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Supplementary Methods, radiologist's reading of CT images, supplementary figure legends, supplementary references

microCT scans

SFigure 1: Histological examination of mouse lungs; SFigure 2: Tissue sections from human lung adenocarcinoma; SFigure 3: Cabozantinib (CBT) and vandetanib (VDT) resistant cells; SFigure 4: Comparison of in vitro inhibition of RET, RETV804L and RETV804M by ponatinib, cabozantinib, vandetanib, and lenvatinib; SFigure 5: Ponatinib treatment schedule and body weight measurements; SFigure 6: Lung section histology from Dox-induced C/KR mice and treated with vehicle or ponatinib for 1 months; SFigure 7: μCT examination of dox-induced C/KR mice with lung tumors before and after one month treatment with vehicle or ponatinib.

This study is aimed to investigate the health-associated benefits of bergamot-dietary fibers (DFs) with a special emphasis on weight loss and lipid-lowering effects, as well as the potential mechanisms involved. The feeding experiment of Sprague-Dawley (SD) rats for 6 weeks showed that DFs had dose-dependent regulatory effects against metabolic syndrome and they controlled obesity by slowing down the rate of weight growth, and reduced body mass index (BMI) and Lee's index without affecting appetite. Furthermore, DFs inhibited increment in TG, TC, LDL-C levels and AI index caused by a high-fat diet, and improved the pathological abnormality of the liver. Western blot results showed that DFs significantly up-regulated the protein expression levels of LXRα and CYP7A1, and down-regulated the levels of SREBP-1c, FAS, ACC and SREBP-2 in the liver. QRT-PCR results showed that DFs up-regulated PGC-1α, PRDM16, UCP-1, and PPARγ in brown adipose tissue. These results suggest that DFs played an effective role in reducing weight and lipids levels by promoting the decomposition and transport of lipids in liver, increasing the energy consumption of brown adipose tissue. DFs intervention reduced the difference in the intestinal microflora between rats fed with a normal diet and those fed with a high-fat diet. Soluble dietary fiber (SDF) and total dietary fiber (TDF) showed better weight loss and hypolipidemic potential compared to insoluble dietary fiber (IDF) at the same dose. In conclusion, bergamot-derived DFs demonstrated the potential to lower blood cholesterol and body weight and could be used to develop novel functional foods for the prevention or treatment of obesity and hyperlipidemia.

Large-scale group decision-making (LSGDM) based on social networks has become an important part of practical decision-making. The trust relationship in social networks has an influence on not only the clustering process but also the consensus reaching process (CRP). Decision-makers (DMs) can take different behaviors by using the trust relationship to influence consensus reaching, so identifying the adjustment behaviors of DMs in CRP is essential. This study considers the influence of the trust relationship on the CRP and proposes a behavior analysis-based consensus model that comprehensively considers the leadership behaviors and non-cooperative behaviors. First, based on the clustering result, the preference similarity of two DMs with the direct trust relationship is calculated to judge whether leadership behavior exists. By judging the leadership behaviors, the number of effective DMs involved in LSGDM will be reduced. Second, based on the identification of leadership behaviors, the non-cooperative or cooperative behaviors are defined by judging whether the adjustment behaviors of effective DMs are conducive to achieving group consensus. Third, the weights of effective DMs and subgroups are punished or rewarded by quantifying the degree of non-cooperative or cooperative behaviors. Finally, the simulation experiments and comparative analysis are presented to illustrate the efficiency of the proposed method.

Soy isoflavones (SI) are known for their beneficial effects in alleviating neurodegenerative diseases, while the mechanism of alleviation of depression-like behaviour by SI remains unclear. In this study, a chronic unpredictable mild stress (CUMS)-induced depression rat model was used to determine the effect of SI in alleviating depression-like behaviour and its possible mechanisms. SI supplements significantly improved the CUMS-induced depression-like behaviour by increasing the monoamine neurotransmitter levels. A specific SI dose significantly modulated the composition of the gut microbiota, which in turn improved the maximum biotransformation ability of SI. Spearman's correlation analysis illustrated that some of the gut microbiota genera were strongly correlated with monoamine neurotransmitters. Moreover, more attention should be paid to gender differences, which may be related to changes in the gut microbiota. These results suggest that SI might affect monoamine neurotransmitters of CUMS rats by reshaping the structure of the gut microbiota, thereby alleviating depression-like behaviour.

Seventy-five wild tilapia samples from six rivers (ten sites) in Guangxi province were collected and analyzed for 53 organochlorine compounds. DDTs, endosulfan, and PCBs were the most dominant compounds found in this study. Tiandong County (TD) and Guigang City (GG) sites were found to be heavily contaminated with high levels of endosulfan (385–925 ng/g lw) and/or DDTs (20.1–422 ng/g lw). The diagnostic ratios indicated that the residues of DDTs and endosulfan in wild tilapia are associated with historical applications as well as the recent introduction of technical DDTs and endosulfan at some sampling sites. The correlation between total length, body mass, and organochlorines (OCs) was higher than the correlation between age and lipid content. There was no significant correlation between organochlorine pesticides (OCPs) and lipid content. Therefore, for organisms, the feeding intensity (related to length and mass) of fish could better reflect degree of pollution than exposure time (age) of fish. The hazardous ratios for the 50th and 95th percentile data of OCPs and PCBs in fish were both below 1, suggesting that daily exposure to OCPs and PCBs yields a lifetime cancer risk lower than 1 in 10,000.

Background Many foods contain proteins and polyphenols, but there is a poor understanding of the nature of the inhibitory effect of protein on the biologic activity of polyphenols. The inhibitory mechanism of the food protein lactoferrin on the antibacterial activity of oligomeric ellagitannin oenothein B (OeB) was investigated using fluorescence quenching, isothermal titration calorimetry (ITC), circular dichroism (CD) measurement and molecular docking. Results The antibacterial activity of OeB against Staphylococcus aureus was inhibited by lactoferrin, which was retained at about 60%. An interaction study revealed that an interaction occurred between OeB and lactoferrin. Thermodynamic analyses indicate that the binding process was spontaneous, and the main driving forces were based on electrostatic interactions that contributed to a high interaction affinity between OeB and lactoferrin. Furthermore, CD spectra provided insights into conformational changes of lactoferrin. Finally, molecular docking analysis provided a visual representation of a single binding site where OeB interacted with specific amino acid residues located at the active site of lactoferrin. In particular, due to the unique macrocyclic structure and rigid ring structure of OeB, a small number of hydroxyl groups in the rigid structure of OeB interacted with the amino acid of lactoferrin while most of the phenolic hydroxyl groups were not associated with lactoferrin. Conclusion Our study provides a theoretical basis for the use of OeB as an antibacterial substance that can be used in nutraceuticals and pharmaceutical products. © 2020 Society of Chemical Industry.

The aim of this study was to investigate the inhibitory mechanism of oenothein B (OeB), a unique oligomer ellagitannin with a rigid structure, on porcine trypsin using fluorescence spectroscopy, isothermal titration calorimetry (ITC), circular dichroism (CD) and molecular docking. Trypsin activity was strongly inhibited by OeB in a competitive way. Fluorescence quenching of trypsin by OeB was a static quenching. The CD spectra showed that binding of OeB to trypsin altered trypsin's conformation. The ITC and docking studies revealed that the inhibitory mechanism of OeB occurred via binding to the interior hydrophobic groups of trypsin and the formation of hydrogen bonds with trypsin through binding to the amino acid residues Asn97, His573, Ser195 and Gln192. This study provides a theoretical and computational basis for the precise control of trypsin in food industry. Based on the results, OeB may be used in food technology research as novel bioactive trypsin inhibitor.

Accurate and highly sensitive detection of viable but nonculturable (VBNC) Vibrio parahaemolyticus is crucial for preventing seafood-related outbreaks. The VBNC state may occur due to bacterial susceptibility to cold shock during food storage. Propidium monoazide (PMA) has been widely applied to detect VBNC foodborne pathogens. In this study, we developed and compared real-time PCR (qPCR) and real-time LAMP (qLAMP) methods combined with an improved propidium monoazide (PMAxx) to detect pathogenic VBNC V. parahaemolyticus. The designed primers and probes were determined to be highly selective for V. parahaemolyticus strains. The combined PMAxx-qPCR method, requiring 100 min, demonstrated a quantification limit of 10.5 colony-forming units (CFU)/mL in pure culture and 28 CFU/g in raw shrimp respectively, which were 10-fold lower than the PMAxx-qLAMP method (45min). When testing mixtures containing different ratios of VBNC to dead V. parahaemolyticus, PMAxx-based methods were notably superior at distinguishing between VBNC and dead bacteria when VBNC cell concentrations were low. Therefore, PMAxx is an effective means for improving the detection and quantification of VBNC V. parahaemolyticus by qPCR and qLAMP. Pretreatment with PMAxx was shown to be suitable for detection of VBNC V. parahaemolyticus in complex food samples, including raw shrimp.

Soy isoflavones (SI) are known for their beneficial effects in alleviating neurodegenerative diseases, while the mechanism of alleviation of depression-like behaviour by SI remains unclear. In this study, a chronic unpredictable mild stress (CUMS)-induced depression rat model was used to determine the effect of SI in alleviating depression-like behaviour and its possible mechanisms. SI supplements significantly improved the CUMS-induced depression-like behaviour by increasing the monoamine neurotransmitter levels. A specific SI dose significantly modulated the composition of the gut microbiota, which in turn improved the maximum biotransformation ability of SI. Spearman's correlation analysis illustrated that some of the gut microbiota genera were strongly correlated with monoamine neurotransmitters. Moreover, more attention should be paid to gender differences, which may be related to changes in the gut microbiota. These results suggest that SI might affect monoamine neurotransmitters of CUMS rats by reshaping the structure of the gut microbiota, thereby alleviating depression-like behaviour.

One immunomagnetic separation (IMS) assay based on immunomagnetic beads (IMBs) has been evaluated as a potential pretreatment tool for the separation and enrichment of target bacteria. In this study, we successfully immobilized antibodies onto magnetic bead surfaces to form IMBs through biotin and a streptavidin (SA) system to capture viable but nonculturable (VBNC) Cronobacter sakazakii (C. sakazakii) from dairy products. Various parameters that affected the capture efficiency (CE) of IMS, including the number of antibodies, IMBs dose, incubation time, magnetic separation time, and immunoreaction temperature, were systematically investigated. We further determined the optimal enrichment conditions for different dairy substrates to ensure maximum enrichment of target pathogens in the system. An IMS technique combining improved propidium monoazide (PMAxx) and droplet digital PCR (ddPCR) was established to detect the pathogenic VBNC C. sakazakii. The IMS-PMAxx-ddPCR method after IMBs enrichment showed higher accuracy when the VBNC C. sakazakii was under 1 Log10 copies/g. The detection limit for this method in a background of powdered infant formula (PIF) was 5.6 copies/g. In summary, the developed IMS-PMAxx-ddPCR method has great potential for the analysis and detection of VBNC bacteria in food.

Equol, which produced from daidzein (one of the principal isoflavones), is recognized to be the most resultful in stimulating an estrogenic and antioxidant response. The daidzein transformation was studied during fermentation of five growth media inoculated with feces from a healthy human, and a daidzein conversion strain was isolated. To enrich the bacterial population involved in daidzein metabolism in a complex mixture, fecal samples were treated with antibiotics. The improved propidium monoazide combined with the quantitative polymerase chain reaction (PMAxx-qPCR) assay showed that the ampicillin treatment of samples did result in a reduction of the total visible bacteria counts by 52.2% compared to the treatment without antibiotics. On this basis, the newly isolated rod-shaped, Gram-positive anaerobic bacterium, named strain Y11 (MN560033), was able to metabolize daidzein to equol under anaerobic conditions, with a conversion ratio (equol ratio: the amount of equol produced/amount of supplemented daizein) of 0.56 over 120 h. The 16S rRNA partial sequence of the strain Y11 exhibited 99.8% identity to that of Slackia equolifaciens strain DZE (NR116295). This study will provide new insights into the biotransformation of equol from daidzein by intestinal microbiota from the strain-level and explore the possibility of probiotic interventions.

This study reports the development and optimization of a real-time loop-mediated isothermal amplification (qLAMP) method for rapid detection of Acetobacter aceti strain in red wine samples. Our results showed that the primers and probes designed for 16S rRNA were effective for A. aceti detection. The quantification limit of real-time polymerase chain reaction (qPCR) and qLAMP in pure culture was 2.05 × 101 colony forming units (CFU) mL−1. qLAMP had a sensitivity of 6.88 × 101 CFU mL−1 in artificially contaminated Changyu dry red wine (CDRW) and Changyu red wine (CRW), and 6.88 × 102 CFU mL−1 in artificially contaminated Greatwall dry red wine (GDRW), which was 10 times higher than that of qPCR. In conclusion, this newly developed qLAMP is a reliable, rapid and accurate method for the detection and quantification of A. aceti species in red wine samples. Furthermore, our work provides a standard reference method for the quantitative detection of A. aceti and other acetic acid bacteria during the fermentation and storage of red wine samples.

OBJECTIVES To evaluate the diagnostic performance of ultrasound (US)-guided fine-needle aspiration with optional core needle biopsy of head and neck lymph nodes and masses, with attention to differences between biopsy of treated squamous cell carcinoma (SCC) and biopsy of other lesions. METHODS Institutional Review Board approval was obtained, and the need for consent was waived for this retrospective study. All 861 US-guided biopsies of head and neck lymph nodes and masses performed between March 1, 2012, and May 16, 2016, were reviewed. RESULTS Of the 861 biopsies, 53 targeted SCC with residual masses after treatment. The biopsy procedures yielded benign or malignant pathologic results in 71.7% (38 of 53) of treated SCC and 90.7% (733 of 808) of all other lesions (P

Renal cell carcinoma (RCC) is a common and devastating disease characterized by a hypoxic microenvironment, epithelial-mesenchymal transition and potent resistance to therapy evidencing the presence of cancer stem cells (CSCs). Various CSC markers have been studied in RCC, but overall there is limited data on their role and most markers studied have been relatively nonspecific. Doublecortin-like kinase 1 (DCLK1) is a validated CSC marker in the gastrointestinal tract and evidence for an equivalent role in other cancers is accumulating. We used bioinformatics, immunohistochemistry, flow cytometry, spheroid self-renewal and chemoresistance assays in combination with overexpression and siRNA-knockdown to study the stem cell-supportive role of DCLK1 alternative splice variants (DCLK1 ASVs) in RCC. To target tumor cells expressing DCLK1 ASVs directly, we developed a novel monoclonal antibody (CBT-15) and delivered it systemically to RCC tumor xenografts. DCLK1 ASVs were overexpressed, enriched together with CSC markers and predictive of overall and recurrence-free survival in RCC patients. In vitro, DCLK1 ASVs were able to directly stimulate essential molecular and functional characteristics of renal CSCs including expression of aldehyde dehydrogenase, self-renewal and resistance to FDA-approved receptor tyrosine kinase and mTOR inhibitors, while targeted downregulation of DCLK1 reversed these characteristics. Finally, targeting DCLK1 ASV-positive cells with the novel CBT-15 monoclonal antibody blocked RCC tumorigenesis in vivo. These findings establish DCLK1 as a CSC marker with implications for therapy, disease progression and survival in RCC and demonstrate the therapeutic value of DCLK1-targeted monoclonal antibodies against renal CSCs.

Cronobacter sakazakii (C. sakazakii), a pathogen that exists in dry and low-moisture environments, such as powder infant formula (PIF), can enter a viable but nonculturable (VBNC) state under harsh conditions, which enables it to escape traditional detection methods and thus poses a potential public health risk. This study aimed at assessing the virulent nature of VBNC C. sakazakii. Our results showed that VBNC C. sakazakii induced intestinal inflammation in neonatal rats. However, the degree of inflammation was significantly lower than that of culturable bacteria due to decreasing endotoxin production, motility, adhesion, and invasion ability in the VBNC state. From the perspective of bacterial translocation, the numbers of C. sakazakii in the blood, liver, and spleen of rats treated with VBNC cells were in the same order of magnitude as those treated with its culturable counterpart and may lead to the same degree of bacteremia. According to the macrophage survival assays, the survival rate of VBNC C. sakazakii within macrophages was 4.7 times higher than that of culturable cells. Based on these findings, we hypothesize that VBNC C. sakazakii evaded the host immune defense system, penetrated the tissue barrier, and translocated to the bloodstream, liver, and spleen through macrophages. Thus, our study reveals that VBNC C. sakazakii could be a potential risk for infants' health.

Objectives Preferential use of fine-needle aspiration (FNA) versus core needle biopsy (CNB) for distinguishing benign from malignant major salivary gland tumors is highly debated. The main disadvantage of FNA is lower sensitivity, whereas arguments against CNB include use of a larger bore needle and greater risk of complications. The aim of this study is to evaluate our experience performing ultrasound-guided (UG) FNA with selective use of CNB based on preliminary cytopathology, and to determine whether our preoperative diagnostic approach is more sensitive and specific than FNA alone—and at least as sensitive and specific as CNB alone. Study Design Retrospective review of UG needle biopsy sampling of lesions arising in or around parotid and submandibular glands. Methods Ultrasounds of 141 needle biopsies were identified. Patient/lesion/needle biopsy characteristics, preliminary cytopathology, final pathology, imaging studies, and subsequent clinical course and treatment were documented. Results Needle biopsies performed according to our protocol provided results that guided clinical decision making in 125 of 135 cases, 92.6% (95% confidence interval [CI], 86.8%–96.4%) of the time. Using 41 cases that had histologic verification, sensitivity was 100% (95% CI, 79.6%–100%), and specificity was 92.3% (95% CI, 75.9%–97.9%) for detecting malignancy. We definitively characterized 120 lesions as benign (84) or malignant (36). Conclusion Preoperative needle biopsy diagnoses allowed clinical management to progress 92.6% of the time. The protocol of FNA with selective use of CNB may potentially reduce patient exposure to risks associated with CNB without the tradeoff of lower sensitivity seen with FNA. Level of Evidence 4. Laryngoscope, 2017

In a viable but nonculturable (VBNC) state, bacteria are no longer culturable on standard laboratory media, but still, remain a pathogenic potential and present possible health risks. In this study, we investigated ampicillin's ability, which is commonly used in dairy cattle disease treatment, to induce Cronobacter sakazakii into the VBNC state. After treatment with ampicillin, the counts of culturable cells decreased from 108 CFU/mL to an undetected level 7–30 days post-treatment. Meanwhile, viable cells were still approximately 104–105 cells/mL, and could be resuscitated under appropriate conditions. Fluorescence microscopy showed that VBNC cell maintained apparent cellular integrity, but that the morphology of VBNC cells differed visibly from that of normal cells. Moreover, the respiratory chain activity of VBNC cells were confirmed by flow cytometry (FCM) analysis, suggesting that cells in a VBNC state were physiologically active. Finally, transcriptomics analysis and real-time PCR (qPCR) validation were used to explore the underlying mechanisms of VBNC cell formation. Over-expression of relA, lon, ppx, and ppk in the toxin-antitoxin (TA) trigger system contributed to VBNC cell formation. In the TA trigger system, RelA and exopolyphosphatases/guanosine pentaphosphate phosphohydrolases (PPX/GPPA) synthesize ppGpp, which activates polyphosphate kinase (PPK), the cellular enzyme that accumulates plyphosphate (PolyP). PolyP combines with and stimulates Lon to degrade the antitoxins, thereby activating the toxins that induce a VBNC state. The results of our research will facilitate a better understanding of the survival strategies that bacteria develop to deal with ampicillin pressure and the health risks associated with VBNC Cronobacter sakazakii induced by antibiotics.

We present an experimental study on non-equilibrium dynamics of a spinor condensate after it is quenched across a superfluid to Mott insulator (MI) phase transition in cubic lattices. Intricate dynamics consisting of spin-mixing oscillations at multiple frequencies are observed in time evolutions of the spinor condensate localized in deep lattices after the quantum quench. Similar spin dynamics also appear after spinor gases in the MI phase are suddenly moved away from their ground states via quenching magnetic fields. We confirm these observed spectra of spin-mixing dynamics can be utilized to reveal atom number distributions of an inhomogeneous system, and to study transitions from two-body to many-body dynamics. Our data also imply the non-equilibrium dynamics depend weakly on the quench speed but strongly on the lattice potential. This enables precise measurements of the spin-dependent interaction, a key parameter determining the spinor physics.

Cronobacter sakazakii (C. sakazakii) in viable but nonculturable (VBNC) state might evade traditional culture detection, and pose a serious threat to food safety. The objective of this study was to establish an improved propidium monoazide (PMAxx), combined with a single intact cell droplet digital PCR (SIC ddPCR) method for rapid and absolute quantification of VBNC C. sakazakii and apply it to the detection of several infant foods. In this study, we optimized the detection conditions for C. sakazakii, and determined the optimal PMAxx final concentration (8 μM), light exposure times (10 min), annealing temperature (58 °C) and elongation time (1 min). In addition, PMAxx-SIC ddPCR demonstrated a higher detection sensitivity and accuracy relative to real-time PCR (qPCR), with 23 CFU/mL in pure culture. Furthermore, PMAxx-SIC ddPCR was used to detect VBNC C. sakazakii in the actual infant food samples. A total of 240 samples were analyzed, out of which 6 samples turned out to be positive by the traditional culture method, whereas 11 samples were found as positive by PMAxx-SIC ddPCR. These results demonstrate that the presence of VBNC may cause the conventional culture-based method to underestimate the size of microbial population.

Oenothein B (OeB), a dimeric macrocyclic ellagitannin isolated from eucalyptus leaves has been demonstrated as a promising natural bioactive compound for its remarkable antitumor, antioxidant, anti-inflammatory and immunomodulating effects. Unfortunately, early study indicates that OeB has quite low bioaccessibility for oral consumption due to their susceptibility to decomposition both in vitro and in vivo. Herein, we report the design and synthesis of food-grade polyelectrolyte complex coacervate using caseinophosphopeptides (CPPs) and chitosan (CS) to encapsulate OeB for enhanced protection through gastrointestinal (GI) tract. Turbidimetric titration, dynamic light scattering (DLS), ζ-potential and scanning electric microscopy (SEM), as well as theoretical calculations based on principle of charge neutralization, were conducted to provide tentative and quantitative description for phase behavior in formation and disassociation of the complex. The optimum fabrication conditions were found to be at pH 5.5, with CPP : CS at 1 : 1, using CS of high molecular weight (980 kDa). The genipin cross-linking protected the system from disassembling in harsh acidic environments. The best cross-linking conditions were found to be 0.6 mg ml−1 genipin addition at 4 h cross-linking reaction time. The particle size and zeta potential of the nanoparticles varied from 200 to 300 nm and +20 to +24.2 mV, respectively. Scanning electron microscopy (SEM) revealed a spherical coacervate phase. Results from in vitro release study proved that controlled release of OeB through GI tract using CPP–CS nanoparticles cross-linked with genipin was achievable.

RET fusions have been found in lung adenocarcinoma, of which KIF5B–RET is the most prevalent. We established inducible KIF5B-RET transgenic mice and KIF5B–RET-dependent cell lines for preclinical modeling of KIF5B–RET-associated lung adenocarcinoma. Doxycycline-induced CCSP-rtTA/tetO-KIF5B-RET transgenic mice developed invasive lung adenocarcinoma with desmoplastic reaction. Tumors regressed upon suppression of KIF5B–RET expression. By culturing KIF5B–RET-dependent BaF3 (B/KR) cells with increasing concentrations of cabozantinib or vandetanib, we identified cabozantinib-resistant RETV804L mutation and vandetanib-resistant-RETG810A mutation. Among cabozantinib, lenvatinib, ponatinib, and vandetanib, ponatinib was identified as the most potent inhibitor against KIF5B–RET and its drug-resistant mutants. Interestingly, the vandetanib-resistant KIF5B-RETG810A mutant displayed gain-of-sensitivity (GOS) to ponatinib and lenvatinib. Treatment of doxycycline-induced CCSP-rtTA/tetO-KIF5B-RET bitransgenic mice with ponatinib effectively induced tumor regression. These results indicate that KIF5B-RET–associated lung tumors are addicted to the fusion oncogene and ponatinib is the most effective inhibitor for targeting KIF5B–RET in lung adenocarcinoma. Moreover, this study finds a novel vandetanib-resistant RETG810A mutation and identifies lenvatinib and ponatinib as the secondary drugs to overcome this vandetanib resistance mechanism. Mol Cancer Ther; 15(10); 2521–9. ©2016 AACR.

// Janani Panneerselvam 1, 4 , Akhil Srivastava 1, 4 , Ranganayaki Muralidharan 1, 4 , Qi Wang 1, 4 , Wei Zheng 1 , Lichao Zhao 1, 4 , Alshine Chen 3, 4 , Yan D. Zhao 3, 4 , Anupama Munshi 2, 4 , Rajagopal Ramesh 1, 4, 5 1 Department of Pathology, The University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104, USA 2 Department of Radiation Oncology, The University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104, USA 3 Department of Biostatistics and Epidemiology, The University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104, USA 4 Stephenson Cancer Center, The University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104, USA 5 Graduate Program in Biomedical Sciences, The University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104, USA Correspondence to: Rajagopal Ramesh, email: rajagopal-ramesh@ouhsc.edu Keywords: IL-24, lung cancer, HMGA1, miR222-3p, metastasis Received: April 21, 2016 Accepted: August 24, 2016 Published: September 02, 2016 ABSTRACT Interleukin (IL)-24, a novel tumor suppressor/cytokine exhibits antitumor activity against a broad-spectrum of human cancer cells. In a recent study, we showed that IL-24 inhibited AKT in lung cancer cells. However, the molecular mechanism of AKT inhibition by IL-24 remains elusive. The high mobility group (HMG) A1 a member of the non-histone chromosomal proteins and commonly referred to as architectural transcription factor, regulates transcription of various genes involved in cell growth and survival. Overexpression of HMGA1 has been shown to be associated with tumor progression and metastasis in several cancers, including human lung cancer. A recent study demonstrated that HMGA1 activates AKT function by reducing the activity of the protein phosphatase, phosphatase 2A subunit B (PPP2R2A) via the oncogenic micro (mi) RNA-222. Based on this report we hypothesized that IL-24-mediated AKT inhibition involved the HMGA1/miR-222 axis. To test our hypothesis, in the present study we used a H1299 lung cancer cell line that expressed exogenous human IL-24 when induced with doxycycline (DOX). Induction of IL-24 expression in the tumor cells markedly reduced HMGA1 mRNA and protein levels. Using a mechanistic approach, we found that IL-24 reduced miR-222-3p and -5p levels, as determined by qRT-PCR. Associated with HMGA1 and miR-222 inhibition was a marked increase in PPP2R2A, with a concomitant decrease in phosphorylated AKT T308/S473 expression. SiRNA-mediated knockdown of HMGA1 in combination with IL-24 significantly reduced AKT T308/S473 protein expression and greatly reduced cell migration and invasion compared with individual treatments. Further combination of IL-24 and a miR-222-3p inhibitor significantly increased PPP2R2A expression. Our results demonstrate for the first time that IL-24 inhibits AKT via regulating the HMGA1/miR-222 signaling node in human lung cancer cells and acts as an effective tumor suppressor. Thus, a therapy combining IL-24 with HMGA1 siRNA or miR-222-3p inhibitor should present effective treatment of lung cancer.

Accurate and rapid identification of the live fraction of pathogenic bacteria in beef food samples has emerged as a pressing issue due to the serious threat posed by pathogens to human health. Escherichia coli O157: H7, one of the most common pathogens in beef, causes hemorrhagic colitis or hemolytic uremic syndrome and subsequently a viable but nonculturable (VBNC) state under low temperatures in which the bacteria do not grow in culture medium but remain potentially virulent. The objective of this study was to establish and evaluate a method that combines improved propidium monoazide (PMAxx) treatment with real-time loop-mediated isothermal amplification (qLAMP) for the detection and quantification of VBNC E. coli O157: H7 in beef. In this study, E. coli O157: H7 was induced to enter the VBNC state by simulating food storage conditions at −20 °C. The respiratory activity of VBNC E. coli O157: H7 was measured by flow cytometry. The optimal PMAxx final concentration was 10 μM with exposure to light for 10 min for photoactivation, which was found to completely inhibit amplification of DNA derived from dead cells. When there were low levels of VBNC microorganisms in beef samples, PMAxx was significantly more accurate and effective at distinguishing between viable and dead E. coli O157: H7 cells. The developed PMAxx-qLAMP assay remarkably exhibited both high sensitivity in pure culture and beef (30 colony-forming units/mL and 300 CFU/g, respectively) and rapidity (60 min). Especially for frozen beef samples, positive ratio was 4.71% (four out of eighty-five were positive) by PMAxx-qLAMP method and the mean measured value of positive samples was 2.04 ± 0.08 Log10 CFU/g. Therefore, PMAxx-qLAMP is an effective means for improving the detection and quantification accuracy of VBNC E. coli O157: H7 in beef.

Immunomagnetic separation (IMS) is an effective method for specific enrichment and purification of target food-borne pathogens from complex food samples. To detect viable but non-culturable (VBNC) Vibrio parahaemolyticus (V. parahaemolyticus) with greater accuracy and sensitivity, we used an improved propidium monoazide (PMAxx) dye to eliminate dead cell interference in an IMS-PMAxx-real-time (quantitative) polymerase chain reaction (IMS-PMAxx-qPCR) assay. We prepared immunomagnetic beads (IMBs) using streptavidin-conjugated magnetic nanoparticles and biotinylated polyclonal antibodies, and optimized the reaction conditions to establish an IMS method for VBNC V. parahaemolyticus. We determined the optimal antibody amount (30 μg), IMBs volume (150 μL), incubation time (45 min), immunomagnetic separation time (4 min), and separation temperature (25 °C). The IMS-PMAxx-qPCR method could detect VBNC V. parahaemolyticus in raw shrimp samples at levels as low as 1.85 CFU/g without any pre-enrichment. The IMS-PMAxx-qPCR assay is highly sensitive, selective, simple, and rapid (

Oenothein B (OeB) is a dimeric macrocyclic ellagitannin isolated from Herbs and fruits that have a variety of biological activities. In order to better understand the effect of OeB on the activity of the digestive enzyme pepsin, interactions between OeB and pepsin were investigated in vitro under simulated physiological conditions based on enzyme inhibition studies, fluorescence, isothermal titration calorimetry, CD, and molecular docking. It was found OeB is an effective inhibitor of pepsin, likely acting in a reversible manner through both competitive and noncompetitive inhibition. Fluorescence quenching of pepsin by OeB was a static quenching. CD spectra showed the addition of OeB causes the main chain of pepsin to loosen and expand and the partial β-sheet structure to be converted to a disordered structure. Isothermal titration calorimetry and docking studies revealed the main binding mechanism of OeB and pepsin was through noncovalent interactions, hydrophobic interactions with OeB and the internal hydrophobic group of pepsin, and then hydrogen bonding between OeB and the Val243 and Asp77 residues of pepsin. Noncovalent bonds between OeB and pepsin change the polarity and structure of enzymes, decreasing enzymatic activity. Compared with small molecular polyphenols, OeB has a weaker hydrophobic interaction with pepsin and less effect on the secondary structure of pepsin. These findings are the first direct elucidation of the interactions between the oligomer ellagitannin OeB and pepsin, further contributing to understanding binding between oligomer ellagitannins and digestive enzymes. PRACTICAL APPLICATION: The results of this study indicate that the interaction between OeB and pepsin has a certain inhibitory effect on pepsin. In order to reduce the impact of OeB on human digestion and its own activities, nano-encapsulation technology can be used in the future to protect oligomeric ellagitannin such as OeB.

To evaluate the diagnostic performance of ultrasound (US)-guided fine-needle aspiration with optional core needle biopsy of head and neck lymph nodes and masses, with attention to differences between biopsy of treated squamous cell carcinoma (SCC) and biopsy of other lesions.Institutional Review Board approval was obtained, and the need for consent was waived for this retrospective study. All 861 US-guided biopsies of head and neck lymph nodes and masses performed between March 1, 2012, and May 16, 2016, were reviewed.Of the 861 biopsies, 53 targeted SCC with residual masses after treatment. The biopsy procedures yielded benign or malignant pathologic results in 71.7% (38 of 53) of treated SCC and 90.7% (733 of 808) of all other lesions (P .001). A reference standard based on subsequent pathologic results or clinical and imaging follow-up was established in 68.4% of procedures. In cases with benign or malignant biopsy results and a subsequent reference standard, the sensitivity values for malignancy were 87.5% (95% confidence interval, 64.0%-96.5%) in treated SCC and 98.3% (95% confidence interval, 96.0%-99.3%) in all other cases (P = .047), and the specificity values were 63.6% (95% confidence interval, 35.4%-84.8%) in treated SCC and 99.5% (95% confidence interval, 97.3%-99.9%) in all other cases (P .001). There were no major complications related to the biopsy procedures.Excluding treated SCC, US-guided fine-needle aspiration with optional core needle biopsy of head and neck lymph nodes and masses has excellent diagnostic performance. Needle biopsy of head and neck SCC with a residual mass after therapy has a high rate of nondiagnostic samples, suboptimal sensitivity, and poor specificity.

BACKGROUND Adequacy assessment for fine-needle aspiration procedures is a standard of care in large medical centers. Although the benefits of this approach include higher adequacy rates with fewer passes, it costs cytopathologist time and affects other clinical responsibilities. The objective of the current study was to evaluate the use of mobile video streaming (FaceTime) technology with the help of smartphone adapters attached to microscopes for remote adequacy assessment of cytologic samples. METHODS The study consisted of 2 phases: Phase 1 was a retrospective assessment of 25 samples by a primary pathologist with simultaneous streaming to a second pathologist using a smartphone (iPhone/iPad) FaceTime connection. Data on the adequacy of each sample and preliminary diagnoses were recorded. In phase 2, live cases were assessed prospectively by an onsite primary pathologist and by a remote pathologist using an iPhone/iPad-FaceTime connection. The testing phase involved prospective assessment of additional samples with a resident or cytotechnologist as the slide driver. RESULTS In phase 1, retrospective evaluation of 25 samples yielded considerable agreement (22 of 25 samples; 88%) between onsite and remote adequacy assessments. Three samples (12%) yielded results that did not agree, including 2 samples that were read as adequate in the onsite evaluation that were assessed as indeterminate using FaceTime. In phase 2 and in the testing phase, 14 samples exhibited considerable agreement on both adequacy and preliminary diagnosis (6 samples in phase 2 and 8 samples in the testing phase) and are currently available for reporting. Problems encountered include software version standardization, camera alignment, and (rarely) comprehension of the audio stream. CONCLUSIONS The current data indicate that iPhone/iPad FaceTime technology can be used to perform remote adequacy assessments of fine-needle aspirations and can help save valuable time for pathologists. Cancer Cytopathol 2016;124:213–220. © 2015 American Cancer Society.

The dynamic vibration absorbers have been applied to attenuate the rotor unbalance and torsional vibrations. The major purpose of this paper is to research the elimination of the fluid-induced vibration in the rotor/seal system using the absorber. The simplified rotor model with the absorber is established, and the Muszynska fluid force model is employed for the nonlinear seal force. The numerical method is used for the solutions of the nonlinear differential equations. The nonlinear responses of the rotor/seal system without and with the absorber are obtained, and then the rotating speed ranges by which the fluid-induced instability can be eliminated completely and partially are presented, respectively. The absorber parameters ranges by which the instability vibration can be eliminated completely and partially are obtained. The results show that the natural frequency vibration due to the fluid-induced instability in the rotor/seal system can be eliminated efficiently using the absorber. The appropriate natural frequency and damping ratio of the absorber can extend the complete elimination region of the instability vibration and postpone the occurrence of the instability vibration.

Renal cell carcinoma (RCC) is a common and devastating disease characterized by a hypoxic microenvironment, epithelial-mesenchymal transition and potent resistance to therapy evidencing the presence of cancer stem cells (CSCs). Various CSC markers have been studied in RCC, but overall there is limited data on their role and most markers studied have been relatively nonspecific. Doublecortin-like kinase 1 (DCLK1) is a validated CSC marker in the gastrointestinal tract and evidence for an equivalent role in other cancers is accumulating. We used bioinformatics, immunohistochemistry, flow cytometry, spheroid self-renewal and chemoresistance assays in combination with overexpression and siRNA-knockdown to study the stem cell-supportive role of DCLK1 alternative splice variants (DCLK1 ASVs) in RCC. To target tumor cells expressing DCLK1 ASVs directly, we developed a novel monoclonal antibody (CBT-15) and delivered it systemically to RCC tumor xenografts. DCLK1 ASVs were overexpressed, enriched together with CSC markers and predictive of overall and recurrence-free survival in RCC patients. In vitro, DCLK1 ASVs were able to directly stimulate essential molecular and functional characteristics of renal CSCs including expression of aldehyde dehydrogenase, self-renewal and resistance to FDA-approved receptor tyrosine kinase and mTOR inhibitors, while targeted downregulation of DCLK1 reversed these characteristics. Finally, targeting DCLK1 ASV-positive cells with the novel CBT-15 monoclonal antibody blocked RCC tumorigenesis in vivo. These findings establish DCLK1 as a CSC marker with implications for therapy, disease progression and survival in RCC and demonstrate the therapeutic value of DCLK1-targeted monoclonal antibodies against renal CSCs.

Androgen ablation is the standard of care prescribed to patients with advanced or metastatic prostate cancer (PCa) to slow down disease progression. Unfortunately, a majority of PCa patients under androgen ablation progress to castration-resistant prostate cancer (CRPC). Several mechanisms including alternative intra-prostatic androgen production and androgen-independent androgen receptor (AR) activation have been proposed for CRPC progression. Aldo-keto reductase family 1 member C3 (AKR1C3), a multi-functional steroid metabolizing enzyme, is specifically expressed in the cytoplasm of PCa cells; and positive immunoreactivity of the type A γ-aminobutyric acid receptor (GABAAR), an ionotropic receptor and ligand-gated ion channel, is detected on the membrane of PCa cells. We studied a total of 72 radical prostatectomy cases by immunohistochemistry, and identified that 21 cases exhibited positive immunoreactivities for both AKR1C3 and GABAAR. In the dual positive cancer cases, AKR1C3 and GABAAR subunit α1 were either expressed in the same cells or in neighboring cells. Among several possible substrates, AKR1C3 reduces 5α-dihydrotesterone (DHT) to form 5α-androstane-3α, 17β-diol (3α-diol). 3α-diol is a neurosteroid that acts as a positive allosteric modulator of the GABAAR in the central nervous system (CNS). We examined the hypothesis that 3α-diol-regulated pathological effects in the prostate are GABAAR-dependent, but are independent of the AR. In GABAAR-positive, AR-negative human PCa PC-3 cells, 3α-diol significantly stimulated cell growth in culture and the in ovo chorioallantoic membrane (CAM) xenograft model. 3α-diol also up-regulated expression of the epidermal growth factor (EGF) family of growth factors and activation of EGF receptor (EGFR) and Src as measured by quantitative polymerase chain reaction and immunoblotting, respectively. Inclusion of GABAAR antagonists reversed 3α-diol-stimulated tumor cell growth, expression of EGF family members, and activation of EGFR and Src to the level observed in untreated cells. Results from the present study suggest that 3α-diol may act as an alternative intra-prostatic neurosteroid that activates AR-independent PCa progression. The involvement of AKR1C3-mediated steroid metabolisms in modulating GABAAR activation and promoting PCa progression requires continued studies.