Hyperactivity of indirect pathway-projecting spiny projection neurons promotes compulsive behavior.

Compulsive behaviors are a hallmark symptom of obsessive compulsive disorder (OCD). Striatal hyperactivity has been linked to compulsive behavior generation in correlative studies in humans and causal studies in rodents. However, the contribution of the two distinct striatal output populations to the generation and treatment of compulsive behavior is unknown. These populations of direct and indirect pathway-projecting spiny projection neurons (SPNs) have classically been thought to promote or suppress actions, respectively, leading to a long-held hypothesis that increased output of direct relative to indirect pathway promotes compulsive behavior. Contrary to this hypothesis, here we find that indirect pathway hyperactivity is associated with compulsive grooming in the Sapap3-knockout mouse model of OCD-relevant behavior. Furthermore, we show that suppression of indirect pathway activity using optogenetics or treatment with the first-line OCD pharmacotherapy fluoxetine is associated with reduced grooming in Sapap3-knockouts. Together, these findings highlight the striatal indirect pathway as a potential treatment target for compulsive behavior.

Organizing activities of axial mesoderm.

For almost a century, developmental biologists have appreciated that the ability of the embryonic organizer to induce and pattern the body plan is intertwined with its differentiation into axial mesoderm. Despite this, we still have a relatively poor understanding of the contribution of axial mesoderm to induction and patterning of different body regions, and the manner in which axial mesoderm-derived information is interpreted in tissues of changing competence. Here, with a particular focus on the nervous system, we review the evidence that axial mesoderm notochord and prechordal mesoderm/mesendoderm act as organizers, discuss how their influence extends through the different axes of the developing organism, and describe how the ability of axial mesoderm to direct morphogenesis impacts on its role as a local organizer.

PI3K/mTOR is a therapeutically targetable genetic dependency in diffuse intrinsic pontine glioma.

Diffuse midline glioma (DMG), including tumors diagnosed in the brainstem (diffuse intrinsic pontine glioma; DIPG), are uniformly fatal brain tumors that lack effective treatment. Analysis of CRISPR/Cas9 loss-of-function gene deletion screens identified PIK3CA and MTOR as targetable molecular dependencies across patient derived models of DIPG, highlighting the therapeutic potential of the blood-brain barrier-penetrant PI3K/Akt/mTOR inhibitor, paxalisib. At the human-equivalent maximum tolerated dose, mice treated with paxalisib experienced systemic glucose feedback and increased insulin levels commensurate with patients using PI3K inhibitors. To exploit genetic dependence and overcome resistance while maintaining compliance and therapeutic benefit, we combined paxalisib with the antihyperglycemic drug metformin. Metformin restored glucose homeostasis and decreased phosphorylation of the insulin receptor in vivo, a common mechanism of PI3K-inhibitor resistance, extending survival of orthotopic models. DIPG models treated with paxalisib increased calcium-activated PKC signaling. The brain penetrant PKC inhibitor enzastaurin, in combination with paxalisib, synergistically extended the survival of multiple orthotopic patient-derived and immunocompetent syngeneic allograft models; benefits potentiated in combination with metformin and standard-of-care radiotherapy. Therapeutic adaptation was assessed using spatial transcriptomics and ATAC-Seq, identifying changes in myelination and tumor immune microenvironment crosstalk. Collectively, this study has identified what we believe to be a clinically relevant DIPG therapeutic combinational strategy.

Optogenetic recruitment of hypothalamic corticotrophin-releasing-hormone (CRH) neurons reduces motivational drive.

Impaired motivational drive is a key feature of depression. Chronic stress is a known antecedent to the development of depression in humans and depressive-like states in animals. Whilst there is a clear relationship between stress and motivational drive, the mechanisms underpinning this association remain unclear. One hypothesis is that the endocrine system, via corticotropin-releasing hormone (CRH) in the paraventricular nucleus of the hypothalamus (PVN; PVNCRH), initiates a hormonal cascade resulting in glucocorticoid release, and that excessive glucocorticoids change brain circuit function to produce depression-related symptoms. Another mostly unexplored hypothesis is that the direct activity of PVNCRH neurons and their input to other stress- and reward-related brain regions drives these behaviors. To further understand the direct involvement of PVNCRH neurons in motivation, we used optogenetic stimulation to activate these neurons 1 h/day for 5 consecutive days and showed increased acute stress-related behaviors and long-lasting deficits in the motivational drive for sucrose. This was associated with increased Fos-protein expression in the lateral hypothalamus (LH). Direct stimulation of the PVNCRH inputs in the LH produced a similar pattern of effects on sucrose motivation. Together, these data suggest that PVNCRH neuronal activity may be directly responsible for changes in motivational drive and that these behavioral changes may, in part, be driven by PVNCRH synaptic projections to the LH.

Coronavirus M Protein Trafficking in Epithelial Cells Utilizes a Myosin Vb Splice Variant and Rab10.

The membrane (M) glycoprotein of coronaviruses (CoVs) serves as the nidus for virion assembly. Using a yeast two-hybrid screen, we identified the interaction of the cytosolic tail of Murine Hepatitis Virus (MHV-CoV) M protein with Myosin Vb (MYO5B), specifically with the alternative splice variant of cellular MYO5B including exon D (MYO5B+D), which mediates interaction with Rab10. When co-expressed in human lung epithelial A549 and canine kidney epithelial MDCK cells, MYO5B+D co-localized with the MHV-CoV M protein, as well as with the M proteins from Porcine Epidemic Diarrhea Virus (PEDV-CoV), Middle East Respiratory Syndrome (MERS-CoV) and Severe Acute Respiratory Syndrome 2 (SARS-CoV-2). Co-expressed M proteins and MYO5B+D co-localized with endogenous Rab10 and Rab11a. We identified point mutations in MHV-CoV M that blocked the interaction with MYO5B+D in yeast 2-hybrid assays. One of these point mutations (E121K) was previously shown to block MHV-CoV virion assembly and its interaction with MYO5B+D. The E to K mutation at homologous positions in PEDV-CoV, MERS-CoV and SARS-CoV-2 M proteins also blocked colocalization with MYO5B+D. The knockdown of Rab10 blocked the co-localization of M proteins with MYO5B+D and was rescued by re-expression of CFP-Rab10. Our results suggest that CoV M proteins traffic through Rab10-containing systems, in association with MYO5B+D.

Accurate staging of chick embryonic tissues via deep learning of salient features.

Recent work shows that the developmental potential of progenitor cells in the HH10 chick brain changes rapidly, accompanied by subtle changes in morphology. This demands increased temporal resolution for studies of the brain at this stage, necessitating precise and unbiased staging. Here, we investigated whether we could train a deep convolutional neural network to sub-stage HH10 chick brains using a small dataset of 151 expertly labelled images. By augmenting our images with biologically informed transformations and data-driven preprocessing steps, we successfully trained a classifier to sub-stage HH10 brains to 87.1% test accuracy. To determine whether our classifier could be generally applied, we re-trained it using images (269) of randomised control and experimental chick wings, and obtained similarly high test accuracy (86.1%). Saliency analyses revealed that biologically relevant features are used for classification. Our strategy enables training of image classifiers for various applications in developmental biology with limited microscopy data.

Hypothalamic corticotrophin releasing hormone neurons in stress-induced psychopathology: Revaluation of synaptic contributions.

Stress has a strong influence on mental health around the world. Decades of research has sought to identify mechanisms through which stress contributes to psychiatric disorders such as depression, to potentially guide the development of therapeutics targeting stress systems. The hypothalamic pituitary adrenal (HPA) axis is the key endocrine system that is responsible for coordinating body-wide changes that are necessary for survival under stress, and much of the research aimed at understanding the mechanisms by which stress contributes to depression has focussed on HPA axis dysfunction. Corticotrophin releasing hormone (CRH) neurons in the paraventricular nucleus of the hypothalamus (PVN) sit at the apex of the HPA axis, integrating signals relevant to stress and external threats, to ensure HPA axis activity is appropriate for the given context. In addition to this, emerging research has demonstrated that neural activity in PVNCRH neurons regulates stress related behaviours via modulation of downstream synaptic targets. This review will summarize convergent evidence from preclinical studies on chronic stress and clinical research in mood disorders demonstrating changes in PVNCRH neural function, consider how this may influence synaptic targets of PVNCRH neurons, and discuss the potential role of these PVNCRH synaptic pathways in the development of maladaptive behaviours following chronic stress that are relevant to depression. We will also highlight important questions for future research aimed at precisely dissecting endocrine and synaptic roles of PVNCRH neurons in chronic stress, their potential interactions, and therapeutic opportunities for the treatment of stress related disorders.

Real-World Comparative Safety and Effectiveness of Irreversible Electroporation and High-Intensity Focused Ultrasound for Prostate Cancer Ablation.

OBJECTIVES: To assess the comparative safety and effectiveness of 2 prostate cancer treatment ablation modalities: irreversible electroporation (IRE) and high-intensity focused ultrasound (HIFU).  METHODS: Two systematic literature reviews (SLRs) and meta-analyses (MAs) on IRE and HIFU were conducted in accordance with PRISMA guidelines. Searches were conducted in PubMed and EMBASE. Independent reviewers assessed literature eligibility and abstracted safety and effectiveness data. Oncological, safety, functional, and quality of life (QOL) outcomes were examined for each technology. MAs were conducted where data quality and availability allowed, using normal methods and a random/mixed effects model, and quality assessments performed. RESULTS: Fifty-five publications (n = 22 IRE; n = 33 HIFU) were included in the SLRs, and MAs were conducted on negative in-field post-procedure biopsy, prostate-specific antigen (PSA) level reduction, potency, urinary continence, and AE rate outcomes. MAs revealed that IRE patients had lower mean percent PSA level reductions, higher mean rates of in-field negative post-treatment biopsy, and higher rates of potency maintenance than HIFU patients. Most adverse events (AEs) reported were comparable and minor (Grades I, II), with urinary tract infection, dysuria, hematuria, and incontinence or urgency most frequently reported. The proportion of patients experiencing a severe AE (≥Grade III) ranged from 0 to 8% after IRE and HIFU. Both modalities were associated with positive functional outcomes as well as maintenance of QOL after treatment. CONCLUSIONS: Both IRE and HIFU were found to produce favorable effectiveness outcomes and have low complication rates while minimally impacting patient urinary and erectile function and maintaining overall QOL. These real-world findings can help guide clinical decision making and improve disease management for patients with prostate cancer.

A neuroepithelial wave of BMP signalling drives anteroposterior specification of the tuberal hypothalamus.

The tuberal hypothalamus controls life-supporting homeostatic processes, but despite its fundamental role, the cells and signalling pathways that specify this unique region of the central nervous system in embryogenesis are poorly characterised. Here, we combine experimental and bioinformatic approaches in the embryonic chick to show that the tuberal hypothalamus is progressively generated from hypothalamic floor plate-like cells. Fate-mapping studies show that a stream of tuberal progenitors develops in the anterior-ventral neural tube as a wave of neuroepithelial-derived BMP signalling sweeps from anterior to posterior through the hypothalamic floor plate. As later-specified posterior tuberal progenitors are generated, early specified anterior tuberal progenitors become progressively more distant from these BMP signals and differentiate into tuberal neurogenic cells. Gain- and loss-of-function experiments in vivo and ex vivo show that BMP signalling initiates tuberal progenitor specification, but must be eliminated for these to progress to anterior neurogenic progenitors. scRNA-Seq profiling shows that tuberal progenitors that are specified after the major period of anterior tuberal specification begin to upregulate genes that characterise radial glial cells. This study provides an integrated account of the development of the tuberal hypothalamus.

Distinct Patterns of Abnormal Lateral Orbitofrontal Cortex Activity During Compulsive Grooming and Reversal Learning Normalize After Fluoxetine.

BACKGROUND: Patients with obsessive-compulsive disorder (OCD) display disrupted performance and abnormal lateral orbitofrontal cortex (LOFC) activity during reversal learning tasks. However, it is unknown whether compulsions and reversal learning deficits share a common neural substrate. To answer this question, we measured neural activity with in vivo calcium imaging in LOFC during compulsive grooming and reversal learning before and after fluoxetine treatment. METHODS: Sapap3 knockout (KO) mice were used as a model for OCD-relevant behaviors. Sapap3 KOs and control littermates were injected with a virus encoding GCaMP6f and implanted with gradient-index lenses to visualize LOFC activity using miniature microscopes. Grooming, reversal learning, and neural activity were measured pre- and post-fluoxetine treatment (18 mg/kg, 4 weeks). RESULTS: Baseline compulsive grooming and reversal learning impairments in KOs improved after fluoxetine treatment. In addition, KOs displayed distinct patterns of abnormal LOFC activity during grooming and reversal learning, both of which normalized after fluoxetine. Finally, reversal learning-associated neurons were distributed randomly among grooming-associated neurons (i.e., overlap is what would be expected by chance). CONCLUSIONS: In OCD, LOFC is disrupted during both compulsive behaviors and reversal learning, but whether these behaviors share common neural underpinnings is unknown. We found that LOFC plays distinct roles in compulsive grooming and impaired reversal learning and their improvement with fluoxetine. These findings suggest that LOFC plays separate roles in pathophysiology and treatment of different perseverative behaviors in OCD.

Differential Effects of Chronic Methamphetamine Treatment on High-Frequency Oscillations and Responses to Acute Methamphetamine and NMDA Receptor Blockade in Conscious Mice.

Dysregulation of high-frequency neuronal oscillations has been implicated in the pathophysiology of schizophrenia. Chronic methamphetamine (METH) use can induce psychosis similar to paranoid schizophrenia. The current study in mice aimed to determine the effect of chronic METH treatment on ongoing and evoked neuronal oscillations. C57BL/6 mice were treated with METH or vehicle control for three weeks and implanted with extradural recording electrodes. Two weeks after the last METH injection, mice underwent three EEG recording sessions to measure ongoing and auditory-evoked gamma and beta oscillatory power in response to an acute challenge with METH (2 mg/kg), the NMDA receptor antagonist MK-801 (0.3 mg/kg), or saline control. A separate group of mice pretreated with METH showed significantly greater locomotor hyperactivity to an acute METH challenge, confirming long-term sensitisation. Chronic METH did not affect ongoing or evoked gamma or beta power. Acute MK-801 challenge reduced ongoing beta power whereas acute METH challenge significantly increased ongoing gamma power. Both MK-801 and METH challenge suppressed evoked gamma power. Chronic METH treatment did not modulate these acute drug effects. There were minor effects of chronic METH and acute METH and MK-801 on selected components of event-related potential (ERP) waves. In conclusion, chronic METH treatment did not exert neuroplastic effects on the regulation of cortical gamma oscillations in a manner consistent with schizophrenia, despite causing behavioural sensitisation.

Distinct mechanisms of planar polarization by the core and Fat-Dachsous planar polarity pathways in the Drosophila wing.

Planar polarity describes the coordinated polarization of cells within a tissue plane, and in animals can be determined by the "core" or Fat-Dachsous pathways. Current models for planar polarity establishment involve two components: tissue-level "global" cues that determine the overall axis of polarity and cell-level feedback-mediated cellular polarity amplification. Here, we investigate the contributions of global cues versus cellular feedback amplification in the core and Fat-Dachsous pathways during Drosophila pupal wing development. We present evidence that these pathways generate planar polarity via distinct mechanisms. Core pathway function is consistent with strong feedback capable of self-organizing cell polarity, which can then be aligned with the tissue axis via weak or transient global cues. Conversely, generation of cell polarity by the Ft-Ds pathway depends on strong global cues in the form of graded patterns of gene expression, which can then be amplified by weak feedback mechanisms.

SHH and Notch regulate SOX9+ progenitors to govern arcuate POMC neurogenesis.

Pro-opiomelanocortin (POMC)-expressing neurons in the hypothalamic arcuate nucleus (ARC) play key roles in feeding and energy homoeostasis, hence their development is of great research interest. As the process of neurogenesis is accompanied by changes in adhesion, polarity, and migration that resemble aspects of epithelial-to-mesenchymal transitions (EMTs), we have characterised the expression and regulation within the prospective ARC of transcription factors with context-dependent abilities to regulate aspects of EMT. Informed by pseudotime meta-analysis of recent scRNA-seq data, we use immunohistochemistry and multiplex in situ hybridisation to show that SOX2, SRY-Box transcription factor 9 (SOX9), PROX1, Islet1 (ISL1), and SOX11 are sequentially expressed over the course of POMC neurogenesis in the embryonic chick. Through pharmacological studies ex vivo, we demonstrate that while inhibiting either sonic hedgehog (SHH) or Notch signalling reduces the number of SOX9+ neural progenitor cells, these treatments lead, respectively, to lesser and greater numbers of differentiating ISL1+/POMC+ neurons. These results are consistent with a model in which SHH promotes the formation of SOX9+ progenitors, and Notch acts to limit their differentiation. Both pathways are also required to maintain normal levels of proliferation and to suppress apoptosis. Together our findings demonstrate that hypothalamic neurogenesis is accompanied by dynamic expression of transcription factors (TFs) that mediate EMTs, and that SHH and Notch signalling converge to regulate hypothalamic cellular homoeostasis.

Rab11FIP1-deficient mice develop spontaneous inflammation and show increased susceptibility to colon damage.

The small GTPase, Rab11a, regulates vesicle trafficking and cell polarity in epithelial cells through interaction with Rab11 family-interacting proteins (Rab11-FIPs). We hypothesized that deficiency of Rab11-FIP1 would affect mucosal integrity in the intestine. Global Rab11FIP1 knockout (KO) mice were generated by deletion of the second exon. Pathology of intestinal tissues was analyzed by immunostaining of colonic sections and RNA-sequencing of isolated colonic epithelial cells. A low concentration of dextran sodium sulfate (DSS, 2%) was added to drinking water for 5 days, and injury score was compared between Rab11FIP1 KO, Rab11FIP2 KO, and heterozygous littermates. Rab11FIP1 KO mice showed normal fertility and body weight gain. More frequent lymphoid patches and infiltration of macrophages and neutrophils were identified in Rab11FIP1 KO mice before the development of rectal prolapse compared with control mice. The population of trefoil factor 3 (TFF3)-positive goblet cells was significantly lower, and the ratio of proliferative to nonproliferative cells was higher in Rab11FIP1 KO colons. Transcription signatures indicated that Rab11FIP1 deletion downregulated genes that mediate stress tolerance response, whereas genes mediating the response to infection were significantly upregulated, consistent with the inflammatory responses in the steady state. Lack of Rab11FIP1 also resulted in abnormal accumulation of subapical vesicles in colonocytes and the internalization of transmembrane mucin, MUC13, with Rab14. After DSS treatment, Rab11FIP1 KO mice showed greater body weight loss and more severe mucosal damage than those in heterozygous littermates. These findings suggest that Rab11FIP1 is important for cytoprotection mechanisms and for the maintenance of colonic mucosal integrity.NEW & NOTEWORTHY Although Rab11FIP1 is important in membrane trafficking in epithelial cells, the gastrointestinal phenotype of Rab11FIP1 knockout (KO) mice had never been reported. This study demonstrated that Rab11FIP1 loss induces mistrafficking of Rab14 and MUC13 and decreases in colonic goblet cells, resulting in impaired mucosal integrity.

An unusual case of comedones.

We present the case of a 71-year-old woman with widespread comedones since adolescence. Histological examination revealed branching hyperpigmented rete ridges and cystically dilated follicular infundibulum containing laminated keratinous debris. We explore the differential diagnosis in the context of other reticulate hyperpigmentation disorders.

Single-cell analysis of early chick hypothalamic development reveals that hypothalamic cells are induced from prethalamic-like progenitors.

The hypothalamus regulates many innate behaviors, but its development remains poorly understood. Here, we used single-cell RNA sequencing (RNA-seq) and hybridization chain reaction (HCR) to profile multiple stages of early hypothalamic development in the chick. Hypothalamic neuroepithelial cells are initially induced from prethalamic-like cells. Two distinct hypothalamic progenitor populations then emerge and give rise to tuberal and mammillary/paraventricular hypothalamic cells. At later stages, the regional organization of the chick and mouse hypothalamus is highly similar. We identify selective markers for major subdivisions of the developing chick hypothalamus and many previously uncharacterized candidate regulators of hypothalamic induction, regionalization, and neurogenesis. As proof of concept for the power of the dataset, we demonstrate that prethalamus-derived follistatin inhibits hypothalamic induction. This study clarifies the organization of the nascent hypothalamus and identifies molecular mechanisms that may control its induction and subsequent development.

Disruption of prepulse inhibition is associated with compulsive behavior severity and nucleus accumbens dopamine receptor changes in Sapap3 knockout mice.

Obsessive compulsive disorder (OCD) is associated with disruption of sensorimotor gating, which may contribute to difficulties inhibiting intrusive thoughts and compulsive rituals. Neural mechanisms underlying these disturbances are unclear; however, striatal dopamine is implicated in regulation of sensorimotor gating and OCD pathophysiology. The goal of this study was to examine the relationships between sensorimotor gating, compulsive behavior, and striatal dopamine receptor levels in Sapap3 knockout mice (KOs), a widely used preclinical model system for OCD research. We found a trend for disruption of sensorimotor gating in Sapap3-KOs using the translational measure prepulse inhibition (PPI); however, there was significant heterogeneity in both PPI and compulsive grooming in KOs. Disruption of PPI was significantly correlated with a more severe compulsive phenotype. In addition, PPI disruption and compulsive grooming severity were associated with reduced dopamine D1 and D2/3 receptor density in the nucleus accumbens core (NAcC). Compulsive grooming progressively worsened in Sapap3-KOs tested longitudinally, but PPI disruption was first detected in high-grooming KOs at 7 months of age. Through detailed characterization of individual differences in OCD-relevant behavioral and neurochemical measures, our findings suggest that NAcC dopamine receptor changes may be involved in disruption of sensorimotor gating and compulsive behavior relevant to OCD.

Long-term effects of young-adult methamphetamine on dorsal raphe serotonin systems in mice: Role of brain-derived neurotrophic factor.

To assess the long-term effects of chronic adolescent methamphetamine (METH) treatment on the serotonin system in the brain, we used serotonin-1A receptor (5-HT1A) and serotonin transporter (SERT) autoradiography, and quantitative tryptophan-hydroxylase 2 (TPH2) immunohistochemistry in the raphe nuclei of mice. Because of the modulatory role of brain-derived neurotrophic factor (BDNF) on the serotonin system and the effects of METH, we included both BDNF heterozygous (HET) mice and wildtype (WT) controls. Male and female mice of both genotypes were treated with an escalating METH dose regimen from the age of 6-9 weeks. At least two weeks later, acute locomotor hyperactivity induced by a 5 mg/kg D-amphetamine challenge was significantly enhanced in METH-pretreated mice, showing long-term sensitisation. METH pretreatment caused a small, but significant decrease of 5-HT1A receptor binding in the dorsal raphe nucleus (DRN) of males independent of genotype, but there were no changes in the median raphe nucleus (MRN) or in SERT binding density. METH treatment reduced the number of TPH2 positive cells in ventral subregions of the rostral and medial DRN independent of genotype. METH treatment selectively reduced DRN cell counts in BDNF HET mice compared to wildtype mice in medial and caudal ventrolateral subregions previously associated with panic-like behaviour. The data increase our understanding of the long-term and selective effects of METH on brain serotonin systems. These findings could be relevant for some of the psychosis-like symptoms associated with long-term METH use.

The inhibitory impact of ammonia on thermally hydrolyzed sludge fed anaerobic digestion.

This study evaluated the impact of ammonia on mesophilic anaerobic digestion (AD) with thermal hydrolysis pretreatment (THP) treating a mixture of primary sludge and waste activated sludge and operated under constant organic loading rate of 9 kg COD/m3 /d. Free ammonia concentrations in the digesters were varied between 37 and 966 mg NH3 -N/L, while maintaining all other operational conditions constant. A decrease in volatile solids reduction from 54 ± 5% (at <554 mg NH3 -N/L) to 35 ± 6% at the maximum free ammonia concentration of 966 mg NH3 -N/L was observed at steady-state conditions. No impact of free ammonia on final dewaterability was detected. Free ammonia thus mostly limited methanogenesis. A free ammonia Monod inhibition constant of 847 ± 222 mg NH3 -N/L for methanogens was estimated based on the digester steady-state methane rates dynamics. This study showed that current THP AD digesters (typically 110-260 mg NH3 -N/L) operate under 12%-18% ammonia inhibition for methanogenesis. Operation under SRT of 15 days, about 2 times more than needed to retain methanogens, can compensate for lower methanogens rates and avoid performance impacts. The later shows a good potential to operate under higher free and total ammonia concentration without jeopardizing performance. PRACTITIONER POINTS: Only from a free ammonia concentration above 554 mg NH3 -N/L, decreased volatile solids reduction and biogas yield were observed. A volatile solids reduction of 35 ± 6% at maximum free ammonia concentration of 966 mg NH3 -N/L was still achieved. A Monod inhibition constant for methanogens of 847 ± 222 mg NH3 -N/L was estimated. It was estimated that current THP AD systems (110-260 mg NH3 -N/L) operate under 12%-18% NH3 inhibition for methanogenesis.