Modeling heritability of temperamental differences, stress reactivity, and risk for anxiety and depression: Relevance to research domain criteria (RDoC).

Animal models provide important tools to study biological and environmental factors that shape brain function and behavior. These models can be effectively leveraged by drawing on concepts from the National Institute of Mental Health Research Domain Criteria (RDoC) Initiative, which aims to delineate molecular pathways and neural circuits that underpin behavioral anomalies that transcend psychiatric conditions. To study factors that contribute to individual differences in emotionality and stress reactivity, our laboratory utilized Sprague-Dawley rats that were selectively bred for differences in novelty exploration. Selective breeding for low versus high locomotor response to novelty produced rat lines that differ in behavioral domains relevant to anxiety and depression, particularly the RDoC Negative Valence domains, including acute threat, potential threat, and loss. Bred Low Novelty Responder (LR) rats, relative to their High Responder (HR) counterparts, display high levels of behavioral inhibition, conditioned and unconditioned fear, avoidance, passive stress coping, anhedonia, and psychomotor retardation. The HR/LR traits are heritable, emerge in the first weeks of life, and appear to be driven by alterations in the developing amygdala and hippocampus. Epigenomic and transcriptomic profiling in the developing and adult HR/LR brain suggest that DNA methylation and microRNAs, as well as differences in monoaminergic transmission (dopamine and serotonin in particular), contribute to their distinct behavioral phenotypes. This work exemplifies ways that animal models such as the HR/LR rats can be effectively used to study neural and molecular factors driving emotional behavior, which may pave the way toward improved understanding the neurobiological mechanisms involved in emotional disorders.

Altered DNA Methylation in the Developing Brains of Rats Genetically Prone to High versus Low Anxiety.

There is growing evidence of abnormal epigenetic processes playing a role in the neurobiology of psychiatric disorders, although the precise nature of these anomalies remains largely unknown. To study neurobiological (including epigenetic) factors that influence emotionality, we use rats bred for distinct behavioral responses to novelty. Rats bred for low novelty response (low responder [LR]) exhibit high levels of anxiety- and depressive-like behavior compared with high novelty responder (HR) rats. Prior work revealed distinct limbic brain development in HR versus LR rats, including altered expression of genes involved in DNA methylation. This led us to hypothesize that DNA methylation differences in the developing brain drive the disparate HR/LR neurobehavioral phenotypes. Here we report altered DNA methylation markers (altered DNA methyltransferase protein levels and increased global DNA methylation levels) in the early postnatal amygdala of LR versus HR male rats. Next-generation sequencing methylome profiling identified numerous differentially methylated regions across the genome in the early postnatal HR/LR amygdala. We also contrasted methylation profiles of male HRs and LRs with a control rat strain that displays an intermediate behavioral phenotype relative to the HR/LR extremes; this revealed that the LR amygdalar methylome was abnormal, with the HR profile more closely resembling that of the control group. Finally, through two methylation manipulations in early life, we found that decreasing DNA methylation in the developing male and female amygdala improves adult anxiety- and depression-like behavior. These findings suggest that inborn DNA methylation differences play important roles in shaping brain development and lifelong emotional behavior.SIGNIFICANCE STATEMENT Epigenetic changes are biological mechanisms that regulate the expression and function of genes throughout the brain and body. DNA methylation, one type of epigenetic mechanism, is known to be altered in brains of psychiatric patients, which suggests a role for DNA methylation in the pathogenesis of psychiatric disorders, such as depression and anxiety. The present study examines brains of rats that display high versus low levels of anxiety- and depression-like behavior to investigate how neural DNA methylation levels differ in these animals and how such differences shape their emotional behavioral differences. Studying how epigenetic processes affect emotional behavior may improve our understanding of the neurobiology of psychiatric disorders and lead to improved treatments.

Treatment of pediatric high-grade central nervous system tumors with high-dose methotrexate in combination with multiagent chemotherapy: A single-institution experience.

BACKGROUND: Effective treatment for pediatric embryonal brain tumors includes dose-intensive multiagent chemotherapy (DIMAC) followed by high-dose chemotherapy with stem cell rescue (HDCSCR). Use of repeated cycles of DIMAC including high-dose methotrexate (HDMTX) without HDCSCR has not been described. PROCEDURE: We retrospectively reviewed the responses/toxicities in 13 patients (aged 2-155 months, median 22 months) with central nervous system (CNS) tumors (atypical teratoid rhabdoid tumors, CNS embryonal tumors not otherwise specified, pineoblastoma, embryonal tumor with multilayered rosettes, and CNS sarcoma) treated over a 12-year period with repeated cycles of HDMTX followed by etoposide, cisplatin, cyclophosphamide, and vincristine. RESULTS: Six patients (46.2%) had disseminated disease at presentation and five (38.5%) had gross total resection. A total of 64 courses of therapy were administered with a median of five courses per patient.  Eight patients (61.5%) received radiation therapy (one at relapse). By completion of therapy, 11 patients (84.6%) achieved a response (six complete, five partial).  Six of the 13 patients (46.2%) remain alive with a median follow-up of 48 months (6-146).  Acute toxicities included fever/neutropenia (70.3%), bacteremia (15.6%), and grade 3 mucositis (18.8%).  Long-term complications included learning disability, seizure disorder, and brain necrosis, without treatment-related deaths. CONCLUSIONS: DIMAC with HDMTX without HDCSCR may be an effective treatment option for selected patients with embryonal or high-grade CNS tumors.

Rats bred for high anxiety exhibit distinct fear-related coping behavior, hippocampal physiology, and synaptic plasticity-related gene expression.

The hippocampus is essential for learning and memory but also regulates emotional behavior. We previously identified the hippocampus as a major brain region that differs in rats bred for emotionality differences. Rats bred for low novelty response (LRs) exhibit high levels of anxiety- and depression-like behavior compared to high novelty responder (HR) rats. Manipulating the hippocampus of high-anxiety LR rats improves their behavior, although no work to date has examined possible HR/LR differences in hippocampal synaptic physiology. Thus, the current study examined hippocampal slice electrophysiology, dendritic spine density, and transcriptome profiling in HR/LR hippocampus, and compared performance on three hippocampus-dependent tasks: The Morris water maze, contextual fear conditioning, and active avoidance. Our physiology experiments revealed increased long-term potentiation (LTP) at CA3-CA1 synapses in HR versus LR hippocampus, and Golgi analysis found an increased number of dendritic spines in basal layer of CA1 pyramidal cells in HR versus LR rats. Transcriptome data revealed glutamate neurotransmission as the top functional pathway differing in the HR/LR hippocampus. Our behavioral experiments showed that HR/LR rats exhibit similar learning and memory capability in the Morris water maze, although the groups differed in fear-related tasks. LR rats displayed greater freezing behavior in the fear-conditioning task, and HR/LR rats adopted distinct behavioral strategies in the active avoidance task. In the active avoidance task, HRs avoided footshock stress by pressing a lever when presented with a warning cue; LR rats, on the other hand, waited until footshocks began before pressing the lever to stop them. Taken together, these findings concur with prior observations of HR rats generally exhibiting active stress coping behavior while LRs exhibit reactive coping. Overall, our current findings coupled with previous work suggest that HR/LR differences in stress reactivity and stress coping may derive, at least in part, from differences in the developing and adult hippocampus.

An Integrated Bioprocess to Recover Bovine Milk Oligosaccharides from Colostrum Whey Permeate.

A major challenge in isolating oligosaccharides from dairy streams is to enrich oligosaccharides while simultaneously reducing the content of simple sugars (mono- and disaccharides) that do not possess the desired prebiotic functions. An integrated approach based on optimized conditions that favor maximum lactose hydrolysis, monosaccharide fermentation and oligosaccharides recovery by nanofiltration was developed. Upon complete lactose hydrolysis and fermentation of the monosaccharides by yeast, nanofiltration of fermented whey permeate from colostrum enabled the recovery of 95% of the oligosaccharides at high purity. While the number of commercially available standards has limited the quantification of only a few sialylated oligosaccharides, the application of both high performance anion-exchange chromatography with pulsed amperometric detection and mass spectrometry provided a complete profile of the final product. Approximately 85% of the oligosaccharides in the final concentrate were sialylated, with the remainder being neutral.

Amygdalar expression of the microRNA miR-101a and its target Ezh2 contribute to rodent anxiety-like behaviour.

A greater understanding of neural mechanisms contributing to anxiety is needed in order to develop better therapeutic interventions. This study interrogates a novel molecular mechanism that shapes anxiety-like behaviour, demonstrating that the microRNA miR-101a-3p and its target, enhancer of zeste homolog 2 (Ezh2) in the amygdala, contribute to rodent anxiety-like behaviour. We utilized rats that were selectively bred for differences in emotionality and stress reactivity, showing that high-novelty-responding (HR) rats, which display low trait anxiety, have lower miR-101a-3p levels in the amygdala compared to low-novelty-responding (LR) rats that characteristically display high trait anxiety. To determine whether there is a causal relationship between amygdalar miR-101a-3p and anxiety behaviour, we used a viral approach to overexpress miR-101a-3p in the amygdala of HR rats and test whether it would increase their typically low levels of anxiety-like behaviour. We found that increasing miR-101a-3p in the amygdala increased HRs' anxiety-like behaviour in the open-field test and elevated plus maze. Viral-mediated miR-101a-3p overexpression also reduced expression of the histone methyltransferase Ezh2, which mediates gene silencing via trimethylation of histone 3 at lysine 27 (H3K27me3). Knockdown of Ezh2 with short-interfering RNA (siRNA) also increased HRs' anxiety-like behaviour, but to a lesser degree than miR-101a-3p overexpression. Overall, our data demonstrate that increasing miR-101a-3p expression in the amygdala increases anxiety-like behaviour and that this effect is at least partially mediated via repression of Ezh2. This work adds to the growing body of evidence implicating miRNAs and epigenetic regulation as molecular mediators of anxiety behaviour.

Role of pH in the recovery of bovine milk oligosaccharides from colostrum whey permeate by nanofiltration.

Milk oligosaccharides are associated with improved health outcomes in infants. Nanofiltration (NF) is used for isolation of bovine milk oligosaccharides (BMO). The study aim was to improve the recovery of BMO from lactose-hydrolyzed colostrum whey permeate. The retention factors of carbohydrates at various pH and transmembrane pressures were determined for a nanofiltration membrane, which was used at pilot scale to purify BMO. Carbohydrates were quantified by liquid chromatography and characterized using nano-LC-Chip-QToF mass spectrometry. BMO purity was improved from an initial 4% in colostrum whey permeate to 98%, with 99.8% permeation of monosaccharides and 96% recovery of oligosaccharides, represented by 23 unique BMO compounds identified in the final retentate. The pH during NF was a determining factor in the selectivity of carbohydrate separation. This NF method can be applied to conventional cheese-whey permeate and other milk types for extraction of bioactive oligosaccharides providing new options for the dairy industry.

Purification of caprine oligosaccharides at pilot-scale.

The purification of caprine milk oligosaccharides (COS) by membrane filtration has been hampered by the low concentration of target COS and high concentration of lactose. In addition, their molecular weight proximity hinders the recovery of a COS fraction with high degree of purity and recovery yield. In this work, the recovery of a high purity COS concentrate was obtained by the optimization of an integrated approach including complete lactose hydrolysis, fermentation of the resulting monosaccharides and nanofiltration. All carbohydrates were quantified using High Performance Anion Exchange Chromatography with Pulsed Amperometric Detection (HPAEC PAD). Defatted goat whey was ultrafiltered with discontinuous diafiltrations to increase the recovery of COS in the whey permeate which was then subsequently concentrated by nanofiltration. COS recovery yields of 75% with negligible amounts of monosaccharides (0.3% of the initial amount of lactose in the whey permeate) were achieved. A final retentate containing 67.6 and 34.4% of acidic and neutral oligosaccharides respectively was obtained from caprine milk.

Validation of a paper-disk approach to facilitate the sensory evaluation of bitterness in dairy protein hydrolysates from a newly developed food-grade fractionation system.

Casein-hydrolysates (NaCaH) are desirable functional ingredients, but their bitterness impedes usage in foods. This study sought to validate a paper-disk approach to help evaluate bitterness in NaCaHs and to develop a food-grade approach to separate a NaCaH into distinct fractions, which could be evaluated by a sensory panel. Membrane filtration generated <0.2-µm and <3-kDa permeates. Further fractionation of the <3-kDa permeate by flash-chromatography generated four fractions using ethanol (EtOH) concentrations of 5, 10, 30 and 50%. As some fractions were poorly soluble in water, the fractions were resolubilzed in EtOH and impregnated into paper-disks for sensory evaluation. Bitterness differences observed in the membrane fractions using this sensory evaluation approach reflected those observed for the same fractions presented as a liquid. The flash-chromatography fractions increased in bitterness with an increase in hydrophobicity, except for the 50% EtOH fraction which had little bitterness. Amino acid analysis of the fractions showed enrichment of different essential amino acids in both the bitter and less bitter fractions. Practical Applications: The developed food-grade fractionation system, allowed for a simple and reasonably scaled approach to separating a NaCaH, into physicochemically different fractions that could be evaluated by a sensory panel. The method of sensory evaluation used in this study, in which NaCaH samples are impregnated into paper-disks, provided potential solutions for issues such as sample insolubility and limited quantities of sample. As the impregnated paper-disk samples were dehydrated, their long storage life could also be suitable for sensory evaluations distributed by mail for large consumer studies. The research, in this study, allowed for a greater understanding of the physicochemical basis for bitterness in this NaCaH. As some essential amino acids were enriched in the less bitter fractions, selective removal of bitter fractions could allow for the incorporation of the less bitter NaCaH fractions into food products for added nutritional value, without negatively impacting sensory properties. There is potential for this approach to be applied to other food ingredients with undesirable tastes, such as polyphenols.

Modeling lactose hydrolysis for efficiency and selectivity: Toward the preservation of sialyloligosaccharides in bovine colostrum whey permeate.

Enzymatic hydrolysis of lactose has been shown to improve the efficiency and selectivity of membrane-based separations toward the recovery of bioactive oligosaccharides. Achieving maximum lactose hydrolysis requires intrinsic process optimization for each specific substrate, but the effects of those processing conditions on the target oligosaccharides are not well understood. Response surface methodology was used to investigate the effects of pH (3.25-8.25), temperature (35-55°C), reaction time (6 to 58 min), and amount of enzyme (0.05-0.25%) on the efficiency of lactose hydrolysis by ß-galactosidase and on the preservation of biologically important sialyloligosaccharides (3'-siallylactose, 6'-siallylactose, and 6'-sialyl-N-acetyllactosamine) naturally present in bovine colostrum whey permeate. A central composite rotatable design was used. In general, ß-galactosidase activity was favored at pH values ranging from 3.25 to 5.75, with other operational parameters having a less pronounced effect. A pH of 4.5 allowed for the use of a shorter reaction time (19 min), lower temperature (40°C), and reduced amount of enzyme (0.1%), but complete hydrolysis at a higher pH (5.75) required greater values for these operational parameters. The total amount of sialyloligosaccharides was not significantly altered by the reaction parameters evaluated, suggesting specificity of ß-galactosidase from Aspergillus oryzae toward lactose as well as the stability of the oligosaccharides at pH, temperature, and reaction time evaluated.

Maternal Style Selectively Shapes Amygdalar Development and Social Behavior in Rats Genetically Prone to High Anxiety.

The early-life environment critically influences neurodevelopment and later psychological health. To elucidate neural and environmental elements that shape emotional behavior, we developed a rat model of individual differences in temperament and environmental reactivity. We selectively bred rats for high versus low behavioral response to novelty and found that high-reactive (bred high-responder, bHR) rats displayed greater risk-taking, impulsivity and aggression relative to low-reactive (bred low-responder, bLR) rats, which showed high levels of anxiety/depression-like behavior and certain stress vulnerability. The bHR/bLR traits are heritable, but prior work revealed bHR/bLR maternal style differences, with bLR dams showing more maternal attention than bHRs. The present study implemented a cross-fostering paradigm to examine the contribution of maternal behavior to the brain development and emotional behavior of bLR offspring. bLR offspring were reared by biological bLR mothers or fostered to a bLR or bHR mother and then evaluated to determine the effects on the following: (1) developmental gene expression in the hippocampus and amygdala and (2) adult anxiety/depression-like behavior. Genome-wide expression profiling showed that cross-fostering bLR rats to bHR mothers shifted developmental gene expression in the amygdala (but not hippocampus), reduced adult anxiety and enhanced social interaction. Our findings illustrate how an early-life manipulation such as cross-fostering changes the brain's developmental trajectory and ultimately impacts adult behavior. Moreover, while earlier studies highlighted hippocampal differences contributing to the bHR/bLR phenotypes, our results point to a role of the amygdala as well. Future work will pursue genetic and cellular mechanisms within the amygdala that contribute to bHR/bLR behavior either at baseline or following environmental manipulations. © 2015 S. Karger AG, Basel.

Amygdala-hippocampus somatostatin interneuron beta-synchrony underlies a cross-species biomarker of emotional state.

Emotional responses arise from limbic circuits including the hippocampus and amygdala. In the human brain, beta-frequency communication between these structures correlates with self-reported mood and anxiety. However, both the mechanism and significance of this biomarker as a readout vs. driver of emotional state remain unknown. Here, we show that beta-frequency communication between ventral hippocampus and basolateral amygdala also predicts anxiety-related behavior in mice, both on long timescales (∼30 min) and immediately preceding behavioral choices. Genetically encoded voltage indicators reveal that this biomarker reflects synchronization between somatostatin interneurons across both structures. Indeed, synchrony between these neurons dynamically predicts approach-avoidance decisions, and optogenetically shifting the phase of synchronization by just 25 ms is sufficient to bidirectionally modulate anxiety-related behaviors. Thus, back-translation establishes a human biomarker as a causal determinant (not just predictor) of emotional state, revealing a novel mechanism whereby interregional synchronization that is frequency, phase, and cell type specific controls emotional processing.

Closed-loop neurostimulation for the treatment of psychiatric disorders.

Despite increasing prevalence and huge personal and societal burden, psychiatric diseases still lack treatments which can control symptoms for a large fraction of patients. Increasing insight into the neurobiology underlying these diseases has demonstrated wide-ranging aberrant activity and functioning in multiple brain circuits and networks. Together with varied presentation and symptoms, this makes one-size-fits-all treatment a challenge. There has been a resurgence of interest in the use of neurostimulation as a treatment for psychiatric diseases. Initial studies using continuous open-loop stimulation, in which clinicians adjusted stimulation parameters during patient visits, showed promise but also mixed results. Given the periodic nature and fluctuations of symptoms often observed in psychiatric illnesses, the use of device-driven closed-loop stimulation may provide more effective therapy. The use of a biomarker, which is correlated with specific symptoms, to deliver stimulation only during symptomatic periods allows for the personalized therapy needed for such heterogeneous disorders. Here, we provide the reader with background motivating the use of closed-loop neurostimulation for the treatment of psychiatric disorders. We review foundational studies of open- and closed-loop neurostimulation for neuropsychiatric indications, focusing on deep brain stimulation, and discuss key considerations when designing and implementing closed-loop neurostimulation.

A multicomponent intervention reduces body weight and cardiovascular risk at a GEICO corporate site.

PURPOSE: To determine whether a multicomponent nutrition intervention program at a corporate site reduces body weight and improves other cardiovascular risk factors in overweight individuals. DESIGN: Prospective clinical intervention study. SUBJECTS/SETTING: Employees of the Government Employees Insurance Company (GEICO) (N = 113), aged 21 to 65 years, with a body mass index > or =25 kg/m(2) and/or previous diagnosis of type 2 diabetes. INTERVENTION: A 22-week intervention including a low-fat, vegan diet. MEASURES: Changes in body weight, anthropometric measures, blood pressure, lipid profile, and dietary intake. ANALYSIS: Multivariate analyses of variance were calculated for clinical and nutrient measures, followed by univariate analyses of variance, to determine the significance of differences between groups in changes over time. RESULTS: Intervention-group participants experienced greater weight changes compared with control-group participants (mean, -5.1 [SE, .6] kg vs. + .1 [SE, .6] kg, p < .0001), as well as greater changes in waist circumference (mean, -4.7 [SE, .6] cm vs. + .8 [SE, .6] cm, p < .0001) and waistratiohip ratio (mean, -.006 [SE, .003] vs. + .014 [SE, .005], p = .0007). Weight loss of 5% of body weight was more frequently observed in the intervention group (48.5%) compared with the control group (11.1%) (chi(2)[1, N = 113] = 16.99, p < .0001). CONCLUSIONS: Among individuals volunteering for a 22-week worksite research study, an intervention using a low-fat, vegan diet effectively reduced body weight and waist circumference.

A worksite vegan nutrition program is well-accepted and improves health-related quality of life and work productivity.

BACKGROUND/AIMS: Vegetarian and vegan diets are effective in preventing and treating several chronic diseases. However, their acceptability outside a clinical trial setting has not been extensively studied. The aim of this study was to determine the acceptability of a worksite vegan nutrition program and its effects on health-related quality of life and work productivity. METHODS: Employees of a major insurance corporation with a body mass index > or =25 kg/m(2) and/or a previous diagnosis of type 2 diabetes received either weekly group instruction on a low-fat vegan diet (n = 68) or received no diet instruction (n = 45) for 22 weeks. RESULTS: The vegan group reported improvements in general health (p = 0.002), physical functioning (p = 0.001), mental health (p = 0.03), vitality (p = 0.004), and overall diet satisfaction (p < 0.001) compared with the control group. The vegan group also reported a decrease in food costs (p = 0.003), and increased difficulty finding foods when eating out (p = 0.04) compared with the control group. The vegan group reported a 40-46% decrease in health-related productivity impairments at work (p = 0.03) and in regular daily activities (p = 0.004). CONCLUSIONS: A worksite vegan nutrition program is well-accepted and can be implemented by employers to improve the health, quality of life, and work productivity of employees.

Effects of Industrial Thermal Treatments on the Release of Bovine Colostrum Glycoprotein N-Glycans by Endo-ß-N-acetylglucosaminidase.

N-Glycans are structurally similar to human milk oligosaccharides, the gold standard prebiotics for infants. Bovine milk N-glycans released by endo-ß-N-acetylglucosaminidase (EndoBI-1) were shown to have similar prebiotic selectivity as human milk oligosaccharides, explaining the interest for N-glycan recovery for use as prebiotics. Industrial thermal treatments such as high-temperature short-time (HTST) and ultra-high-temperature (UHT) might favor the enzymatic deglycosylation of N-glycans through promoting protein denaturation. We investigated the effects of HTST (72 °C for 15 s) and UHT (135 °C for 3 s) on N-glycan release from bovine colostrum glycoproteins by nonimmobilized and amino-immobilized EndoBI-1. A total of 104 N-glycans including isomers/anomers were identified by high-resolution mass spectrometry. In both EndoBI-1 forms, HTST increased the release of N-glycans; however, the impact of UHT on releasing N-glycans was comparable to the nonthermal treatment. Although the amino-immobilized enzyme similarly released neutral N-glycans as the free form, it released fewer sialylated and fucosylated N-glycans.

Successful Resolution of Prominent Somatic Delusions Following Bi-temporal Electroconvulsive Therapy in a patient with Treatment-Resistant Schizoaffective Disorder.

Somatic delusions occur in a variety of psychiatric disorders including schizophrenia, major depressive disorder, and bipolar disorder. Somatization is associated with lower quality of life and greater risk for suicide. Treatment of somatic delusions is extremely challenging. Here we report an interesting case of severe somatic delusions in a 48-year-old African-American female with a long history of treatment resistant schizoaffective disorder, with multiple somatic complaints surrounding constipation, pregnancy, jaw pain, body aches, vaginal itch, malodorous urine, and neck pain, despite normal clinical examinations and negative medical work up. Additionally, she endorsed persistent auditory and visual hallucinations. Her symptoms remained resistant to several trials of psychotropic medications, including clozapine. Chart review of past hospitalizations revealed significant improvement with Electroconvulsive Therapy (ECT), so the team decided to perform a course of six bi-temporal ECT treatments administered over two weeks. Stimulation was applied at a current of 800 mA for 4.5s, with a pulse width of 1 ms and frequency of 60 Hz. This case illustrates the successful use of ECT in treating prominent somatic delusions in a patient with treatment-resistant schizoaffective disorder.

Conversion of Agricultural Streams and Food-Processing By-Products to Value-Added Compounds Using Filamentous Fungi.

The design of new food products and increased agricultural activities have produced a diversity of waste streams or by-products that contain a high load of organic matter. The underutilization of these streams presents a serious threat to the environment and to the financial viability of the agricultural sector and the food industry. Oleaginous microorganisms, such as yeast and microalgae, have been used to convert the organic matter present in many agricultural waste streams into an oil-rich biomass. Filamentous fungi are promising oleaginous microorganisms because of their high lipid accumulation potential and simple biomass recovery, the latter being related to their pellet-like growth morphology in submerged cultivation. This review highlights the use of oleaginous filamentous fungi to convert food by-products into value-added components, including the effect of cultivation conditions on biomass yield and composition. Special attention is given to downstream processing for the commercial production of fungal oil. Also discussed are innovative techniques to optimize the biomass oil yield and to minimize the challenges associated with biomass harvesting and oil extraction at industrial scale.

Immobilization of an Endo-ß-N-acetylglucosaminidase for the Release of Bioactive N-glycans.

As more is learned about glycoproteins' roles in human health and disease, the biological functionalities of N-linked glycans are becoming more relevant. Protein deglycosylation allows for the selective release of N-glycans and facilitates glycoproteomic investigation into their roles as prebiotics or anti-pathogenic factors. To increase throughput and enzyme reusability, this work evaluated several immobilization methods for an endo-ß-N-acetylglucosaminidase recently discovered from the commensal Bifidobacterium infantis. Ribonuclease B was used as a model glycoprotein to compare N-glycans released by the free and immobilized enzyme. Amino-based covalent method showed the highest enzyme immobilization. Relative abundance of N-glycans and enzyme activity were determined using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Kinetic evaluation demonstrated that upon immobilization, both Vmax and the Km decreased. Optimal pH values of 5 and 7 were identified for the free and immobilized enzyme, respectively. Although a higher temperature (65 vs. 45 °C) favored rapid glycan release, the immobilized enzyme retained over 50% of its original activity after seven use cycles at 45 °C. In view of future applications in the dairy industry, we investigated the ability of this enzyme to deglycosylate whey proteins. The immobilized enzyme released a higher abundance of neutral glycans from whey proteins, while the free enzyme released more sialylated glycans, determined by nano-LC Chip Q-ToF MS.