Biotoxins are widely distributed in animals,plants,and microorganisms,functioning as"chemical weapons"of proteins,peptides,or small molecules that evolved for predation,defense,and competition.Compared with general chemical toxins,biotoxins exhibit high potency,strong specificity,and remarkable molecular diversity.While posing potential threats to human health,they also provide u-nique value in elucidating disease mechanisms and inspiring drug development.Representative drugs such as captopril,botulinum toxin,ziconotide,and GLP-1 receptor agonists mark milestones in the ther-apeutic application of toxins.To date,over one hundred biotoxin-derived drug candidates have entered clinical trials across multiple major diseases.However,this field still faces challenges,including low ef-ficiency in resource discovery,limited structural and mechanistic insights,inherent toxicity,and con-straints in synthesis and modification technologies.Looking forward,advances in multi-omics,artificial intelligence,and synthetic biology will drive efficient toxin discovery,detoxification strategies,and preci-sion applications,ultimately promoting a closed-loop progression from basic research to clinical transla-tion.

Lung cancer poses a serious threat to global public health security.Chemotherapy,as the main strategy for cancer treatment,faces challenges such as high toxicity and drug resistance.Anticancer peptides have the potential of being developed into new anticancer drugs due to their advantages of broad-spectrum anticancer activity,rapid action,and difficulty in generating drug resistance,but they also face shortcomings such as weak activity and strong toxic side effects.The weakly acidic microenvironment of tumors(pH 6.5-6.8)provides a good idea for the design of anticancer peptides of high-efficiency and low-toxicity.Previously,we designed the acid-sensitive antibacterial peptide pHly-1 using the wolf spider(Lycosa singoriensis)toxin Lycosin-Ⅰ as a template.In this study,we found that pHly-1 also had acid-sensitive anticancer activity.Further alanine scanning analysis of pHly-1 was carried out,and we ob-tained a mutant pHTP-2 with better acid sensitivity,whose IC50(half maximal inhibitory concentration)against A549 cells was 15.68 μmol/L at pH 6.6 and was greater than 100 μmol/L at pH 7.4.At pH 6.6,pHTP-2 could act on various lung cancer cell lines and induce the death of A549 cells by rapid ly-sis;at pH 7.4,500 μmol/L pHTP-2 had weak toxicity to red blood cells(the hemolysis rate was ap-proximately 38％)and primary myocardial cells(the inhibition rate was 49.7％,with P＜0.05).Analy-sis of its charge,particle size,morphology,and secondary structure showed that at pH 6.6,the histidine in the sequence of pHTP-2 was protonated,increasing the positive charge(P＜0.01),decreasing the hy-drated particle size(P＜0.05)and forming an α-helical structure to induce membrane lysis of A549 cells.At pH 7.4,it was deprotonated,the positive charge decreases,a β-sheet structure was formed and self-aggregation occurred,limiting its effect on the A549 cell membrane and showing weak activity.In summary,pHTP-2 could respond to the weakly acidic microenvironment of tumors to exert selective cyto-toxic activity,effectively overcoming the shortcomings of anticancer peptides such as low efficiency and high toxicity.Our findings suggest that it is a high-quality lead molecule for anticancer drugs.

Kv1.3,a voltage-gated potassium channel,is highly expressed in T lymphocytes,the nervous system,and vascular smooth muscle cells.It plays a critical role in membrane excitability and electrical signal transduction,serving as an important target for studying T-cell function and providing a promising direction for developing therapeutics against autoimmune and inflammatory diseases.Therefore,the de-velopment of specific inhibitors of Kv1.3 channel has emerged as a novel therapeutic strategy for these disorders.In this study,we isolated and purified a novel Kv1.3-inhibitory peptide toxin,LmKTx13,from the venom of the scorpion Lychas mucronatus using reversed-phase high-performance liquid chroma-tography(RP-HPLC).LmKTx13 consists of 38 amino acid residues,including six cysteines that form three disulfide bonds.Whole-cell patch-clamp recordings revealed that LmKTx13 potently inhibited Kv1.3 with an IC50 of 7.92±3.0 nmol/L.Selectivity analysis showed that 2 μmol/L LmKTx13 also in-hibited Kv1.2 and Kv1.7,but exhibited no significant effects on other potassium channel subtypes or voltage-gated sodium channels.Further investigation into the mechanism demonstrated that LmKTx13 acts as a pore-blocking inhibitor of Kv1.3.By analyzing the effects of LmKTx13 on Kv1.3 channel gating ki-netics and performing sequence alignment of the pore regions of Kv1.3 and Kv1.5,we constructed site-directed mutants and identified the pore region of Kv1.3 as the critical binding site for LmKTx13.Key residues involved in the interaction included T425,G427,and H451.In summary,we discovered a no-vel pore-blocking Kv1.3 inhibitor,LmKTx13,from L.mucronatus venom,which exhibits high affinity and selectivity for Kv1.3.These findings highlight its potential as a potential lead molecule for developing Kv1.3-targeted therapeutics.

Wound repair and infection have long posed significant challenges in clinical medicine.Am-phibians,adapting to complex environments through long-term natural selection,have evolved skin sys-tems with remarkable abilities to resist external damage and promote rapid wound healing,making them an important source for discovering candidate molecules for wound healing.In this study,a novel peptide composed of 22 amino acids,with the sequence"VGKAGLETAACKATNSCFNIDW"and a molecular weight of 2299.6 D,was screened from the cDNA library of Odorrana tormota and named PN-VW22.The peptide was synthesized by solid-phase synthesis,and its effects on cell proliferation were evaluated using the CCK-8 assay and scratch wound healing assay in human keratinocytes(HaCaT)and mouse em-bryonic fibroblasts(NIH3T3).Antibacterial activity was assessed by determining the minimum inhibitory concentration(MIC).Results showed that PN-VW22 at various concentrations had no significant effect on the cell viability in NIH3T3 cells(P＞0.05),but significantly enhanced HaCaT cell viability at 0.5μmol/L(P＜0.001).Meanwhile,PN-VW22 induced cell proliferation and promoted wound healing in HaCaT cells,with a healing rate of 64.44％after 24 h incubation at 0.5 μmol/L.Furthermore,PN-VW22 exhibited antibacterial activity against Staphylococcus aureus with an MIC value of 13.92 μmol/L.In sum,this study identified PN-VW22 as a novel bifunctional peptide with both wound repair and anti-infective properties,providing a new toxin peptide template for the development of wound healing drugs.

Pancreatic cancer has emerged as one of the most challenging malignancies worldwide,with its high resistance to chemotherapy being the primary cause of treatment failure.Therefore,enhancing the chemosensitivity of pancreatic cancer has become a major focus of current research.In this study,we in-vestigated how Bufotaline,a bufadienolide extracted from the traditional Chinese medicine toad venom,exhibits its antitumor activity.Specifically,we explored the potential of Bufotaline to enhance the chemo-sensitivity of pancreatic cancer cells to Adriamycin and elucidated its underlying molecular mechanisms.Using CCK-8 and colony formation assays,we demonstrated that Bufotaline enhances the inhibitory effect of Adriamycin on the survival of pancreatic cancer cell lines Patu-8988T,Aspc-1,and Patu-8988S.No-tably,Bufotaline treatment reduced the IC50 of Adriamycin in drug-resistant pancreatic cancer cells to lev-els comparable to those in non-resistant cells.Results from Western blot,immunofluorescence,comet as-say,and TUNEL assays revealed that Bufotaline promotes Adriamycin-induced DNA damage in pancreatic cancer cells.RNA-seq analysis of Patu-8988T cells treated with Adriamycin alone or in combination with Bufotaline showed significant changes in gene expression,and qRT-PCR analysis further confirmed that Bu-fotaline downregulates the expression of DNA damage repair proteins NBS1 and RAD50.Moreover,Western blot analysis revealed that Bufotaline reduces the levels of DNA damage response repair proteins,and Im-munofluorescence experiments indicated that Bufotaline inhibits the activation of the ATM/CHK2 signaling pathway.Finally,in a subcutaneous xenograft mouse model,the combination of Adriamycin and Bufotaline treatment significantly suppressed pancreatic cancer cell growth.In conclusion,Bufotaline enhances Adria-mycin-induced chemosensitivity in pancreatic cancer cells;the combination of Adriamycin and Bufotaline downregulates the expression of DNA damage response repair proteins NBS1 and RAD50,and inhibits the ATM/CHK2-mediated DDR signaling pathway,thereby delaying DNA damage repair.

CRM 197(cross-reacting material 197),a naturally occurring mutant of diphtheria toxin,is a safe and effective vaccine vector and extensively used on developing conjugate or combined vaccines.The mutant loses its enzymatic activity,but fully retains its receptor-binding ability and immunogenicity.In current work,the diphtheria toxin mutant CRM 197 and its fusion proteins with the receptor-binding do-main of botulinum neurotoxin serotype E(EHc)were developed using genetic engineering technology.These recombinant proteins were confirmed by Western blotting and SDS-PAGE.BALB/c mice were im-munized with the CRM197-EHc and EHc-CRM197 fusion proteins,and their immunogenicity was evalua-ted.These two fusion protein molecules,CRM197-EHc and EHc-CRM197,as subunit vaccines,elicited a robust humoral immune response targeting both CRM197 and EHc antigens in the immunized mice.Compared to the mixture of CRM197 and EHc,the mice vaccinated with the fusion proteins(CRM197-EHc and EHc-CRM197)induced higher levels of anti-CRM197 antibodies,and the mice vaccinated with EHc-CRM197 also generated strongest anti-EHc antibodies.Consequently,as a carrier molecule in the fusion protein vaccine,EHc enhances the immunogenicity of CRM197 molecules.Likewise,CRM197 boosts the immunogenicity of EHc in the EHc-CRM197 fusion protein.

The bacterial ADP-ribosylating exotoxins are produced by bacteria and infect different human body tissues.Herein,we investigated the molecular evolution of AB5-type bacterial toxins expressed by Vibrio cholerae and other bacteria with similar invasion mechanisms to interpret the co-evolutionary history of V.cholerae cholera toxin(CT)and their hosts,aiming to reveal the causes of its transdermal immuno-genicity.We elaborated on the intracellular toxicity mechanisms of CT,including ganglioside receptor-mediated endocytosis and hyperactivation of the cAMP pathway,as well as the behavioral traces of related bacteriophages within the genomes of these bacteria.Models such as the relatively decoupled evolution of A and B subunits of CT and the evolutionary coupling of transdermal and mucosal immunity were summa-rized.Furthermore,we described mechanisms including phage-mediated horizontal gene transfer(exem-plified by Vibrio phage CTXΦ),toxin targeting variation,expansion of molecular recognition domains,and functional adaptive evolution of the toxins.In this research,we employed bioinformatic tools to con-struct phylogenetic trees and analyze genetic variations in the amino acid sequences of toxin A/B subunits and proteins of secretion systems.Tajima's test was utilized to quantify genetic distance,diversity,and neutral selection pressure.Key findings include:(1)a"decoupled evolution"mode for the A and B subunits of CT,with the B subunit under stronger negative selection;(2)horizontal gene transfer media-ted by CTXΦ and other phages drives the cross-species spread of toxin genes;(3)the interaction be-tween the toxin co-regulated pilus(TCP)and the TLR-5(Toll-like receptor 5)promotes the transdermal immunogenicity of the CT B subunit.These findings suggest the role of"toxin-host arm race"co-evolu-tion,and are consistent with the hypothesis of intergenerational transmission of immune memory in CT e-volution,thereby providing theoretical support for further research into the biological mechanisms and co-evolutionary history of AB5-type bacterial toxins.

The application of botulinum neurotoxin(BoNT)in cosmetic procedures of the head and neck is well established.In recent years,however,its therapeutic scope has significantly expanded to include functional and pathological disorders within this anatomical region.This article reviews the use of BoNT as an important treatment modality for various head and neck disorders,encompassing movement disor-ders(such as laryngeal,craniocervical,middle ear,oromandibular,and cervical dystonia),peripheral nerve dysfunction(including multiple system atrophy,migraine,synkinesis and spasm following facial nerve palsy),upper aerodigestive tract dysfunction(e.g.,post-laryngectomy complications,cricopharyn-geal dysphagia,pharyngeal pouch,and retrograde cricopharyngeal dysfunction),and autonomic nervous disorders(such as sialorrhea and gustatory sweating syndrome).By leveraging its highly specific neuro-modulatory effects,BoNT provides a reversible,minimally invasive,and potent therapeutic alternative for conditions often refractory to conventional pharmacological or surgical interventions.This review systemat-ically examines the mechanisms of action,injection techniques,clinical efficacy,and safety profiles of BoNT in the above-mentioned diseases,and discusses future applications,aiming to offer comprehensive evidence-based guidance for clinical practice to otolaryngologists-head and neck surgeons.

Lactylation modification is a new type of protein post-translational modification,which medi-ates the covalent binding of lactic acid groups to lysine residues through amide bonds,thus changing pro-tein function and intracellular signal transduction process.Lactylation modifications can be broadly cate-gorized into two types:histone lactylation and non-histone lactylation,both of which are dynamically and precisely regulated by the"Writer-Eraser"enzyme system.Among them,non-histone lactylation,mainly regulated by enzymes such as AARS1 and SIRT3,plays an important role in the progression of many dis-eases,including tumor metabolic reprogramming,ROS stress and signal pathway regulation.Especially in tumors,non-histone lactylation is closely related to tumor proliferation,immune escape and drug re-sistance.Therefore,an in-depth study of the role of non-histone lactylation in the progression of tumors is expected to provide new targets and strategies for the accurate diagnosis and treatment of tumors.It is noteworthy that in the context of non-histone lactylation modification,the interference effect of acetylation modification cannot be ignored.Lactylation and acetylation share similar"writer"and"eraser"enzymes and exhibit overlapping modification sites,suggesting the possibility of functional crosstalk between the two.Due to the current lack of specific editing tools targeting lysine lactylation,it remains challenging to definitively determine whether lactylation plays a predominant regulatory role.This article reviews the re-search progress of non-histone lactylation in tumors in recent years.

Toxin-antitoxin(TA)systems serve as central hubs of bacterial adaptive regulation and play critical roles in the pathogenesis of Mycobacterium tuberculosis(M.tb)and Bordetella pertussis(B.per-tussis).This review summarizes the functional evolution and therapeutic potential of TA systems in M.tb and B.pertussis.It systematically outlines the molecular mechanisms and pathogenic functions of TA sys-tems in these two pathogens.M.tb relies on type Ⅱ TA systems(e.g.,VapBC,MazEF)to drive persis-ter formation and antibiotic tolerance through toxin-mediated ribonuclease activity that cleaves host nucle-ic acids or DarT/DarG-mediated DNA modification.In contrast,B.pertussis utilizes a unique tempera-ture-sensing PhtA/PhtB system to release adenylate cyclase toxin,which targets the host cAMP signaling pathway to achieve immune evasion.Both pathogens employ TA toxins to suppress host defenses-such as VapC cleaving tRNA and RelE degrading NF-κB components.Their high-frequency mutation sites(e.g.,the VapC47-Ser46Leu mutation frequency＞50 000 in M.tuberculosis)reveal strong positive selec-tion pressure,closely associated with persister phenotypes and virulence evolution.This review further discusses therapeutic strategies,including small-molecule inhibitors targeting toxin-antitoxin interactions,TA-deletion attenuated vaccines,and antitoxin-based immunization approaches.Finally,it highlights the need for future research to elucidate TA-host interaction networks and develop nanocarrier delivery tech-nologies to advance breakthroughs in precision therapy for tuberculosis and pertussis.