Shewanella goraebulensis sp. nov., isolated from sea water.

A novel Gram-stain-negative, facultatively anaerobic and rod-shaped bacterial strain, designated as DAU312T, was isolated from the sea water of the eastern coast of the Republic of Korea. Optimal growth was observed at 25 °C, pH 7.0-8.0 and with NaCl concentrations of 2.0 % (w/v). Catalase and oxidase activities were detected. On the basis of 16S rRNA gene sequences, strain DAU312T showed the highest similarity (99.2 %) to the type strain Shewanella electrodiphila MAR441T. The complete genome sequence of strain DAU312T contains 4 893 483 bp and 40.5 mol% G+C. Phylogenetic analyses based on 16S rRNA gene sequences and the up-to-date bacterial core genes showed that strain DAU312T, S. electrodiphila MAR441T and S. olleyana were all part of the same monophyletic clade. Their average nucleotide identity, digital DNA-DNA hybridization and two-way average amino acid identity values with each other and type strains of close Shewanella species were 83.4-77.5 %, 27.3-22.0 % and 89.8-81.2 %, respectively. The major cellular fatty acids (>10 %) were iso-C15 : 0, summed feature 3 (C16 : 1 ω7с and/or C16 : 1 ω6с) and C16 : 0. Phosphatidylethanolamine and phosphatidylglycerol were the main polar lipids. The respiratory quinones were Q-7, Q-8, MK-7 and MMK-7. Based on these polyphasic taxonomic findings, the name Shewanella goraebulensis sp. nov. is suggested for strain DAU312T, which is considered to represent a novel species of the genus Shewanella. The type strain is DAU312T (=KCTC 72427 T=JCM 35744T=KCCM 43478T).

Strategic biomodification for raw plant-based pretreatment biorefining toward sustainable chemistry.

Plant-based pretreatment biorefining is the initial triggering process in biomass-conversion to bio-based chemical products. In view of chemical sustainability, the raw plant-based pretreatment biorefining process is more favorable than the fossil-based one. Its direct use contributes to reducing CO2 emissions and the production cost of the target products by eliminating costly steps, such as the separation and purification of intermediates. Three types of feedstock plant resources have been utilized as raw plant feedstock sources, such as: lignocellulosic, starchy, and inulin-rich feedstock plants. These plant sources can be directly used for bio-based chemical products. To enhance the efficiency of their pretreatment biorefining process, well-designed biomodification schemes are discussed in this review to afford important information on useful biomodification approaches. For lignocellulosic feedstock plants, the enzymes and regulatory elements involved in lignin reduction are discussed using: COMT, GAUT4, CSE, PvMYB4 repressor, etc. For inulin-rich feedstock plants, 1-SST, 1-FFT, 1-FEH, and endoinulinase are illustrated in relation with the reduction of chain length of inulin polymer. For starchy feedstock plants, their biomodification is targeted to enhancing the depolymerization efficiency of starch to glucose monomer units. For this biomodification target, six candidates are discussed. These are SBE I, SBE IIa, SBE IIb, GBSS I, PTSTI, GWD 1, and PTSTI. The biomodification strategies discussed here promise to be conducive to enhancing the efficiency of the plant-based pretreatment biorefining process.

Critical challenges and advances in recombinant adeno-associated virus (rAAV) biomanufacturing.

Gene therapy is a promising therapeutic approach for genetic and acquired diseases nowadays. Among DNA delivery vectors, recombinant adeno-associated virus (rAAV) is one of the most effective and safest vectors used in commercial drugs and clinical trials. However, the current yield of rAAV biomanufacturing lags behind the necessary dosages for clinical and commercial use, which embodies a concentrated reflection of low productivity of rAAV from host cells, difficult scalability of the rAAV-producing bioprocess, and high levels of impurities materialized during production. Those issues directly impact the price of gene therapy medicine in the market, limiting most patients' access to gene therapy. In this context, the current practices and several critical challenges associated with rAAV gene therapy bioprocesses are reviewed, followed by a discussion of recent advances in rAAV-mediated gene therapy and other therapeutic biological fields that could improve biomanufacturing if these advances are integrated effectively into the current systems. This review aims to provide the current state-of-the-art technology and perspectives to enhance the productivity of rAAV while reducing impurities during production of rAAV.

Design space determination to optimize DNA complexation and full capsid formation in transient rAAV manufacturing.

Recombinant adeno-associated virus (rAAV) vectors are a promising platform for in vivo gene therapies. However, cost-effective, well-characterized processes necessary to manufacture rAAV therapeutics are challenging to develop without an understanding of how process parameters (PPs) affect rAAV product quality attributes (PQAs). In this work, a central composite orthogonal experimental design was employed to examine the influence of four PPs for transient transfection complex formation (polyethylenimine:DNA [PEI:DNA] ratio, total DNA/cell, cocktail volume, and incubation time) on three rAAV PQAs related to capsid content (vector genome titer, vector genome:capsid particle ratio, and two-dimensional vector genome titer ratio). A regression model was established for each PQA using partial least squares, and a design space (DS) was defined in which Monte Carlo simulations predicted < 1% probability of failure (POF) to meet predetermined PQA specifications. Of the three PQAs, viral genome titer was most strongly correlated with changes in complexation PPs. The DS and acceptable PP ranges were largest when incubation time and cocktail volume were kept at mid-high setpoints, and PEI:DNA ratio and total DNA/cell were at low-mid setpoints. Verification experiments confirmed model predictive capability, and this work establishes a framework for studying other rAAV PPs and their relationship to PQAs.

Development and Validation of a Convolutional Neural Network Model to Predict a Pathologic Fracture in the Proximal Femur Using Abdomen and Pelvis CT Images of Patients With Advanced Cancer.

BACKGROUND: Improvement in survival in patients with advanced cancer is accompanied by an increased probability of bone metastasis and related pathologic fractures (especially in the proximal femur). The few systems proposed and used to diagnose impending fractures owing to metastasis and to ultimately prevent future fractures have practical limitations; thus, novel screening tools are essential. A CT scan of the abdomen and pelvis is a standard modality for staging and follow-up in patients with cancer, and radiologic assessments of the proximal femur are possible with CT-based digitally reconstructed radiographs. Deep-learning models, such as convolutional neural networks (CNNs), may be able to predict pathologic fractures from digitally reconstructed radiographs, but to our knowledge, they have not been tested for this application. QUESTIONS/PURPOSES: (1) How accurate is a CNN model for predicting a pathologic fracture in a proximal femur with metastasis using digitally reconstructed radiographs of the abdomen and pelvis CT images in patients with advanced cancer? (2) Do CNN models perform better than clinicians with varying backgrounds and experience levels in predicting a pathologic fracture on abdomen and pelvis CT images without any knowledge of the patients' histories, except for metastasis in the proximal femur? METHODS: A total of 392 patients received radiation treatment of the proximal femur at three hospitals from January 2011 to December 2021. The patients had 2945 CT scans of the abdomen and pelvis for systemic evaluation and follow-up in relation to their primary cancer. In 33% of the CT scans (974), it was impossible to identify whether a pathologic fracture developed within 3 months after each CT image was acquired, and these were excluded. Finally, 1971 cases with a mean age of 59 ± 12 years were included in this study. Pathologic fractures developed within 3 months after CT in 3% (60 of 1971) of cases. A total of 47% (936 of 1971) were women. Sixty cases had an established pathologic fracture within 3 months after each CT scan, and another group of 1911 cases had no established pathologic fracture within 3 months after CT scan. The mean age of the cases in the former and latter groups was 64 ± 11 years and 59 ± 12 years, respectively, and 32% (19 of 60) and 53% (1016 of 1911) of cases, respectively, were female. Digitally reconstructed radiographs were generated with perspective projections of three-dimensional CT volumes onto two-dimensional planes. Then, 1557 images from one hospital were used for a training set. To verify that the deep-learning models could consistently operate even in hospitals with a different medical environment, 414 images from other hospitals were used for external validation. The number of images in the groups with and without a pathologic fracture within 3 months after each CT scan increased from 1911 to 22,932 and from 60 to 720, respectively, using data augmentation methods that are known to be an effective way to boost the performance of deep-learning models. Three CNNs (VGG16, ResNet50, and DenseNet121) were fine-tuned using digitally reconstructed radiographs. For performance measures, the area under the receiver operating characteristic curve, accuracy, sensitivity, specificity, precision, and F1 score were determined. The area under the receiver operating characteristic curve was used to evaluate three CNN models mainly, and the optimal accuracy, sensitivity, and specificity were calculated using the Youden J statistic. Accuracy refers to the proportion of fractures in the groups with and without a pathologic fracture within 3 months after each CT scan that were accurately predicted by the CNN model. Sensitivity and specificity represent the proportion of accurately predicted fractures among those with and without a pathologic fracture within 3 months after each CT scan, respectively. Precision is a measure of how few false-positives the model produces. The F1 score is a harmonic mean of sensitivity and precision, which have a tradeoff relationship. Gradient-weighted class activation mapping images were created to check whether the CNN model correctly focused on potential pathologic fracture regions. The CNN model with the best performance was compared with the performance of clinicians. RESULTS: DenseNet121 showed the best performance in identifying pathologic fractures; the area under the receiver operating characteristic curve for DenseNet121 was larger than those for VGG16 (0.77 ± 0.07 [95% CI 0.75 to 0.79] versus 0.71 ± 0.08 [95% CI 0.69 to 0.73]; p = 0.001) and ResNet50 (0.77 ± 0.07 [95% CI 0.75 to 0.79] versus 0.72 ± 0.09 [95% CI 0.69 to 0.74]; p = 0.001). Specifically, DenseNet121 scored the highest in sensitivity (0.22 ± 0.07 [95% CI 0.20 to 0.24]), precision (0.72 ± 0.19 [95% CI 0.67 to 0.77]), and F1 score (0.34 ± 0.10 [95% CI 0.31 to 0.37]), and it focused accurately on the region with the expected pathologic fracture. Further, DenseNet121 was less likely than clinicians to mispredict cases in which there was no pathologic fracture than cases in which there was a fracture; the performance of DenseNet121 was better than clinician performance in terms of specificity (0.98 ± 0.01 [95% CI 0.98 to 0.99] versus 0.86 ± 0.09 [95% CI 0.81 to 0.91]; p = 0.01), precision (0.72 ± 0.19 [95% CI 0.67 to 0.77] versus 0.11 ± 0.10 [95% CI 0.05 to 0.17]; p = 0.0001), and F1 score (0.34 ± 0.10 [95% CI 0.31 to 0.37] versus 0.17 ± 0.15 [95% CI 0.08 to 0.26]; p = 0.0001). CONCLUSION: CNN models may be able to accurately predict impending pathologic fractures from digitally reconstructed radiographs of the abdomen and pelvis CT images that clinicians may not anticipate; this can assist medical, radiation, and orthopaedic oncologists clinically. To achieve better performance, ensemble-learning models using knowledge of the patients' histories should be developed and validated. The code for our model is publicly available online at https://github.com/taehoonko/CNN_path_fx_prediction . LEVEL OF EVIDENCE: Level III, diagnostic study.

Prevalence of Vitamin D Deficiency in Children with Fractures: Before and during the COVID-19 Outbreak.

Background: Although it is generally agreed that vitamin D is important for bone health, the role of vitamin D in preventing fractures in children and adolescents remains unclear. Therefore, this study aimed to investigate the prevalence of vitamin D deficiency and insufficiency in healthy Korean children with fractures. Our secondary aim was to compare serum vitamin D levels before and during the coronavirus disease 2019 (COVID-19) outbreak. Methods: We evaluated 334 patients with fractures who were surgically treated at our institution between 2018 and 2019 before the onset of COVID-19 (group I). In addition, we collected data on the serum 25(OH)D levels of 210 patients who visited our pediatric department for evaluation of short stature (group II) and the serum 25(OH)D levels of the patients with fractures during the COVID-19 pandemic period (group III). A serum 25(OH)D level of <20 ng/mL was considered deficient, between 20 and 32 ng/mL was insufficient, and ≥32 ng/mL was considered sufficient. Results: The mean age was 8.1 ± 3.5 years in group I, 8.2 ± 3.7 years in group II, and 8.6 ± 3.5 years in group III. The prevalence of vitamin D deficiency was 53.0% in group I and 32.9% in group II. The mean serum 25(OH)D level was lower in group I than in group II (20.0 ± 7.3 ng/ml vs. 23.2 ± 6.9 ng/ml, p < 0.001). The mean serum 25(OH)D level of younger patients (<10 years) in group III was lower than that of the younger patients in the prepandemic period (21.4 ± 7.2 ng/mL vs. 19.2 ± 6.8 ng/mL, p=0.037). Conclusions: We observed a high prevalence of vitamin D deficiency/insufficiency in children with fractures who required surgical treatment. During the COVID-19 pandemic, the serum vitamin D levels became even lower, especially in younger children.

Nuclear import of LIKE HETEROCHROMATIN PROTEIN1 is redundantly mediated by importins α-1, α-2 and α-3.

LIKE HETEROCHROMATIN PROTEIN1 (LHP1) encodes the only plant homologue of the metazoan HETEROCHROMATIN PROTEIN1 (HP1) protein family. The LHP1 protein is necessary for proper epigenetic regulation of a range of developmental processes in plants. LHP1 is a transcriptional repressor of flowering-related genes, such as FLOWERING LOCUS T (FT), FLOWERING LOCUS C (FLC), AGAMOUS (AG) and APETALA 3 (AP3). We found that LHP1 interacts with importin α-1 (IMPα-1), importin α-2 (IMPα-2) and importin α-3 (IMPα-3) both in vitro and in vivo. A genetic approach revealed that triple mutation of impα-1, impα-2 and impα-3 resulted in Arabidopsis plants with a rapid flowering phenotype similar to that of plants with mutations in lhp1 due to the upregulation of FT expression. Nuclear targeting of LHP1 was severely impaired in the impα triple mutant, resulting in the de-repression of LHP1 target genes AG, AP3 and SHATTERPROOF 1 as well as FT. Therefore, the importin proteins IMPα-1, -2 and -3 are necessary for the nuclear import of LHP1.

Seagrass-based platform strategies for sustainable hydroxymethylfurfural (HMF) production: toward bio-based chemical products.

Today, sustainable chemistry is a key trend in the chemical manufacturing industry due mainly to concerns over the global environment and resource security. In sustainable chemical manufacture, the choice of a bio-based feedstock plays a pivotal pillar. In terms of feedstock utilization for producing HMF, which is a multivalent platform intermediate easily convertible to valuable chemical products; biopolymers, biofuels, and other important chemicals, seagrass biomasses can be more favorable feedstocks compared with land plant resources due primarily to easy availability and no systematic farming. Moreover, seagrass feedstocks could contribute cost-effectively and sustainably producing HMF by exploiting the beach-cast seagrasses on seagrass-prairies with no feedstock cost, indicating that seagrass biomasses could be a most promising biofeedstock source for sustainable HMF production. We afford a platform bioprocessing technology that has not been attempted before for sustainable HMF production using raw seagrass biomass. This bioprocess can be operated by simple reaction conditions using inorganic Brønsted acids (mainly HCl) and ionic liquid solvents at relatively low temperatures (120-130 °C). In addition, some bioengineering strategies for improving the growth of seagrass biomass and the quantity/quality of nonstructural carbohydrates (starch, sucrose) that can be used as the feeding substrates for HMF production are also discussed. The main aim of this review is to provide some important information about breakthrough bio/technologies conducive to cost-effective and sustainable HMF production.

VprBP has intrinsic kinase activity targeting histone H2A and represses gene transcription.

Histone modifications play important roles in the regulation of gene expression and chromatin organization. VprBP has been implicated in transcriptionally silent chromatin formation and cell-cycle regulation, but the molecular basis underlying such effects remains unclear. Here we report that VprBP possesses an intrinsic protein kinase activity and is capable of phosphorylating histone H2A on threonine 120 (H2AT120p) in a nucleosomal context. VprBP is localized to a large set of tumor suppressor genes and blocks their transcription, in a manner that is dependent on its kinase activity toward H2AT120. The functional significance of VprBP-mediated H2AT120p is further underscored by the fact that RNAi knockdown and small-molecule inhibition of VprBP reactivate growth regulatory genes and impede tumor growth. Our findings establish VprBP as a major kinase responsible for H2AT120p in cancer cells and suggest that VprBP inhibition could be a new strategy for the development of anticancer therapeutics.

Enhanced purification of histidine-tagged carboxymethylcellulase produced by Escherichia coli BL21/LBH-10 and comparison of its characteristics with carboxymethylcellulase without histidine-tag.

To enhance purification yield of the carboxymethylcellulase (CMCase) of P. aquimaris LBH-10, E. coli BL21/LBH-10 was constructed to produce the six histidine-tagged CMCase (CMCase with a His-tag). The purification yield of the CMCase with a His-tag produced by E. coli BL21/LBH-10 was 44.4%. The molecular weight of the CMCase with a His-tag was determined as 56 kDa. Its Km and Vmax were 7.4 g/L and 70.9 g/L min, respectively. The CMCase with a His-tag hydrolyzed avicel, carboxymethylcellulose (CMC), filter paper, pullulan, and xylan but did not hydrolyze cellobiose and p-nitrophenyl-ß-D-glucopyranoside. The optimal temperature for reaction was 50 °C and more than 75% of its original activity was maintained at broad temperatures ranging from 20 to 70 °C after 24 h. The optimal pH was 4.0 and more than 60% of its original activity was maintained at pH ranging from 4.0 to 7.0. The activity of the CMCase with a His-tag was enhanced by CoCl2, KCl, PbCl2, RbCl2, and SrCl2 until the concentration of 100 mM, but inhibited by EDTA, HgCl2, MnCl2, and NiCl2. The characteristics of the CMCase with a His-tag produced by E. coli BL21/LBH-10 were little different from the CMCase without a His-tag, which seemed to resulted from the conformational change in the structure due to a His-tag. The purification yield of the CMCase with a His-tag using affinity chromatography from the cell broth after cell breakdown was proven to be more economic than that from the supernatant with its low concentration of cellulase.

Construction of Escherichia coli BL21/A-53 producing histidine-tagged carboxymethylcellulase and comparison of its characteristics with CMCase without histidine-tag.

To enhance recovery yield of carboxymethylcellulase (CMCase), E. coli BL21/A-53 producing the histidine-tagged CMCase was constructed in this study. The recovery yield of the histidine-tagged CMCase using the His-tag affinity chromatography was 39.8%. The predicted molecular weight of the histidine-tagged CMCase was determined as 56,260 Da. Its Km and Vmax were 9.3 g l-1 and 76.3 g l-1·min-1, respectively. The histidine-tagged CMCase hydrolyzed avicel, carboxymethylcellulose (CMC), filter paper, pullulan, xylan, but there was no detectable activity on cellobiose, p-Nitrophenyl-ß-D-glucopyranoside (pNPG). The optimal temperature and pH for the enzymatic reaction of the histidine-tagged CMCase was 50 °C and 5.0. The histidine-tagged CMCase was enhanced by CoCl2 until the concentration of 100 mM, but inhibited by EDTA, HgCl2, MnCl2, NiCl2, and RbCl2. The characteristics of the histidine-tagged CMCase produced by E. coli BL21/A-53 were compared with those of CMCase without the histidine-tag of Bacillus subtilis subsp. subtilis A-53. The little changed characteristics of the histidine-tagged CMCase compared to the CMCase without a His-tag seemed to be the conformational change in the structure due to a His-tag.

Pneumatosis cystoides intestinalis associated with sunitinib and a literature review.

BACKGROUND: Pneumatosis cystoides intestinalis (PCI) is a rare self-limiting condition characterized by air-filled cysts within intestinal walls. Diagnosis should be prudent because it can mimic pneumoperitoneum leading to unnecessary treatment such as surgical exploration. Although various drugs including anti-neoplastic agents have been suggested as etiologies, cases related to sunitinib are sparse. Because of the rarity of this unusual side effect by sunitinib, we report the case report. CASE PRESENTATION: A 68-year-old female with pancreatic neuroendocrine tumor who was treated with sunitinb for 4 months visited to our hospital complaining of severe diarrhea and mild abdominal discomfort. The abdominal X-ray showed subdiaphragmatic air mimicking intestinal perforation. After the meticulous evaluation including abdomino-pelvic computed tomography, the patient was diagnosed of PCI induced by sunitinib and fully recovered with conservative management. CONCLUSIONS: It is important to note that PCI can develop after treatment with sunitinib because PCI has not been widely known as an adverse event caused by the agent. Furthemore, emergent surgery while sunitinib was administrated without adequate washout period can result in substantial surgical complications which could be avoided with the precise diagnosis.

Is a Price Increase Policy Enough for Adolescent Smokers?: Factors Affecting the Effectiveness of Increasing Cigarette Prices Among Korean Adolescent Smokers.

BACKGROUND: Cigarette pricing policy is one tool for controlling smoking behavior on a national scale. It is unclear, however, what effects such policy has on adolescents and which characteristic subgroups of adolescents are more or less sensitive to cigarette pricing policy. MATERIALS AND METHODS: Our data came from the 2013 Korea Youth Risk Behavior Web-based Survey. The dependent variable was whether or not a participant was classified as a "persistent smokers," defined as a smoker who would continue smoking despite any price increase. Other variables of interest were smoking days (quantity), previous attempts to stop smoking, and previous education on smoking cessation. The statistical analysis was performed using weighted data and the SURVEYFREQ and SURVEYLOGISTIC procedures in SAS 9.3. RESULTS: Among 7094 adolescent smokers (5349 males and 1745 females), 19.9% of males and 25.1% of females reported as persistent smokers. Compared with light smokers, heavy smokers are more likely to be persistent smokers (male: odds ratio [OR] = 2.45, 95% confidence interval [CI] = 2.04-2.95, P value < .001; female: OR = 3.23, 95% CI = 2.44-4.27, P value < .001). When we stratified the data by household income, previous attempts to stop smoking, and previous education on smoking cessation, that trend remained statistically significant. CONCLUSIONS: Because heavier smokers with higher risk of health-related consequences were less sensitive to pricing policy than mild smokers, pricing policy alone is not enough to reduce the societal burden caused by smoking. We suggest that additional cessation policy is needed along with pricing policy for adolescents with heavier smoking behavior in Korea. IMPLICATION: This study shows that heavy smokers are more likely to be persistent smokers despite the cigarette price increase policy, compared with light smokers in Korean adolescents. Because heavier smokers were less sensitive to pricing policy than mild smokers, pricing policy alone is not enough to reduce the societal burden caused by smoking. We suggest that additional tobacco control policies should be evaluated and effective ones implemented in addition to cigarette prices to reduce smoking among regular adolescent smokers.

Differential effects of Best disease causing missense mutations on bestrophin-1 trafficking.

Mutations in bestrophin-1 (Best1) cause Best vitelliform macular dystrophy (BVMD), a dominantly inherited retinal degenerative disease. Best1 is a homo-oligomeric anion channel localized to the basolateral surface of retinal pigment epithelial (RPE) cells. A number of Best1 mutants mislocalize in Madin-Darby canine kidney (MDCK) cells. However, many proteins traffic differently in MDCK and RPE cells, and MDCK cells do not express endogenous Best1. Thus, effects of Best1 mutations on localization in MDCK cells may not translate to RPE cells. To determine whether BVMD causing mutations affect Best1 localization, we compared localization and oligomerization of Best1 with Best1 mutants V9M, W93C, and R218C. In MDCK cells, Best1 and Best1(R218C) were basolaterally localized. Best1(W93C) and Best1(V9M) accumulated in cells. In cultured fetal human retinal pigment epithelium cells (fhRPE) expressing endogenous Best1, Best1(R218C) and Best1(W93C) were basolateral. Best1(V9M) was intracellular. All three mutants exhibited similar fluorescence resonance energy transfer (FRET) efficiencies to, and co-immunoprecipitated with Best1, indicating unimpaired oligomerization. When human Best1 was expressed in RPE in mouse eyes it was basolaterally localized. However, Best1(V9M) accumulated in intracellular compartments in mouse RPE. Co-expression of Best1 and Best1(W93C) in MDCK cells resulted in basolateral localization of both Best1 and Best1(W93C), but co-expression of Best1 with Best1(V9M) resulted in mislocalization of both proteins. We conclude that different mutations in Best1 cause differential effects on its localization and that this effect varies with the presence or absence of wild-type (WT) Best1. Furthermore, MDCK cells can substitute for RPE when examining the effects of BVMD causing mutations on Best1 localization if co-expressed with WT Best1.

Characterization of maltotriose production by hydrolyzing of soluble starch with α-amylase from Microbulbifer thermotolerans DAU221.

A maltotriose-producing α-amylase, AmyA, from a newly isolated bacterial strain Microbulbifer thermotolerans DAU221 was purified and characterized in the heterologous host, Escherichia coli, using the pCold I vector. The amyA gene encoded a 761-residue protein composed of a 33 amino acid secretion signal peptide. The purified α-amylase with a molecular mass of 80 kDa, approximately, shared a sequence motif characteristic of the glycoside hydrolase family 13. The enzyme was optimally active, at 50 °C in sodium phosphate buffer (pH 6.0), by the traditional one factor-at-a-time method. But the optimal conditions of time, temperature, and pH for production of maltotriose from soluble starch were 1.76 h, 44.95 °C, and pH 6.35 by response surface methodology, respectively. Maltotriose, as the major enzyme reaction product, was analyzed by thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC). The enzyme was found to be inhibited by the addition of 10 mM Cu(2+), Fe(3+), Hg(2+), Zn(2+), and EDTA, but exhibited extreme stability toward hexane. The K m and V max values for the hydrolysis of soluble starch were 1.08 mg/mL and 1.736 mmol maltotriose/mg protein/min, respectively.

CXCR2 inhibition enhances sulindac-mediated suppression of colon cancer development.

Small chemical compound sulindac has been approved as a preventive approach against colon cancer for its effectiveness in treatment of precancerous adenoma. Due to its severe toxicities in the cardiovascular, gastrointestinal and renal systems, however, a combination of low-dose sulindac with other chemopreventive agents has been sought after as an alternative therapeutic strategy that could increase its effectiveness, while minimizing its adverse effects. To identify the promising alternative approach, we investigated the therapeutic potential of targeting the interleukin (IL)-8/CXCR2 pathway in colon cancer treatment using both loss-of-function (CXCR2 knockout) and gain-of-function (IL-8 overexpression) mouse models, as the IL-8/CXCR2 pathway has been shown to be activated in intestinal tumors of both human and experimental animals. We found that deletion of CXCR2 gene and ectopic expression of IL-8 suppresses and enhances, respectively, intestinal tumor development caused by a mutation in the APC gene. Moreover, a single copy deletion of CXCR2 gene resulted in abrogation of COX-2 and Gro-α upregulation in intestinal tumors caused by the APC mutation. Moreover, a single copy (heterozygote) deletion of CXCR2 gene was sufficient to synergize with a low-dose sulindac treatment in suppressing APCmin-induced intestinal polyposis. Together, our study provides a therapeutic justification of combined inhibition of CXCR2 and sulindac treatment in colon cancer prevention.

Opposing regulation of PROX1 by interleukin-3 receptor and NOTCH directs differential host cell fate reprogramming by Kaposi sarcoma herpes virus.

Lymphatic endothelial cells (LECs) are differentiated from blood vascular endothelial cells (BECs) during embryogenesis and this physiological cell fate specification is controlled by PROX1, the master regulator for lymphatic development. When Kaposi sarcoma herpes virus (KSHV) infects host cells, it activates the otherwise silenced embryonic endothelial differentiation program and reprograms their cell fates. Interestingly, previous studies demonstrated that KSHV drives BECs to acquire a partial lymphatic phenotype by upregulating PROX1 (forward reprogramming), but stimulates LECs to regain some BEC-signature genes by downregulating PROX1 (reverse reprogramming). Despite the significance of this KSHV-induced bidirectional cell fate reprogramming in KS pathogenesis, its underlying molecular mechanism remains undefined. Here, we report that IL3 receptor alpha (IL3Rα) and NOTCH play integral roles in the host cell type-specific regulation of PROX1 by KSHV. In BECs, KSHV upregulates IL3Rα and phosphorylates STAT5, which binds and activates the PROX1 promoter. In LECs, however, PROX1 was rather downregulated by KSHV-induced NOTCH signal via HEY1, which binds and represses the PROX1 promoter. Moreover, PROX1 was found to be required to maintain HEY1 expression in LECs, establishing a reciprocal regulation between PROX1 and HEY1. Upon co-activation of IL3Rα and NOTCH, PROX1 was upregulated in BECs, but downregulated in LECs. Together, our study provides the molecular mechanism underlying the cell type-specific endothelial fate reprogramming by KSHV.

Disease-causing mutations associated with four bestrophinopathies exhibit disparate effects on the localization, but not the oligomerization, of Bestrophin-1.

BEST1 encodes Bestrophin-1 (Best1), a homo-oligomeric, integral membrane protein localized to the basolateral plasma membrane of the retinal pigment epithelium. Mutations in BEST1 cause five distinct retinal degenerative diseases, including adult vitelliform macular dystrophy (AVMD), autosomal recessive bestrophinopathy (ARB), autosomal dominant vitreoretinochoroidopathy (ADVIRC), and retinitis pigmentosa (RP). The mechanisms underlying these diseases and why mutations cause one disease over another are, for the most part, unknown. To gain insights into these four diseases, we expressed 28 Best1 mutants fused to YFP in polarized MDCK monolayers and, via confocal microscopy and immunofluorescence, live-cell FRET, and reciprocal co-immunoprecipitation experiments, screened these mutants for defects in localization and oligomerization. All 28 mutants exhibited comparable FRET efficiencies to and co-immunoprecipitated with WT Best1, indicating unimpaired oligomerization. RP- and ADVIRC-associated mutants were properly localized to the basolateral plasma membrane of cells, while two AVMD and most ARB mutants were mislocalized. When co-expressed, all mislocalized mutants caused mislocalization of WT Best1 to intracellular compartments. Our current and past results indicate that mislocalization of Best1 is not an absolute feature of any individual bestrophinopathy, occurring in AVMD, BVMD, and ARB. Furthermore, some ARB mutants that do not also cause dominant disease cause mislocalization of Best1, indicating that mislocalization is not a cause of disease, and that absence of Best1 activity from the plasma membrane is tolerated. Lastly, we find that the ARB truncation mutants L174Qfs*57 and R200X can form oligomers with WT Best1, indicating that the first ∼174 amino acids of Best1 are sufficient for oligomerization to occur.

Epigenetic regulation by long noncoding RNAs in plants.

Many eukaryotes, including plants, produce a large number of long noncoding RNAs (lncRNAs).Growing number of lncRNAs are being reported to have regulatory roles in various developmental processes.Emerging mechanisms underlying the function of lncRNAs indicate that lncRNAs are versatile regulatory molecules. They function as potent cis- and trans-regulators of gene expression, including the formation of modular scaffolds that recruit chromatin-modifying complexes to target chromatin. LncRNAs have also been reported in plants. Here, we describe our current understanding on potential roles of lncRNA in plants.

Feasibility of a Novel In-situ Local Tumor Ablation and Recycling Machine Based on Radiofrequency Dielectric Heating: In-depth Review on Research Background and Preliminary Report of an Experimental Study.

Background: In bone sarcomas, chemotherapy has improved the prognosis with advances in diagnostic and surgical technologies, which has led to attempts to save limbs. As early detection and multidisciplinary treatment have improved the survival rate, curative surgery is considered for selected patients with metastatic bone carcinomas. Limb salvage procedures may vary in relation to the reconstruction method, which is accompanied by different complications. To overcome them, we devised a novel concept, in-situ local tumor ablation and recycling machine based on radiofrequency (RF)-induced heating and intended experiments to demonstrate its feasibility. Methods: The fresh femurs of 6-month-old pigs were used after removing the epiphyses; the distal parts were placed in a heating chamber. Fiber-optic temperature sensors were inserted in the metaphysis, meta-diaphysis, and diaphysis. Temperatures were measured six times each during heating at 27.12 MHz at various powers. Additionally, the compressive and bending stiffnesses were measured six times each for the unprocessed, RF-treated, and pasteurized bones, and the results were compared. Results: Under 200 W power output, the temperatures at all measurement sites reached 70 ℃ or higher in 6 minutes, and the temperatures were maintained. The median compressive stiffness of RF-heated bones was 79.2% higher than that of pasteurized bones, but the difference was statistically insignificant. The median bending stiffness of RF-heated bones was approximately 66.3% of that of unprocessed bones, which was 20% higher than that of pasteurized bones. Conclusions: The feasibility to rapidly attain and maintain temperatures for tumor ablation is shown, which favorably preserves bone stiffness through the in-situ local tumor ablation and recycling based on RF heating. The problem of nonuniform temperature distribution might be solved by an optimal design determined from simulation research and additional experiments.