The peripheral terminals of primary nociceptive neurons play an essential role in pain detection mediated by membrane receptors like TRPV1, a molecular sensor of heat and capsaicin. However, the contribution of central terminal TRPV1 in the dorsal horn to chronic pain has not been investigated directly. Combining primary sensory neuron-specific GCaMP3 imaging with a trigeminal neuropathic pain model, we detected robust neuronal hyperactivity in injured and uninjured nerves in the skin, soma in trigeminal ganglion, and central terminals in the spinal trigeminal nucleus. Extensive TRPV1 hyperactivity was observed in central terminals innervating all dorsal horn laminae. The central terminal TRPV1 sensitization was maintained by descending serotonergic (5-HT) input from the brainstem. Central blockade of TRPV1 or 5-HT/5-HT3A receptors attenuated central terminal sensitization, excitatory primary afferent inputs, and mechanical hyperalgesia in the territories of injured and uninjured nerves. Our results reveal new central mechanisms facilitating central terminal sensitization underlying chronic pain.
Spinocerebellar ataxia type 10 (SCA10) is an autosomal dominant neurologic disorder caused by ATTCT expansion in the ATXN10 gene. Previous investigations have identified that depletion of Ataxin-10, the gene product, leads to cellular apoptosis and cytokinesis failure. Herein we identify the mitotic kinase Aurora B as an Ataxin-10 interacting partner. Aurora B interacts with and phosphorylates Ataxin-10 at S12, as evidenced by in vitro kinase and mass spectrometry analysis. Both endogenous and S12-phosphorylated Ataxin-10 localizes to the midbody during cytokinesis, and cytokinetic defects induced by inhibition of ATXN10 expression is not rescued by the S12A mutant. Inhibition of Aurora B or expression of the S12A mutant renders reduced interaction between Ataxin-10 and polo-like kinase 1 (Plk1), a kinase previously identified to regulate Ataxin-10 in cytokinesis. Taken together, we propose a model that Aurora B phosphorylates Ataxin-10 at S12 to promote the interaction between Ataxin-10 and Plk1 in cytokinesis. These findings identify an Aurora B-dependent mechanism that implicates Ataxin-10 in cytokinesis.
The mammary epithelium is organized as a bi-layer of luminal and basal/myoepithelial cells. During pregnancy the luminal compartment expands for milk production, while basal cells are thought to provide structural and contractile support. Here we reveal an unanticipated, pregnancy-specific role of basal epithelia as a central coordinator of lactogenesis. We demonstrate that genetic deletion of the transcription factor p63 (Trp63) gene exclusively within basal cells of the adult gland during pregnancy leads to dramatic defects in luminal cell proliferation and differentiation, resulting in lactation failure. This phenotype is explained by direct transcriptional activation of the EGF-family ligand gene Nrg1 by p63 selectively in basal cells, which is required for luminal ERBB4/STAT5A activation and consequent luminal progenitor cell maturation. Thus, paracrine basal-to-luminal cell signaling, controlled by p63 via NRG1, orchestrates the entire lactation program. Collectively, these findings redefine the paradigm for cellular interactions specifying the functional maturation of the mammary gland.
p63; basal cell; paracrine signaling; lactation; luminal progenitor cells
Megakaryocyte morphogenesis employs a “hypertrophy-like” developmental program, dependent on P-TEFb kinase activation and cytoskeletal remodeling. P-TEFb activation classically occurs by a feedback regulated process of signal-induced, reversible release of active Cdk9-cyclin T modules from large inactive 7SK snRNP complexes. Here we have identified an alternative pathway of irreversible P-TEFb activation in megakaryopoiesis, mediated by dissolution of the 7SK snRNP complex. In this pathway calpain 2 cleavage of the core 7SK snRNP component MePCE promoted P-TEFb release and consequent upregulation of a cohort of cytoskeleton remodeling factors, including α-actinin-1. In a subset of human megakaryocytic leukemias, the transcription factor GATA1 undergoes truncating mutation (GATA1s). Here we linked the GATA1s mutation to defects in megakaryocytic upregulation of calpain 2 and of P-TEFb-dependent cytoskeletal remodeling factors. Restoring calpain 2 expression in GATA1s-mutant megakaryocytes rescued normal development, implicating this morphogenetic pathway as a target in human leukemogenesis.
megakaryopoiesis; P-TEFb; 7SK snRNP; calpain 2; GATA1s mutant
During prion diseases, a normally benign, host protein, denoted PrPC, undergoes alternative folding into the aberrant isoform, PrPSc. We used ELISA assays to identify and confirm hits in order to develop leads that reduce PrPSc in prion-infected dividing and stationary-phase mouse neuroblastoma (ScN2a-cl3) cells. We tested 52,830 diverse small molecules in dividing cells and 49,430 in stationary-phase cells. This led to 3,100 HTS and 970 single point confirmed (SPC) hits in dividing cells, 331 HTS and 55 confirmed SPC hits in stationary-phase cells as well as 36 confirmed SPC hits active in both. Fourteen chemical leads were identified from confirmed SPC hits in dividing cells and three in stationary-phase cells. From more than 682 compounds tested in concentration-effect relationships in dividing cells to determine potency (EC50), 102 had EC50 values between 1–10 µM and 50 had EC50 values of <1 µM; none affected cell viability. We observed an excellent correlation between EC50 values determined by ELISA and Western immunoblotting for 28 representative compounds in dividing cells (R2 = 0.75; p < 0.0001). Of the 55 confirmed SPC hits in stationary-phase cells, 23 were piperazine, indole, or urea leads. The potency (EC50) of one indole in stationary-phase and dividing ScN2a-cl3 cells was 7.5 and 1.6 µM, respectively. Unexpectedly, the number of hits in stationary-phase cells was ~10% of that in dividing cells. The explanation for this difference remains to be determined.
Antiprion compounds; PrPSc; dividing and stationary-phase brain cells
Transient leukemia (TL) is evident in 5–10% of all neonates with Down syndrome (DS) and associated with N-terminal truncating GATA1-mutations (GATA1s). Here we report that TL cell clones generate abundant eosinophils in a substantial fraction of patients. Sorted eosinophils from patients with TL and eosinophilia carried the same GATA1s-mutation as sorted TL-blasts, consistent with their clonal origin. TL-blasts exhibited a genetic program characteristic of eosinophils and differentiated along the eosinophil lineage in vitro. Similarly, ectopic expression of Gata1s, but not Gata1, in wild-type CD34+-hematopoietic stem and progenitor cells induced hyperproliferation of eosinophil promyelocytes in vitro. While GATA1s retained the function of GATA1 to induce eosinophil genes by occupying their promoter regions, GATA1s was impaired in its ability to repress oncogenic MYC and the pro-proliferative E2F transcription network. ChIP-seq indicated reduced GATA1s occupancy at the MYC promoter. Knockdown of MYC, or the obligate E2F-cooperation partner DP1, rescued the GATA1s-induced hyperproliferative phenotype. In agreement, terminal eosinophil maturation was blocked in Gata1Δe2 knockin mice, exclusively expressing Gata1s, leading to accumulation of eosinophil precursors in blood and bone marrow. These data suggest a direct relationship between the N-terminal truncating mutations of GATA1 and clonal eosinophilia in DS patients.
Down syndrome; eosinophilia; GATA1s; MYC; E2F
Cost-effective, solution-processable organic photovoltaics (OPV) present an interesting alternative to inorganic silicon-based solar cells. However, one of the major remaining challenges of OPV devices is their lack of long-term operational stability, especially at elevated temperatures. The synthesis of a fullerene dumbbell and its use as an additive in the active layer of a PCDTBT:PCBM-based OPV device is reported. The addition of only 20 % of this novel fullerene not only leads to improved device efficiencies, but more importantly also to a dramatic increase in morphological stability under simulated operating conditions. Dynamic secondary ion mass spectrometry (DSIMS) and TEM are used, amongst other techniques, to elucidate the origins of the improved morphological stability.
fullerenes; lifetime; organic solar cells; photovoltaics; stability
This study aims to determine the up-to-date prevalence of overweight and obesity, the distributions of body weight perception and weight loss practice in Beijing adults.
A cross-sectional study was conducted in 2011. A total of 2563 men and 4088 women aged 18–79 years from the general population were included. Data were obtained from questionnaire and physical examination.
The prevalence of overweight (BMI 24–27.9 kg/m2) and obesity (BMI≥28 kg/m2) was 42.1% and 20.3% in men and 35.6% and 17.1% in women, respectively. Age was inversely associated with overweight in both sexes, and obesity in women. Education level was negatively associated with overweight and obesity in women but not in men. Only 49.1% men and 58.3% women had a correct perception of their body weight. Underestimation of body weight was more common than overestimation, especially in men, the older people, and those with low education level. The percentage of taking action to lose weight was inversely associated with men and old age, and positively associated with higher education level, higher BMI, and self-perception as “fat” (OR = 3.78 in men, OR = 2.91 in women). Only 26.1% of overweight/obese individuals took action to lose weight. The top two weight loss practices were to reduce the amount of food intake and exercise.
Overweight and obesity were highly prevalent with high incorrect body weight perceptions in the general adult population in Beijing. Weight loss practice was poor in overweight and obese individuals. Actions at multiple levels are needed to slow or control this overweight and obesity epidemic.
Hepatitis B is a major health concern in the Asia-Pacific region, and is endemic in China, Southeast Asia, and Africa. Chronic hepatitis B virus (HBV) infection may cause hepatic cirrhosis and liver cancer. It is estimated that there are more than 350 million chronic HBV carriers worldwide, of whom approximately one quarter will die of chronic hepatitis B-related liver diseases. HBV is transmitted horizontally through blood and blood products or by sexual transmission, and vertically from mother to infant. Perinatal infection is the predominant mode of transmission in countries with a high prevalence of hepatitis B surface antigen (HBsAg) carriage, and perinatal transmission leads to high rates of chronic infection. Therefore, it is important to prevent the mother-to-child transmission (MTCT) of HBV. Research has shown that pregnant women with high HBV DNA levels have an increased risk of MTCT. However, most of the obstetrics guidelines do not make a distinction between pregnant women with high HBV DNA levels and those who are HBsAg positive only. This review addresses the management of pregnant women with high levels of HBV viremia, in terms of antiviral therapy, use of hepatitis B immunoglobulin (HBIG), the combined application of hepatitis B vaccine and HBIG, choice of delivery mode and feeding practices.
Hepatitis B virus; Hepatitis B virus DNA; High level; Management; Pregnancy
Histone deacetylase (HDAC)-inhibitors (HDACis) are well characterized anti-cancer agents with promising results in clinical trials. However, mechanistically little is known regarding their selectivity in killing malignant cells while sparing normal cells. Gene expression-based chemical genomics identified HDACis as being particularly potent against Down syndrome associated myeloid leukemia (DS-AMKL) blasts. Investigating the anti-leukemic function of HDACis revealed their transcriptional and posttranslational regulation of key autophagic proteins, including ATG7. This leads to suppression of autophagy, a lysosomal degradation process that can protect cells against damaged or unnecessary organelles and protein aggregates. DS-AMKL cells exhibit low baseline autophagy due to mTOR activation. Consequently, HDAC inhibition repressed autophagy below a critical threshold, which resulted in accumulation of mitochondria, production of reactive oxygen species, DNA-damage and apoptosis. Those HDACi-mediated effects could be reverted upon autophagy activation or aggravated upon further pharmacological or genetic inhibition. Our findings were further extended to other major acute myeloid leukemia subgroups with low basal level autophagy. The constitutive suppression of autophagy due to mTOR activation represents an inherent difference between cancer and normal cells. Thus, via autophagy suppression, HDACis deprive cells of an essential pro-survival mechanism, which translates into an attractive strategy to specifically target cancer cells.
HDAC inhibitor; autophagy; AML; Down syndrome; ATG7
Naked2 (NKD2) is a member of the Naked family and negatively regulates canonical Wnt signaling. NKD2 may play a role in embryo development and tumor formation by affecting Wnt signaling. In the present study, we describe the establishment of a monoclonal antibody against NKD2 (anti-NKD2 MAb) through the hybridoma method. The purified anti-NKD2 MAb measured a titer of 2.56×105 against NKD2 by indirect ELISA. Western blot analysis, immunoprecipitation, and confocal microscope showed that the anti-NKD2 MAb can specifically combine NKD2 protein in SW480 and LOVO cells. Competitive inhibition assays of Western blot and indirect ELISA showed that the anti-NKD2 MAb can be blocked with NKD21-217 protein. The anti-NKD2 MAb would be helpful for further studies on the structure activity relationship, protein detecting, and cell-signaling pathway of NKD2.
The prion diseases caused by PrPSc, an alternatively folded form of the cellular prion protein (PrPC), are rapidly progressive, fatal, and untreatable neurodegenerative syndromes. We employed HTS ELISA assays to identify compounds that lower the level of PrPSc in prion-infected mouse neuroblastoma (ScN2a-cl3) cells and identified a series of arylamides. SAR studies indicated that small amides with one aromatic, or heteroaromatic ring, on each side of the amide bond are of modest potency. Of note, benzamide (7), with an EC50 of 2200 nM, was one of only a few arylamide hits with a piperazine group on its aniline moiety. The basic piperazine nitrogen can be protonated at physiologic pH, improving solubility, and therefore we wanted to exploit this feature in our search for a drug candidate. An SAR campaign resulted in several key analogs, including a set with biaryl groups introduced on the carbonyl side for improved potency. Several of these biaryl analogs have submicromolar potency, with the most potent analog 17 having an EC50 = 22 nM. More importantly, 17 and several biarylamides (20, 24, 26, 27) were able to traverse the BBB and displayed excellent drug levels in the brains of mice following oral dosing. These biarylamides may represent good starting points for further lead optimization for the identification of potential drug candidates for the treatment of prion diseases.
Neurodegenerative diseases; prion disease; Creutzfeldt-Jakob disease; amide; arylamide; SAR
prion diseases caused by PrPSc, an alternatively
folded form of the cellular prion protein (PrPC), are rapidly
progressive, fatal, and untreatable neurodegenerative disorders. We
employed HTS ELISA assays to identify compounds that lower the level
of PrPSc in prion-infected mouse neuroblastoma (ScN2a-cl3)
cells and identified a series of arylamides. Structure–activity
relationship (SAR) studies indicated that small amides with one aromatic
or heteroaromatic ring on each side of the amide bond are of modest
potency. Of note, benzamide (7), with an EC50 of 2200 nM, was one of only a few arylamide hits with a piperazine
group on its aniline moiety. The basic piperazine nitrogen can be
protonated at physiologic pH, improving solubility, and therefore,
we wanted to exploit this feature in our search for a drug candidate.
An SAR campaign resulted in several key analogues, including a set
with biaryl groups introduced on the carbonyl side for improved potency.
Several of these biaryl analogues have submicromolar potency, with
the most potent analogue 17 having an EC50 = 22 nM. More importantly, 17 and several biarylamides
(20, 24, 26, and 27) were able to traverse the blood–brain barrier (BBB) and
displayed excellent drug levels in the brains of mice following oral
dosing. These biarylamides may represent good starting points for
further lead optimization for the identification of potential drug
candidates for the treatment of prion diseases.
Neurodegenerative diseases; prion disease; Creutzfeldt−Jakob disease; amide; arylamide; SAR
While individuals with insomnia consistently complain of cognitive impairment, previous studies on the effect of insomnia on objective measures of cognitive function have obtained ambiguous results. The relationship between daytime sleepiness and cognitive manifestations in insomnia patients is not clear.
Thirty-six primary insomnia patients (PIPs) and 26 good sleep controls (GSCs) with age and gender matched manner were included in the study. Participants underwent an overnight polysomnography followed by a multiple sleep latency test (MSLT) and an examination of the attention network test (ANT). ANT reflected three attentional networks including alerting, orienting and executive control. According to whether accompanied with excessive daytime sleepiness (EDS), the insomnia group were subdivided into PIPs with EDS (n = 12, score on MSLT<10 min) and PIPs without EDS (n = 24, score on MSLT≥10 min).
PIPs only performed worse on executive control function than GSCs in ANT. PIPs with EDS had longer overall reaction time (RT) related to PIPs without EDS. Further analyses with Pearson correlation analysis showed a significant negative correlation between the overall RT and MSLT latency in insomniacs (r = −0.444, p<0.01), whereas no such correlation was found in controls.
Results suggest that PIPs do show executive control function deficits compared with GSCs. Daytime sleepiness in terms of MSLT latency was associated with poor cognitive manifestations in patients with insomnia.
The Hedgehog (Hh) signaling pathway regulates a variety of tumor-related diseases, including leukemia. The present study aimed to determine whether there was an interaction between the Hh signaling pathway and transforming growth factor (TGF)-β in the KG-1 cell line. KG-1 cells were treated with TGF-β, tumor necrosis factor (TNF)-α and specific inhibitor of smad3 (SIS3). The expression level of Gli family zinc finger 2 (Gli2) was detected by quantitative polymerase chain reaction (qPCR) and western blot analyses. The results revealed that TGF-β significantly decreased the expression level of Gli2 in KG-1 cells, and that TNF-α and TGF-β together further reduced Gli2 expression in KG-1 cells. SIS3 inhibited the effect of TGF-β. These results suggest that Gli2 expression in KG-1 cells is suppressed by TGF-β in a Smad3-dependent manner, TNF-α can enhance the effect of TGF-β on Gli2 expression and that this occurs independently of Hh receptor signaling.
Gli family zinc finger 2; transforming growth factor-β; tumor necrosis factor α; specific inhibitor of smad3
Lineage tracing using Cre/lox transgenic mice provides a powerful tool for studying normal mammary epithelial cell (MEC) development and the cellular origins of mammary tumors under physiological settings. However, generation of new transgenic mice for lineage-tracing purposes is often time consuming. Here, we report a lineage-tracing tool for MECs based on intraductal injection of lineage-specific Cre-expressing adenovirus (Ad-Cre). Using well-characterized promoters for Keratin 8 and Keratin 14, we generated lineage-specific Ad-Cre lines for luminal and basal MECs, respectively. By pulse-chase lineage tracing using these Ad-Cre lines, we showed that luminal and basal lineages are largely self-sustained and that IRS1 and IRS2 are essential for maintaining the basal lineage; we also showed that heterogeneous mammary tumors can be induced from luminal MECs in mice carrying the Etv6-NTRK3 fusion gene. Overall, we validated the Ad-Cre system as a promising and efficient tool for fate mapping of normal and malignant cells in adult tissues.
•Adenovirus-Cre can be used for pulse-chase lineage tracing of adult stem cells•Mammary luminal and basal lineages in adults are largely self-sustained•IRS1 and IRS2 are essential for maintaining the adult mammary basal lineage•Multiple adult mammary luminal cell types may serve as breast cancer cellular origins
In this study, Li and colleagues used cell-type-specific Adeno-Cre viruses for pulse-chase lineage tracing of adult stem cells. In mammary glands, it was found that adult mammary luminal and basal epithelial lineages were largely self-sustained, IRS1 and IRS2 were essential for maintaining the basal linage, and multiple luminal cell subtypes might serve as cellular origins of breast cancer.
We recently found that activation of IL17A signaling promotes the development and progression of acute and chronic pulmonary fibrosis, and that the blockade of IL17A activity attenuates pulmonary fibrosis by promoting the resolution of inflammation and the activation of autophagy. Although the induction of autophagy stimulating the collagen degradation in the fibrotic lung tissue has been identified as a mechanism responsible for the antifibrotic role of targeting IL17A, it remains to be clarified how IL17A signaling suppresses autophagy. Here we report that the phosphorylation of B-cell CLL/lymphoma 2 (BCL2), an apoptosis regulatory protein, was inhibited in the presence of IL17A in lung epithelial cells, and this reduction suppressed the ubiquitination degradation of BCL2, which subsequently attenuated autophagy by promoting the interaction of BCL2 and BECN1. We found that IL17A regulated the phosphorylation of BCL2 through activating the phosphoinositide 3-kinase (PI3K)-glycogen synthase kinase 3 β (GSK3B) signaling cascade. In response to IL17A stimulation, PI3K was activated and resulted in phosphorylation of GSK3B at Ser9, which subsequently attenuated the interaction of GSK3B with BCL2. Interrupting the GSK3B and BCL2 interaction precluded the phosphorylation of BCL2 at Ser70, which could trigger the ubiquitination degradation, and restrained the ubiquitination degradation of BCL2. Consequently, a decrease in the BCL2 degradation induced by IL17A resulted in a suppressed autophagy in lung epithelial cells. These findings indicate that the IL17A-PI3K-GSK3B-BCL2 signaling pathway participates in the attenuation of autophagic activity in lung epithelial cells, which is attributed to be primarily responsible for the development and progression of IL17A-induced pulmonary fibrosis.
inflammatory disease; IL17A signaling pathway; protein stability; protein interaction; pulmonary fibrosis; ubiquitination degradation
Recently, we described the aminothiazole lead (4-biphenyl-4-yl-thiazol-2-yl)-(6-methyl-pyridin-2-yl)-amine (1), which exhibits many desirable properties, including excellent stability in liver microsomes, oral bioavailability of ∼40% and high exposure in the brains of mice. Despite its good pharmacokinetic properties, compound 1 exhibited only modest potency in mouse neuroblastoma cells overexpressing the disease-causing prion protein PrPSc. Accordingly, we sought to identify analogs of 1 with improved antiprion potency in ScN2a-cl3 cells while retaining comparable or superior properties. We now report the discovery of improved lead compounds such as (6-methyl-pyridin-2-yl)-[4-(4-pyridin-3-yl-phenyl)-thiazol-2-yl]-amine (15) and cyclopropanecarboxylic acid (4-biphenyl-thiazol-2-yl)-amide (34), which exhibited brain exposure/EC50 ratios at least ten-fold greater than that of 1.
2-Aminothiazoles; neurological agents; pharmacokinetic optimization; prion disease; structure-activity relationships
We identified the primary symptoms leading to Chinese patients presenting at hospital with suspected obstructive sleep apnea (OSA) and studied the prevalence and characteristics of OSA in confirmed cases.
We collected data on 350 consecutive patients (302 males and 43±11 years old) with suspected OSA who underwent overnight polysomnography (PSG).
Among all patients, rankings of primary symptoms that led to the patients presenting at hospital for PSG were observed apnea (33%), snoring alone (29%), choking/gasping (13%), daytime sleepiness (5%) and other (20%). For severe OSA, prevalence rate was 61%, apnea hypopnea index (AHI) was 64±18, age was 44±10 years old, body mass index (BMI) was 28±3.5 kg/m2, and hypertension rate was 28%.
Self-awareness of symptoms led a majority of the patients to present at hospital in China. Compared to currently available case series studies, our results suggest that OSA patients in East Asian countries are characterized by higher prevalence and more severe apnea, younger age, poorer sleep quality, but less obesity and less comorbidity with hypertension, relative to countries in North America, South America and Europe.
Apnea; symptoms; prevalence; consecutive patients
Prion diseases are a group of fatal neurodegenerative disorders that include Creutzfeldt-Jakob disease (CJD) and kuru in humans, BSE in cattle, and scrapie in sheep. Such illnesses are caused by the conversion and accumulation of a misfolded pathogenic isoform (termed PrPSc) of a normally benign, host cellular protein, denoted PrPC. We employed high-throughput screening (HTS) ELISAs to evaluate compounds for their ability to reduce the level of PrPSc in Rocky Mountain Laboratory (RML) prion-infected mouse neuroblastoma cells (ScN2a-cl3). Arylpiperazines were among the active compounds identified but the initial hits suffered from low potency and poor drug-likeness. The best of those hits, such as 1, 7, 13, and 19, displayed moderate antiprion activity with EC50 values in the micromolar range. Key analogs were designed and synthesized based on the SAR, with analogs 41, 44, 46, and 47 found to have sub-micromolar potency. Analogs 41 and 44 were able to penetrate the blood-brain barrier (BBB) and achieved excellent drug concentrations in the brains of mice after oral dosing. These compounds represent good starting points for further lead optimization in our pursuit of potential drug candidates for the treatment of prion diseases.
Neurodegenerative diseases; prion disease; Creutzfeldt-Jakob disease; piperazine; arylpiperazine; SAR
Creutzfeldt-Jakob disease and kuru
in humans, BSE in cattle, and
scrapie in sheep are fatal neurodegenerative disorders. Such illnesses
are caused by the conversion and accumulation of a misfolded pathogenic
isoform (termed PrPSc) of a normally benign, host cellular
protein, denoted PrPC. We employed high-throughput screening
enzyme-linked immunosorbent assays to evaluate compounds for their
ability to reduce the level of PrPSc in Rocky Mountain
Laboratory prion-infected mouse neuroblastoma cells (ScN2a-cl3). Arylpiperazines
were among the active compounds identified, but the initial hits suffered
from low potency and poor drug-likeness. The best of those hits, such
as 1, 7, 13, and 19, displayed moderate antiprion activity with EC50 values
in the micromolar range. Key analogues were designed and synthesized
on the basis of the structure–activity relationship, with analogues 41, 44, 46, and 47 found
to have submicromolar potency. Analogues 41 and 44 were able to penetrate the blood–brain barrier and
achieved excellent drug concentrations in the brains of mice after
oral dosing. These compounds represent good starting points for further
lead optimization in our pursuit of potential drug candidates for
the treatment of prion diseases.
Neurodegenerative diseases; prion disease; Creutzfeldt-Jakob disease; piperazine; arylpiperazine; structure−activity relationship
To discover drugs lowering PrPSc in prion-infected cultured neuronal cells that achieve high concentrations in brain to test in mouse models of prion disease and then treat people with these fatal diseases.
We tested 2-AMT analogs for EC50 and PK after a 40 mg/kg single dose and 40–210 mg/kg/day doses for 3 days. We calculated plasma and brain AUC, ratio of AUC/EC50 after dosing. We reasoned that compounds with high AUC/EC50 ratios should be good candidates going forward.
We evaluated 27 2-AMTs in single-dose and 10 in 3-day PK studies, of which IND24 and IND81 were selected for testing in mouse models of prion disease. They had high concentrations in brain after oral dosing. Absolute bioavailability ranged from 27–40%. AUC/EC50 ratios after 3 days were >100 (total) and 48–113 (unbound). Stability in liver microsomes ranged from 30–>60 min. Ring hydroxylated metabolites were observed in microsomes. Neither was a substrate for the MDR1 transporter.
IND24 and IND81 are active in vitro and show high AUC/EC50 ratios (total and unbound) in plasma and brain. These will be evaluated in mouse models of prion disease.
antiprion drugs; drug discovery; IND24; IND81; prion disease
Ectopic expression of reprogramming factors has been widely adopted to reprogram somatic nucleus into a pluripotent state (induced pluripotent stem cells [iPSCs]). However, genetic aberrations such as somatic gene mutation in the resulting iPSCs have raised concerns regarding their clinical utility. To test whether the increased somatic mutations are primarily the by-products of current reprogramming methods, we reprogrammed embryonic fibroblasts of inbred C57BL/6 mice into either iPSCs (8 lines, 4 previously published) or embryonic stem cells (ESCs) with somatic cell nuclear transfer (SCNT ESCs; 11 lines). Exome sequencing of these lines indicates a significantly lower mutation load in SCNT ESCs than iPSCs of syngeneic background. In addition, one SCNT-ESC line has no detectable exome mutation, and two pairs of SCNT-ESC lines only have shared preexisting mutations. In contrast, every iPSC line carries unique mutations. Our study highlights the need for improving reprogramming methods in more physiologically relevant conditions.
•Comparison of somatic-coding mutation load in SCNT ESCs and iPSCs in mice•Lower mutational load in SCNT ESCs than iPSCs of syngeneic background
By exome sequencing of pluripotent stem cell lines reprogrammed from embryonic fibroblasts of inbred C57BL/6 mice with somatic cell nuclear transfer-embryonic stem cell (SCNT-ESC) or ectopic expression of reprogramming factors (induced pluripotent stem cells [iPSC]), Zhang, Deng, Xu, and colleagues show that the SCNT-ESC lines harbor a significantly lower somatic-coding mutation load than iPSC lines.
It has been well documented that mutations in the same retinal disease gene can result in different clinical phenotypes due to difference in the mutant allele and/or genetic background. To evaluate this, a set of consanguineous patient families with Leber congenital amaurosis (LCA) that do not carry mutations in known LCA disease genes was characterized through homozygosity mapping followed by targeted exon/whole-exome sequencing to identify genetic variations. Among these families, a total of five putative disease-causing mutations, including four novel alleles, were found for six families. These five mutations are located in four genes, ALMS1, IQCB1, CNGA3, and MYO7A. Therefore, in our LCA collection from Saudi Arabia, three of the 37 unassigned families carry mutations in retinal disease genes ALMS1, CNGA3, and MYO7A, which have not been previously associated with LCA, and 3 of the 37 carry novel mutations in IQCB1, which has been recently associated with LCA. Together with other reports, our results emphasize that the molecular heterogeneity underlying LCA, and likely other retinal diseases, may be highly complex. Thus, to obtain accurate diagnosis and gain a complete picture of the disease, it is essential to sequence a larger set of retinal disease genes and combine the clinical phenotype with molecular diagnosis.
Leber congenital amaurosis; LCA; whole-exome sequencing; SNP; padlock