Stiffness-tunable biomaterials provide a good extracellular matrix environment for axon growth and regeneration

Neuronal growth, extension, branching, and formation of neural networks are markedly influenced by the extracellular matrix—a complex network composed of proteins and carbohydrates secreted by cells. In addition to providing physical support for cells, the extracellular matrix also conveys critical mechanical stiffness cues. During the development of the nervous system, extracellular matrix stiffness plays a central role in guiding neuronal growth, particularly in the context of axonal extension, which is crucial for the formation of neural networks. In neural tissue engineering, manipulation of biomaterial stiffness is a promising strategy to provide a permissive environment for the repair and regeneration of injured nervous tissue. Recent research has fine-tuned synthetic biomaterials to fabricate scaffolds that closely replicate the stiffness profiles observed in the nervous system. In this review, we highlight the molecular mechanisms by which extracellular matrix stiffness regulates axonal growth and regeneration. We highlight the progress made in the development of stiffness-tunable biomaterials to emulate in vivo extracellular matrix environments, with an emphasis on their application in neural repair and regeneration, along with a discussion of the current limitations and future prospects. The exploration and optimization of the stiffness-tunable biomaterials has the potential to markedly advance the development of neural tissue engineering.

Telencephalic stab wound injury induces regenerative angiogenesis and neurogenesis in zebrafish:unveiling the role of vascular endothelial growth factor signaling and microglia

After brain damage,regenerative angiogenesis and neurogenesis have been shown to occur simultaneously in mammals,suggesting a close link between these processes.However,the mechanisms by which these processes interact are not well understood.In this work,we aimed to study the correlation between angiogenesis and neurogenesis after a telencephalic stab wound injury.To this end,we used zebrafish as a relevant model of neuroplasticity and brain repair mechanisms.First,using the Tg(fli1:EGFP×mpeg1.1:mCherry)zebrafish line,which enables visualization of blood vessels and microglia respectively,we analyzed regenerative angiogenesis from 1 to 21 days post-lesion.In parallel,we monitored brain cell proliferation in neurogenic niches localized in the ventricular zone by using immunohistochemistry.We found that after brain damage,the blood vessel area and width as well as expression of the fli1 transgene and vascular endothelial growth factor(vegfaa and vegfbb)were increased.At the same time,neural stem cell proliferation was also increased,peaking between 3 and 5 days post-lesion in a manner similar to angiogenesis,along with the recruitment of microglia.Then,through pharmacological manipulation by injecting an anti-angiogenic drug(Tivozanib)or Vegf at the lesion site,we demonstrated that blocking or activating Vegf signaling modulated both angiogenic and neurogenic processes,as well as microglial recruitment.Finally,we showed that inhibition of microglia by clodronate-containing liposome injection or dexamethasone treatment impairs regenerative neurogenesis,as previously described,as well as injury-induced angiogenesis.In conclusion,we have described regenerative angiogenesis in zebrafish for the first time and have highlighted the role of inflammation in this process.In addition,we have shown that both angiogenesis and neurogenesis are involved in brain repair and that microglia and inflammation-dependent mechanisms activated by Vegf signaling are important contributors to these processes.This study paves the way for a better understanding of the effect of Vegf on microglia and for studies aimed at promoting angiogenesis to improve brain plasticity after brain injury.

Transforming growth factor-beta 1 enhances discharge activity of cortical neurons

Transforming growth factor-beta 1(TGF-β1)has been extensively studied for its pleiotropic effects on central nervous system diseases.The neuroprotective or neurotoxic effects of TGF-β1 in specific brain areas may depend on the pathological process and cell types involved.Voltage-gated sodium channels(VGSCs)are essential ion channels for the generation of action potentials in neurons,and are involved in various neuroexcitation-related diseases.However,the effects of TGF-β1 on the functional properties of VGSCs and firing properties in cortical neurons remain unclear.In this study,we investigated the effects of TGF-β1 on VGSC function and firing properties in primary cortical neurons from mice.We found that TGF-β1 increased VGSC current density in a dose-and time-dependent manner,which was attributable to the upregulation of Nav1.3 expression.Increased VGSC current density and Nav1.3 expression were significantly abolished by preincubation with inhibitors of mitogen-activated protein kinase kinase(PD98059),p38 mitogen-activated protein kinase(SB203580),and Jun NH2-terminal kinase 1/2 inhibitor(SP600125).Interestingly,TGF-β1 significantly increased the firing threshold of action potentials but did not change their firing rate in cortical neurons.These findings suggest that TGF-β1 can increase Nav1.3 expression through activation of the ERK1/2-JNK-MAPK pathway,which leads to a decrease in the firing threshold of action potentials in cortical neurons under pathological conditions.Thus,this contributes to the occurrence and progression of neuroexcitatory-related diseases of the central nervous system.

Activation of adult endogenous neurogenesis by a hyaluronic acid collagen gel containing basic fibroblast growth factor promotes remodeling and functional recovery of the injured cerebral cortex

The presence of endogenous neural stem/progenitor cells in the adult mammalian brain suggests that the central nervous system can be repaired and regenerated after injury.However,whether it is possible to stimulate neurogenesis and reconstruct cortical layers II to VI in non-neurogenic regions,such as the cortex,remains unknown.In this study,we implanted a hyaluronic acid collagen gel loaded with basic fibroblast growth factor into the motor cortex immediately following traumatic injury.Our findings reveal that this gel effectively stimulated the proliferation and migration of endogenous neural stem/progenitor cells,as well as their differentiation into mature and functionally integrated neurons.Importantly,these new neurons reconstructed the architecture of cortical layers II to VI,integrated into the existing neural circuitry,and ultimately led to improved brain function.These findings offer novel insight into potential clinical treatments for traumatic cerebral cortex injuries.

Process mineralogy of copper-nickel sulphide flotation by a cyclonic-static micro-bubble flotation column

In our study we investigated a refractory copper-nickel sulfide ore separation by using a cyclonic-static micro-bubble flotation column (FCSMC). The process mineralogy of the main products was studied. Using a scanning electron microscope-energy dispersive system (SEM-EDS) and an X-ray spectrometer the mineral category and content of samples were analyzed. By using a mineral liberation analyzer (MLA) the mineral liberation characteristics were revealed. It is shown that in roughing feed the monomers liberation degree of nickel pyrite and chalcopyrite take up 84.11% and 88.82%, respectively. In tailings, the lost nickel pyrite and chalcopyrite are mainly monomers. Therefore, strengthening the micro-fine particle recovery capacity is the key to increase recovery.

Experimental study on flow control of the turbulent boundary layer with micro-bubbles

The effect of micro-bubbles on the turbulent boundary layer in the channel flow with Reynolds numbers (Re) ranging from 0.87 × 105 to 1.23 × 105 is experimentally studied by using particle image velocimetry (PIV) measurements.The microbubbles are produced by water electrolysis.The velocity profiles,Reynolds stress and instantaneous structures of the boundary layer,with and without micro-bubbles,are measured and analyzed.The presence of micro-bubbles changes the streamwise mean velocity of the fluid and increases the wall shear stress.The results show that micro-bubbles have two effects,buoyancy and extrusion,which dominate the flow behavior of the mixed fluid in the turbulent boundary layer.The buoyancy effect leads to upward motion that drives the fluid motion in the same direction and,therefore,enhances the turbulence intense of the boundary layer.While for the extrusion effect,the presence of accumulated micro-bubbles pushes the flow structures in the turbulent boundary layer away from the near-wall region.The interaction between these two effects causes the vorticity structures and turbulence activity to be in the region far away from the wall.The buoyancy effect is dominant when the Re is relatively small,while the extrusion effect plays a more important role when Re rises.

Micro-bubble Drag Reduction on a High Speed Vessel Model

Ship hull form of the underwater area strongly influences the resistance of the ship. The major factor in ship resistance is skin friction resistance. Bulbous bows, polymer paint, water repellent paint （highly water-repellent wall）, air injection, and specific roughness have been used by researchers as an attempt to obtain the resistance reduction and operation efficiency of ships. Micro-bubble injection is a promising technique for lowering frictional resistance. The injected air bubbles are supposed to somehow modify the energy inside the turbulent boundary layer and thereby lower the skin friction. The purpose of this study was to identify the effect of injected micro bubbles on a navy fast patrol boat （FPB） 57 m type model with the following main dimensions： L=2 450 ram, B=400 mm, and T=190 mm. The influence of the location of micro bubble injection and bubble velocity was also investigated. The ship model was pulled by an electric motor whose speed could be varied and adjusted. The ship model resistance was precisely measured by a load cell transducer. Comparison of ship resistance with and without micro-bubble injection was shown on a graph as a function of the drag coefficient and Froude number. It was shown that micro bubble injection behind the mid-ship is the best location to achieve the most effective drag reduction, and the drag reduction caused by the micro-bubbles can reach 6%-9%.

Velocity distribution of the flow field in the cyclonic zone of cyclone-static micro-bubble flotation column

Laboratory experiments have been conducted to study the flow field in a cyclone static micro-bubble flotation column. The method of Particle Image Velocimetry (PIV) was used. The flow field velocity distribution in both cross section and longitudinal section within cyclonic zone was studied for different circulating volumes. The cross sectional vortex was also analyzed. The results show that in cross section as the circulating volume increases from 0.187 to 0.350 m 3 /h, the flow velocity ranges from 0 to 0.68 m/s. The flow field is mainly a non-vortex potential flow that forms a free vortex without outside energy input. In the cyclonic region the vortex deviates from the center of the flotation column because a single tangential opening introduces circulating fluid into the column. The tangential component of the velocity plays a defining role in the cross section. In the longitudinal section the velocity ranges from 0 to 0.08 m/s. The flow velocity increases as does the circulating volume. Advantageous mineral separation conditions arise from the combined effects of cyclonic flow in cross and longitudinal section.

Optimization of rheological parameter for micro-bubble drilling fluids by multiple regression experimental design

In order to optimize plastic viscosity of 18 mPa·s circulating micro-bubble drilling fluid formula,orthogonal and uniform experimental design methods were applied,and the plastic viscosities of 36 and 24 groups of agent were tested,respectively.It is found that these two experimental design methods show drawbacks,that is,the amount of agent is difficult to determine,and the results are not fully optimized.Therefore,multiple regression experimental method was used to design experimental formula.By randomly selecting arbitrary agent with the amount within the recommended range,17 groups of drilling fluid formula were designed,and the plastic viscosity of each experiment formula was measured.Set plastic viscosity as the objective function,through multiple regressions,then quadratic regression model is obtained,whose correlation coefficient meets the requirement.Set target values of plastic viscosity to be 18,20 and 22 mPa·s,respectively,with the trial method,5 drilling fluid formulas are obtained with accuracy of 0.000 3,0.000 1 and 0.000 3.Arbitrarily select target value of each of the two groups under the formula for experimental verification of drilling fluid,then the measurement errors between theoretical and tested plastic viscosity are less than 5%,confirming that regression model can be applied to optimizing the circulating of plastic-foam drilling fluid viscosity.In accordance with the precision of different formulations of drilling fluid for other constraints,the methods result in the optimization of the circulating micro-bubble drilling fluid parameters.

基于FP-growth算法的交通事故数据关联规则挖掘研究

为了探寻多种事故影响因素共同作用下诱发交通事故的某种规律以及各因素间的关系,本文采用FP-growth算法对收集到的交通事故数据进行分析研究,挖掘其中的潜在价值信息,找出事故发生的原因,根据分析结果给相关部门提出建议,帮助城市交通管理者制定更有效的管理措施,以达到降低交通事故发生频率的目的。

基于FP-growth的老年行人交通事故损伤致因研究

为研究老年行人交通事故相关因素及其对损伤程度的影响,以中国交通事故深度调查数据库中2013—2023年710起机动车-老年行人事故为分析对象,利用FP-growth算法挖掘出4类影响因素28个类型变量共5594项关联规则。研究结果表明:行人年龄、碰撞速度、事故时间段及事故地点是影响老年行人事故死亡率的显著因素。特别是,行人年龄和碰撞速度对死亡率有显著影响,随着碰撞速度的增加,死亡率显著上升;60岁及以上老年行人年龄每增加1岁,其死亡率提升0.037倍。此外,事故发生在夜间、郊区或村庄的死亡率更高。研究结果可为提高老年行人交通安全,制定相关安全措施提供一定参考。

Effects of thinning on the understory light environment of different stands and the photosynthetic performance and growth of the reforestation species Phoebe bournei

Light levels determine regeneration in stands and a key concern is how to regulate the light environment of different stand types to the requirements of the understory.In this study,we selected three stands typical in south China(a Cryptomeria japonica plantation,a Quercus acutissima plantation,and a mixed stand of both)and three thinning intensities to determine the best understory light environ-ment for 3-year-old Phoebe bournei seedlings.The canopy structure,understory light environment,and photosynthe-sis and growth indicators were assessed following thin-ning.Thinning improved canopy structure and understory light availability of each stand;species composition was the reason for differences in the understory light environ-ment.Under the same thinning intensity,the mixed stand had the greatest light radiation and most balanced spectral composition.P.bournei photosynthesis and growth were closely related to the light environment;all three stands required heavy thinning to create an effective and sustained understory light environment.In a suitable understory light environment,the efficiency of light interception,absorption,and use by seedlings was enhanced,resulting in a higher carbon assimilation the main limiting factor was stomatal conductance.As a shade-avoidance signal,red/far-red radia-tion is a critical factor driving changes in photosynthesis and growth of P.bournei seedlings,and a reduction increased light absorption and use capacity and height:diameter ratios.The growth advantage transformed from diameter to height,enabling seedlings to access more light.Our findings suggest that the regeneration of shade-tolerant species such as P.bournei could be enhanced if a targeted approach to thinning based on stand type was adopted.

GbLMI1 over-expression improves cotton aboveground vegetative growth

Leaves are the main organ for photosynthesis and organic synthesis in cotton.Leaf shape has important effects on photosynthetic efficiency and canopy formation,thereby affecting cotton yield.Previous studies have shown that LMI1(LATE MERISTEM IDENTITY1)is the main gene regulating leaf shape.In this study,the LMI1 gene was inserted into the 35S promoter expression vector,and cotton plants overexpressing LMI1(OE)were obtained through genetic transformation.Statistical analysis of the biological traits of the T_(1) and T_(2) populations showed that compared to the wild type(WT),OE plants had significantly larger leaves,thicker stems and significantly greater dry weight.Furthermore,plant sections of the main vein and petiole showed that the numbers of cells in those tissues of OE plants were significantly greater.In addition,RNA-seq analysis revealed the differential expression of genes related to gibberellin synthesis and NAC gene family(genes containing the NAC domain)between the OE and WT plants,suggesting that LMI1 is involved in secondary wall formation and cell proliferation,which promotes stem thickening.Moreover,Gene Ontology(GO)analysis revealed enrichment in the terms of calcium ion binding,and Kyoto Encyclopedia of Genes and Genomes(KEGG)analysis showed enrichment in the terms of fatty acid degradation,phosphatidylinositol signal transduction system,and c AMP(cyclic adenosine monophosphate)signal pathway.These results suggested that LMI1 OE plants are responsive to gibberellin hormone signals,and have altered messenger signals(c AMP,Ca^(2+))which amplify this function,to promote stronger aboveground vegetative growth.This study found the LMI1 greatly increased the vegetative growth in cotton,which is the basic requirement for higher yield.

Effects of maternal methyl donor intake during pregnancy on ileum methylation and function in an intrauterine growth restriction pig model

Background Intrauterine growth retardation(IUGR)affects intestinal growth,morphology,and function,which leads to poor growth performance and high mortality.The present study explored whether maternal dietary methyl donor(MET)supplementation alleviates IUGR and enhances offspring’s growth performance by improving intestinal growth,function,and DNA methylation of the ileum in a porcine IUGR model.Methods Forty multiparous sows were allocated to the control or MET diet groups from mating until delivery.After farrowing,8 pairs of IUGR and normal birth weight piglets from 8 litters were selected for sampling before suckling colostrum.Results The results showed that maternal MET supplementation tended to decrease the IUGR incidence and increased the average weaning weight of piglets.Moreover,maternal MET supplementation significantly reduced the plasma concentrations of isoleucine,cysteine,urea,and total amino acids in sows and newborn pig-lets.It also increased lactase and sucrase activity in the jejunum of newborn piglets.MET addition resulted in lower ileal methionine synthase activity and increased betaine homocysteine S-methyltransferase activity in the ileum of newborn piglets.DNA methylation analysis of the ileum showed that MET supplementation increased the methyla-tion level of DNA CpG sites in the ileum of newborn piglets.Down-regulated differentially methylated genes were enriched in folic acid binding,insulin receptor signaling pathway,and endothelial cell proliferation.In contrast,up-regulated methylated genes were enriched in growth hormone receptor signaling pathway and nitric oxide biosyn-thetic process.Conclusions Maternal MET supplementation can reduce the incidence of IUGR and increase the weaning litter weight of piglets,which may be associated with better intestinal function and methylation status.

Activation of endogenous neurogenesis and angiogenesis by basic fibroblast growth factor-chitosan gel in an adult rat model of ischemic stroke

Attempts have been made to use cell transplantation and biomaterials to promote cell proliferation,differentiation,migration,and survival,as well as angiogenesis,in the context of brain injury.However,whether bioactive materials can repair the damage caused by ischemic stroke by activating endogenous neurogenesis and angiogenesis is still unknown.In this study,we applied chitosan gel loaded with basic fibroblast growth factor to the stroke cavity 7 days after ischemic stroke in rats.The gel slowly released basic fibroblast growth factor,which improved the local microenvironment,activated endogenous neural stem/progenitor cells,and recruited these cells to migrate toward the penumbra and stroke cavity and subsequently differentiate into neurons,while enhancing angiogenesis in the penumbra and stroke cavity and ultimately leading to partial functional recovery.This study revealed the mechanism by which bioactive materials repair ischemic strokes,thus providing a new strategy for the clinical application of bioactive materials in the treatment of ischemic stroke.

Role of transforming growth factor-βin peripheral nerve regeneration

Injuries caused by trauma and neurodegenerative diseases can damage the peripheral nervous system and cause functional deficits.Unlike in the central nervous system,damaged axons in peripheral nerves can be induced to regenerate in response to intrinsic cues after reprogramming or in a growth-promoting microenvironment created by Schwann cells.However,axon regeneration and repair do not automatically result in the restoration of function,which is the ultimate therapeutic goal but also a major clinical challenge.Transforming growth factor(TGF)is a multifunctional cytokine that regulates various biological processes including tissue repair,embryo development,and cell growth and differentiation.There is accumulating evidence that TGF-βfamily proteins participate in peripheral nerve repair through various factors and signaling pathways by regulating the growth and transformation of Schwann cells;recruiting specific immune cells;controlling the permeability of the blood-nerve barrier,thereby stimulating axon growth;and inhibiting remyelination of regenerated axons.TGF-βhas been applied to the treatment of peripheral nerve injury in animal models.In this context,we review the functions of TGF-βin peripheral nerve regeneration and potential clinical applications.

Dynamic changes of rumen microbiota and serum metabolome revealed increases in meat quality and growth performances of sheep fed bio‑fermented rice straw

Background Providing high-quality roughage is crucial for improvement of ruminant production because it is an essential component of their feed.Our previous study showed that feeding bio-fermented rice straw(BF)improved the feed intake and weight gain of sheep.However,it remains unclear why feeding BF to sheep increased their feed intake and weight gain.Therefore,the purposes of this research were to investigate how the rumen micro-biota and serum metabolome are dynamically changing after feeding BF,as well as how their changes influence the feed intake,digestibility,nutrient transport,meat quality and growth performances of sheep.Twelve growing Hu sheep were allocated into 3 groups:alfalfa hay fed group(AH:positive control),rice straw fed group(RS:negative control)and BF fed group(BF:treatment).Samples of rumen content,blood,rumen epithelium,muscle,feed offered and refusals were collected for the subsequent analysis.Results Feeding BF changed the microbial community and rumen fermentation,particularly increasing(P<0.05)relative abundance of Prevotella and propionate production,and decreasing(P<0.05)enteric methane yield.The histomorphology(height,width,area and thickness)of rumen papillae and gene expression for carbohydrate trans-port(MCT1),tight junction(claudin-1,claudin-4),and cell proliferation(CDK4,Cyclin A2,Cyclin E1)were improved(P<0.05)in sheep fed BF.Additionally,serum metabolome was also dynamically changed,which led to up-regulating(P<0.05)the primary bile acid biosynthesis and biosynthesis of unsaturated fatty acid in sheep fed BF.As a result,the higher(P<0.05)feed intake,digestibility,growth rate,feed efficiency,meat quality and mono-unsaturated fatty acid concentration in muscle,and the lower(P<0.05)feed cost per kg of live weight were achieved by feeding BF.Conclusions Feeding BF improved the growth performances and meat quality of sheep and reduced their feed cost.Therefore,bio-fermentation of rice straw could be an innovative way for improving ruminant production with mini-mizing production costs.

Structural Engineering of Hierarchical Magnetic/Carbon Nanocomposites via In Situ Growth for High-Efficient Electromagnetic Wave Absorption

Materials exhibiting high-performance electromagnetic wave absorption have garnered considerable scientific and technological attention,yet encounter significant challenges.Developing new materials and innovative structural design concepts is crucial for expanding the application field of electromagnetic wave absorption.Particularly,hierarchical structure engineering has emerged as a promising approach to enhance the physical and chemical properties of materials,providing immense potential for creating versatile electromagnetic wave absorption materials.Herein,an exceptional multi-dimensional hierarchical structure was meticulously devised,unleashing the full microwave attenuation capabilities through in situ growth,selfreduction,and multi-heterogeneous interface integration.The hierarchical structure features a three-dimensional carbon framework,where magnetic nanoparticles grow in situ on the carbon skeleton,creating a necklace-like structure.Furthermore,magnetic nanosheets assemble within this framework.Enhanced impedance matching was achieved by precisely adjusting component proportions,and intelligent integration of diverse interfaces bolstered dielectric polarization.The obtain Fe_(3)O_(4)-Fe nanoparticles/carbon nanofibers/Al-Fe_(3)O_(4)-Fe nanosheets composites demonstrated outstanding performance with a minimum reflection loss(RLmin)value of−59.3 dB and an effective absorption bandwidth(RL≤−10 dB)extending up to 5.6 GHz at 2.2 mm.These notable accomplishments offer fresh insights into the precision design of high-efficient electromagnetic wave absorption materials.

Delineation of urban growth boundary based on FLUS model under the perspective of land use evaluation in hilly mountainous areas

With rapid economic development,the size of urban land in China is expanding dramatically.The Urban Growth Boundary(UGB)is an expandable spatial boundary for urban construction in a certain period in order to control the urban sprawl.Reasonable delineation of UGB can inhibit the disorderly spread of urban space and guide the normal development of the city.It is of practical significance for the construction of green urban space.The study utilizes GIS technology to establish a land construction suitability evaluation system for Nankang city,which is experiencing rapid urban expansion,and outlines the preliminary UGB under the future land use simulation(FLUS)model.At the same time,considering the coupled coordination of"Production-Living-Ecological Space",and based on the suitability evaluation,we revised the preliminary UGB by combining the advantages of the patch-generating land use simulation(PLUS)model and the convex hull model to delineate the final UGB.The results show that:1)the comprehensive score of the evaluation of the suitability of the construction of land from high to low shows the distribution of the center of the city to the surrounding circle type spread,the center of the city has the highest suitability score.The results of convex hull model show that the urban expansion type of Nankang is epitaxial.In the future,the urban expansion will mainly occur in the northern part of the city.The PLUS model predicts an increase of 3359.97 hm^(2)of construction land in Nankang by 2035,of which 2022.97 hm^(2)is urban construction land.2)The FLUS model has a prediction accuracy of 86.3%and delineates a preliminary UGB area of 9215.07 hm^(2).3)We used the results of the construction suitability evaluation,PLUS model simulation results,and convex hull model predictions to revise the originally delineated UGB.The final delineated UGB area is 8895.67 hm^(2)and it is capable of meeting the future development of the study area.The results of the delineation can promote sustainable urban development,and the delineation methodology can provide a reference basis for the preparation of territorial spatial planning.

PRaG 3.0 therapy for human epidermal growth factor receptor 2-positive metastatic pancreatic ductal adenocarcinoma:A case report

BACKGROUND Pancreatic ductal adenocarcinoma(PDAC)is a highly fatal disease with limited effective treatment especially after first-line chemotherapy.The human epidermal growth factor receptor 2(HER-2)immunohistochemistry(IHC)positive is associated with more aggressive clinical behavior and shorter overall survival in PDAC.CASE SUMMARY We present a case of multiple metastatic PDAC with IHC mismatch repair proficient but HER-2 IHC weakly positive at diagnosis that didn’t have tumor regression after first-line nab-paclitaxel plus gemcitabine and PD-1 inhibitor treatment.A novel combination therapy PRaG 3.0 of RC48(HER2-antibody-drug conjugate),radio-therapy,PD-1 inhibitor,granulocyte-macrophage colony-stimulating factor and interleukin-2 was then applied as second-line therapy and the patient had confirmed good partial response with progress-free-survival of 6.5 months and overall survival of 14.2 month.She had not developed any grade 2 or above treatment-related adverse events at any point.Percentage of peripheral CD8^(+) Temra and CD4^(+) Temra were increased during first two activation cycles of PRaG 3.0 treatment containing radiotherapy but deceased to the baseline during the maintenance cycles containing no radiotherapy.CONCLUSION PRaG 3.0 might be a novel strategy for HER2-positive metastatic PDAC patients who failed from previous first-line approach and even PD-1 immunotherapy but needs more data in prospective trials.