Molecular and Functional Characterization of Caveolae in Mixed Cultures of Human NT-2 Neurons and Astrocytes

Caveolae are plasma membrane invaginations that are enriched in cholesterol-binding proteins called caveolins. The presence of caveolae and caveolins in mixed cultures of human neurons and glia has not been investigated. Here, we sought to determine the presence of caveolae and caveolins in human NTera-2 (NT2/D1) cells, differentiated with retinoic acid into neuron-like (NT2/N) and astrocyte-like (NT2/A) cells. We found that while caveolin-3 mRNA levels remained relatively constant, caveolin-1 and -2 levels were upregulated in NT2/A and downregulated in NT2/N. No caveolin-1 immunoreactivity was detected in NT2/N. Electron microscopy revealed numerous flask-shaped invaginations (~86–102 nm in diameter) in the plasma membrane of NT2/A and NT2/N cells, while only few were detected in NT2/D1 cells. Immunoelectron microscopy localized caveolin-1 gold particles in the flask-shaped structures on plasmalemma and cytoplasmic vesicles of NT2/A cells. Furthermore, NT2/A endocytosed Alexa 488 conjugated-cholera toxin B subunit (CTX-B) through a caveolae- and clathrin-dependent pathway, whereas NT2/N endocytosed CTX-B through a caveolae-independent pathway. We have established that while NT2/A expressed functional caveolae, the molecular identity of the plasma membrane invaginations in NT2/N is unknown. The expression of caveolin proteins was differentially regulated in these cells. Taken together, our findings support the usefulness of the human NT2 model system to study the role of caveolins in neuron–glia communication, and their involvement in brain health and disease.

Towards A Novel Multi-Epitopes Chimeric Vaccine for Simulating Strong Immune Responses and Protection against Morganella morganii

Morganella morganii is one of the main etiological agents of hospital-acquired infections and no licensed vaccine is available against the pathogen. Herein, we designed a multi-epitope-based vaccine against M. morganii. Predicted proteins from fully sequenced genomes of the pathogen were subjected to a core sequences analysis, followed by the prioritization of non-redundant, host non-homologous and extracellular, outer membrane and periplasmic membrane virulent proteins as vaccine targets. Five proteins (TonB-dependent siderophore receptor, serralysin family metalloprotease, type 1 fimbrial protein, flagellar hook protein (FlgE), and pilus periplasmic chaperone) were shortlisted for the epitope prediction. The predicted epitopes were checked for antigenicity, toxicity, solubility, and binding affinity with the DRB*0101 allele. The selected epitopes were linked with each other through GPGPG linkers and were joined with the cholera toxin B subunit (CTBS) to boost immune responses. The tertiary structure of the vaccine was modeled and blindly docked with MHC-I, MHC-II, and Toll-like receptors 4 (TLR4). Molecular dynamic simulations of 250 nanoseconds affirmed that the designed vaccine showed stable conformation with the receptors. Further, intermolecular binding free energies demonstrated the domination of both the van der Waals and electrostatic energies. Overall, the results of the current study might help experimentalists to develop a novel vaccine against M. morganii.

Efferent and Afferent Connections of Neuropeptide Y Neurons in the Nucleus Accumbens of Mice

Neuropeptide Y (NPY) is a neural peptide distributed widely in the brain and has various functions in each region. We previously reported that NPY neurons in the nucleus accumbens (NAc) are involved in the regulation of anxiety behavior. Anterograde and retrograde tracing studies suggest that neurons in the NAc project to several areas, such as the lateral hypothalamus (LH) and ventral pallidum (VP), and receive afferent projections from the cortex, thalamus, and amygdala. However, the neural connections between accumbal NPY neurons and other brain areas in mice remain unclear. In this study, we sought to clarify these anatomical connections of NPY neurons in the NAc by investigating their neural outputs and inputs. To selectively map NPY neuronal efferents from the NAc, we injected Cre-dependent adeno-associated viruses (AAVs) into the NAc of NPY-Cre mice. This revealed that NAc NPY neurons exclusively projected to the LH. We confirmed this by injecting cholera toxin b subunit (CTb), a retrograde tracer, into the LH and found that approximately 7–10% of NPY neurons in the NAc were double-labeled for mCherry and CTb. Moreover, retrograde tracing using recombinant rabies virus (rRABV) also identified NAc NPY projections to the LH. Finally, we investigated monosynaptic input to the NPY neurons in the NAc using rRABV. We found that NPY neurons in the NAc received direct synaptic connections from the midline thalamic nuclei and posterior basomedial amygdala. These findings provide new insight into the neural networks of accumbal NPY neurons and should assist in elucidating their functional roles.

Modern History of Cholera Vaccines and the Pivotal Role of icddr,b

The rapid spread of the 7 th cholera pandemic over Asia in the 1960s led to several large field studies that revealed that the traditional injectable cholera vaccines had poor efficacy, usually less than 50% for only 3-6 months, which led WHO in the 1970s to stop recommending cholera vaccination. At the same time, it stimulated research that has led to the development of the effective orally administered cholera vaccines (OCVs) that today are a cornerstone in WHO´s strategy for “Ending Cholera – A Global Roadmap to 2030”. The first effective OCV, Dukoral™, containing a mixture of inactivated Vibrio cholerae bacteria and cholera toxin B subunit, was licenced in 1993 and is together with two similar inactivated whole-cell OCVs, Shanchol™ and Euvichol™/Euvichol-Plus™, the OCVs currently prequalified and recommended by WHO. This brief review is a personalized account of the “modern history” of the development of these now universally recognized effective tools for the control and ultimate elimination of cholera, and of the pivotal role of icddr,b and Bangladesh for this development.

Cholera Toxin as a Probe for Membrane Biology

Cholera toxin B-subunit (CTxB) has emerged as one of the most widely utilized tools in membrane biology and biophysics. CTxB is a homopentameric stable protein that binds tightly to up to five GM1 glycosphingolipids. This provides a robust and tractable model for exploring membrane structure and its dynamics including vesicular trafficking and nanodomain assembly. Here, we review important advances in these fields enabled by use of CTxB and its lipid receptor GM1.

Ceramide structure dictates glycosphingolipid nanodomain assembly and function

Gangliosides in the outer leaflet of the plasma membrane of eukaryotic cells are essential for many cellular functions and pathogenic interactions. How gangliosides are dynamically organized and how they respond to ligand binding is poorly understood. Using fluorescence anisotropy imaging of synthetic, fluorescently labeled GM1 gangliosides incorporated into the plasma membrane of living cells, we found that GM1 with a fully saturated C16:0 acyl chain, but not with unsaturated C16:1 acyl chain, is actively clustered into nanodomains, which depends on membrane cholesterol, phosphatidylserine and actin. The binding of cholera toxin B-subunit (CTxB) leads to enlarged membrane domains for both C16:0 and C16:1, owing to binding of multiple GM1 under a toxin, and clustering of CTxB. The structure of the ceramide acyl chain still affects these domains, as co-clustering with the glycosylphosphatidylinositol (GPI)-anchored protein CD59 occurs only when GM1 contains the fully saturated C16:0 acyl chain, and not C16:1. Thus, different ceramide species of GM1 gangliosides dictate their assembly into nanodomains and affect nanodomain structure and function, which likely underlies many endogenous cellular processes.

Expression of Bordetella pertussis Antigens Fused to Different Vectors and Their Effectiveness as Vaccines

Pertussis is an acute respiratory tract infection caused by Bordetella pertussis. Even though its current vaccine coverage is relatively broad, they still have some shortcomings such as short protection time and might be incapable of blocking the spread of the disease. In this study, we developed new pertussis vaccine candidates by separately fusing three pertussis antigens (B. pertussis fimbriae 2 “Fim2”, pertussis toxin S1 subunit “PtxS1”, and filamentous hemagglutinin “FHA1877–2250”) to each of two immune-boosting carrier proteins (B subunits of AB5 toxin family: cholera toxin B subunit “CTB” and shiga toxin B subunit “StxB”). We then immunized mice with these fusion antigens and found that they significantly increased the serum antibody titers and elicited high bactericidal activity against B. pertussis. After CTB-or StxB-fused antigen-immunized mice were challenged with a non-lethal dose of B. pertussis, the bacterial loads in different tissues of these mice were significantly reduced, and their lung damage was nearly invisible. Furthermore, we also demonstrated that these candidate vaccines could provide strong prophylactic effects against a lethal challenge with B. pertussis. Overall, our candidate vaccines conferred better immune protection to mice compared with pertussis antigen alone. This B5 subunit-based vaccine strategy provides a promising option for vaccine design.

An Update on Cholera Immunity and Current and Future Cholera Vaccines

Individual resistance to cholera infection and disease depends on both innate host factors and adaptive immunity acquired by a previous infection or vaccination. Locally produced, intestinal-mucosal secretory IgA (SIgA) antibodies against bacterial surface lipopolysaccharide (LPS) O antigens and/or secreted cholera toxins are responsible for the protective adaptive immunity, in conjunction with an effective mucosal immunologic memory that can elicit a rapid anamnestic SIgA antibody response upon re-exposure to the antigen/pathogen even many years later. Oral cholera vaccines (OCVs), based on inactivated Vibrio cholerae whole-cell components, either together with the cholera toxin B subunit (Dukoral™) or administered alone (Shanchol™/Euvichol-Plus™) were shown to be consistently safe and effective in large field trials in all settings. These OCVs are recommended by the World Health Organisation (WHO) for the control of both endemic cholera and epidemic cholera outbreaks. OCVs are now a cornerstone in WHO’s global strategy found in “Ending Cholera: A Global Roadmap to 2030.” However, the forecasted global demands for OCV, estimated by the Global Alliance for Vaccines and Immunization (GAVI) to 1.5 billion doses for the period 2020–2029, markedly exceed the existing manufacturing capacity. This calls for an increased production capacity of existing OCVs, as well as the rapid introduction of additional and improved vaccines under development.

Spray-Dried Formulation of Epicertin, a Recombinant Cholera Toxin B Subunit Variant That Induces Mucosal Healing

Epicertin (EPT) is a recombinant variant of the cholera toxin B subunit, modified with a C-terminal KDEL endoplasmic reticulum retention motif. EPT has therapeutic potential for ulcerative colitis treatment. Previously, orally administered EPT demonstrated colon epithelial repair activity in dextran sodium sulfate (DSS)-induced acute and chronic colitis in mice. However, the oral dosing requires cumbersome pretreatment with sodium bicarbonate to conserve the acid-labile drug substance while transit through the stomach, hampering its facile application in chronic disease treatment. Here, we developed a solid oral formulation of EPT that circumvents degradation in gastric acid. EPT was spray-dried and packed into enteric-coated capsules to allow for pH-dependent release in the colon. A GM1-capture KDEL-detection ELISA and size-exclusion HPLC indicated that EPT powder maintains activity and structural stability for up to 9 months. Capsule disintegration tests showed that EPT remained encapsulated at pH 1 but was released over 180 min at pH 6.8, the approximate pH of the proximal colon. An acute DSS colitis study confirmed the therapeutic efficacy of encapsulated EPT in C57BL/6 mice upon oral administration without gastric acid neutralization pretreatment compared to vehicle-treated mice (p < 0.05). These results provide a foundation for an enteric-coated oral formulation of spray-dried EPT.