In this research, we constructed the Human IRES Atlas database for a comprehensive understanding of cellular IRESs in humans. First, currently available and suitable IRES prediction tools  were used to obtain transcriptome-wide human IRESs. Then, we collected eight genres of translation initiation–related features to help study the potential molecular mechanisms of each of the putative IRESs. Three functional tests  were devised to evaluate the functionality of the identified putative IRESs. Moreover, an easy-to-use interface and an IRES–translation initiation interaction map for each gene transcript were implemented to help understand the interactions between IRESs and translation initiation–related features. Researchers can easily search/browse an IRES of interest using the web interface and deduce testable mechanism hypotheses of human IRES-driven translation initiation based on the integrated results. In summary, Human IRES Atlas integrates putative IRES elements and translation initiation–related experiments for better usage of these data and deduction of mechanism hypotheses.It is now known that cap-independent translation initiation facilitated by internal ribosome entry sites (IRESs) is vital in selective cellular protein synthesis under stress and different physiological conditions. However, three problems make it hard to understand transcriptome-wide cellular IRES-mediated translation initiation mechanisms: (i) complex interplay between IRESs and other translation initiation–related information, (ii) reliability issue of Database URL: http://cobishss0.im.nuk.edu.tw/Human_IRES_Atlas/ Cells in proliferation adapt their metabolism to environmental stimuli through the regulation of protein synthesis, and translational regulation can provide instant protein level adjustment for handling sudden environmental changes and stress responses . Importatrans-acting factors (ITAFs) to remodel their structures, thus opening the hindrance to ribosome recruitment. Second, upstream open reading frames (uORFs) in the neighborhood of an IRES can retain ribosomes in the uORF regions to transform the IRES structure into an active form using amino acid stress. The resulting active conformed IRES then helps presume the cap-independent translation initiation. Third, IRES can modulate different regulation signals around 5ʹ UTR and recruit internal ribosomes accordingly. Some known translational regulation signals such as cap inhibition, uORF translation, ITAFs or RNA G-quadruplexes can trigger the bypassing of canonical cap-dependent translation initiation. These basic known cellular IRES functions provide clues to select the transcriptome-wide IRES sequences in humans.Currently known cellular IRES elements are mostly found in genes involved in stress, mitosis or apoptosis . They usin silico methods to first narrow down the search space. As a consequence, studying IRES in humans requires the following two advances: (i) evaluate the functionality of the in silico predicted putative IRESs  scores and conditional translation efficiency. For elucidating the complex connections between IRES elements and translation initiation–related features, we integrated eight types of datasets that probe vital translation initiation information to help biologists infer possible IRES-mediated translational regulatory mechanisms. For analyzing the interconnections between IRES and translation initiation–related features for individual gene transcripts, an easy-to-use interface and interaction maps were constructed. Human IRES Atlas provides the functionality to search for an IRES of interest or browse the human transcriptome for putative IRES lists satisfying the user-customized filtering criteria. In summary, this database is designed in an integrative manner for simultaneous consideration of putative IRESs and translation initiation features. Many critical observations and testable mechanism hypotheses can be retrieved from this integration. Human IRES Atlas is available online at P-values. Third, eight genres of translation initiation–related datasets were collected and integrated into the database to facilitate biologists to form testable mechanism hypotheses for IRES-driven translational regulation  in their model training process . Hence, et al.  to systeIt is known that the false discovery rate of the prediction process can be reduced if a potential IRES sequence is identified by more than one IRES screening tool . Hence tAlthough most well-known cellular IRESs were found in the 5ʹ UTRs, some IRESs in CDSs and 3ʹ UTRs were also reported to drive the translation of novel proteins or protein isoforms . IRES scEight genres of translation initiation feature data were gathered and integrated into the database to assist biologists in understanding the interplay between IRES elements and translation initiation. The integration also helps users propose testable molecular mechanism hypotheses for IRES-driven translational regulation. The following contents were collected and integrated in this repository: translation initiation site (TIS) probing experiments, uORF sequences, sequence conservation scores , ribosome profiling data, structure information of the IRES elements, experimentally identified ITAF target sequences, IRES activity bicistronic measurements and literature-curated IRES verification experiment evidence. Details of the deposited datasets are described in the following subsections.In Human IRES Atlas, the putative human IRES elements were systematically identified by computational methods. To form reliable IRES-driven translation initiation mechanism hypotheses, we are required to understand the functionality of these putative IRES identifications. Recent studies on cellular IRES have revealed that known human IRES elements are rich in their GC contents to form some special ITAF interacting sequences or structures , 22. HowP-value against the hypothesis that an IRES element of interest is more conserved than its corresponding transcript in phastCons conservation scores. Multiple hypotheses testing bias was corrected by the FDR control approach for the calculated P-values among all IRES elements.Functional elements are often evolutionary conserved among different closely related species , 32. HenP-values among all IRES elements.Translation initiation in eukaryotes proceeds in a series of complicated steps that lead to the final ribosome complex formation. Hence, the ribosome-binding sites located within the 5ʹ UTR sequences under different cellular conditions can reveal important translational regulation hints for active translation programs . The expStructural features have been found in several well-studied cellular IRESs , 45. Altet al. . The RPI scores among all putative IRESs in Human IRES Atlas were corrected by the FDR multiple hypothesis control approach.Structurally similar non-coding RNA (ncRNA) usually carry out similar roles in RNA–protein complexes. Thus, proteins showing interactions with them tend to work in similar biological processes. For estimating the tendency of a given RNA candidate structure to interact with a protein, we resorted to the human ncRNAs having verified RPIs curated in the RNAInter database . Specifiet al. . The defet al. . In brieCellular IRESs in eukaryotes are reported to cooperate with specialized RNA-binding protein called ITAFs in the regulation of the ribosome translation machinery , 23. By et al. -oligos-eGFP library, representing a lack of identification confidence. Other normal real numbers represent the extent of IRES activity of the regulatory sequences. When values larger than the background signal are reported for a given identified IRES, subsequent verification experiments to rule out cryptic promoters and splicing activity can be designed to confirm the existence of IRES functionality. In total, we collected and mapped the probed human oligo sequences with IRES activity measurement to 39 700 transcript segments  in this database.Recently, Weingarten-Gabbay et al.  devised et al.  for the functional test(s), and the database will show only the potential IRES elements with significant q-values. The resulting IRES list can be downloaded in the browse page. (iii) Transcript detail page: after selecting the transcript of interest in the query results, a detailed translation mechanism deduction page is provided. In this page, the detailed IRES summary and translation initiation–related features are integrated to help understand and infer the testable mechanism hypotheses for each IRES. (iv) Download function: the integrative IRES–translation initiation interaction map and the supplemented experimental datasets can be downloaded as plain text files for subsequent analyses. An easy-to-use web interface was built to facilitate these functions.In Human IRES Atlas, four essential database functions are implemented to help select IRES elements of interest: (i) search function: users can search the putative IRESs for different transcripts of a given gene. (ii) Filter and browse function: users are allowed to browse the deposited transcriptome-wide human IRESs categorized by chromosomes or KEGG pathway annotations of genes regulated by them. The KEGG pathway annotation helps users identify the IRES elements that regulate genes involved in the same biological pathway. This criterion helps biologists check if a particular biological pathway consists of different potential IRES regulation mechanisms. To enable users to select only the putative IRESs with significant cis uORFs and TISs to mediate their activity   is the gene that transcribes and translates to a protein that acts as an autocrine/paracrine growth factor in lung branching morphogenesis , bone de83E-13*) . In the 83E-13*) , suggest83E-13*) . A link or users .By careful consideration of the integrated information, we can deduce that FGF9 transcripts could be translationally regulated through the interplay between the structural IRES, uORF segments and ITAFs see . By the Human IRES Atlas was created for two primary purposes: (i) to provide comprehensive and systematic IRES identifications in the human species and (ii) to integrate IRES elements and translation initiation–related information for biologists to understand the interconnections between them. By constructing a comprehensive IRES–translation initiation interaction map, biologists can get testable hypotheses on cellular IRES elements and devise subsequent verification experiments. Currently, four existing databases and datasets are devoted to similar tasks as Human IRES Atlas and thus are compared in the following discussion.in silico IRES identification and construct a comprehensive IRES–translation initiation interaction map to complement the sparsity of IRES verification experiments.Two IRES databases [IRESite  and IRESAnother related work called IRES Omnibus  studied et al.  of 0.037. The putative IRESs deposited in Human IRES Atlas have a sensitivity of 0.32 and an FPR of 0.226 on the ground truth dataset. We can see from these results that Human IRES Atlas achieves a better sensitivity with a small sacrifice of the FPR. Furthermore, if we merge the potential IRESs in Human IRES Atlas and the results of Weingarten-Gabbay et al., a sensitivity of 0.43 and an FPR of 0.258 are observed. The near linearly independent sensitivity gain means that the two results are mostly complementary. By examining the data of Weingarten-Gabbay et al., 19% of the literature-curated IRESs were found. Thirty-two percent of the literature-curated IRESs could be determined in Human IRES Atlas. While oversampling 5ʹ UTRs can lead to higher chances of overlapping with the true positive set, it also increases the possibility of overlapping the putative IRESs with the true negative set, raising the FPR. There is a compromise between the sensitivity and the FPR when oversampling 5ʹ UTRs. The putative IRES elements deposited in Human IRES Atlas are estimated to have a higher sensitivity than the corresponding FPR. Hence, Human IRES Atlas provides extra insights for IRES-mediated translation regulation in humans.The study by Weingarten-Gabbay et al.  is anothIRESbase  and the   were used for systematic IRES screening. The results of different tools were collected by selecting the nested consensus minimal sequences among different prediction algorithms. Hence the prediction uncertainty and false discovery rate inherent in different tools were minimized during the identification process. To further help evaluate the functionality of the putative IRESs, three tests  were designed and performed. Besides performing the transcriptome-wide human IRES identification, we also integrated eight categories of translation initiation–related datasets. Biologists can further understand the IRES-driven translation regulation and develop testable molecular mechanism hypotheses for subsequent research through the translation initiation–related features. In summary, Human IRES Atlas integrates more data and provides more functionality than existing tools. It specifically aims to perform systematic IRES identification and help deduce the IRES-driven cellular translation regulation mechanisms.in silico IRESs and various translation initiation–related data. Based on the eight gathered categories of translation initiation–related features, we can assess the significance of the putative IRESs and deduce potential IRES-driven translation regulation mechanism hypotheses. However, several issues may exist when interpreting the results generated from the systems-wide data integration process. Researchers usually perturbed cells and conducted distinct high-throughput experiments for gathering genome-wide information (The Human IRES Atlas database is constructed by depositing systematically identified ormation . Howeverormation . Hence, Diverse statistical analysis strategies were applied to the high-throughput experiment datasets. For example, we adopted the eTIS data by thresholding the positional read-count fold change and some other minimum detection requirement between samples treated with the ribosome E-site translation inhibitors CHX and LTM . The higin vitro based on experiments on sequence libraries. Non-canonical TIS and uORF information were identified in silico by investigating comparative genomics and sequence contents. These data reveal the normal status of the translation program. To assist in cell type-specific or condition-specific IRES verification experimental design, one can refer to ribosome profiling experiments conducted under different conditions/cell types or refer to special cellular conditions where the ITAF-binding events were observed. Diverse ribosome profiling sequencing results are fragmentary in the literature. Recently, a database called HPRDviewer (Data integration in multicellular organisms often brings about the issue of cellular systems bias. In metazoa species, correct temporal and spatial gene expression of the same genome is the core control on cell differentiation . While dRDviewer  is constin silico identified IRESs from the human transcriptome. Besides the transcriptome-wide putative IRES identification, eight genres of translation initiation–related datasets were integrated and three novel IRES functionality tests were designed. To facilitate the understanding of the connections between IRES elements and these translation initiation–related features, an easy-to-use interface for the integrated results was implemented for biologists to infer possible molecular mechanisms of human IRES-driven translation initiation. Researchers can easily search the IRES of interest or browse the IRESs on genes involved in specific biological pathways with the database’s user-friendly web-interface. By simultaneously studying the human IRES and the translation initiation–related features, the database can provide testable molecular mechanism hypotheses on human gene translation regulation. We believe that Human IRES Atlas can help biologists better study mechanisms of cellular IRES-driven translation initiation, IRES-related cancer progression and other IRES-related genetic disorders.In this research, a novel database called Human IRES Atlas was constructed to deposit the systematic


Matrix metalloproteinases (MMPs) were considered as targets for the treatment of various cancers. However, initial trials using broad inhibitors to MMPs have failed, partly attributed to the contrasting functions of these proteases acting as tumor promoters and suppressors, among other reasons. Our data now suggest that specific inhibition of MMP14 might represent a more specific approach, as loss of this protease in fibroblasts resulted in reduced growth of grafted melanomas. Here, we found that deletion of MMP14 in fibroblasts generates a matrix-rich environment that reduces tumor vascularization and melanoma cell proliferation. In in vitro and ex vivo assays, we showed that the latter is mediated by stiffening of the tissue due to collagen accumulation. Additionally, in vivo, we show that independently of MMP14 deletion, a collagen-rich stiff matrix inhibits the growth of melanomas.Sf−/−) leads to a fibrotic skin phenotype with the accumulation of collagen type I, resulting from impaired proteolysis. Here, we show that melanoma growth in these mouse fibrotic dermal samples was decreased, paralleled by reduced tumor cell proliferation and vessel density. Using atomic force microscopy, we found increased peritumoral matrix stiffness of early but not late melanomas in the absence of fibroblast-derived MMP14. However, total collagen levels were increased at late melanoma stages in MMP14Sf−/− mice compared to controls. In ex vivo invasion assays, melanoma cells formed smaller tumor islands in MMP14Sf−/− skin, indicating that MMP14-dependent matrix accumulation regulates tumor growth. In line with these data, in vitro melanoma cell growth was inhibited in high collagen 3D spheroids or stiff substrates. Most importantly, in vivo induction of fibrosis using bleomycin reduced melanoma tumor growth. In summary, we show that MMP14 expression in stromal fibroblasts regulates melanoma tumor progression by modifying the peritumoral matrix and point to collagen accumulation as a negative regulator of melanoma.Skin homeostasis results from balanced synthesis and degradation of the extracellular matrix in the dermis. Deletion of the proteolytic enzyme MMP14 in dermal fibroblasts (MMP14 The tumor microenvironment (TME) consists of numerous different stromal cell types, such as fibroblast and cancer-associated fibroblasts (CAFs), vascular and immune cells, and a scaffold of the extracellular matrix (ECM) . Cross-cThe non-cellular part of the TME is a dynamic structural network that consists of a variety of extracellular molecules, including collagens, elastin, fibronectin, laminins, and proteoglycans. These proteins are secreted, deposited, and remodeled mostly by fibroblasts, thus ensuring tissue homeostasis or providing a tumor microenvironment supporting tumor growth. Besides its supportive function, the ECM serves as a scaffold for tissue and cells and regulates cellular processes such as proliferation, cell death, migration, or differentiation in physiological and pathological conditions . The comSf−/−), we demonstrated that MMP14 is the major collagenase in skin homeostasis -thymidine, and then recognized by an antibody conjugate. Cell proliferation is shown as average amount of incorporated BrdU into the DNA of proliferative cells measured at 450 nm wavelength.B16F1 cells were grown in serum-free DMEM for 24 h before plating in pre-coated 96-well plates for a further 24 h. Cellular proliferation was analyzed using the Cell Proliferation ELISASf−/− mice and followed the tumor growth over time. Tumors formed in MMP14Sf−/− mice were significantly smaller than in control animals  coated with either low (0.03 mg/mL) or high (0.3 mg/mL) concentrations of collagen type I. Fibronectin (0.01 mg/mL) was used as a positive control. After 48 h, melanoma cell proliferation was analyzed by quantitating the levels of PCNA a and thrTo ascertain whether the environment’s structure would differently affect melanoma cell proliferation, we cultured melanoma cells as spheroids in a three-dimensional fibrillar collagen gel. We used increasing collagen type I concentrations  and Matrigel as a positive control . On day To confirm that increases in collagen and tissue stiffness levels affect melanoma development in vivo, independently of MMP14 deletion in fibroblasts, we undertook a different in vivo approach. We induced in wild-type mice the formation of fibrotic, collagen-rich skin areas by daily intradermal injections of bleomycin or NaCl as control over four weeks. Melanoma cells were injected intradermally into the fibrotic skin and tumor growth was followed over time . We founSf−/− mice model provided us with the great opportunity to directly ask whether loss of MMP14 and the arising resulting collagen-rich stiff ECM promotes or inhibits the growth of melanomas in vivo. Melanomas grafted in this environment showed lower growth rates than controls and did not display altered infiltration of inflammatory cells. Using in vitro invasion systems devoid of cellular activities, we determined that the structural peritumoral environment generated in the absence of MMP14 in fibroblasts has an inhibitory effect on the growth of melanomas.The homeostatic balance between ECM synthesis and degradation is altered in many skin diseases, including cancer. Several proteases contribute to matrix breakdown, including the matrix metalloproteinases (MMPs) produced by either tumor or stromal cells . MMP14 iSf−/− mice tumors compared to control mice. It is possible that after tumor growth increased and many more cells entered the tumor microenvironment, tumors partly overcame the restraints of the tissue by loosening the ECM and dissipating stiffness around the tumors. Such a function was shown to be controlled by the contractile and remodeling activity of cancer-associated fibroblasts [We detected increased stiffness in the skin and the peritumoral areas of early but not late MMP14roblasts ,39. Usinroblasts . The exproblasts ,41,42.Sf−/− mice, we detected increased collagen content, however the amounts and pattern of collagen cross-links were not altered compared to controls. Comparably, the two enzymes mediating this process at different levels, LH2 and LOX, were not changed. Although we did not detect alterations in the collagen cross-links in the MMP14Sf−/− mice, increased matrix stiffness may result from increased collagen or collagen cross-linking through non-enzymatic glycation [Sf−/− mice may also be due to increased matrix density in peritumoral areas. These data suggest that the effects of matrix density and cross-linkage can have effects independent of each other.During cancer progression, especially in many epithelial types, increased peritumoral collagen accumulation and enhanced cross-linking lead to stiffened tissue ,43. In slycation . These tlycation . As the lycation . Along tSf−/− mice could contributed to altering the mechanical properties of the ECM independently of enhanced cross-linkage. These structural alterations may also hinder cell migration [Sf−/− and control mice dermis samples. This fact was not surprising, as melanoma cells can express MMP14 and navigate small paths generated in the ECM.In agreement with our previous data, the increased collagen density in the skin of MMP14igration ,47. HoweOn the contrary, the expansion of tumor cells from the nests was impaired. These data would suggest that the growth of melanoma cells and the development of tumor nodules formed within the matrix need support from an active stroma providing stimuli or larger paths favoring outgrowth in the surrounding matrices. In support of these possibilities, it was shown that the cross-talk with the tissue stroma generates tracks of proteins that support collective migration through heterologous cell–cell contacts with stromal fibroblasts . FurtherWe have shown that increased collagen density and tissue tension resulting from MMP14 deletion in fibroblasts leads to melanoma growth inhibition. However, it is unclear whether this is the direct consequence of deletion of MMP14 or whether an environment with high collagen, matrix density, and altered mechanics levels would be sufficient to inhibit melanoma growth. To date, although collagen and tissue rigidity are documented players in cancer progression, the role of these factors in the melanoma environment is quite controversial ,34,36. HThe negative effects of increased collagen density in melanoma growth in mice is supported by different observations made in patients. For example, human desmoplastic melanoma is characterized by an abundant fibrous matrix and a more favorable prognosis than nodular melanoma with low matrix accumulation . FurtherAltogether, our data demonstrate that loss of MMP14 activity in fibroblasts leads to enhanced tissue density and tension, thus inhibiting melanoma growth by attenuating angiogenesis and tumor cell proliferation. Furthermore, these data highlight the negative roles of collagen and stiffness in melanoma growth.


Here, we utilized our previously described human stem cell-derived neural model to investigate the effect of lyssavirus infection on microRNA (miRNA) expression in human neural cells and their secreted exosomes. Conventional differential expression analysis identified 25 cellular and 16 exosomal miRNAs that were significantly altered  in response to different lyssavirus strains. Supervised machine learning algorithms determined 6 cellular miRNAs  were indicative of lyssavirus infection (100% accuracy), with the first four miRNAs having previously established roles in neuronal function, or panic and impulsivity-related behaviors. Another 4-miRNA signatures in exosomes  can independently predict lyssavirus infected cells with >99% accuracy. Identification of these robust lyssavirus miRNA signatures offers further insight into neural lineage responses to infection and provides a foundation for utilizing exosome miRNAs in the development of next-generation molecular diagnostics for rabies.Despite being vaccine preventable, rabies (lyssavirus) still has a significant impact on global mortality, disproportionally affecting children under 15 years of age. This neurotropic virus is deft at avoiding the immune system while travelling through neurons to the brain. Until recently, research efforts into the role of non-coding RNAs in rabies pathogenicity and detection have been hampered by a lack of human  Rabies virus  is among the most lethal viruses that invade and infect the central nervous system (CNS), leading to an estimated 59,000 deaths annually worldwide . As rabivia the peripheral nervous system in an asymptomatic pre-clinical phase, typically taking between two weeks and three months to occur  has proven efficacy in combatting the infection in the pre-clinical phase . Whilst An absence of diagnostic tests for pre-clinical rabies infection further compounds the difficulty treating and managing the risk of clinical disease onset. The virus can efficiently evade antigen presentation to the immune system during replication and transit between neurons , so tradin vitro and 24 hours in vivo when challenged with Hendra virus) (MicroRNAs (miRNAs) are central players in cellular development and function. They have established roles in brain development and neuronal communication and are important in the development of neurodegenerative disorders . These sa virus) . By exama virus) .Recent studies have highlighted miRNAs as potential biomarkers for a wide range of infectious diseases, including viral infection . MicroRNFurthermore, miRNAs are often packaged into exosomes (extracellular vesicles 30-100 nm in size) and their abundance changes in a wide range of disease states, including neurodegenerative and infectious diseases . As exosMicroRNA expression profiling has been documented in mouse brain tissue following infection with rabies virus , althougIn this study we have utilized one of these models to investigate the lyssavirus-induced changes in neural miRNAs, providing insights into the molecular mechanisms of pathogenesis of this deadly neurotropic virus. Using this model, we have also examined virus-mediated miRNAs released in exosomes as a putative source of disease biomarkers for future diagnostic applications. Implementation of bioinformatics and advanced machine learning (ML) techniques in this study have aided the discovery, refinement, and validation of these host biomarkers, and allowed a direct comparison to the conventional differential expression (DE) analysis of miRNA signatures. Together, these novel cell culture and computational platforms have provided a new way of assessing the host-virus response of lyssavirus infection in human neurons and provides a modern appraisal on the validity of miRNA signatures as biomarkers of lyssavirus infection.Human ethics: All work using human pluripotent-derived neural stem and progenitor cells (hNPCs) was carried out in accordance with Australia’s National Health and Medical Research Council (NHMRC) ‘National Statement on Ethical Conduct in Human Research’ , the ‘Australian Code for the Responsible Conduct of Research’ , and with approval from the Commonwealth Scientific and Industrial Research Organization (CSIRO) Health and Medical Research Ethics Committee (LR 16/2017 29/11/2017).HDF51i-509 neural progenitor cells (NPCs) were maintained in culture and terminally differentiated as previously described for at least 25 days . LyssaviImmunoblotting was carried out using the BOLT pre-cast electrophoresis system (Invitrogen), where 20 µl of protein lysate was separated on 4-12% pre-cast NuPAGE Bis-Tris Midi Gels (Life Technologies) and transferred to PVDF (in full) membrane (ThermoFisher Scientific). Immunoblots were blocked with 3% w/v BSA in DPBS and incubated with primary antibodies for 1 hr, before detection with HRP-conjugated secondary antibodies , and an ECL detection kit (Pierce). Primary antibodies: rabbit anti-rabies nucleoprotein  as per the manufacturer’s protocol. Briefly, 500 µl of exosome isolation reagent was thoroughly mixed with 1 ml of cell-free supernatant and then incubated for 16 hr at 4°C. The reagent-supernatant mix was then centrifuged at 10,000 x g for 1 hr to pellet the exosomes. This method results the concentration of exosomes, however a small amount of other non-exosomal contaminants and protein aggregates may also be present , the latTotal RNA from human stem cell-derived neural cells and exosomes was isolated using the miRCURY RNA Isolation Kit-Cell and Plant  following the manufacturer’s protocol. RNA was quantitated using a spectrophotometer. cDNA libraries were constructed using the QIAseq miRNA Library Kit and QIAseq miRNA NGS 48 Index IL (Qiagen) as per the manufacturer’s protocol. For cell samples, 200 ng of total input RNA was used as the template with 16 cycles of library amplification. For exosome samples, 5 µl of eluted total RNA was used as the template with 22 cycles of library amplification. All libraries went through pre-sequencing quality control using the High Sensitivity DNA kit on the Bioanalyzer 2100 (Agilent) to ensure appropriate amplicon insert and minimal adapter carryover. Libraries were analyzed at the Australian Genome Research Facility (AGRF) for 100 bp single-end sequencing using the HiSeq 2500 (Illumina).Pre-processing and downstream analysis was conducted as previously described . BrieflyLyssavirus N protein RNA and 18S rRNA was quantified as previously described . BrieflyStatistical analyses were completed using the SciPy v1.6.0  and scikIn line with previous observations , this ne2 > 0.5), indicating that the degree of viral replication does not have a direct relationship with host miRNA expression. Predictably, there was considerable overlap between the miRNAs detected in the cellular and exosomal fractions, however 31 miRNAs were only found in secreted exosomes  raw reads for the cellular samples, and 15-33 million (average 18 million) raw reads for their corresponding exosome samples . Adapters were removed from the raw reads, and then the reads were subject to filtering based on length and quality, leaving 7-13 million cellular reads (average 9 million), and 3-14 million exosomal reads (average 8 million) for further analysis. A total of 832 mature cellular and 556 mature exosomal miRNAs were identified using miRDeep2  cellular miRNAs were found  after infection with at least one lyssavirus strain compared to uninfected mock controls  significantly altered  miRNAs for lyssaviruses.Several studies have previously examined lyssavirus-mediated miRNA changes in brain homogenates of infected mouse models . HoweverIn this study, we also took the opportunity to compare differing bioinformatics approaches: conventional differential expression (DE) and machine learning (ML) analysis. DE is performed by comparing the expression levels of each miRNA between groups and using a statistical test to assess the probability of any difference happening by random chance. As there is usually a large number of comparisons (832 in the cell samples), stringent p-value corrections are often applied to control false positives. This approach effectively identifies large scale changes but can result in false negatives due to overly harsh p-value corrections. This potentially explains why no DE exosomal miRNAs were identified in human neural cultures infected with SHBRV , usually in terms of accuracy, precision, recall or ROC AUC. Although a ML model can be constructed using a single miRNA, it is more often built using multiple miRNAs, utilizing their combined expression pattern, rather than looking at them individually. This will better take into consideration the complexity of the larger system and enable accurate predictive analysis. Unfortunately, ML can be challenging to implement, and its performance is often dependent on the amount of data available . ML is yet to be become a default methodology within the biological sciences, although it is likely to become more widespread. Indeed, some miRNA studies have already implemented this approach .By applying a multivariate, machine learning (ML) data analysis methodology, we have identified unique cellular and exosomal miRNA signatures of lyssavirus pathology that predict infection with 100% and 99.2% accuracy respectively 4D. ThesUnderstanding the roles of these newly identified miRNAs in the host-cell infection response holds the potential to significantly improve our knowledge of the pathology and pathogenesis of viral disease. Here, ML analysis has highlighted both cell-associated and exosomal miRNAs associated with virus infection in neurons, that were not all detected using DE analysis. MicroRNA-5701  regulates neuronal cell death through mitochondrial-lysosomal cross talk , but takFor example, many of the cellular and exosomal miRNAs identified through ML analysis have been previously associated with neuronal cell death and neurodegenerative disorders. MicroRNA-99b-5p and miR-5701 (ML cell signature) have established roles in neuronal cell death , neuroprWhilst lyssavirus does not induce apoptosis in infected neurons, it has been shown that neurons activate a selective and compartmentalized SARM-1-mediated degeneration of their axons and dendrites in response to infection with different field strains of lyssavirus . This reGABRA6, a gene implicated in the etiology of anxiety disorders  is associated with panic disorder and directly targets the gamma-aminobutyric acid receptor isorders . Overexpisorders . MicroRNisorders . Circulaisorders . Finally in mice  and prom in mice . This mi in mice . TogetheInterestingly, linear discriminant analysis (LDA) of the 16 exosome miRNAs obtained from DE analysis was able to highlight strain-specific miRNA changes, especially when comparing the lab-adapted CVS-11 strain and the field isolates. CVS-11 infection affected the expression of more distinct miRNAs than the field isolates, both within neural cell cultures and the exosomes they secrete 2. This Finally, exosome miRNA signatures have the potential to be explored as biomarkers for novel diagnostic tests against pre-clinical rabies infection. As previously mentioned, Rabies has an unusually long asymptomatic incubation period with little to no early immune activation, which has led to a gap in effective ante-mortem diagnostic testing protocols. Prophylactic treatment is effective but costly, and sometimes inaccessible. A tool to stratify infected patients and justify the cost versus benefit of therapeutic intervention would enable more efficient implementation, particularly throughout the developing world.in vivo murine model of infection as opposed to/compared to our in vitro human neural culture system; serum versus cell culture supernatant sampling of putative miRNAs; low sampling numbers from the animal model which displayed extensive heterogeneity in viral RNA and protein expression in infected groups; and the timing of sampling – 1 week versus 72 hours post-inoculation. On this last point it is interesting to note that  in nerve function, damage and repair responses, the contribution of glial miRNAs to viral pathogenesis and neural system damage also warrants a more detailed investigation. Finding these signatures in accessible body fluids is also an essential step toward the establishment of this miRNA infection signature as a biomarker for in vitro diagnostics.From our study, the four exosomal miRNAs  that were identified could be used to predict lyssavirus infection in the neural cells with >99% accuracy. Whilst this is promising foundational data upon which to develop a novel circulating biomarker, further work is still needed to ensure these miRNAs are detectable None-the-less, whilst optimization of novel biomarker signatures for viral diseases like rabies matures, new technologies are emerging in parallel that enable rapid, cost-effective exosome capture and miRNA detection . IdeallyIn conclusion, this study has brought together protocols and experimental techniques across a range of specialisms  to gain further insight into the neural response to rabies infection. We show that applying a machine learning approach to conventional bioinformatic assessment can improve the accuracy and detail of our datasets. In addition, information obtained from DE and ML analysis provides interesting insights into the molecular role and relationship of miRNA clusters during viral infection. It would be interesting, and pertinent, to examine these miRNA changes in other neurotropic infections to assess their specificity to rabies and their role in wider diseases states. Lastly, we have identified a novel exosomal miRNA signature can independently predict lyssavirus infected cells with >99% accuracy and provides a foundation for utilizing exosome miRNAs in the development of next-generation molecular diagnostics for rabies. Although still in the early discovery stage as an “ideal” biomarker for rabies, further research of this signature, or the methodologies used to find it, may eventually become the foundation upon which diagnostics, improved pre-clinical management and potentially new treatments are identified for the disease.https://www.ncbi.nlm.nih.gov/bioproject/PRJNA765814).The original contributions presented in this study are publicly available at the NCBI Sequence Read Archive using BioProject PRJNA765814  Probing Biosystems Future Science Platform.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.


Personalized therapy of non-small cell lung cancer (NSCLC) relies heavily on histopathological analyses that require invasive biopsies that have relatively high costs, provide limited assessment of tumor heterogeneity and are associated with potentially life-threatening complications. This retrospective study is aimed at evaluating the potential benefit of using predictive models that integrate radiomic features extracted from computed tomography (CT) images and commonly assessed clinical predictors to characterize the overall survival (OS) of stage III NSCLC adenocarcinoma patients receiving chemoradiation. Different than previous studies, our proposed approach explicitly accounts for CT parameter heterogeneity, such as presence or lack of intravenous contrast material and differences in CT scanner vendors through feature harmonization. Using a relatively homogeneous population of 110 patients, our results demonstrate that radiomic biomarkers derived using feature harmonization significantly improved the prediction of OS in our cohort when combined with Eastern Cooperative Oncology Group (ECOG) status and age at diagnosis, suggesting their potential in assisting clinical decision making.) upon the baseline model ). Our results suggest that harmonized radiomic phenotypes can significantly improve OS prediction in stage III NSCLC after chemoradiation.We evaluate radiomic phenotypes derived from CT scans as early predictors of overall survival (OS) after chemoradiation in stage III primary lung adenocarcinoma. We retrospectively analyzed 110 thoracic CT scans acquired between April 2012−October 2018. Patients received a median radiation dose of 66.6 Gy at 1.8 Gy/fraction delivered with proton (55.5%) and photon (44.5%) beam treatment, as well as concurrent chemotherapy (89%) with carboplatin-based (55.5%) and cisplatin-based (36.4%) doublets. A total of 56 death events were recorded. Using manual tumor segmentations, 107 radiomic features were extracted. Feature harmonization using ComBat was performed to mitigate image heterogeneity due to the presence or lack of intravenous contrast material and variability in CT scanner vendors. A binary radiomic phenotype to predict OS was derived through the unsupervised hierarchical clustering of the first principal components explaining 85% of the variance of the radiomic features. C-scores and likelihood ratio tests (LRT) were used to compare the performance of a baseline Cox model based on ECOG status and age, with a model integrating the radiomic phenotype with such clinical predictors. The model integrating the radiomic phenotype ) significantly improved ( Lung cancer is the most common cause of cancer-related deaths in the United States , with ovThe development of accurate models to predict disease recurrence in patients with NSCLC treated with definitive chemoradiotherapy, also known as treatment failure, could play a fundamental role in the efficient personalization of treatment. In fact, previous investigators have utilized clinicopathological data to develop predictive models for treatment failures, demonstrating an ability to predict patterns of failure in NSCLC  and to iStandard of care medical images acquired during lung cancer management has a yet untapped wealth of information of in situ tumor architecture, heterogeneity and peritumoral environment, which may have prognostic implications. Radiomics, a quantitative method for assessing diagnostic images, involves detailed mathematical analyses of the images to determine whether or not particular patterns are observed in the data, which could, optimally, be used to assist in predictive analytics and clinical decision making . In NSCLMultiple prior investigators have assessed the use of radiomics in developing predictive models for NSCLC disease control outcomes. Among the most recent related works is the one performed by Wang et al.  in whichIn this study, we investigate the association between OS and radiomic features extracted from chest CT scans of a relatively homogeneous cohort of stage III NSCLC adenocarcinoma patients at our institution using a predictive pipeline that explicitly accounts for image heterogeneity due to image acquisition protocols, vendors and intravenous contrast material.CT scans were retrospectively analyzed from a set of stage III NSCLC patients previously treated at the University of Pennsylvania. This study was compliant with the Health Insurance Portability and Accountability Act, approved by our Institutional Review Board, and in accordance with U.S. Common Rule (Penn IRB protocol #832329). The inclusion criteria were limited to several parameters: (1) histologically confirmed AJCC 7th Edition stage III NSCLC adenocarcinomas, (2) pre-treatment CT scans within 2 weeks of chemoradiation, and (3) first radiation course to primary tumor in the lung. Patients with pre-operative radiotherapy as well as patients with post-operative CT scans were excluded. An initial cohort of 132 consecutive patients treated from April 2012 through October 2018 at our institution was identified for this study. From the initial cohort, 14 patients were excluded due to incomplete or corrupted imaging files, and 3 more patients were ruled out due to images with collapsed lungs or mediastinal primary tumors that would not allow for precise segmentation. Finally, 5 more patients whose treatment was interrupted early because of death or other adverse events were also excluded from the study. Therefore, a total of 110 patients diagnosed with stage III NSCLC adenocarcinoma patients, with available CT scans and consistently treated with chemoradiation, were included in our analysis. All patients were treated with concurrent or sequential chemoradiation with platinum containing regimens, and either proton beam therapy (PBT) or intensity-modulated radiation treatment (IMRT).For each patient, the primary tumor was manually segmented by one out of three experienced radiologists  from a pretreatment, preoperative CT scan. Images were preprocessed using histogram normalization based on z-scores to compensate for outliers due to artifacts within the image. Additionally, since 65% of the studies have slice thickness of 3 mm, an isotropic spline interpolation to 3 mm was applied to all the images to account for possible differences in image spatial resolution. A set of 107 radiomic features was extracted using the software Pyradiomics 3.0.1 , includiIn this study, we collected a set of continuous and categorical predictors of OS reported in the existing literature. The continuous features were age at diagnosis , body map-value significance level ComBat is a harmonization technique originally developed for genomic studies , which h package  for R 4. package  was usedp-value with We used principal component analysis to reduce the dimensionality of the ComBat harmonized features by selecting the first principal components (PCs) explaining 85% of the variance of the features. After that, we performed unsupervised hierarchical clustering, applying the maximum distance linkage. Consensus clustering was used to identify the optimal number of clusters ) and non-harmonized ) features, were assessed by fitting a Cox proportional hazards regression model and then estimating the C-score associated with the fitted models. The 95% confidence interval of the average C-score was calculated using a total of 50,000 bootstrap replicates assuming the standard t-distribution-based approximation. All the analyses were implemented using the SimpleITK , SciPy , scikit-Combinations of subsets of clinical features were used to fit several Cox proportional hazards model to assess their combined predictive performance. The Cox model, which combined the clinical features with the best performance, was then selected as the baseline model. In addition, to assess the predictive performance of the radiomic phenotypes, we fitted Cox proportional hazards models combining the clinical features of the baseline models with Phenotype (ComBat) and Phenotype (non-ComBat). We also compared the predictive performance of the model combining PCs to the baseline variables, but only considered the first PCs whose hazard ratios (HR) were significantly different from 1 in the corresponding Cox proportional hazards model to reduce overfitting.This analysis corresponds to the development and validation of a predictive model using resampling or analysis type 1b as specified by Collins et al. in . FinallyAs in the univariate analysis, 95% confidence intervals of the average performance measurements were calculated using estimates as bootstrapped samples using the standard t-distribution-based approximation with 50,000 bootstrap replicates. Our proposed pipeline for multivariate survival analysis in stage III NSCLC adenocarcinoma is summarized in The characteristics of the 110 patients in the study are provided in In univariate analysis, the ECOG status with C-score = 0.62 ), is the only clinical feature able to predict systematic survival differences in our cohort. Additionally, Phenotype (ComBat) with C-score = 0.61 ) shows good predictive performance. A detailed heatmap showing the resulting phenotype labels across the patient cohort, as well as its qualitative associations with clinical covariates  and outcomes  is shown in In multivariate analysis, after assessing different subsets of the available clinical variables, i.e., age at diagnosis, BMI, pack per year, race, gender, and ECOG status, the results suggest that the model integrating age at diagnosis and ECOG status  showed the best predictive performance with C-score = 0.65 ), therefore such model was used as the baseline model for comparison. A predictive model integrating such clinical variables with the non-harmonized imaging phenotype  + ECOG + Age) did not exhibit a significant improvement  and Phenotype (ComBat) (C-score = 0.61) were able to predict OS differences in our cohort. On multivariate analysis, we found that, while the model based on ECOG + Age (C-score = 0.65) and the model based on 2 PCs + ECOG + Age (C-score = 0.65) each demonstrated an ability to predict for OS, the predictive performance significantly improved further , we conclude that the improvement in the predictive model is due to ComBat’s ability to add to the consistency of radiomic features across the various scans.Prior reports of the use of ComBat in computational medical imaging to solve classification tasks have been published in the literature, mainly to harmonize images acquired in different clinical centers and under different acquisition protocols in classification problems. Regarding feature harmonization to account for batch effects due to multiple centers, Whitney et al.  used a sRegarding the harmonization of features extracted using different image acquisition devices, Shayesteh et al.  implemenWhile prior reports of the use of ComBat exist for breast cancer , colorecThe previous literature on the implementations of ComBat to harmonize radiomic features suggests that the main causes of batch effects in image datasets are mainly differences in image acquisition protocols, as well as device-associated factors , convolutional kernels, scanner model and manufacturer) ,50,51,52In ival see . Further77)) see . Notice The proposed evaluation method is mainly based on conventional inferential statistics to calculate C-scores for survival prediction, as well as confidence intervals, which are not intrinsically high cost, especially given our modest cohort size (A few limitations exist. First, most patients were treated prior to the initial publication of the PACIFIC trial in late 2017 , which iWe report our results using radiomic features, utilizing ComBat feature harmonization, to predict OS using pre-treatment chest CT scans from a relatively homogeneous cohort of stage III NSCLC adenocarcinoma patients managed with definitive chemoradiotherapy. Our results suggest that imaging phenotypes extracted through unsupervised machine learning using ComBat-based feature harmonization can enhance the predictive performance of well-known clinical predictors of OS, which is particularly relevant to stage III NSCLC management, which in recent years has incorporated novel variants of chemotherapy, radiotherapy modalities and immunotherapies to its treatment. Importantly, the additional use and refinement of this model are critical to be able to incorporate predictive modeling safely and responsibly into the clinical decision making.


Plant rhizobiomes consist of microbes that are influenced by the physical, chemical, and biological properties of the plant root system. While plant-microbe interactions are generally thought to be local, accumulating evidence suggests that topologically disconnected bulk soil microbiomes could be linked with plants and their associated rhizospheric microbes through volatile organic compounds (VOCs). While several studies have focused on the effect of soil physicochemical properties for VOC movement, it is less clear how VOC signaling is affected by microbial communities themselves when VOCs travel across soils. To gain a better understanding of this, we propose that soil microbe-plant communities could be viewed as “metarhizobiomes,” where VOC-mediated interactions extend the plant rhizobiome further out through interconnected microbial metapopulation networks. In this minireview, we mainly focus on soil microbial communities and first discuss how microbial interactions within a local population affect VOC signaling, leading to changes in the amount, type, and ecological roles of produced VOCs. We then consider how VOCs could connect spatially separated microbial populations into a larger metapopulation network and synthesize how (i) VOC effects cascade in soil matrix when moving away from the source of origin and (ii) how microbial metapopulation composition and diversity shape VOC-signaling between plants and microbes at the landscape level. Finally, we propose new avenues for experimentally testing VOC movement in plant-microbe metapopulation networks and suggest how VOCs could potentially be used for managing plant health in natural and agricultural soils. Plant-associated microbiomes have received considerable attention from scientists as key components of plant development, growth, and immunity . In part2–10–14–While several studies have focused on the effect of soil physicochemical properties in governing VOC movement in the soil matrix –19, it i–The microbial activity and plant roots are the main sources of VOCs in the soil , 19. Add2 and O2) (While the rhizosphere gradient size for most biotic and abiotic processes has been reported as 0.5 to 4 mm and >20 mm for inorganic gases (CO and O2) , 40, the and O2) , 41. For and O2) . In anot and O2) , 24. TogVerticillium longisporum fungi upregulated the metabolic activity of Paenibacillus polymyxa, while the VOCs of P. polymyxa inhibited the cellular metabolism and growth of V. longisporum, but upregulated genes related to stress responses and the production of antimicrobial VOCs  but at the same time showed a very high bioactivity to Pythium species (oomycetes)  . VOC bione study  and wither study . These fer study  on VOC-mer study , 22, wouer study  and a reommunity . While Vommunity , signal ommunity .Even though the bulk soil acts as an initial microbial pool for plant rhizobiome, the growth, development, and aging of plants cause clear shifts in rhizobiome composition, making it distinct from the bulk soil . As a reFusarium culmorum, the bacterium Serratia plymuthica has been shown to upregulate the production of sodorifen VOC organisms beyond the rhizosphere environment to at least the centimeter scale as suggested by de la Porte et al. . This coHere, we propose that VOCs could coordinate bulk and rhizosphere soil microbiome functioning as a metarhizobiome, superseding the topological range limitation of contact-dependent microbe-microbe-plant interactions. Such plant metarhizobiomes would include microbes residing in the near physical vicinity of the plants (rhizobiome), as well as the VOC-connected populations located further apart in the bulk soil. Such multidirectional long-distance communication could fundamentally change how we perceive microbial ecology in the spatially structured soil matrix, allowing plant-microbe metapopulations to interact and trade information without restrictions imposed by the proximity and cooccurrence of the same local habitat. The attained knowledge could be potentially further used in the management of plant health in the agricultural context and to understand plant-microbe biodiversity and distribution in natural environments. The proposed predictions put forward by our conceptual framework should be rigorously tested in the future. This could be achieved by bringing together interdisciplinary scientists working on microbial ecology and evolution, genetics, biochemistry, and plant biology and by taking advantage of bespoke experimental systems that allow direct manipulation and quantification of microbe-plant communities and emitted VOCs.


Allogeneic islet transplantation has become an effective treatment option for severe Type 1 diabetes with intractable impaired awareness due to hypoglycemic events. Although current immunosuppressive protocols effectively prevent the acute rejection associated with initial T cell activation in recipients, chronic rejection has remained an obstacle for achieving long-term allogeneic islet engraftment. The development of donor-specific immune tolerance to the allograft is the ultimate goal given its potential ability to overcome chronic rejection and disregard the need for maintenance immunosuppression, which may be toxic to islet grafts. Recently, a breakthrough in tolerance induction during allogeneic islet transplantation using apoptotic donor lymphocytes (ADLs) in a non-human primate model had been reported. Several studies have suggested that the clonal depletion, anergy, and expansion of the antigen-specific regulatory immune network are the mechanisms for donor-specific tolerance with ADLs, which act synergistically to induce robust transplant tolerance. This achievement represents a huge step forward toward the clinical application of immune tolerance induction. We herein summarize the reported operational induction therapies in islet transplantation using the ADLs. Moreover, a few obstacles for the engraftment of transplanted islets, such as islet immunogenicity and instant blood-mediated response, which need to be resolved in the future, are also discussed. Allogeneic pancreatic islet transplantation has been established as an effective option for severe Type 1 diabetes with intractable impaired awareness due to hypoglycemic events. Islet transplantation involves the intrahepatic delivery of donor-isolated islet cells that supplement insulin production, promoting the recovery of endogenous insulin secretion. Evidence has shown that the development of established immunosuppression protocol has improved outcomes of allogeneic islet transplantation . A majorWhen considering the adverse effects of immune suppression, some immunosuppressive drugs can be toxic to islet grafts , which cThe development of donor-specific immune tolerance to an allograft is the ultimate goal of any transplantation given its ability to possibly resolve chronic rejection and disregard the need for maintenance immunosuppression. Inducing donor-specific tolerance in animal transplantation models has been met with several challenges. Nonetheless, the accumulation of information has allowed the recent emergence of several key insights for operational tolerance induction, including the role of regulatory B cells (Bregs) for inducing or maintaining immunological tolerance.As recently as 2019, Sigh et al. reported on a breakthrough in the tolerance induction protocol for allogeneic islet transplantation in non-human primate (NHP) models . This apWe herein summarize the reported operational induction therapies in islet transplantation using the ADL protocol and review an essential mechanism of tolerance induction and maintenance based on the current knowledge gained from experimental animal models. A few obstacles hindering immunological tolerance and transplanted islet engraftment that need to be resolved in the future are also discussed.The immune system can learn to discriminate between the self and nonself via a complex set of central and peripheral immune tolerance mechanisms. When considering immunological tolerance to the allograft, the high proportion of MHC alloreactive T cells, which generally range from 5 to 10% , has beeDuring the immune destruction of the islet graft, the initial process of rejection is characterized by a rapid infiltration of innate immune cells to the grafts, which can be followed by an antigen-specific T cell response. The established immunosuppression protocol incorporating T cell depletion or anti-TNFa monoclonal antibodies could achieve T cell activation in allogeneic islet transplantation, which contributes to enhancing short-term graft survival . HoweverTIM-1, which was initially reported to be a T cell costimulatory marker, is a member of the T cell immunoglobulin and mucin domain family of costimulatory molecules. However, an in vivo mice study by Ding et al. showed that TIM-1 was constitutively expressed on B cells rather than T cells and that 6–40% of TIM-1+ B cells express IL-10, including transitional, marginal zone, and follicular B cells . Thus, rMoreover, a mouse islet allograft model found that anti-CD45RB antibody in combination with anti-TIM-1 antibody had a synergistic effect in inducing tolerance in all recipients. The dual antibody treatment significantly expanded regulatory B and T cells depending on the presence of recipient B cells with IL-10 activity . Using BAlthough several promising approaches for tolerance induction in a rodent model of transplantation have been reported, as discussed earlier, very few have been translated to human or NHP transplantation models. Unlike laboratory mice, NHPs and humans already have a large collection of memory T and B cells at the time of transplantation. Heterologous immunity or the cross-reactive immune response has the potential to be alloreactive. For instance, up to 45% of anti-CMV T cell clones have been reported to be alloreactive . ExistinOne of the most advanced approaches for sustained tolerance is the use of hematopoietic cell transplantation to achieve durable chimerism, where the donor and recipient hematopoietic cells coexist at levels detectable by flow cytometry ,25,26. TBased on the evidence of ECDI-fixed cell-based tolerance induction in autoimmune models, this approach has been applied to transplantation models, including islet transplantation. Studies have shown that pre- and post-transplant infusions of donor splenocytes treated with ECDI induce donor-specific tolerance and prolong graft survival in rodent organ transplantation models  and suppression of post-transplant expansion of alloreactive T cells in recipients . In partConsidering the successful results of the ADL-based tolerance induction protocol, the impact of one DRB-matched ADL infusion on the immune system of recipients warrants discussion. Interestingly, the frequency of circulating cells, such as Tregs, Tr1, and Bregs, which make up the regulatory network, is significantly higher in recipients with one-DRB-matched ADL than in those with fully mismatched ADLs . StudiesPre-existing donor-reactive memory T and B cells and donor-specific antibodies have been recognized as a cause of accelerated allograft rejection in sensitized allotransplantation ,52. SeveIslet grafts may be more likely to be resistant to immunological tolerance induction. As mentioned earlier, several studies on operational tolerance induction reported that the islet transplantation model failed the induction challenge, while the same induction protocol was successful in an organ transplantation model, such as cardiac grafts  or kidneEstimates have shown that roughly 50% of transplanted islets are irreversibly damaged during the peritransplant period, which usually spans from hours to days. Over 25% of transplanted islets are reported to be lost immediately after infusion into the portal vein . A signiThe short-term culture of isolated islets may precondition islet grafts for the improvement of islet transplantation outcomes. As described earlier, multiple proinflammatory cytokines are secreted from islets and act as strong stimulators of the host’s innate immune response. Evidence has shown that these molecules may serve as therapeutic targets for enhancing islet transplantation . HoweverApart from suppressing the cytokine secretion of the islets, preconditioning of the islets may affect the status of tissue monocytes therein. Studies have revealed the significance of donor- and recipient-derived DCs in allograft rejection. Accordingly, donor DCs in islet tissues can migrate into the recipient’s secondary lymphatic tissue and circulation. In fact, Paolo et al. demonstrated the rapid migration of donor DCs to recipient lymphoid tissue as early as 3 h after allogeneic islet transplantation . MoreoveThe peritransplant infusion of ADLs under transit immune suppression is a robust protocol for inducing donor-specific tolerance in islet transplantation. This achievement encourages the belief that immunological tolerance can eventually be achieved in humans. Research must continue to address several unanswered questions, including the impact of ADL on sensitized recipients or recurrent alloimmunity as a cause of allogeneic islet failure in Type 1 diabetes or islet transplant recipients. Given the fact that tolerance induction seems to be more challenging in islet transplantation due to islet-specific factors, including the high immunogenicity of the islet itself and IBMIR, developing protocols for alleviating the systemic inflammatory response provoked by transplanted islets are warranted for the successful induction of immunological tolerance.


The surgical management of deep brain lesions is challenging, with significant morbidity. Advances in surgical technology have presented the opportunity to tackle these lesions.We performed a complete resection of a thalamic/internal capsule CM using a tubular retractor system via a parietal trans-sulcal para-fascicular (PTPF) approach without collateral injury to the nearby white matter tracts.PTPF approach to lateral thalamic/internal capsule lesions can be safely performed without injury to eloquent white matter fibres. The paucity of major vessels along this trajectory and the preservation of lateral ventricle integrity make this approach a feasible alternative to traditional approaches.The online version contains supplementary material available at 10.1007/s00701-021-04884-2. Cavernous malformations (CM) involving deep structures, such as basal ganglia and thalamus, account for roughly 5–10% of cerebral CM , 8. The Following the induction of general anaesthesia, transcranial corkscrew electrodes and peripheral needle electrodes were applied for monitoring the cortico-spinal tract (CST), via motor evoked potentials (MEPs) and the spinothalamic tract via somatosensory evoked potentials (SSEPs), respectively. The patient was then positioned in the right lateral position with the head fixed in a 3-point Mayfield clamp.A horse-shoe incision was made, followed by a 5-cm parietal craniotomy, allowing adequate exposure around a pre-planned sulcal entry point for an optimal trajectory to the lesion. A curved durotomy was performed to expose the left parietal lobe. The pre-planned sulcal entry point was identified using intra-operative neuro-navigation . A 1-cm linear arachnoidotomy, centred at the desired entry point, was made within the intra-parietal sulcus . Under cThis approach and technique can be considered for lesions located in the posterolateral thalamus/posterior internal capsule. This location is slightly more lateral compared to regions 3 and 4, and slightly more anterior compared to region 5 of the thalamus as described by Rangel-Castilla and Spetzler . The extThe para-fascicular route to lesions in this region might only be feasible with the availability of a TRS such as the NICO BrainPath® allowing atraumatic passage around fibre tracts. Such a system allows rapid accurate placement of the TRS through a safe surgical pathway determined by fibre tractography. We do not recommend the use of this trajectory with the traditional microsurgical entry and advancement of non-navigable retractor blades because of risk of injury to the surrounding white matter tracts. This approach may not be suitable for lesions located centrally within the thalamus, as this would involve traversing the ventral lateral and ventral posterior nuclei of the thalamus. The suitability of other approaches mentioned earlier should be meticulously considered pre-operatively.Complication avoidance is achieved through a number of factors, starting with precise pre-operative image interpretation and trajectory planning. White matter tractography is a necessity for this approach in order to preserve the integrity of eloquent axonal tracts, particularly, corticospinal tract, arcuate fasciculus, and the optic radiation. Secondly, although surface optical registration is very accurate for neuro-navigation, the senior author prefers fiducials for high fidelity cases such as this. Intraoperative neurophysiological monitoring adds information to that obtained from preoperative tractography, and gives real-time information to the neurosurgeon. During the resection through the tubular retractor, remain within the haemosiderin lined cavity to avoid transgression into nearby white matter fibres, particularly, the corticospinal tract anteriorly within the IC. Meticulous haemostasis is necessary to avoid postoperative haemorrhage.Ample time must be dedicated to preoperative imaging interpretation and meticulous planning. Perioperative lumbar drainage is not advised for this approach as brain shift from cerebrospinal fluid drainage can alter the accuracy of the navigation system. Patients should ideally be evaluated before and after treatment for language and cognitive function, as well as visual field analysis.Preoperatively, all management options including conservative management and stereotactic radiosurgery should be discussed with the patient. Available surgical trajectories and associated risks should also be discussed. The risks to speech, contralateral motor function, and visual fields should be adequately explained. The patient should be informed and prepared for postoperative hemiplegia due to manipulation around the CST which in the majority of patients is transient.Supplementary file1 (MP4 410217 KB)Below is the link to the electronic supplementary material.


A 9-year-old male with history of mixed hearing loss presented with petrous apex lesion following episode of meningitis. Serial imaging revealed persistence of the lesion necessitating biopsy to rule out malignancy. Biopsy revealed inflammatory changes. The management of petrous apex lesions following meningitis can be conservative but repeat imaging is necessary to rule out progression and to rule out neoplastic process. We describe a case of petrous apicitis following bacterial meningitis in a child presenting with profound sensorineural hearing loss and progressive, bony erosions on imaging that prompted surgical intervention. Petrous apicitis is commonly associated with lesions of the fifth and sixth cranial nerves. However, sensorineural hearing loss and facial nerve paralysis are rare but important presenting symptoms for the clinician to remain aware of. Although petrous apicitis is traditionally described as an acute process, it has the potential to take an indolent course. These features, coupled with its capacity for bony destructive changes, may mimic a neoplastic process.A 9-year-old male was admitted to the pediatric intensive care unit with group A beta-hemolytic streptococcal meningitis and right-sided otorrhea. He had a history of recurrent acute otitis media and tympanostomy tubes, the most recent of which were placed 1 month prior to admission. At baseline, the patient had symmetric mild to moderate sensorineural hearing loss secondary to STRC-related non-syndromic deafness.On admission, audiogram revealed new onset profound, right-sided sensorineural hearing loss. On day two of admission, the patient developed right facial nerve weakness, House-Brackmann grade III. magnetic resonance imaging (MRI) taken on day three revealed non-coalescent mastoiditis. Management consisted of intravenous cefepime, metronidazole and vancomycin; ciprofloxacin-dexamethasone ear drops; and oral dexamethasone. He was discharged on Day 8 with a peripherally inserted central catheter (PICC) line and prescribed a 14-day total course of antibiotics, ciprofloxacin-dexamethasone ear drops and 9 days of oral steroids with a taper.Serial MRIs were conducted following discharge. MRI at 1 month showed a contrast-enhancing mass in the right internal auditory canal with enhancement of the right petrous apex and associated labyrinthitis. An MRI at 2 months showed persistence of the lesion, whereas computed tomography (CT) showed new osseous erosion and irregular lucency within the posterior cortex of the petrous apex  and 2. GAt surgery, the petrous apex was accessed using a translabyrinthine approach. Biopsies taken from the right vestibular nerve and the internal auditory canal showed no evidence of malignancy. Intraoperative findings included extensive scar tissue formation in internal auditory canal and labyrinthitis ossificans with a non-patent labyrinth. Gram stain, acid fast bacillus (AFB), fungal cultures and anaerobic cultures were negative. Clinically, the patient’s recovery was unremarkable.Petrous apicitis is suppurative inflammation of the petrous apex with parallel findings in the middle ear and mastoid . AlthougPetrous apicitis is classically associated with Gradenigo’s syndrome, first described in 1907 as otorrhea, ipsilateral sixth nerve palsy and retroorbital pain . This trProfound sensorineural hearing loss, as seen in our patient, is rarely described in older case reports as a prominent feature of petrous apicitis . In the Finally, although petrous apicitis is most commonly described in its acute form, it has the potential to manifest as a chronic, indolent process that mirrors the histopathologic changes seen in chronic middle ear disease , 10–12. Sensorineural hearing loss and facial nerve paralysis are important presenting symptoms of petrous apicitis. Furthermore, petrous apicitis has the potential to mimic a neoplastic process. These characteristics should be considered in the differential diagnosis of pediatric petrous apex lesions when the clinical presentation is appropriate.


We describe a challenging transvenous embolization technique involving a dual-lumen balloon microcatheter (DLBM) and liquid materials for cavernous sinus dural arteriovenous fistula (CSDAVF).DLBM contributed to identifying the shunt point and preventing liquid material leakage to normal venous drainage without treatment-related complications. In a transvenous embolization using liquid materials for CSDAVF complications such as cranial nerve palsy and embolic agent migration into the internal carotid artery due to numerous anastomoses must be considered. The use of angiography during DLBM inflation to characterize the shunt point and DLBM to prevent liquid material leakage to the normal venous drainage might decrease the mass effect due to liquid materials, thereby minimizing the causes of newly occurring cranial nerve palsy.This technique may be helpful for the treatment of CSDAVF in practice, but there is generally a risk in using liquid materials in the cavernous sinus; therefore, further consideration is needed in the future. The natural course of a cavernous sinus dural arteriovenous fistula (CSDAVF) is more benign than that of a dural arteriovenous fistula (DAVF) arising at other sites. These lesions regress spontaneously in 5.3% of cases . HoweverOnyx™  is a liquid mixture of an ethylene vinyl alcohol copolymer suspended in dimethyl sulfoxide (DMSO) and tantalum powder. This non-adhesive liquid exhibits excellent penetration of complex vascular structures, allows user-based control to reduce the risk of accidental embolization, and can be delivered through a single slow injection . TransveAlthough transarterial embolization (TAE) with Onyx has been reported ,11, its We searched a prospectively maintained single-center database for patients who underwent CSDAVF treatment between December 2009 and 2019. All patients were examined in our hospital by an independent neurosurgeon who examined their eyes and measured neurological outcomes using the modified Rankin Scale (mRS), ocular symptoms, and cranial nerve palsy . Four-vessel digital subtraction angiography was performed before and immediately after embolization and at the six-month follow-up. Some patients underwent magnetic resonance angiography at the 6- and 12-month follow-ups.The safety end point was the incidence of treatment-related complications, which were defined as vessel perforations during embolization, hemorrhage or ischemic stroke, death, asystole, or new neurologic or ocular deficits. The efficacy end points were complete angiographic occlusion at 12 months after treatment and improvements in ocular symptoms and cranial nerve palsy at 12 months after treatment.All procedures were performed under general anesthesia, and continuous anticoagulation was maintained with heparin sodium. Of the two embolization strategies, venous embolization was usually the first treatment choice, whereas the arterial approach was indicated if venous embolization could not be achieved. For TVE, the fistula compartment of the cavernous sinus was catheterized through the inferior petrosal sinus (IPS). When embolic materials were used, coiling by venous access was first indicated. Liquid materials were injected through venous access into the cavernous sinus when complete fistula occlusion could not be achieved with coils alone.Onyx was approved in Japan for DAVF treatment on April 25, 2018. However, the following statement was included in the attached document about Onyx: “unless the doctors decide that it is unavoidable, the doctors should not perform with Onyx in the cavernous sinus” . Our hosWe identified five patients to prevent leakage and two patients to indicate the shunt point (SP) Table . Here, wFor control angiography, a 4-Fr catheter was introduced to the external or common carotid artery related to the CSDAVF from a femoral artery. The Flexor Shuttle® 7-Fr Guiding Sheath  was placed into the internal jugular vein from a femoral vein. Next, a 6-Fr guiding catheter was placed into the IPS over a 0.035-inch outer diameter guidewire , and a DMSO-compatible microcatheter  was advanced through the guiding catheter with a 0.014-inch guidewire . A DLBM was then navigated to the SP of the cavernous sinus through the IPS. Once inflated, the point where the balloon appeared to occlude the fistula on angiography was considered the SP. Finally, we performed coil embolization of the SP to provide a scaffold for liquid materials, in which the liquid materials were injected into the SP and minimized the infiltration to the feeding arteries during DLBM inflation. Corticosteroids were administered for several days after the procedure. In the absence of complications, patients were extubated in the operating room and discharged after four to five days.In two cases, DLBM successfully identified the SP without complications. Five patients were treated with DLBM, which prevented liquid material leakage to normal venous drainage. Patients who underwent TVE with a DLBM achieved complete occlusion at the follow-up. And one patient presented with ocular symptoms and cranial nerve palsy at the 12-month follow-up. However, all patients showed improved symptoms at the 12-month follow-up. Treatment-related complications were not identified.We described the performance of TVE with a DLBM and liquid materials (NBCA and Onyx) for CSDAVF.Coil packing of the entire cavernous sinus may induce sinus and ophthalmic vein thrombosis and aggravate symptoms. However, targeted CSDAVF embolization does not exacerbate symptoms because the target is limited to a small compartment of the cavernous sinus . TVE witAlthough TAE with Onyx is a safe and effective alternative for patients with CSDAVF experiencing failed transvenous catheterization, the use of liquid materials carries the risk of dangerous embolization of anastomoses between the dural branches of the external and internal carotid arteries, vasa nervorum, ophthalmic artery, or vertebral artery ,11. TherFor a carotid-cavernous fistula (Barrow type A), intra-arterial balloon-assisted Onyx embolization is a powerful option that prevents inadvertent migration of the embolic material into the arterial system, facilitates visualization of the internal carotid artery, and provides a buttress for coils deployed in the cavernous sinus . ContrasOur technique has some limitations. Although it is useful when the SP is limited to a small compartment, targeted TVE is difficult for a CSDAVF with multiple diffuse shunts. In such cases, TVE involves coil embolization of the cortical venous reflux and SP embolization with liquid embolic agents while protecting the normal venous drainage route. Additionally, DMSO-related toxicity may occur during TVE for CSDAVF treatment with Onyx. Development of trigeminocardiac reflex, inappropriate antidiuretic hormone syndrome, and acute respiratory distress syndrome are potential side effects of DMSO; therefore, slow injection is paramount for preventing toxicity ,20. InfiThis report describes a challenging technique for target embolization with a DLBM and liquid materials for CSDAVF. The DLBM contributes to characterization of the SP for CSDAVF. No new cranial nerve palsy was luckily detected in our cases; thus, this technique might result in decreasing the mass effect of the cavernous sinus and may lead to useful TVE with liquid materials for CSDAVF in the future. However, there is generally a risk in using liquid materials in the cavernous sinus; therefore, further consideration is needed in the future.


Flavivirus genus. We summarized 153 immunoreactive peptides from the Dengue virus, Zika virus, Japanese encephalitis virus, West Nile virus, and tick-borne encephalitis virus described in studies published from 1989 to 2020. We included peptides from structural  and nonstructural (Ns1–5) viral proteins that demonstrated relevant immunoreactivity with antibodies from naturally infected or vaccinated humans. We included peptides that demonstrated relevant reactivity features, such as indicators of disease severity related to immunological or immunopathological outcomes, differential or group diagnostic markers, immunotherapy candidates, and potential for vaccine formulation. The majority of immunoreactive peptides were described for DENV probably due to its long-lasting impact on human health and the lack of efficient vaccines and therapeutic methods. Immune landscape data regarding linear immunoreactive and continuous flavivirus peptides are still scarce, and a complete and more detailed map remains to be elucidated. Therefore, this review provides valuable data for those investigating the antibody response against flavivirus infection.This manuscript is an up-to-date review of experimentally validated linear and continuous epitopes identified from arbovirus members of the  Flavivirus genus belongs to Flaviviridae family and currently comprises 53 recognized viral species, many of which are important human pathogens  with a single linear positive-sense RNA genome of approximately 11 kbp. The genomic RNA presents a single open-reading frame (ORF) that is translated into a single polyprotein precursor of approximately 3,400 amino acids. This precursor polyprotein is co-translationally processed by host and virus-specific proteases to yield three structural proteins (capsid (C), pre-membrane (prM), and envelope (E)), which are responsible for assembling the viral particle, and seven nonstructural proteins , which are primarily involved in viral replication .via its basic residues and with the viral membrane via its hydrophobic cleft  or M (IgM)), such as viral- or group-specific reactivity, a protective or immunopathological outcome, immunological or immunodiagnostic potential, and reactivity with monoclonal antibodies. Based on these criteria, we identified 153 peptides from structural and nonstructural proteins in the DENV (serotypes 1–4), ZIKV, WNV, JEV, and TBEV  of viral infection  that poorly neutralizes the DENV but potentially enhances its infectivity was characterized using a phage-display peptide library . This mAA mapping study based on prM protein identified linear reactive peptides from DENV3 and ZIKV using IgG serum samples from convalescent DENV and ZIKV patients . Among tDuring WNV infection, most neutralizing antibodies are directed against the envelope protein; however, a subset of antibodies have been reported to additionally recognize the prM/M protein . Two linThe vast majority of defined reactive antibodies to flaviviruses are directed against the envelope protein, a surface-exposed structure on the virus particle. Antibodies elicited against the envelope protein have critical roles during protection or ADE to flavivirus infection . TherefoA study of overlapping epitope mapping combined with ELISA screening for DENV1 and ZIKV envelope protein identified two linear peptides from DENV1  that demonstrated higher levels of IgG in convalescent ZIKV patients than those from convalescent DENV patients . In addiA study using molecular dynamics simulations and energy decomposition predictions to analyze the DENV envelope protein identified a number of putative linear epitope sequences . One linA study combining multistep sequence- and structure-based bioinformatic prediction with an overlapping synthetic peptide library of the entire DENV2 envelope protein identified and evaluated a number of potential linear B-cell epitopes . Using aIn addition, another study predicted and selected common peptides among all DENV serotypes showing no similarities with other flaviviruses from the envelope and Ns1 protein regions of DENV2. Subsequently, these peptides were chemically synthesized using a solid-phase synthesis technique and evaluated by ELISA to perform DENV-specific detection of IgM and IgG from the sera of infected patients . This stin vitro lymphoproliferation, with a predominantly Th1-type immune response and a consequently reduced viremia in vivo. Thus, this study provides important insights into multi-epitope peptide-based strategies for vaccine development that are able to induce a long-lasting protective immune response.Another study described two linear peptides from the DENV2 envelope protein  that are able to recognize IgG antibodies from convalescent DENV2 patients compared to those from healthy subjects . These p+ T cells from immunized BALB/c mice after peptide stimulation. Furthermore, another study performed epitope mapping of the DENV3 and ZIKV envelope proteins by ELISA-based screening with overlapping peptides using convalescent sera from ZIKV and DENV patients from Singapore and Thailand  that is recognized by four other highly neutralizing mAbs from ZIKV-infected patients   . This peAnother study performed SPOT array epitope mapping using overlapping synthetic peptides on the full-length TBEV proteome . The autAmong the nonstructural proteins, most immunoreactive peptides have been defined for the Ns1 protein, which is also a target of protective humoral and cellular responses . AntibodA study of ELISA epitope mapping with synthetic peptides from predicted B-cell epitopes of the DENV2 Ns1 protein identified one linear peptide  that specifically reacts with IgM and IgG antibodies from the sera of acute and convalescent dengue fever patients, but does not cross-react with antibodies from JEV patients . AdditioBased on prediction algorithms from the DNASTAR, BCEPRED, and IEDB databases, one study selected eight potential diagnostic DENV2 peptides from the Ns1 protein that are common to all DENV serotypes . These pAnother study characterized linear epitope recognition regions of nine monoclonal antibodies isolated from six acute or convalescent secondarily infected DENV2 patients by epitope mapping with truncated recombinant Ns1 proteins . All of in vitro, potentially correlating with DHF syndrome development  and five peptides from the Ns1 protein of the ZIKV  as potential common linear flavivirus recognition peptides . These pAnother epitope mapping study synthesized 33 linear peptides of 15–30 aa in length covering the whole ZIKV Ns1 protein and screened them for IgG detection using 20 ZIKV-positive convalescent sera samples . Eight lThe antigenic potential of the nonstructural proteins is still not fully understood; thus, serological identification of antibodies directed against immunogenic peptides from these proteins may be intriguing. However, a number of studies have identified immunoreactive antibodies against these proteins, including elevated seroprevalence of anti-Ns4A during secondary infection, with Ns5 used as a specific antigen for serological detection . Antibodin silico study performed B-cell epitope mapping prediction on DENV proteins (excluding similar epitopes from YFV and ZIKV) and identified 9 DENV-conserved and 15 polymorphic epitopes , Oropouche virus (OROV), and CHIKV, identified a specific linear peptide from ZIKV Ns2B protein (Pep147: DITWEKDAEVTGNSPRLDVALD) that is highly specific for ZIKV IgG detection . This peepitopes . These pFlavivirus genus, including the DENV, ZIKV, JEV, WNV, and TBEV, as reported in 29 manuscripts published from 1986 to 2020. Unsurprisingly, the largest number of peptides in the literature was defined for the DENV, especially for the DENV serotype 2 , followed by the Ns1 protein (49 peptides). These proteins are considered to be the most immunogenic targets of the immune system and are therefore the main targets for antiviral drug research, biological marker identification, immunodiagnosis, vaccine candidate development, and studies of pathogenesis . Fewer iely Ns2B , Ns4A  restrictions, and host genetic background. Flavivirus infection can cause a diverse range of disease severity in humans, ranging from mild febrile to severe neuroinvasive or hemorrhagic syndromes that last from days to months. During infection, the humoral immune response produces a variety of biological markers that indicate the possible prognosis of disease severity, including antibodies that recognize specific viral antigens. In this review, we have described a range of linear immunoreactive peptides that may be useful for disease prognosis, such as Pep32 from DIII of the DENV1 envelope protein, which demonstrated a positive IgG detection signal for sera from patients with mild DENV disease. However, patients progressing to a more severe disease state demonstrated very limited IgG detection using this peptide; therefore, it could be used as a biological marker of disease severity for DENV infection . Additioin vivo protection against DENV2 infection due to a cellular immune response, although they induce low titers of neutralizing antibodies  Scholarship number 2018/09383-3 and grant numbers 2019/27333-6 and 2019/26119-0.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.