A comprehensive comparison of molecular and phenotypic profiles between hepatitis B virus (HBV)-infected and non-HBV-infected hepatocellular carcinoma by multi-omics analysis.

Hepatitis B virus (HBV) infection is a major etiology of hepatocellular carcinoma (HCC). An interesting question is how different are the molecular and phenotypic profiles between HBV-infected (HBV+) and non-HBV-infected (HBV-) HCCs? Based on the publicly available multi-omics data for HCC, including bulk and single-cell data, and the data we collected and sequenced, we performed a comprehensive comparison of molecular and phenotypic features between HBV+ and HBV- HCCs. Our analysis showed that compared to HBV- HCCs, HBV+ HCCs had significantly better clinical outcomes, higher degree of genomic instability, higher enrichment of DNA repair and immune-related pathways, lower enrichment of stromal and oncogenic signaling pathways, and better response to immunotherapy. Furthermore, in vitro experiments confirmed that HBV+ HCCs had higher immunity, PD-L1 expression and activation of DNA damage response pathways. This study may provide insights into the profiles of HBV+ and HBV- HCCs, and guide rational therapeutic interventions for HCC patients.

Crosstalk in extrahepatic and hepatic system in NAFLD/NASH.

Non-alcoholic fatty liver disease (NAFLD) has emerged as the most prevalent chronic liver disease globally. Non-alcoholic steatohepatitis (NASH) represents an extremely progressive form of NAFLD, which, without timely intervention, may progress to cirrhosis or hepatocellular carcinoma. Presently, a definitive comprehension of the pathogenesis of NAFLD/NASH eludes us, and pharmacological interventions targeting NASH specifically remain constrained. The aetiology of NAFLD encompasses a myriad of external factors including environmental influences, dietary habits and gender disparities. More significantly, inter-organ and cellular interactions within the human body play a role in the development or regression of the disease. In this review, we categorize the influences affecting NAFLD both intra- and extrahepatically, elaborating meticulously on the mechanisms governing the onset and progression of NAFLD/NASH. This exploration delves into progress in aetiology and promising therapeutic targets. As a metabolic disorder, the development of NAFLD involves complexities related to nutrient metabolism, liver-gut axis interactions and insulin resistance, among other regulatory functions of extraneous organs. It further encompasses intra-hepatic interactions among hepatic cells, Kupffer cells (KCs) and hepatic stellate cells (HSCs). A comprehensive understanding of the pathogenesis of NAFLD/NASH from a macroscopic standpoint is instrumental in the formulation of future therapies for NASH.

Targeting aberrant glycosylation to modulate microglial response and improve cognition in models of Alzheimer's disease.

Altered glycosylation profiles have been correlated with potential drug targets in various diseases, including Alzheimer's disease (AD). In this area, the linkage between bisecting N-acetylglucosamine (GlcNAc), a product of N-acetylglucosaminyltransferase III (GnT-III), and AD has been recognized, however, our understanding of the cause and the causative role of this aberrant glycosylation in AD are far from completion. Moreover, the effects and mechanisms of glycosylation-targeting interventions on memory and cognition, and novel targeting strategies are worth further study. Here, we showed the characteristic amyloid pathology-induced and age-related changes of GnT-III, and identified transcription factor 7-like 2 as the key transcription factor responsible for the abnormal expression of GnT-III in AD. Upregulation of GnT-III aggravated cognitive dysfunction and Alzheimer-like pathologies. In contrast, loss of GnT-III could improve cognition and alleviate pathologies. Furthermore, we found that an increase in bisecting GlcNAc modified ICAM-1 resulted in impairment of microglial responses, and genetic inactivation of GnT-III protected against AD mechanistically by blocking the aberrant glycosylation of ICAM-1 and subsequently modulating microglial responses, including microglial motility, phagocytosis ability, homeostatic/reactive state and neuroinflammation. Moreover, by target-based screening of GnT-III inhibitors from FDA-approved drug library, we identified two compounds, regorafenib and dihydroergocristine mesylate, showing pharmacological potential leading to modulation of aberrant glycosylation and microglial responses, and rescue of memory and cognition deficits.

von Hippel-Lindau-targeting microRNA-143-3p attenuates mitochondrial abnormality via AMPK/PGC-1α axis in Parkinson's disease.

Parkinson's disease (PD) is the second most common neurodegenerative disease characterized by selective loss of dopaminergic neurons. We previously found that inhibition of von Hippel-Lindau (VHL) can alleviate dopaminergic neuron degeneration in PD models via regulation of mitochondrial homeostasis, however, the disease-related alterations of VHL and the regulatory mechanisms of VHL level in PD need to be further investigated. In this study, we found that the levels of VHL were markedly increased in multiple cell models of PD and identified microRNA-143-3p (miR-143-3p) as a promising candidate for regulating VHL expression involved in PD. miR-143-3p directly bound to the 3'untranslated region of human VHL mRNA and inhibited its translation, and exerted neuroprotective effects by improving cell viability, apoptosis and tyrosine hydroxylase abnormality. Furthermore, we demonstrated that miR-143-3p exerted neuroprotection by attenuating mitochondrial abnormality via AMP-activated protein kinase (AMPK)/peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) axis, and AMPK inhibitor abolished the beneficial effects of miR-143-3p on the cell model of PD. Therefore, we identify the dysregulated VHL and miR-143-3p in PD, and propose the therapeutic potential of miR-143-3p to alleviate PD by improving mitochondrial homeostasis via AMPK/PGC-1α axis.

Self-assembled nanoparticles based on DNA origami and a nitrated T helper cell epitope as a platform for the development of personalized cancer vaccines.

Neoantigen vaccines constitute an emerging and promising cancer immunotherapy. However, not all neoantigens have anti-tumor activity, as poor CD4+ epitope recognition can lead to the lack of greatly limit the persistence of the CD8+ T cell response. Therefore, we designed a self-assembled nanoplatform hereinafter referred to as DNA-coupled nitrated T helper cell epitope nanoparticle (DCNP) based on DNA origami containing a nitrated CD4 + T cell epitope, which can facilitate the effective activation of neoantigen-specific CD8+ T cells. Moreover, we embedded the cytidine-phosphate-guanosine oligonucleotide (CpG ODN) motif sequence in the DNA skeleton to function as a built-in adjuvant to activate Toll-like receptor 9. DCNP can markedly improve adjuvant and neoantigen co-delivery to lymphoid organs and promote neoantigen presentation on dendritic cells. Moreover, DCNP induced robust, and long-lived neoantigen-specific CD8+ T cell responses that significantly delayed tumor growth. Further, these effects were largely dependent on the nitrated T cell epitope. Collectively, our findings indicate that DCNP is a promising platform that could improve the development of personalized therapeutic neoantigen vaccines for cancer immunotherapy.

Inhibiting von Hippel‒Lindau protein-mediated Dishevelled ubiquitination protects against experimental parkinsonism.

Dopaminergic neuron degeneration is a hallmark of Parkinson's disease (PD). We previously reported that the inactivation of von Hippel‒Lindau (VHL) alleviated dopaminergic neuron degeneration in a C. elegans model. In this study, we investigated the specific effects of VHL loss and the underlying mechanisms in mammalian PD models. For in vivo genetic inhibition of VHL, AAV-Vhl-shRNA was injected into mouse lateral ventricles. Thirty days later, the mice received MPTP for 5 days to induce PD. Behavioral experiments were conducted on D1, D3, D7, D14 and D21 after the last injection, and the mice were sacrificed on D22. We showed that knockdown of VHL in mice significantly alleviated PD-like syndromes detected in behavioral and biochemical assays. Inhibiting VHL exerted similar protective effects in MPP+-treated differentiated SH-SY5Y cells and the MPP+-induced C. elegans PD model. We further demonstrated that VHL loss-induced protection against experimental parkinsonism was independent of hypoxia-inducible factor and identified the Dishevelled-2 (DVL-2)/ß-catenin axis as the target of VHL, which was evolutionarily conserved in both C. elegans and mammals. Inhibiting the function of VHL promoted the stability of ß-catenin by reducing the ubiquitination and degradation of DVL-2. Thus, in vivo overexpression of DVL-2, mimicking VHL inactivation, protected against PD. We designed a competing peptide, Tat-DDF-2, to inhibit the interaction between VHL and DVL-2, which exhibited pharmacological potential for protection against PD in vitro and in vivo. We propose the therapeutic potential of targeting the interaction between VHL and DVL-2, which may represent a strategy to alleviate neurodegeneration associated with PD.

Influencing factors of nonclinical pharmacists' willingness to transform: a cross-sectional survey in Xinjiang, China.

BACKGROUND: There is a serious shortage of clinical pharmacists in Xinjiang, China. A six-month to one-year on-the-job training programme can rapidly transition nonclinical pharmacists into clinical pharmacists to resolve this issue. However, not all nonclinical pharmacists are willing to become clinical pharmacists, and many factors may influence their willingness. This study aims to assess the transformation intention of nonclinical hospital pharmacists and the contributing elements to make recommendations to accelerate the transformation of hospital pharmacists to clinical pharmacists. METHODS: Cross-sectional survey was conducted in secondary and tertiary hospitals in Xinjiang. Taking 14 prefectures in Xinjiang as a cluster, 34 hospitals were randomly selected. By snowball sampling, the heads of pharmaceutical departments introduced non-clinical pharmacists to participate in an anonymous questionnaire survey, which included 41 questions about basic demographic information, cognition and attitudes towards pharmaceutical care, potential factors and willingness to transform, and it took an average of 10 min to complete. Using multifactor logistic regression, the contributing elements of transformation intention were analysed. RESULTS: The survey was conducted from May to October 2022. 338 valid responses were obtained, with a response rate of 91.85% and a willingness to transform rate of 81.67%. There were significant differences in the willingness to transform among nonclinical pharmacists of different ages (P < 0.05), marital statuses (P < 0.05), years of employment (P < 0.05), and technical titles (P < 0.05). There were significant differences between the two groups in the following four aspects: whether the setting of human resources in the pharmaceutical department was reasonable (P < 0.05), the educational level of clinical pharmacists (P < 0.05), the higher salary level of clinical pharmacists (P < 0.05), and whether they had experience in pharmaceutical care (P < 0.05). There was a significant difference in the total score of the pharmaceutical care attitude scale (P < 0.05) between the willing and unwilling groups. The results of multivariate logistic regression analysis revealed that the experience of providing pharmaceutical care (OR = 4.601, 95% CI: 1.13-18.69, P < 0.05) and attitude towards pharmaceutical care (OR = 3.302, 95% CI: 1.19-9.19, P < 0.05) had a statistically significant influence on the transformation intention of nonclinical pharmacists. CONCLUSIONS: One-fifth of nonclinical pharmacists were unwilling to transition to clinical pharmacists. The attitude towards and experience of pharmaceutical care affected pharmacists' transformation intention, so the suggestion is proposed to promote the transformation of nonclinical pharmacists into clinical pharmacists.

Cytosolic Protein Delivery for Intracellular Antigen Targeting Using Supercharged Polypeptide Delivery Platform.

Despite the well-recognized clinical success of therapeutic proteins, especially antibodies, their inability to penetrate the cell membrane restricts them to secretory extracellular or membrane-associated targets. Developing a direct cytosolic protein delivery system would offer unique opportunities for intracellular target-related therapeutic proteins. Here, we generated a supercharged polypeptide (SCP) with high cellular uptake efficiency, endosomal escape ability, and good biosafety and developed an SCP with an unnatural amino acid containing the phenylboronic acid (PBA) group, called PBA-SCP. PBA-SCP is capable of potently delivering proteins with various isoelectric points and molecular sizes into the cytosol of living cells without affecting their bioactivities. Importantly, cytosolically delivered antibodies remain functional and are capable of targeting, labeling, and manipulating diverse intracellular antigens. This study demonstrates an efficient and versatile intracellular protein delivery platform, especially for antibodies, and provides new possibilities for expanding protein-based therapeutics to intracellular "undruggable" targets.

Association of hospital pharmacy-related knowledge and skills with occupational stress of clinical pharmacists in tertiary hospitals of China.

OBJECTIVE: Occupational stress of health care providers may lower the quality of care. Person-environment fit theory and practical evidence have indicated that various types of knowledge and skills of health care providers are differentially associated with occupational stress. Clinical pharmacists are an indispensable part of medical teams. Clinical pharmacists in China are generally under high occupational stress, but what kind of knowledge and skills can relieve their occupational stress remains unclear. This study aimed to assess the association between the specific knowledge and skills of clinical pharmacists and their occupational stress in China. This study aimed to assess the association between the specific knowledge and skills of clinical pharmacists and their occupational stress in China. METHODS: A field questionnaire survey using a stratified sampling was conducted to gather data on occupational stress, knowledge and skills related to hospital pharmacy, and other factors of occupational stress using the Brief Job Stress Questionnaire and a self-developed instrument. Ordinary least squares regression was used to evaluate the association of the participants' knowledge, skills, and other factors with their occupational stress. RESULTS: A total of 625 clinical pharmacists from 311 tertiary hospitals in China (response rate = 84%) participated. Knowledge of or skills related to pharmaceutical care service provision (P = 0.02), the use of computers and the Internet in pharmacy practice (P = 0.02), interpersonal communication (P = 0.10), or pharmacoepidemiology (P = 0.08) was associated with reduced occupational stress of the participants. Participants who had credentials of nation-level specialized (P = 0.09) and general training (P = 0.04) for clinical pharmacist had lower degrees of occupational stress than those without these credentials. The participants' clinical professions, routine tasks, technical titles, and type of hospital they work in were also associated with their occupational stress. CONCLUSION: Enhancing several aspects of knowledge or skills among clinical pharmacists in tertiary hospitals in China may help reduce their occupational stress. Efforts are needed to improve the education and training system of clinical pharmacists in China.

Light-Controlled Tyrosine Nitration of Proteins.

Tyrosine nitration of proteins is one of the most important oxidative post-translational modifications in vivo. A major obstacle for its biochemical and physiological studies is the lack of efficient and chemoselective protein tyrosine nitration reagents. Herein, we report a generalizable strategy for light-controlled protein tyrosine nitration by employing biocompatible dinitroimidazole reagents. Upon 390 nm irradiation, dinitroimidazoles efficiently convert tyrosine residues into 3-nitrotyrosine residues in peptides and proteins with fast kinetics and high chemoselectivity under neutral aqueous buffer conditions. The incorporation of 3-nitrotyrosine residues enhances the thermostability of lasso peptide natural products and endows murine tumor necrosis factor-α with strong immunogenicity to break self-tolerance. The light-controlled time resolution of this method allows the investigation of the impact of tyrosine nitration on the self-assembly behavior of α-synuclein.

The current status of pharmaceutical care provision in tertiary hospitals: results of a cross-sectional survey in China.

BACKGROUND: Pharmaceutical care is attached with increasing importance around the world due to its clinical and economical effects. Tertiary hospitals are equipped with the richest healthcare resources and pioneer in the implementation of pharmaceutical care. Understanding current status of pharmaceutical care provision in tertiary hospitals not only helps to improve the practice in tertiary hospitals but also guide the development of pharmaceutical care in secondary and primary health institutions. METHOD: Data of a cross-sectional survey were used. The cross-sectional survey was conducted from July 2015 to June 2016, involving 520 hospital directors, 740 clinical pharmacists, 1298 physicians, 778 dispensing pharmacists and 3096 patients from 292 hospitals of 23 provinces, 4 municipalities in mainland China. The survey aimed to comprehensively investigate the current status of pharmaceutical care and health care professional's understanding of clinical pharmacist system in tertiary hospitals. This study reports results pertaining to current status of pharmaceutical care, including pharmacy department practice rules, guidelines and records, application of rational drug use software, staffing and working arrangement of clinical pharmacists and physicians, patients' satisfaction toward pharmaceutical care. RESULTS: A majority of the tertiary hospitals established clinical pharmacist system (84.2%), clinical pharmacist management rules (89%), clinical pharmacists' working ethics (89%) and applied clinical rational drug use software (93.8%). However, a number of hospitals did not establish a performance evaluation system (37%) and payment rules for pharmaceutical care (81.9%). Most of the clinical pharmacists met the educational background set by the government. Averagely there were 8.3 clinical pharmacists per hospital and 90.7% of the tertiary hospitals had at least five full-time clinical pharmacists. Pharmaceutical care services provided include checking prescriptions, making treatment plans and joining clinical rounds and etc. Both physicians and patients were generally satisfied with pharmaceutical care services provided. CONCLUSION: China has made progress in pharmaceutical care provision, but problems such as lack of rules for pharmaceutical care payment and a performance evaluation system, a monotonous variety of pharmaceutical care activities remain unsolved. Policy makers and hospitals directors are suggested to pay more attention to these problems.

Microenvironment-Responsive Delivery of the Cas9 RNA-Guided Endonuclease for Efficient Genome Editing.

Successful and efficient delivery of Cas9 protein and gRNA into cells is critical for genome editing and its therapeutic application. In this study, we developed an improved supercharged polypeptide (SCP) mediated delivery system based on dithiocyclopeptide linker to realize the effective genome editing in tumor cells. The fusion protein Cas9-linker-SCP (Cas9-LS) forms positively charged complexes with gRNA in vitro to provide possibilities for gRNA delivery into cells. Under the microenvironment of tumor cells, the dithiocyclopeptide linker, containing matrix metalloproteinase 2 (MMP-2) sensitive sequence and an intramolecular disulfide bond, can be completely disconnected to promote the release of Cas9 protein with the nuclear localization sequence (NLS) in the cytoplasm and transfer to the cell nucleus for highly efficient genome editing, resulting in an obvious increase of indel efficiency in comparison to fusion protein without dithiocyclopeptide linker (Cas9-SCP). Furthermore, Cas9-LS shows no significant cytotoxicity and minimal hemolytic activity. We envision that the microenvironment-responsive Cas9 protein delivery system can facilitate more efficient genome editing in tumor cells.

Establishment of an HPLC-based method to identify key proteases of proteins in vitro.

Given that the biological functions of proteins may decrease or even be lost due to degradation by proteases, it is of great significance to identify potential proteases that degrade protein drugs during systemic circulation. In this work, we describe a method based on high-performance liquid chromatography (HPLC) to identify key proteases that degrade therapeutic proteins in blood, including endopeptidases and exopeptidases. Here, the degradation of proteins was detected by competition with standard substrates of proteases and is shown as the relative residue rate. Four protein drugs were subjected to this method, and the results suggested that growth hormone was degraded by aminopeptidase N and kallikrein-related peptidase 5, pertuzumab was hardly degraded by the proteases, factor VII was degraded by carboxypeptidase B, neprilysin, dipeptidyl peptidase-4 and peptidyl dipeptidase A, and fibrinogen was degraded by carboxypeptidase B and kallikrein-related peptidase 5, findings consistent with the literature. The results were confirmed by microscale thermophoresis; additionally, activity detection in vitro substantiated that the degradation of factor VII decreased its activity. We demonstrate that this method can be used to identify key proteases of proteins with high accuracy, precision and durability.

Do clerkship schemes effectively improve pharmacy students' understanding of and attitudes regarding pharmaceutical care?--a pre-post study in China Pharmaceutical University.

BACKGROUND: Clerkship is potentially an effective measure of improving the pharmacy students' understanding of and attitudes toward pharmaceutical care. This research aimed to validate whether clerkship schemes in China effectively improve pharmacy students' understanding of and attitudes toward PC, and discuss on how to improve the clerkship schemes for better quality. METHODS: A pre-post and self-administrated questionnaire survey among three continuous years of students was conducted to measure Chinese fifth-year clinical pharmacy students' differences in understanding of pharmaceutical care and attitudes toward pharmaceutical care before and after their clerkship schemes. Statistical analysis was conducted on the retrieved data. RESULTS: 602 respondents completed the post-part survey (drop rate = 1.8%). Correct rates of all statements regarding students' understanding of pharmaceutical care were generally increased, but the rates of respondents holding correct understanding of the primary goal of pharmaceutical care (71.9 to 85.0%), the necessity of drug information support in pharmaceutical care (77.1 to 87.5%) and the patients' role and duty when receiving pharmaceutical care (66.1 to 70.6%) were unsatisfactory before the clerkship and the improvement was not significant. Most statements regarding students' attitudes toward pharmaceutical care were improved in general. However, rates of respondents holding negative attitudes toward students performing pharmaceutical care during their clerkship (3.7 to 7.5%) and choosing pharmaceutical care provider as their career after clerkship schemes (10.8 to 14.8%) had increased after the clerkship schemes. CONCLUSION: Clerkship generally improves clinical pharmacy students' understanding of and attitudes toward pharmaceutical care, yet adjustments are also required in some contents of the clerkship for further improvements in its outcomes.

Potent Protein Delivery into Mammalian Cells via a Supercharged Polypeptide.

The efficient delivery of proteins into cells is needed to fully realize the potential of protein-based therapeutics. Current protein delivery strategies generally suffer from poor endosomal escape and low tolerance for serum. Here, the genetic fusion of a supercharged polypeptide, called SCP, to a protein provides a generic method for intracellular protein delivery. It allows efficient protein endocytosis and endosomal escape and is capable of potently delivering various proteins with a range of charges, sizes, and bioactivities into the nucleus of living cells. SCP is discovered to bind directly to the nuclear import protein importin ß1 and gains access to the nucleus. Furthermore, SCP shows minimal hemolytic activity and stability in serum and lacks toxicity and immunogenicity in vivo. Effective gene editing can be achieved by SCP-mediated delivery of Cas9 protein and guide RNA. This study may provide an efficient and useful tool for the design and development of cell-nuclear-targeted drug delivery.

C-terminal site-specific PEGylated Exendin-4 analog: A long-acting glucagon like Peptide-1 receptor agonist, on glycemic control and beta cell function in diabetic db/db mice.

PEG modification is a common clinical strategy for prolonging the half-life of therapeutic proteins or polypeptides. In a previous work, we have successfully synthesized PEG-modified Exendin-4 (PE) by conjugating a 20 kDa PEG to the C-terminal of Exendin-4. Then, we introduced an integrative characterization for PE to evaluate its hypoglycemic activity and pharmacokinetic properties. The normoglycemic efficacies and therapeutic activity of PE were investigated in db/db mice. The hypoglycemic time after single administration of PE on db/db mice was prolonged from 8.4 h to 54.9 h. In multiple treatment with PE, the fasting blood glucose in various PE dosages (50, 150, and 250 nmol/kg) were remarkably reduced, and the glycosylated hemoglobin level was decreased to 2.0%. When the in vivo single- and multiple-dose pharmacokinetics of PE were examined in Sprague-Dawley rats, the half-life was prolonged to 31.7 h, and no accumulation effect was observed. Overall, this study provided a novel promising therapeutic approach to improving glucose-controlling ability and extending half-life without accumulation in vivo for long-acting treatment of type-2 diabetes.

Rapid de novo generation of antigen specific human B cells with expression of Blimp-1 and AID by in vitro immunization.

In vitro immunization with antigens and cytokines triggers specific human B-cell response in short periods and is therefore superior to conventional in vivo immunization for antibody development. However, this new technology is limited by low efficiency, poor reproducibility, and requirement of pre-immunized lymphocytes. In this study, we demonstrate a novel method for de novo inducing antigen-specific human B cells in vitro. Unlike previous in vitro immunization of unfractionated PBMCs, we firstly optimized the conditions for inducing monocyte-derived dendritic cells (DCs) to efficiently capture, process, and present antigens. Instead of using the conventional method to activate Th2 cells for in vitro immunization, we succeeded to differentiate naïve CD4+ T cells into T follicular helper (Tfh) cells using antigen-sensitized DCs and cytokine cocktail. We discovered the differentiated T cells expressed ICOS, PD-1, BCL-6, and IL-21 at high levels. After 12 days of T-B co-culture, we observed induced T cells efficiently promoted naïve B cells to differentiate into plasmablasts secreting antigen-specific antibodies, with expression of Blimp-1 and AID related to affinity maturation and class switching. Thus, we established a new co-culture system with naïve lymphocyte populations for de novo acquisition of specifically in vitro immunized B cells potentially for development of therapeutic antibodies, which also provides novel insights into understanding the complex interactions among immune cells in lymph nodes.

Selection, purification, and characterization of a HER2-targeting soluble designed ankyrin repeat protein by E. coli surface display using HER2-positive melanoma cells.

Human epidermal growth factor receptor 2 (HER2) is a powerful target for cancer immune therapy. The development of anti-HER2 monoclonal antibodies targeting different domains of HER2 is quite effective. However, the selection and production of multivalent antibodies are complicated. In this study, a mimivirus-based designed ankyrin repeat protein (DARPin) targeting HER2 was selected from an artificial library by bacteria surface display. The selection was performed on HER2-positive B16BL6/E2 melanoma cells and HER2-nagative cells. DARPin selected from the library could be expressed in soluble form with a yield of 70 mg/L. After purified by two continuous and easy steps, the purity of DARPin was 90% as established by SDS-PAGE and RP-HPLC. Selected DARPin showed significant HER2-targeting ability with an affinity of 1.05 ± 0.47 µM. MTT assay demonstrated that at the concentration of 640 nM, the selected DARPin dimer could inhibit the SK-BR-3 growth at a rate of 36.63 and 46.34% in 48 and 72 hr incubation separately, which was similar to trastuzumab (43.12 and 49.14% separately). These findings suggested that it was an effective method to select antibody mimetic DARPin by bacteria surface display combined with live cells sorting and provided a drug candidate for cancer therapy.

The novel α-glucan YCP improves the survival rates and symptoms in septic mice by regulating myeloid-derived suppressor cells.

Sepsis is a life-threatening health condition that is initially characterized by uncontrolled inflammation, followed by the development of persistent immunosuppression. YCP is a novel α-glucan purified from the mycelium of the marine fungus Phoma herbarum YS4108, which has displayed strong antitumor activity via enhancing host immune responses. In this study, we investigated whether YCP could influence the development of sepsis in a mouse model. Caecal ligation and puncture (CLP)-induced sepsis was established in mice that were treated with YCP (20 mg/kg, ip or iv) 2 h before, 4 and 24 h after the CLP procedure, and then every other day. YCP administration greatly improved the survival rate (from 39% to 72% on d 10 post-CLP) and ameliorated disease symptoms in the septic mice. Furthermore, YCP administration significantly decreased the percentage of myeloid-derived suppressor cells (MDSCs) in the lungs and livers, which were dramatically elevated during sepsis. In cultured BM-derived cells, addition of YCP (30, 100 µg/mL) significantly decreased the expansion of MDSCs; YCP dose-dependently decreased the phosphorylation of STAT3 and increased the expression of interferon regulatory factor-8 (IRF-8). When BM-derived MDSCs were co-cultured with T cells, YCP dose-dependently increased the production of arginase-1 (Arg-1) and inducible nitric oxide synthase (iNOS), and activated the NF-κB pathway. In addition, the effects of YCP on MDSCs appeared to be dependent on toll-like receptor (TLR) 4. These results reveal that YCP inhibits the expansion of MDSCs via STAT3 while enhancing their immunosuppressive function, partially through NF-κB. Our findings suggest that YCP protects mice against sepsis by regulating MDSCs. Thus, YCP may be a potential therapeutic agent for sepsis.

Metabolic engineering of Bacillus subtilis for biosynthesis of heparosan using heparosan synthase from Pasteurella multocida, PmHS1.

Heparosan, the capsular polysaccharide discovered in many pathogenic bacteria, is a promising material for heparin preparation. In this study, the Pasteurella multocida heparosan synthase 1 (PmHS1) module was used to synthesize heparosan with controlled molecular weight, while tuaD/gtaB module or gcaD module was responsible for UDP-precursors production in Bacillus subtilis 168. After metabolic pathway optimization, the yield of heparosan was as high as 237.6 mg/L in strain containing PmHS1 module and tuaD/gtaB module, which indicated that these two modules were key factors in heparosan production. The molecular weight of heparosan varied from 39 to 53 kDa, which indicated that heparosan molecular weight could be adjusted by the amount of PmHS1 and the ratio of two UDP precursors. The results showed that it would be possible to produce safe heparosan with appropriate molecular weight which is useful in heparin production.