After the success of immunotherapy in the treatment of advanced metastatic cancer, further evaluation in earlier settings, including high-risk, surgically-resectable disease is underway. Potential benefits of a neoadjuvant immunotherapeutic approach include presurgical tumor shrinkage, reduced surgical morbidity, early eradication of micrometastases and prevention of distant disease, and greater antigen-specific T cell response. For some cancers, pathologic response has been established as a surrogate measure for long-term outcomes, therefore offering the ability for early and objective assessment of treatment efficacy and the potential to inform and personalize adjuvant treatment clinical decision-making. Leveraging the neoadjuvant treatment setting offers the ability to deeply interrogate longitudinal tissue in order to gain translatable, pan-malignancy insights into response and mechanisms of resistance to immunotherapy. Neoadjuvant immunotherapy across cancers was a focus of discussion at the virtual Immunotherapy Bridge meeting . Clinical, biomarker, and pathologic insights from prostate, breast, colon, and non-small-cell lung cancers, melanoma and non-melanoma skin cancers were discussed and are summarized in this report. The development of immunotherapy has revolutionized treatment of metastatic cancer and improved survival for patients across many types of cancer. After demonstrating marked success in the treatment of advanced malignancies, further evaluation of immunotherapy in earlier settings, including high-risk, surgically-resectable disease has been under investigation. Earlier administration of therapies shown to be effective in a metastatic setting may offer further benefits and neoadjuvant therapy has an established role in several cancers. Notable benefits of a neoadjuvant approach include reduced surgical morbidity through tumor shrinkage, and the opportunity for early eradication of micrometastases and prevention of distant disease. Preclinical data suggest neoadjuvant checkpoint inhibition is superior to adjuvant checkpoint inhibition, with increased eradication of distant metastases, improved survival and greater antigen-specific T cell response following primary tumor resection in a murine breast cancer model . For somNeoadjuvant immunotherapy across cancers was a focus of discussion at the virtual Immunotherapy Bridge meeting . Clinical, biomarker, and pathologic insights from prostate, breast, colon, and non-small-cell lung cancers (NSCLC), melanoma and non-melanoma skin cancers were discussed and are summarized in this report.Large numbers of clinical immunotherapy trials are ongoing, which include patients who are polymorphic and with heterogeneous disease. These studies can generate hypotheses in a realistic clinical setting which can then be tested in the laboratory in appropriate models in a reverse translational approach. However, clinical trial design has had to be rethought to obtain appropriate clinical samples for laboratory studies. Presurgical or tissue-based phase Ia or IIa neoadjuvant trials can allow for evaluation of clinical signals in new tumor types, biomarker analysis and mechanistic insights, such as through immune monitoring and gene expression profiling in pre- and post-treatment tumor samples. The first of these neoadjuvant trials was in 2006, prior to any US Food and Drug Administration approvals, and assessed anti-cytotoxic T-lymphocyte-associated antigen (CTLA)-4 in 12 patients with localized bladder cancer . This shIn general, higher tumor mutational burden (TMB) is associated with better response to immune checkpoint blockade. Prostate cancer is typically poorly immunogenic. In patients with metastatic castration-resistant prostate cancer, high intratumoral CD8 T cell density and an interferon (IFN)-γ response gene signature and/or antigen-specific T cell responses were associated with a favorable response to ipilimumab . There wBased on these data, the combination of nivolumab 1 mg/kg plus ipilimumab 3 mg/kg were evaluated in the CheckMate 650 trial in patients with metastatic castration-resistant prostate cancer . ObjectiThere are multiple immune checkpoints, which are dynamic in their expression and so should be evaluated in both pre- and on-treatment tumor samples to guide clinical-decision making. Neoadjuvant trials provide clinical benefit for patients with localized disease, as well as offering mechanistic insights that can be used to develop combination therapies.Immunotherapy with PD-1/PD-L1 blockade was first approved for patients with metastatic triple-negative breast cancer (TNBC) based on the IMpassion 130 trial of atezolizumab and KEYNOTE-355 trial of pembrolizumab , 12. TheSeveral neoadjuvant trials in breast cancer have explored these questions. I-SPY2 was an open-label, multicenter phase II trial in which patients with high-risk stage II/III breast cancer were adaptively randomized to neoadjuvant chemotherapy with or without pembrolizumab . The addIn the phase III IMpassion 031 trial, 333 patients with previously untreated stage II-III TNBC were randomized to neoadjuvant chemotherapy plus atezolizumab or placebo . The addIn contrast to these two studies, pCR rate was not significantly improved by the addition of neoadjuvant atezolizumab to carboplatin and nab-paclitaxel in the NeoTrip trial of 280 patients with TNBC (43.5% vs 40.8% with placebo) . Unlike In the phase II GeparNuevo trial, 174 patients with TNBC were randomized to durvalumab or placebo for 2 weeks before the addition of nab-paclitaxel followed by epirubicin plus cyclophosphamide . The triOverall, the KEYNOTE-522 and IMPassion 031 studies provide consistent and complementary results that are supportive of the role of neoadjuvant immunotherapy, with both studies showing an increase in pCR and benefit observed regardless of PD-L1 status. The role of adjuvant immunotherapy in patients with a pCR remains unclear. The optimal chemotherapy regimen is also still be identified. Optimal patient selection is also important; 35–50% of patients will obtain pCR with chemotherapy alone so there is a need to identify which patients might be able to avoid immunotherapy and its associated toxicities. Biomarkers of response and toxicity are also required for those patients who might benefit from immunotherapy  has been associated with response rates of 30–70% in microsatellite-instability-high (MSI-H) or mismatch-repair-deficient (dMMR) tumors. However, response rates in mismatch-repair-proficient tumors (pMMR) are low. MMR deficient tumors account for only around 4% of all metastatic CRC, and 15% of early-stage CRC, although this still accounts for large numbers of patients.Neoadjuvant immunotherapy may have higher efficacy in early-stage colon cancers. In the exploratory NICHE study, chemotherapy-naïve patients with dMMR or pMMR tumors received a single dose of ipilimumab and two doses of nivolumab before surgery . In prelUnderestimation of pathological response using computed tomography (CT) scans was observed in several patients in the NICHE trial. Inflammation, acellular mucin and fibrosis accounted for the discordance between radiologic and pathologic responses. Potentially, the use of circulating tumor DNA and/or endoscopy may help in identifying patients who could avoid surgery in an organ-sparing approach.The preliminary data from the NICHE study compare favorably with standard of care neoadjuvant chemotherapy in dMMR/MSI colon cancers. In the FOxTROT trial of 1053 patients with CRC, 95% of dMMR/MSI tumors had no response to chemotherapy . HoweverMPR is predictive of long-term survival benefit in neoadjuvant chemotherapy-treated patients with NSCLC –25 and hThe rationale for neoadjuvant immune checkpoint inhibition is provided by several lines of evidence. Preclinical data demonstrated that tumor PD-L1 expression facilitates the development and spread of metastasis in murine models of NSCLC . In thesRuminococcus and Akkermansia spp. was associated with MPR to combination therapy and with favourable T cell receptor clonality at surgery [Coriobacteriaceae [Several trials have indicated that neoadjuvant immunotherapy is safe and effective in patients with NSCLC. Initial studies showed that neoadjuvant nivolumab was well tolerated and induced a MPR in 45% of resected tumors . Neoadju surgery . Interes surgery . NIF waseriaceae . The NIFOther studies have reported that immune checkpoint blockade combined with chemotherapy or radiation has clinical activity in patients with resectable NSCLC. The addition of neoadjuvant nivolumab, atezolizumab or durvalumab to neoadjuvant chemotherapy have all shown clinical promise in small-scale phase II studies, with encouraging MPR and pCR rates and overall good tolerability –33. NeoaImportantly, across all these studies, neoadjuvant immunotherapy did not negatively impact surgery. Delays to surgery were infrequent and resectability rates ranged from 81 to 96%, confirming that planned surgery after neoadjuvant immunotherapy is overall feasible.In the first phase III trial of neoadjuvant immunotherapy to report, the CheckMate-816 trial, patients with resectable NSCLC were randomized to chemotherapy with or without nivolumab . In the In conclusion, neoadjuvant immunotherapy is safe, feasible and active in patients with resectable NSCLC. The combination of nivolumab and ipilimumab induces higher MPR rates than historical controls of neoadjuvant chemotherapy and, in an exploratory analysis, than nivolumab monotherapy, and is also associated with greater tumor immune infiltrate and immunological memory than PD-1 monotherapy. CheckMate-816 is the first phase III trial to show a pCR benefit with neoadjuvant nivolumab plus chemotherapy in resectable NSCLC, with no major impact on surgery feasibility. Preclinical and translational studies are needed to identify biomarkers of response and mechanisms of resistance to these therapies, in order to develop more effective combinatorial strategies for patients with resectable NSCLC.In patients with clinical stage III melanoma, survival outcomes have historically been disappointing. Although adjuvant studies of immune- and targeted therapy have shown improved outcomes, it should be noted that these studies have screening failure rates of 15–25% due to rapid progression of disease after surgery in high-risk stage III patients. It can be postulated that patients with an aggressive disease phenotype may benefit more from a neoadjuvant approach.In an International Neoadjuvant Melanoma Consortium (INMC) pooled analysis of six immunotherapy or BRAF targeted therapy clinical trials, pCR correlated with improved recurrence-free survival (RFS)  and overall survival (OS)  . In patiSeveral studies have investigated the effects of neoadjuvant checkpoint inhibition in melanoma. In a study of 27 patients receiving single-dose neoadjuvant plus adjuvant pembrolizumab, eight had a pCR or MPR . All eigThe open-label OpACIN-neo trial was designed to identify an effective but more tolerable dosing schedule of ipilimumab plus nivolumab, with patients randomized to one of three different ipilimumab plus nivolumab dosing regimens . Of thesIn another recent study, 30 patients with clinical stage III or oligometastatic stage IV melanoma with Response Evaluation Criteria in Solid Tumors (RECIST) v1.1 disease were treated with neoadjuvant followed by adjuvant nivolumab plus the anti-LAG3 antibody relatlimab . The pCROverall, the ipilimumab 1 mg/kg plus nivolumab 3 mg/kg appear to be the most robust regimen, although nivolumab plus relatlimab appears to be comparable. Ongoing questions include whether adjuvant therapy is required after neoadjuvant treatment, especially in patients with robust pathological responses. We also need to know if neoadjuvant therapy can reduce the extent of surgery and whether baseline tumor features can be used to help to delineate which neoadjuvant treatment regimen might be most appropriate Fig. .Fig. 2OvSimilar to melanoma, non-melanoma skin cancer (NMSC) including cutaneous squamous cell carcinoma (CSCC) has a high mutational burden, providing a strong rationale for the use of immunotherapy. Several trials have shown the efficacy of PD-1 inhibition in CSCC –46. HoweIn an MD Anderson Cancer Center trial, 20 patients with primary or recurrent stage III-IVA CSCC of the head and neck (measurable disease of > 1.5 cm per RECIST 1.1) with planned curative-intent surgery and adjuvant radiation received two cycles of neoadjuvant PD-1 inhibition with cemiplimab, . The priAlthough only six patients (30%) had partial imaging responses by RECIST, 15 patients (75%) had either a pCR  or a MPR . This illustrates the underestimation of pathological response by radiographic imaging. Of the 15 pathologic responders, 12 (80%) did not receive adjuvant radiation as planned and at a median follow up of 22.6 months all remain free of disease. An additional 2 (10%) patients had a pPR, were treated with adjuvant therapy and also remain disease free. Three (15%) patients had > 50% viable tumor remaining at the time of surgery and were classified as non-responders. These patients had neither a clinical nor pathologic response, and all recurred. In retrospect, 2 of these patients had borderline resectable disease at presentation, underscoring the importance of careful patient selection. Histopathologic assessments showed significant heterogeneity, even among responders, including in the extent of inflammatory infiltrate. The 12-month DSS, DFS, and overall survival (OS) rates were 95%, 90%, and 95%, respectively.Pre-treatment gene expression profiling revealed an inflamed TME in patients with pCR or MPR, indicating that a favorable immune microenvironment prior to cemiplimab treatment is associated with pathologic response. Mass cytometry CyTOF analysis of pre- and post-treatment tumor samples showed changes in the tumor immune microenvironment after cemiplimab treatment were also associated with pathologic responses, in particular enrichment of memory CD8 + T-cell clusters. A regulatory T cell subset (CD3 + CD4 + FOXP3 +) and a myeloid cell subset expressing inhibitory marker VISTA (CD68 + CD14 + VISTA +) were associated with non-response and may help explain why some patients failed treatment. A confirmatory multicenter phase II trial with 76 patients receiving neoadjuvant cemiplimab followed by surgery and adjuvant cemiplimab, radiation or observation was recently completed .In conclusion, NMSC, in particular CSCC, is highly responsive to immunotherapy and the earlier application of immunotherapy may improve outcomes. Neoadjuvant immunotherapy is well tolerated and demonstrates durable responses in CSCC with no surgical delays and diminished need for adjuvant radiation therapy. Careful patient selection for neoadjuvant immunotherapy remains paramount, and a favorable baseline TME may predict response Fig. .Fig. 3ImPathological assessment is critical for evaluating neoadjuvant therapy and is typically the primary endpoint in clinical trials. It is therefore important that it is performed in a standardized manner; the INMC has developed guidelines for pathological evaluation of specimens to facilitate this. Tumor size assessed by radiology correlates with the tumor bed size but not the extent of residual viable tumor .Reproducibility of pathological response is very important. In the OpACIN-neo trial, patients were recruited in Australia and Europe (the Netherlands and Sweden). Pathological responses were evaluated at the respective centers at which patients were recruited. Lymph node dissection specimens of 83 patients were shared across centers and re-evaluated by pathologists at the other sites, with excellent interobserver reproducibility for pathological response category and percentage pathological response being observed . StandarPathological response is an excellent predictor of clinical outcome in melanoma patients treated with neoadjuvant therapies. As a consequence, pathological response evaluation enables clinicians to provide information to patients on their long-term prognosis after a short period of neoadjuvant therapy and may also inform clinical decision-making. In the PRADO trial of combination neoadjuvant nivolumab and ipilimumab, patients with a pCR or near pCR in the index node were followed-up with observation only, whereas patients with a partial or no response underwent therapeutic lymph node dissection (TLND); patients with no response in their TLND then received adjuvant nivolumab or dabrafenib plus trametinib while partial response patients underwent observation . Thus, pIn OpACIN-neo, almost all patients with recurrence had ≥ 70% residual viable melanoma. Type of pathological response also predicted outcome, with patients with a high fibrosis subtype of treatment response having the strongest association with lack of recurrence and prolonged RFS. These data are being used to develop a predictive score for relapse . PatientIn conclusion, pathological response assessment is reproducible. Radiology tumor size correlates with the tumor bed size but not pathological response. Pathological, peripheral blood, molecular and microbiome-related factors may all be associated with response and prolonged survival.Administration of immunotherapy in a neoadjuvant setting offers the potential to help improve outcomes for patients with high-risk, surgically resectable cancers. Neoadjuvant immunotherapy may improve operability, prime the antitumor immune response at an early stage, minimise micrometastatic disease, and prevent tumor relapse. Additionally, neoadjuvant treatment also provides an opportunity for early assessment of tumor response to therapy using surrogate endpoints of clinical efficacy at surgery. Neoadjuvant immunotherapy rather than upfront surgery and adjuvant therapy has the potential to become a new standard of care in several settings.The Neoadjuvant Immunotherapy Across Cancers at the 2021 Immunotherapy Bridges meeting provided an invaluable opportunity to bring together experts in immunotherapy and neoadjuvant treatment. Highlighted in this session were changes to standard of care as well as ongoing trials which continue to evaluate neoadjuvant immunotherapies in different tumors. This session also highlighted the neoadjuvant platform as a unique, pan-cancer opportunity to leverage biomarker analyses from robust tissue collections to gain novel insights into mechanisms of response and resistance to therapies, potentially informing subsequent development of immunotherapy in other indications. While surrogate endpoint validation for chemotherapy is well established, pathologic response to neoadjuvant immunotherapy is in development and will be critical to placing biomarker insights, clinical response, and outcomes into context. Treatment of surgically resectable cancers with neoadjuvant immunotherapy remains of significant interest to improve outcomes for patients across cancer types and stages.


Because parasites have an inextricable relationship with their host, they have the potential to serve as viral reservoirs or facilitate virus host shifts. And yet, little is known about viruses infecting parasitic hosts except for blood-feeding arthropods that are well-known vectors of zoonotic viruses. Herein, we uncovered viruses of flatworms  that specialize in parasitizing vertebrates and their ancestral free-living relatives. We discovered 115 novel viral sequences, including 1 in Macrostomorpha, 5 in Polycladida, 44 in Tricladida, 1 in Monogenea, 15 in Cestoda, and 49 in Trematoda, through data mining. The majority of newly identified viruses constitute novel families or genera. Phylogenetic analyses show that the virome of flatworms changed dramatically during the transition of neodermatans to a parasitic lifestyle. Most Neodermata viruses seem to codiversify with their host, with the exception of rhabdoviruses, which may switch hosts more often, based on phylogenetic relationships. Neodermata rhabdoviruses also have a position ancestral to vertebrate-associated rhabdo viruses, including lyssaviruses, suggesting that vertebrate-associated rhabdoviruses emerged from a flatworm rhabdovirus in a parasitized host. This study reveals an extensive diversity of viruses in Platyhelminthes and highlights the need to evaluate the role of viral infection in flatworm-associated diseases.IMPORTANCE Little is known about the diversity of parasite-associated viruses and how these viruses may impact parasite fitness, parasite-host interactions, and virus evolution. The discovery of over a hundred viruses associated with a range of free-living and parasitic flatworms, including parasites of economic and clinical relevance, allowed us to compare the viromes of flatworms with contrasting lifestyles. The results suggest that flatworms acquired novel viruses after their transition to a parasitic lifestyle and highlight the possibility that they acquired viruses from their hosts and vice versa. An interesting example is the discovery of flatworm rhabdoviruses that have a position ancestral to rabies viruses and other vertebrate-associated rhabdoviruses, demonstrating that flatworm-associated viruses have emerged in a vertebrate host at least once in history. Therefore, parasitic flatworms may play a role in virus diversity and emergence. The roles that parasite-infecting viruses play in parasite-associated diseases remain to be investigated. Over the past few years, high-throughput sequencing technologies have expanded the virosphere well beyond pathogenic viruses and/or viruses that can be cultured. Therefore, the known taxonomy and understanding of virus evolution have increased dramatically . One sucPlatyhelminthes, also known as flatworms, constitute a diverse phylum estimated to contain up to 100,000 species with diverse body plans, lifestyles, and ecological roles , 5. The 6–Schistosoma, is the second most important neglected tropical disease after malaria, affecting over 200 million people and causing 200,000 deaths annually worldwide , and sometimes, a second intermediate host  –12. Neod–orldwide . Chronicnfection , 15. Humium spp. , 16, 17.Bunyavirales and the family Nyamiviridae  have been reported from Schistosoma japonicum and a mix of Taenia sp.  and Sequence Read Archive (SRA) databanks  and 34 viruses at the family level, indicating that Platyhelminthes host a unique viral diversity. The majority of viruses were classified within the orders Picornavirales (27 viruses), Mononegavirales (21 viruses), Bunyavirales (17 viruses), Martellivirales (14 viruses), Ghabrivirales (11 viruses), and Amarillovirales (8 viruses)  between the number of viruses discovered within an order and the mean read coverage of those viruses  . Other o viruses . It is pPicornavirales, Martellivirales, Ghabrivirales, Bunyavirales and Jingchuvirales, as well as viruses within the families Flaviviridae, Nyamiviridae, and Rhabdoviridae, were found within more than one Platyhelminthes species. We used phylogenetic methods to further investigate their relationships to each other and to the known viral diversity. To build phylogenetic trees, we included representatives of previously characterized families, as well as unassigned viruses that showed high sequence similarity to viruses described here. Phylogenetic analyses revealed that viruses of Platyhelminthes often cluster together, separately from other known viruses, providing evidence that they constitute distinct taxa. Note that newly discovered flatworm-associated viruses that were not phylogenetically related to other viruses of Platyhelminthes were not investigated further because the host remains putative. These viruses could be associated with the host diet or with a coinfecting microorganism or could result from contamination during sample processing. Overall, including only taxa for which viruses were found in at least two different Platyhelminthes species and based on a combination of phylogenetic analyses, genome composition analyses, and shared percentages of identity (see below), our data provide evidence for at least seven new families and 18 new genera of Platyhelminthes-specific viruses that will be submitted to ICTV for evaluation  that contained a picorna-like virus (Beihai razor-shell virus 4) and a bunya-like virus (Beihai bunya-like virus 2) closely related to trematode-associated viruses discovered here. The spotted paddle-tail newt (SRR6291293), within which a Neodermata virus-like rhabdovirus was found (Fujian dimarhabdovirus), appeared infected by trematodes known to infect amphibians and nonfish vertebrates (Mesocoelium sp. and Spirometra sp.). The Wenling sharpspine skate mix (SRR6291349) within which another Neodermata virus-like rhabdovirus was found (Wenling dimarhabdovirus 8) was infected by a cestode from the family Echinobothriidae known to infect Elasmobranchii. Finally, two sample mixes of fish gills (SRR6291357 and SRR6291374) within which more Neodermata virus-like rhabdoviruses were found (Wenling dimarhabdovirus 10 and Beihai dimarhabdovirus 1) contained reads that aligned against monogenean parasite nucleotide sequences, but the low percentage of reads belonging to Platyhelminthes prevented the successful assembly of transcripts. The phylogenetic positions of these viruses and the demonstration that the host organisms were infected by Neodermata parasites at the time of sampling suggest that these viruses could be infecting the parasite rather than the vertebrate or invertebrate host. Additional viruses that were probably associated with tapeworms or flukes but for which we could not conduct the same analysis (either the raw data were not available or samples were processed in a way that eliminated host-associated transcripts) include picornaviruses . FuturePicornavirales and Ghabrivirales in the free-living Rhabditophora than in the parasitic Neodermata , whereas the Provittati virus of the free-living worm Prostheceraeus vittatus clustered most closely with viruses of liver flukes. And yet, the RNA-dependent RNA polymerase (RdRP) proteins of these viruses showed maximums of 36% and 45% amino acid identity to their closest relatives, suggesting that they belong to different taxa. More sampling is needed to help resolve the phylogeny of Platyhelminthes. Nevertheless, our findings indicate that the transition of a protoneodermatan worm from free living to parasitism over 500 mega-annum (Ma) ago  ago  impactedGhabrivirales, viruses of cestodes and trematodes are found on distinct branches and constitute two novel proposed families, suggesting distinct evolutionary origins before diversification within their parasitic hosts  from the viruses associated with the monogenean Eudiplozoon nipponicum and the cestode Triaenophorus nodulosus (Schistosoma japonicum and Trichobillharzia regenti) clustered together, separately from viruses associated with the liver flukes of the family Opisthorchiidae  that clustered together, and separately from viruses associated with the Psilostomidae (Psilotrema simillimum and Sphaeridiotrema pseudoglobulus). Past experimental investigations on the transmission mode of Schistocephalus solidus viruses revealed that most viruses, including SsJV and a Bunya-like virus, are vertically transmitted from parents to offspring , often clustered separately based on their parasitic host’s phylogenetic relationships, suggesting a close association between parasitic hosts and their viruses. Within the order ic hosts . Within l family . Among todulosus . Those tffspring . The codSchistocephalus solidus worms and is transmitted to parasitized hosts. If this characteristic is conserved among Neodermata-associated rhabdoviruses, it would provide an avenue for rhabdovirus host switching between parasites coinfecting the same hosts and between parasites and their intermediate and definitive hosts.Parasites have intimate relationships with their hosts, and they can infect different host individuals or species over the course of their life cycles, two factors that can facilitate virus transmission and spillover . The phyThe genomes of rhabdoviruses of cestodes and trematodes exhibited extensive diversity in genome length . As expected, this genome length variation was associated with variation in the number of predicted genes. The rhabdovirus genome is normally composed of a minimum of five open reading frames (ORFs) encoding five structural proteins—the nucleoprotein (N), the polymerase-associated phosphoprotein (P), the matrix protein (M), the glycoprotein (G), and the RdRP (L). And yet, five of the rhabdoviruses of Neodermata encoded only four proteins, with the loss of the spike glycoprotein. Other Neodermata rhabdovirus genomes contained up to three additional small ORFs between G and L, encoding putative proteins.Alpharhabdovirinae subfamily  manipulates the parasitic wasp’s behavior to increase hyperparasitism—forcing the wasp to lay its eggs in already parasitized hosts—and increase egg load to increase its horizontal transmission , leishmania RNA virus 1 (LRV1) is excreted within exosomes and exacerbates Leishmania’s pathogenicity by causing a hyperinflammatory response and metastatic secondary lesions, known as mucocutaneous leishmaniasis -induced proinflammatory innate immune response is amplified upon antiparasitic treatment due to the release of viruses by dying parasites  sequence database all 45 assembled transcriptomes available, corresponding to 38 flatworm species (Table S1). In addition, we downloaded 104 Sequence Read Archive (SRA) files (Table S1) from trematodes and cestodes for which no assembled transcriptome was available, which allowed us to process data for 28 additional species. All SRA data sets were assembled in-house. For this purpose, raw sequences were trimmed for quality and adapter removal using Trimmomatic version 0.36.0  with def−10) against a viral protein database containing sequences from the NCBI Reference Sequence database . All putative viral transcripts were then translated into proteins to conduct reciprocal BLASTp against GenBank nonredundant (nr) protein database 244, released on 25 June 2021, and confirm virus discovery (Table S2). When partial viral genomes were identified in TSA data sets, corresponding raw reads were downloaded from SRA and reassembled in-house as described above. Next, viral contigs within in-house-reassembled transcriptomes were identified through BLASTx against a viral protein database containing flatworm-associated viral sequences initially discovered from TSA and SRA data and closely related sequences. Reassembly efforts and BLAST searches allowed us to complete or extend the length of partial genome sequences by obtaining longer contigs from in-house assemblies and/or identifying smaller contigs to be used for scaffolding. Viral genome structures and sequences were validated and eventually corrected by mapping all reads and assembled contigs against the newly identified viral genomes as references using Burroughs-Wheeler Aligner (BWA) (version 0.7.8) .Initially, viral contigs larger than 500 bp were discovered in TSA and assembled SRA data by comparing   and weren 0.7.8) , 60. When 0.7.8) . Rounds Open reading frame predictions were obtained using Translate on Expasy and from alignments with related reference virus genomes. Annotation of domains was extracted from comparisons against the Conserved Domain Database (CDD) as implemented by BLASTp against the nr protein database. Initial supergroup assignation was determined from best BLAST matches. Viral RNA-dependent RNA polymerase (RdRP) sequences were aligned, using the E-INS-I algorithm implemented in the program MAFFT (version 7) , to reprSRR6291374, SRR6291293, SRR6291349, SRR6291357, and SRR3401916 were downloaded and assembled as described above using SPAdes. Transcript annotation was conducted using MegaBLAST against GenBank to identify transcripts of Neodermata. In addition, the taxonomic composition from each data set was assessed by comparing 1% of the reads against GenBank. The taxonomic composition was visualized using the Krona chart (Our phylogenetic analysis revealed that some viruses associated with nonparasitic hosts were very closely related to viruses of Neodermata parasites. To determine whether the detection of these previously reported viral sequences resulted from contamination due to parasitized hosts at the time of sampling, we investigated the presence of parasite sequences. To do this, SRA data sets na chart . The preBK059652 to BK059766 as indicated in Table S3.Viral sequences are available under GenBank accession numbers


Chronic opioid use is linked to persistent and severe sleep/wake disturbances in patients. These opioid-related sleep problems increase risk for developing opioid dependence, mood disorders and in turn overdose in chronic pain patients receiving opioid therapy. Despite the well-established link between long-term opioid use and sleep disorders, the mechanism by which opioids perturb sleep remains unclear. Interestingly, animal studies indicate that opioids disrupt sleep/wake behaviors by altering an animal’s ability to synchronize their circadian rhythms to environmental light cycles . A specific subset of retinal cells known as intrinsically photosensitive retinal ganglion cells (ipRGCs) that express μ-opioid receptors are exclusively responsible for transmitting environmental light information to sleep/circadian centers in the brain. Thus, this review will focus on the effect of opioids on ipRGCs and their projection regions that are involved in the photoentrainment of sleep/wake behaviors. Lastly, we discuss the viability of ipRGCs as a potential therapeutic target for treating opioid-related sleep/wake problems. Chronic opioid use has been shown to disrupt healthy sleep/wake behavior, which often manifests as insomnia and daytime fatigue . The slevia activation of the μ-opioid receptor (MOR), with lesser contributions from interactions with the κ- (KOR) and δ-opioid receptors (DOR)  . These ars (DOR) . While trs (DOR) . DORs hars (DOR) . While iMany of the sleep centers in the brain express opioid receptors and thus have the potential to contribute to opioid-induced sleep/wake changes . HomeostGiven the importance of light in regulating sleep/wake behavior, this review assesses how opioid drugs alter the light-mediated regulation of sleep/wake behavior. Specifically, we examine the evidence that light-sensitive sleep/circadian centers within the central nervous system (CNS) contribute to the development of opioid-related sleep disturbances. In mammals, the retina is responsible for conveying environmental light into neuronal signals that can be interpreted by the brain for both image forming and non-image forming visual functions. Non-image forming visual functions, such as the light-mediated regulation of sleep/wake behavior, is dependent on intrinsically photosensitive retinal ganglion cells (ipRGCs) . Six subMany clinical studies have noted the negative impact that chronic opioid use can have on sleep/wake behavior . Opioid In this review, we assess the evidence that opioids could alter sleep/wake behavior by acting directly on M1 ipRGCs and their projection regions involved in the light-mediated regulation of sleep/wake cycles. These projection regions can be distinguished based on their function within the context on sleep/wake regulation through parallel pathways . ipRGCs While there is no clinical evidence that opioids alter image forming visual functions, via modulation of voltage-gated potassium and calcium currents  and the ventral lateral geniculate nucleus (vLGN) of the thalamus .Per1 gene expression within the rat SCN -producing neurons that synapse on the SCN . In rat,M1 ipRGCs have also been shown to the vLGN of the thalamus, another region implicated in circadian photoentrainment . While tAcute light-induced regulation of sleep in mice has been shown to be mediated by ipRGC inputs to the hypothalamus, specifically the preoptic area (POA) and the lateral hypothalamus (LH) .The preoptic area, specifically the ventrolateral preoptic area (VLPO), has been studied extensively in regard to its role in sleep . In rodeNotably, this region contains two separate populations of Gamma Aminobutyric Acid (GABA)-ergic neurons that are thought to be important mediators of sleep. Galanin-expressing GABAergic neurons in the POA are thought to be involved in the homeostatic regulation of sleep , howeverThe lateral hypothalamus (LH) has been heavily implicated in both sleep and opioid use disorders , making persistent sleep disturbances associated with chronic opioid use.Both endogenous opioid peptides and opioid drugs can suppress excitatory inputs to LH neurons as well as directly inhibit the firing of LH orexin/hypocretin neurons . InteresAdditionally, LH neurons receive input from light responsive GABAergic neurons in the POA . As cellFor opioid drugs to able to suppress ipRGC activity, they must deposit in the retina following systemic exposure. Previous studies have also shown that opioids deposit in the mammalian retina/VH following systemic administration . RecentlThis introduces a novel retinal route by which opioids could persistently alter sleep/wake behavior. This is especially exciting because retinal neurons are far more accessible for targeted interventions compared to sleep/wake centers in the brain. Targeting sleep centers in the brain to treat opioid-related sleep problems is exceedingly difficult due to the number of brain regions involved in both sleep/wake as well as other opioid-mediated effects on the body. However, the retina represents a unique CNS structure that lies outside the cranial vault, making it far more accessible for targeted therapeutic interventions.NB wrote the manuscript and created the figures. All authors edited and reviewed the manuscript for final submission.


Fracture patients are frail and have high mortality. We investigated whether introducing a fast-track strategy during post-surgery care and including early rehabilitation protocols may shorten the length of hospital stay (LOS) while improving the overall clinical effectiveness. A training ground was built inside the inpatient area dedicated to trauma settings. Usual postoperative care consists of immobilization during the first day, but patients may start rehabilitation earlier, 24 hours after the surgical procedure, with a fast-track strategy. In general, gait speed, step length, and self-assessment in terms of mobility improve significantly in the first six postoperative weeks in fracture patients. As delayed postoperative mobility during hospitalization was observed, the established training ground may help with this concern. The expert physiotherapist may contribute to ameliorating the indicators showing great potential in postoperative rehabilitation regardless of fracture pattern. The primary outcome was postoperative physical functioning. Secondary outcomes included the patient’s assessment of therapeutic effect , perceived pain intensity, health services utilization, treatment side effects, and adverse events. Data were analyzed by univariate analysis and binary logistic regression showing a reduction of LOS of almost three days. Further, the optimized hip fracture program reduced the rate of in-hospital postoperative complications and mortality. Adding to the schedule, some PROMPTS (Patient-reported outcome measures) could further integrate the patient empowerment perspective into the quality set of values. For this reason, ‘fast track’ may define a crucial policy able to guarantee rapid rehabilitation, becoming a key factor to achieving a good clinical effect. Fast-track rehabilitation facilitates a shortened hospital stay and cost-saving and can be used to optimize the patient’s condition before admission to a rehabilitation facility• Early rehabilitation protocols may shorten the length of hospital stay (LOS) while improving the overall clinical effectiveness.• Human factors and patients empowerments may help.


The important role of pyroptosis in tumor progression has been well characterized in recent years. However, little is known about the impact of tumor pyroptosis characteristics on patient prognosis and tumor microenvironment (TME) as well as efficacy of immunotherapy. In this study, we successfully classified colon cancer samples into three pyroptosis characterizations with different prognosis and TME cell infiltration patterns based on the expression of pyroptosis-related genes. Cluster 2, with the characterizations of immunosuppression, was classified as immune-desert cell infiltration patterns. Cluster 3, with the patterns of immune-inflamed cell infiltration, had the feature of an activated innate and adaptive immunity and significant prolonged survival. The activation of stromal pathways including EMT, angiogenesis and TGF-β in cluster 1 may mediate the impaired immune penetration of this cluster, which was classified as immune-excluded cell infiltration patterns. Our results demonstrated the PyroSig signature was a robust and independent biomarker for predicting patient prognosis. Patients with low PyroSig signature was confirmed to be correlated with treatment advantages and significant prolonged survival in two anti-checkpoint immunotherapy cohorts. This study identified three pyroptosis-related subtypes with distinct molecular features, clinical and microenvironment cell infiltration patterns in colon cancer, which could promote individualized immunotherapy for colon cancer. As one of the most common tumor types in digestive system, the incidence rate and mortality of colon cancer are increasing year by year –3. AlthoBased on the published studies, we in total extracted 31 pyroptosis-related genes from the GEO datasets. We used the STRING to establish the protein-protein interaction network (PPI), and reveal widespread protein interactions among these genes . The GO Considering the detailed clinical annotations in GSE39582 cohorts, we used this cohort for the subsequent analyses. The consensus clustering was utilized to reveal the pyroptosis characterization in colon cancer and classify patients into distinct molecular subtypes. Based on the expression of entire pyroptosis-related genes, we successfully classified all samples into three different pyroptosis characterizations, which was referred to as cluster 1 subtype, cluster 2 subtype and cluster 3 subtype, respectively . PCA metIn order to evaluate the correlation between TME cell infiltration patterns and pyroptosis characterizations, we calculated the infiltration abundance of immune cells in each cluster. We found a high level of immune cell infiltration in the tumor microenvironment of cluster 1, sharing the similar immune cell infiltration abundance with the cluster 3 , 3B. HowWe analyzed the expression of chemokines and cytokines that characterize these three pyroptosis characterization clusters to further explore their characteristics of TME immunoregulation –3G 16]..16]. TheTo further explore the potential biological characteristics of the three pyroptosis characterizations, we investigated the transcriptome differences between the three clusters and a total of 1219 DEGs were determined . We analWe used TCGA-COAD cohort as the validation set. Consistent with the GEO cohort, patients with low PyroSig also experienced an improved overall survival compared to those with high PyroSig . CompareTo further determine the prognostic predictive value of the PyroSig signature, we generalized it to other colon cancer cohorts. Based on the GSE17536, GSE37892 and GSE38832 collected from GEO database, we found that patients with lower PyroSig were consistently associated with improved survival compared with those with higher PyroSig, which further confirmed the potential of PyroSig signature as an independent prognostic biomarker in colon cancer –6C. The in vivo, and its prediction accuracy is often poor. It has become an important means to improve the prediction efficiency to identify novel molecular subtypes and build a multi gene model by combining a group of gene signature with machine learning [Although the diagnosis and treatment of colon cancer have made some progress in recent years with the development of technology, it has not significantly reduced the incidence rate and mortality of colon cancer. In addition, for advanced colon cancer, existing treatment methods are still limited. Therefore, the early diagnosis and treatment as well as the mechanism of recurrence monitoring are particularly important in colon cancer . Due to learning . Pyroptolearning –21. On tlearning . Yang etlearning . Zhang elearning . At the Here, we integrated multi-omics data on colon cancer from the TCGA database to further explore the genomic signatures of cell-coke-death-related genes in colon cancer tissues. Although the copy number variation and mutation frequency of these genes are relatively low, the expression levels of these genes are significantly different between colon cancer tissues and normal tissues. Through consensus clustering of these genes, we revealed three distinct cellular pyroptosis characterizations, implying that colon cancer patients could be divided into three distinct molecular subtypes based on pyroptosis-related genes. GSVA and GO enrichment analysis showed that these three subtypes were highly correlated with immune related signaling pathways. The evaluation of TME immune cell infiltration confirmed that these three pyroptosis related molecular subtypes had significantly different TME cell infiltration patterns, and were consistent with the three immune phenotypes of tumors. In general, innate immune cells and stroma were significantly activated in cluster 1 subtypes, which was consistent with the immune-excluded phenotype. Cluster 2 subtype presented an inhibitory microenvironment with relatively few innate and adaptive immune cells, so it was classified as immune-desert phenotype. Contrary to the former two, the microenvironment in cluster 3 subtype had a large number of immune cells infiltrating, and the immune related signal pathway also showed an activated phenotype, which was consistent with the immune-inflamed phenotype. Although both cluster 1 and cluster 3 subtypes belong to “cold tumors”, their mechanisms were not identical. The stroma in the TME was significantly activated in cluster 1 subtype, mediating tumor immune escape. There were abundant immune cells in both immune-excluded and immune-inflamed tumors. However, unlike the immune-inflamed tumors, the activated stroma retained immune cells around the nests of tumor cells, limiting immune cell infiltration into the parenchyma of the tumor. The interaction between the stroma and the immune cells made the immune cells appear to be present inside the tumor. Although PD-1/PD-L1 inhibitors could stimulate T cell activation and proliferation around the stroma, they could not stimulate infiltration, thus limiting the clinical response rate of this subtype to immunotherapy –29. In aAt present, it has become a hot topic to transform “cold tumor” into “hot tumor” to increase immune infiltration of microenvironment –33. In tThis study identified three pyroptosis characterizations with distinct clinical, molecular characteristics and TME cell infiltration patterns, and constructed PyroSig signature based on these pyroptosis characterizations, which could be served as a robust and independent biomarker for predicting patient outcomes and efficacy of immunotherapy. It might help to promote individualized immunotherapy for colon cancer from the perspective of pyroptosis characterizations.After systematic search of GEO and TCGA database, we collected a total of 5 colon cancer cohorts including the TCGA-COAD, GSE39582, GSE17536, GSE37892, GSE38832, which contain detailed clinical information –37. All The pyroptosis-related genes were derived from published studies. In order to identify distinct pyroptosis characterization clusters in colon cancer, we used ConsensuClusterPlus R package to execute consensus clustering based on the mRNA expression of these pyroptosis-related genes. Consensus clustering is repeated 1000 times to ensure the stability of molecular classification .The Gene Set Variation Analysis (GSVA) and GO enrichment analysis was used to execute functional annotation for uncovering the signaling pathways involved in these pyroptosis characterizations. The “c2.cp.kegg.v6.2.symbols” gene set was downloaded from GSEA database –42. The Multiple algorithms have now been developed for assessing the abundance of various immune cell infiltrates of the tumor microenvironment based on gene expression. In this study, we used single sample gene set enrichment analysis (ssGSEA) to measure the abundance of microenvironmental immune cell infiltration under three pyroptosis characterizations. We obtained the gene sets with 28 immune cells from a previous study , 46.Considering that pyroptosis characterizations played an important role in the prognosis and microenvironment of colon cancer, we constructed pyroptosis related signature. First, a univariate Cox regression model was used to explore the correlation between patient prognosis and the expression of these DEGs. The least absolute shrinkage and selection operator regression method (LASSO) was then performed for the expression of the DEGs with the prognosis P value less than 0.001 . The Pyrwhere i is the expression of prognosis-related genes.We used the Kaplan-Meier method to plot survival curves and used the log-rank tests for significance test based on the survminer R package. In addition, a univariate and multivariate Cox regression models were used to reveal the prognostic value of PyroSig signature as a continuous variable. The MaxStat R package was utilized to determine the optimal cut-off point of PyroSig to classify patients into low and high groups. We executed the Wilcoxon test to calculate the difference significance between two groups. The One-way ANOVA test and Kruskal-Wallis tests were utilized to calculate the difference significance groups of three or more . All dathttp://www.ncbi.nlm.nih.gov/geo) and the GDC portal .All data related to this work can be acquired from the Gene-Expression Omnibus (GEO, Supplementary Figure 1Supplementary Tables


Touch-like phantom limb sensations can be elicited through targeted transcutaneous electrical nerve stimulation (tTENS) in individuals with upper limb amputation. The corresponding impact of sensory stimulation on cortical activity remains an open question. Brain network research shows that sensorimotor cortical activity is supported by dynamic changes in functional connections between relevant brain regions. These groups of interconnected regions are functional modules whose architecture enables specialized function and related neural processing supporting individual task needs. Using electroencephalographic (EEG) signals to analyze modular functional connectivity, we investigated changes in the modular architecture of cortical large-scale systems when participants with upper limb amputations performed phantom hand movements before, during, and after they received tTENS. We discovered that tTENS substantially decreased the flexibility of the default mode network (DMN). Furthermore, we found increased interconnectivity (measured by a graph theoretic integration metric) between the DMN, the somatomotor network (SMN) and the visual network (VN) in the individual with extensive tTENS experience. While for individuals with less tTENS experience, we found increased integration between DMN and the attention network. Our results provide insights into how sensory stimulation promotes cortical processing of combined somatosensory and visual inputs and help develop future tools to evaluate sensory combination for individuals with amputations. Recent studies developed sensory stimulation strategies and showed promising results in reinstating touch-like sensations to these individuals5. The somatosensory information that was provided through stimulation improved individual’s performance in object manipulation and prosthesis control, demonstrating the importance of somatosensory input in the context of upper limb amputation. It is important to note that humans interact with the environment through multiple sensory inputs. Therefore, recent studies developed multisensory stimulation paradigms using combined visual and somatosensory inputs and showed improved sensory perception and prosthesis embodiment8. However, the cortical underpinnings of enhanced sensory perception and motor performance have not been commensurately investigated. Most literature demonstrates the benefits of sensory stimulation through behavioral and functional assessments such as questionnaires, phantom limb telescoping measurements, and proprioceptive drift measurements10. Consequently, understanding the cortical processing of sensory stimulation is crucial, because this knowledge can inform future design of effective stimulation paradigms.For individuals with an upper limb amputation, the lack of afferent somatosensory inputs has been reported as one of the main reasons for decreased prosthesis usage14. These studies showed evoked responses14 and increased activation in the central and centro-parietal regions of EEG signals5. These results validated that sensory stimulation elicited activity in the sensorimotor regions of the brain. Meanwhile, network neuroscience methods have allowed researchers to model cortical activity not as mere activation in the cortex, but as complex and intertwined functional connections between different cortical regions. This approach is suitable to study dynamic activity in the brain such as sensorimotor processing16. In this context, our previous work used dynamic functional connectivity and found that sensory stimulation to individuals with upper limb amputations led to increased functional connectivity between somatosensory, motor, and higher-level processing regions18.Previous work collected electroencephalographic (EEG) recordings when individuals with amputations received sensory stimulation19. These large-scale systems are typically established based on functional engagement of different regions20. As the brain processes internal and external information, cortical regions form several specific cognitive functional networks22. Among common large-scale systems, the DMN has been at the center of substantial research, owing to the conventional thought that the DMN facilitates spontaneous cognitive and stimulus-independent processes23. However, its functional role has not been completely elucidated yet. Recent work proposed that the DMN plays a role in contextualizing external sensory information with internal processes24. Furthermore, studies suggested that neural activity within the DMN may relate to brain activity in other cognitive systems, including those that are active during a task25. In this context, investigating cortical functions from the perspective of large-scale systems allows us to understand how changes resulting from external sensory information occur in the brain.Under the brain network paradigm, previous research involving individuals with upper limb amputations investigated changes in functional connections among cortical large-scale systems such as the somatomotor network (SMN) and the default mode network (DMN)26. As an individual with upper limb amputation performs a daily task, the most common external sensory information entails somatosensory information elicited via sensory stimulation, as well as visual information present in the environment. Previous studies showed that the reintroduced sensory information helped with fulfilling task requirements such as controlling a prosthesis or identifying object compliance12. At the cortical level, as we carry out different daily tasks, some brain regions  dynamically change their interaction patterns with other regions to adapt to different task demands  on large-scale systems and functional modules in the cortex Fig. A. Does sflexibility, integration, and recruitment, which quantify 1) changes among functional modules (flexibility) and 2) interactions within and across different large-scale systems (integration and recruitment)  would be more active over the duration of the task.To address these questions, we recorded EEG from individuals with upper limb amputations, each performing hand movements before, during, and after sensory stimulation that elicited sensations in their phantom hand. We also recruited intact-limb individuals for the same experiment. We investigated how dynamic modular networks characterized changes in interaction between large-scale systems and functional modules in response to sensory stimulation. We used graph theoretic metrics such as Three participants with upper limb amputations Table , with tTAfter sensory mapping, and prior to EEG recording, we asked each participant to associate tTENS elicited sensations with hand movements that were displayed on the computer screen Fig. A. ParticWe first investigated whether each individual’s history with sensory stimulation influenced change in modular architecture. Participant A01 had experience with tTENS for over 1.5 years , while participant A02 had lesser experience  and participant A03 had no experience with tTENS prior to our study  , attention network (ATN), and task-positive network (TPN) . We then calculated the magnitude of percentage change in flexibility of each large-scale system between Pre-Stim and Stim-Move conditions  source localization algorithmflexibility and each individual’s experience with tTENS, we performed the following analyses separately for each individual with upper limb amputations.These results suggest that the more experience an individual has with sensory stimulation via tTENS, the more changes in the brain’s modular architecture are observed. Based on the dependencies we found between changes in flexibility and experience with sensory stimulation, we investigated whether sensory stimulation via tTENS induced increase or decrease in flexibility for each individual. We first compared flexibility between Pre-Stim and Stim-Move conditions and found that for the experienced participant (A01), flexibility decreased for all five large-scale systems , we did not observe significant statistical changes in flexibility between Pre-Stim and Stim-Move conditions. For A03, the greatest change was observed as increase in flexibility for ATN . These two metrics provide insight into whether nodes in the four systems are interacting more with nodes in their own system (high recruitment) or with nodes in other systems (high integration) during sensory stimulation (Stim-Move) and afterwards (Post-Stim). For each participant with upper limb amputations, we first examined overall changes in integration and recruitment. We then computed integration between each pair of the four large-scale systems and recruitment of each system.Our analysis of the integration of the four large-scale systems , while VN is interacting less with itself (decreased recruitment).We then investigated pairwise integration . Overall integration  showed higher ion Fig. , compariIn this study, we aimed to investigate the influence of sensory stimulation on cortical large-scale systems and functional modules. We verified the hypothesis that dynamic modular networks reveal changes in interaction between large-scale systems and functional modules in response to tTENS. Our work provides insight into understanding the cortical processing of combined somatosensory and visual inputs while individuals with upper limb amputations performed phantom hand movements. While subjective or behavioral outcomes from participants with upper limb amputations can provide evidence for the benefits of sensory stimulation, detailed understanding of the cortical impacts is necessary for future design of optimal stimulation strategies and training regimens. Here, our work bridges the gap between the vast behavioral research on tactile sensations for individuals with upper limb amputations and the less explored aspect of cortical activity in response to sensory stimulation.flexibility of DMN decreased in participants A01 (experienced) and A02 (rudimentary) when they performed phantom hand movement and received tTENS  increased integration between SMN, VN, and DMN for participant A01 who is experienced with tTENS ENS Fig. C, D. Theflexibility of all large-scale systems decreased substantially in participant A01 when he made phantom hand movements with sensory stimulation  as opposed to merely hand open or close, which were selected by individuals with less tTENS experience (A02 and A03). The higher complexity of hand movements could be related to our observations. Previous studies on skill learning suggest that flexibility is desirable for large-scale systems in the brain to support new behavior and it decreases in the late stage of a learning process, when requirements of functional adaptability reduce35. In this regard, it is plausible that our observed decrease in flexibility indicates that the motor task of making a hand movement became less challenging and more “intuitive” to complete for participant A01.Our results showed that ion Fig. A. Flexibflexibility provides insights into whether functional modules change dynamically, integration and recruitment focus on the interaction within and between different large-scale systems, namely SMN, DMN, VN, and ATN in this study. Our results from participant A01 show that VN interacts more with SMN and DMN and less with itself during sensory stimulation  are more active. A02 Fig. B. IntereA01 Fig. A, suggesintegration between DMN and ATN of participant A02 during Stim-Move  are less active in participants A02 and A03 during Stim-Move. Rather, both SMN and VN showed trends of increased flexibility of ATN for both A02 and A03 . Further, when such interaction was present, the task of performing a phantom hand movement showed improved outcome. Our previous work showed improved motor decoding accuracy after sensory stimulation5.Our work shows that DMN is actively interacting with other systems for A01 and A02, while ATN is more active for A02 and A03. We postulate that each individual’s experience with tTENS can explain the differences we observe. For the tTENS-experienced individual, increased recruitment of VN suggests that there could be a process of honing in on the crucial requirements for completing a hand movement task. In particular, the tTENS-experienced individual showed decrease in recruitment of VN. These observations could indicate that the tTENS-experienced individual could focus on the task itself, rather than trying to use the presented information to do the task.When participants were less experienced with tTENS, higher-level processing may be required in the brain to incorporate this reintroduced sensation. For participants A02 and A03, increased ATN activity suggests more processing is exerted between large-scale systems that are not directly associated with a sensory modality. Information on The differences we observed based on experience with sensory stimulation could also be related to the fidelity of phantom sensations provided and the complexity of hand movements each individual selected. Sensation fidelity was not directly studied, but it is likely that our participant with extensive tTENS experience (A01) perceived the phantom sensations better. With increased quality of the perceived sensations, the participant could then better associate the location of the sensation with him performing phantom hand movements. We can not yet infer the causality among tTENS experience, sensation fidelity, and movement complexity. Therefore, further research is needed to link these factors and cortical large-scale system interaction together.8. Regardless, the cortical processing of combined somatosensory and visual inputs need to be understood in the context of upper limb amputation. In our work and within the cognitive neuroscience framework, sensory combination constitutes the merging of multiple sensory signals to form an unambiguous percept39. In the context of sensory stimulation for upper limb prostheses, the added artificial somatosensory information helps an individual perceive the environment through multiple sensory inputs. Here, our results showed the combination of somatosensory and visual inputs was manifested in terms of dynamic modular networks in the cortex. In particular, the interaction pattern we observed between SMN, DMN and VN for our tTENS-experienced participant could be an indication of effective combination of multiple sensory inputs. Our work suggests the possibility of using dynamic modular networks to assess the cortical processing of combined sensory inputs. Future studies on cortical large-scale systems could include simultaneous somatosensory and visual feedback to further explore cortical responses with prosthesis usage.It is important to note that while our experiment included somatosensory and visual inputs, the setup was different from previous work on multisensory stimulation, which focused more on providing tactile and visual feedback to improve behavioral and functional outcomesIn conclusion, investigating the influence of sensory stimulation on large-scale systems could serve as a tool for mechanistic exploration of sensory combination. Insights into how well a stimulation approach promotes high interaction of somatosensory and visual networks could be important for designing effective stimulation strategies for improved prosthesis perception and control.This study was approved by the Johns Hopkins Medicine (JHM) Institutional Review Board. All study experiments were performed in accordance with the relevant guidelines and regulations of JHM and the ethical standards of the Declaration of Helsinki. Each participant provided their written informed consent to participate in this study. Additionally, a written informed consent was obtained from each participant to have their images taken for an online open access publication. No known neurological disorders were found or reported in any of the participants.40.Monophasic square wave pulses were provided to each participant by a beryllium copper probe connected to an isolated current stimulator . Sensory mapping was performed to identify stimulation sites that elicited phantom sensations in participants with upper limb amputations and referred sensations in intact-limb participants. Areas of the residual limb were scanned with the beryllium copper probe, targeting underlying nerve bundles, namely the median, ulnar, and radial nerves. During sensory mapping, an outline of a hand was used for each individual to report the activation regions in the phantom hand. The corresponding stimulation site was marked. Amplitudes of 0.5–2.6 mA, frequencies of 2–4 Hz and pulse widths of 1–5 ms were used as stimulation parameters. Details of the aforementioned sensory mapping procedures and parameters were adopted from our published work41 . A modified version of the Virtual Integration Environment (VIE)  was used to provide visual cues of the selected hand movements17.In each condition, one trial refers to the duration in which the visual cue and/or sensory stimulation was presented for 2 seconds followed by a 4-second delay with ±25% jitter. For A01, each movement cue was presented 10 times in the Pre-Stim condition and 20 times in all other conditions. For all other participants, each movement cue was presented 20 times in each condition. Sensory stimulation during the experiment used monophasic square waves with a frequency of 45–50 Hz, a pulse width of 1–2 ms, and an amplitude of 0.5–2.6 mA based on our previous work42. A fifth-order Butterworth filter was applied with a low frequency cutoff of 0.3 Hz and high frequency cutoff of 50 Hz. A notch filter at 60 Hz was then applied on the bandpass-filtered signals to remove power line artifacts. Independent component analysis was used to further reduce artifacts caused by eye, muscle, or other movements during data collection. The data was segmented into epochs starting from 1 second before the start of each trial and ending at 2 seconds after stimulus onset. The 2 seconds from the stimulus start point  were used for analysis. We focused on the alpha (8–13 Hz) frequency band and the Pre-Stim, Stim-Move and Post-Stim conditions.The sixty-four-channel EEG recordings were processed using the EEGLAB toolbox in MATLAB34, then further divided the source-localized EEG data into 80 regions of interest  based on the automated anatomical labeling (AAL) atlas43. Most ROIs were assigned to one of the five predetermined large-scale functional systems based on the cortical area each ROI belongs to and its functional role with respect to task performance. The five large-scale functional systems that are relevant to our study are: SMN, DMN, VN, TPN, and ATN. The remaining non-categorized ROIs were labelled as “Others” 49 [Supplementary Methods Eq. (1)]. For each trial, a total of 12 time windows formed a weighted multilayer graph, which models the dynamic cortical response to sensory stimulation as functional interdependencies between nodes  on the source-localized data for each trial to compute undirected functional connectivity. The functional connectivity metric was the weighted phase lag index (WPLI), which calculates the phase leads and lags normalized by the imaginary component of the instantaneous phase difference between two time series signals51. Since this algorithm is non-deterministic, we repeated community detection 500 times for each trial in each condition. All metrics computed were averaged over the 500 repetitions.To obtain the functional modules, we used the generalized Louvain (GenLouvain) community detection algorithm [Supplementary Methods Eqs. (2) and (3)]flexibility (F), which computes the ratio of the number of times a node changes its functional module (S), to the total number of temporal windows (T), or 52. Nodes that have low flexibility change their corresponding functional modules less frequently than those with high flexibility throughout a trial during a task53  overall integration, which considers the average integration of nodes in all large-scale systems, and (b) pairwise integration, which considers integration between two specific pairs of large-scale systems [Supplementary Methods Eqs. (4)–(6)]33. The third metric is recruitment, which computes the probability that a node co-occurs in functional modules with nodes from the same large-scale system, thereby describing the connections between nodes within the same large-scale system 33. The recruitment and integration metrics shed light on interaction between and within large-scale systems. We also calculated node-to-system density, which computes the average connectivity strength between a node in one system and all nodes in another system [Supplementary Methods Eq. (9)]54.For each repetition of the community detection algorithm, we computed three main dynamic modular network metrics. The first metric is flexibility. Two-tailed paired t-tests were used to compare: 1) Pre-Stim and Stim-Move conditions and 2) Pre-Stim and Post-Stim conditions. Normality of the dynamic modular network metrics were confirmed using an Anderson-Darling test. The metrics for one condition were averaged for participants with repeated trials. False discovery rate (FDR) with statistical significance level 55.56.Pearson’s correlation coefficients were calculated between tTENS experience and magnitude of percentage change in Supplementary Information.


Clinical standardization and calibration training is recommended to increase the reproducibility of periodontal probing, but its impact on manual periodontal probing outcomes has received little attention. This study examined the reproducibility of manual periodontal probing performed by a periodontist after completion of a comprehensive standardization and calibration training program.A newly-educated periodontist was subjected to an individualized periodontal probing standardization and calibration training program involving approximately 24 total hours of lecture, bench-top, and clinical instruction/evaluation. Satisfactory completion of each portion of the training program required ≥ 95% intra-examiner agreement within 1 mm between initial and repeat measurements, and a ≥ 90% level of exact agreement with measurements by a “gold standard” examiner. The periodontist then evaluated bleeding on probing (BOP) and performed duplicate measurements of probing depth (PD) and the distance between the cementoenamel junction and gingival margin (CEJ-GM) with a manual periodontal probe on 567 periodontal sites exhibiting ≥ 5 mm PD with BOP in 39 adults. Clinical periodontal attachment level (CAL) was calculated for each site as (PD) - (CEJ-GM).Intra-examiner measurement error (the standard deviation for a single measurement) was found to be 0.21 mm for PD, 0.15 mm for CEJ-GM, and 0.26 mm for CAL. Replicate assessments of PD and CAL yielded excellent exact agreement kappa scores of 0.86 and 0.87, respectively. Greater intra-examiner measurement error was found at periodontal sites with more gingival inflammation as measured by higher BOP index scores.These findings demonstrate that a rigorous periodontal probing standardization and calibration training program facilitates acquisition of highly reproducible PD and CAL assessments in moderate to deep inflamed periodontal pockets with a manual periodontal probe. Similar formal hands-on training should be incorporated into dental education programs and clinical research studies to improve the diagnostic performance of manual periodontal probing of the periodontium. Periodontal probing provides the foundation for clinical examination of the periodontium, helping determine periodontal diagnosis and evaluate therapeutic outcomes in patients ,2. HowevDiagnosis of periodontitis is dependent upon reliable periodontal probing. Clinical periodontal attachment level (CAL) provides a clinical approximation of connective tissue attachment to tooth root surfaces . Manual CAL measurements are widely recognized as the “gold standard” for identifying progressive periodontitis in patients . Lindhe To increase the reproducibility of periodontal probing, formal standardization and calibration training , also knHowever, the effectiveness of more extensive and rigorous hands-on training programs is not known. To address this issue, the present study examined the reproducibility of periodontal probing measurements attained by a newly-educated periodontist following the completion of a comprehensive periodontal probing standardization and calibration training program.This study involved a secondary retrospective analysis of periodontal probing reproducibility data from one study site  participating in a previous US Food and Drug Administration (FDA)-approved phase-3 product evaluation of 10% doxycycline hyclate in a biodegradable drug delivery system . The repA newly-educated periodontist with no research experience (author BPF) was the single periodontist examiner for the periodontal probing reproducibility study. Because of his relative clinical periodontal inexperience, no baseline reproducibility assessments of his periodontal probing technique were made. Prior to the start of the probing reproducibility study, he underwent individualized pre-study periodontal probing standardization and calibration training involving approximately 24 total hours of lecture, bench-top exercises, clinical instruction, and evaluation ,14. An iAfter completion of the pre-study standardization and calibration training program, the periodontist examiner conducted periodontal probing reproducibility examinations on 39 systemically-healthy adults  years), who presented with localized to generalized severe periodontitis  . Whe. Whe3]. The clinical examinations were conducted by the trained and calibrated periodontist examiner. Cotton roll isolation and air drying were used to establish a dry field during the clinical examination procedures, with measurement values verbally called out to a data recording assistant for transcription. In order to standardize data collection procedures at all periodontal sites irrespective of their intraoral location, as required by the FDA-approved product evaluation protocol, all clinical measurements at both facial and lingual tooth surfaces were obtained using mirror-assisted indirect vision . The cliij at a site level is estimable based on the difference between replicate measurements dij , and where one assumes measurement errors are independent of patient and site-type, i.e., var (yijk) = σ2.Data analysis was performed using a statistical computer software package . Mean values and the SD of differences between initial and replicate periodontal site assessments were calculated for PD, CEJ-GM distance, and CAL. Intra-examiner measurement error (ME) for each of the probing assessments was estimated by calculating the SD for a single measurement as follows :(ME)=σKappa statistics were used to quantify intra-examiner agreement beyond chance for site-based replicate assessments of PD and CAL . Exact kThe influence of gingival inflammation on intra-examiner reproducibility of PD, CEJ- GM distance, and CAL was evaluated by comparing their reproducibility parameters across increasing BOP index scores.Patients in the lowest and highest quintile of patients ranked by the total number of periodontal sites/patient subjected to replicate evaluations were also compared relative to agreement attained within 1 mm for replicate PD, CEJ-GM distance, and CAL measurements.A total of 567 periodontal pockets demonstrating ≥ 5 mm PD and BOP were evaluated in the probing reproducibility examinations, of which 468 (82.5%) were located on interproximal tooth surfaces, with each of the 39 study patients contributing 7–20 periodontal sites from at least two dentition quadrants . Among oAmong specific tooth surfaces, lingual surfaces generally yielded lower intra-examiner measurement error values and higher kappa scores, as compared to buccal tooth surfaces for both PD and CAL  and 3.All PD and CEJ-GM replicate measurements, and all but two CAL replicate measurements (one in each patient group), were within 1 mm of each other in patients where only a few periodontal sites were subjected to replicate evaluations , as well as in patients where a higher number of periodontal sites were scored twice .The present study findings demonstrate that a manual periodontal probe visually read to the nearest whole millimeter may provide highly reproducible PD and CAL measurements on moderate to deep inflamed periodontal pockets when employed by a rigorously trained and calibrated periodontist. These findings agree with and extend previous studies of manual probes ,20–24, wA remarkably low SD for a single CAL assessment of 0.26 mm (intra-examiner error) was attained in the present study. This value is strikingly better than the SD of single CAL assessments, as calculated by Yang et al. , that raSeveral reasons may account for the markedly better CAL reproducibility achieved in the present study as compared to previous investigations of manual periodontal probes –24,26,27It is additionally possible that the periodontist recalled initial measurement values when performing replicate evaluations, particularly in patients where a small number of periodontal sites were examined twice. However, the standardized examination protocol, where assessments of other parts of the dentition and at least 15 minutes transpired between replicate site measurements, helped mitigate against this possibility. The high probe reproducibility found in patients with many sites scored twice , was similar to patients where replicate evaluations were performed on only a few sites , suggesting that examiner performance in the present study was not predominately due to recall bias. However, it remains to be established if other dental professionals can attain and maintain over time similar levels of probing reproducibility when exposed to the standardization and calibration training program employed in the present study. If so, then longitudinal monitoring of CAL in clinical practice and periodontal research studies may be reliably performed with a manual probe, instead of a controlled-force probe with an acrylic occlusal reference stent, since comparable levels of intra-examiner error are found between them . BetImportantly, the excellent probe reproducibility attained by the periodontist in the study patients corresponded as well or better with reproducibility levels achieved in the pre-study standardization and calibration training program. This supports the concept that examiner performance attained during standardization and calibration training is a critical determinant of a clinician’s subsequent reliability in making accurate and consistent periodontal measurements in clinical practice settings and research studies.Of the clinical components used to calculate CAL, there was less intra-examiner error for a single measurement of the CEJ-GM distance 0.15 mm) than for PD (0.21 mm), even though the CEJ may be difficult to locate in subgingival and interproximal locations 5 mm than,26. HoweThe increased degree of reproducibility in PD and CAL measurements on lingual as compared to buccal tooth surfaces in the present study  and 3 isA newly-educated periodontist, after completing a rigorous periodontal probing standardization and calibration training program, was able to obtain highly reproducible PD and CAL assessments in moderate to deep inflamed periodontal pockets using a manual periodontal probe. Similar formal hands-on training should be incorporated to a greater extent into dental education programs and clinical research studies to improve the diagnostic performance of manual periodontal probing of the periodontium.


Our study investigated the feasibility to measure pressure profiles inside the inflation balloon during direct implantation of Edwards Sapien 3 ultra-prostheses using an additional syringe with a digital pressure read-out. Pressure profiles of 15 patients for 26 mm valve size were analyzed. Uniform patterns were found for 5 patients similar to those of previously acquired in vitro curves. 10 patients showed strikingly different pressure profiles compared to the above-mentioned group, marked by an earlier pressure increase, single or multiple pressure drops or higher overall pressure. Measuring the percentage of under-expansion of the prostheses, using calibrated angiographic projections revealed a significant difference between both groups. Our data raises the hypothesis that the acquisition of pressure profiles might help to better understand not only the implantation procedure itself but also the highly individual patient-device interaction, offering new information and a new perspective on optimization of TAVR implantation in the future. Measurements were made using a standardized iso-center calibrated angiographic projections and a pigtail as size reference. The pressure profiles and peak-pressures are reflecting the heterogenic pathology and patient population of aortic stenosis, where a multitude of factors influence the outcome. Like coronary stents, where malaposition affects long-term results, under-expansion of the aortic valve stent [Balloon-expandable transcatheter aortic valves have shown excellent results, even surpassing surgical aortic valve replacement in short-term observation . Yet, thImplantation via fixed volume inflation or judging the amount of volume manipulation manually might prove inadequate to address these points in such a heterogenic pathology, where the amount of calcification is not the only determining factor of stenotic severity and where other factors contribute to maximum inflation pressure.As demonstrated by the pressure profiles in our study, using solely the exerted pressure to guide the implantation would have led to suboptimal results. An inflation pressure caped at 5.5 ATM for 26 mm valves, as proposed by Snir et al. [A high effective orifice area remains a hallmark for any valve replacement. As the diameter (d) and the maximal effective orifice area have a quadratic correlation (Our study for the first time describes different pressure/time profiles during TAVR, suggesting that measurement of pressure profile during implantation is feasible. Our data raises the hypothesis that the acquisition of pressure profiles might help to better understand not only the implantation procedure itself but also the highly individual patient-device interaction, offering additional information and a new perspective on optimization of TAVR. Further studies are warranted to investigate whether such profiles and their interpretation can identify and correlate with acute clinical events such as annular rupture as well as events impeding long-term clinical outcome such as PVL, pacemaker-implantation, or hemodynamic performance affecting long-term valve durability.


Atezolizumab is a programmed death-ligand 1 (PD-L1) targeted antibody that prevents the binding of PD-L1 to specific T-cell receptors, thereby increasing anticancer immunity. It has been regarded as a useful first-line treatment in patients with small-cell lung cancer with a more tolerable side effect profile than chemotherapeutic agents. However, few studies focusing on the severity of adverse effects from immune checkpoint inhibitors (ICPI) have been previously reported, particularly acute fulminant colitis requiring surgical invention. We report a case of fulminant colitis refractory to high dose corticosteroid treatment in a patient with known ulcerative colitis (UC) undergoing treatment for small-cell lung cancer (SCLC) with atezolizumab. The upregulation of PD-L1 expression in patients with ulcerative colitis may play a significant role in an imbalanced T-helper cell response creating a pro-inflammatory state. The use of ICPIs to treat SCLC has been reported to increase the risk of developing inflammatory colitis. Atezolizumab use in a patient with known inflammatory bowel disease (IBD) may predispose this population to a higher risk of developing severe inflammatory colitis. We present an unusual complication associated with medical intervention in an immunocompromised patient without an established pathophysiology. The suspicion of using ICPIs in patients with IBD as a potential cause for the development of fulminant colitis is relevant and essential in the diagnostic workup for this patient population complaining of significant gastrointestinal symptoms. Atezolizumab is an IgG monoclonal antibody used to treat several different cancers including breast, bladder, and hepatocellular carcinomas, as well as, small cell and non-small cell lung cancer . This anWe report a case of a 59-year-old female with a medical history significant for poorly controlled ulcerative colitis treated with adalimumab, mesalamine, and intermittent courses of intravenous (IV) corticosteroids with poor success and a prolonged history of tobacco dependence with diagnosed chronic obstructive pulmonary disease (COPD). The patient was subsequently diagnosed with metastatic small-cell lung cancer (SCLC) with bone metastasis, confirmed by endoscopic ultrasound/fine needle aspiration and positive emission tomography (PET) scan after complaining of several weeks of worsening cough, shortness of breath, malaise, and an unintentional 15 lbs weight loss.The patient completed five cycles of chemotherapy with carboplatin, etoposide, and infliximab over a three-month course, with no further progression of her disease process. The patient’s UC was controlled at this time and she was subsequently started on a twelve-week course of atezolizumab. She developed acute onset of generalized abdominal pain and hematochezia approximately four weeks into her twelve-week course of atezolizumab and was started on vedolizumab with resolution of her hematochezia. At the conclusion of twelve weeks, the patient was complaining of worsening generalized abdominal pain. At this time, she was started on infliximab with no resolution of her symptoms and ultimately underwent a colonoscopy with biopsy confirmed moderate pancolitis characterized by erosions, erythema, granularity, and shallow ulcerations from the anus to the cecum. The patient developed bloody bowel movements and her abdominal discomfort continued to worsen post procedure and was started on oral prednisone with no symptom improvement and she presented to the emergency department (ED) at our hospital.Upon presentation to the ED her hemoglobin (Hgb) was within normal limits at 12.9 g/dL. A CT of the abdomen/pelvis with PO contrast showed pancolitis as demonstrated in Figure The patient underwent an exploratory laparotomy, total abdominal colectomy, and end ileostomy creation. Intraoperative pathology specimens were positive for chronic, active mucosal inflammation, acute cryptitis and crypt abscesses, and reactive/regenerative glandular epithelial changes shown in Figure Immunotherapy using ICPIs is becoming more common to treat solid tumor cancers . There iWhile atezolizumab and similar ICPIs have less complications and a higher survival rate than chemotherapeutic treatments, it is not without immune-related adverse events (irAE) . PD-L1 iThe mechanism(s) associated with the development of inflammatory colitis remains unclear . HoweverThere is no previous literature on patients treated with PD-L1 inhibitors who suffered pancolitis refractory to corticosteroid therapy resulting in a pancolectomy. Whether or not the patient’s prior atezolizumab therapy contributed to colonic inflammatory injury is difficult to determine; however, she had 12 of total doses of atezolizumab. To implicate atezolizumab in the pathological process in our patient is largely speculative. Nevertheless, it is certainly within reason to consider PD-L1 inhibitors capable of producing such an acute, dramatic histological, and clinical outcome, especially when considering the mechanism of action. Our patient developed fulminant pancolitis requiring surgical intervention following 12 recent atezolizumab doses. She was treated with high-dose corticosteroids after a colonoscopy confirmed the findings of fulminant colitis. Fulminant, refractory colitis should be considered a potential complication and included in the differential diagnosis when assessing GI complaints in patients undergoing immune checkpoint inhibitor therapy. Further research is needed to determine whether ICPI therapy should be considered an absolute contraindication in patients with uncontrolled or poorly controlled colitis.