A network of substrates of the E3 ubiquitin ligases MDM2 and HUWE1 control apoptosis independently of p53.

In the intrinsic pathway of apoptosis, cell-damaging signals promote the release of cytochrome c from mitochondria, triggering activation of the Apaf-1 and caspase-9 apoptosome. The ubiquitin E3 ligase MDM2 decreases the stability of the proapoptotic factor p53. We show that it also coordinated apoptotic events in a p53-independent manner by ubiquitylating the apoptosome activator CAS and the ubiquitin E3 ligase HUWE1. HUWE1 ubiquitylates the antiapoptotic factor Mcl-1, and we found that HUWE1 also ubiquitylated PP5 (protein phosphatase 5), which indirectly inhibited apoptosome activation. Breast cancers that are positive for the tyrosine receptor kinase HER2 (human epidermal growth factor receptor 2) tend to be highly aggressive. In HER2-positive breast cancer cells treated with the HER2 tyrosine kinase inhibitor lapatinib, MDM2 was degraded and HUWE1 was stabilized. In contrast, in breast cancer cells that acquired resistance to lapatinib, the abundance of MDM2 was not decreased and HUWE1 was degraded, which inhibited apoptosis, regardless of p53 status. MDM2 inhibition overcame lapatinib resistance in cells with either wild-type or mutant p53 and in xenograft models. These findings demonstrate broader, p53-independent roles for MDM2 and HUWE1 in apoptosis and specifically suggest the potential for therapy directed against MDM2 to overcome lapatinib resistance.

The Adjuvant Activity and Mechanisms of Action for Mastoparan 7 Peptide After Intranasal Immunization in Mice

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Male mice song syntax depends on social contexts and influences female preferences.

In 2005, Holy and Guo advanced the idea that male mice produce ultrasonic vocalizations (USV) with some features similar to courtship songs of songbirds. Since then, studies showed that male mice emit USV songs in different contexts (sexual and other) and possess a multisyllabic repertoire. Debate still exists for and against plasticity in their vocalizations. But the use of a multisyllabic repertoire can increase potential flexibility and information, in how elements are organized and recombined, namely syntax. In many bird species, modulating song syntax has ethological relevance for sexual behavior and mate preferences. In this study we exposed adult male mice to different social contexts and developed a new approach of analyzing their USVs based on songbird syntax analysis. We found that male mice modify their syntax, including specific sequences, length of sequence, repertoire composition, and spectral features, according to stimulus and social context. Males emit longer and simpler syllables and sequences when singing to females, but more complex syllables and sequences in response to fresh female urine. Playback experiments show that the females prefer the complex songs over the simpler ones. We propose the complex songs are to lure females in, whereas the directed simpler sequences are used for direct courtship. These results suggest that although mice have a much more limited ability of song modification, they could still be used as animal models for understanding some vocal communication features that songbirds are used for.

Huntingtin is required for normal excitatory synapse development in cortical and striatal circuits.

Huntington's disease (HD) is a neurodegenerative disease caused by the expansion of a poly-glutamine (poly-Q) stretch in the huntingtin (Htt) protein. Gain-of-function effects of mutant Htt have been extensively investigated as the major driver of neurodegeneration in HD. However, loss-of-function effects of poly-Q mutations recently emerged as potential drivers of disease pathophysiology. Early synaptic problems in the excitatory cortical and striatal connections have been reported in HD, but the role of Htt protein in synaptic connectivity was unknown. Therefore, we investigated the role of Htt in synaptic connectivity in vivo by conditionally silencing Htt in the developing mouse cortex. When cortical Htt function was silenced, cortical and striatal excitatory synapses formed and matured at an accelerated pace through postnatal day 21 (P21). This exuberant synaptic connectivity was lost over time in the cortex, resulting in the deterioration of synapses by 5 weeks. Synaptic decline in the cortex was accompanied with layer- and region-specific reactive gliosis without cell loss. To determine whether the disease-causing poly-Q mutation in Htt affects synapse development, we next investigated the synaptic connectivity in a full-length knock-in mouse model of HD, the zQ175 mouse. Similar to the cortical conditional knock-outs, we found excessive excitatory synapse formation and maturation in the cortices of P21 zQ175, which was lost by 5 weeks. Together, our findings reveal that cortical Htt is required for the correct establishment of cortical and striatal excitatory circuits, and this function of Htt is lost when the mutant Htt is present.

Preexisting compensatory amino acids compromise fitness costs of a HIV-1 T cell escape mutation.

BACKGROUND: Fitness costs and slower disease progression are associated with a cytolytic T lymphocyte (CTL) escape mutation T242N in Gag in HIV-1-infected individuals carrying HLA-B*57/5801 alleles. However, the impact of different context in diverse HIV-1 strains on the fitness costs due to the T242N mutation has not been well characterized. To better understand the extent of fitness costs of the T242N mutation and the repair of fitness loss through compensatory amino acids, we investigated its fitness impact in different transmitted/founder (T/F) viruses. RESULTS: The T242N mutation resulted in various levels of fitness loss in four different T/F viruses. However, the fitness costs were significantly compromised by preexisting compensatory amino acids in (Isoleucine at position 247) or outside (glutamine at position 219) the CTL epitope. Moreover, the transmitted T242N escape mutant in subject CH131 was as fit as the revertant N242T mutant and the elimination of the compensatory amino acid I247 in the T/F viral genome resulted in significant fitness cost, suggesting the fitness loss caused by the T242N mutation had been fully repaired in the donor at transmission. Analysis of the global circulating HIV-1 sequences in the Los Alamos HIV Sequence Database showed a high prevalence of compensatory amino acids for the T242N mutation and other T cell escape mutations. CONCLUSIONS: Our results show that the preexisting compensatory amino acids in the majority of circulating HIV-1 strains could significantly compromise the fitness loss due to CTL escape mutations and thus increase challenges for T cell based vaccines.

Menthol attenuates respiratory irritation and elevates blood cotinine in cigarette smoke exposed mice.

Addition of menthol to cigarettes may be associated with increased initiation of smoking. The potential mechanisms underlying this association are not known. Menthol, likely due to its effects on cold-sensing peripheral sensory neurons, is known to inhibit the sensation of irritation elicited by respiratory irritants. However, it remains unclear whether menthol modulates cigarette smoke irritancy and nicotine absorption during initial exposures to cigarettes, thereby facilitating smoking initiation. Using plethysmography in a C57Bl/6J mouse model, we examined the effects of L-menthol, the menthol isomer added to cigarettes, on the respiratory sensory irritation response to primary smoke irritants (acrolein and cyclohexanone) and smoke of Kentucky reference 2R4 cigarettes. We also studied L-menthol's effect on blood levels of the nicotine metabolite, cotinine, immediately after exposure to cigarette smoke. L-menthol suppressed the irritation response to acrolein with an apparent IC₅₀ of 4 ppm. Suppression was observed even at acrolein levels well above those necessary to produce a maximal response. Cigarette smoke, at exposure levels of 10 mg/m³ or higher, caused an immediate and marked sensory irritation response in mice. This response was significantly suppressed by L-menthol even at smoke concentrations as high as 300 mg/m³. Counterirritation by L-menthol was abolished by treatment with a selective inhibitor of Transient Receptor Potential Melastatin 8 (TRPM8), the neuronal cold/menthol receptor. Inclusion of menthol in the cigarette smoke resulted in roughly a 1.5-fold increase in plasma cotinine levels over those observed in mice exposed to smoke without added menthol. These findings document that, L-menthol, through TRPM8, is a strong suppressor of respiratory irritation responses, even during highly noxious exposures to cigarette smoke or smoke irritants, and increases blood cotinine. Therefore, L-menthol, as a cigarette additive, may promote smoking initiation and nicotine addiction.

Adjunctive β2-agonist treatment reduces glycogen independently of receptor-mediated acid α-glucosidase uptake in the limb muscles of mice with Pompe disease.

Enzyme or gene replacement therapy with acid α-glucosidase (GAA) has achieved only partial efficacy in Pompe disease. We evaluated the effect of adjunctive clenbuterol treatment on cation-independent mannose-6-phosphate receptor (CI-MPR)-mediated uptake and intracellular trafficking of GAA during muscle-specific GAA expression with an adeno-associated virus (AAV) vector in GAA-knockout (KO) mice. Clenbuterol, which increases expression of CI-MPR in muscle, was administered with the AAV vector. This combination therapy increased latency during rotarod and wirehang testing at 12 wk, in comparison with vector alone. The mean urinary glucose tetrasaccharide (Glc4), a urinary biomarker, was lower in GAA-KO mice following combination therapy, compared with vector alone. Similarly, glycogen content was lower in cardiac and skeletal muscle following 12 wk of combination therapy in heart, quadriceps, diaphragm, and soleus, compared with vector alone. These data suggested that clenbuterol treatment enhanced trafficking of GAA to lysosomes, given that GAA was expressed within myofibers. The integral role of CI-MPR was demonstrated by the lack of effectiveness from clenbuterol in GAA-KO mice that lacked CI-MPR in muscle, where it failed to reverse the high glycogen content of the heart and diaphragm or impaired wirehang performance. However, the glycogen content of skeletal muscle was reduced by the addition of clenbuterol in the absence of CI-MPR, as was lysosomal vacuolation, which correlated with increased AKT signaling. In summary, β2-agonist treatment enhanced CI-MPR-mediated uptake and trafficking of GAA in mice with Pompe disease, and a similarly enhanced benefit might be expected in other lysosomal storage disorders.

Assessment of toxicity and biodistribution of recombinant AAV8 vector-mediated immunomodulatory gene therapy in mice with Pompe disease.

A preclinical safety study was conducted to evaluate the short- and long-term toxicity of a recombinant adeno-associated virus serotype 8 (AAV2/8) vector that has been developed as an immune-modulatory adjunctive therapy to recombinant human acid α-glucosidase (rhGAA, Myozyme) enzyme replacement treatment (ERT) for patients with Pompe disease (AAV2/8-LSPhGAApA). The AAV2/8-LSPhGAApA vector at 1.6 × 10(13) vector particles/kg, after intravenous injection, did not cause significant short- or long-term toxicity. Recruitment of CD4(+) (but not CD8(+)) lymphocytes to the liver was elevated in the vector-dosed male animals at study day (SD) 15, and in group 8 animals at SD 113, in comparison to their respective control animals. Administration of the vector, either prior to or after the one ERT injection, uniformly prevented the hypersensitivity induced by subsequent ERT in males, but not always in female animals. The vector genome was sustained in all tissues through 16-week postdosing, except for in blood with a similar tissue tropism between males and females. Administration of the vector alone, or combined with the ERT, was effective in producing significantly increased GAA activity and consequently decreased glycogen accumulation in multiple tissues, and the urine biomarker, Glc4, was significantly reduced. The efficacy of the vector (or with ERT) was better in males than in females, as demonstrated both by the number of tissues showing significantly effective responses and the extent of response in a given tissue. Given the lack of toxicity for AAV2/8LSPhGAApA, further consideration of clinical translation is warranted in Pompe disease.

The Effect of Diet-Induced Obesity and Subsequent Weight Loss on Body Composition, Glucose Clearance, Metabolite Profile and Liver Amp-Activated Protein Kinase in Mice

Obesity, currently an epidemic, is a difficult disease to combat because it is marked by both a change in body weight and an underlying dysregulation in metabolism, making consistent weight loss challenging. We sought to elucidate this metabolic dysregulation resulting from diet-induced obesity (DIO) that persists through subsequent weight loss. We hypothesized that weight gain imparts a change in “metabolic set point” persisting through subsequent weight loss and that this modification may involve a persistent change in hepatic AMP-activated protein kinase (AMPK), a key energy-sensing enzyme in the body. To test these hypotheses, we tracked metabolic perturbations through this period, measuring changes in hepatic AMPK. To further understand the role of AMPK we used AICAR, an AMPK activator, following DIO. Our findings established a more dynamic metabolic model of DIO and subsequent weight loss. We observed hepatic AMPK elevation following weight loss, but AICAR administration without similar dieting was unsuccessful in improving metabolic dysregulation. Our findings provide an approach to modeling DIO and subsequent dieting that can be built upon in future studies and hopefully contribute to more effective long-term treatments of obesity.

Transsynaptic Tracing from Peripheral Targets with Pseudorabies Virus Followed by Cholera Toxin and Biotinylated Dextran Amines Double Labeling.

Transsynaptic tracing has become a powerful tool used to analyze central efferents that regulate peripheral targets through multi-synaptic circuits. This approach has been most extensively used in the brain by utilizing the swine pathogen pseudorabies virus (PRV)(1). PRV does not infect great apes, including humans, so it is most commonly used in studies on small mammals, especially rodents. The pseudorabies strain PRV152 expresses the enhanced green fluorescent protein (eGFP) reporter gene and only crosses functional synapses retrogradely through the hierarchical sequence of synaptic connections away from the infection site(2,3). Other PRV strains have distinct microbiological properties and may be transported in both directions (PRV-Becker and PRV-Kaplan)(4,5). This protocol will deal exclusively with PRV152. By delivering the virus at a peripheral site, such as muscle, it is possible to limit the entry of the virus into the brain through a specific set of neurons. The resulting pattern of eGFP signal throughout the brain then resolves the neurons that are connected to the initially infected cells. As the distributed nature of transsynaptic tracing with pseudorabies virus makes interpreting specific connections within an identified network difficult, we present a sensitive and reliable method employing biotinylated dextran amines (BDA) and cholera toxin subunit b (CTb) for confirming the connections between cells identified using PRV152. Immunochemical detection of BDA and CTb with peroxidase and DAB (3, 3'-diaminobenzidine) was chosen because they are effective at revealing cellular processes including distal dendrites(6-11).

Whole genome sequencing identifies circulating Beijing-lineage Mycobacterium tuberculosis strains in Guatemala and an associated urban outbreak.

Limited data are available regarding the molecular epidemiology of Mycobacterium tuberculosis (Mtb) strains circulating in Guatemala. Beijing-lineage Mtb strains have gained prevalence worldwide and are associated with increased virulence and drug resistance, but there have been only a few cases reported in Central America. Here we report the first whole genome sequencing of Central American Beijing-lineage strains of Mtb. We find that multiple Beijing-lineage strains, derived from independent founding events, are currently circulating in Guatemala, but overall still represent a relatively small proportion of disease burden. Finally, we identify a specific Beijing-lineage outbreak centered on a poor neighborhood in Guatemala City.

Live Imaging of Host-Parasite Interactions in a Zebrafish Infection Model Reveals Cryptococcal Determinants of Virulence and Central Nervous System Invasion.

UNLABELLED: The human fungal pathogen Cryptococcus neoformans is capable of infecting a broad range of hosts, from invertebrates like amoebas and nematodes to standard vertebrate models such as mice and rabbits. Here we have taken advantage of a zebrafish model to investigate host-pathogen interactions of Cryptococcus with the zebrafish innate immune system, which shares a highly conserved framework with that of mammals. Through live-imaging observations and genetic knockdown, we establish that macrophages are the primary immune cells responsible for responding to and containing acute cryptococcal infections. By interrogating survival and cryptococcal burden following infection with a panel of Cryptococcus mutants, we find that virulence factors initially identified as important in causing disease in mice are also necessary for pathogenesis in zebrafish larvae. Live imaging of the cranial blood vessels of infected larvae reveals that C. neoformans is able to penetrate the zebrafish brain following intravenous infection. By studying a C. neoformans FNX1 gene mutant, we find that blood-brain barrier invasion is dependent on a known cryptococcal invasion-promoting pathway previously identified in a murine model of central nervous system invasion. The zebrafish-C. neoformans platform provides a visually and genetically accessible vertebrate model system for cryptococcal pathogenesis with many of the advantages of small invertebrates. This model is well suited for higher-throughput screening of mutants, mechanistic dissection of cryptococcal pathogenesis in live animals, and use in the evaluation of therapeutic agents. IMPORTANCE: Cryptococcus neoformans is an important opportunistic pathogen that is estimated to be responsible for more than 600,000 deaths worldwide annually. Existing mammalian models of cryptococcal pathogenesis are costly, and the analysis of important pathogenic processes such as meningitis is laborious and remains a challenge to visualize. Conversely, although invertebrate models of cryptococcal infection allow high-throughput assays, they fail to replicate the anatomical complexity found in vertebrates and, specifically, cryptococcal stages of disease. Here we have utilized larval zebrafish as a platform that overcomes many of these limitations. We demonstrate that the pathogenesis of C. neoformans infection in zebrafish involves factors identical to those in mammalian and invertebrate infections. We then utilize the live-imaging capacity of zebrafish larvae to follow the progression of cryptococcal infection in real time and establish a relevant model of the critical central nervous system infection phase of disease in a nonmammalian model.

Cell cycle Start is coupled to entry into the yeast metabolic cycle across diverse strains and growth rates.

Cells have evolved oscillators with different frequencies to coordinate periodic processes. Here we studied the interaction of two oscillators, the cell division cycle (CDC) and the yeast metabolic cycle (YMC), in budding yeast. Previous work suggested that the CDC and YMC interact to separate high oxygen consumption (HOC) from DNA replication to prevent genetic damage. To test this hypothesis, we grew diverse strains in chemostat and measured DNA replication and oxygen consumption with high temporal resolution at different growth rates. Our data showed that HOC is not strictly separated from DNA replication; rather, cell cycle Start is coupled with the initiation of HOC and catabolism of storage carbohydrates. The logic of this YMC-CDC coupling may be to ensure that DNA replication and cell division occur only when sufficient cellular energy reserves have accumulated. Our results also uncovered a quantitative relationship between CDC period and YMC period across different strains. More generally, our approach shows how studies in genetically diverse strains efficiently identify robust phenotypes and steer the experimentalist away from strain-specific idiosyncrasies.