Here, we discuss current research progress on MG-derived in vivo neurogenesis in the mouse retina, focusing on the use of stringent fate mapping techniques to evaluate and validate de novo regeneration of retinal neurons through the reprogramming of endogenous MG. Establishing stringent experimental criteria is critical for examining current and future studies on MG-derived regeneration of photoreceptors, retinal inter-neurons, and retinal ganglion cells.Müller glia (MG) are a potential source of stem cells in the mammalian retina that could replenish lost retinal neurons for vision restoration. Unlike their counterpart in zebrafish, mammalian MG are quiescent and they do not spontaneously generate new retinal neurons. In recent years, extensive research efforts have been made to unlock the regenerative capabilities of Müller glia (MG) for  The retina is a highly organized laminar structure composed of specialized nerve cells in the back of the eye. Loss of retinal neurons from either genetic deficiencies or environmental insults leads to vision impairment and blindness. The vast majority of vision loss results from the degeneration of two classes of retinal neurons, the light-sensing photoreceptors that mediate the first step in vision and retinal ganglion cells (RGCs), the output neurons in the retina that send visual information from the eye to the brain via their long axon projections. Degeneration of photoreceptors and RGCs is the leading cause of vision loss in major eye diseases, including age-related macular degeneration, diabetic retinopathy, and glaucoma. Therefore, photoreceptors and RGCs are in great need of being replenished.in vivo. When growth factors  were injected in vivo after NMDA-induced neuronal degeneration in the mouse retina, substantial MG proliferation occurs and some of the MG progeny differentiate into cells that express features of retinal interneurons such as amacrine cells  mouse retina, MG from the NFI-depleted mouse retina can be reactivated by NMDA-induced injury and a subset of them transdifferentiated into bipolar and amacrine-like interneurons  of RGC loss, respectively. Furthermore, both studies showed that a small proportion of reprogrammed MG was converted into amacrine cells.Very recently, two e retina . The sece retina . Strikinin vivo cellular reprogramming ;LNL-tTA;tetO-Ascl1-ires-GFP mice. Ascl1-expressing MG were lineage traced with GFP expression , which was down-regulated shortly after injury but elevated at later stages, Hoang et al. examined tamoxifen-induced Glast-CreER;CAG-LSL-Sun1-GFP;Nfia/b/xlox/lox mice, in which NFIs were selectively deleted in MG. A third MG-specific mouse line, GFAP-Cre, was used by Yao et al. in their two studies. To examine MG cell fate changes after AAV-mediated gene transfer of β-catenin and subsequent gene transfer of three pro-rod transcription factors , Yao et al. generated GFAP-Cre;Ai14 (Rosa26-tdTomato) mice to genetically trace MG lineages independently of the AAV-mediated labeling system (Glast-CreER and Rlbp1-CreER), the GFAP-Cre line is not tamoxifen-inducible. Given the concern of the GFAP promoter driving transgene expression in neurons in the AAV-mediated expression system, selection of the Glast-CreER or Rlbp1-CreER line would be a better choice for fate mapping experiments in MG-derived neurogenesis studies.To examine the role of Ascl1 in MG-derived neurogenesis in the damaged mouse retina, Reh and colleagues performed stringent fate mapping experiments using two MG-specific tamoxifen-inducible Cre mouse lines, pression . In anotpression , Hoang eg system . DifferePtbp1 downregulation, by injecting AAV-GFAP-GFP-Cre into the eyes of Ai9 (Rosa-CAG-LSL-tdTomato-WPRE) reporter mice  to isolate lineage traced GFPrneurons . In theirneurons . Hoang ee status . Identife status , they diin vivo reprogramming process would identify the transitional cellular states of MG-derived neurogenesis. Reh and colleagues showed that the reprogrammed MGs were ‘hybrid’ cells with a morphological appearance similar to MG and bipolar neurons  and the other daughter cell migrating to the outer nuclear layer (ONL) and growing rod outer/inner segments. At the terminal stage, the daughter cell that had migrated to ONL differentiated into a mature rod photoreceptor, while the other MG-derived daughter cell remained in the INL and eventually turned off the expression of the Rhodopsin-tdTomato reporter  can be fairly uniformly overexpressed or conditionally knocked out when a Cre-responsive mouse line expressing the transgene(s) was crossed with a cell-type-specific Cre lineage tracing line. Two main limitations exist for using this tool for gene manipulations that include: 1) transgenic mouse lines are not always available for every gene, and crossing mouse lines could be time-consuming, especially when multiple genes need to be manipulated simultaneously. 2) Mosaic effects could occur when a Cre-responsive transgenic mouse line is crossed with a tamoxifen-induced Cre mouse line. Rueda et al. reported mosaic expression of the Yap5SA transgene in mouse MG when crossing with the Glast-CreER line specific to MG induced by tamoxifen  manipulated genes are not restricted by the availability of mouse lines. 2) AAV vectors can be readily constructed for a wide range of experimental manipulations and purposes, and the preparation of recombinant AAVs is much less time-consuming. However, the weaknesses of using AAV-mediated gene manipulations also exist that include: 1) the limited packaging size of AAVs restricts its application to manipulate large genes. 2) the transduction efficiency, as well as the cell-type-specificity of AAV-mediated gene transfer, can be largely influenced by AAV serotypes . 3) Lastpromoter . More re in vivo , reemphain vivo reprogramming studies on MG-derived neurogenesis in the mouse retina. Having reviewed the methods and criteria used in recent in vivo MG-derived reprogramming studies in mice  Secondly, genetic-based scRNA-seq profiling and visualization of morphological changes will help reveal transitional cellular states when reprogrammed MG undergo neuronal differentiation. 3) Finally, EdU incorporation assays can be performed to examine whether MG-derived neurogenesis is resultant from direct transdifferentiation/conversion of MG in the absence of cell proliferation or from MG-derived progenitors that require MG proliferation before they undergo neuronal differentiation.Performing stringent fate mapping experiments are extremely critical to evaluate  in mice  and discin vivo neurogenesis in the mouse retina, we hope that unlocking the regenerative capacity of mammalian MG will continue to make strides in the important branch of regenerative medicine and visual neuroscience.It is noteworthy that AAV-mediated gene transfer is a powerful tool in reprogramming research. However, AAV-based systems cannot be used exclusively to examine MG-derived neurogenesis due to possible leaky expression in endogenous retinal neurons. With a powerful gene manipulation tool at hand and stringent evaluation criteria to critically examine MG-derived


Some individuals do not return to baseline health following SARS-CoV-2 infection, leading to a condition known as Long COVID. The underlying pathophysiology of Long COVID remains unknown. Given that autoantibodies have been found to play a role in severity of COVID infection and certain other post-COVID sequelae, their potential role in Long COVID is important to investigate. Here we apply a well-established, unbiased, proteome-wide autoantibody detection technology (PhIP-Seq) to a robustly phenotyped cohort of 121 individuals with Long COVID, 64 individuals with prior COVID-19 who reported full recovery, and 57 pre-COVID controls. While a distinct autoreactive signature was detected which separates individuals with prior COVID infection from those never exposed to COVID, we did not detect patterns of autoreactivity that separate individuals with Long COVID relative to individuals fully recovered from SARS-CoV-2 infection. These data suggest that there are robust alterations in autoreactive antibody profiles due to infection; however, no association of autoreactive antibodies and Long COVID was apparent by this assay. Some individuals do not fully return to baseline health following SARS-CoV-2 infection and experience ongoing morbidity following the acute phase of COVID-19 . Immune Acute SARS-CoV-2 infection is associated with the generation of autoreactive antibodies, particularly among individuals with severe disease requiring hospitalization –8. For eAlthough much work has been done exploring the potential virologic and immunologic factors driving Long COVID, the evaluation of autoantibodies in this condition has been more limited. Measurement of anti-nuclear antibodies on standard clinical tests has yielded mixed findings, with some studies identifying a high prevalence of antinuclear antibodies (ANAs) among those with Long COVID –15 and oThe identification of previously reported autoantibodies can be performed using targeted assays. However, identifying the full range of novel autoreactive antibodies, whether they are pathological or not, requires technologies capable of high-throughput, unbiased, proteome-wide screens. In this study, we screened a cohort of individuals with prior SARS-CoV-2 infection, many of whom met clinical criteria for Long COVID, to determine whether a consistent pattern of autoreactivity could be identified. This same technology has been previously utilized to discover novel autoantibodies in a wide range of disease contexts –23.We employed a previously published proteome-wide approach using a T7 phage-display assay with immunoprecipitation and next-generation sequencing (PhiP-Seq) –24. We tTo investigate whether the ARHGAP31 peptide enrichment could be the result of cross-reactivity with antibodies directed against SARS-CoV-2 proteins, we performed a multiple sequence alignment of this 49-amino acid fragment against the full SARS-CoV-2 proteome. A region within the SARS-CoV2 Orf1a polyprotein was identified with considerable physico-chemical similarity to a portion of the autoreactive fragment in ARHGAP31. We were unable to identify any clinical differences between individuals with and without ARHGAP31 autoreactivity, such as differences in frequency of neurologic symptoms.ARHGAP31 is most highly expressed in neuronal cells, Langerhans cells, and endothelial cells . The 20 most enriched proteins with at least 5-fold greater than background (defined as fold-change over mock-IP with protein A/G beads) were compared . SeventeSubgroup analyses were performed to determine whether any of these observed autoreactivities were enriched in particular symptom-defined phenotypes of Long COVID. These include cardiopulmonary , central neurologic symptoms , any neurologic symptom , gastrointestinal , musculoskeletal , and upper respiratory (runny nose and sore throat).The peptide enrichments from individuals with particular Long COVID phenotypes were compared to the enrichments from individuals with Convalescent COVID (no Long COVID symptoms) using a one-sided Kolmogorov-Smirnov test. None of the top 20 autoantibodies were enriched in severe Long COVID, and only 3 autoantibodies were statistically increased in any phenotype: TTF2 and KDM3B in those with cardiopulmonary symptoms and FUCA1 in those with upper respiratory symptoms . HoweverIn addition to analyzing the distribution of post-COVID autoreactive peptide enrichments, we also performed additional analyses to detect those enrichments which might be present only in Long COVID or particular subcategories of Long COVID. To feature-weight enriched peptides, we applied logistic regressions to the degree of enrichment for each individual with Long COVID and each individual Long COVID symptom phenotype versus enrichment from convalescent COVID patients who did not have the particular symptoms. We included all previously discussed symptom phenotypes, as well as individuals with difficulty concentrating, worsened quality of life, new depression, and generalized anxiety disorder. We were unable to identify a set of enriched proteins specific to any of these groups of patients which could effectively distinguish the cohort from controls, as the best receiver operating characteristic (ROC) area under the curve (AUC) was 0.67, and the mean AUC was 0.44 .Autoimmunity has been proposed as one potential mechanism driving Long COVID. We applied an unbiased, proteome-wide, validated approach to assess associations between antibody autoreactivity and clinical phenotype. A clear and robust difference in autoreactivity was detected between those infected with SARS-CoV-2 and pre-COVID controls. This difference was constituted by peptides from diverse and varied proteins, most of which are intracellular, suggesting that the origin of the differential enrichment is due to cross-reactivity with SARS-CoV-2 directed antibodies in those that were exposed. A sequence comparison between a peptide from the most enriched protein, ARHGAP31, and Orf1a of SARS-CoV-2 supports this notion, but orthogonal validation through fine-scale epitope mapping and antibody cloning would be required to demonstrate this conclusively. While the clinical significance of incidental autoreactivity due to the humoral immune response to SARS-CoV-2 remains largely unknown, prior studies have identified cross reactive autoantibodies in severe sequelae of SARS-CoV-2 infection, including those that develop severe neurological symptoms . Understanding the clinical consequences of SARS-CoV-2 driven autoreactivity deserves further attention, perhaps through long-term longitudinal studies.We found no association to support the hypothesis that autoreactivity, as detected in this assay, contributes to Long COVID. Despite numerous successes, PhIP-seq possesses a number of limitations. Because the T7 phage displayed peptides are only 49 amino acids, the assay inherently detects mostly linear epitopes. Therefore, complex conformational, post translationally modified, or multimeric protein configurations are not predicted to be detectable by this assay, and thus these results do not completely rule out autoimmune interactions in Long COVID that are beyond the scope of PhIP-seq.In addition, our classification of Long COVID and its symptom phenotypes was based exclusively on participant self-report of symptoms. It is possible that future analyses in more homogenous cohorts, particularly those with objectively measured physiologic perturbations now associated with certain Long COVID phenotypes  may yet reveal a role of autoantibodies in at least a subset of individuals experiencing Long COVID.Long COVID remains a complex clinical entity. Its causes are likely multifactorial, and there is growing consensus that different phenotypes are driven by different pathophysiologic mechanisms , 3. AddiNCT04362150). The details of study design and measurement have been reported previously (Participants were volunteers in the UCSF Long-term Impact of Infection with Novel Coronavirus (LIINC) study  responses. Symptoms case definitions include: the presence of any new or worsening symptoms since SARS-CoV-2 infection (Long COVID); presence of 5 or more symptoms (severe Long COVID); specific symptom groups according to organ system involvement or phenotypic cluster; and individual symptoms when at least 25 individuals experienced the symptom. For individual and grouped symptom outcomes, we developed 3 potential comparisons between the symptomatic group and  all indiWe defined 6 groups of symptoms (symptom phenotypes) based on either organ system cluster. These include cardiopulmonary , CNS-specific , any neurologic symptom , gastrointestinal , musculoskeletal , and upper respiratory (runny nose and sore throat).Quality-of-life was assessed using the EuroQol-5D (EQ-5D), Patient Health Questionnaire depression scale (PHQ-8), and Generalized Anxiety Disorder scale (GAD-7). Individuals with Long COVID and the lowest overall quality-of-life (QOL) score measured via the visual analogue scale of the EQ-5D were compared to individuals with the highest overall QOL scores among those with and without Long COVID. Individuals with responses categorized as “moderate depression” on the PHQ-8 (score higher than 10) and “moderate anxiety” on the GAD-7 (score higher than 9) were compared to all participants with scores indicating less severe classifications than “moderate depression” and “moderate anxiety” in the following groups:  all partIn addition, we recently demonstrated associations between Long COVID and other chronic latent viral infections, including serologic evidence suggesting recent Epstein-Barr virus (EBV) reactivation . For thiAt each visit, whole blood was collected in EDTA tubes. Plasma was isolated and stored in −80F until the time of analysis.https://www.protocols.io/view/scaled-high-throughput-vacuum-phip-protocol-ewov1459kvr2/v1).PhIP-Seq was performed following our previously published vacuum-based PhIP-Seq protocol   was performed at the gene-level, in which all reads for all peptides mapping to the same gene were summed, and 0.5 reads were added to each gene to allow inclusion of genes with zero reads in mathematical analyses. Within each individual sample, reads were normalized by converting to the percentage of total reads. To normalize each sample against background non-specific binding, a fold-change(FC) over mock-IP was calculated by dividing the sample read percentage for each gene by the mean read-percentage of the same gene for the AG bead only controls. This FC signal was then used for side-by-side comparison between samples and cohorts. Samples which had a FC of 5 or greater were considered enriched for an antibody, and samples with a FC of 6 standard deviations above the mean of pre-COVID controls were considered positive for an autoantibody. FC values were also used to calculate z-scores for each disease category sample by using each respective control (as specified in figures and results), and for each control sample by using all remaining controls. These z-scores were used for the logistic-regression feature weighting. In the case peptide-level analysis, raw reads were normalized by calculating the number of reads per 100,000 reads.All statistical analysis was performed in Python using the Scipy Stats package. A two-way Kolmogorov-Smirnoff(KS) test was used for comparisons of FC PhIP-Seq data between groups of samples, except in the case of specifically looking for those genes with increased signal only in the disease-cohort in which a one-way KS test was employed. The logistic regression machine-learning classifiers were performed using our recently described methods . UtiliziSupplement 1Supplemental Table 1: Characteristics of study participants.Characteristics of all study participants, stratified by Long COVID status.Supplement 2Supplemental Figure 1: PhIP-Seq is unable to distinguish Long COVID symptom phenotypes from controls.(A) Logistic regression receiver operating characteristic (ROC) curves for Long COVID patients with different specified symptom phenotypes relative to patients previously infected with COVID without the phenotype, or (B) with and without EBV laboratory findings.


Vibrio parahaemolyticus type 3 secretion system 2 is a distant homolog of the ToxR/ToxS master regulator of virulence and has evolved beyond confident sequence recognition. Exploiting the newly developed machine learning methods for structure prediction, we observe a VtrC-like lipocalin fold for both the ToxS periplasmic domain and other detected periplasmic sensor components. This structure prediction supports the divergent evolution of VtrA/VtrC-like co-component signal transduction systems and suggests a role for lipid sensing in regulating virulence in enteric bacteria.Using the domain and operon organization of VtrA/VtrC, combined with fold predictions, we identify co-component signal transduction systems in enteric bacteria that likely regulate virulence. We observe that the heterodimeric VtrA/VtrC periplasmic bile acid receptor controlling the  Vibrio parahaemolyticus type III secretion system adopts an obligate heterodimer of two proteins encoded by partially overlapping VtrA and VtrC genes. This co-component signal transduction system binds bile acid using a lipocalin-like domain in VtrC and transmits the signal through the membrane to a cytoplasmic DNA-binding transcription factor in VtrA. Using the domain and operon organization of VtrA/VtrC, we identify a fast-evolving superfamily of co-component systems in enteric bacteria. Accurate machine learning–based fold predictions for the candidate co-components support their homology in the twilight zone of rapidly evolving sequences and provide mechanistic hypotheses about previously unrecognized lipid-sensing functions.Bacterial signal transduction systems sense changes in the environment and transmit these signals to control cellular responses. The simplest one-component signal transduction systems include an input sensor domain and an output response domain encoded in a single protein chain. Alternatively, two-component signal transduction systems transmit signals by phosphorelay between input and output domains from separate proteins. The membrane-tethered periplasmic bile acid sensor that activates the  Escherichia coli transmit environmental signals through a cytoplasmic CheA sensor histidine kinase and CheY receiver as part of a two-component system. These chemoreceptors use a periplasmic ligand binding domain to interact with maltose-, ribose-, and galactose-binding proteins that mediates chemotaxis toward these sugars (2+ (or Ca2+) conditions, PhoQ signals to the receiver domain of its DNA-binding transcriptional regulator PhoP to activate transcription. The PhoP and PhoQ genes are also neighboring  (V. parahaemolyticus infection of the human gut (The ToxR CadC-like HTH family includes another membrane-tethered transcription factor (VtrA) from r (VtrC) . VtrA/Vtuman gut , 26. Theuman gut . Despiteuman gut , 19. GivProtein folds can often be inferred from their evolutionary relationships to experimentally determined structures. These relationships have traditionally been detected at the sequence level by homology-based protein structure prediction methods. However, for fast-evolving domains such as in the periplasmic regions of VtrA/VtrC and ToxR/ToxS, identifying homologs using sequences has proven difficult, even for the most sensitive methods –29. Here30V. parahaemolyticus T3SS2 virulence secretion machinery. The two genes encoding the VtrA/VtrC co-component signal transduction system are neighboring in the V. parahaemolyticus genome and form an operon . Thus, VtrA and ToxR exemplify homologous transcription factors whose periplasmic input domains have evolved beyond sequence recognition.The periplasmic VtrA/VtrC structure adopts an obligate heterodimer that senses the bile acid taurodeoxycholate (TDC) using a lipocalin-like fold adopted by the VtrC component A. The bin operon B. Functin operon B suggestn operon C. DespitSI Appendix, Table S1). Despite this lack of a detectable sequence relationship, the ToxS sequence does identify an alternate lipocalin-like fold with borderline confidence and coverage . This hint at ToxS adopting a similar fold as its VtrC counterpart prompted us to build a structure model using newly developed structure prediction methods, including AlphaFold and ColabFold . PsaF works together with its neighboring membrane-tethered transcription factor PsaE to control the expression of the pH6 antigen adhesin virulence factor in Yersinia . The VtrA/ToxR sequence search identified numerous CadC-like HTH-containing proteins from all represented genomes . These identified sequences were filtered using TMH prediction, resulting in 28 transmembrane-tethered transcription factors from eight species of enterobacteria. To further filter these identified transcription factors for candidate VtrA/VtrC-like co-component signal transduction systems, we inspected their gene neighborhood for a similar potential cotranslated operon organization. Adjacent downstream genes in the same orientation as the identified transcription factors were subjected to transmembrane and signal peptide prediction. One notable candidate co-component system from Yersinia pseudotuberculosis required an N-terminal extension of the predicted start site to include the TMH . This alternate start site was likely misannotated due to its reading frame overlap with the upstream transcription factor (as was seen with the VtrA/VtrC genes) and is extended correctly in close orthologs from other Y. pseudotuberculosis strains.While the periplasmic components of VtrA/VtrC, ToxR/ToxS, and PsaE/PsaF have diverged beyond confident sequence recognition, the CadC-like HTH retains significant sequence similarity and can be used to search for candidate co-component systems in other pathogenic bacteria. Starting from VtrA and ToxR, we searched a database of well-studied bacterial pathogens with complete genomes of high sequence and annotation quality , while the last represents the MarT (FidL cassette from pathogenicity island 3 (Spi-3). The MarT/FidL gene products are cotranscribed and activate expression of the MisL autotransporter, which functions as a host-specific intestinal colonization factor  pathogenicity island in K12 that is fully present in pathogenic strains like E. coli O157:H7 saki, which also encodes YqeI/YqeJ. In addition to this virulence regulator, the pathogenic E. coli O157:H7 saki strain encodes an additional cassette, GrvA (FidL. The GrvA transcription factor activates the locus of enterocyte effacement (LEE)–dependent adherence.Inspection of genomic neighborhoods excluded membrane-tethered CadC one-component transcription factors, for which the VtrA HTH domain is named, and dismissed other pairs that lack input domains or neighboring proteins predicted to be in the periplasm. In addition to the known criteria . Y. pseuthe MarT /FidL casn factor –40. The Shigella unknown protein SF3507 and the V. cholerae TcpH models were less-confident predictions. The final candidate gene cassette, STM0342, was predicted as a four-stranded β-meander that corresponds to half of the barrel and may represent a deterioration of its sequence-related neighbor STM0345. Interestingly, STM0342 includes two Cys residues located too far from one another to form an intrachain disulfide, and the deterioration may form a homodimer suggested by an AlphaFold model  and E. coli ETT2 (YqeI/YqeJ) has been noted  highlight the relationships between family members, where the disulfide-containing proteins FidL, YqeJ, STM0342, STM0345, and SF3507 are confidently related by sequence. The remaining families have diverged beyond confident sequence recognition using the most sensitive sequence comparison methods .The lipocalin-like structure predictions for each of the VtrC-like components support the notion that the second periplasmic component from each gene cassette arose from a common ancestor, despite their sequence divergence. In support of this proposed homologous relationship, the lipocalin-like folds from the en noted . The preTo visualize the relationships between all the VtrC-like families, collected sequence homologs were clustered C. The diPseudomonas species  lacks candidate co-component systems. One of the VtrC-like sequences from Pseudomonas fluorescens strain C1 overlaps with a VtrA-like transcription factor, highlighting the preserved operon organization in this species.Two additional groups of collected homologs diverge from the FidL cluster, including BprQ-like sequences and sequences from various acterium . The Bprei T3SS3 , 47. BprSI Appendix, Fig. S1). Protein–protein complex prediction for many of the other candidate co-components suggests similar interactions, although with generally lower confidence. In support of their heterodimeric interaction, the periplasmic domains from the co-component signal transduction systems PsaE/PsaF from Yersinia and YqeI/YqeJ from E. coli form dimeric complexes when expressed together .The VtrA/VtrC periplasmic bile receptor forms an obligate heterodimer, and many of the identified candidate co-components are known to function together . BecauseThe VtrA/VtrC periplasmic structure forms an obligate heterodimer with a mobile loop covering the lipid binding site in the absence of bile acid B. Upon bTo gauge whether the newly identified periplasmic components might bind lipids, we defined per-residue conservations for several of the VtrC-like clustered sequence groups and mapped them to the structure models to highlight potential functional surfaces. Superposition of the ToxS structure model with the experimental VtrC model bound to TDC E highligConservations in the YP3282 structure model F surrounThe membrane-embedded one-component transcription factor CadC includes an N-terminal HTH, followed by a TMH and a C-terminal periplasmic sensor B. This dYersinia YP3564 model is the only one with the C-terminal helix found in VtrA and ToxR, while its distantly related paralog (YP3383) model lacks the helix and shows a deteriorating N-terminal strand. The models for several of the VtrA-like components  retain a similar overall fold without the C-terminal helix  that contribute to the fold. The experimental VtrA and ToxR periplasmic domain structures include a common set of SSEs (β αβ 3)α) α) 3)α) t YqeI in D. The N- YqeI in D. Finall YqeI in A and 4A,SI Appendix, Fig. S3). The starting Met (atg codon) from the downstream gene tends to overlap with the Cys (tg(t/c) codon) from the disulfide. In this case, the starting Met from the second gene is shifted by one nucleotide upstream with respect to the first gene. The TcpP/TcpH, PsaE/PsaF, YP3564/YP3565, STM0341/STM0342, and STM0344/STM0345 gene overlaps observe this tendency. Alternatively, SF3508/SF3507 and YP3283/YP3282, which are encoded in the complementary strands, include the Cys residues in their overlap. Finally, the MarT/FidL overlap includes an His residue (cat codon) shifted one nucleotide upstream with respect to the MarT overlapping Met (atg codon).Notably, except for VtrA, the co-component transcription factor periplasmic domains contain conserved disulfides. Each of these includes a Cys residue near the C terminus of the protein sequence B and D. SI Appendix, Table S1). The more divergent VtrA-like components . The distance heat map resembles a heat map generated using similarity scores from another structure superposition program . Although it is tempting to speculate that the co-component signal transduction systems replaced the loss of the CadC helical domain with a gained VtrC-like heterodimer, their structure dissimilarity prevents any confident assignment of homology. This questionable relationship extends to the TcpP component, especially given the tendency of domain recombination among bacterial signaling proteins . The E. coli K12 YqeI/YqeJ cassette also appears to be a remnant of the complete ETT2 pathogenicity island present in pathogenic strains like E. coli O157:H7 saki. Pathogenic E. coli O157:H7 saki includes a paralogous YqeI/YqeJ gene cassette (GrvA/FidL) that activates the LEE-dependent adherence , providing a mechanism for its preservation.The similarity in folds between the CadC N-terminal periplasmic domain and the periplasmic domains from the co-component transcription factors points toward their derivation from a common membrane-tethered transcription factor. The Cad system, which is widespread in enteric bacteria, helps maintain a neutral cytoplasmic pH in the acidic environment of the stomach , 12. TheOverall, our studies support a model for a signaling paradigm used by enteric bacteria to sense extracellular cues in the periplasm. The co-component signal transduction system includes an input periplasmic sensor that is tightly associated with its transcription factor, as exhibited by the operon organization, coevolution, and obligate periplasmic domain interaction. This co-component system provides an additional, albeit specialized, mechanism for transmitting an extracellular signal sensed by a periplasmic input through the membrane to a transcription factor output domain, adding to the canonical transmembrane one-component and two-component signaling systems . UnderstV. parahaemolyticus ToxR (BAC59083.1) and VtrA (BAC62675.1) were used as queries with PSI-BLAST . Identified VtrA-related hits were filtered for a predicted TMH using Phobius (VtrA/VtrC), the upstream in-frame region was translated for cases where the adjacent gene was missing a predicted N-terminal TMH or SP.Sequences corresponding to SI-BLAST  . All sequences were clustered with CLANS  using BLCandidate VtrC-like sequences found in tandem with VtrA-like transmembrane transcription factors were submitted to AlphaFold2  structurStarting from candidate co-component protein pairs, we searched for the homologs of each protein encoded by nucleotide sequences in the European Nucleotide Archive database and the Integrated Microbial Genomes and Microbiomes database of the Joint Genome Institute. We used six rounds of iterative HMMER  search wFor each protein pair, their homologs that are encoded next to each other in the nucleotide sequences were extracted. The sequences of these protein pairs were concatenated, and the multiple sequence alignment (MSA) was derived from the pairwise sequence alignments made by HMMER. The MSA was then filtered by sequence identity  and gap ratio in each sequence , and the resulting nonredundant MSA was used as input for complex structure modeling.We deployed AlphaFold , a methoAB) and self  Z-scores were transformed into distances using the following equation: −ln.The experimental structures corresponding to the VtrA periplasmic domain (5kewA), the ToxR periplasmic domain (6uueA), and the CadC N-terminal periplasmic domain  were compared to the models for GrvA (189-270), YqeI (186-269), STM0344 (170-247), SF3808 (170-254), BprP (256-326), PsaE (160-214), YP3564 (162-219), YP3383 (235-348), and TcpH (169-221). All-against-all structures were compared using DaliLte . Pairwis


Youth admitted to the pediatric intensive care unit (PICU) for traumatic brain injury (TBI) commonly struggle with long-term residual effects in the domains of physical, cognitive, emotional, and psychosocial/family functioning. In the cognitive domain, executive functioning (EF) deficits are often observed. The Behavior Rating Inventory of Executive Functioning, Second Edition (BRIEF-2) is a parent/caregiver-completed measure that is regularly utilized to assess caregivers' perspectives of daily EF abilities. Using parent/caregiver-completed measures like the BRIEF-2 in isolation as outcome measures for capturing symptom presence and severity might be problematic given that caregiver ratings are vulnerable to influence from external factors. As such, this study aimed to investigate the association between the BRIEF-2 and performance-based measures of EF in youth during the acute recovery period post-PICU admission for TBI. A secondary aim was to explore associations among potential confounding factors, including family-level distress, injury severity, and the impact of pre-existing neurodevelopmental conditions. Participants included 65 youths, 8–19 years of age, admitted to the PICU for TBI, who survived hospital discharge and were referred for follow-up care. Non-significant correlations were found between BRIEF-2 outcomes and performance-based measures of EF. Measures of injury severity were strongly correlated with scores from performance-based EF measures, but not BRIEF-2. Parent/caregiver-reported measures of their own health-related quality of life were related to caregiver responses on the BRIEF-2. Results demonstrate the differences captured by performance-based versus caregiver-report measures of EF, and also highlight the importance of considering other morbidities related to PICU admission. Caregiver ratings tend to be influenced by factors including stress level, skewed recollections of pre-morbid functioning, and response bias.30–33A multi-modal approach is recommended to assess functioning and track TBI recovery post-PICU admission; however, neuropsychological evaluations indexing EF are seldom completed during the acute recovery phase.34–40 These findings indicate that parent-reported and performance-based EF measures may capture a different set of constructs , particularly when EF challenges are in the more normative or mildly impaired range.34 More research directly evaluating the connection between family distress and parent-reported EF post-TBI is needed to ensure that EF outcome measures (BRIEF/BRIEF-2) are utilized effectively in TBI populations given both the prominence and longevity of executive dysfunction typically present after the acute recovery phase >10 years post-injury.41Further, the literature highlights notable discrepancies between informant-reported and performance-based measures of EF. For instance, the BRIEF/BRIEF-2 has been shown to poorly correlate with performance-based measures of EF across numerous pediatric samples unless TBIs were in the moderate to severe range.42 As such, the research is lacking temporal specificity when it comes to multi-component performance-based assessment of EF, while also accounting for pre-morbid functioning, injury severity, and parent stress. Additionally, pre-existing neurodevelopmental diagnoses are often not reported or addressed, although they are a known risk factor for executive dysfunction beyond injury severity.43–46Further, earlier studies have been agnostic to time post-injury when studying EF in pediatric TBI. This oversight is, in part, attributable to the rarity of neuropsychological evaluations and rehabilitation programs services available.The primary aim of this study was to determine the concordance between parent-reported measures and performance-based measures of EF after neurocritical care during the acute recovery period. A secondary aim was to explore possible confounding factors on EF outcomes, including family-level distress, injury severity, and pre-existing neurodevelopmental conditions. We hypothesized that caregiver-reported measures of EF would not be significantly associated with performance-based measures. Conversely, we hypothesized that parent-reported measures would be associated with caregiver-reported measures of family impact and distress. Additionally, we hypothesized that performance-based measures of EF would relate to injury severity. Finally, we hypothesized that there would be group differences across those with and without pre-existing neurodevelopmental diagnoses.M ] = 51.72 days, [SD] = 16.27; range = 26–85) after date of injury  at Doernbecher Children's Hospital ∼1–3 months  recorded at the time of injury  and ISS.41 The Wide-Range Achievement Test (WRAT), 4th Edition (WRAT-4) was given until April 2021, then replaced with the 5th Edition (WRAT-5) to assess Word Reading and provide a proxy, age-adjusted estimate of pre-morbid cognitive functioning.58–61 Youth completed either the Children's Memory Scale (CMS) Numbers subtest or the Wechsler Adult Intelligence Scale, Fourth Edition (WAIS-IV) Digit Span subtests to capture attention and working memory performance.63 The Child and Adolescent Memory Profile (ChAMP) Lists subtest was given to gauge verbal learning and general memory ability.64The PCCNRP clinic was utilized to determine the presence of cognitive impairment through implementation of a fixed neuropsychological screening battery consisting of multiple performance-based measures .41 The W65 The Verbal Fluency task consisted of a patient's ability to use language and abide by inhibition rules to generate a word based on a single letter or semantic category. For Trail Making, examinees were tasked with connecting and sequencing numbers in order  capturing processing speed, whereas the numbers and letters switching task assessed cognitive flexibility .65 The Wechsler Intelligence Scale for Children, Fifth Edition (WISC-V) or the WAIS-IV Coding and Symbol Search subtests were administered to assess processing speed.66All patients completed parts of the Delis-Kaplan Executive Function System (D-KEFS) producing scaled scores for Verbal Fluency and Trail Making tasks.21 The BRIEF-2 also provides three additional index scores to evaluate specific areas related to EF: Emotion Regulation Index (ERI), Cognitive Regulation Index (CRI), and Behavior Regulation Index (BRI). The Pediatric Quality of Life Inventory (PedsQL) Parent Related Health Quality of Life (HRQL) was used to collect information regarding caregiver functioning after hospital discharge.68Information regarding daily executive skills was collected by the BRIEF-2 as the parent-report measure.69 First, to compare differences between patients with and without pre-injury neurodevelopmental conditions, independent samples t-tests were used.All data analyses were completed using SPSS software .70 The maximum number of convergences was set to 25. All eight neurocognitive measures were retained. The WRAT Word Reading subtest was given to provide a proxy, age-adjusted estimate for pre-morbid cognitive functioning.Given the high correlations across performance-based EF measures , princip72 Using this method, more negative residuals represent worse cognitive functioning at follow-up than expected based on estimated pre-morbid functioning, whereas more positive residuals represent better functioning.Higher NCI scores are reflective of stronger neurocognitive ability. Combining all participant data yielded a single component solution accounting for 51.27% of the total explained variance . The NCIIn order to explore the relationship between caregiver-reported and performance-based measures of EF, bivariate correlations were then conducted assessing the relationships between the residual of Word Reading and NCI and the BRIEF-2 Global Executive Composite (GEC) score  with ISS scores being 1–38  and length of stay 1–33 days .Among the 65 patients who completed the follow-up visit, we evaluated 38 males and 27 females 8–19 years of age assessed ∼1–3 months after critical care hospitalization for TBI . Most paThe following results referring to performance-based neurocognitive outcomes account for the NCI score and estimated pre-morbid functioning with residualized change scores, as described above. Results are similar when original NCI scores are used see .r(64) = −0.02, p = 0.10) or injury severity. However, a strong negative correlation was observed between BRIEF-2 GEC and HRQL total score.Bivariate correlations showed non-significant relationships between NCI and BRIEF-2 GEC and index scores . SimilarOn the other hand, NCI was strongly, negatively correlated with ISS , such thn = 14), exploratory correlations were not conducted.Significant group differences were observed for BRIEF-2 GEC, NCI, Word Reading Score, and the residual of NCI and Word Reading between patients with and without a pre-injury neurodevelopmental condition before admittance to the PICU . Those w33TBI-related EF impairment, coupled with the effects of family distress post-PICU admission, is an important area of consideration when working with youth in the acute phase of TBI recovery. The present study contributes to the pediatric critical care and TBI literature by providing novel information regarding the relationship between caregiver-reported outcomes, performance-based measures, and hospital-based measures of injury severity in a cohort of pediatric TBI survivors. In support of the study hypotheses, parent-reported outcomes on the BRIEF-2 were not associated with performance-based neuropsychological measures. Results indicate that performance-based assessment and the BRIEF-2 capture different constructs associated with EF, which is consistent with previous research suggesting that the two methods capture different components of EF.21 Some researchers have suggested that the behaviors rated by parents on this measure may be more related to emotional and behavioral skills , rather than standardized cognition-based neuropsychological assessments administered in a quiet, calm, and distraction-free setting .73–76 As such, caregiver ratings likely reflect hot EF challenges, such as emotional and behavioral dysregulation, which are observed in daily life. By this metric, greater child-reported EF disturbance may reflect higher family burden.78 In the current study, we found that caregiver-rated EF was strongly correlated with parent-reported distress.The authors of the BRIEF-2 sought to create a measure that describes the day-to-day components and presentations of EF through parent proxy report evaluating a child's ability to modulate behavior, emotions, and metacognitive skills.80 For instance, clinically elevated levels of family distress have been shown to be a barrier to adherence to clinical recommendations.82 In addition, parent distress may interact with recovery by reducing parental resources to alleviate and manage children's emotional, cognitive, and physical symptoms.80–85It is also possible that elevated family distress hinders child recovery, though this cannot be determined by this study given that parent ratings of their own distress and their child's EF were given at the same time. Still, this study's findings are consistent with previous research identifying family distress to impact recovery trajectories.81 In these cases, had performance-based measures of EF not been administered, these children's needs would have gone unidentified and untreated. Therefore, a multi-system approach is warranted to effectively identify the culmination of sequalae and family stress observed in this population.84–88It is also important to consider that some families may under-report symptoms on rating forms both about their own level of distress and about functioning in their children. Research demonstrates that ratings of a child's behavior are likely to be biased because of respondents' perceptions, which are often influenced by contextual factors such as demographic background, societal expectations, and skewed recollections of pre-morbid functioning.89–92 Past work shows that EF is vulnerable post-TBI, with these impairments being present early in the acute recovery phase and persisting long term in youth who presented with complicated mild, moderate, or severe TBI and also experienced hospitalization, critical care, and comorbid orthopedic injury.87 EF deficits can hinder academic achievement and adaptive functioning in daily life.92 The current work emphasizes the importance of evaluating for impairment in EF post-TBI, especially in those with greater overall injury severity. Ideally, these children will benefit from neuropsychological evaluations that include both performance-based measures and informant-reported measures to quantify EF. This approach identifies both the impact of TBI on cognitive skills and how these impairments affect daily functioning.As expected, there was a significant relationship between expert-rated injury severity and performance-based measures of EF. This relationship remained significant even when accounting for pre-morbid individual differences in cognition indexed by word reading scores, a proxy measure known to remain intact after neurological insult.92–95 Secondary exploratory analyses in the present study revealed differences in EF based on pre-morbid developmental diagnoses, suggesting that pre-morbid conditions may exacerbate or result in diminished EF capacities on both parent report and performance-based measures after TBI. Given our small sample size and inability to directly measure pre-morbid functioning, future research is warranted to further elucidate the connection between pre-injury abilities and EF recovery.In addition to performance-based and informant-reported measures of EF when considering TBI outcomes, it is also necessary to consider pre-injury abilities. Though the design of our study did not allow for direct measurement of baseline EF pre-injury, we examined the impact of previously identified neurodevelopmental conditions. The past TBI literature has identified lower pre-injury abilities to be a significant risk factor for recovery.96 Overall, the results underscore the need for researchers and professionals to carefully consider the type of information that can be gleaned from various measurement tools, especially when interpreting outcomes to inform treatment and guide intervention.Together, the results demonstrate the differences captured by performance-based and parent reported measures of EF, and also highlight the importance of considering other morbidities, particularly family distress related to PICU admission. We assert that both types of EF measures have merit and add useful information to research and clinical care; however, consistent with previous research, performance-based and informant-report measures appear to capture different constructs related to EF, and utilization of either of these measures in isolation may inaccurately capture EF outcomes, particularly if not accounting for the impact of family distress in an acute pediatric TBI population.The present study has several limitations. First, this study is a single-institution, retrospective cohort study with a relatively small sample size largely attributable to the unique population of interest. Additionally, patient TBI severity ranged from mild without intracranial injury to severe with multiple intracranial injuries. Yet, heterogeneity is the rule in community samples drawn from patients admitted and treated in the PICU. Further, there are known geographical and institutional differences regarding acute treatment and critical care approaches, which may limit the generalizability of our findings. Relatedly, broad inferences beyond the scope of this pediatric TBI population are limited. This study was a cross-sectional analysis, thus limiting determination of causation. Further, longitudinal studies are needed too given that all outcome measures were collected 1–3 months after hospital discharge. Future research with greater statistical power is needed to explore the contribution of injury characteristics on outcomes.81 Overall, these findings evince that the BRIEF-2 and neuropsychological assessment measure different constructs, with the BRIEF-2 capturing subtle behavioral problems, parental psychological distress, and emotional constructs associated with pediatric TBI recovery. Conversely, performance measures of EF may capture specific functional neurocognitive deficits directly related to injury severity. Moreover, our study demonstrates that BRIEF-2 scores are related to measures of caregiver distress, and therefore using this measure as a primary clinical outcome for cognition requires the caveat that it also reflects other morbidities related to PICU admission.The present study revealed a non-significant association between caregiver-reported measures and performance-based measures of EF in the acute-recovery period after pediatric TBI. The findings demonstrate the importance of multi-method assessment to detect potential acquired deficits to inform rehabilitation. Further, these findings are consistent with previous literature attesting that performance-based measures may underestimate behavioral and emotional impairments associated with EF.


Epidermal growth factor receptor (EGFR)-mutant nonsmall cell lung cancer (NSCLC) patients are less likely to be programmed death-ligand 1 (PD-L1)-positive compared with wild-type EGFR mutant tumors. Given the rarity of actionable driver genes in squamous cell lung cancer (SQCC), the frequency of SQCC patients simultaneously carrying EGFR driver gene mutation and having PD-L1 over-expression is extremely low. Studies on the effectiveness and safety of EGFR-TKIs or immune-checkpoint inhibitors (ICIs) in this subset of patients are lacking.The patient suffered from coughing and chest pain for 1 month. A chest CT revealed a mass with a cavity in the right lung, enlarged mediastinal lymph nodes, diffuse pleural thickening in the right pleura, and pleural effusion of the right chest.A pleural biopsy was performed using a video-assisted thoracoscope. The pathological examination revealed a poorly differentiated squamous cell carcinoma of lung. Further genetic testing identified exon 19 deletion mutation in EGFR with abundance of 0.27%. Meanwhile, immunohistochemical PD-L1 analysis showed a TPS of 90%.The patient was initially resistant to EGFR-TKIs but exhibited a rapid and marked response to pembrolizumab.After 5 cycles of pembrolizumab monotherapy, the patient developed Grade 3 immune-related dermatitis, and ICI therapy was suspended.ICI monotherapy could be an effective therapy in SQCC patients with low-abundance of EGFR mutations and PD-L1 over-expression. However, close attention should be paid to immune-related adverse events. In addition, there is overwhelming evidence demonstrating that a remarkable and durable response to the immune-checkpoint inhibitors (ICIs) targeting the programmed cell death-1 (PD-1) receptor and programmed death-ligand 1 (PD-L1) pathways has been achieved in NSCLC patients, especially those with high PD-L1 expression.–5 However, the majority of clinical trials examining the efficacy of ICIs in NSCLC have excluded patients harboring oncogenic driver mutations.–4 Moreover, data from some reports have suggested that ICI monotherapy is much less effective in NSCLC patients with EGFR driver alterations than those with no driver gene mutations, whether as first-line or subsequent-line therapy.–9 Thus, due to the encouraging effects of EGFR-TKIs but the poor efficacy of single-agent ICI in NSCLC patients harboring the EGFR sensitive mutation, ICI monotherapy is generally not recommended in this subset of patients. However, as only a small percentage of squamous cell lung cancer (SQCC) patients carry oncogenic drivers, almost all the data on this subject are from studies on lung adenocarcinoma, and there is insufficient evidence on the efficacy of ICIs in SQCC with driver mutations.Epidermal growth factor receptor tyrosine-kinase inhibitors (EGFR-TKIs) have been used as the standard first-line treatment for advanced nonsmall-cell lung cancer (NSCLC) with an EGFR sensitive mutation (L858R or exon 19 deletion).We report a patient with advanced SQCC with an EGFR exon 19 deletion mutation and a PD-L1 Tumor Proportion Score (TPS) of 90%. He was initially resistant to EGFR-TKI treatment but showed a marked response to pembrolizumab.Ethics approval for this study was obtained from the Shaoxing People’s Hospital Ethics Committee, and the patient provided informed consent for the publication of this case report and the accompanying images.A 54-year-old man with a 30 pack-year smoking history, who had been suffering from coughing and chest pain for 1 month, was admitted to our hospital in November 2019. A chest CT revealed a 2 cm mass with a cavity in the middle lobe of the right lung, enlarged mediastinal lymph nodes, diffuse pleural thickening in the right pleura, and pleural effusion of the right chest. A subsequent positron emission tomography-computed tomography (PET-CT) scan confirmed an abnormal increase of glucose metabolism in the right lung mass and thick pleura . Deepa et al also reported that PD-L1 ≥ 50% seldom overlaps with the presence of driver oncogene EGFR. Given the rarity of actionable driver genes in SQCC, the frequency of SQCC patients simultaneously carrying the EGFR driver gene mutation and having PD-L1 over-expression is extremely low. To our knowledge, only 1 case report related to this subject has been published so far.SQCC is a distinct histologic subtype of NSCLC and the EGFR mutation rate in this subset of patients is <4%. Nonetheless, previous studies have revealed that the abundance of the EGFR mutation may be related to the efficacy of EGFR-TKIs. NSCLC patients with low-abundance EGFR mutation may experience reduced ORR and shorter PFS time compared to groups with high-abundance EGFR mutation.,16 Data from these studies indicate that treatment with EGFR-TKI for the patient in our case would probably be relatively ineffective, and the patient did indeed experience primary resistance to EGFR-TKI therapy.Multiple clinical studies have already demonstrated that SQCC patients are generally less sensitive to EGFR-TKI therapy than those with adenocarcinoma. It has been reported that the progression-free survival (PFS) for EGFR-TKI treatment in EGFR mutant SQCC is 3 months, and results from a pooled analysis indicated an objective response rate (ORR) and disease control rate of 30% and 70%, respectively.,18 In this case, to exclude co-occurring genetic mutations, we conducted a next-generation sequencing-based gene panel test, but the result was negative. Alternatively, PD-L1 expression could be a progression factor for EGFR-TKI primary resistance. Several studies have retrospectively examined the relationship between PD-L1 expression and the efficacy of EGFR-TKIs, and most of them have revealed that PD-L1 expression has a negative impact on the clinical outcome of EGFR mutant patients.–22It has been reported that approximately 10–30% of EGFR-mutant advanced NSCLC patients do not exhibit an objective response to EGFR-TKIs. Many factors contribute to the primary resistance of NSCLC with the sensitive EGFR mutation to EGFR-TKIs. Firstly, co-occurring genetic alterations in T790M/TP53/RB1/PTEN/PIK3CA may play a role in primary resistance mechanisms to EGFR-TKIs. In addition, a pooled analysis of CheckMate 057, KEYNOTE-010, POPULAR, and OAK clinical trials revealed that ICI monotherapy provided no benefits over docetaxel as second-line treatment for EGFR-mutant NSCLC patients with failed EGFR-TKI therapy. Moreover, data from the ImmunoTarget trial showed that the ORR and PFS of EGFR-mutant NSCLC patients treated with ICI monotherapy at any line of treatment were 12% and 2.1 m, respectively.Indeed, for NSCLC patients with the EGFR mutation, not only did high PD-L1 expression negatively impact the efficacy of EGFR-TKIs, but PD-1 inhibitor therapy was also reported to be less effective in this subgroup of patients compared to those with wild-type EGFR as first-line therapy. A clinical Phase II trial of pembrolizumab in TKI-naïve EGFR-mutant NSCLC patients with positive PD-L1 showed no response.,23 Except high PD-L1 levels, Limited data indicated that smoking history, L858R mutation subtype, or uncommon EGFR mutation type,27 are also associated with better outcomes following treatment with immunotherapy. A recent study retrospectively analyzed advanced NSCLC patients with resistance to EGFR-TKIs who received ICI treatment. The results indicated that patients with a short PFS to EGFR-TKIs showed a better response to subsequent anti-PD-1/PD-L1-based immunotherapy in EGFR-mutation NSCLC. Considering these viewpoints, our patient could benefit from ICI due to his high PD-L1 expression, history of smoking, and short PFS to EGFR-TKIs.Although the efficacy of ICI in NSCLC patients harboring oncogenic alterations is poor, the PD-L1 status is still believed to be the most effective predictive biomarker for identifying patients most likely to benefit from ICI.,30 Conversely, another study has found that erlotinib plus atezolizumab or erlotinib plus nivolumab did not demonstrate excess toxicity. With regard to PD-L1 inhibitor and EGFR-TKI sequential therapy, it has been reported that treatment with sequential PD-L1 blockade followed by osimertinib resulted in a severe immune-related adverse event. Despite this, no severe immune-related adverse events were identified among patients treated with osimertinib followed by PD-L1 or PD-L1 followed by afatinib or erlotinib. However, our patient developed Grade 3 immune-associated dermatitis when receiving pembrolizumab after failed EGFR-TKI therapy. It was difficult to distinguish whether the dermatitis was only related to the immunotherapy or was also associated with the increased risk of adverse events due to the PD-1 inhibitor treatment after EGFR-TKI therapy.In terms of immune-related toxicity, several studies state that PD-L1 inhibitor and EGFR-TKI combination therapy may be associated with an increased risk of toxicity.This report indicates that high PD-L1 expression may correlate with primary resistance to EGFR-TKIs in treating naïve advanced NSCLC with EGFR mutation. For SQCC patients, if they have lower EGFR mutation fractions, but higher PD-L1 expression, ICI monotherapy may be a therapeutic option. During the ICIs treatment, close attention should be paid to immune-related adverse events. However, further investigations into the optimal treatment for SQCC with the EGFR mutation and PD-L1 over-expression are required to confirm this hypothesis.The authors thank the patient and her family for signing informed consent for publication.Qiu-Xia Liu conceptualized the study and drafted the manuscript; Jian-Guo Wei and Yi-Yi Chen collected and analyzed the patient data; Jian-Fang Wang supervised the manuscript preparation. All authors have read and approved the final version to be submitted.


Siniperca chuatsi) is a cultured freshwater fish species that is popular in China because of its high market value. With the development of a high-density cultural mode in mandarin fish, viral diseases have increased and seriously restricted the development of aquaculture, such as ranavirus and rhabdovirus. Herein, the role of mandarin fish DDX41 (scDDX41) and its DEAD and HELIC domains in the antiviral innate immune response were investigated. The level of scDDX41 expression was up-regulated following treatment with poly(dA:dT) or Mandarin fish ranavirus (MRV), suggesting that scDDX41 might be involved in fish innate immunity. The overexpression of scDDX41 significantly increased the expression levels of IFN-I, ISGs, and pro-inflammatory cytokine genes. Co-immunoprecipitation and pull-down assays showed that the DEAD domain of scDDX41 recognized the IFN stimulatory DNA and interacted with STING to activate IFN-I signaling pathway. Interestingly, the HELIC domain of scDDX41 could directly interact with the N-terminal of STING to induce the expression levels of IFN-I and ISGs genes. Furthermore, the scDDX41 could enhance the scSTING-induced IFN-I immune response and significantly inhibit MRV replication. Our work would be beneficial to understand the roles of teleost fish DDX41 in the antiviral innate immune response.DDX41 is an intracellular DNA sensor that evokes type I interferon (IFN-I) production via the adaptor stimulator of interferon gene (STING), triggering innate immune responses against viral infection. However, the regulatory mechanism of the DDX41-STING pathway in teleost fish remains unclear. The mandarin fish ( Asp-Glu-Ala-Asp (DEAD) box 41 (DDX41) is a crucial cytosolic DNA sensor and plays a crucial role in antiviral innate immune response via the adaptor stimulator of interferon gene (STING) to induce type I interferon (IFN-I) response. DDX41 consists of two conserved RecA-like domains (DEAD and HELIC domains), which contain various functional motifs involved in nucleic acid recognition, ATP binding, ATP hydrolysis, and RNA unwinding ,3,4. In Siniperca chuatsi) is a cultured freshwater species that is economically important due to its high market value in China. However, the spread and outbreaks of viral diseases, e.g., Mandarin fish ranavirus (MRV), Infectious spleen and kidney necrosis virus, and Siniperca chuatsi rhabdovirus, have caused remarkable loss to the mandarin fish culture industry . ,6. scDDXscDDX41 could enhance scSTING-induced IFN-I immune response, the expression levels of downstream genes in the cells co-transfected with scDDX41 and scSTING were examined. As shown in scDDX41 significantly enhanced the activities of IFN-β-luc and the expression levels of scIFN-h, scMx, scISG15, and scViperin genes in the cells co-transfected with scSTING. These results suggested that scDDX41 enhanced the scSTING-induced IFN-I immune response.To further investigate whether scDDX1 and interferon stimulatory DNA (ISD) was evaluated using EMSA. As shown in scDDX41 could bind to the labeled probe of ISD to form a retarded band, whereas the retarded bands of the DNA–protein complex were eliminated when 10× and 100× unlabeled probes were added to competitively bind to the scDDX41 protein. By contrast, no retarded band of the DNA–protein complex was observed in the analysis of the control group performed using nuclear proteins extracted from cells transfected with pCMV-myc-GFP or pCMV-myc empty plasmid. These findings confirmed that scDDX41 could bind to ISD. To further determine whether the DEAD domains of scDDX41 could directly bind to dsDNA, the dsDNA-binding abilities of myc-tagged scDDX41 and myc-tagged scDDX41ΔDEAD were assessed by pull-down assays using 5ʹ-biotinylated ISD (bio-ISD). The myc-tagged human DDX41 (hsDDX41) was used as positive control. As shown in hsDDX41 and scDDX41 but not scDDX41ΔDEAD due to the absence of the DEAD domain. Hence, scDDX41 could bind to DNA through the DEAD domain. These results indicated that scDDX41 recognized dsDNA through the DEAD domain.To verify whether teleost fish DDX41 could recognize dsDNAs, the interaction between scDDX41, the transcription level of MRV viral major capsid protein (mcp), ICP-18 and DNA polymerase (DNA pol) genes were evaluated in scDDX41-overexpressing cells. As shown in mcp, ICP-18, and DNA pol genes were significantly inhibited in scDDX41-overexpressing cells (A) compared with that in the cells transfected with the control vector at 24 and 48 h post-infection (p < 0.01). The relative expression levels of scIFN-h (E), scMx (F), scISG15 (G), scViperin (H), and scTNF-α (I) genes in the cells transfected with scDDX41-myc were significantly higher than those in the cells transfected with pCMV-myc at 24 and 48 h (p < 0.01) post-MRV infection. Moreover, the level of virus load and virus titers (TCID50) was also suppressed by scDDX41 at 24 and 48 h post-infection, compared to those in the control group , zebrafish (Danio rerio), orange spotted grouper (Epinephelus coioides), Nile tilapia (Oreochromis niloticus), and grass carp (Ctenopharyngodon idella) for the past few years [P. olivaceus DDX41 directly sensed cyclic di-GMP and triggered an IFN immune response to enhance the antiviral effect [D. rerio DDX41 activated NF-κB and IFN-I signaling pathways through the interaction of the DEAD domain with STING [E. coioides DDX41 enhanced MAVS- and TBK1-induced IFN and inflammatory immune responses [C. idella DDX41 interacted with STING to induce the phosphorylation and nuclear translocation of IRF7 and then initiated IFN-I and ISG15 expressions [scDDX41 is a key region for binding to DNA (ISD) and interaction with STING to trigger IFN and inflammatory immune responses. Hence, scDDX41 is an important DNA pattern-recognition receptor for STING-dependent type I IFN responses in teleost fish.DDX41, a member of the DExD/H-box helicase superfamily, is involved in intracellular DNA recognition and triggers innate immune responses against viral infection ,6,11. Cuew years ,13,14. Pl effect . D. reriesponses . C. idelD. rerio DDX41 might obstruct the DDX41-STING signaling pathways [scSTING contains five TM domains [scDDX41 could directly interact with not only scSTING-NTD but also scSTING-CTD, and the HELIC domain of scDDX41 could also directly interact with scSTING-NTD. Hence, the DEAD and HELIC domains of scDDX41 contributed to the interaction of scDDX41 with scSTING to induce STING-dependent IFN-I and inflammatory immune responses. As such, the activation of the DDX41-STING pathway might differ between mammals and teleost fish. The detailed mechanisms of the DDX41 HELIC domains in synergistically regulating the STING-mediated innate immune response in fish should be further studied.DDX41 can directly bind to the STING-N-segment transmembrane region through the DEAD domain and subsequently promotes the activation of TBK1 and IRF3, leading to IFN-I production; meanwhile, the HELIC domain of DDX41 regulates the binding ability of the DEAD domain to dsDNA but does not interact with STING ,22. Telepathways . Althougpathways ,23. The pathways , and the domains . The TM  domains . Those fscDDX41 activated the IFN-I responses by interacting with scSTING though the DEAD and HELIC domain, and the scDDX41-mediated STING pathway possessed antiviral activity against MRV infection. This work will help us to understand the roles of teleost fish DDX41 and its HELIC domain in innate immune responses.In summary, our study demonstrated that the


Colorectal cancer (CRC) is a heterogeneous disease with one of the highest rates of incidence and mortality among cancers worldwide. Understanding the CRC tumor microenvironment (TME) is essential to improve diagnosis and treatment. Within the CRC TME, tumor-infiltrating lymphocytes (TILs) consist of a heterogeneous mixture of adaptive immune cells composed of mainly anti-tumor effector T cells (CD4+ and CD8+ subpopulations), and suppressive regulatory CD4+ T (Treg) cells. The balance between these two populations is critical in anti-tumor immunity. In general, while tumor antigen-specific T cell responses are observed, tumor clearance frequently does not occur. Treg cells are considered to play an important role in tumor immune escape by hampering effective anti-tumor immune responses. Therefore, CRC-tumors with increased numbers of Treg cells have been associated with promoting tumor development, immunotherapy failure, and a poorer prognosis. Enrichment of Treg cells in CRC can have multiple causes including their differentiation, recruitment, and preferential transcriptional and metabolic adaptation to the TME. Targeting tumor-associated Treg cell may be an effective addition to current immunotherapy approaches. Strategies for depleting Treg cells, such as low-dose cyclophosphamide treatment, or targeting one or more checkpoint receptors such as CTLA-4 with PD-1 with monoclonal antibodies, have been explored. These have resulted in activation of anti-tumor immune responses in CRC-patients. Overall, it seems likely that CRC-associated Treg cells play an important role in determining the success of such therapeutic approaches. Here, we review our understanding of the role of Treg cells in CRC, the possible mechanisms that support their homeostasis in the tumor microenvironment, and current approaches for manipulating Treg cells function in cancer. Colorectal cancer (CRC) is the third most diagnosed cancer worldwide, and the second main cause of cancer-related death . The maiThe TME includes malignant tumor cells together with immune cells, stromal cells, endothelial cells, extracellular matrix (ECM), cytokines, chemokines, and other soluble mediators –17. CancFOXP3+ regulatory T cells, hereafter termed Treg cells, are essential in the maintenance of immunological homeostasis and self-tolerance –43. TheyIn the thymus, tTreg cell differentiation is driven by strong T cell receptor (TCR)-mediated recognition of self-antigens , 63. In FOXP3-expressing CD4+ T cells can be further subdivided into two main subsets: central Treg cells (cTreg cells) and effector Treg cells (eTreg cells). However, in humans, activated Teff cells can transiently express FOXP3 and secrete pro-inflammatory cytokines , however they are considered to be non-suppressive , 72. cTrFoxp3 expression and other Treg cell signature genes including Cd25, Nrp1, and Il1rl1  in Treg cells from CRC tumors and adjacent healthy colon samples demonstrated higher TSDR-DMRs demethylation in TI-Treg cells  and Treg cells  , Il17a (IL-17) and Ifng (IFNγ), repressing their expression. However, in CRC RORγt+ TI-Treg cells, sustained Wnt and/or TCR signaling leads to nuclear translocation of β-catenin where it binds to TCF-1 facilitating the transcription of pro-inflammatory genes. Increased numbers of pathogenic β-catenin(hi) RORγt+ TI-Treg cells correlates with the progression from IBD to CRC in humans and in murine colitis-associated dysplasia models  , 153. ILigenesis , 153–156a models , 157. CRa models . Conversa models , 159.+ Treg cells retain a suppressive phenotype through hypomethylation of Treg-specific signature genes including Foxp3, Ctla-4, Gitr, Eos, and Helios , and the NAD : NADH ratio. These adaptations permit Treg cells to differentiate and work effectively under conditions of low-glucose and high lactate as found in the TME , promoting the functional maturation of TI-Treg cells. SREBP-deficient Treg cells show decreased expression of PD-1 and have been associated with an effective anti-tumor immune response and reduced tumor growth   that drives the expression of anti-inflammatory cytokines, including IL-10 and TGF-β (Other metabolites abundant in the TME include retinoic acid (RA) and adenosine. RA can act as a cofactor to induce Foxp3 expression and promote the conversion of CD4+ T cells into Treg cells while inhibiting the generation of Th17 cells. In the intestine, CD103+ DCs can generate RA from vitamin A and drive Treg cells differentiation in the presence of TGF-β , 208. A nd TGF-β , 211.Despite the metabolic challenges present in the TME such as low glucose and amino acid availability, high lactate and lipid concentration, acidity, and hypoxia, TI-Treg cells show a remarkable ability to engage unique metabolic reprogramming, compared to other Teff cells, to support their bioenergetic and functional needs. Understanding the TI-Treg cell metabolic reprogramming potentially provides novel therapeutic approaches for targeting in the control CRC pathogenesis.Modulating TI-Treg cell function offers an important strategy to improve therapeutic responses for a wide variety of tumors. A variety of approaches are currently under development including Treg cell depletion, suppressing their activity, impeding their recruitment, and preventing their differentiation within the TME. The challenge presented is to specifically target TI-Treg cells without affecting other Treg populations, thereby diminishing the risk of developing unwanted autoimmune responses. In the past decade, the efficacy of checkpoint inhibitors to block immune checkpoint receptors or their ligands has greatly benefited the survival of patients with solid tumors –216. As PD-1 is highly expressed in TI-Treg cells and plays a role in their homeostasis and function. PD-1 signaling also promotes FAO to support their bioenergetics needs in the TME . PD-1-exTreg cells express high levels of cytotoxic T lymphocyte antigen 4 (CTLA-4) in a FOXP3-dependent manner. CTLA-4 binds and inhibits B7 molecules (CD80 and CD86) on the surface of APCs such as DCs with higher affinity than their CD28 co-stimulatory signal. This results in the induction of immunological tolerance . Murine Novel immune checkpoint targets such as TIGIT, LAG-3 and TIM-3 are currently under pre-clinical investigation and are being evaluated for their safety profiles in phase I trials , 245. InTIM-3 is another immune checkpoint molecule expressed on innate immune cells such as DCs and NK cells . pTreg cf)  . CD39+ Tf)  and inhi in vivo . Adoptiv in vivo .CCR8 expression can discriminate between Treg cells infiltrating CRC and other solid tumors, from those found in secondary lymphoid organs . In soliOther broader strategies effective in Treg cell depletion include drugs such as cyclophosphamide (CY). The administration of low-dose CY, a chemotherapeutic agent used to treat cancer, seems to predominantly affect Treg cells over other effector cells (Teff cells or NKs), as has been demonstrated in a variety of human cancers and animal models. Low-dose CY can lead to a reduction in Treg cell numbers, upregulation of pro-inflammatory cytokines, and boosts the innate antitumor immune response –270. In Refinement of combinational therapies targeting Treg cells while promoting anti-tumor immune responses is clearly a promising approach to treat CRC more effectively. However, it remains critical to consider CRC subtypes, and to specifically target TI-Treg cells, rather than systemically depleting all Treg or other Teff cells. These are issues that still require proper evaluation when considering new therapeutic strategies.in vivo or developing valid models for evaluating immune cell-TME interactions in vitro. Immunotherapy approaches can stimulate anti-tumor immune responses of effector cells or inhibit immunosuppressive mechanisms. Refinement of combinational therapies targeting Treg cells together with promoting Teff cell anti-tumor immune responses provides promising strategies to treat CRC more effectively. However, Treg cell-directed immunotherapy for CRC patients has several limitations. To start with, the different CRC molecular subtypes likely have a critical role in determining the success of certain therapeutic approaches. Due to CRC heterogeneity, treatment stratification to the tumor subtype will likely be required. Moreover, a better understanding of the (sub)phenotypes and functional diversity of CRC TI-Treg cells is still needed. This is essential to avoid compromising immune hemostasis and thereby developing unwanted side effects. Utilizing bivalent antibody approaches may help relieve the problem of specifically targeting only TI-Treg cells, as long as appropriate tumor markers are also available .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.


Nursing home residents have been disproportionately affected by COVID-19; older age, comorbidities, and the congregate nature of nursing homes place residents at higher risk for infection and severe COVID-19–associated outcomes, including death  and vaccination data to NHSN in April 2020 and December 2020, respectively, and federal mandates issued by the Centers for Medicare & Medicaid Services (CMS) require CMS-certified nursing homes to report these data weekly.NHSN analyzed weekly COVID-19 case and vaccination status data during October 10, 2022–January 8, 2023, for CMS-certified nursing homes to assess the data collected based on NHSN’s 2022 fourth quarter (October–December) definition of up-to-date COVID-19 vaccination status.Facilities with missing case or vaccination data were excluded from the analysis. NHSN calculated IRRs by up-to-date vaccination status each week. NHSN also analyzed a subset of data from facilities voluntarily reporting dates, types, and number of primary series doses and booster doses received by each resident  to calculate the proportion of up-to-date residents who had received a bivalent booster dose.Among 16,352 nursing homes, 15,049 (92%) in the 50 U.S. states and the District of Columbia reported case and vaccination data for ≥1 week during the 13-week study period.Each week, COVID-19 incidence rates among nursing home residents who were not up to date with COVID-19 vaccination were higher than those among residents who were up to date . IncidenWeekly incidence rates of COVID-19 among nursing home residents who were not up to date with COVID-19 vaccines were 30%–50% higher than were those among residents who were up to date during October 10, 2022–January 8, 2023. Among the subset of nursing homes reporting additional details on vaccine doses for each resident, almost all residents with up-to-date COVID-19 vaccination status had received a bivalent booster dose, suggesting that up-to-date vaccination status can be used to represent bivalent booster dose coverage among nursing home residents. The findings in this report are consistent with other recent studies supporting effectiveness of bivalent booster doses, including a study among adults aged ≥18 years demonstrating that bivalent booster doses maximized protection against symptomatic SARS-CoV-2 infection compared with protection from monovalent vaccination alone  with the up-to-date group.In this study of U.S. nursing home residents during October 2022–January 2023, differences in crude COVID-19 incidence rates among persons who were up to date with COVID-19 vaccinations and those who were not suggest that staying up to date with CDC-recommended vaccinations, which now includes receiving a bivalent booster dose, maximizes protection against COVID-19. Efforts to address barriers and increase bivalent COVID-19 booster dose coverage among nursing home residents are critical to preventing illness, severe disease, and death in this population.COVID-19 vaccines are effective against SARS-CoV-2 infection in nursing home residents; however, the impact of recently recommended vaccinations, including bivalent booster doses, in this population is unknown.Nursing home residents who were not up to date with recommended COVID-19 vaccines had a 30%–50% higher risk for acquiring SARS-CoV-2 infection compared with residents who were up to date with COVID-19 vaccines.This study supports other recent findings that the bivalent booster dose offers additional protection in persons who previously received monovalent vaccines. Nursing home residents can maximize protection against COVID-19 by receiving bivalent COVID-19 booster doses to stay up to date with recommended COVID-19 vaccinations.


The Papua New Guinea (PNG) Health Department retrospectively reported six cases of Zika virus (ZIKV) from a cohort of febrile patients during outbreaks of dengue and malaria in 2016. However, the transmission of ZIKV remains unclear due to lack of testing capability. This study aimed to determine the level of immunity to ZIKV among PNG military personnel (PNGMP) in 2019.+ samples.Sera of 208 PNGMP recruited in April 2019 was tested for the presence of anti-ZIKV immunoglobulin G (IgG) and M (IgM) antibodies using Euroimmun IgG/IgM detection kits, and anti-ZIKV neutralizing antibody (Nab) against a ZIKV African strain on all anti-ZIKV-IgG/IgM+ samples had anti-ZIKV-Nab titres ≥20, as well as an anti-ZIKV-Nab titre ratio ≥4 compared with the Nab titres of four anti-dengue serotypes, so met the criteria of the World Health Organization (WHO) for confirmed ZIKV infection.Anti-ZIKV seropositivity of these sera was as follows: IgG, 67%; IgM, 9%; and Nab, 65%. Five of 19 anti-ZIKV-IgM+ samples met the WHO criteria for confirmed ZIKV infection, suggesting a recent undetected outbreak in PNGMP. These results provide better understanding of the current ZIKV epidemic status in PNGMP.The prevalence of anti-ZIKV-Nab of 65% suggests that there are high levels of ZIKV exposure among PNGMP. Five of the 19 anti-ZIKV-IgM Zika virus (ZIKV) infection is associated with congenital neurological abnormalities, such as fetal microcephaly and Guillain–Barré syndrome . ZIKV inDue to a lack of testing capability, the actual incidence of ZIKV infection in PNG remains largely unknown. Currently, laboratory diagnosis of ZIKV infection depends on the detection of anti-ZIKV-IgM antibody, which normally appears in serum 5–7 days after disease onset. The authors conducted a population-based ZIKV seroprevalence survey on sera obtained from PNG military personnel (PNGMP) in April 2019 to determine the level of immunity to ZIKV among PNGMP.These results are part of an infectious disease surveillance study conducted by the Australian Defence Force in conjunction with the PNG Defence Force in April 2019. Seventy-six PNGMP from Manus Island and 132 PNGMP from Wewak consented voluntarily to participate in this survey. All participants were asked if they had experienced febrile illness and travelled within the 4 weeks preceding blood sampling .Table 1C+ samples were tested for anti-ZIKV neutralizing antibody (Nab) against the prototype strain MR766 (Rhesus/1947/Uganda/MR766). Anti-ZIKV-IgM+ samples were also tested for anti-dengue Nabs with strains of dengue-1Hawaii, dengue-2NGC, dengue-3H-87 and denuge-4H-241. Detailed methods are described in Appendix 1 .Anti-ZIKV, anti-Japanese encephalitis virus (JEV) and anti-dengue NS1 protein specific IgG/IgM antibodies were detected using enzyme-linked immunosorbent assay (ELISA) kits. Anti-ZIKV ELISA+ subjects had anti-ZIKV-Nab to the MR766 strain   and Yap + but anti-ZIKA-Nab− indicates that the current commercially available ELISA detection kits for ZIKV may not be suitable for diagnostic or seroprevalence survey purposes in dengue- and JEV-endemic areas, such as PNG, due to possible serological cross-reactivity among flaviviruses (+ on ELISA should be confirmed by neutralizing assay for diagnostic/surveillance purposes.The higher proportion of samples showing anti-ZIKV-IgG positivity compared with anti-ZIKV-Nab positivity could be due to the endemicity of other flavivirus that are antigenically closely related to ZIKV, such as dengue virus and JEV. The finding of two samples that were anti-ZIKV-IgMiviruses  (Table 2This preliminary finding requires further support from additional investigations, as the present study had a small sample size and only PNGMP were included. The authors intend to expand their arbovirus surveillance programme in PNG to include investigation of circulating ZIKV strains, mosquito behaviours and antibody prevalence amongst the entire population of PNG in order to better understand the potential risk of ZIKV transmission in PNG.


Most bio-inspired antireflective nanostructures are extremely vulnerable and suffer from complicated lithography-based fabrication procedures. To address the issues, we report a scalable and simple non-lithography-based approach to engineer robust antireflective structures, inspired by the longtail glasswing butterfly, in a single step. The resulting two-dimensional randomly arranged 80/130/180 nm silica colloids, partially embedded in a polymeric matrix, generate a gradual refractive index transition at the air/substrate interface to suppress light reflection. Importantly, the randomly arranged subwavelength silica colloids display even better antireflection performance for large incident angles than that of two-dimensional non-close-packed silica colloidal crystals. The biomimetic coating is of considerable technological importance in numerous practical applications. Fresnel reflection occurs when light penetrates through an interface between two optical media with different refractive indices ,2. The lOver four hundred million years of natural selection, living beings have created diverse functional architectures for survival. For example, moth eyes, hawk moth wings, and cicada wings are covered with hexagonally arranged submicrometer-scale protuberances to reduce light scattering as well as to minimize reflectivity in the broadband wavelength region over wide incident angles ,13,14. TBy contrast, spontaneous crystallization of colloids renders an inexpensive and simple alternative for creating high-resolution antireflective structures. The self-assembled colloidal monolayers can serve as templates to pattern demanded architectures ,19. HoweLongtail glasswing butterfly wings are covered with irregular positioning moderate-aspect-ratio dome-shaped structures to produce angle-independent optical transparency ,29. InteSpherical silica colloids with less than 5% diameter variation are synthesized by following the well-established StÖber method . In a stSpherical StÖber silica colloids with tunable diameters are dispersed in photocurable ethoxylated trimethylolpropane triacrylate (ETPTA)  monomers with a colloid volume fraction of 25%. 2-hydroxy-2-methyl-1-phenyl-1-propanone  is added as a photoinitiator. The viscosity of the resulting suspension is determined by the colloidal volume fraction. A higher colloidal volume fraction leads to a more viscous suspension. The as-prepared suspension is deposited onto an RCA-cleaned poly  (PET)  substrate, followed by a spin-coating process (5000 rpm for 2 min) to uniformly spread the silica colloids. The polymerization of ETPTA monomers can be greatly increased by employing Darocur 1173 in a UV curing chamber .To investigate the dependences of silica colloid size and arrangement on antireflection characteristics, non-close-packed 80 nm silica colloidal crystals, non-close-packed 130 nm silica colloidal crystals, and non-close-packed 180 nm silica colloidal crystals are spin-coated on PET substrates, respectively . Insteadberances .To address the issue, the spin-coating technology is utilized to engineer two-dimensional randomly arranged subwavelength silica colloids. Spherical silica colloids with diameters of 80 nm, 130 nm, and 180 nm, synthesized by a standard StÖber method, are dispersed in photocurable ETPTA monomers with a colloid volume fraction of 25%. Bioinspired by the longtail glasswing butterfly wings, the amounts of 80 nm colloids, 130 nm colloids, and 180 nm colloids in the silica colloidal suspension are adjusted in a ratio of 1:2:1. The as-prepared silica colloidal suspension is deposited and spread on a PET substrate in a spin-coating process. The ETPTA monomers are finally UV-polymerized to bring about a monolayered silica colloid/poly(ETPTA) composite.As displayed in To further evaluate their corresponding omnidirectional antireflection characteristics, optical reflection and transmission measurements of all the above-mentioned PET substrates are acquired in the visible spectrum at varied incident angles. The measurements from five different regions of each substrate are averaged and compared in h) is expressed as The antireflective properties are further complemented by plotting calculated effective refractive index  composite using a pencil set with different hardness grades (1H–5H) ,35,36,37To conclude, a scalable approach is developed to engineer longtail glasswing butterfly wing-inspired antireflective structures in a single-step. The resulting subwavelength-scale silica colloid/polymer composite behaves with broadband antireflection characteristics and a competitive coating hardness. Importantly, in comparison with non-close-packed silica colloidal crystals, the randomly arranged silica colloids exhibit improved antireflection performance for large incident angles. It is worth mentioning that randomly arranged silica colloids can be easily fabricated by the scalable and microfabrication-compatible spin-coating technology. Furthermore, commercial silica colloids with large diameter variations are much more inexpensive. This low-cost and simple methodology offers a new opportunity in practical applications. Further structural optimization is still under examination and will be reported on in the near future.


Ubiquitination is a critical type of protein post-translational modification playing an essential role in many cellular processes. To date, more than eight types of ubiquitination exist, all of which are involved in distinct cellular processes based on their structural differences. Studies have indicated that activation of the ubiquitination pathway is tightly connected with inflammation-related diseases as well as cancer, especially in the non-proteolytic canonical pathway, highlighting the vital roles of ubiquitination in metabolic programming. Studies relating degradable ubiquitination through lys48 or lys11-linked pathways to cellular signaling have been well-characterized. However, emerging evidence shows that non-degradable ubiquitination  remains to be defined. In this review, we summarize the non-proteolytic ubiquitination involved in tumorigenesis and related signaling pathways, with the aim of providing a reference for future exploration of ubiquitination and the potential targets for cancer therapies. Ubiquitination, also known as ubiquitylation, refers to the process by which ubiquitin , with the help of a series of special enzymes, classifies proteins in cells, selects target proteins, and modifies those proteins . UbiquitProtein ubiquitination requires three different enzymes: E1 ubiquitin-activating enzymes, E2 ubiquitin-conjugating enzymes, and E3 ubiquitin ligases . E1 enzyThe versatility of ubiquitination is determined by the complex assembly pattern of ubiquitin molecules on the target protein. Ubiquitins can be attached to one or multiple lysine residues with either a single ubiquitin molecule  or ubiquitin polymers (poly-ubiquitination) . Poly-ubPoly-ubiquitin chains occur when a single ubiquitin molecule is repeatedly connected in series with another ubiquitin lysine residue. Substrate proteins can be distinguished by poly-ubiquitin chains by attaching between different types of deubiquition formations, single mono-ubiquitination events, multiple mono-ubiquitinations events, homotypic ubiquitination events and heterotypic ubiquitination events (branched and non-branched ubiquitination) . This alWith the assistance of E1, E2, and E3, mono-ubiquitination occurs when a single ubiquitin is attached to its target proteins, then Ub molecules are added to the model in linear ways one by one . The seqNumerous studies have found that ubiquitin acetylation inhibits poly-ubiquitination elongation, and phosphoubiquitin leads to mitophagy. Any PTM chain can be changed to ubiquitin chains, which may prevent or facilitate ubiquitin interactions. Protein phosphorylation is linked to ubiquitination for proteasomal degradation. Reports have shown that the phosphorylation of ULK1 by MAPK1/3 kinase interacts with BTRC, which leads to subsequent proteasomal degradation and attenuates breast cancer bone metastasis . HoweverEncoding and decoding the ubiquitin code is performed by factors that recognize Ub chains and connect the substrate proteins to the downstream response . RecogniThe simplest version of ubiquitination or mono-ubiquitination confers non-degradative activities including protein localization , endocytOf note, other chains also regulate specific physiological functions: Lys6-linked poly-ubiquitin was supposed to be indirectly linked to DNA damage response with the help of heterodimeric ubiquitin E3 ligase BRCA1–BARD1 . Linear Non-proteolytic ubiquitination, including both mono-ubiquitination and poly-ubiquitination , has become key regulators in a variety of cancers. How they function as signaling entities in the pathogenesis and progression of cancer will be beneficial to gain an in-depth understanding of ubiquitination .Mono-ubiquitination has been suggested to be even more dynamic than previously thought, and its functions have been deciphered by various ubiquitin-binding proteins. It has been demonstrated that the cellular functionality of ubiquitin is mediated by mono-ubiquitin and/or poly-ubiquitin. In fact, mono-ubiquitination is associated with tumorigenesis, not limited to membrane transportation, endocytosis, receptor internalization, degradation in lysosomes, and protein reprocessing . BMI1 inDrosophila and is indispensable for inflammatory responses by activating Imd pathway -specific deubiquitinase (DUB), is a rare member of the OTU family of DUBs. Its proximal ubiquitin moiety cannot break down isopeptide linkages of ubiquitin chains, but can efficiently cleave peptide bonds present in the linear chains . OTULIN β-catenin activity through linear ubiquitination, promoting gastric tumorigenesis . It can be specifically catalyzed by LUBAC. For instance, EGFR recruits PKP2 to the plasma membrane and cooperates with LUBAC (HOIP), activating linear ubiquitination of NEMO, which is critical for tumor cell proliferation . Also, Eigenesis . Interesigenesis . Moreoveigenesis .It has been well established that Lys63 chains (ubiquitin chains topology lysine 63 poly-ubiquitin linkages) regulate and trigger distinct cellular signaling, including kinase activation, signal transduction, protein trafficking, endocytosis and DNA repair . The E2 The Ubc13/Uev1A complex has been shown to conjugate Lys63-linked poly-ubiquitination of substrate proteins, which contribute to breast cancer metastasis via NF-кB signaling regulation ; 2017. IκB signaling  was shown to interact with TRAF6 through Lys63-dependent poly-ubiquitination in order to promote tumor invasion . It has Lys6-linked ubiquitin chains are less abundant in resting cells and their functional implications are unclear . It has It has also been revealed that community-based learning collaborative (CBLC) assembles K6- and K11-linked poly-ubiquitin on EGFR and positively regulates its stability. The sustained activation of EGFR is largely dependent on CBLC-mediated ubiquitination, and its dysregulation is preferentially destined for membrane recycling, which plays an important role in non-small-cell lung carcinoma (NSCLC) progression .Structural findings indicated that USP30 efficiently cleaves Lys6-linked Ub chains, which abrogates parkin-mediated Ub-chain formation in mitochondria. Dysfunction of this novel distal Ub-recognition mechanism is associated with physiological disorders such as hepatocellular carcinoma . Althoug−/− mice were apparently enhanced by hampering Treg development and increasing Th17 response, suggesting its pro-tumor effects  and innate immunity. A previous study demonstrated that RNF168 can target the N-terminal tail of histones H2A/H2A.X, generating Lys27-linked ubiquitin chains. In response to DNA damage, K27 ubiquitination is the major source of PTMs that mark chromatin. Meanwhile, the activition of DDR can be inhibited by mutation of K27, which hinders the localization of 53BP1 and BRCA1 to DDR foci . DDR is Recent proteomic data have identified the role of Lys29-and Lys33-linked ubiquitin chains in various biological processes, including the control of AMPK-mediated mitochondrial function and Wnt-induced transcription signaling . AMPK-reK29-linked ubiquitin chains play important roles in driving cancer invasion and metastasis, as well as in the positive regulation of immunity . SeveralMixed linkage chains send mixed signaling messages that can be identified by different linkage-specific receptors. Our understanding of the mixed linkages and branched poly-ubiquitin is limited. Previous reports have elucidated that Tax in combination with UbcH2, Uhc5c, or UbcH7, can catalyze the construction of free mixed-linkage poly-ubiquitin chains, which are responsible for IKK-NF-κB activation and induction of T cell transformation . FurtherNon-proteolytic ubiquitination, the molecular switch in cell fate regulation, plays a crucial role in post-translational protein modifications. Diverse ubiquitination enzymes are essential for ubiquitination linkages that are necessary for normal metabolism and physiological functions. Meanwhile, it is also the root cause of physiological disorders, such as cancer. The aberrant regulation of the UPS is typically achieved by ubiquitination enzymes, DUBs, 20S proteasome catalytic core particles and 19S proteasome regulatory particles . In gene