Toxicology and safety study of L-tryptophan and its impurities for use in broiler feed.

L-tryptophan has been utilized as a feed additive in animal nutrition to improve growth performance, as well as a dietary supplement to alleviate various emotional symptoms in humans. Despite its benefits, concerns regarding its safety arose following the outbreak of eosinophilia-myalgia syndrome (EMS) among individuals who consumed L-tryptophan. The causative material of EMS was determined to be not L-tryptophan itself, but rather L-tryptophan impurities resulting from a specific manufacturing process. To investigate the effect of L-tryptophan and its impurities on humans who consume meat products derived from animals that were fed L-tryptophan and its impurities, an animal study involving broiler chickens was conducted. The animals in test groups were fed diet containing 0.065%-0.073% of L-tryptophan for 27 days. This study aimed to observe the occurrence of toxicological or EMS-related symptoms and analyze the residues of L-tryptophan impurities in meat products. The results indicated that there was no evidence of adverse effects associated with the test substance in the investigated parameters. Furthermore, most of the consumed EMS-causing L-tryptophan impurities did not remain in the meat of broiler chickens. Thus, this study demonstrated the safety of L-tryptophan and some of its impurities as a feed additive.

Three-year mortality, readmission, and medical expenses in critical care survivors: A population-based cohort study.

BACKGROUND: Due to the increasing number of critical care survivors, population-based studies on the long-term outcomes after discharge are necessary to inform local decision-making. OBJECTIVES: This study aimed to investigate mortality and its risk factors, readmissions, and medical expenses of intensive care unit survivors for 3 years after hospital discharge. METHODS: This retrospective study analysed data from the National Health Insurance Service-National Sample Cohort in Korea. Of the 195,702 patients who survived and were discharged from hospital in 2012, 2693 intensive care unit patients were assigned to the case group for the study, and the remaining 193,009 were assigned to the comparison group. The primary outcome was all-cause mortality for 3 years after discharge. Secondary outcomes were all-cause hospital readmission and medical expenses in 3 years. We analysed risk factors for mortality using the Cox proportional hazard regression. The differences in hospital readmission and medical expenses between the case and comparison groups were analysed by multivariate logistic regression and independent t-tests. RESULTS: The 1-year, 2-year, and 3-year cumulative mortality rates in the case group were 15.9%, 20.5%, and 24.4%, respectively, and older age, disability, medical admission, and longer hospital stay increased mortality. Almost 40% of intensive care unit survivors were readmitted to hospital within 6 months of discharge, and their odds of being readmitted were significantly higher than those of the comparison group. Medical expenses were also significantly higher in the case group, with the highest paid within 6 months. CONCLUSIONS: Mortality, hospital readmission, and medical expenses for intensive care unit survivors were the worst within 6 months of discharge. In light of the long-term recovery trajectory of critical illness, it is necessary to investigate what factors may have contributed to the negative outcome during this period. Further research is needed to determine which services primarily contributed to the increase in medical expenses.

BEAR: A Novel Virtual Screening Method Based on Large-Scale Bioactivity Data.

Data-driven drug discovery exploits a comprehensive set of big data to provide an efficient path for the development of new drugs. Currently, publicly available bioassay data sets provide extensive information regarding the bioactivity profiles of millions of compounds. Using these large-scale drug screening data sets, we developed a novel in silico method to virtually screen hit compounds against protein targets, named BEAR (Bioactive compound Enrichment by Assay Repositioning). The underlying idea of BEAR is to reuse bioassay data for predicting hit compounds for targets other than their originally intended purposes, i.e., "assay repositioning". The BEAR approach differs from conventional virtual screening methods in that (1) it relies solely on bioactivity data and requires no physicochemical features of either the target or ligand. (2) Accordingly, structurally diverse candidates are predicted, allowing for scaffold hopping. (3) BEAR shows stable performance across diverse target classes, suggesting its general applicability. Large-scale cross-validation of more than a thousand targets showed that BEAR accurately predicted known ligands (median area under the curve = 0.87), proving that BEAR maintained a robust performance even in the validation set with additional constraints. In addition, a comparative analysis demonstrated that BEAR outperformed other machine learning models, including a recent deep learning model for ABC transporter family targets. We predicted P-gp and BCRP dual inhibitors using the BEAR approach and validated the predicted candidates using in vitro assays. The intracellular accumulation effects of mitoxantrone, a well-known P-gp/BCRP dual substrate for cancer treatment, confirmed nine out of 72 dual inhibitor candidates preselected by primary cytotoxicity screening. Consequently, these nine hits are novel and potent dual inhibitors for both P-gp and BCRP, solely predicted by bioactivity profiles without relying on any structural information of targets or ligands.

Combination of PD-1 Checkpoint Blockade and Botulinum Toxin Type A1 Improves Antitumor Responses in Mouse Tumor Models of Melanoma and Colon Carcinoma.

BACKGROUND: Tumor innervation has been shown to be utilized by some solid cancers to support tumor initiation, growth, progression, and metastasis, as well as confer resistance to immune checkpoint blockade through suppression of antitumor immunologic responses. Since botulinum neurotoxin type A1 (BoNT/A1) blocks neuronal cholinergic signaling, its potential use as an anticancer drug in combination with anti-PD-1 therapy was investigated in four different syngeneic mouse tumor models. METHODS: Mice implanted with breast (4T1), lung (LLC1), colon (MC38), and melanoma (B16-F10) tumors were administered a single intratumoral injection of 15 U/kg BoNT/A1, repeated intraperitoneal injections of 5 mg/kg anti-PD-1 (RMP1-14), or both. RESULTS: Compared to the single-agent treatments, anti-PD-1 and BoNT/A1 combination treatment elicited significant reduction in tumor growth among B16-F10 and MC38 tumor-bearing mice. The combination treatment also lowered serum exosome levels in these mice compared to the placebo control group. In the B16-F10 syngeneic mouse tumor model, anti-PD-1 + BoNT/A1 combination treatment lowered the proportion of MDSCs, negated the increased proportion of Treg cells, and elicited a higher number of tumor-infiltrating CD4+ and CD8+ T lymphocytes into the tumor microenvironment compared to anti-PD-1 treatment alone. CONCLUSION: Our findings demonstrate the synergistic antitumor effects of BoNT/A1 and PD-1 checkpoint blockade in mouse tumor models of melanoma and colon carcinoma. These findings provide some evidence on the potential application of BoNT/A1 as an anticancer drug in combination with immune checkpoint blockade and should be further explored.

Moral Distress Regarding End-of-Life Care Among Healthcare Personnel in Korean University Hospitals: Features and Differences Between Physicians and Nurses.

BACKGROUND: Healthcare professionals often experience moral distress while providing end-of-life care. This study explored how physicians and nurses experienced moral distress when they cared for critically and terminally ill patients in tertiary hospitals in South Korea. METHODS: This study used semi-structured in-depth interviews. A total of 22 people in two tertiary hospitals were interviewed, nine (40.9%) of which were physicians and 13 (59.1%) were nurses. The recorded interview files and memos were analyzed using grounded theory. RESULTS: Most physicians and nurses encountered similar feelings of anger, helplessness, and burden owing to a lack of appropriate resources for end-of-life care. However, the factors and contexts of their moral distress differed. Nurses mainly addressed poorly organized end-of-life care, intensive labor conditions without support for nurses, and providing care without participation in decision-making. Meanwhile, physicians addressed the prevailing misperceptions on end-of-life care, communication failure between physicians owing to hierarchy and fragmented disciplines, the burden of responsibility in making difficult decisions, and the burden of resource allocation. CONCLUSION: Differences in moral distress between physicians and nurses leave them isolated and can affect communication regarding healthcare. Mutual understanding between job disciplines will enhance their communication and help resolve conflicts in end-of-life care.

Effects of nonpharmacological interventions on sleep improvement and delirium prevention in critically ill patients: A systematic review and meta-analysis.

OBJECTIVE: Sleep disturbance and delirium are common problems experienced by critically ill patients in the intensive care unit (ICU). These interrelated issues increase the length of stay in the ICU but might also negatively affect long-term health outcomes. The objective of this study was to identify the nonpharmacological interventions provided to improve sleep or prevent delirium in ICU patients or both and integrate their effect sizes. REVIEW METHODS: This study was a registered systematic review and meta-analysis. We searched MEDLINE, CINAHL, EMBASE, Web of Science, and Cochrane Library from their inception until December 2021. We included randomised controlled trials and nonrandomised controlled trials-(RCT) that provided nonpharmacological interventions and reported sleep or delirium as outcome variables. Studies not published in English or whose full text was not available were excluded. The quality of the evidence was assessed with version 2 of the Cochrane risk-of-bias tool for RCTs and the Risk Of Bias In Non-randomised Studies of Interventions (ROBINS-I). RESULTS: The systematic review included 118 studies, and the meta-analysis included 100 studies. Overall nonpharmacological interventions had significant effects on subjective sleep quality (standardised mean difference = 0.30, 95% confidence interval [CI] = 0.05 to 0.56), delirium incidence (odds ratio = 0.62, 95% CI = 0.53 to 0.73), and delirium duration (standardised mean difference = -0.68, 95% CI = -0.93 to -0.43). In individual interventions, aromatherapy, music, and massage effectively improved sleep. Exercise, family participation, information giving, cognitive stimulation, bright light therapy, architectural intervention, and bundles/protocols effectively reduced delirium. Light/noise blocking was the only intervention that ensured both sleep improvement and delirium prevention. CONCLUSIONS: Our results suggest nonpharmacological interventions improve sleep and prevent delirium in ICU patients. We recommend that ICU nurses use nonpharmacological interventions that promote person-environment compatibility in their clinical practice. The results of our review can guide nurses in adopting interventions related to sleep and delirium. PROSPERO REFERENCE NUMBER: CRD42021230815.

Exploring uncertainty and use of real-world data in the National Institute for Health and Care Excellence single technology appraisals of targeted cancer therapy.

OBJECTIVES: Dealing with uncertainty is one of the critical topics in health technology assessment. The greater decision uncertainty in appraisals, the less clear the clinical- and cost-effectiveness of the health technology. Although the development of targeted cancer therapies (TCTs) has improved patient health care, additional complexity has been introduced in drug appraisals due to targeting more specific populations. Real-world data (RWD) are expected to provide helpful information to fill the evidence gaps in appraisals. This study compared appraisals of TCTs with those of non-targeted cancer therapies (non-TCTs) regarding sources of uncertainty and reviewed how RWD have been used to supplement the information in these appraisals. METHODS: This study reviews single technology appraisals (STAs) of oncology medicines performed by the National Institute for Health and Care Excellence (NICE) over 11 years up to December 2021. Three key sources of uncertainty were identified for comparison (generalisability of clinical trials, availability of direct treatment comparison, maturity of survival data in clinical trials). To measure the intensity of use of RWD in appraisals, three components were identified (overall survival, volume of treatment, and choice of comparators). RESULTS: TCTs received more recommendations for provision through the Cancer Drugs Fund (27.7, 23.6% for non-TCT), whereas similar proportions were recommended for routine commissioning. With respect to sources of uncertainty, the external validity of clinical trials was greater in TCT appraisals (p = 0.026), whereas mature survival data were available in fewer TCT appraisals (p = 0.027). Both groups showed similar patterns of use of RWD. There was no clear evidence that RWD have been used more intensively in appraisals of TCT. CONCLUSIONS: Some differences in uncertainty were found between TCT and non-TCT appraisals. The appraisal of TCT is generally challenging, but these challenges are neither new nor distinctive. The same sources of uncertainty were often found in the non-TCT appraisals. The uncertainty when appraising TCT stems from insufficient data rather than the characteristics of the drugs. Although RWD might be expected to play a more active role in appraisals of TCT, the use of RWD has generally been limited.

Temporal and spatial goal-directed reaching in upper limb prosthesis users.

Understanding the fundamental characteristics of prosthetic movement control is imperative in improving prosthesis design and training. This study quantified how using an upper limb prosthesis affected performance during goal-directed reaching tasks. Nine prosthesis users with unilateral transradial limb absence and nine healthy controls completed a series of goal-directed reaching movements with different goals: one spatial and three temporal with different goal frequencies. We quantified end-point accuracy, smoothness, and peak speed for the spatial task and temporal accuracy, horizontal distance, and speed for the temporal task. For the temporal task, we also used a goal-equivalent manifold (GEM) approach to decompose variability in movement distance and speed into those perpendicular and tangential to the GEM. Detrended fluctuation analysis (DFA) quantified the temporal persistence of each time series. For the spatial task, movements made with prostheses were less smooth, had larger end-point errors, and had slower peak speed compared to those with control limbs (p < 0.041). For the temporal task, movements made with prostheses and intact limbs of prosthesis users and control limbs were similar in distance and speed and had similar timing errors (p > 0.138). Timing errors, distance, speed, and GEM deviations were corrected similarly between prosthetic limbs and control limbs (p > 0.091). The mean and variability of distance, speed, and perpendicular deviations decreased with increased goal frequency (p < 0.001). Our results suggest that prosthesis users have a sufficient internal model to successfully complete ballistic movements but are unable to accurately complete movements requiring substantial feedback.

Adjustments in Shoulder and Back Kinematics during Repetitive Palletizing Tasks.

Repetitive task performance is a leading cause of musculoskeletal injuries among order-picking workers in warehouses. The repetition of lifting tasks increases the risk of back and shoulder injuries among these workers. While lifting in this industry is composed of loaded and unloaded picking and placing, the existing literature does not address the separate analysis of the biomechanics of the back and shoulder for these events. To that end, we investigated the kinematics of the back and shoulder movements of nine healthy male participants who performed three sessions of a simulated de/palletization task. Their back and shoulder kinematics were sensed using an optical motion capture system to determine the back inclination and shoulder flexion. Comparison of the kinematics between the first and last sessions indicated statistically significant changes in the timings, angles, coordination between the back and shoulder, and moments around the shoulder (p<0.05). The majority of the significant changes were observed during the loaded events, which confirms the importance of the separation of these events for biomechanical analysis. This finding suggests that focusing worker evaluation on the loaded periods can provide important information to detect kinematic changes that may affect musculoskeletal injury risk.

Prediction models for early diagnosis of actinomycotic osteomyelitis of the jaw using machine learning techniques: a preliminary study.

BACKGROUND: This study aimed to develop and validate five machine learning models designed to predict actinomycotic osteomyelitis of the jaw. Furthermore, this study determined the relative importance of the predictive variables for actinomycotic osteomyelitis of the jaw, which are crucial for clinical decision-making. METHODS: A total of 222 patients with osteomyelitis of the jaw were analyzed, and Actinomyces were identified in 70 cases (31.5%). Logistic regression, random forest, support vector machine, artificial neural network, and extreme gradient boosting machine learning methods were used to train the models. The models were subsequently validated using testing datasets. These models were compared with each other and also with single predictors, such as age, using area under the receiver operating characteristic (ROC) curve (AUC). RESULTS: The AUC of the machine learning models ranged from 0.81 to 0.88. The performance of the machine learning models, such as random forest, support vector machine and extreme gradient boosting was significantly superior to that of single predictors. Presumed causes, antiresorptive agents, age, malignancy, hypertension, and rheumatoid arthritis were the six features that were identified as relevant predictors. CONCLUSIONS: This prediction model would improve the overall patient care by enhancing prognosis counseling and informing treatment decisions for high-risk groups of actinomycotic osteomyelitis of the jaw.

The relationship between person-centred care and the intensive care unit experience of critically ill patients: A multicentre cross-sectional survey.

BACKGROUND: Person-centred care has the potential to improve the patient experience in the intensive care unit (ICU). However, the relationship between person-centred care perceived by critically ill patients and their ICU experience has yet to be determined. OBJECTIVES: The aim of this study was to investigate the relationship between person-centred care and the ICU experience of critically ill patients. METHODS: This study was a multicentre, cross-sectional survey involving 19 ICUs of four university hospitals in Busan, Korea. The survey was conducted from June 2019 to July 2020, and 787 patients who had been admitted to the ICU for more than 24 hours participated. We measured person-centred care using the Person-Centered Critical Care Nursing perceived by Patient Questionnaire. Participants' ICU experience was measured by the Korean version of the Intensive Care Experience Questionnaire that consists of four subscales. We analysed the relationship between person-centred care and each area of the ICU experience using multivariate linear regression. RESULTS: Person-centred care was associated with 'awareness of surroundings' (ß = 0.29, p < .001), 'frightening experiences' (ß = -0.31, p < .001), and 'satisfaction with care' (ß = 0.54, p < .001). However, there was no significant association between person-centred care and 'recall of experience'. CONCLUSIONS: We observed that person-centred care was positively related to most of the ICU experiences of critically ill patients except for recall of experience. Further studies on developing person-centred nursing interventions are needed.

The effect of postintensive care syndrome on the quality of life of intensive care unit survivors: A secondary analysis.

BACKGROUND: Despite increasing interest in postintensive care syndrome and the quality of life of intensive care unit survivors, the empirical literature on the relationship between these two variables is limited. OBJECTIVES: This study aimed to examine whether postintensive care syndrome predicts the quality of life of intensive care unit survivors. METHODS: We analysed secondary data, which were collected as part of a larger cross-sectional study. The participants were recruited from six health institutions in Korea. The data of 496 survivors who had been admitted to an intensive care unit for at least 48 h during the past year were analysed. They responded to measures of postintensive care syndrome and quality of life. RESULTS: The participants' mean physical and mental component summary scores (quality of life) were 40.08 ± 8.99 and 40.24 ± 11.19, respectively. Physical impairment (ß = -0.48, p < 0.001), unemployment (ß = -0.19, p < 0.001), low income (ß = -0.11, p = 0.004), older age (ß = -0.08, p = 0.039), and cognitive impairment (ß = -0.11, p = 0.045) predicted lower physical component summary scores. Mental (ß = -0.49, p < 0.001) and cognitive impairment (ß = -0.14, p = 0.005) and low income (ß = -0.09, p = 0.014) predicted mental component summary scores. CONCLUSIONS: The participants reported poor physical and mental health-related quality of life. Postintensive care syndrome, unemployment, low income, and older age were the main predictors of poor quality of life. In addition, postintensive care syndrome was a stronger risk factor for poor quality of life than demographic characteristics and intensive care unit treatment factors.

Risk factors for post-intensive care syndrome: A systematic review and meta-analysis.

OBJECTIVE: The objective of this study was to identify the risk factors for each area of post-intensive care syndrome (PICS) and to determine their effect size. REVIEW METHOD USED: This study used systematic review and meta-analysis. DATA SOURCES: PubMed, CINAHL, EMBASE, PsycINFO, and Cochrane Library were searched. REVIEW METHODS: Eighty-nine studies were selected for the review based on the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines. The PICS areas and risk factors reported in the individual studies were reviewed and categorised. We used the Newcastle-Ottawa Scale to evaluate the quality of studies. The effect size of each risk factor was calculated as odds ratio (OR). RESULTS: There were 33 mental health studies, 15 cognitive impairment studies, 32 physical impairment studies, eight studies on two areas, and one study on all three areas. Sixty risk factors were identified, including 33 personal and 27 intensive care unit (ICU)-related factors. Significant risk factors for mental health included female sex (odds ratio [OR] = 3.37, 95% confidence interval [CI]: 1.12-10.17), previous mental health problems (OR = 9.45, 95% CI: 2.08-42.90), and negative ICU experience (OR = 2.59, 95% CI: 2.04-3.28). The only significant risk factor for cognitive impairment was delirium (OR = 2.85, 95% CI: 1.10-7.38). Significant risk factors for physical impairment included older age (OR = 2.19, 95% CI: 1.11-4.33), female sex (OR = 1.96, 95% CI: 1.32-2.91), and high disease severity (OR = 2.54, 95% CI: 1.76-3.66). CONCLUSIONS: Although PICS is a multidimensional concept, each area has been studied separately. Significant risk factors for PICS included older age, female sex, previous mental health problems, disease severity, negative ICU experience, and delirium. To prevent PICS, the multidisciplinary team should pay attention to modifiable risk factors such as delirium and patients' ICU experience.

Regioselectivity significantly impacts microsomal glucuronidation efficiency of R/S-6, 7-, and 8-hydroxywarfarin.

Coumadin (R/S-warfarin) metabolism plays a critical role in patient response to anticoagulant therapy. Several cytochrome P450s oxidize warfarin into R/S-6-, 7-, 8-, 10, and 4'-hydroxywarfarin that can undergo subsequent glucuronidation by UDP-glucuronosyltransferases (UGTs); however, current studies on recombinant UGTs cannot be adequately extrapolated to microsomal glucuronidation capacities for the liver. Herein, we estimated the capacity of the average human liver to glucuronidate hydroxywarfarin and identified UGTs responsible for those metabolic reactions through inhibitor phenotyping. There was no observable activity toward R/S-warfarin, R/S-10-hydroxywarfarin or R/S-4'-hydroxywarfarin. The observed metabolic efficiencies (Vmax/Km) toward R/S-6-, 7-, and especially 8-hydroxywarfarin indicated a high glucuronidation capacity to metabolize these compounds. UGTs demonstrated strong regioselectivity toward the hydroxywarfarins. UGT1A6 and UGT1A1 played a major role in R/S-6- and 7-hydroxywarfarin glucuronidation, respectively, whereas UGT1A9 accounted for almost all of the generation of the R/S-8-hydroxywarfarin glucuronide. In summary, these studies expanded insights to glucuronidation of hydroxywarfarins by pooled human liver microsomes, novel roles for UGT1A6 and 1A9, and the overall degree of regioselectivity for the UGT reactions.

Effects of repeated waist-pull perturbations on gait stability in subjects with cerebellar ataxia.

BACKGROUND: Damage to the cerebellum can affect neural structures involved in locomotion, causing gait and balance disorders. However, the integrity of cerebellum does not seem to be critical in managing sudden and unexpected environmental changes such as disturbances during walking. The cerebellum also plays a functional role in motor learning. Only a few effective therapies exist for individuals with cerebellar ataxia. With these in mind, we aimed at investigating: (1) corrective response of participants with cerebellar ataxia (CA) to unexpected gait perturbations; and (2) the effectiveness of a perturbation-based training to improve their dynamic stability during balance recovery responses and steady walking. Specifically, we hypothesized that: (1) CA group can show a corrective behavior similar to that of a healthy control group; (2) the exposure to a perturbation-based treatment can exploit residual learning capability, thus improving their dynamic stability during balance recovery responses and steady locomotion. METHODS: Ten participants with cerebellar ataxia and eight age-matched healthy adults were exposed to a single perturbation-based training session. The Active Tethered Pelvic Assist Device applied unexpected waist-pull perturbations while participants walked on a treadmill. Spatio-temporal parameters and dynamic stability were determined during corrective responses and steady locomotion, before and after the training. The ANalysis Of VAriance was the main statistical test used to assess the effects of group (healthy vs CA) and training (baseline vs post) on spatio-temporal parameters of the gait and margin of stability. RESULTS: Data analysis revealed that individuals with cerebellar ataxia behaved differently from healthy volunteers: (1) they retained a wider base of support during corrective responses and steady gait both before and after the training; (2) due to the training, patients improved their anterior-posterior margin of stability during steady walking only. CONCLUSIONS: Our results revealed that participants with cerebellar ataxia could still rely on their learning capability to modify the gait towards a safer behavior. However, they could not take advantage from their residual learning capability while managing sudden and unexpected perturbations. Accordingly, the proposed training paradigm can be considered as a promising approach to improve balance control during steady walking in these individuals.

Effects of a smartphone application for cognitive rehearsal intervention on workplace bullying and turnover intention among nurses.

BACKGROUND: Bullying in nursing workplaces has been considered a serious problem that increases nurse turnover. AIM: To develop a cognitive rehearsal intervention for workplace bullying and examine its effects on nurses' bullying experiences and turnover intentions. METHODS: We developed a smartphone application to cognitively train nurses to handle bullying situations in the workplace. This application included common bullying situations and appropriate non-violent communication scenarios. A cluster quasi-randomized trial was performed with 72 hospital nurses working in a university hospital in South Korea from November 2016 to January 2017. We measured workplace bullying experiences and turnover intention before intervention and 4 and 8 weeks after intervention in both intervention and control groups. RESULTS: The cognitive rehearsal intervention developed in this study was effective for decreasing nurses' person-related bullying, work-related bullying experiences, and turnover intention. However, it had no effects on intimidation-related bullying experiences. CONCLUSIONS: The smartphone application-based cognitive rehearsal intervention can serve as a personal coping measure for person-related and work-related bullying among nurses. It is recommended that the intervention developed in this study be applied as a strategy to reduce nurse turnover.

Abolishment of N-glycan mannosylphosphorylation in glyco-engineered Saccharomyces cerevisiae by double disruption of MNN4 and MNN14 genes.

Mannosylphosphorylated glycans are found only in fungi, including yeast, and the elimination of mannosylphosphates from glycans is a prerequisite for yeast glyco-engineering to produce human-compatible glycoproteins. In Saccharomyces cerevisiae, MNN4 and MNN6 genes are known to play roles in mannosylphosphorylation, but disruption of these genes does not completely remove the mannosylphosphates in N-glycans. This study was performed to find unknown key gene(s) involved in N-glycan mannosylphosphorylation in S. cerevisiae. For this purpose, each of one MNN4 and five MNN6 homologous genes were deleted from the och1Δmnn1Δmnn4Δmnn6Δ strain, which lacks yeast-specific hyper-mannosylation and the immunogenic α(1,3)-mannose structure. N-glycan profile analysis of cell wall mannoproteins and a secretory recombinant protein produced in mutants showed that the MNN14 gene, an MNN4 paralog with unknown function, is essential for N-glycan mannosylphosphorylation. Double disruption of MNN4 and MNN14 genes was enough to eliminate N-glycan mannosylphosphorylation. Our results suggest that the S. cerevisiae och1Δmnn1Δmnn4Δmnn14Δ strain, in which all yeast-specific N-glycan structures including mannosylphosphorylation are abolished, may have promise as a useful platform for glyco-engineering to produce therapeutic glycoproteins with human-compatible N-glycans.

Monoclonal B-cell lymphocytosis in healthy blood donors: an unexpectedly common finding.

Circulating monoclonal B cells may be detected in healthy adults, a condition called monoclonal B-cell lymphocytosis (MBL). MBL has also been identified in donated blood, but no systematic study of blood donors has been reported. Using sensitive and specific laboratory methods, we detected MBL in 149 (7.1%; 95% confidence interval, 6.0% to 8.3%) of 2098 unique donors ages 45 years or older in a Midwestern US regional blood center between 2010 and 2011. Most of the 149 donors had low-count MBL, including 99 chronic lymphocytic leukemia-like (66.4%), 22 atypical (14.8%), and 19 CD5(-) (12.8%) immunophenotypes. However, 5 donors (3.4%) had B-cell clonal counts above 500 cells per µL, including 3 with 1693 to 2887 cells per µL; the clone accounted for nearly all their circulating B cells. Four donors (2.7%) had 2 distinct MBL clones. Of 51 MBL samples in which immunoglobulin heavy chain (IGH)V-D-J genotypes could be determined, 71% and 29% used IGHV3- and IGHV4-family genes, respectively. Sequencing revealed 82% with somatic hypermutation, whereas 18% had >98% germ-line identity, including 5 with entirely germ-line sequences. In conclusion, MBL prevalence is much higher in blood donors than previously reported, and although uncommon, the presence of high-count MBL warrants further investigations to define the biological fate of the transfused cells in recipients.

Comparison of fluorescent tags for analysis of mannose-6-phosphate glycans.

Mannose-6-phosphate (M-6-P) glycan analysis is important for quality control of therapeutic enzymes for lysosomal storage diseases. Here, we found that the analysis of glycans containing two M-6-Ps was highly affected by the hydrophilicity of the elution solvent used in high-performance liquid chromatography (HPLC). In addition, the performances of three fluorescent tags--2-aminobenzoic acid (2-AA), 2-aminobenzamide (2-AB), and 3-(acetyl-amino)-6-aminoacridine (AA-Ac)--were compared with each other for M-6-P glycan analysis using HPLC and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The best performance for analyzing M-6-P glycans was shown by 2-AA labeling in both analyses.