Control of SARS-CoV-2 infection after Spike DNA or Spike DNA+Protein co-immunization in rhesus macaques

The speed of development, versatility and efficacy of mRNA-based vaccines have been amply demonstrated in the case of SARS-CoV-2. DNA vaccines represent an important alternative since they induce both humoral and cellular immune responses in animal models and in human trials. We tested the immunogenicity and protective efficacy of DNA-based vaccine regimens expressing different prefusion-stabilized Wuhan-Hu-1 SARS-CoV-2 Spike antigens upon intramuscular injection followed by electroporation in rhesus macaques. Different Spike DNA vaccine regimens induced antibodies that potently neutralized SARS-CoV-2 in vitro and elicited robust T cell responses. The antibodies recognized and potently neutralized a panel of different Spike variants including Alpha, Delta, Epsilon, Eta and A.23.1, but to a lesser extent Beta and Gamma. The DNA-only vaccine regimens were compared to a regimen that included co-immunization of Spike DNA and protein in the same anatomical site, the latter of which showed significant higher antibody responses. All vaccine regimens led to control of SARS-CoV-2 intranasal/intratracheal challenge and absence of virus dissemination to the lower respiratory tract. Vaccine-induced binding and neutralizing antibody titers and antibody-dependent cellular phagocytosis inversely correlated with transient virus levels in the nasal mucosa. Importantly, the Spike DNA+Protein co-immunization regimen induced the highest binding and neutralizing antibodies and showed the strongest control against SARS-CoV-2 challenge in rhesus macaques.     

Production and evaluation of dry alginate-chitosan microcapsules as an enteric delivery vehicle for probiotic bacteria

This study investigates the production of alginate microcapsules, which have been coated with the polysaccharide chitosan, and evaluates some of their properties with the intention of improving the gastrointestinal viability of a probiotic ( Bifidobacterium breve ) by encapsulation in this system. The microcapsules were dried by a variety of methods, and the most suitable was chosen. The work described in this Article is the first report detailing the effects of drying on the properties of these microcapsules and the viability of the bacteria within relative to wet microcapsules. The pH range over which chitosan and alginate form polyelectrolyte complexes was explored by spectrophotometry, and this extended into swelling studies on the microcapsules over a range of pHs associated with the gastrointestinal tract. It was shown that chitosan stabilizes the alginate microcapsules at pHs above 3, extending the stability of the capsules under these conditions. The effect of chitosan exposure time on the coating thickness was investigated for the first time by confocal laser scanning microscopy, and its penetration into the alginate matrix was shown to be particularly slow. Coating with chitosan was found to increase the survival of B. breve in simulated gastric fluid as well as prolong its release upon exposure to intestinal pH.     

Fixation Biases towards the Index Finger in Almost-Natural Grasping

We use visual information to guide our grasping movements. When grasping an object with a precision grip, the two digits need to reach two different positions more or less simultaneously, but the eyes can only be directed to one position at a time. Several studies that have examined eye movements in grasping have found that people tend to direct their gaze near where their index finger will contact the object. Here we aimed at better understanding why people do so by asking participants to lift an object off a horizontal surface. They were to grasp the object with a precision grip while movements of their hand, eye and head were recorded. We confirmed that people tend to look closer to positions that a digit needs to reach more accurately. Moreover, we show that where they look as they reach for the object depends on where they were looking before, presumably because they try to minimize the time during which the eyes are moving so fast that no new visual information is acquired. Most importantly, we confirmed that people have a bias to direct gaze towards the index finger’s contact point rather than towards that of the thumb. In our study, this cannot be explained by the index finger contacting the object before the thumb. Instead, it appears to be because the index finger moves to a position that is hidden behind the object that is grasped, probably making this the place at which one is most likely to encounter unexpected problems that would benefit from visual guidance. However, this cannot explain the bias that was found in previous studies, where neither contact point was hidden, so it cannot be the only explanation for the bias.     

A novel mitotropic oligolysine nanocarrier: Targeted delivery of covalently bound D-Luciferin to cell mitochondria

New and emerging therapeutic approaches focus on the targeted delivery of therapeutic agents to cell mitochondria with high specificity. Herein we present a novel mitotropic nanocarrier based on an oligolysine scaffold by addition of two triphenylphosphonium cations per oligomer. Although the parent oligolysine failed to enter healthy cells, the triphenylphosphonium modified carrier, with or without d-Luciferin, attached as cargo molecule, demonstrated striking mitochondrial specificity. Furthermore, the oligolysine bound d-Luciferin exhibited chemiluminescence, of lower intensity than free d-Luciferin, yet of remarkably longer steady-state temporal profile.     

Mathematical Modeling of Renal Tubular Glucose Absorption after Glucose Load

A partial differential Progressive Tubular Reabsorption (PTR) model, describing renal tubular glucose reabsorption and urinary glucose excretion following a glucose load perturbation, is proposed and fitted to experimental data from five subjects. For each subject the Glomerular Filtration Rate was estimated and both blood and urine glucose were sampled following an Intra-Venous glucose bolus. The PTR model was compared with a model representing the conventional Renal Threshold Hypothesis (RTH). A delay bladder compartment was introduced in both formulations. For the RTH model, the average threshold for glycosuria varied between 9.90±4.50 mmol/L and 10.63±3.64 mmol/L (mean ± Standard Deviation) under different hypotheses; the corresponding average maximal transport rates varied between 0.48±0.45 mmol/min (86.29±81.22 mg/min) and 0.50±0.42 mmol/min (90.62±76.15 mg/min). For the PTR Model, the average maximal transports rates varied between 0.61±0.52 mmol/min (109.57±93.77 mg/min) and 0.83±0.95 mmol/min (150.13±171.85 mg/min). The time spent by glucose inside the tubules before entering the bladder compartment varied between 1.66±0.73 min and 2.45±1.01 min.The PTR model proved much better than RTH at fitting observations, by correctly reproducing the delay of variations of glycosuria with respect to the driving glycemia, and by predicting non-zero urinary glucose elimination at low glycemias. This model is useful when studying both transients and steady-state glucose elimination as well as in assessing drug-related changes in renal glucose excretion.     

EGFR and HER2 exert distinct roles on colon cancer cell functional properties and expression of matrix macromolecules

Background

ErbB receptors, EGFR and HER2, have been implicated in the development and progression of colon cancer. Several intracellular pathways are mediated upon activation of EGFR and/or HER2 by EGF. However, there are limited data regarding the EGF-mediated signaling affecting functional cell properties and the expression of extracellular matrix macromolecules implicated in cancer progression.

Methods

Functional assays, such as cell proliferation, transwell invasion assay and migration were performed to evaluate the impact of EGFR/HER2 in constitutive and EGF-treated Caco-2 cells. Signaling pathways were evaluated using specific intracellular inhibitors. Western blot was also utilized to examine the phosphorylation levels of ERK1/2. Real time PCR was performed to evaluate gene expression of matrix macromolecules.

Results

EGF increases cell proliferation, invasion and migration and importantly, EGF mediates overexpression of EGFR and downregulation of HER2. The EGF–EGFR axis is the main pathway affecting colon cancer's invasive potential, proliferative and migratory ability. Intracellular pathways (PI3K-Akt, MEK1/2-Erk and JAK-STAT) are all implicated in the migratory profile. Notably, MT1- and MT2-MMP as well as TIMP-2 are downregulated, whereas uPA is upregulated via an EGF–EGFR network. The EGF–EGFR axis is also implicated in the expression of syndecan-4 and TIMP-1. However, glypican-1 upregulation by EGF is mainly mediated via HER2.

Conclusions and general significance

The obtained data highlight the crucial importance of EGF on the expression of both receptors and on the EGF–EGFR/HER2 signaling network, reveal the distinct roles of EGFR and HER2 on expression of matrix macromolecules and open a new area in designing novel agents in targeting colon cancer. This article is part of a Special Issue entitled Matrix-mediated cell behaviour and properties.     

Exploring the nonlinear geometry of protein homology

The explosion of biological data resulting from genomic and proteomic research has created a pressing need for data analysis techniques that work effectively on a large scale. An area of particular interest is the organization and visualization of large families of protein sequences. An increasingly popular approach is to embed the sequences into a low-dimensional Euclidean space in a way that preserves some predefined measure of sequence similarity. This method has been shown to produce maps that exhibit global order and continuity and reveal important evolutionary, structural, and functional relationships between the embedded proteins. However, protein sequences are related by evolutionary pathways that exhibit highly nonlinear geometry, which is invisible to classical embedding procedures such as multidimensional scaling (MDS) and nonlinear mapping (NLM). Here, we describe the use of stochastic proximity embedding (SPE) for producing Euclidean maps that preserve the intrinsic dimensionality and metric structure of the data. SPE extends previous approaches in two important ways: (1) It preserves only local relationships between closely related sequences, thus allowing the map to unfold and reveal its intrinsic dimension, and (2) it scales linearly with the number of sequences and therefore can be applied to very large protein families. The merits of the algorithm are illustrated using examples from the protein kinase and nuclear hormone receptor superfamilies.     

Induction, purification, and characterization of two extracellular alpha-L-arabinofuranosidases from Fusarium oxysporum

In the presence of L-arabinose as sole carbon source, Fusarium oxysporum produces two alpha-L-arabinofuranosidases (ABFs) named ABF1 and ABF2, with molecular masses of 200 and 180 kDa, respectively. The two F. oxysporum proteins have been purified to homogeneity. The purified enzymes are composed of three equal subunits and are neutral proteins with pIs of 6.0 and 7.3 for ABF1 and ABF2, respectively. With p-nitrophenyl alpha-L-arabinofuranoside (pNPA) as the substrate, ABF1 and ABF2 exhibited Km values of 0.39 and 0.28 mmol.L(-1), respectively, and Vmax values of 1.6 and 4.6 micromol.min(-1).(mg of protein)(-1), respectively, and displayed optimal activity at pH 6.0 and 50-60 degrees C. ABFs released arabinose only from sugar beet arabinan and not from wheat soluble and insoluble arabinoxylans. The enzymes were not active on substrates containing ferulic acid ester linked to C-5 and C-2 linkages of pNPA showing that phenolic substituents of pNPA sterically hindered the action of ABFs.