Recursive construction of perfect DNA molecules from imperfect oligonucleotides

Making faultless complex objects from potentially faulty building blocks is a fundamental challenge in computer engineering, nanotechnology and synthetic biology. Here, we show for the first time how recursion can be used to address this challenge and demonstrate a recursive procedure that constructs error‐free DNA molecules and their libraries from error‐prone oligonucleotides. Divide and Conquer (D&C), the quintessential recursive problem‐solving technique, is applied in silico to divide the target DNA sequence into overlapping oligonucleotides short enough to be synthesized directly, albeit with errors; error‐prone oligonucleotides are recursively combined in vitro, forming error‐prone DNA molecules; error‐free fragments of these molecules are then identified, extracted and used as new, typically longer and more accurate, inputs to another iteration of the recursive construction procedure; the entire process repeats until an error‐free target molecule is formed. Our recursive construction procedure surpasses existing methods for de novo DNA synthesis in speed, precision, amenability to automation, ease of combining synthetic and natural DNA fragments, and ability to construct designer DNA libraries. It thus provides a novel and robust foundation for the design and construction of synthetic biological molecules and organisms.     

The Arabidopsis Rho of Plants GTPase AtROP6 Functions in Developmental and Pathogen Response Pathways

How plants coordinate developmental processes and environmental stress responses is a pressing question. Here, we show that Arabidopsis (Arabidopsis thaliana) Rho of Plants6 (AtROP6) integrates developmental and pathogen response signaling. AtROP6 expression is induced by auxin and detected in the root meristem, lateral root initials, and leaf hydathodes. Plants expressing a dominant negative AtROP6 (rop6^DN) under the regulation of its endogenous promoter are small and have multiple inflorescence stems, twisted leaves, deformed leaf epidermis pavement cells, and differentially organized cytoskeleton. Microarray analyses of rop6^DN plants revealed that major changes in gene expression are associated with constitutive salicylic acid (SA)-mediated defense responses. In agreement, their free and total SA levels resembled those of wild-type plants inoculated with a virulent powdery mildew pathogen. The constitutive SA-associated response in rop6^DN was suppressed in mutant backgrounds defective in SA signaling (nonexpresser of PR genes1 [npr1]) or biosynthesis (salicylic acid induction deficient2 [sid2]). However, the rop6^DN npr1 and rop6^DN sid2 double mutants retained the aberrant developmental phenotypes, indicating that the constitutive SA response can be uncoupled from ROP function(s) in development. rop6^DN plants exhibited enhanced preinvasive defense responses to a host-adapted virulent powdery mildew fungus but were impaired in preinvasive defenses upon inoculation with a nonadapted powdery mildew. The host-adapted powdery mildew had a reduced reproductive fitness on rop6^DN plants, which was retained in mutant backgrounds defective in SA biosynthesis or signaling. Our findings indicate that both the morphological aberrations and altered sensitivity to powdery mildews of rop6^DN plants result from perturbations that are independent from the SA-associated response. These perturbations uncouple SA-dependent defense signaling from disease resistance execution.Rho of Plants (ROPs), also known as RACs (for clarity, the ROP nomenclature will be used throughout this article), comprise a plant-specific group of Rho family small G proteins. Like other members of the Ras superfamily of small G proteins, ROPs function as molecular switches, existing in a GTP-bound “on” state and a GDP-bound “off” state. In the GTP-bound state, ROPs interact with specific effectors that transduce downstream signaling or function as scaffolds for interaction with additional effector molecules (Berken and Wittinghofer, 2008). Conserved point mutations in the G1 (P loop) Gly-15 or the G3 (switch II) Gln-64, which abolish GTP hydrolysis, or the G1 Thr-20 or G4 Asp-121 that compromise GDP/GTP exchange, can form either constitutively active or dominant negative mutants, respectively (Feig, 1999; Berken et al., 2005; Berken and Wittinghofer, 2008; Sorek et al., 2010). Primarily based on studies with neomorphic mutants, ROPs have been implicated in the regulation of cytoskeleton organization and dynamics, vesicle trafficking, auxin transport and response, abscisic acid (ABA) response, and response to pathogens (Nibau et al., 2006; Yalovsky et al., 2008; Yang, 2008; Lorek et al., 2010; Wu et al., 2011; and refs. therein).In Arabidopsis (Arabidopsis thaliana), there are 11 ROP proteins (Winge et al., 1997). Assigning specific functions to individual members of this family is difficult, however, because ROPs are functionally redundant. A ROP10 loss-of-function mutant was reported to be ABA hypersensitive (Zheng et al., 2002), displaying enhanced expression of tens of genes in response to ABA treatments (Xin et al., 2005). However, in the absence of exogenous ABA, gene expression in the rop10 mutant was similar to that in wild-type plants (Xin et al., 2005). Loss of leaf epidermis pavement cell polarity was reported for rop4 rop2-RNAi (for RNA interference) double mutant plants (Fu et al., 2005). Mild changes in pavement and hypocotyl cell structure and microtubule (MT) organization were reported for a rop6 loss-of-function mutant (Fu et al., 2009).The involvement of ROPs in auxin-regulated development has been addressed in several studies (Wu et al., 2011). Ectopic expression of a dominant negative ROP2 (rop2^DN) mutant under regulation of the 35S promoter resulted in a loss of apical dominance and a reduction in the number of lateral roots. In contrast, ectopic expression of constitutively active ROP2 (rop2^CA) caused an increase in the number of lateral roots and an enhanced decrease in primary root length in response to auxin. Consistent with these findings, the expression of a constitutively active NtRAC1 in tobacco (Nicotiana tabacum) protoplasts induced the expression of auxin-regulated genes in the absence of auxin and promoted the formation of protein nuclear bodies containing components of the proteasome and COP9 signalosome (Tao et al., 2002, 2005; Wu et al., 2011). The ROP effector ICR1 (for interactor of constitutively active ROP1) regulates polarized secretion and is required for polar auxin transport (Lavy et al., 2007; Bloch et al., 2008; Hazak et al., 2010; Hazak and Yalovsky, 2010). In the root, local auxin gradients induce the accumulation of ROPs in trichoblasts at the site of future root hair formation (Fischer et al., 2006). Recently, it was shown that interdigitation of leaf epidermis pavement cells depends on Auxin-Binding Protein1 (ABP1)-mediated ROP activation (Xu et al., 2010). Taken together, these data indicate that ROPs are involved in both mediating the auxin response and facilitating directional auxin transport. It is still unclear, however, which ROPs function in these processes.ROP function was linked to plant defense responses in several studies. In rice (Oryza sativa), OsRAC1 is a positive regulator of the hypersensitive response, possibly through interactions with the NADPH oxidase RbohB, Required for Mla12 Resistance, and Heat Shock Protein90 (Ono et al., 2001; Thao et al., 2007; Wong et al., 2007). Interestingly, other members of the rice ROP family, namely RAC4 and RAC5, are negative regulators of resistance to the rice blast pathogen Magnaporthe grisea (Chen et al., 2010). Similar to rice, when expressed in tobacco, dominant negative OsRAC1 suppressed the hypersensitive response (Moeder et al., 2005). In barley (Hordeum vulgare), several constitutively active ROP/RAC mutants and a MT-associated ROPGAP1 loss-of-function mutant enhanced susceptibility to the powdery mildew Blumeria graminis f. sp. hordei (Bgh). The activated ROP-enhanced susceptibility to Bgh was attributed to disorganization of the actin cytoskeleton and was shown to depend on Mildew Resistance Locus O (MLO; Schultheiss et al., 2002, 2003; Opalski et al., 2005; Hoefle et al., 2011). In barley, three ROP proteins, HvRACB, HvRAC1, and HvRAC3, were linked to both development and pathogen response (Schultheiss et al., 2005; Pathuri et al., 2008; Hoefle et al., 2011).We have analyzed the function of the Arabidopsis AtROP6 (ROP6) by characterizing its expression pattern and its regulation by auxin and the phenotype of plants that express rop6^DN under the regulation of its endogenous promoter. The utilization of the dominant negative mutant overcame functional redundancy, while expression under the regulation of the endogenous promoter enabled the analysis of ROP6 function in a developmental context. Phenotypic and gene expression analyses indicate that ROP6 functions in developmental, salicylic acid (SA)-dependent, and SA-independent defense response pathways.     

AL101, a gamma-secretase inhibitor,has potent antitumor activity against adenoid cystic carcinoma with activated NOTCH signaling

Adenoid cystic carcinoma (ACC) is an aggressive salivary gland malignancy with limited treatment options for recurrent or metastatic disease. Due to chemotherapy resistance and lack of targeted therapeutic approaches, current treatment options for the localized disease are limited to surgery and radiation, which fails to prevent locoregional recurrences and distant metastases in over 50% of patients. Approximately 20% of patients with ACC carry NOTCH-activating mutations that are associated with a distinct phenotype, aggressive disease, and poor prognosis. Given the role of NOTCH signaling in regulating tumor cell behavior, NOTCH inhibitors represent an attractive potential therapeutic strategy for this subset of ACC. AL101 (osugacestat) is a potent γ-secretase inhibitor that prevents activation of all four NOTCH receptors. While this investigational new drug has demonstrated antineoplastic activity in several preclinical cancer models and in patients with advanced solid malignancies, we are the first to study the therapeutic benefit of AL101 in ACC. Here, we describe the antitumor activity of AL101 using ACC cell lines, organoids, and patient-derived xenograft models. Specifically, we find that AL101 has potent antitumor effects in in vitro and in vivo models of ACC with activating NOTCH1 mutations and constitutively upregulated NOTCH signaling pathway, providing a strong rationale for evaluation of AL101 in clinical trials for patients with NOTCH-driven relapsed/refractory ACC.Subject terms: Head and neck cancer, Targeted therapies     

Adaptor Protein GRB2 Promotes Src Tyrosine Kinase Activation and Podosomal Organization by Protein-tyrosine Phosphatase ? in Osteoclasts

The non-receptor isoform of protein-tyrosine phosphatase ϵ (cyt-PTPe) supports adhesion of bone-resorbing osteoclasts by activating Src downstream of integrins. Loss of cyt-PTPe reduces Src activity in osteoclasts, reduces resorption of mineralized matrix both in vivo and in cell culture, and induces mild osteopetrosis in young female PTPe KO mice. Activation of Src by cyt-PTPe is dependent upon this phosphatase undergoing phosphorylation at its C-terminal Tyr-638 by partially active Src. To understand how cyt-PTPe activates Src, we screened 73 Src homology 2 (SH2) domains for binding to Tyr(P)-638 of cyt-PTPe. The SH2 domain of GRB2 bound Tyr(P)-638 of cyt-PTPe most prominently, whereas the Src SH2 domain did not bind at all, suggesting that GRB2 may link PTPe with downstream molecules. Further studies indicated that GRB2 is required for activation of Src by cyt-PTPe in osteoclast-like cells (OCLs) in culture. Overexpression of GRB2 in OCLs increased activating phosphorylation of Src at Tyr-416 and of cyt-PTPe at Tyr-638; opposite results were obtained when GRB2 expression was reduced by shRNA or by gene inactivation. Phosphorylation of cyt-PTPe at Tyr-683 and its association with GRB2 are integrin-driven processes in OCLs, and cyt-PTPe undergoes autodephosphorylation at Tyr-683, thus limiting Src activation by integrins. Reduced GRB2 expression also reduced the ability of bone marrow precursors to differentiate into OCLs and reduced the fraction of OCLs in which podosomal adhesion structures assume organization typical of active, resorbing cells. We conclude that GRB2 physically links cyt-PTPe with Src and enables cyt-PTPe to activate Src downstream of activated integrins in OCLs.