► Novel polymeric coatings able to modulate capillary walls properties were synthesized. ► High stability of the film is assured by silane and chemically reactive groups. ► Functional monomers allows ...also electroosmotic flow modulation to desired values. ► These coatings provide very efficient protein separations in capillary electrophoresis.
Capillary electrophoresis (CE) is one of the most powerful techniques for the separation of biomolecules. However, the separation efficiency of proteins in CE is often compromised by their tendency to interact with the silanol groups on the surface of the inner capillary and by an uncontrolled electroosmotic flow. Herein, we report on the synthesis of novel hydrophilic polymeric coatings that can modulate the properties of the capillary walls. The novelty of these poly(N,N-dimethylacrylamide)-based copolymers relies on the simultaneous presence of chemically reactive groups (N-acryloyloxysuccinimide and glycidyl methacrylate) and silane groups in the backbone, which results in highly stable films due to the covalent reaction between the polymer and the glass silanols. Although the functional monomers are reactive towards nucleophilic groups in proteins, they can be effectively blocked in the presence of amino modified agents. In addition, after a careful optimization of monomer concentration, it is possible to confer anti-fouling properties to the polymer coatings, and thus allow for highly efficient acidic and alkaline protein separations. Furthermore, the presence of these monomers makes it possible to modulate the electroosmotic flow from negligible to reduced values, depending on the desired application.
AbstractObjectiveTo determine the global capacity (availability, accessibility, quality, and affordability) to deliver kidney replacement therapy (dialysis and transplantation) and conservative ...kidney management.DesignInternational cross sectional survey.SettingInternational Society of Nephrology (ISN) survey of 182 countries from July to September 2018.ParticipantsKey stakeholders identified by ISN’s national and regional leaders.Main outcome measuresMarkers of national capacity to deliver core components of kidney replacement therapy and conservative kidney management.ResultsResponses were received from 160 (87.9%) of 182 countries, comprising 97.8% (7338.5 million of 7501.3 million) of the world’s population. A wide variation was found in capacity and structures for kidney replacement therapy and conservative kidney management—namely, funding mechanisms, health workforce, service delivery, and available technologies. Information on the prevalence of treated end stage kidney disease was available in 91 (42%) of 218 countries worldwide. Estimates varied more than 800-fold from 4 to 3392 per million population. Rwanda was the only low income country to report data on the prevalence of treated disease; 5 (<10%) of 53 African countries reported these data. Of 159 countries, 102 (64%) provided public funding for kidney replacement therapy. Sixty eight (43%) of 159 countries charged no fees at the point of care delivery and 34 (21%) made some charge. Haemodialysis was reported as available in 156 (100%) of 156 countries, peritoneal dialysis in 119 (76%) of 156 countries, and kidney transplantation in 114 (74%) of 155 countries. Dialysis and kidney transplantation were available to more than 50% of patients in only 108 (70%) and 45 (29%) of 154 countries that offered these services, respectively. Conservative kidney management was available in 124 (81%) of 154 countries. Worldwide, the median number of nephrologists was 9.96 per million population, which varied with income level.ConclusionsThese comprehensive data show the capacity of countries (including low income countries) to provide optimal care for patients with end stage kidney disease. They demonstrate substantial variability in the burden of such disease and capacity for kidney replacement therapy and conservative kidney management, which have implications for policy.
In this paper, we report on the postpolymerization modification (PPM) of a polymer to introduce new functionalities that enable click chemistry reactions for microarray applications. The parent ...polymer, named copoly(DMA-NAS-MAPS), is composed of N,N-dimethylacrylamide (DMA), a monomer that self-adsorbs onto different materials through weak interactions such as hydrogen bonding or van der Waals forces, 3-(trimethoxysilyl)propyl methacrylate (MAPS) that strengthens the stability of the coating through the formation of covalent bonds with siloxane groups on the surface to be coated, and N-acryloyloxysuccinimide (NAS), an active ester group, highly reactive toward nucleophiles, which enables bioprobe immobilization. This copolymer has been widely exploited to coat surfaces for microarray applications but exhibits some limitations because of the potential hydrolysis of the active ester (NHS ester). The degradation of the NHS ester hampers the use of this coating in some situations, for example, when probe immobilization cannot be accomplished through a microspotting situation, but in large volumes, for example, in microchannel derivatization or micro-/nanoparticle functionalization. To overcome the limitations of NHS esters, we have developed a family of polymers that originate from the common copolymer precursor, by reacting the active ester contained in the polymer chain with a bifunctional amine. In particular, the functional groups introduced in the polymer using PPM enable click chemistry reactions such as azide/alkyne or thiol/maleimide “click” reactions, with suitably modified biomolecules. The advantages of such reactions are quantitative yields, orthogonality of functional groups, and insensitivity of the reaction to pH. The new click functionalities, inserted with quantitative yields, improve the stability of the coating, enabling the attachment of biomolecules directly from a solution and avoiding the spotting of reduced volumes (pL) of probes. Finally, we have demonstrated the applicability of the click surfaces in a highly effective solid-phase PCR for the genotyping of the G12D KRAS mutation.
The Renal Healthcare Program Uruguay (NRHP-UY) is a national, multidisciplinary program that provides care to chronic kidney disease (CKD) patients. In this study, we report the global results of CKD ...patient outcomes and a comparison between those treated at the NRHP-UY Units, with those patients who were initially included in the program but did not adhere to follow up. A cohort of not-on dialysis CKD patients included prospectively in the NRHP-UY between October 1.sup.st 2004 and September 30.sup.th 2017 was followed-up until September 30.sup.th 2019. Two groups were compared: a) Nephrocare Group: Patients who had at least one clinic visit during the first year on NRHP-UY (n = 11174) and b) Non-adherent Group: Patients who were informed and accepted to be included but had no subsequent data registered after admission (n = 3485). The study was approved by the Ethics Committee and all patients signed an informed consent. Outcomes were studied with Logistic and Cox's regression analysis, Fine and Gray competitive risk and propensity-score matching tests. 14659 patients were analyzed, median age 70 (60-77) years, 56.9% male. The Nephrocare Group showed improved achievement of therapeutic goals, ESKD was more frequent (HR 2.081, CI 95%1.722-2.514) as planned kidney replacement therapy (KRT) start (OR 2.494, CI95% 1.591-3.910), but mortality and the combined event (death and ESKD) were less frequent (HR 0.671, CI95% 0.628-0.717 and 0.777, CI95% 0.731-0.827) (p = 0.000) compared to the Non-adherent group. Results were similar in the propensity-matched group: ESKD (HR 2.041, CI95% 1.643-2.534); planned kidney replacement therapy (KRT) start (OR 2.191, CI95% 1.322-3.631) death (HR 0.692, CI95% 0.637-0.753); combined event (HR 0.801, CI95% 0.742-0.865) (p = 0.000). Multidisciplinary care within the NRHP-UY is associated with timely initiation of KRT and lower mortality in single outcomes, combined analysis, and propensity-matched analysis.
Lactic acid bacteria (LAB) communities shape the sensorial and functional properties of artisanal hard-cooked and long-ripened cheeses made with raw bovine milk like Parmigiano Reggiano (PR) cheese. ...While patterns of microbial evolution have been well studied in PR cheese, there is a lack of information about how this microbial diversity affects the metabolic and functional properties of PR cheese.
To fill this information gap, we characterized the cultivable fraction of natural whey starter (NWS) and PR cheeses at different ripening times, both at the species and strain level, and investigated the possible correlation between microbial composition and the evolution of peptide profiles over cheese ripening.
The results showed that NWS was a complex community of several biotypes belonging to a few species, namely,
,
, and
subsp.
. A new species-specific PCR assay was successful in discriminating the cheese-associated species
,
,
, and
. Based on the resolved patterns of species and biotype distribution,
and
were most frequently isolated after 24 and 30 months of ripening, while the number of biotypes was inversely related to the ripening time. Peptidomics analysis revealed more than 520 peptides in cheese samples. To the best of our knowledge, this is the most comprehensive survey of peptides in PR cheese. Most of them were from β-caseins, which represent the best substrate for LAB cell-envelope proteases. The abundance of peptides from β-casein 38-88 region continuously increased during ripening. Remarkably, this region contains precursors for the anti-hypertensive lactotripeptides VPP and IPP, as well as for β-casomorphins. We found that the ripening time strongly affects bioactive peptide profiles and that the occurrence of
species is positively linked to the incidence of eight anti-hypertensive peptides. This result highlighted how the presence of specific LAB species is likely a pivotal factor in determining PR functional properties.
Mucins are large glycoproteins whose degradation requires the expression of several glycosil hydrolases to catalyze the cleavage of the oligosaccharide chains and release monosaccharides that can be ...assimilated. In this study, we present a characterization on the strains
WC0700,
WC0709, and
WC0705. These three strains were previously isolated from enrichment cultures on mucin of fecal samples from healthy subjects and can use mucin as sole carbon and nitrogen source. Genome analysis and
functional analysis of these strains elucidated their physiological and biochemical features.
WC0700 harbored the highest number of glycosyl hydrolases specific for mucin degradation, while
WC0705 had the least. These predicted differences were confirmed growing the strains on 5 mucin-decorating monosaccharides (L-fucose, N-Acetylneuraminic acid, galactose, N-acetylgalactosamine, and N-acetylglucosamine) as only source of carbon. Fermenting mucin, they all produced formic, acetic, propionic, butyric, isovaleric, and lactic acids, and ethanol; acetic acid was the main primary metabolite. Further catabolic capabilities were investigated, as well as antibiotic susceptibility, biofilm formation, tolerance to oxygen and temperature. The potential pathogenicity of the strains was evaluated through
research of virulence factors. The merge between comparative and functional genomics and biochemical/physiological characterization provided a comprehensive view of these mucin degraders, reassuring on the safety of these species and leaving ample scope for deeper investigations on the relationship with the host and for assessing if some relevant health-promoting effect could be ascribed to these SCFA producing species.
•Extracellular vesicles (EVs) from Pectobacterium sp. were characterized for the first time.•Capillary electrophoresis of EVs was presented.•The CE method was shown to separate vesicles from ...macromolecular aggregates.•The method enables qualitative and quantitative analysis of EVs isolates.
The extracellular vesicles (EVs) released by plant pathogens of the Pectobacterium genus were investigated. The isolates were obtained using differential centrifugation followed by filtration and were characterized in terms of total protein content and particle size distribution. The transmission electron microscopy (TEM) analysis revealed the presence of two morphologically differentiated subpopulations of vesicles in the obtained isolates. The proteomic analysis using matrix-assisted laser desorption ionization mass spectrometry with time of flight detector (MALDI-TOF/TOF-MS) enabled to identify 62 proteomic markers commonly found in EVs of Gram-negative rods from the Enterobacteriaceae family. Capillary electrophoresis (CE) was proposed as a novel tool for the characterization of EVs. The method allowed for automated and fast (<15 min per sample) separation of vesicles from macromolecular aggregates with low sample consumption (about 10 nL per analysis). The approach required simple background electrolyte (BGE) composed of 50 mM BTP and 75 mM glycine (pH 9.5) and standard UV detection. The report presents a new opportunity for quality control of samples containing EVs.
Approximately 11% of people with kidney failure worldwide are treated with peritoneal dialysis (PD). This study examined PD use and practice patterns across the globe.
A cross-sectional survey.
...Stakeholders including clinicians, policy makers, and patient representatives in 182 countries convened by the International Society of Nephrology between July and September 2018.
PD use, availability, accessibility, affordability, delivery, and reporting of quality outcome measures.
Descriptive statistics.
Responses were received from 88% (n=160) of countries and there were 313 participants (257 nephrologists 82%, 22 non-nephrologist physicians 7%, 6 other health professionals 2%, 17 administrators/policy makers/civil servants 5%, and 11 others 4%). 85% (n=156) of countries responded to questions about PD. Median PD use was 38.1 per million population. PD was not available in 30 of the 156 (19%) countries responding to PD-related questions, particularly in countries in Africa (20/41) and low-income countries (15/22). In 69% of countries, PD was the initial dialysis modality for≤10% of patients with newly diagnosed kidney failure. Patients receiving PD were expected to pay 1% to 25% of treatment costs, and higher (>75%) copayments (out-of-pocket expenses incurred by patients) were more common in South Asia and low-income countries. Average exchange volumes were adequate (defined as 3-4 exchanges per day or the equivalent for automated PD) in 72% of countries. PD quality outcome monitoring and reporting were variable. Most countries did not measure patient-reported PD outcomes.
Low responses from policy makers; limited ability to provide more in-depth explanations underpinning outcomes from each country due to lack of granular data; lack of objective data.
Large inter- and intraregional disparities exist in PD availability, accessibility, affordability, delivery, and reporting of quality outcome measures around the world, with the greatest gaps observed in Africa and South Asia.
Natural whey starters (NWS) are undefined bacterial communities produced daily from whey of the previous cheese-making round, by application of high temperature. As a result, in any dairy plant, NWS ...are continuously evolving, undefined mixtures of several strains and/or species of lactic acid bacteria, whose composition and performance strongly depend on the selective pressure acting during incubation. While NWS is critical to assure consistency to cheese-making process, little is known about the composition, functional features, and plant-to-plant fluctuations. Here, we integrated 16S rRNA metabarcoding and culture-dependent methods to profile bacterial communities of 10 NWS sampled in the production area of Parmigiano Reggiano cheese. 16S rRNA metabarcoding analysis revealed two main NWS community types, namely NWS type-H and NWS type-D. Lactobacillus helveticus was more abundant in NWS type-H, whilst Lactobacillus delbrueckii/St. thermophilus in NWS type-D, respectively. Based on the prediction of metagenome functions, NWS type-H samples were enriched in functional pathways related to galactose catabolism and purine metabolism, while NWS type-D in pathways related to aromatic and branched chain amino acid biosynthesis, which are flavor compound precursors. Culture-dependent approaches revealed low cultivability of individual colonies as axenic cultures and high genetic diversity in the pool of cultivable survivors. Co-culturing experiments showed that fermentative performance decreases by reducing the bacterial complexity of inoculum, suggesting that biotic interactions and cross-feeding relationships could take place in NWS communities, assuring phenotypic robustness. Even though our data cannot directly predict these ecological interactions, this study provides the basis for experiments targeted at understanding how selective regime affects composition, bacterial interaction, and fermentative performance in NWS.
PDF
