Platinum-Loaded Cerium Oxide Capable of Repairing Neuronal Homeostasis for Cerebral Ischemia-Reperfusion Injury Therapy.

Effective neuroprotective agents are required to prevent neurological damage caused by reactive oxygen species (ROS) generated by cerebral ischemia-reperfusion injury (CIRI) following an acute ischemic stroke. Herein, it is aimed to develop the neuroprotective agents of cerium oxide loaded with platinum clusters engineered modifications (Ptn-CeO2). The density functional theory calculations show that Ptn-CeO2 could effectively scavenge ROS, including hydroxyl radicals (·OH) and superoxide anions (·O2 -). In addition, Ptn-CeO2 exhibits the superoxide dismutase- and catalase-like enzyme activities, which is capable of scavenging hydrogen peroxide (H2O2). The in vitro studies show that Ptn-CeO2 could adjust the restoration of the mitochondrial metabolism to ROS homeostasis, rebalance cytokines, and feature high biocompatibility. The studies in mice CIRI demonstrate that Ptn-CeO2 could also restore cytokine levels, reduce cysteine aspartate-specific protease (cleaved Caspase 3) levels, and induce the polarization of microglia to M2-type macrophages, thus inhibiting the inflammatory responses. As a result, Ptn-CeO2 inhibits the reperfusion-induced neuronal apoptosis, relieves the infarct volume, reduces the neurological severity score, and improves cognitive function. Overall, these findings suggest that the prominent neuroprotective effect of the engineered Ptn-CeO2 has a significant neuroprotective effect and provides a potential therapeutic alternative for CIRI.

Computed Tomography Imaging Analysis of the MPFL Femoral Footprint Morphology and the Saddle Sulcus: Evaluation of 1094 Knees.

BACKGROUND: The medial patellofemoral ligament (MPFL) has been reported to be anatomically attached from an osseous saddle region (saddle sulcus) between neighboring landmarks on the femur, including the adductor tubercle (AT), medial epicondyle (ME), and medial gastrocnemius tubercle (MGT). However, the position and prevalence of the saddle sulcus remain unknown. PURPOSE: To study the femoral footprint of MPFL and the prevalence of the saddle sulcus with computed tomography (CT) imaging; quantify the position of the saddle sulcus; and determine the relevant factors of the identified position and measuring distances. STUDY DESIGN: Cross-sectional study; Level of evidence, 3. METHODS: A total of 1094 knees in 753 patients were studied. Knees were organized into an anterior cruciate ligament reconstruction (ACLR) group (controls) and a recurrent patellar dislocation (RPD) group. Using 3-dimensionally reconstructed CT images, the authors determined the prevalence of the saddle sulcus and its position relative to the AT, the ME, the Schöttle point (1.3 mm anterior to the distal posterior cortex and 2.5 mm distal to the posterior origin of the medial femoral condyle), and the Fujino point (approximately 10 mm distal to the AT). Analysis of covariance was used to adjust for age, sex, side, and body mass index on the measurements. RESULTS: There were 555 knees in the control group and 539 knees in the RPD group. The MPFL femoral footprint presented as an oblique, oblong, osseous region (saddle sulcus) in 75.7% of knees (75.0%, ACLR group vs 76.4%, RPD group; P < .001). The saddle sulcus was located a mean of 12.2 mm (95% CI, 12.0-12.4 mm) from a line connecting the apex of the AT to the ME (AT-ME) and a mean of 7.6 mm (95% CI, 7.5-7.8 mm) posteriorly perpendicular to that line. The location as a proportion of the AT-ME distance was 63.1% (95% CI, 62.6%-63.7%) in the X direction and 39.8% (95% CI, 39.1%-40.5%) in the Y direction. The Schöttle and Fujino points lay anterior and proximal to the saddle sulcus more than 5 mm away from the center of the saddle sulcus. Women had a higher prevalence of saddle sulcus (odds ratio [OR], 1.33 [95% CI, 1.00-1.75]; P = .046) compared with men. CONCLUSION: The saddle sulcus was identified in 75.7% of knees from the medial femoral aspect, with its center located consistently between the AT and ME.

Retracted: Pancratistatin Inhibits the Growth of Colorectal Cancer Cells by Inducing Apoptosis, Autophagy, and G2/M Cell Cycle Arrest.

An editorial decision has been made to retract this manuscript due to breach of publishing guidelines, following the identification of non-original and manipulated figures.Reference:Yong Xiong, Yi-Jia Xiong, Dong-Yang Liu, Rong-Rong Shen: Pancratistatin Inhibits the Growth of Colorectal Cancer Cells by Inducing Apoptosis, Autophagy, and G2/M Cell Cycle Arrest.Med Sci Monit 2019; 25:6015-6022. 10.12659/MSM.916116.

A short deletion in the DNA-binding domain of STAT3 suppresses growth and progression of colon cancer cells.

In this study, we investigated the effect of a short deletion in the DNA-binding domain of STAT3 (STAT3del) on the transcriptional activation of STAT3 target genes and its relationship with colon carcinogenesis. We used the CRISPR-CAS9 gene editing system to delete a short sequence encoding amino acids 400-411 in the DNA-binding domain (amino acid sequence: 317-567) from STAT3 gene in SW480, SW620 and HCT116 colon cancer cells. ChIP sequencing analysis showed that STAT3del occupancy was significantly reduced in 1029 genes and significantly increased in 475 genes compared to wild-type STAT3. The mutation altered the DNA motifs recognized by STAT3del as compared to the wild-type STAT3. We observed a strong correlation between expression of the STAT3 target genes and the loss or gain of STAT3del binding to their promoters. CCK-8, wound healing, and TUNEL assays showed reduced proliferation, migration, and survival of SW480, SW620 and HCT-116 cells expressing STAT3del as compared to the corresponding controls. These findings demonstrate that a short deletion in the DNA-binding domain of STAT3 alters its genome-wide DNA-binding and transcriptional profile of STAT3-target proteins, and suppresses the growth, progression and survival of colon cancer cells.

Optimizing the Design of Diatom Biosilica-Targeted Fusion Proteins in Biosensor Construction for Bacillus anthracis Detection.

In vivo functionalization of diatom biosilica frustules by genetic manipulation requires careful consideration of the overall structure and function of complex fusion proteins. Although we previously had transformed Thalassiosira pseudonana with constructs containing a single domain antibody (sdAb) raised against the Bacillus anthracis Sterne strain, which detected an epitope of the surface layer protein EA1 accessible in lysed spores, we initially were unsuccessful with constructs encoding a similar sdAb that detected an epitope of EA1 accessible in intact spores and vegetative cells. This discrepancy limited the usefulness of the system as an environmental biosensor for B. anthracis. We surmised that to create functional biosilica-localized biosensors with certain constructs, the biosilica targeting and protein trafficking functions of the biosilica-targeting peptide Sil3T8 had to be uncoupled. We found that retaining the ER trafficking sequence at the N-terminus and relocating the Sil3T8 targeting peptide to the C-terminus of the fusion protein resulted in successful detection of EA1 with both sdAbs. Homology modeling of antigen binding by the two sdAbs supported the hypothesis that the rescue of antigen binding in the previously dysfunctional sdAb was due to removal of steric hindrances between the antigen binding loops and the diatom biosilica for that particular sdAb.

Pancratistatin Inhibits the Growth of Colorectal Cancer Cells by Inducing Apoptosis, Autophagy, and G2/M Cell Cycle Arrest.

BACKGROUND Worldwide, colorectal cancer is ranked as the third most prevalent cancer. The natural compound, pancratistatin, extracted from the spider lily, has previously been shown to target apoptosis in cancer cells lines. This study aimed to investigate the effects of pancratistatin in human colorectal cancer cells in vitro. MATERIAL AND METHODS Human colorectal cancer cell lines, including HTC-15 cells, were compared with a normal human colonic fibroblast cell line, CDD-18Co. Cells were treated with increasing doses of pancratistatin. The MTT assay was used to assess cell viability. Fluorescence microscopy using DAPI and Annexin-V/propidium iodide (PI) was used to detect cell apoptosis. Cell autophagy was detected by electron microscopy. Cell migration was evaluated using a wound healing assay, and Western blot determined the expression levels of cell cycle proteins. RESULTS Pancratistatin inhibited the growth of the colorectal cancer cells with an IC50 ranging from 15-25 µM, but had a limited effect in normal CCD-18Co cells, with an IC50 of >100 µM. Pancratistatin reduced HCT-15 cell migration. Growth inhibition due to pancratistatin was associated with morphological changes of HCT-15 cells and included autophagy and apoptosis, and increased expression the autophagic proteins, LC3II, beclin-1, and Bax. Pancratistatin induced arrest of HCT-15 cells at G2/M of the cell cycle and inhibited phosphorylation of cdc2/cyclin-dependent kinase 1 (CDK1) and Cdc25c and the expression of cyclin B1. CONCLUSIONS Pancratistatin inhibited the growth of colorectal cancer cells in vitro by inducing apoptosis, autophagy, and G2/M cell cycle arrest.

Molecular evidence of a toxic effect on a biofilm and its matrix.

Shewanella oneidensis MR-1 wild-type and a hyper-adhesive mutant CP2-1-S1 are used as model organisms and Cr(vi) is selected as a toxic chemical to study biofilm and toxic chemical interactions. Biofilms are cultured in a microfluidic device for in situ time-of-flight secondary ion mass spectrometry imaging. This approach is viable for studying biofilms' responses to antimicrobial resistance.

Distance-Matched Tagging Sequence Optimizes Live-Cell Protein Labeling by a Biarsenical Fluorescent Reagent AsCy3_E.

Cell permeable biarsenical fluorescent dyes built around a cyanine scaffold (AsCy3) create the ability to monitor the structural dynamics of tagged proteins in living cells. To extend the capability of this photostable and bright biarsenical probe to site-specifically label cellular proteins, we have compared the ability of AsCy3 to label two different tagging sequences (i.e., CCKAEAACC and CCKAEAAKAEAAKCC), which were separately engineered onto enhanced green fluorescent proteins (EGFPs) and expressed in Escherichia coli. The cysteine pairs within the shorter protein tag (i.e., Cy3TAG) are designed to specifically match the 14.5 Å interarsenic atomic separation within AsCy3, whereas the longer protein tag (Cy3TAG+6) was identified using a peptide screening approach and reported to enhance the binding affinity and brightness. We report that AsCy3 binds both the tagged proteins with similar high affinities (Kd < 1 µM) under both in vivo labeling conditions and following isolation and labeling of the tagged EGFP protein. Greater experimental reproducibility and substantially larger AsCy3 labeling stoichiometries are observed under in vivo conditions using the shorter Cy3TAG in comparison to the Cy3TAG+6. These results suggest that the use of the distance-matched and conformationally restricted Cy3TAG avoids nonspecific protein interactions, thereby enabling routine measurements of protein localization and conformational dynamics in living cells.

Detection of unique Ebola virus oligonucleotides using fluorescently-labeled phosphorodiamidate morpholino oligonucleotide probe pairs.

Here we identify a low-cost diagnostic platform using fluorescently-labeled phosphorodiamidate morpholino oligonucleotide (PMO) probe pairs, which upon binding target oligonucleotides undergo fluorescence resonance energy transfer (FRET). Using a target oligonucleotide derived from the Ebola virus (EBOV), we have derivatized PMO probes with either Alexa Fluor488 (donor) or tetramethylrhodamine (acceptor). Upon EBOV target oligonulceotide binding, observed changes in FRET between PMO probe pairs permit a 25 pM lower limit of detection; there is no off-target binding within a complex mixture of nucleic acids and other biomolecules present in human saliva. Equivalent levels of FRET occur using PMO probe pairs for single or double stranded oligonucleotide targets. High-affinity binding is retained under low-ionic strength conditions that disrupt oligonucleotide secondary structures (e.g., stem-loop structures), ensuring reliable target detection. Under these low-ionic strength conditions, rates of PMO probe binding to target oligonucleotides are increased 3-fold relative to conventional high-ionic strength conditions used for nucleic acid hybridization, with half-maximal binding occurring within 10 min. Our results indicate an ability to use PMO probe pairs to detect clinically relevant levels of EBOV and other oligonucleotide targets in complex biological samples without the need for nucleic acid amplification, and open the possibility of population screening that includes assays for the genomic integration of DNA based copies of viral RNA.

Hydrogel Tethering Enhances Interdomain Stabilization of Single-Chain Antibodies.

Here, we identify the importance of molecular crowding agents in the functional stabilization of scFv antibodies. Antibodies were tethered through an engineered calmodulin (CaM)-binding peptide into a stimulus-responsive hydrogel composed of poly(ethylene glycol) (PEG)-functionalized CaM. Macromolecular crowding is modulated by transient heating, which decreases effective pore sizes. Using a fluorescent ligand bound to the scFv, frequency-domain fluorescence spectroscopy was used to assess the structural coupling between the VH and the VL domains and relationships with functional stabilization. There is minimal structural coupling between the VH and the VL domains in solution, as is apparent from the substantial rotational mobility for the bound ligand, that is suggestive of an independent mobility for the VH and the VL domains. In comparison, the hydrogel matrix acts to structurally couple the VH and the VL domains, resulting in a reduction in rotational mobility and a retention of ligand binding in the presence of 8.0 M urea. Under these same conditions, ligand binding is disrupted for scFv antibodies in solution. Increases in the stabilization of scFv antibodies in hydrogels is not simply the result of molecular crowding because decreases in pore size act to destabilize ligand binding. Rather, our results suggest that the functional stabilization of the scFv antibody within the PEG hydrogel matrix includes important factors involving protein solvation that stabilize interdomain interactions between the VH and the VL domains necessary for ligand binding.

Dynamic Stabilization of Expressed Proteins in Engineered Diatom Biosilica Matrices.

Self-assembly of recombinant proteins within the biosilica of living diatoms represents a means to construct functional materials in a reproducible and scalable manner that will enable applications that harness the inherent specificities of proteins to sense and respond to environmental cues. Here we describe the use of a silaffin-derived lysine-rich 39-amino-acid targeting sequence (Sil3T8) that directs a single chain fragment variable (scFv) antibody or an enhanced green fluorescent protein (EGFP) to assemble within the biosilica frustule, resulting in abundance of >200 000 proteins per frustule. Using either a fluorescent ligand bound to the scFv or the intrinsic fluorescence of EGFP, we monitored protein conformational dynamics, accessibility to external quenchers, binding affinity, and conformational stability. Like proteins in solution, proteins within isolated frustules undergo isotropic rotational motion, but with 2-fold increases in rotational correlation times that are indicative of weak macromolecular associations within the biosilica. Solvent accessibilities and high-affinity (pM) binding are comparable to those in solution. In contrast to solution conditions, scFv antibodies within the biosilica matrix retain their binding affinity in the presence of chaotropic agents (i.e., 8 M urea). Together, these results argue that dramatic increases in protein conformational stability within the biosilica matrices arise through molecular crowding, acting to retain native protein folds and associated functionality with the potential to allow the utility of engineered proteins under a range of harsh environmental conditions associated with environmental sensing and industrial catalytic transformations.

Antigen Binding and Site-Directed Labeling of Biosilica-Immobilized Fusion Proteins Expressed in Diatoms.

The diatom Thalassiosira pseudonana was genetically modified to express biosilica-targeted fusion proteins comprising either enhanced green fluorescent protein (EGFP) or single chain antibodies engineered with a tetracysteine tagging sequence. Of interest were the site-specific binding of (1) the fluorescent biarsenical probe AsCy3 and AsCy3e to the tetracysteine tagged fusion proteins and (2) high and low molecular mass antigens, the Bacillus anthracis surface layer protein EA1 or small molecule explosive trinitrotoluene (TNT), to biosilica-immobilized single chain antibodies. Analysis of biarsenical probe binding using fluorescence and structured illumination microscopy indicated differential colocalization with EGFP in nascent and mature biosilica, supporting the use of either EGFP or bound AsCy3 and AsCy3e in studying biosilica maturation. Large increases in the lifetime of a fluorescent analogue of TNT upon binding single chain antibodies provided a robust signal capable of discriminating binding to immobilized antibodies in the transformed frustule from nonspecific binding to the biosilica matrix. In conclusion, our results demonstrate an ability to engineer diatoms to create antibody-functionalized mesoporous silica able to selectively bind chemical and biological agents for the development of sensing platforms.

Two-dimensional and three-dimensional dynamic imaging of live biofilms in a microchannel by time-of-flight secondary ion mass spectrometry.

A vacuum compatible microfluidic reactor, SALVI (System for Analysis at the Liquid Vacuum Interface), was employed for in situ chemical imaging of live biofilms using time-of-flight secondary ion mass spectrometry (ToF-SIMS). Depth profiling by sputtering materials in sequential layers resulted in live biofilm spatial chemical mapping. Two-dimensional (2D) images were reconstructed to report the first three-dimensional images of hydrated biofilm elucidating spatial and chemical heterogeneity. 2D image principal component analysis was conducted among biofilms at different locations in the microchannel. Our approach directly visualized spatial and chemical heterogeneity within the living biofilm by dynamic liquid ToF-SIMS.

Controlled activation of protein rotational dynamics using smart hydrogel tethering.

Stimulus-responsive hydrogel materials that stabilize and control protein dynamics have the potential to enable a range of applications that take advantage of the inherent specificity and catalytic efficiencies of proteins. Here we describe the modular construction of a hydrogel using an engineered calmodulin (CaM) within a poly(ethylene glycol) (PEG) matrix that involves the reversible tethering of proteins through an engineered CaM-binding sequence. For these measurements, maltose binding protein (MBP) was isotopically labeled with (13)C and (15)N, permitting dynamic structural measurements using TROSY-HSQC NMR spectroscopy. The protein dynamics is suppressed upon initial formation of hydrogels, with a concomitant increase in protein stability. Relaxation of the hydrogel matrix following transient heating results in enhanced protein dynamics and resolution of substrate-induced large-amplitude domain rearrangements.

Enzyme design from the bottom up: an active nickel electrocatalyst with a structured peptide outer coordination sphere.

Catalytic, peptide-containing metal complexes with a well-defined peptide structure have the potential to enhance molecular catalysts through an enzyme-like outer coordination sphere. Here, we report the synthesis and characterization of an active, peptide-based metal complex built upon the well-characterized hydrogen production catalyst [Ni(P(Ph)2N(Ph))2](2+) (P(Ph)2N(Ph)=1,3,6-triphenyl-1-aza-3,6-diphosphacycloheptane). The incorporated peptide maintains its ß-hairpin structure when appended to the metal core, and the electrocatalytic activity of the peptide-based metal complex (≈100,000 s(-1)) is enhanced compared to the parent complex ([Ni(P(Ph)2N(APPA))2](2+); ≈50,500 s(-1)). The combination of an active molecular catalyst with a structured peptide provides a scaffold that permits the incorporation of features of an enzyme-like outer-coordination sphere necessary to create molecular electrocatalysts with enhanced functionality.

ALDH1A1 Deficiency in Gorlin Syndrome Suggests a Central Role for Retinoic Acid and ATM Deficits in Radiation Carcinogenesis.

We hypothesize that aldehyde dehydrogenase 1A1 (ALDH1A1) deficiency will result in impaired ataxia-telangiectasia mutated (ATM) activation in a retinoic acid-sensitive fashion. Data supporting this hypothesis include (1) reduced ATM activation in irradiated primary dermal fibroblasts from ALDH1A1-deficient Gorlin syndrome patients (GDFs), relative to ALDH1A1-positive normal human dermal fibroblasts (NHDFs) and (2) increased ATM activation by X-radiation in GDFs pretreated with retinoic acid, however, the impact of donor variability on ATM activation in fibroblasts was not assessed and is a prudent consideration in future studies. Clonogenic survival of irradiated cells showed differential responses to retinoic acid as a function of treatment time. Long-term (5 Day) retinoic acid treatment functioned as a radiosensitizer and was associated with downregulation of ATM protein levels. Short-term (7 h) retinoic acid treatment showed a trend toward increased survival of irradiated cells and did not downregulate ATM protein levels. Using a newly developed IncubATR technology, which defines changes in bulk chemical bond patterns in live cells, we can discriminate between the NHDF and GDF phenotypes, but treatment of GDFs with retinoic acid does not induce reversion of bulk chemical bond patterns associated with GDFs toward the NHDF phenotype. Collectively, our preliminary investigation of the Gorlin phenotype has identified deficient ALDH1A1 expression associated with deficient ATM activation as a possible susceptibility factor that is consistent with the high incidence of spontaneous and radiation-induced carcinogenesis in these patients. The IncubATR technology exhibits sufficient sensitivity to detect phenotypic differences in live cells that may be relevant to radiation health effects.

Synthesis and application of an environmentally insensitive Cy3-based arsenical fluorescent probe to identify adaptive microbial responses involving proximal dithiol oxidation.

Reversible disulfide oxidation between proximal cysteines in proteins represents a common regulatory control mechanism to modulate flux through metabolic pathways in response to changing environmental conditions. To enable in vivo measurements of cellular redox changes linked to disulfide bond formation, we have synthesized a cell-permeable thiol-reactive affinity probe (TRAP) consisting of a monosubstituted cyanine dye derivatized with arsenic (i.e., TRAP_Cy3) to trap and visualize dithiols in cytosolic proteins. Alkylation of reactive thiols prior to displacement of the bound TRAP_Cy3 by ethanedithiol permits facile protein capture and mass spectrometric identification of proximal reduced dithiols to the exclusion of individual cysteines. Applying TRAP_Cy3 to evaluate cellular responses to increases in oxygen and light levels in the photosynthetic microbe Synechococcus sp. PCC7002, we observe large decreases in the abundance of reduced dithiols in cellular proteins, which suggest redox-dependent mechanisms involving the oxidation of proximal disulfides. Under these same growth conditions that result in the oxidation of proximal thiols, there is a reduction in the abundance of post-translational oxidative protein modifications involving methionine sulfoxide and nitrotyrosine. These results suggest that the redox status of proximal cysteines responds to environmental conditions, acting to regulate metabolic flux and minimize the formation of reactive oxygen species to decrease oxidative protein damage.

Magnetic particle detection (MPD) for in-vitro dosimetry.

In-vitro tests intended for evaluating the potential health effects of magnetic nanoparticles generally require an accurate measure of cell dose to promote the consistent use and interpretation of biological response. Here, a simple low-cost inductive sensor is developed for quickly determining the total mass of magnetic nanoparticles that is bound to the plasma membrane and internalized by cultured cells. Sensor operation exploits an oscillating magnetic field (f0=250kHz) together with the nonlinear response of particle magnetization to generate a harmonic signal (f3=750kHz) that varies linearly with particulate mass (R(2)>0.999) and is sufficiently sensitive for detecting ∼100ng of carboxyl-coated iron-oxide nanoparticles in under a second. When exploited for measuring receptor-mediated nanoparticle uptake in RAW 264.7 macrophages, results show that the achieved dosimetric performance is comparable with relatively expensive analytical techniques that are much more time-consuming and labor-intensive to perform. The described sensing is therefore potentially better suited for low-cost in-vitro assays that require fast and quantitative magnetic particle detection.

Optimized design and synthesis of a cell-permeable biarsenical cyanine probe for imaging tagged cytosolic bacterial proteins.

To optimize cellular delivery and specific labeling of tagged cytosolic proteins by biarsenical fluorescent probes built around a cyanine dye (Cy3) scaffold, we have systematically varied the polarity of the N-alkyl chain (i.e., 4-5 methylene groups appended by a sulfonate or methoxy ester moiety) and arsenic capping reagent (ethanedithiol versus benzenedithiol). Optimal live-cell labeling and visualization of tagged cytosolic proteins is reported using an ethanedithiol capping reagent with the uncharged methoxy ester functionalized N-alkyl chains. These measurements demonstrate the general utility of this new class of photostable and highly fluorescent biarsenical probes based on the cyanine dye scaffold for in vivo labeling of tagged cellular proteins for live cell imaging measurements of protein dynamics.

Synthesis of a targeted biarsenical Cy3-Cy5 affinity probe for super-resolution fluorescence imaging.

Photoswitchable fluorescent probes capable of the targeted labeling of tagged proteins are of significant interest due to their ability to enable in situ imaging of protein complexes within native biomolecular assemblies. Here we describe the synthesis of a fluorescent probe (AsCy3Cy5) and demonstrate the targeted labeling and super-resolution imaging of a tagged protein within a supramolecular protein complex.