Biophysical properties of the isolated spike protein binding helix of human ACE2.

The entry of the severe acute respiratory syndrome coronavirus 2 virus in human cells is mediated by the binding of its surface spike protein to the human angiotensin-converting enzyme 2 (ACE2) receptor. A 23-residue long helical segment (SBP1) at the binding interface of human ACE2 interacts with viral spike protein and therefore has generated considerable interest as a recognition element for virus detection. Unfortunately, emerging reports indicate that the affinity of SBP1 to the receptor-binding domain of the spike protein is much lower than that of the ACE2 receptor itself. Here, we examine the biophysical properties of SBP1 to reveal factors leading to its low affinity for the spike protein. Whereas SBP1 shows good solubility (solubility > 0.8 mM), circular dichroism spectroscopy shows that it is mostly disordered with some antiparallel ß-sheet content and no helicity. The helicity is substantial (>20%) only upon adding high concentrations (≥20% v/v) of 2,2,2-trifluoroethanol, a helix promoter. Fluorescence correlation spectroscopy and single-molecule photobleaching studies show that the peptide oligomerizes at concentrations >50 nM. We hypothesized that mutating the hydrophobic residues (F28, F32, and F40) of SBP1, which do not directly interact with the spike protein, to alanine would reduce peptide oligomerization without affecting its spike binding affinity. Whereas the mutant peptide (SBP1mod) shows substantially reduced oligomerization propensity, it does not show improved helicity. Our study shows that the failure of efforts, so far, to produce a short SBP1 mimic with a high affinity for the spike protein is not only due to the lack of helicity but is also due to the heretofore unrecognized problem of oligomerization.

Altered Membrane Mechanics Provides a Receptor-Independent Pathway for Serotonin Action.

Serotonin, an important signaling molecule in humans, has an unexpectedly high lipid membrane affinity. The significance of this finding has evoked considerable speculation. Here we show that membrane binding by serotonin can directly modulate membrane properties and cellular function, providing an activity pathway completely independent of serotonin receptors. Atomic force microscopy shows that serotonin makes artificial lipid bilayers softer, and induces nucleation of liquid disordered domains inside the raft-like liquid-ordered domains. Solid-state NMR spectroscopy corroborates this data at the atomic level, revealing a homogeneous decrease in the order parameter of the lipid chains in the presence of serotonin. In the RN46A immortalized serotonergic neuronal cell line, extracellular serotonin enhances transferrin receptor endocytosis, even in the presence of broad-spectrum serotonin receptor and transporter inhibitors. Similarly, it increases the membrane binding and internalization of oligomeric peptides. Our results uncover a mode of serotonin-membrane interaction that can potentiate key cellular processes in a receptor-independent fashion.

Membrane affinity of individual toxic protein oligomers determined at the single-molecule level.

Oligomers are the key suspects in protein aggregation-linked diseases, such as Alzheimer's and Type II diabetes, and most likely exert their toxicity by interacting with lipid membranes. However, the "which oligomer" question remains an obstacle in understanding the disease mechanism, as the exact identity of the toxic oligomer(s) is not yet known. Oligomers exist as a mixture of species of different sizes (i.e. as different 'n-mers') in a physiological solution, making it difficult to determine the properties of individual species. Here we demonstrate a method based on single-molecule photo-bleaching (smPB) which can provide an answer to the "which oligomer" question, at least as far as membrane affinity is concerned. We calculate the ratio of the oligomer size distribution of human Islet Amyloid Polypeptide (IAPP) in the aqueous phase and that on a coexisting artificial lipid bilayer, and this measures the relative membrane affinity of individual oligomeric species. A problem with smPB measurements is that they can be very sensitive to pre-measurement bleaching. Here we correct for pre-bleaching using a covalently linked multimeric peptide as a bleaching standard. We find that the order of membrane affinity for IAPP n-mers is trimer > dimer > tetramer ≫ monomer. Our results agree well with the average membrane affinity values of oligomeric and monomeric solutions previously measured with Fluorescence Correlation Spectroscopy. The "which oligomer" question, in the context of membrane affinity, can therefore, be solved quantitatively for any membrane-active toxic protein aggregate.

Single-Molecule Maps of Membrane Insertion by Amyloid-ß Oligomers Predict Their Toxicity.

The interaction of small Amyloid-ß (Aß) oligomers with the lipid membrane is an important component of the pathomechanism of Alzheimer's disease (AD). However, oligomers are heterogeneous in size. How each type of oligomer incorporates into the membrane, and how that relates to their toxicity, is unknown. Here, we employ a single molecule technique called Q-SLIP (Quencher-induced Step Length Increase in Photobleaching) to measure the membrane insertion of each monomeric unit of individual oligomers of Aß42, Aß40, and Aß40-F19-Cyclohexyl alanine (Aß40-F19Cha), and correlate it with their toxicity. We observe that the N-terminus of Aß42 inserts close to the center of the bilayer, the less toxic Aß40 inserts to a shallower depth, and the least toxic Aß40-F19Cha has no specific distribution. This oligomer-specific map provides a mechanistic representation of membrane-mediated Aß toxicity and should be a valuable tool for AD research.

A Toxicogenic Interaction between Intracellular Amyloid-ß and Apolipoprotein-E.

Alzheimer's disease (AD) is associated with the aggregation of amyloid ß (Aß) and tau proteins. Why ApoE variants are significant genetic risk factors remains a major unsolved puzzle in understanding AD, although intracellular interactions with ApoE are suspected to play a role. Here, we show that specific changes in the fluorescence lifetime of fluorescently tagged small Aß oligomers in rat brain cells correlate with the cellular ApoE content. An inhibitor of the Aß-ApoE interaction suppresses these changes and concomitantly reduces Aß toxicity in a dose-dependent manner. Single-molecule techniques show changes both in the conformation and in the stoichiometry of the oligomers. Neural stem cells derived from hiPSCs of Alzheimer's patients also exhibit these fluorescence lifetime changes. We infer that intracellular interaction with ApoE modifies the N-terminus of the Aß oligomers, inducing changes in their stoichiometry, membrane affinity, and toxicity. These changes can be directly imaged in live cells and can potentially be used as a rapid and quantitative cellular assay for AD drug discovery.

Determining the Stoichiometry of Amyloid Oligomers by Single-Molecule Photobleaching.

Small oligomers are the initial intermediates in the pathway to amyloid fibril formation. They have a distinct identity from the monomers as well as from the protofibrils and the fibrils, both in their structure and in their properties. In many cases, they play a crucial biological role. However, due to their transient nature, they are difficult to characterize. "Oligomer" is a diffuse definition, encompassing aggregates of many different sizes, and this lack of precise definition causes much confusion and disagreement between different research groups. Here, we define the small oligomers as "n"-mers with n < 10, which is the size range in which the amyloid proteins typically exist at the initial phase of the aggregation process. Since the oligomers dynamically interconvert into each other, a solution of aggregating amyloid proteins will contain a distribution of sizes. A precise characterization of an oligomeric solution will, therefore, require quantification of the relative population of each size. Size-based separation methods, such as size-exclusion chromatography, are typically used to characterize this distribution. However, if the interconversion between oligomers of different sizes is fast, this would not yield reliable results. Single-molecule photobleaching (smPB) is a direct method to evaluate this size distribution in a heterogeneous solution without separation. In addition, understanding the mechanism of action of amyloid oligomers requires knowing the affinity of each oligomer type to different cellular components, such as the cell membrane. These measurements are also amenable to smPB. Here we show how to perform smPB, both for oligomers in solution and for oligomers attached to the membrane.

Single Molecule Measurements of the Accessibility of Molecular Surfaces.

An important measure of the conformation of protein molecules is the degree of surface exposure of its specific segments. However, this is hard to measure at the level of individual molecules. Here, we combine single molecule photobleaching (smPB, which resolves individual photobleaching steps of single molecules) and fluorescence quenching techniques to measure the accessibility of individual fluorescently labeled protein molecules to quencher molecules in solution. A quencher can reduce the time a fluorophore spends in the excited state, increasing its photostability under continuous irradiation. Consequently, the photo-bleaching step length would increase, providing a measure for the accessibility of the fluorophore to the solvent. We demonstrate the method by measuring the bleaching step-length increase in a lipid, and also in a lipid-anchored peptide (both labelled with rhodamine-B and attached to supported lipid bilayers). The fluorophores in both molecules are expected to be solvent-exposed. They show a near two-fold increase in the step length upon incubation with 5 mM tryptophan (a quencher of rhodamine-B), validating our approach. A population distribution plot of step lengths before and after addition of tryptophan show that the increase is not always homogenous. Indeed there are different species present with differential levels of exposure. We then apply this technique to determine the solvent exposure of membrane-attached N-terminus labelled amylin (h-IAPP, an amyloid associated with Type II diabetes) whose interaction with lipid bilayers is poorly understood. hIAPP shows a much smaller increase of the step length, signifying a lower level of solvent exposure of its N-terminus. Analysis of results from individual molecules and step length distribution reveal that there are at least two different conformers of amylin in the lipid bilayer. Our results show that our method ("Q-SLIP", Quenching-induced Step Length increase in Photobleaching) provides a simple route to probe the conformational states of membrane proteins at a single molecule level.

Ordered and Disordered Segments of Amyloid-ß Drive Sequential Steps of the Toxic Pathway.

While the roles of intrinsically disordered protein domains in driving interprotein interactions are increasingly well-appreciated, the mechanism of toxicity of disease-causing disordered proteins remains poorly understood. A prime example is Alzheimer's disease (AD) associated amyloid beta (Aß). Aß oligomers are highly toxic partially structured peptide assemblies with a distinct ordered region (residues ∼10-40) and a shorter disordered region (residues ∼1-9). Here, we investigate the role of this disordered domain and its relation to the ordered domain in the manifestation of toxicity through a set of Aß fragments and stereoisomers designed for this purpose. We measure their effects on lipid membranes and cultured neurons, probing their toxicity, intracellular distributions, and specific molecular interactions using the techniques of confocal imaging, lattice light sheet imaging, fluorescence lifetime imaging, and fluorescence correlation spectroscopy. Remarkably, we find that neither part-Aß10-40 or Aß1-9, is toxic by itself. The ordered part (Aß10-40) is the major determinant of how Aß attaches to lipid bilayers, enters neuronal cells, and localizes primarily in the late endosomal compartments. However, once Aß enters the cell, it is the disordered part (only when it is connected to the rest of the peptide) that has a strong and stereospecific interaction with an unknown cellular component, as demonstrated by distinct changes in the fluorescence lifetime of a fluorophore attached to the N-terminal. This interaction appears to commit Aß to the toxic pathway. Our findings correlate well with Aß sites of familial AD mutations, a significant fraction of which cluster in the disordered region. We conclude that, while the ordered region dictates attachment and cellular entry, the key to toxicity lies in the ordered part presenting the disordered part for a specific cellular interaction.

In Hospital Mortality in Patients with Impaired Fasting Glucose and Acute Myocardial Infarction in a Tertiary Care Centre of Rural Bengal

Introduction : The Cardiovascular mortality in Diabetics is 2-4 times higher than in Non-diabetic population. But there is still controversy regarding Pre-diabetes (IFG and IGT) as a Cardiovascular Risk Factor. Aims and Objectives : In this study we aimed to investigate the early in-hospital mortality among Acute Myocardial Infarction (AMI) patients having Impaired Fasting Glucose (IFG) during the first 7 days of hospitalization. Materials and Methods: A total of 150 AMI patients were evaluated and followed up for their glycemic status and early in hospital mortality (first 7 days) at Burdwan Medical College, Burdwan, West Bengal. Result and Analysis: Mortality in patients having IFG (18%) was higher and as much as in DM (20%) compared to euglycemic (4%) patients but the mortality is not correlated with mean Fasting Plasma Glucose (FPG) level. Conclusion : IFG (ie, pre-diabetes) increases Cardiovascular mortality as much as diabetes. So, IFG may be a marker or risk factor for mortality but lowering FPG in AMI patients is unlikely to yield beneficial effect regarding mortality.

Prevalence Of Fecolith In Clinically Suspected Cases Of Acute Appendicitis.

In this study we wanted to nd out the alliance between the presence of fecolith and appendicitis. The present study was conducted on 100 patients all having an initial clinical diagnosis of acute appendicitis. 96 out of 100 patients were operated on. 4 patients were treated non-operatively. Out of 96 patients operated, 75 had conrmed histopathological diagnosis of acute appendicitis. Fecolith was found in 44% acute appendicitis cases and in 17.6% cases histologically normal appendix, which proves that the presence of fecolith is not characteristic of appendicitis. Gangrenous and perforated cases were more frequently associated with fecolith showing that it plays an aggressive role in the pathophysiology of the disease. Additional line of research on the subject matter is suggested.

Estimation of Stature From Fragment of Femur (Popliteal Length) in Bengalee Population.

Introduction: Of the mathematical methods, regression equations have been successfully used for estimation of stature. Population specific formulae produce more accurate results. The present investigation was designed to estimate stature from fragment of femur obtained from a collection of Bengalee population of the state of West Bengal of India. Materials & Methods: The fragment of the femur (Popliteal length of femur) was measured by a vertical length from the point where the distance between external borders of both linea aspera lips becomes 10 mm (it was considered as the lower end of linea aspera, where the two lips diverges below), to the ground where lower surfaces of both the condyles were in contact. Results: The following regression equation was obtained: Stature in feet = 0.127[20.1184 + 1.6890x]. (‘x’ stands for popliteal length of femur in centimeter.) Discussion: This would help in identification of unknown skeletal remains, as estimation of stature is an important part in establishing the biological profile of skeletal remains.