Senescence marker protein 30 protects intestinal epithelial cells against inflammation-induced cell death by enhancing Nrf2 activity

Senescence marker protein 30 (SMP30) is a calcium-binding protein whose expression decreases during senescence. SMP30 deficiency increases susceptibility to cytokine-induced apoptosis in the liver and to radiation-induced apoptosis in the small intestine. Furthermore, colonic epithelial cell death is associated with the severity of colitis. Therefore, in the present study, we investigated the function of SMP30 during intestinal inflammation. In SMP30 deficient mice, colitis was significantly exacerbated as demonstrated by increased mortality (p?=?0.001), body weight loss (p?=?0.0105 at day 8), rectal bleeding (p?=?0.0047 at day 8) and diarrhea (p?=?0.0030 at day 8), histological scores (ulcers, p?=?0.0002; edema, p?=?0.0125; leukocyte infiltration, p?=?0.0016) and productions of pro-inflammatory cytokines (IL-1α, p?=?0.0452; IL-6, p?=?0.0074; G-CSF, p?=?0.0036). In addition, greater proportions of apoptotic cells and lower levels of anti-apoptotic marker proteins (total PARP-1 and Bcl-2) were observed in the inflamed intestines of SMP30 deficient mice than in wild type controls. In vitro experiments on colonic epithelial cells showed that stable SMP30 expression inhibited but that SMP30 siRNA expression increased TNF-α-induced apoptosis. SMP30 inhibition decreased Nrf2 mRNA expression levels (p?<?0.0001), but SMP30 overexpression increased Nrf2 mRNA expression levels (p?=?0.0495). The underlying mechanism by which SMP30 protected cells appeared to be by inhibiting Nrf2 ubiquitination and Keap1 expression, and thus enhancing Nrf2 activity. Moreover, SMP30 deficiency increased the incidence of colitis-associated colon cancer as determined by increased mortality (p?=?0.0572) and average polyp number (p?=?0.0277). Collectively, these findings suggest that SMP30 protects intestinal epithelial cells from apoptosis and this can contribute to amelioration of colitis and colitis-associated colon cancer.     

Influence of storage condition on exosome recovery

Most of mammalian cells release extracellular vesicles including exosomes which mediate intercellular communication by delivering a variety of molecules. Despite of their importance in normal physiology and disease progression, the standard criteria of storage condition is indefinite and controversial. Therefore, we investigated exosome’s recovery yield and stability by various storage conditions. To investigate the effect of short-term storage temperature on exosome stability, exosomes were incubated at temperatures ranging from -70 to 90°C for 30 min. Immunoblot results showed that all exosome-associated proteins incubated at 90°C were mostly degraded for a short period of time. To examine the effect of long-term storage, isolated exosomes were incubated for 10 days at from -70°C to room temperature (RT), and exosomal protein, RNA and exosome markers were examined. Protein and RNA amounts were most reduced at RT compared with -70 and 4°C. Incubation at 4°C and RT resulted in major loss of CD63, and decreasing level of HSP70 was shown at only RT. In addition, flow cytometry result showed that exosome population became more dispersed after RT incubation for 10 days compared with -70°C incubated or freshly isolated exosomes. In summary, our results indicate that different storage temperature and period influences recovery yield and morphology of exosome, and storage at below -70°C is the favorable condition for preservation of fresh exosomes for clinical application and basic researches.     

Survival Outcomes According to Adjuvant Treatment and Prognostic Factors Including Host Immune Markers in Patients with Curatively Resected Ampulla of Vater Cancer

Background

Ampulla of Vater cancer (AoV Ca) is a rare tumor, and its adjuvant treatment has not been established. The purpose of this study was to find out prognostic factors including host immunity and role of adjuvant treatment in AoV Ca.

Methods and Findings

We reviewed 227 AoV Ca patients with curative resection. Clinical characteristics, adjuvant treatment, disease-free survival (DFS) and overall survival (OS) were analyzed. Among all patients, 63.9, 36.1 and 33.9% had T1/T2, T3/T4 stage and lymph node-positive disease (LN+), respectively. OS of all patients was 90.9 months (95% CI: 52.9–129.0). OS was different according to neutrophil-to-lymphocyte ratio (HR 1.651, 95% CI: 1.11–2.47), platelet-to-lymphocyte ratio (HR 1.488, 95% CI: 1.00–2.21) and systemic inflammatory index (HR 1.669, 95% CI: 1.13–2.47). In multivariate analysis, adverse prognostic factors for OS included vascular invasion (HR 2.571, 95% CI: 1.20–5.53) and elevated CA 19–9 (HR 1.794, 95% CI: 1.07–3.05). A total of 104 patients (46.3%) received adjuvant treatment (25 out of 111of T1/T2 & LN (-), 79 out of 116 of T3/T4 or LN (+)). In T3/T4 or LN (+) stage, adjuvant CCRT with maintenance chemotherapy provided the longest OS (5-year OS rate: 47.0 vs. 41.4%).

Conclusions

Vascular invasion and elevated CA 19–9 were adverse prognostic factors in resected AoV Ca. In T3/T4 or LN (+) stage, adjuvant CCRT with maintenance chemotherapy provided the best survival outcome. Adjuvant treatment should be further defined in AoV Ca, especially with poor prognostic factors.     

Measurement Variability of Persistent Pulmonary Subsolid Nodules on Same-Day Repeat CT: What Is the Threshold to Determine True Nodule Growth during Follow-Up?

Purpose

To assess the measurement variability of subsolid nodules (SSNs) in follow-up situations and to compare the degree of variability between measurement metrics.

Methods

Two same-day repeat-CT scans of 69 patients (24 men and 45 women) with 69 SSNs were randomly assigned as initial or follow-up scans and were read by the same (situation 1) or different readers (situation 2). SSN size and solid portion size were measured in both situations. Measurement variability was calculated and coefficients of variation were used for comparisons.

Results

Measurement variability for the longest and average diameter of SSNs was ±1.3 mm (±13.0%) and ±1.3 mm (±14.4%) in situation 1, and ±2.2 mm (±21.0%) and ±2.1 mm (±21.3%) in situation 2, respectively. For solid portion, measurement variability on lung and mediastinal windows was ±1.2 mm (±27.1%) and ±0.8 mm (±24.0%) in situation 1, and ±3.7 mm (±61.0%) and ±1.5 mm (±47.3%) in situation 2, respectively. There were no significant differences in the degree of variability between the longest and average diameters and between the lung and mediastinal window settings (p>0.05). However, measurement variability significantly increased when the follow-up and initial CT readers were different (p<0.001).

Conclusions

A cutoff of ±2.2 mm can be reliably used to determine true nodule growth on follow-up CT. Solid portion measurements were not reliable in evaluating SSNs’ change when readers of initial and follow-up CT were different.     

More Accurate Prediction of Metastatic Pancreatic Cancer Patients’ Survival with Prognostic Model Using Both Host Immunity and Tumor Metabolic Activity

Introduction

Neutrophil to lymphocyte ratio (NLR) and standard uptake value (SUV) by ¹⁸F-FDG PET represent host immunity and tumor metabolic activity, respectively. We investigated NLR and maximum SUV (SUVmax) as prognostic markers in metastatic pancreatic cancer (MPC) patients who receive palliative chemotherapy.

Methods

We reviewed 396 MPC patients receiving palliative chemotherapy. NLR was obtained before and after the first cycle of chemotherapy. In 118 patients with PET prior to chemotherapy, SUVmax was collected. Cut-off values were determined by ROC curve.

Results

In multivariate analysis of all patients, NLR and change in NLR after the first cycle of chemotherapy (ΔNLR) were independent prognostic factors for overall survival (OS). We scored the risk considering NLR and ΔNLR and identified 4 risk groups with different prognosis (risk score 0 vs 1 vs 2 vs 3: OS 9.7 vs 7.9 vs 5.7 vs 2.6 months, HR 1 vs 1.329 vs 2.137 vs 7.915, respectively; P<0.001). In PET cohort, NLR and SUVmax were independently prognostic for OS. Prognostication model using both NLR and SUVmax could define 4 risk groups with different OS (risk score 0 vs 1 vs 2 vs 3: OS 11.8 vs 9.8 vs 7.2 vs 4.6 months, HR 1 vs 1.536 vs 2.958 vs 5.336, respectively; P<0.001).

Conclusions

NLR and SUVmax as simple parameters of host immunity and metabolic activity of tumor cell, respectively, are independent prognostic factors for OS in MPC patients undergoing palliative chemotherapy.     

RNAi-mediated <Emphasis Type="Italic">Soybean mosaic virus</Emphasis> (SMV) resistance of a Korean Soybean cultivar

Soybean [Glycine max (L.) Merr.] is an important crop for vegetable oil production, and is a major protein source worldwide. Because of its importance as a crop, genetic transformation has been used extensively to improve its valuable traits. Soybean mosaic virus (SMV) is one of the most well-known viral diseases affecting soybean. Transgenic soybean plants with improved resistance to SMV were produced by introducing HC-Pro coding sequences within RNA interference (RNAi) inducing hairpin construct via Agrobacterium-mediated transformation. During an experiment to confirm the response of transgenic plants (T₂) to SMV infection, no T₂ plants from lines #2 (31/31), #5 (35/35) or #6 (37/37) exhibited any SMV symptoms, indicating strong viral resistance (R), whereas NT (non-transgenic wild type) plants and those from lines #1, #3 and #4 exhibited mild mosaic (mM) or mosaic (M) symptoms. The northern blot analysis showed that three resistant lines (#2, #5 and #6) did not show the detection of viral RNA accumulation while NT, EV (transformed with empty vector carrying only Bar) and lines #1, #3 and #4 plants were detected. T₃ seeds from SMV-inoculated T₂ plants were harvested and checked for changes in seed morphology due to viral infection. T₃ seeds of lines #2, #5 and #6 were clear and seed coat mottling was not present, which is indicative of SMV resistance. RT-PCR and quantitative real-time PCR showed that T₃ seeds from the SMV-resistant lines #2, #5 and #6 did not exhibit any detection of viral RNA accumulation (HC-Pro, CP and CI), while the viral RNA accumulation was detected in SMV-susceptible lines #1, #3 and #4 plants. During the greenhouse test for viral resistance and yield components, T₃ plants from the SMV-inoculated transgenic lines #2, #5 and #6 showed viral resistance (R) and exhibited a more favorable average plant height, number of nodes per plant, number of branches per plant, number of pods per plant and total seed weight with statistical significance during strong artificial SMV infection than did other plant lines. In particular, the SMV-resistant line #2 exhibited superior average plant height, pod number and total seed weight with highly significance. According to our results, RNAi induced by the hairpin construct of the SMV HC-Pro sequence effectively confers much stronger viral resistance than did the methods used during previous trials, and has the potential to increase yields significantly. Because of its efficiency, the induction of RNAi-mediated resistance will likely be used more frequently as part of the genetic engineering of plants for crop improvement.     

Complete chloroplast genome sequences of <Emphasis Type="Italic">Solanum commersonii</Emphasis> and its application to chloroplast genotype in somatic hybrids with <Emphasis Type="Italic">Solanum tuberosum</Emphasis>

Key message

Chloroplast genome of Solanum commersonii and S olanum tuberosum were completely sequenced, and Indel markers were successfully applied to distinguish chlorotypes demonstrating the chloroplast genome was randomly distributed during protoplast fusion.

Abstract

Somatic hybridization has been widely employed for the introgression of resistance to several diseases from wild Solanum species to overcome sexual barriers in potato breeding. Solanum commersonii is a major resource used as a parent line in somatic hybridization to improve bacterial wilt resistance in interspecies transfer to cultivated potato (S. tuberosum). Here, we sequenced the complete chloroplast genomes of Lz3.2 (S. commersonii) and S. tuberosum (PT56), which were used to develop fusion products, then compared them with those of five members of the Solanaceae family, S. tuberosum, Capsicum annum, S. lycopersicum, S. bulbocastanum and S. nigrum and Coffea arabica as an out-group. We then developed Indel markers for application in chloroplast genotyping. The complete chloroplast genome of Lz3.2 is composed of 155,525 bp, which is larger than the PT56 genome with 155,296 bp. Gene content, order and orientation of the S. commersonii chloroplast genome were highly conserved with those of other Solanaceae species, and the phylogenetic tree revealed that S. commersonii is located within the same node of S. tuberosum. However, sequence alignment revealed nine Indels between S. commersonii and S. tuberosum in their chloroplast genomes, allowing two Indel markers to be developed. The markers could distinguish the two species and were successfully applied to chloroplast genotyping (chlorotype) in somatic hybrids and their progenies. The results obtained in this study confirmed the random distribution of the chloroplast genome during protoplast fusion and its maternal inheritance and can be applied to select proper plastid genotypes in potato breeding program.

     

Dynamics and Interactions of OmpF and LPS: Influence on Pore Accessibility and Ion Permeability

The asymmetric outer membrane of Gram-negative bacteria is formed of the inner leaflet with phospholipids and the outer leaflet with lipopolysaccharides (LPS). Outer membrane protein F (OmpF) is a trimeric porin responsible for the passive transport of small molecules across the outer membrane of Escherichia coli. Here, we report the impact of different levels of heterogeneity in LPS environments on the structure and dynamics of OmpF using all-atom molecular dynamics simulations. The simulations provide insight into the flexibility and dynamics of LPS components that are highly dependent on local environments, with lipid A being the most rigid and O-antigen being the most flexible. Increased flexibility of O-antigen polysaccharides is observed in heterogeneous LPS systems, where the adjacent O-antigen repeating units are weakly interacting and thus more dynamic, compared to homogeneous LPS systems in which LPS interacts strongly with each other with limited overall flexibility due to dense packing. The model systems were validated by comparing molecular-level details of interactions between OmpF surface residues and LPS core sugars with experimental data, establishing the importance of LPS core oligosaccharides in shielding OmpF surface epitopes recognized by monoclonal antibodies. There are LPS environmental influences on the movement of bulk ions (K⁺ and Cl⁻), but the ion selectivity of OmpF is mainly affected by bulk ion concentration.