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Natural protein assemblies have many sophisticated architectures and functions, creating nanoscale storage containers, motors and pumps. Inspired by these systems, protein monomers have been engineered to self-assemble into supramolecular architectures inc

Introduction: Liver transplantation is an important option for patients of neuroendocrine tumors (NET) with metastases limited to the liver. Peptide receptor radionuclide therapy (PRRNT) is effective in the management of metastatic NET. Aim(s): To assess safety and efficacy of PRRNT after liver transplantation in metastatic NET. Materials and methods: This was a retrospective analysis in five patients (age 52±9 years; four males and one female) of NET with progressive extra hepatic metastatic disease after liver transplantation. None of the patients had evidence of recurrent disease in the transplanted liver. The patients received PRRNT with 3, 2, 2, 1 and 2 cycles respectively with Lu-177 alone, the cumulative administered activity ranging from 7.6 to 22.3 GBq after liver transplantation. Results: Three out of the five patients had grade 1 erythrocytopenia and two of the patients showed evidence of grade 1 renal toxicity. Two of the patients had stable disease, while in two patients it continued to progress. The follow-up is awaited in one patient. Conclusion: PRRNT is a safe and effective therapeutic option for progressive disease after liver transplantation in NET. [ABSTRACT FROM AUTHOR]

Introduction: Hepatic metastases significantly affect the overall prognosis and quality of life in patients with gastroenteropancreatic neuroendocrine tumours (GEP-NET). Radiofrequency ablation (RFA) is one of the important local ablative strategies for hepatic metastases. Peptide receptor radionuclide therapy (PRRNT) is a well-established therapeutic modality for metastatic NET. Aim(s): To assess the efficacy of combined PRRNT and RFA in the management of hepatic metastases of NET. Materials and methods: Nine patients of known metastatic GEP-NET underwent PRRNT (2-5 cycles with Lu-177 and/or Y-90 labeled DOTATATE/DOTATOC) before or after RFA for hepatic metastases. All patients had undergone SSTR PET/CT using Ga-68 DOTATOC before and after the therapies and also for follow-up. Results: In four out of the 10 patients, there was a complete remission over a follow-up period of respectively 36 months, 16 months, nine months and six months, one patient had partial remission over 18 months and one patient stable disease over 30 month follow-up period to date. However in three patients, the disease continued to progress. On retrospective analysis, two out of these patients had intermediate grade NET and one had high grade NET. Conclusion: PRRNT in combination with RFA is effective in the management of hepatic metastases in GEP-NET. [ABSTRACT FROM AUTHOR]

Introduction: Patients with gastroenteropancreatic neuroendocrine tumors (GEP-NET) present frequently with hepatic metastases. Radiofrequency ablation (RFA) is an important local ablative treatment for hepatic metastases. Aim(s): To evaluate the role of Ga-68 somatostatin receptor PET/CT (SSTR PET/CT) in the assessment of response to RFA in hepatic metastases of NET in comparison with the other radiological imaging modalities. Materials and methods: Ten patients of known GEP-NET underwent RFA (in total of 17 segments) for hepatic metastases. All patients had undergone SSTR PET/CT using Ga-68 DOTATOC, 4-phase CT, MRI and USG before, and 3-6 months after RFA. Results: In six out of the 10 patients in 13 liver segments, there was a complete necrosis in the metastases as noted by cold lesions on post-therapy 4-phase CT, MRI and USG, and no uptake on SSTR PET/CT. In the remaining four patients, there was a minor molecular response in the metastases on SSTR PET/CT (>25% decrease in standardized uptake values on post-therapy PET). In two of these four patients, there were no significant detectable changes on USG. In one patient, CT and MRI could not detect any changes to indicate response to RFA. Conclusion: SSTR PET/CT may be better than morphological imaging modalities for restaging of hepatic metastases of GEP-NET after RFA, as it assesses the viability and functional response by accurate quantification of somatostatin receptor expression in the metastases. [ABSTRACT FROM AUTHOR]

Rationale: Neuroendocrine neoplasia (NEN) of small bowel (SBNEN) frequently present with metastatic disease. Theranostics (molecular imaging followed by targeting therapy) allow for personalised medicine. Liquid biopsies enable precise identification of residual disease and real-time monitoring of therapeutic response. Our aim was to determine the clinical utility of a combination of surgery, theranostics, and a multigene blood measurement in metastasised SBNEN. Methods: Inclusion criteria were SBNEN, G1/G2 NEN, initial tumour diagnosis, stage IV NEN, positivity on 68 Ga somatostatin analogue PET/CT, eligible for surgery, and 177 Lu peptide receptor radionuclide therapy (PRRT). Blood samples for NETest were collected longitudinally. Progression-free survival (PFS) and overall survival (OS) were calculated. NETest results were assessed prior to surgery and during clinical follow-up. Results: A surgical cohort of 39 SBNEN patients met eligibility criteria. Thirty-two patients underwent ileal resection and 7 right hemicolectomy. The mean number of 177 Lu PRRT cycles was 4. Mortality was nil. Surgical morbidity was 10.3%. Transient grade 1/2 toxicity occurred in 41% (PRRT). NETest scores (n=9 patients) decreased in 100% following treatment and correlated with diminished tumour volume and disease stabilization following surgery and PRRT. Median follow-up: 78 months. Median PFS and OS: 42.7 and 110 months, respectively. Progression-free survival at 1-, 3-, and 5-years was 79.4%, 57.1% and 40.5%, respectively. Overall survival at 1-, 3-, and 5-years was 97.4%, 97.4%, and 94.1%, respectively. Conclusions: Surgery combined with 177 Lu PRRT is safe and provides favourable PFS and OS in selected patients with advanced SBNEN. Liquid biopsy (NETest) has the potential to accurately delineate disease status., Competing Interests: Competing Interests: MK is an employee of Wren Laboratories. IMM is a consultant to Wren Laboratories. A.F. has received educational grants and research grants from Novartis. A.F. acted as a speaker for Novartis and has received their speaker honorarium, for work not related to the present paper., (© The author(s).)

Azobenzene is a photo-isomerizing molecule whose end-to-end distance changes upon external illumination. When combined with site-specific reactive groups, it can be used as molecular tweezers to remote-control the structure and function of protein targets. The present study gives a brief overview over the rational design strategies that use an azobenzene-based photoswitchable cross-linker to engineer ON/OFF switches into functional proteins or to reprogram proteins for novel functions. The re-engineered proteins may be used as remote controls for cellular pathways, as light-gated drug delivery platforms or as light-powered machinery of synthetic cells and micro-scaled factories., (© 2017 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.)

The F1 sub-complex of ATP synthase is a biological nanomotor that converts the free energy of ATP hydrolysis into mechanical work with an astonishing efficiency of up to 100% (Kinosita et al., 2000). To probe the principal mechanics of the machine, I re-engineered the active site of E.coli F1 ATPase with a structure-based protein design approach: by incorporation of a site-specific, photoswitchable crosslinker, whose end-to-end distance can be modulated by illumination with light of two different wavelengths, a dynamic constraint was imposed on the inter-atomic distances of the α and β subunits. Crosslinking reduced the ATP hydrolysis activity of four designs tested in vitro and in one case created a synthetic ATPase whose activity can be reversibly modulated by subsequent illumination with near UV and blue light. The work is a first step into the direction of the long-term goal to design nanoscaled machines based on biological parts that can be precisely controlled by light.

Controllable protein-based machines and materials are of considerable interest for diverse biotechnological applications. We previously re-engineered an ATP-driven protein machine, a group II chaperonin, to function as a light-gated nanocage. Here we develop and test a model for the molecular mechanism of the re-engineered chaperonin, which undergoes a large-scale closed to open conformational change triggered by reversible photo-isomerization of a site-specifically attached azobenzene crosslinker. In silico experiments using all-atom simulations suggest that rigid body motions of protein subdomains couple the length changes of the crosslinker to rearrangements of the nucleotide-binding pocket, leading to cage opening. We tested this model by designing a mutant for which the orientation of the two protein subdomains forming the nucleotide-binding pocket is directly controlled by the crosslinker, and confirmed successful reversible photoswitching in vitro. The model probes the conformational cycle of group II chaperonins and offers a design principle for engineering other light-driven protein-based molecular machines., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

In recent years, interest in controlling protein function with light has increased. Light offers a number of unique advantages over other methods, including spatial and temporal control and high selectivity. Here, we describe a general protocol for engineering a protein to be controllable with light via reaction with an exogenously introduced photoisomerizable small molecule and illustrate our protocol with two examples from the literature: the engineering of the calcium affinity of the cell-cell adhesion protein cadherin, which is an example of a protein that switches from a native to a disrupted state (Ritterson et al. J Am Chem Soc (2013) 135:12516-12519), and the engineering of the opening and closing of the chaperonin Mm-cpn, an example of a switch between two functional states (Hoersch et al.: Nat Nanotechn (2013) 8:928-932). This protocol guides the user from considering which proteins may be most amenable to this type of engineering, to considerations of how and where to make the desired changes, to the assays required to test for functionality.

Natural protein assemblies have many sophisticated architectures and functions, creating nanoscale storage containers, motors and pumps. Inspired by these systems, protein monomers have been engineered to self-assemble into supramolecular architectures including symmetrical, metal-templated and cage-like structures. The complexity of protein machines, however, has made it difficult to create assemblies with both defined structures and controllable functions. Here we report protein assemblies that have been engineered to function as light-controlled nanocontainers. We show that an adenosine-5'-triphosphate-driven group II chaperonin, which resembles a barrel with a built-in lid, can be reprogrammed to open and close on illumination with different wavelengths of light. By engineering photoswitchable azobenzene-based molecules into the structure, light-triggered changes in interatomic distances in the azobenzene moiety are able to drive large-scale conformational changes of the protein assembly. The different states of the assembly can be visualized with single-particle cryo-electron microscopy, and the nanocages can be used to capture and release non-native cargos. Similar strategies that switch atomic distances with light could be used to build other controllable nanoscale machines.

A characteristic of human gastroenteropancreatic neuroendocrine tumors (GEP-NET) is a minute unobtrusive primary tumor which often cannot be detected by common physical examinations. It therefore remains unidentified until the tumor has spread and space-occupying metastases cause clinical symptoms leading to diagnosis. Cases in which the primary cannot be located are referred to as NET with CUP-syndrome (cancer of unknown primary syndrome). With the help of array-CGH (comparative genomic hybridization, Agilent 105K) and gene expression analysis (Agilent 44K), microdissected primaries and their metastases were compared to identify up- and down-regulated genes which can be used as a marker for tumor progression. In a next analysis step, a hierarchical clustering of 41.078 genes revealed three genes [C-type lectin domain family 13 member A (CD302), peptidylprolyl isomerase containing WD40 repeat (PPWD1) and abhydrolase domain containing 14B (ABHD14B)] which expression levels can categorize the metastases into three groups depending on the localization of their primary. Because cancer therapy is dependent on the localization of the primary, the gene expression level of these three genes are promising markers to unravel the CUP syndrome in NET.

Light-induced activation of the LOV2-Jα domain of the photoreceptor phototropin from oat is believed to involve the detachment of the Jα helix from the central β-sheet and its subsequent unfolding. The dynamics of these conformational changes were monitored by time-resolved emission spectroscopy with 100 ns time resolution. Three transitions were detected during the LOV2-Jα photocycle with time constants of 3.4 μs, 500 μs, and 4.3 ms. The fastest transition is due to the decay of the flavin phosphorescence in the transition of the triplet LOV(L)(660) state to the singlet LOV(S)(390) signaling state. The 500 μs and 4.3 ms transitions are due to changes in tryptophan fluorescence and may be associated with the dissociation and unfolding of the Jα helix, respectively. They are absent in the transient absorption signal of the flavin chromophore. The tryptophan fluorescence signal monitors structural changes outside the chromophore binding pocket and indicates that there are at least three LOV(S)(390) intermediates. Since the 500 μs and 4.3 ms components are absent in a construct without the Jα helix and in the mutant W557S, the fluorescence signal is mainly due to tryptophan 557. The kinetics of the main 500 μs component is strongly temperature dependent with activation energy of 18.2 kcal/mol suggesting its association with a major structural change. In the structurally related PAS domain protein PYP the N-terminal cap dissociates from the central β-sheet and unfolds upon signaling state formation with a similar time constant of ∼1 ms. Using transient fluorescence we obtained a nearly identical activation energy of 18.5 kcal/mol for this transition.