Changing human hair fibre colour and shape from the follicle.

INTRODUCTION: Natural hair curvature and colour are genetically determined human traits, that we intentionally change by applying thermal and chemical treatments to the fibre. Presently, those cosmetic methodologies act externally and their recurrent use is quite detrimental to hair fibre quality and even to our health. OBJECTIVES: This work represents a disruptive concept to modify natural hair colour and curvature. We aim to model the fibre phenotype as it is actively produced in the follicle through the topical delivery of specific bioactive molecules to the scalp. METHODS: Transcriptome differences between curly and straight hairs were identified by microarray. In scalp samples, the most variable transcripts were mapped by in situ hybridization. Then, by using appropriate cellular models, we screened a chemical library of 1200 generic drugs, searching for molecules that could lead to changes in either fibre colour or curvature. A pilot-scale, single-centre, investigator-initiated, prospective, blind, bilateral (split-scalp) placebo-controlled clinical study with the intervention of cosmetics was conducted to obtain a proof of concept (RNEC n.92938). RESULTS: We found 85 genes transcribed significantly different between curly and straight hair, not previously associated with this human trait. Next, we mapped some of the most variable genes to the inner root sheath of follicles, reinforcing the role of this cell layer in fibre shape moulding. From the drug library screening, we selected 3 and 4 hits as modulators of melanin synthesis and gene transcription, respectively, to be further tested in 33 volunteers. The intentional specific hair change occurred: 8 of 14 volunteers exhibited colour changes, and 16 of 19 volunteers presented curvature modifications, by the end of the study. CONCLUSION: The promising results obtained are the first step towards future cosmetics, complementary or alternative to current methodologies, taking hair styling to a new level: changing hair from the inside out.

Long-term experimental evolution reveals purifying selection on piRNA-mediated control of transposable element expression.

BACKGROUND: Transposable elements (TEs) are an almost universal constituent of eukaryotic genomes. In animals, Piwi-interacting small RNAs (piRNAs) and repressive chromatin often play crucial roles in preventing TE transcription and thus restricting TE activity. Nevertheless, TE content varies widely across eukaryotes and the dynamics of TE activity and TE silencing across evolutionary time is poorly understood. RESULTS: Here, we used experimentally evolved populations of C. elegans to study the dynamics of TE expression over 409 generations. The experimental populations were evolved at population sizes of 1, 10 and 100 individuals to manipulate the efficiency of natural selection versus genetic drift. We demonstrate increased TE expression relative to the ancestral population, with the largest increases occurring in the smallest populations. We show that the transcriptional activation of TEs within active regions of the genome is associated with failure of piRNA-mediated silencing, whilst desilenced TEs in repressed chromatin domains retain small RNAs. Additionally, we find that the sequence context of the surrounding region influences the propensity of TEs to lose silencing through failure of small RNA-mediated silencing. CONCLUSIONS: Our results show that natural selection in C. elegans is responsible for maintaining low levels of TE expression, and provide new insights into the epigenomic features responsible.

Individual Limb Muscle Bundles Are Formed through Progressive Steps Orchestrated by Adjacent Connective Tissue Cells during Primary Myogenesis.

Although the factors regulating muscle cell differentiation are well described, we know very little about how differentiating muscle fibers are organized into individual muscle tissue bundles. Disruption of these processes leads to muscle hypoplasia or dysplasia, and replicating these events is vital in tissue engineering approaches. We describe the progressive cellular events that orchestrate the formation of individual limb muscle bundles and directly demonstrate the role of the connective tissue cells that surround muscle precursors in controlling these events. We show how disruption of gene activity within or genetic ablation of connective tissue cells impacts muscle precursors causing disruption of muscle bundle formation and subsequent muscle dysplasia and hypoplasia. We identify several markers of the populations of connective tissue cells that surround muscle precursors and provide a model for how matrix-modifying proteoglycans secreted by these cells may influence muscle bundle formation by effects on the local extracellular matrix (ECM) environment.

Nanoparticles Alter Secondary Metabolism in Plants via ROS Burst.

The particles within the size range of 1 and 100 nm are known as nanoparticles (NPs). NP-containing wastes released from household, industrial and medical products are emerging as a new threat to the environment. Plants, being fixed to the two major environmental sinks where NPs accumulate - namely water and soil, cannot escape the impact of nanopollution. Recent studies have shown that plant growth, development and physiology are significantly affected by NPs. But, the effect of NPs on plant secondary metabolism is still obscure. The induction of reactive oxygen species (ROS) following interactions with NPs has been observed consistently across plant species. Taking into account the existing link between ROS and secondary signaling messengers that lead to transcriptional regulation of secondary metabolism, in this perspective we put forward the argument that ROS induced in plants upon their interaction with NPs will likely interfere with plant secondary metabolism. As plant secondary metabolites play vital roles in plant performance, communication, and adaptation, a comprehensive understanding of plant secondary metabolism in response to NPs is an utmost priority.

Mechanisms of vertebrate embryo segmentation: Common themes in trunk and limb development.

Various ultradian rhythms ensure proper temporal regulations during embryo development. The embryo molecular clock, which was first identified in the presomitic mesoderm (PSM) underlying periodic somite formation, is one among them. Somites are the earliest manifestation of the segmented vertebrate body and they are formed with strict temporal precision. The tetrapod limb is also a segmented structure and the formation of limb bone elements have also been associated with a molecular clock, operating in the distal limb mesenchyme. In both the PSM and the distal limb mesenchyme, the molecular clock (MC) is influenced by FGF, SHH and RA, which are also the key regulators of the development of these tissues. While somitogenesis has been continuously scrutinized to understand the mechanisms of the MC, the limb bud has served as an outstanding paradigm to study how a cohort of undifferentiated cells is organized into functional 3D structures. The fact that both the trunk and limb development are shaped by the MC and by common signaling molecules has prompted the exciting possibility of establishing parallelisms between somitogenesis and limb development. Systematically correlating various parameters during trunk and limb development will help us to appreciate the common principles underlying segmented structure formation and allow the rise of new questions in order to fill the gaps in our present understanding. In this review we have established the parallelisms between somitogenesis and limb development at the level of gene expression patterns and their regulation. Finally, we have also discussed the most evident new avenues this exercise could open to the scientific community.

Getting a handle on embryo limb development: Molecular interactions driving limb outgrowth and patterning.

Development of the vertebrate embryo involves multiple segmentation processes to generate a functional, articulated organism. Cell proliferation, differentiation and patterning involve spatially and temporally regulated gene expression and signal transduction mechanisms. The developing vertebrate limb is an excellent model to study such fine-tuned regulations, whereby cells proliferate and are differentially sculptured along the proximal-distal, anterior-posterior and dorsal-ventral axes to form a functional limb. Complementary experimental approaches in different organisms have enhanced our knowledge on the molecular events underlying limb development. Herein, we summarize the current knowledge of the main signaling mechanisms governing vertebrate limb initiation, outgrowth, specification of limb segments and termination.

PEGylated ofloxacin nanoparticles render strong antibacterial activity against many clinically important human pathogens.

The rise of bacterial resistance against important drugs threatens their clinical utility. Fluoroquinones, one of the most important classes of contemporary antibiotics has also reported to suffer bacterial resistance. Since the general mechanism of bacterial resistance against fluoroquinone antibiotics (e.g. ofloxacin) consists of target mutations resulting in reduced membrane permeability and increased efflux by the bacteria, strategies that could increase bacterial uptake and reduce efflux of the drug would provide effective treatment. In the present study, we have compared the efficiencies of ofloxacin delivered in the form of free drug (OFX) and as nanoparticles on bacterial uptake and antibacterial activity. Although both poly(lactic-co-glycolic acid) (OFX-PLGA) and methoxy poly(ethylene glycol)-b-poly(lactic-co-glycolic acid) (OFX-mPEG-PLGA) nanoformulations presented improved bacterial uptake and antibacterial activity against all the tested human bacterial pathogens, namely, Escherichia coli, Proteus vulgaris, Salmonella typhimurium, Pseudomonas aeruginosa, Klebsiella pneumoniae and Staphylococcus aureus, OFX-mPEG-PLGA showed significantly higher bacterial uptake and antibacterial activity compared to OFX-PLGA. We have also found that mPEG-PLGA nanoencapsulation could significantly inhibit Bacillus subtilis resistance development against OFX.

Delivery as nanoparticles reduces imatinib mesylate-induced cardiotoxicity and improves anticancer activity.

Clinical effectiveness of imatinib mesylate in cancer treatment is compromised by its off-target cardiotoxicity. In the present study, we have developed physically stable imatinib mesylate-loaded poly(lactide-co-glycolide) nanoparticles (INPs) that could sustainably release the drug, and studied its efficacy by in vitro anticancer and in vivo cardiotoxicity assays. MTT (methylthiazolyldiphenyl-tetrazolium bromide) assay revealed that INPs are more cytotoxic to MCF-7 breast cancer cells compared to the equivalent concentration of free imatinib mesylate. Wistar rats orally administered with 50 mg/kg INPs for 28 days showed no significant cardiotoxicity or associated changes. Whereas, increased alanine aminotransferase, aspartate aminotransferase, and alkaline phosphatase levels, and reduced white blood cell, red blood cell, and hemoglobin content were observed in the animals administered with free drug. While the histological sections from hearts of animals that received INPs did not show any significant cardiotoxic symptoms, loss of normal architecture and increased cytoplasmic vacuolization were observed in the heart sections of animals administered with free imatinib mesylate. Based on these results, we conclude that nano-encapsulation of imatinib mesylate increases its efficacy against cancer cells, with almost no cardiotoxicity.

Test anxiety levels of board exam going students in Tamil Nadu, India.

The latest report by the National Crime Records Bureau has positioned Tamil Nadu as the Indian state with highest suicide rate. At least in part, this is happening due to exam pressure among adolescents, emphasizing the imperative need to understand the pattern of anxiety and various factors contributing to it among students. The present study was conducted to analyze the level of state anxiety among board exam attending school students in Tamil Nadu, India. A group of 100 students containing 50 boys and 50 girls from 10th and 12th grades participated in the study and their state anxiety before board exams was measured by Westside Test Anxiety Scale. We found that all board exam going students had increased level of anxiety, which was particularly higher among boys and 12th standard board exam going students. Analysis of various demographic variables showed that students from nuclear families presented higher anxiety levels compared to their desired competitive group. Overall, our results showing the prevalence of state anxiety among board exam going students in Tamil Nadu, India, support the recent attempt taken by Tamil Nadu government to improve student's academic performance in a healthier manner by appointing psychologists in all government schools.

Limb patterning: from signaling gradients to molecular oscillations.

The developing forelimb is patterned along the proximal-distal and anterior-posterior axes by opposing gradients of retinoic acid and fibroblast growth factors and by graded sonic hedgehog signaling, respectively. However, how coordinated patterning along both axes is accomplished with temporal precision remains unknown. The limb molecular oscillator hairy2 was recently shown to be a direct readout of the combined signaling activities of retinoic acid, fibroblast growth factor and sonic hedgehog in the limb mesenchyme. Herein, an integrated time-space model is presented to conciliate the progress zone and two-signal models for limb patterning. We propose that the limb clock may allow temporal information to be decoded into positional information when the distance between opposing signaling gradients is no longer sufficient to provide distinct cell fate specification.

Joint interpretation of AER/FGF and ZPA/SHH over time and space underlies hairy2 expression in the chick limb.

Embryo development requires precise orchestration of cell proliferation and differentiation in both time and space. A molecular clock operating through gene expression oscillations was first described in the presomitic mesoderm (PSM) underlying periodic somite formation. Cycles of HES gene expression have been further identified in other progenitor cells, including the chick distal limb mesenchyme, embryonic neural progenitors and both mesenchymal and embryonic stem cells. In the limb, hairy2 is expressed in the distal mesenchyme, adjacent to the FGF source (AER) and along the ZPA-derived SHH gradient, the two major regulators of limb development. Here we report that hairy2 expression depends on joint AER/FGF and ZPA/SHH signaling. FGF plays an instructive role on hairy2, mediated by Erk and Akt pathway activation, while SHH acts by creating a permissive state defined by Gli3-A/Gli3-R>1. Moreover, we show that AER/FGF and ZPA/SHH present distinct temporal and spatial signaling properties in the distal limb mesenchyme: SHH acts at a long-term, long-range on hairy2, while FGF has a short-term, short-range action. Our work establishes limb hairy2 expression as an output of integrated FGF and SHH signaling in time and space, providing novel clues for understanding the regulatory mechanisms underlying HES oscillations in multiple systems, including embryonic stem cell pluripotency.

Retinoic acid signaling regulates embryonic clock hairy2 gene expression in the developing chick limb.

Embryo development proceeds under strict temporal control and an embryonic molecular clock (EC), evidenced by cyclic gene expression, is operating during somite formation and limb development, providing temporal information to precursor cells. In somite precursor cells, EC gene expression and periodicity depends on Retinoic acid (RA) signaling and this morphogen is also essential for limb initiation, outgrowth and patterning. Since the limb EC gene hairy2 is differentially expressed along the proximal-distal axis as growth proceeds, concomitant with changes in flank-derived RA activity in the mesenchyme, we have interrogated the role of RA signaling on limb hairy2 expression regulation. We describe RA as a positive regulator of limb hairy2 expression. Ectopic supplementation of RA induced hairy2 in a short time period, with simultaneous transient activation of Erk/MAPK, Akt/PI3K and Gli3 intracellular pathways. We further found that FGF8, an inducer of Erk/MAPK, Akt/PI3K pathways, was not sufficient for ectopic hairy2 induction. However, joint treatment with both RA and FGF8 induced hairy2, indicating that RA is creating a permissive condition for p-Erk/p-Akt action on hairy2, most likely by enhancing Gli3-A/Gli3-R levels. Finally, we observed an inhibitory action of BMP4 on hairy2 and propose a model whereby RA shapes limb hairy2 expression during limb development, by activating its expression and counteracting the inhibitory action of BMP4 on hairy2. Overall, our work reports a novel role for RA in the regulation of limb clock hairy2 gene expression and elucidates the temporal response of multiple intracellular pathways to RA signaling in limb development.

Comprehensive analysis of fibroblast growth factor receptor expression patterns during chick forelimb development.

Specific interactions between fibroblast growth factors (Fgf1-22) and their tyrosine kinase receptors (FgfR1-4) activate different signalling pathways that are responsible for the biological processes in which Fgf signalling is implicated during embryonic development. In the chick, several Fgf ligands (Fgf2, 4, 8, 9, 10, 12, 13 and 18) and the four FgfRs (FgfR 1, 2, 3 and 4) have been reported to be expressed in the developing limb. The precise spatial and temporal expression of these transcripts is important to guide the limb bud to develop into a wing/leg. In this paper, we present a detailed and systematic analysis of the expression patterns of FgfR1, 2, 3 and 4 throughout chick wing development, by in situ hybridisation on whole mounts and sections. Moreover, we characterize for the first time the different isoforms of FGFR1-3 by analysing their differential expression in limb ectoderm and mesodermal tissues, using RT-PCR and in situ hybridisation on sections. Finally, isoform-specific sequences for FgfR1IIIb, FgfR1IIIc, FgfR3IIIb and FgfR3IIIc were determined and deposited in GenBank with the following accession numbers: GU053725, GU065444, GU053726, GU065445, respectively.

Poly(D,L-lactic-co-glycolic acid) nanoencapsulation reduces Erlotinib-induced subacute toxicity in rat.

Erlotinib-HCl is a quinazoline derivative used as a drug in the therapy of non-small-cell lung cancer. The present study was conducted to compare the subacute toxicity induced by Erlotinib-HCl delivered to rats as nanoparticles and as free drug. Wistar rats were orally administered with a daily dosage of 200 mg kg(-1) Erlotinib-HCl either as free drug or as Poly(D,L-lactic-co-glycolic acid) (PLGA) encapsulated nanoparticles. After four weeks of treatment, the animals were analyzed for toxicological changes. Although nanoparticulate form of the drug did not induce any toxicity, free drug significantly reduced the levels of white blood cells (WBC), red blood cells (RBC) and haemoglobin, while increasing the levels of neutrophils and corpuscular haemoglobin. Moreover, aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels were significantly increased in the animals administered with free drug. Histopathological studies confirmed significant damage to the internal organs of animals treated with free drug. Whereas, the internal organs of animals treated with the drug encapsulated in PLGA nanoparticles were more or less similar to the healthy organs. Our results show that Erlotinib-HCl delivered in the form of nanoparticles has less toxic effect than the free drug in experimental rats.

Chick Hairy1 protein interacts with Sap18, a component of the Sin3/HDAC transcriptional repressor complex.

BACKGROUND: The vertebrate adult axial skeleton, trunk and limb skeletal muscles and dermis of the back all arise from early embryonic structures called somites. Somites are symmetrically positioned flanking the embryo axial structures (neural tube and notochord) and are periodically formed in a anterior-posterior direction from the presomitic mesoderm. The time required to form a somite pair is constant and species-specific. This extraordinary periodicity is proposed to depend on an underlying somitogenesis molecular clock, firstly evidenced by the cyclic expression of the chick hairy1 gene in the unsegmented presomitic mesoderm with a 90 min periodicity, corresponding to the time required to form a somite pair in the chick embryo. The number of hairy1 oscillations at any given moment is proposed to provide the cell with both temporal and positional information along the embryo's anterior-posterior axis. Nevertheless, how this is accomplished and what biological processes are involved is still unknown. Aiming at understanding the molecular events triggered by the somitogenesis clock Hairy1 protein, we have employed the yeast two-hybrid system to identify Hairy1 interaction partners. RESULTS: Sap18, an adaptor molecule of the Sin3/HDAC transcriptional repressor complex, was found to interact with the C-terminal portion of the Hairy1 protein in a yeast two-hybrid assay and the Hairy1/Sap18 interaction was independently confirmed by co-immunoprecipitation experiments. We have characterized the expression patterns of both sap18 and sin3a genes during chick embryo development, using in situ hybridization experiments. We found that both sap18 and sin3a expression patterns co-localize in vivo with hairy1 expression domains in chick rostral presomitic mesoderm and caudal region of somites. CONCLUSION: Hairy1 belongs to the hairy-enhancer-of-split family of transcriptional repressor proteins. Our results indicate that during chick somitogenesis Hairy1 may mediate gene transcriptional repression by recruiting the Sin3/HDAC complex, through a direct interaction with the Sap18 adaptor molecule. Moreover, since sap18 and sin3a are not expressed in the PSM territory where hairy1 presents cyclic expression, our study strongly points to different roles for Hairy1 throughout the PSM and in the prospective somite and caudal region of already formed somites.