Single-cell spatial transcriptome reveals cell-type organization in the macaque cortex.

Elucidating the cellular organization of the cerebral cortex is critical for understanding brain structure and function. Using large-scale single-nucleus RNA sequencing and spatial transcriptomic analysis of 143 macaque cortical regions, we obtained a comprehensive atlas of 264 transcriptome-defined cortical cell types and mapped their spatial distribution across the entire cortex. We characterized the cortical layer and region preferences of glutamatergic, GABAergic, and non-neuronal cell types, as well as regional differences in cell-type composition and neighborhood complexity. Notably, we discovered a relationship between the regional distribution of various cell types and the region's hierarchical level in the visual and somatosensory systems. Cross-species comparison of transcriptomic data from human, macaque, and mouse cortices further revealed primate-specific cell types that are enriched in layer 4, with their marker genes expressed in a region-dependent manner. Our data provide a cellular and molecular basis for understanding the evolution, development, aging, and pathogenesis of the primate brain.

Spatiotemporal transcriptomic atlas of mouse organogenesis using DNA nanoball-patterned arrays.

Spatially resolved transcriptomic technologies are promising tools to study complex biological processes such as mammalian embryogenesis. However, the imbalance between resolution, gene capture, and field of view of current methodologies precludes their systematic application to analyze relatively large and three-dimensional mid- and late-gestation embryos. Here, we combined DNA nanoball (DNB)-patterned arrays and in situ RNA capture to create spatial enhanced resolution omics-sequencing (Stereo-seq). We applied Stereo-seq to generate the mouse organogenesis spatiotemporal transcriptomic atlas (MOSTA), which maps with single-cell resolution and high sensitivity the kinetics and directionality of transcriptional variation during mouse organogenesis. We used this information to gain insight into the molecular basis of spatial cell heterogeneity and cell fate specification in developing tissues such as the dorsal midbrain. Our panoramic atlas will facilitate in-depth investigation of longstanding questions concerning normal and abnormal mammalian development.

Deciphering spatial domains from spatial multi-omics with SpatialGlue.

Advances in spatial omics technologies now allow multiple types of data to be acquired from the same tissue slice. To realize the full potential of such data, we need spatially informed methods for data integration. Here, we introduce SpatialGlue, a graph neural network model with a dual-attention mechanism that deciphers spatial domains by intra-omics integration of spatial location and omics measurement followed by cross-omics integration. We demonstrated SpatialGlue on data acquired from different tissue types using different technologies, including spatial epigenome-transcriptome and transcriptome-proteome modalities. Compared to other methods, SpatialGlue captured more anatomical details and more accurately resolved spatial domains such as the cortex layers of the brain. Our method also identified cell types like spleen macrophage subsets located at three different zones that were not available in the original data annotations. SpatialGlue scales well with data size and can be used to integrate three modalities. Our spatial multi-omics analysis tool combines the information from complementary omics modalities to obtain a holistic view of cellular and tissue properties.

Complement factor B is critical for sub-RPE deposit accumulation in a model of Doyne honeycomb retinal dystrophy with features of age-related macular degeneration.

EFEMP1 R345W is a dominant mutation causing Doyne honeycomb retinal dystrophy/malattia leventinese (DHRD/ML), a rare blinding disease with clinical pathology similar to age-related macular degeneration (AMD). Aged Efemp1  R345W/R345W knock-in mice (Efemp1ki/ki) develop microscopic deposits on the basal side of retinal pigment epithelial cells (RPE), an early feature in DHRD/ML and AMD. Here, we assessed the role of alternative complement pathway component factor B (FB) in the formation of these deposits. RNA-seq analysis of the posterior eyecups revealed increased unfolded protein response, decreased mitochondrial function in the neural retina (by 3 months of age) and increased inflammatory pathways in both neural retina and posterior eyecups (at 17 months of age) of Efemp1ki/ki mice compared with wild-type littermate controls. Proteomics analysis of eye lysates confirmed similar dysregulated pathways as detected by RNA-seq. Complement activation was increased in aged Efemp1ki/ki eyes with an approximately 2-fold elevation of complement breakdown products iC3b and Ba (P < 0.05). Deletion of the Cfb gene in female Efemp1ki/ki mice partially normalized the above dysregulated biological pathway changes and oral dosing of a small molecule FB inhibitor from 10 to 12 months of age reduced sub-RPE deposits by 65% (P = 0.029). In contrast, male Efemp1ki/ki mice had fewer sub-RPE deposits than age-matched females, no elevation of ocular complement activation and no effect of FB inhibition on sub-RPE deposits. The effects of FB deletion or inhibition on Efemp1ki/ki mice supports systemic inhibition of the alternative complement pathway as a potential treatment of dry AMD and DHRD/ML.

Transcriptome analysis of Kluyveromyces marxianus under succinic acid stress and development of robust strains.

Kluyveromyces marxianus has become an attractive non-conventional yeast cell factory due to its advantageous properties such as high thermal tolerance and rapid growth. Succinic acid (SA) is an important platform molecule that has been applied in various industries such as food, material, cosmetics, and pharmaceuticals. SA bioproduction may be compromised by its toxicity. Besides, metabolite-responsive promoters are known to be important for dynamic control of gene transcription. Therefore, studies on global gene transcription under various SA concentrations are of great importance. Here, comparative transcriptome changes of K. marxianus exposed to various concentrations of SA were analyzed. Enrichment and analysis of gene clusters revealed repression of the tricarboxylic acid cycle and glyoxylate cycle, also activation of the glycolysis pathway and genes related to ergosterol synthesis. Based on the analyses, potential SA-responsive promoters were investigated, among which the promoter strength of IMTCP2 and KLMA_50231 increased 43.4% and 154.7% in response to 15 g/L SA. In addition, overexpression of the transcription factors Gcr1, Upc2, and Ndt80 significantly increased growth under SA stress. Our results benefit understanding SA toxicity mechanisms and the development of robust yeast for organic acid production. KEY POINTS: • Global gene transcription of K. marxianus is changed by succinic acid (SA) • Promoter activities of IMTCP2 and KLMA_50123 are regulated by SA • Overexpression of Gcr1, Upc2, and Ndt80 enhanced SA tolerance.

Cationic proteins from eosinophils bind bone morphogenetic protein receptors promoting vascular calcification and atherogenesis.

AIMS: Blood eosinophil count and eosinophil cationic protein (ECP) concentration are risk factors of cardiovascular diseases. This study tested whether and how eosinophils and ECP contribute to vascular calcification and atherogenesis. METHODS AND RESULTS: Immunostaining revealed eosinophil accumulation in human and mouse atherosclerotic lesions. Eosinophil deficiency in ΔdblGATA mice slowed atherogenesis with increased lesion smooth muscle cell (SMC) content and reduced calcification. This protection in ΔdblGATA mice was muted when mice received donor eosinophils from wild-type (WT), Il4-/-, and Il13-/- mice or mouse eosinophil-associated-ribonuclease-1 (mEar1), a murine homologue of ECP. Eosinophils or mEar1 but not interleukin (IL) 4 or IL13 increased the calcification of SMC from WT mice but not those from Runt-related transcription factor-2 (Runx2) knockout mice. Immunoblot analyses showed that eosinophils and mEar1 activated Smad-1/5/8 but did not affect Smad-2/3 activation or expression of bone morphogenetic protein receptors (BMPR-1A/1B/2) or transforming growth factor (TGF)-ß receptors (TGFBR1/2) in SMC from WT and Runx2 knockout mice. Immunoprecipitation showed that mEar1 formed immune complexes with BMPR-1A/1B but not TGFBR1/2. Immunofluorescence double-staining, ligand binding, and Scatchard plot analysis demonstrated that mEar1 bound to BMPR-1A and BMPR-1B with similar affinity. Likewise, human ECP and eosinophil-derived neurotoxin (EDN) also bound to BMPR-1A/1B on human vascular SMC and promoted SMC osteogenic differentiation. In a cohort of 5864 men from the Danish Cardiovascular Screening trial and its subpopulation of 394 participants, blood eosinophil counts and ECP levels correlated with the calcification scores of different arterial segments from coronary arteries to iliac arteries. CONCLUSION: Eosinophils release cationic proteins that can promote SMC calcification and atherogenesis using the BMPR-1A/1B-Smad-1/5/8-Runx2 signalling pathway.

The Role of Bioactive Small Molecules in COPD Pathogenesis.

Chronic obstructive pulmonary disease (COPD) is recognized as a predominant contributor to mortality worldwide, which causes significant burdens to both society and individuals. Given the limited treatment options for COPD, there lies a critical realization: the imperative for expeditious development of novel therapeutic modalities that can effectively alleviate disease progression and enhance the quality of life experienced by COPD patients. Within the intricate field of COPD pathogenesis, an assortment of biologically active small molecules, encompassing small protein molecules and their derivatives, assumes crucial roles through diverse mechanisms. These mechanisms relate to the regulation of redox balance, the inhibition of the release of inflammatory mediators, and the modulation of cellular functions. Therefore, the present article aims to explore and elucidate the distinct roles played by different categories of biologically active small molecules in contributing to the pathogenesis of COPD.

Early autoimmunity and outcome in virus encephalitis: a retrospective study based on tissue-based assay.

To explore the autoimmune response and outcome in the central nervous system (CNS) at the onset of viral infection and correlation between autoantibodies and viruses. METHODS: A retrospective observational study was conducted in 121 patients (2016-2021) with a CNS viral infection confirmed via cerebrospinal fluid (CSF) next-generation sequencing (cohort A). Their clinical information was analysed and CSF samples were screened for autoantibodies against monkey cerebellum by tissue-based assay. In situ hybridisation was used to detect Epstein-Barr virus (EBV) in brain tissue of 8 patients with glial fibrillar acidic protein (GFAP)-IgG and nasopharyngeal carcinoma tissue of 2 patients with GFAP-IgG as control (cohort B). RESULTS: Among cohort A (male:female=79:42; median age: 42 (14-78) years old), 61 (50.4%) participants had detectable autoantibodies in CSF. Compared with other viruses, EBV increased the odds of having GFAP-IgG (OR 18.22, 95% CI 6.54 to 50.77, p<0.001). In cohort B, EBV was found in the brain tissue from two of eight (25.0%) patients with GFAP-IgG. Autoantibody-positive patients had a higher CSF protein level (median: 1126.00 (281.00-5352.00) vs 700.00 (76.70-2899.00), p<0.001), lower CSF chloride level (mean: 119.80±6.24 vs 122.84±5.26, p=0.005), lower ratios of CSF-glucose/serum-glucose (median: 0.50[0.13-0.94] vs 0.60[0.26-1.23], p=0.003), more meningitis (26/61 (42.6%) vs 12/60 (20.0%), p=0.007) and higher follow-up modified Rankin Scale scores (1 (0-6) vs 0 (0-3), p=0.037) compared with antibody-negative patients. A Kaplan-Meier analysis revealed that autoantibody-positive patients experienced significantly worse outcomes (p=0.031). CONCLUSIONS: Autoimmune responses are found at the onset of viral encephalitis. EBV in the CNS increases the risk for autoimmunity to GFAP.

High-intensity focussed ultrasound and hysteroscopy endo-operative system cold device procedures for treating >4cm diameter FIGO Type 2 uterine myoma and ensuring successful pregnancy.

PURPOSE: Laparotomic or hysteroscopic myomectomy, and high-intensity focussed ultrasound (HIFU), can be used in the treatment of Type 2 myomas with a > 50% intramural component. Hysteroscopic electroresection may damage the remaining endometrium and complete ablation of myomas may not be realised via HIFU treatment. We report and examine the results of 1 case of HIFU treatment, accompanied with the hysteroscopy endo-operative system (HEOS) cold device procedure. MATERIALS AND METHODS: These were used for treating a > 4cm diameter Type 2 uterine myoma and ensuring successful pregnancy. HIFU treatment of uterine myomas can achieve point-by-point ablation of lesions as far as possible without damaging the endometrium. The HEOS cold device procedure was implemented 3 months after HIFU treatment. RESULTS: The patient got pregnant naturally in the third month after receiving treatment, with no complications during her pregnancy. The patient gave birth to a healthy male via full-term cesarean section. CONCLUSIONS: HIFU treatment, accompanied with the HEOS cold device procedure, ensured complete myoma removal. It also preserved the integrity of the myometrium and prevented uterine perforation during surgery.

For the patient who had a large uterine myoma and wanted to achieve pregnancy soon, HIFU accompanied with the HEOS cold device procedure could ensure complete myoma removal, whilst preserving the integrity of myometrium and preventing uterine perforation during surgery.

Discovery and therapeutic implications of bioactive dihydroxylated phenolic acids in patients with severe heart disease and conditions associated with inflammation and hypoxia.

Our initial studies detected elevated levels of 3,4-dihydroxyphenyllactic acid (DHPLA) in urine samples of patients with severe heart disease when compared with healthy subjects. Given the reported anti-inflammatory properties of DHPLA and related dihydroxylated phenolic acids (DPAs), we embarked on an exploratory multi-centre investigation in patients with no urinary tract infections to establish the possible pathophysiological significance and therapeutic implications of these findings. Chinese and Caucasian patients being treated for severe heart disease or those conditions associated with inflammation (WBC ≥ 10 ×109/L or hsCRP ≥ 3.0 mg/L) and/or hypoxia (PaO2 ≤ 75 mmHg) were enrolled; their urine samples were analyzed by HPLC, HPLC-MS, GC-MS and biotransformation assays. DHPLA was detected in urine samples of patients, but undetectable in healthy volunteers. Dynamic monitoring of inpatients undergoing treatment showed their DHPLA levels declined in proportion to their clinical improvement. In DHPLA-positive patients' fecal samples, Proteus vulgaris and P. mirabilis were more abundant than healthy volunteers. In culture, these gut bacteria were capable of reversible interconversion between DOPA and DHPLA. Furthermore, porcine and rodent organs were able to metabolize DOPA to DHPLA and related phenolic acids. The elevated levels of DHPLA in these patients suggest bioactive DPAs are generated de novo as part of a human's defense mechanism against disease. Because DHPLA isolated from Radix Salvia miltiorrhizae has a multitude of pharmacological activities, these data underpin the scientific basis of this medicinal plant's ethnopharmacological applications as well as highlighting the therapeutic potential of endogenous, natural or synthetic DPAs and their derivatives in humans.

Insights into the development of pentylenetetrazole-induced epileptic seizures from dynamic metabolomic changes.

Epilepsy is often considered to be a progressive neurological disease, and the nature of this progression remains unclear. Understanding the overall and common metabolic changes of epileptic seizures can provide novel clues for their control and prevention. Herein, a chronic kindling animal model was established to obtain generalized tonic-clonic seizures via the repeated injections of pentylenetetrazole (PTZ) at subconvulsive dose. Dynamic metabolomic changes in plasma and urine from PTZ-kindled rats at the different kindling phases were explored using NMR-based metabolomics, in combination with behavioral assessment, brain neurotransmitter measurement, electroencephalography and histopathology. The increased levels of glucose, lactate, glutamate, creatine and creatinine, together with the decreased levels of pyruvate, citrate and succinate, ketone bodies, asparagine, alanine, leucine, valine and isoleucine in plasma and/or urine were involved in the development and progression of seizures. These altered metabolites reflected the pathophysiological processes including the compromised energy metabolism, the disturbed amino acid metabolism, the peripheral inflammation and changes in gut microbiota functions. NMR-based metabolomics could provide brain disease information by the dynamic plasma and urinary metabolic changes during chronic epileptic seizures, yielding classification of seizure stages and profound insights into controlling epilepsy via targeting deficient energy metabolism.

Small-molecule factor B inhibitor for the treatment of complement-mediated diseases.

Dysregulation of the alternative complement pathway (AP) predisposes individuals to a number of diseases including paroxysmal nocturnal hemoglobinuria, atypical hemolytic uremic syndrome, and C3 glomerulopathy. Moreover, glomerular Ig deposits can lead to complement-driven nephropathies. Here we describe the discovery of a highly potent, reversible, and selective small-molecule inhibitor of factor B, a serine protease that drives the central amplification loop of the AP. Oral administration of the inhibitor prevents KRN-induced arthritis in mice and is effective upon prophylactic and therapeutic dosing in an experimental model of membranous nephropathy in rats. In addition, inhibition of factor B prevents complement activation in sera from C3 glomerulopathy patients and the hemolysis of human PNH erythrocytes. These data demonstrate the potential therapeutic value of using a factor B inhibitor for systemic treatment of complement-mediated diseases and provide a basis for its clinical development.

Efficient COI barcoding using high throughput single-end 400 bp sequencing.

BACKGROUND: Over the last decade, the rapid development of high-throughput sequencing platforms has accelerated species description and assisted morphological classification through DNA barcoding. However, the current high-throughput DNA barcoding methods cannot obtain full-length barcode sequences due to read length limitations (e.g. a maximum read length of 300 bp for the Illumina's MiSeq system), or are hindered by a relatively high cost or low sequencing output (e.g. a maximum number of eight million reads per cell for the PacBio's SEQUEL II system). RESULTS: Pooled cytochrome c oxidase subunit I (COI) barcodes from individual specimens were sequenced on the MGISEQ-2000 platform using the single-end 400 bp (SE400) module. We present a bioinformatic pipeline, HIFI-SE, that takes reads generated from the 5' and 3' ends of the COI barcode region and assembles them into full-length barcodes. HIFI-SE is written in Python and includes four function modules of filter, assign, assembly and taxonomy. We applied the HIFI-SE to a set of 845 samples (30 marine invertebrates, 815 insects) and delivered a total of 747 fully assembled COI barcodes as well as 70 Wolbachia and fungi symbionts. Compared to their corresponding Sanger sequences (72 sequences available), nearly all samples (71/72) were correctly and accurately assembled, including 46 samples that had a similarity score of 100% and 25 of ca. 99%. CONCLUSIONS: The HIFI-SE pipeline represents an efficient way to produce standard full-length barcodes, while the reasonable cost and high sensitivity of our method can contribute considerably more DNA barcodes under the same budget. Our method thereby advances DNA-based species identification from diverse ecosystems and increases the number of relevant applications.

Pharmacokinetics of 13 active components in a rat model of middle cerebral artery occlusion after intravenous injection of Radix Salviae miltiorrhizae-Lignum dalbergiae odoriferae prescription.

As a representative formulation of Radix Salviae miltiorrhizae (Danshen)-Lignum Dalbergiae odoriferae (Jiangxiang), Xiangdan injection is widely prescribed for cardio- and cerebrovascular diseases in practice. This necessitates a pharmacokinetic investigation of this formulation to make it safer and more broadly applicable. We developed and validated a sensitive, selective, and reliable high-performance liquid chromatography with tandem mass spectrometry method for the simultaneous determination of 11 phenolic compounds including danshensu plus two diterpenoid quinones like cryptotanshinone and tanshinone IIA in rat. We applied this method for the pharmacokinetic studies of the 13 compounds in a rat model of middle cerebral artery occlusion after intravenous injection of Xiangdan injection or Danshen injection. In sham-operated rats, the animals taking Xiangdan injection exhibited significant growth of the area under the curve for danshensu, protocatechuic aldehyde, and tanshinone IIA compared with the changes seen in the data of those administrated with Danshen injection. Such a pattern was also observed in middle cerebral artery occlusion rats, whereas increased the area under the curve values were observed for danshensu, protocatechuic aldehyde, caffeic acid, rosmarinic acid, and tanshinone IIA. These results demonstrated that synergistic interactions occurred between the components of Danshen and the active compounds of Jiangxiang both in sham-operated and middle cerebral artery occlusion rats, increasing the bioavailability of Danshen. The results presented herein can be used to determine a reference dose for the clinical application of Xiangdan injection, and to elucidate the synergistic mechanism of Danshen and Jiangxiang.

A multiplex PCR for the identification of Echinococcus multilocularis, E. granulosus sensu stricto and E. canadensis that infect human.

Echinococcus granulosus sensu stricto (s.s.), Echinococcus multilocularis and Echinococcus canadensis are the common causes of human echinococcosis in China. An accurate species identification tool for human echinococcosis is needed as the treatments and prognosis are different among species. The present work demonstrates a method for the simultaneous detection of these three Echinococcus species based on multiplex polymerase chain reaction (mPCR). Specific primers of this mPCR were designed based on the mitochondrial genes and determined by extensive tests. The method can successfully detect either separated or mixed target species, and generate expected amplicons of distinct size for each species. Sensitivity of the method was tested by serially diluted DNA, showing a detection threshold as less as 0.32 pg for both E. granulosus s.s. and E. canadensis, and 1.6 pg for E. multilocularis. Specificity assessed against 18 other parasites was found to be 100% except weakly cross-react with E. shiquicus. The assay was additionally applied to 69 echinococcosis patients and 38 healthy persons, confirming the high reliability of the method. Thus, the mPCR described here has high application potential for clinical identification purposes, and can further provide a useful tool for evaluation of serology and imaging method.

Metabolite identification of tanshinol borneol ester in rats by high-performance liquid chromatography combined with electrospray ionization quadrupole time-of-flight mass spectrometry.

Tanshinol borneol ester (DBZ) is a potential drug candidate composed of danshensu and borneol. It shows anti-ischemic and anti-atherosclerosis activity. However, little is known about its metabolism in vivo. This research aimed to elucidate the metabolic profile of DBZ through analyzing its metabolites using high-performance liquid chromatography combined with electrospray ionization quadrupole time-of-flight mass spectrometry. Chromatographic separation was performed on an Agilent TC-C18 column (150 × 4.6 mm, 5.0 µm) with gradient elution using methanol and water containing 0.2% (v/v) formic acid as the mobile phase. Metabolite identification involved analyzing the retention behaviors, changes in molecular weights and MS/MS fragment patterns of DBZ and its metabolites. As a result, 20 potential metabolites were detected and tentatively identified in rat plasma, urine and feces after administration of DBZ. DBZ could be metabolized to O-methylated DBZ, DBZ-O-glucuronide, O-methylated DBZ-O-glucuronide, hydroxylated DBZ and danshensu. Danshensu, a hydrolysis product of DBZ, could further be transformed into 12 metabolites. The proposed method was confirmed to be a reliable and sensitive alternative for characterizing metabolic pathways of DBZ and providing valuable information on its druggability.

Small-molecule factor D inhibitors targeting the alternative complement pathway.

Complement is a key component of the innate immune system, recognizing pathogens and promoting their elimination. Complement component 3 (C3) is the central component of the system. Activation of C3 can be initiated by three distinct routes-the classical, the lectin and the alternative pathways-with the alternative pathway also acting as an amplification loop for the other two pathways. The protease factor D (FD) is essential for this amplification process, which, when dysregulated, predisposes individuals to diverse disorders including age-related macular degeneration and paroxysmal nocturnal hemoglobinuria (PNH). Here we describe the identification of potent and selective small-molecule inhibitors of FD. These inhibitors efficiently block alternative pathway (AP) activation and prevent both C3 deposition onto, and lysis of, PNH erythrocytes. Their oral administration inhibited lipopolysaccharide-induced AP activation in FD-humanized mice. These data demonstrate the feasibility of inhibiting the AP with small-molecule antagonists and support the development of FD inhibitors for the treatment of complement-mediated diseases.

Investigation on temperature-induce conformational change of immobilized ß2 adrenergic receptor.

The ß2 adrenergic receptor (ß2-AR) is a prototypical family A G protein-coupled receptor (GPCR) and an excellent model system for studying the mechanism of GPCR activation. Purified ß2-AR was immobilized on macroporous silica gel to obtain liquid chromatographic stationary phase. The resulting phase was packed into a stainless steel column (4.6 × 50 mm, 7 µm) and used for on-line chromatographic system. When column oven temperature increased from 20.0 °C to 40.0 °C, uncomplete separate chromatographic peaks of ephedrine and pseudoephedrine as receptor conformational probe were gradually merged into one peak, meanwhile retention time and resolution of the probes were reduced correspondingly, which suggested that temperature could regulate protein conformation. Temperature-induced conformational change of immobilized ß2-AR, especially changes at higher temperatures, indicated that constructed receptor chromatography could simulate fever disease state of human body and clarify receptor conformation change at pathological condition. At the same time this study could also provide new ideas for screening active components in pathological conditions.

Specific correlation between the major chromosome 10q26 haplotype conferring risk for age-related macular degeneration and the expression of HTRA1.

PURPOSE: A region within chromosome 10q26 has a set of single nucleotide polymorphisms (SNPs) that define a haplotype that confers high risk for age-related macular degeneration (AMD). We used a bioinformatics approach to search for genes in this region that may be responsible for risk for AMD by assessing levels of gene expression in individuals carrying different haplotypes and by searching for open chromatin regions in the retinal pigment epithelium (RPE) that might include one or more of the SNPs. METHODS: We surveyed the PubMed and the 1000 Genomes databases to find all common (minor allele frequency > 0.01) SNPs in 10q26 strongly associated with AMD. We used the HaploReg and LDlink databases to find sets of SNPs with alleles in linkage disequilibrium and used the Genotype-Tissue Expression (GTEx) database to search for correlations between genotypes at individual SNPs and the relative level of expression of the genes. We also accessed Encyclopedia of DNA Elements (ENCODE) to find segments of open chromatin in the region with the AMD-associated SNPs. Predicted transcription factor binding motifs were identified using HOMER, PROMO, and RegulomeDB software programs. RESULTS: There are 34 polymorphisms within a 30-kb region that are in strong linkage disequilibrium (r2>0.8) with the reference SNP rs10490924 previously associated with risk for AMD. The expression of three genes in this region, PLEKHA1, ARMS2, and HTRA1 varies between people who have the low-AMD-risk haplotype compared with those with the high-AMD-risk haplotype. For PLEKHA1, 44 tissues have an expression pattern with the high-AMD-risk haplotype associated with low expression (rs10490924 effect size -0.43, p = 3.8 x 10-5 in ovary). With regard to ARMS2, the variation is most pronounced in testes: homozygotes with the high-AMD-risk haplotype express ARMS2 at lower levels than homozygotes with the low-AMD-risk haplotype; expression in heterozygotes falls in between (rs10490924 effect size -0.79, p = 7.5 x 10-24). For HTRA1, the expression pattern is the opposite; the high-AMD-risk haplotype has higher levels of expression in 27 tissues (rs10490924 effect size 0.40, p = 1.5 × 10-7 in testes). None of the other 22 genes within one megabase of rs10490924, or any gene in the entire genome, have mRNA expression levels that correlate with the high-AMD-risk haplotype. More than 100 other SNPs in the 10q26 region affect the expression of PLEKHA1 and ARMS2 but not that of HTRA1; none of these SNPs affects the risk for AMD according to published genome-wide association studies (GWASs). Two of the AMD-risk SNPs (rs36212732 and rs36212733) affect transcription factor binding sites in proximity to a DNase I hypersensitive region (i.e., a region of open chromatin) in RPE cells. CONCLUSIONS: SNPs in chromosome 10q26 that influence the expression of only PLEKHA1 or ARMS2 are not associated with risk for AMD, while most SNPs that influence the expression of HTRA1 are associated with risk for AMD. Two of the AMD-risk SNPs affect transcription factor binding sites that may control expression of one of the linked genes in the RPE. These findings suggest that the variation in the risk for AMD associated with chromosome 10q26 is likely due to variation in HTRA1 expression. Modulating HTRA1 activity might be a potential therapy for AMD.

Effects of two chronic stresses on mental state and hair follicle melanogenesis in mice.

Growing bodies of data show that psychological stress can be associated with hair loss and vitiligo. Researchers have revealed that stress could indeed inhibit hair growth in vivo, but the relationship between chronic stress and melanogenesis remains unknown. In this study, we established two types of stress models, chronic restraint stress (CRS) and chronic unpredicted mild stress (CUMS) mice models, and explored the possible role of stress in mice hair follicle melanogenesis. We found that stress changed hippocampal morphology, decreased 5-HT level in brain and skin and down-regulated 5-HT1A receptor expression in hippocampal CA1 region and skin. The alterations of 5-HT and 5-HT1A receptor might be a threshold of central stress to associate with the behaviour changes. Both two stresses caused cellular damage of melanocytes and inhibition of keratinocytes proliferation in HF, which made the synthetic pigment loss. CRS which was considered primarily as a "psychological" stressor had the lower melanin production in HF, as well as the level of 5-HT in skin was down-regulated more than those in CUMS group.