Confinement of Monolithic Stationary Phases in Targeted Regions of 3D-Printed Titanium Devices Using Thermal Polymerization

Abstract In this study, we have prepared thermally initiated polymeric monolithic stationary phases within discrete regions of 3D-printed titanium devices. The devices were created with controllable hot and cold regions. The monolithic stationary phases were first locally created in capillaries inserted into the channels of the titanium devices. The homogeneity of the monolith structure and the interface length were studied by scanning a capacitively coupled conductivity contactless detector (C4D) along the length of the capillary. Homogeneous monolithic structures could be obtained within a titanium device equipped with a hot and cold jacket connected to two water baths. The confinement method was optimized in capillaries. The sharpest interfaces (between monolith and empty channel) were obtained with the hot region maintained at 70 °C and the cold region at 4 or 10 °C, with the latter temperature yielding better repeatability. The optimized conditions were used to create monoliths bound directly to the walls of the titanium channels. The fabricated monoliths were successfully used to separate a mixture of four intact proteins using reversed-phase liquid chromatography. Further chromatographic characterization showed a permeability (Kf) of ∼4 × 10–15 m2 and a total porosity of 60%. Since their introduction in the chromatographic world, porous polymer monoliths have proven to be powerful separation media. These chromatographic supports have been widely applied for applications, such as microscale liquid chromatography (LC) of peptides and proteins, but have also been used in capillary electrochromatography (CEC),(1) gas chromatography (GC),(2) sample preparation,(3) and catalysis.(4) The ease of preparation of monoliths, diverse chemistry options, and high permeabilities have made them popular materials for analytical devices, such as microfluidic chips for LC. In the past decade, miniaturization has been realized by developing lab-on-a-chip solutions, where several analytical processes can be integrated within a few square centimeters. In such systems, due to the small channels and articulated geometries, the particle-packing procedure has proven to be challenging.(5) In contrast, monolithic beds are usually created in situ by free-radical polymerization of monomers in the presence of porogens and they are well-suited for chip-based separations. The proliferation of microfluidic devices has spurred new interest in polymer monoliths for applications such as enzymatic reactors(6,7) and microfluidic mixers.(8) This development has been boosted by the advent of additive manufacturing (or 3D-printing), which allows for rapid prototyping of complex structures, converting computer-aided-design (CAD) models into physical objects. Unfortunately, the use of 3D-printed analytical devices for chromatographic analysis is limited by the solvent compatibility of some materials (e.g., acrylate-based polymers) and in some cases by their transparency at the desired wavelength (e.g., UV or IR wavelengths). Several successful steps have been taken to locally photopolymerize monolithic stationary phases in discrete regions of microfluidic devices.(9−12) Heat is an alternative way to transfer energy to the monomer precursors for initiating the polymerization. However, accurate control of temperature in small confined spaces is more difficult to achieve, and so far only few steps have been taken in this direction.(13) In this work, two methods are explored to achieve confined thermal polymerization. The first approach involves direct contact (DC) between Peltier elements and the surface of a titanium device. In the second approach, recirculating jackets are used for localized heating and cooling (heating/cooling jackets, HCJ). The latter approach resembles a recirculation-based freeze–thaw valve.(14) In both approaches, defined hot (HR) and cold (CR) regions are created. We aim to fabricate poly(styrene-co-divinylbenzene) (PS-DVB) monolithic stationary phases within a 3D-printed titanium microfluidic device through polymerization at 70 °C, and to separate intact proteins using this device

The MDM2 antagonist idasanutlin in patients with polycythemia vera:results from a single-arm phase 2 study

Idasanutlin, an MDM2 antagonist, showed clinical activity and a rapid reduction in JAK2 V617F allele burden in patients with polycythemia vera (PV) in a phase 1 study. This open-label phase 2 study evaluated idasanutlin in patients with hydroxyurea (HU)-resistant/-intolerant PV, per the European LeukemiaNet criteria, and phlebotomy dependence; prior ruxolitinib exposure was permitted. Idasanutlin was administered once daily on days 1 through 5 of each 28-day cycle. The primary end point was composite response (hematocrit control and spleen volume reduction > 35%) in patients with splenomegaly and hematocrit control in patients without splenomegaly at week 32. Key secondary end points included safety, complete hematologic response (CHR), patient-reported outcomes, and molecular responses. All patients (n = 27) received idasanutlin; 16 had response assessment (week 32). Among responders with baseline splenomegaly (n = 13), 9 (69%) attained any spleen volume reduction, and 1 achieved composite response. Nine patients (56%) achieved hematocrit control, and 8 patients (50%) achieved CHR. Overall, 43% of evaluable patients (6/14) showed a ≥50% reduction in the Myeloproliferative Neoplasm Symptom Assessment Form Total Symptom Score (week 32). Nausea (93%), diarrhea (78%), and vomiting (41%) were the most common adverse events, with grade ≥ 3 nausea or vomiting experienced by 3 patients (11%) and 1 patient (4%), respectively. Reduced JAK2 V617F allele burden occurred early (after 3 cycles), with a median reduction of 76%, and was associated with achieving CHR and hematocrit control. Overall, the idasanutlin dosing regimen showed clinical activity and rapidly reduced JAK2 allele burden in patients with HU-resistant/- intolerant PV but was associated with low-grade gastrointestinal toxicity, leading to poor long-term tolerability. This trial was registered at www.clinincaltrials.gov as #NCT03287245

A sex-informed approach to improve the personalised decision making process in myelodysplastic syndromes: a multicentre, observational cohort study

Background Sex is a major source of diversity among patients and a sex-informed approach is becoming a new paradigm in precision medicine. We aimed to describe sex diversity in myelodysplastic syndromes in terms of disease genotype, phenotype, and clinical outcome. Moreover, we sought to incorporate sex information into the clinical decision-making process as a fundamental component of patient individuality. Methods In this multicentre, observational cohort study, we retrospectively analysed 13 284 patients aged 18 years or older with a diagnosis of myelodysplastic syndrome according to 2016 WHO criteria included in the EuroMDS network (n=2025), International Working Group for Prognosis in MDS (IWG-PM; n=2387), the Spanish Group of Myelodysplastic Syndromes registry (GESMD; n=7687), or the Dusseldorf MDS registry (n=1185). Recruitment periods for these cohorts were between 1990 and 2016. The correlation between sex and genomic features was analysed in the EuroMDS cohort and validated in the IWG-PM cohort. The effect of sex on clinical outcome, with overall survival as the main endpoint, was analysed in the EuroMDS population and validated in the other three cohorts. Finally, novel prognostic models incorporating sex and genomic information were built and validated, and compared to the widely used revised International Prognostic Scoring System (IPSS-R). This study is registered with ClinicalTrials.gov, NCT04889729. Findings The study included 7792 (58middot7%) men and 5492 (41middot3%) women. 10 906 (82middot1%) patients were White, and race was not reported for 2378 (17middot9%) patients. Sex biases were observed at the single-gene level with mutations in seven genes enriched in men (ASXL1, SRSF2, and ZRSR2 p<0middot0001 in both cohorts; DDX41 not available in the EuroMDS cohort vs p=0middot0062 in the IWG-PM cohort; IDH2 p<0middot0001 in EuroMDS vs p=0middot042 in IWG-PM; TET2 p=0middot031 vs p=0middot035; U2AF1 p=0middot033 vs p<0middot0001) and mutations in two genes were enriched in women (DNMT3A p<0middot0001 in EuroMDS vs p=0middot011 in IWG-PM; TP53 p=0middot030 vs p=0middot037). Additionally, sex biases were observed in co-mutational pathways of founding genomic lesions (splicing-related genes, predominantly in men, p<0middot0001 in both the EuroMDS and IWG-PM cohorts), in DNA methylation (predominantly in men, p=0middot046 in EuroMDS vs p<0middot0001 in IWG-PM), and TP53 mutational pathways (predominantly in women, p=0middot0073 in EuroMDS vs p<0middot0001 in IWG-PM). In the retrospective EuroMDS cohort, men had worse median overall survival (81middot3 months, 95% CI 70middot4-95middot0 in men vs 123middot5 months, 104middot5-127middot5 in women; hazard ratio [HR] 1middot40, 95% CI 1middot26-1middot52; p<0middot0001). This result was confirmed in the prospective validation cohorts (median overall survival was 54middot7 months, 95% CI 52middot4-59middot1 in men vs 74middot4 months, 69middot3-81middot2 in women; HR 1middot30, 95% CI 1middot23-1middot35; p<0middot0001 in the GEMSD MDS registry; 40middot0 months, 95% CI 33middot4-43middot7 in men vs 54middot2 months, 38middot6-63middot8 in women; HR 1middot23, 95% CI 1middot08-1middot36; p<0middot0001 in the Dusseldorf MDS registry). We developed new personalised prognostic tools that included sex information (the sex-informed prognostic scoring system and the sex-informed genomic scoring system). Sex maintained independent prognostic power in all prognostic systems; the highest performance was observed in the model that included both sex and genomic information. A five-to-five mapping between the IPSS-R and new score categories resulted in the re-stratification of 871 (43middot0%) of 2025 patients from the EuroMDS cohort and 1003 (42middot0%) of 2387 patients from the IWG-PM cohort by using the sex-informed prognostic scoring system, and of 1134 (56middot0%) patients from the EuroMDS cohort and 1265 (53middot0%) patients from the IWG-PM cohort by using the sex-informed genomic scoring system. We created a web portal that enables outcome predictions based on a sex-informed personalised approach. Interpretation Our results suggest that a sex-informed approach can improve the personalised decision making process in patients with myelodysplastic syndromes and should be considered in the design of clinical trials including low-risk patients. Copyright (c) 2022 Published by Elsevier Ltd. All rights reserved

Artrite settica nel puledro: valutazione di alcunitra i fattori che influenzano esito e prognosie confronto di diverse strategie di trattamento

L’artrite settica nel puledro è una patologia frequente e grave, che può comprometterne in alcuni casi la stessa sopravvivenza. Si sviluppa in genere in associazione ad altre condizioni patologiche che consentono una diffusione per via ematogena di batteri e la loro conseguente localizzazione a livello articolare. L’esito dipende strettamente dalla rapidità con cui si riesce ad effettuare una diagnosi e ad impostare un’opportuna terapia, dal tipo di terapia, dalla gravità della condizione e dal grado di compromissione sistemica del soggetto. Diverse sono le strategie di trattamento attuabili, ma quelle che prevedono l’esecuzione di lavaggi articolari e somministrazioni locali di antibiotici specifici sembrano avere maggior successo

Micro-flow size-exclusion chromatography for enhanced native mass spectrometry of proteins and protein complexes

Size-exclusion chromatography (SEC) employing aqueous mobile phases with volatile salts at neutral pH combined with native mass spectrometry (nMS) is a valuable tool to characterize proteins and protein aggregates in their native state. However, the liquid-phase conditions (high salt concentrations) frequently used in SEC-nMS hinder the analysis of labile protein complexes in the gas phase, necessitating higher desolvation-gas flow and source temperature, leading to protein fragmentation/dissociation. To overcome this issue, we investigated narrow SEC columns (1.0 mm internal diameter, I.D.) operated at 15-μL/min flow rates and their coupling to nMS for the characterization of proteins, protein complexes and higher-order structures (HOS). The reduced flow rate resulted in a significant increase in the protein-ionization efficiency, facilitating the detection of low-abundant impurities and HOS up to 230 kDa (i.e., the upper limit of the Orbitrap-MS instrument used). More-efficient solvent evaporation and lower desolvation energies allowed for softer ionization conditions (e.g., lower gas temperatures), ensuring little or no structural alterations of proteins and their HOS during transfer into the gas phase. Furthermore, ionization suppression by eluent salts was decreased, permitting the use of volatile-salt concentrations up to 400 mM. Band broadening and loss of resolution resulting from the introduction of injection volumes exceeding 3% of the column volume could be circumvented by incorporating an online trap-column containing a mixed-bed ion-exchange (IEX) material. The online IEX-based solid-phase extraction (SPE) or “trap-and-elute” set-up provided on-column focusing (sample preconcentration). This allowed the injection of large sample volumes on the 1-mm I.D. SEC column without compromising the separation. The enhanced sensitivity attained by the micro-flow SEC-MS, along with the on-column focusing achieved by the IEX precolumn, provided picogram detection limits for proteins

Micro-flow Size-Exclusion Chromatography for enhanced native Mass Spectrometry of proteins and protein complexes

Native Size-exclusion chromatography (SEC) employing aqueous mobile phases with volatile salts at neutral pH combined with native mass spectrometry (nMS) is a useful tool for the characterization of proteins in their native state. However, in many cases the conditions needed to realize the hyphenation of SEC with MS require relative high activation energy and therefore hinder the analysis of labile protein complexes. In this work, we are investigating the advantages of narrow SEC columns (1 mm internal diameter) operated at 15 μL/min flow rates coupled directly to nMS for the characterization of proteins, labile protein complexes and their higher-order structures (HOS). Reducing the flow rate, allowed for a significant increase of the MS sensitivity and ionization efficiency, facilitating detection of low-abundant impurities and HOS (up to the limit of the Orbitrap-MS used, i.e. 230 kDa). More-efficient solvent evaporation could be achieved, allowing using softer MS conditions (e.g. lower gas temperature, lower activation energy) that ensured (little or) no structural alterations or denaturation of the proteins and their HOS during their transfer to the gas phase. Furthermore, high-ionic-strength conditions of volatile salts (200-400 mM), are often necessary to ensure (almost) interaction-free SEC analysis of proteins, such as antibodies (mAbs). With this approach the salt tolerance of the MS was much improved. Because of the reduced column dimensions, band broadening effects resulting from the injection volume became more critical. At high injection volumes (exceeding 3% of the column volume) of more dilute samples, the peak shape and width was affected. Therefore, a new set-up was developed to pre-concentrate the injected proteins on an anion and cation-exchange mixed bed trap column prior to SEC-nMS analysis. This “trap-and-elute” set-up was able to eliminate adverse injection-volume effects in SEC and provide additional desalting, while improving MS detection limits

Serum amyloid A, fibrinogen, and aptoglobin as inflammation markers in the horse: preliminary results

The early detection and monitoring of inflammation is a primary challenge in veterinary medicine. The circulating concentrations of acute-phase proteins may provide an objective measure of both the severity of insult and the intensity of inflammatory responses in the horse. A clinical study on the possible role of serum amyloid A (SAA), fibrinogen, and haptoglobin as inflammation markers in the horse was conducted in 40 horses, with a significant correlation emerging between the patterns of SAA concentrations and clinical assessments of the patients being monitored during their hospitalization. The results of this preliminary study support the use of SAA as an important parameter at diagnostic, prognostic, and therapeutic stages. The sequential levels of SAA appear to be particularly useful in the postintervention monitoring of surgical patients

Serum amyloid A, fibrinogen, and haptoglobin as inflammation markers in the horse: preliminary results

The early detection and monitoring of inflammation is a primary challenge in veterinary medicine. The circulating concentrations of acute-phase proteins may provide an objective measure of both the severity of insult and the intensity of inflammatory responses in the horse. A clinical study on the possible role of serum amyloid A (SAA), fibrinogen, and haptoglobin as inflammation markers in the horse was conducted in 40 horses, with a significant correlation emerging between the patterns of SAA concentrations and clinical assessments of the patients being monitored during their hospitalization. The results of this preliminary study support the use of SAA as an important parameter at diagnostic, prognostic, and therapeutic stages. The sequential levels of SAA appear to be particularly useful in the postintervention monitoring of surgical patients