This study investigated the regulation of thermogenic capacity in classical brown adipose tissue (BAT) and subcutaneous inguinal (SC Ing) white adipose tissue (WAT) and how it affects whole-body ...energy expenditure in sedentary and endurance-trained rats fed ad libitum either low fat or high fat (HF) diets. Analysis of tissue mass, PGC-1α and UCP-1 content, the presence of multilocular adipocytes, and palmitate oxidation revealed that a HF diet increased the thermogenic capacity of the interscapular and aortic brown adipose tissues, whereas exercise markedly suppressed it. Conversely, exercise induced browning of the SC Ing WAT. This effect was attenuated by a HF diet. Endurance training neither affected skeletal muscle FNDC5 content nor circulating irisin, but it increased FNDC5 content in SC Ing WAT. This suggests that locally produced FNDC5 rather than circulating irisin mediated the exercise-induced browning effect on this fat tissue. Importantly, despite reducing the thermogenic capacity of classical BAT, exercise increased whole-body energy expenditure during the dark cycle. Therefore, browning of subcutaneous WAT likely exerted a compensatory effect and raised whole-body energy expenditure in endurance-trained rats. Based on these novel findings, we propose that exercise-induced browning of the subcutaneous WAT provides an alternative mechanism that reduces thermogenic capacity in core areas and increases it in peripheral body regions. This could allow the organism to adjust its metabolic rate to accommodate diet-induced thermogenesis while simultaneously coping with the stress of chronically increased heat production through exercise.
Metastatic breast cancers (mBCs) are largely resistant to immune checkpoint blockade, but the mechanisms remain unclear. Primary breast cancers are characterized by a dense fibrotic stroma, which is ...considered immunosuppressive in multiple malignancies, but the stromal composition of breast cancer metastases and its role in immunosuppression are largely unknown. Here we show that liver and lung metastases of human breast cancers tend to be highly fibrotic, and unlike primary breast tumors, they exclude cytotoxic T lymphocytes (CTLs). Unbiased analysis of the The Cancer Genome Atlas database of human breast tumors revealed a set of genes that are associated with stromal T-lymphocyte exclusion. Among these, we focused on CXCL12 as a relevant target based on its known roles in immunosuppression in other cancer types. We found that the CXCL12 receptor CXCR4 is highly expressed in both human primary tumors and metastases. To gain insight into the role of the CXCL12/CXCR4 axis, we inhibited CXCR4 signaling pharmacologically and found that plerixafor decreases fibrosis, alleviates solid stress, decompresses blood vessels, increases CTL infiltration, and decreases immunosuppression in murine mBC models. By deleting CXCR4 in αSMA⁺ cells, we confirmed that these immunosuppressive effects are dependent on CXCR4 signaling in αSMA⁺ cells, which include cancer-associated fibroblasts as well as other cells such as pericytes. Accordingly, CXCR4 inhibition more than doubles the response to immune checkpoint blockers in mice bearing mBCs. These findings demonstrate that CXCL12/CXCR4-mediated desmoplasia in mBC promotes immunosuppression and is a potential target for overcoming therapeutic resistance to immune checkpoint blockade in mBC patients.
The Bloom’s helicase ortholog, Sgs1, plays central roles to coordinate the formation and resolution of joint molecule intermediates (JMs) during meiotic recombination in budding yeast. Sgs1 can ...associate with type-I topoisomerase Top3 and its accessory factor Rmi1 to form a conserved complex best known for its unique ability to decatenate double-Holliday junctions. Contrary to expectations, we show that the strand-passage activity of Top3-Rmi1 is required for all known functions of Sgs1 in meiotic recombination, including channeling JMs into physiological crossover and noncrossover pathways, and suppression of non-allelic recombination. We infer that Sgs1 always functions in the context of the Sgs1-Top3-Rmi1 complex to regulate meiotic recombination. In addition, we reveal a distinct late role for Top3-Rmi1 in resolving recombination-dependent chromosome entanglements to allow segregation at anaphase. Surprisingly, Sgs1 does not share this essential role of Top3-Rmi1. These data reveal an essential and pervasive role for the Top3-Rmi1 decatenase during meiosis.
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•DNA strand passage by Top3-Rmi1 is pivotal for meiotic recombination•Conditional alleles define temporally and functionally distinct roles of Top3-Rmi1•Top3-Rmi1 partners with RecQ helicase BLM/Sgs1 to prevent abnormal recombination•Top3-Rmi1 is essential for disentangling chromosomes enabling complete separation
Topoisomerases are essential for chromosome metabolism. Tang et al. show that the Top3-Rmi1 complex must decatenate DNA strands to process intermediates during homologous recombination. Top3-Rmi1 partners with BLM/Sgs1 to prevent abnormal recombination. Top3-Rmi1 is also essential for disentangling chromosomes, enabling their complete separation.
Mutations in the leptin gene (ob) result in a metabolic disorder that includes severe obesity
, and defects in thermogenesis
and lipolysis
, both of which are adipose tissue functions regulated by ...the sympathetic nervous system. However, the basis of these sympathetic-associated abnormalities remains unclear. Furthermore, chronic leptin administration reverses these abnormalities in adipose tissue, but the underlying mechanism remains to be discovered. Here we report that ob/ob mice, as well as leptin-resistant diet-induced obese mice, show significant reductions of sympathetic innervation of subcutaneous white and brown adipose tissue. Chronic leptin treatment of ob/ob mice restores adipose tissue sympathetic innervation, which in turn is necessary to correct the associated functional defects. The effects of leptin on innervation are mediated via agouti-related peptide and pro-opiomelanocortin neurons in the hypothalamic arcuate nucleus. Deletion of the gene encoding the leptin receptor in either population leads to reduced innervation in fat. These agouti-related peptide and pro-opiomelanocortin neurons act via brain-derived neurotropic factor-expressing neurons in the paraventricular nucleus of the hypothalamus (BDNF
). Deletion of BDNF
blunts the effects of leptin on innervation. These data show that leptin signalling regulates the plasticity of sympathetic architecture of adipose tissue via a top-down neural pathway that is crucial for energy homeostasis.
The bacterial adaptive immune system CRISPR–Cas9 has been appropriated as a versatile tool for editing genomes, controlling gene expression, and visualizing genetic loci. To analyze Cas9’s ability to ...bind DNA rapidly and specifically, we generated multiple libraries of potential binding partners for measuring the kinetics of nuclease-dead Cas9 (dCas9) interactions. Using a massively parallel method to quantify protein–DNA interactions on a high-throughput sequencing flow cell, we comprehensively assess the effects of combinatorial mismatches between guide RNA (gRNA) and target nucleotides, both in the seed and in more distal nucleotides, plus disruption of the protospacer adjacent motif (PAM). We report two consequences of PAM-distal mismatches: reversal of dCas9 binding at long time scales, and synergistic changes in association kinetics when other gRNA–target mismatches are present. Together, these observations support a model for Cas9 specificity wherein gRNA–DNA mismatches at PAM-distal bases modulate different biophysical parameters that determine association and dissociation rates. The methods we present decouple aspects of kinetic and thermodynamic properties of the Cas9–DNA interaction and broaden the toolkit for investigating off-target binding behavior.
Cancer-associated fibroblasts (CAFs) can either suppress or support T lymphocyte activity, suggesting that CAFs may be reprogrammable to an immunosupportive state. Angiotensin receptor blockers ...(ARBs) convert myofibroblast CAFs to a quiescent state, but whether ARBs can reprogram CAFs to promote T lymphocyte activity and enhance immunotherapy is unknown. Moreover, ARB doses are limited by systemic adverse effects such as hypotension due to the importance of angiotensin signaling outside tumors. To enhance the efficacy and specificity of ARBs in cancer with the goal of revealing their effects on antitumor immunity, we developed ARB nanoconjugates that preferentially accumulate and act in tumors. We created a diverse library of hundreds of acid-degradable polymers and chemically linked ARBs to the polymer most sensitive to tumor pH. These tumor microenvironment-activated ARBs (TMA-ARBs) remain intact and inactive in circulation while achieving high concentrations in tumors, wherein they break down to active ARBs. This tumor-preferential activity enhances the CAF-reprogramming effects of ARBs while eliminating blood pressure-lowering effects. Notably, TMA-ARBs alleviate immunosuppression and improve T lymphocyte activity, enabling dramatically improved responses to immune-checkpoint blockers in mice with primary as well as metastatic breast cancer.
Mechanically interlocked molecules that change their conformation in response to stimuli have been developed by synthetic chemists as building blocks for molecular machines. Here we describe a ...natural product, the lasso peptide benenodin-1, which exhibits conformational switching between two distinct threaded conformers upon actuation by heat. We have determined the structures of both conformers and have characterized the kinetics and energetics of the conformational switch. Single amino acid substitutions to benenodin-1 generate peptides that are biased to a single conformer, showing that the switching behavior is potentially an evolvable trait in these peptides. Lasso peptides such as benenodin-1 can be recognized and cleaved by enzymes called lasso peptide isopeptidases. We show that only the native conformer of benenodin-1 is cleaved by its cognate isopeptidase. Thus, thermally induced conformational switching of benenodin-1 may also be relevant to the biological function of these molecules.
Here, we describe single-tube long fragment read (stLFR), a technology that enables sequencing of data from long DNA molecules using economical second-generation sequencing technology. It is based on ...adding the same barcode sequence to subfragments of the original long DNA molecule (DNA cobarcoding). To achieve this efficiently, stLFR uses the surface of microbeads to create millions of miniaturized barcoding reactions in a single tube. Using a combinatorial process, up to 3.6 billion unique barcode sequences were generated on beads, enabling practically nonredundant cobarcoding with 50 million barcodes per sample. Using stLFR, we demonstrate efficient unique cobarcoding of more than 8 million 20- to 300-kb genomic DNA fragments. Analysis of the human genome NA12878 with stLFR demonstrated high-quality variant calling and phase block lengths up to N50 34 Mb. We also demonstrate detection of complex structural variants and complete diploid de novo assembly of NA12878. These analyses were all performed using single stLFR libraries, and their construction did not significantly add to the time or cost of whole-genome sequencing (WGS) library preparation. stLFR represents an easily automatable solution that enables high-quality sequencing, phasing, SV detection, scaffolding, cost-effective diploid de novo genome assembly, and other long DNA sequencing applications.
This article will look at the recent Hachette decision against the Internet Archive, analyzing how the court's reliance on past authorities with insufficient context distorted their meanings. It will ...focus only on the controlled digital lending (CDL) aspect, not discussing the other claims in the suit or exploring the specific implementation of CDL by the Internet Archive (IA). Since CDL programs can vary widely, IA is better situated than others to identify missing context related to the analysis of the unique components of their efforts. And other libraries engaging in CDL should be able to easily see where their programs differ from the judge's description of IA's. For that reason, the analysis below only delves into the language that might be used to chill all CDL programs or innovation more generally.
This paper looks at the recent Redigi court decision and discusses its impact on controlled digital lending programs (CDL) by libraries. As there are notable differences in facts between Redigi and ...CDL programs, the decision should have minimal impact on the framework designed to digitize and lend library materials within the United States.
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