Heterogeneous pools of adult neural stem cells (NSCs) contribute to brain maintenance and regeneration after injury. The balance of NSC activation and quiescence, as well as the induction of lineage-specific transcription factors, may contribute to diversity of neuronal and glial fates. To identify molecular hallmarks governing these characteristics, we performed single-cell sequencing of an unbiased pool of adult subventricular zone NSCs. This analysis identified a discrete, dormant NSC subpopulation that already expresses distinct combinations of lineage-specific transcription factors during homeostasis. Dormant NSCs enter a primed-quiescent state before activation, which is accompanied by downregulation of glycolytic metabolism, Notch, and BMP signaling and a concomitant upregulation of lineage-specific transcription factors and protein synthesis. In response to brain ischemia, interferon gamma signaling induces dormant NSC subpopulations to enter the primed-quiescent state. This study unveils general principles underlying NSC activation and lineage priming and opens potential avenues for regenerative medicine in the brain.

Quantitative real-time polymerase chain reactions (qRT-PCR) have become the method of choice for rapid, sensitive, quantitative comparison of RNA transcript abundance. Useful data from this method depend on fitting data to theoretical curves that allow computation of mRNA levels. Calculating accurate mRNA levels requires important parameters such as reaction efficiency and the fractional cycle number at threshold (CT) to be used; however, many algorithms currently in use estimate these important parameters. Here we describe an objective method for quantifying qRT-PCR results using calculations based on the kinetics of individual PCR reactions without the need of the standard curve, independent of any assumptions or subjective judgments which allow direct calculation of efficiency and CT. We use a four-parameter logistic model to fit the raw fluorescence data as a function of PCR cycles to identify the exponential phase of the reaction. Next, we use a three-parameter simple exponent model to fit the exponential phase using an iterative nonlinear regression algorithm. Within the exponential portion of the curve, our technique automatically identifies candidate regression values using the P-value of regression and then uses a weighted average to compute a final efficiency for quantification. For CT determination, we chose the first positive second derivative maximum from the logistic model. This algorithm provides an objective and noise-resistant method for quantification of qRT-PCR results that is independent of the specific equipment used to perform PCR reactions. Keywordsquantitative polymerase chain reaction; four-parameter logistic model; three-parameter simple exponent model; noise-resistant algorithm

Near-infrared (NIR) fluorescent sensors have emerged as promising molecular tools for imaging biomolecules in living systems. However, NIR fluorescent sensors are very challenging to be developed. Herein, we describe the discovery of a new class of NIR fluorescent dyes represented by 1a/1c/1e, which are superior to the traditional 7-hydroxycoumarin and fluorescein with both absorption and emission in the NIR region while retaining an optically tunable hydroxyl group. Quantum chemical calculations with the B3LYP exchange functional employing 6-31G(d) basis sets provide insights into the optical property distinctions between 1a/1c/1e and their alkoxy derivatives. The unique optical properties of the new type of fluorescent dyes can be exploited as a useful strategy for development of NIR fluorescent sensors. Employing this strategy, two different types of NIR fluorescent sensors, NIR-H(2)O(2) and NIR-thiol, for H(2)O(2) and thiols, respectively, were constructed. These novel sensors respond to H(2)O(2) or thiols with a large turn-on NIR fluorescence signal upon excitation in the NIR region. Furthermore, NIR-H(2)O(2) and NIR-thiol are capable of imaging endogenously produced H(2)O(2) and thiols, respectively, not only in living cells but also in living mice, demonstrating the value of the new NIR fluorescent sensor design strategy. The new type of NIR dyes presented herein may open up new opportunities for the development of NIR fluorescent sensors based on the hydroxyl functionalized reactive sites for biological imaging applications in living animals.

Highlights d Time-dependent stem cell depletion levels off in the old brain via increased quiescence d Age minimally changes the neural stem cell transcriptome d Once-activated neural stem cells perform similar in the old and young brain d The old niche keeps stem cells quiescent via inflammation and Wnt activity regulation