Early telomerase inactivation accelerates aging independently of telomere length.

Telomerase is required for long-term telomere maintenance and protection. Using single budding yeast mother cell analyses we found that, even early after telomerase inactivation (ETI), yeast mother cells show transient DNA damage response (DDR) episodes, stochastically altered cell-cycle dynamics, and accelerated mother cell aging. The acceleration of ETI mother cell aging was not explained by increased reactive oxygen species (ROS), Sir protein perturbation, or deprotected telomeres. ETI phenotypes occurred well before the population senescence caused late after telomerase inactivation (LTI). They were morphologically distinct from LTI senescence, were genetically uncoupled from telomere length, and were rescued by elevating dNTP pools. Our combined genetic and single-cell analyses show that, well before critical telomere shortening, telomerase is continuously required to respond to transient DNA replication stress in mother cells and that a lack of telomerase accelerates otherwise normal aging.

CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes.

The genetic interrogation and reprogramming of cells requires methods for robust and precise targeting of genes for expression or repression. The CRISPR-associated catalytically inactive dCas9 protein offers a general platform for RNA-guided DNA targeting. Here, we show that fusion of dCas9 to effector domains with distinct regulatory functions enables stable and efficient transcriptional repression or activation in human and yeast cells, with the site of delivery determined solely by a coexpressed short guide (sg)RNA. Coupling of dCas9 to a transcriptional repressor domain can robustly silence expression of multiple endogenous genes. RNA-seq analysis indicates that CRISPR interference (CRISPRi)-mediated transcriptional repression is highly specific. Our results establish that the CRISPR system can be used as a modular and flexible DNA-binding platform for the recruitment of proteins to a target DNA sequence, revealing the potential of CRISPRi as a general tool for the precise regulation of gene expression in eukaryotic cells.

Nodal signaling has dual roles in fate specification and directed migration during germ layer segregation in zebrafish.

During gastrulation, endodermal cells actively migrate to the interior of the embryo, but the signals that initiate and coordinate this migration are poorly understood. By transplanting ectopically induced endodermal cells far from the normal location of endoderm specification, we identified the inputs that drive internalization without the confounding influences of fate specification and global morphogenic movements. We find that Nodal signaling triggers an autocrine circuit for initiating endodermal internalization. Activation of the Nodal receptor directs endodermal specification through sox32 and also induces expression of more Nodal ligands. These ligands act in an autocrine fashion to initiate endodermal cell sorting. Our work defines an 'AND' gate consisting of sox32-dependent endodermal specification and Nodal ligand reception controlling endodermal cell sorting to the inner layer of the embryo at the onset of gastrulation.

High Responsivity Vacuum Nano-Photodiode Using Single-Crystal CsPbBr3 Micro-Sheet.

Field electron emission vacuum photodiode is promising for converting free-space electromagnetic radiation into electronic signal within an ultrafast timescale due to the ballistic electron transport in its vacuum channel. However, the low photoelectric conversion efficiency still hinders the popularity of vacuum photodiode. Here, we report an on-chip integrated vacuum nano-photodiode constructed from a Si-tip anode and a single-crystal CsPbBr3 cathode with a nano-separation of ~30 nm. Benefiting from the nanoscale vacuum channel and the high surface work function of the CsPbBr3 (4.55 eV), the vacuum nano-photodiode exhibits a low driving voltage of 15 V with an ultra-low dark current (50 pA). The vacuum nano-photodiode demonstrates a high photo responsivity (1.75 AW-1@15 V) under the illumination of a 532-nm laser light. The estimated external quantum efficiency is up to 400%. The electrostatic field simulation indicates that the CsPbBr3 cathode can be totally depleted at an optimal thickness. The large built-in electric field in the depletion region facilitates the dissociation of photoexcited electron-hole pairs, leading to an enhanced photoelectric conversion efficiency. Moreover, the voltage drop in the vacuum channel increases due to the photoconductive effect, which is beneficial to the narrowing of the vacuum barrier for more efficient electron tunneling. This device shows great promise for the development of highly sensitive perovskite-based vacuum opto-electronics.

Nanobiochar paper based electrochemical immunosensor for fast and ultrasensitive detection of microcystin-LR.

A portable, cheap and sensitive paper type electrochemical immunosensor was developed with conductive nanobiochar paper as the conductive layer and utilized for sensitive detection of microcystin-LR (MCLR) toxin in water. The paper immunosensor was constructed by coating of highly conductive and dispersible nanobiochar particle (nBC) and anti-MCLR antibody on the filter paper via dipping-drying method. The presence of MCLR could be specifically quantified amperometrically by the nBC-paper immunosensor with the response time of less than 5 min, and the lowest detection limit of 17 pM (0.017 µg/L) was achieved. Moreover, the proposed immunosensor exhibited high selectivity, reproducibility and storage stability, and was also used for environmental water detection with satisfactory recovery. The successful fabrication of low cost and ubiquitous biochar based paper type electrochemical immunosensing system would have significant value for the development of highly cost-effective electrochemical device.

Immobilization of laccase on magnetically separable biochar for highly efficient removal of bisphenol A in water.

Laccase was stably immobilized on a cost effective and nanosized magnetic biochar (L-MBC) by adsorption, precipitation and crosslinking, and it was used for high performance BPA removal. A large amount of enzyme could be immobilized on the magnetic biochar with high activity (2.251 U per mg MBC), and the L-MBC could be magnetically separated from the aqueous solution in 20 seconds. The successful immobilization of laccase was also confirmed via FTIR, SEM, and EDS analyses. The L-MBC presented better storage and stability performances, pH tolerance and thermal stability than the free laccase. It was found that BPA with an initial concentration of 25 mg L-1 could be thoroughly removed within 75 min, where BPA removal was attributed to enzymatic degradation and adsorption. In addition, the BPA removal efficiency by the L-MBC could be maintained above 85% even after seven cycles of repeated use. Due to high stability and efficient recyclability, the L-MBC-based biocatalyst has the potential to be a reliable method for treating BPA in environmental water sources.

Positioning the cleavage furrow: All you need is Rho.

RhoA controls cleavage furrow formation during cell division, but whether RhoA suffices to orchestrate spatiotemporal dynamics of furrow formation is unknown. In this issue, Wagner and Goltzer (2016. J. Cell Biol http://dx.doi.org/10.1083/jcb.201603025) show that RhoA activity can induce furrow formation in all cell cortex positions and cell cycle phases.

Determination of ubiquitin fitness landscapes under different chemical stresses in a classroom setting.

Ubiquitin is essential for eukaryotic life and varies in only 3 amino acid positions between yeast and humans. However, recent deep sequencing studies indicate that ubiquitin is highly tolerant to single mutations. We hypothesized that this tolerance would be reduced by chemically induced physiologic perturbations. To test this hypothesis, a class of first year UCSF graduate students employed deep mutational scanning to determine the fitness landscape of all possible single residue mutations in the presence of five different small molecule perturbations. These perturbations uncover 'shared sensitized positions' localized to areas around the hydrophobic patch and the C-terminus. In addition, we identified perturbation specific effects such as a sensitization of His68 in HU and a tolerance to mutation at Lys63 in DTT. Our data show how chemical stresses can reduce buffering effects in the ubiquitin proteasome system. Finally, this study demonstrates the potential of lab-based interdisciplinary graduate curriculum.