Stretching taxes CRISPR's abilities

Preventing off-target effects of CRISPR/Cas9 remains a major obstacle to the genome editing method's clinical application. In fact, even predicting when and where off-target effects will occur presently remains an imprecise undertaking at best. Researchers from Astrazeneca plc and Imperial College London have developed a molecular-precision tool for DNA manipulation that allowed them to demonstrate that CRISPR's off-target effects increased as DNA was physically stretched. Off-target effects did not occur at random, showing that stretching exposed off-target sites, rather than creating them through physically stressing the DNA. "We propose that duplex DNA destabilization during cellular processes (for example, transcription, replication, etc.) can expose these cryptic off-target sites to Cas9 activity, highlighting the need for improved off-target prediction algorithms," the authors wrote. Their work appeared in the Feb. 25, 2019, issue of Nature Structural and Molecular Biology.

Typhus vaccination broadly alters immune response

Researchers from the Liverpool School of Tropical Medicine and the University of Liverpool have identified broad-spectrum changes in immune response after vaccination with live-attenuated Salmonella typhi. Previous studies have shown that some vaccinations appear to reduce all-cause mortality in addition to protecting against the specific pathogen they target. The team investigated the molecular mechanisms of that effect by comparing the reactions of cells from vaccinated volunteers to controls in cell culture. They discovered that vaccination with S. typhi altered the cytokine responses of both innate and adaptive cells to other pathogen types, including the fungus Candid albicans, the bacterium M. tuberculosis, influenza virus and tetanus toxoid. The strongest responses were seen in monocytes, which are innate immune cells. "The changes that we have observed could influence susceptibility to infection through altered immune responses mounted to subsequently encountered pathogens," the authors wrote, and consequently, "these changes could influence all-cause mortality." Their work appeared in the Feb. 27, 2019, issue of Science Advances.

Optogenetics helps understand TDP-43 aggregates

Scientists at the University of Pittsburgh have developed a method to induce the formation of TDP-43 aggregates through optogenetics, and used it to gain new insights into toxic mechanisms of TDP-43 aggregates in amyotrophic lateral sclerosis (ALS). Protein aggregates are a general feature of neurodegenerative diseases, but the nature of the protein, as well as the location of the aggregates, vary among conditions. TDP-43 is the protein that aggregates in ALS, and the authors demonstrated that the aggregation occurred when TDP-43 binding to RNA was disrupted. Specifically, "our work and other observations suggest that an altered TDP-43:RNA ratio creates an aggregation-prone environment for TDP-43 and serves as an upstream event in TDP-43 proteinopathy. This may occur due to disease-causing mutations... that disrupt nuclear transport dynamics, resulting in elevated cytoplasmic TDP-43 protein and retention of RNA in the nucleus," the authors wrote. Their work was published in the Feb. 27, 2019, online issue of Neuron.

Tackling tendonitis

Researchers at Columbia University have demonstrated that the activation of IKK-beta, a key upstream regulator of NF-kappaB, caused inflammatory tendon damage, and its inhibition could protect mice from overuse-induced tendinitis. NF-kappaB activation has been observed in tendinitis, but whether it contributed to tendon degeneration or was part of repair attempts had not been clear. The researchers showed that NF-kappaB, activated by IKK-beta, was part of the driving force behind tendon degeneration. Genetic deletion of IKK-beta specifically in fibroblasts of the joint protected mice from tendinitis. There are as yet no clinically effective IKK-beta inhibitors, but structural studies that have elucidated new structural details of IKK-beta offer hope for the development of more potent and/or more specific IKK-beta inhibitors. Such inhibitors could help to realize the potential that, as the authors wrote, "targeting of the IKK-beta/NF-kappaB pathway in tendon stromal cells may offer previously unidentified therapeutic approaches in the management of human tendon disorders." Their work appeared in the Feb. 27, 2019, issue of Science Translational Medicine.

Power to the pyrimidines

A team at the University of Michigan has demonstrated that macrophage-released pyrimidines drove resistance to the chemotherapy drug gemcitabine in pancreatic cancer models. Previous work had shown that the presence of tumor-associated macrophages, which are abundant in pancreatic tumors, correlated with resistance to chemotherapy. The team investigated possible mechanisms of that resistance, and showed that pancreatic tumor cells could reprogram macrophage metabolism, leading to the release of pyrimidines. Structurally speaking, gemcitabine is a pyrimidine itself, and the release of structurally related pyrimidines by macrophages led to competitive inhibition of gemcitabine activity. "Together, these findings provide insights into the role of macrophages in pancreatic cancer therapy and have potential to inform the design of future treatments," the authors wrote. "Additionally, we report that pyrimidine release is a general function of alternatively activated macrophage cells, suggesting an unknown physiological role of pyrimidine exchange by immune cells." Their work appeared in the Feb. 28, 2019, online issue of Cell Metabolism.

Newly identified pathway provides fat for macrophages

Fat cells serve as energy reservoirs, and release their bounty via lipolysis, an enzyme-driven process. Scientists at Columbia University have identified an alternate pathway for fat cells to release energy that was particularly important for affecting the differentiation and activity of fat tissue resident macrophages. The team showed that in addition to enzyme-generated release of fatty acids from fat cells, cells also released "exosome-sized, lipid-filled vesicles (AdExos) that become a source of lipid for local macrophages. Adipose tissue from lean mice released ~1% of its lipid content per day via exosomes ex vivo, a rate that more than doubles in obese animals." Macrophages are the most abundant type of immune cells in adipose tissue and can account for 50 percent of cells in obese individuals, and their driving of inflammatory processes contributes to systemic insulin resistance in obese individuals. Understanding their regulation could offer insights into combating metabolic disorders. The team reported its results in the March 1, 2019, issue of Science.

High-throughput, low-stress analysis method separates out brain cells

Methodological advances have made it clear that as far as brain functioning is concerned, neurons are only one of multiple important cell types. However, a thorough understanding of the cellular ensemble that contributes to brain functioning is still hampered by the fact that high-throughput cell sorting and analysis methods tend to degrade the quality of the data they are collecting. Researchers from the Laboratory of Neurotherapies and Neuromodulation at Lausanne University Hospital have developed a method of cellular analysis that was able to achieve high throughput while maintaining low cellular stress. "The samples generated with the new protocol are compatible with non-coding RNA profiling, alternative splicing, and genomic variant and high-throughput protein quantification," the authors wrote. "The opportunity to combine these high-throughput methods with the new procedure will contribute to our understanding of the 'cellular interactome' from the gene to protein activity, which is at the core of individual cell function." They reported their method in the Feb. 26, 2019, issue of Cell Reports.

Prostate cancer cell lineage switch identified

Scientists from Sanford Burnham Prebys Medical Discovery Institute have identified how treatment with anti-androgen therapy can lead to the development of treatment-resistant neuroendocrine prostate cancer (NEPC) in patients. The team showed that treatment with androgen blockers led to down-regulation of protein kinase C lambda/iota, which caused increases in the nonessential amino acid serine through an mTORC1/ATF4-driven pathway, epigenetically reprogramming tumor cells to an NEPC-like state. In addition to identifying targets for NEPC treatment, the authors wrote that their discoveries with respect to the role of protein kinase C lambda/iota have "broader implications in cancer since lineage plasticity has emerged as a mechanism of drug resistance, not only in [prostate cancer], but also in other types of neoplasia." They published their findings In the Feb. 25, 2019, issue of Cancer Cell.

Decreased inhibition, but no hyperexcitation, in multiple autism models

Researchers from the University of California at Berkeley have investigated four single-gene mouse models of autism, and discovered that none of them showed overall increased neuronal excitation that is assumed to be a central feature of autism spectrum disorders (ASD). Instead, though all four mice showed decreased inhibition, the decrease seemed to be a compensatory mechanism that normalized overall firing rates. Modeling experiments showed that changes in excitation and inhibition were matched to each other in each mouse model "to yield stable, not increased, synaptic depolarization for cells near spike threshold. Correspondingly, whisker-evoked spiking was not increased in vivo despite detectably reduced inhibition," the authors wrote. They concluded that the increased inhibition "is a common circuit phenotype but appears to reflect homeostatic stabilization of synaptic drive rather than driving network hyperexcitability in autism." Their work appeared in the Feb. 20, 2019, issue of Neuron after earlier publication online.

The Model T of CAR T cells?

Researchers at Yale University have developed a simplified method of engineering chimeric antigen receptor T (CAR T) cells, paving the way for assembly-line style production of such cells. The team used CRISPR-based engineering to create CAR T cells, but as their nuclease, the team used Cpf1 instead of the more typical Cas9. Compared to Cas9, Cpf1 needs fewer RNA partners to work with, has higher specificity in human cells and is more suitable for precision engineering because of the types of DNA breaks it generates. Using Cpf1, "building a stable CAR T cell with homology-directed repair knock-in and immune-checkpoint knockout (KIKO CAR T cell) was achieved at high efficiency in one step," the authors wrote. "CD22-specific AAV–Cpf1 KIKO CAR T cells have potency comparable to that of Cas9 CAR T cells in cytokine production and cancer cell killing, while expressing lower levels of exhaustion markers. This versatile system opens new capabilities of T-cell engineering with simplicity and precision." The team reported its method in the Feb. 25, 2019, issue of Nature Methods.