An antisense RNA-mediated mechanism eliminates a meiosis-specific copper -regulated transcript in mitotic cells

Significance Statement

Eukaryotic cell division undergoes either mitosis or meiosis.  In the case of meiosis, this process allows diploid precursor cells to produce haploid gametes, which are required for sexual reproduction.  Because several specialized proteins are involved in meiosis-specific mechanisms such as meiotic homologous recombination, meiotic nuclear divisions, and forespore morphogenesis, their presence is detrimental to cells proliferating in mitosis.  Cells have therefore developed different mechanisms to prevent or eliminate meiosis-specific transcripts during mitosis.  One mechanism of mRNA elimination involves genes that are organized into convergent pairs.  In this case two genes are in proximity of one another with their transcription orientated one toward the other.  We have recently discovered that Schizosaccharomyces pombe iss1⁺ and cum1⁺ genes are arranged with their transcription orientated convergently.  The iss1⁺ gene produces two transcript isoforms, including a long antisense mRNA that is complementary to the meiotic cum1⁺ sense transcript.  When the production of the long iss1⁺ antisense transcript was disrupted by insertion of a polyadenylation signal immediately downstream of iss1⁺, results showed high levels of cum1⁺ sense mRNA during mitosis, which were copper starvation- and Cuf1-dependent.  This finding suggested that inhibition of expression of cum1⁺ sense mRNA occurs due to the presence of the long iss1⁺ antisense RNA.  In fission yeast, sense/antisense RNA duplexes accumulate in G1 phase of the cell cycle, especially in regions where convergent genes are present.  In G1, transcription of several convergent genes fails to terminate after their proximal cleavage and polyadenylation sites, thereby resulting in a transcriptional read-through that produces long antisense/sense transcripts.  Accumulation of long antisense/sense RNA duplexes activates the RNAi pathway, which leads to gene silencing and heterochromatin formation over convergent gene regions.  To assess involvement of the RNAi machinery in silencing cum1⁺ transcription, we probed cum1⁺ mRNA levels in four mutants that affect the RNAi pathway.  These mutants were dcr1D (dicer), ago1D (argonaute), rdp1D (RNA-dependent RNA polymerase) and clr4D (histone methyltransferase).  Using G1-synchronized wild-type and dcr1D, ago1D, rpd1D, and clr4D mutant cells, we determined that components of the RNAi pathway are involved in antisense-mediated repression of cum1⁺ transcription, especially during the G1 phase of the cell cycle.  In this study, we have uncovered a mechanism by which a copper- and meiosis-specific transcript is selectively inhibited in growing cells, preventing its untimely expression during mitosis.  To our knowledge, this is the first report to show an antisense transcription control of a copper-regulated transcript under physiological low copper conditions. 

About The Author

Vincent Normant obtained his bachelor’s degree (2012) in Biology and Immunology from the Université de Nantes in France.  He continued his training as a Master I and Master II student in the program of Master Biologie Santé at the Université de Nantes in France.  He then joined the laboratory of Dr. S. Labbé at the Department of Biochemistry at the Université de Sherbrooke (Canada) as a doctoral student in 2013.  His research focuses on metal-dependent mechanisms of meiotic gene regulation in the fission yeast Schizosaccharomyces pombe.  

About The Author

Dr. Simon Labbé is a Professor of Biochemistry in the Faculty of Medicine and Health Sciences at the Université de Sherbrooke in Québec, Canada.  He received his bachelor’s degree in Microbiology from the Université Laval (Québec) in 1987.  He undertook graduate studies at Laval Université and received his Master (1989) and Ph.D. (1995) in Microbiology and Molecular-cellular biology, respectively.  Dr. Labbé continued his training as a post-doctoral fellow with Dr. Dennis J. Thiele at the Department of Biological Chemistry at the University of Michigan in Ann Arbor, MI, USA.  He was supported by Fellowship awards, including the prestigious Centennial Fellowship from the Medical Research Council of Canada.  Over the past sixteen years he has been at the Université de Sherbrooke where he has established his own laboratory.  As an independent investigator, he has received the New Investigator Award from the Canadian Institutes of Health Research, the Junior II and Senior Investigator Awards from the Fonds de la Recherche en Santé du Québec.  His research group has developed a productive yeast model (Schizosaccharomyces pombe) for investigating copper and iron metabolism at the molecular level.  Because S. pombe provides a genetically tractable model that permits the drawing of parallels with other eukaryotic living systems, especially filamentous yeasts, Dr. Labbé’s research has contributed to the identification of molecular mechanisms that regulate copper and iron transport, as well as uncovering different strategies that yeasts have acquired to take up copper and iron from their environment and/or hosts.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure Legend: Illustration of a proposed model for elimination of cum1⁺ meiotic transcripts in mitotic cells.

Journal Reference

J Biol Chem. 2015;290(37):22622-37.

Normant V¹, Beaudoin J¹, Labbé S².

Show Affiliations

1. Département de Biochimie, Faculté de médecine et des sciences de la santé, Université de Sherbrooke, Sherbrooke, QC, J1E 4K8, Canada.
2. Département de Biochimie, Faculté de Médecine et des Sciences de la Santé, Université de Sherbrooke, Sherbrooke, Quebec J1E 4K8, Canada Simon.Labbe@USherbrooke.ca.

Abstract

Sense and antisense transcripts produced from convergent gene pairs could interfere with the expression of either partner gene. In Schizosaccharomyces pombe, we found that the iss1(+) gene produces two transcript isoforms, including a long antisense mRNA that is complementary to the meiotic cum1(+) sense transcript, inhibiting cum1(+) expression in vegetative cells. Inhibition of cum1(+) transcription was not at the level of its initiation because fusion of the cum1(+) promoter to the lacZ gene showed that activation of the reporter gene occurs in response to low copper conditions. Further analysis showed that the transcription factor Cuf1 and conserved copper-signaling elements (CuSEs) are required for induction of cum1(+)-lacZ transcription under copper deficiency. Insertion of a multipartite polyadenylation signal immediately downstream of iss1(+) led to the exclusive production of a shorter iss1(+) mRNA isoform, thereby allowing accumulation of cum1(+) sense mRNA in copper-limited vegetative cells. This finding suggested that the long iss1(+) antisense  mRNA could pair with cum1(+) sense mRNA, thereby producing double-stranded RNA molecules that could induce RNAi. We consistently found that mutant strains for RNAi (dcr1Δ, ago1Δ, rdp1Δ, and clr4Δ) are defective in selectively eliminating cum1(+) sense transcript in the G1 phase of the cell cycle. Taken together, these results describe the first example of a copper-regulated meiotic gene repressed by an antisense transcription mechanism in vegetative cells.

© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Go To J Biol Chem

 

 

 

 

 

 

Impact of graphene oxide on viability of Chinese hamster ovary and mouse hepatoma MH-22A cells

Significance Statement

 Advances in nanoscience and nanotechnology, enabling the synthesis of new nanomaterials, have led to the development of a number of new drug delivery systems. Carbon materials, like graphene oxide (GO), are known to be more environmentally and biologically friendly than inorganic materials, and it is one of the most common elements in our ecosystem. In biomedical applications and nanomedicine, graphene oxide have been utilized over existing drug delivery vectors due to their ability to cross cell membranes easily and their high surface area, which provides multiple attachment sites for drug targeting. Indeed, with the expanding use of graphene oxide in biomedical applications, a broader understanding of its toxicology is of high importance. The molecular basis for translocation and toxicity of graphene oxide is still largely unclear. So, for the successful application of graphene oxide in nanomedicine the knowledge of the influence of graphene oxide on the viability of various cells and the search for the ways to reduce the toxicity of graphene oxide still remains an unsolved problem. In the present work we estimated the influence of graphene oxide and graphene oxide with 10% of bovine serum albumin on Chinese hamster ovary and mouse hepatoma MH-22A cells in vitro and the structural changes of the cells by atomic force microscopy (AFM), distribution of graphene oxide inside the cells by Raman spectroscopy, imaging. Healthy and cancer cells were chosen in order to determine if the impact of graphene oxide on cell viability is different in healthy and cancer cells.  

About The Author

Valentinas Snitka, Prof. dr. habil.

Valentinas Snitka internationally recognized expert in nanobiotechnology. He pioneered Scanning probe microscopy research in Lithuania in 1993; visiting researcher in Campinas University, Brasil, University of Pensilvania, USA, Twente University, The Netherlands, MIMOS Corporation, Kuala Lumpur etc.  His scientific expertise include Scanning Probe Microscopy methods and instrumentation, cell imaging and manipulation by SPM, electroporation of cells, nanoRaman (SERS,TERS) spectromicroscopy, synthesis and physicochemical characterization of nanoparticles, nanotoxicity, – 100+ publication in international journals, international patents, numerous invited lectures.  e was serving EC as FP6 Nanotechnology, Materials and Production committee member, a member of EU Scientific and Technical Research Committee (CREST). During 2007-2008 he was appointed as Chief Scientist at MIMOS Corp. Kuala Lumpur to develop nanotechnology roadmap and infrastructure. During last 3 years he is serving EC Directorate-General for Research, Directorate G: Industrial technologies as Project Technical Advisor for implementation of FP7 projects in Nanobiotechnologies. He participates in National SF project and is a leader of project funded by European Social Fund (2013-current). 

About The Author

Nora Grinceviciute, M.S.

Nora Grinceviciute completed a M.S. degree in the pharmaceutical sciences program from Lithuanian University of Life Sciences. Now she is Ph.D. student at Kaunas University of Technology (Research Center for Microsystems and Nanotechnology) in chemical engineering. She has experience in industrial biotechnology (2010-2012 Sicor Biotech UAB), working with atomic force microscopy, Raman spectroscopy, Supercritical angle fluorescence microscopy, chemical functionalization, lipid bilayer formation, studying impact of nanoparticles on cells and lipid membranes. Participated in research project as younger researcher, has an international collaboration experience working in University of Zurich (Switzerland). 

About The Author

Danute Batiuskaite, Assoc. Prof.  Dr.

Danute Batiuskaite received her PhD degree from Vytautas Magnus University, Kaunas, Lithuania, in 2003. Now she is an associate professor at the same university, the Faculty of Natural Science. She has the experience in teaching, participation in projects, national and international collaboration, doing experiments in vitro and in vivo, working with electropermeabilization of cells and tissues, searching for the optimal electrochemoterapy conditions, cytotoxicity of vitamins C and K3, impact of nanoparticles on cells. 

Figure legend.

a) Raman scattering image of GO distribution in CHO cell. Intensity maps of the ‘G’ band of GO nanoparticles. b) Viability of CHO and mouse hepatoma MH-22A cells after the treatment with BSA, GO and GO–BSA at different concentrations of GO. c) AFM image of CHO control cells.

 

 

Journal Reference

Toxicol In Vitro. 2015;29(5):1195-200.

Batiuskaite D¹, Grinceviciute N², Snitka V³.

Show Affiliations

1. Research Center for Microsystems and Nanotechnology, Kaunas University of Technology, 65 Studentu str., Kaunas LT-51369, Lithuania; Department of Biology, Faculty of Natural Sciences, Vytautas Magnus University, 58 K. Donelaicio str., Kaunas LT-44248, Lithuania.
2. Research Center for Microsystems and Nanotechnology, Kaunas University of Technology, 65 Studentu str., Kaunas LT-51369, Lithuania. Electronic address: nora.grinceviciute@ktu.lt.
3. Research Center for Microsystems and Nanotechnology, Kaunas University of Technology, 65 Studentu str., Kaunas LT-51369, Lithuania. Electronic address: vsnitka@ktu.lt.

Abstract

The evaluation of the cyto- and bio-compatibility is a critical step in the development of graphene oxide (GO) as a new promising material for in vivo biomedical applications. In this study, we report the impact of GO, with and without the addition of bovine serum albumin, on healthy (Chinese hamster ovary) and a cancer (mouse hepatoma MH-22A) cells viability and the estimation of the intracellular distribution of GO inside the cells in vitro. The viability tests were performed using a colony formation assay. The intracellular distribution of GO was estimated using Raman spectroscopy and imaging. The viability of both cell lines decreased with increasing concentration of  graphene  oxide (12.5-50.0 μg/ml): in the case of Chinese hamster ovary cells viability decreased from 44% to 11%, in the case of mouse hepatoma MH-22A cells–from 22% to 3%. These cell lines significantly differed in their response to GO and GO-BSA formulations. The results of viability tests correlate with results of atomic force microscopy and Raman spectroscopy and imaging findings. The GO influence on cell morphology changes, cell structure, cells colony growth dynamics and GO accumulation inside the cells was higher in the case of  mouse  hepatoma MH-22A cells.

Copyright © 2015 Elsevier Ltd. All rights reserved.

Go To Toxicol In Vitro

 

 

3D fibre deposition and stereolithography techniques for the design of multifunctional nanocomposite magnetic scaffolds

Significance Statement

 Osteochondral bone injuries beyond the self-repair threshold represent a great challenge. The combination of stereolithography and 3D fibre deposition modelling represent a valuable tool to manufacture complex scaffolds. Stereolithography allows to produce thin scaffold layer, finely structured, suitable to reproduce the thin cartilage layer. However, this technique prevents to process polymeric materials incorporating a significant particulate reinforcement phase. On the other hand, 3D fibre deposition prevents to finely structure a scaffold, as the stratification thickness depends on the needle diameter. However, this technique allows to process composite materials incorporating a significant particulate reinforcement phase. This requirement is important as bone scaffolds are concerned. Therefore, by combining stereolithography and 3D fibre deposition modelling it is possible to benefit from the advantages of each single technique, and complex multimaterial scaffolds, such as scaffolds for osteochondral tissue regeneration, can be manufactured.

Poly(ethylene glycol) and poly(caprolactone) based nano-composites have been processed through stereolithography and 3D fibre deposition, respectively. Results shows that dynamic mechanical properties (e.g. storage and loss moduli) of the region of the scaffold manufactured through 3D fibre deposition reproduce those of trabecular bone. Instead, dynamic mechanical properties in compression of poly(ethylene glycol) based nano-composite mimic those of the articular cartilage. Also, the Brazilian test suggests a stable interface between the differently processed scaffold regions, and adhesion between these regions relies at least on a micromechanical interlocking.

The incorporation of a superparamagnetic nano-reinforcement phase, based on iron oxide nanoparticles encapsulated in a polymeric shell, allows for further improving the scaffold material design by offering a unique opportunity. Poly(ethylene glycol) and poly(caprolactone) based superparamagnetic scaffolds become magnetised as an external magnetic field is applied, and magnetization goes to zero as the external field is turned off. This peculiarity of superparamagnetic nanocomposite scaffolds allows to attract magnetically functionalised bioaggregates using and external magnetic field, and to release these bioaggregates on demand. This opportunity represents an important tool to guide the regeneration process of complex tissues like the osteochondral defect.

Non-degradable prosthesis based on metals, polymers and ceramics, represent the available solution to restore joint disorders. However, the life span of such approach is very limited, especially when dealing with population aging. Of course, the possibility to regenerate ostechondral bone would be the best solution. By using the reverse engineering approach to define the osteochondral defect and implementing the combination of rapid prototyping techniques, in the future it would be probably possible to manufacture customized polymer based scaffold for joint disorders.

Journal Reference

J Mater Sci Mater Med. 2015;26(10):250.

De Santis R¹, D’Amora U², Russo T², Ronca A², Gloria A², Ambrosio L³.

Show Affiliations

1. Institute of Polymers, Composites and Biomaterials, National Research Council of Italy, V. le J.F. Kennedy 54 – Pad. 20 Mostra d’Oltremare, 80125, Naples, Italy. rosantis@unina.it.
2. Institute of Polymers, Composites and Biomaterials, National Research Council of Italy, V. le J.F. Kennedy 54 – Pad. 20 Mostra d’Oltremare, 80125, Naples, Italy.
3. Department of Chemical Science and Materials Technology, National Research Council of Italy, Piazzale Aldo Moro 7, 00185, Rome, Italy.

Abstract

Magnetic nanocomposite scaffolds based on poly(ε-caprolactone) and poly(ethylene glycol) were fabricated by 3D fibre deposition modelling (FDM) and stereolithography techniques. In addition, hybrid coaxial and bilayer magnetic scaffolds were produced by combining such techniques. The aim of the current research was to analyse some structural and functional features of 3D magnetic scaffolds obtained by the 3D fibre deposition technique and by stereolithography as well as features of multimaterial scaffolds in the form of coaxial and bilayer structures obtained by the proper integration of such methods. The compressive mechanical behaviour of these scaffolds was investigated in a wet environment at 37 °C, and the morphological features were analysed through scanning electron microscopy (SEM) and X-ray micro-computed tomography. The capability of amagnetic scaffold to absorb magnetic nanoparticles (MNPs) in water solution was also assessed. confocal laser scanning microscopy was used to assess the in vitro biological behaviour of human mesenchymal stem cells (hMSCs) seeded on 3D structures. Results showed that a wide range of mechanical properties, covering those spanning hard and soft tissues, can be obtained by 3D FDM and stereolithography techniques. 3D virtual reconstruction and SEM showed the precision with which the scaffolds were fabricated, and a good-quality interface between poly(ε-caprolactone) and poly(ethylene glycol) based scaffolds was observed for bilayer and coaxial scaffolds. Magnetised scaffolds are capable of absorbing water solution of magnetic nanoparticles, and a preliminary information on cell adhesion and spreading of human mesenchymal stem cells was obtained without the application of an external magnetic field.

Go To J Mater Sci Mater Med

 

 

Alternatively Spliced Human TREK-1 Variants Alter TREK-1 Channel Function and Localization

Journal Reference

Biol Reprod. 2015 Nov;93(5):122.

Cowles CL¹, Wu YY¹, Barnett SD¹, Lee MT¹, Burkin HR¹, Buxton IL².

Show Affiliations

1. Myometrial Function Laboratory, Department of Pharmacology, University of Nevada School of Medicine, Reno, Nevada.
2. Myometrial Function Laboratory, Department of Pharmacology, University of Nevada School of Medicine, Reno, Nevada ibuxton@medicine.nevada.edu.

Abstract

TREK-1, an outward-rectifying potassium channel activated by stretch, is found in the myometrium of pregnant women. Decreased expression of TREK-1 near term suggests that TREK-1 may contribute to uterine quiescence during gestation. Five alternatively spliced TREK-1 variants were identified in the myometrium of mothers who delivered spontaneously preterm (<37 wk), leading to the hypothesis that these TREK-1 variants could interfere with TREK-1 function or expression. To investigate a potential role for these variants, immunofluorescence, cell surface assays, Western blots, and patch clamp were employed to study TREK-1 and TREK-1 variants expressed in HEK293T cells. The results of this study demonstrate that coexpression of TREK-1 with TREK-1 variants alters TREK-1 expression and suppresses channel function. Each variant affected TREK-1 in a disparate manner. In HEK293T cells coexpressing TREK-1 and each variant, TREK-1 membrane expression was diminished with compartmentalization inside the cell. When expressed alone, individual  variants displayed channel properties that were significantly decreased compared to full-length TREK-1. In coexpression studies using patch clamp, basal TREK-1 currents were reduced by ∼64% (4.3 vs. 12.0 pA/pF) on average at 0 mV when coexpressed with each variant. TREK-1 currents that were activated by intracellular acidosis were reduced an average of ∼77% (21.4 vs. 94.5 pA/pF) at 0 mV when cells were transfected with TREK-1 and any one of the splice variants. These data correlate the presence of TREK-1 variants to reduced TREK-1 activity, suggesting a pathological role for TREK-1 variants in preterm labor.

© 2015 by the Society for the Study of Reproduction, Inc.

Go To Biol Reprod

 

 

The influence of printing parameters on cell survival rate and printability in microextrusion-based 3D cell printing technology

Significance Statement

High cell survival rate and good printability, commonly conflicts with each other, has always been the pursuit in the practice of cell 3D printing technology. Since both can be influenced by many process parameters, Yao and her coworkers from Tsinghua University, China and Drexel University, USA focused on the establishment of an applicable protocol for microextrusion-based 3D cell printing technology to achieve both high cell survival rate and good printability. Their studies demonstrated that even though the bioink composition and concentration, holding temperature and holding time would all influence bioink rheological property in the printing period, bioink storage modulus was the decisive factor for both cell survival rate and printability. Different process parameter combinations would result in the similar storage modulus and thus showed similar cell survival rate after 3D bioprinting process. When the bioink storage modulus was in the range of 154 Pa to 382 Pa, 3D printing of A549 cell-laden gelatin/alginate construct with both high cell survival rate (>90%) and good printability could be achieved. Researchers in the bioprinting field may find this protocol useful for adjusting the printing process quickly. And the methodology and the protocol in this study may also be useful for the design and development of new temperature-sensitive bioinks for 3D cell printing. Future work will be focused on revealing the detailed mechanism and mathematical modelling of cell survival and printability in microextrusion-based 3D cell printing technology and optimizing the parameter ranges for bioprinting other types of cells, e.g. pluripotent stem cells.

About The Author

Dr. Rui Yao is Assistant Professor of Biomanufacturing Research Center, Department of Mechanical Engineering, Tsinghua University, Beijing, China. She has interdisciplinary background of tissue engineering, biomaterials, biofabrication and stem cells from Tsinghua University, MIT and Peking Union Medical University. Her main focus is Stem Cell-based Biofabrication and applications in Tissue Engineering, 3D Physiology/Pathological Model and Drug Testing Model. She has published over 40 journal papers and conference abstracts, filed over 10 patents and conducted over 10 oral presentations in international conferences in the field of her research.

 

Journal Reference

Biofabrication. 2015 Nov 2;7(4):045002.

Zhao Y, Li Y, Mao S, Sun W, Yao R.

Biomanufacturing Center, Department of Mechanical Engineering, Tsinghua University, Beijing, People’s Republic of China. Biomanufacturing and Rapid Forming Technology Key Laboratory of Beijing, Beijing, People’s Republic of China.

Abstract

Three-dimensional (3D) cell printing technology has provided a versatile methodology to fabricate cell-laden tissue-like constructs and in vitro tissue/pathological models for tissue engineering, drug testing and screening applications. However, it still remains a challenge to print bioinks with high viscoelasticity to achieve long-term stable structure and maintain high cell survival rate after printing at the same time. In this study, we systematically investigated the influence  of 3D cell printing parameters, i.e. composition and concentration of bioink, holding temperature and holding time, on the printability  and  cell survival rate in microextrusion-based 3D cell printing technology. Rheological measurements were utilized to characterize the viscoelasticity of gelatin-based bioinks. Results demonstrated that the bioink viscoelasticity was increased when increasing the bioink concentration, increasing holding time and decreasing holding temperature below gelation temperature. The decline of cell survival rate after 3D cell printing process was observed when increasing the viscoelasticity of the gelatin-based bioinks. However, different process parameter combinations would result in the similar rheological characteristics and thus showed similar cell survival rate after 3D bioprinting process. On the other hand, bioink viscoelasticity should also reach a certain point to ensure good printability and shape fidelity. At last, we proposed a protocol for3D bioprinting of temperature-sensitive gelatin-based hydrogel bioinks with both high cell survival rate and good printability. This research would be useful for biofabrication researchers to adjust the 3D bioprinting process parameters quickly and as a referable template for designing new bioinks.

Go To Biofabrication

 

 

 

The use of a cerebral perfusion and immersion-fixation process for subsequent white matter dissection

Significance Statement

White matter is a fundamental component of the brain architecture which allows the communication and the reciprocal modulation between close or distant cortical territories through a balanced network of axons, fibres and fascicles.

An extensive anatomical knowledge of the major white matter (WM) bundles is mandatory in order to tailor the neurosurgical approach and to optimize the resection of various deep subcortical lesions. Modern techniques such as Magnetic Resonance diffusion tensor tractografy, provided a unique insight into the brain white matter networks, revealing the organization of deep and superficial pathways.

 Because of the intrinsic limitations associated with the DTI, white matter dissection of previously formalin fixed brains still remains one of the most trustable techniques in order to validate the white matter connectivity and at the same time to acquire a comprehensive three-dimensional orientation during neurosurgical-neuroanatomical training.

Anatomists of every century have described many different techniques for specimen preparation and dissection in order to understand the complex WM architectural organization. However it was Josef Klingler, who in the 1935 revolutionized this field of research. He developed a new method of brain fixation, which consisted of freezing already formalin-fixed brains before dissection. Despite the tremendous impact of this technique for both neurosurgical training and research, according to the literature it still presents some intrinsic limitations. Because based on the immersion into 5% formalin, a long time for specimens preparation is needed. Moreover a very variable level of fixation is often reported and this can affect the qualitu of the dissection. From the anatomical point of view, difficult demonstration of superficial white matter architecture or thin white matter bundles within the basal ganglia, difficult identification of deep vascular structures during the same dissection are some of the limitations experienced during the white matter dissection with Klingler’s technique.

We evaluated the quality of a different method for specimen preparation based on an intra-carotidal formalin perfusion fixation process before the freezing stage and the dissection. This technique showed several practical advantages: the time needed for a “physiological” fixation is minimized. The brain can be removed intact 48 hours after the perfusion and is already fixed, thus decreasing the risk of accidental gyral deformation during its removal. The quality of the fixed brains can be maintained for a very long time without the need for additional procedures. The homogeneous and rapid fixation of the brain allowed us to document several fine additional anatomical details.

A more systematic organization of the white matter terminations underneath the sulcal or gyral surface can be demonstrated with this technique. This aspect is quite important and a more comprehensive knowledge of the superficial white matter should be mandatory in order to tailor cortical and subcortical resection with the least invasive possible surgical trajectories.

All the major associative bundles were isolated without technical problems and it was even possible to isolate them en bloc. This aspect is a direct signal of the quality of the specimens and at the same time this can open new possibilities for the research on symmetry, lateralization, and biophysical features of each white matter bundle.

Some of the most complex and thin bundles were dissected without any technical limitation on these specimens. This aspect can lead to a better understanding of the white matter organization of deep regions such as the basal ganglia region with the possibility to identify new surgical targets i.e. for deep brain stimulation based on their connectivity.

A very fundamental advantage of our specimens is the use of the physiological intracranial vascular network for the fixation process, which preserves vascular landmarks at each stage of white matter dissection until their sub-millimetric terminations. These fine and constant details allow correlation between vessels and white matter structures with the obvious advantage in three-dimensional orientation that can be employed during any standard neurosurgical procedure.

 These results provided encouraging data about the great potential of this technique to improve the quality of white matter dissection and the deep anatomical orientation within the brain. A shorter time for preparation, a more homogeneous fixation and no technical limitation for a fine description of superficial and deep white matter anatomy are some of the advantages of our technique. We suggests the more widely use of perfusion-fixation process, which may help in improving the quality of white matter dissection for research, didactic purposes and neurosurgical training.

 

About The Author

Francesco Latini, M.D.

Dr Latini completed his MD degree in 2000 at University of Bologna (Alma Mater). He completed his residency (cum laude) in Neurosurgery at University of Ferrara, Italy and since 2013 is specialist in Neurosurgery. He worked as junior consultant for the division of Neurosurgery at the Dpt of Neuroscience and Rehabilitation, S.Anna University Hospital of Ferrara Italy until 2014 with a specific research project on brain connectivity and patients with lesions in eloquent areas underwent awake surgery.

He did a research fellowship at International Neuroscience Institute , Hannover (Germany)  and a clinical research fellowship with Dr Ali Krisht at the Arkansas Neuroscience Institute at St Vincent’s Infirmary in Little Rock, Arkansas (USA) in 2012. He is currently working as neurosurgeon and visiting researcher at the Department of Neurosurgery, Uppsala University Hospital, Uppsala, Sweden. His main clinical and research focus are: neuroanatomy, neuroimaging, neuropsychology, brain connectivity and the treatment of intracranial tumours.

His current research projects are  “Studies of the tumor to brain interface in meningiomas – Peritumoral edema and invasiveness” and “Neurosurgical anatomy studies of the white matter in the human brain – post mortem fiber dissection and in vivo tractography of magnetic resonance imaging studies”

He is member of the European Association of Neurosurgical Societies (EANS), of the Human Brain Mapping Organization (HBMO), of the Swedish Society for Neuroscience (SSfN) and of the Federation of European Neuroscience Societies (FENS).

About The Author

Mats Hjortberg  PhD 

Senior Lecturer in Anatomy-  Dept. of Medical Cell Biology, Uppsala University

BSc in Biochemistry (1985), MSc in Anatomy (1989), PhD in Anatomy  (1992). Post Doctoral Fellow at Baylor College of Medicine 1993

Junior Lecturer of in Anatomy at Dept. Physiotherapy, Uppsala University 1994-2005

Junior Lecturer in Anatomy at Dept. Medical Cell Biology, Uppsala University 2005-2008

Senior Lecturer in Anatomy and Head of the Anatomy Unit at Dept. Medical Cell Biology 2009-

Among the current research projects there is “Neurosurgical anatomy studies of the white matter in the human brain – post mortem fiber dissection and in vivo tractography of magnetic resonance imaging studies”.

About The Author

Håkan Aldskogius

MD, Uppsala University, 1971

PhD in Anatomy, Karolinska Institutet, 1974

Postdoctoral Fogarty Fellow, Albany Medical College, Neurology, 1977-78

Assistant Professor of Anatomy, Karolinska Institutet, 1978-1984

University Lecturer in Anatomy, Karolinska Institutet, 1984-1995

Professor of Medical Structural Bology, Uppsala University, 1995-2010

Chairman, Department of Neuroscience, Uppsala University, 2006-2010

Professor Emeritus, Uppsala University, 2010-

Field of research: Spinal cord neural degeneration, regeneration and plasticity

137 original and 19 review publications in PubMed, ca 5 000 citations, h-factor 41

Recent publication: Hoeber J, Trolle C, Konig N, Du Z, Gallo A, Hermans E, Aldskogius H, Shortland P, Zhang SC, Deumens R, Kozlova EN. Human embryonic stem cell-derived progenitors assist functional sensory axon regeneration after dorsal root avulsion injury. Scientific Reports. 2015 Jun 8;5:10666. doi: 10.1038/srep10666.

Editor: Glial Interfaces in the Nervous System, IOS Press, 2002; Animal Models of Spinal Cord Repair, Humana Press, 2013

Field of education: Gross Anatomy, Functional Neuroanatomy, Regenerative Neurobiology

Book Chapter: Structure and Function of the Nervous System in Clinical Neuroscience, Liber, 2015 (Swedish)

Commitments of Trust: Evaluator European Commission, Marie Sklodowska-Curie program, 2012-2015; Member Medical Faculty Executive Committee for Medical Education, 1996-2010; Consultant Medical Faculty Executive Committee for Medical Education, 2010-2015

Professional memberships: American Association of Anatomists, Society for Neuroscience, Federations of European Neuroscience Societies, Swedish Society for Physicians.

About The Author

Mats Ryttlefors, M.D., Ph.D.

Dr Ryttlefors completed his MD degree in 2002 and PhD degree in 2009 at Uppsala University, Uppsala, Sweden. He wrote his thesis for the PhD degree on studies of subarachnoid haemorrhage in elderly patients with Prof Per Enblad as main supervisor. He attended residency in Neurosurgery at the Uppsala University Hospital and is since 2010 specialist in Neurosurgery. He did a clinical research fellowship with Dr Ali Krisht at the Arkansas Neuroscience Institute at St Vincent’s Infirmary in Little Rock, Arkansas in 2012. He is currently working as staff neurosurgeon and researcher at the Department of Neurosurgery, Uppsala University Hospital, Uppsala, Sweden. His main clinical and research focus is the treatment of intracranial tumours. He is currently supervisor of two research projects: “Studies of the tumor to brain interface in meningiomas – Peritumoral edema and invasiveness” and “Neurosurgical anatomy studies of the white matter in the human brain – post mortem fiber dissection and in vivo tractography of magnetic resonance imaging studies”. 

Figure Legend 1. Superficial White matter terminations (SWM).

Examples of the superficial white matter terminations in three different regions (the three colored squares) of the brain convexity identified during dissection of a right hemisphere. The white matter terminations terminations demonstrated with high magnification at the microscope, have a parallel direction and end perpendicular to the pial/cortical surface.

AF: Arcuatus Fasciculus (section); CR: corona radiata; SSS: Sagittal stratum of Sachs; Ins: Insula; M1: primary motor area (precentral gyrus). A: Anterior, P: posterior.

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure Legend 2. Major fibre bundles.

1. A) The frontal aslant tract (FASt) has been removed with an “en bloc” dissection from the frontal lobe of this left hemisphere. This bundle connects the Supplementary motor area (SMA) and the region close to it (Pre-SMA) with the frontal operculum (PoP) and the pars triangularis (PT), considered part of the Broca’s area, crucial for speech production.
2. B) The cingulum (Ci) has been dissected and removed “en bloc” from the medial surface of this right hemisphere, showing the three main parts of the corpus callosum (genu, Body and splenium) and the Callosal Radiation (Ca radiation). The three major components of the Ci are marked in the frontal region (Fro), in the retrosplenial and temporal parahippocampal region (PHG). The cingulum is involved in several high order functions, such as attention, emotion, memory, visual and spatial skills among others.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure Legend 3 Deep venous anastomosis.

Example of deep medullary venous system of the brain (called “candelabra” type), which can be identified under high magnification and dissected untils its submillimetric division within the deep white matter layers and periventricular areas.

 

 

 

Journal Reference

J Neurosci Methods. 2015;253:161-9.

Latini F¹, Hjortberg M², Aldskogius H³, Ryttlefors M⁴.

Show Affiliations

1. Department of Neuroscience, Neurosurgery, Uppsala University, Akademiska sjukhuset, 75185 Uppsala, Sweden. Electronic address: francesco.latini@neuro.uu.se.
2. Department of Medical Cell Biology, Education, Uppsala University, Box 571, 75123 Uppsala, Sweden.
3. Department of Neuroscience, Regenerative Neurobiology, Uppsala University, Box 593, 75124 Uppsala, Sweden.
4. Department of Neuroscience, Neurosurgery, Uppsala University, Akademiska sjukhuset, 75185 Uppsala, Sweden.

Abstract

BACKGROUND:

The Klingler’s method for white matter dissection revolutionized the study of deep cerebral anatomy. Although this technique madewhite matter dissection more feasible and widely used, it still presents some intrinsic limitations.

NEW METHOD:

We evaluated the quality of different methods for specimen preparation based on an intra-carotidal formalin perfusion fixationprocess. Ten post-mortem human hemispheres were prepared with this method and dissected in a stepwise manner.

RESULTS:

The homogeneous and rapid fixation of the brain allowed documentation of several fine additional anatomical details. Intra-cortical whitematter terminations were described during the first stage of dissection on each specimen. No limitations were encountered during dissection of the major associative bundles. On the contrary, the quality of the fixation of the specimens made it possible to isolate them en bloc. One of the most complex and deep bundles (accumbo-frontal fasciculus) was dissected without technical limitations. Deep vascular structures were very well preserved and dissected within the white matter until their sub-millimetric terminations.

COMPARISON WITH EXISTING METHOD:

Short time for preparation, a more homogeneous fixation, no technical limitation for a detailed description of superficial and deep white matter anatomy, the possibility to dissect with a single technique the fibre organization and the white mattervascular architecture are the advantages reported with the perfusion fixation.

CONCLUSION:

These results provide encouraging data about the possibility to use a perfusion fixation process, which may help in improving the quality of white matter dissection for research, didactic purposes and surgical training.

Copyright © 2015 Elsevier B.V. All rights reserved.

Go To J Neurosci Methods

 

 

 

 

Methods for Probing Lysosomal Membrane Permeabilization

Significance Statement

The authors report three methods for quantifying and visualizing lysosomal membrane permeabilization:
(i) monitoring lysosomal membrane permeabilization by immunocytochemistry,
(ii) visualizing lysosomal membrane permeabilization by fluorescent dextran release,and
(iii) quantification of lysosomal membrane permeabilization by activity measurements of lysosomal enzymes in digitonin-extracted cytosol. Such methods can help researchers in advancing our knowledge in understanding cell death in normal physiology as well as in disease such as cancer.  

First Report

A Method to Monitor Lysosomal Membrane Permeabilization by Immunocytochemistry

Cold Spring Harb Protoc. 2015;2015(10):pdb.prot086181.

Groth-Pedersen L¹, Jäättelä M², Nylandsted J².

Show Affiliations

1. Department of Pediatrics and Adolescent Medicine, Rigshospitalet University Hospital, DK-2100 Copenhagen, Denmark; Unit for Cell Death and Metabolism, Center for Autophagy, Recycling and Disease, Danish Cancer Society Research Center, DK-2100 Copenhagen, Denmark.
2. Unit for Cell Death and Metabolism, Center for Autophagy, Recycling and Disease, Danish Cancer Society Research Center, DK-2100 Copenhagen, Denmark.

Abstract

Programmed cell death involving lysosomal membrane permeabilization (LMP) is a common phenomenon-more the rule than the exception under various cytotoxic stimuli and stressful cellular conditions. The protocol presented here is based on immunocytochemical staining of cathepsin B or L to visualize translocation from the lysosomal lumen to the cytosol. In healthy cells, cathepsins appear in localized punctate structures representing intact lysosomes, whereas LMP results in a diffuse staining pattern throughout the cytoplasm. LMP can be triggered upstream, downstream, or independently of the classical apoptotic death pathway involving mitochondrial outer membrane permeabilization (MOMP). Co-staining with antibodies recognizing the active form of Bax allows investigation of the order of events between LMP and MOMP in death signaling.

© 2015 Cold Spring Harbor Laboratory Press.

Go To Cold Spring Harb Protoc

 

Second Report

Cold Spring Harb Protoc. 2015;2015(11):pdb.prot086165. 

Quantification of Lysosomal Membrane Permeabilization by Cytosolic Cathepsin and β-N-Acetyl-Glucosaminidase Activity Measurements.

Jäättelä M¹, Nylandsted  J¹.

Abstract
Programmed cell death involving lysosomal membrane permeabilization (LMP) is an alternative cell death pathway induced under various cellular conditions and by numerous cytotoxic stimuli. The method presented here to quantify LMP takes advantage of the detergent digitonin, which creates pores in cellular membranes by replacing cholesterol. The difference in cholesterol content between the plasma membrane (high) and lysosomal membrane (low) allows titration of digitonin to a concentration that permeabilizes the plasma membrane but leaves lysosomal membranes intact. The extent of LMP is determined by measuring the cytosolic activity of lysosomal hydrolases (e.g., cysteine cathepsins) and/or β-N-acetyl-glucosaminidase in the digitonin-extracted cytoplasm and comparing it to the total cellular enzyme activity. Digitonin extraction of the cytosol can be combined with precipitation of protein and/or western blot analysis for detection of lysosomal proteins (e.g., cathepsins).

© 2015 Cold Spring Harbor Laboratory Press.

 

Third Report

Cold Spring Harb Protoc. 2015;2015(10):pdb.prot086173. 

Visualizing Lysosomal Membrane Permeabilization by Fluorescent Dextran Release.

Ellegaard AM¹, Jäättelä M¹, Nylandsted  J¹.

Abstract
Lysosomal membrane permeabilization (LMP) is an effective programmed cell death pathway triggered in response to a variety of cytotoxic stimuli and cellular conditions. In the method presented here, LMP is monitored by first taking advantage of the steady endocytic capacity of cells to load fluorescent dextran into lysosomes, and then simply observing the translocation of lysosomally localized dextran into the cytosol after an LMP-inducing insult. Fluorescent dextran in healthy cells appears in punctate structures representing intact lysosomes, whereas after LMP, a diffuse staining pattern throughout the cytoplasm is observed. Using this method, LMP can be followed in real time using time-lapse imaging. The size of pores formed in the membrane during LMP by size exclusion can also be determined using dextrans of different sizes and colors.

© 2015 Cold Spring Harbor Laboratory Press.

 

Enhanced paracellular transport of insulin can be achieved via transient induction of myosin light chain phosphorylation

Significance Statement

Oral delivery of proteins such as peptide therapeutics remains a major challenge for the pharmaceutical industry. Recently we reported the enhanced transport of oral insulin across rat intestine in vivo using peptides that that were rationally designed to induce myosin light chain (MLC) phosphorylation. These permeable inhibitors of MLC phosphatase (PIP peptides) transiently open intestinal tight junctions that exist between adjunct epithelial cells. In doing so, PIP peptides can be used to dynamically enhance solute uptake through the paracellular route, proving a promising new, mechanism-based approach for enhancing peptide therapeutic absorption following oral administration.

Presently, the medical field and pharmaceutical industry are facing a diabetes epidemic on a global scale with most patients poorly controlling their blood sugar levels, leading to devastating morbidity issues and ultimately advanced mortality. Better management of blood sugar is of paramount concern to limit this ever-increasing burden on health care systems. Oral delivery of insulin, and more recently incretin molecules such as GLP-1 secreted by intestinal epithelial cells in response to food intake, could provide a simpler needle-free method of treating diabetes, with the possibility of an enhanced clinical benefit. Orally administered insulin absorbed across the intestinal mucosa and would be delivered to the liver by the portal circulation, just as insulin is normally delivered to the liver by the pancreas of a non-diabetic human. Thus, successful oral administration of insulin and GLP-1 are now considered more than just a strategy to improve patient compliance and comfort, but essential to effectively combat the diabetes epidemic. PIP peptides will likely lead to exciting opportunities for oral delivery of therapeutic agents such as insulin and GLP-1, and to transformative treatments for diabetic patients.

These peptides act by a defined mechanism of action, without causing any intestinal damage, by direct actions on the epithelium after topical application. Clinical utilization of agents enhancing paracellular permeability without first understanding their mechanisms of permeabilization is challenging both because of potential safety issues and translation to humans from pre-clinical models. PIP peptides overcome previous challenges to clinical development strategies for oral insulin and GLP-1 dosage forms because they act transiently and locally following topical application, and do not appear to affect other tissues prior to their metabolism and elimination from the body.  Most importantly, PIP peptides work through a defined and endogenous pathway, do not appear to incite cell damage or an extensive co-uptake of bacterial components (like lipopolysaccharides) present in the intestinal lumen.

Our initial studies using a simple administration into the intestine of rats have shown 3-4% bioavailability for insulin. We believe that formulation efforts to enhance the stability of the peptides therapeutic to be delivered could increase this level of bioavailability significantly. While our initial studies have used insulin to demonstrate the feasibility of PIP peptides, we are interested in exploring a spectrum of therapeutic peptides for oral delivery. Although only insulin has been tested to date we would expect the PIP peptides to be capable of delivering other peptides and small proteins orally. We are seeking industrial partners for this technology and are keen to validate the PIP peptides in a large animal study.

About The Author

Randy Mrsny currently holds a Professor’s chair of Epithelial Cell Biology at the University of Bath in the Department of Pharmacy and Pharmacology where he studies biological principles associated with normal epithelia cell function and how these are affected in disease states. His work in drug delivery is internationally recognized as evidenced by his election as president of the Controlled Release Society and to co-organize a Gordon Conference on Drug Delivery. 

 

 

 

 

 

 

 

 

 

Journal Reference

J Control Release. 2015;210:189-97.

Taverner A¹, Dondi R¹, Almansour K¹, Laurent F¹, Owens SE², Eggleston IM¹, Fotaki N¹, Mrsny RJ³.

Show Affiliations

1. Department of Pharmacy and Pharmacology, University of Bath, Bath BA2 7AY, UK.
2. Welsh School of Pharmacy, Cardiff University, Cardiff, CF10 3XF, UK.
3. Department of Pharmacy and Pharmacology, University of Bath, Bath BA2 7AY, UK. Electronic address: Rjm37@bath.ac.uk.

Abstract

The intestinal epithelium functions to effectively restrict the causal uptake of luminal contents but has been demonstrated to transiently increase paracellular permeability properties to provide an additional entry route for dietary macromolecules. We have examined a method to emulate this endogenous mechanism as a means of enhancing the oral uptake of insulin. Two sets of stable Permeant Inhibitor of Phosphatase (PIP) peptides were rationally designed to stimulate phosphorylation of intracellular epithelial myosin light chain (MLC) and screened using Caco-2 monolayers in vitro. Apical application of PIP peptide 640, designed to disrupt protein– protein interactions between protein phosphatase 1 (PP1) and its regulator CPI-17, resulted in a reversible and non-toxic transient reduction in Caco-2 monolayer trans-epithelial electric resistance (TEER) and opening of the paracellular route to 4 kDa fluorescent dextran but not 70 kDa dextran in vitro. Apical application of PIP peptide 250, designed to impede MYPT1-mediated regulation of PP1, also decreased TEER in a reversible and non-toxic manner but transiently opened the paracellular route to both 4 and 70 kDa fluorescent dextrans. Direct injection of PIP peptides 640 or 250 with human insulin into the lumen of rat jejunum caused a decrease in blood glucose levels that was PIP peptide and insulin dose-dependent and correlated with increased pMLC levels. Systemic levels of insulin suggested approximately 3–4% of the dose injected into the intestinal lumen was absorbed, relative to a subcutaneous injection. Measurement of insulin levels in the portal vein showed a time window of absorption that was consistent with systemic concentration-time profiles and approximately 50% first-pass clearance by the liver. Monitoring the uptake of a fluorescent form of insulin suggested its uptake occurred via the paracellular route. Together, these studies add validation to the presence of an endogenous mechanism used by the intestinal epithelium to dynamically regulate its paracellular permeability properties and better define the potential to enhance the oral delivery of biopharmaceuticals via a transient regulation of an endogenous mechanism controlling the intestinal paracellular barrier.

Copyright © 2015. Published by Elsevier B.V.

Go To J Control Release.

 

 

 

 

A unilateral medial frontal cortical lesion impairs trial and error learning without visual control

 

 

 

 

 

About The Author

Dr. Céline Amiez is a CNRS (French National Center for Scientific Research) Researcher based at the Stem Cell and Brain Research Institute (SBRI) / INSERM (French National Institute of Health and Medical Research) U846 at the University of Lyon, France. She completed her Ph.D. in electrophysiology with Dr. Jean-Paul Joseph (Bron, France) and her post-doctoral fellowship in functional neuroimaging with Dr. Michael Petrides (McGill University, Montreal QC, Canada). Her research aims to understand the anatomo-functional organization of the frontal cortex in primates.

 

About The Author

Dr. Anne Sophie Champod is an Assistant Professor in the Department of Psychology at Acadia University and a clinical neuropsychologist in Nova Scotia, Canada.  She completed her Ph.D. in Clinical Psychology at McGill University (Montreal, QC, Canada) and her postdoctoral work at Dalhousie University (Halifax, NS, Canada). Her research has aimed to better understand the neuronal basis of attention and working memory processes with the ultimate goal of developing new assessment techniques and rehabilitation interventions to improve cognitive functioning in neurological populations.  Her current research work involves the development of new assessment tools and rehabilitation interventions targeting spatial attention skills that are frequently affected in stroke.  

 

About The Author

Dr Charlie Wilson is a post-doctoral researcher at the Stem Cell and Brain Research Institute (SBRI), INSERM (French National Institute of Health and Medical Research) in the University of Lyon, France. He trained with Dr David Gaffan at the Dept. Experimental Psychology, University of Oxford working on primate neuropsychology, and subsequently with Dr Emmanuel Procyk in Lyon working on electrophysiological techniques. His research focuses on the long-term cognitive and neurophysiological changes that occur whilst human and non-human primate subjects learn to learn, and whilst they use cognitive control to make efficient decisions, focusing on the critical role contributed by specific cortical regions. He also works on the role of dopamine in cognitive control processes, in particular using a primate model of the Parkinsonian brain.  

About The Author

Dr. Emmanuel Procyk is based at the CNRS (National Center for Scientific Research), where he leads an INSERM (French National Institute of Health and Medical Research) lab at the Stem Cell and Brain Research institute in the University of Lyon, France. Trained as a neurophysiologist with Dr. Jean Paul Joseph (Bron, France) and Dr. Patricia Goldman-Rakic (Yale, USA), he now seeks to understand the neural bases of adaptive cognitive functions in primates.

About The Author

Michael Petrides is a James McGill Professor at the Montreal Neurological Institute, Department of Neurology and Neurosurgery and the Department of Psychology at McGill University. He obtained a B.Sc. in Experimental Psychology and a M.Sc. in Neurological Science from the University of London and then a Ph.D. in Behavioural Neuroscience from the University of Cambridge.  He subsequently worked as a post-doctoral research fellow at the Montreal Neurological Institute and as a research fellow at Harvard Medical School. The major aspect of Dr. Petrides’ research work is concerned with understanding the functional and anatomical organization of the primate frontal cortex. He has pursued this work in studies with patients who had excisions from the frontal cortex for the treatment of epilepsy, in studies on monkeys with selective lesions in particular parts of the frontal cortex, and in functional neuroimaging studies in healthy human participants. Based on this work he has proposed an influential theoretical framework to understand the functional organization of the lateral prefrontal cortex. Another aspect of his work has been the comparative cytoarchitectonic analysis of the human and the monkey prefrontal cortex that allows integration of research on nonhuman primates with research on the human brain.  

Journal Reference

Neuropsychologia. 2015;75:314-21.

Amiez C¹, Champod AS², Wilson CR³, Procyk E⁴, Petrides M⁵.

Show Affiliations

1. Institut National de la Santé et de la Recherche Médicale U846, Stem Cell and Brain Research Institute, 69675 Bron, France; Université de Lyon, Lyon 1, Unité Mixte de Recherche S-846, 69003 Lyon, France; Montreal Neurological Institute, McGill University, Montreal, Quebec, Canada H3A2B4.
2. Montreal Neurological Institute, McGill University, Montreal, Quebec, Canada H3A2B4; Brain Repair Centre, Dalhousie University, Life Sciences Research Institute, Halifax, Nova Scotia, Canada B3H 4R2; Acadia University, Department of Psychology, Wolfville, Nova Scotia, Canada B4P 2R6.
3. Institut National de la Santé et de la Recherche Médicale U846, Stem Cell and Brain Research Institute, 69675 Bron, France; Université de Lyon, Lyon 1, Unité Mixte de Recherche S-846, 69003 Lyon, France.
4. Institut National de la Santé et de la Recherche Médicale U846, Stem Cell and Brain Research Institute, 69675 Bron, France; Université de Lyon, Lyon 1, Unité Mixte de Recherche S-846, 69003 Lyon, France. Electronic address: celine.amiez@inserm.fr.
5. Montreal Neurological Institute, McGill University, Montreal, Quebec, Canada H3A2B4.

 

Abstract

There is considerable debate regarding the involvement of the medial frontal cortex in motor and cognitive functions. Recent neuroimaging data suggest a fundamental underlying process that links the motor and cognitive roles of the mid-cingulate cortex (MCC), namely the processing of feedback during trial and error learning in the cingulate motor region that is related to the modality of the feedback. These data suggest that the specific motor context of a task may be a critical determinant of how its outcome is processed in the MCC. We assessed a patient before and after surgery for brain tumour removal in the medial frontal cortex, and a group of matched control subjects. Subjects had to find by trial and error the stimulus associated with the correct feedback amongst four or five similar stimuli. Subjects performed the task in two different visuo-motor contexts: with the response pad and hand visible and with no sight of either pad or hand. The patient showed a selective impairment in this task relative to control subjects in the hardest conditions and the impairment was most marked when the response pad and the hand were not visible. The results support a specific role of the medial frontal cortex in the construction of a sensorimotor representation of choices and related feedback by encoding the contingency between an efference copy of the action and its outcome.

Copyright © 2015 Elsevier Ltd. All rights reserved.

Go To Neuropsychologia

 

 

 

 

CD8 Memory Cells Develop Unique DNA Repair Mechanisms Favoring Productive Division

Significance Statement

Immune responses are efficient because the rare naïve antigen-specific cells proliferate extensively after antigen stimulation. This property raises an important paradox: how CD8 T cells may survive the DNA lesions necessarily induced during their extensive division without undergoing transformation.  In secondary responses in the absence of CD4 help, cells with DNA damages are ensured to die. In the presence of CD4 help DDRs become very efficient, justifying the major amplitude of these secondary responses. These results highlight the fundamental role of DDRs in CD8 immune responses.  

About The Author

Alessia Galgano, PhD

Alessia graduated in Biological Sciences and Biomolecolar Sciences from University of Turin. She went then and did her PhD in Science at ETH Zurich. Afterward she took a post-doctoral fellowship in Paris in the research unit directed by Dr. Prof. Benedita Rocha. Currently she is a senior post-doc at the Pasteur Institut in Paris in the group of Dr. Marc Lecuit.

Journal Reference

PLoS One. 2015;10(10):e0140849.

Galgano A¹, Barinov A¹, Vasseur F¹, de Villartay JP², Rocha B¹.

Show Affiliations

1. INSERM, U1020, CNRS, UMR 8253, Medical Faculty Paris Descartes, Université Paris Descartes Sorbonne Paris Cité, Paris, France.
2. INSERM, UMR 1163, Institut Imagine, Hôpital Necker-Enfants Malades, Paris, France.

Abstract

Immune responses are efficient because the rare antigen-specific naïve cells are able to proliferate extensively and accumulate upon antigen stimulation. Moreover, differentiation into memory cells actually increases T cell accumulation, indicating improved productive division in secondary immune responses. These properties raise an important paradox: how T cells may survive the DNA lesions necessarily induced during their extensive division without undergoing transformation. We here present the first data addressing the DNA damage responses (DDRs) of CD8 T cells in vivo during exponential expansion in primary and secondary responses in mice. We show that during exponential division CD8 T cells engage unique DDRs, which are not present in other exponentially dividing cells, in T lymphocytes after UV or X irradiation or in non-metastatic tumor cells. While in other cell types a single DDR pathway is affected, all DDR pathways and cell cycle checkpoints are affected in dividing CD8 T cells. All DDR pathways collapse in secondary responses in the absence of CD4 help. CD8 T cells are driven to compulsive suicidal divisions preventing the propagation of DNA lesions. In contrast, in the presence of CD4 help all the DDR pathways are up regulated, resembling those present in metastatic tumors. However, this up regulation is present only during the expansion phase; i.e., their dependence on antigen stimulation prevents CD8 transformation. These results explain how  CD8  T cells maintain genome integrity in spite of their extensive division, and highlight the fundamental role of DDRs in the efficiency of CD8 immune responses.

Go To PLoS One

 

 

 

 

PCSK9 deficiency unmasks a sex- and tissue-specific subcellular distribution of the LDL and VLDL receptors in mice

Are PCSK9 antibodies (Praluent, Repatha) for hypercholesterolemia treatment more efficient in men than women?

Significance Statement

 In 2015, monoclonal antibodies (mAbs) directed against PCSK9, and preventing its interaction with the LDLR, were made available as Praluent (alirocumab, Sanofi & Regeneron) or Repatha (evolocumab, Amgen). They are subcutaneously injected every two weeks and lower LDLc to levels never achieved before (by ~60%), making them the biggest weapon against cardiovascular disease since the advent of statins. In 2003, we identified the proprotein convertase subtilisin-kexin 9 (PCSK9) and established that its gene is the 3rd one known to be involved in FH after those encoding the LDL receptor and apoB. Indeed, PCSK9 enhanced degradation of the LDLR in endosomes/lysosomes, with ensuing high LDLc. In agreement, healthy individuals lacking functional PCSK9 exhibited a spectacular ~80% drop in their LDLc (~0.4 mM), thus reinforcing the validity of PCSK9 as an excellent target to treat hypercholesterolemia.

The absence of PCSK9 leads to a sex- and tissue-specific subcellular distribution of the LDLR Like humans, mice lacking PCSK9 exhibit very low levels of cholesterol. In these mice, the fraction of the LDL receptor present at the cell surface of hepatocytes seems to be regulated in a sex-dependent manner. In the liver of male and female mice lacking PCSK9, we observed a similar increase in the total amount of LDL receptor. However, its subcellular distribution differed in a sex-dependent manner: cell surface levels of the LDL receptor were dramatically increased in males, but not females.

Estrogens are responsible for the sex-dependent subcellular distribution of the LDLR Ovariectomized PCSK9-deficient females treated with placebo exhibited typical male patterns, with an accumulation of the LDLR at the hepatocyte cell surface. In contrast, those receiving 17β-estradiol (E2) maintained female patterns. Our working model is that the “eraser” effect of PCSK9 on cell surface LDL receptor is dominant, and that its presence masks the mechanism by which E2 regulates the surface levels of these two receptors.
Does a similar mechanism exist in women? If this E2-mediated regulation has its counterpart in humans and leads to a lower LDLR activity, the LDL uptake by the liver may differ in hypercholesterolemic men and women who receive therapeutic PCSK9 mAbs. Could this treatment have a lower efficacy in pre-menopausal women (with high E2 levels) than in men? Although >100 publications reported on clinical trials that led to the approval of PCSK9 mAbs to treat hypercholesterolemia, none of them compared the sex-dependent efficacy of these mAbs. However, the Clinical Briefing Document for alirocumab (FDA) revealed that, in a long term study, 1,438 men and 872 women (predominantly post-menopausal) responded by 65.5% and 53.4% reductions in LDLc, respectively, thus showing a 22% higher efficacy in men. Moreover, a subgroup analysis revealed that post-menopausal women responded with a 16% higher efficacy than pre-menopausal women (54.8% versus 47.3%). Thus, men seem to respond with a 38.5% higher efficacy than pre-menopausal (65.5% versus 47.3%). Independently, in the Amgen Clinical Briefing Document, a graph comparing the efficacy of evolocumab injection every two weeks versus that of ezetimibe in 46 women and 52 men also indicates a higher efficacy in men.

A thorough analysis of the clinical data accumulated in phase II and III trials concerning the possible sex-dependent efficacy of PCSK9 monoclonal antibodies will hopefully be soon available. If confirmed, the elucidation of the mechanism implicated will certainly be valuable.

About The Author

Anna Stepanova Roubtsova, M.D., M.S. Anna, did her medical studies in Yaroslavl State Madical Academy in Russia in 1986. She completed a Master in Experimental Medicine (McGill University, Canada) in 2008, and a Clinical Research Professional Development Program in 2009. She is now a Research Assistant at the Institut de Recherches Cliniques de Montréal (IRCM).

About The Author

Dr. Annik Prat received her PhD in molecular biology in 1988 from the University Pierre et Marie Curie in Paris, France. After 3 years at the Biozentrum in Basel, Switzerland, as a post-doctoral fellow, she became Associate Professor in Molecular Biology at the University Pierre et Marie Curie. Since 1997, she is a staff scientist at the Clinical Research Institute of Montreal (IRCM) and works in Nabil G. Seidah’s laboratory, in which PCSK9 was discovered.

Model for the E2 regulation of LDLR subcellular trafficking

PCSK9 exerts a dominant “eraser” effect on surface LDLR (left green panel). PCSK9 deficiency (KO) leads to ~3-fold higher LDLR levels in the liver (right red panel). In KO males, both homogenates and plasma membrane (PM) fractions show a ~ 3-fold increase of LDLR levels. In KO females, the increase is seen in homogenates only, not in PM fractions. E2 reduces the access of the LDLR to the plasma membrane or its residence time therein. Ovariectomy (Ovx) leads to a male pattern with high levels of cell surface LDLR. E2 treatment reverts this phenotype to a female one with LDLR being mostly intracellular.

Journal Reference

J Lipid Res. 2015 Nov;56(11):2133-42

Roubtsova A¹, Chamberland A¹, Marcinkiewicz J¹, Essalmani R¹, Fazel A², Bergeron JJ², Seidah NG¹, Prat A¹.

¹Laboratory of Biochemical Neuroendocrinology, Institut de Recherches Cliniques de Montréal (affiliated with the University of Montreal), Montreal, Quebec, Canada.

²Department of Medicine, McGill University Hospital Research Institute, Montreal, Quebec, Canada.

ABSTRACT

Proprotein convertase subtilisin kexin type 9 (PCSK9), the last member of the family of Proprotein Convertases related to Subtilisin and Kexin, regulates LDL-cholesterol by promoting the endosomal/lysosomal degradation of the LDL receptor (LDLR). Herein, we show that the LDLR cell surface levels dramatically increase in the liver and pancreatic islets of PCSK9 KO male but not female mice. In contrast, in KO female mice, the LDLR is more abundant at the cell surface enterocytes, as is the VLDL receptor (VLDLR) at the cell surface of adipocytes. Ovariectomy of KO female mice led to a typical KO male pattern, whereas 17β-estradiol (E2) treatment restored the female pattern without concomitant changes in LDLR adaptor protein 1 (also known as ARH), disabled-2, or inducible degrader of the LDLR expression levels. We also show that this E2-mediated regulation, which is observed only in the absence of PCSK9, is abolished upon feeding the mice a high-cholesterol diet. The latter dramatically represses PCSK9 expression and leads to high surface levels of the LDLR in the hepatocytes of all sexes and genotypes. In conclusion, the absence of PCSK9 results in a sex- and tissue-specific subcellular distribution of the LDLR and VLDLR, which is determined by E2 levels.

Go To Journal of Lipid Research 

 

An inducible transgenic mouse breast cancer model for the analysis of tumor antigen specific CD8+ T-cell responses

Significance Statement

 Immune therapy is a promising approach for improving the treatment of cancer. However, the major obstacles in its successful application, the tumor-induced mechanisms that lead to immune-evasion, have not been satisfactorily resolved. Analysis of the immune status of a given tumor entity and identification of the obstructed immune checkpoints thus are crucial issues for the development of immune-therapeutic anti-cancer strategies. Due to the limited possibilities for analyzing the respective parameters in humans, suitable animal models should be of great value. However, animal models that allow the analysis of how the growth of naturally arising tumors can be controlled by inducing tumor antigen specific immune responses are scarce.

In this study we tested the suitability of our BALB/c mouse based transgenic WAP-T animal model for mammary carcinoma to study tumor antigen specific CD8⁺ T-cell responses during tumor growth and progression, as well as parameters which obstruct a successful immune response. In WAP-T mice, tumorigenesis is induced in the adult mammary gland by expression of viral oncogenes (the SV40 tumor antigens) in parity-induced epithelial mammary gland progenitor cells. Tumorigenesis and the ensuing invasive carcinomas have been well characterized in previous studies, and WAP-T mice have been validated by cross-species comparison as a suitable animal model for human triple-negative mammary carcinomas. For this study we extended our WAP-T mouse lines by WAP-T_NP mice, in which the transgene additionally codes for the NP_118-126-epitope contained within the nucleoprotein of lymphocytic choriomeningitis virus (LCMV), resulting in the expression of a chimeric T-Ag/NP protein (T-Ag_NP). We thus were able to compare immune responses against the “weak” (i.e. low affinity) T-cell epitopes of SV40 T-Ag expressed by WAP-T mice with those against the immune-dominant LCMV NP-epitope in T-Ag_NP expressed by WAP-T_NP mice.

While immunization of WAP-T mice with SV40 did not induce a measurable CTL immune response, immunization of WAP-T_NP mice with LCMV induced a strong response which led to transient tumor cell elimination. Most intriguingly, we found that WAP-T_NP mice mount an endogenous immune response (i.e. without immunization) against the LCMV NP-epitope, as elimination of CD8⁺ T-cells by anti-CD8⁺ antibodies or by irradiation promoted the outgrowth of tumors in WAP-T_NP mice.  WAP-T_NP tumor mice thus contain NP-epitope specific CD8⁺ T-cells, which, however, are only weakly active due to expression of the programmed death-1 protein (PD1), an important player in the PD1/PD-L1 axis of immune checkpoints obstructing CTL activity. Consequently, treatment of WAP-T_NP tumor with anti-PD1 antibodies largely restored their activity. This is demonstrated by the experiment shown in Fig. 1, where we exchanged endogenous lymphocytes of WAP-T_NP tumor mice by lymphocytes derived from different donor mice by adoptive transfer (see scheme in Fig. 1A). Fig. 1B, panel a shows the typical T-Ag_NP expression in nuclei of WAP-T_NP mammary carcinoma cells (red dots). No T-Ag_NP expressing cells can be seen in tumor areas of WAP-T_NP mice that had received lymphocytes from BALB/c mice infected with LCMV due to the presence of a highly active NP-epitope specific CTL population (Fig. 1B, panel b). Similarly, tumor areas from WAP-T_NP mice, which had received lymphocytes from anti-PD1 treated WAP-T_NP mice, were also devoid of T-Ag_NP expressing cells (Fig. 1B, panel c). Thus the anti-tumor activity of exhausted PD1 expressing CD8⁺ T-cells could be largely re-activated.

Comparative analysis of WAP-T and WAP-T_NP mice thus is suited to analyze the parameters leading to immune evasion of tumors expressing “weak” (WAP-T) or “strong” (WAP-T_NP) tumor antigen epitopes, and to test strategies for overcoming blockades in immune response. Furthermore, WAP-T_NP mice show that in the case of tumors expressing a “strong” T-cell epitope, immunization strategies might be developed which, together with strategies blocking immune checkpoints might lead to successful tumor elimination. 

Figure Legend

Elimination of T-Ag_NP expressing WAP-T_NP tumor cells in mammary carcinomas via adoptive transfer of NP-specific CTLs from LCMV infected mice or of re-activated exhausted CTLs from WAP-T_NP tumor mice. (A) Schematic display of immune therapies and (B) immune histologic analysis of T-Ag_NP expression in WAP-T_NP tumor cells in mammary glands of acceptor mice, whose own immune cells were eliminated by sublethal irradiation with 4 Gy. Strong elimination of T-Ag_NP expressing cells is seen after transfer of immune cells either from LCMV infected BALB/c (b) as donors or from WAP-T_NP tumor mice after “therapy” with anti-PD1 antibodies (c) as donors; untreated WAP-T_NP tumor mice served as positive controls (a).

 

 

 

 

 

 

 

 

 

Journal Reference

Oncotarget. 2015 Nov 17;6(36):38487-503.

Bruns M¹, Wanger J¹, Utermöhlen O², Deppert W^1,3. 

Show Affiliations

1.Heinrich-Pette-Institute, Leibniz-Institute for Experimental Virology, Hamburg, Germany.

2. Institute for Medical Microbiology, Immunology and Hygiene, Medical Center and Center for Molecular Medicine Cologne (CMMC), University of Cologne, Cologne, Germany.

3.Institute forTumor Biology, University Medical Center Hamburg-Eppendorf (UKE), University of Hamburg, Hamburg, Germany.

Abstract

In Simian virus 40 (SV40) transgenic BALB/c WAP-T mice tumor development and progression is driven by SV40 tumor antigens encoded byinducible transgenes. WAP-T mice constitute a well characterized mouse model for breast cancer with strong similarities to the corresponding human disease. BALB/c mice mount only a weak cellular immune response against SV40 T-antigen (T-Ag). For studying tumor antigen specific CD8+ T-cellresponses against transgene expressing cells, we created WAP-TNP mice, in which the transgene additionally codes for the NP118-126-epitope contained within the nucleoprotein of lymphocytic choriomeningitis virus (LCMV), the immune-dominant T-cell epitope in BALB/c mice. We then investigated in WAP-TNP mice the immune responses against SV40 tumor antigens and the NP-epitope within the chimeric T-Ag/NP protein (T-AgNP). Analysis of the immune-reactivity against T-Ag in WAP-T and of T-AgNP in WAP-TNP mice revealed that, in contrast to wild type (wt) BALB/c mice, WAP-T and WAP-TNP mice were non-reactive against T-Ag. However, like wtBALB/c mice, WAP-T as well as WAP-TNP mice were highly reactive against the immune-dominant LCMV NP-epitope, thereby allowing the analysis of NP-epitope specific cellular immune responses in WAP-TNP mice. LCMV infection of WAP-TNP mice induced a strong, LCMV NP-epitope specific CD8+ T-cell response, which was able to specifically eliminate T-AgNP expressing mammary epithelial cells both prior to tumor formation (i.e. in cells of lactating mammary glands), as well as in invasive tumors. Elimination of tumor cells, however, was only transient, even after repeated LCMV infections. Further studies showed that already non-infected WAP-TNP tumor mice contained LCMV NP-epitope specific CD8+ T-cells, albeit with strongly reduced, though measurable activity. Functional impairment of these ‘endogenous’ NP-epitope specific T-cells seems to be caused by expression of the programmed death-1 protein (PD1), as anti-PD1 treatment of splenocytes from WAP-TNP tumor mice restored their activity. These characteristics are similar to those found in many tumor patients and render WAP-TNP mice a suitable model for analyzing parameters to overcome the blockade of immune checkpoints in tumor patients.

=5750&pubmed-linkout=1″ target=”blank” ]Go To Oncotarget.