Hepatic transcriptome of the freeze-tolerant Cope's gray treefrog, Dryophytes chrysoscelis: responses to cold acclimation and freezing.

BACKGROUND: Cope's gray treefrog, Dryophytes chrysoscelis, withstands the physiological challenges of corporeal freezing, partly by accumulating cryoprotective compounds of hepatic origin, including glycerol, urea, and glucose. We hypothesized that expression of genes related to cryoprotectant mobilization and stress tolerance would be differentially regulated in response to cold. Using high-throughput RNA sequencing (RNA-Seq), a hepatic transcriptome was generated for D. chrysoscelis, and gene expression was compared among frogs that were warm-acclimated, cold-acclimated, and frozen. RESULTS: A total of 159,556 transcripts were generated; 39% showed homology with known transcripts, and 34% of all transcripts were annotated. Gene-level analyses identified 34,936 genes, 85% of which were annotated. Cold acclimation induced differential expression both of genes and non-coding transcripts; freezing induced few additional changes. Transcript-level analysis followed by gene-level aggregation revealed 3582 differentially expressed genes, whereas analysis at the gene level revealed 1324 differentially regulated genes. Approximately 3.6% of differentially expressed sequences were non-coding and of no identifiable homology. Expression of several genes associated with cryoprotectant accumulation was altered during cold acclimation. Of note, glycerol kinase expression decreased with cold exposure, possibly promoting accumulation of glycerol, whereas glucose export was transcriptionally promoted by upregulation of glucose-6-phosphatase and downregulation of genes of various glycolytic enzymes. Several genes related to heat shock protein response, DNA repair, and the ubiquitin proteasome pathway were upregulated in cold and frozen frogs, whereas genes involved in responses to oxidative stress and anoxia, both potential sources of cellular damage during freezing, were downregulated or unchanged. CONCLUSION: Our study is the first to report transcriptomic responses to low temperature exposure in a freeze-tolerant vertebrate. The hepatic transcriptome of Dryophytes chrysoscelis is responsive to cold and freezing. Transcriptomic regulation of genes related to particular pathways, such as glycerol biosynthesis, were not all regulated in parallel. The physiological demands associated with cold and freezing, as well as the transcriptomic responses observed in this study, are shared with several organisms that face similar ecophysiological challenges, suggesting common regulatory mechanisms. The role of transcriptional regulation relative to other cellular processes, and of non-coding transcripts as elements of those responses, deserve further study.

Incidence and cost of potentially avoidable emergency department visits for urolithiasis.

OBJECTIVES: Urolithiasis represents a leading cause of emergency department (ED) presentation nationally, affecting approximately 10% of Americans. However, most patients require neither hospital admission nor surgical intervention. This study investigates patient and facility factors associated with potentially avoidable ED visits and their economic consequences. STUDY DESIGN: Retrospective analysis. METHODS: Patients presenting to the ED for index urolithiasis events were selected using Florida and New York all-payer data from the 2016 Healthcare Cost and Utilization Project state databases. Avoidable visits were defined as subsequent ED encounters following initial ED presentation that did not result in intervention, admission, or referral to an acute care facility. Utilizing multivariable logistic and linear regression, researchers discerned patient and facility factors predictive of avoidable ED presentations and associated costs. RESULTS: Of the 167,102 ED encounters for urolithiasis, 7.9% were potentially avoidable, totaling $94,702,972 in potential yearly cost savings. Mean encounter-level costs were higher for unavoidable vs avoidable visits ($5885 vs $2098). In contrast, mean episode-based costs were similar for avoidable and unavoidable episodes ($7200 vs $7284). Receiving care in small metropolitan (vs large metropolitan) communities was associated with potentially avoidable visits, whereas increased comorbidities and Hispanic ethnicity were protective against avoidable visits. CONCLUSIONS: The incidence of ED use for subsequent urolithiasis care reveals opportunity for enhanced outpatient availability to reduce hospital-based costs. Several nonclinical factors are associated with potentially avoidable ED visits for urolithiasis, which, if appropriately targeted, may represent an opportunity to reduce health care spending without compromising the quality of care delivery.

#Coronavirus on TikTok: user engagement with misinformation as a potential threat to public health behavior.

Coronavirus disease (COVID)-related misinformation is prevalent online, including on social media. The purpose of this study was to explore factors associated with user engagement with COVID-related misinformation on the social media platform, TikTok. A sample of TikTok videos associated with the hashtag #coronavirus was downloaded on September 20, 2020. Misinformation was evaluated on a scale (low, medium, and high) using a codebook developed by experts in infectious diseases. Multivariable modeling was used to evaluate factors associated with number of views and presence of user comments indicating intention to change behavior. One hundred and sixty-six TikTok videos were identified and reviewed. Moderate misinformation was present in 36 (22%) videos viewed a median of 6.8 million times (interquartile range [IQR] 3.6-16 million), and high-level misinformation was present in 11 (7%) videos viewed a median of 9.4 million times (IQR 5.1-18 million). After controlling for characteristics and content, videos containing moderate misinformation were less likely to generate a user response indicating intended behavior change. By contrast, videos containing high-level misinformation were less likely to be viewed but demonstrated a nonsignificant trend towards higher engagement among viewers. COVID-related misinformation is less frequently viewed on TikTok but more likely to engage viewers. Public health authorities can combat misinformation on social media by posting informative content of their own.

PSA density is complementary to prostate MP-MRI PI-RADS scoring system for risk stratification of clinically significant prostate cancer.

BACKGROUND: While prostate multiparametric-magnetic resonance imaging (MP-MRI) has improved the diagnosis of clinically significant prostate cancer (CSPC), the complementary use of prostate-specific antigen (PSA) levels to risk-stratify for CSPC requires further study. The objective of this project was to determine if prostate MP-MRI and PSA can provide complementary insights into CSPC risk stratification. METHODS: In an IRB-approved study, pathologic outcomes from patients who underwent MR/US fusion-targeted prostate biopsy were stratified by various parameters including PSA, PSA density (PSAD), age, race, and PI-RADS v2 score. CSPC was defined as a Gleason score ≥7. Logistic regression was used to determine odds ratios (OR) with 95% confidence intervals (CI). P values were reported as two-sided with p < 0.05 considered statistically significant. ROC curves were generated for assessing the predictive value of tests and sensitivity + specificity optimization was performed to determine optimal testing cutoffs. RESULTS: A total of 327 patients with 709 lesions total were analyzed. PSAD and PI-RADS scores provided complementary predictive value for diagnosis of CSPC (AUC PSAD: 0.67, PI-RADS: 0.72, combined: 0.78, p < 0.001). When controlling for PI-RADS score, age, and race, multivariate analysis showed that PSAD was independently associated with CSPC (OR 1.03 per 0.01 PSAD increase, 95% CI 1.02-105, p < 0.001). The optimal cutoff of PSAD ≥ 0.1 ng/ml/cc shows that a high versus low PSAD was roughly equivalent to an increase in 1 in PI-RADS score for the presence of CSPC (4% of PI-RADS ≤3 PSAD low, 6% of PI-RADS 3 PSAD high vs. 5% of PI-RADS 4 PSAD low, 22% of PI-RADS 4 PSAD high vs. 29% of PI-RADS 5 PSAD low, 46% of PI-RADS 5 PSAD high were found to have CSPC). CONCLUSIONS: PSAD with a cutoff of 0.1 ng/ml/cc appears to be a useful marker that can stratify the risk of CSPC in a complementary manner to prostate MP-MRI.

A Novel Interactive Virtual Medical Student Clinical Rotation for Ophthalmology.

Objective This article describes a novel clinical rotation that uses technology to create a remote ophthalmology learning experience with the goal of improving virtual exposure to medical and surgical ophthalmic training for medical students. Methods Our unique curriculum incorporates mobile-mounted tablets which allow students to virtually participate in inpatient consults, clinic, and ophthalmic surgery. An adaptable mounting device attached to the slit lamp allows students to observe examinations in real time, enhancing recognition of ocular pathologies. Students participate in a robust curriculum that includes independent learning modules, video lectures, interactive modules, podcasts, and surgical video rounds. Students engage with residents and faculty in interactive-guided lectures and case-based discussions that focus on the American Academy of Ophthalmology white paper teaching objectives. Students are mailed surgical instruments and participate in surgical modules and faculty-led virtual wet laboratories. Results Our unique virtual curriculum combines didactic learning, interactive content, and novel technology applications such as mobile tablets, slit lamp-mounted devices, and faculty-led virtual wet laboratories. Conclusion Virtual technologies can be utilized to enhance ophthalmology medical student education in a safe and effective way during the coronavirus disease 2019 pandemic, and to improve educational access in the future.

The Use of Three-dimensional Visualization Techniques for Prostate Procedures: A Systematic Review.

CONTEXT: As an emerging technique, three-dimensional (3D) visualization has become more popular and can facilitate education, training, surgical planning, and intraoperative guidance for prostate cancer surgery. OBJECTIVE: In this review, we aim to present the impact of 3D printing, virtual reality (VR), and augmented reality (AR) techniques for prostate cancer procedures, specifically prostate biopsy and radical prostatectomy (RP). EVIDENCE ACQUISITION: A systematic review was performed by two investigators according to the Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) criteria. EVIDENCE SYNTHESIS: A total of 541 papers were identified in PubMed, Scopus, and Embase. Of these, 53 studies were identified for detailed review and 25 were qualified. Two more studies were identified from the references; thus, 27 studies were finally included in this systematic review. Nine papers reported on the use of 3D reconstructed models, mainly in education/training and intraoperative guidance; nine reported on VR, focusing on simulation training model and intraoperative guidance; and nine reported on AR technique with its best indication for surgical guidance in robotic RP. CONCLUSIONS: Three-dimensional visualization techniques have gradually been introduced and developed in prostate procedures, and demonstrate potential utility not only for education/training, but also for surgical planning and intraoperative guidance. Prospective studies are needed to demonstrate clinical utility and validation of these technologies. PATIENT SUMMARY: Despite low-quality evidence, promising signals were identified to demonstrate that three-dimensional visualization could help facilitate prostate procedures, in terms of education/training, surgical planning, and intraoperative guidance. It is still in a very early stage, and more studies need to be conducted to justify its widespread use.

Expression and immunolocalization of aquaporins HC-1, -2, and -3 in Cope's gray treefrog, Hyla chrysoscelis.

We have previously identified two aquaporins (HC-1, HC-2) and a glyceroporin (HC-3), homologs, respectively, of mammalian AQP1, AQP2, and AQP3, from the freeze-tolerant treefrog Hyla chrysoscelis. The objective of the present study was to investigate by Western blotting and immunohistofluorescence the expression and localization of these proteins in warm-acclimated, hydrated treefrogs. We hypothesized that patterns of protein expression would reflect unique osmoregulatory roles for the three aquaporins. Western blots revealed a spectrum of protein bands from 28 kDa to 65+ kDa; treatment with N-glycosidase suggested that this reflected variable glycosidation of the aquaporins. HC-1 was expressed in all organs, including dermis of skin, sinusoids and septa of liver, Bowman's capsule of kidney, intestinal lacteal vessels, and perimysium and vasculature of muscle. HC-3 expression was also widespread, but with different localization, including epidermis and dermis of skin, renal collecting ducts, and colonic villous epithelium. HC-2 expression was limited to osmoregulatory organs (renal collecting ducts and epidermis). In many ways, the expression of these proteins paralleled their mammalian homologs. For example, HC-2 and HC-3 expression in collecting ducts appeared similar to the mammalian pattern (the former more apical, the latter more basal). However, some aspects of localization (e.g. HC-1 in Bowman's capsule) were unique, and the ubiquity of HC-3 expression may relate to its facilitation of glycerol transport in this animal that possesses glycerol-dependent freeze tolerance.

Right bundle branch block as a screening test for pulmonary embolism in chronic spinal cord injury.

OBJECTIVES: To evaluate right bundle branch block (RBBB) on electrocardiograms (ECGs) as a screening tool for the diagnosis of pulmonary embolism (PE) in a chronic spinal cord injury (SCI) population and to determine the prevalence of PE. DESIGN: Retrospective analysis. SETTING: Boston Veterans Affairs Healthcare System. PARTICIPANTS: Consecutive SCI participants (N=112) who were followed at this institution until death between 1999 and 2005 at an average age of 71 years, a duration of paralysis of 31 years, with a tetraplegic level in 62%, and no useful motor function in 84%. INTERVENTION: Not applicable. MAIN OUTCOME MEASURES: PE, as a cause of RBBB, was diagnosed by autopsy, a source of thromboembolism, imaging, or other ECG signs of PE. Chronic obstructive lung disease was diagnosed by pulmonary function tests and myocardial infarction by ECG or echocardiogram. RESULTS: Twenty-nine participants (26%) had RBBB (6 with incomplete RBBB). Evidence that PE caused RBBB was found in 21 (72%), with 4 (3 massive) found by autopsy, 4 by the presence of an embolic source, 3 by imaging, 2 by the intermittent course of RBBB, 1 by abrupt onset of RBBB before death, and 7 by other ECG signs. RBBB represented chronic obstructive lung disease or myocardial infarction in the remaining 8. The onset of RBBB occurred either within months of SCI in 5 (1 before SCI) or years after SCI in 24 (6-50y, median 32y). RBBB was constant in 27 and intermittent in 2. The duration of RBBB ranged from 20 minutes to 31 years, median 4 years. CONCLUSIONS: RBBB may be a useful initial screening test for PE complicating chronic SCI. PE, often recurrent and sometimes fatal, is prevalent in chronic SCI.

Postfreeze viability of erythrocytes from Dryophytes chrysoscelis.

Dryophytes chrysoscelis (formerly Hyla chrysoscelis, Cope's gray treefrog) is a freeze-tolerant anuran that accumulates glycerol and urea during cold acclimation and freezing. It is hypothesized that glycerol and urea function as cryoprotectants by minimizing osmotically induced cell damage during freezing and thawing, thereby improving the postfreeze viability of red blood cells (RBCs) when frozen in medium containing those solutes. To test this, erythrocytes were obtained from warm (22°C) and cold-acclimated (4°C) frogs and suspended in 280 mOsM phosphate-buffered saline (PBS). RBCs were frozen in 280 mOsM, isosmotic/isotonic, PBS, or in PBS made hyperosmotic by addition of 150 mM solutes. Postfreeze viability was determined with a hemolysis assay. Postfreeze viability of cells from warm-acclimated frogs improved from 18.9 ± 1.3% in PBS to 47.4 ± 5.2% in PBS with urea ( p < 0.01). The addition of other solutes (glycerol, glucose, NaCl, or sorbitol) had no effect. RBCs from cold-acclimated frogs had 45.8 ± 3.4% viability when frozen in 280 mOsM PBS, and this improved to 71.6 ± 8.9% or 71.9 ± 1.6%, respectively, when frozen with glycerol ( p < 0.01) or urea ( p < 0.001). The viability of RBCs from cold-acclimated frogs was not different between unfrozen cells 86.7-88.4%) and those frozen with glycerol (71.6 ± 8.9%, p > 0.05) or with urea (71.9 ± 1.6%, p > 0.05). These data suggest that (a) cold acclimation induces cellular changes in RBCs that result in improved postfreeze viability, and (b) glycerol and urea are part of a complex cryoprotectant system in D. chrysoscelis.

The cryoprotectant system of Cope's gray treefrog, Dryophytes chrysoscelis: responses to cold acclimation, freezing, and thawing.

Cope's gray treefrog (Dryophytes chrysoscelis) is one of few freeze-tolerant frogs that mobilize glycerol as a cryoprotectant, yet cold and freezing-induced accumulation of this and other osmolytes has received little attention in this species. This study investigated the development of freeze tolerance in D. chrysoscelis, analyzing the response of the cryoprotectant system to cold acclimation, freezing, and thawing. Glycerol production was low and unresponsive to acclimation temperature, or duration of acclimation to 5 °C, except for one cold-acclimated frog that presented elevated glycerol in plasma, liver, and skeletal muscle. Curiously, glycerol concentration in skeletal muscle was higher than that of plasma and liver, in both warm- and cold-acclimated frogs, suggesting glycerol synthesis in muscle. Urea concentration in plasma doubled in response to cold acclimation but did not change during freezing. Freezing induced hepatic glycogen catabolism and an increase in glycerol and glucose in several tissues, although the mobilization dynamics differed between these cryoprotectants, possibly as a result of different transport mechanisms. Although hepatic glucose mobilization was of considerable magnitude, glucose accumulation in peripheral tissues was low and was surpassed by that of glycerol and urea. The muscle production of glycerol and the cold-induced accumulation of urea imply a role for skeletal muscle metabolism in the mobilization of cryoprotective solutes in D. chrysoscelis. The cryoprotectant system of D. chrysoscelis is complex, highly variable, and unique, with glycerol, glucose, and likely urea serving as cryoprotectants.

Expression of the aquaglyceroporin HC-9 in a freeze-tolerant amphibian that accumulates glycerol seasonally.

As ambient temperatures fall in the autumn, freeze-tolerant Cope's gray treefrogs, Dryophytes chrysoscelis (formerly Hyla chrysoscelis), accumulate glycerol as a cryoprotective agent. We hypothesized that these treefrogs express an ortholog of the mammalian aquaglyceroporin AQP9 and that AQP9 expression is upregulated in the cold to facilitate glycerol transport. We sequenced 1790 bp from cloned cDNA that codes for a 315 amino acid protein, HC-9, containing the predicted six transmembrane spanning domains, two Asn-Pro-Ala (NPA) motifs, and five amino acid residues characteristic of aquaglyceroporins. Functional characterization after heterologous expression of HC-9 cRNA in Xenopus laevis oocytes indicated that HC-9 facilitates glycerol and water permeability and is partially inhibited by 0.5 mmol/L phloretin or 0.3 mmol/L HgCl2 HC-9 mRNA (qPCR) and protein (immunoblot) were expressed in most treefrog tissues analyzed (muscle, liver, bladder, stomach, kidney, dorsal skin, and ventral skin) except the protein fraction of red blood cells. Contrary to our prediction, both mRNA and protein expression were either unchanged or downregulated in most tissues in response to cold, freezing, and thawing. A notable exception to that pattern occurred in liver, where protein expression was significantly elevated in frozen (~4-fold over warm) and thawed (~6-fold over warm) conditions. Immunofluorescence labeling of HC-9 protein revealed a signal that appeared to be localized to the plasma membrane of hepatocytes. Our results indicate that gray treefrogs express an AQP9-like protein that facilitates glycerol permeability. Both the transcriptional and translational levels of HC-9 change in response to thermal challenges, with a unique increase in liver during freezing and thawing.

Fibrinogen metabolism in patients with spinal cord injury.

BACKGROUND/OBJECTIVE: Deep venous thrombosis and pulmonary thromboembolism are common within weeks of spinal cord injury (SCI) but clinically uncommon in the chronically paralyzed. Fibrinogen half-life (FHL) and fibrin uptake of the legs (FUT), as indicators of an active thrombotic process, have been used to test this clinical impression. METHODS: Data from the use of autologous preparations of radioiodinated fibrinogen to determine FHL and FUT in 17 men paralyzed at cervical (6), thoracic (10), and lumbar levels (1), at ASIA grades A (15) and C (2) in 1974 to 1976 were reviewed. Group A consisted of 12 subjects 29 +/- 8 years of age and paralyzed 1 week to 5 months (median, 1 month). Group B consisted of 5 subjects 46 +/- 17 years of age and paralyzed 24 to 96 months (median, 36 months). Group B subjects were older and paralyzed longer than Group A. Group C consisted of 4 able-bodied control subjects enrolled at the same time for FHL studies, and these subjects were 34 to 38 years of age. RESULTS: FHL was 61 +/- 14 hours for all SCI subjects and 95 +/- 23 hours for Group C (P = 0.001). Group A FHL was 59 +/- 16 hours, and FUT was positive in 8 of 12 subjects. Group B FHL was 66 +/- 7 hours, and FUT was positive in 3 of 4 subjects (1 FUT not done; P = 0.30 and 1.0, respectively). CONCLUSIONS: Fibrinogen metabolism was abnormal in patients with acute SCI at high risk for pulmonary thromboembolism (PE) but continued to be abnormal beyond the high risk period for PE, possibly because of the greater age of the patients in the long-term paralysis group.

Diabetes mellitus and preventable spinal cord injury.

PURPOSE: A high prevalence of diabetes mellitus (DM) among spinal cord injury (SCI) populations has been noted. A review and follow-up of acute admissions to a SCI service was conducted to explain this high prevalence. METHODS: Records of 166 patients admitted for rehabilitation within 108 days of paralysis were reviewed. Cases of DM were identified by records of treatment or fasting blood glucose levels > 140 mg% or hemoglobin A1c < 7 g%. The onset of DM relative to SCI, the age at injury, and the cause of injury were recorded. New cases of DM and all deaths within a 5-year follow-up were determined for those patients 40 years of age or older at paralysis. RESULTS: The prevalence of DM was 0 of 79 younger patients (aged 16 to 39 years) but 18 of 87 older patients (aged 40 to 86 years), or 21%. All patients with DM had been diagnosed before SCI (16 patients) or during the initial hospitalization (2 patients). The cause of injury was falling in 11 of 18 (61%) DM patients and 31 of 69 (45%) nondiabetic older patients (P = 0.29). Falling was a less common cause of injury in the younger group (13 of 78 [17%], P < 0.001). The cause of paralysis was medical (ie, vascular, infectious) in 5 patients in the DM group (28%) and 5 patients in the non-DM group (6%) (P = 0.03). The 5-year mortality for patients with DM was 7 of 17 (one lost to follow-up) (41%) and 10 of 64 (16%; 5 lost to follow-up) for the nondiabetic older patients (P = 0.04). The 5-year prevalence of DM in the survivors (4 patients developed DM, 1 patient did not survive) was 23%, similar to the initial prevalence. CONCLUSION: DM is a risk factor for SCI. Attention to this risk could prevent some SCIs.

Postural hypotension, hyponatremia, and salt and water intake: case reports.

PURPOSE: Postural hypotension (PH) and hyponatremia are common and often coexistent among patients with severe paralysis secondary to spinal cord injury. Volume depletion could account for these conditions. This study examined whether salt and water intake correlated with the severity of PH. PARTICIPANTS: Participants were 4 patients with tetraplegia, motor and sensory complete, aged 68 to 83 years, who were paralyzed for 9 to 54 years, who had PH. These patients were ranked by the amount of ephedrine prescribed on a daily basis to treat PH over the preceding 2-year period. METHODS: The total urinary output of sodium and water and the effect of orthostasis on urine output rate, osmolality, sodium concentration, and creatinine secretion were determined over a 48-hour period of collection and compared with severity of PH. RESULTS: The ephedrine requirements, in order of decreasing severity of PH, were 100 mg/d, 25 mg/d, 12.5 mg/d, and no ephedrine needed. The 24-hour sodium excretions in that order were 50, 92, 180, and 164 mEq. The urine volumes were 1.4, 3, 2.6, and 5.4 L, respectively. In the same order of decreasing PH severity, the sitting position relative to the recumbent position was characterized by increasing rates of creatinine secretion (ratios of 0.69, 0.74, 0.95, and 0.80), increasing rates of water excretion (ratios of 0.49, 0.28, 0.69, and 0.99), decreasing urine osmolality (ratios of 1.2, 1.8, 1.3, and 0.8), and increasing sodium concentrations (ratios of 0.9, 1.3, 1.2, and 2.6). CONCLUSION: In these individuals with tetraplegia, severe PH was accompanied by avid conservation of water and impaired retention of sodium in the sitting position, as well as limited salt and water intake.

Spinal cord lesions caused by arteriovenous malformation: clinical course and risk of cancer.

PURPOSE: Patients with nontraumatic myelopathy caused by arteriovenous malformation (AVM) are encountered in cohorts of traumatic myelopathy (SCI) patients. The study describes the clinical course of SCI secondary to AVM and compares the incidence of cancer with that in SCI patients. PARTICIPANTS: Twenty-three patients with AVM and 219 patients with SCI patients aged 50 years and older. METHODS: The 2 groups were described by the character of paralysis and compared for the incidence of cancer in a retrospective review of records from the index year 1989. RESULTS: Twenty-three patients with AVM became paralyzed, with onset from age 22 years to 85 years (mean, 53 years). The presenting symptoms were pain (usually back pain), paresthesias, and weakness. The clinical picture varied, with evolution of symptoms reported over a wide period ranging from minutes to 27 years (median, 6 months). Levels of the spinal cord damage were thoracic in 74% of the AVM group vs 40% for the SCI group, P = .004. For patients with AVM, grade of paralysis was American Spinal Injury Association D (some significant use of the legs) in 26% vs 16% for patients with SCI, P = .38. Nine patients with AVM (39%) developed cancer (skin, brain, blood, thyroid, liver, bladder, and prostate at age 66 +/- 7 years) with an incidence of 5.92 cancers/100 patient years. Thirty-five patients with SCI (9%) had cancer (skin, blood, lung, bowel, bladder, and prostate, age 67 +/- 5 years) develop at a rate of 1.70 cancers/100 patient years, 95% confidence interval = 1.89-6.51 for the difference, P = .001. CONCLUSION: Patients with nontraumatic spinal cord lesions caused by AVM often present with pain, may have a protracted clinical course, and have cancer develop more often than do those with traumatic SCI.

Dynamic regulation of aquaglyceroporin expression in erythrocyte cultures from cold- and warm-acclimated Cope's gray treefrog, Hyla chrysoscelis.

Cope's gray treefrog, Hyla chrysoscelis,is a freeze-tolerant anuran which accumulates and distributes glycerol as a cryoprotectant before freezing. We hypothesize that HC-3, an aquaglyceroporin member of the MIP family of water pores, may play an important role in the process of freeze tolerance by mediating transmembrane passage of glycerol and water during cold-acclimation. The objectives of this study were two-fold: to examine HC-3 protein abundance and cellular localization in erythrocytes from cold- and warm-acclimated frogs and to develop and characterize an erythrocyte cell culture system for examining HC-3 gene regulation. Compared with warm-acclimated frogs, erythrocytes from cold-acclimated frogs had higher HC-3 protein expression and enhanced plasma membrane localization. Furthermore, erythrocytes from cold- and warm-acclimated frogs maintained in culture at 4 and 20°C exhibited time- and temperature-dependent regulation of HC-3 expression and an increase in the abundance of high molecular weight immunoreactive species within 24 hr of culture at 20°C. Deglycosylation of erythrocyte proteins resulted in the disappearance of the high molecular weight species, indicating that HC-3 is post-translationally modified by N-linked glycosylation. Erythrocytes cultured in media containing glycerol also showed an increased abundance of the high molecular weight bands and enhanced plasma membrane localization of HC-3, suggesting a role for glycerol in regulating HC-3 subcellular trafficking. Thus, the development of this erythrocyte cell culture system from H. chrysoscelis opened an opportunity to study the properties of cells with changing expression of an aquaglyceroporin, HC-3, and to explore the factors regulating that expression.

Endo-Porter-mediated delivery of phosphorodiamidate morpholino oligos (PMOs) in erythrocyte suspension cultures from Cope's gray treefrog Hyla chrysoscelis.

Cope's gray treefrog, Hyla chrysoscelis, is a freeze-tolerant anuran that accumulates cryoprotective glycerol during cold acclimation. H. chrysoscelis erythrocytes express the aquaglyceroporin HC-3, which facilitates transmembrane glycerol and water movement. Aquaglyceroporins have no pharmacological inhibitors, and no genetic knockout tools currently exist for H. chrysoscelis. A phosphorodiamidate morpholino oligo (PMO)-mediated expression knockdown approach was therefore pursued to provide a model for testing the role of HC-3. We describe a novel procedure optimized for specific, efficient knockdown of HC-3 expression in amphibian erythrocyte suspensions cultured at nonmammalian physiological temperatures using Endo-Porter. Our protocol includes three critical components: pre-incubation at 37°C, two rounds of Endo-Porter and HC-3 PMO administration at ~23°C, and continuous shaking at 190 rpm. This combination of steps resulted in 94% reduction in HC-3 protein expression (Western blot), substantial decrease in HC-3 expression in >65% of erythrocytes, and no detectable expression in an additional 30% of cells (immunocytochemistry).

Glycerol uptake by erythrocytes from warm- and cold-acclimated Cope's gray treefrogs.

Cope's gray treefrogs, Hyla chrysoscelis, accumulate glycerol during the period of cold acclimation that leads to the development of freeze tolerance. Glycerol must cross cell membranes in numerous processes during this time, including exit from hepatocytes where glycerol is synthesized and entry into other tissues, where glycerol is cryoprotective. Thus, we hypothesized that erythrocytes from H. chrysoscelis would be permeable to glycerol and that that permeability would be up-regulated during cold acclimation. Further, we hypothesized that glycerol permeability would be associated with the expression of aquaporins, particularly those from the glyceroporin sub-family. Erythrocytes from warm-acclimated treefrogs had high glycerol permeability at 20°C, as assessed by the time required for osmotic lysis following suspension in 0.2 M glycerol. That osmotic lysis, as well as uptake of radio-labeled glycerol, was inhibited by 0.3 mM HgCl(3). Permeability assessed via osmotic lysis was markedly reduced at 5°C. These properties were similar in animals deriving from northern (Ohio) and southern (Alabama) populations, although suggestive (through statistical interactions) of greater glycerol permeability in northern animals. Erythrocytes expressed mRNA and protein for a previously described glyceroporin, HC-3. In cold-acclimated animals, HC-3 protein expression was up-regulated, but we could not detect a concomitant enhancement of glycerol permeability.