Chaperone-mediated autophagy targeting chimeras (CMATAC) forthe degradation of ERα in breast cancer

Estrogen receptor alpha(ERα/ESR1)is overexpressed in over half of all breast cancers and is considered a valuable therapeutic target in ERαpositive breast cancer.Here,we designed a membrane-permeant Chaperonemediated Autophagy Targeting Chimeras(CMATAC)peptide to knockdown endogenous ERαprotein through chaperone-mediated autophagy.The peptide contains a cell membrane-penetrating peptide(TAT)that allows the peptide to by-pass the plasma membrane,anαI peptide as a protein-binding peptide(PBD)that binds specifically to ERα,and CMA-targeting peptide(CTM)that targeting chaperone-mediated autophagy.We validated that ERαtargeting peptide was able to target and degrade ERαto reduce the viability of ERαpositive breast cancer cells.Taken together,our studies provided a new method to reduce the level of intracellular ERαprotein via CMATAC,and thus may provide a new strategy for the treatment of ERαpositive breast cancer.

The Potential of Rat Inner Cell Mass and Fetal Neural Stem Cells to Generate Chimeras

The rat chimera is an important animal model for the study of complex human diseases. In the present study we evaluated the chimeric potential of rat inner cell masses （ICMs） and fetal neural stem （FNS） cells. In result, three rat chimeras were produced by day 5 （D5） Sprague-Dawley （SD） blastocysts injected with ICMs derived from day 6 （D6） and D5 Dark Agouti （DA） blastocysts; four rat chimeras had been generated by D5 DA blastocyst injected with D5 SD ICMs. For the requirement of gene modification, cultured rat inner cell mass cells were assessed to produce chimeras, but no chimeras were generated from injected embryos. The potential to generate chimeras from rFNS and transfected rFNS cells were tested, but no chimeric pups were produced. Only 2 of 41 fetuses derived from D5 DA blastocyst injection with SD LacZ transfected rFNS cells showed very low number of LacZ positive cells in the section. These results indicate that DA and SD rat ICMs arc able to contribute to chimeras, but their potential decreases significantly after culture in vitro （P〈0.05）, and rFNS cells only have the potential to contribute to early fetal development.

Establishment of Embryonic Stem Cell Lines from C57BL/6J Mice and Generation of Chimeras

Four embryonic stem (ES) cell lines, designated CE1, CE2, CE3 and CE4, were isolated from C57BL/6J blastocysts. The ratio of normal diploid composition of these cell lines is above 70%. To examine the differentiation potential of the ES cells, the CE2 cells were injected subcutaneously into syngenic mice, and many kinds of differentiated cells were observed on the sections of the teratoma derived from this ES cell line. On the other hand, to test the chimeric ability of the ES cells, the CE2 cells were microinjected into the blastocysts of ICR mice, and a chimera was obtained among living pups. These results show that CE2 ES cells are pluripotent stem cells, which can differentiate into many kinds of cell types, and can be used as a cell system for further research.

Controlling chaos and supressing chimeras in a fractional-order discrete phase-locked loop using impulse control

A fractional-order difference equation model of a third-order discrete phase-locked loop(FODPLL) is discussed and the dynamical behavior of the model is demonstrated using bifurcation plots and a basin of attraction. We show a narrow region of loop gain where the FODPLL exhibits quasi-periodic oscillations, which were not identified in the integer-order model. We propose a simple impulse control algorithm to suppress chaos and discuss the effect of the control step. A network of FODPLL oscillators is constructed and investigated for synchronization behavior. We show the existence of chimera states while transiting from an asynchronous to a synchronous state. The same impulse control method is applied to a lattice array of FODPLL, and the chimera states are then synchronized using the impulse control algorithm. We show that the lower control steps can achieve better control over the higher control steps.

Production of chicken chimeras by fusing blastodermal cells with electroporation

Aim: To establish techniques for producing somatic and germline chimeric chicken by transferring blastodermal cellsfused with electroporation. Methods: Stage-X blastodermal cells isolated from freshly laid fertile unincubated whiteLeghorn and Rhode Island red chicken eggs were fused with electroporation. The treated cell suspension was transferredto the recovery medium (DMEM containing 10% FBS) and was injected into the subgenninal cavity of recipient unin-cubated embryos (stage X). Results: Of 177 recipient embryos injected with the fusing blastodermal cells, 6(3.4 %) survived to hatching. Somatic chimerism was examined in the melanocyte of the feather. The presence offeathers originating from the donor cell was observed in 1 bird (16.7%) out of the 6 hatched birds. After 21 days ofincubation two birds out of five embryos were subjected to polymerase chain reaction (PCR) analysis for W-chromo-some-specific DNA for each tissue. One bird possessed W-chromosome-specific DNA in the stomach, and the other ex-hibited the same DNA in the left and right gonads and other tissues, but not the stomach. Conclusion: Recipientembryo having electrofused blastodennal cells yields somatic and germline chimeric chickens more successfully.(Asian J Androl 2000 Dec; 2: 271-275)

Spiral wave chimeras in populations of oscillators coupled to a slowly varying diffusive environment

Chimera states are firstly discovered in nonlocally coupled oscillator systems.Such a nonlocal coupling arises typically as oscillators are coupled via an external environment whose characteristic time scaleτis so small(i.e.,τ→0)that it could be eliminated adiabatically.Nevertheless,whether the chimera states still exist in the opposite situation(i.e.,τ≫1)is unknown.Here,by coupling large populations of Stuart–Landau oscillators to a diffusive environment,we demonstrate that spiral wave chimeras do exist in this oscillator-environment coupling system even whenτis very large.Various transitions such as from spiral wave chimeras to spiral waves or unstable spiral wave chimeras as functions of the system parameters are explored.A physical picture for explaining the formation of spiral wave chimeras is also provided.The existence of spiral wave chimeras is further confirmed in ensembles of FitzHugh–Nagumo oscillators with the similar oscillator-environment coupling mechanism.Our results provide an affirmative answer to the observation of spiral wave chimeras in populations of oscillators mediated via a slowly changing environment and give important hints to generate chimera patterns in both laboratory and realistic chemical or biological systems.

A Proximity-Triggered Strategy toward Transferable Proteolysis Targeting Chimeras

Over the past 20 years,great efforts have been invested in developing site-specific approaches to protein modification to dissect protein functions directly and accurately.Here,we report a proximitytriggered group transfer strategy from a sulfonium warhead to a Cysteine(Cys)residue of the target protein.With a guiding ligand,cargoes could be transferred selectively from a sulfonium center onto the Cys residue in the vicinity of their binding interface.The successful thalidomide transfer of sulfonium 1-X could be applied intracellularly for epidermal growth factor receptor degradation,highlighting the potential of group transfer strategy as a suite of chemical biology studies,including cell imaging,protein profiling,and protein degradation by simply employing different transferrable groups.

Development of PROTACS degrading KRAS and SOS1

The Kirsten rat sarcoma virus—son of sevenless 1(KRAS-SOS1)axis drives tumor growth preferentially in pancreatic,colon,and lung cancer.Now,KRAS G12C mutated tumors can be successfully treated with inhibitors that covalently block the cysteine of the switch II binding pocket of KRAS.However,the range of other KRAS mutations is not amenable to treatment and the G12C-directed agents Sotorasib and Adragrasib show a response rate of only approximately 40%,lasting for a mean period of 8 months.One approach to increase the efficacy of inhibitors is their inclusion into proteolysis-targeting chimeras(PROTACs),which degrade the proteins of interest and exhibit much higher antitumor activity through multiple cycles of activity.Accordingly,PROTACs have been developed based on KRAS-or SOS1-directed inhibitors coupled to either von Hippel-Lindau(VHL)or Cereblon(CRBN)ligands that invoke the proteasomal degradation.Several of these PROTACs show increased activity in vitro and in vivo compared to their cognate inhibitors but their toxicity in normal tissues is not clear.The CRBN PROTACs containing thalidomide derivatives cannot be tested in experimental animals.Resistance to such PROTACS arises through downregulation or inactivation of CRBN or factors of the functional VHL E3 ubiquitin ligase.Although highly active KRAS and SOS1 PROTACs have been formulated their clinical application remains difficult.

An intelligent control method based on artificial neural network for numerical flight simulation of the basic finner projectile with pitching maneuver

In this paper,an intelligent control method applying on numerical virtual flight is proposed.The proposed algorithm is verified and evaluated by combining with the case of the basic finner projectile model and shows a good application prospect.Firstly,a numerical virtual flight simulation model based on overlapping dynamic mesh technology is constructed.In order to verify the accuracy of the dynamic grid technology and the calculation of unsteady flow,a numerical simulation of the basic finner projectile without control is carried out.The simulation results are in good agreement with the experiment data which shows that the algorithm used in this paper can also be used in the design and evaluation of the intelligent controller in the numerical virtual flight simulation.Secondly,combined with the real-time control requirements of aerodynamic,attitude and displacement parameters of the projectile during the flight process,the numerical simulations of the basic finner projectile’s pitch channel are carried out under the traditional PID(Proportional-Integral-Derivative)control strategy and the intelligent PID control strategy respectively.The intelligent PID controller based on BP(Back Propagation)neural network can realize online learning and self-optimization of control parameters according to the acquired real-time flight parameters.Compared with the traditional PID controller,the concerned control variable overshoot,rise time,transition time and steady state error and other performance indicators have been greatly improved,and the higher the learning efficiency or the inertia coefficient,the faster the system,the larger the overshoot,and the smaller the stability error.The intelligent control method applying on numerical virtual flight is capable of solving the complicated unsteady motion and flow with the intelligent PID control strategy and has a strong promotion to engineering application.

Degradation of FAK-targeting by proteolytic targeting chimera technology to inhibit the metastasis of hepatocellular carcinoma

Liver cancer is a prevalent malignant cancer,ranking third in terms of mortality rate.Metastasis and recurrence primarily contribute to the high mortality rate of liver cancer.Hepatocellular carcinoma(HCC)has low expression of focal adhesion kinase(FAK),which increases the risk of metastasis and recurrence.Nevertheless,the efficacy of FAK phosphorylation inhibitors is currently limited.Thus,investigating the mechanisms by which FAK affects HCC metastasis to develop targeted therapies for FAK may present a novel strategy to inhibit HCC metastasis.This study examined the correlation between FAK expression and the prognosis of HCC.Additionally,we explored the impact of FAK degradation on HCC metastasis through wound healing experiments,transwell invasion experiments,and a xenograft tumor model.The expression of proteins related to epithelial-mesenchymal transition(EMT)was measured to elucidate the underlying mechanisms.The results showed that FAK PROTAC can degrade FAK,inhibit the migration and invasion of HCC cells in vitro,and notably decrease the lung metastasis of HCC in vivo.Increased expression of E-cadherin and decreased expression of vimentin indicated that EMT was inhibited.Consequently,degradation of FAK through FAK PROTAC effectively suppressed liver cancer metastasis,holding significant clinical implications for treating liver cancer and developing innovative anti-neoplastic drugs.

Studies of substrate binding region of mEAAC1 and mASCT1 by constructing chimeras

The cDNA chimeras between two subtypes of mouse excitatory amino acid transporter family, mouse excitatory amino acid carrier 1 （mEAAC1） and mouse alanine serine cysteine transporter 1 （mASCT1）, were constructed by recombinant PCR. After transcription in vitro, the cRNA was injected and expressed in Xenopus laevis oocytes. 3H-Glu and 3H-Ser were used as isotopic tracer to measure the flux of amino acids. The results showed that there might not be the key amino acids responsible for substractive specificity in the NH2-terminal and its adjacent regions of these two transporters, which probably supported the formation of the substrata binding sites.

Generation of rat-mouse chimeras by introducing single cells of rat inner cell masses into mouse blastocysts

In the field of developmental biology and regenerative medicine,mammalian interspecific chimeras have been proved very useful for investigating early embryonic development and the immune system establishment,and extended to a promising potential for human organ generation（Rossant et al.,1982）.

Generation of rat blood vasculature and hematopoietic cells in rat-mouse chimeras by blastocyst complementation

Interspecies chimera through blastocyst complementation could be an alternative approach to create human organs in animals by using human pluripotent stem cells.A mismatch of the major histocompatibility complex of vascular endothelial cells between the human and host animal will cause graft rejection in the transplanted organs.Therefore,to achieve a transplantable organ in animals without rejection,creation of vascular endothelial cells derived from humans within the organ is necessary.In this study,to explore whether donor xeno-pluripotent stem cells can compensate for blood vasculature in host animals,we generated rat-mouse chimeras by injection of rat embryonic stem cells(rESCs)into mouse blastocysts with deficiency of Flk-1 protein,which is associated with endothelial and hematopoietic cell development.We found that rESCs could differentiate into vascular endothelial and hematopoietic cells in the rat-mouse chimeras.The whole yolk sac(YS)of Flk-1^EGFP/ECFP rat-mouse chimera was full of rat blood vasculature.Rat genes related to vascular endothelial cells,arteries,and veins,blood vessels formation process,as well as hematopoietic cells,were highly expressed in the YS.Our results suggested that rat vascular endothelial cells could undergo proliferation,migration,and self-assembly to form blood vasculature and that hematopoietic cells could differentiate into B cells,T cells,and myeloid cells in rat-mouse chimeras,which was able to rescue early embryonic lethality caused by Flk-1 deficiency in mouse.

Design and Synthesis of Proteolysis Targeting Chimeras for Inducing BRD4 Protein Degradation

In this paper, we synthesized a series of proteolysis targeting chimeras（PROTACs） using VHL E3 ligase ligands for BRD4 protein degradation. One of the most promising compound 19g exhibited robust potency of BRD4 inhibition with IC50 value of （18.6±1.3） nmol/L, respectively. Furthermore, compound 19g potently inhibited cell proliferation in BRIM-sensitive cell lines RS4;11 with IC50 value of （34.2±4.3） nmol/L and capable of inducing de- gradation of BRD4 protein at 0.4-0.6 μmol/L in the RS4;11 leukemia cells. These data show that compound 19g is a highly potent and efficacious BRIM degrader.

Construction of serial deletants and chimeras of multi-kringle containing molecules and primary analysis of their functions

In comparison of amino acid sequences of 4 kringles of both macrophage stimulating protein (MSP) and hepatocyte growth factor (HGF), consensus motif sequence was determined. According to this consensus sequence, a pair of universal primers were designed. In combination with specific upstream or downstream primer of MSP or HGF respectively, serial fragments containing variant number of kringle (from 1 to 4) can be obtained by once PCR. By ligating the C terminal and N terminal fragments with different combination, serial deletants and chimeras of MSP and HGF were constructed. Sequence analysis showed that the degeneracy for universal primers and the sequences of those constructed deletants and chimeras are desired. Biological assay of these deletants revealed that wild type MSP can inhibit the growth of some tumor cell lines and that kringle 1 of MSP is essential for function as that of HGF.

Entangled chimeras in nonlocally coupled bicomponent phase oscillators:From synchronous to asynchronous chimeras

Chimera states,a symmetry-breaking spatiotemporal pattern in nonlocally coupled identical dynamical units,have been identified in various systems and generalized to coupled nonidentical oscillators.It has been shown that strong heterogeneity in the frequencies of nonidentical oscillators might be harmful to chimera states.In this work,we consider a ring of nonlocally coupled bicomponent phase oscillators in which two types of oscillators are randomly distributed along the ring:some oscillators with natural.frequency w1 and others with w2.In this model,the heterogeneity in frequency is measured by frequency mismatch|w1-w2|between the oscillators in these two subpopulations.We report that the nonlocally coupled bicomponent phase oscillators allow for chimera states no matter how large the frequency mismatch is.The bicomponent oscillators are composed of two chimera states,one supported by oscillators with natural frequency wI and the other by oscillators with natural frequency w2.The two chimera states in two subpopulations are synchronized at weak frequency mismatch,in which the coberent oscillators in thern share similar mean phase velocity,and are desynchronized at large frequency mismatch,in which the coherent oscillators in different subpopulations have distinct mean phase velocities.The synchronization-desynchronization transition between chimera states in these two subpopulations is observed with the increase in the frequency mismatch.The observed phenomena are theoretically analyzed by passing to the continuum limit and using the Ott-Antonsen approach.

Cellular response toβ-amyloid neurotoxicity in Alzheimer's disease and implications in new therapeutics

β-Amyloid(Aβ)is a specific pathological hallmark of Alzheimer's disease(AD).Because of its neurotoxicity,AD patients exhibit multiple brain dysfunctions.Disease-modifying therapy(DMT)is the central concept in the development of AD thera-peutics today,and most DMT drugs that are currently in clinical trials are anti-Aβdrugs,such as aducanumab and lecanemab.Therefore,understanding Aβ's neurotoxic mechanism is crucial for Aβ-targeted drug development.Despite its total length of only a few dozen amino acids,Aβis incredibly diverse.In addition to the well-known Aβ_(1-42),N-terminally truncated,glutaminyl cyclase(QC)catalyzed,and pyroglutamate-modified Aβ(pEAβ)is also highly amyloidogenic and far more cytotoxic.The extracel-lular monomeric Aβ_(x-42)(x=1-11)initiates the aggregation to form fibrils and plaques and causes many abnormal cellular responses through cell membrane receptors and receptor-coupled signal pathways.These signal cascades further influence many cel-lular metabolism-related processes,such as gene expression,cell cycle,and cell fate,and ultimately cause severe neural cell damage.However,endogenous cellular anti-Aβdefense processes always accompany the Aβ-induced microenvironment alterations.Aβ-cleaving endopeptidases,Aβ-degrading ubiquitin-proteasome system(UPS),and Aβ-engulfing glial cell immune responses are all essential self-defense mechanisms that we can leverage to develop new drugs.This review discusses some of the most recent advances in understanding Aβ-centric AD mechanisms and suggests prospects for promising anti-Aβstrategies.

Applications and prospects of cryo-EM in drug discovery

Drug discovery is a crucial part of human healthcare and has dramatically benefited human lifespan and life quality in recent centuries, however, it is usually time-and effort-consuming. Structural biology has been demonstrated as a powerful tool to accelerate drug development. Among different techniques, cryo-electron microscopy(cryo-EM) is emerging as the mainstream of structure determination of biomacromolecules in the past decade and has received increasing attention from the pharmaceutical industry. Although cryo-EM still has limitations in resolution, speed and throughput, a growing number of innovative drugs are being developed with the help of cryo-EM. Here, we aim to provide an overview of how cryo-EM techniques are applied to facilitate drug discovery. The development and typical workflow of cryo-EM technique will be briefly introduced, followed by its specific applications in structure-based drug design, fragment-based drug discovery, proteolysis targeting chimeras, antibody drug development and drug repurposing. Besides cryo-EM, drug discovery innovation usually involves other state-of-the-art techniques such as artificial intelligence(AI), which is increasingly active in diverse areas. The combination of cryo-EM and AI provides an opportunity to minimize limitations of cryo-EM such as automation, throughput and interpretation of mediumresolution maps, and tends to be the new direction of future development of cryo-EM. The rapid development of cryo-EM will make it as an indispensable part of modern drug discovery.

“Rigid-elastic chimera” pore skeleton model and overpressure porosity measurement method for shale: A case study of the deep overpressure siliceous shale of Silurian Longmaxi Formation in southern Sichuan Basin, SW China

Based on analysis of pore features and pore skeleton composition of shale,a“rigid elastic chimeric”pore skeleton model of shale gas reservoir was built.Pore deformation mechanisms leading to increase of shale porosity due to the pore skeleton deformation under overpressure were sorted out through analysis of stress on the shale pore and skeleton.After reviewing the difficulties and defects of existent porosity measurement methods,a dynamic deformed porosity measurement method was worked out and used to measure the porosity of overpressure Silurian Longmaxi Formation shale under real formation conditions in southern Sichuan Basin.The results show:(1)The shale reservoir is a mixture of inorganic rock particles and organic matter,which contains inorganic pores supported by rigid skeleton particles and organic pores supported by elastic-plastic particles,and thus has a special“rigid elastic chimeric”pore structure.(2)Under the action of formation overpressure,the inorganic pores have tiny changes that can be assumed that they don’t change in porosity,while the organic pores may have large deformation due to skeleton compression,leading to the increase of radius,connectivity and ultimately porosity of these pores.(3)The“dynamic”deformation porosity measurement method combining high injection pressure helium porosity measurement and kerosene porosity measurement method under ultra-high variable pressure can accurately measure porosity of unconnected micro-pores under normal pressure conditions,and also the porosity increment caused by plastic skeleton compression deformation.(4)The pore deformation mechanism of shale may result in the"abnormal"phenomenon that the shale under formation conditions has higher porosity than that under normal pressure,so the overpressure shale reservoir is not necessarily“ultra-low in porosity”,and can have porosity over 10%.Application of this method in Well L210 in southern Sichuan has confirmed its practicality and reliability.

Giant teratoma with isolated intestinal duplication in adult: A case report and review of literature

BACKGROUND A combination of diseases is a rare phenomenon.Their clinical manifestations can vary,and the diagnosis can be challenging.Intestinal duplication is a rare congenital malformation,whereas retroperitoneal teratoma is a tumor in the retroperitoneal space,derived from the remaining embryonic tissue.There are relatively few clinical findings on adult retroperitoneal benign tumors.It is hard to believe that these two rare diseases can happen to the same person.CASE SUMMARY A 19-year-old woman complaining of abdominal pain with nausea and vomiting was admitted.Abdominal computed tomography angiography was suggested for invasive teratoma.Intraoperative exploration revealed that the giant teratoma was connected to an isolated intestinal tract in the retroperitoneum.The postoperative pathological examination revealed that mature giant teratoma was present with intestinal duplication.This was a rare intraoperative finding that was successfully treated surgically.CONCLUSION The clinical manifestations of intestinal duplication malformation are various,and difficult to diagnose before the operation.The possibility of intestinal replication should be considered when intraperitoneal cystic lesions are present.