Detection of Glycolytically Active Lacticaseibacillus paracasei Strain Shirota by Flow Cytometry Targeting the Efflux Activity of Fluorescent Dye: a Potential Tool for Quality Assessment of Probiotic Cells in Milk Products.

The rapid and accurate detection of viable probiotic cells in dairy products is important for assessing product quality in manufacturing. Flow cytometry is widely used for the rapid analysis of bacterial cells. However, further investigation is needed into the optimum property to use it for assessing cell viability. Here, we proposed using the efflux activity of a fluorescent dye, carboxyfluorescein (CF), as an indicator of cell viability. CF is generated from 5(6)-carboxyfluorescein diacetate as a result of cleavage by intracellular esterase. It generally accumulates in the cell, but certain bacterial species are known to extrude it. We found here that the probiotic strain Lacticaseibacillus paracasei strain Shirota (LcS) also extruded CF in the presence of energy sources, such as glucose. To investigate the mechanism of its CF-efflux activity, we screened CF-efflux-negative mutants from a random mutagenesis LcS library and examined the whole genome for genes responsible for CF efflux. We identified a base substitution in the pfkA gene in the glycolytic pathway, and we demonstrated that intact pfkA was essential for CF efflux, indicating that CF-efflux-positive cells must have uncompromised glycolytic activity. We also confirmed that there was a good correlation between the rate of CF-efflux-positive cells and that of colony-forming cells of LcS in a fermented milk product, whereas other properties, such as esterase activity and cell membrane integrity, lost their correlation with the colony-forming activity after long storage. We propose that CF-efflux activity could be an appropriate indicator of cell viability in certain probiotic strains. IMPORTANCE To our knowledge, this is the first report to demonstrate that CF efflux requires uncompromised glycolytic activity in certain lactic acid bacteria. Compared with the cell properties currently widely used for cell viability assessment, such as intracellular esterase activity and membrane integrity, CF-efflux activity enables the accurate detection of culturable cells, especially in products stored for long periods at cold temperatures. These results indicate strongly that CF-efflux activity can be an adequate cell-viability indicator and that flow cytometric quantification could be an alternative to conventional CFU counting. Our findings should be especially informative for dairy/probiotic product manufacturing.

Rapid and Selective Photo-degradation of Polymers: Design of an Alternating Copolymer with an o-Nitrobenzyl Ether Pendant.

The development of polymers with on-demand degradability is required to alleviate the current global issues on polymer-waste pollution. Therefore, we designed a vinyl ether monomer with an o-nitrobenzyl (oNBn) group as a photo-deprotectable pendant (oNBnVE) and synthesized an alternating copolymer with an oNBn-capped acetal backbone via cationic copolymerization with p-tolualdehyde (pMeBzA). The resultant alternating copolymer could be rapidly degraded into lower-molecular-weight compounds upon simple exposure to UV irradiation without any reactants or catalysts, while it was sufficiently stable toward heat and ambient light. This degradation proceeds via cleavage of the hemiacetal structure generated upon photo-deprotection of the oNBn pendant. The oNBn-peculiar degradability allowed the exclusive photo-degradation of the oNBnVE/pMeBzA segments in a diblock copolymer composed of oNBnVE/pMeBzA and benzyl vinyl ether (BnVE)/pMeBzA segments.

Evaluation of the QIAstat-Dx Respiratory SARS-CoV-2 panel, a rapid multiplex PCR method for the diagnosis of COVID-19.

INTRODUCTION: Rapid, simple, and accurate methods are required to diagnose coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This study aimed to evaluate the performance of the QIAstat-Dx Respiratory SARS-CoV-2 Panel (QIAstat-SARS-CoV-2), a rapid multiplex PCR assay for SARS-CoV-2 detection. METHODS: Nasopharyngeal swabs (NPS) that were obtained from patients with COVID-19 who were diagnosed at the National Center for Global Health and Medicine were used in this study. When the NPS samples were found to be negative for SARS-CoV-2 after treatment, they were used as negative samples. We evaluated the performance of the QIAstat-SARS-CoV-2 comparing SARS-CoV-2 detection with the National Institute of Infectious Diseases in Japan-recommended real-time polymerase chain reaction (RT-PCR) method (NIID-RT-PCR). RESULTS: In total, 45 NPS samples were analyzed. The proportion of overall agreement between QIAstat-SARS-CoV-2 and NIID-RT-PCR on 45 samples was 91.0% with a sensitivity of 84.0% (21/25), specificity at 100% (20/20), negative predictive value at 83.3% (20/24), and positive predictive value at 100% (21/21). There were no patients with co-infections with pathogens other than SARS-CoV-2. CONCLUSIONS: QIAstat-SARS-CoV-2 showed a high agreement in comparison with the NIID-RT-PCR for the detection of SARS-CoV-2. The QIAstat-SARS-CoV-2 also provided a rapid and accurate diagnosis for COVID-19, even when the concurrent detection of other respiratory pathogens was desired, and therefore, has the potential to direct appropriate therapy and infection control precautions.

Reconstruction of global regulatory network from signaling to cellular functions using phosphoproteomic data.

Cellular signaling regulates various cellular functions via protein phosphorylation. Phosphoproteomic data potentially include information for a global regulatory network from signaling to cellular functions, but a procedure to reconstruct this network using such data has yet to be established. In this paper, we provide a procedure to reconstruct a global regulatory network from signaling to cellular functions from phosphoproteomic data by integrating prior knowledge of cellular functions and inference of the kinase-substrate relationships (KSRs). We used phosphoproteomic data from insulin-stimulated Fao hepatoma cells and identified protein phosphorylation regulated by insulin specifically over-represented in cellular functions in the KEGG database. We inferred kinases for protein phosphorylation by KSRs, and connected the kinases in the insulin signaling layer to the phosphorylated proteins in the cellular functions, revealing that the insulin signal is selectively transmitted via the Pi3k-Akt and Erk signaling pathways to cellular adhesions and RNA maturation, respectively. Thus, we provide a method to reconstruct global regulatory network from signaling to cellular functions based on phosphoproteomic data.

Temporal coding of insulin action through multiplexing of the AKT pathway.

One of the unique characteristics of cellular signaling pathways is that a common signaling pathway can selectively regulate multiple cellular functions of a hormone; however, this selective downstream control through a common signaling pathway is poorly understood. Here we show that the insulin-dependent AKT pathway uses temporal patterns multiplexing for selective regulation of downstream molecules. Pulse and sustained insulin stimulations were simultaneously encoded into transient and sustained AKT phosphorylation, respectively. The downstream molecules, including ribosomal protein S6 kinase (S6K), glucose-6-phosphatase (G6Pase), and glycogen synthase kinase-3ß (GSK3ß) selectively decoded transient, sustained, and both transient and sustained AKT phosphorylation, respectively. Selective downstream decoding is mediated by the molecules' network structures and kinetics. Our results demonstrate that the AKT pathway can multiplex distinct patterns of blood insulin, such as pulse-like additional and sustained-like basal secretions, and the downstream molecules selectively decode secretion patterns of insulin.

Antimicrobial susceptibility surveillance of bacterial isolates recovered in Japan from odontogenic infections in 2013.

We report on the findings of the first antimicrobial susceptibility surveillance study in Japan of isolates recovered from odontogenic infections. Of the 38 facilities where patients representing the 4 groups of odontogenic infections were seen, 102 samples were collected from cases of periodontitis (group 1), 6 samples from pericoronitis (group 2), 84 samples from jaw inflammation (group 3) and 54 samples from phlegmon of the jaw bone area (group 4) for a total of 246 samples. The positivity rates of bacterial growth on culture were 85.3%, 100%, 84% and 88.9%, respectively, for groups 1, 2, 3 and 4. Streptococcus spp. isolation rates according to odontogenic infection group were 22% (group 1), 17.7% (group 3) and 20.7% (group 4). Anaerobic isolation rates were 66.9% (group 1), 71.8% (group 3) and 68.2% (group 4). Drug susceptibility tests were performed on 726 strains excluding 121 strains that were undergrown. The breakdown of the strains subjected to testing was 186 Streptococcus spp., 179 anaerobic gram-positive cocci, 246 Prevotella spp., 27 Porphyromonas spp., and 88 Fusobacterium spp. The isolates were tested against 30 antimicrobial agents. Sensitivities to penicillins and cephems were good except for Prevotella spp. The low sensitivities of Prevotella spp is due to ß-lactamase production. Prevotella strains resistant to macrolides, quinolones, and clindamycin were found. No strains resistant to carbapenems or penems were found among all strains tested. No anaerobic bacterial strain was resistant to metronidazole. Antimicrobial susceptibility testing performed on the S. anginosus group and anaerobic bacteria, which are the major pathogens associated with odontogenic infections, showed low MIC90 values to the penicillins which are the first-line antimicrobial agents for odontogenic infections; however, for Prevotella spp., penicillins combined with ß-lactamase inhibitor showed low MIC90 values.

[A case of gastrointestinal stromal tumor of the duodenum with ruptured liver metastasis during administration of imatinib].

A fifty-year-old man with a liver metastasis of a duodenal gastrointestinal stromal tumor (GIST) previously treated with imatinib. Thirty-three months following initiation of the therapy, he visited the emergency room of our hospital presenting with an upper abdominal pain. Dynamic CT scan revealed a ruptured liver metastasis of duodenal GIST. We used transcatheter arterial embolization to stop the bleeding. Due to the rarity of this condition, we herein report this case with an article review.

[Short-Term and Long-Term Outcomes of Colonic Stenting as a Bridge to Surgery for Obstructive Colorectal Cancer].

We examined the short-term and long-term outcomes in 50 patients who underwent stenting as a bridge to surgery(BTS) for obstructive colorectal cancer. The patients comprised 30 men and 20 women, with a mean age of 74.0 years. Stenting and decompression were successful in all patients, and the mean time to oral intake after stenting was 2.4 days. No serious complications related to stenting occurred. Colonoscopy after stenting was important for the preoperative diagnosis of coexisting lesions and planning of the extent of resection. Elective and one-stage surgeries could be performed in all patients after stenting. Regarding long-term outcomes, the 5-year overall survival rate and disease-free survival rate in the BTS patients with Stage Ⅱ plus Ⅲ cancer were 73.1% and 55.7%, respectively. The results of this study suggest that BTS for obstructive colorectal cancer is an effective treatment strategy for not only short-term but also long-term outcomes.

System identification of signaling dependent gene expression with different time-scale data.

Cells decode information of signaling activation at a scale of tens of minutes by downstream gene expression with a scale of hours to days, leading to cell fate decisions such as cell differentiation. However, no system identification method with such different time scales exists. Here we used compressed sensing technology and developed a system identification method using data of different time scales by recovering signals of missing time points. We measured phosphorylation of ERK and CREB, immediate early gene expression products, and mRNAs of decoder genes for neurite elongation in PC12 cell differentiation and performed system identification, revealing the input-output relationships between signaling and gene expression with sensitivity such as graded or switch-like response and with time delay and gain, representing signal transfer efficiency. We predicted and validated the identified system using pharmacological perturbation. Thus, we provide a versatile method for system identification using data with different time scales.

[Results of Preoperative Colon Stent Placement for Obstructive Colorectal Cancer].

We examined short-term outcomes in 34 patients who had stenting as a bridge to surgery(BTS)for obstructive colorectal cancer during the 5-year period between April 2012 and March 2017.T he patients were 22 men and 12 women with a mean age of 72.6 years. Stenting and decompression were successful in all patients, and the mean time to oral intake after stenting was 2.5 days.No serious complications related to stenting occurred.Elective surgery could be performed in all patients after stenting.The mean number of days to surgery was 24.7 days.Laparoscopic surgery was performed in 14 patients.Postoperative complications included minor leakage in 1 patient, an abdominal wall abscess due to tumor invasion of the abdominal wall in 1 patient, and heart failure and pneumonia, as serious complications, in 1 patient each.Colorectal stenting in patients with obstructive colorectal cancer is a safe and relatively simple procedure.This is an effective treatment strategy in which preoperative colorectal decompression enables a one-stage resection.

[Findings from Total Colonoscopy in Obstructive Colorectal Cancer Patients Who Underwent Stent Placement as a Bridge to Surgery(BTS)].

We clinically investigated 34 patients with obstructive colorectal cancer who underwent placement of a colonic stent as a bridge to surgery(BTS), focusing on endoscopic findings after stent placement.Twenty -nine patients(85.3%)underwent colonoscopy after stent placement, and the entire large intestine could be observed in 28(96.6%).Coexisting lesions were observed in 22(78.6%)of these 28 patients.The lesions comprised adenomatous polyps in 17 patients(60.7%), synchronous colon cancers in 5 patients(17.9%), and obstructive colitis in 3 patients(10.7%), with some overlapping cases.All patients with multiple cancers underwent one-stage surgery, and all lesions were excised at the same time.Colonoscopy after colonic stent placement is important for preoperative diagnosis of coexisting lesions and planning the extent of resection. These considerations support the utility of colonic stenting for BTS.

Investigation of Scattered Radiation Reduction Effect by Use of Collimator Cover Covered with Leaded Sheet in Angiography Equipment.

The purpose of this study was to investigate the effect of scattered radiation reduction to medical staff by attaching the leaded sheet on the collimator cover of the angiography equipment. Ambient dose equivalent was measured to compare the rate of scattered radiation reduction between with and without the leaded sheet. Shielding effect was confirmed for scattered radiation in all directions, especially 27% of shielding ratio in the head and neck area when angiography equipment installed with small detector, and more than 40% of shielding ratio when adjusting a cut portion of leaded sheet to the field size. However, it decreased when the dose area product meter was not attached. Therefore, our proposed leaded sheet can reduce radiation dose to medical staff during angiographic and interventional procedures.

Longitudinal Investigation of Carriage Rates, Counts, and Genotypes of Toxigenic Clostridium difficile in Early Infancy.

UNLABELLED: Asymptomatic infant carriers of toxigenic Clostridium difficile are suggested to play a role in the transmission of C. difficile infection (CDI) in adults. However, the mode of C. difficile carriage in infants remains to be fully elucidated. We investigated longitudinal changes in carriage rates, counts, and strain types of toxigenic C. difficile in infants. Stools collected from 111 healthy infants in Belgium periodically from birth until the age of 6 months were examined by quantitative PCR targeting 16S rRNA and toxin genes. Toxigenic C. difficile was detected in 18 of 111 infants (16%) in the period up to the age of 6 months. The carriage rate of toxigenic C. difficile remained below 5% until the age of 3 months. The carriage rate increased to 13% 1 week after weaning (average age, 143 days) and reached 16% at the age of 6 months. Counts of toxigenic C. difficile bacteria ranged from 10(4) to 10(8) cells/g of stool. Notably, two infants retained >10(8) cells/g of stool for at least several weeks. Average counts in the 18 infants hovered around 10(7) cells/g of stool from the age of 3 days until the age of 6 months, showing no age-related trend. Genotyping of toxigenic C. difficile isolates from the 18 infants revealed that 11 infants each retained a particular monophyletic strain for at least a month. The genotype most frequently identified was the same as that frequently identified in symptomatic adult CDI patients. Thus, toxigenic C. difficile strains-potential causes of CDI in adults-colonized the infants' intestines. IMPORTANCE: Our study provides longitudinal data on counts and strain types of toxigenic C. difficile in infants. We found that considerable numbers of toxigenic C. difficile bacteria colonized the infants' intestines. The results of strain typing suggest that toxigenic C. difficile carried by healthy infants could be potentially pathogenic to adults. These results and findings are informative not only for ecological studies but also for efforts to prevent or control the spread of CDI in adults.

[Cases of Obstructive Colon Cancer for Which Elective Surgery Was Performed after Colonic Stent Placement].

The present study investigated the short-term outcomes of 20 patients with obstructive colon cancer who underwent colonic stent placement as a bridge to surgery (BTS) during the 3-year period between April 2012 and March 2015. Subjects comprised 13 men and 7 women, with a mean age of 68.3 years. Placement and decompression were successfully achieved in all of the patients. Oral ingestion became possible from a mean of 2.7 days after placement. No serious complications associated with placement were encountered. Total colonoscopy was performed after placement in 17 patients (85%), and independent advanced cancer was seen in the proximal portion of the colon in 1 patient. Elective surgery was performed for all of the patients after placement. Excluding the 2 patients for whom preoperative chemotherapy or treatment of another disease was prioritized, the mean interval to surgery for the remaining 18 patients was 23.2 days. The operative procedure performed was laparoscopic surgery in 8 patients (40%). Although minor leakage (n=1) and abdominal wall abscess (n=1) were observed as postoperative complications, the patients generally had an uneventful course. Colonic stent placement for obstructive colon cancer is relatively easy and safe, and may be considered as an effective treatment method that enables favorable intestinal decompression preoperatively and one-stage resection.

[Stage IV Gastric Cancer Made Resectable Following a Chemotherapy Regimen That Included Trastuzumab-A Case Report].

A 70-year-old man was referred to our hospital after anemia was identified by his general practitioner. A detailed examination led to the diagnosis of unresectable StageIV gastric cancer with metastasis to the paraaortic lymph nodes. The cancer was HER2-positive; therefore, the patient was administered trastuzumab, capecitabine, and cisplatin combination therapy. A remarkable reduction in the size of the lymph nodes was observed. The reduction in the size of the metastatic foci was accompanied by enlargement of the primary tumor, which was considered an indication for surgery. He underwent total gastrectomy, and histopathology showed absence of cancerous cells in the resected lymph nodes, indicating a curative resection. In Japan, trastuzumab, capecitabine, and cisplatin combination therapy is currently recommended as the standard therapy for unresectable advanced/recurrent HER2-positive gastric cancer, and the findings of the present case suggest that it may also be useful as neoadjuvant chemotherapy.

The selective control of glycolysis, gluconeogenesis and glycogenesis by temporal insulin patterns.

Insulin governs systemic glucose metabolism, including glycolysis, gluconeogenesis and glycogenesis, through temporal change and absolute concentration. However, how insulin-signalling pathway selectively regulates glycolysis, gluconeogenesis and glycogenesis remains to be elucidated. To address this issue, we experimentally measured metabolites in glucose metabolism in response to insulin. Step stimulation of insulin induced transient response of glycolysis and glycogenesis, and sustained response of gluconeogenesis and extracellular glucose concentration (GLC(ex)). Based on the experimental results, we constructed a simple computational model that characterises response of insulin-signalling-dependent glucose metabolism. The model revealed that the network motifs of glycolysis and glycogenesis pathways constitute a feedforward (FF) with substrate depletion and incoherent feedforward loop (iFFL), respectively, enabling glycolysis and glycogenesis responsive to temporal changes of insulin rather than its absolute concentration. In contrast, the network motifs of gluconeogenesis pathway constituted a FF inhibition, enabling gluconeogenesis responsive to absolute concentration of insulin regardless of its temporal patterns. GLC(ex) was regulated by gluconeogenesis and glycolysis. These results demonstrate the selective control mechanism of glucose metabolism by temporal patterns of insulin.

Design of sec-Benzyl Vinyl Ethers toward the Synthesis of Alternating Copolymers Composed of Vinyl Alcohol and Vinyl Ether Units.

In this work, we designed benzyl vinyl ethers carrying alkyl substituents at the benzyl position (i.e., sec-BnVEs) as bulky, reactive, and transformable monomers to realize the alternating cationic copolymerization with an alkyl vinyl ether (VE). In particular, the isopropyl substitution caused not only the bulkiness to suppress the successive propagation but also an enhancement of the vinyl group reactivity to promote crossover propagation with a less bulky VE comonomer. The isopropyl-substituted BnVE (iPr-BnVE) underwent living cationic alternating copolymerization with n-butyl VE (nBVE), and the alternating propagation was strongly suggested by the reactivity ratios. The subsequent deprotection of the sec-benzyl pendant afforded the vinyl alcohol (VA)-nBVE alternating copolymer, and the corresponding statistical copolymer was also synthesized by using the nonsubstituted monomer (BnVE) instead of iPr-BnVE. The alternating copolymer exhibited a higher glass transition temperature, which likely stems from the uniform and efficient hydrogen-bonding formation due to the periodic sequence.

An Investigation of the Non-selective Etching of Synthetic Polymers by Electrospray Droplet Impact/Secondary Ion Mass Spectrometry (EDI/SIMS).

Among the various types of cluster secondary ion mass spectrometry (SIMS), electrospray droplet impact/secondary ion mass spectrometry (EDI/SIMS) is unique due to its high ionization efficiency and non-selective atomic/molecular-level surface etching ability. In this study, EDI/SIMS was applied to the non-selective etching of synthetic polymers of polystyrene (PS) and poly(9,9-di-n-octylfluonyl-2,7diyl) (PFO) deposited on a silicon substrate. The polymers gave characteristic fragment ions and the mass spectra remained unchanged with prolonged EDI irradiation time, indicating that non-selective etching can be achieved by EDI irradiation, a finding that is consistent with our previous reports based on EDI/X-ray photoelectron spectroscopy analyses. From the irradiation time and film thickness, the etching rates for PS and PFO were roughly estimated to be 0.6 nm/min and 0.15 nm/min, respectively, under the experimental conditions that were used. After the depletion of polymer sample on the surface, ion signals originating from the exposed silicon substrate were observed. This indicates that EDI/SIMS is applicable to the analysis of the interface of multilayered films composed of organic and inorganic materials.

Comparison of hepatic responses to glucose perturbation between healthy and obese mice based on the edge type of network structures.

Interactions between various molecular species in biological phenomena give rise to numerous networks. The investigation of these networks, including their statistical and biochemical interactions, supports a deeper understanding of biological phenomena. The clustering of nodes associated with molecular species and enrichment analysis is frequently applied to examine the biological significance of such network structures. However, these methods focus on delineating the function of a node. As such, in-depth investigations of the edges, which are the connections between the nodes, are rarely explored. In the current study, we aimed to investigate the functions of the edges rather than the nodes. To accomplish this, for each network, we categorized the edges and defined the edge type based on their biological annotations. Subsequently, we used the edge type to compare the network structures of the metabolome and transcriptome in the livers of healthy (wild-type) and obese (ob/ob) mice following oral glucose administration (OGTT). The findings demonstrate that the edge type can facilitate the characterization of the state of a network structure, thereby reducing the information available through datasets containing the OGTT response in the metabolome and transcriptome.

A case of eosinophilic polyangiitis with granulomatosis that evolved to cardiac arrest due to advanced atrioventricular block.

Cardiac manifestations are the major cause of mortality in patients with eosinophilic granulomatosis with polyangiitis (EGPA). Among these manifestations in EGPA patients, in the literature, there are fewer reports describing bradycardia in EGPA patients than those describing tachycardia. A 50-year-old woman with a history of childhood-onset asthma. At age 28, she was diagnosed with eosinophilic gastroenteritis without the diagnosis of EGPA and was started on a systemic steroid and had maintenance daily dose of 2.5 mg after gradually tapered. She had experiencing dizziness and palpitations 2 weeks after discontinuation of the steroid treatment. At emergency visit, electrocardiography revealed an advanced atrioventricular block of 3:1 or less. Forty-eight minutes after the start of electrocardiography, only a P wave was observed and cardiac arrest occurred for 9 s and temporary emergency pacing was performed immediately. She was diagnosed as EGPA presenting leukocyte count, 16,500/µL, 42.8% of which were eosinophils and sinusitis in computed-tomography. She could be survival by treatment of steroid, following the patient to withdraw from an external pacemaker. She received prednisolone of 60 mg, intravenous cyclophosphamide and intravenous immunoglobulin. She had relapsed presenting peripheral eosinophilia, abdominal and numbness in the toes of the left leg pain, but not arrythmia after tapered of prednisolone. Following additional steroid pulse, she had an increase of prednisolone and continued by intravenous cyclophosphamide, intravenous immunoglobulin and started mepolizumab. We presented a severe case of EGPA presenting an advanced atrioventricular block into cardiac arrest.