The additive and synergistic therapeutic effects derived from combinations of chemotherapeutic drugs and radiation have established an indispensable paradigm: cancer must be attacked on multiple fronts. However, the increased antitumor efficacy of such combinational regimens is also associated with severe systemic toxicity, as these drugs cannot selectively target tumor cells. Monoclonal antibodies (mAbs), which have exquisite specificity for their antigens, are becoming an increasingly important class of antitumor agents, as they enhance the efficacy of various therapeutic regimens without significantly increasing systemic toxicity. Furthermore, preclinical and early clinical evidence indicate that combinations of antibody-based drugs provide even greater efficacy with minimal side effects. Unfortunately, the research, manufacturing and regulatory costs of mAb development pose a significant barrier to the use of antibody-based combination therapies. An emerging alternative is the use of dual-targeting bispecific antibodies (BsAbs). BsAbs are derived from the recombination of variable domains of two antibodies with different specificities; BsAbs are thus capable of binding both antigens of their parental antibodies. With the recent progress that has been made in antibody engineering technology, BsAbs that simultaneously target two tumor-associated molecules (eg, growth factor receptors) are being heralded for their potential to deliver two therapeutic moieties in a single molecule.

Characterizing the identity and types of neurons in the brain, as well as their associated function, requires a means of quantifying and comparing 3D neuron morphology. Presently, neuron comparison methods are based on statistics from neuronal morphology such as size and number of branches, which are not fully suitable for detecting local similarities and differences in the detailed structure. We developed BlastNeuron to compare neurons in terms of their global appearance, detailed arborization patterns, and topological similarity. BlastNeuron first compares and clusters 3D neuron reconstructions based on global morphology features and moment invariants, independent of their orientations, sizes, level of reconstruction and other variations. Subsequently, BlastNeuron performs local alignment between any pair of retrieved neurons via a tree-topology driven dynamic programming method. A 3D correspondence map can thus be generated at the resolution of single reconstruction nodes. We applied BlastNeuron to three datasets: (1) 10,000+ neuron reconstructions from a public morphology database, (2) 681 newly and manually reconstructed neurons, and (3) neurons reconstructions produced using several independent reconstruction methods. Our approach was able to accurately and efficiently retrieve morphologically and functionally similar neuron structures from large morphology database, identify the local common structures, and find clusters of neurons that share similarities in both morphology and molecular profiles.

Superresolution imaging techniques based on the precise localization of single molecules, such as photoactivated localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM), achieve high resolution by fitting images of single fluorescent molecules with a theoretical Gaussian to localize them with a precision on the order of tens of nanometers. PALM/STORM rely on photoactivated proteins or photoswitching dyes, respectively, which makes them technically challenging. We present a simple and practical way of producing point localization-based superresolution images that does not require photoactivatable or photoswitching probes. Called bleaching/blinking assisted localization microscopy (BaLM), the technique relies on the intrinsic bleaching and blinking behaviors characteristic of all commonly used fluorescent probes. To detect single fluorophores, we simply acquire a stream of fluorescence images. Fluorophore bleach or blink-off events are detected by subtracting from each image of the series the subsequent image. Similarly, blink-on events are detected by subtracting from each frame the previous one. After image subtractions, fluorescence emission signals from single fluorophores are identified and the localizations are determined by fitting the fluorescence intensity distribution with a theoretical Gaussian. We also show that BaLM works with a spectrum of fluorescent molecules in the same sample. Thus, BaLM extends single molecule-based superresolution localization to samples labeled with multiple conventional fluorescent probes.

Significance: Genetically encoded calcium ion (Ca2+) indicators (GECIs) are powerful tools for monitoring intracellular Ca2+ concentration changes in living cells and model organisms. In particular, GECIs have found particular utility for monitoring the transient increase of Ca2+concentration that is associated with the neuronal action potential. However, the palette of highly optimized GECIs for imaging of neuronal activity remains relatively limited. Expanding the selection of available GECIs to include new colors and distinct photophysical properties could create new opportunities for in vitro and in vivo fluorescence imaging of neuronal activity. In particular, blue-shifted variants of GECIs are expected to have enhanced two-photon brightness, which would facilitate multiphoton microscopy.

Aim: We describe the development and applications of T-GECO1-a high-performance blue-shifted GECI based on the Clavularia sp.-derived mTFP1.

Approach: We use protein engineering and extensive directed evolution to develop T-GECO1. We characterize the purified protein and assess its performance in vitro using one-photon excitation in cultured rat hippocampal neurons, in vivo using one-photon excitation fiber photometry in mice, and ex vivo using two-photon Ca2+ imaging in hippocampal slices.

Results: The Ca2+-bound state of T-GECO1 has an excitation peak maximum of 468 nm, an emission peak maximum of 500 nm, an extinction coefficient of 49,300M−1cm−1, a quantum yield of 0.83, and two-photon brightness approximately double that of EGFP. The Ca2+-dependent fluorescence increase is 15-fold, and the apparent Kd for Ca2+ is 82 nM. With two-photon excitation conditions at 850 nm, T-GECO1 consistently enabled the detection of action potentials with higher signal-to-noise (SNR) than a late generation GCaMP variant.

Conclusions: T-GECO1 is a high-performance blue-shifted GECI that, under two-photon excitation conditions, provides advantages relative to late generation GCaMP variants.

Keywords: blue-shifted fluorescence; genetically encoded calcium ion indicator; neuronal activity imaging; protein engineering; two-photon excitation.

Insulin signaling controls organ growth and final body size in insects. Recent results have begun to clarify how insulin signaling drives organ growth to match nutrient levels, but have not yet elucidated how insulin signaling controls final body size.

Separate recent studies have revealed the physiological changes underlying the evolution of body size in an insect and advanced our understanding of the genetics of insect growth. These studies highlight the gulf between physiological and genetic studies of growth control and the exciting opportunities for unification of these fields.

Manipulation of hematopoietic stem/progenitor cells (HSPCs) ex vivo is of clinical importance for stem cell expansion and gene therapy applications. However, most cultured HSPCs are actively cycling, and show a homing and engraftment defect compared with the predominantly quiescent noncultured HSPCs. We previously showed that HSPCs make contact with osteoblasts in vitro via a polarized membrane domain enriched in adhesion molecules such as tetraspanins. Here we show that increased cell cycling during ex vivo culture of HSPCs resulted in disruption of this membrane domain, as evidenced by disruption of polarity of the tetraspanin CD82. Chemical disruption or antibody-mediated blocking of CD82 on noncultured HSPCs resulted in decreased stromal cell adhesion, homing, and engraftment in nonobese diabetic/severe combined immunodeficiency IL-2γ(null) (NSG) mice compared with HSPCs with an intact domain. Most leukemic blasts were actively cycling and correspondingly displayed a loss of domain polarity and decreased homing in NSG mice compared with normal HSPCs. We conclude that quiescent cells, unlike actively cycling cells, display a polarized membrane domain enriched in tetraspanins that mediates homing and engraftment, providing a mechanistic explanation for the homing/engraftment defect of cycling cells and a potential new therapeutic target to enhance engraftment.

Manipulation of hematopoietic stem/progenitor cells (HSPCs) ex vivo is of clinical importance for stem cell expansion and gene therapy applications. However, most cultured HSPCs are actively cycling, and show a homing and engraftment defect compared with the predominantly quiescent noncultured HSPCs. We previously showed that HSPCs make contact with osteoblasts in vitro via a polarized membrane domain enriched in adhesion molecules such as tetraspanins. Here we show that increased cell cycling during ex vivo culture of HSPCs resulted in disruption of this membrane domain, as evidenced by disruption of polarity of the tetraspanin CD82. Chemical disruption or antibody-mediated blocking of CD82 on noncultured HSPCs resulted in decreased stromal cell adhesion, homing, and engraftment in nonobese diabetic/severe combined immunodeficiency IL-2γ(null) (NSG) mice compared with HSPCs with an intact domain. Most leukemic blasts were actively cycling and correspondingly displayed a loss of domain polarity and decreased homing in NSG mice compared with normal HSPCs. We conclude that quiescent cells, unlike actively cycling cells, display a polarized membrane domain enriched in tetraspanins that mediates homing and engraftment, providing a mechanistic explanation for the homing/engraftment defect of cycling cells and a potential new therapeutic target to enhance engraftment.

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.