In vivo adenine base editing rescues adrenoleukodystrophy in a humanized mouse model.

X-linked adrenoleukodystrophy (ALD), an inherited neurometabolic disorder caused by mutations in ABCD1, which encodes the peroxisomal ABC transporter, mainly affects the brain, spinal cord, adrenal glands, and testes. In ALD patients, very-long-chain fatty acids (VLCFAs) fail to enter the peroxisome and undergo subsequent ß-oxidation, resulting in their accumulation in the body. It has not been tested whether in vivo base editing or prime editing can be harnessed to ameliorate ALD. We developed a humanized mouse model of ALD by inserting a human cDNA containing the pathogenic variant into the mouse Abcd1 locus. The humanized ALD model showed increased levels of VLCFAs. To correct the mutation, we tested both base editing and prime editing and found that base editing using ABE8e(V106W) could correct the mutation in patient-derived fibroblasts at an efficiency of 7.4%. Adeno-associated virus (AAV)-mediated systemic delivery of NG-ABE8e(V106W) enabled robust correction of the pathogenic variant in the mouse brain (correction efficiency: ∼5.5%), spinal cord (∼5.1%), and adrenal gland (∼2%), leading to a significant reduction in the plasma levels of C26:0/C22:0. This established humanized mouse model and the successful correction of the pathogenic variant using a base editor serve as a significant step toward treating human ALD disease.

Amphiregulin normalizes altered circuit connectivity for social dominance of the CRTC3 knockout mouse.

Social hierarchy has a profound impact on social behavior, reward processing, and mental health. Moreover, lower social rank can lead to chronic stress and often more serious problems such as bullying victims of abuse, suicide, or attack to society. However, its underlying mechanisms, particularly their association with glial factors, are largely unknown. In this study, we report that astrocyte-derived amphiregulin plays a critical role in the determination of hierarchical ranks. We found that astrocytes-secreted amphiregulin is directly regulated by cAMP response element-binding (CREB)-regulated transcription coactivator 3 (CRTC3) and CREB. Mice with systemic and astrocyte-specific CRTC3 deficiency exhibited a lower social rank with reduced functional connectivity between the prefrontal cortex, a major social hierarchy center, and the parietal cortex. However, this effect was reversed by astrocyte-specific induction of amphiregulin expression, and the epidermal growth factor domain was critical for this action of amphiregulin. These results provide evidence of the involvement of novel glial factors in the regulation of social dominance and may shed light on the clinical application of amphiregulin in the treatment of various psychiatric disorders.

Generation of genetically engineered mice for lung cancer with mutant EGFR.

Although epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (TKIs) have shown dramatic response and improvement in treating lung cancer with mutant EGFR, the emergence of drug resistance remains a major problem. In particular, some mutations including T790 M and C797S have been recognized as mechanisms of acquired resistance because they weaken binding affinity to drugs. To date, many attempts have been made to develop a new drug for overcoming acquired resistance to EGFR-TKIs, including secondary mutations. However, an appropriate animal model to evaluate in vivo efficacy during novel drug development remains lacking. In this study, we generated a novel transgenic mouse model that conditionally expresses human EGFRL858R/T790M/C797S and firefly luciferase using Cas9-mediated homology-independent targeted integration. Using a lung-specific Sftpc-CreERT2 mouse line, we induced expression of both the human EGFRL858R/T790M/C797S transgene and firefly luciferase in the lungs of adult mice. The expression of these genes and lung cancer occurrence was monitored using an in vivo imaging system and magnetic resonance imaging, respectively. Overall, our mouse model can be utilized to develop new drugs for overcoming C797S-mediated resistance to osimertinib; further, such knock-in systems for expressing oncogenes may be applied to study tumorigenesis and the development of other targeted agents.

Lipid signatures reflect the function of the murine primary placentation†.

The placenta regulates maternal-fetal communication, and its defect leads to significant pregnancy complications. The maternal and embryonic circulations are primitively connected in early placentation, but the function of the placenta during this developmentally essential period is relatively unknown. We thus performed a comparative proteomic analysis of the placenta before and after primary placentation and found that the metabolism and transport of lipids were characteristically activated in this period. The placental fatty acid (FA) carriers in specific placental compartments were upregulated according to gestational age, and metabolomic analysis also showed that the placental transport of FAs increased in a time-dependent manner. Further analysis of two mutant mice models with embryonic lethality revealed that lipid-related signatures could reflect the functional state of the placenta. Our findings highlight the importance of the nutrient transport function of the primary placenta in the early gestational period and the role of lipids in embryonic development. SUMMARY SENTENCE: The placenta is activated characteristically in terms of lipid transport during primary placentation, and the lipid-related signatures closely reflect the functional state of the placenta.

Association between ARID2 and RAS-MAPK pathway in intellectual disability and short stature.

BACKGROUND: ARID2 belongs to the Switch/sucrose non-fermenting complex, in which the genetic defects have been found in patients with dysmorphism, short stature and intellectual disability (ID). As the phenotypes of patients with ARID2 mutations partially overlap with those of RASopathy, this study evaluated the biochemical association between ARID2 and RAS-MAPK pathway. METHODS: The phenotypes of 22 patients with either an ARID2 heterozygous mutation or haploinsufficiency were reviewed. Comprehensive molecular analyses were performed using somatic and induced pluripotent stem cells (iPSCs) of a patient with ARID2 haploinsufficiency as well as using the mouse model of Arid2 haploinsufficiency by CRISPR/Cas9 gene editing. RESULTS: The phenotypic characteristics of ARID2 deficiency include RASopathy, Coffin-Lowy syndrome or Coffin-Siris syndrome or undefined syndromic ID. Transient ARID2 knockout HeLa cells using an shRNA increased ERK1 and ERK2 phosphorylation. Impaired neuronal differentiation with enhanced RAS-MAPK activity was observed in patient-iPSCs. In addition, Arid2 haploinsufficient mice exhibited reduced body size and learning/memory deficit. ARID2 haploinsufficiency was associated with reduced IFITM1 expression, which interacts with caveolin-1 (CAV-1) and inhibits ERK activation. DISCUSSION: ARID2 haploinsufficiency is associated with enhanced RAS-MAPK activity, leading to reduced IFITM1 and CAV-1 expression, thereby increasing ERK activity. This altered interaction might lead to abnormal neuronal development and a short stature.

Multiplex gene targeting in the mouse embryo using a Cas9-Cpf1 hybrid guide RNA.

CRISPR-Cas systems, including Cas9 and Cpf1 (Cas12a), are promising tools for generating gene knockout mouse models. Unlike Cas9, Cpf1 can generate multiple crRNAs from a single concatemeric crRNA precursor, which is favorable for multiplex gene editing. Recently, a hybrid guide RNA (hgRNA) system employing both Cas9 and Cpf1 was developed for multiplex gene editing. As the crRNA of Cpf1 was linked to the 3' end of the sgRNA for Cas9, it can be split into separate guide RNAs by Cpf1. To examine whether this Cas9-Cpf1 hybrid system is suitable for multiplex gene knockouts in the mouse embryo, we generated an hgRNA that simultaneously targets the mouse Il10ra gene by Cas9 and mouse Dr3 (or Tnfrsf25, death receptor3) gene by Cpf1. The expression of hgRNA from a single promoter induced significant indels at each gene in cultured mouse cells upon the co-expression of both Cas9 and Cpf1. Interestingly, the hgRNA exhibited comparable Cas9-mediated indel activity without Cpf1 expression. Similarly, when the hgRNA was co-microinjected with both Cas9 and Cpf1 mRNAs into mouse zygotes at the pronuclear stage, founder mice were generated harboring mutations in both the Il10ra and Dr3 genes. However, when Cas9 mRNA was used alone without Cpf1 mRNA, the mouse Il10ra gene targeting was significantly decreased. These results indicate that the hgRNA system is a possible tool for multiplex gene targeting in the mouse embryo.

The Genetic Basis of Reporter Mouse Strains.

Genetically engineered mouse (GEM) models have been revolutionizing the biomedical studies on deciphering the physiological roles of genes in vivo. In addition to deactivating a gene in mice, diverse strategies have been created to monitor gene expressions and molecular dynamics of specific proteins in vivo. Although gene targeting in mouse embryonic stem (ES) cells was essential for the precise engineering of the mouse genome over almost three decades, this process is a time-consuming, expensive, and laborious one. These days, new technologies that directly apply engineered endonucleases, such as zinc-finger nucleases (ZFNs), Transcription Activator-Like Effector (TALE) Nucleases (TALENs), and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system, into the mouse zygotes are enabling us to rapidly replace conventional gene targeting in mouse ES cells. In this chapter, we will describe the principles of reporter mouse strains and the recent advances in generating them using engineered endonucleases.

Cerebral bioenergetic differences measured by phosphorus-31 magnetic resonance spectroscopy between bipolar disorder and healthy subjects living in two different regions suggesting possible effects of altitude.

AIM: Increased oxidative stress in cerebral mitochondria may follow exposure to the systemic hypobaric hypoxia associated with residing at higher altitudes. Because mitochondrial dysfunction is implicated in bipolar disorder (BD) pathophysiology, this may impact the cerebral bioenergetics in BD. In this study, we evaluated the cerebral bioenergetics of BD and healthy control (HC) subjects at two sites, located at sea level and at moderate altitude. METHODS: Forty-three veterans with BD and 33 HC veterans were recruited in Boston (n = 22) and Salt Lake City (SLC; n = 54). Levels of phosphocreatine, ß nucleoside triphosphate (ßNTP), inorganic phosphate, and pH over total phosphate (TP) were measured using phosphorus-31 magnetic resonance spectroscopy in the following brain regions: anterior cingulate cortex and posterior occipital cortex, as well as bilateral prefrontal and occipitoparietal (OP) white matter (WM). RESULTS: A significant main effect of site was found in ßNTP/TP (Boston > SLC) and phosphocreatine/TP (Boston < SLC) in most cortical and WM regions, and inorganic phosphate/TP (Boston < SLC) in OP regions. A main effect analysis of BD diagnosis demonstrated a lower pH in posterior occipital cortex and right OP WM and a lower ßNTP/TP in right prefrontal WM in BD subjects, compared to HC subjects. CONCLUSION: The study showed that there were cerebral bioenergetic differences in both BD and HC veteran participants at two different sites, which may be partly explained by altitude difference. Future studies are needed to replicate these results in order to elucidate the dysfunctional mitochondrial changes that occur in response to hypobaric hypoxia.

Inhibition of euchromatin histone-lysine N-methyltransferase 2 sensitizes breast cancer cells to tumor necrosis factor-related apoptosis-inducing ligand through reactive oxygen species-mediated activating transcription factor 4-C/EBP homologous protein-death receptor 5 pathway activation.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has been characterized as an anti-cancer therapeutic agent with prominent cancer cell selectivity over normal cells. However, breast cancer cells are generally resistant to TRAIL, thus limiting its therapeutic potential. In this study, we found that BIX-01294, a selective inhibitor of euchromatin histone methyltransferase 2/G9a, is a strong TRAIL sensitizer in breast cancer cells. The combination of BIX-01294 and TRAIL decreased cell viability and led to an increase in the annexin V/propidium iodide-positive cell population, DNA fragmentation, and caspase activation. BIX-01294 markedly increased death receptor 5 (DR5) expression, while silencing of DR5 using small interfering RNAs abolished the TRAIL-sensitizing effect of BIX-01294. Specifically, BIX-01294 induced C/EBP homologous protein (CHOP)-mediated DR5 gene transcriptional activation and DR5 promoter activation was induced by upregulation of the protein kinase R-like endoplasmic reticulum kinase-mediated activating transcription factor 4 (ATF4). Moreover, inhibition of reactive oxygen species by N-acetyl-L-cysteine efficiently blocked BIX-01294-induced DR5 upregulation by inhibiting ATF4/CHOP expression, leading to diminished sensitization to TRAIL. These findings suggest that BIX-01294 sensitizes breast cancer cells to TRAIL by upregulating ATF4/CHOP-dependent DR5 expression with a reactive oxygen species-dependent manner. Furthermore, combination treatment with BIX-01294 and TRAIL suppressed tumor growth and induced apoptosis in vivo. In conclusion, we found that epigenetic regulation can contribute to the development of resistance to cancer therapeutics such as TRAIL, and further studies of unfolded protein responses and the associated epigenetic regulatory mechanisms may lead to the discovery of new molecular targets for effective cancer therapy.

Highly efficient gene knockout in mice and zebrafish with RNA-guided endonucleases.

RNA-guided endonucleases (RGENs), derived from the prokaryotic Type II CRISPR-Cas system, enable targeted genome modification in cells and organisms. Here we describe the establishment of gene-knockout mice and zebrafish by the injection of RGENs as Cas9 protein:guide RNA complexes or Cas9 mRNA plus guide RNA into one-cell-stage embryos of both species. RGENs efficiently generated germline transmittable mutations in up to 93% of newborn mice with minimal toxicity. RGEN-induced mutations in the mouse Prkdc gene that encodes an enzyme critical for DNA double-strand break repair resulted in immunodeficiency both in F0 and F1 mice. We propose that RGEN-mediated mutagenesis in animals will greatly expedite the creation of genetically engineered model organisms, accelerating functional genomic research.

Use of a patterned current blocking layer to enhance the light output power of InGaN-based light-emitting diodes.

We employed a patterned current blocking layer (CBL) to enhance light output power of GaN-based light-emitting diodes (LEDs). Nanoimprint lithography (NIL) was used to form patterned CBLs (a diameter of 260 nm, a period of 600, and a height of 180 nm). LEDs (chip size: 300 × 800 µm2) fabricated with no CBL, a conventional SiO2 CBL, and a patterned SiO2 CBL, respectively, exhibited forward-bias voltages of 3.02, 3.1 and 3.1 V at an injection current of 20 mA. The LEDs without and with CBLs gave series resistances of 9.8 and 11.0 Ω, respectively. The LEDs with a patterned SiO2 CBL yielded 39.6 and 11.9% higher light output powers at 20 mA, respectively, than the LEDs with no CBL and conventional SiO2 CBL. On the basis of emission images and angular transmittance results, the patterned CBL-induced output enhancement is attributed to the enhanced light extraction and current spreading.

An Open-Label Pilot Study of Combined Augmentation With Creatine Monohydrate and 5-Hydroxytryptophan for Selective Serotonin Reuptake Inhibitor- or Serotonin-Norepinephrine Reuptake Inhibitor-Resistant Depression in Adult Women.

PURPOSE: Many women with major depressive disorder (MDD) respond inadequately to standard treatments. Augmentation of conventional antidepressants with creatine monohydrate and 5-hydroxytryptophan (5-HTP) could correct deficits in serotonin production and brain bioenergetics associated with depression in women, yielding synergistic benefit. We describe an open-label study of 5-HTP and creatine augmentation in women with MDD who had failed selective serotonin reuptake inhibitor (SSRI) or serotonin-norepinephrine reuptake inhibitor (SNRI) monotherapy. METHODS: Fifteen women who were adequately adherent to an SSRI or SNRI and currently experiencing MDD, with a 17-item Hamilton Depression Rating Scale (HAM-D) score of 16 or higher, were treated with 5 g of creatine monohydrate daily and 100 mg of 5-HTP twice daily for 8 weeks, with 4 weeks of posttreatment follow-up. The primary outcome was change in mean HAM-D scores. RESULTS: Mean HAM-D scores declined from 18.9 (SD, 2.5) at pretreatment visits to 7.5 (SD, 4.4) (P < 0.00001), a decrease of 60%. Participants did not experience any serious treatment-related adverse events. CONCLUSIONS: Combination treatment with creatine and 5-HTP may represent an effective augmentation strategy for women with SSRI- or SNRI-resistant depression. Given the limitations of this small, open-label trial, future study in randomized, placebo-controlled trials is warranted.

Preliminary Psychometric Evaluation of the Hamilton Depression Rating Scale in Methamphetamine Dependence.

OBJECTIVE: The purpose of this study was to test the initial psychometric properties of the 17-item Hamilton Depression Rating Scale (HAM-D) in individuals with and without major depressive disorder who use methamphetamine. We used data from two completed studies and two ongoing clinical trials. The HAM-D has well established reliability and validity in a variety of populations. However, there are no published reports of reliability and validity of the HAM-D in a methamphetamine-using population. METHODS: HAM-D and depression status data were extracted from four separate studies for this psychometric assessment. Using these data, we evaluated three measures of construct validity: internal consistency, contrasted group validity, and factorial validity. RESULTS: We found potential concerns with the construct validity of the HAM-D in users of methamphetamine. Intercorrelations between items were primarily less than 0.20 and the Cronbach's alpha value in this sample was 0.58, indicating potential issues with internal consistency. The results of two-sample t-tests suggest concerns with contrasted group validity, as no significant difference in average scores were found for nine items. Consistent with previous studies, a principal component analysis indicates that the HAM-D is multidimensional. CONCLUSIONS: The 17-item HAM-D might not reliably and validly measure depression severity in a methamphetamine-using population. Given our small sample, additional research is needed, though, to further test the psychometric properties of the HAM-D in individuals who use methamphetamine.

Creatine target engagement with brain bioenergetics: a dose-ranging phosphorus-31 magnetic resonance spectroscopy study of adolescent females with SSRI-resistant depression.

Major depressive disorder (MDD) often begins during adolescence and is projected to become the leading cause of global disease burden by the year 2030. Yet, approximately 40 % of depressed adolescents fail to respond to standard antidepressant treatment with a selective serotonin reuptake inhibitor (SSRI). Converging evidence suggests that depression is related to brain mitochondrial dysfunction. Our previous studies of MDD in adult and adolescent females suggest that augmentation of SSRI pharmacotherapy with creatine monohydrate (CM) may improve MDD outcomes. Neuroimaging with phosphorus-31 magnetic resonance spectroscopy ((31)P-MRS) can measure the high-energy phosphorus metabolites in vivo that reflect mitochondrial function. These include phosphocreatine (PCr), a substrate for the creatine kinase reaction that produces adenosine triphosphate. As part of the National Institute of Mental Health's experimental medicine initiative, we conducted a placebo-controlled dose-ranging study of adjunctive CM for adolescent females with SSRI-resistant MDD. Participants were randomized to receive placebo or CM 2, 4 or 10 g daily for 8 weeks. Pre- and post-treatment (31)P-MRS scans were used to measure frontal lobe PCr, to assess CM's target engagement with cerebral energy metabolism. Mean frontal lobe PCr increased by 4.6, 4.1 and 9.1 % in the 2, 4 and 10 g groups, respectively; in the placebo group, PCr fell by 0.7 %. There was no group difference in adverse events, weight gain or serum creatinine. Regression analysis of PCr and depression scores across the entire sample showed that frontal lobe PCr was inversely correlated with depression scores (p = 0.02). These results suggest that CM achieves target engagement with brain bioenergetics and that the target is correlated with a clinical signal. Further study of CM as a treatment for adolescent females with SSRI-resistant MDD is warranted.

Enhanced light extraction efficiency in organic light-emitting diode with randomly dispersed nanopattern.

An optical scattering layer composed of randomly dispersed nanopatterns (RDNPs) was introduced in an organic light-emitting diode (OLED) to increase the out-coupling efficiency. An RDNP was fabricated by direct printing on a glass substrate. Owing to its low haze and high transmittance, the RDNP acted as a light extraction layer in the OLED. The RDNP OLEDs showed higher current density and luminance than the reference devices at the same voltage. The current and power efficiencies of the RDNP OLED increased by 25% and 34%, respectively, without electrical degradation. Furthermore, the RDNP devices achieved an external quantum efficiency of 27.5% at 1 mA/cm².

Decreased brain PME/PDE ratio in bipolar disorder: a preliminary (31) P magnetic resonance spectroscopy study.

OBJECTIVES: The aim of the present study was to measure brain phosphorus-31 magnetic resonance spectroscopy ((31) P MRS) metabolite levels and the creatine kinase reaction forward rate constant (kf ) in subjects with bipolar disorder (BD). METHODS: Subjects with bipolar euthymia (n = 14) or depression (n = 11) were recruited. Healthy comparison subjects (HC) (n = 23) were recruited and matched to subjects with BD on age, gender, and educational level. All studies were performed on a 3-Tesla clinical magnetic resonance imaging system using a (31) P/(1) H double-tuned volume head coil. (31) P spectra were acquired without (1) H-decoupling using magnetization-transfer image-selected in vivo spectroscopy. Metabolite ratios from a brain region that includes the frontal lobe, corpus callosum, thalamus, and occipital lobe are expressed as a percentage of the total phosphorus (TP) signal. Brain pH was also investigated. RESULTS: Beta-nucleoside-triphosphate (ß-NTP/TP) in subjects with bipolar depression was positively correlated with kf (p = 0.039, r(2) = 0.39); similar correlations were not observed in bipolar euthymia or HC. In addition, no differences in kf and brain pH were observed among the three diagnostic groups. A decrease in the ratio of phosphomonoesters to phosphodiesters (PME/PDE) was observed in subjects with bipolar depression relative to HC (p = 0.032). We also observed a trend toward an inverse correlation in bipolar depression characterized by decreased phosphocreatine and increased depression severity. CONCLUSIONS: In our sample, kf was not altered in the euthymic or depressed mood state in BD. However, decreased PME/PDE in subjects with bipolar depression was consistent with differences in membrane turnover. These data provide preliminary support for alterations in phospholipid metabolism and mitochondrial function in bipolar depression.

Generation of knockout mice using engineered nucleases.

The use of engineered nucleases in one-cell stage mouse embryos is emerging as an efficient alternative to conventional gene targeting in mouse embryonic stem (ES) cells. These nucleases are designed or reprogrammed to specifically induce double strand breaks (DSBs) at a desired genomic locus, and efficiently introduce mutations by both error-prone and error-free DNA repair mechanisms. Since these mutations frequently result in the loss or alteration of gene function by inserting, deleting, or substituting nucleotide sequences, engineered nucleases are enabling us to efficiently generate gene knockout and knockin mice. Three kinds of engineered endonucleases have been developed and successfully applied to the generation of mutant mice: zinc-finger nuclease (ZFNs), transcription activator-like effector nucleases (TALENs) and RNA-guided endonucleases (RGENs). Based on recent advances, here we provide experimentally validated, detailed guidelines for generating non-homologous end-joining (NHEJ)-mediated mutant mice by microinjecting TALENs and RGENs into the cytoplasm or the pronucleus of one-cell stage mouse embryos.

Gender differences in the effect of tobacco use on brain phosphocreatine levels in methamphetamine-dependent subjects.

BACKGROUND: A high prevalence of tobacco smoking has been observed in methamphetamine users, but there have been no in vivo brain neurochemistry studies addressing gender effects of tobacco smoking in methamphetamine users. Methamphetamine addiction is associated with increased risk of depression and anxiety in females. There is increasing evidence that selective analogues of nicotine, a principal active component of tobacco smoking, may ease depression and improve cognitive performance in animals and humans. OBJECTIVES: To investigate the effects of tobacco smoking and gender on brain phosphocreatine (PCr) levels, a marker of brain energy metabolism reported to be reduced in methamphetamine-dependent subjects. METHODS: Thirty female and 27 male methamphetamine-dependent subjects were evaluated with phosphorus-31 magnetic resonance spectroscopy ((31)P-MRS) to measure PCr levels within the pregenual anterior cingulate, which has been implicated in methamphetamine neurotoxicity. RESULTS: Analysis of covariance revealed that there were statistically significant slope (PCr versus lifetime amount of tobacco smoking) differences between female and male methamphetamine-dependent subjects (p = 0.03). In females, there was also a statistically significant interaction between lifetime amounts of tobacco smoking and methamphetamine in regard to PCr levels (p = 0.01), which suggests that tobacco smoking may have a more significant positive impact on brain PCr levels in heavy, as opposed to light to moderate, methamphetamine-dependent females. CONCLUSION: These results indicate that tobacco smoking has gender-specific effects in terms of increased anterior cingulate high energy PCr levels in methamphetamine-dependent subjects. Cigarette smoking in methamphetamine-dependent women, particularly those with heavy methamphetamine use, may have a potentially protective effect upon neuronal metabolism.

Creatine as a Novel Treatment for Depression in Females Using Methamphetamine: A Pilot Study.

OBJECTIVE: Depression among methamphetamine users is more prevalent in females than males, but gender-specific treatment options for this comorbidity have not been described. Reduced brain phosphocreatine levels have been shown to be lower in female methamphetamine users compared to males, and, of relevance, studies have demonstrated an association between treatment-resistant depression and reduced brain phosphocreatine concentrations. The nutritional supplement creatine monohydrate has been reported to reduce symptoms of depression in female adolescents and adults taking antidepressants, as well as to increase brain phosphocreatine in healthy volunteers. Therefore, the purpose of this pilot study was to investigate creatine monohydrate as a treatment for depression in female methamphetamine users. METHODS: Fourteen females with depression and comorbid methamphetamine dependence were enrolled in an 8-week open label trial of 5 g of daily creatine monohydrate and of these 14, 11 females completed the study. Depression was measured using the Hamilton Depression Rating Scale (HAMD) and brain phosphocreatine levels were measured using phosphorus magnetic resonance spectroscopy pre- and post-creatine treatment. Secondary outcome measures included anxiety symptoms, measured with the Beck Anxiety Inventory (BAI), as well as methamphetamine use, monitored by twice weekly urine drug screens and self-reported use. RESULTS: The results of a linear mixed effects repeated measures model showed significantly reduced HAMD and BAI scores as early as week 2 when compared to baseline scores. This improvement was maintained through study completion. Brain phosphocreatine concentrations were higher at the second phosphorus magnetic resonance spectroscopy scan compared to the baseline scan; Mbaseline = 0.223 (SD = 0.013) vs. Mpost-treatment = 0.233 (SD = 0.009), t (9) = 2.905, p <.01, suggesting that creatine increased phosphocreatine levels. Also, a reduction in methamphetamine positive urine drug screens of greater than 50% was observed by week 6. Finally, creatine was well tolerated and adverse events that were related to gastrointestinal symptoms and muscle cramping were determined as possibly related to creatine. CONCLUSIONS: The current study suggests that creatine treatment may be a promising therapeutic approach for females with depression and comorbid methamphetamine dependence. This study is registered on clinicaltrials.gov (NCT01514630).

Ei24, a novel E2F target gene, affects p53-independent cell death upon ultraviolet C irradiation.

The deficiency of retinoblastoma (Rb) gene deregulates E2F transcription factors and thus induces E2F target genes directly or p53 target genes indirectly via mouse p19(Arf) (or p14(ARF) in humans), an E2F target gene. Here, we identified that etoposide-induced 2.4 mRNA (Ei24)/p53-induced gene 8 (Pig8), a p53 target gene involved in apoptosis and autophagy, was up-regulated in Rb(-/-) mouse embryonic fibroblasts (MEFs). The Ei24 promoter was activated by E2F1 via multiple E2F-responsive elements, independently of the previously reported p53-responsive element. Chromatin immunoprecipitation assays revealed that E2F1 directly acts on the mouse Ei24 promoter. We observed that Ei24 expression was suppressed in p53(-/-) MEFs upon UVC irradiation, which was exacerbated in p53(-/-) E2f1(-/-) MEFs, supporting the positive role of E2F1 on Ei24 transcription. Furthermore, Ei24 knockdown sensitized p53(-/-) MEFs against UVC irradiation. Together, our data indicate that Ei24 is a novel E2F target gene contributing to the survival of p53-deficient cells upon UVC irradiation and thus may have a potential significance as a therapeutic target of certain chemotherapy for treating p53-deficient tumors.