chimeric, spherical VLP (sVLP). sVLP retained certain parvovirus characteristics, such as the ability to agglutinate pig erythrocytes, and structural morphology similar to CPV virions. Immunization with sVLP induced RBD-specific humoral and cellular immune responses in mice. sVLP-specific antisera from these animals were able to prevent pseudotyped MERS-CoV entry into susceptible cells, with neutralizing antibody titers reaching 1: 320. IFN-γ, IL-4 and IL-2 secreting cells induced by the RBD were detected in the splenocytes of vaccinated mice by ELISpot. Furthermore, mice inoculated with sVLP or an adjuvanted sVLP vaccine elicited T-helper 1 (Th1) and T-helper 2 (Th2) cell-mediated immunity. Our study demonstrates that sVLP displaying the RBD of MERS-CoV are promising prophylactic candidates against MERS-CoV in a potential outbreak situation. [ABSTRACT FROM AUTHOR]

• An efficient sulfation-modification system for CSA synthesis was constructed. • The soluble expression of C4ST was increased by 30 times. • The stability of AST IV was significantly improved with addition of glycerol. • CSA with high sulfation degree was produced with protein scaffold strategies. • 15 g CSA was produced within 24 h in 1 L system. Chondroitin sulfate (CS) extracted from animal tissues has been widely used as nutraceutical and pharmaceutical products for osteoarthritis treatment. Here we developed an efficient sulfation-modification system for large scale preparation of CSA in vitro. First, the expression level of C4ST was improved by 30 times with fusion of the chaperone SUMO. Then, glycerol as a protein stabilizer was found to improve rat AST IV stability during the regeneration of cofactor PAPS. Then peptide linkers or protein scaffolds were employed to assemble AST IV and C4ST into artificial complexes to bring the enzymes and PAPS spatially closer and enhance the catalytic efficiency of chondroitin sulfation. Eventually, the system was scaled up to 1 L system and 15 g chondroitin was converted to CSA in 24 h, with a 98 % conversion. The present study made a step further towards the industrial production of CSA with different sulfation degrees. [ABSTRACT FROM AUTHOR]