Conformation-specific anti-Mad2 monoclonal antibodies for the dissection of checkpoint signaling

The spindle assembly checkpoint (SAC) ensures accurate chromosome segregation during mitosis by delaying the activation of the anaphase-promoting complex/cyclosome (APC/C) in response to unattached kinetochores. The Mad2 protein is essential for a functional checkpoint because it binds directly to Cdc20, the mitotic co-activator of the APC/C, thereby inhibiting progression into anaphase. Mad2 exists in at least 2 different conformations, open-Mad2 (O-Mad2) and closed-Mad2 (C-Mad2), with the latter representing the active form that is able to bind Cdc20. Our ability to dissect Mad2 biology in vivo is limited by the absence of monoclonal antibodies (mAbs) useful for recognizing the different conformations of Mad2. Here, we describe and extensively characterize mAbs specific for either O-Mad2 or C-Mad2, as well as a pan-Mad2 antibody, and use these to investigate the different Mad2 complexes present in mitotic cells. Our antibodies validate current Mad2 models but also suggest that O-Mad2 can associate with checkpoint complexes, most likely through dimerization with C-Mad2. Furthermore, we investigate the makeup of checkpoint complexes bound to the APC/C, which indicate the presence of both Cdc20-BubR1-Bub3 and Mad2-Cdc20-BubR1-Bub3 complexes, with Cdc20 being ubiquitinated in both. Thus, our defined mAbs provide insight into checkpoint signaling and provide useful tools for future research on Mad2 function and regulation.     

Alagille Syndrome Candidates for Liver Transplantation: Differentiation from End-Stage Biliary Atresia Using Preoperative CT

Purpose

To compare preoperative CT findings before liver transplantation between patients with Alagille syndrome (AGS) and those with end-stage biliary atresia (BA).

Materials and Methods

The institutional review board approved this retrospective study. Eleven children with AGS (median age, 19.0 ± 13.0 months; male to female ratio, 3:8) and 109 children with end-stage BA (median age, 17.9 ± 25.8 months; male to female ratio, 37:72) who underwent abdomen CT as candidates for liver transplant were included. CT images were reviewed focusing on hepatic parenchymal changes, vascular changes, presence of focal lesions, and signs of portal hypertension.

Results

Hepatic parenchymal changes were present in 27% (3/11) of AGS patients and 100% (109/109) of end-stage BA patients (P < .001). The hepatic artery diameter was significantly smaller (1.9 mm versus 3.6 mm, P = 008), whereas portal vein diameter was larger (6.8 mm versus 5.0 mm, P < .001) in patients with AGS compared with patients with end-stage BA. No focal lesion was seen in patients with AGS, whereas 44% (48/109) of patients with end-stage BA had intrahepatic biliary cysts (39%, 43/109) and hepatic tumors (8%, 9/109) (P = .008). Splenomegaly was commonly seen in both groups (P = .082), and ascites (9% [1/11] versus 50% [54/109], P = .010) and gastroesophageal varix (0% [0/11] versus 80% [87/109], P < .001) were less common in patients with AGS than in patients with end-stage BA.

Conclusion

Fibrotic or cirrhotic changes of the liver, presence of focal lesions, and relevant portal hypertension were less common in patients with AGS than in patients with end-stage BA.     

Simple Scoring System and Artificial Neural Network for Knee Osteoarthritis Risk Prediction: A Cross-Sectional Study

Background

Knee osteoarthritis (OA) is the most common joint disease of adults worldwide. Since the treatments for advanced radiographic knee OA are limited, clinicians face a significant challenge of identifying patients who are at high risk of OA in a timely and appropriate way. Therefore, we developed a simple self-assessment scoring system and an improved artificial neural network (ANN) model for knee OA.

Methods

The Fifth Korea National Health and Nutrition Examination Surveys (KNHANES V-1) data were used to develop a scoring system and ANN for radiographic knee OA. A logistic regression analysis was used to determine the predictors of the scoring system. The ANN was constructed using 1777 participants and validated internally on 888 participants in the KNHANES V-1. The predictors of the scoring system were selected as the inputs of the ANN. External validation was performed using 4731 participants in the Osteoarthritis Initiative (OAI). Area under the curve (AUC) of the receiver operating characteristic was calculated to compare the prediction models.

Results

The scoring system and ANN were built using the independent predictors including sex, age, body mass index, educational status, hypertension, moderate physical activity, and knee pain. In the internal validation, both scoring system and ANN predicted radiographic knee OA (AUC 0.73 versus 0.81, p<0.001) and symptomatic knee OA (AUC 0.88 versus 0.94, p<0.001) with good discriminative ability. In the external validation, both scoring system and ANN showed lower discriminative ability in predicting radiographic knee OA (AUC 0.62 versus 0.67, p<0.001) and symptomatic knee OA (AUC 0.70 versus 0.76, p<0.001).

Conclusions

The self-assessment scoring system may be useful for identifying the adults at high risk for knee OA. The performance of the scoring system is improved significantly by the ANN. We provided an ANN calculator to simply predict the knee OA risk.     

The Tau/A152T mutation,a risk factor for frontotemporal‐spectrum disorders,leads to NR2B receptor‐mediated excitotoxicity

We report on a novel transgenic mouse model expressing human full‐length Tau with the Tau mutation A152T (hTau^AT), a risk factor for FTD‐spectrum disorders including PSP and CBD. Brain neurons reveal pathological Tau conformation, hyperphosphorylation, mis‐sorting, aggregation, neuronal degeneration, and progressive loss, most prominently in area CA3 of the hippocampus. The mossy fiber pathway shows enhanced basal synaptic transmission without changes in short‐ or long‐term plasticity. In organotypic hippocampal slices, extracellular glutamate increases early above control levels, followed by a rise in neurotoxicity. These changes are normalized by inhibiting neurotransmitter release or by blocking voltage‐gated sodium channels. CA3 neurons show elevated intracellular calcium during rest and after activity induction which is sensitive to NR2B antagonizing drugs, demonstrating a pivotal role of extrasynaptic NMDA receptors. Slices show pronounced epileptiform activity and axonal sprouting of mossy fibers. Excitotoxic neuronal death is ameliorated by ceftriaxone, which stimulates astrocytic glutamate uptake via the transporter EAAT2/GLT1. In summary, hTau^AT causes excitotoxicity mediated by NR2B‐containing NMDA receptors due to enhanced extracellular glutamate.     

Quercetin 3,7-O-dimethyl ether from Siegesbeckia pubescens suppress the production of inflammatory mediators in lipopolysaccharide-induced macrophages and colon epithelial cells

Siegesbeckia pubescens (Compositae) is an annual herb indigenous to Korean mountainous regions. Recent reports have been issued on some compounds derived from S. pubescens for its anti-inflammatory activity or mode of action. The quercetin 3,7-O-dimethyl ether (QDE) isolated from the herbs of S. pubescens suppressed the lipopolysaccharide (LPS)-induced nitric oxide and inducible nitric oxide synthase (iNOS) protein production in mouse macrophages. QDE downregulated pro-inflammatory cytokines such as interleukin (IL)-6, IL-1β, tumor necrosis factor -α levels in LPS-stimulated macrophages. Also, QDE decreased the expression of LPS-induced iNOS and cyclooxygenase-2 (COX-2) protein and the production of IL-8 in LPS-induced HT-29 cells. Macrophages and colon epithelial cells are important for regulating the colon immune systems, thus QDE may regulate inflammatory colon disease via LPS-induced inflammation in macrophages and colon epithelial cells. QDE, anti-inflammatory constituent of S. pubescens herbs, can be expected to be a potential candidate for therapeutics against inflammatory bowel disease.     

Morphological changes and increase of resistance to oxidative stress by overexpression of the <Emphasis Type="Italic">LebZIP2</Emphasis> gene in <Emphasis Type="Italic">Nicotiana benthamiana</Emphasis>

The tomato bZIP2-encoding gene was inserted into the Nicotiana benthamiana genome using Agrobacterium-mediated transformation to characterize resistance to oxidative stress and two herbicides, glyphosate and paraquat. We produced transgenic tobacco plants using the LebZIP2 gene, which were then utilized to examine salt stress and herbicide resistance through oxidative mechanisms. Transgenic LebZIP2-overexpressing plants were examined using specific primers for selection marker genes (PCR using genomic DNA) and target genes (RT-PCR). Based on microscopic examination, we observed an increase in leaf thickness and cell number in transgenic plants. The electrolyte leakage of leaves suggested that LebZIP2-overexpressing lines were weak tolerant to NaCl stress and resistant to methyl viologen. During our analysis, transgenic lines were exposed to different herbicides. Transgenic plants showed an increased tolerance based on visual injury, as well as an increased biomass. Based on these results, the LebZIP2 gene may be involved in oxidative stress tolerance and cell development in plants.     

KIN‐4/MAST kinase promotes PTEN‐mediated longevity of Caenorhabditis elegans via binding through a PDZ domain

PDZ domain‐containing proteins (PDZ proteins) act as scaffolds for protein–protein interactions and are crucial for a variety of signal transduction processes. However, the role of PDZ proteins in organismal lifespan and aging remains poorly understood. Here, we demonstrate that KIN‐4, a PDZ domain‐containing microtubule‐associated serine‐threonine (MAST) protein kinase, is a key longevity factor acting through binding PTEN phosphatase in Caenorhabditis elegans. Through a targeted genetic screen for PDZ proteins, we find that kin‐4 is required for the long lifespan of daf‐2/insulin/IGF‐1 receptor mutants. We then show that neurons are crucial tissues for the longevity‐promoting role of kin‐4. We find that the PDZ domain of KIN‐4 binds PTEN, a key factor for the longevity of daf‐2 mutants. Moreover, the interaction between KIN‐4 and PTEN is essential for the extended lifespan of daf‐2 mutants. As many aspects of lifespan regulation in C. elegans are evolutionarily conserved, MAST family kinases may regulate aging and/or age‐related diseases in mammals through their interaction with PTEN.     

The Effect of Probiotics on Halitosis: a Systematic Review and Meta-analysis

Probiotics and Antimicrobial Proteins - Although several studies have evaluated the inhibitory effect of probiotics on halitosis, findings are inconsistent. This systematic review and meta-analysis...     

Cleaved Cochlin Sequesters Pseudomonas aeruginosa and Activates Innate Immunity in the Inner Ear

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