Function and Regulation of Vibrio campbellii Proteorhodopsin: Acquired Phototrophy in a Classical Organoheterotroph

Proteorhodopsins (PRs) are retinal-binding photoproteins that mediate light-driven proton translocation across prokaryotic cell membranes. Despite their abundance, wide distribution and contribution to the bioenergy budget of the marine photic zone, an understanding of PR function and physiological significance in situ has been hampered as the vast majority of PRs studied to date are from unculturable bacteria or culturable species that lack the tools for genetic manipulation. In this study, we describe the presence and function of a horizontally acquired PR and retinal biosynthesis gene cluster in the culturable and genetically tractable bioluminescent marine bacterium Vibrio campbellii. Pigmentation analysis, absorption spectroscopy and photoinduction assays using a heterologous over-expression system established the V. campbellii PR as a functional green light absorbing proton pump. In situ analyses comparing PR expression and function in wild type (WT) V. campbellii with an isogenic ΔpR deletion mutant revealed a marked absence of PR membrane localization, pigmentation and light-induced proton pumping in the ΔpR mutant. Comparative photoinduction assays demonstrated the distinct upregulation of pR expression in the presence of light and PR-mediated photophosphorylation in WT cells that resulted in the enhancement of cellular survival during respiratory stress. In addition, we demonstrate that the master regulator of adaptive stress response and stationary phase, RpoS1, positively regulates pR expression and PR holoprotein pigmentation. Taken together, the results demonstrate facultative phototrophy in a classical marine organoheterotrophic Vibrio species and provide a salient example of how this organism has exploited lateral gene transfer to further its adaptation to the photic zone

RpoS1 as an activator of <i>pR</i> expression.

<p>(a) <i>V. campbellii</i> WT, Δ<i>pR</i>, Δ<i>rpoS1</i> and Δ<i>rpoS2</i> M9 minimal medium stationary phase cultures (36 hr) that were either continuously illuminated or dark were assessed by reverse-transcription real-time PCR for <i>pR</i> expression or (b) <i>rpoS1</i> expression. White bars–continuously illuminated cultures; black bars–continuously dark cultures; n.d. – none detected. Data shown represent means ± SD of two technical replicates from two independent experiments. (c) Comparison of PR-mediated pigmentation in <i>V. campbellii</i> WT, Δ<i>pR</i>, Δ<i>rpoS1</i>, and Δ<i>rpoS2</i> stationary phase cells grown under continuous illumination.</p

Influence of continuous light or dark on <i>V. campbellii</i> WT and Δ<i>pR</i> growth in minimal medium and <i>pR</i> transcription.

<p>White squares–<i>V. campbellii</i> WT in continuous light; black squares–<i>V. campbellii</i> WT in continuous dark; white circles–<i>V. campbellii</i> Δ<i>pR</i> in continuous light; black circles–<i>V. campbellii</i> Δ<i>pR</i> in continuous dark. (a) Optical density measurements (OD₆₀₀) and (b) Flow cytometry-based assessment of bacterial growth in M9 minimal medium cultures. Data shown represent means ± SD of duplicate determinations from two experiments. (c) Time course analysis of <i>V. campbellii</i> WT <i>pR</i> transcription via reverse-transcription real-time PCR from M9 minimal medium cultures grown in continuous light or dark.</p

Joint EANM/SNMMI procedure guideline for the use of 177Lu-labeled PSMA-targeted radioligand-therapy (177Lu-PSMA-RLT).

Prostate-specific membrane antigen (PSMA) is expressed by the majority of clinically significant prostate adenocarcinomas, and patients with target-positive disease can easily be identified by PSMA PET imaging. Promising results with PSMA-targeted radiopharmaceutical therapy have already been obtained in early-phase studies using various combinations of targeting molecules and radiolabels. Definitive evidence of the safety and efficacy of [177Lu]Lu-PSMA-617 in combination with standard-of-care has been demonstrated in patients with metastatic castration-resistant prostate cancer, whose disease had progressed after or during at least one taxane regimen and at least one novel androgen-axis drug. Preliminary data suggest that 177Lu-PSMA-radioligand therapy (RLT) also has high potential in additional clinical situations. Hence, the radiopharmaceuticals [177Lu]Lu-PSMA-617 and [177Lu]Lu-PSMA-I&amp;T are currently being evaluated in ongoing phase 3 trials. The purpose of this guideline is to assist nuclear medicine personnel, to select patients with highest potential to benefit from 177Lu-PSMA-RLT, to perform the procedure in accordance with current best practice, and to prepare for possible side effects and their clinical management. We also provide expert advice, to identify those clinical situations which may justify the off-label use of [177Lu]Lu-PSMA-617 or other emerging ligands on an individual patient basis

Joint EANM/SNMMI procedure guideline for the use of 177Lu-labeled PSMA-targeted radioligand-therapy (177Lu-PSMA-RLT)

Prostate-specific membrane antigen (PSMA) is expressed by the majority of clinically significant prostate adenocarcinomas, and patients with target-positive disease can easily be identified by PSMA PET imaging. Promising results with PSMA-targeted radiopharmaceutical therapy have already been obtained in early-phase studies using various combinations of targeting molecules and radiolabels. Definitive evidence of the safety and efficacy of [177Lu]Lu-PSMA-617 in combination with standard-of-care has been demonstrated in patients with metastatic castration-resistant prostate cancer, whose disease had progressed after or during at least one taxane regimen and at least one novel androgen-axis drug. Preliminary data suggest that 177Lu-PSMA-radioligand therapy (RLT) also has high potential in additional clinical situations. Hence, the radiopharmaceuticals [177Lu]Lu-PSMA-617 and [177Lu]Lu-PSMA-I&T are currently being evaluated in ongoing phase 3 trials. The purpose of this guideline is to assist nuclear medicine personnel, to select patients with highest potential to benefit from 177Lu-PSMA-RLT, to perform the procedure in accordance with current best practice, and to prepare for possible side effects and their clinical management. We also provide expert advice, to identify those clinical situations which may justify the off-label use of [177Lu]Lu-PSMA-617 or other emerging ligands on an individual patient basis