Muscle or liver-specific Sirt3 deficiency induces hyperacetylation of mitochondrial proteins without affecting global metabolic homeostasis

Sirt3 is a mitochondrial sirtuin, predominantly expressed in highly metabolic tissues. Germline ablation of Sirt3 has major metabolic consequences, including increased susceptibility to metabolic damage and oxidative stress after high fat feeding. In order to determine the contribution of liver and skeletal muscle to these phenotypes, we generated muscle-specific Sirt3 (Sirt3skm−/−) and liver-specific Sirt3 (Sirt3hep−/−) knock-out mice. Despite a marked global hyperacetylation of mitochondrial proteins, Sirt3skm−/− and Sirt3hep−/− mice did not manifest any overt metabolic phenotype under either chow or high fat diet conditions. Similarly, there was no evidence for increased oxidative stress in muscle or liver when Sirt3 was ablated in a tissue-specific manner. These observations suggest that the mitochondrial hyperacetylation induced by Sirt3-deletion in a tissue specific manner is not necessarily linked to mitochondrial dysfunction and does not recapitulate the metabolic abnormalities observed in the germline Sirt3 knock-out mice

Automated detection and staging of malaria parasites from cytological smears using convolutional neural networks

Microscopic examination of blood smears remains the gold standard for laboratory inspection and diagnosis of malaria. Smear inspection is, however, time-consuming and dependent on trained microscopists with results varying in accuracy. We sought to develop an automated image analysis method to improve accuracy and standardization of smear inspection that retains capacity for expert confirmation and image archiving. Here, we present a machine learning method that achieves red blood cell (RBC) detection, differentiation between infected/uninfected cells, and parasite life stage categorization from unprocessed, heterogeneous smear images. Based on a pretrained Faster Region-Based Convolutional Neural Networks (R-CNN) model for RBC detection, our model performs accurately, with an average precision of 0.99 at an intersection-over-union threshold of 0.5. Application of a residual neural network-50 model to infected cells also performs accurately, with an area under the receiver operating characteristic curve of 0.98. Finally, combining our method with a regression model successfully recapitulates intraerythrocytic developmental cycle with accurate lifecycle stage categorization. Combined with a mobile-friendly web-based interface, called PlasmoCount, our method permits rapid navigation through and review of results for quality assurance. By standardizing assessment of Giemsa smears, our method markedly improves inspection reproducibility and presents a realistic route to both routine lab and future field-based automated malaria diagnosis

Selective Ion Changes during Spontaneous Mitochondrial Transients in Intact Astrocytes

The bioenergetic status of cells is tightly regulated by the activity of cytosolic enzymes and mitochondrial ATP production. To adapt their metabolism to cellular energy needs, mitochondria have been shown to exhibit changes in their ionic composition as the result of changes in cytosolic ion concentrations. Individual mitochondria also exhibit spontaneous changes in their electrical potential without altering those of neighboring mitochondria. We recently reported that individual mitochondria of intact astrocytes exhibit spontaneous transient increases in their Na+ concentration. Here, we investigated whether the concentration of other ionic species were involved during mitochondrial transients. By combining fluorescence imaging methods, we performed a multiparameter study of spontaneous mitochondrial transients in intact resting astrocytes. We show that mitochondria exhibit coincident changes in their Na+ concentration, electrical potential, matrix pH and mitochondrial reactive oxygen species production during a mitochondrial transient without involving detectable changes in their Ca2+ concentration. Using widefield and total internal reflection fluorescence imaging, we found evidence for localized transient decreases in the free Mg2+ concentration accompanying mitochondrial Na+ spikes that could indicate an associated local and transient enrichment in the ATP concentration. Therefore, we propose a sequential model for mitochondrial transients involving a localized ATP microdomain that triggers a Na+-mediated mitochondrial depolarization, transiently enhancing the activity of the mitochondrial respiratory chain. Our work provides a model describing ionic changes that could support a bidirectional cytosol-to-mitochondria ionic communication

Investigating Feedback on Practice Among Teachers: Coherence of Observed and Perceived Feedback

Despite that benefits of feedback in student learning are reported in much research, little has been reported regarding the use of feedback from teach- ers to other teachers—a key tool in professional development. In this study, we triangulated data from videotaped peer coaching sessions, ques- tionnaires, and interviews regarding 12 primary school teachers in four peer groups in the Netherlands. We focused our research on two issues: the interplay of observed feedback dimensions and elements and perceptions of that feedback. Feedback dimensions were generally effective and the influence of the elements on the dimensions mostly aligned with the expectations. Teachers generally perceived feedback as effective. More- over, effective observed feedback was perceived as effective. Findings indicate that peer coaches should stimulate coached teachers to become goal directed, specific, detailed, and neutral (neither positive nor negative) by using feedback elements so as to optimize feedback processes

Heterodimers of photoreceptor-specific nuclear receptor (PNR/NR2E3) and peroxisome proliferator-activated receptor (PPARγ) are disrupted by retinal disease-associated mutations

Photoreceptor-specific nuclear receptor (PNR/NR2E3) and Tailless homolog (TLX/NR2E1) are human orthologs of the NR2E group, a subgroup of phylogenetically related members of the Nuclear Receptor (NR) superfamily of transcription factors. We assessed the ability of these NRs to form heterodimers with other members of the human NRs representing all major subgroups. The TLX ligand binding domain (LBD) did not appear to form homodimers or interact directly with any other NR tested. The PNR LBD was able to form homodimers, but also exhibited robust interactions with the LBDs of PPARγ/NR1C3 and TRβ/NR1A2. The binding of PNR to PPARγ was specific for this paralog, as no interaction was observed with the LBDs of PPARαNR1C1 or PPARδNR1C2. In support of these findings, PPARγ and PNR were found to be co-expressed in human retinal tissue extracts and could be co-immunoprecipitated as a native complex. Selected sequence variants in the PNR LBD associated with human retinopathies, or a mutation in the dimerization region of PPARγ LBD associated with familial partial lipodystrophy type 3, were found to disrupt PNR/PPARγ complex formation. Wild type PNR, but not a PNR309G mutant, was able to repress PPARγ-mediated transcription in reporter assays. In summary our results reveal novel heterodimer interactions in the NR superfamily, suggesting previously unknown functional interactions of PNR with PPARγ and TRβ that have potential importance in retinal development and disease

Prospective functional classification of all possible missense variants in PPARG.

Clinical exome sequencing routinely identifies missense variants in disease-related genes, but functional characterization is rarely undertaken, leading to diagnostic uncertainty. For example, mutations in PPARG cause Mendelian lipodystrophy and increase risk of type 2 diabetes (T2D). Although approximately 1 in 500 people harbor missense variants in PPARG, most are of unknown consequence. To prospectively characterize PPARγ variants, we used highly parallel oligonucleotide synthesis to construct a library encoding all 9,595 possible single-amino acid substitutions. We developed a pooled functional assay in human macrophages, experimentally evaluated all protein variants, and used the experimental data to train a variant classifier by supervised machine learning. When applied to 55 new missense variants identified in population-based and clinical sequencing, the classifier annotated 6 variants as pathogenic; these were subsequently validated by single-variant assays. Saturation mutagenesis and prospective experimental characterization can support immediate diagnostic interpretation of newly discovered missense variants in disease-related genes.This work was supported by grants from the National Institute of Diabetes and Digestive and Kidney Diseases (1K08DK102877-01, to A.R.M.; 1R01DK097768-01, to D.A.), NIH/Harvard Catalyst (1KL2TR001100-01, to A.R.M.), the Broad Institute (SPARC award, to A.R.M. and T.M.), and the Wellcome Trust (095564, to K.C.; 107064, to D.B.S.).This is the author accepted manuscript. The final version is available from Nature Publishing Group via http://dx.doi.org/10.1038/ng.370

Genome Wide Analysis of Inbred Mouse Lines Identifies a Locus Containing Ppar-γ as Contributing to Enhanced Malaria Survival

The genetic background of a patient determines in part if a person develops a mild form of malaria and recovers, or develops a severe form and dies. We have used a mouse model to detect genes involved in the resistance or susceptibility to Plasmodium berghei malaria infection. To this end we first characterized 32 different mouse strains infected with P. berghei and identified survival as the best trait to discriminate between the strains. We found a locus on chromosome 6 by linking the survival phenotypes of the mouse strains to their genetic variations using genome wide analyses such as haplotype associated mapping and the efficient mixed-model for association. This new locus involved in malaria resistance contains only two genes and confirms the importance of Ppar-γ in malaria infection

A Novel RNAi Lethality Rescue Screen to Identify Regulators of Adipogenesis

Adipogenesis, the differentiation of fibroblast-like mesenchymal stem cells into mature adipocytes, is tightly regulated by a complex cascade of transcription factors, including the nuclear receptor Peroxisome proliferator activator receptor γ (PPARγ). RNAi-mediated knock down libraries may present an atractive method for the identification of additional adipogenic factors. However, using in vitro adipogenesis model systems for high-throughput screening with siRNA libraries is limited since (i) differentiation is not homogeneous, but results in mixed cell populations, and (ii) the expression levels (and activity) of adipogenic regulators is highly dynamic during differentiation, indicating that the timing of RNAi-mediated knock down during differentiation may be extremely critical. Here we report a proof-of-principle for a novel RNAi screening method to identify regulators of adipogenesis that is based on lethality rescue rather than differentiation, using microRNA expression driven by a PPARγ responsive RNA polymerase II promoter. We validated this novel method through screening of a dedicated deubiquitinase knock down library, resulting in the identification of UCHL3 as an essential deubiquitinase in adipogenesis. This system therefore enables the identification of novel genes regulating PPARγ-mediated adipogenesis in a high-throughput setting

AMP-activated protein kinase inhibits NF-κB signaling and inflammation: impact on healthspan and lifespan

Adenosine monophosphate-activated protein kinase (AMPK) is a crucial regulator of energy metabolic homeostasis and thus a major survival factor in a variety of metabolic stresses and also in the aging process. Metabolic syndrome is associated with a low-grade, chronic inflammation, primarily in adipose tissue. A low-level of inflammation is also present in the aging process. There are emerging results indicating that AMPK signaling can inhibit the inflammatory responses induced by the nuclear factor-κB (NF-κB) system. The NF-κB subunits are not direct phosphorylation targets of AMPK, but the inhibition of NF-κB signaling is mediated by several downstream targets of AMPK, e.g., SIRT1, PGC-1α, p53, and Forkhead box O (FoxO) factors. AMPK signaling seems to enhance energy metabolism while it can repress inflammatory responses linked to chronic stress, e.g., in nutritional overload and during the aging process. AMPK can inhibit endoplasmic reticulum and oxidative stresses which are involved in metabolic disorders and the aging process. Interestingly, many target proteins of AMPK are so-called longevity factors, e.g., SIRT1, p53, and FoxOs, which not only can increase the stress resistance and extend the lifespan of many organisms but also inhibit the inflammatory responses. The activation capacity of AMPK declines in metabolic stress and with aging which could augment the metabolic diseases and accelerate the aging process. We will review the AMPK pathways involved in the inhibition of NF-κB signaling and suppression of inflammation. We also emphasize that the capacity of AMPK to repress inflammatory responses can have a significant impact on both healthspan and lifespan