Dissecting embryonic and extraembryonic lineage crosstalk with stem cell co-culture.

Embryogenesis necessitates harmonious coordination between embryonic and extraembryonic tissues. Although stem cells of both embryonic and extraembryonic origins have been generated, they are grown in different culture conditions. In this study, utilizing a unified culture condition that activates the FGF, TGF-ß, and WNT pathways, we have successfully derived embryonic stem cells (FTW-ESCs), extraembryonic endoderm stem cells (FTW-XENs), and trophoblast stem cells (FTW-TSCs) from the three foundational tissues of mouse and cynomolgus monkey (Macaca fascicularis) blastocysts. This approach facilitates the co-culture of embryonic and extraembryonic stem cells, revealing a growth inhibition effect exerted by extraembryonic endoderm cells on pluripotent cells, partially through extracellular matrix signaling. Additionally, our cross-species analysis identified both shared and unique transcription factors and pathways regulating FTW-XENs. The embryonic and extraembryonic stem cell co-culture strategy offers promising avenues for developing more faithful embryo models and devising more developmentally pertinent differentiation protocols.

Melanopsin retinal ganglion cells mediate light-promoted brain development.

During development, melanopsin-expressing intrinsically photosensitive retinal ganglion cells (ipRGCs) become light sensitive much earlier than rods and cones. IpRGCs project to many subcortical areas, whereas physiological functions of these projections are yet to be fully elucidated. Here, we found that ipRGC-mediated light sensation promotes synaptogenesis of pyramidal neurons in various cortices and the hippocampus. This phenomenon depends on activation of ipRGCs and is mediated by the release of oxytocin from the supraoptic nucleus (SON) and the paraventricular nucleus (PVN) into cerebral-spinal fluid. We further characterized a direct connection between ipRGCs and oxytocin neurons in the SON and mutual projections between oxytocin neurons in the SON and PVN. Moreover, we showed that the lack of ipRGC-mediated, light-promoted early cortical synaptogenesis compromised learning ability in adult mice. Our results highlight the importance of light sensation early in life on the development of learning ability and therefore call attention to suitable light environment for infant care.

Kinome-wide siRNA screen identifies a DCLK2-TBK1 oncogenic signaling axis in clear cell renal cell carcinoma.

TANK-binding kinase 1 (TBK1) is a potential therapeutic target in multiple cancers, including clear cell renal cell carcinoma (ccRCC). However, targeting TBK1 in clinical practice is challenging. One approach to overcome this challenge would be to identify an upstream TBK1 regulator that could be targeted therapeutically in cancer specifically. In this study, we perform a kinome-wide small interfering RNA (siRNA) screen and identify doublecortin-like kinase 2 (DCLK2) as a TBK1 regulator in ccRCC. DCLK2 binds to and directly phosphorylates TBK1 on Ser172. Depletion of DCLK2 inhibits anchorage-independent colony growth and kidney tumorigenesis in orthotopic xenograft models. Conversely, overexpression of DCLK2203, a short isoform that predominates in ccRCC, promotes ccRCC cell growth and tumorigenesis in vivo. Mechanistically, DCLK2203 elicits its oncogenic signaling via TBK1 phosphorylation and activation. Taken together, these results suggest that DCLK2 is a TBK1 activator and potential therapeutic target for ccRCC.

Ballistic two-dimensional InSe transistors.

The International Roadmap for Devices and Systems (IRDS) forecasts that, for silicon-based metal-oxide-semiconductor (MOS) field-effect transistors (FETs), the scaling of the gate length will stop at 12 nm and the ultimate supply voltage will not decrease to less than 0.6 V (ref. 1). This defines the final integration density and power consumption at the end of the scaling process for silicon-based chips. In recent years, two-dimensional (2D) layered semiconductors with atom-scale thicknesses have been explored as potential channel materials to support further miniaturization and integrated electronics. However, so far, no 2D semiconductor-based FETs have exhibited performances that can surpass state-of-the-art silicon FETs. Here we report a FET with 2D indium selenide (InSe) with high thermal velocity as channel material that operates at 0.5 V and achieves record high transconductance of 6 mS µm-1 and a room-temperature ballistic ratio in the saturation region of 83%, surpassing those of any reported silicon FETs. An yttrium-doping-induced phase-transition method is developed for making ohmic contacts with InSe and the InSe FET is scaled down to 10 nm in channel length. Our InSe FETs can effectively suppress short-channel effects with a low subthreshold swing (SS) of 75 mV per decade and drain-induced barrier lowering (DIBL) of 22 mV V-1. Furthermore, low contact resistance of 62 Ω µm is reliably extracted in 10-nm ballistic InSe FETs, leading to a smaller intrinsic delay and much lower energy-delay product (EDP) than the predicted silicon limit.

Depolymerization of plastics by means of electrified spatiotemporal heating.

Depolymerization is a promising strategy for recycling waste plastic into constituent monomers for subsequent repolymerization1. However, many commodity plastics cannot be selectively depolymerized using conventional thermochemical approaches, as it is difficult to control the reaction progress and pathway. Although catalysts can improve the selectivity, they are susceptible to performance degradation2. Here we present a catalyst-free, far-from-equilibrium thermochemical depolymerization method that can generate monomers from commodity plastics (polypropylene (PP) and poly(ethylene terephthalate) (PET)) by means of pyrolysis. This selective depolymerization process is realized by two features: (1) a spatial temperature gradient and (2) a temporal heating profile. The spatial temperature gradient is achieved using a bilayer structure of porous carbon felt, in which the top electrically heated layer generates and conducts heat down to the underlying reactor layer and plastic. The resulting temperature gradient promotes continuous melting, wicking, vaporization and reaction of the plastic as it encounters the increasing temperature traversing the bilayer, enabling a high degree of depolymerization. Meanwhile, pulsing the electrical current through the top heater layer generates a temporal heating profile that features periodic high peak temperatures (for example, about 600 °C) to enable depolymerization, yet the transient heating duration (for example, 0.11 s) can suppress unwanted side reactions. Using this approach, we depolymerized PP and PET to their monomers with yields of about 36% and about 43%, respectively. Overall, this electrified spatiotemporal heating (STH) approach potentially offers a solution to the global plastic waste problem.

Glucose-driven TOR-FIE-PRC2 signalling controls plant development.

Nutrients and energy have emerged as central modulators of developmental programmes in plants and animals1-3. The evolutionarily conserved target of rapamycin (TOR) kinase is a master integrator of nutrient and energy signalling that controls growth. Despite its key regulatory roles in translation, proliferation, metabolism and autophagy2-5, little is known about how TOR shapes developmental transitions and differentiation. Here we show that glucose-activated TOR kinase controls genome-wide histone H3 trimethylation at K27 (H3K27me3) in Arabidopsis thaliana, which regulates cell fate and development6-10. We identify FERTILIZATION-INDEPENDENT ENDOSPERM (FIE), an indispensable component of Polycomb repressive complex 2 (PRC2), which catalyses H3K27me3 (refs. 6-8,10-12), as a TOR target. Direct phosphorylation by TOR promotes the dynamic translocation of FIE from the cytoplasm to the nucleus. Mutation of the phosphorylation site on FIE abrogates the global H3K27me3 landscape, reprogrammes the transcriptome and disrupts organogenesis in plants. Moreover, glucose-TOR-FIE-PRC2 signalling modulates vernalization-induced floral transition. We propose that this signalling axis serves as a nutritional checkpoint leading to epigenetic silencing of key transcription factor genes that specify stem cell destiny in shoot and root meristems and control leaf, flower and silique patterning, branching and vegetative-to-reproduction transition. Our findings reveal a fundamental mechanism of nutrient signalling in direct epigenome reprogramming, with broad relevance for the developmental control of multicellular organisms.

CDK11 requires a critical activator SAP30BP to regulate pre-mRNA splicing.

CDK11 is an emerging druggable target for cancer therapy due to its prevalent roles in phosphorylating critical transcription and splicing factors and in facilitating cell cycle progression in cancer cells. Like other cyclin-dependent kinases, CDK11 requires its cognate cyclin, cyclin L1 or cyclin L2, for activation. However, little is known about how CDK11 activities might be modulated by other regulators. In this study, we show that CDK11 forms a tight complex with cyclins L1/L2 and SAP30BP, the latter of which is a poorly characterized factor. Acute degradation of SAP30BP mirrors that of CDK11 in causing widespread and strong defects in pre-mRNA splicing. Furthermore, we demonstrate that SAP30BP facilitates CDK11 kinase activities in vitro and in vivo, through ensuring the stabilities and the assembly of cyclins L1/L2 with CDK11. Together, these findings uncover SAP30BP as a critical CDK11 activator that regulates global pre-mRNA splicing.

Acute cannabinoids impair working memory through astroglial CB1 receptor modulation of hippocampal LTD.

Impairment of working memory is one of the most important deleterious effects of marijuana intoxication in humans, but its underlying mechanisms are presently unknown. Here, we demonstrate that the impairment of spatial working memory (SWM) and in vivo long-term depression (LTD) of synaptic strength at hippocampal CA3-CA1 synapses, induced by an acute exposure of exogenous cannabinoids, is fully abolished in conditional mutant mice lacking type-1 cannabinoid receptors (CB(1)R) in brain astroglial cells but is conserved in mice lacking CB(1)R in glutamatergic or GABAergic neurons. Blockade of neuronal glutamate N-methyl-D-aspartate receptors (NMDAR) and of synaptic trafficking of glutamate α-amino-3-hydroxy-5-methyl-isoxazole propionic acid receptors (AMPAR) also abolishes cannabinoid effects on SWM and LTD induction and expression. We conclude that the impairment of working memory by marijuana and cannabinoids is due to the activation of astroglial CB(1)R and is associated with astroglia-dependent hippocampal LTD in vivo.

An SHR-SCR module specifies legume cortical cell fate to enable nodulation.

Legumes, unlike other plants, have the ability to establish symbiosis with nitrogen-fixing rhizobia. It has been theorized that a unique property of legume root cortical cells enabled the initial establishment of rhizobial symbiosis1-3. Here we show that a SHORTROOT-SCARECROW (SHR-SCR) stem cell program in cortical cells of the legume Medicago truncatula specifies their distinct fate. Regulatory elements drive the cortical expression of SCR, and stele-expressed SHR protein accumulates in cortical cells of M. truncatula but not Arabidopsis thaliana. The cortical SHR-SCR network is conserved across legume species, responds to rhizobial signals, and initiates legume-specific cortical cell division for de novo nodule organogenesis and accommodation of rhizobia. Ectopic activation of SHR and SCR in legumes is sufficient to induce root cortical cell division. Our work suggests that acquisition of the cortical SHR-SCR module enabled cell division coupled to rhizobial infection in legumes. We propose that this event was central to the evolution of rhizobial endosymbiosis.

CD73 contributes to the pathogenesis of fusion-negative rhabdomyosarcoma through the purinergic signaling pathway.

Rhabdomyosarcoma (RMS) is the most common type of soft tissue sarcoma in children and adolescents. Fusion-negative RMS (FN-RMS) accounts for more than 80% of all RMS cases. The long-term event-free survival rate for patients with high-grade FN-RMS is below 30%, highlighting the need for improved therapeutic strategies. CD73 is a 5' ectonucleotidase that hydrolyzes AMP to adenosine and regulates the purinergic signaling pathway. We found that CD73 is elevated in FN-RMS tumors that express high levels of TWIST2. While high expression of CD73 contributes to the pathogenesis of multiple cancers, its role in FN-RMS has not been investigated. We found that CD73 knockdown decreased FN-RMS cell growth while up-regulating the myogenic differentiation program. Moreover, mutation of the catalytic residues of CD73 rendered the protein enzymatically inactive and abolished its ability to stimulate FN-RMS growth. Overexpression of wildtype CD73, but not the catalytically inactive mutant, in CD73 knockdown FN-RMS cells restored their growth capacity. Likewise, treatment with an adenosine receptor A2A-B agonist partially rescued FN-RMS cell proliferation and bypassed the CD73 knockdown defective growth phenotype. These results demonstrate that the catalytic activity of CD73 contributes to the pathogenic growth of FN-RMS through the activation of the purinergic signaling pathway. Therefore, targeting CD73 and the purinergic signaling pathway represents a potential therapeutic approach for FN-RMS patients.

Twist2 amplification in rhabdomyosarcoma represses myogenesis and promotes oncogenesis by redirecting MyoD DNA binding.

Rhabdomyosarcoma (RMS) is an aggressive pediatric cancer composed of myoblast-like cells. Recently, we discovered a unique muscle progenitor marked by the expression of the Twist2 transcription factor. Genomic analyses of 258 RMS patient tumors uncovered prevalent copy number amplification events and increased expression of TWIST2 in fusion-negative RMS. Knockdown of TWIST2 in RMS cells results in up-regulation of MYOGENIN and a decrease in proliferation, implicating TWIST2 as an oncogene in RMS. Through an inducible Twist2 expression system, we identified Twist2 as a reversible inhibitor of myogenic differentiation with the remarkable ability to promote myotube dedifferentiation in vitro. Integrated analysis of genome-wide ChIP-seq and RNA-seq data revealed the first dynamic chromatin and transcriptional landscape of Twist2 binding during myogenic differentiation. During differentiation, Twist2 competes with MyoD at shared DNA motifs to direct global gene transcription and repression of the myogenic program. Additionally, Twist2 shapes the epigenetic landscape to drive chromatin opening at oncogenic loci and chromatin closing at myogenic loci. These epigenetic changes redirect MyoD binding from myogenic genes toward oncogenic, metabolic, and growth genes. Our study reveals the dynamic interplay between two opposing transcriptional regulators that control the fate of RMS and provides insight into the molecular etiology of this aggressive form of cancer.

Developmental trajectory of pluripotent stem cell establishment in Arabidopsis callus guided by a quiescent center-related gene network.

In plant tissue culture, callus formation is induced by a high auxin concentration. Among the three cell layers (the outer, middle and inner cell layers) of the callus, pluripotency acquisition in the middle cell layer is required for the potential ability of the callus to regenerate organs. Here, we reveal the developmental trajectory of middle cell layer initiation and maintenance in callus tissue originating from Arabidopsis thaliana hypocotyls. The S phase of the cell cycle is essential for the expression of quiescent center-related SCARECROW (SCR), PLETHORA1 (PLT1) and WUSCHEL-RELATED HOMEOBOX5 (WOX5) genes during the division of callus founder cells to initiate the callus primordium. After callus initiation, SHOOT-ROOT (SHR) proteins move from the inner to the middle cell layer and act together with SCR to promote the expression of PLT1 and WOX5. WOX5 represses the expression of VASCULAR-RELATED NAC-DOMAIN (VND) genes, thereby preventing callus tissue from differentiating into xylem cells. PLT1 and PLT2 directly activate JACKDAW (JKD), which is necessary for pluripotency acquisition in the middle cell layer. We hypothesize that the middle cell layer could have pluripotent stem cell activity and its establishment requires the quiescent center-related SCR-SHR-WOX5-PLT1/2-JKD gene network.

Assessing next-generation sequencing-based computational methods for predicting transcriptional regulators with query gene sets.

This article provides an in-depth review of computational methods for predicting transcriptional regulators (TRs) with query gene sets. Identification of TRs is of utmost importance in many biological applications, including but not limited to elucidating biological development mechanisms, identifying key disease genes, and predicting therapeutic targets. Various computational methods based on next-generation sequencing (NGS) data have been developed in the past decade, yet no systematic evaluation of NGS-based methods has been offered. We classified these methods into two categories based on shared characteristics, namely library-based and region-based methods. We further conducted benchmark studies to evaluate the accuracy, sensitivity, coverage, and usability of NGS-based methods with molecular experimental datasets. Results show that BART, ChIP-Atlas, and Lisa have relatively better performance. Besides, we point out the limitations of NGS-based methods and explore potential directions for further improvement.

Uptake of oomycete RXLR effectors into host cells by clathrin-mediated endocytosis.

Filamentous (oomycete and fungal) plant pathogens deliver cytoplasmic effector proteins into host cells to facilitate disease. How RXLR effectors from the potato late blight pathogen Phytophthora infestans enter host cells is unknown. One possible route involves clathrin-mediated endocytosis (CME). Transient silencing of NbCHC, encoding clathrin heavy chain, or the endosome marker gene NbAra6 encoding a Rab GTPase in the model host Nicotiana benthamiana, attenuated P. infestans infection and reduced translocation of RXLR effector fusions from transgenic pathogen strains into host cells. By contrast, silencing PP1c isoforms, susceptibility factors not required for endocytosis, reduced infection but did not attenuate RXLR effector uptake. Endosome enrichment by ultracentrifugation and sucrose gradient fractionation revealed co-localization of RXLR effector Pi04314-RFP with clathrin-coated vesicles. Immunopurification of clathrin- and NbAra6-associated vesicles during infection showed that RXLR effectors Pi04314-RFP and AvrBlb1-RFP, but not apoplastic effector PiSCR74-RFP, were co-immunoprecipitated during infection with pathogen strains secreting these effectors. Tandem mass spectrometry analyses of proteins co-immunoprecipitated with NbAra6-GFP during infection revealed enrichment of host proteins associated with endocytic vesicles alongside multiple pathogen RXLR effectors, but not apoplastic effectors, including PiSCR74, which do not enter host cells. Our data show that the uptake of P. infestans RXLR effectors into plant cells occurs via CME.

Author Correction: A new coronavirus associated with human respiratory disease in China.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

A new coronavirus associated with human respiratory disease in China.

Emerging infectious diseases, such as severe acute respiratory syndrome (SARS) and Zika virus disease, present a major threat to public health1-3. Despite intense research efforts, how, when and where new diseases appear are still a source of considerable uncertainty. A severe respiratory disease was recently reported in Wuhan, Hubei province, China. As of 25 January 2020, at least 1,975 cases had been reported since the first patient was hospitalized on 12 December 2019. Epidemiological investigations have suggested that the outbreak was associated with a seafood market in Wuhan. Here we study a single patient who was a worker at the market and who was admitted to the Central Hospital of Wuhan on 26 December 2019 while experiencing a severe respiratory syndrome that included fever, dizziness and a cough. Metagenomic RNA sequencing4 of a sample of bronchoalveolar lavage fluid from the patient identified a new RNA virus strain from the family Coronaviridae, which is designated here 'WH-Human 1' coronavirus (and has also been referred to as '2019-nCoV'). Phylogenetic analysis of the complete viral genome (29,903 nucleotides) revealed that the virus was most closely related (89.1% nucleotide similarity) to a group of SARS-like coronaviruses (genus Betacoronavirus, subgenus Sarbecovirus) that had previously been found in bats in China5. This outbreak highlights the ongoing ability of viral spill-over from animals to cause severe disease in humans.

Mutations in microRNA processing genes in Wilms tumors derepress the IGF2 regulator PLAG1.

Many childhood Wilms tumors are driven by mutations in the microRNA biogenesis machinery, but the mechanism by which these mutations drive tumorigenesis is unknown. Here we show that the transcription factor pleomorphic adenoma gene 1 (PLAG1) is a microRNA target gene that is overexpressed in Wilms tumors with mutations in microRNA processing genes. Wilms tumors can also overexpress PLAG1 through copy number alterations, and PLAG1 expression correlates with prognosis in Wilms tumors. PLAG1 overexpression accelerates growth of Wilms tumor cells in vitro and induces neoplastic growth in the developing mouse kidney in vivo. In both settings, PLAG1 transactivates insulin-like growth factor 2 (IGF2), a key Wilms tumor oncogene, and drives mammalian target of rapamycin complex 1 (mTORC1) signaling. These data link microRNA impairment to the PLAG1-IGF2 pathway, providing new insight into the manner in which common Wilms tumor mutations drive disease pathogenesis.

Paxillin family proteins Hic-5 and LPXN promote lipid storage by regulating the ubiquitination degradation of CIDEC.

Many metabolic diseases are caused by disorders of lipid homeostasis. CIDEC, a lipid droplet (LD)-associated protein, plays a critical role in controlling LD fusion and lipid storage. However, regulators of CIDEC remain largely unknown. Here, we established a homogeneous time-resolved fluorescence (HTRF)-based high-throughput screening method and identified LPXN as a positive regulatory candidate for CIDEC. LPXN and Hic-5, the members of the Paxillin family, are focal adhesion adaptor proteins that contribute to the recruitment of specific kinases and phosphatases, cofactors, and structural proteins, participating in the transduction of extracellular signals into intracellular responses. Our data showed that Hic-5 and LPXN significantly increased the protein level of CIDEC and enhanced CIDEC stability not through triacylglycerol synthesis and FAK signaling pathways. Hic-5 and LPXN reduced the ubiquitination of CIDEC and inhibited its proteasome degradation pathway. Furthermore, Hic-5 and LPXN enlarged LDs and promoted lipid storage in adipocytes. Therefore, we identified Hic-5 and LPXN as novel regulators of CIDEC. Our current findings also suggest intervention with Hic-5 and LPXN might ameliorate ectopic fat storage by enhancing the lipid storage capacity of white adipose tissues.

Transcription factor WOX11 modulates tolerance to cyst nematodes via adventitious lateral root formation.

The transcription factor WUSCHEL-RELATED HOMEOBOX 11 (WOX11) in Arabidopsis (Arabidopsis thaliana) initiates the formation of adventitious lateral roots upon mechanical injury in primary roots. Root-invading nematodes also induce de novo root organogenesis leading to excessive root branching, but it is not known if this symptom of disease involves mediation by WOX11 and if it benefits the plant. Here, we show with targeted transcriptional repression and reporter gene analyses in Arabidopsis that the beet cyst nematode Heterodera schachtii activates WOX11-mediated adventitious lateral rooting from primary roots close to infection sites. The activation of WOX11 in nematode-infected roots occurs downstream of jasmonic acid-dependent damage signaling via ETHYLENE RESPONSE FACTOR109, linking adventitious lateral root formation to nematode damage to host tissues. By measuring different root system components, we found that WOX11-mediated formation of adventitious lateral roots compensates for nematode-induced inhibition of primary root growth. Our observations further demonstrate that WOX11-mediated rooting reduces the impact of nematode infections on aboveground plant development and growth. Altogether, we conclude that the transcriptional regulation by WOX11 modulates root system plasticity under biotic stress, which is one of the key mechanisms underlying the tolerance of Arabidopsis to cyst nematode infections.

Hypoxia exosome derived CEACAM5 promotes tumor-associated macrophages M2 polarization to accelerate pancreatic neuroendocrine tumors metastasis via MMP9.

Exosomes play significant roles in the communications between tumor cells and tumor microenvironment. However, the specific mechanisms by which exosomes modulate tumor development under hypoxia in pancreatic neuroendocrine tumors (pNETs) are not well understood. This study aims to investigate these mechanisms and made several important discoveries. We found that hypoxic exosomes derived from pNETs cells can activate tumor-associated macrophages (TAM) to the M2 phenotype, in turn, the M2-polarized TAM, facilitate the migration and invasion of pNETs cells. Further investigation revealed that CEACAM5, a protein highly expressed in hypoxic pNETs cells, is enriched in hypoxic pNETs cell-derived exosomes. Hypoxic exosomal CEACAM5 was observed to induce M2 polarization of TAM through activation of the MAPK signaling pathway. Coculturing pNETs cells with TAM or treated with hypoxic exosomes enhanced the metastatic capacity of pNETs cells. In conclusion, these findings suggest that pNETs cells generate CEACAM5-rich exosomes in a hypoxic microenvironment, which in turn polarize TAM promote malignant invasion of pNETs cells. Targeting exosomal CEACAM5 could potentially serve as a diagnostic and therapeutic strategy for pNETs.