Oral delivery of therapeutic proteins bioencapsulated in plant cells: Preclinical and clinical advances

Oral delivery of protein drugs (PDs) made in plant cells could revolutionize current approaches to their production and delivery. Expression of PDs reduces their production cost by elimination of prohibitively expensive fermentation, purification, cold transportation/storage, and sterile injections and increases their shelf life for several years. The ability of plant cell wall to protect PDs from digestive acids/enzymes, commensal bacteria to release PDs in gut lumen after lysis of plant cell wall, and the role of gut-associated lymphoid tissue in inducing tolerance facilitate prevention or treatment of allergic, autoimmune diseases or antidrug antibody responses. The delivery of functional proteins facilitates treatment of inherited or metabolic disorders. Recent advances in making PDs free of antibiotic resistance genes in edible plant cells, long-term storage at ambient temperature maintaining their efficacy, production in Current Good Manufacturing Practice (cGMP) facilities, Investigational New Drug (IND)-enabling studies for clinical advancement, and Food and Drug Administration approval of orally delivered PDs augur well for advancing this novel drug delivery platform technology.     

Parsing disease‐relevant protein modifications from epiphenomena: perspective on the structural basis of SOD1‐mediated ALS

Conformational change and modification of proteins are involved in many cellular functions. However, they can also have adverse effects that are implicated in numerous diseases. How structural change promotes disease is generally not well‐understood. This perspective illustrates how mass spectrometry (MS), followed by toxicological and epidemiological validation, can discover disease‐relevant structural changes and therapeutic strategies. We (with our collaborators) set out to characterize the structural and toxic consequences of disease‐associated mutations and post‐translational modifications (PTMs) of the cytosolic antioxidant protein Cu/Zn‐superoxide dismutase (SOD1). Previous genetic studies discovered >180 different mutations in the SOD1 gene that caused familial (inherited) amyotrophic lateral sclerosis (fALS). Using hydrogen–deuterium exchange with mass spectrometry, we determined that diverse disease‐associated SOD1 mutations cause a common structural defect – perturbation of the SOD1 electrostatic loop. X‐ray crystallographic studies had demonstrated that this leads to protein aggregation through a specific interaction between the electrostatic loop and an exposed beta‐barrel edge strand. Using epidemiology methods, we then determined that decreased SOD1 stability and increased protein aggregation are powerful risk factors for fALS progression, with a combined hazard ratio > 300 (for comparison, a lifetime of smoking is associated with a hazard ratio of ~15 for lung cancer). The resulting structural model of fALS etiology supported the hypothesis that some sporadic ALS (sALS, ~80% of ALS is not associated with a gene defect) could be caused by post‐translational protein modification of wild‐type SOD1. We developed immunocapture antibodies and high sensitivity top‐down MS methods and characterized PTMs of wild‐type SOD1 using human tissue samples. Using global hydrogen–deuterium exchange, X‐ray crystallography and neurotoxicology, we then characterized toxic and protective subsets of SOD1 PTMs. To cap this perspective, we present proof‐of‐concept that post‐translational modification can cause disease. We show that numerous mutations (N➔D; Q➔E), which result in the same chemical structure as the PTM deamidation, cause multiple diseases. Copyright © 2017 John Wiley & Sons, Ltd.     

呼吸声分类技术研究及检测系统设计*

患有呼吸问题的病人由于呼吸道腺体不受意识控制、异物或者其本身肺部病变等原因会出现呼吸异常。医护人员通过病人的呼吸状况可以找寻和分析病人出现这种呼吸状况的原因。而以往呼吸声的诊断方式是专业的医护人员通过使用听诊器对病人进行肺部听诊。但是听诊的结果取决于医护人员的经验与相关参数。在初级诊断治疗阶段,初级医生识别听诊声的效率以及准确率很低,一般从20%到80%不等。因此会存在10%到20%的高误诊率（漏诊、错诊和延误）。本文提出了一种利用PVDF薄膜传感器提取语声特征的检测系统,该检测系统根据病人发出声音的不同,提取呼吸声特征值判断病人的呼吸状况。通过PVDF传感器采集的微弱呼吸声再经过KNN算法分类之后其识别率可达90.6%,对于细分种较类多的湿罗声,其识别率在80.2%左右。综上表明相比于传统听诊方式通过PVDF传感器采集识别的结果具有更高的准确性和可靠性,其判断呼吸状况的结果可以为医护人员提供参考,更好的为患有呼吸疾病的病人提供监测。     

Synthesis of voiced sounds using low-dimensional models of the vocal cords and time-varying subglottal pressure

The vocal cords play an important role on voice production. Air coming from the lungs is forced through the narrow space between the two vocal cords that are set in motion in a frequency that is governed by the tension of the attached muscles. The motion of the vocal cords changes the type of flow, that comes from the lungs, to pulses of air, and as the flow passes through the oral and nasal cavities, it is amplified and further modified until it is radiated from the mouth. This complex process can be modeled by a system of integral-differential equations. This paper considers two mechanical models previously used for explaining the dynamics of the vocal cords. It shows that the level of naturalness of the sound generated by these models is rather poor, and it proposes temporal variations of the parameters of the models to increase such level. Examples of synthetic vowels and diphthongs are given to assess the models. In general, the results show that, although the system of voice production is complex, we can achieve satisfactory results with relatively simple low-dimensional models, by suitable temporal variations of the aerodynamic parameters.     

Polyphenols and Fish Oils for Improving Metabolic Health: A Revision of the Recent Evidence for Their Combined Nutraceutical Effects

Polyphenols and omega-3 polyunsaturated fatty acids from fish oils, i.e., eicosapentaenoic and docosahexaenoic acids, are well-recognized nutraceuticals, and their single antioxidant and anti-inflammatory properties have been demonstrated in several studies found in the literature. It has been reported that the combination of these nutraceuticals can lead to three-fold increases in glutathione peroxidase activity, two-fold increases in plasma antioxidant capacity, decreases of 50–100% in lipid peroxidation, protein carbonylation, and urinary 8-isoprotanes, as well as 50–200% attenuation of common inflammation biomarkers, among other effects, as compared to their individual capacities. Therefore, the adequate combination of those bioactive food compounds and their single properties should offer a powerful tool for the design of successfully nutritional interventions for the prevention and palliation of a plethora of human metabolic diseases, frequently diet-induced, whose etiology and progression are characterized by redox homeostasis disturbances and a low-grade of chronic inflammation. However, the certain mechanisms behind their biological activities, in vivo interaction (both between them and other food compounds), and their optimal doses and consumption are not well-known yet. Therefore, we review here the recent evidence accumulated during the last decade about the cooperative action between polyphenols and fish oils against diet-related metabolic alterations, focusing on the mechanisms and pathways described and the effects reported. The final objective is to provide useful information for strategies for personalized nutrition based on these nutraceuticals.     

A column‐switching HPLC‐MS/MS method for mucopolysaccharidosis type I analysis in a multiplex assay for the simultaneous newborn screening of six lysosomal storage disorders

Lysosomal storage disorders comprise a group of rare genetic diseases in which a deficit of specific hydrolases leads to the storage of undegraded substrates in lysosomes. Impaired enzyme activities can be assessed by MS/MS quantification of the reaction products obtained after incubation with specific substrates. In this study, a column‐switching HPLC‐MS/MS method for multiplex screening in dried blood spot of the lysosomal enzymes activities was developed. Mucopolysaccharidosis type I, Fabry, Gaucher, Krabbe, Niemann–Pick A/B and Pompe diseases were simultaneously assayed. Dried blood spots were incubated with substrates and internal standards; thereafter, supernatants were collected with minor manipulations. Samples were injected, trapped into an online perfusion column and, by a six‐port valve, switched online through the C₁₈ analytical column to perform separation of metabolites followed by MS/MS analysis. A total of 1136 de‐identified newborn screening samples were analyzed to determine references for enzymes activity values. As positive controls, we analyzed dried blood spots from three patients with Pompe, one with Fabry, one with Krabbe disease and two with MPS I, and in all cases the enzyme activities were below the cutoff values measured for newborns, except for an MPS I patient after successful hematopoietic stem cell transplantation. Copyright © 2014 John Wiley & Sons, Ltd.