Correlative imaging of the spatio-angular dynamics of biological systems with multimodal instant polarization microscope.

The spatial and angular organization of biological macromolecules is a key determinant, as well as informative readout, of their function. Correlative imaging of the dynamic spatio-angular architecture of cells and organelles is valuable, but remains challenging with current methods. Correlative imaging of spatio-angular dynamics requires fast polarization-, depth-, and wavelength-diverse measurement of intrinsic optical properties and fluorescent labels. We report a multimodal instant polarization microscope (miPolScope) that combines a broadband polarization-resolved detector, automation, and reconstruction algorithms to enable label-free imaging of phase, retardance, and orientation, multiplexed with fluorescence imaging of concentration, anisotropy, and orientation of molecules at diffraction-limited resolution and high speed. miPolScope enabled multimodal imaging of myofibril architecture and contractile activity of beating cardiomyocytes, cell and organelle architecture of live HEK293T and U2OS cells, and density and anisotropy of white and grey matter of mouse brain tissue across the visible spectrum. We anticipate these developments in joint quantitative imaging of density and anisotropy to enable new studies in tissue pathology, mechanobiology, and imaging-based screens.

OpenCell: Endogenous tagging for the cartography of human cellular organization.

Elucidating the wiring diagram of the human cell is a central goal of the postgenomic era. We combined genome engineering, confocal live-cell imaging, mass spectrometry, and data science to systematically map the localization and interactions of human proteins. Our approach provides a data-driven description of the molecular and spatial networks that organize the proteome. Unsupervised clustering of these networks delineates functional communities that facilitate biological discovery. We found that remarkably precise functional information can be derived from protein localization patterns, which often contain enough information to identify molecular interactions, and that RNA binding proteins form a specific subgroup defined by unique interaction and localization properties. Paired with a fully interactive website (opencell.czbiohub.org), our work constitutes a resource for the quantitative cartography of human cellular organization.

Genome-wide programmable transcriptional memory by CRISPR-based epigenome editing.

A general approach for heritably altering gene expression has the potential to enable many discovery and therapeutic efforts. Here, we present CRISPRoff-a programmable epigenetic memory writer consisting of a single dead Cas9 fusion protein that establishes DNA methylation and repressive histone modifications. Transient CRISPRoff expression initiates highly specific DNA methylation and gene repression that is maintained through cell division and differentiation of stem cells to neurons. Pairing CRISPRoff with genome-wide screens and analysis of chromatin marks establishes rules for heritable gene silencing. We identify single guide RNAs (sgRNAs) capable of silencing the large majority of genes including those lacking canonical CpG islands (CGIs) and reveal a wide targeting window extending beyond annotated CGIs. The broad ability of CRISPRoff to initiate heritable gene silencing even outside of CGIs expands the canonical model of methylation-based silencing and enables diverse applications including genome-wide screens, multiplexed cell engineering, enhancer silencing, and mechanistic exploration of epigenetic inheritance.

Cascades of Homeostatic Dysregulation Promote Incubation of Cocaine Craving.

In human drug users, cue-induced drug craving progressively intensifies after drug abstinence, promoting drug relapse. This time-dependent progression of drug craving is recapitulated in rodent models, in which rats exhibit progressive intensification of cue-induced drug seeking after withdrawal from drug self-administration, a phenomenon termed incubation of drug craving. Although recent results suggest that functional alterations of the nucleus accumbens (NAc) contribute to incubation of drug craving, it remains poorly understood how NAc function evolves after drug withdrawal to progressively intensify drug seeking. The functional output of NAc relies on how the membrane excitability of its principal medium spiny neurons (MSNs) translates excitatory synaptic inputs into action potential firing. Here, we report a synapse-membrane homeostatic crosstalk (SMHC) in male rats, through which an increase or decrease in the excitatory synaptic strength induces a homeostatic decrease or increase in the intrinsic membrane excitability of NAc MSNs, and vice versa. After short-term withdrawal from cocaine self-administration, despite no actual change in the AMPA receptor-mediated excitatory synaptic strength, GluN2B NMDA receptors, the SMHC sensors of synaptic strength, are upregulated. This may create false SMHC signals, leading to a decrease in the membrane excitability of NAc MSNs. The decreased membrane excitability subsequently induces another round of SMHC, leading to synaptic accumulation of calcium-permeable AMPA receptors and upregulation of excitatory synaptic strength after long-term withdrawal from cocaine. Disrupting SMHC-based dysregulation cascades after cocaine exposure prevents incubation of cocaine craving. Thus, cocaine triggers cascades of SMHC-based dysregulation in NAc MSNs, promoting incubated cocaine seeking after drug withdrawal.SIGNIFICANCE STATEMENT Here, we report a bidirectional homeostatic plasticity between the excitatory synaptic input and membrane excitability of nucleus accumbens (NAc) medium spiny neurons (MSNs), through which an increase or decrease in the excitatory synaptic strength induces a homeostatic decrease or increase in the membrane excitability, and vice versa. Cocaine self-administration creates a false homeostatic signal that engages this synapse-membrane homeostatic crosstalk mechanism, and produces cascades of alterations in excitatory synapses and membrane properties of NAc MSNs after withdrawal from cocaine. Experimentally preventing this homeostatic dysregulation cascade prevents the progressive intensification of cocaine seeking after drug withdrawal. These results provide a novel mechanism through which drug-induced homeostatic dysregulation cascades progressively alter the functional output of NAc MSNs and promote drug relapse.

Quantitative MRI assessment of a novel direction modulated brachytherapy tandem applicator for cervical cancer at 1.5T.

BACKGROUND AND PURPOSE: The purpose of this work is to quantitatively investigate the artifacts and image distortions induced in the MR images by a recently proposed direction modulated brachytherapy (DMBT) tandem applicator prototype. This new MRI-compatible applicator allows better sparing of organs-at-risk (OAR) for cervical cancer patients, while providing conformal dose distributions to target volumes. MATERIALS AND METHODS: Specific phantom and tools were designed and manufactured for this study. The phantom was filled with a tissue-like solution and MR images were acquired with clinical protocols as per GEC-ESTRO recommendations. Images were obtained at 6 different orientations that mimic possible clinical settings and full-width-at-half-maximum (FWHM) was recorded at multiple locations/angles. The accuracy of detecting the centerline of the tandem was assessed using a novel radial-fiducials mount. RESULTS: FWHM from all line profiles at all angles and all orientations was 6.14±0.7mm (compared to 6mm of the actual DMBT tandem diameter). The in-plane spatial-shift observed at para-axial and para-sagittal views was less than 0.5mm. CONCLUSIONS: This work demonstrated that the novel DMBT tandem applicator prototype has minimal artifact in T2-weighted images employed in clinical practice, suggesting the applicator might be a good candidate for MRI-guided adaptive brachytherapy.

Minimally Invasive Treatment of Temporomandibular Joint Ankylosis and Fibrosis of Temporalis Muscle.

A case of severe mandibular hypomobility due to fibrosis of the left temporalis tendon, combined with ankylosis of the temporomandibular joint, is presented. This case emphasizes the importance of reconstructing the historical timeline to establish a correct diagnosis, ultimately leading to appropriate treatment. The use of minimally invasive surgical techniques and the importance of postoperative rehabilitation are emphasized.

Emphysematous pyelonephritis in a domestic shorthair cat.

A 3-year-old male castrated domestic shorthair cat was presented with an acute history of lethargy and decreased appetite. Pertinent physical examination abnormalities included palpable irregularity of the right kidney and pain on palpation of the left kidney. Ultrasonographic imaging of the abdomen revealed gas present at the corticomedullary junction of the left kidney, consistent with emphysematous pyelonephritis, as well as emphysematous cystitis. While quantitative urine culture via pyelocentesis yielded a negative culture, a sample via cystocentesis was positive for Escherichia coli and emphysematous changes were presumed most likely secondary to an ascending infection. The purpose of this report is to describe the temporary management of ureteral obstruction secondary to emphysematous pyelonephritis using a ureteral stent in a cat.

Pilot study of the suitability of dorsal vulval skin as a transposition flap: vascular anatomic study and clinical application.

OBJECTIVE: To identify the blood supply to the vulval fold and adjacent skin, and evaluate it as a transposition flap for closing perineal wounds in dogs. STUDY DESIGN: Prospective study. ANIMALS OR SAMPLE POPULATION: Five female canine cadavers and 2 cases referred for excision of mast cell tumors adjacent to the vulva. METHODS: Dissection was performed to identify the vascular supply to the vulval fold in two cadavers following arterial injection of red latex and methylene blue, respectively. In three cadavers, barium sulfate mixed 1:1 with water was injected into the terminal aorta. The vulval fold and surrounding perineal skin was excised and radiographed. Transposition flaps using the vulval fold and adjacent skin were used to close skin defects in two dogs presented for wide excision of mast cell tumors situated ventro-lateral and dorso-lateral to the vulva, respectively. RESULTS: The vulval fold and adjacent skin was perfused bilaterally by branches of the ventral perineal and external pudendal arteries, which entered dorsally and ventrally, respectively. As incisions used to create a transposition flaps from the skin surrounding the vulval fold transect these vessels, the flap is dependent on the sub-dermal plexus for survival. There was 100% survival of transposition flaps in the 2 clinical cases and healing proceeded uneventfully with acceptable cosmetic and functional results. CONCLUSIONS: The vulval fold and surrounding skin can be used as a subdermal plexus flap to close large perineal defects in dogs. CLINICAL RELEVANCE: Availability of a defined local skin flap will improve treatment of diseases resulting in large perineal skin defects in female dogs.

Biomechanical evaluation of screw-in femoral implant in cementless total hip system.

OBJECTIVE: To compare (1) proximal femoral axial strains, (2) femoral head deflection, and (3) failure mechanical properties, between Helica head and neck prosthesis implanted femora and normal femora. STUDY DESIGN: In vitro study. SAMPLE POPULATION: Cadaveric canine femora (n = 5 pair). METHODS: Femoral bone strains and head displacement during in vitro simulation of midstance of the gallop were evaluated using cadaveric femurs cyclically loaded in vitro. Strains and displacements were compared within femurs, before and after, prosthesis implantation; and throughout cycling to seek evidence of movement with cyclic loading. Subsequently, implanted femurs and contralateral, intact femurs were loaded to failure to compare failure mechanical properties and modes of failure. RESULTS: Proximal femoral axial strains were significantly different between intact and implanted femora on all 4 cortical surfaces (P < .05). Compressive strains were lower in the implanted femur on all cortical surfaces, except on the caudal surface which was higher. No difference was noted for femoral head angle under an axial load corresponding to gallop (P > .05). Vertical head displacement was ∼0.1 mm greater for implanted femora than intact femora (P < .05). Yield and failure loads and yield energy of implanted femora were 39-54% lower than those for intact femora (P < .05). Mode of failure for both the intact and implanted femora did not appear to be different. CONCLUSION: Helica femoral prosthesis alters strain distribution in the proximal aspect of the femur and exhibits initial micromotion. Failure load in axial compression of the Helica-implanted femur is less than that of the normal femur, but greater than that expected in vivo.

Fosphenytoin may cause hemodynamically unstable bradydysrhythmias.

The prodrug fosphenytoin (FOS) was recently introduced as an alternative to phenytoin (PTN) and has since become a first line therapy for status epilepticus. Unlike PTN, FOS generally has been considered to be safe from cardiac toxicity. To better characterize cardiac toxicity associated with FOS administration, we performed a review of the Food and Drug Administration's Adverse Event Reporting System databank for reports of possible FOS toxicity from 1997-2002. There were 29 applicable reports of adverse cardiac events likely related to FOS infusion, including 10 cardiac deaths. Among survivors, there were four cases of high-grade atrioventricular block, and five cases of transient sinus arrest. Our data suggest that FOS may produce more cardiac toxicity than previously thought. Clinicians should consider administering intravenous FOS in a monitored setting for selected high-risk patients.

Tenecteplase and return of spontaneous circulation after refractory cardiopulmonary arrest.

Even with the benefit of cardiopulmonary resuscitation, the prognosis of cardiac arrest remains poor. Multiple case series describe survival with the use of thrombolytic therapy for refractory cardiac arrest. Presumably thrombolysis treats that subset of cardiac arrest cases resulting from fulminant pulmonary embolism, or perhaps massive myocardial infarctions. Published reports to date have dealt exclusively with streptokinase, urokinase, reteplase, or recombinant tissue plasminogen activator. The authors report the first case of return of spontaneous circulation with the administration of tenecteplase. Tenecteplase is a recently developed reengineered isomer of tissue plasminogen activator that possesses many properties of the ideal cardiac arrest thrombolytic agent. It is bolus dosed, stable at room temperature before reconstitution, and is compatible with most other advanced cardiac life support medications. Because of clinical equivalency and its logistical advantages, tenecteplase should be evaluated as an alternative to other thrombolytics in future trials involving cardiac arrest.