53BP1 interacts with the RNA primer from Okazaki fragments to support their processing during unperturbed DNA replication.

RNA-binding proteins (RBPs) are found at replication forks, but their direct interaction with DNA-embedded RNA species remains unexplored. Here, we report that p53-binding protein 1 (53BP1), involved in the DNA damage and replication stress response, is an RBP that directly interacts with Okazaki fragments in the absence of external stress. The recruitment of 53BP1 to nascent DNA shows susceptibility to in situ ribonuclease A treatment and is dependent on PRIM1, which synthesizes the RNA primer of Okazaki fragments. Conversely, depletion of FEN1, resulting in the accumulation of uncleaved RNA primers, increases 53BP1 levels at replication forks, suggesting that RNA primers contribute to the recruitment of 53BP1 at the lagging DNA strand. 53BP1 depletion induces an accumulation of S-phase poly(ADP-ribose), which constitutes a sensor of unligated Okazaki fragments. Collectively, our data indicate that 53BP1 is anchored at nascent DNA through its RNA-binding activity, highlighting the role of an RNA-protein interaction at replication forks.

Close Ties between the Nuclear Envelope and Mammalian Telomeres: Give Me Shelter.

The nuclear envelope (NE) in eukaryotic cells is essential to provide a protective compartment for the genome. Beside its role in connecting the nucleus with the cytoplasm, the NE has numerous important functions including chromatin organization, DNA replication and repair. NE alterations have been linked to different human diseases, such as laminopathies, and are a hallmark of cancer cells. Telomeres, the ends of eukaryotic chromosomes, are crucial for preserving genome stability. Their maintenance involves specific telomeric proteins, repair proteins and several additional factors, including NE proteins. Links between telomere maintenance and the NE have been well established in yeast, in which telomere tethering to the NE is critical for their preservation and beyond. For a long time, in mammalian cells, except during meiosis, telomeres were thought to be randomly localized throughout the nucleus, but recent advances have uncovered close ties between mammalian telomeres and the NE that play important roles for maintaining genome integrity. In this review, we will summarize these connections, with a special focus on telomere dynamics and the nuclear lamina, one of the main NE components, and discuss the evolutionary conservation of these mechanisms.

53BP1: Keeping It under Control, Even at a Distance from DNA Damage.

Double-strand breaks (DSBs) are toxic lesions that can be generated by exposure to genotoxic agents or during physiological processes, such as during V(D)J recombination. The repair of these DSBs is crucial to prevent genomic instability and to maintain cellular homeostasis. Two main pathways participate in repairing DSBs, namely, non-homologous end joining (NHEJ) and homologous recombination (HR). The P53-binding protein 1 (53BP1) plays a pivotal role in the choice of DSB repair mechanism, promotes checkpoint activation and preserves genome stability upon DSBs. By preventing DSB end resection, 53BP1 promotes NHEJ over HR. Nonetheless, the balance between DSB repair pathways remains crucial, as unscheduled NHEJ or HR events at different phases of the cell cycle may lead to genomic instability. Therefore, the recruitment of 53BP1 to chromatin is tightly regulated and has been widely studied. However, less is known about the mechanism regulating 53BP1 recruitment at a distance from the DNA damage. The present review focuses on the mechanism of 53BP1 recruitment to damage and on recent studies describing novel mechanisms keeping 53BP1 at a distance from DSBs.

Phospho-Ku70 induced by DNA damage interacts with RNA Pol II and promotes the formation of phospho-53BP1 foci to ensure optimal cNHEJ.

Canonical non-homologous end-joining (cNHEJ) is the prominent mammalian DNA double-strand breaks (DSBs) repair pathway operative throughout the cell cycle. Phosphorylation of Ku70 at ser27-ser33 (pKu70) is induced by DNA DSBs and has been shown to regulate cNHEJ activity, but the underlying mechanism remained unknown. Here, we established that following DNA damage induction, Ku70 moves from nucleoli to the sites of damage, and once linked to DNA, it is phosphorylated. Notably, the novel emanating functions of pKu70 are evidenced through the recruitment of RNA Pol II and concomitant formation of phospho-53BP1 foci. Phosphorylation is also a prerequisite for the dynamic release of Ku70 from the repair complex through neddylation-dependent ubiquitylation. Although the non-phosphorylable ala-Ku70 form does not compromise the formation of the NHEJ core complex per se, cells expressing this form displayed constitutive and stress-inducible chromosomal instability. Consistently, upon targeted induction of DSBs by the I-SceI meganuclease into an intrachromosomal reporter substrate, cells expressing pKu70, rather than ala-Ku70, are protected against the joining of distal DNA ends. Collectively, our results underpin the essential role of pKu70 in the orchestration of DNA repair execution in living cells and substantiated the way it paves the maintenance of genome stability.

Increase in lamin B1 promotes telomere instability by disrupting the shelterin complex in human cells.

Telomere maintenance is essential to preserve genomic stability and involves telomere-specific proteins, DNA replication and repair proteins. Lamins are key components of the nuclear envelope and play numerous roles, including maintenance of the nuclear integrity, regulation of transcription, and DNA replication. Elevated levels of lamin B1, one of the major lamins, have been observed in some human pathologies and several cancers. Yet, the effect of lamin B1 dysregulation on telomere maintenance remains unknown. Here, we unveil that lamin B1 overexpression drives telomere instability through the disruption of the shelterin complex. Indeed, lamin B1 dysregulation leads to an increase in telomere dysfunction-induced foci, telomeric fusions and telomere losses in human cells. Telomere aberrations were preceded by mislocalizations of TRF2 and its binding partner RAP1. Interestingly, we identified new interactions between lamin B1 and these shelterin proteins, which are strongly enhanced at the nuclear periphery upon lamin B1 overexpression. Importantly, chromosomal fusions induced by lamin B1 in excess were rescued by TRF2 overexpression. These data indicated that lamin B1 overexpression triggers telomere instability through a mislocalization of TRF2. Altogether our results point to lamin B1 as a new interacting partner of TRF2, that is involved in telomere stability.

Lamin B1 sequesters 53BP1 to control its recruitment to DNA damage.

Double-strand breaks (DSBs) are harmful lesions and a major cause of genome instability. Studies have suggested a link between the nuclear envelope and the DNA damage response. Here, we show that lamin B1, a major component of the nuclear envelope, interacts directly with 53BP1 protein, which plays a pivotal role in the DSB repair. This interaction is dissociated after DNA damage. Lamin B1 overexpression impedes 53BP1 recruitment to DNA damage sites and leads to a persistence of DNA damage, a defect in nonhomologous end joining and an increased sensitivity to DSBs. The identification of interactions domains between lamin B1 and 53BP1 allows us to demonstrate that the defect of 53BP1 recruitment and the DSB persistence upon lamin B1 overexpression are due to sequestration of 53BP1 by lamin B1. This study highlights lamin B1 as a factor controlling the recruitment of 53BP1 to DNA damage sites upon injury.

A Link between Replicative Stress, Lamin Proteins, and Inflammation.

Double-stranded breaks (DSB), the most toxic DNA lesions, are either a consequence of cellular metabolism, programmed as in during V(D)J recombination, or induced by anti-tumoral therapies or accidental genotoxic exposure. One origin of DSB sources is replicative stress, a major source of genome instability, especially when the integrity of the replication forks is not properly guaranteed. To complete stalled replication, restarting the fork requires complex molecular mechanisms, such as protection, remodeling, and processing. Recently, a link has been made between DNA damage accumulation and inflammation. Indeed, defects in DNA repair or in replication can lead to the release of DNA fragments in the cytosol. The recognition of this self-DNA by DNA sensors leads to the production of inflammatory factors. This beneficial response activating an innate immune response and destruction of cells bearing DNA damage may be considered as a novel part of DNA damage response. However, upon accumulation of DNA damage, a chronic inflammatory cellular microenvironment may lead to inflammatory pathologies, aging, and progression of tumor cells. Progress in understanding the molecular mechanisms of DNA damage repair, replication stress, and cytosolic DNA production would allow to propose new therapeutical strategies against cancer or inflammatory diseases associated with aging. In this review, we describe the mechanisms involved in DSB repair, the replicative stress management, and its consequences. We also focus on new emerging links between key components of the nuclear envelope, the lamins, and DNA repair, management of replicative stress, and inflammation.

[Aging: A matter of DNA damage, nuclear envelope alterations and inflammation?] / Le vieillissement - Une histoire de dommages de l'ADN, d'enveloppe nucléaire altérée et d'inflammation ?

The accumulation of senescent cells accompanies organismal aging. Senescent cells produce an inflammatory microenvironment that is conducive to the development of many age-related diseases. Here we describe the different situations leading to cellular senescence and show that these situations are frequently associated with DNA damage. We also discuss the intimate link between cell aging and perturbations in the nuclear envelope, namely in nuclear lamins, as seen in progeroid syndromes. Finally, we present evidence that these alterations are associated with DNA repair defects, the persistence of DNA damage, and an inflammatory phenotype.

TITLE: Le vieillissement - Une histoire de dommages de l'ADN, d'enveloppe nucléaire altérée et d'inflammation ? ABSTRACT: Le vieillissement est associé à une accumulation de cellules sénescentes produisant un environnement cellulaire inflammatoire qui pourrait expliquer différentes maladies liées à l'âge. Diverses situations menant à la sénescence sont liées à la présence de dommages de l'ADN. De plus, de nombreux syndromes progéroïdes sont associés à une instabilité du génome ou de la structure nucléaire. Nous discuterons du lien étroit existant entre l'altération des lamines, composants de l'enveloppe nucléaire, et le vieillissement cellulaire. Nous verrons que l'altération de l'enveloppe nucléaire, comme celle observée dans la Progéria, est aussi associée à des défauts de réparation de l'ADN, à une persistance de dommages de l'ADN et à un phénotype inflammatoire.

Anticancer chemotherapy and radiotherapy trigger both non-cell-autonomous and cell-autonomous death.

Even though cell death modalities elicited by anticancer chemotherapy and radiotherapy have been extensively studied, the ability of anticancer treatments to induce non-cell-autonomous death has never been investigated. By means of multispectral imaging flow-cytometry-based technology, we analyzed the lethal fate of cancer cells that were treated with conventional anticancer agents and co-cultured with untreated cells, observing that anticancer agents can simultaneously trigger cell-autonomous and non-cell-autonomous death in treated and untreated cells. After ionizing radiation, oxaliplatin, or cisplatin treatment, fractions of treated cancer cell populations were eliminated through cell-autonomous death mechanisms, while other fractions of the treated cancer cells engulfed and killed neighboring cells through non-cell-autonomous processes, including cellular cannibalism. Under conditions of treatment with paclitaxel, non-cell-autonomous and cell-autonomous death were both detected in the treated cell population, while untreated neighboring cells exhibited features of apoptotic demise. The transcriptional activity of p53 tumor-suppressor protein contributed to the execution of cell-autonomous death, yet failed to affect the non-cell-autonomous death by cannibalism for the majority of tested anticancer agents, indicating that the induction of non-cell-autonomous death can occur under conditions in which cell-autonomous death was impaired. Altogether, these results reveal that chemotherapy and radiotherapy can induce both non-cell-autonomous and cell-autonomous death of cancer cells, highlighting the heterogeneity of cell death responses to anticancer treatments and the unsuspected potential contribution of non-cell-autonomous death to the global effects of anticancer treatment.

A nonsense mutation in the DNA repair factor Hebo causes mild bone marrow failure and microcephaly.

Inherited bone marrow failure syndromes are human conditions in which one or several cell lineages of the hemopoietic system are affected. They are present at birth or may develop progressively. They are sometimes accompanied by other developmental anomalies. Three main molecular causes have been recognized to result in bone marrow failure syndromes: (1) defects in the Fanconi anemia (FA)/BRCA DNA repair pathway, (2) defects in telomere maintenance, and (3) abnormal ribosome biogenesis. We analyzed a patient with mild bone marrow failure and microcephaly who did not present with the typical FA phenotype. Cells from this patient showed increased sensitivity to ionizing radiations and phleomycin, attesting to a probable DNA double strand break (dsb) repair defect. Linkage analysis and whole exome sequencing revealed a homozygous nonsense mutation in the ERCC6L2 gene. We identified a new ERCC6L2 alternative transcript encoding the DNA repair factor Hebo, which is critical for complementation of the patient's DNAdsb repair defect. Sequence analysis revealed three structured regions within Hebo: a TUDOR domain, an adenosine triphosphatase domain, and a new domain, HEBO, specifically present in Hebo direct orthologues. Hebo is ubiquitously expressed, localized in the nucleus, and rapidly recruited to DNAdsb's in an NBS1-dependent manner.

Control of alternative end joining by the chromatin remodeler p400 ATPase.

Repair of DNA double-strand breaks occurs in a chromatin context that needs to be modified and remodeled to allow suitable access to the different DNA repair machineries. Of particular importance for the maintenance of genetic stability is the tight control of error-prone pathways, such as the alternative End Joining pathway. Here, we show that the chromatin remodeler p400 ATPase is a brake to the use of alternative End Joining. Using specific intracellular reporter susbstrates we observed that p400 depletion increases the frequency of alternative End Joining events, and generates large deletions following repair of double-strand breaks. This increase of alternative End Joining events is largely dependent on CtIP-mediated resection, indicating that it is probably related to the role of p400 in late steps of homologous recombination. Moreover, p400 depletion leads to the recruitment of poly(ADP) ribose polymerase (PARP) and DNA ligase 3 at DNA double-strand breaks, driving to selective killing by PARP inhibitors. All together these results show that p400 acts as a brake to prevent alternative End Joining-dependent genetic instability and underline its potential value as a clinical marker.

Impact of the MRN Complex on Adeno-Associated Virus Integration and Replication during Coinfection with Herpes Simplex Virus 1.

UNLABELLED: Adeno-associated virus (AAV) is a helper-dependent parvovirus that requires coinfection with adenovirus (AdV) or herpes simplex virus 1 (HSV-1) to replicate. In the absence of the helper virus, AAV can persist in an episomal or integrated form. Previous studies have analyzed the DNA damage response (DDR) induced upon AAV replication to understand how it controls AAV replication. In particular, it was shown that the Mre11-Rad50-Nbs1 (MRN) complex, a major player of the DDR induced by double-stranded DNA breaks and stalled replication forks, could negatively regulate AdV and AAV replication during coinfection. In contrast, MRN favors HSV-1 replication and is recruited to AAV replication compartments that are induced in the presence of HSV-1. In this study, we examined the role of MRN during AAV replication induced by HSV-1. Our results indicated that knockdown of MRN significantly reduced AAV DNA replication after coinfection with wild-type (wt) HSV-1 or HSV-1 with the polymerase deleted. This effect was specific to wt AAV, since it did not occur with recombinant AAV vectors. Positive regulation of AAV replication by MRN was dependent on its DNA tethering activity but did not require its nuclease activities. Importantly, knockdown of MRN also negatively regulated AAV integration within the human AAVS1 site, both in the presence and in the absence of HSV-1. Altogether, this work identifies a new function of MRN during integration of the AAV genome and demonstrates that this DNA repair complex positively regulates AAV replication in the presence of HSV-1. IMPORTANCE: Viral DNA genomes trigger a DNA damage response (DDR), which can be either detrimental or beneficial for virus replication. Adeno-associated virus (AAV) is a defective parvovirus that requires the help of an unrelated virus such as adenovirus (AdV) or herpes simplex virus 1 (HSV-1) for productive replication. Previous studies have demonstrated that the cellular Mre11-Rad50-Nbs1 (MRN) complex, a sensor and regulator of the DDR, negatively regulates AAV replication during coinfection with AdV, which counteracts this effect by inactivating the complex. Here, we demonstrate that MRN positively regulates AAV replication during coinfection with HSV-1. Importantly, our study also indicates that MRN also favors integration of AAV genomes within the human AAVS1 site. Altogether, this work indicates that MRN differentially regulates AAV replication depending on the helper virus which is present and identifies a new function of this DNA repair complex during AAV integration.

Is homologous recombination really an error-free process?

Homologous recombination (HR) is an evolutionarily conserved process that plays a pivotal role in the equilibrium between genetic stability and diversity. HR is commonly considered to be error-free, but several studies have shown that HR can be error-prone. Here, we discuss the actual accuracy of HR. First, we present the product of genetic exchanges (gene conversion, GC, and crossing over, CO) and the mechanisms of HR during double strand break repair and replication restart. We discuss the intrinsic capacities of HR to generate genome rearrangements by GC or CO, either during DSB repair or replication restart. During this process, abortive HR intermediates generate genetic instability and cell toxicity. In addition to genome rearrangements, HR also primes error-prone DNA synthesis and favors mutagenesis on single stranded DNA, a key DNA intermediate during the HR process. The fact that cells have developed several mechanisms protecting against HR excess emphasize its potential risks. Consistent with this duality, several pro-oncogenic situations have been consistently associated with either decreased or increased HR levels. Nevertheless, this versatility also has advantages that we outline here. We conclude that HR is a double-edged sword, which on one hand controls the equilibrium between genome stability and diversity but, on the other hand, can jeopardize the maintenance of genomic integrity. Therefore, whether non-homologous end joining (which, in contrast with HR, is not intrinsically mutagenic) or HR is the more mutagenic process is a question that should be re-evaluated. Both processes can be "Dr. Jekyll" in maintaining genome stability/variability and "Mr. Hyde" in jeopardizing genome integrity.

Is non-homologous end-joining really an inherently error-prone process?

DNA double-strand breaks (DSBs) are harmful lesions leading to genomic instability or diversity. Non-homologous end-joining (NHEJ) is a prominent DSB repair pathway, which has long been considered to be error-prone. However, recent data have pointed to the intrinsic precision of NHEJ. Three reasons can account for the apparent fallibility of NHEJ: 1) the existence of a highly error-prone alternative end-joining process; 2) the adaptability of canonical C-NHEJ (Ku- and Xrcc4/ligase IV-dependent) to imperfect complementary ends; and 3) the requirement to first process chemically incompatible DNA ends that cannot be ligated directly. Thus, C-NHEJ is conservative but adaptable, and the accuracy of the repair is dictated by the structure of the DNA ends rather than by the C-NHEJ machinery. We present data from different organisms that describe the conservative/versatile properties of C-NHEJ. The advantages of the adaptability/versatility of C-NHEJ are discussed for the development of the immune repertoire and the resistance to ionizing radiation, especially at low doses, and for targeted genome manipulation.

A role for BLM in double-strand break repair pathway choice: prevention of CtIP/Mre11-mediated alternative nonhomologous end-joining.

The choice of the appropriate double-strand break (DSB) repair pathway is essential for the maintenance of genomic stability. Here, we show that the Bloom syndrome gene product, BLM, counteracts CtIP/MRE11-dependent long-range deletions (>200 bp) generated by alternative end-joining (A-EJ). BLM represses A-EJ in an epistatic manner with 53BP1 and RIF1 and is required for ionizing-radiation-induced 53BP1 focus assembly. Conversely, in the absence of 53BP1 or RIF1, BLM promotes formation of A-EJ long deletions, consistent with a role for BLM in DSB end resection. These data highlight a dual role for BLM that influences the DSB repair pathway choice: (1) protection against CtIP/MRE11 long-range deletions associated with A-EJ and (2) promotion of DNA resection. These antagonist roles can be regulated, according to cell-cycle stage, by interacting partners such as 53BP1 and TopIII, to avoid unscheduled resection that might jeopardize genome integrity.

Oxydative stress alters nuclear shape through lamins dysregulation: a route to senescence.

Progeroid phenotypes are mainly encountered in 2 types of syndromes: in laminopathies, which are characterized by nuclear shape abnormalities due to lamin A alteration, and in DNA damage response defect syndromes. Because lamin A dysregulation leads to DNA damages, it has been proposed that senescence occurs in both types of syndromes through the accumulation of damages. We recently showed that elevated oxidative stress is responsible for lamin B1 accumulation, nuclear shape alteration and senescence in the DDR syndrome, ataxia telangiectasia (A-T). Interestingly, overexpression of lamin B1 in wild type cells is sufficient to induce senescence without the induction of DNA damages. Here, we will discuss the importance of controlling the lamins level in order for maintenance nuclear architecture and we will comment the relationships of lamins with other senescence mechanisms. Finally, we will describe emerging data reporting redox control by lamins, leading us to propose a general mechanism by which reactive oxygen species can induce senescence through lamin dysregulation and NSA.

Terminal deoxynucleotidyl transferase requires KU80 and XRCC4 to promote N-addition at non-V(D)J chromosomal breaks in non-lymphoid cells.

Terminal deoxynucleotidyl transferase (TdT) is a DNA polymerase that increases the repertoire of antigen receptors by adding non-templated nucleotides (N-addition) to V(D)J recombination junctions. Despite extensive in vitro studies on TdT catalytic activity, the partners of TdT that enable N-addition remain to be defined. Using an intrachromosomal substrate, we show here that, in Chinese hamter ovary (CHO) cells, ectopic expression of TdT efficiently promotes N-additions at the junction of chromosomal double-strand breaks (DSBs) generated by the meganuclease I-SceI and that the size of the N-additions is comparable with that at V(D)J junctions. Importantly, no N-addition was observed in KU80- or XRCC4-deficient cells. These data show that, in a chromosomal context of non-lymphoid cells, TdT is actually able to promote N-addition at non-V(D)J DSBs, through a process that strictly requires the components of the canonical non-homologous end-joining pathway, KU80 and XRCC4.

Accumulation of cyclophilin A isoforms in conditioned medium of irradiated breast cancer cells.

Secreted proteins play a key role in cell signaling and communication. We recently showed that ionizing radiations induced a delayed cell death of breast cancer cells, mediated by the death receptor pathways through the expression of soluble forms of "death ligands." Using the same cell model, the objective of our work was the identification of diffusible factors, secreted following cell irradiation, potentially involved in cell death signaling. Differential proteomic analysis of conditioned media using 2DE resulted in detection of numerous spots that were significantly modulated following cell irradiation. The corresponding proteins were identified using MALDI-TOF MS and LC-MS/MS approaches. Interestingly, five isoforms of cyclophilin A were observed as increased in conditioned medium of irradiated cells. These isoforms differed in isoelectric points and in accumulation levels. An increase of cyclophilin A secretion was confirmed by Western blotting of conditioned media of irradiated or radiosentive mammary cells. These isoforms displayed an interesting pattern of protein maturation and post-translational modifications, including an alternating removal of N-terminal methionine, associated with a combination of acetylations and methylations. The role of the protein is discussed in relation with its potential involvement in the mechanisms of intercells relationships and radiosensitivity.

Oxidative stress induces an ATM-independent senescence pathway through p38 MAPK-mediated lamin B1 accumulation.

We report crosstalk between three senescence-inducing conditions, DNA damage response (DDR) defects, oxidative stress (OS) and nuclear shape alterations. The recessive autosomal genetic disorder Ataxia telangiectasia (A-T) is associated with DDR defects, endogenous OS and premature ageing. Here, we find frequent nuclear shape alterations in A-T cells, as well as accumulation of the key nuclear architecture component lamin B1. Lamin B1 overexpression is sufficient to induce nuclear shape alterations and senescence in wild-type cells, and normalizing lamin B1 levels in A-T cells reciprocally reduces both nuclear shape alterations and senescence. We further show that OS increases lamin B1 levels through p38 Mitogen Activated Protein kinase activation. Lamin B1 accumulation and nuclear shape alterations also occur during stress-induced senescence and oncogene-induced senescence (OIS), two canonical senescence situations. These data reveal lamin B1 as a general molecular mediator that controls OS-induced senescence, independent of established Ataxia Telangiectasia Mutated (ATM) roles in OIS.

Paving the way for invasive species: road type and the spread of common ragweed (Ambrosia artemisiifolia).

Roads function as prime habitats and corridors for invasive plant species. Yet despite the diversity of road types, there is little research on the influence of these types on the spread of invaders. Common ragweed (Ambrosia artemisiifolia), a plant producing large amounts of allergenic pollen, was selected as a species model for examining the impact of road type on the spread of invasive plants. We examined this relationship in an agricultural region of Quebec, Canada. We mapped plant distribution along different road types, and constructed a model of species presence. Common ragweed was found in almost all sampling sites located along regional (97%) and local paved (81%) roads. However, verges of unpaved local roads were rarely (13%) colonized by the plant. A model (53% of variance explained), constructed with only four variables (paved regional roads, paved local roads, recently mown road verges, forest cover), correctly predicted (success rate: 89%) the spatial distribution of common ragweed. Results support the hypothesis that attributes associated with paved roads strongly favour the spread of an opportunistic invasive plant species. Specifically, larger verges and greater disturbance associated with higher traffic volume create propitious conditions for common ragweed. To date, emphasis has been placed on controlling the plant in agricultural fields, even though roadsides are probably a much larger seed source. Strategies for controlling the weed along roads have only focused on major highways, even though the considerable populations along local roads also contribute to the production of pollen. Management prioritizations developed to control common ragweed are thus questionable.