A SEVA-based, CRISPR-Cas3-assisted genome engineering approach for Pseudomonas with efficient vector curing.

IMPORTANCE: The CRISPR-Cas3 editing system as presented here facilitates the creation of genomic alterations in Pseudomonas putida and Pseudomonas aeruginosa in a straightforward manner. By providing the Cas3 system as a vector set with Golden Gate compatibility and different antibiotic markers, as well as by employing the established Standard European Vector Architecture (SEVA) vector set to provide the homology repair template, this system is flexible and can readily be ported to a multitude of Gram-negative hosts. Besides genome editing, the Cas3 system can also be used as an effective and universal tool for vector curing. This is achieved by introducing a spacer that targets the origin-of-transfer, present on the majority of established (SEVA) vectors. Based on this, the Cas3 system efficiently removes up to three vectors in only a few days. As such, this curing approach may also benefit other genomic engineering methods or remove naturally occurring plasmids from bacteria.

Extended Q-range small-angle neutron scattering to understand the morphology of proton-exchange membranes: the case of the functionalized syndiotactic-polystyrene model system.

Semi-crystalline polymers exhibit microphase separation into crystalline and amorphous domains characterized by multiple structural levels with sizes ranging from ångströms to hundreds of nanometres. The combination of small-angle (SANS) and wide-angle (WANS) neutron scattering on the same beamline enables reliable in situ characterization of such materials under application-relevant conditions, with the unique advantage of contrast variation by controlled labelling, allowing the structure of such multi-component systems to be resolved in detail. This paper reports a structural analysis performed on deuterated polymer membranes based on syndiotactic polystyrene (sPS) using an extended Q-range SANS and WANS combination, always with the same neutron scattering instrument, either a pinhole SANS diffractometer installed at a research reactor or a 'small- and wide-angle' time-of-flight diffractometer installed at a neutron spallation source. sPS is a semi-crystalline material that becomes hydrophilic and proton conducting when suitable functionalization is achieved by thin film sulfonation, and can form various co-crystalline complexes (clathrates) with small organic molecules stored in the crystalline phase as guests in the vacancies between the polymer helices. Therefore, this material is interesting not only for its conducting properties but also for its versatility as a model system to evaluate the usefulness of extended Q-range neutron scattering in such studies. Variation of neutron contrast was achieved in the amorphous hydrophilic phase by using H2O or D2O to hydrate the membranes and in the crystalline phase by loading the clathrates with deuterated or protonated guest molecules. The experimental approach, the advantages and limitations of the two types of instrumentation used in such analyses, and the main results obtained with respect to the structural characterization of sulfonated sPS membranes under different hydration and temperature conditions are reported, and the potential of this method for similar structural studies on other semi-crystalline polymeric materials is discussed.

Salicylic Acid Release from Syndiotactic Polystyrene Staple Fibers.

Films and fibers of syndiotactic polystyrene (sPS), being amorphous or exhibiting nanoporous crystalline (NC) or dense crystalline phases, were loaded with salicylic acid (SA), a relevant non-volatile antimicrobial molecule. In the first section of the paper, sPS/SA co-crystalline (CC) δ form is characterized, mainly by wide angle X-ray diffraction (WAXD) patterns and polarized Fourier transform infrared (FTIR) spectra. The formation of sPS/SA δ CC phases allows the preparation of sPS fibers even with a high content of the antibacterial guest, which is also retained after repeated washing procedures at 65 °C. A preparation procedure starting from amorphous fibers is particularly appropriate because involves a direct formation of the CC δ form and a simultaneous axial orientation. The possibility of tuning drug amount and release kinetics, by simply selecting suitable crystalline phases of a commercially available polymer, makes sPS fibers possibly useful for many applications. In particular, fibers with δ CC forms, which retain SA molecules in their crystalline phases, could be useful for antimicrobial textiles and fabrics. Fibers with the dense γ form which easily release SA molecules, because they are only included in their amorphous phases, could be used for promising SA-based preparations for antibacterial purposes in food processing and preservation and public health. Finally, using a cell-based assay system and antibacterial tests, we investigated the cellular activity, toxicity and antimicrobial properties of amorphous, δ CC forms and dense γ form of sPS fibers loaded with different contents of SA.

Hydrogen Bonded Dimer of an Alcohol with the Derived Carboxylic Acid Triggering their Sorption by Nanoporous-crystalline PPO Films.

Films exhibiting nanoporous-crystalline (NC) phases of poly(2,6-dimethyl-1,4-phenylene) oxide (PPO), which are highly effective to absorb apolar organic guest molecules, are also able to absorb polar molecules (like alcohols and carboxylic acids) but only from concentrated organic solutions. NC PPO films, which do not absorb alcohols and carboxylic acids from diluted aqueous solutions, exhibits a huge uptake (even above 30 wt %) of benzyl alcohol (BAL) and benzoic acid (BA), if BA is obtained by spontaneous room temperature oxidation of BAL in aqueous solution. This phenomenon is rationalized by an easy uptake, mainly by the PPO intrahelical crystalline empty channels, of a BAL/BA 1/1 hydrogen-bonded dimer. This huge uptake of BAL/BA dimer by NC PPO films, which is also fast for films exhibiting the orientation of the crystalline helices perpendicular to the film plane (c⊥ orientation), can be exploited for purification of water from BAL, when present in traces. High and fast sorption of a hydrogen bonded dimer and negligible sorption of the two separate compounds is possibly unprecedented for absorbent materials.

CEMIP (KIAA1199) regulates inflammation, hyperplasia and fibrosis in osteoarthritis synovial membrane.

Osteoarthritis (OA) synovial membrane is mainly characterized by low-grade inflammation, hyperplasia with increased cell proliferation and fibrosis. We previously underscored a critical role for CEMIP in fibrosis of OA cartilage. However, its role in OA synovial membrane remains unknown. An in vitro model with fibroblast-like synoviocytes from OA patients and an in vivo model with collagenase-induced OA mice were used to evaluate CEMIP-silencing effects on inflammation, hyperplasia and fibrosis. Our results showed that i. CEMIP expression was increased in human and mouse inflamed synovial membrane; ii. CEMIP regulated the inflammatory response pathway and inflammatory cytokines production in vitro and in vivo; iii. CEMIP induced epithelial to mesenchymal transition pathway and fibrotic markers in vitro and in vivo; iv. CEMIP increased cell proliferation and synovial hyperplasia; v. CEMIP expression was increased by inflammatory cytokines and by TGF-ß signaling; vi. anti-fibrotic drugs decreased CEMIP expression. All these findings highlighted the central role of CEMIP in OA synovial membrane development and underscored that targeting CEMIP could be a new therapeutic approach.

Pilot study to investigate the feasibility of conducting a randomised controlled trial that compares Immediate versus Optional Delayed surgical repair for treatment of acute Anterior cruciate ligament injury: IODA pilot trial.

INTRODUCTION: Standard care for anterior cruciate ligament (ACL) injuries includes surgical reconstruction of the ACL. However, two randomised controlled trials (RCTs) concluded that conservative treatment does not result in inferior clinical outcomes compared with immediate ACL reconstruction. More research is needed to in the first place verify these results, and second to assess whether patient-specific parameters determine whether a patient would benefit from one treatment option over the other. However, before running a full RCT, it seems necessary to perform a pilot study that assesses the feasibility of recruiting patients with ACL for such a RCT. This is because recruitment may be challenging as many patients have strong treatment beliefs. Therefore, this pilot study will assess whether a large RCT is feasible with regard to participant recruitment, adherence to the allocated treatment arm and protocol feasibility. These pilot findings will help deciding about progressing to a future full RCT. METHODS AND ANALYSIS: This is a pragmatic, multicentre, randomised controlled pilot trial with two parallel groups. Patients with an acute ACL injury will be recruited from two Belgian hospitals. Patients will be randomised to either conservative treatment or surgical treatment. Patients will be followed-up at 3, 6 and 12 months postrandomisation. Recruitment feasibility will be evaluated by calculating the recruitment rate 4 months after the two sites have been initiated. Clear criteria for progression to a full trial are defined. Adherence to the protocol will be assessed by calculating the proportion of patients who complete the assessments. Furthermore the proportion of patients who cross-over between treatment arms during the follow-up period will be assessed. ETHICS AND DISSEMINATION: The study was approved by the ethical committees: Ethische Commissie Onderzoek UZ/KU Leuven (S62004) and Comité d'Ethique Hospitalo-Facultaire Universitaire de Liège (2020212). Results will be made available to caregivers, researchers and funder. TRIAL REGISTRATION NUMBER: This trial is registered on ClinicalTrials.gov (NCT04408690) on 29 May 2020.

Nanoporous-Crystalline Poly(2,6-dimethyl-1,4-phenylene)oxide Aerogels with Selectively Sulfonated Amorphous Phase for Fast VOC Sorption from Water.

This paper describes the preparation and characterization of poly(2,6-dimethyl-1,4-phenylene)oxide (PPO) highly porous monolithic aerogels with a hydrophobic nanoporous-crystalline phase and a hydrophilic sulfonated amorphous phase. The sulfonated aerogels were obtained by the sulfonation of PPO physical gels, followed by the supercritical CO2 extraction of solvents. WAXD and FTIR analysis showed that the nanoporous-crystalline phase was preserved for a degree of sulfonation up to c.a. 35%, allowing a highly volatile organic compound (VOC) sorption capacity. The sulfonated PPO aerogels exhibited a high water sorption capacity, with a water uptake of up to 500 wt%, and faster VOC sorption kinetics from water with respect to unsulfonated aerogels.

Dependence on Film Thickness of Guest-Induced c Perpendicular Orientation in PPO Films.

For poly(2,6-dimethyl-1,4-phenylene)oxide (PPO) films exhibiting nanoporous-crystalline (NC) phases, c⟂ orientation (i.e., crystalline polymer chain axes being preferentially perpendicular to the film plane) is obtained by crystallization of amorphous films, as induced by sorption of suitable low-molecular-mass guest molecules. The occurrence of c⟂ orientation is relevant for applications of NC PPO films because it markedly increases film transparency as well as guest diffusivity. Surprisingly, we show that the known crystallization procedures lead to c⟂ oriented thick (50-300 µm) films and to unoriented thin (≤20 µm) films. This absence of crystalline phase orientation for thin films is rationalized by fast guest sorption kinetics, which avoid co-crystallization in confined spaces and hence inhibit formation of flat-on lamellae. For thick films exhibiting c⟂ orientation, sigmoid kinetics of guest sorption and of thickening of PPO films are observed, with inflection points associated with guest-induced film plasticization. Corresponding crystallization kinetics are linear with time and show that co-crystal growth is poorly affected by film plasticization. An additional relevant result of this study is the linear relationship between WAXD crystallinity index and DSC melting enthalpy, which allows evaluation of melting enthalpy of the NC α form of PPO (ΔHmο = 42 ± 2 J/g).

Influence of Glucocorticoids on Cellular Senescence Hallmarks in Osteoarthritic Fibroblast-like Synoviocytes.

Osteoarthritis (OA) is recognized as being a cellular senescence-linked disease. Intra-articular injections of glucocorticoids (GC) are frequently used in knee OA to treat synovial effusion but face controversies about toxicity. We investigated the influence of GC on cellular senescence hallmarks and senescence induction in fibroblast-like synoviocytes (FLS) from OA patients and mesenchymal stem cells (MSC). METHODS: Cellular senescence was assessed via the proliferation rate, ß-galactosidase staining, DNA damage and CKI expression (p21, p16INK4A). Experimental senescence was induced by irradiation. RESULTS: The GC prednisolone did not induce an apparent senescence phenotype in FLS, with even higher proliferation, no accumulation of ß-galactosidase-positive cells nor DNA damage and reduction in p21mRNA, only showing the enhancement of p16INK4A. Prednisolone did not modify experimental senescence induction in FLS, with no modulation of any senescence parameters. Moreover, prednisolone did not induce a senescence phenotype in MSC: despite high ß-galactosidase-positive cells, no reduction in proliferation, no DNA damage and no CKI enhancement was observed. CONCLUSIONS: We provide reassuring in vitro data about the use of GC regarding cellular senescence involvement in OA: the GC prednisolone did not induce a senescent phenotype in OA FLS (the proliferation ratio was even higher) and in MSC and did not worsen cellular senescence establishment.

Monomeric and Dimeric Carboxylic Acid in Crystalline Cavities and Channels of Delta and Epsilon Forms of Syndiotactic Polystyrene.

Delta (δ) and epsilon (ε) co-crystalline forms of syndiotactic polystyrene with a carboxylic acid guest were obtained by sorption of liquid hexanoic acid in syndiotactic polystyrene films exhibiting delta and epsilon nanoporous-crystalline forms. The characterization study is facilitated by axially stretched syndiotactic polystyrene films, used both for polarized FTIR spectra and for WAXD fiber patterns. Particularly informative are two carbonyl-stretching FTIR peaks, attributed to monomeric and dimeric hexanoic acid. The dichroism of these carbonyl peaks indicates that both delta and epsilon phases are able to include hexanoic acid as isolated guest molecules, while only the epsilon phase is also able to include dimeric hexanoic acid molecules in its crystalline channels. The inclusion of both isolated and dimeric hexanoic acid species in the epsilon form crystalline channels produces extremely fast hexanoic acid uptakes by syndiotactic polystyrene epsilon form films.

c-Perpendicular Orientation of Poly(ʟ-lactide) Films.

Poly(ʟ-lactide) (PLLA) films, even of high thickness, exhibiting co-crystalline and crystalline α phases with their chain axes preferentially perpendicular to the film plane (c⊥ orientation) have been obtained. This c⊥ orientation, unprecedented for PLLA films, can be achieved by the crystallization of amorphous films as induced by low-temperature sorption of molecules being suitable as guests of PLLA co-crystalline forms, such as N,N-dimethylformamide, cyclopentanone or 1,3-dioxolane. This kind of orientation is shown and quantified by two-dimensional wide-angle X-ray diffraction (2D-WAXD) patterns, as taken with the X-ray beam parallel to the film plane (EDGE patterns), which present all the hk0 arcs centered on the meridian. PLLA α-form films, as obtained by low-temperature guest-induced crystallization, also exhibit high transparency, being not far from those of the starting amorphous films.

Molecular Features Behind Formation of α or ß Co-Crystalline and Nanoporous-Crystalline Phases of PPO.

Guest molecular features determining the formation of α and ß phases of poly(2-6-dimethyl-1,4-phenylene) oxide (PPO) are explored by collecting literature data and adding many new film preparations, both by solution casting and by guest sorption in amorphous films. Independently of the considered preparation method, the α-form is favored by the hydrophobic and bulky guest molecules, while the hydrophilic and small guest molecules favor the ß-form. Furthermore, molecular modeling studies indicate that the ß-form inducer guests establish stronger dispersive interactions with the PPO units than the α-form inducer guests. Thus, the achievement of co-crystalline (and derived nanoporous crystalline) α- and ß-forms would result from differences in energy gain due to the host-guest interactions established at the local scale.

Nanoporous Crystalline Composite Aerogels with Reduced Graphene Oxide.

High-porosity monolithic composite aerogels of syndiotactic polystyrene (sPS) and poly(2,6-dimethyl-1,4-phenylene oxide) (PPO) containing reduced graphene oxide (r-GO) were prepared and characterized. The composite aerogels obtained by supercritical carbon dioxide (scCO2) extraction of sPS/r-GO and PPO/r-GO gels were characterized by a fibrillar morphology, which ensured good handling properties. The polymer nanoporous crystalline phases obtained within the aerogels led to high surface areas with values up to 440 m2 g-1. The role of r-GO in aerogels was studied in terms of catalytic activity by exploring the oxidation capacity of composite PPO and sPS aerogels toward benzyl alcohol in diluted aqueous solutions. The results showed that, unlike sPS/r-GO aerogels, PPO/r-GO aerogels were capable of absorbing benzyl alcohol from the diluted solutions, and that oxidation of c.a. 50% of the sorbed benzyl alcohol molecules into benzoic acid occurred.

Axial Orientation of Co-Crystalline Phases of Poly(2,6-Dimethyl-1,4-Phenylene)Oxide Films.

Films exhibiting co-crystalline (CC) phases between a polymer host and low-molecular-mass guest molecules are relevant for many applications. As is usual for semi-crystalline polymers, axially oriented films can give relevant information on the crystalline structure, both by Wide Angle X-ray diffraction fiber patterns and by polarized Fourier-transform infrared spectroscopy. Axially oriented CC phases of poly(2,6-dimethyl-1,4-phenylene)oxide (PPO) with 1,3,5-trimethylbenzene (mesitylene) can be simply obtained by the stretching of CC PPO films. In fact, due to the plasticization effect of this highly boiling guest, PPO orientation can occur in a stretching temperature range (170-175 °C) nearly 50 °C lower than that generally needed for PPO films (220-230 °C). This low stretching temperature range allows avoidance of polymer oxidation, as well as formation of the mesomorphic dense γ PPO phase. Axially oriented CC phases of PPO with toluene, i.e., with a more volatile guest, can be instead obtained by the stretching (in the same low temperature range: 170-175 °C) of CC PPO blend films with polystyrene.

Total joint replacement improves pain, functional quality of life, and health utilities in patients with late-stage knee and hip osteoarthritis for up to 5 years.

OBJECTIVES: To study and identify the determinants of the impact on pain, function, and quality of life of a prosthetic replacement surgery after 5 years of survival in patients with osteoarthritis (OA) of the lower limb. METHOD: In total, 626 osteoarthritic patients from a University Hospital, divided in 2 groups (according to surgical site), were prospectively followed for 5 years after hip (n = 346) or knee (n = 280) replacement. Validated specific Western Ontario and McMaster Universities Arthritis Index (WOMAC) and generic (SF-36 and EQ) instruments assessing quality of life were used prior to surgery and yearly, thereafter. We defined a good outcome as a clinically relevant improvement in WOMAC greater than or equal to the minimally important difference (MID). Regressions showed the relationships among preoperative, perioperative, and postoperative measures and the evolution of WOMAC scores after 5 years (percent change). We also examined any predictors of good outcomes. RESULTS: The beneficial effect on quality of life observed during the first year after hip and knee arthroplasty (HA and KA) was maintained for up to 5 years. More than 3/4 of the patients in our study experienced a good outcome (86.04% in HA group and 79.91% in KA group). Both the good outcome and the 5-year change in WOMAC are predicted by preoperative (i.e., radiological severity, comorbidities, disability, and level of education), perioperative (i.e., length of hospital stay and place of discharge), and postoperative (i.e., complications) variables in the two groups. CONCLUSIONS: Joint arthroplasty is a highly valuable therapeutic strategy for hip or knee OA patients who do not respond to pharmacological management. These results represent a step towards the collection of robust, scientifically sound data that will facilitate the completion of health economic analyses in the field of OA. KEY POINTS: • This study reports the long term outcomes of hip and knee replacement surgery in late-stage OA.• We identified pre-, per-, and post-operative determinants which contribute to a greater improvement in pain and function, hence increasing patients' satisfaction.• These results could contribute to select an OA population which has a high probability to get an optimal benefit from total joint replacement.

Highly Robust and Selective System for Water Pollutants Removal: How to Transform a Traditional Photocatalyst into a Highly Robust and Selective System for Water Pollutants Removal.

Highly porous monolithic aerogels based on ZnO photocatalyst and syndiotactic polystyrene (s-PS) were obtained by supercritical CO2 treatment of ZnO/s-PS gels. The prepared aerogels were characterized and their photocatalytic activity was evaluated using phenol and toluene as water pollutant models. The s-PS nanoporous crystalline phase, able to absorb pollutant molecules, was proven to be necessary to ensure high photocatalytic efficiency as the aerogel acts not only as a support, but also as pollutant pre-concentrator. The reusability of ZnO/s-PS aerogels is also strong showing no decrease in photocatalytic activity after six consecutive degradation trials. Finally, the aerogel matrix prevents ZnO dissolution occurring under acidic conditions and promotes a selective removal of the pollutants. The synergy between the photocatalyst and the innovative polymeric support provides the composite system with robustness, chemical stability, easy recovery after treatment, high efficiency of pollutant removal with a marked selectivity which make these materials promising for large scale applications.

Early Clinically Relevant Improvement in Quality of Life and Clinical Outcomes 1 Year Postsurgery in Patients with Knee and Hip Joint Arthroplasties.

Objective To measure and identify the determinants of the outcomes after hip/knee arthroplasty (HA/KA) in patients with osteoarthritis during the first postsurgical year. Design In this prospective observational study, we evaluated the preoperative and postoperative (3, 6, and 12 months) outcomes of 626 patients who underwent HA (346 with median age 65 years, 59% female) or KA (280 with median age 66.5 years, 54% female) between 2008 and 2013. Generic and specific tools were used to measure health-related quality of life (HRQoL) and utility. Good outcome was defined as an improvement in WOMAC (Western Ontario and McMaster Universities Osteoarthritis Index) greater than or equal to the minimal important difference (MID). Regressions were performed to evaluate the relationship between preoperative and postoperative measures and evolution of WOMAC/good outcome. Results We observed an almost systematic improvement of all parameters for up to 12 months, but especially at the 3-month follow-up. The low number of comorbidities and the absence of postoperative complications were the common determinants of improvement of WOMAC total score after 12 months. Other parameters (background of the joint, preoperative function and length of hospital stay in KA group; place of discharge in HA group) affected the evolution of WOMAC scores. 87.09% of HA and 73.06% of KA patients experienced a good outcome. A small number of comorbidities, a worse preoperative function, a shortened hospital stay (KA only), and an absence of early postoperative complications (HA only) significantly predicted a good outcome. Conclusions Intermediate HRQoL following HA or KA improved quickly from preoperative levels for all instruments. More than 70% of patients achieved a good outcome defined as improved pain, stiffness and disability and the predictors are slightly close.

Monolithic polymeric aerogels with VOCs sorbent nanoporous crystalline and water sorbent amorphous phases.

Monolithic syndiotactic polystyrene (s-PS) aerogels, formed by highly crystalline nanofibrils with a hydrophobic nanoporous-crystalline phase and a hydrophilic amorphous phase have been prepared and characterized. These aerogels, with a high degree of sulfonation of the amorphous phase and nearly negligible sulfonation of the crystalline phase, are obtained by treating physical gels exhibiting δ-clathrate form. With respect to unsulfonated nanoporous-crystalline polymeric aerogels, these new selectively sulfonated aerogels present the great advantage of a high water diffusivity and water uptake up to 600 wt %. This water uptake increases greatly the sorption kinetics of organic pollutants by the hydrophobic nanopores of the crystalline phase. For instance, for aerogels with a sulfonation of 10%, the diffusivity of a volatile organic compound (1,2-dichloroethane, DCE) from 10 ppm aqueous solution is more than 3 orders of magnitude higher than that for the unsulfonated aerogel and is very close to the DCE diffusivity in water.

Monolithic nanoporous crystalline aerogels.

Monolithic aerogels can be easily obtained by drying physical gels formed by linear uncross-linked polymers. Preparation methods, structure, and properties of these physically cross-linked polymeric aerogels are reviewed, with particular emphasis to those whose cross-linking knots are crystallites and, more in particular, crystallites exhibiting nanoporous-crystalline forms. The latter aerogels present beside disordered amorphous micropores (typical of all aerogels) also all identical nanopores of the crystalline phases. Their outstanding guest transport properties combined with low material cost, robustness, durability, and ease of handling and recycle make these aerogels suitable for applications in chemical separations, purification, and storage as well as in biomedicine. Scientific, technological, and industrial perspectives for monolithic nanoporous-crystalline polymeric aerogels are also discussed.