The hemoglobinopathies, such as β-thalassemia and sickle cell anemia (SCA), are characterized by mutations of the β-globin gene resulting in either decreased or functionally abnormal hemoglobin (Hb) production. As bone marrow transplant is the only curative option for these patients, there is a strong need for new therapeutic approaches. Both β-thalassemia and SCA represent ideal targets for gene therapy since introduction of a normal β-globin gene can ameliorate the phenotype, as we and others have shown previously. Overcoming the developmental silencing of the fetal γ-globin gene represents an additional approach for the treatment of hemoglobinopathies. Here, we directly compare a recently established approach to activate the γ-globin gene using forced chromatin looping with pharmacologic approaches to raise γ-globin expression.

The β-type globin genes are activated through dynamic interactions with a distal upstream enhancer, the locus control region (LCR). The LCR physically contacts the developmental stage appropriate globin gene via chromatin looping, a process partially dependent on the protein Ldb1. Previously, we have shown that tethering Ldb1 to the murine β-globin promoter with a custom designed zinc finger protein (ZF-Ldb1) can induce loop formation and β-globin transcription in an erythroid cell line (Deng et al., 2012). Further work showed that forced chromatin looping can be exploited to potently reactivate fetal globin gene expression in adult human erythroid cells (Deng et al., 2014). Here we compared the efficacy and toxicity of ZF-Ldb1 to pharmacologic compounds that induce HbF in cultured hematopoietic stem progenitor cell-derived erythroid cultures from normal and SCA donors.

ZF-Ldb1 increased HbF synthesis in SCA erythroid cells (N=8) up to 86% and, concurrently, reduced sickle Hb (HbS) below 15%, consistent with previous studies of erythroid cells from normal probands. Preliminary results obtained from treating SCA specimens (N=3) show that the induction of HbF in cells treated with ZF-Ldb1 is twice as high (+35.55% ± 8.34%, at a dose of ~ one ZF-Ldb1 transgene copy per cell) as that observed using pomalidomide (+16.50% ± 14.57%, 20μM) and decitabine (+15.60% ± 12.36%, 0.5μM). Tranylcypromine and hydroxyurea showed the lowest HbF increase (+9.67% ± 3.26% and +5.06 ± 2.82%, 1.5μM and 150μM respectively).

Importantly, decitabine and pomalidomide treatment lowered cell viability to 39% and 26%, respectively, while ZF-Ldb1 expressing cells retained normal viability similar to control populations.

In related experiments, we are comparing the expression of a battery of genes known to regulate HbF levels (BCL11A, SOX6, KLF1 and C-Myb) in normal and SCA derived erythroid cells treated with ZF-Ldb1 or HbF inducers and compared to controls. Preliminary analyses indicate altered expression of KLF1 in SCA versus normal cells, consistent with a superior response of SCA cells to HbF induction.

In conclusion, lentiviral-mediated ZF-Ldb1 gene transfer appears superior to pharmacologic compounds in terms of efficacy and cell viability further supporting suitability for the reactivation of HbF in SCA erythroid cells.

Synaptic connectivity and molecular composition provide a blueprint for information processing in neural circuits. Detailed structural analysis of neural circuits requires nanometer resolution, which can be obtained with serial-section electron microscopy. However, this technique remains challenging for reconstructing molecularly defined synapses. We used a genetically encoded synaptic marker for electron microscopy (GESEM) based on intra-vesicular generation of electron-dense labeling in axonal boutons. This approach allowed the identification of synapses from Cre recombinase-expressing or GAL4-expressing neurons in the mouse and fly with excellent preservation of ultrastructure. We applied this tool to visualize long-range connectivity of AGRP and POMC neurons in the mouse, two molecularly defined hypothalamic populations that are important for feeding behavior. Combining selective ultrastructural reconstruction of neuropil with functional and viral circuit mapping, we characterized some basic features of circuit organization for axon projections of these cell types. Our findings demonstrate that GESEM labeling enables long-range connectomics with molecularly defined cell types.

Obesity is associated with increased blood pressure (BP), which in turn increases the risk of cardiovascular diseases. We found that the increase in leptin levels seen in diet-induced obesity (DIO) drives an increase in BP in rodents, an effect that was not seen in animals deficient in leptin or leptin receptors (LepR). Furthermore, humans with loss-of-function mutations in leptin and the LepR have low BP despite severe obesity. Leptin's effects on BP are mediated by neuronal circuits in the dorsomedial hypothalamus (DMH), as blocking leptin with a specific antibody, antagonist, or inhibition of the activity of LepR-expressing neurons in the DMH caused a rapid reduction of BP in DIO mice, independent of changes in weight. Re-expression of LepRs in the DMH of DIO LepR-deficient mice caused an increase in BP. These studies demonstrate that leptin couples changes in weight to changes in BP in mammalian species.

A subclass of fluorescent proteins (FPs), large Stokes shift (LSS) FP, are characterized by increased spread between excitation and emission maxima. We report a photoswitchable variant of a red FP with an LSS, PSLSSmKate, which initially exhibits excitation and emission at 445 and 622 nm, but violet irradiation photoswitches PSLSSmKate into a common red form with excitation and emission at 573 and 621 nm. We characterize spectral, photophysical, and biochemical properties of PSLSSmKate in vitro and in mammalian cells and determine its crystal structure in the LSS form. Mass spectrometry, mutagenesis, and spectroscopy of PSLSSmKate allow us to propose molecular mechanisms for the LSS, pH dependence, and light-induced chromophore transformation. We demonstrate the applicability of PSLSSmKate to superresolution photoactivated localization microscopy and protein dynamics in live cells. Given its promising properties, we expect that PSLSSmKate-like phenotype will be further used for photoactivatable imaging and tracking multiple populations of intracellular objects.

Staphylococcus aureus responds to changing extracellular environments in part by adjusting its proteome through alterations of transcriptional priorities and selective degradation of the preexisting pool of proteins. In Bacillus subtilis, the proteolytic adaptor protein MecA has been shown to play a role in assisting with the proteolytic degradation of proteins involved in competence and the oxidative stress response. However, the targets of TrfA, the MecA homolog in S. aureus, have not been well characterized. In this work, we investigated how TrfA assists chaperones and proteases to regulate the proteolysis of several classes of proteins in S. aureus. By fusing the last 3 amino acids of the SsrA degradation tag to Venus, a rapidly folding yellow fluorescent protein, we obtained both fluorescence-based and Western blot assay-based evidence that TrfA and ClpCP are the adaptor and protease, respectively, responsible for the degradation of the SsrA-tagged protein in S. aureus. Notably, the impact of TrfA on degradation was most prominent during late log phase and early stationary phase, due in part to a combination of transcriptional regulation and proteolytic degradation of TrfA by ClpCP. We also characterized the temporal transcriptional regulation governing TrfA activity, wherein Spx, a redox-sensitive transcriptional regulator degraded by ClpXP, activates trfA transcription while repressing its own promoter. Finally, the scope of TrfA-mediated proteolysis was expanded by identifying TrfA as the adaptor that works with ClpCP to degrade antitoxins in S. aureus. Together, these results indicate that the adaptor TrfA adds temporal nuance to protein degradation by ClpCP in S. aureus.

The reciprocal hemizygosity test is a straightforward genetic test that can positively identify genes that have evolved to contribute to a phenotypic difference between strains or between species. The test involves a comparison between hybrids that are genetically identical throughout the genome except at the test locus, which is rendered hemizygous for alternative alleles from the two parental strains. If the two reciprocal hemizygotes display different phenotypes, then the two parental alleles must have evolved. New methods for targeted mutagenesis will allow application of the reciprocal hemizygosity test in many organisms. This review discusses the principles, advantages, and limitations of the test.

Muscular hydrostats (such as mollusks), and fluid-filled animals (such as annelids), can exploit their constant-volume tissues to transfer forces and displacements in predictable ways, much as articulated animals use hinges and levers. Although larval insects contain pressurized fluids, they also have internal air tubes that are compressible and, as a result, they have more uncontrolled degrees of freedom. Therefore, the mechanisms by which larval insects control their movements are expected to reveal useful strategies for designing soft biomimetic robots. Using caterpillars as a tractable model system, it is now possible to identify the biomechanical and neural strategies for controlling movements in such highly deformable animals. For example, the tobacco hornworm, Manduca sexta, can stiffen its body by increasing muscular tension (and therefore body pressure) but the internal cavity (hemocoel) is not iso-barometric, nor is pressure used to directly control the movements of its limbs. Instead, fluid and tissues flow within the hemocoel and the body is soft and flexible to conform to the substrate. Even the gut contributes to the biomechanics of locomotion; it is decoupled from the movements of the body wall and slides forward within the body cavity at the start of each step. During crawling the body is kept in tension for part of the stride and compressive forces are exerted on the substrate along the axis of the caterpillar, thereby using the environment as a skeleton. The timing of muscular activity suggests that crawling is coordinated by proleg-retractor motoneurons and that the large segmental muscles produce anterograde waves of lifting that do not require precise timing. This strategy produces a robust form of locomotion in which the kinematics changes little with orientation. In different species of caterpillar, the presence of prolegs on particular body segments is related to alternative kinematics such as "inching." This suggests a mechanism for the evolution of different gaits through changes in the usage of prolegs, rather than, through extensive alterations in the motor program controlling the body wall. Some of these findings are being used to design and test novel control-strategies for highly deformable robots. These "softworm" devices are providing new insights into the challenges faced by any soft animal navigating in a terrestrial environment.

Post-translational histone modifications are highly correlated with transcriptional activity, but the relative timing of these marks and their dynamic interplay during gene regulation remains controversial. To shed light on this problem and clarify the connections between histone modifications and transcription, we demonstrate how FabLEM (Fab-based Live Endogenous Modification labeling) can be used to simultaneously track histone H3 Lysine 9 acetylation (H3K9ac) together with RNA polymerase II Serine 2 and Serine 5 phosphorylation (RNAP2 Ser2ph/Ser5ph) in single living cells and their progeny. We provide a detailed description of the FabLEM methodology, including helpful tips for preparing and loading fluorescently conjugated antigen binding fragments (Fab) into cells for optimal results. We also introduce simple procedures for analyzing and visualizing FabLEM data, including color-coded scatterplots to track correlations between modifications through the cell cycle and temporal cross-correlation analysis to dissect modification dynamics. Using these methods, we find significant correlations that span cell generations, with a relatively strong correlation between H3K9ac and Ser5ph that appears to peak a few hours before mitosis and may reflect the bookmarking of genes for efficient re-initiation following mitosis. The techniques we have developed are broadly applicable and should help clarify how histone modifications dynamically contribute to gene regulation.

Targeting visually identified neurons for electrophysiological recording is a fundamental neuroscience technique; however, its potential is hampered by poor visualization of pipette tips in deep brain tissue. We describe quantum dot-coated glass pipettes that provide strong two-photon contrast at deeper penetration depths than those achievable with current methods. We demonstrated the pipettes' utility in targeted patch-clamp recording experiments and single-cell electroporation of identified rat and mouse neurons in vitro and in vivo.