Investigating the feasibility of mid infrared spectroscopy for monitoring an industrial de-racemization biotransformation process.

Biotransformation processes have become industrially important in recent years as routes to the manufacture of high value chemical intermediates. However, measurements of key process features and analyte concentrations during these processes are still typically carried out using off-line analysis methods. Vibrational spectroscopic techniques have been extensively utilised for the monitoring and control of a variety of industrial processes. Despite the techniques success with a range of challenging biological matrices, including fermentation and cell culture systems, application of this approach to biotransformation systems has been limited. In the present study the potential of mid infrared spectroscopy to monitor an industrially relevant de-racemization biotransformation process has been investigated. This process presents a number of difficulties due to the optically challenging sample media, close structural similarities and stoichiometric relationship between the key analytes of interest. A PLS model based on the mid infrared spectra obtained during three replicates of the biotransformation process was constructed. In order to ensure that co-linearity within the system had been adequately addressed the spectral contributors to the model were examined. External validation of the constructed model was achieved by challenging the model with two previously unseen replicates of the process. The constructed model was able to predict the concentrations of two key analytes in various samples from these unseen replicates without the requirement for any time consuming sample pre-treatment stages, thus demonstrating the feasibility of near real-time mid infrared monitoring of such an industrial de-racemization biotransformation process.

Assessment of near-infrared spectral information for rapid monitoring of bioprocess quality.

Access to real-time process information is desirable for consistent and efficient operation of bioprocesses. Near-infrared spectroscopy (NIRS) is known to have potential for providing real-time information on the quantitative levels of important bioprocess variables. However, given the fact that a typical NIR spectrum encompasses information regarding almost all the constituents of the sample matrix, there are few case studies that have investigated the spectral details for applications in bioprocess quality assessment or qualitative bioprocess monitoring. Such information would be invaluable in providing operator-level assistance on the progress of a bioprocess in industrial-scale productions. We investigated this aspect and report the results of our investigation. Near-infrared spectral information derived from scanning unprocessed culture fluid (broth) samples from a complex antibiotic production process was assessed for a data set that incorporated bioprocess variations. Principal component analysis was applied to the spectral data and the loadings and scores of the principal components studied. Changes in the spectral information that corresponded to variations in the bioprocess could be deciphered. Despite the complexity of the matrix, near-infrared spectra of the culture broth are shown to have valuable information that can be deconvoluted with the help of factor analysis techniques such as principal component analysis (PCA). Although complex to interpret, the loadings and score plots are shown to offer potential in process diagnosis that could be of value in the rapid assessment of process quality, and in data assessment prior to quantitative model development.

The functional anatomy of gaze-evoked tinnitus and sustained lateral gaze.

OBJECTIVE: To identify neural sites associated with gaze-evoked tinnitus (GET), an unusual condition that may follow cerebellar-pontine angle surgery. METHODS: The authors examined eight patients with GET and used PET to map the neural sites activated by lateral gaze in them and seven age- and sex-matched control subjects. RESULTS: In patients with GET, tinnitus loudness and pitch increased with lateral gaze and, to a lesser extent, up and down gaze. Evidence for neural activity related to GET was seen in the auditory lateral pontine tegmentum or auditory cortex. GET-associated nystagmus appears to activate the cuneus and cerebellar vermis. These sites were found in addition to an extensive network that included frontal eye fields and other sites in frontal, parietal, and temporal cortex that were activated by lateral gaze in seven control subjects and the patients. The unilateral deafness in patients with GET was associated with expansion of auditory cortical areas responsive to tones delivered to the good ear. In addition to GET, unilateral deafness, end-gaze nystagmus, and facial nerve dysfunction were common. CONCLUSIONS: Patients with GET have plastic changes in multiple neural systems that allow neural activity associated with eye movement, including those associated with the neural integrator, to stimulate the auditory system. Anomalous auditory activation is enhanced by the failure of cross-modal inhibition to suppress auditory cortical activity. The time course for the development of GET suggests that it may be due to multiple mechanisms.

The functional anatomy of the normal human auditory system: responses to 0.5 and 4.0 kHz tones at varied intensities.

Most functional imaging studies of the auditory system have employed complex stimuli. We used positron emission tomography to map neural responses to 0.5 and 4.0 kHz sine-wave tones presented to the right ear at 30, 50, 70 and 90 dB HL and found activation in a complex neural network of elements traditionally associated with the auditory system as well as non-traditional sites such as the posterior cingulate cortex. Cingulate activity was maximal at low stimulus intensities, suggesting that it may function as a gain control center. In the right temporal lobe, the location of the maximal response varied with the intensity, but not with the frequency of the stimuli. In the left temporal lobe, there was evidence for tonotopic organization: a site lateral to the left primary auditory cortex was activated equally by both tones while a second site in primary auditory cortex was more responsive to the higher frequency. Infratentorial activations were contralateral to the stimulated ear and included the lateral cerebellum, the lateral pontine tegmentum, the midbrain and the medial geniculate. Contrary to predictions based on cochlear membrane mechanics, at each intensity, 4.0 kHz stimuli were more potent activators of the brain than the 0.5 kHz stimuli.

Site-directed mutagenesis of human tissue transglutaminase: Cys-277 is essential for transglutaminase activity but not for GTPase activity.

Transglutaminases (EC 2.3.2.13) catalyze an acyl-transfer reaction between peptide-bound glutamine residues and primary amines, including the epsilon-amino group of lysine residues in protein. Purified human erythrocyte transglutaminase was found to have another activity, i.e., GTP hydrolysis. Treatment of the enzyme with iodoacetamide, a cysteine-directed reagent, caused a 94% loss of TGase activity within 8 min, but no significant loss of GTPase activity. Cys-277, a known residue which is selectively modified by iodoacetamide, was replaced with Ser by site-directed mutagenesis to assess the role of the Cys-277 in the transglutaminase/GTPase activities. Wild-type cDNA, coding for human endothelial cell transglutaminase, and its C277S-mutated cDNA were cloned into a plasmid vector that contained a promoter from phage T7, and then expressed in Escherichia coli. The wild-type recombinant enzyme was indistinguishable from human erythrocyte transglutaminase in mobility on a SDS-polyacrylamide gel, immunoreactivity and catalytic activities for transglutaminase and GTPase. However, the recombinant enzyme was not blocked at the N-terminal alanine residue, as is the case in the naturally occurring erythrocyte enzyme. The C277S mutant enzyme showed no transglutaminase activity, but had Km and kcat values for GTPase activity that were comparable to those of wild-type recombinant and natural erythrocyte enzymes. These results demonstrate that Cys-277 is essential for transglutaminase activity, but not for GTPase activity, and that N-terminal blocking of tissue-type transglutaminase is not critical for either transglutaminase or GTPase activities.

Evoked potentials: computer-automated threshold-tracking procedure using an objective detection criterion.

A computer-automated, threshold-tracking procedure was developed for measuring evoked response thresholds on-line. The procedure utilizes an objective detection criterion (correlation) to evaluate whether a response is absent or present. Sound intensity is adjusted according to a modified PEST (parameter estimation by sequential testing) procedure in order to estimate threshold. The computer-automated procedures were used to obtain objective estimates of evoked response thresholds in normal and hearing-impaired chinchillas. The evoked response waveforms stored in the computer were also used to obtain visual judgments of threshold. The objective thresholds determined by the computer were virtually identical to the visual detection thresholds. Thus, the computer-automated procedure provides a reliable, objective, and efficient method of estimating evoked response thresholds.

Hearing loss in children with congenital heart disease: a preliminary report.

Life expectancy for children with serious congenital heart defects has dramatically increased in recent years due to advances in medical and surgical care. Anatomic studies of temporal bones in patients with non-syndromic congenital heart disease have demonstrated a high incidence of middle and inner ear anomalies. Systematic study of auditory function in these patients has not been reported. Fifty children with serious congenital heart disease have been evaluated with otologic examination and auditory brainstem-evoked response (ABR) testing. The patients ranged in age from 3 days to 17 years-8 months. Eight patients had hearing loss and normal otologic examinations, 14 patients had hearing loss and associated otitis media with effusion, and 28 patients had normal hearing by ABR testing. These results show a high incidence (16%) of hearing loss not associated with otitis media with effusion in the study population. We suggest that children with serious congenital heart disease are at high risk for hearing loss. This patient group will benefit from early identification, and appropriate medical and audiologic management.

Objective versus visual detection of the auditory brain stem response.

The use of objective (mathematical) procedures in auditory brain stem response (ABR) testing has been suggested in order to eliminate the subjectivity of visual judgments and to increase sensitivity of detection. This investigation compared with effectiveness of visual and objective detection of the ABR. Auditory brain stem responses were obtained in normal adults at several click levels. An equal number of waveforms obtained without stimulation were used to measure false-positive responses. Visual judgments of ABR presence were made by four experienced raters. Objective analysis was accomplished using three methods: correlation, variance ratio, and multiple pre-post z tests. The mean sensitivity score was highest for visual detection, followed in order by the correlation, variance ratio, and multiple z test methods. While visual scoring was statistically the most sensitive, the practical difference between measures was small. In addition, there was considerable disagreement between raters in their judgments. Therefore, the most sensitive of the objective measures--correlation, is considered to be a useful detection procedure to eliminate observer bias and to achieve consistency of scoring.