Simple, Scalable Proteomic Imaging for High-Dimensional Profiling of Intact Systems.

Combined measurement of diverse molecular and anatomical traits that span multiple levels remains a major challenge in biology. Here, we introduce a simple method that enables proteomic imaging for scalable, integrated, high-dimensional phenotyping of both animal tissues and human clinical samples. This method, termed SWITCH, uniformly secures tissue architecture, native biomolecules, and antigenicity across an entire system by synchronizing the tissue preservation reaction. The heat- and chemical-resistant nature of the resulting framework permits multiple rounds (>20) of relabeling. We have performed 22 rounds of labeling of a single tissue with precise co-registration of multiple datasets. Furthermore, SWITCH synchronizes labeling reactions to improve probe penetration depth and uniformity of staining. With SWITCH, we performed combinatorial protein expression profiling of the human cortex and also interrogated the geometric structure of the fiber pathways in mouse brains. Such integrated high-dimensional information may accelerate our understanding of biological systems at multiple levels.

Use of Chlorhexidine to Prevent Ventilator-Associated Pneumonia in a Long-term Care Setting: A Retrospective Medical Record Review.

BACKGROUND: The purpose of this study was to explore the use of the oral decontamination solution chlorhexidine (CHX) to reduce ventilator-associated pneumonia (VAP) in a long-term ventilator care setting over time. Most of the research in this area has been conducted in acute and intensive care settings. METHODS: This study was a retrospective medical record review conducted in a long-term care facility with a dedicated ventilator unit. Veterans records (N = 12) were accessed for this study. The study covered 50 months, with a 43-month time period during which CHX was administered. RESULTS: While the sample size was small, many of the veterans on ventilators used CHX for years without an incident of VAP. CONCLUSIONS: These findings support using CHX to significantly reduce the number of days of intravenous antibiotics used to treat VAP with little side effects.

Protection of tissue physicochemical properties using polyfunctional crosslinkers.

Understanding complex biological systems requires the system-wide characterization of both molecular and cellular features. Existing methods for spatial mapping of biomolecules in intact tissues suffer from information loss caused by degradation and tissue damage. We report a tissue transformation strategy named stabilization under harsh conditions via intramolecular epoxide linkages to prevent degradation (SHIELD), which uses a flexible polyepoxide to form controlled intra- and intermolecular cross-link with biomolecules. SHIELD preserves protein fluorescence and antigenicity, transcripts and tissue architecture under a wide range of harsh conditions. We applied SHIELD to interrogate system-level wiring, synaptic architecture, and molecular features of virally labeled neurons and their targets in mouse at single-cell resolution. We also demonstrated rapid three-dimensional phenotyping of core needle biopsies and human brain cells. SHIELD enables rapid, multiscale, integrated molecular phenotyping of both animal and clinical tissues.

In situ expansion of engineered human liver tissue in a mouse model of chronic liver disease.

Control of both tissue architecture and scale is a fundamental translational roadblock in tissue engineering. An experimental framework that enables investigation into how architecture and scaling may be coupled is needed. We fabricated a structurally organized engineered tissue unit that expanded in response to regenerative cues after implantation into mice with liver injury. Specifically, we found that tissues containing patterned human primary hepatocytes, endothelial cells, and stromal cells in a degradable hydrogel expanded more than 50-fold over the course of 11 weeks in mice with injured livers. There was a concomitant increase in graft function as indicated by the production of multiple human liver proteins. Histologically, we observed the emergence of characteristic liver stereotypical microstructures mediated by coordinated growth of hepatocytes in close juxtaposition with a perfused vasculature. We demonstrated the utility of this system for probing the impact of multicellular geometric architecture on tissue expansion in response to liver injury. This approach is a hybrid strategy that harnesses both biology and engineering to more efficiently deploy a limited cell mass after implantation.

Medial amygdala lesions selectively block aversive pavlovian-instrumental transfer in rats.

Pavlovian conditioned stimuli (CSs) play an important role in the reinforcement and motivation of instrumental active avoidance (AA). Conditioned threats can also invigorate ongoing AA responding [aversive Pavlovian-instrumental transfer (PIT)]. The neural circuits mediating AA are poorly understood, although lesion studies suggest that lateral, basal, and central amygdala nuclei, as well as infralimbic prefrontal cortex, make key, and sometimes opposing, contributions. We recently completed an extensive analysis of brain c-Fos expression in good vs. poor avoiders following an AA test (Martinez et al., 2013, Learning and Memory). This analysis identified medial amygdala (MeA) as a potentially important region for Pavlovian motivation of instrumental actions. MeA is known to mediate defensive responding to innate threats as well as social behaviors, but its role in mediating aversive Pavlovian-instrumental interactions is unknown. We evaluated the effect of MeA lesions on Pavlovian conditioning, Sidman two-way AA conditioning (shuttling) and aversive PIT in rats. Mild footshocks served as the unconditioned stimulus in all conditioning phases. MeA lesions had no effect on AA but blocked the expression of aversive PIT and 22 kHz ultrasonic vocalizations in the AA context. Interestingly, MeA lesions failed to affect Pavlovian freezing to discrete threats but reduced freezing to contextual threats when assessed outside of the AA chamber. These findings differentiate MeA from lateral and central amygdala, as lesions of these nuclei disrupt Pavlovian freezing and aversive PIT, but have opposite effects on AA performance. Taken together, these results suggest that MeA plays a selective role in the motivation of instrumental avoidance by general or uncertain Pavlovian threats.

d-Cycloserine augmentation of cognitive remediation in schizophrenia.

d-Cycloserine (DCS) has been shown to enhance memory and, in a previous trial, once-weekly DCS improved negative symptoms in schizophrenia subjects. We hypothesized that DCS combined with a cognitive remediation (CR) program would improve memory of a practiced auditory discrimination task and that gains would generalize to performance on unpracticed cognitive tasks. Stable, medicated adult schizophrenia outpatients participated in the Brain Fitness CR program 3-5 times per week for 8weeks. Subjects were randomly assigned to once-weekly adjunctive treatment with DCS (50mg) or placebo administered before the first session each week. Primary outcomes were performance on an auditory discrimination task, the MATRICS cognitive battery composite score and the Scale for the Assessment of Negative Symptoms (SANS) total score. 36 subjects received study drug and 32 completed the trial (average number of CR sessions=26.1). Performance on the practiced auditory discrimination task significantly improved in the DCS group compared to the placebo group. DCS was also associated with significantly greater negative symptom improvement for subjects symptomatic at baseline (SANS score ≥20). However, improvement on the MATRICS battery was observed only in the placebo group. Considered with previous results, these findings suggest that DCS augments CR and alleviates negative symptoms in schizophrenia patients. However, further work is needed to evaluate whether CR gains achieved with DCS can generalize to other unpracticed cognitive tasks.

Development of an aversive Pavlovian-to-instrumental transfer task in rat.

Pavlovian-to-instrumental transfer (PIT) is an effect whereby a classically conditioned stimulus (CS) enhances ongoing instrumental responding. PIT has been extensively studied with appetitive conditioning but barely at all with aversive conditioning. Although it's been argued that conditioned suppression is a form of aversive PIT, this effect is fundamentally different from appetitive PIT because the CS suppresses, instead of facilitates, responding. Five experiments investigated the importance of a variety of factors on aversive PIT in a rodent Sidman avoidance paradigm in which ongoing shuttling behavior (unsignaled active avoidance or USAA) was facilitated by an aversive CS. Experiment 1 demonstrated a basic PIT effect. Experiment 2 found that a moderate amount of USAA extinction produces the strongest PIT with shuttling rates best at around 2 responses per minute prior to the CS. Experiment 3 tested a protocol in which the USAA behavior was required to reach the 2-response per minute mark in order to trigger the CS presentation and found that this produced robust and reliable PIT. Experiment 4 found that the Pavlovian conditioning US intensity was not a major determinant of PIT strength. Experiment 5 demonstrated that if the CS and US were not explicitly paired during Pavlovian conditioning, PIT did not occur, showing that CS-US learning is required. Together, these studies demonstrate a robust, reliable and stable aversive PIT effect that is amenable to analysis of neural circuitry.