Biomechanical characterization of human tissue is of increasing importance as new technologies gain importance in modern medicine. Without haptical sensation, measurement of forces associated with surgical techniques will be the primary source of feedback in technology assisted procedures (eg. robotic surgery). An important biomechanical parameter is the suture pullout force (SPOF), the maximum safe force that could be applied to a suture before tearing the tissue.The aim of this study is to analyze suture pullout forces of the pancreatic duct. This data could be used for a wide range of applications and will be useful in understanding how biomechanical properties of the biliary tract change with age, sex, and BMI. Donated organs used in this study were tested within 72 hours of death. Cross sections of the pancreas were prepared and 4-0 sutures were looped through one side of the pancreatic duct wall. The pancreas was held in place while the suture was pulled in tension. The tensile force was measured continuously and the peak force before specimen failure was taken as the SPOF. 103 suture pullout tests were performed on pancreatic ducts from 14 donors. The overall SPOF is 2.87 N ± 1.36 N. The mean SPOF for females is 2.12 N and 3.03 N for males (p=0.019). The SPOF of pancreatic ducts from donors with BMI less than 26 averaged 3.22 N while SPOF for donors with BMI greater than 26 averaged 2.51 N (p=0.045). Donors older than 35 averaged 2.69 N while donors younger than 35 averaged 2.87 N (p=0.123). Preliminary results suggest male pancreatic ducts have a higher SPOF than female pancreatic ducts. As BMI increases, the pancreatic duct SPOF decreases. The effect of age is inconclusive at this time.

 Iaccarino et al exposed one hour of light flickering at 40Hz to awake 5XFAD Alzheimer's Disease (AD) mouse models, consequently generating action potentials at 40 Hz and activating mircoglia. Consequent colocalization of microglia with Aß plaque actuely and clearing of Aß plaque after seven days was observed, but only in the visual cortex. We hypothesized transcranially delivered, near diagnostic ultrasound (tnDU) can replicate the results of Iaccarino et al but throughout its area of application, thus not limited to the visual cortex. We exposed sedated 5XFAD mice to tnDU at 40Hz with 400microsecond-long pulses for one hour, targeting one hemisphere of brain centered on its hippocampus. Chronic studies targeted comparable brain in each hemisphere for one hour/day for five days. Histology and EEG recoding revealed acute application of tnDU activated more microglia that colocalized with Aß plaque, relative to the contralateral hemisphere of treated brain with brain activation at 40Hz. Chronic application reduced their Aß plaque burden by nearly half relative to paired sham animals. Our results compare to those of Iaccarino et al but throughout the area of ultrasound-exposed brain. Our results also compare to those achieved by medications that target Aß for a substantially shorter period of time. The proximity of our ultrasound protocol to those shown as safe for non-human primates and humans may motivate its rapid translation to human studies. 

The field of regenerative medicine is approaching the goal of using stem cell therapy to replace part of an organ that has been damaged irreversibly. Our laboratory differentiates kidney organoids, 3D multicellular structures that functionally and compositionally resemble the respective organ they model, from human iPSCs (induced pluripotent stem cells). However, organoids we work with are largely avascular, whilst organs in vivo are highly vascularized. Our goal for this project is to build a microfluidic, vascular platform in which organoids can grow. To accomplish this, we adopted a microfluidic chip which was fabricated using soft lithography. Consequently, PDMS (polydimethylsiloxane), a polymer commonly used in soft lithography, was molded and bound to a glass coverslip using plasma binding. With this platform, we successfully engineered microvascular networks through vasculogenesis and angiogenesis and optimized the protocol of vascularization to sustain the cells by submerging the microfluidic chip in cell culture medium. Human umbilical vein endothelial cells and human lung fibroblasts were suspended in fibrinogen ECM (extracellular matrix), seeded into the microfluidic chips with micropillars to contain the cells within their respective channels, and developed into 3D microvascular networks with visible lumen. We then stained the vasculature with endothelial cell markers (i.e. CD31, CD54, VWF) and tested the perfusability by flowing polysterene beads through the microfluidic chip, observing the retention of polysterene beads within the vessels. Finally, we altered our design, specifically the height and width of the channels, to incorporate kidney organoids. Currently, we are using this platform for vascularizing kidney organoids and simultaneously implementing a flow system to induce shear stress on the microvasculature to attain physiological parameters. Ultimately, we aim to vascularize a kidney organoid to demonstrate the vascularization of stem cell tissue in vitro and see growth of tissue within our system, which would further our process in the translation pathway from bench to bedside for kidney regenerative medicine.

In the US, the number of children with obesity has reached a staggering 13.7 million. Though there are diets to assist weight loss, recent research suggests a neurobiological basis of obesity specifically related to the mediobasal hypothalamus (MBH), a critical brain structure involved in energy homeostasis, metabolism, and appetite. However, proliferation of hypothalamic gliosis, a cellular inflammatory response, disrupts the function and is shown, in rodents, as a key component in diet-induced obesity. Further conclusions reveal that highly caloric and high-fat diets, in rodents, can cause MBH gliosis. This project seeks to investigate the relationship between diet and hypothalamic gliosis in children, the latter assessed by magnetic resonance imaging (MRI). We expect children with an unhealthy diet to have an increased BMI z-score and greater evidence of MBH gliosis. Participants (N=192) were recruited as part of the longitudinal NIH Adolescent Brain Cognitive Development study. Anthropometric and demographic data were collected along with brain MRI T2-weighted images at the baseline visit. MBH gliosis was measured by using the signal ratio for T2 intensity of the mean bilateral MBH/Amygdala signal ratio; Putamen/Amygdala was used as control ratio. At the one year follow-up, the child’s habitual diet in the past year was assessed using a parent-report food frequency questionnaire. Higher total points represented a healthier overall diet. Additionally, follow-up anthropometric data was obtained to determine the child’s adiposity change over time. At baseline, mean age was 9.9±0.6 and 49% were males. Mean BMI z-score was 0.76±1.05, 19% were overweight and 23% with obesity. Preliminary results in a subset of participants (N=60) revealed a trend for an association between an unhealthy diet and evidence of MBH gliosis (t=1.59, P=0.117). By emphasizing the neurobiological basis of obesity, potential insights can inform targeted diet-related treatments of childhood obesity; thus, furthering the understanding of child obesity pathogenesis.

Obesity is an ever-expanding health epidemic. It is estimated that twenty-eight million US citizens die every year from obesity related diseases. We have previously shown that rapamycin, an inhibitor of mTOR, prevents diet-induced obesity in adult mice. Our results suggest that rapamycin may be increasing the activity of C/EBP-β, a transcription factor involved in regulating the expression of several metabolic genes. Adefovir dipivoxil is an anti-retroviral drug that increases the activity of C/EBP-β in vitro. In order to determine whether activation of C/EBP-β was responsible for the resistance to diet-induced obesity in the presence of rapamycin, we conducted a six-week long experiment with mice given either a 60% kcal/fat or a 10% kcal/fat diet. We used two ways of administering doses of adefovir dipivoxil. We injected it daily or added it to the food supply. This was done to test its effectiveness in preventing diet-induced obesity and activating C/EBP-β. Our results show that the mice given adefovir dipivoxil have a lower body weight and a smaller amount of fat gain compared to the untreated mice. Livers of mice treated with adefovir dipivoxil show increased activation of C/EBP-β, similar to our previous results with rapamycin. Additionally, adefovir dipivoxil and rapamycin decrease fatty acid synthesis and increase beta-oxidation in vivo. The levels of proteins involved in fatty acid oxidation, such as Carnitine palmitoyl transferase I and Long-chain-fatty-acid—CoA ligase 1, are increased. Conversely, Acetyl-CoA carboxylase is heavily phosphorylated, consistent with decreased fatty acid synthesis. We are planning to determine the mechanisms of action of adefovir dipivoxil because of its effects similar to rapamycin against diet-induced obesity. Considering that rapamycin increases lifespan in multiple model organisms, further research could determine whether adefovir dipivoxil has similar effects on longevity.

Children have more exposure to screen media today than any other generation. In the U.S., rates of obesity in children have also tripled in 4 decades. A disruption in the brain called gliosis has been described to participate in obesity pathophysiology if it occurs in a brain region important for energy balance, called the mediobasal hypothalamus (MBH). The objective of this study is to explore possible relationships between MBH gliosis and parents’ reports on screen time their child engages in. This study will also look into variables that may mediate these relationships, such as body adiposity and impulsivity. I hypothesize that there will be a positive correlation between screen media use and MBH gliosis with obesity and the level of impulsivity being positively correlated to both gliosis and screen time. The methodology consists of data from participants (N = 192) in the NIH Adolescent Brain Cognitive Development study. The NIH Toolbox Flanker Inhibitory Control and Attention Test was used to measure child’s impulsivity, and the parents were given the Screen time report to measure the average daily screen media time of each child. Along with these measures, participant’s age, sex, race, BMI z-score, waist/height ratio, and T2-weighted MRI will be included. MBH gliosis will be measured by T2 MRI signal intensity (brightness) of the mean bilateral MBH/Amygdala signal ratio; Putamen/Amygdala will be used as control ratio. Participants were 51% female with an average age of 9.9±0.63 years. Within our sample, 57.3%, 19.3%, and 23.4% of subjects were considered healthy weight, overweight, and with obesity, respectively. Anthropometric and behavioral data recorded on average 11.2 months after the initial tests will be included to calculate changes over time. This research will help to further illuminate the relationships between screen time and obesity, and will potentially be helpful in proposing new treatments for children with obesity.

The blood-brain barrier (BBB) is a layer of tight-junction endothelial cells that make up the capillaries in the brain and strictly regulate what molecules can pass from the blood into the brain. Many molecules, including insulin, cannot passively cross this barrier but require an active transport system at the surface of the BBB. Once in the brain, insulin plays a role in memory and cognition. Indeed, Alzheimer’s disease is characterized by decreased sensitivity to insulin, which could be explained by a malfunctioning insulin receptor (IR) or impaired transport at the BBB. However, before we can begin to investigate the IR under disease conditions, we must first understand its standard regulation and function in a healthy system. Specifically, we aim to determine what factors mediate the endocytosis of insulin into the endothelial cells of the BBB. To do this, we focused on clathrin and caveolin, two proteins involved in different endocytic pathways. We performed cardiac perfusions on mice, where we first administered a drug to inhibit either clathrin or caveolin, and then we perfused with radiolabeled insulin. Afterwards, brains were collected and dissected into regions. Radioactivity was measured in the hypothalamus, olfactory bulbs, and whole brain, and the data was graphed over time to determine if there were changes in insulin binding or transport rates. Our results help elucidate the molecular processes necessary for insulin transport and binding at the BBB, which can ultimately help us understand how IR uptake and insulin transport may go awry in Alzheimer’s disease.

Chronic Kidney Disease of Unknown Etiology (CKDu) is a disease that impairs kidney function in individuals without the presence of diabetes or hypertension- common causes of Chronic Kidney Disease. Epidemiological studies find that CKDu is common among residents of agricultural communities in warm climates like Sri Lanka and Central America. In addition to heat stress, other hypothesized contributing factors to CKDu include heavy metals, agrochemicals, and food contamination. Ochratoxin A (OTA), a fungal mycotoxin commonly present in 3rd world food supplies, is cited as a potential contributing factor in CKDu. OTA is present in blood and/or urine of individuals with CKDu and OTA induces acute nephrotoxicity in animal species. To investigate whether heat stress and OTA synergistically increase the risk of CKDu, we conducted a 2D in-vitro study exposing primary human proximal tubule epithelial cells (PTEC) to combinations of OTA (0-10 µM), heat stress (24 hr. at 39℃) and 2,4- Dinitrophenol (DNP-10 µM). DNP is a mitochondrial uncoupling agent that decreases the production of ATP, resulting in energy release as heat. The effects of heat and DNP on the OTA treated cells were measured using a MTT assay to assess cell viability after the treatment period of two and four days. Based on the findings to date, there is evidence of nephrotoxicity because of treatment, but the question of whether or not heat exacerbates damage to the cells after two and four days of treatment remains unclear. The next steps will be to investigate the mechanism(s) of cellular toxicity using RNAseq to assess global changes in gene expression and to quantitate protein biomarkers of nephrotoxicity. This will allow us to delineate the effect(s) of heat stress and OTA as single agents versus the synergistic effects of heat stress/OTA as contributing factors in development of CKDu.