UCSF

Many mechanisms govern both how animals respond to stimuli and how these responses inform physical state and future behavior. In C. elegans, these mechanisms include the generation and spread of various species of small RNAs. Emerging as a potent regulator of gene expression in both a single animal and its progeny, small RNAs are reshaping the fields of behavior and transgenerational inheritance. The rising prominence of small RNA within the context of these fields highlights gaps in the knowledge of essential processes, such as the precise mechanisms of small RNA export. RNA import, thanks to earlier efforts characterizing systemic RNA interference (RNAi), are relatively well understood. However, much less is known about how RNAi and other small RNA exit cells. This manuscript details tools and approaches for identifying mobile small RNAs, members of RNA export pathways, and points of regulation in the export process.

CRISPR/Cas9-based functional genomics have transformed our ability to elucidate mammalian cell biology. However, most previous CRISPR-based screens were conducted in cancer cell lines rather than healthy, differentiated cells such as neurons. Neurons possess unique cell biological properties that enable them to exert highly specialized physiological functions. To enable systematic characterization of neuronal cell biology, we established CRISPR interference (CRISPRi)- and CRISPR activation (CRISPRa)-based platforms in human neurons derived from induced pluripotent stem cells (iPSCs) that allow robust repression or activation of endogenous genes and large-scale genetic screens in human neurons. Using this toolkit, we performed multiple genome-wide screens to identity genes controlling neuronal survival under unstressed and oxidative stress conditions and genes regulating neuronal redox homeostasis. These screens provide numerous novel biological insights. We also demonstrate that our platforms can be coupled with single-cell RNA sequencing and longitudinal high-content imaging to reveal consequences of genetic perturbations on gene expression and neuronal morphology respectively. Our results highlight the power of unbiased genetic screens in iPSC-derived differentiated cell types and provide a platform for systematic interrogation of normal and disease states of neurons.

Purpose: Chemotherapy-induced diarrhea (CID) is a common symptom that occurs in 50% to 80% of patients. Given that the majority of the data on the occurrence and severity of CID is based on physician-rated toxicity criteria, this study’s purposes were to: identify subgroups of patients with distinct CID profiles and determine how these subgroups differ in terms of demographic and clinical characteristics; severity, frequency, and distress of CID; the co-occurrence of common GI symptoms, and QOL.

Methods: Patients (n=1133) completed the Memorial Symptom Assessment Scale six times over two cycles of chemotherapy (CTX). Latent profile analysis was used to identify subgroups of patients with distinct diarrhea profiles. Differences among these subgroups were evaluated using parametric and nonparametric statistics.

Results: Four distinct diarrhea profiles were identified: none (58.3%), decreasing (22.0%), increasing (5.2%), and high (14.5%). Compared to the none class, patients in the high class had a lower functional status, a worse comorbidity profile, were more likely to have gastrointestinal cancer, and were more likely to receive CTX on a 14 day cycle. No differences were found among the classes in the percentages of patients who received CTX with a targeted therapy.

Conclusion: Given that CID occurred in over 40% of the patients, clinicians should assess for this symptom and other common GI symptoms and initiate appropriate pharmacologic and dietary interventions.

Abstract

Background: During alarm fatigue, true alarms can go unnoticed placing patients at risk for untoward outcomes. Patients with a left ventricular assist device (LVAD) may create challenges during electrocardiographic (ECG) monitoring due to technical alarms (i.e., artifact, ECG leads off), noise and vibrations associated with LVADs, and being able to tolerate some arrythmias. Clinical Nurse Specialists play a central role in and developing evidenced based strategies to improve alarm safety with the ultimate goal of improving patient outcomes. Purpose/Aim: In this case series, we analyze three patients being treated with an LVAD device in the cardiac intensive care unit (ICU) and determine: 1) the number and type of audible arrhythmia alarms; 2) the number of true versus false arrhythmias; 3) the number, type and duration of technical alarms; and 4) report alarm burden. Methods: Secondary analysis using data from the University of California, San Francisco (UCSF) Alarm Study. Results: There were a total of 547 arrhythmia alarms and 98% were false. There were 25,232 technical alarms. Of 514 total hours of ECG monitoring, technical alarms occurred for 65.9 (13%) hours. Alarm burden of 50.15 alarms per monitored hour in the ICU. Conclusion: Audible arrhythmia alarms are common in LVAD patients, and the vast majority are false. Importantly, none of the arrhythmia alarms led to an untoward event (i.e., code blue or death). Technical alarms are also very common and occur for hours during routine ECG monitoring. Continuous ECG monitoring creates unique challenges in LVAD patients. Future studies are needed to explore strategies, both clinical and algorithm bases, to improve the accuracy of arrhythmia detection and minimize technical alarms in LVAD patients.

Introduction: Dental implants have become a popular choice for replacing missing teeth. It is estimated that every year, over two million implants are placed throughout the world. However, dental implants may exhibit immediate or delayed complications that could lead to implant failure. The aim of this study was to explore the factors coincidental with removal of failed implants at the UCSF Postgraduate Periodontology Clinic (PGPC) by reviewing electronic health records (EHR) for the last 19 years.

Methods: EHR data from Axium were searched for the dental code D6100 (implant removal) from January 2001 – April 2019. Furthermore, code D7140 (simple tooth removal) was used to include cases that were suspected to be misclassified. Inclusion criteria was focused on patients who had dental implant(s) removed and had sufficient records for analysis. Patient and surgical data were gathered from dental records and analyzed. Exclusion criteria focused on patient data that was incomplete or not accessible.

Results: Upon review of 3,426 simple tooth extractions and 81 implant explantations, it was determined that 114 implants were explanted from 88 patients over 19 years. Fifteen patients were removed from the data analysis based on exclusion criteria. Analysis was performed on 73 patients who had a total of 96 implants removed. Of these, 59% of patients had periodontal disease and 32% were smokers. Amongst the medical conditions reviewed, 32% of patients reported cardiovascular disease, 23% had a bone disorder, and 18% had emotional disorders. In terms of the implants, 41 implants were placed in grafted bone; 54% being allograft. Out of 96 implants removed, 58 were late failures and 33 implants failed before loading. Furthermore, we examined the reasons for tooth extractions at sites with subsequent implant failure and found that 6 teeth had endodontic treatment and 3/6 had periapical abscesses. Forty-five percent of cases that had a late failure did not report regular recall and maintenance appointments.

Conclusion: Implant failure was coincidental with a history of periodontal disease and other risk factors, such as cardiovascular disease and emotional disorders. A lack of consistent maintenance recalls was frequently found in cases with late implant failure. Not every patient is a good candidate for implant therapy and risk factors have to be carefully evaluated and presented to the patient. This research highlights the need for a systematic evaluation of patients planning to receive implants and the need to inform them of possible complications and risks of implant treatment.

Biological macromolecular assemblies play crucial roles in most cellular processes. The determination of their structures, thermodynamics, and kinetics is essential to understand their function, evolution, modulation, and design. Determining such models, however, remains challenging. One particularly powerful approach to constructing models in general is integrative modeling. Integrative modeling aims to maximize the accuracy, precision, and completeness of models, by simultaneously utilizing all available information, including experimental data, physical principles, statistical analyses, and other prior models. The goal of this thesis is to expand the scope of integrative modeling to the inference of spatial, thermodynamic, and kinetic aspects of macromolecular assemblies.

In Chapter I, I introduce the integrative modeling framework for spatiotemporal modeling of biological macromolecular assemblies. In Chapter II, I demonstrate how the synergy between multi-chemistry cross-linking mass spectrometry and integrative modeling can map the structural dynamics of macromolecular assemblies, by application to the human Cop9 signalosome complex. In Chapter III, I present a method for determining structures, free energy differences, and kinetic rates of macromolecular assemblies along their functional cycle, mainly from negative stain electron microscopy (EM). We apply the method to the yeast Hsp90 to estimate the free energy differences and kinetic parameters along its nucleotide hydrolysis cycle, which includes open and closed states of Hsp90. In Chapter IV, I describe a validation of stochastic sampling in integrative modeling. The remaining chapters describe applications of integrative modeling to assemblies of various sizes and scales, using various sources of information, thus illustrating the flexibility of the integrative modeling approach. Specifically, I apply integrative modeling to the human ECM29-Proteasome assembly under oxidative stress (Chapter V), the yeast nuclear pore complex (NPC) cytoplasmic mRNA export platform (Chapter VI), the major membrane ring component of the yeast NPC (Chapter VII), the entire yeast NPC (Chapter VIII), and the reconstruction of 3D structures of MET antibodies (Chapter IX).

IMPORTANCE Time-restricted eating has increased in popularity among people trying to lose weight. However, the efficacy and safety have not been explored in a large, prospective randomized trial.

OBJECTIVE To compare the effects of weight loss in people who are overweight and obese prescribed a time-restricted eating protocol versus standard three meals per day.

DESIGN, SETTING, AND PARTICIPANTS The Time-Restricted Eating (TREAT) study was a 12-week randomized controlled trial of men and women aged 18-64 with a body mass index of 27-43 kg/m2. The study was conducted on a custom mobile study app on the Eureka Research Platform, and participants received a Bluetooth scale for daily use. Participants lived anywhere in the United States, with a subset of participants (n=50) living within 60 miles of San Francisco who underwent comprehensive, in-person metabolic testing.

INTERVENTION Subjects were randomized to one of two eating plans and received daily reminders about their eating windows through the study app. The consistent meal timing (CMT) group was instructed to eat three structured meals per day. The time-restricted eating (TRE) group was instructed to eat ad libitum from 12:00 pm until 8:00 pm and completely abstain from caloric intake from 8:00 pm until 12:00 pm the following day (16h fast:8h eat). Specific recommendations for caloric intake or macronutrient content were not prescribed to either group, and participants were not advised to make any changes to their physical activity.

MAIN OUTCOMES AND MEASURES The primary outcome was weight loss as measured at home on the Bluetooth-connected scale. Secondary outcomes were weight loss in the in-person cohort and changes in fat mass, lean mass, fasting insulin, fasting glucose, HbA1C, total energy expenditure, and resting energy expenditure. Bonferroni-corrected alphas and confidence intervals were used to correct for multiple comparisons for secondary outcomes. All other outcomes were considered exploratory and were not corrected for multiple outcomes.

RESULTS 116 subjects (mean [SD] age, 46.5 [10.5] years), participated in the study. There was a significant decrease in weight in the TRE (-0.94kg, 95% CI, -1.68, -0.20, p=0.013), but no significant change in the CMT group (-0.68kg, 95% CI, -1.41, 0.05, p=0.07) or between groups (-0.26kg, 95% CI, -1.30, 0.78, p=0.63). In the in-person cohort, (n=25 TRE, n=25 CMT), there was a significant within-group decrease in weight in the TRE group (-1.70kg, 95% CI, -2.56, -0.83, p<0.001), but no significant changes in any of the other secondary outcomes. There was a significant within-group decrease in lean mass in the TRE group (-1.10kg, 95% CI, -1.73, -0.48, p<0.001). There was also a significant difference in appendicular lean mass index between groups (-0.162kg/m2, 95% CI, -0.274, -0.050, p=0.005).

CONCLUSIONS AND RELEVANCE Prescribing TRE to people who are overweight and obese led to modest weight loss, but there was no difference in weight loss when compared to those randomized to CMT. Moreover, the majority of weight lost in people randomized to TRE came from loss of lean mass rather than fat mass. The mechanism(s) underlying this phenomenon is unknown, and future studies will be aimed at exploring the relationship between fasting and lean body mass.

Limited information exists on the effects of self-reported exercise on subjective and objective measures of chemotherapy-induced peripheral neuropathy (CIPN). In this study, we evaluated for differences in demographic and clinical characteristics, as well as subjective and objective measures of CIPN and balance among 290 cancer survivors with CIPN who were classified into one of three exercise (Ex) groups (i.e., NoEx, LessEx, RecEX) based on the recommended level of >150 minutes per week. Survivors completed self-report questionnaires and underwent a sensory examination and balance testing. Compared to RecEx group (34.8%), survivors in the NoEx group (20.7%) had less education, were less likely to be married/partnered, had a lower household income, a higher level of comorbidity, and a poorer functional status. No differences were found among the three exercise groups in duration of CIPN, pain intensity scores, or changes in light touch, cold and pain sensations. However, compared to the RecEx group, survivors in the NoEx group had higher vibratory thresholds and worse scores on objective measures of balance. Based on our “real world” findings, clinicians can recommend walking as a therapeutic option, as well as referrals to physical therapy for additional balance and strength training in survivors with CIPN.

Non-invasive techniques to assess metabolic reprogramming during cancer progression can be used to improve therapeutic selection and provide an early assessment of therapeutic response or resistance in individual patients. Prior studies have shown that metabolic reprogramming plays a key role in the development of prostate cancer and renal cell carcinoma (RCC). This dissertation further elucidates the metabolic alterations that occur in treatment-resistant prostate cancer and in patient-derived models of RCC using high-resolution nuclear magnetic resonance (NMR) spectroscopy and hyperpolarized (HP) 13C magnetic resonance imaging (MRI), with the goal of identifying new non-invasive diagnostic imaging tools. Glycolysis, metabolism of pyruvate and glutamate via the tricarboxylic acid (TCA) cycle, glutaminolysis, and glutathione synthesis are upregulated in castration-resistant prostate cancer (CRPC) compared to their androgen-dependent counterparts, using human cell lines as well a treatment-driven transgenic murine model. These metabolic alterations were reversed in castration-resistant murine tumors by treatment with a secondary androgen pathway inhibitor, apalutamide, suggesting that early metabolic responses to treatment can be monitored using non-invasive imaging techniques. Furthermore, treatment-emergent small cell neuroendocrine prostate cancer, a consequence of protracted treatment with primary androgen deprivation therapy and secondary androgen pathway inhibitors, exhibits significantly upregulated glycolysis, TCA cycle metabolism of pyruvate and glutamate, and glutaminolysis, as well as significantly altered redox capacity compared to castration-resistant prostate adenocarcinoma using patient-derived xenograft models. Finally, the metabolic differences associated with the tumor microenvironment were compared between various patient-derived models of RCC, finding that RCC patient-derived xenografts (PDXs) displayed higher redox capacity and were more proliferative than cells and tissue slices derived from the PDXs and maintained ex vivo. The work presented in this dissertation suggests that a combination of HP [1-13C]pyruvate, [2-13C]pyruvate, [5-13C]glutamine, and [1-13C]dehydroascorbate can be used to distinguish advanced prostate cancer and RCC subtypes in future HP 13C MRI of patients for improved treatment selection and monitoring.

Objective: This study evaluates the impact of an oral health and nutrition intervention in children aged 2 to 6 years old in Central and South Vietnam. Associations between early childhood caries, child nutritional status, and level of oral health and nutrition education of parents were evaluated longitudinally.

Methods: A total of 593 parent-child pairs were recruited from 5 primary schools in Ho Chi Minh City and Da Nang, Vietnam in 2011. Children ages 2, 3 and 4 from the baseline year in 2011 were used as the comparison group and followed for two consecutive years from 2011 to 2013. The intervention was comprised of oral health and nutrition education, fluoride varnish application, and dental screenings. Parents completed surveys about children’s dietary habits, oral health practices, and dental history. Children received annual anthropometric assessment and dental examinations.

Results: There was a high prevalence of dental caries (58.5% – 84.8%) and untreated decay (89.9% – 97.5%) in this population. The number of treated teeth and reported children who own their own toothbrushes and toothpaste increased during the course of the study. The proportion of parents giving their children soda, juice, and chips increased each year. In addition, the prevalence of overweight/obese children increased each study year.

Conclusion: This study showed that preschool-aged children in urban/suburban Vietnam experienced a high prevalence of early childhood caries and overweight status. As children grew older, they consumed higher amounts of sugar sweetened beverages and junk food. The high proportion of 2-year-olds with tooth decay conveys the need for pre-natal and infant oral health and nutritional education to begin before age 2. Physical exercise should also be promoted in conjunction with a healthier lifestyle. These programs should place emphasis on less consumption of unhealthy foods and accessible dental care for children in order to prevent early childhood caries.