Split-wrmScarlet and split-sfGFP: tools for faster,easier fluorescent labeling of endogenous proteins in Caenorhabditis elegans

We create and share a new red fluorophore, along with a set of strains, reagents and protocols, to make it faster and easier to label endogenous Caenorhabditis elegans proteins with fluorescent tags. CRISPR-mediated fluorescent labeling of C. elegans proteins is an invaluable tool, but it is much more difficult to insert fluorophore-size DNA segments than it is to make small gene edits. In principle, high-affinity asymmetrically split fluorescent proteins solve this problem in C. elegans: the small fragment can quickly and easily be fused to almost any protein of interest, and can be detected wherever the large fragment is expressed and complemented. However, there is currently only one available strain stably expressing the large fragment of a split fluorescent protein, restricting this solution to a single tissue (the germline) in the highly autofluorescent green channel. No available C. elegans lines express unbound large fragments of split red fluorescent proteins, and even state-of-the-art split red fluorescent proteins are dim compared to the canonical split-sfGFP protein. In this study, we engineer a bright, high-affinity new split red fluorophore, split-wrmScarlet. We generate transgenic C. elegans lines to allow easy single-color labeling in muscle or germline cells and dual-color labeling in somatic cells. We also describe a novel expression strategy for the germline, where traditional expression strategies struggle. We validate these strains by targeting split-wrmScarlet to several genes whose products label distinct organelles, and we provide a protocol for easy, cloning-free CRISPR/Cas9 editing. As the collection of split-FP strains for labeling in different tissues or organelles expands, we will post updates at doi.org/10.5281/zenodo.3993663     

Greater binding affinity of trivalent antimony to a CCCH zinc finger domain compared to a CCHC domain of kinetoplastid proteins

It has been reported recently that Sb(III) competes with Zn(II) for its binding to the CCHC zinc finger domain of the HIV-1 NCp7 protein, suggesting that zinc finger proteins may be molecular targets for antimony-based drugs. Here, the interaction of Sb(III) with a CCCH zinc finger domain, which is considered to play a crucial role in the biology of kinetoplastid protozoa, has been characterized for the first time. The binding characteristics of Sb(III) were compared between a CCCH-type peptide derived from a kinetoplastid protein and two different CCHC-type zinc finger peptides. The formation of 1 : 1 Zn-peptide and Sb-peptide complexes from the different peptides was demonstrated using circular dichroism, UV absorption, fluorescence spectroscopies and ESI-MS. Titration of the Zn-peptide complexes with SbCl(3) was performed at pH 6 and 7, exploiting the intrinsic fluorescence of the peptides. The differential spectral characteristics of the peptides allowed for competition experiments between the different peptides for binding of Zn(II). The present study establishes that Sb(III) more effectively displaces Zn(II) from the CCCH peptide than CCHC ones, as a result of both the greater stability of the Sb-CCCH complex (compared to Sb-CCHC complexes) and the lower stability of the Zn-CCCH complex (compared to Zn-CCHC complexes). Comparison of the binding characteristics of Sb(III) or Zn(II) between the CCHC-type peptides with different amino acid sequences supports the model that not only the conserved zinc finger motif, but also the sequence of non-conserved amino acids determines the binding affinity of Sb(III) and Zn(II). These data suggest that the interaction of Sb(III) with CCCH-type zinc finger proteins may modulate, or even mediate, the pharmacological action of antimonial drugs.     

TheEscherichia coli mannitol permease as a model for transport via the bacterial phosphotransferase system

The bacterial phosphoenolpyruvate-dependent carbohydrate phosphotransferase system (PTS) consists of several proteins whose primary functions are to transport and phosphorylate their substrates. The complexity of the PTS undoubtedly reflects its additional roles in chemotaxis to PTS substrates and in regulation of other metabolic processes in the cell. The PTS permeases (Enzymes II) are the membrane-associated proteins of the PTS that sequentially recognize, transport, and phosphorylate their specific substrates in separate steps, and theEscherichia coli mannitol permease is one of the best studied of these proteins. It consists of two cytoplasmic domains (EIIA and EIIB) involved in mannitol phosphorylation and an integral membrane domain (EIIC) which is sufficient to bind mannitol, but which transports mannitol at a rate that is dependent on phosphorylation of the EIIA and EIIB domains. Recent results show that several residues in a hydrophilic, 85-residue segment of the EIIC domain are important for the binding, transport, and phosphorylation of mannitol. This segment may be at least partially exposed to the cytoplasm of the cell. A model is proposed in which this region of the EIIC domain is crucial in coupling phosphorylation of the EIIB domain to transport through the EIIC domain of the mannitol permease.     

Cardioprotection by HO-4038, a novel verapamil derivative, targeted against ischemia and reperfusion-mediated acute myocardial infarction

Many cardiac interventional procedures, such as coronary angioplasty, stenting, and thrombolysis, attempt to reintroduce blood flow (reperfusion) to an ischemic region of myocardium. However, the reperfusion is accompanied by a complex cascade of cellular and molecular events resulting in oxidative damage, termed myocardial ischemia-reperfusion (I/R) injury. In this study, we evaluated the ability of HO-4038, an N-hydroxypiperidine derivative of verapamil, on the modulation of myocardial tissue oxygenation (Po(2)), I/R injury, and key signaling molecules involved in cardioprotection in an in vivo rat model of acute myocardial infarction (MI). MI was created in rats by ligating the left anterior descending coronary artery (LAD) for 30 min followed by 24 h of reperfusion. Verapamil or HO-4038 was infused through the jugular vein 10 min before the induction of ischemia. Myocardial Po(2) and the free-radical scavenging ability of HO-4038 were measured using electron paramagnetic resonance spectroscopy. HO-4038 showed a significantly better scavenging ability of reactive oxygen radicals compared with verapamil. The cardiac contractile functions in the I/R hearts were significantly higher recovery in HO-4038 compared with the verapamil group. A significant decrease in the plasma levels of creatine kinase and lactate dehydrogenase was observed in the HO-4038 group compared with the verapamil or untreated I/R groups. The left ventricular infarct size was significantly less in the HO-4038 (23 +/- 2%) compared with the untreated I/R (36 +/- 4%) group. HO-4038 significantly attenuated the hyperoxygenation (36 +/- 1 mmHg) during reperfusion compared with the untreated I/R group (44 +/- 2 mmHg). The HO-4038-treated group also markedly attenuated superoxide production, increased nitric oxide generation, and enhanced Akt and Bcl-2 levels in the reperfused myocardium. Overall, the results demonstrated that HO-4038 significantly protected hearts against I/R-induced cardiac dysfunction and damage through the combined beneficial actions of calcium-channel blocking, antioxidant, and prosurvival signaling activities.     

Complex patterns of dopamine‐related gene expression in the ventral tegmental area of male zebra finches relate to dyadic interactions with long‐term female partners

Dopaminergic projections from the ventral tegmental area (VTA) to multiple efferent targets are implicated in pair bonding, yet the role of the VTA in the maintenance of long‐term pair bonds is not well characterized. Complex interactions between numerous neuromodulators modify activity in the VTA, suggesting that individual differences in patterns of gene expression in this region may explain individual differences in long‐term social interactions in bonded pairs. To test this hypothesis we used RNA‐seq to measure expression of over 8000 annotated genes in male zebra finches in established male‐female pairs. Weighted gene co‐expression network analysis identified a gene module that contained numerous dopamine‐related genes with TH found to be the most connected gene of the module. Genes in this module related to male agonistic behaviors as well as bonding‐related behaviors produced by female partners. Unsupervised learning approaches identified two groups of males that differed with respect to expression of numerous genes. Enrichment analyses showed that many dopamine‐related genes and modulators differed between these groups, including dopamine receptors, synthetic and degradative enzymes, the avian dopamine transporter and several GABA‐ and glutamate‐related genes. Many of the bonding‐related behaviors closely associated with VTA gene expression in the two male groups were produced by the male's partner, rather than the male himself. Collectively, results highlight numerous candidate genes in the VTA that can be explored in future studies and raise the possibility that the molecular/genetic organization of the VTA may be strongly shaped by a social partner and/or the strength of the pair bond.     

Photooxidative stress‐induced and abundant small RNAs in Rhodobacter sphaeroides

Exposure to oxygen and light generates photooxidative stress by the bacteriochlorophyll a mediated formation of singlet oxygen (¹O₂) in Rhodobacter sphaeroides. Our study reports the genome‐wide search for small RNAs (sRNAs) involved in the regulatory response to ¹O₂. By using 454 pyrosequencing and Northern blot analysis, we identified 20 sRNAs from R. sphaeroides aerobic cultures or following treatment with ¹O₂ or superoxide (O^–₂). One sRNA was specifically induced by ¹O₂ and its expression depends on the extracytoplasmic function sigma factor RpoE. Two sRNAs induced by ¹O₂ and O^–₂ were cotranscribed with upstream genes preceded by promoters with target sequences for the alternative sigma factors RpoH_I and RpoH_II. The most abundant sRNA was processed in the presence of ¹O₂ but not by O^–₂. From this and a second sRNA a conserved 3′‐segment accumulated from a larger precursor. Absence of the RNA chaperone Hfq changed the half‐lives, abundance and processing of ¹O₂‐affected sRNAs. Orthologues of three sRNA genes are present in different alpha‐proteobacteria, but the majority was unique to R. sphaeroides or Rhodobacterales species. Our discovery that abundant sRNAs are affected by ¹O₂ exposure extends the knowledge on the role of sRNAs and Hfq in the regulatory response to oxidative stress.     

Active site alanine mutations convert deubiquitinases into high‐affinity ubiquitin‐binding proteins

A common strategy for exploring the biological roles of deubiquitinating enzymes (DUBs) in different pathways is to study the effects of replacing the wild‐type DUB with a catalytically inactive mutant in cells. We report here that a commonly studied DUB mutation, in which the catalytic cysteine is replaced with alanine, can dramatically increase the affinity of some DUBs for ubiquitin. Overexpression of these tight‐binding mutants thus has the potential to sequester cellular pools of monoubiquitin and ubiquitin chains. As a result, cells expressing these mutants may display unpredictable dominant negative physiological effects that are not related to loss of DUB activity. The structure of the SAGA DUB module bound to free ubiquitin reveals the structural basis for the 30‐fold higher affinity of Ubp8^C146A for ubiquitin. We show that an alternative option, substituting the active site cysteine with arginine, can inactivate DUBs while also decreasing the affinity for ubiquitin.     

Dancing to the top: How collective action revitalized arts education in Boston

In 2009, a group of local foundations, the school district, arts organizations, and the Mayor's Office launched the Boston Public Schools Arts Education Initiative (BPS-AE)—a multiyear, citywide, public–private initiative aimed at increasing BPS students' access to in-school arts education. Managed by a strong local intermediary with deep experience in education, this initiative used several complementary, overlaid strategies: direct service, system building, and community engagement—all of which were supported by a philanthropic collaborative of local and national funders. Today, 17,000 more BPS students receive arts education; nearly 130 additional full-time certified arts teachers have been hired; and district spending on arts has risen to $26 million annually, compared to $15 million in 2009. There is also growing demand from the larger community for more and better arts education. In 2015, BPS-AE produced a case study that provides a more detailed explication of the multitiered strategy it used to achieve these results. The study offers recommendations as to how other cities can involve diverse key constituencies to advance this kind of initiative, create leadership structures that support effective citywide collaboration, engage community stakeholders in participatory planning processes, leverage private philanthropy to boost public funding, and develop a centralized support system for school principals and arts teachers.     

Tidal exchange of total mercury and methylmercury between a salt marsh and a Chesapeake Bay sub-estuary

We examined the net exchange of total mercury (THg) and methylmercury (MeHg) between a tidal marsh and its adjacent estuary over a 1-year period from August 2007 to July 2008. Our objectives were to estimate the importance of tidal salt marshes as sources and sinks of mercury within the Chesapeake Bay system, and to examine the hydrologic and biogeochemical controls on mercury fate and transport in tidal marshes. Tidal flows and water chemistry were measured at an established tidal flume at the mouth of the principal tidal creek of a 3-ha marsh section at the Smithsonian Environmental Research Center. Fluxes were estimated by combining continuous tidal flow measurement for the entire study year, with discrete, hourly, flow-weighted measurements of filterable and particulate THg and MeHg, dissolved organic carbon (DOC), and suspended particulate matter (SPM) made over 20 tidal cycles during the year. We found that the marsh was a relatively small net tidal source of MeHg, mainly during the warmer growing season. We also confirmed that the marsh was a substantial source of DOC to the adjacent estuary. DOC was a significant predictor of both filterable THg and MeHg fluxes. However, although the marsh was a source of filterable THg, it was overall a net sink for THg because of particulate trapping. The net per-area annual flux of MeHg from tidal marshes is greater than other MeHg pathways within Chesapeake Bay. The annual load of MeHg from tidal marshes into Chesapeake Bay, however, is likely small relative to fluvial fluxes and efflux from bottom sediment. This study suggests that MeHg production within the tidal marsh has greater consequences for biota inhabiting the marsh than for the efflux of MeHg from the marsh.