This report describes the protocol for an ongoing project funded by the National Institutes of Health (R01MH108155) that is focused on effects of childhood maltreatment (MALTX) on neurocircuitry changes associated with adolescent major depressive disorder (MDD). Extant clinical and neuroimaging literature on MDD is reviewed, which has relied on heterogeneous samples that do not parse out the unique contribution of MALTX on neurobiological changes in MDD. Employing a 2 × 2 study design , and based on a cohesive theoretical model that incorporates behavioral, cognitive and neurobiological domains, we describe the multi-modal neuroimaging techniques used to test whether structural and functional alterations in the fronto-limbic and fronto-striatal circuits associated with adolescent MDD are moderated by MALTX. We hypothesize that MDD + MALTX youth will show alterations in the fronto-limbic circuit, with reduced connectivity between the amygdala (AMG) and the prefrontal cortex (PFC), as the AMG is sensitive to stress/threat during development. Participants with MDD will exhibit increased functional connectivity between the AMG and PFC due to self-referential negative emotions. Lastly, MDD + MALTX will only show changes in motivational/anticipatory aspects of the fronto-striatal circuit, and MDD will exhibit changes in motivational and consummatory/outcome aspects of reward-processing. Our goal is to identify distinct neural substrates associated with MDD due to MALTX compared to other causes, as these markers could be used to more effectively predict treatment outcome, index treatment response, and facilitate alternative treatments for adolescents who do not respond well to traditional approaches. Major depressive disorder (MDD) is the leading cause of disability worldwide ,2, and iThe overarching aim of this study is to determine whether two distinct MDD subtypes (with and without MALTX) are warranted through an examination of structural and functional neural circuits in a diverse sample of adolescents. Because adolescence is characterized as the period with the most pronounced neural changes and maturation in grey and white matter since infancy –17, and Aim 1: Test whether structural and functional alterations associated with depression in the fronto-limbic circuit are moderated by a history of abuse and identify structural and functional differences in this circuit between MDD and MDD + MALTX. Structural differences: We hypothesize that MDD + MALTX will have smaller grey matter volume in the HIPP and prefrontal cortex (PFC) but greater AMG volume, and lower fractional anisotropy (FA) in the fronto-limbic tracts (cingulum/uncinate fasciculus), compared to MDD. Functional differences: MDD + MALTX will exhibit reduced functional connectivity between the AMG, HIPP, and PFC, both at rest and in response to a cognitive-emotional task, and MDD will exhibit increased functional connectivity between these regions.Aim 2: Test whether structural and functional alterations associated with depression in the fronto-striatal circuit are moderated by a history of abuse and identify structural and functional differences in this circuit between MDD and MDD + MALTX. Structural differences: We hypothesize that MDD will have smaller grey matter volume in ventral striatum (VS) and lower FA in the fronto-striatal tracts compared to MDD + MALTX. Functional differences: MDD will exhibit increased functional connectivity between the VS and PFC during resting-state as well as reward anticipation and receipt phases in response to a reward-processing task. Additionally, MDD + MALTX will exhibit reduced functional connectivity at rest and only during the anticipation phase of the reward-processing task.Aim 3: Test whether abuse history moderates the associations of specific symptom constructs, such as negative or positive affect, anhedonia, or rumination, with key nodes in the fronto-limbic and fronto-striatal circuits. We hypothesize that a history of abuse will moderate the associations of negative affect and rumination with fronto-limbic circuit, and anhedonia with fronto-striatal circuit. However, we expect that abuse history will not moderate the association between positive affect and fronto-striatal circuit.interconnected. This dynamic, which simultaneously influences emotional and reward processes, leads to decreased functional connectivity in MDD + MALTX and increased connectivity in MDD in both circuits , which are group-matched on age, sex, pubertal stage, race/ethnicity and socioeconomic status (SES).All human subjects research described herein was approved by the Institutional Review Board at University of California, Irvine in 2017 (Protocol #2017–3440). Following a NIH (Sponsor) initial site-visit and approval, recruitment was initiated in February 2018. Participants are recruited from communities throughout Southern California. Adolescents of both sexes/genders and all racial/ethnic groups are eligible to participate if they are between 13 and 17 years of age and in Tanner Stage II or greater of pubertal development. Exclusion criteria include contraindications for imaging procedures , as well as conditions that would affect brain development, including IQ below 80, birth complications or premature birth, maternal substance abuse during pregnancy, neurological disease, or head trauma with loss of consciousness. As previously mentioned, youth taking psychotropic medication that may affect the central nervous system are excluded, unless willing to adhere to standard wash-out periods for certain medications  prior to scanning. Youth who used alcohol and/or drugs in the week prior to study entry (based on self-report or urine drug screen), are suspected to be pregnant, and those who reported experiencing multiple unrelated forms of trauma  are not eligible.Furthermore, participants and/or their biological parents with a history of mania or hypomania are excluded, as unipolar and bipolar depression may have distinct neural markers. To further minimize diagnostic comorbidity, youth are excluded if they meet DSM-5 criteria for moderateto-severe disruptive disorders, substance use disorders or autism spectrum disorder in the previous six months, or were exhibiting psychotic symptoms or active suicidal ideation at recruitment. However, youth determined to meet criteria for anxiety and trauma-based disorders  are eligible, as these diagnoses are often comorbid with MDD, have overlapping symptoms, and likely have shared etiological factors –86. We wThe assessment schedule is presented in Youth and caregivers are separately administered the computerized version of the Kiddie Schedule for Affective Disorders and Schizophrenia—Present and Lifetime Version   by post-The Childhood Adversity Interview (CAI)  is also Participants in the MDD + MALTX group meet DSM-5 criteria for current unipolar MDD based on K-SADS-COMP consensus ratings and meet the threshold for significant abuse history prior to age 10 based on ratings of ≥3 (at least moderate severity) on any of the three abuse items on the CAI. The MDD group meets criteria for current unipolar MDD and does not have any significant abuse history . Adolescents categorized in the MALTX group meet threshold criteria for significant history of abuse but do not have significant current or lifetime psychiatric history, with the exception of phobias. The NC group does not have any significant abuse or psychiatric history, as indicated above.Parental psychopathology is assessed using a semi-structured interview, the Family History-Research Diagnostic Criteria (FH-RDC) , adminisVisit 1 assessments, as indicated in With respect to measures used in exploratory analyses, affect and mood (the Positive and Negative Affect Schedule) , parent At Visit 1, a urine drug screen and MRI safety screen  are administered. Medication and treatment history are also gathered.At visit 2, adolescents complete a neurocognitive battery to test whether executive functioning measures correlate with neural markers or psychiatric symptoms. The battery assesses for verbal and non-verbal reasoning  , workingThe multi-modal imaging techniques include structural MRI (sMRI), diffusion tensor imaging (DTI), resting-state functional MRI (rs-fMRI) and task-based fMRI, all of which amount to approximately 90 min of scanning. Scans are acquired on a 3.0 Tesla Siemens Prisma  scanner, using a standard radiofrequency 12-channel head coil. T2-weighted scans, co-planar to the functional images  and high resolution T1-weighted scans , are acquired for registration purposes. Functional images are acquired with a gradient-echo, EPI sequence: 34 oblique axial slices , oriented to the AC-PC line, and encompassing the entire cerebrum and most of the cerebellum . For rs-fMRI, which measures intrinsic functional connectivity, participants are instructed to remain awake, relax with their eyes open, and look at a fixation cross for approximately 10 min. For task-based fMRI scans, stimulus presentation is completed using E-Prime with the images projected onto an overhead LCD panel and a 5-button box for recording behavioral data. An automated higher-order shim procedure is applied to minimize magnetic field inhomogeneities.FreeSurfer image analysis suite is used to perform cortical reconstruction and volumetric segmentation . Surface2; less than 15 min) [DTI data are corrected for image distortions due to eddy currents  and stat 15 min) . HARDI p 15 min)  to track 15 min) , with th 15 min) ,128.Probabilistic fiber tractography is performed using the FMRIB FDT toolbox, which uses Bayesian techniques to estimate the most probable location of a pathway between two seed points –132. FibWe use tract-based spatial statistics (TBSS) to examine voxel-level differences in FA between groups ,134 whicAdolescents complete an Emotional Go/No-Go fMRI Task (EmoGnG), which targets emotional and cognitive processes, and allows us to measure responses in the fronto-limbic circuit . ParticiParticipants also complete a Monetary Reward Task, which has been shown to reliably elicit striatal and medial PFC responses to anticipation and receipt of reward in both adolescent and adult subjects, including individuals with mood disorders –140. AdoRest and task-based images are aligned using rigid-body co-registration to reduce the effects of head motion and subsequently co-registered to the high-resolution T1-weighted structural image. The transformation of the structural image to atlas space is then applied to the functional images, followed by resampling, yielding functional images at 3mm isotropic resolution in the atlas space. These are spatially smoothed using a 6mm-FWHM Gaussian kernel. After initial pre-processing, time series at each voxel have white matter, cerebrospinal fluid and motion-related signals removed via regression , then arp < 0.05, using random field theory to correct for the multiple voxel comparisons based on spatial smoothness [Resting-state functional connectivity is calculated as Z-transformed correlation coefficient between pre-processed time series, using the CONN toolbox  and connoothness ,154 to iAdult depression studies have shown the significant complimentary benefits of combining structural and functional neuroimaging techniques with respect to deepening our understanding of pathophysiology and treatment responsiveness ,156. TheP-values are then estimated using permutation testing (FMRIB Randomise tool) and corrected for multiple comparisons using a cluster-forming threshold in FSL.Functional connectivity is tested by integrating structural and functional data using two different approaches. First, we compare groups on the correlation between overall strength of the structural connectivity  and functional connectivity  for the fronto-limbic tract  and the fronto-striatal tract . Values for each group are then r-to-z transformed and tested using ANOVA/ANCOVAs, thereby implementing a conservative test of structure-function association for the entire tract. Second, because there may be more specific relationships between structure and function, we use regression analyses to test for differences at each voxel within the path. Functional connectivity values, group, and group × functional connectivity interaction are all used as predictor variables. Data management is completed through the HIPAA-compliant remote electronic data capture (REDCap) system , wherebyTo test for the moderation effects of maltreatment on structural and functional alterations associated with MDD within the fronto-limbic circuit (Hypothesis 1) and fronto-striatal circuit (Hypothesis 2), multiple regressions will be used and will include MDD and MALTX as main effects and MDD × MALTX as an interaction term; demographic and clinical covariates will be included if these differ by group. Focus will be placed on simple main effects when interactions are significant. Subsequently, we will assess for structural and functional differences in the depressed samples between participants in the MDD and MDD + MALTX groups. Secondary analyses will include testing for differences between NC and MALTX (non-depressed adolescents), between MALTX and MDD + MALTX , and between NC and MDD .In the event that analyses do not yield significant interactions, we will focus on the main effects of MDD and MALTX when testing group differences. Accordingly, we aim to answer the following questions with our findings: (a) whether the neural substrates of MDD participants differ from those without MDD, irrespective of abuse history; and (b) whether the neural substrates for MALTX participants differ from those without MALTX, irrespective of depression. Within-group analyses will include the covariates of clinical  and demographic  variables. Similar analyses will be conducted to test for the moderation effects of maltreatment on the association between distinct depressive symptom profiles and structural/functional connectivity measures in fronto-limbic and fronto-striatal circuits (Hypothesis 3). In these analyses, various domains of depression will either be dichotomized  or tested as dimensional variables by mean centering the scores.For exploratory analyses, we will examine the relationship between structural connectivity changes and functional connectivity, and between resting-state and task-based functional connectivity. In finding alterations in the key nodes common to cognitive-emotional and reward circuits, we seek to establish whether they influence both emotion- and reward-processing. Whole-brain analyses will be performed to identify changes in the extended parts of these circuits . Various covariates will be included in our models as moderators on brain changes, such as age of depression onset, PTSD symptoms, and social support, and these will be examined in relation to behavioral and neurocognitive variables.f2 = 0.05, indicating small-to-medium- effect sizes [Based on power analyses utilizing traditional criteria  , our proct sizes . Given tct sizes , as wellThe grant application received an Impact Score of 20 and a 6th Percentile Score. The main weaknesses expressed by the review committee include the unreliability of participants’ recollections about maltreatment history and the cross-sectional study design. Specifically, the cross-sectional design may not be able to reveal the dynamic effects of maltreatment and brain changes. Some reviewers indicated that the approach, in and of itself is not necessarily innovative, but the question is critical and significance of the study substantial. The committee indicated that these weaknesses did not diminish the reviewers’ enthusiasm and they agreed that the neural markers associated with the two depression subtypes may lead to better-informed treatments.We acknowledge that retrospective reports of maltreatment are not very reliable. However, we instituted a multi-informant, multi-method assessment to improve reliability. Both youth and parent are interviewed using a standardized instrument (CAI). Additionally, we obtain information from Child Protective Services, when feasible. Youth also complete a self-report detailing past traumas (CTQ). Furthermore, given the shorter life history, retrospective bias is less in adolescents compared to adult samples.We agree that prospective studies are the gold standard for identifying temporal changes in neurocircuitry in relation to maltreatment history, depression outcome and the associated neural changes, and we gave it considerable thought. However, given our current knowledge in this field and relative costs, we decided that the best course of action is a well-designed, cross-sectional study as the first step to distinguish unique and interactive effects of maltreatment and depression before embarking on a more expensive and intensive longitudinal study.This study’s innovation is primarily conceptual. We felt it was important to use established methods to compare findings from existing data in adolescent/adult samples to distinguish the two depression phenotypes. We also use the latest advances in MRI data collection and analysis methods to provide exceptional resolution to facilitate distinction between the groups.We are currently at the end of Year 2 of a 5-year project. As of March 2020, we recruited and completed 131 participants, with 24 adolescents in MDD + MALTX, 37 in MDD, 13 in MALTX, and 57 in NC. Only seven participants (not counting the 131 completed) were withdrawn thus far following informed consent due to various reasons . Additionally, of the adolescents screened, the most common reasons for ineligibility are MRI incompatibility , current psychotropic medication, MDD history but no current episode, and personal or family history of bipolar disorder.https://chronicleofsocialchange.org/child-welfare-2/californias-surgeon-general-readies-statewide-screening-for-child-trauma/37658). The California Department of Health Care Services has just implemented training to all Medi-Cal (Medicaid) providers on ACEs screening (https://www.dhcs.ca.gov/provgovpart/Pages/TraumaCare.aspx). With these new initiatives, we anticipate that we will be in a better position to recruit the MALTX and MDD + MALTX groups.As per NIH mandate for this Award, we cannot ask for abuse history during the phone screen. We continue to make strides toward recruiting these high-risk youths, including reaching out to over 150 community organizations and agencies across Southern California, including agencies working with victims of trauma. The Governor of California has allocated $45 Million in the 2020–2021 fiscal year budget to reimburse Medicaid providers for screening of Adverse Childhood Experiences (ACEs) and an additional $50 Million to train primary care providers on administering these screenings :105–20 .Van Erp T, Jirsaraie R, Faulkner M, Scambray K, Fong J, Taylor D, Rao U. Dentate gyrus volume is associated with childhood maltreatment and depression severity in adolescents. Presented at the 57th Annual Meeting of the American College of Neuropsychopharmacology; 2018 Dec 9–13; Hollywood, FL, USA .Faulkner M, Jirsaraie R, Zurita T, Fong J, Scambray K, Rao U. Unpredictability in childhood predicts executive function impairment in depressed and non-depressed adolescents. Presented at the 6th Annual Symposium Organized by the Conte Center at UCI; 2019 Mar 12; Irvine, CA, USA .Sharma A, Scambray K, Jirsaraie R, Faulkner M, Rao U. White matter changes in fronto-limbic pathways in adolescent depression. Presented at the 6th Annual Symposium Organized by the Conte Center at UCI; 2019 Mar 12; Irvine, CA, USA .Sharma A, van Erp TGM, Scambray K, Jirsaraie R, Faulkner M, Rao U. Sustained amygdala response to fearful faces in depressed adolescents with childhood maltreatment. Presented at the 66th Annual Meeting of the American Academy of Child and Adolescent Psychiatry, 2019 Oct 14–19; Chicago, IL, USA .Sharma A, van Erp TGM, Faulkner M, Forbes E, Rao U. Decreased striatal response to monetary reward in depressed adolescents. Presented at the 58th Annual Meeting of the American College of Neuropsychopharmacology; 2019 Dec 8–11; Orlando, FL, USA .Millwood SN, Gillespie M, Sharma A, Huszti H, Rao U. Amygdala volume differences in depressed adolescents with and without childhood maltreatment. Presented at the 7th Annual Symposium Organized by the Conte Center at UCI; 2020 Feb 25; Irvine, CA, USA .The current study aims to understand the unique contribution that maltreatment history can have on the neurobiology of adolescent depression, with the goal of identifying two distinct depression subtypes. We address current gaps in the neuroimaging literature by focusing on adolescents, reducing participant heterogeneity by using stringent depression and maltreatment criteria, using comprehensive psychodiagnostic interviews, and implementing state-of-the-art, multi-modal neuroimaging techniques. We expect to differentiate structural and functional alterations in fronto-limbic and fronto-striatal circuits between MDD and MDD + MALTX groups, and determine whether maltreatment history moderates the association between depressive symptom profiles and these neural circuits. Moreover, we expect to discover whether maltreatment-induced neural differences are distinct or whether they reflect associated alterations in brain structures. We will also assess whether individual differences in neural correlates among these groups serve as risk or resiliency markers for depression. Exploratory analyses will allow us to examine the correlations between various clinical and demographic variables and neural changes in the MDD + MALTX group. The findings from this study will broaden and deepen our understanding of the neurobiological correlates of adolescent MDD versus MDD + MALTX, which may have important implications for shaping clinical formulation and treatment guidelines, predicting treatment outcomes more effectively, and indexing treatment response for the two MDD subtypes. Such knowledge will also be helpful in developing new treatments for subgroups that do not respond well to traditional interventions. With well-characterized samples of adolescents and promising results from this cross-sectional study, we hope to follow these cohorts longitudinally with new intramural and/or extramural funding to better characterize the neurobiological vulnerability and resiliency factors associated with depression onset and recurrence during the developmental transition to adulthood.


Monoamine oxidase B (MAOB) is expressed in the mitochondrial membrane and has a key role in degrading various neurologically active amines such as benzylamine, phenethylamine and dopamine with the help of Flavin adenine dinucleotide (FAD) cofactor. The Parkinson’s disease associated symptoms can be treated using inhibitors of MAO-B as the dopamine degradation can be reduced. Currently, many inhibitors are available having micromolar to nanomolar binding affinities. However, still there is demand for compounds with superior binding affinity and binding specificity with favorable pharmacokinetic properties for treating Parkinson’s disease and computational screening methods can be majorly recruited for this. However, the accuracy of currently available force-field methods for ranking the inhibitors or lead drug-like compounds should be improved and novel methods for screening compounds need to be developed. We studied the performance of various force-field-based methods and data driven approaches in ranking about 3753 compounds having activity against the MAO-B target. The binding affinities computed using autodock and autodock-vina are shown to be non-reliable. The force-field-based MM-GBSA also under-performs. However, certain machine learning approaches, in particular KNN, are found to be superior, and we propose KNN as the most reliable approach for ranking the complexes to reasonable accuracy. Furthermore, all the employed machine learning approaches are also computationally less demanding. Parkinson’s disease (PD) is the second common progressive neurodegenerative disorder next to Alzheimer’s disease and it affects about 2% or 5 % of the population above 65 or 85 years of age, respectively, with 5–20 cases per 1,00,000 population every year worldwide [ynuclein . Deficieynuclein . Apart fblongata . MitochoMAO-A is the major isoform in the non-central nervous system organs and therefore MAO-A deaminates tyramine in the gut. MAO-A inhibitors prevent deamination of tyramine in gut, but it is received by norepinephrine nerve terminals, and thereby it acts as false neurotransmitter by displacing stored norepinephrine . This rel-3,4-dihydroxyphenylalanine (l-DOPA- dopamine precursor) [MAO-B exists in dimeric form and each monomer unit has large globular structure (1–488 amino acid residues) exposed to solvent and a short helical domain (489–520 residues) in C-terminal bound to the lipid bilayer of mitochondrial membrane. The solvent exposed globular domain consists of both substrate-binding domain and FAD-binding domain . A largeal helix . The actal helix . Both thal helix . Crystalal helix . The cofal helix . Hydrophal helix . Moreoveal helix . Adenosial helix . These dal helix . These ral helix . Moreoveal helix . Besidesal helix . MAO-B cinolines . Therefoinolines . Therefoecursor) . TherefoThe effective therapy for the motor symptoms in PD includes modulation of dopamine system by dopamine agonists, exogenous dopamine and interfering the dopamine catabolism using monoamine oxidase (MAO-B) inhibitors. MAO-B inhibitors are commonly used for management of both psychiatric disorders and PD. Hence, development of potent MAO-B inhibitors, efficient delivery systems and novel combination drugs are of utmost importance due to the growing prevalence of PD. Given the above considerations, the present study explored the characteristics of established and experimental MAO-B inhibitors used in PD therapy. The objective of the study is to predict the binding affinities of bioactive compounds or drugs retrieved from ChEMBL database  against The structure of MAO-B enzyme in its safinamide bound state is displayed in Firstly, we analyzed the binding free energies predicted using autodock-vina software  and autoHere, we approximated that inhibition constants are approximately equal to IComplexes ,24. In pUsing the above equation, the calculated binding free energies are in the range of −1.7 to −14.9 kcal/mol and the extreme values correspond to the compounds CHEMBL1766622 and CHEMBL348961, respectively. Now, we discuss the performance of molecular docking-based scoring in ranking the MAO-B:ligand complexes. terature . Howeverterature . The curWe also computed the binding free energies using MM-GBSA approach and compared to the experimental binding affinities. Usually, the protein–ligand input structure used for computing MM-GBSA-based binding free energies can be obtained using different approaches. One can use the crystal structure or the structure from molecular docking studies using different softwares such as autodock-vina, autodock4.0 or GLIDE. Here, we carried out force-field molecular dynamics using the protein–ligand complex structures as obtained from docking study using autodock-vina and we computed two sets of MM-GBSA free energies. Set 1 uses the minimum energy structure for the complex as the input configuration. Set 2 uses as many as 100 configurations from molecular dynamics simulations carried out at 30 K. One can as well use the trajectories corresponding to ambient temperature simulations but then it is necessary to include entropic contributions which is computationally very demanding to estimate. In the low temperature simulations, the entropic contributions are negligible and so the binding enthalpies can be approximated directly for binding free energies. Finally, we aimed to test the performance of data driven approaches which are becoming very popular recently for predicting various drug-like properties including the binding affinities. In particular, we employed supervised machine learning approaches and our dataset included various physicochemical properties computed for ligands as the descriptors and the binding free energy as the quantity to be predicted. The dataset was randomly divided into training (80% of the ligands included) and test (20% of the ligands included) datasets while all 3753 ligands make the full dataset. Different machine learning approaches were employed to predict the binding affinities for both the datasets. The ligands from the test dataset were not exposed to the machine learning models and so the correlation between the experimental and predicted binding free energies was expected to be lower when compared to that in the case of training dataset. We employed eight different machine learning approaches and four of the them performed better when compared to the rest. The results of those ML approaches performed better are shown in Currently, there exist various force-field-based scoring functions to rank the protein–ligand complexes. In this study, we explored the performance of scoring functions as implemented in autodock-vina , autodocThe machine learning-based approaches can be used for predicting any properties given that there exists suitable dataset which correlates the property to be predicted to set of molecular descriptors. In this study, we used dataset of SMILES and ICgorithms ,36,37,38The substrate cavity space located next to FAD cofactor is the most attractive binding site for the ligands see . There aWe searched the ChEMBL database  for the it tools . InitialThe geometry optimized structures for the 3753 compounds were used for the molecular docking using autodock-vina  and autoThe most stable binding modes from molecular docking calculations using autodock-vina software were used for generating the input configuration for molecular dynamics and MM-GBSA calculations. The molecular dynamics calculations require the charges and force-fields for the ligands. Thus, the molecular structure as in the most stable binding mode for each of ligands was used for generating electrostatic potential fitted charges using Merz–Kollman–Singh scheme ,49. DensIn total, 3753 biologically active compounds for the target, human MAO-B, were selected for machine learning calculations. The physicochemical descriptors were used to predict the biological activity. For physicochemical descriptors, 1054 molecular descriptors, including Acidic Group Count, ALOGP, APol, Aromatic Atoms Count, Aromatic Bonds Count, Atom Count, Autocorrelation, Basic Group Count, Bond Count, Constitutional, Crippen, Detour Matrix, EState Atom Type, Extended Topochemical Atom, Fragment Complexity, HBond Acceptor Count, HBond Donor Count, Hybridization Ratio, Ring Count, Rotatable Bonds Count, Topological, Topological Charge, Topological Distance Matrix, TPSA, Weight, XLogP, etc., were calculated using PaDEL software . It is kParkinson’s disease is one of the most common neurodegenerative diseases and is associated with accumulation of


Disruption effects of genes encoding CodY, FrlR, RNaseY, and nucleoid-associated protein YlxR, four known regulatory factors for PfrlB, were examined for expression of each fusion construct. Expression of PfrlB-gfp and PfrlB-mCherry, which were located at amyE and its original locus, respectively, was bimodal; and disruption of ylxR resulted in the disappearance of the clear bimodal expression pattern in flow cytometric analyses. This suggested a role for YlxR on the bimodal expression of PfrlB. The data indicated that YlxR acted on the bimodal expression of PfrlB through both transcription and translation. YlxR regulates many genes, including those related to translation, supporting the above notion. Depletion of RNaseY abolished heterogenous expression of transcriptional PfrlB-gfp but not bimodal expression of translational PfrlB-mCherry, suggesting the role of RNaseY in regulation of the operon through mRNA stability control and regulatory mechanism for PfrlB-mCherry at the translational level. Based on these results, we discuss the meaning and possible cause of bimodal PfrlB expression.Bacteria must survive harsh environmental fluctuations at times and have evolved several strategies. “Collective” behaviors have been identified due to recent progress in single-cell analysis. Since most bacteria exist as single cells, bacterial populations are often considered clonal. However, accumulated evidence suggests this is not the case. Gene expression and protein expression are often not homogeneous, resulting in phenotypic heterogeneity. In extreme cases, this leads to bistability, the existence of two stable states. In many cases, expression of key master regulators is bimodal via positive feedback loops causing bimodal expression of the target genes. We observed bimodal expression of metabolic genes for alternative carbon sources. Expression profiles of the  Bacillus subtilis development of genetic competence for uptake of extracellular DNA, bimodal expression of the key master regulator ComK is observed, which leads to the differentiation of a fraction of cells among the cell population into the competent state -modified tRNA  itself requires cshA encoding a DEAD-box RNA helicase  and exhibits non-homogeneous expression . The hetenhanced , b. Furtpromoter . TsaEBD ied tRNA . The t6Aunctions . Expresshelicase . Proteometylated . It has etylated . CshA ac-gfp-R, amyE-RR/PacsA-(SD)-gfp-R, amyE-RR/PcodV-(SD)-gfp-R, and amyE-RR/PilvB-(SD)-gfp-R, respectively. Second, PCR products were amplified from the strain OAM-N32 using the oligonucleotide pair gfp(SD)-F/amyE-FF  strain 168; and among the resultant chloramphenicol resistant colonies, those with amylase non-producing phenotype were selected on the LB agar plate containing 1% starch. To construct OAM913, OAM922, and OAM927, the plasmids pUKM504-frlR, pfrlB-SD-gfp, and pfrlB-mCherry were transformed to 168. To construct strains carrying the /amyE-FF . Each frGrowth conditions and β-galactosidase analysis procedures were previously described .L-lysine . Microscopy was performed with an Olympus BX51 phase contrast and fluorescence microscope with a 100 × PLAN-N objective . Images were captured using a CoolSNAP HQ charge-coupled device camera  and Metavue 4.6r8 software .Cells were picked up from flesh colony on LB agar plate and inoculated to 1 ml of LB medium in L-tube. The tube was shaken for 14 h at 37°C. One hundred microliters of the culture was centrifuged, and 80 μl of the supernatant was removed. The cells were then resuspended in the remaining 20 μl. Portions (2 μl) of each sample were mounted on glass slides treated with 0.1% (wt/vol) poly-All the strains were streaked on LB agar plates supplemented with specific antibiotics and incubated overnight. The resulting single colony was picked up and grown overnight in 1 ml of LB medium in L-tube at 37°C. Cells were washed and resuspended in phosphate-buffered saline (PBS) and directly measured on BD LSRFortessa  with an argon laser (488 nm) and yellow green laser (561 nm). For each sample, the green fluorescent signal or mCherry signal of 30,000 cells was collected by a bandpass (BP) filter . The fluorescent intensity was calculated in arbitrary units (AUs). All the captured data were further analyzed with FlowJo version 7.6.5 software .frlBONMD-yurJ operon is subject to the severe YlxR-dependent transcription repression according to previous YlxR-transcriptome analysis  cells were observed during growth in SM  was used, and thus growth profile showed diauxie probably due to two carbon sources, glucose and Amadori products  and created transcriptional GFP fusions, and then we evaluated their expression by flow cytometry. All three of the promoter fusions showed homogeneous expression  and flow cytometric analyses showed that the disruption of frlR almost completely abolished the expression of both PfrlB-lacZ and PfrlB-gfp  was observed in the codY disruptant (data not shown). However, this enhanced rate of the fusion expression was 500-fold lower than the value reported by According to previous reports, the repressors CodY and FrlR bind to the promoter region of frlB . The Pfr at amyE . To conffrlB-gfp , 5A2. Thpression , 5A3, agpression . We notefrlB (yurP)-lacZ in its original chromosomal context is 10-fold enhanced by codY disruption when cultured in minimal glucose-glutamine medium containing a mixture of 16 additional amino acids , rrnE-16S (encodes an rRNA), and rpsNB . Therefore, it is possible that YlxR acts not only on the transcription of PfrlB but also on its translation. To explore this possibility, we constructed a translational fusion of frlB with the fluorescence protein gene mCherry at its original chromosomal position. In the construct, the mCherry protein was added with three amino acids to the N-terminus and expressed with the frlB SD sequence as required for its translational initiation , which was lower than that observed in the microscopic analysis. This may be due to detection of auto-fluorescence derived from the intracellular molecules like NADPH and aromatic amino acids from cells with no GFP-expressing strain, i.e., the control strain 168, in flow cytometry. Discrimination of weak but significant fluorescence signals from auto-fluorescence is difficult in flow cytometry. Contrary to this, in microscopic analysis, we did not observe auto-fluorescence derived from the strain without the gfp gene under the conditions we used (data not shown). Therefore, we had to explore the possibility using the microscopic analysis. As shown in ylxS-gfp expression; however, a small fraction of mCherry-ON cells did exhibit associated PylxS-gfp expression. Among the mCherry-ON cells, distribution of PylxS-gfp expression was low compared with that among the mCherry-OFF cells with the difference being statistically meaningful . These results indicated that some cells differentiate into PfrlB-expressing cells, even though there is no availability of amino sugars. This means that the Bacillus cells adopt a bet-hedging strategy with respect to nutritional fluctuation. At the transcription level, the observed bimodal or heterogeneous expression of PfrlB located at ectopic or original locus was caused by mRNA degradation triggered by RNaseY; however, this regulation was restricted for the transcriptional fusion expression through mRNA stability control. Based on our current study, we suggest that YlxR bimodal expression may result in the bimodal expression of the frlB operon through regulation at both the transcription and post-transcription levels including translation. Similar to YlxR in Bacillus subtilis, Escherichia coli nucleoid-associated proteins Ler and H-NS cause bimodal expression from the enterocyte effacement (LEE) pathogenicity loci . In addition, flow cytometry analysis using PfrlB-gfp at amyE revealed no GFP fluorescence expression in the cshA mutant . Consequently, we speculated that cshA may positively regulate the expression of frlB, independent of ylxR . Based on these data, we concluded that FrlR functions as an activator and that fructose-arginine is not an inducer of PfrlB expression at least in LB medium.A previous study presented a model where FrlR was a repressor and suggested that the inducer fructosamine-6-phosphate may inhibit the repressor activity of FrlR, leading to expression of the B operon . Our exptivators , which r on FrlR . The profrlB promoter was located in its original chromosomal position as disruption of codY did not affect frlB expression from the amyE locus. However, in an earlier report, PfrlB-lacZ expression at amyE increased in the codY disruptant (frlB (-202/ + 90) than that used in this study (-168/ + 47). The difference may have led to the discrepancy in the results. Under the conditions we used, however, codY disruption influenced frlB expression at its original position, but not its ectopic amyE position. Thus, the function of CodY might be dependent on the chromosomal position of the target gene frlB. This is not unprecedented as several other examples have been reported  results in the bimodal expression from the promoter of the genes encoding the sugar phospho-transfer system (PTS) for cellobiose/lactose (frlB in B. subtilis deserves further study, since the substrate of FrlB is not the most favorable carbon source but is sometimes provided. For example, this is the case in rhizospheres where it is actually preferred by Gram-positive bacteria of the family Bacillaceae.There are few known cases of bimodal expression of metabolic genes. In pulation . In Pseut operon . These a bimodal . In Lact/lactose . Interes/lactose . TherefoThe original contributions presented in the study are included in the article/MO performed experiments and wrote the manuscript. KS performed experiments. YK performed statistical analyses. All authors contributed to the article and approved the submitted version.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.


This study aimed to explore the association between malnutrition, clinical parameters, and health-related quality of life in elderly hospitalized patients with Parkinson’s disease (PD).Cross-sectional study of 92 hospitalized elderly patients with PD (mean age 73.6 ± 6.7 years) without dementia. The Mini Nutritional Assessment (MNA) was used to evaluate nutritional status. Motor impairment and non-motor symptoms burden , depression (Becks Depression Inventory-II), and health-related quality of life  were assessed.Every second patient was malnourished or at risk of malnutrition. In the multivariable analysis, male gender, longer disease duration, higher Hoehn & Yahr and depression were associated with total MNA score. Besides non-motor symptoms and motor impairment, malnutrition was an independent predictor of poor health-related quality of life. In the multivariate analysis, malnutrition had a statistically significant effect on emotional well-being, mobility, social support, stigmatization, and cognition. The strongest association was found between malnutrition and emotional well-being.Elderly male persons with longer PD duration and higher disease stages are more likely to be malnourished or at risk for malnutrition. Malnutrition was mainly associated with poor emotional well-being, suggesting that treatment of depression and anxiety beside diet and physical activity can help improving nutrition status in these subjects. The MNA should not be used independent of other measures of cognition and depression in people with advanced PD. Parkinson’s disease (PD) is a common neurodegenerative disorder characterized by motor impairment and a plethora of non-motor symptoms. People with PD are at a high risk of malnutrition . In PD, A gold standard for the optimal definition of malnutrition is still lacking . The EurMalnutrition is associated with poor health-related quality of life (QoL) and differentially influences distinct domains of health-related QoL, especially well-being and mobility domains , 8, 9. HWhat is the prevalence of malnutrition in hospitalized elderly patients with PD?To what extent does malnutrition predict health-related QoL in elderly patients with PD?Which domains of health-related QoL are mainly affected by malnutrition?This cross-sectional study was approved by the local ethics committee of the Jena University Hospital (4572-10/15), and all patients gave their written informed consent. Data were collected from patients with PD at the beginning of their stay (day 1–3) in the neurological ward of the Department of Neurology at the Jena University Hospital (Germany). All the patients were hospitalized because of the worsening of motor function or complications . All received a multimodal treatment by specialized therapists and medication modifications during their stay in the hospital  . Inclusiwww.mna-elderly.com). It was specifically designed for the elderly and is recommended by the European Society for Clinical Nutrition and Metabolism. It consists of six questions about dietary regime in the last 3 months, weight loss, immobility, recent stress period, neuropsychological disorders (depression and dementia), and body mass index (BMI) or calf circumference  [Several clinical variables were recorded, including the MDS-sponsored revision of the Unified Parkinson's Disease Rating Scale III (MDS-UPDRS III) to assess motor function, the revised Non-Motor Symptoms Questionnaire (NMS-Quest), Hoehn and Yahr staging (H&Y), and levodopa equivalent daily dose (LEDD). Cognition was assessed using the Montreal Cognitive Assessment (MoCa); PD dementia was defined as MoCa < 21 . Beck’s trition) .Epidemiological factors, the MDS-UPDRS III, NMS-Quest, H&Y, LEDD, MoCa and disease duration were derived from the medical records. The BDI and PDQ-39 were self-reports. The MNA was assessed by MT. After exclusion of 8 people with missing data in the BDI or PDQ-39, the data from 92 subjects were used for the following analyses.The SPSS statistical computer package  was used for all statistical analyses. Values are given as mean and standard deviation or median and interquartile range (IQR). Categorical variables are presented as numbers or percentages.Multiple linear regression analysis was subsequently performed to ascertain the independent predictors of the MNA. The clinical parameters / independent variables were derived from the literature and included: age, gender, disease duration, H&Y, MDS-UPDRS III, NMS-Quest, BDI, and LEDD , 16–18. A second linear regression was used to study the association between PDQ-39SI (dependent variable) and MNA and variables known to influence health-related QoL in PD  , 20. BefAnonymized data from this study are available as supplementary material.The detailed clinical characteristics are given in In the linear regression analyses male gender, longer disease duration, higher H&Y stage, and higher BDI were found to be associated with malnutrition .In the next step, we aimed to answer if malnutrition is associated with overall health-related QoL (PDQ-39SI) after controlling for known factors that influence PDQ-39  , 20. In p = 0.016; Wilk's Λ = 0.799, partial η2 = 0.20). However, significant univariate main effects for MNA were only found on emotional well-being , mobility , stigmatization , and social support . As indicated by the partial η2, the strongest association was found between malnutrition and emotional well-being. The MANCOVA was conducted to examine whether these findings could be accounted for by other medical covariates . Here, disease duration , NMS-Quest , and MDS-UPDRS III , but not age (p = 0.13), were significant in the model. In addition, the results did not change after controlling for these variables, as there remained a significant main effect of MNA on the emotional well-being , mobility , stigmatization , social support , and in addition cognition .Given that malnutrition impacts the PDQ-39SI, we then analyzed the relationship between nutrition status and the PDQ-39 domains. A MANOVA was used to study the association between malnutrition and the eight PDQ-39 domains. The MANOVA revealed a significant multivariate main effect for the MNA total score on the eight PDQ-39 subdomains  malnutrition was associated with male gender, longer disease duration, higher H&Y and depressive mood. Three recent studies also investigated the association between MNA and clinical parameters. Fereshtehnejad et al. investigated 150 patients with PD (men age 60.8 ± 10.8) and found in their multivariate linear model that depression, UPDRS total score, LEDD and patients’ sex were significantly associated with the total MNA score . The cohWe found that malnutrition is an independent predictor of the PDQ-39SI in elderly subjects with PD after controlling for other known predictors of health-related QoL, namely NMS which have large impact on QoL in elderly patients , 23–25. The MNA is widely used and is available in multiple languages and many studies in various settings used this tool. A large body of evidence underlines the usefulness of the MNA as screening tool in elderly people. However, despite these benefits of MNA, the sensitivity of the MNA is still debated because it has been related to a high risk of overdiagnosis of malnutrition –32. MoreOur study has some more limitations. The analysis of health-related QoL and malnutrition was restricted to hospitalized patients without dementia, limiting the generalizability of our results. Moreover,—as in the other studies –10—we diThe predictors of malnutrition and the impact on health-related QoL in our cohort of elderly hospitalized patients differ from the existing studies in younger patients. We found that elderly male persons with longer disease duration and higher disease stages are more likely to be malnourished or at risk for malnutrition. Malnutrition was mainly associated with poor emotional well-being. Further studies in elderly patients with PD should therefore answer if the treatment of depression and anxiety beside diet and physical activity can help to improve nutrition status in these subjects. The MNA should not be used independent of other measures of cognition and depression in people with advanced PD.S1 Data(SAV)Click here for additional data file.


T-cell lymphomas are rare malignancies that are often found in older individuals. Here we present the case of a 63 year old female that presented with diffuse cervical, inguinal, and axillary lymphadenopathy with associated weight loss and night sweats. The patient underwent excisional lymph node biopsy which revealed a T-cell lymphoma with findings characteristic of peripheral T-cell lymphoma, not otherwise specified. •EBV may be found positive in PTCL-NOS, Lennert lymphoma variant.•There is frequent loss or decreased expression of CD5 and CD7 in PTCL-NOS.•Excisional biopsy is necessary to diagnose malignancy if there is high suspicion and FNA/core biopsy are negative. There are two main types of T-cell lymphomas including peripheral T-cell lymphomas and T-lymphoblastic lymphoma/leukemia. Peripheral T-cell lymphomas include cutaneous T-cell lymphomas, adult T-cell leukemia/lymphoma, angioimmunoblastic T-cell lymphoma, enteropathy associated intestinal T-cell lymphoma, anaplastic large cell lymphoma, and peripheral T-cell lymphoma, not otherwise specified [2A 63 year old white female with past medical history significant for coronary artery disease with stent placement presented to the hospital with diffuse cervical, inguinal, and axillary lymphadenopathy that had been present for almost two years. The patient reported that her lymph nodes were progressively increasing in size and causing her to have dysphagia to solids and shortness of breath when lying flat. She also endorsed looser fitting clothes indicating weight loss and drenching night sweats.The patient said that she first developed lymphadenopathy on the right side of her neck two years ago while working in a chicken farm with exposure to ammonia. She saw a physician for lymphadenopathy at that time but was told that her swollen lymph nodes were likely reactive from ammonia. The patient quit her job but the lymphadenopathy persisted.Extensive work-up for the patient's lymphadenopathy was done with the highest suspicion for malignancy versus infectious etiology as the cause of the patient's presentation. Computed tomography of the chest showed diffuse cervical, mediastinal, and axillary lymphadenopathy with areas of necrosis and scattered groundglass opacities in the upper and lower lobes with multiple bilateral pulmonary nodules. Computed tomography of the abdomen and pelvis showed extensive abdominal, retroperitoneal, and pelvic lymphadenopathy, splenomegaly, and scattered sclerotic foci throughout the vertebral bodies and pelvis.Infectious work-up including HIV, syphilis, histoplasma, tuberculosis, toxoplasma, blastomyces, brucella, bartonella, coccidioides, and HTLV I and II proved to be negative. However EBV viral capsid antibody, early antigen IgG, and nuclear antigen antibody were all found to be elevated.Next, a fine needle aspiration and core biopsy were performed on the patient's left axillary node. Surprisingly the results proved to be negative for malignancy and the patient was diagnosed with granulomatous lymphadenitis with eosinophilic infiltrate. However, due to the patient's extensive lymphadenopathy an excisional lymph node biopsy of the left inguinal area was performed. The biopsy results showed T-cell lymphoma with Ebstein-Barr virus highlighting rare small-sized cells on immunoperoxidase studies. Polymerase chain reaction of the sample showed T-cell receptor beta and gamma gene rearrangement.3The patient's excisional biopsy results showed T-cell lymphoma with the most likely differentials being peripheral T-cell lymphoma, not otherwise specified (PTCL-NOS) versus angioimmunoblastic T-cell lymphoma (AITL). The pathologist's report described the excised lymph node as completely effaced with increased vascularity and a mixed inflammatory infiltrate with numerous small collections of epithelioid histiocytes distributed throughout the lymph node. AITL, a subtype of mature peripheral T-cell lymphoma was considered due to the increased vascular proliferation and EBV (Ebstein-Barr virus) positive cells in loose clusters in the sample. However, its probability was lowered due to lack of aberrant expression of markers CD10, BCL6, CXCL13, and PD-1 by the neoplastic T cells which have a high sensitivity and specificity for AITL when combinantly detected . The patPTCL-NOS are a group of heterogeneous diseases that involve the lymph nodes and extranodal sites that are diagnosed based on histopathology, aberrant T cell phenotype, and T-cell receptor rearrangement . VariantFurthermore, this patient endorsed that her lymphadenopathy first started while she was working in a chicken farm with exposure to ammonia. It is known that ammonia is a corrosive substance that irritates body areas that it comes in contact with such as the lungs, skin, and eyes. However, ammonia is not known to be a carcinogen and likely was not the cause of this patient's malignancy . She did4The treatment of T-cell lymphoma depends on the type and includes chemotherapy, immunotherapy, targeted therapy, radiation, steroids, or stem cell transplantation. CHOP  or EPOCH  chemotherapies are usually the initial regimens given to patients. Patients with relapsed disease are treated with ICE  therapy . The medNot commissioned, externally peer reviewed.


Background: Myxofibrosarcoma is a type of soft-tissue sarcoma that is associated with high rates of local recurrence and distant metastases. The first-line treatment for metastatic soft-tissue sarcoma has conventionally been doxorubicin-based. Recent evidence suggests that myxofibrosarcoma may be molecularly similar to undifferentiated pleomorphic sarcoma (UPS), which is particularly sensitive to gemcitabine-based therapy. The goal of this study was to evaluate the activity of gemcitabine-containing regimens for the treatment of metastatic myxofibrosarcoma refractory to doxorubicin. Material and Methods: We retrospectively evaluated seven consecutive cases of metastatic myxofibrosarcoma at our institution treated with gemcitabine-based therapy in the second-line setting, after progression on doxorubicin. Baseline clinical and baseline characteristics were collected. Primary endpoints were objective response rate (ORR), progression-free survival (PFS) and overall survival (OS). Results: After progression on first-line doxorubicin, a partial, or complete radiological response was observed in four of seven patients who received gemcitabine-based chemotherapy. With a median follow-up of 14 months, median progression-free and overall survival were 8.5 months and 11.4 months, respectively. Conclusions: Gemcitabine-based chemotherapy was associated with encouraging response rates in this cohort, similar to those seen in UPS. Both entities could be studied together for novel gemcitabine-based regimens. Soft-tissue sarcomas are a relatively rare tumor group and first-line treatment for metastatic disease has conventionally been doxorubicin-based . AlthougGiven the rarity of soft-tissue sarcoma, subgroups have usually been studied altogether for potential therapies. However, recent evidence has shown that systemic therapy response differs depending on histology. Notably, undifferentiated pleomorphic sarcomas (UPS) and leiomyosarcomas are sensitive to regimens containing gemcitabine .Myxofibrosarcoma (MFS) is another subtype of soft-tissue sarcoma which usually arises in the extremities and is associated with high rates of local recurrence and distant metastases . Tumor gBetween 2010 and 2019, a total of 17 cases of metastatic MFS refractory to doxorubicin were identified and retrospectively evaluated at the McGill University Health Centre (MUHC). Inclusion criteria were a diagnosis of metastatic MFS on second- line treatment with gemcitabine-based therapy after progression on doxorubicin; availability of radiological and clinical data for assessment of response. Ten patients were excluded due to lack of follow-up, other agent used as second-line therapy or no further treatment offered after doxorubicin. A total of 7 patients were included in our cohort (The baseline characteristics of all seven patients are summarized in One of seven patients received gemcitabine with docetaxel as a second-line treatment, five patients received gemcitabine and dacarbazine and one received gemcitabine as a single agent. Four of seven patients showed either a partial or a complete radiological response A. One paThe American Joint Committee on Cancer R classification R0: wide negative margin. R1: microscopically positive margin. R2: macroscopically positive margin.Rb and p53, two tumor suppressor genes, from chromosomal deletions or loss-of-function mutations, in both entities [Skp2, an oncogene that promotes cell turnover, to proliferate, and thus Li et al. suggest targeting Skp2 in order to suppress the cancer with novel therapies. Gemcitabine, an antimetabolite that incorporates into DNA and blocks replication, has shown its activity in tumor cells presenting with loss of function for p53 in different tumor subgroups [MFS and UPS, both formerly known as malignant fibrous histiocytoma, can be difficult to distinguish. Some authors have proposed that a threshold of at least 10% of myxoid component be an attribute of MFS . It is tubgroups . A phase II randomized trial comparing gemcitabine monotherapy to gemcitabine with docetaxel in metastatic soft-tissue sarcomas refractory to initial treatment showed superiority in terms of overall survival and progression-free survival with the combination . The resRegarding newer treatments, immunotherapy with pembrolizumab is active in UPS, as shown in a prospective multicenter study . AlthougOn the basis of the above-mentioned relationship between UPS and MFS and the promising response of MFS to gemcitabine, we agree that both entities could be studied together for novel gemcitabine-based regimens, as already done by some investigators .


As the most important organ in our bodies, the brain plays a critical role in deciding sex-related differential features; however, the underlying neural circuitry basis remains unclear. Here, we used a cell-type-specific rabies virus-mediated monosynaptic tracing system to generate a sex differences-related whole-brain input atlas of locus coeruleus noradrenaline (LC-NE) neurons. We developed custom pipelines for brain-wide comparisons of input sources in both sexes with the registration of the whole-brain data set to the Allen Mouse Brain Reference Atlas. Among 257 distinct anatomical regions, we demonstrated the differential proportions of inputs to LC-NE neurons in male and female mice at different levels. Locus coeruleus noradrenaline neurons of two sexes showed general similarity in the input patterns, but with differentiated input proportions quantitatively from major brain regions and diverse sub-regions. For instance, inputs to male LC-NE neurons were found mainly in the cerebrum, interbrain, and cerebellum, whereas inputs to female LC-NE neurons were found in the midbrain and hindbrain. We further found that specific subsets of nuclei nested within sub-regions contributed to overall sex-related differences in the input circuitry. Furthermore, among the totaled 123 anatomical regions with proportion of inputs >0.1%, we also identified 11 sub-regions with significant statistical differences of total inputs between male and female mice, and seven of them also showed such differences in ipsilateral hemispheres. Our study not only provides a structural basis to facilitate our understanding of sex differences at a circuitry level but also provides clues for future sexually differentiated functional studies related to LC-NE neurons. Sex differences, i.e., differences between male and female organisms, are prevalent among a number of organs, including the liver, kidney, muscles, gut, and brain . RecentlLocus coeruleus, a brainstem nucleus, contains at least two different cell types, the thoroughly studied noradrenaline (LC-NE) neurons and the recently emphasized GABAergic neurons (LC-GABA). Locus coeruleus noradrenaline neurons belong to a family of neuromodulatory systems. Although they have a small population, they project to a variety of regions across the brain with ascending projections to the olfactory bulb, cerebral cortex, amygdala, hippocampus, and thalamus, and descending projections to the medulla and spinal cord. Reciprocally, LC-NE neurons receive a broad range of inputs from similar brain regions . Thus, iDue to the key role in regulating mammal neural networks, multiple studies have focused on LC and employed LC-NE neurons as paradigms for elucidating the underlying mechanisms of sex differences with regard to the effects of sex hormones, structural differences, and molecular mechanisms . Among tHerein, we used cell-type-specific rabies virus-mediated monosynaptic tracing systems to map the brain-wide input sources of LC-NE neurons in male and female mice. We registered the whole-brain to Allen Mouse Brain Reference Atlas, made quantitative comparisons of the input data sets at different levels, and generated the sex difference-related whole-brain atlas. We identified multiple presynaptic regions that showed substantial input differences in both ipsilateral and contralateral hemispheres. We further demonstrated that sex difference-related overall differential inputs to LC-NE neurons were derived from specific subsets of nuclei nested within respective sub-regions. Our study thus provided a structural basis for the further sex-involved functional studies targeting LC-NE neurons.RRID: MMRRC_032081-UCD; n = 4 each) with similar weight (21–23 g) were chosen to make faithful comparisons. Adeno-associated virus (AAV) helpers used for rabies virus (RV)-mediated monosynaptic tracing were prepared according to our previous viral co-packaging strategy (our unpublished data)1. All vectors and viruses used in this study were provided by BrainVTA . Two vectors, AAV-EF1α-DIO-EGFP-TVA  and AAV-EF1α-DIO-RG (0.5 μg/μl), were premixed at a ratio of 1:2, and then the mixtures of the two plasmids were co-transfected with Ad helper vector and pAAV-rep/cap vector into HEK293T cells, followed by the standard processes of AAV production /ml. Genetically modified RV (EnvA-SADΔG-DsRed) were prepared as previously described  environment with a 12 h:12 h light:dark cycle.We used 2-month-old male and female dopamine β-hydroxylase-Cre (Dbh-Cre) transgenic mice (Tg(Dbh-cre)KH212Gsat/Mmucd, Stock #032081-UCD, oduction . The resescribed  and the All animal experimental procedures were approved by the Institutional Animal Ethics Committee of Huazhong University of Science and Technology (HUST). The injection protocol was performed as follows. Mice were deeply anesthetized by 3% isoflurane in oxygen. A volume of 100 nl rAAV helpers and lAAV-DIO-GT/RG were injected into the right hemisphere of the LC (anterior/posterior (AP) – 5.4 mm, medial/lateral (ML) – 0.8 mm and dorsal/ventral (DV) – 3.7 mm) of the male and female mice unilaterally using stereotaxic apparatus . Three weeks later, 150 nl of genetically modified RV was injected into the same area of both groups. Viruses were all injected with a pulled glass micropipette at a rate of 10 nl/min for each injection and the micropipette was allowed to stay for an additional 5 min or more before withdrawal. After nine days’ infection of RV, all mice were transcardially perfused with 0.01 M phosphate buffered saline (PBS) followed by 4% paraformaldehyde (PFA) in 0.01 M PBS. The extracted brain samples were embedded with 4% agarose  in 0.01 M PBS and sectioned coronally at 50-μm thickness across the whole brain with VT1200S vibratome . Coronal sections were sequentially collected to the 48-well plate with PBS. Every second section was chosen for analysis and counterstained with 4’,6’-diamidino-2-phenylindole (DAPI) to determine the cortical and laminar border, followed by mounting with covers glass using anti-fade fluorescence mounting medium  and sealing with nail polish for imaging.The whole-brain coronal sections were imaged with a VS120 slide scanner  at 10× magnification. Three channels were used for imaging, with blue channel for DAPI staining, red channel for input cells, and the green channel for starter cells across the injection site. During the process of imaging, parameters were adjusted based on the input signals of specific sections. For sections with dense input labeling, we first imaged the whole coronal plane with a high exposure time to ensure cells with lower signals were captured and further reimaged the dense labeling areas with a much lower exposure time to make the input cells visually identifiable  to the most posterior (−6.36 mm from the bregma)] were sequentially aligned from left to right. All sections were aligned in the same direction  to distinguish the ipsilateral hemispheres (hemispheres with the injection site) and the contralateral hemispheres. Manual rotations were applied to some improperly placed sections. All the processed images were stored in JPEG format for subsequent matching. The online 2D version of the Allen Mouse Brain Reference Atlas (2011 Allen reference atlas)2.Coarse matching: the prerequisite of perfect registration was to apply the appropriate reference plates to the corresponding raw images. To facilitate the registration, we divided the whole brain into five parts based on the large landmarks: (a) Part I (+3.0 to +1.0 mm from the bregma): matching was based on the changing of the cerebral cortex  and the shape of the corpus callosum and anterior forceps (fa); (b) Part II (+1.0 to −1.0 mm from the bregma): matching was based on the changing of the ventral part of each coronal section and the shape of the lateral septal complex (LSX); (c) Part III (−1.0 to −3.8 mm from the bregma): matching was based on the shape of the hippocampus; (d) Part IV (−3.8 to −5.3 mm from the bregma): matching was based on the shape of the periaqueductal gray (PAG); (e) Part V (−5.3 to −6.36 mm from the bregma): matching was based on the shape of the fourth ventricle (V4). The brains were sliced at a 50-μm thickness and every other section was chosen for analysis, which was perfectly in accordance with the 100 μm interval between two adjacent Allen Mouse Brain Reference Atlas plates. Once the distance from the bregma of one section was defined, the residual sections can be defined accordingly. For each section, the size of the raw image was set to resemble the reference plate, making the following fine adjustment easier.3.2. Allen online Nissl staining database was particularly useful for midbrain regions (−4.0 to −5.0 mm from the bregma), where the landmarks defined by DAPI staining were not obvious and the DsRed-labeled input signals were extremely dense. Multiple easily identifiable landmarks were available for the fine adjustment, including the corpus callosum , interal capsule , and cerebal peduncle . For instance, cc is a representative landmark that distinguishes the cortical regions from the subcortical regions; int was used to distinguish the caudoputamen (CP) from the thalamus and cpd was used to distinguish the amygdala from the hypothalamus.Fine adjustment: the coarse matching brain images went through free-form deformation to finely tone down diverse sub-regions by manipulating several grid points. We used the following information to assist the free-form deformation, including landmarks provided by DAPI staining and distribution of DsRed-labeled input signals and Allen online Nissl staining databaseManual identification of the input cells was performed using a multi-point tool in the ImageJ software . Identification of the positive signals, i.e., DsRed-labeled input cells with apparent cell bodies, were counted by the smart judgment of two experienced annotators who were blind to the sex of the mice. Several criteria were used to distinguish the positive signals from artificial signals, including the shape and size of cell bodies, whether with apparent dendrites protruding from cell bodies or not. For example, cell bodies were typically of irregular shapes whereas noise signals were more frequently found with a round shape; cell bodies typically had more than one protruding dendrite whereas noise signals did not. Moreover, the prevalent, brightly labeled dendrites whose cell bodies were cut off due to the mechanical sectioning caused great confusion to cell counts, which, however, could be solved by the size of the structure since cell bodies typically had a larger diameter than dendrites . Input cQuantification of the starter cells was based on the co-expression of EGFP and DsRed. In both male and female groups, the vast majority of the starter cells were located in the LC . A very For the generation of sex differences-related whole-brain input atlas of LC-NE neurons, brain-wide quantitative analysis regarding the proportion of inputs in each anatomical region  were performed for both male and female groups. Anatomical definitions and anatomical classifications of diverse brain regions were all in accordance with the Allen Mouse Brain Reference Atlas. All quantitative data were presented as the mean ± s.e.m. Significance was analyzed using Mann–Whitney U test in GraphPad Prism version 6.0 .n = 4 mice for each group). We next counted DsRed-labeled input cells  across the whole brain in all animals. We found that male mice had a slightly higher number of input cells  than female mice . In both groups, an average of 97 coronal sections were obtained . Overall, LC-NE neurons of the two groups shared similar input sources and exhibited a bilateral symmetrical manner for most brain regions . For exaTo investigate the sex-related different inputs of LC-NE neurons more comprehensively, we aimed to generate the whole-brain input atlas of LC-NE neurons in both sexes to obtain the information of: (i) the precise anatomical localization of input signals and (ii) the number of input neurons in each anatomical area. To address these issues, we initially registered each imaged section within an individual whole-brain data set to the standard Allen Mouse Brain Reference Atlas with the assistance of landmarks afforded by DAPI staining . We furtThe criterion we adopted for anatomical classifications were mainly based on specifications introduced by the Allen Brain Atlas (sunburst mode), similar to previous studies . After rWe first examined inputs of LC-NE neurons on a gross level. The midbrain, motor-related (MBmot) area provided the largest inputs in both groups among 12 major regions, with an average 30.2% out of total inputs for females, a little higher than males (26.8%). Similar to MBmot, we found discriminated distributions of proportion of inputs in the other 11 major regions. Specifically, inputs were dominant in the cerebrum, interbrain, and cerebellum (CB), the former two including the cortical plate (CTXpl), cortical subplate (CTXsp), striatum (STR), pallidum (PAL), thalamus (TH), and hypothalamus (HY) in males; whereas inputs were dominant in the midbrain and hindbrain, which include the sensory-/motor-/behavioral state-related midbrain part (MBsen/MBmot/MBsta), pons (P), and medulla (MY) in females . For insWe next examined the differences between inputs to male and female LC-NE neurons in specific sub-regions of the 12 major brain regions. Among 257 distinct anatomical regions, 123 were identified with proportion of inputs >0.1% (over 40 cells per region) and were used for further direct comparisons . We initP = 0.0286, Mann–Whitney test), which were derived from ipsilateral hemispheres rather than contralateral hemisphere . Across the remaining cortical sub-regions, we observed that visual (VIS), retrosplenial (RSP), and posterior parietal association areas (PTLp) provided more than 2-fold total, ipsilateral, and contralateral proportion of inputs to male mice than females. The other sub-regions, including several subdivisions in medial prefrontal areas , the orbital area (ORB), agranular insular area (AI), gustatory areas, visceral area, auditory areas, and temporal association areas, all shared similar proportion of inputs between male and female mice. Moreover, we also observed that no significant statistical differences were found in the sub-regions of HPF, the hippocampal formation (HIP), and the parahippocampal region (RHP), however, there was a prominent trend toward projecting to male mice, with over 2-fold difference in total, ipsilateral, and contralateral inputs when compared with female mice , which were also derived from the ipsilateral hemisphere, similar to MO and SS in cortical regions , whereas the latter showed a similar proportion. Similarly, behavioral state regions (MY-sat), which contributed the fewest proportion of inputs to the medulla, also showed significant statistical difference of both total and ipsilateral inputs  between the two sexes with over 2-fold more inputs to females.We also examined the differences between the two sexes in the midbrain and interbrain where inputs were dominant in females. Overall, the differentials in these female-dominant input areas were not as apparent as in males. In MBmot  between the two sexes, whereas SI and BST contained similar inputs for both groups , substantia innominate (SI), MSC, and BST took the majority of inputs in the dorsal, ventral, medial, and caudal regions of PAL, respectively. Among these four subdivisions, GPe and MSC in males contained nearly 2-fold more proportion of inputs and showed significant statistical difference of both total and ipsilateral inputs  and ventral group of dorsal thalamus (VENT) accounted for the largest inputs in DORsm and DORpm, respectively. We found that subdivisions in DORsm  and DORpm , all showed averaged 2-fold more proportion of inputs in males, both from ipsilateral and contralateral hemispheres, though statistical difference was not significant , since fP = 0.0286, Mann–Whitney test) when compared with PRNr, which showed similar inputs   .In summary, male and female LC-NE neurons shared similar input regions, but with differentiated inputs from some of the sub-regions, and further, from some of the nuclei within the corresponding sub-regions.Based on these observations, we further compared proportions of total, ipsilateral, and contralateral inputs in both sexes among 123 anatomical regions . We found that 41 of them, which made up over one-third of the anatomical regions, showed a similar proportion of inputs in male and female mice. Furthermore, we identified 11 anatomical regions with significant statistical differences of total inputs between the two sexes, and seven of them also showed such differences in ipsilateral hemispheres, with only one region showing such differences in the contralateral hemisphere . These dOur study provided the brain-wide quantitative analysis of direct inputs to LC-NE neurons in male and female mice and generated the sex-related differential whole-brain input atlas for the first time (to the best of our knowledge). For 257 anatomical areas covering the whole brain, proportions of inputs at different regional, sub-regional, and nuclear levels were analyzed. We found that LC-NE neurons of two sexes shared an overall similarity of input patterns, but with differential inputs from major brain regions and diverse sub-regions . Inputs With brain-wide quantitative analysis of input sources to LC-NE neurons, we identified multiple sexually differentiated anatomical brain regions, including the hippocampus, MSC in basal forebrain, thalamus, PVH in hypothalamus, and cerebellar cortex. The findings are consistent with previous studies in rats showing that somal and dendritic differences exist within different sexes . For theT66) , weight (21–23 g), viruses (co-packaged rAAV helper), and data analysis (comparisons within the same area ranging from Bregma +3.0 to −6.36 mm and exclusion of the areas adjacent to the injection site). However, menstrual cycle was not taken into consideration, which might affect the normal conditions of female animals . Though 66T) . Further66T) , the difIn summary, our study not only provides the structural basis for our understanding of sex differences in LC-NE neurons at the circuitry level but also provides clues for related further functional studies. Further, the strategies employed in our study offer a paradigm applicable for the input networks in other brain regions or neuron types. Therefore, our data and strategies should be helpful for the neuroscience community for a broad range of functional and structural research.All datasets generated for this study are included in the article/The animal study was reviewed and approved by Institutional Animal Ethics Committee of Huazhong University of Science and Technology (HUST).Y-HZ conceived the project. PS, JW, and MZ performed the virus injection experiments. PS performed the imaging with the assistance of YM and performed the whole-brain registrations. PS, JW, YM, YW, XD, and YM performed manual counting of the input cells. PS and JW analyzed the data. FX provided helpful advice for manuscript. Y-HZ, PS, and JW wrote the manuscript with discussion and improvements from all authors. All authors contributed to the article and approved the submitted version.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.


Introduction: Social-distancing due to COVID-19 has led to social isolation, stress, and mental health issues in older adults, while overwhelming healthcare systems worldwide. Telehealth involving phone calls by trained volunteers is understudied and may be a low-cost, scalable, and valuable preventive tool for mental health. In this context, from patient participatory volunteer initiatives, we have adapted and developed an innovative volunteer-based telehealth intervention program for older adults (TIP-OA).Methods and analysis: To evaluate TIP-OA, we are conducting a mixed-methods longitudinal observational study.Participants: TIP-OA clients are older adults (age ≥ 60) recruited in Montreal, Quebec.Intervention: TIP-OA volunteers make weekly friendly phone calls to seniors to check in, form connections, provide information about COVID-19, and connect clients to community resources as needed.Measurements: Perceived stress, fear surrounding COVID-19, depression, and anxiety will be assessed at baseline, and at 4- and 8-weeks. Semi-structured interviews and focus groups will be conducted to assess the experiences of clients, volunteers, and stakeholders.Results: As of October 15th, 2020, 150 volunteers have been trained to provide TIP-OA to 305 older clients. We will consecutively select 200 clients receiving TIP-OA for quantitative data collection, plus 16 volunteers and 8 clinicians for focus groups, and 15 volunteers, 10 stakeholders, and 25 clients for semi-structured interviews.Discussion: During COVID-19, healthcare professionals' decreased availability and increased needs related to geriatric mental health are expected. If successful and scalable, volunteer-based TIP-OA may help prevent and improve mental health concerns, improve community participation, and decrease healthcare utilization.Clinical Trial Registration: ClinicalTrials.gov NCT04523610; https://clinicaltrials.gov/ct2/show/NCT04523610?term=NCT04523610&draw=2&rank=1 The novel SARS-CoV-2 virus causing COVID-19 emerged in late 2019 , 4. GlobA proposed means of increasing access to mental healthcare during COVID-19 is through telehealth, or the use of technology for remote clinical care and health education . TeleheaCurrently, global healthcare systems are overburdened, with professionals increasingly becoming burnt out, reassigned, or infected , 18. AddEvaluating the effects on perceived stress, fear related to COVID-19, symptoms of depression and anxiety in older adults , as well as the effects on mental healthcare utilization.Comparing clients' mental health outcomes based on baseline risk .Analyzing the experiences of clients, clinicians, volunteers, and organizers/stakeholders involved in the program using semi-structured interviews and focus groups.We are offering a large-scale telehealth intervention program, whereby trained volunteers make weekly friendly phone calls to older adults in Montreal, Canada to serve three main purposes:To provide friendly conversation and social connection;To provide information about COVID-19;To connect older adults with services and resources in the community to ensure that the necessities of daily living  are met.The program is a community-based social support intervention that may serve both as prevention and treatment of mental health issues in older adults. In order to evaluate the program and share our findings internationally, we are also conducting a mixed-methods longitudinal study. We are consecutively sampling clients, volunteers, and other implicated parties and collecting both quantitative and qualitative data to understand the impact of this intervention.TM, a widely used video conferencing platform. Selected inclusion criteria are the ability to (1) commit for a minimum of 3-months; (2) provide this service to 3-5 clients; and (3) call each client at least once every week. Applicants with a criminal record or applicants who refuse to submit to a background check are excluded from volunteering.Volunteers are recruited (1) using an advertisement circulated on social media; (2) through direct recruitment of medical, social work, psychology, and nursing students from McGill University; (3) from the research and clinical teams' social networks; and (4) through the Jewish General Hospital (JGH) Foundation and Auxiliary, a volunteer non-profit organization. Interested individuals are rigorously screened and interviewed using ZoomTM, to a maximum of five volunteers per session . The initial virtual training was provided in kind collaboration with the Touch-Volunteers in Partnership (ViP) program at the Jewish General Hospital. The Touch ViP program was created through a participatory mixed-methods study, which engaged persons living with mental health conditions, their families, and mental health professionals over 3 years to support recovery-oriented practices (What connects us-Ce qui nous lie project Dementia Community Investment fund (1920-HQ-000092), to support the aim to create a web of inter-sectoral resources and decrease stigma. In addition to the stratified matching, the program has been further modified for TIP-OA to address the needs of older adults during COVID-19 with feedback from trainers, clinicians, and volunteers.Members of the volunteer management team stratify volunteers according to past volunteering and clinical experience(s) , and common scenarios that may come up during calls. The training also includes basic information about psychotic features and instructs volunteers to reach out to the clinical team if they require extra support in interacting with their client, or if they have concern for the safety and well-being of their client or someone else. The design of this training informs the follow-up methodology used to support volunteers, with an emphasis on stigma reduction as a means toward interpersonal connection. Similar to Touch, the training and implementation are iterative and responsive to the needs of volunteers and clients.Clients are either self-referred or referred from the JGH (COVID-19 inpatient units or the geriatric psychiatry clinic), long-term care facilities, local primary care clinics, or community organizations in the west-central Montreal healthcare/administrative catchment region . Interested clients and clinicians can call our voicemail number, send an email, or fill out a form on our website to refer to our program. Included clients are consenting older adults (age ≥ 60) living in Montreal. Excluded individuals (1) are actively suicidal, (2) have a severe hearing impairment or other disability preventing the use of a phone, or (3) are suffering from moderate or severe dementia, as determined by clinical judgment , or (4) are unable or unwilling to provide informed consent.At intake, the screening clinician or trainee  spends 20–30 min on the phone with each client to collect basic demographic information and perform an informal clinical assessment regarding the client's level of cognitive functioning (coded as having suspected mild dementia or not). In cases where the client was referred from a clinic, our clinical team receives information from the referring clinician regarding the clients' cognitive and overall functioning. The screening clinician also assesses the client's level of risk and the complexity of their case, conducting a brief assessment developed primarily from Culo's paper  and natiMultiple systems are in place for volunteer support and quality assurance:Follow-up meetings: The training team hosts weekly drop-in follow-up meetings for volunteers on ZoomTM. Trainers answer volunteers' inquiries and facilitate learning through group discussion and problem solving. Training and implementation can also be responsive to the emergent concerns and situations from these follow-up sessions. Although we encourage all volunteers to attend a follow-up meeting with the trainers, we emphasize to code red volunteers that they should refer to these sessions for extra support in managing their clients' cases.Volunteer hotline: Volunteers are provided with a phone number to call during working hours  should they have clinical and/or urgent concerns regarding a client. An automatic system connects the volunteer with the clinician on-call for rapid assistance. The clinician support services are provided by professionals and advanced trainees with considerable experience in geriatric mental health .Volunteer email address: Volunteers are given an email address to contact in the case that (1) their client does not wish to receive the service anymore, (2) the volunteer would like to take on another client, or (3) the client/volunteer would like to be paired with someone new. A research assistant checks this email daily.Client voicemail: Upon entry into the telehealth intervention, a clinician provides clients with a voicemail number to contact should they have any queries . The trainers instruct volunteers to additionally give this number to clients during their first contact. A team member checks this voicemail on a daily basis and relays the messages to the appropriate person. This voicemail is also used for clients who self-refer to receive TIP-OA.TIP-OA involves 1–2 times/week friendly phone calls from trained volunteers to adults >60 years old. Although the calls serve primarily to provide friendly conversation, they also grant volunteers the opportunity to inquire about clients' general well-being, provide information about COVID-19, and assess basic needs the client may have. If needed, volunteers refer clients to community resources and services . In this way, the friendly phone calls may prevent against the worsening or development of mental health issues or may treat pre-existing or recent mental health conditions.The first 200 TIP-OA clients who provide informed consent will participate in data collection for research.The client exposure for the quantitative portion of this study is the TIP-OA intervention.All primary and secondary outcomes will be assessed over the phone at baseline, 4-, and 8-weeks (primary study endpoint):The Perceived Stress Scale (PSS) is a validated and commonly used measure of stress  is a 9-item self-report questionnaire used to assess depressive symptom severity. The questionnaire asks individuals how often in the last 2 weeks they have been bothered by problems like “feeling down, depressed, and hopeless” and “poor appetite or overeating.” Scores for each question include 0 , 1 , 2 , and 3 (nearly every day)  is a brief scale that measures symptoms of anxiety present in the previous 2 weeks. Respondents' scores can range from 0  to 3 (nearly every day). Items include “Not being able to stop or control worrying” and “Being so restless that it's hard to sit still”  in the 8 weeks prior to baseline and (2) during 8-week follow-up.talized” . The Patery day) . The Gent still” . Mental Baseline risk: during the screening process, our clinician team codes clients as green (low risk), orange (medium risk), or red (high risk)  and eight clinicians/stakeholders, who will be consecutively selected and invited over the phone to participate. Verbal informed consent will be obtained before the start of the focus groups.Semi-structured interviews: 10 different stakeholders , 15 volunteers, and 25 clients (consecutively selected) will additionally be invited to participate in individual semi-structured interviews, for which verbal informed consent will be obtained. The client interviews will consist of open-ended and guided questions regarding the overall program and its impact on their mental health and well-being  between important subgroups . Analyses will be run using R statistical software.Descriptive statistics for continuous variables will include means and standard deviations, while categorical variables will be summarized using frequencies and proportions. A within-subjects repeated measure analysis of variance (ANOVA) will be conducted for the PSS (primary outcome), COVID Fear Scale, PHQ-9, GAD-7, and mental healthcare utilization (secondary outcomes) to assess differences in measures between baseline and the 8-week follow-up between groups with different baseline risk . Additional analysis of covariance (ANCOVAs) will control for variables that differ between groups at baseline. We will use LOCF to handle missing data that results from study attrition. Although we have found it extremely rare to be missing baseline data when, for example, a participant answers all but one question on a measure, in any such case we will exclude the participant's data for that measure. All ANOVAS will be tested at two-tailed alpha = 0.05. Linear mixed effect regression models, adjusting for age and sex, will analyze the effect of TIP-OA on all outcomes  collected at baseline and 8-week follow-up. We will perform additional linear mixed effect regression models accounting for a larger group of important variables such as client referral source  and cognitive functioning  on mental health outcomes. Variable selection in linear mixed effect model  will be A descriptive phenomenological approach will be used to analyze qualitative data. This descriptive thematic analysis will focus on the lived experiences of clients, volunteers, and stakeholders in order to map out the broader phenomena emerging from TIP-OA. We will code data based on repeated patterns of words, key-words-in-context (KWIC), contrasting information, and by searching for missing data. These patterns will help to identify and organize themes based on frequency, recurrence, and complexity of meanings in the data. Using a gender lens, participant narratives will be analyzed to understand their experiences. Coding will take place in three phases. Phase 1 will be descriptive, classifying the participants' narratives into standardized, predefined codes. In phase 2, descriptive coding will inform categorical coding by helping to further define data categories. In phase 3, analytical coding will allow interpretation of data-grounded emergent themes. To establish and authenticate links between different sources, we will conduct data triangulation by approaching individual narratives from various sources. We will use NVIVO  to estabComparable intervention studies for late-life stress, fear, depression, and anxiety have detected statistical significances and clinically meaningful effects , 34 inclTM focus groups, and 25 clients, 15 volunteers, and 10 stakeholders will be selected in the same manner for semi-structured interviews on ZoomTM.As of October 15th, 2020, 150 volunteers have been trained to provide TIP-OA to 305 older clients. Final research ethics approval has been provided and we will consecutively recruit 200 clients for quantitative data collection. After 8-weeks, 16 volunteers and eight clinicians will be consecutively selected for ZoomThrough regular friendly phone calls and the development of relationships between older adults and trained volunteers during the COVID-19 pandemic, TIP-OA aims to not only improve geriatric mental health symptoms, but also prevent mental illness in a wide sample of older adults. Crucially, many of the design elements  are geared toward long-term sustainability. Our longitudinal mixed-methods evaluation of TIP-OA's effectiveness, viability, and impact will inform future systematic telephone-based support for older adults by identifying roadblocks, thus allowing for rapid mobilization of quality telehealth services in response to future crises. Depression and anxiety symptoms in adults during COVID-19 in China have been estimated to be 15.0 and 30.9%, respectively , compareOlder adults, especially those with pre-existing mental health conditions, have so far fared relatively poorly during COVID-19 , but preTIP-OA has a similar structure to previous telehealth interventions but may, in the context of the COVID-19 pandemic, show a greater positive impact on mental health due to the government enforced social distancing. Compared to similar studies, our mixed-method data collection and our broad variety of mental health outcomes , will give a more comprehensive picture of TIPOA's impact on clients' mental health. Despite taking years to implement, typically therapeutic mental health clinical trials produce evidence for narrow populations  due to extensive exclusion criteria that may disqualify up to 85% of people in depression trials  and up tTIP-OA can be a large-scale low-cost telehealth intervention that leverages lay-volunteers to support geriatric mental health. A foundational component of TIP-OA, the volunteer training, resulted from long-term participatory research with patients and other stakeholders in psychiatry, and thus promotes important aspects of mental health recovery including Connectedness and Empowerment, derived from the CHIME model . FurtherThe main limitation is that this study is non-blinded, non-randomized, and observational. If funding permits, we plan to conduct a pilot randomized controlled trial comparing outcomes of TIP-OA vs. a waitlist control. Another limitation is that our system for categorizing clients  for participation was required for this study in accordance with the national legislation and the institutional requirements. Recorded verbal informed consent to participate in this study will be obtained from the patients/participants, and a copy of the consent form will be mailed or emailed to each participant according to their preference.ElD contributed to the design of the volunteer management protocols, to the direction of the volunteer management team, and to the conception and writing of this manuscript. HS led the research development of this project and played a major role in the writing of this manuscript. AA contributed to the clinical components of this project and to the editing of this manuscript. BV, AG, EmD, IR, and AS developed the clinical protocols for this program and provided feedback on this manuscript. PL-G contributed to the clinical duties of this program and to the revision and submission of this manuscript. HP, RN, JG, KC, and CR contributed to the conception and implementation of the volunteer management, community resource, and research components of TIP-OA. CW developed the volunteer training component and along with NS, JG, HS, KC, and PL-G trained the TIP-OA volunteers. AM, CH, CL, OB, EM, DS, SK, VN, MM, M-AB, GA, KL, and IV all provided important revisions on this manuscript. C-LS determined and verified the analytic methods. SR and SB were responsible for the overall vision, conception, implementation, and supervision of the presented idea. All authors contributed to the article and approved the submitted version.DS is a site investigator on a clinical trial sponsored by Hoffmann La Roche. VN receives research funding from Brain Canada, Eli Lilly, as well as Merck & Biogen Pharmaceutical companies (project completed). He declares no influence or impact on the project under consideration. SK receives research support from Brain and Behavior Foundation, National Institute on Aging, BrightFocus Foundation, Brain Canada, Canadian Institute of Health Research, Center for Aging and Brain Health Innovation, Center for Addiction and Mental Health, University of Toronto. He also receives equipment support from Soterix Medical. GA served on the Advisory Board of Eisai and of Janssen Pharmaceuticals. He also served on the Speakers Bureaus of Allergan, Otsuka, and Takeda-Lundbeck and his work was supported by P50 MH113838. SR receives a junior investigator salary award from the Fonds de Recherche Quebec–Santé and an investigator-initiated research grant from Satellite Healthcare  for an unrelated project. The remaining 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.