TAX1BP1 and FIP200 orchestrate non-canonical autophagy of p62 aggregates for mouse neural stem cell maintenance

Autophagy plays a pivotal role in diverse biological processes,including the maintenance and differentiation of neural stem cells(NSCs).Interestingly,while complete deletion of Fip200 severely impairs NSC maintenance and differentiation,inhibiting canonical autophagy via deletion of core genes,such as Atg5,Atg16l1,and Atg7,or blockade of canonical interactions between FIP200 and ATG13(designated as FIP200-4A mutant or FIP200 KI)does not produce comparable detrimental effects.This highlights the likely critical involvement of the non-canonical functions of FIP200,the mechanisms of which have remained elusive.Here,utilizing genetic mouse models,we demonstrated that FIP200 mediates non-canonical autophagic degradation of p62/sequestome1,primarily via TAX1BP1 in NSCs.Conditional deletion of Tax1bp1 in fip200hGFAP conditional knock-in(cKI)mice led to NSC deficiency,resembling the fip200hGFAP conditional knockout(cKO)mouse phenotype.Notably,reintroducing wild-type TAX1BP1 not only restored the maintenance of NSCs derived from tax1bp1-knockout fip200hGFAP cKI mice but also led to a marked reduction in p62 aggregate accumulation.Conversely,a TAX1BP1 mutant incapable of binding to FIP200 or NBR1/p62 failed to achieve this restoration.Furthermore,conditional deletion of Tax1bp1 in fip200hGFAP cKO mice exacerbated NSC deficiency and p62 aggregate accumulation compared to fip200hGFAP cKO mice.Collectively,these findings illustrate the essential role of the FIP200-TAX1BP1 axis in mediating the non-canonical autophagic degradation of p62 aggregates towards NSC maintenance and function,presenting novel therapeutic targets for neurodegenerative diseases.

Explicit K-symplectic methods for nonseparable non-canonical Hamiltonian systems

We propose efficient numerical methods for nonseparable non-canonical Hamiltonian systems which are explicit,K-symplectic in the extended phase space with long time energy conservation properties. They are based on extending the original phase space to several copies of the phase space and imposing a mechanical restraint on the copies of the phase space. Explicit K-symplectic methods are constructed for two non-canonical Hamiltonian systems. Numerical tests show that the proposed methods exhibit good numerical performance in preserving the phase orbit and the energy of the system over long time, whereas higher order Runge–Kutta methods do not preserve these properties. Numerical tests also show that the K-symplectic methods exhibit better efficiency than that of the same order implicit symplectic, explicit and implicit symplectic methods for the original nonseparable non-canonical systems. On the other hand, the fourth order K-symplectic method is more efficient than the fourth order Yoshida’s method, the optimized partitioned Runge–Kutta and Runge–Kutta–Nystr ¨om explicit K-symplectic methods for the extended phase space Hamiltonians, but less efficient than the the optimized partitioned Runge–Kutta and Runge–Kutta–Nystr ¨om extended phase space symplectic-like methods with the midpoint permutation.

A Study of Non-Canonical Lagrangian

Non-canonical Lagrangian (Lagrangian with non-quadratic kinetic term) has been studied in the context of cosmology. In this work, the non-canonical Lagrangian with potential energy term has been discussed. We have obtained the periodic and solitary wave solutions for certain types of potential. The solutions obtained here may provide some new direction in the theory of phase transition, quantum field theory and related phenomena.

Improving Plant Genome Editing with High-Fidelity xCas9 and Non-canonical PAM-Targeting Cas9-NG

Two recently engineered SpCas9 variants, namely xCas9 and Cas9-NG, show promising potential in improving targeting specificity and broadening the targeting range. In this study, we evaluated these Cas9 variants in the model and crop plant, rice. We first tested xCas9-3.7, the most effective xCas9 variant in mammalian cells, for targeted mutagenesis at 16 possible NGN PAM (protospacer adjacent motif) combinations in duplicates. xCas9 exhibited nearly equivalent editing efficiency to wild-type Cas9 (Cas9-WT) at most canonical NGG PAM sites tested, whereas it showed limited activity at non-canonical NGH (H = A, C, T) PAM sites. High editing efficiency of xCas9 at NGG PAMs was further demonstrated with C to T base editing by both rAPOBECI and PmCDAI cytidine deaminases. With mismatched sgRNAs, we found that xCas9 had improved targeting specificity over the Cas9-WT. Furthermore, we tested two Cas9-NG variants, Cas9-NGv1 and Cas9-NG, for targeting NGN PAMs. Both Cas9-NG variants showed higher editing efficiency at most non-canonical NG PAM sites tested, and enabled much more efficient editing than xCas9 at AT-rich PAM sites such as GAT, GAA, and CAA. Nevertheless, we found that Cas9-NG variants showed significant reduced activity at the canonical NGG PAM sites. In stable transgenic rice lines, we demonstrated that Cas9-NG had much higher editing efficiency than Cas9-NGv1 and xCas9 at NG PAM sites. To expand the base-editing scope, we developed an efficient C to T base-editing system by making fusion of Cas9-NG nickase (D10A version), PmCDAI, and UGI. Taken together, our work benchmarked xCas9 as a high-fidelity nuclease for targeting canonical NGG PAMs and Cas9-NG as a preferred variant for targeting relaxed PAMs for plant genome editing.

Non-canonical scalar implicatures, face/politeness considerations, and neo-Gricean pragmatics

One of the politeness strategies,which has recently been observed,is the use of a semantically weak scalar expression to implicate the meaning of one or more of its stronger alternatives in a face-threatening context-a speech situation in which a loss of face can potentially be induced for the addressee,the speaker or both-out of politeness concerns.I call this type of marked scalar implicatures‘non-canonical scalar implicatures’.This article provides an informed discussion of the linguistic phenomenon in a few languages and presents a new analysis of it within the neo-Gricean pragmatic framework.

Multi-organ site metastatic reactivation mediated by non-canonical discoidin domain receptor 1 signaling

With the support of the National Natural Science Foundation of China and the Ministry of Science and Technology of China,the research teams led by Prof.Gao Hua(高华)from Tongji University,and Prof.Filippo Giancotti at Memorial Sloan Kettering Cancer Center,reported recently on the mechanism of multiorgan site metastatic creactivation,which was published in Cell(2016,166:47—62).

Engineering DNA logic systems with non-canonical DNA-nanostructures:basic principles,recent developments and bio-applications

Engineering DNA logic systems is considered as one of the most promising strategies for next-generation molecular computers.Owing to the inherent features of DNA,such as low cost,easy synthesis,and controllable hybridization,various DNA logic devices with different functions have been developed in the recent decade.Besides,a variety of logic-programmed biological applications are also explored,which initiates a new chapter for DNA logic computing.Although this field has gained rapid developments,a systematical review that could not only elaborate the logic principles of diverse DNA logic devices but also outline recent representative works is urgently needed.In this review,we first elaborate the general classification and logical principle of diverse DNA logic devices,in which the operating strategy of these devices and representative examples are selectively presented.Then,we review state-of-the-art advancements in DNA computing based on different non-canonical DNA-nanostructures during the past decade,in which some classical works are summarized.After that,the innovative applications of DNA computing to logic-controlled bioanalysis,cell imaging,and drug load/delivery are selectively presented.Finally,we analyze current obstacles and suggest appropriate prospects for this area.

Aberrant regulation of Wnt signaling in hepatocellular carcinoma

Hepatocellular carcinoma(HCC) is one of the most lethal malignancies in the world. Several signaling pathways,including the wingless/int-1(Wnt) signaling pathway,have been shown to be commonly activated in HCC. The Wnt signaling pathway can be triggered via both catenin β1(CTNNB1)-dependent(also known as "canonical") and CTNNB1-independent(often referred to as "non-canonical") pathways. Specifically,the canonical Wnt pathway is one of those most frequently reported in HCC. Aberrant regulation from three complexes(the cell-surface receptor complex,the cytoplasmic destruction complex and the nuclear CTNNB1/T-cell-specific transcription factor/lymphoid enhancer binding factor transcriptional complex) are all involved in HCC. Although the non-canonical Wnt pathway is rarely reported,two main non-canonical pathways,Wnt/planar cell polarity pathway and Wnt/Ca2+ pathway,participate in the regulation of hepatocarcinogenesis. Interestingly,the canonical Wnt pathway is antagonized by non-canonical Wnt signaling in HCC. Moreover,other signaling cascades have also been demonstrated to regulate the Wnt pathway through crosstalk in HCC pathogenesis. This review provides a perspective on the emerging evidence that the aberrant regulation of Wnt signaling is a critical mechanism for the development of HCC. Furthermore,crosstalk between different signaling pathways might be conducive to the development of novel molecular targets of HCC.

Deciphering the role of hedgehog signaling in pancreatic cancer

Pancreatic cancer, mostly pancreatic ductal adenocarcinoma （PDAC）, is a leading cause of cancer-related death in the US, with a dismal median survival of 6 months. Thus, there is an urgent unmet need to identify ways to diagnose and to treat this deadly cancer. Although a number of genetic changes have been identified in pancreatic cancer, their mechanisms of action in tumor development, progression and metastasis are not completely understood. Hedgehog signaling, which plays a major role in embryonic development and stem cell regulation, is known to be activated in pancreatic cancer; however, specific inhibitors targeting the smoothened molecule failed to improve the condition of pancreatic cancer patients in clinical trials. Furthermore, results regarding the role of Hh signaling in pancreatic cancer are controversial with some reporting tumor promoting activities whereas others tumor suppressive actions. In this review, we will summarize what we know about hedgehog signaling in pancreatic cancer, and try to explain the contradicting roles of hedgehog signaling as well as the reason（s） behind the failed clinical trials. In addition to the canonical hedgehog signaling, we will also discuss several non-canonical hedgehog signaling mechanisms.

V-ATPase activity is indispensible in chemical-induced noncanonical autophagy

Autophagy is a highly evolutionarily conserved pathway that depends on lysosome to degrade misfolded proteins and damaged organelles. Besides canonical autophagy, studies have shown some chemicals could bypass several core genes to induce autophagy, but the targets and regulatory mechanism is still unclear. In this work one novel chemical, G1, was screened out which could trigger both canonical autophagy and non-canonical autophagy by recruiting Atgl6L1 to pre-autophagosomal site and causing LC3 lipidation. The Gl-induced non-canonical auto- phagy was ULK1, and Beclinl-independent but ubiquitin-like conjugation system-dependent, indicating G1 might target the upstream of Atgl6L1. Moreover, inhibition of V-ATPase by specific V-ATPase inhibitiors could suppress the formation of Gl-indueed autophagosomes in FIP200-defieient MEF cells. While other classic lysosomal inhibi- tors could not block the puneta of Atgl2, Atgl6L1 and LC3, in different stages, suggesting V-ATPase activity in- stead of lysosome function is required for Gl-indueed non-canonical autophagy. These studies broaden the under- standing of different working pattern of autophagy and the crucial roles of V-ATPase in the regulation of different au- tophagy.

The evolving roles of Wnt signaling in stem cell proliferation and differentiation, the development of human diseases, and therapeutic opportunities

The evolutionarily conserved Wnt signaling pathway plays a central role in develop-ment and adult tissue homeostasis across species.Wnt proteins are secreted,lipid-modified signaling molecules that activate the canonical(β-catenin dependent)and non-canonical(β-catenin independent)Wnt signaling pathways.Cellular behaviors such as proliferation,differ-entiation,maturation,and proper body-axis specification are carried out by the canonical pathway,which is the best characterized of the known Wnt signaling paths.Wnt signaling has emerged as an important factor in stem cell biology and is known to affect the self-renewal of stem cells in various tissues.This includes but is not limited to embryonic,hematopoietic,mesenchymal,gut,neural,and epidermal stem cells.Wnt signaling has also been implicated in tumor cells that exhibit stem cell-like properties.Wnt signaling is crucial for bone formation and presents a potential target for the development of therapeutics for bone disorders.Not surprisingly,aberrant Wnt signaling is also associated with a wide variety of diseases,including cancer.Mutations of Wnt pathway members in cancer can lead to unchecked cell proliferation,epithelial-mesenchymal transition,and metastasis.Altogether,advances in the understand-ing of dysregulated Wnt signaling in disease have paved the way for the development of novel therapeutics that target components of the Wnt pathway.Beginning with a brief overview of the mechanisms of canonical and non-canonical Wnt,this review aims to summarize the cur-rent knowledge of Wnt signaling in stem cells,aberrations to the Wnt pathway associated with diseases,and novel therapeutics targeting the Wnt pathway in preclinical and clinical studies.

Site-specific incorporation of reduction-controlled guest amino acids into proteins for cucurbituril recognition

Protein recognition using host-guest recognition approach is of great interest but has been limited mainly to the protein N-terminal residues.Here,we site-specific incorporated two novel non-canonical amino acids containing supramolecular guest motifs into protein via an expanded genetic code.Through Staudinger reduction reactions,the encoded unnatural residues on protein becoming activated and can be specifically recognized by cucurbit[7]uril(CB[7])and cucurbit[8]uril(CB[8]).We demonstrated that enzyme containing guest amino acid incorporated near the active site can be reversibly regulated by CB[7]recognition,and CB[8]recognition induces protein dimerization.These amino acids will make useful addition to the supramolecular toolbox for protein targeting using molecular recognition approaches.

Expanding the targeting scope of CRISPR/Cas9-mediated genome editing by Cas9 variants in Brassica

CRISPR/Cas9,presently the most widely used genome editing technology,has provided great potential for functional studies and plant breeding.However,the strict requirement for a protospacer adjacent motif(PAM)has hindered the application of the CRISPR/Cas9 system because the number of targetable genomic sites is limited.Recently,the engineered variants Cas9-NG,SpG,and SpRY,which recognize non-canonical PAMs,have been successfully tested in plants(mainly in rice,a monocot).In this study,we evaluated the targeted mutagenesis capabilities of these Cas9 variants in two important Brassica vegetables,Chinese cabbage(Brassica rapa spp.pekinensis)and cabbage(Brassica oleracea var.capitata).Both Cas9-NG and SpG induced efficient mutagenesis at NGN PAMs,while SpG outperformed Cas9-NG at NGC and NGT PAMs.SpRY achieved efficient editing at almost all PAMs(NRN>NYN),albeit with some self-targeting activity at transfer(T)-DNA sequences.And SpRY-induced mutants were detected in cabbage plants in a PAM-less fashion.Moreover,an adenine base editor was developed using SpRY and TadA8e deaminase that induced A-to-G conversions within target sites using non-canonical PAMs.Together,the toolboxes developed here induced successful genome editing in Chinese cabbage and cabbage.Our work further expands the targeting scope of genome editing and paves the way for future basic research and genetic improvement in Brassica.

Characterization of heavy-chain antibody gene repertoires in Bactrian camels

Camelids are the only mammals that can produce functional heavy-chain antibodies(HCAbs).Although HCAbs were discovered over 30 years ago,the antibody gene repertoire of Bactrian camels remains largely underexplored.To characterize the diversity of variable genes of HCAbs(VHHs),germline and rearranged VHH repertoires are constructed.Phylogenetics analysis shows that all camelid VHH genes are derived from a common ancestor and the nucleotide diversity of VHHs is similar across all camelid species.While species-specific hallmark sites are identified,the non-canonical cysteines specific to VHHs are distinct in Bactrian camels and dromedaries compared with alpacas.Though low divergence at the germline repertoire between wild and domestic Bactrian camels,higher expression of VHHs is observed in some wild Bactrian camels than that of domestic ones.This study not only adds our understanding of VHH repertoire diversity across camelids,but also provides useful resources for HCAb engineering.

Approximate Conditional Likelihood for Generalized Linear Models with General Missing Data Mechanism

The generalized linear model is an indispensable tool for analyzing non-Gaussian response data, with both canonical and non-canonical link functions comprehensively used. When missing values are present, many existing methods in the literature heavily depend on an unverifiable assumption of the missing data mechanism, and they fail when the assumption is violated. This paper proposes a missing data mechanism that is as generally applicable as possible, which includes both ignorable and nonignorable missing data cases, as well as both scenarios of missing values in response and covariate.Under this general missing data mechanism, the authors adopt an approximate conditional likelihood method to estimate unknown parameters. The authors rigorously establish the regularity conditions under which the unknown parameters are identifiable under the approximate conditional likelihood approach. For parameters that are identifiable, the authors prove the asymptotic normality of the estimators obtained by maximizing the approximate conditional likelihood. Some simulation studies are conducted to evaluate finite sample performance of the proposed estimators as well as estimators from some existing methods. Finally, the authors present a biomarker analysis in prostate cancer study to illustrate the proposed method.

Metabolic features of cancer cells

Cancer cells uniquely reprogram their cellular activities to support their rapid proliferation and migration and to coun-teract metabolic and genotoxic stress during cancer progression.In this reprograming,cancer cells’metabolism and other cellular activities are integrated and mutually regulated,and cancer cells modulate metabolic enzymes spatially and temporally so that these enzymes not only have altered metabolic activities but also have modulated subcellular localization and gain non-canonical functions.This review and several others in this issue of Cancer Communications discuss these enzymes’newly acquired functions and the non-canonical functions of some metabolites as features of cancer cell metabolism,which play critical roles in various cellular activities,including gene expression,anabolism,catabolism,redox homeostasis,and DNA repair.

Genomewide decoupling of H2AK119ub1 and H3K27me3 in early mouse development

Polycomb group(Pc G)proteins are crucial chromatin regulators during development.H2 AK119 ub1(H2 Aub)and H3 K27 me3 are catalyzed by Polycomb-repressive complex 1 and 2(PRC1/2)respectively,and they largely overlap in the genome due to mutual recruitment of the two complexes.However,it is unclear whether PRC1/H2 Aub and PRC2/H3 K27 me3 can also function independently.By developing an ultra-sensitive carrier-DNA-assisted chromatin immunoprecipitation sequencing method termed CATCH-Seq,we generated allelic H2 Aub profiles in mouse gametes and early embryos.Our results revealed an unexpected genomewide decoupling of H2 Aub and H3 K27 me3 in mouse preimplantation embryos,where H2 Aub but not H3 K27 me3 was enriched at Pc G targets while only H3 K27 me3 was deposited in the broad distal domains associated with DNA methylation-independent non-canonical imprinting.These observations suggest that H2 Aub represses future bivalent genes during early embryogenesis without H3 K27 me3,but it is not required for the maintenance of non-canonical imprinting,which is mediated by maternal H3 K27 me3.Thus,our study reveals the distinct depositions and independent functions of H2 Aub and H3 K27 me3 during early mammalian development.

Chemical modifications of proteins and their applications in metalloenzyme studies

Protein chemical modifications are important tools for elucidating chemical and biological functions of proteins.Several strategies have been developed to implement these modifications,including enzymatic tailoring reactions,unnatural amino acid incorporation using the expanded genetic codes,and recognition-driven transformations.These technologies have been applied in metalloenzyme studies,specifically in dissecting their mechanisms,improving their enzymatic activities,and creating artificial enzymes with non-natural activities.Herein,we summarize some of the recent efforts in these areas with an emphasis on a few metalloenzyme case studies.

Expanding beyond canonical metabolism:Interfacing alternative elements,synthetic biology,and metabolic engineering

Metabolic engineering offers an exquisite capacity to produce new molecules in a renewable manner.However,most industrial applications have focused on only a small subset of elements from the periodic table,centered around carbon biochemistry.This review aims to illustrate the expanse of chemical elements that can currently(and potentially)be integrated into useful products using cellular systems.Specifically,we describe recent advances in expanding the cellular scope to include the halogens,selenium and the metalloids,and a variety of metal incorporations.These examples range from small molecules,heteroatom-linked uncommon elements,and natural products to biomining and nanotechnology applications.Collectively,this review covers the promise of an expanded range of elemental incorporations and the future impacts it may have on biotechnology.

Post-transcriptional regulation of miRNA biogenesis and functions

MicroRNAs(miRNAs)are a highly conserved class of small(18–24 nucleotides)non-coding RNAs that regulate a broad spectrum of biological processes.Aberrations or corruptions of miRNA functions may lead to deregulated cell proliferation,tumorigenesis,and ultimately,cancer.Increasing evidences suggested that a large fraction of miRNAs is regulated at the posttranscriptional stage,which impacts on the level and function of miRNAs during cell development and human diseases.Recently,several distinct mechanisms are emerging to regulate the biogenesis,stability and function of miRNAs at post-transcriptional level,such as specific binding to terminal loops of miRNA precursors(primiRNAs or pre-miRNAs)by RNA-binding proteins and 3’-terminal modifications by particular enzymes.Signaling cascades and post-translational modifications of the core components of RNA machinery also take part in the posttranscriptional regulation of miRNAs.