VCP/p97 is essential for maturation of ubiquitin-containing autophagosomes and this function is impaired by mutations that cause IBMPFD.

VCP (VCP/p97) is a ubiquitously expressed member of the AAA(+)-ATPase family of chaperone-like proteins that regulates numerous cellular processes including chromatin decondensation, homotypic membrane fusion and ubiquitin-dependent protein degradation by the proteasome. Mutations in VCP cause a multisystem degenerative disease consisting of inclusion body myopathy, Paget disease of bone, and frontotemporal dementia (IBMPFD). Here we show that VCP is essential for autophagosome maturation. We generated cells stably expressing dual-tagged LC3 (mCherry-EGFP-LC3) which permit monitoring of autophagosome maturation. We determined that VCP deficiency by RNAi-mediated knockdown or overexpression of dominant-negative VCP results in significant accumulation of immature autophagic vesicles, some of which are abnormally large, acidified and exhibit cathepsin B activity. Furthermore, expression of disease-associated VCP mutants (R155H and A232E) also causes this autophagy defect. VCP was found to be essential to autophagosome maturation under basal conditions and in cells challenged by proteasome inhibition, but not in cells challenged by starvation, suggesting that VCP might be selectively required for autophagic degradation of ubiquitinated substrates. Indeed, a high percentage of the accumulated autophagic vesicles contain ubiquitin-positive contents, a feature that is not observed in autophagic vesicles that accumulate following starvation or treatment with Bafilomycin A. Finally, we show accumulation of numerous, large LAMP-1 and LAMP-2-positive vacuoles and accumulation of LC3-II in myoblasts derived from patients with IBMPFD. We conclude that VCP is essential for maturation of ubiquitin-containing autophagosomes and that defect in this function may contribute to IBMPFD pathogenesis.

Anti-AIDS agents 81. Design, synthesis, and structure-activity relationship study of betulinic acid and moronic acid derivatives as potent HIV maturation inhibitors.

In our continuing study of triterpene derivatives as potent anti-HIV agents, different C-3 conformationally restricted betulinic acid (BA, 1) derivatives were designed and synthesized in order to explore the conformational space of the C-3 pharmacophore. 3-O-Monomethylsuccinyl-betulinic acid (MSB) analogues were also designed to better understand the contribution of the C-3' dimethyl group of bevirimat (2), the first-in-class HIV maturation inhibitor, which is currently in phase IIb clinical trials. In addition, another triterpene skeleton, moronic acid (MA, 3), was also employed to study the influence of the backbone and the C-3 modification toward the anti-HIV activity of this compound class. This study enabled us to better understand the structure-activity relationships (SAR) of triterpene-derived anti-HIV agents and led to the design and synthesis of compound 12 (EC(50): 0.0006 microM), which displayed slightly better activity than 2 as a HIV-1 maturation inhibitor.

Boosting high-intensity focused ultrasound-induced anti-tumor immunity using a sparse-scan strategy that can more effectively promote dendritic cell maturation.

BACKGROUND: The conventional treatment protocol in high-intensity focused ultrasound (HIFU) therapy utilizes a dense-scan strategy to produce closely packed thermal lesions aiming at eradicating as much tumor mass as possible. However, this strategy is not most effective in terms of inducing a systemic anti-tumor immunity so that it cannot provide efficient micro-metastatic control and long-term tumor resistance. We have previously provided evidence that HIFU may enhance systemic anti-tumor immunity by in situ activation of dendritic cells (DCs) inside HIFU-treated tumor tissue. The present study was conducted to test the feasibility of a sparse-scan strategy to boost HIFU-induced anti-tumor immune response by more effectively promoting DC maturation. METHODS: An experimental HIFU system was set up to perform tumor ablation experiments in subcutaneous implanted MC-38 and B16 tumor with dense- or sparse-scan strategy to produce closely-packed or separated thermal lesions. DCs infiltration into HIFU-treated tumor tissues was detected by immunohistochemistry and flow cytometry. DCs maturation was evaluated by IL-12/IL-10 production and CD80/CD86 expression after co-culture with tumor cells treated with different HIFU. HIFU-induced anti-tumor immune response was evaluated by detecting growth-retarding effects on distant re-challenged tumor and tumor-specific IFN-gamma-secreting cells in HIFU-treated mice. RESULTS: HIFU exposure raised temperature up to 80 degrees centigrade at beam focus within 4 s in experimental tumors and led to formation of a well-defined thermal lesion. The infiltrated DCs were recruited to the periphery of lesion, where the peak temperature was only 55 degrees centigrade during HIFU exposure. Tumor cells heated to 55 degrees centigrade in 4-s HIFU exposure were more effective to stimulate co-cultured DCs to mature. Sparse-scan HIFU, which can reserve 55 degrees-heated tumor cells surrounding the separated lesions, elicited an enhanced anti-tumor immune response than dense-scan HIFU, while their suppressive effects on the treated primary tumor were maintained at the same level. Flow cytometry analysis showed that sparse-scan HIFU was more effective than dense-scan HIFU in enhancing DC infiltration into tumor tissues and promoting their maturation in situ. CONCLUSION: Optimizing scan strategy is a feasible way to boost HIFU-induced anti-tumor immunity by more effectively promoting DC maturation.

Biomimetic engineered muscle with capacity for vascular integration and functional maturation in vivo.

Tissue-engineered skeletal muscle can serve as a physiological model of natural muscle and a potential therapeutic vehicle for rapid repair of severe muscle loss and injury. Here, we describe a platform for engineering and testing highly functional biomimetic muscle tissues with a resident satellite cell niche and capacity for robust myogenesis and self-regeneration in vitro. Using a mouse dorsal window implantation model and transduction with fluorescent intracellular calcium indicator, GCaMP3, we nondestructively monitored, in real time, vascular integration and the functional state of engineered muscle in vivo. During a 2-wk period, implanted engineered muscle exhibited a steady ingrowth of blood-perfused microvasculature along with an increase in amplitude of calcium transients and force of contraction. We also demonstrated superior structural organization, vascularization, and contractile function of fully differentiated vs. undifferentiated engineered muscle implants. The described in vitro and in vivo models of biomimetic engineered muscle represent enabling technology for novel studies of skeletal muscle function and regeneration.

Tissue-engineered cardiac patch for advanced functional maturation of human ESC-derived cardiomyocytes.

Human embryonic stem cell-derived cardiomyocytes (hESC-CMs) provide a promising source for cell therapy and drug screening. Several high-yield protocols exist for hESC-CM production; however, methods to significantly advance hESC-CM maturation are still lacking. Building on our previous experience with mouse ESC-CMs, we investigated the effects of 3-dimensional (3D) tissue-engineered culture environment and cardiomyocyte purity on structural and functional maturation of hESC-CMs. 2D monolayer and 3D fibrin-based cardiac patch cultures were generated using dissociated cells from differentiated Hes2 embryoid bodies containing varying percentage (48-90%) of CD172a (SIRPA)-positive cardiomyocytes. hESC-CMs within the patch were aligned uniformly by locally controlling the direction of passive tension. Compared to hESC-CMs in age (2 weeks) and purity (48-65%) matched 2D monolayers, hESC-CMs in 3D patches exhibited significantly higher conduction velocities (CVs), longer sarcomeres (2.09 ± 0.02 vs. 1.77 ± 0.01 μm), and enhanced expression of genes involved in cardiac contractile function, including cTnT, αMHC, CASQ2 and SERCA2. The CVs in cardiac patches increased with cardiomyocyte purity, reaching 25.1 cm/s in patches constructed with 90% hESC-CMs. Maximum contractile force amplitudes and active stresses of cardiac patches averaged to 3.0 ± 1.1 mN and 11.8 ± 4.5 mN/mm(2), respectively. Moreover, contractile force per input cardiomyocyte averaged to 5.7 ± 1.1 nN/cell and showed a negative correlation with hESC-CM purity. Finally, patches exhibited significant positive inotropy with isoproterenol administration (1.7 ± 0.3-fold force increase, EC50 = 95.1 nm). These results demonstrate highly advanced levels of hESC-CM maturation after 2 weeks of 3D cardiac patch culture and carry important implications for future drug development and cell therapy studies.

Emi2-mediated inhibition of E2-substrate ubiquitin transfer by the anaphase-promoting complex/cyclosome through a D-box-independent mechanism.

Vertebrate eggs are arrested at Metaphase II by Emi2, the meiotic anaphase-promoting complex/cyclosome (APC/C) inhibitor. Although the importance of Emi2 during oocyte maturation has been widely recognized and its regulation extensively studied, its mechanism of action remained elusive. Many APC/C inhibitors have been reported to act as pseudosubstrates, inhibiting the APC/C by preventing substrate binding. Here we show that a previously identified zinc-binding region is critical for the function of Emi2, whereas the D-box is largely dispensable. We further demonstrate that instead of acting through a "pseudosubstrate" mechanism as previously hypothesized, Emi2 can inhibit Cdc20-dependent activation of the APC/C substoichiometrically, blocking ubiquitin transfer from the ubiquitin-charged E2 to the substrate. These findings provide a novel mechanism of APC/C inhibition wherein the final step of ubiquitin transfer is targeted and raise the interesting possibility that APC/C is inhibited by Emi2 in a catalytic manner.

Reproduction and population growth in free-ranging mantled howling monkeys.

Free-ranging mantled howling monkey (Alouatta palliata Gray) females experienced a regular estrus cycle averaging 16.3 days, demonstrated sexual skin changes, and participated in multiple matings before becoming pregnant. Gestation averaged 186 days. The average interval between births was 22.5 months. Sexual maturity occurred at approximately 36 and 42 months for females and males, respectively. Female age at first birth was about 3 1/2 years. Births were scattered during some years and clustered during others. The age, rank, and parity of the females affected infant survival. More female than male infants survived to one year of age. Increased population size was the result of immigration rather than births.

Mechanical regulation of chondrogenesis.

Mechanical factors play a crucial role in the development of articular cartilage in vivo. In this regard, tissue engineers have sought to leverage native mechanotransduction pathways to enhance in vitro stem cell-based cartilage repair strategies. However, a thorough understanding of how individual mechanical factors influence stem cell fate is needed to predictably and effectively utilize this strategy of mechanically-induced chondrogenesis. This article summarizes some of the latest findings on mechanically stimulated chondrogenesis, highlighting several new areas of interest, such as the effects of mechanical stimulation on matrix maintenance and terminal differentiation, as well as the use of multifactorial bioreactors. Additionally, the roles of individual biophysical factors, such as hydrostatic or osmotic pressure, are examined in light of their potential to induce mesenchymal stem cell chondrogenesis. An improved understanding of biomechanically-driven tissue development and maturation of stem cell-based cartilage replacements will hopefully lead to the development of cell-based therapies for cartilage degeneration and disease.

Interspecies avian brain chimeras reveal that large brain size differences are influenced by cell-interdependent processes.

Like humans, birds that exhibit vocal learning have relatively delayed telencephalon maturation, resulting in a disproportionately smaller brain prenatally but enlarged telencephalon in adulthood relative to vocal non-learning birds. To determine if this size difference results from evolutionary changes in cell-autonomous or cell-interdependent developmental processes, we transplanted telencephala from zebra finch donors (a vocal-learning species) into Japanese quail hosts (a vocal non-learning species) during the early neural tube stage (day 2 of incubation), and harvested the chimeras at later embryonic stages (between 9-12 days of incubation). The donor and host tissues fused well with each other, with known major fiber pathways connecting the zebra finch and quail parts of the brain. However, the overall sizes of chimeric finch telencephala were larger than non-transplanted finch telencephala at the same developmental stages, even though the proportional sizes of telencephalic subregions and fiber tracts were similar to normal finches. There were no significant changes in the size of chimeric quail host midbrains, even though they were innervated by the physically smaller zebra finch brain, including the smaller retinae of the finch eyes. Chimeric zebra finch telencephala had a decreased cell density relative to normal finches. However, cell nucleus size differences between each species were maintained as in normal birds. These results suggest that telencephalic size development is partially cell-interdependent, and that the mechanisms controlling the size of different brain regions may be functionally independent.

Measuring fast gene dynamics in single cells with time-lapse luminescence microscopy.

Time-lapse fluorescence microscopy is an important tool for measuring in vivo gene dynamics in single cells. However, fluorescent proteins are limited by slow chromophore maturation times and the cellular autofluorescence or phototoxicity that arises from light excitation. An alternative is luciferase, an enzyme that emits photons and is active upon folding. The photon flux per luciferase is significantly lower than that for fluorescent proteins. Thus time-lapse luminescence microscopy has been successfully used to track gene dynamics only in larger organisms and for slower processes, for which more total photons can be collected in one exposure. Here we tested green, yellow, and red beetle luciferases and optimized substrate conditions for in vivo luminescence. By combining time-lapse luminescence microscopy with a microfluidic device, we tracked the dynamics of cell cycle genes in single yeast with subminute exposure times over many generations. Our method was faster and in cells with much smaller volumes than previous work. Fluorescence of an optimized reporter (Venus) lagged luminescence by 15-20 min, which is consistent with its known rate of chromophore maturation in yeast. Our work demonstrates that luciferases are better than fluorescent proteins at faithfully tracking the underlying gene expression.

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.