The role of succinate and ROS in reperfusion injury - A critical appraisal.

We critically assess the proposal that succinate-fuelled reverse electron flow (REF) drives mitochondrial matrix superoxide production from Complex I early in reperfusion, thus acting as a key mediator of ischemia/reperfusion (IR) injury. Real-time surface fluorescence measurements of NAD(P)H and flavoprotein redox state suggest that conditions are unfavourable for REF during early reperfusion. Furthermore, rapid loss of succinate accumulated during ischemia can be explained by its efflux rather than oxidation. Moreover, succinate accumulation during ischemia is not attenuated by ischemic preconditioning (IP) despite powerful cardioprotection. In addition, measurement of intracellular reactive oxygen species (ROS) during reperfusion using surface fluorescence and mitochondrial aconitase activity detected major increases in ROS only after mitochondrial permeability transition pore (mPTP) opening was first detected. We conclude that mPTP opening is probably triggered initially by factors other than ROS, including increased mitochondrial [Ca2+]. However, IP only attenuates [Ca2+] increases later in reperfusion, again after initial mPTP opening, implying that IP regulates mPTP opening through additional mechanisms. One such is mitochondria-bound hexokinase 2 (HK2) which dissociates from mitochondria during ischemia in control hearts but not those subject to IP. Indeed, there is a strong correlation between the extent of HK2 loss from mitochondria during ischemia and infarct size on subsequent reperfusion. Mechanisms linking HK2 dissociation to mPTP sensitisation remain to be fully established but several related processes have been implicated including VDAC1 oligomerisation, the stability of contact sites between the inner and outer membranes, cristae morphology, Bcl-2 family members and mitochondrial fission proteins such as Drp1.

Does attendance at anatomy practical classes correlate with assessment outcome? A retrospective study of a large cohort of undergraduate anatomy students.

BACKGROUND: Anatomy in medical curricula is typically taught via pedagogy consisting of didactic lectures combined with a practical component. The practical component often includes traditional cadaveric dissection classes and/or workshops utilizing anatomical models, carefully prosected cadaveric material and radiology. The primary aim of this study was to determine if there is an association between attendance at practical classes in anatomy and student assessment outcomes. A secondary aim was to determine if student assessment outcomes were better when students preferentially attended workshops or prosection style practical classes. METHOD: We retrospectively examined practical attendance records and assessment outcomes from a single large anatomy subject (approx. 450 students) to identify how attendance at anatomy practical classes correlates with assessment outcome. RESULTS: Students who scored above the median mark for each assessment attended significantly more practical classes than students who scored below the median assessment mark (Mann Whitney; p < 0.001), and students who attended more than half the practical classes had significantly higher scores on assessments than students that attended less than half the practical classes (Mann Whitney; P < 0.01). There was a statistically significant positive correlation between attendance at practical classes and outcomes for each assessment (Spearman's correlation; p < 0.01). There was no difference in assessment outcomes for students who preferentially attended more dissection compared to prosection style classes and vice versa (Mann Whitney; p > 0.05). CONCLUSIONS: Our findings show there is an association between student attendance at practical classes and performance on anatomy assessment.

AR-C155858 is a potent inhibitor of monocarboxylate transporters MCT1 and MCT2 that binds to an intracellular site involving transmembrane helices 7-10.

In the present study we characterize the properties of the potent MCT1 (monocarboxylate transporter 1) inhibitor AR-C155858. Inhibitor titrations of L-lactate transport by MCT1 in rat erythrocytes were used to determine the Ki value and number of AR-C155858-binding sites (Et) on MCT1 and the turnover number of the transporter (kcat). Derived values were 2.3+/-1.4 nM, 1.29+/-0.09 nmol per ml of packed cells and 12.2+/-1.1 s-1 respectively. When expressed in Xenopus laevis oocytes, MCT1 and MCT2 were potently inhibited by AR-C155858, whereas MCT4 was not. Inhibition of MCT1 was shown to be time-dependent, and the compound was also active when microinjected, suggesting that AR-C155858 probably enters the cell before binding to an intracellular site on MCT1. Measurement of the inhibitor sensitivity of several chimaeric transporters combining different domains of MCT1 and MCT4 revealed that the binding site for AR-C155858 is contained within the C-terminal half of MCT1, and involves TM (transmembrane) domains 7-10. This is consistent with previous data identifying Phe360 (in TM10) and Asp302 plus Arg306 (TM8) as key residues in substrate binding and translocation by MCT1. Measurement of the Km values of the chimaeras for L-lactate and pyruvate demonstrate that both the C- and N-terminal halves of the molecule influence transport kinetics consistent with our proposed molecular model of MCT1 and its translocation mechanism that requires Lys38 in TM1 in addition to Asp302 and Arg306 in TM8 [Wilson, Meredith, Bunnun, Sessions and Halestrap (2009) J. Biol. Chem. 284, 20011-20021].

The inhibition of monocarboxylate transporter 2 (MCT2) by AR-C155858 is modulated by the associated ancillary protein.

In mammalian cells, MCTs (monocarboxylate transporters) require association with an ancillary protein to enable plasma membrane expression of the active transporter. Basigin is the preferred binding partner for MCT1, MCT3 and MCT4, and embigin for MCT2. In rat and rabbit erythrocytes, MCT1 is associated with embigin and basigin respectively, but its sensitivity to inhibition by AR-C155858 was found to be identical. Using RT (reverse transcription)-PCR, we have shown that Xenopus laevis oocytes contain endogenous basigin, but not embigin. Co-expression of exogenous embigin was without effect on either the expression of MCT1 or its inhibition by AR-C155858. In contrast, expression of active MCT2 at the plasma membrane of oocytes was significantly enhanced by co-expression of exogenous embigin. This additional transport activity was insensitive to inhibition by AR-C155858 unlike that by MCT2 expressed with endogenous basigin that was potently inhibited by AR-C155858. Chimaeras and C-terminal truncations of MCT1 and MCT2 were also expressed in oocytes in the presence and absence of exogenous embigin. L-Lactate Km values for these constructs were determined and revealed that the TM (transmembrane) domains of an MCT, most probably TM7-TM12, but not the C-terminus, are the major determinants of L-lactate affinity, whereas the associated ancillary protein has little or no effect. Inhibitor titrations of lactate transport by these constructs indicated that embigin modulates MCT2 sensitivity to AR-C155858 through interactions with both the intracellular C-terminus and TMs 3 and 6 of MCT2. The C-terminus of MCT2 was found to be essential for its expression with endogenous basigin.

Ultrasound-guided regional anesthesia: how much practice do novices require before achieving competency in ultrasound needle visualization using a cadaver model.

BACKGROUND AND OBJECTIVES: Ultrasound needle visualization is a fundamental skill required for competency in ultrasound-guided regional anesthesia. The primary objective of this study using a cadaver model was to quantify the number of procedures that novices need to perform before competency, using a predefined dynamic scoring system was achieved in ultrasound needle visualization skills. METHODS: Fifteen trainees, novices to ultrasound-guided regional anesthesia, performed 30 simulated sciatic nerve blocks in cadavers. After each procedure, a supervisor provided feedback regarding quality-compromising behaviors. Learning curves were constructed for each individual trainee by calculating cusum statistics. Trainees were categorized into those who were proficient, not proficient, and undetermined. A mathematical model predicted the number of procedures required before an acceptable success rate would be attained. Logistic regression was used to identify factors associated with success. RESULTS: There was wide variability in individual cusum curves. The mean number of trials required to achieve competency in this cohort was 28. Trainees were categorized as proficient (n = 6), not proficient (n = 5), and undetermined (n = 4). With each subsequent procedure, there was a significant increase in the likelihood of success for trainees categorized as not proficient (P = 0.023) or undetermined (P = 0.024) but not for trainees categorized as proficient (P = 0.076). Participants recruited later in the study had an increased likelihood of success (P < 0.001). CONCLUSIONS: Trainees became competent in ultrasound needle visualization at a variable rate. This study estimates that novices would require approximately 28 supervised trials with feedback before competency in ultrasound needle visualization is achieved.

Triplex staples: DNA double-strand cross-linking at internal and terminal sites using psoralen-containing triplex-forming oligonucleotides.

A method has been developed to attach 4'-(hydroxymethyl)-4,5',8-trimethylpsoralen to the 5 position of thymine bases during solid-phase oligonucleotide synthesis. UV irradiation of triplex-forming oligonucleotides (TFOs) containing internally attached psoralens produces photoadducts at TpA steps within target duplexes, thus relaxing the constraints on selection of psoralen target sequences. Photoreaction of TFOs containing two psoralens, located at the 5'- and 3'-ends, has been used to create double-strand cross-links (triplex staples) at both termini of the TFO. Such complexes have no free single-stranded ends. TFOs containing 4'-(hydroxymethyl)-4,5',8-trimethylpsoralen, 3-methyl-2-aminopyridine, and 5-(3-aminoprop-2-ynyl)deoxyuridine formed photoadducts with target duplexes under near-physiological conditions.