Magnesium ascorbyl phosphate vesicular carriers for topical delivery; preparation, in-vitro and ex-vivo evaluation, factorial optimization and clinical assessment in melasma patients.

Ascorbic acid (vitamin C) is an antioxidant that is widely used in cosmetics in skincare products. Due to the excessive low stability of ascorbic acid in cosmetic formulations, the stabilized ascorbic acid derivative, magnesium ascorbyl phosphate (MAP) was formulated as vesicular carriers; ethosomes and niosomes. The aim was to deliver MAP at the intended site of action, the skin, for sufficient time with enhanced permeation to get an effective response. Ethosomes were formulated using a full 32 factorial design to study ethanol and phospholipid concentration effect on ethosomes properties. Niosomes were formulated using 23 factorial designs to study the effect of surfactant type, surfactant concentration and cholesterol concentration on niosomes properties. The prepared formulations were evaluated for their Entrapment efficiency, particle size, polydispersity index, zeta potential and % drug permeated. The optimized ethosomal and niosomal formulations were incorporated into carbopol gel and evaluated for their permeation, skin retention and stability. A comparative split-face clinical study was done between the ethosomal and niosomal formulations for melasma treatment using Antera 3 D® camera. The optimized ethosomal and niosomal gels showed comparable controlled permeation and higher skin retention over their ethosomes and niosomes formulations respectively. Magnesium ascorbyl phosphate ethosomal gel showed clinically and statistically significant melanin level decrease after one month while MAP niosomal gel showed clinically and statistically significant melanin level decrease after six months. A combination of MAP ethosomes and niosomes could be promising skincare formulations for melasma and hyperpigmentation short and long-term treatment.

Efficacy and safety of plasma gel versus platelet-rich plasma in periorbital rejuvenation: a comparative split-face clinical and Antera 3D camera study.

BACKGROUND: Periorbital skin is the thinnest. That is why, it is the easiest to wrinkle and the most challenging to rejuvenate. Platelet-rich plasma (PRP) as well as plasma gel have been used for skin rejuvenation and considered relatively safe and effective. METHODS: This split-face study was conducted on forty female patients seeking periorbital rejuvenation where PRP was injected in the right (Rt) side and plasma gel in the left (Lt) side, two treatment sessions 4 weeks apart (week 0 and week 4). Patients were followed up 2 weeks after each treatment session (week 2 and week 6) as well as 12 weeks after the last session (week 16) using both subjective [physician assessment through Global Aesthetic Improvement score (GAIS) and patient's satisfaction (Likert scale)] and objective [Antera 3D camera] assessment methods. RESULTS: Both modalities yielded a significant improvement of periorbital wrinkles after the 2nd session, with significantly better results on the plasma gel injected side; however, the improvement achieved through both modalities could not be maintained for the following 3 months. Besides, objective assessment could not prove any improvement in periorbital hyperpigmentation. CONCLUSION: Two sessions of both PRP and plasma gel are effective for periorbital rejuvenation, with plasma gel showing significantly better results. However, improvement was not maintained for 3 months.

Persistent Tinnitus after Inhaled N,N-dimethyltryptamine (DMT).

This case report describes a 39-year-old male with remote history of polysubstance use disorder and depression who developed tinnitus after use of inhaled N,N-dimethyltryptamine (DMT). Although development of ear ringing was attributed to use on a single occasion, tinnitus occurred within the context of a larger self-experiment involving weekly microdoses of lysergic acid diethylamide (LSD). Distress and anxiety over the ear ringing prompted evaluation by an audiologist, primary care physician, and consultant psychopharmacologist. Tinnitus persisted for several months, although intensity and ability to cope with symptoms improved over time. A microdose of psilocybin mushrooms exacerbated tinnitus on two separate occasions, after which psychedelics were discontinued. Psychedelics are associated with a range of acute sensory changes including auditory phenomenon, although have not previously been associated with tinnitus in medical literature. Here, we present a probable case of tinnitus associated with DMT use and review potential underlying mechanisms connecting psychedelics and tinnitus.

Analysis of symmetricity in the three different (sagittal, transverse and frontal) planes in generalized nonsegmental vitiligo.

BACKGROUND: Nonsegmental vitiligo is defined as being "often symmetrical", however, no work has tackled the point as to how valid it is to depend upon the concept of symmetricity in generalized nonsegmental vitiligo. AIMS: To investigate vitiligo symmetry, taking into account sites of predilection, the clinical characteristics of patients were studied. METHODS: This multicentric study included 712 nonsegmental vitiligo patients with 2876 examined lesions. Three models were drawn for each patient. Sagittal, transverse and frontal planes were drawn to divide the body into right/left, upper/lower and anterior/posterior halves respectively. Patients were examined by Wood's light and analyzed for symmetry. RESULTS: Bilateral involvement was present in 78% (P < 0.001). Studying the similarity of clinical involvement in the upper and lower body parts revealed that such similarity was present in 38%, with a significant positive association in some areas. Studying clinical similarity in the anteroposterior distribution pattern revealed a significant positive association in 11%. LIMITATIONS: Relatively low number of patients. CONCLUSIONS: We found significant bilateral symmetry in the lesions of 78% of vitiligo patients. Our work could aid in drawing the anticipated vitiligo map in patients with active disease, helping in increasing our understanding of the clinical behaviour of this disease.

Ascorbic acid derivative-loaded modified aspasomes: formulation, in vitro, ex vivo and clinical evaluation for melasma treatment.

Vitamin C (L-Ascorbic acid) has many favourable effects on the skin such as antioxidant, anti-aging and whitening effects. Its instability and low permeability limit its pharmaceutical use in cosmetic and dermatological products. Instead, Mg ascorbyl phosphate (MAP), an ascorbic acid derivative, has the same effect with higher stability is being used. In this work, a vesicular system, aspasomes, containing MAP was developed and evaluated. Aspasomes are multilayered vesicles formed by amphiphiles molecules, Ascorbyl palmitate (ASP), in combination with cholesterol and charged lipids for drug encapsulation. Here, we investigated the use of lecithin instead of the charged lipid dicetyl phosphate for aspasomes development. Nine formulations were prepared and evaluated for their entrapment efficiency, particle size, polydispersity index (PDI) and zeta potential. Their entrapment efficiency ranged from 33.00 ± 2.27 to 95.18 ± 1.06, while their particle size was from 373.34 ± 60.85 to 464.37 ± 93.46 nm with acceptable PDI (from 0.212 ± 0.068 to 0.351 ± 0.061) and zeta potential (from -37.52 ± 2.42 to -50.36 ± 1.82). Three formulations were selected and evaluated for their drug release, permeation and retention into skin. One formulation was selected to be formulated as aspasomal topical cream and gel. The aspasomal cream was found to have enhanced drug permeation and skin retention over the aspasomal gel as well as the aspasomes formulation. MAP aspasomal cream was evaluated clinically as an effective treatment for melasma against 15% trichloroacetic acid (TCA) and the results recorded that the aspasomal cream showed the greatest degree of improvement regarding the hemi-MASI scores with 35% of patients rating it as excellent treatment. The study showed that MAP aspasomal cream can be considered a novel treatment of melasma which is free of side effects. Its efficacy as a monotherapy is superior to that of chemical peeling using 15% TCA.

Rags to riches: Amino acid sensing by the Rag GTPases in health and disease.

The Rags represent a unique family of evolutionarily conserved, heterodimeric, lysosome-localized small GTPases that play an indispensible role in regulating cellular metabolism in response to various amino acid signaling mechanisms. Rapid progress in the field has begun to unveil a picture in which Rags act as central players in translating information regarding cellular amino acid levels by modulating their nucleotide binding status through an ensemble of support proteins localized in and around the lysosomes. By cooperating with other signaling pathways that converge on the lysosomes, Rags promote anabolic processes through positively affecting mTORC1 signaling in the presence of abundant amino acids. Conversely, Rag inactivation plays an indispensible role in switching cellular metabolism into a catabolic paradigm by promoting the activity of the master lysosomal/autophagic transcription factors TFEB and TFE3. Precise control of Rag signaling is necessary for cells to adapt to constantly changing cellular demands and emerging evidence has highlighted their importance in a wide variety of developmental and pathological conditions.

TFEB and TFE3 cooperate in the regulation of the innate immune response in activated macrophages.

The activation of transcription factors is critical to ensure an effective defense against pathogens. In this study we identify a critical and complementary role of the transcription factors TFEB and TFE3 in innate immune response. By using a combination of chromatin immunoprecipitation, CRISPR-Cas9-mediated genome-editing technology, and in vivo models, we determined that TFEB and TFE3 collaborate with each other in activated macrophages and microglia to promote efficient autophagy induction, increased lysosomal biogenesis, and transcriptional upregulation of numerous proinflammatory cytokines. Furthermore, secretion of key mediators of the inflammatory response (CSF2, IL1B, IL2, and IL27), macrophage differentiation (CSF1), and macrophage infiltration and migration to sites of inflammation (CCL2) was significantly reduced in TFEB and TFE3 deficient cells. These new insights provide us with a deeper understanding of the transcriptional regulation of the innate immune response.

TFEB and TFE3 are novel components of the integrated stress response.

To reestablish homeostasis and mitigate stress, cells must activate a series of adaptive intracellular signaling pathways. The participation of the transcription factors TFEB and TFE3 in cellular adaptation to starvation is well established. Here, we show that TFEB and TFE3 also play an important role in the cellular response to ER stress. Treatment with ER stressors causes translocation of TFEB and TFE3 to the nucleus in a process that is dependent on PERK and calcineurin but not on mTORC1. Activated TFEB and TFE3 enhance cellular response to stress by inducing direct transcriptional upregulation of ATF4 and other UPR genes. Under conditions of prolonged ER stress, TFEB and TFE3 contribute to cell death, thus revealing an unexpected role for these proteins in controlling cell fate. This work evidences a broader role of TFEB and TFE3 in the cellular response to stress than previously anticipated and reveals an integrated cooperation between different cellular stress pathways.

Novel Role of TRPML2 in the Regulation of the Innate Immune Response.

TRPMLs (or mucolipins) constitute a family of endosomal cation channels with homology to the transient receptor potential superfamily. In mammals, the TRPML family includes three members: TRPML1-3. Although TRPML1 and TRPML3 have been well characterized, the cellular function of TRPML2 has remained elusive. To address TRPML2 function in a physiologically relevant cell type, we first analyzed TRPML2 expression in different mouse tissues and organs and found that it was predominantly expressed in lymphoid organs and kidney. Quantitative RT-PCR revealed tight regulation of TRPML2 at the transcriptional level. Although TRPML2 expression was negligible in resting macrophages, TRPML2 mRNA and protein levels dramatically increased in response to TLR activation both in vitro and in vivo. Conversely, TRPML1 and TRPML3 levels did not change upon TLR activation. Immunofluorescence analysis demonstrated that endogenous TRPML2 primarily localized to recycling endosomes both in culture and primary cells, in contrast with TRPML1 and TRPML3, which distribute to the late and early endosomal pathway, respectively. To better understand the in vivo function of TRPML2, we generated a TRPML2-knockout mouse. We found that the production of several chemokines, in particular CCL2, was severely reduced in TRPML2-knockout mice. Furthermore, TRPML2-knockout mice displayed impaired recruitment of peripheral macrophages in response to i.p. injections of LPS or live bacteria, suggesting a potential defect in the immune response. Overall, our study reveals interesting differences in the regulation and distribution of the members of the TRPML family and identifies a novel role for TRPML2 in the innate immune response.

Novel roles for the MiTF/TFE family of transcription factors in organelle biogenesis, nutrient sensing, and energy homeostasis.

The MiTF/TFE family of basic helix-loop-helix leucine zipper transcription factors includes MITF, TFEB, TFE3, and TFEC. The involvement of some family members in the development and proliferation of specific cell types, such as mast cells, osteoclasts, and melanocytes, is well established. Notably, recent evidence suggests that the MiTF/TFE family plays a critical role in organelle biogenesis, nutrient sensing, and energy metabolism. The MiTF/TFE family is also implicated in human disease. Mutations or aberrant expression of most MiTF/TFE family members has been linked to different types of cancer. At the same time, they have recently emerged as novel and very promising targets for the treatment of neurological and lysosomal diseases. The characterization of this fascinating family of transcription factors is greatly expanding our understanding of how cells synchronize environmental signals, such as nutrient availability, with gene expression, energy production, and cellular homeostasis.

The nutrient-responsive transcription factor TFE3 promotes autophagy, lysosomal biogenesis, and clearance of cellular debris.

The discovery of a gene network regulating lysosomal biogenesis and its transcriptional regulator transcription factor EB (TFEB) revealed that cells monitor lysosomal function and respond to degradation requirements and environmental cues. We report the identification of transcription factor E3 (TFE3) as another regulator of lysosomal homeostasis that induced expression of genes encoding proteins involved in autophagy and lysosomal biogenesis in ARPE-19 cells in response to starvation and lysosomal stress. We found that in nutrient-replete cells, TFE3 was recruited to lysosomes through interaction with active Rag guanosine triphosphatases (GTPases) and exhibited mammalian (or mechanistic) target of rapamycin complex 1 (mTORC1)-dependent phosphorylation. Phosphorylated TFE3 was retained in the cytosol through its interaction with the cytosolic chaperone 14-3-3. After starvation, TFE3 rapidly translocated to the nucleus and bound to the CLEAR elements present in the promoter region of many lysosomal genes, thereby inducing lysosomal biogenesis. Depletion of endogenous TFE3 entirely abolished the response of ARPE-19 cells to starvation, suggesting that TFE3 plays a critical role in nutrient sensing and regulation of energy metabolism. Furthermore, overexpression of TFE3 triggered lysosomal exocytosis and resulted in efficient cellular clearance in a cellular model of a lysosomal storage disorder, Pompe disease, thus identifying TFE3 as a potential therapeutic target for the treatment of lysosomal disorders.

The RAVE complex is an isoform-specific V-ATPase assembly factor in yeast.

The regulator of ATPase of vacuoles and endosomes (RAVE) complex is implicated in vacuolar H(+)-translocating ATPase (V-ATPase) assembly and activity. In yeast, rav1 mutants exhibit a Vma(-) growth phenotype characteristic of loss of V-ATPase activity only at high temperature. Synthetic genetic analysis identified mutations that exhibit a full, temperature-independent Vma(-) growth defect when combined with the rav1 mutation. These include class E vps mutations, which compromise endosomal sorting. The synthetic Vma(-) growth defect could not be attributed to loss of vacuolar acidification in the double mutants, as there was no vacuolar acidification in the rav1 mutant. The yeast V-ATPase a subunit is present as two isoforms, Stv1p in Golgi and endosomes and Vph1p in vacuoles. Rav1p interacts directly with the N-terminal domain of Vph1p. STV1 overexpression suppressed the growth defects of both rav1 and rav1vph1, and allowed RAVE-independent assembly of active Stv1p-containing V-ATPases in vacuoles. Mutations causing synthetic genetic defects in combination with rav1 perturbed the normal localization of Stv1-green fluorescent protein. We propose that RAVE is necessary for assembly of Vph1-containing V-ATPase complexes but not Stv1-containing complexes. Synthetic Vma(-) phenotypes arise from defects in Vph1p-containing complexes caused by rav1, combined with defects in Stv1p-containing V-ATPases caused by the second mutation. Thus RAVE is the first isoform-specific V-ATPase assembly factor.

Efficacy of Onabotulinum Toxin A (Botox) versus Abobotulinum Toxin A (Dysport) Using a Conversion Factor (1 : 2.5) in Treatment of Primary Palmar Hyperhidrosis.

Background. Two preparations of botulinum A toxin (BTX-A) are commercially available for the treatment of palmar hyperhidrosis (PPH): Botox (Allergan; 100 U/vial) and Dysport (Ipsen Limited; 500 U/vial), which are not bioequivalent. Results regarding an appropriate conversion factor between them are controversial. Objectives. This paper aims to compare the efficacy of Botox and Dysport in PPH using a conversion factor of 1 : 2.5. Methods. Eight patients with severe PPH received intradermal injections of Botox in one palm and Dysport in the other in the same session. Clinical assessment was performed at baseline and posttreatment for 8 months using Minor's iodine starch test, Hyperhidrosis Disease Severity Scale (HDSS), and Dermatology Life Quality Index (DLQI) test. Results. At 3 weeks, a significant decrease in sweating for both preparations was noted which was more pronounced with Dysport compared with Botox. At 8 weeks, this difference turned insignificant. Continued evaluation showed similar improvement in both palms with a nonsignificant difference. Patients with longer disease duration were more liable to relapse. Conclusion. The efficacy and safety of Botox and Dysport injections were similar using a conversion factor of 1 : 2.5. There was a trend towards a more rapid action after Dysport treatment but without significant importance.

Loss of vacuolar H+-ATPase (V-ATPase) activity in yeast generates an iron deprivation signal that is moderated by induction of the peroxiredoxin TSA2.

Vacuolar H(+)-ATPases (V-ATPases) acidify intracellular organelles and help to regulate overall cellular pH. Yeast vma mutants lack V-ATPase activity and allow exploration of connections between cellular pH, iron, and redox homeostasis common to all eukaryotes. A previous microarray study in a vma mutant demonstrated up-regulation of multiple iron uptake genes under control of Aft1p (the iron regulon) and only one antioxidant gene, the peroxiredoxin TSA2 (Milgrom, E., Diab, H., Middleton, F., and Kane, P. M. (2007) Loss of vacuolar proton-translocating ATPase activity in yeast results in chronic oxidative stress. J. Biol. Chem. 282, 7125-7136). Fluorescent biosensors placing GFP under transcriptional control of either an Aft1-dependent promoter (P(FIT2)-GFP) or the TSA2 promoter (P(TSA2)-GFP) were constructed to monitor transcriptional signaling. Both biosensors were up-regulated in the vma2Δ mutant, and acute V-ATPase inhibition with concanamycin A induced coordinate up-regulation from both promoters. PTSA2-GFP induction was Yap1p-dependent, indicating an oxidative stress signal. Total cell iron measurements indicate that the vma2Δ mutant is iron-replete, despite up-regulation of the iron regulon. Acetic acid up-regulated P(FIT2)-GFP expression in wild-type cells, suggesting that loss of pH control contributes to an iron deficiency signal in the mutant. Iron supplementation significantly decreased P(FIT2)-GFP expression and, surprisingly, restored P(TSA2)-GFP to wild-type levels. A tsa2Δ mutation induced both nuclear localization of Aft1p and P(FIT2)-GFP expression. The data suggest a novel function for Tsa2p as a negative regulator of Aft1p-driven transcription, which is induced in V-ATPase mutants to limit transcription of the iron regulon. This represents a new mechanism bridging the antioxidant and iron-regulatory pathways that is intimately linked to pH homeostasis.

Subunit interactions and requirements for inhibition of the yeast V1-ATPase.

Disassembly of the yeast V-ATPase into cytosolic V(1) and membrane V(0) sectors inactivates MgATPase activity of the V(1)-ATPase. This inactivation requires the V(1) H subunit (Parra, K. J., Keenan, K. L., and Kane, P. M. (2000) J. Biol. Chem. 275, 21761-21767), but its mechanism is not fully understood. The H subunit has two domains. Interactions of each domain with V(1) and V(0) subunits were identified by two-hybrid assay. The B subunit of the V(1) catalytic headgroup interacted with the H subunit N-terminal domain (H-NT), and the C-terminal domain (H-CT) interacted with V(1) subunits B, E (peripheral stalk), and D (central stalk), and the cytosolic N-terminal domain of V(0) subunit Vph1p. V(1)-ATPase complexes from yeast expressing H-NT are partially inhibited, exhibiting 26% the MgATPase activity of complexes with no H subunit. The H-CT domain does not copurify with V(1) when expressed in yeast, but the bacterially expressed and purified H-CT domain inhibits MgATPase activity in V(1) lacking H almost as well as the full-length H subunit. Binding of full-length H subunit to V(1) was more stable than binding of either H-NT or H-CT, suggesting that both domains contribute to binding and inhibition. Intact H and H-CT can bind to the expressed N-terminal domain of Vph1p, but this fragment of Vph1p does not bind to V(1) complexes containing subunit H. We propose that upon disassembly, the H subunit undergoes a conformational change that inhibits V(1)-ATPase activity and precludes V(0) interactions.

Association of beta-defensin 1 single nucleotide polymorphism with atopic dermatitis.

Atopic dermatitis (AD) is a chronic relapsing, pruritic, inflammatory skin disease, which results from a complex interplay between genetic and environmental factors. Defensins are broadly dispersed family of antimicrobial peptides which are classified into 2 distinct families: the alpha-defensins and the beta-defensins. The primary function of defensins is to protect the skin from invasion by foreign pathogens. Previous studies suggested that single nucleotide polymorphisms (SNPs) of the beta-defensin 1 gene (DEFB1) could be involved in the development of AD. The Aim of the study is to examine DEFB1 gene to gain a better understanding of their role in the pathophysiology of AD patients and their involvement in AD susceptibility and severity. 35 atopic patients and 10 healthy volunteers as controls were investigated. They were subjected to analysis of absolute eosinophil count, total and specific IgE and detection of Beta-defensin-1 gene polymorphism at position 692 and 1654 using PCR amplification and restriction analysis. We observed significant difference in the distribution of the DEFB1 AIG polymorphism at 692 (P<0.01) in AD patients compared to controls, but not at 1654. A statistical significant association between DEFB1 692 GG genotype and elevated total serum IgE level (P<0.01), and between DEFB1 692 GG and AG genotypes & 1654 AA genotype and high absolute eosinophil count (P<0.05) were found. Concerning Specific IgE there was significant association between DEFB1 692 GG genotype and positive specific IgE to dermatophytes and HDM (House Dust Mite) (P1<0.01) while DEFB1 1654AA genotype shows significant association with positive specific IgE to cockroaches (P<0.05). Regarding SCORAD severity index, there was significant statistical association between DEFB1 692 GG and AG & DEFB1 1654 AA and AG genotype with severe AD disease (P<0.05). The correlation between atopic markers and SCORAD severity index shows that there was a significant statistical relationship between serum levels of total IgE (P<0.01), absolute eosinophil count (P<0.01), specific IgE to cat (P<0.05), HDM (P<0.01) and cockroaches (P<0.01) and SCORAD. Our findings support previously studies suggesting that DEFB1 gene is one of the candidate genes for atopy. G allele at site 692& AA genotype at site 1654 may be useful as markers for AD susceptibility and severity

Loss of vacuolar proton-translocating ATPase activity in yeast results in chronic oxidative stress.

Yeast mutants lacking vacuolar proton-translocating ATPase (V-ATPase) subunits (vma mutants) were sensitive to several different oxidants in a recent genomic screen (Thorpe, G. W., Fong, C. S., Alic, N., Higgins, V. J., and Dawes, I. W. (2004) Proc. Natl. Acad. Sci. U. S. A. 101, 6564-6569). We confirmed that mutants lacking a V(1) subunit (vma2Delta), V(o) subunit, or either of the two V(o) a subunit isoforms are acutely sensitive to H(2)O(2) and more sensitive to menadione and diamide than wild-type cells. The vma2Delta mutant contains elevated levels of reactive oxygen species and high levels of oxidative protein damage even in the absence of an applied oxidant, suggesting an endogenous source of oxidative stress. vma2Delta mutants lacking mitochondrial DNA showed neither improved growth nor decreased sensitivity to peroxide, excluding respiration as the major source of the endogenous reactive oxygen species in the mutant. Double mutants lacking both VMA2 and components of the major cytosolic defense systems exhibited synthetic sensitivity to H(2)O(2). Microarray analysis comparing wild-type and vma2Delta mutant cells grown at pH 5, permissive conditions for the vma2Delta mutant, indicated high level up-regulation of several iron uptake and metabolism genes that are part of the Aft1/Aft2 regulon. TSA2, which encodes an isoform of the cytosolic thioredoxin peroxidase, was strongly induced, but other oxidative stress defense systems were not induced. The results indicate that V-ATPase activity helps to protect cells from endogenous oxidative stress.

Gene expression analysis of peripheral blood leukocytes from discordant sib-pairs with schizophrenia and bipolar disorder reveals points of convergence between genetic and functional genomic approaches.

We performed global RNA transcript analysis and comprehensive gene group analysis of peripheral blood leukocyte (PBL) RNA from two groups of matched sib-pairs that were discordant for either schizophrenia (n = 33 sib-pairs) or bipolar disorder (n = 5 sib-pairs). The pairs chosen for these analyses were selected from families with known patterns of genetic linkage (5q for schizophrenia and 6q for bipolar disorder). At the single gene level, we obtained lists of the transcripts with the most significant changes in expression and from these lists determined those with the highest degree of predictive power for classifying subjects according to diagnosis in these samples. At the gene group level, we comprehensively analyzed pairwise expression changes of more than 4,000 functional groups and cytogenetic locations, and present a novel method of displaying these data that we term "cytogenomic" mapping. Verification of selected changes in expression was performed using quantitative real-time RT-PCR. Our results provide compelling evidence for the utility of analyzing PBL RNA for changes in expression in neuropsychiatric disorders.