Far-red fluorescent proteins (FPs) are desirable for in vivo imaging because less light is scattered, absorbed, or reemitted by endogenous biomolecules. A new class of FP was developed from an allophycocyanin α-subunit (APCα). Native APC requires a lyase to incorporate phycocyanobilin. The evolved FP, named small Ultra-Red FP (smURFP), covalently attaches biliverdin (BV) without a lyase, and has 642/670 nm excitation/emission peaks, a large extinction coefficient (180,000 M−1cm−1) and quantum yield (18%), and comparable photostability to eGFP. SmURFP has significantly increased BV incorporation rate and protein stability compared to the bacteriophytochrome (BPH) FPs. BV supply is limited by membrane permeability, so expression of heme oxygenase-1 with heme precursors increases fluorescence of BPH/APCα FPs. SmURFP (but not BPH FPs) can incorporate a more membrane-permeant BV analog, making smURFP fluorescence in situ comparable to FPs from jellyfish/coral. A far-red/near-infrared fluorescent cell cycle indicator was created with smURFP and a BPH FP.

Purpose Laparoscopic nephrectomy with autotransplantation is a viable option when renal preservation is required or ureteral reconstruction is impossible. In this study we report on our long-term experience with laparoscopic nephrectomy with autotransplantation. Materials and Methods A retrospective review of data from all patients who underwent laparoscopic nephrectomy with autotransplantation since 2000 revealed data for 52 of 59 patients after study exclusions. Indications for laparoscopic nephrectomy with autotransplantation included ureteral stricture disease (41), renal malignancy (7), ptotic kidney (1), chronic flank pain (1), renal artery aneurysm (1) and renovascular hypertension (1). Followup included ultrasonography, nuclear renography and computerized tomography. Complications analyzed were Clavien-Dindo grade III or higher. Results A total of 52 patients (30 women, 57.6%) underwent laparoscopic nephrectomy with autotransplantation at a median age of 48 years (range 12 to 76). At a median followup of 73.5 months 47 patients (90.3%) had long-term function of the autotransplanted renal unit including 3 of 4 (75%) solitary kidneys. There were 5 patients (9.7%) who experienced renal unit failure at a median of 15 months. Of these patients 3 required nephrectomy of autotransplant unit secondary to renal vein thrombosis (1 day), pseudoaneurysm (15 months) and chronic pain (48 months). Overall 4 patients had early complications and 8 had late complications. In the tumor group 4 patients had disease progression and all are alive. Conclusions Laparoscopic nephrectomy with autotransplantation is an excellent long-term surgical option (greater than 90% success rate with longer than 6-year median followup) for complex ureteral and renal conditions that necessitate preservation of renal parenchyma. However, tumor progression is possible after ex vivo tumor excision. Therefore, careful patient selection and followup are mandatory. This report supports the safety, efficacy and durability of laparoscopic nephrectomy with autotransplantation in experienced hands.

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SummaryThe performance of a clinical urinary test-strip reader Clinitek 200 was evaluated for dog and rat urines, in the context of pre-clinical toxicology studies. No major discrepancies were found between data generated by visual estimation or automatic measurement. Analysis of spiked samples showed good agreement between actual concentrations and Clinitek 200 responses for ketone bodies and glucose although a lack of sensitivity was found for the latter. Results for proteins showed over-or underestimation in dog and rat urines respectively at low concentrations, and overestimation at high concentrations in both species. Reproducibility of responses was excellent for ketone bodies, glucose and proteins but was weaker for haemoglobin and bilirubin. High bilirubin concentrations were found to interfere with the haemoglobin reaction. The pH measurements were found to be accurate only around pH 7. Specific gravity measurements were unreliable. Overall, the Clinitek 200 as a screening tool proved sufficiently reliable in the measurement of all parameters tested, with the exception of specific gravity. Keywords: Laboratory animals; Urinalysis; Test strip; Automated analysisThe clinical chemistry analyses performed in the course of pre-clinical toxicology studies, with

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.