Epigenetic changes in stroke may revolutionize cell-based therapies aimed at reducing ischemic stroke risk and damage. Epigenetic changes are a novel therapeutic target due to their specificity and potential for reversal. Possible targets for epigenetic modification include DNA methylation and demethylation, post-translational histone modification, and the actions of non-coding RNAs such as microRNAs. Many of these epigenetic modifications have been reported to modulate atherosclerosis development and progression, ultimately contributing to stroke pathogenesis. Furthermore, epigenetics may play a major role in inflammatory responses following stroke. Stem cells for stroke have demonstrated safety in clinical trials for stroke and show therapeutic benefit in pre-clinical studies. The efficacy of these cell-based interventions may be amplified with adjunctive epigenetic modifications. This review advances the role of epigenetics in atherosclerosis and inflammation in the context of stroke, followed by a discussion on current stem cell studies modulating epigenetics to ameliorate stroke damage. Once termed the “incoming epidemic of the 21st century” by the World Health Organization, stroke is a major worldwide health concern and the 5th leading cause of death in the United States . AvailabThe field of epigenetics has recently expanded to include the development of new treatments to target a variety of multifactorial diseases such as stroke. Epigenetic changes have emerged as a potential area for research because epigenetic aberrations are pharmaceutically reversible . PossiblMany of these epigenetic modifications have been reported to modulate atherosclerosis development and progression, ultimately contributing to stroke pathogenesis . FurtherHyperlipidemia, and by extension atherosclerosis, is a major risk factor associated with stroke . AtherosOne promising therapeutic target for combating epigenetic regulated atherosclerosis in stroke involves the expression of histone deacetylases. For example, in models of histone deacetylase 3 (HDAC3) knockout mice, HDAC3 deficiency promotes the stabilization of atherosclerotic plaques by inducing fibrosis over existing lesion . In humaAside from HDACs, another major player in epigenetics is the pattern of DNA methylation. Methylating certain genes can have significant downstream effects and provide potential therapeutic targets. For example, atherosclerotic arteries have much lower levels of methylated CpG islands in genes associated with angiogenesis, atherogenesis, and inflammation. These include the downregulation of FOXP1, upregulation of the AGE receptor ligand, and activators of NOTCH1 and EGFL7 . On the Common RNA mechanisms studied in epigenetics include the functions of miRNAs and other non-coding RNAs (ncRNAs). Changes in physiological conditions can precipitate alterations in ncRNA function and expression to create domino-like downstream effects. For example, miRNA-10a is downregulated in areas of the cardiovascular system with high levels of shear stress, such as the inner curvature of the aortic arch of rat models. This leads to higher levels of atherogenesis which can be rescued by the co-administration of retinoic acid receptor-α and retinoid X receptor-α agonists . This is−/− atherosclerotic mouse models, and its overexpression inhibits apoptosis in human umbilical vein endothelial cells . Mi. Mi52]. miRNAs exhibit many of their anti-inflammatory properties via microglial modulation. Of major relevance in stroke pathology is miR-124 due to its primary expression in the CNS. In rats, this miRNA is elevated in the plasma following middle cerebral artery occlusion despite no changes in other miRNAs such as miR-122 and miR-133 . This miStem cells have been largely studied as potential therapeutics for ischemic stroke due to their ability to differentiate into various cells of the nervous system. One of the primary objectives of acute stem cell therapy is to mitigate secondary injury following stroke, which is mediated by proinflammatory processes, mitochondrial dysfunction, oxidative damage, and apoptosis. Chronic treatment focuses on regeneration through the stimulation of processes such as vasculogenesis, neurogenesis, angiogenesis, and synaptogenesis . PrimaryThe properties of different sources of stem cells have been identified and studied in the context of stroke. One such class of cells is mesenchymal cells, which have been shown to exhibit neuroprotective and therapeutic properties. Different subtypes of mesenchymal cells show therapeutic potential for stroke. Induced pluripotent derived mesenchymal stem cells were found to exhibit neuroprotective effects on hypoxic PC12 cells through mitochondrial transfer .A subset of mesenchymal cells called Muse cells are capable of specific differentiation into all three germ layers. Muse cell transplantation in experimentally induced stroke rats improves neurological and motor function with a high ratio of neuronal cell differentiation .Embryonic stem cells demonstrate great potential as a therapeutic due to their ability to indefinitely divide and differentiate into cells from all three germ layers . IntraveBone marrow-derived mesenchymal stem cells (BM-MSCs) show promise as a therapeutic for stroke and potentially play a role in physiological neurorestoration. They are capable of differentiation into osteogenic, chondrogenic, and adipogenic cells . During Another type of stem cell found in bone marrow is hematopoietic stem cells (HSCs), which can differentiate into all types of blood cells and are recruited by chemokines and other chemotactic factors . MoleculYet, another class of stem cells which has shown therapeutic potential for ischemic stroke is neural stem cells (NSCs), which can differentiate into neurons and glial cells such as astrocytes and oligodendrocytes . SourcesIn the setting of ischemic stroke models, many recent murine and cell culture studies involving administration of cell products  from stem cells demonstrate promising neurological recuperation. Though it is historically understood that stem cells improve neurological status following cerebral ischemia, the following studies attempt to elicit the epigenetics involved and thus possible treatment modalities. A comprehensive list of the preclinical trials on epigenetic modification to stem cell therapy as a treatment modality for ischemic stroke is outlined in Multipotent BMSCs were epigenetically modified to overproduce miR-133b; exosomes from these cells were then harvested and administered intra-arterially to rats following middle cerebral artery occlusion (MCAO). These rats had improved neurological function and plasticity compared to rats injected with exosomes from BMSCs infected with blank vector, or phosphate-buffered saline. In an additional experiment, cultured astrocytes subjected to the oxygen and glucose deprivation (OGD) model were incubated with exosomes from naïve BMSCs, miR-133b downregulated BMSCs, or miR-133b-overexpressing BMSCs. Compared to naïve treatment, exosomes from miR-133b-overexpressing BMSCs significantly increase the number of exosomes released from the OGD astrocytes, whereas exosomes from miR-133b downregulated BMSCs decrease the number released. Exosomes harvested from the OGD astrocytes (cultured with exosomes from miR-133b-overexpressing BMSCs) also increase neurite branching in cultured cortical embryonic rat neurons .Rats that underwent the MCAO model then intravenously received either miR-17-92 cluster-enriched exosomes (harvested from BMSCs infected with a miR-17-92 cluster plasmid), control BMSC exosomes, or liposomes. Compared to the control liposome group, both exosome-recipient groups demonstrated increased neurological function; however, the miR-17-92 cluster-enriched exosomes were significantly superior. Factors assessed include neurological function, oligodendrogenesis, neurogenesis, and neurite remodeling/dendrite plasticity within the ischemic site. miR-17-92 cluster-enriched exosomes inhibit phosphatase and tensin homolog, thus increasing the activation of the pathway involving PI3K/protein kinase B/mechanistic target of rapamycin/glycogen synthase kinase 3β .MiR-126 acts upon the stromal cell-derived factor 1/C-X-C chemokine receptor type 7 signaling pathway. Mice demonstrate decreased infarction volume and increased angiogenesis following infusion with bone marrow-derived endothelial progenitor cells (EPC) treated with miR-126 .In both rat MCAO and microglia OGD models, administration of exosomes from mesenchymal stem cells overexpressing miR-223-3p decreases infarct volume, and improves neurological deficits, learning, and memory. Within the ischemic zone, proinflammatory agents are suppressed and anti-inflammatory agents are promoted. Similar to the previous study, miR-223-3p promotes M2 microglial transformation .Exosomes were collected from bone marrow-derived mesenchymal stem cells overexpressing miR-138-5p and cultured with astrocytes subjected to OGD. Under these conditions, astrocytes demonstrate increased proliferation, and decreased apoptosis and inflammation. In a separate experiment, the modified exosomes were administered to mice following MCAO, ameliorating neuronal injury . The mecIn the setting of MCAO, mice have decreased miR-542-3p and increased Toll-like receptor 4 (TLR4); of note, these molecules bind each other. Adeno-associated virus packaged miR-542-3p reduces infarction area, number of degenerating neurons, expression of inflammatory agents, and inflammatory cell infiltration. In a separate experiment, astrocytes undergoing OGD and exposed to miR-542-3p packaged in mesenchymal stem cells, show decreased apoptosis and reactive oxygen species. The mechanism involves the inhibition of TLR4 .In the setting of MCAO, rats have decreased miR-150-5p and increased Toll-like receptor 5 (TLR5). Vesicles from bone marrow mesenchymal stem cells were isolated and administered to MCAO rat models, improving neurological function, and decreasing neuronal apoptosis and inflammatory factors. The mechanism involves inhibition of TLR5 .In MCAO rat models, exosomes from bone marrow mesenchymal stem cells transfer miR-23a-3p, which polarizes microglia to their M2 form .Extracellular vesicles from bone marrow stromal cells ameliorate neurological injury and blood–brain barrier permeability in MCAO mice and microglia/astrocyte OGD models. This phenomenon is mediated via miR-124 that may act on peroxiredoxin 1 .Bone marrow mesenchymal stem cell-derived exosomes containing miR-455-3p ameliorate hippocampal neuronal injury in MCAO mice models and OGD cell models. The mechanism is via targeting of PDCD7 .Mesenchymal stem cell-derived extracellular vesicles harboring miR-93 demonstrate improvement in MCAO rat models and OGD hippocampal neuron models. MiR-93 targets histone deacetylase 4 (HDAC4). HDAC4 deacetylates and thus inhibits the antiapoptotic molecule Bcl-2, leading to increased infarct volume and neuron apoptosis, and worsened neurobehavioral impairments .Exosomes from bone marrow mesenchymal stem cells upregulate miR-21-5p and thus improve neurological function and reduce infarct volume in mouse stroke models. In vitro studies involving human umbilical vein endothelial cells demonstrated increased expression of VEGF, VEGFR2, Ang-1, and Tie-2 following exposure to exosomes .A separate study first upregulated miR-126 in adipose derived stem cells, then harvested and intravenously administered exosomes to control and stroke model rats. Following stroke, such rats exhibit improved neurological functional recovery, decreased neuronal death, and increased cell proliferation. These modified exosomes also inhibit microglial activation and expression of proinflammatory agents .Rats demonstrate decreased area of cerebral infarction following infusion with adipose-derived stem cells treated with miR-30d-5p. The mechanism is via the inhibition of autophagy-mediated microglial polarization to M1 .Contrary to the protective findings of miRs thus far, miR-21-3p is elevated in the setting of MCAO and reduces MAT2B expression in neural cells. The adipose-derived mesenchymal stem cell and its exosomes suppress miR-21-3p, thus protecting the blood–brain barrier, and inhibiting inflammation and apoptosis .Neurons were exposed to OGD and then cultured with either adipose-derived mesenchymal stem cells (ADMSC) or ADMSC-secreted vesicles. Both therapies reduce cell death, and the mechanism is via decreased autophagy and p53-BNIP3 activity. Interestingly, ADMSC’s effects are lost when treated with an inhibitor of exosome secretion. Understanding that miR-25-3p is the most expressed miRNA in ADMSC-vesicles involved with the p53 pathway, the investigators discovered that an oligonucleotide mimic decreases cell death, whereas an anti-oligonucleotide increases autophagy and death. This phenomenon is observed similarly in mice models .Vesicles from adipose-derived stem cells overexpressing miR-31 improve neurological function and reduce infarct volume and neuronal apoptosis in MCAO mice models. In neuron OGD models, the vesicle therapy decreases apoptotic factors caspase-3 and Bax and increased survival. The mechanism is via miR-31 binding to the 3′-untranslated region of TRAF6, thus inhibiting TRAF6 and subsequently inhibiting IRF5 (IRF5 promotes neuron apoptosis in the setting of OGD) .ADMSCs and ADMSC-derived vesicles were cultured with neurons subjected to OGD. Consistent with the literature, this results in decreased neuronal apoptosis. Unique to this study, it was found that miR-22-3p is upregulated in ASC-vesicles; treatment with miR-22-3p inhibitor reverses the beneficial effects, increasing apoptosis. Mechanistically, miR-22-3p binds KDM6B, thus inhibiting its downstream effects on BMP2/BMF .Similarly, in mice MCAO models, intravenous injection of vesicles from adipose-derived stem cells promotes M2 polarization. The latter study also demonstrates decreased brain atrophy volume, improved neuromotor and cognitive functions, attenuated loss of oligodendrocytes, increased angiogenesis, and proliferation of endothelial cells. MiRNA in these vesicles target STAT1 and PTEN, adjusting microglial polarization .Exosomes from human umbilical cord mesenchymal stem cells expressing miR-146a-5p reduce microglia-mediated inflammation in OGD models, and both reduce infarct volume and ameliorate behavioral deficits and microglia activation in murine stroke models. The mechanism is via inhibition of the IRAK1/TRAF6 pathway .When exosomes are harvested from urine-derived stem cells and administered intravenously to rat stroke models, the rats show enhanced neurogenesis and recovery. An additional experiment subjected neural stem cells to OGD along with the exosomes; the therapy promotes neuronal proliferation and differentiation. The mechanism may involve exosomal miR-26a’s inhibitory effect on histone deacetylase 6 .The role of epigenetic modifications in stroke pathophysiology has resulted in a plethora of intriguing and groundbreaking new research. The specificity of some of these epigenetic changes, such as with miRNAs and ncRNAs, allow for targeted therapeutic intervention. With a better understanding of how these epigenetic modifications influence inflammation and atherosclerosis, cell-based therapies can be developed to directly modulate these changes and mitigate stroke risk and damage.


However, COVID-19 patients with cirrhosis were associated with higher rates of worsening hepatic encephalopathy, ascites and esophageal varices. Patients with cirrhosis receiving mRNA vaccines had significantly better survival rates compared to viral vector or inactivated vaccines. Primary vaccination with the BNT162b2 vaccine was the most effective in preventing acute hepatic decompensating events, COVID-19 infection requiring hospital admission and in-hospital mortality.Patients with cirrhosis are vulnerable to hepatic decompensation events and death following COVID-19 infection. Therefore, primary vaccination with COVID-19 vaccines is fundamental to reducing the risk of COVID-19 related deaths in patients with cirrhosis. However, limited data are available about the effectiveness of mRNA vaccines compared to other vaccines. The aim of our study was to investigate the efficacy of mRNA vaccines versus other vaccines in cirrhosis. In this retrospective study, we compared clinical characteristics and vaccine effectiveness of 399 COVID-19 patients without cirrhosis (GROUP A) to 52 COVID-19 patients with cirrhosis (GROUP B). 54 hospitalised cirrhosis controls without COVID-19 (GROUP C) were randomly sampled 1:1 and matched by gender and age. Of the cirrhosis cases, we found no difference ( Severe acute respiratory syndrome SARS-CoV-2) causing COVID-19 disease has emerged as a pandemic around the globe. Although comorbid conditions such as diabetes, hypertension, chronic lung diseases (particularly COPD), chronic kidney disease and cardiovascular disease have been reported as major risk factors for COVID-19 mortality, the prognostic value of chronic liver disease (in particular liver cirrhosis) is still undefined  causing ,4,5. FurTherefore, we aimed to investigate the impact of primary vaccination with different SARS-CoV-2 vaccines on patients with cirrhosis. Furthermore, the aim of our study was to evaluate the effectiveness of mRNA vaccines, as compared to other COVID-19 vaccines, in cirrhosis.We performed a retrospective multicentre study by reviewing data of COVID-19 patients hospitalized in several centres of Semmelweis University between March 2020 and May 2022. Diagnosis of SARS-CoV-2 required a positive reverse transcription-polymerase chain reaction  test on nasopharyngeal swabs based on the protocol by the World Health Organization . Further2) <60 mm Hg or peripheral oxygen saturation (SpO2) <90% at room air .,25.24,25A study of a COVID-19 cohort of 220,727 US patients reported that hospital mortality rates of COVID-19 patients with cirrhosis and those without cirrhosis were 8.9% and 3.9%, respectively . RegardiAs for the etiology of cirrhosis, alcohol-related liver cirrhosis was found to be a leading cause and patients with alcohol-use disorder are notably more susceptible to hepatic decompensation following COVID-19 infection, in line with recently published studies ,31,32.The Clinical Practice Guidelines of the European Association for the Study of the Liver (EASL) emphasized the significance of regular clinical screening to the prompt detection and treatment of the complications of cirrhosis . SurveilA North American matched cohort of 762 patients reported that COVID-19-related in-hospital mortality was lower in patients with cirrhosis after receiving one or two mRNA vaccines in comparison with unvaccinated patients with liver cirrhosis . MoreoveApart from mRNA vaccines, the Hungarian vaccination campaign included the AZD1222-AstraZeneca, Gam-COVID-Vac-Sputnik V and HB02-Sinopharm vaccines. In a nationwide, retrospective study investigating 3 740 066 Hungarian citizens, the overall estimated effectiveness of AstraZeneca, Sputnik-V and Sinopharm against COVID-19-related death was lower compared to Pfizer-BioNTech and Moderna . Our stuAltogether, our data provide evidence that COVID-19 infection is associated with the progression of liver cirrhosis and the development of acute hepatic decompensation events possibly due to aggravated immune dysfunction. Moreover, our findings demonstrate the effectiveness of primary vaccination with mRNA vaccines in patients with liver cirrhosis, predominantly in those receiving the Pfizer-BioNTech vaccine.The limitations of our data are mostly due to the retrospective study design and relatively small sample size. First, the number of enrolled patients with liver cirrhosis and those following COVID-19 infection were limited in our study. The higher mortality in GROUP C compared to GROUP B supports the notion that patients in GROUP C were admitted in a more vulnerable condition owing to advanced disease progression of cirrhosis. Second, the single-dose Ad26.COV2.S-Janssen vaccine was excluded from our investigation. As the cases were recorded over a prolonged time interval, a potential bias is expected as different cohorts were exposed to different contagion conditions. The study period overlapped with different waves of multiple variants of SARS-CoV-2, which may have influenced the effectiveness of the COVID-19 vaccines investigated in our analysis. Third, some vaccines were categorically indicated for elderly patients or patients with comorbidities, hence confounding patient characteristics could lead to perceived variation in the effectiveness of COVID-19 vaccines. Therefore, further prospective studies are needed to evaluate the effectiveness of all approved COVID-19 vaccines immunizing against new, upcoming variants in patients with cirrhosis.On the other hand, the strengths of our study include its multicentre matched cohort design and its focus on a susceptible population group with a higher risk of COVID-19-related mortality. Although limited data are available about the impact of COVID-19 vaccination regimens on the clinical outcome of patients with liver cirrhosis, our methodology exclusively allows better interpretation to evaluate the impact of different COVID-19 vaccines on hospital outcomes in COVID-19 infection and liver cirrhosis.Patients with cirrhosis experiencing a COVID-19 infection requiring hospitalization were significantly coupled with acute hepatic decompensation events. However, baseline liver cirrhosis severity is a major determinant of in-hospital mortality. Hence, primary vaccination with mRNA vaccines was significantly associated with better a prognosis in patients with cirrhosis. Outstandingly, primary vaccination with the BNT162b2 vaccine was the most effective in preventing acute hepatic decompensating events, COVID-19-related adverse outcomes and in-hospital mortality.


Current methods for profiling DNA methylation require costly reagents, sequencing, or labor time. We introduce FML-seq, a sequencing library protocol that greatly reduces all these costs. Relative to other techniques tested on the same human cell lines, FML-seq produces similar measurements of absolute and differential cytosine methylation at a fraction of the price. FML-seq enables inexpensive, high-throughput experimental designs for large-scale epigenetics research projects. DNA methylation is a crucial and well-studied epigenetic mark in many eukaryotes . HoweverBase-conversion sequencing produces a dual-channel signal, analogous to single-nucleotide polymorphism profiling: each base is read several times, sometimes converted and sometimes not, and percent methylation is calculated as the proportion of total reads in which that base was not converted. Some alternative methods produce a single-channel signal, analagous to RNA-seq, ChIP-seq, or ATAC-seq. Methylated DNA immunoprecipitation (MeDIP-seq) , like ChHere we introduce FML-seq, a method that combines methylation-dependent restriction digestion with an alternative adapter ligation scheme to greatly reduce the time, labor, and cost compared with previous similar protocols. FML-seq fills the gap for rapid, high-throughput, and cost-effective DNA methylation profiling.The protocol for fragmentation at methylated loci and sequencing (FML-seq) comprises only three steps . First, FML-seq’s specificity for methylated cytosine depends on the endonuclease. Concordant with previous reports , digestiThe other factor in FML-seq’s specificity is whether other kinds of gDNA fragmentation produce ligatable ends. To fit the adapters, a fragment must have a 5′ overhang of 4nt with a terminal phosphate, which is produced by the endonuclease but unlikely to result from other kinds of fragmentation. Accordingly, no-endonuclease control libraries using gDNA from either fresh cells or degraded archival tissue showed no detectable library product unless greatly overamplified .To compare FML-seq with other methods, we prepared libraries from four well-studied human cell lines . The seqAt a variety of functional genome elements FML-seq signal was unimodal and log-distributed, unlike measurements from WGBS which tended toward the extremes of 0% and 100% methylation, and the greatest dynamic range of FML-seq signal was among gene promoters . PromoteSerial dilutions from 60ng gDNA input showed successful but lower-quality results down to 6ng . BecauseBy profiling genome-wide DNA methylation more cost-effectively than current methods, FML-seq will allow new studies with a larger scale of samples than financially feasible with previous methods, while the short protocol in 96-well plates will significantly reduce labor and risks of error . RecentlWhereas dual-channel signals like base conversion have roughly equal sensitivity at all loci they measure, FML-seq’s single-channel signal is more precise when the signal is high and less certain when the signal is low. In particular the FML-seq read scores contain more zeroes than observed from base-conversion sequencing methods. These drop-outs could represent loci with low methylation but also ascertainment biases such as loci with few copies of the restriction motif, loci with low accessibility to sequencing (e.g. unmappable duplicated sequences), or low sequencing depth of the library overall. This greater uncertainty at lower-signal sites is analogous to the heteroskedastic RNA-seq signal from both lower-abundance and shorter transcripts, whose ascertainment bias similarly varies from one transcript to the next but can be assumed equal for a given transcript across different samples, and the accuracy of both FML-seq’s methylation scores and its differential methylation ratios appears to be similarly maintained by normalizations. In typical experiments the goal is not to quantify the absolute methylation state of each locus but rather to identify loci whose methylation differs between biological conditions of interest, just as RNA-seq typically searches for differentially expressed transcripts rather than for the absolute molecular copy number of each transcript. If the loci under examination are individual cytosine positions in the genome, base-conversion methods provide precise quantification but FML-seq measures too few positions at realistic sequencing depth; however, a biological question about DNA methylation is likely to consider entire genome regions, such as gene bodies or regulatory elements. Here we have shown that FML-seq quantitatively detects differentially methylated genome regions.mCNNR is very common. We cautiously restricted our data analysis to mCGNR as methylation in the human genome typically occurs at the CpG dinucleotide, but the lambda bacteriophage experiment showed that FML-seq is equally compatible with other genomes in which 5mC occurs in other contexts. Other endonucleases related to MspJI produce the same 5′ 4-base overhang compatible with FML-seq’s adapter design  before the matrices were concatenated. Hierarchical clustering was perfomed by UPGMA on Pearson distances (1 – r) of the concatenated matrix.A subset of 9,876 promoters were measurable by every method . From this subset, the 500 promoters with the greatest variation among cell lines were selected by the greatest Supplement 1Supplement 2Supplement 3Supplement 4Supplement 5


Although cardiac 123I-mIBG imaging has been studied in many cardiac and neurological diseases, it has extensively been studied in ischemic and non-ischemic CHF. Therefore, this review will focus on the role of 123I-mIBG imaging in CHF. This non-invasive, widely available technique has been established to evaluate the prognosis in CHF. Standardization, especially among various combinations of gamma camera and collimator, is important for identifying appropriate thresholds for adequate risk stratification. Interestingly, in contrast to the linear relationship between 123I-mIBG-derived parameters and overall prognosis, there seems to be a “bell-shape” curve for 123I-mIBG-derived parameters in relation to ventricular arrhythmia or appropriate implantable cardioverter defibrillator (ICD) therapy in patients with ischemic CHF. In addition, there is a potential clinical role for cardiac 123I-mIBG imaging in optimizing patient selection for implantation of expensive devices such as ICD and cardiac resynchronization therapy (CRT). Based on cardiac 123I-mIBG data risk models and machine learning, models have been developed for appropriate risk assessment in CHF.Cardiac sympathetic upregulation is one of the neurohormonal compensation mechanisms that play an important role in the pathogenesis of chronic heart failure (CHF). In the past decades, cardiac  Chronic heart failure (CHF) is a clinical syndrome with a growing incidence and prevalence. In addition to the activated renin–angiotensin–aldosterone system and natriuretic peptides, myocardial sympathetic innervation is increased in patients with CHF. Initially, these neurohormonal systems are able to compensate for the impaired myocardial function. However, the long-term activation of these compensation mechanisms has detrimental effects on myocardial structure and function resulting in heart failure (HF) progression.Cardiac sympathetic innervation has several cardiovascular actions including heart rate acceleration (positive chronotropic effect) and increase in myocardial contractility (positive inotropic effect). Norepinephrine (NE) is the neurotransmitter of myocardial sympathetic innervation and is stored in vesicles in the presynaptic nerve terminals. Via exocytosis, NE is released into the synaptic cleft. Most of the released NE undergoes re-uptake into the presynaptic sympathetic terminal nerve axons via the uptake-1 mechanism. This transport system, the so-called norepinephrine transporter (NET), is responsible for approximately 70–90% of the NE re-uptake from the myocardial sympathetic synaptic cleft. As a consequence, only a small amount of the released NE will be available to stimulate the post-synaptic β-adrenergic receptors (β-AR) of the myocytes .In CHF, the increased cardiac sympathetic activity is caused by an increased release of NE into the synaptic cleft. In addition, NE re-uptake via the NET is decreased resulting in elevated synaptic levels of NE. These elevated NE levels result in a spillover into the plasma with consequently increased plasma levels of NE concomitant with the severity of left ventricular dysfunction ,3. InitiClinical assessment of myocardial sympathetic innervation can be performed by measuring NE plasma levels. Although increased NE plasma levels are associated with mortality in CHF , these l123I-meta-iodobenzylguanidine (123I-mIBG) for planar and SPECT imaging and 11C-hydroxyephedrine (HED) for PET imaging. Both tracers are NE analogs resistant to metabolic enzymes and show high affinity for NET allowing the visualization of presynaptic sympathetic nerve function. Other presynaptic PET tracers include 11C-epinephrine, 11C-phenylephrine, and 18F-flubrobenguana . Th. Th123I- imaging  88]. It. It123I-s in TCM . In tota imaging . After 3recovers . Of intee by NET . This le123I-mIBG assessed cardiac sympathetic activity as a measure of response to pharmaceutical or device therapy [123I-mIBG assessed cardiac sympathetic activity in HF [123I-mIBG imaging suggested the patient’s true arrhythmic event risk was extremely low [Despite the numerous studies showing changes of  therapy ,38,59,67ty in HF ,45,86 moty in HF  even if mely low .123I-mIBG-guided therapy improves outcomes in CHF subject. Without such data, cardiologists have little incentive to order cardiac 123I-mIBG scintigraphy for clinical decision making, Eventual approval of a cardiac PET agent capable of quantifying sympathetic innervation will probably make the challenge of convincing the cardiologist to use cardiac 123I-mIBG scintigraphy for clinical decision making even more daunting. In contrast to locations where 123I-mIBG is relatively inexpensive and can be used as a binary diagnostic test agent, it seems unlikely there will be significant growth in cardiac 123I-mIBG imaging in the foreseeable future. However, given the increasing medical costs associated with CHF, a better selection of subjects for expensive device therapy, such as ICD and CRT, is mandatory. The current selection criteria fail to make a proper selection of patients that benefit from these devices [123I-mIBG imaging in CHF, it will be in guiding the selection of these CHF subjects. Although currently available data show promising result for cardiac 123I-mIBG imaging, none of these studies was designed to demonstrate that 123I-mIBG-guided findings can be used to improve patient outcomes.Currently, there are no randomized clinical trials that have evaluated cardiac  devices . If ther123I-mIBG imaging is a non-invasively, widely available imaging technique and has been established to evaluate the prognosis in CHF. Standardization, especially among various gamma camera–collimator combinations is important for identifying appropriate thresholds for adequate risk stratification and extrapolation of data to other institutions. Most importantly, in contrast to the linear relationship between 123I-mIBG-derived parameters and the overall prognosis in CHF, there seems a “bell-shape” curve for 123I-mIBG-derived parameters in relation to fatal arrhythmias. These new insights could be helpful, especially in the optimization of patient selection for expensive device implantation such as ICD and CRT.Cardiac


The ongoing COVID-19 pandemic caused by SARS-CoV-2 infections has quickly developed into a global public health threat. COVID-19 patients show distinct clinical features, and in some cases, during the severe stage of the condition, the disease severity leads to an acute respiratory disorder. In spite of several pieces of research in this area, the molecular mechanisms behind the development of disease severity are still not clearly understood. Recent studies demonstrated that SARS-CoV-2 alters the host cell splicing and transcriptional response to overcome the host immune response that provides the virus with favorable conditions to replicate efficiently within the host cells. In several disease conditions, aberrant splicing could lead to the development of novel chimeric transcripts that could promote the functional alternations of the cell. As severe SARS-CoV-2 infection was reported to cause abnormal splicing in the infected cells, we could expect the generation and expression of novel chimeric transcripts. However, no study so far has attempted to check whether novel chimeric transcripts are expressed in severe SARS-CoV-2 infections. In this study, we analyzed several publicly available blood transcriptome datasets of severe COVID-19, mild COVID-19, other severe respiratory viral infected patients, and healthy individuals. We identified 424 severe COVID-19 -specific chimeric transcripts, 42 of which were recurrent. Further, we detected 189 chimeric transcripts common to severe COVID-19 and multiple severe respiratory viral infections. Pathway and gene enrichment analysis of the parental genes of these two subsets of chimeric transcripts reveals that these are potentially involved in immune-related processes, interferon signaling, and inflammatory responses, which signify their potential association with immune dysfunction leading to the development of disease severity. Our study provides the first detailed expression landscape of chimeric transcripts in severe COVID-19 and other severe respiratory viral infections. The ongoing COVID-19 pandemic is caused by SARS-CoV-2, a recently identified member of the Coronaviridae family ,4,5. DesAberrant splicing promotes the increase in non-canonical splicing events, resulting in the generation of novel chimeric transcripts ,17,18,19SARS-CoV-2 induces the widespread alternation of host cell-splicing machinery and transcriptional dynamics in severe COVID-19 infection, which raises the possibility of the generation and expression of novel chimeric transcripts in severe COVID-19-infected cells. However, no study so far has attempted to check if SARS-CoV-2-induced aberrant splicing in severe COVID-19 infections could generate novel chimeric transcripts, nor the potential functional impact of these chimeric transcripts’ generation. In this study, we integrated publicly available RNA-seq datasets of blood from 85 severe COVID-19 patients, 10 mild COVID-19 patients, 84 other severe respiratory viral infected patients, 54 healthy persons, and 199 samples from 32 different tissues and identified the chimeric transcripts unique to severe COVID-19 infections.RNA-seq data from several sources were collected for this study. A total of 44 blood samples of RNA-seq data from severe COVID-19 patients and 10 samples from healthy donors were obtained from the Gene Expression Omnibus (GEO) database (GSE171110) . RNA-seqUsing our in-house reference-based method ChiTaH , all thePadj ≤ 0.05, a gene was deemed significantly upregulated, and when log2foldchange was negative and Padj ≤ 0.05, it was deemed significantly downregulated. The Benjamini–Hochberg method was used to adjust the p-values and the false discovery rate (FDR) [STAR aligner  was usedte (FDR) .p-value cutoff < 0.05 was set to select the enriched processes and pathways. The KEGG [Metascape  was usedThe KEGG  and REACThe KEGG  pathway We identified a total of 957 chimeric transcripts from 78 bulk RNA-seq and 7 single-cell RNA (scRNA)-seq data from severe COVID-19 patients. In total, 1175 chimeric transcripts were identified in mild COVID-19 patients from 10 bulk RNA-seq samples, and 1281 chimeric transcripts were identified in various severe respiratory viral infections from 84 bulk RNA-seq samples. Moreover, we also identified 1061 chimeric transcripts from 54 healthy samples, while 2066 chimeric transcripts were identified in 199 samples from 32 normal tissues. Chimeric transcripts were observed to be abundant in all samples analyzed ). HoweveChimeric transcripts expressed in multiple severe COVID-19 patient blood samples could be associated with the development of the severity of COVID-19. We have identified 42 chimeric transcripts as recurrent, which are expressed in at least 3 severe COVID-19 patient samples . The genSeveral chimeric transcripts could be generated by the non-canonical splicing-based mechanisms, such as the trans-splicing and cis-splicing of adjacent genes (cis-SAGe) . Trans-sIf the emergence of chimeric transcripts can regulate the functionality of their parental genes, then we might expect the differential expression of at least one of their parental genes. Interestingly, we observed in several instances that at least one of the parental genes of recurrent severe COVID-19 specific chimeric transcripts from different datasets were differentially expressed . This fiCommon chimeric transcripts from multiple severe respiratory viral infections and severe COVID-19 could be associated with the immune response common to the development of disease severity caused by most respiratory viral infections. With this aim, we have identified 189 common chimeric transcripts unique to both severe COVID-19 and multiple other severe respiratory viral infections . Then, wIn this study, we analyzed several publicly available RNA-seq datasets containing 85 severe COVID-19 patients’ blood samples, 10 mild COVID-19 patients’ blood samples, 84 other respiratory viral infected patients’ blood samples, and 253 healthy human samples from 32 different tissues, including blood. We identified 424 severe COVID-19 specific chimeric transcripts; among these, 42 chimeric transcripts were recurrent which were mapped in at least 3 samples. The important enriched biological processes and pathways of the parental genes of these recurrent severe COVID-19 specific chimeric transcripts highlighted that they are involved in cellular stress response, phagocytosis, and interferon signaling. The genes associated with the cellular stress response pathway could be involved in the activation of innate and adaptive immune responses and play a pivotal role in host defenses and inflammation . Next, wp-value criteria selected to screen the differentially expressed genes. We chose the strict p-value, p <0.05, as the criterion for detection of differential gene expression, so there is a chance that we could have missed some differentially expressed genes in some datasets because of the quality of the sequencing data and read count bias. Interestingly, instead of the difference of sequence quality in different datasets, we mapped all the recurrent severe COVID-19 specific chimeric transcripts detected in the E-MTAB-10926 samples in the single-cell high-quality (scRNA)-seq dataset from the PRJNA633393 samples. Therefore, the production of the chimeric transcripts could be the important signature behind the generation of stress responses leading to COVID-19 severity development. Furthermore, we found 189 common chimeric transcripts unique to both severe COVID-19 and other severe respiratory viral infections whose parental genes were enriched in adaptive immune response and inflammatory response. This could signify these chimeric transcripts might be associated with stress responses associated with dysfunctional immune responses common to multiple respiratory viral severe infections.If the chimeric transcripts alter the functionality of their parental genes, then we should expect at least one of their parental genes could be differentially expressed. Interestingly, we observed in several instances that one of the parental genes of recurrent severe COVID-19 specific chimeric transcripts was differentially expressed. We observed consistent differential gene expression patterns for some chimeric transcript’s parental genes in all datasets; however, the chimeric transcripts mapped in some datasets, but not all . We chosOur study first identified the chimeric transcripts unique to severe COVID-19 infections and demonstrated their potential functional importance . During The findings of this present study suggest the potential role of recurrent severe COVID-19 specific chimeric transcripts in alternations of immune-related processes, activating the processes that favor the viral transcription, gene expression, and alternations of various biological and metabolic processes that could lead to the development of severe disease. Furthermore, we identified common chimeric transcripts found in severe COVID-19 and other severe respiratory viral infections. The parental genes of these common chimeric transcripts are functionally enriched for adaptive immune response and inflammatory response. These observations suggest these chimeric transcripts could be associated with different stresses associated with the dysfunctional immune response generated during multiple severe respiratory viral infections, including COVID-19. The limitation of our study was the lack of experimental validation, as we relied on publicly available RNA-seq data from patients. Further, the study’s use of publicly available datasets had varying experimental designs, and the patient samples came from diverse populations which were collected at different time points during the development of COVID-19 severity. Despite the limitations inherent to computational analysis, this study successfully uncovered the hidden layers of the blood transcriptome from the various respiratory viral infected patients and identified several potential functionally relevant chimeric transcripts unique to severe COVID-19 and common to multiple severe respiratory virus infections. Further experimental studies are necessary to elucidate the specific functions of these chimeric transcripts in COVID-19 pathogenesis.


Case Report. Case 1. A 36-year-old woman with a history of right hemithyroidectomy for a benign thyroid nodule, on a stable dose of levothyroxine with euthyroid labs, presented with progressively worsening left neck pain, episodic palpitations, and heat intolerance after the second dose of mRNA1273 (Moderna) vaccine. Examination revealed an enlarged and tender left lobe of the thyroid with suppressed TSH but normal free T4 and ESR, signifying subacute thyroiditis. She was managed conservatively without corticosteroids or beta-blockers, and her symptoms resolved. A follow-up revealed increasing TSH, and levothyroxine was restarted. Case 2. A 33-year-old man with a history of anxiety disorder on Sertraline, presented with a two-week history of palpitations, heat intolerance, and 10-pound weight loss after the second dose of BNT162b2 (Pfizer-BioNTech) vaccine. Examination revealed a normal thyroid gland with no tenderness with elevated thyroid peroxidase and thyroglobulin antibodies. Ultrasound showed a diffusely heterogeneous thyroid with increased vascularity, suggesting silent thyroiditis. Follow-up revealed a hypothyroid phase with high TSH for which levothyroxine supplementation was started. Discussion. COVID-19 vaccine-associated subacute and silent thyroiditis have occurred following all three kinds of available vaccines, characterized by an initial thyrotoxic phase, followed by a hypothyroid phase and a recovery phase. Hypotheses include an immune response triggering thyroid inflammation or cross-reactivity with viral proteins.  SARS-CoV-2 has been known to cause multisystemic involvement, gaining entry through ACE-2 and TMPRSS2 receptors. COVID-19 vaccine-associated thyroiditis cases are now being reported.  COVID-19 vaccine-associated thyroiditis is rare, but long-term monitoring of these patients is essential to ensure appropriate diagnosis and management of the potential hypothyroid phase. The novel coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has affected more than 250 million people worldwide .Several effective vaccines have been introduced, and currently, mass vaccination drives are underway. The inactivated vaccines use adjuvants such as aluminum salts and toll-like receptor (TLR) agonists to increase the immunogenic response to vaccination . HoweverWe report two cases of thyroiditis that developed soon after receiving the second dose of the mRNA COVID-19 vaccine. The article's objective is to create awareness about a possible association between thyroiditis and the COVID-19 mRNA vaccines.A 36-year-old woman with a history of right hemithyroidectomy for a benign thyroid nodule six years ago, presented with a 6-week history of progressively worsening left-sided neck pain accompanied by palpitations and heat intolerance. She developed transient myalgia and neck pain after her first dose of mRNA1273 (Moderna COVID-19 vaccine). Her current symptoms started 2 weeks after receiving the second dose of the vaccine. She had been euthyroid on a stable dose of levothyroxine for the last 6 years. She denied any history of viral or respiratory illness prior to the onset of symptoms. She had no family history of thyroid disorders or autoimmune conditions. Laboratory evaluation revealed suppressed TSH at urgent care triggering a consultation with an endocrinologist, which took place in a few weeks, by when her symptoms were spontaneously resolving.On physical examination, her heart rate was 88/min, the temperature was 37.7°C, her blood pressure was 110/65, and her respiratory rate was 20/min. A neck examination revealed an enlarged and tender left thyroid lobe. There was no palpable lymphadenopathy, exophthalmos, pharyngeal erythema, irregular heart rhythm, hepatosplenomegaly, edema, tremors, or skin lesions. Laboratory investigations revealed a TSH of less than 0.01 mIU/L with a free T4 of 1.8 ng/dl on the upper limit of normal . AntithyA 33-year-old man with a past history of anxiety disorder treated with sertraline presented with a two-week history of persistent palpitations, heat intolerance, and 10 pounds of weight loss. He reported no past or family history of thyroid disorders. He did not have any history of COVID-19 infection and was vaccinated with both doses of BNT162b2 (Pfizer-BioNTech COVID-19 vaccine) 4 weeks apart. Soon after receiving the second vaccine dose, he developed a high-grade fever at 102.5 F, myalgia, and sore throat, which persisted for 2 weeks. Subsequently, he experienced palpitations, diaphoresis, and heat intolerance along with generalized weakness. An endocrinology referral was triggered as he was found to have suppressed TSH along with elevated CRP at his primary care provider's office . On physWe describe two cases of thyroiditis associated with the COVID-19 vaccine, subacute thyroiditis for the first case and silent thyroiditis for the second case. Both patients had an initial thyrotoxic phase followed by a hypothyroid phase which required levothyroxine supplementation.Subacute and silent thyroiditis is a self-limiting thyroid disorder. They are more prevalent in women. Viral infections and immune-modulatory drugs have been implicated as etiological factors. Characteristically, they have an initial destructive thyrotoxic phase, followed by a hypothyroid phase and a recovery phase, leading to the normalization of thyroid functions .The endocrine system has been found to be involved in COVID-19, causing hypopituitarism, adrenal necrosis, pancreatic beta-cell damage, gonadal dysfunction, and thyroiditis . The exaAlong with the common viruses such as CMV and HSV, cases of thyroiditis have emerged in association with COVID-19. The ACE-2 and TMPRSS2 that allow SARS-CoV-2 to infiltrate human cells are highly expressed in the thyroid follicular cells [A recent study by Muller et al. suggested silent thyroiditis induced by COVID-19 occurs in an underlying setting of nonthyroidal illness in critically ill male patients. This was usually followed by a permanent hypothyroid phase . HoweverAdditionally, an increasing number of cases of COVID-19 vaccine-associated thyroiditis are now being reported. These cases have occurred following all three kinds of available vaccines, inactivated SARS-CoV-2 vaccine, mRNA-based vaccine, and vector-based vaccine. Most cases that developed after the inactivated vaccine have been attributed to adjuvants, triggering adverse reactions in genetically predisposed individuals, and causing ASIA syndrome –15. In aA few other cases have been described, including a 42-year-old female who developed painful hyperthyroid symptoms five days after the first dose of the BNT162b2 vaccine and required a short course of steroids and beta-blockers with which her symptoms improved . SimilarAdenovirus-vector vaccines stimulate innate immunity sensors whereas mRNA vaccines encode the viral spike S glycoprotein, causing a potent immunological response. The vaccine is formulated in lipid particles which enable the delivery of RNA into the host cells to allow expression of the SARS-CoV-2 S antigen. This process elicits an immune response to the S antigen, which protects against COVID-19 infection .The mechanism of postvaccination-associated thyroiditis remains unknown. It has been hypothesized that vaccinations may be responsible for producing an immune response that triggers thyroid inflammation in predisposed patients. Another hypothesis includes the SARS-CoV-2 spike protein, nucleoprotein, and membrane protein which cross-reacts with thyroid peroxidase .There has been some recent understanding regarding the potential immune upheaval due to mRNA COVID-19 vaccination and its role in causing myocarditis, particularly in young men. The mechanism may include molecular mimicry between the spike protein of SARS-CoV-2 and self-antigens, increased cytokine expression secondary to dysregulated immune pathways, or an exaggerated immune response to viral mRNA .In these two cases of thyroiditis after the mRNA COVID-19 vaccination, although a direct causal relationship could not be established, however, no other causes could be identified and temporality with the administration of the mRNA COVID-19 vaccine is very strongly suggestive. Up to 10% of patients with subacute thyroiditis develop permanent hypothyroidism. Hence, it is important that these patients are followed long-term . OverallCOVID-19 vaccine-induced thyroiditis can range from subacute to silent presentation and is a rare phenomenon. The purpose of this report is to raise awareness among providers about the same. It is important for clinicians to be aware of this presentation to recognize COVID-19 vaccine-related immune effects in the thyroid to accurately manage the relevant stages of thyroiditis. Long-term monitoring of these patients is essential to ensure an appropriate diagnosis of the potential hypothyroid phase in them. Further investigation is needed to determine possible predisposing factors and mechanisms leading to thyroiditis after the COVID-19 vaccine.


This study investigated longitudinal clinical, structural and functional brain alterations in Parkinson’s disease patients with freezing of gait (PD-FoG) and in those developing (PD-FoG-converters) and not developing FoG (PD-non-converters) over two years. Moreover, this study explored if any clinical and/or MRI metric predicts FoG development. Thirty PD-FoG, 11 PD-FoG-converters and 11 PD-non-converters were followed for two years. Thirty healthy controls were included at baseline. Participants underwent clinical and MRI visits. Cortical thickness, basal ganglia volumes and functional network graph metrics were evaluated at baseline and over time. In PD groups, correlations between baseline MRI and clinical worsening were tested. A ROC curve analysis investigated if baseline clinical and MRI measures, selected using a stepwise model procedure, could differentiate PD-FoG-converters from PD-non-converters. At baseline, PD-FoG patients had widespread cortical/subcortical atrophy, while PD-FoG-converters and non-converters showed atrophy in sensorimotor areas and basal ganglia relative to controls. Over time, PD-non-converters accumulated cortical thinning of left temporal pole and pallidum without significant clinical changes. PD-FoG-converters showed worsening of disease severity, executive functions, and mood together with an accumulation of occipital atrophy, similarly to PD-FoG. At baseline, PD-FoG-converters relative to controls and PD-FoG showed higher global and parietal clustering coefficient and global local efficiency. Over time, PD-FoG-converters showed reduced parietal clustering coefficient and sensorimotor local efficiency, PD-non-converters showed increased sensorimotor path length, while PD-FoG patients showed stable graph metrics. Stepwise prediction model including dyskinesia, postural instability and gait disorders scores and parietal clustering coefficient was the best predictor of FoG conversion. Combining clinical and MRI data, ROC curves provided the highest classification power to predict the conversion . Structural MRI is a useful tool to monitor PD progression, while functional MRI together with clinical features may be helpful to identify FoG conversion early. Despite the great research effort, the pathophysiology underlying FoG is still debated. Current evidence supports the multifactorial nature of FoG, suggesting that the alteration of motor, cognitive and behavioral resources might contribute to FoG manifestation as a consequence of fronto-striatal failure7.Freezing of gait (FoG) is a disabling phenomenon affecting many Parkinson’s disease (PD) patients with the progression of the disease14. Consistent findings have widely demonstrated fronto-parietal atrophy in FoG patients relative to those without and healthy controls resulting in executive-attentive and visuospatial dysfunctions, and temporo-parietal cortical alterations being responsible for altered self-body sensorimotor perception and motor planning16. Moreover, evidence reported that reduced volume of basal ganglia in PD-FoG patients is responsible for impaired gait automaticity and motor control8. Many studies have reported reduced resting-state functional connectivity (FC) in default-mode, executive-attentive, visual and sensorimotor networks in PD-FoG patients relative to patients without FoG and healthy controls20. Both increased and decreased FC was found between cortico-subcortical and cerebellar structures suggesting that FC changes might be related both to neurodegeneration and compensatory mechanisms23. Graph analysis is a relatively recent tool useful to characterize the functional brain network topology in terms of global and local network organization, which has been found to be disrupted in PD25. Few recent studies using graph analysis26 suggested an altered FC network organization of frontal areas and of the dorsal attention network in PD-FoG patients relative to PD without FoG and healthy controls.Several MRI studies reported the presence of widespread brain cortical and subcortical grey matter (GM) damage and functional alterations in PD-FoG patients30. On the other hand, very few studies provided longitudinal MRI data to explore the progression of PD-FoG patients and even fewer MRI studies monitored over time PD patients developing FoG in order to identify predictive brain signs of FoG onset32. Preliminary findings showed that baseline white matter hyperintensities and midbrain dysfunction might predispose for higher risk of developing FoG over time32. Moreover, one study has explored the progression of both structural and functional brain alterations in PD-FoG focusing on the thalamus and suggested that a model including thalamic local volume inflation measures predicts FoG conversions with good accuracy33. Within this framework, the aims of this study were: 1) to investigate whole-brain cortical and subcortical GM changes and functional network properties at baseline and over 1- and 2-year follow-up in PD-FoG patients, in patients developing FoG (PD-FoG-converters) and in PD-non-converters; and 2) to identify the best prediction model of FoG development combining both clinical and MRI features.To date, several studies suggested the presence of clinical motor and non-motor predictors of FoG development, including for instance gait and balance deficits, cognitive changes, mood alterations, hyposmia and REM sleep behavior disordersAt baseline, patients and healthy controls were similar for age, sex, education, and handedness. The three groups of patients were comparable for all the above-mentioned variables and also for age at onset, disease duration, HY, UPDRS-III, NMS-Q, LEDD and for all cognitive measures at baseline Table . RelativLongitudinal clinical results showed a worsening of motor symptoms (UPDRS-III beta=9.64), language functions (semantic fluency beta = −4.15), and mood  in PD-FoG-converters, and a worsening of motor symptoms (UPDRS-III beta=6.11), global cognition (ACE tot beta=5.18), executive-attentive functions , memory  and depressive symptoms (HDRS beta=3.90) in PD-FoG. Moreover, the latter group showed significantly higher LEDD over time (beta = 167.28). No significant differences were found in PD-non-converters over time and in group-by-time interaction between groups.PD-FoG patients showed reduced mean thickness of bilateral superior temporal sulci, left sensorimotor  and right supramarginal areas Fig.  and reduOver time, PD-non-converters showed a significant mean cortical thinning of left temporal pole and a trend toward a significant reduced volume of the left pallidum Fig. . PD-FoG-Correlation analysis showed that in PD-FoG patients cortical atrophy of left precentral gyrus at baseline correlated with worsening of executive functions at 2-year follow-up; lower global local efficiency and global clustering at baseline correlated with worsening of visuo-spatial functions at two years; and lower parietal local efficiency and clustering coefficient at baseline correlated with worsening of global cognition and executive functions at 1-year follow-up Fig. .Fig. 4Cot = −2.03; p = 0.05) and PIGD score , and parietal clustering coefficient  was the best predictor of FoG conversion. Hereafter, ROC curve analysis showed that clinical variables resulting from the stepwise model at baseline distinguished PD-FoG-converters from PD-non-converters with a good accuracy   Fig. . MRI var–1) Fig. . Combini–1) Fig. . ConsideIn this study, we investigated the clinical, cortical and subcortical GM and functional brain network alterations over 2-year follow-up in PD-FoG patients, PD-FoG-converters and PD-non-converters. Moreover, we assessed the role of MRI variables together with clinical features in identifying PD-FoG-converters over two years of follow-up. Our study has the main strengths to include PD subgroups matched for confounding variables at baseline and to monitor FoG progression and conversion using both clinical measures and multiparametric MRI metrics longitudinally. Our findings support the important role of structural and functional MRI to monitor disease progression and to detect brain changes that might precede the development of specific clinical features such as FoG.33 investigated the role of thalamic structural changes in predicting FoG development. Both the global volume and sub-regional shape variations of subcortical structures were investigated suggesting that specific local shape inflation of the thalamus predicted conversion with a good accuracy (AUC = 0.87). In our study, we only performed a global volume analysis and we confirmed that the global volume of the thalamus is not altered in PD-FoG-converters relative to PD-non-converters. At baseline, we observed that PD-FoG patients had a widespread cortical/subcortical atrophy, while both PD-FoG-converters and non-converters showed patterns of GM atrophy only in sensorimotor areas and basal ganglia. These results are in line with the clinical characteristics of the patients. Indeed, despite our PD groups were statistically matched for disease severity and disease duration, PD-FoG patients were in a more advanced stage of the disease presenting a slightly higher UPDRS III score relative to PD-non-converters and cognitive/behavioral alterations relative to healthy controls  and cognitive characteristics at baseline, the pattern of progression that we observed in PD-FoG-converters might represent a more aggressive disease spreading according to the Braak model6. The fast structural changes of posterior cortical areas might be associated with the accumulation of executive-attentive deficits and with the development of FoG, supporting once again the multifactorial nature of this phenomenon34. Cortical thickness changes in PD-FoG-converters are similar to the pattern of posterior cortical thinning that we observed in PD-FoG patients (i.e. thinning of the isthmus cingulate). PD-FoG patients also continued to worsen in all cognitive domains and mood. Interestingly, the reduced cortical thickness of the motor cortex in PD-FoG at baseline correlated with the accumulation of executive deficits over 2 years, suggesting the strong interaction between motor and cognitive alterations in PD-FoG patients as previously reported8.One recent studyls Table . Accordi36. The pattern of increased parietal clustering coefficient might also represent an attempt to compensate for the incremental structural alteration of posterior cortical areas in PD-FoG-converters34. Interestingly the higher parietal clustering coefficient at baseline contributed to differentiate PD-FoG–converters from PD-non-converters (AUC = 0.78), suggesting that a topological functional alteration of the parietal cortex might be considered a red flag preceding FoG development. We also found that specific clinical variables such as a higher PIGD score at the UPDRS and more frequent dyskinesia might contribute to identify PD-FoG-converters relative to PD-non-converters at baseline (AUC = 0.91). The higher PIGD and dyskinesia scores might represent a reduced response to pharmacological therapy that is known to be associated with FoG development. Previous studies already suggested these clinical features as potential predictors of FoG38. Other studies suggested that also non-motor symptoms including REM sleep disorders, cognitive and mood alterations might contribute to FoG development30, however we did not observe any difference between PD-FoG-converters and PD-non-converters at baseline in our sample. Our study has the main strength to combine clinical and MRI data showing that the highest accuracy of the prediction model (AUC = 0.95) metric could be obtained combining clinical and functional topological MRI data.Our study investigated also functional topological brain network alterations underlying FoG development. Graph metrics are useful to understand how the brain is topologically organized to support efficient information processes at a local and global level. At baseline, PD-FoG-converters showed higher global and parietal local efficiency and clustering coefficient relative to PD-FoG cases and healthy controls, suggesting an increased functional communication in the parietal network and globally in the brain in PD-FoG-converters. These findings are in line with recent evidence supporting the presence of a cholinergic up-regulation in specific gait control networks  as a compensatory mechanism to counteract aberrant dopaminergic function in PD24. This hypothesis is supported also by the significant correlations that we found between the lower global local efficiency, global clustering and parietal clustering at baseline and the worsening of attentive/executive functions at 1-year and 2-year follow-up in PD-FoG patients. On the contrary, the increased parietal clustering coefficient at baseline could be also considered a maladaptive process as a consequence of neuronal loss. In any case, the central role of parietal structural and functional alterations in identifying most severe PD cases and PD-FoG is well-established40. This area is crucial for the sensory integration of spatio-temporal inputs, for working memory processes and for movement programming43. Thus, it is easy to understand how the structural/functional disruption of the parietal lobe could contribute to FoG manifestation and to executive-attentive dysfunctions44. Our findings suggest the importance of studying the parietal functional topological characteristics from the initial stages of the disease in order to detect early functional reorganization that might help foresee FoG development.Over 2 years, both PD-non-converters and PD-FoG-converters showed a functional topological disruption. The increased sensorimotor path length in the PD-non-converter group and the decreased sensorimotor local efficiency in PD-FoG-converters might represent a reduced ability to transfer information within the sensorimotor network and between the sensorimotor and other networks as a consequence of the disease progression. In addition, PD-FoG-converters likely lost the ability to compensate showing a reduction of parietal clustering coefficient. One might speculate that the progressive loss of this compensatory mechanism at the parietal level could contribute to FoG development. This is in line with recent evidence suggesting that PD-FoG patients showed reduced global and local brain functional efficiency relative to healthy controls and PD without FoGp = 0.05). Second, we did not have longitudinal MRI data in healthy subjects that could be useful to control for the aging effects. Third, we used a 1.5 T MRI scanner, which is characterized by a lower spatial resolution and BOLD signal to noise ratio resolution compared with higher field strength scanners. Fourth, PD patients were evaluated in ON status. However, our analysis accounted for both LEDD at baseline and LEDD change over time in order to mitigate the effect of this potential confounding factor. Fourth, a longer follow-up might improve the possibility to monitor FoG conversion and progression; however, to the best of our knowledge, this is the first longitudinal study exploring brain correlates underlying FoG development in PD patients. Fifth, given the small sample size of the PD-FoG-converter and PD-non-converter groups, the prediction model might suffer from overfitting of data. Future studies should verify our preliminary findings in an independent sample.This study is not without limitations. First, the samples of PD-FoG-converters and PD-non-converters were quite small and this could have contributed to the absence of group-by-time interaction in the clinical and MRI findings. Moreover, the three groups of PD patients were statistically matched for UPDRS-III although bordering statistical significance . PD-FoG patients had FoG at baseline (according to the criteria reported in the next paragraph), while PD-FoG-converters developed FoG over the 2-year follow-up. Patients received a comprehensive evaluation in ON medication state including neurological, cognitive/behavioral and MRI assessments at study entry and at 1-year and 2-year follow-up. MRI scans were acquired to evaluate regional cortical thickness, basal ganglia volumes and functional network graph metrics alterations. Patients were excluded if they had: Hoehn and Yahr (HY) score > 4 and/or dementia47 because they are usually less cooperative and may have some difficulties to stay still into the MRI scanner and to participate to all the study visits; moderate/severe head tremor at rest; cerebrovascular disorders (including vascular parkinsonism) or intracranial masses on routine MRI; history of traumatic brain injury; any other major neurological and medical condition; and MR images with artefacts. Thirty healthy controls matched for age, sex and education, without any neurological, psychiatric, or other disorders, were also recruited among friends and non-consanguineous relatives of patients and by word of mouth and performed clinical cognitive evaluation and MRI assessments at baseline as comparison with PD groups.A sample of 52 idiopathic PD patientsEthical standard committees on human experimentation of IRCCS Ospedale San Raffaele, Milan, Italy and Clinic of Neurology, Faculty of Medicine, University of Belgrade, Serbia approved the study protocol; all participants provided written informed consent prior to study inclusion.48 was calculated. Disease severity was defined using the HY stage score49 and the UPDRS-III50. The presence and the severity of FoG was defined according to both the observation of FoG by a neurologist (item 3.11 UPDRS-III ≥ 1) and the FoG Questionnaire (item 3 of FoG-Q ≥ 2)51. Motor experiences of daily living were assessed using UPDRS-II50. Non-motor complications such as dyskinesia and fluctuations were evaluated using UPDRS-IV50. Non-Motor Symptoms Questionnaire (NMS-Q) was used to assess non-motor symptoms 52. The most prominent clinical features of REM Sleep Behavior Disorder were assessed with the RBD Screening Questionnaire (RBDSQ)53.At study entry and each follow-up visit, an experienced neurologist blinded to MRI results performed clinical assessments. Patients were examined in ON state . Demographic, general clinical and family data  were obtained using a semi-structured interview. Levodopa equivalent daily dose (LEDD)11.At study entry and each follow-up visit, patients performed neuropsychological and behavioral evaluations within 48 h from MRI by expert neuropsychologists, blinded to the neurological data and MRI results. All the neuropsychological and behavioral variables were acquired at each time point. The same test battery was applied in healthy controls at study entry. The assessment evaluated global cognition with the Addenbrooke’s Cognitive Examination-revised (ACE-R); memory with the Rey Auditory Verbal Learning Test (RAVLT), and the pattern (PRM) and spatial (SRM) recognition memory tests from the Cambridge Neuropsychological Test Automated Battery (CANTAB); executive functions with the digit span backward, Intra/Extra Dimensional Set Shift test (IED) from the CANTAB, and the Stroop color-word test; attention and working memory with the digit ordering test and the letter cancellation test; language with the Boston Naming Test (BNT) and the language subtest of ACE-R; language fluency with semantic and phonemic fluencies; visuospatial abilities with the Hooper Visual Organization test and the visuospatial subtest of ACE-R. Mood was evaluated with the Hamilton Depression Rating Scale score (HDRS), Hamilton Anxiety Rating scale score (HAMA) and Apathy Evaluation Scale. For neuropsychological references, see our previous works2; slice thickness=4 mm, 200 sets of 30 contiguous axial slices). During RS fMRI scanning, subjects were instructed to remain motionless, to keep their eyes closed, and not to think about anything in particular; 2) high resolution 3D sagittal T1-weighted Turbo Field Echo (TFE) ; and 3) dual-echo (DE) turbo spin-echo .Baseline and follow-up brain MRI scans were acquired on the same 1.5 Tesla Philips Medical System Achieva machine. Subjects were scanned between 10 and 11 a.m., i.e., treated PD patients were 90–120 min after their regular morning dopaminergic therapy administration in order to be sure they were in a stable ON phase. The following MR sequences were obtained in the following order: 1) gradient-echo (GRE) echo planar imaging (EPI) for RS fMRI 55 as previously described11 and the mean cortical thickness measures of each ROI were calculated.Cortical reconstruction and estimation of mean cortical thickness (34 regions of interest [ROIs] per hemisphere) based on gyral and sulcal structureshttps://fsl.fmrib.ox.ac.uk/fsl/fslwiki/FIRST) was applied to 3D T1-weighted TFE images of each subject at each visit and used to automatically segment basal ganglia bilaterally. Mean basal ganglia volumes were calculated and multiplied by the normalization factor derived from SIENAx to correct for subject head size (http://www.fmrib.ox.ac.uk/fsl/sienax/index.html).FMRIB’s Integrated Registration and Segmentation Tool (FIRST) in FSL (http://www.brain-connectivity-toolbox.net). Eighty-three ROIs from both hemispheres were grouped into six anatomical macro-areas: temporal, parietal, occipital, fronto-insular, basal ganglia, and sensorimotor areas57. Network metrics  were assessed to characterize the topological organization of global brain and lobar networks20. The strength of a node is defined as the sum of weights of links connected to the node. Path length is the average shortest path to reach another node. Clustering coefficient is how well a node tends to cluster and so to be segregated in the network or in the brain.Global and mean lobar functional network characteristics were explored using the Brain Connectivity Matlab toolbox , resulting in 83 areas, which were used to define the brain nodes for the network analysis. Briefly, the images were automatically processed following the standard freesurfer procedure, which includes brain extraction, segmentation of grey matter (GM) and white matter (WM), and parcellation in cortical and subcortical regions. GM was parcellated according to a standardized atlas54 and segmented in 68 cortical areas. Segmentations of basal ganglia were obtained from the Freesurfer pipeline and added to the previous 68 areas.T1-weighted images were processed and parcellated using the Freesurfer suite (V 5.3 http://www.fmrib.ox.ac.uk/fsl/melodic/)58. The following FSL-standard preprocessing pipeline was applied: (1) motion correction using MCFLIRT; (2) high-pass temporal filtering (lower frequency: 0.01 Hz); (3) spatial smoothing (Gaussian Kernel of FWHM 6 mm). Head motion parameters (mean absolute cumulative translation and mean rotation) are reported in Supplementary Table RS fMRI data processing was carried out using the FMRIB software library (FSLv5.0). First, T1-weighted images were skull stripped using the Brain Extraction Tool and segmented in GM, WM, and cerebrospinal fluid (CSF) maps using the FMRIB’s Automated Segmentation Tool. Resulting images were registered into the RS fMRI native space of each subject through a 7 degree-of freedom (DOF) linear affine transformation using FMRIB’s Linear Image Registration Tool. The first four volumes of the fMRI data were removed to reach complete magnet signal stabilization. Then, individual RS fMRI images were processed using MELODIC  of the matrix. Pearson’s correlation coefficients were then converted to z-scores using Fisher’s r-to-z transformation. Negative values were set as ‘NaN’ to mark these brain regions as unconnected58. Functional connections of each subject were required to be present in each structural connectivity matrix, i.e., we measured functional interactions only where an anatomical connection between two areas occurs in the independent healthy control sample. Indeed, functional connectome matrices are dense and we need to apply a threshold in order to avoid spurious functional connections. Since neuron-to-neuron transmission along network connections is the most likely mechanism for the pattern of pathological spread in neurodegenerative diseases, we decided to mask functional matrix with structural data. Consequently, we measured functional interactions only where anatomical connection is present. The construction of the white matter network in an independent healthy control group  has been already described in our previous paper20.Functional connectivity matrices were obtained on the basis of correlation analysis. Mean time series were extracted from each region of interest by averaging the signal from all voxels within each region. RS fMRI data were masked with subject’s GM map in order to consider only voxels corresponding to GM and avoid the effect of atrophy. Cortical GM was segmented using SPM12, while basal ganglia  maps were obtained using FIRST in FSL. The Pearson’s correlation coefficient between the mean time-series of each node pair, indicating the level of functional connectivity between regions Socio-demographic and clinical variables, the mean cortical thickness of 34 ROIs per hemisphere, the mean basal ganglia volumes and graph metrics were compared between groups at the study entry using the Kruskal-Wallis test, followed by Dunn’s post-hoc test for continuous data, or Fischer’s exact test for categorical data. Linear mixed effect models were estimated in PD groups and group-by-time interaction was assessed to evaluate longitudinal between-group differences using time as a continuous variable. Random effect of subject for each model has been considered. Such models were adjusted for age, sex, LEDD at baseline and LEDD change over time.vs PD-FoG-converters; (ii) PD-non-converters vs PD-FoG-converters; and (ii) PD-non-converters vs PD-FoG. The approach used for the stepwise model was a bidirectional elimination, a combination of forward selection and backward elimination. We tested the multicollinearity between different predictors.Baseline MRI metrics which were found to be significantly different between groups were correlated with clinical data showing statistically significant changes over 1- and 2-years using Spearman’s correlation. Moreover, a ROC curve was calculated to identify the accuracy of clinical and MRI metrics in distinguishing PD-FoG-converters from PD-non-converters. Stepwise model selection procedure was applied to candidate baseline clinical and MRI metrics chosen a priori on the basis of variables that were significantly different in the following comparisons: (i) controls p values were Bonferroni-corrected for the number of group comparisons at p < 0.05.All statistical analyses were performed using SPSS (version 26) and R Statistical Software (version 4.0.3); Further information on research design is available in the Supplementary materialReporting Summary


In order to assess SARS-CoV-2 real time quantitative polymerase chain reaction (RT-qPCR) results in a real-life setting, three independent laboratories in Graz (Austria) set up a continuous cross comparison schedule. The following test systems were used: The QIAGEN NeuMoDx SARS-CoV-2 Assay, the Allplex™ 2019-nCoV Assay (Seegene) on a MicroLab Nimbus (Hamilton) platform combined with RealStar SARS-CoV-2 RT-PCR Assay (Altona Diagnostics GmbH), and the cobas SARS-CoV-2 test on a fully automated cobas 6800 system (Roche). A total of 200 samples were analysed, 184 (92%) were found to be concordant with all testing platforms, 14 (7%) discordant. Two (1%) samples tested invalid on a single platform and were excluded from further analysis. Discordant results were distributed randomly across the assays. The Ct values from all assays correlated closely with each other. All discordant samples showed Ct values ≥ 26. SARS-CoV-2 RT-qPCR assays may show considerable variability, especially in samples with low viral RNA concentrations. Decision makers should thus balance the advantages and disadvantages of RT-qPCR for mass screening and adopt suitable strategies that ensure a rational management of positive samples with high Ct values. SARS-CoV-2 testing by real time quantitative polymerase chain reaction (RT-qPCR) has been pivotal for patients, health care providers, and political decision makers to manage the ongoing COVID-19 pandemic. Due to its high sensitivity and specificity, SARS-CoV-2 RT-qPCR testing allows the early identification of infected individuals regardless of clinical symptoms. This aspect is of particular importance, as infected individuals are already contagious during the incubation period, and long before the development of SARS-CoV-2 specific antibodies . These fUsing a highly sensitive method, such as RT-qPCR, for large scale SARS-CoV-2 testing is particularly critical as minimal amounts of viral RNA can produce a positive result. In fact, some CE-IVD certified SARS-CoV-2 RT-qPCR tests run up to 45 cycles in order to maximize sensitivity. However, in vitro experiments suggest that cycle threshold (Ct) values of 26 and higher indicate a low viral load, which is insufficient to infect cultured cells [In order to assess SARS-CoV-2 RT-qPCR results in a real-life setting, three independent laboratories in the city of Graz (Austria) set up a quality assurance initiative establishing a continuous cross comparison scheme, where samples were exchanged daily for several months. The participating laboratories were all involved in routine SARS-CoV-2 RT-qPCR testing throughout the pandemic and used different analytical systems.A total of 200 samples were tested with three different testing platforms . Two samFor all concordant results, Ct values obtained were compared . DespiteAll discordant samples are shown in Here we show that the parallel use of different SARS-CoV-2 RT-qPCR systems, which was often necessary during the COVID-19 pandemic, harbors a significant risk of discordant or non-reproducible results, especially when testing low risk populations. Importantly, discordant results were obtained with all assays included. The potential implications of variable results especially in samples with low amounts of viral RNA should motivate decision makers to adapt existing RT-qPCR based screening programs for SARS-CoV-2, especially in asymptomatic individuals.In this study, the SARS-CoV-2 RT-qPCR assays from QIAGEN, Roche, and Altona agreed reasonably well. Recently published data from the National Austrian External Quality Assurance (EQA) scheme showed that 1–7% of the recorded results for individual samples included in a proficiency panel were reported false negative . The higWhile the issue of false negative SARS-CoV-2 RT-qPCR results has been discussed widely in the scientific community, much less is known about false positives ,9. WithoCauses of false positives SARS-CoV-2 RT-qPCR tests include contamination during sampling, RNA extraction, and PCR amplification ,11. TherThe present results provide clear evidence that SARS-CoV-2 RT-qPCR results vary when viral load is low. We and others have shown that individuals with low viral load are usually not infectious, as shown by cell culture-based infection assays ,15. TherA major strength of the present study is that the initial analysis by QIAGEN and retesting with another assay were performed using the same sample, which rules out sample collection errors. In addition, blinded retesting was usually performed within 24 h, but not later than 72 h (on weekends). Moreover, the tested samples were rather diverse as they were derived from symptomatic patients as well as from asymptomatic hospital employees. This ensures that the results account for a broad range of variables that are encountered in real life. Without clinical information and the possibility to recollect additional samples, a further characterization of discordant results was impossible in this study. Discordant results can occur for various reasons including use of different target genes, different nucleic acid extraction and amplification efficiencies, and the number of manipulations involved in sample processing . DespiteIn conclusion, commercial SARS-CoV-2 RT-qPCR assays show considerable variability, which has an important impact on results and may lead to inappropriate isolation decisions. Therefore, decision makers should balance the advantages and disadvantages of RT-qPCR for mass screening and adopt suitable strategies that ensure a rational management of weakly positive results. Alternatively, rapid antigen testing may be considered as a cost-effective alternative to identify infectious individuals in low-risk populations.The Clinical Institute of Medical and Chemical Laboratory Diagnostics (CIMCL) at the Medical University of Graz  selected daily two swab samples for SARS-CoV-2 RT-qPCR cross-comparison with two other laboratories. One sample was SARS-CoV-2 positive and the other one negative based on the QIAGEN NeuMoDx SARS-CoV-2 Assay . Oropharyngeal or nasopharyngeal swabs were collected using the Copan eSwab™  collection and transport system. From each sample, two aliquots were produced. One aliquot was sent to the Diagnostic and Research Institute of Hygiene, Microbiology, and Environmental Medicine (IHMU) at the MUG, the other one to the Institute of Hospital Hygiene and Microbiology (IKM) at the University Hospital Graz, Austria. Both laboratories were blinded to the primary results produced by the CIMCL. All methods used are CE-IVD labeled.https://www.qcmd.org/, accessed on 20 September 2022). The IKM tested the samples with a semi-automated RT-qPCR method. Viral RNA was extracted from 200 μL of sample with the StarMag viral DNA/RNA 200 C Kit  on a MicroLab Nimbus  automated extraction platform. Extracted RNA was eluted in 100 µL of buffer. Then, 10 µL of the eluate was used for SARS-CoV-2 amplification and detection with the RealStar SARS CoV-2 RT-PCR Assay . Amplification and detection were performed on a Light Cycler 480 real-time thermal cycler . The IKM laboratory performs daily quality control measurements and participates successfully in an external quality assurance program .All molecular tests used in this study were performed according to the manufacturers’ protocols. Characteristics of tests are shown in Qualitative results and Ct values were recorded in a Microsoft Excel data base. First, we analyzed the qualitative agreement of all methods by plotting the number of positive and negative results for each method with the initial analysis as reference. The qualitative results of the NeuMoDx 288 were compared with those of the other methods by chi-square test for categorical variables. Next, we identified samples with discordant results, where at least one method produced a discordant result compared to the initial analysis. With SARS-CoV-2 RT-qPCR, qualitative results are generated; however, many countries obliged laboratories to report Ct values as an estimate of viral load. Therefore, positive results were used to perform Spearman correlation analyses between the Ct values of the NeuMoDx 288 method and each of the other methods. The Ct value differences were rounded to whole numbers. Samples with at least one negative result were classified as discordant. Statistical calculations were performed with SPSS Statistics 28.0.1 .