Screening forCronobacterSpecies in Powdered and Reconstituted Infant Formulas and from Equipment Used in Formula Preparation in Maternity Hospitals

Background/Aims: Cronobacter spp. have been identified as being of considerable risk to neonates. The occurrence of organism in infant formulas is therefore of considerable interest. Methods: The occurrence of Cronobacter spp. in infant feeds (formulas and fortified cow’s milk) was determined using most probable number (MPN) analysis, and from formula preparation utensils. Ninety nine samples were analyzed, of which 42 were unopened cans of powdered infant formula (PIF), 25 reconstituted infant formulas in feeding bottles, 27 utensils used from the preparation of infant formula, and 5 samples of fortified cow’s milk. Presumptive Cronobacter spp. isolates were identified using the 7 allele multilocus sequence typing (MLST) scheme. Results: C. sakazakii, C. malonaticus and C. muytjensii were recovered from PIF. Although the incidence of Cronobacter in PIF was 29% (12/42), the level was low with an average of 0.54 MPN/100g. According to MLST profiling, C. sakazakii was the most frequently isolated Cronobacter species, and C. sakazakii ST4 (associated with neonatal meningitis) was recovered from 2/42 PIF samples at 0.51 and 0.92 MPN/100g. Conclusions: Cronobacter spp. can be isolated from PIF and therefore strict hygienic practices during PIF preparation are important to minimize neonate exposure and reduce the risk of severe infections

Engineering modular half-antibody conjugated nanoparticles for targeting CD44v6-expressing cancer cells

Gastric cancer (GC) remains a major cause of death worldwide mainly because of the late detection in advanced stage. Recently, we proposed CD44v6 as a relevant marker for early detection of GC, opening new avenues for GC-targeted theranostics. Here, we designed a modular nanoscale system that selectively targets CD44v6-expressing GC cells by the site-oriented conjugation of a new-engineered CD44v6 half-antibody fragment to maleimide-modified polystyrene nanoparticles (PNPs) via an efficient bioorthogonal thiol-Michael addition click chemistry. PNPs with optimal particle size (200 nm) for crossing a developed biomimetic CD44v6-associated GC stromal model were further modified with a heterobifunctional maleimide crosslinker and click conjugated to the novel CD44v6 half-antibody fragment, obtained by chemical reduction of full antibody, without affecting its bioactivity. Collectively, our results confirmed the specific targeting ability of CD44v6-PNPs to CD44v6-expressing cells (1.65-fold higher than controls), highlighting the potential of CD44v6 half-antibody conjugated nanoparticles as promising and clinically relevant tools for the early diagnosis and therapy of GC. Additionally, the rational design of our nanoscale system may be explored for the development of several other nanotechnology-based disease-targeted approaches.This work was supported by Norte Portugal Regional Operational Programme (NORTE2020) under the PORTUGAL 2020 Partnership Agreement through the European Regional Development Fund (ERDF) projects Norte-01-0145-FEDER-000012 and NORTE-07-0124-FEDER-000029, through COMPETE 2020-Operational Programme for Competitiveness and Internationalization (POCI) Portugal 2020 and Portuguese Foundation for Science and Technology (FCT) in the framework of the projects POCI-01-0145-FEDER-007274, POCI-01-0145-FEDER-016390, and PTDC/CTMNAN/120958/2010, B.N.L. doctoral grant (SFRH/BD/87400/2012) and postdoctoral grant (PTDC/MEC-GIN/29232/2017). R.F.P. was supported by Institute of Network Bioengineering for Healthy Aging (0245_IBEROS_1_E)

Norbornene-chitosan spray-dried microspheres for peptide conjugation using thiol-ene “photoclick” chemistry

This work was financed by Portuguese funds through FCT/MCTES (Fundação para a Ciência e a Tecnologia/Ministério da Ciência, Tecnologia e Inovação) in the framework of the projects 2022.06048.PTDC (i3S), UIDB/50006/2020 (LAQV-REQUIMTE), LA/P/0045/2020 (ALiCE) and UIDB/00511/2020 (LEPABE). P.A. (SFRH/BD/145471/2019) and D.F. (SFRH/ BD/146890/2019) doctoral grants, were financially supported by national (FCT/Norte 2020 Framework) and European Union (ESF – European Social Fund) funds. B.E. acknowledges FCT for the contract based on the “Lei do Emprego Científico” (DL 57/2016). Maria Cristina L. Martins also acknowledges FCT (LA/P/0070/2020), project Bio2Skin Advanced (2021-24):NORTE-01-0247-FEDER-047225; and MOBILIsE Project, which has received funding from the European Union’s Horizon 2020 research and innovation program under grant agreement no. 951723.The action of bioactive peptides, such as antimicrobial peptides (AMP), in the human body is often compromised by limited residence time and stability in the target site. Bioconjugation of peptides to biomaterial surfaces is one of the strategies that may overcome these limitations. Herein, norbornene-chitosan (NorChit) microspheres were engineered to react with thiolated peptides by thiolene “photoclick” chemistry. NorChit microspheres were produced by spray drying and crosslinked with dithiothreitol (DTT) to prevent their solubilization. Microspheres with a diameter of 5 ± 2 µm showed round and smooth morphology with pockets over the surface that could be related with hydrophobic interactions between internal norbornene groups. Thiol-ene bioconjugation carried out using a fluorescent model peptide, showed a yield of 45%, whereas using the peptide but without UV exposure indicated a maximum of peptide adsorption of 30%. Altogether, NorChit microspheres show the potential for carrying bioactive peptides, which may open avenues for AMP activity onto harsh environments in the bod

Accuracy of prediction models for long-term type 2 diabetes remission after gastric bypass

AimTo evaluate the accuracy of DiaBetter, DiaRem, Ad-DiaRem and 5y-Ad-DiaRem scores' at predicting T2D remission 10 or more years after surgery.MethodsPatients with obesity and T2D (n = 126) submitted to RYGB with 10 or more years of follow-up. It was a unicentric trial. Pre-operative anthropometric and clinical data was retrieved to calculate DiaRem, DiaBetter, Ad-DiaRem and 5y-Ad-DiaRem scores, while a hospital visit was conducted to assess current diabetes status. The area under the receiver operating characteristic (AUROC) curve was calculated as estimate of the scores' accuracy to predict long-term T2D remission.ResultsAmong the entire cohort (n = 126), 70 subjects (55.6%) achieved and maintained T2D remission 10 or more years after RYGB. The 5y-Ad-DiaRem score was the one that depicted the highest discriminative power (AUROC = 0.838) to predict long-term T2D remission when compared to DiaBetter (AUROC = 0.735), DiaRem (AUROC = 0.721) and Ad-DiaRem (AUROC = 0.720).ConclusionThe score with highest accuracy to predict long-term T2D remission after RYGB surgery was the 5y-Ad-DiaRem. Yet, the available scores accuracy to predict T2D remission in the long term is still suboptimal, highlighting the unmet need for a better scoring system

Single-Molecule Super-Resolution Imaging of T-Cell Plasma Membrane CD4 Redistribution upon HIV-1 Binding

The first step of cellular entry for the human immunodeficiency virus type-1 (HIV-1) occurs through the binding of its envelope protein (Env) with the plasma membrane receptor CD4 and co-receptor CCR5 or CXCR4 on susceptible cells, primarily CD4+ T cells and macrophages. Although there is considerable knowledge of the molecular interactions between Env and host cell receptors that lead to successful fusion, the precise way in which HIV-1 receptors redistribute to sites of virus binding at the nanoscale remains unknown. Here, we quantitatively examine changes in the nanoscale organisation of CD4 on the surface of CD4+ T cells following HIV-1 binding. Using singlemolecule super-resolution imaging, we show that CD4 molecules are distributed mostly as either individual molecules or small clusters of up to 4 molecules. Following virus binding, we observe a local 3-to-10-fold increase in cluster diameter and molecule number for virus-associated CD4 clusters. Moreover, a similar but smaller magnitude reorganisation of CD4 was also observed with recombinant gp120. For one of the first times, our results quantify the nanoscale CD4 reorganisation triggered by HIV-1 on host CD4+ T cells. Our quantitative approach provides a robust methodology for characterising the nanoscale organisation of plasma membrane receptors in general with the potential to link spatial organisation to function

Tariff-Mediated Network Effects versus Strategic Discounting: Evidence from German Mobile Telecommunications

Mobile telecommunication operators routinely charge subscribers lower prices for calls on their own network than for calls to other networks (on-net discounts). Studies on tariff-mediated network effects suggest this is due to large operators using on-net discounts to damage smaller rivals. Alternatively, research on strategic discounting suggests small operators use on-net discounts to advertise with low on-net prices. We test the relative strength of these effects using data on tariff setting in German mobile telecommunications between 2001 and 2009. We find that large operators are more likely to offer tariffs with on-net discounts but there is no consistently significant difference in the magnitude of discounts. Our results suggest that tariff-mediated network effects are the main cause of on-net discounts

Simcluster: clustering enumeration gene expression data on the simplex space

Transcript enumeration methods such as SAGE, MPSS, and sequencing-by-synthesis EST "digital northern", are important high-throughput techniques for digital gene expression measurement. As other counting or voting processes, these measurements constitute compositional data exhibiting properties particular to the simplex space where the summation of the components is constrained. These properties are not present on regular Euclidean spaces, on which hybridization-based microarray data is often modeled. Therefore, pattern recognition methods commonly used for microarray data analysis may be non-informative for the data generated by transcript enumeration techniques since they ignore certain fundamental properties of this space.

Here we present a software tool, Simcluster, designed to perform clustering analysis for data on the simplex space. We present Simcluster as a stand-alone command-line C package and as a user-friendly on-line tool. Both versions are available at: http://xerad.systemsbiology.net/simcluster.

Simcluster is designed in accordance with a well-established mathematical framework for compositional data analysis, which provides principled procedures for dealing with the simplex space, and is thus applicable in a number of contexts, including enumeration-based gene expression data