Biliary Leaks: Multidisciplinary Approach to Diagnosis and Treatment.

Bile leaks arise from various causes such as trauma, complications after hepatobiliary surgery, and intrahepatic malignancies or their associated liver-directed treatments. Bile leaks can result in significant morbidity and mortality. Delayed diagnosis is not uncommon due to nonspecific manifestations; therefore, a high clinical suspicion is needed. A multidisciplinary approach for treatment of biliary leaks with prompt referral to tertiary care centers with experienced hepatobiliary surgeons, advanced endoscopists, and interventional radiologists is needed to address these challenging complications. Management of biliary leaks can range from conservative management to open surgical repair. Minimally invasive procedures play a crucial role in biliary leak treatment, and the interventional radiologist can help guide appropriate management on the basis of a clear understanding of the pathophysiology of biliary leaks and a current knowledge of the armamentarium of treatment options. In most cases, a simple diversion of bile to decompress the biliary system may prove effective. However, persistent and high-output biliary leaks require delineation of the source with tailored treatment options to control the leak. This may be done by additional diversions, occluding the source, reestablishing connections, or using a combination of therapies to bridge to more definitive surgical interventions. The authors describe the different treatment options and emphasize the role of interventional radiology. ©RSNA, 2024.

Evaluation of the Catheter Clamp over Hydrophilic Guide Wire Central Venous Catheter Exchange Technique for Air Embolism Prophylaxis in an In Vitro Model.

PURPOSE: To develop a reproducible in vitro model simulating central venous catheter (CVC) exchange with high potential for air embolization and test the hypothesis that a closed catheter clamp over hydrophilic guide wire exchange technique will significantly reduce the volume of air introduced during CVC exchange. MATERIALS AND METHODS: The model consisted of a 16-F valved sheath, 240-mL container, and pressure transducer submerged in water in a 1,200-mL suction canister system. Continuous wall suction was applied to the canister to maintain negative pressure at -7 mm Hg or -11 mm Hg. Each trial consisted of 0.035-inch hydrophilic guide wire introduction, over-the-wire catheter exchange, and wire removal following clinical protocol. A total of 256 trials were performed, 128 trials at each pressure with the catheter clamp open (n = 64) or closed (n = 64) around the hydrophilic guide wire. RESULTS: There was a statistically significant lower volume of air introduced with closed clamp over-the-wire exchanges than with open clamp exchanges at both pressures (2-tailed t-test, P < .001). At -7 mm Hg, a mean of 48.0 mL (SD ± 9.3) of air was introduced with open clamp and 20.6 mL (SD ± 4.7) of air was introduced with closed clamp. At -11 mm Hg, 97.8 mL (SD ± 11.9) of air was introduced with open clamp and 37.8 mL (SD ± 6.3) of air was introduced with closed clamp. CONCLUSIONS: This study demonstrated the use of a reproducible in vitro model mimicking conditions causing air embolism during CVC exchange. Results showed that CVC exchange using closed catheter clamp over hydrophilic guide wire exchange technique significantly reduced the volume of air introduced per exchange.

Generation of precision preclinical cancer models using regulated in vivo base editing.

Although single-nucleotide variants (SNVs) make up the majority of cancer-associated genetic changes and have been comprehensively catalogued, little is known about their impact on tumor initiation and progression. To enable the functional interrogation of cancer-associated SNVs, we developed a mouse system for temporal and regulatable in vivo base editing. The inducible base editing (iBE) mouse carries a single expression-optimized cytosine base editor transgene under the control of a tetracycline response element and enables robust, doxycycline-dependent expression across a broad range of tissues in vivo. Combined with plasmid-based or synthetic guide RNAs, iBE drives efficient engineering of individual or multiple SNVs in intestinal, lung and pancreatic organoids. Temporal regulation of base editor activity allows controlled sequential genome editing ex vivo and in vivo, and delivery of sgRNAs directly to target tissues facilitates generation of in situ preclinical cancer models.

Drag-and-drop genome insertion of large sequences without double-strand DNA cleavage using CRISPR-directed integrases.

Programmable genome integration of large, diverse DNA cargo without DNA repair of exposed DNA double-strand breaks remains an unsolved challenge in genome editing. We present programmable addition via site-specific targeting elements (PASTE), which uses a CRISPR-Cas9 nickase fused to both a reverse transcriptase and serine integrase for targeted genomic recruitment and integration of desired payloads. We demonstrate integration of sequences as large as ~36 kilobases at multiple genomic loci across three human cell lines, primary T cells and non-dividing primary human hepatocytes. To augment PASTE, we discovered 25,614 serine integrases and cognate attachment sites from metagenomes and engineered orthologs with higher activity and shorter recognition sequences for efficient programmable integration. PASTE has editing efficiencies similar to or exceeding those of homology-directed repair and non-homologous end joining-based methods, with activity in non-dividing cells and in vivo with fewer detectable off-target events. PASTE expands the capabilities of genome editing by allowing large, multiplexed gene insertion without reliance on DNA repair pathways.

Creation of an Ex Vivo Renal Perfusion Model to Investigate Microwave Ablation.

This study hypothesized that an ex vivo renal perfusion model can create smaller microwave ablation (MWA) measurements during perfused states compared with nonperfused states across multiple device settings. Nine bovine kidneys, a fluoroscopic compatible perfusion model, and a commercially-available clinical MWA system were used to perform 72 ablations (36 perfused and 36 nonperfused) at 9 different device settings. Comparing perfused and nonperfused ablations at each device setting, significant differences in volume existed for 6 of 9 settings (P < .05). Collapsed across time settings, the ablation volumes by power were the following (perfused and nonperfused, P value): 60 W, 2.3 cm3 ± 1.0 and 7.2 cm3 ± 2.7, P < .001; 100 W, 5.4 cm3 ± 2.1 and 11.5 cm3 ± 5.6, P < .01; and 140 W, 11.2 cm3 ± 3.7 and 18.7 cm3 ± 6.3, P < .01. Applied power correlated with ablation volume: perfused, 0.021 cm3/W and R = 0.462, P = .004, and nonperfused, 0.029 cm3/W and R = 0.565, P < .001. These results support that an ex vivo perfused organ system can evaluate MWA systems and demonstrate heat sink perfusion effects of decreased ablation size.

Broad-spectrum CRISPR-mediated inhibition of SARS-CoV-2 variants and endemic coronaviruses in vitro.

A major challenge in coronavirus vaccination and treatment is to counteract rapid viral evolution and mutations. Here we demonstrate that CRISPR-Cas13d offers a broad-spectrum antiviral (BSA) to inhibit many SARS-CoV-2 variants and diverse human coronavirus strains with >99% reduction of the viral titer. We show that Cas13d-mediated coronavirus inhibition is dependent on the crRNA cellular spatial colocalization with Cas13d and target viral RNA. Cas13d can significantly enhance the therapeutic effects of diverse small molecule drugs against coronaviruses for prophylaxis or treatment purposes, and the best combination reduced viral titer by over four orders of magnitude. Using lipid nanoparticle-mediated RNA delivery, we demonstrate that the Cas13d system can effectively treat infection from multiple variants of coronavirus, including Omicron SARS-CoV-2, in human primary airway epithelium air-liquid interface (ALI) cultures. Our study establishes CRISPR-Cas13 as a BSA which is highly complementary to existing vaccination and antiviral treatment strategies.

Comparative outcomes of Inferior Vena Cava filters placed at bedside using digital radiography versus conventional fluoroscopy.

PURPOSE: To retrospectively assess the outcomes of Inferior Vena Cava (IVC) filters placed in critically ill patients in the ICU at bedside using digital radiograph (DR) guidance with previous cross-sectional imaging for planning, compared to IVC filters placed by conventional fluoroscopy (CF). METHOD AND MATERIALS: The cohort consisted of 129 IVC filter placements; 48 placed at bedside and 81 placed conventionally from July 2015 to September 2016. Patient demographics, indication, radiation exposures, access site, procedural duration, dwell time, and complications were identified by the EMR. IVC Filter positioning with measurements of tip to renal vein distance and lateral filter tilt were performed when cavograms or post placement CTs were available for review. Statistical analysis was performed using Stata IC 11.2. RESULTS: Technical success of the procedure was 100% in both groups. Procedural duration was longer at the bedside lasting 14.5 +/- 10.2 versus 6.7 +/- 6.0 â€‹min (p<0.0001). The bedside DR group had a median radiation exposure of 25 â€‹mGy (15-35) and the CF group had mean radiation exposure of 256.94 â€‹mGy +/- 158.6. There was no significant difference in distance of IVC tip to renal vein (p=0.31), mispositioning (p=0.59), degree of filter tilt (p=0.33), or rate of complications (p=0.65) between the two groups. CONCLUSION: IVCF placement at the bedside using DR is comparable to CF with no statistical difference in outcomes based on IVCF positioning, degree of lateral tilt or removal issues. It decreased radiation dose, but with overall increased procedural time.

In vivo adenine base editing of PCSK9 in macaques reduces LDL cholesterol levels.

Most known pathogenic point mutations in humans are C•G to T•A substitutions, which can be directly repaired by adenine base editors (ABEs). In this study, we investigated the efficacy and safety of ABEs in the livers of mice and cynomolgus macaques for the reduction of blood low-density lipoprotein (LDL) levels. Lipid nanoparticle-based delivery of mRNA encoding an ABE and a single-guide RNA targeting PCSK9, a negative regulator of LDL, induced up to 67% editing (on average, 61%) in mice and up to 34% editing (on average, 26%) in macaques. Plasma PCSK9 and LDL levels were stably reduced by 95% and 58% in mice and by 32% and 14% in macaques, respectively. ABE mRNA was cleared rapidly, and no off-target mutations in genomic DNA were found. Re-dosing in macaques did not increase editing, possibly owing to the detected humoral immune response to ABE upon treatment. These findings support further investigation of ABEs to treat patients with monogenic liver diseases.

Prime editing in mice reveals the essentiality of a single base in driving tissue-specific gene expression.

BACKGROUND: Most single nucleotide variants (SNVs) occur in noncoding sequence where millions of transcription factor binding sites (TFBS) reside. Here, a comparative analysis of CRISPR-mediated homology-directed repair (HDR) versus the recently reported prime editing 2 (PE2) system was carried out in mice over a TFBS called a CArG box in the Tspan2 promoter. RESULTS: Quantitative RT-PCR showed loss of Tspan2 mRNA in aorta and bladder, but not heart or brain, of mice homozygous for an HDR-mediated three base pair substitution in the Tspan2 CArG box. Using the same protospacer, mice homozygous for a PE2-mediated single-base substitution in the Tspan2 CArG box displayed similar cell-specific loss of Tspan2 mRNA; expression of an overlapping long noncoding RNA was also nearly abolished in aorta and bladder. Immuno-RNA fluorescence in situ hybridization validated loss of Tspan2 in vascular smooth muscle cells of HDR and PE2 CArG box mutant mice. Targeted sequencing demonstrated variable frequencies of on-target editing in all PE2 and HDR founders. However, whereas no on-target indels were detected in any of the PE2 founders, all HDR founders showed varying levels of on-target indels. Off-target analysis by targeted sequencing revealed mutations in many HDR founders, but none in PE2 founders. CONCLUSIONS: PE2 directs high-fidelity editing of a single base in a TFBS leading to cell-specific loss in expression of an mRNA/long noncoding RNA gene pair. The PE2 platform expands the genome editing toolbox for modeling and correcting relevant noncoding SNVs in the mouse.

Can Dynamic Contrast-Enhanced CT Quantify Perfusion in a Stimulated Muscle of Limited Size? A Rat Model.

BACKGROUND: Muscle injury may result in damage to the vasculature, rendering it unable to meet the metabolic demands of muscle regeneration and healing. Therefore, therapies frequently aim to maintain, restore, or improve blood supply to the injured muscle. Although there are several options to assess the vascular outcomes of these therapies, few are capable of spatially assessing perfusion in large volumes of tissue. QUESTIONS/PURPOSES: Can dynamic contrast-enhanced CT (DCE-CT) imaging acquired with a clinical CT scanner be used in a rat model to quantify perfusion in the anterior tibialis muscle at spatially relevant volumes, as assessed by (1) the blood flow rate and tissue blood volume in the muscle after three levels of muscle stimulation (low, medium, and maximum) relative to baseline as determined by the non-stimulated contralateral leg; and (2) how do these measurements compare with those obtained by the more standard approach of microsphere perfusion? METHODS: The right anterior tibialis muscles of adult male Sprague Dawley rats were randomized to low- (n = 10), medium- (n = 6), or maximum- (n = 3) level (duty cycles of 2.5%, 5.0%, and 20%, respectively) nerve electrode coupled muscle stimulation directly followed by DCE-CT imaging. Tissue blood flow and blood volume maps were created using commercial software and volumetrically measured using NIH software. Although differences in blood flow were detectable across the studied levels of muscle stimulation, a review of the evidence suggested the absolute blood flow quantified was underestimated. Therefore, at a later date, a separate set of adult male Sprague Dawley rats were randomized for microsphere perfusion (n = 7) to define blood flow in the animal model with an accepted standard. With this technique, intra-arterial particles sized to freely flow in blood but large enough to lodge in tissue capillaries were injected. Simultaneously, blood sampling at a fixed flow rate was simultaneously performed to provide a fixed blood flow rate sample. The tissues of interest were then explanted and assessed for the total number of particles per tissue volume. Tissue blood flow rate was then calculated based on the particle count ratio within the reference sample. Note that a tissue's blood volume cannot be calculated with this method. Comparison analysis to the non-stimulated baseline leg was performed using two-tailed paired student t-test. An ANOVA was used to compare difference between stimulation groups. RESULTS: DCE-CT measured (mean ± SD) increasing tissue blood flow differences in stimulated anterior tibialis muscle at 2.5% duty cycle (32 ± 5 cc/100 cc/min), 5.0% duty cycle (46 ± 13 cc/100 cc/min), and 20% duty cycle (73 ± 3 cc/100 cc/min) compared with the paired contralateral non-stimulated anterior tibialis muscle (10 ± 2 cc/100 cc/min, mean difference 21 cc/100 cc/min [95% CI 17.08 to 25.69]; 9 ± 1 cc/100 cc/min, mean difference 37 cc/100 cc/min [95% CI 23.06 to 50.11]; and 11 ± 2 cc/100 cc/min, mean difference 62 cc/100 cc/min [95% CI 53.67 to 70.03]; all p < 0.001). Similarly, DCE-CT showed increasing differences in tissue blood volumes within the stimulated anterior tibialis muscle at 2.5% duty cycle (23.2 ± 4.2 cc/100 cc), 5.0% duty cycle (39.2 ± 7.2 cc/100 cc), and 20% duty cycle (52.5 ± 13.1 cc/100 cc) compared with the paired contralateral non-stimulated anterior tibialis muscle (3.4 ± 0.7 cc/100 cc, mean difference 19.8 cc/100 cc [95% CI 16.46 to 23.20]; p < 0.001; 3.5 ± 0.4 cc/100 cc, mean difference 35.7 cc/100 cc [95% CI 28.44 to 43.00]; p < 0.001; and 4.2 ± 1.3 cc/100 cc, mean difference 48.3 cc/100 cc [95% CI 17.86 to 78.77]; p = 0.010). Microsphere perfusion measurements also showed an increasing difference in tissue blood flow in the stimulated anterior tibialis muscle at 2.5% duty cycle (62 ± 43 cc/100 cc/min), 5.0% duty cycle (89 ± 52 cc/100 cc/min), and 20% duty cycle (313 ± 269 cc/100 cc/min) compared with the paired contralateral non-stimulated anterior tibialis muscle (8 ± 4 cc/100 cc/min, mean difference 55 cc/100 cc/min [95% CI 15.49 to 94.24]; p = 0.007; 9 ± 9 cc/100 cc/min, mean difference 79 cc/100 cc/min [95% CI 33.83 to 125.09]; p = 0.003; and 18 ± 18 cc/100 cc/min, mean difference 295 cc/100 cc/min [95% CI 8.45 to 580.87]; p = 0.023). Qualitative comparison between the methods suggests that DCE-CT values underestimate tissue blood flow with a post-hoc ANOVA showing DCE-CT blood flow values within the 2.5% duty cycle group (32 ± 5 cc/100 cc/min) to be less than the microsphere perfusion value (62 ± 43 cc/100 cc/min) with a mean difference of 31 cc/100 cc/min (95% CI 2.46 to 60.23; p = 0.035). CONCLUSIONS: DCE-CT using a clinical scanner is a feasible modality to measure incremental changes of blood flow and tissue blood volume within a spatially challenged small animal model. Care should be taken in studies where true blood flow values are needed, as this particular small-volume muscle model suggests true blood flow is underestimated using the specific adaptions of DCE-CT acquisition and image processing chosen. CLINICAL RELEVANCE: CT perfusion is a clinically available modality allowing for translation of science from bench to bedside. Adapting the modality to fit small animal models that are relevant to muscle healing may hasten time to clinical utility.

Impact of Required Versus Self-Directed Use of Virtual Patient Cases on Clerkship Performance: A Mixed-Methods Study.

PURPOSE: To explore how students use and benefit from virtual patient cases (VPCs). METHOD: In academic years 2013-2014 and 2014-2015, cohorts of students in pediatrics (Peds), family medicine (FM), and internal medicine (IM) clerkships were allocated to either core required use (CRU) or self-directed use (SU) of MedU VPCs. Outcomes included number and time of case review, student perception of learning from VPCs, National Board of Medical Examiners (NBME) subject examination scores, and summative clinical ratings for medical knowledge and differential diagnoses/problem solving. Focus groups were conducted each year. Mean differences were compared by t test. RESULTS: A total of 255 students participated in the study. Mean number of cases completed by the CRU group was significantly higher than that by the SU group (13.9 vs. 3.1 for FM, 16.1 vs. 3.9 for Peds, and 10.4 vs. 1.2 for IM) (P < .001). Student-perceived value ratings of VPCs were similar between groups. Students described VPCs as time consuming but useful for supplementing clinical conditions not seen in person. Mean scores on NBME subject examinations for CRU versus SU groups were not different between groups in any clerkship, nor were there significant differences in the summative clinical ratings for medical knowledge or differential diagnosis/clinical reasoning. CONCLUSIONS: Although VPCs continue to serve an important role in exposing students to clinical conditions not seen in person, the optimal employment of this technology in clerkship pedagogy requires further exploration.

A Closer Look at the Hippocampus and Memory.

Current interpretations of hippocampal memory function are blind to the fact that viewing behaviors are pervasive and complicate the relationships among perception, behavior, memory, and brain activity. For example, hippocampal activity and associative memory demands increase with stimulus complexity. Stimulus complexity also strongly modulates viewing. Associative processing and viewing thus are often confounded, rendering interpretation of hippocampal activity ambiguous. Similar considerations challenge many accounts of hippocampal function. To explain relationships between memory and viewing, we propose that the hippocampus supports the online memory demands necessary to guide visual exploration. The hippocampus thus orchestrates memory-guided exploration that unfolds over time to build coherent memories. This new perspective on hippocampal function harmonizes with the fact that memory formation and exploratory viewing are tightly intertwined.

Macular Carotenoids, Aerobic Fitness, and Central Adiposity Are Associated Differentially with Hippocampal-Dependent Relational Memory in Preadolescent Children.

OBJECTIVES: To examine the associations of macular pigment carotenoids (lutein, meso-zeaxanthin, and zeaxanthin), aerobic fitness, and central adiposity with hippocampal-dependent relational memory in prepubescent children. STUDY DESIGN: Children between 7 and 10 years of age (n = 40) completed a task designed to assess relational memory performance and participated in aerobic fitness, adiposity, and macular pigment optical density (MPOD) assessment. Aerobic fitness was assessed via a modified Balke treadmill protocol designed to measure maximal oxygen volume. Central adiposity was assessed via dual-energy x-ray absorptiometry. MPOD was measured psychophysically by the use of customized heterochromatic flicker photometry. Statistical analyses included correlations and hierarchical linear regression. RESULTS: Aerobic fitness and MPOD were associated negatively with relational memory errors (P < .01), whereas central adiposity was associated positively with relational memory errors (P < .05). Hierarchical regression analysis revealed that MPOD accounted for a significant amount of the variance in relational memory performance even after we accounted for aerobic fitness (ß = -0.388, P = .007). CONCLUSIONS: Even after we adjusted for aerobic fitness and central adiposity, factors known to relate to hippocampal-dependent memory, MPOD positively and significantly predicted hippocampal-dependent memory performance. TRIAL REGISTRATION: ClinicalTrials.gov: NCT01619826.

Hippocampal structure predicts cortical indices of reactivation of related items.

One of the key components of relational memory is the ability to bind together the constituent elements of a memory experience, and this ability is thought to be supported by the hippocampus. Previously we had shown that these relational bindings can be used to reactivate the cortical processors of an absent item in the presence of a relationally bound associate (Walker et al., 2014). Specifically, we recorded the event-related optical signal (EROS) when presenting the scene of a face-scene pair during a preview period immediately preceding a test display, and demonstrated reactivation of a face-processing cortical area (the superior temporal sulcus, STS) for scenes that had been previously paired with faces, relative to scenes that had not. Here we combined the EROS measures during the same preview paradigm with anatomical estimates of hippocampal integrity (structural MRI measures of hippocampal volume and diffusion tensor imaging measures of mean fractional anisotropy and diffusivity) to provide evidence that the hippocampus is mediating this reactivation phenomenon. The study was run in a sample of older adults aged 55-87, taking advantage of the high amount of hippocampal variability present in aging. We replicated the functional reactivation of STS during the preview period, specific to scenes previously paired with faces. Crucially, we also found that this phenomenon is correlated with structural hippocampus integrity. Both STS reactivation and hippocampal structure predicted subsequent recognition performance. These data support the theory that relational memory is sustained by an interaction between hippocampal and cortical sensory processing regions, and that these functions may be at the basis of episodic memory changes in normal aging.

Implementation of a Mini-CEX Requirement Across All Third-Year Clerkships.

PROBLEM: Direct observation of medical students performing clinical tasks, such as eliciting a patient history or examining a patient, and the provision of feedback, are foundational to student improvement but have been reported to occur infrequently. The mini clinical evaluation exercise (mini-CEX) is a tool that can facilitate direct observation and feedback. This study assessed the impact of a mini-CEX requirement across all 3rd-year clerkships on student report of direct observation by faculty and objectively measured clinical skills. INTERVENTION: A mini-CEX requirement across all 3rd-year clerkships was implemented in the 2012-2013 academic year. The impact of the mini-CEX requirement on student report of direct observation was assessed by end-of-clerkship surveys and Association of American Medical Colleges (AAMC) Graduation Questionnaire (GQ) items on direct observation. The impact on students' clinical skills was assessed by a summative Objective Structured Clinical Examination (OSCE). Pre/post comparisons were assessed with chi-square and Fisher's exact tests. CONTEXT: A mini-CEX requirement had been in place for the internal medicine clerkship, and student reports of direct observation were historically higher for the internal medicine clerkship than for other clerkships. Faculty, residents, and students at each of the clinical sites across all 6 clerkships were oriented to the use of the mini-CEX; the feasibility of its use during usual patient interaction settings and the importance of direct observation and feedback for student improvement were emphasized during these sessions. OUTCOME: Adherence to the mini-CEX requirement was high: 92% of required forms were completed, and 78% of completed forms indicated that specific feedback was given. The proportion of students reporting direct observation of physical examination significantly increased in all clerkships, with the largest relative increase occurring in surgery (from 49% to 87%), χ2(1, N = 225) = 37.70, p < .0001. Significant increases were seen in faculty observation of history taking in pediatrics, surgery, and psychiatry. Direct observation rates also increased on the AAMC GQ items for history taking and physical exam for all clerkships. Failures on the summative OSCE decreased from 12% preintervention to 2% postintervention (p = .0046). LESSONS LEARNED: Institution of a mini-CEX requirement was feasible across all 3rd-year clerkships and was associated with a significant increase in student report of direct observation by faculty and a decrease in summative OSCE failure rates.

When memory leads the brain to take scenes at face value: face areas are reactivated at test by scenes that were paired with faces at study.

In the first use of the event-related optical signal as a brain imaging tool for the study of long-term memory, we examined relational or associative aspects of memory, widely presumed to involve the interplay among multiple brain regions in representing and reactivating different elements of a given event. Here, we found that a brain region known to be involved in face processing (the posterior superior temporal sulcus) was active not only when viewing faces during the study phase but also when viewing scenes at test that, through prior learning, were associated with specific faces. These findings, demonstrating the activation of stimulus-specific cortical regions in the absence of stimuli of that type, based on learned relations, reveal cortical substrates of the reactivation of relational memories.

Hydroxyapatite scaffold pore architecture effects in large bone defects in vivo.

To examine the effect of scaffold pore size on bone regeneration within hydroxyapatite scaffolds in large segmental defects, this study evaluated two porous interconnected architectures having similar porosity and strut thickness but different pore sizes. Using a 10 mm segmental rabbit radius defect model, a bilayer scaffold architecture mimicking the cortical-cancellous organization of bone (pore size 200 µm outer layer, 450 µm inner layer) was compared to a purely trabecular-like architecture (pore size 340 µm) and an untreated defect. Bone regeneration was measured using micro-computed tomography and histology after four and eight weeks of in vivo implantation, and the mechanical strength of the defect site after eight weeks' implantation was assessed using flexural testing. Although both bilayer and trabecular architectures promoted bone growth, the trabecular scaffolds were observed to have more uniform new bone distribution within the scaffold interior at four weeks and greater bone regeneration overall after eight weeks' implantation (149 ± 9 mm³ compared to 121 ± 8 mm³ in the bilayer and 66 ± 14 mm³ in the defect). Additionally, the trabecular scaffolds were observed to exhibit significantly greater flexural strength (124% increase) and toughness (388% increase) when compared to the empty defects after eight weeks' implantation. It was concluded from this study that a larger uniform pore size led to greater functional bone regeneration over a longer implantation period for large segmental defects.

Variation in salamander tail regeneration is associated with genetic factors that determine tail morphology.

Very little is known about the factors that cause variation in regenerative potential within and between species. Here, we used a genetic approach to identify heritable genetic factors that explain variation in tail regenerative outgrowth. A hybrid ambystomatid salamander (Ambystoma mexicanum x A. andersoni) was crossed to an A. mexicanum and 217 offspring were induced to undergo metamorphosis and attain terrestrial adult morphology using thyroid hormone. Following metamorphosis, each salamander's tail tip was amputated and allowed to regenerate, and then amputated a second time and allowed to regenerate. Also, DNA was isolated from all individuals and genotypes were determined for 187 molecular markers distributed throughout the genome. The area of tissue that regenerated after the first and second amputations was highly positively correlated across males and females. Males presented wider tails and regenerated more tail tissue during both episodes of regeneration. Approximately 66-68% of the variation in regenerative outgrowth was explained by tail width, while tail length and genetic sex did not explain a significant amount of variation. A small effect QTL was identified as having a sex-independent effect on tail regeneration, but this QTL was only identified for the first episode of regeneration. Several molecular markers significantly affected regenerative outgrowth during both episodes of regeneration, but the effect sizes were small (<4%) and correlated with tail width. The results show that ambysex and minor effect QTL explain variation in adult tail morphology and importantly, tail width. In turn, tail width at the amputation plane largely determines the rate of regenerative outgrowth. Because amputations in this study were made at approximately the same position of the tail, our results resolve an outstanding question in regenerative biology: regenerative outgrowth positively co-varies as a function of tail width at the amputation site.

Salamander Hox clusters contain repetitive DNA and expanded non-coding regions: a typical Hox structure for non-mammalian tetrapod vertebrates?

Hox genes encode transcription factors that regulate embryonic and post-embryonic developmental processes. The expression of Hox genes is regulated in part by the tight, spatial arrangement of conserved coding and non-coding sequences. The potential for evolutionary changes in Hox cluster structure is thought to be low among vertebrates; however, recent studies of a few non-mammalian taxa suggest greater variation than originally thought. Using next generation sequencing of large genomic fragments (>100 kb) from the red spotted newt (Notophthalamus viridescens), we found that the arrangement of Hox cluster genes was conserved relative to orthologous regions from other vertebrates, but the length of introns and intergenic regions varied. In particular, the distance between hoxd13 and hoxd11 is longer in newt than orthologous regions from vertebrate species with expanded Hox clusters and is predicted to exceed the length of the entire HoxD clusters (hoxd13-hoxd4) of humans, mice, and frogs. Many repetitive DNA sequences were identified for newt Hox clusters, including an enrichment of DNA transposon-like sequences relative to non-coding genomic fragments. Our results suggest that Hox cluster expansion and transposon accumulation are common features of non-mammalian tetrapod vertebrates.

Pharmacist-led workshops to enhance pharmacotherapy knowledge for medical students.

BACKGROUND: Graduating medical students have reported concern regarding inadequate training in pharmacotherapy. Teaching by clinical pharmacists may improve medical students' pharmacotherapy knowledge. PURPOSE: To assess the impact of pharmacist led workshops on 4th year medical students' knowledge of pharmacotherapy and satisfaction. METHODS: Senior medical students enrolled in intensive care unit rotations at a US medical school were randomized to an intervention of pharmacist led case-based workshops or a control group without an explicit pharmacotherapy curriculum. Intervention group students attended four weekly 1-hour workshops that covered topics in pharmacokinetics, pharmacodynamics, drug interactions and toxicity. A multiple-choice test of clinical vignettes assessed students' knowledge of pharmacotherapy. An end of clerkship survey assessed student satisfaction with teaching. RESULTS: Of 176 medical students eligible, 148 agreed to participate and were randomized to the intervention (n = 63) or control groups (n = 85). Student satisfaction with pharmacist led workshops was high. End of clerkship performance on clinical vignettes (minimum score 0, maximum 100) was similar between the groups (mean score 47 (SD = 12.2) for intervention vs 44 (SD = 13.0) for control group, p = 0.16). On end of clerkship survey, only 8% of control group students agreed or strongly agreed that the standard curriculum provided sufficient teaching in pharmacotherapy. The majority of students (82%) felt that pharmacotherapy should be taught formally in the clinical years. CONCLUSION: Pharmacist led workshops on pharmacotherapy were well received by senior medical students but did not improve performance on a test of pharmacotherapy knowledge. Further study is needed to define optimal strategies for improving medical students' pharmacotherapy knowledge.