Personalized Oncogenomics: Clinical Experience with Malignant Peritoneal Mesothelioma Using Whole Genome Sequencing

Peritoneal mesothelioma is a rare and sometimes lethal malignancy that presents a clinical challenge for both diagnosis and management. Recent studies have led to a better understanding of the molecular biology of peritoneal mesothelioma. Translation of the emerging data into better treatments and outcome is needed. From two patients with peritoneal mesothelioma, we derived whole genome sequences, RNA expression profiles, and targeted deep sequencing data. Molecular data were made available for translation into a clinical treatment plan. Treatment responses and outcomes were later examined in the context of molecular findings. Molecular studies presented here provide the first reported whole genome sequences of peritoneal mesothelioma. Mutations in known mesothelioma-related genes NF2, CDKN2A, LATS2, amongst others, were identified. Activation of MET-related signaling pathways was demonstrated in both cases. A hypermutated phenotype was observed in one case (434 vs. 18 single nucleotide variants) and was associated with a favourable outcome despite sarcomatoid histology and multifocal disease. This study represents the first report of whole genome analyses of peritoneal mesothelioma, a key step in the understanding and treatment of this disease.     

Deficient and Null Variants of SERPINA1 Are Proteotoxic in a Caenorhabditis elegans Model of α1-Antitrypsin Deficiency

α1-antitrypsin deficiency (ATD) predisposes patients to both loss-of-function (emphysema) and gain-of-function (liver cirrhosis) phenotypes depending on the type of mutation. Although the Z mutation (ATZ) is the most prevalent cause of ATD, >120 mutant alleles have been identified. In general, these mutations are classified as deficient (<20% normal plasma levels) or null (<1% normal levels) alleles. The deficient alleles, like ATZ, misfold in the ER where they accumulate as toxic monomers, oligomers and aggregates. Thus, deficient alleles may predispose to both gain- and loss-of-function phenotypes. Null variants, if translated, typically yield truncated proteins that are efficiently degraded after being transiently retained in the ER. Clinically, null alleles are only associated with the loss-of-function phenotype. We recently developed a C. elegans model of ATD in order to further elucidate the mechanisms of proteotoxicity (gain-of-function phenotype) induced by the aggregation-prone deficient allele, ATZ. The goal of this study was to use this C. elegans model to determine whether different types of deficient and null alleles, which differentially affect polymerization and secretion rates, correlated to any extent with proteotoxicity. Animals expressing the deficient alleles, Mmalton, Siiyama and S (ATS), showed overall toxicity comparable to that observed in patients. Interestingly, Siiyama expressing animals had smaller intracellular inclusions than ATZ yet appeared to have a greater negative effect on animal fitness. Surprisingly, the null mutants, although efficiently degraded, showed a relatively mild gain-of-function proteotoxic phenotype. However, since null variant proteins are degraded differently and do not appear to accumulate, their mechanism of proteotoxicity is likely to be different to that of polymerizing, deficient mutants. Taken together, these studies showed that C. elegans is an inexpensive tool to assess the proteotoxicity of different AT variants using a transgenic approach.     

The Effector Domain of MARCKS Is a Nuclear Localization Signal that Regulates Cellular PIP2 Levels and Nuclear PIP2 Localization

Translocation to the nucleus of diacylglycerol kinase (DGK)– ζ is dependent on a sequence homologous to the effector domain of Myristoylated Alanine Rich C-Kinase Substrate (MARCKS). These data would suggest that MARCKS could also localize to the nucleus. A single report demonstrated immunofluorescence staining of MARCKS in the nucleus; however, further experimental evidence confirming the specific domain responsible for this localization has not been reported. Here, we report that MARCKS is present in the nucleus in GBM cell lines. We then over-expressed wild-type MARCKS (WT) and MARCKS with the effector domain deleted (ΔED), both tagged with V5-epitope in a GBM cell line with low endogenous MARCKS expression (U87). We found that MARCKS-WT localized to the nucleus, while the MARCKS construct without the effector domain remained in the cytoplasm. We also found that over-expression of MARCKS-WT resulted in a significant increase in total cellular phosphatidyl-inositol (4,5) bisphosphate (PIP₂) levels, consistent with prior evidence that MARCKS can regulate PIP₂ levels. We also found increased staining for PIP₂ in the nucleus with MARCKS-WT over-expression compared to MARCKS ΔED by immunofluorescence. Interestingly, we observed MARCKS and PIP₂ co-localization in the nucleus. Lastly, we found changes in gene expression when MARCKS was not present in the nucleus (MARCKS ΔED). These data indicate that the MARCKS effector domain can function as a nuclear localization signal and that this sequence is critical for the ability of MARCKS to regulate PIP₂ levels, nuclear localization, and gene expression. These data suggests a novel role for MARCKS in regulating nuclear functions such as gene expression.     

Testing for Odor Discrimination and Habituation in Mice

This video demonstrates a technique to establish the presence of a normally functioning olfactory system in a mouse. The test helps determine whether the mouse can discriminate between non-social odors and social odors, whether the mouse habituates to a repeatedly presented odor, and whether the mouse demonstrates dishabituation when presented with a novel odor. Since many social behavior tests measure the experimental animal’s response to a familiar or novel mouse, false positives can be avoided by establishing that the animals can detect and discriminate between social odors. There are similar considerations in learning tests such as fear conditioning that use odor to create a novel environment or olfactory cues as an associative stimulus. Deficits in the olfactory system would impair the ability to distinguish between contexts and to form an association with an olfactory cue during fear conditioning. In the odor habitation/dishabituation test, the mouse is repeatedly presented with several odors. Each odor is presented three times for two minutes. The investigator records the sniffing time directed towards the odor as the measurement of olfactory responsiveness. A typical mouse shows a decrease in response to the odor over repeated presentations (habituation). The experimenter then presents a novel odor that elicits increased sniffing towards the new odor (dishabituation). After repeated presentation of the novel odor the animal again shows habituation. This protocol involves the presentation of water, two or more non-social odors, and two social odors. In addition to reducing experimental confounds, this test can provide information on the function of the olfactory systems of new knockout, knock-in, and conditional knockout mouse lines.     

Scalable 96-well Plate Based iPSC Culture and Production Using a Robotic Liquid Handling System

Continued advancement in pluripotent stem cell culture is closing the gap between bench and bedside for using these cells in regenerative medicine, drug discovery and safety testing. In order to produce stem cell derived biopharmaceutics and cells for tissue engineering and transplantation, a cost-effective cell-manufacturing technology is essential. Maintenance of pluripotency and stable performance of cells in downstream applications (e.g., cell differentiation) over time is paramount to large scale cell production. Yet that can be difficult to achieve especially if cells are cultured manually where the operator can introduce significant variability as well as be prohibitively expensive to scale-up. To enable high-throughput, large-scale stem cell production and remove operator influence novel stem cell culture protocols using a bench-top multi-channel liquid handling robot were developed that require minimal technician involvement or experience. With these protocols human induced pluripotent stem cells (iPSCs) were cultured in feeder-free conditions directly from a frozen stock and maintained in 96-well plates. Depending on cell line and desired scale-up rate, the operator can easily determine when to passage based on a series of images showing the optimal colony densities for splitting. Then the necessary reagents are prepared to perform a colony split to new plates without a centrifugation step. After 20 passages (~3 months), two iPSC lines maintained stable karyotypes, expressed stem cell markers, and differentiated into cardiomyocytes with high efficiency. The system can perform subsequent high-throughput screening of new differentiation protocols or genetic manipulation designed for 96-well plates. This technology will reduce the labor and technical burden to produce large numbers of identical stem cells for a myriad of applications.     

Place and Response Learning in the Open-field Tower Maze

This protocol describes how the Open-field Tower Maze (OFTM) paradigm is used to study spatial learning in rodents. This maze is especially useful for examining how rats learn to use a place- or response-learning to successfully navigate in an open-field arena. Additionally, this protocol describes how the OFTM differs from other behavioral maze paradigms that are commonly used to study spatial learning in rodents. The OFTM described in this article was adapted from the one previously described by Cole, Clipperton, and Walt (2007). Specifically, the OFTM was created to test spatial learning in rodents without the experimenter having to consider how “stress” might play a role as a confounding variable. Experiments have shown that stress-alone can significantly affect cognitive function¹. The representative results section contains data from an experiment that used the OFTM to examine the effects of estradiol treatment on place- and response-learning in adult female Sprague Dawley rats². Future studies will be designed to examine the role of the hippocampus and striatum in place- and response-learning in the OFTM.     

Inter-Individual Differences in Neurobehavioural Impairment following Sleep Restriction Are Associated with Circadian Rhythm Phase

Although sleep restriction is associated with decrements in daytime alertness and neurobehavioural performance, there are considerable inter-individual differences in the degree of impairment. This study examined the effects of short-term sleep restriction on neurobehavioural performance and sleepiness, and the associations between individual differences in impairments and circadian rhythm phase. Healthy adults (n = 43; 22 M) aged 22.5 ± 3.1 (mean ± SD) years maintained a regular 8:16 h sleep:wake routine for at least three weeks prior to laboratory admission. Sleep opportunity was restricted to 5 hours time-in-bed at home the night before admission and 3 hours time-in-bed in the laboratory, aligned by wake time. Hourly saliva samples were collected from 5.5 h before until 5 h after the pre-laboratory scheduled bedtime to assess dim light melatonin onset (DLMO) as a marker of circadian phase. Participants completed a 10-min auditory Psychomotor Vigilance Task (PVT), the Karolinska Sleepiness Scale (KSS) and had slow eye movements (SEM) measured by electrooculography two hours after waking. We observed substantial inter-individual variability in neurobehavioural performance, particularly in the number of PVT lapses. Increased PVT lapses (r = -0.468, p < 0.01), greater sleepiness (r = 0.510, p < 0.0001), and more slow eye movements (r = 0.375, p = 0.022) were significantly associated with later DLMO, consistent with participants waking at an earlier circadian phase. When the difference between DLMO and sleep onset was less than 2 hours, individuals were significantly more likely to have at least three attentional lapses the following morning. This study demonstrates that the phase of an individual’s circadian system is an important variable in predicting the degree of neurobehavioural performance impairment in the hours after waking following sleep restriction, and confirms that other factors influencing performance decrements require further investigation.     

Sawfishes and Other Elasmobranch Assemblages from the Mio-Pliocene of the South Caribbean (Urumaco Sequence,Northwestern Venezuela)

The Urumaco stratigraphic sequence, western Venezuela, preserves a variety of paleoenvironments that include terrestrial, riverine, lacustrine and marine facies. A wide range of fossil vertebrates associated with these facies supports the hypothesis of an estuary in that geographic area connected with a hydrographic system that flowed from western Amazonia up to the Proto-Caribbean Sea during the Miocene. Here the elasmobranch assemblages of the middle Miocene to middle Pliocene section of the Urumaco sequence (Socorro, Urumaco and Codore formations) are described. Based on new findings, we document at least 21 taxa of the Lamniformes, Carcharhiniformes, Myliobatiformes and Rajiformes, and describe a new carcharhiniform species (†Carcharhinus caquetius sp. nov.). Moreover, the Urumaco Formation has a high number of well-preserved fossil Pristis rostra, for which we provide a detailed taxonomic revision, and referral in the context of the global Miocene record of Pristis as well as extant species. Using the habitat preference of the living representatives, we hypothesize that the fossil chondrichthyan assemblages from the Urumaco sequence are evidence for marine shallow waters and estuarine habitats.     

Impact of an Interdisciplinary Deep Brain Stimulation Screening Model on Post-Surgical Complications in Essential Tremor Patients

Objective

To investigate the relationship of our interdisciplinary screening process on post-operative unintended hospitalizations and quality of life.

Background

There are currently no standardized criteria for selection of appropriate Deep Brain Stimulation candidates and little hard data exists to support the use of any singular method.

Methods

An Essential Tremor cohort was selected from our institutional Deep Brain Stimulation database. The interdisciplinary model utilized seven specialties who pre-operatively screened all potential Deep Brain Stimulation candidates. Concerns for surgery raised by each specialty were documented and classified as none, minor, or major. Charts were reviewed to identify unintended hospitalizations and quality of life measurements at 1 year post-surgery.

Results

Eighty-six percent (44/51) of the potential screened candidates were approved for Deep Brain Stimulation. Eight (18%) patients had an unintended hospitalization during the follow-up period. Patients with minor or major concerns raised by any specialty service had significantly more unintended hospitalizations when compared to patients without concerns (75% vs. 25%, p < 0.005). The rate of hospitalization revealed a direct relationship to the “level of concern”; ranging from 100% if major concerns, 42% if minor concerns, and 7% if no concerns raised, p = 0.001. Quality of life scores significantly worsened in patients with unintended hospitalizations at 6 (p = 0.046) and 12 months (p = 0.027) when compared to baseline scores. No significant differences in tremor scores between unintended and non-unintended hospitalizations were observed.

Conclusions

The number and level of concerns raised during interdisciplinary Deep Brain Stimulation screenings were significantly related to unintended hospitalizations and to a reduced quality of life. The interdisciplinary evaluation may help to stratify risk for these complications. However, data should be interpreted with caution due to the limitations of our study. Further prospective comparative and larger studies are required to confirm our results.     

Surfactant Protein D Binds to Coxiella burnetii and Results in a Decrease in Interactions with Murine Alveolar Macrophages

Coxiella burnetii is a Gram-negative, obligate intracellular bacterium and the causative agent of Q fever. Infections are usually acquired after inhalation of contaminated particles, where C. burnetii infects its cellular target cells, alveolar macrophages. Respiratory pathogens encounter the C-type lectin surfactant protein D (SP-D) during the course of natural infection. SP-D is a component of the innate immune response in the lungs and other mucosal surfaces. Many Gram-negative pulmonary pathogens interact with SP-D, which can cause aggregation, bactericidal effects and aid in bacterial clearance. Here we show that SP-D binds to C. burnetii in a calcium-dependent manner with no detectable bacterial aggregation or bactericidal effects. Since SP-D interactions with bacteria often alter macrophage interactions, it was determined that SP-D treatment resulted in a significant decrease in C. burnetii interactions to a mouse alveolar macrophage model cell line MH-S indicating SP-D causes a significant decrease in phagocytosis. The ability of SP-D to modulate macrophage activation by C. burnetii was tested and it was determined that SP-D does not alter the correlates measured for macrophage activation. Taken together these studies support those demonstrating limited activation of alveolar macrophages with C. burnetii and demonstrate interactions with SP-D participate in reduction of phagocyte attachment and phagocytosis.