水稻悬浮细胞中稻瘟菌激发子诱导性受体类似激酶cDNA的克隆及特征分析

采用改进的差异显示方法由稻瘟菌激发子处理的水稻悬浮培养细胞中得到７个诱导表达的蛋白激酶 ｃＤＮＡ 片段。为获得这些蛋白激酶的编码全长序列，构建了稻瘟菌激发子处理的水稻细胞ｃＤＮＡ 文库。以上述一个 ｃＤＮＡ 片段作为探针筛选文库，得到一个含有完整阅读框的 ｃＤＮＡ 克隆ＯｓＥＰＫ１。分析ＯｓＥＰＫ１的推定氨基酸序列的结果表明：它由Ｎ 端信号肽、胞外区域、跨膜结构和胞内蛋白激酶结构域等几部分组成，可能为一受体类似蛋白激酶。对比 ＯｓＥＰＫ１与其它植物受体类似蛋白激酶在胞外区域的相似性，结果显示ＯｓＥＰＫ１与小麦叶锈激酶家族同源性较高，推测它们可能属于同一类植物受体类似蛋白激酶。此外，ｎｏｒｔｈｅｒｎ 杂交显示，ＯｓＥＰＫ１可由稻瘟菌激发子迅速瞬时诱导，因而它的诱导可能也是水稻对稻瘟菌侵染做出的一种早期防卫反应。

毛地黄皂苷诱导的水稻悬浮细胞防卫基因表达的信号传导

用带水稻苯丙氨酸解氨酶PAL 2启动子和GUS读码框嵌合基因的水稻悬浮细胞证明毛地黄皂苷能诱导水稻PAL 2的转录。NAD-PH、NADH、过氧化氢、超氧歧化酶、过氧化氢酶、甘露醇、1,2-羟基苯-3,5-二磺酸(Tiron)、paraquat、精氨酸、还原型和氧化型的谷胱甘肽、N-乙酰-L-半胱氨酸、二硫苏糖醇等都不影响毛地黄皂苷诱导的PAL2转导。只有在超氧歧化酶和过氧化氢酶或超氧歧化酶和Tiron一起时才微弱地抑制这种诱导。毛地黄皂苷能在缺钙培养基中诱导胞外介质碱性化,但它只在含钙离子的条件下才诱导PAL 2转录。钙离子载体A 23187及Stauros-poline不诱导PAL 2基因转录和胞外介质碱性化。钙调素的拮抗剂N-(6-Aminohexy1)-5-chloro-1-naphthalenesulfonamide(W7)和trifluoperazine(TFP)能抑制毛地黄皂苷诱导的PAL 2转录,但一些蛋白激酶的激活剂和抑制剂包括phorbol-12-myristate(TPA)、1-(5-Isoquinoline sulfony1)-2-methylpiperaz-ine(H7)、staurosporine及genestein不干扰它对PAL 2转录的诱导,G蛋白激活剂GTP-γS和抑制剂百日咳毒素也不影响这种诱导。毛地黄皂苷不诱导几丁酶转录。二硫苏糖醇能抑制几丁酶转录。伴刀豆球蛋白及麦胚凝集素能微弱地促进几丁酶基因转录,但对PAL 2表达没有影响。看来毛地黄皂苷诱导的水稻悬浮细胞PAL 2转录与活性氧激活没有简单和直接的关系。但它依赖于胞外介质碱性化和钙-钙调素,它的信号传导途径与诱导几丁酶转录的信号传导途径是不同的。

受稻瘟病原真菌侵染的水稻早期表达基因的cDNA克隆(英文)

以亲和性与非亲和性两个稻瘟病原真菌小种 (Magnaporthegrisea (Hebert)Barr)感染同一水稻品种 (OryzasativaL .cv .ShenxianggengNo .4)的植株产生明显不同的致病和抗病反应 ,由此建立了有效的感染系统。应用区分显示技术获得两个在侵染早期具有诱导表达特征的cDNA克隆 ,其中一个同时在致病和抗病反应中进行早期诱导表达 ,但在抗病反应中的诱导相对早于其在致病反应中的诱导表达。而另一克隆则只在抗病反应中进行早期诱导表达。基因组DNA杂交表明这两个cDNA克隆均来自水稻的基因组。顺序分析表明在水稻的基因数据库中均没有它们的同源序列 ,它们在水稻对稻瘟病原菌侵染的抗性反应中可能起重要作用。

Tyrosyl radical in haemoglobin and haptoglobin-haemoglobin complex:how does haptoglobin make haemoglobin less toxic?

One of the difficulties in creating a blood substitute on the basis of human haemoglobin(Hb) is the toxic nature of Hb when it is outside the safe environment of the red blood cells.The plasma protein haptoglobin(Hp) takes care of the Hb physiologically leaked into the plasma-it binds Hb and makes it much less toxic while retaining the Hb’s high oxygen transporting capacity.We used Electron Paramagnetic Resonance(EPR) spectroscopy to show that the protein bound radical induced by H2O2 in Hb and Hp-Hb complex is formed on the same tyrosine residue(s),but,in the complex,the radical is found in a more hydrophobic environment and decays slower than in unbound Hb,thus mitigating its oxidative capacity.The data obtained in this study might set new directions in engineering blood substitutes for transfusion that would have the oxygen transporting efficiency typical of Hb,but which would be non-toxic.

Going deep:Roots,carbon,and analyzing subsoil carbon dynamics

Agricultural practices currently contribute to the emission of approximately 10%–12%atmospheric greenhouse gasses,with tillage agriculture accelerating soil disruption and carbon dioxide release.Nevertheless,various options exist,such as converting new lands for agriculture,promoting healthier plant-based diets,adopting advanced management technologies,and minimizing food loss.However,these measures alone are insufficient to ensure environmental sustainability within planetary boundaries.

Differential stem cell aging kinetics in Hutchinson-Gilford progeria syndrome and Werner syndrome

progeria syndrome （HGPS） and Wemer syndrome （WS） are two of the best characterized human progeroid syndromes. HGPS is caused by a point mutation in lamin A （LMNA） gene, resulting in the production of a truncated protein product-progerin. WS is caused by mutations in 14/RN gem）, encoding a loss-of-function RecQ DNA helicase. Here, by gene editing we created isogenic human embryonic stem cells （ESCs） with heterozygous （G608G/＋） or homozygous （G608G/G608G） LMNA mutation and biallelic WRN knockout, for modeling HGPS and WS pathogenesis, respectively. While ESCs and endothelial cells （ECs） did not present any features of premature senescence, HGPS- and WS-mesenchymal stem cells （MSCs） showed aging-associated phenotypes with different kinetics. WS-MSCs had early-onset mild premature aging phenotypes while HGPS-MSCs exhibited iate-onset acute premature aging characterisitcs. Taken together, our study compares and contrasts the distinct pathologies underpinning the two premature aging disorders, and provides reliable stem-cell based models to identify new therapeutic strategies for pathological and physiological aging.

Arabidopsis FtsZ2-1 and FtsZ2-2 Are Functionally Redundant, But FtsZ-Based Plastid Division Is Not Essential for Chloroplast Partitioning or Plant Growth and Development

FtsZ1 and FtsZ2 are phylogenetically distinct families of FtsZ in plants that co-localize to mid-plastid rings and facilitate division of chloroplasts. In plants, altered levels of either FtsZ1 or FtsZ2 cause dose-dependent defects in chloroplast division; thus, studies on the functional relationship between FtsZgenes require careful manipulation of FtsZ levels in vivo. To define the functional relationship between the two FtsZ2 genes in Arabidopsis thaliana, FtsZ2-1 and FtsZ2-2, we expressed FtsZ2-1 in an ftsZ2-2 null mutant, and vice versa, and determined whether the chloroplast division defects were rescued in plants expressing different total levels of FtsZ2. Full rescue was observed when either the FtsZ2-1 or FtsZ2-2 level approximated total FtsZ2 levels in wild-type （WT）. Additionally, FtsZ2-2 interacts with ARC6, as shown previously for FtsZ2- 1. These data indicate that FtsZ2-1 and FtsZ2-2 are functionally redundant for chloroplast division in Arabidopsis. To rigorously validate the requirement of each FtsZ family for chloroplast division, we replaced FtsZ1 with FtsZ2 in vivo, and vice versa, while maintaining the FtsZ level in the transgenic plants equal to that of the total level in WT. Chloroplast division defects were not rescued, demonstrating conclusively that FtsZ1 and FtsZ2 are non-redundant for maintenance of WT chloroplast numbers. Finally, we generated ftsZtriple null mutants and show that plants completely devoid of FtsZ protein are viable and fertile. As plastids are presumably essential organelles, these findings suggest that an FtsZ-independent mode of plastid partitioning may occur in higher plants.

Human induced pluripotent stem cells derived hepatocytes:rising promise for disease modeling,drug development and cell therapy

Recent advances in the study of human hepatocytes derived from induced pluripotent stem cells(iPSC)represent new promises for liver disease study and drug discovery.Human hepatocytes or hepatocyte-like cells differentiated from iPSC recapitulate many func-tional properties of primary human hepatocytes and have been demonstrated as a powerful and efficient tool to model human liver metabolic diseases and fa-cilitate drug development process.In this review,we summarize the recent progress in this field and discuss the future perspective of the application of human iPSC derived hepatocytes.

Establishment of hepatic and neural differentiation platforms of Wilson’s disease specific induced pluripotent stem cells

The combination of disease-specific human induced pluripotent stem cells(iPSC)and directed cell differentiation offers an ideal platform for modeling and studying many inherited human diseases.Wilson’s disease(WD)is a monogenic disorder of toxic copper accumulation caused by pathologic mutations of the ATP7B gene.WD affects multiple organs with primary manifestations in the liver and central nervous system(CNS).In order to better investigate the cellular pathogenesis of WD and to develop novel therapies against various WD syndromes,we sought to establish a comprehensive platform to differentiate WD patient iPSC into both hepatic and neural lineages.Here we report the generation of patient iPSC bearing a Caucasian population hotspot mutation of ATP7B.Combining with directed cell differentiation strategies,we successfully differentiated WD iPSC into hepatocyte-like cells,neural stem cells and neurons.Gene expression analysis and cDNA sequencing confirmed the expression of the mutant ATP7B gene in all differentiated cells.Hence we established a platform for studying both hepatic and neural abnormalities of WD,which may provide a new tool for tissue-specific disease modeling and drug screening in the future.

Compound screening platform using human induced pluripotent stem cells to identify small molecules that promote chondrogenesis

Articular cartilage,which is mainly composed of collagen Ⅱ,enables smooth skeletal movement.Degeneration of collagen Ⅱ can be caused by various events,such as injury,but degeneration especially increases over the course of normal aging.Unfortunately,the body does not fully repair itself from this type of degeneration,resulting in impaired movement.Microfracture,an articular cartilage repair surgical technique,has been commonly used in the clinic to induce the repair of tissue at damage sites.Mesenchymal stem cells(MSC)have also been used as cell therapy to repair degenerated cartilage.However,the therapeutic outcomes of all these techniques vary in different patients depending on their age,health,lesion size and the extent of damage to the cartilage.The repairing tissues either form fibrocartilage or go into a hypertrophic stage,both of which do not reproduce the equivalent functionality of endogenous hyaline cartilage.One of the reasons for this is inefficient chondrogenesis by endogenous and exogenous MSC.Drugs that promote chondrogenesis could be used to induce self-repair of damaged cartilage as a non-invasive approach alone,or combined with other techniques to greatly assist the therapeutic outcomes.The recent development of human induced pluripotent stem cell(iPSCs),which are able to self-renew and differentiate into multiple cell types,provides a potentially valuable cell resource for drug screening in a“more relevant”cell type.Here we report a screening platform using human iPSCs in a multi-well plate format to identify compounds that could promote chondrogenesis.

Mutations in foregut SOX2^+ cells induce efficient proliferation via CXCR2 pathway

Identification of the precise molecular pathways involved in oncogene-induced transformation may help us gain a better understanding of tumor initiation and promotion. Here, we demonstrate that SOX2^+ foregut epithelial cells are prone to oncogenic transformation upon mutagenic insults, such as Kras^G12D and p53 deletion. GFP-based lineage-tracing experiments indicate that SOX2^+ cells are the cells-of-origin of esophagus and stomach hyperplasia. Our observations indicate distinct roles for oncogenic KRAS mutation and P53 deletion. p53 homozygous deletion is required for the acquisition of an invasive potential, and Kras^G12D expression, but not p53 deletion, suffices for tumor formation. Global gene expression analysis reveals secreting factors upregulated in the hyperplasia induced by oncogenic KRAS and highlights a crucial role for the CXCR2 pathway in driving hyperplasia. Collectively, the array of genetic models presented here demonstrate that stratified epithelial cells are susceptible to oncogenic insults, which may lead to a better understanding of tumor initiation and aid in the design of new cancer therapeutics.

The AFB1 auxin receptor controls the cytoplasmic auxin response pathway in Arabidopsis thaliana

The phytohormone auxin triggers root growth inhibition within seconds via a non-transcriptionalpathway.Among members of the TIR1/AFB auxin receptor family,AFB1 has a primary role in this rapidresponse. However, the unique features that confer this specific function have not been identified.Here we show that the N-terminal region of AFB1, including the F-box domain and residues thatcontribute to auxin binding,is essential and sufficient for its specific role in the rapid response. Substitutionof the N-terminal region of AFB1 with that of TIR1 disrupts its distinct cytoplasm-enriched localizationand activity in rapid root growth inhibition by auxin. Importantly, the N-terminal region of AFB1 isindispensable for auxin-triggered calcium influx, which is a prerequisite for rapid root growth inhibition.Furthermore, AFB1 negatively regulates lateral root formation and transcription of auxin-induced genes,suggesting that it plays an inhibitory role in canonical auxin signaling. These results suggest that AFB1may buffer the transcriptional auxin response, whereas it regulates rapid changes in cell growth thatcontributeto rootgravitropism.

Single-cell transcriptomic atlas of mouse cochlear aging

Progressive functional deterioration in the cochlea is associated with age-related hearing loss(ARHL).However,the cellular and molecular basis underlying cochlear aging remains largely unknown.Here,we established a dynamic single-cell transcriptomic landscape of mouse cochlear aging,in which we characterized aging-associated transcriptomic changes in 27 different cochlear cell types across five different time points.Overall,our analysis pinpoints loss of proteostasis and elevated apoptosis as the hallmark features of cochlear aging,highlights unexpected age-related transcriptional fluctuations in intermediate ceils localized in the stria vascularis(SV)and demonstrates that upregulation of endoplasmic reticulum(ER)chaperon protein HSP90AA1 mitigates ER stress-induced damages associated with aging.Our work suggests that targeting unfolded protein response pathways may help alleviate aging-related sVatrophyand hencedelay theprogressionofARHL.

Distinctive and complementary roles of E2F transcription factors during plant replication stress responses

Survival of living organisms is fully dependent on their maintenance of genome integrity,being permanently threatened by replication stress in proliferating cells.Although the plant DNA damage response(DDR)regulator SOG1 has been demonstrated to cope with replication defects,accumulating evidence points to other pathways functioning independent of SOG1.Here,we report the roles of the Arabidopsis E2FA and EF2B transcription factors,two well-characterized regulators of DNA replication,in plant response to replication stress.Through a combination of reverse genetics and chromatin immunoprecipitation approaches,we show that E2FA and E2FB share many target genes with SOG1,providing evidence for their involvement in the DDR.Analysis of double-and triple-mutant combinations revealed that E2FB,rather than E2FA,plays the most prominent role in sustaining plant growth in the presence of replication defects,either operating antagonistically or synergistically with SOG1.Conversely,SOG1 aids in overcoming the replication defects of E2FA/E2FB-deficient plants.Collectively,our data reveal a complex transcriptional network controlling the replication stress response in which E2Fs and SOG1 act as key regulatory factors.

Neural stem cells:mechanisms and modeling

In the adult brain,neural stem cells have been found in two major niches:the hippocampus and the olfactory bulb.Neurons derived from these stem cells contribute to learning,memory,and the autonomous repair of the brain under pathological conditions.Hence,the physi-ology of adult neural stem cells has become a signifi-cant component of research on synaptic plasticity and neuronal disorders.In addition,the recently developed induced pluripotent stem cell technique provides a powerful tool for researchers engaged in the patho-logical and pharmacological study of neuronal disor-ders.In this review,we briefly summarize the research progress in neural stem cells in the adult brain and in the neuropathological application of the induced pluripotent stem cell technique.

Modeling neuropsychiatric disorders using human induced pluripotent stem cells

Neuropsychiatric disorders are complex disorders characterized by heterogeneous genetic variations,variable symptoms,and widespread changes in anatomical pathology.In the context of neuropsychiatric disorders,limited access to relevant tissue types presents challenges for understanding disease etiology and developing effective treatments.Induced pluripotent stem cells(iPSCs)reprogrammed from patient somatic cells offer an opportunity to recapitulate disease development in relevant cell types,and they provide novel approaches for understanding disease mechanisms and for development of effective treatments.Here we review recent progress and challenges in differentiation paradigms for generating disease-relevant cells and recent studies of neuropsychiatric disorders using human pluripotent stem cell(hPSC)models where cellular phenotypes linked to disease have been reported.The use of iPSC-based disease models holds great promise for understanding disease mechanisms and supporting discovery of effective treatments.

nflammasomes in cancer： a double-edgecl sword

Chronic inflammatory responses have long been observed to be associated with various types of cancer and play decisive roles at different stages of cancer development. Inflammasomes, which are potent induc- ers of interleukin （IL）-I~ and IL-18 during infammation, are large protein complexes typically consisting of a Nod-like receptor （NLR）, the adapter protein ASC, and Caspase-1. During malignant transformation or cancer therapy, the inflammasomes are postulated to become activated in response to danger signals arising from the tumors or from therapy-induced damage to the tumor or healthy tissue. The activation of inflammasomes plays diverse and sometimes contrasting roles in cancer promotion and therapy depending on the specific con- text. Here we summarize the role of different inflamma- some complexes in cancer progression and therapy. Inflammasome components and pathways may provide novel targets to treat certain types of cancer; however, using such agents should be cautiously evaluated due to the complex roles that inflammasomes and pro- inflammatory cytokines play in immunity.

Modeling CADASIL vascular pathologies with patient-derived induced pluripotent stem cells

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy(CADASIL)is a rare hereditary cerebrovascular disease caused by a NOTCH3 mutation.However,the underlying cellular and molecular mechanisms remain unidentified.Here,we generated non-integrative induced pluripotent stem cells(iPSCs)from fibroblasts of a CADASIL patient harboring a heterozygous NOTCH3 mutation(c.3226C>T,p.R1076C).Vascular smooth muscle cells(VSMCs)differentiated from CADASIL-specific iPSCs showed gene expression changes associated with disease phenotypes,including activation of the NOTCH and NF-kB signaling pathway,cytoskeleton disorganization,and excessive cell proliferation.In comparison,these abnormalities were not observed in vascular endothelial cells(VECs)derived from the patients iPSCs.Importantly,the abnormal upregulation of NF-kB target genes in CADASIL VSMCs was diminished by a NOTCH pathway inhibitor,providing a potential therapeutic strategy for CADASIL.Overall,using this iPSCbased disease model,our study identified clues for studying the pathogenic mechanisms of CADASIL and developing treatment strategies for this disease.

Modeling xeroderma pigmentosum associated neurological pathologies with patients-derived iPSCs

Xeroderma pigmentosum （XP） is a group of genetic disorders caused by mutations of XP-associated genes, resulting in impairment of DNA repair. XP patients frequently exhibit neurological degeneration, but the underlying mechanism is unknown, in part due to lack of proper disease models. Here, we generated patientspecific induced pluripotent stem cells （iPSCs） harboring mutations in five different XP genes including XPA, XPB, XPC, XPG, and XPV. These iPSCs were further differentiated to neural cells, and their susceptibility to DNA damage stress was investigated. Mutation of XPA in either neural stem cells （NSCs） or neurons resulted in severe DNA damage repair defects, and these neural cells with mutant XPA were hyper-sensitive to DNA damage-induced apoptosis. Thus, XP-mutant neural cells represent valuable tools to clari the molecular mechanisms of neurological abnormalities in the XP patients.