A Broad Phenotypic Screen Identifies Novel Phenotypes Driven by a Single Mutant Allele in Huntington’s Disease CAG Knock-In Mice

Huntington’s disease (HD) is an autosomal dominant neurodegenerative disorder caused by the expansion of a CAG trinucleotide repeat in the HTT gene encoding huntingtin. The disease has an insidious course, typically progressing over 10-15 years until death. Currently there is no effective disease-modifying therapy. To better understand the HD pathogenic process we have developed genetic HTT CAG knock-in mouse models that accurately recapitulate the HD mutation in man. Here, we describe results of a broad, standardized phenotypic screen in 10-46 week old heterozygous HdhQ111 knock-in mice, probing a wide range of physiological systems. The results of this screen revealed a number of behavioral abnormalities in HdhQ111/+ mice that include hypoactivity, decreased anxiety, motor learning and coordination deficits, and impaired olfactory discrimination. The screen also provided evidence supporting subtle cardiovascular, lung, and plasma metabolite alterations. Importantly, our results reveal that a single mutant HTT allele in the mouse is sufficient to elicit multiple phenotypic abnormalities, consistent with a dominant disease process in patients. These data provide a starting point for further investigation of several organ systems in HD, for the dissection of underlying pathogenic mechanisms and for the identification of reliable phenotypic endpoints for therapeutic testing

Poly(I:C) Enhances the Susceptibility of Leukemic Cells to NK Cell Cytotoxicity and Phagocytosis by DC

α Active specific immunotherapy aims at stimulating the host's immune system to recognize and eradicate malignant cells. The concomitant activation of dendritic cells (DC) and natural killer (NK) cells is an attractive modality for immune-based therapies. Inducing immunogenic cell death to facilitate tumor cell recognition and phagocytosis by neighbouring immune cells is of utmost importance for guiding the outcome of the immune response. We previously reported that acute myeloid leukemic (AML) cells in response to electroporation with the synthetic dsRNA analogue poly(I:C) exert improved immunogenicity, demonstrated by enhanced DC-activating and NK cell interferon-γ-inducing capacities. To further invigorate the potential of these immunogenic tumor cells, we explored their effect on the phagocytic and cytotoxic capacity of DC and NK cells, respectively. Using single-cell analysis, we assessed these functionalities in two- and three-party cocultures. Following poly(I:C) electroporation AML cells become highly susceptible to NK cell-mediated killing and phagocytosis by DC. Moreover, the enhanced killing and the improved uptake are strongly correlated. Interestingly, tumor cell killing, but not phagocytosis, is further enhanced in three-party cocultures provided that these tumor cells were upfront electroporated with poly(I:C). Altogether, poly(I:C)-electroporated AML cells potently activate DC and NK cell functions and stimulate NK-DC cross-talk in terms of tumor cell killing. These data strongly support the use of poly(I:C) as a cancer vaccine component, providing a way to overcome immune evasion by leukemic cells

A consensus protocol for functional connectivity analysis in the rat brain

Task-free functional connectivity in animal models provides an experimental framework to examine connectivity phenomena under controlled conditions and allows for comparisons with data modalities collected under invasive or terminal procedures. Currently, animal acquisitions are performed with varying protocols and analyses that hamper result comparison and integration. Here we introduce StandardRat, a consensus rat functional magnetic resonance imaging acquisition protocol tested across 20 centers. To develop this protocol with optimized acquisition and processing parameters, we initially aggregated 65 functional imaging datasets acquired from rats across 46 centers. We developed a reproducible pipeline for analyzing rat data acquired with diverse protocols and determined experimental and processing parameters associated with the robust detection of functional connectivity across centers. We show that the standardized protocol enhances biologically plausible functional connectivity patterns relative to previous acquisitions. The protocol and processing pipeline described here is openly shared with the neuroimaging community to promote interoperability and cooperation toward tackling the most important challenges in neuroscience

Specialized Yeast Ribosomes: A Customized Tool for Selective mRNA Translation

Evidence is now accumulating that sub-populations of ribosomes - so-called specialized ribosomes - can favour the translation of subsets of mRNAs. Here we use a large collection of diploid yeast strains, each deficient in one or other copy of the set of ribosomal protein (RP) genes, to generate eukaryotic cells carrying distinct populations of altered ‘specialized’ ribosomes. We show by comparative protein synthesis assays that different heterologous mRNA reporters based on luciferase are preferentially translated by distinct populations of specialized ribosomes. These mRNAs include reporters carrying premature termination codons (PTC) thus allowing us to identify specialized ribosomes that alter the efficiency of translation termination leading to enhanced synthesis of the wild-type protein. This finding suggests that these strains can be used to identify novel therapeutic targets in the ribosome. To explore this further we examined the translation of the mRNA encoding the extracellular matrix protein laminin ?3 (LAMB3) since a LAMB3-PTC mutant is implicated in the blistering skin disease Epidermolysis bullosa (EB). This screen identified specialized ribosomes with reduced levels of RP L35B as showing enhanced synthesis of full-length LAMB3 in cells expressing the LAMB3-PTC mutant. Importantly, the RP L35B sub-population of specialized ribosomes leave both translation of a reporter luciferase carrying a different PTC and bulk mRNA translation largely unaltered

Sapovirus Outbreaks in Long-Term Care Facilities, Oregon and Minnesota, USA, 2002–2009

Sapovirus gives new meaning to the phrase “cradle to grave.” Historically, sapovirus has been associated with gastrointestinal illness in children living in group settings such as hospitals, shelters, or refugee camps. But now, sapovirus outbreaks are occurring among elderly residents of long-term care and similar facilities. These elderly residents are especially vulnerable to rapidly transmitted gastrointestinal viruses and serious complications. This virus has been making the rounds in long-term care facilities since 2002, and outbreaks started increasing in 2007. Sapovirus testing should be added to routine diagnostic workups for gastrointestinal infections, regardless of patient age group. Results can be used to develop prevention, control, and treatment guidelines, especially for vulnerable elderly populations

Incidence of Norovirus and Other Viral Pathogens That Cause Acute Gastroenteritis (AGE) among Kaiser Permanente Member Populations in the United States, 2012–2013

<div><p>Noroviruses and other viral pathogens are increasingly recognized as frequent causes of acute gastroenteritis (AGE). However, few laboratory-based data are available on the incidence of AGE caused by viral pathogens in the U.S. This study examined stool specimens submitted for routine clinical diagnostics from patients enrolled in Kaiser Permanente (KP) health plans in metro Portland, OR, and the Maryland, District of Columbia, and northern Virginia geographic areas to estimate the incidence of viral enteropathogens in these populations. Over a one-year study period, participating laboratories randomly selected stools submitted for routine clinical diagnostics for inclusion in the study along with accompanying demographic and clinical data. Selected stools were tested for norovirus, rotavirus, sapovirus, and astrovirus using standardized real-time RT-PCR protocols. Each KP site provided administrative data which were used in conjunction with previously published data on healthcare utilization to extrapolate pathogen detection rates into population-based incidence rates. A total of 1,099 specimens collected during August 2012 to September 2013 were included. Mean age of patients providing stool specimens was 46 years (range: 0–98 years). Noroviruses were the most common viral pathogen identified among patients with AGE (n = 63 specimens, 6% of specimens tested). In addition, 22 (2%) of specimens were positive for rotavirus; 19 (2%) were positive for sapovirus; and 7 (1%) were positive for astrovirus. Incidence of norovirus-associated outpatient visits was 5.6 per 1,000 person-years; incidence of norovirus disease in the community was estimated to be 69.5 per 1,000 person-years. Norovirus incidence was highest among children <5 years of age (outpatient incidence = 25.6 per 1,000 person-years; community incidence = 152.2 per 1,000 person-years), followed by older adults aged >65 years (outpatient incidence = 7.8 per 1,000 person-years; community incidence = 75.8 per 1,000 person-years). Outpatient incidence rates of rotavirus, sapovirus, and astrovirus were 2.0, 1.6, 0.6 per 1,000 person-years, respectively; community incidence rates for these viruses were 23.4, 22.5, and 8.5 per 1,000 person-years, respectively. This study provides the first age-group specific laboratory-based community and outpatient incidence rates for norovirus AGE in the U.S. Norovirus was the most frequently detected viral enteropathogen across the age spectrum with the highest rates of norovirus disease observed among young children and, to a lesser extent, the elderly. These data provide a better understanding of the norovirus disease burden in the United States, including variations within different age groups, which can help inform the development, targeting, and future impacts of interventions, including vaccines.</p></div

Outpatient and community incidence of viral pathogens that cause AGE, by site.

<p>Outpatient and community incidence of viral pathogens that cause AGE, by site.</p