Background The androgen-receptor isoform encoded by splice variant 7 lacks the ligand-binding domain, which is the target of enzalutamide and abiraterone, but remains constitutively active as a transcription factor. We hypothesized that detection of androgen-receptor splice variant 7 messenger RNA (AR-V7) in circulating tumor cells from men with advanced prostate cancer would be associated with resistance to enzalutamide and abiraterone. Methods We used a quantitative reverse-transcriptase–polymerase-chain-reaction assay to evaluate AR-V7 in circulating tumor cells from prospectively enrolled patients with metastatic castration-resistant prostate cancer who were initiating treatment with either enzalutamide or abiraterone. We examined associations between AR-V7 status (positive vs. negative) and prostate-specific antigen (PSA) response rates (the primary end point), freedom from PSA progression (PSA progression–free survival), clinical or radiographic progression–free survival, and overall survival. Results A total of 31 enzalutamide-treated patients and 31 abiraterone-treated patients were enrolled, of whom 39% and 19%, respectively, had detectable AR-V7 in circulating tumor cells. Among men receiving enzalutamide, AR-V7–positive patients had lower PSA response rates than AR-V7–negative patients (0% vs. 53%, P = 0.004) and shorter PSA progression–free survival (median, 1.4 months vs. 6.0 months; P<0.001), clinical or radiographic progression–free survival (median, 2.1 months vs. 6.1 months; P<0.001), and overall survival (median, 5.5 months vs. not reached; P = 0.002). Similarly, among men receiving abiraterone, AR-V7–positive patients had lower PSA response rates than AR-V7–negative patients (0% vs. 68%, P = 0.004) and shorter PSA progression–free survival (median, 1.3 months vs. not reached; P<0.001), clinical or radiographic progression–free survival (median, 2.3 months vs. not reached; P<0.001), and overall survival (median, 10.6 months vs. not reached, P = 0.006). The association between AR-V7 detection and therapeutic resistance was maintained after adjustment for expression of full-length androgen receptor messenger RNA. Conclusions Detection of AR-V7 in circulating tumor cells from patients with castration-resistant prostate cancer may be associated with resistance to enzalutamide and abiraterone. These findings require large-scale prospective validation. (Funded by the Prostate Cancer Foundation and others.)

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CRISPR/Cas9 genome targeting systems have been applied to a variety of species. However, most CRISPR/Cas9 systems reported for plants can only modify one or a few target sites. Here, we report a robust CRISPR/Cas9 vector system, utilizing a plant codon optimized Cas9 gene, for convenient and high-efficiency multiplex genome editing in monocot and dicot plants. We designed PCR-based procedures to rapidly generate multiple sgRNA expression cassettes, which can be assembled into the binary CRISPR/Cas9 vectors in one round of cloning by Golden Gate ligation or Gibson Assembly. With this system, we edited 46 target sites in rice with an average 85.4% rate of mutation, mostly in biallelic and homozygous status. We reasoned that about 16% of the homozygous mutations in rice were generated through the non-homologous end-joining mechanism followed by homologous recombination-based repair. We also obtained uniform biallelic, heterozygous, homozygous, and chimeric mutations in Arabidopsis T1 plants. The targeted mutations in both rice and Arabidopsis were heritable. We provide examples of loss-of-function gene mutations in T0 rice and T1 Arabidopsis plants by simultaneous targeting of multiple (up to eight) members of a gene family, multiple genes in a biosynthetic pathway, or multiple sites in a single gene. This system has provided a versatile toolbox for studying functions of multiple genes and gene families in plants for basic research and genetic improvement.

Recent studies have demonstrated that fatty acids induce insulin resistance in skeletal muscle by blocking insulin activation of insulin receptor substrate-1 (IRS-1)-associated phosphatidylinositol 3-kinase (PI3-kinase). To examine the mechanism by which fatty acids mediate this effect, rats were infused with either a lipid emulsion (consisting mostly of 18:2 fatty acids) or glycerol. Intracellular C18:2 CoA increased in a timedependent fashion, reaching an ϳ6-fold elevation by 5 h, whereas there was no change in the concentration of any other fatty acyl-CoAs. Diacylglycerol (DAG) also increased transiently after 3-4 h of lipid infusion. In contrast there was no increase in intracellular ceramide or triglyceride concentrations during the lipid infusion. Increases in intracellular C18:2 CoA and DAG concentration were associated with protein kinase C (PKC)-activation and a reduction in both insulin-stimulated IRS-1 tyrosine phosphorylation and IRS-1 associated PI3-kinase activity, which were associated with an increase in IRS-1 Ser 307 phosphorylation. These data support the hypothesis that an increase in plasma fatty acid concentration results in an increase in intracellular fatty acyl-CoA and DAG concentrations, which results in activation of PKC-leading to increased IRS-1 Ser 307 phosphorylation. This in turn leads to decreased IRS-1 tyrosine phosphorylation and decreased activation of IRS-1-associated PI3-kinase activity resulting in decreased insulin-stimulated glucose transport activity.

The emergence and spread of COVID-19 since December, 2019, has brought great challenges to global public health. As of April 23, 2020, more than 2•5 million confirmed cases and more than 175 000 deaths had been reported globally. 1 Respiratory tract manifestations such as fever and cough are the most commonly reported symptoms in patients with COVID-19. 2 Evidence of digestive system involvement in patients with COVID-19 was first reported by a group in China. 3 Emerging data showed that the gastrointestinal tract and liver might also represent target organs of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on the basis of the findings that angiotensin-converting enzyme 2 (ACE2), the major receptor of SARS-CoV-2, is expressed in the gastro intestinal tract as well as liver cells. 4 The detection of SARS-CoV-2 viral RNA in patients' stool and the potential for faecal-oral transmission has raised

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Fluorescence correlation spectroscopy (FCS) is generally used to obtain information about the number of fluorescent particles in a small volume and the diffusion coefficient from the autocorrelation function of the fluorescence signal. Here we demonstrate that photon counting histogram (PCH) analysis constitutes a novel tool for extracting quantities from fluorescence fluctuation data, i.e., the measured photon counts per molecule and the average number of molecules within the observation volume. The photon counting histogram of fluorescence fluctuation experiments, in which few molecules are present in the excitation volume, exhibits a super-Poissonian behavior. The additional broadening of the PCH compared to a Poisson distribution is due to fluorescence intensity fluctuations. For diffusing particles these intensity fluctuations are caused by an inhomogeneous excitation profile and the fluctuations in the number of particles in the observation volume. The quantitative relationship between the detected photon counts and the fluorescence intensity reaching the detector is given by Mandel's formula. Based on this equation and considering the fluorescence intensity distribution in the two-photon excitation volume, a theoretical expression for the PCH as a function of the number of molecules in the excitation volume is derived. For a single molecular species two parameters are sufficient to characterize the histogram completely, namely the average number of molecules within the observation volume and the detected photon counts per molecule per sampling time epsilon. The PCH for multiple molecular species, on the other hand, is generated by successively convoluting the photon counting distribution of each species with the others. The influence of the excitation profile upon the photon counting statistics for two relevant point spread functions (PSFs), the three-dimensional Gaussian PSF conventionally employed in confocal detection and the square of the Gaussian-Lorentzian PSF for two photon excitation, is explicitly treated. Measured photon counting distributions obtained with a two-photon excitation source agree, within experimental error with the theoretical PCHs calculated for the square of a Gaussian-Lorentzian beam profile. We demonstrate and discuss the influence of the average number of particles within the observation volume and the detected photon counts per molecule per sampling interval upon the super-Poissonian character of the photon counting distribution.

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