Deficiency in origin licensing proteins impairs cilia formation: implications for the aetiology of meier-gorlin syndrome

Mutations in ORC1, ORC4, ORC6, CDT1, and CDC6, which encode proteins required for DNA replication origin licensing, cause Meier-Gorlin syndrome (MGS), a disorder conferring microcephaly, primordial dwarfism, underdeveloped ears, and skeletal abnormalities. Mutations in ATR, which also functions during replication, can cause Seckel syndrome, a clinically related disorder. These findings suggest that impaired DNA replication could underlie the developmental defects characteristic of these disorders. Here, we show that although origin licensing capacity is impaired in all patient cells with mutations in origin licensing component proteins, this does not correlate with the rate of progression through S phase. Thus, the replicative capacity in MGS patient cells does not correlate with clinical manifestation. However, ORC1-deficient cells from MGS patients and siRNA-mediated depletion of origin licensing proteins also have impaired centrosome and centriole copy number. As a novel and unexpected finding, we show that they also display a striking defect in the rate of formation of primary cilia. We demonstrate that this impacts sonic hedgehog signalling in ORC1-deficient primary fibroblasts. Additionally, reduced growth factor-dependent signaling via primary cilia affects the kinetics of cell cycle progression following cell cycle exit and re-entry, highlighting an unexpected mechanism whereby origin licensing components can influence cell cycle progression. Finally, using a cell-based model, we show that defects in cilia function impair chondroinduction. Our findings raise the possibility that a reduced efficiency in forming cilia could contribute to the clinical features of MGS, particularly the bone development abnormalities, and could provide a new dimension for considering developmental impacts of licensing deficiency

Cascade of Neural Events Leading from Error Commission to Subsequent Awareness Revealed Using EEG Source Imaging

The goal of the present study was to shed light on the respective contributions of three important action monitoring brain regions (i.e. cingulate cortex, insula, and orbitofrontal cortex) during the conscious detection of response errors. To this end, fourteen healthy adults performed a speeded Go/Nogo task comprising Nogo trials of varying levels of difficulty, designed to elicit aware and unaware errors. Error awareness was indicated by participants with a second key press after the target key press. Meanwhile, electromyogram (EMG) from the response hand was recorded in addition to high-density scalp electroencephalogram (EEG). In the EMG-locked grand averages, aware errors clearly elicited an error-related negativity (ERN) reflecting error detection, and a later error positivity (Pe) reflecting conscious error awareness. However, no Pe was recorded after unaware errors or hits. These results are in line with previous studies suggesting that error awareness is associated with generation of the Pe. Source localisation results confirmed that the posterior cingulate motor area was the main generator of the ERN. However, inverse solution results also point to the involvement of the left posterior insula during the time interval of the Pe, and hence error awareness. Moreover, consecutive to this insular activity, the right orbitofrontal cortex (OFC) was activated in response to aware and unaware errors but not in response to hits, consistent with the implication of this area in the evaluation of the value of an error. These results reveal a precise sequence of activations in these three non-overlapping brain regions following error commission, enabling a progressive differentiation between aware and unaware errors as a function of time elapsed, thanks to the involvement first of interoceptive or proprioceptive processes (left insula), later leading to the detection of a breach in the prepotent response mode (right OFC)

Temporal Variations of Skin Pigmentation in C57Bl/6 Mice Affect Optical Bioluminescence Quantitation

ABSTRACT PURPOSE: Depilation-induced skin pigmentation in C57Bl/6 mice is a known occurrence, and presents a unique problem for quantitative optical imaging of small animals, especially for bioluminescence. The work reported here quantitatively investigated the optical attenuation of bioluminescent light due to melanin pigmentation in the skin of transgenic C57B1/6 mice, modified such that luciferase expression is under the transcription control of a physiologically and pharmacologically inducible gene. PROCEDURE: Both in vivo and ex vivo experiments were performed to track bioluminescence signal attenuation through different stages of the mouse hair growth cycle. Simultaneous reflectance measurements were collected in vivo to estimate melanin levels. RESULTS: Biological variability of skin pigmentation was found to dramatically affect collected bioluminescent signal emerging through the skin of the mice. When compared to signal through skin with no pigmentation, the signal through highly-pigmented skin was attenuated an average of 90%. Correlation of reflectance signals to bioluminescence signal loss forms the basis of the proposed correction method. We observed, however, that variability in tissue composition, which results in inconsistent reflectance spectra, limits the accuracy of the correction method but can be improved by incorporating more complex analysis. CONCLUSION: Skin pigmentation is a significant variable in bioluminescent imaging, and should be considered in experimental design and implementation for longitudinal studies, and especially when sensitivity to small signal changes, or differences among animals, is required

Identification of molecular mechanisms for cellular drug resistance by combining drug activity and gene expression profiles

Acquired drug resistance is a major problem in cancer treatment. To explore the genes involved in chemosensitivity and resistance, 10 human tumour cell lines, including parental cells and resistant subtypes selected for resistance against doxorubicin, melphalan, teniposide and vincristine, were profiled for mRNA expression of 7400 genes using cDNA microarray technology. The drug activity of 66 cancer agents was evaluated on the cell lines, and correlations between drug activity and gene expression were calculated and ranked. Hierarchical clustering of drugs based on their drug–gene correlations yielded clusters of drugs with similar mechanism of action. Genes correlated with drug sensitivity and resistance were imported into the PathwayAssist software to identify putative molecular pathways involved. A substantial number of both proapoptotic and antiapoptotic genes such as signal transducer and activator of transcription 1, mitogen-activated protein kinase 1 and focal adhesion kinase were found to be associated to drug resistance, whereas genes linked to cell cycle control and proliferation, such as cell division cycle 25A and signal transducer of activator of transcription 5A, were associated to general drug sensitivity. The results indicate that combined information from drug activity and gene expression in a resistance-based cell line panel may provide new knowledge of the genes involved in anticancer drug resistance and become a useful tool in drug development

Comparison of dissolved and particulate arsenic distributions in shallow aquifers of Chakdaha, India, and Araihazar, Bangladesh

International audienceBackground The origin of the spatial variability of dissolved As concentrations in shallow aquifers of the Bengal Basin remains poorly understood. To address this, we compare here transects of simultaneously-collected groundwater and aquifer solids perpendicular to the banks of the Hooghly River in Chakdaha, India, and the Old Brahmaputra River in Araihazar, Bangladesh. Results Variations in surface geomorphology mapped by electromagnetic conductivity indicate that permeable sandy soils are associated with underlying aquifers that are moderately reducing to a depth of 10–30 m, as indicated by acid-leachable Fe(II)/Fe ratios 5 mg L-1. More reducing aquifers are typically capped with finer-grained soils. The patterns suggest that vertical recharge through permeable soils is associated with a flux of oxidants on the banks of the Hooghly River and, further inland, in both Chakdaha and Araihazar. Moderately reducing conditions maintained by local recharge are generally associated with low As concentrations in Araihazar, but not systematically so in Chakdaha. Unlike Araihazar, there is also little correspondence in Chakdaha between dissolved As concentrations in groundwater and the P-extractable As content of aquifer particles, averaging 191 ± 122 ug As/L, 1.1 ± 1.5 mg As kg-1 (n = 43) and 108 ± 31 ug As/L, 3.1 ± 6.5 mg As kg-1 (n = 60), respectively. We tentatively attribute these differences to a combination of younger floodplain sediments, and therefore possibly more than one mechanism of As release, as well as less reducing conditions in Chakdaha compared to Araihazar. Conclusion Systematic dating of groundwater and sediment, combined with detailed mapping of the composition of aquifer solids and groundwater, will be needed to identify the various mechanisms underlying the complex distribution of As in aquifers of the Bengal Basin

Altered Ultrasonic Vocalization and Impaired Learning and Memory in Angelman Syndrome Mouse Model with a Large Maternal Deletion from Ube3a to Gabrb3

Angelman syndrome (AS) is a neurobehavioral disorder associated with mental retardation, absence of language development, characteristic electroencephalography (EEG) abnormalities and epilepsy, happy disposition, movement or balance disorders, and autistic behaviors. The molecular defects underlying AS are heterogeneous, including large maternal deletions of chromosome 15q11–q13 (70%), paternal uniparental disomy (UPD) of chromosome 15 (5%), imprinting mutations (rare), and mutations in the E6-AP ubiquitin ligase gene UBE3A (15%). Although patients with UBE3A mutations have a wide spectrum of neurological phenotypes, their features are usually milder than AS patients with deletions of 15q11–q13. Using a chromosomal engineering strategy, we generated mutant mice with a 1.6-Mb chromosomal deletion from Ube3a to Gabrb3, which inactivated the Ube3a and Gabrb3 genes and deleted the Atp10a gene. Homozygous deletion mutant mice died in the perinatal period due to a cleft palate resulting from the null mutation in Gabrb3 gene. Mice with a maternal deletion (m−/p+) were viable and did not have any obvious developmental defects. Expression analysis of the maternal and paternal deletion mice confirmed that the Ube3a gene is maternally expressed in brain, and showed that the Atp10a and Gabrb3 genes are biallelically expressed in all brain sub-regions studied. Maternal (m−/p+), but not paternal (m+/p−), deletion mice had increased spontaneous seizure activity and abnormal EEG. Extensive behavioral analyses revealed significant impairment in motor function, learning and memory tasks, and anxiety-related measures assayed in the light-dark box in maternal deletion but not paternal deletion mice. Ultrasonic vocalization (USV) recording in newborns revealed that maternal deletion pups emitted significantly more USVs than wild-type littermates. The increased USV in maternal deletion mice suggests abnormal signaling behavior between mothers and pups that may reflect abnormal communication behaviors in human AS patients. Thus, mutant mice with a maternal deletion from Ube3a to Gabrb3 provide an AS mouse model that is molecularly more similar to the contiguous gene deletion form of AS in humans than mice with Ube3a mutation alone. These mice will be valuable for future comparative studies to mice with maternal deficiency of Ube3a alone

Renal Failure Affects the Enzymatic Activities of the Three First Steps in Hepatic Heme Biosynthesis in the Acute Intermittent Porphyria Mouse

Chronic kidney disease is a long-term complication in acute intermittent porphyria (AIP). The pathophysiological significance of hepatic overproduction of the porphyrin precursors aminolevulinate acid (ALA) and porphobilinogen (PBG) in chronic kidney disease is unclear. We have investigated the effect of repetitive acute attacks on renal function and the effect of total or five-sixth nephrectomy causing renal insufficiency on hepatic heme synthesis in the porphobilinogen deaminase (PBGD)-deficient (AIP) mouse. Phenobarbital challenge in the AIP-mice increased urinary porphyrin precursor excretion. Successive attacks throughout 14 weeks led to minor renal lesions with no impact on renal function. In the liver of wild type and AIP mice, 5/6 nephrectomy enhanced transcription of the first and rate-limiting ALA synthase. As a consequence, urinary PBG excretion increased in AIP mice. The PBG/ALA ratio increased from 1 in sham operated AIP animals to over 5 (males) and over 13 (females) in the 5/6 nephrectomized mice. Total nephrectomy caused a rapid decrease in PBGD activity without changes in enzyme protein level in the AIP mice but not in the wild type animals. In conclusion, high concentration of porphyrin precursors had little impact on renal function. However, progressive renal insufficiency aggravates porphyria attacks and increases the PBG/ALA ratio, which should be considered a warning sign for potentially life-threatening impairment in AIP patients with signs of renal failure

Immaturity of the Oculomotor Saccade and Vergence Interaction in Dyslexic Children: Evidence from a Reading and Visual Search Study

Studies comparing binocular eye movements during reading and visual search in dyslexic children are, at our knowledge, inexistent. In the present study we examined ocular motor characteristics in dyslexic children versus two groups of non dyslexic children with chronological/reading age-matched. Binocular eye movements were recorded by an infrared system (mobileEBT®, e(ye)BRAIN) in twelve dyslexic children (mean age 11 years old) and a group of chronological age-matched (N = 9) and reading age-matched (N = 10) non dyslexic children. Two visual tasks were used: text reading and visual search. Independently of the task, the ocular motor behavior in dyslexic children is similar to those reported in reading age-matched non dyslexic children: many and longer fixations as well as poor quality of binocular coordination during and after the saccades. In contrast, chronological age-matched non dyslexic children showed a small number of fixations and short duration of fixations in reading task with respect to visual search task; furthermore their saccades were well yoked in both tasks. The atypical eye movement's patterns observed in dyslexic children suggest a deficiency in the visual attentional processing as well as an immaturity of the ocular motor saccade and vergence systems interaction

Elevated Peripheral Neutrophils and Matrix Metalloproteinase 9 as Biomarkers of Functional Outcome Following Subarachnoid Hemorrhage

There is growing evidence supporting the role of inflammation in early brain injury and cerebral vasospasm following subarachnoid hemorrhage (SAH). Matrix metalloproteinases (MMPs) are released by inflammatory cells and can mediate early brain injury via disruption of the extracellular matrix and mediate vasospasm by cleaving endothelin-1 into vasoactive fragments. We hypothesize that inflammation marked by neutrophil elevation and MMP-9 release in human SAH is associated with vasospasm and with poor clinical outcome. We enrolled consecutive SAH subjects (N = 55), banked serial blood and cerebrospinal fluid (CSF) samples, and evaluated their 3-month modified Rankin scores (mRS). Vasospasm was defined as >50% vessel caliber reduction on angiography 6–8 days post-SAH. A poor outcome was defined as mRS > 2. We compared blood leukocyte and neutrophil counts during post-SAH days 0–14 with respect to vasospasm and 3-month outcome. In a subset of SAH subjects (N = 35), we compared blood and CSF MMP-9 by enzyme-linked immunosorbent assay (ELISA) on post-SAH days 0–1, 2–3, 4–5, 6–8, and 10–14 with respect to vasospasm and to 3-month outcome. Persistent elevation of blood leukocyte (p = 0.0003) and neutrophil (p = 0.0002) counts during post-SAH days 0–14 are independently associated with vasospasm after adjustment for major confounders. In the same time period, blood neutrophil count (post-SAH days 2–3, p = 0.018), blood MMP-9 (post-SAH days 4–5, p = 0.045), and CSF MMP-9 (post-SAH days 2–3, p = 0.05) are associated with poor 3-month SAH clinical outcome. Neutrophil count correlates with blood MMP-9 (post-SAH days 6–8, R = 0.39; p = 0.055; post-SAH days 10–14, R = 0.79; p < 0.0001), and blood MMP-9 correlates with CSF MMP-9 (post-SAH days 4–5, R = 0.72; p = 0.0002). Elevation of CSF MMP-9 during post-SAH days 0–14 is associated with poor 3-month outcome (p = 0.0078). Neither CSF nor blood MMP-9 correlates with vasospasm. Early rise in blood neutrophil count and blood and CSF MMP-9 are associated with poor 3-month SAH clinical outcome. In blood, neutrophil count correlates with MMP-9 levels, suggesting that neutrophils may be an important source of blood MMP-9 early in SAH. Similarly, CSF and blood MMP-9 correlate positively early in the course of SAH, suggesting that blood may be an important source of CSF MMP-9. Blood and CSF MMP-9 are associated with clinical outcome but not with vasospasm, suggesting that MMP-9 may mediate brain injury independent of vasospasm in SAH. Future in vitro studies are needed to investigate the role of MMP-9 in SAH-related brain injury. Larger clinical studies are needed to validate blood and CSF MMP-9 as potential biomarkers for SAH outcome

Retardation of arsenic transport through a Pleistocene aquifer

Groundwater drawn daily from shallow alluvial sands by millions of wells over large areas of south and southeast Asia exposes an estimated population of over a hundred million people to toxic levels of arsenic1. Holocene aquifers are the source of widespread arsenic poisoning across the region2, 3. In contrast, Pleistocene sands deposited in this region more than 12,000 years ago mostly do not host groundwater with high levels of arsenic. Pleistocene aquifers are increasingly used as a safe source of drinking water4 and it is therefore important to understand under what conditions low levels of arsenic can be maintained. Here we reconstruct the initial phase of contamination of a Pleistocene aquifer near Hanoi, Vietnam. We demonstrate that changes in groundwater flow conditions and the redox state of the aquifer sands induced by groundwater pumping caused the lateral intrusion of arsenic contamination more than 120 metres from a Holocene aquifer into a previously uncontaminated Pleistocene aquifer. We also find that arsenic adsorbs onto the aquifer sands and that there is a 16–20-fold retardation in the extent of the contamination relative to the reconstructed lateral movement of groundwater over the same period. Our findings suggest that arsenic contamination of Pleistocene aquifers in south and southeast Asia as a consequence of increasing levels of groundwater pumping may have been delayed by the retardation of arsenic transport.National Science Foundation (U.S.) (NSF grant EAR09-11557)Swiss Agency for Development and Cooperation (Grant NAFOSTED 105-09-59-09 to CETASD, the Centre for Environmental Technology and Sustainable Development (Vietnam))National Institute of Environmental Health Sciences (NIEHS grant P42 ES010349)National Institute of Environmental Health Sciences (NIEHS grant P42 ES016454