Propranolol reduced physiological tremor in control mice and reduced pathophysiological tremor in wdl/wdlCar8 mice to control levels. Open field and footprinting assays showed that propranolol did not correct ataxia in wdl/wdlCar8 mice. In vivo recordings in awake mice revealed that propranolol modulates the spiking activity of control and wdl/wdlCar8 Purkinje cells. Recordings in cerebellar nuclei neurons, the targets of Purkinje cells, also revealed altered activity in propranolol-treated control and wdl/wdlCar8 mice. Next, we tested whether propranolol reduces tremor through β1 and β2 adrenergic receptors. Propranolol did not change tremor amplitude or cerebellar nuclei activity in β1 and β2 null mice or wdl/wdlCar8 mice lacking β1 and β2 receptor function. These data show that propranolol can modulate cerebellar circuit activity through β-adrenergic receptors and may contribute to tremor therapeutics.Tremor is the most common movement disorder. Several drugs reduce tremor severity, but no cures are available. Propranolol, a β-adrenergic receptor blocker, is the leading treatment for tremor. However, the in vivo circuit mechanisms by which propranolol decreases tremor remain unclear. Here, we test whether propranolol modulates activity in the cerebellum, a key node in the tremor network. We investigated the effects of propranolol in healthy control mice and  Tremor is the most common movement disorder, affecting an estimated 70 million people worldwide . The maiDespite its prevalence, the mechanisms that initiate tremor within the central nervous system remain poorly understood, and as a result, treatment options are limited. For severe cases of essential tremor, surgeries including thalamotomy or deep brain stimulation (DBS) are often the only effective ways to relieve symptoms ,23,24,251 and β2 adrenergic receptors throughout the body [Propranolol is a non-selective β-blocker that counters the action of adrenaline and noradrenaline at both βthe body ,35. Propthe body , and it the body . Propranthe body ,39,40,41the body ,45,46,47the body ,49,50,51the body , raisingThe cerebello-thalamo-cortical pathway is likely a key circuit for mediating tremor ,54,55, a1 and β2 adrenergic receptors. We used wdl/wdlCar8 mutant mice to test how cerebellar dysfunction responds to propranolol in a genetic mouse model with severe tremor. Importantly, the pathology and dysfunction of wdl/wdlCar8 mice involves a functional defect within the cerebellum, but is free from gross anatomical disruptions [Car8) gene results in loss of the CAR8 protein. CAR8 expression is initiated during development and is predominantly expressed in cerebellar Purkinje cells. wdl/wdlCar8 mice have an action tremor with an amplitude that worsens with age, peaks at ~8–12 Hz [wdl/wdlCar8 mice is accompanied by ataxia and dystonia [wdl/wdlCar8 mice. Our studies aim to uncover how propranolol operates at the cellular and circuit levels in the cerebellum while reducing tremor symptoms.Here, we investigated whether propranolol reduces tremor through changes in cerebellar circuit activity, by testing whether propranolol’s action is mediated through βruptions . Mutatio~8–12 Hz , and is ~8–12 Hz . The tredystonia ,65,66. WMouse husbandry and experiments were performed under an approved Institutional Animal Care and Use Committee (IACUC) protocol at Baylor College of Medicine (BCM). Male and female mouse genetic models  were obtained from The Jackson Laboratory  and a colony was established and thereafter maintained at BCM. Mice of both sexes were studied. All mice used in this study were mature adults, with their ages ranging between 4 and 12 months old. Detailed sample sizes of animals and data points of all mouse genotypes and conditions used in this study are described in the figure legends and wdl/wdlCar8 mutants, and −/−;β2-AR−/−β1-AR mutants were purchased from The Jackson Laboratory . The waddles mutation in the wdl/wdlCar8 mutants arose spontaneously in 1995 and has been maintained by The Jackson Laboratory since. For −/−;β2-AR−/−β1-AR double mutant mice, the original strain was created as described by Rohrer et al., 1999 [−/−β1-AR homozygous mutant mice with −/−β2-AR homozygous mutant mice to generate compound heterozygotes, the offspring of which were then mated to obtain compound homozygotes. −/−Β1-AR mice were created using a targeting vector containing a neomycin resistance gene driven by the mouse phosphoglycerate kinase promoter to disrupt most of the Adrb1 coding region . −/−β2-AR mutant mice were created in a similar fashion using a targeting vector, again containing a neomycin resistance gene driven by the mouse phosphoglycerate kinase promoter to disrupt Adrb2 such that the end of the fourth transmembrane segment is absent, rendering the receptors nonfunctional. To generate wdl/wdlCar8 mice lacking β-adrenergic receptors, wdl/wdlCar8 mice were crossed with −/−;β2-AR−/−β1-AR mice to generate −/−;β2-AR−/−;Car8wdl/wdlβ1-AR triple allele mutants. We used a standard PCR genotyping protocol to differentiate the mutants from the controls, using the primer sequences listed in C57BLKS/J controls, l., 1999 , by matiAdult mice were administered a 20 mg/kg propranolol hydrochloride  solution dissolved in sterilized distilled water via intraperitoneal injection (IP) with a 31-gauge 6 mm syringe. A total of 20 mg/kg is a clinically moderate dosage for human patients on propranolol treatment for tremor. A reduction in tremor consistently developed between 10 and 20 min post-injection, and data recordings were made no shorter than 15 min and no longer than 6 h after injection. Mice were given one dose of propranolol, one time only, to avoid any potential cumulative effects.Tremor was detected using a custom-built tremor monitor. For each experiment, mice are placed into a translucent plastic box with an open top. The box is held firmly in midair by eight elastic cords. An accelerometer is attached to the bottom, allowing for signal detection of oscillatory movements in two dimensions. Further details can be found in our previous publication .Mice were allowed to acclimate to the tremor monitor for 300 s before recordings of tremor were made. All tremor monitor recordings were sampled at 10 kHz and lowpass filtered at 5 kHz. Power spectrums of tremor were made using a fast Fourier transform (FFT) with a Hanning window. An offset was applied if the tremor waveform was not centered on zero and the recordings were downsampled using the Spike2 software ‘interpolate’ channel process in order to produce frequency bins aligned to whole numbers. FFT frequency resolution was targeted to either ~0.25 Hz or ~0.5 Hz per bin. Sonogram plots of tremor were made using the Spike2 software ‘sonogram’ channel draw mode with a Hanning window. We report frequency ranges in order to account for natural variability in the tremor signals. The entirety of each 300 s post-acclimation tremor recording was analyzed for each animal in each group.2 of power was set so that only strong, sudden movements–such as footsteps and rearing–would be captured as non-tremor movements. Each event surpassing the detection threshold occurring at least 0.1 s apart from the prior or following event was counted as a single movement, and the total number of such detected movements over the entire 300 s tremor monitor recording was analyzed for each animal in each condition.To examine whether non-tremor movements may affect overall tremor readings, non-tremor movement information was extracted from each tremor recording by applying a power spectrum analysis using an FFT to the raw tremor monitor data to determine the power within the 50–100 Hz range. An offset was applied to the raw waveform before analysis if it was not centered on zero. FFT frequency resolution was targeted to 19.54 Hz with a window size of 0.0512 s. Because clinical tremors are categorized at frequencies below 30 Hz, the 50–100 Hz range of data collected by the tremor monitor represents non-tremor movements. Furthermore, a detection threshold of 50 mVwdl/wdlCar8 baseline (no propranolol) conditions, the data analyzed and represented in this study are taken from the database used in Miterko et al., 2021, from which we extracted data for control and wdl/wdlCar8 mice at baseline to make the direct comparison between the two genotypes by pooling baseline data across different “frequency” groups published in Miterko et al., 2021. For the propranolol-treated conditions of control and wdl/wdlCar8 mice, new data was collected; since mice quickly acclimate to the open field assay, the same mouse cannot be recorded from twice. Baseline and propranolol-treated data are, therefore, from two separate cohorts of mice of each genotype. Mice were treated with propranolol, then acclimated to the open field room for 1.5 h. Immediately following the acclimation period, mice were placed in the open field apparatus, where their movement was recorded for 30 min. White noise was provided in the background during the acclimation and the recording periods. The movement time, number of movement events, and number of ambulatory events of each mouse were analyzed .General locomotor function was assessed with the open field assay using the open field locomotion system , as described in Miterko et al., 2021 . For botwdl/wdlCar8, and −/−;β2-AR−/−β1-AR mice before and after propranolol treatment were assessed using the footprinting assay as described in Carter et al., 1999 [The gait characteristics of control, l., 1999 . To tracWe have previously described our general surgical techniques in detail . In brieDuring surgeries for awake neural recording experiments, the dorsal aspect of the skull was exposed, and a circular craniotomy of about 2 mm in diameter was performed dorsal to the interposed nucleus . A custom-built 3D-printed chamber was placed around the craniotomy and filled with antibiotic ointment. A custom headplate used to stabilize the mouse’s head during recordings was affixed over bregma, and a skull screw was secured into an unused region of the skull. All implanted items were secured using C and B Metabond Adhesive Luting Cement  followed by a layer of dental cement  to completely enclose the area.w/v NaCl solution. Electrodes had an impedance of 4–13 MΩ and were made of tungsten . Electrodes were connected to a preamplifier headstage . The headstage was attached to a motorized micromanipulator . Headstage output was amplified and bandpass filtered at 0.3–13 kHz  before being digitized , recorded, and analyzed using Spike2 software . Electrical activity was additionally monitored aurally using an audio monitor  connected to the output of the amplifier.Single-unit extracellular recordings were performed as previously described ,72,73,74Purkinje cells were identified by their firing rate and the presence of both complex and simple spike activity. Accordingly, cerebellar nuclei cells were identified by their relatively deep location within the cerebellum, approximately 2.5–3 mm deep, and their firing rate. The surface of the tissue was determined based on the significant reduction in electrical noise that occurs when the electrode, initially suspended in air, touches the tissue. During recording sessions, the experimenter monitored the sound of the electrical activity to determine whether tissue membranes were being breached, white matter tracts were being traversed, or whether sound quality deviated significantly from traditional cerebellar recordings. Using these criteria, we have consistently been able to target the cerebellar nuclei . Only tr−1 was calculated by taking the inverse of the interspike intervals (ISI−1) and distributing these in 10 Hz bins. We then found the bin with the largest proportion of spikes and defined the mode ISI−1 as the center of that bin . Our measures of global regularity or burstiness (CV) and regularity (CV2) were based on the interspike intervals (ISI) between two adjacent spikes (in seconds). CV = standard deviation(ISI)/mean(ISI), and CV2 = mean(2*|ISIn − ISIn−1|/(ISIn + ISIn−1)) [Firing properties were analyzed using Spike2 , Microsoft Excel , custom MATLAB code , and GraphPad Prism  software. All electrophysiological recording data were spike sorted in Spike2. We sorted out three types of spikes: Purkinje cell simple spikes, Purkinje cell complex spikes, and cerebellar nuclei spikes. Complex spikes were characterized by their large amplitude, and post-spike depolarization and the smaller spikelets that follow. All other Purkinje cell action potentials were characterized as simple spikes, and all spikes from cerebellar nuclei cells were characterized as such. After spike sorting our traces in Spike2, we calculated several parameters to describe the firing rate and regularity in MATLAB  as described in Van der Heijden et al., 2021 . For thiISIn−1)) .Perfusion and tissue fixation were performed as previously described . Briefly1 receptor  and anti-β2 receptor  antibodies were used to label both adrenergic receptor subtypes in the cerebellum. Mouse monoclonal anti-calbindin D28  was used to label the morphology of adult Purkinje cells. Calbindin D28 encodes a calcium binding protein that is expressed exclusively within Purkinje cells of the cerebellum [2O2. The DAB reaction was stopped with PBS when the optimal color intensity was reached. Staining for fluorescent immunohistochemistry was performed using donkey anti-mouse or anti-rabbit antibodies conjugated to Alexa-488 and -555 fluorophores . Tissue sections were coverslipped using either Entellan mounting media  or FLUORO-GEL with Tris buffer . Sample size was not determined using a priori power analysis but was based on the criteria for significance in observations. A total of 8 cerebella from control, 5 from wdl/wdlCar8, and 5 from −/−;β2-AR−/−β1-AR mice were used in this study, which were processed for immunohistochemistry to examine the expression of β1 and β2 adrenergic receptor expression throughout the cerebellar cortex.Rabbit polyclonal anti-βPhotomicrographs of stained tissue sections were captured with Zeiss AxioCam MRm (fluorescence) and AxioCam MRc5 (DAB-reacted tissue sections) cameras mounted on a Zeiss Axio Imager.M2 microscope or on a Zeiss AXIO Zoom.V16 microscope. Apotome imaging  of tissue sections was performed and images acquired and analyzed using either Zeiss AxioVision software (release 4.8) or Zeiss ZEN software . After imaging, the raw data was imported into Photoshop CC 2022  and corrected for brightness and contrast levels.p < 0.05) followed by a Tukey’s multiple comparisons test to compare data between control and wdl/wdlCar8 mice, before and after propranolol treatment. We used a paired t-test (p < 0.05) to compare −/−;β2-AR−/−β1-AR and −/−;β2-AR−/−;Car8wdl/wdlβ1-AR mice before and after propranolol treatment. We used PRISM for two-way ANOVA and t-test analyses. We fitted a linear regression model between the mean cerebellar nuclei neuron firing parameters  and mean average tremor power in each group. We used the MATLAB function ‘fitlm’ to calculate the fit and statistical significance of the fit for each parameter separately. The number of animals tested is represented by ‘N’; the number of cells included in the analyses of electrophysiological recordings is represented by ‘n’. ‘Control’ refers to C57BLKS/J mice. p value > 0.05 = ns, ≤0.05 = *, ≤0.01 = **, ≤0.001 = ***, <0.0001 = ****.We used a repeated measures two-way ANOVA  in the previous 0.5 s. The height of the bar-graphs represents the mean for each group and the individual replicates are visualized using dots, squares, or triangles. Most figure colorizations in this article are from the Wong, 2011  palette wdl/wdlCar8 mice also exhibit a tremor between 8–10 Hz  reflect the overall mobility of a subject, with reduced lengths representing reduced mobility. Stance distance (the hypotenuse of stride and sway) represents posture during gait. Sway distances (the distance between left and right paw placement) indicate the stability of a subject’s balance in gait. We found that ng” gait D, right.wdl/wdlCar8 mice, this may suggest that any changes in neural circuit activity that propranolol induces are also likely specific to hallmarks of tremor pathophysiology. We sought to find potential cerebellar circuit mechanisms of propranolol by conducting awake, single-unit in vivo electrophysiological recordings of both Purkinje cells and cerebellar nuclei neurons in control and wdl/wdlCar8 mice before and after propranolol treatment  of each cell to better compare the patterns of control and wdl/wdlCar8 Purkinje cells during active firing periods and minimize the skewing effects of inter-burst pauses in wdl/wdlCar8 cells. Interestingly, the mode ISI−1 of wdl/wdlCar8 simple spikes after propranolol administration was significantly reduced to the same level of the control animals at baseline, mirroring the tremor findings in wdl/wdlCar8 Purkinje cells is significantly higher than in controls; however, CV is not significantly changed by propranolol in both control and wdl/wdlCar8 (wdl/wdlCar8 mice. Altogether, this data shows that propranolol affects firing rates, but not firing irregularity, in Purkinje cell simple spikes.The neural mechanisms of propranolol are unclear. Since propranolol specifically affects tremor but not ataxic gaits in reatment A. Purkin pattern B. Purkin pattern A. We reaecreased B,C. Desp8wdl/wdl C. CV2 is−1 were significantly decreased in control and wdl/wdlCar8 mice after propranolol treatment (wdl/wdlCar8 mice only. The reduced wdl/wdlCar8 complex spike CV following propranolol is not significantly different from the control complex spike CV at baseline. CV2 measures are not changed by propranolol in either genotype. These quantifications indicate that propranolol reduces the firing rate and corrects some features of irregularity of Purkinje cell complex spikes.For Purkinje cell complex spikes, both the firing rate and mode ISIreatment D. The cowdl/wdlCar8 mice (wdl/wdlCar8 cerebellar nuclei neurons exhibit a high-frequency bursting pattern similar to their Purkinje cells (−1 and CV compared to controls. Propranolol corrects both the high mode ISI−1 and CV, which are significantly reduced in wdl/wdlCar8 cerebellar nuclei neurons to control levels after treatment (wdl/wdlCar8 mice following propranolol treatment.We next examined the electrophysiological effects of propranolol on cerebellar nuclei neurons A, which wdl mice C. Car8wdje cells B, which reatment C. CV2 me−1, CV, and CV2, and group mean in maximal tremor power. Only the model fit between mode ISI−1 and maximal tremor power was significant. This analysis indicates that propranolol may reduce the power of tremor by reducing the predominant firing rate of cerebellar nuclei neurons.Finally, we investigated which of the parameters that describe the firing patterns in cerebellar nuclei neurons could best predict tremor severity. We fitted a linear regression model between the group means in firing rate, mode ISI1 and β2 adrenergic receptors, the primary molecular targets of propranolol, are expressed throughout many regions of human and rodent brains [1 and β2 receptors in the cerebellum on a broad scale [1 and β2 adrenergic receptor antibodies to understand the possible localization of propranolol’s targets throughout the cerebellar cortex. We found antibody binding throughout the cerebellar cortex (1 and β2 antibody signal is present in Purkinje cell bodies and dendrites (wdl/wdlCar8 mice exhibit differences in β1 and β2 antibody binding compared to control mice, given their known cerebellar pathology. Comparison of control and wdl/wdlCar8 tissue show no disparity in β1 and β2 signal, with expression also seen in the granular layer, Purkinje cell layer, and molecular layers for wdl/wdlCar8 mice as in the controls (1 and β2 adrenergic receptor antibody binding in the cerebellum supports our findings that propranolol may modulate neural activity at key nodes of the cerebellar circuit during tremor reduction in this model.Previous studies have indicated that βt brains  and are t brains ,87. Radiad scale ,89; hower cortex A,E, presendrites B–H″. We controls . The loc1 and β2 receptors during propranolol treatment. To study this, we used −/−;β2-AR−/−β1-AR double homozygous mutant mice with non-functional β1 and β2 receptors [1β and 2β receptor complexes, suggesting that the receptors are rendered as non-functional [1β and 2β receptor antibodies used here may detect the truncated, mutant 1β and 2β receptor in −/−;β2-AR−/−β1-AR, which could explain the persistent signal we observed when comparing the mutants to control and wdl/wdlCar8 mice (We sought to determine whether our tremor and electrophysiology findings were dependent on βeceptors . The gennctional ,90,91. Twdl mice . For our−/−;β2-AR−/−β1-AR mice, we conducted tremor, footprinting, and electrophysiological measurements. We reasoned that if propranolol acts through β1 and β2 receptors to elicit the reduction in tremor, then −/−;β2-AR−/−β1-AR mice would have no change in tremor amplitude measurements following propranolol treatment. Indeed, tremor monitor recordings show that −/−;β2-AR−/−β1-AR mice have no significant difference in tremor before or after propranolol (wdl/wdlCar8 mice (−/−;β2-AR−/−β1-AR mice (−/−;β2-AR−/−β1-AR mice in the footprinting assay, mirroring the finding that propranolol does not alter gait parameters in control or wdl/wdlCar8 mice (Using pranolol A–C, whicwdl mice . Non-tre−/− mice D. Furthewdl mice .−/−;β2-AR−/−β1-AR mice show that propranolol is able to alter some aspects of Purkinje cell activity despite the deletion of functional domains in 1β and 2β receptor in the mutant mice (−/−;β2-AR−/−β1-AR Purkinje cell simple spikes, the firing rate is significantly decreased after propranolol, although mode ISI−1, CV, and CV2 measures are unchanged after propranolol (−/−;β2-AR−/−β1-AR mice, with firing rate and mode ISI−1 significantly decreased while CV and CV2 are significantly increased (−1, CV, or CV2 after propranolol treatment (Curiously, our electrophysiological recordings in ant mice G–I. In βpranolol H. Complencreased I. Nevertreatment J,K. Thes1 and β2 receptor function in propranolol-mediated tremor reduction, we crossed our wdl/wdlCar8 and −/−;β2-AR−/−β1-AR lines to generate −/−;β2-AR−/−;Car8wdl/wdlβ1-AR triple mutant mice. These mice lack CAR8 protein in their Purkinje cells but additionally lack β1 and β2 receptor function. We reasoned that examining the effects of propranolol in these −/−;β2-AR−/−;Car8wdl/wdlβ1-AR triple mutant mice would provide further evidence of propranolol acting through β1 and β2 receptors to reduce tremor, since these mice persist with the pathophysiological wdl/wdlCar8 tremor. We found that in −/−;β2-AR−/−;Car8wdl/wdlβ1-AR mice, propranolol has no effect on tremor or non-tremor movements (−/−;β2-AR−/−;Car8wdl/wdlβ1-AR mice shows no change in firing rate, mode ISI−1, CV, and CV2 following propranolol treatment (−/−;β2-AR−/−β1-AR double mutants and −/−;β2-AR−/−;Car8wdl/wdlβ1-AR triple mutants indicate that propranolol modulates cerebellar function, and tremor severity, through β1 and β2 receptors.To further understand the involvement of βovements A–C. Addireatment D,E. Altowdl/wdlCar8 mice that have pathophysiological tremor that arises as a result of Purkinje cell dysfunction. We then uncovered that propranolol changes cerebellar circuit activity patterns during tremor reduction in wdl/wdlCar8 mice, mainly by slowing firing rates and correcting firing irregularity in Purkinje cells and cerebellar nuclei neurons. Our findings provide evidence that propranolol’s potential tremor reduction mechanism of action is through influencing central nervous system activity. We show that the cerebellum, which has critical roles in tremor pathophysiology, is impacted by propranolol treatment on a circuit level.The continued use of propranolol as a treatment for tremor necessitates investigations into its in vivo neural mechanisms. In this study, we found that propranolol reduces tremor in wdl/wdlCar8 mice, we also found that propranolol reduces physiological tremor in control mice. This result corresponds with the known effects and usages of propranolol in humans [wdl/wdlCar8 and physiological tremors are action tremors, which means that there is an increase in tremor severity with movement [wdl/wdlCar8 and control mice have unchanged open field assay measurements after propranolol administration and are similar to each other at baseline, the only difference being the ambulatory activity count, which is reduced in wdl/wdlCar8 mice at baseline, relative to control mice. If a change in movement alone accounted for the increased tremor we find in wdl/wdlCar8 mice relative to control mice at baseline, we would expect that wdl/wdlCar8 mice would move more than control mice at baseline. Similarly, if a change in movement alone accounted for the decreased tremor severity we find in wdl/wdlCar8 mice after propranolol administration relative to baseline, we would expect that wdl/wdlCar8 mice with propranolol would move less than wdl/wdlCar8 mice at baseline. However, with the open field assay we see the opposite between the genotypes at baseline and no change within the genotypes between baseline and with propranolol, suggesting that changes in movement are not driving the differences in tremor severity. This is supported with our non-tremor movement analysis. Frequencies from 50 to 100 Hz include sharp deflections of acceleration indicative of initiation of movement or footfalls in addition to possible harmonics of underlying tremor. Therefore, we use this range as an estimate of movement within the tremor monitor. Similar to our open field findings, we see no difference between wdl/wdlCar8 and control mice at baseline. Additionally, we see no difference between wdl/wdlCar8 mice at baseline and wdl/wdlCar8 mice after propranolol administration. Taken together, these data suggest that our tremor findings are not driven by changes in overall movement. Furthermore, we found that β1 and β2 adrenergic receptors, thought to be the primary molecular targets of propranolol, are broadly labeled throughout the cerebellar cortex and are required for propranolol to reduce tremor in mice. These data provide evidence that β1 and β2 receptor expression help define the tremor circuit, adding to our understanding of which brain regions mediate tremor.In addition to treating pathophysiological tremor in n humans . Proprann humans ,94,95. Tn humans ,97. Addimovement ,98. The wdl/wdlCar8 mice are in agreement with previous studies showing propranolol’s efficacy in treating human patients with tremor [–/–α1 mouse model with depleted GABAA receptor α1 subunits [Our findings that propranolol significantly reduces tremor in h tremor ,45,99 ansubunits ,101,102.wdl/wdlCar8, or −/−;β2-AR−/−β1-AR mice in open field and footprinting assays—and especially that propranolol does not affect ataxic gait parameters in wdl/wdlCar8 mice—supports the hypothesis that propranolol and its mechanisms are specific to tremor pathophysiology. However, additional motor assays that are able to differentially assess ataxic phenotypes in wdl/wdlCar8 mice, such as the rotarod or parallel floor rod assays, could be conducted to fully evaluate this possibility. Still, the source of ataxia, dystonia, and tremor may all intersect at a shared anatomical circuit in the cerebellum [wdl/wdlCar8 mice specifically, ataxia, dystonia, and tremor result from a single mutation affecting cerebellar Purkinje cells [wdl/wdlCar8 mice support this hypothesis. The pathophysiological baseline firing activity of wdl/wdlCar8 mice has been well-documented and is characterized by high-frequency burst periods [wdl/wdlCar8 tremor while reducing the firing rate, mode ISI−1, and CV irregularity of Purkinje cells and cerebellar nuclei neurons within the high-frequency bursts, is interesting in the context of those studies. Yet, it also adds to the growing body of evidence showing that the targeted therapeutic manipulation of cerebellar circuit activity patterns, such as with DBS, has strong potential for treating motor disorders [Previous studies have also examined the effects of propranolol in human patients with ataxia and dystonia, which is interesting given the potential common origins of these diseases from within the cerebellum. Propranolol was found to temporarily alleviate ataxia in a few clinical reports ,104. Addrebellum ,111,112.je cells . Interesje cells ,66,72,77 periods ,66,69. C periods . Our finisorders ,69. FurtAlthough we provide additional insight for how the cerebellar circuit contributes to tremor, our study focused on testing how propranolol reduces tremor. Despite being a foremost tremor treatment, a debate still continues about how propranolol might affect the central nervous system. Some studies have postulated that propranolol may have a generally depressive effect in the central nervous system, based on clinical observations and self-reported patient measures of energy and mood levels ,48 as weIn addition to altering a single component of the cerebellar circuit, it is also possible that propranolol can directly affect several components of the circuit at the source, including the inferior olive and cerebellar nuclei, or other regions of the motor circuit. Climbing fiber signals originating from the inferior olive may be dampened by the effects of propranolol within the olive itself, which in turn may contribute to the reduction in complex spike activity seen in our recorded Purkinje cells. Complex spike activity patterns and synchrony are increasingly understood to contribute to tremor outcomes, a hypothesis that is supported by evidence in which heightened inferior olive coupling and complex spike synchrony may underlie tremor in several drug-based and genetic mouse models ,120,121.While considering the sites of propranolol’s action, it is also important to consider how the cerebellar cortex and cerebellar nuclei interact. Indeed, several studies have shown that cerebellar nuclei neuron activity is often both correlated and anti-correlated to Purkinje cell activity, contrary to the parsimonious assumption that the firing rates of cerebellar nuclei neurons are always the inverse of their Purkinje cell inputs ,123,124.wdl/wdlCar8 tremor treatment. However, the current consensus is that tremor is a disease of the central nervous system, originating within the brain [Previous studies that examined the tremor-reducing mechanisms of propranolol focused on measures in the peripheral nervous system, suggesting that propranolol may be active at β-adrenergic receptors in the muscle spindles during tremor attenuation ,46,47. The brain ,129,130.1 and β2 receptor antibody signal is heavily expressed in the cerebellum circuit provides an anatomical framework for understanding where propranolol may be active. Although numerous studies have examined the distribution and role of β-adrenergic receptors in the rest of the body [1 and β2 receptors in the cerebellum to appreciate their cellular resolution, uncovering their distribution across the cerebellar cortical layers and in relation to Purkinje cells. Our findings that these receptors are necessary for propranolol to reduce tremor in −/−;β2-AR−/−β1-AR double-mutant mice and −/−;β2-AR−/−;Car8wdl/wdlβ1-AR triple mutant mice provide evidence that propranolol acts through β1 and β2 receptors, and is likely not working through secondary affinities for β3 or 5-HT receptors for its therapeutic effects. Our electrophysiology data showing that −/−;β2-AR−/−β1-AR Purkinje cells do change firing activity with propranolol, may however be explained by propranolol’s secondary affinity for 5-HT receptors. These receptors are abundant in the afferent fibers of the cerebellum that directly modulate Purkinje cell activity [−/−;β2-AR−/−β1-AR mice.Our immunohistochemistry data showing that the βthe body ,133,134,the body ,89. In oactivity , and we −/−;β2-AR−/−;Car8wdl/wdlβ1-AR triple mutant mice, we surmise that the electrophysiological responses to propranolol would resemble a combination of the Purkinje cell effects seen independently in wdl/wdlCar8 baseline cells, and −/−;β2-AR−/−β1-AR double mutant cells. The characteristic high-frequency bursting signature of wdl/wdlCar8 Purkinje cells is likely to be preserved, but there may be reductions in overall simple and complex spike firing rates such as those seen in −/−;β2-AR−/−β1-AR Purkinje cells following propranolol. Ultimately, our tremor monitor and cerebellar nuclei electrophysiology data in the −/−;β2-AR−/−β1-AR and −/−;β2-AR−/−;Car8wdl/wdlβ1-AR mice support the hypothesis that the mechanisms underlying propranolol’s tremor reducing effects require intact β1 and β2 receptors in the cerebellum. Our genetic crosses between the −/−;β2-AR−/−β1-AR and wdl/wdlCar8 mutants generated triple mutant mice that were difficult to breed and maintain especially for in vivo electrophysiological experiments. For these reasons, for the mice that we were able to salvage, we chose to focus our recordings on the cerebellar nuclei given their critical roles in cerebellar output; any potential defects could, in theory, be uncovered by examining this node in the circuit. We found that propranolol does not induce changes in our measurements of firing mode in cerebellar nuclei neurons of triple mutants, suggesting the possibility that β1 and β2 receptors are functionally necessary for tremor reduction in the wdl/wdlCar8 model. In accordance with these data, based on our cerebellar nuclei neuron electrophysiology data from control and wdl/wdlCar8 subjects, we have confidence that the firing mode remains a reliable predictor of tremor outcomes, and this is reflected in the recorded tremor and electrophysiology data from the triple mutants. Still, additional electrophysiological data from −/−;β2-AR−/−;Car8wdl/wdlβ1-AR mice would offer additional insights into the intersecting roles of β-adrenergic receptor function within the wdl/wdlCar8 model. Although propranolol seems to require β1 and β2 receptors for tremor reduction, the mechanism may involve a non-canonical signaling pathway in Purkinje cells.Although we did not record from Purkinje cells in Our findings from this study add to the growing body of evidence that propranolol has prominent effects in the central nervous system, particularly in the cerebellar circuit. Prior studies have found that propranolol inhibits Purkinje cell GABAergic neurotransmission in slice preparations, suggesting that monoaminergic facilitation of cerebellar activity may play a role in synaptic plasticity and motor coordination ,136. Ind


Aging is a process that affects almost all multicellular organisms and since our population ages with increasing prevalence of age-related diseases, it is important to study basic processes involved in aging. Many studies have been published so far using different and often single age markers to estimate the biological age of organisms or different cell culture systems. However, comparability of studies is often hampered by the lack of a uniform panel of age markers. Consequently, we here suggest an easy-to-use biomarker-based panel of classical age markers to estimate the biological age of cell culture systems that can be used in standard cell culture laboratories. This panel is shown to be sensitive in a variety of aging conditions. We used primary human skin fibroblasts of different donor ages and additionally induced either replicative senescence or artificial aging by progerin overexpression. Using this panel, highest biological age was found for artificial aging by progerin overexpression. Our data display that aging varies depending on cell line and aging model and even from individual to individual showing the need for comprehensive analyses. If we wish to consider physiological health state, the age of the donor at biopsy should be his or her estimated biological age. In practice, however, this biological age is rarely available and it must be substituted by the donor’s chronological age, which we call “donor age” in the following. In principle, the two main contributors cannot easily be distinguished with respect to their effects on the physiological “health” state of the cells. Thus, for the purpose of this paper, we define the age of cells in a cell culture  as the donor age plus the effects of continued culturing expressed as a time period in terms of donor age. This biological age can be estimated by biomarkers  and it corresponds to a physiological “health” state which is predictive of the future behaviour of the cells. This affords a more precise description of the respective cell models used in scientific studies concerning their biological age, before they are used for experiments, which is recommended for a better characterization of the cells.Aging, usually described as a time-dependent functional decline , is a phin vitro cell cultures are the most commonly used systems. There are a variety of cell models for aging and all types of (age-related) diseases including simple cell models with heterogeneous expression systems , patient-derived primary cells  as well as induced-pluripotent stem cell (iPSC) models. Since the discovery of the latter, cell models of disease and/or aging have become of even greater interest. While these technologies enable to investigate patient-derived cell models of so far not available origins , these seem to significantly alter general aspects of cell physiology including their biological age. Especially in research on age-related diseases, it is therefore important to also consider the effects of such rejuvenation, e.g., by the process of iPSC generation, on various age markers.Aging processes have been studied in a variety of model systems. In addition to the various mouse models, An age marker is a measurable characteristic for estimating biological age or a related condition  in a bioThere are different aging hallmarks, and for some of them there are established markers to characterize biological age. For example, López-Otín described nine hallmarks of aging in three different main categories . The priin vitro cell culture systems.Many putative age markers have thus been described and the age markers investigated in different studies often differ significantly . For thiThe epigenome, especially DNA methylation, thereby is the most often reported singular factor to quantify an individual’s age. DNA methylation marks are the basis of so called “aging clocks” and often correlate well with the chronological age of the donor from which the tissue was derived. Furthermore, there is evidence that they can be used to estimate biological age. In 2013, Hovarth developed one of the first “aging clocks” that allows to estimate the chronological age by DNA methylation markers, for many tissues and some cell types. In this study, a collection of available DNA methylation data sets was used to define and evaluate a specific age predictor, based on CpG islands whose DNA methylation levels correlate with the chronological age of individuals, referred to as DNAm age. The difference between the predicted chronological age and the actual chronological age is then considered to be an estimate of biological age. Accordingly, DNAm age showed high accuracy in estimating chronological age . Of notein vitro cellular aging resembled tissue aging in vivo. This study established an algorithm using replicative aged mouse embryonic fibroblasts and subsequently validated it in multiple murine tissues. Interestingly, it was sensitive to detect rejuvenation during cellular reprogramming, but is was reported that this progressive kind of cellular aging is different from non-replicative senescence  and reported a lack of correlation of SA-β-Galactosidase with the CultureAge score  at 37°C and 5% CO2. Fibroblasts were subcultured after trypsinisation . Culture medium was changed every 3–4 days.Human skin fibroblasts from apparently healthy young, mid-age and old as well as diseased (Progeria) donors were purchased from Coriell Institute or prepared in our lab as described before , 2021. C5 per well. When fibroblasts reached confluency, cells were counted and again seeded at a density of 2x105. This was continued over 75–150 days. PDL was calculated as PDL = 3.2*(logharvested cells—logseeded cells) + actual passage number.For determination of PDL, human fibroblasts were seeded into 6-well-plates  at a density of 2x104 per well in an 8-well µ-Slide  and incubated for 48 h. Cells were fixed with 4% PFA at 37°C for 20 min followed by permeabilization with 0.2% of TritonX and blocked with Blocking Buffer (Pierce Protein-Free T20 (TBS) Blocking Buffer, ThermoFisher Scientific, #37571) at RT for 1 h. Primary antibodies (Anti-phospho-Histone H2A.X (Ser139), #05-636-25ug, 1:500, Millipore; Anti-Histone H3 (di methyl K9), #ab1220, 1:500, abcam; Anti-Lamin B1, #ab16048, 1:500, abcam; Anti-Vimentin, #PA1-16759, 1:300, Thermo Fisher Scientific) were incubated overnight at 4°C. After washing, secondary antibodies (Goat anti-Mouse IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Invitrogen; Goat anti-Rabbit IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Invitrogen) were incubated for 90 min. Finally, after repeated washing, cells were mounted with 4.6-diamidino-2-phenylindole (DAPI) Fluoromount-G  and images were taken with a LSM900 confocal microscope (Zeiss). Analysis was done with Fiji software. γH2A.X foci were counted per nucleus . Corrected total cell fluorescence (CTCF) was calculated as following: (CTCF) = Integrated Density—. Nucleus size was determined with area (pixel units) of nucleus with Fiji software.Fibroblasts were seeded at a density of 2.5x10Telg, Telc) and 300 nM of each single copy gene primer . Following primer sequences were used: Telg 5′ACACTAAGGTTTGGGTTTGGGTTTGGGTTTGGGTTAGTGT′3; Telc 5′TGTTAGGTATCCCT ATCCCTATCCCTATCCCTATCCCTAACA′3; Albu 5′CGGCGGCGGGCGGCGCGGGCTGGGCGGAAATGCTGCACAGA ATCCTTG′3; Albd 5′GCCCGGCCCGCCGCGCCCGTCCCGCCGGAAAAGCATGGTCGCCTGTT′3. Ratio of telomere to single-copy gene content (TLR) is taken as relative measurement of telomere length and expressed in arbitrary units. The intra-assay coefficients of variation were <0.3 for all samples.A standard extraction kit  was used for DNA extraction. Mean telomere length was determined using the modified MM-qPCR as described previously . DNA samCDKN1A(p21)-Fwd 5′-GACACCACTGGAGGGTGACT-3′, CDKN1A(p21)-Rev 5′-CAGGTCCACATGGTCT TCCT-3′, CDKN2A(p16)-Fwd 5′-CTCGTGCTGATGCTACTGAGGA-3′, CDKN2A(p16)-Rev 5′-GGTCGGCGCAGTT GGGCTCC-3′. qPCRs were performed in duplicates for each sample (n = 1 correspond to 2 replicates). Data sets were normalized relative to GAPDH  using delta-delta-CT method.Total RNA was isolated using the “Quick RNA Miniprep” Kit  according to the manufacturer’s protocol and cDNA was generated from 200 ng isolated RNA with High-Capacity cDNA Reverse Transcription Kit . mRNA expression levels were determined using Rotor Gene (QIAGEN) with Rotor-Gene SYBR® Green PCR Kit  using the following primers: Activity of SA-β-Gal was determined using the “Senescence β-Galaktosidase staining” Kit  according to the manufacturer’s protocol and referred to as X-Gal staining in the figures. Cells with cytoplasmic staining were scored as positive. Relative amount to total cell numbers was quantified.n = 1 correspond to 2 replicates).ELISA from cell culture supernatants was performed using “Human IL-6/Interleukin-6 ELISA Kit PicoKine™”  or “Human IL-8/Interleukin-8 ELISA Kit PicoKine™”  according to the manufacturer’s recommended procedures. ELISA were performed in duplicates for each sample , <0.01 (∗∗), <0.001 (∗∗∗). Data were plotted using GraphPad Prism 8 (LaJolla) showing mean and standard error of the mean.Statistical analyses were performed using GraphPad Prism 8 (LaJolla). Experimental groups were compared using one-way-ANOVA (followed by Dunnett’s multiple comparison test). Statistical significance was set up at 2) , mid-age (34–48 years), and old individuals (78–96 years) ; Table 12) . Primary2) .Next, the primary hallmarks of aging  were examined. DNA damage is a driving force of aging because it has a number of molecular consequences that are also hallmarks of aging, such as genomic instability, telomere attrition, epigenetic changes, and/or impaired mitochondrial function . To analFollowing, single antagonistic age markers were systematically investigated, including induction of cell cycle arrest, expression of SA-βGal, activation of senescence-associated secretory phenotype (SASP), Lamin B1 expression and morphological changes . Cell cycle arrest in senescence is largely mediated by activation of one or both of the tumor suppressor pathways p53/p21 and p16/pRB . These tIn summary, when systematically investigating above mentioned well known single age markers, there was a remarkable heterogeneity between single human donors of different ages and it might thus be hard to judge upon the biological age if only investigating one single aging marker alone. While SA-βGAL was the best single age marker to depict old donor age, it did not perfectly distinguish midage from young donor age. Therefore, we went on to develop a strategy to use above mentioned markers together in one panel to finally yield an integrated AgeScore result.R = 0.86  and high passages (p= > 30 except Old 2–4) of young, mid-age and old age donor-derived human fibroblasts. From every age group we choose two different fibroblast lines that display sufficient growth to enable passaging until high passage.For replicative aging  we systehigh) displayed significant increase in DSBs measured by γH2A.X foci in comparison to the mean of all low passages (low) of young donor cells   . Fibrobl2) . Further2) . In comp2) .Secondly, we checked antagonistic age markers. Activation of cell cycle arrest was detected in WS by upregulated RNA expression levels of p16 . FurtherInterestingly, the pattern of individual markers within the AgeScore was different between HGPS cells and the cells of artificial aging, which were aged by overexpression of Progerin . FurtherDespite the existence of a variety of putative age markers, comparison of different studies on aging cells is hampered by the fact that often only single markers are used . Here, wCell models of disease and/or aging are of even greater importance since the discovery of iPSC and other “induced” cell technologies. While these technologies enable to investigate patient-derived cell models of previously unavailable origin , they significantly alter also general aspects of the cell including their biological age. Different age scores/clocks have thus been proposed in the last years which are estimating biological age with various degrees of accuracy . It is wper se, which is based, amongst others, on the genetic heterogeneity of donors  of the respective donors themselves. Ideally this would mean that the biological age of the respective donor has also been estimated using an age score. In practice, however, this biological age of the donors is often not available. This heterogeneity was less pronounced in case of replicative senescence and induced accelerated aging. In order to minimize this heterogeneity in the latter, we compared the replicative senescence and the progerin overexpression induced aging not to the respective donor cell (=isogenic background) but to the mean of all control conditions. Nevertheless, replicative and progerin induced senescence experimental settings might be more defined than aging in an individual prior to cell isolation with or without premature aging disease.In addition, we examined the pattern of the individual markers within the age-associated marker panel of the cells and in the conditions studied . This adin vivo and in vitro. In addition, the expression of specific markers, e.g., p16 and p21, is tissue dependent. Consequently, the AgeScore might be affected as well by the cell origin. Furthermore, some markers investigated only seldomly became positive in aged conditions despite having been described as established age markers (A limitation of our study is the exclusive use of human dermal fibroblasts. It is well accepted that cellular aging differs across tissues  markers . Thus fuin vitro cell culture and demonstrated its validity in estimating donor age, replicative and artificial aging as well as premature aging. This AgeScore can be easily investigated in standard cell culture laboratories yielding both an individual value but also marker patterns that allow good comparability of the diversity of the aging processes. Further studies are needed to further validate the use of the AgeScore proposed here, also for other in vitro and in vivo applications.In summary, we here propose the use of an age-associated marker panel, which considers key age markers for application in


Results: An increase in symptom severity resulted in worsening of the “participation in society” , “life activities—household” , and “getting along with people”  WHO-DAS 2.0 domains. Positive symptoms (13.89 ± 3.48) correlated strongly with “getting along with people” , “life activities—household” , and “participation in society” , and negative symptoms (14.25 ± 4.16) with “participation in society”  and “life activities—household” . Symptoms of disorganization (15.67 ± 4.16) had the highest impact on “life activities—household” , “getting along with people” , and “participation in society” . Episodic memory  was remotely related to comprehension and communication. The information processing speed , visual memory , and focused executive functions showed moderate correlations with all domains on the WHO-DAS 2.0 scale . Attention  was moderately related to community activities. Semantic  and literal  verbal fluency demonstrated weak correlations with “cognition—understanding”, “getting along with people”, and “participation in society”. Conclusion: Symptoms of disorganization and disturbed executive functions contribute most to disability in patients with schizophrenia through impairment of real-world functioning, especially in social interactions and communication. Severe clinical symptoms (negative and disorganization-related ones) as well as deficits in executive function, verbal memory, and verbal fluency cause the biggest problems in the functional domains of interaction with other people and participation in society.The study aimed to assess the impact of clinical symptoms and cognitive impairment on disability in patients with paranoid schizophrenia (PS). Methods: 108 patients with schizophrenia were included . Their average age was 38.86 ± 10.02 years and the disease duration was 12.80 ± 8.20 years, with mean disease onset of 24 years. Clinical symptoms were assessed with the PANSS, and cognitive performance was measured using a seven-item neurocognitive battery. The disability level of the subjects was assessed using the World Health Organization—Disability Assessment Schedule 2.0 (WHO-DAS 2.0). The relation between the variables studied was assessed using Spearman’s rank correlation coefficient ( Schizophrenia is a severe mental illness that, despite its relatively low prevalence of approximately 1% , is rankSocio-occupational decline in schizophrenia patients is mostly due to negative  and disoBased on the limited research in this area in Bulgaria, the present study aims to assess the impact of clinical symptoms and cognitive impairment on disability in a clinical sample of patients with paranoid schizophrenia.We examined 108 patients with paranoid schizophrenia (PS)  with an average age of 38.86 ± 10.02 years, an average duration of the disease 12.80 ± 8.20 years, and an average age of onset of the disease of 24.00 years  treated during the period 2017–2019 at the Psychiatry Department of Pleven University Hospital, Bulgaria. Participants fulfilled the following inclusion criteria: (1) ICD-10 [The subjects’ disability level was assessed using the full version of World Health Organization—Disability Assessment Schedule 2 (WHO–DAS 2.0) . It contThe following neurocognitive battery was used in the sequence indicated:Ten words Luria test: a verbal memory (VM) test that measures fixation , retention, and reproduction .Trail making yest parts A and B (TMT A&B): a standardized assessment test of processing speed, attention switching, cognitive flexibility, distributed and selective attention, ability to form concepts, and executive functions (part B only).Logical memory test: measures both episodic memory and disorders of thinking.Digit symbol substitution test (DSST): assesses visuo-perceptual functions (including scanning), attention, executive functions (including working memory), and motor speed.Verbal fluency test (VF): measures executive functions and semantic memory.Benton visual retention test (BVRT): designed to measure visual perception, visual memory as well as basic visual abilities, visuo-motor coordination, and perceptual-motor integration.A detailed description of the abovementioned neurocognitive tools can be found elsewhere  along wiAll patients were capable of providing informed consent for participation, which they all did before their enrolment in the study. The study itself was approved by the Research Ethics Committee of Medical University of Pleven. All participants were native Bulgarian language speakers having at least eight years of education. Additionally, all of them were right-handed according to the Edinburgh Handedness Inventory—Short Form .R) and Spearman’s rank correlation coefficient (sr) at probability p < 0.05. All WHO-DAS 2.0 results were calculated using the complex IRT (item–response–theory) method. Multiple regression analysis was conducted to examine the relationship of symptomatology and cognitive impairment with individual domains of WHO-DAS 2.0. Our primary task was to (1) determine whether a relationship between different symptom groups  and cognitive impairment on the one hand and WHO-DAS 2.0 domains on the other objectively exists and (2) to measure the specific quantitative relationships by assessing the influence of the factor variables on the outcome variable.The exploration of the relation between social functioning, severity of clinical symptoms, and neurocognitive test battery performance was carried out by means of correlation analysis for linear dependence—Pearson correlation coefficient , “life activities—household” , and “getting along with people” . Positive symptoms (13.89 ± 3.48) correlated higher with “getting along with people” , “life activities—household” , and “participation in society” , and negative symptoms (14.25 ± 4.16) with “participation in society”  and “life activities—household” . Symptoms of disorganization (15.67 ± 4.16) had the highest impact on “life activities—household” , “getting along with people” , and “participation in society” . All correlations were significant at p = 0.01. “Getting around” had no relation with disease severity.The average severity of the disease based on PANSS score was 71.80 ± 5.10. The increase of symptom severity resulted in worsening of “participation in society” —χr = −0.28, p = 0.003), excluding “getting around”. The retention of verbal material  had weak correlations with domains “self-care” , “getting along with people” , “life-activities—household” , and “participation in society” . Reproduction (10 words Luria) correlated slightly only with the “self-care” domain . Verbal fluency—semantic  and literal —also showed slight correlation with “cognition—understanding”, “self-care”, “getting along with people”, “life activities—household”, and “participation in society”. Psychomotor speed (measured using TMT-A) , focused attention (assessed using DSST) , and executive functions (graded using TMT-B)  showed moderate correlations with all domains of the WHO-DAS 2.0. Moderately pronounced correlations were found with the number of wrong answers in visual memory , measured using BVRT, and weak correlations with the number of correct answers .Fixation on auditory verbal memory (10 words Luria test) had weak correlations with all domains of the WHO-DAS 2.0 scale  and “participation in society” . A positive correlation was found between executive functions and “life activities—household”  and “participation in society” . Further, regression analysis (Tabl. 8) revealed that negative symptoms and executive functions significantly influence “life activities—household” with 65.0% coefficient of determination  and “participation in society” with 60.0% coefficient of determination .The correlation analysis revealed a significant positive correlation between negative symptoms and “life activities—household” , “life activities—household” , and “participation in society” . Regression analysis (Tabl. 8) revealed that disorganized symptoms and verbal fluency, may influence “cognition—understanding and communicating life” with 56.0% coefficient of determination . Disorganized symptoms alone influenced “life activities—household” with a 63.0% coefficient of determination  and “participation in society” with a 58.0% coefficient of determination .We observed a significant positive correlation between disorganized symptoms and “cognition—understanding and communicating life”  as well as the relationship with others (item D4). In addition to that, we observed a strong correlation with social participation D6 . Verbal memory especially significantly determines the impairment degree in community activities, communication, and interpersonal relationships. Verbal fluency deficit in its turn also causes difficulties in communication, which hampers establishing and keeping friendships [6). In their review of the neural correlates of the perception of socio-emotional stimuli, Green & Leitman (2008) [Out results show that verbal memory  and verbendships . Individn (2008)  raise thn (2008) .3) and daily living activities (D5) are basic and are the last to be lost with advancing age or in the course of illness, because they are built first in the course of individual development. We found that mobility (D2) was slightly correlated with psychomotor speed measured using TMT-A and DSST, but not using TMT-B  are not taken into account. In addition, in our analysis, we did not take into account the influence of protective factors such as availability of a partner, higher income, or stronger social support.Poor occupational functioning is a hallmark of schizophrenia contributing to the burden of disease on individuals, their families, and society. WHODAS 2.0 is a clinically useful tool for the assessment of psychosocial functioning in patients with schizophrenia. Symptoms of disorganization and disturbed executive functions contribute the most to disability in patients with schizophrenia through impairment of real-world functioning, especially in social interactions and communication. Severe clinical symptoms (negative and disorganization-related ones) as well as deficits in executive function, verbal memory, and verbal fluency cause the biggest problems in the functional domains of interaction with other people and participation in society.


Despite the presence of COVID-19 epidemiologic data in Africa, there are gaps in the understanding of healthcare workers’ concerns and fears early in the pandemic.A retrospective cross-sectional multi-country pan-African qualitative survey case study on the perceived effects of the COVID-19 pandemic on healthcare workers in the continent focused specifically on personal safety and misinformation. The survey was distributed to 13 countries via snowball sampling of practitioners between April 22 and May 15, 2020. The survey solicited free-form answers, resulting in a large spectrum of responses. Qualitative analysis included open and axial coding methods for thematic emergence.A total of 489 analyzable responses were recorded. The majority of respondents  highlighted personal safety concerns including lack of resources and training to prevent infection (33%); fear of infection and transmission (24%); lack of public awareness and compliance with regulations (12%); governmental concerns (9%) and economic insecurity (11%) amongst others. 328 respondents (67%) reported having heard misinformation about COVID-19. Responses included misinformation regarding origin of the virus (11%), false modes of transmission (6%), differential effect for specific groups (30%), unproven cures (35%), and disbelief in existence (11%). Responses for misinformation and fears revealed categorical associations between certain countries.Addressing fears and concerns of frontline healthcare workers facilitates their essential role in combating community misinformation, and further understanding could provide essential insight to institutions and governments to direct resource allotment and community education. •African healthcare workers had personal safety concerns with the COVID-19 pandemic.•Healthcare workers heard a variety of misinformation in their communities.•Surveying healthcare workers contextualizes lived experience on the frontline.•Surveying healthcare workers can identify resource needs and community education. AlthougHere we describe a retrospective cross-sectional multi-country pan-African qualitative survey case study on the perceived effects of the COVID-19 pandemic on HCW in Africa. We first describe the methods of seeking qualitative data from HCW participants across the continent followed by results collating HCW input in their own words. The qualitative questions in this study surveyed HCW about personal safety concerns and misinformation about COVID-19. Thereafter, we explore the practical implications of the qualitative responses and emergent themes on current considerations across Africa in regards to managing the COVID-19 pandemic and its effects on HCW.2UIN 7932  testing capacity,” “oxygen cylinders, concentrators, mechanical ventilators, N95s and surgical face masks, lab investigation (RT PCR), and drugs.” The most common concern was a lack of personal protective equipment (PPE), with some respondents reporting having “no PPE at all.”Additionally, medical professionals voiced concern about a lack of proper training or protocol in place to successfully navigate the health crisis, noting “inadequate training on safety measure[s],” “compromised teaching with no leadership or guidance,” and “we don't apply [the] triage system perfectly.”3.1.3Due to the reported “infectious character,” “deadly” nature, and “rapid spread” of the novel coronavirus, participants expressed fear of becoming infected with SARS-CoV-2. Two HCWs attributed their concerns to having personal pre-existing health conditions. There were concerns about unsafe work environments—many respondents worried about contracting the virus due to the possibility of transmission to patients, families, and communities. Community transmission through overcrowding was a problem in respondents’ workplaces, roads, and public transportation.3.1.4Concerns in this category were related to variable public adherence with COVID-19 mitigation strategies. Respondents reported that many in their communities did not “abide by the lockdown,” “observe recommended precautionary measures,” or “adhere to ministry of health instructions.” Respondents attributed public noncompliance to a lack of understanding in their communities, detailing “the community is not aware of the magnitude and seriousness of the pandemic and measures of prevention.” One respondent ascribed “society … not strictly abiding by the lockdown” to “sociocultural habits [being] deep rooted,” and another commented “people are not willing to listen to the health care workers.”3.1.5Survey participants expressed dissatisfaction with their governments’ responses to COVID-19 including a lack of reliable and regularly updated case reporting from governments. Respondents reported governmental responses were poorly-organized or nonexistent, describing “no coordinated effort to enforce … laws,” no “national restraint on movement of people,” and “poor … government policies.” Some were frustrated with a lack of government transparency, specifically “no daily updates on tests, number of cases or complications from the virus by the government.” Finally, a few respondents were concerned about the failure of their governments to provide supplies, shelter, and transportation to healthcare workers including basic needs and palliative care to the people.3.1.6Primarily, respondents were concerned about poverty, since many in their countries lived “hand to mouth” and were prevented from working due to the pandemic. They also noted that theft and hunger were on the rise. Some respondents cited general economic instability, such as inflation or a possible market crash, as the root of their concern. Finally, three respondents highlighted HCW were being “underpaid” or not paid at all.3.1.7Respondents (n = 10) voiced concerns about transportation, such as HCW being “required to go to work during a lockdown when they don't have private cars … [and] aren't being paid.” HCW (n = 9) were also worried about the lack of accessibility and availability of essential resources such as food and supplies. Survey participants drew attention to social stigmatization of HCW, COVID-19 patients, and COVID-19 mitigation strategies.3.23.2.1The majority of respondents  reported to have heard misinformation about COVID-19 and 16% left this question blank. Out of all respondents who indicated hearing misinformation, 89% (n = 293/328) provided specific example(s) of misinformation. Classification as misinformation was solely based on survey respondent categorization as such in response to this survey question, rather than factual accuracy. The categories of misinformation clustered around origin of the virus (11%), false modes of transmission (6%), severity (10%), differential effect (30%), unproven cures (35%), disbelief in existence (11%), and case reporting (7%). While there was much crossover between African countries in response categories to the personal safety question, responses to this misinformation question revealed associations with certain countries .Fig. 2Mi3.33.3.1Respondents reported hearing misinformation that COVID-19 had a religious origin , had a man-made origin (n = 6), was sarin gas , or came from goats .3.3.2Respondents heard COVID-19 was transmitted “sexually,” by “mosquitoes,” by “animal carriers,” “over the phone,” and by “5G internet.” Four respondents believed “COVID-19 is airborne” to be misinformation. Remaining respondents confirmed misinformation about the mode of transmission of the virus but did not specify further.3.3.3The great majority of respondents in this category (n = 23) reported misinformation that COVID-19 was not very serious, deadly, or contagious, or was similar to the common cold or flu.3.3.4Many respondents (n = 34) described misinformation about SARS-CoV-2 having little to no effect on Africans, such as the disease not being “as severe or prevalent in blacks,” as “fatal as seen in other nations,” or not affecting Africans at all. One HCW heard “Africans are immune to COVID-19 based on previous exposure to diverse tropical infections.”Respondents (n = 20) heard about a differential effect of COVID-19 in relation to climate: “The disease [has a] lower transmission rate in hot temperature[s],” and “The warm climate in our country prevents us from getting COVID.” Finally, respondents referred to SARS-CoV-2 having little to no effect in youth (n = 14) and religious countries or people .3.3.5Nearly half (n = 59/103) of the respondents in this category heard traditional medicines or natural remedies protected against or treated COVID-19. Common responses were garlic , ginger , lemon , and onion  as home remedies. Other subcategories of misinformation of cures or protective methods against COVID-19 were drinking alcohol (n = 12); hydroxychloroquine/chloroquine (n = 9); hot drinks such as warm water or tea (n = 7); sunlight, ultraviolet light, and/or heat (n = 7); and steam inhalation .3.3.6This category was mainly associated with Nigeria (n = 12/32). Respondents stated many in their communities believed COVID-19 to be a “hoax,” “rumor,” “political conspiracy,” and “scam by [the] government to siphon public funds.” One respondent related hearing “the swabs being imported are tainted with the virus so we all test positive.” Because of disbelief in the existence of COVID-19, one respondent shared that community members “throw cautions to the wind.”3.3.7This category was associated with Sudan. Many respondents believed there was a “false record of confirmed cases” and “misinformation about [the] number affected by COVID-19.” One Sudanese respondent stated, “in Sudan, we are in [a] transition period so many people get benefits from mobilizing [the] wrong information like increasing the number of new cases, number of deaths, etc.” A Tanzanian HCW shared “the president [is] giving false information to the nation regarding [the] statistics of COVID-19.” Other respondents noted a misperception in their communities that COVID-19 had disappeared due to actions that had been taken. Due to “reopening of transport, market, other activities, and fewer … daily positive case reports [from] the ministry,” a HCW said COVID-19 was “perceived as decreased/disappeared in our country.”4Our qualitative survey uncovered variability in the lived experience of HCW across the African continent early in the COVID-19 pandemic. Notably, 57% of respondents confirmed concerns about their personal safety and 67% heard misinformation in their communities.This data contributes to the current literature on HCW perceptions and experience of the COVID-19 pandemic. Several studies evaluated knowledge, attitude and practices in individual cross-sectional studies, for example, in Egypt, Nigeria, Uganda, Cameroon, South Africa, Ethiopia and Mozambique . The menOur survey results identify societal needs as a core component of personal safety through its notable intersection with governmental public policies. The pandemic had a dramatic personal effect on HCWs, including lack of resources, social stigma, economic insecurity, fears of infection spreading, challenges with governmental policies, and community misinformation. Participants identified PPE, HCW compensation, and unified public health messaging and adherence as critical to personal safety.Surveying misinformation is a key component to understanding the beliefs behind health behaviors and regional patterns of information dissemination. Roozenbeek et al. investigated COVID-19 misinformation across the UK, Ireland, USA, Spain and Mexico, finding a link between misinformation susceptibility, vaccine hesitancy and lower likelihood of complying with public health guidance . Early iLimitations. Our study had a number of limitations. First, although the survey included respondents from 13 African countries, each country was not equally represented in the data. More respondents from countries with lower representation would have provided comprehensive information for stronger associations. Demographically, respondents were largely male and majority physicians, which is not representative of the entire workforce across Africa. Rural areas were underrepresented in the data since respondents were largely residents of urban areas working in medical centers. Future studies would benefit from respondent pools with greater diversity in country, gender, health profession, political preference, and setting. A further challenge of qualitative analysis is a limited ability to capture the original intention of a respondent. It is possible that responses could have been misinterpreted or important feedback missed due to lack of clarity during the analysis. The survey was only distributed in English; this language barrier may have led to a misinterpretation of the data and limited the depth of the answers in the absence of respondents’ preferred language. Conducting future surveys in multiple languages would be mutually beneficial to respondents and researchers.4.1Qualitative research seeks to explore a group in their context and setting . InstituHennepin Healthcare Institutional Review Board and Ethics Committee.10.13039/100006090University of Minnesota Center for Global Health and Social Responsibility; 10.13039/100007249UMN COVID-19 Rapid Response Grants and 10.13039/100000867Robert Wood Johnson Foundation to Jose Daniel Debes.Funding was received through the Allison Benjamin: Data analysis, Writing the Paper. Amir Sultan: Study concept, Data Collection, Writing the Paper. Mirghani Yousif: Study concept, Data Collection, Writing the Paper. Abdelmajeed Moussa: Study concept, Data Collection, Writing the Paper. Ehab Fawzy Abdo: Study concept, Data Collection, Writing the Paper. Johnstone Kayandabila: Study concept, Data Collection, Writing the Paper. Kenneth Ssebambulidde: Study concept, Data Collection, Writing the Paper.Lucy Ochola: Study concept, Data Collection, Writing the Paper. Ifeorah Ijeoma: Study concept, Data Collection, Writing the Paper. Nasreen Syeda Quadri: Study concept, Data Collection, Writing the Paper. Jose Daniel Debes: Securing funding, Study concept, Data Analysis, Data Collection, Writing the Paper.1. Name of the registry: N/A.2. Unique Identifying number or registration ID: N/A.3. Hyperlink to your specific registration (must be publicly accessible and will be checked): N/A.Jose D. Debes.Consent to participate:Participation in our anonymous survey was entirely voluntary; written consent was not requested by participants.The corresponding author may be contacted for all material used and data obtained from this study.Not commissioned, externally peer-reviewed.N/A.


Controlled hypotension is a reversible procedure in which the patient’s baseline mean arterial blood pressure is reduced by 30% and sustained at 60–70 mmHg during the procedure. It decreases blood loss and provides clear surgical field during the procedures.The purpose of this study was to compare the efficacy of controlled hypotension agents esmolol, remifentanil, and nitroglycerin in functional endoscopic sinus surgery, in terms of hemodynamic changes and impact on the surgical efficiency.The research was carried out as a cohort study. Patients who underwent functional endoscopic sinus surgery were randomized into 3 groups. Controlled hypotension was achieved with remifentanil (Group R), esmolol (Group E) and nitroglycerin (Group N). The efficacy of the drugs was tested by comparing the length of time with the targeted mean arterial pressure, the amount of anesthetics used, surgical field bleeding score and surgeon’s satisfaction.p = 0.01) and Group N (p = 0.14). The amount of volatile anesthetics used was 25.0 mL (15–51), 43.0 mL (21–105) and 40.0 mL (26–97) in Groups R, E and N, respectively (p < 0.001). While there was more bleeding with nitroglycerin, surgical field bleeding scores were lower in Group R when compared with esmolol (p = 0.001) and nitroglycerin (p < 0.001). The analysis of surgeon’s satisfaction scores concluded that surgeons were more satisfied with the group R (100%), when compared with group E (60%) and group N (30%) (p < 0.001).Between May to December 2015, 60 patients were included and randomized equally into 3 different study groups. The median of the length of time with the targeted mean arterial pressure was shorter in the Group R when compared with Group E (Less volatile agent, short time to achieve controlled hypotension, stable blood pressure, lower surgical field bleeding scores and larger length of time with the targeted mean arterial pressure were found as the advantages of Remifentanil. Less costly, efficiency of achieving the targeted median arterial pressure and less postoperative complications were the advantages of nitroglycerin. In functional endoscopic sinus surgery procedures, appropriate controlled hypotensive agents should be selected according to the patients’ characteristics and advantages/disadvantages of the drugs. Functional Endoscopic Sinus Surgery (FESS) is an important way of approach, mostly used in pathologies obstructing the sinuses. In addition to its efficacy, the procedure provides drainage of sinuses without damaging the normal physiology and anatomy of the nasal cavity.Controlled Hypotension (CH) at a moderate level is defined as a reversible and controlled reduction of Mean Arterial Pressure (MAP) to 60‒70 mmHg or a 30% reduction of baseline MAP.The purpose of this study is to compare the CH agents Esmolol, Remifentanil, and Nitroglycerin for FESS, in terms of hemodynamic changes and impacts on surgical efficacy.The study was conducted in Keciören Education and Research Hospital, Department of Anesthesiology and Reanimation. Institutional Ethics Committee approved the study protocol (nº B.10.4.İSM.4.06.68.49) and the study was in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki. All participants gave their informed consent prior to their inclusion in the study.Between 2015 May and 2015 December, ASA I–II patients, between the ages of 18‒50 and who were scheduled for FESS were included in the study. The patients with ASA score of III and more, medications including β-blockers, opioids and ones having impact on the cardiovascular system, chronic hypertension, coronary artery disease, arrhythmias, chronic obstructive pulmonary disease, chronic renal or hepatic failure, history of cerebrovascular disease, diabetes, severe anemia (less than 7 gr/dL), coagulopathy, history of sinus surgery, history of allergy to one of the study drugs and who were pregnant or lactating were excluded from the study. Sealed envelopes were used to assign the patients to treatment groups; Group R (remifentanil), Group E (esmolol) and Group N (nitroglycerin). The patients were randomized after signed a written informed consent just before the procedure. Two anesthesiologists followed the patients: one for randomization and preparation of the drug, one for monitoring and documentation of the results. Thus the observer was blinded.2) and Bispectral Index (BIS)  were recorded. After recording preoperative measurements, induction of anesthesia was performed with 1 mg/kg lidocaine (2%), propofol 2–3 mg/kg and fentanyl 1 μg/kg IV. Patients were intubated with 0.6 mg/kg rocuronium and maintenance of anesthesia was sustained by with sevoflurane 2–4%, nitrous oxide 50% and oxygen 50% to keep the BIS in the 40‒60 range. In addition, mechanical ventilation was continued to provide an end-tidal carbon dioxide level of 32–36  mmHg. All the patients were in 45 degrees supine position. Before starting the procedure CH was performed in study groups. In Group R, remifentanil was used with a loading dose of 1 mcg/kg in 60 sec. The maintenance of anesthesia was sustained with 0.1 mcg/kg/min remifentanil and the dose increased to provide a MAP of 60‒65 mmHg. In Group E, esmolol was used with a loading dose of 1 mg/kg in 60 sec. The maintenance was sustained with 0.4 mg/kg/h esmolol and the dose increased to provide a MAP of 60–65 mmHg. In Group N, nitroglycerin was used with a maintenance dose of 2 mcg/min to provide a MAP of 60–65 mmHg. During the procedure HR less than 50 beats/min were defined as bradycardia and it was treated with 0.015 mg/kg atropine. In addition, if the MAP decreased to less than 60 mmHg for more than 60 s, the dosage of the drug is halved and followed further. If the intolerable hypotension continued, then the CH medication was terminated. After FESS was completed and the study drug was terminated, the patients were followed up to opening their eyes and extubation time.The patients’ gender, age, weight, ASA score and indications of the FESS were recorded. Patients were monitored by ECG, pulse oximetry, invasive/noninvasive blood pressure and Bispectral Index (BIS). Thus Heart Rate (HR), systolic arterial pressure, diastolic arterial pressure, Mean Arterial Pressure (MAP), Oxygen Saturation  were declared by the primary surgeon. . The total amount of the volatile agent (Sevoflurane) used was documented. The efficacy of the drugs was evaluated with LTMAP, Surgical Field Bleeding Score (SFBS) and Surgeon’s Satisfaction (SS). SS were graded as excellent, good, moderate, bad, and very bad. For analysis, SS were grouped as satisfied (good and excellent) and not satisfied . To increase the efficacy and optimal subjectivity of the evaluation process, only 2 surgeons were included in the study.During procedures; HR, SAP, DAP, MAP, SpOt-test and non-parametric tests for repeated measures (Friedman Test). Differences between non-parametric variables were analyzed with Mann–Whitney U test. The Chi-Square or Fisher exact tests were used to compare categorical variables. All analyses were performed using SPSS 17.0 for Windows . The p-value of less than 0.05 was considered as statistically significant.Baseline characteristics of the patient group were described using proportions for dichotomous and categorical variables. Differences between continuous variables were assessed with the Student p = 0.43). In 95% of the patients, CH goals were achieved. In all study groups success rate was more than 90% and it was 100% in Group N. The median LTMAP was 10 (5–20) minutes in Group R, 15 (4–40) minutes in Group E and 15 (5–40) minutes in Group N (p = 0.052) (p = 0.01) and Group N (p = 0.14). The analysis of HR concluded that the maximum HR during procedures were similar in all study arms groups (p = 0.90) and Group R were exposed to more bradycardia (p < 0.001)  a. It was< 0.001)  Fig. 1bbp = 0.43p = 0.051) for hemodynamic changes (p < 0.001). There were postoperative complications in 10% of Group R, 5% of Group E and none of the Group N patients (p = 0.34). Bronchospasm was observed as a complication in these groups.During the perioperative period, extra medications were used in 10 (50%) patients in Group N, 7 (35%) in Group R and 3 (15%) patients in Group E ( changes . In addip < 0.001). While there was more bleeding with nitroglycerin, SFBS scores were less in Group R when compared with esmolol (p = 0.001) and nitroglycerin (p < 0.001). The analysis of SS scores concluded that surgeons were more satisfied with Group R (100%), when compared with Group E (60%) and Group N (30%) (p < 0.001). There was a positive correlation with SFBS and SS scores .The analysis of efficacy was performed by comparing LTMAP, SFBS, and SS in study arms. The SFBS were 2 (1–2) in Group R, 2 (2–3) in Group E and 3 (2–4) in Group N (In the present study, we aimed to compare the efficacy of three CH agents, Remifentanil, esmolol, nitroglycerin. We concluded that less volatile agent, short time to approach CH, stable blood pressure, less SFBS and short operation time length were the advantages of Remifentanil. Efficacy of approaching targeted MAP and less postoperative complications were the advantages of Nitroglycerin.Functional endoscopic sinus surgery is widely used because of its feasibility and low complication rates. Bleeding is the most common complication of FESS.In the literature, there are numerous studies comparing 2 drugs, especially esmolol and nitroglycerin. However, there are limited studies comparing three agents for CH. Srivastava et al. concluded that nitroglycerin was superior to esmolol with its shorter LTMAP, lower bleeding scores and producing less reflex tachycardia.Esmolol is a β-adrenergic receptor blocker and has been used for CH for many years. In addition, vasoconstriction in arterioles and precapillary sphincters provides less bleeding and better operation field.Esmolol and nitroglycerin provide hypotension by directly acting on cardiovascular structures. However, Remifentanil is an ultra-short-acting µ-agonist opioid receptor. Its most important advantages are short half-life and not having effects on microcirculation.The study had some inevitable limitations. The evaluation of CH efficacy was performed by assessing SS and SFBS. However, those are subjective parameters. We tried to decrease the bias related with subjectivity by working with 2 surgeons. Objective parameters could provide a more efficient analysis. The inclusion of more patients could enable us to do further analysis. The patients were older in the Group N when compared with others. However, because the renal, hepatic functions and performance scores of the patients were similar, we ignored the age difference between groups.Remifentanil provides a lower surgical field bleeding score, stable blood pressure and short time to targeted mean arterial blood pressure with the use of less volatile anesthetic agent. However the nitroglycerin group produces easy control of blood pressure with less postoperative complications. As a result, appropriate CH agents should be selected according to patients’ characteristics and advantages/disadvantages of drugs during FESS procedures.The authors declare no conflicts of interest.


Cuproptosis is a fresh form of the copper-elesclomol-triggered, mitochondrial tricarboxylic acid (TCA) dependent cell death. Yet, the subsumed mechanism of cuproptosis-associated lncRNAs in carcinoma is not wholly clarified. Here, We appraised 580 cuproptosis-associated lncRNAs in sarcoma and thereafter construed a module composing of 6 cuproptosis lncRNAs, entitled CuLncScore, utilizing a machine learning methodology. It could outstandingly discern the prognosis of patients in parallel with discriminating tumor immune microenvironment traits. Moreover, we simulate the classification system of cuproptosis lncRNAs by unsupervised learning method to facilitate differentiation of clinical denouement and immunotherapy modality options. Notably, Our Taizhou cohort validated the stability of CuLncScore and the classification system. Taking a step further, we checked these 6 cuproptosis lncRNAs by Quantitative real-time polymerase chain reaction (qRT-PCR) to ascertain their authenticity. All told, our investigations highlight that cuproptosis lncRNAs are involved in various components of sarcoma and assist in the formation of the tumor immune microenvironment. These results provide partial insights to further comprehend the molecular mechanisms of cuproptosis lncRNAs in sarcoma and could be helpful for the development of personalized therapeutic strategies targeting cuproptosis or cuproptosis lncRNAs. Sarcoma is not a singular entity, but a set of well over 100 subtypes of malignant growths derived from the wider mesenchymal stroma of bone and soft tissue, with a spectrum of mutable biological and clinical traits . Given tCuproptosis, a novel form of the copper-elesclomol-triggered, mitochondrial tricarboxylic acid (TCA) dependent cell death, occurs by a mechanism independent of well-known apoptosis and pyroptosis . JuxtapolncRNAs are non-protein-coding RNA species with multifarious bio functions that have been documented to be dysregulated in a wide range of pathologies and to be pivotal agents in the ontogenesis of carcinogenesis . In sarcTo systematically understand cuproptosis lncRNAs and unravel their mystery in sarcoma, we unearthed genomic information of TCGA-SARC samples in an attempt to disentangle the presumable role of cuproptosis lncRNAs in sarcoma. We were excited to detect a significant impact of cuproptosis lncRNAs on the tumor microenvironment. Moreover, We constructed a new risk-prognosis model, called CuLncScore, to facilitate the easy assessment of patient prognosis and therapeutic response. Moving further, we partitioned patients into 4 clusters to delineate their variable immune status and prognosis. Of notable is that the CuLncScore and categories were validated in our Taizhou cohort. Our study provides significant insight into the function of cuproptosis lncRNAs in sarcoma.p < .001 were accepted as cuproptosis-associated lncRNAs.Peter et al. proposed new insights into cuproptosis, from which we extracted 12 key genes, covering FDX1, LIAS, LIPT1, DLD, DLAT, PDHA1, PDHB, MTF1, GLS, CDKN2A, ATP7B, and SLC31A1, named cuproptosis genes . PearsonIn this investigation, we enrolled RNA sequencing data, SNV data, CNV data, and clinical data from the TCGA-SARC cohort, Data above were divided equally into train and test groups based on random assignment. To be first, a cuproptosis lncRNAs score model, entitled CuLncScore, was constructed using LASSO regression. AC131056.3, AP001476.2, CTC-453G23.8, CTC-550B14.7, CTD-2651B20.7, and RP11-351I21.11 opted for model configuration. Based on the linear combination of gene expression values and regression coefficients, risk scores were calculated for SARC patients. The formula was as follows:The gene set enrichment analysis (GSEA) was performed to check potential biological processes in high and low-risk groups. The “CIBERSORT” R package was used to estimate the proportion of immune cells.We retrospectively enrolled 40 patients who had surgical treatment at Taizhou Hospital from 1 February 2014, to 30 December 2021, which we named the Taizhou cohort. The study followed the guiding principles of the Declaration of Helsinki and was approved by the ethical review committee of Taizhou Hospital. All participants gave written informed consent. A total of 12 tumor samples  and 6 paracancerous normal control samples were submitted for transcriptome sequencing. Total RNA was purified from FFPE samples and only samples with DV200 ≥ 30% were selected for subsequent RNA sequence analysis. See our previous report for more details on the method .via PrimeScriptTM RT kit (Takara). qRT-PCR analysis was run on the SYBR Premix Ex Taq (Takara). Normalization of all expression databases to GAPDH (as an endo-controller gene) using the 2−ΔΔCT method. Primer sequences referred to the following.After paraffin tissue was dewaxed, RNA was isolated by Trizol reagent . Then, complementary DNA (cDNA) was synthesized AC131056.3_Forward: 5′- GCTGTCAAATGAGGCAGGTTG-3'; Reverse: 5′-GTAAGAGCCTGATGCGTGGA-3'. AP001476.2_Forward: 5′-GTTTCATAAGTGAAATGCCTGCT-3'; Reverse: 5′-ACCCTCACTGGATACACCAAAA-3'. CTC-453G23.8_Forward: 5′-ATCTTTGGCGTGTCAGCACT-3'; Reverse: 5′-CCAAGTCTCCAGACTCTTGCTT -3'. CTC-550B14.7_Forward: 5′-TCTTCTGGTTGTTCTGAAGGG-3'; Reverse: 5′-ACTCTTCCTTTGGGGAAGCAA-3'. CTD-2651B20.7_Forward: 5′-TGATGAGGGAGTCGCAAGCA-3'; Reverse: 5′-ACAAGTCAGGCTTCTTGACCC-3'. RP11-351I21.11_Forward: 5′-CATTCTGGTGGCCGAGAGAC-3'; Reverse: 5′-TTGGGCTGAGATGGCTAGTATT-3'. GAPDH_Forward: 5′- GGAGCGAGATCCCTCCAAAAT-3'; Reverse: 5′- GGCTGTTGTCATACTTCTCATGG-3'.R software (version 3.6.1) was used for all statistical analyses. Wilcoxon test was used for comparison between groups if not otherwise stated. Kruskal test was employed for overall comparison of multiple sets and Pearson correlation coefficient was for correlation analysis.Cuproptosis is a fresh conception, and the underlying role of cuproptosis-associated lncRNAs in sarcoma captured our attention particularly well, both in terms of their expression patterns and potential prognostic impact. For starters, in TCGA-SARC, we authenticated 580 cuproptosis lncRNAs by co-expression analysis algorithm . As a fuTo check the stability of the model, we randomly grouped the TCGA-SARC cohort into two equally, named the train group and the test group. Soon after, based on the median score, patients were ranked as high-risk and low-risk, respectively. By Kaplan-Meier analysis, we showed that the CuLncScore could accurately predict prognosis, those with higher risk had a worse prognosis. This was confirmed in both the train group  and testTo dig deeper, we aimed to use GSEA to explore the differences in intrinsic molecular mechanisms comparing the high- and low-risk groups and revealed that pathways associated with the high-risk group included basal cell carcinoma, cell cycle, and hedgehog signaling pathway , while pThe tumor immune microenvironment is firmly linked to patient prognosis and immunotherapeutic response . To furtCategorizing patients to draw a line between their unique immune statuses would allow for easy clinical guidance in tailoring individualized immunotherapy regimens. Hence, we classified the TCGA-SARC into four new clusters using an unsupervised algorithm, nominated cluster 1, cluster 2, cluster 3, and cluster 4 . Kaplan-The principal component analysis (PCA) plots present the distribution of patients in different CuLncScore groups  and fourVia profiling of the data, we constructed a neoteric CuLncScore system with superior prognostic predictive capabilities. This called for more practical testing, so we enrolled a cohort containing 12 cases of sarcoma ( sarcoma . This re sarcoma . Patient sarcoma . This ou sarcoma , which w sarcoma .The newborn copper-associated form of cell death, cuproptosis, is setting off a burning wave of research that could shed new light on oncotherapy . lncRNAsp-value <.001 were our selection thresholds, and we ultimately yielded 580 lncRNAs, defined as cuproptosis lncRNAs.At the outset of this work, cuproptosis lncRNA and sarcoma were the centerpiece agents. Through a literature search, no precise definition of cuproptosis lncRNA has been yet reported. Mindful of the correlation between the expression levels of mRNAs and lncRNAs, we characterized lncRNAs tightly associated with cuproptosis genes using the pearson correlation analysis method, which has been broadly applied in the field of computational genomics of tumors . In the + T cells and CD56+ NK cell infiltration in the tumor, and those cells are correlated with the anti-tumor immune response, resulting in a favorable prognosis (In this case, we noted the anomalous phenomenon that RP11-351I21.11 was highly expressed in the tumor, whereas the cox analysis showed that it might work as a protective factor. Likewise, CTC-453G23.8 and CTC-550B14.7 were hyper expressed in tumors, while a cox analysis realistically concluded that it could be a risk element. It seems to contradict the conventional notion that genes with high expression in tumors are oncogenes, instead, low expression is oncogenes. Actually, we missed an essential point that neoplasms are the outcomes of staged development. For instance, TGF-beta, which plays a part at different stages of cancer, is probably oncogenic in the very early stage and pro-cancer in the advanced . Moreoverognosis .Sarcoma, as one of the tumors with extremely poor outcomes , urgentlAt a time when tumor immunotherapy is in full swing, there is a consensus that differentiating the state of different immune microenvironments could better guide the choice-making of treatment modalities . SarcomaSarcomas typically have reoccurring driver genomic events, covering amplification, mutations, or translocations . For insWe must admit that the present study contains certain limitations. Primarily, the validation cohort of this report is a single-center retrospective study and still awaits further validation by multi-center clinical cohort support. Despite the implementation of several immunohistochemical experiments, our findings imply that certain cuproptosis lncRNAs may play an essential role in sarcoma, but more in-depth mechanisms lack exploration. Our team is handling further diligent work on this topic.Profoundly, the CuLncScore model and classification system based on cuproptosis lncRNAs are excellent in assessing the prognosis and immune microenvironment status of sarcoma patients and hold prospective for further clinical application.