BLOC-2 Subunit HPS6 Deficiency Affects the Tubulation and Secretion of Von Willebrand Factor from Mouse Endothelial Cells

Publication date: Available online 21 October 2016
Source:Journal of Genetics and Genomics
Author(s): Jing Ma, Zhe Zhang, Lin Yang, Janos Kriston-Vizi, Daniel F. Cutler, Wei Li
Hermansky-Pudlak syndrome (HPS) is a recessive disorder with bleeding diathesis, which has been linked to platelet granule defects. Both platelet granules and endothelial Weibel-Palade bodies (WPBs) are members of lysosome-related organelles (LROs) whose formation is regulated by HPS protein associated complexes such as BLOC (biogenesis of lysosome organelles complex) -1, -2, -3, AP-3 (adaptor protein complex-3) and HOPS (homotypic fusion and protein sorting complex). Von Willebrand factor (VWF) is critical to hemostasis, which is stored in a highly-multimerized form as tubules in the WPBs. In this study, we found the defective, but varying, release of VWF into plasma after desmopressin (DDAVP) stimulation in HPS1 (BLOC-3 subunit), HPS6 (BLOC-2 subunit), and HPS9 (BLOC-1 subunit) deficient mice. In particular, VWF tubulation, a critical step in VWF maturation, was impaired in HPS6 deficient WPBs. This likely reflects a defective endothelium, contributing to the bleeding tendency in HPS mice or patients. The differentially defective regulated release of VWF in these HPS mouse models suggests the need for precise HPS genotyping before DDAVP administration to HPS patients.



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cdc-25.4, a Caenorhabditis elegans Ortholog of cdc25, Is Required for Male Mating Behavior

Cell division cycle 25 (cdc25) is an evolutionarily conserved phosphatase that promotes cell cycle progression. Among the four cdc25 orthologs in Caenorhabditis elegans, we found that cdc-25.4 mutant males failed to produce outcrossed progeny. This was not caused by defects in sperm development, but by defects in male mating behavior. The cdc-25.4 mutant males showed various defects during male mating, including contact response, backing, turning, and vulva location. Aberrant turning behavior was the most prominent defect in the cdc-25.4 mutant males. We also found that cdc-25.4 is expressed in many neuronal cells throughout development. The turning defect in cdc-25.4 mutant males was recovered by cdc-25.4 transgenic expression in neuronal cells, suggesting that cdc-25.4 functions in neurons for male mating. However, the neuronal morphology of cdc-25.4 mutant males appeared to be normal, as examined with several neuronal markers. Also, RNAi depletion of wee-1.3, a C. elegans ortholog of Wee1/Myt1 kinase, failed to suppress the mating defects of cdc-25.4 mutant males. These findings suggest that, for successful male mating, cdc-25.4 does not target cell cycles that are required for neuronal differentiation and development. Rather, cdc-25.4 likely regulates non-canonical substrates in neuronal cells.

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The Interaction of Genotype and Environment Determines Variation in the Maize Kernel Ionome

Plants obtain soil-resident elements that support growth and metabolism from the water- flow facilitated by transpiration and active transport processes. The availability of elements in the environment interacts with the genetic capacity of organisms to modulate element uptake through plastic adaptive responses, such as homeostasis. These interactions should cause the elemental contents of plants to vary such that the effects of genetic polymorphisms will be dramatically dependent on the environment in which the plant is grown. To investigate genotype by environment interactions underlying elemental accumulation, we analyzed levels of elements in maize kernels of the Intermated B73 x Mo17 (IBM) recombinant inbred population grown in 10 different environments spanning a total of six locations and five different years. In analyses conducted separately for each environment, we identified a total of 79 quantitative trait loci controlling seed elemental accumulation. While a set of these QTL were found in multiple environments, the majority were specific to a single environment, suggesting the presence of genetic by environment interactions. To specifically identify and quantify QTL by environment interactions (QEIs), we implemented two methods: linear modeling with environmental covariates and QTL analysis on trait differences between growouts. With these approaches, we found several instances of QEI, indicating that elemental profiles are highly heritable, interrelated, and responsive to the environment.

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Micro-environmental Gene Expression Plasticity Among Individual Drosophila melanogaster

Differences in phenotype among genetically identical individuals exposed to the same environmental condition are often noted in genetic studies. Despite this commonplace observation, little is known about the causes of this variability, which has been termed microenvironmental plasticity. One possibility is that stochastic or technical sources of variance produce these differences. A second possibility is that this variation has a genetic component. We have explored gene expression robustness in the transcriptomes of 730 individual Drosophila melanogaster of 16 fixed genotypes, 9 of which are infected with Wolbachia. Three replicates of flies were grown controlling for food, day/night cycles, humidity, temperature, sex, mating status, social exposure, and circadian timing of RNA extraction. Despite the use of inbred genotypes and carefully controlled experimental conditions, thousands of genes were differentially expressed, revealing a unique and dynamic transcriptional signature for each individual fly. We found that 23% of the transcriptome was differentially expressed among individuals, and that the variability in gene expression among individuals is influenced by genotype. This transcriptional variation originated from specific gene pathways, suggesting a plastic response to the micro-environment; but there was also evidence of gene expression differences due to stochastic fluctuations. These observations reveal previously unappreciated genetic sources of variability in gene expression among individuals, which has implications for complex trait genetics and precision medicine.

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Robust Transgene Expression from Bicistronic mRNA in the Green Alga Chlamydomonas reinhardtii

The unicellular green alga Chlamydomonas reinhardtii is a model organism that provides an opportunity to understand the evolution and functional biology of the lineage that includes the land plants, as well as aspects of the fundamental core biology conserved throughout the eukaryotic phylogeny. Although many tools are available to facilitate genetic, molecular biological, biochemical, and cell biological studies in Chlamydomonas, expression of unselected transgenes of interest (GOIs) has been challenging. In most methods used previously, the GOI and a selectable marker are expressed from two separate mRNAs, so that their concomitant expression is not guaranteed. In this study, we developed constructs that allow expression of an upstream GOI and downstream selectable marker from a single bicistronic mRNA. Although this approach in other systems has typically required a translation-enhancing element such as an internal ribosome-entry site for the downstream marker, we found that a short stretch of unstructured junction sequence was sufficient to obtain adequate expression of the downstream gene, presumably through post-termination reinitiation. With this system, we obtained robust expression of both endogenous and heterologous GOIs, including fluorescent proteins and tagged fusion proteins, in the vast majority of transformants, thus eliminating the need for tedious secondary screening for GOI-expressing transformants. This improved efficiency should greatly facilitate a variety of genetic and cell-biological studies in Chlamydomonas and also enable new applications such as expression-based screens and large-scale production of foreign proteins.

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The role of endobronchial ultrasonography for mediastinal lymphadenopathy in cases with extrathoracic malignancy

Abstract

Introduction

Many extrathoracic malignancies can metastasize to lungs and mediastinal lymph nodes. Whether mediastinal lesions are metastasis in these patients changes staging, prognosis, and treatment strategy. In this study, we aimed to find out the contribution of EBUS-TBNA to the diagnosis in cases with extrathoracic malignancy.

Materials and methods

Patients who had been previously diagnosed as extrapulmonary solid organ malignancy and in whom mediastinal or hilar lymphadenopathy developed during their follow-up and EBUS-TBNA was applied for diagnostic purposes were retrospectively included in this study.

Results

A total of 91 patients consisting of 35 females (38.5 %) and 56 males (61.5 %) were included in the study. The mean age of the patients was 60.5 (±11.4). Malignancy was not observed in 54 (59.3 %) patients; primary malignancy metastasis was detected in 33 (36.3 %) patients, and primary lung cancer was detected in 4 (4.4 %) patients with EBUS-TBNA. The sensitivity of EBUS-TBNA in extrathoracic malignancies was determined as 90.2 %; its specificity was determined as 100 %, its negative predictive value as 92.5 %, its positive predictive value as 100 %, and its diagnostic accuracy as 95.6 %. The highest rate was determined in the left lower paratracheal lymph node when they were examined in terms of malignancy detection rate in lymph node stations.

Conclusion

EBUS-TBNA is a minimally invasive method with quite a low complication rate that does not require general anesthesia. It should be the first step method to be used in the diagnosis of mediastinal and hilar lymphadenopathies seen in extrathoracic malignancies since it has high diagnostic accuracy, sensitivity, and specificity. EBUS-TBNA significantly reduces the need for surgical intervention. Further surgical interventions can be planned in patients in whom diagnostic competence is not ensured.

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Simulation-based camera navigation training in laparoscopy—a randomized trial

Abstract

Background

Inexperienced operating assistants are often tasked with the important role of handling camera navigation during laparoscopic surgery. Incorrect handling can lead to poor visualization, increased operating time, and frustration for the operating surgeon—all of which can compromise patient safety. The objectives of this trial were to examine how to train laparoscopic camera navigation and to explore the transfer of skills to the operating room.

Materials and methods

A randomized, single-center superiority trial with three groups: The first group practiced simulation-based camera navigation tasks (camera group), the second group practiced performing a simulation-based cholecystectomy (procedure group), and the third group received no training (control group). Participants were surgical novices without prior laparoscopic experience. The primary outcome was assessment of camera navigation skills during a laparoscopic cholecystectomy. The secondary outcome was technical skills after training, using a previously developed model for testing camera navigational skills. The exploratory outcome measured participants' motivation toward the task as an operating assistant.

Results

Thirty-six participants were randomized. No significant difference was found in the primary outcome between the three groups (p = 0.279). The secondary outcome showed no significant difference between the interventions groups, total time 167 s (95% CI, 118–217) and 194 s (95% CI, 152–236) for the camera group and the procedure group, respectively (p = 0.369). Both interventions groups were significantly faster than the control group, 307 s (95% CI, 202–412), p = 0.018 and p = 0.045, respectively. On the exploratory outcome, the control group for two dimensions, interest/enjoyment (p = 0.030) and perceived choice (p = 0.033), had a higher score.

Conclusions

Simulation-based training improves the technical skills required for camera navigation, regardless of practicing camera navigation or the procedure itself. Transfer to the clinical setting could, however, not be demonstrated. The control group demonstrated higher interest/enjoyment and perceived choice than the camera group.

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