The cerebellum plays a crucial role in sensorimotor and associative learning. However, the contribution of molecular layer interneurons (MLIs) to these processes is not well understood. We used two-photon microscopy to study the role of ensembles of cerebellar MLIs in a go-no go task where mice obtain a sugar water reward if they lick a spout in the presence of the rewarded odorant and avoid a timeout when they refrain from licking for the unrewarded odorant. In naive animals the MLI responses did not differ between the odorants. With learning, the rewarded odorant elicited a large increase in MLI calcium responses, and the identity of the odorant could be decoded from the differential response. Importantly, MLIs switched odorant responses when the valence of the stimuli was reversed. Finally, mice took a longer time to refrain from licking in the presence of the unrewarded odorant and had difficulty becoming proficient when MLIs were inhibited by chemogenetic intervention. Our findings support a role for MLIs in learning valence in the cerebellum. This study shows that cerebellar molecular layer interneurons (MLIs) develop responses encoding for identity of the stimulus in an associative learning task. Chemogenetic inhibition of MLIs decreased the ability of mice to discriminate stimuli suggesting that MLIs encode for stimulus valence. Importantly, subtle changes in this PC excitatory–inhibitory balance generate robust, bidirectional changes in the output of PCs6.The cerebellum plays a pivotal role in coordinating movements through sensorimotor integration. It receives massive input through mossy fiber synapses onto granule cells (GCs) to form a circuit efficient in complex pattern separation2+ in PCs elicited by motor error signals conveyed by climbing fibers (CFs) is a classical model of plasticity10. However, recent studies indicate that CFs also signal reward prediction12 or decision-making errors13, and the cerebellum modulates association pathways in the ventral tegmental area (VTA) contributing to reward-based learning and social behavior14. Furthermore, although LTD at the PF–PC synapse is often considered as the substrate for cerebellar dependent learning10, such learning can occur in the absence of LTD and may therefore involve other forms of plasticity15. A potential substrate for plasticity is the PF-MLI synapse16 where LTP can be induced in slices by pairing MLI depolarization with PF stimulation17 and in vivo by conjunctive stimulation of PFs and CFs18, believed to underlie changes in the size of cutaneous receptive fields20. In addition, high frequency stimulation of PFs alters subunit composition of AMPA receptors, rendering them calcium impermeable21, a long-lasting change linked to behavioral modifications22. Furthermore, MLIs have been proposed to participate in cerebellar plasticity24, and Rowan et al. found graded control of PC plasticity by MLI inhibition25, suggesting that MLI inhibition is a gate for learning stimulus valence, which conveys information as to whether the stimulus is rewarded. However, whether there is a causal participation of MLIs in reward-associated learning is unknown.Plasticity in cerebellar circuit activity plays an important role in generation of adequate output. Indeed, long-term depression (LTD) mediated by dendritic increases in Ca27. We applied two-photon microscopy28 to record Ca2+ changes in ensembles of MLIs and utilized chemogenetics to explore the functional role of MLI activity in learning.Here we explored whether MLI activity plays a role in reward-associated learning in a go–no go task where mice learn to lick to obtain a water reward2+ indicators GCaMP6/7. MLIs were imaged within the superficial 50 μm of the ML in head-fixed mice through a 2 × 2 mm glass window implanted above the cerebellar vermis   Fig. , and theF/F, Fig. 31. The average diameter of the ROIs in this field of view (FOV) was 10.5 ± 4 μm  and reached a peak when the animal was rewarded  for the last second of odorant application increased as a function of percent correct performance for the S+ condition, whereas it remained stable for the S− condition  and for the interaction between performance and the odorant (S+ vs. S−) . The bootstrapped 95% confidence interval (CI) for an LDA trained after shuffling stimulus was used as a control. The time course for LDA decoding accuracy for the session with the learning curve in Fig. p values <0.01 and <0.05, 24 observations, 18 d.f., n = 4 sessions, 4 mice, GLM F-statistic 16.4, p < 0.001). Post-hoc tests corrected for multiple comparison using false discovery rate (FDR)33 yielded a statistically significant difference for decoding accuracy for either reinforcement or odorant vs. shuffled for proficient . The LDA analysis revealed that the accuracy for decoding the odorant identity from ΔF/F during the odorant period increases as the animal learns to differentiate between odorants.We utilized a linear discriminant analysis (LDA) to determine whether a hyperplane placed in the multidimensional space of Δp < 0.001, 72 observations, 54 d.f., n = 4 sessions, 4 mice, GLM F-statistic = 23.6, p < 0.001). Thus, MLI activity encodes for the stimulus even when a single ROI is analyzed indicating that stimulus information encoded by MLI activity is highly redundant.Since dimensionality of MLI neural activity is low  with an increase in ΔF/F before reversal , maintained the odorant increase in ΔF/F to what was then the unrewarded odorant  immediately following reversal  and switched responses with increases to the new rewarded stimulus  when they became proficient in the reversal task   when the valence was reversed . Thus, after successful reversal the ΔF/F time course switched for the two odorants: the stimulus-induced increase in ΔF/F took place for the reinforced odorant, not for the chemical identity of the odorant.In order to determine whether the MLIs responded to the chemical identity of the odorant, as opposed to responding to the valence , we reversed odorant reinforcement. When the reward was reversed for a proficient mouse the animal kept licking for the previous rewarded odorant resulting in a fall in percent correct below 50%, and as the animal learned the new valence the percent correct raised back above 80% Fig. . We analsed Fig. . GLM anaP < 0.001), but not between forward and reverse . These results indicated that for the proficient mouse it is possible to decode contextual identity, suggesting that MLI activity encodes for valence.Next we computed the accuracy for decoding the reinforced odorant for trials when the animal was proficient in either the forward or reverse trials using LDA analysis. Figure To gain a better understanding of the information on odorant valence present in the responses of the MLI ensemble we asked whether stimulus decoding accuracy calculated with LDA for proficient mice differed between correct (Hits and CRs) and incorrect trials (Miss and FAs). If information in MLI activity reflects the outcome of the trial, we would expect that decoding accuracy would be lower for incorrect trials. On the other hand, if information encoded by MLI activity reflects the stimulus regardless of trial outcome decoding accuracy would not differ between correct and incorrect trials.F/F during the odorant application regardless whether the trial was a correct response (Hit or CR) or an error  . In contrast, ΔF/F did not differ between Hits vs. Miss and CR vs. FA , while Hits/Miss differ from CR/FA . To survey the information encoded in MLI activity in trials with different outcomes in proficient mice, we utilized LDA analysis to decode the stimulus . In addition, GLM analysis did not find a significant difference between outcomes  or time period (odorant vs. reinforcement) indicating that MLI activity reflects the stimulus regardless of trial outcome . This analysis determined that odorant-induced MLI Ca2+ changes carry information on the stimulus, as opposed to the outcome.We performed this analysis for sessions that included at least one error trial (Miss or FA) when the animals were proficient. In these time series the majority of the ROIs exhibited changes in ΔA) Table . In addi35, we examined the correlation between ΔF/F and the LR. When the animal was proficient the animal licked at least once in each of the two lick segments during application of the S+ odorant, increased licking after receiving the water reward and refrained from licking for the S− trials  and the derivative of the lick rate (DtLR)34 and plotted their relationship during different time periods for proficient mice. Figure F/F and the LR. Examples of these correlations for a single session for a proficient mouse are shown in Fig. F/F and the LR was larger during the reinforcement period  compared to the odorant  and pre-odorant  periods , and was smaller for both the pre-odorant  and odorant  periods  and between the reinforced and odorant periods for DtΔF/F vs. DtLR . These data suggested that changes in ΔF/F during the reinforcement period reflect changes in lick activity, while changes in ΔF/F during the odorant period are less dependent on licks, and maybe dependent on multiple variables.In order to explore the relationship of MLI activity to licking we proceeded to examine the correlation between Δods Fig. . Similarods Fig. . We proc LR Fig.  and DtΔFtLR Fig.  were postLR Fig. . A two-sF/F in CR trials when the mouse did not lick and for time courses aligned to the beginning of ΔF/F changes after odorant addition. We compared ΔF/F and lick frequency during the odorant application period for CR trials when the animal did not lick during the two 2 s odorant response periods vs. CR trials when the animal licked .We performed complementary studies of ΔF/F and lick frequency in the time period shortly after odorant application when ΔF/F increases for both S+ and S−, before ΔF/F decreases for S− (and keeps increasing for S+). In Crus II, where neural activity of MLIs is thought to reflect licks, ΔF/F increases whenever there is an increase in licking frequency35. We aligned the traces to the point where the time derivative for ΔF/F increased above 0.03. We found that ΔF/F increased for both S+ and S− . In contrast, in this time period there was no increase in lick frequency . The data on the relationship between lick frequency and ΔF/F indicate that although there is a dependence between these two variables, the dependence is not consistent with a direct relationship between ΔF/F and lick frequency, as found in Crus II.Furthermore, we analyzed the relationship between the time course of ΔF/F time course and the LR . In contrast, a GLM analysis of the changes in ΔF/F as a function of volume delivered in dry and wet lick conditions did not yield statistically significant changes . Finally, if MLI activity reflected reinforcement value ΔF/F should show a positive correlation with volume of sugar water delivered . We did not find a significant correlation between lick frequency and ΔF/F for either dry  or wet licking . This experiment indicated that MLI activity does not reflect reward value, and is consistent with MLI activity reflecting valence.The correlation between Δ LR Fig.  raises tF/F on the different behavioral and stimulus variables37. We included event variables, whole trial variables, and continuous variables , we found that GLM explained a substantial percent of the average ΔF/F variance ranging from 23.5 to 95% . Furthermore, variables describing the licks contributed to the GLM fit during the outcome period . In contrast, the other two variables (body kinematics and reinforcement history) did not differ in contribution during the different periods . These data indicate that odorant identity contributes to modeling MLI activity during the odorant application period while licks contribute to MLI activity during the reinforcement period.Black traces in Fig. 95% Fig. . We next38. To control for off-target effects of clozapine-N-oxide (CNO)39, we injected PV-Cre mice with Cre-dependent mCherry AAV virus in another group of six mice (control group)  40 min before the start of the session (control-CNO and hM4Di-CNO).In order to determine whether activity of MLIs plays a role in behavioral responses in the go–no go task we used a Cre-dependent AAV virus to express the inhibitory DREADDs receptor hM4Di in MLIs in six PV-Cre mice (hM4Di group)p < 0.01, 24 observations, 20 d.f., n = 6 mice, GLM F-statistic = 13.7, p < 0.001) and post-hoc tests indicated that the hM4Di expressing group differs between CNO and saline , while there are no significant differences between CNO and saline for control mice , indicating an effect of CNO-induced inhibition of MLIs expressing hM4Di on behavioral output and the absence of off-target CNO effects.Mice in all groups with the exception of the hM4Di-CNO attained proficiency (≥80% percent correct) Fig. . GLM anap > 0.05, 60 observations, 56 d.f., n = 6 mice, GLM F-statistic = 2.96, p > 0.05). Yet GLM found a statistically significant difference for S− for the interaction between CNO and hM4Di expression . We obtained a similar impairment of performance in a separate set of experiments with two hM4Di mice and two controls where animals discriminated between 1% Iso and MO, and CNO was applied when the animal was naive, and we reversed the reward . Simulation of S+ trials showed that odorant stimulus though PF inputs increases the activity of SCs that inhibit PC firing eliciting an increase in the LR   and CNO (p < 0.05) and for the interactions between S+ vs. S− and CNO .To model the effect of inhibitory chemogenetics Fig. , we consThe model is simple. For example, it does not include basket cells. Furthermore, we did not model the large increase we find in the LR when the mouse receives the sugar water reward. In addition, we did not perform an exhaustive study of how the variables affect the changes in lick strength. Therefore, other explanations should be explored in future studies with alternate computational models and an exhaustive search of the input variables. Regardless, our model provides plausible mechanisms for the results that can be tested in future experiments with slice electrophysiology and awake behaving recording.We found that vermal MLIs developed a differential response to odorants in the go–no go task that switched when the valence was reversed. Decoding analysis revealed that when the animal was proficient the contextual identity of the odorant could be decoded from MLI responses. GLM analysis revealed that contextual identity made a large contribution to the fit of MLI activity during the odorant application period. Chemogenetic inhibition of MLIs impaired achievement of proficient discrimination of odorants. These data indicate that MLIs play a role in associative learning by encoding valence.43. CFs carrying error signals make profuse synaptic connections on the dendrites of PCs and elicit powerful excitatory dendritic Ca2+ spikelets47. Furthermore, CFs also signal reward prediction12 or decision-making errors13, and the cerebellum modulates association pathways in VTA enabling a cerebellar contribution to reward-based learning and social behavior14. The increase in Ca2+ mediates LTD in subsets of synapses innervated by co-activated GC PFs carrying sensorimotor information relevant to learning49. However, recent studies by Rowan et al.25 revealed that increasing feedforward inhibition by MLIs can switch the valence of plasticity from LTD to LTP . In addition, adaptive changes in the vestibulo-ocular reflex elicited by CF optogenetic activation switched from increase to decrease depending on whether MLIs were co-activated25. Finally, MLIs gate supralinear CF-evoked Ca2+ signaling in the PC dendrite51. These studies suggest that the valence of learning is graded by MLI activity.The cerebellum has been implicated in mediating supervised learning through an iterative process whereby the response to an input is evaluated against a desired outcome, and errors are used to adjust adaptive elements within the systemF/F responses and stimulus decoding for correct and incorrect behavioral response trials  mice and wild-type C57BL/6J mice. The animals were housed in a vivarium with a 14/10 h light/dark cycle. Food was available ad libitum. Access to water was restricted in for the behavioral training sessions according to approved protocols, all mice were weighed daily and received sufficient water during behavioral training to maintain ≥80% of original body weight. Animal are housed at 72 ± 2 °F and a humidity of 40 ± 10%.To perform immunostaining, mice were sacrificed and transcardially perfused with ice cold 4% paraformaldehyde , followed by incubation in 30% sucrose . After the brain was incubated in the sucrose solution, 60-μm-thick slices were cut with a cryostat. The slices were imaged using a confocal laser scanning microscope  to determine the GCaMP expression patterns in the cerebellum. The slices were counterstained with DAPI .−1). A craniotomy was made over the vermis of cerebellum centered at midline 6.8 mm posterior to Bregma leaving the dura intact (lobule VI). A square glass window (2 mm × 2 mm) of No. 1 cover glass  was placed over the craniotomy and the edges were sealed with cyanoacrylate glue . The window was further secured with Metabond , and a custom-made steel head bracket was glued to the skull.Adult mice (8 weeks or older) were first exposed to isoflurane (2.5%) and then maintained anesthetized by intraperitoneal ketamine–xylazine injection  . The viral infection method we use has been reported to express GCaMP in MLIs, and not in PCs65. Supplementary Fig. 2+ indicator65. In one C57BL/6 animal each, we used AAV5-Syn-GCaMP6s or AAVrg-Syn-jGCaMP7f  with similar results to those obtained with GCaMP6f . The floor in an olfactometer that controlled valves to deliver a 1:40 dilution of odorant at a rate of 2 L min−1. The LR was calculated from the lick records and the time course was convolved with a 2 s Gaussian for the experiments where we performed multiphoton calcium imaging. We did not convolve the LR records for the experiments with chemogenetics. The water-deprived mice started the trial by licking on the water port. The odorant was delivered after a random time interval ranging from 1 to 1.5 s. In S+ trials, the mice needed to lick at least once in two 2 s lick segments to obtain a reward (0.1 g ml−1 sucrose water) er) Fig. . The int67 . Measurement of body movement with a single camera gives limited information. Five mice were used for the go–no go experiments. Four mice were imaged when they were naive. The window became opaque for two of the mice preventing MLI imaging for the reversal experiment.Movement of the mouse was imaged in the infrared to prevent light interference with the non-descanned detection in the visible using a 1 Megapixel NIR security camera  at 30 frames/s. Velocity of body movement was estimated using the Farneback algorithm coded in MatlabFor the experiment in Supplementary Fig. 39, 1.2 μl of AAV8-hSyn-DIO-hM4D(Gi)-mCherry virus  was bilaterally injected into six PV-Cre animals at ±0.5 mm lateral to midline, 6.8 mm posterior to Bregma and 200–400 μm below the brain surface. For control, an AAV8-hSyn-DIO-mCherry virus  was injected in the same position in 6 PV-Cre animals. This viral infection method results in expression of the protein in MLIs, and not in PCs65. We performed two separate experiments: (1) For the experiments in Fig. −1 CNO . (2) For the experiment in Supplementary Fig. −1 CNO  40 min before starting the session and were trained to discriminate between 1% Iso (S+) and MO (S−) for 6 sessions for a total of 500–600 trials. The reinforcement was then reversed and the animals were again injected with CNO 40 min before the sessions and were trained to discriminate between MO (S+) and 1% Iso (S−) for 5 sessions for a total of 400–500 trials.For chemogenetic inhibition of MLI activity30. The head-fixed two-photon imaging system consisted of a movable objective microscope  paired with a 80 MHz, ~100 fs laser  centered at 920 nm. The MOM was fitted with a single photon epifluorescence eGFP filter path (475 nm excitation/500–550 nm emission) used for initial field targeting followed by switching to the two-photon laser scanning path for imaging GCaMP at the depth of the MLIs. The galvometric laser scanning system was driven by SlideBook 6.0 . The two-photon time lapses were acquired at 256 × 256 pixels using a 1.0 NA/20x water emersion objective  at 5.3 Hz. On the day of initial imaging, a FOV was selected to image a large number of active cerebellar neurons located in the most superficial planes of the molecular layer  including mostly SCs, and several batches of 6000 frames (a time series) were collected in each training session. After two-photon imaging a second image of the vasculature was captured with wide field epifluorescence to reconfirm the field.All the animals were first habituated to the setup to minimize stress during the imaging experiments. All the imaging sessions started at least 10 min after mice had been head fixed. We searched for active MLIs while imaging zones in the vermis of lobule VI, between the midline and the paravermal vein, an area of the cerebellum where GCs acquire a predictive feedback signal or expectation reward2+-independent68. Supplementary Fig. 31. CaImAn identifies different spatial components (addressed here as ROIs) and a component representing the background and neuropil signals. Baseline of intensity (F0) was defined as the mean fluorescence intensity before trial start, defined as the time when the animal first licked. This was when fluorescence started to increase above baseline and the odorant was added at a random time 1–1.5 s after trial start. Intensity traces (F) were normalized according to the formula ΔF/F  = (F − F0)/F0. After CaImAn analysis, the ΔF/F traces of the spatial components were sorted and we assigned trial traces to different behavioral events  and aligned them to trial start, odorant onset or water delivery. Finally, the time course for the average ΔF/F did not differ greatly between the different GCaMP variants as would be expected for a fast firing interneuron with small increases in Ca2+ per action potential  to exclude image sequences exhibiting axial movement. We did not find evidence of axial movement while the animal was engaged in the go–no go task. In addition, we performed control imaging where we excited GCaMP6f at 820 nm, a two-photon excitation wavelength where fluorescence emission is Ca33. The post-hoc comparisons between pairs of data were performed either with a two-sided t test, or a ranksum test, depending on the result of an Anderson–Darling test of normality. 95% CIs shown in the figures as vertical black lines or shading bounding the lines were estimated by bootstrap analysis of the mean by sampling with replacement 1000 times using the bootci function in MATLAB.Statistical analysis was performed in Matlab 9.6 . Statistical significance for changes in measured parameters for factors such as learning and odorant identity (S+ vs. S−) was estimated using a GLM, with post-hoc tests for all data pairs corrected for multiple comparisons using FDRF/F measured from all components in the FOV was accomplished via LDA in Matlab. ΔF/F for all components for every trial except one were used to train the LDA, and the missing trial was classified by its fit into the pre-existing dataset. This was repeated for all trials and was performed separately for analysis where the identity of the odorants was shuffled. Fluorescence intensity traces, LRs, and kinematics in Fig. PCA was calculated using the Matlab Statistics Toolbox. Classification of trials using Δ69, we defined the dimension of the system (dim) with M inputs as the square of the sum of the eigenvalues of the covariance matrix of the measured ΔF/F for all ROIs in the FOV divided by the sum of each eigenvalue squared:i are the eigenvalues of the covariance matrix of ΔF/F computed over the distribution of ΔF/F signals measured in the FOV. If the components of ΔF/F are independent and have the same variance, all the eigenvalues are equal and dim(ΔF/F) = M. Conversely, if the ΔF/F components are correlated so that the data points are distributed equally in each dimension of an m-dimensional subspace of the full M-dimensional space, only m eigenvalues will be nonzero and dim(ΔF/F) = m.Following Litwin-Kumar et al.37. We used the Matlab fitglm function to fit the per trial ΔF/F time course for mice proficient in the go–no go task with a GLM. We included event variables, whole trial variables and continuous variables. Continuous variables quantified kinematics including the LR, the derivative of the LR and the velocity and acceleration of movements made by base of the tail of the head-fixed animal during the trial .For stellate cell simulation we used reconstructed mouse SC morphology available in Neuromorpho  using NLMorphologyViewer 0.3.0 (http://www.neuronland.org). We proceeded to create the electrical compartmental model with passive and active properties of the SC membrane. The passive parameters of the SC model were adapted mainly from Molineux et al.71. We set the specific membrane resistivity Rm = 20 kΩ cm2, the specific membrane capacitance Cm = 1.5 μF cm−2 71, and the intracellular resistivity Ri = 115 Ω cm72. The input resistance Rin = 571.39 MΩ and membrane time constant τm = 40.30 ms were obtained injecting a hyperpolarizing current into the soma . The time constant was obtained by a double exponential fit of membrane voltage decay. Those values are within the range of experimental values measured in SCs73.We removed the axons from the original swc morphology file and exported the reconstructed morphology into a NEURON 7.5 hoc file , delayed rectifier potassium currents (KDR), A-type potassium currents (KA) and transient calcium currents (CaT)75 .All simulations were performed on the NEURON 7.5 simulatorFurther information on research design is available in the Supplementary InformationPeer Review FileReporting SummaryDescription of Additional Supplementary FilesSupplementary Movie 1Supplementary Movie 2


Forced degradation study is a systemic characterization of degradation products of active pharmaceutical ingredient (API) at conditions which posses more harsh environment that accelerates degradation of API. Forced degradation and stability studies would be useful in selection of proper, packaging material and storage conditions of the API. These are also useful to demonstrate degradation pathways and degradation products of the API and further characterisation of the degradation products using mass spectrometry. TGR5 is a G protein-coupled receptor, activation of which promotes secretion of glucagon-like peptide-1 (GLP-1) and modulates insulin secretion. The potent and orally bioavailable TGR5 agonist, ZY12201, shows activation of TGR5 which increase secretion of GLP-1 and help in lowering blood glucose level in animal models. Hence it is necessary to establish and study degradation pathway and stability of API for better handling and regulatory approval. Force degradation studies of ZY12201 have shown presence of one oxidative impurity during oxidative degradation in HPLC analysis. The oxidized product is further characterized by LC–MS to elucidate structure of impurity and characterize its degradation pathway.The online version contains supplementary material available at 10.1007/s42452-021-04660-y. Type 2 diabetes mellitus (T2DM) is a metabolic disorder sparked by insulin resistance and dysfunction of the β cells. Type-2 diabetes is generally characterized by increase in the resistant to insulin which leads to higher blood glucose level . Impaire50 of 57 pM and mTGR EC50 of 62 pM with a favorable pharmacokinetic properties and demonstrated in-vivo glucose lowering effects in animal models (ED50 of 7.9 mg/kg and ED90 of 29.2 mg/kg) [Bile acids plays a significance role in the emulsification lipids in the body and absorption of vitamins A, D, E, K . Along w2 mg/kg) . This woAbdelhameed et al. –20 have Standards and samples of ZY12201 were synthesized at Zydus Research Center, Cadila Healthcare Ltd.  , 11. HPLSee Table Test solutions of ZY12201 with 1000 µg/mL concentration were prepared in diluents and further sonicated for 5 min to dissolve the compound, which were further analyzed by HPLC.A chromatography was performed on alliance waters separation module e2695 with YMC Triart C18 150 mm, 4.6 mm and 3 µm particle size column using mobile phase includes, Solvent A 10 mM ammonium acetate buffer pH (8.5 ± 0.05) and solvent B was mixture of 0.1% ammonia in acetonitrile with pump flow rate of 1.2 mL/min. The HPLC gradient mode ratios have been mentioned in Table An impurity obtained in the oxidation was further characterized and identified by LC–MS system. An electrospray LC–MS system  was used for the identification of degradation impurities formed during stress testing studies. Chromatography was performed on YMC Triart C18 150 mm, 4.6 mm and 3 µm particle size column from YMC co. Ltd. Japan using mobile phase consisting of mobile phase A (10 mM ammonium acetate (pH 8.5) with ammonia solution) and mobile phase B (0.1% ammonia in acetonitrile) at a flow rate of 1.2 mL/min. The LC gradient program was applied as per Table Forced degradation studies have been proved as an useful tool in analyzing stability of pharmaceutical products in different environmental conditions. Stability data and force degradation studies are very crucial for necessary regulatory aprovals . Even ICChromatogram of the control sample for the reference in the degradation study has been given in Figure S-1.Test sample of 50 mg weighed in 50 mL volumetric flask, added 2–3 mL of diluent to dissolve followed by 1 mL of 5.0 M hydrochloric acid solution and heated at 60 °C for 120 min. Cool it at room temperature and neutralize the solution with 5.0 M sodium hydroxide solution with help of pH paper. The chromatogram obtained from the sample after acidic hydrolysis reaction of ZY12201 has shown a satisfactory separation of compound and the degradation products .Test sample of 50 mg weighed in 50 mL volumetric flask, add 2–3 mL of diluent to dissolve then add 1 mL of 5.0 M sodium hydroxide solution and heated at 60 °C for 120 min. Cool it at room temperature and neutralize the solution with 5.0 M hydrochloric acid solution with help of pH paper. The chromatogram for the alkali treated ZY12201 sample was achieved satisfactory .Hydrogen peroxide has been used for the oxidative stress degradation. Electron transfer serves as basic mechanism for the oxidative forced degradation of drug substance . ZY122012 light; 300–800 nm wavelengths are commonly used to cause the photolytic degradation [ICH guidelines have recommended the conditions protocols for photo stability studies . Accordiradation . Free raradation . ZY12201Sample was exposed at 105 °C for 24 h to study the thermal decomposition or thermolysis caused by heat . HPLC chForce degradation studies have concluded that there was no significant changes have been found during the degradation study except oxidative degradation. Summary of force degradation study has been shown in Table Peak purity is a comparison of the reference standard to the API in the sample stressed by forced degradation to confirm that no impurity is eluted. Peak threshold is used as a parameter for determining peak purity in HPLC. For the acceptance of the peak purity, angle should be less than a purity threshold. Peak purity results are summarized in Table Identification of degradation related impurities for ZY12201 was done in oxidation treated sample through LC–MS technique. Total eight impurities were formed during oxidation degradation, among all impurity in ZY12201 oxidation treated sample, one impurity was confirmed and identified through mass spectral analysis. The degraded impurities mass and retention time has been reported in Table +) suggesting the possibility of Molecular formula C31H31FN4O4S, which confirms the theoretical molecular weight of Impurity-1.The positive ion mass spectral analysis of impurity-1 was observed at 575 Below is the link to the electronic supplementary material.


The research results show that greater repeatability of the technique (lower RE) has a significant impact on the length of the shot put.The purpose of the study was to analyze the variability of performance and kinematics of different shot put techniques in elite athletes  and sub-elite athletes . Each athlete performed 6 trials. Only 34 trials in group A and 27 trials in group B were analyzed. Two high-speed digital cameras were positioned 8 m from the center of the shot put throwing circle. All throws performed during international and national competitions were analyzed using the Ariel Performance Analysis System. To estimate variability of kinematic parameters, the value of relative error was calculated. The average relative error generally showed low variability for the analyzed indicators. In only 4 analyzed cases, variability was high (>20%). Statistical analysis was used to find indicators which have a significant influence on the distance of the throw (according to the sports level and technique). Significant inverse correlations (at  Several throwing techniques of the shot put  have been trialed over the years. The gliding technique was introduced in the 1950s and was prevalent for many years. The gliding technique involves a linear push out of the back to the front of the circle while facing away from the sector. Then, shot putters rotate their body toward the throwing section, putting the shot for maximum distance. The rotational style appeared in the 1970s and is the most popular nowadays . The iniThe use of 3D video motion analysis helped athletes and their coaches to analyze the technique they use  and to find ways to increase efficiency and improve performance.Kinematics of motion is not the same, even during repeated execution of the same movement. Natural variations in the position, velocity and acceleration of the body limbs take place even during apparently identical movements and influence the variability of the throw kinematics . Consequ2) were slightly taller (3 cm on average), had greater weight and BMI (7.2 kg and 0.6 kg/m2) and were a little older (1.3 years) than shot putters who used the rotational technique in the same group . A similar dependence (with slightly greater differences between the two techniques) was observed in athletes in group B. Athletes who used the glide technique , were 3.7 years older, 3 cm taller, 12.7 kg heavier and had BMI about 2.4 kg/m2 greater than athletes who used the rotational technique . There were no significant differences (p < 0.05) for the height of the athletes’ center of gravity depending on the group and technique  = 0.7484, p = 0.416).Fourteen elite and sub-elite shot putters (right-handed) took part in this analysis. Eight elite participants, including 5 athletes using the rotational technique and 3 athletes using the glide technique, performed trials during an international competition (group A), whereas six sub-elite participants  performed trials during national championships (group B). In the year of the analyzed competition, all athletes from group A belonged to the group of thirty world-leading shot putters (places from 18th to 37th), with the best results in the range between 20.38 m and 20.64 m. National ranking of 100 best results of the year in the shot put included performances of 6 competitors from group B. The best results of the athletes taking part in the analyzed national competition (group B) ranged from 20.32 m to 18.77 m . The shot putters from group A who used the glide technique  were analyzed. In group A, 5 athletes who used the rotational technique performed 21 measured trials, while 3 athletes who used the glide technique performed 13 measured trials. In group B, 3 athletes who used the rotational technique performed 13 measured trials, while 3 athletes who used the glide technique performed 14 measured trials. All participants gave their informed consent and were informed of the benefits and risks of the investigation prior to signing an institutionally approved informed consent document to take part in the study. The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the Ethics Committee of Józef Piłsudski University of Physical Education in Warsaw (SKE 01–02/2013).Two high-speed digital cameras  were positioned perpendicular to each other and 8 m from the center of the shot put throwing circle.All throws performed during international and national competitions were recorded and then analyzed using the Ariel Performance Analysis System (APAS). Eighteen points were digitized. Sixteen points were placed on the athlete’s body, including the big toe, ankle, knee, hip, wrist, elbow, and shoulder for the left and right side of the body as well as the right hand, the chin and the top of the head. The 17th point was placed in the center of the shot, and the last (18th) was placed on the right-side edge of the toe-board of the throwing circle. The analyzed area of the throwing circle was calibrated with a 1.5 m × 2 m × 1.5 m reference scaling frame. The calibration was performed before and after the competition session. Synchronized data sequences from all camera views were utilized.The kinematic parameters of the athlete and the shot during release (RLS–the last contact of the athlete) presented in To estimate variability of kinematic parameters, the value of relative error (RE) was calculated.Two-factor analysis of variance (ANOVA) was used to analyze significant differences depending on the two factors: the GROUP (sports level) and/or the TECHNIQUE .http://www.statsoft.com (accessed on 1 December 2019)) was used for statistical analysis. The significance level α = 0.05 was used to assess the significance of differences and correlations.Correlations between selected indicators were determined using Pearson’s correlation coefficient. All statistical analyses were based on RE of selected indicators. The use of RE made it possible to perform a statistical analysis of all shot-put measured trials. The values of the distance of the throws were used in the Pearson’s correlation analysis with RE of the selected indicators to find the most important correlations between them. Statistica 10.0  STATISTICA , Version 10. The angle indicators  like β_pThe greatest differences were found for the S-H as the effect of GROUP and TECHNIQUE. Other indicators of the athlete and the shot had low RE value .p ≤ 0.0001), λ_r (F(1.57) = 14.890, p ≤ 0.001) φ_r (F(1.57) = 16.306, p = 0.002), S-H (F(1.57) = 9.545, p = 0.003), γ (F(1.57) = 9.552, p = 0.003), VxCG (F(1.57) = 7.547, p = 0.008), VxS, VyS, VrS (F(1.57) = 10.657, p = 0.002, F(1.57) = 16.897, p ≤ 0.001, F(1.57) = 28.846, p ≤ 0.001, respectively) and also for h (F(1.57) = 13.0, p ≤ 0.001). Statistically significant influence of the TECHNIQUE was observed for mean RE of the 3 indicators: φ_r (F(1.57) = 5.461, p = 0.002), S-H (F(1.57) = 10.462, p = 0.002), γ (F(1.57) = 6.950, p = 0.011) and for RE of VxS (F(1.57) = 6.052, p = 0.017). Both GROUP and the TECHNIQUE factors significantly influenced mean RE of φ_r (F(1.57) = 18.377, p ≤ 0.001), δ_r (F(1.57) = 4.574, p = 0.037), VyS (F(1.57) = 4.766, p = 0.332), γ (F(1.57) = 4.887, p = 0.031), and D (F(1.57) = 14.417, p ≤ 0.001).Statistically significant influence of the GROUP factor  was noted for mean RE: δ_r (F(1.57) = 35.432, The highest level of RE (about 20%) was found for the horizontal velocity of CG in both groups and techniques and for the vertical velocity of CG except for the rotational technique in group A . Levels p = 0.006) and z_S (F(1.57) = 6.042, p = 0.017). In the case of the technique (without the division into sports levels), significant differences were found for RE of x_CG, y_CG, z_CG and R_S (F(1.57) = 5.795, p = 0.019, F(1.57) = 11.670, p = 0.001, F(1.57) = 17.312, p ≤ 0.001, F(1.57) = 9.464, p = 0.003, respectively) and also for RE of z_S (F(1.57) = 16.414, p ≤ 0.001). Both the GROUP and the TECHNIQUE factors significantly influenced RE level of x_CG (F(1.57) = 4.982, p = 0.030), y_CG ((F(1.57) = 4.982, p = 0.030), and R_S, (F(1.57) = 8.205, p = 0.006).Depending on the GROUP factor  only, statistically significant differences were noted for RE level of R_S (F(1.57) = 8.107, All the statistically significant correlations between the distance of the throw and RE of selected release indicators had inverse correlations, which meant that the lower the variability of the indicator, the longer the distance was . The invThe most important observation of the study was the identification of variability in performance and kinematics for elite and sub-elite shot putters. This study fills gaps that have not been previously addressed in research on the shot put. Release indicators have been frequently examined in the competition like the shot put ,5. AccorIn general, in our research, low RE characterized RLS indicators and the indicators of the path of athletes’ center of gravity and the center of the shot. Moreover, lower RE was found in the trials of higher-level athletes (group A). The greatest differences between groups A and B were found in the rotational technique for S-H, Vy_CG and Vx_CG. The greatest differences in the glide technique were also observed for S-H, Vy_CG and Vy_S. It was only in the case of Vx_CG that general average RE  in group A was found to be greater than in group B. The average RE for the 8 finalists from our study group A–international level  compared to group in the study of Shaa , also wip < 0.05) linear dependence was found between the longest trials and the relative errors of release indicators for the longest distance of each athlete. It was observed that while the distance was increasing, the value of RE was decreasing linearly for 4 release indicators  in the glide technique in group A, for 2 indicators  in the glide technique in group B, and for 3 indicators  in the rotational technique in group B. The lowest average values of the relative error for the longest trials were noted in 16 cases in the glide technique in group A, in 6 cases in the glide technique in group B, in 5 cases in the rotational technique in group A and in 7 cases in the rotational technique in group B. The longest trial in the rotational technique, without the division into sports level and in relation to the other distances, had the lowest average value of the relative error for 3 indicators . In the glide technique, without the division into sports level, the longest trial had the lowest average value of the relative error for 7 cases . The results of this study expanded the findings of previous optimization modeling attempts for elite men’s shot put [Taking into account all the analyzed athletes, without the division into sports levels and techniques used, significant  had the lowest mean values of RE, especially in the elite group. A significant influence of the resultant shot velocity on the distance was confirmed. Also, the right knee joint angle and the left hip joint angle had a significant effect on the distance of the throw. The greatest differences between the elite and sub-elite group were found for S-H and Vy_CG. Further research on the topic is necessary to better understand all of the relationships both between these variables and with performance. The observations of this study provide useful information for the technical development of male throwers and may provide an insight into new training methods that should mainly be focused on the power development associated with the mode of the shot put technique. The research results show that greater repeatability of the technique (lower RE) has a significant impact on the length of the shot put. Along with the decrease in RE, the distance of the shot put increased. The relationship between the sports level and greater repeatability confirms the importance of the technique automatism and stabilization of the technique in sport.


Cancer can cause physical changes and affect satisfaction with a persons’ physical appearance, which in turn can affect overall quality of life. Previous studies have primarily focused on women with breast cancer and few is known about body image in patients with other cancers and especially men. The present study compares satisfaction with body image of patients with different types of cancer with the general population and across sexes and identifies risk factors for diminished body image. Additionally, patients that were diagnosed within the last year and those living with cancer for longer are compared.N = 531 cancer patients answered the German Self-Image Scale to assess body image. One sample t-tests are utilized to compare the body image of cancer patients with the general population. Stepwise regression analyses were used to identify factors associated with body image and ANOVAs with posthoc tests as well as t-tests were used to examine group differences.In this cross-sectional study, Cancer patients showed diminished body image compared to the general population. For men, higher relationship satisfaction and lower cancer-specific distress were associated with more positive body self-acceptance (SA), whereas younger age, higher relationship satisfaction, and lower cancer-specific distress resulted in better perceived partner-acceptance of one’s body (PA). In women, higher education, lower anxiety and cancer-specific distress were associated with more positive SA. Female cancer patients with breast/gynecological cancer reported better SA than those with visceral cancers. Higher relationship satisfaction and lower cancer-specific distress were found to be associated with more satisfactory PA in females. Time since diagnosis did not affect body image in this study.Results indicate that cancer patients regardless of sex tend to have decreased body image satisfaction. Future research directions include examination of additional entities of cancer, deeper research in men and the role of time since diagnosis. With approximately 19.3 million new cancer diagnoses and an estimated 9.9 million deaths from cancer worldwide in 2020, cancer is currently one of the leading global health concerns , sleep dMost of the body image-related research in the field of psycho-oncology focuses on the consequences of irreversible body alterations through surgical treatment (e.g. scars and amputations). Thus, researchers have assessed breast cancer surgery and its side effects , the impMost studies that investigate body image in cancer patients and survivors are exclusively conducted with female participants and focus nearly entirely on the most common cancer entities like breast cancer . StudiesAnother area that has hardly been explored so far in psycho-oncological body image research is intimate relationships. Nonetheless, close relationships have been shown to be important sources of coping with cancer  and spoufirst goal was to add to the currently still ambivalent literature on body image comparisons between cancer patients and the general population. Thus, the German version of the Self Image Scale (SIS-D) was utilized as a measure for body image satisfaction as it provides normative scale scores for the German general population )  α = .94  and Cronthe problem does not apply to me) to 5 (the problem applies to me and is a very big problem). The FBK-R23’s α for the total score was .92 in the present study which is equally good as in the original study . Total scores range from 0 to 115 and the cut-off for clinical distress related to cancer is 34.Cancer-specific concerns and distress were assessed with the German version of the Questionnaire on Stress in Cancer Patients (FBK-R23 ). The FBnever) to 5 (very often). All item scores are added up to a sum score with higher values indicating higher levels of fear of progression (range: 5–60). A cut-off of 34 indicates a dysfunctional fear of progression. The PA-F-KF had an α of .91 in the present study and an α of .87 in the validation study ),The present study adds to the existing literature on body image in cancer patients and suggests that satisfaction with one’s body in cancer patients is generally impaired in a statistically significant way. This indicates that the consideration of the satisfaction with the physical appearance should be an important part of psycho-oncological care regardless of patients’ sex or type of cancer. Possible body image difficulties then need to be addressed by psycho-oncological professionals. Questionnaires such as the SIS could ideally be used to identify body image problems. However, if this is not possible, body image problems should also be asked about in the medical or psycho-oncological interview. As this often also impacts sexual satisfaction, this is an important area to consider in clinical care. Since sexuality is often not addressed, or addressed inadequately, body image issues might also not be raised. It is likely that many patients may also have inhibitions about addressing this topic on their own. Therefore, the recommendation is that the initiative should come from the professional staff. Besides individually tailored interventions on the clinician-patient level, structured and evaluated interventions for individuals as well as groups exist, especially for breast cancer survivors e.g. , 82)..82]).The results of the present study suggest that cancer and cancer treatment can potentially lead to a decreased sense of SA and PA not only in women with breast cancer but also in cancer patients across the sexes and with a variety of different cancers entities. Future research is necessary to further examine group differences and predictors of satisfaction with one’s body with longitudinal studies in patients with different kinds of cancers and to investigate the effect of time since diagnosis more deeply. Additionally, research in larger samples that allow differentiation between specific cancer diagnoses and in populations with other chronic medical conditions like transplantation, heart disease, diabetes, and multiple sclerosis should be conducted in the future.S1 Dataset(SAV)Click here for additional data file.


Background: In renal transplantation, chronic transplant dysfunction (CTD) is associated with increased PCSK9 and dyslipidemia. PCSK9 is an enzyme that increases plasma cholesterol levels by downregulating LDLR expression. We recently showed increased PCSK9–syndecan-1 interaction in conditions of proteinuria and renal function loss. Treatment with heparin(oids) might be a therapeutic option to improve dyslipidemia and CTD. We investigated the effects of (non-)anticoagulant heparin(oids) on serum lipids, syndecan-1 and PCSK9 levels, and CTD development.Methods: Kidney allotransplantation was performed from female Dark Agouti to male Wistar Furth recipients. Transplanted rats received daily subcutaneous injections of saline, unfractionated heparin, and RO-heparin or NAc-heparin (2 mg heparin(oid)/kg BW) until sacrifice after 9 weeks of treatment.Results: Saline-treated recipients developed hypertension, proteinuria, and loss of creatinine clearance , along with glomerulosclerosis and arterial neo-intima formation. Saline-treated recipients showed significant increase in plasma triglycerides (p < 0.05), borderline increase in non-HDLc/HDLc (p = 0.051), and ∼10-fold increase in serum syndecan-1 (p < 0.05), without significant increase in serum PCSK9 at 8 weeks compared to baseline. Heparin and non-anticoagulant RO-heparin administration in transplanted rats completely prevented an increase in triglycerides compared to saline-treated recipients at 8 weeks (both p < 0.05). Heparin(oids) treatment did not influence serum total cholesterol (TC), plasma syndecan-1 and PCSK9 levels, creatinine clearance, proteinuria, glomerulosclerosis, and arterial neo-intima formation, 8 weeks after transplantation. Combining all groups, increased syndecan-1 shedding was associated with TC  and glomerulosclerosis , whereas the non-HDLc/HDLc ratio was associated with the neo-intimal score in the transplanted kidneys .Conclusion: Prevention of triglyceridemia by (non-)anticoagulant heparin(oids) neither influenced PCSK9/syndecan-1 nor precluded CTD, which however did associate with the shedding of lipoprotein clearance receptor syndecan-1 and the unfavorable cholesterol profile. Chronic transplant dysfunction (CTD) is a functional decline of the transplanted kidney characterized by a progressive increase in plasma creatinine levels, proteinuria, and hypertension . About 3The pathogenesis of CTD is complex and multifactorial, and involves immune and non-immune factors. Human leukocyte antigen (HLA) mismatches between donor and recipient give rise to cytotoxic T cells, NK cells, and donor-specific antibodies, causing immune-related injury to the graft, whereas hypertension, ischemia, infections, dyslipidemia, diabetes, and drug toxicity cause non-immune-related graft injury . Among tvia their negatively charged sulfated sugar groups and therefore bear the potential to be developed as PCSK9 inhibitors for the treatment of dyslipidemia (Dyslipidemia is characterized by increased serum triglycerides (TGs), total cholesterol (TC), low-density lipoprotein cholesterol (LDLc), very-low-density lipoprotein cholesterol (VLDLc), and normal or reduced high-density lipoprotein cholesterol (HDLc) . Dyslipiipidemia .Interestingly, treatment with heparin(oids) is found to improve serum lipid levels (TG and TC) in patients on renal replacement therapy . The effTherefore, in this study, we investigated 1) the effect of heparin and non-anticoagulant heparinoids on plasma values of lipids, syndecan-1, and PCSK9; 2) the efficacy of heparin and non-anticoagulant heparinoids to prevent the development of glomerulosclerosis and arterial neo-intima; and 3) the association of plasma lipid levels with plasma syndecan-1, PCSK9, and degree of glomerulosclerosis and arterial neo-intima formation in a rat CTD model.Agouti (DA) rats (donors) and 38 10-week-old male inbred Wistar Furth (WF) rats (recipients) were used. DA and WF rats were obtained from Harlan Nederland  and Charles River Laboratories , respectively. The local animal Ethics Committee of the University of Groningen approved all the procedures used in the study, and the principles of Laboratory Animal Care  were followed.In this study, 38 10-week-old female inbred Dark Kidney allotransplantation was performed from female DA donors to male WF recipients according to standard procedures as described previously  and immun = 9) and two non-anticoagulant heparinoids derived from regular heparin: N-desulfated, N-reacetylated heparin  and periodate-oxidized, borohydride-reduced heparin  as reported before by n = 10) received daily saline injections.In this study, interventions were performed by daily (s.c.) injections of 2 mg/kg BW/day with regular unfractionated heparin  as previously described  with minIdentification and quantification of glomerulosclerosis and neo-intima formation in kidneys were determined with periodic acid-Schiff (PAS) and Verhoeff’s staining, respectively, and blindly scored as mentioned previously by The sections were semi-quantitatively scored for focal glomerulosclerosis in a blinded fashion by determining the level of mesangial expansion and focal adhesion in each quadrant in a glomerulus and expressed on a scale from 0 to 4. If the glomerulus was unaffected, it was scored as 0; if one quadrant of the glomerulus was affected, it was scored as 1, two affected quadrants as 2, three affected quadrants as 3, and 4 affected quadrants as 4. In total, 50 glomeruli per kidney were analyzed, and the total FGS score was calculated by multiplying the score by the percentage of glomeruli with the same FGS score. The sum of these scores gives the total FGS score with a maximum of 400.Neo-intima formation was scored at 200× magnification  by determining the percentage of luminal occlusion. All the identifiable elastin positive intrarenal vessels were evaluated in a blinded fashion. The lumen of vessels was the mean length of two straight lines drawn from the internal elastic lamina (IEL) and passing through the center of the vessel. The areas enclosed by the lumen, internal elastic lamina, and external lamina were measured. The area between the lumen and internal elastic lamina was described as the neo-intimal area. The percentage of neo-intimal area to the area enclosed by IEL  is described as the percentage of luminal occlusion.p value of <0.05 was considered statistically significant.Analyses were performed using GraphPad version 8.0.1 (GraphPad software). The one-way ANOVA test and the Kruskal Wallis test were used to compare differences between the groups. When significant differences were observed between the means, Dunnett’s multiple comparison test, corrected for multiple comparisons, or Dunn’s multiple comparison test, corrected for multiple comparisons, was used as post-test to identify which specific means were significant from the others . Data are given as means ± SEM. The non-parametric Spearman correlation was used to analyze the association between parameters. For all experiments, a n = 8), heparin (n = 8), N-acetyl heparin (n = 8), and RO-heparin (n = 8). In the plasmas of the rats taken at 8 weeks after renal transplantation, 4 h after heparin(oid) injection, we measured the activated partial thromboplastin time. In the saline-treated transplanted rats, this was 75 s . In the regular heparin group, this time was 173 s , 73 s in the RO-heparin group, and 69 s in the N-acetyl heparin group  (not shown). These data show that both chemically modified heparin preparations indeed were non-anticoagulant, resulting in a similar activated partial thromboplastin time compared with saline-treated rats.This study included 38 male WF rats that were transplanted with a female DA kidney as reported before . Renal gTreatment with heparin and non-anticoagulant heparins had no effect on body weight , food anSaline-treated groups developed CTD-related renal failure as evidenced by reduced creatinine clearance and a rise in both serum creatinine and urinary protein excretion . Serum cp < 0.05; To investigate the effects of heparin and non-anticoagulant heparinoids on serum lipid levels, serum TG and TC levels were measured. Serum TG levels were significantly increased in saline- and NAc-heparin-treated groups over time, whereas heparin and RO-heparin treatment prevented the increase in serum TG levels over time . At 8 weNo significant differences in serum TC levels were observed in saline-treated group over time, although values tend to increase over time . All trep < 0.05). VLDLc levels were increased in saline and NAc-heparin (p < 0.01) treated groups, whereas heparin and RO-heparin treatment prevented the increase in VLDLc levels over time  (not shown).To get more insight into cholesterol profiles, we further profiled serum lipoproteins levels . VLDLc lver time . At 8 weLDLc and HDLc levels were increased in all groups over time . At 8 weAltogether, these data indicate that heparin and RO-heparin treatment prevented an increase in serum TG and VLDLc levels without affecting the LDLc, HDLc, and non-HDlc/HDLc ratio in the CTD model.Serum PCSK9 levels in the RO-heparin treated group and the NAc-heparin treated group were significantly lower than those in the saline-treated group at baseline . Becauser = 0.5, p = 0.03; r = 0.49, p = 0.03; and r = 0.49, p = 0.03, respectively) but not with TG, VLDLc, and renal function parameters (not shown).We previously reported syndecan-1 shedding in the same transplant model as used here . Serum sr = 0.53, p = 0.021)    and negactively) .r = 0.64, p < 0.001)  . All treated groups showed variable lumen occlusion due to neo-intima formation. No significant differences were observed in neo-intima formation in any of the treatment groups compared to the saline-treated group . Interes< 0.001) , suggest= 0.046) .In a rat model of CTD, we show that heparin and non-anticoagulant heparinoid (RO-heparin) could prevent the increase in serum TG and the TG-rich VLDL particles. None of the heparin(oids), however, could prevent a progressive increase in TC, LDLc, HDLc, serum PCSK9, and serum syndecan-1 levels. Similarly, transplant glomerulosclerosis and arterial neo-intima formation could not be attenuated by heparin(oid) treatment. Arterial neo-intimal scores were positively associated with unfavorable cholesterol profile (non-HDLc/HDLc ratio). Transplant glomerulosclerosis was positively associated with shedding of the lipoprotein clearance receptor syndecan-1. These data suggest that development and progression of CTD are independent of changes in TGs and VLDLc levels, rather related to syndecan-1 shedding and cholesterol profile.Although we did not study the lipoprotein lipase (LPL) activity in our model, the effects of heparin in reducing plasma TG and VLDLc levels might be attributed to the release/activation of lipoprotein lipase and hepatic lipase as reported before .We observed >10-fold increase in serum syndecan-1 levels in saline as well as heparin/non-anticoagulant heparin-treated groups over time. Furthermore, we observed no effects of heparin and non-anticoagulant heparinoids on serum syndecan-1 levels despite strong reductions in TG and VLDLc levels. It is worthwhile to mention that serum syndecan-1 levels at 8 weeks are strongly positively associated with TC, LDLc, and HDLc levels, but also with glomerulosclerosis. These data indicate that increased syndecan-1 shedding leading to reduced lipoprotein clearance might be a cause underlying dyslipidemia and glomerulosclerosis in our model. Although reported by others , neitherTissue remodeling and invasion of the neo-intima into the vascular lumen due to inflammatory processes inside the walls of arteries are important hallmarks of CTD. The interaction of lipids such as oxidized LDL and immune cells is thought to be a driving force behind chronic inflammation of the arterial wall during atherogenesis. Accumulation of lipid particles in vessel walls and subsequent immunological response cause plaque formation, which gets aggravated in dyslipidemic situations . Elevateversus LMW heparin/non-anticoagulant heparin derivatives.Apart from the role of dyslipidemia in transplant vasculopathy, dyslipidemia also induces glomerulosclerosis. Various studies have shown the accumulation of lipoproteins in glomerular mesangium, which accelerates matrix production in several rat models of renal diseases . MoreoveIn conclusion, the efficacy of heparin and non-anticoagulant heparins in lipid reduction is heterogeneous, controversial, and context dependent. Furthermore, complete relying on heparin and non-anticoagulant heparins in preventing CTD and CTD-related tissue remodeling might not be warranted by our results, at least not in the transplantation setting. Future investigations on non-immunological proteinuric animal models and human nephrotic diseases might be superior to target PCSK9/syndecan-1/cholesterol axis.


Candida mannan in blood sera samples for the diagnosis of invasive candidiasis. To reinvestigate carbohydrate specificity of EBCA-1, a panel of biotinylated oligosaccharides structurally related to distinct fragments of Candida mannan were loaded onto a streptavidin-coated plate to form a glycoarray. Its use demonstrated that EBCA-1 recognizes the trisaccharide β-Man-(1→2)-α-Man-(1→2)-α-Man and not homo-α-(1→2)-linked pentamannoside, as was reported previously.Monoclonal antibody EBCA-1 is used in the sandwich immune assay for the detection of circulating  Candida are the most common agents causing nosocomial fungal infections, and the fourth most common cause of nosocomial bloodstream infections (BSI) overall. Invasive candidiasis (IC) affects about 750,000 people worldwide with a case fatality rate of ~30–55%  wer wer22] wThe wells of 96 streptavidin-coated plates  were coated with biotin-tagged oligosaccharides 1–18  -α-Man-(1→2)-α-Man trisaccharide sequence from the “reducing” end by one 2)-α-Man unit (12→13) or its dimer 2)-α-Man-(1→2)-α-Man (12→14), or that elongation from the “non-reducing” end by one β-Man-(1→2)-unit (12→15) have not influenced the recognition by mAb EBCA-1. On the contrary, the elongation of the β-Man-(1→2)-α-Man-(1→2)-α-Man trisaccharide sequence from the “non-reducing” end by dimer β-Man-(1→2)-β-Man-(1→2)-(12→16) remarkably decreased the binding with mAb EBCA-1, while the attachment of one additional β-Man-(1→2)- unit (16→17) practically blocked the binding to mAb.Aspergillus fumigatus galactomannan [The knowledge of fine carbohydrate specificity of the monoclonal antibody used in a diagnostic kit is important for understanding the molecular basis of observed possible false-positive and false-negative results. Previously, we reported the reinvestigation of carbohydrate specificity of EB-A2 monoclonal antibody used for the immune detection of the tomannan , where ttomannan ,20,21 ortomannan  for mAb tomannan .Candida strains lacking such fragment in the structure of their mannans [The reinvestigated trisaccharide epitope is abundantly present in many yeast mannans, however there are  mannans . This maCandida mannans reported previously -manno-fragments of e papers ,20,21,47 strains . It can  strains .Candida mannan would be very promising for high performance diagnostics of invasive candidiasis. The generation of mAbs which are capable of recognizing such fragments are still faced with a challenge to test their applicability for clinical diagnostic needs.To the best of our knowledge, there is no diagnostic antibody with an undoubtedly proven ability to recognize homo-α-(1→2)-linked oligomanoside chains. The raising of such mAbs is a rather complex task due to the tolerance of the mammalian immune system to α-mannosides, as opposed to higher immunogenicity of β-mannosides. However, antibodies against α-(1→2)-linked oligomanosides containing branch points of


Asthmatics do not appear to have increased susceptibility to COVID-19.Uncontrolled severe asthma may be associated with worsened COVID-19 outcomes, especially in asthmatics managed with oralcorticosteroids. Risk mitigation measures such as hand hygiene, social distancing and wearing of face masks must be observed at all times. Asthma should be managed as outlined in local and international guidelines.Ensure an adequate supply of medication, and inhaled corticosteroids should not be withdrawnChronic obstructive pulmonary disease (COPD) is associated with severe COVID-19 disease and poor outcomes. Maintenance of background medication is important to avoid exacerbations of COPD.Vaccination against influenza is strongly advised for all patients with asthma and COPDVaccination against pneumococcal infection is advisable for patients with COPD. Patients with obstructive airway disease on oral corticosteroids and/or with impaired lung function should take stringent safety precautions.This statement will be updated when more data become availableAsthma and COPD occur commonly in South Africa. SARS-CoV-2 is a novel coronavirus, which can result in COVID-19-associated severerespiratory infection with respiratory failure and the need for mechanical ventilation. The South African Thoracic Society has prepared a guidancestatement to assist clinicians and patients with asthma and COPD during the current epidemic. Thus, while asthma was not documented as a risk factor/underlying comorbidity for COVID-19 infection in earlier studies, somemore recent studies, particularly from the USA, have shown that asthmamay be a significant comorbid condition.3] Furthermore, asthma wasalso found to be relatively common among intensive care unit cases withCOVID-19 and in patients who died. Therefore, while mild to moderateasthma that is well controlled may not be a risk factor for COVID-19,people with uncontrolled and severe asthma, those receiving repeatedor regular doses of oral corticosteroids (OCS) and those with underlyingstructural lung damage may be at risk of more severe COVID-19.Asthmatics do not appear to have increased susceptibility to COVID-19.[4] Asthma with recent use of OCS was associated with highermortality compared with non-asthmatics when fully adjusted for othervariables  1.13 (1.01 - 1.26)).[4]By contrast, the International Severe Acute Respiratory and EmergingInfection Consortium (ISARIC) study reported that 14% of hospitalisedpatients with asthma have no increased risk of death.[5] Data fromSpain[3] suggest that a few patients with asthma were infected withSARS-CoV-2 and this was not associated with asthma exacerbationRecently published data from the UK primary care and COVID-19system notification databases have indicated that asthma (withoutcorticosteroid use) was not associated with higher mortality whenadjusting for confounders in 10 926 COVID-19-associated deaths inthe UK.7]Although data from the previous severe acute respiratory syndromeand Middle East respiratory syndrome outbreaks did not demonstrate any increased risk of infection nor severe disease in asthmatics, this maybe different for COVID-19, and further data are awaited.12] Data fromprevious influenza outbreaks and the 2009 H1N1 pandemic suggestthat asthma was associated with hospitalisation,[13] but interestingly,protective for mortality.[14]Regarding other asthma phenotypes and patients from other settings,there is little data available, and thus recommendations cannot be made.Few asthma patients are recorded in published cohorts of SARS-CoV-2infections in China, and it has not been linked to poor outcomes.[15] Inhaled or OCS should not bewithheld if indicated to treat exacerbations of asthma.Based on these limited data, severe asthmatics  seem to be at higher risk of death than non-asthmatics. There is noevidence to suggest that inhaled steroids should be stopped as they areprotective against asthma exacerbations.et al.[14]reported on 1 592 patients and demonstrated a significant associationbetween COPD and poor outcomes in COVID-19 (odds ratio 5.69 (2.49 -13.00)). The second meta-analysis included 15 studies with 2 473 patients.The patients with COPD had a relative risk of 1.88 (1.4 - 2.4) for severeCOVID-19 compared with non-COPD patients.[16] These data providesufficient evidence establishing an association between COPD and riskof severe COVID-19. Tobacco smoking has also been associated withsevere COVID-19 and adverse outcomes.[17] It is unclear, however, ifsmoking is an additive risk factor in addition to COPD. It is worthnoting that data are discordant and in both the UK[17] and China,[18]COPD did appear to confer increased risk of death among hospitalisedand confirmed cases, respectively.19]Current evidence summarised in two meta-analyses indicate that chronicobstructive pulmonary disease (COPD) patients are at a significantlyhigher risk of severe COVID-19 and adverse outcomes. Lippi [20] Inhaled steroids area risk factor for bacterial pneumonia and should be avoided unlessindicated[19] and where dual bronchodilators are not accessible.Influenza and pneumococcal vaccination are recommended as wellas a regular supply of background medication.


Patients with obstructive lung diseases may be at risk of hospitalization and/or death due to COVID-19.To estimate the frequency of severe COVID-19, and COVID-19-related mortality in a well-defined large population of patients with asthma and chronic inflammatory lung disease (COPD). Further to assess the frequency of asthma and COPD as registered comorbidities at discharge from hospital, and in death certificates.At the start of the pandemic, the Swedish National Airway Register (SNAR) included 271,404 patients with a physician diagnosis of asthma and/or COPD. In September 2020, after the first COVID-19 wave in Sweden, the database was linked with the National Patient Register (NPR), the Swedish Intensive Care Register and the Swedish Cause of Death Register, which all provide data about COVID-19 based on International Classification of Diseases (ICD-10) codes. Severe COVID-19 was defined as hospitalization and/or intensive care or death due to COVID-19.Among patients in SNAR, 0.5% with asthma, and 1.2% with COPD were identified with severe COVID-19. Among patients  < 18 years with asthma, only 0.02% were severely infected. Of hospitalized adults, 14% with asthma and 29% with COPD died. Further, of patients in SNAR, 56% with asthma and 81% with COPD were also registered in the NPR, while on death certificates the agreement was lower (asthma 24% and COPD 71%).The frequency of severe COVID-19 in asthma and COPD was relative low. Mortality for those hospitalized was double as high in COPD compared to asthma. Comorbid asthma and COPD were not always identified among patients with severe COVID-19. Although most cases have no or very mild symptoms, the infection can also present as a severe disease with pneumonia, respiratory failure and multi-organ failure, with substantial mortality. After the first wave, in early September 2020, The National Board of Health and Welfare in Sweden stated that 22,260 patients had been hospitalized with COVID-19 and 5,981 had died.At the end of December 2019, the first cases of coronavirus disease 2019 (COVID-19) were reported from China. Since then, COVID-19 has evolved as a major worldwide health crisis. previous studies have reported an increased risk of severe disease with need for hospitalization and worse clinical outcomes in patients with underlying comorbidities such as hypertension, cardiovascular disease and diabetes mellitus.6 However, the reported frequency of hospitalized COVID-19 patients with underlying respiratory diseases varies between countries. Studies from both China and Italy have reported low rates (less than 5%) of respiratory diseases among hospitalized patients with COVID-19,8 while higher figures (14%-18%) have been reported from the United Kingdom and United States.10 In Sweden, the proportion of patients with chronic lung diseases was 14% in the intensive care units (ICU) during the first months of the pandemic.In addition to older age and male sex, chronic obstructive pulmonary diseases (COPD), on the contrary, has shown to be associated with an increasing need for intensive care treatment and risk of death. However, most studies are based on patients with COVID-19 identified with asthma or COPD as comorbidities, rather than settings using high-quality nationwide registers for identification of COVID-19 in patients with obstructive lung diseases. The aim of this study was to estimate the frequency of severe COVID-19, and COVID-19-related mortality in a well-defined large population of Swedish patients with asthma and COPD. A secondary aim was to assess the frequency of asthma and COPD as registered comorbidities at discharge from hospital and in death certificates.Overall, asthma patients do not seem to represent a risk group,15 The development and design of SNAR have been described in detail and the current study was approved by the Swedish Ethical Review Authority .The participants were identified in SNAR, which was initiated in 2013 and includes data on patients with a current physician diagnosis of asthma (children and adults) and/or COPD from primary and secondary care, as well as data on hospitalized COPD patients. In Sweden, the diagnostic criteria for asthma and COPD follow international guidelines.In August 2020, SNAR included in total 271,404 patients, of whom 198,113 (73%) had asthma, 55,942 (21%) had COPD, and 17,349 (6%) had both diagnoses. Patients who had been included in SNAR, but died before January 2020 were excluded from the study population.17 These registers provide data about COVID-19 based on International Classification of Diseases, version 10 (ICD-10) codes U07.1 (COVID-19 confirmed by laboratory testing) and U07.2 . The registration of inpatient care, intensive care and causes of death have coverage of nearly 100%.17 A severe COVID-19 was defined as hospitalization (inpatient care identified as primary discharge diagnosis in NPR and/or ICU registration in SIR) or death due to COVID-19 (registered as an underlying or contributing cause of death in SCDR). Patients in SNAR with severe COVID-19 were defined as cases and patients in SNAR without severe COVID-19 as controls. Data extraction from SNAR was conducted on August 17, 2020, and linked with data from the NPR (inpatient care), the Swedish Intensive Care Registry (SIR), and the Swedish Cause of Death Register (SCDR) on 11 September.The frequency of asthma and COPD as underlying comorbidities to COVID-19, registered as secondary discharge diagnoses, was retrieved from the NPR at discharge by using ICD-10 codes for asthma J45 (asthma with/ without acute exacerbation) and COPD J44 (COPD with/without acute exacerbation). Report of chronic lung disease among patients receiving intensive care for COVID-19 were analysed from the SIR. From the SCDR, J44 and J45 were used to identify asthma and COPD reported as underlying or contributing causes of death together with COVID-19.SAS 9.4 for Windows  was used for statistical analyses. Frequencies, proportions, means and standard deviations (SD) were used to describe data. For relevant estimates and differences, 95% confidence intervals (CI) were calculated to assess statistical precision.Among children with asthma in SNAR, only 0.02% (8 of 46 123 patients) had been hospitalized due to COVID-19. In adults with asthma in SNAR, 0.5% (784 of 168 488 patients) were hospitalized due to COVID-19% and 0.1% (175 of 168 488) died of COVID-19, making the frequency of severe COVID-19% to 0.5%  .versus 61%, respectively. Regardless of sex, cases with severe COVID-19 were substantially older than controls, with an average age of 66 years, compared with 52 years (n = 784), 98 patients (13%) received intensive care with an average stay of 13 days at the ICU   .n = 721) a total of 54 (7%) cases required intensive care with an average stay at the ICU for 10 days  were hospitalized, and 0.5% (357 of 72 421) died of COVID-19. The frequency of severe COVID-19 in patients with COPD from SNAR was 1.2 % (870 out of 72 421) . The pro 10 days , and 210 10 days . No sex- 10 days .In 81% of the cases with COPD diagnosis in SNAR, J44 was recorded in NPR as secondary discharge diagnosis after COVID-19-related hospitalization. Among COPD cases attending the ICU, 85% were identified having a chronic lung disease in SIR. In 71% of the cases, J44 was recorded as an underlying or contributing cause of death in the SCDR .This study describes the frequency of severe COVID-19 in a cohort of over 270 000 Swedish asthma and COPD patients retrieved from the SNAR, a national quality register. Severe COVID-19 was more common in COPD than in asthma, and after hospitalization, 14% of asthma patients and 29% of COPD patients died. Increasing age was associated with severe COVID-19, and men with COPD were more prone to a poor outcome (death). Despite the fact that we identified patients with known diagnosed asthma or COPD who developed severe COVID-19, their underlying comorbid obstructive lung disease was not always registered as a secondary discharge diagnosis or in death certificates, however more often in COPD than asthma.versus 0.3% in the general population ⩾ 18 years), but with unadjusted data, it is difficult to generalize our results. Today, COPD is well recognized as a comorbidity associated with an increased risk of poor outcome in COVID-19. As far as asthma is concerned, there is no convincing evidence of asthma as a risk of severe COVID-19,18 and COVID-19 does not appear to induce severe asthma exacerbations or increase the risk of worse clinical outcome of COVID-19. However, our results showed that a higher proportion of patients with asthma required intensive care than patients with COPD, which also is shown in another Swedish study based on ICU patients in relation to COVID-19. In the latter study, it was speculated that more severe COPD patients could be subject to limitation for care. In our study, patients with COPD were older than those with asthma, and older age may also be a reason for limitation of intensive care. Contrary in US, a higher level of patients seemed to be admitted to ICU due to COVID-19, which may reflect different management of COVID-19 between countries.The proportion of a severe COVID-19 among patients with COPD registered in SNAR seems to be somewhat higher than in the Swedish general population  children with asthma in SNAR had been hospitalized due to COVID-19. However, up to date, it is still unknown if asthma is associated with an increased risk of worse outcome in COVID-19 among children.Our findings are consistent with previous reports that age influences the risk of worse outcome in COVID-19. Children seem to present milder symptoms when infected with COVID-19, and the need for hospitalization is low.30 and an increased vulnerability in elderly patients suffering from comorbidities such as COPD. There are reports about elderly patients being at higher risk of pulmonary and cardiac complications and increased mortality in COVID-19. This may be the reason for the high mortality rate (nearly 30%) in hospitalized patients with COPD in our study. One explanation to increased risk of poor outcome in the elderly could be the natural aging process of respiratory and immune systems, which might increase the susceptibility to viral infections and lead to more serious clinical outcome.3335We have shown that adult patients with severe COVID-19 were significantly older than those without a severe infection, both in asthma and COPD and regardless of sex. At an early stage in the pandemic, reports indicated a poor prognosis in elderly infected with COVID-1937 male sex seems to be associated with severe COVID-19. There are various possible explanations for worse clinical outcomes and higher mortality for COVID-19 infected men than women. The risk of infection after exposure seems to be equal between sexes, but women seem to have better interferon and Toll-like receptor (TLR) mediated anti-viral response and viral clearance. Besides, higher mortality can be associated with increased cytokine concentrations and dysregulated inflammatory response in men, leading to respiratory and cardiovascular complications.As in other studies, and the management of severe COVID-19 is heterogenic which may affect the external generalization of our results.A major strength is the large database of SNAR with well-characterized physician-diagnosed patients with asthma and COPD. The possibility to link SNAR with other national registers allows monitoring the frequency of a severe COVID-19 infection among asthma and COPD patients in Sweden. Linked together, the current data are a unique resource for respiratory research in patients with obstructive lung diseases. Importantly, the hospital care of patients with COVID-19 varies between countries. For example, the ICU beds are in general fewer in Sweden than in other European countries41 a limitation is a difficulty to reach a 100% coverage in SNAR. However, of all counties in Sweden, health care units in Stockholm and Västra Götaland transmit most frequently data into SNAR, and these counties also have had the highest incident of COVID-19 in Sweden. Thus, over 250 000 patients included from these two counties, together with smaller counties in Sweden, are estimated to be a sample size, large enough, to identify the frequency of a severe COVID-19. Importantly, transmitted data from healthcare systems to the NPR and the National Causes of Death Register are related to a delay. As a consequence of data delay, it may be a risk that our data do not include all cases with severe COVID-19 in Sweden until September. A severe infection may therefore have been under-estimated instead of over-estimated in our study.As asthma and COPD are chronic diseases with a high prevalence in the population, in Sweden estimated to 10% in asthma and 7% in COPD,Further limitations are no information on the severity of asthma and COPD, and no age-matched control group of non-asthma and non-COPD in this manuscript. Future research will be aimed to study severe COVID-19 in asthma and COPD and the associations with disease severity, pharmacological treatment and comorbidities.In Sweden, 0.5% of adults with asthma and 1.2 % of those with COPD in SNAR had a severe COVID-19 during the first wave of the pandemic. Of patients being hospitalized due to COVID-19, 14% of those with asthma and 29% of those with COPD died. Asthma and COPD are not always registered in Swedish health care as comorbidities together with COVID-19, nor as underlying or contributing causes of death. However, COPD is reported more often than asthma, suggesting that physicians in Sweden assess COPD to play a more significant role in the disease course of a severe COVID-19 than asthma.


The fatality rates and factors associated with death from coronavirus disease 2019 (COVID-19) in hemodialysis patients have been extensively investigated. However, data on peritoneal dialysis (PD) patients remain scarce.In this nationwide cohort study, we assessed the 28-day COVID-19-related fatality rate in PD patients between August 2021 and July 2022 using data from the InCov19-PD registry. Predictors associated with death were evaluated using a multivariable Cox regression model. Changes in functional status before and during COVID-19 were also examined.p < 0.01 for all). Conversely, the number of COVID-19 vaccines administered  and receiving corticosteroid therapy during COVID-19  were associated with a decreased risk of death within 28 days after COVID-19 diagnosis. The number of functionally independent PD patients dropped from 94% at baseline to 63% during COVID-19 (p < 0.01).A total of 1,487 eligible participants were evaluated. During the study period, 196 participants died within 28 days after COVID-19 diagnosis . In a multivariable Cox regression model, an increased risk of death within 28 days after COVID-19 diagnosis among PD patients was independently associated with functional impairment during COVID-19 , SARS-CoV-2 infection with the Delta variant , and the need for respiratory support  (The COVID-19-related 28-day fatality rate was high among PD patients. The predictors of COVID-19-related death in PD patients were similar to those in hemodialysis patients. During COVID-19, PD patients commonly experienced functional deterioration. The coronavirus disease 2019 (COVID-19) pandemic posed unprecedented challenges for patients with end-stage kidney disease (ESKD) requiring kidney replacement therapy (KRT). Numerous studies have demonstrated that the case fatality rates of COVID-19 in ESKD patients are significantly higher than in the general population . To mitiAmong ESKD patients diagnosed with COVID-19, those on chronic PD therapy have received much less attention than those on the other dialysis modalities. The majority of the COVID-19 mortality data and clinical outcomes are derived from the HD and kidney transplant (KT) populations , 8, 9. AHerein, we sought to determine the 28-day case fatality rate and predictors of COVID-19-related death among patients on chronic PD therapy. Alterations in patients’ functional status and pattern of routine PD care, focusing on PD bag exchange, were also investigated.This is a prospective observational nationwide cohort using data from the InCov19-PD registry, a national surveillance registry that assessed the clinical outcomes and health impacts of SARS-CoV-2 infection in Thai PD patients. Under the auspices of the Nephrology Society of Thailand (NST), the InCov19-PD registry prospectively collected data from August 2021 to July 2022 on PD patients diagnosed with COVID-19. Patients were included in the study if they were PD patients who tested positive for SARS-CoV-2, were at least 12 years old, and had received chronic PD treatment for at least 1 month before COVID-19 diagnosis. For PD patients who had multiple COVID-19 episodes, only the initial COVID-19 episodes were included in the study. Any PD patients who had no documented clinical outcomes at day 28 after COVID-19 diagnosis, aged less than 12 years old, or commencing PD treatment for acute kidney injury were excluded from the study. A COVID-19 diagnosis was confirmed either by a positive SARS-CoV-2 real-time reverse transcription polymerase chain reaction test or a rapid antigen test kit (ATK) on samples obtained from the nasopharyngeal swab.The Institutional Review Board of the Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand approved this study (IRB No. 0298/65). All participants provided written informed consent before enrollment. The study was conducted following the principles laid out in the declaration of Helsinki.The primary outcome was the 28-day case fatality rate of PD patients with confirmed COVID-19 in the InCov19-PD registry. The COVID-19 confirmation date was utilized as the index date when calculating the 28-day case fatality rate. Survivors were participants who were still alive 28 days following the COVID-19 confirmation date. The remaining individuals who died within 28 days as a result of COVID-19 were classified as non-survivors.Secondary objectives included identifying predictors of COVID-19-related death within 28 days following a confirmed COVID-19 diagnosis. The impacts of COVID-19 on hospitalization rate, the need for respiratory support, and changes in patient functional status and patterns of PD bag exchange during COVID-19 were also examined. The baseline functional status of patients before and at the time of COVID-19 diagnosis was evaluated and categorized as independent, partially dependent, or totally dependent on a caregiver.via an online database system. Clinical outcomes were evaluated at baseline and monitored until the patient died or until day 28 after COVID-19 diagnosis, whichever came first. The baseline functional status of PD patients and assistance with PD bag exchange before and during COVID-19 were obtained from the index patient and/or their family members using a semi-structured questionnaire. The COVID-19 confirmation date was utilized as the index date for each participant’s baseline characteristics, laboratory data, functional status, and vaccination record. All participants were followed until death, recovery, or hospital discharge.At the end of July 2021, physicians and PD nurses from all NST-registered PD facilities were invited to voluntarily participate in this study. The InCov19-PD registry collected patient-level and facility-level data from all voluntarily participating facilities and study participants using a standard process and standardized data collection instruments. The NST verified the index case by contacting the treating physicians and reference PD nurses. Responsible physicians or PD nurses provided the NST with demographic data, comorbidities, details of PD prescriptions, COVID-19 vaccination status and regimens, laboratory parameters at the time of COVID-19 diagnosis, identified SARS-CoV-2 strains, initial and subsequent treatments, and COVID-19-related complications t-test for continuous variables. The 28-day case fatality rate of the overall cohort was computed. Fatality outcome was analyzed by survival analyses using Kaplan–Meier curves with a Log-rank test. All included patients had a complete follow-up, thus there was no censoring for missing outcome data. The associations between the covariates and the 28-day case fatality outcome were first evaluated using univariable Cox proportional hazard regression and subsequently adjusted for age. The multivariable Cox regression model was conducted using backward elimination and list-wise exclusion of missing data. All variables with age-adjusted p-values of less than 0.10 were candidates for the multivariable model. The least significant variables were removed repeatedly from the model until all variables had p-values below 0.05. The proportional hazards assumption was verified using Schoenfeld residuals and plots. Data were analyzed using R 4.0.5 . P-values less than 0.05 were statistically significant.The categorical variables were described as the frequency with percentage, while the continuous variables were presented as mean with standard deviation (SD). Baseline demographic data and laboratory parameters between the survivor and non-survivor groups were compared using the Chi-square test and Fisher’s exact test for categorical factors, and the independent The InCov19-PD registry documented a total of 1,660 PD patients diagnosed with COVID-19 from August 2021 to July 2022. Of those, 1,487 eligible participants with a complete clinical record of outcomes on day 28 after COVID-19 diagnosis and meeting other inclusion criteria were included in the analysis . Baselinp < 0.001], had a higher prevalence of diabetes mellitus , and were more functionally dependent  at baseline (p < 0.0001) (p < 0.0001)  study participants died (non-survivor group), while 1,291 (87%) survived (survivor group). Patients in the non-survivor group were significantly older . At baseline, most (94%) PD patients in this cohort received continuous ambulatory PD (CAPD). During COVID-19, 46 (6%) patients switched from CAPD to automated PD (APD), while all APD patients continued their initial dialysis modality.Of 1,487 PD patients diagnosed with COVID-19, a total of 921 (62%) patients were hospitalized. Approximately 64% of these patients needed oxygen or other invasive respiratory support . More pap < 0.001). Furthermore, the impaired functional status during COVID-19, but not at baseline, was associated with the risk of dying within 28 days from a COVID-19 diagnosis (p < 0.001) in the multivariable model. Similarly, COVID-19 impacted the bag exchange patterns of PD patients. At baseline, more than half (59%) of PD patients could independently perform PD bag exchange without assistance. During COVID-19, 10% of patients who were previously independent of PD bag exchange required additional assistance from caregivers or nurses. In the multivariate model, however, the need for PD bag exchange support during COVID-19 illness was not associated with the risk of dying within 28 days from a COVID-19 diagnosis.The functional status of PD patients with COVID-19 was altered. The number of PD patients who were classified as independent in their activities of daily living (ADLs) declined from 94% at baseline to 63% after COVID-19 diagnosis . The patients/participants provided their written informed consent to participate in this study.PC, PD, SB, SK, SS, and TK conceptualized and designed the study. PC, SB, and TK collected the data and drafted the manuscript. PC, SB, TH, TN, and TK analyzed the data. All authors reviewed and approved a final version of the manuscript.