Candida albicans, Candida tropicalis, Candida grabrata, and Cryptococcus neoformans are causative pathogens of opportunistic diseases in immunocompromised human patients. Silkworms are killed by injection of these pathogenic fungi into their hemolymph. In this paper, we describe recent results by our laboratory and other researchers using gene-deficient strains of these pathogenic fungi. The silkworm is considered to be a useful model animal for understanding the pathogenicity of these fungi. Silkworms are also beneficial for evaluating therapeutically active anti-fungal reagents.

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Innate immunity acts as a front-line barrier against invading pathogens, and the majority of the components are widely conserved among species. Regulation of innate immunity is important for overcoming infections and preventing self-damaging sepsis. Using the silkworm (Bombyx mori) as an animal model, we elucidated the activation processes of innate immunity with emphasis on a multifunctional insect cytokine called paralytic peptide. Moreover, we established an ex vivo system using silkworm larval specimens to quantitatively evaluate the immunostimulatory activity of natural compounds. We observed that overactivation of innate immunity in silkworms induces tissue damage followed by host death, resembling sepsis-induced multi-organ failure in humans. Here, we summarize our recent findings and propose the usefulness of the silkworm as an animal model for studying immune regulation and for evaluating compounds with the potential to regulate innate immunity.

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Oesophageal Squamous Cell Carcinoma (ESCC) is one of the two main subtypes of oesophageal cancer, affecting mainly populations in Asia. Though there have been great efforts to develop methods for a better prognosis, there is still a limitation in the staging of this affection. As a result, ESCC is detected at advances stages, when the interventions on the patient do not have such a positive outcome, leading in many cases to recurrence and to a very low 5-year survival rate, causing high mortality. A way to decrease the number of deaths is the use of biomarkers that can trace the advance of the disease at early stages, when surgical or chemotherapeutic methodologies would have a greater effect on the evolution of the subject. The new high throughput omics technologies offer an unprecedented chance to screen for thousands of molecules at the same time, from which a new set of biomarkers could be developed. One of the most convenient types of samples is saliva, an accessible body fluid that has the advantage of being non-invasive for the patient, being easy to store or to process. This review will focus on the current status of the new omics technologies regarding salivaomics in ESCC, or when not evaluated yet, the achievements in related diseases.

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The beneficial antithrombotic effect of some dietary components may offer the most promising approach of prevention of cardiovascular diseases and arterial thrombosis. The major stumbling block in finding effective dietary components is the lack of physiologically relevant techniques which can detect potential antithrombotic effect in humans. The presently used platelet function and coagulation tests do not allow the assessment of global thrombotic status and their value in screening dietary components for antithrombotic effect is questionable. Most of these in vitro tests ignore the effect of flow and shear stress, thrombin generation and vascular endothelium, the major contributors to arterial thrombogenesis in humans. As a gold standard, we employed the helium-neon (He-Ne) laser-induced thrombosis test in murine carotid artery and mesenteric microvessels, as the pathomechanism of this test closely reflects arterial thrombogenesis in humans. Results obtained with laser thrombosis test were compared with various shear-induced in vitro platelet function tests which use native blood (Haemostatometry, Thrombotic Status Analyser, Global Thrombosis Test-GTT). Contribution of vascular endothelium to thrombogenesis was assessed by measuring flow-mediated vasodilation (FMV) in vivo. The combination of the two shear-induced ex vivo thrombosis tests (Haemostatometry and GTT) with FMV correlated most closely with the laser-thrombosis test. Our findings suggest that combining the commercially available point-of-care GTT with the FMV test could provide a better assessment of the overall thrombotic status than either of the two tests alone.

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This study attempts to identify whether and why the difference in corruption progress exists between organic and conventional farm products by conducting two corruption experiments of farm products and separation experiment of bacteria, as well as farmer survey. The results of corruption experiments for Wenzhou mandarin oranges (Citrus unshiu) and polished rice showed that conventional farm products demonstrated fast-growing corruption with strong unpleasant smell distinctively different from organic farm products. The separation experiment of bacteria indicated a high possibility of fungus appearance in organic farm products and coccus or bacillus appearance in conventional farm products, which are significantly consistent with the results of two corruption experiments and the fact that organic farmers are strongly conscious of the use of fermented organic fertilizers with effective microorganism in their cultivation. These results offer empirical evidences for supporting the development of organic agriculture and the consumption expansion of organic farm products, but further works are necessary.

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Increasing studies suggest that gestational diabetes mellitus (GDM) and pre-gestational diabetes mellitus (PGDM) may be associated with an increased risk of major congenital malformations (MCM) in the offspring. To determine whether GDM or PGDM is associated with an increased risk of congenital malformations, we performed a meta-analysis of cohort studies. We systematically searched the PubMed, Web of Science and Cochrane Library (from January, 1990 to October, 2014) and reviewed the reference lists of included papers to search for additional studies. Meta-analysis tools were used to summarize results. Summary relative risks (RRs) with 95% confidence intervals (CIs) were calculated with random-effects models or fixed-effects models. Study quality was assessed using the Newcastle-Ottawa scale. A total of 21 cohort studies were included in the meta-analysis. Analysis of all studies showed that both PGDM and GDM were associated with an increased risk of MCM (RR = 2.44, 95% CI = 1.92-3.10, I² = 78.3%, p = 0.342; RR = 1.11, 95% CI = 1.11-1.27, I² = 9.9%, p < 0.001, respectively). There is a slightly higher risk of major congenital malformations in women with GDM than in the reference group. However, this risk is much lower than in women with PGDM. Further large-scale prospective cohort studies are needed to test the effect of PGDM and GDM on specific congenital malformations risk.

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Some peptides that are highly conserved between insects and mammals have anti-tumor action. Screening for inhibitors of cell growth from animal fluids may provide useful clues to anti-tumor drugs. Inducers of autophagy also have anti-tumor activity. The current authors recently studied a protein found in silkworm hemolymph, Niemann-Pick disease type C2 (NPC2). This protein, which is highly conserved among eukaryotes, was found to have anti-proliferative action on a silkworm cell line. The current study found that the silkworm NPC2 protein also inhibits the growth of FM3A murine breast cancer cells. In FM3A cells, silkworm NPC2 increased phosphorylation of AMP-activated protein kinase and decreased phosphorylation of Akt and mammalian target of rapamycin, which are regulators of autophagy. This study also found that NPC2 increased the amount of microtubule-associated protein light chain 3 (LC3)-II, an autophagosome marker, in FM3A cells. Silkworm NPC2 also induced an increase in the number of LC3-dots, a marker of pre-autophagic endosomes, in FM3A cells. When silkworm NPC2 was used to inhibit FM3A cell growth, that inhibition was attenuated by chloroquine, which inhibits autophagic activity by preventing lysosomal acidification. Murine NPC2 also inhibited growth and induced autophagy in FM3A cells. These findings suggest that NPC2 is involved in the induction and/or maintenance of autophagy and may help to elucidate the mechanisms underlying other neurodegenerative disorders such as Niemann-Pick disease.

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Cucurbitacin I (CBI) is a triterpene from a bitter melon called Goya grown in Okinawa, Japan, and directly inhibits both the Tyr-kinase JAK2 and the G protein RAC, leading to the inactivation of PAK1 (RAC/CDC42-activated kinase 1). Bio 30, a propolis produced in New Zealand, contains CAPE (caffeic acid phenethyl ester) as the major anti-cancer ingredient which directly down-regulates RAC, leading to the inactivation of PAK1. Since PAK1 is essential for the growth of RAS cancer cells such as A549 cell line which carry an oncogenic K-RAS mutant, and the melanogenesis in skin cells, here using these PAK1-blockers as model compounds, we introduce a new approach to the quick assessment of PAK1-blockers in cell culture. First, combining the immuno-precipitation (IP) of PAK1 from cell lysate and the in vitro ATP_Glo kinase assay kit (called "Macaroni-Western" assay), we confirmed that both CBI and Bio 30 inactivate PAK1 in A549 lung cancer cells in 24 h, and inhibit their PAK1-dependent growth in 72 h. Furthermore, we verified that CBI inhibits the PAK1/PAK4-dependent melanogenesis in melanoma cells by far more than 50%, while Bio 30 inhibits the melanogenesis only by 50%, with only a merginal effect on their growth per se. Since the "Macaroni-Western" kinase assay and melanogenesis are both rather simple and quick, the combination of these two cell culture assays would be highly useful for selecting both "potent" (highly cell-permeable) and "safe" (non-toxic) natural or synthetic PAK1-blockers.

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In order to investigate whether the hemodynamic indices, including stroke volume variation (SVV) and pulse pressure variation (PPV) could predict fluid responsiveness in patients undergoing protective one-lung ventilation. 60 patients scheduled for a combined thoracoscopic and laparoscopic esophagectomy were enrolled and randomized into two groups. The patients in the protective group (Group P) were ventilated with a tidal volume of 6 mL/kg, an inspired oxygen fraction (FiO₂) of 80%, and a positive end expiratory pressure (PEEP) of 5 cm H₂O. Patients in the conventional group (Group C) were ventilated with a tidal volume of 8 mL/kg and a FiO₂ of 100%. Dynamic variables were collected before and after fluid loading (7 mL/kg hydroxyethyl starch 6%, 0.4 mL/kg/min). Patients whose stroke volume index (SVI) increased by more than 15% were defined as responders. Data collected from 45 patients were finally analyzed. Twelve of 24 patients in Group P and 10 of 21 patients in Group C were responders. SVV and PPV significantly changed after the fluid loading. The receive operating characteristic (ROC) analysis showed that the thresholds for SVV and PPV to discriminate responders were 8.5% for each, with a sensitivity of 66.7% (SVV) and 75% (PPV) and a specificity of 50% (SVV) and 83.3% (PPV) in Group P. However, the thresholds for SVV and PPV were 8.5% and 7.5% with a sensitivity of 80% (SVV) and 90% (PPV) and a specificity of 70% (SVV) and 80% (PPV) in Group C. We found SVV and PPV could predict fluid responsiveness in protective one-lung ventilation, but the accuracy and ability of SVV and PPV were weak compared with the role they played in a conventional ventilation strategy.

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Porous ethyl cellulose (EC) microspheres were prepared from the acetone-glycerin-water ternary system using an oil/water (O/W)-type emulsion solvent extraction method. The O/ W type emulsion was prepared using acetone dissolved ethyl cellulose as an oil phase and aqueous glycerin as a water phase. The effects of the different solvent extraction modes on the porosity of the microspheres were investigated. The specific surface area of the porous EC microspheres was estimated by the gas adsorption method. When the solvent was extracted rapidly by mixing the emulsion with water instantaneously, porous EC microspheres with a maximum specific surface area of 40.7 ± 2.1 m²/g were obtained. On the other hand, when water was added gradually to the emulsion, the specific surface area of the fabricated microspheres decreased rapidly with an increase in the infusion period, with the area being 25-45% of the maximum value. The results of an analysis of the ternary phase diagram of the system suggested that the penetration of water and glycerin from the continuous phase to the dispersed phase before solidification affected the porosity of the fabricated EC microspheres.

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