Dynamics of an incoherent feedforward loop drive ERK-dependent pattern formation in the early Drosophila embryo.

Positional information in development often manifests as stripes of gene expression, but how stripes form remains incompletely understood. Here, we use optogenetics and live-cell biosensors to investigate the posterior brachyenteron (byn) stripe in early Drosophila embryos. This stripe depends on interpretation of an upstream ERK activity gradient and the expression of two target genes, tailless (tll) and huckebein (hkb), that exert antagonistic control over byn. We find that high or low doses of ERK signaling produce transient or sustained byn expression, respectively. Although tll transcription is always rapidly induced, hkb converts graded ERK inputs into a variable time delay. Nuclei thus interpret ERK amplitude through the relative timing of tll and hkb transcription. Antagonistic regulatory paths acting on different timescales are hallmarks of an incoherent feedforward loop, which is sufficient to explain byn dynamics and adds temporal complexity to the steady-state model of byn stripe formation. We further show that 'blurring' of an all-or-none stimulus through intracellular diffusion non-locally produces a byn stripe. Overall, we provide a blueprint for using optogenetics to dissect developmental signal interpretation in space and time.

Preformation and epigenesis converge to specify primordial germ cell fate in the early Drosophila embryo.

A critical step in animal development is the specification of primordial germ cells (PGCs), the precursors of the germline. Two seemingly mutually exclusive mechanisms are implemented across the animal kingdom: epigenesis and preformation. In epigenesis, PGC specification is non-autonomous and depends on extrinsic signaling pathways. The BMP pathway provides the key PGC specification signals in mammals. Preformation is autonomous and mediated by determinants localized within PGCs. In Drosophila, a classic example of preformation, constituents of the germ plasm localized at the embryonic posterior are thought to be both necessary and sufficient for proper determination of PGCs. Contrary to this longstanding model, here we show that these localized determinants are insufficient by themselves to direct PGC specification in blastoderm stage embryos. Instead, we find that the BMP signaling pathway is required at multiple steps during the specification process and functions in conjunction with components of the germ plasm to orchestrate PGC fate.

Multi-spectral SWIR lidar for imaging and spectral discrimination through partial obscurations.

We have developed a multi-spectral SWIR lidar system capable of measuring simultaneous spatial-spectral information for imaging and spectral discrimination through partial obscurations. We employ objects in the presence and absence of a series of obscurants to evaluate the capability of the system in classifying the objects of interest based on spectral and range information. We employ a principal component analysis-based algorithm in classifying the objects and quantifying the accuracy of detection under various obscured scenarios. The merits of multi-spectral lidar over hyperspectral imaging are highlighted for target identification in the presence of obscurants.

Cis-effects on gene expression in the human prenatal brain associated with genetic risk for neuropsychiatric disorders.

The majority of common risk alleles identified for neuropsychiatric disorders reside in noncoding regions of the genome and are therefore likely to impact gene regulation. However, the genes that are primarily affected and the nature and developmental timing of these effects remain unclear. Given the hypothesized role for early neurodevelopmental processes in these conditions, we here define genetic predictors of gene expression in the human fetal brain with which we perform transcriptome-wide association studies (TWASs) of attention deficit hyperactivity disorder (ADHD), autism spectrum disorder, bipolar disorder, major depressive disorder, and schizophrenia. We identify prenatal cis-regulatory effects on 63 genes and 166 individual transcripts associated with genetic risk for these conditions. We observe pleiotropic effects of expression predictors for a number of genes and transcripts, including those of decreased DDHD2 expression in association with risk for schizophrenia and bipolar disorder, increased expression of a ST3GAL3 transcript with risk for schizophrenia and ADHD, and increased expression of an XPNPEP3 transcript with risk for schizophrenia, bipolar disorder, and major depression. For the protocadherin alpha cluster genes PCDHA7 and PCDHA8, we find that predictors of low expression are associated with risk for major depressive disorder while those of higher expression are associated with risk for schizophrenia. Our findings support a role for altered gene regulation in the prenatal brain in susceptibility to various neuropsychiatric disorders and prioritize potential risk genes for further neurobiological investigation.

Guide Swap enables genome-scale pooled CRISPR-Cas9 screening in human primary cells.

CRISPR-Cas9 screening allows genome-wide interrogation of gene function. Currently, to achieve the high and uniform Cas9 expression desirable for screening, one needs to engineer stable and clonal Cas9-expressing cells-an approach that is not applicable in human primary cells. Guide Swap permits genome-scale pooled CRISPR-Cas9 screening in human primary cells by exploiting the unexpected finding that editing by lentivirally delivered, targeted guide RNAs (gRNAs) occurs efficiently when Cas9 is introduced in complex with nontargeting gRNA. We validated Guide Swap in depletion and enrichment screens in CD4+ T cells. Next, we implemented Guide Swap in a model of ex vivo hematopoiesis, and identified known and previously unknown regulators of CD34+ hematopoietic stem and progenitor cell (HSPC) expansion. We anticipate that this platform will be broadly applicable to other challenging cell types, and thus will enable discovery in previously inaccessible but biologically relevant human primary cell systems.

Implications for research and clinical use from a Rasch analysis of the HOOS-12 and KOOS-12 instruments.

OBJECTIVE: To evaluate the structural validity of the 12-item Hip disability and Osteoarthritis Outcome Score (HOOS-12) and 12-item Knee injury and Osteoarthritis Outcome Score (KOOS-12) using Rasch analysis and consider psychometric implications for research and clinical use. METHOD: Individual-level HOOS-12 and KOOS-12 data from the Australian Orthopaedic Association National Joint Replacement Registry, collected before and after primary total hip and knee replacement, were used for this analysis. Using the Rasch analytic approach, overall model fit and item fit were examined, together with potential reasons for misfit including response threshold ordering, differential item functioning, internal consistency, unidimensionality and item targeting. RESULTS: Overall misfit to the Rasch model was evident for both instruments. A degree of item misfit was also observed, although most items demonstrated logical sequencing of response options. Only two items (hip/knee pain frequency and awareness of hip/knee problems) displayed disordered response thresholds. The pain, function, and quality of life domains of the HOOS-12 and KOOS-12 demonstrated excellent internal consistency reliability (person separation index: 0.80-0.93) and unidimensionality. A mismatch between item difficulty and person ability scores at the highest end of the HOOS-12 and KOOS-12 scales contributed to post-operative ceiling effects (mean logit for HOOS-12: 3.57; KOOS-12: 2.58; ≈0 indicates well-targeted scale). CONCLUSION: We found evidence to support the structural validity of the three HOOS-12 and KOOS-12 domains for evaluating joint replacement outcomes. However, there may be missing content in both instruments particularly for high-functioning patients. Minor refinement of some response options may be warranted to improve item performance.

Performance of the HOOS-12 and KOOS-12 instruments for evaluating outcomes from joint replacement surgery.

OBJECTIVE: To evaluate the psychometric properties of the 12-item Hip disability and Osteoarthritis Outcome Score (HOOS-12) and Knee injury and Osteoarthritis Outcome Score (KOOS-12) for use in evaluating outcomes after joint replacement for osteoarthritis. DESIGN: Patient-reported outcomes data collected by the Australian Orthopaedic Association National Joint Replacement Registry were used for this analysis. HOOS-12 and KOOS-12 domain (pain, function, quality of life) and summary impact data were available. The Oxford Hip Score (OHS), Oxford Knee Score (OKS) and EQ-5D-5L were used as comparators. Instruments were administered pre-operatively and at 6 months post-operatively. Internal consistency reliability, floor and ceiling effects, convergent validity, known groups validity, and responsiveness were evaluated using standard psychometric techniques. RESULTS: Baseline HOOS-12 and KOOS-12 data were available for 3,023 patients undergoing primary total hip replacement and 4,010 patients undergoing primary total knee replacement. At baseline, high internal consistency was demonstrated for all domains and summary scores (Cronbach's alpha: HOOS-12 = 0.81-0.93; KOOS-12 = 0.82-0.92). Post-operative ceiling effects (>15% of patients scoring the best possible score) were identified for the HOOS-12 pain (46%), function (39%) and quality of life domains (26%) and summary score (17%), and for the KOOS-12 pain (21%) and function domains (18%). The HOOS-12 and KOOS-12 could differentiate between two known groups (lowest/highest OHS or OKS quartiles post-operatively; p < 0.001) and were highly responsive to change (effect sizes for HOOS-12: 2.20-2.83; KOOS-12: 1.82-2.35). CONCLUSION: The HOOS-12 and KOOS-12 have good psychometric properties for capturing joint replacement outcomes including excellent responsiveness, although ceiling effects may limit monitoring of post-operative improvement.

Neural correlates of the production effect: An fMRI study.

Recognition memory is improved for items produced at study (e.g., by reading them aloud) relative to a non-produced control condition (e.g., silent reading). This production effect is typically attributed to the extra elements in the production task (e.g., motor activation, auditory perception) enhancing item distinctiveness. To evaluate this claim, the present study examined the neural mechanisms underlying the production effect. Prior to a recognition memory test, different words within a study list were read either aloud, silently, or while saying "check" (as a sensorimotor control condition). Production improved recognition, and aloud words yielded higher rates of both recollection and familiarity judgments than either silent or control words. During encoding, fMRI revealed stronger activation in regions associated with motor, somatosensory, and auditory processing for aloud items than for either silent or control items. These activations were predictive of recollective success for aloud items at test. Together, our findings are compatible with a distinctiveness-based account of the production effect, while also pointing to the possible role of other processing differences during the aloud trials as compared to silent and control.

The role of the motor system in generating creative thoughts.

Neurocognitive research is pertinent to developing mechanistic models of how humans generate creative thoughts. Such models usually overlook the role of the motor cortex in creative thinking. The framework of embodied or grounded cognition suggests that creative thoughts (e.g. using a shoe as a hammer, improvising a piano solo) are partially served by simulations of motor activity associated with tools and their use. The major hypothesis stemming from the embodied or grounded account is that, while the motor system is used to execute actions, simulations within this system also support higher-order cognition, creativity included. That is, the cognitive process of generating creative output, not just executing it, is deeply embedded in motor processes. Here, we highlight a collection of neuroimaging research that implicates the motor system in generating creative thoughts, including some evidence for its functionally necessary role in generating creative output. Specifically, the grounded or embodied framework suggests that generating creative output may, in part, rely on motor simulations of possible actions, and that these simulations may by partially implemented in the motor regions themselves. In such cases, action simulations (i.e. reactivating or re-using the motor system), do not result in overt action but instead are used to support higher-order cognitive goals like generating creative uses or improvising.

Investigating grounded conceptualization: motor system state-dependence facilitates familiarity judgments of novel tools.

Theories of embodied cognition propose that we recognize tools in part by reactivating sensorimotor representations of tool use in a process of simulation. If motor simulations play a causal role in tool recognition then performing a concurrent motor task should differentially modulate recognition of experienced vs. non-experienced tools. We sought to test the hypothesis that an incompatible concurrent motor task modulates conceptual processing of learned vs. non-learned objects by directly manipulating the embodied experience of participants. We trained one group to use a set of novel, 3-D printed tools under the pretense that they were preparing for an archeological expedition to Mars (manipulation group); we trained a second group to report declarative information about how the tools are stored (storage group). With this design, familiarity and visual attention to different object parts was similar for both groups, though their qualitative interactions differed. After learning, participants made familiarity judgments of auditorily presented tool names while performing a concurrent motor task or simply sitting at rest. We showed that familiarity judgments were facilitated by motor state-dependence; specifically, in the manipulation group, familiarity was facilitated by a concurrent motor task, whereas in the spatial group familiarity was facilitated while sitting at rest. These results are the first to directly show that manipulation experience differentially modulates conceptual processing of familiar vs. unfamiliar objects, suggesting that embodied representations contribute to recognizing tools.

Lamellipodia are crucial for haptotactic sensing and response.

Haptotaxis is the process by which cells respond to gradients of substrate-bound cues, such as extracellular matrix proteins (ECM); however, the cellular mechanism of this response remains poorly understood and has mainly been studied by comparing cell behavior on uniform ECMs with different concentrations of components. To study haptotaxis in response to gradients, we utilized microfluidic chambers to generate gradients of the ECM protein fibronectin, and imaged the cell migration response. Lamellipodia are fan-shaped protrusions that are common in migrating cells. Here, we define a new function for lamellipodia and the cellular mechanism required for haptotaxis - differential actin and lamellipodial protrusion dynamics lead to biased cell migration. Modest differences in lamellipodial dynamics occurring over time periods of seconds to minutes are summed over hours to produce differential whole cell movement towards higher concentrations of fibronectin. We identify a specific subset of lamellipodia regulators as being crucial for haptotaxis. Numerous studies have linked components of this pathway to cancer metastasis and, consistent with this, we find that expression of the oncogenic Rac1 P29S mutation abrogates haptotaxis. Finally, we show that haptotaxis also operates through this pathway in 3D environments.

Effect of UVB Light Exposure on Tensile Behavior of Carbon Nanotube Sheet.

The effects of ultraviolet B (UVB) exposure on the tensile behavior of CNT sheet was investigated in this study. Two types of CNT sheet, one acid treated and one un-treated, were directly exposed to UVB light for 500 hours. The exposure was done using a UVB lamp inside a dark chamber under room temperature ambient condition. The microstructure of the CNT sheets were studied both prior to and after UVB exposure using a scanning electron microscope. Upon completion of the exposure duration, the CNT sheet test coupons were tested mechanically in tension using a microtester to evaluate the tensile strength and behavior. The results were compared to those of the CNT sheet test coupons that were not exposed to UVB light. It was observed that the strength of the acid treated CNT sheet decreased after UVB exposure while the strength of the un-treated CNT sheet increased. Apart from slight changes in stiffness, the overall mechanical behavior with increasing applied load did not show much change after the exposure to UVB light. The microscopic analysis showed evidences of morphological changes in the CNT microstructure upon UVB exposure. These changes supported the change in mechanical strength as a result of the UVB exposure.

Modeled De Facto Reuse and Contaminants of Emerging Concern in Drinking Water Source Waters.

De facto reuse is the percentage of drinking water treatment plant (DWTP) intake potentially composed of effluent discharged from upstream wastewater treatment plants (WWTPs). Results from grab samples and a De Facto Reuse in our Nation's Consumable Supply (DRINCS) geospatial watershed model were used to quantify contaminants of emerging concern (CECs) concentrations at DWTP intakes to qualitatively compare exposure risks obtained by the two approaches. Between nine and 71 CECs were detected in grab samples. The number of upstream WWTP discharges ranged from 0 to >1,000; comparative de facto reuse results from DRINCS ranged from <0.1 to 13% during average flow and >80% during lower streamflows. Correlation between chemicals detected and DRINCS modeling results were observed, particularly DWTPs withdrawing from midsize water bodies. This comparison advances the utility of DRINCS to identify locations of DWTPs for future CEC sampling and treatment technology testing.

Differential Tuning of Ventral and Dorsal Streams during the Generation of Common and Uncommon Tool Uses.

Our use of tools is situated in different contexts. Prior evidence suggests that diverse regions within the ventral and dorsal streams represent information supporting common tool use. However, given the flexibility of object concepts, these regions may be tuned to different types of information when generating novel or uncommon uses of tools. To investigate this, we collected fMRI data from participants who reported common or uncommon tool uses in response to visually presented familiar objects. We performed a pattern dissimilarity analysis in which we correlated cortical patterns with behavioral measures of visual, action, and category information. The results showed that evoked cortical patterns within the dorsal tool use network reflected action and visual information to a greater extent in the uncommon use group, whereas evoked neural patterns within the ventral tool use network reflected categorical information more strongly in the common use group. These results reveal the flexibility of cortical representations of tool use and the situated nature of cortical representations more generally.

Optimization of an Enzymatic Antibody-Drug Conjugation Approach Based on Coenzyme A Analogs.

Phosphopantetheine transferases (PPTases) can be used to efficiently prepare site-specific antibody-drug conjugates (ADCs) by enzymatically coupling coenzyme A (CoA)-linker payloads to 11-12 amino acid peptide substrates inserted into antibodies. Here, a two-step strategy is established wherein in a first step, CoA analogs with various bioorthogonal reactivities are enzymatically installed on the antibody for chemical conjugation with a cytotoxic payload in a second step. Because of the high structural similarity of these CoA analogs to the natural PPTase substrate CoA-SH, the first step proceeds very efficiently and enables the use of peptide tags as short as 6 amino acids compared to the 11-12 amino acids required for efficient one-step coupling of the payload molecule. Furthermore, two-step conjugation provides access to diverse linker chemistries and spacers of varying lengths. The potency of the ADCs was largely independent of linker architecture. In mice, proteolytic cleavage was observed for some C-terminally linked auristatin payloads. The in vivo stability of these ADCs was significantly improved by reduction of the linker length. In addition, linker stability was found to be modulated by attachment site, and this, together with linker length, provides an opportunity for maximizing ADC stability without sacrificing potency.

Are Filopodia Privileged Signaling Structures in Migrating Cells?

Filopodia are thin, fingerlike structures that contain bundled actin filaments and project from the cell periphery. These structures are dogmatically endowed with the ability to sense cues in the microenvironment, implying that filopodia foster local signal transduction, yet their small diameter hampers the imaging of dynamic processes therein. To overcome this challenge, we analyzed total internal reflection fluorescence images of migrating fibroblasts coexpressing either a plasma membrane marker or tagged AktPH domain, a translocation biosensor for signaling through the phosphoinositide 3-kinase pathway, along with a cytosolic volume marker. We devised a scheme to estimate the radii of filopodia using either the membrane marker or volume marker data, and we used that information to account for geometry effects in the biosensor data. With conservative estimates of relative target molecule abundance, it is revealed that filopodia typically harbor higher densities of 3' phosphoinositides than adjacent regions at the cell periphery. In this context at least, the analysis supports the filopodial signaling hypothesis.

Efficient Preparation of Site-Specific Antibody-Drug Conjugates Using Phosphopantetheinyl Transferases.

Post-translational modification catalyzed by phosphopantetheinyl transferases (PPTases) has previously been used to site-specifically label proteins with structurally diverse molecules. PPTase catalysis results in covalent modification of a serine residue in acyl/peptidyl carrier proteins and their surrogate substrates which are typically fused to the N- or C-terminus. To test the utility of PPTases for preparing antibody-drug conjugates (ADCs), we inserted 11 and 12-mer PPTase substrate sequences at 110 constant region loop positions of trastuzumab. Using Sfp-PPTase, 63 sites could be efficiently labeled with an auristatin toxin, resulting in 95 homogeneous ADCs. ADCs labeled in the CH1 domain displayed in general excellent pharmacokinetic profiles and negligible drug loss. A subset of CH2 domain conjugates underwent rapid clearance in mouse pharmacokinetic studies. Rapid clearance correlated with lower thermal stability of the particular antibodies. Independent of conjugation site, almost all ADCs exhibited subnanomolar in vitro cytotoxicity against HER2-positive cell lines. One selected ADC was shown to induce tumor regression in a xenograft model at a single dose of 3 mg/kg, demonstrating that PPTase-mediated conjugation is suitable for the production of highly efficacious and homogeneous ADCs.

Structure and function of the DUF2233 domain in bacteria and in the human mannose 6-phosphate uncovering enzyme.

DUF2233, a domain of unknown function (DUF), is present in many bacterial and several viral proteins and was also identified in the mammalian transmembrane glycoprotein N-acetylglucosamine-1-phosphodiester α-N-acetylglucosaminidase ("uncovering enzyme" (UCE)). We report the crystal structure of BACOVA_00430, a 315-residue protein from the human gut bacterium Bacteroides ovatus that is the first structural representative of the DUF2233 protein family. A notable feature of this structure is the presence of a surface cavity that is populated by residues that are highly conserved across the entire family. The crystal structure was used to model the luminal portion of human UCE (hUCE), which is involved in targeting of lysosomal enzymes. Mutational analysis of several residues in a highly conserved surface cavity of hUCE revealed that they are essential for function. The bacterial enzyme (BACOVA_00430) has ∼1% of the catalytic activity of hUCE toward the substrate GlcNAc-P-mannose, the precursor of the Man-6-P lysosomal targeting signal. GlcNAc-1-P is a poor substrate for both enzymes. We conclude that, for at least a subset of proteins in this family, DUF2233 functions as a phosphodiester glycosidase.

Protection from outpatient sudden cardiac death following ICD removal using a wearable cardioverter defibrillator.

BACKGROUND: An implantable cardioverter defibrillator (ICD) is effective in preventing sudden cardiac death (SCD). Once an ICD is removed and reimplantation is not feasible, a wearable cardioverter defibrillator (WCD) may be an alternative option. We determined the effectiveness of WCD for SCD prevention in patients who were discharged after ICD removal. METHODS: A retrospective study was conducted on all WCD (LifeVest, ZOLL, Pittsburgh, PA, USA) patients who underwent ICD removal due to cardiac device infections (CDIs) at two referral centers between January 1, 2005 and December 31, 2009. Clinical characteristics, device information, and WCD data were analyzed. Sudden cardiac arrest was defined as all sustained ventricular tachycardia (VT) and ventricular fibrillation occurring within a single 24-hour period. RESULTS: Ninety-seven patients (mean age 62.8 ± 13.3, male 80.4%) were included in the study. The median duration of antibiotic use was 14.7 days (interquartile range [IQR] 10-30). The median daily WCD use was 20 hours/day and the median length of use was 21 days (IQR 5-47). A total of three patients were shocked by WCD. Two patients had four episodes of sustained VT, successfully terminated by the WCD. A third patient experienced two inappropriate treatments due to oversensitivity of the signal artifact. Three patients experienced sudden death outside the hospital while not wearing the device. Five patients died while hospitalized. CONCLUSION: WCD can prevent SCD, until ICD reimplantation is feasible in patients who underwent device removals for CDI. However, patient compliance is essential for the effective use of this device.

A test of the embodied simulation theory of object perception: potentiation of responses to artifacts and animals.

Theories of embodied object representation predict a tight association between sensorimotor processes and visual processing of manipulable objects. Previous research has shown that object handles can 'potentiate' a manual response (i.e., button press) to a congruent location. This potentiation effect is taken as evidence that objects automatically evoke sensorimotor simulations in response to the visual presentation of manipulable objects. In the present series of experiments, we investigated a critical prediction of the theory of embodied object representations that potentiation effects should be observed with manipulable artifacts but not non-manipulable animals. In four experiments we show that (a) potentiation effects are observed with animals and artifacts; (b) potentiation effects depend on the absolute size of the objects and (c) task context influences the presence/absence of potentiation effects. We conclude that potentiation effects do not provide evidence for embodied object representations, but are suggestive of a more general stimulus-response compatibility effect that may depend on the distribution of attention to different object features.