Towards More Human and Humane Testing: The Role of the Device Supplier Industry.

In vivo testing has been the gold standard for preclinical drug development and toxicology. However, animal-based methods often lack human relevance and have a low predictability rate, not to mention the enormous ethical and financial concerns associated with their use. For instance, according to the US Congressional Budget Office (cbo.gov), it takes an average of 10.5 years to take a compound from the preclinical phase to the market, with a cost of US$1-US$2 billion. 90% of drugs that are tested in animals and enter clinical trials fail due to lack of safety and efficacy - this fact questions the significance of in vivo testing. Cells in culture can recapitulate certain aspects of physiology and disease, as well as indicate drug responses and toxicity. Thus, they represent a sophisticated human-relevant and humane alternative. With advances in the in vitro field, it is anticipated that confidence will be gained towards a move away from traditional in vivo models. Specialised supplier industries have been a driving force in the transition to non-animal research, by translating new approach methodologies into scalable products that have been adopted by the regulatory and testing industries - but we are still at the beginning. In this article, we introduce the perspective of the device supplier industry on the current challenges and opportunities surrounding the adoption of new in vitro methods, with the goal of promoting effective co-operation with scientists and other stakeholders. In addition, we highlight some examples of where non-animal approaches have been used in regulatory submissions, as well as listing some educational and training resources that can help when selecting the most appropriate assay.

Inhibition of protein misfolding and aggregation by natural phenolic compounds.

Protein misfolding and aggregation into fibrillar deposits is a common feature of a large group of degenerative diseases affecting the central nervous system or peripheral organs, termed protein misfolding disorders (PMDs). Despite their established toxic nature, clinical trials aiming to reduce misfolded aggregates have been unsuccessful in treating or curing PMDs. An interesting possibility for disease intervention is the regular intake of natural food or herbal extracts, which contain active molecules that inhibit aggregation or induce the disassembly of misfolded aggregates. Among natural compounds, phenolic molecules are of particular interest, since most have dual activity as amyloid aggregation inhibitors and antioxidants. In this article, we review many phenolic natural compounds which have been reported in diverse model systems to have the potential to delay or prevent the development of various PMDs, including Alzheimer's and Parkinson's diseases, prion diseases, amyotrophic lateral sclerosis, systemic amyloidosis, and type 2 diabetes. The lower toxicity of natural compounds compared to synthetic chemical molecules suggest that they could serve as a good starting point to discover protein misfolding inhibitors that might be useful for the treatment of various incurable diseases.

Natural Products as Modulators of the Proteostasis Machinery: Implications in Neurodegenerative Diseases.

Proteins play crucial and diverse roles within the cell. To exert their biological function they must fold to acquire an appropriate three-dimensional conformation. Once their function is fulfilled, they need to be properly degraded to hamper any possible damage. Protein homeostasis or proteostasis comprises a complex interconnected network that regulates different steps of the protein quality control, from synthesis and folding, to degradation. Due to the primary role of proteins in cellular function, the integrity of this network is critical to assure functionality and health across lifespan. Proteostasis failure has been reported in the context of aging and neurodegeneration, such as Alzheimer's and Parkinson's disease. Therefore, targeting the proteostasis elements emerges as a promising neuroprotective therapeutic approach to prevent or ameliorate the progression of these disorders. A variety of natural products are known to be neuroprotective by protein homeostasis interaction. In this review, we will focus on the current knowledge regarding the use of natural products as modulators of different components of the proteostasis machinery within the framework of age-associated neurodegenerative diseases.

Delaying aging in Caenorhabditis elegans with protein aggregation inhibitors.

Recent evidence suggests that during aging there is widespread accumulation of aggregated insoluble proteins, even in the absence of pathological conditions. Pharmacological manipulation of protein aggregation might be helpful to unveil the involvement of protein aggregates during aging, as well as to develop novel strategies to delay aging. Here we investigated the effect of known protein aggregation inhibitors on the lifespan and health-span of Caenorhabditis elegans. For this purpose, we selected various structurally diverse anti-aggregation compounds and screened them in liquid and solid medium for their ability to alter the rate of aging in vivo. Our results show that treatment of C. elegans with diverse aggregation inhibitors significantly increases the animal lifespan and health-span. These findings indicate that protein misfolding and aggregation may play an important role in cellular dysfunction during aging, opening a novel approach to increase longevity and enhance the quality of life during aging.

Targeting the Human Influenza a Virus: The Methods, Limitations, and Pitfalls of Virtual Screening for Drug-like Candidates Including Scaffold Hopping and Compound Profiling.

In this study, we describe the input data and processing steps to find antiviral lead compounds by a virtual screen. Two-dimensional and three-dimensional filters were designed based on the X-ray crystallographic structures of viral neuraminidase co-crystallized with substrate sialic acid, substrate-like DANA, and four inhibitors (oseltamivir, zanamivir, laninamivir, and peramivir). As a result, ligand-receptor interactions were modeled, and those necessary for binding were utilized as screen filters. Prospective virtual screening (VS) was carried out in a virtual chemical library of over half a million small organic substances. Orderly filtered moieties were investigated based on 2D- and 3D-predicted binding fingerprints disregarding the "rule-of-five" for drug likeness, and followed by docking and ADMET profiling. Two-dimensional and three-dimensional screening were supervised after enriching the dataset with known reference drugs and decoys. All 2D, 3D, and 4D procedures were calibrated before execution, and were then validated. Presently, two top-ranked substances underwent successful patent filing. In addition, the study demonstrates how to work around reported VS pitfalls in detail.

The leaf-area shrinkage effect can bias paleoclimate and ecology research.

PREMISE OF THE STUDY: Leaf area is a key trait that links plant form, function, and environment. Measures of leaf area can be biased because leaf area is often estimated from dried or fossilized specimens that have shrunk by an unknown amount. We tested the common assumption that this shrinkage is negligible. METHODS: We measured shrinkage by comparing dry and fresh leaf area in 3401 leaves of 380 temperate and tropical species and used phylogenetic and trait-based approaches to determine predictors of this shrinkage. We also tested the effects of rehydration and simulated fossilization on shrinkage in four species. KEY RESULTS: We found that dried leaves shrink in area by an average of 22% and a maximum of 82%. Shrinkage in dried leaves can be predicted by multiple morphological traits with a standard deviation of 7.8%. We also found that mud burial, a proxy for compression fossilization, caused negligible shrinkage, and that rehydration, a potential treatment of dried herbarium specimens, eliminated shrinkage. CONCLUSIONS: Our findings indicate that the amount of shrinkage is driven by variation in leaf area, leaf thickness, evergreenness, and woodiness and can be reversed by rehydration. The amount of shrinkage may also be a useful trait related to ecologically and physiological differences in drought tolerance and plant life history.

Recognizing pitfalls in virtual screening: a critical review.

The aim of virtual screening (VS) is to identify bioactive compounds through computational means, by employing knowledge about the protein target (structure-based VS) or known bioactive ligands (ligand-based VS). In VS, a large number of molecules are ranked according to their likelihood to be bioactive compounds, with the aim to enrich the top fraction of the resulting list (which can be tested in bioassays afterward). At its core, VS attempts to improve the odds of identifying bioactive molecules by maximizing the true positive rate, that is, by ranking the truly active molecules as high as possible (and, correspondingly, the truly inactive ones as low as possible). In choosing the right approach, the researcher is faced with many questions: where does the optimal balance between efficiency and accuracy lie when evaluating a particular algorithm; do some methods perform better than others and in what particular situations; and what do retrospective results tell us about the prospective utility of a particular method? Given the multitude of settings, parameters, and data sets the practitioner can choose from, there are many pitfalls that lurk along the way which might render VS less efficient or downright useless. This review attempts to catalogue published and unpublished problems, shortcomings, failures, and technical traps of VS methods with the aim to avoid pitfalls by making the user aware of them in the first place.

Development of a novel pharmacophore model to screen specific inhibitors for the serine-threonine protein phosphatase calcineurin.

Calcineurin (CaN) is a calcium/calmodulin-dependent serine/threonine phosphatase with a crucial role in cellular homeostasis. It is also the target of the Food and Drug Administration (FDA) approved immunosuppressant drugs FK506 and cyclosporine A. Recent work from our group and others indicated that an uncontrolled increase in CaN activity causes synaptic dysfunction and neuronal death in various models of neurodegenerative diseases associated with calcium dysregulation. Furthermore, pharmacological normalization of CaN activity can prevent disease progression in animal models. However, none of the FDA-approved CaN inhibitors bind CaN directly, leading to adverse side effects. The development of direct CaN inhibitors is required to reduce off-target effects, but its highly conserved active site and similar mechanism of action with other protein serine/threonine phosphatases impose a significant challenge. In this work, we developed a novel pharmacophore model to screen for CaN-specific inhibitors. Then, we performed a virtual screen for molecules having the pharmacophore model. We also show that the molecules identified in this screen can inhibit CaN with a low micromolar IC50. Interestingly, the inhibitors identified from the screen do not inhibit phosphoprotein phosphatase 2A, a member of the serine/threonine phosphatase family that shares 43% sequence identity with the CaN active site. The pharmacophore model that we developed and validated in this work may help to accelerate the development of specific CaN inhibitors.

Extensive accumulation of misfolded protein aggregates during natural aging and senescence.

Accumulation of misfolded protein aggregates is a hallmark event in many age-related protein misfolding disorders, including some of the most prevalent and insidious neurodegenerative diseases. Misfolded protein aggregates produce progressive cell damage, organ dysfunction, and clinical changes, which are common also in natural aging. Thus, we hypothesized that aging is associated to the widespread and progressive misfolding and aggregation of many proteins in various tissues. In this study, we analyzed whether proteins misfold, aggregate, and accumulate during normal aging in three different biological systems, namely senescent cells, Caenorhabditis elegans, and mouse tissues collected at different times from youth to old age. Our results show a significant accumulation of misfolded protein aggregates in aged samples as compared to young materials. Indeed, aged samples have between 1.3 and 2.5-fold (depending on the biological system) higher amount of insoluble proteins than young samples. These insoluble proteins exhibit the typical characteristics of disease-associated aggregates, including insolubility in detergents, protease resistance, and staining with amyloid-binding dye as well as accumulation in aggresomes. We identified the main proteins accumulating in the aging brain using proteomic studies. These results show that the aged brain contain large amounts of misfolded and likely non-functional species of many proteins, whose soluble versions participate in cellular pathways that play fundamental roles in preserving basic functions, such as protein quality control, synapsis, and metabolism. Our findings reveal a putative role for protein misfolding and aggregation in aging.

Soft-Lithographic Patterning of Luminescent Carbon Nanodots Derived from Collagen Waste.

Luminescent carbon dots (Cdots) synthesized using inexpensive precursors have inspired tremendous research interest because of their superior properties and applicability in various fields. In this work, we report a simple, economical, green route for the synthesis of multifunctional fluorescent Cdots prepared from a natural, low-cost source: collagen extracted from animal skin wastes. The as-synthesized metal-free Cdots were found to be in the size range of ∼1.2-9 nm, emitting bright blue photoluminescence with a calculated Cdot yield of ∼63%. Importantly, the soft-lithographic method used was inexpensive and yielded a variety of Cdot patterns with different geometrical structures and significant cellular biocompatibility. This novel approach to Cdot production highlights innovative ways of transforming industrial biowastes into advanced multifunctional materials which offer exciting potential for applications in nanophotonics and nanobiotechnology using a simple and scalable technique.

Role of protein misfolding and proteostasis deficiency in protein misfolding diseases and aging.

The misfolding, aggregation, and tissue accumulation of proteins are common events in diverse chronic diseases, known as protein misfolding disorders. Many of these diseases are associated with aging, but the mechanism for this connection is unknown. Recent evidence has shown that the formation and accumulation of protein aggregates may be a process frequently occurring during normal aging, but it is unknown whether protein misfolding is a cause or a consequence of aging. To combat the formation of these misfolded aggregates cells have developed complex and complementary pathways aiming to maintain protein homeostasis. These protective pathways include the unfolded protein response, the ubiquitin proteasome system, autophagy, and the encapsulation of damaged proteins in aggresomes. In this paper we review the current knowledge on the role of protein misfolding in disease and aging as well as the implication of deficiencies in the proteostasis cellular pathways in these processes. It is likely that further understanding of the mechanisms involved in protein misfolding and the natural defense pathways may lead to novel strategies for treatment of age-dependent protein misfolding disorders and perhaps aging itself.

Localización inusual de lesiones líticas de mieloma múltiple con resonancia magnética. Reporte de dos casos / Unusual location of multiple myeloma lytic lesions with magnetic resonance imaging. Report of two cases

Resumen: El mieloma múltiple (MM) es una enfermedad maligna hematológica que se caracteriza por la proliferación de células plasmáticas monoclonales en la médula ósea. La prueba de diagnóstico estándar de oro para MM es un aspirado y/o biopsia de médula ósea (MO), que define la cantidad de células plasmáticas atípicas y constituye la base del sistema de clasificación diagnóstica del Grupo de Trabajo Internacional del Mieloma. Las lesiones del MM en tejido óseo son líticas y su localización más frecuente es en la columna vertebral, pelvis, cráneo y costillas. Aunque las lesiones óseas predominan en estas regiones del esqueleto y en las extremidades proximales también se presentan en menor proporción en codos, rodillas y escápula. En los estudios por imagen la radiografía simple es el principal estudio diagnóstico en la detección de cambios óseos destructivos por MM, sin embargo tiene baja sensibilidad. El estudio de resonancia magnética (RM) es el estudio de elección por tener mayor sensibilidad y especificidad para el diagnóstico de esta enfermedad. Objetivo: Informar 2 casos con infiltración por mieloma múltiple en regiones inusuales.

Abstract: Multiple myeloma (MM) is an hematologic malignancy characterized by the proliferation of malignant monoclonal plasma cells in the bone marrow. The diagnostic test for MM is a bone marrow aspirate or biopsy to define the amount of atypical plasma cells and it is the basis of the diagnostic classification system of the International Working Group Myeloma. MM lesions are lytic bone tissue and are most frequently located in the spine, pelvis, skull and ribs. Bone lesions predominate in these regions of the skeleton and proximal extremities but may occur to a lesser extent on elbows, knees and scapula. In imaging studies plain radiography remains the primary diagnostic study in detecting destructive bone changes multiple myeloma, however the MRI study is the study of choice because the sensitivity and specificity for the diagnosis of this disease. Objectives: Report two cases with multiple myeloma infiltration in unusual regions.

Effect of glucose and fatty acid availability on neonatal and adult heart contractility.

Changes in contractility of newborn and adult hearts with different substrates were studied under oxygenation and hypoxia. Under oxygenation, insulin (50 mU/l) and low doses of fatty acids (sodium palmitate 0.12 mM) increased the differential ventricular pressure (DVP), while higher doses of fatty acids (from 0.3 to 1.5 mM) decreased it. However, when both low doses of fatty acids and insulin were added simultaneously, tension development decreased. Hypoxia reduced DVP, and low doses of fatty acids restored cardiac force. The contractile response to extracellular glucose concentrations changed during development, and sensitivity to high doses of fatty acids increased with age. In adult cardiomyocytes, glucose uptake was also inhibited by sodium palmitate under oxygenation when cardiac metabolism is fatty acid dependent, but not under hypoxia, when it consumes carbohydrates. Newborn cardiomyocytes consumed more glucose than adult cells, they did not respond to insulin, and palmitate did not completely inhibit glucose uptake neither under oxygenation nor under hypoxia. Substrate availability modified glucose uptake and contractility by independent mechanisms.