Synthesis and Bioluminescence of 'V'-Shaped Firefly Luciferin Analogues Based on A Novel Benzobisthiazole Core.

The design of π-extended conjugation 'V'-shaped red shifted bioluminescent D-luciferin analogues based on a novel benzobisthiazole core is described. The divergent synthetic route allowed access to a range of amine donor substituents through an SN Ar reaction. In spectroscopic studies, the 'V'-shaped luciferins exhibited narrower optical band gaps, more red-shifted absorption and emission spectra than D-luciferin. Their bioluminescence characteristics were recorded against four different luciferases (PpyLuc, FlucRed, CBR2 and PLR3). With native luciferase PpyLuc, the 'V'-shaped luciferins demonstrated more red-shifted emissions than D-luciferin (λbl =561 nm) by 60 to 80 nm. In addition, the benzobisthiazole luciferins showed a wide range of bioluminescence spectra from the visible light region (λbl =500 nm) to the nIR window (>650 nm). The computational results validate the design concept which can be used as a guide for further novel D-luciferin analogues based upon other 'V'-shaped heterocyclic cores.

Bioluminescence, photophysical, computational and molecular docking studies of fully conformationally restricted enamine infraluciferin.

A new rationally designed fully rotationally restricted luciferin has been synthesised. This synthetic luciferin, based upon the structure of infraluciferin, has two intramolecular H-bonds to reduce degrees of freedom, an amine group to enhance ICT process, and an alkenyl group to increase π-conjugation. In the spectroscopic measurements and computational calculations, enamine luciferin showed more red-shifted absorption and fluorescence emission than LH2 and iLH2. With PpyWT luciferase enamine luciferin gave bioluminescence at 564 nm which is similar to LH2 at 561 nm. Further investigation by docking studies revealed that the emission wavelength of enamine luciferin might be attributed to the unwanted twisted structure caused by Asp531 within the enzyme. With mutant luciferase FlucRed, the major emission peak was shifted to 606 nm, a distinct shoulder above 700 nm, and 21% of its spectrum located in the nIR range.

A higher spectral range of beetle bioluminescence with infraluciferin.

Coleopteran bioluminescence is unique in that beetle luciferases emit colors ranging between green (ca.550 nm) and red (ca.600 nm), including intermediate colors such as yellow and orange, allowing up to 3 simultaneous parameters to be resolved in vitro with natural luciferin (D-LH2). Here, we report a more than doubling of the maximum bioluminescence wavelength range using a single synthetic substrate, infraluciferin (iLH2). We report that different luciferases can emit colors ranging from visible green to near-infrared (nIR) with iLH2, including in human cells. iLH2 was designed for dual color far-red to nIR bioluminescence imaging (BLI) in small animals and has been utilized in different mouse models of cancer (including a metastatic hepatic model showing detailed hepatic morphology) and for robust dual parameter imaging in vivo (including in systemic hematological models). Here, we report the properties of different enzymes with iLH2: Lampyrid wild-type (WT) Photinus pyralis (Ppy) firefly luciferase, Ppy-based derivatives previously engineered to be thermostable with D-LH2, and also color-shifted Elaterid-based enzymes: blue-shifted Pyrearinus termitilluminans derivative Eluc (reported D-LH2 λmax = 538 nm) and red-shifted Pyrophorus plagiopthalamus derivative click beetle red (CBR) luciferase (D-LH2 λmax = 618 nm). As purified enzyme, in bacteria or in human cells, Eluc emitted green light (λmax = 536 nm) with DL-iLH2 whereas Ppy Fluc (λmax = 689 nm), x2 Fluc (λmax = 704 nm), x5 Fluc (λmax = 694 nm), x11 Fluc (λmax = 694 nm) and CBR (λmax = 721 nm) produced far-red to nIR peak wavelengths. Therefore, with iLH2, enzyme λmaxes can be separated by ca.185nm, giving almost non-overlapping spectra. This is the first report of single-substrate bioluminescence color emission ranging from visible green to nIR in cells and may help shed light on the color tuning mechanism of beetle luciferases. We also report on the reason for the improvement in activity of x11 Fluc with iLH2 and engineer an improved infraluciferase (iluc) based on this mutant.

Applications of bioluminescence in biotechnology and beyond.

Bioluminescence is the fascinating natural phenomenon by which living creatures produce light. Bioluminescence occurs when the oxidation of a small-molecule luciferin is catalysed by an enzyme luciferase to form an excited-state species that emits light. There are over 30 known bioluminescent systems but the luciferin-luciferase pairs of only 11 systems have been characterised to-date, whilst other novel systems are currently under investigation. The different luciferin-luciferase pairs have different light emission wavelengths and hence are suitable for various applications. The last decade or so has seen great advances in protein engineering, synthetic chemistry, and physics which have allowed luciferins and luciferases to reach previously uncharted applications. The bioluminescence reaction is now routinely used for gene assays, the detection of protein-protein interactions, high-throughput screening (HTS) in drug discovery, hygiene control, analysis of pollution in ecosystems and in vivo imaging in small mammals. Moving away from sensing and imaging, the more recent highlights of the applications of bioluminescence in biomedicine include the bioluminescence-induced photo-uncaging of small-molecules, bioluminescence based photodynamic therapy (PDT) and the use of bioluminescence to control neurons. There has also been an increase in blue-sky research such as the engineering of various light emitting plants. This has led to lots of exciting multidisciplinary science across various disciplines. This review focuses on the past, present, and future applications of bioluminescence. We aim to make this review accessible to all chemists to understand how these applications were developed and what they rely upon, in simple understandable terms for a graduate chemist.

Shining light on the electronic structure and relaxation dynamics of the isolated oxyluciferin anion.

Firefly bioluminescence is exploited widely in imaging in the biochemical and biomedical sciences; however, our fundamental understanding of the electronic structure and relaxation processes of the oxyluciferin that emits the light is still rudimentary. Here, we employ photoelectron spectroscopy and quantum chemistry calculations to investigate the electronic structure and relaxation of a series of model oxyluciferin anions. We find that changing the deprotonation site has a dramatic influence on the relaxation pathway following photoexcitation of higher lying electronically excited states. The keto form of the oxyluciferin anion is found to undergo internal conversion to the fluorescent S1 state, whereas we find evidence to suggest that the enol and enolate forms undergo internal conversion to a dipole bound state, possibly via the fluorescent S1 state. Partially resolved vibrational structure points towards the involvement of out-of-plane torsional motions in internal conversion to the dipole bound state, emphasising the combined electronic and structural role that the microenvironment plays in controlling the electronic relaxation pathway in the enzyme.

Diagnostic and Interventional Radiology Case Volume and Education in the Age of Pandemics: Impact Analysis and Potential Future Directions.

RATIONALE AND OBJECTIVES: To assess the immediate impact of the COVID-19 pandemic on Diagnostic and Interventional Radiology education, and to propose measures to preserve and augment trainee education during future crises. MATERIALS AND METHODS: Diagnostic Radiology (DR) studies and Interventional Radiology (IR) procedures at a single tertiary-care teaching institution between 2015 and 2020 were reviewed. DR was divided by section: body, cardiothoracic, musculoskeletal (MSK), neuroradiology, nuclear medicine, pediatrics, and women's imaging. IR was divided by procedural types: arterial, venous, lymphatic, core, neuro, pediatrics, dialysis, cancer embolization or ablation, noncancer embolization, portal hypertension, and miscellaneous. Impact on didactic education was also assessed. ANOVA, t test, and multiple comparison correction were used for analysis. RESULTS: DR and IR caseloads decreased significantly in April 2020 compared to April of the prior 5 years (both p < 0.0001). Case volumes were reduced in body (49.2%, p < 0.01), MSK (54.2%, p < 0.05), neuro (39.3%, p < 0.05), and women's imaging (75.5%, p < 0.05) in DR, and in arterial (62.6%, p < 0.01), neuro IR (57.6%, p < 0.01) and core IR (42.6%, p < 0.05) in IR. IR trainee average caseload in April 2020 decreased 51.9% compared to April of the prior 5 years (p < 0.01). Utilization of online learning increased in April. Trainees saw significant increases in overall DR didactics (31.3%, p = 0.02) and no reduction in IR didactics, all online. Twelve major national and international DR and IR meetings were canceled or postponed between March and July. CONCLUSION: Decreases in caseload and widespread cancellation of conferences have had significant impact on DR/IR training during COVID-19 restrictions. Remote learning technologies with annotated case recording, boards-style case reviews, procedural simulation and narrated live cases as well as online lectures and virtual journal clubs increased during this time. Whether remote learning can mitigate lost opportunities from in-person interactions remains uncertain. Optimizing these strategies will be important for potential future restricted learning paradigms and can also be extrapolated to augment trainee education during unrestricted times.

Acute Improvement in Intraoperative EMG During Common Fibular Nerve Decompression in Patients with Symptomatic Diabetic Sensorimotor Peripheral Neuropathy: EMG and Clinical Attribute Interrelations.

STUDY AIMS: Electromyographic (EMG) recordings of the fibularis longus and tibialis anterior muscles were performed intraoperatively during nerve decompression (ND) of the common fibular nerve (CFN) in patients with symptomatic diabetic sensorimotor peripheral neuropathy. Patient demographics and clinical attributes were compared against changes in EMG after ND and analyzed for possible correlations. METHODS: Intraoperative changes in CFN EMG were analyzed for correlations against sex, age, body mass index (BMI), hemoglobin A1c (A1c), and type and duration of diabetes. RESULTS: Statistically significant changes were found between EMG changes and patient attributes, but no individual correlations were established. Significant EMG improvement was observed for both men and women (p < 0.0001 and p < 0.05, respectively), age groups (4th decade: p < 0.05; 5th decade: p < 0.05; 6th decade: p < 0.01; 7th decade: p < 0.005), diabetes duration (0-9 years: p = 0.002; 10-19 years: p = 0.002; 20-29 years: p = 0.03), and for type 1 and 2 diabetes (type 1: p < 0.005; type 2: p < 0.001). EMG improvement was greater in patients with the highest BMI levels (30-34.9: p = 0.014; 35-39.9: p = 0.013; > 39.9: p = 0.043), and highest A1c levels (> 6.4%; p < 0.0001). CONCLUSION: Although long-term clinical studies are needed, these results provide insight into which patients might benefit most from this surgery. These results also suggest that surgical ND can produce an acute improvement in nerve function for both men and women, for people with type 1 and 2 diabetes, and across a wide range of ages, BMI, A1c levels, and disease duration.

Correlation Between Radiology ACGME Case Logs Values and ABR Core Exam Pass Rate.

RATIONALE AND OBJECTIVES: There is discordance between the American Board of Radiology (ABR) and many radiology trainees with respect to the most appropriate means to prepare for the ABR Core Examination. Whereas the ABR suggests that participation in routine clinical examination interpretation best prepares a trainee for the practical material of the test, residents, and many program directors feel that time away from clinical service for study and review courses are necessary. This study examines the relationship between studies interpreted in the first three years of residency as reported in the Accreditation Council for Graduate Medical Education case logs and performance of first-time test takers on the ABR Core Examination. MATERIALS AND METHODS: Accreditation Council for Graduate Medical Education case log data was anonymized for a single year cohort of residents in all accredited radiology residencies. This was then provided to the ABR and matched with performance on the Core Examination. A random effects logistic regression model was used to evaluate for a relationship between the number of examinations read and the pass/fail status of the Core Exam. RESULTS: Modeling using a linear and a quadratic term yields a significant relationship between case log values and Core Exam performance. There is a positive correlation until an inflection point of approximately 11,000 examinations, at which point a negative correlation develops. CONCLUSION: The data supports that active engagement in clinical duties is associated with better performance on the ABR Core Examination, with the caveat that there appears to be a point at which service outweighs educational value. Beyond this, performance on the examination declines.

Near-infrared dual bioluminescence imaging in mouse models of cancer using infraluciferin.

Bioluminescence imaging (BLI) is ubiquitous in scientific research for the sensitive tracking of biological processes in small animal models. However, due to the attenuation of visible light by tissue, and the limited set of near-infrared bioluminescent enzymes, BLI is largely restricted to monitoring single processes in vivo. Here we show, that by combining stabilised colour mutants of firefly luciferase (FLuc) with the luciferin (LH2) analogue infraluciferin (iLH2), near-infrared dual BLI can be achieved in vivo. The X-ray crystal structure of FLuc with a high-energy intermediate analogue, 5'-O-[N-(dehydroinfraluciferyl)sulfamoyl] adenosine (iDLSA) provides insight into the FLuc-iLH2 reaction leading to near-infrared light emission. The spectral characterisation and unmixing validation studies reported here established that iLH2 is superior to LH2 for the spectral unmixing of bioluminescent signals in vivo; which led to this novel near-infrared dual BLI system being applied to monitor both tumour burden and CAR T cell therapy within a systemically induced mouse tumour model.

A Divergent Synthetic Route to the Vallesamidine and Schizozygine Alkaloids: Total Synthesis of (+)-Vallesamidine and (+)-14,15-Dehydrostrempeliopine.

The total synthesis of representative members of the schizozygine alkaloids, (+)-vallesamidine and (+)-14,15-dehydrostrempeliopine, were completed from a late-stage divergent intermediate. The synthesis took advantage of efficient nitro-group reactions with the A/B/C ring skeleton constructed concisely on a gram scale through an asymmetric Michael addition, nitro-Mannich/lactamisation, Tsuji-Trost allylation, and intramolecular C-N coupling reaction. Other key features of the synthesis are a novel [1,4] hydride transfer/Mannich-type cyclisation to build ring E and a diastereoselective ring-closing metathesis reaction to construct ring D. This approach gave access to a late-stage C14,C15 alkene divergent intermediate that could be simply transformed into (+)-vallesamidine, (+)-14,15-dehydrostrempeliopine, and potentially other schizozygine alkaloids and unnatural derivatives.

Role of Photoisomerization on the Photodetachment of the Photoactive Yellow Protein Chromophore.

The photocycle of photoactive yellow protein (PYP) is initiated by a photoinduced trans-cis isomerization around a C═C bond in the chromophore that lies at the heart of the protein; however, in addition to the desired photochemical pathway, the chromophore can undergo competing electronic relaxation processes. Here we combine gas-phase anion photoelectron spectroscopy and quantum chemistry calculations to investigate how locking the C═C bond in the chromophore controls the competition between these electronic relaxation processes following photoexcitation in the range 400-310 nm. We find evidence to suggest that preventing trans-cis isomerization effectively turns off internal conversion to the ground electronic state and enhances electron emission from the first electronically excited state.

Radiology Residency Match: The Cost of Being in the Dark.

RATIONALE AND OBJECTIVES: The Electronic Resident Application Service (ERAS) publishes monthly statistics before the match and the National Resident Matching Program publishes the match outcomes. We sought to determine whether early ERAS data influences applicant behavior and correlates with match outcomes. MATERIALS AND METHODS: We searched the 2007-2017 ERAS archives for the applicant pool size (PS), the average number of applications per program (AP), and the average number of applications per applicant (AA) in November, before radiology match, and the 2007-2017 National Resident Matching Program archives for the average number of ranked applicants needed to fill each position (ANRA) and the number of unfilled positions (UP) in radiology match. Correlation coefficients were calculated for each pair. RESULTS: PS correlated very strongly with AP (r = 0.80, p = 0.001708), UP (r = -0.92, p = 0.000063) and ANRA (r = -0.90, p = 0.000164). UP correlated strongly with ANRA (r = 0.76, p = 0.006349) and AP (r = -0.77, p = 0.005339). A trend to moderate correlation between AP and ANRA (r = 0.58, p = 0.062686) and AA (r = 0.53, p = 0.074395) did not reach statistical significance. There was no correlation between AA and PS in the same (r = -0.05, p = 0.878585) or the following year (r = 0.35, p = 0.297166), and AA and UP in the same (r = 0.13, p = 0.701983) or the following year (r = 0.32, p = 0.336136). CONCLUSION: The real-time data reported by ERAS in November, before match, is a predictor of radiology match outcomes and can be used by all participants to limit their application and recruitment costs. Medical students applying to radiology do not consider either the real-time or historic data when submitting ERAS applications.

ΔFlucs: Brighter Photinus pyralis firefly luciferases identified by surveying consecutive single amino acid deletion mutations in a thermostable variant.

The bright bioluminescence catalyzed by Photinus pyralis firefly luciferase (Fluc) enables a vast array of life science research such as bio imaging in live animals and sensitive in vitro diagnostics. The effectiveness of such applications is improved using engineered enzymes that to date have been constructed using amino acid substitutions. We describe ΔFlucs: consecutive single amino acid deletion mutants within six loop structures of the bright and thermostable ×11 Fluc. Deletion mutations are a promising avenue to explore new sequence and functional space and isolate novel mutant phenotypes. However, this method is often overlooked and to date there have been no surveys of the effects of consecutive single amino acid deletions in Fluc. We constructed a large semi-rational ΔFluc library and isolated significantly brighter enzymes after finding ×11 Fluc activity was largely tolerant to deletions. Targeting an "omega-loop" motif (T352-G360) significantly enhanced activity, altered kinetics, reduced Km for D-luciferin, altered emission colors, and altered substrate specificity for redshifted analog DL-infraluciferin. Experimental and in silico analyses suggested remodeling of the Ω-loop impacts on active site hydrophobicity to increase light yields. This work demonstrates the further potential of deletion mutations, which can generate useful Fluc mutants and broaden the palette of the biomedical and biotechnological bioluminescence enzyme toolbox.

Electronic structure and dynamics of torsion-locked photoactive yellow protein chromophores.

The photocycle of photoactive yellow protein (PYP) begins with small-scale torsional motions of the chromophore leading to large-scale movements of the protein scaffold triggering a biological response. The role of single-bond torsional molecular motions of the chromophore in the initial steps of the PYP photocycle are not fully understood. Here, we employ anion photoelectron spectroscopy measurements and quantum chemistry calculations to investigate the electronic relaxation dynamics following photoexcitation of four model chromophores, para-coumaric acid, its methyl ester, and two analogues with aliphatic bridges hindering torsional motions around the single bonds adjacent to the alkene group. Following direct photoexcitation of S1 at 400 nm, we find that both single bond rotations play a role in steering the PYP chromophore through the S1/S0 conical intersection but that rotation around the single bond between the alkene moiety and the phenoxide group is particularly important. Following photoexcitation of higher lying electronic states in the range 346-310 nm, we find that rotation around the single bond between the alkene and phenoxide groups also plays a key role in the electronic relaxation from higher lying states to the S1 state. These results have potential applications in tuning the photoresponse of photoactive proteins and materials with chromophores based on PYP.

Photoelectron spectroscopy of isolated luciferin and infraluciferin anions in vacuo: competing photodetachment, photofragmentation and internal conversion.

The electronic structure and excited-state dynamics of the ubiquitous bioluminescent probe luciferin and its furthest red-shifted analogue infraluciferin have been investigated using photoelectron spectroscopy and quantum chemistry calculations. In our electrospray ionization source, the deprotonated anions are formed predominantly in their phenolate forms and are directly relevant to studies of luciferin and infraluciferin as models for their unstable oxyluciferin and oxyinfraluciferin emitters. Following photoexcitation in the range 357-230 nm, we find that internal conversion from high-lying excited states to the S1(1ππ*) state competes efficiently with electron detachment. In infraluciferin, we find that decarboxylation also competes with direct electron detachment and internal conversion. This detailed spectroscopic and computational study defines the electronic structure and electronic relaxation processes of luciferin and infraluciferin and will inform the design of new bioluminescent systems and applications.

Nerve Decompression and Restless Legs Syndrome: A Retrospective Analysis.

INTRODUCTION: Restless legs syndrome (RLS) is a prevalent sleep disorder affecting quality of life and is often comorbid with other neurological diseases, including peripheral neuropathy. The mechanisms related to RLS symptoms remain unclear, and treatment options are often aimed at symptom relief rather than etiology. RLS may present in distinct phenotypes often described as "primary" vs. "secondary" RLS. Secondary RLS is often associated with peripheral neuropathy. Nerve decompression surgery of the common and superficial fibular nerves is used to treat peripheral neuropathy. Anecdotally, surgeons sometimes report improved RLS symptoms following nerve decompression for peripheral neuropathy. The purpose of this retrospective analysis was to quantify the change in symptoms commonly associated with RLS using visual analog scales (VAS). METHODS: Forty-two patients completed VAS scales (0-10) for pain, burning, numbness, tingling, weakness, balance, tightness, aching, pulling, cramping, twitchy/jumpy, uneasy, creepy/crawly, and throbbing, both before and 15 weeks after surgical decompression. RESULTS: Subjects reported significant improvement among all VAS categories, except for "pulling" (P = 0.14). The change in VAS following surgery was negatively correlated with the pre-surgery VAS for both the summed VAS (r = -0.58, P < 0.001) and the individual VAS scores (all P < 0.01), such that patients who reported the worst symptoms before surgery exhibited relatively greater reductions in symptoms after surgery. CONCLUSION: This is the first study to suggest improvement in RLS symptoms following surgical decompression of the common and superficial fibular nerves. Further investigation is needed to quantify improvement using RLS-specific metrics and sleep quality assessments.

Convergent synthesis and optical properties of near-infrared emitting bioluminescent infra-luciferins.

Infra-luciferin, an alkene linked analogue of luciferin, gives bioluminescence emission >700 nm and has the potential to be used for multiparametric in vivo imaging. We report here a high yielding, scalable and convergent synthesis of infra-luciferin which will allow the synthesis of other conjugated luciferins for investigation in near-infrared bioluminescence imaging. We demonstrated this potential by using the new route to synthesise a diene linked analogue of luciferin, the fluorescent and bioluminescent properties of which were compared to those of d-luciferin and infra-luciferin. We found that extension of conjugation to a diene linker resulted in the specific bioluminescence activity being reduced by 3-4 orders of magnitude compared to d-luciferin. Analogous to its fluorescence emission spectrum, the diene linked analogue exhibited two peaks in its bioluminescence spectrum, the major one being slightly blue-shifted compared to natural d-luciferin, and a minor peak at ca. 800 nm. The fluorescence quantum yield and pH dependence of fluorescence were also determined.