G6P-capturing molecules in the periplasm of Escherichia coli accelerate the shikimate pathway.

Escherichia coli, the most studied prokaryote, is an excellent host for producing valuable chemicals from renewable resources as it is easy to manipulate genetically. Since the periplasmic environment can be easily controlled externally, elucidating how the localization of specific proteins or small molecules in the periplasm affects metabolism may lead to bioproduction development using E. coli. We investigated metabolic changes and its mechanisms occurring when specific proteins are localized to the E. coli periplasm. We found that the periplasmic localization of ß-glucosidase promoted the shikimate pathway involved in the synthesis of aromatic chemicals. The periplasmic localization of other proteins with an affinity for glucose-6-phosphate (G6P), such as inactivated mutants of Pgi, Zwf, and PhoA, similarly accelerated the shikimate pathway. Our results indicate that G6P is transported from the cytoplasm to the periplasm by the glucose transporter protein EIICBGlc, and then captured by ß-glucosidase.

Metabolic engineering of E. coli for improving mevalonate production to promote NADPH regeneration and enhance acetyl-CoA supply.

Microbial production of mevalonate from renewable feedstock is a promising and sustainable approach for the production of value-added chemicals. We describe the metabolic engineering of Escherichia coli to enhance mevalonate production from glucose and cellobiose. First, the mevalonate-producing pathway was introduced into E. coli and the expression of the gene atoB, which encodes the gene for acetoacetyl-CoA synthetase, was increased. Then, the deletion of the pgi gene, which encodes phosphoglucose isomerase, increased the NADPH/NADP+ ratio in the cells but did not improve mevalonate production. Alternatively, to reduce flux toward the tricarboxylic acid cycle, gltA, which encodes citrate synthetase, was disrupted. The resultant strain, MGΔgltA-MV, increased levels of intracellular acetyl-CoA up to sevenfold higher than the wild-type strain. This strain produced 8.0 g/L of mevalonate from 20 g/L of glucose. We also engineered the sugar supply by displaying ß-glucosidase (BGL) on the cell surface. When cellobiose was used as carbon source, the strain lacking gnd displaying BGL efficiently consumed cellobiose and produced mevalonate at 5.7 g/L. The yield of mevalonate was 0.25 g/g glucose (1 g of cellobiose corresponds to 1.1 g of glucose). These results demonstrate the feasibility of producing mevalonate from cellobiose or cellooligosaccharides using an engineered E. coli strain.

A Teleophthalmology Support System Based on the Visibility of Retinal Elements Using the CNNs.

This paper proposes a teleophthalmology support system in which we use algorithms of object detection and semantic segmentation, such as faster region-based CNN (FR-CNN) and SegNet, based on several CNN architectures such as: Vgg16, MobileNet, AlexNet, etc. These are used to segment and analyze the principal anatomical elements, such as optic disc (OD), region of interest (ROI) composed by the macular region, real retinal region, and vessels. Unlike the conventional retinal image quality assessment system, the proposed system provides some possible reasons about the low-quality image to support the operator of an ophthalmoscope and patient to acquire and transmit a better-quality image to central eye hospital for its diagnosis. The proposed system consists of four steps: OD detection, OD quality analysis, obstruction detection of the region of interest (ROI), and vessel segmentation. For the OD detection, artefacts and vessel segmentation, the FR-CNN and SegNet are used, while for the OD quality analysis, we use transfer learning. The proposed system provides accuracies of 0.93 for the OD detection, 0.86 for OD image quality, 1.0 for artefact detection, and 0.98 for vessel segmentation. As the global performance metric, the kappa-based agreement score between ophthalmologist and the proposed system is calculated, which is higher than the score between ophthalmologist and general practitioner.

Physiologically Based Pharmacokinetic Model of the CYP2D6 Probe Atomoxetine: Extrapolation to Special Populations and Drug-Drug Interactions.

Physiologically based pharmacokinetic (PBPK) modeling of drug disposition and drug-drug interactions (DDIs) has become a key component of drug development. PBPK modeling has also been considered as an approach to predict drug disposition in special populations. However, whether models developed and validated in healthy populations can be extrapolated to special populations is not well established. The goal of this study was to determine whether a drug-specific PBPK model validated using healthy populations could be used to predict drug disposition in specific populations and in organ impairment patients. A full PBPK model of atomoxetine was developed using a training set of pharmacokinetic (PK) data from CYP2D6 genotyped individuals. The model was validated using drug-specific acceptance criteria and a test set of 14 healthy subject PK studies. Population PBPK models were then challenged by simulating the effects of ethnicity, DDIs, pediatrics, and renal and hepatic impairment on atomoxetine PK. Atomoxetine disposition was successfully predicted in 100% of healthy subject studies, 88% of studies in Asians, 79% of DDI studies, and 100% of pediatric studies. However, the atomoxetine area under the plasma concentration versus time curve (AUC) was overpredicted by 3- to 4-fold in end stage renal disease and hepatic impairment. The results show that validated PBPK models can be extrapolated to different ethnicities, DDIs, and pediatrics but not to renal and hepatic impairment patients, likely due to incomplete understanding of the physiologic changes in these conditions. These results show that systematic modeling efforts can be used to further refine population models to improve the predictive value in this area.

Identification of amino acid determinants in CYP4B1 for optimal catalytic processing of 4-ipomeanol.

Mammalian CYP4B1 enzymes are cytochrome P450 mono-oxygenases that are responsible for the bioactivation of several exogenous pro-toxins including 4-ipomeanol (4-IPO). In contrast with the orthologous rabbit enzyme, we show here that native human CYP4B1 with a serine residue at position 427 is unable to bioactivate 4-IPO and does not cause cytotoxicity in HepG2 cells and primary human T-cells that overexpress these enzymes. We also demonstrate that a proline residue in the meander region at position 427 in human CYP4B1 and 422 in rabbit CYP4B1 is important for protein stability and rescues the 4-IPO bioactivation of the human enzyme, but is not essential for the catalytic activity of the rabbit CYP4B1 protein. Systematic substitution of native and p.S427P human CYP4B1 with peptide regions from the highly active rabbit enzyme reveals that 18 amino acids in the wild-type rabbit CYP4B1 protein are key for conferring high 4-IPO metabolizing activity. Introduction of 12 of the 18 amino acids that are also present at corresponding positions in other human CYP4 family members into the p.S427P human CYP4B1 protein results in a mutant human enzyme (P+12) that is as stable and as active as the rabbit wild-type CYP4B1 protein. These 12 mutations cluster in the predicted B-C loop through F-helix regions and reveal new amino acid regions important to P450 enzyme stability. Finally, by minimally re-engineering the human CYP4B1 enzyme for efficient activation of 4-IPO, we have developed a novel human suicide gene system that is a candidate for adoptive cellular therapies in humans.

Generation of specific monoclonal antibodies against the extracellular loops of human claudin-3 by immunizing mice with target-expressing cells.

Human claudin-3 (CLDN3) is a tetraspanin transmembrane protein of tight junction structures and is known to be over-expressed in some malignant tumors. Although a specific monoclonal antibody (MAb) against the extracellular domains of CLDN3 would be a valuable tool, generation of such MAbs has been regarded as difficult using traditional hybridoma techniques, because of the conserved sequence homology of CLDN3s among various species. In addition, high sequence similarity is shared among claudin family members, and potential cross-reactivity of MAb should be evaluated carefully. To overcome these difficulties, we generated CLDN3-expressing Chinese hamster ovary and Sf9 cells to use an immunogens and performed cell-based screening to eliminate cross-reactive antibodies. As a result, we generated MAbs that recognized the extracellular loops of CLDN3 but not those of CLDN4, 5, 6, or 9. Further in vitro studies suggested that the isolated MAbs possessed the desired binding properties for the detection or targeting of CLDN3.

Ocular cytochrome P450s and transporters: roles in disease and endobiotic and xenobiotic disposition.

Drug metabolism and transport processes in the liver, intestine and kidney that affect the pharmacokinetics and pharmacodynamics of therapeutic agents have been studied extensively. In contrast, comparatively little research has been conducted on these topics as they pertain to the eye. Recently, however, catalytic functions of ocular cytochrome P450 enzymes have gained increasing attention, in large part due to the roles of CYP1B1 and CYP4V2 variants in primary congenital glaucoma and Bietti's corneoretinal crystalline dystrophy, respectively. In this review, we discuss challenges to ophthalmic drug delivery, including Phase I drug metabolism and transport in the eye, and the role of three specific P450s, CYP4B1, CYP1B1 and CYP4V2 in ocular inflammation and genetically determined ocular disease.

A case of recurrent laryngeal nerve paralysis caused by radiofrequency ablation for mediastinal recurrence of lung cancer.

Radiofrequency ablation (RFA) has emerged as a potent therapeutic modality for tumor treatment, and offers benefits such as reduced recovery time and minimal damage to nearby tissues. However, RFA is not devoid of complications, notably nerve damage during intrathoracic lesion treatments, which can significantly impact patients' quality of life. This report describes the unique case of a 71-year-old male who experienced hoarseness attributed to injury to the recurrent nerve after RFA for a locally recurrent lung cancer lesion in the mediastinum near the aortic arch. Although RFA has the advantages of a minimally invasive nature and positive outcomes, its risk of nerve injury, specifically in the thoracic region, highlights the need for improved techniques and preventive measures.

A case of percutaneous deep pelvic abscess drainage using CT fluoroscopic guided cranio-caudal puncture technique.

A 52-year-old male patient presented with complaints of abdominal and back pain. CT revealed a deep pelvic abscess extending into the anterior sacral space. Since puncture via the conventional transgluteal approach cannot reach a deep abscess, percutaneous pelvic abscess drainage was performed under CT fluoroscopy using the cranio-caudal puncture technique. The cranio-caudal puncture requires needle insertion perpendicular to the CT cross-section. This method advances the CT gantry deeper than the needle tip and follows the CT cross-section with the needle tip. This series of images and movements continues until the needle reaches the target. The procedure was successful without complications, the abscess was reduced in size, and blood test data improved. The cranio-caudal puncture technique provides an alternative for the drainage of deep pelvic abscesses that avoids the complications associated with gluteal muscle puncture. Percutaneous drainage of pelvic abscesses under CT fluoroscopy-guided cranio-caudal puncture offers a safe option as a puncture route for deep pelvic abscesses.

Percutaneous transhepatic sclerotherapy for ascending colonic varices due to left-sided portal hypertension.

Left-sided portal hypertension (LSPH) causes varices and splenomegaly due to splenic vein issues. Colonic varices are rare and lack standardized treatment. We report the successful treatment of colonic varices caused by LSPH, by addressing both the afferent and efferent veins. A 70-year-old man with distal cholangiocarcinoma had surgery without splenic vein resection, leading to proximal splenic vein stenosis and varices at multiple locations. Percutaneous transhepatic splenic venography revealed that collateral veins flowed into the ascending colonic varices and returned to the portal vein. Complete thrombosis of the varices was achieved by injecting sclerosants and placing coils in both the afferent and efferent veins. The procedure was safe and effective, with no variceal recurrence. This approach provides a minimally invasive option for treating colonic varices associated with LSPH.

[Specimen misidentification in pathology laboratory: trends and measures].

Specimen misidentification in pathology laboratories may have serious consequences. Reports on the frequency of errors in pathology laboratories in Japan are rare. We reviewed near-miss and incident reports over 7 years in our laboratory, extracted those associated with misidentification, analyzed annual changes in numbers and content, and discussed the problems faced and measures taken to prevent misidentification. Of 113,447 pathological cases, 88 (0.078%) reports were associated with misidentification. Of these 88 misidentification cases, 19% occurred before and during accessioning, 16% during dissecting and sectioning, 30% during embedding, 13% during tissue cutting and slide mounting, 19% during slide submitting, and 3% during diagnosis. Two cases (2.3%) of misidentification were detected after diagnosis; however, misidentification did not appear to cause adverse effects in any patient. The frequency of events is similar to that reported in the literature; specimen misidentification was noted in 0.1-0.2% of cases in a modern pathology laboratory. Two-thirds of misidentification events occurred associated with gross specimens, similar to findings in other studies. With the introduction of new technologies that minimize the possibility of human errors (e.g., barcode reading, digital imaging of every specimen, and installation of a glass slide printer), education on medical safety, and the use of multiple safety nets (e.g., diagnosis cancelling and slide checking), errors have decreased, but have not been eliminated completely. Recording errors and reporting them to the hospital and social community, and maintaining a sustainable quality improvement system is very important to reduce errors in pathology.

Triacetic acid lactone production using 2-pyrone synthase expressing Yarrowia lipolytica via targeted gene deletion.

An environmentally sustainable world can be realized by using microorganisms to produce value-added materials from renewable biomass. Triacetic acid lactone (TAL) is a high-value-added compound that is used as a precursor of various organic compounds such as food additives and pharmaceuticals. In this study, we used metabolic engineering to produce TAL from glucose using an oleaginous yeast Yarrowia lipolytica. We first introduced TAL-producing gene 2-pyrone synthase into Y. lipolytica, which enabled TAL production. Next, we increased TAL production by engineering acetyl-CoA and malonyl-CoA biosynthesis pathways by redirecting carbon flux to glycolysis. Finally, we optimized the carbon and nitrogen ratios in the medium, culminating in the production of 4078 mg/L TAL. The strategy presented in this study had the potential to improve the titer and yield of polyketide biosynthesis.

Coil-assisted retrograde transvenous obliteration of gastric varices by an inverted catheter tip technique via the pericardiophrenic vein.

A 70-year-old woman with liver cirrhosis was admitted to our hospital for treatment of growing gastric varices in the fundus. Computed tomography showed gastric varices continuously draining the pericardiophrenic vein via the inferior phrenic vein. Balloon-occluded retrograde transvenous obliteration by a transjugular approach was planned. However, a conventional balloon catheter or microballoon catheter could not be inserted into the efferent vein near the varices because of the narrowness and tortuosity of the vein. Hence, coil-assisted retrograde transvenous obliteration was performed by an inverted catheter tip technique using a single conventional microcatheter. This technique might be useful for cases in which it is difficult to insert a balloon catheter into the efferent vein.

Afferent vein embolization via retrograde approach as a potential treatment strategy for bleeding duodenal varices.

The standard treatment for ruptured duodenal varices remains to be established. Emergency balloon-occluded retrograde transvenous obliteration is challenging in patients with bleeding because re-rupture of varices can occur due to increased pressure when using the retrograde approach. Herein, we describe a case in which a catheter was retrogradely advanced to the afferent vein beyond bleeding duodenal varices; however, the varices re-ruptured during coil embolization, and a part of the catheter was deviated into the intestinal tract. The rupture site was embolized by liquid embolic materials from the microcatheter. Embolization via retrograde approach needs to be carefully performed.

CYP4V2 in Bietti's crystalline dystrophy: ocular localization, metabolism of ω-3-polyunsaturated fatty acids, and functional deficit of the p.H331P variant.

Bietti's crystalline corneoretinal dystrophy (BCD) is a recessive degenerative eye disease caused by germline mutations in the CYP4V2 gene. More than 80% of mutant alleles consist of three mutations, that is, two splice-site alterations and one missense mutation, c.992C>A, which translates to p.H331P. In the present study, we analyzed the expression of CYP4 family members in human tissues and conducted functional studies with the wild-type and p.H331P enzymes, to elucidate the link between CYP4V2 activity and BCD. Expression analysis of 17 CYP1 to CYP4 genes showed CYP4V2 to be a major cytochrome P450 in ARPE-19 cells (a human cell line spontaneously generated from normal human retinal pigmented epithelium) and the only detectable CYP4 transcript. Immunohistochemical analyses demonstrated that CYP4V2 protein was present in epithelial cells of the retina and cornea and the enzyme was localized to endoplasmic reticulum. Recombinant reconstituted CYP4V2 protein metabolized eicosapentaenoic acid and docosahexaenoic acid (an important constituent of the retina) to their respective ω-hydroxylated products at rates similar to those observed with purified CYP4F2, which is an established hepatic polyunsaturated fatty acid (PUFA) hydroxylase. The disease-associated p.H331P variant was undetectable in Western blot analyses of HepG2 cells stably transduced with lentiviral expression vectors. Finally, overexpression of functional CYP4V2 in HepG2 cells altered lipid homeostasis. We demonstrated that CYP4V2 protein is expressed at high levels in ocular target tissues of BCD, that the enzyme is metabolically active toward PUFAs, and that the functional deficit among patients with BCD who carry the H331P variant is most likely a consequence of the instability of the mutant protein.

An early fire detection algorithm using IP cameras.

The presence of smoke is the first symptom of fire; therefore to achieve early fire detection, accurate and quick estimation of the presence of smoke is very important. In this paper we propose an algorithm to detect the presence of smoke using video sequences captured by Internet Protocol (IP) cameras, in which important features of smoke, such as color, motion and growth properties are employed. For an efficient smoke detection in the IP camera platform, a detection algorithm must operate directly in the Discrete Cosine Transform (DCT) domain to reduce computational cost, avoiding a complete decoding process required for algorithms that operate in spatial domain. In the proposed algorithm the DCT Inter-transformation technique is used to increase the detection accuracy without inverse DCT operation. In the proposed scheme, firstly the candidate smoke regions are estimated using motion and color smoke properties; next using morphological operations the noise is reduced. Finally the growth properties of the candidate smoke regions are furthermore analyzed through time using the connected component labeling technique. Evaluation results show that a feasible smoke detection method with false negative and false positive error rates approximately equal to 4% and 2%, respectively, is obtained.

Measurements of the binding of a large protein using a substrate density-controlled DNA chip.

The DNA chip that immobilizes DNA oligonucleotides on a solid plate surface is used for many diagnostic applications. For maximizing the detection sensitivity and accuracy, it is important to control the DNA density on a chip surface and establish a convenient method for optimizing the density. Here, the binding of DNA mismatch-binding protein MutS to the DNA substrate on the chip was investigated, which can be applied for high-throughput single-nucleotide polymorphism analysis in a genome. We prepared the DNA chips where the DNA substrate density was changed simply by using a mixed DNA solution. The binding of MutS was significantly influenced by the amount of DNA substrate on the chip as a consequence of steric crowding, and the moderate density that gave the distance between the DNA substrates greater than the size of the protein was appropriate to obtain accurate kinetic parameters. The substrate density-controlled DNA chip prepared using the mixed DNA solution has distinctive advantages for maximizing the detection capability and kinetic analysis of the binding of MutS and probably also other large proteins.

[Safety management in pathology laboratory: from specimen handling to confirmation of reports].

Medical errors in pathological diagnosis give a huge amount of physical and psychological damage to patients as well as medical staffs. We discussed here how to avoid medical errors in surgical pathology laboratory through our experience. Handling of surgical specimens and diagnosing process requires intensive labor and involves many steps. Each hospital reports many kinds of accidents or incidents, however, many laboratories share common problems and each process has its specific risk for the certain error. We analyzed the problems in each process and concentrated on avoiding misaccessioning, mislabeling, and misreporting. We have made several changes in our system, such as barcode labels, digital images of all specimens, putting specimens in embedding cassettes directly on the endoscopic biopsied specimens, and using a multitissue control block as controls in immunohistochemistry. Some problems are still left behind, but we have reduced the errors by decreasing the number of artificial operation as much as possible. A pathological system recognizing the status of read or unread the pathological reports by clinician are now underconstruction. We also discussed about quality assurance of diagnosis, cooperation with clinicians and other comedical staffs, and organization and method. In order to operate riskless work, it is important for all the medical staffs to have common awareness of the problems, keeping careful observations, and sharing all the information in common. Incorporation of an organizational management tool such as ISO 15189 and utilizing PDCA cycle is also helpful for safety management and quality improvement of the laboratory.

Incidentally identified ductus arteriosus aneurysm in eight adults: a case series.

Ductus arteriosus aneurysm (DAA) in adulthood is a rare entity. We retrospectively reviewed our medical records from the past 10 years and identified 8 cases of adult DAA (6 males and 2 females aged between 69 and 89 years; mean, 76 years), using multiplanar reconstruction and three-dimensional reconstruction CT images. The aneurysm was suspected incidentally in all cases based on the results of chest radiographic screening or post-operative follow-up CT for lung or colon cancer. All eight patients were asymptomatic but had a history of or concurrent hypertension (n = 5, 62.5%), diabetes mellitus (n = 3, 37.5%), cerebrovascular disease (n = 3, 37.5%), ischemic heart disease (n = 1, 12.5%), and cardiac failure (n = 1). All patients had no history of trauma (n = 8, 100%). Six had a history of cigarette smoking. The aneurysm size ranged from 2.0 × 4.0 to 6.3 × 5.3 cm (mean, 3 × 5 cm). The surgical procedures used were four cases of total arch replacement and two cases of thoracic endovascular aortic repair. Two patients were not surgically treated. The median follow-up was 14.5 months (range, 2 months to 9 years). In the two patients who were not surgically treated, the aneurysm enlarged in one, and remained unchanged in the other. Of the six surgically managed cases, one was lost to follow-up, and another patient died of an unrelated cause. The remaining four cases had no enlargement of the aneurysm. No ruptures were reported in any of the cases. DAA should be considered when a saccular aneurysm is located in the minor curvature of the aortic arch and extending toward the left pulmonary trunk in adult patients. Differentiating adult DAA is important, because it is associated with a high risk of rupture due to the fragile nature of true aneurysms.

Splenic Vein Diameter is a Risk Factor for the Portal Venous System Thrombosis After Partial Splenic Artery Embolization.

PURPOSE: Portal venous system thrombosis is a complication of partial splenic artery embolization, and pre-treatment risk assessment is thus important. The purpose of this study was to identify the risk factors for portal venous system thrombosis after partial splenic artery embolization. MATERIALS AND METHODS: We retrospectively analyzed 67 consecutive patients who underwent contrast-enhanced computed tomography before and after first partial splenic artery embolization between July 2007 and October 2018. As risk factors, we investigated age, sex, hematological data, liver function, steroid use, heparin use, and findings from pre- and post-treatment computed tomography. Uni- and multivariate analyses were performed to evaluate the relationship between thrombus appearance or growth and these factors. Values of p < 0.05 were considered significant. RESULTS: Partial splenic artery embolization was technically successful in all 67 patients. Nine patients showed appearance or growth of thrombus. Univariate analysis showed maximum diameter of the splenic vein before treatment (p = 0.0076), percentage of infarcted spleen (p = 0.017), and volume of infarcted spleen (p = 0.022) as significant risk factors. Multivariate analysis showed significant differences in maximum diameter of the splenic vein before treatment (p = 0.041) and percentage of infarcted spleen (p = 0.023). According to receiver operating characteristic analysis, cutoffs for maximum diameter of the splenic vein and percentage of infarcted spleen for distinguishing the appearance or growth of thrombus were 17 mm and 58.2%. CONCLUSION: Large maximum diameter of the splenic vein before partial splenic artery embolization and high percentage of infarcted spleen after partial splenic artery embolization were identified as risk factors for portal venous system thrombosis. LEVEL OF EVIDENCE: Level 4, Case Series.