Self-organization process in newborn skin organoid formation inspires strategy to restore hair regeneration of adult cells.

Organoids made from dissociated progenitor cells undergo tissue-like organization. This in vitro self-organization process is not identical to embryonic organ formation, but it achieves a similar phenotype in vivo. This implies genetic codes do not specify morphology directly; instead, complex tissue architectures may be achieved through several intermediate layers of cross talk between genetic information and biophysical processes. Here we use newborn and adult skin organoids for analyses. Dissociated cells from newborn mouse skin form hair primordia-bearing organoids that grow hairs robustly in vivo after transplantation to nude mice. Detailed time-lapse imaging of 3D cultures revealed unexpected morphological transitions between six distinct phases: dissociated cells, cell aggregates, polarized cysts, cyst coalescence, planar skin, and hair-bearing skin. Transcriptome profiling reveals the sequential expression of adhesion molecules, growth factors, Wnts, and matrix metalloproteinases (MMPs). Functional perturbations at different times discern their roles in regulating the switch from one phase to another. In contrast, adult cells form small aggregates, but then development stalls in vitro. Comparative transcriptome analyses suggest suppressing epidermal differentiation in adult cells is critical. These results inspire a strategy that can restore morphological transitions and rescue the hair-forming ability of adult organoids: (i) continuous PKC inhibition and (ii) timely supply of growth factors (IGF, VEGF), Wnts, and MMPs. This comprehensive study demonstrates that alternating molecular events and physical processes are in action during organoid morphogenesis and that the self-organizing processes can be restored via environmental reprogramming. This tissue-level phase transition could drive self-organization behavior in organoid morphogenies beyond the skin.

iNOS inhibits hair regeneration in obese diabetic (ob/ob) mice.

Previous studies have shown that androgenic alopecia is associated with metabolic syndrome and diabetes. However, the detailed mechanism whereby diabetes causes alopecia still remains unclear. We focused on the inflammatory response that is caused by diabetes or obesity, given that inflammation is a risk factor for hair loss. Inducible nitric oxide synthase (iNOS) is known to be upregulated under conditions of acute or chronic inflammation. To clarify the potential role of iNOS in diabetes-related alopecia, we generated obese diabetic iNOS-deficient (ob/ob; iNOS-KO mice). We observed that ob/ob; iNOS-KO mice were potentiated for the transition from telogen (rest phase) to anagen (growth phase) in the hair cycle compared with iNOS-proficient ob/ob mice. To determine the effect of nitric oxide (NO) on the hair cycle, we administered an iNOS inhibitor intraperitoneally (compound 1400 W, 10 mg/kg) or topically (10% aminoguanidine) in ob/ob mice. We observed that iNOS inhibitors promoted anagen transition in ob/ob mice. Next, we administered an NO donor (S-nitrosoglutathione, GSNO), to test whether NO has the telogen elongation effects. The NO donor was sufficient to induce telogen elongation in wild-type mice. Together, our data indicate that iNOS-derived NO plays a role in telogen elongation under the inflammatory conditions associated with diabetes in mice.

Characterization of follicular minoxidil sulfotransferase activity in a cohort of pattern hair loss patients from the Indian Subcontinent.

Several studies have established that sulfotransferase enzyme activity in the outer root sheath of plucked hair follicles predicts response to topical minoxidil in the treatment of pattern hair loss. However, the prevalence of this enzyme activity among Indian patients has not been studied. Additionally, no reports in the literature characterize sulfotransferase activity based on sex, age, duration of hair loss, grade of hair loss, and family history. In this study we utilized a sulfotransferase activity assay first reported by Goren et al. We characterize the follicular sulfotransferase activity of 120 pattern hair loss patients visiting a dermatology outpatient clinic in India. Overall, 40.8% of patients with pattern hair loss had low levels of sulfotransferase. Surprisingly, 49.3% of men had low levels of sulfotransferase compared to 26.6% of women. No correlation was found between sulfotransferase activity and age, duration of hair loss, grade of hair loss, or family history. A sub-analysis of patient reported outcomes (PRO) validated previous findings that sulfotransferase enzyme activity is a predictive marker for minoxidil response in pattern hair loss patients.

Diverse selective regimes shape genetic diversity at ADAR genes and at their coding targets.

A-to-I RNA editing operated by ADAR enzymes is extremely common in mammals. Several editing events in coding regions have pivotal physiological roles and affect protein sequence (recoding events) or function. We analyzed the evolutionary history of the 3 ADAR family genes and of their coding targets. Evolutionary analysis indicated that ADAR evolved adaptively in primates, with the strongest selection in the unique N-terminal domain of the interferon-inducible isoform. Positively selected residues in the human lineage were also detected in the ADAR deaminase domain and in the RNA binding domains of ADARB1 and ADARB2. During the recent history of human populations distinct variants in the 3 genes increased in frequency as a result of local selective pressures. Most selected variants are located within regulatory regions and some are in linkage disequilibrium with eQTLs in monocytes. Finally, analysis of conservation scores of coding editing sites indicated that editing events are counter-selected within regions that are poorly tolerant to change. Nevertheless, a minority of recoding events occurs at highly conserved positions and possibly represents the functional fraction. These events are enriched in pathways related to HIV-1 infection and to epidermis/hair development. Thus, both ADAR genes and their targets evolved under variable selective regimes, including purifying and positive selection. Pressures related to immune response likely represented major drivers of evolution for ADAR genes. As for their coding targets, we suggest that most editing events are slightly deleterious, although a minority may be beneficial and contribute to antiviral response and skin homeostasis.

Normal fur development and sebum production depends on fatty acid 2-hydroxylase expression in sebaceous glands.

2-Hydroxylated fatty acid (HFA)-containing sphingolipids are abundant in mammalian skin and are believed to play a role in the formation of the epidermal barrier. Fatty acid 2-hydroxylase (FA2H), required for the synthesis of 2-hydroxylated sphingolipids in various organs, is highly expressed in skin, and previous in vitro studies demonstrated its role in the synthesis of HFA sphingolipids in human keratinocytes. Unexpectedly, however, mice deficient in FA2H did not show significant changes in their epidermal HFA sphingolipids. Expression of FA2H in murine skin was restricted to the sebaceous glands, where it was required for synthesis of 2-hydroxylated glucosylceramide and a fraction of type II wax diesters. Absence of FA2H resulted in hyperproliferation of sebocytes and enlarged sebaceous glands during hair follicle morphogenesis and anagen (active growth phase) in adult mice. This was accompanied by a significant up-regulation of the epidermal growth factor receptor ligand epigen in sebocytes. Loss of FA2H significantly altered the composition and physicochemical properties of sebum, which often blocked the hair canal, apparently causing a delay in the hair fiber exit. Furthermore, mice lacking FA2H displayed a cycling alopecia with hair loss in telogen. These results underline the importance of the sebaceous glands and suggest a role of specific sebaceous gland or sebum lipids, synthesized by FA2H, in the hair follicle homeostasis.

Lesch-Nyhan syndrome: rapid detection of heterozygotes by use of hair follicles.

A method is described which permits rapid phenotypic diagnosis of the Lesch-Nyhan heterozygote by direct assay of hypoxanthine guanine phosphori-bosyltransferase activity in single hair follicles obtained from the scalp.

Dissecting the non-canonical functions of telomerase.

It is now well established that the canonical function of telomerase protects the telomere repeats from erosion and the consequent induction of replicative senescence or apoptosis. In the absence of key cell cycle checkpoint proteins, the canonical function of telomerase also prevents chromosome fusions and immortalizes human cells. The canonical function of telomerase requires both the telomerase reverse transcriptase enzyme (TERT) which adds telomere (TTAGGG) repeats to the chromosome ends and the telomerase RNA component (TERC), which provides the template for TERT. However, there is growing evidence that telomerase has other (non-canonical) functions. These functions can be divided further into those that require telomerase activity but not telomere lengthening (non-canonical I or NC I) and those that require neither telomerase activity nor telomere lengthening (non-canonical II or NC II). NC I functions are associated with the induction of neoplasia in both epidermis and mammary gland, the correct response to DNA damage, and insensitivity to transforming growth factor beta. In contrast, NC II functions are not sufficient for the induction of neoplasia and are associated with the activation of the WNT and MYC signaling pathways in keratinocytes and a more general resistance to the induction of apoptosis by a variety of stimuli. The overexpression of either TERT or TERC appears to be capable of providing NC I functions but NC II functions require neither TERC nor the integrity of the TERT catalytic site. The molecular mechanisms underpinning both NC I and NC II are largely obscure but transcriptional profile changes have been reported by some groups. In this article, we will discuss the proposed mechanisms of NC I and NC II and their relevance to normal and neoplastic cell functions.

Alcohol Biomarkers in Clinical and Forensic Contexts.

BACKGROUND: Biomarkers of alcohol consumption are important not only in forensic contexts, e.g., in child custody proceedings or as documentation of alcohol abstinence after temporary confiscation of a driver's license. They are increasingly being used in clinical medicine as well for verification of abstinence or to rule out the harmful use of alcohol. METHODS: This review is based on pertinent publications that were retrieved by a selective literature search in PubMed concerning the direct and indirect alcohol markers discussed here, as well as on the authors' experience in laboratory analysis and clinical medicine. RESULTS: Alongside the direct demonstration of ethanol, the available markers of alcohol consumption include the classic indirect markers carbohydrate-deficient transferrin (CDT), gamma-glutamyltransferase (GGT), and mean corpuscular volume (MCV) as well as direct alcohol markers such as ethyl glucuronide (EtG) and ethyl sulfate (EtS) in serum and urine and EtG and fatty acid ethyl esters (FAEE) in hair. Phosphatidylethanol (PEth) is a promising parameter that com - plements the existing spectrum of tests with high specificity (48-89%) and sensi - tivity (88-100%). In routine clinical practice, the demonstration of positive alcohol markers often leads patients to admit previously denied alcohol use. This makes it possible to motivate the patient to undergo treatment for alcoholism. CONCLUSION: The available alcohol biomarkers vary in sensitivity and specificity with respect to the time period over which they indicate alcohol use and the minimum extent of alcohol use that they can detect. The appropriate marker or combination of markers should be chosen in each case according to the particular question that is to be answered by laboratory analysis.

Temperature-sensitive tyrosinase associated with peripheral pigmentation in oculocutaneous albinism.

Several types of autosomal recessive oculocutaneous albinism (OCA) are associated with abnormal tyrosinase function and a generalized reduction in or absence of cutaneous and eye melanin. Each is thought to result from a different mutant allele at the tyrosinase locus, with the mutation producing an enzyme with little or no activity in all involved tissues. In this paper, we report a new type of OCA that results from a tyrosinase allele producing a temperature-sensitive enzyme. The proband had white hair in the warmer areas (scalp and axilla) and progressively darker hair in the cooler areas (extremities) of her body. Melanocyte and melanosome architecture were normal. Quantitative hairbulb tyrosinase (dopa oxidase) assay demonstrated a loss of activity above 35-37 degrees C. Plasma pheomelanin and urine eumelanin intermediates were reduced and correlated with hair melanin content. This is the first temperature-sensitive tyrosinase mutation to be reported in humans and is analogous to the Siamese mutation in the cat and the Himalayan mutation in the mouse.

Molecular beacon genotyping for globoid cell leukodystrophy from hair roots in the twitcher mouse and rhesus macaque.

Rapid and accurate genotype determination is ideal for the maintenance of breeding colonies of laboratory animal models of genetic disease. The rhesus macaque and murine (twitcher) models of globoid cell leukodystrophy have a dinucleotide deletion or single nucleotide substitution, respectively, which abolish ceramide beta-galactosidase activity and are authentic models of Krabbe disease. We report a molecular beacon PCR assay for each species which allows unambiguous determination of the genotype in under 4h. The assay works reliably with DNA extracted from hair roots using Chelex-100 in a 20 min, 100 degrees C incubation. We demonstrate that genotyping from hair roots is a preferred alternative to collecting blood or tissue for DNA extraction because it reduces animal distress, uses an inexpensive reagent, and is simpler and faster. Following amplification on a standard thermocycler with a 96-well plate format, these molecular beacon assays can be read on a standard laboratory fluorescent plate reader, eliminating the need to use a real-time thermocycler or to open the plate for subsequent restriction enzyme digestion and gel electrophoresis. The multiplexed ratio of fluorescence from wild-type- and mutant-specific beacons reporting at 560 nm and 535 nm wavelengths is distinct for each genotype.

Functional barrel cortex in 'hairless', nude mice.

Although nude mice are not truly hairless, they demonstrate abnormal hair structure and growth patterns, which are related to their genetic state. Whereas wild-type mice are born with visible vibrissae, nude mice are distinguishable at birth by the lack of visible vibrissae, which do not appear until approximately postnatal day 6. Additionally, adult nude mice have abnormal whisker cycling patterns in which structurally normal whisker follicles produce fragile whiskers which break or fallout leaving follicles whiskerless for several days before a fine replacement whisker appears and develops. The current study shows that despite these abnormal periods of whisker deprivation, the barrel cortex of nude mice develops a normal structural appearance viewed with cytochrome oxidase staining. Additionally, intrinsic optical imaging studies of barrel cortex responses to single whisker stimulation do not appear altered from normal despite periodic loss of adjacent whiskers.

The skin and hair as surrogate tissues for measuring the target effect of inhibitors of phosphoinositide-3-kinase signaling.

BACKGROUND: The purpose of the study was to evaluate the use of phospho-Akt in mouse and human skin as a surrogate target for tumor phospho-Akt to measure the effect of antitumor inhibitors of phosphatidylinositol-3-kinase (PI-3-K)/Akt (protein kinase B) signaling. METHOD: The expression of phosphoSer473-Akt was quantitatively assessed by Western blotting in human HT-29 colon, MCF-7 breast, A-549 non small cell lung tumor xenografts in mice, and by immunohistochemistry in mouse skin and human hair. RESULTS: The pattern of PI-3-K isoforms in human hair keratinocytes was similar to that in tumor but mouse hair keratinocytes showed a different pattern. A high level of phospho-Akt staining was present in keratinocytes of the external root sheath of the hair and was inhibited by the PI-3-K inhibitor PX-866 administered to mice, and in human hair exposed to PX-866 in culture. The inhibition of phospho-Akt by PX-866 in mouse hair keratinocytes was greater than inhibition of phospho-Akt in HT-29 and A-549 xenografts in the same mice. Phospho-Akt in mouse hair keratinocytes was inhibited by the Akt inhibitor PX-316 to a lesser degree than in MCF-7 tumor xenografts. CONCLUSIONS: Hair offers a way of measuring the effects of PI-3-K signaling inhibitors and, in cancer patients, may provide a readily obtainable surrogate tissue for assessing PI-3-K and phospho-Akt inhibition in tumor.

Expression of gamma-glutamyl transpeptidase activity in the developing mouse tooth, intervertebral disc, and hair follicle.

Expression of gamma-glutamyl transpeptidase (GGT) in the developing mouse tooth, intervertebral disc, and hair follicle was investigated in terms of its localization during ontogenic stages and its association or lack of association with cell proliferation (labeled nuclei after [3H]thymidine injection or metaphase-arrested cells after colchicine injection). The data demonstrate that (a) GGT expression followed a program of activity and localization changes that correlated with the progressive emergence of developmental stages and (b) GGT activity in developing tissues derived either from epithelium (enamel-producing cells and hair follicle cells) or from mesenchyme (intervertebral disc cells) was localized only in mitotically quiescent cellular layers or regions associated with the production of specialized tissue products; however, not all postmitotic regions expressed GGT activity. Although further research is needed to clarify the role of GGT in normal and neoplastic tissues, we conclude that increasing evidence from this and other laboratories implicates GGT as a marker of cell differentiation, cell aging, and/or reduced cell proliferation.

Alternative Sampling Strategies for Cytochrome P450 Phenotyping.

Interindividual variability in the expression and function of drug metabolizing cytochrome P (CYP) 450 enzymes, determined by a combination of genetic, non-genetic and environmental parameters, is a major source of variable drug response. Phenotyping by administration of a selective enzyme substrate, followed by the determination of a specific phenotyping metric, is an appropriate approach to assess the in vivo activity of CYP450 enzymes as it takes into account all influencing factors. A phenotyping protocol should be as simple and convenient as possible. Typically, phenotyping metrics are determined in traditional matrices, such as blood, plasma or urine. Several sampling strategies have been proposed as an alternative for these traditional sampling techniques. In this review, we provide a comprehensive overview of available methods using dried blood spots (DBS), hair, oral fluid, exhaled breath and sweat for in vivo CYP450 phenotyping. We discuss the relation between phenotyping metrics measured in these samples and those in conventional matrices, along with the advantages and limitations of the alternative sampling techniques. Reliable phenotyping procedures for several clinically relevant CYP450 enzymes, including CYP1A2, CYP2C19 and CYP2D6, are currently available for oral fluid, breath or DBS, while additional studies are needed for other CYP450 isoforms, such as CYP3A4. The role of hair analysis for this purpose remains to be established. Being non- or minimally invasive, these sampling strategies provide convenient and patient-friendly alternatives for classical phenotyping procedures, which may contribute to the implementation of CYP450 phenotyping in clinical practice.

Rat hair follicle and epidermal transglutaminases. Biochemical and immunochemical isoenzymes.

Epidermal and hair follicle transglutaminases (1,4-alpha-D-glucan: orthophosphate alpha-D-glucosyltransferase EC 2.4.1.1) were differentially isolated and subsequently purified from newborn or 4-5-day-old rats. Both enzymes migrated identically on ion-exchange chromatography but were widely separated by block electrophoresis, with the epidermal enzyme migrating further toward the anode. Each enzyme was finally purified by gel filtration. Epidermal transglutaminase had an apparent molecular weight of 56 000-58 000 in this medium and in gels containing sodium dodecyl sulfate (SDS), while hair follicle transglutaminase had a molecular weight of 52 000-54 000 and was reduced to two apparently identical subunits of a molecular weight of 27 000 by denaturing media. Antiserum specific to each transglutaminase was produced in chickens; when conjugated to fluorescein these antisera localized the enzymes to the granular layer of epidermis and the inner root sheath of follicles, respectively.

Combined biochemical and immunochemical comparison of peptidylarginine deiminases present in various tissues.

We have performed a combined biochemical and immunochemical study on the identity of peptidylarginine deiminases (EC 3.5.3.15) present in various mammalian tissues. First, we purified peptidylarginine deiminase from rat skeletal muscle. It gave a single band of molecular weight 83,000 in sodium dodecyl sulfate polyacrylamide gel electrophoresis. Next we immunized rabbits with the purified enzyme. The resulting antibodies reacted specifically with the antigen in Western blot assay. Most of the enzyme activities present in rat skeletal muscle, brain, spinal cord, submaxillary gland and spleen could be characterized as the same muscle-type enzyme by immunoprecipitation and Western blot assay. The antibodies did not react with enzyme samples obtained from rat hair follicles and bovine epidermis. The lack of immunoreactivity of the epidermal enzyme could not be accounted for by the species difference, since the antibodies reacted with a 83 kDa polypeptide of bovine brain, which was thought to represent a bovine counterpart of the muscle-type enzyme. The epidermal enzyme could be distinguished from the other enzyme samples by its high activity towards benzoylarginine. These data suggest the existence of at least three types of peptidylarginine deiminase in mammalian tissues, i.e., a muscle type, a hair follicle type, and an epidermal type.

Dynamic expression of ornithine decarboxylase in hair growth.

Ornithine decarboxylase (ODC) is the key enzyme in the synthesis of polyamines, small cationic molecules believed to have a role in many cellular processes such as cell migration, proliferation and differentiation. We show that ODC expression is associated with cell proliferation and commitment in hair follicle development and hair growth. In embryonic epidermis, ODC is expressed in ectodermal cells at sites where follicles develop, and persists in cells at the leading edge of the follicle placode. ODC is abundantly expressed in proliferating bulb cells of anagen follicles, except for a pocket of cells at the base of the bulb. Entry of the follicle into catagen is accompanied by a down-regulation of ODC expression, which is not resumed until a new follicle is initiated. In vibrissae, ODC expression is more complex. ODC is expressed not only in the bulb but also in the hair shaft, presenting a striking biphasic pattern. Additionally, ODC is expressed in a group of outer root sheath cells in the vicinity of the follicle bulge, the putative site of hair follicle stem cells.

Correlation between 25 element contents in mane hair in riding horses and atrioventricular block.

The influence of atrioventricular block (AV-block) on the trace elemental status in a horse hair was studied. The particle-induced X-ray emission (PIXE) method has provided a reliable, rapid, easy, and relatively inexpensive diagnostic method. Twenty-five elements (Al, Br, Ca, Cl, Co, Cu, Cr, Fe, Hg, K, Mg, Mn, Na, Nb, Ni, P, Pb, Rb, S, Se, Si, Sr, Ti, Y, and Zn) in mane hair and serum were measured by the PIXE method. A horse hair with first- and second-degree AV-block contained significantly greater amounts of Br, Ca, Sr, and Zn than those of horses without electrocardiographic abnormalities, whereas there was no significant differences in the elemental contents of the serum of the both groups. Those results in contents of a horse hair suggest that the evaluation of the degree of ionic imbalance by this method might be used to predict the susceptibility of a horse to heart disease much before symptoms appear.

Collagenase IV plays an important role in regulating hair cycle by inducing VEGF, IGF-1, and TGF-ß expression.

BACKGROUND: It has been reported that collagenases (matrix metalloproteinase 2 [MMP-2] and matrix metalloproteinase 9 [MMP-9]) are associated with hair cycle, whereas the mechanism of the association is largely unknown. METHODS: The mice were randomly allocated into four groups: saline, and 5, 10, and 15 nM SB-3CT. Immunohistochemical analysis was employed to examine MMP-2 and MMP-9 protein. Real-time polymerase chain reaction and enzyme-linked immunosorbent assay were performed to determine mRNA and protein levels of VEGF, IGF-1, TGF-ß, and GAPDH. Growing hair follicles from anagen phase III-IV were scored based on hematoxylin and eosin staining. Hair regrowth was also evaluated. RESULTS: Results showed that mRNA expressions of enzymes changed with a peak at late anagen and a trough at telogen after depilation. Immunostaining showed that the highest expression of MMP-2 was more than that of MMP-9, and the highest expression of enzymes changed during anagen. The localizations of MMP-2 changed from dermal papilla, keratinocyte strand, out of root sheath, and basal plate at early anagen, to hair bulb, inner root sheath, and outer root sheath at late anagen. The localization of MMP-9 changed from partial keratinocyte to dermal papilla at early anagen and to outer root sheath at late anagen. VEGF, IGF-1, and TGF-ß have been shown to regulate hair growth. We found mRNA and protein expressions of VEGF and IGF-1 fluctuated with a peak at anagen and a decrease at catagen to telogen. In contrast, mRNA and protein expressions of TGF-ß changed with highest and lowest levels at anagen and telogen, respectively. With selective inhibitor of collagenase IV, SB-3CT, mice showed significant suppressed hair growth and decreased expression of VEGF, IGF-1, and TGF-ß. The MMPs agonist also significantly increased expression of VEGF, IGF-1, and TGF-ß. Meanwhile, SB-3CT treatment significantly suppressed hair growth. CONCLUSION: All these data suggest that the type IV collagenases, MMP-2 and MMP-9, play important roles in hair cycle, and this could be mediated by induced expression of VEGF, IGF-1, and TGF-ß.

Localization of minoxidil sulfotransferase in rat liver and the outer root sheath of anagen pelage and vibrissa follicles.

The precise biochemical mechanism and site(s) of action by which minoxidil stimulates hair growth are not yet clear. Minoxidil sulfate is the active metabolite of minoxidil, with regard to smooth muscle vasodilation and hair growth. Formation of minoxidil sulfate is catalyzed by specific PAPS-dependent sulfotransferase(s) and minoxidil-sulfating activities have been previously reported to be present in liver and hair follicles. One of these minoxidil-sulfating enzymes has been purified from rat liver (rat minoxidil sulfotransferase, MST) and a rabbit anti-MST antibody has been prepared. Using this anti-MST antibody, we have immunohistochemically localized minoxidil sulfotransferase in the liver and anagen hair follicles from rat. In rat pelage and vibrissa follicles, this enzyme is localized within the cytoplasm of epithelial cells in the lower outer root sheath. Although the immunolocalization of MST might not necessarily correlate with the MST activity known to be present in anagen follicles, the results of this study strongly suggest that the lower outer root sheath of the hair follicle may serve as a site for the sulfation of topically applied minoxidil.