Goal: We set to record and analyze for the first time concurrent electroencephalographic (EEG), electrocardiographic (ECG), and unrestrained whole-body plethysmographic (Pleth) signals from control (WT - wild type) and SUDEP-prone mice . Methods: Employing multivariate autoregressive models (MVAR) we measured all tri-organ effective directional interactions by the generalized partial directed coherence (GPDC) in the frequency domain over time (hours). Results: When compared to the control (WT) animals, the SUDEP-prone (KO) animals exhibited (p < 0.001) reduced afferent and efferent interactions between the heart and the brain over the full frequency spectrum (0-200Hz), enhanced efferent interactions from the brain to the lungs and from the heart to the lungs at high (>90 Hz) frequencies , and decreased feedback from the lungs to the brain at low (<40 Hz) frequencies. Conclusions: These results show that impairment in the afferent and efferent pathways in the holistic neuro-cardio-respiratory network could lead to SUDEP, and effective connectivity measures and their dynamics could serve as novel biomarkers of susceptibility to SUDEP and seizures respectively.Sudden unexpected death in epilepsy (SUDEP) is the leading cause of epilepsy-related mortality and its pathophysiological mechanisms remain unknown.  I.Sudden unexpected death in epilepsy (SUDEP), the leading cause of death in epilepsy with an incidence rate of roughly 0.5% of the epilepsy patient population per year, has only recently begun to be addressed The cardiorespiratory system consists of strongly coupled and interdependent organs and tissues that deliver oxygen and other nutrients to the rest of the body Biosignal analysis for measuring both direct and indirect interactions among brain, heart and lungs has been attempted in the past. In 2005, a non-linear measure of cardiorespiratory coupling was introduced to capture really low-frequency stochastic oscillatory behavior between heart and lung activity Kcna1 gene knockout (KO) mouse. The Kcna1 gene encodes axonal membrane voltage-gated Kv1.1 potassium channel Kcna1 gene (–/–Kcna1) are frequently utilized to investigate genetic and pathophysiological mechanisms underlying SUDEP since about 75% of animals exhibit seizure-related sudden death before the age of 2 months (SUDEP-prone) mice to identify novel biomarkers associated with increased SUDEP risk. In addition to providing advanced warning for susceptibility to SUDEP and possibly to status epilepticus (SE), these findings could be found useful in identifying seizure severity, as well as enabling development of reliable biomarkers for evaluating and monitoring the efficacy of different epilepsy treatments.Due to lack of reliable biomarkers, and since SUDEP occurs suddenly and unexpectedly, it is very difficult to reliably and prospectively identify patients at risk and then monitor them in the long term. Thus, SUDEP investigations in humans tend to be hindered by smaller sample sizes that prevent high statistical power. Preclinical animal models can overcome this limitation and provide a suitable system to identify novel biomarkers for future validation in focused patient cohorts. One of the most prominent mammalian SUDEP models currently in use is the II.A.Kcna1 knockout (KO) allele (Kcna1–/–) was generated by targeted deletion of the entire open reading frame of the Kcna1 gene on mouse chromosome 6 leading to complete loss of Kv1.1 function, as previously described The B.WT (n=7) and KO mice (n=8) of both sexes (ages 4–6 weeks) were surgically implanted with bilateral silver wire electrodes (0.005-inch diameter) attached to a microminiature connector for recording in a tethered configuration, as described previously in Four EEG electrodes (four channels) were inserted into the subdural space through cranial burr holes overlying left and right temporal and parietal cortices, and two electrodes were inserted above the left and right frontal cortices for a reference and ground, respectively. For ECG, two thoracic electrodes (one channel) were tunneled subcutaneously on either side and sutured in place to record cardiac activity. Mice were allowed to recover for 3 days and then placed in an unrestrained whole-body plethysmography (Pleth) chamber  to record respiratory waveforms  modeling over concurrent short-time epochs (windows), each from multiple time series, is recommended for network connectivity analysis assuming that these time series  are recorded from different parts of a multi-dimensional, linear and wide-sense stationary system. MVAR modelling reduces computational cost and allows exploratory insight into a system. For each time window, the estimated array of MVAR model coefficients can then be further analyzed in the frequency domain and, depending on different types of normalization utilized, provide frequency-specific measures of directional functional connectivity  between the nodes of the assumed network configuration of the system. One such directed connectivity measure is the Generalized Partial Directed Coherence (GPDC) that provides an accurate measure of effective direct inflow from one node to another at particular frequencies III.A.Table S1.We first sought to uncover general trends in neuro-cardio-respiratory connectivity per interaction between organs within each genotype and then across the two genotypes (WT and KO). Towards this goal, we integrated the statistically significant GPDC (ssGPDC) values between any of the three organs  across time  per frequency band for all animals of each genotype. Thus, the profiles of the mean and the standard error of the mean (SEM) of the ssGPDC values for both genotypes were calculated per frequency band for each of the six inter-organ interactions. Profiles of the ssGPDC values for both genotypes and each directional interaction are shown in the frequency domain in A)Trends in connectivity as function of frequency for each individual interaction between organs: The HeartBrain interactions were higher in the high frequency band (above 40 Hz) than in the low frequency band (below 40 Hz), while the BrainHeart interactions were relatively constant within the broad frequency band of 20-180 Hz Relative strength of each interaction with respect to the other interactions: The observed relative strengths of the interactions overall  were similar in both WT and KO mice, with the exception of HeartBrain interactions. The order of interaction pairs with respect to their magnitude was: [BrainLungs] >[HeartLungs]⋍[HeartBrain] > rest of interactions. Statistical comparison between all possible interaction pairs is provided in Table S2.C)Comparison of each inter-organ interaction between KO and WT animals: Across genotypes, several important differences per interaction between organs emerged. The most prominent difference was found to be in the HeartBrain  interactions where the KO mice exhibited reduced functional connectivity in both afferent and efferent directions and almost over the full frequency band (20-200 Hz)  were found elevated in KO vs. WT mice at frequencies above 100 Hz  were found lower in KO vs. WT mice within narrow frequency bands below 100 Hz , illustrates the significantly reduced HeartBrain, BrainHeart and LungsBrain interactions  how does seizure occurrence affect the differences in the inter-organ interactions between WT and SUDEP-prone animals, and (ii) how do inter-organ interactions in the KO animals change over time in the presence of seizures.Two out of the eight KO mice (Kv1.1 966 and Kv1.1 967) experienced seizures during their respective 5.5-hour and 7.5-hour recordings of concurrent EEG, ECG and Pleth. We included the first 4-h portion of the recordings from both Kv1.1 966 and Kv1.1 967 in our 4-h analysis of the previous (i) above, we repeated the analysis described in Brain⇒Lungs (EEG ⇒ Pleth) and Heart⇒Lungs (ECG ⇒ Pleth) interactions. This implies that the presence or absence of seizures does not change significantly the pre-existing differences in those interactions in the KO animals. In particular, the Heart⇒Lungs interaction (Brain⇒Lungs (S3).To address question (ii) above we analyzed separately the dynamics of the inter-organ interactions over time  and frequency. For this analysis, the inter-organ interactions that exhibited a) a high percentage of ssGPDC values  and b) statistically significant differences  between the WT and KO groups (see BrainHeart), and the efferent interactions from the heart and brain to the lungs (BrainLungs and HeartLungs) were considered for dynamical analysis. To maximize the significance of dynamical trends we were attempting to capture, we considered the connectivities in the frequency band for which there was the maximum difference in the respective ssGPDC profiles between the WT and KO groups, that is, the frequency band with the minimum P-value for the Wilcoxon rank sum test on equality of medians.To address question oups see  across tThe smoothed ssGPDC values for these interactions over time for the two KO mice (A966 and A967) in the presence of seizures are shown in blue color) and all KO animals (HB in red color) that had no seizures during their 4-h recordings. In Kv1.1 966 (HeartBrain interaction (frequency sub-band: 80-90 Hz) in this KO mouse was severely impaired during the period with seizures  is also impaired in the same way as the HeartBrain interaction . It is noteworthy that BrainHeart recovers much faster than the HeartBrain interaction after the end of the seizures, with a trend towards recovery starting even after the fifth seizure in this cluster of 6 seizures, indicating an early recovery of central cardiac control (BrainLungs connectivity (frequency sub-band: 130-140 Hz) was abnormally high (even beyond the red HB) during pre-ictal periods, and decreases to values within the blue HB for a short time following each seizure's end and permanently after the last seizure (HeartLungs connectivity (frequency sub-band: 150-160 Hz) is lower peri-ictally throughout the recording compared to the other KO and WT animals  with seizures during the recording session are given in Figure 7IV.HeartLungs, BrainLungs and Brain⇔Heart interactions in SUDEP-prone KO mice versus WT mice  did not exhibit highly statistically significant differences between the KO and WT animals except for a very narrow frequency band around 80 and 40 Hz respectively   and/or seizure clusters (mostly displayed in this study by the BrainLungs and BrainHeart interactions), a concept that we postulated and have shown in the past by spatiotemporal analysis of scalp and intracranial EEG from patients and epileptic rats b) the potential use of the dynamics of specific interactions  for prediction of seizures; studies have shown increased inflammatory processes in the larynx in cases of the similarly infrequent and sudden condition, the Sudden Infant Death Syndrome (SIDS), suggesting the involvement of abnormal hyper-reactivity of the reflexes and apnea c) identifying periods of seizure susceptibility  From a dynamics perspective, we studied the inter-organ interactions with respect to the timing of events that were very well-defined, the epileptic seizures, and compared their pre- and post-ictal values to referential values of inter-organ interactions during seizure-free periods from WT and SUDEP-prone KO animals. The general pre-ictal trend was an elevated input to the lungs from the brain and a reduced input to the heart from the brain; post-ictally (after seizure end), the opposite changes  were observed in those interactions. These changes were most prominent at high frequencies. Furthermore, the most distinct dynamics occurred over larger timescales (2 hours), that is, when comparing the period with intense seizure activity to the subsequent seizure-free (recovery) period . These fFirst, the interactions we investigated showed frequency dependence, especially interactions that included the respiratory system. Such intricate frequency dependencies may be better quantified via a cross-frequency analysis Second, in theory, linear modelling can accurately model only linear systems. However, linear models can approximate nonlinear systems and nonlinear behavior under specific constraints . In the case of a biological signal, where the underlying system is typically nonlinear and the constraints are relatively unknown, the linear modelling approach should always constitute a first approximation that could lead to learning of the constraints for a subsequent better linear approximation and so on Third, despite the experiments being state-of-the-art with the appropriate controls, it remains to be seen if our results are general to SUDEP or specific only to the Kcna1 KO animal model of SUDEP.To our knowledge, this study is the first attempt to assess the communication between the brain, heart and lungs together in controlled experiments centered on a neurodegenerative disease. This study is also a rare comparison of control and diseased groups in epilepsy, as typical studies investigating mechanisms of epilepsy and SUDEP lack control subjects due to the nature of human data collection in clinical epilepsy research. Even though important and statistically very significant results were generated through our analysis of the experimental data, we would like to acknowledge the following experimental and theoretical limitations of the present study that could guide future studies. V.BrainLungs efferent connections appears to be a key feature preceding seizure manifestation, implying hyper-reactivity in this branch of the system involving either the respiratory sensory centers in the brain, the pathway between the lungs and the brain, or the sensory system in the lungs. In addition, decreased Heart⇔Brain activity seems to also play a key role in the manifestation of seizures and possibly SUDEP. These novel findings, following further validation in additional datasets from animals and humans, may be incorporated in seizure detection and prediction algorithms within intelligent neuromodulation devices for seizure control and for detection of periods of susceptibility to seizures and risk of SUDEP.In this study, we employed directed network connectivity analysis between the brain, heart and lungs on a rare dataset of concurrently recorded EEG, ECG and Pleth signals in WT and SUDEP-prone KO animals. The results of this network analysis suggest important frequency-dependent abnormalities in the neuro-cardio-respiratory system of KO mice. Connections between the brain and heart, and the brain and lungs showed the most statistically significant abnormalities in the SUDEP-prone KO animals. Further analysis of their dynamics showed that they may play an important role in the generation of seizures (ictogenesis). Pre-ictal elevation of Figure S1. The results from our analysis of the impact of the order of the MVAR model on the values of inter-organ connectivities are given in Figure S2. In Tables S1, S3 and S4, using non-parametric statistical tests, the p-values of the differences in the medians of connectivity per interaction, respectively, between: 1) WT  and KO  averaged across all frequencies (as depicted in 2) KO  and KO  at each individual frequency band for validation of conclusions from 3) WT  and KO  at each individual frequency band in order to select the frequency bands for the dynamic analysis depicted in Table S2 depicts p-values for each inter-organ interaction with each of the rest of the other inter-organ interactions per animal genotype (WT and KO).In the supplementary material, the estimation of directed connectivities by MVAR and the assessment of their statistical significance are briefly described. The percentage of GPDC values rendered not statistically significant (p>0.05) per interaction and frequency band for each of the two genotypes (KO and WT) is illustrated in


ABSTRACT IMPACT: This work provides supporting evidence for the development of a novel immunosuppression therapy for transplant patients. OBJECTIVES/GOALS: Our laboratory reported that inhibition of the kinase DNA-PK(cs) in mice delays allogeneic graft rejection in part by mitigating the induction of certain cytokines. We hypothesized that this was due to an inhibition of intracellular signaling programs in T cells and designed studies to identify the mechanism(s) by which this occurs. METHODS/STUDY POPULATION: The immortalized Jurkat T cell line was used to evaluate the effect of the DNA-PK(cs) inhibitor NU7441 on T cell activation by PMA/Ionomycin or PMA/PHA. Mouse primary splenocytes also were used to demonstrate the universality and reproducibility of our observations. Initially, protein mass spectrometry of lysates from untreated and NU7441-treated Jurkat cells identified proteins of interest regulated by DNA-PK(cs) that play a role in T cell activation and cytokine production. CRISPR genome editing was used to validate a potential downstream target of DNA-PK(cs). Western blot, ELISA, and flow cytometry were used to document changes in protein levels with respect to treatments. RESULTS/ANTICIPATED RESULTS: We observed that expression of the transcription factor Egr1 was highly induced after activation but attenuated after treatment with NU7441 in both Jurkat T cells and mouse splenocytes. Phosphorylated serine 301 of Egr1 was identified by mass spectrometry in stimulated cells and fits the kinase consensus sequence for DNA-PK(cs). Both an endogenous CRISPR-generated serine 301 to alanine mutant and expression of a plasmid-based S301A mutant resulted in an unstable form of Egr1 that was barely detectable. In contrast, expression of either a S301 to D or E phospho-mimetic mutant resulted in a stable form of the protein detectable by Western blot. Further evaluation of these mutants and Egr1 phosphorylation is underway to determine the mechanism by which DNA-PK(cs) kinase regulates protein stability. DISCUSSION/SIGNIFICANCE OF FINDINGS: We previously reported a role for DNA-PK(cs) in immunomodulation. We now have evidence that this occurs in part through stabilization of Egr1. We believe this novel finding will lead to uncovering a broader role for DNA-PK(cs) as a mediator of protein stability in T cells and provide support for targeting DNA-PK(cs) in immunosuppression therapy.


Prolonged use of opioids can cause opioid-induced hyperalgesia (OIH). The impact of alternative splicing on OIH remains partially characterized. A study of the absolute and relative modes of action of alternative splicing further the understanding of the molecular mechanisms underlying OIH. Differential absolute and relative isoform profiles were detected in the trigeminal ganglia and nucleus accumbens of mice presenting OIH behaviors elicited by chronic morphine administration relative to control mice. Genes that participate in glutamatergic synapse , myelin protein processes , and axon guidance presented absolute and relative splicing associated with OIH. Splicing of genes in the gonadotropin-releasing hormone receptor pathway was detected in the nucleus accumbens while splicing in the vascular endothelial growth factor, endogenous cannabinoid signaling, circadian clock system, and metabotropic glutamate receptor pathways was detected in the trigeminal ganglia. A notable finding was the prevalence of alternatively spliced transcription factors and regulators  in the trigeminal ganglia. Insights into the nociceptive and antinociceptive modulatory action of Hnrnpk were gained. The results from our study highlight the impact of alternative splicing and transcriptional regulators on OIH and expose the need for isoform-level research to advance the understanding of morphine-associated hyperalgesia.  Chronic pain affects 11% to 40% of the United States population . A commoRodent models of OIH have advanced the understanding of the central nervous system (CNS) regions and molecular mechanisms underlying OIH ,9,10,11.Alternative splicing can regulate gene expression and action through the production of transcript isoforms that differ in their capability to generate biologically active products ,16. DiffDespite the established effects of alternative splicing on genes annotated to pathways associated with OIH , the impact of alternative splicing in OIH remains partially characterized. The overarching goal of this study is to gain a comprehensive understanding of the effects of alternative splicing on OIH. The supporting objectives are, a) the characterization of alternative splicing action modes associated with OIH that are region-dependent or ubiquitous of the region, and b) to uncover pathways predominantly impacted by alternative splicing. The findings from our study aid in the identification of transcript isoforms associated with OIH, therefore improving the accuracy of detecting effective therapeutic targets to ameliorate OIH.Animal experimental protocols were approved by the Office of Animal Care and Institutional Biosafety Committee at the University of Illinois at Chicago in accordance with AALAC guidelines, the Animal Care Policies of the University of Illinois at Chicago, and the NIH Public Health Service Policy on the Humane Care and Use of Animals.A bulk RNA-sequencing experiment was undertaken to measure the level of gene expression in the NAc and TG of male adult C57BL6/J mice administered with morphine to elicit OIH (OIH group) relative to control mice (CON group) . The aniThe sequenced reads from each sample were aligned to the C57Bl/6J mouse genome (version GRCm38)  using STA comprehensive characterization of the role of alternative splicing on OIH was gained by studying absolute and relative mode of actions. The absolute differential analysis was implemented on a per-transcript isoform basis and enabled us to assess the overall splicing effect that resulted in the change in the expression of individual transcript isoforms between the OIH and control groups for each region. However, the quantification of changes in absolute transcript isoform expression cannot offer information into changes in the transcript isoform proportion between treatments. Therefore, a second analysis was undertaken to test for relative differential isoform expression.P-value for multiple testing [P-value and minimum log2(fold change between OIH and control groups).The absolute differential isoform expression between the OIH and control groups within region was tested using the edgeR (v.3.14.0)  in the R testing . The criP-value per gene intron cluster [P-value and minimum ΔPSI between OIH and control groups. The detection of significant alternative splicing encompassed the simultaneous consideration of the overall P-value and minimum ΔPSI of a gene. The series of introns representing transcript isoforms within a cluster and the corresponding mapped genes were visualized using Gencode M18 mouse genome reference annotation [The relative differential isoform expression between the OIH and control groups within region was tested using LeafCutter (v.0.2.9) .The clus cluster ,12. Multnotation . The complementary insights gained on OIH stem from the distinct description of alternative splicing offered by the two analyses considered. The absolute analysis models each annotated isoform profile independent from the other isoforms in the gene and allows testing for significant changes between OIH and control. The absolute analysis of annotated isoforms employs known exon usage information, therefore enabling the alignment and benchmarking of the present isoform-level analysis against the previously published gene-level analysis . The relhttp://www.webgestalt.org/, accessed on 1 May 2021) enrichment routines [P-value < 0.0005, irrespectively of the profile sign (over- or under-expression in OIH relative to control) were considered in the analysis. The ORA approach was also used to identify enrichment of Gene Ontology Biological Processes [The impact of OIH on the absolute and relative alternative splicing of genes within PANTHER pathways  in the Nroutines . Two comroutines ,36, and rocesses  in the lP-value) of absolute or relative differential expression. The GSEA of absolute differential expression between OIH and control uses the sign of each transcript isoform, where a positive log2(fold change) indicates over-expression, and a negative log2(fold change) indicates under-expression in OIH relative to control mice.The GSEA considered the profile of all transcript isoforms tested, and the input score was the signed –log10 of the transcript isoforms in a cluster encompasses both signs because some transcript isoforms are over-abundant relative to the remaining isoforms that are under-abundant in OIH versus control mice. The sign assigned to the GSEA input score for each transcript isoform cluster was the sign of the most extreme ΔPSI between OIH and control. Therefore, a positive GSEA score sign indicates that the transcript isoform presenting the most extreme change was over-expressed in OIH relative to control mice, while a negative GSEA sign indicates that the most extreme transcript isoform in the cluster was under-expressed in OIH relative to control mice.P-value and FDR-adjusted P-value of the pathway enrichment. ORA uses hypergeometric test and computes an enrichment ratio (observed versus expected). GSEA computes the normalized enrichment score (NES) based on the maximum deviation of the cumulative sum based on the signed fold-change divided by the average of the permutated enrichment scores (based on 2000 permutations). Mus musculus was used as the reference genome, and the enrichment analysis limited the pathways with a minimum of 5 detected genes and a maximum of 2000 genes per category. The enrichment tests of the absolute and relative analysis and the background genome were implemented at the gene level with the goal of identifying pathways with prevalent alternative splicing processes. Both ORA and GSEA provide a P-value < 0.005, |fold change| > 2) between OIH and control mice. To facilitate the visualization of the networks, only direct interactions (edges) connecting two genes (nodes) that have significant differential isoform expression or splicing are depicted. While the reconstructed network enables the detection of molecular relationship between alternative spliced or expressed, the hub genes presenting high connectivity with other genes that may play an important role in the development or maintenance of OIH. To understand the interaction between the genes presenting absolute or relative alternative splicing mode of action associated with OIH within region, we reconstructed networks depicting known relationships between the genes using the BisoGenet plug-in  within CP-value < 0.005, |fold change| > 2) were queried for enriched transcription factors (TFs) using the iRegulon [In addition to studying the relationship between alternatively spliced genes associated with OIH based on protein-protein interactions, the relationships between these genes through common transcription factor were also investigated. Genes that presented absolute or relative differential splicing between OIH and control  between OIH and control mice in the NAc and TG, respectively. The absolute differentially gene expression between OIH and control mice at FDR-adjusted P-value < 5.0 × 10–5 and |fold change| > 2 in NAc and TG are presented in P-value < 0.05 in NAc and TG are provided in Overall, 263 transcript isoforms from 250 genes and 363 transcript isoforms from 319 genes present absolute differential expression  relative differential expression of isoforms between OIH and control mice. P-value < 0.1) between OIH and control mice including the most extreme positive and negative ΔPSI between the two groups in the NAc and TG. A positive ΔPSI identifies a transcript isoform present in higher proportion in OIH relative to control mice, while a negative ΔPSI identifies a transcript isoform present in lower proportion in OIH relative to control mice. Relative differential splicing was detected at P-value < 0.0005 in 40 genes in the NAc and 32 genes in the TG  in the TG. The relative differential splicing identified in the TG for Fryl, was characterized by the over-expression of an isoform that extends across two exons in OIH relative to control mice with the exon-skipping isoform presenting a proportion ratio of 0.87(OIH):0.36(control). The significant differential splicing identified in the TG for Eml6 was characterized by the under-expression of an isoform that extends across two exons in OIH relative to control mice with a proportion ratio 0.32(OIH):0.83(control). The significant relative differential splicing identified in the TG for Ktn1, was characterized by the under-expression of an isoform that extends across two exons in OIH relative to control mice with a proportion ratio 0.18(OIH):0.28(control). The significant relative differential splicing identified in the TG for AW554918, was characterized by the under-expression of an isoform that extends across two 3′ exons in OIH relative to control mice with a proportion ratio 0.28 (OIH):0.42 (control).P-value < 0.001) associated with the effects of OIH on absolute and relative differential isoform expression between OIH and control mice in the NAc and TG identified using the complementary ORA and GSEA analyses are listed in Enriched PANTHER pathways . The study of simultaneous changes in absolute and relative transcript isoform expression enabled us to understand the predominant alternative splicing mode of action. The majority of genes had one alternative splicing mode of action  and the splicing was expressed in one region . Nine ouThe enrichment of mRNA metabolic and RNA splicing processes were detected among the genes that presented two splicing mode of actions in one region or one mode of action across two regions . The enrThe connectivity among genes that presented absolute or relative differential splicing was higher in NAc than in TG . In the P-values of the effects of gestational immune activation  by weaning group (nursed or weaned), and sex on the chemistry analyte and cortisol concentrations and body weights of 22-day-old pigs.Highly connected genes in the TG network include Ras/Rap GTPase-activating protein SynGAP (Syngap1), E3 ubiquitin-protein ligase NEDD4-like (Nedd4l), and sodium channel protein type 8 subunit alpha (Scn8a) that presented both absolute and relative differential splicing B. Other P-value <0.005 and |fold change between treatments| >1).The TG network B includeAlthough morphine is typically prescribed for the analgesic effect, repeated morphine administration can trigger the transition from analgesia to hyperalgesia and hypersensitivity to stimuli . Repeated administration of morphine elicits OIH in mice, characterized by lower mechanical stimuli threshold ,10. StudAltogether, the results of the absolute and relative analysis in The majority of the extreme differentially expressed transcript isoforms in the NAc were under-expressed in OIH relative to control mice , whereasP-value < 5.0 × 10–5) in OIH relative to control mice (Transcript isoforms of Fxyd2 (ENSMUST000000213158.1), Pirt (ENSMUST000000123434.2), Acpp (ENSMUST00000062723.13), Avil (ENSMUST00000026500.11), Ntng1 (ENSMUST000000128219.8) were under-expressed  and this profile was also reported in the gene level analysis [Two transcript isoforms of Mpz (ENSMUST00000070758.9 and ENSMUST000000111334.1) were under-expressed in OIH relative to control mice  was under-expressed in OIH relative to control mice. This pattern could be linked to the under-expression of Akap11 in human induced pluripotent sensory neurons exposed to Bortezomib, an anti-cancer medication that induces painful peripheral neuropathy [P-value < 6.2 × 10–9). The detected association between the Mbnl1 transcript isoform and OIH is complementary to the identification of Mbnl1 as a candidate gene for inflammatory nociception [A transcript isoform of the A kinase (PRKA) anchor protein 11 gene . The detected association between Dmxl2 and OIH could be associated with reports of up-regulation of the Dmxl2 protein in the spinal cord of a mouse model of neuropathic pain [Many transcript isoforms that presented absolute differential expression between OIH and control mice in the TG, are alternative forms within the same gene . Two trahic pain . LikewisP-value < 0.07 and P-value < 0.01, respectively, A transcript isoform (ENSMUST00000211368.1) belonging to phosphodiesterase 4 (Pde2a), an isoform ENSMUST00000196469.4 belonging to Nfkb1, and an isoform (ENSMUST00000151045.2) belonging to phosphodiesterase 4 (Gria1) were under-expressed in OIH relative to control mice in the TG  that was over-expressed in OIH relative to control mice. The higher expression in OIH relative to control mice could be linked to reports of reduced acute nociception and chronic pain in a Shank2 knockout line relative to wild-type [The under-expression of a Gria1 transcript isoform could be aligned with the established role of Gria1 in neuropathic pain signaling among dorsal horn neurons, glutamate receptors signaling, and synaptic long-term potentiation . Shank2 ild-type . Moreoveild-type , and thiP-value < 0.006 and P-value < 0.0004, respectively, The Ophn1 transcript isoform ENSMUST00000147805 and the Ogt transcript isoform ENSMUST00000155792.1 were over-expressed in OIH relative to control mice  in OIH, the isoforms of Dnm2 and Dnm1 were under-expressed  in OIH relative to control mice. The dynamin gene family is known for experiencing extensive alternative splicing and participate in synaptic vesicle recycling, post-synaptic receptor internalization, neurosecretion and neuronal process extension, processes associated with stimuli transmission [Transcript isoforms in the dynamin genes Dnm3 (ENSMUST00000193110.1), Dnm2 (ENSMUST00000169194.8), Dnm1 (ENSMUST00000230022.1) presented absolute differential expression between OIH and control mice in the TG. While the Dnm3 isoform was over-expressed  had opposite expression profiles between OIH and control mice . SplicinThe relative differential expression of St8sia1 isoforms associated with OIH identified in the NAc was characterized by the over-expression of a transcript isoform that just includes a 3′ exon in OIH relative to control mice . The assThe alternative splicing of Cadps was characterized by the under-expression of an isoform that excludes an intermediate exon in OIH relative to control mice in the NAc . DysreguThe alternative splicing of Mief1 in the NAc associated with OIH was characterized by the under-expression of a transcript isoform that includes a lengthy 5′ exon in OIH relative to control mice . AlthougP-value < 0.05, P-value < 0.0005 than TG . These differences in splicing profiles suggest that the role of alternative splicing underlying OIH is region-dependent.Notable finding from the regional comparison of the differential alternative splicing between OIH and control mice was that TG presented more extreme splicing characterized by more extreme differences in the proportion of transcript isoforms between treatments relative to NAc  yet this transcript was under-expressed in the TG of OIH relative to control mice. The opposite profiles of absolute and relative Hnrnpk transcript isoform across brain regions are could be linked to reports of morphine-induced accumulation of HNRNPK protein in mouse brain regions correlated with nociceptive and antinociceptive modulatory systems [Our study of absolute and relative action modes further the understanding of the association between OIH and alternative splicing in Hnrnpk. Hnrnpk presented significant relative splicing in the NAc, and the most extreme DPSI was positive. This pattern indicates that a Hnrnpk transcript was relatively more abundant in the NAc of OIH relative to control mice . Also, a systems .One module of the NAc gene network A encompaAll the genes in the TG network were associated with the NMDA receptor system. In the sensory neurons, the NMDA receptor system plays a key role in the generation and maintenance of central sensitization during pain states . DysfuncThe identification of transcription factors pre B cell leukemia homeobox 1 (Pbx1) and aryl hydrocarbon receptor nuclear translocator like 2 (Arntl2), that could potentially target many genes presenting alternative splicing events enhanced the understanding of molecular mechanisms associated with OIH . The rolThe study of alternative splicing processes in a genome-wide scale using complementary approaches pointed to more than 150 isoforms and genes associated with OIH in two CNS regions. While the isoform findings were not experimentally validated, filtering criteria aimed at securing adequate isoform depth that would strengthen the findings. Also, the promising leads were discussed in the context of selective statistical and biological significance and supporting literature review in relevant pain and opioid-associated pathways while references at the gene level inidicate the need for further study at the isoform level across sexes, pre-existing pain conditions, and CNS regions.The present study offers insights into the prevalent role of alternative splicing in the molecular mechanisms associated with OIH elicited by chronic morphine exposure. Both, changes in the absolute level of the transcript isoform and in the relative proportion of the transcript isoforms within a gene were detected in association with OIH in CNS regions that participate in stimuli signal transmission and opioid dependency. These findings can augment the accuracy in the identification of effect targets to reduce the effects of OIH.


Antineutrophil cytoplasmic antibodies (ANCA) vasculitis is common after the age of 50 years but it can occur at any age. There is a slight male preponderance and it is more common in Whites than Blacks but the black race confers a worse prognosis. The clinical features of ANCA vasculitis vary considerably. The manifestation of the disease depends on the organs affected, the chronicity of the disease, and how quiescent it is. Non-specific symptoms of malaise, fatigue, fever, and weight loss are common. Crescentic glomerulonephritis with focal necrosis is usually the pathology underlying renal disease. Manifestations of renal disease include hematuria and proteinuria which may progress to renal failure.We present a case of a 75-year-old female who presented with acute worsening of renal function and nephrotic-range proteinuria with positive testing for p-ANCA after the recent commencement of treatment with tofacitinib. This prompted a suspicion of ANCA-vasculitis. The patient was started on pulse dose steroids and rituximab after kidney biopsy confirmation of ANCA-vasculitis with crescentic glomerulonephritis. Tofacitinib is a specific inhibitor of Janus-associated kinases . In the United States, it is approved for the treatment of moderate to severe active rheumatoid arthritis, psoriatic arthritis, and moderate to severe ulcerative colitis . MultiplWe present a case report of a 75-year-old female who had started tofacitinib for the treatment of refractory rheumatoid arthritis. The patient had been unsuccessfully treated with adalimumab and etanercept with recurrence of symptoms after about a month of treatment with each of these medications. She had also been on variable doses of prednisone throughout the course of her treatment.Two months after starting treatment with tofacitinib, her creatinine was noted to have increased from 1.9 mg/dL to 2.9 mg/dL together with 9 gm/day of proteinuria. At this time she also was p-ANCA positive though prior testing for p-ANCA was negative, four years ago. She was then referred to a nephrologist by her rheumatologist.Tofacitinib was stopped and she was admitted to the hospital for acute kidney injury. She was started on pulse dose steroids, followed by 60 mg of prednisone daily. A kidney biopsy was done on admission which showed p-ANCA mediated pauci-immune focal necrotizing and focal sclerosing glomerulonephritis with 23% partial crescents. Rituximab was subsequently added to her treatment regimen and further serology workup was negative for anti-Smith antigen, anti-histone antigen, and anti-ribonucleoprotein antigen. ANA was positive and C3 was low.She was readmitted a month after her first admission for shortness of breath, dyspnea on exertion, progressive leg swelling and treated for congestive heart failure and cardiorenal syndrome. A month after her second admission, she was readmitted for respiratory distress. On physical examination, she had tachycardia and tachypnea and had bilateral crackles at her lung bases with bilateral pitting pedal edema. Computerized tomography scan of the chest showed extensive bilateral interstitial pneumonitis which had progressed from a previous study. She was placed on pulse dose steroids and high flow oxygen and subsequently 40 mg of prednisone 12 hourly and IV Cyclophosphamide for interstitial pneumonitis secondary to ANCA-vasculitis. The treatment with Cyclophosphamide was complicated by the development of painful mouth ulcers. The patient was also placed on trimethoprim-sulfamethoxazole for Pneumocystis jirovecii pneumonia prophylaxis but switched to Atovaquone because of hyperkalemia. Although she was on high flow oxygen, the patient began to desaturate and the treatment course was complicated by bacterial infections and sepsis. Her oxygen saturation continued to get worse until she developed cardiopulmonary arrest and died.Our patient presented with acute worsening of renal function and nephrotic-range proteinuria with positive testing for p-ANCA after the recent commencement of treatment with tofacitinib. This prompted a suspicion of ANCA-vasculitis. The patient was placed on pulse dose steroids and rituximab after kidney biopsy confirmation of ANCA-vasculitis with crescentic glomerulonephritis.ANCA-associated vasculitis (AAV) is one of the causes of small vessel vasculitis. Other causes of small vessel vasculitis include anti-glomerular basement membrane disease, IgA vasculitis, and cryoglobulinemic vasculitis. ANCA-associated vasculitis is pauci-immune necrotizing vasculitis with few or no immune deposits in the walls of small vessels affected. A distinguishing feature of AAV is the presence of necrotizing inflammation Figures -5 10-12-1210-12.AAV is categorized based on clinical and pathological characteristics into granulomatosis with polyangiitis (GPA), formerly known as Wegener’s granulomatosis, eosinophilic granulomatosis with polyangiitis (EGPA), and microscopic polyangiitis (MPA). Granulomatous inflammation is common to both GPA and EGPA, however, EGPA is associated with asthma and eosinophilia unlike GPA. Granulomas are absent in MPA. According to the revised International Chapel Hill Consensus Conference Nomenclature of Vasculitides, the classification of AAV should include ANCA-specificity, for example, PR3-ANCA or MPO-ANCA because this confers more specificity to the disease entity -15.It is clear that ANCA plays a role in the pathogenesis of pauci-immune small vessel vasculitis as there is a strong correlation between the presence of antineutrophil cytoplasmic antibodies and disease development . The devThe relationship between ANCA titers and disease activity is unclear and not well understood. Though high ANCA titers usually correlate with worse disease activity, the absence of ANCA does not mean the absence of disease activity. However, ANCA-negative vasculitis is more often associated with renal-limited disease while ANCA serotype positivity is a strong marker for the underlying genetic pathogenesis . It is aAlthough the relationship between ANCA titers and disease activity is unclear, the relationship between ANCA type and disease relapse is well established. PR3 ANCA confers double the risk of disease relapse compared to MPO-ANCA. Bone destruction or saddle-nose deformity is invariably associated with PR3-ANCA while most patients with kidney-limited disease are positive for MPO-ANCA. More than half of the patients with Churg-Strauss syndrome test negative for ANCA ,16.ANCA vasculitis is more common after the age of 50 years but it can occur at any age. There is a slight male preponderance and it is more common in Whites than Blacks but the black race confers a worse prognosis. The clinical features of ANCA vasculitis vary considerably. The manifestation of the disease depends on the organs affected, the chronicity of the disease, and how quiescent it is. Non-specific symptoms of malaise, fatigue, fever, and weight loss are common. Crescentic glomerulonephritis with focal necrosis is usually the pathology underlying renal disease. Manifestations of renal disease include hematuria and proteinuria which may progress to renal failure. Renal disease may also manifest as rapidly proliferative glomerulonephritis. Skin manifestations of vasculitis typically present as palpable purpura that begin as recurrent crops in the lower extremities which may become ulcerated. Necrotizing inflammation in the upper respiratory tract could lead to bone destruction and manifest as saddle-nose deformity. This is more common in patients who are PR3-ANCA positive . Lung inThe diagnosis of AAV is based on clinicopathologic features. ANCA testing aids in the diagnosis but has some limitations. Testing for ANCA includes indirect immunofluorescence (IIF) assay and enzyme immunoassay (EIA). IIF shows two distinct patterns of staining. A diffuse pattern of staining throughout the cytoplasm and around the nucleus is described as cytoplasmic, c-ANCA and perinuclear, p-ANCA. Conventional practice has been to do initial testing by IIF and confirmation of positive ANCA by EIA, however recent studies have shown significant variability in results by standard tests for ANCA by IIA and there is now a trend to do ANCA screening by EIA methods only, especially because PR3 and MPO-ANCA not only have a well-defined genetic basis but also because it confers a disease specificity that IIF testing lacks. Not all patients who have pathological evidence of ANCA vasculitis are ANCA-positive. About 9-17% of patients with AAV test negative for ANCA and more than 50% of patients with Churg-Strauss syndrome are ANCA-negative .The treatment of AAV typically involves an induction phase, maintenance of remission phase, and treatment of relapses. The standard for induction therapy for AAV has traditionally been a combination of corticosteroids with cyclophosphamide for a period of 3-6 months. However, the toxicity of cumulative doses of cyclophosphamide has always been a source of concern. The Pulse Versus Daily Oral Cyclophosphamide for Induction of Remission in Antineutrophil Cytoplasmic Antibody-Associated Vasculitis Study (CYCLOPS) demonstrated that monthly intravenous pulse doses of cyclophosphamide achieved similar remission rates when compared to daily oral cyclophosphamide while reducing cumulative total dose . RituximIn non-organ threatening AAV, methotrexate and mycophenolate mofetil (MMF) have been shown to be equally effective in inducing remission. For maintenance of remission, azathioprine is the medication of choice in combination with low-dose steroids according to EULAR/ERA-EDTA . Other mWe hypothesize that the occurrence of new-onset ANCA-vasculitis in our patient is associated with tofacitinib because there have been published case reports of increased risk of inducing vasculitis in patients treated with tyrosine-kinase inhibitors -23. Our Tofacitinib has increasingly been used in the United States since it was first approved for use in 2012. The common side effects of neutropenia, infections are well known and expected. This case of vasculitis may have been induced by tofacitinib, but a clear causality can't be proven based on this case report. It is unique because tofacitinib is used to treat an over-active immune system but in our patient, it also induced another disease of the immune system. It also raises the question of whether the mechanism of activation of tofacitinib by myeloperoxidase in neutrophils has some relationship with the occurrence of new-onset ANCA-vasculitis. Clinicians should therefore have a high index of suspicion with the onset or worsening of proteinuria and/or hematuria in a patient being treated with tofacitinib.


A global outbreak of severe acute respiratory syndrome (SARS) caused by a new coronavirus (CoV-2) began in December 2019. It induced a novel identified sickness named coronavirus disease 2019 (COVID-19). The rapid emergence of COVID-19 along with the considerable illness and mortality of the disease made it a major public health issue. Due to the lack of effective treatments, an extensive attempt is under way to provide and examine antiviral drugs.2 The created endosome, where a slightly acidic environment is required for optimum fusion of its membrane and virus envelope, followed by the stages of fusion, uncoating and release of the genome into the host cell cytoplasm.4 The protonation of critical residues on a viral envelope glycoprotein alters its conformation and exposes a hydrophobic “fusion peptide” domain to facilitate the fusion.5 Infection of SARS-CoV-2 may be prevented by treatment of cells with inhibitors of endosome acidification.Coronaviruses are enveloped positive-stranded RNA viruses that replicate in the host cell cytoplasm. To release their nucleocapsid into the cell, they rely on the binding to the cellular receptors, angiotensin converting enzyme 2 (ACE2) for SARS-CoV-2, by major conformational changes of the virus spike glycoprotein (S) and entry into the cells by endocytosis.3), like endosomotropic weak bases ammonium chloride (NH4Cl), chloroquine (CLQ)/ hydroxychloroquine (CLQ-OH) as well as bafilomycin A (BAF), may raise the pH in vesicle/endosomes and would be expected to inhibit nucleocapsid release of a SARS-CoV-2 which required an acidic endosomal environment for uncoating  by the patients with confirmed COVID-19, several times during the day, probably able to stop the fusion and uncoating stages. In this case, the replication phase will also remain barren and eventually the respiratory infection will be controlled. Prospective controlled trials are needed to evaluate this method efficacy.Not applicable.The authors declare that there is no conflict of interests.


We report a case report regarding the eradication of isolated lymph-nodal para-aortic recurrence in the aortic region down the left renal vein (LRV) in a patient treated two years earlier in another hospital for a FIGO stage IC2 high-grade serous ovarian carcinoma with a video showing the para-aortic space after eradication of the metastatic tissue. A 66 year-old woman was admitted 24 months after the initial surgical procedure for an increased Ca 125 level and CT scan that revealed a 3 cm para-aortic infrarenal lymph-nodal recurrence that was confirmed by PET/CT scan. A secondary cytoreductive surgery (SCS) with a para-aortic lymph-nodal dissection of the tissue down the LRV and radical omentectomy were performed: during the cytoreduction, the right hemicolon was mobilized. The anterior surface of the inferior vena cava (IVC), aorta and LRV were exposed. The metastatic lymph nodes were detected in the para-ortic space down the proximal part of the LRV and eradicated; an en bloc infrarenal lymph-node dissection from the aortocaval region was performed. The operative time during the surgical procedure was 212 min with a blood loss of 120 mL. No intra- and postoperative complications, including ureteral or vascular injury or renal dysfunction, occurred. At histological examination, three dissected lymph nodes were positive for metastasis, and the patient was discharged five days after laparotomy without side effects and underwent chemotherapy 3 weeks later; after a follow-up of 42 months, no recurrence was detected. In conclusion, secondary debulking surgery can be considered a safe and effective therapeutic option for the management of recurrences, although long-term follow-ups are necessary to evaluate the overall oncologic outcomes of this procedure. A secondary debulking surgery with resection of a suspicious recurrence should be considered in women with a high-grade serous ovarian carcinoma to complete cytoreduction ,2,3; secDisease relapse is the primary cause of death from ovarian carcinoma. Isolated lymph node relapse (ILNR) is a rare pattern of ovarian carcinoma recurrence, with a reported median post-recurrence survival of 2.5 to four years . In this article, we reported a case regarding the eradication of isolated lymph-nodal para-aortic recurrence with a video showing the para-aortic space after eradication of the metastatic tissue without any postoperative complications in a patient treated two years earlier in another hospital for a high-grade serous ovarian carcinoma.A 66 year-old postmenopausal patient, with a history of debulking surgery for a high-grade serous ovarian carcinoma two years earlier in a secondary hospital, was admitted at our department for a suspected isolated lymph-nodal para-aortic recurrence.The patient was managed for FIGO stage IC2 two years earlier with a type A radical hysterectomy (according to the Querleu–Morrow classification) , bilater24 months after the initial surgical procedure, an increased Ca 125 level and CT scan performed at follow-up revealed a 3 cm para-aortic infrarenal lymph-nodal recurrence that was confirmed by PET/CT scan.A secondary debulking open surgery with a para-aortic lymph-nodal radical dissection of the tissue down the LRV and a radical omentectomy were performed: during the cytoreduction, the right hemicolon was separated from the retroperitoneum and was mobilized. The anterior surface of the inferior vena cava (IVC), aorta and LRV were exposed. The metastatic lymph nodes were detected in the para-ortic space down the proximal part of the LRV and eradicated; an en bloc resection of the infrarenal lymph nodes in the aortocaval region was performed, as shown in No intra- or postoperative complications, including ureteral or vascular injury or renal dysfunction, occurred. At histological examination, three dissected lymph nodes were positive for metastasis, and the patient was regularly discharged five days after the laparotomic procedure without any significative side effects and underwent chemotherapy three weeks later; actually, after a follow-up of 42 months no recurrence was detected.Chemotherapy has always been considered the treatment of choice for recurrent epithelial ovarian cancer (EOC), but its use in isolated lymph-nodal relapse (ILNR) is still debated. Secondary cytoreductive surgery (SCS) involves high risks of vascular injury due to the proximity of major intra-abdominal and pelvic blood vessels. Vascular repairs during gynecologic oncologic surgery are often performed for iatrogenic injuries or during extensive oncologic resections and are increasing in frequency ,5,6,7.Pelvic and aortic lymphadenectomy are performed during surgical treatment of gynecological malignancies for their prognostic and therapeutic significance . These pSeveral retrospective studies confirmed the advantages of SCS in patients with a long disease-free interval, resectable disease (based on imaging), an absence of ascites, a limited number of metastatic sites and a good performance status . HoweverBlanchard et al. reported their experience with 27 patients with an ILNR among 640 patients. They observed that after initial surgical treatment, the median progression-free survival (PFS) was 26 months. There was no difference in the two-year survival after ILNR between the groups with early relapse (before 24 months) and late relapse (after 24 months). The time to relapse may not have its usual prognostic value and may not have the same value as in other sites of relapse. The prognosis of ILNR seems better than the prognosis of metastatic recurrence of EOC at other sites. Immediate or delayed therapy should be discussed in cases of asymptomatic ILNR .Santillan et al analyzed the outcomes of SCS for the isolated lymph-nodal relapse of EOC and the subsequent survival rate. Twenty-five patients with epithelial ovarian cancer who underwent SCS for isolated lymph node relapse were identified from tumor registry databases. All patients received platinum-based chemotherapy following primary surgery. With SCS, a residual disease ≤1 cm was achieved in 100% of patients. At a median post-recurrence follow-up time of 19 months, eight patients (32%) have died of the disease, seven (28%) are alive with disease, and 10 (40%) patients are alive without evidence of disease. The median post-recurrence OS after SCS for recurrent nodal disease was 37 months. They concluded that an optimal SCS for recurrent epithelial ovarian cancer presenting as isolated node metastases is feasible in the majority of cases with a favorable long-term survival outcome .In their retrospective study, Legge et al. analyzed 301 patients with recurrence after optimal cytoreductive surgery for epithelial ovarian cancer, of whom 32 had isolated lymph node recurrence and were identified from tumor registry databases. They observed that, although these results need to be confirmed with long-term multicenter studies, ILNR seems to represent a less aggressive pattern of disease relapse; however, peritoneal spreading after the first ILNR documentation is frequently associated with a poor outcome .p = 0.036), no residual tumor after the first surgery and the absence of ascites; these could predict a complete resection in 79% of patients. They concluded that only a complete resection was associated with prolonged survival in recurrent ovarian cancer (1). In the DESKTOP II prospective trial, all consecutive patients with platinum-sensitive first or second relapse were enrolled. The score was applied to all patients, and a total of 129 patients with a positive score and first relapse were operated on. The rate of complete resection was 76%, thus confirming the validity of this score regarding the positive prediction of complete resectability. They concluded that this score was the first validated instrument for predicting the surgical outcome in recurrent ovarian cancer  who received SCS and CHT resulted in showing a significantly longer PFS compared with those who received CHT alone . The primary endpoint analysis showed a median overall survival (OS) of 53.7 months with and 46.0 months without surgery (Our experience confirms that SCS can be considered a safe and effective therapeutic option for the management of isolated lymph-nodal recurrence of ovarian cancers, with a significantly low morbidity and postoperative hospitalization, although it should be reserved for oncologic surgeons trained in extensive surgical procedures; furthermore, large multicenter randomized clinical trials with longer follow-ups are necessary to confirm the overall oncologic outcomes of this procedure.


S. aureus) remains a serious cause of infections in the United States and worldwide. Methicillin susceptible S. aureus (MSSA) is the cause of half of all health care–associated staphylococcal infections, and Methicillin Resistant S. aureus (MRSA) is the leading cause of community onset skin and soft tissue infections in the US. This study looks at a 15-year trend of community onset (CO)-MRSA and MSSA infections and determines ‘best’ to ‘worst’ infection trends. We identified distinct groups of CO-MRSA and MSSA infection rate trajectories by grouping census tracts of the 20 county Atlanta Metropolitan Statistical Area (MSA) between 2002 to 2016 with similar temporal trajectories. Staphylococcus aureus (S. aureus infections and relevant US census data (at the census tract level). A group based trajectory model was applied to generate community onset S. aureus trajectory infection groups  by census tract and were mapped using ArcGIS. This is a retrospective study from 2002-2016, using electronic health records of children living in Atlanta, Georgia with S.aureus occurrence during this time period belonged to low infection rate groups for both MRSA and MSSA, with a higher proportion occurring in the less densely populated counties. Census tracts in DeKalb County, one of Atlanta’s most densely populated areas, had the highest proportion of the worst infection trend patterns . Three CO-MSSA infection groups  and two CO-MRSA infection groups  were detected among 909 census tracts in the 20 counties. We found ~74% of all the census tracts with Trends of Community-Onset MRSA and MSSA Infection Rates Based on Group-based Trajectory ModelsSpatial patterns for CO-MRSA and CO-MSSA Trajectory Trends in the Atlanta Metropolitan Area Between 2002 to 2016S. aureus infection patterns, stratified by antibiotic resistance over geographic areas and time, identify communities with higher risks for MRSA infection compared to MSSA infection. Further investigation of the determinants of the trajectory groupings and the geographic outliers identified by this study may be a way to target prevention strategies aimed to prevent S. aureus infections. Trends of All Authors: No reported disclosures


Background: Immune checkpoint inhibitors have been successfully used in a variety of tumors, however, the efficacy of immune checkpoint blockade therapy for patients with glioma is limited. In this study, we tried to clarify gene expression signatures related to the prognosis of gliomas and construct a signature to predict the survival of patients with gliomas.Methods: Calcium-related differential expressed genes (DEGs) between gliomas and normal brain tissues were comprehensively analyzed in two independent databases. Univariate, multivariate Cox regression analysis and proportional hazards model were used to identify the prognostic of calcium-related risk score signature. The CIBERSORT algorithm and association analysis were carried out to evaluate the relationship between calcium-related signature and characteristic clinical features, tumor-infiltrating immune cell signatures as well as immune checkpoint molecules in glioma. A nomogram model was developed for predicting the overall survival for patients with gliomas.Results: We found the intersection of 415 DEGs between gliomas and normal brain tissues, and identified that an eighteen calcium-related gene panel was significantly enriched in these DEGs. A calcium-related signature derived risk score was developed to divide patients into high- and low-risk groups. Low levels of calcium-related gene expression in high-risk score cases were accompanied with worse outcomes of patients. Calcium-related risk scores were significantly associated with characteristic clinical features, immune infiltrating signatures of tumor microenvironment, and exhausted T cell markers including programmed cell death 1 (PD-1), lymphocyte activating 3 (LAG3), and T cell membrane protein 3 (TIM-3), which contribute to an adverse therapeutic effect of immunotherapy. Calcium-related signature risk score was considered as an independent prognostic parameter to predict the of overall survival of patients with gliomas in nomogram model.Conclusion: Our study demonstrated that calcium signaling pathway is highly associated with immunosuppression of gliomas and overall survival of patients. Targeting the calcium signaling pathway might be a new strategy to reverse the immunosuppressive microenvironment of gliomas and improve the efficacy of glioma immunotherapy. Gliomas remain the most prevalent and incurable primary intracranial tumors of brain cancer , with anAlthough the central nervous system (CNS) has been recognized as an “immune privileged” site , accumulIn the last decade, immunotherapy, a revolution in cancer treatments and therapies, has become one of the most promising strategies with the ability to penetrate the BBB. Unfortunately, the remarkable success of immune checkpoint blockade (ICB) in other malignancies such as advanced melanoma , non-smaIn this study, we explored intersection differential expressed genes (DEGs) between glioma tumors and normal tissues from The Cancer Genome Atlas (TCGA) cohort  and a public RNA sequencing data described in 1 The processed genomic data, proteome normalized data, and clinical characteristics of 99 GBM cases and 10 normal samples were downloaded from the 2) database, including 268 GBM patients in accession GSE16011 and 100 GBM patients in accession GSE4271. Patients without clinical evidence were excluded from this study.The gene expression RNA-seq data and clinical information of 691 gliomas (contains 525 LGG cases and 166 GBM cases) and 5 normal brain tissues were downloaded from TCGA.p-value less than 0.01 and the absolute log2 fold change greater than 1 was used for defining DEGs. Then we selected the intersection of DEGs (iDEGs) in these 2 datasets and explored the biological functions of the iDEGs by the Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis and the gene ontology (GO) analysis. Only pathways with a p-value less than 0.05 were considered to be significantly enriched pathways. Gene set enrichment analysis (GSEA) was applied for identifying statistically gene sets.R package “limma” was utilized to identify the DEGs between gliomas and normal brain samples in two transcriptional datasets including the TCGA and the dataset from We selected iDEGs related to the calcium signaling pathway and performed univariate cox regression analysis to determine the gene with prognostic significance in the TCGA datasets. Then the Cox proportional hazards model was adopted for the construction of the optimal gene set with R package “glmnet.” Subsequently, the linear combination of gene expression weighted by regression coefficients (Coeffs) was developed to calculate the risk scores of each patient in the TCGA dataset with the following formula:i meant the 18 calcium-related genes and Exp meant the mRNA expression level.where Meanwhile, this formula was used to calculate the risk scores for the other 4 datasets of patients with gliomas. The R package “survival” and “survminer” was used to determine the optimal cut-off value of risk score to divide patients into high- or low-risk groups. The package “survivalROC” was used to evaluate the predictive efficiency of the gene signature by developing the time-dependent receiver operating characteristics (ROC) curve with area under the curve (AUC) value.p-value < 0.01 as statistical significance.The pathologic feature information was obtained along with the transcriptome data in the TCGA and CGGA datasets. The grade of the gliomas in the TCGA database was not allowed to download anymore, so we chose the other two datasets from GEO to study the association between the risk score and the grade of gliomas instead. To illuminate the association between the risk score and the IDH-mutation and the grade of gliomas, we used the Mann-Whitney Wilcoxon test and considered a 3 to estimate the relative abundance of 22 types of immune cells in gliomas from the TCGA database. CIBERSORT is a deconvolution algorithm, which uses a set of reference gene expression values as the minimum representation of each cell type, and estimates the cell composition of complex tissues through supporting vector regression based on the gene expression profiles of a large number of tumor samples  and consensus heatmap were used to assess the optimal K.The clinical characters considering age, chemotherapy, IDH-mutation, and risk score were selected to construct a visualized prognostic nomogram model. R packages “rms” and “survival” were utilized to predict the probability of 1-, 2-, and 3-year overall survival for patients with gliomas. The predictive capability of the nomogram was evaluated through measuring discrimination and calibration utilizing a bootstrap approach under 1,000 resampling. Discrimination was accessed via the Concordance index (C-index), which is used to evaluate the predictive value of a nomogram. The predictive ability of the nomogram model is more accurate when the C-index of the nomogram is closer to 1. Calibration curves were applied to evaluate the consistency between the predicted survival probability of the nomogram and the actually observed possibility.We first analyzed different mRNA expressions among glioma tissues and normal tissues. 2009 DEGs were detected in the TCGA database, while 799 DEGs were identified in the dataset from We next tried to find out which iDEGs were involved in the calcium signaling pathway. As a result, 19 genes were identified  and wereWe then performed cross-validation tuning parameter selection by use of a proportional hazards model to confirm the association of calcium-related gene panel with the best prognostic value . As a reCalcium signaling serves as a ubiquitous second messenger and controls numerous cellular processes. We performed principal component analysis (PCA) for all glioma samples in the TCGA cohort and showed that the high- and low-risk score cases based on calcium-related signatures were largely consistent with discrete sections of glioma patients stratified by PCA . Then weWe verified the association between calcium signal and immune responses in the other way. We stratified 691 gliomas patients from TCGA database and took an unbiased approach using consensus clustering of the gene expression profile. Hierarchical clustering of 2487 immune-related genes identified two main subtypes  , assessi+ T cells, Tregs, NK cells, macrophages , T cell membrane protein 3 (TIM-3), cytotoxic T-lymphocyte associated protein 4 (CTLA-4), PD-1, B- and T-lymphocyte-associated protein (BTLA), B7-H4, and B7-H3. There was a significantly positive correlation between risk score and the expression of these immune checkpoints  to reconstruct calcium-related signatures of glioma in the TCGA cohort at the immune cell types level. As shown in Univariate Cox regression analysis was applied for assessing the contribution of a calcium-related signature as an independent prognostic parameter to the overall survival of glioma cases in the TCGA cohort. As shown in In this study, we systematically evaluated the prognostic values of our calcium-related gene signature in patients with gliomas and proposed a calcium-related risk score system. The higher calcium-related risk score was tightly associated with the immunosuppressive tumor microenvironment and predicted a worse prognosis of glioma patients, suggesting that the calcium-related risk score provided an appropriate tool for pretreatment stratification and post-trial evaluation of patients.+ channels and its stores in the endoplasmic reticulum, controlling a large array of principle physiological and pathological processes  can be found in the article/XL and CL: conception and design, writing, review, and revision of the manuscript. CL, JC, ZS, PX, and GL: development of methodology. CL, PL, ZLiu, CZ, DX, and ZLin: acquisition of data. XL: study supervision. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Chronic liver disease is a major public threat and the second leading cause of loss of working life years in Europe . It is oAlcoholic liver disease (ALD) is the leading cause of liver death and the responsible mechanisms were reviewed by Liu et al. . The manThis Special Issue pays close attention to the role and potential of microRNAs (miRNAs), short non-coding RNAs involved in intra- and extracellular signaling pathways and post-transcriptional regulation of gene expression . MiRNAs Gsk3b) and repressed sirtuin 1-mediated mitochondrial biogenesis. These events were associated with alleviated mitochondrial proteostatic stress [In addition to both review articles, Yang et al. directly demonstrated the usefulness of a miRNA induction in mice fed with a methionine-choline-deficient diet (MCD) that is used as a model of fatty liver disease. This is particularly relevant since non-alcoholic fatty liver disease (NAFLD) represents not only a leading, but also a rapidly growing cause of chronic liver disease and is sometimes referred to as the “first pandemic” of the 21st century . Comparec stress . While tIn another attempt to characterize the factors responsible for NAFLD development, Hohenester et al. turned to the “American lifestyle-induced obesity syndrome” (ALiOS) diet mouse model and examined the contribution of the NLRP3 inflammasome and its effectors IL-1 and IL-18. NLRP3 is a so-called pattern recognition receptor that detects danger signals released from damaged cells/bacteria and activates downstream cytokines such as IL-1 and IL-18 ,8. HohenAn experimental paper from Lunova et al. assessed the functional relevance of two different interferon lambda genes, IFNL3 and IFNL4 . InterfeLast but not least, Rabekova et al. took advantage of a large cohort of patients with liver cirrhosis and studied whether genetic variants in the alpha1-antitrypsin gene affect the susceptibility to development of HCC . In lineAltogether, this Special Issue accurately illustrates the current developments in Hepatology such as a focus on personalized medicine with individualized prediction of a disease course as well as the need for stratification of patients into different risk groups. It also reflects the importance of miRNAs, the complexity of inflammatory responses, as well as the growing attention to alcoholic/non-alcoholic liver disease as the leading causes of liver-related mortality.


Fibrillar adhesins are long multidomain proteins that form filamentous structures at the cell surface of bacteria. They are an important yet understudied class of proteins composed of adhesive and stalk domains that mediate interactions of bacteria with their environment. This study aims to characterize fibrillar adhesins in a wide range of bacterial phyla and to identify new fibrillar adhesin-like proteins to improve our understanding of host-bacteria interactions.Through careful literature and computational searches, we identified 82 stalk and 27 adhesive domain families in fibrillar adhesins. Based on the presence of these domains in the UniProt Reference Proteomes database, we identified and analysed 3,542 fibrillar adhesin-like proteins across species of the most common bacterial phyla. We further enumerate the adhesive and stalk domain combinations found in nature and demonstrate that fibrillar adhesins have complex and variable domain architectures, which differ across species. By analysing the domain architecture of fibrillar adhesins, we show that in Gram positive bacteria, adhesive domains are mostly positioned at the N-terminus and cell surface anchors at the C-terminus of the protein, while their positions are more variable in Gram negative bacteria. We provide an open repository of fibrillar adhesin-like proteins and domains to enable further studies of this class of bacterial surface proteins.This study provides a domain-based characterization of fibrillar adhesins and demonstrates that they are widely found in species across the main bacterial phyla. We have discovered numerous novel fibrillar adhesins and improved our understanding of pathogenic adhesion and invasion mechanisms.The online version contains supplementary material available at (10.1186/s12864-021-07586-2). Studying how bacteria interact with their host is essential for understanding bacterial infection processes and characterizing interactions with commensal bacteria. Adhesion to the host cell is the first step of a bacterial infection. Interfering in the adhesion process by Anti-adhesion therapies, primarily by stimulating a humoral immune response, is an effective strategy to prevent infections and has been widely used for the development of vaccines targeting host binding proteins . For exaFibrillar adhesins are long filamentous surface proteins that share similar functions and are expressed by a variety of different bacteria . BesidesFA-like proteins are extracellular.FA-like proteins possess an adhesive region usually at the protein terminus.FA-like proteins possess a stalk-like region that forms a rigid rod-like structure.FA-like proteins usually possess a surface anchoring motif or domain.FA-like proteins are composed of one or a small number of identical fibrillar chains, where each monomer extends through the whole length of the fibril.Few fibrillar adhesins have been studied so far and these come from a limited range of organisms. To our knowledge, no comprehensive review or study of fibrillar adhesins across bacterial species has been performed before. It would be infeasible to experimentally characterise these proteins at large scale and determine their fibrillar nature. In this work, we aim to computationally identify fibrillar adhesin proteins using a sequence-based perspective. Thus, we define a new class of proteins that we call fibrillar adhesin-like (FA-like) proteins based on the common features of known fibrillar adhesins. These proteins have the following properties: Fibrillar adhesins appear to be composed of a relatively limited number of domain families, which facilitates their computational identification. We search for FA-like proteins based on a well studied collection of adhesive and stalk domains and we use Pfam domain definitions to identify and characterize FA-like proteins. This study aims to create a comprehensive survey of fibrillar adhesins across bacterial phyla to gain understanding of their prevalence, distribution and composition. We hope that this study will guide researchers to investigate interesting new fibrillar adhesins, leading to an improvement in our understanding of microbial interactions in health and disease.Fibrillar adhesins are composed of multiple stalk domains, commonly repeated in tandem, and at least one adhesive domain. We gathered a set of 27 adhesive Pfam domain families from known fibrillar adhesins in the literature and 82 stalk domain families from proteins with tandem sequence repeats, as described in the “The majority (19 out of 27) of the adhesive domains in our set bind to protein ligands, while 7 out of 27 bind to carbohydrates (supplementary table S2). The Ice_binding domain Pfam: PF11999) differs from the other adhesive domains in that it doesn’t bind to host cells or to other bacteria, but to ice crystals, allowing organisms to survive in extremely cold environments [999 diffeListeria monocytogenes. Different combinations of adhesive and stalk domains are found across FA-like proteins. The total number of stalk domains in FA-like proteins varies from 1 to 73. Figure We identified a total of 3,542 FA-like proteins composed of at least one adhesive and at least one stalk domain from our sets. These represent 0.013% of all proteins in the UniProt bacterial reference proteomes. Species-wise, FA-like proteins are found in 26% of the bacterial reference proteomes . The larDespite some of the FA-like proteins identified having a low number of annotated stalk domains, the actual number might be higher, since some stalk domains are not yet known and therefore not annotated. In addition, the expression of FA-like proteins with a variety of stalk domain numbers leads to length variation, which is suggested to play a role in the regulation of the bacterial interactions with its binding partner . NeverthBacteria use a wide range of different protein domains to mediate adherence to their host or other organisms. To date, it is not known which of these domains are the most common within fibrillar adhesins. Our domain based screen for FA-like proteins Fig. b shows tWe were further interested to investigate if there is a common domain grammar in FA-like proteins. In the detected FA-like proteins, we found combinations of 76 different stalk domains with 23 different adhesive domains. SlpA (Pfam: PF03217), I-set (Pfam: PF07679) Fil_haemagg (Pfam: PF05594), Fil_haemagg_2 (Pfam: PF13332), DUF5649 (Pfam: PF18886) and Beta_helix_3 (Pfam: PF18889) were the only stalk domains not found in combination with a known adhesive domain and the adhesive domains SabA_adhesion (Pfam: PF18304), SSURE (Pfam: PF11966), FadA (Pfam: PF09403) and FimH_man-bind (Pfam: PF09160) were not found with any known stalk domains.Streptococcus mutans adhesin P1 protein [We plotted the combinations of adhesive and stalk domains as a heatmap in Fig.  protein .Fig. 3SThe pair YadA_head and YadA_stalk (Pfam: PF05662) are the most prevalent domain combination, found in 770 FA-like proteins. YadA_stalk is by far the most commonly occurring stalk domain with annotations in 772 of the 3,542 FA-like proteins, followed by SpaA (Pfam: PF17802), with 668 FA-like protein hits (supplementary figure S1). Collagen_bind (Pfam: PF05737) and SpaA are the second most prevalent domain combination found in 303 FA-like proteins.S. aureus (UniProt: D2JAN8) with AlphaC_N and AlphaC_N2 adhesive domains in combination with Big_6 stalk domains. SAP077A_019 is not part of the UniProt Reference Proteomes and its adhesive domains are not captured by the current Pfam models.There are likely more adhesive and stalk domains not yet known and therefore not included in this study. Furthermore, we only used proteins from the UniProt Reference Proteomes, so potential FA-like proteins and novel domain combinations might be missing in this study. Moreover, imperfect annotation of stalk or adhesive domains by the Pfam hidden Markov models (HMMs) may lead to further missing FA-like proteins. One example is the SAP077A_019 fibrillar adhesin of Besides combinations of adhesive and stalk domains, which occur together in the same protein, there are also stalk domains that are commonly found together (supplementary figure S2). SpaA and Cna_B (Pfam: PF05738) are the stalk domains most often found together, in a total of 99 FA-like proteins. Both domain families belong to the Transthyretin clan (Pfam: CL0287), indicating that they are homologous and possess the same fold. SpaA is found together with a wide range of stalk domains, several of which belong to the Transthyretin clan and others belong to different clans such as the Big_9 domain of the E-set clan (Pfam: CL0159). Out of the 76 stalk domains which are found in combination with an adhesive domain, there are six domains which are not found with another known stalk domain on the same protein (in the Reference Proteomes). These five domains are Rib (Pfam: PF08428), QPE (Pfam: PF18874), Big_3 (Pfam: PF07523), Big_3_2 (Pfam: PF12245), DUF5977 (Pfam: PF19404) and AIDA (Pfam: PF16168).Domain pairs that are annotated together in the same protein and that function together are called supra-domains . In 222 Another example of a supra-domain, present on the heatmap in Fig. Fibrillar adhesins are surface proteins that are attached to the bacterial cell surface. The attachment mechanism can take many forms, such as non-covalent attachment by S-layer homology domains , or covaTo better characterise FA-like proteins we created a list of known anchoring motifs, signals and domains (supplementary table S3). The frequency of each of these different types of anchoring methods is shown in Fig. The Gram positive anchor motif is the most prevalent one in the detected FA-like proteins with 668 hits in total, of which 489 FA-like proteins belong to Firmicutes and 164 belong to the Actinobacteria phylum. In addition to the LPxTG motif, the alternative Gram positive anchor motifs LPxTA, NPxTG, LPxGA, LAxTGand NPQTN were included .The S-layer homology domain was found in 72 detected FA-like proteins and again mostly in the generally Gram positive Firmicutes. The anchor domain CHU_C is found in 134 FA-like proteins, all of which belong to the Bacteroidetes phylum. Por_Secre_tail is found in 124 FA-like proteins and mostly in Bacteroidetes. Four proteins with LysM domains (Pfam: PF01476) were detected, two proteins with Choline_bind_1 (Pfam: PF01473), two proteins with Choline_bind_3 (Pfam: PF19127), two with IAT_beta and one with GW domain. In Proteobacteria the YadA anchor (Pfam: PF03895) domain, Autotransporter domain (Pfam: PF03797) and the LapG + HemolysinCabind anchor were the most prevalent. The LapG motif is the protease cleavage site at the N-terminus of RTX adhesins, where the proteins are cleaved by the LapG protease and subsequently inserted into a beta barrel of an outer membrane pore . To be cWe ran the protein subcellular localization predictor PSORTb to identify that 871 of the 3,542 FA-like proteins were predicted to be localised at the cell wall, 547 to be at the outer membrane, 451 to be extracellular and 1,428 of these proteins had no localisation predicted . PSORTb S. aureus [It is generally assumed that, in fibrillar adhesins, adhesive domains are projected away from the bacterial cell surface by the elongated structure composed of stalk domains, promoting its interaction with the host cell. This architecture is similar to multiprotein pili adhesins, where the protein with the binding function is the membrane distal protein of all pili subunits . In fibr. aureus , 27. In In the Gram negative phyla Bacteroidetes and Proteobacteria, the distribution of domains is more variable. For Proteobacteria, the adhesive as well as the stalk domains show a relatively even distribution along the length of the FA-like proteins with a weak preference for adhesive domains at the termini Fig. c. In BacS. mutans , where the adhesive domain Adhesin_P1_N is annotated at the N-terminus and a second adhesive domain GbpC is annotated in the middle of the protein sequence. Adhesin_P1_N folds then to interact with the stalk domains Antigen_C and AgI_II_C2 at the C-terminus, which results in having GbpC at the top of the protein [There are known examples of centrally located adhesive domains. One example is adhesin P1 protein in  protein . Adhesin protein .Listeria monocytogenes. The number of FA-like proteins per bacterial reference proteome varies: proteomes with the highest prevalence commonly contain in the order of 10 FA-like proteins, reaching in the case of Streptobacillus moniliformis up to 21 FA-like proteins (1.47%).To get a deeper understanding on the origins and evolution of FA-like proteins, we looked at the distribution of adhesive and stalk domains across the bacterial phyla see Fig. . FA-likeSucciniclasticum, Acidaminococcus, Dialister, Veillonella and Megasphaera, whose domain composition are more similar to the Proteobacteria. This underlines the importance of the cell surface composition for the architecture of FA-like proteins. In Proteobacteria the FA-like proteins are mainly built out of YadA_head and YadA_stalk domains. For nearly all of the Firmicutes genera and many Actinobacteria FA-like proteins composed of Collagen_bind, VWA or TED (Pfam: PF08341) in combination with the SpaA stalk domain could be detected. The Gram negative Bacteroidetes use a wider range of domains compared to the Proteobacteria, of which many adhesive domains are the same as in Gram positive bacteria.The adhesive and stalk domains in FA-like proteins mapped to the genera in Fig. There are adhesive and stalk domains found in a wide range of genera, as well as adhesive and stalk domains limited to a few specific genera. VWA, for example, is an adhesive domain widely distributed and found in combination with different stalk domains in Firmicutes, Proteobacteria, Bacteroidetes, Planctomycetes and Actinobacteria. On the other hand, Intimin_C (Pfam: PF07979), seems to be adapted to Proteobacteria as it was only detected in this phylum in combination with the Big_1 (Pfam: PF02369) and Big_2 (Pfam: PF02368) stalk domains.We observed that some of the domains classified as adhesive in our list can also be found in repeats, leading us to question the limits between adhesive and stalk domain functions, and whether they can be interchangeable. Multiple adhesive domains repeated in tandem might form stalks, and stalk domains might evolve adhesion function over time to increase the binding avidity to host cells.Haemophilus influenzae Hap adhesin [S. aureus [E. coli [In Fig.  adhesin , but for. aureus . Similar[E. coli , suggestListeria monocytogenes, but also in poorly characterised pathogens, like Sneathia amnii.In this paper we aimed to comprehensively identify FA-like proteins and describe their characteristic domain composition, architecture and taxonomic distribution. Our identification approach is based on Pfam family HMMs of known adhesive and stalk domains, and has resulted in the discovery of many novel FA-like proteins from a wide range of bacterial genera. These proteins include new putative binding proteins in well studied pathogens like Our results show the various different adhesive and stalk domain combinations that exist in nature and also how frequently they are found. The information can facilitate the identification of new fibrillar adhesins. For instance, when analysing a new FA-like protein, a potential annotated adhesive domain can infer the existence of a specific stalk domain and vice versa. Domain grammar approaches have shown that a knowledge of the domain grammar of proteins can enhance sequence similarity searching , 33. ForOne of the more striking results of our domain grammar analysis was the presence of adhesive domains at the N-terminus of Firmicutes FA-like proteins. This preference is largely due to the presence of a C-terminal sortase motif in these proteins. For many of the analysed adhesive domains their relative position in the FA-like proteins stays constant (supplementary figure S4). This observation can be partly explained by the low number of FA-like proteins in which adhesive domains are found, for example in the case of AlphaC_N. There might be further limitations of adhesive domains to be found at either side of the stalk in FA-like proteins, such as effective binding orientation. The Bact_lectin domain is mostly found in the N-terminal third of FA-like proteins (supplementary figure S4) and binds sucrose at the N-terminal end of the adhesive domain in the SraP adhesin . It is qS. aureus [The investigation of the taxonomic distribution of adhesive and stalk domains show a wide range of different domains in Gram positive bacteria. One reason for this variety might be that the majority of fibrillar adhesins already studied belong to Firmicutes genera, as for example . aureus , and sub. aureus , but the. aureus  for the In our results, we describe that the adhesive domain SSURE is not found in combination with other known stalk domains, and that it is found on average in 3 copies per protein. We additionally know that SSURE is tandemly repeated. This leads to the hypothesis that some adhesive domains might be able to function as the stalk itself. Several adhesive domains are indeed found repeated. A reason for repeating adhesive domains could be to increase the avidity of binding to the host cells. Thus, duplications of these domains may lead to both extending the stalk length and increasing binding efficiency. In particular, when the host ligand is a collagen, the adhesive domain might bind to different positions simultaneously on the host ligand. The adhesive domain Collagen_bind binds collagen with the very specific collagen hug mechanism which requires the supra-domain of Big_8 domain N-terminal to the Collagen_bind domain. Therefore, it would be interesting to find out why Collagen_bind occurs in repeats and whether all repeats contribute to the adhesion process.The greatest limitation of our FA-like protein discovery process is that it requires a protein to contain a combination of known stalk and adhesive domains. There are undoubtedly numerous fibrillar adhesive proteins missed by our approach. There are several reasons for these missed proteins. Firstly, there may be FA-like proteins that have missing domain annotations for either the stalk or adhesive domain or both. This could be because there is no domain family represented within Pfam currently. A second possibility is that the current Pfam model misses true examples of the stalk or adhesive domain. And thirdly, simply that there are adhesive and stalk domains we did not yet identify. An estimation of the completeness of the stalk domain list for known adhesive domains is given in supplementary table S4. Nevertheless, the information about fibrillar adhesins gained in this study will be important for further improvements in future identification of these proteins.This study presents to our knowledge the most comprehensive characterisation of fibrillar adhesins to date. We have used a protein domain based screen to identify over 3,000 fibrillar adhesin-like proteins and characterised their domain content, architecture and taxonomic distribution. The results underline the complexity of fibrillar adhesins, with highly variable domain organisations as well as with various different domain combinations found in nature. The detected FA-like proteins are widespread across the bacterial taxonomic tree. The set of FA-like proteins and associated stalk and adhesive domains identified in this study set a foundation stone to discover further fibrillar adhesins.We searched in the literature for experimentally confirmed bacterial adhesins, for example by a keyword search for adhesins in PubMed. We analysed the domain architecture of the relevant proteins and identified the adhesive domains by searching for the binding regions. We also identified numerous adhesive domains through investigation of structures in the Protein Data Bank. In each case we identified the relevant entries in the Pfam database and recorded the relevant identifiers.Stalk domains were detected by searching for tandem repeat domains in the Pfam database, whereby repeats are counted as tandem repeats when the sequence separation between two domains from the same Pfam family is shorter than 35 residues. The further putative stalk domain families were checked manually in Pfam to ensure their description, species distribution and domain architecture matched the stalk domain definition. Domain families found only in eukaryotes, and non-surface and enzymatic proteins, were excluded.Tandem sequence repeats were identified in proteins without known Pfam annotations with the T-REKS tool using 70% sequence identity and length between 50-200 residues as parameters . Six newIn this study all bacterial proteins from the UniProt Reference Proteomes (release 2020_03) were used . The 7,5The number of stalk domains per protein in combination with one adhesive domain were counted, regardless of whether the stalk domains belonged to the same Pfam family or not. The frequency of adhesive and stalk domains as well as the combinations of adhesive and stalk domains and different stalk domains found on the same FA-like proteins were counted, whereby each domain was only counted once even if multiple domains of the same family were annotated on the protein.https://www.uniprot.org/uploadlists/).For the generation of the density plots representing the domain positions within the proteins, the protein length and the envelope domain location information were used from the HMMER search output file. The fractional start and end positions for the domains were calculated. Furthermore, sortase motifs were identified by regular expression in the C-terminal 50 amino acids of the FA-like protein sequences. For the analysis of the domain architecture between the different phyla, the taxonomic phyla were mapped to the UniProt protein identifier by using Retrieve/ID mapping from the UniProt website (Marinobacter hydrocarbonoclasticus (UniProt: H8W6K8), the described secondary structure motifs were not found so we only searched for the protease cleavage motif AAG within the first 150 amino acids in the FA-like protein sequences. To increase the probability that the found hits are indeed RTX adhesins, we additionally searched for the HemolysinCabind domain, which is characteristic for the RTX adhesins. In total 62 LapG cleavage motifs and HemolysinCabind domains could be found together in a single protein. In 31 of these 62 FA-like protein hits the short coil motif GxDPT could be found, whereas the alpha-helical element motif xxIQxAIAA was only found in two of these proteins. For comparison, the PSORTb webserver (version 3.0.2) was used to predict the localization of the 3,542 FA-like proteins [To find out how FA-like proteins are attached to the bacterial cell, HMMs of known anchor domains were downloaded from Pfam. We used the following models: Gram_pos_anchor (Pfam: PF00746), SLH (Pfam: PF00395), CHU_C (Pfam: PF13585), Por_Secre_Tail (Pfam: PF18962), LysM (Pfam: PF01476), Choline_bind_1 (Pfam: PF01473), Choline_bind_2 (Pfam: PF19085), Choline_bind_3 (Pfam: PF19127), YadA_anchor (Pfam: PF03895) IAT_beta (Pfam: PF11924), GW (Pfam: PF13457), PG_binding_1 (Pfam: PF01471), PG_binding_2 (Pfam: PF08823) and PG_binding_3 (Pfam: PF09374) . By HMMEFor taxonomic analysis, additional to the phyla, the genera were also mapped by using Retrieve/ID mapping from the UniProt website. The 16s rRNA sequences of representative bacterial genera and of genera with a minimum of 5 detected FA-like proteins were aligned in JalView using Muscle with the default settings , 44. AftProtein sequences containing known adhesive domains were downloaded from Pfam version 33.1 . ProteinThe HMMER search results of the long bacterial proteins with known adhesive domains against the Pfam database from the “For each adhesive domain found in the FA-like proteins we calculated if its mid point is within the N-terminal, middle or C-terminal third of the FA-like protein. We then calculated the percentage of occurrence in each of the three relative positions for each adhesive domain. The results are presented in the heatmap in supplementary figure S4.Additional file 1 Supplementary figures and tables.