Carotid cavernous fistulae: indications for urgent treatment

Angiographic and clinical data from 155 patients with carotid cavernous fistulae were retrospectively reviewed to determine angiographic features associated with increased risk of morbidity and mortality. These features included presence of a pseudoaneurysm, large varix of the cavernous sinus, venous drainage to cortical veins, and thrombosis of venous outflow pathways distant from the fistula. Clinical signs and symptoms that characterized a hazardous carotid cavernous fistula included increased intracranial pressure, rapidly progressive proptosis, diminished visual acuity, hemorrhage, and transient ischemic attacks. Cortical venous drainage from the carotid cavernous fistula is secondary to occlusion or absence of the normal venous outflow pathways and is associated with signs and symptoms of increased intracranial pressure and an increased risk of intraparenchymal hemorrhage. Angiographic demonstration of a cavernous sinus varix, with extension of the sinus into the subarachnoid space, is associated with an increased risk of fatal subarachnoid hemorrhage. Identification of these high-risk features provides a basis for making decisions about treatment.     

Pooled protein tagging,cellular imaging,and in situ sequencing for monitoring drug action in real time

The levels and subcellular localizations of proteins regulate critical aspects of many cellular processes and can become targets of therapeutic intervention. However, high-throughput methods for the discovery of proteins that change localization either by shuttling between compartments, by binding larger complexes, or by localizing to distinct membraneless organelles are not available. Here we describe a scalable strategy to characterize effects on protein localizations and levels in response to different perturbations. We use CRISPR-Cas9-based intron tagging to generate cell pools expressing hundreds of GFP-fusion proteins from their endogenous promoters and monitor localization changes by time-lapse microscopy followed by clone identification using in situ sequencing. We show that this strategy can characterize cellular responses to drug treatment and thus identify nonclassical effects such as modulation of protein–protein interactions, condensate formation, and chemical degradation.

Currently available mass-spectrometry methods (Rix and Superti-Furga 2009; Martinez Molina et al. 2013; Savitski et al. 2014; Huber et al. 2015; Drewes and Knapp 2018) for monitoring the effects of cellular perturbations on proteomes cannot be scaled efficiently to monitor time-dependent effects in high throughput. A different approach to study drug action is live-cell imaging of protein dynamics in cells expressing a protein of interest fused to a fluorescent tag. Traditionally, such reporter cells are generated either by overexpression to nonphysiologic levels, by oligonucleotide-directed homologous recombination in yeast, or by using CRISPR-Cas9 and homology-directed repair (HDR) to endogenously tag proteins in human cells (Ghaemmaghami et al. 2003; Huh et al. 2003; Chong et al. 2015; Leonetti et al. 2016). In addition to those targeted approaches, “gene trapping” or “CD-tagging” strategies, which rely on the random, viral integration of fluorescent tags as synthetic exons, have been used for analyzing dynamic changes in response to drugs (Jarvik et al. 1996; Morin et al. 2001; Cohen et al. 2008; Kang et al. 2016), but they are limited by integration site biases and require the isolation and characterization of clones before using them in an arrayed format. Recently, a strategy combining genome engineering and gene trapping using homology-independent CRISPR-Cas9 editing to place a fluorescent tag as a synthetic exon into introns of individual target genes has been described (Serebrenik et al. 2019). The strategy relies on a generic sgRNA excising a fluorescent tag flanked by splice acceptor and donor sites from a generic donor plasmid, which is coexpressed with a gene-specific intron-targeting sgRNA specifying the integration site. Here we show the scalability of that strategy to enable pooled protein tagging of more than 900 metabolic enzymes and epigenetic modifiers. Exposing the GFP-tagged cells to compounds allows us to monitor drug effects on the localization and levels of hundreds of proteins in real time in a pooled format, followed by identification of responding clones by in situ sequencing of the expressed intron-targeting sgRNA that corresponds to the tagged protein (Fig. 1A).Open in a separate windowFigure 1.Pooled GFP intron-tagging of metabolic enzymes. (A) Schematic outline of the approach. (B) Identification of targetable introns within metabolic genes. (C) FACS sorting of clones with successful GFP-tagging by signal enrichment over background mCherry intensity used as control for autofluorescence. (D) Representative image of sorted GFP-tagged cell pool. Scale bar, 25 µm. (E) Comparison of RNA-seq expression in HAP1 cells between genes for which GFP-tagged cells could be isolated and genes that were targeted in the sgRNA library but did not result in successful clone isolation.     

Infrared-spectrometric determination of D2O in biological fluids

The determination of D₂O in biological fluids by means of infrared spectrometry was reinvestigated. When the temperature of a solution, containing D₂O in the range from natural abundance to 5 ml·1^–1 increases, its absorbance decreases and the wavenumber of maximum absorption shifts to a higher value. Both changes are linearly related to the change in temperature. Storage for 17 d in either glass or polyethylene tubes does not affect the D₂O concentration. Purification of biological fluids by vacuum-sublimation removes all substances which also absorb at the O-D vibration band and the recovery of D₂O from plasma and urine is complete. The partition ratio of D₂O between plasma water and red cell water equals unity, and the same holds for plasma water and urine water over a wide range of urine flows and osmolalities. The arterial and urinary disappearance curves of D₂O, measured over several days, both permit the calculation of the total amount of body water (V ^bw), the daily water turn-over (F) and the half-time of water in the body (t _1/2), but the data derived from arterial disappearance curves are more precise. In 16 male mongrel dogs (25–32 kg body mass) the following results were obtained:V ^bw=626±28 ml·kg^–1,F=12.0±3.2% andt _1/2=6.21±1.78 d.     

Factors Influencing Nursing Career Choices and Choice of Study Program

In advance of a recruitment campaign, Israeli first-year nursing students of all ethnicities were surveyed to elucidate what factors had influenced them to make nursing their career and what sort of training track they preferred. The responses made it clear that different factors influence different groups differently. There were noticeable differences by gender, age, and ethnicity. Overall, training institutions were chosen for their closeness to the student's home but other factors also operated among particular groups, such as institutional prestige and flexible entry criteria. There was a blatant preference for academic, particularly university-sited, programs over diploma programs.     

Comparison of conventional and computed arthrotomography with MR imaging in the evaluation of the shoulder

To compare conventional arthrography and computed arthrotomography (CAT) with magnetic resonance (MR) imaging in the evaluation of the shoulder, we studied 18 patients who underwent conventional double contrast arthrography and CAT, and T1-, balanced, and T2-weighted MR imaging. The arthrograms were independently reviewed by two of the authors and the MR images were independently reviewed by three other authors in a systematic fashion with the aid of a prewritten evaluation form. The findings were compared among reviewers and between imaging methods. We found MR comparable to conventional arthrography in the evaluation of the rotator cuff; however, MR also enabled evaluation of tendonitis, which could not be accomplished with conventional arthrography. Because of MR's superior soft tissue imaging capability, we were able to stage the impingement syndrome. Magnetic resonance also allowed evaluation of the glenoid labrum and capsuloligamentous structures and assessment of instability in a fashion similar to CAT. In most cases, information obtained from MR equaled or exceeded that obtained from conventional arthrography and CAT. With refinement in technique and increased experience, we believe that MR may replace arthrography in the evaluation of the shoulder.     

High-resolution magnetic resonance imaging of the knee joint: normal anatomy

Excellent morphologic detail was depicted in thin-section, high-resolution magnetic resonance (MR) images obtained with the use of a solenoid surface coil specifically designed for the knee joint. The multiplanar anatomy of the knee was determined by correlating MR images of six fresh cadavers and 10 normal adult knees with corresponding photographs of cryoplaned specimens and by a cross-referencing multiplanar imaging technique.     

Meniscal injuries: detection using MR imaging

Both retrospective and blinded analyses of thin-section, high-resolution magnetic resonance (MR) images of the knee joint, produced using a solenoid surface coil, indicate that MR imaging is an effective technique for evaluating meniscal injuries. Images of 49 patients were evaluated, and the results were correlated with those of subsequent arthroscopy. A grading scale was developed to rate the index of suspicion of a meniscal tear based on the MR images. Overall, approximately 80% of menisci rated grade 4 (definite tear) or 3 (probable tear) were found to have corresponding tears at arthroscopy. In many other patients with a grade 4 or 3 meniscus in whom a corresponding tear was not found arthroscopically, meniscal tears at other sites or other abnormalities were correctly diagnosed using MR. A majority of the false-positive MR images involved the posterior horns of the menisci, the sites of most false-negative arthroscopic diagnoses. The predictive value of a negative MR image was almost 100%. Even in patients with moderate-to-large effusions, the menisci were accurately evaluated. The results imply that MR imaging is useful in the preoperative evaluation of suspected meniscal tears.     

Cholinergic cerebral vasodilator effect of ketamine in rabbits

To analyze the mechanism of the cerebral vasodilator effect of ketamine in anesthetized rabbits, we measured the internal carotid blood flow with an electromagnetic flowmeter, the arterial pressure, intracranial pressure, end-tidal CO2, and the electroencephalogram. Ketamine injection (1 mg/kg) induced a significant cerebral vasodilatation that was blocked by scopolamine, a cholinergic antagonist. In contrast, the increase in cerebral blood flow after ketamine was additive to the cerebral vasodilator actions of inhaled CO2 and of physostigmine infusion, two procedures that activate cholinergic mechanisms. These observations suggest that in rabbits, ketamine activates a cholinergic cerebral vasodilator system.     

Solenoid surface coils in magnetic resonance imaging

A nonplanar solenoidal surface radiofrequency coil is used as a receiver with a conventional transmitter coil in a magnetic resonance imaging system. The improved signal-to-noise ratio, compared with that of conventional fixed saddle or solenoid receiver coils, permits higher resolution imaging and thinner image sections. In addition, the problem of signal dropoff that occurs in deep structures with planar and other noncircumferential surface coils is eliminated. Solenoid surface coils are particularly useful in imaging deep structures in anatomic regions that do not fit standard head and body coils, such as the neck, knees, and other smaller body parts.