Engineering yeast genomes and populations

The field of synthetic biology seeks to use design principles of life to create new genes, organisms and populations to both better understand biology as well as generate species with useful properties. Budding yeast has been a workhorse for synthetic biology, as well as an important model organism in the broader fields of molecular biology and genetics. This thesis aimed to create genome engineering tools for the manipulation of genomes, with direct applications in yeast. I focused developing high-throughput and highly efficient methods for making genomic modifications in yeast to allow for the generation of large libraries of precisely modified yeast genomes. By manipulation of endogenous DNA recombinases and mismatch repair enzymes in yeast, we were able to develop an oligonucleotide only method for genome engineering to generate libraries as large as 10^5 individuals with a frequency of modification as high as 1%. Additionally, we validated the use of RNA-guided CRISPR/Cas9 endonucleases to make changes in yeast genomes, resulting in frequencies of genome modification >90% in transformed populations. We further optimized this method to generate larger libraries as high as 10^5 individuals and explored a proof of concept epistasis experiment involving thermotolerance. Lastly, the propagation of changes to successive generations is useful when engineering organisms on the population level. To this end we explored the use of RNA-guided gene drives to bias inheritance in S. cerevisiae. We show that inheritance of these selfish elements can be biased to over 99% and is reversible

Optogenetic and pharmacological suppression of spatial clusters of face neurons reveal their causal role in face gender discrimination

Neurons that respond more to images of faces over nonface objects were identified in the inferior temporal (IT) cortex of primates three decades ago. Although it is hypothesized that perceptual discrimination between faces depends on the neural activity of IT subregions enriched with “face neurons,” such a causal link has not been directly established. Here, using optogenetic and pharmacological methods, we reversibly suppressed the neural activity in small subregions of IT cortex of macaque monkeys performing a facial gender-discrimination task. Each type of intervention independently demonstrated that suppression of IT subregions enriched in face neurons induced a contralateral deficit in face gender-discrimination behavior. The same neural suppression of other IT subregions produced no detectable change in behavior. These results establish a causal link between the neural activity in IT face neuron subregions and face gender-discrimination behavior. Also, the demonstration that brief neural suppression of specific spatial subregions of IT induces behavioral effects opens the door for applying the technical advantages of optogenetics to a systematic attack on the causal relationship between IT cortex and high-level visual perception.National Institutes of Health (U.S.) (Grant K99 EY022924)National Institutes of Health (U.S.) (Grant R21 EY023053)National Institutes of Health (U.S.) (Grant R01 EY14970

Clinical significance of ventricular fibrillation-flutter induced by ventricular programmed stimulation

Two hundred twenty-four patients underwent ventricular programmed stimulation (VPS) without prior documentation of the clinical occurrence of sustained ventricular tachycardia (VT) or ventricular fibrillation-flutter (VF). Indications for VPS were: palpitations or nonsustained VT during ambulatory monitoring (85 patients), syncope or presyncope (137 patients), and a family history of sudden death (two patients). Sustained VF requiring transthoracic defibrillation was initiated by VPS in 18 patients (8.0%). Four patients were treated for inducible VF with antiarrhythmic agents directed by electropharmacologic testing; five patients were treated empirically; nine patients received no therapy. No patient has had a cardiac arrest or sudden death during a follow-up period of 25.2 +/- 13.8 months (mean +/- standard deviation). VF was initiated by two ventricular extrastimuli in three patients and by three extrastimuli in 15 patients. The incidence of VF was similar in patients with and without previous symptoms (8.8% vs 6.9%) or heart disease (7.1% vs 9.6%). It was significantly higher when VPS at three ventricular sites with a current of 5 mA (pulse width 2 msec) was compared to programmed stimulation at two ventricular sites with a current twice diastolic threshold (pulse width 2 msec) (15.2% vs 3.0%, p < 0.05). VF initiated by VPS in patients without prior VT or VF appears to be a nonspecific finding. Antiarrhythmic therapy for VF may not be necessary in these patients.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/25695/1/0000249.pd

Safety of procuring research tissue during a clinically indicated kidney biopsy from patients with lupus: data from the Accelerating Medicines Partnership RA/SLE Network

Objectives In lupus nephritis the pathological diagnosis from tissue retrieved during kidney biopsy drives treatment and management. Despite recent approval of new drugs, complete remission rates remain well under aspirational levels, necessitating identification of new therapeutic targets by greater dissection of the pathways to tissue inflammation and injury. This study assessed the safety of kidney biopsies in patients with SLE enrolled in the Accelerating Medicines Partnership, a consortium formed to molecularly deconstruct nephritis.Methods 475 patients with SLE across 15 clinical sites in the USA consented to obtain tissue for research purposes during a clinically indicated kidney biopsy. Adverse events (AEs) were documented for 30 days following the procedure and were determined to be related or unrelated by all site investigators. Serious AEs were defined according to the National Institutes of Health reporting guidelines.Results 34 patients (7.2%) experienced a procedure-related AE: 30 with haematoma, 2 with jets, 1 with pain and 1 with an arteriovenous fistula. Eighteen (3.8%) experienced a serious AE requiring hospitalisation; four patients (0.8%) required a blood transfusion related to the kidney biopsy. At one site where the number of cores retrieved during the biopsy was recorded, the mean was 3.4 for those who experienced a related AE (n=9) and 3.07 for those who did not experience any AE (n=140). All related AEs resolved.Conclusions Procurement of research tissue should be considered feasible, accompanied by a complication risk likely no greater than that incurred for standard clinical purposes. In the quest for targeted treatments personalised based on molecular findings, enhanced diagnostics beyond histology will likely be required