Development of an online authentic radiology viewing and reporting platform to test the skills of radiology trainees in Low- and Middle-Income Countries.

BACKGROUND: Diagnostic radiology residents in low- and middle-income countries (LMICs) may have to provide significant contributions to the clinical workload before the completion of their residency training. Because of time constraints inherent to the delivery of acute care, some of the most clinically impactful diagnostic radiology errors arise from the use of Computed Tomography (CT) in the management of acutely ill patients. As a result, it is paramount to ensure that radiology trainees reach adequate skill levels prior to assuming independent on-call responsibilities. We partnered with the radiology residency program at the Aga Khan University Hospital in Nairobi (Kenya) to evaluate a novel cloud-based testing method that provides an authentic radiology viewing and interpretation environment. It is based on Lifetrack, a unique Google Chrome-based Picture Archiving and Communication System, that enables a complete viewing environment for any scan, and provides a novel report generation tool based on Active Templates which are a patented structured reporting method. We applied it to evaluate the skills of AKUHN trainees on entire CT scans representing the spectrum of acute non-trauma abdominal pathology encountered in a typical on-call setting. We aimed to demonstrate the feasibility of remotely testing the authentic practice of radiology and to show that important observations can be made from such a Lifetrack-based testing approach regarding the radiology skills of an individual practitioner or of a cohort of trainees. METHODS: A total of 13 anonymized trainees with experience from 12 months to over 4 years took part in the study. Individually accessing the Lifetrack tool they were tested on 37 abdominal CT scans (including one normal scan) over six 2-hour sessions on consecutive days. All cases carried the same clinical history of acute abdominal pain. During each session the trainees accessed the corresponding Lifetrack test set using clinical workstations, reviewed the CT scans, and formulated an opinion for the acute diagnosis, any secondary pathology, and incidental findings on the scan. Their scan interpretations were composed using the Lifetrack report generation system based on active templates in which segments of text can be selected to assemble a detailed report. All reports generated by the trainees were scored on four different interpretive components: (a) acute diagnosis, (b) unrelated secondary diagnosis, (c) number of missed incidental findings, and (d) number of overcalls. A 3-score aggregate was defined from the first three interpretive elements. A cumulative score modified the 3-score aggregate for the negative effect of interpretive overcalls. RESULTS: A total of 436 scan interpretations and scores were available from 13 trainees tested on 37 cases. The acute diagnosis score ranged from 0 to 1 with a mean of 0.68 ± 0.36 and median of 0.78 (IQR: 0.5-1), and there were 436 scores. An unrelated secondary diagnosis was present in 11 cases, resulting in 130 secondary diagnosis scores. The unrelated secondary diagnosis score ranged from 0 to 1, with mean score of 0.48 ± 0.46 and median of 0.5 (IQR: 0-1). There were 32 cases with incidental findings, yielding 390 scores for incidental findings. The number of missed incidental findings ranged from 0 to 5 with a median at 1 (IQR: 1-2). The incidental findings score ranged from 0 to 1 with a mean of 0.4 ± 0.38 and median of 0.33 (IQR: 0- 0.66). The number of overcalls ranged from 0 to 3 with a median at 0 (IQR: 0-1) and a mean of 0.36 ± 0.63. The 3-score aggregate ranged from 0 to 100 with a mean of 65.5 ± 32.5 and median of 77.3 (IQR: 45.0, 92.5). The cumulative score ranged from - 30 to 100 with a mean of 61.9 ± 35.5 and median of 71.4 (IQR: 37.4, 92.0). The mean acute diagnosis scores and SD by training period were 0.62 ± 0.03, 0.80 ± 0.05, 0.71 ± 0.05, 0.58 ± 0.07, and 0.66 ± 0.05 for trainees with ≤ 12 months, 12-24 months, 24-36 months, 36-48 months and > 48 months respectively. The mean acute diagnosis score of 12-24 months training was the only statistically significant greater score when compared to ≤ 12 months by the ANOVA with Tukey testing (p = 0.0002). We found a similar trend with distribution of 3-score aggregates and cumulative scores. There were no significant associations when the training period was categorized as less than and more than 2 years. We looked at the distribution of the 3-score aggregate versus the number of overcalls by trainee, and we found that the 3-score aggregate was inversely related to the number of overcalls. Heatmaps and raincloud plots provided an illustrative means to visualize the relative performance of trainees across cases. CONCLUSION: We demonstrated the feasibility of remotely testing the authentic practice of radiology and showed that important observations can be made from our Lifetrack-based testing approach regarding radiology skills of an individual or a cohort. From observed weaknesses areas for targeted teaching can be implemented, and retesting could reveal their impact. This methodology can be customized to different LMIC environments and expanded to board certification examinations.

Chemical-Shift Perturbations Reflect Bile Acid Binding to Norovirus Coat Protein: Recognition Comes in Different Flavors.

Bile acids have been reported as important cofactors promoting human and murine norovirus (NoV) infections in cell culture. The underlying mechanisms are not resolved. Through the use of chemical shift perturbation (CSP) NMR experiments, we identified a low-affinity bile acid binding site of a human GII.4 NoV strain. Long-timescale MD simulations reveal the formation of a ligand-accessible binding pocket of flexible shape, allowing the formation of stable viral coat protein-bile acid complexes in agreement with experimental CSP data. CSP NMR experiments also show that this mode of bile acid binding has a minor influence on the binding of histo-blood group antigens and vice versa. STD NMR experiments probing the binding of bile acids to virus-like particles of seven different strains suggest that low-affinity bile acid binding is a common feature of human NoV and should therefore be important for understanding the role of bile acids as cofactors in NoV infection.

Simulation of Mixed Self-Assembled Monolayers on Gold: Effect of Terminal Alkyl Anchor Chain and Monolayer Composition.

Self-assembling monolayers provide a reproducible synthetic microenvironment for tethering lipid bilayers to incorporate proteins and lay the ground for numerous applications in nanotechnology and biomedical engineering. Although the structure of single-component monolayers is well investigated, there is far less insight into the molecular behavior at the interface of mixed monolayers at different mole fractions. Here, we present and apply a novel procedure to simulate and analyze multicomponent self-assemblies of alkanethiols over a wide range of mole concentrations of anchoring compounds. In particular, the structural features of monolayers consisting of a matrix compound and either a short (C8) or a long (C16) anchor compound on Au(111)-like surfaces were investigated first using coarse-grained and subsequently full-atomistic molecular dynamics simulations. Different scenarios of spatial distributions (random vs clustering) of anchoring molecules on flat surfaces were probed. The results of the simulations are in excellent agreement with the experimental data from ellipsometry and infrared reflection absorption spectroscopy. For short anchoring molecules, a random spatial distribution in the matrix is obtained. At low, experimentally relevant anchor compound mole fractions < 0.1, only for long-chain (C16)-terminal alkyls, phase segregation and self-association of the anchoring molecules can be observed, which are also seen in experiment.

Membrane localization and dynamics of geranylgeranylated Rab5 hypervariable region.

The small GTPase Rab5 is a key regulator of endosomal trafficking processes and a marker for the early endosome. The C-terminal hypervariable region (HVR) of Rab5 is post-translationally modified at residues Cys212 and Cys213 to accommodate two geranylgeranyl anchors (C20 carbon chain length) in order to associate Rab5 with the membrane. The structural role of the HVR regarding protein-early endosome membrane recruitment is not resolved due to its high degree of flexibility and lack of crystallographic information. Here, full-atomistic and coarse-grained molecular dynamics simulations of the truncated Rab5 HVR206-215 in three model membranes of increasing complexity (pure phospholipid bilayer, ternary membrane with cholesterol, six-component early endosome) were performed. Specific electrostatic interactions between the HVR206-215 Arg209 residue and the phosphate group of the inositol ring of PI(3)P were detected. This shows that PI(3)P acts as a first contact site of protein recruitment to the early endosome. The free energy change of HVR206-215 extraction from the bilayer was largest for the physiological negatively charged membrane. 5µs coarse-grained simulations revealed an active recruitment of PI(3)P to the HVR206-215 supporting the formation of Rab5- and PI(3)P enriched signaling platforms.

Are the Crystal Structures of Enantiopure and Racemic Mandelic Acids Determined by Kinetics or Thermodynamics?

Mandelic acids are prototypic chiral molecules where the sensitivity of crystallized forms (enantiopure/racemic compound/polymorphs) to both conditions and substituents provides a new insight into the factors that may allow chiral separation by crystallization. The determination of a significant number of single crystal structures allows the analysis of 13 enantiopure and 30 racemic crystal structures of 21 (F/Cl/Br/CH3/CH3O) substituted mandelic acid derivatives. There are some common phenyl packing motifs between some groups of racemic and enantiopure structures, although they show very different hydrogen-bonding motifs. The computed crystal energy landscape of 3-chloromandelic acid, which has at least two enantiopure and three racemic crystal polymorphs, reveals that there are many more possible structures, some of which are predicted to be thermodynamically more favorable as well as slightly denser than the known forms. Simulations of mandelic acid dimers in isolation, water, and toluene do not differentiate between racemic and enantiopure dimers and also suggest that the phenyl ring interactions play a major role in the crystallization mechanism. The observed crystallization behavior of mandelic acids does not correspond to any simple "crystal engineering rules" as there is a range of thermodynamically feasible structures with no distinction between the enantiopure and racemic forms. Nucleation and crystallization appear to be determined by the kinetics of crystal growth with a statistical bias, but the diversity of the mandelic acid crystallization behavior demonstrates that the factors that influence the kinetics of crystal nucleation and growth are not yet adequately understood.

Stat3 signaling regulates embryonic stem cell fate in a dose-dependent manner.

Stat3 is essential for mouse embryonic stem cell (mESC) self-renewal mediated by LIF/gp130 receptor signaling. Current understanding of Stat3-mediated ESC self-renewal mechanisms is very limited, and has heretofore been dominated by the view that Stat3 signaling functions in a binary "on/off" manner. Here, in contrast to this binary viewpoint, we demonstrate a contextual, rheostat-like mechanism for Stat3's function in mESCs. Activation and expression levels determine whether Stat3 functions in a self-renewal or a differentiation role in mESCs. We also show that Stat3 induces rapid differentiation of mESCs toward the trophectoderm (TE) lineage when its activation level exceeds certain thresholds. Stat3 induces this differentiation phenotype via induction of Tfap2c and its downstream target Cdx2. Our findings provide a novel concept in the realm of Stat3, self-renewal signaling, and pluripotent stem cell biology. Ultimately, this finding may facilitate the development of conditions for the establishment of authentic non-rodent ESCs.

Pleiotropy of glycogen synthase kinase-3 inhibition by CHIR99021 promotes self-renewal of embryonic stem cells from refractory mouse strains.

BACKGROUND: Inhibition of glycogen synthase kinase-3 (GSK-3) improves the efficiency of embryonic stem (ES) cell derivation from various strains of mice and rats, as well as dramatically promotes ES cell self-renewal potential. ß-catenin has been reported to be involved in the maintenance of self-renewal of ES cells through TCF dependent and independent pathway. But the intrinsic difference between ES cell lines from different species and strains has not been characterized. Here, we dissect the mechanism of GSK-3 inhibition by CHIR99021 in mouse ES cells from refractory mouse strains. METHODOLOGY/PRINCIPAL FINDINGS: We found that CHIR99021, a GSK-3 specific inhibitor, promotes self-renewal of ES cells from recalcitrant C57BL/6 (B6) and BALB/c mouse strains through stabilization of ß-catenin and c-Myc protein levels. Stabilized ß-catenin promoted ES self-renewal through two mechanisms. First, ß-catenin translocated into the nucleus to maintain stem cell pluripotency in a lymphoid-enhancing factor/T-cell factor-independent manner. Second, ß-catenin binds plasma membrane-localized E-cadherin, which ensures a compact, spherical morphology, a hallmark of ES cells. Further, elevated c-Myc protein levels did not contribute significantly to CH-mediated ES cell self-renewal. Instead, the role of c-Myc is dependent on its transformation activity and can be replaced by N-Myc but not L-Myc. ß-catenin and c-Myc have similar effects on ES cells derived from both B6 and BALB/c mice. CONCLUSIONS/SIGNIFICANCE: Our data demonstrated that GSK-3 inhibition by CH promotes self-renewal of mouse ES cells with non-permissive genetic backgrounds by regulation of multiple signaling pathways. These findings would be useful to improve the availability of normally non-permissive mouse strains as research tools.

Germline competent embryonic stem cells derived from rat blastocysts.

Rats have important advantages over mice as an experimental system for physiological and pharmacological investigations. The lack of rat embryonic stem (ES) cells has restricted the availability of transgenic technologies to create genetic models in this species. Here, we show that rat ES cells can be efficiently derived, propagated, and genetically manipulated in the presence of small molecules that specifically inhibit GSK3, MEK, and FGF receptor tyrosine kinases. These rat ES cells express pluripotency markers and retain the capacity to differentiate into derivatives of all three germ layers. Most importantly, they can produce high rates of chimerism when reintroduced into early stage embryos and can transmit through the germline. Establishment of authentic rat ES cells will make possible sophisticated genetic manipulation to create models for the study of human diseases.

Dual-energy digital subtraction chest radiography: technical considerations.

In the evaluation of asbestos-related pulmonary and pleural abnormalities, conventional chest radiography has been shown to have a low sensitivity for the detection of lung nodules and subtle interstitial disease. Pleural plaques may simulate pulmonary nodules, and interstitial processes can be masked by adjacent pleural abnormalities. Dual-energy digital subtraction chest radiography may enable investigators to characterize asbestos-related pulmonary and pleural abnormalities with greater accuracy. "Soft-tissue" images, designed to remove pleural calcifications, may allow for better evaluation of the lung parenchyma. "Bone" images, designed to remove soft-tissue structures, may enhance the detection of pleural calcifications. In this pictorial essay we illustrate the methods, technical considerations, and limitations of dual-energy digital subtraction chest radiography performed with global subtraction weighting factors.