Structure-based discovery of low molecular weight compounds that stimulate neurite outgrowth and substitute for nerve growth factor

Olanzapine, an atypical antipsychotic drug, was previously shown to protect neuronal cells against nutrient deprivation and to enhance neurite outgrowth. In an effort to identify small molecules with greater potency, the structure of olanzapine was used as a template to search commercially available chemical inventories for compounds with similar features. These compounds were evaluated for their ability to protect cells against glutamine deprivation and low-serum conditions. Positive compounds, 'hits' from initial screening, were then tested for stimulation of neurite outgrowth, alone and in combination with suboptimum concentrations of nerve growth factor (NGF). Numerous neuroprotective compounds (mw < 550 Da) were identified that significantly stimulated neurite outgrowth in PC12 cells. These included 4', 6'-diamidino-2-phenylindole, a nuclear stain; staurosporine, an antibiotic and kinase inhibitor; and 2-phenylamino-adenosine, an adenosine analog. The small molecules were comparable with NGF, and in fact, replaced NGF in outgrowth assays. Pharmacophore analysis of the hits led to the design and synthesis of an active compound, LSU-D84, which represented an initial lead for drug discovery efforts.     

Dynamic Mutual Adaptation: Human-Animal Interaction in Reindeer Herding Pastoralism

Most of the existing anthropological literature that recognizes human-animal interaction as being at the core of nomadic pastoralism focuses on nomads as the only active agents of this interaction. Nomads interact with their animals by either adapting their actions to animal behavior or by changing this behavior in ways to suit them. Based on empirical material from two groups of reindeer herding nomads in northern Russia, we suggest that human-animal interaction in nomadic pastoralism can be better understood as being the result of a dynamic mutual behavioral adaptation. In the process of this adaptation, animals change their behavior in response to the herders’ actions, which in turn leads to a responsive change to herders’ patterns of actions, etc. We argue that this approach can account for the differences in both animal behavior and herding technologies across nomadic pastoralist cultures, as well as for some of the divergent developments within these cultures.     

Three-dimensional cancer cell culture in high-yield multiscale scaffolds by shear spinning

Polymeric scaffolds comprising two size scales of microfibers and submicron fibers can better support three-dimensional (3D) cell growth in tissue engineering, making them an important class of healthcare material. However, a major manufacturing barrier hampers their translation into wider practical use: scalability. Traditional production of two-scale scaffolds by electrospinning is slow and costly. For day-to-day cell cultures, the scaffolds need to be affordable, made in high yield to drive down cost. Combining expertise from academia and industry from the United Kingdom and United States, this study uses a new series of high-yield, low-cost scaffolds made by shear spinning for tissue engineering. The scaffolds comprise interwoven submicron fibers and microfibers throughout as observed under scanning electron microscopy and demonstrate good capability to support cell culturing for tumor modeling. Three model human cancer cell lines (HEK293, A549 and MCF-7) with stable expression of GFP were cultured in the scaffolds and found to exhibit efficient cell attachment and sustained 3D growth and proliferation for 30 days. Cryosection and multiphoton fluorescence microscopy confirmed the formation of compact 3D cell clusters throughout the scaffolds. In addition, comparative growth curves of 2D and 3D cultures show significant cell-type-dependent differences. This work applies high-yield shear-spun scaffolds in mammalian tissue engineering and brings practical, affordable applications of multiscale scaffolds closer to reality. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2750, 2019.     

Centrosome defects cause microcephaly by activating the 53BP1‐USP28‐TP53 mitotic surveillance pathway

Mutations in centrosome genes deplete neural progenitor cells (NPCs) during brain development, causing microcephaly. While NPC attrition is linked to TP53‐mediated cell death in several microcephaly models, how TP53 is activated remains unclear. In cultured cells, mitotic delays resulting from centrosome loss prevent the growth of unfit daughter cells by activating a pathway involving 53BP1, USP28, and TP53, termed the mitotic surveillance pathway. Whether this pathway is active in the developing brain is unknown. Here, we show that the depletion of centrosome proteins in NPCs prolongs mitosis and increases TP53‐mediated apoptosis. Cell death after a delayed mitosis was rescued by inactivation of the mitotic surveillance pathway. Moreover, 53BP1 or USP28 deletion restored NPC proliferation and brain size without correcting the upstream centrosome defects or extended mitosis. By contrast, microcephaly caused by the loss of the non‐centrosomal protein SMC5 is also TP53‐dependent but is not rescued by loss of 53BP1 or USP28. Thus, we propose that mutations in centrosome genes cause microcephaly by delaying mitosis and pathologically activating the mitotic surveillance pathway in the developing brain.     

Development of a novel strategy to target CD39 antithrombotic activity to the endothelial-platelet microenvironment in kidney ischemia–reperfusion injury

Kidney ischemia–reperfusion injury (IRI) is common during transplantation. IRI is characterised by inflammation and thrombosis and associated with acute and chronic graft dysfunction. P-selectin and its ligand PSGL-1 are cell adhesion molecules that control leukocyte-endothelial and leukocyte-platelet interactions under inflammatory conditions. CD39 is the dominant vascular nucleotidase that facilitates adenosine generation via extracellular ATP/ADP-phosphohydrolysis. Adenosine signalling is protective in renal IRI, but CD39 catalytic activity is lost with exposure to oxidant stress. We designed a P-selectin targeted CD39 molecule (rsol.CD39-PSGL-1) consisting of recombinant soluble CD39 that incorporates 20 residues of PSGL-1 that bind P-selectin. We hypothesised that rsol.CD39-PSGL-1 would maintain endothelial integrity by focusing the ectonucleotidase platelet-inhibitory activity and reducing leukocyte adhesion at the injury site. The rsol.CD39-PSGL-1 displayed ADPase activity and inhibited platelet aggregation ex vivo, as well as bound with high specificity to soluble P-selectin and platelet surface P-selectin. Importantly, mice injected with rsol.CD39-PSGL-1 and subjected to renal IRI showed significantly less kidney damage both biochemically and histologically, compared to those injected with solCD39. Furthermore, the equivalent dose of rsol.CD39-PSGL-1 had no effect on tail template bleeding times. Hence, targeting recombinant CD39 to the injured vessel wall via PSGL-1 binding resulted in substantial preservation of renal function and morphology after IRI without toxicity. These studies indicate potential translational importance to clinical transplantation and nephrology.     

Decreased Left Caudate Volume Is Associated with Increased Severity of Autistic-Like Symptoms in a Cohort of ADHD Patients and Their Unaffected Siblings

Autism spectrum disorder (ASD) symptoms frequently occur in individuals with attention-deficit/hyperactivity disorder (ADHD). While there is evidence that both ADHD and ASD have differential structural brain correlates, knowledge of the structural brain profile of individuals with ADHD with raised ASD symptoms is limited. The presence of ASD-like symptoms was measured by the Children''s Social Behavior Questionnaire (CSBQ) in a sample of typically developing controls (n = 154), participants with ADHD (n = 239), and their unaffected siblings (n = 144) between the ages of 8 and 29. Structural magnetic resonance imaging (MRI) correlates of ASD ratings were analysed by studying the relationship between ASD ratings and grey matter volumes using mixed effects models which controlled for ADHD symptom count and total brain volume. ASD ratings were significantly elevated in participants with ADHD relative to controls and unaffected siblings. For the entire group (participants with ADHD, unaffected siblings and TD controls), mixed effect models revealed that the left caudate nucleus volume was negatively correlated with ASD ratings (t = 2.83; P = 0.005). The current findings are consistent with the role of the caudate nucleus in executive function, including the selection of goals based on the evaluation of action outcomes and the use of social reward to update reward representations. There is a specific volumetric profile associated with subclinical ASD-like symptoms in participants with ADHD, unaffected siblings and controls with the caudate nucleus and globus pallidus being of critical importance in predicting the level of ASD-like symptoms in all three groups.     

The demonstration of an extramembranous location for the putative amphipathic helix of the acetylcholine receptor

It has been demonstrated that the lysyl residues at the centers of the potential amphipathic helices of the alpha- and beta-subunits could be readily labeled in native acetylcholine receptor with anionic electrophiles. This result indicates that the regions in the sequences of the alpha- and beta-polypeptides which patterns of hydropathy are those of amphipathic helices are fully exposed on the surface of the native protein and do not span the membrane.     

Immediate exercise hyperemia in humans is contraction intensity dependent: evidence for rapid vasodilation.

We tested the hypothesis that rapid vasodilation proportional to contraction intensity contributes to the immediate (first cardiac cycle after initial contraction) exercise hyperemia. Ten healthy subjects performed single 1-s isometric forearm contractions at 5, 10, 15, 20, 30, 50, and 70% maximal voluntary contraction intensity (MVC) in arm above heart (AH) and below heart (BH) positions. Forearm blood flow (FBF; brachial artery mean blood velocity, Doppler ultrasound), mean arterial pressure (arterial tonometry), and heart rate (electrocardiogram) were measured beat by beat. Venous emptying (measured with a forearm strain gauge) was already maximized at 5% MVC, indicating that increases in contraction intensity did not further empty the forearm veins. Immediate increases in FBF were linearly proportional to contraction intensity from 5 to 70% MVC in AH (slope = 4.4 +/- 0.5%DeltaFBF/%MVC). In BH, the immediate increase in FBF demonstrated a curvilinear relationship with increasing contraction intensity and was greater than AH at 15, 20, 30, and 50% MVC (P < 0.05). Peak changes in FBF were greater in BH vs. AH from 10 to 50% MVC, even when venous refilling was complete (P < 0.05). These data support the existence of a rapid-acting vasodilatory mechanism(s) at the onset of human forearm exercise.