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Small molecules that increase the presynaptic function of aminergic cells may provide neuroprotection in Parkinson's disease (PD) as well as treatments for attention deficit hyperactivity disorder (ADHD) and depression. Model genetic organisms such as Drosophila melanogaster may enhance the detection of new drugs via modifier or 'enhancer/suppressor' screens, but this technique has not been applied to processes relevant to psychiatry. To identify new aminergic drugs in vivo, we used a mutation in the Drosophila vesicular monoamine transporter (dVMAT) as a sensitized genetic background and performed a suppressor screen. We fed dVMAT mutant larvae ∼ 1000 known drugs and quantitated rescue (suppression) of an amine-dependent locomotor deficit in the larva. To determine which drugs might specifically potentiate neurotransmitter release, we performed an additional secondary screen for drugs that require presynaptic amine storage to rescue larval locomotion. Using additional larval locomotion and adult fertility assays, we validated that at least one compound previously used clinically as an antineoplastic agent potentiates the presynaptic function of aminergic circuits. We suggest that structurally similar agents might be used to development treatments for PD, depression and ADHD, and that modifier screens in Drosophila provide a new strategy to screen for neuropsychiatric drugs. More generally, our findings demonstrate the power of physiologically based screens for identifying bioactive agents for select neurotransmitter systems.

Monoamine neurotransmitters such as noradrenalin are released from both synaptic vesicles (SVs) and large dense-core vesicles (LDCVs), the latter mediating extrasynaptic signaling. The contribution of synaptic versus extrasynaptic signaling to circuit function and behavior remains poorly understood. To address this question, we have previously used transgenes encoding a mutation in the Drosophila Vesicular Monoamine Transporter ( dVMAT ) that shifts amine release from SVs to LDCVs. To circumvent the use of transgenes with non-endogenous patterns of expression, we have now used CRISPR-Cas9 to generate a trafficking mutant in the endogenous dVMAT gene. To minimize disruption of the dVMAT coding sequence and a nearby RNA splice site, we precisely introduced a point mutation using single-stranded oligonucleotide repair. A predicted decrease in fertility was used as a phenotypic screen to identify founders in lieu of a visible marker. Phenotypic analysis revealed a defect in the ovulation of mature follicles and egg retention in the ovaries. We did not detect defects in the contraction of lateral oviducts following optogenetic stimulation of octopaminergic neurons. Our findings suggest that release of mature eggs from the ovary is disrupted by changing the balance of VMAT trafficking between SVs and LDCVs. Further experiments using this model will help determine the mechanisms that sensitize specific circuits to changes in synaptic versus extrasynaptic signaling., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2023 The Authors.)

Neurotransmitters are crucial for the relay of signals between neurons and their target. Monoamine neurotransmitters dopamine (DA), serotonin (5-HT), and histamine are found in both invertebrates and mammals and are known to control key physiological aspects in health and disease. Others, such as octopamine (OA) and tyramine (TA), are abundant in invertebrates. TA is expressed in both Caenorhabditis elegans and Drosophila melanogaster and plays important roles in the regulation of essential life functions in each organism. OA and TA are thought to act as the mammalian homologs of epinephrine and norepinephrine respectively, and when triggered, they act in response to the various stressors in the fight-or-flight response. 5-HT regulates a wide range of behaviors in C. elegans including egg-laying, male mating, locomotion, and pharyngeal pumping. 5-HT acts predominantly through its receptors, of which various classes have been described in both flies and worms. The adult brain of Drosophila is composed of approximately 80 serotonergic neurons, which are involved in modulation of circadian rhythm, feeding, aggression, and long-term memory formation. DA is a major monoamine neurotransmitter that mediates a variety of critical organismal functions and is essential for synaptic transmission in invertebrates as it is in mammals, in which it is also a precursor for the synthesis of adrenaline and noradrenaline. In C. elegans and Drosophila as in mammals, DA receptors play critical roles and are generally grouped into two classes, D1-like and D2-like based on their predicted coupling to downstream G proteins. Drosophila uses histamine as a neurotransmitter in photoreceptors as well as a small number of neurons in the CNS. C. elegans does not use histamine as a neurotransmitter. Here, we review the comprehensive set of known amine neurotransmitters found in invertebrates, and discuss their biological and modulatory functions using the vast literature on both Drosophila and C. elegans . We also suggest the potential interactions between aminergic neurotransmitters systems in the modulation of neurophysiological activity and behavior., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Rosikon, Bone and Lawal.)

Objectives: To reliably quantify the radiographic severity of COVID-19 pneumonia with the Radiographic Assessment of Lung Edema (RALE) score on clinical chest X-rays among inpatients and examine the prognostic value of baseline RALE scores on COVID-19 clinical outcomes., Setting: Hospitalised patients with COVID-19 in dedicated wards and intensive care units from two different hospital systems., Participants: 425 patients with COVID-19 in a discovery data set and 415 patients in a validation data set., Primary and Secondary Outcomes: We measured inter-rater reliability for RALE score annotations by different reviewers and examined for associations of consensus RALE scores with the level of respiratory support, demographics, physiologic variables, applied therapies, plasma host-response biomarkers, SARS-CoV-2 RNA load and clinical outcomes., Results: Inter-rater agreement for RALE scores improved from fair to excellent following reviewer training and feedback (intraclass correlation coefficient of 0.85 vs 0.93, respectively). In the discovery cohort, the required level of respiratory support at the time of CXR acquisition (supplemental oxygen or non-invasive ventilation (n=178); invasive-mechanical ventilation (n=234), extracorporeal membrane oxygenation (n=13)) was significantly associated with RALE scores (median (IQR): 20.0 (14.1-26.7), 26.0 (20.5-34.0) and 44.5 (34.5-48.0), respectively, p<0.0001). Among invasively ventilated patients, RALE scores were significantly associated with worse respiratory mechanics (plateau and driving pressure) and gas exchange metrics (PaO2/FiO2 and ventilatory ratio), as well as higher plasma levels of IL-6, soluble receptor of advanced glycation end-products and soluble tumour necrosis factor receptor 1 (p<0.05). RALE scores were independently associated with 90-day survival in a multivariate Cox proportional hazards model (adjusted HR 1.04 (1.02-1.07), p=0.002). We replicated the significant associations of RALE scores with baseline disease severity and mortality in the independent validation data set., Conclusions: With a reproducible method to measure radiographic severity in COVID-19, we found significant associations with clinical and physiologic severity, host inflammation and clinical outcomes. The incorporation of radiographic severity assessments in clinical decision-making may provide important guidance for prognostication and treatment allocation in COVID-19., Competing Interests: Competing interests: Dr. Kitsios has received research funding from Karius, Inc. Dr. McVerry receives research funding from Bayer Pharmaceuticals, Inc. All other authors disclosed no conflict of interest, (© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Drosophila melanogaster express vesicular transporters for the storage of neurotransmitters acetylcholine, biogenic amines, GABA, and glutamate. The large array of powerful molecular-genetic tools available in Drosophila enhances the use of this model organism for studying transporter function and regulation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

BACKGROUND The diagnosis of systemic sclerosis sine scleroderma (ssSSc) with renal crisis is difficult because of its unusual presentation and rarity. CASE REPORT A 45-year-old man presented to the Emergency Department with worsening nausea, vomiting, and exertional dyspnea for 3 weeks. Initial examination showed blood pressure 182/108 mmHg without skin thickening or other skin manifestations. Laboratory investigations showed serum creatinine level 21.73 mg/dL and diffuse airspace opacities on chest radiography. He was admitted to the intensive care unit and started on emergent hemodialysis. He was anemic and became gradually hypoxic, requiring supplemental oxygen. Computed tomography of the chest showed bilateral infiltrates. Antinuclear antibodies (ANA) were positive for centromere pattern with titer of 320. Antineutrophil cytoplasmic antibodies and antiglomerular basement membrane antibodies were negative. He was started on therapeutic plasmapheresis (TP) and captopril, which resulted in significant improvement of respiratory symptoms. The kidney biopsy revealed thrombotic microangiopathy. Anticentromere, anti-Scl-70, and antiribonucleic acid polymerase III antibodies, drawn after 4 sessions of TP, were not detected. CONCLUSIONS Here we report a rare case of ssSSc with renal crisis in a patient who presented with acute renal failure requiring hemodialysis and suspected pulmonary hemorrhage. Clinical improvement was achieved by TP and angiotensin-converting enzyme inhibitor. The diagnosis of ssSSc was difficult and required an ANA pattern and kidney biopsy.

The neurotransmitter acetylcholine (ACh) is involved in critical organismal functions that include locomotion and cognition. Importantly, alterations in the cholinergic system are a key underlying factor in cognitive defects associated with aging. One essential component of cholinergic synaptic transmission is the vesicular ACh transporter (VAChT), which regulates the packaging of ACh into synaptic vesicles for extracellular release. Mutations that cause a reduction in either protein level or activity lead to diminished locomotion ability whereas complete loss of function of VAChT is lethal. While much is known about the function of VAChT, the direct role of altered ACh release and its association with either an impairment or an enhancement of cognitive function are still not fully understood. We hypothesize that point mutations in Vacht cause age-related deficits in cholinergic-mediated behaviors such as locomotion, and learning and memory. Using Drosophila melanogaster as a model system, we have studied several mutations within Vacht and observed their effect on survivability and locomotive behavior. Here we report for the first time a weak hypomorphic Vacht allele that shows a differential effect on ACh-linked behaviors. We also demonstrate that partially rescued Vacht point mutations cause an allele-dependent deficit in lifespan and defects in locomotion ability. Moreover, using a thorough data analytics strategy to identify exploratory behavioral patterns, we introduce new paradigms for measuring locomotion-related activities that could not be revealed or detected by a simple measure of the average speed alone. Together, our data indicate a role for VAChT in the maintenance of longevity and locomotion abilities in Drosophila and we provide additional measurements of locomotion that can be useful in determining subtle changes in Vacht function on locomotion-related behaviors., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Acetylcholinergic (ACh) neurotransmission is essential for key organismal functions such as locomotion and cognition. However, the mechanism through which ACh is regulated in the central nervous system is not fully understood. The vesicular acetylcholine transporter (VAChT) mediates the packaging and transport of ACh for exocytotic release and is a critical component of the ACh release machinery. Yet its precise role in the maintenance of cholinergic tone remains a subject of active investigation. Here we use the overexpression of VAChT as a tool to investigate the role of changes in ACh exocytosis on the regulation of synaptic activity and its downstream consequences. We measured the effect of an increase in VAChT expression on locomotion and cognitive performance as well as on organismal survival across the lifespan. We report the surprising finding that increased VAChT expression results in a significantly shorter lifespan in comparison to control flies. Moreover, constructs overexpressing VAChT demonstrate an age-dependent decrease in locomotion performance. Importantly, we report clear deficits in learning and memory which we measured through a courtship conditioning assay. Together, these data provide evidence for the adverse effects of overexpression of the vesicular acetylcholine transporter in the maintenance of normal behavioral abilities in Drosophila and demonstrates for the first time a role for ACh in the regulation of organismal survival., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The role of the vesicular acetylcholine transporter (VAChT) in the regulation of cholinergic neurotransmission has not been fully elucidated. Here we sought to develop a tool for studying vesicular acetylcholine transporter function, and we present data on the validation of our new anti-VAChT antibody. We show that the immunoreactivity of the antibody is not due to an artifact of secondary antibody staining, and we present two additional validation data. First, the peptide epitope used to generate the antibody is able to block the binding of the anti-VAChT antibody in vivo . Further, RNA interference (RNAi) -mediated knockdown of VAChT function in cholinergic neurons drastically reduces anti-VAChT staining in those constructs. Additional evidence for the antibody functionality is presented in our research article on the subject (Boppana et al., 2017) [1].

The neurodegenerative effects of Parkinson's disease (PD) are marked by a selective loss of dopaminergic (DA) neurons. Epidemiological studies suggest that chronic exposure to the pesticide paraquat may increase the risk for PD and DA cell loss. However, combined exposure with additional fungicide(s) including maneb and/or ziram may be required for pathogenesis. To explore potential pathogenic mechanisms, we have developed a Drosophila model of chronic paraquat exposure. We find that while chronic paraquat exposure alone decreased organismal survival and motor function, combined chronic exposure to both paraquat and maneb was required for DA cell death in the fly. To initiate mechanistic studies of this interaction, we used additional genetic reagents to target the ubiquitin proteasome system, which has been implicated in some rare familial forms of PD and the toxic effects of ziram. Genetic inhibition of E1 ubiquitin ligase, but not the proteasome itself, increased DA cell death in combination with maneb but not paraquat. These studies establish a model for long-term exposure to multiple pesticides, and support the idea that pesticide interactions relevant to PD may involve inhibition of protein ubiquitination., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Monoamine neurotransmitters are stored in both synaptic vesicles (SVs), which are required for release at the synapse, and large dense-core vesicles (LDCVs), which mediate extrasynaptic release. The contributions of each type of vesicular release to specific behaviors are not known. To address this issue, we generated mutations in the C-terminal trafficking domain of the Drosophila vesicular monoamine transporter (DVMAT), which is required for the vesicular storage of monoamines in both SVs and LDCVs. Deletion of the terminal 23 aa (DVMAT-Δ3) reduced the rate of endocytosis and localization of DVMAT to SVs, but supported localization to LDCVs. An alanine substitution mutation in a tyrosine-based motif (DVMAT-Y600A) also reduced sorting to SVs and showed an endocytic deficit specific to aminergic nerve terminals. Redistribution of DVMAT-Y600A from SV to LDCV fractions was also enhanced in aminergic neurons. To determine how these changes might affect behavior, we expressed DVMAT-Δ3 and DVMAT-Y600A in a dVMAT null genetic background that lacks endogenous dVMAT activity. When expressed ubiquitously, DVMAT-Δ3 showed a specific deficit in female fertility, whereas DVMAT-Y600A rescued behavior similarly to DVMAT-wt. In contrast, when expressed more specifically in octopaminergic neurons, both DVMAT-Δ3 and DVMAT-Y600A failed to rescue female fertility, and DVMAT-Y600A showed deficits in larval locomotion. DVMAT-Y600A also showed more severe dominant effects than either DVMAT-wt or DVMAT-Δ3. We propose that these behavioral deficits result from the redistribution of DVMAT from SVs to LDCVs. By extension, our data suggest that the balance of amine release from SVs versus that from LDCVs is critical for the function of some aminergic circuits., (Copyright © 2014 the authors 0270-6474/14/346924-14$15.00/0.)

The exocytotic release of neurotransmitters requires active transport into synaptic vesicles and other types of secretory vesicles. Members of the SLC18 family perform this function for acetylcholine (SLC18A3, the vesicular acetylcholine transporter or VAChT) and monoamines such as dopamine and serotonin (SLC18A1 and 2, the vesicular monoamine transporters VMAT1 and 2, respectively). To date, no specific diseases have been attributed to a mutation in an SLC18 family member; however, polymorphisms in SLC18A1 and SLC18A2 may confer risk for some neuropsychiatric disorders. Additional members of this family include SLC18A4, expressed in insects, and SLC18B1, the function of which is not known. SLC18 is part of the Drug:H(+) Antiporter-1 Family (DHA1, TCID 2.A.1.2) within the Major Facilitator Superfamily (MFS, TCID 2.A.1)., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

To investigate the regulation of Drosophila melanogaster behavior by biogenic amines, we have exploited the broad requirement of the vesicular monoamine transporter (VMAT) for the vesicular storage and exocytotic release of all monoamine neurotransmitters. We used the Drosophila VMAT (dVMAT) null mutant to globally ablate exocytotic amine release and then restored DVMAT activity in either individual or multiple aminergic systems, using transgenic rescue techniques. We find that larval survival, larval locomotion, and female fertility rely predominantly on octopaminergic circuits with little apparent input from the vesicular release of serotonin or dopamine. In contrast, male courtship and fertility can be rescued by expressing DVMAT in octopaminergic or dopaminergic neurons, suggesting potentially redundant circuits. Rescue of major aspects of adult locomotion and startle behavior required octopamine, but a complementary role was observed for serotonin. Interestingly, adult circadian behavior could not be rescued by expression of DVMAT in a single subtype of aminergic neurons, but required at least two systems, suggesting the possibility of unexpected cooperative interactions. Further experiments using this model will help determine how multiple aminergic systems may contribute to the regulation of other behaviors. Our data also highlight potential differences between behaviors regulated by standard exocytotic release and those regulated by other mechanisms.

Dopamine is cytotoxic and may play a role in the development of Parkinson's disease. However, its interaction with environmental risk factors such as pesticides remains poorly understood. The vesicular monoamine transporter (VMAT) regulates intracellular dopamine content, and we have tested the neuroprotective effects of VMAT in vivo using the model organism Drosophila melanogaster. We find that Drosophila VMAT (dVMAT) mutants contain fewer dopaminergic neurons than wild type, consistent with a developmental effect, and that dopaminergic cell loss in the mutant is exacerbated by the pesticides rotenone and paraquat. Overexpression of DVMAT protein does not increase the survival of animals exposed to rotenone, but blocks the loss of dopaminergic neurons caused by this pesticide. These results are the first to demonstrate an interaction between a VMAT and pesticides in vivo, and provide an important model to investigate the mechanisms by which pesticides and cellular DA may interact to kill dopaminergic cells., ((c) 2010 Elsevier Inc. All rights reserved.)

The 'rate of living' theory predicts that longevity should be inversely correlated with the rate of mitochondrial respiration. However, recent studies in a number of model organisms, including mice, have reported that interventions that retard the aging process are, in fact, associated with an increase in mitochondrial activity. To better understand the relationship between energy metabolism and longevity, we supplemented the endogenous respiratory chain machinery of the fruit fly Drosophila melanogaster with the alternative single-subunit NADH-ubiquinone oxidoreductase (Ndi1) of the baker's yeast Saccharomyces cerevisiae. Here, we report that expression of Ndi1 in fly mitochondria leads to an increase in NADH-ubiquinone oxidoreductase activity, oxygen consumption, and ATP levels. In addition, exogenous Ndi1 expression results in increased CO2 production in living flies. Using an inducible gene-expression system, we expressed Ndi1 in different cells and tissues and examined the impact on longevity. In doing so, we discovered that targeted expression of Ndi1 in fly neurons significantly increases lifespan without compromising fertility or physical activity. These findings are consistent with the idea that enhanced respiratory chain activity in neuronal tissue can prolong fly lifespan.

We conducted a study to determine how accurate various ENT specialists were in estimating the size of 100 tympanic membrane (TM) perforations with standard otoscopy. The specialists included, in descending order of rank, 2 Consultant Surgeons, 2 Senior Registrars, and 2 Registrars, all of whom had confirmed good vision. We compared their estimates, which were made independently and expressed as a percentage of the total area of the TM, with exact measurements calculated with computer-based video-otoscopy. We found that the video-otoscopic calculations were far superior to the estimates of the specialists, even the most experienced Consultants (p < 0.01). We recommend that video-otoscopy be used whenever possible.

While studying the developmental functions of the Drosophila dopamine synthesis pathway genes, we noted interesting and unexpected mutant phenotypes in the developing trachea, a tubule network that has been studied as a model for branching morphogenesis. Specifically, Punch (Pu) and pale (ple) mutants with reduced dopamine synthesis show ectopic/aberrant migration, while Catecholamines up (Catsup) mutants that over-express dopamine show a characteristic loss of migration phenotype. We also demonstrate expression of Punch, Ple, Catsup and dopamine in tracheal cells. The dopamine pathway mutant phenotypes can be reproduced by pharmacological treatments of dopamine and a pathway inhibitor 3-iodotyrosine (3-IT), implicating dopamine as a direct mediator of the regulatory function. Furthermore, we show that these mutants genetically interact with components of the endocytic pathway, namely shibire/dynamin and awd/nm23, that promote endocytosis of the chemotactic signaling receptor Btl/FGFR. Consistent with the genetic results, the surface and total cellular levels of a Btl-GFP fusion protein in the tracheal cells and in cultured S2 cells are reduced upon dopamine treatment, and increased in the presence of 3-IT. Moreover, the transducer of Btl signaling, MAP kinase, is hyper-activated throughout the tracheal tube in the Pu mutant. Finally we show that dopamine regulates endocytosis via controlling the dynamin protein level.