Comparative analysis of diverse toxins from a new pharmaceutical centipede,Scolopendra mojiangica

As the oldest venomous animals,centipedes use their venom as a weapon to attack prey and for protection.Centipede venom,which contains many bioactive and pharmacologically active compounds,has been used for centuries in Chinese medicine,as shown by ancient records.Based on comparative analysis,we revealed the diversity of and differences in centipede toxin-like molecules between Scolopendra mojiangica,a substitute pharmaceutical material used in China,and S.subspinipes mutilans.More than 6000 peptides isolated from the venom were identified by electrospray ionization-tandem mass spectrometry(ESI-MS/MS)and inferred from the transcriptome.As a result,in the proteome of S.mojiangica,246 unique proteins were identified:one in five were toxin-like proteins or putative toxins with unknown function,accounting for a lower percentage of total proteins than that in S.mutilans.Transcriptome mining identified approximately 10 times more toxin-like proteins,which can characterize the precursor structures of mature toxinlike peptides.However,the constitution and quantity of the toxin transcripts in these two centipedes were similar.In toxicity assays,the crude venom showed strong insecticidal and hemolytic activity.These findings highlight the extensive diversity of toxin-like proteins in S.mojiangica and provide a new foundation for the medical-pharmaceutical use of centipede toxin-like proteins.

Preliminary optimization of a Chinese herbal medicine formula based on the neuroprotective effects in a rat model of rotenone-induced Parkinson's disease

Objective： The main objective of this study was to preliminarily determine the optimum formulation of a Chinese herbal formula that may have neuroprotective effects against rotenone-induced Parkinson＇s disease （PD）. Methods： Seven recipes were made from Dihuang （DH, Rehmanniaglutinosa Libosch）, Roucongrong （RCR, Cistanche deserticola Y.C.Ma）, Niuxi （NX, Achyranthes bidentata BI.） and Shanzhuyu （SZY, Comus officinalis Sieb. et Zucc） in different proportions, according to the principles of uniform design （4 factors 7 levels）. Tyrosine hydroxylase （TH）-positive neurons in substantia nigra pars compacta （SNpc） were detected by immunohistochemistry and rotenone-exposure days necessary to induce PD symptoms were recorded. To probe one likely mechanism of the formulas, echinacoside （ECH） concentrations of all seven recipes were determined by high-performance liquid chromatography and related to number of TH-positive neu- rons. Results： The data showed that recipe 4 （DH：RCR：SZY：NX = 1：1：1：1 ） and recipe 7 （DH：RCR：SZY：NX = 7：5：3： 1） partially reversed rotenone-induced death of TH-positive neurons in the SNpc and significantly increased rotenone-e^posed days compared with model group. Pharmacologically, there was not a strong correlation between ECH concentration and TH-positive neurons. Conclusion： The investigated formulations of Chinese herbs had neuroprotective effects against PD mod- els, and the neuroprotective effects were weakly related to the proportion of key herbs. However the neu- roprotective effects of the formula may not result from a single active constituent.

Discovery of a first-in-class ANXA3 degrader for the treatment of triple-negative breast cancer

Triple-negative breast cancer(TNBC)is a nasty disease with extremely high malignancy and poor prognosis.Annexin A3(ANXA3)is a potential prognosis biomarker,displaying an excellent correlation of ANXA3 overexpression with patients'poor prognosis.Silencing the expression of ANXA3effectively inhibits the proliferation and metastasis of TNBC,suggesting that ANXA3 can be a promising therapeutic target to treat TNBC.Herein,we report a first-in-class ANXA3-targeted small molecule(R)-SL18,which demonstrated excellent anti-proliferative and anti-invasive activities to TNBC cells.(R)-SL18 directly bound to ANXA3 and increased its ubiquitination,thereby inducing ANXA3 degradation with moderate family selectivity.Importantly,(R)-SL18 showed a safe and effective therapeutic potency in a high ANXA3-expressing TNBC patient-derived xenograft model.Furthermore,(R)-SL18 could reduce theβ-catenin level,and accordingly inhibit the Wnt/β-catenin signaling pathway in TNBC cells.Collectively,our data suggested that targeting degradation of ANXA3 by(R)-SL18 possesses the potential to treat TNBC.

Strategic design of lysine-targeted irreversible covalent NDM-1 inhibitors

New Delhi metallo-β-lactamase 1(NDM-1) can hydrolyze most β-lactam antibiotics, which is the major factor for drug resistance of Gram-negative bacteria. The binding of most reversible inhibitors to NDM-1 is relatively weak due to the shallow active pocket of NDM-1. Alternatively, irreversible covalent inhibitors can prevent their dissociation from the target, leading to permanent inactivation of the protein.Herein, we report a series of irreversible covalent inhibitors of NDM-1 targeting the conserved Lys211 in the active pocket. Several methods, including mass spectrometry, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, fluorescent labeling, and coumarin probe were used to demonstrate that pentafluorophenyl ester formed a covalent bond with Lys211. Moreover, our target inhibitor, in combination with meropenem, achieved an antibacterial effect on drug-resistant bacteria, along with an excellent safety profile. Our new strategy in designing lysine-targeted irreversible covalent NDM-1 inhibitors provides a potential option for the clinical treatment of Gram-negative bacteria.

Acupuncture Regulates the Balance of CD4^+ T Cell Subtypes in Experimental Asthma Mice

Objective: To evaluate the involvement of different CD4+ T cell subtypes in the anti-asthmatic effects of acupuncture in asthmatic mice. Methods: BALB/c mice were challenged by ovalbumin (OVA) for the establishment of experimental asthma model. Mice were divided into 4 groups by a random number table including the normal control, asthma model, acupuncture and sham acupuncture groups (14 per group). Acupoints Dazhui (GV 14), bilateral Fengmen (BL 12) and Feishu (BL 13) were selected for manual acupuncture treatment every other day for 4 weeks and Huantiao (GB 30) was selected for sham acupuncture. Airway hyperresponsiveness was examined by Buxco Pulmonary System. Pulmonary histopathology analysis was performed for inflammatory cell infiltration and mucus hypersecretion by haematoxylin eosin staining and periodic acid-Schiff staining. Inflammatory mediators assays of serum were investigated by enzyme-linked immunosorbent assay and Bio-Plex. CD4+ T cell subpopulations including the expression levels of important factors in T lymphocyte polarization in lung tissue were examined by flow cytometric and Western blot analyses. Related pathways were detected by Western blot assay. Results: Compared with the OVA-induced asthma model group, acupuncture could attenuate airway hyperresponsiveness, inhibit inflammatory cell infiltration and mucus hypersecretion (P<0.05 or P<0.01). Furthermore, acupuncture increased the expressions of T-bet and Foxp3+, the cell numbers of CD4+ interferon gamma (IFN-γ)+ and CD4+ Foxp3+ in lung tissue and the level of Treg type cytokine interleukin (IL)-10 in serum (P<0.05 or P<0.01). Meanwhile, acupuncture reduced the RAR-related orphan receptor gamma t (RORγ t) level, the cell numbers of CD4+ IL-17A+ as well as the levels of IL-5, IL-13 and IL-17A in serum (P<0.05 or P<0.01). In addition, both acupuncture and sham acupuncture could inhibit the phosphorylation of p38 and p44/42 (P<0.01). Conclusion: Acupuncture could alleviate allergic airway inflammation by strengthening the activities of Th1 and Treg, thus regulating the balance of CD4+ T cell subtypes in experimental asthmatic mice.

Enhanced glioma-targeting and stability of ~LGICP peptide coupled with stabilized peptide ~DA7R

Malignant glioma is usually accompanied by vigorous angiogenesis to provide essential nutrients. An effective glioma targeting moiety should include excellent tumor-cell homing ability as well as good neovasculature-targeting efficiency, and should be highly resistant to enzyme degradation in the bloodstream. The phage display-selected heptapeptide, the glioma-initiating cell peptide(GICP), was previously reported as a ligand for the VAV3 protein(a Rho-GTPase guanine nucleotide exchange factor),which is mainly expressed on glioma cells; the stabilized heptapeptide ~DA7R has been shown to be the ligand of both vascular endothelial growth factor receptor 2(VEGFR2) and neuropilin-1(NRP-1), and has demonstrated good neovasculature-targeting ability. By linking ~DA7R and GICP, a multi-receptor targeting molecule was obtained. The stability of these three peptides was evaluated and their targeting efficiency on tumor-related cells and models was compared. The ability of these peptides to cross the blood–tumor barrier(BTB) was also determined. The results indicate that the coupled Y-shaped peptide ~DA7R–GICP exhibited improved tumor and neovasculature targeting ability and had higher efficiency in crossing the BTB than either individual peptide.

Surface modification of PGP for a neutrophil–nanoparticle co-vehicle to enhance the anti-depressant effect of baicalein

Exploiting cells as vehicles combined with nanoparticles combined with therapy has attracted increasing attention in the world recently. Red blood cells, leukocytes and stem cells have been used for tumor immunotherapy, tissue regeneration and inflammatory disorders, and it is known that neutrophils can accumulate in brain lesions in many brain diseases including depression. N-Acetyl Pro–Gly–Pro(PGP) peptide shows high specific binding affinity to neutrophils through the CXCR2 receptor. In this study, PGP was used to modify baicalein-loaded solid lipid nanoparticles(PGP-SLNs) to facilitate binding to neutrophils in vivo. Brain-targeted delivery to the basolateral amygdala(BLA) was demonstrated by enhanced concentration of baicalein in the BLA. An enhanced anti-depressant effect was observed in vitro and in vivo. The mechanism involved inhibition of apoptosis and a decrease in lactate dehydrogenase release. Behavioral evaluation carried out with rats demonstrated that anti-depression outcomes were achieved. The results indicate that PGP-SLNs decrease immobility time, increase swimming time and climbing time and attenuate locomotion in olfactory-bulbectomized(OB) rats. In conclusion, PGP modification is a strategy for targeting the brain with a cell–nanoparticle delivery system for depression therapy.

King cobra peptide OH-CATH30 as a potential candidate drug through clinic drug-resistant isolates

Cationic antimicrobial peptides （AMPs） are considered as important candidate therapeutic agents, which exert potent microbicidal properties against bacteria, fungi and some viruses. Based on our previous findings king cobra cathelicidin （OH-CATH） is a 34-amino acid peptide that exerts strong antibacterial and weak hemolytic activity. The aim of this research is to evaluate the efficacy of both OH-CATH30 and its analog D-OH-CATH30 against clinical isolates comparing with routinely utilized antibiotics in vitro. In this study, 584 clinical isolates were tested （spanning 2013-2016） and the efficacy of the candidate peptides and antibiotics were determined by a broth microdilution method according to the CLSI guidelines. Among the 584 clinical isolates, 85% were susceptible to OH-CATH30 and its analogs. Both L- and D-OH-CATH30 showed higher efficacy against （toward） Gram-positive bacteria and stronger antibacterial activity against nearly all Gram-negative bacteria tested compare with antibiotics. The highest bactericidal activity was detected against Acinetobacter spp., including multi-drug-resistant Acinetobacter baumannfi （MRAB） and methicillin-resistant Staphylococcus aureus （MRSA）. The overall efficacy of OH-CATH30 and its analogs was higher than that of the 9 routinely used antibiotics. OH-CATH30 is a promising candidate drug for the treatment of a wide variety of bacterial infections which are resistant to many routinely used antimicrobial agents.

Inhibition of post-trabeculectomy fibrosis via topically instilled antisense oligonucleotide complexes co-loaded with fluorouracil

Trabeculectomy is the mainstay of surgical glaucoma treatment,while the success rate was unsatistying due to postoperative scarring of the filtering blebs.Clinical countermeasures for scar prevention are intraoperative intervention or repeated subconjunctival injections.Herein,we designed a codelivery system capable of transporting fluorouracil and anti-TGF-β2 oligonucleotide to synergistically inhibit fibroblast proliferation via topical instillation.This co-delivery system was built based on a cationic dendrimer core(PAMAM),which encapsulated fluorouracil within hydrophobic cavity and condensed oligonucleotide with surface amino groups,and was further modified with hyaluronic acid and cell-penetrating peptide penetratin.The co-delivery system was self-assembled into nanoscale complexes with increased cellular uptake and enabled efficient inhibition on proliferation of fibroblast cells.In vivo studies on rabbit trabeculectomy models further confirmed the anti-fibrosis efficiency of the complexes,which prolonged survival time of filtering blebs and maintained their height and extent during wound healing process,exhibiting an equivalent effect on scar prevention compared to intraoperative infiltration with fluorouracil.Qualitative observation by immunohistochemistry staining and quantitative analysis by Western blotting both suggested that TGF-β2 expression was inhibited by the co-delivery complexes.Our study provided a potential approach promising to guarantee success rate of trabeculectomy and prolong survival time of filtering blebs.

Characteristics of Lymphocyte Nuclear Factor-κB Signal Transduction Kinase Expression in Aging Process and Regulatory Effect of Epimedium Flavonoids

Objective：To study the characteristics of lymphocyte nuclear factor kappa B（NF-κB） signal transduction kinase-related molecular mRNA differential expressions at various month age segments in aging process and the intervening effect of Epimedium flavonoids（EF） on it.Methods：Sixty SD rats were divided into six groups,according to animals＇ age,i.e.,the 3 days（d） group,the 4 months（m） group,the 10 m group,the 18 m group,the 27 m group,and the 27 m＋EF group.RNA was extracted from separated splenic lymphocytes. Adopting NF-κB signal path functional genome oligonucleotide gene-chip（128 related genes）,the integral characteristics and differences of NF-κB signal transduction kinase-related mRNA expressions were determined, and the intervening effect of EF was examined.Results：The mean level of the NF-κB signal transduction kinase-related mRNA expressions in rats＇ splenic lymphocytes lowered with aging;the highest expression was presented at 3 d after birth,and then,it lowered gradually,with the lowest level at 18 m or 27 m.After EF intervention,the expression level was raised to the 10-18 m level in the aged rats.Conclusion：The changing rules of lymphocyte NF-κB-signal-transduction-kinase-related mRNA expressions in various stages of aging are helpful for selecting the well time for preventing and intervening aging,and will also give a hint to the molecular index for assessment of senility retarding researches.

Kidney-Qi deficiency diagnosed by Deng's diagnosis standard was not correlated with aging based on clinical observation of 90 participants

OBJECTIVE: To verify the Traditional Chinese Medicine(TCM) theory that kidney-Qi deficiency(KQD)is considered to be the main cause of aging using cross-sectional study.METHODS: Demographic and lifestyle characteristics of 90 healthy participants were collected with a self-administered questionnaire. KQD syndrome was diagnosed according to Deng's diagnosis standard. Creatinine-adjusted urinary 8-hydroxy-2'-deoxyguanosine(8-OH-dG) and 8-isomeric-prostaglandin2α(8-iso-PGF2α), salivary advanced oxidation protein products(AOPPs), malondialdehyde(MDA) and dehydroepiandrosterone-sulfate(DHEA-S) were selected as aging markers and measured using enzyme-linked immunosorbent assay.RESULTS: No significant differences were observed in participant characteristics between the KQD group and non-KQD(NKQD) group(P > 0.05). Levels of 8-OH-dG, 8-iso-PGF2α, AOPPs, and MDA increased with age, except for a slight decrease in8-OH-dG in the older group. The increase in8-iso-PGF2α was significant(P < 0.05). DHEA-S significantly decreased with increasing age(P < 0.01).8-OH-dG levels were higher in the KQD group compared with the NKQD group. Levels of urinary8-iso-PGF2α, salivary AOPPs, and MDA in the KQD group were lower than in the NKQD group. Salivary DHEA-S was higher in the KQD group compared with the NKQD group. However, differences between KQD group and NKQD group were not significant.CONCLUSION: The current results suggested that KQD syndrome, as diagnosed by Deng's standard,does not underlie the aging phenotype.

All-stage targeted therapy for the brain metastasis from triple-negative breast cancer

Brain metastasis is a common and serious complication of breast cancer,which is commonly associated with poor survival and prognosis.In particular,the treatment of brain metastasis from triplenegative breast cancer(BM-TNBC)has to face the distinct therapeutic challenges from tumor heterogeneity,circulating tumor cells(CTCs),blood-brain barrier(BBB)and blood-tumor barrier(BTB),which is in unmet clinical needs.Herein,combining with the advantages of synthetic and natural targeting moieties,we develop a“Y-shaped”peptide pVAP-decorated platelet-hybrid liposome drug delivery system to address the all-stage targeted drug delivery for the whole progression of BM-TNBC.Inherited from the activated platelet,the hybrid liposomes still retain the native affinity toward CTCs.Further,the peptide-mediated targeting to breast cancer cells and transport across BBB/BTB are demonstrated in vitro and in vivo.The resultant delivery platform significantly improves the drug accumulation both in orthotopic breast tumors and brain metastatic lesions,and eventually exhibits an outperformance in the inhibition of BM-TNBC compared with the free drug.Overall,this work provides a promising prospect for the comprehensive treatment of BMTNBC,which could be generalized to other cell types or used in imaging platforms in the future.

Liposomes and lipid disks traverse the BBB and BBTB as intact forms as revealed by two-step F?rster resonance energy transfer imaging

The blood–brain barrier(BBB) and the blood–brain tumor barrier(BBTB) prevent drug and nano-drug delivery systems from entering the brain. However, ligand-mediated nano-drug delivery systems have significantly enhanced the therapeutic treatment of glioma. In this study we investigated the mechanism especially the integrity of liposomes and lipid disks while traversing the BBB and BBTB both in vitro and in vivo. Fluorophores(DiO, DiI and DiD) were loaded into liposomes and lipid disks to form F?rster resonance energy transfer(FRET) nano-drug delivery systems. Using brain capillary endothelial cells as a BBB model, we show that liposomes and disks are present in the cytoplasm as their intact forms and traverse the BBB with a ratio of 0.68‰ and 1.67‰, respectively. Using human umbilical vein endothelial cells as BBTB model, liposomes and disks remained intact and traversed the BBTB with a ratio of 2.31‰and 8.32‰ at 3 h. Ex vivo imaging and immunohistochemical results revealed that liposomes and disks could traverse the BBB and BBTB in vivo as intact forms. In conclusion, these observations explain in part the mechanism by which nano-drug delivery systems increase the therapeutic treatment of glioma.

Engineered platelets-based drug delivery platform for targeted thrombolysis

Thrombolytic agents have thus far yielded limited therapeutic benefits in the treatment of thrombotic disease due to their short half-life,low targeting ability,and association with serious adverse reactions,such as bleeding complications.Inspired by the natural roles of platelets during thrombus formation,we fabricated a platelet-based delivery system(NO@uPA/PLTs)comprising urokinase(uPA)and arginine(Arg)for targeted thrombolysis and inhibition of re-embolism.The anchoring of uPA to the platelet surface by lipid insertion increased the thrombotic targeting and in vivo circulation duration of uPA without disturbing platelet functions.Nitric oxide(NO)generated by the loaded Arg inhibited platelet aggregation and activation at the damaged blood vessel,thereby inhibiting re-embolism.NO@uPA/PLTs effectively accumulated at the thrombi in pulmonary embolism and carotid artery thrombosis model mice and exerted superior thrombolytic efficacy.In addition,the platelet delivery system showed excellent thrombus recurrence prevention ability in a mouse model of secondary carotid artery injury.The coagulation indicators in vivo showed that the platelet-based uPA and NO co-delivery system possessed a low hemorrhagic risk,providing a promising tool for rapid thrombolysis and efficient inhibition of posttreatment re-embolism.

A pentapeptide enabled AL3810 liposome-based glioma-targeted therapy with immune opsonic effect attenuated

AL3810,a molecular dual inhibitor of the vascular endothelial growth factor receptor(VEGFR)and fibroblast growth factor receptor(FGFR),has earned the permission of phase II clinical trial for tumor treatment by China FDA.As a reversible ATP-competitive inhibitor,AL3810 targets ATP-binding site on intracellular region of VEGFR and FGFR,whereas,AL3810 lacking interplay with extracellular region of receptors rendered deficient bloodebrain tumor barrier(BBTB)recognition,poor brain penetration and unsatisfactory anti-glioma efficacy.Integrin avb3 overexpressed on capillary endothelial cells of BBTB as well as glioma cells illuminated ligand-modified liposomes for pinpoint spatial delivery into glioma.The widely accepted peptide c(RGDyK)-modified liposome loading AL3810 of multiple dosing caused hypothermia,activated anti-c(RGDyK)-liposome IgG and IgM antibody and pertinent complements C3 b and C5 b-9,and experienced complement-dependent opsonization.We newly proposed a pentapeptide mn with superb avb3-binding affinity and tailored AL3810-loaded mn-modified liposome that afforded impervious blood circulation,targeting ability,and glioma therapeutic expertise as vastly alleviated immune opsonization on the underpinning of the finite antibodies and complements assembly.Stemming from attenuated immunogenicity,peptide mn strengthened liposome functions as a promising nanocarrier platform for molecular targeting agents.

Insights into Stem Cell Therapy for Premature Ovarian Insufficiency

Hormone therapy,assisted reproductive technology,and regenerative medicine have been used to treat infertility due to premature ovarian insufficiency(POI),with limited success.It is timely to survey the field by outlining the controversies and promising prospects of evolving stem cell(SC)therapy for patients with POI.We first discuss several strategies of tissue-derived SC therapy and induced/engineered SC therapy and then enumerate mechanisms,including cellular regenerability induced in reproductive tissues and paracrine effects induced by various chemokines.Next,we evaluate the potential benefits of SC-based tissue engineering in reversing ovarian aging.Finally,we discuss the clinical feasibility of SC therapy and generalized regenerative medicine for the treatment of POI.In summary,SCs and SC-derived exosomes,induced pluripotent SCs,engineered SCs,and tissue engineering could start a new chapter for fertility rehabilitation in patients with POI.Uncovering the underlying molecular mechanisms and biological efficacy could be facilitated not only by animal experiments but also by security screening and clinical trials to validate SC-based therapy for POI.

Mature dendritic cell-derived dendrosomes swallow oxaliplatin-loaded nanoparticles to boost immunogenic chemotherapy and tumor antigen-specific immunotherapy

The cytomembrane-derived delivery platform represents a promising biomimetic strategy in oncotherapy.To achieve durable and reliable tumor inhibition,mature dendrosomes(mDs),which were isolated from bone marrow-derived dendritic cells undergoing CT26 tumor antigen(TA)stimulation,were fused with redox-responsive nanoparticles(NPs)that were composed of poly(disulfide ester amide)polymers with an intensified disulfide density and hydrophobic oxaliplatin(OXA)prodrugs with the ability to potentiate immunogenicity.In vitro and in vivo results revealed that NP/mDs could induce tumor cell death through mitochondrial pathway and thus created immunogenic microenvironments,but also elicited immunocyte differentiation by TA cross-dressing and infiltration by direct presentation.By further neutralizing immune-regulatory interaction,the administration of PD-L1 antibody(αPD-L1)greatly improved antitumor efficiency of NP/mDs.Furthermore,the effectors of host immune systems effectively inhibited the growth and metastasis of distal tumors,likely because the autologous TA evoked by OXA and allogeneic TA delivered by mDs acted as additional stimuli to reinforce the immune response of tumor-specific T cells and immunosurveillance toward oncogenesis.These results demonstrated that NP/mDs could simultaneously realize immunogenic chemotherapeutics and specific TA delivery.In combination withαPD-L1,the antitumor effect was further enhanced.Therefore,NP/mDs provide a promising strategy for the comprehensive treatment of malignancy.