Clonal dynamics in early human embryogenesis inferred from somatic mutation.

Cellular dynamics and fate decision in early human embryogenesis remain largely unknown owing to the challenges of performing studies in human embryos1. Here, we explored whole-genomes of 334 single-cell colonies and targeted deep sequences of 379 bulk tissues obtained from various anatomical locations of seven recently deceased adult human donors. Using somatic mutations as an intrinsic barcode, we reconstructed early cellular phylogenies that demonstrate (1) an endogenous mutational rate that is higher in the first cell division but decreases to approximately one per cell per cell division later in life; (2) universal unequal contribution of early cells to embryo proper, resulting from early cellular bottlenecks that stochastically set aside epiblast cells within the embryo; (3) examples of varying degrees of early clonal imbalances between tissues on the left and right sides of the body, different germ layers and specific anatomical parts and organs; (4) emergence of a few ancestral cells that will substantially contribute to adult cell pools in blood and liver; and (5) presence of mitochondrial DNA heteroplasmy in the fertilized egg. Our approach also provides insights into the age-related mutational processes and loss of sex chromosomes in normal somatic cells. In sum, this study provides a foundation for future studies to complete cellular phylogenies in human embryogenesis.

Estimation of intrafamilial DNA contamination in family trio genome sequencing using deviation from Mendelian inheritance.

With the increasing number of sequencing projects involving families, quality control tools optimized for family genome sequencing are needed. However, accurately quantifying contamination in a DNA mixture is particularly difficult when genetically related family members are the sources. We developed TrioMix, a maximum likelihood estimation (MLE) framework based on Mendel's law of inheritance, to quantify DNA mixture between family members in genome sequencing data of parent-offspring trios. TrioMix can accurately deconvolute any intrafamilial DNA contamination, including parent-offspring, sibling-sibling, parent-parent, and even multiple familial sources. In addition, TrioMix can be applied to detect genomic abnormalities that deviate from Mendelian inheritance patterns, such as uniparental disomy (UPD) and chimerism. A genome-wide depth and variant allele frequency plot generated by TrioMix facilitates tracing the origin of Mendelian inheritance deviations. We showed that TrioMix could accurately deconvolute genomes in both simulated and real data sets.

Persistence and enrichment of dominant T cell clonotypes in expanded tumor-infiltrating lymphocytes of breast cancer.

BACKGROUND: Adoptive cell therapy using tumor-infiltrating lymphocytes (TILs) has shown promising results in cancer treatment, including breast cancer. However, clonal dynamics and clinical significance of TIL expansion ex vivo remain poorly understood. METHODS: We investigated T cell receptor (TCR) repertoire changes in expanded TILs from 19 patients with breast cancer. We compared TCR repertoire of TILs at different stages of expansion, including initial (2W TILs) and rapid expansion (REP TILs), and their overlap with formalin fixed paraffin embedded (FFPE) and peripheral blood. Additionally, we examined differences in TCR repertoire between CD4+ and CD8+ REP TILs. RESULTS: In descending order of proportion, average of 60% of the top 10% clonotypes of FFPE was retained in 2W TIL (60% in TRB, 64.7% in TRA). Among the overlapped clonotypes between 2W TILs and REP TILs, 69.9% was placed in top 30% of 2W TIL. The proportion of clonotypes in 2W TIL and REP TIL showed a significant positive correlation. CD4+ and CD8+ T cells show similar results in diversity and CDR3 length. CONCLUSIONS: Our study traces the changes in TILs repertoire from FFPE to 2W TIL and REP TIL and confirmed that clonotypes with high frequencies in TILs have a high likelihood of maintaining their priority throughout culture process.

Comparison of the Effect of Landmark-Based Midline and Paramedian Approaches on Spinal Anesthesia-Related Complications in Adult Patients: A Meta-Analysis of Randomized Controlled Trials.

Background and Objectives: Spinal anesthesia is widely used in various types of surgery. However, several complications can occur afterward. This study aimed to identify differences in the incidence of anesthesia-related complications according to the approach methods (midline versus paramedian) for landmark-based spinal anesthesia. Materials and Methods: We searched electronic databases, including PubMed, EMBASE, CENTRAL, Scopus, and Web of Science, for eligible randomized controlled trials. The primary outcome was post-dural puncture headache (PDPH) incidence, and secondary outcomes were low back pain (LBP) incidence and success rate in the first trial of spinal anesthesia. We estimated the odds ratio (OR) with 95% confidence intervals (CI) using a random-effects model. Results: In total, 2280 patients from 13 randomized controlled trials were included in the final analysis. The incidence rates of PDPH were 5.9% and 10.4% in the paramedian and midline approach groups, respectively. The pooled effect size revealed that the incidence of PDPH (OR: 0.43, 95% CI [0.22-0.83]; p = 0.01; I2 = 53%) and LBP (OR: 0.27, 95% CI [0.16-0.44]; p < 0.001; I2 = 16%) decreased, and the success rate in the first attempt was higher (OR: 2.30, 95% CI [1.36-3.87]; p = 0.002; I2 = 35%) with the paramedian than with the midline approach. Conclusions: Paramedian spinal anesthesia reduced PDPH and LBP and increased the success rate of the first attempt.

Personalised circulating tumour DNA assay with large-scale mutation coverage for sensitive minimal residual disease detection in colorectal cancer.

BACKGROUND: Postoperative minimal residual disease (MRD) detection using circulating-tumour DNA (ctDNA) requires a highly sensitive analysis platform. We have developed a tumour-informed, hybrid-capture ctDNA sequencing MRD assay. METHODS: Personalised target-capture panels for ctDNA detection were designed using individual variants identified in tumour whole-exome sequencing of each patient. MRD status was determined using ultra-high-depth sequencing data of plasma cell-free DNA. The MRD positivity and its association with clinical outcome were analysed in Stage II or III colorectal cancer (CRC). RESULTS: In 98 CRC patients, personalised panels for ctDNA sequencing were built from tumour data, including a median of 185 variants per patient. In silico simulation showed that increasing the number of target variants increases MRD detection sensitivity in low fractions (<0.01%). At postoperative 3-week, 21.4% of patients were positive for MRD by ctDNA. Postoperative positive MRD was strongly associated with poor disease-free survival (DFS) (adjusted hazard ratio 8.40, 95% confidence interval 3.49-20.2). Patients with a negative conversion of MRD after adjuvant therapy showed significantly better DFS (P < 0.001). CONCLUSION: Tumour-informed, hybrid-capture-based ctDNA assay monitoring a large number of patient-specific mutations is a sensitive strategy for MRD detection to predict recurrence in CRC.

Inactivation of Salmonella Typhimurium and Listeria monocytogenes on buckwheat seeds through combination treatment with plasma, vacuum packaging, and hot water.

AIMS: The objectives of this study were to evaluate the effect of combination treatment with cold plasma (CP), vacuum packaging (VP), and hot water (HW) on the inactivation of foodborne pathogens on buckwheat seeds, and determined the germination rates of seeds and the quality of sprouts following combination treatment. METHODS AND RESULTS: Buckwheat seeds inoculated with Salmonella Typhimurium and Listeria monocytogenes were treated with CP, HW, CP + HW, VP + HW, or CP + VP + HW. The germination rates of the HW-, CP + HW-, VP + HW-, and CP + VP + HW-treated seeds and the antioxidant activities and rutin contents of the CP + HW- and CP + VP + HW-treated sprouts were determined. HW, CP + HW, and CP + VP + HW were found to reduce the levels of the two pathogens to below the detection limit (1.0 log CFU g-1) at 70°C. However, HW and CP + HW significantly reduced the germination rate of buckwheat seeds. CP + VP + HW did not affect the germination rate of seeds nor the antioxidant activities and rutin content of buckwheat sprouts. CONCLUSIONS: These results indicate that CP + VP + HW can be used as a novel control method to reduce foodborne pathogens in seeds without causing quality deterioration.

The effect of intranasal dexmedetomidine administration on emergence agitation or delirium in pediatric patients after general anesthesia: A meta-analysis of randomized controlled trials.

BACKGROUND: Emergence agitation or delirium can occur in pediatric patients after anesthesia. Dexmedetomidine is known to reduce the impairment of postoperative cognitive function. AIMS: This study aimed to identify the role of intranasal administration of dexmedetomidine in lowering the development of emergence agitation or emergence delirium in pediatric patients after general anesthesia. METHODS: Electronic databases, including PubMed, EMBASE, CENTRAL, Scopus, and Web of Science, were searched to identify studies. The primary outcome was the proportion of patients who underwent emergence agitation or emergence delirium after the surgery. Secondary outcomes included emergence time and incidence of postoperative nausea and/or vomiting. We estimated the odds ratio and mean difference with 95% confidence intervals for the determination of effect size using a random-effects model. RESULTS: In total, 2103 pediatric patients from 20 randomized controlled trials were included in the final analysis. The incidence of emergence agitation or emergence delirium was 13.6% in the dexmedetomidine group and 33.2% in the control group. The pooled effect size revealed that intranasal dexmedetomidine administration significantly reduced the incidence of postoperative emergence agitation or emergence delirium in pediatric patients undergoing surgery under general anesthesia (odds ratio 0.25, 95% confidence interval 0.18-0.34; p = .0000; I2 = 37.74%). Additionally, significant difference was observed in emergence time between the two groups (mean difference 2.42, 95% confidence interval 0.37-4.46; p = .021; I2 = 98.40%). Children in the dexmedetomidine group had a significantly lower incidence of postoperative nausea and/or vomiting than those in the control group (odds ratio 0.39, 95% confidence interval 0.24-0.64; p = .0002; I2 = 0.00%). CONCLUSIONS: Intranasal dexmedetomidine reduced the incidence of emergence agitation or emergence delirium in pediatric patients after general anesthesia.

Anticancer Effect of Cold Atmospheric Plasma in Syngeneic Mouse Models of Melanoma and Colon Cancer.

Cold atmospheric plasma (CAP) may have applications in treating various types of malignant tumors. This study assessed the anticancer effects of CAP using melanoma and colon cancer cell lines. CAP treatment significantly reduced the in vitro viability of melanoma and colon cancer cell lines and had a negligible effect on the viability of normal human melanocytes. Additionally, CAP and epidermal growth factor receptor (EGFR) inhibitor had an additive anticancer effect in a CAP-resistant melanoma cell line. Reactive oxygen and nitrogen species known to be generated by CAP enhanced the anticancer effects of CAP and EGFR inhibitors. The in vivo anticancer activities of CAP were evaluated by testing its effects against syngeneic tumors induced in mice by melanoma and colon cancer cells. CAP treatment reduced tumor volume and weight in both cancer models, with the extent of tumor reduction dependent on the duration and number of CAP treatments. Histologic examination also revealed the tumoricidal effects of CAP in both tumor models. In conclusion, CAP inhibits the growth of mouse melanoma and colon cancer cell lines in vitro and shows tumoricidal effects against mouse models of melanoma and colon cancer in vivo.

Protein interactome and cell-type expression analyses reveal that cytoplasmic FMR1-interacting protein 1 (CYFIP1), but not CYFIP2, associates with astrocytic focal adhesion.

The two members of the cytoplasmic FMR1-interacting protein family, CYFIP1 and CYFIP2, are evolutionarily conserved multifunctional proteins whose defects are associated with distinct types of brain disorders. Even with high sequence homology between CYFIP1 and CYFIP2, several lines of evidence indicate their different functions in the brain; however, the underlying mechanisms remain largely unknown. Here, we performed reciprocal immunoprecipitation experiments using CYFIP1-2 × Myc and CYFIP2-3 × Flag knock-in mice and found that CYFIP1 and CYFIP2 are not significantly co-immunoprecipitated with each other in the knock-in brains compared with negative control wild-type (WT) brains. Moreover, CYFIP1 and CYFIP2 showed different size distributions by size-exclusion chromatography of WT mouse brains. Specifically, mass spectrometry-based analysis of CYFIP1-2 × Myc knock-in brains identified 131 proteins in the CYFIP1 interactome. Comparison of the CYFIP1 interactome with the previously identified brain region- and age-matched CYFIP2 interactome, consisting of 140 proteins, revealed only eight common proteins. Investigations using single-cell RNA-sequencing databases suggested non-neuronal cell- and neuron-enriched expression of Cyfip1 and Cyfip2, respectively. At the protein level, CYFIP1 was detected in both neurons and astrocytes, while CYFIP2 was detected only in neurons, suggesting the predominant expression of CYFIP1 in astrocytes. Bioinformatic characterization of the CYFIP1 interactome, and co-expression analysis of Cyfip1 with astrocytic genes, commonly linked CYFIP1 with focal adhesion proteins. Immunocytochemical analysis and proximity ligation assay suggested partial co-localization of CYFIP1 and focal adhesion proteins in cultured astrocytes. Together, these results suggest a CYFIP1-specific association with astrocytic focal adhesion, which may contribute to the different brain functions and dysfunctions of CYFIP1 and CYFIP2. Cover Image for this issue: https://doi.org/10.1111/jnc.15410.

Dynamic changes in longitudinal circulating tumour DNA profile during metastatic colorectal cancer treatment.

BACKGROUND: Circulating tumour DNA (ctDNA) has been spotlighted as an attractive biomarker because of its easy accessibility and real-time representation of tumour genetic profile. However, the clinical utility of longitudinal ctDNA monitoring has not been clearly defined. METHODS: Serial blood samples were obtained from metastatic colorectal cancer patients undergoing first-line chemotherapy. ctDNA was sequenced using a targeted next-generation sequencing platform which included 106 genes. Changes in ctDNA profile and treatment outcome were comprehensively analysed. RESULTS: A total of 272 samples from 62 patients were analysed. In all, 90.3% of patients had detectable ctDNA mutation before treatment. ctDNA clearance after chemotherapy was associated with longer progression-free survival which was independent of radiological response (adjusted hazard ratio 0.22, 95% confidence interval 0.10-0.46). Longitudinal monitoring was able to detect ctDNA progression which preceded radiological progressive disease (PD) in 58.1% (median 3.3 months). Diverse resistant mutations (34.9%) and gene amplification (7.0%) at the time of PD were discovered. For 16.3% of the PD patients, the newly identified mutations could be potential candidates of targeted therapy or clinical trial. CONCLUSION: ctDNA profile provided a more accurate landscape of tumour and dynamic changes compared to radiological evaluation. Longitudinal ctDNA monitoring may improve personalised treatment decision-making.

USP37 Deubiquitinates CDC73 in HPT-JT Syndrome.

The CDC73/HRPT2 gene, a defect which causes hyperparathyroidism-jaw tumor (HPT-JT) syndrome, encodes CDC73/parafibromin. We aimed to investigate whether CDC73 would be a target for ubiquitin-proteasome degradation. We cloned full-length cDNAs encoding a family of 58 ubiquitin-specific deubiquitinating enzymes (DUBs), also known as ubiquitin-specific proteases (USPs). Use of the yeast two-hybrid system then enabled us to identify USP37 as interacting with CDC73. The biochemical interaction between the USP37 and CDC73 and their reciprocal binding domains were studied. Co-localization of CDC73 and USP37 was observed in cells. CDC73 was found to be polyubiquitinated, and polyubiquitination of CDC73 was prominent in mutants. CDC73 was deubiquitinated via K48-specific ubiquitin chains by USP37, but not by the catalytically inactive USP37C350S mutant. Observation of the binding between deletion mutants of CDC73 and USP37 revealed that the ß-catenin binding site of CDC73 and the ubiquitin-interacting motifs 2 and 3 (UIM2 and 3) of USP37 were responsible for the interaction between the two proteins. Moreover, these two enzymes co-existed within the nucleus of COS7 cells. We conclude that USP37 is a DUB for CDC73 and that the two proteins interact through specific domains, suggesting that USP37 is responsible for the stability of CDC73 in HPT-JT syndrome.

Cohnella cholangitidis sp. nov., a novel species of the genus Cohnella isolated from a clinical specimen in Korea.

A Gram-positive, aerobic, rod-shaped bacterium, designated as strain 1605-214T, was isolated from the blood sample of a patient with cholangitis. Based on its 16S rRNA gene sequence, the strain 1605-214T belonged to the genus Cohnella and exhibited 97.9% sequence identity with Cohnella luojiensis DSM 24270T (GQ214052). DNA-DNA hybridization, digital DNA-DNA hybridization, and average nucleotide identity values between the two species were 23% ± 1.9, 21.1%, and 77.2%, respectively. The cellular fatty acids of strain 1605-214T were mainly comprised of anteiso-C15:0 (36.1%), iso-C16:0 (16.5%), and C16:0 (15.1%). The predominant quinone was menaquinone-7; predominant polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, and aminophospholipid-1. The cell wall peptidoglycan of strain 1605-214T contained meso-diaminopimelic acid. DNA G + C content of strain 1605-214T was 50.6 mol%. 5187 genes out of a total of 5413 (94.6%) were assigned putative functions using eggNOG v5.0. Based on genotypic characteristics and genomic sequence analysis results, strain 1605-214T was confirmed to represent a novel species of genus Cohnella, for which the name Cohnella cholangitidis sp. nov., was proposed.

Microstructural evolution in self-catalyzed GaAs nanowires during in-situ TEM study.

The microstructural evolutions in self-catalyzed GaAs nanowires (NWs) were investigated by using in situ heating transmission electron microscopy (TEM). The morphological changes of the self-catalyst metal gallium (Ga) droplet, the GaAs NWs, and the atomic behavior at the interface between the self-catalyst metal gallium and GaAs NWs were carefully studied by analysis of high-resolution TEM images. The microstructural change of the Ga-droplet/GaAs-NWs started at a low temperature of ∼200 °C. Formation and destruction of atomic layers were observed at the Ga/GaAs interface and slow depletion of the Ga droplet was detected in the temperature range investigated. Above 300 °C, the evolution process dramatically changed with time: The Ga droplet depleted rapidly and fast growth of zinc-blende (ZB) GaAs structures were observed in the droplet. The Ga droplet was completely removed with time and temperature. When the temperature reached ∼600 °C, the decomposition of GaAs was detected. This process began in the wurtzite (WZ) structure and propagated to the ZB structure. The morphological and atomistic behaviors in self-catalyzed GaAs NWs were demonstrated based on thermodynamic considerations, in addition to the effect of the incident electron beam in TEM. Finally, GaAs decomposition was demonstrated in terms of congruent vaporization.

Accelerated decomposition of Bi2S3 nanorods in water under an electron beam: a liquid phase transmission electron microscopy study.

Evaluating the stability of semiconductor photocatalysts is critical in the development of efficient catalysts. The morphological and microstructural behaviors of nanorod-shaped Bi2S3 semiconductors in aqueous solution were studied using a liquid cell transmission electron microscopy (TEM) technique. The rapid decomposition of Bi2S3 in water was observed under electron beam irradiation during TEM. Rounded bright spots due to a reduction in thickness were observed on the Bi2S3 nanorods at the initial stage of the decomposition, and rounded dark particles appeared outside of the nanorods in the solution, continuing the decomposition. This was confirmed by analyzing the atomic structure of the newly formed small particles, which consisted of an orthorhombic Bi2S3 phase. The stability-related decomposition of the Bi2S3 nanorods was demonstrated by considering the reduction and oxidation potentials of Bi2S3 in an aqueous solution. The effect of water radiolysis by the incident electron during TEM observations on the decomposition process was also determined by considering the time-dependent concentration behavior of the chemical species. Our study therefore reflects a novel route to evaluate the stabilities of semiconductor photocatalysts, which could ultimately solve a range of energy and environmental pollution problems.

The MicroRNA-92a/Sp1/MyoD Axis Regulates Hypoxic Stimulation of Myogenic Lineage Differentiation in Mouse Embryonic Stem Cells.

Hypoxic microenvironments exist in developing embryonic tissues and determine stem cell fate. We previously demonstrated that hypoxic priming plays roles in lineage commitment of embryonic stem cells. In the present study, we found that hypoxia-primed embryoid bodies (Hyp-EBs) efficiently differentiate into the myogenic lineage, resulting in the induction of the myogenic marker MyoD, which was not mediated by hypoxia-inducible factor 1α (HIF1α) or HIF2α, but rather by Sp1 induction and binding to the MyoD promoter. Knockdown of Sp1 in Hyp-EBs abrogated hypoxia-induced MyoD expression and myogenic differentiation. Importantly, in the cardiotoxin-muscle injury mice model, Hyp-EB transplantation facilitated muscle regeneration in vivo, whereas transplantation of Sp1-knockdown Hyp-EBs failed to do. Moreover, we compared microRNA (miRNA) expression profiles between EBs under normoxia versus hypoxia and found that hypoxia-mediated Sp1 induction was mediated by the suppression of miRNA-92a, which directly targeted the 3' untranslated region (3' UTR) of Sp1. Further, the inhibitory effect of miRNA-92a on Sp1 in luciferase assay was abolished by a point mutation in specific sequence in the Sp1 3' UTR that is required for the binding of miRNA-92a. Collectively, these results suggest that hypoxic priming enhances EB commitment to the myogenic lineage through miR-92a/Sp1/MyoD regulatory axis, suggesting a new pathway that promotes myogenic-lineage differentiation.

5-HTTLPR-rs25531 and Antidepressant Treatment Outcomes in Korean Patients with Major Depression.

INTRODUCTION: Despite the ethnic differences in 5-HTTLPR (S allele relates to better antidepressant response in Korean and Japanese people, while L allele with better response in Caucasians), it is unclear whether 5-HTTLPR and its high expression locus rs25531 are interactively associated with antidepressant treatment outcome. We investigated the individual and interaction effects of these polymorphisms on antidepressant treatment outcomes in the Korean population. METHODS: A total of 464 Korean subjects with major depressive disorder completed 6 weeks of antidepressant monotherapy. Venous blood was extracted for genotyping 5-HTTLPR and rs25531 by polymerase chain reaction and DNA sequencing. We used logistic regression analyses to verify the main and interaction effects of 5-HTTLPR and rs25531 on response and remission after antidepressant treatment. RESULTS: After adjusting for covariates, the SS genotype of 5-HTTLPR was significantly associated with better treatment outcomes (p<0.001, odds ratio [OR] [95% confidence interval (CI)]=2.435 [1.551, 3.823] in response; p<0.001, OR [95% CI]=2.912 [1.730, 4.903] in remission), while G-containing genotype (AG+GG) of rs25531 was only associated with remission (p=0.034, OR [95% CI]=2.104 [1.058, 4.181]). The interaction effect of 5-HTTLPR and rs25531 on response and remission was insignificant (all p>0.05). DISCUSSION: Our findings suggest variations in allelic frequency and functionality of 5-HTTLPR and rs25531 among the different ethnicities. We found a minor advantage of rs25531 in achieving remission. However, there was no interaction effect with 5-HTTLPR.

High-yield synthesis and purification of recombinant human GABA transaminase for high-throughput screening assays.

Many studies have focussed on modulating the activity of γ-aminobutyric acid transaminase (GABA-T), a GABA-catabolizing enzyme, for treating neurological diseases, such as epilepsy and drug addiction. Nevertheless, human GABA-T synthesis and purification have not been established. Thus, biochemical and drug design studies on GABA-T have been performed by using porcine GABA-T mostly and even bacterial GABA-T. Here we report an optimised protocol for overexpression of 6xHis-tagged human GABA-T in human cells followed by a two-step protein purification. Then, we established an optimised human GABA-T (0.5 U/mg) activity assay. Finally, we compared the difference between human and bacterial GABA-T in sensitivity to two irreversible GABA-T inhibitors, gabaculine and vigabatrin. Human GABA-T in homodimeric form showed 70-fold higher sensitivity to vigabatrin than bacterial GABA-T in multimeric form, indicating the importance of using human GABA-T. In summary, our newly developed protocol can be an important first step in developing more effective human GABA-T modulators.

Evaluation of the Inhibitory Effects of Pyridylpyrazole Derivatives on LPS-Induced PGE2 Productions and Nitric Oxide in Murine RAW 264.7 Macrophages.

A series of thirteen triarylpyrazole analogs were investigated as inhibitors of lipopolysaccharide (LPS)-induced prostaglandin E2 (PGE2) and nitric oxide (NO) production in RAW 264.7 macrophages. The target compounds 1a-m have first been assessed for cytotoxicity against RAW 264.7 macrophages to determine their non-cytotoxic concentration(s) for anti-inflammatory testing to make sure that the inhibition of PGE2 and NO production would not be caused by cytotoxicity. It was found that compounds 1f and 1m were the most potent PGE2 inhibitors with IC50 values of 7.1 and 1.1 µM, respectively. In addition, these compounds also showed inhibitory effects of 11.6% and 37.19% on LPS-induced NO production, respectively. The western blots analysis of COX-2 and iNOS showed that the PGE2 and NO inhibitory effect of compound 1m are attributed to inhibition of COX-2 and iNOS protein expression through inactivation of p38.

Guanine Deaminase in Human Epidermal Keratinocytes Contributes to Skin Pigmentation.

Epidermal keratinocytes are considered as the most important neighboring cells that modify melanogenesis. Our previous study used microarray to show that guanine deaminase (GDA) gene expression is highly increased in melasma lesions. Hence, we investigated the role of GDA in skin pigmentation. We examined GDA expression in post-inflammatory hyperpigmentation (PIH) lesions, diagnosed as Riehl's melanosis. We further investigated the possible role of keratinocyte-derived GDA in melanogenesis by quantitative PCR, immunofluorescence staining, small interfering RNA-based GDA knockdown, and adenovirus-mediated GDA overexpression. We found higher GDA positivity in the hyperpigmentary lesional epidermis than in the perilesional epidermis. Both UVB irradiation and stem cell factor (SCF) plus endothelin-1 (ET-1) were used, which are well-known melanogenic stimuli upregulating GDA expression in both keratinocyte culture alone and keratinocyte and melanocyte coculture. GDA knockdown downregulated melanin content, while GDA overexpression promoted melanogenesis in the coculture. When melanocytes were treated with UVB-exposed keratinocyte-conditioned media, the melanin content was increased. Also, GDA knockdown lowered SCF and ET-1 expression levels in keratinocytes. GDA in epidermal keratinocytes may promote melanogenesis by upregulating SCF and ET-1, suggesting its role in skin hyperpigmentary disorders.

Comparison and correlation among in vitro and in vivo assays to assess cord blood quality according to delivery temperature and time after collection.

Cord blood (CB) has been used as an alternative source for unrelated allogeneic hematopoietic stem cell transplantation. To determine which assay was useful for predicting the successful outcome of CB transplantation, CBs were grouped according to the temperature (4 °C, 24 °C, and 37 °C) and time (24, 48, and 72 h) after collection. The viability, early apoptosis, and colony forming units (CFUs) were ascertained for the total nucleated cells (TNCs) and CD34+ cells; in addition, the engraftment of infused CD34+ cells in NSG mice was determined. The viability of the TNCs and CD34+ cells and total CFUs were significantly decreased whereas the early apoptosis was significantly increased in the 72 h group at 37 °C compared to that of the 24 h group at 24 °C. The viability and early apoptosis of the TNCs correlated with those of CD34+ cells. In addition, the viability and early apoptosis correlated with the number of granulocyte/monocyte progenitor CFUs. In transplanted NSG mice, the frequency of human CD45+ cells decreased in the 72 h group at 24 °C compared to that of the 24 h group at 24 °C and was negatively correlated with early apoptosis of TNCs and CD34+ cells. This study demonstrated that the early apoptosis of TNCs and CD34+ cells constitutes a useful marker for predicting the engraftment of HSCs and may provide helpful data for standard assessment regarding CB quality by analyzing the correlation between in vitro and in vivo assays using NSG mice.