Insertion-trigger residues differentially modulate endosomal escape by cytotoxic necrotizing factor toxins.

The cellular specificity, potency, and modular nature of bacterial protein toxins enable their application for targeted cytosolic delivery of therapeutic cargo. Efficient endosomal escape is a critical step in the design of bacterial toxin-inspired drug delivery (BTIDD) vehicles to avoid lysosomal degradation and promote optimal cargo delivery. The cytotoxic necrotizing factor (CNF) family of modular toxins represents a useful model for investigating cargo-delivery mechanisms due to the availability of many homologs with high sequence identity, their flexibility in swapping domains, and their differential activity profiles. Previously, we found that CNFy is more sensitive to endosomal acidification inhibitors than CNF1 and CNF2. Here, we report that CNF3 is even less sensitive than CNF1/2. We identified two amino acid residues within the putative translocation domain (E374 and E412 in CNFy, Q373 and S411 in CNF3) that differentiate between these two toxins. Swapping these corresponding residues in each toxin changed the sensitivity to endosomal acidification and efficiency of cargo-delivery to be more similar to the other toxin. Results suggested that trafficking to the more acidic late endosome is required for cargo delivery by CNFy but not CNF3. This model was supported by results from toxin treatment of cells in the presence of NH4Cl, which blocks endosomal acidification, and of small-molecule inhibitors EGA, which blocks trafficking to late endosomes, and ABMA, which blocks endosomal escape and trafficking to the lysosomal degradative pathway. These findings suggest that it is possible to fine-tune endosomal escape and cytosolic cargo delivery efficiency in designing BTIDD platforms.

Electric Field Strength From Prefrontal Transcranial Direct Current Stimulation Determines Degree of Working Memory Response: A Potential Application of Reverse-Calculation Modeling?

BACKGROUND: Transcranial direct current stimulation (tDCS) for working memory is an enticing treatment, but there is mixed evidence to date. OBJECTIVES: We tested the effects of electric field strength from uniform 2 mA dosing on working memory change from prestimulation to poststimulation. Second, we statistically evaluated a reverse-calculation method of individualizing tDCS dose and its effect on normalizing electric field at the cortex. MATERIALS AND METHODS: We performed electric field modeling on a data set of 28 healthy older adults (15 women, mean age = 73.7, SD = 7.3) who received ten sessions of active 2 mA tDCS (N = 14) or sham tDCS (N = 14) applied over bilateral dorsolateral prefrontal cortices (DLPFC) in a triple-blind design. We evaluated the relationship between electric field strength and working memory change on an N-back task in conditions of above-median, high electric field from active 2 mA (N = 7), below-median, low electric field from active 2 mA (N = 7), and sham (N = 14) at regions of interest (ROI) at the left and right DLPFC. We then determined the individualized reverse-calculation dose to produce the group average electric field and measured the electric field variance between uniform 2 mA doses vs individualized reverse-calculation doses at the same ROIs. RESULTS: Working memory improvements from pre- to post-tDCS were significant for the above-median electric field from active 2 mA condition at the left DLPFC (mixed ANOVA, p = 0.013). Furthermore, reverse-calculation modeling significantly reduced electric field variance at both ROIs (Levene's test; p < 0.001). CONCLUSIONS: Higher electric fields at the left DLPFC from uniform 2 mA doses appear to drive working memory improvements from tDCS. Individualized doses from reverse-calculation modeling significantly reduce electric field variance at the cortex. Taken together, using reverse-calculation modeling to produce the same, high electric fields at the cortex across participants may produce more effective future tDCS treatments for working memory.

Coping in the Clinic: Effects of Clinically Elevated Anxiety on Dynamic Neurophysiological Mechanisms of Escape/Avoidance Preparation.

BACKGROUND: Treatments for anxiety and related disorders target exaggerated escape/avoidance as a core feature, but current methods fail to improve escape/avoidance habits for many treatment-seeking individuals. To support developing tools that increase treatment efficacy by targeting mechanisms more directly, the current work examined potential distinctions in the neurophysiologies of escape and avoidance and tested how clinical anxiety affects these neurophysiologies. METHODS: Twenty-five treatment-seeking individuals with varied principal diagnoses (e.g., generalized anxiety disorder, posttraumatic stress disorder) and 20 non-treatment-seeking control subjects participated. In the study task, approximately 5.25-second cues predicted aversive images that could be avoided (blocked by a button press before image onset), escaped (ended by a button press after image onset), or not controlled. To examine neural processing and defensive response modulation, anticipatory event-related potentials were derived, and startle reflexes were probed throughout each cue. RESULTS: Multidimensional profiles were observed such that 1) anticipatory event-related potential enhancement was only reliable during avoidance preparation, and event-related potentials potentially reflected perceived/instrumental control; and 2) startle reflexes were inhibited during avoidance preparation, relatively enhanced during escape preparation, and further enhanced during uncontrollable anticipation, thus potentially reflecting fear-related activation. Treatment-seeking status, then, did not affect cortical processing, but it did moderate context-dependent fear (if individuals with severe depression were excluded) such that treatment-seeking individuals without depression showed exaggerated startle during escape, but not avoidance, preparation. CONCLUSIONS: Data suggest a specific effect of anxiety on fear system activation during preparation to escape aversion. This effect warrants further investigation as a precision target for interventions that directly modulate the specific underlying neural circuitry.

Brain stimulation in zero gravity: transcranial magnetic stimulation (TMS) motor threshold decreases during zero gravity induced by parabolic flight.

We are just beginning to understand how spaceflight may impact brain function. As NASA proceeds with plans to send astronauts to the Moon and commercial space travel interest increases, it is critical to understand how the human brain and peripheral nervous system respond to zero gravity. Here, we developed and refined head-worn transcranial magnetic stimulation (TMS) systems capable of reliably and quickly determining the amount of electromagnetism each individual needs to detect electromyographic (EMG) threshold levels in the thumb (called the resting motor threshold (rMT)). We then collected rMTs in 10 healthy adult participants in the laboratory at baseline, and subsequently at three time points onboard an airplane: (T1) pre-flight at Earth gravity, (T2) during zero gravity periods induced by parabolic flight and (T3) post-flight at Earth gravity. Overall, the subjects required 12.6% less electromagnetism applied to the brain to cause thumb muscle activation during weightlessness compared to Earth gravity, suggesting neurophysiological changes occur during brief periods of zero gravity. We discuss several candidate explanations for this finding, including upward shift of the brain within the skull, acute increases in cortical excitability, changes in intracranial pressure, and diffuse spinal or neuromuscular system effects. All of these possible explanations warrant further study. In summary, we documented neurophysiological changes during brief episodes of zero gravity and thus highlighting the need for further studies of human brain function in altered gravity conditions to optimally prepare for prolonged microgravity exposure during spaceflight.

Identification of Common Neural Circuit Disruptions in Emotional Processing Across Psychiatric Disorders.

OBJECTIVE: Disrupted emotional processing is a common feature of many psychiatric disorders. The authors investigated functional disruptions in neural circuitry underlying emotional processing across a range of tasks and across psychiatric disorders through a transdiagnostic quantitative meta-analysis of published neuroimaging data. METHODS: A PubMed search was conducted for whole-brain functional neuroimaging findings published through May 2018 that compared activation during emotional processing tasks in patients with psychiatric disorders (including schizophrenia, bipolar or unipolar depression, anxiety, and substance use) to matched healthy control participants. Activation likelihood estimation (ALE) meta-analyses were conducted on peak voxel coordinates to identify spatial convergence. RESULTS: The 298 experiments submitted to meta-analysis included 5,427 patients and 5,491 control participants. ALE across diagnoses and patterns of patient hyper- and hyporeactivity demonstrated abnormal activation in the amygdala, the hippocampal/parahippocampal gyri, the dorsomedial/pulvinar nuclei of the thalamus, and the fusiform gyri, as well as the medial and lateral dorsal and ventral prefrontal regions. ALE across disorders but considering directionality demonstrated patient hyperactivation in the amygdala and the hippocampal/parahippocampal gyri. Hypoactivation was found in the medial and lateral prefrontal regions, most pronounced during processing of unpleasant stimuli. More refined disorder-specific analyses suggested that these overall patterns were shared to varying degrees, with notable differences in patterns of hyper- and hypoactivation. CONCLUSIONS: These findings demonstrate a pattern of neurocircuit disruption across major psychiatric disorders in regions and networks key to adaptive emotional reactivity and regulation. More specifically, disruption corresponded prominently to the "salience" network, the ventral striatal/ventromedial prefrontal "reward" network, and the lateral orbitofrontal "nonreward" network. Consistent with the Research Domain Criteria initiative, these findings suggest that psychiatric illness may be productively formulated as dysfunction in transdiagnostic neurobehavioral phenotypes such as neurocircuit activation.