Endosomal egress and intercellular transmission of hepatic ApoE-containing lipoproteins and its exploitation by the hepatitis C virus.

Liver-generated plasma Apolipoprotein E (ApoE)-containing lipoproteins (LPs) (ApoE-LPs) play central roles in lipid transport and metabolism. Perturbations of ApoE can result in several metabolic disorders and ApoE genotypes have been associated with multiple diseases. ApoE is synthesized at the endoplasmic reticulum and transported to the Golgi apparatus for LP assembly; however, the ApoE-LPs transport pathway from there to the plasma membrane is largely unknown. Here, we established an integrative imaging approach based on a fully functional fluorescently tagged ApoE. We found that newly synthesized ApoE-LPs accumulate in CD63-positive endosomes of hepatocytes. In addition, we observed the co-egress of ApoE-LPs and CD63-positive intraluminal vesicles (ILVs), which are precursors of extracellular vesicles (EVs), along the late endosomal trafficking route in a microtubule-dependent manner. A fraction of ApoE-LPs associated with CD63-positive EVs appears to be co-transmitted from cell to cell. Given the important role of ApoE in viral infections, we employed as well-studied model the hepatitis C virus (HCV) and found that the viral replicase component nonstructural protein 5A (NS5A) is enriched in ApoE-containing ILVs. Interaction between NS5A and ApoE is required for the efficient release of ILVs containing HCV RNA. These vesicles are transported along the endosomal ApoE egress pathway. Taken together, our data argue for endosomal egress and transmission of hepatic ApoE-LPs, a pathway that is hijacked by HCV. Given the more general role of EV-mediated cell-to-cell communication, these insights provide new starting points for research into the pathophysiology of ApoE-related metabolic and infection-related disorders.

Characterization of a multipurpose NS3 surface patch coordinating HCV replicase assembly and virion morphogenesis.

The hepatitis C virus (HCV) life cycle is highly regulated and characterized by a step-wise succession of interactions between viral and host cell proteins resulting in the assembly of macromolecular complexes, which catalyse genome replication and/or virus production. Non-structural (NS) protein 3, comprising a protease and a helicase domain, is involved in orchestrating these processes by undergoing protein interactions in a temporal fashion. Recently, we identified a multifunctional NS3 protease surface patch promoting pivotal protein-protein interactions required for early steps of the HCV life cycle, including NS3-mediated NS2 protease activation and interactions required for replicase assembly. In this work, we extend this knowledge by identifying further NS3 surface determinants important for NS5A hyperphosphorylation, replicase assembly or virion morphogenesis, which map to protease and helicase domain and form a contiguous NS3 surface area. Functional interrogation led to the identification of phylogenetically conserved amino acid positions exerting a critical function in virion production without affecting RNA replication. These findings illustrate that NS3 uses a multipurpose protein surface to orchestrate the step-wise assembly of functionally distinct multiprotein complexes. Taken together, our data provide a basis to dissect the temporal formation of viral multiprotein complexes required for the individual steps of the HCV life cycle.

Hepatitis B Virus Targets Lipid Transport Pathways to Infect Hepatocytes.

BACKGROUND & AIMS: A single hepatitis B virus (HBV) particle is sufficient to establish chronic infection of the liver after intravenous injection, suggesting that the virus targets hepatocytes via a highly efficient transport pathway. We therefore investigated whether HBV uses a physiological liver-directed pathway that supports specific host-cell targeting in vivo. METHODS: We established the ex vivo perfusion of intact human liver tissue that recapitulates the liver physiology to investigate HBV liver targeting. This model allowed us to investigate virus-host cell interactions in a cellular microenvironment mimicking the in vivo situation. RESULTS: HBV was rapidly sequestered by liver macrophages within 1 hour after a virus pulse perfusion but was detected in hepatocytes only after 16 hours. We found that HBV associates with lipoproteins in serum and within machrophages. Electron and immunofluorescence microscopy corroborated a co-localization in recycling endosomes within peripheral and liver macrophages. Recycling endosomes accumulated HBV and cholesterol, followed by transport of HBV back to the cell surface along the cholesterol efflux pathway. To reach hepatocytes as final target cells, HBV was able to utilize the hepatocyte-directed cholesterol transport machinery of macrophages. CONCLUSIONS: Our results propose that by binding to liver targeted lipoproteins and using the reverse cholesterol transport pathway of macrophages, HBV hijacks the physiological lipid transport pathways to the liver to most efficiently reach its target organ. This may involve transinfection of liver macrophages and result in deposition of HBV in the perisinusoidal space from where HBV can bind its receptor on hepatocytes.

Convergent use of phosphatidic acid for hepatitis C virus and SARS-CoV-2 replication organelle formation.

Double membrane vesicles (DMVs) serve as replication organelles of plus-strand RNA viruses such as hepatitis C virus (HCV) and SARS-CoV-2. Viral DMVs are morphologically analogous to DMVs formed during autophagy, but lipids driving their biogenesis are largely unknown. Here we show that production of the lipid phosphatidic acid (PA) by acylglycerolphosphate acyltransferase (AGPAT) 1 and 2 in the ER is important for DMV biogenesis in viral replication and autophagy. Using DMVs in HCV-replicating cells as model, we found that AGPATs are recruited to and critically contribute to HCV and SARS-CoV-2 replication and proper DMV formation. An intracellular PA sensor accumulated at viral DMV formation sites, consistent with elevated levels of PA in fractions of purified DMVs analyzed by lipidomics. Apart from AGPATs, PA is generated by alternative pathways and their pharmacological inhibition also impaired HCV and SARS-CoV-2 replication as well as formation of autophagosome-like DMVs. These data identify PA as host cell lipid involved in proper replication organelle formation by HCV and SARS-CoV-2, two phylogenetically disparate viruses causing very different diseases, i.e. chronic liver disease and COVID-19, respectively. Host-targeting therapy aiming at PA synthesis pathways might be suitable to attenuate replication of these viruses.

Online proprioception feeds plasticity of arm representation following tool-use in healthy aging.

Following tool-use, the kinematics of free-hand movements are altered. This modified kinematic pattern has been taken as a behavioral hallmark of the modification induced by tool-use on the effector representation. Proprioceptive inputs appear central in updating the estimated effector state. Here we questioned whether online proprioceptive modality that is accessed in real time, or offline, memory-based, proprioception is responsible for this update. Since normal aging affects offline proprioception only, we examined a group of 60 year-old adults for proprioceptive acuity and movement's kinematics when grasping an object before and after tool-use. As a control, participants performed the same movements with a weight-equivalent to the tool-weight-attached to their wrist. Despite hampered offline proprioceptive acuity, 60 year-old participants exhibited the typical kinematic signature of tool incorporation: Namely, the latency of transport components peaks was longer and their amplitude reduced after tool-use. Instead, we observed no kinematic modifications in the control condition. In addition, online proprioception acuity correlated with tool incorporation, as indexed by the amount of kinematics changes observed after tool-use. Altogether, these findings point to the prominent role played by online proprioception in updating the body estimate for the motor control of tools.

Gesture Classification Using LSTM Recurrent Neural Networks.

The classification of human hand gestures has gained widespread recognition as a natural and powerful way to interact intuitively and efficiently with computers. Specifically, this approach has facilitated the development of a number of important applications in the medical training field, specially as a way to objectively evaluate surgical tasks of novices compared to an expert surgeon. In this paper, 3D medical gestures, acquired by an instrumented laparoscopic forceps, were classified using Long Short Term Memory (LSTM) recurrent neural networks (RNN). Recognition results are based on gesture dynamics and a comparison of gesture trajectories between novices to an expert motion are presented. Using LSTM RNN, we were able to achieve a recognition rate of 99.1%.

Gesture segmentation and classification using affine speed and energy.

The characterization and analysis of hand gestures are challenging tasks with an important number of applications in human-computer interaction, machine vision and control, and medical gesture recognition. Specifically, several researchers have tried to develop objective evaluation methods of surgical skills for medical training. As a result, the adequate selection and extraction of similarities and differences between experts and novices have become an important challenge in this area. In particular, some of these works have shown that human movements performed during surgery can be described as a sequence of constant affine-speed trajectories. In this article, we will show that affine speed can be used to segment medical hand movements and present how the mechanical energy computed in the segment is analyzed to compare surgical skills. The position and orientation of the instrument end effectors are determined by six video photographic cameras. In addition, two laparoscopic instruments are capable of measuring simultaneously the forces and torques applied to the tool. Finally, we will report the results of these experiments and present a correlation between the mechanical energy values, dissipated during a procedure, and the surgical skills.

Design of a new instrumented forceps: application to safe obstetrical forceps blade placement.

Today, medical simulators are increasingly gaining appeal in clinical settings. In obstetrics childbirth simulators provide a training and research tool for comparing various techniques that use obstetrical instruments or validating new methods. Especially in the case of difficult deliveries, the use of obstetrical instruments-such as forceps, spatulas, and vacuum extractors-has become essential. However, such instruments increase the risk of injury to both the mother and fetus. Only clinical experience acquired in the delivery room enables health professionals to reduce this risk. In this context, we have developed, in collaboration with researchers and physicians, a new type of instrumented forceps that offers new solutions for training obstetricians in the safe performance of forceps deliveries. This paper focuses on the design of this instrumented forceps, coupled with the BirthSIM simulator. This instrumented forceps allows to study its displacement inside the maternal pelvis. Methods for analyzing the operator repeatability and to compare forceps blade placements to a reference one are developed. The results highlight the need of teaching tools to adequately train novice obstetricians.

A new obstetric forceps for the training of junior doctors: a comparison of the spatial dispersion of forceps blade trajectories between junior and senior obstetricians.

OBJECTIVE: The purpose of this study was to create a new instrument for the training of doctors in the use of forceps and to compare the trajectories of forceps blades between junior and senior obstetricians. STUDY DESIGN: We equipped a simulator and forceps with spatial location sensors. The head of the fetus was in an occipitoanterior location, at a "+5" station. Forceps blade trajectories were analyzed subjectively with the 3-dimensional spatial graph and objectively based on 3 points of special interest. Each obstetrician performed 4 forceps blades placements. We compared the trajectories of junior and senior obstetricians. RESULTS: For senior operators, spatial dispersion was "excellent," "very good," or "good" in 92% of cases, whereas this was the case for only 38% of junior doctors (92% vs 38%; P < .001). CONCLUSION: A new instrument has been designed to demonstrate the trajectory of forceps blades during application in a simulator. The instrument captures the difference in experience between senior and junior clinicians.

Automatic gesture analysis using constant affine velocity.

Hand human gesture recognition has been an important research topic widely studied around the world, as this field offers the ability to identify, recognize, and analyze human gestures in order to control devices or to interact with computer interfaces. In particular, in medical training, this approach is an important tool that can be used to obtain an objective evaluation of a procedure performance. In this paper, some obstetrical gestures, acquired by a forceps, were studied with the hypothesis that, as the scribbling and drawing movements, they obey the one-sixth power law, an empirical relationship which connects path curvature, torsion, and euclidean velocity. Our results show that obstetrical gestures have a constant affine velocity, which is different for each type of gesture and based on this idea this quantity is proposed as an appropriate classification feature in the hand human gesture recognition field.

An arc-length warping algorithm for gesture recognition using quaternion representation.

This paper presents a new algorithm, called Dynamic Arc-Length Warping algorithm (DALW) for hand gesture recognition based on the orientation data. In this algorithm, after calculating the quaternion for each orientation measurement, we use DALW algorithm to obtain a similarity measure between different trajectories. We present the benefits of using quaternion alongside the implementation of Dynamic Arc Length Warping to present an optimized tool for gesture recognition.We show the advantages of this approach compared with other techniques. This tool can be used to distinguish similar and different gestures. An experimental validation is carried out to classify a series of simple human gestures.

Simulation of an instrumental childbirth for the training of the forceps extraction: control algorithm and evaluation.

This paper presents the control algorithm implanted on the childbirth simulator BirthSIM in order to provide training to novice obstetricians. The forceps extraction is an obstetric manipulation learned by experience. However, nowadays the training is mainly provided during real childbirths. This kind of training could lead to dramatic consequences due to the lack of experience of some operators. This paper explains the approach that has been used to simulate the dynamic process of a childbirth on the BirthSIM simulator. We especially focus on one procedure that reproduces a difficult instrumental delivery. The recorded tractive force to extract the fetus corresponds to the literature results that confirm the realism of the simulator. The novice results emphasize the need of a childbirth simulator in order to gain initial experience without any risks.

Does forceps training on a birth simulator allow obstetricians to improve forceps blade placement?

OBJECTIVE: The aim of this study was to evaluate whether forceps training on a birth simulator allows obstetricians to improve forceps blade placement. STUDY DESIGN: Analysis was based on 600 forceps blade placements performed by ten trainees on a simulator. The trajectories used by the trainees were assessed using reference spheres that reflected an optimal bimalar placement. Three definitions of success were used: small-sphere success, medium-sphere success and large-sphere success were respectively defined by the forceps blade tip being within 5, 10 or 15mm of the center of the sphere (the small-sphere being nested within the medium-sphere and the small and medium being nested within the large-sphere). Wilcoxon paired analysis was performed to compare the first (50 trajectories) and final (50 trajectories) sets of five forceps placements. Graphical representation and linear regression were used to visualize the learning process. RESULTS: 596 trajectories were available for analysis. During the last set of five forceps the success rate was respectively 28%, 72% and 86% for small-sphere, medium-sphere and large-sphere success with the right blade and 8%, 32% and 70% for the left blade. Wilcoxon analysis showed a highly significant improvement for all kinds of success in the right blade and for large-sphere success in the left blade. Linear regression slopes were significant. Using a projection, the theoretical numbers of placements needed to achieve a 100% success rate for small-sphere, medium-sphere and large-sphere were respectively 80, 45 and 35. CONCLUSION: These results strongly suggest that performing forceps blade placement on birth simulator allows obstetricians to improve their skills.

A new kind of training for obstetric residents: simulator training.

This paper presents work resulting from a collaboration between obstetricians and researchers. It shows the benefits from the use of an instrumented childbirth simulator for the training of obstetricians and midwives. This new tool allows to surpass the constraints linked to the traditional training in a childbirth ward. This simulator training is designed to complete the traditional training used in teaching hospitals. Such a training allows residents to acquire a beginning experience before training in a childbirth ward but it also allows instructors to improve the teaching gestures without constraints. A clinical study of the forceps blades placement gesture with several residents who trained on a childbirth simulator is made. The results clearly show the progress in the obstetric gestures of all the residents who have used the simulator.