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Bioluminescent sensor proteins have recently gained popularity in both basic research and point-of-care diagnostics. Sensor proteins based on intramolecular complementation of split NanoLuc are particularly attractive because their intrinsic modular design enables for systematic tuning of sensor properties. Here we show how the sensitivity of these sensors can be enhanced by the introduction of catalytically inactive variants of the small SmBiT subunit (DarkBiTs) as intramolecular inhibitors. Starting from previously developed bioluminescent antibody sensor proteins (LUMABS), we developed single component, biomolecular switches with a strongly reduced background signal for the detection of three clinically relevant antibodies, anti-HIV1-p17, cetuximab (CTX), and an RSV neutralizing antibody (101F). These new dark-LUMABS sensors showed 5-13-fold increases in sensitivity which translated into lower limits of detection. The use of DarkBiTs as competitive intramolecular inhibitor domains is not limited to the LUMABS sensor family and might be used to boost the performance of other bioluminescent sensor proteins based on split luciferase complementation.

Linker engineering is rapidly gaining prominence as protein engineers and synthetic biologists construct increasingly sophisticated protein assemblies capable of executing complex molecular functions in the context of biosensing, biocatalysis, or biotherapeutics. Depending on the application, the structural and functional requirements imposed on the underlying linkers can differ vastly. At the same time, there is a distinct lack of methods to effectively code linkers at the level of DNA and tailor them to the functional requirements of different fusion proteins. Addressing these limitations, a scalable framework is presented to compose co-polymeric linkers of variable lengths and amino acid composition based on a limited number of linker fragments stored in sequence-verified entry plasmids. The assembly process is exemplified for Pro-rich linkers in the context of a Zn2+-responsive dual-readout BRET/FRET sensor while examining how linker composition impacts key functional properties such as ligand affinity, dynamic range, and their ability to separate structurally distinct domains.

Linkers play critical roles in the construction of synthetic protein switches and sensors as they functionally couple a receptor with an actuator. With an increasing number of molecular toolboxes and experimental strategies becoming available that can be applied to engineer protein switches and sensors with tailored response functions, optimising the connecting linkers remains an idiosyncratic and empiric process. This review aims to provide an in-depth analysis of linker motifs, the biophysical properties they confer, and how they impact the performance of synthetic protein switches and sensors while identifying trends, mechanisms, and strategies that underlie the most potent switches and sensors.

Linker engineering constitutes a critical, yet frequently underestimated aspect in the construction of synthetic protein switches and sensors. Notably, systematic strategies to engineer linkers by predictive means remain largely elusive to date. This is primarily due to our insufficient understanding how the biophysical properties that underlie linker functions mediate the conformational transitions in artificially engineered protein switches and sensors. The construction of synthetic protein switches and sensors therefore heavily relies on experimental trial-and-error. Yet, methods for effectively generating linker diversity at the genetic level are scarce. Addressing this technical shortcoming, iterative functional linker cloning (iFLinkC) enables the combinatorial assembly of linker elements with functional domains from sequence verified repositories that are developed and stored in-house. The assembly process is highly scalable and given its recursive nature generates linker diversity in a combinatorial and exponential fashion based on a limited number of linker elements.

Small 17(33), 2101066 (2021). doi:10.1002/smll.202101066, Published by Wiley-VCH, Weinheim

Linker engineering constitutes a critical, yet frequently underestimated aspect in the construction of synthetic protein switches and sensors. Notably, systematic strategies to engineer linkers by predictive means remain largely elusive to date. This is primarily due to our insufficient understanding how the biophysical properties that underlie linker functions mediate the conformational transitions in artificially engineered protein switches and sensors. The construction of synthetic protein switches and sensors therefore heavily relies on experimental trial-and-error. Yet, methods for effectively generating linker diversity at the genetic level are scarce. Addressing this technical shortcoming, iterative functional linker cloning (iFLinkC) enables the combinatorial assembly of linker elements with functional domains from sequence verified repositories that are developed and stored in-house. The assembly process is highly scalable and given its recursive nature generates linker diversity in a combinatorial and exponential fashion based on a limited number of linker elements.

Proteases have emerged as a promising class of enzymes to build post-translationally regulated signalling functions in diverse organisms and cell types ranging from simple prokaryotes to higher eukaryotes and in reconstituted systems in vitro. An expanding repertoire of proteases can now be readily configured to build tailored sensors, switches and transducers, and is increasingly facilitating the construction of complex sensory systems for a variety of biotechnological and biomedical applications. This is complemented by an increasing understanding of the fundamental design principles underlying biological signal processing at both protein-level and circuit-level that is now actively probed through synthesis. This review thus aims to summarize and analyse the most promising conceptual and experimental approaches that can be applied to build artificial signalling functions with proteases while highlighting advances, drawbacks and limitations.

We present the case of a 49-year-old woman with sudden onset of severe headaches and a ruptured aneurysm located inside the fenestration of the infraclinoid part of the internal carotid artery in the segments C4 and C5 distal to the origin of the ophthalmic artery. An interdisciplinary approach enabled the successful treatment of the aneurysm by wrapping and stent-assisted coiling. We discuss this rare congenital anomaly of a fenestrated internal carotid artery together with the 12 other cases published worldwide.

Objective: Sophisticated shunt valves provide the possibility of pressure adjustment and antisiphon control but have a higher probability of valve dysfunction especially in a posthemorrhagic setting. The aim of the present study is to analyze the clinical outcome of patients with shunt dependent posthemorrhagic[for full text, please go to the a.m. URL], 67. Jahrestagung der Deutschen Gesellschaft für Neurochirurgie (DGNC), 1. Joint Meeting mit der Koreanischen Gesellschaft für Neurochirurgie (KNS)

Cell-free expression allows to synthesize membrane proteins in completely new formats that can relatively easily be customized for particular applications. Amphiphilic superstructures such as micelles, lipomicelles, or nanodiscs can be provided as nano-devices for the solubilization of membrane proteins. Defined empty bilayers in the form of nanodiscs offer native like environments for membrane proteins, supporting functional folding, proper oligomeric assembly as well as stability. Even very difficult and detergent-sensitive membrane proteins can be addressed by the combination of nanodisc technology with efficient cell-free expression systems as the direct co-translational insertion of nascent membrane proteins into supplied preassembled nanodiscs is possible. This chapter provides updated protocols for the synthesis of membrane proteins in presence of preassembled nanodiscs suitable for emerging applications such as screening of lipid effects on membrane protein function and the modulation of oligomeric complex formation.

Recent years have witnessed increasing efforts to engineer artificial biological functions through recombination of modular-organized toolboxes of protein scaffolds and parts. A critical, yet frequently neglected aspect concerns the identity of peptide linkers or spacers connecting individual domains which remain poorly understood and challenging to assemble. Addressing these limitations, iFlinkC comprises a highly scalable DNA assembly process that facilitates the combinatorial recombination of functional domains with linkers of varying length and flexibility, thereby overcoming challenges with high GC-content and the repeat nature of linker elements. The capacity of iFLinkC is demonstrated in the construction of synthetic protease switches featuring PDZ-FN3-based affinity clamps and single-chain FKBP12-FRB receptors as allosteric inputs. Library screening experiments demonstrate that linker space is highly plastic as the induction of allosterically regulated protease switches can vary from >150-fold switch-ON to >13-fold switch-OFF solely depending on the identity of the connecting linkers and relative orientation of functional domains. In addition, Pro-rich linkers yield the most potent switches contradicting the conventional use of flexible Gly-Ser linkers. Given the ease and efficiency how functional domains can be readily recombined with any type of linker, iFLinkC is anticipated to be widely applicable to the assembly of any type of fusion protein.

Cell-free expression allows to synthesize membrane proteins in completely new formats that can relatively easily be customized for particular applications. Amphiphilic superstructures such as micelles, lipomicelles, or nanodiscs can be provided as nano-devices for the solubilization of membrane proteins. Defined empty bilayers in the form of nanodiscs offer native like environments for membrane proteins, supporting functional folding, proper oligomeric assembly as well as stability. Even very difficult and detergent-sensitive membrane proteins can be addressed by the combination of nanodisc technology with efficient cell-free expression systems as the direct co-translational insertion of nascent membrane proteins into supplied preassembled nanodiscs is possible. This chapter provides updated protocols for the synthesis of membrane proteins in presence of preassembled nanodiscs suitable for emerging applications such as screening of lipid effects on membrane protein function and the modulation of oligomeric complex formation.

ZusammenfassungZiel: Retrospektive Analyse über Nutzen und Risiken der Carmustin-Therapie in der multimodalen Therapie des Glioblastoma multiforme.Material und Methoden: Patienten mit Glioblastoma multiforme wurden zwischen 2008 und 2011 unter Einsatz von Carmustin operiert. Es wurden unerwünschte Ereignisse protokolliert und retrospektiv die Sterbedaten eingeholt.Ergebnisse: Von 88 operierten Patienten wurde bei 30 Carmustin-Wafer implantiert. 69 unerwünschte Ereignisse traten in beiden Therapiegruppen im postoperativen Verlauf bis zum Versterben der Patienten oder bis zum Untersuchungszeitpunkt auf. Die durchschnittliche Überlebenszeit in der Gruppe der mit Carmustin therapierten Patienten lag bei 385 Tagen versus 343 Tage ohne Carmustin-Therapie (p = 0,41).Schlussfolgerung und klinische Relevanz: Durch den Einsatz von Carmustin konnte kein Überlebensvorteil, jedoch auch keine erhöhte Toxizität innerhalb des multimodalen Ansatzes für dieses Patientenkollektiv nachgewiesen werden. Trotz des zu erwartenden Nutzens für weniger als 20% aller Glioblastompatienten scheint es sinnvoll, die multimodale Therapie mit Carmustin-Wafern bei selektierten Patientengruppen weiter prospektiv zu erforschen.

Modern diagnostics are inconceivable without quick and robust tests. With today’s lab-on-a-chip applications, cell-free expression comes back into focus. Two German student teams from the Universities of Bielefeld and Freiburg developed new diagnostic tools based on cell-free expression. Freiburg established a label-free screening for diseases; Bielefeld focused on the detection of harmful substances in water. Both systems offer robust and user-friendly alternatives to current diagnostic tools.

Hintergrund: Nach Shunt-Implantation von konventionellen Differentialdruckventilen bei Patienten mit Hydrocephalus internus oder mit Normaldruckhydrozephalus ist in der Mehrzahl der Falle eine postoperative Abnahme der Ventrikelweite zu sehen. Patienten und Methoden: Von September 1997 bis Mai 2001 wurden im Unfallkrankenhaus Berlin 60 Patienten mit einem Normaldruckhydrozephalus durch Implantation eines hydrostatischen Ventils (Miethke-Dual-Switch-Ventil) operativ behandelt. Bei allen Patienten erfolgten in einer prospektiven Studie die klinische und eine CT-Untersuchung praoperativ und 1 Jahr nach der Operation. Dabei wurde die Ventrikelgrose mittels Bestimmung des Evans-Index verifiziert. Ergebnisse: 77% der Patienten weisen nur eine geringe oder gar keine Anderung der Ventrikelgrose nach der Operation auf, wobei in dieser Gruppe 65% einen exzellenten bis guten, 13% einen befriedigenden und 22% einen schlechten Krankheitsverlauf haben. Bei 17% der Patienten wurde eine masige Ruckbildung der Ventrikelweite 1 Jahr nach der Shunt-Operation sichtbar. In dieser Patientengruppe fanden wir in 40% ein exzellentes bis gutes, in 20% ein zufrieden stellendes und in 40% ein schlechtes Outcome. Bei der Patientengruppe mit deutlicher Reduktion der Ventrikelgrose (6%) sind zu je 50% exzellente bis gute bzw. schlechte Krankheitsverlaufe zu eruieren. Schlussfolgerung: Nach Implantation eines hydrostatischen Ventils ist ein guter postoperativer Krankheitsverlauf bei Patienten mit einem Normaldruckhydrozephalus in der Mehrzahl der Falle nicht mit einer Reduktion der Ventrikelweite 1 Jahr nach der Operation vergesellschaftet. Bei Patienten mit einem Normaldruckhydrozephalus ist nach unseren Erfahrungen die Implantation eines Miethke-Dual-Switch-Ventils zu empfehlen.

Neurosurgical therapy aims to minimize secondary brain damage after a severe head injury. This includes the evacuation of intracranial space-occupying hematomas, the reduction of intracranial volumes, external ventricular drainage, and aggressive therapy in order to influence increased intracranial pressure (ICP) and decreased P(ti)O2. When conservative treatment fails, a decompressive craniectomy might be successful in lowering ICP.

It is well known that in patients with communicating hydrocephalus or normal pressure hydrocephalus, ventricular volume decreases following implantation of differential pressure valved shunts. Hydrostatic valves (Miethke dual-switch valves) were implanted in 60 normal pressure hydrocephalus patients at Unfallkrankenhaus Berlin between September 1997 and September 2001. One year postoperatively, these patients underwent CT scan, and their ventricular size was ascertained using the Evans index. Although 77% of these patients showed no postoperative change in ventricular volume, 65% nonetheless showed good to excellent clinical improvement, 13% satisfactory improvement and 22% no improvement. A moderate reduction in ventricular size was observed in 17% of the patients in our cohort. 40% of these patients showed good to excellent clinical improvement, 20% satisfactory improvement, and 40% unsatisfactory improvement. A marked reduction in ventricular size was observed in 6% of our patients. Of these latter patients, 50% showed good to excellent outcomes, while 50% had unsatisfactory outcomes. The favorable outcomes following implantation of a hydrostatic shunt in patients with normal pressure hydrocephalus did not correlate with decreased ventricular volume one year after operation. Better clinical outcomes were observed in patients with little or no alteration in ventricular size than in patients with a marked decrease in ventricular size. Postoperative change in ventricular volume should be assessed differently for patients with normal pressure hydrocephalus than in patients with hypertensive hydrocephalus.

Neurosurgical therapy aims to minimize the secondary brain damage after a severe head injury. This includes the evacuation of an intracranial space occupying bleeding, the reduction of intracranial volumes, in hematocephalus an external ventricular drainage, and the conservative therapy in order to influence an increased intracranial pressure (ICP) and a decreased p(ti)02. When conservative treatment fails to act a decompressive craniectomy might be successful in lowering ICP. From September 1997 until August 2001 we operated on 439 patients with severe head injuries. 50 patients (11%) were treated by means of a decompressive craniectomy.