Partial Neuronal Reprogramming via Modified Yamanaka Cocktail

Animal behaviours that are superficially similar can express different intents in different contexts, but how this flexibility is achieved at the level of neural circuits is not understood. For example, males of many species can exhibit mounting behaviour towards same- or opposite-sex conspecifics1, but it is unclear whether the intent and neural encoding of these behaviours are similar or different. Here we show that female- and male-directed mounting in male laboratory mice are distinguishable by the presence or absence of ultrasonic vocalizations (USVs)2-4, respectively. These and additional behavioural data suggest that most male-directed mounting is aggressive, although in rare cases it can be sexual. We investigated whether USV+ and USV- mounting use the same or distinct hypothalamic neural substrates. Micro-endoscopic imaging of neurons positive for oestrogen receptor 1 (ESR1) in either the medial preoptic area (MPOA) or the ventromedial hypothalamus, ventrolateral subdivision (

Factor-induced reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) is inefficient, complicating mechanistic studies. Here, we examined defined intermediate cell populations poised to becoming iPSCs by genome-wide analyses. We show that induced pluripotency elicits two transcriptional waves, which are driven by c-Myc/Klf4 (first wave) and Oct4/Sox2/Klf4 (second wave). Cells that become refractory to reprogramming activate the first but fail to initiate the second transcriptional wave and can be rescued by elevated expression of all four factors. The establishment of bivalent domains occurs gradually after the first wave, whereas changes in DNA methylation take place after the second wave when cells acquire stable pluripotency. This integrative analysis allowed us to identify genes that act as roadblocks during reprogramming and surface markers that further enrich for cells prone to forming iPSCs. Collectively, our data offer new mechanistic insights into the natur

BACKGROUND: Accurately assessing individual ambient air pollution exposure is a crucial part of epidemiological studies looking at the adverse health effect of poor air quality. This is particularly challenging in developing countries with high levels of air pollution, mostly due to sparse monitoring networks with a lack of consistent data. METHODS: We evaluated the performance of six different machine learning algorithms in predicting fine particulate matter (PM2.5) concentrations in Ulaanbaatar, Mongolia using data between 2010 and 2018. We found that the algorithms produce robust results based on performance metrics. RESULTS: Random forest (RF) and gradient boosting models performed the best with leave-one-location-out cross-validated R2 of 0.82 for when using data from the entire study period. After applying tuned models on the hold-out test set, R2 increased to 0.96 for the RF and 0.90 for the gradient boosting model. We also predicted PM2.5 concentrations for each administrative

Ketogenic diets recapitulate certain metabolic aspects of dietary restriction such as reliance on fatty acid metabolism and production of ketone bodies. We investigated whether an isoprotein ketogenic diet (KD) might, like dietary restriction, affect longevity and healthspan in C57BL/6 male mice. We find that Cyclic KD, KD alternated weekly with the Control diet to prevent obesity, reduces midlife mortality but does not affect maximum lifespan. A non-ketogenic high-fat diet (HF) fed similarly may have an intermediate effect on mortality. Cyclic KD improves memory performance in old age, while modestly improving composite healthspan measures. Gene expression analysis identifies downregulation of insulin, protein synthesis, and fatty acid synthesis pathways as mechanisms common to KD and HF. However, upregulation of PPARα target genes is unique to KD, consistent across tissues, and preserved in old age. In all, we show that a non-obesogenic ketogenic diet improves survival, memory, and h

PD-1, a receptor expressed by T cells, B cells, and monocytes, is a potent regulator of immune responses and a promising therapeutic target. The structure and interactions of human PD-1 are, however, incompletely characterized. We present the solution nuclear magnetic resonance (NMR)-based structure of the human PD-1 extracellular region and detailed analyses of its interactions with its ligands, PD-L1 and PD-L2. PD-1 has typical immunoglobulin superfamily topology but differs at the edge of the GFCC' sheet, which is flexible and completely lacks a C" strand. Changes in PD-1 backbone NMR signals induced by ligand binding suggest that, whereas binding is centered on the GFCC' sheet, PD-1 is engaged by its two ligands differently and in ways incompletely explained by crystal structures of mouse PD-1 · ligand complexes. The affinities of these interactions and that of PD-L1 with the costimulatory protein B7-1, measured using surface plasmon resonance, are significantly weaker than expecte

Epigenetic modification of the mammalian genome by DNA methylation (5-methylcytosine) has a profound impact on chromatin structure, gene expression and maintenance of cellular identity. The recent demonstration that members of the Ten-eleven translocation (Tet) family of proteins can convert 5-methylcytosine to 5-hydroxymethylcytosine raised the possibility that Tet proteins are capable of establishing a distinct epigenetic state. We have recently demonstrated that Tet1 is specifically expressed in murine embryonic stem (ES) cells and is required for ES cell maintenance. Using chromatin immunoprecipitation coupled with high-throughput DNA sequencing, here we show in mouse ES cells that Tet1 is preferentially bound to CpG-rich sequences at promoters of both transcriptionally active and Polycomb-repressed genes. Despite an increase in levels of DNA methylation at many Tet1-binding sites, Tet1 depletion does not lead to downregulation of all the Tet1 targets. Interestingly, although Tet1-

Implementation of polygenic risk scores (PRS) may improve disease prevention and management but poses several challenges: the construction of clinically valid assays, interpretation for individual patients, and the development of clinical workflows and resources to support their use in patient care. For the ongoing Veterans Affairs Genomic Medicine at Veterans Affairs (GenoVA) Study we developed a clinical genotype array-based assay for six published PRS. We used data from 36,423 Mass General Brigham Biobank participants and adjustment for population structure to replicate known PRS-disease associations and published PRS thresholds for a disease odds ratio (OR) of 2 (ranging from 1.75 (95% CI: 1.57-1.95) for type 2 diabetes to 2.38 (95% CI: 2.07-2.73) for breast cancer). After confirming the high performance and robustness of the pipeline for use as a clinical assay for individual patients, we analyzed the first 227 prospective samples from the GenoVA Study and found that the frequency

Aging is a complex progression of changes best characterized as the chronic dysregulation of cellular processes leading to deteriorated tissue and organ function. Although aging cannot currently be prevented, its impact on life- and healthspan in the elderly can potentially be minimized by interventions that aim to return these cellular processes to optimal function. Recent studies have demonstrated that partial reprogramming using the Yamanaka factors (or a subset; OCT4, SOX2, and KLF4; OSK) can reverse age-related changes in vitro and in vivo. However, it is still unknown whether the Yamanaka factors (or a subset) are capable of extending the lifespan of aged wild-type (WT) mice. In this study, we show that systemically delivered adeno-associated viruses, encoding an inducible OSK system, in 124-week-old male mice extend the median remaining lifespan by 109% over WT controls and enhance several health parameters. Importantly, we observed a significant improvement in frailty scores in

Transcription factors (TFs) regulate gene expression through chromatin where nucleosomes restrict DNA access. To study how TFs bind nucleosome-occupied motifs, we focused on the reprogramming factors OCT4 and SOX2 in mouse embryonic stem cells. We determined TF engagement throughout a nucleosome at base-pair resolution in vitro, enabling structure determination by cryo-electron microscopy at two preferred positions. Depending on motif location, OCT4 and SOX2 differentially distort nucleosomal DNA. At one position, OCT4-SOX2 removes DNA from histone H2A and histone H3; however, at an inverted motif, the TFs only induce local DNA distortions. OCT4 uses one of its two DNA-binding domains to engage DNA in both structures, reading out a partial motif. These findings explain site-specific nucleosome engagement by the pluripotency factors OCT4 and SOX2, and they reveal how TFs distort nucleosomes to access chromatinized motifs.

Oct4, Sox2, Klf4, and cMyc (OSKM) reprogram somatic cells to pluripotency. To gain a mechanistic understanding of their function, we mapped OSKM-binding, stage-specific transcription factors (TFs), and chromatin states in discrete reprogramming stages and performed loss- and gain-of-function experiments. We found that OSK predominantly bind active somatic enhancers early in reprogramming and immediately initiate their inactivation genome-wide by inducing the redistribution of somatic TFs away from somatic enhancers to sites elsewhere engaged by OSK, recruiting Hdac1, and repressing the somatic TF Fra1. Pluripotency enhancer selection is a stepwise process that also begins early in reprogramming through collaborative binding of OSK at sites with high OSK-motif density. Most pluripotency enhancers are selected later in the process and require OS and other pluripotency TFs. Somatic and pluripotency TFs modulate reprogramming efficiency when overexpressed by altering OSK targeting, somatic

Recent studies have shown that defined sets of transcription factors can directly reprogram differentiated somatic cells to a different differentiated cell type without passing through a pluripotent state, but the restricted proliferative and lineage potential of the resulting cells limits the scope of their potential applications. Here we show that a combination of transcription factors (Brn4/Pou3f4, Sox2, Klf4, c-Myc, plus E47/Tcf3) induces mouse fibroblasts to directly acquire a neural stem cell identity-which we term as induced neural stem cells (iNSCs). Direct reprogramming of fibroblasts into iNSCs is a gradual process in which the donor transcriptional program is silenced over time. iNSCs exhibit cell morphology, gene expression, epigenetic features, differentiation potential, and self-renewing capacity, as well as in vitro and in vivo functionality similar to those of wild-type NSCs. We conclude that differentiated cells can be reprogrammed directly into specific somatic stem c

Using mouse skin, where bountiful reservoirs of synchronized hair follicle stem cells (HF-SCs) fuel cycles of regeneration, we explore how adult SCs remodel chromatin in response to activating cues. By profiling global mRNA and chromatin changes in quiescent and activated HF-SCs and their committed, transit-amplifying (TA) progeny, we show that polycomb-group (PcG)-mediated H3K27-trimethylation features prominently in HF-lineage progression by mechanisms distinct from embryonic-SCs. In HF-SCs, PcG represses nonskin lineages and HF differentiation. In TA progeny, nonskin regulators remain PcG-repressed, HF-SC regulators acquire H3K27me3-marks, and HF-lineage regulators lose them. Interestingly, genes poised in embryonic stem cells, active in HF-SCs, and PcG-repressed in TA progeny encode not only key transcription factors, but also signaling regulators. We document their importance in balancing HF-SC quiescence, underscoring the power of chromatin mapping in dissecting SC behavior. Our

The identification of distinct tissue-specific natural killer (NK) cell populations that apparently mature from local precursor populations has brought new insight into the diversity and developmental regulation of this important lymphoid subset. NK cells provide a necessary link between the early (innate) and late (adaptive) immune responses to infection. Gaining a better understanding of the processes that govern NK cell development should allow us to harness better NK cell functions in multiple clinical settings, as well as to gain further insight into how these cells undergo malignant transformation. In this review, we summarize recent advances in understanding sites and cellular stages of NK cell development in humans and mice.

Successful reprogramming of differentiated human somatic cells into a pluripotent state would allow creation of patient- and disease-specific stem cells. We previously reported generation of induced pluripotent stem (iPS) cells, capable of germline transmission, from mouse somatic cells by transduction of four defined transcription factors. Here, we demonstrate the generation of iPS cells from adult human dermal fibroblasts with the same four factors: Oct3/4, Sox2, Klf4, and c-Myc. Human iPS cells were similar to human embryonic stem (ES) cells in morphology, proliferation, surface antigens, gene expression, epigenetic status of pluripotent cell-specific genes, and telomerase activity. Furthermore, these cells could differentiate into cell types of the three germ layers in vitro and in teratomas. These findings demonstrate that iPS cells can be generated from adult human fibroblasts.

Disruption of the nucleotide excision repair (NER) pathway by mutations can cause xeroderma pigmentosum, a syndrome predisposing affected individuals to development of skin cancer. The xeroderma pigmentosum C (XPC) protein is essential for initiating global genome NER by recognizing the DNA lesion and recruiting downstream factors. Here we show that inhibition of the deacetylase and longevity factor SIRT1 impairs global genome NER through suppressing the transcription of XPC in a SIRT1 deacetylase-dependent manner. SIRT1 enhances XPC expression by reducing AKT-dependent nuclear localization of the transcription repressor of XPC. Finally, we show that SIRT1 levels are significantly reduced in human skin tumors from Caucasian patients, a population at highest risk. These findings suggest that SIRT1 acts as a tumor suppressor through its role in DNA repair.

Alzheimer's disease (AD) is a devastating neurodegenerative disorder and a major medical problem. Here, we have investigated the impact of amyloid-β (Aβ) oligomers, AD-related neurotoxins, in the brains of rats and adult nonhuman primates (cynomolgus macaques). Soluble Aβ oligomers are known to accumulate in the brains of AD patients and correlate with disease-associated cognitive dysfunction. When injected into the lateral ventricle of rats and macaques, Aβ oligomers diffused into the brain and accumulated in several regions associated with memory and cognitive functions. Cardinal features of AD pathology, including synapse loss, tau hyperphosphorylation, astrocyte and microglial activation, were observed in regions of the macaque brain where Aβ oligomers were abundantly detected. Most importantly, oligomer injections induced AD-type neurofibrillary tangle formation in the macaque brain. These outcomes were specifically associated with Aβ oligomers, as fibrillar amyloid deposits were

Mutations in BRCA1 and BRCA2 predispose individuals to certain cancers1-3, and disease-specific screening and preventative strategies have reduced cancer mortality in affected patients4,5. These classical tumour-suppressor genes have tumorigenic effects associated with somatic biallelic inactivation, although haploinsufficiency may also promote the formation and progression of tumours6,7. Moreover, BRCA1/2-mutant tumours are often deficient in the repair of double-stranded DNA breaks by homologous recombination8-13, and consequently exhibit increased therapeutic sensitivity to platinum-containing therapy and inhibitors of poly-(ADP-ribose)-polymerase (PARP)14,15. However, the phenotypic and therapeutic relevance of mutations in BRCA1 or BRCA2 remains poorly defined in most cancer types. Here we show that in the 2.7% and 1.8% of patients with advanced-stage cancer and germline pathogenic or somatic loss-of-function alterations in BRCA1/2, respectively, selective pressure for biallelic i

Recent advances have suggested that direct induction of neural stem cells (NSCs) could provide an alternative to derivation from somatic tissues or pluripotent cells. Here we show direct derivation of stably expandable NSCs from mouse fibroblasts through a curtailed version of reprogramming to pluripotency. By constitutively inducing Sox2, Klf4, and c-Myc while strictly limiting Oct4 activity to the initial phase of reprogramming, we generated neurosphere-like colonies that could be expanded for more than 50 passages and do not depend on sustained expression of the reprogramming factors. These induced neural stem cells (iNSCs) uniformly display morphological and molecular features of NSCs, such as the expression of Nestin, Pax6, and Olig2, and have a genome-wide transcriptional profile similar to that of brain-derived NSCs. Moreover, iNSCs can differentiate into neurons, astrocytes, and oligodendrocytes. Our results demonstrate that functional NSCs can be generated from somatic cells b

iPSCs (induced pluripotent stem cells) offer an unparalleled opportunity to generate and study physiologically relevant cell types in culture. iPSCs can be generated by reprogramming almost any somatic cell type using pluripotency factors such as Oct4, SOX2, Nanog and Klf4. By reprogramming cells from patients carrying disease-associated mutations, and subsequent differentiation into the cell type of interest, researchers now have the opportunity to study disease-specific cell types which were previously inaccessible. In the case of PD (Parkinson's disease), reprogramming is advancing rapidly, and cell lines have been generated from patients carrying mutations in several disease-associated genes, including SNCA (α-synuclein), PARK2 (parkin), PINK1 (phosphatase and tensin homologue deleted on chromosome 10-induced putative kinase 1), PARK7 (DJ-1) and LRRK2 (leucine-rich repeat kinase 2), as well as idiopathic cases. Functional dopaminergic neurons have been differentiated from these cel