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Background The combination of sodium bisulfite treatment with highly-parallel sequencing is a common method for quantifying DNA methylation across the genome. The power to detect between-group differences in DNA methylation using bisulfite-sequencing approaches is influenced by both experimental (e.g. read depth, missing data and sample size) and biological (e.g. mean level of DNA methylation and difference between groups) parameters. There is, however, no consensus about the optimal thresholds for filtering bisulfite sequencing data with implications for the reproducibility of findings in epigenetic epidemiology. Results We used a large reduced representation bisulfite sequencing (RRBS) dataset to assess the distribution of read depth across DNA methylation sites and the extent of missing data. To investigate how various study variables influence power to identify DNA methylation differences between groups, we developed a framework for simulating bisulfite sequencing data. As expected, sequencing read depth, group size, and the magnitude of DNA methylation difference between groups all impacted upon statistical power. The influence on power was not dependent on one specific parameter, but reflected the combination of study-specific variables. As a resource to the community, we have developed a tool, POWEREDBiSeq, which utilizes our simulation framework to predict study-specific power for the identification of DNAm differences between groups, taking into account user-defined read depth filtering parameters and the minimum sample size per group. Conclusions Our data-driven approach highlights the importance of filtering bisulfite-sequencing data by minimum read depth and illustrates how the choice of threshold is influenced by the specific study design and the expected differences between groups being compared. The POWEREDBiSeq tool, which can be applied to different types of bisulfite sequencing data (e.g. RRBS, whole genome bisulfite sequencing (WGBS), targeted bisulfite sequencing and amplicon-based bisulfite sequencing), can help users identify the level of data filtering needed to optimize power and aims to improve the reproducibility of bisulfite sequencing studies.

For the fatigue life of thin-walled components, not only fatigue crack initiation, but also crack growth is decisive. The phase-field method for fracture is a powerful tool to simulate arbitrary crack phenomena. Recently, it has been applied to fatigue fracture. Those models pose an alternative to classical fracture-mechanical approaches for fatigue life estimation. In the first part of this paper, the parameters of a phase-field fatigue model are calibrated and its predictions are compared to results of fatigue crack growth experiments of aluminium sheet material. In the second part, compressive residual stresses are introduced into the components with the help of laser shock peening. It is shown that those residual stresses influence the crack growth rate by retarding and accelerating the crack. In order to study these fatigue mechanisms numerically, a simple strategy to incorporate residual stresses in the phase-field fatigue model is presented and tested with experiments. The study shows that the approach can reproduce the effects of the residual stresses on the crack growth rate.

Fatty acid synthase (FASN) is the only human lipogenic enzyme available for de novo fatty acid synthesis and is often highly expressed in cancer cells. We found that FASN mRNA levels were significantly higher in acute myeloid leukemia (AML) patients than in healthy granulocytes or CD34+ hematopoietic progenitors. Accordingly, FASN levels decreased during all-trans retinoic acid (ATRA)-mediated granulocytic differentiation of acute promyelocytic leukemia (APL) cells, partially via autophagic degradation. Furthermore, our data suggest that inhibition of FASN expression levels using RNAi or (-)-epigallocatechin-3-gallate (EGCG) accelerated the differentiation of APL cell lines and significantly re-sensitized ATRA refractory non-APL AML cells. FASN reduction promoted translocation of transcription factor EB (TFEB) to the nucleus, paralleled by activation of CLEAR network genes and lysosomal biogenesis. Together, our data demonstrate that inhibition of FASN expression in combination with ATRA treatment facilitates granulocytic differentiation of APL cells and may extend differentiation therapy to non-APL AML cells.

We developed a new and injectable poly-dicalcium phosphate dihydrate (P-DCPD) forming cement. The key structural difference between P-DCPD and classical DCPD is that P-DCPD is composed of interconnected P-DCPD crystals by interlocking to the polyphosphate chains. In contrast, DCPD is composed of a package of DCPD crystals with weak mutual ionic bonding. The purpose of this continuing study was to compare the physicochemical properties between P-DCPD and DCPD cement particles. Data collected from SEM, X-ray diffraction, and Raman Spectroscopy approaches demonstrated that P-DCPD has a more stable chemical structure than DCPD as evidenced by much less transformation to hydroxyapatite (HA) during setting. Nanoindentation showed a similar hardness while the elastic modulus of P-DCPD is much lower than DCPD that might be due to the much less HA transformation of P-DCPD. P-DCPD has much lower zeta potential and less hydrophilicity than DCPD because of its entangled and interconnected polyphosphate chains. It is expected that superhydrophilic DCPD undergoes faster dissolution than P-DCPD in an aqueous environment. Another interesting finding is that the pH of eluent from P-DCPD is more neutral (6.6-7.1) than DCPD (5.5-6.5). More extensive experiments are currently underway to further evaluate the potential impacts of the different physiochemical performance observed of P-DCPD and DCPD cement particles on the biocompatibility, degradation behavior and bone defect healing efficacy both in vivo and in vitro.

BACKGROUND: Short bowel syndrome resulting from small bowel resection (SBR) is associated with significant morbidity and mortality. Many adverse sequelae including steatohepatitis and bacterial overgrowth are thought to be related to increased bacterial translocation, suggesting alterations in gut permeability. We hypothesized that after intestinal resection, the intestinal barrier is altered via toll-like receptor 4 (TLR4) signaling at the intestinal level. METHODS: B6 and intestinal-specific TLR4 knockout (iTLR4 KO) mice underwent 50% SBR or sham operation. Transcellular permeability was evaluated by measuring goblet cell associated antigen passages via two-photon microscopy. Fluorimetry and electron microscopy evaluation of tight junctions (TJ) were used to assess paracellular permeability. In parallel experiments, single-cell RNA sequencing measured expression of intestinal integral TJ proteins. Western blot and immunohistochemistry confirmed the results of the single-cell RNA sequencing. RESULTS: There were similar number of goblet cell associated antigen passages after both SBR and sham operation (4.5 versus 5.0, P > 0.05). Fluorescein isothiocyanate–dextran uptake into the serum after massive SBR was significantly increased compared with sham mice (2.13 ± 0.39 ng/μL versus 1.62 ± 0.23 ng/μL, P < 0.001). SBR mice demonstrated obscured TJ complexes on electron microscopy. Single-cell RNA sequencing revealed a decrease in TJ protein occludin (21%) after SBR (P < 0.05), confirmed with immunostaining and western blot analysis. The KO of iTLR4 mitigated the alterations in permeability after SBR. CONCLUSIONS: Permeability after SBR is increased via changes at the paracellular level. However, these alterations were prevented in iTLR4 mice. These findings suggest potential Intestinal barrier protein targets for restoring the intestinal barrier and obviating the adverse sequelae of short bowel syndrome.

Over the past decade, genomic and transcriptomic analyses have uncovered promising tumor antigens including immunotherapeutic targets in bladder cancer (BCa). Conventional tumor antigens are proteins expressed on the plasma membrane of tumor cells such as EGFR, FGFR3, and ERBB2 in BCa, which can be targeted by antibodies or similar epitope-specific binding reagents. The cellular proteome consists of ∼100,000 proteins but the expression of these proteins is rarely unique to tumor cells. Many tumor-associated proteins are post-translationally modified with phosphorylation, glycosylation, ubiquitination, or SUMOylation moieties. Although these modifications expand the complexity, they potentially offer novel targeting opportunities across tumor sub-populations. Experimental targeting of cancer-specific post-translational modifications (PTMs) has shown encouraging results in pre-clinical models of BCa, which could potentially overcome issues with inherent intra-tumor heterogeneity due to simultaneous expression on different proteins. Here, we review current knowledge on post-translational modifications in BCa and highlight recent efforts in experimental targeting strategies.

Environmental science and pollution research : ESPR (2020). doi:10.1007/s11356-020-11729-5, Published by Springer, Berlin

The AAA-ATPase VCP/p97 cooperates with the SEP-domain adapters p37, UBXN2A and p47 in stripping inhibitor-3 (I3) from protein phosphatase-1 (PP1) for activation. In contrast to p97-mediated degradative processes, PP1 complex disassembly is ubiquitin-independent. It is therefore unclear how selective targeting is achieved. Using biochemical reconstitution and crosslink mass spectrometry, we show here that SEP-domain adapters use a multivalent substrate recognition strategy. An N-terminal sequence element predicted to form a helix, together with the SEP-domain, binds and engages the direct target I3 in the central pore of p97 for unfolding, while its partner PP1 is held by a linker between SHP box and UBX domain locked onto the peripheral N-domain of p97. Although the I3-binding element is functional in p47, p47 in vitro requires a transplant of the PP1-binding linker from p37 for activity stressing that both sites are essential to control specificity. Of note, unfolding is then governed by an inhibitory segment in the N-terminal region of p47, suggesting a regulatory function. Together, this study reveals how p97 adapters engage a protein complex for ubiquitin-independent disassembly while ensuring selectivity for one subunit.

Nanofibrillated cellulose (NFC) extracted from biomass has potential applications in material science and biomedical engineering. In this study, NFC was obtained from bleached eucalyptus Kraft pulp (BEKP) using two commercial enzyme cocktails with cellulolytic and hemicellulolytic activities and non-catalytic protein (swollenin), followed by ultrasonication. This work represents an initial study of the implementation of non-catalytic proteins along with enzymes to extract NFC from biomass. Enzymatic pretreatment was performed to partially remove hemicellulose while enhancing cellulose accessibility for NFC extraction. Cellulase pretreatment with xylanase and swollenin supplementation increased cellulose accessibility and fiber swelling due to extensive hemicellulose removal (> 80%) and fiber morphology changes. Subsequent ultrasonication was performed for cellulose nanofibrillation resulting in high NFC yields (61–97%), while keeping NFC properties almost unchanged. Through this process, cellulose nanofibers with diameters ranging from 3 nm to 10 nm were effectively isolated from BEKP, which allows to produce high quality NFC for further applications.

This study aims at quantifying the steric shielding effect of multivalent glycoconjugates targeting pathogens by blocking their carbohydrate binding sites. Specifically, PEGylated and non-PEGylated glycoconjugates are studied as inhibitors of lectins and bacterial adhesins evaluating the steric repulsion effect of the nonbinding PEG chains. We use the soft colloidal probe (SCP) adhesion assay to monitor the change in the adhesion energy of mannose (Man)-decorated hydrogel particles on a layer of concanavalin A (ConA) in the presence of sequence-defined multivalent glycoconjugate inhibitors over time. The results show that PEGylated glycoconjugates achieve a stronger adhesion inhibition when compared to non-PEGylated glycoconjugates although the dissociation constants (KD) of the PEGgylated compounds to ConA were larger. These results appear in line with Escherichia coli adhesion inhibition assays showing a small increase of bacteria detachment by PEGgylated glycoconjugates compared to non-PEGylated compounds. This suggests that an increase of sterical shielding via PEGylation may help reduce the invasiveness of pathogens even after they have adhered. Adhesion studies based on electrostatic interactions using amine-linked PEG of varying molecular weight confirm that such sterical shielding effect is not limited to carbohydrate-mediated adhesion.

For improved cost-effectiveness and temperature-stability, a ready to use lateral flow assay (LFA) is developed in this work for detecting inflammation/infection biomarker C-reactive protein (CRP) in human patient samples on the basis of aptamers. In prescreening investigations, an aptamer with CRP affinity was immobilized on microarray chips in forward and sandwich formats to optimize assay conditions. We suggest these microarray techniques as a resource-sparing and fast-screening instrument for evaluation of various conditions. The capability of the aptamer to detect CRP was shown. Optimized assay conditions were consequently transferred to the LFA-platform. Here we could demonstrate for the first time an aptamer-based LFA for the detection of CRP in human patient samples in pathologically relevant concentrations. The cutoff for CRP detection is set at 10 mg/L, providing a distinctive "yes" (≥10 mg/L CRP) or "no" (

Baloxavir marboxil (BXM) was approved in 2018 for treating influenza A and B virus infections. It is a first-in-class inhibitor targeting the endonuclease activity of the virus polymerase acidic (PA) protein. Clinical trial data revealed that PA amino acid substitutions at residue 38 (I38T/F/M) reduced BXM potency and caused virus rebound in treated patients, although the fitness characteristics of the mutant viruses were not fully defined. To determine the fitness impact of the I38T/F/M substitutions, we generated recombinant A/California/04/2009 (H1N1)pdm09, A/Texas/71/2017 (H3N2), and B/Brisbane/60/2008 viruses with I38T/F/M and examined drug susceptibility in vitro, enzymatic properties, replication efficiency, and transmissibility in ferrets. Influenza viruses with I38T/F/M substitutions exhibited reduced baloxavir susceptibility, with 38T causing the greatest reduction. The I38T/F/M substitutions impaired PA endonuclease activity as compared to that of wild-type (I38-WT) PA. However, only 38T/F A(H3N2) substitutions had a negative effect on polymerase complex activity. The 38T/F substitutions decreased replication in cells among all viruses, whereas 38M had minimal impact. Despite variable fitness consequences in vitro, all 38T/M viruses disseminated to naive ferrets by contact and airborne transmission, while 38F-containing A(H3N2) and B viruses failed to transmit via the airborne route. Reversion of 38T/F/M to I38-WT was rare among influenza A viruses in this study, suggesting stable retention of 38T/F/M genotypes during these transmission events. BXM reduced susceptibility-associated mutations had variable effects on in vitro fitness of influenza A and B viruses, but the ability of these viruses to transmit in vivo indicates a risk of their spreading from BXM-treated individuals.

A key aspect of the transformation of the economic sector towards a sustainable bioeconomy is the development of environmentally friendly alternatives for hitherto used chemicals, which have negative impacts on environmental health. However, the implementation of an ecotoxicological hazard assessment at early steps of product development to elaborate the most promising candidates of lowest harm is scarce in industry practice. The present article introduces the interdisciplinary proof-of-concept project GreenToxiConomy, which shows the successful application of a Green Toxicology strategy for biosurfactants and a novel microgel-based pesticide release system. Both groups are promising candidates for industrial and agricultural applications and the ecotoxicological characterization is yet missing important information. An iterative substance- and application-oriented bioassay battery for acute and mechanism-specific toxicity within aquatic and terrestrial model species is introduced for both potentially hazardous materials getting into contact with humans and ending up in the environment. By applying in silico QSAR-based models on genotoxicity, endocrine disruption, skin sensitization and acute toxicity to algae, daphnids and fish individual biosurfactants resulted in deviating toxicity, suggesting a pre-ranking of the compounds. Experimental toxicity assessment will further complement the predicted toxicity to elaborate the most promising candidates in an efficient pre-screening of new substances.

Patients with the complex congenital heart disease (CHD) are usually associated with right ventricular outflow tract dysfunction and typically require multiple surgical interventions during their lives to relieve the right ventricular outflow tract abnormality. Transcatheter pulmonary valve replacement was used as a non-surgical, less invasive alternative treatment for right ventricular outflow tract dysfunction and has been rapidly developing over the past years. Despite the current favorable results of transcatheter pulmonary valve replacement, many patients eligible for pulmonary valve replacement are still not candidates for transcatheter pulmonary valve replacement. Therefore, one of the significant future challenges is to expand transcatheter pulmonary valve replacement to a broader patient population. This review describes the limitations and problems of existing techniques and focuses on decellularized tissue engineering for pulmonary valve stenting.

International audience; Background: Rasagiline, a monoamine oxidase B inhibitor with neuroprotective potential in Parkinson's disease, has shown a disease-modifying effect in the SOD1-Gly93Ala low-expressing mouse model of amyotrophic lateral sclerosis, both alone and in combination with riluzole. We sought to test whether or not rasagiline 1 mg/day can prolong survival in patients with amyotrophic lateral sclerosis also receiving riluzole.Methods: Patients with possible, probable, or definite amyotrophic lateral sclerosis were enrolled to our randomised, placebo-controlled, parallel-group, double-blind, phase 2 trial from 15 German network for motor neuron diseases (MND-NET) centres (university hospitals or clinics). Eligible patients were aged at least 18 years, had onset of progressive weakness within the 36 months before the study, had disease duration of more than 6 months and less than 3 years, and had a best-sitting slow vital capacity of at least 50%. After a 4-week screening period, eligible patients were randomly assigned (1:1) to receive either rasagiline (1 mg/day) or placebo in addition to riluzole (100 mg/day), after stratification for site of onset (bulbar or spinal) and study centre. Patients and all personnel assessing outcome parameters were masked to treatment allocation. Patients were followed up 2, 6, 12, and 18 months after randomisation. The primary endpoint was survival time, defined as the time to death or time to study cutoff date (ie, the last patient's last visit plus 14 days). Analyses of primary outcome and safety measures were done in all patients who received at least one dose of trial treatment (intention-to-treat population). The trial is registered with ClinicalTrials.gov, number NCT01879241.Findings: Between July 2, 2013, and Nov 11, 2014, 273 patients were screened for eligibility, and 252 patients were randomly assigned to receive rasagiline (n=127) or placebo (n=125). 126 patients taking rasagiline and 125 taking placebo were included in the intention-to-treat analysis. For the primary outcome, the survival probability at the end of the study was 0·43 (95% CI 0·25-0·59) in the rasagiline group (n=126) and 0·53 (0·43-0·62) in the placebo group (n=125). The estimated effect size (hazard ratio) was 0·91 (one-sided 97·5% CI -infinity to 1·34; p=0·31). Rasagiline was well tolerated, and most adverse events were due to amyotrophic lateral sclerosis disease progression rather than treatment; the most frequent of these were dysphagia (32 [25%] taking rasagiline vs 24 [19%] taking placebo) and respiratory failure (25 [20%] vs 31 [25%]). Frequency of adverse events were comparable between both groups.Interpretation: Rasagiline was safe in patients with amyotrophic lateral sclerosis. There was no difference between groups in the primary outcome of survival, although post-hoc analysis suggested that rasagiline might modify disease progression in patients with an initial slope of Amyotrophic Lateral Sclerosis Functional Rating Scale Revised greater than 0·5 points per month at baseline. This should be confirmed in another clinical trial.Funding: Teva Pharmaceutical Industries.

PURPOSE: To evaluate corneal stiffening in porcine eyes induced by corneal cross-linking (CXL) using riboflavin dissolved in either aqueous dextran or hydroxypropyl methylcellulose (HPMC) solution. METHODS: Fifty-one porcine corneas were divided into three groups of 17 each. After deepithelialization, the first (Dresden) group was treated for 30 minutes with 0.1% riboflavin (riboflavin-5-monophosphate in 0.9% NaCl) dissolved in hypertonic 20% dextran and the second (HPMC) group for 30 minutes with isotonic solution containing 0.1% riboflavin and 1.1% HPMC. Thereafter, corneas of both groups were irradiated using 5.4 J/cm 2 (irradiance of 9 mW/cm 2 for 10 minutes; 10*9). After CXL, all corneas were kept in an isotonic 16% dextran bath for 2 hours to obtain an equal hydration state. The third group served as the control group. Stress-strain measurements were performed on 5-mm–wide strips. Corneal thickness was monitored throughout the entire course of the experiments. RESULTS: The required stress for a 10% strain was increased by 83% in the Dresden group and 35% in the HPMC group compared to the control group. Resultant Young's modulus (at 10% strain) was 2.53 ± 0.73, 1.87 ± 0.50, and 1.47 ± 0.44 Pa for the Dresden, HPMC, and control groups, respectively. The differences between the Dresden and HPMC groups ( P = .006), the Dresden and control groups ( P < .001), and the HPMC and control groups ( P = .014) were statistically significant. Pachymetry measurements showed a significantly increased corneal thickness after application of HPMC compared with the Dresden group ( P = .002) and control group ( P = .041). CONCLUSIONS: The biomechanical stiffening of the cornea by CXL can be achieved using dextran- and HPMC-based riboflavin solutions in porcine corneas with an application time of 30 minutes. Dextran-based riboflavin solutions seem to induce a slightly stronger biomechanical response in this setting. HPMC solutions induce less thinning than dextran solutions. [ J Refract Surg . 2021;37(9):631–635.]

Retinal degeneration is a leading cause of vision impairment and blindness worldwide and medical care for advanced disease does not exist. Stem cell-derived retinal organoids (RtOgs) became an emerging tool for tissue replacement therapy. However, existing RtOg production methods are highly heterogeneous. Controlled and predictable methodology and tools are needed to standardize RtOg production and maintenance. In this study, we designed a shear stress-free micro-millifluidic bioreactor for nearly labor-free retinal organoid maintenance. We used a stereolithography (SLA) 3D printer to fabricate a mold from which Polydimethylsiloxane (PDMS) was cast. We optimized the chip design using in silico simulations and in vitro evaluation to optimize mass transfer efficiency and concentration uniformity in each culture chamber. We successfully cultured RtOgs at three different differentiation stages (day 41, 88, and 128) on an optimized bioreactor chip for more than 1 month. We used different quantitative and qualitative techniques to fully characterize the RtOgs produced by static dish culture and bioreactor culture methods. By analyzing the results from phase contrast microscopy, single-cell RNA sequencing (scRNA seq), quantitative polymerase chain reaction (qPCR), immunohistology, and electron microscopy, we found that bioreactor-cultured RtOgs developed cell types and morphology comparable to static cultured ones and exhibited similar retinal genes expression levels. We also evaluated the metabolic activity of RtOgs in both groups using fluorescence lifetime imaging (FLIM), and found that the outer surface region of bioreactor cultured RtOgs had a comparable free/bound NADH ratio and overall lower long lifetime species (LLS) ratio than static cultured RtOgs during imaging. To summarize, we validated an automated micro-millifluidic device with significantly reduced shear stress to produce RtOgs of comparable quality to those maintained in conventional static culture.

Transpiration cooling has been theoretically identified as a more efficient thermal protection system, compared to regenerative and film cooling techniques. The availability of porous light-weight high-temperature materials, such as ceramic matrix composites (CMCs), is making transpiration cooling an attractive solution for use in future re-usable rocket thrust chambers for cheaper access to space. In this study, a numerical framework, utilizing the industry standard computational fluid dynamics (CFD) code ANSYS CFX, for the simulation of transpiration cooled rocket thrust chambers is presented. The framework is shown to be suitable for predicting the performance, both in-terms of thermal protection and thrust produced, of thrust chambers that utilise transpiration cooling systems. A liquid oxygen (LOX) and methane (CH 4 ) oxidiser and fuel combination is used, although the method is not limited to these substances. Foreign gas transpiration is modelled by a multi-component model. Different porous liner geometries and reservoir conditions are studied to demonstrate their effect on the performance of the thrust chamber. The numerical framework was developed to design a sub-scale transpiration cooled rocket thrust chamber ground test.

Employing novel 2D materials with topologically protected current-carrying edge states is promising to boost the on-current in electronic devices. Using nanoribbons is essential to reduce the contribution of the 2D bulk states to the current. Making the nanoribbon widths narrower allows one to put more current-carrying edge states under the gate of a fixed width thus boosting the current. However, the edge states start to interact in narrow nanoribbons. Based on an effective k·p model, we analyze the topologically protected edge states and their conductance for several 2D materials as a function of the normal electric field. We compare the 2D materials MoS 2 , MoSe 2 , WS 2 , and WSe 2 in the topological 1T′ phase and find the largest electric field-induced conductance modulation is in MoS 2 nanoribbons.

Recent advancements in medical imaging, virtual surgical planning (VSP), and three-dimensional (3D) printing have potentially changed how today’s craniomaxillofacial surgeons use patient information for customized treatments. Over the years, polyetheretherketone (PEEK) has emerged as the biomaterial of choice to reconstruct craniofacial defects. With advancements in additive manufacturing (AM) systems, prospects for the point-of-care (POC) 3D printing of PEEK patient-specific implants (PSIs) have emerged. Consequently, investigating the clinical reliability of POC-manufactured PEEK implants has become a necessary endeavor. Therefore, this paper aims to provide a quantitative assessment of POC-manufactured, 3D-printed PEEK PSIs for cranial reconstruction through characterization of the geometrical, morphological, and biomechanical aspects of the in-hospital 3D-printed PEEK cranial implants. The study results revealed that the printed customized cranial implants had high dimensional accuracy and repeatability, displaying clinically acceptable morphologic similarity concerning fit and contours continuity. From a biomechanical standpoint, it was noticed that the tested implants had variable peak load values with discrete fracture patterns and failed at a mean (SD) peak load of 798.38 ± 211.45 N. In conclusion, the results of this preclinical study are in line with cranial implant expectations, however, specific attributes have scope for further improvements.

End-stage age-related macular degeneration (AMD) and retinitis pigmentosa (RP) are two major retinal degenerative (RD) conditions that result in irreversible vision loss. Permanent eye damage can also occur in battlefields or due to accidents. This suggests there is an unmet need for developing effective strategies for treating permanent retinal damages. In previous studies, co-grafted sheets of fetal retina with its retinal pigment epithelium (RPE) have demonstrated vision improvement in rat retinal disease models and in patients, but this has not yet been attempted with stem-cell derived tissue. Here we demonstrate a cellular therapy for irreversible retinal eye injuries using a “total retina patch” consisting of retinal photoreceptor progenitor sheets and healthy RPE cells on an artificial Bruch’s membrane (BM). For this, retina organoids (ROs) (cultured in suspension) and polarized RPE sheets (cultured on an ultrathin parylene substrate) were made into a co-graft using bio-adhesives [gelatin, growth factor-reduced matrigel, and medium viscosity (MVG) alginate]. In vivo transplantation experiments were conducted in immunodeficient Royal College of Surgeons (RCS) rats at advanced stages of retinal degeneration. Structural reconstruction of the severely damaged retina was observed based on histological assessments and optical coherence tomography (OCT) imaging. Visual functional assessments were conducted by optokinetic behavioral testing and superior colliculus electrophysiology. Long-term survival of the co-graft in the rat subretinal space and improvement in visual function were observed. Immunohistochemistry showed that co-grafts grew, generated new photoreceptors and developed neuronal processes that were integrated into the host retina. This novel approach can be considered as a new therapy for complete replacement of a degenerated retina.

International audience; The effect of enzymatic modification of polysaccharides on the formed supramolecular structures was studied. Gum Arabic and chitosan were modified by covalent grafting of ferulic acid oxidation products so that they could establish new interactions such as π interactions. The stoichiometry of the interaction was studied by ITC measurements, as well as its strength and related thermodynamic parameters. These experiments were supported by Zeta potential and turbidity measurements and optical macroscopy observations. Grafting of phenolic compounds onto the polymers increased their ability to exhibit π-π stacking and π-cation interactions and led to the formation of various supramolecular structures. For native gum Arabic and chitosan assemblies, the stoichiometry value determined by ITC was consistent with optical microscopy, macroscopic observations and Zeta potential (around a molar ratio gum Arabic:chitosan of 3). The structures formed were coacervates. For the modified polymers, the optimal molar ratio determined by Zeta potential measurements (3.27) differed from the one determined by ITC (6.3). This meant the formation aggregates was not only due to electrostatic interactions but also to other type of interactions (new π-π stacking and π-cation interactions). It was concluded that by modifying the polymers, it was possible to control the supramolecular structures formed

Background: TNBC is associated with higher percentages of pathological complete response (pCR) to neoadjuvant chemotherapy (NAC), and women with a pCR have a favorable prognosis. However, previous studies have found that women with residual disease have a substantially higher risk of recurrence than women with other subtypes of breast cancer. Additionally, other recent studies have found that therapeutic blockade of PD-L1 binding by atezolizumab has resulted in relevant anti-tumor efficacy. Design This is a phase III, double blind, placebo-control trial evaluating neoadjuvant atezolizumab with NAC followed by adjuvant atezolizumab in TNBC. Pts are stratified by region (North America; Europe), tumor size (1.1-3.0cm; >3.0cm), AC/EC schedule (q2w; q3w), nodal status (positive; negative), and PD-L1 status (positive; negative or indeterminate) then randomized 1:1 to receive atezolizumab/placebo 1200 mg IV every 3 wks concurrently with both sequential regimens of weekly paclitaxel 80 mg/m2 IV for 12 doses with every 3-wk carboplatin AUC of 5 IV for 4 doses followed by AC/EC every 2-3 wks (per investigator discretion) for 4 cycles. Following surgery, pts resume atezolizumab/placebo 1200 mg IV every 3 wks as adjuvant therapy for 6 months. Radiotherapy based on local standards is co-administered with atezolizumab/placebo. Eligibility criteria Centrally-confirmed ER-neg, PR-neg, HER2-neg invasive breast cancer by ASCO/CAP guidelines. Primary tumor must be stage T2 or T3 if cN0 or cN1 with negative biopsy or T1c, T2, or T3 if cN1 with positive biopsy or cN2 or cN3. LVEF >55% and no significant cardiac history. Statistical methods Co-primary endpoints are event-free survival (EFS) and pCR breast/nodes. Secondary endpoints include pCR breast, overall survival, distant disease-free survival, safety and toxicity. Trial is an academic collaboration between NSABP and GBG with support from Genentech/Roche. As of 7-8-19, 279/1,520 pts have been randomized. NCT03281954 Support: Genentech/Roche. Citation Format: Charles E Geyer Jr., Sibylle Loibl, Priya Rastogi, Sabine Seiler, Joseph P Costantino, Valentina Nekljudova, Patricia Cortazar, Peter C Lucas, Carsten Denkert, Eleftherios P Mamounas, Christian Jackish, Norman Wolmark. A randomized double-blind phase III clinical trial of neoadjuvant chemotherapy (NAC) with atezolizumab or placebo in patients (pts) with triple negative breast cancer (TNBC) followed by adjuvant atezolizumab or placebo: NSABP B-59/GBG 96-GeparDouze [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr OT2-04-08.

BACKGROUND Obesity is a risk factor for colorectal cancer, yet metabolic distinctions between healthy right and left colon tissue, before cancer is diagnosed, remains largely unknown. This study compared right-ascending and left-descending colon tissue metabolomes to identify differences from the stool metabolome in normal weight, overweight, and obese adults. AIM To examine right and left colon tissue metabolites according to body mass index that may serve as mechanistic targets for interventions and biomarkers for colon cancer risk. METHODS Global, non-targeted metabolomics was applied to assess right-ascending and left-descending colon tissue collected from healthy adults undergoing screening colonoscopies to test the hypothesis that BMI differentially impacts colon tissue metabolite profiles. The colon tissue and stool metabolome of healthy adults (n = 24) was analyzed for metabolite signatures and metabolic pathway networks implicated in progression of colorectal cancer. RESULTS Ascending and descending colon contained 504 host, food, and microbiota-derived metabolites from normal weight, overweight and obese adults grouped according to body mass index. Amino acids, lipids, and nucleotides were among the chemical types that further differentiated from the stool metabolite profiles. Normal weight adults had 46 significantly different metabolites between ascending and descending colon tissue locations, whereas there were 37 metabolite differences in overweight and 28 metabolite differences for obese adults (P < 0.05). Obese adults had trimethylamine N-oxide, endocannabinoids and monoacylglycerols with different relative abundances identified between ascending and descending colon. Primary and secondary bile acids, vitamins, and fatty acids also showed marked relative abundance differences in colon tissue from overweight/obese adults. CONCLUSION There were metabolite profile differences between right-ascending and left-descending colon tissue in healthy adults. Colon lipids and other metabolites in obese and overweight adults were distinguished from normal weight participants and associated with gut inflammation, nutrient absorption, and products of microbiota metabolism.

Retinal organoid technology enables generation of an inexhaustible supply of three-dimensional retinal tissue from human pluripotent stem cells (hPSCs) for regenerative medicine applications. The high similarity of organoid-derived retinal tissue and transplantable human fetal retina provides an opportunity for evaluating and modeling retinal tissue replacement strategies in relevant animal models in the effort to develop a functional retinal patch to restore vision in patients with profound blindness caused by retinal degeneration. Because of the complexity of this very promising approach requiring specialized stem cell and grafting techniques, the tasks of retinal tissue derivation and transplantation are frequently split between geographically distant teams. Delivery of delicate and perishable neural tissue such as retina to the surgical sites requires a reliable shipping protocol and also controlled temperature conditions with damage-reporting mechanisms in place to prevent transplantation of tissue damaged in transit into expensive animal models. We have developed a robust overnight tissue shipping protocol providing reliable temperature control, live monitoring of the shipment conditions and physical location of the package, and damage reporting at the time of delivery. This allows for shipping of viable (transplantation-competent) hPSC-derived retinal tissue over large distances, thus enabling stem cell and surgical teams from different parts of the country to work together and maximize successful engraftment of organoid-derived retinal tissue. Although this protocol was developed for preclinical in vivo studies in animal models, it is potentially translatable for clinical transplantation in the future and will contribute to developing clinical protocols for restoring vision in patients with retinal degeneration.

The application of molded interconnected devices (MID) holds great potential to reduce volume and weight of electronic circuit carriers. The material for laser-based manufacturing usually contents additives inside the matrix, which are required for the activation process. This paper presents an alternative without any additives in the mold. The main focus is the experimental investigation of laser fluence as well as pulse and line distance. The process parameters, that lead to a homogeneous metallization, are described and evaluated in order to define the boundaries of the new laser activation process.

The recently resolved crystal structure of the neuropeptide Y Y1 receptor (Y1R), co-crystallized with the high-affinity (pKi: 10.11), argininamide-type Y1R antagonist UR-MK299 (2), revealed that the Nω-carbamoyl substituent (van der Waals volume: 139 A3) is deeply buried in the receptor, occupying a hydrophobic pocket. We synthesized and characterized a series of argininamides, structurally related to 2. Y1R affinity decreased with increasing size of the carbamoyl residue (minimal pKi: 5.67). Exceeding a critical size of the substituent (van der Waals volume: 212 A3), the ligands bound in an inverted mode with the carbamoyl side chain located at the surface of the receptor, as suggested by induced-fit docking and MD simulations.

Cellulose processing remains a challenge as it is insoluble in water and common organic solvents. Ionic liquids (ILs) are organic salts with a melting point below 100 °C and are known for their excellent solvent properties. Unlike common organic solvents, which can form toxic or flammable vapours due to their high volatility, ILs can be considered as more environmentally friendly due to their negligible vapour pressure and flame retardant properties. We found that N-butyl-N-methylpyrrolidinium hydroxide enables rapid dissolution of up to 20 wt% Avicel® cellulose at 25 °C in aqueous solution (50 wt% water), making it the first pyrrolidinium-based salt capable of dissolving cellulose. Furthermore, solubility studies are currently carried out mainly with the naked eye, microscopy or spectroscopy. The former is a subjective method because it depends on the observer, and particles at the micro-level cannot be seen with the human eye. Microscopic and spectroscopic analyses are suitable for the verification of solubility; however, the acquisition costs of the instruments are high, and sample preparation is time-consuming. We propose that turbidity is a suitable measure for solubility, and investigated a simple and fast method to evaluate cellulose solubility in aqueous N-butyl-N-methylpyrrolidinium hydroxide by employing a turbidimeter which was compared with microscopy and ocular (eye) observation. In this study, we have not only found a promising new solvent for cellulose processing, but also offer a reliable solubility analysis.

In this work, we present spiropyran derivatives (SP) involving a halogen bonded network with the addition of inorganic building blocks, i.e. inorganic acids and bivalent metal salts. In solution, a ring-opening isomerization of the spiropyran results in the colored merocyanine isomer (MC), which is only rarely observed in the solid state. By detaining the phenolate oxygen atom of the merocyanine in a hydrogen (HB) or halogen bond (XB) we can successfully obtain this form and access a variation in chromic properties. The O atom of the open MC form unfortunately only represents a weak XB acceptor which led us to introduce supplementary building blocks to strengthen this type of interaction. Fourteen new crystal forms were determined with 3 spiropyran derivatives (1,3,3-trimethylindolinospiropyran (SPH/MCH), 1,3,3-trimethylindolino-6′-nitrobenzopyrylospiran (SPNO2/MCNO2), 1,3,3-trimethylindolino-β-naphthopyrylospiran (SPBenz/MCBenz)) and several di- and tritopic XB donors (1,3,5-triiodotrifluorobenzene (135tfib), 1,4-diiodotetrafluorobenzene (14tfib), 1,3-diiodotetrafluorobenzene (13tfib), 1,2-diiodotetrafluorobenzene (12tfib), abd 1,2-diiodotetrafluoroethan (12tfe)) as well as a polymorphic form of the parent compound with hydrochloric acid. We successfully expanded the XB interaction possibilities with hydrochloric acid or zinc/cobalt chloride and found proof of the tunability of the chromic properties in the solid state using these building blocks.

UDP-3-O-((R)-3-hydroxymyristoyl)-N-glucosamine deacetylase (LpxC) is as an attractive target for the discovery and development of novel antibacterial drugs to address the critical medical need created by multidrug resistant Gram-negative bacteria. By using a scaffold hopping approach on a known family of methylsulfone hydroxamate LpxC inhibitors, several hit series eliciting potent antibacterial activities against Enterobacteriaceae and Pseudomonas aeruginosa were identified. Subsequent hit-to-lead optimization, using cocrystal structures of inhibitors bound to Pseudomonas aeruginosa LpxC as guides, resulted in the discovery of multiple chemical series based on (i) isoindolin-1-ones, (ii) 4,5-dihydro-6H-thieno[2,3-c]pyrrol-6-ones, and (iii) 1,2-dihydro-3H-pyrrolo[1,2-c]imidazole-3-ones. Synthetic methods, antibacterial activities and relative binding affinities, as well as physicochemical properties that allowed compound prioritization are presented. Finally, in vivo properties of lead molecules which belong to the most promising pyrrolo-imidazolone series, such as 18d, are discussed.

We studied the ability of D-phenyglycinamide to form diastereomeric salts with a series of dicarboxylic acids, i.e., malic, methylsuccinic, camphoric, and a drug naproxen in order to investigate its potential as an agent for chiral resolution. D-phenylglycinamide readily formed salts with the aforementioned compounds – we discovered 21 salts in total. In the case of malic and methylsuccinic acids, besides the salts with the 1:1 stoichiometry, we also found 2:1 salts and a two-step reaction mechanism was suggested, with the 2:1 salts being an intermediate preceding the formation of 1:1 salts. In the case of camphoric acid, only hydrated 1:1 salts were identified. In the Dphenylglicinamide/naproxen system, multiple 1:1 salts were identified, including hydrated D-PGA salts with S- and RS-naproxen. Variable-temperature in situ synchrotron X-ray diffraction was used to study selected samples. In the naproxen system, it gave access to two additional unhydrated polymorphs. The possibility of chiral resolution in solid state was tested via liquid-assisted grinding of D-PGA with the racemic forms of all the compounds: 1:1 combinations of all the salts with dicarboxylic acids yielded 1:1 diastereomeric salts, whereas 2:1 combinations for malic and methylsuccinic acids resulted in the 2:1 salts with the racemate. Unlike, dicarboxylic acids, when DPGA was ground with RS-naproxen it directly led to the salts with racemic naproxen, thereby showing no chiral resolution in solid state. We also report the structure of D-phenylglycinamide itself for the first time. The results of this work showed that D-phenylglycinamide is a viable base for diastereomeric salt formation with carboxylic acids and uncovered some pitfalls that can be encountered when it is used for chiral resolution

The lymphostins are a family of closely related pyrrolo[4,3,2-de]quinoline natural products produced by Streptomyces and Salinispora actinobacteria. Neolymphostin A was recently shown to strongly inhibit phosphoinositide 3-kinase (PI3K) and the mammalian target of rapamycin (mTOR) in a covalent manner via conjugation to a catalytic lysine residue in the ATP-binding pocket of the enzymes, making this metabolite the first reported covalent kinase inhibitor from a bacterium. A flexible and efficient synthetic route toward these alkaloids would allow for improvements in their solubility, stability, and selectivity and help to deliver a viable drug candidate. We have since established a short synthesis to methyl 8-bromo-1,3,4,5-tetrahydropyrrolo[4,3,2-de]quinoline-4-carboxylate via a conjugate addition/intramolecular Ullman reaction sequence. However, attempts to oxidize this intermediate to the pyrrolo[4,3,2-de]quinoline characteristic of the lymphostins resulted in formation of either a 2-oxo-1,2-dihydropyrrolo[4,3,2-de]quinoline or an unusual N,C-linked tetrahydropyrroloquinoline-pyrroloquinoline heterodimer. We expect that key modifications to the tetrahydropyrroloquinoline intermediate prior to oxidation should prevent these side reactions and pave the way for the completion of the synthesis.

Lipopolysaccharide (LPS) is a bacterial endotoxin present in cigarette smoke. LPS is known to induce inflammation and to increase the size and the multiplicity of lung tumors induced by tobacco-specific nitrosamines. However, the means by which LPS contributes to pulmonary carcinogenesis are not known. One possible mechanism includes LPS-mediated epigenetic deregulation, which leads to aberrant expression of genes involved in DNA repair, tumor suppression, cell cycle progression, and cell growth. In the present work, epigenetic effects of LPS were examined in alveolar type II lung cells of A/J mice. Type II cells were selected because they serve as progenitors of lung adenocarcinomas in smoking induced lung cancer. A/J mice were intranasally treated with LPS, followed by isolation of alveolar type II cells from the lung using cell panning. Global levels of DNA methylation and histone acetylation were quantified by mass spectrometry, while genome-wide transcriptomic changes were characterized by RNA-Seq. LPS treatment was associated with epigenetic changes including decreased cytosine formylation and reduced histone H3K14 and H3K23 acetylation, as well as altered expression levels of genes involved in cell adhesion, inflammation, immune response, and epigenetic regulation. These results suggest that exposure to inflammatory agents in cigarette smoke leads to early epigenetic changes in the lung, which may collaborate with genetic changes to drive the development of lung cancer.

Identification of hazardous compounds, as the first step of water protection and regulation, is still challenged by the difficulty to establish a linkage between toxic effects and suspected contaminants. Genotoxic compounds are one type of highly relevant toxicants in surface water, which may attack the DNA and lead to cancer in individual organism, or even damaged germ cells to be passed on to future generations. Thus, the establishment of a linkage between genotoxic effects and genotoxicant is important for environmental toxicologists and chemists. For this purpose, in the present study in silico methods were integrated with bioassays, chemical analysis and literature information to identify genotoxicants in surface water. Large volume water samples from 22 sampling sites of the Danube were collected and subjected to biological and chemical analysis. Samples from the most toxic sites (JDS32, JDS44 and JDS63) induced significant genotoxic effects in the micronucleus assay, and two of them caused mutagenicity in the Ames fluctuation assay. Chemical analysis showed that 68 chemicals were detected in these most toxic samples. Literature findings and in silico techniques using the OECD QSAR Toolbox and the ChemProp software package revealed genotoxic potentials for 29 compounds out of 68 targeted chemicals. To confirm the integrative technical data, the micronucleus assay and the Ames fluctuation assay were applied with artificial mixtures of those compounds and the raw water sample extracts. The results showed that 18 chemicals explained 48.5% of the genotoxicity in the micronucleus assay. This study highlights the capability of in silico techniques in linking adverse biological effect to suspicious hazardous compounds for the identification of toxicity drivers, and demonstrates the genotoxic potential of pollutants in the Danube.

Coherent and incoherent long-range atomic motions accompany singlet exciton fission in pentacene., Singlet exciton fission (SEF) is a key process for developing efficient optoelectronic devices. An aspect rarely probed directly, yet with tremendous impact on SEF properties, is the nuclear structure and dynamics involved in this process. Here, we directly observe the nuclear dynamics accompanying the SEF process in single crystal pentacene using femtosecond electron diffraction. The data reveal coherent atomic motions at 1 THz, incoherent motions, and an anisotropic lattice distortion representing the polaronic character of the triplet excitons. Combining molecular dynamics simulations, time-dependent density-functional theory, and experimental structure factor analysis, the coherent motions are identified as collective sliding motions of the pentacene molecules along their long axis. Such motions modify the excitonic coupling between adjacent molecules. Our findings reveal that long-range motions play a decisive part in the electronic decoupling of the electronically correlated triplet pairs and shed light on why SEF occurs on ultrafast time scales.

Avoiding damage of the endothelial cells, especially in thin corneas, remains a challenge in corneal collagen crosslinking (CXL). Knowledge of the riboflavin gradients and the UV absorption characteristics after topical application of riboflavin in concentrations ranging from 0.1% to 0.5% could optimize the treatment. In this study, we present a model to calculate the UV-intensity depending on the corneal thickness. Ten groups of de-epithelialized porcine corneas were divided into 2 subgroups. Five groups received an imbibition of 10 min and the other five groups for 30 min. The applied riboflavin concentrations were 0.1%, 0.2%, 0.3%, 0.4% and 0.5% diluted in a 15% dextran solution for each subgroup. After the imbibition process, two-photon fluorescence microscopy was used to determine fluorescence intensity, which was compared to samples after saturation, yielding the absolute riboflavin concentration gradient of the cornea. The extinction coefficient of riboflavin solutions was measured using a spectrophotometer. Combining the obtained riboflavin concentrations and the extinction coefficients, a depth-dependent UV-intensity profile was calculated for each group. With increasing corneal depth, the riboflavin concentration decreased for all imbibition solutions and application times. The diffusion coefficients of 10 min imbibition time were higher than for 30 min. A higher RF concentration and a longer imbibition time resulted in higher UV-absorption and a lower UV-intensity in the depth of the cornea. Calculated UV-transmission was 6 percentage points lower compared to the measured transmission. By increasing the riboflavin concentration of the imbibition solution, a substantially higher UV-absorption inside the cornea is achieved. This offers a simple treatment option to control the depth of crosslinking e.g. in thin corneas, resulting in a lower risk of endothelial damage.

Cellulose is the main component of biomass and is the most abundant biopolymer on earth, it is a non-toxic, low-cost material that is biocompatible and biodegradable. Cellulose gels are receiving increasing attention as medical products, e.g., as wound dressings. However, the preparation of cellulose hydrogels employing unmodified cellulose is scarcely reported because of the cumbersome dissolution of cellulose. In previous studies, we developed the new promising cellulose solvent N-butyl-N-methylpyrrolidinium hydroxide in an aqueous solution, which can dissolve up to 20 wt% cellulose within a short time at room temperature. In this study, we employed this solvent system and investigated the gelation behavior of cellulose after crosslinker addition. The swelling behavior in water (swelling ratio, water uptake), the mechanical properties under compression, and the antibacterial activity against Escherichia coli and Bacillus subtilis were investigated. We have developed a simple and fast one-pot method for the preparation of cellulose gels, in which aqueous pyrrolidinium hydroxide solution was acting as the solvent and as an antibacterial reagent. The pyrrolidinium hydroxide content of the gels was controlled by adjustment of the water volume employed for swelling. Simple recovery of the solvent system was also possible, which makes this preparation method environmentally benign.

Aims Total haemoglobin mass (tot-Hb) increases during high-altitude acclimatization. Normalization of tot-Hb upon descent is thought to occur via neocytolysis, the selective destruction of newly formed erythrocytes. Because convincing experimental proof of neocytolysis is lacking, we performed a prospective study on erythrocyte survival after a stay at the Jungfraujoch Research Station (JFJRS; 3450 m). Methods Newly formed erythrocytes of 12 male subjects (mean age 23.3 years) were age cohort labelled in normoxia (110 m) and during a 19-day high-altitude sojourn by ingestion of 13 C2- and 15 N-labelled glycine respectively. Elimination dynamics for erythrocytes produced in normoxia and at high altitude were measured by isotope ratio mass spectrometry of haem, by determining tot-Hb, reticulocyte counts, erythrocyte membrane protein 4.1a/4.1b ratio and by mathematical modelling. Results Tot-Hb increased by 4.7% ± 2.7% at high altitude and returned to pre-altitude values within 11 days after descent. Elimination of 13 C- (normoxia) and 15 N- (high altitude) labelled erythrocytes was not different. Erythropoietin levels and counts of CD71-positive reticulocytes decreased rapidly after descent. The band 4.1a/4.1b ratio decreased at altitude and remained low for 3-4 days after descent and normalized slowly. There was no indication of haemolysis. Conclusion We confirm a rapid normalization of tot-Hb upon descent. Based on the lack of accelerated removal of age cohorts of erythrocytes labelled at high altitude, on patterns of changes in reticulocyte counts and of the band 4.1a/4.1b ratio and on modelling, this decrease did not occur via neocytolysis, but by a reduced rate of erythropoiesis along with normal clearance of senescent erythrocytes.

We present a photonic BiCMOS coherent receiver dedicated to the O-band and achieve detection at up to 56 GBd using quadrature-phase shift-keying (QPSK) at various optical signal-to-noise ratios (OSNRs). Transmission experiments are performed and potential applications within that power budget are discussed.

International audience; Maintaining the integrity of the mitotic spindle in metaphase is essential to ensure normal cell division. We show here that depletion of microtubule-associated protein ATIP3 reduces metaphase spindle length. Mass spectrometry analyses identi-ied the microtubule minus-end depolymerizing kinesin Kif2A as an ATIP3 binding protein. We show that ATIP3 controls metaphase spindle length by interacting with Kif2A and its partner Dda3 in an Aurora kinase A-dependent manner. In the absence of ATIP3, Kif2A and Dda3 accumulate at spindle poles, which is consistent with reduced poleward microtubule lux and shortening of the spindle. ATIP3 silencing also limits Aurora A localization to the poles. Transfection of GFP-Aurora A, but not kinase-dead mutant, rescues the phenotype, indicating that ATIP3 maintains Aurora A activity on the poles to control Kif2A targeting and spindle size. Collectively, these data emphasize the pivotal role of Aurora kinase A and its mutual regulation with ATIP3 in controlling spindle length.

Purpose Photochemical crosslinking of the sclera is an emerging technique that may prevent excessive eye elongation in pathologic myopia by stiffening the scleral tissue. To overcome the challenge of uniform light delivery in an anatomically restricted space, we previously introduced the use of flexible polymer waveguides. We presently demonstrate advanced waveguides that are optimized to deliver light selectively to equatorial sclera in the intact orbit. Methods Our waveguides consist of a polydimethylsiloxane cladding and a polyurethane core, coupled to an optical fiber. A reflective silver coating deposited on the top and side surfaces of the waveguide prevents light leakage to nontarget, periorbital tissue. Postmortem rabbits were used to test the feasibility of in situ equatorial sclera crosslinking. Tensometry measurements were performed on ex vivo rabbit eyes to confirm a biomechanical stiffening effect. Results Metal-coated waveguides enabled efficient light delivery to the entire circumference of the equatorial sclera with minimal light leakage to the periorbital tissues. Blue light was delivered to the intact orbit with a coefficient of variation in intensity of 22%, resulting in a 45 ± 11% bleaching of riboflavin fluorescence. A 2-fold increase in the Young's modulus at 5% strain (increase of 92% P < 0.05, at 25 J/cm2) was achieved for ex vivo crosslinked eyes. Conclusions Flexible polymer waveguides with reflective, biocompatible surfaces are useful for sclera crosslinking to achieve targeted light delivery. We anticipate that our demonstrated procedure will be applicable to sclera crosslinking in live animal models and, potentially, humans in vivo.

The increasing concern over bisphenol A (BPA) has directed much attention toward bisphenol F (BPF) and bisphenol S (BPS) as BPA alternatives for the development of "BPA-free" products. Consequently, BPS and BPF were frequently detected in surface water, sediment, sewage effluent, indoor dust, and even in food and biological fluids in humans. Thus, environmental researches start to focus on the potential environmental risks of BPA alternatives. While the estrogenically active metabolites and the specific estrogenically active structure are still unknown. In this study, the MTT assay on acute cytotoxicity and the recombinant transactivation assay were carried out to determine whether BPF and BPS are suitable alternatives to BPA. Our results show that the cytotoxic and estrogenic activities of BPS and BPF are lower than those of BPA. However, after the addition of a rat liver homogenate to simulate mammal metabolism, BPF exhibited higher estrogenic activity than BPA. To identify the chemical structures and estrogen receptor binding affinities of active estrogenic metabolites, LC-MS, MetaPrint2D(-React), and VirtualToxLab were integrated. The observed results indicated that the para-hydroxylated BPF and BPF-OCH3 might have strong ER binding affinities. These results demonstrate that metabolization is important to consider upon investigating endocrine disruption of chemicals getting into contact with humans, such as in dental sealing or food packaging. Alternatives to potentially hazardous substances should be thoroughly tested prior to use.

A coupled accelerator mass spectrometer–gas interface system has been successfully operating at the Hertelendi Laboratory of Environmental Studies, Debrecen, Hungary, since 2013. Over the last 6 years more than 500 gas targets were measured below 100 µg carbon content for carbon isotopic composition. The system was tested with blanks, OxII, IAEA-C1, IAEA-C2, and IAEA-C7 standards. The performance of our instrumentation shows good agreement with other published gas-interface system data and also shows a quite good agreement with the nominal value of international standard samples. There is a measurable but quite small memory effect after modern samples, but this does not significantly affect the final results. Typical ion currents at the low energy side were between 10–15 µA with a 5% CO2 in He mixing ratio. The relative errors average ±6% for samples greater than or equal to 10 µgC sample with mean count rates of 300 counts per microgram C for OxII. The blank is comparable with other systems, which is 0.0050 ± 0.0018 F14C or 34,000–47,000 yr BP, which allows for the routine measurement of both of small environmental and archeological samples.

Ultrasmall gold nanoparticles (diameter about 2 nm) were surface‐functionalized with cysteine‐carrying precision macromolecules. These consisted of sequence‐defined oligo(amidoamine)s (OAAs) with either two or six cysteine molecules for binding to the gold surface and either with or without a PEG chain (3400 Da). They were characterized by 1H NMR spectroscopy, 1H NMR diffusion‐ordered spectroscopy (DOSY), small‐angle X‐ray scattering (SAXS), and high‐resolution transmission electron microscopy. The number of precision macromolecules per nanoparticle was determined after fluorescent labeling by UV spectroscopy and also by quantitative 1H NMR spectroscopy. Each nanoparticle carried between 40 and 100 OAA ligands, depending on the number of cysteine units per OAA. The footprint of each ligand was about 0.074 nm2 per cysteine molecule. OAAs are well suited to stabilize ultrasmall gold nanoparticles by selective surface conjugation and can be used to selectively cover their surface. The presence of the PEG chain considerably increased the hydrodynamic diameter of both dissolved macromolecules and macromolecule‐conjugated gold nanoparticles., Ultrasmall gold nanoparticles (diameter about 2 nm) were surface‐functionalized with cysteine‐carrying precision macromolecules. These consisted of sequence‐defined oligo(amidoamine)s (OAAs) with either two or six cysteine molecules for binding to the gold surface and either with or without a PEG chain (3400 Da). The presence of the PEG chain considerably increased the hydrodynamic diameter of both dissolved macromolecules and macromolecule‐conjugated gold nanoparticles.

Nucleic acid and histone modifications critically depend on central metabolism for substrates and co-factors. Although a few enzymes related to the formation of these required metabolites have been reported in the nucleus, the corresponding metabolic pathways are considered to function elsewhere in the cell. Here we show that a substantial part of the mitochondrial tricarboxylic acid (TCA) cycle, the biosynthetic hub of epigenetic modification factors, is operational also in the nucleus. Using13C-tracer analysis, we identified activity of glutamine-to-fumarate, citrate-to-succinate, and glutamine-to-aspartate routes in the nuclei ofHeLacells. Proximity labeling mass-spectrometry revealed a spatial vicinity of the involved enzymes with core nuclear proteins, supporting their nuclear location. We further show nuclear localization of aconitase 2 and 2-oxoglutarate dehydrogenase in mouse embryonic stem cells. Together, our results demonstrate operation of an extended metabolic pathway in the nucleus warranting a revision of the canonical view on metabolic compartmentalization and gene expression regulation.

Mitochondrial respiratory chain disorders are empirically managed with variable antioxidant, cofactor and vitamin ‘cocktails’. However, clinical trial validated and approved compounds, or doses, do not exist for any single or combinatorial mitochondrial disease therapy. Here, we sought to pre-clinically evaluate whether rationally designed mitochondrial medicine combinatorial regimens might synergistically improve survival, health and physiology in translational animal models of respiratory chain complex I disease. Having previously demonstrated that gas-1(fc21) complex I subunit ndufs2−/− C. elegans have short lifespan that can be significantly rescued with 17 different metabolic modifiers, signaling modifiers or antioxidants, here we evaluated 11 random combinations of these three treatment classes on gas-1(fc21) lifespan. Synergistic rescue occurred only with glucose, nicotinic acid and N-acetylcysteine (Glu + NA + NAC), yielding improved mitochondrial membrane potential that reflects integrated respiratory chain function, without exacerbating oxidative stress, and while reducing mitochondrial stress (UPRmt) and improving intermediary metabolic disruptions at the levels of the transcriptome, steady-state metabolites and intermediary metabolic flux. Equimolar Glu + NA + NAC dosing in a zebrafish vertebrate model of rotenone-based complex I inhibition synergistically rescued larval activity, brain death, lactate, ATP and glutathione levels. Overall, these data provide objective preclinical evidence in two evolutionary-divergent animal models of mitochondrial complex I disease to demonstrate that combinatorial Glu + NA + NAC therapy significantly improved animal resiliency, even in the face of stressors that cause severe metabolic deficiency, thereby preventing acute neurologic and biochemical decompensation. Clinical trials are warranted to evaluate the efficacy of this lead combinatorial therapy regimen to improve resiliency and health outcomes in human subjects with mitochondrial disease.

BackgroundShort bowel syndrome (SBS) results from significant loss of small intestinal length. In response to this loss, adaptation occurs, with Epidermal Growth Factor Receptor (EGFR) being a key driver. Besides enhanced enterocyte proliferation, we have revealed that adaptation is associated with angiogenesis. Further, we have found that small bowel resection (SBR) is associated with diminished oxygen delivery and elevated levels of hypoxia-inducible factor 1-alpha (HIF1α).MethodsWe ablated EGFR in the epithelium and endothelium as well as HIF1α in the epithelium, ostensibly the most hypoxic element. Using these mice, we determined the effects of these genetic manipulations on intestinal blood flow after SBR using photoacoustic microscopy (PAM), intestinal adaptation and angiogenic responses. Then, given that endothelial cells require a stromal support cell for efficient vascularization, we ablated EGFR expression in intestinal subepithelial myofibroblasts (ISEMFs) to determine its effects on angiogenesis in a microfluidic model of human small intestine.ResultsDespite immediate increased demand in oxygen extraction fraction measured by PAM in all mouse lines, were no differences in enterocyte and endothelial cell EGFR knockouts or enterocyte HIF1α knockouts by POD3. Submucosal capillary density was also unchanged by POD7 in all mouse lines. Additionally, EGFR silencing in ISEMFs did not impact vascular network development in a microfluidic device of human small intestine.ConclusionsOverall, despite the importance of EGFR in facilitating intestinal adaptation after SBR, it had no impact on angiogenesis in three cell types-enterocytes, endothelial cells, and ISEMFs. Epithelial ablation of HIF1α also had no impact on angiogenesis in the setting of SBS.