The rod synapse in aging wildtype and Dscaml1 mutant mice.

The retina is an intricately organized neural tissue built on cone and rod pathways for color and night vision. Genetic mutations that disrupt the proper function of the rod circuit contribute to blinding diseases including retinitis pigmentosa and congenital stationary night blindness (CSNB). Down Syndrome cell adhesion molecule like 1 (Dscaml1) is expressed by rods, rod bipolar cells (RBCs), and sub-populations of amacrine cells, and has been linked to a middle age onset of CSNB in humans. However, how Dscaml1 contributes to this visual deficit remains unexplored. Here, we probed Dscaml1's role in the maintenance of the rod-to-RBC synapse using a loss of function mouse model. We used immunohistochemistry to investigate the anatomical formation and maintenance of the rod-to-RBC synapse in the young, adult, and aging retina. We generated 3D reconstructions, using serial electron micrographs, of rod spherules and RBCs to measure the number of invaginating neurites, RBC dendritic tip number, and RBC mitochondrial morphology. We find that while rod-to-RBC synapses form and are maintained, similar to wildtype, that there is an increase in the number of invaginating neurites in rod spherules, a reduction in RBC dendritic tips, and reduced mitochondrial volume and complexity in the Dscaml1 mutant retina compared to controls. We also observed precocious sprouting of RBC dendrites into the outer nuclear layer (ONL) of the Dscaml1 mutant retina compared to controls. These results contribute to our knowledge of Dscaml1's role in rod circuit development and maintenance and give additional insight into possible genetic therapy targets for blinding diseases and disorders like CSNB.

Repurposing Clemastine to Target Glioblastoma Cell Stemness.

Brain tumor-initiating cells (BTICs) and tumor cell plasticity promote glioblastoma (GBM) progression. Here, we demonstrate that clemastine, an over-the-counter drug for treating hay fever and allergy symptoms, effectively attenuated the stemness and suppressed the propagation of primary BTIC cultures bearing PDGFRA amplification. These effects on BTICs were accompanied by altered gene expression profiling indicative of their more differentiated states, resonating with the activity of clemastine in promoting the differentiation of normal oligodendrocyte progenitor cells (OPCs) into mature oligodendrocytes. Functional assays for pharmacological targets of clemastine revealed that the Emopamil Binding Protein (EBP), an enzyme in the cholesterol biosynthesis pathway, is essential for BTIC propagation and a target that mediates the suppressive effects of clemastine. Finally, we showed that a neural stem cell-derived mouse glioma model displaying predominantly proneural features was similarly susceptible to clemastine treatment. Collectively, these results identify pathways essential for maintaining the stemness and progenitor features of GBMs, uncover BTIC dependency on EBP, and suggest that non-oncology, low-toxicity drugs with OPC differentiation-promoting activity can be repurposed to target GBM stemness and aid in their treatment.

An Accurate and Automated Method for Adenoma Detection Rate and Report Card Generation Utilizing Common Electronic Health Records.

GOALS: To develop an automated method for Adenoma Detection Rate (ADR) calculation and report card generation using common electronic health records (EHRs). BACKGROUND: ADR is the most widely accepted colonoscopy quality indicator and is inversely associated with interval colorectal cancer incidence and mortality. However, ADR is difficult to efficiently measure and disseminate, due to need for data integration from distinct electronic databases. METHODS: We migrated data from an endoscopy reporting software (Endosoft) to Epic Reporting Servers where it was combined with anatomic pathology data (Beaker Lab Information System, EPIC Systems). A natural language processing expression was developed to search Beaker pathology reports for accurate identification of adenomatous polyps. A blinded physician manually validated a final cohort of 200 random procedures. ADR report cards were automatically generated utilizing the Crystal Reports feature within EPIC. RESULTS: Validation of the natural language processing algorithm for ADR showed a sensitivity, specificity, and accuracy of 100%. ADR was automatically calculated for 12 endoscopists over a calendar year. Two thousand two hundred seventy-six screening colonoscopies were performed with 775 procedures having a least one adenoma detected, for a total ADR of 34%. Report cards were successfully generated within the EPIC EHR and distributed to endoscopists by secure e-mail. CONCLUSION: We describe an accurate, automated and scalable process for ADR calculation and reporting utilizing commonly adopted EHRs and data integration methods. By integrating the process of ADR collection and streamlining dissemination of reports, this methodology is poised to enhance colonoscopy quality care across health care networks that use it.

Developmental deficits of MGE-derived interneurons in the Cntnap2 knockout mouse model of autism spectrum disorder.

Interneurons are fundamental cells for maintaining the excitation-inhibition balance in the brain in health and disease. While interneurons have been shown to play a key role in the pathophysiology of autism spectrum disorder (ASD) in adult mice, little is known about how their maturation is altered in the developing striatum in ASD. Here, we aimed to track striatal developing interneurons and elucidate the molecular and physiological alterations in the Cntnap2 knockout mouse model. Using Stereo-seq and single-cell RNA sequencing data, we first characterized the pattern of expression of Cntnap2 in the adult brain and at embryonic stages in the medial ganglionic eminence (MGE), a transitory structure producing most cortical and striatal interneurons. We found that Cntnap2 is enriched in the striatum, compared to the cortex, particularly in the developing striatal cholinergic interneurons. We then revealed enhanced MGE-derived cell proliferation, followed by increased cell loss during the canonical window of developmental cell death in the Cntnap2 knockout mice. We uncovered specific cellular and molecular alterations in the developing Lhx6-expressing cholinergic interneurons of the striatum, which impacts interneuron firing properties during the first postnatal week. Overall, our work unveils some of the mechanisms underlying the shift in the developmental trajectory of striatal interneurons which greatly contribute to the ASD pathogenesis.

A Second Wave for the Neurokinin Tac2 Pathway in Brain Research.

Despite promising advances in basic research of the neurokinin B/Tac2 pathway in both animals and humans, clinical applications are yet to be implemented. This is likely because of our limited understanding of the action of the pathway in the brain. While this system controls neuronal activity in multiple regions, the precise impact of Tac2-induced cellular responses on behavior remains unclear. Recently, elegant studies revealed a key contribution to stress-related behaviors and memory. Here, we discuss the crucial importance of bridging the gap between the Tac2 pathway's involvement in cell physiology and cognition to comprehend its role in health and disease. We propose that a better understanding of the Tac2 pathway in the brain could provide an essential perspective for basic investigations, which in turn will feed clinical research.

OFF bipolar cell density varies by subtype, eccentricity, and along the dorsal ventral axis in the mouse retina.

The neural retina is organized along central-peripheral, dorsal-ventral, and laminar planes. Cellular density and distributions vary along the central-peripheral and dorsal-ventral axis in species including primates, mice, fish, and birds. Differential distribution of cell types within the retina is associated with sensitivity to different types of damage that underpin major retinal diseases, including macular degeneration and glaucoma. Normal variation in retinal distribution remains unreported for multiple cell types in widely used research models, including mouse. Here we map the distribution of all known OFF bipolar cell (BC) populations and horizontal cells. We report significant variation in the distribution of OFF BC populations and horizontal cells along the dorsal-ventral and central-peripheral axes of the retina. Distribution patterns are much more pronounced for some populations of OFF BC cells than others and may correspond to the cell type's specialized functions.

Evidence that knock down of GSK-3ß in Chronic Myelogenous Leukemia cells augments IFN-γ-induced apoptosis.

The role of interferon-gamma (IFN-γ) in Chronic Myelogenous/Myeloid Leukemia (CML) and in the treatment of CML remains unclear; specifically, the effect of IFN-γ on apoptosis. There is reported interplay between IFN-γ and glycogen synthase kinase-3 (GSK-3), a kinase which has been implicated in both cell death and, conversely, cell survival. Thus, we utilized the CML-derived HAP1 cell line and a mutant HAP1 GSK-3ß knocked-down cell line (GSK-3ß 31bp) to investigate whether GSK-3 modulates IFN-γ's action on CML cells. Significantly less GSK-3ß 31bp cells, relative to HAP1 cells, were present after 48 h treatment with IFN-γ. IFN-γ treatment significantly decreased GSK-3ß 31bp substrate adhesiveness (relative to HAP1 cells); an observation often correlated with cell death. Fluorescence microscopy revealed that IFN-γ induces a modest level of apoptosis in the HAP1 cells and that IFN-γ induced apoptosis is significantly enhanced in GSK-3ß 31bp cells. Utilizing a complementary GSK-3ß knocked-down cell line (8bp) we found, via flow cytometric analysis, that IFN-γ induced apoptosis is significantly enhanced in GSK-3ß 8bp cells relative to HAP1 cells. Combined, our findings suggest that IFN-γ induces apoptosis of CML cells and that loss of GSK-3ß significantly augments IFN-γ-induced apoptosis.

Galectin-1 Influences Breast Cancer Cell Adhesion to E-selectin Via Ligand Intermediaries.

INTRODUCTION: Invasion of other tissues during bloodborne metastasis in part requires adhesion of cancer cells to vascular endothelium by specific fluid shear-dependent receptor-ligand interactions. This study investigates the hypothesis that the adhesion is mediated by ligands shared between endothelial E-selectin and Galectin-1 (Gal-1), both of which are upregulated during inflammation and cancer. METHODS: Flow chamber adhesion and dynamic biochemical tissue analysis (DBTA) assays were used to evaluate whether Gal-1 modulates E-selectin adhesive interactions of breast cancer cells and tissues under dynamic flow conditions, while immunocytochemistry, immunohistochemistry, western blotting, and fluorescence anisotropy were used to study molecular interactions under static conditions. RESULTS: Dynamic adhesion assays revealed a shear-dependent binding interaction between Gal-1hFc treated breast cancer cells and tissues and E-selectin-coated beads, causing ~ 300% binding increase of the beads compared to negative controls. Immunocyto- and immunohistochemical analyses showed that Gal-1 and E-selectin fluorescent signals colocalized on cells and tissues at ~ 75% for each assay. Immunoprecipitation and Western blotting of Mac-2BP from breast cancer cell lysates revealed that Gal-1 and E-selectin share Mac-2BP as a ligand, while fluorescence anisotropy and circulating tumor cell model systems exhibited competitive or antagonistic binding between Gal-1 and E-selectin for shared ligands, including Mac-2BP. Furthermore, Mac-2BP functional blockade inhibited the effects of Gal-1 on E-selectin binding. CONCLUSIONS: In summary, this investigation reveals a shear-dependent interaction between E-selectin and Gal-1 that may be due to intermediation by a similar or shared ligand(s), including Mac-2BP, which may provide a rational basis for development of novel diagnostics or therapeutics for breast cancer.

Isolation and characterization of chimeric human Fc-expressing proteins using protein a membrane adsorbers and a streamlined workflow.

Laboratory scale to industrial scale purification of biomolecules from cell culture supernatants and lysed cell solutions can be accomplished using affinity chromatography. While affinity chromatography using porous protein A agarose beads packed in columns is arguably the most common method of laboratory scale isolation of antibodies and recombinant proteins expressing Fc fragments of IgG, it can be a time consuming and expensive process. Time and financial constraints are especially daunting in small basic science labs that must recover hundreds of micrograms to milligram quantities of protein from dilute solutions, yet lack access to high pressure liquid delivery systems and/or personnel with expertise in bioseparations. Moreover, product quantification and characterization may also excessively lengthen processing time over several workdays and inflate expenses (consumables, wages, etc.). Therefore, a fast, inexpensive, yet effective protocol is needed for laboratory scale isolation and characterization of antibodies and other proteins possessing an Fc fragment. To this end, we have devised a protocol that can be completed by limited-experience technical staff in less than 9 hr (roughly one workday) and as quickly as 4 hr, as opposed to traditional methods that demand 20+ work hours. Most required equipment is readily available in standard biomedical science, biochemistry, and (bio)chemical engineering labs, and all reagents are commercially available. To demonstrate this protocol, representative results are presented in which chimeric murine galectin-1 fused to human Fc (Gal-1hFc) from cell culture supernatant was isolated using a protein A membrane adsorber. Purified Gal-1hFc was quantified using an expedited Western blotting analysis procedure and characterized using flow cytometry. The streamlined workflow can be modified for other Fc-expressing proteins, such as antibodies, and/or altered to incorporate alternative quantification and characterization methods.

Mac-2 binding protein is a novel E-selectin ligand expressed by breast cancer cells.

Hematogenous metastasis involves the adhesion of circulating tumor cells to vascular endothelium of the secondary site. We hypothesized that breast cancer cell adhesion is mediated by interaction of endothelial E-selectin with its glycoprotein counter-receptor(s) expressed on breast cancer cells. At a hematogenous wall shear rate, ZR-75-1 breast cancer cells specifically adhered to E-selectin expressing human umbilical vein endothelial cells when tested in parallel plate flow chamber adhesion assays. Consistent with their E-selectin ligand activity, ZR-75-1 cells expressed flow cytometrically detectable epitopes of HECA-452 mAb, which recognizes high efficiency E-selectin ligands typified by sialofucosylated moieties. Multiple E-selectin reactive proteins expressed by ZR-75-1 cells were revealed by immunoprecipitation with E-selectin chimera (E-Ig chimera) followed by Western blotting. Mass spectrometry analysis of the 72 kDa protein, which exhibited the most prominent E-selectin ligand activity, corresponded to Mac-2 binding protein (Mac-2BP), a heretofore unidentified E-selectin ligand. Immunoprecipitated Mac-2BP expressed sialofucosylated epitopes and possessed E-selectin ligand activity when tested by Western blot analysis using HECA-452 mAb and E-Ig chimera, respectively, demonstrating that Mac-2BP is a novel high efficiency E-selectin ligand. Furthermore, silencing the expression of Mac-2BP from ZR-75-1 cells by shRNA markedly reduced their adhesion to E-selectin expressing cells under physiological flow conditions, confirming the functional E-selectin ligand activity of Mac-2BP on intact cells. In addition to ZR-75-1 cells, several other E-selectin ligand positive breast cancer cell lines expressed Mac-2BP as detected by Western blot and flow cytometry, suggesting that Mac-2BP may be an E-selectin ligand in a variety of breast cancer types. Further, invasive breast carcinoma tissue showed co-localized expression of Mac-2BP and HECA-452 antigens by fluorescence microscopy, underscoring the possible role of Mac-2BP as an E-selectin ligand. In summary, breast cancer cells express Mac-2BP as a novel E-selectin ligand, potentially revealing a new prognostic and therapeutic target for breast cancer.

Enantioselective protonation of catalytically generated chiral enolates as an approach to the synthesis of alpha-chloroesters.

Treatment of alpha,alpha-dichloroaldehydes with various phenols in the presence of chiral triazolium salt catalysts and excess base results in the synthesis of alpha-chloro aryl esters in good yield and enantioselectivity. The reaction is tolerant of various functionality on the aldehyde as well as several electronically diverse phenols. The product chloroesters were further transformed into chloroacid, chlorohydrin, and azidoesters with nearly complete retention of enantioselectivity.

Conversion of alpha-haloaldehydes into acylating agents by an internal redox reaction catalyzed by nucleophilic carbenes.

Reactivity umpolung allows us to consider nontraditional bond disconnections. We report herein that treatment of an alpha-haloaldehyde with a nucleophile in the presence of catalytic amounts of nucleophilic carbenes results in an internal redox reaction giving rise to a dehalogenated acylating agent as an intermediate by a new reaction manifold. A brief illustration of the scope of this reaction is presented along with evidence supporting the direct intervention of the carbene in the acylation step.

Stereoretentive O-to-C rearrangement of vinyl acetals: solvent cage effects as a stereocontrol element.

The Lewis acid-mediated rearrangement of chiral vinyl acetals may be induced to provide the product of stereoretention using Me3Al and BF3.OEt2 in concert. The selectivities obtained in this reaction (86:14 to 96:4) are complementary to that observed when relying on oxocarbenium facial bias to control the newly formed stereocenter. Evidence is presented that this reaction occurs by tight ion-pair binding in the solvent cage. The relay of C-O bond stereochemistry to a C-C bond stereocenter via ionic intermediates is an addition to similar established methods such as the Claisen rearrangement.