Comprehensive modeling of cell culture profile using Raman spectroscopy and machine learning.

Chinese hamster ovary (CHO) cells are widely utilized in the production of antibody drugs. To ensure the production of large quantities of antibodies that meet the required specifications, it is crucial to monitor and control the levels of metabolites comprehensively during CHO cell culture. In recent years, continuous analysis methods employing on-line/in-line techniques using Raman spectroscopy have attracted attention. While these analytical methods can nondestructively monitor culture data, constructing a highly accurate measurement model for numerous components is time-consuming, making it challenging to implement in the rapid research and development of pharmaceutical manufacturing processes. In this study, we developed a comprehensive, simple, and automated method for constructing a Raman model of various components measured by LC-MS and other techniques using machine learning with Python. Preprocessing and spectral-range optimization of data for model construction (partial least square (PLS) regression) were automated and accelerated using Bayes optimization. Subsequently, models were constructed for each component using various model construction techniques, including linear regression, ridge regression, XGBoost, and neural network. This enabled the model accuracy to be improved compared with PLS regression. This automated approach allows continuous monitoring of various parameters for over 100 components, facilitating process optimization and process monitoring of CHO cells.

Combination index of the concentration and in vivo antagonism activity of racemic warfarin and its metabolites to assess individual drug responses.

The present study was undertaken to examine whether in vivo vitamin K epoxide reductase complex 1 (VKOR) "actual" antagonism activity, calculated by the concentrations and the reported anticoagulant activities of the R- and S-warfarin enantiomers and their metabolites, correlates with the weekly dose of warfarin. Five patients under palliative care were enrolled in our study and 20 serum samples were analyzed by an enantioselective high-performance liquid chromatography-ultraviolet detection method. In vivo VKOR inhibition activities of S-warfarin, R-warfarin, 7- and 10-hydroxywarfarin were calculated as the ratio of drug or metabolite concentration to the IC50. The mean drug concentrations (± SD) of S- and R-warfarin, 7-hydroxywarfarin and 10-hydroxywarfarin were 334 ± 154 ng/ml, 370 ± 115 ng/ml, 42 ± 15 ng/ml and 80 ± 44 ng/ml, respectively. Then, in vivo VKOR actual antagonism activities of S- and R-warfarin, 7-hydroxywarfarin and 10-hydroxywarfarin were calculated. Good correlation (R2 = 0.69-0.72) was obtained between the weekly warfarin dose and the ratios of INR/actual antagonism activity, while poor correlation was observed between the weekly warfarin dose and INR (R2 = 0.32) or the activities (R2 = 0.17-0.21). Actual antagonism activities along with the INR correlated well with the warfarin dose. This parameter may be useful for predicting or altering warfarin doses, although further verification in a larger study is required.

Disaster Nursing Knowledge and Competencies Among Nursing University Students Participated in Relief Activities Following the 2016 Kumamoto Earthquakes.

OBJECTIVE: This study examined disaster nursing knowledge and competencies among university nursing students who participated in relief activities following the 2016 Kumamoto earthquakes. METHODS: Participants were university nursing students involved in disaster relief activities. Of the 260 individuals to whom a self-report questionnaire was distributed at nursing universities and hospitals located in Kyushu, southwestern Japan, 201 participants returned the questionnaires by mail (response rate 77.3%), of which 108 questionnaires were complete (valid response rate 41.5%). Questions involved experiences related to their volunteer activities, their motive for volunteering, type of relief activities performed, and use of disaster nursing knowledge and competencies. RESULTS: We identified four categories of disaster nursing competencies: "understanding and implementation of assistance to victims in collaboration with other members of the disaster response team," "understanding the natural disaster's influence on victims," "ethical practice in a disaster recovery area," and "understanding of their role within the disaster relief organization." Those who had completed a disaster nursing program had superior measures of performance across all four competencies compared with those who had not yet completed the program. CONCLUSIONS: Four domains of disaster nursing knowledge and competencies used by university nursing students during the 2016 Kumamoto earthquake relief activities were identified. Those who had completed a disaster nursing program had higher competencies than were those who were either currently or not yet enrolled in it.

Molecular-shape selectivity by naphthalimido-modified silica stationary phases: Insight into the substituents effect of naphthalene on shape recognition and π-π interactions via electrostatic potential.

This report describes the molecular-shape selectivity of four naphthalimido-modified silica (NIM) stationary phases. To investigate the selectivity, several alkylbenzenes and polycyclic aromatic hydrocarbons (PAHs) were tested using capillary electrochromatography (CEC) with each NIM stationary phase. Results revealed that nitro group-substituted NIM phases had outstanding molecular-shape recognition ability toward PAHs; a strong negative correlation existed between the k' value and L/B ratio that is opposite those observed for C18 phases, in addition, one possessed 10-fold greater molecular-shape recognition ability compared to existing stationary phases. In addition, the position of the nitro group on the naphthalimide moiety modulated the π-stacking interaction due to steric hindrance. Furthermore, structure optimization and electrostatic potential (ESP) surfaces were used to clarify the mechanism of recognition. Based on this high recognition ability, separations of several positional isomers were examined. Results indicated that structure optimization and ESP maps could reveal the effect of functional groups on π-π interactions, which will aid in the design of new NIM stationary phases with shape selectivity, while taking advantage of electrostatic interactions.

Effect of weak acid hypochlorous solution on selected viruses and bacteria of laboratory rodents.

Weak acid hypochlorous solution (WAHS) is known to have efficacy for inactivating pathogens and to be relatively safe with respect to the live body. Based on these advantages, many animal facilities have recently been introducing WAHS for daily cleaning of animal houses. In this study, we determined the effect of WAHS in inactivating specific pathogens of laboratory rodents and pathogens of opportunistic infection. WAHS with an actual chloride concentration of 60 ppm and a pH value of 6.0 was generated using purpose-built equipment. One volume of mouse hepatitis virus (MHV), Sendai virus, lymphocytic choriomeningitis virus, Bordetella bronchiseptica, Pasteurella pneumotropica, Corynebacterium kutscheri, Staphylococcus aureus, and Pseudomonas aeruginosa was mixed with 9 or 99 volumes of WAHS (×10 and ×100 reaction) for various periods (0.5, 1, and 5 min) at 25°C. After incubation, the remaining infectious viruses and live bacteria were determined by plaque assay or culture. In the ×100 reaction mixture, infectious viruses and live bacteria could not be detected for any of the pathogens examined even with the 0.5-min incubation. However, the effects for MHV, B. bronchiseptica, and P. aeruginosa were variable in the ×10 reaction mixture with the 0.5- and 1-min incubations. Sufficient effects were obtained by elongation of the reaction time to 5 min. In the case of MHV, reducing organic substances in the virus stock resulted in the WAHS being completely effective. WAHS is recommended for daily cleaning in animal facilities but should be used properly in order to obtain a sufficient effect, which includes such things as using a large enough volume to reduce effects of organic substances.

Effect of hypochlorite-based disinfectants on inactivation of murine norovirus and attempt to eliminate or prevent infection in mice by addition to drinking water.

We evaluated the in vitro efficacy of weak acid hypochlorous solution (WAHS) against murine norovirus (MNV) by plaque assay and compared the efficacy with diluted NaOCl (Purelox) and 70% ethanol. WAHS was as effective as 70% ethanol and diluted Purelox for 0.5-min reactions. For 0.5-min reactions in the presence of mouse feces emulsion, the efficacy of WHAS and 1:600 diluted Purelox was decreased, reducing the virus titers by 2.3 and 2.6 log10, respectively, while 70% ethanol reduced the titer by more than 5 log10. However, WAHS showed more than 5 log10 reductions for the 5-min reaction even in the presence of feces emulsion. Since WAHS showed enough efficacy in inactivating MNV in vitro, we tried to eliminate MNV from MNV-infected mice by substituting WAHS for their drinking water. However, MNV was found to be positive in feces of mice drinking WAHS by an RT-nested PCR and plaque assay. To investigate whether hypochlorite-based disinfectants could prevent infection of a mouse with MNV, WAHS or 1:6,000 diluted Purelox was substituted for the drinking water of mice for 2 or 4 weeks, and then the mice were placed in a cage with an MNV-infected mouse. The supply of disinfectants was continued after cohabitation, but MNV was detected in the feces of all the mice at 1 week after cohabitation. In this study, we tried to eliminate and prevent MNV infection from mice by supplying hypochlorite-based disinfectants as an easy and low-cost method. Unfortunately, drinking disinfectants was ineffective, so it is important to keep the facility environment clean by use of effective disinfectants. Also, animals introduced into facilities should be tested as MNV free by quarantine and periodically confirmed as MNV free by microbiological monitoring.

Isolation and characterization of toxigenic Corynebacterium ulcerans from 2 closed colonies of cynomolgus macaques (Macaca fascicularis) in Japan.

The aim of the present study was to determine the prevalence of infection by toxigenic Corynebacterium ulcerans in cynomolgus macaques (Macaca fascicularis) housed in an animal facility in Japan. Samples from the pharynges of animals from 2 closed colonies (colony A, n = 47; colony B, n = 21) were cultured. C. ulcerans grew from 43% and 47% of the samples from colonies A and B, respectively. The toxigenicity of these isolates was assessed by using PCR analysis for the diphtheria toxin gene and the Elek test and Vero cytotoxicity assay to detect diphtheria toxin. The proportion of macaques harboring toxigenic C. ulcerans was 6% in colony A and 29% in colony B. Analysis of diphtheria antitoxin neutralization titers in the sera revealed that 23% and 33% of macaques from colonies A and B, respectively, had a history of infection with toxigenic C. ulcerans. Pulsed-field gel electrophoresis of the toxigenic isolates showed that all of those recovered from macaques in colony B showed an identical genotype, suggesting that transmission of the organism occurred within the colony. However, isolates from colony A macaques showed 3 different genotypes, one of which was identical to the isolate from colony B. Additional studies evaluating the prevalence and transmission of toxigenic C. ulcerans within colonies of nonhuman primates are necessary to help control the spread of the infection. The current study is the first description of the isolation and characterization of toxigenic C. ulcerans from nonhuman primates in Japan.

Role of mouse hepatitis virus (MHV) receptor murine CEACAM1 in the resistance of mice to MHV infection: studies of mice with chimeric mCEACAM1a and mCEACAM1b.

Although most inbred mouse strains are highly susceptible to mouse hepatitis virus (MHV) infection, the inbred SJL line of mice is highly resistant to its infection. The principal receptor for MHV is murine CEACAM1 (mCEACAM1). Susceptible strains of mice are homozygous for the 1a allele of mCeacam1, while SJL mice are homozygous for the 1b allele. mCEACAM1a (1a) has a 10- to 100-fold-higher receptor activity than does mCEACAM1b (1b). To explore the hypothesis that MHV susceptibility is due to the different MHV receptor activities of 1a and 1b, we established a chimeric C57BL/6 mouse (cB61ba) in which a part of the N-terminal immunoglobulin (Ig)-like domain of the mCeacam1a (1a) gene, which is responsible for MHV receptor function, is replaced by the corresponding region of mCeacam1b (1b). We compared the MHV susceptibility of these chimeric mice to that of SJL and B6 mice. B6 mice that are homozygous for 1a are highly susceptible to MHV-A59 infection, with a 50% lethal dose (LD(50)) of 10(2.5) PFU, while chimeric cB61ba mice and SJL mice homozygous for 1ba and 1b, respectively, survived following inoculation with 10(5) PFU. Unexpectedly, cB61ba mice were more resistant to MHV-A59 infection than SJL mice as measured by virus replication in target organs, including liver and brain. No infectious virus or viral RNA was detected in the organs of cB61ba mice, while viral RNA and infectious virus were detected in target organs of SJL mice. Furthermore, SJL mice produced antiviral antibodies after MHV-A59 inoculation with 10(5) PFU, but cB61ba mice did not. Thus, cB61ba mice are apparently completely resistant to MHV-A59 infection, while SJL mice permit low levels of MHV-A59 virus replication during self-limited, asymptomatic infection. When expressed on cultured BHK cells, the mCEACAM1b and mCEACAM1ba proteins had similar levels of MHV-A59 receptor activity. These results strongly support the hypothesis that although alleles of mCEACAM1 are the principal determinants of mouse susceptibility to MHV-A59, other as-yet-unidentified murine genes may also play a role in susceptibility to MHV.

Colorimetric method for enzymatic screening assay of ATP using Fe(III)-xylenol orange complex formation.

In hygiene management, recently there has been a significant need for screening methods for microbial contamination by visual observation or with commonly used colorimetric apparatus. The amount of adenosine triphosphate (ATP) can serve as the index of a microorganism. This paper describes the development of a colorimetric method for the assay of ATP, using enzymatic cycling and Fe(III)-xylenol orange (XO) complex formation. The color characteristics of the Fe(III)-XO complexes, which show a distinct color change from yellow to purple, assist the visual observation in screening work. In this method, a trace amount of ATP was converted to pyruvate, which was further amplified exponentially with coupled enzymatic reactions. Eventually, pyruvate was converted to the Fe(III)-XO complexes through pyruvate oxidase reaction and Fe(II) oxidation. As the assay result, yellow or purple color was observed: A yellow color indicates that the ATP concentration is lower than the criterion of the test, and a purple color indicates that the ATP concentration is higher than the criterion. The method was applied to the assay of ATP extracted from Escherichia coli cells added to cow milk.

Detection of the antibody to lymphocytic choriomeningitis virus in sera of laboratory rodents infected with viruses of laboratory and newly isolated strains by ELISA using purified recombinant nucleoprotein.

An enzyme-linked immunosorbent assay (ELISA) was developed to detect the antibody against lymphocytic choriomeningitis virus (LCMV) in sera of laboratory animals. In this ELISA system, LCMV-nucleoprotein (NP) expressed by recombinant baculovirus and purified with high molar urea was used as the antigen. Sera from laboratory animals experimentally infected with the Armstrong strain or the newly isolated M1 strain of LCMV were examined to detect anti-LCMV antibody by the ELISA system, and the reactivity was compared with that of IFA test. Regardless of LCMV strain, all the sera of adult mice infected with LCMV were positive with very high optical density (OD). Also, the sera from mice neonatally infected with LCMV M1 strain were positive with slightly lower OD than adult mice. In contrast, all the sera of uninfected mice were negative to LCMV-NP antigen. Similarly, anti-LCMV antibodies were detected in all the sera of hamsters, mastomyses, and gerbils infected with the LCMV Armstrong strain. The results of the ELISA were in complete agreement with those of IFA, and indicate the high sensitivity and specificity of the ELISA system in the detection of anti-LCMV antibody. Because this ELISA system does not require handling infectious LCMV in the course of the antigen preparation and serological assay, there is no risk of contamination in the laboratory or nearby animal facility. In addition, by using negative control antigen in parallel with positive antigen in ELISA, we can exactly check the LCMV contamination in laboratory animals.

Lymphocytic choriomeningitis infection undetected by dirty-bedding sentinel monitoring and revealed after embryo transfer of an inbred strain derived from wild mice.

Persistent LCMV infection in wild-derived MAI/Pas mice housed under conventional conditions remained undetected for a decade, despite periodic health monitoring using dirty-bedding sentinels. When MAI/Pas mice were rederived by embryo transfer, recipient mothers produced antiLCMV antibodies, which first revealed the presence of the virus in the colony. Before this information was obtained, MAI/Pas mice had been shipped to another facility, undergone cesarean rederivation there, and been introduced into the recipient barrier. The foster mothers of rederived pups were LCMV-negative according to enzyme-linked immunosorbent assay, but sera of both cesarean-rederived MAI/Pas mice and their foster mothers were positive for LCMV infection by immunofluorescent assay (IFA). LCMV was isolated from the MAI/Pas mice, and its genomic RNA was sequenced. Examination of animal technicians in contact with LCMV-infected mice and of other mouse samples by IFA or a reverse transcriptase-polymerase chain reaction test (or both) revealed that neither the workers nor other animals had been infected with LCMV. Experimental data showed that LCMV transmission from persistently infected mice to naïve ones occurred only after direct contact of animals housed in the same cage. This experience demonstrates the importance of careful viral monitoring in the transfer of laboratory rodents between institutions, the limitation of dirty-bedding sentinels for detection of LCMV infection, and the inadequacy of cesarean rederivation for elimination of enzootic LCMV infection. 111

Detection of 14 alleles derived from the MHC class I A locus in cynomolgus monkeys.

A basic understanding of the major histocompatibility complex (MHC) class I, which, together with T-cell receptors, is a key player in antigen recognition by cytotoxic T lymphocytes, is necessary to study the cellular immune response to intracellular pathogens. The MHC has hardly been reported in cynomolgus monkeys ( Macaca facicularis), although cynomolgus monkeys have been frequently used as the surrogate animal model. We attempted to determine the nucleotide sequences of the MHC class I A locus of cynomolgus monkeys ( Mafa-A) and eventually 34 independent sequences of Mafa-A were obtained from 29 cynomolgus monkeys. These 34 sequences were classified into 14 Mafa-A alleles according to the results of phylogenetic analyses using the neighbor-joining method. One to three Mafa-A alleles were obtained from a single animal. We also tried to establish a multiplex PCR-SSP method for convenient typing of Mafa-A alleles. cDNA from a family of cynomolgus monkeys, which is composed of four sirs and four dams, were examined by multiplex PCR-SSP. The result of multiplex PCR-SSP showed that an individual cynomolgus monkey had two or three Mafa-A alleles, suggesting that the A locus of cynomolgus monkeys might be duplicated.

Detection of MHV-RNAs in mouse intestines and in filter dust in mouse room ventilation duct by a modified RT-nested PCR.

We applied RT-nested PCR for the detection of MHV genomic RNA in a modified manner to obtain RNA from the intestines of mice and from filter dust in the ventilation ducts of the room in which a contaminated mouse colony was kept. Since the sequences of MHV-RNA that were extracted from the intestine of a serologically MHV-positive mouse in room No. 2 (MS2) and from the filter dust in a ventilation duct in the same room (FD2) were identical, amplified product from filter dust was demonstrated to come from the MHV contaminated room. Furthermore, sequences of FD2 and of filter dust from another contaminated mouse room, No. 7 (FD7) showed 38 nucleotide exchanges among 368-bp (10.3%), suggesting that two different MHV strains were contaminating our facilities. SSCP analysis of Dra I-digested PCR product of 393 bp also showed different patterns in FD2 and FD7 samples.

[A surgical case of recurrent gastric cancer successfully treated by TS-1 plus low-dose consecutive administration of CDDP following TS-1 monotherapy].

The patient was a 65-year-old woman who was discovered to have recurrent gastric cancer involving para-aortic lymph nodes 21 months after postoperative low-dose FP therapy for gastric cancer. The recurrent cancer was judged to be unresectable, and TS-1 chemotherapy (one course consisting of 4-week administration of 100 mg/day and a 2-week withdrawal period) was performed. Although a complete response occurred in the para-aortic lymph nodes and a partial response in the residual stomach (i.e., only a shallow erosion remained) after the end of course 2, histological examination showed "no change". The following regimen was therefore used in courses 3 and 4: 6-day administration of CDDP at a dose of 15 mg/body (10 mg/m2)/day the first week, concomitant administration of 90 mg/body (60 mg/m2) of CDDP plus 100 mg/day of TS-1 the next three weeks, and two-week withdrawal of chemotherapy. Since down-staging was observed at the completion of course 4, total gastrectomy of the residual stomach was performed. No noteworthy adverse reactions to chemotherapy were observed, and good patient QOL (e.g., appetite) was achieved. Based on these findings, this chemotherapy regimen appears to be an effective treatment modality for far advanced gastric cancer, particularly involving the abdominal para-aortic lymph nodes.

Oxo-Centered Mixed-Ligand Triruthenium Complexes Having Redox-Active N-Methyl-4,4'-bipyridinium Ions (mbpy(+)). Reversible Multistep Electrochemical Properties of [Ru(III)(2)Ru(II)(&mgr;(3)-O)(&mgr;-CH(3)CO(2))(6)(mbpy(+))(2)(CO)](2+) and [Ru(III)(3)(&mgr;(3)-O)(&mgr;-CH(3)CO(2))(6)(mbpy(+))(2)(L)](3+) (L = H(2)O and N-Heterocyclic Ligands).

A new series of oxo-centered acetate-bridged triruthenium comlexes having two redox-active N-methyl-4,4'-bipyridinium ions (mbpy(+)) have been prepared, and their reversible multistep and multielectron electrochemical properties are reported: [Ru(III)(2)Ru(II)(&mgr;(3)-O)(&mgr;-CH(3)CO(2))(6)(mbpy(+))(2)(CO)](2+) and [Ru(III)(3)(&mgr;(3)-O)(&mgr;-CH(3)CO(2))(6)(mbpy(+))(2)(L)](3+) (L = H(2)O, pyrazine (pz), pyridine (py), imidazole (Him), and 4-(dimethylamino)pyridine (dmap)). Among these series, the CO complex, [Ru(III)(2)Ru(II)(&mgr;(3)-O)(&mgr;-CH(3)CO(2))(6)(mbpy(+))(2)(CO)](ClO(4))(2).2DMF (1b.2DMF) was structurally characterized by X-ray crystallography. 1b.2DMF crystallizes in the monoclinic space group P2(1)/m (No. 11) with a = 8.740(6) Å, b = 32.269(6) Å, c = 10.276(4) Å, beta = 103.37(5) degrees, V = 2820(2) Å(3), Z = 2, d(calcd) = 1.636 g cm(-)(3), and R = 0.071 (R(w) = 0.074) for 5277 independent reflections (|F(o)| > 3sigma(|F(o)|). The (CO)Ru.Ru distance (3.410(2) Å) is appreciably longer than the other Ru.Ru distance (3.276(2) Å), indicating that the trinuclear core is in the valence-trapped Ru(III)(2)Ru(II)(CO) oxidation state. The cyclic voltammogram of [Ru(III)(2)Ru(II)(&mgr;(3)-O)(&mgr;-CH(3)CO(2))(6)(mbpy(+))(2)(CO)](PF(6))(2) (1a) shows a total of seven reversible one-electron redox steps at E(1/2) = +0.90, +0.26, -1.07, -1.17, -1.56, -1.97, and -2.32 V and one irreversible step at E(pc) = -2.99 V vs Fc/Fc(+) in a 0.1 M [(n-C(4)H(9))(4)N]PF(6)-CH(3)CN solution (M = mol dm(-)(3)). All of the waves are clearly assignable to the triruthenium "Ru(3)(&mgr;(3)-O)" core-based or mbpy(+) ligand-based processes. The splitting of each ligand-based redox processes (mbpy(+)/mbpy(*) and mbpy(*)/mbpy(-)) into two one-electron steps indicates that electronic interactions between two terminal ligands occur through the triruthenium cluster core. Other mixed-ligand Ru(III)(3) analogs also show multistep redox behavior involving a total of eight or nine electrons. While the extent of interactions between ligands is much smaller than that found in the CO complex, it is systematically changed by the nature of L; with more basic L, interactions between two mbpy(+) ligands become larger.