Tumor-Derived PD-L1+ Exosomes with Natural Inflammation Tropism for Psoriasis-Targeted Treatment.

Psoriasis is a chronic inflammatory disease whose etiology is directly related to the dysregulation of cutaneous immune homeostasis. However, how to finely modulate the skin immune microenvironment to restore homeostasis remains an important challenge. Inspired by the natural attribute of tumor exosomes in the immune escape, the tumor-derived exosomes as an active targeting nanoplatform for the effective treatment of inflammatory skin disorder were first reported. As keratinocytes and immune cells express high PD-1 during the onset of psoriasiform skin inflammation, the PD-L1-positive exosomes derived from melanoma cells carrying pristimerin with extremely anti-inflammatory potential were yielded to treat psoriasis. The PD-L1+ exosomes carrying pristimerin were characterized, and the cellular uptake was performed to evaluate the PD-1 target capability. The anti-inflammatory action of PD-L1+ exosomes carrying pristimerin was observed in both in vitro and in vivo models of psoriasis. Our exosomes substantially increased pristimerin uptake with CD4+ T cells and keratinocytes, significantly inhibited the proliferation of Th17 cells, and promoted Treg differentiation in a psoriasis-like model. Obviously, PD-L1+ exosomes carrying pristimerin significantly and safely reversed imiquimod (IMQ)-induced psoriasis in mice, indicated by reducing epidermal thickness, decreasing plaque formation, and suppressed excessive inflammatory response, due to its dual targeting of both CD4+ T cells and keratinocytes gathering around the lesion. The inflammatory cell infiltration and pro-inflammatory cytokine production in psoriasis were suppressed by our engineered exosomes. Besides, PD-L1+ exosomes carrying pristimerin treatment alleviated ferroptosis-related changes in psoriatic skin, thereby dampening excessive inflammation and, in turn, decreasing the abnormal proliferation of keratinocytes in psoriatic lesions. This study demonstrates that our engineered exosomes can not only act as a treat-to-target strategy for psoriasis treatment but also provide insight in clinical application of inflammatory disorders.

Fosaprepitant for postoperative nausea and vomiting in patients undergoing laparoscopic gastrointestinal surgery: a randomised trial.

BACKGROUND: Postoperative nausea and vomiting (PONV) is a major problem after surgery. Even with double prophylactic therapy including dexamethasone and a 5-hydroxytryptamine-3 receptor antagonist, the incidence is still high in many at-risk patients. Fosaprepitant, a neurokinin-1 receptor antagonist, is an effective antiemetic, but its efficacy and safety in combination antiemetic therapy for preventing PONV remain unclear. METHODS: In this randomised, controlled, double-blind trial, 1154 participants at high risk of PONV and undergoing laparoscopic gastrointestinal surgery were randomly assigned to either a fosaprepitant group (n=577) receiving fosaprepitant 150 mg i.v. dissolved in 0.9% saline 150 ml, or a placebo group (n=577) receiving 0.9% saline 150 ml before anaesthesia induction. Dexamethasone 5 mg i.v. and palonosetron 0.075 i.v. mg were each administered in both groups. The primary outcome was the incidence of PONV (defined as nausea, retching, or vomiting) during the first 24 postoperative hours. RESULTS: The incidence of PONV during the first 24 postoperative hours was lower in the fosaprepitant group (32.4% vs 48.7%; adjusted risk difference -16.9% [95% confidence interval: -22.4 to -11.4%]; adjusted risk ratio 0.65 [95% CI: 0.57 to 0.76]; P<0.001). There were no differences in severe adverse events between groups, but the incidence of intraoperative hypotension was higher (38.0% vs 31.7%, P=0.026) and intraoperative hypertension (40.6% vs 49.2%, P=0.003) was lower in the fosaprepitant group. CONCLUSIONS: Fosaprepitant added to dexamethasone and palonosetron reduced the incidence of PONV in patients at high risk of PONV undergoing laparoscopic gastrointestinal surgery. Notably, it increased the incidence of intraoperative hypotension. CLINICAL TRIAL REGISTRATION: NCT04853147.

Macrophages-derived exosomal lncRNA LIFR-AS1 promotes osteosarcoma cell progression via miR-29a/NFIA axis.

BACKGROUND: Osteosarcoma (OS) is the most common primary malignant bone tumor in young people. Tumor-associated macrophages (TAMs) have been reported to play an important role in the development of osteosarcoma. However, the detailed molecular mechanisms remain largely unknown and need to be elucidated. Recently, exosomes have been reported as the crucial mediator between tumor cells and the tumor microenvironment. And a lot of lncRNAs have been reported to act as either oncogenes or tumor suppressors in osteosarcoma. In this research, we aim to explore the role of macrophages-derived exosomal lncRNA in osteosarcoma development and further elucidated the potential molecular mechanisms involved. METHODS: TAMs were differentiated from human mononuclear cells THP-1, and a high-throughput microarray assay was used to analyze the dysregulated lncRNAs and miRNAs in osteosarcoma cells co-cultured with macrophages-derived exosomes. Western blot, qRT-PCR assays, and Dual-luciferase reporter assay were used to verify the interaction among LIFR-AS1, miR-29a, and NFIA. Cck-8, EdU, colony formation assay, wound-healing, and transwell assay were performed to explore the characterize the proliferation and metastasis ability of OS cells. And qPCR, Western blots, immunohistochemistry, and cell immunofluorescence were used to detect the expression of relative genes or proteins. RESULTS: In this study, we found that THP-1-induced macrophage-derived exosomes could facilitate osteosarcoma cell progression both in vitro and in vivo. Then, the results of the high-throughput microarray assay showed that LIFR-AS1 was highly expressed and miR-29a was lowly expressed. Furthermore, LIFR-AS1 was identified as a miR-29a sponge, and NFIA was validated as a direct target of miR-29a. Functional assays demonstrated that knockdown of exosomal LIFR-AS1 could attenuate the promotion effects of macrophages-derived exosomes on osteosarcoma cell progression and miR-29a inhibition could reserve the effect of LIFR-AS1-knockdown exosomes. Correspondingly, NFIA-knockdown could partially reverse the tumor inhibition effect of miR-29a on osteosarcoma cells. CONCLUSIONS: Taken together, macrophages-derived exosomal lncRNA LIFR-AS1 can promote osteosarcoma cell proliferation, invasion, and restrain cell apoptosis via miR-29a/NFIA axis, which can act as a potential novel therapeutic target for osteosarcoma therapy.

HER4 promotes the growth and metastasis of osteosarcoma via the PI3K/AKT pathway.

Osteosarcoma is the most common primary malignant bone tumor, which occurs in adolescents. As reported by our previous studies, HER4 indicates a poor prognosis of primary osteosarcoma. However, its mechanisms in the pathogenesis of osteosarcoma have not yet been studied. The purpose of this study was to investigate the role of HER4 in osteosarcoma and whether the PI3K/AKT pathway is involved. In this study, western blot analysis was used to investigate the expression of HER4 protein in osteosarcoma tissues and cell lines. CCK8 and transwell assays were used to detect the effects of HER4 on the proliferation, migration, and invasion of osteosarcoma cells in vitro. The effects of HER4 on the growth and metastasis of osteosarcoma in vivo were detected by tumor formation and immunofluorescence in nude mice. The role of the PI3K/AKT pathway in HER4 regulation of the growth and metastasis of osteosarcoma was examined by western blot analysis and immunofluorescence assay. We found that HER4 protein was highly expressed in clinical osteosarcoma specimens and osteosarcoma cells. HER4 markedly promoted the proliferation, migration, and invasion of osteosarcoma cells in vitro as well as the growth and metastasis of osteosarcoma in vivo. HER4 overexpression upregulated the expression of phosphorylated protein kinase B (pAKT), proliferation marker antigen Ki67, and metastasis cell marker matrix metalloproteinase 9 (MMP9). Notably, PI3K/AKT inhibitor LY294002 significantly inhibited the effects of HER4 via the downregulation of pAKT, Ki67, and MMP9. Moreover, LY294002 markedly blocked the effects of HER4-induced upregulation of tumor malignancy. The present study suggests that HER4 may promote the growth and metastasis of osteosarcoma via the PI3K/AKT pathway. The HER4/PI3K/AKT pathway could serve as a potential target for the treatment of osteosarcoma.

Clinicopathological and prognostic values of ErbB receptor family amplification in primary osteosarcoma.

Osteosarcoma is a malignant bone tumor with extremely high invasion, metastasis and mortality. The prognosis of patients with osteosarcoma remains poor. The ErbB receptor family was found to be overexpressed in human cancers and associated with poor prognosis. However, the role of ErbB receptor family in osteosarcoma has not been fully understood. The present study aimed to investigate the clinicopathological and prognostic significances of ErbB receptors in primary osteosarcoma. Western blot (WB), reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and fluorescence in situ hybridization (FISH) were used to detect the protein and gene expression of ErbB receptors in 60 primary osteosarcoma specimens and 30 non-neoplastic bone tissues. WB and RT-qPCR analyses showed that the protein and mRNA expression levels of EGFR, ErbB3 and ErbB4 in osteosarcoma specimens were significantly higher than those in non-neoplastic bone tissues. Seventeen (28.33%), 15 (25.00%) and 15 (25.00%) osteosarcoma specimens presented with amplification of EGFR, ErbB3 and ErbB4 gene, respectively, which were significantly higher compared with non-neoplastic bone tissues. The amplification of ErbB3 and ErbB4 in osteosarcoma was associated with advanced surgical stage. The amplification of EGFR, ErbB3, ErbB4 and the co-amplification of EGFR-ErbB3, EGFR-ErbB4, ErbB3-ErbB4 was linked with poor response to chemotherapy and distant metastasis. The amplification of EGFR, ErbB3 and ErbB4, as well as their co-amplification demonstrated independent prognostic values for reduced survival time of osteosarcoma patients and may serve as potential therapeutic targets for osteosarcoma patients in the future.

Arginine side chain stacking with peptide plane stabilizes the protein helix conformation in a cooperative way.

A combined experimental and computational study is performed for arginine side chain stacking with the protein α-helix. Theremostability measurements of Aristaless homeodomain, a helical protein, suggest that mutating the arginine residue R106, R137 or R141, which has the guanidino side chain stacking with the peptide plane, to alanine, destabilizes the protein. The R-PP stacking has an energy of ∼0.2-0.4 kcal/mol. This stacking interaction mainly comes from dispersion and electrostatics, based on MP2 calculations with the energy decomposition analysis. The calculations also suggest that the stacking stabilizes 2 backbone-backbone h-bonds (i→i-4 and i-3→i-7) in a cooperative way. Desolvation and electrostatic polarization are responsible for cooperativity with the i→i-4 and i-3→i-7 h-bonds, respectively. This cooperativity is supported by a protein α-helices h-bond survey in the pdb databank where stacking shortens the corresponding h-bond distances.

Mir-1287 suppresses the proliferation, invasion, and migration in hepatocellular carcinoma by targeting PIK3R3.

Mature microRNAs (miRNAs) are a class of small noncoding RNA molecules involved in regulation of post-translational gene expression. Although aberrant levels of miRNAs have been found in various tumor tissues, their importance in tumor development and the molecular basis of their regulatory role remain unclear. Our bioinformatic analysis on The Cancer Genome Atlas database and microarray-based comparison of miRNA in different cell lines revealed that the level of mir-1287 is suppressed in hepatocellular carcinoma (HCC) cells. When upregulated, mir-1287 can reduce the tumorigenesis phenotypes of HCC cells in several in vitro models. We further found that mir-1287 directly targets messenger RNA encoding PIK3R3, which is a tumor-promoting factor acting in several pathways linked to tumorigenesis. Our study suggests that aberrant suppression of mir-1287 is potentially responsible for the development of HCC, and miRNA-based strategies may be developed for efficient detection and treatment of HCC.

Entropy Drives the Formation of Salt Bridges in the Protein GB3.

Salt bridges are very common in proteins. But what drives the formation of protein salt bridges is not clear. In this work, we determined the strength of four salt bridges in the protein GB3 by measuring the ΔpKa values of the basic residues that constitute the salt bridges with a highly accurate NMR titration method at different temperatures. The results show that the ΔpKa values increase with temperature, thus indicating that the salt bridges are stronger at higher temperatures. Fitting of ΔpKa values to the van't Hoff equation yields positive ΔH and ΔS values, thus indicating that entropy drives salt-bridge formation. Molecular dynamics simulations show that the protein and solvent make opposite contributions to ΔH and ΔS. Specifically, the enthalpic gain contributed from the protein is more than offset by the enthalpic loss contributed from the solvent, whereas the entropic gain originates from the desolvation effect.

Improving the activity of Trichoderma reesei cel7B through stabilizing the transition state.

Trichoderma reesei (Tr.) cellulases, which convert cellulose to reducing sugars, are a promising catalyst used in the lignocellulosic biofuel production. Improving Tr. cellulases activity, though very difficult, is highly desired due to the recalcitrance of lignocellulose. Meanwhile, it is preferable to enhance the cellulase's promiscuity so that substrates other than cellulose can also be hydrolyzed. In this work, an attempt is made to improve the catalytic activity of a major endogluanase Tr. Cel7B against xylan which crosslinks with cellulose in lignocellulose. By using quantum mechanics/molecular mechanics (QM/MM) molecular dynamics (MD) simulations, the transition state of the xylo-oligosaccharide hydrolysis is identified. Then, mutations are introduced and their effect on the transition state stabilization is ranked based on the free energy calculations. Seven top ranked mutants are evaluated experimentally. Three mutants A208Q, A222D, and G230R show a higher activity than the wild-type Tr. Cel7B in the hydrolysis of xylan (by up to 47%) as well as filter paper (by up to 50%). The combination of the single mutants can further improve the enzyme activity. Our work demonstrates that the free energy method is effective in engineering the Tr. Cel7B activity against xylan and cellulose, and thus may also be useful for improving the activity of other Tr. cellulases. Biotechnol. Bioeng. 2016;113: 1171-1177. © 2015 Wiley Periodicals, Inc.

Controlled release by novel lysostaphin-loaded hydroxyapatite/chitosan composites.

Lysostaphin is highly effective on eliminating methicillin resistant Staphylococcus aureus (MRSA). In order to achieve controlled release of lysostaphin, a biocompatible drug carrier is needed. Hydroxyapatite/chitosan (HA/CS) composites were chosen to carry lysostaphin and sample composites with different weight ratios of HA to CS, including 80/20, 70/30, 60/40, and 40/60, were prepared. Multiple analyses were performed to determine the structural and physicochemical properties of the composites, including scanning electron microscopy, X-ray diffraction and Fourier transform infrared spectroscopy. We immersed HA/CS composites loaded with 1 wt% lysostaphin to test in vitro release activity and cultured MC3T3-E1 cells to carry out biocompatibility test. The result of the release behavior of the composites revealed that the controlled release of lysostaphin from 60/40 HA/CS composites was the highest release rate of (87.4 ± 2.8)%, which lasted for 120 hours. In biocompatibility testing, MC3T3-E1 cells were able to proliferate on the surface of these composites, and the extract liquid from the composites could increase the growth of the cells. These results demonstrate the controlled release of lysostaphin from HA/CS composites and their biocompatibility, suggesting the potential application of these composites to bone injury and infection applications.

Hydrogen/deuterium exchange mass spectrometry and site-directed disulfide cross-linking suggest an important dynamic interface between the two lysostaphin domains.

Lysostaphin is a peptidoglycan hydrolase secreted by Staphylococcus simulans. It can specifically lyse Staphylococcus aureus and is being tested as a novel antibacterial agent. The protein contains an N-terminal catalytic domain and a C-terminal cell wall targeting domain. Although the two domains from homologous enzymes were structurally determined, the structural organization of lysostaphin domains remains unknown. We used hydrogen/deuterium exchange mass spectrometry (H/DX-MS) and site-directed disulfide cross-linking to probe the interface between the lysostaphin catalytic and targeting domains. H/DX-MS-mediated comparison of peptides from full-length lysostaphin and the separated domains identified four peptides of lower solvent accessibility in the full-length protein. Cross-linking analysis using cysteine pair substitutions within those peptides showed that two pairs of cysteines can form disulfide bonds, supporting the domain association role of the targeted peptides. The cross-linked mutant exhibited a binding capacity to S. aureus that was similar to that of the wild-type protein but reduced bacteriolytic activity probably because of restraint in conformation. The diminished activity was further reduced with increasing NaCl concentrations that can cause contractions of bacterial peptidoglycan. The lytic activity, however, could be fully recovered by reducing the disulfide bonds. These results suggest that lysostaphin may require dynamic association of the two domains for coordinating substrate binding and target cleavage on the elastic peptidoglycan. Our study will help develop site-specific PEGylated lysostaphin to treat systemic S. aureus infections.

The malignancy of chordomas is enhanced via a circTLK1/miR-16-5p/Smad3 positive feedback axis.

CircRNAs play crucial roles in various malignancies via an increasing number of reported regulatory mechanisms, including the classic sponging mechanism between circRNAs and micro RNAs (miRNAs). We performed bioinformatic analyses and identified circTLK1 as a regulator of malignant chordoma progression. Moreover, we observed that circTLK1 showed high expression in chordoma cells and tissues, while circTLK1 interference suppressed chordoma cell proliferation and invasion. In addition, circTLK1 directly interacted with miR-16-5p, which has previously been shown to repress chordoma, and circTLK1 knockdown suppressed Smad3 expression. Chromatin immunoprecipitation sequencing further demonstrated that Smad3 acts as a positive regulator by interacting with TLK1, thereby mediating the circTLK1/miR-16-5p/Smad3 positive feedback axis. Taken together, our findings suggested that the disruption of the circTLK1/miR-16-5p/Smad3 positive feedback pathway, particularly via the Smad3 inhibitor SIS3, could be a promising therapeutic strategy.

The COPS3-FOXO3 positive feedback loop regulates autophagy to promote cisplatin resistance in osteosarcoma.

Chemotherapy is an important treatment modality for osteosarcoma (OS), but the development of chemoresistance limits the therapeutic efficacy of OS and results in a poor prognosis. Thus, a better understanding of the mechanisms underlying chemoresistance in OS is essential. We previously demonstrated that COPS3/CSN3 (COP9 signalosome subunit 3) functions as an oncogene to promote OS cells lung metastasis, which is closely related to chemoresistance. Here, we showed that COPS3 was significantly upregulated in OS tissues with poor response to preoperative chemotherapy. Moreover, COPS3 depletion made OS cells more sensitive to cisplatin treatment in vitro and in vivo, implicating COPS3 as a driver of cisplatin resistance. Mechanistic investigations showed that COPS3 induced a cytoprotective macroautophagy/autophagy in response to cisplatin. Specifically, we identified FOXO3 as a critical target of COPS3, as high expression of COPS3 enhanced the nuclear abundance of FOXO3 and increased the expression of FOXO3-responsive genes, promoting autophagosome formation and maturation. In turn, FOXO3 regulated COPS3 levels by inhibiting ubiquitin-mediated degradation and attenuating SKP2-mediated COPS3 inhibition, cooperatively maintaining a high level of COPS3. In both COPS3-expressing OS cells and a murine xenograft model, inhibition of autophagy could also overcome resistance to cisplatin. Collectively, our results offer insights into the mechanisms of cisplatin resistance and suggest that targeting COPS3-mediated autophagy is a promising therapeutic strategy for overcoming the cisplatin resistance of OS.Abbreviations: 3-MA: 3-methyladenine; BECN1: beclin 1; ChIP: chromatin immunoprecipitation; CHX: cycloheximide; COPS3/CSN3: COP9 signalosome subunit 3; CQ: chloroquine; DEGs: differentially expressed genes; FOXO3: forkhead box O3; GFP: green fluorescent protein; IC50: 50% inhibitory concentration; LAMP1: lysosomal associated membrane protein 1; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MTOR: mechanistic target of rapamycin kinase; mRFP: monomeric red fluorescent protein; OS: osteosarcoma; PBS: phosphate-buffered saline; qRT-PCR: quantitative real-time PCR; RAB7: RAB7, member RAS oncogene family; RPS6KB1/p70S6K1: ribosomal protein S6 kinase B1; SEM: standard error of the mean; shRNA: short hairpin RNA; siRNA: small interfering RNA; SKP2: S-phase kinase associated protein 2; TEM: transmission electron microscopy; UPS: ubiquitin-proteasome system.

Effects of remote ischaemic preconditioning on myocardial injury after major abdominal surgery in patients at high risk for cardiovascular adverse events in China (RIPC-MAS): protocol for a randomised, sham-controlled, observer-blinded trial.

INTRODUCTION: Myocardial injury after non-cardiac surgery (MINS) caused by an ischaemic mechanism is common and is associated with adverse short-term and long-term prognoses. However, MINS is a recent concept, and few studies have prospectively used it as a primary outcome. Remote ischaemic preconditioning (RIPC) is a non-invasive procedure that induces innate cardioprotection and may reduce MINS. METHODS AND ANALYSIS: This is a multicentre, randomised, sham-controlled, observer-blinded trial. Patients with a high clinical risk of cardiovascular events who are scheduled to undergo major abdominal surgery will be enrolled. A total of 766 participants will be randomised (1:1 ratio) to receive RIPC or control treatment before anaesthesia. RIPC will comprise four cycles of cuff inflation for 5 min to 200 mm Hg and deflation for 5 min. In the controls, an identical-looking cuff will be placed around the arm but will not be actually inflated. The primary outcome will be MINS, defined as at least one postoperative cardiac troponin (cTn) concentration above the 99th percentile upper reference limit of the cTn assay as a result of a presumed ischaemic mechanism. This trial will test the concentration of high-sensitivity cardiac troponin T (hs-cTnT). The secondary outcomes will be hs-cTnT levels reaching/above the prognostically important thresholds, peak hs-cTnT and total hs-cTnT release during the initial 3 days after surgery, length of hospital stay after surgery, length of stay in the intensive care unit, myocardial infarction, major adverse cardiovascular events, cardiac-related death, all-cause death within 30 days, 6 months, 1 year and 2 years after surgery, and postoperative complications and adverse events within 30 days after surgery. ETHICS AND DISSEMINATION: This study protocol (version 5.0 on 7 April 2023) was approved by the Ethics Committee of Sixth Affiliated Hospital of Sun Yat-sen University. The findings will be published in peer-reviewed journals. TRIAL REGISTRATION NUMBER: NCT05733208.

Absence of associations with prefrontal cortex and cerebellum may link to early language and social deficits in preschool children with ASD.

Introduction: Autism spectrum disorder (ASD) is a complex developmental disorder, characterized by language and social deficits that begin to appear in the first years of life. Research in preschool children with ASD has consistently reported increased global brain volume and abnormal cortical patterns, and the brain structure abnormalities have also been found to be clinically and behaviorally relevant. However, little is known regarding the associations between brain structure abnormalities and early language and social deficits in preschool children with ASD. Methods: In this study, we collected magnetic resonance imaging (MRI) data from a cohort of Chinese preschool children with and without ASD (24 ASD/20 non-ASD) aged 12-52 months, explored group differences in brain gray matter (GM) volume, and examined associations between regional GM volume and early language and social abilities in these two groups, separately. Results: We observed significantly greater global GM volume in children with ASD as compared to those without ASD, but there were no regional GM volume differences between these two groups. For children without ASD, GM volume in bilateral prefrontal cortex and cerebellum was significantly correlated with language scores; GM volume in bilateral prefrontal cortex was significantly correlated with social scores. No significant correlations were found in children with ASD. Discussion: Our data demonstrate correlations of regional GM volume with early language and social abilities in preschool children without ASD, and the absence of these associations appear to underlie language and social deficits in children with ASD. These findings provide novel evidence for the neuroanatomical basis associated with language and social abilities in preschool children with and without ASD, which promotes a better understanding of early deficits in language and social functions in ASD.

EnzyBase: a novel database for enzybiotic studies.

BACKGROUND: Enzybiotics are becoming increasingly recognized as potential alternative therapies for drug-resistant bacteria. Although only a few enzybiotics are currently well characterized, much information is still missing or is unavailable for researchers. The construction of an enzybiotics database would therefore increase efficiency and convenience in investigating these bioactive proteins and thus help reduce or delay the recent increase in antibiotic resistance. DESCRIPTION: In the present manuscript, we describe the development of a novel and original database called EnzyBase, which contains 1144 enzybiotics from 216 natural sources. To ensure data quality, we limited the source of information to authoritative public databases and published scientific literature. The interface of EnzyBase is easy to use and allows users to rapidly retrieve data according to their desired search criteria and blast the database for homologous sequences. We also describe examples of database-aided enzybiotics discovery and design. CONCLUSION: EnzyBase serves as a unique tool for enzybiotic studies. It has several potential applications, e.g. in silico enzybiotic combination as cocktails, and novel enzybiotic design, in response to continuously emerging drug-resistant pathogens. This database is a valuable platform for researchers who are interested in enzybiotic studies. EnzyBase is available online at http://biotechlab.fudan.edu.cn/database/EnzyBase/home.php.

Expression, purification and characterization of recombinant human serine proteinase inhibitor Kazal-type 6 (SPINK6) in Pichia pastoris.

Human serine proteinase inhibitor Kazal-type 6 (SPINK6) belongs to the medically important SPINK family. Malfunctions of SPINK members are linked to many diseases, including pancreatitis, skin barrier defects, and cancer. SPINK6 has been shown to selectively inhibit Kallikrein-related peptidases (KLKs) in human skin. As a SPINK protein, it contains a typical Kazal domain, which requires three intramolecular disulfide bonds for correct folding and activity. Preparation of functional protein is a prerequisite for studying this important human factor. Here, we report the successful generation of tagless SPINK6 using a yeast expression system. The recombinant protein was secreted and purified by cation exchange and size-exclusion chromatography. The protein identity was confirmed by MALDI-TOF MS and N-terminal sequencing. Pichia pastoris-derived recombinant human SPINK6 (rhSPINK6) showed higher inhibitory activity against Kallikrein-related peptidase 14 (KLK14) (K(i)=0.16 nM) than previously reported Escherichia coli-derived rhSPINK6 (K(i)=0.5 nM). This protein also exhibited moderate inhibition of bovine trypsin (K(i)=33 nM), while previous E. coli-derived rhSPINK6 did not. The results indicate that P. pastoris is a better system to generate active rhSPINK6, warranting further studies on this medically important SPINK family candidate.

PI3K inhibitor impairs tumor progression and enhances sensitivity to anlotinib in anlotinib-resistant osteosarcoma.

Despite recent improvements in the therapeutic management of osteosarcoma (OS), the ongoing challenges in overcoming resistance to tyrosine kinase inhibitors (TKIs) warrant new strategies to improve overall patient survival. In this study, we established four anlotinib-resistant OS cell lines and demonstrated that the mechanism of anlotinib resistance is due to the loss of PTEN and reactivation of the MAPK pathway. Reduced PTEN expression was also observed in tumor samples from patients with OS and lung metastasis. We investigated the effects of an orally active PI3K inhibitor, either alone or in combination with anlotinib, on the progression of resistant cells and a xenograft nude mouse model. Notably, PI3K inhibitor suppressed anlotinib-resistant OS cell proliferation, migration, invasion, and cytoskeleton formation, and induced apoptosis. Combined treatment with anlotinib augmented these effects by restoring PTEN expression and decreasing MAPK and PI3K/AKT/mTOR signaling. PI3K inhibitors could reverse anlotinib resistance in OS, limiting OS cell development in combination with anlotinib. Our findings rationalize further studies on the applications of PI3K inhibitors that can be clinically used in anlotinib-refractory OS management.

Controlled release of minocycline in hydroxyapatite/chitosan composite for periodontal bone defect repair.

The aim was to obtain bone repair materials with sustained release of minocycline and evaluate the effect in periodontal bone defect repair. Two complex material, hydroxyapatite/chitosan (HA/CS) and minocycline-hydroxyapatite/chitosan (Mino-HA/CS), were prepared by the co-precipitation method. The physical and chemical property, cytotoxicity, release of minocycline and the bacteriostasis examination of the materials were evaluated, they were applied to the rabbit model of mandible bone defect to evaluate their effects on the regeneration of periodontal bone defect. After minocycline was added to HA/CS, the setting time of the material was prolonged, the compressive strength was reduced and the pore size and porosity were increased significantly. The pH value did not change obviously and stayed in the neutral range. Mino-HA/CS could promote the growth of osteoblasts effectively compared with control medium. In vivo, Mino-HA/CS material showed better effect of promoting periodontal bone formation.

Development of a Nomogram for Predicting the Efficacy of Preoperative Chemotherapy in Osteosarcoma.

BACKGROUND: Due to the obvious heterogeneity of osteosarcoma, many patients are not sensitive to neoadjuvant chemotherapy. In this study, the clinical characteristics and auxiliary examinations of patients with osteosarcoma were used to predict the effect of preoperative chemotherapy, so as to guide the clinical adjustment of the treatment plan to improve the prognosis of patients. METHODS: In this study, 90 patients with pathologically confirmed osteosarcoma were included, and they were randomly divided into training cohort (n=45) and validation cohort (n=45). A prediction model of preoperative chemotherapy efficacy for osteosarcoma was established by multivariate logistic regression analysis, and a nomogram was used as the visualization of the model. The ROC curve and C-index were used to evaluate the accuracy of the nomogram. Decision curve analysis (DCA) was used to evaluate the net benefit of the nomogram in predicting the efficacy of neoadjuvant chemotherapy under different threshold probabilities. RESULTS: In the study, the age, gender, location, tumor volume, metastasis at the first visit, MSTS staging, C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), alkaline phosphatase (ALP), and lactate dehydrogenase (LDH) were used in the multivariate logistic regression analysis and the construction of the nomogram. The AUC and C-index of the training cohort were 0.793 (95% CI: 0.632, 0.954) and 0.881 (95% CI: 0.776, 0.986), respectively. The AUC and C-index in the validation cohort were 0.791 (95% CI: 0.644, 0.938) and 0.813 (95% CI: 0.679, 0.947), respectively, which were close to the training cohort. DCA showed that the model had good clinical application value. CONCLUSION: Based on the clinical characteristics of patients and auxiliary examinations, the nomogram can be good used to predict the efficacy of preoperative chemotherapy for osteosarcoma.