A novel method for multiple phenotype association studies based on genotype and phenotype network.

Joint analysis of multiple correlated phenotypes for genome-wide association studies (GWAS) can identify and interpret pleiotropic loci which are essential to understand pleiotropy in diseases and complex traits. Meanwhile, constructing a network based on associations between phenotypes and genotypes provides a new insight to analyze multiple phenotypes, which can explore whether phenotypes and genotypes might be related to each other at a higher level of cellular and organismal organization. In this paper, we first develop a bipartite signed network by linking phenotypes and genotypes into a Genotype and Phenotype Network (GPN). The GPN can be constructed by a mixture of quantitative and qualitative phenotypes and is applicable to binary phenotypes with extremely unbalanced case-control ratios in large-scale biobank datasets. We then apply a powerful community detection method to partition phenotypes into disjoint network modules based on GPN. Finally, we jointly test the association between multiple phenotypes in a network module and a single nucleotide polymorphism (SNP). Simulations and analyses of 72 complex traits in the UK Biobank show that multiple phenotype association tests based on network modules detected by GPN are much more powerful than those without considering network modules. The newly proposed GPN provides a new insight to investigate the genetic architecture among different types of phenotypes. Multiple phenotypes association studies based on GPN are improved by incorporating the genetic information into the phenotype clustering. Notably, it might broaden the understanding of genetic architecture that exists between diagnoses, genes, and pleiotropy.

Joint analysis of multiple phenotypes for extremely unbalanced case-control association studies.

In genome-wide association studies (GWAS) for thousands of phenotypes in biobanks, most binary phenotypes have substantially fewer cases than controls. Many widely used approaches for joint analysis of multiple phenotypes produce inflated type I error rates for such extremely unbalanced case-control phenotypes. In this research, we develop a method to jointly analyze multiple unbalanced case-control phenotypes to circumvent this issue. We first group multiple phenotypes into different clusters based on a hierarchical clustering method, then we merge phenotypes in each cluster into a single phenotype. In each cluster, we use the saddlepoint approximation to estimate the p value of an association test between the merged phenotype and a single nucleotide polymorphism (SNP) which eliminates the issue of inflated type I error rate of the test for extremely unbalanced case-control phenotypes. Finally, we use the Cauchy combination method to obtain an integrated p value for all clusters to test the association between multiple phenotypes and a SNP. We use extensive simulation studies to evaluate the performance of the proposed approach. The results show that the proposed approach can control type I error rate very well and is more powerful than other available methods. We also apply the proposed approach to phenotypes in category IX (diseases of the circulatory system) in the UK Biobank. We find that the proposed approach can identify more significant SNPs than the other viable methods we compared with.

Joint analysis of multiple phenotypes for extremely unbalanced case-control association studies using multi-layer network.

MOTIVATION: Genome-wide association studies is an essential tool for analyzing associations between phenotypes and single nucleotide polymorphisms (SNPs). Most of binary phenotypes in large biobanks are extremely unbalanced, which leads to inflated type I error rates for many widely used association tests for joint analysis of multiple phenotypes. In this article, we first propose a novel method to construct a Multi-Layer Network (MLN) using individuals with at least one case status among all phenotypes. Then, we introduce a computationally efficient community detection method to group phenotypes into disjoint clusters based on the MLN. Finally, we propose a novel approach, MLN with Omnibus (MLN-O), to jointly analyse the association between phenotypes and a SNP. MLN-O uses the score test to test the association of each merged phenotype in a cluster and a SNP, then uses the Omnibus test to obtain an overall test statistic to test the association between all phenotypes and a SNP. RESULTS: We conduct extensive simulation studies to reveal that the proposed approach can control type I error rates and is more powerful than some existing methods. Meanwhile, we apply the proposed method to a real data set in the UK Biobank. Using phenotypes in Chapter XIII (Diseases of the musculoskeletal system and connective tissue) in the UK Biobank, we find that MLN-O identifies more significant SNPs than other methods we compare with. AVAILABILITY AND IMPLEMENTATION: https://github.com/Hongjing-Xie/Multi-Layer-Network-with-Omnibus-MLN-O.

Efficient markerless genetic manipulation of Pasteurella multocida using lacZ and pheSm as selection markers.

Pasteurella multocida is a zoonotic conditional pathogen that infects multiple livestock species, causing substantial economic losses in the animal husbandry industry. An efficient markerless method for gene manipulation may facilitate the investigations of P. multocida gene function and pathogenesis of P. multocida. Herein, a temperature-sensitive shuttle vector was constructed using lacZ as a selection marker, and markerless glgB, opa, and hyaE mutants of P. multocida were subsequently constructed through blue-white colony screening. The screening efficiency of markerless deletion strains was improved by the lacZ system, and the method could be used for multiple gene deletions. However, the fur mutant was unavailable via this method. Therefore, we constructed a pheSm screening system based on mutated phenylalanine tRNA synthetase as a counterselection marker to achieve fur deletion mutant. The transformed strain was sensitive to 20 mM p-chloro-phenylalanine, demonstrating the feasibility of pheSm as a counter-selective marker. The pheSm system was used for markerless deletions of glgB, opa, and hyaE as well as fur that could not be screened by the lacZ system. A comparison of screening efficiencies of the system showed that the pheSm counterselection system was more efficient than the lacZ system and broadly applicable for mutant screening. The methods developed herein may provide valuable tools for genetic manipulation of P. multocida.IMPORTANCEPasteurella multocida is a highly contagious zoonotic pathogen. An understanding of its underlying pathogenic mechanisms is of considerable importance and requires efficient species-specific genetic tools. Herein, we propose a screening system for P. multocida mutants using lacZ or pheSm screening markers. We evaluated the efficiencies of both systems, which were used to achieve markerless deletion of multiple genes. The results of this study support the use of lacZ or pheSm as counterselection markers to improve counterselection efficiency in P. multocida. This study provides an effective genetic tool for investigations of the virulence gene functions and pathogenic mechanisms of P. multocida.

A new hip fracture risk index derived from FEA-computed proximal femur fracture loads and energies-to-failure.

Hip fracture risk assessment is an important but challenging task. Quantitative CT-based patient-specific finite element (FE) analysis (FEA) incorporates bone geometry and bone density in the proximal femur. We developed a global FEA-computed fracture risk index to increase the prediction accuracy of hip fracture incidence. PURPOSE: Quantitative CT-based patient-specific finite element (FE) analysis (FEA) incorporates bone geometry and bone density in the proximal femur to compute the force (fracture load) and energy necessary to break the proximal femur in a particular loading condition. The fracture loads and energies-to-failure are individually associated with incident hip fracture, and provide different structural information about the proximal femur. METHODS: We used principal component analysis (PCA) to develop a global FEA-computed fracture risk index that incorporates the FEA-computed yield and ultimate failure loads and energies-to-failure in four loading conditions of 110 hip fracture subjects and 235 age- and sex-matched control subjects from the AGES-Reykjavik study. Using a logistic regression model, we compared the prediction performance for hip fracture based on the stratified resampling. RESULTS: We referred the first principal component (PC1) of the FE parameters as the global FEA-computed fracture risk index, which was the significant predictor of hip fracture (p-value < 0.001). The area under the receiver operating characteristic curve (AUC) using PC1 (0.776) was higher than that using all FE parameters combined (0.737) in the males (p-value < 0.001). CONCLUSIONS: The global FEA-computed fracture risk index increased hip fracture risk prediction accuracy in males.

Saponin and Ribosome-Inactivating Protein Synergistically Trigger Lysosome-Dependent Apoptosis by Inhibiting Lysophagy: Potential to Become a New Antitumor Strategy.

The susceptibility of lysosomal membranes in tumor cells to cationic amphiphilic drugs (CADs) enables CADs to induce lysosomal membrane permeabilization (LMP) and trigger lysosome-dependent cell death (LDCD), suggesting a potential antitumor therapeutic approach. However, the existence of intrinsic lysosomal damage response mechanisms limits the display of the pharmacological activity of CADs. In this study, we report that low concentrations of QS-21, a saponin with cationic amphiphilicity extracted from Quillaja Saponaria tree, can induce LMP but has nontoxicity to tumor cells. QS-21 and MAP30, a type I ribosome-inactivating protein, synergistically induce apoptosis in tumor cells at low concentrations of both. Mechanistically, QS-21-induced LMP helps MAP30 escape from endosomes or lysosomes and subsequently enter the endoplasmic reticulum, where MAP30 downregulates the expression of autophagy-associated LC3 proteins, thereby inhibiting lysophagy. The inhibition of lysophagy results in the impaired clearance of damaged lysosomes, leading to the leakage of massive lysosomal contents such as cathepsins into the cytoplasm, ultimately triggering LDCD. In summary, our study showed that coadministration of QS-21 and MAP30 amplified the lysosomal disruption and can be a new synergistic LDCD-based antitumor therapy.

A high hydrophobic moment arginine-rich peptide screened by a machine learning algorithm enhanced ADC antitumor activity.

Cell-penetrating peptides (CPPs) with better biomolecule delivery properties will expand their clinical applications. Using the MLCPP2.0 machine algorithm, we screened multiple candidate sequences with potential cellular uptake ability from the nuclear localization signal/nuclear export signal database and verified them through cell-penetrating fluorescent tracing experiments. A peptide (NCR) derived from the Rev protein of the caprine arthritis-encephalitis virus exhibited efficient cell-penetrating activity, delivering over four times more EGFP than the classical CPP TAT, allowing it to accumulate in lysosomes. Structural and property analysis revealed that a high hydrophobic moment and an appropriate hydrophobic region contribute to the high delivery activity of NCR. Trastuzumab emtansine (T-DM1), a HER2-targeted antibody-drug conjugate, could improve its anti-tumor activity by enhancing targeted delivery efficiency and increasing lysosomal drug delivery. This study designed a new NCR vector to non-covalently bind T-DM1 by fusing domain Z, which can specifically bind to the Fc region of immunoglobulin G and effectively deliver T-DM1 to lysosomes. MTT results showed that the domain Z-NCR vector significantly enhanced the cytotoxicity of T-DM1 against HER2-positive tumor cells while maintaining drug specificity. Our results make a useful attempt to explore the potential application of CPP as a lysosome-targeted delivery tool.

Exploring biomarkers associated with severity of knee osteoarthritis in Southern China using widely targeted metabolomics.

BACKGROUND: Metabolomics is a tool to study the pathogenesis of diseases and their associated metabolites, but there are still insufficient metabolomic studies on severe knee osteoarthritis.To investigate the differences in serum metabolites between healthy populations and knee osteoarthritis (KOA) patients in Southern China using widely targeted metabolomics, and to explore biomarkers and their metabolic pathways that could be associated with the severity of KOA. METHODS: There were 10 healthy individuals in the control group and 32 patients with KOA. According to the Kellgren-Lawrence (KL) grading system, KOA was further divided into mild (n = 13, KL grade 1 and 2) and severe (n = 19, KL grade 3 and 4). Serum samples from all participants were collected and analyzed metabolomics based on ultra-performance liquid chromatography/electrospray ionization/tandem mass spectrometry. We screened for differential metabolites between patients and controls, and between mild and severe KOA. We explored the metabolic pathways involved in differential metabolism using the Kyoto Encyclopedia of Genes and Genomes database. RESULTS: Sixty-one metabolites were differentially expressed in the sera of the patient group compared with the control group (45 upregulated and 16 downregulated). Analysis of the mild and severe KOA groups showed a total of 12 differential metabolites. Receiver operating characteristic curve analysis showed N-alpha-acetyl-L-asparagine was a good predictor of advanced osteoarthritis(OA).Differential metabolites are enriched in multiple pathways such as arachidonic acid metabolism. CONCLUSION: Widely targeted metabolomics found that upregulation of the amino acid metabolite N-α-acetyl-L-asparagine was significantly associated with severe KOA and could be a biomarker for predicting severity of KOA. Arachidonic acid metabolism may play an important role in patients with severe KOA.

Raddeanin A synergistically enhances the anti-tumor effect of MAP30 in multiple ways, more than promoting endosomal escape.

Biomacromolecules such as proteins and nucleic acids are very attractive due to their high efficiency and specificity as cancer therapeutics. In fact, the endocytosed macromolecules are often trapped in the endosomes and cannot exhibit pharmacological effects well. Many strategies have been used to address this bottleneck, and one promising approach is to exploit the endosomal escape-promoting effect of triterpenoid saponins to aid in the release of biomacromolecules. Here, Raddeanin A (RA, an oleanane-type triterpenoid saponin) was proved to significantly promote endosomal escape as it recruited Galectin-9, an endosomal escape event reporter. As expected, RA effectively enhanced the anti-tumor effect of MAP30 (a type I ribosome-inactivating protein derived from Momordica charantia). However, based on the results of fluorescent colocalization, RA did not significantly promote MAP30 release from endosomes, suggesting that RA enhances MAP30 activity not only by promoting endosomal escape. Furthermore, it was found that the inhibitors of micropinocytosis and caveolae could almost completely inhibit the cytotoxicity of MAP30 combined with RA without affecting the cytotoxicity of MAP30 alone, indicating that RA may regulate the endocytic pathway of MAP30. Meanwhile, the effect of RA is related to the intra vesicular pH and cholesterol content on cell membrane, and is also cell-type dependent. Therefore, RA enhanced the anti-tumor effect of MAP30 in multiple ways, not just by promoting endosomal escape. Our findings will help to further decipher the possible mechanisms by which triterpenoid saponins enhance drug activity, and provide a new perspective for improving the activity of endocytosed drugs.

Tandem fusion of albumin-binding domains promoted soluble expression and stability of recombinant trichosanthin in vitro and in vivo.

Trichosanthin (TCS), as a type 1 ribosome-inactivating protein, has a very high cytoplasmic activity in vitro and can quickly kill cancer cells. However, it is easily filtered and cleared by the kidney, which results in the short half-life and severely limits its application. In this study, we constructed several recombinant proteins by fusing the albumin binding domain mutant ABD035(abbreviated as ABD) to the N- or C-terminus of TCS to endow the recombinant TCS fusion protein with a longer half-life property binding with endogenous human serum albumin (HSA) via ABD to effectively exert its anti-tumor activity in vivo. Pull down, Dynamic light scattering and ELISA assays all showed that TCS fused with two ABD sequences at the C-terminus of TCS, has stronger binding capacity to HSA in vitro than TCS with one ABD. In vivo studies in BALB/C mice were performed and the elimination half-life of TCS-ABD-ABD is about 15-fold longer compared to TCS and anti-tumor activity is about 30% higher than that of TCS alone in BALB/C mouse experiments. Moreover, we found that TCS with two ABDs in tandem have the highest soluble expression level, more than 5 times higher than that of TCS, and the yield of purified protein of TCS-ABD-ABD was as high as 68.9 mg/L culture solution, which was about 7-fold higher than that of TCS. Furthermore, MTT assay showed that the anti-tumor activity of TCS-ABD-ABD was significantly higher than TCS fused with only one ABD sequence, indicating that the repeated ABD sequences facilitated the biological activity of TCS. In this paper, the fusion of the albumin-binding domain in tandem with TCS can effectively improve its stability in vivo and also significantly increase its soluble expression, expanding the application of the albumin-binding domain in the high soluble expression and stability of protein drugs.

Primary hepatic mucosa-associated lymphoid tissue lymphoma with HP and previous HBV infection: A case report and literature review.

Primary hepatic mucosa-associated lymphoid tissue (MALT) lymphoma is a relatively rare disease with low malignancy, and its aetiology is unclear. A 65-year-old man presented with abdominal pain. Hepatitis virus examination revealed a previous hepatitis B virus (HBV) infection, and a carbon-13 urea breath test result was positive for the patient. Abdominal contrast-enhanced computed tomography revealed a patch of abnormal density in the right posterior lobe of the liver. The patient underwent VI segment hepatectomy and was pathologically diagnosed with hepatic MALT lymphoma. After the operation, he received quadruple anti-Helicobacter pylori (HP) therapy and refused other treatments. He has been followed up by telephone for 20 months after discharge and is now in a stable condition. In this study, we counted 105 cases of hepatic MALT lymphomas reported in English or Chinese since 1995 and summarised the clinical characteristics and concomitant diseases in this condition. Based on the literature review, we speculated that chronic infectious diseases, especially viral infections (including hepatitis C virus (HCV) and HBV) and HP infection, are associated with the pathogenesis of primary hepatic MALT lymphoma. In addition, autoimmune diseases might also play a role in this condition.

Excellent outcomes with Oxford Uni-compartmental knee arthroplasty in anteromedial osteoarthritis patients (≤60 years) at mid-term follow-up.

BACKGROUND: The use of Oxford uni-compartmental knee arthroplasty (UKA) has rapidly increased worldwide,however,the relevance of younger patients for postoperative function after Oxford UKA remains unclear. The main purpose of our study is to clarify the effectivemess of Oxford UKA in the younger Chinese patients with anteromedial osteoarthritis (AMOA). METHODS: We retrospectively enrolled 252 consecutive patients who underwent Oxford UKA for AMOA with a minimum follow-up of 5 years between March 2013 and December 2016. The patients were divided into the younger (≤60 years) and elderly (> 60 years) age groups. The demographic data and surgery variables were recorded and compared. Patient satisfaction grade, range of motion (ROM), Oxford knee score (OKS), Hospital for Special Surgery (HSS) score, Western Ontario and McMaster (WOMAC) Universities Osteoarthritis Index score and postoperative complications were recorded. The 5-year survival of the implants were also compared with TKA revision as the endpoint. RESULTS: A total of 252 consecutive patients were recruited, including 96 aged 60 years or less and 156 aged over 60 years. The mean follow-up duration in the younger and elderly groups were 73.6 months (SD,standard deviation, 4.1) and 74.7 months (SD 6.2) respectively. Patient satisfaction rate was high in both groups (P = 0.805). Furthermore, no significant differences were observed in postoperative ROM(P = 0.299), OKS(P = 0.117), HSS(P = 0.357) and WOMAC scores(P = 0.151) between the younger and elderly groups (P>0.05). However, the incidence of joint stiffness (P = 0.033) and delayed wound dehiscence (P = 0.026) were significantly different between both groups. Five-year implant survival without revision were also similar in both groups (96.9% vs 97.4%, P = 0.871), and that for the entire cohort was 97.2% (95% CI 95.4-99.6). CONCLUSION: Oxford UKA for AMOA demonstrated favorable results in younger patients aged ≤60 years at a minimum 5-year follow-up in terms of patient satisfaction, functional outcomes, implant survival and postoperative complications. Therefore, younger patients might not be considered as an absolute contraindication to Oxford UKA.

Magnitude and Spatial Distribution Control of the Supercurrent in Bi2O2Se-Based Josephson Junction.

Many proposals for exploring topological quantum computation are based on superconducting quantum devices constructed on materials with strong spin-orbit coupling (SOC). For these devices, full control of both the magnitude and the spatial distribution of the supercurrent is highly demanded, but has been elusive up to now. We constructed a proximity-type Josephson junction on nanoplates of Bi2O2Se, a new emerging semiconductor with strong SOC. Through electrical gating, we show that the supercurrent can be fully turned ON and OFF, and its real-space pathways can be configured either through the bulk or along the edges. Our work demonstrates Bi2O2Se as a promising platform for constructing multifunctional hybrid superconducting devices as well as for searching for topological superconductivity.

Wound closure position in total knee arthroplasty: flexion versus extension-a meta-analysis of randomized controlled trials.

BACKGROUND: Total knee arthroplasty (TKA) is one of the most commonly used procedures in orthopedics. However, whether different would closure positions affect the clinical outcomes after TKA remains controversial. We conducted a meta-analysis of randomized controlled trials (RCT) to assess the effect of wound closure position on clinical TKA outcomes. METHODS: Embase, PubMed, and the Cochrane Library databases were systematically searched. A systematic review and meta-analysis of all RCTs were performed to prove the role of different wound closure positions on TKA. RESULTS: Five RCTs containing 389 patients were included. Surgical closure of 90° flexion in TKA was associated with higher post-operative range-of-motion (ROM) at post-operative 4 weeks, lower VAS post-operative pain scores 4 weeks and 3 months, better peak torque difference of flexor muscle strength at 60 and 180°/s angular velocities between the flexion and the extension groups, and better total work difference of flexor muscle strength at 180°/s angular velocity. The American Knee Society Score did not show any significant difference between two closure techniques. No complications were described in the literature review. CONCLUSIONS: Wound closure in 90° flexion during TKA may provide better postoperative ROM, higher pain relief, preferable muscle strength improvement in short-term follow-up, and no increase in the risks of wound complications. LEVEL OF EVIDENCE: Level II.

Effective Therapeutic Drug Delivery by GALA3, an Endosomal Escape Peptide with Reduced Hydrophobicity.

Endosomal escape is a rate-limiting step in the cytosolic delivery of therapeutic drugs. Overcoming this barrier is crucial to achieve an effective biological based therapy. In this work, we evaluated the ability of a synthetic biomimetic peptide derived from the GALA to facilitate endosomal escape of protein drugs. Our results showed that the cytoplasmic distribution of GALA fusion proteins changed according to the hydrophobicity of GALA. One of the synthetic peptides, GALA3, significantly enhanced the endosomal escape efficiency of protein drugs. The cytosolic delivery capacity of GALA3 was significantly higher than that of several previously reported endosomal escape peptides, including hemagglutinin 2 (HA2). Moreover, when GALA3 was fused to BLF1-HBP, a ribosome-inactivating protein with cell-penetrating peptide HBP, the cytotoxicity of the fusion protein was significantly increased in various cell lines, including H460, HeLa, A549, and SMCC-7721. The growth inhibition effect of GALA3-BLF1-HBP was at least 20 times greater than that of BLF1-HBP alone in different tumor cell lines. GALA3 effectively promoted the endosomal escape of BLF1-HBP in a pH-dependent manner and greatly enhanced the apoptotic activity of BLF1-HBP. Taken together, our data show that by adjusting the hydrophobicity of GALA we obtained a more effective endosomal escape peptide. Therefore, GALA3-fusions can improve the efficiency of therapeutic protein drugs.

Analysis of Triterpenoid Saponins Reveals Insights into Structural Features Associated with Potent Protein Drug Enhancement Effects.

Plant-based saponins are amphipathic glycosides composed of a hydrophobic aglycone backbone covalently bound to one or more hydrophilic sugar moieties. Recently, the endosomal escape activity of triterpenoid saponins has been investigated as a potentially powerful tool for improved cytosolic penetration of protein drugs internalized by endocytic uptake, thereby greatly enhancing their pharmacological effects. However, only a few saponins have been studied, and the paucity in understanding the structure-activity relationship of saponins imposes significant limitations on their applications. To address this knowledge gap, 12 triterpenoid saponins with diverse structural side chains were screened for their utility as endosomolytic agents. These compounds were used in combination with a toxin (MAP30-HBP) comprising a type I ribosome-inactivating protein fused to a cell-penetrating peptide. Suitability of saponins as endosomolytic agents was assessed on the basis of cytotoxicity, endosomal escape promotion, and synergistic effects on toxins. Five saponins showed strong endosomal escape activity, enhancing MAP30-HBP cytotoxicity by more than 106 to 109 folds. These saponins also enhanced the apoptotic effect of MAP30-HBP in a pH-dependent manner. Additionally, growth inhibition of MAP30-HBP-treated SMMC-7721 cells was greater than that of similarly treated HeLa cells, suggesting that saponin-mediated endosomolytic effect is likely to be cell-specific. Furthermore, the structural features and hydrophobicity of the sugar side chains were analyzed to draw correlations with endosomal escape activity and derive predictive rules, thus providing new insights into structure-activity relationships of saponins. This study revealed new saponins that can potentially be exploited as efficient cytosolic delivery reagents for improved therapeutic drug effects.

Screening and characterization of a novel high-efficiency tumor-homing cell-penetrating peptide from the buffalo cathelicidin family.

Targeted delivery of antitumor drugs is especially important for tumor therapy. Cell-penetrating peptides (CPPs) have been shown to be very effective drug carriers for tumor therapy. However, most CPPs lack tumor cell specificity. Here, we identified a highly efficient CPP, CAT, from the newly identified buffalo-derived cathelicidin family, which exhibits a preferential binding capacity for multiple tumor cell lines and delivers carried drug molecules into cells. CAT showed an approximately threefold to sixfold higher translocation efficiency than some reported cell-penetrating antimicrobial peptides, including the well-known classical CPP TAT. Moreover, the delivery efficiency of CAT was greater in a variety of tested tumor cells than in normal cells, especially for the human hepatoma cell line SMMC-7721, for which delivery was 7 times more efficient than the normal human embryonic lung cell line MRC-5, according to fluorescent labeling experiment results. CAT was conjugated to the Momordica charantia-derived type-I ribosome-inactivating protein MAP 30, and the cytotoxicity of the MAP 30-CAT fusion protein in the tumor cell line SMMC-7721 was significantly enhanced compared with that of the unconjugated MAP 30. The IC50 value of MAP 30-CAT was approximately 83 times lower than the IC50 value of the original MAP 30. Interestingly, the IC50 value of MAP 30 alone for MRC-5 was approximately twofold higher than the value for SMMC-7721, showing a small difference. However, when MAP 30 was conjugated to CAT, the difference in IC50 values between the two cell lines was significantly increased by 38-fold. The results of the flow cytometric detection of apoptosis revealed that the increase in cytotoxicity after CAT conjugation was mainly caused by the increased induction of apoptosis by the fusion protein. These results suggest that CAT, as a novel tumor-homing CPP, has great potential in drug delivery applications in vivo and will be beneficial to the development of tumor therapeutics.

Effectively enhancing cytotoxic and apoptotic effects of alpha-momorcharin by integrating a heparin-binding peptide.

Alpha-momorcharin (α-MMC), a type I ribosome-inactivating protein, has attracted a great deal of attention because of its antitumor activity. However, the cytotoxicity of α-MMC is limited due to insufficient cellular internalization in cancer cells. To enhance the cytotoxicity of α-MMC, a heparin-binding domain derived from heparin-binding epidermal growth factor (named heparin-binding peptide [HBP]) was used as a cell-penetrating peptide and fused to the C-terminus of α-MMC. This novel α-MMC-HBP fusion protein was expressed and purified with a Ni2+ -resin. The N-glycosidase activity and DNase activity assay indicated that the introduction of HBP did not interfere with the intrinsic bioactivities of α-MMC. HBP was able to efficiently carry α-MMC into the tested cancer cells and significantly enhance the cytotoxic effects of α-MMC in a dose-dependent manner. This enhanced cytotoxic ability occurred due to the higher level of cell apoptosis induced by α-MMC-HBP, which was demonstrated in western blot analysis in which α-MMC-HBP triggered caspase 8, caspase 9, casapase 3, and PARP more intensely than α-MMC alone. α-MMC-HBP led to an upregulation of cleaved PARP and an increase in the Bax/Bcl-2 ratio. Our study provided a new practical way to greatly improve the antitumor activity of α-MMC, which could significantly expand the pharmaceutical applications of α-MMC.

A novel trichosanthin fusion protein with increased cytotoxicity to tumor cells.

OBJECTIVE: To evaluate the anti-tumor effects of trichosanthin after fusion with a cell penetrating peptide, heparin-binding peptide (HBP), derived from human heparin-binding EGF-like growth factor (HB-EGF). RESULTS: The fusion protein of trichosanthin-HBP was expressed in Escherichia coli BL21 and purified by Ni-NTA affinity chromatography. The HBP domain had no influence on the topological inactivation activity and N-glycosidase activity of trichosanthin. Trichosanthin-HBP significantly inhibited the growth of tested cancer cells which are impervious to trichosanthin. Tumor cell apoptosis and both the mitochondrial- and death receptor-mediated apoptotic signaling pathways induced by trichosanthin-HBP were more significant than those induced by trichosanthin in HeLa cells. CONCLUSION: HBP is an efficient intracellular delivery vehicle for trichosanthin and makes trichosanthin-HBP become a promising agent for cancer therapy.

The heparin-binding domain of HB-EGF as an efficient cell-penetrating peptide for drug delivery.

Cell-penetrating peptides (CPPs) have been shown to be potential drug carriers for cancer therapy. The inherently low immunogenicity and cytotoxicity of human-derived CPPs make them more suitable for intracellular drug delivery compared to other delivery vehicles. In this work, the protein transduction ability of a novel CPP (termed HBP) derived from the heparin-binding domain of HB-EGF was evaluated. Our data shows, for the first time, that HBP possesses similar properties to typical CPPs and is a potent drug delivery vector for improving the antitumor activity of impermeable MAP30. The intrinsic bioactivities of recombinant MAP30-HBP were well preserved compared to those of free MAP30. Furthermore, HBP conjugated to the C-terminus of MAP30 promoted the cellular uptake of recombinant MAP30-HBP. Moreover, the fusion of HBP to MAP30 gave rise to significantly enhanced cytotoxic effects in all of the tumor cell lines tested. In HeLa cells, this cytotoxicity was mainly caused by the induction of cell apoptosis. Further investigation revealed that HBP enhanced MAP30-induced apoptosis through the activation of the mitochondrial- and death receptor-mediated signaling pathways. In addition, the MAP30-HBP fusion protein caused more HeLa cells to become arrested in S phase compared to MAP30 alone. These results highlight the MAP30-HBP fusion protein as a promising drug candidate for cancer therapy and demonstrate HBP, a novel CPP derived from human HB-EGF, as a new potential vector for antitumor drug delivery. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.