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The human genome contains millions of candidatecis-regulatory elements (CREs) with cell-type-specific activities that shape both health and myriad disease states. However, we lack a functional understanding of the sequence features that control the activity and cell-type-specific features of these CREs. Here, we used lentivirus-based massively parallel reporter assays (lentiMPRAs) to test the regulatory activity of over 680,000 sequences, representing a nearly comprehensive set of all annotated CREs among three cell types (HepG2, K562, and WTC11), finding 41.7% to be functional. By testing sequences in both orientations, we find promoters to have significant strand orientation effects. We also observe that their 200 nucleotide cores function as non-cell-type-specific ‘on switches’ providing similar expression levels to their associated gene. In contrast, enhancers have weaker orientation effects, but increased tissue-specific characteristics. Utilizing our lentiMPRA data, we develop sequence-based models to predict CRE function with high accuracy and delineate regulatory motifs. Testing an additional lentiMPRA library encompassing 60,000 CREs in all three cell types, we further identified factors that determine cell-type specificity. Collectively, our work provides an exhaustive catalog of functional CREs in three widely used cell lines, and showcases how large-scale functional measurements can be used to dissect regulatory grammar.

Nucleotide changes in gene regulatory elements are important determinants of neuronal development and disease. Using massively parallel reporter assays in primary human cells from mid-gestation cortex and cerebral organoids, we interrogated thecis-regulatory activity of 102,767 sequences, including differentially accessible cell-type specific regions in the developing cortex and single-nucleotide variants associated with psychiatric disorders. In primary cells, we identified 46,802 active enhancer sequences and 164 disorder-associated variants that significantly alter enhancer activity. Activity was comparable in organoids and primary cells, suggesting that organoids provide an adequate model for the developing cortex. Using deep learning, we decoded the sequence basis and upstream regulators of enhancer activity. This work establishes a comprehensive catalog of functional gene regulatory elements and variants in human neuronal development.One Sentence SummaryWe identify 46,802 enhancers and 164 psychiatric disorder variants with regulatory effects in the developing cortex and organoids.

Autism spectrum disorder (ASD) is a common, complex, and highly heritable condition with contributions from both common and rare genetic variations. While disruptive, rare variants in protein-coding regions clearly contribute to symptoms, the role of rare non-coding remains unclear. Variants in these regions, including promoters, can alter downstream RNA and protein quantity; however, the functional impacts of specific variants observed in ASD cohorts remain largely uncharacterized. Here, we analyzed 3600 de novo mutations in promoter regions previously identified by whole-genome sequencing of autistic probands and neurotypical siblings to test the hypothesis that mutations in cases have a greater functional impact than those in controls. We leveraged massively parallel reporter assays (MPRAs) to detect transcriptional consequences of these variants in neural progenitor cells and identified 165 functionally high confidence de novo variants (HcDNVs). While these HcDNVs are enriched for markers of active transcription, disruption to transcription factor binding sites, and open chromatin, we did not identify differences in functional impact based on ASD diagnostic status.

Human Accelerated Regions (HARs) are conserved genomic loci that evolved at an accelerated rate in the human lineage and may underlie human-specific traits. We generated HARs and chimpanzee accelerated regions with the largest alignment of mammalian genomes to date. To facilitate exploration of accelerated evolution in other lineages, we implemented an open-source Nextflow pipeline that runs on any computing platform. Combining deep-learning with chromatin capture experiments in human and chimpanzee neural progenitor cells, we discovered a significant enrichment of HARs in topologically associating domains (TADs) containing human-specific genomic variants that change three-dimensional (3D) genome organization. Differential gene expression between humans and chimpanzees at these loci in multiple cell types suggests rewiring of regulatory interactions between HARs and neurodevelopmental genes. Thus, comparative genomics together with models of 3D genome folding revealed enhancer hijacking as an explanation for the rapid evolution of HARs.One-Sentence SummaryHuman-specific changes to 3D genome organization may have contributed to rapid evolution of mammalian-conserved loci in the human genome.

The Neanderthal and Denisovan genomes enabled the discovery of sequences that differ between modern and archaic humans, the majority of which are noncoding. However, our understanding of the regulatory consequences of these differences remains limited, in part due to the decay of regulatory marks in ancient samples. Here, we used a massively parallel reporter assay in embryonic stem cells, neural progenitor cells, and bone osteoblasts to investigate the regulatory effects of the 14,042 single-nucleotide modern human-specific variants. Overall, 1791 (13%) of sequences containing these variants showed active regulatory activity, and 407 (23%) of these drove differential expression between human groups. Differentially active sequences were associated with divergent transcription factor binding motifs, and with genes enriched for vocal tract and brain anatomy and function. This work provides insight into the regulatory function of variants that emerged along the modern human lineage and the recent evolution of human gene expression.

Gene regulatory elements play a key role in orchestrating gene expression during cellular differentiation, but what determines their function over time remains largely unknown. Here, we performed perturbation-based massively parallel reporter assays at seven early time points of neural differentiation to systematically characterize how regulatory elements and motifs within them guide cellular differentiation. By perturbing over 2,000 putative DNA binding motifs in active regulatory regions, we delineated four categories of functional elements, and observed that activity direction is mostly determined by the sequence itself, while the magnitude of effect depends on the cellular environment. We also find that fine-tuning transcription rates is often achieved by a combined activity of adjacent activating and repressing elements. Our work provides a blueprint for the sequence components needed to induce different transcriptional patterns in general and specifically during neural differentiation.

Massively parallel reporter assays (MPRAs) functionally screen thousands of sequences for regulatory activity in parallel. Although MPRAs have been applied to address diverse questions in gene regulation, there has been no systematic comparison of how differences in experimental design influence findings. Here, we screen a library of 2,440 sequences, representing candidate liver enhancers and controls, in HepG2 cells for regulatory activity using nine different approaches (including conventional episomal, STARR-seq, and lentiviral MPRA designs). We identify subtle but significant differences in the resulting measurements that correlate with epigenetic and sequence-level features. We also test this library in both orientations with respect to the promoter, validating en masse that enhancer activity is robustly independent of orientation. Finally, we develop and apply a novel method to assemble and functionally test libraries of the same putative enhancers as 192-mers, 354-mers, and 678-mers, and observe surprisingly large differences in functional activity. This work provides a framework for the experimental design of high-throughput reporter assays, suggesting that the extended sequence context of tested elements, and to a lesser degree the precise assay, influence MPRA results.

The Neanderthal and Denisovan genomes enabled the discovery of sequences that differ between modern and archaic humans, the majority of which are noncoding. However, our understanding of the regulatory consequences of these differences remains limited, in part due to the decay of regulatory marks in ancient samples. Here, we used a massively parallel reporter assay in embryonic stem cells, neural progenitor cells and bone osteoblasts to investigate the regulatory effects of the 14,042 single-nucleotide modern human-specific variants. Overall, 1,791 (13%) of sequences containing these variants showed active regulatory activity, and 407 (23%) of these drove differential expression between human groups. Differentially active sequences were associated with divergent transcription factor binding motifs, and with genes enriched for vocal tract and brain anatomy and function. This work provides insight into the regulatory function of variants that emerged along the modern human lineage and the recent evolution of human gene expression.

Massively parallel reporter assays (MPRAs) can simultaneously measure the function of thousands of candidate regulatory sequences (CRSs) in a quantitative manner. In this method, CRSs are cloned upstream of a minimal promoter and reporter gene, alongside a unique barcode, and introduced into cells. If the CRS is a functional regulatory element, it will lead to the transcription of the barcode sequence, which is measured via RNA sequencing and normalized for cellular integration via DNA sequencing of the barcode. This technology has been used to test thousands of sequences and their variants for regulatory activity, to decipher the regulatory code and its evolution, and to develop genetic switches. Lentivirus-based MPRA (lentiMPRA) produces ‘in-genome’ readouts and enables the use of this technique in hard-to-transfect cells. Here, we provide a detailed protocol for lentiMPRA, along with a user-friendly Nextflow-based computational pipeline—MPRAflow—for quantifying CRS activity from different MPRA designs. The lentiMPRA protocol takes ~2 months, which includes sequencing turnaround time and data processing with MPRAflow.

Massively parallel reporter assays (MPRAs) functionally screen thousands of sequences for regulatory activity in parallel. To date, there has been no systematic comparison of differences in MPRA design. Here, we screen a library of 2,440 candidate liver enhancers and controls for regulatory activity in HepG2 cells using nine different MPRA designs. We identify subtle but significant differences that correlate with epigenetic and sequence-level features, as well as differences in dynamic range and reproducibility. We also validate en masse that enhancer activity is robustly independent of orientation, at least for our library and designs. Finally, with a new method, we assemble and test the same enhancers as 192-mers, 354-mers, and 678-mers, and observe surprisingly large differences. This work provides a framework for the experimental design of high-throughput reporter assays, suggesting that the extended sequence context of tested elements, and to a lesser degree the precise assay, influence MPRA results.

Pyridoxine-dependent epilepsy (PDE) is a rare autosomal-recessive disorder typically presenting with neonatal seizures and is sometimes difficult to diagnose, because the clinical features mimic those of birth asphyxia. A Japanese newborn boy presented with pulmonary haemorrhage and convulsions on the day of birth. Brain computed tomography showed diffuse, but mild, low-density cerebral white matter and a thin subdural hematoma in the posterior fossa. He did not have thrombocytopenia or coagulopathy. His respiratory status improved with conservative treatment, but his convulsions were persistent even after prescription of several antiepileptic drugs. His serum and cerebrospinal fluid showed decreased vitamin B6 vitamers and increased upstream metabolites of α-aminoadipic semialdehyde dehydrogenase, strongly suggesting a diagnosis of PDE; the epileptic spasms ceased after administration of intravenous pyridoxal phosphate hydrate. Gene analysis revealed novel compound heterozygous mutations in ALDH7A1 that included NM_001182.4:[c.1196G > T] and [c.1200 + 1G > A]. Atypical birth asphyxia with persistent neonatal seizure should prompt vitamin B6/metabolite screening.

Background: Hepcidin, an iron-regulatory hormone, plays a key role in preventing iron overload. Few studies have investigated the regulation of hepcidin in low-birth-weight (LBW) infants who are vulnerable to iron imbalance. Objectives: To identify perinatal factors associated with serum hepcidin levels in LBW infants. Methods: Ninety-two LBW infants with a median gestational age (GA) of 32.6 weeks and birth weight of 1,587 g were prospectively enrolled. Serum hepcidin-25 (Hep25) levels were measured from umbilical cord blood using liquid chromatography-tandem mass spectrometry. The relationship between Hep25 levels and prematurity or other possible hepcidin-regulatory factors was evaluated. Results: The median Hep25 level was 7.3 ng/mL (interquartile range: 2.85-16.38). log(Hep25) correlated with birth weight (r = 0.229, p = 0.028), log(interleukin-6 [IL-6]) (r = 0.408, p < 0.001), log(erythropoietin) (r = -0.302, p = 0.004), transferrin saturation (r = 0.29, p = 0.005), soluble transferrin receptor (r = -0.500, p < 0.001), and log(ferritin) (r = 0.696, p < 0.001). Serum iron and hemoglobin levels did not correlate with log(Hep25). Hep25 levels were higher among infants with chorioamnionitis and infants born vaginally and lower among infants born to mothers with pregnancy-induced hypertension than among infants without the respective characteristics. Stepwise multiple linear regression analysis confirmed the significant association of log(Hep25) with GA, log(IL-6), log(erythropoietin), and soluble transferrin receptor. Conclusions: Among LBW infants, GA, IL-6, erythropoietin, and soluble transferrin receptor were associated with Hep25 levels. Therefore, prematurity, inflammation, hypoxia, and erythropoietic activity may be important perinatal factors that affect hepcidin levels.

Pyridoxine-dependent epilepsy (PDE) is a rare autosomal-recessive disorder typically presenting with neonatal seizures and is sometimes difficult to diagnose, because the clinical features mimic those of birth asphyxia. A Japanese newborn boy presented with pulmonary haemorrhage and convulsions on the day of birth. Brain computed tomography showed diffuse, but mild, low-density cerebral white matter and a thin subdural hematoma in the posterior fossa. He did not have thrombocytopenia or coagulopathy. His respiratory status improved with conservative treatment, but his convulsions were persistent even after prescription of several antiepileptic drugs. His serum and cerebrospinal fluid showed decreased vitamin B6 vitamers and increased upstream metabolites of α-aminoadipic semialdehyde dehydrogenase, strongly suggesting a diagnosis of PDE; the epileptic spasms ceased after administration of intravenous pyridoxal phosphate hydrate. Gene analysis revealed novel compound heterozygous mutations in ALDH7A1 that included NM_001182.4:[c.1196G > T] and [c.1200 + 1G > A]. Atypical birth asphyxia with persistent neonatal seizure should prompt vitamin B6/metabolite screening.

Epigenomic regulation and lineage-specific gene expression act in concert to drive cellular differentiation, but the temporal interplay between these processes is largely unknown. Using neural induction from human pluripotent stem cells (hPSCs) as a paradigm, we interrogated these dynamics by performing RNA sequencing (RNA-seq), chromatin immunoprecipitation sequencing (ChIP-seq), and assay for transposase accessible chromatin using sequencing (ATAC-seq) at seven time points during early neural differentiation. We found that changes in DNA accessibility precede H3K27ac, which is followed by gene expression changes. Using massively parallel reporter assays (MPRAs) to test the activity of 2,464 candidate regulatory sequences at all seven time points, we show that many of these sequences have temporal activity patterns that correlate with their respective cell-endogenous gene expression and chromatin changes. A prioritization method incorporating all genomic and MPRA data further identified key transcription factors involved in driving neural fate. These results provide a comprehensive resource of genes and regulatory elements that orchestrate neural induction and illuminate temporal frameworks during differentiation.

The integrative analysis of high-throughput reporter assays, machine learning, and profiles of epigenomic chromatin state in a broad array of cells and tissues has the potential to significantly improve our understanding of noncoding regulatory element function and its contribution to human disease. Here, we report results from the CAGI 5 regulation saturation challenge where participants were asked to predict the impact of nucleotide substitution at every base pair within five disease-associated human enhancers and nine disease-associated promoters. A library of mutations covering all bases was generated by saturation mutagenesis and altered activity was assessed in a massively parallel reporter assay (MPRA) in relevant cell lines. Reporter expression was measured relative to plasmid DNA to determine the impact of variants. The challenge was to predict the functional effects of variants on reporter expression. Comparative analysis of the full range of submitted prediction results identifies the most successful models of transcription factor binding sites, machine learning algorithms, and ways to choose among or incorporate diverse datatypes and cell-types for training computational models. These results have the potential to improve the design of future studies on more diverse sets of regulatory elements and aid the interpretation of disease-associated genetic variation.

The majority of common variants associated with common diseases, as well as an unknown proportion of causal mutations for rare diseases, fall in noncoding regions of the genome. Although catalogs of noncoding regulatory elements are steadily improving, we have a limited understanding of the functional effects of mutations within them. Here, we perform saturation mutagenesis in conjunction with massively parallel reporter assays on 20 disease-associated gene promoters and enhancers, generating functional measurements for over 30,000 single nucleotide substitutions and deletions. We find that the density of putative transcription factor binding sites varies widely between regulatory elements, as does the extent to which evolutionary conservation or integrative scores predict functional effects. These data provide a powerful resource for interpreting the pathogenicity of clinically observed mutations in these disease-associated regulatory elements, and comprise a rich dataset for the further development of algorithms that aim to predict the regulatory effects of noncoding mutations., Interpreting genetic variation in the noncoding genome remains challenging, with functional effects difficult to predict. Here, the authors perform saturation mutagenesis combined with massively parallel reporter assays for 20 disease-associated regulatory elements, quantifying the effects of over 30,000 variants.

Genome wide association studies (GWAS) in obesity have identified a large number of noncoding loci located near genes expressed in the central nervous system. However, due to the difficulties in isolating and characterizing specific neuronal subpopulations, few obesity-associated SNPs have been functionally characterized. Leptin responsive neurons in the hypothalamus are essential in controlling energy homeostasis and body weight. Here, we combine FACS-sorting of leptin-responsive hypothalamic neuron nuclei with genomic and epigenomic approaches (RNA-seq, ChIP-seq, ATAC-seq) to generate a comprehensive map of leptin-response specific regulatory elements, several of which overlap obesity-associated GWAS variants. We demonstrate the usefulness of our leptin-response neuron regulome, by functionally characterizing a novel enhancer near Socs3, a leptin response-associated transcription factor. We envision our data to serve as a useful resource and a blueprint for functionally characterizing obesity-associated SNPs in the hypothalamus.

Massively parallel reporter assays (MPRAs) functionally screen thousands of sequences for regulatory activity in parallel. Although MPRAs have been applied to address diverse questions in gene regulation, there has been no systematic comparison of how differences in experimental design influence findings. Here, we screen a library of 2,440 sequences, representing candidate liver enhancers and controls, in HepG2 cells for regulatory activity using nine different approaches (including conventional episomal, STARR-seq, and lentiviral MPRA designs). We identify subtle but significant differences in the resulting measurements that correlate with epigenetic and sequence-level features. We also test this library in both orientations with respect to the promoter, validatingen massethat enhancer activity is robustly independent of orientation. Finally, we develop and apply a novel method to assemble and functionally test libraries of the same putative enhancers as 192-mers, 354-mers, and 678-mers, and observe surprisingly large differences in functional activity. This work provides a framework for the experimental design of high-throughput reporter assays, suggesting that the extended sequence context of tested elements, and to a lesser degree the precise assay, influence MPRA results.

The majority of common variants associated with common diseases, as well as an unknown proportion of causal mutations for rare diseases, fall in noncoding regions of the genome. Although catalogs of noncoding regulatory elements are steadily improving, we have a limited understanding of the functional effects of mutations within them. Here, we performed saturation mutagenesis in conjunction with massively parallel reporter assays on 20 disease-associated gene promoters and enhancers, generating functional measurements for over 30,000 single nucleotide substitution and deletion mutations. We find that the density of putative transcription factor binding sites varies widely between regulatory elements, as does the extent to which evolutionary conservation or various integrative scores predict functional effects. These data provide a powerful resource for interpreting the pathogenicity of clinically observed mutations in these disease-associated regulatory elements, and also comprise a gold-standard dataset for the further development of algorithms that aim to predict the regulatory effects of noncoding mutations.

The molecular components governing neural induction remain largely unknown. Here, we applied a suite of genomic and computational tools to comprehensively identify these components. We performed RNA-seq, ChIP-seq (H3K27ac, H3K27me3) and ATAC-seq on human embryonic stem cells (hESCs) at seven early neural differentiation time points (0-72 hours) and identified thousands of induced genes and regulatory regions. We analyzed the function of ~2,500 selected regions using massively parallel reporter assays at all time points. We found numerous temporal enhancers that correlated with similarly timed epigenetic marks and gene expression. Development of a prioritization method that incorporated all genomic data identified key transcription factors (TFs) involved in neural induction. Individual overexpression of eleven TFs and several combinations in hESCs found novel neural induction regulators. Combined, our results provide a comprehensive map of genes and functional regulatory elements involved in neural induction and identify master regulator TFs that are instrumental for this process.One Sentence SummaryUsing numerous genomic assays and computational tools we characterized the dynamic changes that take place during neural induction.

To examine the relationship between chorionicity and neurodevelopmental outcomes in very low birth weight twins at 3 years of age. A retrospective cohort study of 3538 twins who were admitted to 91 tertiary perinatal centers in the Neonatal Research Network of Japan between 2003 and 2012. In a comparison of the followed-up 796 monochorionic twins and 786 dichorionic twins, the overall rate of neurodevelopmental impairment was higher in monochorionic twins; specifically, the rate of disability in the language-social area of the Japanese standardized developmental test was higher in monochorionic twins than in dichorionic twins (adjusted odds ratio, 1.55; 95% confidence interval, 1.07–2.25; P = 0.02). Chorionicity was associated with neurodevelopmental outcome (especially for language and social development) in a large cohort of very low birth weight twins who had a high rate of perinatal morbidity and neurodevelopmental impairment.

Using machine learning (ML), we interrogated the function of all human-chimpanzee variants in 2,645 Human Accelerated Regions (HARs), some of the fastest evolving regions of the human genome. We predicted that 43% of HARs have variants with large opposing effects on chromatin state and 14% on neurodevelopmental enhancer activity. This pattern, consistent with compensatory evolution, was confirmed using massively parallel reporter assays in human and chimpanzee neural progenitor cells. The species-specific enhancer activity of assayed HARs was accurately predicted from the presence and absence of transcription factor footprints in each species. Despite these striking cis effects, activity of a given HAR sequence was nearly identical in human and chimpanzee cells. These findings suggest that HARs did not evolve to compensate for changes in the trans environment but instead altered their ability to bind factors present in both species. Thus, ML prioritized variants with functional effects on human neurodevelopment and revealed an unexpected reason why HARs may have evolved so rapidly.

Enhancers control the timing, location and expression levels of their target genes. Nucleotide variation in enhancers has been shown to lead to numerous phenotypes, including human disease. While putative enhancer sequences and nucleotide variation within them can now be detected in a rapid manner using various genomic technologies, the understanding of the functional consequences of these variants still remains largely unknown. Massively parallel reporter assays (MPRAs) can overcome this hurdle by providing the ability to test thousands of sequences and nucleotide variants within them for enhancer activity en masse. Here, we describe this technology and specifically focus on how it is being used to obtain an increased understanding of enhancer regulatory code and grammar.

Candidate enhancers can be identified on the basis of chromatin modifications, the binding of chromatin modifiers and transcription factors and cofactors, or chromatin accessibility. However, validating such candidates as bona fide enhancers requires functional characterization, typically achieved through reporter assays that test whether a sequence can drive expression of a transcriptional reporter via a minimal promoter. A longstanding concern is that reporter assays are mainly implemented on episomes, which are thought to lack physiological chromatin. However, the magnitude and determinants of differences incis-regulation for regulatory sequences residing in episomes versus chromosomes remain almost completely unknown. To address this question in a systematic manner, we developed and applied a novel lentivirus-based massively parallel reporter assay (lentiMPRA) to directly compare the functional activities of 2,236 candidate liver enhancers in an episomal versus a chromosomally integrated context. We find that the activities of chromosomally integrated sequences are substantially different from the activities of the identical sequences assayed on episomes, and furthermore are correlated with different subsets of ENCODE annotations. The results of chromosomally-based reporter assays are also more reproducible and more strongly predictable by both ENCODE annotations and sequence-based models. With a linear model that combines chromatin annotations and sequence information, we achieve a Pearson’s R2of 0.347 for predicting the results of chromosomally integrated reporter assays. This level of prediction is better than with either chromatin annotations or sequence information alone and also outperforms predictive models of episomal assays. Our results have broad implications for howcis-regulatory elements are identified, prioritized and functionally validated.

Background Despite the early use of phototherapy and exchange transfusion in premature infants based on total serum bilirubin (TSB), the reemergence of kernicterus has been reported. The aim of this study was to assess the validity of using TSB as the criterion for phototherapy in extremely low-birthweight infants (ELBWI). Methods We reviewed the medical charts of 43 ELBWI admitted to hospital between January 2009 and December 2010, and analyzed the relationship between TSB and unbound bilirubin (UB). Results No infant underwent exchange transfusion or developed acute bilirubin encephalopathy. There was a significant correlation between TSB and UB measured immediately before phototherapy during the first 7 days of life (r = 0.657, P < 0.001), but none thereafter (r = 0.120, P = 0.213). Thirty-seven percent of infants who underwent phototherapy during the first 7 days of life had suprathreshold USB but subthreshold TSB, whereas this rose to 97% thereafter. Conclusions No correlation was observed between TSB and UB in ELBWI after the first 7 days of life, and almost all phototherapy sessions were initiated based on the UB criterion, even though TSB was below the accepted threshold. UB may be high if jaundice is evaluated solely on the basis of TSB.

Background The feeding interval is an important factor in enteral feeding of premature infants. We investigated postprandial intestinal blood flow in stable very-low-birthweight infants fed at 2-h and 3-h intervals. Methods We used pulsed wave Doppler ultrasound to measure blood flow velocity of the superior mesenteric artery (SMA) before feeding and at 15, 30, 45, and 60 min after feeding. Measurements were made on the day of starting enteral nutrition (1 or 2 days of age), and at 3 and 5 days of age. A total of 21 studies were performed in seven infants fed every 2 h, and 54 studies were performed in 18 infants fed every 3 h. Results In infants fed every 2 h, SMA blood flow velocity increased from before feeding to 30 min after feeding and then decreased at 60 min after feeding. In infants fed every 3 h, SMA blood flow velocity increased after feeding, reaching a peak at 30 min. The correlation coefficients between the volume of milk per feed and the postprandial increase in time-averaged mean blood flow velocity were 0.398 (P = 0.074, n = 21) and 0.597 (P = 0.000, n = 54) in infants fed at 2-h and 3-h intervals, respectively. Conclusions SMA blood flow velocity significantly increased after feeding in infants fed at 2-h and 3-h intervals. The volume of milk per feed might affect the postprandial increase in SMA blood flow velocity.

We have analyzed Emx2 enhancers to determine how Emx2 functions during forebrain development are regulated. The FB (forebrain) enhancer we identified immediately 3′ downstream of the last coding exon is well conserved among tetrapods and unexpectedly directed all the Emx2 expression in forebrain: caudal forebrain primordium at E8.5, dorsal telencephalon at E9.5-E10.5 and the cortical ventricular zone after E12.5. Otx, Tcf, Smad and two unknown transcription factor binding sites were essential to all these activities. The mutant that lacked this enhancer demonstrated that Emx2 expression under the enhancer is solely responsible for diencephalon development. However, in telencephalon, the FB enhancer did not have activities in cortical hem or Cajal-Retzius cells, nor was its activity in the cortex graded. Emx2 expression was greatly reduced, but persisted in the telencephalon of the enhancer mutant, indicating that there exists another enhancer for Emx2 expression unique to mammalian telencephalon.

In vertebrate embryos, positioning of the boundary between the midbrain and hindbrain (MHB) and subsequent isthmus formation are dependent upon the interaction between the Otx2 and Gbx genes. In zebrafish, sequential expression of gbx1 and gbx2 in the anterior hindbrain contributes to this process, whereas in mouse embryos, a single Gbx gene (Gbx2) is responsible for MHB development. In the present study, to investigate the regulatory mechanism of gbx2 in the MHB/isthmic region of zebrafish embryos, we cloned the gene and showed that its organization is conserved among different vertebrates. Promoter analyses revealed three enhancers that direct reporter gene expression after the end of epiboly in the anterior-most hindbrain, which is a feature of the zebrafish gbx2 gene. One of the enhancers is located upstream of gbx2 (AMH1), while the other two enhancers are located downstream of gbx2 (AMH2 and AMH3). Detailed analysis of the AMH1 enhancer showed that it directs expression in the rhombomere 1 (r1) region and the dorsal thalamus, as has been shown for gbx2, whereas no expression was induced by the AMH1 enhancer in other embryonic regions in which gbx2 is expressed. The AMH1 enhancer is composed of multiple regulatory subregions that share the same spatial specificity. The most active of the regulatory subregions is a 291-bp region that contains at least two Pax2-binding sites, both of which are necessary for the function of the main component (PB1-A region) of the AMH1 enhancer. In accordance with these results, enhancer activity in the PB1-A region, as well as gbx2 expression in r1, was missing in no isthmus mutant embryos that lacked functional pax2a. In addition, we identified an upstream conserved sequence of 227bp that suppresses the enhancer activity of AMH1. Taken together, these findings suggest that gbx2 expression during the somitogenesis stage in zebrafish is regulated by a complex mechanism involving Pax2 as well as activators and suppressors in the regions flanking the gene.

We investigated the usefulness of the intracoronary administration of nicorandil (NIC) compared with that of papaverine (PAP) in the evaluation of coronary flow reserve (CFR) in 17 patients, including 10 patients with old myocardial infarction and 7 patients with angina pectoris. CFR was measured with a Doppler guidewire inserted into the distal site of the left anterior descending coronary artery during intracoronary administration of 10 mg PAP, and of 0.5 mg, 1.0 mg, 2.0 mg, and 3.0 mg NIC. We examined the changes in heart rate (HR), mean blood pressure (mBP), the total score of QTc interval on a 12-lead electrocardiogram (sigma QTc), and ST-T segment, before and after drug administration. CFR was significantly lower during administration of 0.5 mg (1.9 +/- 0.9) and 1.0 mg (2.2 +/- 0.9) NIC than during administration of PAP (2.6 +/- 1.1) (P0.01). There was no significant difference in the CFR during administration of 2.0 mg (2.6 +/- 1.0) or 3.0 mg (2.5 +/- 1.0) NIC and that observed during administration of PAP. The CFR during administration of PAP was significantly correlated with that during administration of 2.0 mg NIC (r2 = 0.72, P0.0001) and 3.0 mg NIC (r2 = 0.70, P0.0001). PAP, but not NIC, significantly altered the HR, mBP, and sigma QTc. Inverted T waves were observed in 14 patients, and elevation of the ST segment was observed in 4 patients during administration of PAP (including 1 patient with ventricular tachycardia). The administration of 0.5 mg to 2.0 mg NIC was not associated with ST-T segment changes, except in 1 patient, but inverted T waves were observed in 2 patients and depression of the ST segment was observed in 2 patients during administration of 3.0 mg NIC. Intracoronary administration of NIC is useful and safe for evaluating the CFR. The appropriate dose for measuring CFR is 2.0 mg nicorandil.

We present the case of a 44-year-old man with abnormal myocardial fatty acid metabolism who exhibited no myocardial uptake of 123I-beta-methyl- iodophenyl pentadecanoic acid (123I-BMIPP). This patient presented to our hospital with an ECG abnormality detected during a medical check-up. He felt no chest discomfort, but a 12-lead ECG at rest showed flat T-waves in leads I, V5, and V6 with no marked ischemic changes during exercise. A left ventriculogram and coronary angiograms were normal. Thallium-201 single photon emission computed tomography imaging revealed a normal myocardial uptake, but 123I-BMIPP imaging showed no such uptake. However, 18F-labelled fluorodeoxyglucose positron emission tomography imaging after an overnight fast showed a marked increase in myocardial uptake. It appears that myocardial uptake of 123I-BMIPP was totally lacking and that energy production by the myocardium during fasting depended on the metabolism of glucose rather than of fatty acids.

Despite continual progress in the cataloging of vertebrate regulatory elements, little is known about their organization and regulatory architecture. Here we describe a massively parallel experiment to systematically test the impact of copy number, spacing, combination and order of transcription factor binding sites on gene expression. A complex library of ~5,000 synthetic regulatory elements containing patterns from 1 2 liver-specific transcription factor binding sites was assayed in mice and in HepG2 cells. We find that certain transcription factors act as direct drivers of gene expression in homotypic clusters of binding sites, independent of spacing between sites, whereas others function only synergistically. Heterotypic enhancers are stronger than their homotypic analogs and favor specific transcription factor binding site combinations, mimicking putative native enhancers. Exhaustive testing of binding site permutations suggests that there is flexibility in binding site order. Our findings provide quantitative support for a flexible model of regulatory element activity and suggest a framework for the design of synthetic tissue-specific enhancers.

Despite the early use of phototherapy and exchange transfusion in premature infants based on total serum bilirubin (TSB), the reemergence of kernicterus has been reported. The aim of this study was to assess the validity of using TSB as the criterion for phototherapy in extremely low-birthweight infants (ELBWI).We reviewed the medical charts of 43 ELBWI admitted to hospital between January 2009 and December 2010, and analyzed the relationship between TSB and unbound bilirubin (UB).No infant underwent exchange transfusion or developed acute bilirubin encephalopathy. There was a significant correlation between TSB and UB measured immediately before phototherapy during the first 7 days of life (r = 0.657, P0.001), but none thereafter (r = 0.120, P = 0.213). Thirty-seven percent of infants who underwent phototherapy during the first 7 days of life had suprathreshold USB but subthreshold TSB, whereas this rose to 97% thereafter.No correlation was observed between TSB and UB in ELBWI after the first 7 days of life, and almost all phototherapy sessions were initiated based on the UB criterion, even though TSB was below the accepted threshold. UB may be high if jaundice is evaluated solely on the basis of TSB.

A 69-year-old postmenopausal woman who was prescribed anastrozole for 10 months after surgical removal of her breast cancer, was referred to our hospital for acute renal failure. Because it was possible that her renal failure was related to her treatment with anastrozole, the treatment was immediately discontinued. After renal biopsy was performed to examine her renal failure, she was diagnosed as crescentic glomerulonephritis, probably related with the treatment of anastrozole. Twenty mg of oral prednisolone was administered daily after methylprednisolone pulse therapy(500 mg/day intravenous administration for three days). Her renal dysfunction was gradually improved. Renal dysfunction was considered to be a rare complication of anastrozole. Patients who are prescribed anastrozole should be watched carefully for the development of renal dysfunction.

The feeding interval is an important factor in enteral feeding of premature infants. We investigated postprandial intestinal blood flow in stable very-low-birthweight infants fed at 2-h and 3-h intervals.We used pulsed wave Doppler ultrasound to measure blood flow velocity of the superior mesenteric artery (SMA) before feeding and at 15, 30, 45, and 60 min after feeding. Measurements were made on the day of starting enteral nutrition (1 or 2 days of age), and at 3 and 5 days of age. A total of 21 studies were performed in seven infants fed every 2 h, and 54 studies were performed in 18 infants fed every 3 h.In infants fed every 2 h, SMA blood flow velocity increased from before feeding to 30 min after feeding and then decreased at 60 min after feeding. In infants fed every 3 h, SMA blood flow velocity increased after feeding, reaching a peak at 30 min. The correlation coefficients between the volume of milk per feed and the postprandial increase in time-averaged mean blood flow velocity were 0.398 (P = 0.074, n = 21) and 0.597 (P = 0.000, n = 54) in infants fed at 2-h and 3-h intervals, respectively.SMA blood flow velocity significantly increased after feeding in infants fed at 2-h and 3-h intervals. The volume of milk per feed might affect the postprandial increase in SMA blood flow velocity.

Otx2 plays essential roles in rostral brain development, and its counteraction with Gbx2 has been suggested to determine the midbrain-hindbrain boundary (MHB) in vertebrates. We previously identified the FM enhancer that is conserved among vertebrates and drives Otx2 transcription in forebrain/midbrain from the early somite stage. In this study, we found that the POU homeodomain of class III POU factors (Brn1, Brn2, Brn4, and Oct6) associates with noncanonical target sequence TAATTA in the FM enhancer. MicroRNA-mediated knockdown of Brn2 and Oct6 diminished the FM enhancer activity in anterior neural progenitor cells (NPCs) differentiated from P19 cells. The class III POU factors associate with the FM enhancer in forebrain and midbrain but not in hindbrain. We also demonstrated that the Gbx2 homeodomain recognizes the same target TAATTA in the FM enhancer, and Gbx2 associates with the FM enhancer in hindbrain. Gbx2 misexpression in the anterior NPCs repressed the FM enhancer activity and inhibited Brn2 association with the enhancer, whereas Gbx2 knockdown caused ectopic Brn2 association in the posterior NPCs. These results suggest that class III POU factors and Gbx2 share the same target site, TAATTA, in the FM enhancer and that their region-specific binding restricts Otx2 expression at the MHB.