Highway traffic monitoring on medium resolution satellite images

International audienceThese last years, earth observation imagery has significantly improved. Public satellites such as WorldView-3 can now produce images with a Ground Sample Distance of 31cm, reaching an equivalent resolution than aerial images. Perhaps more importantly, the revisit frequency has also been greatly enhanced: providers such as Planet can now acquire images of an area on a daily basis. These major improvements are fueled by an increasing demand for frequent objects detection. An application generating a particular interest is vehicle detection. Indeed, vehicle detection can give to public and private actors valuable data such as traffic monitoring and parking occupancy rate estimations. Several datasets, such as DOTA or VehSat, already exist, allowing researchers to train machine learning algorithms to detect vehicles. However, these datasets focus on relatively high definition and expensive aerial and satellite images. In this paper, we will present a method for detecting vehicles on medium resolution satellite images, with a GSD comprised between 1 and 5 meters. This approach can notably be used on Planet images, allowing to monitor traffic of an area on a daily basis

Novel human liver-tropic AAV variants define transferable domains that markedly enhance the human tropism of AAV7 and AAV8

Recent clinical successes have intensified interest in using adeno-associated virus (AAV) vectors for therapeutic gene delivery. The liver is a key clinical target, given its critical physiological functions and involvement in a wide range of genetic diseases. Here, we report the bioengineering of a set of next-generation AAV vectors, named AAV-SYDs (where “SYD” stands for Sydney, Australia), with increased human hepato-tropism in a liver xenograft mouse model repopulated with primary human hepatocytes. We followed a two-step process that staggered directed evolution and domain-swapping approaches. Using DNA-family shuffling, we first mapped key AAV capsid regions responsible for efficient human hepatocyte transduction in vivo. Focusing on these regions, we next applied domain-swapping strategies to identify and study key capsid residues that enhance primary human hepatocyte uptake and transgene expression. Our findings underscore the potential of AAV-SYDs as liver gene therapy vectors and provide insights into the mechanism responsible for their enhanced transduction profile

GTP binding regulates cellular localization of Parkinso\u144s disease-associated LRRK2

Mutations in LRRK2 comprise the most common cause of familial Parkinso\u144s disease (PD), and sequence variants modify risk for sporadic PD. Previous studies indicate that LRRK2 interacts with microtubules and alters microtubule-mediated vesicular transport processes. However, the molecular determinants within LRRK2 required for such interactions have remained unknown. Here we report that most pathogenic LRRK2 mutants cause relocalization of LRRK2 to filamentous structures which colocalize with a subset of microtubules, and an identical relocalization is seen upon pharmacological LRRK2 kinase inhibition. The pronounced colocalization with microtubules does not correlate with alterations in LRRK2 kinase activity, but rather with increased GTP binding. Synthetic mutations which impair GTP binding, as well as LRRK2 GTP-binding inhibitors profoundly interfere with the abnormal localization of both pathogenic mutant as well as kinase-inhibited LRRK2. Conversely, addition of a non-hydrolyzable GTP analog to permeabilized cells enhances the association of pathogenic or kinase-inhibited LRRK2 with microtubules. Our data elucidate the mechanism underlying the increased microtubule association of select pathogenic LRRK2 mutants or of pharmacologically kinase-inhibited LRRK2, with implications for downstream MT-mediated transport events

Attenuation of Heparan Sulfate Proteoglycan Binding Enhances In Vivo Transduction of Human Primary Hepatocytes with AAV2

Use of the prototypical adeno-associated virus type 2 (AAV2) capsid delivered unexpectedly modest efficacy in an early liver-targeted gene therapy trial for hemophilia B. This result is consistent with subsequent data generated in chimeric mouse-human livers showing that the AAV2 capsid transduces primary human hepatocytes in vivo with low efficiency. In contrast, novel variants generated by directed evolution in the same model, such as AAV-NP59, transduce primary human hepatocytes with high efficiency. While these empirical data have immense translational implications, the mechanisms underpinning this enhanced AAV capsid transduction performance in primary human hepatocytes are yet to be fully elucidated. Remarkably, AAV-NP59 differs from the prototypical AAV2 capsid by only 11 aa and can serve as a tool to study the correlation between capsid sequence/structure and vector function. Using two orthogonal vectorological approaches, we have determined that just 2 of the 11 changes present in AAV-NP59 (T503A and N596D) account for the enhanced transduction performance of this capsid variant in primary human hepatocytes in vivo, an effect that we have associated with attenuation of heparan sulfate proteoglycan (HSPG) binding affinity. In support of this hypothesis, we have identified, using directed evolution, two additional single amino acid substitution AAV2 variants, N496D and N582S, which are highly functional in vivo. Both substitution mutations reduce AAV2's affinity for HSPG. Finally, we have modulated the ability of AAV8, a highly murine-hepatotropic serotype, to interact with HSPG. The results support our hypothesis that enhanced HSPG binding can negatively affect the in vivo function of otherwise strongly hepatotropic variants and that modulation of the interaction with HSPG is critical to ensure maximum efficiency in vivo. The insights gained through this study can have powerful implications for studies into AAV biology and capsid development for preclinical and clinical applications targeting liver and other organs

High-Throughput In Vitro, Ex Vivo, and In Vivo Screen of Adeno-Associated Virus Vectors Based on Physical and Functional Transduction

Adeno-associated virus (AAV) vectors are quickly becoming the vectors of choice for therapeutic gene delivery. To date, hundreds of natural isolates and bioengineered variants have been reported. While factors such as high production titer and low immunoreactivity are important to consider, the ability to deliver the genetic payload (physical transduction) and to drive high transgene expression (functional transduction) remains the most important feature when selecting AAV variants for clinical applications. Reporter expression assays are the most commonly used methods for determining vector fitness. However, such approaches are time consuming and become impractical when evaluating a large number of variants. Limited access to primary human tissues or challenging model systems further complicates vector testing. To address this problem, convenient high-throughput methods based on next-generation sequencing (NGS) are being developed. To this end, we built an AAV Testing Kit that allows inherent flexibility in regard to number and type of AAV variants included, and is compatible with in vitro, ex vivo, and in vivo applications. The Testing Kit presented here consists of a mix of 30 known AAVs where each variant encodes a CMV-eGFP cassette and a unique barcode in the 3′-untranslated region of the eGFP gene, allowing NGS-barcode analysis at both the DNA and RNA/cDNA levels. To validate the AAV Testing Kit, individually packaged barcoded variants were mixed at an equal ratio and used to transduce cells/tissues of interest. DNA and RNA/cDNA were extracted and subsequently analyzed by NGS to determine the physical/functional transduction efficiencies. We were able to assess the transduction efficiencies of immortalized cells, primary cells, and induced pluripotent stem cells in vitro, as well as in vivo transduction in naïve mice and a xenograft liver model. Importantly, while our data validated previously reported transduction characteristics of individual capsids, we also identified novel previously unknown tropisms for some AAV variants

The rat suprachiasmatic nucleus: the master clock ticks at 30Hz.

KEY POINTS: Light‐responsive neurones in the rat suprachiasmatic nucleus discharge with a harmonic distribution of interspike intervals, whereas unresponsive neurones seldom do. This harmonic patterning has a fundamental frequency of close to 30 Hz, and is the same in light‐on cells as in light‐off cells, and is unaffected by exposure to light. Light‐on cells are more active than light‐off cells in both subjective day and subjective night, and both light‐on cells and light‐off cells respond more strongly to changes in light intensity during the subjective night than during the subjective day. Paired recordings indicate that the discharge of adjacent light‐responsive cells is very tightly synchronized. The gap junction inhibitor carbenoxolone increases the spontaneous activity of suprachiasmatic nucleus neurones but does not block the harmonic discharge patterning. ABSTRACT: The suprachiasmatic nucleus (SCN) of the hypothalamus has an essential role in orchestrating circadian rhythms of behaviour and physiology. In the present study, we recorded from single SCN neurons in urethane‐anaesthetized rats, categorized them by the statistical features of their electrical activity and by their responses to light, and examined how activity in the light phase differs from activity in the dark phase. We classified cells as light‐on cells or light‐off cells according to how their firing rate changed in acute response to light, or as non‐responsive cells. In both sets of light‐responsive neurons, responses to light were stronger at subjective night than in subjective day. Neuronal firing patterns were analysed by constructing hazard functions from interspike interval data. For most light‐responsive cells, the hazard functions showed a multimodal distribution, with a harmonic sequence of modes, indicating that spike activity was driven by an oscillatory input with a fundamental frequency of close to 30 Hz; this harmonic pattern was rarely seen in non‐responsive SCN cells. The frequency of the rhythm was the same in light‐on cells as in light‐off cells, was the same in subjective day as at subjective night, and was unaffected by exposure to light. Paired recordings indicated that the discharge of adjacent light‐responsive neurons was very tightly synchronized, consistent with electrical coupling

Changes in the Circadian Rhythm in Patients with Primary Glaucoma

Purpose The current study was undertaken to investigate whether glaucoma affects the sleep quality and whether there is any difference between patients with primary glaucoma (primary open angle glaucoma, POAG and primary angle-closure glaucoma, PACG) and healthy subjects, using a validated self-rated questionnaire, the Pittsburgh Sleep Quality Index (PSQI). Methods The sleep quality of patients with POAG and PACG was tested against normal controls. Subjects were divided into three sub-groups according to age. Differences in the frequency of sleep disturbances (PSQI score >7) were assessed. The differences of sleep quality within the three groups and within the POAG group depending on the patients’ intraocular pressure (IOP) and impairment of visual field (VF) were also studied. Results 92 POAG patients, 48 PACG patients and 199 controls were included. Sleep quality declined with age in control and POAG group (tendency chi-square, P0.05). No significant differences were found in POAG group between patients with a highest IOP in daytime and at nighttime (χ2-test, P>0.05). Conclusions The prevalence of sleep disorders was higher in patients with POAG and PACG than in controls. PACG patients seemed to have a more serious problem of sleep disorders than POAG patients between 61 to 80 years old. No correlation was found between the prevalence of sleep disorders and impairment of VF or the time when POAG patients showed a highest IOP

PAK6-mediated phosphorylation of PPP2R2C regulates LRRK2-PP2A complex formation

Mutations in leucine-rich repeat kinase 2 (LRRK2) are a common cause of inherited and sporadic Parkinson’s disease (PD) and previous work suggests that dephosphorylation of LRRK2 at a cluster of heterologous phosphosites is associated to disease. We have previously reported subunits of the PP1 and PP2A classes of phosphatases as well as the PAK6 kinase as regulators of LRRK2 dephosphorylation. We therefore hypothesized that PAK6 may have a functional link with LRRK2’s phosphatases. To investigate this, we used PhosTag gel electrophoresis with purified proteins and found that PAK6 phosphorylates the PP2A regulatory subunit PPP2R2C at position S381. While S381 phosphorylation did not affect PP2A holoenzyme formation, a S381A phosphodead PPP2R2C showed impaired binding to LRRK2. Also, PAK6 kinase activity changed PPP2R2C subcellular localization in a S381 phosphorylation-dependent manner. Finally, PAK6-mediated dephosphorylation of LRRK2 was unaffected by phosphorylation of PPP2R2C at S381, suggesting that the previously reported mechanism whereby PAK6-mediated phosphorylation of 14-3-3 proteins promotes 14-3-3-LRRK2 complex dissociation and consequent exposure of LRRK2 phosphosites for dephosphorylation is dominant. Taken together, we conclude that PAK6-mediated phosphorylation of PPP2R2C influences the recruitment of PPP2R2C to the LRRK2 complex and PPP2R2C subcellular localization, pointing to an additional mechanism in the fine-tuning of LRRK2 phosphorylation