Extracellular Albumin and Endosomal Ions Prime Enterovirus Particles for Uncoating That Can Be Prevented by Fatty Acid Saturation.

There is limited information about the molecular triggers leading to the uncoating of enteroviruses under physiological conditions. Using real-time spectroscopy and sucrose gradients with radioactively labeled virus, we show at 37°C, the formation of albumin-triggered, metastable uncoating intermediate of echovirus 1 without receptor engagement. This conversion was blocked by saturating the albumin with fatty acids. High potassium but low sodium and calcium concentrations, mimicking the endosomal environment, also induced the formation of a metastable uncoating intermediate of echovirus 1. Together, these factors boosted the formation of the uncoating intermediate, and the infectivity of this intermediate was retained, as judged by end-point titration. Cryo-electron microscopy reconstruction of the virions treated with albumin and high potassium, low sodium, and low calcium concentrations resulted in a 3.6-Å resolution model revealing a fenestrated capsid showing 4% expansion and loss of the pocket factor, similarly to altered (A) particles described for other enteroviruses. The dimer interface between VP2 molecules was opened, the VP1 N termini disordered and most likely externalized. The RNA was clearly visible, anchored to the capsid. The results presented here suggest that extracellular albumin, partially saturated with fatty acids, likely leads to the formation of the infectious uncoating intermediate prior to the engagement with the cellular receptor. In addition, changes in mono- and divalent cations, likely occurring in endosomes, promote capsid opening and genome release.IMPORTANCE There is limited information about the uncoating of enteroviruses under physiological conditions. Here, we focused on physiologically relevant factors that likely contribute to opening of echovirus 1 and other B-group enteroviruses. By combining biochemical and structural data, we show that, before entering cells, extracellular albumin is capable of priming the virus into a metastable yet infectious intermediate state. The ionic changes that are suggested to occur in endosomes can further contribute to uncoating and promote genome release, once the viral particle is endocytosed. Importantly, we provide a detailed high-resolution structure of a virion after treatment with albumin and a preset ion composition, showing pocket factor release, capsid expansion, and fenestration and the clearly visible genome still anchored to the capsid. This study provides valuable information about the physiological factors that contribute to the opening of B group enteroviruses.

Tyrosinase-Mediated Bioconjugation. A Versatile Approach to Chimeric Macromolecules.

We present a method for tyrosine-selective and reversible bioconjugation; tyrosines are enzymatically converted into catechols and in situ "clicked" onto boronic acids. Importantly, our process selectively produces catechols and avoids quinones, thereby improving the control over the chemical identity of the products. We have conjugated boronic acid-containing hyaluronic acid (HyA) to peptides bearing tyrosines in variable number and position; the use of tagging peptides for the provision of well exposed tyrosine residues-in our case the hemagglutinin-derived HA-tag-makes our approach applicable to virtually any protein; we have demonstrated this concept by conjugating HA-tagged ovalbumin to HyA, thereby also showing the feasibility of producing chimeric proteoglycans. A caveat of this appproach is that, although the formation of boronic esters does not affect the biological recognition of substrates (ovalbumin and HyA), the introduction of catechols may alter some of their biological properties: for example, only after tyrosinase treatment ovalbumin directly induced dendritic cell maturation, either alone or as a HyA conjugate.

Chitosan/Hyaluronic Acid Nanoparticles: Rational Design Revisited for RNA Delivery.

Chitosan/hyaluronic acid (HA) nanoparticles can be used to deliver an RNA/DNA cargo to cells overexpressing HA receptors such as CD44. For these systems, unequivocal links have not been established yet between chitosan macromolecular (molecular weight; degree of deacetylation, i.e., charge density) and nanoparticle variables (complexation strength, i.e., stability; nucleic acid protection; internalization rate) on one hand, and transfection efficiency on the other hand. Here, we have focused on the role of avidity on transfection efficiency in the CD44-expressing HCT-116 as a cellular model; we have employed two differently sized payloads (a large luciferase-encoding mRNA and a much smaller anti-Luc siRNA), and a small library of chitosans (variable molecular weight and degree of deactylation). The RNA avidity for chitosan showed-as expected-an inverse relationship: higher avidity-higher polyplex stability-lower transfection efficiency. The avidity of chitosan for RNA appears to lead to opposite effects: higher avidity-higher polyplex stability but also higher transfection efficiency. Surprisingly, the best transfecting particles were those with the lowest propensity for RNA release, although this might be a misleading relationship: for example, the same macromolecular parameters that increase avidity can also boost chitosan's endosomolytic activity, with a strong enhancement in transfection. The performance of these nonviral vectors appears therefore difficult to predict simply on the basis of carrier- or payload-related variables, and a more holistic consideration of the journey of the nanoparticle, from cell uptake to cytosolic bioavailability of payload, is needed. It is also noteworthy that the nanoparticles used in this study showed optimal performance under slightly acidic conditions (pH 6.4), which is promising for applications in a tumoral extracellular environment. It is also worth pointing out that under these conditions we have for the first time successfully delivered mRNA with chitosan/HA nanoparticles.

Enteral Nutrition Support Reduces the Necessity of Total Parenteral Nutrition to Reach Patient-Specific Caloric Goals Postpancreaticoduodenectomy.

OBJECTIVES: Pancreaticoduodenectomy (PD) is associated with significant rates of postoperative complications. Although there is evidence that enteral nutrition support (ENS) may reduce postoperative sepsis, the true value of ENS in the abrogation of septic complications remains controversial. The aim of our study is to investigate the postoperative outcome of patients post-PD with and without ENS. METHODS: Using our prospective institutional database, we identified 202 patients from 2001 through 2009 who underwent PD. Of the 202 patients, 121 matched our inclusion criteria. In total, 67 of 121 (55.4%) patients received ENS, whereas 54 (44.6%) patients had no ENS and served as controls. Postoperative morbidity and mortality were recorded and analyzed. RESULTS: No significant differences were found in the postoperative morbidity of the patients. The anastomotic leak rate was 13% in both the ENS and control groups (P = 0.846). There was no difference in mortality within the two groups (4% vs 5%, P = 0.881). Significantly more patients in the control group received total parenteral nutrition (P = 0.033). CONCLUSIONS: ENS is not associated with lower rates of postoperative morbidity and mortality. It does, however, reduce the necessity of additional total parenteral nutrition to reach patient-specific caloric goals.

Medical occurrence and safety of SARS-CoV-2 vaccination outside of the hospital setting.

During COVID-19 pandemic, vaccination has been strongly recommended and advocated to prevent COVID-19 infection and adverse outcomes, particularly among at-risk populations. The vaccination against SARS-CoV-2 (COVAC) occurred at off-site locations capable of accommodating large crowds, distinct from the hospital setting, where a team of intensivists, emergency physicians, and nurses, ensuring prompt medical attention (medical occurrences, MO) in cases of adverse event following immunization. Our aims were to estimate the incidence of MO, and to assess its association with demographics, and vaccine characteristics. Our retrospective cohort study included all subject aged 12 years and older who received vaccinations at two large out-of-hospital vaccination hubs (Fiera Milano City, Palazzo delle Scintille), between April 12th and August 31st, 2021. Nine hundred and ninety-five thousand and twenty-eight vaccinations were administrated. MOs incidence rate was 278/100,000 doses (95% confidence interval (CI) 268-289). Most MOs were mild (86.27%) and mainly observed in subjects who received the Comirnaty vaccine; 92 MOs (3.32%) were severe and mostly occurred in recipients of the Vaxzeria vaccine. The incidence rate for hospital transfers following vaccination was 4.7/100,000 doses (95% CI 3.5-6.2) and any level of anaphylaxis occurred in 0.4 cases per 100,000 administrated doses (95% CI 0.3.-0.7). Sex, age, type of vaccine and first dose were associated with incidence of MO. Our results showed a low incidence rate in MOs after COVAC, mainly mild and support the feasibility, effectiveness and safety of vaccinations administered in hubs with a dedicated SEU located outside of the hospital setting.

Role of the nutrition professional in high-risk obstetrics inpatient teams.

Beyond the management of gestational diabetes and weight control, limited literature exists on nutritional management in high-risk pregnancies. This study is a starting point for understanding the role of the registered dietitian (RD) in high-risk obstetrics (HRO) inpatient teams. Demographic information was gathered on Ontario HRO inpatient units and patient characteristics, and the RD's role in this setting was explored. Representatives from all six HRO units in Ontario completed a questionnaire. Five of the HRO units had an RD on the team. The RDs stated that their primary role in the unit involved providing nutrition education and support, screening patients for nutritional risk, and writing vitamin and mineral orders. This was the first study in which RDs' role in HRO inpatient units was examined. Existing literature supports the role of good nutrition in preventing and treating the conditions seen in HRO units; however, RDs' specific role remains unclear. Future studies are needed to analyze the effect of nutrition on these HRO conditions, and to support the development of best practice guidelines.

Coating of nitrocellulose for colorimetric DNA microarrays.

We report on the modification of a nitrocellulose film with copoly(DMA-NAS-MAPS), a tercopolymer based on N,N-dimethylacrylamide (DMA), N-acryloyloxysuccinimide (NAS), and 3-(trimethoxysilyl)propyl-methacrylate (MAPS). The chains of this polymer, interacting with nitrocellulose fibers, introduce active ester functionalities that promote the covalent binding of short oligonucleotide fragments to the nitrocellulose thin film. Using colorimetric detection, naked eye visible DNA microarrays are developed for easy identification of foodborne pathogens. The fast and robust procedure of nitrocellulose functionalization opens the opportunity to implement this material in disposable analytical microdevices that do not require sophisticated readout systems.

The different ways to chitosan/hyaluronic acid nanoparticles: templated vs direct complexation. Influence of particle preparation on morphology, cell uptake and silencing efficiency.

This study is about linking preparative processes of nanoparticles with the morphology of the nanoparticles and with their efficiency in delivering payloads intracellularly. The nanoparticles are composed of hyaluronic acid (HA) and chitosan; the former can address a nanoparticle to cell surface receptors such as CD44, the second allows both for entrapment of nucleic acids and for an endosomolytic activity that facilitates their liberation in the cytoplasm. Here, we have systematically compared nanoparticles prepared either A) through a two-step process based on intermediate (template) particles produced via ionotropic gelation of chitosan with triphosphate (TPP), which are then incubated with HA, or B) through direct polyelectrolyte complexation of chitosan and HA. Here we demonstrate that HA is capable to quantitatively replace TPP in the template process and significant aggregation takes place during the TPP-HA exchange. The templated chitosan/HA nanoparticles therefore have a mildly larger size (measured by dynamic light scattering alone or by field flow fractionation coupled to static or dynamic light scattering), and above all a higher aspect ratio (R g/R H) and a lower fractal dimension. We then compared the kinetics of uptake and the (antiluciferase) siRNA delivery performance in murine RAW 264.7 macrophages and in human HCT-116 colorectal tumor cells. The preparative method (and therefore the internal particle morphology) had little effect on the uptake kinetics and no statistically relevant influence on silencing (templated particles often showing a lower silencing). Cell-specific factors, on the contrary, overwhelmingly determined the efficacy of the carriers, with, e.g., those containing low-MW chitosan performing better in macrophages and those with high-MW chitosan in HCT-116.

Binding and Internalization in Receptor-Targeted Carriers: The Complex Role of CD44 in the Uptake of Hyaluronic Acid-Based Nanoparticles (siRNA Delivery).

CD44 is an endocytic hyaluronic acid (HA) receptor, and is overexpressed in many carcinomas. This has encouraged the use of HA to design CD44-targeting carriers. This paper is about dissecting the mechanistic role of CD44. Here, HA-decorated nanoparticles are used to deliver siRNA to both tumoral (AsPC-1, PANC-1, HT-29, HCT-116) and non-tumoral (fibroblasts, differently polarized THP-1 macrophages, HUVEC) human cell lines, evaluating the initial binding of the nanoparticles, their internalization rate, and the silencing efficiency (cyclophilin B (PPIB) gene). Tumoral cells internalize faster and experience higher silencing than non-tumoral cells. This is promising as it suggests that, in a tumor, HA nanocarriers may have limited off-target effects. More far-reaching is the inter-relation between the four parameters of the study: CD44 expression, HA binding on cell surfaces, internalization rate, and silencing efficiency. No correlation is found between binding (an early event) and any of the other parameters, whereas silencing correlates both with speed of the internalization process and CD44 expression. This study confirms on one hand that HA-based carriers can perform a targeted action, but on the other it suggests that this may not be due to a selective binding event, but rather to a later recognition leading to selective internalization.

Patchy Amphiphilic Dendrimers Bind Adenovirus and Control Its Host Interactions and in Vivo Distribution.

The surface of proteins is heterogeneous with sophisticated but precise hydrophobic and hydrophilic patches, which is essential for their diverse biological functions. To emulate such distinct surface patterns on macromolecules, we used rigid spherical synthetic dendrimers (polyphenylene dendrimers) to provide controlled amphiphilic surface patches with molecular precision. We identified an optimal spatial arrangement of these patches on certain dendrimers that enabled their interaction with human adenovirus 5 (Ad5). Patchy dendrimers bound to the surface of Ad5 formed a synthetic polymer corona that greatly altered various host interactions of Ad5 as well as in vivo distribution. The dendrimer corona (1) improved the ability of Ad5-derived gene transfer vectors to transduce cells deficient for the primary Ad5 cell membrane receptor and (2) modulated the binding of Ad5 to blood coagulation factor X, one of the most critical virus-host interactions in the bloodstream. It significantly enhanced the transduction efficiency of Ad5 while also protecting it from neutralization by natural antibodies and the complement system in human whole blood. Ad5 with a synthetic dendrimer corona revealed profoundly altered in vivo distribution, improved transduction of heart, and dampened vector sequestration by liver and spleen. We propose the design of bioactive polymers that bind protein surfaces solely based on their amphiphilic surface patches and protect against a naturally occurring protein corona, which is highly attractive to improve Ad5-based in vivo gene therapy applications.

Self-replicating RNA vaccine functionality modulated by fine-tuning of polyplex delivery vehicle structure.

The major limitations with large and complex self-amplifying RNA vaccines (RepRNA) are RNase-sensitivity and inefficient translation in dendritic cells (DCs). Condensing RepRNA with polyethylenimine (PEI) gave positive in vitro readouts, but was largely inferior to virus-like replicon particles (VRP) or direct electroporation. In the present study, we improved such polyplex formulation and determined that fine-tuning of the polyplex structure is essential for ensuring efficacious translation. Thereby, three parameters dominate: (i) PEI molecular weight (MW); (ii) RepRNA:PEI (weight:weight) ratio; and (iii) inclusion of cell penetrating peptides (CPPs). Seven commercially available linear PEIs (MW 2,500-250,000) were classified as strong, intermediate or low for their aptitude at complexing and protecting RepRNA for delivery into porcine blood DCs. Inclusion of (Arg)9 or TAT(57-57) CPPs further modified the translation readouts, but varied for different gene expressions. Dependent on the formulation, translation of the gene of interest (GOI) inserted into the RepRNA (luciferase, or influenza virus hemagglutinin or nucleoprotein) could decrease, while the RepRNA structural gene (E2) translation increased. This was noted in the porcine SK6 cell line, as well as both porcine and, for the first time, human DCs. Two formulations - [Rep/PEI-4,000 (1:3)] and [Rep/PEI-40,000 (1:2)/(Arg)9] were efficacious in vivo in mice and pigs, where specific CD8+ T and CD4+ T-cell responses against the GOI-encoded antigen were observed for the first time. The results demonstrate that different polyplex formulations differ in their interaction with the RepRNA such that only certain genes can be translated. Thus, delivery of these large self-replicating RNA molecules require definition with respect to translation of different genes, rather than just the GOI as is the norm, for identifying optimal delivery for the desired immune activation in vivo.

Mannosylation Allows for Synergic (CD44/C-Type Lectin) Uptake of Hyaluronic Acid Nanoparticles in Dendritic Cells, but Only upon Correct Ligand Presentation.

The selective targeting of dendritic cells (DCs) can lead to more efficacious vaccines. Here, materials have been designed for a synergic DC targeting: interacting with CD44 through the use of hyaluronic acid (HA), and with mannose-binding lectins (typical DC pattern recognition receptors) through HA mannosylation. Negatively charged, HA-displaying nanoparticles are produced via polyelectrolyte complexation of (mannosylated) HA and high- or low- molecular-weight chitosan (CS, 36 and 656 kDa). Using CS36, HA is better exposed and the particles have a higher affinity for HA receptors; this means a higher number of receptors clustered around each particle and, due to the rather limited CD44 availability, an overall lower uptake per cell. Employing Langerhans-like XS106 cells, all particles show negligible toxicity or inflammatory activation. The cellular uptake kinetics are qualitatively similar to other leukocytic models and thus considered to be CD44-dominated; the uptake increases with increasing HA mannosylation and with the use of adjuvants (LPS, mannan) for CS36/HA but not for CS656//HA particles; this indicates that the interactions with mannose-binding receptors requires a correct ligand presentation, and only in that case can they be enhanced by appropriate adjuvants. In summary, mannose-binding receptors can be used to enhance the internalization of HA-based carriers, although this positive synergy depends on the mode of ligand presentation.

Hyaluronan/Tannic Acid Nanoparticles Via Catechol/Boronate Complexation as a Smart Antibacterial System.

Nanoparticles based on hyaluronic acid (HA) are designed to deliver tannic acid (TA) as an antimicrobial agent. The presence of HA makes these particles potentially useful to target bacteria that colonize cells presenting HA membrane receptors (e.g. CD44), such as macrophages. HA bearing 3-aminophenyl boronic acid groups (HA-APBA) is reacted with TA, yielding nanoparticles with a size that decreases with decreasing HA molecular weight (e.g. 200 nm for 44 kDa, 400 nm for 737 kDa). The boronate esters make the nanoparticles stable at physiological pH, but their hydrolysis in an acidic environment (pH = 5) leads to swelling/solubilization, therefore potentially allowing TA release in endosomal compartments. We have assessed the nanoparticle toxicity profile (on RAW 264.7 macrophages) and their antimicrobial activity (on E. coli and on both methicillin-sensitive and -resistant S. aureus). The antibacterial effect of HA-APBA/TA nanoparticles was significantly higher than that of TA alone, and has very similar activity to TA coformulated with a reducing agent (ascorbic acid), which indicates both the nanoparticles to protect TA catechols from oxidation, and the effective release of TA after nanoparticle internalization. Therefore, there is potential for these nanoparticles to be used in stable, effective, and potentially targetable nanoparticle-based antimicrobial formulations.

Additives for vaccine storage to improve thermal stability of adenoviruses from hours to months.

Up to 80% of the cost of vaccination programmes is due to the cold chain problem (that is, keeping vaccines cold). Inexpensive, biocompatible additives to slow down the degradation of virus particles would address the problem. Here we propose and characterize additives that, already at very low concentrations, improve the storage time of adenovirus type 5. Anionic gold nanoparticles (10-8-10-6 M) or polyethylene glycol (PEG, molecular weight ∼8,000 Da, 10-7-10-4 M) increase the half-life of a green fluorescent protein expressing adenovirus from ∼48 h to 21 days at 37 °C (from 7 to >30 days at room temperature). They replicate the known stabilizing effect of sucrose, but at several orders of magnitude lower concentrations. PEG and sucrose maintained immunogenicity in vivo for viruses stored for 10 days at 37 °C. To achieve rational design of viral-vaccine stabilizers, our approach is aided by simplified quantitative models based on a single rate-limiting step.

Priority persistent contaminants in people dwelling in critical areas of Campania Region, Italy (SEBIOREC biomonitoring study).

To investigate if protracted living in degraded environments of the Caserta and Naples provinces (Campania Region, Italy) had an impact on exposure of local people, highly toxic persistent contaminants were measured in blood, blood serum, and human milk of a large number of healthy donors. Sampling was carried out from 2008 to 2009. Blood was collected from over 850 20-64-year old donors; by pooling, 84 blood and 84 serum samples were obtained. Milk was donated by 52 mothers: specimens were pooled into six samples. Polychlorodibenzodioxins (PCDDs), polychlorodibenzofurans (PCDFs), and polychlorobiphenyls (PCBs, dioxin-like (DL) and non-dioxin-like (Σ6PCBs)), arsenic (As), cadmium (Cd), mercury (Hg), and lead (Pb) were measured in serum (organic biomarkers) and blood (metals); these chemicals and polybromobiphenyl ethers (Σ9PBDEs) were analyzed in milk. PCDD+PCDF, DL-PCB, TEQTOT, and Σ6PCB concentration ranges (medians) in serum were 6.26-23.1 (12.4), 3.42-31.7 (11.5), 10.0-52.8 (23.9) pgTEQ97/g fat, and 55.5-647 (219) ng/g fat, respectively, while in milk concentration ranges were 5.99-8.77, 4.02-6.15, 10.0-14.2 pgTEQ97/g fat, and 48.7-74.2 ng/g fat. Likewise, As, Cd, Hg, and Pb findings in blood spanned 2.34-13.4 (5.83), 0.180-0.930 (0.475), 1.09-7.60 (2.60), 10.2-55.9 (28.8) µg/L, respectively; only Pb could be measured in milk (2.78-5.99 µg/L). Σ9PBDE levels in milk samples were 0.965-6.05 ng/g fat. Biomarkers' concentrations were found to be compatible with their current values in European countries and in Italy, and consistent with an exposure primarily determined by consumption of commercial food from the large distribution system. Based on relatively higher biomarker values within the hematic biomonitoring database, the following municipalities were flagged as possibly deserving attention for health-oriented interventions: Brusciano and Caivano (As), Giugliano (Hg), Pianura (PCDDs+PCDFs), and Qualiano-Villaricca (As, Hg). The analysis of samples' qualitative variability indicated that biomarker composition was sensitive at municipality level, a feature that can potentially drive interventions for future local risk assessment and/or management measures.