GORAB scaffolds COPI at the trans-Golgi for efficient enzyme recycling and correct protein glycosylation

COPI is a key mediator of protein trafficking within the secretory pathway. COPI is recruited to the membrane primarily through binding to Arf GTPases, upon which it undergoes assembly to form coated transport intermediates responsible for trafficking numerous proteins, including Golgi-resident enzymes. Here, we identify GORAB, the protein mutated in the skin and bone disorder gerodermia osteodysplastica, as a component of the COPI machinery. GORAB forms stable domains at the trans-Golgi that, via interactions with the COPI-binding protein Scyl1, promote COPI recruitment to these domains. Pathogenic GORAB mutations perturb Scyl1 binding or GORAB assembly into domains, indicating the importance of these interactions. Loss of GORAB causes impairment of COPI-mediated retrieval of trans-Golgi enzymes, resulting in a deficit in glycosylation of secretory cargo proteins. Our results therefore identify GORAB as a COPI scaffolding factor, and support the view that defective protein glycosylation is a major disease mechanism in gerodermia osteodysplastica.Peer reviewe

Guidelines and Recommendations for Laboratory Analysis in the Diagnosis and Management of Diabetes Mellitus

Background: Multiple laboratory tests are used to diagnose and manage patients with diabetes mellitus. The quality of the scientific evidence supporting the use of these tests varies substantially. Approach: An expert committee compiled evidence-based recommendations for the use of laboratory testing for patients with diabetes. A new system was developed to grade the overall quality of the evidence and the strength of the recommendations. Draft guidelines were posted on the Internet and presented at the 2007 Arnold O. Beckman Conference. The document was modified in response to oral and written comments, and a revised draft was posted in 2010 and again modified in response to written comments. The National Academy of Clinical Biochemistry and the Evidence-Based Laboratory Medicine Committee of the American Association for Clinical Chemistry jointly reviewed the guidelines, which were accepted after revisions by the Professional Practice Committee and subsequently approved by the Executive Committee of the American Diabetes Association. Content: In addition to long-standing criteria based on measurement of plasma glucose, diabetes can be diagnosed by demonstrating increased blood hemoglobin A$_{1c}$ (HbA$_{1c}$) concentrations. Monitoring of glycemic control is performed by self-monitoring of plasma or blood glucose with meters and by laboratory analysis of HbA$_{1c}$. The potential roles of noninvasive glucose monitoring, genetic testing, and measurement of autoantibodies, urine albumin, insulin, proinsulin, C-peptide, and other analytes are addressed. Summary: The guidelines provide specific recommendations that are based on published data or derived from expert consensus. Several analytes have minimal clinical value at present, and their measurement is not recommended

On-line monitoring of Phaffia rhodozyma fed-batch process with in situ dispersive raman spectroscopy

Since the yeast Phaffia rhodozyma was first described some 35 yr ago, there has been significant interest in the development of com. processes to exploit its ability to produce carotenoids (.apprx.80% astaxanthin). However, the optimal conditions for carotenoid prodn. are not well understood. A key limitation has been the lack of an appropriate sensor for online carotenoid quantification. In this study, an in situ Raman spectroscopy probe was used to monitor intracellular carotenoid prodn. for three consecutive P. rhodozyma fed-batch expts. Raman spectroscopy is particularly well suited to the study of carotenoids due to a resonance effect, which greatly enhances the intensity of the three fundamental carotenoid bands, n1 (1513 cm-1, C=C stretch), n2 (1154 cm-1, C-C stretch), and n3 (1003 cm-1, CH3 rock). For all three cultures, the peak height of these bands was linearly correlated with intracellular carotenoid content (1 to 45 mg/L) to a precision of better than 5%, and the correlation from one expt. was directly applicable to others. [on SciFinder (R)

Adaptive control of a G. xylinus fed-batch fermentation using in situ mid-IR spectroscopy

Bacterial exopolysaccharides (EPS) have unique rheol. properties due to their high purity and regular structure. Thus, the food industry frequently uses EPS as thickening, gelling or stabilizing agents. The strain Gluconacetobacter xylinus I 2281 is capable of producing with a very high yield a new sol. EPS named gluconacetan, which is composed of rhamnose, glucose, mannose and glucuronic acid. Growth of these bacteria presents many features that represent a real challenge for both monitoring and control. High EPS concns. in the culture broth cannot be filtered and thus, classical online monitoring tools, such as \"Flow Injection Anal.\" or chromatog. methods, are no longer suitable to follow the fermn. Another problem is related to the ethanol concn. in the reactor. This compd. is the main substrate for biomass prodn. Cells convert it to acetate and metabolize this last to form biomass. When ethanol concn. is above a certain threshold, the enzymic system that digests acetate is inhibited. As a result, biomass growth is stopped and acetate accumulates in the reactor. It is thus necessary to maintain the ethanol concn. in the reactor at a very low concn. In this work fed-batch fermn. of G. xylinus was performed. A concd. ethanol soln. was used as the feed soln. A very simple and novel adaptive control strategy was used to maintain the acetate concn. in the reactor const. Consequently ethanol concn. was forced to remain very low. In situ MIR spectroscopy, a non-invasive optical sensor system that is insensitive to the viscosity changes in the broth was used to monitor simultaneously ethanol, acetate, ammonium, phosphates and fructose concns. Both the control strategy and the exptl. results will be presented. [on SciFinder (R)

Experimental versus simulated calibration mdels for in situ FTIR monitoring of bioprocesses

Over the past decade the interest for bioprocess monitoring using non-invasive IR spectroscopic sensors has increased considerably. This is mainly due to their rapid simultaneous multi-analyte detn. ability, in-situ sterilizability, and low need for maintenance during operation. Most of the components in culture media absorb in the MID-IR region. The complex spectra obtained can be deconvoluted using numerical methods such as Partial Least-Squares. In order to do this, a calibration model for each component needs to be generated. This model is based on an exptl. obtained calibration matrix obtained by taking the spectra of mixts. contg. different concns. of the pure components involved in the reaction. According to ASTM guidelines [1], the no. of different mixts. used in such calibration models must be at least 6 times the no. of absorbing components. This is usually very time-consuming for bioprocesses, since the no. of spectra that need to be collected is very large. One of the main advantages of using Mid-IR spectra compared to NIR is in the linear features the first present. According to Beer's law, it is possible to scale the spectrum of a pure component to obtain its spectra for different concns. Furthermore, the spectrum of a mixt. of components can be theor. achieved by adding up the spectrum of the different pure components involved. It is thus interesting to study if a calibration matrix such as the one proposed by the ASTM guidelines can be generated artificially. In this work the predictions obtained using two different calibration matrixes will be studied. The first calibration matrix contains spectra of different mixts. collected manually, while in the second matrix the spectra were generated artificially from the different pure components present in the reaction system. The two models were used to monitor a fed-batch fermn. of G. xylinus. The results obtained as well as a discussion on the role of noise will be presented. [1] Std. practices for IR quant. anal. (e 1655-97). Annual book of ASTM stds. 1999. [on SciFinder (R)

Bioprocess monitoring and control using the combination of fluorescence, mid-IR, and Raman spectroscopy

Spectroscopic monitoring techniques have many advantages for bioprocess monitoring and control, including simultaneous multi-analyte detns., in-situ sterilizability, and low maintenance during operation. This study discusses the combined application of fluorescence, mid-IR, and Raman spectroscopic techniques for a comprehensive monitoring of Phaffia rhodozyma yeast cultures during batch and fed-batch operation for the prodn. of astaxanthin, a carotenoid of industrial importance. Reliability of the different techniques will be detailed along with their utility to control the growth rate at an optimum value. [on SciFinder (R)

Real-time in situ monitoring of freely suspended and immobilized cell cultures based on mid-infrared spectroscopic measurements

Glucose and lactate profiles in Chinese hamster ovary cell cultures were accurately monitored in real time and in situ during three bioreactor batch cultures lasting 11, 15, and 15 days performed within a 60-day period. Monitoring was accomplished using in situ-collected mid-IR spectra analyzed with a priori one-time established partial least-squares regression models. The robustness of the technique was demonstrated by application of these models without modification after 2.3 yr. Neither recalibration nor instrument maintenance was required during the 2.3-yr period, except for the daily filling of liq. nitrogen for detector cooling during operation. The lactate calibration model yielded accurate abs. concn. estns. during each of the batch cultures with std. errors of est. from 1 to 3 mM. The a priori-established glucose calibration model yielded concn. estns. with an off-set, which was const. throughout a culture. Adjustment of the off-set before inoculation resulted in accurate concn. estns. with Std. errors of est. of approx. 1 mM for each of the bioreactor cultures. Sensitivity in detecting differences of 0.5 mM and selectivity against variation of one metabolite while the other was kept const. was demonstrated during std. addns. of either glucose or lactate. The sensor system proved to be reliable, simple, accurate, sterile, and capable of long-term automatic operation and is considered to be mature enough to be routinely applied for in situ (online) cell culture monitoring. [on SciFinder (R)

Methodology for real-time, multianalyte monitoring of fermentations using an in-situ mid-infrared sensor

An in-situ, mid-IR sensor was used to monitor the major analyte concns. involved in the cultivation of Gluconacetobacter xylinus and the prodn. of gluconacetan, a food-grade exopolysaccharide. To predict the analyte concns., three different sets of std. spectra were used to develop calibration models, applying partial least-squares regression. It was possible to build a valid calibration model to predict the 700 spectra collected during the complete time course of the cultivation, using only 12 spectra collected every 10 h as stds. This model was used to reprocess the concn. profiles from 0 to 15 g/L of nine different analytes with a mean std. error of validation of 0.23 g/L. However, this calibration model was not suitable for real-time monitoring as it was probably based on non-specific spectral features, which were correlated only with the measured analyte concns. Valid calibration models capable of real-time monitoring could be established by supplementing the set of 12 fermn. spectra with 42 stds. of measured analytes. A pulse of 5 g/L ethanol showed the robustness of the model to sudden disturbances. The prediction of the models drifted, however, toward the end of the fermn. The most robust calibration model was finally obtained by the addn. of 34 std. spectra of non-measured analytes. Although the spectra did not contain analyte-specific information, it was believed that this addn. would increase the variability space of the calibration model. Therefore, an expanded calibration model contg. 88 spectra was used to monitor, in real time, the concn. profiles of fructose, acetic acid, ethanol and gluconacetan and allowed std. errors of prediction of 1.11, 0.37, 0.22, and 0.79 g/L, resp. [on SciFinder (R)

The influence of correlated calibration samples on the prediction performance of multivariate models based on mid-infrared spectra of animal cell cultures

The effect of the presence of metabolism-induced concentration correlations in the calibration samples on the prediction performance of partial least-squares regression (PLSR) models and mid-infrared spectra from Chinese hamster ovary cell cultures was investigated. Samples collected from batch cultures contained highly correlated metabolite concentrations as a result of metabolic relations. Calibrations based on such samples could only be used to predict concentrations in new samples if a similar correlation structure was present and failed when the new samples were randomly spiked with the analytes. On the other hand, such models were able to predict glucose correctly even if they were based on a spectral range in which glucose does not absorb, provided that the correlations in the calibration and in the new samples were similar. If however, samples from a calibration culture were randomly spiked with the main analytes, much more robust PLSR models resulted. It was possible to predict analyte concentrations in new samples irrespective of whether the correlation structure was maintained or not. Validity of all established models for any given use could be predicted a priori by computing the space inclusion and observer conditions. Predictions from these computations agreed in all cases with the experimental test of model validity. [on SciFinder (R)