Fluorescence polarization microscopy (FPM) can visualize the dipole orientation of fluorescent molecules and has been used for analyzing architectural dynamics of biomolecules including cytoskeletal proteins. To monitor the orientation of target molecules by FPM, target molecules need to be labeled with fluorophores in a sterically constrained manner, so that the fluorophores do not freely rotate. Recently, a versatile probe for such labeling using fluorescent proteins, POLArIS (Probe for Orientation and Localization Assessment, recognizing specific Intracellular Structures of interest), was reported. POLArIS is a fusion protein consisting of a non-immunoglobulin-based recombinant binder Affimer and a green fluorescent protein (GFP), where the Affimer and GFP are rigidly connected to each other. POLArIS probe for molecules of interest can be developed through phage display screening of Affimer. This screening is followed by the rigid connection of fluorescent proteins to the selected Affimers. The Affimer-based POLArIS, however, cannot be used with animal immune libraries for selecting specific binder clones. In addition, multi-color FPM by POLArIS was not available due to the lack of color variations of POLArIS. In this study, we have developed new versions of POLArIS with nanobodies, which are compatible with animal immune libraries, and expanded color variations of POLArIS with cyan/green/yellow/red fluorescent proteins, enabling multi-color orientation imaging for multiple targets. Using nanobody-based POLArIS orientation probes, we performed two-color FPM of F-actin and vimentin in living cells. Furthermore, we made nanobody-based POLArIS probes that have different dipole orientations for adjusting the orientation of fluorescence polarization with respect to the target molecules. These nanobody-based POLArIS with options of colors and dipole orientations will enhance the performance of this probe for broader applications of fluorescence polarization imaging in living cells, tissues, and whole organisms.

01 Mar 2014·Journal of NeurosurgeryQ1 · MEDICINE

Hydrocephalus shunt technology: 20 years of experience from the Cambridge Shunt Evaluation Laboratory

Q1 · MEDICINE

Article

Author: Pickard, John D ; Richards, Hugh K ; Czosnyka, Zofia H ; Chari, Aswin ; Czosnyka, Marek

ObjectThe Cambridge Shunt Evaluation Laboratory was established 20 years ago. This paper summarizes the findings of that laboratory for the clinician.MethodsTwenty-six models of valves have been tested long-term in the shunt laboratory according to the expanded International Organization for Standardization 7197 standard protocol.ResultsThe majority of the valves had a nonphysiologically low hydrodynamic resistance (from 1.5 to 3 mm Hg/[ml/min]), which may result in overdrainage related to posture and during nocturnal cerebral vasogenic waves. A long distal catheter increases the resistance of these valves by 100%–200%. Drainage through valves without a siphon-preventing mechanism is very sensitive to body posture, which may result in grossly negative intracranial pressure. Siphon-preventing accessories offer a reasonable resistance to negative outlet pressure; however, accessories with membrane devices may be blocked by raised subcutaneous pressure. In adjustable valves, the settings may be changed by external magnetic fields of intensity above 40 mT (exceptions: ProGAV, Polaris, and Certas). Most of the magnetically adjustable valves produce large distortions on MRI studies.ConclusionsThe behavior of a valve revealed during testing is of relevance to the surgeon and may not be adequately described in the manufacturer's product information. The results of shunt testing are helpful in many circumstances, such as the initial choice of shunt and the evaluation of the shunt when its dysfunction is suspected.

01 Mar 2011·Journal of Zoo and Wildlife MedicineQ4 · AGRICULTURAL & FORESTRY SCIENCE

Intermediate Syndrome of Chlorpyriphos Toxicity (Polaris®) in a Caracal (Caracal caracal)

Q4 · AGRICULTURAL & FORESTRY SCIENCE

Article

Author: Merbl, Yael ; Kelmer, Efrat ; Aizenberg, Zachi ; Aroch, Itamar ; Klainbart, Sigal

A 5-yr-old spayed female caracal (Caracal caracal) was presented with complaints of acute onset of mental dullness, ataxia, and ventroflexion of 24-hr duration. The animal's garden territory was sprayed a day earlier with an organophosphate (OP) insecticide (chlorpyriphos-methyl). The caracal was treated for OP toxicosis and mildly improved. It was discharged a day later at the owner's request, although clinical signs did not resolve. During the following week, the caracal was confined to prevent further toxin exposure but did not improve and was presented 8 days later with similar clinical signs. Serum butyril-cholinesterase activity was markedly low. The relatively long interval from OP exposure, along with the duration of clinical signs, suggested an intermediate syndrome of OP toxicity. The caracal was treated symptomatically and progressively improved. It was discharged after 8 days of hospitalization and made full recovery 30 days later. This is the first report of OP toxicity in a caracal, suspected to progress to an intermediate syndrome of OP poisoning.

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