A far-red fluorescent protein evolved from a cyanobacterial phycobiliprotein.

Far-red fluorescent proteins (FPs) are desirable for in vivo imaging because with these molecules less light is scattered, absorbed, or re-emitted by endogenous biomolecules compared with cyan, green, yellow, and orange FPs. We developed a new class of FP from an allophycocyanin α-subunit (APCα). Native APC requires a lyase to incorporate phycocyanobilin. The evolved FP, which we named small ultra-red FP (smURFP), covalently attaches a biliverdin (BV) chromophore without a lyase, and has 642/670-nm excitation-emission peaks, a large extinction coefficient (180,000 M(-1)cm(-1)) and quantum yield (18%), and photostability comparable to that of eGFP. smURFP has significantly greater BV incorporation rate and protein stability than the bacteriophytochrome (BPH) FPs. Moreover, BV supply is limited by membrane permeability, and smURFPs (but not BPH FPs) can incorporate a more membrane-permeant BV analog, making smURFP fluorescence comparable to that of FPs from jellyfish or coral. A far-red and near-infrared fluorescent cell cycle indicator was created with smURFP and a BPH FP.

Parental Preference Assessment for Vesicoureteral Reflux Management in Children.

PURPOSE: Parents of children with vesicoureteral reflux are presented with a variety of management options, which in many cases offer a similar risk-benefit ratio. To facilitate shared decision making, parental preferences regarding vesicoureteral reflux treatment options need to be acknowledged. We aimed to characterize the clinical experience of parents and elicit core themes affecting decision making in regard to managing vesicoureteral reflux in their child. MATERIALS AND METHODS: A semistructured, qualitative interview script was developed and vetted by 25 pediatric urologists to discuss treatment options for vesicoureteral reflux. Additional patient interviews were conducted until new themes failed to arise. Content analysis was performed to extract all statements that described treatment options. Similar statements were combined until a final list of unique themes emerged. RESULTS: A total of 26 interviews were performed, yielding 689 statements about overall parent experiences with managing vesicoureteral reflux in the child and 450 statements (65%) pertaining to treatment options. Of the 13 themes that emerged, those most commonly considered were the prevention of future urinary tract infections by 85% of parents, the efficacy rate of treatment options by 85%, the burden of daily maintenance or compliance by 77%, antibiotic resistance by 69%, chronic kidney damage by 62% and invasiveness by 58%. CONCLUSIONS: Our study emphasizes that when choosing a treatment option for vesicoureteral reflux in their child, parent preferences regarding risks and benefits are variable. However, their chief concerns include whether a method decreases the risk of urinary tract infections, has an acceptable efficacy rate and aligns itself with the capabilities of the family. These themes help frame discussions between families and clinicians regarding vesicoureteral reflux management, and they can facilitate shared decision making.

Directed Evolution of Fluorescent Proteins in Bacteria.

Directed evolution has revolutionized the way scientists create new biomolecules not found in nature. Error-prone polymerase chain reaction (PCR) introduces random mutations and was used to evolve jellyfish and coral fluorescent proteins in bacteria. We describe a novel method for the directed evolution of a far-red fluorescent protein in E. coli. The new method used genes to produce fluorophores inside E. coli and allowed changing the native fluorophore, phycocyanobilin, for a second small-molecule fluorophore, biliverdin. The directed evolution blueshifted the fluorescence, which enhanced the quantum yield to produce a brighter fluorescent protein. Finally, the evolution selected fluorescent proteins that expressed in large quantities in E. coli. The evolved fluorescent protein was named the small ultra-red fluorescent protein (smURFP) and was biophysically as bright as the enhanced green fluorescent protein (EGFP). This chapter describes the materials and methods used to evolve a far-red fluorescent protein in bacteria. While the focus is a fluorescent protein, the protocol is adaptable for the evolution of other biomolecules in bacteria when using a proper selection strategy.

Structural and photophysical characterization of the small ultra-red fluorescent protein.

The small Ultra-Red Fluorescent Protein (smURFP) represents a new class of fluorescent protein with exceptional photostability and brightness derived from allophycocyanin in a previous directed evolution. Here, we report the smURFP crystal structure to better understand properties and enable further engineering of improved variants. We compare this structure to the structures of allophycocyanin and smURFP mutants to identify the structural origins of the molecular brightness. We then use a structure-guided approach to develop monomeric smURFP variants that fluoresce with phycocyanobilin but not biliverdin. Furthermore, we measure smURFP photophysical properties necessary for advanced imaging modalities, such as those relevant for two-photon, fluorescence lifetime, and single-molecule imaging. We observe that smURFP has the largest two-photon cross-section measured for a fluorescent protein, and that it produces more photons than organic dyes. Altogether, this study expands our understanding of the smURFP, which will inform future engineering toward optimal FPs compatible with whole organism studies.

Magnetic Resonance Imaging-Ultrasound Fusion Biopsy During Prostate Cancer Active Surveillance.

BACKGROUND: Fusion biopsy using multiparametric magnetic resonance imaging (MRI) and transrectal ultrasound has demonstrated favorable detection rates of high-grade prostate cancer (PCa) among previously undiagnosed men. However, the diagnostic yield among men with active surveillance (AS) remains undefined. OBJECTIVE: To determine the utility of MRI-ultrasound fusion biopsy during AS by reporting rates of PCa upgrading and comparing findings with systematic biopsy. DESIGN, SETTING, AND PARTICIPANTS: We identified patients with low- and intermediate-risk PCa enrolled in AS who received MRI-ultrasound fusion surveillance biopsies. All completed prostate multiparametric MRI with 3-T and endorectal coil reviewed by radiologists selecting regions of interest, and all underwent MRI-ultrasound fusion biopsy with concurrent systematic biopsy. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: We report MRI-ultrasound fusion biopsy findings, rates of Gleason score (GS) upgrading to ≥3 + 4 (any upgrading) and to ≥4 + 3 (major upgrading), tumor involvement estimates using descriptive statistics, McNemar's test of symmetry, and multivariate logistic regression. RESULTS AND LIMITATIONS: Overall, 207 men underwent MRI-ultrasound fusion biopsy following radiologic suspicion on multiparametric MRI and met inclusion criteria. Agreement between systematic and MRI-ultrasound fusion biopsy GS was borderline statistically significant (p<0.047). In total, 83 men (40%) experienced any upgrading, including 49 (24%) on systematic sampling, 30 (14%) on MRI-targeted cores, and four (2%) on both. Among those with negative results on MRI-ultrasound fusion biopsy, seven (9%) exhibited major upgrading with systematic biopsy. MRI suspicion scores were high (4/5) for all but two patients with any upgrading and for all who experienced major upgrading. On multivariate analysis, older age was associated with higher odds of any upgrading for men with GS ≤3 + 3 on previous biopsy (odds ratio: 1.10; 95% confidence interval, 1.01-1.20; p=0.03). CONCLUSIONS: MRI-ultrasound fusion biopsy resulted in upgrading otherwise undetected by systematic biopsy among a proportion of men with PCa managed with AS. However, upgrading also occurred in areas outside targeted biopsy, suggesting that systematic sampling should be offered to men with AS even with history of extended sextant biopsy. PATIENT SUMMARY: This study examined the role of magnetic resonance imaging (MRI)-ultrasound fusion biopsy for men with prostate cancer managed with active surveillance (AS). In some patients, MRI-ultrasound fusion biopsy resulted in the detection of upgrade otherwise missed with systematic sampling. The findings indicate that MRI-ultrasound fusion biopsy may help with better sampling during AS.