Small but mighty: Case report and practical guidance for peripheral blood stem cell collection in small infants.

Modern apheresis devices, with increased procedural precision, automation, and monitoring, have been shown to allow for safe delivery of apheresis therapies in young children. Medical advances are increasing demand for apheresis procedures like mononuclear cell collection in infants <10 kg, including stem-cell supported chemotherapy, cell collection for chimeric antigen receptor T cell development, and now ex vivo gene therapies for rare genetic diseases. Nevertheless, safe delivery in small infants involves a range of unique considerations and challenges, beyond just size, and experience will vary between centers. In this case report we describe our experience performing mononuclear cell collection in our smallest patient to date and outline a practice guideline developed following a literature review and discussion with both international experts and device representatives. This case may help to inform other clinicians aiming to provide apheresis care to very small infants in their own centers.

Recent Advances in Modeling Membrane ß-Barrel Proteins Using Molecular Dynamics Simulations: From Their Lipid Environments to Their Assemblies.

Spurred by advances in AI-driven modeling and experimental methods, molecular dynamics simulations are now acting as a platform to integrate these different approaches. This combination of methods is especially useful to understand ß-barrel proteins from the molecular level, e.g., identifying specific interactions with lipids or small molecules, up to assemblies comprised of hundreds of proteins and thousands of lipids. In this minireview, we will discuss recent advances, mainly from the last 5 years, in modeling ß-barrel proteins and their assemblies. These approaches require specific kinds of modeling and potentially different model resolutions that we will first describe in Subheading 1. We will then focus on different aspects of ß-barrel protein modeling: how different types of molecules can diffuse through ß-barrel proteins (Subheading 2); how lipids can interact with these proteins (Subheading 3); how ß-barrel proteins can interact with membrane partners (Subheading 4) or periplasmic extensions and partners (Subheading 5) to form large assemblies.

LipIDens: simulation assisted interpretation of lipid densities in cryo-EM structures of membrane proteins.

Cryo-electron microscopy (cryo-EM) enables the determination of membrane protein structures in native-like environments. Characterising how membrane proteins interact with the surrounding membrane lipid environment is assisted by resolution of lipid-like densities visible in cryo-EM maps. Nevertheless, establishing the molecular identity of putative lipid and/or detergent densities remains challenging. Here we present LipIDens, a pipeline for molecular dynamics (MD) simulation-assisted interpretation of lipid and lipid-like densities in cryo-EM structures. The pipeline integrates the implementation and analysis of multi-scale MD simulations for identification, ranking and refinement of lipid binding poses which superpose onto cryo-EM map densities. Thus, LipIDens enables direct integration of experimental and computational structural approaches to facilitate the interpretation of lipid-like cryo-EM densities and to reveal the molecular identities of protein-lipid interactions within a bilayer environment. We demonstrate this by application of our open-source LipIDens code to ten diverse membrane protein structures which exhibit lipid-like densities.

Direct determination of oligomeric organization of integral membrane proteins and lipids from intact customizable bilayer.

Hierarchical organization of integral membrane proteins (IMP) and lipids at the membrane is essential for regulating myriad downstream signaling. A quantitative understanding of these processes requires both detections of oligomeric organization of IMPs and lipids directly from intact membranes and determination of key membrane components and properties that regulate them. Addressing this, we have developed a platform that enables native mass spectrometry (nMS) analysis of IMP-lipid complexes directly from intact and customizable lipid membranes. Both the lipid composition and membrane properties (such as curvature, tension, and fluidity) of these bilayers can be precisely customized to a target membrane. Subsequent direct nMS analysis of these intact proteolipid vesicles can yield the oligomeric states of the embedded IMPs, identify bound lipids, and determine the membrane properties that can regulate the observed IMP-lipid organization. Applying this method, we show how lipid binding regulates neurotransmitter release and how membrane composition regulates the functional oligomeric state of a transporter.

Mesoscale simulations: An indispensable approach to understand biomembranes.

Computer simulation techniques form a versatile tool, a computational microscope, for exploring biological processes. This tool has been particularly effective in exploring different features of biological membranes. In recent years, thanks to elegant multiscale simulation schemes, some fundamental limitations of investigations by distinct simulation techniques have been resolved. As a result, we are now capable of exploring processes spanning multiple scales beyond the capacity of any single technique. In this perspective, we argue that mesoscale simulations require more attention and must be further developed to fill evident gaps in a quest toward simulating and modeling living cell membranes.

Rare EIF4A2 variants are associated with a neurodevelopmental disorder characterized by intellectual disability, hypotonia, and epilepsy.

Eukaryotic initiation factor-4A2 (EIF4A2) is an ATP-dependent RNA helicase and a member of the DEAD-box protein family that recognizes the 5' cap structure of mRNAs, allows mRNA to bind to the ribosome, and plays an important role in microRNA-regulated gene repression. Here, we report on 15 individuals from 14 families presenting with global developmental delay, intellectual disability, hypotonia, epilepsy, and structural brain anomalies, all of whom have extremely rare de novo mono-allelic or inherited bi-allelic variants in EIF4A2. Neurodegeneration was predominantly reported in individuals with bi-allelic variants. Molecular modeling predicts these variants would perturb structural interactions in key protein domains. To determine the pathogenicity of the EIF4A2 variants in vivo, we examined the mono-allelic variants in Drosophila melanogaster (fruit fly) and identified variant-specific behavioral and developmental defects. The fruit fly homolog of EIF4A2 is eIF4A, a negative regulator of decapentaplegic (dpp) signaling that regulates embryo patterning, eye and wing morphogenesis, and stem cell identity determination. Our loss-of-function (LOF) rescue assay demonstrated a pupal lethality phenotype induced by loss of eIF4A, which was fully rescued with human EIF4A2 wild-type (WT) cDNA expression. In comparison, the EIF4A2 variant cDNAs failed or incompletely rescued the lethality. Overall, our findings reveal that EIF4A2 variants cause a genetic neurodevelopmental syndrome with both LOF and gain of function as underlying mechanisms.

Cardiolipin, and not monolysocardiolipin, preferentially binds to the interface of complexes III and IV.

The mitochondrial electron transport chain comprises a series of protein complexes embedded in the inner mitochondrial membrane that generate a proton motive force via oxidative phosphorylation, ultimately generating ATP. These protein complexes can oligomerize to form larger structures called supercomplexes. Cardiolipin (CL), a conical lipid, unique within eukaryotes to the inner mitochondrial membrane, has proven essential in maintaining the stability and function of supercomplexes. Monolysocardiolipin (MLCL) is a CL variant that accumulates in people with Barth syndrome (BTHS). BTHS is caused by defects in CL biosynthesis and characterised by abnormal mitochondrial bioenergetics and destabilised supercomplexes. However, the mechanisms by which MLCL causes pathogenesis remain unclear. Here, multiscale molecular dynamics characterise the interactions of CL and MLCL with yeast and mammalian mitochondrial supercomplexes containing complex III (CIII) and complex IV (CIV). Coarse-grained simulations reveal that both CL and MLCL bind to sites at the interface between CIII and CIV of the supercomplex. Free energy perturbation calculations show that MLCL interaction is weaker than that of CL and suggest that interaction with CIV drives this difference. Atomistic contact analyses show that, although interaction with CIII is similar for CL and MLCL, CIV makes more contacts with CL than MLCL, demonstrating that CL is a more successful "glue" between the two complexes. Simulations of the human CIII2CIV supercomplex show that this interface site is maintained between species. Our study suggests that MLCL accumulation in people with BTHS disrupts supercomplex stability by formation of relatively weak interactions at the interface lipid binding site.

The guidance and adhesion protein FLRT2 dimerizes in cis via dual small-X3-small transmembrane motifs.

Fibronectin Leucine-rich Repeat Transmembrane (FLRT 1-3) proteins are a family of broadly expressed single-spanning transmembrane receptors that play key roles in development. Their extracellular domains mediate homotypic cell-cell adhesion and heterotypic protein interactions with other receptors to regulate cell adhesion and guidance. These in trans FLRT interactions determine the formation of signaling complexes of varying complexity and function. Whether FLRTs also interact at the surface of the same cell, in cis, remains unknown. Here, molecular dynamics simulations reveal two dimerization motifs in the FLRT2 transmembrane helix. Single particle tracking experiments show that these Small-X3-Small motifs synergize with a third dimerization motif encoded in the extracellular domain to permit the cis association and co-diffusion patterns of FLRT2 receptors on cells. These results may point to a competitive switching mechanism between in cis and in trans interactions, which suggests that homotypic FLRT interaction mirrors the functionalities of classic adhesion molecules.

What have molecular simulations contributed to understanding of Gram-negative bacterial cell envelopes?

Bacterial cell envelopes are compositionally complex and crowded and while highly dynamic in some areas, their molecular motion is very limited, to the point of being almost static in others. Therefore, it is no real surprise that studying them at high resolution across a range of temporal and spatial scales requires a number of different techniques. Details at atomistic to molecular scales for up to tens of microseconds are now within range for molecular dynamics simulations. Here we review how such simulations have contributed to our current understanding of the cell envelopes of Gram-negative bacteria.

PyLipID: A Python Package for Analysis of Protein-Lipid Interactions from Molecular Dynamics Simulations.

Lipids play important modulatory and structural roles for membrane proteins. Molecular dynamics simulations are frequently used to provide insights into the nature of these protein-lipid interactions. Systematic comparative analysis requires tools that provide algorithms for objective assessment of such interactions. We introduce PyLipID, a Python package for the identification and characterization of specific lipid interactions and binding sites on membrane proteins from molecular dynamics simulations. PyLipID uses a community analysis approach for binding site detection, calculating lipid residence times for both the individual protein residues and the detected binding sites. To assist structural analysis, PyLipID produces representative bound lipid poses from simulation data, using a density-based scoring function. To estimate residue contacts robustly, PyLipID uses a dual-cutoff scheme to differentiate between lipid conformational rearrangements while bound from full dissociation events. In addition to the characterization of protein-lipid interactions, PyLipID is applicable to analysis of the interactions of membrane proteins with other ligands. By combining automated analysis, efficient algorithms, and open-source distribution, PyLipID facilitates the systematic analysis of lipid interactions from large simulation data sets of multiple species of membrane proteins.

Anatomy of the Breast Fascial System: A Systematic Review of the Literature.

BACKGROUND: Understanding the anatomy of the fascial and ligamentous structures of the breast is important in both aesthetic and reconstructive breast surgery. Several structures have been identified that play a significant role in the aesthetic qualities and support of the breast warranting consideration in the context of breast reconstruction. METHODS: The authors performed a systematic review of anatomical, clinical, histologic, and radiologic studies that have described, characterized, and named these structures. The authors have summarized and critically appraised prior research to clarify and define the key fascial structures of the breast, their anatomical function, and their clinical significance in aesthetic and reconstructive breast surgery. RESULTS: Through their review, six distinct breast fascial structures were encountered consistently in the literature. The authors have organized them into intraglandular and extraglandular structures and have reviewed their significance in the context of reconstructive breast surgery. CONCLUSIONS: The primary fascial structures of the breast are important anatomical landmarks with numerous clinical applications. Cooper ligaments divide the breast parenchyma. The superficial and deep layers of the superficial fascia encase the breast in a "pocket," condensing into one thickened layer of fascia along the peripheral breast footprint. The inframammary fold supports and defines the inferior pole. The horizontal septum is a reliable neurovascular landmark. The vertical septum is a newly discovered fascial structure. There are certainly clinical implications that have yet to be described because of the relatively limited and disputed information on the fascia of the female breast and, ultimately, more research is warranted.

Identification and assessment of cardiolipin interactions with E. coli inner membrane proteins.

Integral membrane proteins are localized and/or regulated by lipids present in the surrounding bilayer. While bacteria have relatively simple membranes, there is ample evidence that many bacterial proteins bind to specific lipids, especially the anionic lipid cardiolipin. Here, we apply molecular dynamics simulations to assess lipid binding to 42 different Escherichia coli inner membrane proteins. Our data reveal an asymmetry between the membrane leaflets, with increased anionic lipid binding to the inner leaflet regions of the proteins, particularly for cardiolipin. From our simulations, we identify >700 independent cardiolipin binding sites, allowing us to identify the molecular basis of a prototypical cardiolipin binding site, which we validate against structures of bacterial proteins bound to cardiolipin. This allows us to construct a set of metrics for defining a high-affinity cardiolipin binding site on bacterial membrane proteins, paving the way for a heuristic approach to defining other protein-lipid interactions.

Modulation of adenosine A2a receptor oligomerization by receptor activation and PIP2 interactions.

GPCRs have been shown to form oligomers, which generate distinctive signaling outcomes. However, the structural nature of the oligomerization process remains uncertain. We have characterized oligomeric configurations of the adenosine A2a receptor (A2aR) by combining large-scale molecular dynamics simulations with Markov state models. These oligomeric structures may also serve as templates for studying oligomerization of other class A GPCRs. Our simulation data revealed that receptor activation results in enhanced oligomerization, more diverse oligomer populations, and a more connected oligomerization network. The active state conformation of the A2aR shifts protein-protein association interfaces to those involving intracellular loop ICL3 and transmembrane helix TM6. Binding of PIP2 to A2aR stabilizes protein-protein interactions via PIP2-mediated association interfaces. These results indicate that A2aR oligomerization is responsive to the local membrane lipid environment. This, in turn, suggests a modulatory effect on A2aR whereby a given oligomerization profile favors the dynamic formation of specific supramolecular signaling complexes.

Unique variants in CLCN3, encoding an endosomal anion/proton exchanger, underlie a spectrum of neurodevelopmental disorders.

The genetic causes of global developmental delay (GDD) and intellectual disability (ID) are diverse and include variants in numerous ion channels and transporters. Loss-of-function variants in all five endosomal/lysosomal members of the CLC family of Cl- channels and Cl-/H+ exchangers lead to pathology in mice, humans, or both. We have identified nine variants in CLCN3, the gene encoding CIC-3, in 11 individuals with GDD/ID and neurodevelopmental disorders of varying severity. In addition to a homozygous frameshift variant in two siblings, we identified eight different heterozygous de novo missense variants. All have GDD/ID, mood or behavioral disorders, and dysmorphic features; 9/11 have structural brain abnormalities; and 6/11 have seizures. The homozygous variants are predicted to cause loss of ClC-3 function, resulting in severe neurological disease similar to the phenotype observed in Clcn3-/- mice. Their MRIs show possible neurodegeneration with thin corpora callosa and decreased white matter volumes. Individuals with heterozygous variants had a range of neurodevelopmental anomalies including agenesis of the corpus callosum, pons hypoplasia, and increased gyral folding. To characterize the altered function of the exchanger, electrophysiological analyses were performed in Xenopus oocytes and mammalian cells. Two variants, p.Ile607Thr and p.Thr570Ile, had increased currents at negative cytoplasmic voltages and loss of inhibition by luminal acidic pH. In contrast, two other variants showed no significant difference in the current properties. Overall, our work establishes a role for CLCN3 in human neurodevelopment and shows that both homozygous loss of ClC-3 and heterozygous variants can lead to GDD/ID and neuroanatomical abnormalities.

COVID-19: neonatal-perinatal perspectives.

The coronavirus disease 2019 (COVID-19) pandemic, resulting from infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has caused severe and widespread illness in adults, including pregnant women, while rarely infecting neonates. An incomplete understanding of disease pathogenesis and viral spread has resulted in evolving guidelines to reduce transmission from infected mothers to neonates. Fortunately, the risk of neonatal infection via perinatal/postnatal transmission is low when recommended precautions are followed. However, the psychosocial implications of these practices and racial/ethnic disparities highlighted by this pandemic must also be addressed when caring for mothers and their newborns. This review provides a comprehensive overview of neonatal-perinatal perspectives of COVID-19, ranging from the basic science of infection and recommendations for care of pregnant women and neonates to important psychosocial, ethical, and racial/ethnic topics emerging as a result of both the pandemic and the response of the healthcare community to the care of infected individuals.

Heterozygous Variants in KDM4B Lead to Global Developmental Delay and Neuroanatomical Defects.

KDM4B is a lysine-specific demethylase with a preferential activity on H3K9 tri/di-methylation (H3K9me3/2)-modified histones. H3K9 tri/di-demethylation is an important epigenetic mechanism responsible for silencing of gene expression in animal development and cancer. However, the role of KDM4B on human development is still poorly characterized. Through international data sharing, we gathered a cohort of nine individuals with mono-allelic de novo or inherited variants in KDM4B. All individuals presented with dysmorphic features and global developmental delay (GDD) with language and motor skills most affected. Three individuals had a history of seizures, and four had anomalies on brain imaging ranging from agenesis of the corpus callosum with hydrocephalus to cystic formations, abnormal hippocampi, and polymicrogyria. In mice, lysine demethylase 4B is expressed during brain development with high levels in the hippocampus, a region important for learning and memory. To understand how KDM4B variants can lead to GDD in humans, we assessed the effect of KDM4B disruption on brain anatomy and behavior through an in vivo heterozygous mouse model (Kdm4b+/-), focusing on neuroanatomical changes. In mutant mice, the total brain volume was significantly reduced with decreased size of the hippocampal dentate gyrus, partial agenesis of the corpus callosum, and ventriculomegaly. This report demonstrates that variants in KDM4B are associated with GDD/ intellectual disability and neuroanatomical defects. Our findings suggest that KDM4B variation leads to a chromatinopathy, broadening the spectrum of this group of Mendelian disorders caused by alterations in epigenetic machinery.

De novo variants in MPP5 cause global developmental delay and behavioral changes.

Membrane Protein Palmitoylated 5 (MPP5) is a highly conserved apical complex protein essential for cell polarity, fate and survival. Defects in cell polarity are associated with neurologic disorders including autism and microcephaly. MPP5 is essential for neurogenesis in animal models, but human variants leading to neurologic impairment have not been described. We identified three patients with heterozygous MPP5 de novo variants (DNV) and global developmental delay (GDD) and compared their phenotypes and magnetic resonance imaging (MRI) to ascertain how MPP5 DNV leads to GDD. All three patients with MPP5 DNV experienced GDD with language delay/regression and behavioral changes. MRI ranged from normal to decreased gyral folding and microcephaly. The effects of MPP5 depletion on the developing brain were assessed by creating a heterozygous conditional knock out (het CKO) murine model with central nervous system (CNS)-specific Nestin-Cre drivers. In the het CKO model, Mpp5 depletion led to microcephaly, decreased cerebellar volume and cortical thickness. Het CKO mice had decreased ependymal cells and Mpp5 at the apical surface of cortical ventricular zone compared with wild type. Het CKO mice also failed to maintain progenitor pools essential for neurogenesis. The proportion of cortical cells undergoing apoptotic cell death increased, suggesting that cell death reduces progenitor population and neuron number. Het CKO mice also showed behavioral changes, similar to our patients. To our knowledge, this is the first report to show that variants in MPP5 are associated with GDD, behavioral abnormalities and language regression/delay. Murine modeling shows that neurogenesis is likely altered in these individuals, with cell death and skewed cellular composition playing significant roles.