Introduction: The ability of synapses to maintain physiological levels of evoked neurotransmission is essential for neuronal stability. A variety of perturbations can disrupt neurotransmission, but synapses often compensate for disruptions and work to stabilize activity levels, using forms of homeostatic synaptic plasticity. Presynaptic homeostatic potentiation (PHP) is one such mechanism. PHP is expressed at the Drosophila melanogaster larval neuromuscular junction (NMJ) synapse, as well as other NMJs. In PHP, presynaptic neurotransmitter release increases to offset the effects of impairing muscle transmitter receptors. Prior Drosophila work has studied PHP using different ways to perturb muscle receptor function—either acutely (using pharmacology) or chronically (using genetics). Some of our prior data suggested that cytoplasmic calcium signaling was important for expression of PHP after genetic impairment of glutamate receptors. Here we followed up on that observation.Methods: We used a combination of transgenic Drosophila RNA interference and overexpression lines, along with NMJ electrophysiology, synapse imaging, and pharmacology to test if regulators of the calcium/calmodulin-dependent protein phosphatase calcineurin are necessary for the normal expression of PHP.Results: We found that either pre- or postsynaptic dysregulation of a Drosophila gene regulating calcineurin, sarah (sra), blocks PHP. Tissue-specific manipulations showed that either increases or decreases in sra expression are detrimental to PHP. Additionally, pharmacologically and genetically induced forms of expression of PHP are functionally separable depending entirely upon which sra genetic manipulation is used. Surprisingly, dual-tissue pre- and postsynaptic sra knockdown or overexpression can ameliorate PHP blocks revealed in single-tissue experiments. Pharmacological and genetic inhibition of calcineurin corroborated this latter finding.Discussion: Our results suggest tight calcineurin regulation is needed across multiple tissue types to stabilize peripheral synaptic outputs. Normal synapse function permits a pre-determined physiological range of activity. Challenges to synapse function can force activity beyond setpoint limits. To maintain stability, synaptic and circuit level homeostatic mechanisms exist in vertebrates and invertebrates Davis, . DefectsDrosophila melanogaster neuromuscular junction (NMJ). The Drosophila NMJ is a well-studied synapse , IP3 receptors, and ryanodine receptors were necessary for the expression of PHP after genetic glutamate receptor loss, but not after acute pharmacological PhTx challenge  encodes the only known ortholog of DSCR1 in Drosophila  has been shown to physically associate with the catalytic subunit of calcineurin  blocks of PHP compared to single tissue impairments. Consistent with this latter finding, global pharmacological inhibition of calcineurin via FK506 increases the homeostatic capacity of the NMJ following a genetic PHP challenge.Here we report that either increases or decreases in Sra are present in multiple synaptic tissues; if those factors are manipulated in tissue-specific ways, there can be profound defects in overall synapse stability. But conversely, global losses (or gains) of function may escape the most severe phenotypes. Our data set a framework for testing how individual molecules contribute to homeostatic synaptic plasticity from different synaptic tissues.Taken together, our data highlight a novel idea for homeostasis at the synapse. Regulatory proteins like Drosophilamelanogaster fruit flies were raised on a standard cornmeal and molasses medium prepared according to the recipe from the Bloomington Drosophila Stock Center . Drosophila husbandry was performed according to standard practices -GAL4  and in conjunction with the deficiency line Df(3R)sbd104 (BDSC:1920). The CaNBRNAi line that was used was TRiP.JF02616 , 10 mM MgCl2, pH 7.2  using the following calculation  BX51WI microscope using a 10× objective and acquired using an Axoclamp 900A amplifier, Digidata 1440A acquisition system, and pClamp10.7  software. Data were analyzed using MiniAnalysis  and the Clampfit  programs. To generate mEPSP and EPSP traces for figures,  coordinates were pulled from the Clampfit program and imported into GraphPad Prism  software. All traces display the recording that was at or closest to the calculated average of each specific condition.Pharmacological agents were bath applied in recording saline at the final concentrations indicated in the text and figures. The agents included Philanthotoxin-433 , Cyclosporin A (Sigma-Aldrich), and FK506 .Wandering third instar larvae were dissected on a Sylgard Petri plate in HL3 and fixed using Bouin’s fixative for 10 min or 4% paraformaldehyde in PBS for 30 min. Larvae were washed with PBS containing 0.2% Triton X-100 (PBST) for 40 min, blocked for an hour with 2.5% BSA, and incubated overnight with primary antibodies at 4°C. This was followed with another 40 min of washes, and incubation in secondary antibodies for 1.5 h at room temperature. The primary antibodies used were mouse anti-Synapsin  1:50 ; rabbit anti-Dlg 1:15,000  application to test the rapid expression of PHP  by about 60%  binding sites for GAL4 transcriptional control upstream of the sra gene; this allows for conditional tissue-specific overexpression of sra. Overexpression of sra neuronally using the pan-neuronal driver, elaV, and sraEY07182 has been shown to result in transcript levels that were approximately twofold higher than controls  + GluRIII RNAi synapses compared to neuron + muscle GAL4 ≫ sraEY07182 alone, and there was only a small—but not statistically significant—increase in NLS quantal content . We found no significant differences in EPSP amplitude or NLS quantal content between any groups . Somewhat surprisingly, sraMi06435 and sraMi06435/Df showed decreased mEPSP amplitude and EPSP amplitude compared to wild type, and no increase in NLS quantal content  and anti-Discs Large .Since the postsynaptic overexpression phenotype was the strongest, we checked if it correlated with NMJ developmental defects. In principle, synaptic developmental defects could explain the physiological phenotypes. We immunostained third instar larval sra resulted in a significant increase compared to other genotypes  levels and glutamate receptor signal intensity. Genetic glutamate receptor challenges normally result in decreased levels of pCaMKII immunofluorescence -Gal4 ≫ sraEY07182 synapses and a non-significant NLS quantal content increase  of that molecule. In principle, challenging a synapse with single-tissue genetic manipulation could disrupt this process.We were puzzled why the single-tissue genetic manipulations of sra was overexpressed postsynaptically  postsynaptically should decrease calcineurin activity in muscle. If the above model is correct, then impairing calcineurin globally via a pharmacological calcineurin inhibitor could reverse the chronic PHP block.Our earlier experiments showed a strong chronic PHP block when ptically . Sra is Drosophila larval neuromuscular junctions has been reported to increase the endocytosis of synaptic vesicles while having no effect on the frequency of spontaneous events or the mean amplitude of evoked events  is a calcineurin-inhibiting drug that binds to FK-binding protein l , and two genes that encode the regulatory subunit, CN B . The core finding is that a single gene encoding a calcineurin regulator can regulate PHP in multiple ways, in different tissues, and over various periods of time throughout the life of the synapse. Our work offers a way to consider how overall synaptic stability is maintained through different kinds of challenges to synapse function.sra, a gene encoding a calcineurin regulator, does not strongly impair synaptic homeostatic compensation mechanisms . We also showed that the maintenance of PHP can be further potentiated via FK506, especially when the system is crippled by low extracellular calcium  leads to a PHP block as it can disrupt the relative amounts of calcineurin activity between the two sides of the synapse. This idea could explain why a combined pre- and postsynaptic sra impairment  may not block PHP. Even though calcineurin activity is changed, the changes are reciprocal between both neuron and muscle.The full complement of pre- and postsynaptic data is complex. One model is that the relative balance of calcineurin function at NMJ tissues may control homeostatic signaling capacity. It is possible that impairing To our knowledge, this type of idea  is new to the field of homeostatic plasticity. But it is reminiscent of prior ideas that have emerged from studies of Hebbian plasticity. One example is the Bienenstock-Cooper-Munro (BCM) rule for visual cortex learning. The BCM rule dictates that either potentiation or depression can occur, but the direction of plasticity ultimately depends upon the level of postsynaptic activity after a presynaptic neuron fires  consistently increase the number of vesicles that are released, and failure to mobilize enough vesicles in response to a challenge may lead to a failure for compensatory NLS quantal content increase and impair evoked release.Calcineurin has several presynaptic roles in various models that have been shown including affecting synaptic vesicle exocytosis and endocytosis, axon remodeling, and regulating neurotransmitter release  and in turn result in defective PHP  by facilitating the transport of synaptic proteins and mitochondria  is necessary for synapse function and plasticity at the Drosophila NMJ  size and CaCaNB knockdown data also support the separation of the acute and chronic phases of PHP as global knockdown led to a weak impairment in acute PHP while leaving chronic PHP intact. These data support the separation of acute and chronic PHP as sra and CaNB expression are likely regulating different processes at different timepoints that integrate to produce functional PHP.Our Other work has suggested that PHP pathways converge onto the same targets  result in similar PHP expression mechanisms  and chronic genetic manipulations (via Sarah is consistent with any of them. Intriguingly, misexpression of Sarah in the postsynaptic muscle causes an increase in the levels of pCaMKII at boutons. By contrast, the downregulation of pCaMKII has been shown by other groups to be a sentinel of successful PHP expression apposing type-Ib boutons, specifically when genetically impairing glutamate receptors (Newman et al., Importantly, these models are not mutually exclusive. Our data regarding calcineurin regulation The original contributions presented in the study are included in the article/NSA and CAF designed the research, analyzed the data, and wrote the manuscript. NSA performed the research. Both authors contributed to the article and approved the submitted version.


Despite its importance, abnormal interactions between the proximal and distal upper extremity muscles of stroke survivors and their impact on functional task performance has not been well described, due in part to the complexity of upper extremity tasks. In this pilot study, we elucidated proximal–distal interactions and their functional impact on stroke survivors by quantitatively delineating how hand and arm movements affect each other across different phases of functional task performance, and how these interactions are influenced by stroke. Fourteen subjects, including nine chronic stroke survivors and five neurologically-intact subjects participated in an experiment involving transport and release of cylindrical objects between locations requiring distinct proximal kinematics. Distal kinematics of stroke survivors, particularly hand opening, were significantly affected by the proximal kinematics, as the hand aperture decreased and the duration of hand opening increased at the locations that requires shoulder abduction and elbow extension. Cocontraction of the extrinsic hand muscles of stroke survivors significantly increased at these locations, where an increase in the intermuscular coherence between distal and proximal muscles was observed. Proximal kinematics of stroke survivors was also affected by the finger extension, but the cocontraction of their proximal muscles did not significantly increase, suggesting the changes in the proximal kinematics were made voluntarily. Our results showed significant proximal-to-distal interactions between finger extension and elbow extension/shoulder abduction of stroke survivors exist during their functional movements. Increased cocontraction of the hand muscles due to increased neural couplings between the distal and proximal muscles appears to be the underlying mechanism. The human upper extremity is capable of performing a myriad of functional tasks due to its biomechanical structure and its abundant motor and sensory neural innervation. Unfortunately, this dexterity is often compromised by stroke, potentially affecting self-care, employment opportunities, and societal interactions .While difficulties occurring at the joint or muscle level, such as hypertonicity , spasticObject manipulation, a common component of task performance, requires further interjoint coordination as the arm must position, orient, and stabilize the hand while the fingers interact with the external object. Separate neural pathways have been suggested for control of the arm and hand  and the In this pilot study, therefore, we aimed to elucidate the impact of abnormal intersegmental interactions of stroke survivors on their function. In particular, we examined in details the kinematics and muscle coordination patterns across all phases of complex functional task performance of chronic stroke survivors and control subjects. In order to overcome aforementioned difficulties, we adopted a within-subject design that varies task conditions  to induce systematic changes in proximal kinematics of stroke survivors during functional tasks, thereby allowing us to examine the effects of proximal movements on the distal mechanics of stroke survivors  even with the high between-subject variability in their task mechanics. Conversely, distal-to-proximal impact was examined by comparing proximal kinematics during the phases that involved different distal movements (hand open vs. grip) within each subject. We also examined intermuscular coherence between the distal and proximal UE muscles of stroke survivors during task performance to gauge the neural coupling between these muscles, which could indicate underlying mechanisms of functional impairment following stroke . The goaWe expected to observe that the proximal movements of stroke survivors will significantly affect the kinematics and muscle activation of the distal tasks. Here, we anticipated that the abnormal distal-proximal interactions would be detected by a significant increase in the common neural inputs to the distal and proximal muscles. We also expected that the distal movements will also change the proximal kinematics, albeit to a lesser degree, as cortical projection to the arm muscles was found stronger after stroke in comparison to its connections to the hand muscles .A total of 14 subjects, including nine chronic stroke survivors (S1–S9) and five neurologically-intact control subjects (C1–C5), participated in the study. Three stroke survivors had severe/severe-moderate upper-limb impairments as indicated by their Fugl-Meyer Assessment of Motor Recovery for the Upper Extremity (FMUE) scores (mean ± SD = 29.7 ± 2.9) and six stroke survivors exhibited moderate-mild UE impairments (FMUE mean ± SD = 44.2 ± 1.9) see . All fivThe experimental protocol was approved by the Institutional Review Boards of MedStar Health Research Institute, and written informed consent was obtained from each subject prior to participation.An eight-camera motion capture system  captured joint kinematics with the sampling rate of 60 Hz. Reflective markers were placed on the following bony landmarks: metacarpophalangeal (MCP), proximal interphalangeal (PIP), and distal interphalangeal (DIP) joints of the index and middle fingers, interphalangeal (IP), MCP, and carpometacarpal (CMC) joints of the thumb, radial and ulnar styloid processes, lateral epicondyle of the elbow, acromion process of the shoulder, and torso .Seven pairs of disposable, self-adhesive Ag/AgCl surface electrodes were placed on the following muscles: extensor digitorum communis (EDC), flexor digitorum superficialis (FDS), first dorsal interosseous (FDI), biceps brachii (BB), lateral head of the triceps brachii (TB), anterior deltoid (AD), and lateral deltoid (LD). During the experiment, EMG signals were amplified, band-pass filtered between 5 and 400 Hz, and sampled at 1 kHz .Subjects were first asked to create maximum activations of seven muscles, which were used to normalize EMG for subsequent analyses. Subjects were first asked to perform isometric contraction of the target muscle while placing each electrode so that muscle palpation can be used to identify the muscle location, which were finger extension (EDC), proximal interphalangeal joint flexion (FDS), index finger abduction (FDI), elbow flexion (BB) and extension (TB), and shoulder flexion (AD) and abduction (LD). The same set of tasks were performed for approximately 2 s to record maximum voluntary contraction of each muscle.0) was marked on the table at waist level in front of the torso and three target locations (L1–L3) were marked in three different movement directions: L1 , L2 (midline), and L3 . Two other targets were located at shoulder level by using height-adjustable platforms: L4  and L5   . ReachinSubjects first performed reaching movements in each of the five movement directions to determine maximum voluntary reaching distance in each direction. Stroke survivors used their more-impaired arm while control subjects used their dominant arm. The target location in each direction was then positioned at 50% of the maximum distance each subject could reach for a given target direction in order to make the task difficulty comparable across subjects with different voluntary ranges of motion.0) to the target location (“transport” task: T1), released the object, and then returned the hand to the location L0. After a brief rest (2–3 s), the subjects repeated the movement, this time retrieving the object from the target location and returning it to L0 (“retrieve” task: T2). Subjects were instructed not to use compensatory strategies such as torso movements during reaching movements.Once the five target locations were determined, for each of these locations, subjects transported a cylindrical object  from the origin  were computed. For the fingers, flexion angles at the MCP and PIP joints of the index and middle fingers were computed and averaged across the two fingers to yield MCP23F and PIP23F. For the arm, elbow flexion (ELF), forearm supination (FAS), shoulder flexion (SHF), and shoulder abduction (SHA) were estimated from marker data.From the motion capture data, joint angles for 11 DOFs were computed. For the thumb, flexion angle at the IP, MCP, and CMC joints, denoted by IPElectromyography signals recorded from the seven target muscles were rectified and low-pass filtered at 5 Hz to create activation profiles. These activation profiles were then normalized by the EMG at maximum contraction to compute normalized activation level of these muscles.1The intermuscular coherence was computed between the following distal and proximal muscles: distal muscles: EDC, FDS, and FDI; proximal muscles: BB, TB, AD, and LD, resulting in 12 pairs. Time-varying coherence values were computed between these muscle pairs to examine the degree of distal-proximal neural coupling throughout the entire movements. For each distal-proximal muscle pair, the time-varying EMG-EMG coherence values were estimated from the raw EMG values using a sliding window of duration 500 ms with an increment of 100 ms, within the MATLAB environment  employing a script by Neurospec .1z-transformed to yield values normally distributed with a standard deviation of approximately 1 , commonly used to represent common neural inputs associated with corticospinal pathways  of the six hand submovement periods, From the angular trajectories of the hand and arm joints, initial and final time points  , and theval (T2) , were idpost-hoc pairwise comparisons, the p-values were corrected for multiple comparisons with a Bonferroni adjustment.The hand task kinematics (joint angles), hand muscle activation levels, and the peak intermuscular coherence values were compared between the following hand submovement periods that involved different arm mechanics, using repeated measures analyses of variance . The arm posture (at different time periods) was set as the within-subject variable, and the subject group (controls vs. stroke) as the between-subject variable. For the (1)Distal kinematics: hand aperture (finger extension) and grip posture were compared between the time points that involve different proximal kinematics.•tf of tf of Hand aperture for object grip: at near-body (•tf of tf of Hand aperture for object release: at near-body (•tiof tiof Grip posture: at near-body ((2)Distal muscle activation: mean activation level of three hand muscles  were compared between the following periods with different proximal kinematics.•Activation during hand open for object grip: at near-body (•Activation during hand open for object release: at near-body ((3)Intermuscular coherence: peak intermuscular coherence values of the 12 distal-proximal muscle pairs were compared between the following periods.•Coherence during hand open for object grip: at near-body (•Coherence during hand open for object release: at near-body ((4)f-tit) were compared.Movement duration: Durations of the following periods  and arm muscle activation levels were compared between the following arm submovement periods that involved different hand mechanics, using repeated measures analyses of variance. The distal task (hand open vs. grip) at different periods was set as the within-subject variable, and the subject group (controls vs. stroke) as the between-subject variable. Again, for the (1)Proximal kinematics: arm joint angles were compared between the following conditions .•n): with grip (tf of tf of Arm posture at target (L(2)Proximal muscle activation: mean activation level of four arm muscles  were compared between the following conditions.•Mean activation during reaching: with grip ((3)f-tit) were compared.Movement duration: Durations of the following periods (•Duration of reaching: with grip (0). Thus, only their proximal kinematics and EMG data were included in the analysis. Six stroke survivors with moderate impairment and five neurologically intact control subjects were able to complete the tasks, and all of their data were used for consequent analyses.Inability to open their hands prevented a subset of stroke survivors (S1–S3) from voluntarily completing the task; three subjects with severe UE impairments could not voluntarily perform the task, as they failed to extend the thumb/fingers enough to grip the object at the near-body location (L0) (during 5) or elbow extension (L2). Control subjects, in contrast, increased their hand aperture at these target locations with respect to the near-body location L0 .Across participants, movements of two proximal DOFs  significantly affected distal movements, particularly the maximum hand aperture for object grip  , but not in control subjects .Similar group × proximal task interactions were observed in the muscle activations. When two hand open movements were compared  was significantly higher at the target locations away from the body than the near body location (L0) for both subject groups , coherence between distal and proximal muscles increased at the early phase of the reaching movements, which was followed by the increase in the activation of the distal muscles  and the agonist/antagonist muscles of each proximal movement .The type of distal movements (hand open vs. grip) also significantly affected coordination of proximal DOFs (shoulder abduction and elbow extension) during outward movements  (p < 0.01) during approach   and smalk: grip) .p < 0.01). Conversely, the difference in elbow flexion between approach (p = 0.06).The distal-to-proximal impact on other proximal DOFs, however, differed between the groups . Shoulder abduction of stroke survivors was greater during approach (p = 0.01). No significant group differences or group × distal task interactions were found.When the arm muscle activation patterns were compared between the two hand movements (hand open vs. grip), however, there was no significant between-task differences in the activation level of most arm muscles, except for the elbow extensor muscle (TB), which exhibited a higher activation level when reaching with hand open   and proximal (reaching) movements (both s ≤0.01) .p = 0.03), and the amount of increase in movement duration was greater at certain target locations (L2 and L5) when compared to the near-body location (L0) . Similar trends  were observed in both groups, although the amount of increase in movement duration due to proximal task was greater for stroke subjects (p = 0.08).A similar impact of proximal movements on the movement duration of distal segments was observed in both subject groups. Hand open duration was longer when subjects approach the object , mainly observed during the tasks that involved hand opening. For the stroke survivors with severe impairments (Fugl-Meyer UE score <25), an inability to open their hands prevented them from performing the reach-transport-release task, as they were unable to grip the object. This is consistent with previous studies that showed successful grip of objects by stroke survivors can only be achieved when they restore their ability to extend the fingers and thumb .Even for the participants with moderate impairment, although they were able to complete the task, significant changes in the task mechanics were observed, especially during movements requiring finger/thumb extension. First, the duration of the hand opening phase was found to be significantly longer than that for neurologically-intact subjects. In contrast, the difference between the two groups in movement duration while gripping (transporting) the object was much smaller. Unlike neurologically-intact subjects, the hand aperture of stroke survivors during retrieval was significantly smaller for distant targets than for the near-body locations, likely contributing to the observed delays in hand openings. Finger/thumb extension of stroke survivors also affected their proximal mechanics; the shoulder abduction of stroke survivors during object approach (with hand open) was significantly greater than that during object transport (with grip).Proper adjustment of hand aperture during object retrieval is an important motor skill developed over time . In neurIn accordance with the previous studies, in our experiments, control subjects increased their hand aperture during retrieval of the object at the target locations (farther from the body), possibly to compensate for potential directional errors in reaching movements or due to reduced visual information of the hand aperture due to the distance. Stroke survivors, however, had decreased hand aperture when retrieving objects from target locations that require shoulder abduction or elbow extension. This decrease in aperture could have significantly hampered their ability to place/orient the hand to be able to grip the object, as well as increasing the task duration.0 to L2/L5: EDC = 20.4 ± 7.8%, FDS = 19.2 ± 22.8%), thereby resulting in the fingers and thumb being more flexed when reaching the target, in a manner similar to the typical flexion synergy patterns observed in stroke survivors .The observed reduction in the hand aperture of stroke survivors at these target locations resulted from global increases in the activation levels of the hand muscles, particularly those of the extrinsic muscles. As the cross-sectional areas of the extrinsic flexor muscles  are much greater than those of extrinsic extensor muscles (EDC or extensor indicis proprius) , equal curvivors . In contThe increase in the activation levels of the hand muscles appears to be related to the increase in the intermuscular coherence at these target locations. Previous studies observed that significant increase in the intermuscular coherence of the hand muscles was accompanied by a concurrent increase in all muscle activation levels  . AbnormaA significant distal-to-proximal impact on task mechanics was also observed across groups, as subjects extended their elbow further during object release  appear to be different from the proximal-to-distal effects, which resulted from a significant increase in distal muscle cocontraction. The observed changes in the proximal movement pattern of stroke survivors  is likely due to their compensatory strategy. For instance, during approach , it was observed that some stroke survivors had to move their hands laterally to grip the object from the side due to the decreased hand aperture. This required greater shoulder abduction, which could lead to further hand closing in accordance with abnormal flexion synergy patterns , while deficits in hand opening required compensatory changes in proximal kinematics. Therefore, restoration of the ability to extend the impaired fingers and thumb should be a primary focus for stroke rehabilitation, even for those who retain the ability to open the hand. More importantly, hand opening should be practiced in conjunction with proximal (reaching) movements so that stroke survivors can learn to overcome the abnormal distal-proximal coupling that affects their task performance.p-values were greater than 0.05. Observational studies are required to examine the impact of abnormal proximal-distal interactions in daily activities of stroke survivors.The number of subjects in this pilot study was small as we aimed to test feasibility of the detailed analysis of kinematic/physiological data throughout the complex upper extremity functional task. Therefore, the results from this study need to be replicated in a future study with a larger number of subjects in order for its findings to be generalized. The small subject number could also have led to type-II errors in the changes in the kinematic (EMG) and physiological (muscle activation) measures, whose Between-subject variability in the impairment of stroke survivors could have contributed to the variability in the data. The level of spasticity of stroke subjects, as well as the degree of their sensory deficits, was neither measured nor controlled, which could have contributed to the between-subject variability observed in the data.The control subjects were much younger than the stroke survivors tested in this study, which could have contributed to the observed between-group differences, in particular the movement time. However, previous studies showed that, while older subjects make slower movements during reaching, their movement coordination patterns remain similar to that of younger subjects .Some stroke subjects adopted compensatory strategies, involving torso movements , during reaching movements. Experimenters instructed subjects not to use such strategy, whenever detected, but it is possible that stroke survivors used torso movements to a certain degree during reaching; this could have reduced the requirements in their arm extension, or could have affected the hand orientation.In this pilot study, we delineated how abnormal coupling between the movements of distal and proximal UE segments of chronic stroke survivors affect their functional movements. In particular, hand open  and elbow extension and shoulder abduction  movements influence each other, resulting in a significant reduction in the distal range of motion (finger extension) and an abnormal proximal synergy pattern (increased elbow flexion/shoulder abduction) that can degrade task performance. Increased cocontraction of the hand muscles due to higher neural couplings between the distal and proximal muscles, indicated by the significant increase in the intermuscular coherence, appears to be the underlying mechanism of the observed functional deficits. The outcome of this study suggests that the finger/thumb extension of chronic stroke survivors should be practiced in conjunction with specific proximal movements (elbow extension/shoulder abduction) to help them overcome the abnormal distal-proximal coupling affecting task performance.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the Institutional Review Boards of MedStar Health Research Institute. The patients/participants provided their written informed consent to participate in this study.SL and BV designed the study. TP, HN, and BV collected the data. TP, HN, and SL analyzed the data. All authors interpreted the data, wrote the manuscript, and read and approved the final manuscript.


Lymphovascular invasion is a hallmark of malignant neoplasms, however the presence of benign epithelium within vessels has been reported in benign processes, albeit infrequently. The proposed mechanism to explain this phenomenon entails mechanical displacement of benign epithelium into the vascular spaces during surgical manipulation or diagnostic interventions. We report a case of numerous benign epithelial vascular emboli in a cholecystectomy specimen. A 29-year-old male presented with acute calculous cholecystitis. Surgery was delayed for several weeks due to COVID-19 infection. Histologic examination of the gallbladder showed subacute cholecystitis, widespread vascular epithelial emboli with associated fibrin deposition and bile embolism supporting an in vivo process. The epithelial emboli were localized in small veins and arterioles with D2–40−/CD31+/CD34+ endothelium. The displaced epithelium showed benign cytologic features, was negative for p53 expression, and had a Ki-67 labelling index like the benign background mucosa, supporting a benign process. There was no evidence of dysplasia or malignancy in the specimen after thorough sampling. Persistent inflammation, mucosal ulceration, transmural mucosal herniation (Rokitansky-Aschoff sinuses), and protracted surgical manipulation secondary to adhesive disease are favored to be the underlying causes of this unusual histologic finding. Although we presume an uneventful outcome, clinical follow up was recommended. COVID-19 infection likely contributed to this phenomenon by causing a delay in the surgical management. In surgical pathology, the microscopic presence of epithelial cells in vascular or lymphatic spaces is almost invariably associated with malignant processes and reported as lymphovascular, lymphatic, and/or angiovascular invasion. For surgeons and oncologists, reporting of this finding by pathologists is considered a risk factor for tumor relapse and metastatic disease, and often an indication for adjuvant therapy. However, the presence of benign epithelium in vessels has also been rarely reported in non-malignant processes. We describe the presence of numerous intravascular deposits of benign gallbladder epithelium in a cholecystectomy specimen of a patient who underwent a delayed cholecystectomy due to a COVID-19 positive test.A 29-year-old male presented to the emergency department with a one-week history of abdominal pain accompanied by nausea and emesis. The pain was intermittent, located in the right upper quadrant with radiation to his back, and was exacerbated by food intake. He had presented several times in the past 6 years with similar episodes. An 11 mm cholelith impacted within the gallbladder neck and multiple additional choleliths were identified on abdominal CT scan along with thickening of the gallbladder wall, consistent with acute calculous cholecystitis. The patient was started on intravenous antibiotics, morphine, and antiemetics. Surgical intervention was postponed due to a positive pre-operative COVID-19 test and the patient was discharged on oral antibiotics. Elective laparoscopic cholecystectomy was performed 4 weeks after initial presentation. During the procedure dissection was tedious due to dense adhesions and fibrous changes. Gross examination of the gallbladder revealed multiple choleliths up to 2.2 cm. The wall thickness ranged from 0.1 cm to 0.8 cm and the mucosa was roughened and focally disrupted but without discrete lesions.Microscopic examination showed transmural thickening and fibrosis, prominent Rokitansky-Aschoff sinuses, subacute inflammatory infiltrate and focal ulceration. Within a localized area of the wall, numerous intramural vessels containing intraluminal fragments of biliary epithelium were discovered. Several epithelial groups were closely associated with fibrin thrombi and bile emboli supporting an in vivo process rather than a grossing artifact. The intravascular epithelium appeared benign, lacking cytologic atypia or architectural complexity. Immunostains for p53 and Ki-67 showed no difference between the intravascular epithelium and the benign background mucosa, further supporting a benign process. Generous sampling of the gallbladder performed after identifying this abnormality showed additional foci of intravascular epithelium but did not reveal evidence of dysplasia or malignancy. Some vessels within the involved regions showed organizing thrombosis and fibrin emboli. The involved submucosal vessels included thin-walled and thick-walled muscular vessels Fig. . ImmunosThe presence of numerous intravascular deposits of biliary epithelium in this case was striking and unexpected, and elicited concern for an occult malignancy which prompted thorough sampling of the specimen and review of the case by several pathologists. The lack of cytologic atypia or architectural complexity in the intravascular epithelium and absence of dysplasia or precursor lesions in the specimen after thorough sampling were strong arguments against a malignant process. Additionally, the demographics of this patient: male, 29-y.o., did not conform to the epidemiology of gallbladder cancer   (McFarland et al. Gallbladders are common surgical specimens; pathologists are fully aware of the possibility of discovering an incidental carcinoma and the need to extensively sample specimens with any unusual histologic findings. A recent meta-analysis describes an incidence of incidental adenocarcinoma in cholecystectomy specimens of only 0.6–0.7% (Pyo et al. Several possible explanations for the observed epithelial intravascular emboli were considered: First, we considered artifactual angioinvasion: the emboli were limited to a specific region of the gallbladder wall, they were present in multiple blocks and levels and there was no epithelial displacement in non-vascular spaces to suggest that they represented displaced epithelium during grossing. The epithelium often conformed to the shape of the vascular spaces and was associated with fibrin thrombi and bile emboli supporting an in vivo process.Second, true angioinvasion was considered: vascular infiltration not related to medical intervention, in the context of benign processes, has been described in benign proliferative processes of the breast (Eusebai and Azzopardi Finally, mechanical pseudoangioinvasion due to manipulation of the gallbladder during surgery was a much logical explanation for the observed pathologic findings. Artefactual displacement of benign epithelium into lymphovascular spaces after invasive procedures has been reported in other organs such as breast (Koo et al. In summary, we describe a case of pseudoangiovascular invasion by benign gallbladder epithelium in a cholecystectomy specimen of a patient who had a delayed cholecystectomy due to a COVID-19 positive test. We favor that this extremely uncommon phenomenon, not previously described, is the result of mechanical, iatrogenically caused displacement of epithelium into abnormally dilated vessels due to an unusually prolonged and difficult surgical manipulation of the gallbladder, in the context of abnormal intraluminal and intravascular pressures. COVID-19 infection likely contributed to this phenomenon by causing a delay in the surgical management.


It has been shown that less consolidated sleep with more arousals or transitions to lighter sleep stages is common in the elderly. In a shorter term, aggravated sleep instability may lead to cognitive decline; in a longer term, it may cause neurodegeneration or fatal disorders.1 Moreover, optogenetic stimulation of Hcrt neurons triggered a shorter latency time of sleep-to-wake transition in aged mice.1 These findings integrally characterize the electrophysiology properties of Hcrt neurons in the aging-related sleep instability. It is likely that altered membrane threshold and spontaneous firing activities of Hcrt neurons may contribute to the excitation-inhibition imbalances in Hcrt connectomics, thus disturbing the coordination of wake/sleep-promoting neuronal circuits. Li’s study emphasizes the necessity of repressing the hyperexcitability of aged Hcrt neurons to maintain sleep quality.Does the hyperexcitability of Hcrt neurons contribute to sleep instability during aging? Li et al. raised this question and linked Hcrt neuronal circuits to aging-related sleep impairment. In their study, aged mice exhibited more significant fragmented sleep correlated with a substantial loss (~38%) of Hcrt neurons in the hypothalamus as compared with young mice. Fiber photometry recording in aged mice showed that Hcrt neurons have more frequent firing patterns highly relevant to wake bouts and sleep fragmentation. Data from patch-clamp recording further verified the elevated intrinsic excitability of Hcrt neurons in aged mice with more depolarized membrane potentials (RMPs).1 They discovered dysfunction of repolarizing M-current mediated by Kcnq2/3 channels and deficiency of Kcnq2 through array tomography at the ultrastructural resolution, and identified the dysregulated Kcnq family subtypes Kcnq1/2/3/5 by single-nucleus RNA-sequencing (snRNA-seq) in aged Hcrt neurons.1 Furthermore, they found that exerting CRISPR/SaCas9–mediated disruption of Kcnq2/3 genes in Hcrt neurons of young mice can mimic the aging-related sleep fragmentation features. Moreover, administration of Kcnq2/3-selective activator, flupirtine, significantly inhibited spontaneous firing activities in Hcrt neurons and increased the sleep stability in aged mice.1 These findings may provide a possible strategy to improve aging-related sleep disorders, and identifying more specific pharmacological targets acting on Hcrt neurons may shed light on conquering the possible unanticipated side effects of Hcrt receptor antagonists.In addition, Li et al. explored the molecular mechanisms underlying the hyperexcitability in Hcrt neurons during aging.1 suggesting that the hyperexcitability in Hcrt neurons specifically emerges in aging. In addition, beyond normative aging, sleep disruption is especially pervasive in neurodegenerative diseases, such as Alzheimer’s disease.4 The activated pathway of Hcrt neurons-to-basal forebrain increases acetylcholine release and activity of cortical neurons, affecting cognitive function.5 Besides, the amyloid-β-induced hyperexcitation is identified to be correlated with the expression of Kcnq2/3 in hippocampal neurons.5 The hyperexcitability of Hcrt neurons in aged mice of this study may provide a theoretical basis for neurodegenerative diseases-associated sleep instability.Hcrt neurons not only participate in regulating aging-related sleep impairment but also related to several other sleep disorders. Among them, the most noteworthy is narcolepsy, a sleep disorder at the early onset with sudden sleep disruption and cataplexy caused by a massive loss of Hcrt neurons or defects in Hcrt receptor 2. Li et al. pointed out that the basic electrophysiological properties and underlying mechanisms in narcolepsy mice are different from those in healthy aged mice,4 It is currently unknown whether the circadian rhythm of Hcrt neurons is involved in aging-related sleep impairment, which deserves further investigation in the future. Besides, although this study has sufficient data to demonstrate the vital role of Hcrt neuronal activity in regulating sleep stability in aged mice, it still lacks evidence from human studies. Therefore, it is of importance to carry out the study on older adults who suffer from sleep disorders to verify whether there is an altered level of Kcnq2/3 in the Hcrt neuron. Furthermore, molecular imaging to probe the change of Hcrt neurons in humans may help clarify the correlations between the Hcrt neuronal activity and sleep stability.Sleep regulation involves a complex of neural pathways and networks in the brain. In addition to the Hcrt system, there are several other interacted sleep-wake regulation circuits, involving the noradrenergic, cholinergic, and dopaminergic systems. Furthermore, the circadian rhythm regulated by a group of clock genes also plays a vital role in controlling the sleep-wake cycle.Overall, Li et al. innovatively discovered an essential role of the arousal-promoting Hcrt system in regulating sleep during aging. Their findings indicate that the hyperexcitability of Hcrt neurons might be an important culprit contributing to the sleep instability in the aged. The pharmacological remedy of sleep disruption by targeting Kcnq2/3 channels could be a promising therapeutic strategy for improving sleep quality and preventing aging-related sleep disorders.


We present an interesting case of a patient with a large scalp defect following Mohs surgery of basal cell carcinoma (BCC) and squamous cell carcinoma (SCC) that was successfully reconstructed with tissue expansion utilizing Orticochea flap, with the addition of an acellular dermal matrix as an adjunct in such hostile scalp reconstruction.Reconstruction of a large scalp defect following oncologic surgical resection is a challenging task. The defect size, location, and elasticity of the soft tissue overlying the calvarium are important factors to be considered when exploring available reconstructive options. When primary closure is not feasible with a large defect, a skin flap or graft is utilized. Skin flap is advantageous as it produces a similar color and texture as the surrounding areas, thus being the favorable method. Wounds involving exposed bone, tendon, and cartilage cannot support grafts due to poor vascularity and thus require a skin flap. One of the multi-flap closure modalities, the Orticochea flap, is an excellent choice for scalp reconstruction on large defects greater than 50 cm We present the case of a 53-year-old female who presented with basal cell carcinoma (BCC) of the scalp and required scalp construction following Mohs surgery. Scalp basal cell carcinoma is one of the rarer types of basal cell carcinomas.A 53-year-old Caucasian female with a past medical history of numerous actinic keratoses, basal cell carcinoma (BCC), and squamous cell carcinoma (SCC) throughout the body presented with a history of extensive crusting lesions on the superior central scalp measuring 12 × 6.5 cm  on the right parietal scalp and two 100-mL Mentor smooth crescent-shaped tissue expanders on the left parietal scalp were placed under the epicranial aponeurosis and expanded with 8-30 mL of saline weekly Figure . FollowiDuring the one-month postoperative follow-up period, the patient’s scalp healed well without any evidence of wound breakdown, infection, or necrosis.Tissue expanders allow for primary closure in procedures involving large skin defects. The expansion of the native tissue maintains vascularity and provides adequate skin needed for proper tension-free closure ,2. In th2 throughout. The galea aponeurotica was scored in a horizontal fashion, perpendicular to the tension line, to enhance elasticity and extend the flaps for closure (Figures The scalp reconstruction began with two large defects post-Mohs surgery by the dermatologist Figure . The ski Figures , 3d. The Figures , 3f. TheLarge skin defects created by wide local excision expose the cranial bone, leading to the disruption of blood supply and slowed healing. Dermal substitute is an acellular bovine tissue matrix made of collagen fibrils and elastin upon which dermal regeneration can occur for improved healing, hemostasis, and elasticity of the skin . CommonlSuccessful reconstruction of the scalp requires a comprehensive preoperative plan and meticulous intraoperative execution. The employment of tissue expanders and Orticochea flap provided additional scalp tissue for advancement flap closure. Our case demonstrates the use of both techniques as an effective method for scalp reconstruction by utilizing existing tissue to repair large scalp defects without extensive anesthesia, prolonged operative times, and potential surgical complications.


While trait-based approaches have provided critical insights into general plant functioning, we lack a comprehensive quantitative view on plant strategies in flooded conditions. Plants adapted to flooded conditions have specific traits  to cope with the environmental stressors including anoxic sediments, and the subsequent presence of phytotoxic compounds. In flooded habitats, plants also respond to potential nutrient and light limitations, e.g. through the expression of leaf economics traits and size-related traits, respectively. However, we do not know whether and how these trait dimensions are connected.Based on a trait dataset compiled on 131 plant species from 141 studies in flooded habitats, we quantitatively analysed how flooding-induced traits are positioned in relation to the other two dominant trait dimensions: leaf economics traits and size-related traits. We evaluated how these key trait components are expressed along wetness gradients, across habitat types and among plant life forms.We found that flooding-induced traits constitute a trait dimension independent from leaf economics traits and size-related traits, indicating that there is no generic trade-off associated with flooding adaptations. Moreover, individual flooding-induced traits themselves are to a large extent decoupled from each other. These results suggest that adaptation to stressful environments, such as flooding, can be stressor specific without generic adverse effects on plant functioning (e.g. causing trade-offs on leaf economics traits).The trait expression across multiple dimensions promotes plant adaptations and coexistence across multifaceted flooded environments. The decoupled trait dimensions, as related to different environmental drivers, also explain why ecosystem functioning  are species and habitat specific. Thus, our results provide a backbone for applying trait-based approaches in wetland ecology by considering flooding-induced traits as an independent trait dimension. By definition, flooding encompasses the hydrological conditions of waterlogging, partial or complete submergence . In respIn addition to dealing with flooding-induced stressors, plants may have to adapt to habitat resources mainly including nutrients and light. The leaf economics spectrum . Size-reAs flooding-induced traits play important but distinct roles compared with leaf economics traits and size-related traits in plant functioning, understanding whether and how these different groups of traits position relative to each other will improve our knowledge of plant strategies that cope with flooding stresses, in combination with differences in nutrient and light availability. If flooding-induced traits are decoupled from leaf economics traits, this suggests that flooding-induced traits are cheap to develop without trade-offs in nutrient acquisition or allocation . Plants So far, some arguments are available that support the case of decoupled relationships .We compiled functional traits on plants recorded in flooded habitats, following the definition of the international Ramsar Convention  and the Root porosity was quantified mainly as either the percentage of the hollow area in the root cross-section or the ratio of hollow volume to the whole root volume. These two methods generally show agreement in air-filled root porosity . Root/shTo evaluate potential drivers of trait–trait relationships, we included habitat wetness, habitat type and growth form in our analysis.The Ellenberg moisture indicator values provide insights into the extent to which species are known to occur at different extents of habitat wetness . These iTo obtain more comprehensive insights into the relationships between the traits of the species and the ecological backgrounds relevant to wetland conditions, we also recorded the habitat type for each trait observation according to a modified Ramsar classification as presented in For this study, we took species mean trait values to allow analysis of trait–trait relationships, as individual studies did not provide all traits for the same situation  in R . Due to R2) were calculated using the sma function in the SMATR package  analysis  on groups of Ellenberg moisture indicator values, is presented in Finally, we ran an ordinary linear regression to examine how each flooding-induced trait relates to habitat wetness affinities . Data of the root/shoot ratio and shoot elongation were logThe PCA on each of the flooding-induced traits with the other two trait dimensions showed largely decoupled patterns. Leaf economics traits mainly occupied PCA axis 1, with size-related and flooding-induced traits on the other axes. The PCA loading scores on the first three PCA axes are shown in Root porosity was to a large extent decoupled from the leaf economics trait axis , but partly covaries with leaf N. The size-related trait (as represented by plant height) was positioned on a third trait axis. The first two PCA axes accounted for 42.0 and 22.8 % of the total variation, respectively . Also, tR2 = 0.22, P < 0.05), root porosity–SLA  and SLA–plant height   . In the The other flooding-induced traits (root/shoot ratio and shoot elongation) did not relate significantly to any leaf economics or size-related trait.The SMA analysis confirmed the significant and strong relationships between the leaf economics traits  . This suP > 0.05), and the R2 values of effect sizes were only 0.00–0.02 . However, there was no relationship between the Ellenberg moisture indicator and root/shoot ratio , or between the Ellenberg moisture indicator and shoot elongation . Hence, among the three flooding-induced traits, variation in root porosity significantly contributed to habitat affinities of plant species along a wetness gradient. Even though the root/shoot ratio and shoot elongation are considered important flooding-induced traits, they were not directly related to the distribution of plants along a wetness gradient.To understand how an individual flooding-induced trait contributes to the generic habitat affinities for wetness of plant species, we further tested the relationships between the Ellenberg moisture indicator and individual flooding-induced traits . Among t2 and O2 gained by shoot elongation may compensate the costs thereof . Moreover, while root porosity significantly contributes to the preference of plant species along a wetness gradient, root/shoot ratio and shoot elongation are not directly linked to the Ellenberg moisture indicator, life form or habitat type  and leaf N–leaf P , and the extremely weak correlations of root porosity–leaf P  and root porosity–plant height , the trade-off between root porosity and leaf N does not fundamentally change our interpretation that root porosity is in a trait dimension decoupled from leaf economics traits . Our study indicates that no integral general strategy exists that perfectly explains the adaptation of plants to complex flooded environments. Instead, the multiple facets of flood tolerance plant strategies, as shown by the combination of functional traits including flooding-induced traits, leaf economics traits and size-related traits, together contribute to the survival of plants in complex flooded environments and help them prosper globally across a wide range of habitat fertilities. These insights provide a trait-based foundation towards understanding the general flood tolerance plant strategies and the functioning of flooded ecosystems, as well as adaptations to habitat stress in different ecosystems.https://academic.oup.com/aob and consist of the following. Appendix A: deriving Ellenberg moisture indicator values for plant species in the analysis. Appendix B: a list of plant species name and traits analysed in this study. Appendix C: principal component analysis (PCA) of leading trait dimension and ANOVA.Supplementary data are available online at mcac031_suppl_Supplementary_Appendix_S1Click here for additional data file.mcac031_suppl_Supplementary_Appendix_S2Click here for additional data file.mcac031_suppl_Supplementary_Appendix_S3Click here for additional data file.


After identifying potential crucial hub proteins, drug–proteome interaction signatures were ranked by the computational analysis of novel drug opportunities (CANDO) platform for multiscale therapeutic discovery to identify potentially repurposable drugs. Subsequently, a literature-based knowledge graph was utilized to rank combinations of drugs that most likely ameliorate inflammatory processes. Proteomic network analysis demonstrated that 233 of the >1800 proteins identified in the BALF were significantly differentially expressed in COPD versus control. Functional annotation of the differentially expressed proteins was used to detail canonical pathways containing the differential expressed proteins. Topological network analysis demonstrated that four putative proteins act as central node proteins in COPD. The drugs with the most similar interaction signatures to approved COPD drugs were extracted with the CANDO platform. The drugs identified using CANDO were subsequently analyzed using a knowledge-based technique to determine an optimal two-drug combination that had the most appropriate effect on the central node proteins. Network analysis of the BALF proteome identified critical targets that have critical roles in modulating COPD pathogenesis, for which we identified several drugs that could be repurposed to treat COPD using a multiscale shotgun drug discovery approach.Bronchoalveolar lavage of the epithelial lining fluid (BALF) can sample the profound changes in the airway lumen milieu prevalent in chronic obstructive pulmonary disease (COPD). We compared the BALF proteome of ex-smokers with moderate COPD who are not in exacerbation status to non-smoking healthy control subjects and applied proteome-scale translational bioinformatics approaches to identify potential therapeutic protein targets and drugs that modulate these proteins for the treatment of COPD. Proteomic profiles of BALF were obtained from (1) never-smoker control subjects with normal lung function (n = 10) or (2) individuals with stable moderate (GOLD stage 2, FEV Chronic obstructive pulmonary disease (COPD) is a leading cause of mortality and morbidity in the US ,4,5,6,7.Although structural changes in the airways, parenchyma, and pulmonary vessels are typical in patients with COPD, the lower airways and the alveoli are the initial sites of the inflammatory process ,18. The There has been a keen interest in understanding the genetic determinants of COPD, as the interaction between genes and the environment leads to protein expression, ultimately resulting in either healthy or disease states. However, genomic data alone does not predict protein abundance or activity; proteins are the ultimate participants in integrated biological processes that lead to healthy physiological function or pathology. Proteome-based analysis of bronchoalveolar lavage fluid (BALF) in COPD can identify tissue-specific markers of inflammation that can lead to understanding the mechanisms of COPD progression.We sought to determine an unbiased proteome-based analysis of BALF in COPD under stable conditions (not in exacerbation status) to identify a broad series of molecules involved in COPD pathogenesis. A label-free proteomics mass spectroscopy method was utilized. The differentially expressed proteins were analyzed using multiple bioinformatics tools to identify critical pathways that were altered in these ex-smoker patients with COPD compared to never-smoker healthy controls and important “hub” proteins implicated in COPD etiology, and identify putative drug candidates that can be repurposed to treat COPD.p-value < 0.05) to identify 423 individual proteins with 76 proteins expressed differently between COPD and controls [p-value < 0.05) that identified 1831 individual proteins and 233 differentially expressed proteins between the two groups. The latter, more practical approach provides important information for biomarker and therapeutic target discovery that may be utilized in future research to discover valuable interventions.The raw proteomic data used in this manuscript were initially detailed in a previously published methodology manuscript using strict criteria  had a significant differential expression in BALF samples from patients with COPD versus healthy ex-smokers; 138 proteins were decreased in COPD while 95 proteins were increased , CCAAT/enhancer-binding protein β-CEBPB (109), paired box gene 2-PAX2 (113), and activating transcription factor 2-CREBP1 (95).Transcription factors  associatThe protein expression datasets were imported into IPA (Ingenuity Systems) and projected onto the relevant biological pathways; processes linked to the differentially expressed proteins were analyzed with the IPA’s manually curated knowledge database. Of the 233 differentially expressed proteins, 217 matched the IPA-curated database and were analyzed. Sixteen pathways were noted to have several proteins associated with the differentially expressed BALF dataset , includiOne-hundred-and-thirty out of 233 BALF differentially expressed proteins were identified in the CANDO human protein library. This subset of proteins within the CANDO platform was used to predict 189 putative drug candidates with the most similar protein interaction signatures to the set of known drugs used to treat COPD . Many ofA total of 233 proteins were identified as differentially expressed between COPD patients and healthy controls by mass spectrometry. Of these, 214 were represented in the Elsevier knowledge graph , with thFour nodes representing proteins in the network stood out based on the normalized betweenness centrality values representing a greater than linear baseline increase from the next lower-ranking node: fibronectin, vimentin, intercellular adhesion molecule 1 (ICAM1), and galectin-3. These potentially key signaling mediators had a betweenness centrality of at least 25% of the maximum.Analysis of the initial data reveals fibronectin and ICAM1 are reduced in COPD patients relative to healthy controls; thus, any candidate therapeutic should target an increase in their activity. The reverse is true for vimentin and galectin-3. We, therefore, sought drugs or combinations of drugs predicted to accomplish the appropriate activation or inhibition of the four most central nodes. Specifically, drugs that will promote central node proteins that were downregulated in the COPD cohort and inhibition of central node proteins that were overabundant in COPD. Therefore, the idealized drug vector constitutes interactions leading to desirable modulation of the central hub protein. CANDO identified 189 distinct drugs  with relWe additionally conducted a targeted query to assess the predicted effects of drugs commonly applied in pulmonary disease treatment on the most central proteins of this regulatory network . While sOur investigation of the COPD BALF proteome utilizing novel bioinformatic techniques revealed significant differences in proteins involved in multiple biological processes, including lung-specific mechanisms, protease/anti-protease homeostasis, immunoregulation, and the extracellular matrix. Proteomic profiling of the complex pathways implicated in COPD provides broader physiological exploration not provided by studying one entity at a time. We identified several differentially expressed proteins in COPD versus controls that, based on a review of published literature, have not been previously implicated in COPD etiology. This preliminary analysis illustrates how our BALF proteomic analysis represents a powerful approach to elucidate COPD pathogenesis and identify novel biomarkers.Employing the bioinformatics tool DAVID and IPA, putative pathway networks were constructed based on the differentially expressed proteins in the BALF proteome that implicated multiple transcription factor pathways and disparate biological processes, such as extracellular space, proteolysis, extracellular matrix, cell adhesion, cytoskeleton, defense response, cell migration, and oxidation reduction.The CANDO platform identified 189 drug candidates with similar protein interaction signatures based on the BALF proteome compared to known drugs used to treat COPD. However, while most putative drug and protein interactions are likely inhibitors, the induction or inhibition of a target protein is indeterminable with solely the binding potential between drug and protein pairs.Topological analysis of the interaction network connecting 233 proteins differentially expressed in COPD through regulatory interactions documented in the literature suggested that ICAM1  and galeIn contrast, the corticosteroid fluocinolone acetonide ,45 and tThe documented actions of these immunomodulators were predicted here to substantially counteract the observed dysregulation of centrally connected proteins in COPD patients. The relatively high representation of immunomodulators among the candidate agents and the increased centrality of fibrosis-related proteins is consistent with the paradigm of airway remodeling as central to COPD pathology . With adOur approach does have some limitations. The variability in how much BALF is recovered from each aliquot of saline infused in to the lower airway in COPD vs. control subjects is inherent in most BALF proteomic analyses. However, the BALF proteins were normalized to albumin BALF concentrations to account for the variability. The examination of protein levels without accounting for post-translational modifications, such as phosphorylation, may neglect important differences in protein interactions and activity, despite no significant differences in protein levels. Also, the BALF samples were from subjects in the COPD group who were ex-smokers. This exclusion limits the generalizability of our findings, particularly in current smokers, since the acute effects of tobacco smoke were excluded in our study design.However, we confined our analysis to ex-smokers with moderate COPD to obtain some uniformity of the COPD phenotype and to avoid the acute inflammatory effects of current smoking. Future work on proteomic profiles will inform us of the difference between such profiles in current smokers and different stages of COPD.A sputum proteomics study endeavored to identify COPD severity biomarkers by employing 2D gel electrophoresis and revealed 15 proteins that were significantly differentially expressed between healthy smoker controls and subjects with GOLD stage II; subsequently, 9 of the 15 candidate proteins were validated with Western Blot. Of the nine candidate proteins validated with Western Blot, seven were statistically significantly different between groups, specifically albumin, alpha-2-HS glycoprotein, transthyretin, PSP94, apolipoprotein A1, lipocalin-1, and PLUNC . EmployiA 2D differential gel electrophoresis study and subsequent mass spectroscopy were performed by Ohlmeier et al., which compared healthy smokers, non-smokers, and smokers with GOLD stage II COPD and revealed a different set of 15 proteins that were differentially expressed between the groups . Of thesIn Lee et al., tumor-free lung tissue harvested from patients with lung cancer resection, when examined via 2D gel electrophoresis/MALDI-TOF-MS, revealed 8 proteins that were upregulated in subjects with COPD compared to non-smokers and 10 significantly differentially expressed proteins between subjects with COPD and smoking subjects without COPD . Two of https://github.com/ram-compbio/CANDO Accessed on 1 December 2021) to identify FDA-approved drugs that could be repurposed to COPD based on their putative interaction with the differentially expressed proteins. Using topological network analysis, we identified putative hub proteins that modulate the cellular pathways associated with COPD. Using the medical literature to predict the repurposed drugs effects on the most important hub protein, we created a refined list of drugs predicted to modulate the cellular pathway to impede COPD pathogenesis and generate proteomic interaction signatures for the compounds.We analyzed the protein quantifications derived from the BALF of subjects with COPD and healthy ex-smoker control subjects via liquid chromatography and mass spectroscopy. We then used pathway analysis tools to identify the relevant cellular pathways associated with differentially expressed proteins quantified from the BALF analysis. We subsequently employed the Computational Analysis of Novel Drug Opportunities (CANDO) platform ; 50–80% predicted with the ratio of FEV1 over forced vital capacity (FEV1/FVC) < 0.70. All ex-smokers had ceased smoking for at least 1 year.We selected BALF obtained from 10 ex-smokers with moderate COPD and 10 healthy non-smoking controls for proteomic analysis, respectively. To minimize variability due to the effects of acute smoking and disease severity, we confined this analysis to ex-smokers and moderate stage 2 disease per the Global Obstructive Lung Disease (GOLD)  criteriaThe research bronchoscopy and BALF sample preparation were performed as described previously .To investigate the soluble molecules in the epithelial lining fluid that may participate in COPD pathogenesis, unbiased proteomic analysis of BALF commenced without protein depletion or fractionation. Details of the methodology were published  and are p-value < 0.05 are included as part of the differentially expressed BALF proteome.The complete separation of the complex peptide mixture utilized a nano-LC/nanospray setup ; the ionhttp://david.abcc.ncifcrf.gov/ Accessed on 21 March 2021) [Gene ontology, transcription factors, and expression locations were determined by uploading the protein expression dataset onto a web-based tool, the NIH’s Database for Annotation, Visualization, and Integrated Discovery (DAVID) v6.7 (ch 2021) ,23. BiolIn addition to annotating differentially expressed proteins with the manually curated pathways cataloged in IPA, a network of protein–protein interactions was created using known regulatory relationships extracted from published scientific literature using the MedScan text-mining engine  as well We used the CANDO platform ,67,68,69This study calculated the interaction scores between 2450 United States Federal Drug Administration (FDA)-approved drugs from the CANDO version 2.3 compound library and a curated human library of 8385 proteins, including 5316 solved X-ray crystallography structures and 3069 computed protein structures modeled by I-TASSER ,71. The For this analysis, we focused on the differentially expressed proteins in the BALF proteome (as described) and drugs used to treat COPD (“MESH:D029424”) . We selebetweenness centrality has been used to describe how a node might serve as an important mediator of information flow in a regulatory network. In this work, n of a network was calculated using the Brandes algorithm [n reflects the amount of control that this node exerts over the interaction between communities of neighboring nodes in the network [s and t are the source and target nodes in the network different from n, s to all t, and s to t that must pass through node n. Here, unweighted betweenness centralities were calculated for each node in the literature-informed protein–protein network. The betweenness centrality scores for all nodes were expressed as fractions of the maximum betweenness centrality present in the network. All calculations were conducted in R version 4.0.2  [Although not a complete descriptor, the topological location and aspects of the connectivity linking a node to a broader biological network can inform the node’s function in mediating network behavior. Among the measures of a node’s importance or centrality, lgorithm . The bet network  and can Austria) .cS, between each drug vector and the idealized intervention vector is calculated [D is the drug vector and M is the idealized intervention. Higher cS indicates a closer match between the drug vector and the idealized vector. An cS of 1 means the two vectors are identical, and −1 indicates that the two are exactly opposed. For multidrug combinations, the net-effect of the individual drug vectors is calculated as:n is the total number of drugs in the combination, iD is the vector corresponding to the ith drug, and sgn is the sign function. The cosine similarity of the net-effect vector and idealized vector is then calculated.Using putative drugs ranked by CANDO and further analyzed via the Elsevier knowledge graph , a drug lculated . Cosine p-values are estimated asr is the number of null cS values greater than the observed cS, and n is the total number of null cS values.The statistical significance of these enrichment scores is determined empirically from an estimated null distribution of cosine similarities. This null distribution uses a set of model-relevant background drugs for which each interacts with at least one entity in the network. All CANDO drugs of interest were included in the background. Empirical Statistical analysis was performed with SPSS/19. Demographic values were depicted as mean ± SEM.In summary, our work provides a valuable pipeline for identifying many proteins associated with COPD pathogenesis that illustrate the complexity of the development of this disease and identify putative therapeutic treatment options using cutting-edge bioinformatics approaches. Identifying differentially expressed proteins will form the basis for future mechanistic studies of critical pathways and novel treatment discovery. The validation of our proposed therapeutic approach in animal models and human pilot studies are important next steps.


Mental health remains a highly stigmatized area of healthcare, and people often conceal their concerns rather than seek assistance or treatment. The Women Support Initiative Forum (WSIF) is a social media platform established in 2018 to provide expert and peer-led psychosocial support services to women of all ages in Bangladesh. The anonymous nature of the forum means that mental health concerns can be aired without fear of identification.th March 2020 and 7th July 2022. Around 1457 posts were initially selected for analysis which was reduced to 1006 after removing duplicates and non-relevant posts, such as queries about the addresses of the doctors and other non-mental health-related issues. A thematic analysis of the data was conducted using an inductive approach.A content analysis was conducted on the anonymous posts retrieved from the WSIF platform between 8n = 1006; 100%). Most also mentioned reasons for seeking mental healthcare , healthcare-seeking behavior , and barriers to seeking mental healthcare . The majority of women described symptoms of stress, depression, and anxiety-like symptoms, which were aggregated under common mental health conditions. Mental health symptoms were ascribed to various external influences, including marital relationship, intrafamilial abuse, and insecurities related to the COVID-19 pandemic. A large proportion of posts were related to women seeking information about mental healthcare services and service providers (psychologists or psychiatrists). The analysis found that most women did not obtain mental healthcare services despite their externalized mental health symptoms. The posts identified clear barriers to women accessing mental health services, including low mental health literacy, the stigma associated with mental healthcare-seeking behavior, and the poor availability of mental health care services.The 1006 posts generated four themes and nine sub-themes. All the women mentioned mental health symptoms (The study revealed that raising mass awareness and designing culturally acceptable evidence-based interventions with multisectoral collaborations are crucial to ensuring better mental healthcare coverage for women in Bangladesh.The online version contains supplementary material available at 10.1186/s12888-022-04414-z. Mental health issues have been identified as one of the most significant contributors to the global burden of disease and disability . More thFactors contributing to mental health issues experienced by women are financial dependency, reduced autonomy in decision-making, interpersonal violence, and the social role of women as child-bearers and caregivers , 8–11. IThe main problems affecting men’s and women’s access to treatment are the scarcity of mental health professionals, the mal-distribution of facilities to urban areas, and the improper implementation of national mental health policy , 15. In The COVID-19 pandemic also had an indirect, adverse impact on mental health inequalities among women –26. The Understanding the challenges of women with mental health problems is key to improving the nature, quality and reach of services. Unfortunately, for many researchers, policy-makers and mental health professionals, the information available about women’s challenges typically comes from clinical settings  or community samples . There is no ready access to women’s spontaneous, every day and personal observations about their mental health challenges.https://www.facebook.com/groups/182300659137448). Eighty percent of members are aged between 20 and 28 years and are distributed across all eight divisions (administrative units) of Bangladesh. WSIF provides online psychotherapy and counseling via expert psychologists and psychiatrists. In addition, it promotes psychosocial wellness and mental healthcare-seeking behavior among women through disseminating mental health-related educational content [The Women Support Initiative Forum (WSIF) is an online women-led initiative that offers a wide range of online psychosocial services through it’s website, LinkedIn, and Facebook group and pages . Establi content .The WSIF closed Facebook group also has an “anonymous post” feature in which women can share their challenges anonymously and receive support. The psychologists and psychosocial supporters respond in the comment section of the posts sharing their opinion and guidance in a non-judgemental manner. Other group members who have had similar experiences also respond in the comment section to support to these women. The posters’ identity is anonymous, known only to the group administrators, who maintain strict confidentiality. This feature allows women to express their problems in detail without fearing they will be identified and socially judged.The anonymous posts provide a unique, unsolicited insight into women’s mental health concerns and treatment-seeking behavior outside a formal healthcare setting. Therefore, our study objective was to explore the pattern of mental healthcare-seeking behavior among women in Bangladesh through social media content analysis of the anonymous posts shared on the WSIF platform. The findings would help to generate evidence on the care-seeking behavior and barriers faced by the women and potentially guide better approaches to service delivery.A thematic content analysis was conducted of the anonymous posts by the Facebook group members of WSIF. Codes were inductively derived from the text data, and themes were defined during analysis. This method paid close attention to the members’ points of view and analyzed their genuine expressions in the posts to better understand the phenomena . We follBangladesh has a population of 164 million, and 49.4% are female. According to a Polish-based social media management platform, around 16,868,448 Bangladeshi women actively use Facebook, of which the majority are between 18 and 24 years old , 35. Theth March 2020 to 7th July 2022 since the first case of COVID-19 was identified on 8th March 2020 in Bangladesh [Two research team members collated the anonymous posts, storing each post as a separate, uniquely identifiable file. The study timeline was from 8ngladesh . And alsngladesh . MoreoveThematic content analysis was utilized to analyze the anonymous posts . In addiAmong the 1006 anonymous posts, 667 (66.30%) were about mental healthcare-seeking behavior, and 552 (54.87%) were related to barriers to seeking mental health support. The findings were summarized in four themes and nine sub-themes in Table n = 1006; 100%) described mental health symptoms, which could be summarized into two major sub-themes: (i) common mental health conditions  and (ii) severe mental illnesses . The posts in which women expressed low mood, hopelessness, lack of concentration, fear, worry, distress were categorized under common mental health conditions as per the evidence [The anonymous posts . Now I am continuously afraid of losing other family members too. My husband does not let me visit anyone, even my siblings. I also lost my job during the early pandemic and could not sleep well. Sometimes I have terrible nightmares.- Anonymous post 144The ongoing pandemic was observed to have a direct effect on the mental health of some women. The women commonly shared fear of infection and tension after being infected with COVID-19. Women also reported different issues related to the pandemic, such as job insecurity and restriction of social activities. One of the posts illustrated the magnitude of the impact.My mother passed away last year, and I have been trying to cope with this trauma ever since. I cannot concentrate on my daily activities, and no one understands how I feel. This pain seems unbearable to me. I am not even able to help myself. I am constantly thinking about suicide. I feel helpless.- Anonymous post 392In addition, some women’s posts indicated severe mental illness, including suicidal thoughts and suicidal attempts. They reported traumatic events as the triggers, such as the death of people close to them or the experience of constant abusive behavior by extended family members.n = 818; 81.31%) revealed that women sought mental healthcare when dealing with various issues related to marital relationships  and different kinds of abuse  they endured.The majority of the posts ”. I cannot forget those incidents no matter how much I try.- Anonymous post 38A few women disclosed that they experienced sexual abuse including improper touching and sexual assault by outsiders. The experience had a longer-term impact on their lives and affected their well-being.n = 667; 66.30%) described the pattern of mental healthcare-seeking of the women. The coded posts were summarized into two sub-themes (i) past history of seeking mental health care  and (ii) visiting mental healthcare professionals .A majority of posts  directly and indirectly reflected on different barriers to seeking mental healthcare. Analyzing these posts, three major sub-themes emerged as: (i) low mental health literacy  (ii) stigma related to seeking mental healthcare , and (iii) lack of availability of mental healthcare services  . Supplementary Table 2. Table of Quotes.