Comparisons of ELISA and Western blot assays for detection of autophagy flux

We analyzed autophagy/mitophagy flux in vitro (C2C12 myotubes) and in vivo (mouse skeletal muscle) following the treatments of autophagy inducers (starvation, rapamycin) and a mitophagy inducer (carbonyl cyanide m-chlorophenylhydrazone, CCCP) using two immunodetection methods, ELISA and Western blotting, and compared their working range, accuracy, and reliability. The ELISAs showed a broader working range than that of the LC3 Western blots (Table 1). Table 2 showed that data value distribution was tighter and the average standard error from the ELISA was much smaller than those of the Western blot, directly relating to the accuracy of the assay. Test-retest reliability analysis showed good reliability for three individual ELISAs (interclass correlation, ≥ 0.7), but poor reliability for three individual Western blots (interclass correlation, ≤ 0.4) (Table 3). Keywords: Autophagy, Mitophagy, ELISA, Western blot, Skeletal muscl

The Characteristics of Metallo-β-Lactamase-Producing Gram-Negative Bacilli Isolated from Sputum and Urine: A Single Center Experience in Korea

Metallo-β-lactamase (MBL) production usually results in high-level resistance to most β-lactams, and a rapid spread of MBL producing major gram-negative pathogens is a matter of particular concern worldwide. However, clinical data are scarce and most studies compared MBL producer (MP) with MBL non-producer (MNP) strains which included carbapenem susceptible isolates. Therefore, we collected clinical data of patients in whom imipenem-nonsusceptible Pseudomonas aeruginosa (PA) and Acinetobacter baumannii (AB) were isolated from sputum or urine, and investigated MBL production and the risk factors related with MBL acquisition. The antimicrobial susceptibility patterns were also compared between MPs and imipenem-nonsusceptible MNPs (INMNP). Among the 176 imipenem-nonsusceptible isolates, 12 MPs (6.8%) were identified. There was no identifiable risk factor that contributed to the acquisition of MPs when compared to INMNPs, and case-fatalities were not different between the two groups. The percentage of susceptible isolates was higher among MPs for piperacilin/tazobactam and fluoroquinolones while that of ceftazidime was higher in INMNPs (p < 0.05). As regards to aztreonam, which has been known to be a uniquely stable β-lactam against MBLs, susceptibility was preserved in only two isolates (16.7%) among MPs, and was not higher than that of INMNPs (23.2%). In conclusion, the contribution of MBLs to imipenem non-susceptibility in PA/ABs isolated from sputum and urine was relatively limited, and there was no significant risk factor associated with acquisition of MPs compared with INMNPs. However, limited susceptibility to aztreonam implies that MPs may hold additional resistance mechanisms, such as extended spectrum β-lactamases, AmpC β-lactamases, or other non-enzymatic mechanisms

High white blood cell count and erythrocyte sedimentation rate are associated with perforated appendicitis in children

Purpose To investigate the predictors of perforated appendicitis (PA) in pediatric patients with appendicitis seen in the emergency department. Methods We retrospectively reviewed 564 pediatric patients ( 13.5 × 109/L (odds ratio [OR], 3.27; confidence interval [CI], 1.49-7.18; P = 0.003) and ESR > 15 mm/h (OR, 3.18; 95% CI, 2.13-4.74; P < 0.001) are independent predictors of PA. Conclusion WBC count and ESR might be better predictors of PA in pediatric patients with appendicitis in the emergency department than the Alvarado score and CRP concentration

Factors associated with administration of analgesics for children with forearm fracture

Purpose Analgesia is essential for the treatment of children’s fracture. We aimed to investigate the factors associated with administration of analgesics in children with forearm fracture. Methods We retrospectively reviewed medical records of children (< 20 years) with forearm fracture who visited 2 tertiary hospital emergency departments from 2014 to 2015. We analyzed factors, such as gender, age, whether the mother accompanied the visit, visiting time and route, mechanism of injury, duration of symptoms, complicated fracture, manual reduction, surgery, and type and route of analgesics. We also performed logistic regression analysis to identify the factors associated with administration of analgesics. Results Of 179 children with forearm fracture, 48 (26.8%) were administered analgesics. These children showed older age, shorter duration of symptoms, and more frequent visit with their mothers, visit during the day, use of emergency medical services, and surgery. After logistic regression analysis, we found use of emergency medical service (adjusted odds ratio [OR], 8.73; 95% confidence interval [CI], 3.16-24.08; P < 0.001), visit with the mother (OR, 6.23; 95% CI, 1.68-23.09; P = 0.006), age (OR, 1.18; 95% CI, 1.05-1.32; P = 0.004), and duration of symptoms (OR, 0.99; 95% CI, 0.986-0.999; P = 0.035) as the factors associated with administration of analgesics. Conclusion The factors associated with administration of analgesics might be communicating skill-related factors, such as older age and shorter duration of symptoms. Children with poor communicating skill may need more aggressive analgesia in the emergency department

The Usefulness of Maximum Standardized Uptake Value at the Delayed Phase of Tc-99m sestamibi single-photon emission computed tomography/computed tomography for Identification of Parathyroid Adenoma and Hyperplasia

Tc-99m sestamibi single-photon emission computed tomography/computed tomography (SPECT/CT) has been used to help surgeons explore the location of parathyroid diseases, but quantitative parameters have not been systemically investigated for this purpose. We aimed to establish objective criteria for adenoma and hyperplasia using the standardized uptake value (SUV) in patients with hyperparathyroidism. Thirty-nine hyperparathyroid patients (male/female: 17/22, age: 58.33 +/- 11.69 years) with at least 1 uptake-positive lesion of any degree by visual assessment in preoperative Tc-99m sestamibi quantitative SPECT/CT were included from Oct 2015 to Oct 2017. Pathologically, 44 lesions (32 adenomas and 12 hyperplasia) were identified. All patients experienced normalized levels of intact parathyroid hormone immediately after surgery. Quantitative SPECT/CT was performed at 10 minute and 2 hour post injection of Tc-99m sestabmibi (dose = 740 MBq), and maximum SUV (SUVmax) was measured for the parathyroid lesions. Experienced pathologists evaluated the percentage cellular proportions of chief cells, oxyphil cells, and clear cells. SUVmax (g/mL) of adenomas, hyperplasia, and reference thyroid tissue were 12.92 +/- 6.68, 7.90 +/- 5.49, and 7.01 +/- 2.62 at 10min (early phase), decreasing to 7.46 +/- 5.66, 4.65 +/- 3.14, and 2.21 +/- 1.07 at 2 hour (delayed phase), respectively. The adenomas showed significantly higher SUVmax than both the hyperplasia (P = .0131) and reference thyroid tissue (P &lt; .0001) along the early and delayed phases, but the SUVmax of the hyperplasia did not differ from that of the reference thyroid tissue (P = .4196). The adenomas and hyperplasia were discriminated from the reference thyroid tissue using a cutoff SUVmax of 3.26 at the delayed phase. The adenomas had lower %proportions of oxyphil cells than the hyperplasia (P = .0054), but its SUVmax at the delayed phase was positively correlated with the %proportions of mitochondria-abundant oxyphil cells (rho = 0.418,P = .0173). The hyperplasia showed no correlation between SUVmax and cellular proportions. SUVmax at the delayed phase in the Tc-99m sestamibi quantitative SPECT/CT was useful for the identification and differentiation of parathyroid lesions causing hyperparathyroidism.Y

Co-option of neurotransmitter signaling for inter-organismal communication in C. elegans

Biogenic amine neurotransmitters play a central role in metazoan biology, and both their chemical structures and cognate receptors are evolutionarily conserved. Their primary roles are in cell-to-cell signaling, as biogenic amines are not normally recruited for communication between separate individuals. Here, we show that in the nematode C. elegans, a neurotransmitter-sensing G protein-coupled receptor, TYRA-2, is required for avoidance responses to osas#9, an ascaroside pheromone that incorporates the neurotransmitter, octopamine. Neuronal ablation, cell-specific genetic rescue, and calcium imaging show that tyra-2 expression in the nociceptive neuron, ASH, is necessary and sufficient to induce osas#9 avoidance. Ectopic expression in the AWA neuron, which is generally associated with attractive responses, reverses the response to osas#9, resulting in attraction instead of avoidance behavior, confirming that TYRA-2 partakes in the sensing of osas#9. The TYRA-2/osas#9 signaling system represents an inter-organismal communication channel that evolved via co-option of a neurotransmitter and its cognate receptor

Application of self-assembly peptides targeting the mitochondria as a novel treatment for sorafenib-resistant hepatocellular carcinoma cells

Currently, there is no appropriate treatment option for patients with sorafenib-resistant hepatocellular carcinoma (HCC). Meanwhile, pronounced anticancer activities of newly-developed mitochondria-accumulating self-assembly peptides (Mito-FF) have been demonstrated. This study intended to determine the anticancer effects of Mito-FF against sorafenib-resistant Huh7 (Huh7-R) cells. Compared to sorafenib, Mito-FF led to the generation of relatively higher amounts of mitochondrial reactive oxygen species (ROS) as well as the greater reduction in the expression of antioxidant enzymes (P &lt; 0.05). Mito-FF was found to significantly promote cell apoptosis while inhibiting cell proliferation of Huh7-R cells. Mito-FF also reduces the expression of antioxidant enzymes while significantly increasing mitochondrial ROS in Huh7-R cells. The pro-apoptotic effect of Mito-FFs for Huh7-R cells is possibly caused by their up-regulation of mitochondrial ROS, which is caused by the destruction of the mitochondria of HCC cells