Diagnosis and clinical management of red cell membrane disorders

Heterogeneous red blood cell (RBC) membrane disorders and hydration defects often present with the common clinical findings of hemolytic anemia, but they may require substantially different management, based on their pathophysiology. An accurate and timely diagnosis is essential to avoid inappropriate interventions and prevent complications. Advances in genetic testing availability within the last decade, combined with extensive foundational knowledge on RBC membrane structure and function, now facilitate the correct diagnosis in patients with a variety of hereditary hemolytic anemias (HHAs). Studies in patient cohorts with well-defined genetic diagnoses have revealed complications such as iron overload in hereditary xerocytosis, which is amenable to monitoring, prevention, and treatment, and demonstrated that splenectomy is not always an effective or safe treatment for any patient with HHA. However, a multitude of variants of unknown clinical significance have been discovered by genetic evaluation, requiring interpretation by thorough phenotypic assessment in clinical and/or research laboratories. Here we discuss genotype-phenotype correlations and corresponding clinical management in patients with RBC membranopathies and propose an algorithm for the laboratory workup of patients presenting with symptoms and signs of hemolytic anemia, with a clinical case that exemplifies such a workup.

Lipids in Pathophysiology and Development of the Membrane Lipid Therapy: New Bioactive Lipids

Membranes are mainly composed of a lipid bilayer and proteins, constituting a checkpoint for the entry and passage of signals and other molecules. Their composition can be modulated by diet, pathophysiological processes, and nutritional/pharmaceutical interventions. In addition to their use as an energy source, lipids have important structural and functional roles, e.g., fatty acyl moieties in phospholipids have distinct impacts on human health depending on their saturation, carbon length, and isometry. These and other membrane lipids have quite specific effects on the lipid bilayer structure, which regulates the interaction with signaling proteins. Alterations to lipids have been associated with important diseases, and, consequently, normalization of these alterations or regulatory interventions that control membrane lipid composition have therapeutic potential. This approach, termed membrane lipid therapy or membrane lipid replacement, has emerged as a novel technology platform for nutraceutical interventions and drug discovery. Several clinical trials and therapeutic products have validated this technology based on the understanding of membrane structure and function. The present review analyzes the molecular basis of this innovative approach, describing how membrane lipid composition and structure affects protein-lipid interactions, cell signaling, disease, and therapy (e.g., fatigue and cardiovascular, neurodegenerative, tumor, infectious diseases).

The Role of RBC Oxidative Stress in Sickle Cell Disease: From the Molecular Basis to Pathologic Implications

Sickle cell disease (SCD) is an inherited monogenic disorder and the most common severe hemoglobinopathy in the world. SCD is characterized by a point mutation in the β-globin gene, which results in hemoglobin (Hb) S production, leading to a variety of mechanistic and phenotypic changes within the sickle red blood cell (RBC). In SCD, the sickle RBCs are the root cause of the disease and they are a primary source of oxidative stress since sickle RBC redox state is compromised due to an imbalance between prooxidants and antioxidants. This imbalance in redox state is a result of a continuous production of reactive oxygen species (ROS) within the sickle RBC caused by the constant endogenous Hb autoxidation and NADPH oxidase activation, as well as by a deficiency in the antioxidant defense system. Accumulation of non-neutralized ROS within the sickle RBCs affects RBC membrane structure and function, leading to membrane integrity deficiency, low deformability, phosphatidylserine exposure, and release of micro-vesicles. These oxidative stress-associated RBC phenotypic modifications consequently evoke a myriad of physiological changes involved in multi-system manifestations. Thus, RBC oxidative stress in SCD can ultimately instigate major processes involved in organ damage. The critical role of the sickle RBC ROS production and its regulation in SCD pathophysiology are discussed here.

Synergistic Action of Membrane-Bound and Water-Soluble Antioxidants in Neuroprotection

Prevention of neurodegeneration during aging, and support of optimal brain function throughout the lifespan, requires protection of membrane structure and function. We review the synergistic action of different classes of dietary micronutrients, as well as further synergistic contributions from exercise and stress reduction, in supporting membrane structure and function. We address membrane-associated inflammation involving reactive oxygen species (ROS) that produce immune regulators from polyunsaturated fatty acids (PUFAs) of membrane phospholipids. The potential of dietary micronutrients to maintain membrane fluidity and prevent chronic inflammation is examined with a focus on synergistically acting membrane-soluble components (zeaxanthin, lutein, vitamin E, and omega-3 PUFAs) and water-soluble components (vitamin C and various phenolics). These different classes of micronutrients apparently operate in a series of intertwined oxidation-reduction cycles to protect membrane function and prevent chronic inflammation. At this time, it appears that combinations of a balanced diet with regular moderate exercise and stress-reduction practices are particularly beneficial. Effective whole-food-based diets include the Mediterranean and the MIND diet (Mediterranean-DASH Intervention for Neurodegenerative Delay diet, where DASH stands for Dietary Approaches to Stop Hypertension).

Laminin Polymerization and Inherited Disease: Lessons From Genetics

The laminins (LM) are a family of basement membranes glycoproteins with essential structural roles in supporting epithelia, endothelia, nerves and muscle adhesion, and signaling roles in regulating cell migration, proliferation, stem cell maintenance and differentiation. Laminins are obligate heterotrimers comprised of α, β and γ chains that assemble intracellularly. However, extracellularly these heterotrimers then assemble into higher-order networks via interaction between their laminin N-terminal (LN) domains. In vitro protein studies have identified assembly kinetics and the structural motifs involved in binding of adjacent LN domains. The physiological importance of these interactions has been identified through the study of pathogenic point mutations in LN domains that lead to syndromic disorders presenting with phenotypes dependent on which laminin gene is mutated. Genotype-phenotype comparison between knockout and LN domain missense mutations of the same laminin allows inferences to be drawn about the roles of laminin network assembly in terms of tissue function. In this review, we will discuss these comparisons in terms of laminin disorders, and the therapeutic options that understanding these processes have allowed. We will also discuss recent findings of non-laminin mediators of laminin network assembly and their implications in terms of basement membrane structure and function.

Metabolomic and Inflammatory Signatures of Symptom Dimensions in Major Depression

Background: Major depressive disorder (MDD) is a highly heterogenous disease, both in terms of clinical profiles and pathobiological alterations. Recently, immunometabolic dysregulations were shown to be correlated with atypical, energy-related symptoms but less so with the Melancholic or Anxious distress symptom dimensions of depression in The Netherlands Study of Depression and Anxiety (NESDA) study. In this study, we aimed to replicate these immunometabolic associations and to characterize the metabolomic correlates of each of the three MDD dimensions. Methods: Using three clinical rating scales, Melancholic, and Anxious distress, and Immunometabolic (IMD) dimensions were characterized in 158 patients who participated in the Predictors of Remission to Individual and Combined Treatments (PReDICT) study and from whom plasma and serum samples were available. The NESDA-defined inflammatory index, a composite measure of interleukin-6 and C-reactive protein, was measured from pre-treatment plasma samples and a metabolomic profile was defined using serum samples analyzed on three metabolomics platforms targeting fatty acids and complex lipids, amino acids, acylcarnitines, and gut microbiome-derived metabolites among other metabolites of central metabolism. Results: The IMD clinical dimension and the inflammatory index were positively correlated (r=0.19, p=.019) after controlling for age, sex, and body mass index, whereas the Melancholic and Anxious distress dimensions were not, replicating the previous NESDA findings. The three symptom dimensions had distinct metabolomic signatures using both univariate and set enrichment statistics. IMD severity correlated mainly with gut-derived metabolites and a few acylcarnitines and long chain saturated free fatty acids. Melancholia severity was significantly correlated with several phosphatidylcholines, primarily the ether-linked variety, lysophosphatidylcholines, as well as several amino acids. Anxious distress severity correlated with several medium and long chain free fatty acids, both saturated and polyunsaturated ones, sphingomyelins, as well as several amino acids and bile acids. Conclusion: The IMD dimension of depression is reliably associated with markers of inflammation. Metabolomics provides powerful tools to inform about depression heterogeneity and molecular mechanisms related to clinical dimensions in MDD, which include a link to gut microbiome and lipids implicated in membrane structure and function.

Membrane Profiling by Free Flow Electrophoresis and SWATH-MS to Characterize Subcellular Compartment Proteomes in Mesembryanthemum crystallinum

The study of subcellular membrane structure and function facilitates investigations into how biological processes are divided within the cell. However, work in this area has been hampered by the limited techniques available to fractionate the different membranes. Free Flow Electrophoresis (FFE) allows for the fractionation of membranes based on their different surface charges, a property made up primarily of their varied lipid and protein compositions. In this study, high-resolution plant membrane fractionation by FFE, combined with mass spectrometry-based proteomics, allowed the simultaneous profiling of multiple cellular membranes from the leaf tissue of the plant Mesembryanthemum crystallinum. Comparisons of the fractionated membranes’ protein profile to that of known markers for specific cellular compartments sheds light on the functions of proteins, as well as provides new evidence for multiple subcellular localization of several proteins, including those involved in lipid metabolism.

Genetic Basis of Type IV Collagen Disorders of the Kidney

The glomerular basement membrane is a vital component of the filtration barrier of the kidney and is primarily composed of a highly structured matrix of type IV collagen. Specific isoforms of type IV collagen, the α3(IV), α4(IV), and α5(IV) isoforms, assemble into trimers that are required for normal glomerular basement membrane function. Disruption or alteration in these isoforms leads to breakdown of the glomerular basement membrane structure and function and can lead to progressive CKD known as Alport syndrome. However, there is wide variability in phenotype among patients with mutations affecting type IV collagen that depends on a complex interplay of sex, genotype, and X-chromosome inactivation. This article reviews the genetic basis of collagen disorders of the kidney as well as potential treatments for these conditions, including direct alteration of the DNA, RNA therapies, and manipulation of collagen proteins.

Construction of a complete set of Neisseria meningitidis mutants and its use for the phenotypic profiling of this human pathogen

The bacterium Neisseria meningitidis causes life-threatening meningitis and sepsis. Here, we construct a complete collection of defined mutants in protein-coding genes of this organism, identifying all genes that are essential under laboratory conditions. The collection, named NeMeSys 2.0, consists of individual mutants in 1584 non-essential genes. We identify 391 essential genes, which are associated with basic functions such as expression and preservation of genome information, cell membrane structure and function, and metabolism. We use this collection to shed light on the functions of diverse genes, including a gene encoding a member of a previously unrecognised class of histidinol-phosphatases; a set of 20 genes required for type IV pili function; and several conditionally essential genes encoding antitoxins and/or immunity proteins. We expect that NeMeSys 2.0 will facilitate the phenotypic profiling of a major human bacterial pathogen.