The CUL3A–LFH1–UBC15 ubiquitin ligase complex mediates SHORT VEGETATIVE PHASE degradation to accelerate flowering at high ambient temperature

Ambient temperature affects flowering time in plants,and the MADS-box transcription factor SHORT VEGETATIVE PHASE(SVP)plays a crucial role in the response to changes in ambient temperature.SVP protein stability is regulated by the 26S proteasome pathway and decreases at high ambient temperature,but the details of SVP degradation are unclear.Here,we show that SVP degradation at high ambient temperature is mediated by the CULLIN3–RING E3 ubiquitin ligase(CRL3)complex in Arabidopsis thaliana.We identified a previously uncharacterized protein that interacts with SVP at high ambient temperature and contains a BTB/POZ domain.We named this protein LATE FLOWERING AT HIGH TEMPERATURE 1(LFH1).Single mutants of LFH1 or CULLIN3A(CUL3A)showed late flowering specifically at 27C.LFH1 protein levels increased at high ambient temperature.We found that LFH1 interacts with CUL3A in the cytoplasm and is important for SVP–CUL3A complex formation.Mutations in CUL3A and/or LFH1 led to increased SVP protein stability at high ambient temperature,suggesting that the CUL3–LFH1 complex functions in SVP degradation.Screening E2 ubiquitin-conjugating enzymes(UBCs)using RING-BOX PROTEIN 1(RBX1),a component of the CRL3 complex,as bait identified UBC15.ubc15 mutants also showed late flowering at high ambient temperature.In vitro and in vivo ubiquitination assays using recombinant CUL3A,LFH1,RBX1,and UBC15 showed that SVP is highly ubiquitinated in an ATP-dependent manner.Collectively,these results indicate that the degradation of SVP at high ambient temperature is mediated by a CRL3 complex comprising CUL3A,LFH1,and UBC15.

Cancer-associated fibroblast-derived secreted phosphoprotein 1 contributes to resistance of hepatocellular carcinoma to sorafenib and lenvatinib

Background:Cancer-associated fibroblasts(CAFs)play an important role in the induction of chemo-resistance.This study aimed to clarify the mechanism underlying CAF-mediated resistance to two tyrosine kinase inhibitors(TKIs),sorafenib and lenvatinib,and to identify a novel therapeutic target for overcoming TKI resistance in hepatocellular carcinoma(HCC).Methods:We performed a systematic integrative analysis of publicly available gene expression datasets and whole-transcriptome sequencing data from 9 pairs of CAFs and para-cancer fibroblasts isolated from human HCC and para-tumor tissues,respectively,to identify key molecules that might induce resistance to TKIs.We then performed in vitro and in vivo experiments to validate selected targets and related mechanisms.The associations of plasma secreted phosphoprotein 1(SPP1)expression levels before sorafenib/lenvatinib treatment with progression-free survival(PFS)and overall survival(OS)of 54 patients with advanced HCC were evaluated using Kaplan-Meier and Cox regression analysis.Results:Bioinformatic analysis identified CAF-derived SPP1 as a candidate molecule driving TKI resistance.SPP1 inhibitors reversed CAF-induced TKI resistance in vitro and in vivo.CAF-derived SPP1 activated rapidly accelerated fibrosarcoma(RAF)/mitogen-activated protein kinase(MAPK)and phosphatidylinositol 3-kinase(PI3K)/protein kinase B(AKT)/mammalian target of rapamycin(mTOR)through the integrin-protein kinase C-alpha(PKCα)signaling pathway and promoted epithelial-to-mesenchymal transition(EMT).A high plasma SPP1 level before TKI treatment was identified as an independent predictor of poor PFS(P=0.026)and OS(P=0.047)in patients with advanced HCC after TKI treatment.Conclusions:CAF-derived SPP1 enhances TKI resistance in HCC via bypass activation of oncogenic signals and EMT promotion.Its inhibition represents a promising therapeutic strategy against TKI resistance inHCC.Moreover,plasma SPP1 level before TKI treatment represents a potential biomarker for treatment response prediction.

Plasmonic bacteria on a nanoporous mirror via hydrodynamic trapping for rapid identification of waterborne pathogens

A rapid,precise method for identifying waterborne pathogens is critically needed for effective disinfection and better treatment.However,conventional methods,such as culture-based counting,generally suffer from slow detection times and low sensitivities.Here,we developed a rapid detection method for tracing waterborne pathogens by an innovative optofluidic platform,a plasmonic bacteria on a nanoporous mirror,that allows effective hydrodynamic cell trapping,enrichment of pathogens,and optical signal amplifications.We designed and simulated the integrated optofluidic platform to maximize the enrichment of the bacteria and to align bacteria on the nanopores and plasmonic mirror via hydrodynamic cell trapping.Gold nanoparticles are self-assembled to form antenna arrays on the surface of bacteria,such as Escherichia coli and Pseudomonas aeruginosa,by replacing citrate with hydroxylamine hydrochloride in order to amplify the signal of the plasmonic optical array.Owing to the synergistic contributions of focused light via the nanopore geometry,self-assembled nanoplasmonic optical antennas on the surface of bacteria,and plasmonic mirror,we obtain a sensitivity of detecting E.coli as low as 102 cells/ml via surface-enhanced Raman spectroscopy.We believe that our label-free strategy via an integrated optofluidic platform will pave the way for the rapid,precise identification of various pathogens.