Plant Milking Technology-An Innovative and Sustainable Process to Produce Highly Active Extracts from Plant Roots.

We have used an original technology (Plant Milking Technology) based on aeroponic cultivation of plants associated with the gentle recovery of active ingredients from roots. Extraction of bioactive molecules was achieved by soaking the roots, still attached to the living plants, into a nontoxic solvent for a 2 h period. This nondestructive recovery process allows using the same root biomass for successive harvesting dates, in a recyclable way. We have applied this technology to Morus alba L. (mulberry tree), an emblematic tree of the Traditional Chinese Medicine (TCM). Trees were aeroponically grown in large-scale devices (100 m2) and were submitted to nitrogen deprivation to increase the content in active molecules (prenylated flavonoids). The Plant Milking technology applied to Morus alba L. allowed to produce an extract enriched in prenylated compounds (18-fold increase when compared to commercial root extract). Prenylated flavonoids (moracenin A and B, kuwanon C, wittiorumin F, morusin) presented a high affinity for the aged-associated collagenase enzyme, which was confirmed by activity inhibition. In accordance, M. alba extract presents efficient properties to regulate the skin matrisome, which is critical during skin aging. The benefits have been especially confirmed in vivo on wrinkle reduction, in a clinical study that involved aged women. Plant Milking technology is an optimal solution to produce active ingredients from plant roots, including trees, that meet both customer expectations around sustainability, as well as the need for an efficient production system for biotechnologists.

nc886, a non-coding RNA, inhibits UVB-induced MMP-9 and COX-2 expression via the PKR pathway in human keratinocytes.

nc886, a long non-coding RNA (ncRNA) of 101 nucleotides in length, is known as a vault RNA or microRNA precursor. Despite the recent discovery that ncRNAs in the nucleus play a crucial role in regulating chromosomal transformation and transcription, only a few studies have focused on the function of ncRNAs in the cytoplasm, such as nc886. Several studies have investigated the function of nc886 as a suppressor of carcinogenesis and inflammation in different cancer cell types; however, its role in the skin has yet to be clearly elucidated. The two RNA binding sites for protein kinase RNA-activated (PKR) are located in the central region of the stable structure of nc886, which competes with other double-stranded RNA species. Successful binding results in decreased PKR activity. Among changes in skin cells induced by ultraviolet B (UVB) radiation, nc886 expression decreases, whereas PKR phosphorylation via mitogen-activated protein kinases (MAPKs) increases. Reduced nc886 expression leads to uncontrolled PKR activity and increases in the expression of inflammatory cytokines, matrix metalloproteinase-9 (MMP-9), type IV collagenase, and cyclooxygenase (COX-2), which ultimately accelerate inflammatory responses and skin aging. The present study investigated the regulatory mechanism associated with PKR activity and nc886-PKR binding in skin cell aging and inflammation. These results suggest a role for nc886 in controlling photoaging and inflammation in skin cells.

Quantitative imaging and semiotic phenotyping of mitochondrial network morphology in live human cells.

The importance of mitochondria in tissue homeostasis, stress responses and human diseases, combined to their ability to transition between various structural and functional states, makes them excellent organelles for monitoring cell health. There is therefore a need for technologies to accurately analyze and quantify changes in mitochondrial organization in a variety of cells and cellular contexts. Here we present an innovative computerized method that enables accurate, multiscale, fast and cost-effective analysis of mitochondrial shape and network architecture from confocal fluorescence images by providing more than thirty features. In order to facilitate interpretation of the quantitative results, we introduced two innovations: the use of Kiviat-graphs (herein named MitoSpider plots) to present highly multidimensional data and visualization of the various mito-cellular configurations in the form of morphospace diagrams (called MitoSigils). We tested our fully automated image analysis tool on rich datasets gathered from live normal human skin cells cultured under basal conditions or exposed to specific stress including UVB irradiation and pesticide exposure. We demonstrated the ability of our proprietary software (named MitoTouch) to sensitively discriminate between control and stressed dermal fibroblasts, and between normal fibroblasts and other cell types (including cancer tissue-derived fibroblasts and primary keratinocytes), showing that our automated analysis captures subtle differences in morphology. Based on this novel algorithm, we report the identification of a protective natural ingredient that mitigates the deleterious impact of hydrogen peroxide (H2O2) on mitochondrial organization. Hence we conceived a novel wet-plus-dry pipeline combining cell cultures, quantitative imaging and semiotic analysis for exhaustive analysis of mitochondrial morphology in living adherent cells. Our tool has potential for broader applications in other research areas such as cell biology and medicine, high-throughput drug screening as well as predictive and environmental toxicology.

Senotherapeutic-like effect of Silybum marianum flower extract revealed on human skin cells.

Cellular senescence causes irreversible growth arrest of cells. Prolonged accumulation of senescent cells in tissues leads to increased detrimental effects due to senescence associated secretory phenotype (SASP). Recent findings suggest that elimination of senescent cells has a beneficial effect on organismal aging and lifespan. In this study, using a validated replicative senescent human dermal fibroblasts (HDFs) model, we showed that elimination of senescent cells is possible through the activation of an apoptotic mechanism. We have shown in this replicative senescence model, that cell senescence is associated with DNA damage and cell cycle arrest (p21, p53 markers). We have shown that Silybum marianum flower extract (SMFE) is a safe and selective senolytic agent targeting only senescent cells. The elimination of the cells is induced through the activation of apoptotic pathway confirmed by annexin V/propidium iodide and caspase-3/PARP staining. Moreover, SMFE suppresses the expression of SASP factors such as IL-6 and MMP-1 in senescent HDFs. In a co-culture model of senescent and young fibroblasts, we demonstrated that senescent cells impaired the proliferative capacities of young cells. Interestingly, when the co-culture is treated with SMFE, the cell proliferation rate of young cells is increased due to the decrease of the senescent burden. Moreover, we demonstrated in vitro that senescent fibroblasts trigger senescent process in normal keratinocytes through a paracrine effect. Indeed, the conditioned medium of senescent HDFs treated with SMFE reduced the level of senescence-associated beta-galactosidase (SA-ß-Gal), p16INK4A and SASP factors in keratinocytes compared with CM of senescent HDFs. These results indicate that SMFE can prevent premature aging due to senescence and even reprograms aged skin. Indeed, thanks to its senolytic and senomorphic properties SMFE is a candidate for anti-senescence strategies.

Mitochondrial morphology is associated with respiratory chain uncoupling in autism spectrum disorder.

Autism spectrum disorder (ASD) is a neurodevelopmental disorder that is associated with unique changes in mitochondrial metabolism, including elevated respiration rates and morphological alterations. We examined electron transport chain (ETC) complex activity in fibroblasts derived from 18 children with ASD as well as mitochondrial morphology measurements in fibroblasts derived from the ASD participants and four typically developing controls. In ASD participants, symptoms severity was measured by the Social Responsiveness Scale and Aberrant Behavior Checklist. Mixed-model regression demonstrated that alterations in mitochondrial morphology were associated with both ETC Complex I+III and IV activity as well as the difference between ETC Complex I+III and IV activity. The subgroup of ASD participants with relative elevation in Complex IV activity demonstrated more typical mitochondrial morphology and milder ASD related symptoms. This study is limited by sample size given the invasive nature of obtaining fibroblasts from children. Furthermore, since mitochondrial function is heterogenous across tissues, the result may be specific to fibroblast respiration. Previous studies have separately described elevated ETC Complex IV activity and changes in mitochondrial morphology in cells derived from children with ASD but this is the first study to link these two findings in mitochondrial metabolism. The association between a difference in ETC complex I+III and IV activity and normal morphology suggests that mitochondrial in individuals with ASD may require ETC uncoupling to function optimally. Further studies should assess the molecular mechanisms behind these unique metabolic changes.Trial registration: Protocols used in this study were registered in clinicaltrials.gov as NCT02000284 and NCT02003170.

Inhibition of UVB-Induced Inflammation by Laminaria japonica Extract via Regulation of nc886-PKR Pathway.

Continuous exposure to ultraviolet B (UVB) can cause photodamage of the skin. This photodamage can be inhibited by the overexpression of the non-coding RNA, nc886, via the protein kinase RNA-activated (PKR) pathway. The study aims to identify how UVB inhibits nc886 expression, and it also seeks to determine whether substances that can control nc886 expression can influence UV-induced inflammation, and the mechanisms involved. The results suggest that UVB irradiation accelerates the methylation of the nc886 gene, therefore, reducing its expression. This induces the activation of the PKR, which accelerates the expression of metalloproteinase-9 (MMP-9) and cyclooxygenase (COX-2), and the production of MMP-9, prostaglandin-endoperoxide synthase (PGE2), and certain pro-inflammatory cytokines, specifically interleukin-8 (IL-8), and tumor necrosis factor- (TNF-). Conversely, in a model of nc886 overexpression, the expression and production of those inflammatory factors are inhibited. In addition, Laminaria japonica extract (LJE) protect the levels of nc886 against UVB irradiation then subsequently inhibit the production of UV-induced inflammatory factors through the PKR pathway.

Complementation assays adapted for DNA repair-deficient keratinocytes.

Genetic alterations affecting nucleotide excision repair, the most versatile DNA-repair mechanism responsible for removal of bulky DNA adducts including ultraviolet (UV) light-induced DNA lesions, may result in the rare, recessively inherited autosomal syndromes xeroderma pigmentosum (XP), Cockayne syndrome (CS), or trichothiodystrophy (TTD). Classical approaches such as somatic cell fusions or microinjection assays have formalized the genetic complexity of these related but clinically distinct syndromes, and contributed to the determination of seven, five, and three complementation groups for XP, CS, and TTD, respectively. XP patients are highly susceptible to photoinduced cutaneous cancers of epidermal origin. To better study the responses to UV irradiation of XP keratinocytes, and to objectively determine the extent to which cutaneous gene therapy may be realized, we set up experimental procedures adapted to ex vivo genetic complementation of keratinocytes from XP patients. We provide here detailed rationales and procedures for these approaches.

Deregulated DNA polymerase beta strengthens ionizing radiation-induced nucleotidic and chromosomal instabilities.

DNA polymerase beta (Pol beta) is an error-prone enzyme which has been found to be overexpressed in several human tumors. By using a couple of recombinant CHO cells differing only from the exogenous expression of Pol beta, we showed here that cells overexpressing Pol beta are much more sensitive to IR treatments by increasing apoptosis. We also found that the surviving cells displayed an hypermutator phenotype which could be explained by different pathways involving Pol beta, such as (i) an increased capacity to incorporate into DNA the mutagenic dGTP analog, 8-oxo-dGTP, one of the most abundant purine-derived nucleotides exposed to gamma-irradiation, (ii) the induction of IR-induced DNA breaks and (iii) accumulation of chromosome aberrations induced by radiation. Alteration of Pol beta expression in irradiated cells thus appears to strengthen both cell death and genetic changes associated with a malignant phenotype. These data provide new insights into the cellular response to radiations and the associated carcinogenic consequences.

Reconstruction of DNA repair-deficient xeroderma pigmentosum skin in vitro: a model to study hypersensitivity to UV light.

Xeroderma pigmentosum (XP) is a rare, recessive, photosensitive and cancer-prone syndrome, the biochemical hallmark of which is a defect in nucleotide excision repair of ultraviolet (UV)-induced mutagenic lesions. After isolation and amplification of several strains of XP-C keratinocytes and fibroblasts, a three-dimensional skin model in vitro comprising both epidermis and a dermal equivalent could be obtained. XP dermal tissues and XP epidermis displayed specific morphological and biochemical characteristics compared with tissues obtained with normal cells. One of the major features was the formation of epidermal invaginations into the dermal equivalent. After UV-B exposure, and contrary to repair of DNA lesions in normal cells, the XP model displayed repair deficiency with long-lasting persistence of UV-induced DNA damage and p53 positive nuclei. Recent data obtained after genetic correction leading to functional XPC gene in keratinocytes and fibroblasts revealed that several abnormal features could be normalized. In conclusion, reconstruction of XP skin in vitro provides a very promising system to study genetic hyperphotosensitivity and opens a rational perspective to XP tissue therapy.

Evidence of finely tuned expression of DNA polymerase beta in vivo using transgenic mice.

DNA polymerase (Pol) is an error-prone repair DNA polymerase that has been shown to create genetic instability and tumorigenesis when overexpressed by only 2-fold in cells, suggesting that a rigorous regulation of its expression may be essential in vivo. To address this question, we have generated mice which express a transgene (Tg) bearing the Pol cDNA under the control of the ubiquitous promoter of the mouse H-2K gene from the major histocompatibility complex. These mice express the Tg only in thymus, an organ which normally contains the most abundant endogenous Pol mRNA and protein, supporting the idea of a tight regulation of Pol in vivo. Furthermore, we found no tumor incidence, suggesting that the single Pol overexpression event is not sufficient to initiate tumorigenesis in vivo.

Terminally differentiated muscle cells are defective in base excision DNA repair and hypersensitive to oxygen injury.

The differentiation of skeletal myoblasts is characterized by permanent withdrawal from the cell cycle and fusion into multinucleated myotubes. Muscle cell survival is critically dependent on the ability of cells to respond to oxidative stress. Base excision repair (BER) is the main repair mechanism of oxidative DNA damage. In this study, we compared the levels of endogenous oxidative DNA damage and BER capacity of mouse proliferating myoblasts and their differentiated counterpart, the myotubes. Changes in the expression of oxidative stress marker genes during differentiation, together with an increase in 8-hydroxyguanine DNA levels in terminally differentiated cells, suggested that reactive oxygen species are produced during this process. The repair of 2-deoxyribonolactone, which is exclusively processed by long-patch BER, was impaired in cell extracts from myotubes. The repair of a natural abasic site (a preferred substrate for short-patch BER) also was delayed. The defect in BER of terminally differentiated muscle cells was ascribed to the nearly complete lack of DNA ligase I and to the strong down-regulation of XRCC1 with subsequent destabilization of DNA ligase IIIalpha. The attenuation of BER in myotubes was associated with significant accumulation of DNA damage as detected by increased DNA single-strand breaks and phosphorylated H2AX nuclear foci upon exposure to hydrogen peroxide. We propose that in skeletal muscle exacerbated by free radical injury, the accumulation of DNA repair intermediates, due to attenuated BER, might contribute to myofiber degeneration as seen in sarcopenia and many muscle disorders.