Model-based control system design to manage process parameters in mammalian cell culture for biopharmaceutical manufacturing.

To enhance the robustness and flexibility of biopharmaceutical manufacturing, a paradigm shift toward methods of continuous processing, such as perfusion, and fundamental technologies for high-throughput process development are being actively investigated. The continuous upstream process must establish an advanced control strategy to ensure a "State of Control" before operation. Specifically, feedforward and feedback control must address the complex fluctuations that occur during the culture process and maintain critical process parameters in appropriate states. However, control system design for industry-standard mammalian cell culture processes is still often performed in a laborious trial-and-error manner. This paper provides a novel control approach in which controller specifications to obtain desired control characteristics can be determined systematically by combining a culture model with control theory. In the proposed scheme, control conditions, such as PID parameters, can be specified mechanistically based on process understanding and control requirements without qualitative decision making or specific preliminary experiments. The effectiveness of the model-based control algorithm was verified by control simulations assuming perfusion Chinese hamster ovary culture. As a tool to assist in the development of control strategies, this study will reduce the high operational workload that is a serious problem in continuous culture and facilitate the digitalization of bioprocesses.

Gelation of konjac glucomannan by acetylmannan esterases from Aspergillus oryzae.

Konjac glucomannan (KGM) is a principal component of the gelatinous food Konjac. Konjac production through alkali treatment releases an undesirable amine-odor. Two acetylesterases (AME1 and AME2) active against konjac glucomannan (polymer or oligomer) were purified from the supernatant of Aspergillus oryzae RIB40 culture. We cloned the genes encoding AME1 and AME2 based on the genomic information of A. oryzae, constructed their expression systems in A. oryzae, and obtained the recombinant enzymes (rAME1 and rAME2). rAME1 did not act on the KGM polymer but only on the KGM oligomer, releasing approximately 60% of the acetic acid in the substrate. However, rAME2 was active against both KGM substrates, releasing approximately 80% and 100% of acetic acid from the polymer and oligomer, respectively. Both enzymes were active against xylan and exhibited a trace activity on ethyl ferulate. The acetyl group position specificities of both enzymes were analyzed via heteronuclear single quantum correlation NMR using oligosaccharides of glucomannan prepared from Aloe vera (AGM), which has a higher acetyl group content than KGM. rAME1 acted specifically on single-substituted acetyl groups and not on double-substituted ones. In contrast, rAME2 appeared to act on all the acetyl groups in AGM. Treatment of 3% KGM with rAME2 followed by heating to 90 °C resulted in gel formation under weakly acidic conditions. This is the first study to induce gelation of KGM under these conditions. A comparison of the breaking and brittleness properties of gels formed by alkaline and enzymatic treatments revealed similar texture of the two gels. Furthermore, scanning electron microscopy of the surface structure of both gels revealed that both formed a fine mesh structure. Our findings on enzymatic gelation of KGM should lead to the development of new applications in food manufacturing industry.

The mycobacterial desaturase DesA2 is associated with mycolic acid biosynthesis.

Mycolic acids are critical for the survival and virulence of Mycobacterium tuberculosis, the causative agent of tuberculosis. Double bond formation in the merochain of mycolic acids remains poorly understood, though we have previously shown desA1, encoding an aerobic desaturase, is involved in mycolic acid desaturation. Here we show that a second desaturase encoded by desA2 is also involved in mycolate biosynthesis. DesA2 is essential for growth of the fast-growing Mycobacterium smegmatis in laboratory media. Conditional depletion of DesA2 led to a decrease in mycolic acid biosynthesis and loss of mycobacterial viability. Additionally, DesA2-depleted cells also accumulated fatty acids of chain lengths C19-C24. The complete loss of mycolate biosynthesis following DesA2 depletion, and the absence of any monoenoic derivatives (found to accumulate on depletion of DesA1) suggests an early role for DesA2 in the mycolic acid biosynthesis machinery, highlighting its potential as a drug target.

MadR mediates acyl CoA-dependent regulation of mycolic acid desaturation in mycobacteria.

Mycobacterium tuberculosis has a lipid-rich cell envelope that is remodeled throughout infection to enable adaptation within the host. Few transcriptional regulators have been characterized that coordinate synthesis of mycolic acids, the major cell wall lipids of mycobacteria. Here, we show that the mycolic acid desaturase regulator (MadR), a transcriptional repressor of the mycolate desaturase genes desA1 and desA2, controls mycolic acid desaturation and biosynthesis in response to cell envelope stress. A madR-null mutant of M. smegmatis exhibited traits of an impaired cell wall with an altered outer mycomembrane, accumulation of a desaturated α-mycolate, susceptibility to antimycobacterials, and cell surface disruption. Transcriptomic profiling showed that enriched lipid metabolism genes that were significantly down-regulated upon madR deletion included acyl-coenzyme A (aceyl-CoA) dehydrogenases, implicating it in the indirect control of ß-oxidation pathways. Electromobility shift assays and binding affinities suggest a unique acyl-CoA pool-sensing mechanism, whereby MadR is able to bind a range of acyl-CoAs, including those with unsaturated as well as saturated acyl chains. MadR repression of desA1/desA2 is relieved upon binding of saturated acyl-CoAs of chain length C16 to C24, while no impact is observed upon binding of shorter chain and unsaturated acyl-CoAs. We propose this mechanism of regulation as distinct to other mycolic acid and fatty acid synthesis regulators and place MadR as the key regulatory checkpoint that coordinates mycolic acid remodeling during infection in response to host-derived cell surface perturbation.

Structural and functional analysis of gum arabic l-rhamnose-α-1,4-d-glucuronate lyase establishes a novel polysaccharide lyase family.

Gum arabic (GA) is widely used as an emulsion stabilizer and coating in several industrial applications, such as foods and pharmaceuticals. GA contains a complex carbohydrate moiety, and the nonreducing ends of the side chains are often capped with l-rhamnose; thus, enzymes that can remove these caps are promising tools for the structural analysis of the carbohydrates comprising GA. In this study, GA-specific l-rhamnose-α-1,4-d-glucuronate lyase from the fungus Fusarium oxysporum 12S (FoRham1) was cloned and characterized. FoRham1 showed the highest amino acid sequence similarity with enzymes belonging to the glycoside hydrolase family 145; however, the catalytic residue on the posterior pocket of the ß-propeller fold protein was not conserved. The catalytic residues of FoRham1 were instead conserved with ulvan lyases belonging to polysaccharide lyase family 24. Kinetic analysis showed that FoRham1 has the highest catalytic efficiency for the substrate α-l-rhamnose-(1→4)-d-glucuronic acid. The crystal structures of ligand-free and α-l-rhamnose-(1→4)-d-glucuronic acid -bound FoRham1 were determined, and the active site was identified on the anterior side of the ß-propeller. The three-dimensional structure of the active site and mutagenesis analysis revealed the detailed catalytic mechanism of FoRham1. Our findings offer a new enzymatic tool for the further analysis of the GA carbohydrate structure and for elucidating its physiological functions in plants. Based on these results, we renamed glycoside hydrolase family 145 as a new polysaccharide lyase family 42, in which FoRham1 is included.

Biochemical and structural characterization of a novel 4-O-α-l-rhamnosyl-ß-d-glucuronidase from Fusarium oxysporum.

In this study, we have isolated the novel enzyme 4-O-α-l-rhamnosyl-ß-d-glucuronidase (FoBGlcA), which releases α-l-rhamnosyl (1→4) glucuronic acid from gum arabic (GA), from Fusarium oxysporum 12S culture supernatant, and for the first time report an enzyme with such catalytic activity. The gene encoding FoBGlcA was cloned and expressed in Pichia pastoris. When GA was subjected to the recombinant enzyme, > 95% of the l-rhamnose (Rha) and d-glucuronic acid in the substrate were released, which indicates that almost all Rha binds to the glucuronic acid at the end of the GA side chains. The crystal structure of FoBGlcA was determined using a single-wavelength anomalous dispersion at 1.51 Å resolution. FoBGlcA consisted of an N-terminal (ß/α)8 -barrel domain and a C-terminal antiparallel ß-sheet domain. This configuration is characteristic of glycoside hydrolase (GH) family 79 proteins. A structural similarity search showed that FoBGlcA mostly resembled GH79 ß-d-glucuronidase (AcGlcA79A) of Acidobacterium capsulatum; however, the root-mean-square deviation value was 3.2 Å, indicating that FoBGlcA has a high structural divergence. FoBGlcA had a low sequence identity with AcGlcA79A (19%) and differed from other GH79 ß-glucuronidases. The structures of FoBGlcA and AcGlcA79A also differed in terms of the loop structure location near subsite -2 of their catalytic sites, which may account for the unique substrate specificity of FoBGlcA. The amino acid residues involved in the catalytic activity of this enzyme were determined by evaluating the activity levels of various mutant enzymes based on the crystal structure analysis of the FoBGlcA reaction product complex. DATABASE: Atomic coordinates and structure factors (codes 7DFQ and 7DFS) have been deposited in the Protein Data Bank (http://wwpdb.org/).

Analysis of phosphatidylethanolamine, phosphatidylcholine, and plasmalogen molecular species in food lipids using an improved 2D high-performance liquid chromatography system.

Phospholipids are an important class of lipids in cell membranes and food. Several high-performance liquid chromatography (HPLC) methods have been developed to analyze phospholipids at the molecular species level. We developed a two-dimensional HPLC system with a charged aerosol detector and mass spectrometry (MS) to analyze phosphatidylethanolamine (PE), phosphatidylcholine (PC), and their plasmalogens (pls) extracted from food materials. Accordingly, the phospholipid molecular species can be analyzed in a single step despite using smaller samples. We confirmed that chromatogram peaks from soybean lecithin are mostly baseline separated, assigned, and quantified (24 molecular species for PE and 27 for PC). In addition, it was confirmed that chromatograms of lipids extracted from chicken breast meat include plasmalogen peaks. The PE fraction in lipids extracted from chicken breast meat contained 17 types of ethanolamine plasmalogens, corresponding to approximately 57% of the total by weight. The PC fraction contained only four choline plasmalogens, corresponding to approximately 11% of the total weight. The composition of the pls-PC molecular species differed from that of pls-PEs. The polyunsaturated fatty acids connected at the sn-2 positions of the pls-PEs consisted of 20.5% 20:4 fatty acid and were independent of the carbon chain at the sn-1 position. However, the 18:1 fatty acid at the sn-2 position was dependent on the carbon chain at the sn-1 position.

Clinical validity of the Japanese version of WAIS-III short forms: Adaptation for patients with mild neurocognitive disorder and dementia.

We investigated the Japanese WAIS-III short form utility in mild neurocognitive disorder and dementia. Our sample consisted of 108 old patients (ages: 65-89; mean age = 78.3). Fifteen short forms (SFs) and full-scale (FS) IQs were compared. The SFs included Dyads (SF1, SF2), Triads (SF3), Tetrads (SF4, SF5, SF6, SF7), Pentad (SF8), Six-subtest (SF9), Seven-subtests (SF10(a)(b), SF11(a)(b), SF12), and Nine-subtest (SF13). Correlations between SFIQs and FSIQ were all significant. Significant differences also were found in paired t-test between FSIQ and 5 SFIQs (SF2: t = -4.16, SF5: t = -7.06, SF7; t = 2.59, SF10(a): t = 2.56, SF12: t = -4.82; p < .05). On the point of clinical accuracy, two SFs led to an appropriate estimated IQ (SF11(a): 84.3%, SF13: 91.7%; within 95% confidence interval and 2 standard error of measurements of FSIQ). However, SF13 was considered to still have a long administration time. The present results suggest that SF11(a) could be the most useful to estimate IQ for Japanese speaking patients with mild neurocognitive disorder and dementia. SF11(a) consists of seven subtests of Similarities, Arithmetic, Digit Span, Information, Picture Completion, Digit Symbol-Coding, and Matrix Reasoning (Ryan & Ward, 1999), and the formula (Axelrod et al., 2001) should be adopted to convert scaled scores into estimated IQ scores.

Sense of coherence and personality traits related to depressive state.

Aims. The current study aims to examine the influence of job stress, SOC, and personality traits on depressive state. Methods. A self-reported survey was conducted among 347 female nurses in a general hospital. Job stress was measured using the Japanese version of the Brief-Job Stress Questionnaire scale. Depressive state was assessed by the K6 scale. We used 13-item SOC scale. Personality traits were assessed by the Japanese version of Ten-Item Personality Inventory. Multiple liner regression analyses were conducted to examine predictors that significantly affect depressive state. Results. Job and life satisfaction and SOC negatively related to the depressive state (ß = -0.76, P < 0.01; ß = -0.18, P < 0.001, resp.) while neuroticism was positively correlated (ß = 0.49, P < 0.001). Also, intrinsic rewards tended to negatively relate (ß = -0.80, P < 0.1). Conclusions. From a practical perspective, the possible influence of SOC and neurotic personality on depressive state should be considered for health care professionals.

Relationship between depressive state, job stress, and sense of coherence among female nurses.

BACKGROUND: People with a strong sense of coherence (SOC) have a high ability to cope with stress and maintain good physical and mental health. AIMS: The aim of this study was to investigate the relationship between depressive state, job stress, and SOC among nurses in a Japanese general hospital. MATERIALS AND METHODS: A self-reporting survey was conducted among 348 female nurses in a general hospital. Job stress was measured using the Japanese version of the effort-reward imbalance (ERI) scale. Depressive state was assessed by the K6 scale. SOC was assessed with the SOC scale, which includes 29 items. Stepwise multiple regression analysis was conducted to examine factors that significantly affect depressive state. RESULTS: SOC, over-commitment, effort-esteem ratio, and age were significantly correlated with the depressive state (ß = -0.46, P < 0.001; ß = 0.27, P < 0.001; ß = 0.16, P < 0.001; ß = -0.10, P < 0.001, respectively). CONCLUSIONS: SOC may have a major influence on the depressive state among female nurses in a Japanese general hospital. From a practical perspective, health care professionals should try to enhance the SOC of nurses.

[The specificity of cognitive reactivity in recurrent major depressive episodes].

The present study examined cognitive vulnerability to relapses of depression by clarifying the characteristics of "cognitive reactivity" in people with recurrent major depressive episodes. Study 1-1 and 1-2 developed a Japanese version of the Leiden Index of Depression Sensitivity-Revised (LEIDS-R), which assessed cognitive reactivity, and evaluated the reliability and validity of the scale. Study 2 examined the characteristics of cognitive reactivity which differentiate people with recurrent major depressive episodes from people with a single episode or none. The Japanese version of the LEIDS-R was shown to have reasonable reliability and validity. Participants with recurrent major depressive episodes showed more repetitive thoughts about negative issues and avoidance from internal and external aversive events when depressive mood was induced, compared to participants with only a single episode of depression. These results suggest that the characteristics of cognitive reactivity are important considerations for preventing relapse of depression.

Relationship between job stress, temperament and depressive symptoms in female nurses.

OBJECTIVES: A casual relationship between temperament, job stress and depressive symptoms has not been established yet. The purpose of this study was to assess the relationships between job stress, temperament and depressive symptoms in female nurses at a Japanese general hospital. MATERIAL AND METHODS: A self-report survey was conducted among 706 nurses. We measured job stress, temperament, and depressive symptoms using the Brief-Job Stress Questionnaire, the TEMPS-A and a screening scale of items from the Ministry of Health, Labour and Welfare of Japan. In order to examine the causal relationship between the measures the stepwise multiple regression and path analyses were used. RESULTS: Depressive symptoms were modestly correlated with job stress (γ = -0.23-0.30). Except for hyperthymic temperament measures, the correlations between depressive symptoms and temperament types were significant and moderate (γ = 0.36-0.50). Overtime, job control as well as depressive and cyclothymic types of temperament were significantly correlated with depressive symptoms (ß = 0.15, p < 0.05; ß = 0.19, p < 0.01; ß = 0.26, p < 0.001; ß = 0.32, p < 0.001, respectively). Path-analysis revealed that depressive and cyclothymic types of temperament influenced depressive symptoms both directly (ß = 0.67, p < 0.001) and indirectly via job stress (ß = 0.35, p < 0.001 from temperament to job stress; ß = 0.20, p < 0.05 from job stress to depressive symptoms). Irritable and anxious types of temperament and quantitative job overload did not contribute to the path-analytic model. CONCLUSIONS: Health care professionals should consider temperament, especially depressive and cyclothymic types, in order to help employees cope better with job stress factors. We need further research about the effective intervention to help employees better cope with their job stress.

Hypolipidemic effects of starch and γ-oryzanol from wx/ae double-mutant rice on BALB/c.KOR-Apoe(shl) mice.

waxy/amylose-extender (wx/ae) double-mutant japonica rice (Oryza sativa L.) produces resistant starch (RS) and a large amount of γ-oryzanol. Our previous study has shown the hypolipidemic effect of wx/ae brown rice on mice. To identify the functional constituents of the hypolipidemic activity in wx/ae rice, we prepared pure wx/ae starch and γ-oryzanol from wx/ae rice and investigated their effect on the lipid metabolism in BALB/c.KOR/Stm Slc-Apoe(shl) mice. The mice were fed for 3 weeks a diet containing non-mutant rice starch, non-mutant rice starch plus γ-oryzanol, wx/ae starch, or wx/ae starch plus γ-oryzanol. γ-Oryzanol by itself had no effect on the lipid metabolism, and wx/ae starch prevented an accumulation of triacylglycerol (TAG) in the liver. Interestingly, the combination of wx/ae starch plus γ-oryzanol not only prevented a TAG accumulation in the liver, but also partially suppressed the rise in plasma TAG concentration, indicating that wx/ae starch and γ-oryzanol could have a synergistic effect on the lipid metabolism.

Wx/ae double-mutant brown rice prevents the rise in plasma lipid and glucose levels in mice.

A wx/ae double-mutant rice is generated by crossing waxy mutant and amylose-extender mutant in rice. Wx/ae brown rice contains highly beneficial nutrients for lipid and glucose metabolism, including resistant starch, dietary fiber, and γ-oryzanol, when compared to Koshihikari brown rice, the non-waxy japonica cultivar. To examine the effects of wx/ae brown rice on glucose and lipid metabolism, type 2 diabetic NSY/Hos mice were fed a high-fat diet containing 25% of wx/ae brown rice or Koshihikari brown rice for 10 weeks. The plasma total cholesterol, non-high-density lipoprotein cholesterol, triglyceride, and non-esterified fatty acid levels of the wx/ae group were significantly lower than those of the Koshihikari group. Moreover, the fasting blood glucose level and pathological score of glycosuria of the wx/ae group were also significantly lower than those of the Koshihikari group. These results indicate that wx/ae brown rice has the potential to prevent the rise in plasma lipid and glucose levels.

Structure, physical, and digestive properties of starch from wx ae double-mutant rice.

Amylopectin is the principal component of starch. The amylose extender (ae) gene encodes the starch-branching enzyme IIb, which is critical in determining the fine structure of endosperm starch. To determine the relationship between the fine structure of amylopectin and its physical properties, rice mutant lines defective in the ae function with altered fine structure of amylopectin and in combination with the waxy (wx) background were selected for comparative studies with primary wild-type and ae starches. The ae mutant endosperms accumulated a high amylose content starch with long amylopectin chains. The ae and wx ae starches showed no significant difference in the unit chain-length distribution of amylopectin and starch granule morphology. The wx ae starch displayed a higher pasting temperature and higher peak viscosity. The gelatinization peak temperatures of the wx, ae, and wx ae starches were 2.2, 13.1, and 17.1 degrees C higher, respectively, than that of the wild-type starch, and the wx ae starch showed a retrogradation peak with a shorter cooling period than that of ae starch. The raw ae and wx ae starches were almost indigestible by alpha-amylase in vitro. Rats fed the wx ae starch showed slowly increasing blood glucose at a lower level than the rats fed the wx or wild-type starch. These results indicate that the primary structure of the rice wx ae amylopectin with enriched long chains changes the granular structure of the starch, including its crystal structure, and results in resistance to in vitro or in vivo degradation.

Rapid species identification of cooked poisonous mushrooms by using real-time PCR.

Species-specific identification of the major cooked and fresh poisonous mushrooms in Japan was performed using a real-time PCR system. Specific fluorescence signals were detected, and no nonspecific signals were detected. Therefore, we succeeded in developing a species-specific test for the identification of poisonous mushrooms within 1.5 h.

A two-factor structure for the Schedule for the Deficit Syndrome in schizophrenia.

The deficit subtype is hypothesized to constitute a homogeneous subgroup of schizophrenia patients. The Schedule for the Deficit Syndrome (SDS) was developed to categorize schizophrenia patients into deficit and non-deficit subtypes. Only one report, however, has suggested the use of a two-factor SDS. The present study used a two-factor SDS and examined the relationships between the factors and the demographic and clinical variables in deficit-type schizophrenia patients. Principal component analysis was performed on data obtained from 70 schizophrenia patients with the deficit syndrome. The two-factor SDS was replicated. Factor 1, avolition, was significantly correlated only with the relational disorder domain, which consisted of two negative items from the Positive and Negative Syndrome Scale. Factor 2, poor emotional expression, was significantly correlated with both the negative symptom domain, which consisted of three negative items, and the relational disorder domain. Since the SDS has two factors, there appear to be different underlying processes for these two factors.

Kraepelinian subtype and deficit syndrome in chronic schizophrenia.

The present study examined the clinical overlap between the Kraepelinian and deficit subtypes of schizophrenia. A total of 103 patients with schizophrenia were divided into four groups on the basis of the presence or absence of the two classifications, and the demographic and clinical characteristics of the groups were statistically compared. There was a significant overlap of Kraepelinian and deficit status, but nevertheless the retention of both classifications appears to be justified.