Quantitation of Host Cell Proteins by Capillary LC/IMS/MS/MS in Combination with Rapid Digestion on Immobilized Trypsin Column Under Native Conditions.

Host cell protein (HCP) impurities are considered a critical quality attribute of biopharmaceuticals because of their potential to compromise safety and efficacy, and LC/MS-based analytical methods have been developed to identify and quantify individual proteins instead of employing enzyme-linked immunosorbent assay to assess total HCP levels. Native digestion enables highly sensitive detection of HCPs but requires overnight incubation to generate peptides, limiting the throughput of sample preparation. In this study, we developed an approach employing native digestion on a trypsin-immobilized column to improve the sensitivity and throughput. We examined suitable databases for the identification of HCPs derived from Chinese hamster ovary (CHO) cells and selected RefSeq's Chinese Hamster as the optimal database. Then, we investigated methods to identify HCPs with greater efficiency than that of denatured in-solution digestion. Native in-column digestion not only reduced the digestion time from overnight to 10 min but also increased the number of quantified HCPs from 154 to 226. In addition to this rapid digestion methodology, we developed high-throughput LC/MS/MS with a monolithic silica column and parallel reaction monitoring-parallel accumulation-serial fragmentation. The optimized system was validated with synthetic peptides derived from high-risk HCPs, confirming excellent linearity, precision, accuracy, and low limit of detection (LOD) and limit of quantification (LOQ) (1-3 ppm). The optimized digestion and analysis method enabled high-throughput quantification of HCPs, and is expected to be useful for quality control and characterization of HCPs in antibody drugs.

Role of follow-up gallium scintigraphy in the evaluation of malignant external otitis (skull base osteomyelitis): A case report.

Malignant otitis externa (skull base osteomyelitis) can be fatal and long-term antibiotic therapy is recommended. Despite being potentially fatal, this infection causes minor changes in inflammatory biomarkers (white blood cell count and C-reactive protein levels) upon blood testing. Computed tomography and magnetic resonance imaging changes persist over a long period. Therefore, it is difficult to determine the optimal time for the discontinuation of antibiotics. We present a 77-year-old male whose medical history included type 2 diabetes mellitus who suffered from chronic otitis media with Pseudomonas aeruginosa infection. His condition did not improve with proper treatment, and imaging revealed malignant otitis media. Intravenous cefepime treatment was administered. Antibiotic treatment was de-escalated to oral levofloxacin treatment after Gallium-67 scintigraphy showed less accumulation after 6 weeks of Cefepime administration; accumulation almost disappeared after 1 year. In this report, we describe the usefulness of gallium scintigraphy in the evaluation of malignant otitis externa.

Maxillary Sinus NUT Carcinoma: A Case Report.

Background/Aim: Nuclear protein in testis (NUT) carcinoma is extremely rare, occurs in the midline of the body, progresses rapidly and is refractory to treatment; most patients die within a year. Here, we describe a case of maxillary sinus NUT carcinoma presenting with epistaxis and nasal obstruction that was treated as a standard head and neck carcinoma. Case Report: The patient was a 41-year-old male with a left buccal swelling; the diagnosis was made of primary NUT carcinoma of the left maxillary sinus and bone metastasis in the cervical spine. After induction chemotherapy with docetaxel plus cisplatin and 5-fluorouracil, the tumor decreased in size, and the patient was further treated with cisplatin and radiation therapy. One month after that, the tumor remained small, however, lung metastasis was observed. Therefore, nivolumab was administered. Cetuximab and paclitaxel were administered after the lung metastasis worsened, but the patient developed progressive disease and died 11 months after diagnosis. Conclusion: Effective treatments for NUT carcinoma have not yet been established. However, early testing to establish the diagnosis may provide useful insights to guide clinical decisions to improve patient outcomes.

Extending the Coverage of Lys-C/Trypsin-Based Bottom-up Proteomics by Cysteine S-Aminoethylation.

To improve the coverage in bottom-up proteomics, S-aminoethylation of cysteine residues (AE-Cys) was carried out with 2-bromoethylamine, followed by cleavage with lysyl endopeptidase (Lys-C) or Lys-C/trypsin. A model study with bovine serum albumin showed that the C-terminal side of AE-Cys was successfully cleaved by Lys-C. The frequency of side reactions at amino acids other than Cys was less than that in the case of carbamidomethylation of Cys with iodoacetamide. Proteomic analysis of A549 cell extracts in the data-dependent acquisition mode after AE-Cys modification afforded a greater number of identified protein groups, especially membrane proteins. In addition, label-free quantification of proteins in mouse nonsmall cell lung cancer (NSCLC) tissue in the data-independent acquisition mode after AE-Cys modification showed improved NSCLC pathway coverage and greater reproducibility. Furthermore, the AE-Cys method could identify an epidermal growth factor receptor peptide containing the T790 M mutation site, a well-established lung-cancer-related mutation site that has evaded conventional bottom-up methods. Finally, AE-Cys was found to fully mimic Lys in terms of collision-induced dissociation fragmentation, ion mobility separation, and cleavage by Lys-C/trypsin, except for sulfoxide formation during sample preparation.

Postoperative Pharyngeal Fistula Diagnosed by Prone Position Computed Tomography With Oral Contrast Agent: A Case Report.

BACKGROUND/AIM: A pharyngeal fistula is the most common complication of total laryngectomy; thus, accurate diagnosis and treatment are important. Diagnosis is usually made by the finding of leakage of the contrast agent outside the pharynx during swallowing contrast examination. Herein, we encountered a case in which fine leaks not detected on contrast examination during swallowing were visualized and diagnosed by computed tomography (CT) imaging with oral contrast media with the patient in a prone position. CASE REPORT: During imaging in a prone position, the contrast agent entered the sutures on the cephalocaudal and ventral sides of the surgical site, which were particularly prone to leaks due to gravity, and it was possible to diagnose minute leaks. When there is a high risk of postoperative pharyngeal fistula, such as in reconstructive cases with a pedunculated flap or with overlapping risk factors such as preoperative irradiation, CT imaging with contrast medium in a prone position is considered useful when swallowing contrast examination does not provide a clear diagnosis. However, suture failure is possible, and this should be evaluated. CONCLUSION: This case suggests that routine prone CT may lead to the early detection of postoperative pharyngeal fistula in high-risk cases. Further accumulation of cases is required to confirm our findings.

The actual state of and factors for speech perception ability in adult cochlear implant wearers.

PURPOSE: To elucidate the status of speech perception ability in cochlear implant wearers with unknown deafness causes. MATERIALS AND METHODS: We extracted 1095 patients between January 1, 1986, and December 31, 2019; among them, there were 418 first-surgery adults who wore implants made by Cochlear. Finally, we included 204 patients (69 males and 135 women) without cochlear morphological abnormalities. All electrodes were inserted into the cochlea, without major intraoperative and postoperative problems. The minimum, maximum, and average ages of surgery were 17 years, 85 years, and 56.5 years, respectively. The participants were divided according to the electrode (Group A, CI22 straight electrode; Group B, CI24 straight electrode; Group C, modiolar hugging electrode type electrode; and Group D, slim straight electrode). We evaluated the following parameters: cochlear implant threshold and single-syllable, word, and single-sentence hearing ability. Further, we investigated impactful background factors. RESULTS: There was a decreased cochlear implant threshold in Groups B and C. Group B had a better ability to hear single syllables, words, and sentences than Group A. Groups C and D had significantly better ability to hear words than Group B. Low hearing aid threshold, good hearing ability with the 67S hearing aid, and short duration of hearing loss were associated with enhanced hearing ability. CONCLUSION: In this study, it was suggested that the listening ability may have improved because of the progress of the electrodes. However, the possibility of a ceiling effect was also suggested.

Preoperative Evaluation of Substernal Goiter by Computed Tomography in the Extended Neck Position.

Sternotomy is indicated when a goiter cannot be resected via a cervical incision, such as in the case of a substernal goiter extending beyond the aortic arch. In this article, we report a case of a large substernal goiter that was successfully removed using the cervical approach only. This is a case of a 68-year-old woman, diagnosed with goiter 20 years ago, who complained of a neck mass enlargement with associated cough. Pathological examination revealed no malignancy. Computed tomography (CT) scan showed an 11-cm thyroid mass reaching the level of the aortic arch. Preoperatively, we evaluated the substernal extent of the goiter via CT in the extended neck position to decide whether sternotomy was necessary. With the patient's neck extended, the goiter withdraws cranially above the aortic arch. The mass was then removed via the cervical approach without sternotomy. Preoperative CT in the extended neck position was thus deemed helpful in deciding whether or not sternotomy was required.

Transoral Endoscopic Examination of the Oropharynx With Tongue Protrusion, Phonation, and Open Mouth.

Background/Aim: We examined the diagnostic performance of the tongue protrusion with phonation and open mouth (TOPPOM) method for visualizing structures of the oropharynx. Patients and Methods: Transoral endoscopy was performed on 20 healthy participants to evaluate 12 oropharynx subsites under three conditions: open mouth (OM), phonation with open mouth (POM), and TOPPOM. Each subsite was scored from 0 to 2 depending on subsite visualization, and the scores were summed. Images of subsite-adjacent mucosa were similarly scored. Results: The total scores were significantly higher for TOPPOM than for POM and for POM than for OM. Such scores were observed for both the palatine arches, both palatine tonsils, the left lingual tonsillar sulcus, and the vallecula. Conclusion: TOPPOM enables visualization of the oropharynx through transoral endoscopic examination, and TOPPOM with conventional transnasal endoscopy may enable early detection of oropharyngeal carcinomas and lesions and improve the performance of pre- and post-treatment evaluations.

Pituitary-adrenal dysfunction caused by nivolumab for head and neck cancer.

Nivolumab exerts antitumor effects by inhibiting binding of PD-L1 to PD-1, and offers proven effectiveness in various disease areas, including cancers of the head and neck. The mechanisms of action lead nivolumab to induce immune-related adverse events (irAE). We report a case of pituitary-adrenal dysfunction to isolated adrenocorticotropic hormone (ACTH) deficiency as an irAE of nivolumab in a patient treated for head and neck cancer. This is the first report of an irAE of nivolumab in the field of head and neck squamous cell cancer. The patient was a man in his 50s with cancer of the tongue and hypopharynx that recurred after chemoradiotherapy, surgery and chemotherapy. After starting nivolumab, irAEs developed after 8 courses. The case was managed from the early stages in collaboration with the endocrinology department. Pituitary-adrenal hypofunction due to isolated ACTH deficiency was diagnosed on the basis of endocrine tests. The patient responded to hydrocortisone replacement therapy and has been able to continue treatment with nivolumab while continuing oral hydrocortisone. Although irAEs involving pituitary gland disorders are rare, these events can become life-threatening when severe. Early diagnosis and treatment are essential and require regular blood sampling and collaboration with specialists from an early stage.

New Vocal Fold-shortening Procedure: External Approach to Anterior Vocal Fold Plication.

INTRODUCTION: We believe that our new procedure offers a useful external approach to anterior vocal fold plication. Vocal cord shortening by laryngomicrosurgery (LMS) involves plication of the anterior commissure, but this difficult procedure requires high-level skills. Dehiscence of the plicated location may occur postoperatively. METHODS: In the present case, we applied an innovative procedure combining vocal cord shortening by LMS with cervical incision. This facilitated plication, and no postoperative dehiscence was encountered. RESULTS: Speaking fundamental frequency (SFF) was 110 Hz at the first visit, 170 Hz after type 4 thyroplasty, 167 Hz after thyroarytenoid muscle myectomy, and 208 Hz after the present procedure, representing a good outcome. CONCLUSIONS: This procedure could be useful when the SFF is not sufficiently raised by existing procedures. The risk of postoperative dehiscence of the plicated location is low compared with LMS. CASE DETAILS: The patient consulted with a major complaint of low vocal pitch. At the first visit, SFF was 110 Hz, suggesting a male voice. In April 201X, type 4 thyroplasty was performed and successfully raised SFF to 170 Hz. As the patient did not readily pass as a woman, thyroarytenoid muscle myectomy was performed in February 201X +1 but only achieved an SFF of 167 Hz, which was considered unsatisfactory. As a result, the procedure was performed again in September 201X +1.

Theoretical and Experimental Analyses of Tensor-Based Regression and Classification.

We theoretically and experimentally investigate tensor-based regression and classification. Our focus is regularization with various tensor norms, including the overlapped trace norm, the latent trace norm, and the scaled latent trace norm. We first give dual optimization methods using the alternating direction method of multipliers, which is computationally efficient when the number of training samples is moderate. We then theoretically derive an excess risk bound for each tensor norm and clarify their behavior. Finally, we perform extensive experiments using simulated and real data and demonstrate the superiority of tensor-based learning methods over vector- and matrix-based learning methods.

Predicting disease progression from short biomarker series using expert advice algorithm.

Well-trained clinicians may be able to provide diagnosis and prognosis from very short biomarker series using information and experience gained from previous patients. Although mathematical methods can potentially help clinicians to predict the progression of diseases, there is no method so far that estimates the patient state from very short time-series of a biomarker for making diagnosis and/or prognosis by employing the information of previous patients. Here, we propose a mathematical framework for integrating other patients' datasets to infer and predict the state of the disease in the current patient based on their short history. We extend a machine-learning framework of "prediction with expert advice" to deal with unstable dynamics. We construct this mathematical framework by combining expert advice with a mathematical model of prostate cancer. Our model predicted well the individual biomarker series of patients with prostate cancer that are used as clinical samples.

Large-scale EEG/MEG source localization with spatial flexibility.

We propose a novel approach to solving the electro-/magnetoencephalographic (EEG/MEG) inverse problem which is based upon a decomposition of the current density into a small number of spatial basis fields. It is designed to recover multiple sources of possibly different extent and depth, while being invariant with respect to phase angles and rotations of the coordinate system. We demonstrate the method's ability to reconstruct simulated sources of random shape and show that the accuracy of the recovered sources can be increased, when interrelated field patterns are co-localized. Technically, this leads to large-scale mathematical problems, which are solved using recent advances in convex optimization. We apply our method for localizing brain areas involved in different types of motor imagery using real data from Brain-Computer Interface (BCI) sessions. Our approach based on single-trial localization of complex Fourier coefficients yields class-specific focal sources in the sensorimotor cortices.

Localization of class-related mu-rhythm desynchronization in motor imagery based brain-computer interface sessions.

We localize the sources of class-dependent event-related desynchronisation (ERD) of the mu-rhythm related to different types of motor imagery in Brain-Computer Interfacing (BCI) sessions. Our approach is based on localization of single-trial Fourier coefficients using sparse basis field expansions (S-FLEX). The analysis reveals focal sources in the sensorimotor cortices, a finding which can be regarded as a proof for the expected neurophysiological origin of the BCI control signal. As a technical contribution, we extend S-FLEX to the multiple measurement case in a way that the activity of different frequency bins within the mu-band is coherently localized.

Modeling sparse connectivity between underlying brain sources for EEG/MEG.

We propose a novel technique to assess functional brain connectivity in electroencephalographic (EEG)/magnetoencephalographic (MEG) signals. Our method, called sparsely connected sources analysis (SCSA), can overcome the problem of volume conduction by modeling neural data innovatively with the following ingredients: 1) the EEG/MEG is assumed to be a linear mixture of correlated sources following a multivariate autoregressive (MVAR) model; 2) the demixing is estimated jointly with the source MVAR parameters; and 3) overfitting is avoided by using the group lasso penalty. This approach allows us to extract the appropriate level of crosstalk between the extracted sources and, in this manner, we obtain a sparse data-driven model of functional connectivity. We demonstrate the usefulness of SCSA with simulated data and compare it to a number of existing algorithms with excellent results.

A regularized discriminative framework for EEG analysis with application to brain-computer interface.

We propose a framework for signal analysis of electroencephalography (EEG) that unifies tasks such as feature extraction, feature selection, feature combination, and classification, which are often independently tackled conventionally, under a regularized empirical risk minimization problem. The features are automatically learned, selected and combined through a convex optimization problem. Moreover we propose regularizers that induce novel types of sparsity providing a new technique for visualizing EEG of subjects during tasks from a discriminative point of view. The proposed framework is applied to two typical BCI problems, namely the P300 speller system and the prediction of self-paced finger tapping. In both datasets the proposed approach shows competitive performance against conventional methods, while at the same time the results are easier accessible to neurophysiological interpretation. Note that our novel approach is not only applicable to Brain imaging beyond EEG but also to general discriminative modeling of experimental paradigms beyond BCI.

Multivariate analysis of noise in genetic regulatory networks.

Stochasticity is an intrinsic property of genetic regulatory networks due to the low copy numbers of the major molecular species, such as, DNA, mRNA, and regulatory proteins. Therefore, investigation of the mechanisms that reduce the stochastic noise is essential in understanding the reproducible behaviors of real organisms and is also a key to design synthetic genetic regulatory networks that can reliably work. We use an analytical and systematic method, the linear noise approximation of the chemical master equation along with the decoupling of a stoichiometric matrix. In the analysis of fluctuations of multiple molecular species, the covariance is an important measure of noise. However, usually the representation of a covariance matrix in the natural coordinate system, i.e. the copy numbers of the molecular species, is intractably complicated because reactions change copy numbers of more than one molecular species simultaneously. Decoupling of a stoichiometric matrix, which is a transformation of variables, significantly simplifies the representation of a covariance matrix and elucidates the mechanisms behind the observed fluctuations in the copy numbers. We apply our method to three types of fundamental genetic regulatory networks, that is, a single-gene autoregulatory network, a two-gene autoregulatory network, and a mutually repressive network. We have found that there are multiple noise components differently originating. Each noise component produces fluctuation in the characteristic direction. The resulting fluctuations in the copy numbers of the molecular species are the sum of these fluctuations. In the examples, the limitation of the negative feedback in noise reduction and the trade-off of fluctuations in multiple molecular species are clearly explained. The analytical representations show the full parameter dependence. Additionally, the validity of our method is tested by stochastic simulations.