A SARS-CoV-2 RBD219-N1C1 (RBD219-N1C1) recombinant protein antigen formulated on Alhydrogel® has recently been shown to elicit a robust neutralizing antibody response against SARS-CoV-2 pseudovirus ...in mice. The antigen has been produced under current good manufacturing practices (cGMPs) and is now in clinical testing. Here, we report on process development and scale-up optimization for upstream fermentation and downstream purification of the antigen. This includes production at the 1-L and 5-L scales in the yeast,
Pichia pastoris
, and the comparison of three different chromatographic purification methods. This culminated in the selection of a process to produce RBD219-N1C1 with a yield of >400 mg per liter of fermentation with >92% purity and >39% target product recovery after purification. In addition, we show the results from analytical studies, including SEC-HPLC, DLS, and an ACE2 receptor binding assay that were performed to characterize the purified proteins to select the best purification process. Finally, we propose an optimized upstream fermentation and downstream purification process that generates quality RBD219-N1C1 protein antigen and is fully scalable at a low cost.
Key points
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Yeast fermentation conditions for a recombinant COVID-19 vaccine were determined
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Three purification protocols for a COVID-19 vaccine antigen were compared
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Reproducibility of a scalable, low-cost process for a COVID-19 vaccine was shown
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Graphical abstract
The BRD4 protein is associated with various diseases, which has been an attractive target for the treatment of cancer and inflammation. This paper is a follow-up to our previous studies, in which we ...report the structure-based design, synthesis, and evaluation of a new class of small-molecule BRD4 bromodomain inhibitors bearing a benzo
d
isoxazol scaffold. The SARs focused on exploration of the 2′ or 3′ position to afford novel inhibitors that may avoid potential metabolically unstable site. The most potent inhibitor
13f
exhibited high binding affinity to BRD4(1) with a ΔT
m
value of 7.8 °C as evaluated in thermal shift assay (TSA). The potent activity was also demonstrated by a peptide competition assay with an IC
50
value of 0.21 μM. The docking studies revealed the binding mode of the compounds with the active site of BRD4(1). In addition, in silico predictions indicated that these compounds possessed good drug-likeness and pharmacokinetic profile.
Graphic abstract
This paper is a follow-up to our previous studies, in which we report the structure-based design, synthesis, and evaluation of a new class of small-molecule BRD4 bromodomain inhibitors bearing a benzodisoxazol scaffold.
This paper is a follow-up to our previous studies, in which we report the structure-based design, synthesis, and evaluation of a new class of small-molecule BRD4 bromodomain inhibitors bearing a benzodisoxazol scaffold.
Summary
Small RNA molecules play key regulatory roles in many bacterial species. However, little mechanistic data exists for the action of small regulatory RNAs in the human pathogen group A ...Streptococcus (GAS). Here, we analysed the relationship between a putative GAS sRNA and production of the secreted virulence factor streptokinase (SKA). SKA promotes GAS dissemination by activating conversion of host plasminogen into the fibrin‐degrading protease plasmin. Homologues of the putative sRNA‐encoding gene fibronectin/fibrinogen‐binding/haemolytic‐activity/streptokinase‐regulator‐X (fasX) were identified in four different pyogenic streptococcal species. However, despite 79% fasX nucleotide identity, a fasX allele from the animal pathogen Streptococcus zooepidemicus failed to complement a GAS fasX mutant. Using a series of precisely constructed fasX alleles we discovered that FasX is a bona‐fide sRNA that post‐transcriptionally regulates SKA production in GAS. By base‐pairing to the 5′ end of ska mRNA, FasX enhances ska transcript stability, resulting in a ∼10‐fold increase in SKA activity. Our data provide new insights into the mechanisms used by small regulatory RNAs to activate target mRNAs, and enhances our understanding of the regulation of a key GAS virulence factor.
Esophageal varices (EV) is the most common complication of portal hypertension in cirrhosis patients. Radiomics has been progressing remarkably for quantifying the state of diseases. However, there ...are few studies on EV severity prediction by applying radiomics and machine learning. Besides, most of the existing methods apply only single organ for radiomics feature extraction. In this study, we propose a radiomics algorithm based on light gradient boosting machine (LightGBM) to identify high-risk and low-risk EV patients by fusing the radiomics features of liver, spleen and esophagus from the CT images. This approach involves 188 patients, including 151 cirrhotic patients (84 patients with severe EV and 67 patients with mild or no EV) registered in Qilu Hospital of Shandong University and 37 cirrhotic patients (20 patients with severe EV and 17 patients with mild or no EV) retrospectively registered in Jinan Central Hospital from January 2018 to August 2020. Specifically, the radiomics features of liver, spleen and esophagus are extracted after manual segmentation. Then the features of the three organs are fused by linear combination where the weights are estimated by the feature distribution. Finally, classification models are established by cross-combination of multiple feature selection methods (e.g., least absolute shrinkage and selection operator, Boruta, eXtreme gradient boosting (XGBoost) and LightGBM) with multiple classifiers (e.g., support vector machine, random forests, XGBoost and LightGBM) to discriminate the high-risk EV patients from the low-risk EV patients at the individual level. In addition, we propose a classifier ensemble strategy by combining the prediction probability of each organ for final classification. Experimental results demonstrate that the feature of esophageal makes a greater contribution on the diagnosis of EV compared with that of spleen and liver. The proposed multi-organ fusion and LightGBM based radiomics framework has better classification performance compared with the state-of-the-art radiomics approaches.
There is an urgent need for an accessible and low-cost COVID-19 vaccine suitable for low- and middle-income countries. Here, we report on the development of a SARS-CoV-2 receptor-binding domain (RBD) ...protein, expressed at high levels in yeast (Pichia pastoris), as a suitable vaccine candidate against COVID-19. After introducing two modifications into the wild-type RBD gene to reduce yeast-derived hyperglycosylation and improve stability during protein expression, we show that the recombinant protein, RBD219-N1C1, is equivalent to the wild-type RBD recombinant protein (RBD219-WT) in an in vitro ACE-2 binding assay. Immunogenicity studies of RBD219-N1C1 and RBD219-WT proteins formulated with Alhydrogel® were conducted in mice, and, after two doses, both the RBD219-WT and RBD219-N1C1 vaccines induced high levels of binding IgG antibodies. Using a SARS-CoV-2 pseudovirus, we further showed that sera obtained after a two-dose immunization schedule of the vaccines were sufficient to elicit strong neutralizing antibody titers in the 1:1,000 to 1:10,000 range, for both antigens tested. The vaccines induced IFN-γ IL-6, and IL-10 secretion, among other cytokines. Overall, these data suggest that the RBD219-N1C1 recombinant protein, produced in yeast, is suitable for further evaluation as a human COVID-19 vaccine, in particular, in an Alhydrogel® containing formulation and possibly in combination with other immunostimulants.
AGS-30, a new andrographolide derivative, showed significant anticancer and anti-angiogenic characteristics. However, its role in controlling macrophage polarization and tumor immune response is ...unknown. Thus, the main goals of this study are to investigate how AGS-30 regulates macrophage polarization and how it suppresses breast cancer metastasis. AGS-30 inhibited IL-4 and IL-13-induced RAW 264.7 and THP-1 macrophages into M2-like phenotype. However, AGS-30 did not affect the LPS and IFN-γ-induced polarization of M1-like macrophages. AGS-30 reduced the mRNA expressions of CD206, Arg-1, Fizz-1, Ym-1, VEGF, IL-10, MMP2, and MMP9 in M2-like macrophages in a concentration-dependent manner. In contrast, andrographolide treatment at 5 μM did not affect M1-like and M2-like macrophage polarization. The conditioned medium from M2-like macrophages increased 4T1 breast cancer cell migration and invasion, whereas AGS-30 inhibited these effects. In the 4T1 breast tumor xenograft mice, the tumor volume and weight were reduced without affecting body weight after receiving AGS-30. AGS-30 treatment also reduced lung and liver metastasis, with reduced STAT6, CD31, VEGF, and Ki67 protein expressions. Moreover, the tumors had considerably fewer M2-like macrophages and Arg-1 expression, but the proportion of M1-like macrophages and iNOS expression increased after AGS-30 treatment. Same results were found in the tail vein metastasis model. In conclusion, this study shows that AGS-30 inhibits breast cancer growth and metastasis, probably through inhibiting M2-like macrophage polarization. Our findings suggest that AGS-30 may be a potential immunotherapeutic alternative for metastatic breast cancer.
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•AGS-30 shows stronger effects than Andro in suppressing M2-like macrophage polarization•AGS-30 suppresses M2 macrophages-induced angiogenesis and cancer cell mobility in vitro•AGS-30 suppresses breast cancer metastasis by regulating macrophage polarization and activating T cells
Summary
Bacterial pathogens use cell surface‐associated adhesion molecules to promote host attachment and colonization, and the ability to modulate adhesion expression is critical to pathogen ...success. Here, we show that the human‐specific pathogen the group A Streptococcus (GAS) uses a small regulatory RNA (sRNA) to regulate the expression of adhesive pili. The fibronectin/fibrinogen‐binding/haemolytic‐activity/streptokinase‐regulator‐X (FasX) sRNA, previously shown to positively regulate expression of the secreted virulence factor streptokinase (SKA), negatively regulates the production of pili on the GAS cell surface. FasX base pairs to the extreme 5′ end of mRNA from the pilus biosynthesis operon, and this RNA:RNA interaction reduces the stability of the mRNA, while also inhibiting translation of at least the first gene in the pilus biosynthesis operon (cpa, which encodes a minor pilin protein). The negative regulation of pilus expression by FasX reduces the ability of GAS to adhere to human keratinocytes. Our findings cement FasX sRNA as an important regulator of virulence factor production in GAS and identify that FasX uses at least three distinct mechanisms, positive (ska mRNA) and negative (pilus operon mRNA) regulation of mRNA stability, and negative regulation of mRNA translation (cpa mRNA), to post‐transcriptionally regulate target mRNAs during infection.
Alzheimer's disease (AD) is a neurodegenerative disease characterized by progressive deterioration of memory and cognition. Mild cognitive impairment (MCI) has been implicated as a prodromal phase of ...AD. Although abnormal functional connectivity (FC) has been demonstrated in AD and MCI, the clinical differentiation of AD, MCI, and normal aging remains difficult, and the distinction between MCI and normal aging is especially problematic. We hypothesized that FC between the hippocampus and other brain structures is altered in AD and MCI, and that measurement of abnormal FC could have diagnostic utility for the classification of different AD stages.
Elderly adults aged 60-85 years were assigned to AD, MCI, or normal control (NC) groups based on clinical criteria. Functional magnetic resonance scanning was completed by 119 subjects. Five dimension reduction/classification methods were applied, using hippocampus-derived FC strengths as input features. Classification performance of the five dimensionality reduction methods was compared between AD, MCI, and NC groups.
FCs between the hippocampus and left insula, left thalamus, cerebellum, right lingual gyrus, posterior cingulate cortex, and precuneus were significantly reduced in AD and MCI. Support vector machine learning coupled with sparse principal component analysis demonstrated the best discriminative performance, yielding classification accuracies of 82.02% (AD vs. NC), 81.33% (MCI vs. NC), and 81.08% (AD vs. MCI).
Hippocampus-seed-based FCs were significantly different between AD, MCI, and NC groups. FC assessment combined with widely used machine learning methods can improve AD differential diagnosis, and may be especially useful to distinguish MCI from normal aging.
Ascaris lumbricoides is one of the three major soil-transmitted gastrointestinal helminths (STHs) that infect more than 440 million people in the world, ranking this neglected tropical disease among ...the most common afflictions of people living in poverty. Children infected with this roundworm suffer from malnutrition, growth stunting as well as cognitive and intellectual deficits. An effective vaccine is urgently needed to complement anthelmintic deworming as a better approach to control helminth infections. As37 is an immunodominant antigen of Ascaris suum, a pig roundworm closely related to the human A. lumbricoides parasite, recognized by protective immune sera from A. suum infected mice. In this study, the immunogenicity and vaccine efficacy of recombinant As37 were evaluated in a mouse model.
As37 was cloned and expressed as a soluble recombinant protein (rAs37) in Escherichia coli. The expressed rAs37 was highly recognized by protective immune sera from A. suum egg-infected mice. Balb/c mice immunized with 25 μg rAs37 formulated with AddaVax™ adjuvant showed significant larval worm reduction after challenge with A. suum infective eggs when compared with a PBS (49.7%) or adjuvant control (48.7%). Protection was associated with mixed Th1/2-type immune responses characterized by high titers of serological IgG1 and IgG2a and stimulation of the production of cytokines IL-4, IL-5, IL-10 and IL-13. In this experiment, the AddaVax™ adjuvant induced better protection than the Th1-type adjuvant MPLA (38.9%) and the Th2-type adjuvant Alhydrogel (40.7%). Sequence analysis revealed that As37 is a member of the immunoglobulin superfamily (IgSF) and highly conserved in other human STHs. Anti-As37 antibodies strongly recognized homologs in hookworms (Necator americanus, Ancylostoma ceylanicum, A. caninum) and in the whipworm Trichuris muris, but there was no cross-reaction with human spleen tissue extracts. These results suggest that the nematode-conserved As37 could serve as a pan-helminth vaccine antigen to prevent all STH infections without cross-reaction with human IgSF molecules.
As37 is an A. suum expressed immunodominant antigen that elicited significant protective immunity in mice when formulated with AddaVax™. As37 is highly conserved in other STHs, but not in humans, suggesting it could be further developed as a pan-helminth vaccine against STH co-infections.
Background and aims
Highly accurate noninvasive methods for predicting gastroesophageal varices needing treatment (VNT) are desired. Radiomics is a newly emerging technology of image analysis. This ...study aims to develop and validate a novel noninvasive method based on radiomics for predicting VNT in cirrhosis.
Methods
In this retrospective–prospective study, a total of 245 cirrhotic patients were divided as the training set, internal validation set and external validation set. Radiomics features were extracted from portal-phase computed tomography (CT) images of each patient. A radiomics signature (Rad score) was constructed with the least absolute shrinkage and selection operator algorithm and tenfold cross-validation in the training set. Combined with independent risk factors, a radiomics nomogram was built with a multivariate logistic regression model.
Results
The Rad score, consisting of 14 features from the gastroesophageal region and 5 from the splenic hilum region, was effective for VNT classification. The diagnostic performance was further improved by combining the Rad score with platelet counts, achieving an AUC of 0.987 (95% CI 0.969–1.00), 0.973 (95% CI 0.939–1.00) and 0.947 (95% CI 0.876–1.00) in the training set, internal validation set and external validation set, respectively. In efficacy and safety assessment, the radiomics nomogram could spare more than 40% of endoscopic examinations with a low risk of missing VNT (< 5%), and no more than 8.3% of unnecessary endoscopic examinations still be performed.
Conclusions
In this study, we developed and validated a novel, diagnostic radiomics-based nomogram which is a reliable and noninvasive method to predict VNT in cirrhotic patients.
Clinical trials registration
NCT04210297.
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