Publications by Year: 2020

BACKGROUND: The majority of eukaryotic promoters utilize multiple transcription start sites (TSSs). How multiple TSSs are specified at individual promoters across eukaryotes is not understood for most species. In Saccharomyces cerevisiae, a pre-initiation complex (PIC) comprised of Pol II and conserved general transcription factors (GTFs) assembles and opens DNA upstream of TSSs. Evidence from model promoters indicates that the PIC scans from upstream to downstream to identify TSSs. Prior results suggest that TSS distributions at promoters where scanning occurs shift in a polar fashion upon alteration in Pol II catalytic activity or GTF function.

RESULTS: To determine the extent of promoter scanning across promoter classes in S. cerevisiae, we perturb Pol II catalytic activity and GTF function and analyze their effects on TSS usage genome-wide. We find that alterations to Pol II, TFIIB, or TFIIF function widely alter the initiation landscape consistent with promoter scanning operating at all yeast promoters, regardless of promoter class. Promoter architecture, however, can determine the extent of promoter sensitivity to altered Pol II activity in ways that are predicted by a scanning model.

CONCLUSIONS: Our observations coupled with previous data validate key predictions of the scanning model for Pol II initiation in yeast, which we term the shooting gallery. In this model, Pol II catalytic activity and the rate and processivity of Pol II scanning together with promoter sequence determine the distribution of TSSs and their usage.

OBJECTIVES: To analyze factors during early stage of urinary tract infection (UTI) that are associated with development of chronic UTI.

METHODS: Mice were inoculated with Uropathogenic Escherichia coli (UPEC) 2 times 24 hours apart. At 1, 3, 7, 10, 14, 21 and 28 days post infection (dpi), urine bacterial loads and voiding behavior (voiding spot assay, VSA) were measured. At 1 and 28 dpi, 32 urine inflammatory cytokines/chemokines were measured using enzyme-linked immunosorbent assay (ELISA). Bladder and kidney cytokines/chemokines were measured on 28 dpi. Mice that had no more than 1 episode of urine bacterial load < 104 colony forming unit/ml during the entire 4 weeks were defined as susceptible to chronic UTI, otherwise, mice were considered resistant.

RESULTS: At 28 dpi, 64.3% mice developed chronic UTI (susceptible group) and 35.7% mice did not (resistant group). Factors at 1 dpi that were predictive of chronic UTI included increased urine IL-2 (OR 11.9, 95%CI 1.1-130.8, P = .043) and increased urine IL-10 (OR 14.0, 95%CI 1.0-201.2, P = .052). At 28 dpi, there were several significant differences between the susceptible vs resistant groups including urine/tissue bacterial loads and certain urine/tissue cytokines/chemokines.

CONCLUSIONS: Higher urine IL-2 and IL-10 at 1 dpi predicted chronic UTI infection in this model. There have been recent publications associating both of these cytokines to UTI susceptibility. Further explorations into IL-2 and IL-10 mediated pathways could shed light on the biology of recurrent and chronic UTI which are difficult to treat.

Diabetic bladder dysfunction is a frequent complication of diabetes. Although many mouse models of diabetes now exist, there has been little systematic effort to characterize them for the timing of onset and severity of bladder dysfunction. We monitored metabolic status and tested bladder function by void spot assay and limited anesthetized cystometry in both male and female mice of three models of obesity and diabetes: a type 1 diabetes model (the Akita mouse) and two type 2 diabetes models [the diet-induced obese (DIO) model and the ob/ob mouse]. Akita mice had insulin pellets implanted subcutaneously every 3 mo to mimic poorly controlled type 1 diabetes in humans. Mice were hyperglycemic by 48 days after implants. Female mice exhibited no bladder dysfunction at any age up to 20 mo and gained weight normally. In contrast, by 7 mo, male Akita mice developed a profound polyuria and failed to show normal weight gain. There were no observable signs of bladder dysfunction in either sex. DIO mice on high/low-fat diets for 16 mo exhibited mild hyperglycemia in female mice (not in male mice), mild weight gain, and no evidence of bladder dysfunction. Ob/ob mice were followed for 8 mo and became extremely obese. Male and female mice were glucose intolerant, insulin intolerant, and hyperinsulinemic at 4 mo. By 8 mo, their metabolic status had improved but was still abnormal. Urine volume increased in male mice but not in female mice. Bladder dysfunction was observed in the spotting patterns of female mice at 4 and 6 mo of age, resolving by 8 mo. We conclude there are dramatic sex-related differences in lower urinary tract function in these models. Male Akita mice may be a good model for polyuria-related bladder remodeling, whereas female ob/ob mice may better mimic storage problems related to loss of outlet control in a setting of type 2 diabetes complicated by obesity.

BACKGROUND: Our understanding of the neural circuits controlling micturition and continence is constrained by a paucity of techniques for measuring voiding in awake, behaving mice.

NEW METHOD: To facilitate progress in this area, we developed a new, non-invasive assay, micturition video thermography (MVT), using a down-facing thermal camera above mice on a filter paper floor.

RESULTS: Most C57B6/J mice void infrequently, with a stereotyped behavioral sequence, and usually in a corner. The timing of each void is indicated by the warm thermal contrast of freshly voided urine. Over the following 10-15 min, urine cools to ∼3 °C below the ambient temperature and spreads radially in the filter paper. By measuring the area of cool contrast comprising this "thermal void spot," we can derive the initially voided volume. Thermal videos also reveal mouse behaviors including a home-corner preference apart from void spots, and a stereotyped, seconds-long pause while voiding.

COMPARISON WITH EXISTING METHODS AND CONCLUSIONS: MVT is a robust, non-invasive method for measuring the timing, volume, and location of voiding. It improves on an existing technique, the void spot assay, by adding timing information, and unlike the cystometrogram preparation, MVT does not require surgical catheterization. Combining MVT with current neuroscience techniques will improve our understanding of the neural circuits that control continence, which is important for addressing the growing number of patients with urinary incontinence as the population ages.

The general anesthetic ketamine has been repurposed by physicians as an anti-depressant and by the public as a recreational drug. However, ketamine use can cause extensive pathological changes, including ketamine cystitis. The mechanisms of ketamine's anti-depressant and adverse effects remain poorly understood. Here we present evidence that ketamine is an effective L-type Ca2+ channel (Cav1.2) antagonist that directly inhibits calcium influx and smooth muscle contractility, leading to voiding dysfunction. Ketamine prevents Cav1.2-mediated induction of immediate early genes and transcription factors, and inactivation of Cav1.2 in smooth muscle mimics the ketamine cystitis phenotype. Our results demonstrate that ketamine inhibition of Cav1.2 signaling is an important pathway mediating ketamine cystitis. In contrast, Cav1.2 agonist Bay k8644 abrogates ketamine-induced smooth muscle dysfunction. Indeed, Cav1.2 activation by Bay k8644 decreases voiding frequency while increasing void volume, indicating Cav1.2 agonists might be effective drugs for treatment of bladder dysfunction.