Properties of salivary mucins: MUCSB protein expression by isolation of C-terminal constructs in the cystine knot motif

Thesis (MSD)--Boston University, Goldman School of Dental Medicine, 2005.Includes bibliographical references: leaves 73-77.The present study of salivary mucin MUC5B has sought to isolate and evaluate the properties of peptide constructs derived from within the cystine-knot motif. Three peptide constructs of different lengths were designed from the C-terminal sequence of MUC5B and each construct incorporated restriction sites for the endonucleases BamHI, at their 5' end, and Xhol at their 3' end. Amplification was accomplished using RT-PCR, with cDNA for the RT reaction derived from RNA isolated from frozen human submandibular glands. Constructs were cloned in to E. coli using a TOPO vector and cultured in order to isolate plasmid inserts that were then ligated into a pGEX-5X-2 vector. BL21 E. coli were utilized as host cells, using the pGEX-5X-2 vector to transform the construct plasmids. Protein expression was then induced in the host cells using IPTG and the GST fusion-protein product collected via centrifugation of the sonicated cell pellets. Fusion proteins were then isolated using Sepharose 4B affinity column purification. Isolates were finally dialyzed against deionized water and lyophilized; the lyophilate was then intended for immunization of rabbits. Fusion protein isolation required extensive optimization, including the reduction of sonication cycles and the addition of protease inhibitors in order to reduce the action of host cell enzymes on the protein product. Attempts to cleave constructs from their GST fusion partners were unsuccessful due to the possible effects of intramolecular folding during expression that may have blocked the enzyme recognition site. Denaturants were employed in order to improve enzyme-protein interaction, however, colorimetric assays revealed that even suggested levels of these chemicals significantly reduced the activity of the cleavage enzyme

The effect of cannabidiol (CBD) on behavioral and neuroinflammatory consequences of comorbid AUD and PTSD in a rat model

Alcohol use disorder (AUD) and post-traumatic stress disorder (PTSD) are debilitating conditions that often co-occur, with an estimated 41-79% comorbidity rate. A major concern with the co-occurrence of these disorders is the tendency for one to exacerbate the other. Specifically, symptoms related to PTSD are a significant risk factor for the development of AUD, and alcohol abuse worsens PTSD symptoms. This cycle, along with a lack of effective pharmacological treatment options, leads to significant behavioral and physiological deficits. Additionally, remission for comorbid AUD and PTSD is much more difficult to attain due to exacerbated symptomology and a lack of FDA-approved medications. In recent years, cannabidiol (CBD), a non-psychoactive compound found in cannabis, has been a focus of study due to its therapeutic potential. Researchers have demonstrated the anxiolytic and anti-inflammatory effects of CBD in both humans and animals, showing its promise as a novel therapeutic agent in the treatment of psychiatric disorders. The purpose of this study is to investigate the hypothesis that CBD will reduce fear-related behaviors and neuroinflammation in a rat model of comorbid AUD and PTSD. Our AUD/PTSD model utilized restraint stress and chronic intermittent ethanol exposure procedures. To investigate changes in future stress sensitivity all animals were exposed to a contextual fear conditioning paradigm, which was used to train the animals to associate environmental and auditory cues (environment appearance and tone) with an aversive stimulus (mild foot-shock). 30 minutes prior to each conditioning session, rats received an intraperitoneal injection of CBD (20mg/kg) or 0.9% Saline. Once the animals learned to associate the cues with a shock, they were exposed to an extinction learning procedure that involved presentation of the cue alone (no shock). This procedure parallels exposure therapy in humans, allowing for the assessment adaptations to fear learning. The amount of time the rats remain still (freezing) during the tone represents fear-related behavior. Our current results indicate rats with a history of stress and alcohol exposure displayed significantly higher freezing behaviors and this effect was significantly decreased with CBD treatment. This suggests that when CBD is administered during fear learning, it is able to attenuate heightened stress sensitivity associated with AUD/PTSD. To evaluate how CBD mediates the neuroinflammatory response associated with AUD and PTSD, brains from the rats were extracted and analyzed for the inflammatory cytokine tumor necrosis factor a (TNF-a). Specific regions of interest included the medial prefrontal cortex and hippocampus, areas associated with anxiety, memory, and addiction. Neuroinflammation analyses are still ongoing, however it is predicted that rats who received CBD will show a reduction in inflammation in the medial prefrontal cortex and hippocampus. Taken together, the current results show promise for CBD to reduce enhanced fear-related behavior associated with comorbid AUD and PTSD

INTERPERSONAL HAND-ENERGY REGISTRATION: EVIDENCE FOR IMPLICIT PERFORMANCE AND PERCEPTION

Treatments such as Non-Contact Therapeutic Touch are based on the hypothesis that the hands can send and receive energy and information. From a dynamical energy systems perspective, the hands can be viewed as rich generators of complex patterns of biophysical energy and information, including electrostatic movement effects, electromagnetic potentials (muscular, cardiac, skin potentials, etc.), and temperature (e.g. infrared). Two experiments (n = 20 and n = 41) were conducted to determine whether blindfolded subjects could detect the presence of an menter's hand placed a few inches above one of their hands. A 2 x 2 within subject counterbalanced design (left and right subject's hands by left and right experimenter's hands) was employed. The average correct detections was 66% (chance = 50%, P < .00001). Subject's average estimate of their performance was 54%. Poor and low performance subjects correctly estimated their actual performance, whereas medium and high performance subjects seriously underestimated their performance. However, even for subjects who performed at or below chance, their ratings of confidence per trial were significantly higher for correct versus incorrect trials, suggesting that they had implicit perception of hand-energy. Subjects who spontaneously reported some awareness of temperature (but not tingling or pressure) reported higher overall estimates of performance. Implications for future research on interpersonal hand-energy registration are considered

The probability density function tail of the Kardar-Parisi-Zhang equation in the strongly non-linear regime

An analytical derivation of the probability density function (PDF) tail describing the strongly correlated interface growth governed by the nonlinear Kardar-Parisi-Zhang equation is provided. The PDF tail exactly coincides with a Tracy-Widom distribution i.e. a PDF tail proportional to $\exp( - c w_2^{3/2})$, where $w_2$ is the the width of the interface. The PDF tail is computed by the instanton method in the strongly non-linear regime within the Martin-Siggia-Rose framework using a careful treatment of the non-linear interactions. In addition, the effect of spatial dimensions on the PDF tail scaling is discussed. This gives a novel approach to understand the rightmost PDF tail of the interface width distribution and the analysis suggests that there is no upper critical dimension.Comment: 17 pages, 2 figure

Isolated bladder metastasis causing large bowel obstruction: a case report of an atypical presentation of intussusception

Intussusception of the large bowel is a rare clinical entity. In adults, this pathology is usually associated with a malignant lead point and often requires operative management. Reported is the case of an 83-year-old female who was recently diagnosed with superficial bladder cancer (T1) treated by partial cystectomy. She presented 3 months post-operatively with an isolated mucosal metastasis of the transverse colon causing intussusception and large bowel obstruction. The patient was successfully treated by colonic resection with primary anastomosis. Histology was significant for a pedunculated sarcomatoid bladder carcinoma originating from the colonic mucosa with incomplete invasion of the bowel wall. An isolated mucosal metastasis of this variety has not been reported in the literature to date