IMCellXMBD: A statistical approach for robust cell identification and quantification from imaging mass cytometry images

ImagingMass Cytometry (IMC) has become a useful tool in biomedical research due to its capability to measure over 100 markers simultaneously. Unfortunately, some protein channels in IMC images can be very noisy, whichmay significantly affect the phenotyping results without proper data processing. We developed IMCellXMBD, a highly effective and generalizable cell identification and quantification method for IMC images. IMCell performs denoising by subtracting an estimated background noise value from pixel values for each individual protein channel, identifies positive cells from negative cells by comparing the distribution between segmented cells and decoy cells, and normalize the protein expression levels of the identified positive cells for downstream data analysis. Experimental results demonstrate that our method significantly improves the reliability of cell phenotyping which is essential for using IMC in biomedical studies.

The cryo-EM structure of an ERAD protein channel formed by tetrameric human Derlin-1

Endoplasmic reticulum–associated degradation (ERAD) is a process directing misfolded proteins from the ER lumen and membrane to the degradation machinery in the cytosol. A key step in ERAD is the translocation of ER proteins to the cytosol. Derlins are essential for protein translocation in ERAD, but the mechanism remains unclear. Here, we solved the structure of human Derlin-1 by cryo–electron microscopy. The structure shows that Derlin-1 forms a homotetramer that encircles a large tunnel traversing the ER membrane. The tunnel has a diameter of about 12 to 15 angstroms, large enough to allow an α helix to pass through. The structure also shows a lateral gate within the membrane, providing access of transmembrane proteins to the tunnel, and thus, human Derlin-1 forms a protein channel for translocation of misfolded proteins. Our structure is different from the monomeric yeast Derlin structure previously reported, which forms a semichannel with another protein.

Stable Polymer Bilayers for Protein Channel Recordings at High Guanidinium Chloride Concentrations

Use of chaotropic reagents is common in biophysical characterization of biomolecules. When the study involves transmembrane protein channels, the stability of the protein channel and supporting bilayer membrane must be considered. In this letter we show that planar bilayers composed of poly(1,2-butadiene)-b-poly(ethylene oxide) diblock copolymer are stable and leak-free at high guanidinium chloride concentrations, in contrast to diphytanoyl phosphatidylcholine bilayers which exhibit deleterious leakage under similar conditions. Further, insertion and functional analysis of channels such as α-hemolysin and MspA are straightforward in these polymer membranes. Finally, we demonstrate that α-hemolysin channels maintain their structural integrity at 2M guanidinium chloride concentrations using blunt DNA hairpins as molecular reporters.

Computational Study of Ions and Water Permeation and Transportation Mechanisms of the SARS-CoV-2 Pentameric E Protein Channel

Coronavirus disease 2019 (COVID-19) is caused by a novel coronavirus (SARS-CoV-2) and represents the causative agent of a potentially fatal disease that is of public health emergency of international concern. Coronaviruses, including SARS-CoV-2, encode an envelope (E) protein, which is a small, hydrophobic membrane protein; the E protein of SARS-CoV-2 has high homology with that of severe acute respiratory syndrome coronavirus. (SARS-CoV) In this study, we provide insights into the function of the SARS-CoV-2 E protein channel and the ion and water permeation mechanisms on the basis of combined in silico methods. Our results suggest that the pentameric E protein promotes the penetration of monovalent ions through the channel. Analysis of the potential mean force (PMF), pore radius and diffusion coefficient reveals that Leu10 and Phe19 are the hydrophobic gates of the channel. In addition, the pore demonstrated a clear wetting/dewetting transition with monovalent cation selectivity under transmembrane voltage, which indicates that it is a hydrophobic voltage-dependent channel. Overall, these results provide structural-basis insights and molecular-dynamic information that are needed to understand the regulatory mechanisms of ion permeability in the pentameric SARS-CoV-2 E protein channel.

Elaborate mechanism for the synthesis of lipopolysaccharide points to its functional importance in Gram-negative bacteria

Gram-negative bacteria such as Escherichia coli and Salmonella enterica possess two phospholipid bilayer membranes in the cell envelope. Known as the inner and outer membrane, proteins encased in the outer membrane as well as surface moieties displayed by the membrane play important roles in host-cell recognition, cell-cell interactions and triggering of immune response in host. One such molecule that partakes in triggering immune responses in human is lipopolysaccharides that constitute the outer leaflet of the outer membrane of Gram-negative bacteria. Specifically, recent research has added new details and mechanisms for the elaborate choreographed enzymatic reactions that synthesize lipopolysaccharides. In particular, lipopolysaccharides are synthesized in the inner membrane and transported to the outer membrane through a dedicated protein channel formed by proteins of the same lipopolysaccharide synthesis pathway. In short, significant amount of proteins and cellular resources are expended in the synthesis and transport of lipopolysaccharides which suggests important evolutionary significance and functionality of the molecule. Specifically, evolutionary significance of lipopolysaccharide can be gleaned from the organisation of the pathway that mediate its synthesis, where dedicated channels are constructed from proteins to help the unidirectional transfer of the molecule from the inner to outer membrane. Such dedicated channels are not of high occurrence in cells, which suggests critical functional importance of lipopolysaccharides to Gram-negative bacteria. Perhaps, signalling mechanisms responsible for cell-cell interactions are mediated by lipopolysaccharides, or the molecule might play important roles in cellular recognition between different bacterial species in community assemblage such as biofilm. Collectively, mechanistic studies aimed at understanding the formation of the outer membrane of Gram-negative bacteria has opened our eyes to the elaborate mechanism by which lipopolysaccharides on the outer leaflet of the outer membrane are synthesized. Comprising a synthetic machinery and a dedicated protein channel for transporting the synthesized lipopolysaccharides from the inner to outer membrane, a dedicated pathway of proteins mediates the synthesis of this molecule; thereby, pointing to its functional importance to the bacterial cell. While lipopolysaccharides are known to trigger immune responses in humans, its potential broader role in cell-cell communications such as those important for maintaining organisation and community structure in biofilm communities remain unappreciated. Overall, given the significant amount of energy and cellular resources dedicated to its synthesis, lipopolysaccharides should have broader functional roles and might partake in many as-yet unknown signalling and metabolic activities at the cellular and community level.

Elaborate mechanism for the synthesis of lipopolysaccharide points to its functional importance in Gram-negative bacteria

Gram-negative bacteria such as Escherichia coli and Salmonella enterica possess two phospholipid bilayer membranes in the cell envelope. Known as the inner and outer membrane, proteins encased in the outer membrane as well as surface moieties displayed by the membrane play important roles in host-cell recognition, cell-cell interactions and triggering of immune response in host. One such molecule that partakes in triggering immune responses in human is lipopolysaccharides that constitute the outer leaflet of the outer membrane of Gram-negative bacteria. Specifically, recent research has added new details and mechanisms for the elaborate choreographed enzymatic reactions that synthesize lipopolysaccharides. In particular, lipopolysaccharides are synthesized in the inner membrane and transported to the outer membrane through a dedicated protein channel formed by proteins of the same lipopolysaccharide synthesis pathway. In short, significant amount of proteins and cellular resources are expended in the synthesis and transport of lipopolysaccharides which suggests important evolutionary significance and functionality of the molecule. Specifically, evolutionary significance of lipopolysaccharide can be gleaned from the organisation of the pathway that mediate its synthesis, where dedicated channels are constructed from proteins to help the unidirectional transfer of the molecule from the inner to outer membrane. Such dedicated channels are not of high occurrence in cells, which suggests critical functional importance of lipopolysaccharides to Gram-negative bacteria. Perhaps, signalling mechanisms responsible for cell-cell interactions are mediated by lipopolysaccharides, or the molecule might play important roles in cellular recognition between different bacterial species in community assemblage such as biofilm. Collectively, mechanistic studies aimed at understanding the formation of the outer membrane of Gram-negative bacteria has opened our eyes to the elaborate mechanism by which lipopolysaccharides on the outer leaflet of the outer membrane are synthesized. Comprising a synthetic machinery and a dedicated protein channel for transporting the synthesized lipopolysaccharides from the inner to outer membrane, a dedicated pathway of proteins mediates the synthesis of this molecule; thereby, pointing to its functional importance to the bacterial cell. While lipopolysaccharides are known to trigger immune responses in humans, its potential broader role in cell-cell communications such as those important for maintaining organisation and community structure in biofilm communities remain unappreciated. Overall, given the significant amount of energy and cellular resources dedicated to its synthesis, lipopolysaccharides should have broader functional roles and might partake in many as-yet unknown signalling and metabolic activities at the cellular and community level.