Bidirectional interplay between the peripheral immune and nervous systems plays a crucial role in maintaining homeostasis and responding to noxious stimuli. This crosstalk is facilitated by a variety of cytokines, inflammatory mediators and neuropeptides. Dysregulation of this delicate physiological balance is implicated in the pathological mechanisms of various skin disorders and peripheral neuropathies. The skin is a highly complex biological structure within which peripheral sensory nerve terminals and immune cells colocalise. Herein, we provide an overview of the sensory innervation of the skin and immune cells resident to the skin. We discuss modulation of cutaneous immune response by sensory neurons and their mediators , and sensory neuron regulation by cutaneous immune cells . In particular, we discuss recent findings concerning neuroimmune communication in skin infections, psoriasis, allergic contact dermatitis and atopic dermatitis. We then summarize evidence of neuroimmune mechanisms in the skin in the context of peripheral neuropathic pain states, including chemotherapy-induced peripheral neuropathy, diabetic polyneuropathy, post-herpetic neuralgia, HIV-induced neuropathy, as well as entrapment and traumatic neuropathies. Finally, we highlight the future promise of emerging therapies associated with skin neuroimmune crosstalk in neuropathic pain. No longer is the skin regarded a purely physical barrier separating an organism from its environment . Modern Far from operating in isolation, tremendous crosstalk occurs between the peripheral sensory nervous system and the immune system. Upon activation, immune cells release a myriad of inflammatory mediators and intercellular messenger chemicals, which can stimulate sensory nerve terminals in the skin. Analogously, nerve terminals can release a cocktail of neuropeptides which modulate immune cell function .While cutaneous neuroimmune interactions are critical in healthy host physiology, their dysregulation is implicated in many disease states, notably psoriasis , atopic The skin is predominately innervated by nerves that convey sensory information from the environment to the spinal cord and the brain. These primary afferents are responsible for detecting various sensory inputs including non-harmful stimuli, such as warmth, cooling and light touch, and harmful noxious or painful stimuli. Nerve endings within the skin arise from primary sensory neuron cell bodies in the DRG for body sensations and the trigeminal ganglia for facial sensations . MorpholStructurally, the skin can be divided into two main components: the outer epidermis and the deeper dermis. Both layers contain primary sensory afferents, which can be categorized according to degree of myelination, conduction velocity, axonal diameter and nerve ending type Figure 1Aβ, Aδ and C fibers can be further distinguished based on their distal terminals within the skin. Aβ fibers generally terminate within the dermis, usually forming close association with hair follicles in hairy skin and various specialized end organs, such as Meissner’s corpuscles, Merkel cells, Ruffini endings and Pacinian corpuscles in glabrous skin. Aδ fibers terminate within the epidermis as free nerve endings or in the dermis of haired skin as circumferential endings . UnmyeliC fibers can be broadly classified, neurochemically, into two groups depending on expression of neuropeptides: peptidergic or non-peptidergic . PeptideIn the skin, the most superficial layer of the epidermis, the stratum corneum, is the first barrier against hazardous environmental threats. This layer is comprised of flattened keratinocytes that have become denucleated; these cells are known as corneocytes. Lipids fill intercellular spaces between corneocytes to form a physical and chemical barrier, commonly referred to as ‘bricks (corneocytes) and mortar (lipids)’. This hydrophobic layer, which is largely unbroken  except by skin appendages, such as hair follicles and tear ducts, restricts water movement . Deep tovia keratinocyte-sensory neuron purinergic signalling , dermal dendritic cells (dDCs), macrophages, and mast cells, in addition to lymphocyte cells such as T cells and B cells Figure 2Langerhans cells are the sole resident myeloid cell of the epidermis, contributing to the physical and immunological skin barrier. LCs have a dendritic phenotype; their projections propagate through keratinocyte tight junctions in the epidermis, allowing them to detect antigens throughout this layer without disrupting its permeability . These cDermal dendritic cells reside in the dermal layer and, similar to LCs, traffic foreign antigens to lymph nodes to induce adaptive immune response during pathogen invasion . In factvia cytokine release  synthesized mediators  and recirculating T cells   and regulatory T (Treg) cells  cells . Th cellor cells , 62. Trenfection . They arnfection , vitilignfection  and allenfection  and a kenfection .via eccrine sweat glands has been implicated in the regulation of microbial environments on the skin surface (regs) via eccri surface , 74. Rese (regs) . Bregs sic cells . UltimatIn addition to their primary role of transmitting signals from the periphery to the spinal cord and brain, somatosensory neurons mediate immune responses through the release of neuropeptides and neurotransmitters from peripheral terminals. Innate and adaptive immunocytes express receptors for sensory neuron-derived factors, facilitating direct communication between nervous and immune cells. Sensory neurons have been shown to modulate immune response in models of infection and inflammation –79. For Modulation of immune response by sensory neurons has been implicated in skin infections and in several inflammatory skin conditions, including ACD, AD and psoriasis.via specific receptors on neuronal surfaces. Activation of these receptors modulates ion channel activity and enhances action potential generation, resulting in pain sensation -mediated degranulation of mast cells and subsequent histamine release , TNF and interleukins at the site of injury, contributing to nociceptor sensitization  receptors located on itch-sensing fibers. Recent evidence has suggested that macrophages play a role in resolution of pain by adopting an anti-inflammatory phenotype. Macrophages express GPR37, a receptor of neuroprotectin D1, a specialized pro-resolving mediator involved in inflammation resolution (via increased release of IL-10 and transforming growth factor β (TGF-β) and attenuated release of IL-1β and TNF  . ResolutThese models have demonstrated that neuropathic pain is characterized by dysregulation of the pro-/anti-inflammatory balance resulting in excessive neuroinflammation at both the site of nerve injury and at the sensory nerve terminals. In this setting, sensitized nociceptor terminals can undergo deleterious plastic transformation, characterized by altered expression of receptors, channels, transporters and neurotransmitters . A varieCIPN is a common, dose-limiting side-effect of chemotherapy use , 133 andNumerous studies in rodents and skin biopsy of CIPN patients have demonstrated loss of IENFs indicating IENF degeneration due to chemotherapy-induced neurotoxicity –148. In Diabetes is set to reach 366 million affected individuals by 2030, reaching pandemic levels . Diabetidb/db models of type 2 diabetes  is a chronic condition characterized by neuropathic pain persisting for 3 months or more after an outbreak of shingles . ShingleAs VSV-specific cell-mediated immunity falls, anterograde transport  of the dormant virus to the affected cutaneous dermatome triggers an array of cellular responses in both infected and non-infected resident skin cells. Skin biopsies of affected dermatomes reveal a reduction in cutaneous sensory afferent innervation . Loss ofPeripheral mechanisms of PHN are not fully elucidated and are subject to debate . MicroRNAntiretroviral therapy has converted human immunodeficiency virus (HIV) infection from a progressive, fatal illness to a manageable chronic condition . NeuropaA hallmark of HIV-induced sensory neuropathy is a loss of IENF density  althoughNucleoside reverse transcriptase inhibitors (NRTIs) are key antiretroviral drugs for the treatment of HIV infection however, as with chemotherapy, the development of sensory neuropathy is a dose-limiting side effect . A mouseThe complex interplay between the cutaneous sensory nervous system and immune system may also contribute to various clinical manifestations of entrapment and traumatic neuropathies. The following section addresses the evidence suggestive of cutaneous neuroimmune interactions in several subtypes of compressive/traumatic neuropathy .Carpal tunnel syndrome (CTS) is the most common entrapment neuropathy caused by a chronic, focal compression of the median nerve in the carpal tunnel. Patients with CTS experience sensory abnormalities, such as painful paraesthesia, mechanical allodynia, hyperalgesia and loss of thermal sensitivity, which are associated with an altered function of both myelinated and unmyelinated sensory axons –199. In Trigeminal trophic syndrome (TTS) is a rare complication of a trigeminal nerve insult that is characterized by persistent facial ulceration and loss of sensation along the distribution of the affected trigeminal dermatome  is a devastating complication of minor or moderate tissue injury in the extremities and is characterized by sensory, motor, and autonomic dysfunction  patches, and BTX-A –241. Whiexplants . Howeverexplants . Lidocaiexplants . To dateexplants  was discexplants . TargetiGiven the limited effectiveness of current neuropathic pain treatments, further therapeutic strategies are needed to meet this clinical need. A better understanding of the neuroimmune molecular mechanisms underpinning neuropathic pain pathogenesis may illuminate new avenues of therapeutic research. Given the dysregulation of the cutaneous immune system in neuropathic pain states, one could hypothesize that targeted cutaneous immunomodulatory therapies may present an effective treatment for neuropathic pain, specifically when it is localized. Effectiveness of immunotherapy in treating skin disorders is varied. The successful modulation of pathogenic inflammatory cytokine pathways by targeted immunotherapies to treat psoriasis has shown encouraging results . ContrasAdditional research potential may lie in the emerging field of optogenetics. Optogenetics involves the manipulation of cellular activity by introducing and stimulating light-sensitive transmembrane ion channels, commonly in neurons . This stGM-T and DL conceived and designed the study. DL drafted the manuscript and prepared the figures. PM contributed to writing sections of the manuscript. All authors contributed to the article and approved the submitted version.This study was supported in part by a grant from the National Health and Medical Research Council of Australia to GM-T (ID # APP1162060). The funder had no role in study design, decision to publish or preparation of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be constructed as a potential conflict of interest.


It is already known that DNA alterations do not fully recapitulate the complex nature of a tumor or its potential interaction with specific treatments. Therefore, in order to establish more precise and effective therapeutic approaches for non-small cell lung cancer, tumors will have to be characterized in a more accurate and comprehensive way. In this regard, transcription profiling has already demonstrated its utility in further stratifying patients in a much more refined way than genomic alterations. Examples of this include the definition of intrinsic subtypes in colorectal cancer, breast, or non-small cell lung cancer tumors based on their expression patterns. Moreover, the characterization of the activity levels of the pathways involved in tumor progression and development is bound to better predict the specific response to a certain therapy than isolated biomarkers such as specific DNA alterations or the expression of single genes. This is especially relevant in the context of patients not harboring targetable alterations or those developing resistance after treatment. Recent technological advances and the application of high-throughput mutation and transcriptome analyses have improved our understanding of cancer diseases, including non-small cell lung cancer. For instance, genomic profiling has allowed the identification of mutational events which can be treated with specific agents. However, detection of DNA alterations does not fully recapitulate the complexity of the disease and it does not allow selection of patients that benefit from chemo- or immunotherapy. In this context, transcriptional profiling has emerged as a promising tool for patient stratification and treatment guidance. For instance, transcriptional profiling has proven to be especially useful in the context of acquired resistance to targeted therapies and patients lacking targetable genomic alterations. Moreover, the comprehensive characterization of the expression level of the different pathways and genes involved in tumor progression is likely to better predict clinical benefit from different treatments than single biomarkers such as PD-L1 or tumor mutational burden in the case of immunotherapy. However, intrinsic technical and analytical limitations have hindered the use of these expression signatures in the clinical setting. In this review, we will focus on the data reported on molecular classification of non-small cell lung cancer and discuss the potential of transcriptional profiling as a predictor of survival and as a patient stratification tool to further personalize treatments.  Lung cancer ranks first in the number of cancer-related deaths worldwide . Lung caThe 2015 World Health Organization (WHO) Classification of Lung Tumors divides lung cancer into two main histological types: epithelial and neuroendocrine tumors. Epithelial tumors, often also called non-small cell lung cancer (NSCLC), account for 80–85% of all cancer diagnoses and are subsequently subdivided into two major subtypes: non-squamous cell carcinomas, further divided in lung adenocarcinoma (LUAD) and large cell carcinoma, and squamous cell carcinomas (LUSC) . The TNMTechnological advances within the last decades have led to a significant improvement in our understanding of tumor biology through the implementation of large-scale genomic and transcriptomic analyses. In this way, whole-transcriptome gene expression profiling, coupled with bioinformatics analyses, has been used to identify distinct NSCLC molecular subtypes . HoweverIn this review, we attempt to analyze the current state of transcription-based classifications in the context of NSCLC and lay the groundwork for future studies aiming to define a transcriptional classification of NSCLC with clinical value, both as a predictor of survival and as a patient stratification tool to further personalize treatments. This is particularly relevant since a significant proportion of patients lack actionable targets and are currently being treated with chemotherapy alone or in combination with immunotherapy. Gene expression is intimately linked to cellular phenotype and tumor behavior. Messenger RNA (mRNA) expression has been extensively used to distinguish biologically homogeneous subtypes of a disease. In lung cancer, seminal studies on transcriptional profiling included different morphological types  in order to classify them based on their differential gene expression ,7. TheseFollowing the evidence of LUAD’s higher molecular heterogeneity, a number of studies developed between 2001 and 2016 sought to generate a classification of lung LUAD based on gene-expression profiling ,22,24,25EGFR alterations were mostly present on bronchioid tumors, whereas KRAS and TP53 were more common within the magnoid subtype. In addition, magnoid tumors exhibited the highest chromosomal instability, copy number alterations, DNA hypermethylation, and genome-wide mutation rates. To date, the most accepted classification was described back in 2006 . Hayes eKRAS mutation and STK11 inactivation frequency, while the PI subtype was enriched for NF-1 and TP53 co-mutation. On the other hand, activating EGFR mutations or ALK rearrangements were more prevalent within the TRU subtype. After all, this classification did not contribute much to the previous one but served as an independent validation of Hayes et al.’s and Wilkerson et al.’s intrinsic subtypes in a larger set of tumors. Hayes et al.’s and Wilkerson et al.’s ,20 classt-test, sensitivity analyses, self-organizing map (SOM) neural networks, and hierarchical clustering methods and reported a new classification into four intrinsic lung LUAD subtypes [EGFR alteration was enriched in subtype 4, while TP53 mutations tended to be more frequent in subtypes 1 and 2. In another study, Chen et al. reported a novel classification consisting of six lung LUAD subtypes , integrating gene expression, DNA methylation, copy number alterations, and protein expression data [Over the years, the TCGA project has been incorporating new tumor cases, which means a doubling increase in the number of lung LUAD cases with available molecular data. This opened the opportunity for further research on the establishment of accurate and clinically relevant tumor molecular subtypes. Hu et al. combined k-means clustering, subtypes , named 1ion data . In agreAlthough most of the previously mentioned studies focused on lung LUAD, some studies also reported intrinsic transcriptional subtypes for LUSC . BetweenIn a more comprehensive study published in 2010, Wilkerson et al. proposed four intrinsic LUSC subtypes: primitive, secretory, basal, and classical . These gKEAP1, NFE2L2, and PTEN alterations frequency, as well as pronounced hypermethylation and chromosomal instability. By contrast, primitive tumors more commonly exhibited RB1 and PTEN alterations, while basal tumors showed NF1 alterations. Interestingly, CDKN2A deletions, which are a common event in LUSC, were not associated with any subtype.In 2012, Wilkerson et al.’s LUSC transcriptional subtypes’ were tested in the TCGA LUSC dataset, along with other available molecular data, to classify and characterize 178 lung LUSC samples . Again, In 2017, Chen et al. took advantage of the incorporation of new LUSC tumors within the TCGA resource and delved into the study of LUSC subtypes combining omics layers other than transcriptomics . Three LOverall, different classifications have been proposed for both lung LUAD and LUSC but have never been adopted within the clinical practice. Gene expression signatures derived from these studies, generally conducted on a limited number of patients, sometimes included large sets of genes that passed the significance threshold. This often makes it difficult to select the most relevant genes, leading to less reproducible results or identification of the relevant biological processes that these genes represent . In factIn this regard, in addition to the signatures derived from lung cancer molecular subtypes studies, several groups designed studies whose main objective was to identify prognostic signatures, some of them also able to predict clinical benefit from specific treatments, using gene expression profiling in surgically resected tumor samples. In 2017, Tang et al. evaluated 42 of these signatures for their prognostic potential via a meta-analysis on almost 2000 early-stage NSCLC patients collected from 15 studies . AlthougAlthough distant in time, both studies agreed on the limitations that can be encountered when evaluating and proposing prognostic signatures for their clinical use. Some of these caveats, which also apply to recent studies , includeNSCLC is not a single entity but is, in fact, multiple pathologies, each with unique molecular features, which we are only beginning to understand ,38. EvenCurrent biomedical research is focused on developing treatments that specifically target abnormally activated regulatory pathways of cancer cells, such as signal transduction, cell cycle, DNA repair, metabolism, and apoptosis resistance. Significant advances in understanding the genomic landscape of lung cancer through comprehensive genomic profiling have allowed physicians to tailor treatment options in many cancer types, including lung cancer ,41. Around 38% of patients with NSCLC present an oncogenic driver mutation, which enables the use of targeted treatment options that improve survival outcomes and reduce toxicity compared to standard chemotherapy ,43,44. EEGFR and KRAS mutations, the transcriptional signatures allowed further refinement of disease subclassification. In this regard, only two of these studies found specific transcriptional profiling patterns for patients harboring EGFR or ALK alterations, indicating that the presence of these alterations may confer a very specific gene expression pattern [EGFR mutated tumors are still considered to be a relatively homogeneous entity for therapeutic decisions in NSCLC, despite drug responses and time to progression are relatively variable among patients. Moreover, gene expression profiles of tumors harboring mutations in other key genes such as KRAS or TP53 showed significant heterogeneity [KRAS is the most common gain-of-function mutation in NSCLC, accounting for approximately 30% of lung AC in Western countries and about 10% in Asian countries [KRAS mutated tumors [Gene expression analysis could become a powerful tool to further stratify patients for targeted therapies and to predict potential differences in response to treatment among patients harboring the same genomic alteration. In this regard, there are only a few studies concerning the comparison of driver alterations and defined transcriptional subtypes in the context of NSCLC ,46,47,48 pattern ,26,27,47ogeneity . In partountries . Recent d tumors . Given tIn summary, this evidence supports the fact that DNA alterations do not fully recapitulate the complex nature of lung tumors. Therefore, further stratification of these patients based on their transcriptional profiling might improve the selection of individuals that would benefit from existing targeted therapies. Moreover, advances in this direction could lead to the development of novel combinations with standard care to target alternative signaling cascades in patients suspected to be non-responders or the anticipation of resistance mechanisms and the design of combination treatments upfront. Targeted DNA sequencing has recently been demonstrated to be a comprehensive tool to detect multiple types of oncogenic alterations, including relevant oncogenic kinase fusions in NSCLC . Currently available DNA-based panels, such as FDA-cleared MSK-IMPACT large panel, are designed to detect rearrangements via tiling of the appropriate introns known to likely harbor the genomic alteration . HoweverOn the other hand, a significant percentage of NSCLC patients do not harbor any known actionable genomic alterations, especially in LUSC, for whom there are currently no available targeted therapies. For this reason, there is a need to unravel novel vulnerabilities other than genomic oncogenic alterations and to combine new technologies that allow a deeper understanding of cancer biology, such as those centered on gene expression. In this regard, the WINTHER and CoPPO trials evaluated the value of transcriptomics in addition to genomic sequencing to guide treatment decisions in advanced malignancies that have progressed following standard treatments ,58. BothOverall, these trials showed that transcriptomic profiling beyond classical tumor DNA sequencing is useful to make treatment decisions in patients with solid tumors. Therefore, future research should seriously consider combining genomic and transcriptomic data.Although the use of targeted therapies has led to clinical benefit in patients with NSCLC, responses to these treatments are rarely complete and inevitably temporary because of the appearance of treatment resistance mechanisms. There are two main types of resistance mechanisms to targeted drug therapy: primary resistance or acquired resistance. Additionally, these processes can be further subclassified as on-target and off-target . RegardiEGFR-TKIs are widely used against NSCLC with EGFR mutations. Thus, preventing and delaying EGFR-TKI resistance is a critical clinical concern involving the treatment of EGFR-mutant NSCLC . In the PD-1) or its ligand PD-L1 axis, either alone, in the case of patients with PD-1 expression ≥ 50%, or in combination with chemotherapy, in the case of PD-1 expression < 50%, has become the standard of care in advanced NSCLC lacking driver oncogenic alterations [Until recently, the standard first-line therapy for patients with metastatic or advanced NSCLC has been doublet platinum chemotherapy . Nowadayerations . HoweverPreviously established biomarkers for predicting clinical outcomes of immunotherapy in NSCLC, such as PD-L1 expression, tumor mutational burden (TMB) or single genomic alterations in STK11, KEAP1, or PTEN genes, cannot accurately predict clinical benefit in all patients . In thisGene expression profiling, interrogating several immune-related genes, is becoming an attractive approach to characterize tumor immune microenvironment and predict immunotherapy response, going beyond the measurement of single genes such as PD-L1 . In partA recent study demonstrated that multigene signatures constitute a reliable tool to identify T-cell pro-inflammatory phenotypes across different solid tumors, including NSCLC, likely to respond to PD-(L)1 inhibition therapy outperforming PD-L1 immunohistochemistry in PD-L1 unselected patients . Of noteOverall, these two studies showed that gene expression profiling constitutes a powerful tool for predicting response to immunotherapy outperforming previously single analyte biomarkers. However, both used multiplexed panels containing a few selected genes, thus limiting the amount of information that can be captured compared to whole transcriptome sequencing. In this regard, we believe that further studies introducing whole transcriptome sequencing, with the subsequent development of standardized single-cell RNA sequencing analysis pipelines to properly analyze the composition of complex mixtures of cells, would be useful not only to distinguish which patients will derive long-term clinical benefit from immunotherapy, but also to characterize the mechanisms of acquired resistance to anti-PD-(L)1 treatment, and to progress towards a more precise immunotherapy. Given the lack of accurate biomarkers for immune therapy response in NSCLC, gene expression profiling was explored for lung LUAD and LUSC. Faruki et al.  assessedTranscriptomics has not historically had an excessively relevant role in the diagnosis and treatment of NSCLC. Intrinsic technical and analytical difficulties , in addition to the weight of genomics in the management of NSCLC have contributed to hinder its use within the clinical practice.Future clinical development of targeted therapies relies on the integration of all the accumulated knowledge on NSCLC biology. The definition and characterization of molecularly homogeneous NSCLC subtypes is bound to result in the design of effective therapeutic strategies. To this end, it is crucial to move away from characterizing tumors just on the basis of single biomarkers, which are so far unable to fully predict the complexity of the disease and drug response. In this way, we should evolve towards the integration of different technologies that allow us to take into account clonal heterogeneity, the development of resistance mechanisms, and interactions with the tumor microenvironment. In this context, transcriptome profiling has been demonstrated to be a powerful tool for patient stratification and drug selection, especially in the context of acquired drug resistance and patients with advanced NSCLC lacking actionable drivers. The relevance of transcriptional profiling will likely increase with the progress of immune therapy and the need for predictive markers. Moreover, the recognition of signaling pathways altered upon tumor progression through gene expression profiling has conferred new opportunities for drug development and has increased the number of patients that can be matched to a treatment. In addition, the identification of associations between the altered pathways and the presence of specific mutational patterns responsible for acquired resistance and tumor progression might unravel new therapeutic targets. In this context, there have recently been important developments in the field of transcriptomics in order to increase resolution and specificity. For instance, single-cell RNA-Seq allows the determination of the gene expression profiles of individual cells. In this way, it provides information about tumor heterogeneity and cellular interactions. Moreover, it also provides insights into which cells might be responsible for resistance development and through which transcriptional processes, exposing new targets for drug development.Nevertheless, the implementation of whole-transcriptome interrogation techniques into NSCLC management frameworks will mainly depend on prospective validation studies of the identified signatures to assess whether they are able to improve treatment decisions and recapitulate the heterogeneity of the disease beyond currently available strategies.


Cosmetic dermal fillers are a sought-after procedure globally. However, despite the safety claims of filler materials by the manufacturers, adverse reactions still occur.This case report is of a 66-year-old female who presented with a late-onset complication of a hyaluronic acid dermal filler that clinically mimicked a salivary gland neoplasm. The patient presented with firm peri-oral swellings of short duration that clinically mimicked a pleomorphic adenoma and mucoepidermoid carcinoma. The diagnosis was that of a foreign-body granulomatous response to dermal fillers. Although other mimics of a similar nature are reported a knowledgeable clinician, careful choice of filler material, knowledge of the product, thorough pre-procedural history taking and post-procedural patient follow-up can drastically minimize a possible misdiagnosis. The reaction was treated with a combination of hyaluronidase, betamethasone and 5-flurouracil intra-lesional injections monthly for 11 consecutive months, with total clinical resolution.Patient education of the procedure, product name and the possibility of an adverse reaction occurring, even years later or at a site distant to the initial site of placement, is vital. Further, we review the recent reported adverse association of the new mRNA COVID-19 vaccines and dermal filler placement. Both the increasing demand for facial aesthetic procedures and the growing market for dermal filler products have resulted in a parallel spike in complications  show that soft tissue filler rose rapidly in popularity and has been the second most performed minimally invasive cosmetic procedure every single year since 2007  biopsy from the upper left lip showed abundant extracellular pale pink staining mucinous-type matrix material with interspersed foamy cells, reported as being suggestive of a mucoepidermoid carcinoma or pleomorphic adenoma. She was a non-smoker, with no allergies nor significant family history. There was no history of trauma, difficulty with filler placement, prior dental procedures, local or generalized infection, including COVID-19 infection prior to the development of the inflammatory response nor vaccination. In the absence of a history of dermal filler placement, the presence of multiple indurated nodules, which appeared clinically infiltrative, the rapid development of the lesions, the FNA findings and the frequent and similar occurrence of salivary gland neoplasia at this site the clinical differential diagnosis was that of a salivary gland neoplasm.An incision biopsy of the left side of the upper lip done under local anesthesia comprised three portions of soft tissue measuring 25 × 10 x 5 mm. Histologic examination showed fibrous connective tissue with bundles of skeletal muscle fibers and lobules of adipocytes, devoid of surface epithelium/epidermis and almost entirely effaced by large pools of pale staining eosinophilic material that elicited a prominent foreign-body giant cell granulomatous response  Allergan Pharmaceutical (Pty) Ltd. placed 10 months prior, to the midface and peri-oral areas. The midface filler remained soft and impalpable with no adverse reaction to this area. The reaction was treated with a combination of hyaluronidase , betamethasone  and 5-flurouracil  by intra-lesional injection using a fanning technique with a 30G needle , WITS, Johannesburg, South Africa, granted ethics clearance (M220282) in accordance with the Declaration of Helsinki (2013).The placement of any filler product is associated with the risk of an adverse reaction  or synthetic fillers  , infection, hypersensitivity, lumps/nodules, asymmetry, defects in contour, skin discoloration, vascular compromise and tissue necrosis , while purely cytologic/histologic mimics include liposarcoma (n = 12) and mucoepidermoid carcinoma (n = 2) , high-frequency ultrasonography and spectroscopy  in the diagnosis of foreign-body granulomas to dermal fillers is controversial. While some reports show FNAC to be a less invasive, useful method to confirm clinical suspicions of adverse reactions to dermal fillers . This may contribute to an increase in adverse reactions to dermal fillers. Even though this is very infrequent, temporary and treatable with corticosteroid therapy, knowledge and recognition of this possible reaction by both the clinician and patient is important .Interestingly, patients with a history of dermal filler use have demonstrated side effects with the mRNA COVID-19 vaccine. The Centers for Disease Control and Preventions (CDC) have thus highlighted clinical considerations for mRNA COVID-19 vaccine use and its likely consequence in patients with a history of dermal filler use . No specific filler type was associated with this side effect. The patient with the lip filler reported a similar reaction previously following a flu vaccine. This reaction may be because most dermal fillers are composed of hyaluronic acid, a natural substance and a component of our collagen, which retains fluid. In an attempt to create natural immunity to a virus following vaccination, the immune system reacts by sending healing cells to the area, resulting in swelling. In a patient with dermal fillers undergoing an immune response, the increased hyaluronic acid in the dermal filler could result in swelling due to the ability of hyaluronic acid to retain water .In the phase 3 trial of 30 000 mRNA-vaccinated subjects (Moderna COVID-19 vaccine), three patients with dermal fillers  showed moderate facial swelling as an adverse reaction (Knowledge of facial anatomy, different injection techniques, the various properties of the different filler products and their indications, history of filler placement and the patients’ needs and expectations are essential in reducing the risk of adverse outcomes (Koli and Davda The global demand for dermal filler placement necessitates a focus on patient safety. The recently updated 10-point plan for procedural safety in soft tissue filler treatments provides treating physicians with methodical strategy for avoiding and managing adverse reaction (Heydenrych et al. The clinical and histological diagnosis of an adverse granulomatous reaction to a dermal filler is often challenging for diverse reasons, including COVID-19 vaccinations. An awareness of this manifestation mimicking a salivary gland neoplasm is significant to ensure the correct diagnosis and treatment. Patient education regarding the name and nature of the injected filler material and the possibility of adverse reactions is important.


Peptide nucleic acids have emerged as promisingchemical agents in antigene or antisense therapeutics, as splicingmodulators or in gene editing. Their main benefits, compared to DNAor RNA agents, are their biochemical stability and the lack of negativecharges throughout the backbone, leading to negligible electrostaticinteraction with the strand with which they are hybridizing. As aresult, hybridization of PNA strands with DNA or RNA strands leadsto higher binding energies and melting temperatures. A lack of naturaltransporters, however, necessitates the formation of PNA-containingchimeras or the formulation of nanoparticular cell delivery methods.Here, we set out to explore the progress made in using imaging agentsbased on PNAs in diagnostic applications and highlight selected developmentsand challenges. As a result, PNA strands can be preparedusing well-established solid-phase peptide synthesis (SPPS). In addition,variations of the original AEG backbone have been reported, whichmodify properties of the PNA strand. Peptidenucleic acids (PNAs), a class of xeno-nucleic acids (XNAs),have received significant amounts of academic interest over recentyears.2 Due to the fact that PNA strands do notseem to occur in nature, no efficient pathway to break them down hasevolved in biological systems. As a result, they are easier to handleand possess in vivo kinetic stability higher than that of DNA- orRNA-based agents.3 The lack of negativecharges from the AEG backbone of a PNA strand results in little tono electrostatic repulsion with complementary DNA or RNA single strands.As a result, PNA/DNA or PNA/RNA double strands display hybridizationenergies and melting temperatures significantly higher than thoseof DNA or RNA double strands. In addition, the lack of negative chargesallows for a more efficient formation of triple helices (triplex formation),which are involved in DNA recombination and gene editing.4Gupta et al. as well as Sharma et al. reviewedvarious derivativesand selected aspects, such that these will not be discussed furtherhereby.6 There are a few reports of their use in gene-editing applications,although the exact mechanism of this has not been elucidated yet.4 As a result of their broad applicability, theyhave been evaluated as agents for the treatment of various cancers,hereditary diseases, such as Huntington’s disease, and as novelantivirals.11There have been numerous in vitro studies,along with some promisingpreclinical studies in animal models, to investigate their potentialin the treatment and management of various diseases. PNA-based agentsmay be used as antisense or antigene agents or as splicing modulators1.5,6 The12 As such,PNAs without additional vectors or delivery mechanisms show littleto no pharmacological effect.12 In addition,they typically distribute swiftly and are excreted quickly comparedto larger biomolecules.14 To counteract thesefeatures, a delivery mechanism must be implemented. This can be realizedby formation of a PNA–vector chimera or by developing appropriatenanoparticle formulations.Apart from exploring their therapeutic use, some researchgroupshave also demonstrated and reported their potential as diagnosticmarkers, essentially using PNAs as retention enhancers for a conjugatedimaging motif, thus enabling PNAs to act as theranostics. Mammaliancell lines lack transport mechanisms for them and uptake of nativePNAs in eukaryotic cells is low.Here, we present an overview of theuse of PNAs as synthetic buildingblocks for imaging agents, highlighting the state of the art and challengesinvolved in probe design and testing and underlining the current progressbeing made in both basic research and clinical settings. The termcontrast agent in this context refers to compounds which may be usedin any imaging modality.14 Such chimeras are hypothesized to improve signal-to-background inimaging applications by acting as retention enhancers .One way to achieve this is the formulation of a chimera-type imagingagent. A PNA chimera comprises the actual PNA fragment, a functionalitywhich facilitates molecular imaging , and a vector for targeting transporters or receptorson the cell surface, typically in the form of a peptide (fragment).Compounds of this nature cannot be classified as small molecules,as their masses typically range from 4 to 6 kDa. As such, characterizationis primarily done by HPLC/MS and binding assays on complementary immobilizedDNA, as well as by variable-temperature UV/vis spectroscopy.nhancers 2. In a t1499mTc to enable single photon emission computed tomography (SPECT).17 Small linkers were introduced between the differentfunctionalities to minimize interactions between them.The particular benefit of developingchimera-type imaging agentsthen is the high versatility and modularity, which can be achievedwith such agents. The same PNA fragment can, at least theoretically,be combined with any vector and any imaging modality. Similarly, thePNA fragment in such a design can be changed as needed, without affectingthe overall methodology used to produce the resulting chimera. Inaddition, such constructs have been shown, despite their size, toexhibit rapid biodistribution.18 To assess the effect of a PNA-based probe, experiments with a mismatchedanalogue and ideally a PNA-free analogue are necessary, and a numberof further experiments are possible to demonstrate that binding tocomplementary mRNA, rather than other factors, improves retentionin the targeted tissue. For instance, Tian et al. developed WT-4433, an analogue of TW-4185, in whichthe ligand moiety is replaced by fluorescein. The fluorescent probeswere used in cell-imaging assays and indicated promising results whencompared to the mismatched derivative WT-4361.18 A xenograft study in mice showed that the PNA chimeras accumulatedin the tumor and could still be detected after 24 h. Control experimentsfeaturing a PNA-free peptide–ligand conjugate (WT-990) showed little to no signal at the time points chosen in this study(WT-4172) and a different cyclizedpeptide )showed reduced tumor-to-background ratios.17 These results suggest that the PNA may, in fact, lead to increasedretention in cells. Furthermore, the results obtained with PNA mismatchprobes indicate that binding to mRNA indeed plays a role in the enhancedretention and in vivo distribution. Western blots of the CCD1 proteinshowed reduced quantities of protein in mice treated with the antisensePNA, compared to the mismatched or PNA-free analogues, further indicatingthat binding between PNA and mRNA occurred in vivo.17 Noticeably, the probes featuring the 99mTc ligandand d(cys-ser-lys-cys) show significant kidney uptake. This is likelya feature of both functionalities, rather than an inherent featureof PNA chimeras. Other 99mTc tetrapeptide ligands are well-knownfor facilitating urinary excretion.20 Similarly,expression of IGF1R in mice kidneys may be high, as even a PNA-freepeptide–radioligand combination shows considerable uptake,while the peptide mismatch shows little specific uptake. Other authorsworking with biomolecular IGF1R imaging agents, such as affibodies,likewise observed considerable kidney uptake.21Typical choices of linkers includedin the constructs highlightedin is study3. Simila18Therefore, a more thoroughuse of bioinformatics and mRNA expressiondata, not only for the development of the antisense PNA but also inthe choice of vectors, may be important for future progress. Chimerasfeaturing other ligands show lower renal and instead increased hepaticuptake.d-Tat57–49 fragment for cell targeting, antisensePNA to DsRed2, DOTA for ligation of gadolinium(III) for magnetic resonanceimaging (MRI), and fluorescein isothiocyanate (FITC) for fluorescenceimaging (FI).14 The comparison of the antisensePNA probe to a mismatch derivative in vitro showed no significantdifference in fluorescence intensity after 18 h of incubation, likelydue to endosomal entrapment. As noted by other authors, entrapmentin endosomes is a commonly observed issue in the delivery of theseagents.14 Many cell-penetrating peptides (CPPs) willnot be able to effectively translocate from endosomes or lysosomesinto the cytosol.22 Shiraishi et al. investigatedthe influence of conjugates of a PNA molecule and various CPPs invitro, using a luciferase assay in which an aberrant splicing sitewas targeted, such that binding of PNA led to the translation of functionalluciferase protein.23 Their results indicatednot only that the efficiency of the treatment was highly dependenton the exact CPP used but also that treatment of cells with Ca2+ (from CaCl2) improved delivery significantly.23This indicates that the distributionand clearance behavior canlikely be optimized either by changing vectors or linkers or by adjustingmolecular charge and solubility. A central challenge in the designand optimization of chimeras for diagnostic purposes is the distributionon the cellular level, specifically in terms of cytosol release. Mishraet al. developed a multimodal imaging agent, comprising the 25 While mostof these studies showed increased cytosol release in vitro, such deliverymechanisms were also suggested to have certain drawbacks such as alack of the desired selectivity for the targeted cells, instead relyingexclusively on the PNA-based retention enhancement, potentially limitingthe achievable contrast. As such, development of release mechanismsinto the cytoplasm is of utmost importance to develop effective PNA-basedtherapeutic or imaging agents, giving rise to further challenges intheir development. There are numerous unexplored avenues for the developmentof PNA-based chimeras. Only a small number of vectors and imagingagents have been explored so far in terms of their diagnostic capabilityin disease models in vivo.Recently, it was also demonstrated thatthis may be addressed bydeveloping cyclic CPPs of the general formulas dCys-CPP-Cys, cCPP,or CPP-hemagglutinin conjugates.Optimization of clearance and biodistributionbehavior in vivois of great importance to make such constructs applicable to imagingof cancers or following in real time disease biomarkers. Developmentof reliable delivery methods into the cytoplasm is necessary to realizethe full potential of this class of probes. Finding general syntheticand imaging strategies to meet these challenges would allow for thedevelopment of new multimodal imaging agents, agents for nuclear therapyor gadolinium or boron neutron capture therapy, which could capitalizeon the enhanced retention successfully demonstrated by some authors.27 A schematic representation canbe found in trans-cyclooctene and tetrazine,28 or on thesupramolecular recognition of a host and a guest, such as the interactionbetween adamantane and cyclodextrin.29 Incontrast to other methods, pretargeting approaches rely on presentationof the recognition motif on the cell surface. As such, successfulapproaches usually rely on large biomolecules, such as monoclonalantibodies or affibodies, to avoid the quick internalization typicallyobserved for small molecules and peptides. This alleviates the needfor developing endosomal escape strategies and allows one to focuson other challenges, like the overall biodistribution and excretionof unbound probes. Several authors already successfully demonstratedthe use of such a strategy involving PNAs.30As an alternativeto the use of PNA chimeras, PNAs may also beused as motifs for supramolecular recognition in pretargeting approaches.31 More recent work performedby Leonidova et al. demonstrated the use of a cetuximab–PNAconjugate for targeting of cancers overexpressing the endothelialgrowth factor receptor (EGFR).30 Similarly,the use of a trastuzumab–affibody fusion protein, synthesizedusing UV click chemistry and featuring a PNA strand conjugated tothe affibody, has been explored.27To the best of our knowledge,the first report of PNA-based pretargetingwas published in 1997 and demonstrated its potential in both infectionand tumor models.19 After allowing for initial biodistribution ofthe affibody conjugate, lutetium-177-labeled PNA constructs of variouslength were injected. SPECT/CT images obtained as part of this studyare depicted in Tano et al. synthesized a conjugate of a PNA molecule and a HER2-targetingaffibody, expanding the investigated biomolecules further.19 As with some other pretargeting approaches,considerable washout was observed, potentially limiting the therapeuticapplicability. Nevertheless, for diagnostic purposes, such a strategyseems promising. Other biomolecules or fragments are, to the bestof our knowledge, hitherto unexplored for PNA-based pretargeting.However, the use of single domain antibodies (nanobodies), diabodies,or isolated antigen-binding fragments (Fab) could be of interest togenerate conjugates with better biodistribution and excretion dynamics.Noticeably, the shorterPNA constructs produced higher tumor-to-kidneyratios in xenograft studies, presumably due to favorable pharmacokinetics.30 Alternative PNA derivatives with improved solubility and/or reduced self-aggregationbehavior may further improve excretion of an unbound PNA-based probe.Similarly, the rapid clearance of unbound radiolabeled PNA constructsin vivo is desirable. Several authors have investigated the distributionbehavior of these constructs in the absence of biomolecules decoratedwith the complementary PNA fragment. The available results suggestthat shorter or PEGylated derivatives are more rapidly cleared, withclearance predominantly occurring via the urinary tract.Although PNA-based chimeras are relatively straightforward to synthesize,thanks to progress in technologies such as microwave-based peptideand nucleic acid synthesizers and the highly modular aspects in thenature of building blocks relevant to the molecular design, thereare aforementioned challenges regarding the delivery and subsequentcytosol distribution and biodistribution, which thus far slowed downtheir development for disease diagnostic purposes. In PNA constructscapable of avoiding or escaping endosomal entrapment post-cellulardelivery, the binding to (overexpressed) RNA must slow down the effluxsufficiently relative to unbound PNA to contribute to contrast.34Overall, the PNA constructs reported to date typically showed littleto no toxicity, both in vitro and in vivo, and are straightforwardto functionalize and adapt to specific targets. Despite some promisingadvances, no PNA chimera has made the transition into a clinical trialyet. Effective translocation across the blood–brain barrieris a problem observed with many drug molecules and has only been demonstratedfor a few of the PNA-based probes developed so far.The considerablerenal or hepatic retention would likely rendermost PNA chimeras unsuitable for imaging applications in the lowerabdomen if similar distribution patterns were found in humans. However,this may be addressed by choosing more suitable vectors and adjustinglinkers and/or overall charge of the molecule. The development ofPNA constructs for pretargeting applications may be a straightforwardway to address this issue. Similarly, to other pretargeting approaches,PNA-based constructs have great potential for further developmentand clinical translation, due to the specificity of their interactionwith a complementary PNA strand.35An alternative that may beable to address some of these problemsmay be the formulation of nanoparticulate PNA delivery systems orPNA nanomedicines. Synthetic developments in this area would relyheavily on the advances that have already been made in deliveringRNA into the cytosol, such as in mRNA vaccines, and in designing cancer-targetingnanoparticles.36 While endosomalentrapment is usually not a problem that is commonly observed withnanoparticles, a PNA-based agent which is encapsulated would stillhave to be released into the cytosol to take effect. One option toachieve this is the use of lipid nanoparticles. A limitation of nanoparticle-basedapproaches may be that they typically show slow biodistribution, asthey move through the lymphatic system. Due to this, and the significantamount of time needed for washout of unbound probe, some long-livednonstandard radioisotopes such as 89Zr or 52Mn may be of interest for nuclear imaging applications. Alternatively,other imaging modes could be considered, such as photoacoustic imaging,fluorescence imaging, or MRI, although the lower sensitivity requireslarger quantities.Many nanoparticlesare known to accumulate in cancerous tissues,which is typically explained through the debated enhanced retentionand permeation hypothesis.37 for diagnostic purposes. It is likely thatthere is a great number of different siRNAs and miRNAs yet to be discovered.Apart from targeting mRNAs, there are opportunitiesto target thevarious forms of noncoding RNAs, such as small interfering (si)RNAsor miRNAs, as demonstrated by some authors,40 For example,using a fluorophore/quencher system, Shigeto et al. were able to differentiatedifferent lung cancer cell lines by fluorescence microscopy.41 Using PNA-based imaging agents in this contextmay aid in the development of nucleic-acid-based therapies, by visualizingsuccessful delivery of active agents in preclinical development.Regarding optical imaging approaches capable of delivering informationon PNA probes’ behavior with high resolution at the in vitrolevel, several modalities have been successfully demonstrated, suchas the delivery of PNA/DNA binary Förster resonance energytransfer probes for visualization of splicing patterns, mRNA expressionlevels, or visualizing siRNA delivery into cells.Overall, PNA-based imaging agents have a number of potential diagnosticimaging applications, additional to therapeutic applications, bothin vitro and in vivo. Their high stability renders these probes ratherstraightforward to handle in drug delivery approaches, unlike RNAmolecules which are readily degraded by the virtually ubiquitous RNases,and their high hybridization energies with complementary PNA, RNA,or DNA strands leads to a plethora of therapeutic and diagnostic opportunities.


Knee osteoarthritis is the most common joint disease. It causes pain and suffering for affected patients and is the source of major economic costs for healthcare systems. Despite ongoing research, there is a lack of knowledge regarding disease mechanisms, biomarkers, and possible cures. Current treatments do not fulfill patients’ long-term needs, and it often requires invasive surgical procedures with subsequent long periods of rehabilitation. Researchers and companies worldwide are working to find a suitable cell source to engineer or regenerate a functional and healthy articular cartilage tissue to implant in the damaged area. Potential cell sources to accomplish this goal include embryonic stem cells, mesenchymal stem cells, or induced pluripotent stem cells. The differentiation of stem cells into different tissue types is complex, and a suitable concentration range of specific growth factors is vital. The cellular microenvironment during early embryonic development provides crucial information regarding concentrations of signaling molecules and morphogen gradients as these are essential inducers for tissue development. Thus, morphogen gradients implemented in developmental protocols aimed to engineer functional cartilage tissue can potentially generate cells comparable to those within native cartilage. In this review, we have summarized the problems with current treatments, potential cell sources for cell therapy, reviewed the progress of new treatments within the regenerative cartilage field, and highlighted the importance of cell quality, characterization assays, and chemically defined protocols. Osteoarthritis (OA) is the most common form of chronic joint disease, affecting all joints in the body, resulting in progressive cartilage degeneration. Risk factors associated with OA include age, obesity, family history, or trauma that has caused damage to the cartilage . It involves harvesting the patient’s cells from a healthy and non-weight-bearing donor site, the isolation of chondrocytes, i.e., ECM removal, and cell expansion One-stage 4th generation ACI techniques are emerging, and they are increasingly being implemented. Examples include mixing directly isolated chondrocytes with either directly isolated autologous bone marrow stem cells or allogeneic stem cells  such as microfracture. This arthroscopic technique creates small microfractures in the bone under the cartilage defect to trigger a regenerative response from mesenchymal stem cells (MSCs) in the bone marrow. The method is best suited for smaller defects created by trauma and not for OA , MSCs, and iPSC are potential cell sources for understanding OA disease mechanisms and use in a cell therapy-based treatment. ESCs are pluripotent and can divide infinitely , and fluorescent-activated cell sorting (FACS) are commonly used in combination with well-known chondrocyte markers such as collagen type II, SOX9, and aggrecan , but all have a high potential for cartilage regeneration. We have reviewed different cell-based products for cartilage regeneration to summarize their current market status and ongoing clinical trials with current methods and problems in mind.Worldwide, companies are focused on developing cell-based products that repair or regenerate cartilage to amend defects caused by, e.g., OA or trauma. Different strategies have been applied to accomplish this. The well-known ACI method has evolved to include a supporting matrix or scaffold product, aka matrix-associated autologous chondrocyte implantation (MACI). Recently, products that involved the administration of autologous or allogeneic stems cells through intra-articular injection have emerged, either with or without a supporting matrix. Another strategy is to surgically implant 3D biocompatible cell-seeded scaffolds, as described earlier in the review. However, cell-based therapies have been subject to strict regulation by authorities  where arthroscopically harvested chondrocytes are seeded within a matrix or scaffold material before implantation during a second procedure. These products have largely replaced previous product generations, which involved a liquid cell suspension and the use of a peristomal flap or a collagen membrane, such as Carticel and ChondroCelect   (Australian New Zealand Clinical Trial Registry, in vivo, including development, inflammation, wound healing, and cancer metastasis. These processes can be studied in vitro using quantifiable and controllable gradients to mimic those present in vivo. In stem cell differentiation and development, the gradients are essential inducers of tissue structure generation and functionality (Zecca et al., in vivo or in vitro (Teleman & Cohen, For decades, researchers have known about the importance of gradients in developmental biology (Zecca et al., Regarding stem cell differentiation towards chondrocytes, little research is published around biomolecular gradients and their influence on differentiation despite the evident importance during tissue development (Jullien & Gurdon, in vivo conditions with matrix-bound cells, where local concentrations influence them (Fig. Nano-gradient technology offers a platform with an extensive range of biomolecule binding possibilities, providing a broad potential to gain knowledge of differentiation and cell-protein interactions. Moreover, the technology provides new opportunities to elucidate dose-dependent events, such as inducing migratory behavior. The nano-gradients are gradients of activator molecules bound to gold nanoparticles precisely distributed on a surface. They provide a unique chemically and physically defined substrate for controlled culture systems with a highly reproducible capacity (Andreasson et al., hem Fig. . These phem Fig. .Fig. 1Thin vivo conditions, combining other materials and technologies, and forming a step in the differentiation protocol from where the cells can be removed and further cultured. The nano-gradient technology allows for screening an optimal growth factor density providing a robust differentiation protocol due to a precise and controlled stimulation, compared to, e.g., a solution-based gradient where growth factors are constantly moving (Minchiotti et al., The nano-gradients also allow the opportunity to combine factors, e.g., a growth factor with an ECM protein, further mimicking in vivo conditions. Stem cell cultures for therapy require high cell quality and a homogeneous cell population; however, traditional 2D cultures provide limited expansion and differentiation capacity (Zhang et al., in vivo enable regulation of cell responses, which are necessary for the function and structure during tissue generation in embryonic development (Zecca et al., All stem cell research and therapeutic applications, such as tissue regeneration, require defined and stable protocols to precisely control the cells during differentiation, but also to maintain required cellular properties and simultaneously mimic Current treatments of local cartilage lesions and OA focus on reducing pain and inflammation with insufficient long-term results. Today, no treatment is focused on disease-modifying mechanisms, and cell-based therapies struggle to generate high-quality cartilage. MSCs have become a commonly used cell source in developing approved and generally accepted stem cell therapy. Many companies have ongoing or completed clinical trials with promising results despite possible drawbacks, such as MSCs tending to form hypertrophic chondrocytes and bone instead of high-quality hyaline cartilage during chondrogenic differentiation. iPSC-derived chondrocytes have emerged as a potential alternative to MSCs, overcoming many of their drawbacks. However, issues, such as safety, have not been fully investigated to successfully commercialize iPSC-based treatments. To our knowledge, only one iPSC-based therapy for OA is in the clinical phase, currently undergoing a significant phase III trial. Biomolecular gradients are a potential aid to overcome problems with the differentiation of iPSCs. Gradients are essential in embryonic development. By utilizing gradients in the differentiation protocols, it is possible to provide a defined molecular stimulation to the cells and increase robustness compared to earlier protocols. A stable and more robust gradient would theoretically aid in generating a defined cell population for implantation into the damaged cartilage area. Further research, however, is required to accomplish this. Nonetheless, the research and development in this area are rapidly evolving in the quest to use stem cell-based therapies to treat cartilage damage.


Splenosis is a rare disease, which is often discovered incidentally years after surgical procedures on the spleen or traumatic splenic lesions. Through injury of the splenic capsule, splenic cells are able to spread and autoimplant in a fashion similar to the process of metastatic cancer. Here we present the case of a 62-year-old female patient with a palpable tumor of the lower abdomen. Her medical history was unremarkable, except for splenectomy after traumatic splenic lesion in her childhood. Clinical examination and diagnostic imaging raised the suspicion of advanced ovarian cancer, which was further substantiated by the typical presentation of adnexal masses and disseminated peritoneal metastases during the following staging laparotomy. Surprisingly, we also found peritoneal implants macroscopically similar to splenic tissue. Microscopic examination of tissue specimens by intrasurgical frozen section confirmed the diagnosis of intra-abdominal splenosis. The patient then underwent cytoreductive surgery with complete resection of all cancer manifestations, sparing the remaining foci of splenosis to avoid further morbidity. This case demonstrates the rare coincidence of intra-abdominal carcinoma and splenosis, which could lead to intraoperative difficulties by misinterpreting benign splenic tissue. Therefore, splenosis should be considered in patients with medical history of splenic lesions and further diagnostic imaging like Tc-99m-tagged heat-damaged RBC scan could be used for presurgical distinguishing between tumor spread in the abdominal cavity and disseminated splenosis. The presented case report should not only raise awareness for the rare disease splenosis, but also emphasize the need to consider the possibility of simultaneous incidence of benign and malignant intra-abdominal lesions, as to our knowledge this is the first published case of simultaneous peritoneal carcinomatosis and splenosis. Splenosis is a rare disease, which is often discovered incidentally years after surgical procedures on the spleen or traumatic splenic lesions with subsequent splenectomy. Through injury of the splenic capsule, splenic cells are able to spread into the abdominal cavity comparable to disseminated peritoneal metastases. In the majority of cases with reported splenosis, the splenic tissue organizes itself like a congenital accessory spleen on various ectopic intra-abdominal locations. There are rare cases, especially with additional laceration of the diaphragm, where splenic tissue is even found in the thoracic cavity.A 62-year-old patient (II Gravida/II Para) presented with diarrhea for several weeks and a palpable tumor of the lower abdomen. Her medical history was unremarkable, except for having had a splenectomy after traumatic splenic lesion in her childhood. Clinical examination showed a palpable mass in the lower abdomen without any further anomalies. Using vaginal ultrasound, we detected ascites and suspicious adnexal masses. On the right ovary, there was a 38 × 28 mm large, homogeneous, solid, hypervascular tumor  and on Further diagnostic workup included a coloscopy and gastroscopy without any pathological findings and a computer tomography (CT) scan of the abdomen and thorax . In theThe intraoperative situs showed the typical appearance of advanced ovarian cancer with disseminated peritoneal metastases. In addition to superficial tumor spread in the abdominal cavity and adnexal masses, there were multiple peritoneal implants, involving the mesentery and serosa of the small intestine, macroscopically similar to splenic tissue . AlthouWith the intrasurgical confirmation of splenosis, the remaining foci of splenosis could be spared and surgery was limited to clinically suspected cancer manifestations. Cytoreductive surgery comprised hysterectomy, bilateral adnexectomy, deperitonealization of the recto-uterine pouch, omentectomy, appendectomy, and resection of several peritoneal cancer lesions from the mesentery and resulted in macroscopic complete resection. Histology showed a high grade serous ovarian cancer, pT3b, N0, M0, L0, V0, Pn0 (FIGO IIIb). The patient was hospitalized for 12 days and afterwards underwent chemotherapy with six cycles of carboplatin/paclitaxel, as well as a maintenance therapy with bevacizumab for 1 year. After surgery and the first cycle of chemotherapy, CA125 levels returned to normal (10.5 U/mL) and did not increase again during therapy. We could discharge the patient into oncological follow-up care without any clinical evidence of remaining or recurrent cancer (follow-up time so far 17 months after surgery).One of the most important goals in treatment of advanced epithelial ovarian cancer— determining overall survival and progression free survival—is complete resection of the tumor mass in primary surgery.Despite its rare occurrence, there are other cases described in literature, where splenosis led to complications during surgery. Nicolas et al for example presented a case of a 30-year-old patient, who showed excessive weight gain after gastric band surgery and was therefore indicated for conversion into a gastric sleeve.Another pitfall was described by Sorensen et al, which shows the consequences of misdiagnosis for the individual patient and strongly emphasizes the importance of awareness.These cases emphasize the importance of diagnosing splenosis prior to surgery. However, it is a big challenge in clinical routine and especially awareness of this disease is limited. Although the lesions are visible in CT scans with similarity to splenic tissue in density and enhancement of contrast agent, reliable diagnosis is difficult.After imaging diagnostics, several patients were misdiagnosed with malignomas like hepatocellular carcinoma,One method is the Tc-99m-tagged heat-damaged RBC scan (Tc-99m-DRCB), where heat denaturated red blood cells are tagged with Tc-99m. These denaturated cells accumulate in the red pulp and can be used to detect splenic tissue.111Indium-tagged thrombocytes, which gather in the reticuloendothelial system of splenic tissue.Another technique is using scintigraphy with111Indium-tagged thrombocytes was performed in the presented case.Since there are no distinct symptoms of splenosis, patients who underwent splenectomy should generously undergo prior examination for remaining splenic tissue. Especially in the absence of Howell-Jolly-Bodies, the lack of increase in reticulocyte count and the presence of antipneumococcal antibodies in nonvaccinated patientsTo our knowledge, this is the first published case of simultaneous incidence of peritoneal carcinosis and splenosis. Most of the cases in the literature only report splenosis as a differential diagnosis to peritoneal carcinomatosis.In conclusion, intra-abdominal splenosis is a rare event in patients who underwent surgical procedures of the spleen or traumatic splenic lesions. Coincidence of splenosis with malignancies can mimic peritoneal cancer spread to the abdominal cavity and may lead to misinterpretation of the tumor extent. However, reliable discrimination of cancer masses and splenosis is essential for the prevention of unnecessary surgery and avoiding further morbidity, as well as for the correct classification of clinical tumor stage and the appropriate treatment scheduling. Consideration of splenosis as a potential differential diagnosis for intra-abdominal tumor lesions is important, especially in patients with corresponding medical history, and physicians should be aware of this rare event also in cases of simultaneous malignancies.


Many recent studies highlight the fundamental importance of viruses. Besides their important role as human and animal pathogens, their beneficial, commensal or harmful functions are poorly understood. By developing and applying tailored bioinformatical tools in important virological models, the Marie Skłodowska-Curie Initiative International Training Network VIROINF will provide a better understanding of viruses and the interaction with their hosts. This will open the door to validate methods of improving viral growth, morphogenesis and development, as well as to control strategies against unwanted microorganisms. The key feature of VIROINF is its interdisciplinary nature, which brings together virologists and bioinformaticians to achieve common goals. The Innovative Training Network (ITN) is one of the Marie Skłodowska-Curie Actions of the European Commission, which aims to train a new generation of creative, entrepreneurial and innovative early-stage researchers, able to face current and future challenges and to convert knowledge and ideas into products and services for economic and social benefit. Within the ITN VIROINF, we aim to understand virus-host interactions, by bridging virology and bioinformatics being an emerging field, see The main themes of VIROINF are: (i) Modelling virus-host interactions, including virus identification, host prediction, virus-host interactions, virus regulation, and virus products; and (ii) Modelling virus evolution in hosts, including microevolution (virus quasispecies) and macroevolution  in Jena in 2017, a broad range of applied bioinformaticians and virologists met to guide developments in virus bioinformatics. The interactions between the members resulted in the identification of a broad range of opportunities for collaboration between the research groups. In order to create a framework for collaboration between these research groups we propose the implementation of the VIROINF ITN  and requThis part of the ITN aims at developing fundamental bioinformatical tools for modelling virus-host interactions and understanding these interactions mechanistically. In order to describe virus-host interactions, interacting viruses need to be identified and hosts need to be determined.en masse, but different research groups use different protocols to analyse these samples and prepare them for sequencing. All the current approaches result in relative distributions, which lack absolute quantitation. Sample preparation, methods and standardisation are prerequisites for meaningful and comparative work. On the bioinformatical side, the most frequently used similarity-based virus identification approaches are largely limited by available databases, which are incomplete and often poorly annotated. VIROINF aims to set up a multiple layer database for phages and their hosts, which with the help of the European Virus Bioinformatics Center will be performed in the first months of VIROINF.Despite large ongoing efforts to identify viruses in humans, animals and the environment, there are still multiple challenges remaining in the identification of novel viruses. These challenges concern both the wet-lab as well as the bioinformatical side. On the wet-lab side, divergent viruses can now be identified from an infected sample VIROINF aims to quantify viruses by the following four approaches: (i) spiking in known amounts of selected viruses into a sample as internal controls; (ii) counting virus particles using flow cytometric approaches in collaboration with Corina Brussaard from the Netherlands Institute for Sea Research; (iii) test Minion sequencing technology on unamplified extracted viral nucleic acids, and (iv) NGS-based quantification developed in collaboration with non-academic partner Baseclear. The novel wet-lab approaches will be combined with newly developed advanced bioinformatical tools to identify viruses from metagenomics datasets (ESR-UMG), using sequence dependent and independent approaches. Motif searches, k-mer frequencies, and genome organisation are de novo approaches that will complement homology-based methods, together allowing the detection of known and unknown viruses, yielding a comprehensive detection pipeline of all types of viruses ranging from well-studied human pathogens to completely novel bacteriophages. A range of available tools for taxonomic classification will be combined into a rapid pipeline that will be used from many beneficiaries: ESR-FU, ESR-HMGU, ESR-JMU, ESR-ETH, ESR-RKI, ESR-UZH and ESR-UL. These tools will be made available in the public domain by VIROINF. Together, these developed wet-lab and bioinformatical approaches will be used by ESR-UL to provide an in depth in silico and in vivo characterization of the bacteriophage population infecting the most important members of the honeybee gut microbiota, as well as studying their role in honey bee health and development.ESR-UL—Development of more quantitative viral metagenomics approaches to investigate the role of bacteriophages in the honey bee gut and their effects on health and development. Very few is known about the role of the gut virus population (the virome) in health and disease of honey bees. Recently the NetoVIR protocol was developed to purify viral particles from biological samples, before deep sequencing using Illumina NGS technology [chnology . Using tThe third objective of this project is to provide an in depth in silico and in vivo characterization of the bacteriophage population infecting the most important members of the honeybee gut microbiota, as well as studying their role in honey bee health and development. Both the bacteriome (16 s) and the virome (shotgun) inhabiting the digestive tract of these honey bees will be characterized. Furthermore, bacteria will be isolated and characterized from the crop, midgut and hindgut of the collected honeybees, and finally these bacterial isolates will be used to isolate bacteriophages followed by their genome sequencing and functional gene annotation. Depending on the available time, we may also start with inoculation experiments of life honey bees see .ESR-UMG—A machine learning pipeline to identify and characterise novel viruses in metagenome data. The wide use of deep sequencing technology in biology and medicine has led to huge publicly available sequencing data bases, offering novel insights into the genetic and genomic makeup of life. Similarly, metagenome sequencing projects offer a deep view into microbiomes. Only a minuscule fraction of the virome has so far been identified; however, many hitherto unknown viral sequences lie hidden in already available data. The objective is to develop efficient computational pipelines to search for novel viruses in genome or metagenome sequencing data, as well as in public DNA and RNA sequencing databases such as NCBI Genbank/SRA. Template-based search strategies will be developed to search for unknown viruses that are related to a known virus family which is used as search template [template . We willHosts of viruses infecting higher eukaryotes have been well described in the past, whereas the specific hosts of bacteria-infecting viruses are usually unknown. The aims of VIROINF are to investigate how viruses of Bacteria or Archaea (bacteriophages) are functionally linked to each other and to their unicellular hosts. This is crucial for understanding how phages impact the microbiome as they act as key regulators of bacterial population size , and canStaphylococcus aureus. Phages are important mediators of bacterial virulence and hence impact human health. Understanding how bacterial gene content is influenced by this lateral gene transfer mechanism is important for understanding how viruses impact both the specific bacteria and the human host [VIROINF will, for the first time, address this topic using a combined experimental and computational approach: ESR-HMGU develops methods to experimentally link phages to their hosts as described by Li Deng , as wellman host . The tooESR-HMGU—Bacteriophage–host relationships prediction. This project seeks to understand how (bacterio)phages are functionally linked to their Bacteria or Archaea hosts both using experimental and bioinformatics approach. Phages identified using cultivation independent viral metagenomic sequencing approach lack sequence similarity to any of previously sequenced ones and can hardly be assigned to their hosts. Different methods exist to predict hosts for phage sequence, e.g., single or multiple features , or using these features in machine learning models. Therefore, we aim to integrate features of our unique experimental identified, host-linked viral metagenomic data in a deep learning model to improve the prediction performance. Particular attention will be given to phages of anaerobic bacteria which are underrepresented comparing to those infecting aerobic bacteria [bacteria . ESR-HMGbacteria ,10,11. IESR-UG—Computational prediction of virus-host interactions in the microbiome. Virus-host interactions are highly specific as viruses (including bacteriophages/phages) have co-evolved with the systems they infect and depend on for replication. As a consequence, phylogenetic relatedness and shared genomic properties of host species are a strong predictors of host infectivity. However, our knowledge of host relationships remains sparse and is unknown for many viruses and phages, particularly for metagenomically assembled data. In this project, existing resources and genomic signals will be leveraged to infer missing/unobserved and probable hosts, e.g., where infection has not been observed due to host immunity. Machine learning models will be trained on this data to predict virus-host/ phage-bacteria interactions at the species and strain levels  . A new aESR-JMU—Conservation of regulatory elements and effector mechanisms in lytic and latent cytomegalovirus infection. The human cytomegalovirus (HCMV) is a herpesvirus prevalent worldwide responsible for life-threatening infections in individuals with an impaired or immature immune system. During the first 1–2 h after virus entry into a cell, a decision is made whether this results in lytic, latent or abortive infection. Previous single cell RNA-seq (scRNA-seq) approaches did neither provide the necessary temporal resolution nor sensitivity to analyse this crucial phase of virus-host interaction. To overcome these limitations, we recently developed single-cell SLAM-seq ESR-RUMC—Impact of host immune pathways on virus evolution Evolution of viruses is strongly affected by antagonistic co-evolution of virus and host. Host immune pathways select for viruses that evade, avoid, or suppress the immune response, which in turn may drive counter-adaptations in host immune genes. For example, the antiviral RNA interference (RNAi) mechanism is actively suppressed by many insect viruses [Drosophila melanogaster and other invertebrates [Drosophila C virus, widespread pathogens of Drosophila that suppress RNAi at different stages [ viruses , likely tebrates ,21. Whett stages ,23.ESR-WZ—Elucidating the role of RNA modification during cytomegalovirus infection. N6-methyladenosine (m6A) plays critical roles in RNA metabolism and function. In addition to the internal m6A, N6,2′-O-dimethyladenosine (m6Am) is present at the transcription start nucleotide of capped mRNAs. The methyltransferase that adds this modification, PCIF1, was recently identified but the functional importance of this modification still remained enigmatic.Human cytomegalovirus (HCMV) is an important human pathogen that replicates in the nucleus exploiting cellular machineries. We will investigate if HCMV utilises the m6Am modification by conducting m6A-IP and high throughput sequencing using primary fibroblasts and investigate its functional importance especially to viral titers in the absence of PCIF1. Our objectives are: (a) We will examine m6Am modification landscape during viral infection  by conducting m6A-IP on HCMV infected primary fibroblasts and analysing the enrichment of the first base compared to input control. (b) We will dissect the effect of this modification on viral propagation by generating PCIF1 KO cells. (c) We will analyse how m6Am modification affects viral RNA lifecycle using RNA-seq , 4sU-seq  and Ribo-seq (RNA translation) applied on infected PCIF1 KO and control cells.Viruses circulate predominantly in their natural reservoir host but frequently also infect other species. However, to establish a new lineage the virus has to adapt to the new environment by acquiring advantageous new mutations. These mutations also influence the secondary structure of the viral DNA and RNA, in turn affecting the replication process. ESR-RKI, will study the fundamental correlation between virus evolution and RNA secondary structures. Being more specific, in case of segmented genomes ) packaging is influenced by the secondary structure of the virus genomic RNA segments. In cells co-infected with different viruses, new variants with mixed genomes can emerge that have the potential to overcome the natural host species barrier and to cause a devastating pandemic. Mixing of viral segments, also designated genetic reassortment, is possible due to a very efficient incorporation of different genome segments into one virus particle. ESR-CNRS will systematically generate reassortant viruses between two human IAVs and select those that replicate most efficiently for further functional studies. Additionally, ESR-CNRS will use chemical probing to compare the genome structure in parental and those reassortant viruses. ESR-FSU will study genetic reassortment events of IAV and especially the role of RNA-RNA interaction that are supposed to coordinate the interaction of the different genomes which are finally incorporated into viral particles. The focus of ESR-FSU is to develop a computational tool that can predict the complex interplay of the RNA-RNA interaction required for genome packing between quite diverse IAV based on experimental data.ESR-FSU—Deciphering the RNA genome packaging code of influenza A viruses. Currently, bioinformatical tools are not specifically designed for viruses. However, viruses bring unique features, which require specific bioinformatical tools to trace virus-host interaction. For example the number of sequences in a quasispecies is massively high due to their high mutation rate, but only a few interact again with the host cells. Some viruses, such as IAV, are segmented RNA viruses, which urgently require tools with specific features: RNA viruses should include standardised secondary structure predictions, leading to RNA-RNA interaction prediction necessary for the packaging of segmented RNA viruses, as preliminary depicted in [icted in ,25. TherESR-CNRS—Mechanism of coordinated incorporation of segmented virus genomes into viral particles. Genetic reassortment between divergent IAVs in the host is a highly biased process in which only a small fraction of all possible genotypes are efficiently produced. This restriction is due to the limited compatibility between packaging signals of divergent IAVs and the limited conservation of RNA-RNA interactions required for genome packaging, but might also be influenced by the host as environment. Thus, analyses of genetic reassortment not only shed light on how viruses with pandemic potential are produced, but also provides crucial information about the coordinated incorporation of the segmented IAV genome. Therefore, we will (a) generate reassortants between two human IAVs that will contain seven segments from one parental virus and one segment from the second parental virus. (b) Compare the secondary structure of the genomic RNA segments in the two parental and up to eight selected reassortant viruses grown in human cells using chemical probing. This analysis will be performed with ESR FU1 and highlights differences in RNA-RNA interactions between parental and reassortant viruses  metagenomics samples. We aim for large-scale identification of phage sequences, functional annotation and in vitro validation of phages as treatment against harmful bacteria.ESR-UZH—Generation of experimental datasets for phage-bacterial interactions in complex communities and mutational spectra analysis following exposure to selective pressure to support artificial intelligence applications for R&D of bacteriophage products. Bacteriophages are the most abundant organisms on Earth, making it difficult to select which ones have potential for therapeutic or other commercial applications. Current methods rely on non-iterative in vitro elaboration for product development, requiring >6 months per phage product. In the current project, we aim to generate input data from experiments to build deep learning algorithms that will guide and support phage product development by (a) revealing which phages compete best under which specific conditions and combinations, (b) identifying mutations important for phage activity and survival under environmental conditions , and (c) demonstrating the population effects of phage infection in complex community structures. In this project, we will use a top-down approach to characterise the resulting community composition and mutational spectra from in vitro assays and/or clinical samples to measure: (i) competition between multiple phages; (ii) phage-interaction in complex communities to simulate microbiome and polymicrobial infection settings; and (iii) exposition of phage to multiple environmental conditions using genomic re-sequencing and/or cycling temperature capillary electrophoresis (CTCE) to identify regions of mutations associated with specific stimuli.ESR-UV—Computational methods for the analysis of metagenomic datasets to extract viral sequences within the context of commercial data-mining/R&D support for bacteriophage products. Bacteriophages are increasingly being developed for the treatment of bacterial infections, and their role as modulators of the microbiome is of a growing area of extension of phage use. The sheer number and diversity of phage particles makes it impossible to isolate and test each of them individually. This project aims to identify and harness phage sequences for commercial use by developing a method for large-scale identification of phage sequences, functional annotation, and in vitro validation of findings in a bottom-up approach. We have four specific aims: (i) Identification of phage sequences within >50 metagenomic datasets . Reconstruction of viral genomes sequences from metagenomic data ; Reconstruction of cellular genome sequences (MAGs) from the same/related metagenomic datasets, in order to extract potential phage-host pairs from co-occurrence patterns; Prediction of prophage sequences in MAG in order to facilitate host prediction of phage sequences (in collaboration with ESR-UMG). (ii) Extraction and testing of sequences to validate WP 1 findings ie., hypothesis testing by either isolating the phages themselves or having their genome synthesised (in collaboration with ESR-UZH). (iii) Sub-analysis of geographical and temporal variation in metagenomic datasets that could justify product composition or modification analysis of geographic and temporal variation of phage sequences according to the local epidemiology  is examined in the viral population, also known as ‘virus quasispecies’. In the area of macroevolution, the long term evolution (millions of years) is examined to determine the relation of virus families.Viruses display high genetic diversity both within and among viral species, as well as within and among infected hosts. The composition of mixed samples can be assessed by metagenomics approaches , such asVIROINF will search for patterns which can emerge when host affiliation is projected onto viral taxonomy. Branch permutation techniques will be used to statistically determine at which level the viruses and hosts co-evolve. These patterns will reveal the evolution of virus-host associations for different classes of viruses. The associations will be validated by analysing both, known and predicted, host-associations independently. Moreover, virus-host associations will be used to validate the co-evolutionary signal.ESR-ETH—Development of new computational methods for characterisation and analysis of intra-host viral populations. Viruses exist in their hosts as populations of genetically heterogeneous particles, often referred to as viral quasispecies. Intra-host genomic diversity can result in phenotypic heterogeneity, and it has been linked to viral pathogenesis and virulence. NGS can be used to assess the genomic heterogeneity of virus populations in a cost-efficient manner, but this approach is challenging due to sequencing errors and short read length. In this project, we will develop improved tools for the reconstruction of viral genomic diversity from both short-read and long-read NGS data, and we will leverage the power of the inferred population structure to inform models of virus micro-evolution, to detect selection, and to correlate population features to viral and host phenotypes.ESR-RKI—Genotype-Phenotype mapping and inference of epistatic interactions driving adaptation in viral hosts. Viruses hijack the host cellular machinery for replication. This hijacking is driven by the interaction of viral proteins and non-coding RNAs with host-cellular components. Viral genomic sites instrumental to these interactions are thus conserved through evolution, yet bioinformatics analysis of evolutionary conservation cannot tell apart and quantify the functional relevance of genomic sites under selection. We have previously shown using the Mutational Interference Mapping Experiment (MIME) that in vitro [in vitro  and in vin vitro  evolutioin vitro  to identDrosophila virus) to predict phenotypic contributions to virus growth, mortality and host transcriptional responses.On the technical side, (a) we will learn maximum entropy models  from nucleotide abundances in functionally selected and de-selected virus quasispecies to identify single- and interacting sites. (b) As an alternative approach and a means to validate a), we investigate the application of deep learning approaches to identify higher-order epistasis in collaboration with secondment Bernhard Renard (HPI). (c) We then derive algebraic expressions from kinetic modelling of the investigated selection process to interpret the derived coupling terms mechanistically . This allows to characterise the complex (and possibly highly constraining) fitness landscape on which adaptation takes place. In the development phase our computational methods will be benchmarked by simulating the selection experiments and the corresponding NGS data sets, as well as with existing NGS datasets and phenotypic endpoints from previous work . Subsequently, we will apply the tools to NGS data generated by in vitro evolution experiments of ESR-CNRS/secondment R. Smyth (HIRI) to investigate domains in the influenza genomic RNA, which are responsible for viral packaging/re-assortment, in collaboration with AllGenetics (AG). Moreover, we will apply the methods to in vivo evolution experiments conducted by ESR-FU (deformed wing virus), as well as ESR-RUMC , but thESR-FU—Evolution of Deformed wing virus (DWV) in bees.Varroa destructor, which can vector the virus. In this project, we will carry out experiments with the aim of understanding the genomic basis of virus virulence (see This project seeks to understand the evolution and impact of virus virulence. The honeybee virus Deformed wing virus (DWV) is an emerging pathogen that is responsible for widespread declines of honeybees in the Northern hemisphere. The impact of the virus has been exacerbated by the arrival of an ectoparasitic mite: ence see . TogetheLayer 1: project specific training (bioinformatics and virology) and scientific and transferable skill courses at the host institution; Layer 2: training through project-specific ESR-ESR partnerships , and through secondments; Layer 3: training through network-wide events. Yellow stars indicate additional training for the ESRs ).We hope to be a nucleus that not only conducts fundamental research, but also reaches out into both research communities—virologists and bioinformaticians—to foster further collaborations. We are open to discussions about integrating your appreciated research in any way possible, from joint talks or conferences, to pair-wise collaborations, to association in our consortium. Please do not hesitate to contact us (


While osseointegration has become already clinically established concept in healthy patients, clinicians are facing a growing number of implant-supported rehabilitations on pathophysiologically impacted bone.locus minoris resistenziae. In fact, a high-performance surface might be requested for a long-standing integration of the fixture.In these scenarios, moderately rough surfaces/traditional protocols might represent a Bioactive surfaces with increased surface energy might respond to this requisite without encountering possible higher risk of bacterial contamination as rough-surfaced implants demonstrated.Actually, the surface energy directly correlates with hydrophilicity and (on the contrary) indirectly with the presence of contaminants on the surface. In fact, it decreases with increased surface deposition of atmospheric elements or pollutants (present even on the “sterile” new implants).As demonstrated, decontamination of the implant surface is an essential prerequisite for cell adhesion. However, even in optimal conditions of surface decontamination, the titanium fixture still remains hydrophobic and, then, less “tissue friendly.” This is correlated to the oxidation of the external titanium layers due to the presence of oxygen into the implant sterile package.The bioactivation through chemical or biophysical methods increases fixture surface energy and then wettability, removing the oxidized external layers.The biological advantage of such activation is both qualitative (higher number of adhered cells) and quantitative (flat vs. spreaded arrangement), with a stronger adhesion, and this implies a faster cell adhesion and better cell stratification.Translated to the clinics, this strategy promises to result in stronger osseintegration even in the initial stages of the treatment in physiological quality bone or after the traditional timing in compromised bone-quality patients.Luigi CanulloHenriette LernerPaolo Pesce