TEAD4 regulates trophectoderm differentiation upstream of CDX2 in a GATA3-independent manner in the human preimplantation embryo.

STUDY QUESTION: What is the role of transcriptional-enhanced associate (TEA) domain family member 4 (TEAD4) in trophectoderm (TE) differentiation during human embryo preimplantation development in comparison to mouse? SUMMARY ANSWER: TEAD4 regulates TE lineage differentiation in the human preimplantation embryo acting upstream of caudal-type homeobox protein 2 (CDX2), but in contrast to the mouse in a GATA-binding protein 3 (GATA3)-independent manner. WHAT IS KNOWN ALREADY: Tead4 is one of the earliest transcription factors expressed during mouse embryo preimplantation development and is required for the expression of TE-associated genes. Functional knock-out studies in mouse, inactivating Tead4 by site-specific recombination, have shown that Tead4-targeted embryos have compromised development and expression of the TE-specific Cdx2 and Gata3 is downregulated. Cdx2 and Gata3 act in parallel pathways downstream of Tead4 to induce successful TE differentiation. Downstream loss of Cdx2 expression, compromises TE differentiation and subsequent blastocoel formation and leads to the ectopic expression of inner cell mass (ICM) genes, including POU Class 5 homeobox 1 (Pou5f1) and SRY-box transcription factor (Sox2). Cdx2 is a more potent regulator of TE fate in mouse as loss of Cdx2 expression induces more severe phenotypes compared with loss of Gata3 expression. The role of TEAD4 and its downstream effectors during human preimplantation embryo development has not been investigated yet. STUDY DESIGN, SIZE, DURATION: The clustered regularly interspaced short palindromic repeats-clustered regularly interspaced short palindromic repeats (CRISPR)-associated genes (CRISPR-Cas9) system was first introduced in pronuclei (PN)-stage mouse zygotes aiming to identify a guide RNA (gRNA), yielding high editing efficiency and effective disruption of the Tead4 locus. Three guides were tested (gRNA1-3), each time targeting a distinct region of Exon 2 of Tead4. The effects of targeting on developmental capacity were studied in Tead4-targeted embryos (n = 164-summarized data from gRNA1-3) and were compared with two control groups; sham-injected embryos (n = 26) and non-injected media-control embryos (n = 51). The editing efficiency was determined by next-generation sequencing (NGS). In total, n = 55 (summarized data from gRNA1-3) targeted mouse embryos were analysed by NGS. Immunofluorescence analysis to confirm successful targeting by gRNA1 was performed in Tead4-targeted embryos, and non-injected media-control embryos. The downregulation of secondary TE-associated markers Cdx2 and Gata3 was used as an indirect confirmation of successful Tead4-targeting (previously shown to be expressed downstream of Tead4). Additional groups of gRNA1 Tead4-targeted (n = 45) and media control (n = 36) embryos were cultured for an extended period of 8.5 days, to further assess the developmental capacity of the Tead4-targeted group to develop beyond implantation stages. Following the mouse investigation, human metaphase-II (MII) oocytes obtained by IVM were microinjected with gRNA-Cas9 during ICSI (n = 74) to target TEAD4 or used as media-control (n = 33). The editing efficiency was successfully assessed in n = 25 TEAD4-targeted human embryos. Finally, immunofluorescence analysis for TEAD4, CDX2, GATA3 and the ICM marker SOX2 was performed in TEAD4-targeted (n = 10) and non-injected media-control embryos (n = 29). PARTICIPANTS/MATERIALS, SETTING, METHODS: A ribonucleoprotein complex consisting of a gRNA-Cas9 mixture, designed to target Exon 2 of Tead4/TEAD4, was microinjected in mouse PN stage zygotes or human IVM MII oocytes along with sperm. Generated embryos were cultured in vitro for 4 days in mouse or 6.5 days in human. In mouse, an additional group of Tead4-targeted and media-control embryos was cultured in vitro for an extended period of 8.5 days. Embryonic development and morphology were assessed daily, during culture in vitro of mouse and human embryos and was followed by a detailed scoring at late blastocyst stage. Targeting efficiency following gRNA-Cas9 introduction was assessed via immunostaining and NGS analysis. MAIN RESULTS AND THE ROLE OF CHANCE: NGS analysis of the Tead4-targeted locus revealed very high editing efficiencies for all three guides, with 100% of the mouse embryos (55 out of 55) carrying genetic modifications resulting from CRISPR-Cas9 genome editing. More specifically, 65.22% (15 out 23) of the PN zygotes microinjected with gRNA1-Cas9, which exhibited the highest efficiency, carried exclusively mutated alleles. The developmental capacity of targeted embryos was significantly reduced (data from gRNA1), as 44.17% of the embryos arrested at the morula stage (2.5 days post coitum), coincident with the initiation of TE lineage differentiation, compared with 8.51% in control and 12.50% in sham control groups. High-quality blastocyst formation rates (Grade 3) were 8.97% in the gRNA1-targeted group, compared with 87.23% in the media-control and 87.50% in the sham group. Immunofluorescence analysis in targeted embryos confirmed downregulation of Tead4, Cdx2, and Gata3 expression, which resulted from successful targeting of the Tead4 locus. Tead4-targeted mouse embryos stained positive for the ICM markers Pou5f1 and Sox2, indicating that expression of ICM lineage markers is not affected. Tead4-targeted embryos were able to cavitate and form a blastocoel without being able to hatch. Extended embryo culture following zona pellucida removal, revealed that the targeted embryos can attach and form egg-cylinder-like structures in the absence of trophoblast giant cells. In human embryos, Exon 2 of TEAD4 was successfully targeted by CRISPR-Cas9 (n = 74). In total, 25 embryos from various developmental stages were analysed by NGS and 96.00% (24 out of 25) of the embryos carried genetic modifications because of gRNA-Cas9 editing. In the subgroup of the 24 edited embryos, 17 (70.83%) carried only mutant alleles and 11 out of these 17 (64.70%) carried exclusively frameshift mutations. Six out of 11 embryos reached the blastocyst stage. In contrast to mice, human-targeted embryos formed blastocysts at a rate (25.00%) that did not differ significantly from the control group (23.81%). However, blastocyst morphology and TE quality were significantly compromised following TEAD4-targeting, showing grade C TE scores, with TE containing very few cells. Immunofluorescence analysis of TEAD4-targeted embryos (n = 10) confirmed successful editing by the complete absence of TEAD4 and its downstream TE marker CDX2, but the embryos generated retained expression of GATA3, which is in contrast to what we have observed and has previously been reported in mouse. In this regard, our results indicate that GATA3 acts in parallel with TEAD4/CDX2 towards TE differentiation in human. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: CRISPR-Cas9 germline genome editing, in some cases, induces mosaic genotypes. These genotypes are a result of inefficient and delayed editing, and complicate the phenotypic analysis and developmental assessment of the injected embryos. We cannot exclude the possibility that the observed differences between mouse and human are the result of variable effects triggered by the culture conditions, which were however similar for both mouse and human embryos in this study. Furthermore, this study utilized human oocytes obtained by IVM, which may not fully recapitulate the developmental behaviour of in vivo matured oocytes. WIDER IMPLICATIONS OF THE FINDINGS: Elucidation of the evolutionary conservation of molecular mechanisms that regulate the differentiation and formation of the trophoblast lineage can give us fundamental insights into early implantation failure, which accounts for ∼15% of human conceptions. STUDY FUNDING/COMPETING INTEREST(S): The research was funded by the FWO-Vlaanderen (Flemish fund for scientific research, Grant no. G051516N), and Hercules funding (FWO.HMZ.2016.00.02.01) and Ghent University (BOF.BAS.2018.0018.01). G.C. is supported by FWO-Vlaanderen (Flemish fund for scientific research, Grant no. 11L8822N). A.B. is supported by FWO-Vlaanderen (Flemish fund for scientific research, Grant no. 1298722 N). We further thank Ferring Pharmaceuticals (Aalst, Belgium) for their unrestricted educational grant. The authors declare no competing interests. TRIAL REGISTRATION NUMBER: N/A.

Real-Time Monitoring of Doxorubicin Release from Hybrid Nanoporous Anodic Alumina Structures.

This work demonstrates an advanced approach to fabricate Hybrid nanoporous anodic alumina gradient-index filters (Hy-NAA-GIFs) through a heterogeneous anodization process combining sinusoidal current-density anodization and constant potential anodization. As a result, the hybrid structure obtained reveals a single photonic stopband (PSB), which falls within the absorption region of the drug molecule and the intensity of the spectrum that are far from such absorption range. The prepared structures were loaded with the doxorubicin (DOX) drug through the drop-casting method, which allows for evaluating the maximum reflectance of the relative height of the PSB with the average reflectance of the spectrum intensity. Thereafter, this property has been applied in a flow cell setup connected to a reflectance spectrophotometer where different drug-loaded samples were placed to study the behavior and kinetics of the drug release in real-time by varying two parameters, i.e., different pore length and flow rates. As such, obtained results were analyzed with a model that includes a sum of two inverted exponential decay functions with two different characteristic time releases. Overall, this study opens up several possibilities for the Hy-NAA-GIFs to study the drug kinetics from nanoporous structures.

An optical biosensor for the determination of cathepsin B as a cancer-associated enzyme using nanoporous anodic alumina modified with human serum albumin-thionine.

An interferometric reflectance spectroscopy-based biosensor for the determination of cathepsin B (Cat B) as a cancer-related enzyme has been fabricated. For this purpose, the nanoporous anodic alumina (NAA) was fabricated electrochemically. The NAA was then modified with the amino-silane coupling agent. After that, human serum albumin (HSA) was immobilized into the NAA pores by using glutaraldehyde as a cross-linking agent. Subsequently, the carboxylic group of HSA was activated with N-ethyl-N'-(3-(dimethylamino)propyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) to attach to thionine (TH) as a photoprobe to fabricate the labeled HSA (HSA-TH). HSA-TH plays a significant role in this sensor to determine cathepsin B as a model analyte for the development of the interferometric reflectance spectroscopy-based biosensor for the measurement of protease. The attached TH adsorbed the illuminated white light on NAA modified with HSA-TH. Therefore, the intensity of the reflected light to the charge-coupled device (CCD) detector decreased in the wavelength range 450-1050 nm. In the presence of Cat B, HAS-TH cleaved into short peptide fragments and washed away by flow cell system. Since TH was removed from NAA, the intensity of the reflected light increased. The peak area has a logarithmic relationship with the concentration of Cat B in the range 0.5 to 64.0 nM. The limit of detection of the biosensor sensor was 0.08 nM. The optical sensor was used for the determination of Cat B in a human serum sample. Graphical abstract Schematic presentation of biosensor for the determination of the cathepsin B which is based on nanoporous anodic alumina modified with HSA-thionine. The principle response of the optical biosensor is based on detecting changes in the intensity of the reflected light after cleaving the immobilized HSA-thionine by cathepsin B into short peptide fragments.

Advances in Optical Biosensors and Sensors Using Nanoporous Anodic Alumina.

This review paper focuses on recent progress in optical biosensors using self-ordered nanoporous anodic alumina. We present the fabrication of self-ordered nanoporous anodic alumina, surface functionalization, and optical sensor applications. We show that self-ordered nanoporous anodic alumina has good potential for use in the fabrication of antibody-based (immunosensor), aptamer-based (aptasensor), gene-based (genosensor), peptide-based, and enzyme-based optical biosensors. The fabricated optical biosensors presented high sensitivity and selectivity. In addition, we also showed that the performance of the biosensors and the self-ordered nanoporous anodic alumina can be used for assessing biomolecules, heavy ions, and gas molecules.

A photoelectrochemical sandwich immunoassay for protein S100ß, a biomarker for Alzheimer's disease, using an ITO electrode modified with a reduced graphene oxide-gold conjugate and CdS-labeled secondary antibody.

A sandwich-type photoelectrochemical immunoassay is described for the protein S100ß which is an Alzheimer's disease biomarker found in the astrocytes of the brain. Antibody against S100ß (anti-S100ß) was labeled with CdS quantum dots and then acted as a secondary antibody. The labeled antibody was characterized by FTIR, ultraviolet-visible and fluorescence spectroscopy. An indium-tin oxide (ITO) electrode was modified with a nanocomposite prepared from reduced graphene oxide and gold nanoparticles. Then, a sol-gel film containing isocyanate functional groups (-N=C=O) was cast on the surface of the electrode. The NCO group reacts with amino groups of the labeled antibody to covalently bind them to the surface. The S100ß was bound by the primary immobilized antibody on the rGO-Au/ITO electrode and then sandwiched with the labeled secondary antibody. Cyclic voltammetry and electrochemical impedance spectroscopy were applied to confirm the stepwise changes in the electrochemical properties of the electrode surface. The photoelectrochemical immunoassay, typically operated at a potential of +0.2 V (vs. Ag|AgClsat) gives a signal that is related to the logarithm of the S100ß concentration in the range from 0.25 to 10 ng·mL-1 with a lower detection limit of 0.15 pg·mL-1. The method was successfully applied to the determination of S100ß in human serum samples. Graphical abstract Schematic presentation of an immunosensor which is based on an indium tin oxide modified with reduced graphene oxide decorated with gold nanocomposite and antibody. The immunosensor was applied for the determination of S100ß biomarker by using in the labeled antibody.

Aptamer-Based Nanoporous Anodic Alumina Interferometric Biosensor for Real-Time Thrombin Detection.

Aptamer biosensors are one of the most powerful techniques in biosensing. Achieving the best platform to use in aptamer biosensors typically includes crucial chemical modifications that enable aptamer immobilization on the surface in the most efficient manner. These chemical modifications must be well defined. In this work we propose nanoporous anodic alumina (NAA) chemically modified with streptavidin as a platform for aptamer immobilization. The immobilization of biotinylated thrombin binding aptamer (TBA) was monitored in real time by means of reflective interferometric spectroscopy (RIfS). The study has permitted to characterize in real time the path to immobilize TBA on the inner pore walls of NAA. Furthermore, this study provides an accurate label-free method to detect thrombin in real-time with high affinity and specificity.

Optical Monitoring of the Capillary Filling Dynamics Variation in Nanoporous Anodic Alumina toward Sensing Applications.

Fluid imbibition-coupled laser interferometry (FICLI) is a technique in which the kinetics of a fluid infiltrating a nanoporous anodic alumina (NAA) membrane is monitored by the interference of a laser beam at the membrane top and bottom surfaces. Further processing of the measured data results in an estimate of the pore radius. In this work, we study the accuracy of FICLI in the detection of small changes in pore radius, and we evaluate the possibility of using such detection as a sensing paradigm. The accuracy is estimated by measuring samples with increasing pore radius, obtained by successive wet etching steps, and repeatability is evaluated by using different liquids. For decreasing pore radius, samples obtained by the successive deposition of polyelectrolyte double layers are used. With the aim of evaluating the possibility of the FICLI method to sense biological binding events, BSA attachment detection is demonstrated by applying FICLI to samples before and after immobilization of the protein. Results show that the technique permits an accurate estimation of the pore radius, the pore-etching rate (with a radius variation of retch,DI = 1.05 nm/min ± 0.11 nm/min), and the polyelectrolyte double layer thickness (with a radius variation of rPAH/PSS = 3.2 nm ± 0.2 nm per polyelectrolyte double layer). Furthermore, the pore radius reduction measured after BSA immobilization (dBSA = 4.9 nm ± 1.1 nm) is in good agreement with the protein size, as reported in the literature. With these results, we provide a sound basis for the applicability of FICLI as a sensitive technique for the characterization of NAA pore radius modifications.

Silica Nanopills for Targeted Anticancer Drug Delivery.

Multifunctional SiO2 microtubes for targeted drug delivery are produced with precise control over shape and size by combining lithography and electrochemical etching. The hollow core is loaded with a lipophilic anticancer drug generating nanopills and an antibody is conjugated to the external surface for cancer cell targeting. Results demonstrate selective killing of neuroblastoma cells that express the cognate receptor.

Structural and optical nanoengineering of nanoporous anodic alumina rugate filters for real-time and label-free biosensing applications.

In this study, we report about the structural engineering and optical optimization of nanoporous anodic alumina rugate filters (NAA-RFs) for real-time and label-free biosensing applications. Structurally engineered NAA-RFs are combined with reflection spectroscopy (RfS) in order to develop a biosensing system based on the position shift of the characteristic peak in the reflection spectrum of NAA-RFs (Δλpeak). This system is optimized and assessed by measuring shifts in the characteristic peak position produced by small changes in the effective medium (i.e., refractive index). To this end, NAA-RFs are filled with different solutions of d-glucose, and the Δλpeak is measured in real time by RfS. These results are validated by a theoretical model (i.e., the Looyenga-Landau-Lifshitz model), demonstrating that the control over the nanoporous structure makes it possible to optimize optical signals in RfS for sensing purposes. The linear range of these optical sensors ranges from 0.01 to 1.00 M, with a low detection limit of 0.01 M of d-glucose (i.e., 1.80 ppm), a sensitivity of 4.93 nm M(-1) (i.e., 164 nm per refractive index units), and a linearity of 0.998. This proof-of-concept study demonstrates that the proposed system combining NAA-RFs with RfS has outstanding capabilities to develop ultrasensitive, portable, and cost-competitive optical sensors.

CPE-Na-Based Hole Transport Layers for Improving the Stability in Nonfullerene Organic Solar Cells: A Comprehensive Study.

Organic photovoltaic (OPV) cells have experienced significant development in the last decades after the introduction of nonfullerene acceptor molecules with top power conversion efficiencies reported over 19% and considerable versatility, for example, with application in transparent/semitransparent and flexible photovoltaics. Yet, the optimization of the operational stability continues to be a challenge. This study presents a comprehensive investigation of the use of a conjugated polyelectrolyte polymer (CPE-Na) as a hole layer (HTL) to improve the performance and longevity of OPV cells. Two different fabrication approaches were adopted: integrating CPE-Na with PEDOT:PSS to create a composite HTL and using CPE-Na as a stand-alone bilayer deposited beneath PEDOT:PSS on the ITO substrate. These configurations were compared against a reference device employing PEDOT:PSS alone, as the HTL increased efficiency and fill factor. The instruments with CPE-Na also demonstrated increased stability in the dark and under simulated operational conditions. Device-based PEDOT:PSS as an HTL reached T80 after 2500 h while involving CPE-Na in the device kept at T90 in the same period, evidenced by a reduced degradation rate. Furthermore, the impedance spectroscopy and photoinduced transient methods suggest optimized charge transfer and reduced charge carrier recombination. These findings collectively highlight the potential of CPE-Na as a HTL optimizer material for nonfluorine OPV cells.

Tailoring Tamm Plasmon Resonances in Dielectric Nanoporous Photonic Crystals.

The fields of plasmonics and photonic crystals (PCs) have been combined to generate model light-confining Tamm plasmon (TMM) cavities. This approach effectively overcomes the intrinsic limit of diffraction faced by dielectric cavities and mitigates losses associated with the inherent properties of plasmonic materials. In this study, nanoporous anodic alumina PCs, produced by two-step sinusoidal pulse anodization, are used as a model dielectric platform to establish the methodology for tailoring light confinement through TMM resonances. These model dielectric mirrors feature highly organized nanopores and narrow bandwidth photonic stopbands (PSBs) across different positions of the spectrum. Different types of metallic films (gold, silver, and aluminum) were coated on the top of these model dielectric mirrors. By structuring the features of the plasmonic and photonic components of these hybrid structures, the characteristics of TMM resonances were studied to elucidate effective approaches to optimize the light-confining capability of this hybrid TMM model system. Our findings indicate that the coupling of photonic and plasmonic modes is maximized when the PSB of the model dielectric mirror is broad and located within the midvisible region. It was also found that thicker metal films enhance the quality of the confined light. Gas sensing experiments were performed on optimized TMM systems, and their sensitivity was assessed in real time to demonstrate their applicability. Ag films provide superior performance in achieving the highest sensitivity (S = 0.038 ± 0.001 nm ppm-1) based on specific binding interactions between thiol-containing molecules and metal films.

The Spanish Pancreatic Club recommendations for the diagnosis and treatment of chronic pancreatitis: part 1 (diagnosis).

Chronic pancreatitis (CP) is a relatively uncommon, complex and heterogeneous disease. The absence of a gold standard applicable to the initial phases of CP makes its early diagnosis difficult. Some of its complications, particularly chronic pain, can be difficult to manage. There is much variability in the diagnosis and treatment of CP and its complications amongst centers and professionals. The Spanish Pancreatic Club has developed a consensus on the management of CP. Two coordinators chose a multidisciplinary panel of 24 experts on this disease. A list of questions was drafted, and two experts reviewed each question. Then, a draft was produced and shared with the entire panel of experts and discussed in a face-to-face meeting. This first part of the consensus addresses the diagnosis of CP and its complications.

The Spanish Pancreatic Club's recommendations for the diagnosis and treatment of chronic pancreatitis: part 2 (treatment).

Chronic pancreatitis (CP) is a complex disease with a wide range of clinical manifestations. This range comprises from asymptomatic patients to patients with disabling symptoms or complications. The management of CP is frequently different between geographic areas and even medical centers. This is due to the paucity of high quality studies and clinical practice guidelines regarding its diagnosis and treatment. The aim of the Spanish Pancreatic Club was to give current evidence-based recommendations for the management of CP. Two coordinators chose a multidisciplinary panel of 24 experts on this disease. These experts were selected according to clinical and research experience in CP. A list of questions was made and two experts reviewed each question. A draft was later produced and discussed with the entire panel of experts in a face-to-face meeting. The level of evidence was based on the ratings given by the Oxford Centre for Evidence-Based Medicine. In the second part of the consensus, recommendations were given regarding the management of pain, pseudocysts, duodenal and biliary stenosis, pancreatic fistula and ascites, left portal hypertension, diabetes mellitus, exocrine pancreatic insufficiency, and nutritional support in CP.

Optofluidic characterization of nanoporous membranes.

An optofluidic method that accurately identifies the internal geometry of nanochannel arrays is presented. It is based on the dynamics of capillary-driven fluid imbibition, which is followed by laser interferometry. Conical nanochannel arrays in anodized alumina are investigated, which present an asymmetry of the filling times measured from different sides of the membrane. It is demonstrated by theory and experiments that the capillary filling asymmetry only depends on the ratio H of the inlet to outlet pore radii and that the ratio of filling times vary closely as H(7/3). Besides, the capillary filling of conical channels exhibits striking results in comparison to the corresponding cylindrical channels. Apart from these novel results in nanoscale fluid dynamics, the whole method discussed here serves as a characterization technique for nanoporous membranes.

Generation of Tamm Plasmon Resonances for Light Confinement Applications in Narrowband Gradient-Index Filters Based on Nanoporous Anodic Alumina.

Gold-coated gradient-index filters based on nanoporous anodic alumina (Au-coated NAA-GIFs) were used as model platforms to elucidate how Tamm plasmons can be tailored by engineering the geometric features of the plasmonic and photonic components of these hybrid structures. NAA-GIFs with well-resolved, intense photonic stopbands at two positions of the visible spectrum were fabricated through sinusoidal pulse anodization. These model photonic crystals were used to assess how the quality of Tamm plasmon resonances can be enhanced by tuning the features of the dielectric mirror and the thickness of the porous gold coating layer. It is found that the highest value of the quality factor of Tamm resonance (Q Tamm = 237) is obtained for 11 nm of gold on a dielectric mirror with low porosity corresponding to the resonant spectral position of λTamm of ∼698 nm. Our analysis indicates that Tamm resonances in as-produced Au-coated NAA-GIFs are weak due to the constrained range of wavelengths (narrow bands) at which these photonic crystal structures reflect light. However, after broadening of their photonic stopband upon pore widening, Tamm resonances become better resolved, with higher intensity. It is also observed that the quality of light confinement worsens progressively with the thickness of the porous gold coating layer after a critical value. In contrast to conventional surface plasmon resonance systems, this hybrid Tamm porous system does not require complex coupling systems and provides a nanoporous structure that can be readily tailored for a range of photonic technologies such as sensing and lasing.

Allogenic Stem Cells Carried by Porous Silicon Scaffolds for Active Bone Regeneration In Vivo.

To date, bone regeneration techniques use many biomaterials for bone grafting with limited efficiencies. For this purpose, tissue engineering combining biomaterials and stem cells is an important avenue of development to improve bone regeneration. Among potentially usable non-toxic and bioresorbable scaffolds, porous silicon (pSi) is an interesting biomaterial for bone engineering. The possibility of modifying its surface can allow a better cellular adhesion as well as a control of its rate of resorption. Moreover, release of silicic acid upon resorption of its nanostructure has been previously proved to enhance stem cell osteodifferentiation by inducing calcium phosphate formation. In the present study, we used a rat tail model to experiment bone tissue engineering with a critical size defect. Two groups with five rats per group of male Wistar rats were used. In each rat, four vertebrae were used for biomaterial implantation. Randomized bone defects were filled with pSi particles alone or pSi particles carrying dental pulp stem cells (DPSC). Regeneration was evaluated in comparison to empty defect and defects filled with xenogenic bone substitute (Bio-Oss®). Fluorescence microscopy and SEM evaluations showed adhesion of DPSCs on pSi particles with cells exhibiting distribution throughout the biomaterial. Histological analyzes revealed the formation of a collagen network when the defects were filled with pSi, unlike the positive control using Bio-Oss®. Overall bone formation was objectivated with µCT analysis and showed a higher bone mineral density with pSi particles combining DPSC. Immunohistochemical assays confirmed the increased expression of bone markers (osteocalcin) when pSi particles carried DPSC. Surprisingly, no grafted cells remained in the regenerated area after one month of healing, even though the grafting of DPSC clearly increased bone regeneration for both bone marker expression and overall bone formation objectivated with µCT. In conclusion, our results show that the association of pSi with DPSCs in vivo leads to greater bone formation, compared to a pSi graft without DPSCs. Our results highlight the paracrine role of grafted stem cells by recruitment and stimulation of endogenous cells.

Ionic Field Screening in MAPbBr3 Crystals Revealed from Remnant Sensitivity in X-ray Detection.

Research on metal halide perovskites as absorbers for X-ray detection is an attractive subject due to the optimal optoelectronic properties of these materials for high-sensitivity applications. However, the contact degradation and the long-term instability of the current limit the performance of the devices, in close causality with the dual electronic-ionic conductivity of these perovskites. Herein, millimeter-thick methylammonium-lead bromide (MAPbBr3) single and polycrystalline samples are approached by characterizing their long-term dark current and photocurrent under X-ray incidence. It is shown how both the dark current and the sensitivity of the detectors follow similar trends at short-circuit (V = 0 V) after biasing. By performing drift-diffusion numerical simulations, it is revealed how large ionic-related built-in fields not only produce relaxations to equilibrium lasting up to tens of hours but also continue to affect the charge kinetics under homogeneous low photogeneration rates. Furthermore, a method is suggested for estimating the ionic mobility and concentration by analyzing the initial current at short-circuit and the characteristic diffusion times.

Significant Stability Improvement of Fullerene Organic Photovoltaics via ZnO Film Modification through the Intermittent Spray Pyrolysis Technique.

Morphological control of the layers within the bulk heterojunction organic photovoltaics (BHJ-OPVs) is a key feature that governs their performance. In the present work, we demonstrate that zinc oxide-ZnO-interlayers sprayed via the intermittent spray pyrolysis technique, employing a low-concentration precursor solution, can yield inverted BHJ-OPVs as efficient as the standard reported ones using the conventional laboratory-scale spin-coating technique. However, we record a pioneer stability behavior of the fabricated inverted fullerene organic photovoltaics (iF-OPVs) with various sprayed ZnO conditions. Thus, after optimizing the sprayed ZnO interfacial layer morphology for the inverted PTB7-Th:PC70BM devices, by carefully inspecting the interdependence between the sprayed ZnO thin film morphology and the figures of merit of the optimized iF-OPVs, we conducted a distinct analysis on the optical and electronic properties of the fresh and degraded devices using external quantum efficiency measurements and impedance spectroscopy. Hence, we showed that the most proper ZnO microstructural morphology was obtained by spraying 25 running cycles (25R). Remarkably, we observed that 25R-ZnO-based iF-OPV devices showed a stunning stability behavior and maintained 85% of their initial power conversion efficiency even after 16.7 months without encapsulation in a dry nitrogen glovebox, demonstrating an excellent shelf stability. Accordingly, this approach might facilitate the scalability of inverted OPVs for industrial production visibility.

Prediction of peritoneal soiling in acute appendicitis with simple clinical and laboratory data. Prospective, multicenter, cohort study of 2,645 adult patients nationwide.

BACKGROUND AND AIMS: In order to facilitate the preoperative prediction of complicated appendicitis, we propose a complementary approach by selecting an endpoint defined by the intraoperative finding of peritoneal soiling (PS). METHODS: Over a 6-month period, 38 centers (5% of all public hospitals) attending emergency general surgery patients on a 24-h, 7-days a week basis, enrolled consecutive adult patients requiring appendectomy. Patients were stratified according to the absence or the finding of PS during the surgical procedure. RESULTS: A total of 2645 patients were included; median age (IQR) was 35 (22-51) years, 44.3% were female. The laparoscopic approach was used in 70.8% of appendectomies. In a third of patients (31.7%), there was PS with pus around the appendix, or bowel contents, free pus, or blood in the peritoneal cavity. To develop the prediction model, 1764 patients were randomly selected for the derivation cohort and the remaining 881 patients were assigned to the validation cohort. On multivariable logistic regression analysis of all patients, two clinical variables (age, and pulse) and three laboratory variables (serum urea, serum sodium, and white blood cell count) were individually associated (P < .05) with a greater probability of having PS (Hosmer-Lemeshow chi, 1.63; P = .99; C-statistic, 0.7). Based on the multivariable regression model, both static and dynamic nomograms were developed for the prediction of PS in patients with acute appendicitis. CONCLUSIONS: The entry of simple clinical and laboratory variables in the dynamic nomogram may be useful in guiding the initial management of patients with acute appendicitis in resource-limited settings.

Memory T Cell Subpopulations as Early Predictors of Remission to Vedolizumab in Ulcerative Colitis.

Background: Vedolizumab is a humanized monoclonal antibody targeting the α4ß7 integrin used for the treatment of ulcerative colitis. Few biomarkers related to vedolizumab response have been identified. The aim of this work was to assess whether baseline circulating CD4+ and CD8+ memory T-lymphocyte subpopulations could help to identify patients with response to vedolizumab treatment in ulcerative colitis. Methods: Prospective pilot study in 15 patients with active ulcerative colitis and previous failure to anti-TNFα starting vedolizumab treatment. Peripheral blood samples were obtained before the first dose of vedolizumab and at week 6 and 14 of treatment. Clinical remission was defined as a Mayo Clinic partial score of ≤2 points without any concomitant dose of steroids. Biochemical remission or endoscopic improvement was defined as fecal calprotectin <250 mcg/g or Mayo endoscopic subscore ≤1. Results: At week 14, nine patients achieved clinical remission and eight patients achieved biochemical remission or endoscopic improvement. Patients in clinical remission presented higher baseline CD8 α4ß7 + memory T cells concentration when compared with patients with no remission. In addition, patients with biochemical remission or endoscopic improvement at week 14 presented higher baseline concentration of CD8 α4ß7 + memory T cells. No differences were identified according to flare severity, extent of disease or type of anti-TNFα failure. There were no significant differences regarding changes in T cell subsets during vedolizumab induction. Conclusion: CD8+ α4ß7 + memory T cells before starting vedolizumab therapy could be an early predictor of remission in ulcerative colitis patients and therefore help to select a subset of responders.