The story of the network of genes that controls fruit patterning in Arabidopsis is still unfolding, and here we have seen familiar players take on different parts and we have identified new regulators that may play important roles. Arabidopsis fruit are patterned into three major regions: the valves, the replum, and the valve margin. Previous studies have shown that the FRUITFULL (FUL) and REPLUMLESS (RPL) genes are responsible for patterning the valve and replum, respectively. To identify new regulators, we screened for mutants that showed rescued replum development in the sensitized rpl ful double mutant background. We identified one suppressor (m33) that likely identifies a new fruit patterning gene as well as another suppressor (m413) that corresponds to the well-known floral homeotic gene APETALA2 (AP2). Although previous studies have characterized the role of AP2 during flower development and its regulation by miR172, little is known about its function during fruit morphogenesis. Here we used promoter-GUS fusions to analyze the expression patterns of the five miR172 genes in reproductive and vegetative tissues and found that miR172 may be controlling AP2 activity in these tissues. In addition, we found that FUL may be an upstream regulator of miR172 in the fruit. These studies have allowed us to incorporate the role for miR172 into current models of fruit patterning. Although the final chapter to this story has yet to be written, our studies have added new insights that will undoubtedly contribute to the long-term goal of understanding the complex network of gene interactions that underlies fruit morphogenesis

Dopamine is a key catecholamine in the brain and kidney, where it is involved in a number of physiological functions such as locomotion, cognition, emotion, endocrine regulation, and renal function. As a membrane-impermeant hormone and neurotransmitter, dopamine is thought to signal by binding and activating dopamine receptors, members of the G protein coupled receptor (GPCR) family, only on the plasma membrane. Here, using novel nanobody-based biosensors, we demonstrate for the first time that the dopamine D1 receptor (D1DR), the primary mediator of dopaminergic signaling in the brain and kidney, not only functions on the plasma membrane but becomes activated at the Golgi apparatus in the presence of its ligand. We present evidence that activation of the Golgi pool of D1DR is dependent on organic cation transporter 2 (OCT2), a dopamine transporter, providing an explanation for how the membrane-impermeant dopamine accesses subcellular pools of D1DR. We further demonstrate that dopamine activates Golgi-D1DR in murine striatal medium spiny neurons, and this activity depends on OCT2 function. We also introduce a new approach to selectively interrogate compartmentalized D1DR signaling by inhibiting Gαs coupling using a nanobody-based chemical recruitment system. Using this strategy, we show that Golgi-localized D1DRs regulate cAMP production and mediate local protein kinase A activation. Together, our data suggest that spatially compartmentalized signaling hubs are previously unappreciated regulatory aspects of D1DR signaling. Our data provide further evidence for the role of transporters in regulating subcellular GPCR activity.

Cells interpret a variety of signals through G-protein-coupled receptors (GPCRs) and stimulate the generation of second messengers such as cyclic adenosine monophosphate (cAMP). A long-standing puzzle is deciphering how GPCRs elicit different physiological responses despite generating similar levels of cAMP. We previously showed that some GPCRs generate cAMP from both the plasma membrane and the Golgi apparatus. Here we demonstrate that cardiomyocytes distinguish between subcellular cAMP inputs to elicit different physiological outputs. We show that generating cAMP from the Golgi leads to the regulation of a specific protein kinase A (PKA) target that increases the rate of cardiomyocyte relaxation. In contrast, cAMP generation from the plasma membrane activates a different PKA target that increases contractile force. We further validated the physiological consequences of these observations in intact zebrafish and mice. Thus, we demonstrate that the same GPCR acting through the same second messenger regulates cardiac contraction and relaxation dependent on its subcellular location.

The reliable forward engineering of genetic systems remains limited by the ad hoc reuse of many types of basic genetic elements. Although a few intrinsic prokaryotic transcription terminators are used routinely, termination efficiencies have not been studied systematically. Here, we developed and validated a genetic architecture that enables reliable measurement of termination efficiencies. We then assembled a collection of 61 natural and synthetic terminators that collectively encode termination efficiencies across an ∼800-fold dynamic range within Escherichia coli. We simulated co-transcriptional RNA folding dynamics to identify competing secondary structures that might interfere with terminator folding kinetics or impact termination activity. We found that structures extending beyond the core terminator stem are likely to increase terminator activity. By excluding terminators encoding such context-confounding elements, we were able to develop a linear sequence-function model that can be used to estimate termination efficiencies (r = 0.9, n = 31) better than models trained on all terminators (r = 0.67, n = 54). The resulting systematically measured collection of terminators should improve the engineering of synthetic genetic systems and also advance quantitative modeling of transcription termination.

Tuberculous meningitis (TBM) is the most severe form of tuberculous with substantial mortality. In May 2015, 54 researchers from 10 countries met in Da Lat, Vietnam, to discuss advances in TBM. Among the attendees were researchers involved in pivotal studies on the use of Xpert MTB/Rif for TBM diagnosis. Attendees discussed the 2014 World Health Organization strong recommendation favoring the use of Xpert "in preference to conventional microscopy and culture as the initial diagnostic test for cerebrospinal fluid (CSF) if the sample volume is low or if additional specimens cannot be obtained to make a quick diagnosis." Attendees were concerned that the limitations of Xpert testing for TBM are not emphasized. Clear guidance is needed for the investigational pathway for TBM, including recommendations on the diagnostic package of investigations, which does not stop with Xpert testing. Second, emphasis on the large CSF volumes (ideally 8-10 mL) needed for Xpert testing is required. Guidelines should also emphasize that TBM is a medical emergency and early treatment reduces mortality.