Alzheimer's disease (AD) is the leading cause of dementia in the elderly, affecting over 27 million people worldwide. AD represents a complex neurological disorder which is best understood as the consequence of a number of interconnected genetic and lifestyle variables, which culminate in multiple changes to brain structure and function. These can be observed on a gross anatomical level in brain atrophy, microscopically in extracellular amyloid plaque and neurofibrillary tangle formation, and at a functional level as alterations of metabolic activity. At a molecular level, metal dyshomeostasis is frequently observed in AD due to anomalous binding of metals such as Iron (Fe), Copper (Cu), and Zinc (Zn), or impaired regulation of redox-active metals which can induce the formation of cytotoxic reactive oxygen species and neuronal damage. Metal chelators have been administered therapeutically in transgenic mice models for AD and in clinical human AD studies, with positive outcomes. As a result, neuroimaging of metals in a variety of intact brain cells and tissues is emerging as an important tool for increasing our understanding of the role of metal dysregulation in AD. Several imaging techniques have been used to study the cerebral metallo-architecture in biological specimens to obtain spatially resolved data on chemical elements present in a sample. Hyperspectral techniques, such as particle-induced X-ray emission (PIXE), energy dispersive X-ray spectroscopy (EDS), X-ray fluorescence microscopy (XFM), synchrotron X-ray fluorescence (SXRF), secondary ion mass spectrometry (SIMS), and laser ablation inductively coupled mass spectrometry (LA-ICPMS) can reveal relative intensities and even semi-quantitative concentrations of a large set of elements with differing spatial resolution and detection sensitivities. Other mass spectrometric and spectroscopy imaging techniques such as laser ablation electrospray ionization mass spectrometry (LA ESI-MS), MALDI imaging mass spectrometry (MALDI-IMS), and Fourier transform infrared spectroscopy (FTIR) can be used to correlate changes in elemental distribution with the underlying pathology in AD brain specimens. Taken together, these techniques provide new techniques to probe the pathobiology of AD and pave the way for identifying new therapeutic targets. The current review aims to discuss the advantages and challenges of using these emerging elemental and molecular imaging techniques, and highlight clinical achievements in AD research using bioimaging techniques. Alzheimer's disease (AD) is the most common progressive age-related neurodegenerative disorder, affecting about 2% of the population in the developed world Mattson, . ClinicaAD is a complex multifactorial disorder associated with irregular protein aggregation  ablation of material off the surface that is then directed into an elemental analyzer, (b) ion generation within the sample, and (c) ion generation and ablation from the sample surface. Most of these techniques create spatially resolved elemental data by moving a flat specimen on a stage in precise intervals under the incident beam, and recording the change in analyte flux  that is associated with a specific element.A pulsed laser can be used to ablate material from a selected area of the sample surface and the gaseous plume swept into another instrument for elemental analysis. A laser ablation (LA) sampling accessory can be integrated with a more traditional atomic spectroscopy system for sensitive multi-elemental analysis. These are usually inductively coupled plasma (ICP) systems using mass spectrometry (LA-ICPMS), or optical emission spectroscopy (LA-ICPOES) for detection of the elements , onto a tissue surface under vacuum  measures the energy loss due to scattering processes when a low energy, monoenergetic electron beam interacts with a sample. When used in a transmission electron microscope, EELS can provide atomic-scale resolution with excellent detection limits although biological applications are limited  remains the most widely used metal imaging technique in the clinical setting . More recently a novel metal imaging PET approach has been developed, using radioactive coordination bis(thiosemicarbazonato)copper complex of 64Cu. This targets copper homeostasis and has been designed to bind selectively to amyloid plaques  is another technique which facilitates Complementary information regarding the role, uptake, transport, and storage of redox active metals associated with irregular protein abnormalities can be obtained using a combination of elemental imaging techniques, such as LA-ICPMS, and other biomolecular mass spectrometry imaging techniques such as laser ablation coupled with electrospray ionization mass spectrometry (LA-ESI-MS) or MALDI-IMS. While LA-ICPMS can be employed to identify the specific protein-bound metals, ESI-MS/MALDI enables the identification of the structure, dynamics and biological function of metal-protein complexes  which arises from the C=O stretching vibration, and the Amide II band (~1540 cm−1) which is due to the N-H bending and C-N stretching vibrations of the peptide backbone. The vibrational frequency of an aggregated protein is about 1620–1625 cm−1, owing to its hydrophobic environment  is another molecular imaging tool that can be combined with LA-ICPMS. These tools have been used to image the secondary structure of metal-protein complexes  and a mutant human presenilin 1 (PS1-dE9) both directed to CNS neurons. This mouse model develops amyloid pathology as well as learning and memory deficits by 6 months of age, independent of signs of neurodegeneration  compared to a young (2 month) mice  and Aβ-(1–42) are the most abundant amyloid peptides in APP23 transgenic mice encoding the hAPP751 with Swedish mutation  and nuclear magnetic resonance (NMR) to show the structural conversion of Aβ from a soluble α-helical protein to a fibrillar β-sheet protein (Barrow et al., In AD, Aβ plaques are formed by the transformation of Aβ from a soluble form through to an oligomeric intermediate, culminating in the formation of an aggregated, fibrillary structure Ii, . The molIn vitro FTIR analysis provided confirmatory evidence that soluble tau protein is natively unfolded and composed of random coil structures, whilst PHFs which are present in the AD brain have a greater level of β-structure (Berriman et al., Apart from the Aβ protein, FTIR spectroscopy has also been used to gain a greater understanding of the structural conformation of the tau protein, which is hyperphosphorylated in AD, leading to the formation of NFTs. in vivo analysis, such as MRI and PET, are invaluable in obtaining further understanding on the molecular mechanisms involved in the pathogenesis of AD and to confirm the diagnosis of AD through the identification of unique biomarkers present in the metabolome, lipidome and/or proteome. Additionally, the techniques described in this review have the potential to follow disease progression in AD patients from early to severe stages and assess the effect of novel therapeutic interventions which may retard, stop or reverse progressive neurodegeneration, the ultimate goal being a cure for this debilitating neurodegenerative disorder.Metal imaging techniques are currently primed to facilitate an understanding of the pathobiology of AD, as well as identifying novel diagnostics and therapeutics. Bioimaging techniques are important for elucidating the role of metals in neurodegenerative diseases generally and AD in particular. Advancements in methodology and improved spatial resolution and detection sensitivities are essential for greater insight into the localization and distribution of metal ions at the cellular and tissue level, and their role in disease development and progression. A combination of other imaging techniques such as ESI- and MALDI-IMS, FTIR spectroscopy, and clinical techniques allowing Nady Braidy, Christopher Marjo, Anne Poljak, Tharusha Jayasena, Nibaldo C. Inestrosa, and Perminder Sachdev wrote the draft, reviewed and interpreted the bioimages. Helen Rutlidge and Anne Rich processed the images. Nibaldo C. Inestrosa and Perminder Sachdev provided the conceptual foundation of the review, writing of drafts and interpretation of data.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Molecular dynamics simulations reveal different solvent shells and distance-dependent mobility of ions and solvent molecules. From this, we provide a molecular rationale of effective particle size, net charge and polarizability of the nanoparticles in solution.Molecular models of 5 nm sized ZnO/Zn(OH)2 core-shell nanoparticles in ethanolic solution were derived as scale-up models (based on an earlier model created from ion-by-ion aggregation and self-organization) and subjected to mechanistic analyses of surface stabilization by block-copolymers. The latter comprise a poly-methacrylate chain accounting for strong surfactant association to the nanoparticle by hydrogen bonding and salt-bridges. While dangling poly-ethylene oxide chains provide only a limited degree of sterical hindering to nanoparticle agglomeration, the key mechanism of surface stabilization is electrostatic shielding arising from the acrylates and a halo of Na Nanoparticle stabilization and functionalization constitute key steps to nanomaterial production. To account for the hindering of nanoparticle agglomeration, the role of stabilizing molecules has been characterized by two intuitive concepts: the uptake of solutes may be avoided by i) sterical hinderance and ii) electrostatic shielding. The same mechanistic concepts are commonly discussed to account for stabilizing dispersions of nanoparticles in solution. Both mechanistic pictures are well-established from experiments, e.g. by comparing particle size and hydrodynamic radius and by assessing the net charge of colloids from zeta-potentials ,2.In-depth understanding of the underlying processes on the molecular scale, that is the mechanisms of surfactant association to the particle, the structuring of solvent shells and the distribution of counter ions near a nanoparticle, is rather sparse. While hard to access experimentally, molecular simulations in principle appear very suited to tackle this issue. However, even when employing computationally efficient molecular models (rather than quantum chemistry approaches) the limited time- and length scales still inherent to molecular simulations call for smart simulation techniques. Such an approach was recently presented by Vlugt and coworkers using the example of metal nanoparticles stabilzed by alkylthiols ,4. While2 core/shell nanoparticles with more complex structures. To provide realistic models we build on a recent simulation study related to zinc and hydroxide ion association in ethanolic solution [2 core/shell nanoparticle was prepared as described in detail as supplementary information (8-b-MAA8)8-. While the polyethylenoxide chain (PEO) is uncharged, each of the acrylate units exhibits a -1 charge, thus resulting in a net charge of -8 per surfactant molecule. The MAA8 block is chosen for its good binding affinity to ZnO, whilst the EO8 block shows poor affinity to the nanoparticle (and is thus expected to be dangling into the solution).Molecular simulations related to ZnO nanoparticles synthesized from the vapor may be started from simply cutting hexagonal rods off the single crystal structure . Howeversolution , ripeninormation . The sur3 comprising 8442 ethanol molecules and subjected to periodic boundary conditions.Molecular dynamics simulations were performed on the basis of empirical interaction potentials adopted from earlier studies of ZnO nucleation from solution  and bism+ counter ions) to relax. The sodium ions are initially placed randomly. Subsequently, their positions are propagated by performing nanosecond-scale molecular dynamics simulations to ensure convergence of the ionic distribution in the simulation cell. Three independent runs of this kind were performed to provide at least qualitative account for the abundance of possible nanoparticle-surfactant structures possible even for a single ZnO/Zn(OH)2 nanorod model.The association of surfactant molecules to our nanocrystal models was treated in full analogy to the uptake of ions to a forming aggregate as reported earlier ,10,11. T8-b-MAA8)8- polymers that stably bind to the ZnO/Zn(OH)2 nanoparticle. Surfactant association is dominated by the acrylate units which form a COO- Zn2+ salt bridge and up to 1–2 COO- OH- hydrogen bonds per methylacrylate monomer. On the other hand, the PEO chains were found to not bind to the ZnO surface, but fluctuate considerably and are best described as dangling into the embedding solution  were placed in the solution and allowed to diffuse freely. While ion distribution was found to reach a dynamic equilibrium after a few tens of nanoseconds, additional 100 ns runs were used for the analyses described in the following. While the number of sodium ions was chosen to fully compensate the colloid charges, only a fraction form stable COO- Na+ salt bridges and thus ‘behave’ akin to the surfactant molecules. The remaining sodium ions were found to be loosely associated and are best described as a dynamic equilibrium of Na+ association to surfactant molecules, migration within a nm-sized halo around the nanoparticle and dispersion into the bulk solution . In-between, that is from 0 to 1.8 nm distance, Na+ ions are loosely associated and constitute a positively charged halo which also reduces the effective charge of the colloid.The counter ions :a. The same switching function is applied to attribute a weighted charge to the effective charge of the colloid:We suggest to use the distance dependence of ionic mobility for quantification of the degree of NaThis allows a reasonable estimate of the effectively colloid charge including contributions from its halo of loosely associated counter ions . Moreove+ countercharges  only, from a qualitative viewpoint it is nevertheless intuitive to suggest the observed mechanisms to be of general relevance for rationalizing electrostatic nanoparticle shielding. We would be enthusiastic to offer our ZnO-polymer data for comparison with experiments—or to provide similar modelling work for other systems.S1 FigAll data was averaged over 100 ns.(TIF)Click here for additional data file.S2 FigThe width of the Gaussian fits is used to estimate the polarizability of the halo of counterions.(TIF)Click here for additional data file.S3 FigNumbering of oxygen and hydrogen in (TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S1 Text(DOC)Click here for additional data file.


We aimed at investigating prospective memory and its socio-demographic and neurocognitive correlates in non-psychotic, first-degree relatives (FDRs) of patients with schizophrenia compared to patients with first episode schizophrenia (FES), and healthy controls (HCs).Forty-seven FES patients, 50 non-psychotic FDRs (23 offspring and 27 siblings) of patients with chronic schizophrenia (unrelated to the FES group) and 51 HCs were studied. The Chinese version of the Cambridge Prospective Memory Test (C-CAMPROMPT) was used to measure time-based prospective memory (TBPM) and event-based prospective memory (EBPM) performance. Other cognitive functions (involving respective memory and executive functions) were evaluated with standardized tests. = 10.4, p<0.001) and EBPM  = 10.8, p<0.001). Multiple linear regression analyses revealed that lower scores of the Hopkins Verbal Learning Test-Revised (HVLT-R) and the STROOP Word-Color Test (SWCT) contributed to TBPM impairment, while lower educational level and higher scores of the Color Trails Test-2 (CTT-2) contributed to EBPM deficit in FDRs.After controlling for basic demographic characteristics including age, gender and educational level, there was a significant difference between FDRs, FES and HCs with respect to both TBPM (FFDRs share similar but attenuated prospective memory impairments with schizophrenia patients, suggesting that prospective memory deficits may represent an endophenotype of schizophrenia. Schizophrenia is a frequently debilitating illness, manifesting primarily with positive and negative psychotic symptoms and cognitive impairment. While a wide range of neurocognitive deficits occur in schizophrenia, the impairments are especially prominent in psychomotor speed, memory, attention, reasoning, and social cognition. Memory impairment has been studied extensively PM, defined as “memory for activities to be performed in the future”, is usually subdivided into time-based PM (TBPM) and event-based PM (EBPM) according to the nature of the cue associated with the future intention. TBPM is the ability to remember carrying out an intention at a specific time in the future. EBPM is defined as the ability to remember performing an intended action when a certain cue appears PM deficits have been consistently found in both chronic and first episode schizophrenia Schizophrenia has a strong heritable component The aims of the current study were (1) to determine whether schizophrenia patients’ first-degree relatives (including siblings and offspring) show impairment in TBPM and EBPM compared to healthy controls and to FES; and (2) to clarify the relationship between PM performance and demographic as well as clinical variables.The first hypothesis was that FDRs would share similar but attenuated PM impairments with FES. The second hypothesis was that RM and executive functions would be significantly correlated with PM tasks in FDRs.FDRs (23 offspring and 27 siblings) of schizophrenia patients were recruited as unaffected family members. Further, 47 first-episode schizophrenia patients from either outpatient or inpatient facilities in Beijing Anding Hospital (unrelated to the FDR group) were recruited. FES and FDRs were consecutively referred to the research team for screening of eligibility by their treating psychiatrists (for FES) or their psychotic relatives’ treating psychiatrist (for FDRs). A group of 51 healthy controls (HCs) matched with the FES group by age (±2 years) and education (±2 years) was recruited from the community through advertisements.Familial high-risk individuals matched to the FES group by age (±2 years) and education (±2 years) and fulfilling the following study criteria were invited to participate in the study. (1) Offspring or siblings of patients with schizophrenia according to DSM-IV All patients with first-episode schizophrenia fulfilled the criteria of the Diagnostic and Statistical Manual of Mental Disorder-fourth edition (DSM-IV) HCs without history of or current psychiatric disorders, medical and neurological conditions were also recruited. The absence of psychiatric disorders was established with SCID-DSM-IV. The study protocol was approved by the Clinical Research Ethics Committee of Beijing Anding Hospital. Written informed consent was obtained from each participant.PM was assessed with the validated Chinese version of the Cambridge PM Test (C-CAMPROMPT) Other neuropsychological assessments, targeting RM, verbal fluency, visual attention, and cognitive flexibility included:Hopkins Verbal Learning Test-revised, Chinese version . The CTT Stroop Color Word Test (SCWT), Chinese version;Wisconsin Card Sorting Test (WCST-64). The computerized version of the WCST-64 was adopted to assess the prefrontal lobe function of reactive flexibility All participants were tested in Beijing Anding Hospital. Neuropsychological tests were administered first in a randomized order to minimize the possibility of order effects. After a short break, the C-CAMPROMPT was administered.The whole assessment lasted 1–2 hours and was completed on the same day for controls and relatives and within two consecutive days for patients, as some patients could not complete both tests and tasks on the same day.All participants were asked to repeat the instructions before beginning the C-CAMPROMPT to ensure that they understood what was expected of them. The instructions were repeated and explained to participants who seemed uncertain about the nature of the test. Upon completion of the C-CAMPROMPT, participants were again asked to repeat the requirements of the test to make sure they remembered and understood the instructions. Two psychiatrists (FCZ and WMH) who were trained to assess PM and other cognitive functions administered the PM and other neurocognitive tests.The data were analyzed using SPSS, Version 20.0 for Windows. Comparisons between FDRs, FES and HCs with regard to socio-demographic characteristics and mean scores of all neurocognitive tests were performed using independent sample one-way ANOVA, ANCOVA, Mann-Whitney U tests and chi-square tests, as appropriate. Comparisons between siblings and offspring regarding TBPM and EBPM were performed using independent-samples t-tests. Pearson’s or Spearman rank correlation analysis was performed to determine the associations between performance on TBPM and EBPM tasks and socio-demographic and other neurocognitive tests. Factors independently associated with the performance on the TBPM and EBPM in FDRs were identified with stepwise multiple linear regression analyses. The normality of distributions for the continuous variables was checked with the one-sample Kolmogorov-Smirnov test. Two-tailed tests were used in all analyses with the significance level set at 0.05. = 10.4, p<0.001) and EBPM   = 10.8, p<0.001). In addition, we compared TBPM and EBPM between the siblings and offspring and did not find any significant difference between these two subgroups  independently contributed to poorer TBPM performance, while lower educational level, and higher scores of CTT-2 contributed to worse EBPM performance .Confirming our first hypothesis, FDRs performed significantly poorer on both TBPM and EBPM compared to HCs, and shared a similar pattern of PM performance with schizophrenia patients Previous studies found that FDRs of schizophrenia patients are a heterogeneous group regarding their risk for developing schizophrenia. The risk in offspring is approximately 13%, much higher than that in schizophrenia patients’ parents (6%) and siblings (9%) Neurocognitive impairments have been observed in both siblings Neuroimaging studies have detected prefrontal and temporal grey matter deficits in monozygotic twins discordant for schizophrenia, siblings and other close relatives of participants with schizophrenia; and these deficits may be even progressive Regression analyses for the FDRs group showed that HVLT scores independently contributed to TBPM in FDRs. This may be related to the fact that PM involves a retrospective component In this study, multivariate analyses revealed that prefrontal dysfunction plays a key role in the PM impairment in FDRs of schizophrenia patients. This finding is supported by neuroimaging studies. Increased activity during PM tasks is seen in the anterior and lateral prefrontal cortex, while decreased activity is observed in the medial prefrontal cortex using functional MRI Regression analyses also indicated that although PM deficits could be attributed to dysfunctions in the frontal cortex in FDRs, TBPM and EBPM may depend on different neurocognitive processes. While TBPM was predicted by SCWT, EBPM was predicted by CTT-2. Brain imaging studies have shown that the anterior cingulate cortex and the dorsolateral prefrontal cortex are activated by the Stroop test. Specifically, while the left dorsolateral prefrontal cortex activation is related to the expectation regarding the conflicting nature of the upcoming trial, the right dorsolateral prefrontal cortex is activated when attempting to reduce the attentional conflict The results of our regression analysis, together with the aforementioned line of evidence, suggest that the prefrontal and temporal cortices may act as an integrated circuit during PM tasks in FDRs of schizophrenia. This hypothesis seems to be supported by a previous study, which showed that the intermediate CA1 region of the hippocampus and the medial prefrontal cortex interact in coordinating RM and PM processes in anticipation of obtaining a remote goal The strengths of this study include the relatively large sample size that comprised all types of FDRs and pertinent cognitive assessment battery. However, due to methodological limitations, the results should be interpreted with caution. First, controls and relatives were matched with patients separately in terms of age and education, but the three groups still had significant differences in education level. Nevertheless, findings remained significant even after controlling for the potentially confounding effect of educational level. Second, in multivariate analyses the significant variables only accounted for 30.2% and 17.0% of the variance in TBPM and EBPM, respectively. In order to explore the impact of more variables on PM, a larger sample size will be required in future studies. Third, the cross-sectional design could not explore causal relationships between PM and other variables. Fourth, analyses comparing siblings and offspring of schizophrenia patients were limited by the small sample size of these subgroups.In conclusion, the major findings of this study are: (1) FDRs of schizophrenia patients showed significant impairment in PM; (2) RM deficits and neurocognitive markers of prefrontal dysfunction independently correlated with PM impairment; and (3) both FES and FDRs of schizophrenia showed PM impairment suggesting that PM may be an endophenotype for schizophrenia.


Multiple myeloma is the second most common hematologic malignancy. The treatment of this disease has changed considerably over the last two decades with the introduction to the clinical practice of novel agents such as proteasome inhibitors and immunomodulatory drugs. Basic research efforts towards better understanding of normal and missing immune surveillence in myeloma have led to development of new strategies and therapies that require the engagement of the immune system. Many of these treatments are under clinical development and have already started providing encouraging results. We, for the second time in the last two decades, are about to witness another shift of the paradigm in the management of this ailment. This review will summarize the major approaches in myeloma immunotherapies. Several attempts to control the disease using variety of immune modulation have been made. In the following review, we will summarize the current knowledge of the immune dysfunction in MM and the development of various immune modalities to eradicate the disease. We will review the current uses of immunomodulatory drugs, monoclonal antibodies, various vaccination strategies, autologous activated NK and T cells, engineered T cells and the evolving role of checkpoint inhibitors.Multiple myeloma (MM) is a malignant disease of plasma cells that accounts for about 1% of new cancers and 10% of all hematological malignancies. It affects elderly patients with a median age of 67 years at presentation. The introduction of several novel agents and targeted therapies has significantly improved outcomes for patients with MM. Although few remain in long-term remissions, the majority of patients relapse and die from the disease, usually with a resistant clone. Immune therapy, in the context of an allogeneic graft, has for years been considered the only curative approach for the disease. Unfortunately, allografts are associated with high mortality and overall poor quality of life due to the development of graft It is well established today that all MM patients have a pre-existing none-malignant stage known as monoclonal gammopathy of unknown significance (MGUS) . The mecAlthough myeloma is primarily a disorder of the B cell lineage, the T cell compartment is frequently affected . This deet al. have demonstrated via a population based study that the infection risk even at preclinical stage ie MGUS was increased two folds in 5 and 10 year follow up periods including both bacterial and viral infections [MM immune dysregulation affects other aspects of the immune system as well, directly affecting antigen presentation and up-regulation of inhibitory antigens that promotes immune escape and growth advantage for malignant clones. On the antigen presenting side, elaborate studies on different aspects of dendritic cell (DC) biology have revealed somewhat conflicting results. Some studies have reported defects in peripheral blood DCs such as decreased numbers of circulating peripheral blood monocytes, plasmacytoid DCs (pDCs) and myeloid DCs (mDCs), lower expression levels of both MHC class II (HLA-DR) and costimulatory molecules CD40, CD80) as well as decreased alloreactivity against lymphocytes particularly in the setting of IL-6 inhibition . Other s0 as wellfections .versus-myeloma”; a proof of principle that the immune system can eradicate a malignant clone has unfortunately limited clinical success primarily due to lack of a predictable uniform and potent response as well as to complications of the procedure including high treatment related mortality [Standard treatments for MM include standard and high-dose chemotherapy, proteasome inhibitors and IMiDS which usually are given in combinations in conjunction with corticosteroids in the absence or presence of stem cell support. These treatments have radically changed the disease history and improved overall response rates and survival. However, the disease remains incurable and relapse is inevitable in majority of patients. Immunotherapy for 30 years, in the form of an allogeneic stem cell transplant , has been the only treatment modality associated with long-term complete remissions and possibly cures in MM . An effeortality ,12.versus-myeloma” effect that can benefit the majority of MM patients have been explored. In the following paragraphs we will review immunotherapies as a promising strategy in eliminating the malignant clone in MM focusing on three approaches:(1)Immunomodulatory drugs .By blocking inhibitory molecules or, alternatively, activating stimulatory molecules, these treatments are intended to enhance pre-existing anti-myeloma immune responses. .Cytokines .Reverse tumor mediated immune paralysis(2)MM Vaccines .Adoptive T Cell Transfer  T cells using BCMA and CD-19 or TCR cells engineered to target the NY-ESO-1 antigen).Stimulate myeloma specific immune responses(3)Monoclonal antibodies .Selectively eliminate the malignant clone.Several innovative approaches to enhance the immune system to fight MM and stimulate a “host-IMiDs is a class of drugs that directly affect MM cells and bone marrow microenvironment leading to modulation of cytokines, inhibition of angiogenesis, and augmentation of immune effector numbers and function . Recently interaction of IMiDs with cereblon, a ubiquitin ligase component responsible for substrate binding was shown to be crucial for direct cytotoxic and immune related effects . T-cell ex vivo with idiotypic proteins (Mylovenge) in a way similar to the FDA approved prostate cancer treatment sipuleucel (Provenge) reported an almost 2 year overall survival benefit yet had study limitations [Two separate vaccination approaches have been developed. The first approach is peptide-based vaccines. The pioneer of these approaches, use of idiotype proteins (Id), an attractive concept as it is, did not meet expectations as potential targets possibly secondary to poor immunogenic nature of the protein as well as low expression of these proteins on the plasma cell surface . There hitations . There aOn the other hand, subsequent identification of tumor associated antigens such as MAGE, NY-ESO1, WT-1, RHAMM-R3 and XBP-1 and their use as targets was able to generate cellular responses when used individually and/or in combination in preclinical studies. Clinically, initial results using a single peptide based vaccine demonstrated that such vaccines can be used with few adverse effects and can elicit immune responses but with modest effect on disease control . ParticuThe second vaccination approach involves dendritic cell/myeloma cell fusion (DC/MM). This strategy takes advantage of the ability of the DC to present several antigens from the cell to the host . DC/MM fThe development of effective cytotoxic mAb therapies in MM has been hindered by the lack of distinctively and constitutively expressed target molecules on malignant plasma cells. Studies early after the turn of millennium demonstrated only minimal activity of anti-CD20 rituximab which is expressed on 20% of plasma cells. This was followed by several mAbs . For the purpose of this review we will focus on 2 mAbs that have already demonstrated promising clinical activity in MM.in vitro activity against myeloma cells, killing myeloma cells via antibody dependent cellular cytotoxicity (ADCC) while using the same receptor for activation of NK cells [CS-1 is a transmembrane glycoprotein expressed on normal and malignant plasma cell membranes as well as on NK cells . An IgG1NK cells ,26. ThisNK cells . The iniNK cells , howeverNK cells . RecentlNK cells . InteresNK cells . It is aCD38 is a type II transmembrane glycoprotein with multiple proposed functions in cell adhesion, signaling and enzymatic (cellular nucleic acid metabolism) activity and is expressed on a multiple hematopoietic and non-hematopoietic cell types. Among many hematopoietic cells that harbor this antigen are medullary thymocytes, subpopulations of both activated B and T lymphocytes, NK cells and dendritic cells . Daratumet al., impressive clinical responses were seen in heavily pretreated patient population with 64% double refractory to PIs and IMiDs and had undergone ASCT in 76% [In phase I/II study recently published by Lokhorst T in 76% . DaratumTwo additional antibodies that target CD38 are SAR650984 (isatuximab) and MOR03087  which are under clinical development as monotherapy or in various combinations.Several new mAbs are under development for various cell member targets and others are in early stages but few are worth mentioning. The first is B cell maturation antigen (BCMA), a protein of TNF receptor superfamily which is crucial for long term survival of plasma cells through its binding B cell activating (BAFF) and proliferating (APRIL) factors . SeveralA new area of antibody research has recently focused on bispecific T cell engagers (BiTEs) that combine specificities of two antibodies by simultaneous binding to multiple epitopes, one of which involves the engagement and activation of T cells via their CD3 molecules  The firsImmune checkpoint inhibitors targeting PD-1  on T cells or its cognate ligand, PD-L1 on tumor cells have established activities in many different types of cancers . Whereasex vivo co-stimulation of autologous T-cells via immunomagnetic beads (anti-CD3/CD28 beads) which in the presence of interleukin-2 led to significant activation and expansion of T cells. Infusion of these cells after myeloablative bone marrow conditioning and autologous stem cell transplantation led to early lymphocytosis. In trials using peripheral blood there is no clear evidence for a tumor specific T cell enhancement effect and no impact on outcome. This was probably related to nonspecific stimulation of the entire T cell repertoire including regulatory T cells. However, it was also noted during early trials that vaccination responses were enhanced in patients receiving ex vivo expanded T cells. This led to a series of trials using various antibodies  all of which were associated with enhanced antibody and cellular immunity against the vaccine. In the MAGE antibody trial, vaccine-specific cytokine-producing T cells were detected in 19 of 25 patients (76%), however, the clinical outcomes were not correlated with the generated immunity [Early studies suggested that early lymphocyte count recovery after auto-SCT correlated with improved disease control ,47. Seveimmunity . This maRecently, Borrello and colleagues have shown for the first time that expanding the subset of marrow-infiltrating T lymphocytes (MILs) can led to clinical antitumor immunity . The resin vivo expansion and memory cell generation confers these cells a clear advantage over naked antibodies or adoptively transferred tumor-reactive T cells. A successful example of CD19 targeted CAR—T cell approach was recently published suggesting activity of this therapy [et al. have observed a low, nevertheless a more frequent expression than previously reported on malignant plasma cells and targeting this population via the use of CTL019 cells (lentivirus transduced autologous T cells harboring CD3-zeta/CD137 based anti-CD19 chimeric receptor), reported remission in a 43 year old with 9 prior lines of treatment [etc.) will be needed to eliminate the malignant clone.Single chimeric antigen receptor T-cell based therapy (CART), represents a huge leap in immune therapy. CART cells, constructed by fusing the single chain variable fragment (scFv) of a monoclonal antibody (mAb) specific for a surface antigen with an intracellular signaling domain have shown activity in several CD-19 related disease. The MHC-independent tumor recognition,  therapy . Even threatment . In thisreatment . Again, reatment . AdditioInfusion of auto-engineered T cells with affinity enhanced TCR specific for a common peptide shared between two cancer testis antigens (NY-ESO-1 and LAGE-1) in MM was recently reported . PatientInterleukin-6 (IL-6) is a cytokine that has long been under spotlight in myeloma since late 80s as a driver for myeloma which raised the question of benefit of targeting this cytokine for theurapeutic purposes . A phaseInterleukin-15 (IL-15) is a cytokine that has a critical role in CD8 memory cell and NK cell development, proliferation and activation, making it an attractive target. A complex with superagonistic activity against this cytokine, ALT-803 was recently developed which in preclinical models was able to employ specifically CD8 memory cells . Whereas−5 malignant plasma cell burden which we are not currently able to treat [Myeloma is a disease which has seen four decades of treatment with alkylating agents and steroids with barely any improvement in response statistics. Following the advances in basic and translational research, introduction of novel agents, particularly combination therapies, improved indicators of quality of life and survival significantly. With a median survival of over 5 years and counting, finally our attentions are turned to different strategies to obtain deeper and more durable remissions by starting treatment at asymptomatic stages and incorporating minimal residual disease (MRD) to IMWG response criteria. However, it is important to realize that even at the MRD stage, one still has about one in 10to treat . It is p


Protein-RNA complexes provide a wide range of essential functions in the cell. Their atomic experimental structure solving, despite essential to the understanding of these functions, is often difficult and expensive. Docking approaches that have been developed for proteins are often challenging to adapt for RNA because of its inherent flexibility and the structural data available being relatively scarce. In this study we adapted the RosettaDock protocol for protein-RNA complexes both at the nucleotide and atomic levels. Using a genetic algorithm-based strategy, and a non-redundant protein-RNA dataset, we derived a RosettaDock scoring scheme able not only to discriminate but also score efficiently docking decoys. The approach proved to be both efficient and robust for generating and identifying suitable structures when applied to two protein-RNA docking benchmarks in both bound and unbound settings. It also compares well to existing strategies. This is the first approach that currently offers a multi-level optimized scoring approach integrated in a full docking suite, leading the way to adaptive fully flexible strategies. Protein-RNA interactions often play a major role in the cell. They are involved in many processes such as replication, mRNA transcription or regulation of RNA levels and control the operation of key cellular machineries such as the RNA induced silencing complex (RISC). They are thus good candidates for therapeutic studies http://capri.ebi.ac.uk) challenge The structural modelling of large biomolecules and their interactions is a challenging task. A large number of methods for both predicting and evaluating the results have been developed Protein-RNA complexes are especially difficult to predict and model for two reasons: the inherent flexibility of RNA molecules and the electrostatics driving the binding as the RNA molecule is negatively charged. Progress in RNA structure prediction and folding The availability of structural data is essential for machine learning based strategies for scoring in docking experiments. Various machine learning strategies have been developed in the past for protein-protein complexes Protein-RNA native X-ray structures for learning were downloaded from the Protein-RNA Interface Database (PRIDB) We also used the two protein-RNA benchmarks From all the native structures from both the PRIDB and the benchmarks, near-native and decoy conformations are generated using the Rosetta perturbation protocol The RosettaDock protocol is two-level docking search: low resolution and high resolution. The low resolution stage uses a coarse-grained representation of the partners to quickly sample the search space for candidates. The high resolution stage rebuilds the all-atom partners from the low resolution candidates to perform a refined atomic search possibly including rotamer search and loop optimization.ContactS represents the number of interface residues being defined by having a centroid less than 6 Å away from a centroid in the other partner; BumpS is a distance-based penalty for steric clashes; EnvS defines the probability of finding a residue in a specific environment (buried/exposed and interface/non interface); PairS is a pair potential defining the propensity of residues to be found in interaction in given environments and AlignS is an optional term to match a specific alignment pattern (e.g. antibodies).The low resolution scoring function uses the backbone of the molecule and one centroid per residue These five terms of the low resolution score can be computed for protein-RNA complexes in the same way they were for proteins. For RNA, the backbone is chosen to include the sugar ring and the centroid is taken to be the center of mass of the base. All the parameters for the low resolution scoring terms are computed on the PRIDB reference set.VdWS is a Van der Waals term (Lennard-Jones based), ElecS is a Coulomb term, SolvS a solvent term based on the Lazaridis-Karplus model, HbondS is a H-bond 10–12 potential term, SASAS is the solvent accessible surface area term (often omitted), PairS is a pair potential defining the propensity of residues to be found in interaction in given environments and RotamerS is a probability of finding a specific rotamer. Exactly like for the previous low resolution scores, all the terms can be computed for RNA. The rotamer term and loop optimization are switched off for RNA such as in The high resolution scoring function uses all the atoms of the molecules, including the hydrogen atoms, and is made of seven weighted terms:The low resolution representation for each residue/nucleotide is made of the backbone atoms and one pseudo-atom called centroid to represent the side-chain. For the residues, the location of the centroid is taken from RosettaDock (average over a reference set of PDB structures). For RNA nucleotides, the centroid is taken as the averaged position See . The lowWe performed the optimization by supervised learning. To ensure an accurate learning phase, the perturbation was split in two categories for learning labelled near-native (Irmsd<5 Å) and decoy (Lrsmd>8 Å). The assessment was performed using slightly different categories so as to mimic the CAPRI context: near-native (Irmsd<5 Å) and non-native (Irmsd≥5 Å). While these rmsd range are certainly not always likely to accurately represent a correct RNA binding mode, especially considering the variability in size of the RNA molecules, they represent a reachable goal not yet attained by the CAPRI community.Weights for the all atom scoring function described above were optimized in the [0∶1] interval within the ROC-based Genetic LearneR (ROGER) framework using logistic regression and Receiver Operating Characteristic (ROC) based genetic algorithm as previously described for protein-protein docking pdb-out approach. Inspired by the leave-one-out procedure in statistics, we previously used this strategy for machine learning of protein-protein docking scoring functions The first evaluation of the whole scoring procedure is made using cross-validation and a leave-one-To also avoid biasing the samples towards a category while learning, learning is performed with 30 near-native and 30 decoy structures for each of the 120 pdb file leading to a total size of 7,200 structures for the learning set . Test is performed on the 10,000 candidates of each test pdb file. The global procedure flowchart is available in topE10% is the top 10% scoring and topR10% the best 10% rmsd structures. By looking at the degree of overlap between the two categories, the enrichment score provides insight on how good the scoring is ES<1 corresponds to bad scoring, ES = 1corresponds to random scoring and ES = 10 is perfect scoring. Even if what can be considered good scoring is not obvious, the comparison of ES values between 1 and 10 provides good information on how well the strategy performs on different targets.The learning procedure is initially assessed using standard machine learning criteria: analysis of the ROC curve, ROC-AUC in a cross-validation setting and precision for the top 10 structures. CAPRI/Critical Assessment of protein Structure Prediction (CASP) inspired biological criteria are used for the final assessment: Energy vs. Irmsd curve and Enrichment Score (ES). Interface root mean square deviation (Irmsd) is taken from Lensink et al. A data based docking procedure for protein-RNA complexes should first be able to recover the native and close-to-native states for a reference set of complexes. This is assessed by a careful cross-validation setting. In this study we assessed the performance of our learning procedure by plotting Energy vs. Irmsd and checking the enrichment scores of our procedure relatively to the Rosetta CAPRI default. pdb-out setting. The machine learning procedure was also assessed separately by plotting the ROC curves in the leave-one-The strategy was then further evaluated on the Benchmark I and Benchmark II protein-RNA complex structures in a bound setting. The RosettaDock perturbation generation for the conformations ensures that the packing at the interface is relatively correct. Visual inspection shows that the conformations of best energy conformations are relevant from a biological perspective . In a bound setting, for protein-RNA, the relative influence of the parameters shows that the H-bond network is extremely important and must be maintained. st and 3rd quartile ROC performance were assessed in a full docking run unbound setting . Results can be found in The scoring function was then assessed in both bound and unbound (protein and RNA when available) settings. Perturbation runs were performed in a bound setting on the 40 complexes of the benchmarks not in the reference set. Only the 6 pdb files corresponding to median, 1A current limit of our approach is the way RNA flexibility is handled. Handling RNA flexibility for RNA during docking is a very difficult task Protein-RNA complexes are undoubtedly a real challenge for the design of good docking scoring functions. Using a well curated dataset and a well-designed optimization strategy, we show that we could set up of an efficient protein-docking scoring function that can be used in RosettaDock and that can perform better than the existing option in both bound and unbound settings. While scoring can be improved, the nature of RNA makes the prediction experiment still difficult. Electrostatics plays a large role in RNA interactions and ions have to be modelled. Like ours, the data based approaches are limited by the relatively small number of structures available to take ions into account carefully. RNA flexibility modelling for docking is then the next challenge: while some strategies allow for conformation sampling, selection of one or several putative bound states for large cross-docking experiments are still out of reach for both modelling and computational reasons.http://albios.saclay.inria.fr/rosettadockrnaThe source code and files needed to modify RosettaDock 3.4 are available at: Figure S1Model of a nucleic acid (uracile). The phosphate group and the sugar heavy atoms are depicted in gray: (a) coarse-grained level with the centroid atom in red and (b) full-atom level with the base atoms in blue. The centroid is the geometric center of the heavy atoms.(TIFF)Click here for additional data file.Figure S2Energy vs Irmsd for the whole reference dataset in a leave-one-pdb-out setting.(PDF)Click here for additional data file.Figure S3Energy vs Irmsd for the benchmark set in a bound setting. The 10,000 conformations evaluated for our optimized Rosetta scoring function are shown in black. On each plot, the bottom left panel shows the equivalent non-optimized Rosetta result.(PDF)Click here for additional data file.Figure S4Energy vs Irmsd for the unbound test set. The 10,000 conformations evaluated for our optimized Rosetta scoring function are shown in black. On each plot, the bottom left panel show the equivalent non-optimized Rosetta result.(PDF)Click here for additional data file.Figure S5Structure of the mRNA binding domain of elongation factor SelB from E.coli in complex with SECIS RNA (PDB code 2pjp). Mg2+ ions (shown in yellow) are located at the interface and mediate the interaction.(TIFF)Click here for additional data file.Table S1Protein-RNA complexes reference set from the PRIDB. The rightmost column indicates putative redundancy with the docking benchmarks. The Type column refers to the structural family of the RNA molecule: single strand RNA (ssRNA), double strand RNA or single-stranded RNA of helical/paired structure (dsRNA) or transfer RNA (tRNA).(PDF)Click here for additional data file.Table S2Protein-RNA complexes for the test set. For each complex the unbound column for protein and RNA reports the PDB code of the unbound structures when available. The difficulty codes are taken from (PDF)Click here for additional data file.Table S3Leave-one-pdb-out scoring statistics for the reference dataset. Enrichment Score, 10 best energy candidates, 100 best energy candidates, number of near-native structures and Area Under the ROC Curve are reported for each native structure both using the non-optimized RosettaDock scoring function (Default) and our optimized scoring function (ROGER).(PDF)Click here for additional data file.Table S4Scoring results on the bound benchmark test set. Enrichment Score, 10 best energy candidates, 100 best energy candidates, number of near-native structures and Area Under the ROC Curve are reported for each native structure both using the non-optimized RosettaDock scoring function (Default) and our optimized scoring function (ROGER).(PDF)Click here for additional data file.


The VHH domain is the smallest antigen-binding functional fragment of camelid heavy chain-only antibodies that can be manufactured in microbial expression systems. In the current study we evaluated the efficacy of half-life extended anti-rabies VHH in combination with vaccine for PEP in an intranasal rabies infection model in mice. The PEP combination therapy of systemic anti-rabies VHH and intramuscular vaccine significantly delayed the onset of disease compared to treatment with anti-rabies VHH alone, prolonged median survival time (35 versus 14 days) and decreased mortality , when treated 24 hours after rabies virus challenge. Vaccine alone was unable to rescue mice from lethal disease. As reported also for immune globulins, some interference of anti-rabies VHH with the antigenicity of the vaccine was observed, but this did not impede the synergistic effect. Post exposure treatment with vaccine and human anti-rabies immune globulins was unable to protect mice from lethal challenge. Anti-rabies VHH and vaccine act synergistically to protect mice after rabies virus exposure, which further validates the possible use of anti-rabies VHH for rabies PEP.Post-exposure prophylaxis (PEP) against rabies infection consists of a combination of passive immunisation with plasma-derived human or equine immune globulins and active immunisation with vaccine delivered shortly after exposure. Since anti-rabies immune globulins are expensive and scarce, there is a need for cheaper alternatives that can be produced more consistently. Previously, we generated potent virus-neutralising VHH, also called Nanobodies, against the rabies glycoprotein that are effectively preventing lethal disease in an  Rabies is an infectious disease causing 59,000 deaths and millions are exposed each year worldwide. Post-exposure prophylaxis (PEP) against rabies consists of a combination of passive (immune globulins) and active immunisation (vaccine) directly after viral exposure. Currently used plasma-derived anti-rabies immune globulins are expensive and scarce, urging the development of alternatives. Nanobodies or VHH are the smallest antigen-binding fragments of camelid heavy chain antibodies and are easy to produce with intrinsic good thermal stability and solubility. Combined treatment with anti-rabies VHH and vaccine gave significantly better protection than either compound alone in an intranasal rabies challenge model in mice, which validates the potential use of anti-rabies VHH as replacement of immune globulins in PEP. Rhabdoviridae, Genus Lyssavirus [Rabies virus ultimately causes an aggressive and lethal infection in the brain of humans and other mammals. Rabies virus is a model neurotropic RNA virus that belongs to the family ssavirus ;2. The vssavirus ;4. Each ssavirus .Passive antibody therapy with anti-rabies immune globulins (RIG) plays a major role in rabies post-exposure prophylaxis after high risk exposure . TogetheRabies antibodies can be either from equine (ERIG) or human (HRIG) origin. Due to adverse effects, such as serum sickness, equine antibodies are now used under the form of pepsin-digested Fab fragments, but if available, HRIG is still preferred over ERIG . The proCamelidae, and represent the antigen-binding variable domain. By nature VHH are hydrophilic and do not require hydrophobic interactions with a light chain, which allows high solubility, physicochemical stability and high-yield production in Escherichia coli, yeast or mammalian expression systems. The single domain nature and the small size of VHH also allows for easy formatting by genetic fusion into multimeric and multispecific constructs [VHH or Nanobodies (a trade-name by Ablynx) are the smallest functional fragments (15 kDa) of heavy chain-only antibodies naturally occurring in nstructs –14.Previously, we generated a potent neutralizing anti-rabies VHH recognising two epitopes on the rabies glycoprotein, fused to an anti-albumin VHH to extend its serum half-life (HLE). The Rab-E8/H7-ALB11 was able to neutralize the virus at picomolar doses . Post exThe main aim of this study was to examine whether the combined treatment with anti-rabies VHH and vaccine  after exposure to rabies virus has added value compared to single treatment with either compound in the intranasal rabies virus challenge model, which is very well suited to study intervention strategies for prevention and prophylaxis . Via theVHH directed against the rabies virus glycoprotein G were described previously . BrieflyHuman rabies immune globulins (HRIG)  are gammaglobulins purified from plasma of vaccinated human donors.Rabipur  was reconstituted according to the manufacturer’s instructions and was administered via intraperitoneal or intramuscular injection. The vaccine contains at least 2.5 antigenic units (AU)/ml. It contains the inactivated Flury LEP strain produced on purified chick embryo cells.2.5 50% cell culture infectious doses (CCID50) was used.Challenge Virus Standard (CVS)-11 is a virulent classical rabies virus obtained from the American Type Culture Collection (ATCC reference VR959) and was grown in baby hamster kidney (BHK)-21 cells. For virus inoculation in mice, a dose of 10ad libitum and exposed to a natural day/night light cycle. Intranasal (IN) inoculation procedures are described in detail by Rosseels et al. [et al. (2011) [Six-to-eight weeks old female Swiss outbred mice  were used. Mice were kept in filter top cages, water and feed provided s et al. . The ints et al. . For int. (2011) .Three retro-orbital bleedings were performed under isoflurane anaesthesia during the 28 day immunization period.et al. [Mice were observed daily for signs of disease until 35 days post virus inoculation. Mice develop a typical disease pattern, which progresses as follows: isolation from the group (score 1), slow/less vivid movement (score 2), paresis in paws (score 3), uncoordinated movement (score 4), absence of spontaneous movement (score 5), no response to stimuli (score 6) and the end-stage characterized by mice burying their heads in cage bedding and slow breathing (score 7). The score per mouse ranges thus from 0 (no disease) to 7 (severe nervous disease). Disease progression was represented by plotting the daily score in function of the days post inoculation (DPI). The incubation period was defined as the period between virus inoculation and the first appearance of disease signs. In our experience, mice with a disease score of 6 or more die within 24 hours. Therefore, mice were euthanized by cervical dislocation when they reached a score of ≥ 6. Results were expressed with Kaplan-Meier survival curves. Rabies virus infection in the brain was confirmed using real-time reverse transcriptase polymerase chain reaction (RT-qPCR) as described by Suin et al. , and by ref−Cq, with Cqref equal to 45, the number of cycles in this program.The viral RNA load in the brain of mice was determined using RT-qPCR, as previously described ;22. BrieThe virus-neutralizing titer of serum, antibody and VHH preparations was determined with the Rapid Fluorescent Focus Inhibition Test (RFFIT), according to the Manual of Diagnostic Tests and Vaccine for Terrestrial Animals . The neutralizing potency is expressed in international units (IU)/ml in reference to "The Second International Standard for Anti-Rabies Immunoglobulin", purchased from the United Kingdom National Institute for Biological Standards and Control.in vivo data. Differences in survival times were tested using the Log-Rank test with a Bonferroni post-test, differences in Δ Cq values were tested using a Student’s t-test after normalization to the house-keeping gene. Differences in antibody titers were also tested using a Student’s t-test.GraphPad Prism was used for statistical analyses of All experimental procedures were approved by the Ethical Commission of the WIV-ISP and CODA-CERVA (advice number 070515–05) and were performed according to the EU Directive 2010/63/EU for animal experiments.To validate the protective effect of rabies vaccine  in the intranasal rabies mouse model, mice were vaccinated with two intramuscular vaccine doses (0.25 AU/mouse), with a 3-day interval, following the schedule also used later on for PEP. This vaccination schedule is schematically represented in in vitro (RFFIT) in blood collected at different time points. In The mounting of the humoral immune response in the blood after vaccination was monitored by assessing the rabies neutralization activity To verify if the efficacy of the vaccine would be affected by the simultaneous administration of anti-rabies VHH, an interference phenomenon which is well known for anti-rabies immune globulins , a groupMice were challenged by intranasal virus inoculation 4 weeks after the start of the vaccination (day 0). The survival of mice that received the combination regime 4 weeks before viral challenge was substantially reduced compared to the mice that received only the vaccine (11% versus 50%). The median survival time of these mice was not significantly different from the control group (10 days versus 9.5 days), despite the presence of relatively high neutralizing antibody titers at the moment of challenge. As expected, survival rates of mice that received anti-rabies VHH were comparable to the control group. The presence of the anti-rabies VHH in the circulation hence seems to reduce the vaccine efficacy. This may indicate that in absence of the virus, the binding of the anti-rabies VHH to the vaccine may interfere with the induction of an effective humoral immune response.in vivo studies, post-exposure treatment with the anti-rabies VHH one day after virus challenge was shown to provide protection from disease and death in a dose-dependent manner [In previous t manner . The samt manner . SimilarIn the second experiment, the same vaccination schedule was applied, but instead of anti-rabies VHH, mice were treated with human rabies immune globulins  at 24h after virus challenge. This is the highest volume and dose of the commercial HRIG product which could be administered to mice. Control mice were treated with HRIG alone. A schematic overview of both experiments can be found in The survival curves of the different treatment groups in the post-exposure prophylaxis setting are depicted in Figs The viral RNA load in the brain of mice was also assessed . Mice thTogether these data show that in the post-exposure setting anti-rabies VHH acts synergistically with a standard vaccination regime to protect mice from disease after virus exposure.in vitro [Post exposure prophylaxis (PEP) for rabies consists of a combination of passive (human or equine immune globulins) and active immunisation (vaccine) soon after exposure. Anti-rabies immune globulins are expensive, scarce and often not available or affordable for people in developing countries, that are typically most at risk ;25. Alsoin vitro . We alsoIn humans, rabies can have incubation periods as short as 4–6 days, especially if the virus is deposited in highly innervated facial tissues, as is often the case in children . Failureet al., who also showed that PEP with vaccine only was unable to prevent lethal disease [Compared to anti-rabies VHH or vaccine alone, the combination therapy in a post-exposure setting significantly delayed the onset of disease, prolonged median survival time and decreased mortality. Sixty per cent of mice treated with anti-rabies VHH and vaccine survived the infection, in contrast to 0% with vaccine only and 19% with anti-rabies VHH only. This is in agreement with the observations from Servat  disease . Post-ex disease . We assu disease . This hy disease , we founPre-exposure treatment with vaccine (IM) and VHH (IP) seemed to partially reduce the immunogenicity of the vaccine, a phenomenon that is also described for the combination of RIG and vaccine ;33;34. MPre-exposure vaccination offered only partial protection upon intranasal virus challenge (50% survivors). Half of the mice that were actively immunized with (inactivated) vaccine, both at 28 and 25 days before challenge, still developed lethal brain infection. This incomplete protection, even with high antigenic doses (2 x 0.25 AU/mouse), is also described by other researchers, using similar models . NeverthThe incomplete protection in the post-exposure setting may be explained by the aggressive nature of the used intranasal challenge model, in which virus is inoculated directly on a site that contains a high concentration of olfactory neuronal cells, providing a direct portal of entry to the central nervous system. In earlier studies we found spread of the virus in the olfactory bulbs of the brain already at the first day after inoculation . Once in5−6 CCID50) and yields variable inter-assay results, limiting its use for experimental comparison of intervention strategies [The intranasal challenge model is our preferred experimental model because of the high reproducibility, practicability, safety and animal wellbeing issues . It may rategies .et al. also found that the host immune response has a clear impact on the development of, what they refer to as, either “encephalitic” or “paralytic” disease in mice. Rabies virus challenge in immunocompetent mice resulted in “paralytic disease”, with relatively low viral loads and a high extent of inflammation and damage in the brain. The same challenge in cyclophosphamide-treated mice resulted in the absence of an immune response and “encephalitic disease”, with severe general depression, only minor paralysis, high viral loads, and less neuronal cell damage [et al., whereas the naïve mice evolved comparably to the cyclophosphamide-treated mice. In human cases, the average survival time of paralytic rabies is twice as long, compared to the encephalitic (furious) form [Another remarkable finding was the different clinical picture observed depending on the vaccination status of the mouse prior to virus challenge. Naïve mice typically showed signs of depression, such as isolation from the group, inactivity and unresponsiveness to stimulation. In contrast, pre-immunised mice remained alert and vivid, but developed ascending paresis, resulting in paralysis of all limbs, requiring euthanasia. Vaccinated mice developed disease after a longer incubation period (13.7 instead of 9 days) and had a longer morbidity period (3 instead of 1.5 days), which resulted in a longer median survival time (27 instead of 9 days), compared to naïve mice. They also had lower viral loads in the brain at the peak of disease. The vaccine-induced immune response thus had a clear effect on pathogenesis and symptomatology. Iwasaki l damage . In our us) form . Patientus) form . Also inus) form . Our obsIn humans, passive immunisation with anti-rabies antibodies is expected to bridge the immunity gap between virus exposure and onset of the active antibody production induced by the vaccine. The half-life extension of the anti-rabies VHH is based on the addition an anti-albumin VHH component. In mice, addition of anti-albumin VHH extends the half-life to 0.5–1.9 days , while iThese results provide evidence for the possible use of anti-rabies VHH together with vaccine for post exposure prophylaxis of rabies. Early treatment with anti-rabies VHH can delay the incubation period of the disease, which allows more time for the vaccine-induced immunity to control the infection. The ease of production and high thermal stability of VHH are important advantages over the currently used anti-rabies immune globulins.S1 VideoThis video shows typical symptoms of late stage rabies disease in mice. Animals show signs of apathy and depression.(AVI)Click here for additional data file.S2 VideoThis video shows the contrasting disease symptoms in mice with pre-existing immunity. In contrast to “naïve” mice, mice shows symptoms of incoordination rather than apathy. In these animals disease progression is slower than in naïve mice.(AVI)Click here for additional data file.S1 FigThe control group consisted of mice receiving the vaccine without VHH. Blood was collected at day -28 (prior to vaccination and VHH administration), day -14 and day 0. Mice that received rabies vaccination had high antibody titers from day -14 onwards whereas mice that received the pre-incubated mix of HLE VHH + vaccine or non-HLE VHH + vaccine had significantly lower antibody titers on both days . Error bars represent the standard deviation.(TIF)Click here for additional data file.


The high incidence of armed conflicts in biodiverse regions poses significant challenges in achieving international conservation targets. Because attitudes towards risk vary, we assessed different strategies for protected area planning that reflected alternative attitudes towards the risk of armed conflicts. We find that ignoring conflict risk will deliver the lowest return on investment. Opting to completely avoid conflict-prone areas offers limited improvements and could lead to species receiving no protection. Accounting for conflict by protecting additional areas to offset the impacts of armed conflicts would not only increase the return on investment (an effect that is enhanced when high-risk areas are excluded) but also increase upfront conservation costs. Our results also demonstrate that fine-scale estimations of conflict risk could enhance the cost-effectiveness of investments. We conclude that achieving biodiversity targets in volatile regions will require greater initial investment and benefit from fine-resolution estimates of conflict risk. Armed conflicts pose significant problems for ensuring that regional conservation plans are implemented. Here, Hammill et al. show that offsetting the impacts of protected area loss through protection of additional areas during conflicts provides the greatest return on investment for conservation programs. The establishment of effective protected areas remains at the heart of global conservation efforts171314At a minimum, systematic conservation planning combines data on conservation features  and management costs to identify areas for protection1821Initial attempts at systematic conservation planning utilized coarse national-scale cost and biodiversity data24262729To understand how conflict has affected conservation spending historically, we correlated domestic spending and international aid2 (7%) of its landmass protected35We used Africa as a test case to estimate how incorporating conflict risk affects conservation outcomes. Africa currently has ∼2.2 million km2 planning units. The amount of each conservation feature within each planning unit was calculated along with costs of purchasing and maintenance. Each planning unit was also assigned a conflict risk using two different methods: national-scale estimates from published sources , fewer targets are met than under a conflict-ignorant strategy, regardless of whether national-scale or fine-scale conflict-risk data are used . When naAdopting a conflict-accounting strategy would meet the majority (∼95%) of conservation targets , irrespeThe highest overall return on investment could be achieved under a conflict-sensitive strategy using fine-scale conflict-risk data , when thWhen the upper 95% confidence limit of the fine-scale risk estimate is used to identify areas for protection, the calculated number of targets met and overall return on investment for conflict-accounting and conflict-sensitive strategies are still greater than when the national-scale estimates of risk are employed . The proHistorical conservation spending patterns suggest that high biodiversity nations have been favoured regardless of the conflict risk, indicating a willingness to invest in conflict-prone nations when rewards are high22Our spatial prioritization analysis seeks to identify areas that would protect 30% of each conservation feature's range for the minimum overall cost (a ‘minimum set' problemGorilla beringei graueri), would be essentially abandoned.Choosing to avoid conflict-prone areas would lead to the majority of conservation features not receiving adequate protection. The negative effects of areas being excluded are exaggerated when national-scale estimates of conflict risk are used. When species are endemic to a single, conflict-prone nation, excluding that nation from a protected area network would lead to these species receiving zero protection. For example, if maximum tolerable risk was set at <40%, several iconic conservation features, including the eastern lowland gorilla , increasing return on investment. The conflict-accounting strategy may increase overall conservation success through the following two mechanisms: (1) high-conflict-risk areas are avoided where possible; and (2) additional areas containing conservation features are protected to offset losses predicted to be incurred if conflicts occur32When national-scale conflict risk estimates are used, a strategy of avoiding the most conflict-prone regions and accounting for risk in the remainder (conflict-sensitive) yields a similar return on investment to conflict-accounting. Conversely, when fine-scale risk estimates are used, between risk thresholds of 30% and 55%, a conflict-sensitive strategy yields a greater return on investment than conflict-accounting. Although a conflict-sensitive strategy yields the highest return on investment, it also leads to the exclusion of high-risk areas. This exclusion of high-risk areas may be appealing to conservation managers wishing to limit the exposure of staff to conflict, but means any conservation feature existing predominantly in high-risk areas may be abandoned when maximum tolerable risk is low. The decision to opt for a conflict-accounting versus a conflict-sensitive strategy therefore depends on whether the goal is to achieve the highest return on investment or to save the most species, and be influenced by the risk tolerance of conservation investors and managersFor the two strategies that account for risk (conflict-accounting and conflict sensitive), the fine-scale risk estimate is predicted to outperform national-scale estimates in terms of targets met, cost and returns on investment  value that included the following three core components: the cost of purchasing the land, the foregone agricultural revenue over 5 years and the cost of protected area managementhttps://disturbance.s3.amazonaws.com/EcoregionalRollup.zip). To determine the biodiversity value of each planning unit, we calculated the proportion of the total area of each conservation feature represented in each planning unit. When a planning unit was selected to be part of the protected area network, it contributed in meeting the protection target, which is 30% of the current extent of that conservation featureSpatial data were obtained describing the distributions of Africa's 236 terrestrial mammal species classified as >threatened or data-deficient according to the IUCN red list of endangered specieset al.2), and were obtained from the Armed Conflict Location and Event Dataset (ACLED)et al.The definition of ‘risk' we employed incorporates the following two dimensions: the chances a conflict incident will occur and the loss or damage associated with the event26z=37.44, n=335,694, P<0.001), the number of fatalities  and years since the last incident . The logistic model generated probabilistic outputs (conflict-risk estimates between 1 and 100%) for each planning unit. The model accounted for 35% of the total variation in the data . Full parameter estimates for the logistic model and relationships between the descriptive parameters and conflict risk are given in We built a statistical model to calculate the planning unit-specific conflict risk using local history of conflicts. To calibrate the model, we used the data from 10 years of the ACLED database (1999–2008) to ‘predict' risk in the subsequent 5 years using the presence/absence of an incident in each planning unit between 2009 and 2014 as a response variable. The model fitted a logistic curve using the following three significant descriptive variables (bounded by the years 1999–2008): the number of previous incidents in that planning unit  was calculated and compared with the conservation target. If 30% or more of the conservation feature's original area was contained in the remaining planning units, the conservation target was deemed met. For the best protected area network selected under each risk strategy (out of the 100 Marxan runs), we repeated this procedure 1,000 times. The 1,000 simulations enabled us to produce a distribution of the estimated total number of targets met under each attitude towards risk, allowing the reporting of medians and confidence limits. To estimate return on investment, the predicted performance of the protected area network (number of conservation targets met) was divided by the cost of the protected area.Because our fine-scale estimate of conflict risk has an error estimate associated with it, we re-ran the analyses using the upper and lower confidence intervals. Continental-scale risk estimates using the upper and lower confidence limits, and the results of using these estimates in terms of number of conservation targets met, cost and return on investment are given in The national-level estimates of conflict risk we obtained from published sources did not contain an error calculationHow to cite this article: Hammill, E. et al. Factoring attitudes towards armed conflict risk into selection of protected areas for conservation. Nat. Commun. 7:11042 doi: 10.1038/ncomms11042 (2016).Supplementary Figures 1-6, Supplementary Table 1, Supplementary Notes 1-3 and Supplementary References.


The endosomal sorting complexes required for transport (ESCRT) are needed for three distinct cellular functions in higher eukaryotes: (i) Multivesicular body formation for the degradation of transmembrane proteins in lysosomes, (ii) midbody abscission during cytokinesis and (iii) retroviral budding. Not surprisingly, loss of ESCRT function has severe consequences, which include the failure to down-regulate growth factor receptors leading to deregulated mitogenic signaling. While it is clear that the function of the ESCRT machinery is important for embryonic development, its role in cancer is more controversial. Various experimental approaches in different model organisms arrive at partially divergent conclusions regarding the contribution of ESCRTs to tumorigenesis. Therefore the aim of this review is to provide an overview on different model systems used to study the role of the ESCRT machinery in cancer development, to highlight common grounds and present certain controversies in the field. How loss of ESCRT function and the subsequent block in EGFR degradation affects downstream signaling, appears to be surprisingly difficult to predict. Early on, mouse fibroblast cells with a mutation causing low expression of the ESCRT-I protein Tsg101 were reported to rapidly recycle EGFR back to the cell surface. As a result, they had increased receptor signaling and higher activity of mitogen-activated protein kinase (MAPK) signaling reflected by prolonged ERK1/2 phosphorylation  knockdown caused prolonged EGFR activation as well as hyper-activation of downstream AKT and MAPK signaling  loss-of-function mutants suffer from early pupal lethality. Cells lacking functional Hrs fail to degrade active EGF and Torso tyrosine kinase receptor, which leads to enhanced signaling and defects in embryonic patterning. These data suggest that Hrs and MVB formation function to down-regulate receptor tyrosine kinase signaling , a similar scenario with non-cell autonomous neoplastic transformation of wild-type cells surrounding ESCRT mutants due to continuous Notch signaling could be shown. As for Tsg101 (ESCRT-I), increased Notch activity led to production of the mitogenic JAK-Stat pathway ligand unpaired , Vps32 (Snf7) and Vps2 (Chmp2)] in Drosophila can promote neoplastic transformation but also lead to loss of cell polarity and render the cells prone to apoptosis. Sustained growth factor signaling boosts non-cell autonomous over-proliferation in the adjacent wild-type cells. Only when cell death is blocked in the ESCRT mutants, they overgrow wild-type tissue. Therefore it seems that the MVB pathway is required to balance cell survival and proliferation in developing tissues.Taken together, loss of ESCRT function in Danio rerio), a vertebrate model system, knockdown of ESCRT subunits led to severe developmental defects, like in other multicellular organisms:ESCRT-I: Knockdown of ESCRT-I subunit Vps37A with morpholinos yielded a phenotype with significantly reduced mobility  null embryos developed with their ventral region outside of the yolk sac, had two independent bilateral heart tubes (cardia bifida), lacked a foregut and died around embryonic day E11. Enlarged early endosomes were detected in the mutant cells of several tissues including definitive endoderm, suggesting that a deficiency in vesicular transport via early endosomes underlies the mutant phenotype  embryos displayed embryonic lethality and failed to develop past day E6.5. The mutant embryos were reduced in size and did not form a mesoderm. Tsg101 was essential for the proliferative burst before the onset of gastrulation and its knockout caused an accumulation of the tumor suppressor p53  did not result in breast cancer development in mice but impaired proliferation of the Tsg101 deficient cells siRNA had a negative impact on tumor cell growth . Each of them can be a component of ESCRT-I together with Tsg101, Vps28 and hMVB12A/B. Overexpression of Vps37A inhibited both anchorage-dependent and -independent cell growth shRNA enhanced the tumorigenic potential by increasing anchorage-independent growth in HEK 293T cells and led to tumor formation in a xenograft assay in athymic mice. Conversely, Chmp1A overexpression was accompanied by p53 accumulation and inhibited tumor xenograft growth of human pancreas carcinoma PanC-1 cells. This is indicative of Chmp1A as a tumor suppressor protein in pancreas   in comparison to lower grade tumors (Stage II–III). Moreover, low Vps4B mRNA levels correlated with high EGFR protein levels and high Vps4B mRNA levels with low EGFR abundance  from the same studies.Overall, Hrs (ESCRT-0) and Tsg101 (ESCRT-I) seem to be mostly up- and Vps37A (ESCRT-I), Chmp1A and Vps4B mostly down-regulated in tumor samples. This pattern is frequently reflected by Drosophila experiments indicate that ESCRT mutations generally tend to render cells prone to apoptosis, particularly in competition with wild-type cells. It is therefore tempting to speculate that loss of ESCRT function, although initially not beneficial for the cell, could become advantageous in a subset of tumor cells that have already accumulated anti-apoptotic mutations. An up-regulation of ESCRT proteins on the other hand could speed up transmembrane protein turnover and thereby ease the adaption to changes in the cellular environment.There are examples for certain tumor entities showing preferentially up- or down-regulation of ESCRT components suggesting that tumors cells indeed benefit from alterations in the ESCRT machinery. The role of ESCRTs in tumorigenesis is generally attributed to defective transmembrane protein homeostasis and the resulting consequences on cell signaling. Nevertheless, it is also conceivable that tumorigenic potential could be accounted for by cytokinetic defects that result in polyploidy or even a synergistic effect of both, defects in the MVB pathway and cytokinesis. Potential mechanisms and examples for cell cycle and cytokinesis defects in human cancers have been recently reviewed , Y444-B12 and SFB021 (F21) to DT.