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We recently developed adeno-associated virus (AAV) capsids to facilitate efficient and noninvasive gene transfer to the central and peripheral nervous systems. However, a detailed protocol for generating and systemically delivering novel AAV variants was not previously available. In this protocol, we describe how to produce and intravenously administer AAVs to adult mice to specifically label and/or genetically manipulate cells in the nervous system and organs, including the heart. The procedure comprises three separate stages: AAV production, intravenous delivery, and evaluation of transgene expression. The protocol spans 8 d, excluding the time required to assess gene expression, and can be readily adopted by researchers with basic molecular biology, cell culture, and animal work experience. We provide guidelines for experimental design and choice of the capsid, cargo, and viral dose appropriate for the experimental aims. The procedures outlined here are adaptable to diverse biomedical applications, from anatomical and functional mapping to gene expression, silencing, and editing. Having developed AAV capsids that target sites throughout the body, here the authors describe how to produce and systemically administer these AAVs to rodents to label and/or genetically manipulate cells in the nervous system and visceral organs.

\"Your Future Is Bright! After all, your mother is a robot, your father has joined the alien hive mind, and your dinner will be counterfeit 3D-printed steak. Even though your worker bots have staged a mutiny, and your tour guide speaks only in memes, you can always sell your native language if you need some extra cash.\" -- From publisher's description.

Epigenetic deregulation of gene expression has a role in the initiation and progression of prostate cancer (PCa). The histone methyltransferase MMSET/WHSC1 (Multiple Myeloma SET domain) is overexpressed in a number of metastatic tumors, but its mechanism of action has not been defined. In this work, we found that PCa cell lines expressed significantly higher levels of MMSET compared with immortalized, non-transformed prostate cells. Knockdown experiments showed that, in metastatic PCa cell lines, dimethylation of lysine 36 and trimethylation of lysine 27 on histone H3 (H3K36me2 and H3K27me3, respectively) depended on MMSET expression, whereas depletion of MMSET in benign prostatic cells did not affect chromatin modifications. Knockdown of MMSET in DU145 and PC-3 tumor cells decreased cell proliferation, colony formation in soft agar and strikingly diminished cell migration and invasion. Conversely, overexpression of MMSET in immortalized, non-transformed RWPE-1 cells promoted cell migration and invasion, accompanied by an epithelial–mesenchymal transition (EMT). Among a panel of EMT-promoting genes analyzed, TWIST1 expression was strongly activated in response to MMSET. Chromatin immunoprecipitation analysis demonstrated that MMSET binds to the TWIST1 locus and leads to an increase in H3K36me2, suggesting a direct role of MMSET in the regulation of this gene. Depletion of TWIST1 in MMSET-overexpressing RWPE-1 cells blocked cell invasion and EMT, indicating that TWIST1 was a critical target of MMSET, responsible for the acquisition of an invasive phenotype. Collectively, these data suggest that MMSET has a role in PCa pathogenesis and progression through epigenetic regulation of metastasis-related genes.

The authors report two new engineered AAV capsids that efficiently deliver genes throughout the adult central and peripheral nervous systems after intravenous administration. Complementing these capsids is an AAV toolbox that enables cell morphology and genetic manipulation studies of defined neural cell types in transgenic or wild-type animals. Adeno-associated viruses (AAVs) are commonly used for in vivo gene transfer. Nevertheless, AAVs that provide efficient transduction across specific organs or cell populations are needed. Here, we describe AAV-PHP.eB and AAV-PHP.S, capsids that efficiently transduce the central and peripheral nervous systems, respectively. In the adult mouse, intravenous administration of 1 × 10 11 vector genomes (vg) of AAV-PHP.eB transduced 69% of cortical and 55% of striatal neurons, while 1 × 10 12 vg of AAV-PHP.S transduced 82% of dorsal root ganglion neurons, as well as cardiac and enteric neurons. The efficiency of these vectors facilitates robust cotransduction and stochastic, multicolor labeling for individual cell morphology studies. To support such efforts, we provide methods for labeling a tunable fraction of cells without compromising color diversity. Furthermore, when used with cell-type-specific promoters and enhancers, these AAVs enable efficient and targetable genetic modification of cells throughout the nervous system of transgenic and non-transgenic animals.

Spin-valley locking in monolayer transition metal dichalcogenides has attracted enormous interest, since it offers potential for valleytronic and optoelectronic applications. Such an exotic electronic state has sparsely been seen in bulk materials. Here, we report spin-valley locking in a Dirac semimetal BaMnSb 2 . This is revealed by comprehensive studies using first principles calculations, tight-binding and effective model analyses, angle-resolved photoemission spectroscopy measurements. Moreover, this material also exhibits a stacked quantum Hall effect (QHE). The spin-valley degeneracy extracted from the QHE is close to 2. This result, together with the Landau level spin splitting, further confirms the spin-valley locking picture. In the extreme quantum limit, we also observed a plateau in the z -axis resistance, suggestive of a two-dimensional chiral surface state present in the quantum Hall state. These findings establish BaMnSb 2 as a rare platform for exploring coupled spin and valley physics in bulk single crystals and accessing 3D interacting topological states. Valley dependent spin polarization called spin-valley locking appears in absence of magnetism but it is limited to rare examples of transition metal dichalcogenides. Here, the authors report evidence of spin-valley locking and stacked quantum Hall effect in a bulk Dirac semimetal BaMnSb 2 .

There is a major unmet need for effective treatments in patients with squamous cell carcinoma of the lung. LUX-Lung 8 compared afatinib (an irreversible ErbB family blocker) with erlotinib (a reversible EGFR tyrosine kinase inhibitor), as second-line treatment for patients with advanced squamous cell carcinoma of the lung. We did this open-label, phase 3 randomised controlled trial at 183 cancer centres in 23 countries worldwide. We enrolled adults with stage IIIB or IV squamous cell carcinoma of the lung who had progressed after at least four cycles of platinum-based-chemotherapy. Participants were randomly assigned (1:1) to receive afatinib (40 mg per day) or erlotinib (150 mg per day) until disease progression. The randomisation was done centrally with an interactive voice or web-based response system and stratified by ethnic origin (eastern Asian vs non-eastern Asian). Clinicians and patients were not masked to treatment allocation. The primary endpoint was progression-free survival assessed by independent central review (intention-to-treat population). The key secondary endpoint was overall survival. This trial is registered with ClinicalTrials.gov, NCT01523587. 795 eligible patients were randomly assigned (398 to afatinib, 397 to erlotinib). Median follow-up at the time of the primary analysis of progression-free survival was 6·7 months (IQR 3·1–10·2), at which point enrolment was not complete. Progression free-survival at the primary analysis was significantly longer with afatinib than with erlotinib (median 2·4 months [95% CI 1·9–2·9] vs 1·9 months [1·9–2·2]; hazard ratio [HR] 0·82 [95% CI 0·68–1·00], p=0·0427). At the time of the primary analysis of overall survival (median follow-up 18·4 months [IQR 13·8–22·4]), overall survival was significantly greater in the afatinib group than in the erloinib group (median 7·9 months [95% CI 7·2–8·7] vs 6·8 months [5·9–7·8]; HR 0·81 [95% CI 0·69–0·95], p=0·0077), as were progression-free survival (median 2·6 months [95% CI 2·0–2·9] vs 1·9 months [1·9–2·1]; HR 0·81 [95% CI 0·69–0·96], p=0·0103) and disease control (201 [51%] of 398 patients vs 157 [40%] of 397; p=0·0020). The proportion of patients with an objective response did not differ significantly between groups (22 [6%] vs 11 [3%]; p=0·0551). Tumour shrinkage occurred in 103 (26%) of 398 patients versus 90 (23%) of 397 patients. Adverse event profiles were similar in each group: 224 (57%) of 392 patients in the afatinib group versus 227 (57%) of 395 in the erlotinib group had grade 3 or higher adverse events. We recorded higher incidences of treatment-related grade 3 diarrhoea with afatinib (39 [10%] vs nine [2%]), of grade 3 stomatitis with afatinib (16 [4%] vs none), and of grade 3 rash or acne with erlotinib (23 [6%] vs 41 [10%]). The significant improvements in progression-free survival and overall survival with afatinib compared with erlotinib, along with a manageable safety profile and the convenience of oral administration suggest that afatinib could be an additional option for the treatment of patients with squamous cell carcinoma of the lung. Boehringer Ingelheim.

Background: Some studies have provided the possibility that adipose tissue may mediate air pollution-induced lung dysfunction. Studies using quantified fat mass data are needed to understand the biological mechanisms between adipocyte and air pollution in lung function. We aimed to investigate whether abdominal adiposity measured by computed tomography (CT) modifies the effects of air pollution on lung function in Korean men. Methods: A total of 1876 men who visited one of two health checkup centers were recruited for this study. Adiposity traits such as visceral adipose tissue (VAT), subcutaneous adipose tissue (SAT) and total adipose tissue (TAT) areas were measured by CT. We used the annual mean concentrations of ambient air pollutants including nitrogen dioxide (NO 2 ) and particulate matter with an aerodynamic diameter ⩽10 μm (PM 10 ). Results: Interquartile range (IQR) increase in annual mean concentration of NO 2 was significantly associated with a 2.5% lower forced expiratory volume in 1 s (FEV 1 ) and 2.9% lower forced vital capacity (FVC) (both P <0.05). The decrease in lung function was more strongly associated with adiposity traits than with body mass index. In a stratified analysis of adiposity, compared with subjects with low-VAT area (VAT⩽200 cm 2 ), those with high-VAT area (VAT>200 cm 2 ) showed a rapid decrease in FEV 1 with each IQR increase in PM 10 ( β =–0.0812; 95% confidence interval (CI) =–0.1590, –0.0035) and NO 2 ( β =–0.0979; 95% CI=–0.1611, –0.0346). In the high-VAT group, each IQR increase in NO 2 content was significantly associated with a 10.6% decrease ( β =–0.1056; 95% CI=–0.1770, –0.0343) in FVC. SAT and TAT areas showed similar patterns. Conclusions: We report the first finding that abdominal adiposity intensifies the inverse relationship between air pollution and lung function.

Recombinant adeno-associated viruses (rAAVs) are efficient gene delivery vectors via intravenous delivery; however, natural serotypes display a finite set of tropisms. To expand their utility, we evolved AAV capsids to efficiently transduce specific cell types in adult mouse brains. Building upon our Cre-recombination-based AAV targeted evolution (CREATE) platform, we developed Multiplexed-CREATE (M-CREATE) to identify variants of interest in a given selection landscape through multiple positive and negative selection criteria. M-CREATE incorporates next-generation sequencing, synthetic library generation and a dedicated analysis pipeline. We have identified capsid variants that can transduce the central nervous system broadly, exhibit bias toward vascular cells and astrocytes, target neurons with greater specificity or cross the blood–brain barrier across diverse murine strains. Collectively, the M-CREATE methodology accelerates the discovery of capsids for use in neuroscience and gene-therapy applications. M-CREATE is an in vivo screening strategy for identifying recombinant AAVs with desired tropism. The approach involves both positive and negative selection and yields vectors with diversified cell-type tropism that can cross the blood–brain barrier in adult mice across strains when delivered intravenously.

Crossing the blood–brain barrier in primates is a major obstacle for gene delivery to the brain. Adeno-associated viruses (AAVs) promise robust, non-invasive gene delivery from the bloodstream to the brain. However, unlike in rodents, few neurotropic AAVs efficiently cross the blood–brain barrier in non-human primates. Here we report on AAV.CAP-Mac, an engineered variant identified by screening in adult marmosets and newborn macaques, which has improved delivery efficiency in the brains of multiple non-human primate species: marmoset, rhesus macaque and green monkey. CAP-Mac is neuron biased in infant Old World primates, exhibits broad tropism in adult rhesus macaques and is vasculature biased in adult marmosets. We demonstrate applications of a single, intravenous dose of CAP-Mac to deliver functional GCaMP for ex vivo calcium imaging across multiple brain areas, or a cocktail of fluorescent reporters for Brainbow-like labelling throughout the macaque brain, circumventing the need for germline manipulations in Old World primates. As such, CAP-Mac is shown to have potential for non-invasive systemic gene transfer in the brains of non-human primates. Crossing the blood–brain barrier in primates is a major obstacle to gene delivery in the brain. Here an adeno-associated virus variant (AAV.CAP-Mac) is identified and demonstrated for crossing the blood–brain barrier and delivering gene sequences to the brain of different non-human primates species.