Bioremediation of Hazardous Pollutants Using Enzyme-Immobilized Reactors.

Bioremediation uses the degradation abilities of microorganisms and other organisms to remove harmful pollutants that pollute the natural environment, helping return it to a natural state that is free of harmful substances. Organism-derived enzymes can degrade and eliminate a variety of pollutants and transform them into non-toxic forms; as such, they are expected to be used in bioremediation. However, since enzymes are proteins, the low operational stability and catalytic efficiency of free enzyme-based degradation systems need improvement. Enzyme immobilization methods are often used to overcome these challenges. Several enzyme immobilization methods have been applied to improve operational stability and reduce remediation costs. Herein, we review recent advancements in immobilized enzymes for bioremediation and summarize the methods for preparing immobilized enzymes for use as catalysts and in pollutant degradation systems. Additionally, the advantages, limitations, and future perspectives of immobilized enzymes in bioremediation are discussed.

Sequestosome 1 (p62) accumulation in breast cancer cells suppresses progesterone receptor expression via argonaute 2.

Sequestosome 1 (p62) is a multifunctional adapter protein involved in various physiological functions, such as selective autophagy and oxidative stress response. Hence, aberrant expression and defective regulation of p62 are thought to lead to the onset of various diseases, including cancer. The expression of p62 has been shown to be increased in breast cancer tissues, and is correlated with a poor prognosis. However, the role of p62 in the breast cancer pathophysiology is still unclear. Here, we aimed to analyze the effect of changes in p62 expression on breast cancer cell lines. DNA microarray analysis revealed that the expression of progesterone receptor (PR), which is one of the indices for the classification of breast cancer subtypes, was markedly suppressed by forced expression of p62. The protein expression of PR was also decreased by forced expression of p62, but increased by knockdown of p62. Moreover, we found that p62 knockdown induced the protein expression of argonaute 2 (AGO2). Luciferase reporter assay results showed that the gene expression of PR was promoted by AGO2. Furthermore, results revealed that overexpression of AGO2 partially rescued the decrease in PR expression induced by forced expression of p62. Collectively, our findings indicated that p62 accumulation suppressed the expression of AGO2, which in turn decreased the expression of PR, suggesting that p62 may serve as a marker of aggressive breast cancer and poor prognosis. Moreover, the p62-AGO2-PR axis was identified as a crucial signaling cascade in breast cancer progression.

Hepatic Arterial Infusion Chemotherapy of 5-Fluorouracil for Patients with Unresectable Liver Metastases from Colorectal Cancer Refractory to Standard Systemic Chemotherapy: A Multicenter Retrospective Study.

INTRODUCTION: Hepatic arterial infusion chemotherapy (HAIC) is a feasible treatment for patients with colorectal cancer (CRC) with unresectable liver metastases. OBJECTIVE: The aim of this retrospective study was to assess HAIC of 5-fluorouracil (5FU) in patients with unresectable liver metastases from CRC refractory to standard systemic chemotherapy. METHODS: A total of 137 patients (85 men, 52 women; median age, 62 years; with KRAS mutation, n = 57) were recruited from seven institutions from September 2008 to December 2015. These patients were refractory to systemic chemotherapy including three cytotoxic agents (fluoropyrimidine, oxaliplatin, and irinotecan) with two molecular-targeted agents (bevacizumab and epidermal growth factor receptor antibody [cetuximab or panitumumab]). All patients underwent HAIC of continuous 5FU for unresectable liver metastases. Overall survival time, time to treatment failure, objective response rate, disease control rate, and incidence of adverse events to HAIC were assessed retrospectively. RESULTS: The median overall survival time was 4.8 months (95% confidence interval [CI], 4.0-5.7 months), whereas time to treatment failure was 2.4 months (95% CI, 2.0-2.8 months). The objective overall response rate and disease control rate were 12.4 and 64%, respectively. Grade 3 or 4 adverse events were observed in 2.9% of the patients (hyperbilirubinemia in 2, liver abscess in 1, and myelosuppression in 1). CONCLUSIONS: There were few incidences of severe adverse events to HAIC of 5FU for liver metastases from CRC refractory to standard systemic chemotherapy. Therefore, it might present as a treatment option as last-line chemotherapy.

A Simple, Low Cost, Sensitive, and Portable Electrochemical Immunochromatography Sensing Device to Measure Estrone-3-Sulfate.

In livestock production, point-of-care testing (POCT) technology that enables easy on-site analysis of sex hormones is desired to improve reproductive efficiency. In this context, low-molecular-weight endogenous steroids are particularly important for perinatal management. Therefore, we attempted to use a simple method that combines electrochemical techniques with immunochromatography to measure estrone-3-sulfate (E1S), one of the low-molecular-weight endogenous steroids that is an estrogen ester. The limit of detection (LOD) for E1S achieved by electrochemical immunochromatography was 570.5 ng/mL, which was one to two orders of magnitude lower than that of small molecule compounds analyzed by other POCT techniques (Primpray et al., Anal. Chim. Acta, 2019). In addition, it was indicated by a colorimetric analysis that the sensitivity of the electrochemical immunochromatographic technique could be enhanced by improving the method of application of the antibodies on the nitrocellulose membrane and the contact between the electrochemical detector and the nitrocellulose membrane.

Autopsy findings in the early stage of amyotrophic lateral sclerosis with "dropped head" syndrome.

Dropped head syndrome (DHS) has been rarely observed in amyotrophic lateral sclerosis (ALS), and the neuropathological findings of this condition have almost never been described. The identification of transactivation response DNA-binding protein 43 kDa (TDP-43), which binds to RNA/DNA has provided a new method for studying ALS and frontotemporal lobar degeneration (FTLD). Post-mortem examination of an adult sudden death case of a 71-year-old patient who complained of DHS exhibited severe loss of anterior motor neurons in the cervical cord (C4-6). Loss of nerve fibers of the anterior roots was striking compared with posterior roots, together with marked neurogenic atrophy of posterior muscles semispinalis cervicis. Bunina bodies were found in large neurons of Betz giant cells, but not in the motor neurons of spinal cords, or neurons of bulbar regions. Phosphorylated TDP-43 (p-TDP-43)-positive structures were detected in the residual neurons of the cervical, thoracic and lumber cords, hypoglossal nucleus, cerebellar dentate nucleus and parahippocampal cortex, together with ubiquitin-positive inclusions. Phosphorylated Tau positive structures in neuronal cytoplasm were found in the amygdala, entorhinal cortex and parahippocampal cortex, some of which co-expressed p-TDP-43. The medial zone of cervical cords may be the first onset site, and that is the cause of head drop in the early stage of ALS. In spite of detailed examination, the direct cause of sudden death was not verified. This autopsy report revealed the relation of DHS which is a rare clinical manifestation of ALS, and neuropathological findings.

[A case of cerebral salt wasting syndrome caused by minor head injury].

An 80-year-old woman who was hospitalized due to small subarachnoid hemorrhaging caused by a bruise in the left temporal region of the brain. Nausea/vomiting and malaise appeared after dinner on the fourth day of the illness. Head computed tomography showed that the post-traumatic status was almost normal; however, the sodium ion (Na+) level was 114 mEq/L, indicating severe hyponatremia. Syndrome of inappropriate antidiuretic hormone secretion (SIADH) following a head injury was initially suspected, and water restriction and saline fluid replacement were initiated. However, the Na+ level did not improve, and signs of dehydration emerged. On the seventh day of the illness, drinking water restriction was discontinued, and 3% sodium chloride fluid replacement was initiated. The patient subsequently followed a favorable course, and the Na+ level remained normal even after fluid replacement was discontinued. It is important to differentiate between SIADH and cerebral salt wasting syndrome (CSWS), as the treatment of the two are diametrically opposite. However, distinguishing between these two diseases at an early onset can be difficult, as they have very similar laboratory findings. CSWS can occur in patients with minor head injury, as in the present case, so we should bear this disease in mind as a differential diagnosis, even when imperceptible graduations are recognized in patients.

Stepwise Mechanism of Temperature-Dependent Coacervation of the Elastin-like Peptide Analogue Dimer, (C(WPGVG)3)2.

Elastin-like peptides (ELPs) are distinct, repetitive, hydrophobic sequences, such as (VPGVG) n, that exhibit coacervation, the property of reversible, temperature-dependent self-association and dissociation. ELPs can be found in elastin and have been developed as new scaffold biomaterials. However, the detailed relationship between their amino acid sequences and coacervation properties remains obscure because of the structural flexibility of ELPs. In this study, we synthesized a novel, dimeric ELP analogue (H-C(WPGVG)3-NH2)2, henceforth abbreviated (CW3)2, and analyzed its self-assembly properties and structural factors as indicators of coacervation. Turbidity measurements showed that (CW3)2 demonstrated coacervation at a concentration much lower than that of its monomeric form and another ELP. In addition, the coacervate held water-soluble dye molecules. Thus, potent and distinct coacervation was obtained with a remarkably short sequence of (CW3)2. Furthermore, fluorescence microscopy, dynamic light scattering, and optical microscopy revealed that the coacervation of (CW3)2 was a stepwise process. The structural factors of (CW3)2 were analyzed by molecular dynamics simulations and circular dichroism spectroscopy. These measurements indicated that helical structures primarily consisting of proline and glycine became more disordered at high temperatures with concurrent, significant exposure of their hydrophobic surfaces. This extreme change in the hydrophobic surface contributes to the potent coacervation observed for (CW3)2. These results provide important insights into more efficient applications of ELPs and their analogues, as well as the coacervation mechanisms of ELP and elastin.

Enhancement of Self-Aggregation Properties of Linear Elastin-Derived Short Peptides by Simple Cyclization: Strong Self-Aggregation Properties of Cyclo[FPGVG] n, Consisting Only of Natural Amino Acids.

Elastin-like peptides (ELPs) consist of distinctive repetitive sequences, such as (VPGVG) n, exhibit temperature-dependent reversible self-assembly (coacervation), and have been considered to be useful for the development of thermoresponsive materials. Further fundamental studies evaluating coacervative properties of novel nonlinear ELPs could present design concepts for new thermoresponsive materials. In this study, we prepared novel ELPs, cyclic (FPGVG) n (cyclo[FPGVG] n, n = 1-5), and analyzed their self-assembly properties and structural characteristics. Cyclo[FPGVG] n ( n = 3-5) demonstrated stronger coacervation capacity than the corresponding linear peptides. The coacervate of cyclo[FPGVG]5 was able to retain water-soluble dye molecules at 40 °C, which implied that cyclo[FPGVG]5 could be employed as a base material of DDS (drug delivery system) matrices and other biomaterials. The results of molecular dynamics simulations and circular dichroism measurements suggested that a certain chain length was required for cyclo[FPGVG] n to demonstrate alterations in molecular structure that were critical to the exhibition of coacervation.

Bioluminescence Image as a Quantitative Imaging Biomarker for Preclinical Evaluation of Cryoablation in a Murine Model.

PURPOSE: To employ bioluminescence imaging (BLI) as a quantitative imaging biomarker to assess preclinical evaluation of cryoablation in a murine model. MATERIALS AND METHODS: In vitro, Colon26-Luc (C26-Luc) cells were seeded at 6 different concentrations in 35-mm dishes. These were divided into 6 groups: group 0 (G0), a control group without treatment; and groups 1-5 (G1-G5) according to the number of freeze-thaw cycles, with each cycle consisting of freezing at -80°C for 10 min followed by thawing at room temperature for 5 minutes. BLI and flow-cytometric analysis were performed after cryotherapy. In vivo, 20 tumor-bearing mice with C26-Luc cells were divided into 4 groups: group 0 (G0), a control group; and groups 1-3 (G1-G3) according to the number of freeze-thaw cycles. Each cryoablation procedure was performed for 30 seconds with liquid nitrogen (-170°C) applied with cotton-tipped applicators. BLI was acquired at 6 hours and 1, 3, and 7 days after treatments. RESULTS: In vitro, BLI signal showed a negative correlation with the number of freeze-thaw cycles (r = -0.86, P = .02). In vivo, there was no difference in tumor volume at 1 day after cryoablation among all groups, but the BLI signals were significantly different between G0 and G2/G3 (P = .03 and P = .02, respectively) and between G1 and G3 (P = .04). BLI signals reflected tumor growth speed and survival ratio. CONCLUSIONS: This study demonstrates the direct validation of BLI as a quantitative tool for the early assessment of therapeutic effects of cryoablation.

Branchial cleft-like cysts in Hashimoto's thyroiditis: A case report and literature review.

We report an extremely rare case of branchial cleft-like cysts in Hashimoto's thyroiditis. The patient was a 77-year-old man with a growing mass in the anterior neck. Ultrasonography and computed tomography revealed a cystic lesion with septum in the left thyroid and multiple small cystic lesions in the right thyroid. Lymph node swelling of the cervical region, supraclavicular fossa and submandibular region was also observed. Left thyroidectomy and lymph node dissection were performed. Histologically, cysts were lined by stratified squamous epithelium and dense lymphoid tissue having conspicuous follicle formation surrounded the epithelial lining. Solid cell nest (SCN)-like aggregations were seen in the thyroid parenchyma adjacent to the cyst walls and a small number of thyroid follicles were observed in the fibrous wall. Immunohistochemically, it is suggested that both the cyst lining and SCN-like aggregations are originally from thyroid follicles. Although, the exact histogenesis of branchial cleft-like cysts remains unclear, there are probably two different processes for its development, one is of branchial cleft origin and the other is mere squamous metaplasia, while in our case the latter is suggested. Herein, we report our new case and update information about branchial cleft-like cysts that appears in the literature.

Insulin receptor activation with transmembrane domain ligands.

Complementary surfaces are buried when peptide hormones, growth factors, or cytokines bind and activate cellular receptors. Although these extended surfaces provide high affinity and specificity to the interactions, they also present great challenges to the design of small molecules that might either mimic or antagonize the process. We show that the insulin receptor (IR) and downstream signals can be activated by targeting a site outside of its ligand-binding domain. A 24-residue peptide having the IR transmembrane (TM) domain sequence activates IR, but not related growth factor receptors, through specific interactions with the receptor TM domain. Like insulin-dependent activation, IR-TM requires that IR have a competent ATP-binding site and kinase activation loop. IR-TM also activates mutated receptors from patients with severe insulin resistance, which do not respond to insulin. These results show that IR can be activated through a pathway that bypasses its canonical ligand-binding domain.

A method of cryoprotection for protein crystallography by using a microfluidic chip and its application for in situ X-ray diffraction measurements.

We demonstrate a seamless and contactless method from protein crystallization to X-ray analysis using a microfluidic chip with the aim of obtaining a complete crystallographic data set of a protein crystal under cryogenic conditions. Our microfluidics-based approach did not require direct manipulation of the protein crystal. Therefore, the microfluidic chip approach is suitable for novices of X-ray analysis of protein crystals. We also investigated the effect of stepwise cryoprotection on the quality of protein crystals. Protein crystals with cryoprotection via on-chip manipulation did not show deterioration of crystallographic quality of the protein crystal. The complete diffraction data set of a protein crystal, which is required for determining the 3D structure of the target protein, is obtainable by a simple manipulation.

Discovery of DS-5272 as a promising candidate: A potent and orally active p53-MDM2 interaction inhibitor.

We have published p53-MDM2 interaction inhibitors possessing a novel dihydroimidazothiazole scaffold. Although our lead compound 1 showed strong antitumor activity with single oral administration on a xenograft model using MV4-11 cells harboring wild-type p53, it needed a higher dose (200mg/kg) for distinct efficacy. We executed further optimization with the aim of improvement of potency and physicochemical properties. Thus optimal compounds were furnished by introducing fluorine moieties onto the phenyl ring at the C-6 position and the pyrrolidine part at the C-2 substituent; and modifying the terminal piperazine to 4,7-diazaspiro[2,5]octane variants. Furthermore, replacing 4-chlorophenyl on the C-5 position with pyridyl variant decreased nonspecific cytotoxicity significantly. Our exploration afforded DS-5272 indicating excellent antitumor efficacy from a dose of 25mg/kg on SJSA-1 xenografted models with high safety and good PK profiles, which has appropriate potency as a clinical candidate.

Controlling protein crystal nucleation by droplet-based microfluidics.

Herein, we demonstrate the potential of droplet-based microfluidics for controlling protein crystallization and generating single-protein crystals. We estimated the critical droplet size for obtaining a single crystal within a microdroplet and investigated the crystallization of four model proteins to confirm the effect of protein molecular diffusion on crystallization. A single crystal was obtained in microdroplets smaller than the critical size by using droplet-based microfluidics. In the case of thaumatin crystallization, a single thaumatin crystal was obtained in a 200 µm droplet even with high supersaturation. In the case of ferritin crystallization, the nucleation profile of ferritin crystals had a wider distribution than the nucleation profiles of lysozyme, thaumatin, and glucose isomerase crystallization. We found that the droplet-based microfluidic approach was able to control the nucleation of a protein by providing control over the crystallization conditions and the droplet size, and that the diffusion of protein molecules is a significant factor in controlling the nucleation of protein crystals in droplet-based microfluidics.

Immunohistochemical evaluation of O6 -methylguanine DNA methyltransferase (MGMT) expression in 117 cases of glioblastoma.

Temozolomide (TMZ) is an oral alkylating agent which is widely used in the treatment of glioblastoma (GBM) and is composed of astrocytic and/or oligodendroglial tumors, and the evaluation of O(6) -methylguanine DNA methyltransferase (MGMT) expression is important to predict the response to TMZ therapy. In this study, we conducted immunohistochemical analysis of 117 cases of Japanese GBM including 19 cases of GBM with oligodendroglioma component (GBMO), using a scoring system for quantitative evaluation of staining intensity and proportion of MGMT, and performed survival analysis of these patients. Immunohistochemically, 55 cases (47%) were positive for MGMT with various intensities and proportions (total score (TS) ≥ 2), while 62 cases (53%) were negative (TS = 0). The distribution of MGMT expression pattern was not affected by any clinicopathological parameters such as the histological subtype (GBM vs. GBMO), age and gender. The survival analysis of these patients revealed that the minimal expression of MGMT (TS ≥ 2) was a significant unfavorable prognostic factor (P < 0.001) as well as resectability (P = 0.004). Moreover, multivariate analysis showed that minimal MGMT expression in GBM was the most potent independent predictor for progression free survival (P < 0.001) and also overall patient survival (P < 0.001). This is the first report employing the scoring system for both staining intensity and proportion to evaluate immunohistochemical MGMT expression in GBM. In addition, our results emphases the clinicopathological values of the immunohistochemical approach for MGMT expression in glioma patients as a routine laboratory examination.

Thermodynamic study on hydrated bilayers of ether-linked phosphatidylcholines with terminal perfluorobutyl group.

Novel terminally perfluorobutyl group-containing ether-linked phosphatidylcholines with different alkyl chain lengths (di-O-F4-Cn-PCs, n = 14,16 and 18) were developed as possible materials for stable liposomes aiming at applications of structural and functional analyses of membrane proteins. Differential scanning calorimetric investigations of the thermotropic transition of hydrated di-O-F4-Cn-PC bilayers demonstrated that the transition temperature of every di-O-F4-Cn-PC decreases by ~20 °C compared to their corresponding non-fluorinated PCs, di-O-Cn-PCs. With the elongation of the hydrophobic chain, on the other hand, the transition enthalpy (ΔH) and entropy (ΔS) increased in a linear manner. Comparison of ΔH and ΔS values against the net hydrocarbon chain length between di-O-F4-Cn-PCs and di-O-Cn-PCs strongly suggests that in the thermotropic transition of the di-O-F4-Cn-PC membrane, the perfluorobutyl segments undergo very limited structural changes; therefore, the hydrocarbon segments are mainly responsible for the phase transition.

Enzyme-immobilized reactors for rapid and efficient sample preparation in MS-based proteomic studies.

Proteolysis is a key step in proteomic studies integrated with MS analysis but the conventional method of in-solution digestion is limited by time-consuming procedures and low sensitivity. Furthermore, obtaining reliable peptide maps and meaningful sequence data using MS analysis requires not only the separation of the digested peptides but also strictly defined proteolysis conditions. Recently, various immobilized-enzyme reactors have been developed for highly efficient proteolysis in MS-based proteomic analysis. This review focuses on the proteolysis step using protease-immobilized reactors and rapid analysis of protein sequences. We describe the preparation of enzyme reactors by several techniques and protein digestion under unusual conditions. Analysis of posttranslational modifications by enzyme reactors prepared using our immobilization method is presented as a model application. Analysis systems using immobilized-enzyme reactors are expected to become useful tools for proteomic studies and diverse applications in biotechnology.

NS1-binding protein abrogates the elevation of cell viability by the influenza A virus NS1 protein in association with CRKL.

The influenza A virus non-structural protein 1 (NS1) is a multifunctional virulence factor consisting of an RNA binding domain and several Src-homology (SH) 2 and SH3 binding motifs, which promotes virus replication in the host cell and helps to evade antiviral immunity. NS1 modulates general host cell physiology in association with various cellular molecules including NS1-binding protein (NS1-BP) and signaling adapter protein CRK-like (CRKL), while the physiological role of NS1-BP during influenza A virus infection especially in association with NS1 remains unclear. In this study, we analyzed the intracellular association of NS1-BP, NS1 and CRKL to elucidate the physiological roles of these molecules in the host cell. In HEK293T cells, enforced expression of NS1 of A/Beijing (H1N1) and A/Indonesia (H5N1) significantly induced excessive phosphorylation of ERK and elevated cell viability, while the over-expression of NS1-BP and the abrogation of CRKL using siRNA abolished such survival effect of NS1. The pull-down assay using GST-fusion CRKL revealed the formation of intracellular complexes of NS1-BP, NS1 and CRKL. In addition, we identified that the N-terminus SH3 domain of CRKL was essential for binding to NS1-BP using GST-fusion CRKL-truncate mutants. This is the first report to elucidate the novel function of NS1-BP collaborating with viral protein NS1 in modulation of host cell physiology. In addition, an alternative role of adaptor protein CRKL in association with NS1 and NS1-BP during influenza A virus infection is demonstrated.

Lead optimization of novel p53-MDM2 interaction inhibitors possessing dihydroimidazothiazole scaffold.

With the aim of discovering potent inhibitors of the p53-MDM2 interaction and thus obtaining a potent anticancer drug, we have pursued synthesis and optimization of dihydroimidazothiazole derivatives, which have been discovered via scaffold hopping by mimicing the mode of interaction between MDM2 and Nutlins. Upon the discovery we encountered a problem involving the chemical instability of the scaffold, that is, susceptibility to oxidation which led to imidazothiazole. In order to solve this problem and to obtain further potent compounds, we executed medicinal research and thus furnished the optimal compounds by incorporating the methyl group onto the C-6 position to avoid the oxidation, and by modifying the C-2 moiety of the additional proline motif, which furnished high potency. The incorporation of the pyrrolidine moiety at the C-2 position raised another hydrophobic interaction site with MDM2 protein, which was generated by the induced-fitting observed by co-crystal structure analysis. These optimal molecules showed significant improvement in potency when compared with the early lead (+)-1 or Nutlin-3a.

Percutaneous transhepatic biliary drainage assisted by real-time virtual sonography: a retrospective study.

BACKGROUND: Real-time virtual sonography (RVS) is a diagnostic imaging support system that can synchronize with ultrasound images in conjunction with computed tomography or magnetic resonance images using magnetic navigation system. RVS has been applied in clinical practice to perform such procedures as radiofrequency ablation and biopsy; however, the application of RVS for percutaneous transhepatic biliary drainage (PTBD) is rare. METHODS: Between 2007 and 2012, RVS-assisted PTBD was performed for 30 patients (19 males and 11 females; age range, 41 to 89 years; mean age, 66.9 years) with obstructive jaundice. The targeted bile duct was determined using the RVS system before the procedure. The intervention was considered to be successful when the targeted bile duct was punctured and the drainage catheter was placed in the bile duct. Complications were evaluated according to the Society of Interventional Radiology Clinical Practice Guidelines. RESULTS: A total of 37 interventions were performed for 30 patients. The interventions were successful in 35 (95%) of 37 interventions. The targeted bile ducts were: B3 (n = 24), B5 (n = 7), B8 (n = 3), B6 (n = 1), and the anterior (n = 1) and posterior (n = 1) branches of the right bile duct. The mean targeted bile duct diameter was 4.9 mm (1.9 to 8.2 mm). PTBD was able to be accomplished in all patients because the non-targeted bile ducts were successfully punctured alternatively. No major complications were observed in relation to the interventional procedure. CONCLUSIONS: RVS-assisted PTBD is a feasible and safe procedure. Accurate puncture of targeted bile ducts can be achieved using this method.