Monocytes prevent apoptosis of iPSCs and promote differentiation of kidney organoids.

BACKGROUND: Induced pluripotent stem cells (iPSCs)-derived kidney organoids are a promising model for studying disease mechanisms and renal development. Despite several protocols having been developed, further improvements are needed to overcome existing limitations and enable a wider application of this model. One of the approaches to improve the differentiation of renal organoids in vitro is to include in the system cell types important for kidney organogenesis in vivo, such as macrophages. Another approach could be to improve cell survival. Mesodermal lineage differentiation is the common initial step of the reported protocols. The glycogen synthase kinase-3 (GSK-3) activity inhibitor, CHIR99021 (CHIR), is applied to induce mesodermal differentiation. It has been reported that CHIR simultaneously induces iPSCs apoptosis that can compromise cell differentiation. We thought to interfere with CHIR-induced apoptosis of iPSCs using rapamycin. METHODS: Differentiation of kidney organoids from human iPSCs was performed. Cell survival and autophagy were analyzed using Cell counting kit 8 (CCK8) kit and Autophagy detection kit. Cells were treated with rapamycin or co-cultured with human monocytes isolated from peripheral blood or iPSCs-macrophages using a transwell co-culture system. Monocyte-derived extracellular vesicles (EVs) were isolated using polyethylene glycol precipitation. Expression of apoptotic markers cleaved Caspase 3, Poly [ADP-ribose] polymerase 1 (PARP-1) and markers of differentiation T-Box Transcription Factor 6 (TBX6), odd-skipped related 1 (OSR1), Nephrin, E-Cadherin, Paired box gene 2 (Pax2) and GATA Binding Protein 3 (Gata3) was assessed by RT-PCR and western blotting. Organoids were imaged by 3D-confocal microscopy. RESULTS: We observed that CHIR induced apoptosis of iPSCs during the initial stage of renal organoid differentiation. Underlying mechanisms implied the accumulation of reactive oxygen species and decreased autophagy. Activation of autophagy by rapamacin and by an indirect co-culture of differentiating iPSCs with iPSCs-macrophages and human peripheral blood monocytes prevented apoptosis induced by CHIR. Furthermore, monocytes (but not rapamycin) strongly promoted expression of renal differentiation markers and organoids development via released extracellular vesicles. CONCLUSION: Our data suggest that co-culturing of iPSCs with human monocytes strongly improves differentiation of kidney organoids. An underlying mechanism of monocytic action implies, but not limited to, an increased autophagy in CHIR-treated iPSCs. Our findings enhance the utility of kidney organoid models.

The G-alpha Gpa1 directs septin localization in the mating projection of Saccharomyces cerevisiae through its Ubiquitination Domain and Endocytic Machinery.

The yeast mating response uses a G-protein coupled receptor (GPCR), Ste2, to detect mating pheromone and initiate mating projection morphogenesis. The septin cytoskeleton plays a key role in the formation of the mating projection, forming structures at the base of the projection. Desensitization of the Gα, Gpa1, by the Regulator of G-protein Signaling (RGS), Sst2, is required for proper septin organization and morphogenesis. In cells where the Gα is hyperactive, septins are mislocalized to the site of polarity, and the cells are unable to track a pheromone gradient. We set out to identify the proteins that mediate Gα control of septins during the Saccharomyces cerevisiae mating response by making mutations to rescue septin localization in cells expressing the hyperactive Gα mutant gpa1G302S. We found that single deletions of the septin chaperone Gic1, the Cdc42 GAP Bem3, and the epsins Ent1 and Ent2 rescued the polar cap accumulation of septins in the hyperactive Gα. We created an agent-based model of vesicle trafficking that predicts how changes in endocytic cargo licensing alters localization of endocytosis that mirrors the septin localization we see experimentally. We hypothesized that hyperactive Gα may increase the rate of endocytosis of a pheromone responsive cargo, thereby altering where septins are localized. Both the GPCR and the Gα are known to be internalized by clathrin-mediated endocytosis during the pheromone response. Deletion of the GPCR C-terminus to block internalization partially rescued septin organization. However, deletion of the Gpa1 ubiquitination domain required for its endocytosis completely abrogated septin accumulation at the polarity site. Our data support a model where the location of endocytosis serves as a spatial mark for septin structure assembly and that desensitization of the Gα delays its endocytosis sufficiently that septins are placed peripheral to the site of Cdc42 polarity.

Viewpoint on the Second Transatlantic GPCR Symposium for Early Career Investigators.

The second Transatlantic Early Career Investigator (ECI) G Protein-Coupled Receptor (GPCR) Symposium was an online scientific meeting geared at young GPCR investigators, with the primary goal of expanding opportunities for sharing research and networking among trainees in North America and Europe. Here, we discuss the format of our meeting, its impact, and the challenges and opportunities facing meetings like it in the future.

Phosphorylation of RGS regulates MAP kinase localization and promotes completion of cytokinesis.

Yeast use the G-protein-coupled receptor signaling pathway to detect and track the mating pheromone. The G-protein-coupled receptor pathway is inhibited by the regulator of G-protein signaling (RGS) Sst2 which induces Gα GTPase activity and inactivation of downstream signaling. G-protein signaling activates the MAPK Fus3, which phosphorylates the RGS; however, the role of this modification is unknown. We found that pheromone-induced RGS phosphorylation peaks early; the phospho-state of RGS controls its localization and influences MAPK spatial distribution. Surprisingly, phosphorylation of the RGS promotes completion of cytokinesis before pheromone-induced growth. Completion of cytokinesis in the presence of pheromone is promoted by the kelch-repeat protein, Kel1 and antagonized by the formin Bni1. We found that RGS complexes with Kel1 and prefers the unphosphorylatable RGS mutant. We also found overexpression of unphosphorylatable RGS exacerbates cytokinetic defects, whereas they are rescued by overexpression of Kel1. These data lead us to a model where Kel1 promotes completion of cytokinesis before pheromone-induced polarity but is inhibited by unphosphorylated RGS binding.

Early expression of mature αß TCR in CD4-CD8- T cell progenitors enables MHC to drive development of T-ALL bearing NOTCH mutations.

During normal T cell development in mouse and human, a low-frequency population of immature CD4-CD8- double-negative (DN) thymocytes expresses early, mature αß T cell antigen receptor (TCR). We report that these early αß TCR+ DN (EADN) cells are DN3b-DN4 stage and require CD3δ but not major histocompatibility complex (MHC) for their generation/detection. When MHC - is present, however, EADN cells can respond to it, displaying a degree of coreceptor-independent MHC reactivity not typical of mature, conventional αß T cells. We found these data to be connected with observations that EADN cells were susceptible to T cell acute lymphoblastic leukemia (T-ALL) transformation in both humans and mice. Using the OT-1 TCR transgenic system to model EADN-stage αß TCR expression, we found that EADN leukemogenesis required MHC to induce development of T-ALL bearing NOTCH1 mutations. This leukemia-driving MHC requirement could be lost, however, upon passaging the tumors in vivo, even when matching MHC was continuously present in recipient animals and on the tumor cells themselves. These data demonstrate that MHC:TCR signaling can be required to initiate a cancer phenotype from an understudied developmental state that appears to be represented in the mouse and human disease spectrum.

Flipping a Single Lecture in a Survey Course to Active Learning: Do the Benefits Justify the Costs?

Health education has seen a surge of interest in active learning strategies like the flipped classroom. In response to the need for physical distancing in the age of COVID-19, schools are rapidly shifting to web-based and video technology, sometimes without being able to predict the outcomes of this change. The objectives of this pilot experiment were to (1) compare active learning (AL) methods versus traditional lecture for transmitting and retaining knowledge in the introductory pre-clinical medical school curriculum and (2) weigh whether the costs required to flip instruction were justified by learning gains. The authors took a 2 h lecture for first-year medical students and converted half of it into an AL format. In-person lecture and active learning groups were compared in terms of student knowledge at pre-intervention, immediately post-intervention, and 6 months post-intervention. Costs for first-time delivery and anticipated costs for repeat delivery of each format were calculated. Students' gains in knowledge increased in both groups, though more by lecture (control) than via AL. Delivering a single hour of new AL costs 3.4 times that of a new lecture. Repeat offerings of the AL intervention were estimated to cost 5.4 times that of the repeat lecture. The 1 h AL session was less effective than the 1 h lecture for knowledge acquisition and retention at 6-month follow-up. The AL was more expensive to produce and to repeat. Future research needs to evaluate the impact of AL with a larger N, control group, structured faculty/resident procedures, and assessment of gaining and applying attitudes and skills in addition to knowledge.

Neuropathy and neural plasticity in the subcutaneous white adipose depot.

The difficulty in obtaining as well as maintaining weight loss, together with the impairment of metabolic control in conditions like diabetes and cardiovascular disease, may represent pathological situations of inadequate neural communication between the brain and peripheral organs and tissues. Innervation of adipose tissues by peripheral nerves provides a means of communication between the master metabolic regulator in the brain (chiefly the hypothalamus), and energy-expending and energy-storing cells in the body (primarily adipocytes). Although chemical and surgical denervation studies have clearly demonstrated how crucial adipose tissue neural innervation is for maintaining proper metabolic health, we have uncovered that adipose tissue becomes neuropathic (ie: reduction in neurites) in various conditions of metabolic dysregulation. Here, utilizing both human and mouse adipose tissues, we present evidence of adipose tissue neuropathy, or loss of proper innervation, under pathophysiological conditions such as obesity, diabetes, and aging, all of which are concomitant with insult to the adipose organ as well as metabolic dysfunction. Neuropathy is indicated by loss of nerve fiber protein expression, reduction in synaptic markers, and lower neurotrophic factor expression in adipose tissue. Aging-related adipose neuropathy particularly results in loss of innervation around the tissue vasculature, which cannot be reversed by exercise. Together with indications of neuropathy in muscle and bone, these findings underscore that peripheral neuropathy is not restricted to classic tissues like the skin of distal extremities, and that loss of innervation to adipose may trigger or exacerbate metabolic diseases. In addition, we have demonstrated stimulation of adipose tissue neural plasticity with cold exposure, which may ameliorate adipose neuropathy and be a potential therapeutic option to re-innervate adipose and restore metabolic health.

The Importance of Peripheral Nerves in Adipose Tissue for the Regulation of Energy Balance.

Brown and white adipose tissues are essential for maintenance of proper energy balance and metabolic health. In order to function efficiently, these tissues require both endocrine and neural communication with the brain. Brown adipose tissue (BAT), as well as the inducible brown adipocytes that appear in white adipose tissue (WAT) after simulation, are thermogenic and energy expending. This uncoupling protein 1 (UCP1)-mediated process requires input from sympathetic nerves releasing norepinephrine. In addition to sympathetic noradrenergic signaling, adipose tissue contains sensory nerves that may be important for relaying fuel status to the brain. Chemical and surgical denervation studies of both WAT and BAT have clearly demonstrated the role of peripheral nerves in browning, thermogenesis, lipolysis, and adipogenesis. However, much is still unknown about which subtypes of nerves are present in BAT versus WAT, what nerve products are released from adipose nerves and how they act to mediate metabolic homeostasis, as well as which cell types in adipose are receiving synaptic input. Recent advances in whole-depot imaging and quantification of adipose nerve fibers, as well as other new research findings, have reinvigorated this field of research. This review summarizes the history of research into adipose innervation and brain⁻adipose communication, and also covers landmark and recent research on this topic to outline what we currently know and do not know about adipose tissue nerve supply and communication with the brain.

A peroxidized omega-3-enriched polyunsaturated diet leads to adipose and metabolic dysfunction.

Consumption of diets that differ in fat type and amount, and sequestration of various fatty acids to tissues and organs likely have effects on overall physiology and metabolic health. However, the contributions of dietary lipids to brain-adipose communication and adipose tissue function are poorly understood. We designed six custom diets that differed only in amount and type of dietary fat, with high or low levels of saturated fatty acids (SFA), omega-6 polyunsaturated fatty acids (n-6 PUFA) or omega-3 (n-3) PUFA. Mice fed the n-3 PUFA diet for 16 weeks displayed a striking reduction in weight gain accompanied by smaller adipose depots and improved glucose sensitivity. Reduced body weight occurred despite lowered energy expenditure and no difference in food intake. Despite the apparent beneficial effects to whole body physiology, we have demonstrated for the first time that a peroxidized n-3-enriched diet led to lipotoxicity of white adipose tissue, as evidenced by increased fibrosis, lipofuscin, reduced anti-inflammatory markers and loss of proper nerve supply. While healthful, n-3 fats are prone to peroxidation, and we observed peroxidated lipid metabolites in the adipose tissue of mice on these diets. Furthermore, using a lipidomics approach, we have observed that brain, white adipose tissue and brown adipose tissue accumulate lipid metabolites differently. The brain remained mostly shielded from changes in dietary fat type and amount, but differences in adipose lipid metabolites across these six diets may have affected metabolic function and brain-adipose communication, as observed in this study.

Development of subcortical volumes across adolescence in males and females: A multisample study of longitudinal changes.

The developmental patterns of subcortical brain volumes in males and females observed in previous studies have been inconsistent. To help resolve these discrepancies, we examined developmental trajectories using three independent longitudinal samples of participants in the age-span of 8-22 years (total 216 participants and 467 scans). These datasets, including Pittsburgh (PIT; University of Pittsburgh, USA), NeuroCognitive Development (NCD; University of Oslo, Norway), and Orygen Adolescent Development Study (OADS; The University of Melbourne, Australia), span three countries and were analyzed together and in parallel using mixed-effects modeling with both generalized additive models and general linear models. For all regions and across all samples, males were found to have significantly larger volumes as compared to females, and significant sex differences were seen in age trajectories over time. However, direct comparison of sample trajectories and sex differences identified within samples were not consistent. The trajectories for the amygdala, putamen, and nucleus accumbens were most consistent between the three samples. Our results suggest that even after using similar preprocessing and analytic techniques, additional factors, such as image acquisition or sample composition may contribute to some of the discrepancies in sex specific patterns in subcortical brain changes across adolescence, and highlight region-specific variations in congruency of developmental trajectories.

Identification and Validation of a PD-L1 Binding Peptide for Determination of PDL1 Expression in Tumors.

Blocking the interaction between Programmed Death Ligand 1 (PD-L1) and its receptor, PD-1, is an effective method of treating many types of cancers. Certain tumors overexpress PD-L1, causing host immune cells that express PD-1 to bind PD-L1 and cease killing the tumor. Inhibition of PD-L1 and PD-1 binding can restore host immunity towards tumor killing, and many new drugs have been developed to target this interaction. Current methods of PD-L1 diagnosis have shown to vary based on the antibody, detection kit brand, antigen retrieval method, and clinically defined methods by the FDA. To refine detection of PD-L1, we have identified a peptide, RK-10, and used it to detect PD-L1 expressing tumors with immunohistochemistry or flow cytometry. Flow cytometry was performed on cell lines and patient tissues using a fluorescent peptide (RK-10-Cy5). Immunohistochemistry using a biotin-modified peptide (RK-10-Biotin) was tested against the FDA-approved SP263 clone on biopsied patient tissues. For this study, we evaluated specificity of RK-10 using IHC in over 200 patient tissues, including NSCLC and Hodgkin's Lymphoma. RK-10 shows staining in the tumor regions of FFPE tissues where the SP263 kit does not. RK-10-Cy5 peptide also demonstrates PD-L1 detection in NSCLC, breast, squamous cell carcinoma, and melanoma.

Platelet adenosine diphosphate inhibition in trauma patients by thromboelastography correlates with paradoxical increase in platelet dense granule content by flow cytometry.

BACKGROUND: The mechanism of platelet dysfunction in acute traumatic coagulopathy is unknown. Traumatic brain injury is hypothesized as a cause, while some investigators presume platelets become "exhausted." We hypothesized that platelet hyperstimulation and consumption resulting from trauma leads to decreased platelet function secondary to depletion of platelet granules. METHODS: Twenty-five trauma patients were divided into traumatic brain injury and no traumatic brain injury groups. Healthy volunteers served as controls. All had thromboelastography with platelet mapping and flow cytometric assays of mepacrine performed. Mepacrine uptake in unstimulated platelets was used for quantification of platelet content of dense granules. RESULTS: Twelve patients with traumatic brain injury and 13 patients without traumatic brain injury were enrolled. Twenty-one trauma patients showed adenosine diphosphate inhibition (>30%) on thromboelastography with platelet mapping compared with the healthy volunteers who served as controls (P < .01). Mepacrine assay showed a difference in mean fluorescent intensity for all trauma patients of 4,259 ± 1,341 compared with controls of 3,143 ± 709 (P = .044), correlating with greater quantities of dense granules. Neither adenosine diphosphate inhibition nor average difference in mean fluorescent intensity between traumatic brain injury and no traumatic brain injury groups were significant (P = .2). CONCLUSION: Trauma patients maintain their dense granule, contradicting the theory of platelet granule exhaustion as the etiology for platelet dysfunction in traumatic brain injury.

Environmentally induced heritable changes in flax.

Some flax varieties respond to nutrient stress by modifying their genome and these modifications can be inherited through many generations. Also associated with these genomic changes are heritable phenotypic variations. The flax variety Stormont Cirrus (Pl) when grown under three different nutrient conditions can either remain inducible (under the control conditions), or become stably modified to either the large or small genotroph by growth under high or low nutrient conditions respectively. The lines resulting from the initial growth under each of these conditions appear to grow better when grown under the same conditions in subsequent generations, notably the Pl line grows best under the control treatment indicating that the plants growing under both the high and low nutrients are under stress. One of the genomic changes that are associated with the induction of heritable changes is the appearance of an insertion element (LIS-1) while the plants are growing under the nutrient stress. With respect to this insertion event, the flax variety Stormont Cirrus (Pl) when grown under three different nutrient conditions can either remain unchanged (under the control conditions), have the insertion appear in all the plants (under low nutrients) and have this transmitted to the next generation, or have the insertion (or parts of it) appear but not be transmitted through generations (under high nutrients). The frequency of the appearance of this insertion indicates that it is under positive selection, which is also consistent with the growth response in subsequent generations. Leaves or meristems harvested at various stages of growth are used for DNA and RNA isolation. The RNA is used to identify variation in expression associated with the various growth environments and/or t he presence/absence of LIS-1. The isolated DNA is used to identify those plants in which the insertion has occurred.

Factors influencing cord blood viability assessment before cryopreservation.

BACKGROUND: Cord blood (CB) viability determines product quality and varies with time and temperature of exposure before cryopreservation. Global viability assessment may not reflect viability of white blood cell (WBC) subsets, CD34+ cell viability, or hematopoietic stem/progenitor cells function. STUDY DESIGN AND METHODS: We compared trypan blue (TB) and acridine orange/propidium iodide (AO/PI) staining with flow-cytometric (7-aminoactinomycin D [7-AAD]) viability in total WBCs (Tot-AAD), granulocytes, monocytes, lymphocytes, and CD34+ cells and total nucleated cell, CD34+, and colony-forming cell (CFC) recovery as a function of time and temperature (4, 24, and 37 degrees C) before cryopreservation. RESULTS: TB, AO/PI, and Tot-AAD viability was concordant up to 72 hours (4 degrees C) and 48 hours (24 degrees C) postcollection; however, CD34+ viability was significantly higher due to loss of viable granulocytes. In contrast, at "physiologic" temperature (37 degrees C), the decline in TB, AO/PI, and Tot-AAD viability was significantly lower than the rate of viable CD34+ and CFC loss. At all times and temperatures, CFC recovery correlated best with CD34+ viability and recovery. CONCLUSIONS: CB cell populations exhibit differential time- and temperature-dependent susceptibility to in vitro cell death; consequently, global viability measurements using TB, AO/PI, or 7-AAD (Tot-AAD) significantly underestimate (4-24 degrees C) or overestimate (24-37 degrees C) CD34+ viability and CFC recovery. Our results demonstrate the limitations of global viability assessment with TB, AO/PI, and total AAD; endorse the routine use of CD34+ cell viability measurements; emphasize the importance of temperature control during shipment; and have implications with regard to establishing acceptable "cutoff" values for viability measurements and CB collection through processing time.

The effects of gender, age, ethnicity, and liver cirrhosis on cytochrome P450 enzyme activity in human liver microsomes and inducibility in cultured human hepatocytes.

We have measured cytochrome P450 (CYP) activity in nearly 150 samples of human liver microsomes and 64 samples of cryopreserved human hepatocytes, and we have performed induction studies in over 90 preparations of cultured human hepatocytes. We have analyzed these data to examine whether the expression of CYP enzyme activity in liver microsomes and isolated hepatocytes or the inducibility of CYP enzymes in cultured hepatocytes is influenced by the gender, age, or ethnicity of the donor (the latter being limited to Caucasians, African Americans, and Hispanics due to a paucity of livers from Asian donors). In human liver microsomes, there were no statistically significant differences (P > 0.05) in CYP activity as a function of age, gender, or ethnicity with one exception. 7-Ethoxyresorufin O-dealkylase (CYP1A2) activity was greater in males than females, which is consistent with clinical observation. Liver microsomal testosterone 6beta-hydroxylase (CYP3A4) activity was slightly greater in females than males, but the difference was not significant. However, in cryopreserved human hepatocytes, the gender difference in CYP3A4 activity (females = twice males) did reach statistical significance, which supports the clinical observation that females metabolize certain CYP3A4 substrates faster than do males. Compared with those from Caucasians and African Americans, liver microsomes from Hispanics had about twice the average activity of CYP2A6, CYP2B6, and CYP2C8 and half the activity of CYP1A2, although this apparent ethnic difference may be a consequence of the relatively low number of Hispanic donors. Primary cultures of hepatocytes were treated with beta-naphthoflavone, an inducer of CYP1A2, phenobarbital or rifampin, both of which induce CYP2B6, CYP2C9, CYP2C19, and CYP3A4, albeit it to different extents. Induction of these CYP enzymes in freshly cultured hepatocytes did not appear to be influenced by the gender or age of the donor. Furthermore, CYP3A4 induction in hepatocytes isolated from cirrhotic liver was comparable to that in normal hepatocytes, which supports the "healthy hepatocyte, sick environment" hypothesis of liver cirrhosis. This review summarizes these findings and discusses their implications for the use of human liver microsomes and hepatocytes for in vitro studies of drug metabolism and enzyme induction, which play a key role in drug development.