MYD88L265P augments proximal B-cell receptor signaling in large B-cell lymphomas via an interaction with DOCK8.

Diffuse large B-cell lymphoma (DLBCL) is a clinically and genetically heterogeneous disease with at least 5 recognized molecular subtypes. Cluster 5 (C5)/MCD tumors frequently exhibit concurrent alterations in the toll-like receptor (TLR) and B-cell receptor (BCR) pathway members, MYD88L265P and CD79B, and have a less favorable prognosis. In healthy B cells, the synergy between TLR and BCR signaling pathways integrates innate and adaptive immune responses and augments downstream NF-κB activation. In addition, physiologic TLR9 pathway engagement via MYD88, protein tyrosine kinase 2 (PYK2), and dedicator of cytokinesis 8 (DOCK8) increases proximal BCR signaling in healthy murine B cells. Although C5/MCD DLBCLs are selectively sensitive to Bruton tyrosine kinase (BTK) inhibition in in vitro studies and certain clinical trials, the role of mutated MYD88 in proximal BCR signaling remains undefined. Using engineered DLBCL cell line models, we found that concurrent MYD88L265P and CD79B alterations significantly increased the magnitude and duration of proximal BCR signaling, at the level of spleen tyrosine kinase and BTK, and augmented PYK2-dependent DOCK8 phosphorylation. MYD88L265P DLBCLs have significantly increased colocalization of DOCK8 with both MYD88 and the proximal BCR-associated Src kinase, LYN, in comparison with MYD88WT DLBCLs, implicating DOCK8 in MYD88L265P/proximal BCR cross talk. Additionally, DOCK8 depletion selectively decreased proximal BCR signaling, cellular proliferation, and viability of DLBCLs with endogenous MYD88L265P/CD79BY196F alterations and increased the efficacy of BTK blockade in these lymphomas. Therefore, MYD88L265P/DOCK8-enhanced proximal BCR signaling is a likely mechanism for the increased sensitivity of C5/MCD DLBCLs to BTK blockade.

DTX3L E3 ligase targets p53 for degradation at poly ADP-ribose polymerase-associated DNA damage sites.

P53 is a master transcriptional regulator and effector of the DNA damage response (DDR) that localizes to DNA damage sites, in part, via an interaction with PARP1. However, the mechanisms that regulate p53 abundance and activity at PARP1-decorated DNA damage sites remain undefined. The PARP9 (BAL1) macrodomain-containing protein and its partner DTX3L (BBAP) E3 ligase are rapidly recruited to PARP1-PARylated DNA damage sites. During an initial DDR, we found that DTX3L rapidly colocalized with p53, polyubiquitylated its lysine-rich C-terminal domain, and targeted p53 for proteasomal degradation. DTX3L knockout significantly increased and prolonged p53 retention at PARP-decorated DNA damage sites. These findings reveal a non-redundant, PARP- and PARylation-dependent role for DTX3L in the spatiotemporal regulation of p53 during an initial DDR. Our studies suggest that targeted inhibition of DTX3L may augment the efficacy of certain DNA-damaging agents by increasing p53 abundance and activity.

The Drosophila Duox maturation factor is a key component of a positive feedback loop that sustains regeneration signaling.

Regenerating tissue must initiate the signaling that drives regenerative growth, and sustain that signaling long enough for regeneration to complete. How these key signals are sustained is unclear. To gain a comprehensive view of the changes in gene expression that occur during regeneration, we performed whole-genome mRNAseq of actively regenerating tissue from damaged Drosophila wing imaginal discs. We used genetic tools to ablate the wing primordium to induce regeneration, and carried out transcriptional profiling of the regeneration blastema by fluorescently labeling and sorting the blastema cells, thus identifying differentially expressed genes. Importantly, by using genetic mutants of several of these differentially expressed genes we have confirmed that they have roles in regeneration. Using this approach, we show that high expression of the gene moladietz (mol), which encodes the Duox-maturation factor NIP, is required during regeneration to produce reactive oxygen species (ROS), which in turn sustain JNK signaling during regeneration. We also show that JNK signaling upregulates mol expression, thereby activating a positive feedback signal that ensures the prolonged JNK activation required for regenerative growth. Thus, by whole-genome transcriptional profiling of regenerating tissue we have identified a positive feedback loop that regulates the extent of regenerative growth.

A rapid, gentle and scalable method for dissociation and fluorescent sorting of imaginal disc cells for mRNA sequencing.

Dissociation of imaginal disc cells has been carried out previously to enable flow cytometry and cell sorting to analyze cell cycle progression, cell size, gene expression, and other aspects of imaginal tissues. However, the lengthy dissociation protocols employed may alter gene expression, cell behavior and overall viability. Here we describe a new rapid and gentle method of dissociating the cells of wing imaginal discs that significantly enhances cell viability and reduces the likelihood of gene expression changes. Furthermore, this method is scalable, enabling collection of large amounts of sample for high-throughput experiments without the need for data-distorting amplifications.

Trithorax regulates systemic signaling during Drosophila imaginal disc regeneration.

Although tissue regeneration has been studied in a variety of organisms, from Hydra to humans, many of the genes that regulate the ability of each animal to regenerate remain unknown. The larval imaginal discs of the genetically tractable model organism Drosophila melanogaster have complex patterning, well-characterized development and a high regenerative capacity, and are thus an excellent model system for studying mechanisms that regulate regeneration. To identify genes that are important for wound healing and tissue repair, we have carried out a genetic screen for mutations that impair regeneration in the wing imaginal disc. Through this screen we identified the chromatin-modification gene trithorax as a key regeneration gene. Here we show that animals heterozygous for trithorax are unable to maintain activation of a developmental checkpoint that allows regeneration to occur. This defect is likely to be caused by abnormally high expression of puckered, a negative regulator of Jun N-terminal kinase (JNK) signaling, at the wound site. Insufficient JNK signaling leads to insufficient expression of an insulin-like peptide, dILP8, which is required for the developmental checkpoint. Thus, trithorax regulates regeneration signaling and capacity.

Histomorphometry of preterm and term human placentas / Histomorfometría de placentas de pretérmino y término

The intra-uterine existence of foetus is dependent on placenta, a major organ of nutrition and homeostasis.The present study was carried out to compare morphometric and histological changes in preterm and term human placentas. Eighty placentas collected from Department of Obstetrics and Gynecology, JNMCH, AMU, Aligarh, were divided into group first of preterm placentas up to 36 weeks (n =30) and second group of full term placentas i.e. 37 to 40 weeks ( n = 50) respectively. The samples were fixed in 10 percent formol-saline solution. The gross morphological variables of placentas were studied. There was a significant increase in the placental weight, decidual area and umbilical cord diameter of term placenta as compared to that of the preterm ones. From each placenta whole thickness tissue blocks were taken and processed for paraffin sectioning. Five µ-thick sections were stained with Haematoxylin-eosin and Van Gieson stains and processed for light microscopy. A total of 200 villi were studied in each sample under high power field and occurrence of different features was expressed as percentages for each parameter. The appearance of microvilli and syncytial bud on the syncytium were almost absent in the villi of term placentas. It was concluded that with increasing gestational age there was a gradual increase in the number of capillaries in villi from preterm to term placenta.There was a significant increase in syncytial knot count, fibrinoid necrosis, vasculosyncytial membrane and decrease in the percentage of villi showing cytotrophoblastic cells and number of Hofbauer cells in term group as compared to preterm group.

La existencia intrauterina del feto depende de la placenta, el mayor órgano de nutrición y homeostasis. El estudio se llevó a cabo para comparar los cambios morfométricos e histológicos de la placenta humana de término y pretérmino. Ochenta placentas fueron obtenidas del Departamento de Obstetricia y Ginecología, JNMCH, AMU, Aligarh y se dividieron en grupos, el primer grupo de placentas de pretérmino hasta 36 semanas (n = 30) y el segundo grupo de placentas de término, de 37 a 40 semanas (n = 50 ). Las muestras fueron fijadas en solución de formol-salina al 10 por ciento. Se estudiaron las variables morfológicas macroscópicas de las placentas. Hubo un aumento significativo en el peso de la placenta, el área de decidua y el diámetro del cordón umbilical de la placenta a término en comparación con la de los prematuros. De cada placenta se tomaron y se procesaron bloques de tejido para incluirlos en parafina. Cortes de 5 µm fueron teñidos con HE y Van Gieson para microscopía óptica. De cada muestra fueron estudiadas 200 vellosidades, bajo campo de alta resolución y la aparición de diferentes características se expresó como porcentajes para cada parámetro. La aparición de las microvellosidades y brote sincitial en el sincitio estaban casi ausente en las vellosidades de las placentas de término. Se puede concluir que al aumentar la edad gestacional hubo un aumento gradual en el número de capilares en las vellosidades de la placenta de término. Existe un aumento significativo en el recuento de nudo sincitial, necrosis fibrinoide, membrana vasculosincisial y disminución en el porcentaje de las vellosidades que muestran células citotrofoblástica y número de células de Hofbauer en las placentas del término de grupo en comparación con el grupo de pretérmino.

Epithelial neoplasia in Drosophila entails switch to primitive cell states.

Only select cell types in an organ display neoplasia when targeted oncogenically. How developmental lineage hierarchies of these cells prefigure their neoplastic propensities is not yet well-understood. Here we show that neoplastic Drosophila epithelial cells reverse their developmental commitments and switch to primitive cell states. In a context of alleviated tissue surveillance, for example, loss of Lethal giant larvae (Lgl) tumor suppressor in the wing primordium induced epithelial neoplasia in its Homothorax (Hth)-expressing proximal domain. Transcriptional profile of proximally transformed mosaic wing epithelium and functional tests revealed tumor cooperation by multiple signaling pathways. In contrast, lgl(-) clones in the Vestigial (Vg)-expressing distal wing epithelium were eliminated by cell death. Distal lgl(-) clones, however, could transform when both tissue surveillance and cell death were compromised genetically and, alternatively, when the transcription cofactor of Hippo signaling pathway, Yorkie (Yki), was activated, or when Ras/EGFR signaling was up-regulated. Furthermore, transforming distal lgl(-) clones displayed loss of Vg, suggesting reversal of their terminal cell fate commitment. In contrast, reinforcing a distal (wing) cell fate commitment in lgl(-) clones by gaining Vg arrested their neoplasia and induced cell death. We also show that neoplasia in both distal and proximal lgl(-) clones could progress in the absence of Hth, revealing Hth-independent wing epithelial neoplasia. Likewise, neoplasia in the eye primordium resulted in loss of Elav, a retinal cell marker; these, however, switched to an Hth-dependent primitive cell state. These results suggest a general characteristic of "cells-of-origin" in epithelial cancers, namely their propensity for switch to primitive cell states.

Spatial and temporal variation in indicator microbe sampling is influential in beach management decisions.

Fecal indicator microbes, such as enterococci, are often used to assess potential health risks caused by pathogens at recreational beaches. Microbe levels often vary based on collection time and sampling location. The primary goal of this study was to assess how spatial and temporal variations in sample collection, which are driven by environmental parameters, impact enterococci measurements and beach management decisions. A secondary goal was to assess whether enterococci levels can be predictive of the presence of Staphylococcus aureus, a skin pathogen. Over a ten-day period, hydrometeorologic data, hydrodynamic data, bather densities, enterococci levels, and S. aureus levels including methicillin-resistant S. aureus (MRSA) were measured in both water and sand. Samples were collected hourly for both water and sediment at knee-depth, and every 6 h for water at waist-depth, supratidal sand, intertidal sand, and waterline sand. Results showed that solar radiation, tides, and rainfall events were major environmental factors that impacted enterococci levels. S. aureus levels were associated with bathing load, but did not correlate with enterococci levels or any other measured parameters. The results imply that frequencies of advisories depend heavily upon sample collection policies due to spatial and temporal variation of enterococci levels in response to environmental parameters. Thus, sampling at different times of the day and at different depths can significantly impact beach management decisions. Additionally, the lack of correlation between S. aureus and enterococci suggests that use of fecal indicators may not accurately assess risk for some pathogens.

Resistance to the antilipolytic effect of insulin in adipocytes of African-American compared to Caucasian postmenopausal women.

High fatty acid (FA) flux is associated with systemic insulin resistance, and African-American (AA) women tend to be more insulin resistant. We assessed possible depot and race difference in the antilipolytic effect of insulin in adipocytes isolated from abdominal (Abd) and gluteal (Glt) subcutaneous (sc) adipose tissue of overweight, postmenopausal AA and Caucasian (C) women. Percent body fat, fasting insulin, visceral adiposity, and adipocyte size was higher in AA women. Disinhibited lipolysis (presence of adenosine deaminase) per unit adipocyte surface area was similar in Abd and Glt and in AA and C. However, rates of 'basal' [submaximal phenylisopropyl adenosine (PIA)-suppressed] and insulin-suppressed lipolysis were higher in Abd of AA compared with C women even after adjustment for percent fat and visceral fat area. The race difference in rates of PIA- and insulin-suppressed lipolysis in AA were correlated with their hyperinsulinemia, but AA race, independent of fasting insulin, was associated with lower responsiveness (percent suppression) to submaximal insulin concentrations, although sensitivity (ED50) was not affected. Overall, these data are consistent with the hypothesis that decreased responsiveness of Abd adipocytes to antilipolytic effectors may contribute to higher FA availability and thereby to racial differences in insulin resistance.