Organizing principles of astrocytic nanoarchitecture in the mouse cerebral cortex.

Astrocytes are increasingly understood to be important regulators of central nervous system (CNS) function in health and disease; yet, we have little quantitative understanding of their complex architecture. While broad categories of astrocytic structures are known, the discrete building blocks that compose them, along with their geometry and organizing principles, are poorly understood. Quantitative investigation of astrocytic complexity is impeded by the absence of high-resolution datasets and robust computational approaches to analyze these intricate cells. To address this, we produced four ultra-high-resolution datasets of mouse cerebral cortex using serial electron microscopy and developed astrocyte-tailored computer vision methods for accurate structural analysis. We unearthed specific anatomical building blocks, structural motifs, connectivity hubs, and hierarchical organizations of astrocytes. Furthermore, we found that astrocytes interact with discrete clusters of synapses and that astrocytic mitochondria are distributed to lie closer to larger clusters of synapses. Our findings provide a geometrically principled, quantitative understanding of astrocytic nanoarchitecture and point to an unexpected level of complexity in how astrocytes interact with CNS microanatomy.

A Bio-Conjugated Chlorin-Based Metal-Organic Framework for Targeted Photodynamic Therapy of Triple Negative Breast and Pancreatic Cancers.

The field of photodynamic therapy (PDT) has continued to show promise as a potential method for treating tumors. In this work a photosensitizer (PS) has been delivered to cancer cell lines for PDT by incorporation into the metal-organic framework (MOF) as an organic linker. By functionalizing the surface of MOF nanoparticles with maltotriose the PS can efficiently target cancer cells with preferential uptake into pancreatic and breast cancer cell lines. Effective targeting overcomes some current problems with PDT including long-term photosensitivity and tumor specificity. Developing a PS with optimal absorption and stability is one of the foremost challenges in PDT and the synthesis of a chlorin which is activated by long-wavelength light and is resistant to photo-bleaching is described. This chlorin-based MOF shows anti-cancer ability several times higher than that of porphyrin-based MOFs with little toxicity to normal cell lines and no dark toxicity.

Fluorinated Cycloplatinated(II) Complexes Bearing Bisphosphine Ligands as Potent Anticancer Agents.

A family of cationic cycloplatinated(II) complexes [Pt(dfppy)(P^P)]Cl, dfppy = 2-(2,4-difluorophenyl)pyridine, incorporating bisphosphine ligands, P^P = bis(diphenylphosphino)methane (1, dppm), 1,2-bis(diphenylphosphino)ethane (2, dppe) and 1,2-bis(diphenylphosphino)benzene (3, dppbz), was prepared. The complexes were characterized by means of several analytical and spectroscopic methods. These complexes displayed acceptable stability in the biological environments which was confirmed by NMR, HR ESI-MS and UV-vis techniques. The antiproliferative properties of these complexes were evaluated by National Cancer Institute (NCI) at National Institutes of Health (NIH) against 60 different human tumor cell lines such as leukemia, melanoma, lung, colon, brain, ovary, breast, prostate and kidney. These complexes showed higher cytotoxicity than cisplatin against a wide variety of cancer cell lines such as K-562 (leukemia), HOP-92 (lung), HCT-116 (colon), OVCAR-8 (ovarian), PC-3 (prostate), MDA-MB-468 (breast), and melanoma cancer cell lines. Complex 3 as the most potent compound in this study furnished an excellent anti-proliferative activity compared to the cisplatin against Hela, SKOV3, and MCF-7 cancer cell lines. The main mode of the interaction of 1-3 with DNA was also determined using molecular docking studies.

Depolarizing GABA Transmission Restrains Activity-Dependent Glutamatergic Synapse Formation in the Developing Hippocampal Circuit.

γ-Aminobutyric acid (GABA) is the main inhibitory neurotransmitter in the mature brain but has the paradoxical property of depolarizing neurons during early development. Depolarization provided by GABAA transmission during this early phase regulates neural stem cell proliferation, neural migration, neurite outgrowth, synapse formation, and circuit refinement, making GABA a key factor in neural circuit development. Importantly, depending on the context, depolarizing GABAA transmission can either drive neural activity or inhibit it through shunting inhibition. The varying roles of depolarizing GABAA transmission during development, and its ability to both drive and inhibit neural activity, makes it a difficult developmental cue to study. This is particularly true in the later stages of development when the majority of synapses form and GABAA transmission switches from depolarizing to hyperpolarizing. Here, we addressed the importance of depolarizing but inhibitory (or shunting) GABAA transmission in glutamatergic synapse formation in hippocampal CA1 pyramidal neurons. We first showed that the developmental depolarizing-to-hyperpolarizing switch in GABAA transmission is recapitulated in organotypic hippocampal slice cultures. Based on the expression profile of K+-Cl- co-transporter 2 (KCC2) and changes in the GABA reversal potential, we pinpointed the timing of the switch from depolarizing to hyperpolarizing GABAA transmission in CA1 neurons. We found that blocking depolarizing but shunting GABAA transmission increased excitatory synapse number and strength, indicating that depolarizing GABAA transmission can restrain glutamatergic synapse formation. The increase in glutamatergic synapses was activity-dependent but independent of BDNF signaling. Importantly, the elevated number of synapses was stable for more than a week after GABAA inhibitors were washed out. Together these findings point to the ability of immature GABAergic transmission to restrain glutamatergic synapse formation and suggest an unexpected role for depolarizing GABAA transmission in shaping excitatory connectivity during neural circuit development.

Preparation and Applications of Metal-Organic Frameworks (MOFs): A Laboratory Activity and Demonstration for High School and/or Undergraduate Students.

The chemistry of metal-organic frameworks (MOFs), a new class of emerging crystalline porous solids with three-dimensional (3D) networks composed of metals and multidentate organic molecules, was introduced by using three differently-shaped crystals. We reported new and mild MOF synthesis methods that are simple and devised to be performed in high school or primarily undergraduate school settings. MOF applications were demonstrated by use of our synthesized MOFs in the capture of iodine as a potentially hazardous molecule from solution and as a drug delivery system. These applications can be visually confirmed in minutes. Students can gain knowledge on advanced topics, such as drug delivery systems, through these easy-to-prepare MOFs. Furthermore, students can gain an understanding of powder X-ray analysis and ultraviolet-visible near-infrared spectroscopy. This laboratory experience is practical, including synthesis and application of MOFs. The entire experiment has also been recorded as an educational video posted on YouTube as a free public medium for students to watch and learn. In this article we first report the steps we took to synthesize and analyze the MOFs, followed by a description of a simple demonstration that we verified to effectively exhibit adsorption by MOFs. We conclude with a description of how the laboratory activity and demonstration was implemented in an undergraduate chemistry laboratory.

Peripherally derived macrophages modulate microglial function to reduce inflammation after CNS injury.

Infiltrating monocyte-derived macrophages (MDMs) and resident microglia dominate central nervous system (CNS) injury sites. Differential roles for these cell populations after injury are beginning to be uncovered. Here, we show evidence that MDMs and microglia directly communicate with one another and differentially modulate each other's functions. Importantly, microglia-mediated phagocytosis and inflammation are suppressed by infiltrating macrophages. In the context of spinal cord injury (SCI), preventing such communication increases microglial activation and worsens functional recovery. We suggest that macrophages entering the CNS provide a regulatory mechanism that controls acute and long-term microglia-mediated inflammation, which may drive damage in a variety of CNS conditions.

Physiologic assessment of moderate coronary lesions: a step towards complete revascularization in coronary artery bypass grafting.

BACKGROUND: An accurate diagnostic assessment of coronary artery disease is crucial for patients undergoing coronary artery bypass grafting (CABG). Fractional flow reserve (FFR) and instantaneous wave-free ratio (iFR) to guide complete revascularization have not been adequately studied in patients prior to CABG. We compared an anatomic to a physiologic assessment of moderate coronary lesions (40-70% stenosis) in patients referred for CABG. METHODS: We retrospectively reviewed 109 medical records of patients who underwent CABG at Tulane Medical Center from 2014 to 2016. Patients were divided into an FFR/iFR-guided and an angiography-guided group. Clinical characteristics, procedural outcomes, and clinical outcomes for the two groups were compared over an 18-month follow-up period. RESULTS: There were significantly higher rates of three-vessel anastomoses (85.7% vs. 74.7%, P<0.05) and venous grafting (85.7% vs. 76.8%, P<0.05) in the FFR/iFR group. The FFR/iFR group had a lower rate of grafts placed to the left anterior descending artery (LAD) distribution than the angiography group (7.1% vs. 29.5%, P<0.05). The FFR/iFR group had a higher rate of grafts placed to the left circumflex (LCx) artery distribution than the angiography group (28.6% vs. 9.5%, P<0.05). We observed a trend toward reduction in major adverse cardiac events (MACEs) (7.1% vs. 11.6%, P=0.369) and angina (0.0% vs. 6.3%, P=0.429) in the FFR/iFR group compared to the angiography group over 18 months. CONCLUSIONS: Physiologic assessment of coronary lesions can effectively guide complete revascularization in patients undergoing CABG. Moreover, FFR/iFR-guided CABG was associated with significantly higher rates of three-vessel anastomoses, venous grafting, and graft distribution to the circumflex system.

Detection of activity-dependent vasopressin release from neuronal dendrites and axon terminals using sniffer cells.

Our understanding of neuropeptide function within neural networks would be improved by methods allowing dynamic detection of peptide release in living tissue. We examined the usefulness of sniffer cells as biosensors to detect endogenous vasopressin (VP) release in rat hypothalamic slices and from isolated neurohypophyses. Human embryonic kidney cells were transfected to express the human V1a VP receptor (V1aR) and the genetically encoded calcium indicator GCaMP6m. The V1aR couples to Gq11, thus VP binding to this receptor causes an increase in intracellular [Ca2+] that can be detected by a rise in GCaMP6 fluorescence. Dose-response analysis showed that VP sniffer cells report ambient VP levels >10 pM (EC50 = 2.6 nM), and this effect could be inhibited by the V1aR antagonist SR 49059. When placed over a coverslip coated with sniffer cells, electrical stimulation of the neurohypophysis provoked a reversible, reproducible, and dose-dependent increase in VP release using as few as 60 pulses delivered at 3 Hz. Suspended sniffer cells gently plated over a slice adhered to the preparation and allowed visualization of VP release in discrete regions. Electrical stimulation of VP neurons in the suprachiasmatic nucleus caused significant local release as well as VP secretion in distant target sites. Finally, action potentials evoked in a single magnocellular neurosecretory cell in the supraoptic nucleus provoked significant VP release from the somatodendritic compartment of the neuron. These results indicate that sniffer cells can be used for the study of VP secretion from various compartments of neurons in living tissue. NEW & NOTEWORTHY The specific functional roles of neuropeptides in neuronal networks are poorly understood due to the absence of methods allowing their real-time detection in living tissue. Here, we show that cultured "sniffer cells" can be engineered to detect endogenous release of vasopressin as an increase in fluorescence.

Patient and clinician perspectives on the outpatient after-visit summary: a qualitative study to inform improvements in visit summary design.

OBJECTIVE: We explored patients' and clinicians' perspectives on electronic health record (EHR)-generated outpatient after-visit summaries (AVSs) to inform efforts to maximize the document's utility. MATERIALS AND METHODS: This qualitative study involved focus groups and semistructured interviews with patients ( n = 39) and clinicians ( n = 56) in adult primary care practices serving socioeconomically diverse communities in New York City; Long Island, New York; and Chicago, Illinois. Focus group and interview transcripts were coded and analyzed following standard qualitative methods. RESULTS: Core themes included the use and purpose of the AVS, content modification and prioritization, formatting improvements, customization, privacy and accuracy concerns, and clinician workflow concerns. While most patients valued the document as a visit summary, others considered it a general summary of their health and health care issues, useful for sharing with family or clinicians even if they had access to their health records via web portals. Patients expressed a preference for the order of content items, and many wanted the reasons for medications and referrals stated. Additionally, some patients were confused by multiple medication lists indicating started, stopped, and modified medications, and a single "current" medication list was preferred by both patients and doctors. Concerns were raised about the risk of violating patient privacy and challenges to clinician workflow. DISCUSSION: The AVS is valued by patients and clinicians. Both groups have identified numerous ways it can be improved, but also several obstacles to improvement and effective use. CONCLUSION: EHR vendors should work with stakeholder groups to improve the AVS to ensure that this important communication device achieves its patient-centered potential.

Characteristics of outpatient clinical summaries in the United States.

In the United States, federal regulations require that outpatient practices provide a clinical summary to ensure that patients understand what transpired during their appointment and what to do before the next visit. To determine whether clinical summaries are appropriately designed to achieve these objectives, we examined their content and formatting and their usability. We obtained a convenience sample of clinical summaries from 13 diverse practices across the U.S. and assessed their characteristics using validated measures. We also interviewed key informants at these practices to assess their views of the documents. The summaries were generated by seven different electronic health record platforms. They had small font sizes (median, 10 point) and high reading grade levels (median, 10). Suitability, measured with the Suitability Assessment of Materials was low (median score, 61%) and understandability and actionability, measured with the Patient Education Materials Assessment Test, were fair to moderate (65% and 78%, respectively). Content and order of content were inconsistent across the summaries. Among physicians, 46% found the summaries helpful for clarifying medications while 38% found them helpful for conveying follow-up information. Results suggest that clinical summaries in the U.S. may often be suboptimally designed for communicating important information with patients. A patient-centered approach to designing them is warranted.

Arginase-1 is expressed exclusively by infiltrating myeloid cells in CNS injury and disease.

Resident microglia and infiltrating myeloid cells play important roles in the onset, propagation, and resolution of inflammation in central nervous system (CNS) injury and disease. Identifying cell type-specific mechanisms will help to appropriately target interventions for tissue repair. Arginase-1 (Arg-1) is a well characterised modulator of tissue repair and its expression correlates with recovery after CNS injury. Here we assessed the cellular localisation of Arg-1 in two models of CNS damage. Using microglia specific antibodies, P2ry12 and Fc receptor-like S (FCRLS), we show the LysM-EGFP reporter mouse is an excellent model to distinguish infiltrating myeloid cells from resident microglia. We show that Arg-1 is expressed exclusively in infiltrating myeloid cells but not microglia in models of spinal cord injury (SCI) and experimental autoimmune encephalomyelitis (EAE). Our in vitro studies suggest that factors in the CNS environment prevent expression of Arg-1 in microglia in vivo. This work suggests different functional roles for these cells in CNS injury and repair and shows that such repair pathways can be switched on in infiltrating myeloid cells in pro-inflammatory environments.