Neurological complications due to reactivation of varicella-zoster virus (VZV) are very uncommon in immunocompetent patients. Generally a vesicular rash is present on one or more dermatomes, preceding or following the main manifestation. Few cases are reported in the international literature, but they concern mainly adult or elderly patients.A 12-year-old girl was referred to our hospital for persisting headache, cough and rhinitis for six days. After first examination, diagnosis of anterior sinusitis was made by nasal endoscopy. The day after, the girl developed psychotic symptoms and altered mental status. Computed tomography (CT) scan was immediately performed but was unremarkable; lumbar puncture revealed leukocytosis with lymphocytic predominance and cerebrospinal fluid polymerase chain reaction (PCR) detected varicella-zoster virus DNA. The diagnosis of acute VZV encephalitis was made. The patient was promptly treated with acyclovir infused intravenously and her clinical conditions rapidly improved. Tests made did not show any condition of immunosuppression.Although if rare, reactivation of VZV can occur in immunocompetent children and its complications can involve central nervous system. Among these complications, meningitis is more common, but cerebral parenchyma can also be involved leading to a severe medical condition that is defined meningoencephalitis. In rare cases vesicular rash may be absent; therefore high level of suspicion is required even in those patients in which suggestive clinical features are not present to guide the diagnosis. Intravenous acyclovir represents the treatment of choice to obtain a fast clinical response and to prevent the onset of late-term complications. Varicella-zoster virus (VZV) is a neurotrophic alphaherpesvirus infecting exclusively humans. VZV causes varicella (chickenpox) during the first infection  and then becomes latent in cranial-nerve and dorsal-root ganglia. It can reactivate after several years causing zoster (shingles), sometimes followed by post-herpetic neuralgia. Reactivation generally occurs in elder population or in immunocompromised patients . VZV canA 12-year-old girl was admitted to our hospital for persisting headache, started six days before. At the admission she presented also cough and rhinitis; she referred mild fever (axillary temperature 38 °C) during the previous days and three episodes of vomit. If at the beginning the pain was controlled by paracetamol, during the last days the headache had become deep, localized in the frontal region and associated to photophobia, even though at the beginning the pain was controlled by paracetamol. She had no personal or familial history of headache. Her medical history was unremarkable except for an episode of chickenpox during her infancy. On physical examination vital signs were stable , the girl appeared suffering but her general conditions were good. Central nervous system examination didn’t show any neurological deficit, Kernings and Brudzinski signs were negative, she had no neck stiffness. Deep tendons reflexes were normal. Pupils were equal, round and reactive to light; accommodation reflex was present. Nor nystagmus or diplopia was noticed. Clinical examination of heart, lungs and abdomen was negative. On the skin no lesions were present. Blood count was in normal range, reactive C-protein (CRP) and procalcitonin (PCT) were negative, liver and renal functions were preserved. Analgesic and antipyretic treatment was administered. Nasal endoscopy was performed on the second day of hospitalization and highlighted a picture of acute bacterial sinusitis. Antibiotic therapy was started. The day after the girl developed altered mental status and psychomotor agitation; the headache worsened and was accompanied by three episodes of vomiting events; in few hours the girl developed drowsiness and became less responsive to stimuli. Blood tests were repeated but values were still in normal range. Computed tomography (CT) was performed immediately but was unremarkable. Electroencephalogram (EEG) showed “severe alteration of cortical electrogenesis with exacerbation of diffuse slow cortical activity, with fronto-temporal predominance”. Cerebrospinal fluid examination showed increased protein concentration (72 mg/dl), normal glucose concentration , lymphocytic pleocytosis . The findings were suggestive for viral infection of CNS. Varicella-zoster virus was detected using polymerase chain reaction (PCR). The research of genome of herpes simplex virus 1 and 2, human herpes virus 6, cytomegalovirus and Ebstein-Barr virus was negative. Serological tests showed the presence of IgG vs. VZV and absence of IgM vs. VZV, confirming the reactivation of VZV. Immunological screening for HIV was negative, serum immunoglobulins, T and B lymphocytes counts, complement proteins levels were within normal range for age.The girl was treated with intravenous acyclovir 10 mg/kg three times a day. During the first day of antiviral therapy her clinical conditions rapidly improved and the following day she was well oriented, smiling and quiet, not remembering the events of the days before. Her headache disappeared completely after two days. After 2 weeks of treatment, she was dismissed with oral acyclovir to be continued for 2 weeks more.EEG was performed again after 3 and 12 days from the onset of encephalitis, showing progressive improvement of cortical activity, but a persistent slow focal activity in the right temporal region was registered. Magnetic resonance was performed after 2 days from the diagnosis of encephalitis and one month after the discharge and was unremarkable in both cases. Lumbar puncture was performed again after 14 days of therapy: protein concentration and glucose level were in normal range, a mild leukocytosis was still present . PCR for VZV at that time was negative.Varicella-zoster virus (VZV) causes two clinically distinct diseases. Varicella (or chickenpox) is the result of primary infection, it is extremely contagious and occurs mainly among preschool and school-aged children; the main characteristic is a generalized itchy vesicular rash. Although in most cases the evolution of varicella is benign, complications and atypical presentations can occur in previously healthy people, but their incidence is greater in immunocompromised patients . After pIt is known that varicella-zoster virus can cause several CNS disorders, such as meningitis, cerebellitis, myelitis, vasculitis and stroke-related syndromes. It was recognized as the second most common virus causing viral encephalitis, after herpes simplex virus. This frequency is declining in recent years in some countries after the introduction of VZV vaccination. VZV infections of the central nervous system are generally more frequent in immunocompromised individuals, but are not exclusive of this group of patients. In children, the most common CNS complication is cerebellitis that can develop during primary infection. In adults, encephalitis and meningitis have a higher incidence than in children and are usually subsequent to the reactivation of VZV . In pediThe absence of vesicular rash preceding or following CNS involvement is rare. In a study of CNS complications of VZV carried in 84 children with neurological symptoms associated with VZV infection, only two patients had no rash, despite the diagnosis of VZV meningitis, confirmed by PCR analysis of the CFS .The case we described presents peculiar medical features: encephalitis was caused by reactivation of VZV in an immunocompetent child, who did not present the typical rash. Consequently, we decided to perform a systematic review of the literature on PubMed database matching words “encephalitis”, “central nervous system”, “immunocompetent”, and “varicella zoster”. Of 103 results obtained, we analyzed title and abstract of all relevant papers, regardless of language. We found only two reported cases of encephalitis due to VZV reactivation in pediatric patients in absence of waning of immune system function. A comparison between our case and previous described ones is showed in Table In our opinion, it is significant to highlight that none of the three patients discussed showed the vesicular rash, therefore we should keep a high level of suspicion of VZV infection of central nervous system even in the absence of suggestive diagnostic clinical features. In the suspicion of VZV encephalitis, polymerase chain reaction (PCR) allows the detection of viral DNA and RNA in CSF samples with high sensitivity and specificity , 20. BecIntravenous acyclovir 10–15 mg/kg every 8 h is the treatment of choice; the recommended duration is 14 days but if compromised immune system function is suspected or known, the treatment should be extended to 21 days. Many experts recommend adjunct corticosteroids if CNS vasculitis is present .Even if rare, reactivation of VZV with subsequent involvement of central nervous system can occur in previous healthy people, including children. Vesicular rash could be absent; therefore high level of suspicion for VZV infection has to be kept in patients with clinical, biochemical and instrumental findings suggestive for viral meningoencephalitis. PCR of cerebrospinal fluid is the faster tool to achieve a correct diagnosis and start a prompt therapy. Intravenous acyclovir is the drug of choice.


Adoptive immunotherapy with tumour-infiltrating lymphocytes (TIL) may benefit from the use of selective markers, such as PD-1, for tumour-specific T-cell enrichment, and the identification of predictive factors that help identify those patients capable of rendering tumour-reactive TILs. We have investigated this in ovarian cancer (OC) patients as candidates for TIL therapy implementation.− and PD-1+ CD8 TILs were isolated from ovarian tumours and expanded cells were tested against autologous tumour cells. Baseline tumour samples were examined using flow cytometry, multiplexed immunofluorescence and Nanostring technology, for gene expression analyses, as well as a next-generation sequencing gene panel, for tumour mutational burden (TMB) calculation.PD-1+ fraction. Importantly, a high TIL density in the fresh tumour, the presence of CD137+ cells within the PD-1+CD8+ TIL subset and their location in the tumour epithelium, together with a baseline T-cell-inflamed genetic signature and/or a high TMB, are features that identify patients rendering tumour-reactive TIL products.Tumour-reactive TILs were detected in half of patients and were exclusively present in cells derived from the PD-1+ TILs.We have demonstrated that PD-1 identifies ovarian tumour-specific CD8 TILs and has uncovered predictive factors that identify OC patients who are likely to render tumour-specific cells from PD-1 Accumulating evidence supports ovarian cancer (OC) as a candidate for TIL therapy.4 However, attempts to treat OC patients with TILs have been generally disappointing.7The adoptive transfer of tumour-infiltrating T lymphocytes (TILs) has shown clinical efficacy in patients with metastatic melanoma8 During the TIL expansion process, there is an inter-clonal competition with different T-cell clones increasing or decreasing in frequency. Therefore, the more tumour-specific clones there are in the starting culture, the greater the chance of maintaining tumour-specific clones at an appreciable frequency in the final product. This may explain why the TIL therapy has yielded objective responses in cancers with high numbers of tumour-specific TILs, such as melanoma10 and virus-associated tumours,2 but not in cancers with lower numbers of tumour-specific TILs, such as OC.7An important factor that determines the success of this therapy is the antitumour reactivity of the infused cellular product.12 The CD8+CD137+ population is minority and largely confined to the CD8+PD-1+ TIL subset. For this reason, PD-1 has been proposed as the marker that more comprehensively identifies the repertoire of tumour-specific TILs.11 Recently, we have shown that the enrichment and separate amplification of PD-1+ CD8 TILs improves the antitumour efficacy of TIL therapy in mouse models of solid tumours.13 Our results were further confirmed by Jing et al. in haematological tumours.14Surface markers, such as a programmed cell death protein 1 (PD-1) and CD137, have been proven to be able to identify melanoma-specific CD8 TILs.15 Prior studies have shown that NY-ESO-1-specific CD8+ TILs from OC patients expresses PD-1.16 In addition, ovarian tumours with deficient homologous recombination (HR) have a higher predicted neoantigen (NeoAg) load and infiltrating CD8 T cells in these tumours have increased PD-1 expression.17Overall, these findings have important implications since PD-1 may enable the isolation of rare tumour-specific lymphocytes and allow TIL therapy to be extended to less immunogenic tumours. Several pieces of evidence suggest that PD-1 may also demarcate tumour-specific CD8 T cells in ovarian tumours. Thus, the expression of PD-1 in intraepithelial CD8 TILs has been associated with a favourable prognosis in high-grade serous ovarian carcinomas.19 However, little is known about the factors in the original tumour that may help identify patients able to render tumour-reactive TIL products and, therefore, that might be eligible for this therapy.Recently, investigation of potential biomarkers that may predict sensitivity to immunotherapy has become an area of active research. In the case of TIL therapy, a number of promising biomarkers have been discovered in the infused cell product that could predict clinical response.In this study, we have investigated the role of PD-1 as a selective marker for the pre-enrichment, before expansion, of tumour-specific CD8 TILs in OC. Using multiple molecular and cellular analyses, we have also examined tumours at baseline to identify parameters that help us predict from which patients we can expect to obtain tumour-specific TILs.n = 2–3, 0.7–1 cm3) were mechanically and enzymatically disrupted and the single-cell suspension was used to obtain tumour and non-tumour cell-enriched fractions  and, subsequently, were stained with fluorochrome-conjugated monoclonal antibodies (mAbs) against EPCAM, CD45, CD3, CD4, CD8, PD-1 and CD137. Information on mAb clones and fluorochromes is found in −) and PD-1 high (PD-1hi) CD8+ T cells using a FACSAria cell sorter (BD Biosciences). Isolated cells were separately expanded by Rapid expansion protocol (REP) in T-cell media containing soluble anti-CD3 mAb (OKT3) , human IL-2  and 3 × 107 irradiated peripheral blood leucocytes pooled from 3 different donors. Detailed information is found in Non-tumour cell-enriched fractions were thawed and rested overnight in T-cell media . The next day, cells were stained with fluorochrome-conjugated mAbs against CD8 and PD-1, and 7-amino-actinomycin D (dead cell marker), and were sorted into PD-1-negative (PD-14 cells/well) were co-cultured either alone or with target cells  (105 cells/well) in ELIIP plates (Millipore) coated with purified anti-human IFN-γ mAb (Mabtech), in the presence or absence of HLA-I blocking mAb . Thirty-six hours later, plates were developed as described in + spots. When autologous tumour-enriched cells were available, TIL reactivity was confirmed by assessment of CD137. Briefly, TILs (1 × 105 cells/well) were cultured, either alone or with target cells (0.5–1 × 105 cells/well), in T-cell media, and 24 h later, cells were analysed for CD137 expression by FC.Expanded CD8 TILs and autologous tumour-enriched cells were thawed and separately rested overnight in T-cell media without IL-2. The next day, TILs  and sections mounted with Faramount Aqueous Mounting Medium (Dako). Multiplexed immunofluorescence slides were scanned on a Vectra-Polaris Automated Quantitative Pathology Imaging System (Akoya Biosciences). Tissue imaging, spectral unmixing and phenotyping were performed using inForm software , as described previously.20Multiplex immunofluorescence (IF) staining, validation and analysis are detailed in 21 only SNVs were considered for the mutational load estimation. Tumour mutational burden (TMB) was calculated as the number of somatic SNVs per 0.524 megabase pairs (the targeted genomic regions of TST170). For more detailed information, see The mutational load was estimated using the Trusight Tumour 170 panel (TST170) from Illumina. FFPE-tumour DNA extraction, library preparation and sequencing (NextSeq 500 using high-output cartridge and v2 chemistry) were executed by Macrogen (Korea) according to Illumina’s instructions. Sequence alignment and variant calling were performed using the TST170 BaseSpace application. Variant annotation was performed using Annovar . Positions with depth <100, variant call quality <40, NC  ≥0.03 (for SNVs), strandbias score > −80 and variant allele frequency (VAF) <0.05 were ignored. Germline polymorphism filtering was performed by filtering variant allele frequency (VAF) between 0.4–0.6 and > 0.8. Polymorphisms were excluded if their minor allele frequencies (MAF) in European non-finnish (ENF) population (the ethnic group of our patients) according to gnomAD were > 0. Mutations with MAF(ENF) = 0 or not found in gnomad were also not found in 1000G database. All putative somatic SNVs and INDELs were carefully checked by manual inspection of the sequenced reads using the Integrative Genome Browser (IGV). About 100% of SNVs were “truly SNVs”, whereas 60% of identified somatic indels were “False positive”. Due to the high rate of false-positive Indels and the lower proportion of these variants in OC (~3% of the number of indels + SNVs),p values < 0.05 were considered to identify differentially expressed genes (DEG). Volcano plots and unbiased clustering of DEG were generated using R (v3.5.3). For analysis of immune signatures, after HK normalisation, a log10 transformation was applied, and the signature score was calculated by averaging the expression level of those genes included in the IFN-γ signature, Expanded immune signature and T-cell inflamed signature.22 For more information, see FFPE-tumour RNA was extracted using the RNeasy FFPE kit (QIAGEN). nCounter PanCancerImmune Profiling panel analysis (Nanostring) was performed by IMIBIC (Spain) following the manufacturer’s instructions. Analyses of expression were performed using nSolver Analysis Software (v 4.0), with normalisation utilising positive and negative control probes, as well as the most stable housekeeping (HK) genes across samples. The statistical tests used are detailed in each figure legend. For detailed information, see + cells and EpCAM+ cancer cells   Fig. . Express− and PD-1hi cells, from 10 resected ovarian tumours and expanded them separately. The number of isolated PD-1− and PD-1hi CD8 TILs varied among patients. Both subsets expanded efficiently  cells, as determined by IFN-γ secretion  and patients with non-tumour-reactive (NTR) TILs .To determine if PD-1 may enrich tumour-specific T cells in OC, we isolated CD8 TILs with extreme expression of PD-1, namely PD-1ion Fig.  and CD13ion Fig. . We founion Fig. . Remarkated Fig. . Only PDted Fig. . Recognited Fig. . Our dat+ cells between both groups  and CD137+PD-1+CD4+ and CD137+PD-1+CD8+ TIL subsets were quantified ; thus, CD137+PD-1+CD8+ cells represented a very small cell population in the tumour. Patients with TR TIL products exhibited a slightly higher, but statistically significant, percentage of CD137+PD-1+CD8+ TILs in the tumour epithelium   Fig. .Fig. 5Pa23 were contained in the TST170 panel  system. Defects in these pathways may accumulate an enormous number of mutations due to genome instability during the carcinogenic process and may account for the different mutational load found in our patients. Twenty-eight genes out of about 120 genes involved in direct DDR22 In order to determine if the gene expression profile (GEP) may also predict patients capable of eliciting TR TIL products, we analysed the expression of 770 immune-related genes in 8/10 patients with FFPE-tumour RNA available. Four ovaries with non-malignant disease were included as a control. Transcriptome analysis revealed a set of 36 DEG between the TR and NTR groups  included in the panel. No differentially expressed CTA genes were detected between the TR and the NTR groups , whereas the large majority are cells activated long ago (PD-1+CD137−). The ratio between bystander and tumour-specific TILs varies between tumours,28 and this could explain why in OC, with smaller numbers of tumour-specific lymphocytes in comparison to melanoma,11 only CD137+ cells12 or PD-1+ cells containing a high proportion of CD137+ cells (our study) were able to render TR TILs. A more exhausted state of PD-1+ tumour-specific TILs in OC may also account for the difficulty of obtaining TR TIL products from this population. Patient-related factors may also be implicated. Thus, similar to what we have found in OC, the ability of PD-1 to enrich for tumour-specific TILs in melanoma patients has also been shown to be inconsistent.25However, only in 5 out of 10 patients studied, the PD-1− fraction. A recent study in colorectal and lung cancer has shown that CD39 accurately distinguishes between tumour-specific (PD-1+CD39+) and bystander (PD-1+CD39−) TILs.27 In another study, co-expression of PD-1 with markers of tissue-resident memory cells, such as CD39 and CD103, seemed to identify tumour-specific TILs.29 Presumably, combinations of PD-1 with other markers would be necessary to accurately identify the full repertoire of tumour-specific TILs, especially in tumours in which these cells are rare.Therefore, the use of PD-1 as a single marker to isolate tumour-specific cells appears to be controversial. On the other hand, the expression of CD137 is transitory. Consequently, a large proportion of tumour-specific T cells may be missed in the CD137+PD-1+CD8+ TILs located in the tumour epithelium, suggests that their tumours may have special features that have enabled the activation of T cells, e.g., the presence of relevant antigens or/and an immune-permissive tumour microenvironment (TME). TMB provides an indirect assessment of tumour antigenicity because a high level of mutations will increase the likelihood of neoAg generation. The TMB values in our patients, as estimated using the TST170 panel, ranged from 0 to 15.3 SNVs/mpb. These values were within the range of those found for OC using whole-exome sequencing (WES), although the median values (3.8 SNVs/Mbp) were higher than those estimated with this method (~1.5 mutations/Mb).31 The use of a gene panel enriched in known cancer genes, which are more likely to be mutated in tumours, often leads to TMB overestimation.32 The precise estimation of the mutational load may also have been affected by the low genomic coverage of the TST170 panel (0.53 Mbp), a problem that most seriously affects tumours with low/moderate TMB levels, as is the case of OC.34 However, previous studies have shown that the numbers of mutations detected with the TST170 panel strongly correlate (Pearson correlation 0.97) with those observed with the gold standard WES.34 In addition, exploratory analyses demonstrated that the TST170 panel-based TMB discriminates between immunotherapy responders and non-responders.36 Therefore, although the panel used cannot provide an accurate estimation of the mutational load, it can still be valid to distinguish between patients. Remarkably, those patients with the highest SNV numbers (P02 and P06) belonged to the TR group. Although our data need to be confirmed in a larger cohort of patients using a higher panel size, they suggest that, in some patients at least, the existence of possible neoAgs might account for the capability of rendering TR TILs.The finding that those patients rendering TR TILs had a higher frequency of CD13737 Interestingly, patient P02 showed deleterious mutations in ATM and FBXW7 genes, involved in HR and non-homologous end-joining pathways, respectively, whereas patient P06 presented a likely pathogenic mutation in ERCC2, entailed in the nucleotide excision repair pathway. ATM gene defects strongly correlate with spontaneous TIL infiltration in breast cancer and OC.38 Recently, it has been shown that bladder cancer patients with mutated ATM had better benefits from immune checkpoint inhibitor (ICI) therapy.39 Inactivation of FBXW7 causes genomic instability.40 Importantly, mutations in ATM and FBXW7 genes correlate with higher TMB levels in several cancer types, including OC.47 Mutated ERCC2 has also been associated with high TMB values.41 Although our findings require further investigation, they suggest that deleterious alterations in DDR genes may influence the competence to render TR TILs by leading to an increased TMB, and subsequently increased number of neoAgs.Several intrinsic mechanisms, including deficiencies in DDR genes, have been implicated in contributing to neoAg load.49 A similar GEP has also been associated with high tumour T-cell infiltration in OC patients.51 In line with the transcriptomic data, FC analysis of tumour infiltrates revealed a significantly higher frequency of CD8 and CD4 T cells in those patients who rendered TR TILs. Likewise, a high expression of genes involved in wound healing, mesenchymal transition, extracellular matrix remodelling, angiogenesis and invasiveness, was associated with the lack of antitumour reactivity of the final TIL product. The concurrent overexpression of genes involved in these processes has been associated with the presence of immunosuppressive TME,52 T-cell dysfunctionality53 and resistance to anti-PD-1 therapy.48The finding that some patients from the TR group showed relatively low mutational load suggests that mechanisms independent of neoantigenicity might also account for the capability of rendering TR TILs. The expression profile of immune-related genes reflects the nature of the TME, which is also an important feature of cancer immunobiology. Interestingly, tumours from patients with TR TIL products showed a high expression of genes involved in antigen processing/presentation, T-cell activation, proliferation/differentiation of T cells, cytotoxic activity, IFNγ-signalling, chemoattraction as well as mechanisms to regulate T-cell response. This GEP is characteristic of immune-reactive tumours with prolonged survival and response to ICIs.22 Importantly, patients rendering TR TIL products exhibited significantly higher scores for the three mentioned signatures, in particular for the T-cell inflamed GEP. These findings suggest that GEP may represent a useful tool to identify patients who are likely to render TR TILs and, ultimately, who may benefit from TIL therapy.Three multigene expression signatures  have been associated with clinical benefit from anti-PD-1 treatment in several cancer types.55 The ability of these two parameters to reflect overlapping but not always coincident aspects of tumour immunology may explain their lack of correlation.Interestingly, while the TMB values did not correlate with the GEP score, both parameters stratified TR and NTR groups. Thereby, patients with high values of either TMB and/or GEP rendered TR TIL products, whereas those with low values of both TMB and GEP did not. Our data show some similarities with the predictive value of TMB and GEP to anti-PD-1 therapy across different tumour types.56 and hot-spot mutations,57 as well as for CTA.16 Interestingly, the patient who in our study stood out for rendering highly TR TILs (P05), exhibited a high expression of very potent CTA, such as NY-ESO-1, MAGEC2 and TPTE. In the future, it will be interesting to determine what type of antigens account for the antitumour reactivity of TIL products in OC.The fact that patients with relatively low TMB rendered TR TILs suggests that the quality of neoAgs, but not the quantity, or the presence of relevant shared tumour-associated antigens, may have determined the antitumour reactivity of the final TIL product. In the case of OC, several groups have identified TILs specific for private+ cells in the fresh tumour together with the GEP and the TMB, could guide future steps to defining an extensive predictive biomarker panel to identify patients eligible for TIL therapy. The use of such a predictive biomarker panel together with newer ways to select tumour-specific TILs, will help to extend this cell therapy to less immune-reactive tumours, such as OC.The present study has notable limitations, namely the low number of patients and the inclusion of distinct histological subtypes of epithelial OC. Although our findings warrant additional investigation and need to be validated in larger-scale studies, they provide a rationale for further exploring the utility of biomarkers to (i) identify and selectively expand tumour-specific TILs, and (ii) serve as guides for selecting patients eligible for TIL therapy. In order to increase the tumour reactivity of the final TIL product, other selective markers may be considered together with PD-1 to distinguish tumour-specific from bystander T cells. The combination of several parameters, such as TIL density and the presence of intraepithelial CD137Supplementary Methods, Supplementary Figures and Supplementary Tables S1 and S2Supplementary Table S3


Radiotherapy (RT) is a highly effective anti-cancer therapy delivered to around 50–60% of patients. It is part of therapy for around 40% of cancer patients who are cured of their disease. Until recently, the focus of this anti-tumour efficacy has been on the direct tumour cytotoxicity and RT-induced DNA damage. Recently, the immunomodulatory effects of RT on the tumour microenvironment have increasingly been recognized. There is now intense interest in potentially using RT to induce an anti-tumour immune response, which has led to rethinking into how the efficacy of RT could be further enhanced. Following the breakthrough of immune check point inhibitors (ICIs), a new era of immuno-oncology (IO) agents has emerged and established immunotherapy as a routine part of cancer treatment. Despite ICI improving outcomes in many cancer types, overall durable responses occur in only a minority of patients. The immunostimulatory effects of RT make combinations with ICI attractive to potentially amplify anti-tumour immunity resulting in increased tumour responses and improved outcomes. In contrast, tumours with profoundly immunosuppressive tumour microenvironments, dominated by myeloid-derived cell populations, remain a greater clinical challenge and RT may potentially further enhance the immunosuppression. To harness the full potential of RT and IO agent combinations, further insights are required to enhance our understanding of the role these immunosuppressive myeloid populations play, how RT influences these populations and how they may be therapeutically manipulated in combination with RT to improve outcomes further. These are exciting times with increasing numbers of IO targets being discovered and IO agents undergoing clinical evaluation. Multidisciplinary research collaborations will be required to establish the optimal parameters for delivering RT  in combination with IO agents, including scheduling to achieve maximal therapeutic efficacy. Radiotherapy (RT) is a highly effective anti-cancer therapy known to induce direct DNA damage to tumour cells. More recently, the immunomodulatory effects of RT on the tumour microenvironment (TME) has encouraged investigations into how RT efficacy might be enhanced. RT is able to induce a local anti-tumour immune response, potentially leading to systemic anti-tumour immunity and contributing to tumour regression outside of the local radiation field, termed the “abscopal effect” . Though The immunomodulatory potential of both RT and ICIs has provided rationale for combining RT and IO agents to further improve overall response rates and the duration of responses. Rare abscopal effects are now increasingly reported as case reports in patients receiving combined treatment, but they are still the rare minority and further prospective clinical trials are required –11. WhilThe immune contexture of the TME is now understood to be a significant predictive biomarker of response to immunotherapies. Tumours with an abundance of infiltrating T-cells appear to be most likely to respond to ICI, whereas tumours with an abundance of immunosuppressive myeloid cells and few infiltrating T-cells fail to exhibit a durable response –21. RT mThis review will therefore describe our current understanding of the complex interaction of RT with the TME, how IO agents may modulate this and the challenges of translating combined therapies into the clinic.The therapeutic success of RT has been shown to be dependent on effector T-cells and their ability to respond to tumour antigens . IrradiaIrradiated cells release DAMP signals which enhance the function of DCs. The release of adenosine triphosphate recruits DCs to the tumour. Calreticulin is translocated to the surface of dying cells where it is recognized by DCs, promoting phagocytosis. The passive release of HMGB1 enables DCs to efficiently process and cross-present antigens through toll-like receptor (TLR) 4 dependent signaling, inducing an effective T-cell mediated immune response –28. TherDepletion studies in murine lymphoma models indicate that DCs—not B cells or macrophages—are indeed the major antigen presenting cell (APC) required for durable anti-tumour immunity, demonstrated when RT is combined with stimulatory CD40 mAb . CD40 agThe presence of cytosolic DNA and micronuclei in irradiated tumour cells also activates cGas/STING signalling pathways which stimulate downstream production of immunogenic type I interferons (IFN), responsible for the maturation of DCs amongst other immunostimulatory events –34. ThisRT has also been described to activate mTOR signalling as part of DDR pathways , 40. ThiBoth resident and infiltrating T-cell populations appear to be required for effective control of irradiated tumours and distal tumour sites following RT and anti-PD1 combinations . Local RT-cell recruitment to the TME is mediated by adaptive and innate immune responses induced by RT. Once matured, DCs migrate to tumour draining lymph nodes (LNs) where they prime naïve T-cells towards an effector phenotype. These effector T-cells, along with CD4 + T helper cells, are trafficked to the tumour site via chemokine gradients , 47. RT Work from our own laboratory investigating T-cell receptor clonality has suggested that following RT and anti-PD1 combinations, the immune response in the local TME is dominated by polyclonal expansion of pre-existing T-cell clones . The eraPre-clinical studies of RT-anti-PD1 combinations suggest that irradiating draining LNs impacts T-cell infiltration into the primary tumour, modifies intra-tumoural chemokine expression and reduces overall survival . Since tFurther studies are also needed to clarify the significance of T-cell trafficking in phenotypically immunosuppressive tumours, such as prostate cancers. These may benefit from IO agents which stimulate leukocyte trafficking, such as anti-CD40. The administration of agonistic CD40 alone or in combination with RT leads to a significant increase in CD8+ and CD4 + T-cells infiltrating the tumour, and the combined approach may successfully induce T-cell dependent immunity –52.The infiltration of T-cells with a memory phenotype  into the TME correlates with improved overall survival in lung and ovarian tumours , 54. ResTumours with a high proportion of myeloid and other suppressive effector cells within the TME can hinder anti-tumour immune responses. These tumours are radioresistant and lack clinical responsiveness to ICI , 57–60. Immediately following RT, there is a relative increase in MDSCs, tumour associated macrophages (TAMs) and regulatory T-cells (Tregs) within the TME, as these appear to be more radioresistant than T lymphocytes . MDSCs aRT stimulates the recruitment of suppressive cells to the TME by increasing the expression of inflammatory chemokines such as CXCL5 and CXCL2; these appear important in recruiting MDSCs and Tregs via STING-activated CCR2 pathways, as well as stimulating the production of TGFβ , 69. TGFMDSC have been shown to contribute to patient resistance to ICI –79. RT aThe utilization and development of therapeutic agents which manipulate or reprogram myeloid populations provide another exciting opportunity to improve clinical responses to RT-IO combinations. Stimulatory IO agents employed to activate anti-tumour T-cell responses can also reprogram suppressive cell populations. CD40 agonists manipulate macrophages to acquire an M1 phenotype, thereby upregulating pathways associated with effector T-cell priming . MacrophTargeting TGFβ represents another potential strategy to enhance combination therapies. TGFβ inhibitors in combination with anti-PD1 mAbs demonstrated CD8+ T-cell infiltration and improved tumour control in a subset of urothelial cancer patients and poorly immunogenic breast cancer models , 91. ProPhosphodiesterase-5 (PDE5) inhibitors are currently approved for non-malignant conditions and have been shown to reduce the immunosuppressive capacity of MDSCs in murine tumour models. PDE5 inhibitors increase T-cell infiltration and activation through Arg-1 and NOS down-regulation resulting in improved efficacy of adoptive T-cell therapies . In clinHypoxia has long been established as a significant factor in radioresistance . More reDue to the complex immunological interplay between RT and IO agents Table , there aThe dose and number of fractions of RT will likely play a critical role in the immunomodulation of the TME. High-doses of 12–16 Gy delivered in a single fraction induce protective DDR pathways within the tumour which hinder T-cell response, whereas lower doses have been shown to optimally induce the production of IFNβ required for DC activation . It is cCurrently the majority of clinical trials investigating RT-IO combinations employ single site irradiation, which may not be optimal. In the situation of multiple metastases, multi-site irradiation may improve therapeutic outcomes by reducing disease burden and also by increasing RT-induced immune stimulation, as shown in Fig. Scheduling of the IO agent relative to the delivery of RT is also likely to affect the generation of systemic immune responses. The optimal schedule is likely to depend on disease group, tumour site and the RT-IO combination employed. Research from our laboratory in murine models of CRC evaluated three regimes of RT with anti-PD1: concurrent delivery at the start of RT cycle; concurrent delivery at the end of RT cycle; sequential delivery 7 days after RT completion. Acquired resistance to radiotherapy was overcome by concurrent delivery of anti-PD1 with effective anti-tumour immunity and tumour control; sequential delivery was less effective . HoweverFurther investigations are also needed to identify biomarkers which inform on RT-induced immunological changes, which will guide decision making in the clinic. Predictive biomarkers to identify the most appropriate IO agent for each patient would increase the frequency of responders. PD-L1 expression has emerged as a potential prognostic biomarker correlated with improved survival after RT, though its value as a predictive biomarker for RT-IO agent combinations remains to be seen –120. TheThe ability of RT to reprogram the TME has complex local and systemic consequences. The immunostimulatory effects of RT make combinations with IO agents attractive to amplify effective anti-tumour immunity and improve outcomes. In contrast, immunosuppressive TMEs dominated by MDSC populations remain a greater clinical challenge, as RT may potentially enhance immunosuppression. To harness the full potential of RT-IO agent combinations, further insights are required to understand the role of these immunosuppressive myeloid populations, how RT influences them and the optimal ways to therapeutically manipulate them to improve clinical outcomes. These are exciting times with increasing numbers of IO targets being discovered and undergoing clinical evaluation –127. The


Varicella-zoster virus (VZV) infection can be diagnosed clinically once classical rash occurs but the diagnosis is challenging when typical rash is absent. We reported a case of fulminant central nervous system (CNS) VZV infection in a human immunodeficiency virus (HIV)-infected patient without typical VZV-related rash. CNS VZV infection was unexpected identified by metagenomic next-generation sequencing (mNGS).A 28-year-old HIV-infected patient presented with neurological symptoms for 3 days. The patient, who was not suspected of VZV infection at admission, quickly progressed to deep coma during the first 24 h of hospitalization. An unbiased mNGS was performed on DNA extract from 300 μL cerebrospinal fluid (CSF) with the BGISEQ-50 platform. The sequencing detection identified 97,248  sequence reads uniquely aligned to the VZV genome, and these reads covered a high percentage (99.91%) of the VZV. Presence of VZV DNA in CSF was further verified by VZV-specific polymerase chain reaction and Sanger sequencing. Altogether, those results confirmed CNS VZV infection.This study suggests that mNGS may be a useful diagnostic tool for CNS VZV infection. As mNGS could identify all pathogens directly from CSF sample in a single run, it has the promise of strengthening our ability to diagnose CNS infections in HIV-infected patients. Primary varicella-zoster virus (VZV) infection causes varicella (chickenpox), after which VZV becomes latent in ganglionic neurons. In the case of impaired immunity, virus reactivation leads to zoster (shingles), which manifests as unilateral dermatomal rash , 2. VZV A 28-year-old Chinese male was admitted to our hospital because of headache, nausea and vomiting for 3 days. He was confirmed HIV and hepatitis B virus (HBV) co-infection 2 years ago and did not receive anti-viral therapy. In the preceding month, he had been hospitalized in another hospital due to pneumocystis pneumonia that was resolved with cotrimoxazole and corticosteroids. One week before his admission to our hospital, a skin rash developed on his left foot which did not receive much attention. At the time of his admission to our hospital, this rash appears as superficial ulcer with partial necrosis and black crust surrounded by a cluster of blisters Fig. , which w9, N 0.67; (2) plasma procalcitonin<0.1 ng/mL; C reaction protein: (3) negative serum cryptococcal antigen test; negative serum Treponema pallidum particle agglutination assay; negative serum tuberculosis antibodies; negative interferon-γ release assay; negative blood culture;(4) plasma CMV load: serum HBV load: 2.18 × 108 copies/mL; (5) CD4 cell count: 137 cells/uL; plasma HIV load: 1.2 × 106 copies/mL. His clinical condition was not improved with the above mentioned combinatory anti-infective therapy. On the third day of the hospitalization, a lumbar puncture was performed. The CSF opening pressure was 15 cm of H2O. CSF analysis demonstrated a white blood cell count of 91 cells/ mm3, glucose level of 3.28 mmol/L, total protein level of 9.6 g/L, negative India ink staining, negative cryptococcal antigen test, negative gene Xpert MTB/RIF assay and negative culture result. Also, CSF sample was sent for mNGS analysis. The patient was deceased on the fifth day of the hospitalization. The next day, mNGS analysis was completed which revealed high burden of VZV in the CSF. A follow-up telephone visit of the patient’s family member revealed that the patient had a history of chickenpox in childhood. VZV DNA was detected from stored serum sample.After his admission to our hospital, the main laboratory tests from blood samples were as follows: (1) white blood cell count: 9.41 × 10mNGS was performed in BGI-Shenzhen exactly following our previous protocol , includiPstI site located within gene 38 of VZV, with VZV-specific primers VZV-F (AAGTTCCCCCCGTTCGC) and VZV-R (TGGACTTGAAGATGAACTTAATGAAGC) [To verify the results of mNGS, PCR was performed to amplify the ATGAAGC) . The 89-To etiologically diagnose CNS infection, conventional methods generally need massive volume of CSF sample to perform a battery of tests, which include smear, culture, nucleic acid amplification assays and serological tests. It is not possible to test all the CNS pathogens using those microbiologic methodologies due to limited amount of CSF sample and commercial test kits available. Recently developed mNGS has the capability to overcome limitations of traditional diagnostic tests. This new technology could identify all pathogens directly from CSF sample with a single run in a hypothesis-free and culture-independent manner . StudiesCNS infections are closely associated with morbidity and mortality in HIV-infected patients . Common The clinical practice in our patient suggests that mNGS has the promise of strengthening our ability to diagnose CNS infections in HIV-infected patients. Firstly, HIV-infected patients could suffer from infections that are not commonly seen in general populations. The commercial kits for testing those relatively rare pathogens are often unavailable in many regions. Secondly, HIV-infected patients may have atypical presentations of common pathogens. For example, zoster that due to reactivation of VZV infection is a common clinical condition in HIV-infected patients. However, due to unaware of the atypical infection, CNS VZV infection was not known and not interrogated by traditional methods until mNGS demonstrated the presence of high burden VZV in patient’s CSF. Lastly, CNS infections may be simultaneously caused by multiple pathogens in HIV-infected patients as they are immunocompromised . Those CIt should be noted that mNGS also has some limitations such as human genome contamination and possibly environmental microbial contamination . The vasIn conclusions, in the current study, a fulminant CNS VZV infection without typical rash in an HIV-infected patient was unexpectedly diagnosed by mNGS. This study suggests that mNGS may be a useful diagnostic tool for CNS VZV infection. More importantly, as mNGS does not require a prior knowledge of causative pathogens and could identify all pathogens directly from CSF sample in a single run, it has the promise of strengthening our ability to diagnose CNS infections in HIV-infected patients.


It is noticeable that the highest average nitrogen concentration was observed in tumour 12 ± 0.4% and the lowest in healthy tissues 8 ± 0.9% (p < 0.00001). The highest average carbon content was observed in healthy tissues 57 ± 2.2% and the lowest in tumour 46 ± 1.3% (p < 0.00001). Moreover, values of 15N/14N expressed in delta notation were the highest in healthy tissues 9.84 ± 0.61 and the lowest in tumour 8.92 ± 0.58. Values of 13C/12C tended to be higher in tumour −22.2 ± 0.89 and the lowest in healthy tissues −23.7 ± 1.2. Tumour tissues differ in isotopic composition from tissues taken from margin and healthy tissues taken from distant oral mucosa.Overall prognosis for patients with oral squamous cell carcinomas (OSSC) is still unfavourable. However, there is a hope that a novel diagnostic method may establish better cancer biology characteristics. The aim of this study was to evaluate the isotope ratio of nitrogen and carbon in OSSC as compared to margin and healthy tissue. A total of 18 patients with OSSC were included in the study. Specimens collected covered: four tumour, four margin and two healthy oral mucosa samples. The samples underwent further procedures: lyophilization and isotope ratio mass spectrometry. Measurements of the ratio of stable isotopes of nitrogen  Worldwide data show that lip, oral cavity and pharyngeal cancer is one of the most noteworthy problems among oral diseases due to its significant effects on the quality of life of the patients. In 2017, among all cases of this type of cancer, more than half 57.4% are concerned lip and oral cavity cancers [The worldwide database showed that 630,000 new patients with head and neck cancer are diagnosed per year and almost 350,000 patients died. In total, 90% of head and neck cancers are squamous cell carcinoma raised from the mucosal surface of the oral cavity [In 2012 in Europe, 140,000 new cases of head and neck cancers and 63,500 new deaths were reported. It was noted that men have a four times higher risk having this type of cancer then women [In the United States head and neck cancers constitutes 3% of all malignancies. A total of 60,000 new cases are reported every year with approximately 12,000 resulting deaths.From 2002 to 2012, incidence of cancer in men decreased 0.29% per year and the incidence in women decreased 0.38% per year .In Poland, about 5.5–6.2% of all malignant tumours are head and neck cancers, which is manifested by 5500 to 6000 of new cases per year . CancersFive-year age-standardised relative survival rates in Europe in 1999–2007 have been calculated for 39% for oropharynx cancer, 43% for tongue cancer, 45% for oral cavity cancer and 49% for nasopharynx [Additionally, in Europe in 2012, mortality rate caused by oral cavity and pharynx cancer was 34,200 per 100,000 among male and 9400 per 100,000 among female .Generally, mortality rate in OSCC is more than 50% . InterveThe most common method of treatment mainly depends on the tumour’s stage and grade ,11. HoweA method used for evaluating molecular content of samples is mass spectrometry. One of its forms is isotope ratio mass spectrometry (IRMS) . IsotopeThe IRMS method is based on the measurement of the ratio of a heavier stable isotope to a lighter one, allowing one to detect either the enrichment or depletion in an examined sample (an increase or reduction in the heavier isotope content). The variances in the isotopic ratios of elements are expressed as delta values (δ). Relative measurements are used, by alternating measurements of a sample and a standard within a single measurement cycle and referring the measured isotopic content of the sample to the isotopic content of the standard.13C and 15N isotope abundance may vary between healthy and breast cancer biopsy tissues. The isotope mass content has been related to lipid metabolism, anaplerosis and urea cycle, and thus to three pathways known to be altered in malignant cells. These findings may suggest that isotope balance of 13C and 15N is a good method of metabolism evaluation, because it reflects modifications in C partitioning and N excretion altogether [This method allows to determine the ratio of isotopic composition of elements, which reflects the occurrence of physical or chemical reactions and metabolic processes. Data are used to obtain information on biological material through analysis of organic and non-organic compounds . The mostogether .Scientific research supports the statement that it is possible to characterise the origin of damaged or healthy cells by means of isotope ratio mass spectrometry ,22. CurrIt should be emphasized that this method needs to be thoroughly validated before clinical implementation is warranted.The aim of the study was to evaluate isotope ratio of nitrogen and carbon in oral squamous cell carcinomas (OSCC) as compared to margin and healthy tissue.A total of 18 patients of the Maxillofacial Department treated surgically due to malignant neoplasms of oral cavity were included in the study. Among them were 7 women and 11 men, aged 46–77 years (mean age 68.2 ± 7.8 SD). Smoking was identified in 6 of 11 of men and 4 of 7 of women.The study group consisted consecutive patients that fulfilled inclusion criteria.Primary tumor of oral cavity, located in areas, such as labial or buccal mucosa, the anterior two thirds of tongue, the floor of the mouth, gingiva, alveolus and palate;The result of the histopathological examination confirmed that it is a planoepithelial cancer;Loco-regional advancement of the tumor enabling radical surgical treatment .Including criteria were:Presence of distant metastases;Patients after previous radio and chemotherapy treatment of the head and neck region;Loco-regional advancement of the tumor, not allowing to perform radical surgery.Excluding criteria were:Additional epidemiological and clinical features are presented in For study purposes, a total of 180 tissue samples were collected from patients with oral squamous cell carcinomas at different stages of tumour.The study was approved by the Bioethics Committee (RNN/185/18/KE).During the surgery consisting of removing the primary tumor, collected from each patient were: 4 tumour samples, 4 margin samples and 2 healthy oral mucosa samples (40 mm away from the border of the tumour) with dimensions of ca. 2 × 2 mm. Four of them (2 from tumour and 2 from margin) were fixed in formalin for later histopathological verification. All samples were routinely reviewed, confirmed and evaluated by an experienced specialist in pathomorphology. The following features were assessed: depth of cancer infiltration (mm), angioinvasion, local lymph nodes metastases, nodal capsule infiltration and neuroinvasion with conventional hematoxylin and eosin staining, it is impossible to distinguish healthy tissue from a margin without neoplastic infiltration. However, it was possible to differentiate tissue with neoplastic infiltration from tissue without infiltration, i.e., healthy tissue.A part of tissue samples, 108 out of 180 intended for isotope ratio mass spectrometry (IRMS) procedure were kept at −70 °C before performing further procedures. The obtained tissue sections were frozen at −70 °C for another 48 h and freeze dried . Subsequently, lyophilization procedure was performed.For the IRMS measurements, three samples sized 3 ± 1 mg were prepared from each material. On average, three samples were prepared from each tissue specimen. Each one was weighted into a tin capsule to which 1 mg of vanadium pentoxide was added as a sulphur oxidation catalyst. All were rolled up carefully.IRMS procedure was performed with use of Sercon SL20–22 Continous Flow Isotope Ratio Mass Spectrometer connected with a Sercon SL elemental analyser for simultaneous carbon-nitrogen-sulphur (NCS) analysis. The primary reference standard used was thiobarbituric acid.15N/14N nitrogen isotopic composition, 13C/12C carbon isotopic composition and carbon-to-nitrogen mass C/N ratio were made. Isotopic ratios were described by delta values. Delta is a value that characterizes the ratio of heavier to lighter isotopes in relation to international standards for nitrogen  and carbon . The results were also described by minimum and maximum values, mean value, median and standard deviation. The achievable precision of 15N and 13C spectrometer measurements was ±0.1–0.2.Measurements of p < 0.05.One-way analysis of variance was used for the detection of differences in tumour, margin and healthy tissues. Due to the presence of non-normal data distributions, the Kruskal–Wallis test was applied. The difference is considered significant if A comparison between dependences of clinical, histopathological features and data obtained from spectrometry was performed. The normality of the distributions was assessed by the Shapiro–Wilk test. Pearson correlation analysis  or Spearman’s correlation analysis (otherwise) was used. Spearman’s correlation analysis, Student’s t-test and the Mann–Whitney test were used to compare the data. Stargraphics Centurion XVI, StarPoint Technologies. INC., The Plains, VA, USA, was used to perform statistical analyses.During the study, the percentage content of nitrogen and carbon was obtained in all samples. The results are presented as minimum, maximum, mean, standard deviations and median values. Details of conducted assessments are presented in p-values of all of these observations were less than 0.00001.It is noticeable that the highest content of nitrogen was observed in tumour tissues, medium in margin and the lowest in healthy tissues. The average nitrogen concentration in tumour tissue was 12 ± 0.4%, in margin 10 ± 0.8% and 8 ± 0.9% in healthy tissue. Average content of nitrogen turned out to be statistically significant. p < 0.00001).When it comes to the percentage of carbon content, the results were exactly the opposite. The highest content was observed in healthy tissue, medium in margin and the lowest in tumour tissue. The average values were, respectively, 57 ± 2.2%; 51 ± 3.1% and 46 ± 1.3%. This content turned out to be statistically significant  and carbon .p values were, respectively, p < 0.001, p < 0.0001, p < 0.001.It turned out that values of delta Air were the highest in healthy tissues, ranging from 8.35 (minimum) to 10.78 (maximum), with average 9.84 ± 0.61 and median 9.86, lower in margin tissues: 8.19 (minimum) to 11.38 (maximum), with average 9.53 ± 0.64 and median 9.49, and the lowest in tumour tissues 8.10 (minimum) to 10.88 (maximum), with average 8.93 ± 0.58 and median 8.71. The maximum, mean and median values were statistically significant when results of healthy tissue-tumour and margin-tumour were compared. p-values less than 0.01.In addition, statistically significant differences in the minimum of delta Air were observed between healthy tissue-tumour, with Values of delta PDB tended to be higher in tumour and ranged from −24.36 (minimum) to −19.99 (maximum), with average −22.21 ± 0.89 and median −22.33; in margin −24.93 (minimum) to −21.54 (maximum), with average −23.36 ± 0.92 and median −23.43, and in healthy tissues −25.26 (minimum) to −21.07 (maximum), with average −23.68 ± 1.18 and median −24.32.p-values were, respectively, p < 0.0001, p < 0.0001, and p < 0.001.The mean, standard deviation and median values were statistically significant when the results of healthy tissue-tumour and margin-tumour were compared. p-value, maximum value between margin and tumour turned out to be at the level of statistical tendency.Besides Furthermore, a statistically significant difference of minimum value in all tissues was observed.The isotopic picture of tumour, margin and healthy tissues is summarised in Comparison of clinical features with data obtained from spectroscopy revealed significant dependences in the case of body mass index (BMI) and alcohol consumption.p value was less than 0.05.It turned out that correlations between BMI and nitrogen content in margin tissues and the mean value of delta PDB tumour were statistically significant. In the case of patients with higher BMI, the nitrogen content in margin tissues was also higher. The p value was less than 0.001.Observations were inverse for values of delta PDB. The higher BMI index, the lower mean value of delta PDB tumour. The p value was less than 0.05.Additionally, in patients who confirmed alcohol consumption, higher mean values of delta PDB tumour were noticed. The p < 0.05).Comparison of histopathological features demonstrated a correlation between the occurrence of angioinvasion and higher mean value of delta PDB tumour (p < 0.05) and a lower N/C ratio in tumour tissues (p < 0.05). Details of conducted assessments are presented in Moreover, nodal capsule infiltration was associated with lower values of nitrogen content in tumour tissues (To the best knowledge of this research team, until now there has been no research concerning the use of spectrometry for the assessment of head and neck cancers. It has never been used to evaluate oral squamous cell cancer tissues. This study is a pioneering attempt at filling this gap in science. The head and neck cancers’ occurrence keeps rising so it is essential to establish if spectrometry, this new possibility of evaluation, can be as helpful as other types of diagnosing malignancies. Some studies have already indicated usefulness of IRMS in case of Wilms’ tumour, hepatoblastoma or rhabdomyosarcoma ,22,32. NThe strategy behind the study was to find the possible relation between composition of isotopes or the ratio between them, and histological and clinical characteristics of malignant tumours. These factors were compared in healthy human tissues, the cancer’s margin and neoplastic tissues. Through analysis of the data, it was established that each of the samples had distinctive parameters when it came to their origin, this way indicating the cancerous tissue. The highest average content of nitrogen was found in cancerous tissues. It has been demonstrated in previous studies that the phenomenon of isotope fractionation during various processes of synthesis, mostly deamination and transamination, may constitute an explanation ,35,36,37The results of this study managed to depict the correlation between the isotopic content of different tissues and their origin—tumour, margin or healthy tissue. Various isotopic parameters that were measured could lead to the information on where the specimen was collected. The data seem sufficient to show the potential of the method to differentiate tumor from tumor margin and healthy tissue. The same phenomenon was observed by other researchers, who proved the possible use of IRMS for obtaining information on the biochemical processes and metabolism of affected cells which at this state showed abnormalities ,39. The There are some important factors that may affect the final outcome when it comes to IRMS, but, if the procedure is carried out properly, they do not pose a real problem. For spectrometry, a tumour sample as small as 0.5 mg is enough for analysis; samples tested under this study were bigger. Moreover, the method of collecting the specimens is crucial—for example, the needle aspiration biopsy is not appropriate for this purpose; the correct method is cutting out pieces of tumour, as was done here. Samples collected for this study were properly preserved from decomposition and contamination, in accordance with regulations . The teaIsotope ratio mass spectrometry has proven itself to be a novel, very sensitive method of analysis with promising perspectives. It should be adapted, developed and used for personalised therapies. This study provided some relevant information on an area that has never been covered before—the use of isotope ratio mass spectrometry for analysis of head and neck cancers, providing an opportunity for a better-aimed treatment for patients with head and neck cancers thanks to specifying the risk and prognosis of their disease.Tumour tissues differ in isotopic composition from tissues taken from margin and healthy tissues taken from distant oral mucosa. Additionally, a correlation between the content of isotope composition in oral cancer tissue compared with clinical and histopathological features was revealed.


White-handed gibbons produce loud and acoustically complex songs when interacting with their neighbours or when encountering predators. In both contexts, songs are assembled from a small number of units although their composition differs in context-specific ways. Here, we investigated whether wild gibbons could infer the ‘meaning’ when hearing exemplars recorded in both contexts (i.e. ‘duet songs’ vs. ‘predator songs’). We carried out a playback experiment by which we simulated the presence of a neighbouring group producing either its duet or a predator song in order to compare subjects’ vocal and locomotor responses. When hearing a recording of a duet song, subjects reliably responded with their own duet song, which sometimes elicited further duet songs in adjacent groups. When hearing a recording of a predator song, however, subjects typically remained silent, apart from one of six groups which replied with its own predator song. Moreover, in two of six trials, playbacks of predator songs elicited predator song replies in non-adjacent groups. Finally, all groups showed strong anti-predator behaviour to predator songs but never to duet songs. We concluded that white-handed gibbons discriminated between the two song types and were able to infer meaning from them. We discuss the implications of these findings in light of the current debate on the evolutionary origins of syntax. Chlorocebus pygerythrus) alarm call system, with acoustically distinct call types given to different predator types  rough grunts varies depending on the perceived quality of the food resource (Macaca sylvanus) barks differ in call duration and mean frequency range according to specific external disturbances  alarm roars depends on the nature of the danger (Cercopithecus campbelli) alarm calling, with variation in call rates (Cercopithecus nictitans martini)  , 2006b. martini) .A relevant primate example of complex combinatorial structure is gibbon song. In most species, mated pairs produce morning duets that appear to serve territorial and mate defence functions  and prodHylobates lar), there is also evidence for context-specific song types: duet songs are produced by the mated pair as part of their daily routine while predator songs are given when facing a predator, such as a clouded leopard or python , by broadcasting natural singing events of a neighbouring group simulated from a concealed speaker. We predicted that if gibbons discriminated between predator and duet songs then they should respond with the matching song types and with behaviour adequate to the situation. Specifically, in response to predator songs we predicted increased vigilance, increased defaecation rates and any other type of anti-predator behaviour already reported in the literature . In respN = 53 individuals at the time of the study. Due to a number of constraints, it was only possible to conduct playback experiments with six of the 13 groups , 130 km North-East of Bangkok. Data were collected from December 2012 to August 2014. Thirteen fully habituated groups of white-handed gibbons were monitored, each comprising a primary male, his mated female with her offspring and (in 5 cases) a secondary male, totalling 3 groups .Following The same three sequence types can also be found in predator songs although, overall, they differ in length and are produced with the contribution of most group members. When comparing predator songs with duet songs, for the introductory sequence the initial ‘hoo’ notes series are longer and contain more ‘hoo’ notes, followed by fewer ‘leaning wa’ notes and more ‘hoo’ notes . The greNeofelis nebulosa) model to each group following an established protocol . We then broadcasted songs such that subjects always heard recordings from one of their direct neighbours , with playback conditions randomly counterbalanced . StimuliPlayback trials were carried out from spatially realistic locations, that is, from the home range of the song-providing group towards the home range of the target group. In doing so, we took a number of precautions such that the song-providing group could not overhear its own song. Before each trial, we ensured that the song-providing group was not in the vicinity of the speaker (>100 m radius). We then monitored the area for a period of 1 h to further ensure that the song-providing group was not nearby. For each trial, the speaker was positioned in the overlapping zone between the song-providing and target group, such that it was facing away from the home range centre of the song-providing group towards the target group.https://doi.org/10.6084/m9.figshare.12363050.v1).Due to the difficult visual conditions in the forest, it was impossible to continuously video-tape the entire duration of trials nor to film all group members simultaneously. We therefore decided to restrict observations to the primary male of each group. Males are easily identifiable by their body hair colouration, facial features and genitals. Primary males were video recorded as long as possible (i.e. until they moved out of sight) using a Panasonic SDR-S26 Camcorder. Videos were coded using ELAN software (ELAN (V5.2) Nijmegen: Max Planck Institute for Psycholinguistics). Because the speaker location was not visible on the video clips (outside camera range) it was necessary for the experimenter to comment on the male’s gazing direction during filming, which made blind coding redundant. All video recordings are available on figshare  and proximities to their female partner (m) during 1 h after each trial . For the introductory sequence, we determined the duration of the initial ‘hoo’ notes series (s) and the corresponding number of ‘hoo’ notes, the type of the first ten notes following the ‘hoo’ series, and the duration of the introductory sequence . We measured the interval between the female great call and the male coda reply (s), the total song duration (s), and determined whether a neighbouring group also produced a song and its type. Finally, we identified the presence of ‘sharp wow’ notes and we measured the latency to the first ‘sharp wow’ note (i.e. time elapsed in seconds between the onset of the song bout and the first ‘sharp wow’ emitted).This study was approved by the School of Psychology Ethics Committee of St. Andrews University. Approval was given on the understanding that the ASAB guidelines for the Treatment of Animals in Behavioural research and Teaching are adhered to (n°16112011). The research permit was delivered by the National Research Council of Thailand .We compared behavioural responses within subjects and across playback conditions; the primary male’s latency and duration of first looks towards the speaker, the occurrence of defecations/urinations and branch droppings, the average distance to their female mate and the canopy heights  qualified as regular duet songs, we compared them to both spontaneously produced duet songs and experimentally induced predator songs  = 33.90, P < 0.001, N = 82, Kruskal–Wallis rank sum test). Here as well, post-hoc analyses revealed that males gave earlier replies to female great calls in the predatory context  than in spontaneous duets  or playback duet responses  (P < 0.001 in both cases), with no difference between spontaneous and playback duet responses .Second, male latencies to reply to their female’s great calls also differed significantly between song types . Significant differences were found between song types regarding their early note composition in ‘hoo’ and ‘leaning wa’, but also in ‘wa-oo’ notes (χ2(4) = 96.86, P < 0.001, Pearson’s Chi-squared test). Predator songs contained more ‘hoo’ notes and fewer ‘leaning wa’ notes than duet songs, with no differences between spontaneous and playback duet responses. However, ‘wa-oo’ notes were more common in playback duet responses than spontaneous duet songs, and again in spontaneous duet songs than predator songs  and predator songs (to clouded leopards and other predators). The two song types differ in the overall duration, frequency and distribution of specific notes  and in the location of the female great calls and male replies within each song. In this study, we investigated whether individuals discriminated between these two structurally different song types and whether they could infer meaning from them. We found several lines of evidence in favour of such an ability. First, playbacks of duet songs reliably elicited natural duet song replies  in neighbouring groups and in more distant groups, similar to how natural duet song spread throughout the forest .In related research , we have shown that spatial proximity between two neighbouring groups tends to lead to duet song overlap, due to the fact that the second group refuses to delay singing until the first group has finished their duet song. This behaviour is attenuated by kinship, to the effect that related individuals are more likely to respect each other’s duets, even if produced at close distances. In the current study, all study groups started producing duet songs while the playback duet song was still being broadcast, suggesting that the manipulation was perceived as a territorial threat. Unfortunately, we could not statistically analyse the effect of genetic relatedness in this study because the sample size was too small  with individual contributions not systematically studied. Traditional acoustic analysis may not suffice to make meaningful progress, suggesting that automated call extraction and categorisation techniques may offer more promise to explore the full combinatorial, hierarchical and compositional capacity of gibbon song .10.7717/peerj.9477/supp-1Supplemental Information 1§Nduet songs = 3. Groups A, W and T did not produce any ‘sharp wow’ note in their duet songs. ** means ± SDClick here for additional data file.10.7717/peerj.9477/supp-2Supplemental Information 2Click here for additional data file.10.7717/peerj.9477/supp-3Supplemental Information 3#Negative values can emerge because all vocal responses to duet playbacks temporally overlapped the stimulus. ** means ± SD.Click here for additional data file.10.7717/peerj.9477/supp-4Supplemental Information 4** means ± SD.Click here for additional data file.10.7717/peerj.9477/supp-5Supplemental Information 5¤ As there are ties present in the data, Dunn’s test with Benjamini & Hochberg corrections was used to perform multiple pairwise comparisons between song types on the number of ‘sharp wow’ notes so as to correct z-quantiles for ties. (*P < 0.05).Predator songs were found to be introduced by a longer ‘hoo’ note series, that also contains more ‘hoo’ notes than spontaneous duet songs and duet playback responses . Furthermore, predator songs were found to be longer in duration with a delayed first great call production compared to spontaneous duet songs and duet playback responses .Additionally, differences emerged when analysing the production latency of the first ‘sharp wow’ and in the number of ‘sharp wow’ notes produced, with predator songs containing more ‘sharp wow’ notes with a delayed production .Click here for additional data file.10.7717/peerj.9477/supp-6Supplemental Information 6P < 0.05)..However, songs in response to duet playbacks contained more ‘wa-oo’ notes than spontaneous duet songs and predator songs, with spontaneous duet songs containing also more ‘wa-oo’ notes than predator songs .Click here for additional data file.10.7717/peerj.9477/supp-7Supplemental Information 7Sheet 1: Peak intensity of female great call climaxes in spontaneous duet songsSheet 2: Stimuli characteristicsSheet 3: Response overview of the different tested groups following playback experiments of a duet song or a predator songSheet 4: Singing responses spreading into non-tested neighbouring groups.Sheet 5: Vocal data set for comparison of song responses to duet playback treatments, spontaneous duet songs and predator songs.Sheet 6: Male reply latency to their female mate great calls in song responses to duet playback treatments, spontaneous duet songs and predator songsSheet 7: First ten notes produced in song responses to duet playback treatments, spontaneous duet songs and predator songsSheet 8: Primary male scan sampling data following duet song and predator song playback treatments.Sheet 9: all occurrence behaviours following duet song and predator song playback treatments.Sheet 10: Primary male immediate behaviour following duet song and predator song playback treatments (Latency and duration of first look towards the speaker).Click here for additional data file.10.7717/peerj.9477/supp-8Supplemental Information 8Groups can either overlap another group’s ongoing duet (counter-singing) or await the end of their song that starts their own duet song. We found that group composition , relatedness and spatial proximity all played significant roles in whether a group will engage in respectful or counter-singing exchange with a previous duetting group.Click here for additional data file.10.7717/peerj.9477/supp-9Supplemental Information 9We found consistent differences at all levels of song organisation, i.e. at the note, figure, phrase and sequence level, especially during the early part of song, that could potentially allow signallers to reliably convey external events, allowing listeners to make inference about them.Click here for additional data file.


Among 52 serum metabolites that were identified, 21 were significantly increased in S and 14 and 19 decreased in the Wi and Wa groups, respectively. The most impacted pathways by this metabolic alteration were related by amino acid biosynthesis and metabolism, with an upregulation in S group and downregulation in Wi and Wa groups. Taken together, our results suggest that the increase in metabolic profile in ageing rats is associated with the higher muscle protein degradation that releases several metabolites, especially amino acids into the serum. On the other hand, we hypothesise that the majority of metabolites released by muscle catabolism are used by tumours to sustain rapid cell proliferation and tumorigenesis.Sarcopenia among the older population has been growing over the last few years. In addition, the incidence of cancers increases with age and, consequently, the development of cachexia related cancer. Therefore, the elucidation of the metabolic derangements of sarcopenia and cachexia are important to improve the survival and life quality of cancer patients. We performed the  Cancer-associated cachexia is a multifactorial syndrome characterised by the depletion of skeletal muscle and fat mass and cannot be fully reversed by conventional nutritional support . CachexiThe elucidation of the molecular mechanisms of sarcopenia and cachexia is important to prevent the process of skeletal muscle atrophy and, consequently, improve the survival of cancer patients ,5. In thMetabolomics studies have revealed a distinct plasma, serum, and urine metabolic profile of cancer cachexia patients ,7. AdditThe serum metabolic disturbances associated with enhanced citrate cycle and amino acid metabolism were the main characteristics of cachexia in tumour-bearing mice . FurtherOur previous studies have shown that the Walker 256 tumour growth—an experimental model of cachexia—led to metabolomic  and metaSarcopenia, imposed by the ageing process, led to a significant decrease in gastrocnemius muscle in the ageing control (S) group in comparison to the adult control (A) group. No reduction in gastrocnemius muscle tissue was found in intermediated (Wi) or advanced (Wa) tumour-bearing ageing groups when compared to S group. The liver tissue did not change in groups. Nevertheless, the body weight was significantly decreased in Wa in comparison to S group. As expected, the tumour weight was increased in Wa in comparison to Wi group showing an exponential evolution .Among 52 serum metabolites that were identified , 21 wereThe most impacted pathways in S in comparison to A group by the metabolic alteration were the aminoacyl-tRNA biosynthesis (match status of 15 out of 48 metabolites); alanine, aspartate and glutamate metabolism (match status of 7 out of 48 metabolites); glyoxylate and dicarboxylate metabolism (match status of 6 out of 32 metabolites); glycine, serine and threonine metabolism (match status of 5 out of 34 metabolites); valine, leucine and isoleucine biosynthesis (match status of 3 out of 8 metabolites); arginine biosynthesis (match status of 3 out of 14 metabolites) and phenylalanine, tyrosine and tryptophan biosynthesis (match status of 2 out of 4 metabolites) .Among 52 serum metabolites that were identified , 14 and The most impacted pathways by the metabolic alteration in Wi in comparison to S group were the aminoacyl-tRNA biosynthesis (match status of 9 out of 48 metabolites); alanine, aspartate and glutamate metabolism (match status of 6 out of 28 metabolites) and glyoxylate and dicarboxylate metabolism (match status of 5 out of 32 metabolites). And for Wa in comparison to S group the most impacted pathways were the aminoacyl-tRNA biosynthesis (match status of 13 out of 48 metabolites); alanine, aspartate and glutamate metabolism (match status of 8 out of 28 metabolites); glyoxylate and dicarboxylate metabolism (match status of 6 out of 32 metabolites); glycine, serine and threonine metabolism (match status of 4 out of 34 metabolites) and arginine biosynthesis (match status of 4 out of 14 metabolites) .No significant difference was observed in a comparison of serum metabolic profile between Wi and Wa groups .In this study, we aimed to study the muscle atrophy age-related process, termed as sarcopenia, and the changes in ageing tumour-bearing rats. The Walker 256 tumour experimental model was used to induce muscle atrophy related to tumour growth, termed as cancer cachexia.As expected, sarcopenia decreased the gastrocnemius muscle mass according to other studies ,18,19. H1H-NMR spectroscopy, comparing aged rats to the adult ones, and the negative effects of tumour evolution—intermediary and advanced growth, Wi and Wa groups—to the elderly group.In the context of better understanding the mechanisms that contribute to muscle wasting in sarcopenia and cancer cachexia, we performed a serum metabolic profile, using 1D Among the total of 52 serum metabolites that were identified, 22 were changed between S and A groups, including an increase in asparagine, glutamate, leucine, lysine, methionine, proline, and valine that are involved in amino acid metabolism and biosynthesis pathways. Corroborating these results, Fazelzadeh and collaborators  observedIn order to evaluate if tumour growth would enhance this muscle metabolic alteration in sarcopenic rats, we analysed the serum metabolic profile of ageing tumour-bearing rats in different stages of tumour evolution, intermediated and advanced.Indeed, the evolution of cancer led to important changes in the metabolomic profile. We observed here among the total of 52 serum metabolites identified a reduction in 14 metabolites in the intermediate growth of the tumour (Wi group). As a consequence of the severe tumour growth (Wa group), 19 metabolites decreased, being directly proportional to the serum metabolic alterations. According to our findings, the serum metabolomics clinical studies revealed decreased levels of carnitine, citrate, and valine in cancer cachexia , and alsAdditionally, it is important to emphasise that fumarate changed only in Wa group compared to non-tumour-bearing ageing rats. Since fumarate is a key molecule in the tricarboxylic cycle, especially in neoplastic cells, it is considered one of the oncometabolites, because is found in high concentrations in cancer and adjacent cells, suggesting that this metabolite could be decreased in the serum. This condition modifies the tumour microenvironment, sustaining the invasive neoplastic phenotype . The rolMoreover, an opposite metabolic profile was observed between sarcopenia and sarcopenia associated with cancer cachexia. The metabolic alterations in Wa group impacted mainly the aminoacyl-tRNA biosynthesis and alanine, aspartate and glutamate metabolism pathways, clearly related to the higher tumour cells activities. Similarly, Cala and colleagues  observedTaken together, our results suggest that the increase in the metabolic profile in ageing rats is associated with the higher muscle protein degradation that releases several metabolites, especially amino acids into the serum, associated with a decrease kidney function, which likely explains the gastrocnemius muscle and body weight loss. On the other hand, we hypothesise as a severe effects that the majority of metabolites released by muscle catabolism are used by tumour to sustain the rapid cell proliferation and tumorigenesis, showing the inverse relation between tumour growth and body weight.Adult Wistar rats (approximately 90 days old with 250 g of body weight) and ageing Wistar rats (approximately 400 days old with 650 g of body weight), obtained from the Animal Facilities at the State University of Campinas, UNICAMP, Brazil. All animals were housed in collective cages under controlled environmental conditions . The general guidelines of the UKCCCR  regarding animal welfare were followed, and the experimental protocols were approved by the Institutional Committee for Ethics in Animal Research .n = 4); ageing control (S) (n = 4) and ageing Walker 256 tumour-bearing rats (W), which were euthanised in different days of tumour evolution: approximately with 14 days, W intermediated (Wi) (n = 10), and approximately with 21 days, W advanced (Wa) (n = 10). The tumour implant was performed by a single subcutaneous inoculation of 2 × 106 viable neoplastic cells from Walker 256 tumour in the right flank of W groups. All animals were monitored daily, weighed three times/week, and received food and water ad libitum. The tumour dimensions  were measured once a week using a slide calliper, to verify the properly tumour growth (data not shown). After each specific period of tumour evolution and after 21 days for both control groups (A and S), the rats were euthanised and the blood collected by cardiac puncture. Then, gastrocnemius muscle, liver and tumour tissue were weighed, and blood samples were centrifuged at 1000× g at 4 °C for 10 min, and serum was stored at −80 °C for metabolomic analyses.The animals were distributed into the following different experimental groups: adult control (A) (v/v) deuterium oxide , phosphate buffer  and 0.5 mM TMSP-d4 (3-(trimethylsilyl)-2,2’,3,3’-tetradeuteropropionic acid from Sigma-Aldrich) , then transferred to a 5-mm nuclear magnetic resonance (NMR) tube  for immediate acquisition.Serum samples were filtered through a Microcon YM-3 column  with a 3-kDa membrane centrifuge filter for serum recovery (at 4 °C). Filtered serum (0.2 mL) was diluted in an aqueous solution (0.6 mL) containing 10% (1H-NMR) spectra acquisition was performed using the Inova Agilent NMR spectrometer  operating at a frequency of 600 MHz, equipped with a triple resonance cold probe at a constant temperature of 298 K (25 °C). A total of 256 scans were collected with 32-k data points over a spectral width of 8000 Hz. An acquisition time of 4 s and 1.5 s relaxation delay incorporated between scans [The 1D proton nuclear magnetic resonance . Data were expressed as mean ± standard deviation (SD) [P < 0.05 was considered significant. The statistical analyses were performed using the software Graph Pad Prism 6.0 .The morphometric parameters were presented as absolute and relative values. The delta body weight was calculated as  for non-tumour-bearing groups and as —initial body weight) for tumour-bearing groups. The relative values were calculated dividing gastrocnemius muscle, liver and tumour weights by the respective initial body weight of each animal. The data analyses were performed by one-way ANOVA, followed by post-hoc Tukey’s honestly significant difference (HSD)  and by ion (SD)  and P < P < 0.05 was considered significant. The statistical analyses were performed using the software Graph Pad Prism 6.0 .The serum metabolic profile data analyses were performed by one-way ANOVA, followed by post-hoc Tukey’s HSD. Data were expressed as mean ± SD , and P <The heatmaps and VIP scores data were expressed as abundance ratio (high and low) of the corresponding metabolite. The statistical analyses were performed using the online MetaboAnalyst 4.0 platform , more specifically in the “Statistical Analysis” tool .versus A; Wi versus S; Wa versus S). Data were expressed as match status and regulation (upregulation or downregulation) and adjusted P < 0.01 by false discovery rate (FDR) was considered significant [The metabolic pathways were analysed with a list of metabolites—that were significantly different in the comparisons by one-way ANOVA, followed by post-hoc Tukey’s HSD—by over-representation analysis, using the hypergeometric tests (comparison to S nificant ,37. The nificant ,38.


Page 2: Lines 3 and 4 of Acknowledgments should read as follows. “This work was supported by a Howard Hughes Medical Institute Professor grant awarded to Mark Goldman (grant number 52008137).Volume 9, no. 8, e01540-19, 2020, We thank Jonathan Eisen, Katherine Dahlhausen, Mark Goldman, Ashley Vater, and David Coil for help with course design and planning.”


However, the transformation from the Itô process in the data frame to the corresponding Stratonovich process shifts the drift velocity of the transformed Lagrangian fluid trajectory out of the data frame into a non-inertial frame obtained from the Itô correction. The issue is, ‘Will non-inertial forces arising from this transformation of reference frames make a difference in the interpretation of the solution behaviour of the resulting stochastic equations?’ This issue will be resolved by elementary considerations.Suppose the observations of Lagrangian trajectories for fluid flow in some physical situation can be modelled sufficiently accurately by a spatially correlated Itô stochastic process (with zero mean) obtained from data which is taken in fixed Eulerian space. Suppose we also want to apply Hamilton’s principle to derive the stochastic fluid equations for this situation. Now, the variational calculus for applying Hamilton’s principle requires the Stratonovich process, so we must transform from Itô noise in the  The key element of fluid dynamics is the Kelvin circulation theorem, which is a statement of Newton’s force law for mass distributed on closed material loops t denotes explicit time dependence. That is, the material loops move with the Lagrangian transport velocity vector x at time t. For the fluid situation, Newton’s law states that the time rate of change of the circulation integral—around the material loop dx.ut(x) is the time-dependent momentum per unit mass measured in a fixed Eulerian frame. Since momentum per unit mass and velocity have the same dimensions and because momentum and force are defined in Newton’s force law to be measured in an inertial reference frame, one may refer to ut(x)  in  may be racceleration of velocity distributions ut is a nicety, except for their transformations under smooth maps. Therefore, in transforming to a non-inertial frame such as a rotating frame, the distinction becomes important even in relative to the reference frame moving at velocity R(x), while total momentum per unit mass, relative to the fixed frame after being parallel transported to the coordinate system x in the moving frame by using the connection 1-formWe stress that the Kelvin–Newton relation  is a starelative velocity in the moving reference frame with coordinates x fixed on the surface of the rotating Earth. The Coriolis parameter is curlR(x) = 2Ω(x) where Ω(x) is the angular velocity of the Earth in the moving frame, relative to the fixed stars. Newton’s force law for the rate of change of total momentum in . In total, more than 10 000 drifters have been deployed since 1979, representing nearly 30 million data points of positions along the Lagrangian paths of the drifters at six-hour intervals. An important feature of this data is that the ocean currents show up as spatial correlations, easily recognized visually by the concentrations of colours representing individual paths in also want to apply Hamilton’s principle to derive the stochastic fluid equations for this situation. Now, the variational calculus for applying Hamilton’s principle requires the chain rule and the product rule from vector calculus. The Stratonovich process respects these calculus rules, but Itô calculus is another matter. Thus, to use these rules of calculus to apply Hamilton’s principle, we should transform from Itô noise with zero mean in the data frame to the equivalent Stratonovich noise.Suppose we Problem statement. The question is, ‘Will the transformation transform from Itô noise in the data frame to the equivalent Stratonovich noise make a difference in the solution behaviour of the resulting stochastic equations?’Framework for resolving this issue. We know the transformation from the Itô process in the data frame to the corresponding Stratonovich process shifts the drift velocity of the transformed Lagrangian fluid trajectory out of the data frame into a non-inertial frame obtained from the Itô correction. We know the Itô correction explicitly, since the spatial correlations of the Itô noise have been obtained from the observed data. So, perhaps all is well, even though the spatial correlations depend upon location.data frame. (The data frame is the fixed Eulerian frame in which the Itô drift velocity was defined.) Now, such a shift of reference frame would introduce a non-inertial force into the motion equation for ut, whose derivation using variational calculus requires the Stratonovich representation of the noise. According to oceanographic experience, this non-inertial force can generate circulation of the Eulerian velocity [Thus, we have seen that the Itô correction shifts the Stratonovich drift velocity of the fluid into a spatially-dependent non-inertial frame relative to the velocity ,6. The q3 In this case, including the non-inertial force produces a ‘vortex force’ analogous to the Coriolis force. Upon inspection, we will recognize the derived equations as a version of the Craik–Leibovich (CL) equations [To demonstrate the resolution of this issue, we apply Hamilton’s principle to derive the equations of motion in the example of the stochastic Euler–Boussinesq equations (SEB) for the incompressible flow of a stratified fluid under gravity.quations ,6, alterOceanographic background. The ‘vortex force’ of the deterministic Craik–Leibovich (DCL) theory derived in [prescribed quantity denoted as rived in ,6 was inrived in –11. The ility in  and for ility in . In factrelative velocity, uL, generated simply because the equations for uL are not written in the inertial frame of the data.The three-dimensional results of having transformed the SEB fluid equations into a stochastic version of CL equations have yet to be investigated. However, it would not be surprising if the solutions of the Craik–Leibovich (SCL) equations were interpreted as possessing Langmuir circulations generated by the Itô correction to the stochastic drift velocity. Such an interpretation should be received with care, though, since they would represent circulations of the Objective of the paper. The present note investigates how to deal with non-inertial forces in stochastic dynamics which arise from Itô corrections as changes of frame when applying mixed Itô and Stratonovich stochastic modelling in 3D SEB fluid dynamics.data frame with ‘velocity’ Lagrangian fluid parcels with velocity F = ma only applies in an inertial frame. Undergraduate physics students will recognize this point as the analogue of the familiar Coriolis force felt in a rotating frame. They may also recall that the canonical momentum is not necessarily the mass times the velocity in a rotating frame, or in an external magnetic field. Although the reasoning in the remainder of the paper is elementary, we hope the explicit stochastic fluid dynamical calculations which demonstrate the resolution of this issue for 3D SEB fluid dynamics below may be illuminating.The resolution of this issue has already been given above in the comparison between equations  and 1.41.4. Name(a)This section describes the background for the Itô correction in stochastic fluid dynamics.t denotes explicit time dependence, i.e. not partial time derivative. In this notation, dBt denotes a Brownian motion in time, t, whose divergence-free vector amplitude ξ(xt) depends on the Eulerian spatial position xt = ϕtx0 with initial condition x0. The differential notation (d ) in equation (Multi-time homogenization for fluid dynamics in  was usedThe Stratonovich representation (denoted with symbol °) of the Itô trajectory in  is givenxt(x) in (xt in (4The quantity dxt(x) in  may be rn (xt in  represenuS(x) (without overline) is the Itô correction, while uS(x) in  . The Stou¯E . See [26ut is the Eulerian momentum per unit mass in Newton’s second law and uS(x) between these two quantities in  to vary with the seasons in geophysical applications, say, without interfering with the conclusions of the SCL model.In the present notation, ities in  with theThe effects of uncertainty in the statistics of the Stokes mean drift velocity eated in , as wellons, e.g.–31.ut(x) is the Eulerian velocity at a fixed spatial position The Kelvin circulation integral for the Eulerian representation of the Lagrangian trajectory in  is definor field  to find or field .Lie derivative with respect to the vector field In the Stratonovich representation of the transport velocity vector field dxtL in , we may dxtL in . For thiIn the next section, we will show how passing from the Itô representation of the Lagrangian trajectory in  to its e2.(a)δS = 0 with the following action integral b and the density Dd3x for stochastic advection equations with transport velocity b and Dd3x remain invariant along flow given by the stochastic Lagrangian trajectory in  derivative of functionals, e.g. L2 pairing. Finally, Roman In general, with the constraints in  Hamiltonformulas  and , since the quantity D is a stochastic quantity. As explained in [p) is a semimartingale.5 See remark 2.1 below for the semimartingale formula which determines the pressure. To finish the notation, g in the Lagrangian  one imposes preservation of divut = 0 on the divergence of the motion equation  in (uS(x) in  in  ensures uS(x) in  also hasuS(x) because uS(x) in equation (uS(x) comprise a stochastic version of the ‘vortex force’ in DCL and an added stochastic contribution to the pressure. This vortex force appears in the corresponding Kelvin theorem as a source of circulation of the velocity Equation  may be eequation  is time-n (WCI), –11. The ility in . The resut(x) is linear in the fluid transport velocity a = F/m version of Newton’s law which arises in this special case. Thus, the stochastic ‘vortex force’ in equation total vorticitytotal potential vorticity, defined by The curl of the SCL motion equation  yields t3.The central theorem for fluid dynamics (the Kelvin theorem) involves two frames in which velocities are measured. As we have seen, one velocity is a vector and the other is a co-vector. The integrand is in a fixed inertial frame and the circulation loop is in the moving frame of the Lagrangian fluid parcels. The frame of the specific momentum in the integrand is Eulerian and the frame of the moving loop is Lagrangian. Likewise the data observation frame and the fluid motion frame will differ, if one is modelled as Itô and the other as Stratonovich. Thus, it makes sense that the shifts between frames which occur in transforming a Lagrangian trajectory from Itô to Stratonovich form would introduce non-inertial forces in the motion equations. This was already clear from the Coriolis force and the CL vortex force in the deterministic modelling of fluid dynamics.F, plus the non-inertial force FCoriolis.Similarly, waves are Eulerian while fluid motion is Lagrangian: waves move relative to fixed space through the moving fluid, while the motion of the fluid Doppler shifts the wave frequency. In the CL model, the Eulerian velocity  is given by es as in  for the es as in  and (1.4(a)data frame. However, if one seeks the dynamics of the Lagrangian relative transport velocity, ut, then non-inertial forces will arise due to the Itô-Stokes correction, What does all this mean for the original problem of comparing Itô data with Stratonovich equations of motion derived from Hamilton’s principle for stochastic fluid equations in the Euler-Poincaré form ? It mean